ORCID Profile
0000-0002-3051-5676
Current Organisation
Garvan Institute of Medical Research
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Publisher: Cold Spring Harbor Laboratory
Date: 12-06-2020
DOI: 10.1101/2020.06.09.140921
Abstract: Resistance to endocrine therapy is a major clinical challenge in the management of estrogen receptor (ER)-positive breast cancer. In this setting p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment resistant ER-positive breast cancer. We used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate antitumor effects in p53 wildtype and p53 mutant ER positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and -resistant ER positive breast cancer. We demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant resistant patient derived xenograft model. We conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programs.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/1078-0432.22551388.V1
Abstract: Supplementary Figure S1. The majority of Pb-Cre PTENfl/fl Trp53fl/fl mice are de novo resistant to ADT. Supplementary Figure S2. ADT/PI3Ki combination therapy halts prostate tumor growth up to 14 days, followed by development of resistance in majority of Pb-Cre PTENfl/fl Trp53fl/fl mice. Supplementary Figure S3. PI3Ki treatment with concurrent androgen depletion does not alter proliferation and survival of PTEN 53-deficient murine PC cells in vitro. Supplementary Figure S4. ADT/PI3K inhibitor combination increases MHC-II and PD-1 expression on TAM within the TME of PTEN 53-deficient murine PC. Supplementary Figure S5. PD-1 upregulation suppresses phagocytic capacity of activated TAM. Supplementary Figure S6. Ex vivo AD + PI3Ki + PD-1 antibody treatment activates MHCIIlo TAM when co-cultured with PTEN 53-deficient murine prostate tumor cells. Supplementary Figure S7. The addition of PD-1 blockade to androgen depletion/PI3Ki therapy does not alter phagocytic capacity of PD-1 lo macrophages. Supplementary Figure S8. The combination of androgen depletion, PI3Ki and aPD-1 blockade does not alter phagocytic checkpoint expression on PTEN 53-deficient prostate tumor cells. Supplementary Figure S9. Androgen depletion, singly and in combination with aPD-1, did not alter phagocytosis activity of inactivated MHC-IIlo/PD-1 lo and MHC-IIlo/PD-1 hi TAM subsets. Supplementary Figure S10. Androgen depletion, not PI3Ki or aPD1, directly enhances TAM activation within the TME of PTEN 53-deficient PC. Supplementary Figure S11. PI3Ki does not alter phagocytosis/histone lactylation status of MHC-IIlo/PD-1 lo TAM and MHC-IIlo/PD-1 hi TAM. Supplementary Figure S12. PI3Ki inhibits lactate secretion from PTEN 53-deficient prostate tumor cells within TME. Supplementary Figure S13. Direct ex vivo treatment of TAM with PI3Ki, singly and in combination with PD-1 antibody and/or androgen depletion does not alter their histone lactylation profile. Supplementary Figure S14. ADT + PI3Ki + aPD-1 induces tumor control in 60% of Pb-Cre PTENfl/fl TP53fl/fl mice. Supplementary Figure S15. Depletion of activated TAM abrogates anti-cancer response elicited by ADT + PI3Ki + PD-1 antibody treatment in the PTEN 53-deficient murine prostate GEMM tumors. Supplementary Figure S16. Long-term treatment of ADT + PI3Ki + aPD-1 activates Wnt/βcatenin pathway in murine PTEN 53-deficient GEMM-derived SC1 cells. Supplementary Figure S17. Feedback Wnt/β-catenin-pathway activation within murine PTEN 53-deficient GEMM-derived PC cells following long-term ADT + copanlisib + aPD1 treatment suppresses phagocytosis via increased histone lactylation within bone marrow derived macrophages (BMDM).
Publisher: Springer Science and Business Media LLC
Date: 16-09-2023
Publisher: Informa UK Limited
Date: 08-2013
DOI: 10.4161/ONCI.25409
Publisher: Elsevier BV
Date: 08-2014
DOI: 10.1038/MT.2014.83
Publisher: American Association for Cancer Research (AACR)
Date: 14-03-2013
DOI: 10.1158/0008-5472.CAN-12-1472
Abstract: Basal breast cancer cells feature high expression of the Src family kinase Lyn that has been implicated in the pathogenicity of this disease. In this study, we identified novel Lyn kinase substrates, the most prominent of which was the atypical kinase SgK269 (PEAK1). In breast cancer cells, SgK269 expression associated with the basal phenotype. In primary breast tumors, SgK269 overexpression was detected in a subset of basal, HER2-positive, and luminal cancers. In immortalized MCF-10A mammary epithelial cells, SgK269 promoted transition to a mesenchymal phenotype and increased cell motility and invasion. Growth of MCF-10A acini in three-dimensional (3D) culture was enhanced upon SgK269 overexpression, which induced an abnormal, multilobular acinar morphology and promoted extracellular signal–regulated kinase (Erk) and Stat3 activation. SgK269 Y635F, mutated at a major Lyn phosphorylation site, did not enhance acinar size or cellular invasion. We show that Y635 represents a Grb2-binding site that promotes both Stat3 and Erk activation in 3D culture. RNA interference–mediated attenuation of SgK269 in basal breast cancer cells promoted acquisition of epithelial characteristics and decreased anchorage-independent growth. Together, our results define a novel signaling pathway in basal breast cancer involving Lyn and SgK269 that offers clinical opportunities for therapeutic intervention. Cancer Res 73(6) 1969–80. ©2012 AACR.
Publisher: Public Library of Science (PLoS)
Date: 30-12-2015
Publisher: Springer Science and Business Media LLC
Date: 13-06-2018
DOI: 10.1038/S41467-018-04619-5
Abstract: Understanding the dynamics of endogenous protein–protein interactions in complex networks is pivotal in deciphering disease mechanisms. To enable the in-depth analysis of protein interactions in chromatin-associated protein complexes, we have previously developed a method termed RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins). Here, we present a quantitative multiplexed method (qPLEX-RIME), which integrates RIME with isobaric labelling and tribrid mass spectrometry for the study of protein interactome dynamics in a quantitative fashion with increased sensitivity. Using the qPLEX-RIME method, we delineate the temporal changes of the Estrogen Receptor alpha (ERα) interactome in breast cancer cells treated with 4-hydroxytamoxifen. Furthermore, we identify endogenous ERα-associated proteins in human Patient-Derived Xenograft tumours and in primary human breast cancer clinical tissue. Our results demonstrate that the combination of RIME with isobaric labelling offers a powerful tool for the in-depth and quantitative characterisation of protein interactome dynamics, which is applicable to clinical s les.
Publisher: Springer Science and Business Media LLC
Date: 07-11-2022
DOI: 10.1038/S41467-022-34041-X
Abstract: Cancers evade the immune system through the process of cancer immunoediting. While immune checkpoint inhibitors are effective for reactivating tumour immunity in some cancer types, many other solid cancers, including breast cancer, remain largely non-responsive. Understanding how non-responsive cancers evade immunity and whether this occurs at the clonal level will improve immunotherapeutic design. Here we use DNA barcoding to track murine mammary cancer cell clones during immunoediting and determine clonal transcriptional profiles that allow immune evasion following anti-PD1 plus anti-CTLA4 immunotherapy. Clonal ersity is significantly restricted by immunotherapy treatment in both primary tumours and metastases, demonstrating selection for pre-existing breast cancer cell populations and ongoing immunoediting during metastasis and treatment. Immunotherapy resistant clones express a common gene signature associated with poor survival of basal-like breast cancer patient cohorts. At least one of these genes has an existing small molecule that can potentially be used to improve immunotherapy response.
Publisher: Cold Spring Harbor Laboratory
Date: 11-01-2021
DOI: 10.1101/2021.01.11.426174
Abstract: Cancers evade the immune system in order to grow or metastasise through the process of cancer immunoediting. While immune checkpoint inhibitors have been effective for reactivating tumour immunity in some types of cancer, many other solid cancers, including breast cancer, remain largely non-responsive. Understanding the way non-responsive cancers evolve to evade immunity, what resistance pathways are activated and whether this occurs at the clonal level will improve immunotherapeutic design. We tracked cancer cell clones during the immunoediting process and determined clonal transcriptional profiles that allow immune evasion in murine mammary tumour growth in response to immunotherapy with anti-PD1 and anti-CTLA4. Clonal ersity was significantly restricted by immunotherapy treatment in both primary tumours and metastases. These findings demonstrate that immunoediting selects for pre-existing breast cancer cell populations and that immunoediting is not static, it is ongoing during metastasis and immunotherapy treatment. Isolation of immunotherapy resistant clones revealed unique and overlapping transcriptional signatures. The overlapping gene signature was associated with poor survival of basal-like breast cancer patients in two cohorts. At least one of these overlapping genes has an existing small molecule that can potentially be used to improve immunotherapy response.
Publisher: Wiley
Date: 13-01-2016
DOI: 10.1111/HIS.12904
Abstract: Triple-negative breast cancer (TNBC) patients generally have a poor outcome there is a pressing need to identify more effective therapeutic strategies. Clinical trials targeting programmed death 1 rogrammed death ligand 1 (PD1/PDL1) in melanoma and non-small-cell lung cancer have reported high response rates, and tumoral PDL1 expression has been suggested as a potential biomarker to enrich for patient response to these treatments. There are only very limited data to date reporting the expression of PDL1 in TNBC. PDL1 immunohistochemistry was performed on 161 primary TNBCs and assessed in the tumour as well as immune cells in the stromal compartment. PDL1 expression was very common in TNBC, expressed in the tumour cell membrane (64%), cytoplasm (80%) and stromal (93%) cellular compartments. Cytoplasmic tumoral expression of PDL1 was associated with a lower risk of breast cancer-specific death [hazard ratio (HR) 0.45, P = 0.035] while stromal PDL1 expression was associated with a lower rate of deaths from all causes (HR 0.305, P = 0.0042). Membranous expression of PDL1 was not associated with outcome. While both PDL1 expression and tumour-infiltrating lymphocytes were associated with a better outcome, only lymphovascular invasion and high tumour-infiltrating lymphocytes were independently prognostic for breast cancer-specific death. While PDL1 expression is frequent in TNBC, it was not independently prognostic. There were differences in outcome depending on the cellular compartment of PDL1 expression. These data provide further impetus for investigating the utility of immune checkpoint therapies in TNBC, given the clinical significance of tumour-infiltrating lymphocytes (TILs) and PDL1 expression in this cohort.
Publisher: Springer Science and Business Media LLC
Date: 23-09-2013
DOI: 10.1038/ONC.2013.368
Abstract: The HER2 (ERBB2) and MYC genes are commonly lified in breast cancer, yet little is known about their molecular and clinical interaction. Using a novel chimeric mammary transgenic approach and in vitro models, we demonstrate markedly increased self-renewal and tumour-propagating capability of cells transformed with Her2 and c-Myc. Coexpression of both oncoproteins in cultured cells led to the activation of a c-Myc transcriptional signature and acquisition of a self-renewing phenotype independent of an epithelial-mesenchymal transition programme or regulation of conventional cancer stem cell markers. Instead, Her2 and c-Myc cooperated to induce the expression of lipoprotein lipase, which was required for proliferation and self-renewal in vitro. HER2 and MYC were frequently co lified in breast cancer, associated with aggressive clinical behaviour and poor outcome. Lastly, we show that in HER2(+) breast cancer patients receiving adjuvant chemotherapy (but not targeted anti-Her2 therapy), MYC lification is associated with a poor outcome. These findings demonstrate the importance of molecular and cellular context in oncogenic transformation and acquisition of a malignant stem-like phenotype and have diagnostic and therapeutic consequences for the clinical management of HER2(+) breast cancer.
Publisher: MDPI AG
Date: 04-09-2021
Abstract: Breast cancer is a heterogenous disease with variability in tumor cells and in the surrounding tumor microenvironment (TME). Understanding the molecular ersity in breast cancer is critical for improving prediction of therapeutic response and prognostication. High-plex spatial profiling of tumors enables characterization of heterogeneity in the breast TME, which can holistically illuminate the biology of tumor growth, dissemination and, ultimately, response to therapy. The GeoMx Digital Spatial Profiler (DSP) enables researchers to spatially resolve and quantify proteins and RNA transcripts from tissue sections. The platform is compatible with both formalin-fixed paraffin-embedded and frozen tissues. RNA profiling was developed at the whole transcriptome level for human and mouse s les and protein profiling of 100-plex for human s les. Tissue can be optically segmented for analysis of regions of interest or cell populations to study biology-directed tissue characterization. The GeoMx Breast Cancer Consortium (GBCC) is composed of breast cancer researchers who are developing innovative approaches for spatial profiling to accelerate biomarker discovery. Here, the GBCC presents best practices for GeoMx profiling to promote the collection of high-quality data, optimization of data analysis and integration of datasets to advance collaboration and meta-analyses. Although the capabilities of the platform are presented in the context of breast cancer research, they can be generalized to a variety of other tumor types that are characterized by high heterogeneity.
Publisher: Frontiers Media SA
Date: 17-07-2020
Publisher: American Society of Clinical Oncology (ASCO)
Date: 2017
Abstract: Clinical guidelines recommend that people at high risk of melanoma receive regular surveillance to improve survival through early detection. A specialized High Risk Clinic in Sydney, Australia was found to be effective for this purpose however, wider implementation of this clinical service requires evidence of cost-effectiveness and data addressing potential overtreatment of suspicious skin lesions. A decision-analytic model was built to compare the costs and benefits of specialized surveillance compared with standard care over a 10-year period, from a health system perspective. A high-risk standard care cohort was obtained using linked population data, comprising the Sax Institute’s 45 and Up cohort study, linked to Medicare Benefits Schedule claims data, the cancer registry, and hospital admissions data. Benefits were measured in quality-adjusted life-years gained. Sensitivity analyses were undertaken for all model parameters. Specialized surveillance through the High Risk Clinic was both less expensive and more effective than standard care. The mean saving was A$6,828 (95% CI, $5,564 to $8,092) per patient, and the mean quality-adjusted life-year gain was 0.31 (95% CI, 0.27 to 0.35). The main drivers of the differences were detection of melanoma at an earlier stage resulting in less extensive treatment and a lower annual mean excision rate for suspicious lesions in specialized surveillance (0.81 95% CI, 0.72 to 0.91) compared with standard care (2.55 95% CI, 2.34 to 2.76). The results were robust when tested in sensitivity analyses. Specialized surveillance was a cost-effective strategy for the management of in iduals at high risk of melanoma. There were also fewer invasive procedures in specialized surveillance compared with standard care in the community.
Publisher: Springer Science and Business Media LLC
Date: 08-04-2014
DOI: 10.1038/BJC.2014.181
Publisher: American Association for Cancer Research (AACR)
Date: 14-09-2014
DOI: 10.1158/0008-5472.CAN-14-0053
Abstract: Regulators of differentiated cell fate can offer targets for managing cancer development and progression. Here, we identify Runx2 as a new regulator of epithelial cell fate in mammary gland development and breast cancer. Runx2 is expressed in the epithelium of pregnant mice in a strict temporally and hormonally regulated manner. During pregnancy, Runx2 genetic deletion impaired alveolar differentiation in a manner that disrupted alveolar progenitor cell populations. Conversely, exogenous transgenic expression of Runx2 in mammary epithelial cells blocked milk production, suggesting that the decrease in endogenous Runx2 observed late in pregnancy is necessary for full differentiation. In addition, overexpression of Runx2 drove epithelial-to-mesenchymal transition–like changes in normal mammary epithelial cells, whereas Runx2 deletion in basal breast cancer cells inhibited cellular phenotypes associated with tumorigenesis. Notably, loss of Runx2 expression increased tumor latency and enhanced overall survival in a mouse model of breast cancer, with Runx2-deficient tumors exhibiting reduced cell proliferation. Together, our results establish a previously unreported function for Runx2 in breast cancer that may offer a novel generalized route for therapeutic interventions. Cancer Res 74(18) 5277–86. ©2014 AACR.
Publisher: Springer Science and Business Media LLC
Date: 09-09-2014
Publisher: Wiley
Date: 25-05-2021
DOI: 10.1111/IMR.12976
Abstract: Mesenchymal stromal cells in solid tumors have emerged as important mediators of immune function and response to immunotherapies. As such, comprehensive insights into their biology may reveal new predictors of drug response and new drug targets. While our understanding of mesenchymal biology in cancer is nascent, it is rapidly evolving, driven by advances in single‐cell technologies. These studies reveal distinct subclasses of cancer‐associated fibroblasts (CAFs) with unique properties for immune regulation and control of leukocyte activity. While these studies have revealed several similarities across distinct types of cancer, they still face key challenges in nomenclature. Single‐cell analysis of tumors has also revealed an abundance of perivascular cells with unique biology and associations with immune infiltration. They are often misclassified, likely confounding previous bulk studies, revealing a distinct lineage of cells that remain to be fully characterized. These studies have also shed light on the discrete cell types or transient cell states that shape mesenchymal heterogeneity in tumors, offering insights into new therapeutic strategies to modulate stromal cell differentiation. In this review, we will address how recent advances in single‐cell technologies have shaped our understanding of stromal heterogeneity and their coordination of immune responses in cancer.
Publisher: Cold Spring Harbor Laboratory
Date: 24-09-2018
DOI: 10.1101/424945
Abstract: High-throughput single-cell RNA-Sequencing is a powerful technique for gene expression profiling of complex and heterogeneous cellular populations such as the immune system. However, these methods only provide short-read sequence from one end of a cDNA template, making them poorly suited to the investigation of gene-regulatory events such as mRNA splicing, adaptive immune responses or somatic genome evolution. To address this challenge, we have developed a method that combines targeted long-read sequencing with short-read based transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning. We use Repertoire And Gene Expression sequencing (RAGE-seq) to accurately characterize full-length T cell (TCR) and B cell (BCR) receptor sequences and transcriptional profiles of more than 7,138 lymphocytes s led from the primary tumour and draining lymph node of a breast cancer patient. With this method we show that somatic mutation, alternate splicing and clonal evolution of T and B lymphocytes can be tracked across these tissue compartments. Our results demonstrate that RAGE-Seq is an accessible and cost-effective method for high-throughput deep single cell profiling, applicable to a wide range of biological challenges.
Publisher: Cold Spring Harbor Laboratory
Date: 24-08-2022
DOI: 10.1101/2022.08.23.505054
Abstract: FFPE (formalin-fixed, paraffin-embedded) tissue archives are the largest repository of clinically annotated human specimens. Despite numerous advances in technology, current methods for sequencing of FFPE-fixed single-cells are slow, labour intensive, insufficiently sensitive and have a low resolution, making it difficult to fully exploit their enormous research and clinical potential. Here we introduce single nuclei pathology sequencing (snPATHO-Seq), a sensitive and efficient high-throughput platform to profile the transcriptome of single nuclei extracted from formalin-fixed paraffin-embedded (FFPE) s les. snPATHO-Seq combines an optimised nuclei extraction protocol from archival s les with 10x Genomics probe-based technology targeting the whole transcriptome. We performed direct comparison of the Fixed RNA Profiling (FRP) and established 3’ single cell RNA-Sequencing (scRNA-Seq) workflows through a comprehensive bioinformatics analysis of matched fresh and fixed s les derived from the LNCaP prostate cancer cell line. FRP detected 2.1 times more transcripts in the fixed s le than the 3’ kit did in the fresh s le. Low mitochondrial genes detection using the FRP was translated into 99.9 percent of cells passing the QC filters, compared to 81.6 percent of cells using the v3.1 chemistry. We then optimized snPATHO-Seq and applied it to a human breast cancer metastasis to the liver collected at autopsy and preserved in FFPE, a particularly challenging s le type. Remarkably, at 28,000 reads/cell snPATHO-Seq was able to detect a median of 1850 genes/cell and 3,216 UMI counts/cell. Comparison of snPATHO-Seq with spatial transcriptomics data (10x Genomics Visium FFPE v1) derived from an adjacent section of the same s le revealed a strong correlation, validating the accuracy of the snPATHO-Seq data. Gene expression data from snPATHO-Seq was used to predict cell type composition within each spatial transcriptomic location via deconvolution. Overall, snPATHO-Seq enables high quality and sensitivity snRNA-Seq from preserved tissue s les, unlocking the vast archives of FFPE tissues and thereby allowing extensive retrospective clinical genomic studies.
Publisher: Public Library of Science (PLoS)
Date: 02-01-2020
Publisher: BMJ
Date: 22-02-2013
DOI: 10.1136/JCLINPATH-2012-201361
Abstract: Outcomes have improved significantly for many women diagnosed with breast cancer. For the heterogeneous group of tumours lacking expression of the oestrogen, progesterone and HER2 receptors, 'triple negative' breast cancers (TNBC), the prognosis overall has remained quite poor. When TNBC recurs, there is often little response to chemotherapy, and there are a few treatment options in this setting. Thus, there is an urgent clinical need to identify new therapeutic targets in order to improve the outlook for these patients. This review highlights the most promising therapeutic targets identified through new sequencing technologies, as well as through studies of apoptosis. We also present mounting evidence that the developmental signalling pathways Wnt/β-catenin, NOTCH and Hedgehog play an important role in the pathogenesis and progression of TNBC with new therapeutic approaches inhibiting these pathways in advanced preclinical studies or early clinical trials.
Publisher: EMBO
Date: 21-04-2020
Publisher: Springer Science and Business Media LLC
Date: 24-09-2022
DOI: 10.1186/S13058-022-01540-0
Abstract: Breast cancer cell lines (BCCLs) and patient-derived xenografts (PDXs) are the most frequently used models in breast cancer research. Despite their widespread usage, genome sequencing of these models is incomplete, with previous studies only focusing on targeted gene panels, whole exome or shallow whole genome sequencing. Deep whole genome sequencing is the most sensitive and accurate method to detect single nucleotide variants and indels, gene copy number and structural events such as gene fusions. Here we describe deep whole genome sequencing (WGS) of commonly used BCCL and PDX models using the Illumina X10 platform with an average ~ 60 × coverage. We identify novel genomic alterations, including point mutations and genomic rearrangements at base-pair resolution, compared to previously available sequencing data. Through integrative analysis with publicly available functional screening data, we annotate new genomic features likely to be of biological significance. CSMD1 , previously identified as a tumor suppressor gene in various cancer types, including head and neck, lung and breast cancers, has been identified with deletion in 50% of our PDX models, suggesting an important role in aggressive breast cancers. Our WGS data provides a comprehensive genome sequencing resource of these models.
Publisher: EMBO
Date: 30-10-2023
Publisher: Society of Nuclear Medicine
Date: 05-04-2013
DOI: 10.2967/JNUMED.112.111534
Abstract: Triple-negative breast cancer (TNBC) is associated with poor survival. Chemotherapy is the only standard treatment for TNBC. The prevalence of BRCA1 inactivation in TNBC has rationalized clinical trials of poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors. Similarly, the overexpression of epidermal growth factor receptor (EGFR) rationalized anti-EGFR therapies in this disease. However, clinical trials using these 2 strategies have not reached their promise. In this study, we used EGFR as a target for radioimmunotherapy and hypothesized that EGFR-directed radioimmunotherapy can deliver a continuous lethal radiation dose to residual tumors that are radiosensitized by PARP inhibitors and chemotherapy. We analyzed EGFR messenger RNA in published gene expression array studies and investigated EGFR protein expression by immunohistochemistry in a cohort of breast cancer patients to confirm EGFR as a target in TNBC. Preclinically, using orthotopic and metastatic xenograft models of EGFR-positive TNBC, we investigated the effect of the novel combination of (177)Lu-labeled anti-EGFR monoclonal antibody, chemotherapy, and PARP inhibitors on cell death and the survival of breast cancer stem cells. In this first preclinical study of anti-EGFR radioimmunotherapy in breast cancer, we found that anti-EGFR radioimmunotherapy is safe and that TNBC orthotopic tumors and established metastases were eradicated in mice treated with anti-EGFR radioimmunotherapy combined with chemotherapy and PARP inhibitors. We showed that the superior response to this triple-agent combination therapy was associated with apoptosis and eradication of putative breast cancer stem cells. Our data support further preclinical investigations toward the development of combination therapies using systemic anti-EGFR radioimmunotherapy for the treatment of recurrent and metastatic TNBC.
Publisher: Informa UK Limited
Date: 04-2000
Publisher: Elsevier BV
Date: 10-2001
Publisher: Springer Science and Business Media LLC
Date: 16-07-2013
DOI: 10.1038/ONC.2012.303
Abstract: Protein kinase Cα (PKCα) can phosphorylate the epidermal growth factor receptor (EGFR) at threonine 654 (T654) to inhibit EGFR tyrosine phosphorylation (pY-EGFR) and the associated activation of downstream effectors. However, upregulation of PKCα in a large variety of cancers is not associated with EGFR inactivation, and factors determining the potential of PKCα to downregulate EGFR are yet unknown. Here, we show that ectopic expression of annexin A6 (AnxA6), a member of the Ca(2+) and phospholipid-binding annexins, strongly reduces pY-EGFR levels while augmenting EGFR T654 phosphorylation in EGFR overexpressing A431, head and neck and breast cancer cell lines. Reduced EGFR activation in AnxA6 expressing A431 cells is associated with reduced EGFR internalization and degradation. RNA interference (RNAi)-mediated PKCα knockdown in AnxA6 expressing A431 cells reduces T654-EGFR phosphorylation, but restores EGFR tyrosine phosphorylation, clonogenic growth and EGFR degradation. These findings correlate with AnxA6 interacting with EGFR, and elevated AnxA6 levels promoting PKCα membrane association and interaction with EGFR. Stable expression of the cytosolic N-terminal mutant AnxA6(1-175), which cannot promote PKCα membrane recruitment, does not increase T654-EGFR phosphorylation or the association of PKCα with EGFR. AnxA6 overexpression does not inhibit tyrosine phosphorylation of the T654A EGFR mutant, which cannot be phosphorylated by PKCα. Most strikingly, stable plasma membrane anchoring of AnxA6 is sufficient to recruit PKCα even in the absence of EGF or Ca(2+). In summary, AnxA6 is a new PKCα scaffold to promote PKCα-mediated EGFR inactivation through increased membrane targeting of PKCα and EGFR/PKCα complex formation.
Publisher: Oxford University Press (OUP)
Date: 06-2014
DOI: 10.1093/JNCI/DJU113
Abstract: Patients with neuroblastoma due to the lification of a 130-kb genomic DNA region containing the MYCN oncogene have poor prognoses. Bioinformatics data were used to discover a novel long noncoding RNA, lncUSMycN, at the 130-kb licon. RNA-protein pull-down assays were used to identify proteins bound to lncUSMycN RNA. Kaplan-Meier survival analysis, multivariable Cox regression, and two-sided log-rank test were used to examine the prognostic value of lncUSMycN and NonO expression in three cohorts of neuroblastoma patients (n = 47, 88, and 476, respectively). Neuroblastoma-bearing mice were treated with antisense oligonucleotides targeting lncUSMycN (n = 12) or mismatch sequence (n = 13), and results were analyzed by multiple comparison two-way analysis of variance. All statistical tests were two-sided. Bioinformatics data predicted lncUSMycN gene and RNA, and reverse-transcription polymerase chain reaction confirmed its three exons and two introns. The lncUSMycN gene was co lified with MYCN in 88 of 341 human neuroblastoma tissues. lncUSMycN RNA bound to the RNA-binding protein NonO, leading to N-Myc RNA upregulation and neuroblastoma cell proliferation. High levels of lncUSMycN and NonO expression in human neuroblastoma tissues independently predicted poor patient prognoses (lncUSMycN: hazard ratio [HR] = 1.87, 95% confidence interval [CI] = 1.06 to 3.28, P = .03 NonO: HR = 2.48, 95% CI = 1.34 to 4.57, P = .004). Treatment with antisense oligonucleotides targeting lncUSMycN in neuroblastoma-bearing mice statistically significantly hindered tumor progression (P < .001). Our data demonstrate the important roles of lncUSMycN and NonO in regulating N-Myc expression and neuroblastoma oncogenesis and provide the first evidence that lification of long noncoding RNA genes can contribute to tumorigenesis.
Publisher: American Association for Cancer Research (AACR)
Date: 14-11-2010
DOI: 10.1158/0008-5472.CAN-10-0911
Abstract: To identify therapeutic targets and prognostic markers for basal breast cancers, breast cancer cell lines were subjected to mass spectrometry–based profiling of protein tyrosine phosphorylation events. This revealed that luminal and basal breast cancer cells exhibit distinct tyrosine phosphorylation signatures that depend on pathway activation as well as protein expression. Basal breast cancer cells are characterized by elevated tyrosine phosphorylation of Met, Lyn, EphA2, epidermal growth factor receptor (EGFR), and FAK, and Src family kinase (SFK) substrates such as p130Cas. SFKs exert a prominent role in these cells, phosphorylating key regulators of adhesion and migration and promoting tyrosine phosphorylation of the receptor tyrosine kinases EGFR and Met. Consistent with these observations, SFK inhibition attenuated cellular proliferation, survival, and motility. Basal breast cancer cell lines exhibited differential responsiveness to small molecule inhibitors of EGFR and Met that correlated with the degree of target phosphorylation, and reflecting kinase coactivation, inhibiting two types of activated network kinase (e.g., EGFR and SFKs) was more effective than single agent approaches. FAK signaling enhanced both proliferation and invasion, and Lyn was identified as a proinvasive component of the network that is associated with a basal phenotype and poor prognosis in patients with breast cancer. These studies highlight multiple kinases and substrates for further evaluation as therapeutic targets and biomarkers. However, they also indicate that patient stratification based on expression/activation of drug targets, coupled with use of multi-kinase inhibitors or combination therapies, may be required for effective treatment of this breast cancer subgroup. Cancer Res 70(22) 9391–401. ©2010 AACR.
Publisher: Research Square Platform LLC
Date: 09-2020
DOI: 10.21203/RS.3.RS-62718/V1
Abstract: Antagonistic sex hormone activity occurs in mammary gland development, whereby estrogen stimulates and androgen inhibits post-pubertal growth, but the mechanistic basis of this is largely unknown. Whether sex hormone antagonism occurs in the context of breast cancer is also unclear. The estrogen receptor alpha (ER) unequivocally drives the majority of breast malignancies, but the role of the androgen receptor (AR) is controversial, particularly in the context of ER-positive (ER+) tumours resistant to standard-of-care ER targeting therapies. The controversy has constrained clinical implementation of new drugs that influence AR activity for treatment of this disease. Using a erse panel of cell line and patient-derived models of ER+ breast cancer, we demonstrate that activation, not suppression, of AR activity exerts potent anti-tumour activity in multiple clinically relevant contexts, including tumours resistant to ER targeting therapy. We also show that AR agonists can be combined with old and new (i.e. Palbociclib, a CDK4/6 inhibitor) standard-of-care agents to enhance therapeutic efficacy. Mechanistically, agonist activation of AR altered the distribution of ER and its coactivators (p300, SRC-3) on chromatin, resulting in repression of ER-regulated cell cycle genes and up-regulation of AR target genes, including known tumour suppressors. Consistent with the mechanistic findings, a gene signature of AR activity derived from in vivo models positively predicted disease survival in multiple large, well-annotated clinical cohorts of ER+ breast cancer, outperforming existing pan-cancer or breast cancer specific signatures. These findings provide compelling evidence that AR has a tumour suppressor role in ER+ breast cancer and resolves an important clinical controversy concerning the optimal AR-directed treatment strategy, revealing a rational therapeutic opportunity.
Publisher: Elsevier BV
Date: 09-2022
DOI: 10.1016/J.CCELL.2022.08.021
Abstract: Spatial transcriptomics, with other spatial technologies, has enabled scientists to dissect the organization and interaction of different cell types within the tumor microenvironment. We asked experts to discuss some aspects of this technology from revealing the tumor microenvironment and heterogeneity, to tracking tumor evolution, to guiding tumor therapy, to current technical challenges.
Publisher: Cold Spring Harbor Laboratory
Date: 06-06-2020
DOI: 10.1101/2020.06.04.135277
Abstract: High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity amongst complex human cancers. scRNA-Seq studies using fresh human surgical tissue is logistically difficult, precludes histopathological triage of s les and limits the ability to perform batch processing. This hinderance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers, and on high throughput scRNA-Seq platforms. We show that the viable cryopreservation of human cancers provides high quality single-cell transcriptomes using the Chromium 10X platform. We sequenced a total of ∼120,000 cells from fresh and cryopreserved replicates across three breast cancers, two prostate cancers and a cutaneous melanoma. Importantly, tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We then show that cryopreservation had minimal impacts on results of downstream analyses such as biological pathway enrichment. Further, we demonstrate the advantage of cryopreserving whole-cells for immunophenotyping methods such as CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.
Publisher: Springer Science and Business Media LLC
Date: 04-06-1998
Abstract: The G1 cyclins, cyclin D1 and E, are rate limiting for progression through G1 phase of the cell cycle in breast epithelial cells and are oncogenic when expressed in the mammary epithelium of transgenic mice. These genes are frequently overexpressed in clinical breast cancer where overexpression appears to be associated with specific disease phenotypes, altered responsiveness to therapeutic intervention and patient survival. In order to investigate the functional correlates of cyclin D1 and cyclin E overexpression we employed a panel of normal, immortalized and neoplastic breast epithelial cell lines to examine the relationships between cyclin gene expression, cyclin-CDK complex formation and CDK activity. In agreement with earlier studies cyclin D1 and E expression varied over an approximately tenfold range among the 18 cell lines studied. There was no apparent relationship, however, between cyclin D1 expression and the in vitro activity of its major kinase partner, Cdk4, although MDA-MB-134 cells displayed the highest level of both cyclin D1 expression and Cdk4 activity. Similarly, there was no significant relationship between cyclin E expression and cyclin E-Cdk2 activity. Fractionation of whole cell lysates by gel filtration chromatography revealed that approximately 90% of the cyclin E protein was present in inactive complexes containing the CDK inhibitors p21 and p27. Much of the small fraction of active cyclin E protein was of very high apparent molecular mass, >400 kDa, suggesting that formation of these complexes is a more important determinant of cyclin E-Cdk2 activity than cyclin E abundance. These data suggest that properties of cyclins D1 and E in addition to their ability to activate Cdk4 and Cdk2 may contribute to the effects of overexpression on the breast cancer phenotype.
Publisher: Springer Science and Business Media LLC
Date: 13-06-2015
Publisher: Springer Science and Business Media LLC
Date: 08-2022
Publisher: Springer Science and Business Media LLC
Date: 28-03-2013
DOI: 10.1186/BCR3401
Publisher: Public Library of Science (PLoS)
Date: 03-08-2010
Publisher: Bioscientifica
Date: 02-2019
DOI: 10.1530/ERC-18-0333
Abstract: The role of androgen receptor (AR) in endocrine-resistant breast cancer is controversial and clinical trials targeting AR with an AR antagonist (e.g., enzalutamide) have been initiated. Here, we investigated the consequence of AR antagonism using in vitro and in vivo models of endocrine resistance. AR antagonism in MCF7-derived tamoxifen-resistant (TamR) and long-term estrogen-deprived breast cancer cell lines were achieved using siRNA-mediated knockdown or pharmacological inhibition with enzalutamide. The efficacy of enzalutamide was further assessed in vivo in an estrogen-independent endocrine-resistant patient-derived xenograft (PDX) model. Knockdown of AR inhibited the growth of the endocrine-resistant cell line models. Microarray gene expression profiling of the TamR cells following AR knockdown revealed perturbations in proliferative signaling pathways upregulated in endocrine resistance. AR loss also increased some canonical ER signaling events and restored sensitivity of TamR cells to tamoxifen. In contrast, enzalutamide did not recapitulate the effect of AR knockdown in vitro , even though it inhibited canonical AR signaling, which suggests that it is the non-canonical AR activity that facilitated endocrine resistance. Enzalutamide had demonstrable efficacy in inhibiting AR activity in vivo but did not affect the growth of the endocrine-resistant PDX model. Our findings implicate non-canonical AR activity in facilitating an endocrine-resistant phenotype in breast cancer. Unlike canonical AR signaling which is inhibited by enzalutamide, non-canonical AR activity is not effectively antagonized by enzalutamide, and this has important implications in the design of future AR-targeted clinical trials in endocrine-resistant breast cancer.
Publisher: MDPI AG
Date: 08-09-2020
DOI: 10.3390/BIOM10091295
Abstract: The basic helix-loop-helix (bHLH) transcription factors inhibitor of differentiation 1 (Id1) and inhibitor of differentiation 3 (Id3) (referred to as Id) have an important role in maintaining the cancer stem cell (CSC) phenotype in the triple-negative breast cancer (TNBC) subtype. In this study, we aimed to understand the molecular mechanism underlying Id control of CSC phenotype and exploit it for therapeutic purposes. We used two different TNBC tumor models marked by either Id depletion or Id1 expression in order to identify Id targets using a combinatorial analysis of RNA sequencing and microarray data. Phenotypically, Id protein depletion leads to cell cycle arrest in the G0/G1 phase, which we demonstrate is reversible. In order to understand the molecular underpinning of Id proteins on the cell cycle phenotype, we carried out a large-scale small interfering RNA (siRNA) screen of 61 putative targets identified by using genomic analysis of two Id TNBC tumor models. Kinesin Family Member 11 (Kif11) and Aurora Kinase A (Aurka), which are critical cell cycle regulators, were further validated as Id targets. Interestingly, unlike in Id depletion conditions, Kif11 and Aurka knockdown leads to a G2/M arrest, suggesting a novel Id cell cycle mechanism, which we will explore in further studies. Therapeutic targeting of Kif11 to block the Id1–Kif11 axis was carried out using small molecular inhibitor ispinesib. We finally leveraged our findings to target the Id/Kif11 pathway using the small molecule inhibitor ispinesib in the Id+ CSC results combined with chemotherapy for better response in TNBC subtypes. This work opens up exciting new possibilities of targeting Id targets such as Kif11 in the TNBC subtype, which is currently refractory to chemotherapy. Targeting the Id1–Kif11 molecular pathway in the Id1+ CSCs in combination with chemotherapy and small molecular inhibitor results in more effective debulking of TNBC.
Publisher: CSIRO Publishing
Date: 2003
DOI: 10.1071/SRB03AB97
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/1078-0432.22551385.V1
Abstract: Supplementary Table S1. ADT + PI3Ki + PD-1 antibody leads to TAM activation within TME of PTEN 53-deficient prostate tumors.
Publisher: Public Library of Science (PLoS)
Date: 12-03-2013
Publisher: Springer Science and Business Media LLC
Date: 26-09-2010
DOI: 10.1038/NM.2227
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/1078-0432.22551388
Abstract: Supplementary Figure S1. The majority of Pb-Cre PTENfl/fl Trp53fl/fl mice are de novo resistant to ADT. Supplementary Figure S2. ADT/PI3Ki combination therapy halts prostate tumor growth up to 14 days, followed by development of resistance in majority of Pb-Cre PTENfl/fl Trp53fl/fl mice. Supplementary Figure S3. PI3Ki treatment with concurrent androgen depletion does not alter proliferation and survival of PTEN 53-deficient murine PC cells in vitro. Supplementary Figure S4. ADT/PI3K inhibitor combination increases MHC-II and PD-1 expression on TAM within the TME of PTEN 53-deficient murine PC. Supplementary Figure S5. PD-1 upregulation suppresses phagocytic capacity of activated TAM. Supplementary Figure S6. Ex vivo AD + PI3Ki + PD-1 antibody treatment activates MHCIIlo TAM when co-cultured with PTEN 53-deficient murine prostate tumor cells. Supplementary Figure S7. The addition of PD-1 blockade to androgen depletion/PI3Ki therapy does not alter phagocytic capacity of PD-1 lo macrophages. Supplementary Figure S8. The combination of androgen depletion, PI3Ki and aPD-1 blockade does not alter phagocytic checkpoint expression on PTEN 53-deficient prostate tumor cells. Supplementary Figure S9. Androgen depletion, singly and in combination with aPD-1, did not alter phagocytosis activity of inactivated MHC-IIlo/PD-1 lo and MHC-IIlo/PD-1 hi TAM subsets. Supplementary Figure S10. Androgen depletion, not PI3Ki or aPD1, directly enhances TAM activation within the TME of PTEN 53-deficient PC. Supplementary Figure S11. PI3Ki does not alter phagocytosis/histone lactylation status of MHC-IIlo/PD-1 lo TAM and MHC-IIlo/PD-1 hi TAM. Supplementary Figure S12. PI3Ki inhibits lactate secretion from PTEN 53-deficient prostate tumor cells within TME. Supplementary Figure S13. Direct ex vivo treatment of TAM with PI3Ki, singly and in combination with PD-1 antibody and/or androgen depletion does not alter their histone lactylation profile. Supplementary Figure S14. ADT + PI3Ki + aPD-1 induces tumor control in 60% of Pb-Cre PTENfl/fl TP53fl/fl mice. Supplementary Figure S15. Depletion of activated TAM abrogates anti-cancer response elicited by ADT + PI3Ki + PD-1 antibody treatment in the PTEN 53-deficient murine prostate GEMM tumors. Supplementary Figure S16. Long-term treatment of ADT + PI3Ki + aPD-1 activates Wnt/βcatenin pathway in murine PTEN 53-deficient GEMM-derived SC1 cells. Supplementary Figure S17. Feedback Wnt/β-catenin-pathway activation within murine PTEN 53-deficient GEMM-derived PC cells following long-term ADT + copanlisib + aPD1 treatment suppresses phagocytosis via increased histone lactylation within bone marrow derived macrophages (BMDM).
Publisher: American Association for Cancer Research (AACR)
Date: 15-04-2008
DOI: 10.1158/0008-5472.CAN-07-3079
Abstract: Overexpression of the helix-loop-helix (HLH) protein Id1 has been associated with metastasis in breast cancer, but its role in models of early breast tumorigenesis is not well characterized. We show that the down-regulation of endogenous Id1 via proteosomal degradation and relocalization from the nucleus to the cytoplasm is an early event in the formation of mammary epithelial acini. Overexpression of Id1 in both human MCF-10A and primary mouse mammary epithelial cells disrupted normal acinar development by increasing acinar volume. This occurred in an HLH domain–dependent fashion via an increase in S phase. Id1 overexpression also increased apoptosis leading to accelerated luminal clearance, and this was reversed by coexpression of the proto-oncogene Bcl2, leading to large, disorganized structures with filled lumina. Id1 overexpression was unable to increase the volume of cyclin D1−/− acini, indicating that Id1 is dependent on cyclin D1 for its proliferative effects. In summary, Id1 may contribute to early breast cancer by promoting excessive proliferation through cyclin D1. [Cancer Res 2008 (8):3026–36]
Publisher: Springer Science and Business Media LLC
Date: 27-03-2015
DOI: 10.1038/NCOMMS7548
Abstract: Basal-like breast cancer (BLBC) is a heterogeneous disease with poor prognosis however, its cellular origins and aetiology are poorly understood. In this study, we show that inhibitor of differentiation 4 (ID4) is a key regulator of mammary stem cell self-renewal and marks a subset of BLBC with a putative mammary basal cell of origin. Using an ID4GFP knock-in reporter mouse and single-cell transcriptomics, we show that ID4 marks a stem cell-enriched subset of the mammary basal cell population. ID4 maintains the mammary stem cell pool by suppressing key factors required for luminal differentiation. Furthermore, ID4 is specifically expressed by a subset of human BLBC that possess a very poor prognosis and a transcriptional signature similar to a mammary stem cell. These studies identify ID4 as a mammary stem cell regulator, deconvolute the heterogeneity of BLBC and link a subset of mammary stem cells to the aetiology of BLBC.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/1078-0432.22551385
Abstract: Supplementary Table S1. ADT + PI3Ki + PD-1 antibody leads to TAM activation within TME of PTEN 53-deficient prostate tumors.
Publisher: Cold Spring Harbor Laboratory
Date: 11-02-2022
DOI: 10.1101/2022.02.11.480027
Abstract: The association between cancer and autoimmune disease is unexplained, exemplified by T-cell large granular lymphocytic leukemia (T-LGL) where gain-of-function somatic mutations in STAT3 correlate with co-existing autoimmunity. To resolve whether these mutations are the cause or consequence of CD8 clonal expansions and autoimmunity, here we analyse patients with germline STAT3 GOF syndrome and mice with the T-LGL mutation STAT3 K658N or the most common germline mutation, STAT3 T716M . STAT3 GOF mutations drove accumulation of effector CD8 T cell clones highly expressing the NKG2D receptor for MHC-I-related molecules expressed on stressed cells, and the genes for inflammatory/cytotoxic granzymes, perforin, interferon-γ and Ccl5 /Rantes. CD8 cells were essential to lethal disease in Stat3 K658N mice and their accumulation required NKG2D and the receptor for IL-15 and IL-2, IL2RB. These results demonstrate that STAT3 GOF mutations cause effector CD8 T cell oligoclonal accumulation and that these rogue T cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease. Leukemia and autoimmune-associated STAT3 gain-of-function mutations dysregulate CD8 T cells to cause autoimmune pathology and oligoclonal expansion of cytotoxic killer CD8 T cells, that depend upon NKG2D and IL2RB receptors for signals displayed on stressed, damaged, infected, or mutated tissues.
Publisher: Springer Science and Business Media LLC
Date: 03-09-2018
Publisher: Oxford University Press (OUP)
Date: 16-11-2017
DOI: 10.1093/NAR/GKX1072
Publisher: American Association for Cancer Research (AACR)
Date: 15-05-2023
DOI: 10.1158/1078-0432.C.6563227
Abstract: AbstractPurpose: Phosphatase and tensin homolog (PTEN) loss of function occurs in approximately 50% of patients with metastatic castrate-resistant prostate cancer (mCRPC), and is associated with poor prognosis and responsiveness to standard-of-care therapies and immune checkpoint inhibitors. While PTEN loss of function hyperactivates PI3K signaling, combinatorial PI3K/AKT pathway and androgen deprivation therapy (ADT) has demonstrated limited anticancer efficacy in clinical trials. Here, we aimed to elucidate mechanism(s) of resistance to ADT/PI3K-AKT axis blockade, and to develop rational combinatorial strategies to effectively treat this molecular subset of mCRPC. Experimental Design: Prostate-specific PTEN 53-deficient genetically engineered mice (GEM) with established 150–200 mm sup /sup tumors, as assessed by ultrasound, were treated with either ADT (degarelix), PI3K inhibitor (copanlisib), or anti–PD-1 antibody (aPD-1), as single agents or their combinations, and tumors were monitored by MRI and harvested for immune, transcriptomic, and proteomic profiling, or i ex vivo /i co-culture studies. Single-cell RNA sequencing on human mCRPC s les was performed using 10X Genomics platform. Results: Coclinical trials in PTEN 53-deficient GEM revealed that recruitment of PD-1–expressing tumor-associated macrophages (TAM) thwarts ADT/PI3Ki combination–induced tumor control. The addition of aPD-1 to ADT/PI3Ki combination led to TAM-dependent approximately 3-fold increase in anticancer responses. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their anticancer phagocytic activation, which was augmented by ADT/aPD-1 treatment and abrogated by feedback activation of Wnt/β-catenin pathway. Single-cell RNA-sequencing analysis in mCRPC patient biopsy s les revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression. Conclusions: Immunometabolic strategies that reverse lactate and PD-1–mediated TAM immunosuppression, in combination with ADT, warrant further investigation in patients with PTEN-deficient mCRPC. /
Publisher: Springer Science and Business Media LLC
Date: 14-10-2021
DOI: 10.1038/S41467-021-26271-2
Abstract: In the past decades, transcriptomic studies have revolutionized cancer treatment and diagnosis. However, tumor sequencing strategies typically result in loss of spatial information, critical to understand cell interactions and their functional relevance. To address this, we investigate spatial gene expression in HER2-positive breast tumors using Spatial Transcriptomics technology. We show that expression-based clustering enables data-driven tumor annotation and assessment of intra- and interpatient heterogeneity from which we discover shared gene signatures for immune and tumor processes. By integration with single cell data, we spatially map tumor-associated cell types to find tertiary lymphoid-like structures, and a type I interferon response overlapping with regions of T-cell and macrophage subset colocalization. We construct a predictive model to infer presence of tertiary lymphoid-like structures, applicable across tissue types and technical platforms. Taken together, we combine different data modalities to define a high resolution map of cellular interactions in tumors and provide tools generalizing across tissues and diseases.
Publisher: Springer Science and Business Media LLC
Date: 19-11-2019
DOI: 10.1038/S41598-019-52000-3
Abstract: Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA s les were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein ( AIP ) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of % and precision of %. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy.
Publisher: Springer Science and Business Media LLC
Date: 04-2014
DOI: 10.1186/BCR3654
Publisher: Springer Science and Business Media LLC
Date: 12-08-2020
DOI: 10.1186/S13058-020-01318-2
Abstract: Resistance to endocrine therapy is a major clinical challenge in the management of oestrogen receptor (ER)-positive breast cancer. In this setting, p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment-resistant ER-positive breast cancer. We used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate anti-tumour effects in p53 wildtype and p53 mutant ER-positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and endocrine-resistant ER-positive breast cancer. We demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant-resistant patient-derived xenograft model. We conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine-resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programmes.
Publisher: Cold Spring Harbor Laboratory
Date: 06-06-2020
DOI: 10.1101/2020.06.04.135327
Abstract: The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single cell RNA-sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states, the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell-signalling predictions, we provide evidence that stromal-immune crosstalk acts via a erse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.
Publisher: Springer Science and Business Media LLC
Date: 18-10-2005
Abstract: The helix-loop-helix protein Id1 has been implicated in regulating mammary epithelial cell proliferation and differentiation but the underlying molecular mechanisms are not well characterized. Under low serum conditions, ectopic expression of Id1, but not Id2, allowed continued proliferation of immortalized mammary epithelial cells and breast cancer cells. Conversely, downregulation of Id1 impaired proliferation. The effects of short interfering RNA (siRNA)-mediated downregulation of Id1 were the same as those following downregulation of c-Myc: decreased expression of cyclins D1 and E, reduced phosphorylation of pRb at Ser780 (a site targeted by cyclin D1-Cdk4) and reduced cyclin E-Cdk2 activity. Decreased cyclin D1 expression was an early response to Id1 antisense oligonucleotide treatment. Inhibition of c-Myc function by siRNA, antisense oligonucleotides or a dominant repressor resulted in downregulation of Id1, while ectopic expression of c-Myc resulted in rapid induction of Id1, suggesting that Id1 may be downstream of c-Myc. These data indicate that in mammary epithelial cells, Id1 has cell cycle regulatory functions that are similar to those of c-Myc, and suggest that cyclin D1 may be involved in Id1 regulation of cell cycle progression.
Publisher: Elsevier BV
Date: 06-2021
Publisher: Springer New York
Date: 2008
DOI: 10.1007/978-0-387-78818-0_12
Abstract: Loss of normal growth control is a hallmark of cancer. Thus, understanding the mechanisms of tissue-specific, normal growth regulation and the changes that occur during tumorigenesis may provide insights of both diagnostic and therapeutic importance. Control of cell proliferation in the normal mammary gland is steroid hormone (estrogen and progestin)-dependent, involves complex interactions with other hormones, growth factors and cytokines and ultimately converges on activation of three proto-oncogenes (c-Myc, cyclin D1 and cyclin E1) that are rate limiting for the G1 to S phase transition during normal cell cycle progression. Mammary epithelial cell-specific overexpression of these genes induces mammary carcinoma in mice, while cyclin D1 null mice have arrested mammary gland development and are resistant to carcinoma induced by the neu/erbB2 and ras oncogenes. Furthermore, c-Myc, cyclins D1, E1 and E2 are commonly overexpressed in primary breast cancer where elevated expression is often associated with a more aggressive disease phenotype and an adverse patient outcome. This may be due in part to overexpression of these genes conferring resistance to endocrine therapies since in vitro studies provide compelling evidence that overexpression of c-Myc and to a lesser extent cyclin D1 and cyclin E1, attenuate the growth inhibitory effects of SERMS, antiestrogens and progestins in breast cancer cells. Thus, abnormal regulation of the expression of cell cycle molecules, involved in the steroidal control of cell proliferation in the mammary gland, are likely to be directly involved in the development, progression and therapeutic responsiveness of breast cancer. Furthermore, a more detailed understanding of these pathways may identify new targets for therapeutic intervention particularly in endocrine-unresponsive and endocrine-resistant disease.
Publisher: Elsevier BV
Date: 2021
Publisher: Wiley
Date: 09-01-2020
DOI: 10.1111/FEBS.15186
Abstract: Annexin A6 (AnxA6), a member of the calcium (Ca
Publisher: Springer Science and Business Media LLC
Date: 11-06-2020
DOI: 10.1186/S13058-020-01306-6
Abstract: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical s les demonstrates that ID4 is lified and overexpressed at a higher frequency in BRCA1 -mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.
Publisher: Springer Science and Business Media LLC
Date: 10-05-2021
DOI: 10.1186/S13073-021-00885-Z
Abstract: High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of s les, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer s le cryopreserved as solid tissue fragments. Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.
Publisher: Proceedings of the National Academy of Sciences
Date: 08-04-2008
Abstract: Recent evidence demonstrates that senescence acts as a barrier to tumorigenesis in response to oncogene activation. Using a mouse model of breast cancer, we tested the importance of the senescence response in solid cancer and identified genetic pathways regulating this response. Mammary expression of activated Ras led to the formation of senescent cellular foci in a majority of mice. Deletion of the p19 ARF , p53, or p21 WAF1 tumor suppressors but not p16 INK4a prevented senescence and permitted tumorigenesis. Id1 has been implicated in the control of senescence in vitro , and elevated expression of Id1 is found in a number of solid cancers, so we tested whether overexpression of Id1 regulates senescence in vivo . Although overexpression of Id1 in the mammary epithelium was not sufficient for tumorigenesis, mice with expression of both Id1 and activated Ras developed metastatic cancer. These tumors expressed high levels of p19 Arf , p53, and p21 Waf1 , demonstrating that Id1 acts to make cells refractory to p21 Waf1 -dependent cell cycle arrest. Inactivation of the conditional Id1 allele in established tumors led to widespread senescence within 10 days, tumor growth arrest, and tumor regression in 40% of mice. Mice in which Id1 expression was inactivated also exhibited greatly reduced pulmonary metastatic load. These data demonstrate that established tumors remain sensitive to senescence and that Id1 may be a valuable target for therapy.
Publisher: Elsevier BV
Date: 12-2001
Publisher: American Association for Cancer Research (AACR)
Date: 31-05-2011
DOI: 10.1158/0008-5472.CAN-10-3738
Abstract: Hedgehog (Hh) signaling plays an important role in several malignancies but its clinical significance in breast cancer is unclear. In a cohort of 279 patients with invasive ductal carcinoma of the breast, expression of Hh ligand was significantly associated with increased risk of metastasis, breast cancer-specific death, and a basal-like phenotype. A paracrine signature, encompassing high epithelial Hh ligand and high stromal Gli1, was an independent predictor for overall survival in multivariate analysis. In 2 independent histological progression series (n = 301), Hh expression increased with atypia. Hh ligand overexpression in a mouse model of basal breast cancer increased growth, induced a poorly differentiated phenotype, accelerated metastasis, and reduced survival. A stromal requirement for these effects was supported by the lack of similar Hh-mediated changes in vitro, and by stromal-specific expression of Hh target genes in vivo. Furthermore, inhibition of Hh ligand with a monoclonal antibody (5E1) inhibited tumor growth and metastasis. These data suggest that epithelial–stromal Hh signaling, driven by ligand expression in carcinoma cells, promotes breast cancer growth and metastasis. Blockade of Hh signaling to peritumoral stromal cells may represent a novel therapeutic approach in some basal-like breast cancers. Cancer Res 71(11) 4002–14. ©2011 AACR.
Publisher: Springer Netherlands
Date: 31-07-2012
Publisher: Springer Science and Business Media LLC
Date: 17-02-2019
DOI: 10.1038/S41467-019-11049-4
Abstract: High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly erse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells s led from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer.
Publisher: Elsevier BV
Date: 02-2021
Publisher: Springer Science and Business Media LLC
Date: 24-07-2018
DOI: 10.1038/S41467-018-05220-6
Abstract: The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hedgehog ligand produced by neoplastic cells reprograms cancer-associated fibroblasts (CAFs) to provide a supportive niche for the acquisition of a chemo-resistant, cancer stem cell (CSC) phenotype via FGF5 expression and production of fibrillar collagen. Stromal treatment of patient-derived xenografts with smoothened inhibitors (SMOi) downregulates CSC markers expression and sensitizes tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. These studies identify Hedgehog signaling to CAFs as a novel mediator of CSC plasticity and an exciting new therapeutic target in TNBC.
Publisher: American Society of Hematology
Date: 04-07-2019
Abstract: The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of in idual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.
Publisher: Cold Spring Harbor Laboratory
Date: 14-07-2020
DOI: 10.1101/2020.07.14.200600
Abstract: In the past decades, transcriptomic studies have revolutionized cancer treatment and diagnosis. However, tumor sequencing strategies typically result in loss of spatial information, critical to understand cell interactions and their functional relevance. To address this, we investigate spatial gene expression in HER2-positive breast tumors using Spatial Transcriptomics technology. We show that expression-based clustering enables data-driven tumor annotation and assessment of intra-and interpatient heterogeneity from which we discover shared gene signatures for immune and tumor processes. We integrate and spatially map tumor-associated types from single cell data to find: segregated epithelial cells, interactions between B and T-cells and myeloid cells, co-localization of macrophage and T-cell subsets. A model is constructed to infer presence of tertiary lymphoid structures, applicable across tissue types and technical platforms. Taken together, we combine different data modalities to define novel interactions between tumor-infiltrating cells in breast cancer and provide tools generalizing across tissues and diseases.
Publisher: Springer Science and Business Media LLC
Date: 06-08-2022
DOI: 10.1038/S41467-022-32255-7
Abstract: The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.
Publisher: EMBO
Date: 13-08-2020
Publisher: American Association for Cancer Research (AACR)
Date: 15-05-2023
DOI: 10.1158/1078-0432.22820230.V1
Abstract: Supplementary Figure S1. The majority of Pb-Cre PTENfl/fl Trp53fl/fl mice are de novo resistant to ADT.Supplementary Figure S2. ADT/PI3Ki combination therapy halts prostate tumor growth up to 14 days, followed by development of resistance in majority of Pb-Cre PTENfl/fl Trp53fl/fl mice.Supplementary Figure S3. PI3Ki treatment with concurrent androgen depletion does not alter proliferation and survival of PTEN 53-deficient murine PC cells in vitro.Supplementary Figure S4. ADT/PI3K inhibitor combination increases MHC-II and PD-1 expression on TAM within the TME of PTEN 53-deficient murine PC.Supplementary Figure S5. PD-1 upregulation suppresses phagocytic capacity of activated TAM.Supplementary Figure S6. Ex vivo AD + PI3Ki + PD-1 antibody treatment activates MHCIIlo TAM when co-cultured with PTEN 53-deficient murine prostate tumor cells.Supplementary Figure S7. The addition of PD-1 blockade to androgen depletion/PI3Ki therapy does not alter phagocytic capacity of PD-1 lo macrophages.Supplementary Figure S8. The combination of androgen depletion, PI3Ki and aPD-1 blockade does not alter phagocytic checkpoint expression on PTEN 53-deficient prostate tumor cells.Supplementary Figure S9. Androgen depletion, singly and in combination with aPD-1, did not alter phagocytosis activity of inactivated MHC-IIlo/PD-1 lo and MHC-IIlo/PD-1 hi TAM subsets.Supplementary Figure S10. Androgen depletion, not PI3Ki or aPD1, directly enhances TAM activation within the TME of PTEN 53-deficient PC.Supplementary Figure S11. PI3Ki does not alter phagocytosis/histone lactylation status of MHC-IIlo/PD-1 lo TAM and MHC-IIlo/PD-1 hi TAM.Supplementary Figure S12. PI3Ki inhibits lactate secretion from PTEN 53-deficient prostate tumor cells within TME.Supplementary Figure S13. Direct ex vivo treatment of TAM with PI3Ki, singly and in combination with PD-1 antibody and/or androgen depletion does not alter their histone lactylation profile.Supplementary Figure S14. ADT + PI3Ki + aPD-1 induces tumor control in 60% of Pb-Cre PTENfl/fl TP53fl/fl mice.Supplementary Figure S15. Depletion of activated TAM abrogates anti-cancer response elicited by ADT + PI3Ki + PD-1 antibody treatment in the PTEN 53-deficient murine prostate GEMM tumors.Supplementary Figure S16. Long-term treatment of ADT + PI3Ki + aPD-1 activates Wnt/βcatenin pathway in murine PTEN 53-deficient GEMM-derived SC1 cells.Supplementary Figure S17. Feedback Wnt/β-catenin-pathway activation within murine PTEN 53-deficient GEMM-derived PC cells following long-term ADT + copanlisib + aPD1 treatment suppresses phagocytosis via increased histone lactylation within bone marrow derived macrophages (BMDM).
Publisher: Springer Science and Business Media LLC
Date: 24-05-2022
Publisher: Cold Spring Harbor Laboratory
Date: 06-04-2020
DOI: 10.1101/2020.04.06.026963
Abstract: Differentiation of stem cells embedded within the mammary epithelium is orchestrated by lineage-specifying transcription factors. Unlike the well-defined luminal hierarchy, dissection of the basal lineage has been hindered by a lack of specific markers. Inhibitor of Differentiation 4 (ID4) is a basally-restricted helix-loop-helix (HLH) transcription factor essential for mammary development. Here we show that ID4 is highly expressed in basal stem cells and decreases during myoepithelial differentiation. By integrating transcriptomic, proteomic, and ChIP-sequencing data, we reveal that ID4 is required to suppress myoepithelial gene expression and cell fate. We identify the bHLH protein HEB as a direct binding partner of ID4, and describe a previously-unknown role for this regulator in mammary development. HEB binds to E-boxes in regulatory elements of developmental genes, negatively regulated by ID4, involved in extracellular matrix synthesis and cytoskeletal contraction. Together our findings support a model whereby ID4 binds to HEB and blocks it from promoting myoepithelial specialisation. These new insights expand our current understanding into control of myoepithelial differentiation and mammary gland morphogenesis.
Publisher: Wiley
Date: 11-2007
DOI: 10.1111/J.1550-7408.1997.TB05957.X
Abstract: The cytidine triphosphate synthetase genes from three erse strains of Giardia duodenalis have been sequenced and found to vary significantly from one another. The isolates were chosen as representatives of three demes as determined by several criteria including ergence in the rDNA repeat unit. Inserts in the genes and protein are conserved in length but are the most ergent regions among the three sequences examined. Variation in the rest of the gene occurs primarily in the third base position resulting in many silent mutations. One of the isolates (1709) was found to contain two genes with high sequence homology.
Publisher: Public Library of Science (PLoS)
Date: 27-12-2012
Publisher: Elsevier BV
Date: 12-2015
DOI: 10.1016/J.IJROBP.2015.08.046
Abstract: Local recurrence and distant failure after adjuvant radiation therapy for breast cancer remain significant clinical problems, incompletely predicted by conventional clinicopathologic markers. We had previously identified microRNA-139-5p and microRNA-1274a as key regulators of breast cancer radiation response in vitro. The purpose of this study was to investigate standard clinicopathologic markers of local recurrence in a contemporary series and to establish whether putative target genes of microRNAs involved in DNA repair and cell cycle control could better predict radiation therapy response in vivo. With institutional ethics board approval, local recurrence was measured in a contemporary, prospectively collected series of 458 patients treated with radiation therapy after breast-conserving surgery. Additionally, independent publicly available mRNA/microRNA microarray expression datasets totaling >1000 early-stage breast cancer patients, treated with adjuvant radiation therapy, with >10 years of follow-up, were analyzed. The expression of putative microRNA target biomarkers--TOP2A, POLQ, RAD54L, SKP2, PLK2, and RAG1--were correlated with standard clinicopathologic variables using 2-sided nonparametric tests, and to local/distant relapse and survival using Kaplan-Meier and Cox regression analysis. We found a low rate of isolated local recurrence (1.95%) in our modern series, and that few clinicopathologic variables (such as lymphovascular invasion) were significantly predictive. In multiple independent datasets (n>1000), however, high expression of RAD54L, TOP2A, POLQ, and SKP2 significantly correlated with local recurrence, survival, or both in univariate and multivariate analyses (P<.001). Low RAG1 expression significantly correlated with local recurrence (multivariate, P=.008). Additionally, RAD54L, SKP2, and PLK2 may be predictive, being prognostic in radiation therapy-treated patients but not in untreated matched control in iduals (n=107 P<.05). Biomarkers of DNA repair and cell cycle control can identify patients at high risk of treatment failure in those receiving radiation therapy for early breast cancer in independent cohorts. These should be further investigated prospectively, especially TOP2A and SKP2, for which targeted therapies are available.
Publisher: Springer Science and Business Media LLC
Date: 03-08-2021
DOI: 10.1186/S13058-021-01461-4
Abstract: Particular breast cancer subtypes pose a clinical challenge due to limited targeted therapeutic options and/or poor responses to the existing targeted therapies. While cell lines provide useful pre-clinical models, patient-derived xenografts (PDX) and organoids (PDO) provide significant advantages, including maintenance of genetic and phenotypic heterogeneity, 3D architecture and for PDX, tumor–stroma interactions. In this study, we applied an integrated multi-omic approach across panels of breast cancer PDXs and PDOs in order to identify candidate therapeutic targets, with a major focus on specific FGFRs. MS-based phosphoproteomics, RNAseq, WES and Western blotting were used to characterize aberrantly activated protein kinases and effects of specific FGFR inhibitors. PDX and PDO were treated with the selective tyrosine kinase inhibitors AZD4547 (FGFR1-3) and BLU9931 (FGFR4). FGFR4 expression in cancer tissue s les and PDOs was assessed by immunohistochemistry. METABRIC and TCGA datasets were interrogated to identify specific FGFR alterations and their association with breast cancer subtype and patient survival. Phosphoproteomic profiling across 18 triple-negative breast cancers (TNBC) and 1 luminal B PDX revealed considerable heterogeneity in kinase activation, but 1/3 of PDX exhibited enhanced phosphorylation of FGFR1, FGFR2 or FGFR4. One TNBC PDX with high FGFR2 activation was exquisitely sensitive to AZD4547. Integrated ‘omic analysis revealed a novel FGFR2-SKI fusion that comprised the majority of FGFR2 joined to the C-terminal region of SKI containing the coiled-coil domains. High FGFR4 phosphorylation characterized a luminal B PDX model and treatment with BLU9931 significantly decreased tumor growth. Phosphoproteomic and transcriptomic analyses confirmed on-target action of the two anti-FGFR drugs and also revealed novel effects on the spliceosome, metabolism and extracellular matrix (AZD4547) and RIG-I-like and NOD-like receptor signaling (BLU9931). Interrogation of public datasets revealed FGFR2 lification, fusion or mutation in TNBC and other breast cancer subtypes, while FGFR4 overexpression and lification occurred in all breast cancer subtypes and were associated with poor prognosis. Characterization of a PDO panel identified a luminal A PDO with high FGFR4 expression that was sensitive to BLU9931 treatment, further highlighting FGFR4 as a potential therapeutic target. This work highlights how patient-derived models of human breast cancer provide powerful platforms for therapeutic target identification and analysis of drug action, and also the potential of specific FGFRs, including FGFR4, as targets for precision treatment.
Publisher: Springer Science and Business Media LLC
Date: 04-2022
DOI: 10.1038/S41556-022-00881-4
Abstract: Skeletal muscle has long been recognized as an inhospitable site for disseminated tumour cells (DTCs). Yet its antimetastatic nature has eluded a thorough mechanistic examination. Here, we show that DTCs traffic to and persist within skeletal muscle in mice and in humans, which raises the question of how this tissue suppresses colonization. Results from mouse and organotypic culture models along with metabolomic profiling suggested that skeletal muscle imposes a sustained oxidative stress on DTCs that impairs their proliferation. Functional studies demonstrated that disrupting reduction-oxidation homeostasis via chemogenetic induction of reactive oxygen species slowed proliferation in a more fertile organ: the lung. Conversely, enhancement of the antioxidant potential of tumour cells through ectopic expression of catalase in the tumour or host mitochondria allowed robust colonization of skeletal muscle. These findings reveal a profound metabolic bottleneck imposed on DTCs and sustained by skeletal muscle. A thorough understanding of this biology could reveal previously undocumented DTC vulnerabilities that can be exploited to prevent metastasis in other more susceptible tissues.
Publisher: Wiley
Date: 15-09-2005
DOI: 10.1002/JCP.20199
Abstract: Growth factor receptor bound (Grb)14 is a member of the Grb7 family of src homology (SH)2 domain-containing proteins. These proteins perform both adaptor and modulatory roles in receptor tyrosine kinase (RTK) signaling, although their regulation is poorly understood. In this study, a positive correlation between Grb14 protein expression and ER alpha status in breast cancer cell lines led us to investigate regulation of Grb14 by estradiol and insulin, which synergize in the regulation of breast cancer cell proliferation. In MCF-7 cells maintained in charcoal-stripped serum, Grb14 expression was downregulated by estradiol and increased by the pure anti-estrogen ICI 182780. Under serum-free conditions, insulin enhanced Grb14 expression but this effect was repressed by estradiol when both hormones were used in combination. Using a system in which c-Myc induction drives cell cycle progression independently of estradiol, we demonstrated that Grb14 regulation was specific to estradiol treatment. Finally, we demonstrated a novel functional role for Grb14 whereby its overexpression inhibited not only insulin- but also estrogen-induced cell cycle progression. This was associated with decreased extracellular signal-regulated kinase (Erk)1/2 activation in insulin-stimulated Grb14-overexpressing cells. These data represent the first demonstration of regulation of Grb14 expression levels in response to hormonal stimuli, and are consistent with its role as a repressor of insulin signaling where it is induced as a negative feedback mechanism. A role for Grb14 is also shown in estrogen/insulin crosstalk since estradiol blocks the insulin-induced induction of this protein.
Publisher: American Association for Cancer Research (AACR)
Date: 2015
DOI: 10.1158/2159-8290.CD-14-0621
Abstract: Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their antiresorptive effects on the skeleton. However, because bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their antitumor action, particularly on cells outside of bone, remain unknown. Here, we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumors, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumor-associated macrophages (TAM), but not tumor cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumor of a patient and showed the resected tumor to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumors outside the skeleton and that their antitumor activity is likely to be mediated via TAMs. Significance: Bisphosphonates are assumed to act solely in bone. However, mouse models and clinical trials show that they have surprising antitumor effects outside bone. We provide unequivocal evidence that bisphosphonates target TAMs, but not tumor cells, to exert their extraskeletal effects, offering a rationale for use in patients with early disease. Cancer Discov 5(1) 35–42. ©2014 AACR. See related commentary by Sterling, p. 14 This article is highlighted in the In This Issue feature, p. 1
Publisher: Springer Science and Business Media LLC
Date: 18-05-2018
DOI: 10.1038/S41598-018-26050-Y
Abstract: Docetaxel and cabazitaxel are taxane chemotherapy treatments for metastatic castration-resistant prostate cancer (CRPC). However, therapeutic resistance remains a major issue. MicroRNAs are short non-coding RNAs that can silence multiple genes, regulating several signalling pathways simultaneously. Therefore, synthetic microRNAs may have therapeutic potential in CRPC by regulating genes involved in taxane response and minimise compensatory mechanisms that cause taxane resistance. To identify microRNAs that can improve the efficacy of taxanes in CRPC, we performed a genome-wide screen of 1280 microRNAs in the CRPC cell lines PC3 and DU145 in combination with docetaxel or cabazitaxel treatment. Mimics of miR-217 and miR-181b-5p enhanced apoptosis significantly in PC3 cells in the presence of these taxanes. These mimics downregulated at least a thousand different transcripts, which were enriched for genes with cell proliferation and focal adhesion functions. In idual knockdown of a selection of 46 genes representing these transcripts resulted in toxic or taxane sensitisation effects, indicating that these genes may be mediating the effects of the microRNA mimics. A range of these genes are expressed in CRPC metastases, suggesting that these microRNA mimics may be functional in CRPC. With further development, these microRNA mimics may have therapeutic potential to improve taxane response in CRPC patients.
Publisher: Springer Science and Business Media LLC
Date: 18-01-2021
DOI: 10.1038/S41591-020-01168-7
Abstract: The role of the androgen receptor (AR) in estrogen receptor (ER)-α-positive breast cancer is controversial, constraining implementation of AR-directed therapies. Using a erse, clinically relevant panel of cell-line and patient-derived models, we demonstrate that AR activation, not suppression, exerts potent antitumor activity in multiple disease contexts, including resistance to standard-of-care ER and CDK4/6 inhibitors. Notably, AR agonists combined with standard-of-care agents enhanced therapeutic responses. Mechanistically, agonist activation of AR altered the genomic distribution of ER and essential co-activators (p300, SRC-3), resulting in repression of ER-regulated cell cycle genes and upregulation of AR target genes, including known tumor suppressors. A gene signature of AR activity positively predicted disease survival in multiple clinical ER-positive breast cancer cohorts. These findings provide unambiguous evidence that AR has a tumor suppressor role in ER-positive breast cancer and support AR agonism as the optimal AR-directed treatment strategy, revealing a rational therapeutic opportunity.
Publisher: Springer Science and Business Media LLC
Date: 21-09-2012
DOI: 10.1186/BCR3209
Publisher: American Association for Cancer Research (AACR)
Date: 15-05-2023
DOI: 10.1158/1078-0432.22820230
Abstract: Supplementary Figure S1. The majority of Pb-Cre PTENfl/fl Trp53fl/fl mice are de novo resistant to ADT.Supplementary Figure S2. ADT/PI3Ki combination therapy halts prostate tumor growth up to 14 days, followed by development of resistance in majority of Pb-Cre PTENfl/fl Trp53fl/fl mice.Supplementary Figure S3. PI3Ki treatment with concurrent androgen depletion does not alter proliferation and survival of PTEN 53-deficient murine PC cells in vitro.Supplementary Figure S4. ADT/PI3K inhibitor combination increases MHC-II and PD-1 expression on TAM within the TME of PTEN 53-deficient murine PC.Supplementary Figure S5. PD-1 upregulation suppresses phagocytic capacity of activated TAM.Supplementary Figure S6. Ex vivo AD + PI3Ki + PD-1 antibody treatment activates MHCIIlo TAM when co-cultured with PTEN 53-deficient murine prostate tumor cells.Supplementary Figure S7. The addition of PD-1 blockade to androgen depletion/PI3Ki therapy does not alter phagocytic capacity of PD-1 lo macrophages.Supplementary Figure S8. The combination of androgen depletion, PI3Ki and aPD-1 blockade does not alter phagocytic checkpoint expression on PTEN 53-deficient prostate tumor cells.Supplementary Figure S9. Androgen depletion, singly and in combination with aPD-1, did not alter phagocytosis activity of inactivated MHC-IIlo/PD-1 lo and MHC-IIlo/PD-1 hi TAM subsets.Supplementary Figure S10. Androgen depletion, not PI3Ki or aPD1, directly enhances TAM activation within the TME of PTEN 53-deficient PC.Supplementary Figure S11. PI3Ki does not alter phagocytosis/histone lactylation status of MHC-IIlo/PD-1 lo TAM and MHC-IIlo/PD-1 hi TAM.Supplementary Figure S12. PI3Ki inhibits lactate secretion from PTEN 53-deficient prostate tumor cells within TME.Supplementary Figure S13. Direct ex vivo treatment of TAM with PI3Ki, singly and in combination with PD-1 antibody and/or androgen depletion does not alter their histone lactylation profile.Supplementary Figure S14. ADT + PI3Ki + aPD-1 induces tumor control in 60% of Pb-Cre PTENfl/fl TP53fl/fl mice.Supplementary Figure S15. Depletion of activated TAM abrogates anti-cancer response elicited by ADT + PI3Ki + PD-1 antibody treatment in the PTEN 53-deficient murine prostate GEMM tumors.Supplementary Figure S16. Long-term treatment of ADT + PI3Ki + aPD-1 activates Wnt/βcatenin pathway in murine PTEN 53-deficient GEMM-derived SC1 cells.Supplementary Figure S17. Feedback Wnt/β-catenin-pathway activation within murine PTEN 53-deficient GEMM-derived PC cells following long-term ADT + copanlisib + aPD1 treatment suppresses phagocytosis via increased histone lactylation within bone marrow derived macrophages (BMDM).
Publisher: American Association for Cancer Research (AACR)
Date: 15-05-2023
DOI: 10.1158/1078-0432.22820227.V1
Abstract: Supplementary Table S1. ADT + PI3Ki + PD-1 antibody leads to TAM activation within TME of PTEN 53-deficient prostate tumors.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/1078-0432.C.6563227.V1
Abstract: AbstractPurpose: Phosphatase and tensin homolog (PTEN) loss of function occurs in approximately 50% of patients with metastatic castrate-resistant prostate cancer (mCRPC), and is associated with poor prognosis and responsiveness to standard-of-care therapies and immune checkpoint inhibitors. While PTEN loss of function hyperactivates PI3K signaling, combinatorial PI3K/AKT pathway and androgen deprivation therapy (ADT) has demonstrated limited anticancer efficacy in clinical trials. Here, we aimed to elucidate mechanism(s) of resistance to ADT/PI3K-AKT axis blockade, and to develop rational combinatorial strategies to effectively treat this molecular subset of mCRPC. Experimental Design: Prostate-specific PTEN 53-deficient genetically engineered mice (GEM) with established 150–200 mm sup /sup tumors, as assessed by ultrasound, were treated with either ADT (degarelix), PI3K inhibitor (copanlisib), or anti–PD-1 antibody (aPD-1), as single agents or their combinations, and tumors were monitored by MRI and harvested for immune, transcriptomic, and proteomic profiling, or i ex vivo /i co-culture studies. Single-cell RNA sequencing on human mCRPC s les was performed using 10X Genomics platform. Results: Coclinical trials in PTEN 53-deficient GEM revealed that recruitment of PD-1–expressing tumor-associated macrophages (TAM) thwarts ADT/PI3Ki combination–induced tumor control. The addition of aPD-1 to ADT/PI3Ki combination led to TAM-dependent approximately 3-fold increase in anticancer responses. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their anticancer phagocytic activation, which was augmented by ADT/aPD-1 treatment and abrogated by feedback activation of Wnt/β-catenin pathway. Single-cell RNA-sequencing analysis in mCRPC patient biopsy s les revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression. Conclusions: Immunometabolic strategies that reverse lactate and PD-1–mediated TAM immunosuppression, in combination with ADT, warrant further investigation in patients with PTEN-deficient mCRPC. /
Publisher: American Association for Cancer Research (AACR)
Date: 15-05-2023
DOI: 10.1158/1078-0432.C.6563227.V2
Abstract: AbstractPurpose: Phosphatase and tensin homolog (PTEN) loss of function occurs in approximately 50% of patients with metastatic castrate-resistant prostate cancer (mCRPC), and is associated with poor prognosis and responsiveness to standard-of-care therapies and immune checkpoint inhibitors. While PTEN loss of function hyperactivates PI3K signaling, combinatorial PI3K/AKT pathway and androgen deprivation therapy (ADT) has demonstrated limited anticancer efficacy in clinical trials. Here, we aimed to elucidate mechanism(s) of resistance to ADT/PI3K-AKT axis blockade, and to develop rational combinatorial strategies to effectively treat this molecular subset of mCRPC. Experimental Design: Prostate-specific PTEN 53-deficient genetically engineered mice (GEM) with established 150–200 mm sup /sup tumors, as assessed by ultrasound, were treated with either ADT (degarelix), PI3K inhibitor (copanlisib), or anti–PD-1 antibody (aPD-1), as single agents or their combinations, and tumors were monitored by MRI and harvested for immune, transcriptomic, and proteomic profiling, or i ex vivo /i co-culture studies. Single-cell RNA sequencing on human mCRPC s les was performed using 10X Genomics platform. Results: Coclinical trials in PTEN 53-deficient GEM revealed that recruitment of PD-1–expressing tumor-associated macrophages (TAM) thwarts ADT/PI3Ki combination–induced tumor control. The addition of aPD-1 to ADT/PI3Ki combination led to TAM-dependent approximately 3-fold increase in anticancer responses. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their anticancer phagocytic activation, which was augmented by ADT/aPD-1 treatment and abrogated by feedback activation of Wnt/β-catenin pathway. Single-cell RNA-sequencing analysis in mCRPC patient biopsy s les revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression. Conclusions: Immunometabolic strategies that reverse lactate and PD-1–mediated TAM immunosuppression, in combination with ADT, warrant further investigation in patients with PTEN-deficient mCRPC. /
Publisher: Informa UK Limited
Date: 04-1998
Abstract: COVID-19 disease is spread worldwide and diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and a low-cost alternative especially in low and middle-income countries, which lack structure to perform certain diagnostic techniques. Here we evaluate the sensitivity and specificity of eleven different IC tests in 145 serum s les from confirmed cases of COVID-19 using RT-PCR and 100 negative serum s les from blood donors collected in February 2019. We also evaluated the cross-reactivity with dengue using 20 serum s les from patients with confirmed diagnosis for dengue collected in early 2019 through four different tests. We found high sensitivity (92%), specificity (100%) and an almost perfect agreement (Kappa 0.92) of IC assay, especially when we evaluated IgG and IgM combined after 10 days from the onset of symptoms with RT-PCR. However, we detected cross-reactivity between dengue and COVID-19 mainly with IgM antibodies (5 to 20% of cross-reaction) and demonstrated the need for better studies about diagnostic techniques for these diseases.
Publisher: Bioscientifica
Date: 09-2016
DOI: 10.1530/ERC-16-0196
Abstract: Inhibitor of differentiation (ID) proteins are key regulators of development and tumorigenesis. One member of this family, ID4, controls lineage commitment during mammary gland development by acting upstream of key developmental pathways. Recent evidence suggests an emerging role for ID4 as a lineage-dependent proto-oncogene that is overexpressed and lified in a subset of basal-like breast cancers (BLBCs), conferring poor prognosis. Several lines of evidence suggest ID4 may suppress BRCA1 function in BLBC and in doing so, define a subset of BLBC patients who may respond to therapies traditionally used in BRCA1 -mutant cancers. This review highlights recent advances in our understanding of the requirement for ID4 in mammary lineage commitment and the role for ID4 in BLBC. We address current shortfalls in this field and identify important areas of future research.
Publisher: Cambridge University Press (CUP)
Date: 2008
DOI: 10.1017/S1470903108006688
Abstract: L. Ma, J. Teruya-Feldstein, R. A. Weinberg. Nature 2007 449 (7163): 682–688 Epub 26 September 2007. MicroRNAs have been implicated in regulating erse cellular pathways. Although there is emerging evidence that some microRNAs can function as oncogenes or tumour suppressors, the role of microRNAs in mediating cancer metastasis remains unexplored. Here we show, using a combination of mouse and human cells, that microRNA-10b (miR-10b) is highly expressed in metastatic breast cancer cells and positively regulates cell migration and invasion. Overexpression of miR-10b in otherwise non-metastatic breast tumours initiates robust invasion and metastasis. Expression of miR-10b is induced by the transcription factor Twist, which binds directly to the putative promoter of miR-10b (MIRN10B). The miR-10b induced by Twist proceeds to inhibit translation of the messenger RNA encoding homeobox D10, resulting in increased expression of a well-characterized pro-metastatic gene, RhoC. Significantly, the level of miR-10b expression in primary breast carcinomas correlates with clinical progression. These findings suggest the workings of an undescribed regulatory pathway, in which a pleiotropic transcription factor induces expression of a specific microRNA, which suppresses its direct target and in turn activates another pro-metastatic gene, leading to tumour cell invasion and metastasis.
Publisher: Informa UK Limited
Date: 07-08-2009
DOI: 10.1517/14728220903130612
Abstract: The Hedgehog (Hh) signalling pathway is a highly conserved developmental pathway, which plays critical roles in patterning of the embryo through epithelial to mesenchymal signalling and the maintenance of stem cells in the adult organism. There is increasing evidence that this pathway is dysregulated in many malignancies, including breast cancer. While there has been a significant decrease in mortality from breast cancer, a number of treatment challenges remain, particularly in those tumours which develop resistance to endocrine-based therapy, or which lack expression of hormone or c-erbB2/HER2 receptors. Therapeutic manipulation of the Hh pathway as a potential cancer therapy is attracting great interest, with preclinical studies and clinical trials underway in a range of malignancies. This review highlights important recent developments that affect the potential of the Hh pathway as a novel therapeutic target in early breast cancer.
Publisher: MDPI AG
Date: 13-12-2020
Abstract: We aimed to determine the clinical significance of tumour stroma ratio (TSR) in luminal and triple negative breast cancer (TNBC) using digital image analysis and machine learning algorithms. Automated image analysis using QuPath software was applied to a cohort of 647 breast cancer patients (403 luminal and 244 TNBC) using digital H& E images of tissue microarrays (TMAs). Kaplan–Meier and Cox proportional hazards were used to ascertain relationships with overall survival (OS) and breast cancer specific survival (BCSS). For TNBC, low TSR (high stroma) was associated with poor prognosis for both OS (HR 1.9, CI 1.1–3.3, p = 0.021) and BCSS (HR 2.6, HR 1.3–5.4, p = 0.007) in multivariate models, independent of age, size, grade, sTILs, lymph nodal status and chemotherapy. However, for luminal tumours, low TSR (high stroma) was associated with a favourable prognosis in MVA for OS (HR 0.6, CI 0.4–0.8, p = 0.001) but not for BCSS. TSR is a prognostic factor of most significance in TNBC, but also in luminal breast cancer, and can be reliably assessed using quantitative image analysis of TMAs. Further investigation into the contribution of tumour subtype stromal phenotype may further refine these findings.
Publisher: American Association for Cancer Research (AACR)
Date: 15-05-2023
DOI: 10.1158/1078-0432.22820227
Abstract: Supplementary Table S1. ADT + PI3Ki + PD-1 antibody leads to TAM activation within TME of PTEN 53-deficient prostate tumors.
Publisher: Springer Science and Business Media LLC
Date: 09-2021
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/1078-0432.CCR-22-3350
Abstract: Phosphatase and tensin homolog (PTEN) loss of function occurs in approximately 50% of patients with metastatic castrate-resistant prostate cancer (mCRPC), and is associated with poor prognosis and responsiveness to standard-of-care therapies and immune checkpoint inhibitors. While PTEN loss of function hyperactivates PI3K signaling, combinatorial PI3K/AKT pathway and androgen deprivation therapy (ADT) has demonstrated limited anticancer efficacy in clinical trials. Here, we aimed to elucidate mechanism(s) of resistance to ADT/PI3K-AKT axis blockade, and to develop rational combinatorial strategies to effectively treat this molecular subset of mCRPC. Prostate-specific PTEN 53-deficient genetically engineered mice (GEM) with established 150–200 mm3 tumors, as assessed by ultrasound, were treated with either ADT (degarelix), PI3K inhibitor (copanlisib), or anti–PD-1 antibody (aPD-1), as single agents or their combinations, and tumors were monitored by MRI and harvested for immune, transcriptomic, and proteomic profiling, or ex vivo co-culture studies. Single-cell RNA sequencing on human mCRPC s les was performed using 10X Genomics platform. Coclinical trials in PTEN 53-deficient GEM revealed that recruitment of PD-1–expressing tumor-associated macrophages (TAM) thwarts ADT/PI3Ki combination–induced tumor control. The addition of aPD-1 to ADT/PI3Ki combination led to TAM-dependent approximately 3-fold increase in anticancer responses. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their anticancer phagocytic activation, which was augmented by ADT/aPD-1 treatment and abrogated by feedback activation of Wnt/β-catenin pathway. Single-cell RNA-sequencing analysis in mCRPC patient biopsy s les revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression. Immunometabolic strategies that reverse lactate and PD-1–mediated TAM immunosuppression, in combination with ADT, warrant further investigation in patients with PTEN-deficient mCRPC.
Publisher: Cold Spring Harbor Laboratory
Date: 06-04-2023
DOI: 10.1101/2023.04.06.535805
Abstract: Spatial transcriptomic technologies are powerful tools for resolving the spatial heterogeneity of gene expression in tissue s les. However, little evidence exists on relative strengths and weaknesses of the various available technologies for profiling human tumour tissue. In this study, we aimed to provide an objective assessment of two common spatial transcriptomics platforms, 10X Genomics’ Visium and Nanostring’s GeoMx DSP. The abilities of the DSP and Visium platforms to profile transcriptomic features were compared using matching cell line and primary breast cancer tissue s les. A head-to-head comparison was conducted using data generated from matching s les and synthetic tissue references. Platform specific features were also assessed according to manufacturers’ recommendations to evaluate the optimal usage of the two technologies. We identified substantial variations in assay design between the DSP and Visium assays such as transcriptomic coverage and composition of the transcripts detected. When the data was standardised according to manufacturers’ recommendations, the DSP platform was more sensitive in gene expression detection. However, its specificity was diminished by the presence of non-specific detection. Our results also confirmed the strength and weakness of each platform in characterising spatial transcriptomic features of tissue s les, in particular their application to hypothesis generation versus hypothesis testing. In this study, we share our experience on both DSP and Visium technologies as end users. We hope this can guide future users to choose the most suitable platform for their research. In addition, this dataset can be used as an important resource for the development of new analysis tools.
Publisher: Informa UK Limited
Date: 08-1997
Publisher: Research Square Platform LLC
Date: 13-05-2020
DOI: 10.21203/RS.3.RS-16182/V2
Abstract: Background Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of Differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC, through unknown mechanisms. Methods Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. Results These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical s les demonstrates that ID4 is lified and overexpressed at a higher frequency in BRCA1 -mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. Conclusions These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.
No related grants have been discovered for Alexander Swarbrick.