ORCID Profile
0000-0003-3804-1150
Current Organisation
Garvan Institute of Medical Research
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Publisher: Frontiers Media SA
Date: 21-02-2023
Abstract: Introduction: Dysregulated alternative splicing is a prominent feature of cancer. The inhibition and knockdown of the SR splice factor kinase SRPK1 reduces tumour growth in vivo . As a result several SPRK1 inhibitors are in development including SPHINX, a 3-(trifluoromethyl)anilide scaffold. The objective of this study was to treat two leukaemic cell lines with SPHINX in combination with the established cancer drugs azacitidine and imatinib. Materials and Methods: We selected two representative cell lines Kasumi-1, acute myeloid leukaemia, and K562, BCR-ABL positive chronic myeloid leukaemia. Cells were treated with SPHINX concentrations up to 10μM, and in combination with azacitidine (up to 1.5 μg/ml, Kasumi-1 cells) and imatinib (up to 20 μg/ml, K562 cells). Cell viability was determined by counting the proportion of live cells and those undergoing apoptosis through the detection of activated caspase 3/7. SRPK1 was knocked down with siRNA to confirm SPHINX results. Results: The effects of SPHINX were first confirmed by observing reduced levels of phosphorylated SR proteins. SPHINX significantly reduced cell viability and increased apoptosis in Kasumi-1 cells, but less prominently in K562 cells. Knockdown of SRPK1 by RNA interference similarly reduced cell viability. Combining SPHINX with azacitidine augmented the effect of azacitidine in Kasumi-1 cells. In conclusion, SPHINX reduces cell viability and increases apoptosis in the acute myeloid leukaemia cell line Kasumi-1, but less convincingly in the chronic myeloid leukaemia cell line K562. Conclusion: We suggest that specific types of leukaemia may present an opportunity for the development of SRPK1-targeted therapies to be used in combination with established chemotherapeutic drugs.
Publisher: Springer Science and Business Media LLC
Date: 16-08-2021
DOI: 10.1038/S41388-021-01992-2
Abstract: Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, and is plagued by a paucity of targeted treatment options and tumour resistance to chemotherapeutics. The causal link between chronic inflammation and PDAC suggests that molecular regulators of the immune system promote disease pathogenesis and/or therapeutic resistance, yet their identity is unclear. Here, we couple endoscopic ultrasound-guided fine-needle aspiration, which captures tumour biopsies from all stages, with whole transcriptome profiling of PDAC patient primary tumours to reveal enrichment of the innate immune Toll-like receptor 2 (TLR2) molecular pathway. Augmented TLR2 expression associated with a 4-gene "TLR2 activation" signature, and was prognostic for survival and predictive for gemcitabine-based chemoresistance. Furthermore, antibody-mediated anti-TLR2 therapy suppressed the growth of human PDAC tumour xenografts, independent of a functional immune system. Our results support TLR2-based therapeutic targeting for precision medicine in PDAC, with further clinical utility that TLR2 activation is prognostic and predictive for chemoresponsiveness.
Publisher: Springer Science and Business Media LLC
Date: 15-04-2021
DOI: 10.1038/S41589-021-00783-W
Abstract: CUB domain-containing protein 1 (CDCP1) is an oncogenic orphan transmembrane receptor and a promising target for the detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a new approach for the rapid identification of proteases responsible for key proteolytic events using a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl diphenyl phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe, we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, which acts both by directly cleaving CDCP1 and by activating CDCP1-cleaving plasmin. We show that coexpression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.
Publisher: MyJove Corporation
Date: 22-12-2010
DOI: 10.3791/2055
Publisher: Springer Science and Business Media LLC
Date: 16-03-2015
DOI: 10.1038/NATURE14215
Publisher: Springer International Publishing
Date: 2020
Publisher: Elsevier BV
Date: 06-2021
Publisher: Oxford University Press (OUP)
Date: 09-03-2011
DOI: 10.1093/HMG/DDR090
Abstract: Budding uninhibited by benzimidazole-related 1 (BUBR1) is a central molecule of the spindle assembly checkpoint. Germline mutations in the budding uninhibited by benzimidazoles 1 homolog beta gene encoding BUBR1 cause premature chromatid separation (mosaic variegated aneuploidy) [PCS (MVA)] syndrome, which is characterized by constitutional aneuploidy and a high risk of childhood cancer. Patients with the syndrome often develop Dandy-Walker complex and polycystic kidneys implying a critical role of BUBR1 in morphogenesis. However, little is known about the function of BUBR1 other than mitotic control. Here, we report that BUBR1 is essential for the primary cilium formation, and that the PCS (MVA) syndrome is thus a novel ciliopathy. Morpholino knockdown of bubr1 in medaka fish also caused ciliary dysfunction characterized by defects in cerebellar development and perturbed left-right asymmetry of the embryo. Biochemical analyses demonstrated that BUBR1 is required for ubiquitin-mediated proteasomal degradation of cell ision cycle protein 20 in the G0 phase and maintains anaphase-promoting complex/cyclosome-CDC20 homolog 1 activity that regulates the optimal level of dishevelled for ciliogenesis.
Publisher: Elsevier BV
Date: 2019
Publisher: Springer Science and Business Media LLC
Date: 02-04-2018
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1016/J.GENE.2018.05.095
Abstract: Alternative splicing is a key process required for the regulation of gene expression in normal development and physiology. It is regulated by splice factors whose activities are in turn regulated by splice factor kinases and phosphatases. The CDC-like protein kinases are a widespread family of splice factor kinases involved in normal physiology and in several diseases including cancer. In humans they include the CLK1, CLK2, CLK3 and CLK4 genes. The expression of CLK1 is regulated through alternative splicing producing both full-length catalytically active and truncated catalytically inactive isoforms, CLK
Publisher: Elsevier BV
Date: 12-2020
Publisher: Springer Science and Business Media LLC
Date: 25-06-2020
DOI: 10.1038/S41416-020-0951-2
Abstract: The ERG oncogene, a member of the ETS family of transcription factor encoding genes, is a genetic driver of prostate cancer. It is activated through a fusion with the androgen-responsive TMPRSS2 promoter in 50% of cases. There is therefore significant interest in developing novel therapeutic agents that target ERG . We have taken an antisense approach and designed morpholino-based oligonucleotides that target ERG by inducing skipping of its constitutive exon 4. We designed antisense morpholino oligonucleotides (splice-switching oligonucleotides, SSOs) that target both the 5′ and 3′ splice sites of ERG’s exon 4. We tested their efficacy in terms of inducing exon 4 skipping in two ERG-positive cell lines, VCaP prostate cancer cells and MG63 osteosarcoma cells. We measured their effect on cell proliferation, migration and apoptosis. We also tested their effect on xenograft tumour growth in mice and on ERG protein expression in a human prostate cancer radical prostatectomy s le ex vivo. In VCaP cells, both SSOs were effective at inducing exon 4 skipping, which resulted in a reduction of overall ERG protein levels up to 96 h following a single transfection. SSO-induced ERG reduction decreased cell proliferation, cell migration and significantly increased apoptosis. We observed a concomitant reduction in protein levels for cyclin D1, c-Myc and the Wnt signalling pathway member β-catenin as well as a marker of activated Wnt signalling, p-LRP6. We tested the 3′ splice site SSO in MG63 xenografts in mice and observed a reduction in tumour growth. We also demonstrated that the 3′ splice site SSO caused a reduction in ERG expression in a patient-derived prostate tumour tissue cultured ex vivo. We have successfully designed and tested morpholino-based SSOs that cause a marked reduction in ERG expression, resulting in decreased cell proliferation, a reduced migratory phenotype and increased apoptosis. Our initial tests on mouse xenografts and a human prostate cancer radical prostatectomy specimen indicate that SSOs can be effective for oncogene targeting in vivo. As such, this study encourages further in vivo therapeutic studies using SSOs targeting the ERG oncogene.
Publisher: Frontiers Media SA
Date: 29-11-2021
Abstract: There is an increasing worldwide incidence of patients under 50 years of age presenting with oral squamous cell carcinoma (OSCC). The molecular mechanisms driving disease in this emerging cohort remain unclear, limiting impactful treatment options for these patients. To identify common clinically actionable targets in this cohort, we used whole genome and transcriptomic sequencing of OSCC patient s les from 26 in iduals under 50 years of age. These molecular profiles were compared with those of OSCC patients over 50 years of age (n=11) available from TCGA. We show for the first time that a molecular signature comprising of EGFR lification and increased EGFR RNA abundance is specific to the young subset of OSCC patients. Furthermore, through functional assays using patient tumor-derived cell lines, we reveal that this EGFR lification results in increased activity of the EGFR pathway. Using a panel of clinically relevant EGFR inhibitors we determine that an EGFR - lified patient-derived cell line is responsive to EGFR inhibition, suggesting EGFR lification represents a valid therapeutic target in this subset of OSCC patients. In particular, we demonstrate sensitivity to the second-generation EGFR tyrosine kinase inhibitor afatinib, which offers a new and promising therapeutic avenue versus current EGFR-targeting approaches. We propose that testing for EGFR lification could easily be integrated into current diagnostic workflows and such measures could lead to more personalized treatment approaches and improved outcomes for this younger cohort of OSCC patients.
Publisher: Spandidos Publications
Date: 28-08-2017
DOI: 10.3892/OL.2017.6841
Publisher: No publisher found
Date: 2010
DOI: 10.3791/1937
Publisher: Humana Press
Date: 2011
DOI: 10.1007/978-1-61779-210-6_8
Abstract: The medaka, Oryzias latipes, a small egg-laying freshwater fish, is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens have been carried out. Despite a number of large-scale screens in zebrafish, a substantial number of mutants with new distinct phenotypes were identified in similar large-scale screens in the medaka. This observed difference in phenotype is due to the two species having a unique combination of genetic, biological and evolutional properties. The two genetic models share a whole-genome duplication event over that of tetrapods however, each has independently specialized or lost the function of one of the two paralogues. The two fish species complement each other as genetic systems as straightforward comparison of phenotypes, ease of side-by-side analysis using the same techniques and simple and inexpensive husbandry of mutants make these small teleosts quite powerful in combination. Furthermore, both have draft genome sequences and bioinformatic tools available that facilitate further genetic dissection including whole-genome approaches. Together with the gene-driven approach to generate gene knockout mutants of the fish models, the two fish models complement the mouse in genetically dissecting vertebrate genome functions. The external embryogenesis and transparent embryos of the fish allow systematic isolation of embryonic lethal mutations, the most difficult targets in mammalian mutant screens. This chapter will describe how to work with both medaka and zebrafish almost as one species in a lab, focusing on medaka and highlighting the differences between the medaka and zebrafish systems.
Publisher: MDPI AG
Date: 13-03-2018
DOI: 10.3390/GENES9030161
Publisher: Elsevier BV
Date: 12-2016
DOI: 10.1016/J.CUB.2016.09.037
Abstract: In epithelial tissues, cells expressing oncogenic Ras (hereafter RasV12 cells) are detected by normal neighbors and as a result are often extruded from the tissue [1-6]. RasV12 cells are eliminated apically, suggesting that extrusion may be a tumor-suppressive process. Extrusion depends on E-cadherin-based cell-cell adhesions and signaling to the actin-myosin cytoskeleton [2, 6]. However, the signals underlying detection of the RasV12 cell and triggering extrusion are poorly understood. Here we identify differential EphA2 signaling as the mechanism by which RasV12 cells are detected in epithelial cell sheets. Cell-cell interactions between normal cells and RasV12 cells trigger ephrin-A-EphA2 signaling, which induces a cell repulsion response in RasV12 cells. Concomitantly, RasV12 cell contractility increases in an EphA2-dependent manner. Together, these responses drive the separation of RasV12 cells from normal cells. In the absence of ephrin-A-EphA2 signals, RasV12 cells integrate with normal cells and adopt a pro-invasive morphology. We also show that Drosophila Eph (DEph) is detected in segregating clones of RasV12 cells and is functionally required to drive segregation of RasV12 cells in vivo, suggesting that our in vitro findings are conserved in evolution. We propose that expression of RasV12 in single or small clusters of cells within a healthy epithelium creates ectopic EphA2 boundaries, which drive the segregation and elimination of the transformed cell from the tissue. Thus, deregulation of Eph/ephrin would allow RasV12 cells to go undetected and expand within an epithelium.
Publisher: Springer Science and Business Media LLC
Date: 12-04-2021
DOI: 10.1038/S41598-021-86908-6
Abstract: Dysregulation of alternative splicing is a feature of cancer, both in aetiology and progression. It occurs because of mutations in splice sites or sites that regulate splicing, or because of the altered expression and activity of splice factors and of splice factor kinases that regulate splice factor activity. Recently the CDC2-like kinases (CLKs) have attracted attention due to their increasing involvement in cancer. We measured the effect of the CLK inhibitor, the benzothiazole TG003, on two prostate cancer cell lines. TG003 reduced cell proliferation and increased apoptosis in PC3 and DU145 cells. Conversely, the overexpression of CLK1 in PC3 cells prevented TG003 from reducing cell proliferation. TG003 slowed scratch closure and reduced cell migration and invasion in a transwell assay. TG003 decisively inhibited the growth of a PC3 cell line xenograft in nude mice. We performed a transcriptomic analysis of cells treated with TG003. We report widespread and consistent changes in alternative splicing of cancer-associated genes including CENPE , ESCO2 , CKAP2 , MELK , ASPH and CD164 in both HeLa and PC3 cells. Together these findings suggest that targeting CLKs will provide novel therapeutic opportunities in prostate cancer.
Publisher: EDP Sciences
Date: 10-2019
No related grants have been discovered for Sean Porazinski.