ORCID Profile
0000-0003-3214-3527
Current Organisations
Monash University
,
QIMR Berghofer
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Publisher: Elsevier BV
Date: 11-2022
Publisher: BMJ
Date: 06-07-2022
DOI: 10.1136/GUTJNL-2020-322166
Abstract: Sessile serrated lesions (SSLs) are common across the age spectrum, but the BRAF mutant cancers arising occur predominantly in the elderly. Aberrant DNA methylation is uncommon in SSL from young patients. Here, we interrogate the role of ageing and DNA methylation in SSL initiation and progression. We used an inducible model of Braf mutation to direct recombination of the oncogenic Braf V637E allele to the murine intestine. BRAF mutation was activated after periods of ageing, and tissue was assessed for histological, DNA methylation and gene expression changes thereafter. We also investigated DNA methylation alterations in human SSLs. Inducing Braf mutation in aged mice was associated with a 10-fold relative risk of serrated lesions compared with young mice. There were extensive differences in age-associated DNA methylation between animals induced at 9 months versus wean, with relatively little differential Braf- specific methylation. DNA methylation at WNT pathway genes scales with age and Braf mutation accelerated age-associated DNA methylation. In human SSLs, increased epigenetic age was associated with high-risk serrated colorectal neoplasia. SSLs arising in the aged intestine are at a significantly higher risk of spontaneous neoplastic progression. These findings provide support for a new conceptual model for serrated colorectal carcinogenesis, whereby risk of Braf- induced neoplastic transformation is dependent on age and may be related to age-associated molecular alterations that accumulate in the ageing intestine, including DNA methylation. This may have implications for surveillance and chemopreventive strategies targeting the epigenome.
Publisher: Springer Science and Business Media LLC
Date: 23-05-2022
DOI: 10.1038/S41418-022-01016-W
Abstract: Colorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1 . Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC.
Publisher: Impact Journals, LLC
Date: 15-12-2017
Publisher: BMJ
Date: 17-04-2019
DOI: 10.1136/GUTJNL-2017-315920
Abstract: Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF , and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not Braf V600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.
Publisher: MDPI AG
Date: 14-10-2021
Abstract: Background. The typical methylation patterns associated with cancer are hypermethylation at gene promoters and global genome hypomethylation. Aberrant CpG island hypermethylation at promoter regions and global genome hypomethylation have not been associated with histological colorectal carcinomas (CRC) subsets. Using Illumina’s 450 k Infinium Human Methylation beadchip, the methylome of 82 CRCs were analyzed, comprising different histological subtypes: 40 serrated adenocarcinomas (SAC), 32 conventional carcinomas (CC) and 10 CRCs showing histological and molecular features of microsatellite instability (hmMSI-H), and, additionally, 35 normal adjacent mucosae. Scores reflecting the overall methylation at 250 bp, 1 kb and 2 kb from the transcription starting site (TSS) were studied. Results. SAC has an intermediate methylation pattern between CC and hmMSI-H for the three genome locations. In addition, the shift from promoter hypermethylation to genomic hypomethylation occurs at a small sequence between 250 bp and 1 Kb from the gene TSS, and an asymmetric distribution of methylation was observed between both sides of the CpG islands (N vs. S shores). Conclusion. These findings show that different histological subtypes of CRC have a particular global methylation pattern depending on sequence distance to TSS and highlight the so far underestimated importance of CpGs aberrantly hypomethylated in the clinical phenotype of CRCs.
Publisher: F1000Research
Date: 2016
Publisher: American Association for Cancer Research (AACR)
Date: 07-2017
DOI: 10.1158/1538-7445.AM2017-5375
Abstract: BACKGROUND: The serrated colorectal neoplasia pathway describes the progression of morphologically serrated polyps to cancer and accounts for approximately one third of all colorectal cancer cases. Human serrated polyps are characterised by activating mutation of the BRAF oncogene and widespread DNA methylation changes termed the CpG Island Methylator Phenotype. A causative versus synergistic relationship between BRAF mutation and this methylator phenotype has not been determined. We aimed to address this by developing a murine model for serrated neoplasia driven by BRAF mutation. METHODS: BrafV637E conditionally active mice were crossed with intestine-specific, inducible Villin-CreERT2 mice to direct the BRAF mutation to the intestine at 2 weeks of age. The proximal ilieum or proximal colon were s led at defined time points including 10 days, 10 weeks, 5 months, 8 months, 10 months, 12 months and 14 months. Macroscopic lesions larger than 10mm were bisected for molecular and histological assessment. The entire remaining intestine was fixed and examined histologically. DNA methylation was investigated for 94 genes known to by methylated in colorectal cancer using Epitect MethylII Complete PCR Arrays (Qiagen). RESULTS: Braf mutant mice displayed histologic changes analogous to the human serrated neoplasia pathway. Extensive intestinal hyperplasia developed by 10 days post induction of the BRAF mutation. By 10 weeks, 50% mice had developed areas of crypt dilation reminiscent of human sessile serrated adenomas. By 8 months, the majority of mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months, one of which metastasised to the liver. Compared to age-matched control mice, Braf mutant mice showed significant, gene-specific increases in DNA methylation from 5 months (p& .0001). CONCLUSIONS: Using an in vivo model we observed the temporal accumulation of DNA methylation changes in hyperplastic epithelium in direct response to mutation of the BRAF oncogene. This murine model morphologically and molecularly recapitulates the human serrated neoplasia pathway and establishes a causative role for BRAF mutation in establishing a methylator phenotype. Note: This abstract was not presented at the meeting. Citation Format: Vicki Whitehall, Catherine Bond, Cheng Liu, Futoshi Kawamata, Diane McKeone, Saara Jamieson, Sally Pearson, Susan Woods, Tamsin Lannagan, Lochlan Fennell, Winnie Fernando, Mark Bettington, Daniel Worthley, Barbara Leggett. Oncogenic BRAF mutation induces widespread DNA hypermethylation in a murine model for human serrated colorectal neoplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017 2017 Apr 1-5 Washington, DC. Philadelphia (PA): AACR Cancer Res 2017 (13 Suppl):Abstract nr 5375. doi:10.1158/1538-7445.AM2017-5375
Publisher: Springer Science and Business Media LLC
Date: 06-2017
Publisher: Elsevier BV
Date: 02-2020
Publisher: Springer Science and Business Media LLC
Date: 14-06-2019
Publisher: Informa UK Limited
Date: 02-01-2018
Publisher: University of Queensland Library
Date: 2021
DOI: 10.14264/4E1B52A
Publisher: Elsevier BV
Date: 03-2018
DOI: 10.1038/MODPATHOL.2017.150
Abstract: Conventional adenomas are initiated by APC gene mutation that activates the WNT signal. Serrated neoplasia is commonly initiated by BRAF or KRAS mutation. WNT pathway activation may also occur, however, to what extent this is owing to APC mutation is unknown. We examined aberrant nuclear β-catenin immunolocalization as a surrogate for WNT pathway activation and analyzed the entire APC gene coding sequence in serrated and conventional pathway polyps and cancers. WNT pathway activation was a common event in conventional pathway lesions with aberrant nuclear immunolocalization of β-catenin and truncating APC mutations in 90% and 89% of conventional adenomas and 82% and 70% of BRAF wild-type cancers, respectively. WNT pathway activation was seen to a lesser extent in serrated pathway lesions. It occurred at the transition to dysplasia in serrated polyps with a significant increase in nuclear β-catenin labeling from sessile serrated adenomas (10%) to sessile serrated adenomas with dysplasia (55%) and traditional serrated adenomas (9%) to traditional serrated adenomas with dysplasia (39%) (P=0.0001). However, unlike the conventional pathway, truncating APC mutations were rare in the serrated pathway lesions especially sessile serrated adenomas even when dysplastic (15%) and in the BRAF mutant cancers with microsatellite instability that arise from them (8%). In contrast, APC missense mutations that were rare in conventional pathway adenomas and cancers (3% in BRAF wild-type cancers) were more frequent in BRAF mutant cancers with microsatellite instability (32%). We conclude that increased WNT signaling is important in the transition to malignancy in the serrated pathway but that APC mutation is less common and the spectrum of mutations is different than in conventional colorectal carcinogenesis. Moderate impact APC mutations and non-APC-related causes of increased WNT signaling may have a more important role in serrated neoplasia than the truncating APC mutations common in conventional adenomas.
Publisher: Elsevier BV
Date: 2019
Publisher: Springer Science and Business Media LLC
Date: 05-01-2018
Publisher: American Association for Cancer Research (AACR)
Date: 31-01-2017
DOI: 10.1158/1538-7445.CRC16-PR04
Abstract: The WNT and MAPK signaling pathways are central to colorectal homeostasis. Mutation of the APC tumor suppressor gene activates the WNT pathway to initiate most conventional adenomas. By contrast, oncogenic BRAF mutation initiates development of sessile serrated adenomas (SSA) via activation of the MAPK pathway. The purpose of this study was to investigate the frequency, timing and mechanism of WNT pathway activation in serrated colorectal neoplasia. Methods: The WNT pathway was examined in a range of colorectal polyp and cancer subtypes. Aberrant nuclear immuno-localization of β-catenin was used as a surrogate for WNT pathway activation, compared to the normal membranous staining pattern. The entire coding region of the APC gene was sequenced using a custom enrichment panel that included 242 primer pairs (GeneRead DNAseq Targeted Panel, Qiagen) and the constructed library was sequenced using Illumina's HT TruSeq Kit. As another mechanism of activating WNT, mutations were also assessed in the E3 ubiquitin ligase genes RNF43 and ZNRF3 using Sanger sequencing. To test the interaction of the WNT and MAPK pathways in vivo, a conditional BRAF mutant mouse model was crossed with an inducible Cre driven by the murine Villin gene promoter, to restrict Cre expression and induction of the BRAF mutation to the intestine at 2 weeks. The chemical carcinogen azoxymethane was administered weekly for 6 weeks to activate the WNT pathway from 3 weeks of age. The potential therapeutic implication of targeting these pathways was tested in vitro using the porcupine inhibitor LGK974 and MEK inhibitor PD0325901. Results: The majority of conventional pathway polyps and cancers had nuclear localization of β-catenin (22/28, 79% adenomas and 36/42, 86% BRAF wildtype cancers) and mutations in APC (17/20, 85% adenomas and 19/30, 63% BRAF wild type cancers). Sessile serrated adenomas infrequently showed nuclear staining of β-catenin and 0/20 SSA had mutations. By contrast, the WNT signal was increased in the dysplastic component of SSA with a focus of dysplasia (76/137, 55%), however APC mutations were uncommon (3/20, 15%). Similarly, BRAF mutant cancers had nuclear β-catenin in 36/92 (39%) cancers compared to APC mutation in only 9/80 (11%) s les. This did not significantly differ when s les were stratified by microsatellite instability status. RNF43 and ZNRF3 were commonly mutated in BRAF mutant / microsatellite unstable cancers (47/54, 87% and 16/54, 30%, respectively) as well as BRAF mutant / microsatellite stable cancers (8/33, 24% and 5/33, 15%) compared to only 3/79 (4%) RNF43 mutations and 0/27 ZNRF3 mutations in BRAF wildtype cancers. This was accompanied by lower transcript expression for both RNF43 and ZNRF3 in BRAF mutant compared to BRAF wildtype cancers (P& .0001). We further demonstrated the importance of the WNT pathway in progression of serrated neoplasia by administering azoxymethane to mice with BRAF mutant-induced intestinal hyperplasia. This accelerated the serrated phenotype from 0.6 polyps per mouse to 5.9 per mouse (P& .001). These all showed typical murine serrated adenoma morphology. To investigate potential therapeutic implications we treated colorectal cancer cell lines with the porcupine inhibitor LGK974 and showed a 50% reduction in growth only in RNF43 and/or ZNRF3 mutant cell lines. In the BRAF / RNF43 / ZNRF3 mutant cell line RKO, LGK974 synergized with MEK inhibition to dramatically reduce growth by approximately 95%. Conclusions: The WNT pathway is commonly activated in serrated neoplasia at the transition to dysplasia. This is not due APC mutation but rather mutation of the upstream RNF43 and/or ZNRF43 genes or other WNT pathway targets. The combination of WNT and MAPK pathway inhibition offers potential for improving therapy for patients with serrated pathway cancers. This abstract is also being presented as Poster A21. Citation Format: Vicki Whitehall, Jennifer Borowsky, Catherine Bond, Mark Bettington, Sally-Ann Pearson, Murugan Kalimutho, John Liu, Troy Dumenil, Diane McKeone, Saara Jamieson, Winnie Fernando, Lochlan Fennell, Andrew Clouston, Christophe Rosty, Ian Brown, Neal Walker, Barbara Leggett. Wnt and MAPK pathway activation in conventional and serrated colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes 2016 Sep 17-20 T a, FL. Philadelphia (PA): AACR Cancer Res 2017 (3 Suppl):Abstract nr PR04.
Start Date: 2022
End Date: 2024
Funder: National Health and Medical Research Council
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