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1.
Cancers (Basel) ; 15(8)2023 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-37190303

RESUMO

Colorectal cancer is one of the world's most prevalent and lethal cancers. Mutations of the KRAS gene occur in ~40% of metastatic colorectal cancers. While this cohort has historically been difficult to manage, the last few years have shown exponential growth in the development of selective inhibitors targeting KRAS mutations. Their foremost mechanism of action utilizes the Switch II binding pocket and Cys12 residue of GDP-bound KRAS proteins in G12C mutants, confining them to their inactive state. Sotorasib and Adagrasib, both FDA-approved for the treatment of non-small cell lung cancer (NSCLC), have been pivotal in paving the way for KRAS G12C inhibitors in the clinical setting. Other KRAS inhibitors in development include a multi-targeting KRAS-mutant drug and a G12D mutant drug. Treatment resistance remains an issue with combination treatment regimens including indirect pathway inhibition and immunotherapy providing possible ways to combat this. While KRAS-mutant selective therapy has come a long way, more work is required to make this an effective and viable option for patients with colorectal cancer.

2.
Cancers (Basel) ; 15(3)2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36765763

RESUMO

Colorectal cancer is an important cause of morbidity and mortality worldwide. The current treatment landscape includes chemotherapy, targeted therapy, immunotherapy, radiotherapy, and surgery. A key challenge to improving patient outcomes is the significant inter-patient heterogeneity in treatment response. Tumour organoids derived from the patients' tumours via surgically resected or endoscopically biopsied tissue, have emerged as promising models for personalised medicine. This review synthesises the findings, to date, of studies which have explored the efficacy of ex vivo organoid sensitivity testing for predicting treatment response. Most studies have focused on predicting the response to standard-of-care radiotherapy and chemotherapy options. There is strong evidence to support organoid sensitivity testing of ionising radiation, 5-fluorouracil, and irinotecan, and to a lesser extent, oxaliplatin and TAS-102. Fewer studies have used organoids to identify patients who are likely to benefit from novel treatment options that otherwise remain in clinical trials. This review also summarises recent advancements in organoid culture to include non-epithelial components of the tumour microenvironment, to allow testing of immunotherapy and certain targeted therapy options. Overall, further prospective trials will support the implementation of organoid-based personalised medicine for colorectal cancer patients in the future.

3.
Cell Rep Methods ; 2(11): 100323, 2022 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-36452869

RESUMO

Researching the murine epigenome in disease models has been hampered by the lack of appropriate and cost-effective DNA methylation arrays. Here we perform a comprehensive, comparative analysis between the Mouse Methylation BeadChip (MMB) and reduced-representation bisulfite sequencing (RRBS) in two murine models of colorectal carcinogenesis. We evaluate the coverage, variability, and ability to identify differential DNA methylation of RRBS and MMB. We show that MMB is an effective tool for profiling the murine methylome that performs comparably with RRBS, identifying similar differentially methylated pathways. Although choice of technology is experiment dependent and will be predicated on the underlying biology being probed, these analyses provide insights into the relative strengths and weaknesses of each approach.


Assuntos
Metilação de DNA , Sulfitos , Animais , Camundongos , Metilação de DNA/genética , Análise de Sequência de DNA , Epigenoma
4.
Cell Death Differ ; 29(11): 2288-2302, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35606410

RESUMO

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.


Assuntos
Neoplasias Colorretais , Fatores de Transcrição , Animais , Camundongos , Neoplasias Colorretais/genética , Epigênese Genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Cancers (Basel) ; 13(20)2021 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-34680315

RESUMO

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.

6.
Br J Cancer ; 124(11): 1820-1827, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33782564

RESUMO

BACKGROUND: Aspirin reduces the incidence of conventional adenomas driven by APC mutation and thus colorectal cancer. The effect of aspirin on the ~20% of colorectal cancers arising via BRAF mutation is yet to be established. METHODS: BrafV637E/+;Villin-CreERT2/+ mice were allocated to a control (n = 86) or aspirin-supplemented (n = 83) diet. After 14 months the incidence of murine serrated lesions, carcinoma and distant metastases were measured by histological examination. RNA was extracted from carcinomas from each cohort and subjected to sequencing to identify differentially expressed genes and molecular pathways. RESULTS: Aspirin did not reduce the incidence of murine serrated lesions or carcinoma when compared to control, however, did significantly reduce lesion size (P = 0.0042). Among the mice with carcinoma there was a significant reduction in the incidence of distant metastasis with aspirin treatment (RR 0.69, 95% CI 0.48-0.90, P = 0.0134). Key pathways underlying metastasis of carcinoma cells include NOTCH, FGFR and PI3K signalling, were significantly downregulated in carcinomas sampled from mice on an aspirin-supplemented diet. CONCLUSIONS: Aspirin reduces the incidence of metastatic Braf mutant carcinoma, although this is not due to a reduction in primary disease. The reduction in metastasis could be attributed to a delay or prevention of molecular changes within the primary site driving metastatic growth.


Assuntos
Adenoma , Aspirina/uso terapêutico , Neoplasias Colorretais , Adenoma/tratamento farmacológico , Adenoma/epidemiologia , Adenoma/genética , Adenoma/patologia , Animais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Feminino , Incidência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Instabilidade de Microssatélites/efeitos dos fármacos , Mutação , Metástase Neoplásica , Proteínas Proto-Oncogênicas B-raf/genética
7.
Clin Cancer Res ; 27(10): 2816-2826, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33632927

RESUMO

PURPOSE: While evidence indicates that Fusobacterium nucleatum (F. nucleatum) may promote colorectal carcinogenesis through its suppressive effect on T-cell-mediated antitumor immunity, the specific T-cell subsets involved remain uncertain. EXPERIMENTAL DESIGN: We measured F. nucleatum DNA within tumor tissue by quantitative PCR on 933 cases (including 128 F. nucleatum-positive cases) among 4,465 incident colorectal carcinoma cases in two prospective cohorts. Multiplex immunofluorescence combined with digital image analysis and machine learning algorithms for CD3, CD4, CD8, CD45RO (PTPRC isoform), and FOXP3 measured various T-cell subsets. We leveraged data on Bifidobacterium, microsatellite instability (MSI), tumor whole-exome sequencing, and M1/M2-type tumor-associated macrophages [TAM; by CD68, CD86, IRF5, MAF, and MRC1 (CD206) multimarker assay]. Using the 4,465 cancer cases and inverse probability weighting method to control for selection bias due to tissue availability, multivariable-adjusted logistic regression analysis assessed the association between F. nucleatum and T-cell subsets. RESULTS: The amount of F. nucleatum was inversely associated with tumor stromal CD3+ lymphocytes [multivariable OR, 0.47; 95% confidence interval (CI), 0.28-0.79, for F. nucleatum-high vs. -negative category; P trend = 0.0004] and specifically stromal CD3+CD4+CD45RO+ cells (corresponding multivariable OR, 0.52; 95% CI, 0.32-0.85; P trend = 0.003). These relationships did not substantially differ by MSI status, neoantigen load, or exome-wide tumor mutational burden. F. nucleatum was not significantly associated with tumor intraepithelial T cells or with M1 or M2 TAMs. CONCLUSIONS: The amount of tissue F. nucleatum is associated with lower density of stromal memory helper T cells. Our findings provide evidence for the interactive pathogenic roles of microbiota and specific immune cells.


Assuntos
Neoplasias Colorretais/etiologia , Infecções por Fusobacterium/complicações , Infecções por Fusobacterium/imunologia , Infecções por Fusobacterium/microbiologia , Fusobacterium nucleatum/fisiologia , Subpopulações de Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Adulto , Idoso , Biomarcadores , Biomarcadores Tumorais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/metabolismo , Suscetibilidade a Doenças , Feminino , Imunofluorescência , Infecções por Fusobacterium/epidemiologia , Humanos , Imuno-Histoquímica , Incidência , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pessoa de Meia-Idade , Vigilância da População , Subpopulações de Linfócitos T/metabolismo , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Estados Unidos/epidemiologia
8.
Dig Dis Sci ; 66(12): 4326-4332, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33387125

RESUMO

BACKGROUND: Colorectal cancer is a leading cause of cancer-related death worldwide and approximately 20% of cases can be attributed to a mutation in the BRAF oncogene. Curcumin is a promising chemopreventive agent with various anti-cancer benefits. Although curcumin has been reported to have poor bioavailability, this limitation has been overcome by the formulation of nano-carriers. In this preclinical study, we investigated the ability of an improved formulation of curcumin to reduce the incidence of Braf mutant carcinoma. AIM: To investigate curcumin as a chemopreventive for Braf mutant colorectal cancer in a preclinical study utilizing a murine model of serrated neoplasia. METHODS: An intestine-specific Braf mutant murine model (BrafV637E/+/Villin-CreERT2/+) was administered curcumin micelles (240 mg/kg, n = 69) in normal drinking water. Mice in the control group consumed normal drinking water (n = 83). Mice were euthanized at 14 months and the incidence of murine serrated lesions and carcinoma in each cohort were determined by histologic examination. RESULTS: At completion of the study (14 months), it was found that curcumin did not reduce the incidence or multiplicity of murine serrated lesions but did significantly reduce the number of invasive carcinomas (RR 0.83, 95% CI 0.69-0.9985, P = 0.0360) compared to control. CONCLUSIONS: We have performed the first long-term study assessing curcumin's effect on the development of serrated neoplasia. We found that curcumin significantly reduces the risk of developing Braf mutant colorectal cancer. Our data supports further investigation of curcumin as a chemopreventive to reduce the risk of colorectal cancer arising via the serrated pathway.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma/prevenção & controle , Neoplasias Colorretais/prevenção & controle , Curcumina/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Quimioprevenção , Neoplasias Colorretais/genética , Curcuma , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Camundongos , Fitoterapia
9.
J Nucl Med ; 61(11): 1576-1579, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32358088

RESUMO

At diagnosis, 22% of colorectal cancer (CRC) patients have metastases, and 50% later develop metastasis. Peptide receptor radionuclide therapy (PRRT), such as 177Lu-PSMA-617, is used to treat metastatic prostate cancer. 177Lu-PSMA-617 targets prostate-specific membrane antigen (PSMA), a cell-surface protein enriched in prostate cancer and the neovasculature of other solid tumors, including CRC. We performed 68Ga-PSMA-11 PET/CT imaging of 10 patients with metastatic CRC to assess metastasis avidity. Eight patients had lesions lacking avidity, and 2 had solitary metastases exhibiting very low avidity. Despite expression of PSMA in CRC neovasculature, none of the patients exhibited tumor avidity sufficient to be considered for 177Lu-PSMA-617 PRRT.


Assuntos
Neoplasias Colorretais/diagnóstico por imagem , Ácido Edético/análogos & derivados , Oligopeptídeos/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Dipeptídeos/uso terapêutico , Ácido Edético/farmacocinética , Isótopos de Gálio , Radioisótopos de Gálio , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Humanos , Lutécio , Metástase Neoplásica , Antígeno Prostático Específico , Radioisótopos
10.
Cancers (Basel) ; 12(5)2020 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-32384699

RESUMO

BACKGROUND: WNT activation is a hallmark of colorectal cancer. BRAF mutation is present in 15% of colorectal cancers, and the role of mutations in WNT signaling regulators in this context is unclear. Here, we evaluate the mutational landscape of WNT signaling regulators in BRAF mutant cancers. METHODS: we performed exome-sequencing on 24 BRAF mutant colorectal cancers and analyzed these data in combination with 175 publicly available BRAF mutant colorectal cancer exomes. We assessed the somatic mutational landscape of WNT signaling regulators, and performed hotspot and driver mutation analyses to identify potential drivers of WNT signaling. The effects of Apc and Braf mutation were modelled, in vivo, using the Apcmin/+ and BrafV637/Villin-CreERT2/+ mouse, respectively. RESULTS: RNF43 was the most frequently mutated WNT signaling regulator (41%). Mutations in the beta-catenin destruction complex occurred in 48% of cancers. Hotspot analyses identified potential cancer driver genes in the WNT signaling cascade, including MEN1, GNG12 and WNT16. Truncating APC mutation was identified in 20.8% of cancers. Truncating APC mutation was associated with early age at diagnosis (p < 2 × 10-5), advanced stage (p < 0.01), and poor survival (p = 0.026). Apcmin/+/BrafV637 animals had more numerous and larger SI and colonic lesions (p < 0.0001 and p < 0.05, respectively), and a markedly reduced survival (median survival: 3.2 months, p = 8.8 × 10-21), compared to animals with Apc or Braf mutation alone. CONCLUSIONS: the WNT signaling axis is frequently mutated in BRAF mutant colorectal cancers. WNT16 and MEN1 may be novel drivers of aberrant WNT signaling in colorectal cancer. Co-mutation of BRAF and APC generates an extremely aggressive neoplastic phenotype that is associated with poor patient outcome.

11.
Neoplasia ; 22(2): 120-128, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31935636

RESUMO

The serrated neoplasia pathway gives rise to a distinct subgroup of colorectal cancers distinguished by the presence of mutant BRAFV600E and the CpG Island Methylator Phenotype (CIMP). BRAF mutant CRC are commonly associated with microsatellite instability, which have an excellent clinical outcome. However, a proportion of BRAF mutant CRC retain microsatellite stability and have a dismal prognosis. The molecular drivers responsible for the development of this cancer subgroup are unknown. To address this, we established a murine model of BRAFV600E mutant microsatellite stable CRC and comprehensively investigated the exome and transcriptome to identify molecular alterations in signaling pathways that drive malignancy. Exome sequencing of murine serrated lesions (mSL) and carcinomas identified frequent hot spot mutations within the gene encoding ß-catenin (Ctnnb1). Immunohistochemical staining of ß-catenin indicated that these mutations led to an increase in the presence of aberrant nuclear ß-catenin that resulted in gene expression changes in targets of ß-catenin transcription. Gene expression profiling identified a significant enrichment for transforming growth factor-ß (TGF-ß) signaling that was present in mSL and carcinomas. Early activation of TGF-ß suggests that this pathway may be an early cue directing mSL to microsatellite stable carcinoma. These findings in the mouse model support the importance of alterations in WNT and TGF-ß signaling during the transition of human sessile serrated lesions to malignancy.


Assuntos
Neoplasias Colorretais/genética , Proteínas Proto-Oncogênicas B-raf/genética , Fator de Crescimento Transformador beta/genética , beta Catenina/genética , Animais , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Metilação de DNA/genética , Modelos Animais de Doenças , Humanos , Camundongos , Instabilidade de Microssatélites , Repetições de Microssatélites/genética , Mutação/genética , Sequenciamento do Exoma , Via de Sinalização Wnt/genética
12.
Hum Pathol ; 97: 19-28, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31917154

RESUMO

Patients with inflammatory bowel disease (IBD) have an increased risk of colorectal carcinoma. The significance of serrated lesions resembling traditional serrated adenoma (TSA) in IBD patients is unclear. In this retrospective study, we analyzed 52 TSA-like lesions arising in 30 IBD patients and diagnosed in colectomy or endoscopic specimens. The 27 colectomy lesions presented predominantly as ill-defined areas with granular appearance, with a median size of 15 mm, located throughout the large bowel and associated with synchronous advanced colorectal lesions in 58%. Low-grade serrated dysplasia was present in 56%, high-grade serrated dysplasia in 37%, and TSA-type cytology in 7%. Increased Ki-67 immunostaining and abnormal p53 expression were identified in 96% and 48%, respectively; 74% had a KRAS mutation, and 4% had a BRAF mutation. Endoscopically resectable TSA-like lesions were all discrete polypoid lesions, smaller in size (median 9 mm), predominantly in the distal large bowel, with an adjacent precursor polyp in 24%, and associated with synchronous and metachronous advanced colorectal lesions in 6%. Most (92%) show TSA-type cytology. p53 overexpression was present in 4%, KRAS mutation in 41%, and BRAF mutation in 32%. None of the 52 TSA-like lesions demonstrated loss of MLH1 or SATB2 expression by immunohistochemistry. On follow-up, 4 patients were diagnosed with colorectal carcinoma or high-grade adenomatous IBD-associated dysplasia. None of the patients with lesions showing TSA-type cytology only developed an advanced lesion. Our findings suggest that some TSA-like lesions, essentially from colectomy, may represent a form of IBD-associated dysplasia associated with an increased risk of advanced neoplasia.


Assuntos
Pólipos Adenomatosos/patologia , Carcinoma/patologia , Colite Ulcerativa/patologia , Pólipos do Colo/patologia , Neoplasias Colorretais/patologia , Doença de Crohn/patologia , Lesões Pré-Cancerosas/patologia , Pólipos Adenomatosos/química , Pólipos Adenomatosos/genética , Pólipos Adenomatosos/cirurgia , Adulto , Idoso , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Carcinoma/química , Carcinoma/genética , Carcinoma/cirurgia , Colectomia , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Colite Ulcerativa/cirurgia , Pólipos do Colo/química , Pólipos do Colo/genética , Pólipos do Colo/cirurgia , Colonoscopia , Neoplasias Colorretais/química , Neoplasias Colorretais/genética , Neoplasias Colorretais/cirurgia , Doença de Crohn/genética , Doença de Crohn/metabolismo , Doença de Crohn/cirurgia , Progressão da Doença , Feminino , Humanos , Antígeno Ki-67/análise , Masculino , Pessoa de Meia-Idade , Mutação , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/cirurgia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Estudos Retrospectivos , Resultado do Tratamento , Proteína Supressora de Tumor p53/análise , Adulto Jovem
13.
Clin Epigenetics ; 11(1): 90, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31200767

RESUMO

BACKGROUND: Sessile serrated adenomas (SSAs) are common polyps which give rise to 20-30% of colorectal cancer (CRC). SSAs display clinicopathologic features which present challenges in surveillance, including overrepresentation in young patients, proclivity for the proximal colon and rarity of histologic dysplasia (referred to then as SSAs with dysplasia, SSADs). Once dysplasia develops, there is rapid progression to CRC, even at a small size. There is therefore a clinical need to separate the "advanced" SSAs at high risk of progression to SSAD and cancer from ordinary SSAs. Since SSAs are known to accumulate methylation over time prior to the development of dysplasia, SSAD backgrounds (the remnant SSA present within an SSAD) likely harbour additional methylation events compared with ordinary SSAs. We therefore performed MethyLight and comprehensive methylation array (Illumina MethylationEPIC) on 40 SSAD backgrounds and 40 matched ordinary SSAs, and compared the methylation results with CRC methylation, CRC expression and immunohistochemical data. RESULTS: SSAD backgrounds demonstrated significant hypermethylation of CpG islands compared with ordinary SSAs, and the proportion of hypermethylated probes decreased progressively in the shore, shelf and open sea regions. Hypomethylation occurred in concert with hypermethylation, which showed a reverse pattern, increasing progressively away from the island regions. These methylation changes were also identified in BRAF-mutant hypermethylated CRCs. When compared with CRC expression data, SV2B, MLH1/EPM2AIP1, C16orf62, RCOR3, BAIAP3, OGDHL, HDHD3 and ATP1B2 demonstrated both promoter hypermethylation and decreased expression. Although SSAD backgrounds were histologically indistinguishable from ordinary SSAs, MLH1 methylation was detectable via MethyLight in 62.9% of SSAD backgrounds, and focal immunohistochemical MLH1 loss was seen in 52.5% of SSAD backgrounds. CONCLUSIONS: Significant hyper- and hypomethylation events occur during SSA progression well before the development of histologically identifiable changes. Methylation is a heterogeneous process within individual SSAs, as typified by MLH1, where both MLH1 methylation and focal immunohistochemical MLH1 loss can be seen in the absence of dysplasia. This heterogeneity is likely a generalised phenomenon and should be taken into account in future methylation-based studies and the development of clinical methylation panels.


Assuntos
Adenoma/genética , Neoplasias Colorretais/genética , Metilação de DNA , Proteína 1 Homóloga a MutL/genética , Adenoma/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Neoplasias Colorretais/metabolismo , Ilhas de CpG , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Proteína 1 Homóloga a MutL/metabolismo , Regiões Promotoras Genéticas
14.
Cancer Immunol Res ; 7(4): 559-571, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30894377

RESUMO

CD96 is a novel target for cancer immunotherapy shown to regulate NK cell effector function and metastasis. Here, we demonstrated that blocking CD96 suppressed primary tumor growth in a number of experimental mouse tumor models in a CD8+ T cell-dependent manner. DNAM-1/CD226, Batf3, IL12p35, and IFNγ were also critical, and CD96-deficient CD8+ T cells promoted greater tumor control than CD96-sufficient CD8+ T cells. The antitumor activity of anti-CD96 therapy was independent of Fc-mediated effector function and was more effective in dual combination with blockade of a number of immune checkpoints, including PD-1, PD-L1, TIGIT, and CTLA-4. We consistently observed coexpression of PD-1 with CD96 on CD8+ T lymphocytes in tumor-infiltrating leukocytes both in mouse and human cancers using mRNA analysis, flow cytometry, and multiplex IHF. The combination of anti-CD96 with anti-PD-1 increased the percentage of IFNγ-expressing CD8+ T lymphocytes. Addition of anti-CD96 to anti-PD-1 and anti-TIGIT resulted in superior antitumor responses, regardless of the ability of the anti-TIGIT isotype to engage FcR. The optimal triple combination was also dependent upon CD8+ T cells and IFNγ. Overall, these data demonstrate that CD96 is an immune checkpoint on CD8+ T cells and that blocking CD96 in combination with other immune-checkpoint inhibitors is a strategy to enhance T-cell activity and suppress tumor growth.


Assuntos
Antígenos CD/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Transferência Adotiva , Animais , Antígenos CD/genética , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/terapia
15.
Gut ; 68(4): 684-692, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29666172

RESUMO

OBJECTIVE: 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. DESIGN: 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. RESULTS: 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 BrafV600E 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. CONCLUSION: 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.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Organoides/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Adenocarcinoma/metabolismo , Alelos , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Ilhas de CpG/genética , Reparo de Erro de Pareamento de DNA , Análise Mutacional de DNA , Progressão da Doença , Epigenômica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Modelos Genéticos , Mutação , Organoides/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas B-raf/metabolismo
16.
Gastroenterology ; 155(5): 1362-1365.e2, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30009818

RESUMO

Among sessile serrated adenomas (SSAs) with identical histologic features, some never progress, whereas others become dysplastic and develop into invasive cancers. Development of the CpG island methylator phenotype is a feature of SSA progression; we examined the CIMP status of 448 SSAs and examined the association with patient clinical data. Overall, 190 SSAs were CpG island methylator phenotype-positive. CpG island methylator phenotype positivity was associated with older patient age (P < .001) and proximal polyp site (P < .001), but not with patient sex (P = .94) or polyp size (P = .34). These results might be used to improve SSA surveillance guidelines.


Assuntos
Adenoma/genética , Ilhas de CpG , Metilação de DNA , Neoplasias/etiologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/genética
17.
Fam Cancer ; 17(1): 63-69, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28573495

RESUMO

The WNT signaling pathway is commonly altered during colorectal cancer development. The E3 ubiquitin ligase, RNF43, negatively regulates the WNT signal through increased ubiquitination and subsequent degradation of the Frizzled receptor. RNF43 has recently been reported to harbor frequent truncating frameshift mutations in sporadic microsatellite unstable (MSI) colorectal cancers. This study assesses the relative frequency of RNF43 mutations in hereditary colorectal cancers arising in the setting of Lynch syndrome. The entire coding region of RNF43 was Sanger sequenced in 24 colorectal cancers from 23 patients who either (i) carried a germline mutation in one of the DNA mismatch repair genes (MLH1, MSH6, MSH2, PMS2), or (ii) showed immunohistochemical loss of expression of one or more of the DNA mismatch repair proteins, was BRAF wild type at V600E, were under 60 years of age at diagnosis, and demonstrated no promoter region methylation for MLH1 in tumor DNA. A validation cohort of 44 colorectal cancers from mismatch repair germline mutation carriers from the Australasian Colorectal Cancer Family Registry (ACCFR) were sequenced for the most common truncating mutation hotspots (X117 and X659). RNF43 mutations were found in 9 of 24 (37.5%) Lynch syndrome colorectal cancers. The majority of mutations were frameshift deletions in the G659 G7 repeat tract (29%); 2 cancers (2/24, 8%) from the one patient harbored frameshift mutations at codon R117 (C6 repeat tract) within exon 3. In the ACCFR validation cohort, RNF43 hotspot mutations were identified in 19/44 (43.2%) of samples, which was not significantly different to the initial series. The proportion of mutant RNF43 in Lynch syndrome related colorectal cancers is significantly lower than the previously reported mutation rate found in sporadic MSI colorectal cancers. These findings identify further genetic differences between sporadic and hereditary colorectal cancers. This may be because Lynch Syndrome cancers commonly arise in colorectal adenomas already bearing the APC mutation, whereas sporadic microsatellite unstable colorectal cancers arise from serrated polyps typically lacking APC mutation, decreasing the selection pressure on other WNT signaling related loci in Lynch syndrome.


Assuntos
Adenoma/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Instabilidade de Microssatélites , Proteínas Oncogênicas/genética , Proteína da Polipose Adenomatosa do Colo/genética , Adulto , Idoso , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Seleção Genética , Ubiquitina-Proteína Ligases , Via de Sinalização Wnt/genética
18.
Gastroenterol Res Pract ; 2018: 9250757, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30598662

RESUMO

The BRAF oncogene is an integral component of the MAP kinase pathway, and an activating V600E mutation occurs in 15% of sporadic colorectal cancer. This is an early event in serrated pathway tumourigenesis, and the BRAF V600E has been commonly associated with the CpG island methylator phenotype, microsatellite instability (MSI), and a consistent clinical presentation including a proximal location and predilection for elderly females. A proportion of the BRAF mutant lesions remain as microsatellite stable (MSS), and in contrast to the MSI cancers, they have an aggressive phenotype and correlate with poor patient outcomes. Recent studies have found that they have clinical and molecular features of both the BRAF mutant/MSI and the conventional BRAF wild-type cancers and comprise a distinct colorectal cancer subgroup. This review highlights the importance of the BRAF mutation occurring in colorectal cancer stratified for molecular background and discusses its prognostic and clinical significance.

19.
Epigenetics ; 13(1): 40-48, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29235923

RESUMO

Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.


Assuntos
Neoplasias Colorretais/genética , Metilação de DNA , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Neoplasias Colorretais/patologia , Reparo de Erro de Pareamento de DNA , Humanos , Hiperplasia/genética , Hiperplasia/patologia , Intestino Delgado/patologia , Camundongos Endogâmicos C57BL , Instabilidade de Microssatélites , Neoplasias Experimentais/etiologia , Proteínas Proto-Oncogênicas B-raf/metabolismo
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