transforming-growth-factor-beta and Microsatellite-Instability

It is sometimes difficult but finally possible to distinguish colitis-associated neoplasms from sporadic neoplasms. The frequency of detection of precursor lesions of carcinoma (e.g. dysplasia, intraepithelial neoplasia and adenoma) has increased in recent years, which is most probably due to better endoscopic detection and thus improved histological diagnosis. Carcinogenesis of colitis-associated neoplasms is different from carcinogenesis in sporadic neoplasms because mutations and epigenetic changes are different or may occur at a different point in time. In the present article, these differences will be described and placed in context with carcinogenesis in ulcerative colitis.

Topics: Adenoma; Carcinoma in Situ; Cell Transformation, Neoplastic; Chromosome Aberrations; Colitis, Ulcerative; Colonic Neoplasms; DNA Methylation; DNA Mutational Analysis; Epigenesis, Genetic; Humans; Intestinal Mucosa; Microsatellite Instability; Neoplasm Invasiveness; Neoplasm Staging; Oxidative Stress; Precancerous Conditions; Reactive Oxygen Species; Transforming Growth Factor beta

Recent progress in the field of molecular biology has allowed us to identify at least two different molecular mechanisms implicated in colorectal carcinogenesis (CRC): chromosomal instability (CIN) and genetic instability. Even though the two molecular mechanisms differ, their signalling pathways, implicated in malignant transformation of colonic epithelial cells, appear to be similar. The most frequent group of CRC, which represents 80% of sporadic CRC, is characterized by allelic losses on the short arm of chromosome 17 and 8 and on the long arm of chromosome 5, 18 and 22. These allelic losses are associated with mutations in TP53, APC, SMAD2 and SMAD4 genes. All of these alterations are grouped under the phenotype CIN. A genetic instability termed MSI (microsatellite instability), which results from a mismatch repair (MMR) deficiency, appears in 12-15% of CRC cases. The presence of MMR deficiency leads to the accumulation of mutations in genes controlling cell cycle and apoptosis (TGFBRII, BAX or CASPASE5). More recently, the existence of a third phenotype was suggested. The main alteration associated with this group of tumors is the hypermethylation of the promoter region of numerous genes, leading to their inactivation. An activating mutation of BRAF is frequently associated with this phenotype. As described above, CRC shows genetic heterogeneity, however the consequences in terms of signalling pathway alterations are similar. For example, the activation of Wnt signalling pathways can result from the inactivation of the APC gene in the CIN phenotype or from an activating mutation in the β-catenin gene in MSI tumors. The inactivation of TGFβ pathways is also present in both tumor types and is driven by SMAD4, and more rarely by a SMAD2 inactivating mutation in CIN tumors, or by the existence of a frame-shift mutation occurring in a polyG coding track of the TGFβ (transforming growth factor) receptor type II in MSI tumors. The RAS-MAP kinase pathway is activated by KRAS mutations in CIN tumors or by BRAF mutations in MSI tumors. The p53 pathway is inactivated by TP53 inactivation in CIN tumors or by BAX inactivating mutations in MSI tumors.

Topics: beta Catenin; Chromosomal Instability; Colorectal Neoplasms; DNA Methylation; DNA Mismatch Repair; Elafin; ErbB Receptors; Genes, p53; Genes, Tumor Suppressor; Humans; Microsatellite Instability; Phenotype; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Signal Transduction; Transforming Growth Factor beta

Colorectal cancer (CRC) is the third most common malignancy in the United States. Advances in molecular biology have enhanced the understanding of colorectal carcinogenesis. Approximately 75% of CRCs are sporadic; the rest are hereditary or belong to a familial syndrome. Identification of familial forms of CRC have enabled the development of several models of carcinogenesis and made CRC a well-studied malignancy in terms of molecular pathogenesis. Pathways containing multiple mutations and genetic alterations that play a role in hereditary CRC pathogenesis have been elucidated. Many of the molecular changes seen in these pathways also are involved in the development of sporadic cancers.

Topics: Antineoplastic Agents; Cell Proliferation; Chemotherapy, Adjuvant; Chromosomal Instability; Colorectal Neoplasms; Disease Progression; Humans; Mass Screening; Microsatellite Instability; Molecular Biology; Neovascularization, Pathologic; Positron-Emission Tomography; Prognosis; Tomography, X-Ray Computed; Transforming Growth Factor beta

It has been well documented that there are two major pathways in colorectal carcinogenesis. One is the chromosomal instability pathway (adenoma-carcinoma sequence), which is characterized by allelic losses on chromosome 5q (APC), 17p (p53), and 18q (DCC/SMAD4), and the other is a pathway that involves microsatellite instability. Recent progress in molecular biology, however, has shown that colorectal carcinogenesis is not necessarily clearly divided into these two pathways, but is in fact more complicated. Other routes, including the transforming growth factor-beta/SMAD pathway, the serrated pathway, and the epigenetic pathway, have been reported. Cross talk among these pathways has also been reported. In the invasion and metastasis steps of colorectal cancers, many more genes have now been identified as being involved in proteolysis, adhesion, angiogenesis, and cell growth. Recently accumulated evidence indicates that colorectal cancer is a genetically heterogeneous and complicated disease.

Topics: Adenocarcinoma; Carcinoma; Cell Transformation, Neoplastic; Chromosomal Instability; Colorectal Neoplasms; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Genes, ras; Genes, Tumor Suppressor; Genetic Predisposition to Disease; Humans; Microsatellite Instability; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Suppressor Proteins

The serrated neoplasia pathway gives rise to a distinct subgroup of colorectal cancers distinguished by the presence of mutant BRAF

Topics: Animals; beta Catenin; Colorectal Neoplasms; CpG Islands; Disease Models, Animal; DNA Methylation; Exome Sequencing; Humans; Mice; Microsatellite Instability; Microsatellite Repeats; Mutation; Proto-Oncogene Proteins B-raf; Transforming Growth Factor beta; Wnt Signaling Pathway

Topics: Colorectal Neoplasms; DNA Mismatch Repair; Female; Gene Expression Profiling; Humans; Immune Checkpoint Inhibitors; Male; Microsatellite Instability; Proteomics; Transforming Growth Factor beta

Solid tumour growth is the consequence of a complex interplay between cancer cells and their microenvironment. Recently, a new global transcriptomic immune classification of solid tumours has identified six immune subtypes (ISs) (C1-C6). Our aim was to specifically characterise ISs in colorectal cancer (CRC) and assess their interplay with the consensus molecular subtypes (CMSs).. Clinical and molecular information, including CMSs and ISs, were obtained from The Cancer Genome Atlas (TCGA) (N = 625). Immune cell populations, differential gene expression and gene set enrichment analysis were performed to characterise ISs in the global CRC population by using CMSs.. Only 5 ISs were identified in CRC, predominantly C1 wound healing (77%) and C2 IFN-γ dominant (17%). CMS1 showed the highest proportion of C2 (53%), whereas C1 was particularly dominant in CMS2 (91%). CMS3 had the highest representation of C3 inflammatory (7%) and C4 lymphocyte depleted ISs (4%), whereas all C6 TGF-β dominant cases belonged to CMS4 (2.3%). Prognostic relevance of ISs in CRC substantially differed from that reported for the global TCGA, and ISs had a greater ability to stratify the prognosis of CRC patients than CMS classification. C2 had higher densities of CD8, CD4 activated, follicular helper T cells, regulatory T cells and neutrophils and the highest M1/M2 polarisation. C2 had a heightened activation of pathways related to the immune system, apoptosis and DNA repair, mTOR signalling and oxidative phosphorylation, whereas C1 was more dependent of metabolic pathways.. The correlation of IS and CMS allows a more precise categorisation of patients with relevant clinical and biological implications, which may be valuable tools to improve tailored therapeutic interventions in CRC patients.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Aged; CD8-Positive T-Lymphocytes; Cell Proliferation; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Genes, MHC Class I; Humans; Inflammation; Interferon-gamma; Lymphocytes; Macrophages; Male; Microsatellite Instability; Monocytes; Neovascularization, Pathologic; Prognosis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins p21(ras); Receptors, Antigen, T-Cell; Signal Transduction; Th1 Cells; Th17 Cells; Th2 Cells; Transforming Growth Factor beta; Tumor Microenvironment; Wnt Signaling Pathway; Wound Healing

Mucinous adenocarcinoma is a distinct subtype of colorectal cancer (CRC) with a worse prognosis when compared with non-mucinous adenocarcinoma. The aim of this study was to compare somatic mutations and copy number alteration (CNA) between mucinous and non-mucinous CRC.. Data from The Cancer Genome Atlas-colon adenocarcinoma and rectum adenocarcinoma projects were utilized. Mucinous and non-mucinous CRC were compared with regard to microsatellite status, overall mutation rate, the most frequently mutated genes, mutations in genes coding for mismatch repair (MMR) proteins and genes coding for mucin glycoproteins. CNA analysis and pathway analysis was undertaken.. Mucinous CRC was more likely to be microsatellite instability-high (MSI-H) and hypermutated. When corrected for microsatellite status the single-nucleotide variation and insertion-deletion rate was similar between the two cohorts. Mucinous adenocarcinoma was more likely to have mutations in genes coding for MMR proteins and mucin glycoproteins. Pathway analysis revealed further differences between the two histological subtypes in the cell cycle, RTK-RAS, transforming growth factor-β, and TP53 pathways.. Mucinous CRC has some distinct genomic aberrations when compared with non-mucinous adenocarcinoma, many of which are driven by the increased frequency of MSI-H tumors. These genomic aberrations may play an important part in the difference seen in response to treatment and prognosis in mucinous adenocarcinoma.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Cohort Studies; Colonic Neoplasms; Datasets as Topic; DNA Copy Number Variations; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genomics; Humans; INDEL Mutation; Microsatellite Instability; Mucins; Mutation; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Rectal Neoplasms; Smad4 Protein; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Although the prognostic biomarkers associated with colorectal cancer (CRC) survival are well known, there are limited data on the association between the molecular characteristics and survival after recurrence (SAR). The purpose of this study was to assess the association between pathway mutations and SAR.. Of the 516 patients with stage III or high risk stage II CRC patients treated with surgery and adjuvant chemotherapy, 87 who had distant recurrence were included in the present study. We analyzed the association between SAR and mutations of 40 genes included in the five critical pathways of CRC (WNT, P53, RTK-RAS, TGF-β, and PI3K).. Mutation of genes within the WNT, P53, RTK-RAS, TGF-β, and PI3K pathways were shown in 69(79.3%), 60(69.0%), 57(65.5%), 21(24.1%), and 19(21.8%) patients, respectively. Patients with TGF-β pathway mutation were younger and had higher incidence of mucinous adenocarcinoma (MAC) histology and microsatellite instability-high. TGF-β pathway mutation (median SAR of 21.6 vs. 44.4 months, p = 0.021) and MAC (20.0 vs. 44.4 months, p = 0.003) were associated with poor SAR, and receiving curative resection after recurrence was associated with favorable SAR (Not reached vs. 23.6 months, p <  0.001). Mutations in genes within other critical pathways were not associated with SAR. When MAC was excluded as a covariate, multivariate analysis revealed TGF-β pathway mutation and curative resection after distant recurrence as an independent prognostic factor for SAR. The impact of TGF-β pathway mutations were predicted using the PROVEAN, SIFT, and PolyPhen-2. Among 25 mutations, 23(92.0%)-24(96.0%) mutations were predicted to be damaging mutation.. Mutation in genes within TGF-β pathway may have negative prognostic role for SAR in CRC. Other pathway mutations were not associated with SAR.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Capecitabine; Chemotherapy, Adjuvant; Colon; Colorectal Neoplasms; Deoxycytidine; Female; Fluorouracil; Follow-Up Studies; Humans; Leucovorin; Male; Microsatellite Instability; Middle Aged; Neoplasm Recurrence, Local; Organoplatinum Compounds; Oxaloacetates; Palliative Care; Prognosis; Rectum; Signal Transduction; Survival Analysis; Transforming Growth Factor beta

Programmed cell death protein 1(PD-1) blocking antibodies have been used to enhance immunity in solid tumors and achieve durable clinical responses with an acceptable safety profile in multiple types of cancer. However, only a subset of patients could benefit from PD-1 blockade therapy. Prognostic information including PD-1 ligand (PD-L1) expression, IFN-γ expression signature, tumor mutational burden, and microsatellite instability (MSI) have been evaluated for patients who are selected to receive immune checkpoint therapeutic treatment. Yet the relationship of those biomarkers in determining immune checkpoint therapy is largely unknown.. Immune-profiles of MSI subtype colon cancer were identified from integrating published MSI associated gene expression data. The enriched pathways and transcription factors were analyzed by GSEA assay. The infiltrations of immune cell types into MSI subtype colon cancer tissues were determined by CIBESORT assay.. In the MSI subtype colon cancer patients, PD-L1, IFN-γ and IFN-γ associated genes are highly expressed. And all those genes are favorable effects in colon cancer progress. In addition, we find that Wnt-β-catenin and TGFβ signaling pathways which are two important factors inhibiting PD-1 checkpoint blockade therapy are negatively related with MSI status. We also identify that the immune-profiles in MSI subtype colon cancer are contributed by M1 macrophage infiltration in the tumor environment.. Our results provide the detailed underlying mechanisms of MSI subtype cancer patients are sensitive to PD-1 checkpoint blockade.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Colonic Neoplasms; Gene Expression; Humans; Immunotherapy; Interferon Regulatory Factor-1; Interferon-gamma; Macrophages; Microsatellite Instability; MutL Protein Homolog 1; Phthalazines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prognosis; Programmed Cell Death 1 Receptor; Transcriptome; Transforming Growth Factor beta; Tumor Microenvironment; Wnt Signaling Pathway

Stromal content heavily impacts the transcriptional classification of colorectal cancer (CRC), with clinical and biological implications. Lineage-dependent stromal transcriptional components could therefore dominate over more subtle expression traits inherent to cancer cells. Since in patient-derived xenografts (PDXs) stromal cells of the human tumour are substituted by murine counterparts, here we deploy human-specific expression profiling of CRC PDXs to assess cancer-cell intrinsic transcriptional features. Through this approach, we identify five CRC intrinsic subtypes (CRIS) endowed with distinctive molecular, functional and phenotypic peculiarities: (i) CRIS-A: mucinous, glycolytic, enriched for microsatellite instability or KRAS mutations; (ii) CRIS-B: TGF-β pathway activity, epithelial-mesenchymal transition, poor prognosis; (iii) CRIS-C: elevated EGFR signalling, sensitivity to EGFR inhibitors; (iv) CRIS-D: WNT activation, IGF2 gene overexpression and amplification; and (v) CRIS-E: Paneth cell-like phenotype, TP53 mutations. CRIS subtypes successfully categorize independent sets of primary and metastatic CRCs, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances.

Topics: Animals; Antineoplastic Agents, Immunological; Cell Lineage; Cetuximab; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gene Expression Profiling; Genes, p53; Glycolysis; Heterografts; Humans; Insulin-Like Growth Factor II; Male; Mice; Microsatellite Instability; Mutation; Prognosis; Signal Transduction; Stromal Cells; Transcriptome; Transforming Growth Factor beta

The Cancer Genome Atlas data on colorectal carcinoma have provided a comprehensive view of the tumor's genomic alterations and their tumorigenic roles. Tumor morphology, however, has not been fully integrated into the analysis. The aim of this study was to explore relevant associations between tumor morphology and the newly characterized genomic alterations in colorectal carcinoma. Two hundred and seven colorectal carcinomas that had undergone whole-exome sequencing as part of The Cancer Genome Atlas project and had adequate virtual images in the cBioPortal for Cancer Genomics constituted our study population. Upon analysis, a tight association between 'microsatellite instability-high histology' and microsatellite instability-high (P<0.001) was readily detected and helped validate our image-based histology evaluation. Further, we showed, (1) among all histologies, the not otherwise specified type had the lowest overall mutation count (P<0.001 for entire cohort, P<0.03 for the microsatellite-instable group), and among the microsatellite-instable tumors, this type also correlated with fewer frameshift mutations in coding mononucleotide repeats of a defined set of relevant genes (P<0.01); (2) cytosine phosphate guanine island methylator phenotype-high colorectal cancers with or without microsatellite instability tended to have different histological patterns: the former more often mucinous and the latter more often not otherwise specified; (3) mucinous histology was associated with more frequent alterations in BRAF, PIK3CA, and the transforming growth factor-β pathway when compared with non-mucinous histologies (P<0.001, P=0.01, and P<0.001, respectively); and (4) few colorectal cancers (<9%) exhibited upregulation of immune-inhibitory genes including major immune checkpoints; these tumors were primarily microsatellite-instable (up to 43%, vs <3% in microsatellite-stable group) and had distinctly non-mucinous histologies with a solid growth. These morphology-molecular associations are interesting and propose important clinical implications. The morphological patterns associated with alterations of immune checkpoint genes bear the potential to guide patient selection for clinical trials that target immune checkpoints in colorectal cancer, and provide directions for future studies.

Topics: Carcinoma; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; DNA Methylation; Humans; Microsatellite Instability; Proto-Oncogene Proteins B-raf; Transforming Growth Factor beta

Downregulation of betaglycan (β-glycan) [transforming growth factor β receptor type III (TGFβR3)], which belongs to co-receptors of the TGFβ pathway, occurs in a broad spectrum of primary human malignancies. However, in the case of endometrial cancer (EC), the mechanisms responsible for genetic alterations are still unknown. Therefore, we investigated allelic imbalance at the TGFBR3 locus (1p33‑p32) in the context of β-glycan mRNA and protein expression, as a possible genetic event determining β-glycan deregulation in EC patients. Study of β-glycan allelic imbalance in 48 primary human ECs was performed with the use of three different microsatellite markers, spanned within or in direct proximity to the TGFBR3 locus. Real‑time PCR and western blotting were used for β-glycan mRNA and protein quantification methods, respectively. Altogether, 25 of 39 (64%) informative cases and 25 of 48 (52%) of all specimens showed allelic imbalance in at least one microsatellite marker, concomitantly with decrease at both the β-glycan transcript and protein levels. Interestingly, 54% (15/28), 36% (8/22) and 35% (7/20) of informative ECs displayed allelic loss in D1S188, D1S435 and D1S1588 microsatellite markers, respectively. It is worth pointing out that 5 out of 39 (13%) informative cases showed loss of heterozygosity (LOH) at two microsatellite markers. Microsatellite instability (MSI) was found in two markers, but to a very strictly limited extent. None of the clinicoprognostic features was found to be of significance. Our results suggest that LOH in the TGFBR3 locus may be one of the mechanisms responsible for loss of β-glycan expression. No correlation of LOH at the TGFBR3 locus with clinicopathological parameters suggests that allelic imbalance may be an early genetic event during neoplastic transformation of human endometrium.

Topics: Aged; Blotting, Western; Endometrial Neoplasms; Female; Humans; Loss of Heterozygosity; Microsatellite Instability; Middle Aged; Proteoglycans; Real-Time Polymerase Chain Reaction; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Most colorectal cancer (CRC) cells with high levels of microsatellite instability (MSI-H) accumulate mutations at a microsatellite sequence in the gene encoding transforming growth factor β receptor II (TGFBR2). TGFβ signaling therefore is believed to be defective in these tumors, although CRC cells with TGFBR2 mutations have been reported to remain sensitive to TGFβ. We investigated how TGFβ signaling might continue in MSI-H CRC cells.. We sequenced the 10-adenines microsatellite sequence in the TGFBR2 gene of 32 MSI-H colon cancer tissues and 6 cell lines (HCT116, LS180, LS411N, RKO, SW48, and SW837). Activation of TGFβ signaling was detected by SMAD2 phosphorylation and through use of a TGFβ-responsive reporter construct in all CRC cell lines. Transcripts of TGFBR2 were knocked-down in CRC cells using short hairpin RNA. Full-length and mutant forms of TGFBR2 were expressed in LS411N cells, which do not respond to TGFβ, and their activities were measured.. SMAD2 was phosphorylated in most MSI-H CRC tissues (strong detection in 44% and weak detection in 34% of MSI-H tumors). Phosphorylation of SMAD2 in MSI-H cells required TGFBR2—even the form encoding a frameshift mutation. Transcription and translation of TGFBR2 with a 1-nucleotide deletion at its microsatellite sequence still produced a full-length TGFBR2 protein. However, protein expression required preservation of the TGFBR2 microsatellite sequence; cells in which this sequence was replaced with a synonymous nonmicrosatellite sequence did not produce functional TGFBR2 protein.. TGFβ signaling remains active in some MSI-H CRC cells despite the presence of frameshift mutations in the TGFBR2 gene because the mutated gene still expresses a functional protein. Strategies to reactivate TGFβ signaling in colorectal tumors might not be warranted, and the functional effects of mutations at other regions of microsatellite instability should be evaluated.

Topics: Colorectal Neoplasms; Frameshift Mutation; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; HCT116 Cells; HEK293 Cells; Humans; Microsatellite Instability; Phenotype; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Smad2 Protein; Transcription, Genetic; Transfection; Transforming Growth Factor beta

Topics: Colorectal Neoplasms; Frameshift Mutation; Humans; Microsatellite Instability; Protein Serine-Threonine Kinases; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

In colorectal cancer (CRC), genetic alterations are involved in disease progression from adenoma to carcinoma and metastatic disease. Three different carcinogenesis mechanisms exist: chromosomal instability phenotype (CIN), microsatellite instability phenotype (MSI) and the hypermetylator phenotype (CIMP for CpG Island methylation phenotype). These molecular alterations lead to signal transduction dysfunction, and signaling pathways alterations are involved in cancer mechanisms, such as EGFR pathway, WNT/APC pathway, TGFB pathway and p53 pathway. Recently, molecular signatures have been established, allowing distinction of 4 different types of CCR. Finally, circulating biomarkers are investigated for molecular characterization.

Topics: Chromosomal Instability; Colorectal Neoplasms; CpG Islands; DNA Methylation; ErbB Receptors; Humans; Microsatellite Instability; Transforming Growth Factor beta

Colorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression-based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMSs) with distinguishing features: CMS1 (microsatellite instability immune, 14%), hypermutated, microsatellite unstable and strong immune activation; CMS2 (canonical, 37%), epithelial, marked WNT and MYC signaling activation; CMS3 (metabolic, 13%), epithelial and evident metabolic dysregulation; and CMS4 (mesenchymal, 23%), prominent transforming growth factor-β activation, stromal invasion and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intratumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC-with clear biological interpretability-and the basis for future clinical stratification and subtype-based targeted interventions.

Topics: Carcinoma; Colorectal Neoplasms; Consensus; CpG Islands; DNA Copy Number Variations; DNA Methylation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Information Dissemination; Microsatellite Instability; Mutation; Neovascularization, Pathologic; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); ras Proteins; Transforming Growth Factor beta; Wnt Signaling Pathway

TGFbeta is an important cell growth regulator which may have a role in metastasis formation. Microsatellite unstable (MSI-H) colon cancer serves as a unique model to demonstrate this as most MSI-H colon cancers have a mutation in the transforming growth factor beta receptor II (TGFbetaRII) gene and a low metastatic rate.. To demonstrate an increase in invasion and metastasis in a MSI-H colorectal cancer cell line with a known mutation in TGFbetaRII.. By restoring the wild-type TGFbetaRII gene in the KM12C MSI-H colorectal carcinoma cell line with a known mutation in TGFbetaRII, we have demonstrated that both invasion and metastasis in this cell line was significantly increased. A mouse metastatic model have shown that liver metastases were increased in mice inoculated with cells containing a wild-type TGFbetaRII gene (42% for the transfected group compared with 15% for the control group; p = 0.0379), despite a reduction in the size of primary tumours.. This study highlights an important mechanism which may contribute to the low metastatic rate of MSI-H colon cancers and demonstrates the importance of TGFbeta signalling in metastasis formation. Previous studies involving breast cancer cell lines have shown that blocking TGFbeta signalling results in a reduction in metastasis formation. This study is the first study to use a cell line with a low metastatic rate and TGFbetaRII mutations to demonstrate that restoring TGFbeta signalling increases the metastatic rate.

Topics: Animals; Cell Line, Tumor; Collagen; Colorectal Neoplasms; Drug Combinations; Humans; Laminin; Mice; Microsatellite Instability; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transfection; Transforming Growth Factor beta

The mutational inactivation of transforming growth factor beta receptor type II (TGFBR2) occurs in approximately 30% of colon cancers and promotes the formation of colon cancer by inhibiting the tumor suppressor activity of the TGFB signaling pathway. TGFBR2 mutations occur in >90% of microsatellite unstable (MSI) colon cancers and affect a polyadenine tract in exon 3 of TGFBR2, called BAT-RII, which is vulnerable to mutation in the setting of DNA mismatch repair (MMR) system deficiency. In light of the vulnerable nature of the BAT-RII tract in the setting of MMR inactivation and the favorable effects of TGFBR2 inactivation in colon cancer, analysis of TGFBR2 inactivation provides an opportunity to assess the roles of genomic instability vs. clonal selection in cells acquiring TGFBR2 BAT-RII tract mutations in MSI colon cancer formation. The contribution of genomic instability and/or clonal evolution to the mutational inactivation of TGBFR2 in MSI colon cancers has not been studied in a systematic way that would allow a determination of the relative contribution of these two mechanisms in the formation of MSI colon cancer. It has not been demonstrated whether the BAT-RII tract mutations are strictly a consequence of the BAT-RII region being hypermutable in the setting of MMR deficiency or if the mutations are rather a consequence of clonal selection pressure against the TGFB receptor. Through the use of defined cell line systems, we show that both genomic instability and clonal selection of TGFB resistant cells contribute to the high frequency of TGFBR2 mutations in MSI colon cancer.

Topics: Amino Acid Substitution; Cell Line; Cell Line, Tumor; Colonic Neoplasms; DNA Mutational Analysis; Drug Resistance, Neoplasm; Gene Frequency; Gene Silencing; Growth Inhibitors; HCT116 Cells; Humans; Microsatellite Instability; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

The finding of bone morphogenetic protein (BMP) receptor 1a mutations in juvenile polyposis suggests that BMPs are important in colorectal cancer (CRC). We investigated the BMP pathway in sporadic CRC.. We investigated BMP receptor (BMPR) expression using immunoblotting and sequenced BMPR2 in CRC cell lines. We assessed the expression of BMPRs, SMAD4, and pSMAD1/5/8 in 72 sporadic CRCs using a tissue microarray and immunohistochemistry. We assessed the effect of reintroduction of wild-type BMPR2 on BMP pathway activity and the effect of wild-type or mutated BMPR2 3' untranslated region (UTR) sequences on protein expression by attachment to pCMV-Luc.. BMPR2 and SMAD4 protein expression is abrogated in microsatellite unstable (MSI) and microsatellite stable (MSS) cell lines, respectively. BMPR2 3'UTR is mutated in all MSI and in none of the MSS cell lines. Mutant BMPR2 3'UTR sequences reduced luciferase expression 10-fold compared with wild-type BMPR2 3'UTR. BMPR2 expression is impaired more frequently in MSI CRCs than MSS (85% vs 29%; P < .0001) and shows a mutually exclusive pattern of impaired expression compared with SMAD4. Nine of 11 MSI cancers with impaired expression of BMPR2 have microsatellite mutations. The BMP pathway is inactivated, as judged by nuclear pSMAD1/5/8 expression, in 70% of CRCs, and this correlates with BMPR and SMAD4 loss.. Our data suggest that the BMP pathway is inactivated in the majority of sporadic CRCs. In MSI CRC this is associated predominantly with impaired BMPR2 expression and in MSS CRC with impaired SMAD4 expression.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Line, Tumor; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Immunohistochemistry; Male; Microsatellite Instability; Microsatellite Repeats; Middle Aged; Mutation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Smad4 Protein; Transforming Growth Factor beta

Disruptions to the TGFbeta signalling pathway have been implicated in most human adenocarcinomas. As cancers progress, many acquire resistance to the growth-suppressing properties of TGFbeta while retaining sensitivity to its tumour-promoting effects. Microsatellite unstable colorectal cancers (MSI-H CRCs) possess truncating mutations in the type II TGFbeta receptor (TGFbetaRII) gene that have been assumed to render these tumours insensitive to TGFbeta. However, numerous reports of TGFbetaRII bypass exist and this study was thus undertaken in order to clarify the true extent of TGFbeta sensitivity in MSI-H CRCs. Using stimulation with exogenous TGFbeta, we demonstrated that, while MSI-H CRCs are capable of binding soluble TGFbeta, two out of three cell lines examined remain refractory to its signalling effects. In contrast, use of a specific inhibitor of the type I TGFbeta receptor (TGFbetaRI) revealed that all remain sensitive to signalling by endogenously produced TGFbeta. Specifically, autocrine signalling via TGFbetaRI mediates constitutive activation of Smad2 as well as repression of Erk signalling. Real-time PCR confirmed that these effects are sufficient to affect the expression level of various TGFbeta-modulated genes. An invasion assay revealed that autocrine TGFbetaRI signalling also promotes the invasion capacity of MSI-H CRCs to an extent similar to that seen in their non-MSI-H counterparts. Independent TGFbetaRI signalling, however, has no effect on the rate of proliferation of MSI-H CRC cells. Together, these results demonstrate that MSI-H CRC cell lines are not completely refractory to TGFbeta, despite lacking functional TGFbetaRII. In addition to clarifying the true consequences of natural TGFbetaRII loss and the independent function of TGFbetaRI, our results highlight the selective nature of TGFbeta resistance developed by cancers.

Topics: Autocrine Communication; Benzamides; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Dioxoles; Drug Resistance, Neoplasm; Humans; Loss of Heterozygosity; Microsatellite Instability; Mutation; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta