• Activin Receptors, Type II/genetics
• Adenomatous Polyposis Coli/genetics
• Adenomatous Polyposis Coli/pathology
• Adenomatous Polyposis Coli Protein/genetics
• Colorectal Neoplasms/genetics
• Colorectal Neoplasms/pathology
• Humans
• INDEL Mutation
• MutS Homolog 3 Protein/genetics
• Polymorphism, Single Nucleotide

• the Volkswagenstiftung
• the Center for Molecular Medicine Cologne (CMMC)
• German Cancer Aid
• German Research Foundation

• Colorectal carcinoma
• Cytokine
• Single nucleotide polymorphism
• TGF-ΒR2 gene
• TGF-ΒR2G[-875]A

• Breast cancer
• Cancer immunotherapy
• Immune checkpoint proteins
• PD-1
• T cell exhaustion
• TOX

• Breast Neoplasms/genetics
• Breast Neoplasms/mortality
• Breast Neoplasms/pathology
• Cell Line, Tumor
• Epigenomics/methods
• Female
• High Mobility Group Proteins/metabolism
• Humans
• Survival Analysis
• Transcriptome/genetics

• 2019-2914 Indian Council of Medical Research

• DNA repair
• EMAST
• MSI
• colorectal cancer
• inflammation
• mismatch repair
• neoantigens
• tumor immunology
• tumor microenvironment

• DNA Mismatch Repair
• EMAST
• Frameshift Mutation
• MSH3
• Tetranucleotide Microsatellites

• Cell Separation/methods
• Colorectal Neoplasms/diagnosis
• Colorectal Neoplasms/genetics
• Colorectal Neoplasms/immunology
• DNA Mismatch Repair/genetics
• DNA Mutational Analysis/methods
• Flow Cytometry/methods
• Frameshift Mutation
• Genes, Reporter/genetics
• Genetic Loci/genetics
• Genetic Markers
• Green Fluorescent Proteins/chemistry
• Green Fluorescent Proteins/genetics
• HCT116 Cells
• Humans
• Microsatellite Instability
• Microsatellite Repeats/genetics
• Mutation Rate
• Plasmids/genetics
• Transfection

• R01 CA206010 NCI NIH HHS
• R01 DK067287 NIDDK NIH HHS
• U01 CA162147 NCI NIH HHS

Colorectal cancer has become a serious problem, especially in highly developed countries. As reported by the World Health Organization, the number of colon cancer cases in the world in 2012 amounted to 1.36 million. It is the second most common cancer in females (614,000 cases, 9.2% of the total) and the third in males (746,000 cases, 10.0% of the total) worldwide. It is believed that TGFβ pathway elements are involved in the pathogenesis of colorectal cancer. This study assessed one of these elements, the ACVR2A gene. Qualitative and quantitative analyses of the ACVR2A gene in 84 patients with colorectal cancer was performed. There was no statistically significant association between ACVR2A gene expression and age, gender, histological type, grading of tumor, vascular invasion, and presence of lymphocytes in tumor tissue. No association was observed between the ACVR2A gene expression level and the presence of metastases in regional lymph nodes and distant metastases. In this study, larger tumors (T3 and T4) were characterized by higher ACVR2A expression compared to smaller tumors (T1 and T2). This may indicate an association between ACVR2A expression and the severity of pathological changes in the tumor growth process.

• 3′-untranslated region
• TGFBR2
• microRNA
• protective factor
• seed region
• single nucleotide polymorphisms
• sporadic colorectal cancer

• Constitutional Mismatch Repair Deficiency syndrome
• DNA microsatellites
• DNA mismatch repair
• DNA repair
• EMAST (elevated microsatellelite alterations at selected tetranucleotide repeats)
• Lynch syndrome
• Lynch-like syndrome
• MLH1 hypermethylation
• MSH3
• colorectal cancer
• dinucleotide repeats
• familial
• microsatellite instability
• mononucleotide repeats
• patient outcome
• patient survival
• sporadic
• tetranucleotide repeats
• trinucleotide repeats

• Genetic Techniques
• Mutation
• Poly A/genetics
• Poly A/metabolism
• Promoter Regions, Genetic
• Protein Biosynthesis
• Proteins/genetics
• Proteins/metabolism
• RNA Stability

• R01 GM072778 NIGMS NIH HHS
• R01 GM112824 NIGMS NIH HHS
• R01 NS036570 NINDS NIH HHS
• T32 GM007067 NIGMS NIH HHS

• 5-fluorouracil
• Colorectal cancer
• DNA mismatch repair
• MBD4
• cytotoxicity
• frameshift mutation
• truncated protein

• Colorectal Neoplasms/drug therapy
• Colorectal Neoplasms/genetics
• DNA Mismatch Repair
• Endodeoxyribonucleases/genetics
• Endodeoxyribonucleases/metabolism
• Fluorouracil/therapeutic use
• Frameshift Mutation
• Humans
• MutL Protein Homolog 1/genetics
• MutL Protein Homolog 1/metabolism
• Transfection

• R01 CA206010 NCI NIH HHS
• R01 DK067287 NIDDK NIH HHS
• U01 CA162147 NCI NIH HHS

• Colorectal Neoplasms
• Colorectal Neoplasms, Hereditary Nonpolyposis/genetics
• DNA Mismatch Repair
• Germ-Line Mutation
• Humans
• Microsatellite Instability
• MutS Homolog 3 Protein/genetics

• R01 DK067287 NIDDK NIH HHS
• U01 CA162147 NCI NIH HHS

Mismatch repair (MMR) plays a pivotal role in keeping the genome stable. MMR dysfunction can lead to carcinogenesis by gene mutation accumulation. HMSH2 and hMLH1 are two key components of MMR. High or low expression of them often mark the status of MMR function. Mutations (EGFR, KRAS, etc) are common in non-small cell lung cancer (NSCLC). However, it is not clear what role MMR plays in NSCLC gene mutations. The expression of MMR proteins hMSH2 and hMLH1, and the proliferation markers PCNA and Ki67 were measured by immunohistochemistry in 181 NSCLCs. EGFR and KRAS mutations were identified by high resolution melting analysis. Stronger hMLH1 expression correlated to a higher frequency of EGFR mutations in exon 19 and 21 (p<0.0005). Overexpression of hMLH1 and the adenocarcinoma subtype were both independent factors that related to EGFR mutations in NSCLCs (p=0.013 and p<0.0005). The expression of hMLH1, hMSH2 and PCNA increased, while Ki67 expression significantly decreased (p=0.030) in NSCLCs with EGFR mutations. Overexpression of hMLH1 could be a new molecular marker to predict the response to EGFR-TKIs in NSCLCs. Furthermore, EGFR mutations might be an early event of NSCLC that occur before MMR dysfunction.

• Adaptor Proteins, Signal Transducing/genetics
• Adenocarcinoma/genetics
• Adult
• Aged
• Aged, 80 and over
• Base Pair Mismatch/genetics
• Carcinoma, Non-Small-Cell Lung/genetics
• DNA Mismatch Repair/genetics
• DNA Repair/genetics
• ErbB Receptors/genetics
• Exons/genetics
• Female
• Humans
• Ki-67 Antigen/genetics
• Lung Neoplasms/genetics
• Male
• Middle Aged
• MutL Protein Homolog 1
• Nuclear Proteins/genetics
• Proliferating Cell Nuclear Antigen/genetics
• Proto-Oncogene Proteins/genetics
• Proto-Oncogene Proteins p21(ras)
• ras Proteins/genetics

Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene hMLH1’s promoter is causal, the precise cause of EMAST is not clearly defined but points towards hMSH3 deficiency.

To examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency.

We measured −4 bp framshifts at D8S321 and D20S82 loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, hMLH1^−/−, hMSH6^−/−, and hMSH3^−/− CRC cells. We observed the subcellular location of hMSH3 with oxidative stress.

D8S321 mutations occurred 31-and 40-fold higher and D20S82 mutations occurred 82-and 49-fold higher in hMLH1^−/− and hMSH3^−/− cells, respectively, than in hMSH6^−/− or MMR-proficient cells. hMSH3 knockdown in MMR-proficient cells caused higher D8S321 mutation rates (18.14 and 11.14×10⁻⁴ mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10⁻⁴ mutations/cell/generation; p<0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H₂O₂ to examine its effect on hMSH3. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H₂O₂ treatment.

EMAST is dependent upon the MMR background, with hMSH3^−/− more prone to frameshift mutations than hMSH6^−/−, opposite to frameshift mutations observed for mononucleotide repeats. hMSH3^−/− mimics complete MMR failure (hMLH1^−/−) in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, hMSH3-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3’s subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.

• Active Transport, Cell Nucleus/genetics
• Cell Line, Tumor
• Cell Nucleus/metabolism
• Colorectal Neoplasms/genetics
• Colorectal Neoplasms/metabolism
• Colorectal Neoplasms/pathology
• Cytosol/metabolism
• DNA Mismatch Repair/genetics
• DNA-Binding Proteins/deficiency
• DNA-Binding Proteins/genetics
• DNA-Binding Proteins/metabolism
• Frameshift Mutation/genetics
• Gene Knockdown Techniques
• Genetic Loci/genetics
• Humans
• Microsatellite Repeats/genetics
• MutS Homolog 3 Protein
• Oxidative Stress/genetics

• R01 DK067287 NIDDK NIH HHS
• CA132384 NCI NIH HHS
• U54 CA132384 NCI NIH HHS
• CA132379 NCI NIH HHS
• DK067287 NIDDK NIH HHS
• U01 CA162147 NCI NIH HHS
• CA162147 NCI NIH HHS
• U54 CA132379 NCI NIH HHS

• inherited disease
• human genome
• disease genes
• mutational mechanisms
• mutation hotspots
• dna sequence motifs
• genome architecture
• non-b dna structures

• Alu Elements
• Base Sequence
• DNA Copy Number Variations
• DNA Repeat Expansion
• Genes
• Genetic Diseases, Inborn/genetics
• Genetic Predisposition to Disease
• Genome, Human
• Genome, Mitochondrial
• Humans
• Microsatellite Repeats
• Mutation/genetics
• Polymorphism, Genetic
• Recombination, Genetic
• Retroelements/genetics

• HHSN261200800001C NCI NIH HHS
• HHSN261200800001E NCI NIH HHS
• Z99 CA999999 Intramural NIH HHS

The activin type II receptor (ACVR2) contains 2 identical microsatellites in exon 3 and 10, but only the exon 10 microsatellite is frameshifted in MMR-defective colonic tumors. The reason for this selectivity is not known. We hypothesized that ACVR2 frameshifts were influenced by DNA sequences surrounding the microsatellite. We constructed plasmids in which exon 3 or 10 of ACVR2 were cloned +1bp out-of-frame of EGFP, allowing −1bp frameshift to express EGFP. Plasmids were stably-transfected into MMR-deficient cells, subsequent non-fluorescent cells sorted, cultured, and harvested for mutation analysis. We swapped DNA sequences flanking the exon 3 and 10 microsatellites to test our hypothesis. Native ACVR2 exon 3 and 10 microsatellites underwent heteroduplex formation (A₇/T₈) in hMLH1^−/− cells, but only exon 10 microsatellites fully mutated (A₇/T₇) in both hMLH1^−/− and hMSH6^−/− backgrounds, showing selectivity for exon 10 frameshifts and inability of exon 3 heteroduplexes to fully mutate. Substituting nucleotides flanking the exon 3 microsatellite for nucleotides flanking the exon 10 microsatellite significantly reduced heteroduplex and full mutation in hMLH1^−/− cells. When the exon 3 microsatellite was flanked by nucleotides normally surrounding the exon 10 microsatellite, fully-mutant exon 3 frameshifts appeared. Mutation selectivity for ACVR2 lies partly with flanking nucleotides surrounding each microsatellite.

Activin receptor 2 (ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers.

Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 were sequenced in primary cancers and all exons in colon cancer cell lines. Loss of heterozygosity (LOH) was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2′-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria.

Of 51 MSS colon tumors, 7(14%) lost ACVR2, 2 (4%) ACVR1, and 5(10%) pSMAD2 expression. No somatic ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05).

Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer.