Molecular Signatures for Microbe-Associated Colorectal Cancers.

Background: Genetic factors and microbial imbalances play crucial roles in colorectal cancers (CRCs), yet the impact of infections on cancer initiation remains poorly understood. While bioinformatic approaches offer valuable insights, the rising incidence of CRCs creates a pressing need to precisely identify early CRC events. We constructed a network model to identify continuum states during CRC initiation spanning normal colonic tissue to pre-cancer lesions (adenomatous polyps) and examined the influence of microbes and host genetics. Methods: A Boolean network was built using a publicly available transcriptomic dataset from healthy and adenoma affected patients to identify an invariant Microbe-Associated Colorectal Cancer Signature (MACS). We focused on Fusobacterium nucleatum ( Fn ), a CRC-associated microbe, as a model bacterium. MACS-associated genes and proteins were validated by RT-qPCR, RNA seq, ELISA, IF and IHCs in tissues and colon-derived organoids from genetically predisposed mice ( CPC-APC Min+/- ) and patients (FAP, Lynch Syndrome, PJS, and JPS). Results: The MACS that is upregulated in adenomas consists of four core genes/proteins: CLDN2/Claudin-2 (leakiness), LGR5/leucine-rich repeat-containing receptor (stemness), CEMIP/cell migration-inducing and hyaluronan-binding protein (epithelial-mesenchymal transition) and IL8/Interleukin-8 (inflammation). MACS was induced upon Fn infection, but not in response to infection with other enteric bacteria or probiotics. MACS induction upon Fn infection was higher in CPC-APC Min+/- organoids compared to WT controls. The degree of MACS expression in the patient-derived organoids (PDOs) generally corresponded with the known lifetime risk of CRCs. Conclusions: Computational prediction followed by validation in the organoid-based disease model identified the early events in CRC initiation. MACS reveals that the CRC-associated microbes induce a greater risk in the genetically predisposed hosts, suggesting its potential use for risk prediction and targeted cancer prevention.

Biallelic Mismatch Repair Deficiency in an Adolescent Female.

Constitutional (Biallelic) Mismatch Repair Deficiency is a rare autosomal recessive disorder characterized by numerous cancers presenting as early as the first decade of life. Biallelic germline variants in one of four mismatch repair genes (MLH1, MSH2, MSH6, or PMS2) cause this devastating disease. Given the rarity of the syndrome, often-asymptomatic tumors, and overlap with neurofibromatosis-1, diagnosis is frequently unrecognized or delayed. We present a unique case of a 14-year-old female with minimal gastrointestinal symptoms diagnosed with invasive adenocarcinoma secondary to biallelic PMS2 variants.

Lynch syndrome: a pediatric perspective.

Colorectal cancer is a rare disease in the pediatric age group and, when present, suggests an underlying genetic predisposition. The most common hereditary colon cancer susceptibility condition, Lynch syndrome (LS), previously known as hereditary nonpolyposis colorectal cancer, is an autosomal dominant condition caused by a germline mutation in 1 of 4 DNA mismatch repair (MMR) genes: MLH1, MSH2, MSH6, or PMS2. The mutation-prone phenotype of this disorder is associated with gastrointestinal, endometrial, and other cancers and is now being identified in both symptomatic adolescents with malignancy as well in asymptomatic mutation carriers who are at risk for a spectrum of gastrointestinal and other cancers later in life. We review the DNA MMR system, our present understanding of LS in the pediatric population, and discuss the newly identified biallelic form of the disease known as constitutional mismatch repair deficiency syndrome. Both family history and tumor characteristics can help to identify patients who should undergo genetic testing for these cancer predisposition syndromes. Patients who carry either single allele (LS) or double allele (constitutional mismatch repair deficiency syndrome) mutations in the MMR genes benefit from cancer surveillance programs that target both the digestive and extraintestinal cancer risk of these diseases. Because spontaneous mutation in any one of the MMR genes is extremely rare, genetic counseling and testing are suggested for all at-risk family members.

Evidence for an hMSH3 defect in familial hamartomatous polyps.

BACKGROUND: Patients with hamartomatous polyposis syndromes have increased risk for colorectal cancer (CRC). Although progression of polyps to carcinoma is observed, pathogenic mechanisms remain unknown. The authors examined whether familial hamartomatous polyps harbor defects in DNA mismatch repair (MMR), and assayed for somatic mutation of PTEN, a gene inactivated in the germline of some hamartomatous polyposis syndrome patients. METHODS: Ten hamartomatous polyposis syndrome patients were genotyped for germline mutations. Epithelial and nonepithelial polyp DNA were assayed for microsatellite instability (MSI) and PTEN frameshift mutation. DNA MMR and PTEN protein expression were assessed in all polyps by immunohistochemistry. In addition, 99 MSI-high sporadic CRCs and 50 each of hMLH1(-/-) and hMSH3(-/-) cell clones were examined for PTEN frameshifts. RESULTS: Twenty-five (58%) of 43 hamartomatous polyposis syndrome polyps demonstrated dinucleotide or greater MSI in polyp epithelium, consistent with hMSH3 deficiency. MSI domains lost hMSH3 expression, and PTEN expression was lost in polyps from germline PTEN patients; sporadic hamartomatous polyps did not show any of these findings. PTEN analysis revealed wild type exon 7 and 8 sequences suggestive of nonexistent or rare events for PTEN frameshifts; however, MSI-high sporadic CRC showed 11 (11%) of 99 frameshifts within PTEN, with 4 tumors having complete loss of PTEN expression. Subcloning hMLH1(-/-) and hMSH3(-/-) cells revealed somatic PTEN frameshifts in 4% and 12% of clones, respectively. CONCLUSIONS: Nondysplastic epithelium from hamartomatous polyposis syndrome polyps harbors hMSH3 defects, which may prime neoplastic transformation. Polyps from PTEN(+/-) patients lose PTEN expression, but loss is not a universal early feature of all hamartomatous polyposis syndrome. However, PTEN frameshifts can occur in hMSH3-deficient cells, suggesting that hMSH3 deficiency could drive hamartomatous polyposis syndrome tumorigenesis.

Large intron 14 rearrangement in APC results in splice defect and attenuated FAP.

Familial adenomatous polyposis [FAP (OMIM 175100)] is an autosomal dominant colorectal cancer predisposition syndrome characterized by hundreds to thousands of colonic polyps and, if untreated by a combination of screening and/or surgical intervention, an approximately 99% lifetime risk of colorectal cancer. A subset of FAP patients develop an attenuated form of the condition characterized by lower numbers of colonic polyps (highly variable, but generally less than 100) and a lower lifetime risk of colorectal cancer, on the order of 70%. We report the diagnosis of three attenuated FAP families due to a 1.4-kb deletion within intron 14 of APC, originally reported clinically as a variant of unknown significance (VUS). Sequence analysis suggests that this arose through an Alu-mediated recombination event with a locus on chromosome 6q22.1. This mutation is inherited by family members who presented with an attenuated FAP phenotype, with variable age of onset and severity. Sequence analysis of mRNA revealed an increase in the level of aberrant splicing of exon 14, resulting in the generation of an exon 13-exon 15 splice-form that is predicted to lead to a frameshift and protein truncation at codon 673. The relatively mild phenotypic presentation and the intra-familial variation are consistent with the leaky nature of exon 14 splicing in normal APC. The inferred founder of these three families may account for as yet undetected affected branches of this kindred. This and similar types of intronic mutations may account for a significant proportion of FAP cases where APC clinical analysis fails because of the current limitations of testing options.

Pediatric juvenile polyposis syndromes: an update.

Colon polyps are a common finding in pediatrics and can present with rectal bleeding, abdominal pain, or polyp prolapse from the rectum. Histologically classified as hamartomas, these isolated pediatric polyps lack epithelial dysplasia and have no cancer risk. However, when polyps are present in greater numbers, or are associated with a family history of polyps or colon or other cancers, a polyposis or hereditary colorectal cancer syndrome should be considered. Using a case-based format, this article reviews the clinical features and provides updates on the three most common hamartomatous polyp syndromes of childhood: juvenile polyposis syndrome, Peutz-Jeghers syndrome, and the PTEN hamartoma tumor syndrome. Each syndrome has distinctive intestinal and extra-intestinal findings that, when present, can guide genetic counseling and testing. Lifelong cancer surveillance is crucial to disease prevention and the long-term health of these patients and their families.

Mutation rates of TGFBR2 and ACVR2 coding microsatellites in human cells with defective DNA mismatch repair.

Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFbeta family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1-/-, hMSH6-/-, hMSH3-/-, and MMR-proficient) to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP) gene, allowing a -1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7-35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a -1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2) in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes) and M2 (bright, representing full mutants) were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91 x 10(-4)) and 15 (2.18 x 10(-4)) times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was approximately 3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The -1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background.

Cyclooxygenase-2 expression in polyps from a patient with juvenile polyposis syndrome with mutant BMPR1A.

OBJECTIVES: Cyclooxygenase-2 (COX-2) expression is increased in colorectal cancers and has been reported to be upregulated in Peutz-Jeghers polyps. To determine whether germline and somatic loss of BMPR1A in polyps from a patient with juvenile polyposis syndrome have altered COX-2 expression, we characterized a patient with juvenile polyposis syndrome for BMPR1A germline mutations and examined the polyps for BMPR1A expression and COX-2 expression. PATIENTS AND METHODS: DNA analysis for BMPR1A was performed on a patient with juvenile polyposis syndrome. Multiple polypectomies were performed, and several polyps showed adenomatous change. Genomic DNA was extracted from polyp material for loss of heterozygosity (LOH) analyses with microsatellite markers. Immunohistochemistry was performed on sections using antibodies for BMPR1A and COX-2. RESULTS: The kindred possessed a germline BMPR1A missense mutation. In polyp domains containing cystic and adenomatous epithelium, no LOH was observed using markers near the BMPR1A locus. Immunostaining indicated decreased expression of phospho-SMAD1 (pSMAD1), functionally downstream of the mutant BMPR1A receptor in the cystic epithelium, with further reduction in adenomatous portions within the polyp. COX-2 protein, normally not expressed in the colon, was present and increased in polyp epithelium. CONCLUSIONS: Decreased expression of pSMAD1 in the cystic epithelium with further reduction in the adenomatous area, and increase in COX-2 expression within polyps from the BMPR1A heterozygote, suggest a potential mechanism for adenomatous pathogenesis in these hamartomatous polyps. This may imply that COX-2 inhibitors could be a means for chemoprevention in this syndrome.

Bone morphogenetic protein signaling and growth suppression in colon cancer.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily, which utilize BMP receptors and intracellular SMADs to transduce their signals to regulate cell differentiation, proliferation, and apoptosis. Because mutations in BMP receptor type IA (BMPRIA) and SMAD4 are found in the germline of patients with the colon cancer predisposition syndrome juvenile polyposis, and because the contribution of BMP in colon cancers is largely unknown, we examined colon cancer cells and tissues for evidence of BMP signaling and determined its growth effects. We determined the presence and functionality of BMPR1A by examining BMP-induced phosphorylation and nuclear translocation of SMAD1; transcriptional activity via a BMP-specific luciferase reporter; and growth characteristics by cell cycle analysis, cell growth, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolic assays. These assays were also performed after transfection with a dominant negative (DN) BMPR1A construct. In SMAD4-null SW480 cells, we examined BMP effects on cellular wound assays as well as BMP-induced transcription in the presence of transfected SMAD4. We also determined the expression of BMPR1A, BMP ligands, and phospho-SMAD1 in primary human colon cancer specimens. We found intact BMP signaling and modest growth suppression in HCT116 and two derivative cell lines and, surprisingly, growth suppression in SMAD4-null SW480 cells. BMP-induced SMAD signaling and BMPR1A-mediated growth suppression were reversed with DN BMPR1A transfection. BMP2 slowed wound closure, and transfection of SMAD4 into SW480 cells did not change BMP-specific transcriptional activity over controls due to receptor stimulation by endogenously produced ligand. We found no cell cycle alterations with BMP treatment in the HCT116 and derivative cell lines, but there was an increased G1 fraction in SW480 cells that was not due to increased p21 transcription. In human colon cancer specimens, BMP2 and BMP7 ligands, BMPRIA, and phospho-SMAD1 were expressed. In conclusion, BMP signaling is intact and growth suppressive in human colon cancer cells. In addition to SMADs, BMP may utilize SMAD4-independent pathways for growth suppression in colon cancers.