FAK loss reduces BRAFV600E-induced ERK phosphorylation to promote intestinal stemness and cecal tumor formation.

BRAFV600E mutation is a driver mutation in the serrated pathway to colorectal cancers. BRAFV600E drives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a 'just-right' level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in BRAFV600E-mutant adenomas/polyps in mice and patients. In Vil1-Cre;BRAFLSL-V600E/+;Ptk2fl/fl mice, Fak deletion maximized BRAFV600E's oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAFV600E-induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation increased the level of Lgr4, promoting intestinal stemness and cecal tumor formation. Our findings show that a 'just-right' ERK signaling optimal for BRAFV600E-induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation.

Cecal Tumorigenesis in Aryl Hydrocarbon Receptor-Deficient Mice Depends on Cecum-Specific Mitogen-Activated Protein Kinase Pathway Activation and Inflammation.

The aryl hydrocarbon receptor (AhR) is a transcription factor known as a dioxin receptor. Recently, Ahr-/- mice were revealed to develop cecal tumors with inflammation and Wnt/ß-catenin pathway activation. However, whether ß-catenin degradation is AhR dependent remains unclear. To determine whether other signaling pathways function in Ahr-/- cecal tumorigenesis, we investigated histologic characteristics of the tumors and cytokine/chemokine production in tumors and Ahr-/- peritoneal macrophages. AhR expression was also assessed in human colorectal carcinomas. Of the 28 Ahr-/- mice, 10 developed cecal lesions by 50 weeks of age, an incidence significantly lower than previously reported. Cecal lesions of Ahr-/- mice developed from serrated hyperplasia to adenoma/dysplasia-like neoplasia with enhanced proliferation. Macrophage and neutrophil infiltration into the lesions was also observed early in serrated hyperplasia, although adjacent mucosa was devoid of inflammation. Il1b, Il6, Ccl2, and Cxcl5 were up-regulated at lesion sites, whereas only IL-6 production increased in Ahr-/- peritoneal macrophages after lipopolysaccharide + ATP stimulation. Neither Myc (alias c-myc) up-regulation nor ß-catenin nuclear translocation was observed, unlike previously reported. Interestingly, enhanced phosphorylation of extracellular signal-regulated kinase, Src, and epidermal growth factor receptor and Amphiregulin up-regulation at Ahr-/- lesion sites were detected. In human serrated lesions, however, AhR expression in epithelial cells was up-regulated despite morphologic similarity to Ahr-/- cecal lesions. Our results suggest novel mechanisms underlying Ahr-/- cecal tumorigenesis, depending primarily on cecum-specific mitogen-activated protein kinase pathway activation and inflammation.

Cytokeratin 7-positive/cytokeratin 20-negative cecal adenocarcinoma metastatic to the uterine cervix: a case report.

BACKGROUND: The vast majority of uterine cervical malignancies are primary carcinomas, and secondary neoplasms that metastasize to the uterine cervix from a distant organ are uncommon. Although relatively rare, metastases to the uterine cervix from a primary colon cancer have been reported. We report a rare case of metastatic carcinoma originating from a cecal adenocarcinoma with an unusual cytokeratin 7/cytokeratin 20 immunophenotype. CASE PRESENTATION: A 74-year-old postmenopausal Japanese woman was referred to our hospital for the evaluation of a uterine tumor. She had a past medical history of cecal cancer and had undergone laparoscopically assisted right hemicolectomy at the age of 69 years. During follow-up, she was found to have elevated levels of the tumor markers carbohydrate antigen 19-9 (179.7 IU/mL) and carcinoembryonic antigen (26.9 µg/L). Positron emission tomography/computed tomography showed a focus of high 18F-fluorodeoxyglucose uptake in her uterus. Examination of a cervical biopsy found a poorly differentiated adenocarcinoma that was immunopositive for cytokeratin (CK)7 and caudal-related homeobox 2 (CDX2) expression and immunonegative for cytokeratin 20 expression. The patient underwent radical hysterectomy and bilateral salpingo-oophorectomy. Histopathological examination found invasive growth of irregular and atypical ductal hyperplasia. Immunohistochemical staining of the tumor specimen revealed the same immunophenotype as the biopsy specimen. The cecal cancer specimen from her previous surgery was also examined and found to have the same immunophenotype. The histopathological diagnosis was cecal adenocarcinoma metastatic to the uterine cervix. The patient is currently receiving adjuvant chemotherapy and to date is without evidence of recurrent disease. CONCLUSIONS: Our report illustrates the importance of immunohistochemistry for the correct diagnosis of the origin of a uterine cervical adenocarcinoma in a patient with a medical history of colorectal cancer. Re-examination of a previous oncological specimen is critical for cases with a uterine lesion that is difficult to identify as primary or metastatic cancer.

Cadherin-17 and SATB2 are sensitive and specific immunomarkers for medullary carcinoma of the large intestine.

CONTEXT: Distinction of medullary carcinoma of the large intestine from other cytokeratin (CK) 7⁻/CK20⁻ carcinomas can be challenging when working on a tumor of unknown primary because the majority of medullary carcinomas are negative for CK7, CK20, and CDX2. OBJECTIVE: To investigate the expression of cadherin-17 and SATB-2 and other markers in medullary carcinomas of the large intestine and cadherin-17 and SATB2 in a large number of carcinomas and normal tissues from various organs to further test their diagnostic specificity. DESIGN: This study evaluated cadherin-17 and SATB2 expression in 18 medullary carcinoma cases and 1941 tumors and 358 normal tissues from various organs. Other immunomarkers, including MLH1, PMS2, MSH2, MSH6, CDX2, CK7, CK20, TFF3, MUC4, calretinin, p504S, villin, and synaptophysin, were also tested on the 18 medullary carcinoma cases. RESULTS: The results demonstrated (1) loss of MLH1 and PMS2 in more than 80% of medullary carcinomas; (2) expression of cadherin-17 and SATB2 in 89% of medullary carcinomas; (3) focal expression of TFF3, MUC4, calretinin, CDX2, CK20, and synaptophysin in 72%, 72%, 67%, 67%, 28%, and 17% of 18 medullary carcinoma cases, respectively; and (4) expression of SATB2 and cadherin-17 in 97% and 98% of the colorectal adenocarcinomas, respectively, whereas their expression was seen in 3.6% and 3.3% of nongastrointestinal tumors, respectively. CONCLUSION: We concluded that SATB2 and cadherin-17 were highly sensitive and specific markers for colorectal carcinomas and propose including MLH1, cadherin-17, and SATB2 in a routine immunostaining panel when working on a tumor of unknown primary, especially in an elderly patient with a CK7⁻/CK20⁻ carcinoma.

Synchronous triple cancers of the pancreas, stomach, and cecum treated with S-1 followed by pancrelipase treatment of pancreatic exocrine insufficiency.

CONTEXT: Pancreatic cancer is frequently complicated by malignancies in other organs. However, synchronous triple cancers including pancreatic cancer have been seldom reported in the English language literature. CASE REPORT: We describe the rare case of a 77-year-old man with triple cancers of the pancreas, stomach, and cecum. Biopsies revealed that all three tumors were adenocarcinomas. The pancreatic and gastric tumors were positive for cytokeratin 7 and negative for cytokeratin 20, whereas the cecal tumor was negative for cytokeratin 7 and positive for cytokeratin 20. K-ras mutations were present at codon 12 in the pancreatic tumor and at codon 13 in the cecal tumor, but were absent from the gastric tumor. Since the three tumors had different characteristics, the patient was diagnosed with synchronous triple cancers. Because invasive surgery was required to remove all three tumors and the patient had risk factors for surgery, we elected to treat him with chemotherapy. All three cancers were markedly reduced in size by treatment with cycles of 100 mg/day S-1 for 2 weeks, followed by a 1-week rest. The patient later developed hypoproteinemia and anasarca, which was diagnosed as pancreatic exocrine insufficiency due to pancreatic head cancer. Treatment with pancrelipase resulted in dramatic improvements in hypoproteinemia and anasarca. CONCLUSIONS: This is the first case report in which S-1 was effective in triple cancers of the pancreas, stomach, and cecum. Patients with pancreatic head cancer should be monitored for pancreatic exocrine insufficiency.

[Vasoactive intestinal polypeptide-secreting diffuse ganglioneuromatosis affecting the small intestine and the colon in an infant: an exceptional inaugural manifestation of NF1]. / Ganglioneuromatose iléo-colique diffuse avec hypersécrétion de peptide vaso-intestinal chez un nourrisson : une manifestation inaugurale exceptionnelle d'une NF1.

Diffuse ganglioneuromatosis of the digestive tract is a rare condition, especially in children. It is frequently associated with multiple endocrine neoplasia type 2b and less commonly with neurofibromatosis type 1 (NF1). We report the case of an 8-month-old baby presenting with vasoactive intestinal polypeptide (VIP)-secreting diffuse ganglioneuromatosis affecting the small intestine and the colon and responsible for severe hydric diarrhea. Postoperatively the infant's symptoms resolved and the serum VIP level was normal. NF1 was clinically suspected and then confirmed through genetic testing. Two years later, the child developed an optic pathway glioma, another tumor frequently associated with NF1.

[A case of stage IV AFP producing cecal cancer responding to mFOLFOX6 leading to a partial response].

We report a case of stage IV AFP producing cecal cancer responding to mFOLFOX6 leading to a partial response. The patient was a 72-year-old female with multiple liver metastases of cecal cancer. She underwent a right hemicolectomy and right salpingo-oophorectomy in a non-curative resection. Final findings revealed cecal cancer, type 3, 60 x 55 mm, pSI (right ovary), pN3, sH3, sP0, cM0, fStage IV, respectively. After surgery, chemotherapy with mFOLFOX6 was performed. After 4 courses, a significant tumor reduction (PR) was obtained. She died 1 year 2 months after surgery. This case suggests that chemotherapy with mFOLFOX6 is a potential regimen for AFP producing colon cancer.

Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands.

Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of beta-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent beta-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in Apc(Min/+) mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.

In vivo effect of imatinib on progression of cecal GIST-like tumors in exon 17-type c-kit knock-in mice.

Two families with a germline Asp820Tyr mutation at exon 17 of the c-kit gene and multiple gastrointestinal stromal tumors (GISTs) have been reported. Recently, we generated a knock-in mouse model of the family, and mice with KIT-Asp818Tyr corresponding to human KIT-Asp820Tyr showed a cecal GIST-like tumor. In this report, we examined the in vivo effect of imatinib on tumor progression in knock-in mice. Imatinib of 100 microg/g body weight was administered to heterozygous (KIT-Asp818Tyr/+) mice orally for 7, 14 and 28 days, and cecal tumors were dissected. Both macroscopic size and the measured volume of cecal tumors were not significantly reduced after a 7-, 14- and 28-day administration of imatinib when compared with those before imatinib administration. Cell proliferation was assessed by Ki-67 immunohistochemistry and the labeling index significantly decreased after imatinib administration, but the value of the index after imatinib was only about half compared with that before imatinib. Western blotting and real-time PCR revealed that KIT expression was almost equivalent, but KIT phosphorylation was significantly but not completely inhibited in tumor tissues after 7, 14 and 28 days of imatinib administration when compared with that before imatinib administration. Phosphorylation of Akt and Stat1 was accordingly inhibited after imatinib administration. Thus, imatinib seemed to inhibit in vivo tumor proliferation but not decrease tumor volume on this mouse model, probably because of an insufficient inhibition of phosphorylation of KIT and its downstream signaling molecules. These results suggested that progression of multiple GISTs in patients with germline Asp820Tyr might be partially controlled by imatinib and that model mice provide an opportunity to examine the effect of various other targeted drugs on in vivo tumor progression.

Loss of Rb1 in the gastrointestinal tract of Apc1638N mice promotes tumors of the cecum and proximal colon.

To examine the role of Rb1 in gastrointestinal (GI) tumors, we generated mice with an Apc(1638N) allele, Rb(tm2brn) floxed alleles, and a villin-cre transgene (RBVCA). These animals had exon 19 deleted from Rb1 throughout the GI tract. We have shown previously that Rb1 deficiency is insufficient for GI tumor initiation, with inactivation of an Apc allele capable of overcoming the insufficiency. In this study we demonstrate that RBVCA mice have reduced median survival because of an increase in tumor incidence and multiplicity in the cecum and the proximal colon. Large intestinal tumors are predominantly adenomas, whereas the tumors of the small intestine are a mixture of adenomas and adenocarcinomas. We find truncation mutations to the second Apc allele in tumors of both the large and small intestine. Expression profiles of duodenal and cecal tumors relative to each other show unique gene subsets up and down regulated. Substantial expression patterns compare to human colorectal cancer, including recapitulation of embryonic genes. Our results indicate that Rb1 has significant influence over tumor location in the GI tract, and that both cecal and duodenal tumors initiate through inactivation of Apc. Expression profile analysis indicates the two tumor types differentially regulate distinct sets of genes that are over-expressed in a majority of human colorectal carcinomas.

A case of relapsing secondary bladder adenocarcinoma after right colonic cancer.

BACKGROUND: A 71-year-old woman was referred to a surgical oncology clinic after CT raised suspicion for a bladder neoplasm. She had previously undergone right hemicolectomy and received adjuvant chemotherapy for pT3N1MX cancer of the cecum. A retroperitoneal recurrence had been deemed unsuitable for surgical resection, and had instead been treated with chemoradiation therapy. Follow-up CT raised suspicion for a possible bladder neoplasm. INVESTIGATIONS: CT, physical examination, urinalysis, cystoscopy with biopsy, pathological analysis and immunohistochemical analysis. DIAGNOSIS: Adenocarcinoma of the cecum metastatic to the bladder. MANAGEMENT: The patient underwent open bladder resection with total excision of the neoplasm and was administered adjuvant chemotherapy consisting of irinotecan and cetuximab. Subsequent recurrences at the same site were treated with transurethral resection, while chemotherapy was still in progress. At 7 months' follow-up, the patient remained alive, with no evidence of further recurrence.

Differential gene expression in colon cancer of the caecum versus the sigmoid and rectosigmoid.

BACKGROUND AND AIMS: There are epidemiological, morphological, and molecular differences between normal mucosa as well as between adenocarcinomas of the right and left side of the large bowel. The aim of this study was to investigate differences in gene expression. METHODS: Oligonucleotide microarrays (GeneChip) were used to compare gene expression in 45 single samples from normal mucosa and sporadic colorectal carcinomas (Dukes' B and C) of the caecum compared with the sigmoid and rectosigmoid. Findings were validated by real time polymerase chain reaction. RESULTS: Fifty eight genes were found to be differentially expressed between the normal mucosa of the caecum and the sigmoid and rectosigmoid (p<0.01), including pS2, S100P, and a sialyltransferase, all being expressed at higher levels in the caecum. A total of 118 and 186 genes were differentially expressed between normal and right or left sided tumours of the colon, showing more pronounced differences in Dukes' C than B tumours. Thirty genes differentially expressed in tumour tissue were common to adenocarcinomas of both sides, including known tumour markers such as the matrix metalloproteinases. Keratins 8, 19, and 20 as well as carbonic anhydrases (II, IV, VII) showed side specific expression and were downregulated in left sided tumours whereas teratocarcinoma growth factor and cyclooxygenase 2 (COX-2) were upregulated in left sided adenocarcinomas. Immunohistochemical analysis confirmed differences in side specific expression for cytokeratin 20 and COX-2. CONCLUSIONS: Differences in gene expression between normal mucosa as well as between adenocarcinomas of the caecum and sigmoid or rectosigmoid exist and should be taken into account when examining new targeted therapeutic regimens.

Colliding / concomitant tumors of the intestine: report of 3 cases.

Collision/concomitant tumors of the intestine involving lymphomas are very rare. For these cases molecular genetic analyses are valuable diagnostic adjuncts. We report one collision tumor of the rectum (adenocarcinoma and peripheral T-cell lymphoma, unspecified), and two cases of concomitant tumors (carcinoma in the cecum and lymphoma in the ileum; carcinoma in the sigmoid and lymphoma in the ileum). The collision tumor (poorly differentiated adenocarcinoma and a peripheral T-cell lymphoma, unspecified) showed a variable proportion of the anaplastic tumor cells expressing lymphatic markers as well as cytokeratin. Only polymerase chain reaction (PCR) analysis revealing T-cell monoclonality of the anaplastic part of the colliding tumor allowed the correct diagnosis. In the second case, a moderately differentiated adenocarcinoma in the cecum with a concomitant B-cell Non-Hodgkin lymphoma in the ileum, PCR analysis was non contributory. In the third case (adenocarcinoma in the sigmoid colon and a follicular center lymphoma in the ileum) PCR analysis revealed gene rearrangement of the immunoglobulin heavy chain gene. We would like to emphasize that collision and concomitant tumors of the gut are rare and that molecular genetic analysis may be mandatory for correct diagnosis. It is our impression, that these tumors may be diagnosed more often in the intestinal tract if molecular genetic analysis and immunohistochemistry are used routinely, at least for all anaplastic tumors.

Thymidine and hypoxanthine protection patterns of the folic acid-enhanced synergies for combinations of trimetrexate plus a polyglutamylatable inhibitor of purine or thymidylate synthesis against human ileocecal HCT-8 cells.

In order to examine the intracellular locus of the folic acid (PteGlu)-enhanced synergies of trimetrexate (TMQ) plus the thymidylate synthase (TS) inhibitor, raltitrexed (RTX), and TMQ plus the glycinamide ribonucleotide formyltransferase (GARFT) inhibitor, AG2034, comprehensive protection studies with thymidine (dThd) and hypoxanthine (HX) were conducted in a 96-well plate cell growth inhibition (sulforhodamine B) assay. Current modeling techniques were extended to characterize these protection patterns involving multiple-agent interaction. Wild-type human ileocecal HCT-8 cells and DW2, a subline deficient in folylpoly-gamma-glutamate synthetase (FPGS) were individually treated for 96 h with TMQ, AG2034 and a 1:1 mixture of TMQ:AG2034 or with TMQ, RTX, and a 1:1 mixture of TMQ:RTX in the presence of PteGlu (2.3 or 40 micro M) and the protection agents (10 micro M dThd and/or 100 micro M HX). Drug treatments were randomly assigned to wells. Both isobols and 3-dimensional concentration-effect surfaces were used to assess the nature and the intensity of drug interactions. The structural Hill model was fitted to data with weighted non-linear regression for most cases. A so-called 'double Hill' model was sometimes more appropriate when a plateau in the middle of the concentration-effect curve was found. In HCT-8 and DW2 cells at 2.3 and 40 micro M PteGlu, inhibition of DHFR by TMQ induced antithymidylate and antipurine effects; AG2034 and RTX selectively inhibited de novo purine or thymidine synthesis, respectively. dThd protection increased the PteGlu-enhancement of the TMQ + AG2034 synergy, whereas HX protection increased the PteGlu-enhancement of the TMQ + RTX synergy. The PteGlu-enhanced synergies of TMQ + AG2034 and TMQ + RTX occur primarily through inhibition of purine synthesis and inhibition of thymidylate synthesis, respectively. These results further substantiate the hypothesis that the nonpolyglutamylatable DHFR inhibitor, TMQ, acts as a modulator by decreasing the protection by PteGlu of cells against the polyglutamylatable AG2034 and RTX.

Aberrant regulation of human intestinal proglucagon gene expression in the NCI-H716 cell line.

Despite interest in understanding glucagon-like peptide-1 (GLP-1) production, the factors important for GLP-1 biosynthesis remain poorly understood. We examined control of human proglucagon gene expression in NCI-H716 cells, a cell line that secretes GLP-1 in a regulated manner. Insulin, phorbol myristate acetate, or forskolin, known regulators of rodent proglucagon gene expression, had no effect, whereas sodium butyrate decreased levels of NCI-H716 proglucagon mRNA transcripts. The inhibitory effect of sodium butyrate was mimicked by trichostatin A but was not detected with sodium acetate or isobutyrate. The actions of butyrate were not diminished by the ERK1/2 inhibitor PD98059, p38 inhibitor SB203580, or soluble guanylate cyclase inhibitor LY83583 or following treatment of cells with KT5823, a selective inhibitor of cGMP-dependent protein kinase. NCI-H716 cells expressed multiple proglucagon gene transcription factors including isl-1, pax-6, pax-2, cdx-2/3, pax-4, hepatocyte nuclear factor (HNF)-3 alpha, HNF-3beta, HNF-3 gamma, and Nkx2.2. Nevertheless, the butyrate-dependent inhibition of proglucagon gene expression was not associated with coordinate changes in transcription factor expression and both the human and rat transfected proglucagon promoters were transcriptionally inactive in NCI-H716 cells. Hence, NCI-H716 cells may not be a physiologically optimal model for studies of human enteroendocrine proglucagon gene transcription.

Different beta-catenin immunoexpression in carcinoid tumors of the appendix in comparison to other gastrointestinal carcinoid tumors.

Carcinoid tumor of the appendix is an endocrine tumor that is histologically similar to, but biologically less aggressive than carcinoids arising from other parts of the gastrointestinal tract. In this study, we examined E-cadherin, beta-catenin, DCC, p53 and Ki67 immunoexpression in cases of carcinoid of the appendix and made a comparison with non-appendiceal carcinoid tumors. Nine cases of appendiceal carcinoid and 11 biopsies of carcinoid of other parts of the gastrointestinal tract, five cases of the small intestine and six of the stomach were immunohistochemically evaluated for Ki67, p53, DCC, E-cadherin and beta-catenin. Two main patterns of beta-catenin staining were observed. The first pattern was characterized as membranous and cytoplasmic, and was seen mainly in the peripheral cells of the nests. The second pattern was diffuse, predominantly membranous. Most (five of seven) appendiceal carcinoids and only three of 11 non-appendiceal cases showed the first staining pattern (p < 0.05). Immunoexpression of E-cadherin and DCC was similar in both groups. p53 and Ki-67 immunostaining revealed stronger nuclear positivity in the non-appendiceal carcinoid tumors (statistically not significant). We found a pattern of beta-catenin immunostaining in typical carcinoid tumors of the appendix that was different from the pattern seen in non-appendiceal carcinoid tumors. This alteration suggests that carcinoid of the appendix may represent a different subtype of carcinoid tumors with different immunohistochemical and biological behavior.