Samornmas Kanngurn

Inactivation of TGF-β signaling and loss of PTEN cooperate to induce colon cancer in vivo.

The accumulation of genetic and epigenetic alterations mediates colorectal cancer (CRC) formation by deregulating key signaling pathways in cancer cells. In CRC, one of the most commonly inactivated signaling pathways is the transforming growth factor-beta (TGF-β) signaling pathway, which is often inactivated by mutations of TGF-β type II receptor (TGFBR2). Another commonly deregulated pathway in CRC is the phosphoinositide-3-kinase (PI3K)-AKT pathway. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of PI3K-AKT signaling and is silenced in ∼30% of CRC. The combination of TGFBR2 inactivation and loss of PTEN is particularly common in microsatellite-unstable CRCs. Consequently, we determined in vivo if deregulation of these two pathways cooperates to affect CRC formation by analyzing tumors arising in mice that lack Tgfbr2 and/or Pten specifically in the intestinal epithelium. We found that lack of Tgfbr2 (Tgfbr2IEKO) alone is not sufficient for intestinal tumor formation and lack of Pten (PtenIEKO) alone had a weak effect on intestinal tumor induction. However, the combination of Tgfbr2 inactivation with Pten loss (PtenIEKO;Tgfbr2IEKO) led to malignant tumors in both the small intestine and colon in 86% of the mice and to metastases in 8% of the tumor-bearing mice. Moreover, these tumors arose via a β-catenin-independent mechanism. Inactivation of TGF-β signaling and loss of Pten in the tumors led to increased cell proliferation, decreased apoptosis and decreased expression of cyclin-dependent kinase inhibitors. Thus, inactivation of TGF-β signaling and loss of PTEN cooperate to drive intestinal cancer formation and progression by suppressing cell cycle inhibitors.

RET is a potential tumor suppressor gene in colorectal cancer

Cancer arises as the consequence of mutations and epigenetic alterations that activate oncogenes and inactivate tumor suppressor genes. Through a genome-wide screen for methylated genes in colon neoplasms, we identified aberrantly methylated RET in colorectal cancer. RET, a transmembrane receptor tyrosine kinase and a receptor for the glial cell-derived neurotrophic factor family ligands, was one of the first oncogenes to be identified, and has been shown to be an oncogene in thyroid cancer and pheochromocytoma. However, unexpectedly, we found RET is methylated in 27% of colon adenomas and in 63% of colorectal cancers, and now provide evidence that RET has tumor suppressor activity in colon cancer. The aberrant methylation of RET correlates with decreased RET expression, whereas the restoration of RET in colorectal cancer cell lines results in apoptosis. Furthermore, in support of a tumor suppressor function of RET, mutant RET has also been found in primary colorectal cancer. We now show that these mutations inactivate RET, which is consistent with RET being a tumor suppressor gene in the colon. These findings suggest that the aberrant methylation of RET and the mutational inactivation of RET promote colorectal cancer formation, and that RET can serve as a tumor suppressor gene in the colon. Moreover, the increased frequency of methylated RET in colon cancers compared with adenomas suggests RET inactivation is involved in the progression of colon adenomas to cancer.

NTRK3 is a potential tumor suppressor gene commonly inactivated by epigenetic mechanisms in colorectal cancer.

NTRK3 is a member of the neurotrophin receptor family and regulates cell survival. It appears to be a dependence receptor, and thus has the potential to act as an oncogene or as a tumor suppressor gene. NTRK3 is a receptor for NT-3 and when bound to NT-3 it induces cell survival, but when NT-3 free, it induces apoptosis. We identified aberrantly methylated NTRK3 in colorectal cancers through a genome-wide screen for hypermethylated genes. This discovery led us to assess whether NTRK3 could be a tumor suppressor gene in the colon. NTRK3 is methylated in 60% of colon adenomas and 67% of colon adenocarcinomas. NTRK3 methylation suppresses NTRK3 expression. Reconstitution of NTRK3 induces apoptosis in colorectal cancers, if NT-3 is absent. Furthermore, the loss of NTRK3 expression associates with neoplastic transformation in vitro and in vivo. We also found that a naturally occurring mutant NTRK3 found in human colorectal cancer inhibits the tumor suppressor activity of NTRK3. In summary, our findings suggest NTRK3 is a conditional tumor suppressor gene that is commonly inactivated in colorectal cancer by both epigenetic and genetic mechanisms whose function in the pathogenesis of colorectal cancer depends on the expression status of its ligand, NT-3.

Single Nucleotide Polymorphisms in the Gc Gene for Vitamin D Binding Protein in Common Cancers in Thailand

BACKGROUND This case-control study aimed to determine if there were any associations between the two single nucleotide polymorphisms (SNPs) in Gc, rs7041 (Asp416Glu) and rs4588 (Thr420Lys) and 3 common cancers (breast, lung and colorectal) in Thai patients. MATERIALS AND METHODS Two hundred and eighty two colorectal, 101 breast and 113 lung cancer patients were recruited from one institute during 2011-2013. The controls were age-matched volunteers who had a negative history of index cancers. In addition, vitamin D levels were compared among different genotypes in the 2 SNPs. RESULTS The minor allele frequencies of rs7041 (G) and rs4588 (A) were 0.32 and 0.24, respectively. Under the dominant model, the study found significant associations between minor-allele genotypes of the SNP rs7041 (TG/GG) and lung cancer (odds ratio [OR] 1.78, 95% CI 1.05-3.03). When subgroup analysis was performed according to sex and age at diagnosis, the study found that the minor- allele genotypes of rs7041 (TG/GG) were significantly associated with colorectal cancer in patients whose age at diagnosis was more than 60 years (OR 1.67, 95%CI 1.06-2.61) and the minor-allele genotypes of rs4588 (CA/AA) were significantly associated with colorectal cancer in males aged 60 years or less (OR 2.34, 95%CI 1.25-4.37). When SNP combinations (rs7041-rs4588) were examined, the TT-CA combination had a significant protective association with lung cancer (OR 0.44, 95% CI 0.22-0.85). On evaluation of serum 25(OH)D levels in 205 individuals without cancer (males 144, females 61), the proportion of subjects with low serum vitamin D (< 20 ng/ml) in those harboring CA or AA genotypes of rs4588 (41.7%) was significantly higher than the CC genotype (15.5%, p-value < 0.01). CONCLUSIONS Genetic polymorphisms in Gc were associated with lung and colorectal cancers in Thai patients. Lower serum 25(OH)D in minor variants of rs4588 may explain this association.

Clinical outcomes of gastrointestinal stromal tumor in southern Thailand

AIM To review a single institutional experience in clinical management of gastrointestinal stromal tumors (GIST) and analyze for factors determining treatment outcome. METHODS Clinicopathological data of patients with a diagnosis of GIST who were treated at our institute during November 2004 to September 2009 were retrospectively reviewed. RESULTS Ninety-nine cases were included in the analysis. Primary tumor sites were at the stomach in and small bowel in 44% and 33%, respectively. Thirty-one cases already had metastasis at presentation and the most common metastatic site was the liver. Sixty-four cases (65%) were in the high-risk category. Surgical treatment was performed in 77 cases (78%), 3 of whom received upfront targeted therapy. Complete resection was achieved in 56 cases (73% of operative cases) and of whom 27 developed local recurrence or distant metastasis at a median duration of 2 years. Imatinib was given as a primary therapy in unresectable cases (25 cases) and as an adjuvant in cases with residual tumor (21 cases). Targeted therapy gave partial response in 7 cases (15%), stable disease in 27 cases (57%) and progressive disease in 13 cases (28%). Four-year overall survival was 74% (95% CI: 61%-83%). Univariate survival analysis found that low-risk tumor, gastric site, complete resection and response to imatinib were associated with better survival. CONCLUSION The overall outcomes of GIST can be predicted by risk-categorization. Surgery alone may not be a curative treatment for GIST. Response to targeted therapy is a crucial survival determinant in these patients.

Cushing's Syndrome in an Infant Secondary to Malignant Adrenocortical Tumors with Somatic Mutation of Beta-Catenin

A role of beta-catenin (CTNNB1) in the molecular pathogenesis of adrenocortical carcinoma (ACC) has been suspected in adult ACC and pediatric pigmented nodular adrenocortical disease, but it has never been reported in pediatric ACC. We present the case of a 4-month-old Thai infant who had Cushings syndrome secondary to bilateral adrenal tumors with hepatic metastasis. The child was successfully treated with a bilateral adrenalectomy and wedge resection of the liver nodule. Histopathology revealed bilateral adrenocortical tumors with different histologic grades; the right tumor had a higher score, according to modified Weiss criteria. On molecular study, a deletion mutation of beta-catenin involving codons 44 to 45 was detected in the right adrenal tumor. The same mutation was found in peripheral blood before treatment; this mutation disappeared after tumor removal. The left tumor harbored wild-type beta-catenin. Immunohistochemistry showed nuclear accumulation of beta-catenin on the right adrenal tumor and the metastatic nodule. In summary, we present evidence that supports the role of the Wnt-signaling pathway in the carcinogenesis of pediatric adrenocortical carcinoma.

Expression of BMP6 is Associated with its Methylation Status in Colorectal Cancer Tissue but Lacks Prognostic Significance

BACKGROUND The study aimed to evaluate the incidence of CpG island promoter methylation of BMP6, a member of the transforming growth factor beta family, in tissue samples from colorectal cancers (CRC) and look for its association with BMP6 expression and clinicopathological correlation. MATERIALS AND METHODS Methylation specific PCR for the BMP6 promoter region was performed with 85 frozen tissue samples of CRC and 45 of normal colon. Methylation status of MLH1 was also determined by the same method. Expression of BMP6 was evaluated by immunohistochemistry (IHC), using Allreds scoring system. The methylation status was analyzed against clinical and pathological parameters in CRC. RESULTS The study revealed BMP6 hypermethylation in 34 of 85 tumor specimens (40%), and 15 out of 45 normal tissue samples from CRC (33%). The incidence of hypermethylation was inversely correlated with IHC score. Allreds scores of 7 or more were correlated with lower frequency of BMP6 hypermethylation (29% compared to 50% in the remaining, p-value 0.049). However, there was no association between hypermethylation status and any clinicopathological parameters. The methylation status of BMP6 was not correlated with that of MLH1, a key methylation determinant in CRC. On survival analysis, there was no significant difference in progress-free survival (PFS) between the cases with and without hypermethylation (2-year PFS 74% and 76%, respectively). CONCLUSIONS CpG island methylation of BMP6 is found in high frequency in CRC and this epigenetic event is associated with suppressed protein expression in the tumor tissue. However, the marker is not associated with tumor progression of the disease.

Chalkley Microvessel but not Lymphatic Vessel Density Correlates with Axillary Lymph Node Metastasis in Primary Breast Cancers

This study aimed to investigate tumor microvessel density (MVD) and lymphatic vessel density (LVD) using the Chalkley method as predictive markers for the risk of axillary lymph node metastasis and their relationship to other clinicopathological parameters in primary breast cancer cases. Forty two node-positive and eighty node-negative breast cancers were immunostained for CD34 and D2-40. MVD and LVD were counted by the Chalkley method at x400 magnification. There was a positive significant correlation of the MVD with the tumor size, coexisting ductal carcinoma in situ (DCIS) and lymph node metastases (P<0.05). In multivariate analysis, the MVD (2.86-4: OR 5.87 95%CI 1.05-32; >4: OR 20.03 95%CI 3.47-115.6), lymphovascular invasion (OR 3.46, 95% CI 1.13-10.6), and associated DCIS (OR 3.1, 95%CI 1.04-9.23) independently predicted axillary lymph node metastasis. There was no significant relationship between LVD and axillary lymph node metastasis. However, D2-40 was a good lymphatic vessel marker to enhance the detection of lymphatic invasion compared to H and E staining. In conclusion, MVD by the Chalkley method, lymphovascular invasion and associated DCIS can be additional predictive factors for axillary lymph node metastases in breast cancer. No relationship was identified between LVD and clinicopathological variables, including axillary lymph node metastasis.

Immunohistological evidence for Wnt-signaling activation in Peutz-Jeghers polyposis

ObjectiveMolecular pathogenesis of gastrointestinal polyposis in Peutz-Jegher’s syndrome (PJS) has been linked to the loss-of-function mutation of LKB1. Recent functional genetic studies have pointed out that LKB1 plays a physiological role in controlling the Wnt-signaling pathway and activation of the pathway as a consequence of LKB1 haploinsufficiency might be responsible for the development of harmatomatous polyps. This study aimed to look for immunohistochemical evidence of Wnt-signaling activation in PJS polyps.MethodBeta-catenin immunohistochemistry patterns were evaluated in gastrointestinal polyps from five cases of PJS. All patients were also evaluated for germline mutations of LKB1 and somatic mutations of beta-catenin in the polyps.ResultsFour of the five cases had germline mutations of LKB1, including two novel mutations, a one-base insertion at codon 53 and a large deletion encompassing exon 3 (codon 136–155). PJS polyps from all patients showed generalized membrane and cytoplasmic localizations of beta-catenin along the mucosal endothelium. Polyps from two cases with LKB1 mutations revealed moderate-intensity nuclear staining in approximately 20 and 70% of the polyps.ConclusionThe study offers additional evidence of Wnt-signaling activation in PJS polyp development at the tissue level, although the degree of up-regulation was not as high as has been found in Wnt-associated neoplasms.

Abstract 2415: TGF-ß signaling inactivation inhibits the formation of hepatocellular carcinomas induced by loss of p53

Background: Hepatocellular carcinoma (HCC) is one of the most deadly forms of cancer and results from the accumulation of mutated or altered tumor suppressor genes and/or oncogenes. The tumor suppressor and oncogenes commonly affected include growth factors, receptors and downstream signaling components. Inactivation of the TGF-s signaling pathway and mutation of TP53, the gene for p53, are common in HCCs suggesting they may cooperate in HCC formation. Thus, we assessed whether inactivation of TGF-s signaling, by deletion of the TGF-s receptor type II, TGFBR2, affects the formation of HCCs arising secondary to loss of p53. Aim: To determine if loss of TGFBR2 and p53 cooperate in vivo to affect HCC formation. Methods and Results: Albumin-cre (Alb-Cre) transgenic mice were crossed with mice homozygous for floxed Tgfbr2 (Tgfbr2flx/flx) and/or floxed Trp53 (Trp53flx/flx) to generate mice lacking TGFBR2 and/or p53 in the liver. Deletion of Tgfbr2 alone did not induce liver tumors, while inactivation of both Tgfbr2 and Trp53 resulted in a subset of mice (19%) that developed liver tumors by 65 weeks of age. Surprisingly, deletion of p53 alone, in the context of intact TGF-s receptors, resulted in an increased number of mice developing tumors (38%), as well as increased morbidity (42 weeks of age). Interestingly, in both genotypes, Alb-Cre;Trp53flx/flx;Tgfbr2flx/flx and Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt, the mice develop both hepatocellular carcinomas (HCC) and cholangiocarcinomas (CC), suggesting these tumors originate from a common liver stem cell population. Quantitative RT-PCR analysis of tumor and non-tumor tissue demonstrated a subset of Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt (p53 null) tumors express significantly increased levels of alpha-fetoprotein mRNA, a clinical marker for HCC. Additionally, tumors from the Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt displayed increased TGFβ-1 protein levels as compared to tumors from Trp53 and Tgfbr2 null mice. Furthermore, increased phosphorylated ERK1/2 expression was also present in the tumors from the p53 null mice and correlated with a slight up-regulation in beta-catenin expression, another common molecular event observed in human HCC. Conclusions: The Alb-Cre;Trp53flx/flx;Tgfbr2flx/flx mouse model recapitulates many molecular features of human HCC and demonstrates that TGF-s signaling is oncogenic in the setting of loss of p53 in the liver. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2415. doi:10.1158/1538-7445.AM2011-2415