Search

ABSTRACTSecreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of SFRP3in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate SFRP3expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, SFRP3mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of SFRP3in LUAD and LUSC patients. Moreover, DNA hypermethylation of SFRP3was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of SFRP3expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and in vitrodemethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of CyclinD1expression in vitro. Our results indicate that SFRP3acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression and DNA methylation, as well as its potential clinical impact in non-small-cell lung carcinoma (NSCLC). We examined ITIH5mRNA expression in tumor and adjacent normal lung tissue specimens of NSCLC patients. In addition, methylation frequency of the ITIH5promoter was investigated using methylation-specific PCR and pyrosequencing. Significance of our data was validated by independent data sets from The Cancer Genome Atlas and the Kaplan-Meier Plotter platform. Furthermore, ITIH5 protein expression was evaluated by immunohistochemistry utilizing a tissue microarray with 385 distinct lung tissue samples. Based on our tissue collections, ITIH5mRNA expression was significantly decreased in NSCLC compared to normal lung tissue in line with an increased methylation frequency in lung cancer tissue. Independent TCGA data confirmed significant expression loss of ITIH5in lung cancer concordant with ITIH5promoter hypermethylation in NSCLC. Of interest, low ITIH5mRNA expression was particularly found in the magnoid and squamoid ADC expression subtype, concordant with an unfavorable patients' outcome in squamoid as well as tobacco smoking ADC patients. In conclusion, ITIH5may be a novel putative tumor suppressor gene in NSCLC with a potential molecular significance in the squamoid ADC subtype and further clinical impact for risk stratification of adenocarcinoma patients. In addition, ITIH5may serve as a novel biomarker for prognosis of tobacco smoking ADC patients.

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression as well as its potential clinical and functional impact in colon cancer. Based on 30 tumor and 30 adjacent normal tissues we examined ITIH5mRNA expression and promoter methylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA) platform. In addition, ITIH5 protein expression was evaluated using immunohistochemistry. ITIH5mRNA expression loss was significantly associated (P< 0.001) with hypermethylation of the ITIH5promoter in primary colon tumors. In addition, treatment of tumor cell lines with demethylating (DAC) and histone acetylating (TSA) agents induced ITIH5expression. In line, independent TCGA data revealed a significant expression loss of ITIH5,particularly in the MSI-high and CIMP-positive phenotype concordant with an increased ITIH5hypermethylation in CIMP-positive colon tumors (P< 0.001). In proximal, i.e., right-sided tumors, abundant ITIH5expression was associated with longer overall survival (OS, P= 0.049) and the CIMP-positive (P= 0.032) subgroup. Functionally, ITIH5 re-expression mediated a reduced proliferation in HCT116 and CaCo2 cells. In conclusion, our results indicate that ITIH5is a novel putative tumor suppressor gene in colon cancer with a potential impact in the CIMP-related pathway. ITIH5may serve as a novel epigenetic-based diagnostic biomarker with further clinical impact for risk stratification of CIMP-positive colon cancer patients.