Transmembrane Prostate Androgen-induced Protein Research Articles

P68: Novel monoclonal antibodies to C-terminal end of transmembrane prostate androgen-induced protein (TMEPAI) for evaluation of its role in gastric cancer prognosis

Background Transmembrane prostate androgen-induced protein 1 (TMEPAI) is a membrane protein that has attracted significant attention of many researchers its involvement in TGF- β signaling pathway which involved in malignant transformation and metastatic tumor progression. We investigated the TMEPAI expression level in gastric adenocarcinomas in comparison to non-tumor mucosa samples and determined its potential prognostic significance. Materials and methods Fresh and paraffin-embedded gastric adenocarcinoma samples and paired adjacent normal tissues were collected from gastric cancer patients. Evaluation of the PMEPA 1 gene expression was carried out using RT-PCR. For evaluation of TMEPAI protein expression, monoclonal antibodies (mAbs) were developed by using hybridoma techniques. Specificity of prepared monoclonal antibodies against recombinant TMEPAI and evaluation of its expression in the clinical samples using selected mAbs were performed using immunoblotting and immunohistochemistry. Results We have identified more than two-fold increase in gene expression of PMEPA1 in tumor tissue in 44% of patients. The monoclonal antibodies have shown the capacity to specifically recognize the recombinant TMEPAI in HEK293T cell lysates. We also evaluate the ability of the selected antibodies to recognize the target protein in fixed cells by immunocytochemistry. The evaluation of TMEPAI in adenocarcinoma samples collected from gastric cancer patients revealed decreased protein expression. We have observed pronounced expression of TMEPAI in normal gastric epithelial cells, while tumor cells from gastric adenomas and adenocarcinomas samples were mostly negative for target protein expression. We found that gastric epithelium cells lose the TMEPAI expression concurrent with severe dysplasia. Conclusion Apparently, the TMEPAI may be a potential biomarker of malignant transformation risk of the stomach epithelium. The presented study was financially supported by Grants from the Russian Fund for Basic Research ( 14-04-31500 ) and Tomsk State University Competitiveness Improvement Program.

TMEPAI inhibits TGF-β signaling by promoting lysosome degradation of TGF-β receptor and contributes to lung cancer development

Transforming growth factor-β (TGF-β) signaling plays important roles in embryogenesis and tumorigenesis by controlling cell growth, differentiation and migration. The transmembrane prostate androgen-induced protein (TMEPAI) is elevated in several cancers. TMEPAI expression is induced by TGF-β signaling, and in turn, expression of TMEPAI negatively regulates TGF-β signaling, but the molecular mechanisms of TMEPAI induced TGF-β signaling inhibition are not well understood. Here we report that TMEPAI is localized to the lysosome and late endosome, and that association of TMEPAI with the E3 ubiquitin ligase Nedd4 is required for its transport to the lysosome. TMEPAI associates with the TGF-β type I receptor (TβRI) and promotes its degradation in the lysosome. Depletion of TMEPAI in A549 lung cancer cells inhibits cell proliferation, migration and invasion, while TMEPAI expression in nude mice promotes tumorigenesis. These results reveal a novel function for TMEPAI in regulating TGF-β signaling through the modulation of TβRI levels, which has important implications for cancer development in vivo.

Methylation of the PMEPA1 gene, a negative regulator of the androgen receptor in prostate cancer

The prostate transmembrane protein androgen induced 1 (PMEPA1) gene is highly expressed in prostate epithelial cells and is a direct transcriptional target for the androgen receptor (AR). AR protein levels are controlled by the AR-PMEPA1 negative feedback loop through NEDD4-E3 ligase. Reduced expression of PMEPA1 observed in prostate tumors, suggests that loss of PMEPA1 may play critical roles in prostate tumorigenesis. This study focuses on epigenetic mechanisms of reduced PMEPA1 expression in the cancer of the prostate (CaP). Benign (n = 77) and matched malignant (n = 77) prostate epithelial cells were laser capture micro-dissected from optimum cutting temperature embedded frozen prostate sections from 42 Caucasian American (CA) and 35 African American (AA) cases. Purified DNA specimens were analyzed for CpG methylation of the PMEPA1 gene. PMEPA1 mRNA expression levels were evaluated by qRT-PCR. Analysis of PMEPA1 methylation and mRNA expression in the same tumor cell populations indicated a significant inverse correlation between mRNA expression and methylation in CaP (P = 0.0115). We noted higher frequency of CpG methylation within the evaluated first intronic region of the PMEPA1 gene in prostate tumors of CA men as compared with AA. In CaP cell lines, PMEPA1 expression was induced and AR protein levels were diminished in response to treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (decitabine). Cell culture-based studies demonstrated that decitabine restores PMEPA1 expression in AR-positive CaP cell lines. This report reveals the potential role of PMEPA1 gene methylation in the regulation of AR stability. Thus, downregulation of PMEPA1 may result in increased AR protein levels and function in CaP cells, contributing to prostate tumorigenesis.

P2-11-04: TMEPAI Is a Feedback Regulator of TGF-b Signaling during Breast Cancer Progression.

Abstract Background: Despite lack of hormone receptors (ER/PR) and HER2, many triple negative breast cancers (TNBC) depend on transforming growth factor beta (TGF-β) signaling activity for late growth and metastasis. Thus TGF-β signaling is a major potential target to treat TNBC patients. However, inhibition of TGF-β signaling pathway carries the risk of disturbing the tumor suppressive activity of TGF-β in normal tissues and early cancers. Hence, present study was undertaken with a goal to identify suitable markers which will enable to develop drugs that retard only the “oncogenic activity” of TGF-β while preserving its growth suppressive activities. Materials and Methods: All cell lines were cultured according to the recommended standard procedures. Lentiviral mediated expression vector was used to stably knockdown endogenous TMEPAI expression. DNA transfections and luciferase assays were performed according to vendor instructions. Cell proliferation was measured by quantitation of total cellular DNA. Immunoblotting and immunohistochemical analysis were performed using standard methods. Results: Previously we have shown that Transmembrane prostate androgen-induced protein (TMEPAI), a TGF-β inducible gene has the potential to convert tumor suppressive TGF-β into a tumor promoter. In the present study, we show that Smad binding elements driven luciferase reporter activity, a measure of TGF-β signaling, was dramatically increased in TMEPAI knockdown cells. While wild type cancer cells showed transient raise in phosphorylation of Smad2 and Smad3 with TGF-β treatment, TMEPAI knockdown resulted in increased and sustained levels of phosphorylated Smad2 and Smad3. In normal mammary epithelial cells, continuous presence of TGF-β blocked their growth. In contrast, breast cancer cells showed a biphasic growth response (moderate inhibition up to 72h followed by strong growth stimulation) to TGF-β, while TMEPAI-deficiency blocked this response to TGF-β. Furthermore, exogenous expression of TMEPAI in normal mammary epithelial cells and TMEPAI-knockdown cells resulted in reduced TGF-β-dependent Smad activity. Additionally, we found that TMEPAI subverts tumor suppressive TGF-β dependent Smad signaling into tumor promotive non-Smad signaling through stimulation of stress activated protein kinases. Inhibition of stress kinases also reduced the growth of cancer cells similar to TMEPAI deficiency. To evaluate the translational importance of TMEPAI as predictive marker, immunohistochemical analysis on human breast cancer specimens revealed the gain of TMEPAI expressions in aggressive human breast tumor samples but not in normal human breast tissue specimens. Conclusions: Our results show that TMEPAI, which subverts tumor suppressive Smad signaling, may serve as a novel prognostic and predictive marker for aggressive and TGF-β dependent of metastatic breast cancers. These studies will further provide new clues to develop an effective and decisive anti-TGF-β therapy against aggressive breast cancers without disturbing the growth suppression by TGF-β in normal tissues and early tumors. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-04.

Identification of Human Gene Products Containing Pro-Pro-x-Tyr (PY) Motifs that Enhance Glutathione and Endocytotic Marker Uptake in Yeast

In an attempt to identify genes involved in glutathione (GSH) transport, a human mammary gland cDNA library was screened for clones capable of complementing a defect in GSH uptake in yeast cells that lack Hgt1p, the primary yeast GSH uptake transporter. Five genes capable of rescuing growth on sulfur-deficient GSH-containing medium were identified: prostate transmembrane protein, androgen induced 1 (PMEPA1); lysosomal-associated protein transmembrane 4 alpha (LAPTM4α); solute carrier family 25, member 1 (SLC25A1); lipopolysaccharide-induced TNF factor (LITAF); and cysteine/tyrosine-rich-1 (CYYR1). All of these genes encode small integral membrane proteins of unknown function, although none appear to encode prototypical GSH transporters. Nevertheless, they all increased both intracellular glutathione levels and [³H]GSH uptake rates. [³H]GSH uptake was uniformly inhibited by high concentrations of unlabeled GSH, GSSG, and ophthalmic acid. Interestingly, each protein is predicted to contain Pro-Pro-x-Tyr (PY) motifs, which are thought to be important for regulating protein cell surface expression. Uptake of the endocytotic markers lucifer yellow and FM4-64 was also enhanced by each of the five genes. Mutations of the PY motifs in LITAF largely abolished all of its effects. In summary, although the results do not reveal novel GSH transporters, they identify five PY-containing human gene products that may influence plasma membrane transport activity.