Transmembrane Prostate Androgen-induced Protein Research Articles

Oncogenic role of PMEPA1 and its association with immune exhaustion and TGF-β activation in HCC

Background & AimsTransforming growth factor β (TGF-β) has an oncogenic role in advanced cancer by promoting cell proliferation, metastasis and immunosuppression. PMEPA1 (prostate transmembrane protein androgen induced 1) has been shown to promote TGF-β oncogenic effects in other tumour types. Thus, we aimed to explore the role of PMEPA1 in hepatocellular carcinoma (HCC). MethodsWe analysed n=1097 tumours from HCC patients, including a discovery (n=228) and validation cohort (n=361) with genomic and clinicopathological data. PMEPA1 levels were assessed by qPCR (n=228), gene expression data (n=869) and at the single-cell level (n=54). Genetically engineered mouse models overexpressing MYC+PMEPA1 compared to MYC were generated and HCCs obtained were molecularly analysed. ResultsPMEPA1 was overexpressed in 18% of HCC samples [FC>2; n=201/1097], a feature associated with TGF-β signalling activation (p<0.05) and absence of gene body hypomethylation (p<0.01). HCCs showing both TGF-β signalling and high PMEPA1 levels (12% of cases) were linked to immune exhaustion, Late TGF-β activation, aggressiveness and higher recurrence rates after resection, in contrast to HCCs with only TGF-β signalling (8%) or PMEPA1 overexpression (9%). Single-cell RNA-sequencing analysis identified PMEPA1 expression in HCC and stromal cells. PMEPA1-expressing tumoural cells were predicted to interact with CD4+ Tregs and CD4+ CXCL13+ and CD8+ exhausted T cells. In vivo, overexpression of MYC+PMEPA1 led to HCC development in ∼60% of mice and a decreased survival compared to mice overexpressing MYC alone (p=0.014). MYC+PMEPA1 tumours were enriched in TGF-β signalling, paralleling our human data. ConclusionsIn human HCC, PMEPA1 upregulation is linked to TGF-β activation, immune exhaustion, and an aggressive phenotype. Overexpression of PMEPA1+MYC led to tumoural development in vivo, demonstrating the oncogenic role of PMEPA1 in HCC for the first time. Impact and implicationsPMEPA1 can enhance the tumour-promoting effects of TGF-β in cancer. In this study, we describe that PMEPA1 is highly expressed in ∼18% of HCC patients, a feature associated with poor prognosis, TGF-β activation and exhaustion of immune cells. Similarly, in mouse models, PMEPA1 overexpression promotes HCC development, which demonstrates its oncogenic role. The identification of PMEPA1 as oncogenic driver in HCC and its role in immune exhaustion and poor clinical outcomes enhances our understanding of HCC pathogenesis and opens new avenues for targeted therapeutic interventions.

Unraveling the immune landscape and therapeutic biomarker PMEPA1 for oxaliplatin resistance in colorectal cancer: A comprehensive approach

Oxaliplatin (OXA) is a platinum-based chemotherapeutic agent with promising applications in the treatment of various malignancies, particularly colorectal cancer (CRC). However, the management of OXA resistance remains an ongoing obstacle in CRC therapy. This study aims to comprehensively investigate the immune landscape, targeted therapeutic biomarkers, and mechanisms that influence OXA resistance in CRC. Our results demonstrated that our OXA- resistant CRC prognostic model not only provides risk assessment for patients but also reflects the immune landscape of patients. Additionally, we identified prostate transmembrane protein, androgen-induced1 (PMEPA1) as a promising molecular targeted therapeutic biomarker for patients with OXA-resistant CRC. The mechanism of PMEPA1 may involve cell adhesion, pathways in cancer, and the TGF-β signaling pathway. Furthermore, analysis of CRC clinical samples indicated that patients resistant to OXA exhibited elevated serum levels of TGF-β1, increased expression of PMEPA1 in tumors, a lower proportion of CD8+ T cell positivity, and a higher proportion of M0 macrophage positivity, in comparison to OXA-sensitive individuals. Cellular experiments indicated that selective silencing of PMEPA1, alone or in combination with OXA, inhibited proliferation and metastasis in OXA-resistant CRC cells, HCT116R. Animal experiments further confirmed that PMEPA1 silencing suppressed subcutaneous graft tumor growth and liver metastasis in mice bearing HCT116R and synergistically enhanced the efficacy of OXA. These data highlight the potential of leveraging the therapeutic biomarker PMEPA1, CD8+ T cells, and M0 macrophages as innovative targets for effectively addressing the challenges associated with OXA resistance. Our findings hold promising implications for further clinical advancements in this field.

Exploring the role of PMEPA1 in gastric cancer

Although there are several treatments available for gastric cancer (GC), the prognosis of the disease is still poor due to many factors, such as late diagnosis and tumor heterogeneity. To identify potential therapeutic targets, bioinformatics techniques and clinical sample validation were employed and prostate transmembrane protein androgen induced 1 (PMEPA1) was selected for further study. In the present study, we found that elevated PMEPA1 expression correlates with a worse prognosis and weaker anti-tumor immunity in GC patients. Moreover, our study showed that PMEPA1 not only influences cell proliferation, clone formation, invasion, and migration in vitro, but also plays an important role in GC progression in vivo. Mechanically, PMEPA1 exerts its oncogenic effects through activating the Wnt/β-catenin signaling pathway. Therefore, PMEPA1 is a potential target for treating GC effectively.

Abstract A006: Loss of PMEPA1 gene isoform facilitates the development of hormone and radiation therapies resistance in prostate cancer cells

Abstract Introduction: Nearly all the patients receiving hormone treatment eventually progress into castration resistance stage. In addition, approximately 30% to 40% of patients receiving radiation treatment will develop resistance. The aberrant alterations of androgen receptor (AR) and transforming growth factor-β (TGF-β) signaling play critical roles in such processes. Our studies identified five PMEPA1 (Prostate Transmembrane Protein Androgen Induced 1) gene isoforms in prostate cancer cells. PMEPA1-b isoform was androgen responsive and inhibited androgen signaling. In contrast, PMEPA1 isoforms a and d were TGF-β inducible and suppressed TGF-β signaling. Loss or reduced expressions of PMEPA1 was detected in over 60% of prostate cancer samples. In this study, we investigated the impacts of PMEPA1 isoforms (a, b and d) on the development of hormone and radiation therapies resistance in prostate cancer. Methods: RNA-seq data of unmatched 50 benign and 499 malignant prostate samples from The Cancer Genome Atlas (TCGA) dataset, as well as 101 metastatic castration resistant prostate cancer (mCRPC) biopsy samples from Quigley lab at UCSF (WCDT dataset) were used to study the transcript levels of PMEPA1 isoforms (a, b and d) and their correlations to disease progression. Hormone dependent LNCaP cells and androgen independent PC-3, DU-145 cells were utilized to investigate the impacts of PMEPA1 isoforms (a, b and d) on the responses to of androgen inhibitors (enzalutamide and bicalutamide, 0, 5.0 and 25.0 nM) and radiation (0, 1.0 and 5.0 Gy). The vectors of pcDNA3.1-PMEPA1-a, -b and -d were transfected into prostate cancer cells via Lipofectamine, and endogenous PMEPA1 isoforms were knocked down by isoforms specific siRNAs. The cell growth was assessed by cell counting, BrdU incorporation and cell plating efficiency assays. The protein levels of AR and PSA were evaluated by immunoblotting. The transcript levels of PSA, NKX3.1, NEDD9 and THBS1 were assessed by RT-PCR technology. Results: The reduced transcript levels of PMEPA1 isoforms (a, b and d) were detected in mCRPC group. Lower expression of PMEPA1-b was significantly associated with earlier biochemical recurrence. The growth inhibition and androgen signaling blockage mediated by androgen inhibitors were explicitly reversed by depletion of PMEPA1-b in LNCaP cells. Ectopic PMEPA1-b in isoform b depleted LNCaP cells reinstituted the growth inhibition induced by androgen inhibitors. Knockdown of isoforms a and d conspicuously rescued the decreased cell proliferation and suppressed TGF-β signaling resulted from radiation treatment in PC-3 and DU-145 cells. Similarly, over-expression of PMEPA1-a and -d in siRNA treated cells recapitulated the growth inhibitory effects of radiation treatments. Conclusion: Loss of PMEPA1 gene isoforms (a, b and d) induced the development of resistance to hormone ablation and radiation treatments in prostate cancer cells in vitro through activating androgen and TGF-β signaling. Our research provided a new insight into treatment resistance mechanism of prostate cancer cells. Citation Format: Shashwat Sharad, Hua Li. Loss of PMEPA1 gene isoform facilitates the development of hormone and radiation therapies resistance in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A006.

The impact of conventional smoking versus electronic cigarette on the expression of VEGF, PEMPA1, and PTEN in rat prostate.

The use of electronic cigarettes (e-cigarettes), the alternative to conventional smoking, is increasing considerably worldwide; however, their safety is a matter of debate. Several studies have demonstrated their toxic effects, but no study assessed their effects on the prostate. The current study aimed at evaluating e-cigarettes and conventional smoking prostate toxicity and effects on the expression of vascular endothelial growth factor A (VEGFA), phosphatase and tensin (PTEN), and prostate transmembrane protein androgen induced 1 (PMEPA1). 30 young Wistar rats were categorized into three groups (n=10) as follows: the control group, the conventional smoking group, and the e-cigarette group. The case groups were exposed to cigarettes or e-cigarettes for 40minutes, 3 times a day for four months. Serum parameters, prostate pathology, and gene expression were measured at the end of the intervention. Data were analyzed by Graph Pad prism 9. Histopathological findings presented that both types of cigarette-induced hyperemia and induced inflammatory cell infiltration and hypertrophy of smooth muscle of the vascular wall in the e-cigarette group. Expression of PMEPA1, and VEGFA genes significantly increased in conventional (2.67-fold; P=0.0108, 1.80-fold; P=0.0461 respectively) and e-cigarettes (1.98-fold; P=0.0127, 1.34-fold; P=0.938, respectively) groups compared to the control group. Expression of the PTEN gene non-significantly decreased in the case of groups compared to the control group. We found no significant differences between the two groups in terms of PTEN and PMEPA1 expression, whereas VEGFA was significantly more expressed in a conventional smoking group compared to the e-cigarette group. Therefore, it seems that e-cigarettes could not be taken into account as a better option than conventional smoking, and quitting smoking still is the optimal option.

TMEPAI promotes degradation of the NF-κB signaling pathway inhibitory protein IκBα and contributes to tumorigenesis

The transmembrane prostate androgen-induced protein (TMEPAI) is known to be highly expressed in various types of cancer and promoted oncogenic abilities. However, the mechanisms whereby TMEPAI facilitates tumorigenesis are not fully understood. Here we reported that expression of TMEPAI activated the NF-κB signaling. TMEPAI showed direct interaction with NF-κB pathway inhibitory protein IκBα. Though ubiquitin ligase Nedd4 (neural precursor cell expressed, developmentally down-regulated 4) did not interact with IκBα directly, TMEPAI recruited Nedd4 for ubiquitination of IκBα, leading to IκBα degradation through the proteasomal and lysosomal pathway, and promoted activation of NF-κB signaling. Further study indicated NF-κB signaling is involved in TMEPAI-induced cell proliferation and tumor growth in immune deficient mice. This finding helps to further understand the mechanism of TMEPAI on tumorigenesis and suggests TMEPAI is potential target for cancer treatment.

336 (PB116) - PMEPA1 has an oncogenic role in hepatocellular carcinoma in the context of TGFβ signaling: data from single cell RNAseq and transgenic models

Background: Transforming growth factor β (TGFβ) acts as a tumor suppressor in early stages of carcinogenesis, but promotes invasiveness, angiogenesis and immunosuppression in advanced stages. Prostate Transmembrane Protein Androgen Induced 1 (PMEPA1) negatively regulates TGFβ signaling by interacting with SMADs, and promotes TGFβ oncogenic effects in other cancers. Here, we explored the role of PMEPA1 in hepatocellular carcinoma (HCC).

Abstract PR02: PMEPA1 has an oncogenic role in the context of TGF-β signaling in hepatocellular carcinoma

Abstract Background: Transforming growth factor β (TGFβ) has a dual role in cancer, including hepatocellular carcinoma (HCC). It acts as a tumor suppressor in early stages of hepatocarcinogenesis, but promotes epithelial-to-mesenchymal transition, angiogenesis and immunosuppression in advanced stages. PMEPA1 (prostate transmembrane protein androgen induced 1) negatively regulates TGFβ signaling by interacting with SMAD proteins, and has been shown to promote TGFβ oncogenic effects in other cancers. Thus, we aimed to explore the role of PMEPA1 in HCC pathogenesis. Methods: We analyzed transcriptomic, genomic, epigenomic and clinicopathological data of a discovery cohort comprising 228 HCCs and a validation cohort of 361 HCCs. PMEPA1 levels were quantified by qPCR in the discovery cohort. PMEPA1 overexpression was validated in 6 independent publicly available cohorts (n = 916) with either microarray or RNAseq data. We also evaluated PMEPA1 expression at the single-cell level using a publicly available single-cell RNA sequencing (scRNAseq) dataset comprising 22 HCC samples. Genetically engineered mouse models were generated by performing hydrodynamic tail-vein injection of equal amounts of plasmids overexpressing MYC and MYC+PMEPA1. Molecular and histopathological analyses of the murine tumors are currently ongoing. Results: PMEPA1 was overexpressed in 18% of human HCCs [FC &amp;gt; 2; n = 203/1144], a feature associated with TGFβ signaling (p &amp;lt; 0.05). Overexpression of PMEPA1 was associated with gene body hypermethylation (p &amp;lt; 0.0001). HCCs displaying both TGFβ signaling and high PMEPA1 levels (11% of cases) were significantly enriched in signatures of immune exhaustion, late TGFβ activation, tumor microenvironment (TME) response to TGFβ and active stroma (p &amp;lt; 0.05) when compared with HCCs with TGFβ signalling alone (8% of cases) or PMEPA1 overexpression alone (11% of cases). Analyses of PMEPA1 expression at the single-cell level revealed that this gene is not only expressed by tumoral cells but also by the TME cell populations, including endothelial cells, pericytes, fibroblasts, and dendritic cells. In vivo, overexpression of MYC+PMEPA1 led to HCC development in 9/16 mice and was associated with a significantly reduced survival when compared to mice overexpressing MYC alone (p = 0.014), which did not develop tumors. Compared to healthy liver controls, MYC+PMEPA1 tumors presented upregulation of AKT. Conclusion: PMEPA1 upregulation is linked to TGF-β activation and an aggressive phenotype in human HCC. Overexpression of PMEPA1 in combination with MYC in vivo led to HCC development, showing for the first time the oncogenic role of PMEPA1 in this cancer type. Citation Format: Marta Piqué-Gili, Carmen Andreu-Oller, Roser Pinyol, Roger Esteban, Marina Bárcena-Varela, Judit Peix, Jordi Abril-Fornaguera, Katherine E. Lindblad, Miguel Torres-Martin, Júlia Huguet-Pradell, Daniela Sia, Amaia Lujambio, Josep M Llovet. PMEPA1 has an oncogenic role in the context of TGF-β signaling in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PR02.

Abstract 840: PMEPA1 is an oncogene linked to TGF-β signaling in hepatocellular carcinoma

Abstract Background: Transforming growth factor β (TGFβ) has a dual role in cancer, including hepatocellular carcinoma (HCC). It acts as a tumor suppressor in early stages of hepatocarcinogenesis, but promotes epithelial-to-mesenchymal transition, angiogenesis and immunosuppression in advanced stages. PMEPA1 (prostate transmembrane protein androgen induced 1), a direct target gene of the TGFβ pathway that negatively regulates TGFβ signaling by interacting with SMAD proteins, has been shown to promote TGFβ oncogenic effects in other cancers and we decided to explore its role in HCC pathogenesis. Methods: We analyzed transcriptomic, genomic, epigenomic and clinicopathological data of a discovery cohort of 228 HCCs and a validation cohort of 361 HCCs. PMEPA1 levels were quantified by qPCR in the discovery cohort. PMEPA1 overexpression was validated in 6 independent cohorts (n = 916) using microarray and RNAseq data. Genetically engineered mouse models were generated through hydrodynamic tail-vein injection. Equal amounts of plasmids overexpressing PMEPA1, TGFB1 and MYC were injected in mice to evaluate survival. Molecular and histopathological analyses of the murine tumors are currently ongoing. Results: PMEPA1 was overexpressed in 18% of human HCCs [FC &amp;gt; 2; n = 203/1144], a feature associated with TGFβ signaling (p &amp;lt; 0.05). Overexpression of PMEPA1 was associated with gene body hypermethylation (p &amp;lt; 0.0001). HCCs displaying both TGFβ signaling and high PMEPA1 levels (11% of cases) were significantly enriched in signatures of immune exhaustion, late TGFβ activation, TME response to TGFβ and active stroma (p &amp;lt; 0.05) compared to HCCs with TGFβ signalling alone (8% of cases) or PMEPA1 high levels alone (11% of cases). In vivo, overexpression of MYC+PMEPA1 led to HCC development in 9/16 mice and showed a lower survival when compared to the control arm, overexpression of MYC alone (p = 0.014). On the contrary, only 1/12 mice in the MYC+PMEPA1+TGFβ1 condition developed HCC and none of the MYC+TGFβ1 mice. Conclusion: PMEPA1 upregulation is linked to TGF-β activation and an aggressive phenotype in human HCC. Overexpression PMEPA1 in combination with MYC in vivo led to HCC development, showing for the first time the oncogenic role of PMEPA1 in this cancer. Citation Format: Marta Piqué-Gili, Carmen Andreu-Oller, Roser Pinyol, Marina Bárcena-Varela, Roger Esteban-Fabró, Judit Peix, Jordi Abril-Fornaguera, Katherine E. Lindblad, Miguel Torres-Martin, Daniela Sia, Amaia Lujambio, Josep M. Llovet. PMEPA1 is an oncogene linked to TGF-β signaling in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 840.

PMEPA1 Serves as a Prognostic Biomarker and Correlates with Immune Infiltrates in Cervical Cancer.

Emerging studies have demonstrated that Prostate transmembrane protein androgen induced 1 (PMEPA1) plays crucial roles in the carcinogenesis of many developing human tumors. However, the clinical significance of PMEPA1 expression in cervical cancer (CC) and its contribution to cancer immunity have not been investigated. In this study, we identified PMEPA1 as a survival-related gene in CC based on TCGA datasets. Univariate and multivariate analysis showed that PMEPA1 expression was an independent predictor for overall survival in CC patients. We could observe a strong negative correlation between PMEPA1 expression and PMEPA1 methylation. Two CpG sites of PMEPA1 were associated with overall survival, and one CpG site of PMEPA1 was associated with progression-free survival. The low level of PMEPA1 methylation was associated with advanced clinical stage of CC patients. KEGG assays revealed the genes associated with PMEPA1 expression were mainly enriched in several tumor-related pathways. Increased PMEPA1 expressions were observed to be positively related to high immune infiltration levels in several immune cells. Finally, the pan-cancer assays revealed that PMEPA1 expression was associated with the overall survival of UVM, PAAD, LUSC, BLCA, CESC, and LUAD. Taken together, PMEPA1 is a prognosis-related biomarker for multiple cancer types, especially CC. PMEPA1 is involved in tumor immunity, suggesting PMEPA1 may be a potential immunotherapeutic target in CC.

PMEPA1 Is a Prognostic Biomarker That Correlates With Cell Malignancy and the Tumor Microenvironment in Bladder Cancer.

Prostate transmembrane protein androgen induced 1 (PMEPA1) has been reported to promote cancer progression, but the potential role of PMEPA1 in bladder cancer (BLCA) remains elusive. We assess the role of PMEPA1 in BLCA, via a publicly available database and in vitro study. PMEPA1 was identified from 107 differentially expressed genes (DEGs) to have prognostic value. GO, KEGG, and GSEA analysis indicated that PMEPA1 was involved in cancer progression and the tumor microenvironment (TME). Then bioinformatical analysis in TCGA, GEO, TIMER, and TISIDB show a positive correlation with the inflammation and infiltration levels of three tumor-infiltrating immune cells (TAMs, CAFs, and MDSCs) and immune/stromal scores in TME. Moreover, in vitro study revealed that PMEPA1 promotes bladder cancer cell malignancy. Immunohistochemistry and survival analysis shed light on PMEPA1 potential to be a novel biomarker in predicting tumor progression and prognosis. At last, we also analyzed the role of PMEPA1 in predicting the molecular subtype and the response to several treatment options in BLCA. We found that PMEPA1 may be a novel potential biomarker to predict the progression, prognosis, and molecular subtype of BLCA.

PMEPA1/TMEPAI Is a Unique Tumorigenic Activator of AKT Promoting Proteasomal Degradation of PHLPP1 in Triple-Negative Breast Cancer Cells.

Simple SummaryTMEPAI/PMEPA1 is known to be highly expressed in various types of cancer, including triple-negative breast cancer (TNBC). Here, we found that TMEPAI-knockout (KO) in TNBC cells showed less tumorigenic abilities and upregulated PHLPP1, which dephosphorylates AKT at Ser473. Additionally, PHLPP1 depletion in TMEPAI-KO cells partially but significantly recovered AKT Ser473 phosphorylation, as well as in vitro and in vivo tumorigenic activities. Moreover, we demonstrated that TMEPAI PPxY (PY) motifs are essential for binding to NEDD4-2, an E3 ubiquitin ligase, and the complex formation of PHLPP1 with NEDD4-2, which leads to the polyubiquitination and proteasomal degradation of PHLPP1. These findings indicate that the PY motifs of TMEPAI induce the degradation of PHLPP1 and maintain AKT Ser473 phosphorylation at high levels to enhance the tumorigenic potentiality of TNBC. Therefore, the putative oncogenic role of TMEPAI may lead to developing TMEPAI based targeted therapy and/or diagnosis of TNBC in future.Transmembrane prostate androgen-induced protein (TMEPAI), also known as PMEPA1, is highly expressed in many types of cancer and promotes oncogenic abilities. However, the mechanisms whereby TMEPAI facilitates tumorigenesis are not fully understood. We previously established TMEPAI-knockout (KO) cells from human triple-negative breast cancer (TNBC) cell lines and found that TMEPAI-KO cells showed reduced tumorigenic abilities. Here, we report that TMEPAI-KO cells upregulated the expression of pleckstrin homology (PH) domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and suppressed AKT Ser473 phosphorylation, which was consistent with TCGA dataset analysis. Additionally, the knockdown (KD) of PHLPP1 in TMEPAI-KO cells partially but significantly rescued AKT Ser473 phosphorylation, as well as in vitro and in vivo tumorigenic activities, thus showing that TMEPAI functions as an oncogenic protein through the regulation of PHLPP1 subsequent to AKT activation. Furthermore, we demonstrated that TMEPAI PPxY (PY) motifs are essential for binding to NEDD4-2, an E3 ubiquitin ligase, and PHLPP1-downregulatory ability. Moreover, TMEPAI enhanced the complex formation of PHLPP1 with NEDD4-2 and PHLPP1 polyubiquitination, which leads to its proteasomal degradation. These findings indicate that the PY motifs of TMEPAI suppress the amount of PHLPP1 and maintain AKT Ser473 phosphorylation at high levels to enhance the tumorigenic potentiality of TNBC.

TGF-β-Induced TMEPAI Promotes Epithelial–Mesenchymal Transition in Doxorubicin-Treated Triple-Negative Breast Cancer Cells via SMAD3 and PI3K/AKT Pathway Alteration

IntroductionEpithelial–mesenchymal transition (EMT) and overexpression of drug efflux transporters have been reported to cause doxorubicin resistance. Our previous study indicated that TMEPAI (transmembrane prostate androgen-induced protein) attenuated doxorubicin sensitivity in triple-negative breast cancer cells. However, how TMEPAI contributes to doxorubicin resistance in TNBC remains unclear. Thus, the present study aimed to elucidate the mechanism of TMEPAI in doxorubicin resistance in triple-negative breast cancer cells.MethodsWe used BT549, triple-negative cells wild type (WT), and BT549 TMEPAI knock-out. Both cells were treated with TGF-β 2 ng/mL for 24 hours, followed by TGF-β 2 ng/mL and doxorubicin 12.9 nM for another 24 hours. Afterward, the cells were harvested and counted. Cells were further lysed and used for RT-PCR and Western blot analysis. We determined the expression levels of proliferation, apoptosis, EMT markers, and drug efflux transporters. Additionally, we investigated the expressions of PI3K as well as SMAD3 and AKT phosphorylation.ResultsTNBC cells were shown to be less sensitive to doxorubicin in the presence of TMEPAI. TMEPAI was shown to alleviate the mRNA expressions of apoptosis markers: Bax, Bcl2, Caspase-3, and Caspase-9. Our results indicated that the presence of TMEPAI greatly amplifies EMT and increases drug efflux transporter expressions after doxorubicin treatment. Furthermore, our findings demonstrated that TMEPAI reduced the action of doxorubicin in inhibiting SMAD3 phosphorylation. TMEPAI was also shown to modify the effect of doxorubicin by reducing PI3K expressions and Akt phosphorylation in triple-negative breast cancer cells.ConclusionOur findings indicate that TMEPAI promotes EMT and drug efflux transporters at least in part by shifting doxorubicin action from SMAD3 phosphorylation reduction to PI3K/AKT inhibition in triple-negative breast cancer cells.

Linc00941 regulates esophageal squamous cell carcinoma via functioning as a competing endogenous RNA for miR-877-3p to modulate PMEPA1 expression.

Esophageal squamous cell carcinoma (ESCC) represents one of the most common malignancies and is the fifth leading cause of cancer-related deaths. Long intergenic non-coding RNAs (lincRNAs) have been suggested to be dysregulated in various types of cancers, and a growing number of lincRNAs have been implicated to be functional in the ESCC progression. In this study, we examined the role of linc00941 in the ESCC progression and explored the underlying molecular mechanisms. The bioinformatics analysis identified the up-regulation of linc00941 in the ESCC tissues. Further in vitro studies showed that linc00941 was up-regulated in ESCC cell lines. The loss-of-function studies demonstrated that linc00941 knockdown suppressed ESCC cell proliferation, invasion and migration, and also suppressed the in vivo tumor growth. Furthermore, bioinformatics prediction along with luciferase reporter assay and RNA immunoprecipitation assay implied that linc00941 acted as a competing endogenous RNA for miR-877-3p, and linc00941 regulated ESCC cell progression via at least targeting miR-877-3p. Subsequently, miR-877-3p targeted prostate transmembrane protein, androgen induced 1 (PMEPA1) 3’ untranslated region and repressed PMEPA1 expression in ESCC cells; overexpression of PMEPA1 attenuated the inhibitory effects of linc00941 knockdown on the ESCC cell progression. Linc00941 knockdown suppressed epithelial-mesenchymal transition (EMT) via targeting miR-877-3p/PMEPA1 axis in ESCC cells. In conclusion, our results indicated the oncogenic role of linc00941 in ESCC, and knockdown of linc00941 suppressed ESCC cell proliferation, invasion, migration and EMT via interacting with miR-877-3p/PMEPA1 axis.

PMEPA1 facilitates non-small cell lung cancer progression via activating the JNK signaling pathway.

Prostate transmembrane protein androgen-induced 1 (PMEPA1), a critical checkpoint of multiple signaling pathways, has been demonstrated to play a crucial role in various types of cancers. However, little is known about its function in non-small cell lung cancer (NSCLC). Our objective is to explore the function of PMEPA1 and its potential mechanisms in NSCLC progression. PMEPA1 expression and prognostic significance in adenocarcinoma of lung cancer (LUAD) and squamous cell carcinoma of lung cancer (LUSC) were determined using Gene Expression Profiling Interactive Analysis (GEPIA). Next, a series of cell assays were performed to examine whether overexpression or depletion of PMEPA1 affected the malignant behaviors of NSCLC H1299 cells, such as proliferation and migration. Luciferase reporter gene assays and SP600125 (a JNK inhibitor) were employed to ascertain the regulatory relationship between PMEPA1 and JNK. PMEPA1 is overexpressed in LUAD and LUSC tissues and portends a worse prognosis for cancer patients. Gain and loss of function experiments demonstrated that PMEPA1 executes oncogenetic function in H1299 cells. Mechanism studies elucidated that PMEPA1 stimulated the transcriptional activity of the JNK pathway. PMEPA1 increased the H1299 cell viability, proliferation, and migration which works, at least partially, by triggering the JNK activity. Hence, our findings support that the PMEPA1/JNK axis might be a promising therapeutic target for this challenging disease.

Diagnostic and therapeutic values of PMEPA1 and its correlation with tumor immunity in pan-cancer

AimsThe prostate transmembrane protein, androgen induced 1 (PMEPA1) is differentially expressed in pan-cancer. However, PMEPA1 specific role in cancers has not been fully clarified. This study aims to explore the potential role of Pmepa1 in pan-cancer and specific cancer, with a view to deepening the research on the pathological mechanism of cancer. Main methodsThe Perl language and R language were used to identify the correlation between PMEPA1 expression level and clinical indicators, prognosis values, tumor microenvironment, immune cells' infiltration, immune checkpoint genes, TMB and MSI. The Therapeutic Target Database was used for identifying potential therapeutic drugs that target the pathways that are significantly affected by PMEPA1 expression. Key findingsPMEPA1 differential expression significantly correlated with patients' age, race, tumors' stage and status. PMEPA1 high expression was closely correlated with poor prognosis in many cancer types, excluding prostate adenocarcinoma. PMEPA1 expression was closely related to tumor cells and the immune microenvironment in stromal and immune cells' level, immune cells' infiltration, immune checkpoint genes, tumor mutational burden and microsatellite instability. We also found that the activity of the olfactory transduction pathway was closely related to PMEPA1 expression. In pan-cancer, Trifluoperazine and Halofantrine have the potential to reduce PMEPA1 expression. SignificanceThis study integrated existing data to explore PMEPA1 potential function in cancers, provided insights for the future cancer-related studies.

PMEPA1 Gene Isoforms: A Potential Biomarker and Therapeutic Target in Prostate Cancer

The identification of prostate transmembrane protein androgen induced 1 (PMEPA1), an androgen responsive gene, came initially from the studies of androgen regulatory gene networks in prostate cancer. It was soon followed by the documentation of the expression and functional analysis of transmembrane prostate androgen-induced protein (TMEPAI)/PMEPA1 in other solid tumors including renal, colon, breast, lung, and ovarian cancers. Further elucidation of PMEPA1 gene expression and sequence analysis revealed the presence of five isoforms with distinct extracellular domains (isoforms a, b, c, d, and e). Notably, the predicted amino acid sequences of PMEPA1 isoforms show differences at the N-termini, a conserved membrane spanning and cytoplasmic domains. PMEPA1 serves as an essential regulator of multiple signaling pathways including androgen and TGF-β signaling in solid tumors. Structure-function studies indicate that specific motifs present in the cytoplasmic domain (PY, SIM, SH3, and WW binding domains) are utilized to mediate isoform-specific functions through interactions with other proteins. The understanding of the “division of labor” paradigm exhibited by PMEPA1 isoforms further expands our knowledge of gene’s multiple functions in tumorigenesis. In this review, we aim to summarize the most recent advances in understanding of PMEPA1 isoform-specific functions and their associations with prostate cancer progression, highlighting the potentials as biomarker and therapeutic target in prostate cancer.

PMEPA1/TMEPAI isoforms function via its PY and Smad-interaction motifs for tumorigenic activities of breast cancer cells.

PMEPA1 (prostate transmembrane protein, androgen-induced 1)/TMEPAI (transmembrane prostate androgen-induced protein) is highly expressed in diverse cancers, including breast, lung and prostate cancers. It consists of four isoforms with distinct extracellular regions (isoforms a-d). The expression and function of these isoforms are still poorly understood. Hence, we aimed to identify the preferentially expressed isoforms in breast cancer cells and analyze possible differences in tumorigenic functions. In this study, we used 5' Rapid Amplification of cDNA Ends (RACE) and Western blot analyses to identify the mRNA variants and protein isoforms of TMEPAI and found that TMEPAI isoform d as the major isoform expressed by TGF-β stimulation in breast cancer cells. We then generated CRISPR/Cas9-mediated TMEPAI knockout (KO) breast cancer cell lines and used a lentiviral expression system to complement each isoform individually. Although there were no clear functional differences between isoforms, double PPxY (PY) motifs and a Smad-interaction motif (SIM) of TMEPAI were both essential for colony and sphere formation. Collectively, our results provide a novel insight into TMEPAI isoforms in breast cancer cells and showed that coordination between double PY motifs and a SIM of TMEPAI are essential for colony and sphere formation but not for monolayer cell proliferation.

Abstract P3-03-11: Notch inhibition promotes triple negative breast cancer growth

Abstract Notch1 and Transforming Growth Factor-beta (TGF-β) signaling pathways in cancer have a paradoxical roles as they were shown to have both tumor suppressive and tumor promoting activities. The interaction between these two signaling pathways in cancer progression was addressed in a systematic way. In these studies, we utilized primary human mammary epithelial cells (HMEC) and various triple negative breast cancer (TNBC) cells and examined the molecular cross-talk between TGF-β and Notch signaling pathways using biochemical, molecular and cell biology techniques. First, we noticed lower expression of Notch1 in most of the TNBC cells compared to normal cells. Second, most of these TNBC cells overexpressed TMEPAI (transmembrane prostate androgen induced) protein, which was shown to switch TGF-β from a tumor suppressor to a tumor promoter role in breast cancer. Surprisingly, treatment of TNBC cells with Notch inhibitors increased their growth rate both in the absence or presence of TGF-β. Moreover, Notch inhibition relieved the growth suppression by TGF-β in normal cells. Both normal and TNBC cells showed increased growth in the presence of Notch inhibitors with decreased TGF-β signaling activity. Normal cells that express very low levels of TMEPAI both at mRNA and protein levels, overexpressed TMEPAI protein in the presence of various Notch inhibitors, suggesting that Notch negatively regulates TMEPAI expression. Correspondingly, knockdown of TMEPAI in TNBC cell lines showed increased expression of Notch1 and Jagged 1 suggesting a negative regulation of Notch1 by TMEPAI. Consistent with these observations, TMEPAI, a known binding partner of E3 ligases, causes increased turnover of Notch1 protein. In fact, over expression of active Notch1 in MDA-MB-231 cells inhibited tumor cell growth both in vitro and in vivo. Our results clearly suggest that a crosstalk exists between TGF-β and Notch1 signaling through TMEPAI. Therefore, our studies clearly indicate that use of Notch inhibitors is contraindicated in triple negative breast cancer patients. Citation Format: Pothana Saikumar, Prajjal K Singha, Manjeri A Venkatachalam. Notch inhibition promotes triple negative breast cancer growth [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-11.

Characterization of unique PMEPA1 gene splice variants (isoforms d and e) from RNA Seq profiling provides novel insights into prognostic evaluation of prostate cancer.

Prostate cancer is a disease with heterogeneity of multiple gene transcriptomes and biological signaling pathways involved in tumor development. The prostate transmembrane protein, androgen induced 1 (PMEPA1), a multifunctional protein played critical roles in prostate tumorigenesis. The pleiotropic nature of PMEPA1 in modulating androgen and TGF-β signaling as well as splice variants mechanisms for functional regulations of cancer-associated genes prompted us to investigate the biological roles of PMEPA1 isoforms in prostate cancer. In addition to 4 reported PMEPA1 isoforms (a, b, c and d), one novel isoform PMEPA1-e was identified with RNA Seq analysis of hormone responsive VCaP, LNCaP cells and human prostate cancer samples from The Cancer Genome Atlas (TCGA) dataset. We analyzed the structures, expressions, biological functions and clinical relevance of PMEPA1-e isoform and less characterized isoforms c and d in the context of prostate cancer and AR/TGF-β signaling. The expression of PMEPA1-e was induced by androgen and AR. In contrast, PMEPA1-d was responsive to TGF-β and inhibited TGF-β signaling. Both PMEPA1-d and PMPEA1-e promoted the growth of androgen independent prostate cancer cells. Although PMEPA1-c was responsive to TGF-β, it was found to have no impacts on cell growth and androgen/TGF-β signaling. The TCGA data analysis from 499 patients showed higher expression ratios of PMEAP1-b versus -d or -e strongly associated with enhanced Gleason score. Taken together, our findings first time defined the prostate tumorigenesis mediated by PMEPA1-d and -e isoforms, providing novel insights into the new strategies for prognostic evaluation and therapeutics of prostate tumor.