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-ß 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.

C18 ORF1, a novel negative regulator of transforming growth factor-ß signaling.

Transforming growth factor (TGF)-ß signaling is deliberately regulated at multiple steps in its pathway from the extracellular microenvironment to the nucleus. However, how TGF-ß signaling is activated or attenuated is not fully understood. We recently identified transmembrane prostate androgen-induced RNA (TMEPAI), which is involved in a negative feedback loop of TGF-ß signaling. When we searched for a family molecule(s) for TMEPAI, we found C18ORF1, which, like TMEPAI, possesses two PY motifs and one Smad-interacting motif (SIM) domain. As expected, C18ORF1 could block TGF-ß signaling but not bone morphogenetic protein signaling. C18ORF1 bound to Smad2/3 via its SIM and competed with the Smad anchor for receptor activation for Smad2/3 binding to attenuate recruitment of Smad2/3 to the TGF-ß type I receptor (also termed activin receptor-like kinase 5 (ALK5)), in a similar fashion to TMEPAI. Knockdown of C18ORF1 prolonged duration of TGF-ß-induced Smad2 phosphorylation and concomitantly potentiated the expression of JunB, p21, and TMEPAI mRNAs induced by TGF-ß. Consistently, TGF-ß-induced cell migration was enhanced by the knockdown of C18ORF1. These results indicate that the inhibitory function of C18ORF1 on TGF-ß signaling is similar to that of TMEPAI. However, in contrast to TMEPAI, C18ORF1 was not induced upon TGF-ß signaling. Thus, we defined C18ORF1 as a surveillant of steady state TGF-ß signaling, whereas TMEPAI might help C18ORF1 to inhibit TGF-ß signaling in a coordinated manner when cells are stimulated with high levels of TGF-ß.

Cooperative induction of transmembrane prostate androgen induced protein TMEPAI/PMEPA1 by transforming growth factor-ß and epidermal growth factor signaling.

TMEPAI/PMEPA1 (transmembrane prostate androgen induced-RNA/prostate transmembrane protein, androgen induced 1) is a pro-tumorigenic factor induced by TGF-ß signaling and constitutive TMEPAI expression in lung cancer cells depends on activated autocrine TGF-ß signaling. Here we demonstrate a novel mechanism of TMEPAI transcriptional co-regulation by EGF signaling. Interestingly, we found that ELK-1, downstream of EGFR/Ras/MAPK pathway, modulates TMEPAI expression. ELK-1 binds to the first intron (+1037 to +1294) of the TMEPAI gene together with TGF-ß activated Smad3 and enhances the transcription of TMEPAI. Furthermore, TMEPAI gene activation by EGF and TGF-ß signaling was reduced by the MEK inhibitor U0126. Together, EGF signaling collaboratively regulates TGF-ß-induced TMEPAI expression.

TMEPAI/PMEPA1 enhances tumorigenic activities in lung cancer cells.

TMEPAI/PMEPA1 is a transmembrane protein that was originally identified as a prostatic RNA, the synthesis of which is induced by testosterone or its derivatives. We have recently identified TMEPAI as a direct target gene of transforming growth factor-ß (TGF-ß)/Smad signaling that participates in negative feedback control of the duration and intensity of TGF-ß/Smad signaling. TMEPAI is constitutively and highly expressed in many types of cancer and is associated with poor prognosis. Here, we report that TMEPAI is highly expressed in the lung adenocarcinoma cell lines Calu3, NCI-H23, and RERF-LC-KJ. Expression of TMEPAI in these cancer cells was significantly suppressed by a TGF-ß receptor kinase antagonist, SB208, and by TGF-ß neutralizing antibodies. These results suggest that constitutive expression of TMEPAI in these cancer cells depends on autocrine TGF-ß stimulation. Knockdown of TMEPAI in Calu3 and NCI-H23 cells enhanced levels of Smad2 phosphorylation and significantly suppressed cell proliferation in the presence of TGF-ß, indicating that highly expressed TMEPAI suppresses levels of Smad phosphorylation in these cancer cells and reduces the growth inhibitory effects of TGF-ß/Smad signaling. Furthermore, knockdown of TMEPAI in Calu3 and NCI-H23 cells suppressed sphere formation in vitro and tumor formation in s.c. tissues and in lungs after tail vein injection in NOD-SCID mice in vivo. Together, these experiments indicate that TMEPAI promotes tumorigenic activities in lung cancer cells.

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.

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

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-ß-Induced TMEPAI Attenuates the Response of Triple-Negative Breast Cancer Cells to Doxorubicin and Paclitaxel.

PURPOSE: Triple-negative breast cancer (TNBC) is a refractory type of breast cancer with poor prognosis and limited choice for treatment. Previous studies had shown that TNBC has high expressions of transmembrane prostate androgen-induced protein (TMEPAI). TMEPAI was known to be induced by TGF-ß/Smad signaling and have tumorigenic functions that converting TGF-ß from tumor suppressor to tumor promoter and inducing epithelial-mesenchymal transition (EMT). Therefore, we aimed to define the role of TMEPAI in triple-negative breast cancer cells treatment using several anti-cancers in the presence of TGF-ß. METHODS: TMEPAI-knock out (KO) was carried out in a triple-negative breast cancer cell, BT549. TMEPAI editing was developed using the CRISPR-Cas9 system using two combinations of sgRNA to remove exon 4 of the TMEPAI gene entirely. Genotyping and proteomic analysis were performed to check the establishment of the TMEPAI-KO cells. Wild type (WT) and KO cells were used to determine inhibitory concentration 50% (IC50) of several anti-cancers: doxorubicin, cisplatin, paclitaxel, and bicalutamide in the presence of TGF-ß treatment. RESULTS: KO cells were successfully established by completely removing the TMEPAI gene, which was proven in genomic and proteomic analysis. Further, in TMEPAI-KO cells, we found a significant reduction of IC50 for doxorubicin and paclitaxel, and minimal effects were seen for cisplatin and bicalutamide. Our findings suggest that TGF-ß-induced TMEPAI attenuates the response of TNBC to doxorubicin and paclitaxel, but not to cisplatin and bicalutamide. CONCLUSION: TGF-ß induced TMEPAI contributes to the reduced response of TNBC treatment to doxorubicin and paclitaxel, but minimal on cisplatin and bicalutamide. Further study is needed to confirm our findings in other growth factor-induced cells, as well as in in vivo model.

TMEPAI/PMEPA1 Is a Positive Regulator of Skeletal Muscle Mass.

Inhibition of myostatin- and activin-mediated SMAD2/3 signaling using ligand traps, such as soluble receptors, ligand-targeting propeptides and antibodies, or follistatin can increase skeletal muscle mass in healthy mice and ameliorate wasting in models of cancer cachexia and muscular dystrophy. However, clinical translation of these extracellular approaches targeting myostatin and activin has been hindered by the challenges of achieving efficacy without potential effects in other tissues. Toward the goal of developing tissue-specific myostatin/activin interventions, we explored the ability of transmembrane prostate androgen-induced (TMEPAI), an inhibitor of transforming growth factor-ß (TGF-ß1)-mediated SMAD2/3 signaling, to promote growth, and counter atrophy, in skeletal muscle. In this study, we show that TMEPAI can block activin A, activin B, myostatin and GDF-11 activity in vitro. To determine the physiological significance of TMEPAI, we employed Adeno-associated viral vector (AAV) delivery of a TMEPAI expression cassette to the muscles of healthy mice, which increased mass by as much as 30%, due to hypertrophy of muscle fibers. To demonstrate that TMEPAI mediates its effects via inhibition of the SMAD2/3 pathway, tibialis anterior (TA) muscles of mice were co-injected with AAV vectors expressing activin A and TMEPAI. In this setting, TMEPAI blocked skeletal muscle wasting driven by activin-induced phosphorylation of SMAD3. In a model of cancer cachexia associated with elevated circulating activin A, delivery of AAV:TMEPAI into TA muscles of mice bearing C26 colon tumors ameliorated the muscle atrophy normally associated with cancer progression. Collectively, the findings indicate that muscle-directed TMEPAI gene delivery can inactivate the activin/myostatin-SMAD3 pathway to positively regulate muscle mass in healthy settings and models of disease.

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-ß 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.

TMEPAI/PMEPA1 inhibits Wnt signaling by regulating ß-catenin stability and nuclear accumulation in triple negative breast cancer cells.

Transmembrane prostate androgen-induced protein (TMEPAI) is a type I transmembrane protein induced by several intracellular signaling pathways such as androgen, TGF-ß, EGF, and Wnt signaling. It has been reported that TMEPAI functions to suppress TGF-ß and androgen signaling but here, we report a novel function of TMEPAI in Wnt signaling suppression. First, we show that TMEPAI significantly inhibits TCF/LEF transcriptional activity stimulated by Wnt3A, LiCl, and ß-catenin. Mechanistically, TMEPAI overexpression prevented ß-catenin accumulation in the nucleus and TMEPAI knockout in triple negative breast cancer cell lines promoted ß-catenin stability and nuclear accumulation together with increased mRNA levels of Wnt target genes AXIN2 and c-MYC. The presence of TGF-ß type I receptor kinase inhibitor did not affect the enhanced mRNA expression of AXIN2 in TMEPAI knockout cells. These data suggest that TMEPAI suppresses Wnt signaling by interfering with ß-catenin stability and nuclear translocation in a TGF-ß signaling-independent manner.

Increased Smad3 and reduced Smad2 levels mediate the functional switch of TGF-ß from growth suppressor to growth and metastasis promoter through TMEPAI/PMEPA1 in triple negative breast cancer.

Screening of several TNBC cell lines showed altered Smad2 and Smad3 protein levels compared to normal mammary epithelial cells, suggesting the possibility that it could play an important role in the escape of cancer cells from TGF-ß mediated growth inhibition. To assess the functional relevance of these endogenous molecules, Smad2 or Smad3 expression was knocked down individually and assessed their effects on pro-oncogenic properties of TGF-ß. Smad3 deficiency reduced growth and invasion capacity of breast cancer cells in comparison to Smad2 which had no effect. Smad3 deficiency was also found to be associated with a reduction in the expressions of TMEPAI/PMEPA1 and EMT inducing transcription factors, E-Cadherin and increased expression of cell cycle inhibitors and Vimentin. On the other hand, Smad2 deficiency had opposite effect on these regulators. Interestingly, the decreased growth, invasion and associated gene expressions were largely reversed by overexpressing TMEPAI in Smad3 knockdown cells, suggesting that Smad3-TMEPAI axis may be involved in subverting growth suppressive effects of TGF-ß into growth promotion. Similarly, altered levels of Smad proteins and TMEPAI were also noted in primary TNBC tumor tissues. Analysis of the existing databases provided additional support in terms of TMEPAI and Smad2 expression impacting the survival of TNBC patients. Taken together, our data demonstrate a novel role for Smad3 in cancer transformation and cancer progression through TMEPAI and further suggest that selective targeting of TGF-ß-Smad3-TMEPAI axis may be beneficial in triple negative breast cancer therapy and prevention.

PMEPA1/TMEPAI knockout impairs tumour growth and lung metastasis in MDA-MB-231 cells without changing monolayer culture cell growth.

Prostate transmembrane protein androgen-induced 1 (PMEPA1)/transmembrane prostate androgen-induced protein (TMEPAI), a direct target and a negative regulator of transforming growth factor beta signalling, has an oncogenic role in many cancers. We observed that knockout (KO) of PMEPA1 in human breast cancer cell line MDA-MB-231 using a CRISPR-Cas9 system resulted in reduction of in vivo tumour growth and lung metastasis but not of in vitro monolayer growth capacity of these KO cell lines. This phenomenon was associated with PMEPA1 KO-mediated downregulation of the key proangiogenic factors vascular endothelial growth factor alpha (VEGFA) and interleukin-8 (IL8) that are essential for in vivo but not in vitro growing cells and are also substantial for initiation of lung metastasis.

Development of Novel Monoclonal Antibodies for Evaluation of Transmembrane Prostate Androgen-Induced Protein 1 (TMEPAI) Expression Patterns in Gastric Cancer.

Transmembrane prostate androgen-induced protein 1 (TMEPAI) is a single-span membrane protein, functionally involved in transforming growth factor beta signaling pathway. The particular protein presented in cells in three isoforms, which differs in the length of the soluble N-terminal extracellular domain, making it challenging for the immunochemical recognition. By using quantitative real-time polymerase chain reaction, we identified significant upregulation of PMEPA1 gene expression in malignant tissues of patients with gastric adenocarcinoma. The main part of commercially available anti-TMEPAI antibodies are having polyclonal nature or not suitable for immunocytochemical localization of target protein in tissue specimens. Hence, we decide to generate a set of novel rat monoclonal antibodies (mAb) directed against conservative C-terminal cytoplasmic epitope. Immunoblotting analysis showed that monoclonal antibodies, 2E1, 6C6, and 10A7 were able to recognize specifically target protein in transiently transfected HEK293T and CHO-K1 cells. Especially established mAb, named 10A7, showed the excellent binding ability to target protein in immunohistochemistry. By using developed antibodies, we observed pronounced expression of TMEPAI in normal gastric epithelial cells while tumor cells from gastric adenomas, and adenocarcinoma samples were mostly negative for target protein expression. Also, we found that gastric epithelium cells lose the TMEPAI expression concurrently with severe dysplasia progression, which probably caused by a mechanism involving specific microRNA.

TMEPAI increases lysosome stability and promotes autophagy.

Autophagy is emerging as a critical response of normal and cancer cells to environmental changes and plays an important role in cell metabolism and maintenance of damaged organelles. Transmembrane prostate androgen-induced protein (TMEPAI) is a pro-tumorigenic factor with high expression in tumor cells. In this study, we showed that depletion of TMEPAI leads to lysosomal labilization and inhibits autophagy. Further study showed that the inhibition of autophagy induced by the depletion of TMEPAI is involved in regulation of Beclin-1. Depletion of TMEPAI increases the sensitivity of cancer cells to chemotherapeutic drugs. Our study reveals the role of TMEPAI in promoting lysosome stability and autophagy, which might be used as a target for cancer chemotherapeutic treatment.

Regulation of the TMEPAI promoter by TCF7L2: the C-terminal tail of TCF7L2 is essential to activate the TMEPAI gene.

We previously found that TCF7L2 could activate the TMEPAI gene efficiently, whereas LEF1 could not nearly augment its transcription. When we comprehended the functional difference(s) between TCF7L2 and LEF1 with respect to the activation of the TMEPAI gene, the C-terminal tail of TCF7L2 was needed to reveal its transcriptional activity as well as its interaction with Smad3. Consistently, both TCF7/TCF7L2 and LEF1/TCF7L2 chimeric proteins exhibited an activity similar to TCF7L2 in transcription and Smad3 binding in contrast with LEF1 and TCF7. Our data elaborated on the diverse activity among TCF/LEF family members with respect to the transcriptional regulation of the TMEPAI gene.

Cutting the brakes and flooring the gas: how TMEPAI turns TGF-ß into a tumor promoter.

In normal or nonmalignant cells, TGF-ß inhibits cellular proliferation through activation of the SMAD-dependent canonical signaling pathway. Recent findings demonstrate that the protein TMEPAI1 can block the cytostatic effects of the canonical TGF-ß signaling pathway, while activating cellular proliferation through the noncanonical, SMAD-independent TGF-ß signaling pathway. As TMEPAI1 shows increased expression in the poor prognosis basal and HER2 intrinsic subtypes of breast cancer, these findings point to a new avenue of targeted therapy with considerable therapeutic potential.

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

Transforming growth factor-ß (TGF-ß) 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-ß signaling, and in turn, expression of TMEPAI negatively regulates TGF-ß signaling, but the molecular mechanisms of TMEPAI induced TGF-ß 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-ß type I receptor (TßRI) 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-ß signaling through the modulation of TßRI levels, which has important implications for cancer development in vivo.

TGF-ß induced TMEPAI/PMEPA1 inhibits canonical Smad signaling through R-Smad sequestration and promotes non-canonical PI3K/Akt signaling by reducing PTEN in triple negative breast cancer.

TMEPAI (transmembrane prostate androgen-induced) is amplified at genomic, transcript and protein levels in triple-negative breast cancers and promotes TGF-ß dependent growth, motility and invasion. Tumor promotion by TMEPAI depends on two different but related actions on TGF-ß signaling. Firstly, TMEPAI binds and sequesters regulatory Smads2/3 and thereby decreases growth suppressive signaling by TGF-ß. Secondly, increased expression of TMEPAI decreases PTEN (phosphatase and tensin homolog) abundance, and thereby increases TGF-ß dependent tumor promotive PI3K/Akt signaling. These actions of TMEPAI give rise to increased cell proliferation and motility. Moreover, signaling alterations produced by high TMEPAI were associated with oncogenic Snail expression and lung metastases. Finally, an inverse correlation between TMEPAI and PTEN levels was confirmed in triple negative breast cancer tumor samples. Together, our findings suggest that TMEPAI has dually critical roles to promote TGF-ß dependent cancer cell growth and metastasis. Thus, redirected TGF-ß signaling through TMEPAI may play a pivotal role in TGF-ß mediated tumor promotion.