PMEPA1/TMEPAI is an Independent Tumorigenic Coactivator of AKT Working Through Proteasomal Degradation of PHLPP1 in Triple Negative Breast Cancer Cells

概要

[Purposes]
Transmembrane prostate androgen-induced protein (TMEPAI) is a transforming growth factor-β (TGF-β) induced oncoprotein. It is particularly expressed in various types of cancer and activates multiple oncogenic pathways. However, there is no specific study regarding how and which motifs of TMEPAI have such oncogenic functions in triple negative breast cancer (TNBC). Therefore, I studied the oncogenic function of TMEPAI in TNBC cells and aimed to elucidate the molecular mechanism behind such activities. Later on, I focused to delineate the TMEPAI- mediated regulation of pleckstrin homology leucine rich repeat protein phosphatase 1 (PHLPP1).

[Methods and Materials]
In this study, I used well controlled and established methods of molecular biology to elucidate the oncogenic functions and molecular mechanisms of TMEPAI in TNBC progression. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing technique, small interfering RNA and short hairpin RNA systems were used to knockout (KO) or knockdown (KD) of target proteins. Colony formation, cell migration, scratch wound healing, tumor sphere formation and xenograft tumor formation assays were performed to investigate the oncogenic functions of TMEPAI and PHLPP1. Polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), and Western blotting were used to examine targeted genes and proteins expression. Co-immunoprecipitations were performed to detect the protein-protein interaction. BALB/cAJcl-nu/nu mice were used to evaluate the xenograft tumor formation. Additionally, the cancer genome atlas (TCGA) database of invasive breast cancer (BRCA) patients were analyzed to find the correlation between TMEPAI and phosphoinositide 3-kinase (PI3K)/AK strain transforming (AKT) signaling in the patients.

[Results]
TMEPAI expression is inversely correlated with patients’ survival rate. I found that TMEPAI KO cells remarkably reduced colony formation, cell migration and tumor sphere formation. TCGA clinical data set analysis showed a positive correlation between TMEPAI and PI3K/AKT signaling activity specially in AKT phosphorylation on S473. My Western blot data also demonstrated that TMEPAI enhanced S473 phosphorylation of AKT by suppressing PHLPP1. I also found that KD of PHLPP1 from both parental and TMEPAI KO cells enhanced colony formation, cell migration and tumor sphere formation by promoting PI3K/AKT signaling. This correlation was further confirmed by in vivo experiments.

To investigate which motifs of TMEPAI downregulates PHLPP1, I performed TMEPAI re- expression experiments in TMEPAI KO cells using lentivirus expression system. My results clearly showed that a PPxY motifs (PY motifs) mutant could not suppress PHLPP1 unlike TMEPAI wild type and a Smad interacting motif (SIM) mutant, indicating the PHLPP1 suppressive role of TMEPAI through its PY motifs. Colony formation assay and Western blotting results also corroborated the role of TMEPAI PY motifs for regulation of colony formation and promotion of PI3K/AKT signaling.

Additionally, I also found that TMEPAI binds NEDD4-2, a E3 ubiquitin ligase, through its PY motifs, enhanced the complex formation of NEDD4-2 with PHLPP1, and hence promotes PHLPP1 polyubiquitination and proteasomal degradation. Moreover, I found the KD of NEDD4-2 recovered PHLPP1 protein amount, suggesting a cumulative role of TMEPAI and NEDD4-2 is essential for the proteasomal degradation of PHLPP1 in TNBC cells.

[Discussion]
Since the overactivation of PI3K and/or inactivation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a suppressor of PI3K/AKT signaling, are frequently observed in cancer cells, activation of PI3K/AKT signaling is considered as one of the main drivers of cancer initiation and progression. In this study, I identified a unique oncogenic role of TMEPAI in TNBC cells. I found that TMEPAI promotes colony formation, migration and tumor sphere formation in breast cancer cells. Mechanistically, PY motifs of TMEPAI activates PI3K/AKT signaling specially by enhancing the phosphorylation at serine 473 site of AKT through the down regulation of PHLPP1. These findings are in line with a clinical data (TCGA) analysis from invasive BRCA patients. PHLPP1 is one of the negative regulators of PI3K/AKT signaling and has a tumor suppressive role which was proved by various in vitro and in vivo studies. Here, I also elucidated that PHLPP1 dephosphorylates serine 473 of AKT and suppresses colony formation, cell migration, tumor sphere formation and in vivo tumor formation. These results indicate that activation of PI3K and inactivation of PTEN are not enough for the oncogenic activation of AKT. Degradation of PHLPP1 by TGF-β-induced TMEPAI is essential for the tumorigenic AKT activaion. To clarify the TMEPAI-mediated regulation of PHLPP1, I observed that PHLPP1 ubiquitination was enhanced in the presence of TMEPAI by facilitating complex formation with NEDD4-2. These data suggest a putative oncogenic functions of TMEPAI in TNBC progression via activation of PI3K/AKT signaling.

[Conclusion]
Here, I revealed a novel oncogenic role of TMEPAI via the activation of PI3K/AKT signaling through the downregulation of PHLPP1. Therefore, TGF-β-induced TMEPAI is considered as an essential oncogenic coactivator of AKT working together with genomic mutations of PI3K and PTEN, and an independent biomarker and a promising target to establish molecular targeted therapy for the management and treatment of TNBC patients.