Glycoprotein nonmetastatic melanoma protein B impacts the malignant potential of bladder cancer cells through its hem-immunoreceptor tyrosine-based activation motif.

Bladder cancer is one of the most common cancers among men worldwide. Although multiple genomic mutations and epigenetic alterations have been identified, an efficacious molecularly targeted therapy has yet to be established. Therefore, a novel approach is anticipated. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein that is highly expressed in various cancers. In this study, we evaluated bladder cancer patient samples and found that GPNMB protein abundance is associated with high-grade tumors, and both univariate and multivariate analyses showed that GPNMB is a prognostic factor. Furthermore, the prognosis of patients with high GPNMB levels was significantly poorer in those with nonmuscle invasive bladder cancer (NMIBC) than in those with muscle invasive bladder cancer (MIBC). We then demonstrated that knockdown of GPNMB in MIBC cell lines with high GPNMB inhibits cellular migration and invasion, whereas overexpression of GPNMB further enhances cellular migration and invasion in MIBC cell lines with originally low GPNMB. Therefore, we propose that GPNMB is one of multiple driver molecules in the acquisition of cellular migratory and invasive potential in bladder cancers. Moreover, we revealed that the tyrosine residue in the hemi-immunoreceptor tyrosine-based activation motif (hemITAM) is required for GPNMB-induced cellular motility.

Promotion of squamous cell carcinoma tumorigenesis by oncogene-mediated THG-1/TSC22D4 phosphorylation.

Carcinoma cells possess high proliferative and invasive potentials and exhibit a resilience against stresses, metabolic disorder, and therapeutic efforts. These properties are mainly acquired by genetic alterations including driver gene mutations. However, the detailed molecular mechanisms have not been fully elucidated. Here, we provide a novel mechanism connecting oncogenic signaling and the tumorigenic properties by a transforming growth factor-ß1-stimulated clone 22 (TSC-22) family protein, THG-1 (also called as TSC22D4). THG-1 is localized at the basal layer of normal squamous epithelium and overexpressed in squamous cell carcinomas (SCCs). THG-1 knockdown suppressed SCC cell proliferation, invasiveness, and xenograft tumor formation. In contrast, THG-1 overexpression promoted the EGF-induced proliferation and stratified epithelium formation. Furthermore, THG-1 is phosphorylated by the receptor tyrosine kinase (RTK)-RAS-ERK pathway, which promoted the oncogene-mediated tumorigenesis. Moreover, THG-1 involves in the alternative splicing of CD44 variants, a regulator of invasiveness, stemness, and oxidative stress resistance under the RTK pathway. These findings highlight the pivotal roles of THG-1 as a novel effector of SCC tumorigenesis, and the detection of THG-1 phosphorylation by our established specific antibody could contribute to cancer diagnosis and therapy.

The liver kinase B1 supports mammary epithelial morphogenesis by inhibiting critical factors that mediate epithelial-mesenchymal transition.

The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor ß (TGFß) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional (3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFß, including TGFß auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFß signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFß type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFß family members.

Recent Update on PCSK9 and Platelet Activation Experimental Research Methods: In Vitro and In Vivo Studies.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a crucial factor in the development and progression of cardiovascular diseases. PCSK9 has been demonstrated to modify LDL plasma levels and increase platelet activation, which promotes atherosclerosis, a defining feature of nearly all cardiovascular diseases. Platelet activation has been shown to promote and maintain the response to atherosclerosis development, from beginning to progression and exacerbation, which can lead to advanced cardiovascular events including myocardial infarction (MI) or death. Research on PCSK9 and platelet activation is currently underway with the main goal of reducing the risk of advanced cardiovascular events by preventing or slowing down atherosclerosis progression. Both in vitro and in vivo studies have been used to explore PCSK9 functions to develop new drugs targeting PCSK9. Finding the most suitable study models that represent the pathological and physiological systems found in humans is very important to achieving the goal. This review aimed to present a current and comprehensive overview of the experimental models that have been used to investigate the role of PCSK9 in platelet activation-induced atherosclerotic cardiovascular diseases.

Glycoprotein NMB promotes tumor formation and malignant progression of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC), although one of the most common head and neck cancers, has a static or slightly decreased survival rate because of difficulties in early diagnosis, lack of effective molecular targeting therapy, and severe dysfunction after radical surgical treatments. Therefore, a novel therapeutic target is crucial to increase treatment efficacy and survival rates in these patients. Glycoprotein NMB (GPNMB), whose role in LSCC remains elusive, is a type 1 transmembrane protein involved in malignant progression of various cancers, and its high expression is thought to be a poor prognostic factor. In this study, we showed that GPNMB expression levels in LSCC samples are significantly higher than those in normal tissues, and GPNMB expression is observed mostly in growth-arrested cancer cells. Furthermore, knockdown of GPNMB reduces monolayer cellular proliferation, cellular migration, and tumorigenic growth, while GPNMB protein displays an inverse relationship with Ki-67 levels. Therefore, we conclude that GPNMB may be an attractive target for future LSCC therapy.

TGFß selects for pro-stemness over pro-invasive phenotypes during cancer cell epithelial-mesenchymal transition.

Transforming growth factor ß (TGFß) induces epithelial-mesenchymal transition (EMT), which correlates with stemness and invasiveness. Mesenchymal-epithelial transition (MET) is induced by TGFß withdrawal and correlates with metastatic colonization. Whether TGFß promotes stemness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E-cadherin promoter. In 2D cultures, TGFß induced EMT, generating RFPlow cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGFß withdrawal. RFPlow cells generated robust mammospheres, with epithelio-mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGFß receptor kinase inhibitor. Further stimulation of RFPlow mammospheres with TGFß suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat-pad-transplanted mammospheres, in the absence of exogenous TGFß treatment, established lung metastases with evident MET (RFPhigh cells). In contrast, TGFß-treated mammospheres revealed high tumour-initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGFß to differentiate between pro-stemness and pro-invasive phenotypes.

PCSK9 Promotes Cardiovascular Diseases: Recent Evidence about Its Association with Platelet Activation-Induced Myocardial Infarction.

Cardiovascular diseases are the leading cause of death worldwide, with the majority of the cases being heart failure due to myocardial infarction. Research on cardiovascular diseases is currently underway, particularly on atherosclerosis prevention, to reduce the risk of myocardial infarction. Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been reported to play a role in lipid metabolism, by enhancing low-density lipoprotein (LDL) receptor degradation. Therefore, PCSK9 inhibitors have been developed and found to successfully decrease LDL plasma levels. Recent experimental studies have also implicated PCSK9 in platelet activation, having a key role during atherosclerosis progression. Although numerous studies have addressed the role of PCSK9 role in controlling hypercholesterolemia, studies and discussions exploring its involvement in platelet activation are still limited. Hence, here, we address our current understanding of the pathophysiological process involved in atherosclerosis-induced myocardial infarction (MI) through platelet activation and highlight the molecular mechanisms used by PCSK9 in regulating platelet activation. Undoubtedly, a deeper understanding of the relationship between platelet activation and the underlying molecular mechanisms of PCSK9 in the context of MI progression will provide a new strategy for developing drugs that selectively inhibit the most relevant pathways in cardiovascular disease progression.

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 Promotes Epithelial-Mesenchymal Transition in Doxorubicin-Treated Triple-Negative Breast Cancer Cells via SMAD3 and PI3K/AKT Pathway Alteration.

INTRODUCTION: Epithelial-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. METHODS: We used BT549, triple-negative cells wild type (WT), and BT549 TMEPAI knock-out. Both cells were treated with TGF-ß 2 ng/mL for 24 hours, followed by TGF-ß 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. RESULTS: TNBC 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. CONCLUSION: Our 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.

Glycoprotein non-metastatic melanoma protein B functions with growth factor signaling to induce tumorigenesis through its serine phosphorylation.

Breast cancer is the most common cancer among women. Glycoprotein non-metastatic melanoma protein B (GPNMB), a type I transmembrane protein that is highly expressed in many cancers, including breast cancer, has been shown to be a prognostic factor. We previously reported that GPNMB overexpression confers tumorigenic potential, as evidenced by invasive tumor growth in vivo, sphere formation, and cellular migration and invasion to non-tumorigenic mammary epithelial cells. In this study, we focused on the serine (S) residue in the intracellular domain of GPNMB (S530 in human isoform b and S546 in mouse), which is predicted to be a phosphorylation site. To investigate the roles of this serine residue, we made an antibody specific for S530-phosphorylated human GPNMB and a point mutant in which S530 is replaced by an alanine (A) residue, GPNMB(SA). Established GPNMB(SA) overexpressing cells showed a significant reduction in sphere formation in vitro and tumor growth in vivo as a result of decreased stemness-related gene expression compared to that in GPNMB(WT)-expressing cells. In addition, GPNMB(SA) impaired GPNMB-mediated cellular migration. Furthermore, we found that tyrosine kinase receptor signaling triggered by epidermal growth factor or fibroblast growth factor 2 induces the serine phosphorylation of GPNMB through activation of downstream oncoproteins RAS and RAF.

Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway.

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.

Polyvinyl alcohol scaffolds and supplementation support 3D and sphere culturing of human cancer cell lines by reducing apoptosis and promoting cellular proliferation.

Three-dimensional (3D) culturing mimics the heterogeneous cellular conditions of the in vivo tumor microenvironment compared to 2D monolayer-cultured cells and 3D cultures of established cancer cell lines (sphere culture) or patient-derived cancer cells (organoid culture) are frequently used for cancer research or drug screening and evaluation. To establish more cost and time-efficient 3D culture methods for cancer cell lines, we supplemented sphere culture medium with polyvinyl alcohol (PVA) and found that 3D sphere cultures of breast and pancreatic cancer cell lines were significantly increased. Mechanistically, we found that PVA prevented cell death and promoted cellular proliferation while maintaining levels of stemness-related gene expression. Furthermore, we showed that polyvinyl formal resin (PVF) 3D scaffolds made by cross-linked PVA can function in serum-free, long-term 3D cultures to support maintenance of sphere- or tumor-like cell masses for diverse cancer cell types. Taken together, we demonstrate the effectiveness of PVA and PVF in human cancer cell line culture protocols.

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.

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.

Promotion of cellular senescence by THG-1/TSC22D4 knockout through activation of JUNB.

Induction of cellular senescence in cancerous cells is an important strategy which is used in the treatment of cancer. However, cancer cells are capable of exhibiting resistance to cellular senescence through inactivation of tumor suppressors. Because of this, establishment of a route to cellular senescence induction in cancer cells is a crucial direction for developing future cancer therapies. In this study, we demonstrate the involvement of TSC-22 homologous gene-1 (THG-1, also called TSC22D4) in the suppression of cellular senescence. CRISPR/Cas9 gene editing was used to establish THG-1 knockout (KO) cells in a THG-1 positive esophageal tumor cell line. It was found that THG-1 KO cells exhibited delayed cell proliferation as well as cellular senescence. The elevated expression of the CDK inhibitor P21(CDKN1A) was also identified in senescent cells. Through the investigation of the upstream pathway for induction of P21(CDKN1A), the JUNB pathway was identified to play a critical role in P21(CDKN1A) transcription; in fact, the siRNA-mediated knockdown of JUNB reduced the abundance of P21(CDKN1A) mRNA and cellular senescence in THG-1 KO cells. These findings provide a novel insight into the induction of cellular senescence in THG-1 positive cancer cells.

THG-1 suppresses SALL4 degradation to induce stemness genes and tumorsphere formation through antagonizing NRBP1 in squamous cell carcinoma cells.

Knockdown of THG-1 in TE13 esophageal squamous cell carcinoma (ESCC) cells is known to suppress tumorsphere growth. THG-1 was identified as an NRBP1 binding protein, and NRBP1 was reported to downregulate an stemness-related transcriptional factor SALL4, so we decided to examine the possibility that tumorigenic function of THG-1 is achieved by the competition to the tumor-suppressive function of NRBP1. SALL4 was decreased in THG-1 deficient TE13 cells with reduced tumorsphere formation, while exogenous SALL4 expression in THG-1 deficient TE13 cells recovered expression of stemness genes (NANOG and OCT4) and partially, but significantly, recovered tumorsphere formation ability. Additionally, we found that NRBP1 induced ubiquitination of SALL4, and THG-1 interrupted the ubiquitination of SALL4 by antagonizing NRBP1 binding to SALL4. These results suggest that THG-1 promotes tumorsphere growth of ESCC cells by the stabilization of SALL4 protein and induction of the target stemness genes through competitive binding to NRBP1.

Generation of non-standard macrocyclic peptides specifically binding TSC-22 homologous gene-1.

Transforming growth factor-ß 1 (TGFß1)-stimulated clone 22 (TSC22) family includes proteins containing a leucine zipper domain and a TSC-box that are highly conserved during evolution. Currently, limited data are available on the function of this protein family, especially of TSC-22 homologous gene-1 (THG-1)/TSC22 domain family member 4 (TSC22D4). Similar to other family members, THG-1 functions depending on its interaction with the partner proteins and it is suggested to mediate a broad range of biological processes. THG-1-specific binding molecules will be instrumental for elucidating its functions. Therefore, the Random non-standard Peptide Integrated Discovery (RaPID) system was modified using commercially available materials and used for selecting macrocyclic peptides (MCPs) that bind to THG-1. Several MCPs were identified to bind THG-1. Fluorescein- and biotin-tagged MCPs were synthesized and employed as THG-1 detection probes. Notably, a fluorescein-tagged MCP specifically detected THG-1-expressing cells. Biotin-tagged MCPs can be successfully used for Enzyme-Linked Protein Sorbent Assay (ELISA) like assay of THG-1 protein and affinity-precipitation of purified THG-1 and endogenous THG-1 in esophageal squamous cell carcinoma cell lysates. The modified RaPID system rapidly and successfully identified THG-1-binding MCPs in vitro and the synthesized THG-1 binding MCPs are useful alternatives acting for antibodies.

Role of the kringle-like domain in glycoprotein NMB for its tumorigenic potential.

Glycoprotein NMB (GPNMB) is highly expressed in many types of malignant tumors and thought to be a poor prognostic factor in those cancers, including breast cancer. Glycoprotein NMB is a type IA transmembrane protein that has a long extracellular domain (ECD) and a short intracellular domain (ICD). In general, the ECD of a protein is involved in protein-protein or protein-carbohydrate interactions, whereas the ICD is important for intracellular signaling. We previously reported that GPNMB contributes to the initiation and malignant progression of breast cancer through the hemi-immunoreceptor tyrosine-based activation motif (hemITAM) in its ICD. Furthermore, we showed that the tyrosine residue in hemITAM is involved in induction of the stem-like properties of breast cancer cells. However, the contribution of the ECD to its tumorigenic function has yet to be fully elucidated. In this study, we focused on the region, the so-called kringle-like domain (KLD), that is conserved among species, and made a deletion mutant, GPNMB(ΔKLD). Enhanced expression of WT GPNMB induced sphere and tumor formation in breast epithelial cells; in contrast, GPNMB(ΔKLD) lacked these activities without affecting its molecular properties, such as subcellular localization, Src-induced tyrosine phosphorylation at least in overexpression experiments, and homo-oligomerization. Additionally, GPNMB(ΔKLD) lost its cell migration promoting activity, even though it reduced E-cadherin expression. Although the interaction partner binding to KLD has not yet been identified, we found that the KLD of GPNMB plays an important role in its tumorigenic potential.

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.

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.