Tm4sf19 deficiency inhibits osteoclast multinucleation and prevents bone loss.

BACKGROUND: Multinucleation is a hallmark of osteoclast formation and has a unique ability to resorb bone matrix. During osteoclast differentiation, the cytoskeleton reorganization results in the generation of actin belts and eventual bone resorption. Tetraspanins are involved in adhesion, migration and fusion in various cells. However, its function in osteoclast is still unclear. In this study, we identified Tm4sf19, a member of the tetraspanin family, as a regulator of osteoclast function. MATERIALS AND METHODS: We investigate the effect of Tm4sf19 deficiency on osteoclast differentiation using bone marrow-derived macrophages obtained from wild type (WT), Tm4sf19 knockout (KO) and Tm4sf19 LELΔ mice lacking the large extracellular loop (LEL). We analyzed bone mass of young and aged WT, KO and LELΔ mice by µCT analysis. The effects of Tm4sf19 LEL-Fc fusion protein were accessed in osteoclast differentiation and osteoporosis animal model. RESULTS: We found that deficiency of Tm4sf19 inhibited osteoclast function and LEL of Tm4sf19 was responsible for its function in osteoclasts in vitro. KO and LELΔ mice exhibited higher trabecular bone mass compared to WT mice. We found that Tm4sf19 interacts with integrin αvß3 through LEL, and that this binding is important for cytoskeletal rearrangements in osteoclast by regulating signaling downstream of integrin αvß3. Treatment with LEL-Fc fusion protein inhibited osteoclast function in vitro and administration of LEL-Fc prevented bone loss in an osteoporosis mouse model in vivo. CONCLUSION: We suggest that Tm4sf19 regulates osteoclast function and that LEL-Fc may be a promising drug to target bone destructive diseases caused by osteoclast hyper-differentiation.

ESRP1-regulated isoform switching of LRRFIP2 determines metastasis of gastric cancer.

Although accumulating evidence indicates that alternative splicing is aberrantly altered in many cancers, the functional mechanism remains to be elucidated. Here, we show that epithelial and mesenchymal isoform switches of leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) regulated by epithelial splicing regulatory protein 1 (ESRP1) correlate with metastatic potential of gastric cancer cells. We found that expression of the splicing variants of LRRFIP2 was closely correlated with that of ESRP1. Surprisingly, ectopic expression of the mesenchymal isoform of LRRFIP2 (variant 3) dramatically increased liver metastasis of gastric cancer cells, whereas deletion of exon 7 of LRRFIP2 by the CRISPR/Cas9 system caused an isoform switch, leading to marked suppression of liver metastasis. Mechanistically, the epithelial LRRFIP2 isoform (variant 2) inhibited the oncogenic function of coactivator-associated arginine methyltransferase 1 (CARM1) through interaction. Taken together, our data reveals a mechanism of LRRFIP2 isoform switches in gastric cancer with important implication for cancer metastasis.

Degradation of DRAK1 by CUL3/SPOP E3 Ubiquitin ligase promotes tumor growth of paclitaxel-resistant cervical cancer cells.

Despite favorable responses to initial chemotherapy, drug resistance is a major cause limiting chemotherapeutic efficacy in many advanced cancers. However, mechanisms that drive drug-specific resistance in chemotherapy for patients with advanced cancers are still unclear. Here, we report a unique role of death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK1) associated with paclitaxel resistance in cervical cancer cells. Interestingly, DRAK1 protein level was markedly decreased in paclitaxel-resistant cervical cancer cells without affecting its mRNA expression, which resulted in an increase in tumor necrosis factor receptor-associated factor 6 (TRAF6) expression, as well as an activation of TRAF6-mediated nuclear factor-kappa B (NF-κB) signaling cascade, thereby promoting tumor progression. DRAK1 depletion markedly increased the chemotherapeutic IC50 values of paclitaxel in cervical cancer cells. Ectopic expression of DRAK1 inhibited growth of paclitaxel-resistant cervical cancer cells in vitro and in vivo. Furthermore, DRAK1 was markedly underexpressed in chemoresistant cervical cancer patient tissues compared with chemosensitive samples. We found that DRAK1 protein was destabilized through K48-linked polyubiquitination promoted by the Cullin scaffold protein 3 (CUL3) / speckle-type POZ (poxvirus and zinc finger protein) protein (SPOP) E3 ubiquitin ligase in paclitaxel-resistant cells. Collectively, these findings suggest that DRAK1 may serve as a potential predictive biomarker for overcoming paclitaxel resistance in cervical cancer.

Tetraarsenic hexoxide enhances generation of mitochondrial ROS to promote pyroptosis by inducing the activation of caspase-3/GSDME in triple-negative breast cancer cells.

Although tetraarsenic hexoxide is known to exert an anti-tumor effect by inducing apoptosis in various cancer cells, its effect on other forms of regulated cell death remains unclear. Here, we show that tetraarsenic hexoxide induces the pyroptotic cell death through activation of mitochondrial reactive oxygen species (ROS)-mediated caspase-3/gasdermin E (GSDME) pathway, thereby suppressing tumor growth and metastasis of triple-negative breast cancer (TNBC) cells. Interestingly, tetraarsenic hexoxide-treated TNBC cells exhibited specific pyroptotic characteristics, including cell swelling, balloon-like bubbling, and LDH releases through pore formation in the plasma membrane, eventually suppressing tumor formation and lung metastasis of TNBC cells. Mechanistically, tetraarsenic hexoxide markedly enhanced the production of mitochondrial ROS by inhibiting phosphorylation of mitochondrial STAT3, subsequently inducing caspase-3-dependent cleavage of GSDME, which consequently promoted pyroptotic cell death in TNBC cells. Collectively, our findings highlight tetraarsenic hexoxide-induced pyroptosis as a new therapeutic strategy that may inhibit cancer progression of TNBC cells.

Destablilization of TRAF6 by DRAK1 Suppresses Tumor Growth and Metastasis in Cervical Cancer Cells.

The adaptor protein TNF receptor-associated factor 6 (TRAF6) is a key mediator in inflammation. However, the molecular mechanisms controlling its activity and stability in cancer progression remain unclear. Here we show that death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK1) inhibits the proinflammatory signaling pathway by targeting TRAF6 for degradation, thereby suppressing inflammatory signaling-mediated tumor growth and metastasis in advanced cervical cancer cells. DRAK1 bound directly to the TRAF domain of TRAF6, preventing its autoubiquitination by interfering with homo-oligomerization, eventually leading to autophagy-mediated degradation of TRAF6. Depletion of DRAK1 in cervical cancer cells resulted in markedly increased levels of TRAF6 protein, promoting activation of the IL1ß signaling-associated pathway and proinflammatory cytokine production. DRAK1 was specifically underexpressed in metastatic cervical cancers and inversely correlated with TRAF6 expression in mouse xenograft model tumor tissues and human cervical tumor tissues. Collectively, our findings highlight DRAK1 as a novel antagonist of inflammation targeting TRAF6 for degradation that limits inflammatory signaling-mediated progression of advanced cervical cancer. SIGNIFICANCE: Serine/threonine kinase DRAK1 serves a unique role as a novel negative regulator of the inflammatory signaling mediator TRAF6 in cervical cancer progression.

RNF208, an estrogen-inducible E3 ligase, targets soluble Vimentin to suppress metastasis in triple-negative breast cancers.

The development of triple-negative breast cancer (TNBC) negatively impacts both quality of life and survival in a high percentage of patients. Here, we show that RING finger protein 208 (RNF208) decreases the stability of soluble Vimentin protein through a polyubiquitin-mediated proteasomal degradation pathway, thereby suppressing metastasis of TNBC cells. RNF208 was significantly lower in TNBC than the luminal type, and low expression of RNF208 was strongly associated with poor clinical outcomes. Furthermore, RNF208 was induced by 17ß-estradiol (E2) treatment in an estrogen receptor alpha (ΕRα)-dependent manner. Overexpression of RNF208 suppresses tumor formation and lung metastasis of TNBC cells. Mechanistically, RNF208 specifically polyubiquitinated the Lys97 residue within the head domain of Vimentin through interaction with the Ser39 residue of phosphorylated Vimentin, which exists as a soluble form, eventually facilitating proteasomal degradation of Vimentin. Collectively, our findings define RNF208 as a negative regulator of soluble Vimentin and a prognostic biomarker for TNBC cells.

Erratum: Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation.

[This corrects the article on p. 1 in vol. 23, PMID: 29629343.].

Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation.

BACKGROUND: Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. METHODS: We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. RESULTS: In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2, SNAI1, and ZEB1 in response to TGF-ß1 in human pancreatic and prostate cancer cell lines. We identified GADD45B, CTGF, and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. CONCLUSIONS: These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B, CTGF, and JUNB genes in various cancers.

Co-chaperone BAG2 Determines the Pro-oncogenic Role of Cathepsin B in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is considered incurable with currently available treatments, highlighting the need for therapeutic targets and predictive biomarkers. Here, we report a unique role for Bcl-2-associated athanogene 2 (BAG2), which is significantly overexpressed in TNBC, in regulating the dual functions of cathepsin B as either a pro- or anti-oncogenic enzyme. Silencing BAG2 suppresses tumorigenesis and lung metastasis and induces apoptosis by increasing the intracellular mature form of cathepsin B, whereas BAG2 expression induces metastasis by blocking the auto-cleavage processing of pro-cathepsin B via interaction with the propeptide region. BAG2 regulates pro-cathepsin B/annexin II complex formation and facilitates the trafficking of pro-cathespin-B-containing TGN38-positive vesicles toward the cell periphery, leading to the secretion of pro-cathepsin B, which induces metastasis. Collectively, our results uncover BAG2 as a regulator of the oncogenic function of pro-cathepsin B and a potential diagnostic and therapeutic target that may reduce the burden of metastatic breast cancer.

Galangin enhances TGF-ß1-mediated growth inhibition by suppressing phosphorylation of threonine 179 residue in Smad3 linker region.

Smad3 linker phosphorylation is a candidate target for several kinases that play important roles in cancer cell initiation, proliferation and progression. Also, Smad3 is an essential intracellular mediator of TGF-ß1-induced transcriptional responses during carcinogenesis. Therefore, it is highly advantageous to identify and develop inhibitors targeting Smad3 linker phosphorylation for the treatment of cancers. Galangin (3,5,7-trihydroxyflavone) has been known to be an active flavonoid showing a cytotoxic effect on several cancer cells. However, the mechanism of action of galangin in various cancers remains unclear, and there has been no report concerning regulation of Smad3 phosphorylation by galangin. In the present study, we show that galangin significantly induced apoptosis and inhibited cell proliferation in the presence of TGF-ß1 in both human prostate and pancreatic cancer cell lines. Particularly, galangin effectively inhibits phosphorylation of the Thr-179 site at Smad3 linker region through suppression of CDK4 phosphorylation. Thus, galangin can be a promising candidate as a selective inhibitor to suppress phosphorylation of Smad3 linker region.