Hongmei Yang

Persistent c-FLIP(L) Expression Is Necessary and Sufficient to Maintain Resistance to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand–Mediated Apoptosis in Prostate Cancer

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in a variety of tumorigenic and transformed cell lines but not in many normal cells. Hence, TRAIL has the potential to be an ideal cancer therapeutic agent with minimal cytotoxicity. FLICE inhibitory protein (c-FLIP) is an important regulator of TRAIL-induced apoptosis. Here, we show that persistent expression of c-FLIP(Long) [c-FLIP(L)] is inversely correlated with the ability of TRAIL to induce apoptosis in prostate cancer cells. In contrast to TRAIL-sensitive cells, TRAIL-resistant LNCaP and PC3-TR (a TRAIL-resistant subpopulation of PC3) cells showed increased c-FLIP(L) mRNA levels and maintained steady protein expression of c-FLIP(L) after treatment with TRAIL. Ectopic expression of c-FLIP(L) in TRAIL-sensitive PC3 cells changed their phenotype from TRAIL sensitive to TRAIL resistant. Conversely, silencing of c-FLIP(L) expression by small interfering RNA in PC3-TR cells reversed their phenotype from TRAIL resistant to TRAIL sensitive. Therefore, persistent expression of c-FLIP(L) is necessary and sufficient to regulate sensitivity to TRAIL-mediated apoptosis in prostate cancer cells.

miR-141 Is a Key Regulator of Renal Cell Carcinoma Proliferation and Metastasis by Controlling EphA2 Expression

Purpose: Although microRNAs (miRNA) have been revealed as crucial modulators of tumorigenesis, our understanding of their roles in renal cell carcinoma (RCC) is limited. Here we sought to identify human miRNAs that act as key regulators of renal carcinogenesis. Experimental Design: We performed microarray-based miRNA profiling of clear cell RCC (ccRCC) and adjacent normal tissues and then explored the roles of miR-141 both in vitro and in vivo, which was the most significantly downregulated in ccRCC tissues. Results: A total of 74 miRNAs were dysregulated in ccRCC compared with normal tissues. miR-141 was remarkably downregulated in 92.6% (63/68) ccRCC tissues and would serve as a promising biomarker for discriminating ccRCC from normal tissues with an area under the receiver operating characteristics curve of 0.93. Overexpression of miR-141 robustly impaired ccRCC cell migratory and invasive properties and suppressed cell proliferation by arresting cells at G0–G1 phase in vitro and in human RCC orthotopic xenografts. Significantly, the antitumor activities of miR-141 were mediated by its reversal regulation of erythropoietin-producing hepatocellular (Eph) A2 (EphA2), which then relayed a signaling transduction cascade to attenuate the functions of focal adhesion kinase (FAK), AKT, and MMP2/9. In addition, a specific and inverse correlation between miR-141 and EphA2 expression was obtained in human ccRCC samples. Finally, miR-141 could be secreted from the ccRCC donor cells, and be taken up and function moderately in the ccRCC recipient cells. Conclusion: miR-141 serves as a potential biomarker for discriminating ccRCC from normal tissues and a crucial suppressor of ccRCC cell proliferation and metastasis by modulating the EphA2/p-FAK/p-AKT/MMPs signaling cascade. Clin Cancer Res; 20(10); 2617–30. ©2014 AACR.

c-Fos as a proapoptotic agent in TRAIL-induced apoptosis in prostate cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L promotes apoptosis in cancer cells while sparing normal cells. Although many cancers are sensitive to TRAIL-induced apoptosis, some evade the proapoptotic effects of TRAIL. Therefore, differentiating molecular mechanisms that distinguish between TRAIL-sensitive and TRAIL-resistant tumors are essential for effective cancer therapies. Here, we show that c-Fos functions as a proapoptotic agent by repressing the antiapoptotic molecule c-FLIP(L). c-Fos binds the c-FLIP(L) promoter, represses its transcriptional activity, and reduces c-FLIP(L) mRNA and protein levels. Therefore, c-Fos is a key regulator of c-FLIP(L), and activation of c-Fos determines whether a cancer cell will undergo cell death after TRAIL treatment. Strategies to activate c-Fos or inhibit c-FLIP(L) may potentiate TRAIL-based proapoptotic therapies.

miR-200c Targets CDK2 and Suppresses Tumorigenesis in Renal Cell Carcinoma.

miRNA expression profiles are widely investigated in the major cancers, but their specific roles and functions in cancers have not yet to be fully elucidated. In this study, miRNA expression profiles were determined in clear cell renal cell carcinomas (ccRCC) and in matched normal kidney tissues by using a miRNA microarray platform which covers a total of 851 human miRNAs. Differential expression of 74 miRNAs were identified between ccRCC specimens and their matched adjacent noncancerous tissues, of which 30 were significantly upregulated in ccRCCs, and the other 44 were downregulated (fold change ≥ 2, P < 0.05). Interestingly, miR-200c was commonly downregulated in ccRCC specimens and ccRCC cell lines with significant functional consequences. Growth curve and FACS assay indicated that overexpression of miR-200c suppressed cell growth and induced cell-cycle arrest at G0–G1 phases in SN12-PM6 and 786-O cells. Furthermore, miR-200c could suppress in vivo tumor growth of SN12-PM6 cells in mice. Bioinformatics exposed cyclin-dependent kinase 2 (CDK2) as a potential target of miR-200c, which was validated using a luciferase reporter assay. Mechanistic investigations revealed that miR-200c was directly responsible for suppressing the expression of CDK2 in ccRCC cell lines and xenografts. Taken together, miR-200c plays an antioncogenic role in ccRCC, through controlling cell growth and cell-cycle progression by downregulating the G1–S regulator CDK2. Implications: miR-200c exerts its novel antioncogenic function in renal cell carcinoma by controlling CDK2-dependent cell growth and cell-cycle progression. Mol Cancer Res; 13(12); 1567–77. ©2015 AACR.

Hepatitis C virus NS3 protein enhances hepatocellular carcinoma cell invasion by promoting PPM1A ubiquitination and degradation

BackgroundGrowing evidence suggests that hepatitis C virus (HCV) contributes to hepatocellular carcinoma (HCC) by directly modulating oncogenic signaling pathways. Protein phosphatase magnesium-dependent 1A (PPM1A) has recently emerged as an important tumor suppressor as it can block a range of tumor-centric signaling pathways through protein dephosphorylation. However, the role and regulatory mechanisms of PPM1A in HCV-infected cells have not been reported.MethodsTotal, cytoplasmic, and nuclear PPM1A protein after HCV infection or overexpression of HCV nonstructural protein 3 (NS3) were detected by western blotting. The expression of PPM1A in normal liver and HCV-related HCC tissues was quantified by immunohistochemistry. The effects of HCV infection and NS3 expression on the PPM1A protein level were systematically analyzed, and the ubiquitination level of PPM1A was determined by precipitation with anti-PPM1A and immunoblotting with either anti-ubiquitin or anti-PPM1A antibody. Finally, the roles of NS3 and PPM1A in hepatoma cell migration and invasion were assessed by wound healing and transwell assays, respectively.ResultsHCV infection and replication decreased PPM1A abundance, mediated by NS3, in hepatoma cells. Compared to normal liver tissues, the expression of PPM1A was significantly decreased in the HCC tumor tissues and adjacent non-tumor tissues. NS3 directly interacted with PPM1A to promote PPM1A ubiquitination and degradation, which was dependent on its protease domain. Blockade of PPM1A through small interfering RNA significantly promoted HCC cell migration, invasion, and epithelial mesenchymal transition (EMT), which were further intensified by TGF-β1 stimulation, in vitro. Furthermore, restoration of PPM1A abrogated the NS3-mediated promotion of HCC migration and invasion to a great extent, which was dependent on its protein phosphatase function.ConclusionsOur findings demonstrate that the HCV protein NS3 can downregulate PPM1A by promoting its ubiquitination and proteasomal degradation, which might contribute to the migration and invasion of hepatoma cells and may represent a new strategy of HCV in carcinogenesis.

miR-144-3p as a novel plasma diagnostic biomarker for clear cell renal cell carcinoma.

OBJECTIVES Clear cell renal cell carcinoma (ccRCC) is the most frequent and lethal subtype of renal cell carcinoma, whose most effective measure of curing remains diagnosis and nephrectomy in its early phase. However, there is no feasible and recognized plasma biomarker for the clinical diagnosis of ccRCC. The objective of this study is to identify a novel plasma microRNA (miRNA) acting as an efficient diagnostic plasma biomarker in ccRCC. METHODS AND MATERIALS The plasma miRNA expression profile was quantified by miRNA microarray. Validation of miRNA levels of plasmas and tissues were performed by quantitative reverse transcription polymerase chain reaction in 106 ccRCC, 28 renal angiomyolipomas (AML), and 123 healthy control plasmas and in 110 ccRCC tissues. RESULTS We found that plasma miR-144-3p levels in 106 ccRCC plasmas were remarkably up-regulated compared with that in healthy individuals and in patients with AML. miR-144-3p served as a promising plasma biomarker for yielding an area under the receiver operating characteristic curve of 0.91 with 87.10% sensitivity and 83.02% specificity in discriminating ccRCC from healthy individuals, and an area under the curve of 0.82 with 75.00% sensitivity and 71.70% specificity in discriminating ccRCC from patients with AML. In addition, plasma miR-144-3p levels were significantly decreased after surgery in 106 patients with ccRCC. Next, we examined miR-144-3p levels in 110 human ccRCC tissues, and found that miR-144-3p levels in ccRCC tissues were increased compared with adjacent normal tissues. Pearson correlation analysis revealed that miR-144-3p levels in tumor tissues were positively correlated with preoperative plasma miR-144-3p levels in the matched samples from patients with ccRCC. In addition, the miR-144-3p levels in ccRCC plasmas and tissues were increased in patients with advanced pT stage. CONCLUSIONS Our data indicate that miR-144-3p, which is significantly up-regulated in ccRCC plasmas and tissues, particularly with advanced pT stage, is a novel and excellent plasma biomarker for the diagnosis of ccRCC.

Protein phosphatase 1 suppresses androgen receptor ubiquitylation and degradation

The phosphoprotein phosphatases are emerging as important androgen receptor (AR) regulators in prostate cancer (PCa). We reported previously that the protein phosphatase 1 catalytic subunit (PP1α) can enhance AR activity by dephosphorylating a site in the AR hinge region (Ser650) and thereby decrease AR nuclear export. In this study we show that PP1α increases the expression of wildtype as well as an S650A mutant AR, indicating that it is acting through one or more additional mechanisms. We next show that PP1α binds primarily to the AR ligand binding domain and decreases its ubiquitylation and degradation. Moreover, we find that the PP1α inhibitor tautomycin increases phosphorylation of AR ubiquitin ligases including SKP2 and MDM2 at sites that enhance their activity, providing a mechanism by which PP1α may suppress AR degradation. Significantly, the tautomycin mediated decrease in AR expression was most pronounced at low androgen levels or in the presence of the AR antagonist enzalutamide. Consistent with this finding, the sensitivity of LNCaP and C4–2 PCa cells to tautomycin, as assessed by PSA synthesis and proliferation, was enhanced at low androgen levels or by treatment with enzalutamide. Together these results indicate that PP1α may contribute to stabilizing AR protein after androgen deprivation therapies, and that targeting PP1α or the AR-PP1α interaction may be effective in castration-resistant prostate cancer (CRPC).

Mir-144-3p Promotes Cell Proliferation, Metastasis, Sunitinib Resistance in Clear Cell Renal Cell Carcinoma by Downregulating ARID1A

Background/Aims: We previously performed microRNA (miRNA) microarray to identify effective indicators of clear cell renal cell carcinoma (ccRCC) tissue samples and preoperative/postoperative plasma in which we identified miR-144-3p as an oncomiRNA. However, the molecular mechanism of miR-144-3p remains unclear. This study aims to explore the roles of miR-144-3p in the invasion, migration and Sunitinib-resistance in ccRCC and to elucidate the underlying mechanisms. Methods: Gain and loss of function approaches were used to investigate the cell proliferation, cycle distribution, clonogenicity, migration, invasion, chemosensitivity of miR-144-3p in vitro. The xenograft model was used to assess the effects of miR-144-3p overexpression on tumorigenesis. Bioinformatics analysis and dual-luciferase reporter assay were used to indentify AT-rich interactive domain 1A (ARID1A) as a direct target gene of miR-144-3p. Quantitative RT-PCR, Western blotting, and immunohistochemical (IHC) staining were used to explore ARID1A expression level of the mRNA and protein. Results: We found that miR-144-3p overexpression enhanced cell proliferation, clonogenicity, migration, invasion, and chemoresistance in ccRCC cells. Notably, the oncotumor activities of miR-144-3p were mediated by repressing the expression of ARID1A. The downregulation of ARIDIA could promote the function of miR-144-3p in cell proliferation, metastasis and chemoresistance. Consistently, ARID1A mRNA and protein levels were decreased in ccRCC and in nude mice, and they negatively correlated with miR-144-3p. Conclusion: Higher miR-144-3p may enhance malignancy and resistance to Sunitinib in ccRCC by targeting ARID1A, the observations may uncover novel strategies of ccRCC treatment.

Valproic acid attenuates skeletal muscle wasting by inhibiting C/EBPβ-regulated atrogin1 expression in cancer cachexia

Muscle wasting is the hallmark of cancer cachexia and is associated with poor quality of life and increased mortality. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has important biological effects in the treatment of muscular dystrophy. To verify whether VPA could ameliorate muscle wasting induced by cancer cachexia, we explored the role of VPA in two cancer cachectic mouse models [induced by colon-26 (C26) adenocarcinoma or Lewis lung carcinoma (LLC)] and atrophied C2C12 myotubes [induced by C26 cell conditioned medium (CCM) or LLC cell conditioned medium (LCM)]. Our data demonstrated that treatment with VPA increased the mass and cross-sectional area of skeletal muscles in tumor-bearing mice. Furthermore, treatment with VPA also increased the diameter of myotubes cultured in conditioned medium. The skeletal muscles in cachectic mice or atrophied myotubes treated with VPA exhibited reduced levels of CCAAT/enhancer binding protein beta (C/EBPβ), resulting in atrogin1 downregulation and the eventual alleviation of muscle wasting and myotube atrophy. Moreover, atrogin1 promoter activity in myotubes was stimulated by CCM via activating the C/EBPβ-responsive cis-element and subsequently inhibited by VPA. In contrast to the effect of VPA on the levels of C/EBPβ, the levels of inactivating forkhead box O3 (FoxO3a) were unaffected. In summary, VPA attenuated muscle wasting and myotube atrophy and reduced C/EBPβ binding to atrogin1 promoter locus in the myotubes. Our discoveries indicate that HDAC inhibition by VPA might be a promising new approach for the preservation of skeletal muscle in cancer cachexia.

Inhibition of pp2a activity confers a trail-sensitive phenotype during malignant transformation

TRAIL is a promising anticancer agent because it induces apoptosis in the majority of human cancer cells but spares the normal cells. To determine the mechanistic nature of how normal cells acquire a TRAIL-sensitive phenotype during the process of malignant transformation, an experimental cell system was developed by sequential introduction of human telomerase reverse transcriptase and SV40 T antigens (large and small) into normal human prostatic epithelial cells (PrEC). This model system demonstrated that inhibition of protein phosphatase 2A (PP2A), either by SV40 small T antigen, okadaic acid, Calyculin A, or PP2A catalytic subunit siRNA, sensitized normal human PrEC and immortalized cells to TRAIL-induced apoptosis. Moreover, sensitization occurred during the premalignant period of tumorigenesis and PP2A exerted its antiapoptotic activity by negatively regulating c-Fos/AP-1. In addition, low-dose okadaic acid treatment sensitized TRAIL-resistant cancer cells to TRAIL, suggesting that PP2A inhibitors could be used as an enhancer of apoptosis induced by TRAIL or TRAIL-like agents. These data indicate that downregulation of PP2A activity is a critical step for normal cells to acquire a TRAIL-sensitive phenotype during tumorigenesis and that the level of PP2A activity may foretell cellular sensitivity to TRAIL-induced apoptosis. Implications: Inhibition of PP2A is a key determinant in acquiring TRAIL sensitivity during tumorigenesis, with c-Fos/AP-1 as an essential mediator. Mol Cancer Res; 12(2); 217–27. ©2013 AACR.