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Bladder cancer (BC) ranks as the sixth most common cancer in the United States and is the leading cause of death in patients with urinary malignancies. p63 is a member of the p53 family and is believed to function as a tumor suppressor in human BCs. Our most recent studies revealed a previously unknown function of the RING of XIAP in promoting microRNA 4295 (miR-4295) transcription, thereby reducing p63α protein translation and enhancing normal urothelial transformation, whereas p63α upregulates hsp70 transcription, subsequently activating the HSP70/Wasf3/Wave3/matrix metalloproteinase 9 (MMP-9) axis and promoting BC cell invasion via initiating the transcription factor E2F1. In this study, we found that p63α inhibited cyclin D1 protein expression, subsequently decreasing the ability of BC cell anchorage-independent growth in vitro and tumorigenicity in vivo Mechanistic studies demonstrated that p63α expression is able to downregulate cyclin D1 gene transcription through attenuation of c-Myc mRNA stability. We further show that the reduction of miR-141-3p expression by p63α directly releases its inhibition of 3' untranslated region (UTR) activity of AU-rich element RNA-binding factor 1 (AUF1) mRNA, thereby increasing AUF1 protein translation and further resulting in degradation of c-Myc mRNA, which, in turn, reduces cyclin D1 gene transcription and BC cell anchorage-independent growth. Collectively, our results demonstrate that p63α is a negative regulator of BC cell tumorigenic growth, a distinctly different function than its promotion of BC invasion, thus providing further new insight into the "two faces" of p63α in regulation of BC cell tumorigenic growth and progression/invasion., (Copyright © 2018 American Society for Microbiology.)

Bladder cancer (BC) is the sixth most common cancer in the United States and is the number one cause of death among patients with urinary system malignancies. This makes the identification of invasive regulator(s)/effector(s) as the potential therapeutic targets for managing BC a high priority. p63 is a member of the p53 family of tumor suppressor genes/proteins, plays a role in the differentiation of epithelial tissues, and is believed to function as a tumor suppressor. However, it remains unclear whether and how p63 functions in BC cell invasion after tumorigenesis. Here, we show that p63α protein levels were much higher in mouse high-invasive BC tissues than in normal tissues. Our results also revealed that p63α is crucial for heat shock protein 70 (Hsp70) expression and subsequently increases the ability of BC invasion. Mechanistic experiments demonstrated that p63α can transcriptionally up-regulate Hsp70 expression, thereby promoting BC cell invasion via the Hsp70/Wasf3/Wave3/MMP-9 axis. We further show that E2F transcription factor 1 (E2F1) mediates p63α overexpression-induced Hsp70 transcription. We also found that p63α overexpression activates E2F1 transcription, which appears to be stimulated by p63α together with E2F1. Collectively, our results demonstrate that p63α is a positive regulator of BC cell invasion after tumorigenesis, providing significant insights into the biological function of p63α in BC and supporting the notion that p63α might be a potential target for invasive BC therapy.

The X-linked inhibitor of apoptosis protein (XIAP) contains three N-terminal BIR domains that mediate anti-apoptosis and one C-terminal RING finger domain whose function(s) are not fully defined. Here we show that the RING domain of XIAP strongly inhibits the expression of p63α, a known tumor suppressor. XIAP knockdown in urothelial cells or RING deletion in knockin mice markedly upregulates p63α expression. This RING-mediated p63α downregulation is critical for the malignant transformation of normal urothelial cells following EGF treatment. We further show that the RING domain promotes Sp1-mediated transcription of miR-4295 which targets the 3'UTR of p63α mRNA and consequently inhibits p63α translation. Our results reveal a previously unknown function of the RING of XIAP in promoting miR-4295 transcription, thereby reducing p63α translation and enhancing urothelial transformation. Our data offer novel insights into the multifunctional effects of the XIAP RING domain on urothelial tumorigenesis and the potential for targeting this frequently overexpressed protein as a therapeutic alternative., Competing Interests: The authors declare no conflicts of interest.

p27 is an atypical tumor suppressor that can regulate the activity of cyclin-dependent kinases and G(0)-to-S phase transitions. More recent studies reveal that p27 may also exhibit its tumor-suppressive function through regulating many other essential cellular events. However, the molecular mechanisms underlying these anticancer effects of p27 are largely unknown. In this study, we found that depletion of p27 expression by either gene knock-out or knockdown approaches resulted in up-regulation of both Hsp27 and Hsp70 expression at mRNA- and promoter-derived transcription as well as protein levels upon arsenite exposure, indicating that p27 provides a negative signal for regulating the expression of Hsp27 and Hsp70. Consistently, arsenite-induced activation of JNK2/c-Jun and HSF-1 pathways was also markedly elevated in p27 knock-out (p27(-/-)) and knockdown (p27 shRNA) cells. Moreover, interference with the expression or function of JNK2, c-Jun, and HSF-1, but not JNK1, led to dramatic inhibition of arsenite-induced Hsp27 and Hsp70 expression. Collectively, our results demonstrate that p27 suppresses Hsp27 and Hsp70 expression at the transcriptional level specifically through JNK2/c-Jun- and HSF-1-dependent pathways upon arsenite exposure, which provides additional important molecular mechanisms for the tumor-suppressive function of p27.

The chemopreventive properties of edible berries have been demonstrated both in vitro and in vivo, however, the specific molecular mechanisms underlying their anti-cancer effects are largely unknown. Our previous studies have shown that a methanol extract fraction of freeze-dried black raspberries inhibits benzoapyrene (BaP)-induced transformation of Syrian hamster embryo cells. This fraction also blocks activation of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB) induced by benzoapyrene diol-epoxide (BaPDE) in mouse epidermal JB6 Cl 41 cells. To determine if different berry types exhibit specific mechanisms for their anti-cancer effects, we compared the effects of extract fractions from both black raspberries and strawberries on BaPDE-induced activation of various signaling pathways in Cl 41 cells. Black raspberry fractions inhibited the activation of AP-1, NF-kappaB, and nuclear factor of activated T cells (NFAT) by BaPDE as well as their upstream PI-3K/Akt-p70(S6K) and mitogen-activated protein kinase pathways. In contrast, strawberry fractions inhibited NFAT activation, but did not inhibit the activation of AP-1, NF-kappaB or the PI-3K/Akt-p70(S6K) and mitogen-activated protein kinase pathways. Consistent with the effects on NFAT activation, tumor necrosis factor-alpha (TNF-alpha) induction by BaPDE was blocked by extract fractions of both black raspberries and strawberries, whereas vascular endothelial growth factor (VEGF) expression, which depends on AP-1 activation, was suppressed by black raspberry fractions but not strawberry fractions. These results suggest that black raspberry and strawberry components may target different signaling pathways in exerting their anti-carcinogenic effects., ((c) 2007 Wiley-Liss, Inc.)

Berries have attracted attention for their chemopreventive activities in last a few years. Dietary freeze-dried blackberries have been shown to reduce esophagus and colon cancer development induced by chemical carcinogen in rodents. To elucidate molecular mechanisms involved in chemoprevention by berry extracts, we employed mouse epidermal Cl 41 cell line, a well-characterized in vitro model in tumor promotion studies. Pretreatment of Cl 41 cells with methanol-extracted blackberry fraction RO-ME resulted in a dramatical inhibition of B(a)PDE-induced activation of AP-1 and NFkB, and expression of VEGF and COX-2. The inhibitory effects of RO-ME on B(a)PDE-induced activation of AP-1 and NFkappaB appear to be mediated via inhibition of MAPKs and IkappaBalpha phosphorylation, respectively. In view of the important roles of AP-1, NFkappaB, VEGF and COX-2 in tumor promotion/progression, and VEGF and COX-2 are target of AP-1 and NFkappaB, we anticipate that the ability of black raspberries to inhibit tumor development may be mediated by impairing signal transduction pathways leading to activation of AP-1 and NFkappaB, subsequently resulting in down-regulation of VEGF and COX-2 expression. The RO-ME fraction appears to be the major fraction responsible for the inhibitory activity of black raspberries.

Nickel compounds are a somewhat unique class of carcinogens. Previous studies have demonstrated that NiCl(2) exposure leads to marked induction of hypoxia inducible factor 1 (HIF-1) in human osteosarcoma and BALB/c 3T3 cells, a transcription factor that has been considered to play an important role in tumor promotion and progression. However, the signal transduction pathways leading to HIF-1 induction are not well understood. The present study indicated that exposure of mouse epidermal Cl41 cells to either Ni(3)S(2) or NiCl(2) resulted in activation of phosphatidylinositol 3-kinase (PI-3K), Akt, and p70 S6 kinase (p70(S6k)). Inhibition of PI-3K, Akt, and p70(S6k) by overexpression of a dominant-negative mutant of PI-3K (Deltap85) impaired nickel-induced HIF-1 transactivation. Furthermore, an overexpression of the dominant-negative Akt mutant (Akt-T308A/S473A) blocked nickel-induced Akt phosphorylation and HIF-1 transactivation, whereas inhibition of p70(S6k) activation by pretreatment of cells with rapamycin did not show significant inhibitory effects on HIF-1 transactivation induced by nickel compounds. Consistent with HIF-1 transactivation, inhibition of the PI-3K/Akt pathway by either overexpression of Deltap85 or Akt-T308A/S473A caused dramatic inhibition of Cap43 protein expression induced by nickel compounds, whereas pretreatment of cells with rapamycin did not exhibit inhibition of Cap43 induction. These results demonstrated that nickel compounds induce HIF-1 transactivation and Cap43 protein expression through a PI-3K/Akt-dependent and p70(S6k)-independent pathway. This study should help us understand the signal transduction pathways involved in the carcinogenic effects of nickel compounds.

Activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) are two important transcription factors responsible for the regulation of cytokines, which are involved in cell proliferation and inflammation. Coal workers' pneumoconiosis (CWP) is an occupational lung disease that may be related to chronic inflammation caused by coal dust exposure. In the present study, we demonstrate that coal from the Pennsylvania (PA) coalmine region, which has a high prevalence of CWP, can activate both AP-1 and NFAT in JB6 mouse epidermal cells. In contrast, coal from the Utah (UT) coalmine region, which has a low prevalence of CWP, has no such effects. The PA coal stimulates mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-Jun-NH(2)-terminal kinases, as determined by the phosphorylation assay. The increase in AP-1 by the PA coal was completely eliminated by the pretreatment of cells with PD98059, a specific MAPK kinase inhibitor, and SB202190, a p38 kinase inhibitor, further confirming that the PA coal-induced AP-1 activation is mediated through ERKs and p38 MAPK pathways. Deferoxamine (DFO), an iron chelator, synergistically enhanced the PA coal-induced AP-1 activity, but inhibited NFAT activity. For comparison, cells were treated with ferrous sulfate and/or DFO. We have found that iron transactivated both AP-1 and NFAT, and DFO further enhanced iron-induced AP-1 activation but inhibited NFAT. These results indicate that activation of AP-1 and NFAT by the PA coal is through bioavailable iron present in the coal. These data are in agreement with our previous findings that the prevalence of CWP correlates well with levels of bioavailable iron in coals from various mining regions.

The present study investigated the role of reactive oxygen species (ROS) in activation of nuclear factor of activated T cells (NFAT), a pivotal transcription factor responsible for regulation of cytokines, by asbestos in mouse embryo fibroblast PW cells. Exposure of cells to asbestos led to the transactivation of NFAT in a time- and dose-dependent manner. Scavenging of asbestos-induced H2O2 with N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H2O2 inhibitor) resulted in inhibition of NFAT activation. In contrast, an increase in H2O2 generation by the addition of superoxide dismutase (SOD) slightly enhanced asbestos-induced NFAT activation. In addition, pretreatment of cells with sodium formate did not exhibit any inhibition of NFAT activity induced by asbestos. These results demonstrated that H2O2 appeared to play an important role in asbestos-induced NFAT transactivation. Furthermore, it was observed that incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) not only resulted in NFAT activation by itself, but also enhanced asbestos-induced NFAT induction. Pretreatment of cells with cyclosporin A (CSA), a pharmacological inhibitor of the phosphatase calcineurin, blocked both asbestos- and TPA plus asbestos-induced NFAT activation. These data suggest that asbestos is able to induce NFAT activation through H2O2-dependent and CSA-sensitive pathways, which may be involved in asbestos-induced carcinogenesis.

Growth arrest and DNA damage (GADD) 45α is a member of GADD inducible gene family, and is inducible in cell response to oxidative stress. GADD45α upregulation induces MKK4/JNK activation in some published experimental systems. However, we found here that the depletion of GADD45α (GADD45α−/−) in mouse embryonic fibroblasts (MEFs) resulted in an increase in the phosphorylation of MKK4/7, MKK3/6 and consequently specific up-regulated the activation of JNK/p38 and their downstream transcription factors, such as c-Jun and ATF2, in comparison to those in GADD45α+/+ MEFs cell following nickel exposure. This up-regulation of MKK-JNK/p38 pathway in GADD45α−/− cell could be rescued by the reconstitutional expression of HA-GADD45α in GADD45α−/− MEFs, GADD45α−/−(HA-GADD45α). Subsequent studies indicated that GADD45α deletion repressed expression of PP2Cα, the phosphotase of MKK3/6 and MKK4/7, whereas ectopic expression of HA-PP2Cα in GADD45α−/− cells attenuated activation of MKK3/6-p38 and MKK4/7-JNK pathways. Collectively, our results demonstrate a novel function and mechanism responsible for GADD45α regulation of MKK/MAPK pathway, further provides insight into understanding the big picture of GADD45α in the regulation of cellular responses to oxidative stress and environmental carcinogens. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor-1α (HIF-1α) has been reported to regulate over 100 gene expressions in response to hypoxia and other stress conditions. In the present study, we found that arsenite could induce HIF-1α protein accumulation in both mouse epidermal Cl41 cells and mouse embryonic fibroblasts (MEFs). Knockout of p85α, a regulatory subunit of PI-3K, in MEFs (p85α) dramatically decreased the arsenite-induced HIF-1α accumulation, indicating that p85α is crucial for arsenite effects on the stabilization of HIF-1α protein. Our further studies suggest that arsenite could induce inducible Hsp70 expression, and transfection of inducible Hsp70 into p85α MEFs could restore HIF-1α protein accumulation. Moreover, the results using EMSA and Supershift assays indicate that p85α is crucial for arsenite-induced activation of the heat-shock transcription factor 1 (HSF-1), which is responsible for transcription of inducible Hsp70. Taken together, p85α-mediated HIF-1α stabilization upon arsenite exposure is specifically through HSF-1 activation and subsequent up-regulation of the inducible Hsp70 expression. [ABSTRACT FROM AUTHOR]

The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including cell differentiation, proliferation, apoptosis and oncogenic transformation. It is thought that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 activity is because of the activation of the PKC/MAPK/AP-1 pathway, although the detailed molecular mechanism has not been fully characterized. The tyrosine kinases of epidermal growth factor receptor (EGFR) lie at the head of a complex of signal transduction cascade that modulates cell proliferation, survival, adhesion, migration and differentiation. Currently, little is known about whether EGFR or its tyrosine kinase is necessary for TPA-induced AP-1 activation. In the present study, we investigated this issue using a well-characterized mouse fibroblast B82 cell line, which is devoid of the EGFR, and its stable transfectants with either wild-type EGFR (B82L) or tyrosine kinase-deficient EGFR (mutation at Lys-721) (B82M721). We demonstrated that the TPA or epidermal growth factor (EGF) induced AP-1 activation in the B82L cells that express wild-type EGFR, but not in the B82 cell, whereas autophosphorylation at tyrosine1173 of EGFR in B82L cells was only induced by EGF, but not TPA. The expression of tyrosine kinase-deficient EGFR (mutation at Lys-721) (B82M721) resulted in deficiency of AP-1 induction in cellular response to EGF, while TPA treatment led to fully AP-1 activation. Furthermore, the mutation at Lys-721 of EGFR resulted in impairing of EGFR autophosphorylation at tyrosine1173 induced by EGF. Based on these results, we conclude that TPA-induced AP-1 activation requires the basal level-EGFR protein, but not EGFR tyrosine kinase and EGFR autophosphorylation at tyrosine1173, whereas both EGFR tyrosine kinase and EGFR autophosphorylation at Y1173 play a critical role in EGF-induced AP-1 activation.Oncogene (2003) 22, 211–219.... [ABSTRACT FROM AUTHOR]

Programmed cell death protein 1 (PD-1) and its ligand PD-L1 blockade have been identified to target immune checkpoints to treat human cancers with durable clinical benefit. Several studies reveal that the response to PD-1-PD-L1 blockade might correlate with PD-L1 expression levels in tumor cells. However, the mechanistic pathways that regulate PD-L1 protein expression are not understood. Here, we reported that PD-L1 protein is regulated by ATG7-autophagy with an ATG7-initiated positive feedback loop in bladder cancer (BC). Mechanistic studies revealed that ATG7 overexpression elevates PD-L1 protein level mainly through promoting autophagy-mediated degradation of FOXO3a, thereby inhibiting its initiated miR-145 transcription. The lower expression of miR-145 increases pd-l1 mRNA stability due to the reduction of its direct binding to 3′-UTR of pd-l1 mRNA, in turn leading to increasing in pd-l1 mRNA stability and expression, and finally enhancing stem-like property and invasion of BC cells. Notably, overexpression of PD-L1 in ATG7 knockdown cells can reverse the defect of autophagy activation, FOXO3A degradation, and miR-145 transcription attenuation. Collectively, our results revealed a positive feedback loop to promoting PD-L1 expression in human BC cells. Our study uncovers a novel molecular mechanism for regulating pd-l1 mRNA stability and expression via ATG7/autophagy/FOXO3A/miR-145 axis and reveals the potential for using combination treatment with autophagy inhibitors and PD-1/PD-L1 immune checkpoint blockade to enhance therapeutic efficacy for human BCs. [ABSTRACT FROM AUTHOR]

Epithelial mesenchymal transition (EMT) is highly correlated with metastasis during cancer development. Although previous studies have revealed that ISO is able to inhibit cancer cell invasion and stem-cell properties, little is known about the effects of ISO on EMT markers. The present study explores the potential regulation of ISO on EMT, leading to the inhibition of migration and invasion of bladder cancer cells. We found that ISO inhibited Vimentin, one of the EMT markers, in the invasive bladder cancer cell lines U5637 and T24T. ISO reduced Vimentin protein level by increasing the expression of METTL14. On the other hand, ISO upregulated the METTL14 mRNA by activating the transcription factor FOXO3a. The results demonstrate that ISO inhibits invasion by affecting the EMT marker and offer a novel insight into understanding the upregulation of METTL14 by ISO. • The effect on EMT and its potential mechanisms of ISO is first explored. • ISO treatment significantly upregulated the protein expression of METTL14 via activating its promoter. • ISO treatment inhibited the Vimentin expression by increasing the expression of METTL14. • The study of the effect on anti-cancer by ISO provides novel insight against BC. [ABSTRACT FROM AUTHOR]