Pomegranate extract inhibits EMT in clear cell renal cell carcinoma in a NF-κB and JNK dependent manner.

OBJECTIVE: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma (RCC) and is characterized by biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. One effect of VHL inactivation is hypoxia inducible factor alpha (HIFα)-independent constitutive activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK). Both NF-κB and JNK drive ccRCC growth and epithelial to mesenchymal transition (EMT). The purpose of this study was to determine the biochemical effects of pomegranate juice extracts (PE) on RCC cell lines. METHODS: The pre-clinical effects of PE on NF-κB, JNK, and the EMT phenotype were assayed, including its effect on proliferation, anchorage-independent growth, and invasion of pVHL-deficient RCCs. RESULTS: PE inhibits the NF-κB and JNK pathways and consequently inhibits the EMT phenotype of pVHL-deficient ccRCCs. The effects of PE are concentration-dependent and affect not only biochemical markers of EMT (i.e., cadherin expression) but also functional manifestations of EMT, such as invasion. These effects are manifested within days of exposure to PE when diluted 2000-fold. Highly dilute concentrations of PE (106 dilution), which do not impact these pathways in the short term, were found to have NF-κB and JNK inhibitory effects and ability to reverse the EMT phenotype following prolonged exposure. CONCLUSION: These findings suggest that PE may mediate inhibition growth of pVHL-deficient ccRCCs and raises the possibility of its use as a dietary adjunct to managing patients with active surveillance for small, localized, incidentally identified renal tumors so as to avoid more invasive procedures such as nephrectomy.

Hyperactivated JNK is a therapeutic target in pVHL-deficient renal cell carcinoma.

Clear cell renal cell carcinomas (RCC), the major histologic subtype of RCC accounting for more than 80% of cases, are typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Although accumulation of hypoxia-inducible factor alpha (HIF-α) is the most well-studied effect of VHL inactivation, direct inhibition of HIFα or restoration of wild-type pVHL protein expression has not proved readily feasible, given the limitations associated with pharmacologic targeting of transcription factors (i.e., HIF-α) and gene replacement therapy of tumor suppressor genes (i.e., VHL). Here, we have established that phosphorylated c-Jun, a substrate of the c-Jun-NH(2)-kinase (JNK), is selectively activated in clear cell RCC patient specimens. Using multiple isogenic cell lines, we show that HIF-α-independent JNK hyperactivation is unique to the pVHL-deficient state. Importantly, pVHL-deficient RCCs are dependent upon JNK activity for in vitro and in vivo growth. A multistep signaling pathway that links pVHL loss to JNK activation involves the formation of a CARD9/BCL10/TRAF6 complex as a proximal signal to sequentially stimulate TAK1 (MAPKKK), MKK4 (MAPKK), and JNK (MAPK). JNK stimulates c-Jun phosphorylation, activation, and dimerization with c-Fos to form a transcriptionally competent AP1 complex that drives transcription of the Twist gene and induces epithelial-mesenchymal transition. Thus, JNK represents a novel molecular target that is selectively activated in and drives the growth of pVHL-deficient clear cell RCCs. These findings can serve as the preclinical foundation for directed efforts to characterize potent pharmacologic inhibitors of the JNK pathway for clinical translation.

p120-catenin is transcriptionally downregulated by FOXC2 in non-small cell lung cancer cells.

p120-catenin (p120ctn) plays a major role in cell adhesion and motility through the regulation of E-cadherin and interaction with RhoGTPase and Rac1. p120ctn is downregulated in several malignancies including non-small cell lung cancer (NSCLC). Here, we investigated transcriptional regulation of p120ctn in NSCLC. We cloned a 1,400-bp amplicon of chromosome 11 from position -1,082 to +320 relative to the transcription start site into a firefly luciferase reporter vector and prepared serial deletion constructs to pinpoint cis-acting elements involved in the regulation of p120ctn. We transfected NSCLC cell lines and immortalized normal human respiratory epithelial cells with the abovementioned constructs. We found reduced p120ctn promoter activity, protein level, and mRNA message in lung cancer cells compared with noncancerous immortalized lung epithelial cells. Serial deletion analysis of p120ctn promoter identified a region between positions +267 and +282, which mediated the transcriptional repression of p120ctn. This region harbored putative binding sites for FOXC2 and FOXL1 transcription factors. Direct binding of FOXC2 to the p120ctn promoter between positions +267 and +282 was confirmed by electromobility shift assay. RNAi-mediated silencing of FOXC2 in A549, H157, and H358 cells resulted in increasing p120ctn promoter activity as well as mRNA and protein levels. Finally, silencing FOXC2 in these NSCLC cells enhanced E-cadherin level, which was reversed by simultaneous silencing of p120ctn. In summary, our data support the notion that FOXC2 mediates the transcriptional repression of p120ctn in NSCLC.

NF-kappaB-dependent plasticity of the epithelial to mesenchymal transition induced by Von Hippel-Lindau inactivation in renal cell carcinomas.

The critical downstream signaling consequences contributing to renal cancer as a result of loss of the tumor suppressor gene von Hippel-Lindau (VHL) have yet to be fully elucidated. Here, we report that VHL loss results in an epithelial to mesenchymal transition (EMT). In studies of paired isogenic cell lines, VHL silencing increased the levels of N-cadherin and vimentin and reduced the levels of E-cadherin relative to the parental VHL(+) cell line, which displayed the opposite profile. VHL(+) cells grew as clusters of cuboidal and rhomboid cells, whereas VHL-silenced cells took on an elongated, fibroblastoid morphology associated with a more highly invasive character in Matrigel chamber assays. Based on earlier evidence that VHL loss can activate NF-kappaB, a known mediator of EMT, we tested whether NF-kappaB contributed to VHL-mediated effects on EMT. On pharmacologic or molecular inhibition of NF-kappaB, VHL-silenced cells regained expression of E-cadherin, lost expression of N-cadherin, and reversed their highly invasive phenotype. Introducing a pVHL-resistant hypoxia-inducible factor 1alpha (HIF1alpha) mutant (HIFalpha(M)) into VHL(+) cells heightened NF-kappaB activity, phenocopying EMT effects produced by VHL silencing. Conversely, inhibiting the heightened NF-kappaB activity in this setting reversed the EMT phenotype. Taken together, these results suggest that VHL loss induces an EMT that is largely dependent on HIFalpha-induced NF-kappaB. Our findings rationalize targeting the NF-kappaB pathway as a therapeutic strategy to treat renal tumors characterized by biallelic VHL inactivation.

Inactivation of the CYLD deubiquitinase by HPV E6 mediates hypoxia-induced NF-kappaB activation.

The biochemical mechanisms that underlie hypoxia-induced NF-kappaB activity have remained largely undefined. Here, we find that prolonged hypoxia-induced NF-kappaB activation is restricted to cancer cell lines infected with high-risk human papillomavirus (HPV) serotypes. The HPV-encoded E6 protein is necessary and sufficient for prolonged hypoxia-induced NF-kappaB activation in these systems. The molecular target of E6 in the NF-kappaB pathway is the CYLD lysine 63 (K63) deubiquitinase, a negative regulator of the NF-kappaB pathway. Specifically, hypoxia stimulates E6-mediated ubiquitination and proteasomal degradation of CYLD. Given the established role of NF-kappaB in human carcinogenesis, these findings provide a potential molecular/viral link between hypoxia and the adverse clinical outcomes observed in HPV-associated malignancies.

Pomegranate extract inhibits androgen-independent prostate cancer growth through a nuclear factor-kappaB-dependent mechanism.

Constitutive nuclear factor-kappaB (NF-kappaB) activation is observed in androgen-independent prostate cancer and represents a predictor for biochemical recurrence after radical prostatectomy. Dietary agents such as pomegranate extract (PE) have received increasing attention as potential agents to prevent the onset or progression of many malignancies, including prostate cancer. Here, we show that PE inhibited NF-kappaB and cell viability of prostate cancer cell lines in a dose-dependent fashion in vitro. Importantly, maximal PE-induced apoptosis was dependent on PE-mediated NF-kappaB blockade. In the LAPC4 xenograft model, PE delayed the emergence of LAPC4 androgen-independent xenografts in castrated mice through an inhibition of proliferation and induction of apoptosis. Moreover, the observed increase in NF-kappaB activity during the transition from androgen dependence to androgen independence in the LAPC4 xenograft model was abrogated by PE. Our study represents the first description of PE as a promising dietary agent for the prevention of the emergence of androgen independence that is driven in part by heightened NF-kappaB activity.

Epidermal growth factor receptor inhibition sensitizes renal cell carcinoma cells to the cytotoxic effects of bortezomib.

In renal cell carcinoma (RCC) models, maximal cytotoxicity of the proteasome inhibitor bortezomib is dependent on efficient blockade of constitutive nuclear factor kappaB (NF-kappaB) activity. Signaling through the epidermal growth factor receptor (EGFR) has been shown to result in NF-kappaB activation. Thus, we sought to investigate whether inhibition of the EGFR sensitizes RCC cells to the cytotoxic effects of bortezomib. We first established that constitutive NF-kappaB activity is dependent on signaling through the EGFR in RCC cells. Indeed, blockade of EGFR signaling with an EGFR tyrosine kinase inhibitor (TKI) resulted in inhibition of NF-kappaB activity. Using pharmacologic and genetic approaches, we also showed that EGFR-mediated NF-kappaB activation occurs through the phosphotidylinositol-3-OH kinase/AKT pathway. Combinations of the EGFR-TKI and bortezomib resulted in synergistic cytotoxic effects when RCC cells were pretreated with the EGFR-TKI, but an antagonistic interaction was observed with bortezomib pretreatment. Evaluation of the effects of drug sequencing on inhibition of NF-kappaB activity revealed that EGFR-TKI pretreatment markedly augmented the NF-kappaB inhibitory effect of bortezomib, whereas bortezomib preexposure resulted in suboptimal NF-kappaB blockade and thus provides a biochemical explanation for the drug interaction results. We conclude that the constitutive NF-kappaB activity observed in RCC cells is mediated, at least in part, through an EGFR/phosphotidylinositol-3-OH kinase/AKT signaling cascade. Pretreatment with an EGFR-TKI sensitizes to bortezomib-mediated cytotoxicity by inhibiting constitutive NF-kappaB activity. The combination of bortezomib and a currently approved EGFR inhibitor warrants clinical investigation.

Mechanism of von Hippel-Lindau protein-mediated suppression of nuclear factor kappa B activity.

Biallelic inactivating mutations of the von Hippel-Lindau tumor suppressor gene (VHL) are a hallmark of clear cell renal cell carcinoma (CCRCC), the most common histologic subtype of RCC. Biallelic VHL loss results in accumulation of hypoxia-inducible factor alpha (HIFalpha). Restoring expression of the wild-type protein encoded by VHL (pVHL) in tumors with biallelic VHL inactivation (VHL(-)(/)(-)) suppresses tumorigenesis, and pVHL-mediated degradation of HIFalpha is necessary and sufficient for VHL-mediated tumor suppression. The downstream targets of HIFalpha that promote renal carcinogenesis have not been completely elucidated. Recently, VHL loss was shown to activate nuclear factor kappa B (NF-kappaB), a family of transcription factors that promotes tumor growth. Here we show that VHL loss drives NF-kappaB activation by resulting in HIFalpha accumulation, which induces expression of transforming growth factor alpha, with consequent activation of an epidermal growth factor receptor/phosphatidylinositol-3-OH kinase/protein kinase B (AKT)/IkappaB-kinase alpha/NF-kappaB signaling cascade. We also show that components of this signaling pathway promote the growth of VHL(-)(/)(-) tumor cells. Members of this pathway represent viable drug targets in VHL(-)(/)(-) tumors, such as those associated with CCRCC.

VHL expression in renal cell carcinoma sensitizes to bortezomib (PS-341) through an NF-kappaB-dependent mechanism.

In renal cell carcinomas (RCC), NF-kappaB blockade is required for maximal bortezomib-induced apoptosis, and expression of the von Hippel-Lindau (VHL) tumor suppressor protein downregulates NF-kappaB. Thus, we hypothesized that expression of wild-type (wt) VHL sensitizes RCC cells to bortezomib by reducing constitutive NF-kappaB activity. Using isogenic paired cell lines with and without expression of wtVHL, we have confirmed that VHL expression reduces constitutive NF-kappaB activity. Moreover, VHL expression confers markedly heightened sensitivity to the growth inhibitory effects of bortezomib in vitro. The bortezomib IC50 values were greater than two logs lower in the VHL-expressing cell lines compared to the VHL-deficient counterparts. By manipulating the level of constitutive NF-kappaB activity in an isogenic pair of RCC cell lines independently of VHL expression, we were able to demonstrate that the VHL sensitization effect is due to downregulation of NF-kappaB activity. These findings offer the enticing possibility of using VHL status as a molecular marker to identify RCC patients who may be sensitive to bortezomib. In particular, RCC patients who have non-clear-cell histologies as well as approximately 25% of clear-cell RCCs manifest expression of wtVHL and represent a subpopulation of patients that is apt to respond to bortezomib.

Maximal apoptosis of renal cell carcinoma by the proteasome inhibitor bortezomib is nuclear factor-kappaB dependent.

Advanced renal cell carcinoma (RCC) is resistant to cytotoxic chemotherapy, and immunotherapy has modest activity. Proteasome inhibitors represent a novel class of anticancer agents that have activity across a wide spectrum of tumor types. We investigated the efficacy of the proteasome inhibitor bortezomib (VELCADE, formerly known as PS-341) in RCC and found that bortezomib potently induces apoptosis of RCC cell lines. Blockade of the nuclear factor-kappaB (NF-kappaB) pathway is considered a crucial effect in bortezomib-induced apoptosis, but the dependence on NF-kappaB inhibition for bortezomib-mediated death has not been formally demonstrated. Thus, we also studied the contribution of NF-kappaB inhibition as a mechanism of bortezomib-induced apoptosis in RCC cells, which display constitutive NF-kappaB activation. Ectopic expression of the NF-kappaB family members, p65 (Rel A) and p50 (NF-kappaB1), markedly reduced bortezomib-induced apoptosis. However, when we used selective genetic and chemical inhibitors of NF-kappaB, we found that NF-kappaB blockade was not sufficient to induce apoptosis of RCC cells. Thus, we conclude that maximal bortezomib-induced apoptosis is dependent on its NF-kappaB inhibitory effect, but NF-kappaB-independent effects also play a critical role in the induction of apoptosis by bortezomib. This represents the first report to formally demonstrate that bortezomib-induced NF-kappaB blockade is required to achieve the maximum degree of apoptosis by this drug.

Transcriptional coactivation of c-Jun by the KSHV-encoded LANA.

The Kaposi sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) modulates viral and cellular gene expression, including interleukin 6 (IL-6), a growth factor for KSHV-associated diseases. LANA-driven IL-6 expression is dependent on the activator protein 1 (AP1) response element (RE) within the IL-6 promoter. We show that LANA activates the AP1 RE in a Jun-dependent fashion and that LANA enhances the transcriptional activity of a GAL4-Jun fusion protein. Coimmunoprecipitation studies documented a physical interaction between LANA and c-Jun in transiently transfected 293 cells as well as the KSHV-infected BCBL-1 primary effusion lymphoma (PEL) cell line. Taken together, these data indicate that LANA is a transcriptional coactivator of c-Jun. In addition, electrophoretic mobility shift assays demonstrated that LANA induces binding of a c-Jun-Fos heterodimer to the AP1 RE, but does not itself bind to the AP1 RE. RNA interference experiments confirmed that LANA activates the AP1 RE, stimulates binding of a c-Jun-Fos heterodimer to the AP1 RE, and induces expression of IL-6. These data indicate that LANA is a transcriptional coactivator of c-Jun, a function that may have implications for the pathogenesis of KSHV-associated diseases.

Drug interactions between the proteasome inhibitor bortezomib and cytotoxic chemotherapy, tumor necrosis factor (TNF) alpha, and TNF-related apoptosis-inducing ligand in prostate cancer.

PURPOSE: Proteasome inhibition has been shown to be an effective anticancer therapy in many tumor models, including prostate cancer. We sought to identify drug interactions between the proteasome inhibitor bortezomib and other apoptotic stimuli, including cytotoxic chemotherapy and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In addition, we wanted to gain insight into the role of nuclear factor kappaB inhibition as a mediator of bortezomib cytotoxic effects. EXPERIMENTAL DESIGN: Prostate cancer cell lines (LNCaP, LAPC4, CL1, and DU145) were treated with bortezomib and apoptotic stimuli (TRAIL, chemotherapy, and tumor necrosis factor alpha), alone or in combination. Apoptosis and cell viability were measured, and median effect/combination index analyses were used to quantitate drug interactions. Nuclear factor kappaB activity at baseline and in response to drug treatment was determined by gel shift and reporter gene assays. RESULTS: Bortezomib induced cell death of androgen-dependent (LNCaP and LAPC4) and androgen-independent (CL1 and DU145) prostate cancer cell lines, although androgen-dependent cells were more sensitive to proteasome inhibition. Bortezomib synergized with TRAIL and tumor necrosis factor alpha to induce death in both androgen-dependent and androgen-independent cells. CONCLUSIONS: Bortezomib and TRAIL represent a synergistic drug combination that warrants further evaluation in in vivo models of prostate cancer.

Kaposi's sarcoma-associated herpesvirus encoded vFLIP induces cellular IL-6 expression: the role of the NF-kappaB and JNK/AP1 pathways.

The Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a FADD-like interferon converting enzyme or caspase 8 (FLICE) inhibitory protein (vFLIP) that prevents death receptor-mediated apoptosis by inhibiting the recruitment and activation of FLICE. Since vFLIP physically interacts with tumor necrosis factor receptor associated factor 2 (TRAF2) and TRAF2 mediates activation of the jun NH(2)-terminal kinase (JNK)/activation protein 1 (AP1) pathway, we hypothesized that vFLIP might also activate this pathway. To evaluate this hypothesis, we transiently and stably transfected a vFLIP expression construct and performed several complementary assays to document that vFLIP activates the JNK/AP1 pathway and does so in a TRAF-dependent fashion. As vFLIP also activates the nuclear factor kappaB (NF-kappaB) signaling pathway and the NF-kappaB and JNK/AP1 pathways both modulate cellular interleukin-6 (cIL-6) expression, we postulated that vFLIP induces expression of this cytokine. We show that vFLIP induces cIL-6 expression and activates the cIL-6 promoter, and maximal activation of the cIL-6 promoter by vFLIP requires NF-kappaB and AP1 activation. In addition, vFLIP and latency-associated nuclear antigen (LANA), another KSHV-encoded latent protein, potentiate each other's ability to activate the cIL-6 promoter. Gene silencing experiments by RNA interference demonstrate that vFLIP in BCBL-1 endogenously infected primary effusion lymphoma (PEL) cells mediates JNK/AP1 activation and cIL-6 expression. Thus, we conclude that vFLIP, in addition to its known effects on NF-kappaB activation, also modulates the JNK/AP1 pathway and induces gene expression from the cIL-6 promoter in a JNK/AP1-dependent fashion.

The Kaposi sarcoma-associated herpesvirus (KSHV) induces cellular interleukin 6 expression: role of the KSHV latency-associated nuclear antigen and the AP1 response element.

Cellular interleukin 6 (IL-6) is an important growth factor for Kaposi sarcoma- associated herpesvirus (KSHV)-associated neoplasms, which include human immunodeficiency virus (HIV)-related and -unrelated cases of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Increased IL-6 levels are found in tissues affected with these diseases, and KSHV exists in a latent state in the majority of virally infected cells. In addition, acute infection with KSHV up-regulates IL-6 expression in endothelial cells. Thus, the hypothesis was considered that a latent KSHV gene product up-regulates IL-6 expression. To evaluate this hypothesis, the KSHV latency-associated nuclear antigen (LANA) was expressed in human embryonal kidney 293 cells and a bone marrow stromal cell line. LANA up-regulates IL-6 expression by inducing transcription from the IL-6 promoter, and the AP1 response element within the IL-6 promoter is necessary for and mediates IL-6 up-regulation by LANA. Thus, LANA may play a key pathophysiologic role in KSHV-associated neoplasms by functioning to up-regulate expression of IL-6.