CRISPR/Cas9 genome-wide loss-of-function screening identifies druggable cellular factors involved in sunitinib resistance in renal cell carcinoma.

BACKGROUND: Multi-targeted tyrosine kinase inhibitors (TKIs) are the standard of care for patients with advanced clear cell renal cell carcinoma (ccRCC). However, a significant number of ccRCC patients are primarily refractory to targeted therapeutics, showing neither disease stabilisation nor clinical benefits. METHODS: We used CRISPR/Cas9-based high-throughput loss of function (LOF) screening to identify cellular factors involved in the resistance to sunitinib. Next, we validated druggable molecular factors that are synthetically lethal with sunitinib treatment using cell and animal models of ccRCC. RESULTS: Our screening identified farnesyltransferase among the top hits contributing to sunitinib resistance in ccRCC. Combined treatment with farnesyltransferase inhibitor lonafarnib potently augmented the anti-tumour efficacy of sunitinib both in vitro and in vivo. CONCLUSION: CRISPR/Cas9 LOF screening presents a promising approach to identify and target cellular factors involved in the resistance to anti-cancer therapeutics.

Charon Mediates Immune Deficiency-Driven PARP-1-Dependent Immune Responses in Drosophila.

Regulation of NF-κB nuclear translocation and stability is central to mounting an effective innate immune response. In this article, we describe a novel molecular mechanism controlling NF-κB-dependent innate immune response. We show that a previously unknown protein, termed as Charon, functions as a regulator of antibacterial and antifungal immune defense in Drosophila Charon is an ankyrin repeat-containing protein that mediates poly(ADP-ribose) polymerase-1 (PARP-1)-dependent transcriptional responses downstream of the innate immune pathway. Our results demonstrate that Charon interacts with the NF-κB ortholog Relish inside perinuclear particles and delivers active Relish to PARP-1-bearing promoters, thus triggering NF-κB/PARP-1-dependent transcription of antimicrobial peptides. Ablating the expression of Charon prevents Relish from targeting promoters of antimicrobial genes and effectively suppresses the innate immune transcriptional response. Taken together, these results implicate Charon as an essential mediator of PARP-1-dependent transcription in the innate immune pathway. Thus, to our knowledge, our results are the first to describe the molecular mechanism regulating translocation of the NF-κB subunit from cytoplasm to chromatin.

LDL cholesterol counteracts the antitumour effect of tyrosine kinase inhibitors against renal cell carcinoma.

BACKGROUND: Treatment with tyrosine kinase inhibitors (TKIs) significantly improves survival of patients with renal cell carcinoma (RCC). However, about one-quarter of the RCC patients are primarily refractory to treatment with TKIs. METHODS: We examined viability of RCC and endothelial cells treated with low-density lipoprotein (LDL) and/or TKIs. Next, we validated the potential role of PI3K/AKT signalling in LDL-mediated TKI resistance. Finally, we examined the effect of a high-fat/high-cholesterol diet on the response of RCC xenograft tumours to sunitinib. RESULTS: The addition of LDL cholesterol increases activation of PI3K/AKT signalling and compromises the antitumour efficacy of TKIs against RCC and endothelial cells. Furthermore, RCC xenograft tumours resist TKIs in mice fed a high-fat/high-cholesterol diet. CONCLUSIONS: The ability of renal tumours to maintain their cholesterol homoeostasis may be a critical component of TKI resistance in RCC patients.

Piperlongumine inhibits NF-κB activity and attenuates aggressive growth characteristics of prostate cancer cells.

BACKGROUND: Elevated NF-κB activity has been previously demonstrated in prostate cancer cell lines as hormone-independent or metastatic characteristics develop. We look at the effects of piperlongumine (PL), a biologically active alkaloid/amide present in piper longum plant, on the NF-κB pathway in androgen-independent prostate cancer cells. METHODS: NF-κB activity was evaluated using Luciferase reporter assays and Western blot analysis of p50 and p65 nuclear translocation. IL-6, IL-8, and MMP-9 levels were assessed using ELISA. Cellular adhesion and invasiveness properties of prostate cancer cells treated with PL were also assessed. RESULTS: NF-κB DNA-binding activity was directly down-regulated with increasing concentrations of PL, along with decreased nuclear translocation of p50 and p65 subunits. Expression of IL-6, IL-8, MMP-9, and ICAM-1 was attenuated, and a decrease of cell-to-matrix adhesion and invasiveness properties of prostate cancer cells were observed. CONCLUSIONS: PL-mediated inhibition of NF-κB activity decreases aggressive growth characteristics of prostate cancer cells in vitro.

Targeting NPC1 in Renal Cell Carcinoma.

Rapidly proliferating cancer cells have a greater requirement for cholesterol than normal cells. Tumor cells are largely dependent on exogenous lipids given that their growth requirements are not fully met by endogenous pathways. Our current study shows that ccRCC cells have redundant mechanisms of cholesterol acquisition. We demonstrate that all major lipoproteins (i.e., LDL, HDL, and VLDL) have a comparable ability to support the growth of ccRCC cells and are equally effective in counteracting the antitumor activities of TKIs. The intracellular trafficking of exogenous lipoprotein-derived cholesterol appears to be distinct from the movement of endogenously synthesized cholesterol. De novo synthetized cholesterol is transported from the endoplasmic reticulum directly to the plasma membrane and to the acyl-CoA: cholesterol acyltransferase, whereas lipoprotein-derived cholesterol is distributed through the NPC1-dependent endosomal trafficking system. Expression of NPC1 is increased in ccRCC at mRNA and protein levels, and high expression of NPC1 is associated with poor prognosis. Our current findings show that ccRCC cells are particularly sensitive to the inhibition of endolysosomal cholesterol export and underline the therapeutic potential of targeting NPC1 in ccRCC.

Piperlongumine induces rapid depletion of the androgen receptor in human prostate cancer cells.

BACKGROUND: Androgen receptor (AR) signaling is regarded as the driving force in prostate carcinogenesis, and its modulation represents a logical target for prostate cancer (PC) prevention and treatment. Natural products are the most consistent source of small molecules for drug development. In this study, we investigate the functional impact of piperlongumine (PL), a naturally occurring alkaloid present in the Long pepper (Piper longum), on AR expression in PC cells and delineate its mechanism of action. METHODS: Expression and transcriptional activity of AR was examined by western blotting and luciferase reporter assay, respectively. CellTiter Blue assay was utilized to quantify cell proliferation. Reactive oxygen species (ROS) generation was examined by staining cells with a ROS indicator CM-H(2) DCFDA, followed by flow cytometry analysis. RESULTS: The results of our experiments demonstrate that PL rapidly reduces AR protein levels in PC cells via proteasome-mediated ROS-dependent mechanism. Moreover, PL effectively depletes a modified AR lacking the ligand-binding domain, shedding light on a new paradigm in the treatment approach to prostatic carcinoma that expresses mutated constitutively active AR. Importantly, PL effectively depletes AR in PC cells at low micromolar concentrations, while concurrently exerting a significant inhibitory effect on AR transcriptional activity and proliferation of PC cells. CONCLUSIONS: Our investigation demonstrates for the first time that PL induces rapid depletion of the AR in PC cells. As such, PL may afford novel opportunities for both prevention and treatment of prostatic malignancy.

Examining the Effect of PARP-1 Inhibitors on Transcriptional Activity of Androgen Receptor in Prostate Cancer Cells.

Since the early 1940s, androgen ablation has been the cornerstone of treatment for prostate cancer (PC). Importantly, androgen receptor (AR) signaling is vital not only for the initiation of PC, which is initially androgen-dependent, but also for castration-resistant disease. Recent studies demonstrated clear promise of the poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors for targeting prostate cancer cells harboring mutations in DNA damage-repair genes. In addition, it has been established that PARP-1 inhibition suppresses growth of AR-positive prostate cancer cells in cell and animal models. Thus, prostate cancer represents a particularly promising disease site for targeting PARP-1, given that both DNA repair and AR-mediated transcription depend on PARP-1 function. Here, we describe the development and use of cell-based assay to evaluate the impact of PARP-1 inhibitors on the AR signaling in prostate cancer cells.

Co-administration of piperine and docetaxel results in improved anti-tumor efficacy via inhibition of CYP3A4 activity.

BACKGROUND: Docetaxel is the mainline treatment approved by the FDA for castration-resistant prostate cancer (CRPC) yet its administration only increases median survival by 2-4 months. Docetaxel is metabolized in the liver by hepatic CYP3A4 activity. Piperine, a major plant alkaloid/amide, has been shown to inhibit the CYP3A4 enzymatic activity in a cell-free system. Thus, we investigated whether the co-administration of piperine and docetaxel could increase docetaxel's pharmacokinetic activity in vitro and in vivo. METHODS: Liver CYP3A4 enzymatic activity was measured by fluorescence. In vivo docetaxel pharmacokinetic activity was analyzed by liquid chromatography. An in vivo xenograft model of human CRPC was utilized to assess the anti-tumor effect of docetaxel when co-administered with piperine. RESULTS: Inhibition of hepatic CYP3A4 activity resulted in an increased area under the curve, half-life and maximum plasma concentration of docetaxel when compared to docetaxel alone administration. The synergistic administration of piperine and docetaxel significantly improved the anti-tumor efficacy of docetaxel in a xenograft model of human CRPC. CONCLUSIONS: Docetaxel is one of the most widely used cytotoxic chemotherapeutic agents and is currently the mainstay treatment for metastatic CRPC. Dietary constituents are important agents modifying drug metabolism and transport. In our studies, dietary consumption of piperine increases the therapeutic efficacy of docetaxel in a xenograft model without inducing more adverse effects on the treated mice.

Acetyl-CoA Counteracts the Inhibitory Effect of Antiandrogens on Androgen Receptor Signaling in Prostate Cancer Cells.

The commonly used therapeutic management of PC involves androgen deprivation therapy (ADT) followed by treatment with AR signaling inhibitors (ARSI). However, nearly all patients develop drug-resistant disease, with a median progression-free survival of less than 2 years in chemotherapy-naïve men. Acetyl-coenzyme A (acetyl-CoA) is a central metabolic signaling molecule with key roles in biosynthetic processes and cancer signaling. In signaling, acetyl-CoA serves as the acetyl donor for acetylation, a critical post-translational modification. Acetylation affects the androgen receptor (AR) both directly and indirectly increasing expression of AR dependent genes. Our studies reveal that PC cells respond to the treatment with ARSI by increasing expression of ATP-citrate lyase (ACLY), a major enzyme responsible for cytosolic acetyl-CoA synthesis, and up-regulation of acetyl-CoA intracellular levels. Inhibition of ACLY results in a significant suppression of ligand-dependent and -independent routes of AR activation. Accordingly, the addition of exogenous acetyl-CoA, or its precursor acetate, augments AR transcriptional activity and diminishes the anti-AR activity of ARSI. Taken together, our findings suggest that PC cells respond to antiandrogens by increasing activity of the acetyl-coA pathway in order to reinstate AR signaling.

Reversal of epigenetic silencing of AP-2alpha results in increased zinc uptake in DU-145 and LNCaP prostate cancer cells.

Zinc accumulation is lost during prostate carcinogenesis. Recent studies reveal a strong association between prostate cancer progression and the downregulation of the zinc uptake transporters hZip1 and hZip3. The aim of this work was to assess the involvement of epigenetic processes in the disruption of zinc uptake homeostasis in prostate adenocarcinoma. In this report, we demonstrate an increase in hZip1 and hZip3 zinc transporters' expression and zinc uptake by the prostate cancer cells DU-145 and LNCaP in response to 5-aza-2'-deoxycytidine. This effect is due to demethylation of the promoter region of the activator protein (AP)-2alpha protein, which is crucial for hZip1 and hZip3 genes expression. Loss of AP-2alpha expression in DU-145 and LNCaP prostate cancer cells is due to hypermethylation of its promoter region. Similarly, we found higher AP-2alpha promoter methylation levels in clinical samples of early-stage prostate adenocarcinoma when compared with adjacent non-malignant prostate tissue. Taken together, our findings provide a better understanding of the epigenetic mechanisms that are involved in the loss of AP-2alpha protein in prostate cancer cells which lead to decreased cellular zinc uptake-a sine qua non of prostate cancer development.

Docetaxel-mediated apoptosis in myeloid progenitor TF-1 cells is mitigated by zinc: potential implication for prostate cancer therapy.

BACKGROUND: Docetaxel-based combination chemotherapy is approved by the FDA for the treatment of metastatic castration-resistant prostate cancer. Unfortunately, docetaxel's efficacy is significantly limited by its considerable toxicity on hematopoietic progenitor cells, thus necessitating dose reduction or even discontinuation of the chemotherapy. Induction of pre-mitotic arrest protects cells against docetaxel-mediated toxicity and affords therapeutic opportunities. METHODS: Cell cycle progression was examined by propidium iodide staining. Zinc uptake was determined by FluoZin-3 AM staining. Apoptotic DNA fragmentation was detected using APO-BRDU kit. RESULTS: In the course of our current work, we treated the myeloid progenitor TF-1 cells and the castration-resistant PC-3 and DU-145 prostate cancer cells with physiologically relevant concentrations of zinc. In doing so, we were able to prevent docetaxel-mediated mitotic arrest in zinc accumulating myeloid progenitor TF-1 cells but not in castration-resistant PC-3 and DU-145 prostate cancer cells. Moreover, pre-treatment with zinc abolished docetaxel-induced apoptosis in TF-1 cells, whereas such treatment had no effect on apoptosis in PC-3 and DU-145 prostate cancer cells. CONCLUSIONS: Our results suggest that zinc can protect myeloid progenitor cells against docetaxel-induced toxicity without compromising the drug's anti-tumor activity.

Are all multi-targeted tyrosine kinase inhibitors created equal? An in vitro study of sunitinib and pazopanib in renal cell carcinoma cell lines.

OBJECTIVES: We examined the in vitro cellular effects of the multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and pazopanib on a series of human renal cell carcinoma (RCC) cell lines. METHODS: The human RCC cell lines 769-P, 786-O, HRC-24, HRC-31, HRC-45, HRC-78, SK-26B, and SK-45 were treated with varying concentrations of sunitinib and pazopanib. Cellular proliferation and cellular death were assessed using the CellTiter-Blue Cell Viability Assay and the TUNEL assay, respectively. Effective doses (ED) for inhibition of cellular proliferation or induction of apoptosis were calculated for sunitinib and pazopanib in each RCC cell line. RESULTS: Both sunitinib and pazopanib exhibited anti-proliferative activity to varying degree against all human RCC cell lines; however, sunitinib's effects were achieved at significantly lower concentrations. Moreover, sunitinib had a direct pro-apoptotic effect on all tested cell lines, while pazopanib failed to induce apoptosis in any of the examined human RCC cell lines even at maximal concentrations. CONCLUSIONS: Although sunitinib and pazopanib are often used interchangeably in the clinical setting, our results suggest that in-vitro biological activity of the two agents differs. Sunitinib exhibits a cytotoxic effect on RCC cell lines, while pazopanib's activity is solely cytostatic. These data may be clinically relevant given the current lack of comparative in-vivo studies between the two agents.

Histone-dependent PARP-1 inhibitors: A novel therapeutic modality for the treatment of prostate and renal cancers.

Clinical interest in poly(ADP-ribose) polymerase 1 (PARP-1) has increased over the past decade with the recognition of its roles in transcription regulation, DNA repair, epigenetic bookmarking, and chromatin restructuring. A number of PARP-1 inhibitors demonstrating clinical efficacy against tumors of various origins have emerged in recent years. These inhibitors have been essentially designed as nicotinamide adenine dinucleotide (NAD+) mimetics. However, because NAD+ is utilized by many enzymes other than PARP-1, NAD+ competitors tend to produce certain off-target effects. To overcome the limitation of NAD-like PARP-1 inhibitors, we have developed a new class of PARP-1 inhibitors that specifically targets the histone-dependent route of PARP-1 activation, a mechanism of activation that is unique to PARP-1. Novel histone-dependent inhibitors are highly specific for PARP-1 and demonstrate promising in vitro and in vivo efficacy against prostate and renal tumors. Our findings suggest that novel PARP-1 inhibitors have strong therapeutic potential for the treatment of urological tumors.

Poly(ADP)-Ribosylation Inhibition: A Promising Approach for Clear Cell Renal Cell Carcinoma Therapy.

Poly(ADP-ribose) polymerase 1 (PARP-1) and glycohydrolase (PARG) enzymes regulate chromatin structure, transcription activation, and DNA repair by modulating poly(ADP-ribose) (pADPr) level. Interest in PARP-1 inhibitors has soared recently with the recognition of their antitumor efficacy. We have shown that the development of clear cell renal cell carcinoma (ccRCC) is associated with extreme accumulation of pADPr caused by the enhanced expression of PARP-1 and decreased PARG levels. The most severe misregulation of pADPr turnover is found in ccRCC specimens from metastatic lesions. Both, classical NAD-like and non-NAD-like PARP-1 inhibitors reduced viability and clonogenic potential of ccRCC cell lines and suppressed growth of ccRCC xenograft tumors. However, classical NAD-like PARP-1 inhibitors affected viability of normal kidney epithelial cells at high concentrations, while novel non-NAD-like PARP-1 inhibitors exhibited activity against malignant cells only. We have also utilized different approaches to reduce the pADPr level in ccRCC cells by stably overexpressing PARG and demonstrated the prominent antitumor effect of this "back-to-normal" intervention. We also generated ccRCC cell lines with stable overexpression of PARG under doxycycline induction. This genetic approach demonstrated significantly affected malignancy of ccRCC cells. Transcriptome analysis linked observed phenotype with changes in gene expression levels for lipid metabolism, interferon signaling, and angiogenesis pathways along with the changes in expression of key cancer-related genes.

Cadmium down-regulates expression of XIAP at the post-transcriptional level in prostate cancer cells through an NF-kappaB-independent, proteasome-mediated mechanism.

BACKGROUND: Cadmium has been classified as a human carcinogen, affecting health through occupational and environmental exposure. Cadmium has a long biological half-life (>25 years), due to the flat kinetics of its excretion. The prostate is one of the organs with highest levels of cadmium accumulation. Importantly, patients with prostate cancer appear to have higher levels of cadmium both in the circulation and in prostatic tissues. RESULTS: In the current report, we demonstrate for the first time that cadmium down-regulates expression of the X-linked inhibitor of apoptosis protein (XIAP) in prostate cancer cells. Cadmium-mediated XIAP depletion occurs at the post-transcriptional level via an NF-kappaB-independent, proteasome-mediated mechanism and coincides with an increased sensitivity of prostate cancer cells to TNF-alpha-mediated apoptosis. Prolonged treatment with cadmium results in selection of prostate cancer cells with apoptosis-resistant phenotype. Development of apoptosis-resistance coincides with restoration of XIAP expression in cadmium-selected PC-3 cells. CONCLUSIONS: Selection of cadmium-resistant cells could represent an adaptive survival mechanism that may contribute to progression of prostatic malignancies.

Overexpression of the zinc uptake transporter hZIP1 inhibits nuclear factor-kappaB and reduces the malignant potential of prostate cancer cells in vitro and in vivo.

PURPOSE: Intracellular zinc levels and expression of the zinc uptake transporter, hZIP1, are markedly down-regulated in prostate adenocarcinomatous tissue compared with normal prostate tissue. Our previous studies have shown that zinc inhibits nuclear factor-kappaB (NF-kappaB) activity and reduces the malignant potential of prostate cancer cells in vitro. In this study, we investigate the functional effect of hZIP1 overexpression on NF-kappaB activity and tumorigenic potential in human prostate cancer cells in vitro and in vivo. EXPERIMENTAL DESIGN: NF-kappaB activity in PC-3 prostate cancer cells was examined by Western blotting and luciferase assay. ELISA was used to examine the expression of tumorigenic cytokines. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, adhesion, and invasiveness assays were used to assess the malignant potential of tumor cells. The effect of hZIP1 overexpression on prostate tumor progression in vivo was assessed using a xenograft model. RESULTS: Overexpression of the hZIP1 transporter in PC-3 cells results in significant inhibition of NF-kappaB activity in the presence of physiologic levels of zinc. NF-kappaB inhibition coincides with a reduction in expression of several NF-kappaB controlled prometastatic and antiapoptotic factors as well as sensitization of the cells to etoposide and tumor necrosis factor-mediated apoptosis-inducing ligand-mediated cell death. Moreover, overexpression of the hZIP1 transporter induces regression of prostate tumor growth in a xenograft model. CONCLUSIONS: Our results show that hZIP1 overexpression has a functional effect on the malignant potential of prostate cancer cells via inhibition of NF-kappaB-dependent pathways and support the concept that hZIP1 may function as a tumor suppressor gene in prostate cancer.

Non-NAD-like PARP-1 inhibitors in prostate cancer treatment.

In our previous studies of the molecular mechanisms of poly(ADP-ribose) polymerase 1 (PARP-1)-mediated transcriptional regulation we identified a novel class of PARP-1 inhibitors targeting the histone-dependent route of PARP-1 activation. Because histone-dependent activation is unique to PARP-1, non-NAD-like PARP-1 inhibitors have the potential to bypass the off-target effects of classical NAD-dependent PARP-1 inhibitors, such as olaparib, veliparib, and rucaparib. Furthermore, our recently published studies demonstrate that, compared to NAD-like PARP-1 inhibitors that are used clinically, the non-NAD-like PARP-1 inhibitor 5F02 exhibited superior antitumor activity in cell and animal models of human prostate cancer (PC). In this study, we further evaluated the antitumor activity of 5F02 and several of its novel analogues against PC cells. In contrast to NAD-like PARP-1 inhibitors, non-NAD-like PARP-1 inhibitors demonstrated efficacy against androgen-dependent and -independent routes of androgen receptor signaling activation. Our experiments reveal that methylation of the quaternary ammonium salt and the presence of esters were critical for the antitumor activity of 5F02 against PC cells. In addition, we examined the role of a related regulatory protein of PARP-1, called Poly(ADP-ribose) glycohydrolase (PARG), in prostate carcinogenesis. Our study reveals that PARG expression is severely disrupted in PC cells, which is associated with decreased integrity and localization of Cajal bodies (CB). Overall, the results of our study strengthen the justification for using non-NAD-like PARP-1 inhibitors as a novel therapeutic strategy for the treatment of advanced prostate cancer.

Expression of total and phospho 4EBP1 in metastatic and non-metastatic renal cell carcinoma.

Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) is phosphorylated and activated by mammalian target of rapamycin complex 1, which serves as a regulator of cell growth, cell survival, metastasis and angiogenesis in many types of cancer. The aim of this study was to evaluate the role of phosphorylated 4EBP1 (p4EBP1) in primary renal cell carcinoma (RCC) as a biomarker in metastatic RCC (mRCC) and non-mRCC cohorts. Primary tumor tissue from 254 non-mRCC and 60 mRCC patients were immunohistochemically stained for t4EBP1 and p4EBP1. The disease-free interval (DFI) categorized by the expressions and clinical parameters were assessed by univariate and multivariate analysis in the non-mRCC cohort. Then, the cause-specific survival (CSS) was assessed in the mRCC cohort by the same methods as used in the non-mRCC cohort. In the non-mRCC cohort, patients with t4EBP1 expression had no RCC recurrence. Patients with p4EBP1 expression had the shorter DFI in univariate analysis (P=0.037). p4EBP1 and pT1b-4 expression levels were independent predictors for de novo metastasis. In the mRCC cohort, intermediate/poor MSKCC risk, non-clear cell RCC, and no p4EBP1 expression were correlated with poor CSS on multivariate analysis. Expression of p4EBP1 could be a predictive biomarker for de novo metastasis in non-mRCC patient cohort. By contrast, mRCC patients showing no p4EBP1 expression had shorter CSS than patients with p4EBP1 expression.

PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55α/PP2A reconstitution that triggers centrosome destabilization.

The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here, we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase/anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity.

Depletion of intracellular zinc increases expression of tumorigenic cytokines VEGF, IL-6 and IL-8 in prostate cancer cells via NF-kappaB-dependent pathway.

BACKGROUND: Zinc accumulation diminishes early in the course of prostate malignancy and continues to decline during progression toward hormone-independent growth. In contrast, constitutive levels of NF-kappaB activity increase during progression of prostate cells toward greater tumorigenic potential. We have reported previously that physiological levels of zinc suppress NF-kappaB activity in prostate cancer cells and reduce expression of pro-angiogenic and pro-metastatic cytokines VEGF, IL-6, IL-8, and MMP-9 associated with negative prognostic features in prostate cancer. METHODS: Intracellular zinc levels were examined by atomic absorption spectroscopy. NF-kappaB activity was examined by TransAm and Luciferase reporter assays, and Western blot analysis of p50 nuclear translocation. VEGF, IL-6 and IL-8 levels were assessed by ELISA. RESULTS: Selective zinc deficiency induced by the membrane-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) increases activation of NF-kappaB and up-regulates expression of the NF-kappaB controlled pro-angiogenic and pro-metastatic cytokines VEGF, IL-6 and IL-8 in androgen-independent PC-3 and DU-145 prostate cancer cells. Pre-incubation with I kappaB alpha dominant mutant adenovirus efficiently blocks expression of these cytokines in zinc deficient cells indicating that the observed effects are NF-kappaB dependent. CONCLUSIONS: Our findings suggest that zinc deficiency may contribute to the tumor progression via augmented expression of the NF-kappaB-dependent pro-tumorigenic cytokines.