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.

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.

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.

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.

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.

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.

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.

Resistance to Systemic Therapies in Clear Cell Renal Cell Carcinoma: Mechanisms and Management Strategies.

Renal cell carcinoma (RCC) is the most common form of kidney cancer. It is categorized into various subtypes, with clear cell RCC (ccRCC) representing about 85% of all RCC tumors. The lack of sensitivity to chemotherapy and radiation therapy prompted research efforts into novel treatment options. The development of targeted therapeutics, including multi-targeted tyrosine kinase inhibitors (TKI) and mTOR inhibitors, has been a major breakthrough in ccRCC therapy. More recently, other therapeutic strategies, including immune checkpoint inhibitors, have emerged as effective treatment options against advanced ccRCC. Furthermore, recent advances in disease biology, tumor microenvironment, and mechanisms of resistance formed the basis for attempts to combine targeted therapies with newer generation immunotherapies to take advantage of possible synergy. This review focuses on the current status of basic, translational, and clinical studies on mechanisms of resistance to systemic therapies in ccRCC. Mol Cancer Ther; 17(7); 1355-64. ©2018 AACR.

The convergent roles of NF-κB and ER stress in sunitinib-mediated expression of pro-tumorigenic cytokines and refractory phenotype in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common form of kidney cancer. While cure remains exceptionally infrequent in RCC patients with systemic or recurrent disease, current targeted molecular strategies, including multi-targeted tyrosine kinase inhibitors (TKIs), notably changed the treatment paradigm of advanced renal cancer. Yet, complete and durable responses have been noted in only a few cases. Our studies reveal that sunitinib triggers two resistance-promoting signaling pathways in RCC cells, which emanate from the endoplasmic reticulum (ER) stress response: a PERK-driven ER stress response that induces expression of the pro-tumorigenic cytokines IL-6, IL-8, and TNF-α, and a TRAF2-mediated NF-κB survival program that protects tumor cells against cell death. PERK blockade completely prevents sunitinib-induced expression of IL-6, IL-8 and TNF-α, whereas NF-κB inhibition reinstates sensitivity of RCC cells to sunitinib both in vitro and in vivo. Taken together, our findings indicate that ER stress response may contribute to sunitinib resistance in RCC patients.

Testing PARP Inhibitors Using a Murine Xenograft Model.

Animal models are indispensable research tools for various drug discovery and development applications. In cancer research, the growth of tumors in vivo is necessary in order to closely modulate a potential response in humans. Ectopic (subcutaneous) xenografting of human cancer cells into immunodeficient mice has long been the standard model for preclinical evaluation of novel antitumor therapeutic agents. Here, we describe the establishment and use of subcutaneous xenograft mouse model to evaluate the antitumor activity of PARP-1 inhibitors.

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 and its analogs down-regulate expression of c-Met in renal cell carcinoma.

The c-Met protein, a transmembrane receptor tyrosine kinase, is the product of a proto-oncogene. Its only known ligand, hepatocyte growth factor (HGF), regulates cell growth, motility, migration, invasion, proliferation, and angiogenesis. The aberrant expression of c-Met is often associated with poor prognosis in multiple cancers, including renal cell carcinoma (RCC). Silencing or inactivation of c-Met leads to decreased viability of cancer cells, thereby making ablation of c-Met signaling an attractive concept for developing novel strategies for the treatment of renal tumors. Naturally-occurring products or substances are the most consistent source of drug development. As such, we investigated the functional impact of piperlongumine (PL), a naturally occurring alkaloid present in the Long pepper (Piper longum) on c-Met expression in RCC cells and demonstrated that PL and its analogs rapidly reduce c-Met protein and RNA levels in RCC cells via ROS-dependent mechanism. PL-mediated c-Met depletion coincided with the inhibition of downstream c-Met signaling; namely Erk/MAPK, STAT3, NF-κB and Akt/mTOR. As such, PL and PL analogs hold promise as potential therapeutic agents for the treatment of metastatic RCC and the prevention of postoperative RCC recurrence.

Minor grove binding ligands disrupt PARP-1 activation pathways.

PARP-1 is a nuclear enzyme regulating transcription, chromatin restructuring, and DNA repair. PARP-1 is activated by interaction with NAD+, DNA, and core histones. Each route of PARP-1 activation leads to somewhat different outcomes. PARP-1 interactions with core histones control PARP-1 functions during transcriptional activation in euchromatin. DNA-dependent regulation of PARP-1 determines its localization in heterochromatin and PARP-1-dependent silencing. Here we address the biological significance of DNA-dependent PARP-1 regulation in vitro and in vivo. We report that minor grove binding ligands (MGBLs) specifically target PARP-1 interaction with DNA, and, hence, the DNA-dependent pathway of PARP-1 activation. By obstructing its interaction with DNA molecules, MGBLs block PARP-1 activity in vitro and in vivo, as we demonstrate using Drosophila, as well as human cancer-derived cells. We also demonstrate synergistic inhibition of PARP-1, combining MGBLs with conventional NAD+-dependent inhibitors in human cancer cells. These results suggest that combining different classes of PARP-1 inhibitors can precisely modulate PARP-1 activity in living cells, thus holding promise for new avenues of cancer treatment.

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.

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.

The effect of 5-aminolevulinic acid and its derivatives on protoporphyrin IX accumulation and apoptotic cell death in castrate-resistant prostate cancer cells.

OBJECTIVE: To examine whether pharmacologically relevant zinc-binding agents are capable of depleting X-linked inhibitor of apoptosis protein in tumor cells. Our prior work reveals that treatment with zinc-chelating agents induces selective downregulation of the X-linked inhibitor of apoptosis protein in cancer cells of various origins. A precursor of the heme synthetic pathway, 5-aminolevulinic acid, is metabolized to protoporphyrin IX, which is highly reactive with zinc. We assessed whether modified versions of 5-aminolevulinic acid with lipophilic side chains can enhance efficacy and selectivity with respect to protoporphyrin IX accumulation, X-linked inhibitor of apoptosis protein depletion, and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human castration-resistant prostate cancer cells. METHODS: Seven modified versions of 5-aminolevulinic acid (5 esters and 2 amides) were synthesized. Levels of endogenous protoporphyrin IX were examined by flow cytometry. X-linked inhibitor of apoptosis protein expression was examined by Western blotting. terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay was used to assess cell apoptosis. Results were compared qualitatively. RESULTS: Accumulation of endogenous protoporphyrin IX by castration-resistant prostate cancer cells was shown to be directly related to the carbon chain length of the esterified 5-aminolevulinic acid derivatives. In fact, treatment with 5-aminolevulinic acid-HE was superior to that achieved by 5-aminolevulinic acid with respect to X-linked inhibitor of apoptosis protein downregulation. 5-aminolevulinic acid and 5-aminolevulinic acid-HE in combination with tumor necrosis factor-related apoptosis-inducing ligand significantly enhanced apoptotic cell death in castration-resistant prostate cancer cell lines. CONCLUSION: Esterified derivatives of 5-aminolevulinic acid alone or in combination with other agents may provide therapeutic opportunities in the treatment of castration-resistant prostate cancer by harnessing apoptotic pathways that are triggered by cellular zinc imbalance.

Modulation of Akt/mTOR signaling overcomes sunitinib resistance in renal and prostate cancer cells.

Tyrosine kinase inhibitors exhibit impressive activity against advanced renal cell carcinoma. However, recent clinical studies have shown an equivocal response to sunitinib in patients with castration-resistant prostate cancer. The tumor suppressor PTEN acts as a gatekeeper of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR cell-survival pathway. Our experiments showed that PTEN expression inversely correlates with sunitinib resistance in renal and prostate cancer cells. Restoration of PTEN expression markedly increases sensitivity of tumor cells to sunitinib both in vitro and in vivo. In addition, pharmacologic manipulation of PI3K/Akt/mTOR signaling with PI3K/mTOR inhibitor, GDC-0980, mTOR inhibitor, temsirolimus, or pan-Akt inhibitor, GSK690693, was able to overcome sunitinib resistance in cancer cells. Our findings underscore the importance of PTEN expression in relation to sunitinib resistance and imply a direct cytotoxic effect by sunitinib on tumor cells in addition to its antiangiogenic actions.

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.

Interleukin-6: a potential biomarker of resistance to multitargeted receptor tyrosine kinase inhibitors in castration-resistant prostate cancer.

OBJECTIVE: To determine if cellular interleukin-6 production predicts response to tyrosine kinase inhibitors (TKIs). As clinical experience using TKIs in patients with castration-resistant prostate cancer (CRPC) matures, Phase II trials show a heterogeneous response to sunitinib in CRPC patients. Change in serum prostate-specific antigen (PSA) level has proven unreliable for prediction of CRPC response to TKIs. Interleukin-6 (IL-6), a critical mediator of prostate cancer pathogenesis, has been shown to rise in patients with disease progression. As such, we investigated whether cellular IL-6 production can predict TKI response in both in vitro and in vivo models. METHODS: IL-6 mRNA levels and protein expression were examined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Apoptosis was examined using the terminal dUTP nick-end labeling assay. For in vivo studies, a CRPC xenograft model in C.B17/Icr-scid mice was used. RESULTS: PC-3 and DU-145 CRPC cell lines exhibited a heterogeneous response to sunitinib and pazopanib. Dose-dependent reduction of IL-6 was observed in TKI-sensitive DU-145 cells. In contrast, the TKI-resistant PC-3 cells failed to suppress IL-6 secretion. Instead, in the presence of tumor necrosis factor-alpha, IL-6 rose significantly upon administration of TKIs. Findings of in vitro experiments were confirmed in an in vivo mouse model of CRPC. CONCLUSION: Sensitivity of CRPC cells to TKIs is heterogeneous. These findings are consistent with results of recently published Phase II clinical trials using sunitinib in patients with CRPC. A substantial rise in IL-6 occurs both in vitro and in vivo in the presence of TKIs in resistant PC-3 cells but not in TKI-sensitive DU-145 cells. These findings suggest that IL-6 may represent a biomarker for TKI resistance in patients with CRPC.

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.