Haitao Zhang

Castration-resistant prostate cancer: Adaptive responses in the androgen axis

The androgen signaling axis in prostate cancer is associated with multiple adaptive mechanisms in response to castration. Herein we review these adaptations with an emphasis on recent molecular insights into the growth and development of castration resistant prostate cancer (CRPC). Alterations include both conventional and novel intracrine androgen synthesis pathways and androgen transport as well as androgen receptor (AR) overexpression, mutation, and splice variation. Each of these underlying mechanisms are potentially linked to post-castration growth, especially after treatment with newer hormonal agents such as abiraterone and enzalutamide. Post-translational AR modifications are well documented and these can affect receptor activity, stability, localization, and interaction with other proteins. Changes in recruitment of androgen receptor associated co-activators/repressors and a distinct AR-induced transcriptional program can dramatically alter proliferation, invasion, and metastasis in a ligand and context-dependent manner. Numerous previously uncharacterized non-coding RNAs, some of which are androgen regulated, may also have important biological function in this disease. Taken together, the view of CRPC has changed dramatically in the last several years. This has occurred not only within the setting of multiple treatment paradigm changes, but also as a multiplicity of potential molecular mechanisms underlying this disease state have been explored and discovered.

Androgen receptor splice variants dimerize to transactivate target genes

Constitutively active androgen receptor splice variants (AR-V) lacking the ligand-binding domain have been implicated in the pathogenesis of castration-resistant prostate cancer and in mediating resistance to newer drugs that target the androgen axis. AR-V regulates expression of both canonical AR targets and a unique set of cancer-specific targets that are enriched for cell-cycle functions. However, little is known about how AR-V controls gene expression. Here, we report that two major AR-Vs, termed AR-V7 and AR(v567es), not only homodimerize and heterodimerize with each other but also heterodimerize with full-length androgen receptor (AR-FL) in an androgen-independent manner. We found that heterodimerization of AR-V and AR-FL was mediated by N- and C-terminal interactions and by the DNA-binding domain of each molecule, whereas AR-V homodimerization was mediated only by DNA-binding domain interactions. Notably, AR-V dimerization was required to transactivate target genes and to confer castration-resistant cell growth. Our results clarify the mechanism by which AR-Vs mediate gene regulation and provide a pivotal pathway for rational drug design to disrupt AR-V signaling as a rational strategy for the effective treatment of advanced prostate cancer.

Berberine Suppresses Androgen Receptor Signaling in Prostate Cancer

The androgen receptor (AR) is critical in the normal development and function of the prostate, as well as in prostate carcinogenesis. Androgen deprivation therapy is the mainstay in the treatment of advanced prostate cancer; however, after an initial response, the disease inevitably progresses to castration-resistant prostate cancer (CRPC). Recent evidence suggests that continued AR activation, sometimes in a ligand-independent manner, is commonly associated with the development of CRPC. Thus, novel agents targeting the AR are urgently needed as a strategic step in developing new therapies for this disease state. In this study, we investigated the effect of berberine on AR signaling in prostate cancer. We report that berberine decreased the transcriptional activity of AR. Berberine did not affect AR mRNA expression, but induced AR protein degradation. Several ligand-binding, domain-truncated AR splice variants have been identified, and these variants are believed to promote the development of CRPC in patients. Interestingly, we found that these variants were more susceptible to berberine-induced degradation than the full-length AR. Furthermore, although the growth of LNCaP xenografts in nude mice was inhibited by berberine, and AR expression was reduced in the tumors, the morphology and AR expression in normal prostates were not affected. This study is the first to show that berberine suppresses AR signaling and suggests that berberine, or its derivatives, presents a promising agent for the prevention and/or treatment of prostate cancer. Mol Cancer Ther; 10(8); 1346–56. ©2011 AACR.

Methylseleninic Acid Enhances Paclitaxel Efficacy for the Treatment of Triple-Negative Breast Cancer

A major challenge in breast cancer therapy is the lack of an effective therapeutic option for a particularly aggressive subtype of breast cancer, triple-negative breast cancer. Here we provide the first preclinical evidence that a second-generation selenium compound, methylseleninic acid, significantly enhances the anticancer efficacy of paclitaxel in triple-negative breast cancer. Through combination-index value calculation, we demonstrated that methylseleninic acid synergistically enhanced the growth inhibitory effect of paclitaxel in triple-negative breast cancer cells. The synergism was attributable to more pronounced induction of caspase-mediated apoptosis, arrest of cell cycle progression at the G2/M checkpoint, and inhibition of cell proliferation. Treatment of SCID mice bearing MDA-MB-231 triple-negative breast cancer xenografts for four weeks with methylseleninic acid (4.5 mg/kg/day, orally) and paclitaxel (10 mg/kg/week, through intraperitoneal injection) resulted in a more pronounced inhibition of tumor growth compared with either agent alone. The attenuated tumor growth correlated with a decrease in tumor cell proliferation and an induction of apoptosis. The in vivo study also indicated the safety of using methylseleninic acid in the combination regime. Our findings thus provide strong justification for the further development of methylseleninic acid and paclitaxel combination therapy for the treatment of triple-negative breast cancer.

Subverting ER-Stress towards Apoptosis by Nelfinavir and Curcumin Coexposure Augments Docetaxel Efficacy in Castration Resistant Prostate Cancer Cells

Despite its side-effects, docetaxel (DTX) remains a first-line treatment against castration resistant prostate cancer (CRPC). Therefore, strategies to increase its anti-tumor efficacy and decrease its side effects are critically needed. Targeting of the constitutive endoplasmic reticulum (ER) stress in cancer cells is being investigated as a chemosensitization approach. We hypothesized that the simultaneous induction of ER-stress and suppression of PI3K/AKT survival pathway will be a more effective approach. In a CRPC cell line, C4-2B, we observed significant (p<0.005) enhancement of DTX-induced cytotoxicity following coexposure to thapsigargin and an AKT-inhibitor. However, since these two agents are not clinically approved, we investigated whether a combination of nelfinavir (NFR) and curcumin (CUR), known to target both these metabolic pathways, can similarly increase DTX cytotoxicity in CRPC cells. Within 24 hrs post-exposure to physiologic concentrations of NFR (5 µM) and CUR (5 µM) a significantly (p<0.005) enhanced cytotoxicity was evident with low concentration of DTX (10 nM). This 3-drug combination rapidly increased apoptosis in aggressive C4-2B cells, but not in RWPE-1 cells or in primary prostate epithelial cells (PrEC). Comparative molecular studies revealed that this 3-drug combination caused a more pronounced suppression of phosphorylated-AKT and higher induction in phosphorylated-eIF2α in C4-2B cells, as compared to RWPE-1 cells. Acute exposure (3–9 hrs) to this 3-drug combination intensified ER-stress induced pro-apoptotic markers, i.e. ATF4, CHOP, and TRIB3. At much lower concentrations, chronic (3 wks) exposures to these three agents drastically reduced colony forming units (CFU) by C4-2B cells. In vivo studies using mice containing C4-2B tumor xenografts showed significant (p<0.05) enhancement of DTX’s (10 mg/kg) anti-tumor efficacy following coexposure to NFR (20 mg/kg) & CUR (100 mg/kg). Immunohistochemical (IHC) analyses of tumor sections indicated decreased Ki-67 staining and increased TUNEL intensity in mice exposed to the 3-drug combination. Therefore, subverting ER-stress towards apoptosis using adjuvant therapy with NFR and CUR can chemosensitize the CRPC cells to DTX therapy.

AKR1C3 overexpression may serve as a promising biomarker for prostate cancer progression

BackgroundAldo-keto reductase family 1 member C3 (AKR1C3) is a key steroidogenic enzyme that is overexpressed in prostate cancer (PCa) and is associated with the development of castration-resistant prostate cancer (CRPC). The aim of this study was to investigate the correlation between the expression level of AKR1C3 and the progression of PCa.MethodsSixty human prostate needle biopsy tissue specimens and ten LNCaP xenografts from intact or castrated male mice were included in the study. The relationship between the level of AKR1C3 expression by immunohistochemistry and evaluation factors for PCa progression, including prostate-specific antigen (PSA), Gleason score (GS) and age, were analyzed.ResultsLow immunoreactivity of AKR1C3 was detected in normal prostate epithelium, benign prostatic hyperplasia (BPH) and prostatic intraepithelial neoplasia (PIN). Positive staining was gradually increased with an elevated GS in PCa epithelium and LNCaP xenografts in mice after castration. The Spearman’s r values (rs) of AKR1C3 to GS and PSA levels were 0.396 (P = 0.025) and -0.377 (P = 0.036), respectively, in PCa biopsies. The rs of AKR1C3 to age was 0.76 (P = 0.011). No statistically significant difference was found with other variables.ConclusionOur study suggests that the level of AKR.1C3 expression is positively correlated with an elevated GS, indicating that AKR1C3 can serve as a promising biomarker for the progression of PCa.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7748245591110149.

Survivin gene silencing sensitizes prostate cancer cells to selenium growth inhibition

BackgroundProstate cancer is a leading cause of cancer-related death in men worldwide. Survivin is a member of the inhibitor of apoptosis (IAP) protein family that is expressed in the majority of human tumors including prostate cancer, but is barely detectable in terminally differentiated normal cells. Downregulation of survivin could sensitize prostate cancer cells to chemotherapeutic agents in vitro and in vivo. Selenium is an essential trace element. Several studies have shown that selenium compounds inhibit the growth of prostate cancer cells. The objective of this study is to investigate whether survivin gene silencing in conjunction with selenium treatment could enhance the therapeutic efficacy for prostate cancer and to elucidate the underlying mechanisms.MethodsExpression of survivin was analyzed in a collection of normal and malignant prostatic tissues by immunohistochemical staining. In vitro studies were conducted in PC-3M, C4-2B, and 22Rv1 prostate cancer cells. The effect of selenium on survivin expression was analyzed by Western blotting and semi-quantitative RT-PCR. Survivin gene knockdown was carried out by transfecting cells with a short hairpin RNA (shRNA) designed against survivin. Cell proliferation was quantitated by the 3-(4,5-Dimethylthiazol-2-yl)- 2,5-Diphenyltetrazolium Bromide (MTT) assay and apoptosis by propidium iodide staining followed by flow cytometry analysis. Finally, in vivo tumor growth assay was performed by establishing PC-3M xenograft in nude mice and monitoring tumor growth following transfection and treatment.ResultsWe found that survivin was undetectable in normal prostatic tissues but was highly expressed in prostate cancers. Survivin knockdown or selenium treatment inhibited the growth of prostate cancer cells, but the selenium effect was modest. In contrast to what have been observed in other cell lines, selenium treatment had little or no effect on survivin expression in several androgen-independent prostate cancer cell lines. Survivin knockdown sensitized these cells to selenium growth inhibition and apoptosis induction. In nude mice bearing PC-3M xenografts, survivin knockdown synergizes with selenium in inhibiting tumor growth.ConclusionsSelenium could inhibit the growth of hormone-refractory prostate cancer cells both in vitro and in vivo, but the effects were modest. The growth inhibition was not mediated by downregulating survivin expression. Survivin silencing greatly enhanced the growth inhibitory effects of selenium.

Kruppel-Like Factor 4 Is a Novel Mediator of Selenium in Growth Inhibition

A previous prevention trial showed that selenium supplementation was effective in reducing (by 50%) the incidence of prostate cancer. Selenium has been reported to inhibit the growth of prostate cancer cells in vitro. Multiple mechanisms are likely to be operative in the underlying effect of selenium. Here, we report that Krüppel-like factor 4 (KLF4), a transcription factor of the KLF family, is an important target of selenium. We found that selenium up-regulates KLF4 expression and increases the DNA-binding activity of KLF4 in both the androgen-dependent LNCaP and the androgen-independent PC-3 human prostate cancer cells. The increase of KLF4 mRNA is accounted for primarily by enhanced transcription, although the contribution of a slight abatement in mRNA degradation cannot be ruled out. KLF4 knockdown using short interference RNA significantly weakens the effects of selenium on DNA synthesis inhibition, apoptosis induction, and the expression of three KLF4 target genes, cyclin D1, p21/WAF1, and p27/Kip1. In addition, the overexpression of KLF4 not only leads to an induction of apoptosis in the control cells, but also enhances the DNA synthesis–suppressive and–proapoptotic activities of selenium. Taken together, our results suggest that KLF4 plays a key role in mediating the growth-inhibitory effect of selenium in prostate cancer cells. (Mol Cancer Res 2008;6(2):306–13)

A Whole Blood Assay for AR-V7 and ARv567es in Patients with Prostate Cancer

PURPOSE Most prostate cancer mortality can be attributed to metastatic castration resistant prostate cancer, an advanced stage that remains incurable despite recent advances. The AR (androgen receptor) signaling axis remains active in castration resistant prostate cancer. Recent studies suggest that expression of the AR-V (AR splice variant) AR-V7 may underlie resistance to abiraterone and enzalutamide. However, controversy exists over the optimal assay. Our objective was to develop a fast and sensitive assay for AR-Vs in patients. MATERIALS AND METHODS Two approaches were assessed in this study. The first approach was based on depletion of leukocytes and the second one used RNA purified directly from whole blood preserved in PAXgene® tubes. Transcript expression was analyzed by quantitative reverse transcription-polymerase chain reaction. RESULTS Through a side-by-side comparison we found that the whole blood approach was suitable to detect AR-Vs. The specificity of the assay was corroborated in a cancer-free cohort. Using the PAXgene assay samples from a cohort of 46 patients with castration resistant prostate cancer were analyzed. Overall, AR-V7 and ARv567es were detected in 67.53% and 29.87% of samples, respectively. Statistical analysis revealed a strong association of AR-V positivity with a history of second line hormonal therapies. CONCLUSIONS To our knowledge this is the first study to demonstrate that PAXgene preserved whole blood can be used to obtain clinically relevant information regarding the expression of 2 AR-Vs. These data on a castration resistant prostate cancer cohort support a role for AR-Vs in resistance to therapies targeting the AR ligand-binding domain.

20(S)-Protopanaxadiol-aglycone Downregulation of the Full-length and Splice Variants of Androgen Receptor

As a public health problem, prostate cancer engenders huge economic and life‐quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high‐grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively active AR splice variants (AR‐Vs) lacking the ligand‐binding domain. Thus, novel agents targeting the receptor, preferentially both the full‐length and AR‐Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)‐protopanaxadiol‐aglycone (PPD) effectively downregulates the expression and activity of both the full‐length AR and AR‐Vs. The effects of PPD on AR and AR‐Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome‐mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full‐length AR and AR‐Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer.