Receptor-positive Prostate Cancer Cells Research Articles

Androgen Receptor-Directed Molecular Conjugates for Targeting Prostate Cancer.

Due to its central role in the cellular biology of prostate cancer (PC), androgen receptor (AR) still remains an important therapeutic target for fighting this tumor. Several drugs targeting AR have been reported so far, and many new molecules are expected for the future. In spite of their antitumor efficacy, these drugs are not selective for malignant cells and are subjected to AR-mediated activation of drug resistance mechanisms that are responsible for several drawbacks, including systemic toxicity and disease recurrence and metastasis. Among the several strategies considered to overcome these drawbacks, very appealing appears the design of hybrid small-molecule conjugates targeting AR to drive drug action on receptor-positive PC cells. These compounds are designed around on an AR binder, which selectively engages AR with high potency, coupled with a moiety endowed with different pharmacological properties. In this review we focus on two classes of compounds: a) small-molecules and AR-ligand based conjugates that reduce AR expression, which allow down-regulation of AR levels by activating its proteasome-mediated degradation, and b) AR-ligand-based conjugates for targeting small-molecules, in which the AR binder tethers small-molecules, including conventional antitumor drugs (e.g., cisplatin, doxorubicin, histone deacetylase inhibitors, as well as photo-sensitizers) and selectively directs drug action toward receptor-positive PC cells.

Abstract 1825: ONC201 induces cell death in androgen receptor positive prostate cancer cells and shows synergistic effect with anti-prostate cancer drugs

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer related death among men in the United States. Current therapy for advanced prostate cancer is mainly based on androgen deprivation, though in many cases tumors become androgen-independent and resistant to available treatments. Thus, accessing novel therapies for prostate cancer and resistant tumors remains an important goal in the field. ONC201 is a first-in-class small molecule anti-cancer drug, shown to selectively induce cell death in most cancer cells tested in contrast to matched normal cells. In this study we investigated the single-agent and combination efficacy of ONC201 on a panel of prostate cancer cell lines with varied androgen receptor (AR) and receptor tyrosine kinases (RTKs) expression status. Our preliminary data suggests that the pro-apoptotic effects of ONC201 correlate with expression of androgen receptor and other receptor tyrosine kinases (RTKs). Specifically, high AR and low RTK (c-MET, EGFR, HER2 and IGFR) expression demonstrated higher sensitivity to ONC201 as compared to low AR and high RTK expression as evaluated by cell viability using the Cell-Titer Glo assay. We further modeled these findings in ONC201-sensitive 22Rv1 and ONC201-resistant DU145 cell lines. ONC201 induced robust apoptosis (cleaved PARP) in ONC201-sensitive 22Rv1 cells as compared to ONC201-resistant DU145 cells In addition, ONC201-sensitive 22RV1 cells showed abrogation of total AR expression 72 hours following treatment with ONC201. Thus, AR and RTK status seem to be emerging efficacy markers for response of ONC201 in prostate cancer cells. We are further investigating the involvement of ONC201 in the signal transduction pathway of AR and other RTKs in prostate cancer. Lastly, our initial screening for treatment of cells with a combination of ONC201 and FDA-approved therapies for prostate cancer showed synergistic potential of ONC201 with everolimus and docetaxel in both AR-dependent and independent cells. As expected, synergism of anti-androgen MDV3100 (enzalutamide) with ONC201 was restricted to AR positive prostate cancer cell lines. Our results indicate that ONC201 has therapeutic potential both as a single agent and in combination therapy for prostate cancer. Our long-term goal is to integrate our pre-clinical studies and translate the findings to single agent/combination trials of ONC201 for prostate cancer patients. Citation Format: Avital Lev, Amriti R. Lulla, David T. Dicker, Wafik S. El-Deiry. ONC201 induces cell death in androgen receptor positive prostate cancer cells and shows synergistic effect with anti-prostate cancer drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1825.

Effect of lipidated gonadotropin-releasing hormone peptides on receptor mediated binding and uptake into prostate cancer cells in vitro

Gonadotropin-releasing hormone (GnRH) receptors are overexpressed on many cancer cells but not on primary cell lines. This study was designed to investigate the targeting ability and uptake of dendritic lipidated [Gln1]-GnRH peptide analogues on receptor-positive prostate cancer PC-3 cells relative to receptor-negative ovarian carcinoma SKOV-3 cells for potential application in drug delivery. Direct antiproliferative effect of these was investigated on three GnRH-receptor positive cancer cells, PC-3, LNCaP and DU145. A significant dose dependent growth inhibitory effect was produced in DU145 cells by 5 dendrimers giving an IC50 value of 22–35μM. All compounds were non-toxic to the normal peripheral blood mononuclear cells. From the Clinical EditorThis study demonstrates the use of specific dendritic lapidated GnRH analogues in growth inhibition of GnRH receptor positive prostate cancer cell lines, suggesting potential future clinical use of this or similar strategies to address GnRH receptor positive cancer cells.

Apoptotic Effect of STAMP2 Transfection to PC3 Cells and Caspase Induction with Hydrogen Peroxide Incubation at Lncap Cells- Role of P53

STAMP genes STAMP1/STEAP2 and STAMP2/STEAP4 are expressed in androgen receptor positive-prostate cancer cell line LNCaP, and this regulation was reported, previously. The androgen induction was done at LNCaP cells. STAMP2/STEAP4 was silenced for 1, 2 and 5 days. Using real-time RT-PCR, an apoptosis panel including proapoptotic and/or apoptotic genes was investigated for gene expression alterations in STAMP expressing LNCaP cells in comparison to P53-null PC3 cells in which STAMP2 gene was transfected. In addition, promoter region of the STAMP2 gene was analyzed and putative p53 and androgen receptor (AR) response elements were identified. Thus, p53 gene was silenced using siRNA approach and was induced by hydrogen peroxide. Taken together, prostate-specific STAMP2 gene and its regulation with the p53- and caspase-related pathway was characterized. These results will shed light on understanding the control of proliferation in neoplastic transformation in prostate cancer.

Modulators of Prostate Cancer Cell Proliferation and Viability Identified by Short-Hairpin RNA Library Screening

There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

Abstract 2101: PMEPA1 co-operates with the androgen responsive gene, NEDD4-1, to control the androgen receptor levels in human prostate cancer cells

Abstract Androgen receptor (AR) is a male hormone receptor and a nuclear transcriptional factor that plays central role in the differentiation and growth of the prostate gland and alterations of the AR network contribute to prostate tumorigenesis. The stability of the AR protein has been known to be regulated by the MDM2 E3 ubiquitin ligase. Recent investigations from our group suggest the role of PMEPA1-NEDD4-1 negative feedback loop in the regulation of AR protein levels in prostate cancer cells. In the PMEPA1-NEDD4-1 feedback loop, AR transcriptionally activates PMEPA1, which in turn provides a docking platform to recruit NEDD4-1 E3 ubiquitin ligase, to facilitate the proteasome dependent degradation of the AR. We hypothesize that decreased or absent PMPEA1 expression observed in more than 65% for prostate tumors may lead to the gain of AR functions which contribute to prostate tumorigenesis. Here we further investigate how PMEPA1 recruits NEDD4-1 to alter AR protein levels and AR signaling in prostate cancer cells. Androgen receptor positive prostate cancer cells lines, LNCaP and VCaP, were treated with synthetic androgen R1881 for 24 hours. The androgen receptor, PMEPA1 and NEDD4-1 were knocked down with specific small interfering RNA (siRNA) molecules. Wild-type AR and mt AR (T877A) were ectopically expressed in LNCaP cells with adenoviral expression vectors. Protein levels of AR, PMEPA1, NEDD4-1, as well as AR responsive gene PSA were assessed by immunoblot assays. Our new observations revealed that that NEDD4-1 was androgen inducible in both LNCaP and VCaP cells. Over-expression of wild-type and mutant AR (T877A) resulted in the upregulation of NEDD4-1 in LNCaP cells. Consistent with these findings, inhibition of AR by siRNA lead to down-regulation of NEDD4-1. As expected, loss of PMEPA1 in LNCaP cells induced up-regulation of AR and NEDD4-1 in an androgen dose-dependent manner. Knockdown of NEDD4-1, rather than NEDD4-2, resulted in elevation of androgen receptor levels and decreases of PMEPA1 expression in LNCaP cells. Our data suggest that NEDD4-1 is an androgen inducible gene and a complex inter-relationship between PMEPA1, NEDD4-1 and AR. Intriguingly, the AR degradation function of NEDD4-1 depends on the availability of PMEPA1. Thus PMEPA1-NEDD4-1 co-operation is critical for the regulation of androgen receptor protein levels. In conclusion, loss of PMEPA1 in human prostate cancer may also lead to the activation of NEDD4-1 through elevated AR signaling, representing a feed-forward event in tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2101. doi:10.1158/1538-7445.AM2011-2101

623 PMEPA1 CO-OPERATES WITH THE ANDROGEN RESPONSIVE GENE, NEDD4-1, TO CONTROL THE ANDROGEN RECEPTOR LEVELS IN HUMAN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011623 PMEPA1 CO-OPERATES WITH THE ANDROGEN RESPONSIVE GENE, NEDD4-1, TO CONTROL THE ANDROGEN RECEPTOR LEVELS IN HUMAN PROSTATE CANCER CELLS Hua Li, Albert Dobi, and Shiv Srivastava Hua LiHua Li Rockville, MD More articles by this author , Albert DobiAlbert Dobi Rockville, MD More articles by this author , and Shiv SrivastavaShiv Srivastava Rockville, MD More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1476AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen receptor (AR) is a male hormone receptor and a nuclear transcriptional factor that plays central role in the differentiation and growth of the prostate gland and alterations of the AR network contribute to prostate tumorigenesis. The stability of the AR protein has been known to be regulated by the MDM2 E3 ubiquitin ligase. Recent investigations suggest the role of PMEPA1-NEDD4-1 negative feedback loop in the regulation of AR protein levels in prostate cancer cells. In the PMEPA1-NEDD4-1 feedback loop, AR transcriptionally activates PMEPA1, which in turn provides a docking platform to recruit NEDD4-1 E3 ubiquitin ligase, to facilitate the proteasome dependent degradation of the AR. We hypothesize that decreased or absent PMPEA1 expression observed in more than 65% for prostate tumors may lead to the gain of AR functions which contribute to prostate tumorigenesis. Here we further investigate how PMEPA1 recruits NEDD4-1 to alter AR protein levels and AR signaling in prostate cancer cells. METHODS Androgen receptor positive prostate cancer cells lines, LNCaP and VCaP, were treated with synthetic androgen R1881 for 24 hours. The androgen receptor, PMEPA1 and NEDD4-1 were knocked down with specific small interfering RNA (siRNA) molecules. Wild-type AR and mt AR (T877A) were ectopically expressed in LNCaP cells with adenoviral expression vectors. Protein levels of AR, PMEPA1, NEDD4-1, as well as AR responsive gene PSA were assessed by immunoblot assays. RESULTS Our new observations revealed that that NEDD4-1 was androgen inducible in both LNCaP and VCaP cells. Over-expression of wild-type and mutant AR (T877A) resulted in the upregulation of NEDD4-1 in LNCaP cells. Consistent with these findings, inhibition of AR by siRNA lead to down-regulation of NEDD4-1. As expected, loss of PMEPA1 in LNCaP cells induced up-regulation of AR and NEDD4-1 in an androgen dose-dependent manner. Knockdown of NEDD4-1, rather than NEDD4-2, resulted in elevation of androgen receptor levels and decreases of PMEPA1 expression in LNCaP cells. Our data suggest that NEDD4-1 is an androgen inducible gene and a complex inter-relationship between PMEPA1, NEDD4-1 and AR. Intriguingly, the AR degradation function of NEDD4-1 depends on the availability of PMEPA1. Thus PMEPA1-NEDD4-1 co-operation is critical for the regulation of androgen receptor protein levels. CONCLUSIONS In conclusion, loss of PMEPA1 in human prostate cancer may also lead to the activation of NEDD4-1 through elevated AR signaling, representing a feed-forward event in tumorigenesis. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e251 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hua Li Rockville, MD More articles by this author Albert Dobi Rockville, MD More articles by this author Shiv Srivastava Rockville, MD More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Altered endocytosis of epidermal growth factor receptor in androgen receptor positive prostate cancer cell lines

Although androgens and the androgen receptor (AR) are involved in tumorigenesis of prostate cancer (PC) in initial phases, less clear is the role played in advanced androgen-independent (AI) stages of the disease. Several recent reports indicated that re-expression of AR in PC-derived cell lines determines a less aggressive phenotype of the cells. We have previously demonstrated that re-expression of AR decreases the invasion ability of PC3 cells in vitro by affecting signalling and internalization processes of epidermal growth factor receptor (EGFR). Here, we show that reduced EGFR internalization is also a characteristic of AR positive PC cell lines LNCaP and 22Rv1. Reduced internalization in PC3-AR cells is associated to a defective interaction between the EGFR and two adaptor proteins which mediate the endocytotic process, Grb2 and c-Cbl. As a consequence of such reduced interaction, ubiquitination of the receptor, which is mainly mediated by c-Cbl, is also altered. In addition, we show that internalized EGFR co-localizes with early endosome antigen-1, a marker of clathrin-mediated endocytosis, in PC3-Neo cells but not in AR positive cell lines. Conversely, EGFR maintains co-localization with caveolin-1 after EGF stimulation in PC3-AR cells. These data suggest that expression of AR affects clathrin-mediated endocytosis pathway of EGFR, which, according to recent findings, plays an essential role in the completeness of signalling of the receptor. Taken together, these data emphasize the role of AR in the regulation of EGFR endocytotic trafficking and active signalling in PC cells. In view of the role of EGFR signalling in invasion of carcinoma cells, our data may explain the lower invasive phenotype observed in AR-positive cell lines.

Mechanisms of Cell Death Induced by Histone Deacetylase Inhibitors in Androgen Receptor–Positive Prostate Cancer Cells

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.

Radio sensitivity of human prostate cancer and malignant melanoma cell lines

The relative radioresponsiveness of human prostate cancer compared to malignant melanoma is well known. The effects of β-estradiol or testosterone on the X-irradiation survival of several human cell lines were studied, including: human prostate carcinoma cell lines PC3 and DU145 and human malignant melanoma cell lines A375 and A875. Lines PC3 and DU 145 demonstrated 55–61 fmol per 10 6 cells of androgen receptor with no detectable estrogen or progesterone receptor. Cells were irradiated at 120 cGy/min dose rate. There was no detectable toxicity of up to 10 −4 M testosterone or β-estradiol on PC3 or DU 145 cells in the absence of X-irradiation. At plating efficiencies from 11–13%, and plating densities of 1 × 10 4 cells per 60 cm 2 flask, cell lines PC3 and DU145 demonstrated a D o of 108.5 ± 6.5, n 2.1 ± 0.7 cGy, and D o of 143.5 ± 1.5 cGy, n 2.4 ± 0.5, respectively. The addition of testosterone or β-estradiol at 10 −4 to 10 −10 M prior to or after, X-irradiation did not alter radiosensitivity. At the same dose rate of 120 cGy/min, malignant melanoma cell lines A375 and A875 had a D o of 125 ± 2.5 cGy, n 1.56 ± 0.8 SF 2 0.65 ± 0.03 and line A875 demonstrated a D o of 129 ± 4.5 cGy, n 1.58 ± 0.4 SF 2 0.55 ± 0.04, respectively. The radiosensitivity of melanoma cell lines did not decrease at low dose rate 5 cGy/min. Thus, the in vitro radiosensitivity of androgen receptor positive prostate cancer cell lines is not necessarily altered by the presence of androgen before or after irradiation. The data support the concept that all malignant melanoma cell lines do not show a broad-shouldered cell survival curve in vitro and intrinsic cellular radioresistance.