Ruth L. Vinall

Glycomic Approach for Potential Biomarkers on Prostate Cancer: Profiling of N-Linked Glycans in Human Sera and pRNS Cell Lines

Prostate cancer is a leading cause of cancer death among men. Currently available screening test measures prostate-specific antigen (PSA) to detect prostate cancer. However, this test produces false positive values that often lead to negative biopsies. Therefore, a more reliable diagnostic tool is needed. Glycans in serum are of particular interest as around half of all proteins are glycosylated. In this study, N-linked glycans were enzymatically released by PNGase F from prostate epithelial cell lines (pRNS) expressing wild type or mutant androgen receptors and a small set of human serum samples. Released glycans were purified and partitioned into neutral and acidic components by solid phase extraction (SPE) using graphitized carbon cartridges. The SPE fractions were analyzed by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FT-ICR MS). Significant changes in some high-mannose and fucosylated biantennary complex N-linked glycans were observed in the serum of prostate cancer patients.

MicroRNAs and their potential for translation in prostate cancer

OBJECTIVE Patients die of prostate cancer (CaP) because predictably after a period of response to androgen withdrawal, their CaP becomes castrate resistant. In this paper, we discuss the role that microRNAs (miRNAs) may play in this process. METHODS miRNAs are a group of endogenous, small non-coding RNA molecules that are thought to be responsible for the regulation of up to 30% of gene expression. The miRNA expression profile between androgen responsive and castrate resistant CaP cell lines is compared. Functional studies were carried out to identify the importance of the miRNA targets in controlling this process. RESULTS There were 17 differentially expressed miRNAs found, 10 up-regulated and 7 down-regulated. Among these, miRNA-125b was found to have the ability of rendering LNCaP cells resistant to androgen withdrawal. It was found to be androgen regulated and one of its targets, BAK1, was identified as being involved in how these CaP cells undergo apoptosis functionally. CONCLUSION miRNA-125b, at least in the CaP cell lines tested, is involved in the development of castrate resistance. While clearly this miRNA is only part of the answer, miRNAs may lead us in a new direction in trying to solve the central problem in CaP.

MiR-34a chemosensitizes bladder cancer cells to cisplatin treatment regardless of p53-Rb pathway status.

MiR‐34a is a downstream effector of p53 that has been shown to target several molecules associated with cell cycle and cell survival pathways. As alterations in these pathways are frequent in muscle invasive transitional cell carcinoma of the bladder (MI‐TCC), for example mutation or loss of p53 and Rb, the goal of this study was to determine whether manipulation of miR‐34a expression levels could abrogate the effect of these alterations and sensitize bladder cancer cells to chemotherapy. We demonstrate that transfection of T24, TCCSUP and 5637 with pre‐miR‐34a followed by cisplatin treatment results in a dramatic reduction in clonogenic potential and induction of senescence compared to treatment with cisplatin alone. Molecular analyses identified Cdk6 and sirtuin (SIRT)‐1 as being targeted by miR‐34a in MI‐TCC cells, however, inhibition of Cdk6 and SIRT‐1 was not as effective as pre‐miR‐34a in mediating chemosensitization. Analysis of 27 preneoadjuvant chemotherapy patient samples revealed many of the patients who subsequently did not respond to treatment (based on surgical resection postchemotherapy and 5‐year survival data) express lower levels of miR‐34a, however, a statistically significant difference between the responder and nonresponder groups was not observed (p = 0.1174). Analysis of eight sets of pre‐ and postneoadjuvant chemotherapy patient samples determined miR‐34a expression increased postchemotherapy in only two of the eight patients. The combined data indicate that elevation of miR‐34a expression levels before chemotherapy would be of benefit to MI‐TCC patients, particularly in a setting of low miR‐34a expression.

Dual EGFR/HER2 Inhibition Sensitizes Prostate Cancer Cells to Androgen Withdrawal by Suppressing ErbB3

Purpose: Patients with recurrent prostate cancer are commonly treated with androgen withdrawal therapy (AWT); however, almost all patients eventually progress to castration resistant prostate cancer (CRPC), indicating failure of AWT to eliminate androgen-sensitive prostate cancer. The overall goal of these studies is to determine whether dual inhibition of the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and HER2 would prolong the effectiveness of this treatment in prostate cancer. Experimental Design: We used androgen-dependent LNCaP cells and its CRPC sublines LNCaP-AI and C4-2. Additional data were collected in pRNS-1-1 cells stably expressing a mutant androgen receptor (AR-T877A), and in nude mice harboring CWR22 tumors. Studies utilized EGFR inhibitors erlotinib and AG1478, and HER2 inhibitors trastuzumab and AG879. Results: Dual EGFR/HER2 inhibition induced apoptosis selectively in androgen-sensitive prostate cancer cells undergoing AWT, but not in the presence of androgens, or in CRPC cells. We show that AWT alone failed to induce significant apoptosis in androgen-dependent cells, due to AWT-induced increase in HER2 and ErbB3, which promoted survival by increasing Akt phosphorylation. AWT-induced ErbB3 stabilized the AR and stimulated PSA, while it was inactivated only by inhibition of both its dimerization partners EGFR and HER2 (prostate cancer cells do not express ErbB4); but not the inhibition of any one receptor alone, explaining the success of dual EGFR/HER2 inhibition in sensitizing androgen-dependent cells to AWT. The effectiveness of the inhibitors in suppressing growth correlated with its ability to prevent Akt phosphorylation. Conclusion: These studies indicate that dual EGFR/HER2 inhibition, administered together with AWT, sensitize prostate cancer cells to apoptosis during AWT. Clin Cancer Res; 17(19); 6218–28. ©2011 AACR.

Inappropriate activation of androgen receptor by relaxin via Β-catenin pathway

We have previously demonstrated that human H2-relaxin can mediate androgen-independent growth of LNCaP through a mechanism that involves the activation of the androgen receptor (AR) signaling pathway. The goal of the current study is to elucidate the mechanism(s) by which H2-relaxin causes activation of the AR pathway. Our data indicate that there is cross-talk between AR and components of the Wnt signaling pathway. Addition of H2-relaxin to LNCaP cells resulted in increased phosphorylation of protein kinase B (Akt) and inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β) with subsequent cytoplasmic accumulation of β-catenin. Immunoprecipitation and immunocytochemical studies demonstrated that the stabilized β-catenin formed a complex with AR, which was then translocated into the nucleus. Chromatin immunoprecipitation analysis determined that the AR/β-catenin complex binds to the proximal region of the prostate-specific antigen promoter. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, using LY294002, prevented both H2-relaxin-mediated phosphorylation of Akt and GSK-3β and translocation of β-catenin/AR into the nucleus. Knockdown of β-catenin levels using a β-catenin-specific small interfering RNA inhibited H2-relaxin-induced AR activity. The combined data demonstrate that PI3K/Akt and components of the Wnt pathway can facilitate H2-relaxin-mediated activation of the AR pathway.

Nrdp1-mediated regulation of ErbB3 expression by the androgen receptor in androgen-dependent but not castrate-resistant prostate cancer cells.

Patients with advanced prostate cancer (PCa) are initially susceptible to androgen withdrawal (AW), but ultimately develop resistance to this therapy (castration-resistant PCa, CRPC). Here, we show that AW can promote CRPC development by increasing the levels of the receptor tyrosine kinase ErbB3 in androgen-dependent PCa, resulting in AW-resistant cell cycle progression and increased androgen receptor (AR) transcriptional activity. CRPC cell lines and human PCa tissue overexpressed ErbB3, whereas downregulation of ErbB3 prevented CRPC cell growth. Investigation of the mechanism by which AW augments ErbB3, using normal prostate-derived pRNS-1-1 cells, and androgen-dependent PCa lines LNCaP, PC346C, and CWR22 mouse xenografts, revealed that the AR suppresses ErbB3 protein levels, whereas AW relieves this suppression, showing for the first time the negative regulation of ErbB3 by AR. We show that AR activation promotes ErbB3 degradation in androgen-dependent cells, and that this effect is mediated by AR-dependent transcriptional upregulation of neuregulin receptor degradation protein-1 (Nrdp1), an E3 ubiquitin ligase that targets ErbB3 for degradation but whose role in PCa has not been previously examined. Therefore, AW decreases Nrdp1 expression, promoting ErbB3 protein accumulation, and leading to AR-independent proliferation. However, in CRPC sublines of LNCaP and CWR22, which strongly overexpress the AR, ErbB3 levels remain elevated due to constitutive suppression of Nrdp1, which prevents AR regulation of Nrdp1. Our observations point to a model of CRPC development in which progression of PCa to castration resistance is associated with the inability of AR to transcriptionally regulate Nrdp1, and predict that inhibition of ErbB3 during AW may impair CRPC development.

Combination Treatment of Prostate Cancer Cell Lines with Bioactive Soy Isoflavones and Perifosine Causes Increased Growth Arrest and/or Apoptosis

Purpose: To determine whether targeting the androgen receptor (AR) and Akt pathways using a combination of genistein combined polysaccharide (GCP) and perifosine is more effective at inducing growth arrest/apoptosis in prostate cancer cells compared with treatment with GCP or perifosine as single agents. Experimental Design: The effect of GCP and perifosine treatment was assessed in five prostate cancer cell lines: LNCaP (androgen sensitive), LNCaP-R273H, C4-2, Cds1, and PC3 (androgen insensitive). A clonogenic assay assessed the long-term effects on cell growth and survival. Flow cytometry and Western blot analysis of poly(ADP)ribose polymerase cleavage were used to assess short-term effects. Preliminary studies to investigate mechanism of action included Western blot for P-Akt, Akt, P-p70S6K, p70S6K, p53, and p21; prostate-specific antigen analysis; and the use of myristoylated Akt and AR-specific small interfering RNA. Results: Combination treatment with GCP and perifosine caused a decrease in clonogenic potential in all cell lines. In short-term assays, growth arrest was observed in the majority of cell lines, as well as increased inhibition of Akt activity and induction of p21 expression. Increased apoptosis was only observed in LNCaP. Knockdown of AR caused a further increase in apoptosis. Conclusion: Combination treatment with GCP and perifosine targets the Akt pathway in the majority of the prostate cancer cell lines and causes increased inhibition of cell growth and clonogenicity. In LNCaP, combination treatment targets both the Akt and AR pathways and causes increased apoptosis. These data warrant clinical validation in prostate cancer patients.

Dual Blockade of PKA and NF–κB Inhibits H2 Relaxin-Mediated Castrate-Resistant Growth of Prostate Cancer Sublines and Induces Apoptosis

We previously demonstrated that H2 relaxin (RLN2) facilitates castrate-resistant (CR) growth of prostate cancer (CaP) cells through PI3K/Akt/β-catenin-mediated activation of the androgen receptor (AR) pathway. As inhibition of this pathway caused only ~50% reduction in CR growth, the goal of the current study was to identify additional RLN2-activated pathways that contribute to CR growth. Next-generation sequencing-based transcriptome and gene ontology analyses comparing LNCaP stably transfected with RLN2 versus LNCaP-vector identified differential expression of genes associated with cell proliferation (12.7% of differentially expressed genes), including genes associated with the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and nuclear factor–kappaB (NF–κB) pathways. Subsequent molecular analyses confirmed that the cAMP/PKA and NF–κB pathways play a role in facilitating H2 relaxin-mediated CR growth of CaP cells. Inhibition of PKA-attenuated RLN2-mediated AR activity inhibited proliferation and caused a small but significant increase in apoptosis. Combined inhibition of the PKA and NF–κB signaling pathways via inhibition of PKA and Akt induced significant apoptosis and dramatically reduced clonogenic potential, outperforming docetaxel, the standard of care treatment for CR CaP. Immunohistochemical analysis of tissue microarrays in combination with multispectral quantitative imaging comparing RLN2 levels in patients with benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia, and CaP determined that RLN2 is significantly upregulated in CaP vs BPH (p = 0.002). The combined data indicate RLN2 overexpression is frequent in CaP patients and provides a growth advantage to CaP cells. A near-complete inhibition of RLN2-induced CR growth can be achieved by simultaneous blockade of both pathways.

The Role of EGFR Family Inhibitors in Muscle Invasive Bladder Cancer: A Review of Clinical Data and Molecular Evidence

PURPOSE Conventional platinum based chemotherapy for advanced urothelial carcinoma is plagued by common resistance to this regimen. Several studies implicate the EGFR family of RTKs in urothelial carcinoma progression and chemoresistance. Many groups have investigated the effects of inhibitors of this family in patients with urothelial carcinoma. This review focuses on the underlying molecular pathways that lead to urothelial carcinoma resistance to EGFR family inhibitors. MATERIALS AND METHODS We performed a PubMed® search for peer reviewed literature on bladder cancer development, EGFR family expression, clinical trials of EGFR family inhibitors and molecular bypass pathways. Research articles deemed to be relevant were examined and a summary of original data was created. Meta-analysis of expression profiles was also performed for each EGFR family member based on data sets accessible via Oncomine®. RESULTS Many clinical trials using inhibitors of EGFR family RTKs have been done or are under way. Those that have concluded with results published to date do not show an added benefit over standard of care chemotherapy in an adjuvant or second line setting. However, a neoadjuvant study using erlotinib before radical cystectomy demonstrated promising results. CONCLUSIONS Clinical and preclinical studies show that for reasons not currently clear prior treatment with chemotherapeutic agents rendered patients with urothelial carcinoma with muscle invasive bladder cancer resistant to EGFR family inhibitors as well. However, EGFR family inhibitors may be of use in patients with no prior chemotherapy in whom EGFR or ERBB2 is over expressed.

Genistein combined polysaccharide enhances activity of docetaxel, bicalutamide and Src kinase inhibition in androgen-dependent and independent prostate cancer cell lines.

To determine the benefit of genistein combined polysaccharide (GCP) in combination with the androgen receptor antagonist bicalutamide, the antimicrotubule taxane docetaxel, and the Src kinase inhibitor pp2 as part of a treatment regimen for advanced prostate cancer (CaP).