Lingru Xue

An androgen-regulated miRNA suppresses Bak1 expression and induces androgen-independent growth of prostate cancer cells

Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor and the second leading cause of cancer deaths in American men, the mechanisms explaining the development and progression of CaP remain largely unknown. Recent studies have shown that some aberrantly expressed microRNAs (miRNAs) are involved in tumorigenesis. Although aberrant expression of certain miRNAs has been discovered in CaP, their function in this disease has not yet been defined. In this study, we found differential expression of miR-125b in androgen-dependent and independent CaP cells, as well as in benign and malignant prostate tissues. Furthermore, androgen signaling was able to up-regulate the expression of miR-125b. In addition, transfection of synthetic miR-125b stimulated androgen-independent growth of CaP cells and down-regulated the expression of Bak1. Our results suggest that miR-125b acts as an oncogene, contributing to the pathogenesis of CaP.

miR-125b promotes growth of prostate cancer xenograft tumor through targeting pro-apoptotic genes.

Increasing evidence demonstrates that aberrantly regulated microRNAs (miRNAs) contribute to the initiation and progression of human cancer. We previously have demonstrated that miR‐125b stimulated the growth of prostate cancer (CaP) cells. In this study, we further determined the influence of miR‐125b on the pathogenesis of CaP.

Tumor suppressive miR-124 targets androgen receptor and inhibits proliferation of prostate cancer cells

Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor in American men, the mechanisms underlying the development and progression of CaP remain largely unknown. Recent studies have shown that downregulation of the microRNA miR-124 occurs in several types of human cancer, suggesting a tumor suppressive function of miR-124. Until now, however, it has been unclear whether miR-124 is associated with CaP. In the present study, we completed a series of experiments to understand the functional role of miR-124 in CaP. We detected the expression level of miR-124 in clinical CaP tissues, evaluated the influence of miR-124 on the growth of CaP cells and investigated the mechanism underlying the dysregulation of miR-124. We found that (i) miR-124 directly targets the androgen receptor (AR) and subsequently induces an upregulation of p53; (ii) miR-124 is significantly downregulated in malignant prostatic cells compared to benign cells, and DNA methylation causes the reduced expression of miR-124; and (iii) miR-124 can inhibit the growth of CaP cells in vitro and in vivo. Data from this study revealed that loss of miR-124 expression is a common event in CaP, which may contribute to the pathogenesis of CaP. Our studies also suggest that miR-124 is a potential tumor suppressive gene in CaP, and restoration of miR-124 expression may represent a novel strategy for CaP therapy.

Oncomir miR-125b Suppresses p14ARF to Modulate p53-Dependent and p53-Independent Apoptosis in Prostate Cancer

MicroRNAs are a class of naturally occurring small non-coding RNAs that target protein-coding mRNAs at the post-transcriptional level and regulate complex patterns of gene expression. Our previous studies demonstrated that in human prostate cancer the miRNA miR-125b is highly expressed, leading to a negative regulation of some tumor suppressor genes. In this study, we further extend our studies by showing that miR-125b represses the protein product of the ink4a/ARF locus, p14ARF, in two prostate cancer cell lines, LNCaP (wild type-p53) and 22Rv1 (both wild type and mutant p53), as well as in the PC-346C prostate cancer xenograft model that lentivirally overexpressed miR-125b. Our results highlight that miR-125b modulates the p53 network by hindering the down-regulation of Mdm2, thereby affecting p53 and its target genes p21 and Puma to a degree sufficient to inhibit apoptosis. Conversely, treatment of prostate cancer cells with an inhibitor of miR-125b (anti-miR-125b) resulted in increased expression of p14ARF, decreased level of Mdm2, and induction of apoptosis. In addition, overexpression of miR-125b in p53-deficient PC3 cells induced down-regulation of p14ARF, which leads to increased cell proliferation through a p53-independent manner. Thus, we conclude that miR-125b acts as an oncogene which regulates p14ARF/Mdm2 signaling, stimulating proliferation of prostate cancer cells through a p53-dependent or p53-independent function. This reinforces our belief that miR-125b has potential as a therapeutic target for the management of patients with metastatic prostate cancer.

The R273H p53 mutation can facilitate the androgen-independent growth of LNCaP by a mechanism that involves H2 relaxin and its cognate receptor LGR7

Mutations in p53 occur at a rate of approximately 70% in hormone-refractory prostate cancer (CaP), suggesting that p53 mutations facilitate the progression of CaP to androgen-independent (AI) growth. We have previously reported that transfection of p53 gain of function mutant alleles into LNCaP, an androgen-sensitive cell line, allows for AI growth of LNCaP in vitro. We herein confirm the in vivo relevance of those findings by demonstrating that the R273H p53 mutation (p53R273H) facilitates AI growth in castrated nude mice. In addition, we demonstrate that H2 relaxin is responsible for facilitating p53R273H-mediated AI CaP. H2 relaxin is overexpressed in the LNCaP-R273H subline. Downregulation of H2 relaxin expression results in significant inhibition of AI growth, whereas addition of recombinant human H2 relaxin to parental LNCaP promotes AI growth. Inhibition of AI growth was also achieved by blocking expression of LGR7, the cognate receptor of H2 relaxin. Chromatin immunoprecipitation analysis was used to demonstrate that p53R273H binds directly to the relaxin promoter, further confirming a role for H2 relaxin signaling in p53R273H-mediated AI CaP. Lastly, we used a reporter gene assay to demonstrate that H2 relaxin can induce the expression of prostate-specific antigen via an androgen receptor-mediated pathway.

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.

miR-124 and androgen receptor signaling inhibitors repress prostate cancer growth by downregulating androgen receptor splice variants, EZH2 and Src

miR-124 targets the androgen receptor (AR) transcript, acting as a tumor suppressor to broadly limit the growth of prostate cancer. In this study, we unraveled the mechanisms through which miR-124 acts in this setting. miR-124 inhibited proliferation of prostate cancer cells in vitro and sensitized them to inhibitors of androgen receptor signaling. Notably, miR-124 could restore the apoptotic response of cells resistant to enzalutamide, a drug approved for the treatment of castration-resistant prostate cancer. We used xenograft models to examine the effects of miR-124 in vivo when complexed with polyethylenimine-derived nanoparticles. Intravenous delivery of miR-124 was sufficient to inhibit tumor growth and to increase tumor cell apoptosis in combination with enzalutamide. Mechanistic investigations revealed that miR-124 directly downregulated AR splice variants AR-V4 and V7 along with EZH2 and Src, oncogenic targets that have been reported to contribute to prostate cancer progression and treatment resistance. Taken together, our results offer a preclinical rationale to evaluate miR-124 for cancer treatment.

Prolonged androgen receptor loading onto chromatin and the efficient recruitment of p160 coactivators contribute to androgen-independent growth of prostate cancer cells.

Growth of most ablation‐resistant prostate cancers (CaPs) is dependent on androgen receptor (AR) activity in chromatin, but cancer cells in these tumors have acquired altered AR activation. It is unclear how the aberrantly activated AR loads onto regulatory regions of AR‐targeted genes. The purpose of this study was to assess the AR chromatin loading in an androgen‐depleted environment.

Development of a stress response therapy targeting aggressive prostate cancer

The PERK-eIF2α pathway is activated in aggressive prostate cancer and associated with patient outcome, providing a therapeutic target for the disease. Stressing out prostate cancer As tumors grow, they undergo a variety of metabolic changes that facilitate their proliferation. Protein synthesis is one of the cellular processes that is altered in cancer cells, because its continued activation helps drive cancer growth. This is not a benign adaptation, however, and unchecked up-regulation of protein synthesis can be toxic to the cells because it promotes cellular stress. As Nguyen et al. discovered, prostate cancer cells with a specific combination of mutations can override this stress by activating a protein called eIF2α, which protects them from excessive protein synthesis. To target this pathway, the authors identified an inhibitor of eIF2α that blocks this protective mechanism and has therapeutic activity against aggressive and otherwise untreatable prostate cancer. Oncogenic lesions up-regulate bioenergetically demanding cellular processes, such as protein synthesis, to drive cancer cell growth and continued proliferation. However, the hijacking of these key processes by oncogenic pathways imposes onerous cell stress that must be mitigated by adaptive responses for cell survival. The mechanism by which these adaptive responses are established, their functional consequences for tumor development, and their implications for therapeutic interventions remain largely unknown. Using murine and humanized models of prostate cancer (PCa), we show that one of the three branches of the unfolded protein response is selectively activated in advanced PCa. This adaptive response activates the phosphorylation of the eukaryotic initiation factor 2–α (P-eIF2α) to reset global protein synthesis to a level that fosters aggressive tumor development and is a marker of poor patient survival upon the acquisition of multiple oncogenic lesions. Using patient-derived xenograft models and an inhibitor of P-eIF2α activity, ISRIB, our data show that targeting this adaptive brake for protein synthesis selectively triggers cytotoxicity against aggressive metastatic PCa, a disease for which presently there is no cure.

Influence of Short Polyglutamine Tracts and p160 Coactivators on the Transactivation of the Androgen Receptor

The androgen receptor (AR) acting as a transcription factor plays a pivotal role in the occurrence and progression of prostate cancer (CaP). Several AR-related factors or modulators have been reported to influence AR activity. Whether and how these factors cooperatively modulate the AR activity has not been well defined. In the present study, the combined effect of p160 coactivators, short CAG length (encoding a short polyQ tract), and AR mutations on AR transactivation in a yeast system was evaluated. It was found that the short polyQ tract can upregulate the transactivation of the wild-type (WT) AR and partial-function (PF) AR mutants in response to a physiological level (10(-9) M) of dihydrotestosterone. Addition of a p160 coactivator (SRC-1 or TIF2) to the above systems resulted in a significant increase in the ligand-stimulated transactivation. Although the androgen antagonist bicalutamide could suppress the activity of androgen-activated WT or PF ARs, it was unable to do so for gain-of-function AR mutants. A combination of the short polyQ tract and coactivator TIF2 acted cooperatively on the WT AR and PF AR mutants to enhance their transactivation in response to either a low level of dihydrotestosterone (10(-10) M) or adrenal dehydroepiandrosterone. Taken together, this finding suggests that the modulated AR activity may involve early in the carcinogenesis of CaP. Additionally, these data support the concept that a given CaP in which the AR activity is modulated by multiple AR modulators may progress more readily to castrate resistance.