Rongbin Ge

Personalized Medicine for the Management of Benign Prostatic Hyperplasia

PURPOSE Benign prostatic hyperplasia affects more than 50% of men by age 60 years, and is the cause of millions of dollars in health care expenditure for the treatment of lower urinary tract symptoms and urinary obstruction. Despite the widespread use of medical therapy, there is no universal therapy that treats all men with symptomatic benign prostatic hyperplasia. At least 30% of patients do not respond to medical management and a subset require surgery. Significant advances have been made in understanding the natural history and development of the prostate, such as elucidating the role of the enzyme 5α-reductase type 2, and advances in genomics and biomarker discovery offer the potential for a more targeted approach to therapy. We review the current understanding of benign prostatic hyperplasia progression as well as the key genes and signaling pathways implicated in the process such as 5α-reductase. We also explore the potential of biomarker screening and gene specific therapies as tools to risk stratify patients with benign prostatic hyperplasia and identify those with symptomatic or medically resistant forms. MATERIALS AND METHODS A PubMed® literature search of current and past peer reviewed literature on prostate development, lower urinary tract symptoms, benign prostatic hyperplasia pathogenesis, targeted therapy, biomarkers, epigenetics, 5α-reductase type 2 and personalized medicine was performed. An additional Google Scholar™ search was conducted to broaden the scope of the review. Relevant reviews and original research articles were examined, as were their cited references, and a synopsis of original data was generated with the goal of informing the practicing urologist of these advances and their implications. RESULTS Benign prostatic hyperplasia is associated with a state of hyperplasia of the stromal and epithelial compartments, with 5α-reductase type 2 and androgen signaling having key roles in the development and maintenance of the prostate. Chronic inflammation, multiple growth factor and hormonal signaling pathways, and medical comorbidities have complex roles in prostate tissue homeostasis as well as its evolution into the clinical state of benign prostatic hyperplasia. Resistance to medical therapy with finasteride may occur through silencing of the 5α-reductase type 2 gene by DNA methylation, leading to a state in which 30% of adult prostates do not express 5α-reductase type 2. Novel biomarkers such as single nucleotide polymorphisms may be used to risk stratify patients with symptomatic benign prostatic hyperplasia and identify those at risk for progression or failure of medical therapy. Several inhibitors of the androgen receptor and other signaling pathways have recently been identified which appear to attenuate benign prostatic hyperplasia progression and may offer alternative targets for medical therapy. CONCLUSIONS Progressive worsening of lower urinary tract symptoms and bladder outlet obstruction secondary to benign prostatic hyperplasia is the result of multiple pathways including androgen receptor signaling, proinflammatory cytokines and growth factor signals. New techniques in genomics, proteomics and epigenetics have led to the discovery of aberrant signaling pathways, novel biomarkers, DNA methylation signatures and potential gene specific targets. As personalized medicine continues to develop, the ability to risk stratify patients with symptomatic benign prostatic hyperplasia, identify those at higher risk for progression, and seek alternative therapies for those in whom conventional options are likely to fail will become the standard of targeted therapy.

Inhibition of TNF-α Improves the Bladder Dysfunction That Is Associated With Type 2 Diabetes

Diabetic bladder dysfunction (DBD) is common and affects 80% of diabetic patients. However, the molecular mechanisms underlying DBD remain elusive because of a lack of appropriate animal models. We demonstrate DBD in a mouse model that harbors hepatic-specific insulin receptor substrate 1 and 2 deletions (double knockout [DKO]), which develops type 2 diabetes. Bladders of DKO animals exhibited detrusor overactivity at an early stage: increased frequency of nonvoiding contractions during bladder filling, decreased voided volume, and dispersed urine spot patterns. In contrast, older animals with diabetes exhibited detrusor hypoactivity, findings consistent with clinical features of diabetes in humans. The tumor necrosis factor (TNF) superfamily genes were upregulated in DKO bladders. In particular, TNF-α was upregulated in serum and in bladder smooth muscle tissue. TNF-α augmented the contraction of primary cultured bladder smooth muscle cells through upregulating Rho kinase activity and phosphorylating myosin light chain. Systemic treatment of DKO animals with soluble TNF receptor 1 (TNFRI) prevented upregulation of Rho A signaling and reversed the bladder dysfunction, without affecting hyperglycemia. TNFRI combined with the antidiabetic agent, metformin, improved DBD beyond that achieved with metformin alone, suggesting that therapies targeting TNF-α may have utility in reversing the secondary urologic complications of type 2 diabetes.

F-box protein 10, an NF-κB-dependent anti-apoptotic protein, regulates TRAIL-induced apoptosis through modulating c-Fos/c-FLIP pathway

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces selective apoptotic death of human cancer cells while sparing normal human cells. Although TRAIL holds great promise as a potential anticancer agent, some tumors develop resistance to TRAIL. Previously, we have shown that the activator protein 1 (AP-1) family member, c-Fos, is an important modulator of apoptosis. Although F- box protein 10 (FBXL10) has been implicated to regulate an AP-1 family protein, c-Jun, its role in mediating apoptotic pathways has not been previously investigated. Here, we report that FBXL10 is a transcriptional repressor of c-Fos and a target gene of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-p65 in human cancers. We demonstrate that FBXL10 is an important anti-apoptotic molecule, which directly binds and represses c-Fos promoter in order for cancer cells to resist TRAIL-induced apoptosis. FBXL10 indirectly regulates c-FLIP(L) levels via c-Fos-dependent pathways. Silencing of FBXL10 sensitizes resistant cells to TRAIL, while, overexpression of FBXL10 represses TRAIL-induced apoptosis. Moreover, our results indicate that expression of FBXL10 functions via an NF-κB-dependent pathway, and TRAIL or proteasome inhibitors downregulate FBXL10 via inhibiting NF-κB signaling. Taken together, we find a novel functional role for FBXL10 as an anti-apoptotic molecule, and describe a new apoptotic-related pathway that involves NF-κB/FBXL10/c-Fos/c-FLIP. Therefore, silencing FBXL10 can help overcome resistant cancer cells for pro-apoptotic therapies.

5α-Reductase inhibitors and risk of high-grade or lethal prostate cancer.

IMPORTANCE 5α-Reductase inhibitors (5ARIs) are widely used for benign prostatic hyperplasia despite controversy regarding potential risk of high-grade prostate cancer with use. Furthermore, the effect of 5ARIs on progression and prostate cancer death remains unclear. OBJECTIVE To determine the association between 5ARI use and development of high-grade or lethal prostate cancer. DESIGN, SETTING, AND PARTICIPANTS Prospective observational study of 38,058 men followed up for prostate cancer diagnosis and outcomes between 1996 and 2010 in the Health Professionals Follow-up Study. EXPOSURES Use of 5ARIs between 1996 and 2010. MAIN OUTCOMES AND MEASURES Cox proportional hazards models were used to estimate risk of prostate cancer diagnosis or development of lethal disease with 5ARI use, adjusting for possible confounders including prostate specific antigen testing. RESULTS During 448,803 person-years of follow-up, we ascertained 3681 incident prostate cancer cases. Of these, 289 were lethal (metastatic or fatal), 456 were high grade (Gleason sum [GS] 8-10), 1238 were GS 7, and 1600 were low grade (GS 2-6). A total of 2878 (7.6%) men reported use of 5ARIs between 1996 and 2010. After adjusting for confounders, men who reported ever using 5ARIs over the study period had a reduced risk of overall prostate cancer (hazard ratio [HR], 0.77; 95% CI, 0.65-0.91). 5ARI users had a reduced risk of GS 7 (HR, 0.67; 95% CI, 0.49-0.91) and low-grade (GS 2-6) prostate cancer (HR, 0.74; 95% CI, 0.57-0.95). 5ARI use was not associated with risk of high-grade (GS 8-10) prostate cancer (HR, 0.97; 95% CI, 0.64-1.46) or lethal disease (HR, 0.99; 95% CI, 0.58-1.69). Increased duration of use was associated with significantly lower risk of overall prostate cancer (HR for 1 year of additional use, 0.95; 95% CI, 0.92-0.99), localized (HR, 0.95; 95% CI, 0.90-1.00), and low-grade disease (HR, 0.92; 95% CI, 0.85-0.99). There was no association for lethal, high-grade, or grade 7 disease. CONCLUSIONS AND RELEVANCE While 5ARI use was not associated with developing high-grade or lethal prostate cancer, it was associated with a reduction in low-grade, GS 7, and overall prostate cancer. Because the number of patients with high-grade or lethal prostate cancer in our cohort was limited, we cannot rule out potential risk of harm with 5ARI use.

Reduced levels of 5-α reductase 2 in adult prostate tissue and implications for BPH therapy

5‐α reductase 2 (5‐AR 2) is a key enzyme that is responsible of proper development of prostate tissue. Inhibition of 5‐AR 2 has proven to be efficacious for management of urinary symptoms secondary benign prostatic hyperplasia (BPH). However, some patients are resistant to the therapeutic effects of 5‐AR 2 inhibitor. We wished to determine why some benign non‐cancerous adult human prostates do not express 5‐AR 2, and hypothesized that methylation of 5‐AR 2 promoter region correlated with low expression of 5‐AR 2 protein.

Metformin represses cancer cells via alternate pathways in N-cadherin expressing vs. N-cadherin deficient cells

Metformin has emerged as a potential anticancer agent. Here, we demonstrate that metformin plays an anti-tumor role via repressing N-cadherin, independent of AMPK, in wild-type N-cadherin cancer cells. Ectopic-expression of N-cadherin develops metformin-resistant cancer cells, while suppression of N-cadherin sensitizes cancer to metformin. Manipulation of AMPK expression does not alter sensitivity of cancer to metformin. We show that NF-kappaB is a downstream molecule of N-cadherin and metformin regulates NF-kappaB signaling via suppressing N-cadherin. Moreover, we also suggest that TWIST1 is an upstream molecule of N-cadherin/NF-kappaB signaling and manipulation of TWIST1 expression changes the sensitivity of cancer cells to metformin. In contrast to the cells that express N-cadherin, in N-cadherin deficient cells, metformin plays an anti-tumor role via activation of AMPK. Ectopic expression of N-cadherin makes cancer more resistant to metformin. Therefore, we suggest that metformins anti-cancer therapeutic effect is mediated through different molecular mechanism in wild-type vs. deficient N-cadherin cancer cells. At last, we selected 49 out of 984 patients’ samples with prostatic cancer after radical prostatectomy (selection criteria: Gleason score ≥ 7 and all patients taking metformin) and showed levels of N-cadherin, p65 and AMPK could predict post-surgical recurrence in prostate cancer after treatment of metformin.

Age and Obesity Promote Methylation and Suppression of 5α-Reductase 2: Implications for Personalized Therapy of Benign Prostatic Hyperplasia

PURPOSE In men with symptomatic benign prostatic hyperplasia 5α-reductase inhibitors are a main modality of treatment. More than 30% of men do not respond to the therapeutic effects of 5α-reductase inhibitors. We have found that a third of adult prostate samples do not express 5α-reductase type 2 secondary to epigenetic modifications. We evaluated whether 5α-reductase type 2 expression in benign prostatic hyperplasia specimens from symptomatic men was linked to methylation of the 5α-reductase type 2 gene promoter. We also identified associations with age, obesity, cardiac risk factors and prostate specific antigen. MATERIALS AND METHODS Prostate samples from men undergoing transurethral prostate resection were used. We determined 5α-reductase type 2 protein expression and gene promoter methylation status by common assays. Clinical variables included age, body mass index, hypertension, hyperlipidemia, diabetes, prostate specific antigen and prostate volume. Univariate and multivariate statistical analyses were performed followed by stepwise logistic regression modeling. RESULTS Body mass index and age significantly correlated with methylation of the 5α-reductase type 2 gene promoter (p <0.05) whereas prostate volume, prostate specific antigen or benign prostatic hyperplasia medication did not correlate. Methylation highly correlated with 5α-reductase protein expression (p <0.0001). In a predictive model increasing age and body mass index significantly predicted methylation status and protein expression (p <0.01). CONCLUSIONS Increasing age and body mass index correlate with increased 5α-reductase type 2 gene promoter methylation and decreased protein expression in men with symptomatic benign prostatic hyperplasia. These results highlight the interplay among age, obesity and gene regulation. Our findings suggest an individualized epigenetic signature for symptomatic benign prostatic hyperplasia, which may be important to choose appropriate personalized treatment options.

Metformin inhibits the proliferation of benign prostatic epithelial cells

Objective Benign prostatic hyperplasia (BPH) is the most common proliferative abnormality of the prostate affecting elderly men throughout the world. Epidemiologic studies have shown that diabetes significantly increases the risk of developing BPH, although whether anti-diabetic medications preventing the development of BPH remains to be defined. We have previously found that stromally expressed insulin-like growth factor 1 (IGF-1) promotes benign prostatic epithelial cell proliferation through paracrine mechanisms. Here, we seek to understand if metformin, a first line medication for the treatment of type 2 diabetes, inhibits the proliferation of benign prostatic epithelial cells through reducing the expression of IGF-1 receptor (IGF-1R) and regulating cell cycle. Methods BPE cell lines BPH-1 and P69, murine fibroblasts3T3 and primary human prostatic fibroblasts were cultured and tested in this study. Cell proliferation and the cell cycle were analyzed by MTS assay and flow cytometry, respectively. The expression of IGF-1R was determined by western-blot and immunocytochemistry. The level of IGF-1 secretion in culture medium was measured by ELISA. Results Metformin (0.5-10mM, 6-48h) significantly inhibited the proliferation of BPH-1 and P69 cells in a dose-dependent and time-dependent manner. Treatment with metformin for 24 hours lowered the G2/M cell population by 43.24% in P69 cells and 24.22% in BPH-1 cells. On the other hand, IGF-1 (100ng/mL, 24h) stimulated the cell proliferation (increased by 28.81% in P69 cells and 20.95% in BPH-1 cells) and significantly enhanced the expression of IGF-1R in benign prostatic epithelial cells. Metformin (5mM) abrogated the proliferation of benign prostatic epithelial cells induced by IGF-1. In 3T3 cells, the secretion of IGF-1 was significantly inhibited by metformin from 574.31pg/ml to 197.61pg/ml. The conditioned media of 3T3 cells and human prostatic fibroblasts promoted the proliferation of epithelial cells and the expression of IGF-1R in epithelial cells. Metformin abrogated the proliferation of benign prostatic epithelial cells promoted by 3T3 conditioned medium. Conclusions Our study demonstrates that metformin inhibits the proliferation of benign prostatic epithelial cells by suppressing the expression of IGF-1R and IGF-1 secretion in stromal cells. Metformin lowers the G2/M cell population and simultaneously increases the G0/G1 population. Findings here might have significant clinical implications in management of BPH patients treated with metformin.

MP17-07 ANDROGENIC TO ESTROGENIC SWITCH IN PROSTATE GLAND AS A RESULT OF EPIGENETIC SILENCING OF STEROID 5-? REDUCTASE 2

P1⁄4.133; Q4:1.221 (1.049-1.420), P1⁄4.010). However, the odds ratio for Qmax<10 mL/sec was not significantly different between free T4 quartile groups after adjusting confounding factors. In propensity score matched analysis (matched for age, metabolic syndrome, testosterone, and body mass index at a 1:1 ratio), 1362 cases (Q4 of free T4) and 1362 control subjects (Q1, Q2, and Q3 of free T4) were included for comparison. The ratio of prostate volume 30 mL (15.1% vs. 19.3%, P 1⁄4 .004) and mean prostate volume (23.7 6.7 vs. 24.6 7.3 mL, P1⁄4.001) was higher in the case group than in the control group. Qmax and IPSS were not different between case and control groups. TSH was not significantly related to IPSS, Qmax, and total prostate volume in univariate and multivariate analyses. CONCLUSIONS: Prostate volume, IPSS, and Qmax are significantly related to free T4, and prostate volume is significantly and independently related to total prostate volume in this study. We found a potential role of thyroid hormone in developing BPH.

Regulation of steroid-5-alpha-reductase 2 (SRD5A2) in human prostate by epigenetic modifications.

204 Background: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth. We have found that many aging prostate tissues do not express the enzyme. We previously showed that expression of SRD5A2 is not static and epigenetic modulations by DNA methyltransferase and pro-inflammatory cytokines play important roles in silencing of SRD5A2. Here we wished to define the methylation pattern and identify specific CpG dinucleotides that are methylated in the SRD5A2promoter region. Methods: Ninety six prostate samples from patients were obtained by transurethral resection of prostate (TURP). Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). Bisulfide conversion of Genomic DNA was performed using Epimark bisulfide conversion kit. Primers for bisulfide sequencing were designed using MethPrimer software and bisulfide sequencing was performed using 3730XL DNA Sequencers. Binding of histone demethylase to promoter DNA was analyzed by C...