Germline and somatic DNA repair gene alterations in prostate cancer.

BACKGROUND: Emerging evidence has suggested that DNA repair gene alterations may be important in prostate cancer pathogenesis. In the current study, the authors sought to characterize alterations in DNA repair pathway genes in both primary and metastatic prostate tumors with attention to tissue distribution as well as specific genomic alterations. METHODS: The authors studied the distribution and type of alterations in 24 genes that are considered important for DNA repair in 944 prostate cancers harvested from localized and metastatic tumors. Tumor DNA underwent hybrid capture for all coding exons of 287 or 395 cancer-related genes plus select introns from 19 or 31 genes frequently rearranged in cancer. Captured libraries were sequenced to a median exon coverage depth of >×500. Specific genomic alterations were characterized and the frequencies of mutations by tissue site (prostate vs metastases) were compared using logistic regression. RESULTS: A total of 152 patients from the cohort of 944 men (16%) harbored a germline or somatic mutation in ≥1 DNA repair genes. The most frequently mutated genes were BRCA2 (11.4%) and ATM (5.8%), followed by MSH6 (2.5%) and MSH2 (2.1%). Mutations were identified in approximately 20.1% of primary prostate tumors compared with 18.8% of bone metastases. When stratified by tissue site, the highest rates of DNA repair mutations were found in solid organ metastases, including brain and visceral metastases, compared with prostate. CONCLUSIONS: DNA repair gene mutations are more common in metastatic than localized prostate tumors. Visceral and other solid organ metastases appear enriched for these mutations compared with localized tumors or bone and lymph node metastases.

Bioengineered Noncoding RNAs Selectively Change Cellular miRNome Profiles for Cancer Therapy.

Noncoding RNAs (ncRNAs) produced in live cells may better reflect intracellular ncRNAs for research and therapy. Attempts were made to produce biologic ncRNAs, but at low yield or success rate. Here we first report a new ncRNA bioengineering technology using more stable ncRNA carrier (nCAR) containing a pre-miR-34a derivative identified by rational design and experimental validation. This approach offered a remarkable higher level expression (40%-80% of total RNAs) of recombinant ncRNAs in bacteria and gave an 80% success rate (33 of 42 ncRNAs). New FPLC and spin-column based methods were also developed for large- and small-scale purification of milligrams and micrograms of recombinant ncRNAs from half liter and milliliters of bacterial culture, respectively. We then used two bioengineered nCAR/miRNAs to demonstrate the selective release of target miRNAs into human cells, which were revealed to be Dicer dependent (miR-34a-5p) or independent (miR-124a-3p), and subsequent changes of miRNome and transcriptome profiles. miRNA enrichment analyses of altered transcriptome confirmed the specificity of nCAR/miRNAs in target gene regulation. Furthermore, nCAR assembled miR-34a-5p and miR-124-3p were active in suppressing human lung carcinoma cell proliferation through modulation of target gene expression (e.g., cMET and CDK6 for miR-34a-5p; STAT3 and ABCC4 for miR-124-3p). In addition, bioengineered miRNA molecules were effective in controlling metastatic lung xenograft progression, as demonstrated by live animal and ex vivo lung tissue bioluminescent imaging as well as histopathological examination. This novel ncRNA bioengineering platform can be easily adapted to produce various ncRNA molecules, and biologic ncRNAs hold the promise as new cancer therapeutics.

Image-guided photo-therapeutic nanoporphyrin synergized HSP90 inhibitor in patient-derived xenograft bladder cancer model.

Photodynamic therapy is a promising and effective non-invasive therapeutic approach for the treatment of bladder cancers. Therapies targeting HSP90 have the advantage of tumor cell selectivity and have shown great preclinical efficacy. In this study, we evaluated a novel multifunctional nanoporphyrin platform loaded with an HSP90 inhibitor 17AAG (NP-AAG) for use as a multi-modality therapy against bladder cancer. NP-AAG was efficiently accumulated and retained at bladder cancer patient-derived xenograft (PDX) over 7 days. PDX tumors could be synergistically eradicated with a single intravenous injection of NP-AAG followed by multiple light treatments within 7 days. NP-AAG mediated treatment could not only specifically deliver 17AAG and produce heat and reactive oxygen species, but also more effectively inhibit essential bladder cancer essential signaling molecules like Akt, Src, and Erk, as well as HIF-1α induced by photo-therapy. This multifunctional nanoplatform has high clinical relevance and could dramatically improve management for bladder cancers with minimal toxicity.

The Promise and Disappointment of Neoadjuvant Chemotherapy and Transurethral Resection for Muscle Invasive Bladder Cancer: Updated Results and Long-Term Followup.

INTRODUCTION: Radical cystectomy with neoadjuvant chemotherapy is the standard of care for patients with localized muscle invasive urothelial carcinoma of the bladder. One of the strongest predictors of survival in these patients is pathological response to initial treatment. Our objective was to determine whether we could stratify the need for radical cystectomy based on pathological response to neoadjuvant chemotherapy. METHODS: We present a cohort of patients with muscle invasive urothelial carcinoma of the bladder to whom surveillance and bladder preservation were offered if complete response was achieved following neoadjuvant chemotherapy. Descriptive statistics and survival analysis were performed to assess overall, cancer specific and metastasis-free survival. Patients were stratified based on pathological response to neoadjuvant chemotherapy. RESULTS: A total of 60 patients were included in the cohort, of whom 32 (55%) had absence of residual disease on post-neoadjuvant chemotherapy transurethral resection and 27 (45%) had persistent disease. Of patients undergoing surveillance 52% maintained the bladder without evidence of recurrence. By comparison, of those with recurrence only 20% preserved the bladder and were without evidence of disease. CONCLUSIONS: Long-term followup shows a subset of patients achieving good outcomes while preserving the bladder. However, we also observed an inability to reliably identify this subset of patients given current clinical and pathological markers. Until we are able to achieve that goal, the safest oncologic approach remains neoadjuvant chemotherapy followed by radical cystectomy.

The Phosphatidylinositol 3-Kinase Pathway as a Potential Therapeutic Target in Bladder Cancer.

Purpose: Activation of the PI3K pathway occurs in over 40% of bladder urothelial cancers. The aim of this study is to determine the therapeutic potential, the underlying action, and the resistance mechanisms of drugs targeting the PI3K pathway.Experimental Design: Urothelial cancer cell lines and patient-derived xenografts (PDXs) were analyzed for alterations of the PI3K pathway and for their sensitivity to the small-molecule inhibitor pictilisib alone and in combination with cisplatin and/or gemcitabine. Potential predictive biomarkers for pictilisib were evaluated, and RNA sequencing was performed to explore drug resistance mechanisms.Results: The bladder cancer cell line TCCSUP, which harbors a PIK3CA E545K mutation, was sensitive to pictilisib compared to cell lines with wild-type PIK3CA Pictilisib exhibited stronger antitumor activity in bladder cancer PDX models with PI3KCA H1047R mutation or amplification than the control PDX model. Pictilisib synergized with cisplatin and/or gemcitabine in vitro, significantly delayed tumor growth, and prolonged survival compared with single-drug treatment in the PDX models. The phosphorylation of ribosomal protein S6 correlated with response to pictilisib both in vitro and in vivo, and could potentially serve as a biomarker to predict response to pictilisib. Pictilisib activated the compensatory MEK/ERK pathway that likely contributed to pictilisib resistance, which was reversed by cotreatment with the RAF inhibitor sorafenib. RNA sequencing of tumors resistant to treatment suggested that LSP1 downregulation correlated with drug resistance.Conclusions: These preclinical results provide new insights into the therapeutic potential of targeting the PI3K pathway for the treatment of bladder cancer. Clin Cancer Res; 23(21); 6580-91. ©2017 AACR.

Co-targeting of DNA, RNA, and protein molecules provides optimal outcomes for treating osteosarcoma and pulmonary metastasis in spontaneous and experimental metastasis mouse models.

Metastasis is a major cause of mortality for cancer patients and remains as the greatest challenge in cancer therapy. Driven by multiple factors, metastasis may not be controlled by the inhibition of single target. This study was aimed at assessing the hypothesis that drugs could be rationally combined to co-target critical DNA, RNA and protein molecules to achieve "saturation attack" against metastasis. Independent actions of the model drugs DNA-intercalating doxorubicin, RNA-interfering miR-34a and protein-inhibiting sorafenib on DNA replication, RNA translation and protein kinase signaling in highly metastatic, human osteosarcoma 143B cells were demonstrated by the increase of γH2A.X foci formation, reduction of c-MET expression and inhibition of Erk1/2 phosphorylation, respectively, and optimal effects were found for triple-drug combination. Consequently, triple-drug treatment showed a strong synergism in suppressing 143B cell proliferation and the greatest effects in reducing cell invasion. Compared to single- and dual-drug treatment, triple-drug therapy suppressed pulmonary metastases and orthotopic osteosarcoma progression to significantly greater degrees in orthotopic osteosarcoma xenograft/spontaneous metastases mouse models, while none showed significant toxicity. In addition, triple-drug therapy improved the overall survival to the greatest extent in experimental metastases mouse models. These findings demonstrate co-targeting of DNA, RNA and protein molecules as a novel therapeutic strategy for the treatment of metastasis.

Effect of Neoadjuvant Chemotherapy on Renal Function following Radical Cystectomy: Is there a Meaningful Impact?

Objective: To evaluate the patterns of impact of neoadjuvant chemotherapy (NAC) on renal function across the initial year following treatment for muscle-invasive bladder cancer (MIBC) with radical cystectomy (RC). Methods: We reviewed the charts of 241 patients who underwent RC for urothelial carcinoma of the bladder between 2003-14 at our institution. Renal function was evaluated at multiple time points (pre-chemotherapy, pre-operatively, post-operatively, 6-12 months follow-up), and then classified by CKD staging. Univariable and multivariable logistic regression analyses were performed to determine relationship between NAC and change in CKD stage. Results: Of the 241 patients who underwent RC for urothelial carcinoma of the bladder, 66 (27%) received NAC and 175 (73%) did not. In multivariable analysis, NAC was significantly associated with a decrease of at least one CKD stage from baseline to post-op (p = 0.009), but not to the 6-12 months follow-up time point (p = 0.050). The loss of GFR in the NAC cohort occurs up-front with chemotherapy, but the peri-operative course is similar to those who underwent cystectomy alone. Of the 15 NAC patients (26.8%) who were Stage 3 CKD prior to chemotherapy, none progressed to a higher stage CKD. Conclusion: NAC is associated with an initial decline in GFR, which then remains stable through the first year following RC. Despite an initial insult, patients receiving NAC are not vulnerable to further deterioration. When appropriately selected, NAC does not appear to result in a clinically significant deterioration of renal function.

Decreased expression of let-7c is associated with non-response of muscle-invasive bladder cancer patients to neoadjuvant chemotherapy.

The identification and development of biomarkers which predict response of muscle invasive bladder cancer (MIBC) patients to neoadjuvant chemotherapy would likely increase usage of this treatment option and thereby improve patient survival rates. MiRNA array and qRT-PCR validation was used to identify miRNA which are associated with response to neoadjuvant chemotherapy. RNA was extracted from a total of 41 archival, fully annotated, MIBC patient diagnostic biopsies (20 chemo-responders and 21 non-responders (response is defined as > 5 year survival rate and being pT0 post-chemotherapy)). Microarray and qPCR identified let-7c as being differentially expressed in chemo-responder versus non-responder patients. Patients with higher let-7c expression levels had significantly higher odds of responding to chemotherapy (p = 0.023, OR 2.493, 95% CI 1.121, 5.546), and assessment of let-7c levels allowed for prediction of patient response (AUC 0.72, positive predictive value 59%). Decreased let-7c was associated with MIBC incidence (p < 0.001), and significantly correlated with other related miRNA including those that were not differentially expressed between responders and non-responders. The combined data indicate let-7c plays a role in mediating chemoresistance to neoadjuvant chemotherapy in MIBC patients, and is a modest, yet clinically meaningful, predictor of patient response.

Perioperative Outcomes following Neoadjuvant Chemotherapy and Radical Cystectomy-Is There Room for Improvement?

INTRODUCTION: We identify the impact of neoadjuvant chemotherapy before open radical cystectomy on perioperative outcomes and identify actionable areas for improvement. METHODS: The impact of neoadjuvant chemotherapy on perioperative outcomes after radical cystectomy for muscle invasive bladder cancer from 2003 to 2014 was assessed using an institutional database. Individual outcomes (venous thromboembolism, surgical site infection, cardiac event) and a composite score using the Clavien-Dindo classification were identified. Univariable and multivariable logistic regression models were used to identify predictors of perioperative complication and 30-day readmission rates. RESULTS: A total of 241 patients were included in the study, of whom 175 underwent radical cystectomy alone (72.6%) and 66 were treated with neoadjuvant chemotherapy plus radical cystectomy (27.4%). The 30-day readmission rate for the neoadjuvant chemotherapy cohort was 30.5% compared to 17.2% for radical cystectomy alone. Multivariable logistic regression analysis identified neoadjuvant chemotherapy as an independent predictor of 30-day readmission (OR 3.47, p=0.01). Of the patients on neoadjuvant chemotherapy readmitted within 30 days 72.2% were readmitted with infections. All other outcomes were not significantly associated with neoadjuvant chemotherapy. CONCLUSIONS: While the administration of neoadjuvant chemotherapy did not significantly increase perioperative complications, patients receiving neoadjuvant chemotherapy had an increased rate of 30-day readmission, with infection being the most common etiology. This increased readmission rate has not been previously identified in this patient population to our knowledge and is an important focus for quality improvement.

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.

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.

No improvement noted in overall or cause-specific survival for men presenting with metastatic prostate cancer over a 20-year period.

BACKGROUND: Prostate cancer mortality in the United States has declined by nearly 40% over the last 25 years. However, to the authors' knowledge, the contribution of prostate-specific antigen (PSA) screening for the early detection of prostate cancer remains unclear and controversial. In the current study, the authors attempted to determine whether improvements in survival over time among patients with metastatic prostate cancer have contributed to the decline in mortality. METHODS: Men aged ≥ 45 years who presented with de novo metastatic prostate cancer from 1988 to 2009 were identified within the California Cancer Registry. Overall survival and disease-specific survival were estimated using the Kaplan-Meier method. A multivariate analysis with Cox proportional hazards modeling was performed to adjust for different distributions of variables between groups. RESULTS: A total of 19,336 men presented with de novo metastatic prostate cancer during the study period. On multivariate analysis, overall survival was found to be better for men diagnosed from 1988 through 1992 and 1993 through 1998 than for men diagnosed in the most recent era (hazards ratio, 0.78; 95% confidence interval, 0.72-0.85 [P < .001] and HR, 0.79; 95% confidence interval, 0.74-0.86 [P < .001]). There was no improvement in disease-specific survival observed when comparing the most contemporary men (those diagnosed between 2004 and 2009) with those diagnosed between 1988 and 1997. CONCLUSIONS: In this analysis of men presenting with de novo metastatic prostate cancer, no consistent improvement in overall or disease-specific survival could be demonstrated over time. These data suggest that improvements in survival for patients with advanced disease have not contributed substantially to the observed drop in prostate cancer mortality over the PSA era and that stage migration secondary to PSA screening plays a more prominent role.

Oncomir miR-125b suppresses p14(ARF) 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, p14(ARF), 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 p14(ARF), decreased level of Mdm2, and induction of apoptosis. In addition, overexpression of miR-125b in p53-deficient PC3 cells induced down-regulation of p14(ARF), which leads to increased cell proliferation through a p53-independent manner. Thus, we conclude that miR-125b acts as an oncogene which regulates p14(ARF)/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.

Functional p53 determines docetaxel sensitivity in prostate cancer cells.

BACKGROUND: Docetaxel is the first line treatment for castration resistant prostate cancer (CRPC). However, docetaxel resistance rapidly develops. Identifying the critical mechanisms giving rise to docetaxel resistance is the major challenge in advanced prostate cancer. METHODS: The effects of docetaxel on human DU145, PC3, LNCaP, and C4-2 prostate cancer cells were examined in cell culture, and p53 expression were analyzed by Western blot analysis. The potential role of p53 in docetaxel sensitivity in prostate cancer cells was tested by either p53 silencing using shRNA or p53 overexpression by introducing wild-type p53. RESULTS: We found that DU145 (mutant p53) and PC3 (p53 null) cells were less sensitive than LNCaP and C4-2 cells expressing functional p53 in response to docetaxel. Docetaxel treatment induces considerably higher apoptosis in LNCaP and C4-2 cells than in DU145 and PC3 cells in a dose dependent manner. Docetaxel increases the levels of ser15 phosphorylation of p53 in a dose dependent manner in both LNCaP and C4-2 cells, while has no effect on the levels of ser15 phosphorylation of p53 in DU145 cells. These results suggest that p53 phosphorylation is associated with docetaxel sensitivity in prostate cancer cells. To further confirm whether p53 activation can induce cell sensitivity to docetaxel treatment, we used p53 shRNA to knock down p53 expression in C4-2 cells and determined the cells response to docetaxel treatment. Knockdown of p53 significantly down regulated p53 phosphorylation and blocked docetaxel induced apoptotic cell death compared to the vector control. To further confirm this observation, we established a stable knock out p53 in C4-2 cells. Down regulation of p53 in the stable p53 knock out C4-2 cells significantly inhibited docetaxel induced apoptotic cell death. We also used wild-type (WT) p53 to over express p53 in DU145 cells, and found that expression of WT-p53 in DU145 cells increased their sensitivity to docetaxel. CONCLUSIONS: These results demonstrate that docetaxel induces p53 phosphorylation and that p53 status is a crucial determinant of docetaxel sensitivity in prostate cancer cells.

Expression of microRNAs in urinary bladder samples obtained from dogs with grossly normal bladders, inflammatory bladder disease, or transitional cell carcinoma.

OBJECTIVE: To determine expression of microRNA (miRNA) in urinary bladder samples obtained from dogs with grossly normal urinary bladders, inflammatory bladder disease, or transitional cell carcinoma (TCC) and in cells of established canine TCC cell lines. SAMPLE: Samples of grossly normal bladders (n = 4) and bladders from dogs with inflammatory bladder disease (13) or TCC (18), and cells of 5 established canine TCC cell lines. PROCEDURES: Expression of 5 miRNAs (miR-34a, let-7c, miR-16, miR-103b, and miR-106b) that target p53, Rb, or Bcl-2 protein pathways was determined for bladder samples and cells via quantitative real-time PCR assay. Effects of cisplatin (5µM) on proliferation and miRNA expression of cells were determined. RESULTS: Expression of miR-34a and miR-106b was significantly higher in TCC samples than it was in samples of grossly normal bladders. Expression of miR-34a, miR-16, miR-103b, and miR-106b was higher in TCC samples than it was in bladder samples from dogs with inflammatory bladder disease. Cells of established canine TCC cell lines that had the lowest growth after cisplatin treatment had increased miR-34a expression after such treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Findings of this study indicated results of miRNA expression assays can be used to distinguish between samples of grossly normal bladders and bladders of dogs with inflammatory bladder disease or TCC. This finding may have clinical relevance because currently available diagnostic tests cannot be used to differentiate these tissues, and inflammatory bladder disease and TCC are both prevalent in dogs. Validation of miRNA expression assays as diagnostic tests may be warranted.

Enhancing the effectiveness of androgen deprivation in prostate cancer by inducing Filamin A nuclear localization.

As prostate cancer (CaP) is regulated by androgen receptor (AR) activity, metastatic CaP is treated with androgen deprivation therapy (ADT). Despite initial response, patients on ADT eventually progress to castration-resistant CaP (CRPC), which is currently incurable. We previously showed that cleavage of the 280 kDa structural protein Filamin A (FlnA) to a 90 kDa fragment, and nuclear localization of the cleaved product, sensitized CRPC cells to ADT. Hence, treatment promoting FlnA nuclear localization would enhance androgen responsiveness. Here, we show that FlnA nuclear localization induced apoptosis in CRPC cells during ADT, identifying it as a treatment tool in advanced CaP. Significantly, the natural product genistein combined polysaccharide (GCP) had a similar effect. Investigation of the mechanism of GCP-induced apoptosis showed that GCP induced FlnA cleavage and nuclear localization and that apoptosis resulting from GCP treatment was mediated by FlnA nuclear localization. Two main components of GCP are genistein and daidzein: the ability of GCP to induce G2 arrest was due to genistein whereas sensitivity to ADT stemmed from daidzein; hence, both were needed to mediate GCP's effects. FlnA cleavage is regulated by its phosphorylation; we show that ADT enhanced FlnA phosphorylation, which prevented its cleavage, whereas GCP inhibited FlnA phosphorylation, thereby sensitizing CaP cells to ADT. In a mouse model of CaP recurrence, GCP, but not vehicle, impeded relapse following castration, indicating that GCP, when administered with ADT, interrupted the development of CRPC. These results demonstrate the efficacy of GCP in promoting FlnA nuclear localization and enhancing androgen responsiveness in CaP.

Initiation of prostate cancer in mice by Tp53R270H: evidence for an alternative molecular progression.

Tp53 mutations are common in human prostate cancer (CaP), occurring with a frequency of ∼30% and ∼70% in localized and metastatic disease, respectively. In vitro studies have determined several common mutations of Tp53 that have specific gain-of-function properties in addition to loss of function, including the ability to promote castration-resistant (CR) growth of CaP cells in some contexts. To date, a lack of suitable mouse models has prohibited investigation of the role played by Tp53 mutations in mediating CaP progression in vivo. Here, we describe the effects of conditional expression of a mutant Tp53 (Tp53(R270H); equivalent to the human hotspot mutant R273H) in the prostate epithelium of mice. Heterozygous "Tp53(LSL-R270H/+)" [129S4(Trp53(tm3Tyj))] and "Nkx3.1-Cre" [129S(Nkx3-1(tm3(cre)Mms))] mice with prostate-specific expression of the Tp53(R270H) mutation (p53(R270H/+) Nkx3.1-Cre mice) were bred onto an FVB/N background via speed congenesis to produce strain FVB.129S4(Trp53(tm3Tyj/wt)); FVB.129S(Nkx3-1(tm3(cre)Mms/wt)) and littermate genotype negative control mice. These mutant mice had significantly increased incidences of prostatic intraepithelial neoplasia (PIN) lesions, and these appeared earlier, compared with the Nkx3.1 haploinsufficient (Nkx3.1-Cre het) littermate mice, which did not express the Tp53 mutation. PIN lesions in these mice showed consistent progression and some developed into invasive adenocarcinoma with a high grade, sarcomatoid or epithelial-mesenchymal transition (EMT) phenotype. PIN lesions were similar to those seen in PTEN conditional knockout mice, with evidence of AKT activation concomitant with neoplastic proliferation. However, the invasive tumor phenotype is rarely seen in previously described mouse models of prostatic neoplasia. These data indicate that the Tp53(R270H) mutation plays a role in CaP initiation. This finding has not previously been reported. Further characterization of this model, particularly in a setting of androgen deprivation, should allow further insight into the mechanisms by which the Tp53(R270H) mutation mediates CaP progression.

MicroRNA let-7c is downregulated in prostate cancer and suppresses prostate cancer growth.

PURPOSE: Prostate cancer (PCa) is characterized by deregulated expression of several tumor suppressor or oncogenic miRNAs. The objective of this study was the identification and characterization of miR-let-7c as a potential tumor suppressor in PCa. EXPERIMENTAL DESIGN: Levels of expression of miR-let-7c were examined in human PCa cell lines and tissues using qRT-PCR and in situ hybridization. Let-7c was overexpressed or suppressed to assess the effects on the growth of human PCa cell lines. Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts. RESULTS: We identified miR-let-7c as a potential tumor suppressor in PCa. Expression of let-7c is downregulated in castration-resistant prostate cancer (CRPC) cells. Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro. Suppression of let-7c expression enhanced the ability of androgen-sensitive PCa cells to grow in androgen-deprived conditions in vitro. Reconstitution of Let-7c by lentiviral-mediated intratumoral delivery significantly reduced tumor burden in xenografts of human PCa cells. Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens. CONCLUSIONS: These results demonstrate that microRNA let-7c is downregulated in PCa and functions as a tumor suppressor, and is a potential therapeutic target for PCa.