Irene Casanova-Salas

Identification of miR-187 and miR-182 as Biomarkers of Early Diagnosis and Prognosis in Patients with Prostate Cancer Treated with Radical Prostatectomy

PURPOSE miRNAs are noncoding RNAs that negatively regulate target mRNA gene expression. Aberrant miRNA expression is associated with prostate cancer pathogenesis. We identified miRNAs as potential biomarkers for prostate cancer diagnosis and prognosis. MATERIALS AND METHODS Total RNA was obtained from 10 normal prostate and 50 prostate cancer samples, and analyzed using the GeneChip® miRNA 2.0 Array. At a median followup of 92 months (range 2 to 189) an independent cohort of 273 paraffin embedded prostate cancer samples was used for validation by quantitative reverse transcriptase-polymerase chain reaction. Another 92 urine samples from patients undergoing prostate biopsy were evaluated for these miRNAs. RESULTS miR-182 and 187, the miRNAs most differentially expressed between normal and tumor tissue, were selected for further validation. miR-187 inversely correlated with cT (p = 0.125) and pT (p = 0.0002) stages, Gleason score (p = 0.003) and TMPRSS2-ERG status (p = 0.003). The log rank test showed associations of miR-182 with biochemical (p = 0.026) and clinical (p = 0.043) progression-free survival, as also noted on multivariate analysis. A significant independent improvement in the definition of risk of progression was achieved by combining miR-182 expression with Gleason score (p <0.0001). miR-187 detection in urine provided an independent predictive value for positive biopsy. A prediction model including serum prostate specific antigen, urine PCA3 and miR-187 provided 88.6% sensitivity and 50% specificity (AUC 0.711, p = 0.001). CONCLUSIONS Results show that miR-182 and 187 are promising biomarkers for prostate cancer prognosis to identify patients at risk for progression and for diagnosis to improve the predictive capability of existing biomarkers.

MiR-187 Targets the Androgen-Regulated Gene ALDH1A3 in Prostate Cancer.

miRNAs are predicted to control the activity of approximately 60% of all protein-coding genes participating in the regulation of several cellular processes and diseases, including cancer. Recently, we have demonstrated that miR-187 is significantly downregulated in prostate cancer (PCa) and here we propose a proteomic approach to identify its potential targets. For this purpose, PC-3 cells were transiently transfected with miR-187 precursor and miRNA mimic negative control. Proteins were analyzed by a two-dimensional difference gel electrophoresis (2D-DIGE) and defined as differentially regulated if the observed fold change was ±1.06. Then, MALDI-TOF MS analysis was performed after protein digestion and low abundance proteins were identified by LC–MS/MS. Peptides were identified by searching against the Expasy SWISS PROT database, and target validation was performed both in vitro by western blot and qRT-PCR and in clinical samples by qRT-PCR, immunohistochemistry and ELISA. DIGE analysis showed 9 differentially expressed spots (p<0.05) and 7 showed a down-regulated expression upon miR-187 re-introduction. Among these targets we identified aldehyde dehydrogenase 1A3 (ALDH1A3). ALDH1A3 expression was significantly downregulated in PC3, LNCaP and DU-145 cells after miR-187 re-introduction. Supporting these data, the expression of ALDH1A3 was found significantly (p<0.0001) up-regulated in PCa samples and inversely correlated (p<0.0001) with miR-187 expression, its expression being directly associated with Gleason score (p = 0.05). The expression of ALDH1A3 was measured in urine samples to evaluate the predictive capability of this biomarker for the presence of PCa and, at a signification level of 10%, PSA and also ALDH1A3 were significantly associated with a positive biopsy of PCa. In conclusion, our data illustrate for the first time the role of ALDH1A3 as a miR-187 target in PCa and provide insights in the utility of using this protein as a new biomarker for PCa.

Clinico-pathological significance of the molecular alterations of the SPOP gene in prostate cancer

AIMS Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor recently described to be mutated in prostate cancer (PCa). Hence, studying the gene expression profile and the presence of SPOP mutations in PCa and understanding its clinico-pathological significance as prognostic and therapeutic biomarker are important to further understand its role in PCa development. PATIENTS AND METHODS A cohort of 265 paraffin-embedded PCa samples from patients with more than 5 years of follow-up and treated with radical prostatectomy were collected at our institution for SPOP evaluation. RT-qPCR analysis was performed for expression studies while mutations were assessed by next generation sequencing. Relationship with prognosis was analysed using log-rank analysis and multivariable Cox regression. RESULTS SPOP was found to be strongly down-regulated in PCa (median=0.24; range=0.04-9.98) and its expression was associated with both, biochemical (p=0.003) and clinical progression free survival (p=0.023), the very low SPOP expression levels being associated to the worst prognosis. Multivariate analysis demonstrated that low levels of SPOP independently predicted a worse prognosis for both, biochemical (Hazard ratio (HR)=0.5; confidence interval (CI) 95% [0.4-0.9], p=0.011) and clinical progression (HR=0.6; IC 95% [0.4-1], p=0.046). SPOP mutations were found in 10% of TMPRSS2-ERG (T2E)-negative cases. Log-rank tests showed that mutations were significantly associated with biochemical progression free survival (BPFS) (p=0.009) and also were significant in the multivariable analysis (HR=3.4; IC 95% [1.5-7.6], p=0.004). CONCLUSIONS The present study demonstrates that prognosis varies depending on SPOP expression and mutational status, hence, defining a new biotype of PCa associated with a worse prognosis.

Abstract 2863: TMPRSS2-ERG influences IGF-1R expression and affects its prognostic value in prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA INTRODUCTION: Insulin like growth factor 1 receptor (IGF-1R) plays a critical role in different tumors including prostate cancer (PCa). Recently, a fusion gene involving the androgen receptor-regulated gene TMPRSS2 and one of the ETS transcription factors family member, predominantly ERG, has been described in half of PCa patients. TMPRSS2-ERG results in the overproduction of a truncated ERG protein and cooperates in the establishment of a neoplastic phenotype in prostate cells. The aim of this study was to evaluate TMPRSS2-ERG impact on IGF-1R expression and to verify the value of IGF-1R as biomarker of PCa progression. MATERIAL AND METHODS: IGF-1R expression was evaluated in a panel of six prostate cell lines, including malignant VCaP, DU-145, PC-3, LNCaP, 22RV1 and non malignant RWPE-1 prostate cells by qRT-PCR, western blot and immunofluorescence. ERG siRNA was transiently transfected in VCaP cells and ERG down-regulation was assessed by western blot. Total RNA was extracted from a cohort of 270 paraffin-embedded PCa samples with more than 5 years of follow-up and IGF-1R expression was analysed by qRT-PCR. The prognostic value of IGF-1R was evaluated both in the univariate and multivariate analysis. The clinicopathological informations as well as the TMPRSS2-ERG fusion gene status were considered in the statistical analysis. RESULTS: The IGF-1R expression profile analysis was performed in PCa cells and pointed out that VCaP cell line, harboring the TMPRSS2-ERG fusion gene, carries the highest IGF-1R expression levels both considering mRNA, total protein and membrane expression levels. Interestingly, IGF-1R protein levels were found to be down-regulated upon ERG silencing in VCaP cells. PCa tumors did not show any remarkable difference in the expression of IGF-1R when compared with normal tissues (median=1.04) but IGF-1R was directly correlated with TMPRSS2-ERG status (p=0.027) in tumors. Log-rank tests showed an association between higher IGF-1R expression and better biochemical progression free survival (p=0.047).When dividing the population according to the TMPRSS2-ERG status we found higher fold-change in IGF-1R expression in tumors expressing TMPRSS2-ERG (median=1.25) compared to non-expressors (median=0.91) and a statistically stronger association between IGF-1R and prognosis in the TMPRSS2-ERG negative tumors (p=0.012). Moreover, multivariate analysis confirmed that higher expression of IGF-1R is an independent indicator of better prognosis in this subgroup (HR:0.48.IC95% [0.25-0.90].p=0.023). CONCLUSIONS: In this study we demonstrated that TMPRSS2-ERG affects IGF-1R expression both in vitro and in clinical samples. High expression of IGF-1R is significantly associated with a better clinical outcome of PCa patients. (Grants: FIRB RBAP11884M_005 to KS; FIS PI10/01206 and FPI11/00505 from ISCIII) Citation Format: Caterina Mancarella, Irene Casanova-Salas, Jose Antonio Lopez-Guerrero, Katia Scotlandi. TMPRSS2-ERG influences IGF-1R expression and affects its prognostic value in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2863. doi:10.1158/1538-7445.AM2014-2863