A porcine model system of BRCA1 driven breast cancer

BRCA1 is a breast and ovarian tumor suppressor. Hereditary mutations in BRCA1 result in a predisposition to breast cancer, and BRCA1 expression is down-regulated in ~30% of sporadic cases. The function of BRCA1 remains poorly understood, but it appears to play an important role in DNA repair and the maintenance of genetic stability. Mouse models of BRCA1 deficiency have been developed in an attempt to understand the role of the gene in vivo. However, the subtle nature of BRCA1 function and the well-known discrepancies between human and murine breast cancer biology and genetics may limit the utility of mouse systems in defining the function of BRCA1 in cancer and validating the development of novel therapeutics for breast cancer. In contrast to mice, pig biological systems and cancer genetics appear to more closely resemble their human counterparts. To determine if BRCA1 inactivation in pig cells promotes their transformation and may serve as a model for the human disease, we developed an immortalized porcine breast cell line and stably inactivated BRCA1 using miRNA. The cell line developed characteristics of breast cancer stem cells and exhibited a transformed phenotype. These results validate the concept of using pigs as a model to study BRCA1 defects in breast cancer and establish the first porcine breast tumor cell line

Micro-RNA-186-5p inhibition attenuates proliferation, anchorage independent growth and invasion in metastatic prostate cancer cells

Abstract Background Dysregulation of microRNA (miRNA) expression is associated with hallmarks of aggressive tumor phenotypes, e.g., enhanced cell growth, proliferation, invasion, and anchorage independent growth in prostate cancer (PCa). Methods Serum-based miRNA profiling involved 15 men diagnosed with non-metastatic (stage I, III) and metastatic (stage IV) PCa and five age-matched disease-free men using miRNA arrays with select targets confirmed by quantitative real-time PCR (qRT-PCR). The effect of miR-186-5p inhibition or ectopic expression on cellular behavior of PCa cells (i.e., PC-3, MDA-PCa-2b, and LNCaP) involved the use bromodeoxyuridine (BrdU) incorporation, invasion, and colony formation assays. Assessment of the impact of miR-186-5p inhibition or overexpression on selected targets entailed microarray analysis, qRT-PCR, and/or western blots. Statistical evaluation used the modified t-test and ANOVA analysis. Results MiR-186-5p was upregulated in serum from PCa patients and metastatic PCa cell lines (i.e., PC-3, MDA-PCa-2b, LNCaP) compared to serum from disease-free individuals or a normal prostate epithelial cell line (RWPE1), respectively. Inhibition of miR-186-5p reduced cell proliferation, invasion, and anchorage-independent growth of PC-3 and/or MDA-PCa-2b PCa cells. AKAP12, a tumor suppressor target of miR-186-5p, was upregulated in PC-3 and MDA-PCa-2b cells transfected with a miR-186-5p inhibitor. Conversely, ectopic miR-186-5p expression in HEK 293 T cells decreased AKAP12 expression by 30%. Both pAKT and β-catenin levels were down-regulated in miR-186-5p inhibited PCa cells. Conclusions Our findings suggest miR-186-5p plays an oncogenic role in PCa. Inhibition of miR-186-5p reduced PCa cell proliferation and invasion as well as increased AKAP12 expression. Future studies should explore whether miR-186-5p may serve as a candidate prognostic indicator and a therapeutic target for the treatment of aggressive prostate cancer

Additional file 5 of Micro-RNA-186-5p inhibition attenuates proliferation, anchorage independent growth and invasion in metastatic prostate cancer cells

Figure S2. Up-regulation of AKAP12 in PC-3 cells and pAKT in HEK 293 T cells. A) Total RNA was collected from PC-3 cells transfected with miR-186-5p inhibitor and scramble control 72 h post-transfection. AKAP12 transcript expression was increased by 1.7-fold in transient miR-186-5p inhibited PC-3 cells relative to negative controls (p = 0.0597). B) Protein lysate (35 μg) was collected from HEK 293 T cells transfected with miR-186-5p mimic and scramble control 72 h post-transfection. pAKT expression was enhanced by 1.67-fold increase via miR-186-5p overexpression in HEK 293 T cells (p = 0.196). Data was quantitated from at least 2–3 independent experiments and are represented as mean ± S.D. (TIFF 457 kb

Additional file 3 of Micro-RNA-186-5p inhibition attenuates proliferation, anchorage independent growth and invasion in metastatic prostate cancer cells

Figure S1. Transient and stable miR-186-5p inhibition and overexpression in prostate cancer and normal epithelial cells. miR-186-5p expression was measured following 24–48 h transient post-transfection and stable transfection using qRT-PCR. A) Following transient transfection of cell models with miR-186-5p inhibitor, miR-186-5p was reduced by 33–73% in PC-3 (p = 0.0002), MDA-PCA-2b (p = 0.0061) and LNCaP (p = 0.0381) cells. B) Ectopic expression of miR-186-5p in PC-3 (p = 0.0002), MDA-PCA-2b (p = 0.0061) and LNCaP (p = 0.0381) cells transiently transfected with miR-186-5p mimic. C) MiR-186-5p expression was reduced by 30% in PC-3 cells stably transfected with pcDNA-DEST-47-anti-miR-186 compared to pcDNA-DEST-47 (empty vector) (p = 0.0022). MiR-186-5p was up-regulated by 2.55-fold in RWPE1 cells stably transfected with pcDNA-DEST-47-miR-186 mimic construct compared to the empty vector control (p = 0.0019). Data was quantitated from three independent experiments and are represented as mean fold change ± S.D. (**p-value < 0.005, ***p-value < 0.007). (TIFF 1824 kb

Additional file 1 of Micro-RNA-186-5p inhibition attenuates proliferation, anchorage independent growth and invasion in metastatic prostate cancer cells

Table S1. De-identified demographic and clinico-pathological data. Clinical data and serum from 15 men diagnosed with prostate cancer and five disease-free individuals were obtained from the BioServe Biotechnologies Biorepository (Beltsville, MD). Subjects were self-identified as European American males. There were no significant differences in median age between cases and controls (p = 0.726). Among men diagnosed with prostate cancer, 60% were diagnosed with adenocarcinoma, 66.7% were smokers, and 73.3% received two or more therapies. Relative to controls, cases had higher median serum PSA level (ng/ml) (p = 0.048) and BMI (p = 0.225) values. (DOCX 63 kb