African-American Prostate Cancer Cell Research Articles

Establishment and characterization of a highly tumorigenic African American prostate cancer cell line, E006AA-hT.

Genuine racial differences in prostate cancer (PCa) biology have been considered among the potential reasons to explain PCa disparities. There is no animal model to represent all aspects of human PCa and, more specifically, to be used for PCa disparity research. The lack of a spontaneously transformed in vitro cell-based model system has been a significant impediment to investigating and understanding potential molecular mechanisms, and the hormonal, genetic, and epigenetic factors underlying the biological and clinical aggressiveness of PCa in African American (AA) men.In this study, we established and characterized the E006AA-hT cell line as a highly tumorigenic subline of the previously characterized primary AA-PCa cell line, E006AA. Extensive characterization of the E006AA-hT cell line was accomplished using cytodifferentiation and prostate-specific markers, spectral karyotyping, cell line authentication assays, cell proliferation and migration assays, and in vitro tumorigenesis assays.Spectral karyotyping of E006AA-hT showed a hypertriploid chromosome complement and shared cytogenetic changes similar to its parental cells such as diploid X, absence of Y-chromosomes, numerical gains in chromosomes 5,6,8,10,17,20,21, and marker chromosomes of unknown origin. In addition, E006AA-hT also presented numerous clonal and structural aberrations such as insertion, deletion, duplication, and translocations in chromosomes 1-5, 8, 9, 11, 13, 14, 17, and 18. The E006AA-hT cell line was shown to be highly tumorigenic and produced tumors at an accelerated growth rate in both athymic nude and triple-deficient SCID mice.Silencing the mutated androgen receptor (AR-599 Ser>Gly) did not affect proliferation (loss-of-function), but decreased migration (gain-of-function) in E006AA-hT and its parental cell type. These data support that AR-point mutations may lead simultaneously to different “loss-of-function” and “gain-of-function” phenotypes in PCa cells. E006AA-Par and its subline as the only available spontaneously transformed low- and highly-tumorigenic primary AA-PCa cell lines could be used for basic and translational research aimed in supporting prostate cancer disparity research.

Androgen Receptor-Target Genes in African American Prostate Cancer Disparities

The incidence and mortality rates of prostate cancer (PCa) are higher in African American (AA) compared to Caucasian American (CA) men. To elucidate the molecular mechanisms underlying PCa disparities, we employed an integrative approach combining gene expression profiling and pathway and promoter analyses to investigate differential transcriptomes and deregulated signaling pathways in AA versus CA cancers. A comparison of AA and CA PCa specimens identified 1,188 differentially expressed genes. Interestingly, these transcriptional differences were overrepresented in signaling pathways that converged on the androgen receptor (AR), suggesting that the AR may be a unifying oncogenic theme in AA PCa. Gene promoter analysis revealed that 382 out of 1,188 genes contained cis-acting AR-binding sequences. Chromatin immunoprecipitation confirmed STAT1, RHOA, ITGB5, MAPKAPK2, CSNK2A,1 and PIK3CB genes as novel AR targets in PCa disparities. Moreover, functional screens revealed that androgen-stimulated AR binding and upregulation of RHOA, ITGB5, and PIK3CB genes were associated with increased invasive activity of AA PCa cells, as siRNA-mediated knockdown of each gene caused a loss of androgen-stimulated invasion. In summation, our findings demonstrate that transcriptional changes have preferentially occurred in multiple signaling pathways converging (“transcriptional convergence”) on AR signaling, thereby contributing to AR-target gene activation and PCa aggressiveness in AAs.

Abstract B38: Profiling miR-152 expression in African American prostate cancer cell lines.

Abstract Purpose: African American men have almost twice the incidence and death rates related to prostate cancer compared to Caucasian men. Several studies have suggested that some of these differences may be attributed to the elevated expression of different genes. Recently, a new class of genetic regulators has emerged known as microRNAs. Many reports have found microRNAs to be implicated in cell growth, cell differentiation and the onset of many diseases including prostate cancer. To date, there are only three commercially available African American prostate cancer cell line models. We were able to establish five African American prostate cell lines, representing each stage of prostate cancer progression. With the help of these models, we sought out to investigate the difference of microRNA expression in African American and Caucasian prostate cancer. Methods: We analyzed the expression of over 300 microRNAs in 11 African American and Caucasian cell lines utilizing DiscovArray microRNA Microarray Chip. Validations of the most significant miRNAs were done by qRT-PCR. Characterization of the microRNA was carried out by performing MTT assay, Flow Cytometry, Western Blot, and Imunoflourescence. Results: We identified 15 microRNAs that were differentially expressed between the African American and Caucasian cell lines. Further validations led to the identification of miR-152 which is significantly expressed among both groups. MiR-152 is thought to be responsible for inhibiting DNA methyltransferase 1 (DNMT1). Our results showed that miR-152 reduced cellular proliferation in PC-3, DU-145 and LNCaP prostate cancer cell lines. In addition, miR-152 inhibited DNMT1 protein expression as well as initiated G2-M cell cycle arrest. Conclusion: Validation of these results indicates a significant difference in microRNA expression between African American and Caucasian prostate cancer. Furthermore, these results suggest that microRNAs may play a role in the advance progression seen in prostate cancer in African Americans. These findings are currently being validated in African American and Caucasian tissue samples. Research supported by G12 RR03059-21A1(NIH/RCMI), and CA118623 (NIH/NCI). Citation Format: Shaniece Crystal Theodore, Melissa Davis, Rhim Jhong, Timothy Turner, Clayton Yates. Profiling miR-152 expression in African American prostate cancer cell lines. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B38.

Abstract A66: βarrestin2 contributes to aggressive tumor growth in African American prostate cancer cells

Abstract Prostate cancer (PCa) is the second leading cause of death in African American males (AA). Studies have found the incidence of PCa to be 60% higher in AA men than their Caucasian American counterparts (CA). Poorer prognosis in AA men is exacerbated by lack of adequate access to healthcare and non financial factors such as distrust of the healthcare system. However, these social and socioeconomic factors fail to fully account for this disparity. AA men are diagnosed at younger ages and often present with more advanced tumor stages and higher Gleason scores. Thus, elucidating possible biological causes of prostate cancer disparity is of utmost importance. βarrestin2 (βarr2) is a scaffold protein that associates with G protein coupled receptors (GPCRs) and leads to the desensitization and internalization of these receptors. In addition, more recent studies have implicated βarr2 in a variety of cellular processes including androgen metabolism. We have shown increased βarrestin2 expression in prostate cell lines from AA men (MDA PCa 2B) when compared to those from CA men (LNCap). Furthermore, overexpression of βarr2 in LNCap cells or knockdown of βarr2 in MDA PCa 2B cells was sufficient to alter the growth properties of these cells. These results were seen both in vitro and in vivo. Thus preliminary data indicates that βarr2 may contribute to differences between tumor growth in our model system. Further studies are needed to ascertain the cause and mechanism of βarr2 overexpression in AA men, as well as the clinical significance of these findings. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A66.

Establishment and characterization of a pair of non-malignant and malignant tumor derived cell lines from an African American prostate cancer patient

Research into molecular and genetic mechanisms underlying prostate carcinogenesis in high-risk African American men would be greatly advanced by in vitro models of African American prostate tumors representing primary tumors. However, the generation of immortalized primary African American prostate cancer cells that will accurately reflect the in situ characteristics of malignant epithelium is currently limited but is greatly needed. We have successfully established immortalized cell lines of a pair of non-malignant and malignant tumors derived from an African American prostate cancer patient with HPV-16E6E7 (RC-77N/E and RC-77T/E). RC-77N/E and RC-77T/E cells are currently growing well at passage 40. Both cells exhibit epithelial morphology and are androgen sensitive. The RC-77T/E cells produced tumors in SCID mice whereas the RC-77N/E cells produced no tumor in SCID mice. These cells expressed androgen-regulated prostate-specific homobox gene, NKX 3.1, epithelial cell specific cytokeratn 8, androgen receptor (AR), prostate specific antigen (PSA), and p16. Chromosome analysis showed that both cell lines are similar; near diploid human male (XY) with most chromosome counts in the 45-48 range. However, RC-77T/E cell line has new marker chromosomes: M1B=del/t(4;?)(q28;?), M5=16q+ in addition to those observed in the RC-77N/E cell line (M1=del(4)(q28q34)+hsr in some, M1A=t(4q;?),M2=der(9?),M2A=del(M2p-),M3=iso(?), M4=der(22?)). This is the first documented case of the establishment of pair of non-malignant and malignant tumors derived from an African American prostate cancer patient. These models will provide novel tools to study the molecular and genetic mechanisms of prostate carcinogenesis, especially for high-risk African American men.

Establishment and characterization of a primary androgen-responsive African-American prostate cancer cell line, E006AA.

Establishment of human prostate cancer cell lines is essential to advance our understanding of complex processes associated with the initiation and progression of the disease. In the present study, we report the establishment of a primary African-American prostate cancer cell line (E006AA) as well as its associated stromal cells (S006AA). E006AA cell line was established as a spontaneously immortalized cells from a patient with a clinically localized prostate cancer. Extensive characterization of the cells was accomplished using androgen-dependent growth and sensitivity assays, Western analyses, RT-PCR/real-time PCR, cytogenetic analyses, and tumorigenicity in nude mice. E006AA cell line shows androgen-dependent growth, expresses PSA and the androgen receptor (AR) with 26 CAG repeats in exon 1 of AR. Cytogenetic analyses revealed a hypertriploid karyotype with additional numerical gains in chromosomes 5, 6, 8, 10, 17, 20, 21 and a marker chromosome of unknown origin as well as structural abnormalities in chromosomes 4, 5, 8, 9, 11-14, 18, and 20. This cell line is not tumorigenic in nude mice. S006AA cell line, isolated from the same tumor specimen, also expresses AR and shows the morphological characteristics of smooth muscle cells of prostatic stroma. These cell lines are the first available primary epithelial and stromal cells derived from an African-American patient with organ-confined prostate cancer and in conjunction with other established cell lines, could provide an in vitro model system to investigate early transforming events in prostate cancer.