To address the underlying genetic cause of prostate cancer burden in African Americans, we previously obtained a cohort of normal tumor paired samples from African American and Caucasian men, and looked for differential gene expression within each group. Utilizing a strict filtering criteria, we observed over 97 differentially expressed genes in our African American vs. Caucasian sample set [36
]. To further narrow the list to genes of utmost importance in prostate disease onset, we used a genome-wide association approach that allowed us to rapidly scan for SNPs in the genome of healthy Yorubai population (Yorubai from Ibaden, Nigeria) that might possibly be associated with our 85/97 prostate candidate genes. We utilized the HapMap database of YBI, Nigerian lymphoblastoid cell lines, since there are a limited number of SNP databases containing African American patients. Most importantly the Yorubai population rather than the Caucasian (CEU) population was utilized because the close ancestral ties [54
] between West African Africans and African Americans would increase our chances of finding similar genetic variants associated with prostate disease in men of African descent (Nigerians and African Americans). Approximately 536 genes were identified in our association study as described in methods section (85 candidate genes + 451 cis-regulatory genes) that are associated with the 85/97 candidate genes. Herein we report that a significant number of direct gene-gene interactions were found, however the most significant interactions were observed on chromosome 1 and chromosome 12. Given the importance of these chromosomes in prostate disease, it was appropriate to investigate ABCD3 gene involvement in African American prostate cancer. Our analysis of gene-gene interactions on chromosome 1 and 12 revealed that ABCD3/RanGAP1 and STXBP and TMTC2 gene showed the strongest associations. Of these the ABCD3/RanGAP1 genes were predicted to have the most significant interactions. As such neither ABCD3 or RanGAP1 expression has been implicated in prostate cancer, therefore we chose to focus on ABCD3. The A
assette genes consist of various subfamilies, are typically expressed in both normal and cancer cells. Their functions have been implicated in acquired Multidrug Resistance, MDR, in cancer cell lines. For example, recent reports have shown that MCF-7/AdVp3000 cells that were derived by selection for growth in the presence of doxorubicin, exhibit a 459-fold overexpression of ABCC3 relative to the parental cell line [55
]. A similar situation in prostate cancer cell lines has been observed as MDR1/Pgp/ABCB1 and multidrug resistance-associated protein-1(MRP1/ABCC1), with the half ABC transporter, breast cancer resistance protein BCRP/ABCG2, is able to selectively isolate the putative prostate stem cells from the prostate tissue microenvironment through constitutive efflux of androgen and protects the putative tumor stem cells from androgen deprivation, hypoxia, or adjuvant chemotherapy [56
]. These findings are supported by unpublished data, from Yates laboratory, that suggest that fluorescence-activated cell sorting, FACS isolation of the (SP) cells, selective for functional ABC transporter pumps, have higher in vivo tumorigenicity compared to other cell surface markers (unpublished data). Thus, it is possible that ABCD3 could possibly contribute to aggressive prostate cancer.
Multiple ABC family genes have been implicated to play a role in chemoresistance and progression of prostate and breast cancer, however given the large number of family subtypes only a few have been associated with prostate cancer progression to aggressive disease. Since ABCD3 gene expression has not been identified previously in prostate cancer, we verified this in commonly utilized prostate cancer cell lines, as well as in a normal and primary tumor cell line pair derived from an African American prostate cancer patient [45
]. As in prostate patient samples, ABCD3 was consistently overexpressed in the RC-77N/E/RC-77T/E, LNCaP/C4-2B models, with MDA-PC-2B cells exhibiting the highest expression (10-fold). This data is consistent with the expression of other members of the ABC gene family that have been implicated in prostate cancer, and serves as proof-of-principle evidence that ABCD3 overexpression is indeed correlated with prostate cancer progression. That MDA-2PC-2B cells exhibited the highest expression levels, further implicates a role for ABCD3 in African American prostate cancers.
To further substantiate the link of ABCD3 with other cell signaling molecules that contribute to prostate cancer, we utilized an indirect in silico ingenuity pathway analysis. ABCD3, showed a high probability of being found within three growth factor initiated network hubs involving ERK, MAPK and NFkB proteins. The ERK MAPK has been implicated in a number of pathophysiological events including androgen receptor signaling [58
] and the epithelial-to-mesenchymal (EMT) [59
] that occurs as cancer cells acquire the property to metastasize. That we observed a 4 fold increase in ABCD3 expression in an EGFR overexpressing DU-145 WT cell line compared to non-transfected DU-145 cells, highlights a putative novel regulator of ABCD3. EGFR is overexpressed in African American Prostate patients [60
] and a robust activator of MAPK ERK in normal and cancer cell [61
]. Furthermore overexpression is sufficient to increase proliferation, invasion related EMT, and metastasis [49
]. Thus, it appears that ABDC3 is a novel prostate cancer associated gene, that could, in part, be regulated by EGFR signaling.
Although further studies to investigate the ABCD3/RanGAP1 relationship need to be conducted, our results clearly illustrate the utility of high density SNP analysis, in conjunction with appropriate cell lines that represent the clinical conditions, to identify regulatory genes in prostate cancer. Although a more in-depth analysis of the exact role of ABCD3 in such events as cell proliferation, and chemoresistance is warranted and are underway. The results of this study provide a rationale for use of DU-145/DU-145WT and MDA-PC-2B as culture models to study molecular mechanisms associated with the health disparity in African American prostate cancer patients.