Our studies on profiling and validation of miRNA expressions during transition of AD LNCaP-104S cells to AI and CDX resistant cells revealed activation and inactivation of several signaling networks. We noted a difference in miRNA expressions between the AI subline LNCaP-104R1, and freshly generated CDX resistant LNCaP-104S cells, which includes some of the up regulated (miR-146a) and down-regulated miRNAs (miR-15b-3p and miR-18b). Although we noted differential expression of miRNAs between CS-FBS and CDX treated LNCaP cells we selected only miRNAs that showed either up regulation or down regulation in all treated samples for analysis of their putative targets. Despite similar expression profile of specific miRNAs in CS-FBS and CDX treated samples, some the targets such as DOK4 and VEGF showed differential expression in CSFBS and CDX treated cells. Presumably, this could be the effect of regulation of multiple targets by a given miRNA, which may indirectly affect the net expression of DOK4 and VEGF.
Over expression of miR-146a was noted in all treated cells contrary to the study showing loss of expression of miR-146a in CRPC . Increased miR-146a expression was substantiated by down regulation of its two bona fide targets TRAF6 and IRAK1 in both -104R1 and 3wks treated -104S cells. MiR-146a expression is induced by NF-κB  and acts in a negative feedback loop through degradation of TRAF6 and IRAK1 to reduce NF-κB signaling and inflammatory response. An increase in transcription of miR-146a, as a result of elevated NF-κB activity is noted in thyroid cancer  and down regulation of miR-146a is associated with hyperactivation of NF-kB . Despite this association, an increase in NF-kB1 expression could be predicted in treated -104S cells, as NF-kB1 is a direct target of the down-regulated miRNA miR-9 . Increased expression of the other subunit RelA, which heterodimerizes with NF-kB1 also could be predicted as it is a direct target of the down-regulated miR-7 . It appears that the NF-kB signaling pathway is activated in the early stages of gaining resistance to CDX/androgen blockade and the increased expression of miR-146a is a secondary effect of the activation of the NFkB pathway. As a result, in the initial stages of anti-androgen drug resistance there is decreased inflammatory response but down regulation of tumor suppressor targets of miR-146a, BCORL1  and RNASEL , which may not be detected in fully developed CRPC.
The EGFR signaling pathway could be activated also in the early stages of androgen blockade and CDX treatment. The evidence of EGFR pathway activation in the treated -104S cells is from our results showing an increased expression of EGFR, down regulation of miR-7 and up-regulation miR-222, which are the miRNA regulators of EGFR. Decreased expression of p27Kip1 and Cbl, as two other targets of the up-regulated miR-222, further aid activation of EGFR signaling. C-Cbl, an E3 ubiquitin ligase, inactivates ligand-bound EGFR through EGFR-Cbl complex formation leading to its degradation . C-Cbl activation mediates the tumor suppressive effects of EPhB6 and inhibits cancer cell invasiveness . C-Cbl is also targeted by the up regulated miRNA miR-136 in all treated cells. Down regulation of c-Cbl in treated -104S and untreated -104R1 cells possibly promotes antiandrogen resistance through EGFR stabilization. A loss of expression of AR was noted in these cells (data not shown), which supports the report showing an inverse relationship between expression of AR and EGFR in prostate cancer patients . Up regulation of miR-136 in treated -104S cells was substantiated by the loss of expression of the miR-136 target ZFAND1, an uncharacterized AN1 type zinc finger domain 1 containing protein.
Activation of PI3K/AKT signaling axis also could be predicted in treated -104S cells, as a result of down regulation of miR-7, which inhibits tumor growth and metastasis through inhibition of PI3K/AKT pathways [85, 140]. Down regulation of miR-7 in cancer cells including glioblastoma and its inhibitory effects on EMT and metastasis is well documented . Activation of this pathway could be further aided by over expression of MiR-22 in all treated cells, which exerts a proto oncogenic effect through down regulation of PTEN in AI prostate cancer cells . Additionally, activation of VEGF and DOK4 could be predicted, as these proteins are over expressed in treated -104S cells possibly as a result of down regulation of their regulatory miRNA, miR-205. Earlier studies showed an association between poor prognosis of localized prostate cancer and epigenetic repression of miR-205 , and thus confirms the relevance of the loss of miR-205 in development of CDX resistance. DOK4 is a newly identified substrate of ligand-bound insulin receptor (IRS-5), which upon phosphorylation translocates to mitochondria and recruits c-Src kinase to the mitochondria. Up regulation of DOK4 is also noted in renal cell carcinoma . VEGFA is a target of miR-15b-5p also, which showed 2-10-fold reduction in expression in treated -104S cells and in chemotherapy-resistant squamous cell carcinoma .
Other than modulation of specific signaling axis, altered expression of miRNA clusters is also evident in our study. Members of the miR-17-92 and its paralogous miR-106a-363 clusters, miR-17, miR-18a, miR-18b, miR-20a and miR-106a showed ~9-10-fold down regulation upon CDX treatment and androgen blockade. In support of our observation, loss of expression of miR-17 , miR-18a , miR-20a  and miR-106a  are reported in breast and other cancers. Loss of expression of miR-106a, miR-17 and miR-20a are further supported by an increased expression of their target protein FGD4 in these cells. Contrary to the published study , over expression of miR-34b was noted in AI and CDXR cells, however, an inverse relationship between miR-34b expression and disease free survival of triple negative breast cancer has been reported , which suggests that miR-34b expression may be dependent on the status of hormone responsiveness. Among the other up regulated miRNAs, over expression of let-7f1 , miR-143 , miR-218 , miR-29a , miR-302a , miR-3144 , miR-493  and miR-664  in cancer cells has been reported earlier. Our study also identified a number of miRNAs with > 2-fold difference in expression such as miR-3138, miR-3192, miR-3199, and a subset of miR-548 series, which are not yet known to be involved in development of CRPC.
Additional miRNAs such as miR-518b, miR-205 and miR-596 showed > 10-fold loss of expression upon CDX treatment or androgen withdrawal. In support of our observation, loss of expression and tumor suppressor functions of mir-518b and miR-596 has been documented in other cancers . MiR-1244 and miR-759 are two other miRNAs that are significantly down regulated in all treated cells. This is substantiated by an increased expression of their common target ABHD3 in these cells . Among the other down regulated miRNAs, miR-9 and miR-422a are known to have tumor suppressor roles in various cancer cells [81, 86], whereas miRNAs -454, -3131 and -3185 are noted for the first time to be deregulated during progression of CRPC.
Functional contribution of some of the identified microRNAs in development of CRPC has been previously reported. Over expression of miR-222/221 in AI LAPC4 cells was shown to promote androgen independent cell growth, which was abrogated upon expression of anti-miR-222/221 inhibitors . Down regulation of miR-205 has been correlated with advanced prostate cancer and ectopic expression of miR-205 suppressed AR and MAPK signaling and inhibited cell growth . Down regulation of miR-17 in AI prostate cancer cells also has been demonstrated. Expression of pre-miR-17 in these cells prevented AR induced gene transcription and inhibited cell proliferation .
Analysis of the altered cellular processes during progression towards CDX resistance and androgen independence showed a decreased percentage of miRNAs involved in cancer but an increased percentage in reproductive system, endocrine system, hepatic system and metabolic diseases. It can be speculated that up regulated oncomirs at earlier stages aid in transformation of cells through suppression of tumor suppressors. Whereas, at later stages accumulation of abnormal cellular events triggers expression of additional sets of miRNA, which inhibit key regulatory proteins involved in metabolic process, hormone response and other cellular events. Our qRT-PCR FC data indicate differential expression of miRNAs between 1wk and 3wks treatment, which would have been undetected had the profiling been done only in CDX sensitive/AD and –resistant/AI cells. In silico analysis identified a number of targets that are potentially regulated by one or more altered miRNAs. This includes, two mitosis regulatory proteins CCNJ and CHAMP1 (ZNF828) [120, 121], two oncogenic proteins PIK3CD  and MYB that are over expressed in CRPC , a protein trafficking regulatory protein RAB9B, a ubiquitination promoting protein SPOPL involved in the Hedgehog/Gli signaling pathway  and E2F1 transcription factor .
In summary, our results and in silico network analysis suggest that inhibition of expression of TP53, BRCA, Toll like receptors, IRAK1, STAT1, CHUK and FADD by the up regulated miRNAs, and increased synthesis of EGFR, NFkB1/RelA, E2F family members, BCL2L2, ZBTB7A, EGO2 (EIF2C2) and ZEB2 as the targets of down regulated miRNAs are part of the events that support growth and survival of AI LNCaP cells. During treatment with AR antagonist in androgen-deprived condition, additional inhibition of expression of DDX20, URF1, IRF5 and CDKN3 as the targets of the up regulated miRNAs and increased expression of PRDM1, DOK4, TNFSF9 and NOTCH2 as a result of down regulated miRNAs may provide additional protection against CDX induced cell death. In-depth studies are needed to accurately determine the activation and inactivation of specific signaling pathways during development of insensitivities of prostate cancer cells to AR antagonistic drugs.