**3. Results**

### *3.1. LNCaP95-DR Cells Were Cross-Resistant to Cabazitaxel*

To evaluate the inhibitory effect of docetaxel and cabazitaxel on prostate cancer cell lines, the MTT assay was performed (Figure 1A,B). LNCaP cells were highly sensitive to docetaxel and cabazitaxel, whereas LNCaP95 cells were less sensitive than LNCaP cells. A docetaxel resistant LNCaP95 cell line, LNCaP95-DR, was obtained by exposing parental cells to gradually increasing concentrations of docetaxel. As shown in Figure 1C, LNCaP95-DR cells were significantly less sensitive to docetaxel than LNCaP95-C cells. Furthermore, LNCaP95-DR cells were less sensitive to cabazitaxel than LNCaP95-C cells (Figure 1D). A table showing the IC50s of all these cell lines is provided in Figure 1E. These data sugges<sup>t</sup> that the acquired resistance to docetaxel results in the cross-resistance to cabazitaxel.

**Figure 1.** LN95-DR shows cross-resistance to cabazitaxel. Dose responses for docetaxel (**A**) and cabazitaxel (**B**) on the viability of prostate cancer cell lines (DU145, PC3, LNCaP, and LN95-P) assessed by the MTT assay; Dose responses for docetaxel (**C**) or cabazitaxel (**D**) on the viability of LN95-C and LN95-DR after 72 h; (**E**) A table showing IC50 values and 95% confidence intervals for docetaxel and cabazitaxel on prostate cancer cell lines. LN95-P: parental LNCaP95; LN95-C: time-matched parental LNCaP95 cells treated with DMSO as a vehicle control; LN95-DR: LNCaP95 with acquired resistance to docetaxel.

### *3.2. P-gp Was Overexpressed in LNCaP95-DR Cells and Tariquidar Restored Sensitivity to Docetaxel and Cabazitaxel*

Consistent with a known mechanism of acquired resistance to taxanes, P-gp was overexpressed in LNCaP95-DR cells as measured by the Western blot analysis (Figure 2A). To test whether this high level of P-gp protein in LNCaP95-DR cells played a direct role in the resistance to docetaxel and cabazitaxel, a P-gp inhibitor was tested. Tariquidar is a potent P-gp antagonist that inhibits P-gp mediated drug efflux [30–33]. We found that the monotherapy with tariquidar showed no effect on the proliferation of LNCaP95-DR (data not shown), whilst tariquidar restored the sensitivity of LNCaP95-DR cells to both docetaxel and cabazitaxel (Figure 2B–D). These data indicated that the cross-resistance between docetaxel and cabazitaxel in LNCaP95-DR cells was mainly mediated by P-gp.

**Figure 2.** Tariquidar restored the sensitivity of LNCaP95-DR to docetaxel and cabazitaxel. (**A**) Levels of P-gp protein in LN95-P, LN95-C, and LN95-DR cell lysates using b-actin as a loading control; Effects of inhibition of p-gp on the viability of LN95-C and LN95-DR cells incubated with DMSO or a combination of tariquidar (50 nM, inhibitor of P-gp) and increasing concentrations of docetaxel (**B**) or cabazitaxel (**C**); (**D**) Table showing the IC50s of docetaxel and cabazitaxel in LN95-DR cells incubated with a combination of 50 nM tariquidar.

### *3.3. Expression of AR-V7-Regulated Genes Was Increased in LNCaP95-DR*

To elucidate other potential contributing factors involved in the mechanism of taxane resistance and provide clues for possible intervention, we compared the levels of expression of several key genes in LNCaP95-DR cells using Western blot analysis and real-time RT-qPCR. LNCaP95-DR cells had higher levels of glucocorticoid receptor (GR), UBE2C, and phosphorylated S6 (pS6), but lower levels of BRN-2 proteins as compared to levels in LNCaP95-C (Figures 3A,B and A1C).

**Figure 3.** Levels of proteins suspected to play a role in the resistance to therapies for castration-resistant prostate cancer (CRPC). Western blot analyses using whole cell lysates from cell lines. Expression of proteins in androgen receptor (AR) and AR-V7 signaling (**A**), Jak/STAT, PI3K/AKT/mTOR, Ras/MAPK pathway and neuroendocrine markers (**B**). The ratio among the three cell lines was described, which was normalized to that in LN95-P, comparing the protein expression values of FL-AR and AR-V7 regulated molecules normalized to beta-actin as an internal control (**A**).

Real-time RT-qPCR revealed that the transcript levels of FL-AR and its target gene KLK3 in LNCaP95-DR cells did not differ from those in LNCaP-C cells, whereas the transcript levels of AR-V7 and its target genes, UBE2C and CDC20, were all increased in LNCaP95-DR cells as compared to levels in LNCaP-C cells (Figure 4). Interestingly, neither docetaxel nor cabazitaxel suppressed the

expression of genes regulated by FL-AR (Figure 4A,B). FL-AR was functional in LNCaP95-DR cells as indicated by the induction of PSA-luciferase activity, as well as KLK3 and FKBP5 gene expression in response to the synthetic androgen, R1881 (Figure 4). Neither docetaxel nor cabazitaxel reduced the transcriptional activity of FL-AR in LNCaP95-C or LNCaP95-DR when measuring a PSA-luciferase reporter or endogenous expression of KLK3 and FKBP5 in response to R1881 (Figure 4).

**Figure 4.** Increased expression of AR-V7 target genes in LNCaP95-DR. Levels of mRNA for FL-AR, AR-V7 and their target genes plus PSA (6.1 kb)-luciferase activities in LN95-C and LN95-DR in response to the synthetic androgen R1881 and taxanes. Transcript levels of FL-AR, KLK3, FKBP5, AR-V7, UBE2C, and CDC20 normalized to transcript levels of GAPDH. LN95-C and LN95-DR were treated with DMSO, docetaxel (5 nM) (**A**) or cabazitaxel (10 nM) (**B**) for 1 h prior to the addition of R1881 (1 nM) or EtOH for 48 h. For the luciferase assay (**C**), LN95-C and LN95-DR were treated with DMSO, enzalutamide (10 μM), docetaxel (5 nM), or cabazitaxel (10 nM) for 1 h prior to treatment with R1881 (1 nM) or EtOH for 48 h. n.s.: not significant; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.

BRN2 is a transcription factor that is proposed to play a role in enzalutamide-induced neuroendocrine transdifferentiation [34]. LNCaP95-DR cells had reduced expression of this transcription factor (Figure 3). Therefore, we tested whether the altered expression of BRN2 might correlate to increased sensitivity to enzalutamide. Unfortunately, no difference in cell viability was measured in response to enzalutamide in LNCaP95-DR cells (Figure A1A). All cell lines remained resistant to enzalutamide.

Glucocorticoid receptor (GR), is proposed to play a role in CRPC as an alternative steroid receptor for AR [35]. Although levels of GR protein were elevated in the LNCaP95 cells (Figure 3), the GR agonist, dexamethasone, did not affect the proliferation of the LNCaP95 cells (Figure A1B). This suggested that the GR is not driving proliferation in this model.

Alterations in expression of components of the PI3K/Akt/mTOR occur in 42% of primary prostate tumors and 100% of metastatic tumors [36]. Thus, targeting the PI3K/Akt/mTOR pathway is considered a promising approach for the treatment of CRPC [15,37,38]. To determine a possible role of this pathway, western blot analysis was performed to assess the status of this pathway in LNCaP95-DR cells. We found no significant change in the expression levels of proteins related to the PI3K/Akt/mTOR pathway, with the exception of pS6 (Figures 3B and A1C). However, the mTOR inhibitor, everolimus, did not mediate differential effects in the different cell lines (Figure A1D), which could sugges<sup>t</sup> that an increased pS6 expression was not important for the acquired resistance to docetaxel.

### *3.4. Knockdown of AR-V7 Has No Effect on Sensitivity to Docetaxel and Cabazitaxel*

Given the expression of AR-V7, and its target genes UBE2C and CDC20 being increased in LNCaP95-DR, we examined whether AR-V7 may contribute to the acquisition of resistance to taxanes and whether the targeting of AR-V7 might be a good intervention strategy. To test these we used two approaches, knockdown of AR-V7 and an inhibitor of the AR-Vs transcriptional activities. When AR-V7 expression was transiently knocked down in LNCaP95-DR cells using small interfering RNA (siRNA) (Figure 5A), proliferation of LNCaP95-DR cells was decreased by 37% (Figure 5B). AR-V7 knockdown did not restore the sensitivity of LNCaP95-DR cells to docetaxel or cabazitaxel (Figure 5C).

AR-NTD targeting drugs are a potential treatment strategy for CRPC represented by LNCaP95-DR cells which have acquired resistance to enzalutamide, docetaxel, and cabazitaxel. This is because AR-NTD is essential for the transcriptional activities exerted by both the FL-AR and AR-Vs. Thus, antagonists of AR-NTD, such as EPI-002, could have a therapeutic effect on LNCaP95 driven by AR-V7. Importantly, EPI-002 had an inhibitory effect on the proliferation of LNCaP95-DR cells that was similar to the effect measured with the parental LNCaP95 cells (Figure 5D). Together, the data revealed that AR-NTD-targeting drugs are a feasible intervention for taxane-resistant prostate cancers that are driven by AR-Vs.

**Figure 5.** EPI-002 inhibits proliferation of LN95-DR. (**A**) Levels of proteins of FL-AR and AR-V7 and transcripts of FL-AR, AR-V7, UBE2C, and CDC20 in LN95-DR that were transfected with AR-V7 siRNA. After 48 h of transfection with 5 nM AR-V7 siRNA, LN95-DR cells were incubated in serum-free conditions for 48 h prior to collecting the proteins and for 96 h prior to collecting RNA. Transfection with AR-V7 siRNA sufficiently decreased the expression of AR-V7 and the target genes (UBE2C and CDC20) without affecting the level of the FL-AR; (**B**) Knockdown of AR-V7 decreased the proliferation of LN95-DR by 37%. After 48 h of transfection with 5 nM AR-V7 siRNA, LN95-DR cells were incubated in serum-free conditions for 72 h prior to measuring proliferation; (**C**) Dose response curves for docetaxel or cabazitaxel on the viability of LN95-DR cells treated 48 h after transfection with 5 nM AR-V7 siRNA (AR-V7KD). The table shows IC50s of docetaxel and cabazitaxel in LN95-DR cells after knockdown of AR-V7; (**D**) Dose response curve for EPI-002 on proliferation of both LN95-P and LN95-DR cells as measured by the BrdU ELISA assay. The Table shows IC50 values of EPI-002 in LN95-P and LN95-DR cells. n.s.: not significant; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.
