Overcoming ABCB1 mediated multidrug resistance in castration resistant prostate cancer

Abstract

Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. PCa that relapses after hormonal therapies, referred to as castration resistant PCa (CRPC), often presents with metastases (mCRPC) that are the major cause of mortality. The few available therapies for mCRPC patients include taxanes docetaxel (DTX) and cabazitaxel (CBZ). However, development of resistance limits their clinical use. Mechanistically, resistance arises through upregulation of multidrug resistance (MDR) proteins such as MDR1/ABCB1, making ABCB1 an attractive therapeutic target. Yet, ABCB1 inhibitors failed to be clinically useful due to low specificity and toxicity issues. To study taxanes resistance, we produced CBZ resistant C4-2B cells (RC4-2B) and documented resistance to both CBZ and DTX in cell culture and in 3D prostaspheres settings. RNAseq identified increased expression of ABCB1 in RC4-2B, that was confirmed by immunoblotting and immunofluorescent analysis. ABCB1-specific inhibitor elacridar reversed CBZ and DTX resistance in RC4-2B cells, confirming ABCB1-mediated resistance mechanism. In a cell-based screen using a curated library of cytotoxic drugs, we found that DNA damaging compounds Camptothecin (CPT) and Cytarabine (Ara-C) overcame resistance as seen by similar cytotoxicity in parental C4-2B and resistant RC4-2B. Further, these compounds were cytotoxic to multiple PC cells resistant to taxanes with high ABCB1 expression and, therefore, can be used to conquer the acquired resistance to taxanes in PCa. Finally, inhibition of cyclin-dependent kinases 4/6 (CDK4/6) with small molecule inhibitors (CDK4/6i) potentiated cytotoxic effect of CPT or Ara-C in both parental and resistant cells. Overall, our findings indicate that DNA damaging agents CPT and Ara-C alone or in combination with CDK4/6i can be suggested as a new treatment regimen in CRPC patients, including those that are resistant to taxanes.

Fig. 1. Production and characterization of CBZ-resistant cells.

A Experimental scheme outlining production of CBZ-resistant cells. CRPC C4-2B cells were plated in 50% confluency and treated with CBZ 24 h later. After 72 h of CBZ treatment, cells were recovered for one to several weeks, re-plated, and cycle was repeated with increased concentration of CBZ ranging from 0.1 to 20 nM. B C4-2B (parental) and RC4-2B CBZ-resistant cells were treated with indicated concentrations of CBZ. Results of cell viability assay (left), representative phase contrast images (right) and colony formation assay (bottom). IC50 for CBZ is ~10× higher in RC4-2B cells compared to the parental C4-2B cells. C C4-2B and RC4-2B cells were treated with indicated concentrations of taxane DTX. Results of cell viability assay (left) and colony formation assay (right). IC50 for CBZ is ~300× higher in RC4-2B cells compared to the parental C4-2B cells. CNT control.

Fig. 2. Characterization of CBZ and DTX resistance of RC4-2B cells by 3D prostasphere model.

C4-2B (parental) and RC4-2B CBZ-resistant cells were set up in Matrigel and treated at day 3 with indicated concentrations of taxanes CBZ or DTX for 24 h; at day 21, prostaspheres were stained with cell viability marker Calcein AM and documented. Left: representative images of prostaspheres treated with CBZ (A) or DTX (B). Right: individual area of prostaspheres was analyzed by ImageJ and calculated by Graph PAD Prism. RC4-2B cells are more resistant to CBZ in prostaspheres settings compared with C4-2B parental cells. C Table shows mean size of C4-2B and RC4-2B prostaspheres after treatment with CBZ or DTX. CNT control.

Fig. 3. Characterization of taxanes treatment in PC cells.

A Proliferation (analyzed by cells count in triplicates) is similar in C4-2B and RC4-2B cells. B, C Morphological analysis of CBZ treatment. Cells were treated for 48 h with indicated concentrations of CBZ and stained with Hoechst (pseudo colored green). B, left: representative images of interphase, mitosis, mitotic catastrophe (micronucleated, MN) cells. 5 independent fields (at least 500 cells) were analyzed for their morphology; C4-2B (B, middle) and RC4-2B (B, right). C4-2B and RC4-2B cells have similar mitotic index (~12%) in control conditions. C4-2B cells activate mitotic catastrophe (result of slippage from the CBZ-induced mitotic block) starting at 1 nM of CBZ. RC4-2B cells are more resistant to induction of both mitotic block and mitotic catastrophe. C Representative images after indicated treatment of C4-2B (top) and RC4-2B (bottom). Arrow: mitotic cells; arrowheads: MN cells. D Expression analysis (RNAseq, volcano plot) of C4-2B and RC4-2B cells. Numbers in figure legends correspond to genes that are up- (red) or downregulated (green) in RC4-2B cells compared to C4-2B cells (p-adjusted < 0.05). X: expression, log2; Y: P-value, log10. ABCB1 expression is significantly upregulated in RC4-2 cells compared to C4-2B cells (log2 = 15.2; P-value = 2.8E−37; adjusted P-value = 7E−34). E Immunoblot analysis of ABCB1 protein levels in: C4-2B (parental) and C4-2B derived RC4-2B (CBZ resistant) and TaxR (DTX-resistant) cells, DU145 (parental) and DU145-derived DTXR (DTX-resistant), CTXR (CBZ/DTX-resistant) cells. ABCB1 is elevated in all four resistant cell lines compared with parental cells. The highest levels of ABCB1 protein were documented in RC4-2B and TaxR, intermediate in CTXR (CBZ/DTX-resistant DU145-DTXR), and low in DU145-DTXR (DTX-resistant DU145). Actin: loading control. Numbers below: the relative levels of ABCB1 normalized to actin. F Microscopy analysis of ABCB1 in C4-2B (left) and RC4-2B (right) cells. Top: overlay of ABCB1 (green) and DNA (blue). Bottom: ABCB1 (green). ABSB1 is expressed at the background levels in C4-2B, and is highly expressed in RC4-2B, with expected localization at the plasma membrane. G IC50 (determined by Alamar Blue assay) for CBZ and DTX in C4-2B, RC4-2B, TaxR, DU-145, DTXR and CTXR cell lines. Taxanes resistance in these cell lines is proportional to the levels of ABCB1 expression documented in E. CNT control.

Fig. 4. ABCB1-specific inhibitor elacridar reverses CBZ and DTX resistance in RC4-2B cells.

C4-2B and RC4-2B CBZ-resistant cells were treated with indicated concentrations of CBZ (left) or DTX (right) alone, in combination with 0.5 uM of ABCB1 specific inhibitor elacridar, or with 0.5 uM of elacridar alone. After 72 h of treatment, survival was analyzed by Alamar Blue (top) or colony formation assays (bottom). Inhibition of ABCB1 reversed resistance to both CBZ and DTX in RC4-2B cells. Treatment by elacridar alone has minimal effect on C4-2B or RC4-2B cells. CNT control.

Fig. 5. DNA damage drugs camptothecin (CPT) and cytarabine (Ara-C) are active in taxanes resistant RC4-2B cells.

C4-2B (parental) and RC4-2B taxanes-resistant cells were treated with indicated concentrations of doxorubicin (Dox, top), CPT (middle) and Ara-C (bottom). Survival (Alamar Blue assay, A) and colony formation assay (B representative images [left], colonies area analysis [right]). RC4-2B cells are resistant to doxorubicin. CPT and Ara-C eradicated the taxanes-resistant RC4-2B cells at similar concentrations as those for the taxanes-responsive parental C4-2B cells. C Western blot analysis of PARP-1 in C4-2B and RC4-2B cells after indicated concentrations of doxorubicin and CPT treatment for 24 h. CPT induces PARP-1 cleavage (marker of apoptosis) at the same concentrations in both cell lines, while doxorubicin is effective only in parental C4-2B cells. Actin: loading control. CNT: control. Dashed lines: IC50.

Fig. 6. CPT and Ara-C are active in multiple taxanes resistant PC cell lines.

C4-2B and DU145 (parental), TaxR (C4-2B derived DTX-resistant), DTXR (DU145 derived DTX resistant), and CTXR (DTXR derived DTX/CBZ resistant) cells were treated with indicated concentrations of CPT or Ara-C. After 72 h, cell survival was measured by Alamar Blue assay. CPT eradicated the taxanes-resistant cells at similar concentrations as those for the taxanes-responsive C4-2B and DU145 cells, and Ara-C is more cytotoxic on taxanes-resistant cells. CNT control.

Fig. 7. CDK4/6i Ribociclib potentiates cytotoxic activity of Ara-C and CPT in parental and taxane- resistant cells.

C4-2B and RC4-2B cells were treated with single agents (Ribociclib [1 uM, 7d], Ara-C [1 uM, 24 h, top], or CPT [6 uM, 24 h, bottom]), with Ribociclib (24 h) followed by Ara-C or CPT (24 h), followed by Ribociclib 5d (R → A or R → C), or by Ara-C or CPT (24 h) followed by Ribociclib 6d (A → R or C → R). Effect was analyzed by colony formation assay and quantified by Image J. The error bars represent the standard devisation of three independent experiments. ^nsp > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. CNT control.

Fig. 8. Model.

Taxanes CBZ or DTX induce mitotic block in cells with low ABCB1 levels; they exit mitotic block by mitotic catastrophe and eventually die. Cells with high levels of ABCB1 are resistant to taxanes and complete mitosis. Treatment with Ara-C followed by CDK4/6i induces cell death, thus bypassing taxanes resistance.