Novel actions of next-generation taxanes benefit advanced stages of prostate cancer

Abstract

Purpose: To improve the outcomes of patients with castration-resistant prostate cancer (CRPC), there is an urgent need for more effective therapies and approaches that individualize specific treatments for patients with CRPC. These studies compared the novel taxane cabazitaxel with the previous generation docetaxel, and aimed to determine which tumors are most likely to respond.

Experimental design: Cabazitaxel and docetaxel were compared via in vitro modeling to determine the molecular mechanism, biochemical and cell biologic impact, and cell proliferation, which was further assessed ex vivo in human tumor explants. Isogenic pairs of RB knockdown and control cells were interrogated in vitro and in xenograft tumors for cabazitaxel response.

Results: The data herein show that (i) cabazitaxel exerts stronger cytostatic and cytotoxic response compared with docetaxel, especially in CRPC; (ii) cabazitaxel induces aberrant mitosis, leading to pyknotic and multinucleated cells; (iii) taxanes do not act through the androgen receptor (AR); (iv) gene-expression profiling reveals distinct molecular actions for cabazitaxel; and (v) tumors that have progressed to castration resistance via loss of RB show enhanced sensitivity to cabazitaxel.

Conclusions: Cabazitaxel not only induces improved cytostatic and cytotoxic effects, but also affects distinct molecular pathways, compared with docetaxel, which could underlie its efficacy after docetaxel treatment has failed in patients with CRPC. Finally, RB is identified as the first potential biomarker that could define the therapeutic response to taxanes in metastatic CRPC. This would suggest that loss of RB function induces sensitization to taxanes, which could benefit up to 50% of CRPC cases.

Figure 1. Cabazitaxel shows enhanced anti-tumor effects in CRPC

A. Dose-dependent response to 48h cabazitaxel (CBTX) and docetaxel (DCTX) treatment and 48h fresh media without drugs was assessed in hormone therapy sensitive (LNCaP) and resistant prostate cancer cells (C4-2) by trypan blue exclusion, and cell numbers were normalized to EtOH treatment. B. Bivariate flow cytometry analyses of LNCaP and C4-2 cells treated with 1nM CBTX or control for 16 or 48h. In the top graphs, the x-axis represents relative DNA content as indicated by propidium iodide (PI) staining; the y axis shows cells undergoing active S-phase as indicated by 2h BrdU labeling. Inset values: % BrdU incorporation in viable cells (mean±s.d., from an experiment performed in biological triplicate). The bottom graphs represent the corresponding PI traces only, showing a G2M arrest, followed by cell death after CBTX exposure. C. Quantification of % BrdU+ cells in (B) and Suppl Fig 1, significant reduction is observed in taxane over control (LNCaP p<0.05; C4-2 p<0.0005 for all conditions). CBTX appears to have a mild effect on proliferation of C4-2, although not significand, whereas DCTX does not. D. Taxanes induce significant cell death after 48h of CBTX or DCTX over CTRL in both LNCaP and C4-2 cells (p<0.01, detected as an increase in Sub-G1 content by flow cytometry (PI in B), with enhanced effects in the CRPC cells. Asterisks indicate significant differences between CBTX and DCTX at the same treatment duration, showing no significance in LNCaP, but improved efficacy for CBTX in C4-2 cells (16h 0.0042; 48h p=0.0092).

Figure 2. Cabazitaxel treatment results in increased multinucleated cells and aberrant nuclei

A. The nuclei of fixed LNCaP and C4-2 cells that were treated for 16hrs with taxanes were visualized with DAPI staining (cyan) on a confocal microscope (40x), demonstrating defective mitosis and pyknotic nuclei (yellow and orange arrows). B. F-Actin stained with Alexa Fluor 488 labeled phalloidin shows changes in cytoplasmic volume and cell morphology, as induced by taxanes after 16hrs compared to control. The pink arrows indicate multinucleate cells, which are quantified in the whisker plots in (C). C. Quantification of mitotic figures, pyknotic cells and multinucleate cells after CBTX treatment in at least quadruplicate shows significant elevation of multinucleate LNCaP and C4-2 cells after 16h (p=0.0055 and p=0.0042, respectively) and 48h (p<0.0001 in both cell lines), and pyknotic cells after 48h (p=0.0107; p=0.0096). C4-2 cells show a modest, however non-significant increase in mitotic figures after 16h.

Figure 3. The effects of taxanes are independent of altered AR localization

A. Immunofluorescence after 16h 1nM CBTX, DCTX or CTRL treatment demonstrates nuclear localization of AR (red) is unaffected in presence of steroids. DAPI staining (cyan) denotes cell nuclei. B. In androgen deprivation, AR resides in the cytoplasm of LNCaP cells, but remains nuclear in C4-2 CRPC cells (steroid depleted), even after 16h cabazitaxel exposure.

Figure 4. Cabazitaxel exerts enhanced anti-tumor effects in human tumors

A. Schematic overview of the ex vivo explant assay. Fresh tissue obtained from radical prostatectomy is subdivided and cultured for 6 days on dental sponges submerged in cell culture media with different treatments (CTRL, 50nM CBTX or DCTX), after which tissue is formalin-fixed, paraffin-embedded, and analyzed by standardized immunohistochemistry (IHC) methods. B. H&E and IHC on explant tissues demonstrate that taxane treatment reduces tumor cell proliferation (Ki67) and induces cell death, (Caspase-3). C. Quantification of Ki67 (top) in three independent explant assays, performed on tissue from three patients, shows reduced proliferation in cabazitaxel treated samples compared to control (p<0.05 by Kruskal-Wallis non-parametric multiple comparison analysis). Cabazitaxel demonstrates improved drug efficacy compared to docetaxel ex vivo. Quantification of Caspase-3 in the three explant assays (bottom) indicates a moderate, yet non-significant increase in cell death, as induced by taxane treatment. D. AR (IHC) remains nuclear in tumor specimens after 6 days of taxane treatment.

Figure 5. Tumors that progress to CRPC by RB loss show hypersensitivity to cabazitaxel

A. RB knockdown LNCaP cells (shRB1) show a modest sensitization in vitro to 48h cabazitaxel and 96h in fresh media (no CBTX) in culture conditions mimicking androgen deprivation therapy (ADT), but not in presence of hormones (in serum), as measured cell viability in a trypan blue exclusion assay. B. Treatment schematic for nude athymic mice, subcutaneously injected with LNCaP shRB1 or LNCaP shCon1 cells to obtain xenograft tumors in vivo (n=5 per group). Growth analysis of xenograft tumors was monitored over time of CBTX treatment (start t=0). The graph on the right denotes the sizes for each tumor at t=14 relative to the size at start of treatment (t=0), showing that only LNCaP shRB1 tumors respond to CBTX (p=0.014). C. RB proficient LNCaP xenograft tumors display decreased Ki67 staining (unpaired ttest: p<0.0001), and elevated numbers of mitotic figures after CBTX treatment. AR remains nuclear after CBTX exposure, irrespective of RB status. D. Quantification of mitotic figures (top) and Ki67 positive cells (bottom) in 10 High Power Fields (HPF, 400x) per tumor show elevated % of mitotic figures in shCon1 tumors, and a reduction in proliferating cells in shRB1 tumors.

Figure 6. Expression profiling reveals novel action(s) of cabazitaxel in ADT-sensitive and CRPC prostate cancer cells

A. Venn diagrams of two microarray analyses of p<0.05 after background filtering reveal differential response of castrate-sensitive LNCaP cells and castrate-resistant C4-2 cells to 16hrs 1nM CBTX compared to DCTX, dependent on culture conditions: steroid rich (+) versus depleted (-) media, in triplicate. B. David Bioinformatics GO analyses with p<0.05 (p-values in table) reveal common and distinct pathways for the two taxanes, dependent on cell context and steroid conditions. C. Top 25 significant hits of CBTX unique in androgen deprived (blue) or steroid replete (yellow) conditions are displayed for each cell line, with the corresponding mean fold change over control treated samples. Asterisks highlight common genes between LNCaP and C4-2 cells.