p300 is upregulated by docetaxel and is a target in chemoresistant prostate cancer

Abstract

Administration of the microtubule inhibitor docetaxel is a common treatment for metastatic castration-resistant prostate cancer (mCRPC) and results in prolonged patient overall survival. Usually, after a short period of time chemotherapy resistance emerges and there is urgent need to find new therapeutic targets to overcome therapy resistance. The lysine-acetyltransferase p300 has been correlated to prostate cancer (PCa) progression. Here, we aimed to clarify a possible function of p300 in chemotherapy resistance and verify p300 as a target in chemoresistant PCa. Immunohistochemistry staining of tissue samples revealed significantly higher p300 protein expression in patients who received docetaxel as a neoadjuvant therapy compared to control patients. Elevated p300 expression was confirmed by analysis of publicly available patient data, where significantly higher p300 mRNA expression was found in tissue of mCRPC tumors of docetaxel-treated patients. Consistently, docetaxel-resistant PCa cells showed increased p300 protein expression compared to docetaxel-sensitive counterparts. Docetaxel treatment of PCa cells for 72 h resulted in elevated p300 expression. shRNA-mediated p300 knockdown did not alter colony formation efficiency in docetaxel-sensitive cells, but significantly reduced clonogenic potential of docetaxel-resistant cells. Downregulation of p300 in docetaxel-resistant cells also impaired cell migration and invasion. Taken together, we showed that p300 is upregulated by docetaxel, and our findings suggest that p300 is a possible co-target in treatment of chemoresistant PCa.

Keywords: chemotherapy resistance; colony formation ability; docetaxel; p300; prostate cancer.

Figure 1

Expression of p300 is increased upon docetaxel treatment. (A) Quantification of p300 immunoreactivity scores (IRS) after IHC staining (Mann–Whitney U test; scatter dot blot with line at mean + s.e.m.). (B) IHC staining of p300 (nuclear localization). Upregulation of p300 expression in cancerous tissue of docetaxel-treated patients compared to control patients. (C) p300 and CBP mRNA expression were determined in samples of docetaxel-treated and docetaxel-untreated mCRPC patients (Mann–Whitney U test; box whisker plot with 5–95 percentile).

Figure 2

p300 expression is increased in docetaxel-resistant prostate cancer cells. (A) Comparison of p300 protein expression between docetaxel-sensitive and docetaxel-resistant (DR) PC3 (n = 4), DU145 (n = 5), and CWR22RV1 (n = 4). Data represent mean + s.e.m. (t-test). (B) IHC staining for p300 in docetaxel-resistant PC3-DR, DU145-DR, and CWR22RV1-DR compared to docetaxel-sensitive counterparts. Magnification 40×. (C) PC3 (n = 5), DU145 (n = 5), and CWR22RV1 (n = 4) were treated with the indicated concentrations of docetaxel for 72 h, and p300 protein expression was analyzed by Western blot, and one representative Western blot is shown. Values represent mean + s.e.m. (one-way ANOVA).

Figure 3

Establishment and validation of doxycycline-inducible p300 knockdown cell lines using a non-targeting control (shCtrl) sequence and two specific p300-shRNA sequences (shp300-1 and shp300-2). Docetaxel-sensitive PC3 and docetaxel-resistant PC3-DR are shown here, representing all mentioned cell lines. For better visualization in the following experiments, only doxycycline-treated shCtrl, shp300-1, and shp300-2 replicates are shown. Confirmation of (A) uniform expression of shRNA-constructs by fluorescence microscopy (BF = bright field, GFP = green fluorescent protein) and magnification 40× and (B) p300 knockdown by Western blot analysis after activation of shp300-sequences with 100 ng/mL doxycycline for 72 h. Values indicated are mean + s.e.m. (one-way ANOVA, n = 3). (C) Decreased p300 activity was confirmed by analysis of histone h3 acetylation on lysine18 in PC3 and PC3-DR cells. One representative Western blot out of three independent experiments is shown. (D) Cumulative population doubling levels (PDL, n = 1) of PC3 and PC3-DR cells over time with downregulated p300 were calculated by cell number measurement upon each passage (linear fit regression and test for significance of slopes and intercepts).

Figure 4

p300 inhibition decreases colony formation ability of docetaxel-resistant cells. Representative images and quantification of high-resolution colony formation assays of docetaxel-sensitive PC3 and CWR22RV1 (A) and docetaxel-resistant PC3-DR and CWR22RV1-DR (B). Colony numbers were analyzed by the software CATCH-colonies. Data represent mean + s.e.m. (one-way ANOVA, n = 3).

Figure 5

p300 downregulation decreases cell migration and invasion. (A) Wound scratch assays were performed on confluent layers of PC3-DR shCtrl, shp300-1, and shp300-2 treated with 100 ng/mL doxycycline. Images were taken 48 h after scratch and analyzed by ImageJ (n = 6). (B) PC3-DR were seeded in Boyden chambers and shp300 sequences were activated with 100 ng/mL doxycycline for 72 h. Cell migration was measured after staining with calcein and visualized by fluorescence microscopy (n = 4). (C) Invasion assays were conducted as in B, except that Boyden chambers were pre-coated with Matrigel (n = 3). Values indicated in A–C denote mean + s.e.m. (one-way ANOVA). (D) Wound scratch assays on PC3-DR treated with 10 µM CPI-637. Data represent mean + s.e.m. (t-Test, n = 3). (E) Migration (n = 4) and (F) invasion assays (n = 5) of PC3-DR treated with 20 µM CPI-637. Values indicated are mean + s.e.m. (t-test). (G) Immunofluorescence staining for vimentin (red) and quantification relative to counterstaining of nuclei (blue). Original magnification 630×. (H) mRNA (qPCR) and protein expression (Western blot) of vimentin. Data in G-H represent mean + s.e.m. (one-way ANOVA, n = 5).