Gene expression profiling of the androgen independent prostate cancer cells demonstrates complex mechanisms mediating resistance to docetaxel

Abstract

The molecular mechanisms conferring resistance to docetaxel in prostate cancer patients remain partially understood. We generated docetaxel resistant derivatives of the androgen independent prostate cancer cell lines PC-3 and DU-145. Docetaxel rapidly induces DU-145 cell death via apoptosis and the drug resistant cells were produced by periodically exposing proliferating DU-145 cultures to small doses of docetaxel. In PC-3 cells docetaxel induces delayed cell death via mitotic catastrophe evident by profound multinucleation and formation of giant cells. Mononucleated progeny of the giant PC-3 cells shows significant resistance to docetaxel. Gene expression profiling of these docetaxel resistant PC-3 cells revealed sets of docetaxel inducible and constitutively expressed genes associated with major cancer pathways. A contradictory overlap with DU-145 docetaxel resistant cells was also found. Analyses suggested significant changes associated with apoptotic function, DNA repair, cell growth, survival and proliferation, metabolism, maintenance of cytoskeleton and extracellular matrix formation. These cellular processes often contribute to drug resistance and our study identified a set of genes managing this phenotype. Additional analyses of the drug resistant PC-3 cells using shRNA constructs determined direct relevance of Cyclin G2 to docetaxel resistance as well as prevention of multinucleation, whereas the knockdown of upregulated CYP1B1 showed no effect on either of these processes. Downregulated GBP1 was explored by ectopic overexpression and even though GBP1 has a potential to mediate resistance to docetaxel, it was not utilized in PC-3 cells. The results suggest complex combination of gene expression pattern changes that enables resistance to docetaxel while preventing death via multinucleation.

Figure 1

Dose response curves showing 3 fold difference in relative level of resistance to docetaxel in drug-resistant DU145 T2 cells (IC₅₀ = 1.18329 ng/ml, p = 1.0812 E-06) compared to parental DU145 cultures (IC₅₀ = 0.38624 ng/ml, p = 2.9165 E-06).

Figure 2

(A) Dose response curves showing relative level of resistance to docetaxel in the shCCNG2 transfected 8B2 PC-3 cells (clone #1, IC₅₀ = 2.30325 ng/ml, p = 3.061 E-06) compared to drug sensitive PC-3 cells (IC₅₀ = 0.66182 ng/ml, p = 4.7827 E-015), resistant parental cells 8B2 PC-3 (IC₅₀ = 3.62045 ng/ml, p = 1.5035 E-010) and scTR3007 vector control (IC₅₀ = 3.27586 ng/ml, p = 3.6625 E-07). Drug sensitivity in shCCNG2 transfected cells increased by 1.57 fold compared to parental 8B2 cultures. Inset: Immunoblot confirming knockdown of cyclin G₂. Lanes (1) parental PC-3 cells; (2) sc TR3007 vector control; (3) shCCNG2 clone #1; (4) shCCNG2 clone #4; (5) shCCNG2 clone #2; (6) shCCNG2 clone #7. (B) examples of limited utility of the MTT cell proliferation assay to determine cytotoxicity profile of the giant multinucleated cells derived from cyclin G₂ and CYP1B1 shRNA transfectants. See details in the text. (C) Dose response curves showing relative level of resistance to docetaxel in the shCYP1B1 transfected 8B2 PC-3 cells (IC₅₀ = 2.82571 ng/ml, p = 7.0043 E-016) compared to drug sensitive PC-3 cells (IC₅₀ = 0.66182 ng/ml, p = 4.7827 E-015), resistant parental cells 8B2 PC-3 (IC₅₀ = 3.62045 ng/ml, p = 1.5035 E-010) and scTR3007 vector control (IC₅₀ = 3.27586 ng/ml, p = 3.6625 E-07). an 1.28 upward change in drug sensitivity upon shCYP1B1 transfection compared to 8B2 parental cells was considered significant but insufficient to fully explain robust drug resistance in 8B2 cells. Inset: Immunoblot confirming knockdown of CYP1B1 (lane 3) compared to vector control (lane 2) and parental PC-3 cells (lane 1). (D) Dose response curves showing relative level of resistance to docetaxel in the pCMV GBP1 transfected 8B2 PC-3 cells (IC₅₀ = 2.17439 ng/ml, p = 4.9621 E-013) compared to drug sensitive PC-3 cells (IC₅₀ = 0.66182 ng/ml, p = 4.7827 E-015), resistant parental cells 8B2 PC-3 (IC₅₀ = 3.62045 ng/ml, p = 1.5035 E-010) and pCMV vector control (IC₅₀ = 3.45328 ng/ml, p = 3.022 E-009). Drug sensitivity in pCMV GBP1 transfected cells increased by 1.67 fold compared to 8B2 parental cultures. Inset: Immunoblot confirming successful overexpression of GBP1 (lane 3) compared to the vector control (lane 2) and parental PC-3 cells (lane 1).