Androgen deprivation induces selective outgrowth of aggressive hormone-refractory prostate cancer clones expressing distinct cellular and molecular properties not present in parental androgen-dependent cancer cells

PURPOSE The mechanism of progression of human prostate cancer (CaP) cells u nder androgen ablation therapy remains unclear. To study the alternative pa thways of CaP cell growth under conditions of androgen deprivation, androge n-independent CaP variants were selected and expanded from an androgen-depe ndent CaP line via an in vitro androgen deprivation treatment. Cellular and molecular properties of these androgen-independent variants were character ized both in vitro and in vivo and compared with those of their parental an drogen-dependent cells. METHODS Androgen deprivation treatment of an androgen-dependent CaP cell li ne, LNCaP, was carried out by replacing culture medium with RPMI 1640 mediu m plus 10% charcoal-stripped serum. Cells that survived through the androge n deprivation treatment were harvested and expanded in the androgen-deficie nt culture medium and were designated CL-1. The CL-1 cells were also recult ured in androgen-containing medium and designated CL-2. The growth (cell cy cle analysis, H-3-thymidine incorporation assay, growth expansion, and colo nization efficiency), expression of CaP-associated markers (semiquantitativ e reverse transcriptase polymerase chain reaction), interaction with endoth elial and bone marrow stromal cells, sensitivity to anticancer agents and r adiation (growth inhibition), and tumorigenicity of CL-1 and CL-2 cells wer e determined and compared with these characteristics in parental LNCaP cell s. RESULTS CL-1 and CL-2 cells are fast-growing cells when compared with paren tal LNCaP cells. They were capable of potentiating the growth of endothelia l and bone marrow stromal cells in coculture experiments and acquired signi ficant resistance to radiation and to anticancer cytotoxic agents (Taxol(R) paclitaxel, vinblastine, and etoposide). In contrast to the poorly tumorig enic parental LNCaP cells, CL-1 and CL-2 lines proved highly tumorigenic, e xhibiting invasive and metastatic characteristics In intact and castrated m ice or In female mice within a short period of 3 to 4 weeks. No growth supp lements (e.g., Matrigel) were needed. When transfected with the green fluor escence protein (GFP) gene and transplanted orthotopically in the accessory sex gland, extensive metastatic disease from the primary CL tumor could be identified in bone, lymph nodes, lung, liver, spleen, kidney, and brain. S emiquantitative reverse transcriptase polymerase chain reaction analysis re vealed a markedly distinct molecular expression profile in the CL lines: ov erexpression of basic fibroblast growth factor, interleukin-6. interleukin- 8, vascular endothelial growth factor, transforming growth factor-beta, epi dermal growth factor receptor, caveolin, and bcl-2 messenger RNAs and marke d downregulation of E-cadherin, p-53, and pentaerythritol tetranitrate. CONCLUSIONS Early administration of hormonal therapy after failure of first -line treatment is associated with a profound clonal selection of aggressiv e Al variants, such as CL-1 and CL-2 lines. These tumor lines, with their p arental counterparts, can serve as valuable tools for studying the cellular and molecular mechanisms of CaP progression and metastasis under hormonal therapy. CL-1 and CL-2 offer a unique and reproducible model for the evalua tion of drug sensitivity and for other therapeutic modalities for advanced prostate cancer.