Targeting cell cycle and hormone receptor pathways in cancer

Abstract

The cyclin/cyclin-dependent kinase (CDK)/retinoblastoma (RB)-axis is a critical modulator of cell cycle entry and is aberrant in many human cancers. New nodes of therapeutic intervention are needed that can delay or combat the onset of malignancies. The antitumor properties and mechanistic functions of PD-0332991 (PD; a potent and selective CDK4/6 inhibitor) were investigated using human prostate cancer (PCa) models and primary tumors. PD significantly impaired the capacity of PCa cells to proliferate by promoting a robust G1-arrest. Accordingly, key regulators of the G1-S cell cycle transition were modulated including G1 cyclins D, E and A. Subsequent investigation demonstrated the ability of PD to function in the presence of existing hormone-based regimens and to cooperate with ionizing radiation to further suppress cellular growth. Importantly, it was determined that PD is a critical mediator of PD action. The anti-proliferative impact of CDK4/6 inhibition was revealed through reduced proliferation and delayed growth using PCa cell xenografts. Finally, first-in-field effects of PD on proliferation were observed in primary human prostatectomy tumor tissue explants. This study shows that selective CDK4/6 inhibition, using PD either as a single-agent or in combination, hinders key proliferative pathways necessary for disease progression and that RB status is a critical prognostic determinant for therapeutic efficacy. Combined, these pre-clinical findings identify selective targeting of CDK4/6 as a bona fide therapeutic target in both early stage and advanced PCa and underscore the benefit of personalized medicine to enhance treatment response.

Figure 1

CDK4/6-specific inhibition suppresses proliferation of androgen-dependent PCa cells. The impact of the CDK4/6-specific inhibitor (PD) on proliferation and cell cycle components was characterized in multiple androgen-dependent PCa cell model systems. (a) Bivariate flow cytometry analyses of: LNCaP (upper), LAPC4 (middle) and VCaP (lower) cells treated 24 h with 0.1% dimethylsulphoxide control (left column) or 0.5 μM PD (right column). Profiles are representative of three independent experiments. The x axis denotes relative DNA content as indicated by propidium iodide (PI) staining. The y axis denotes cells undergoing active S-phase as indicated by 2-h pulse-label of BrdU. Inset values: % BrdU incorporation (mean±s.d., from an experiment performed in biological triplicate). (b) Crystal violet staining at day 7 (d₇) relative to plating at day 0 (d₀) from LNCaP, LAPC4, and VCaP cells initially treated with control or 0.5 μM PD for 24 h. Data are representative of three independent experiments. (c) Immunoblot analyses, from parallel treated cells in panel (a) for the indicated prostate and cell cycle components (left panels) and quantified by LI-COR image analyses (right panels). Loading and quantification are relative to Ran. Grey and black bars=control and PD treatments, respectively. * ** *** indicates P-values: <0.05; 0.01; 0.001, respectively.

Figure 2

AR-directed therapies are effective in the presence of CDK4/6-specific inhibition. To assess AR activity, androgen-dependent PCa cells: (a) LNCaP (b) LAPC4 and (c) VCaP were cultured 72 h in media containing steroid-deprived serum (5% charcoal-dextran treated (CDT)) then stimulated 24 h with (or without) DHT (1 nM) in the presence of PD (0.5 μM), Csdx (10 μM) or combination of PD and Csdx. Relative mRNA expression normalized to glyceraldehyde 3-phosphate dehydrogenase was determined by quantitative PCR (qPCR) for the known AR-target genes: KLK3/PSA (left), TMPRSS2 (middle) and KLK2 (right). Indicated treatments for each gene are relative to non-DHT and non-drug treated cells. * ** *** indicates P-values: <0.05; 0.01; 0.001, respectively.

Figure 3

CDK4/6-specific inhibition cooperates with IR to attenuate PCa cell growth. IR was administered in conjunction with PD to evaluate the effect of combinatorial therapy, as described in the Material and methods section. (a) Cell growth analyses of LNCaP, LAPC4 and VCaP cells treated with single-dose IR (2 Gy), PD (0.5 μM) or a combination of both. Cell number, for the indicated times and treatments, was determined by Trypan blue exclusion and was normalized to the initial day of treatment (day 1). **Indicates a P-value <0.01 relative to individual treatments alone. Data shown are representative of three independent experiments. (b) Clonogenic assay using LNCaP cells treated with the indicated dose (Gy) of IR or in combination with PD (0.5 μM). Colonies were stained with crystal violet and counted 14 days post-treatment. Studies were performed in biological triplicate and are representative of three independent experiments. *Indicates a P-value <0.05 relative to IR treatment alone.

Figure 4

CRPC cells are sensitive to CDK4/6-specific inhibition, dependent on RB status. The relevance of PD treatment to advanced PCa was determined using multiple CRPC cell model systems. Flow cytometry was performed on CRPC cells: (a) PC3M; (b) LNCaP shRB1 (stable LNCaP cells depleted of the RB protein); (c) 22Rv1; and (d) C4-2 treated with control (left panels) or PD (right panels), as described in Figure 1a. Representative flow cytometry profiles for each cell model system are shown. The y axis denotes the number of fixed cells stained with propidium iodide (PI). Inset values: % PI-stained cells (mean±s.d., from an experiment performed in biological triplicate) in the G₁-, S-, G₂M-phases as determined using the cell cycle algorithm in FlowJo. Note: 22Rv1 and C4-2 cells were treated in media containing 5% charcoal-dextran treated (CDT) serum, as described in the Materials and methods section. (e) Gene expression analyses from C4-2 cells was performed, as described in Figure 2, to assess the impact of indicated therapies on AR activity. Representative AR target genes: KLK3/PSA (left) and TMPRSS2 (right) are shown. * ** indicates P-values: <0.05; 0.01, respectively.

Figure 5

CDK4/6-specific inhibition suppresses proliferation of xenografts in vivo and primary human prostate tissue ex vivo. The efficacy of PD on PCa was determined by: (a) Proliferative Ki-67 marker analysis from VCaP xenografts, grown in SCID mice, treated with control (n=4 mice) or PD (n=4 mice). Mice were treated with 150 mg/kg of PD, as described detail in the Materials and methods section. Representative images (20 × ) are shown (left panel) and Aperio-based quantification of % Ki-67 (right panel). *Indicates a P-value <0.05. (b) Representative images from histo- and immunohistological analyses of human prostate tissues used for ex vivo tissue explant culture: left panel, images (20 × ) from hematoxylin and eosin (H&E) and RB-stained tissue before processing for culture; right panel, H&E images (20 × , upper row) and Ki-67 images (20 × , lower row) from ex vivo explant tissues treated 6 days with control (left column) and 0.5 (middle column) or 1 μM PD (right column). (c) Aperio-based quantification of % Ki-67 from human explant tissues cultured ex vivo for a short (day 2) and long (day 6) treatment with control (C) or 0.5 or 1 μM PD. Bars represent (mean±s.e.m.) from five patient specimens. *Indicates a P-value <0.05.