GP88 (PC-Cell Derived Growth Factor, progranulin) stimulates proliferation and confers letrozole resistance to aromatase overexpressing breast cancer cells

Abstract

Background: Aromatase inhibitors (AI) that inhibit breast cancer cell growth by blocking estrogen synthesis have become the treatment of choice for post-menopausal women with estrogen receptor positive (ER+) breast cancer. However, some patients display de novo or acquired resistance to AI. Interactions between estrogen and growth factor signaling pathways have been identified in estrogen-responsive cells as one possible reason for acquisition of resistance. Our laboratory has characterized an autocrine growth factor overexpressed in invasive ductal carcinoma named PC-Cell Derived Growth Factor (GP88), also known as progranulin. In the present study, we investigated the role GP88 on the acquisition of resistance to letrozole in ER+ breast cancer cells

Methods: We used two aromatase overexpressing human breast cancer cell lines MCF-7-CA cells and AC1 cells and their letrozole resistant counterparts as study models. Effect of stimulating or inhibiting GP88 expression on proliferation, anchorage-independent growth, survival and letrozole responsiveness was examined.

Results: GP88 induced cell proliferation and conferred letrozole resistance in a time- and dose-dependent fashion. Conversely, naturally letrozole resistant breast cancer cells displayed a 10-fold increase in GP88 expression when compared to letrozole sensitive cells. GP88 overexpression, or exogenous addition blocked the inhibitory effect of letrozole on proliferation, and stimulated survival and soft agar colony formation. In letrozole resistant cells, silencing GP88 by siRNA inhibited cell proliferation and restored their sensitivity to letrozole.

Conclusion: Our findings provide information on the role of an alternate growth and survival factor on the acquisition of aromatase inhibitor resistance in ER+ breast cancer.

Figures

Figure 1

GP88 induces AC1 cell proliferation in vitro. 5 × 104 cells/plate (35 mm well plates) MCF-7AC1 cells were plated in PRF medium supplemented with 5% Charcoal treated FBS overnight. The next day, medium was removed and serum free medium was added without or with increasing concentration of GP88: 150 ng/ml (P-150), 300 ng/ml (P-300) and 600 ng/ml (P-600) or 25 nM androstenedione (AD). After 6 days, cells were trypsinized and cell number was determined by counting cells with a hemocytometer. Experiment was performed three times in triplicate.

Figure 2

GP88 confers AC1 cells Letrozole resistance in vitro. 2000 cells/well (96 well plates) MCF-7AC1 cells were plated in PRF medium supplemented with 5% ChX FBS overnight The next day, medium was removed and serum free medium was added with and without 300 ng/ml GP88 (GP88 (P)), 25 nM androstenedione (AD) and letrozole (L) 50 nM and 100 nM, 200 nM. Cell proliferation was measured after 6 days using Cell Titer-Glo assay. Experiment was performed in triplicates with p < 0.01.

Figure 3

GP88 induces anchorage independent colony formation and confers letrozole resistance comparable to E2. A: AC1 cells (1 × 104 cells/well in 6-well plate) were plated in soft agar plates as described in the Method section in (A) PRF-ChX with and without 300 ng/ml GP88 (P), 25 nM androstenedione (AD) and 200 nM letrozole (L) for 2 weeks. (B) in DMEM/F12 supplemented with 5%FBS, E2 = 10-8 M, 25 nM AD, +/- 300 ng/ml GP88 cultured for 3weeks. Fresh medium was added every 3 to 4 days. At the end of the experiment, colonies were fixed with crystal violet and counted, p < 0.01

Figure 4

Overexpression of GP88 in AC1 cells and MCF-7CA cells confer letrozole resistance. A: GP88 overexpressing AC1 cells (AC1-P) cells were developed by stable transfection of GP88 cDNA expression vector as described in the method section. As control, AC1-EV cells were developed by transfecting empty vector (AC1-EV). For measuring anchorage-independent growth of stably transfected AC1-P cells, AC1-P cells were plated at a density of 5 × 103 cells/well in a 12 wells plate and cultivated for 2 weeks with/without 200 nM letrozole, 25 nM AD, 300 ng/ml GP88 for the AC1 and CA1-EV in 5% ChX-FBS supplemented medium containing soft agar. Medium was changed every 3-4 days. B: Stimulation of anchorage independent growth of MCF-7-CA cells overexpressing GP88. The aromatase-overexpressing cells MCF-7CA were transfected with expression vector containing GP88 cDNA (CA-P) or with empty vector (CA-EV). Anchorage independent growth of stable transfectants was examined as described above. C: Comparison of aromatase enzymatic activity in AC1 and AC1-P cells. AC1 and AC1-P cells were plated in 6-well plates at a density of 1.5 × 105 cells/well. Measurement of aromatase activity assay was performed following the protocol described in the method section.

Figure 5

Bcl-2 upregulation by GP88 in AC1 cells. A: The ability of letrozole to down regulate bcl-2 expression was examined for AC1. AC1 cells were plated at 1.5 × 105 cells/well in 6-well plate and incubated in PRF medium with 5% ChX-FBS. The next day, the medium was removed and fresh PRF-5%ChX-FBS medium was added with the indicated treatments 25 nM androstenedione (AD) or AD with 200 nM letrozole (ADL) alone or in the presence of GP88 (150 ng/ml and 300 ng/ml). After 48 hours, total RNA was extracted with Trizol™. Bcl-2 mRNA expression was determined by RT-PCR and examined by agarose gel electrophoresis and staining by ethidium bromide as described in the method section. GAPDH mRNA expression was examined as internal control for equal loading. B: Bcl-2 and GAPDH band intensities of triplicate experiments were determined by densitometry scanning using a UVP gel densitometer. Relative average intensity of bcl-2 bands from the triplicate experiments in each experimental group were then normalized using GAPDH as an internal standard and expressed as bars ± SD, p < 0.05.

Figure 6

Effect of silencing GP88 expression on cell proliferation and letrozole responsiveness. A: Top panel: Comparison of GP88 expression in MCF-7CA cells (letrozole sensitive) and LTLT cells (letrozole resistant cells). Bottom panel: LTLT-CA cells were treated with either siRNA control or with GP88 siRNA for 48 hours before harvesting the cells to determine GP88 expression. GP88 level was determined by IP-Western analysis and normalized to cell number as described previously (Lu and Serrero, 2001). (B) LTLT-CA cells were transfected with GP88 siRNA or control siRNA according to manufacturer's instructions one day before being plated at 2 × 103 cells/well in 96 well plates in PRF-5%ChX medium and incubated for the time indicated. Cell proliferation was then measured by using the cellTiter-GloR assay using a luminometer (Molecular Devices). (C) LTLT-CA cells were transfected with either GP88 siRNA or Control siRNA as described above. The next day, cells were plated at 2 × 103 cells/well in 96-well plate and incubated in PRF-ChX medium for 24 hrs more. After 24 hrs, the medium was removed and cells were washed with PRF medium twice. Cells were then incubated in serum free fresh medium with the indicated concentration of letrozole for 3 days. After 3 days cells were counted using cellTiter-Glo assay, p < 0.05. D) Increase of GP88 expression in AC1-LetR cells derived from AC1 cells rendered letrozole resistant by long term culture in the presence of 5 nM letrozole. E) Effect of silencing GP88 on proliferation of AC1-LetR cells. AC1-LetR cells were transfected with either control siRNA or GP88 siRNA as described in the method section. Proliferation was measured after 24 and 48 hours exposure by cellTiter-GloR assay, p < 0.05.