A genetically defined mouse ovarian carcinoma model for the molecular characterization of pathway-targeted therapy and tumor resistance

Abstract

Cell lines and tumors with defined genetic alterations provide ideal systems in which to test the molecular mechanisms of tumor sensitivity to pathway-targeted therapy. We have generated mouse ovarian epithelial tumor cell lines that contain various combinations of genetic alterations in the p53, c-myc, K-ras and Akt genes. Using both in vitro and in vivo approaches, we investigated the effect of rapamycin on cell proliferation, tumor growth, and the accumulation of peritoneal ascites. We demonstrated that rapamycin effectively inhibits the growth of tumors that rely on Akt signaling for proliferation, whereas tumors in which Akt signaling is not the driving force in proliferation are resistant to rapamycin. The introduction of activated Akt to the rapamycin-resistant cells does not render the cells susceptible to rapamycin if they can use alternative pathways for survival and proliferation. Accordingly, the rapamycin-sensitive tumors develop resistance to rapamycin when presented with alternative survival pathways, such as the mitogen-activated extracellular kinase signaling pathway. The combination of rapamycin and the mitogen-activated extracellular kinase inhibitor PD98059 is required to diminish proliferation in these cell lines. Our results indicate that mammalian target of rapamycin inhibitors may be effective in a subset of tumors that depend on Akt activity for survival but not effective in all tumors that exhibit Akt activation. Tumors with alternative survival pathways may require the inactivation of multiple individual pathways for successful treatment.

Fig. 1.

Rapamycin inhibits proliferation of cell lines with genetic alterations in p53, c-myc, and Akt (C2 and T2) and p53, K-ras, and Akt (C3 and T3) but does not inhibit proliferation of cell lines with genetic alterations in p53, c-myc, and K-ras (C1 and T1). (A–C) C1, T1, C2, T2, C3, and T3 cells were seeded in equal amounts into six-well dishes. Two days after the cells were seeded, the medium was replaced with medium containing 100 ng/ml rapamycin dissolved in ethanol or ethanol alone (vehicle). The culture medium with rapamycin or vehicle was changed every second day. To monitor cell proliferation, rapamycin- and vehicle-treated cells were harvested every second day and stained with crystal-violet dye. The numbers on the y axis represent relative absorbance of cell-associated dye at 590 nm or the cell number. The error bars indicate standard deviation in triplicate cultures. (D) Representative images of nude mice (n = 8) that were injected i.p. with T1 cells. Seven days after injection, the mice were randomized for i.p. injections of a 5 mg/kg dose of rapamycin (n = 4) or vehicle (n = 4) every second day for 20 days. The tumor burden and extensive ascites accumulation were equivalent in rapamycin-treated and vehicle-treated mice. (E) Representative images of nude mice (n = 10) that were injected i.p. with T2 cells. Seven days after injection, the mice were randomized for i.p. injections ofa5mg/kg dose of rapamycin (n = 5) or vehicle (n = 5) every second day for 17 days. Rapamycin-treated mice were free of tumors and ascites, whereas vehicle-treated mice displayed i.p. carcinomatosis with ascites.

Fig. 2.

Rapamycin inhibits ascites production but does not inhibit the proliferation of rapamycin-resistant C1 cells with constitutively activated Akt. (A) Effect of rapamycin on in vitro proliferation of mC1+Akt cells. Two days after an equal number of cells were seeded into six-well dishes, the medium was replaced with medium containing 100 ng/ml rapamycin dissolved in ethanol or ethanol alone (vehicle). The culture medium with rapamycin or vehicle was changed every second day. To monitor cell proliferation, rapamycin- and vehicle-treated cells were harvested every second day and stained with crystal-violet dye. The numbers on the y axis represent relative absorbance of the cell-associated dye at 590 nm. The error bars indicate standard deviation in triplicate cultures. (B) Representative images of nude mice (n = 15) that were injected i.p. with mC1+Akt cells. Seven days after injection, the mice were randomized for treatment with a 5 mg/kg dose of rapamycin or vehicle every second day for 11 days. Rapamycin-treated mice (n = 8) and vehicle-treated mice (n = 7) displayed i.p. carcinomatosis. However, ascitic fluid was only present in the vehicle-treated mice. (C) The volume of ascites isolated from mice in B. The error bars indicate standard deviation. *, P < 0.01. (D) Levels of VEGF secreted in cell culture supernatant by mC1+Akt cells treated with rapamycin or vehicle. Equal numbers of cells were seeded in six-well dishes for VEGF secretion experiments. After cell attachment, the medium was replaced with a low-serum medium containing 100 ng/ml rapamycin dissolved in ethanol or ethanol alone. Cell culture supernatants were collected 48 h after treatment and subjected to ELISA for mouse VEGF. The error bars indicate standard deviation in six cultures. *, P < 0.01. (E) Western blot showing effective inhibition of the mTOR pathway in rapamycin-resistant and rapamycin-sensitive cell lines. C1, C2, and mC1+Akt cell lines were grown in vitro for 2 days, after which they were treated with 100 ng/ml rapamycin or vehicle for 1 day. Total cell lysates were immunoblotted by using an antibody against the hemagglutinin (HA) tag to determine the levels of the HA-tagged Akt protein. The activity of the mTOR pathway was assessed by comparing the protein levels of phosphorylated p70S6K in vehicle- and rapamycin-treated cells. Phosphorylated p70S6K (Thr-389) was detected with an antibody that recognizes phospho-p70S6K and phospho-p85S6K.

Fig. 3.

Introduction of alternative survival pathways into the rapamycin-sensitive ovarian cell lines induces resistance to rapamycin. (A) Equal numbers of T2, T2+H-ras, T2+K-ras, and T2+Her-2 cells were seeded into six-well dishes. After 2 days, the medium was replaced with medium containing 100 ng/ml rapamycin dissolved in ethanol or ethanol alone (vehicle). The culture medium with rapamycin or vehicle was changed every second day. To monitor cell proliferation, rapamycin- and vehicle-treated cells were harvested every second day and stained with crystal-violet dye. The numbers on the y axis represent relative absorbance of the cell-associated dye at 590 nm or the cell number. The error bars indicate standard deviation in triplicate cultures. (B) Western blot showing effective inhibition of the mTOR pathway in T2, T2+H-ras, T2+K-ras, and T2+Her-2 cell lines. The cells were grown in vitro for 2 days, after which they were treated with 100 ng/ml rapamycin or vehicle for 1 day. The activity of the mTOR pathway was assessed by comparing the protein levels of phosphorylated p70S6K in vehicle- and rapamycin-treated cells. Phosphorylated p70S6K (Thr-389) was detected with an antibody that recognizes phospho-p70S6K and phospho-p85S6K. (C) Representative images of immunocompetent FVB mice that were injected with T2+K-ras (n = 10) or T2+Her-2 (n = 8) cells and subjected to treatment with rapamycin or vehicle. Seven days after cell injection, the mice were randomly selected for treatment with 5 mg/kg of rapamycin or vehicle every second day. After 20 days of treatment, rapamycin- (n = 6) and vehicle-treated (n = 4) T2+K-ras-injected mice displayed i.p. carcinomatosis with an extensive accumulation of ascites. Similarly, rapamycin- (n = 4) and vehicle-treated (n = 4) T2+Her-2-injected mice displayed extensive i.p. carcinomatosis and ascites accumulation 20 days after treatment.

Fig. 4.

Effect of rapamycin, PD98059, and combination treatment on the proliferation of ovarian cancer cell lines that contain multiple survival pathways. Ovarian cancer cell lines T1, T2, mC1+Akt, and T2+K-ras were seeded into six-well dishes in triplicate. After 2 days of growth in culture, the medium was removed and replaced with a medium that contained one of the following: 100 ng/ml rapamycin dissolved in ethanol and 50 μM PD98059 dissolved in DMSO, 100 ng/ml rapamycin with ethanol and DMSO, 50 μM PD98059 with ethanol and DMSO, or ethanol and DMSO. The cells were treated for 8 days, during which time the culture medium was changed every second day. To monitor cell proliferation, drug- and vehicle-treated cells were harvested every second day and stained with crystal-violet dye. The numbers on the y axis represent relative absorbance of the cell-associated dye at 590 nm. The error bars indicate standard deviation in triplicate cultures.