Inhibition of PI3K/mTOR leads to adaptive resistance in matrix-attached cancer cells

Abstract

The PI3K/mTOR-pathway is the most commonly dysregulated pathway in epithelial cancers and represents an important target for cancer therapeutics. Here, we show that dual inhibition of PI3K/mTOR in ovarian cancer-spheroids leads to death of inner matrix-deprived cells, whereas matrix-attached cells are resistant. This matrix-associated resistance is mediated by drug-induced upregulation of cellular survival programs that involve both FOXO-regulated transcription and cap-independent translation. Inhibition of any one of several upregulated proteins, including Bcl-2, EGFR, or IGF1R, abrogates resistance to PI3K/mTOR inhibition. These results demonstrate that acute adaptive responses to PI3K/mTOR inhibition in matrix-attached cells resemble well-conserved stress responses to nutrient and growth factor deprivation. Bypass of this resistance mechanism through rational design of drug combinations could significantly enhance PI3K-targeted drug efficacy.

Figure 1

Ovarian cancer cell lines form acini-like structures in reconstituted basement membrane and matrix-attached cells show resistance to BEZ235-induced apoptosis. (A) Ovarian cancer cell lines (PIK3CA mutations indicated in parenthesis) MCAS (H1047R), SKOV3 (H1047R), OVCA432 (unknown) and OV2008 (E545K) were cultured in Matrigel for 6d and stained for Ki67 (red) or cleaved caspase-3 (red) and counterstained with DAPI (blue). (Mutation information provided by S. Jones and V. Velculescu, personal communication). One representative phase or confocal section is shown. (B) OV2008, MCAS and OVCA432 cells were cultured in Matrigel for 4d and 1μM BEZ235 was added for 48h before the cells were fixed, stained for cleaved caspase-3 (red) and DAPI (blue) and imaged by confocal microscopy. Confocal scale bar 50μm, phase contrast scale bar 200μm. See also Figure S1.

Figure 2

RPPA reveals multiple proteins upregulated in BEZ235 treated cells. (A) OV2008 and SKOV3 cells were cultured in Matrigel (3D) or monolayer (2D) and treated with BEZ235 or DMSO and the protein lysates were analyzed by RPPA (red: increased signal and green: decreased signal, upon BEZ235 treatment). Samples are normalized against DMSO-treated controls. Proteins with significant differences (p<0.05, Student’s T-Test) between BEZ235- and DMSO-treated OV2008 cells in an experiment performed in triplicate are shown for all experiments. (B) Several of the up- and down-regulated proteins from OV2008 RPPA were validated by Western blot analysis. (C) Bcl-2 inhibition abrogates outer cells resistance to PI3K/mTOR inhibition. MCAS and OV2008 cell lines were cultured in 3D and treated with DMSO, BEZ235 or ABT737 alone or in combination for 48h. Cells were imaged by phase contrast, fixed, stained for cleaved caspase-3 (red) and imaged by phase contrast or confocal microscopy. Confocal scale bar 50μm, phase contrast scale bar 200μm. See also Figure S2.

Figure 3

Many of the induced proteins are upregulated at the mRNA level through FOXO-dependent transcription. (A) Heatmaps showing relative levels of mRNA of proteins that were upregulated in the RPPA in OV2008 and MCAS cells; additional RTK’s not present in RPPA were also selected for analysis. mRNA values were normalized relative to DMSO-treated cells, red: upregulation in response to BEZ235 treatment and green: downregulation. ~60% (12/20 for OV2008 and 11/19 for MCAS) of the proteins that are detectably upregulated in response to BEZ235 in RPPAs and detected in the mRNA expression array show elevated mRNA at either timepoint (p=0.000385 for OV2008 and p=0.041 for MCAS, hypergeometric probability distribution). Proteins validated by western blots are marked by asterisk and the direction of change is indicated in parenthesis. (B) Heatmap showing relative levels of mRNAs previously reported as FOXO targets; out of 117 reported FOXO targets 31 are upregulated in at least two conditions (p<.01), showing a significant enrichment (p=0.00335, hypergeometric probability distribution). (C) FOXO1 and FOXO3 were downregulated in OV2008 cells by siRNAs targeting FOXO1 or FOXO3 (left panel) or transduction of a lentiviral vector encoding shRNA for FOXO3, together with FOXO1 siRNAs. Lysates were probed with antibodies to Bcl-2 and IGF1Rβ to monitor upregulation upon 18h BEZ235 treatment. The efficacy of the knock-downs was verified by Western blotting of FOXO1 and FOXO3.

Figure 4

Inhibition of 4E-BP1 phosphorylation correlates with the upregulation of Bcl-2 and IGF1R and disintegration of 3D spheroids. (A) OV2008 cells were grown in 3D cultures for four days and treated with indicated inhibitors targeting PI3K and/or mTOR in combination with ABT-737 for 48h. The cells were fixed, stained for cleaved caspase 3 (red) and DAPI (blue) and imaged with phase and confocal microscopy. Confocal scale bar 50μm, phase contrast scale bar 200μm. (B) OV2008 cells were grown in 3D and treated with indicated drugs and quantitated for structural integrity after 48h drug treatment as described in Methods. Representative images of scored structures (intact, semi-disintegrated, disintegrated) are shown in lower panel, scale bar 100μm. (C) Lysates were harvested from structures cultured in 3D and analyzed for Bcl-2 and IGF1Rβ p-4E-BP1^T37/46, p-S6^S240 and p-AKT^S473 expression after the indicated 48h drug treatments.

Figure 5

Cap-independent translation increases in response to BEZ235 treatment. (A) A dual luciferase reporter (Renilla luciferase expression mediated by cap-dependent translation and firefly luciferase expression by CrPV IRES), was used to monitor cap-independent translation in OV2008 and MCAS cells in response to BEZ235 or Torin1 treatment. The fold increase in the ratio of the firefly/Renilla luciferase levels was calculated as described in Methods. *p<0.05, **p<0.01, ***p<0.001. (B) BCL-2 IRES translational activity was monitored by a reporter containing the BCL2 IRES sequence upstream of firefly luciferase. Cap-dependent translation was monitored using a reporter containing a short unstructured 5’UTR fused to firefly luciferase. Luciferase expression from both reporters was normalized to luciferase mRNA expression and shown as fold change compared to DMSO control. Cartoon modified from Suo et al., 2010. (C) Upregulation of eIF2α phosphorylation was monitored in response to BEZ235 treatment over time in OV2008 and MCAS cells. Error bars shown as +/- SEM. See also Figure S3.

Figure 6

Analysis of BEZ235-treated normal breast epithelial cells and breast tumor cell lines. (A) RPPA analysis of breast cancer cell line spheroids and MCF10A cell monolayers treated with BEZ235 for 48h (red-increased signal upon BEZ235 treatment, green-decreased signal upon BEZ235 treatment). Samples are normalized against DMSO-treated controls. (B): T-47D, MDA-MB-468 and HCC-1569 breast cancer cells lines and (C) non-transformed immortalized MCF10A breast epithelial cells were cultured in Matrigel and treated with DMSO, BEZ235 or ABT737 alone or in combination for 48h. Cells were imaged by phase contrast, fixed, stained by EtBr (red) to mark dead cells and imaged by wide-field Phase contrast microscopy. Scale bar 200μm. (D) Immunoblots of MCF-10A cells treated with DMSO or BEZ235 for 48h. See also Figure S4.

Figure 7

Dual inhibition of Bcl-2 and PI3K/mTOR in an in vivo xenograft model and in primary patient samples causes decreased tumor growth and enhanced cell death. (A) MCAS and OV2008 cells were injected subcutaneously into female nod/scid mice, and after tumors were palpable, mice were treated every 24h with vehicle, GNE493 (mTOR inhibitor) (7mg/kg), ABT-737 (70mg/kg) or a combination of both. Tumors were measured on indicated days and the data is reported as the fold change relative to size of the same tumor on d1. The data, represented as the average +/- SEM, was derived from experiments in which 72 tumors were monitored in 43 mice for MCAS and for OV2008, 40 tumors in 20 mice. *** p<0.001, ** p<0.005. (B) Tumor sections from MCAS xenografts were stained for H&E and imaged at 4x; representative images of tumors of similar size (~300mm³) for each treatment are shown. Scale bar 100μm. (C) Western blot analysis of vehicle- and GNE493-treated MCAS tumors. Tumors were harvested 4h after the last dosing on d7 and probed with the indicated antibodies. Numbers above each lane indicates tumor number. (D) Tumor cells isolated from peritoneal or pleural fluid exudates were cultured in Matrigel for 8d and treated with the indicated inhibitors (ABT-263, an identical Bcl-2 inhibitor to ABT-737). Cell death was quantified by analysis of dead cells (as marked by EtBr) over total cell number (as analyzed by Hoechst stain). *p<0.05, **p<0.01, ***p<0.005. Red boxes indicate average data distribution, highest and lowest value are indicated with connected red caps. Single blue caps indicate SD and connected blue caps +/-SEM.