Dataset Information

An increase in oxidative stress downstream of mTORC1 but not AKT causes a proliferative defect in cancer cells with acquired resistance to PI3K/mTOR inhibitors

ABSTRACT: Proteomics and phosphoproteomics analysis of MCF7 cells sensitive and resistant to the PI3K inhibitor GDC-0941. The analysis was done for parental and 3 resistant cell lines maintained in the presence or the absence of the drug. One of the resistant cell lines (G2) was also treated with vehicle or the kinase inhibitors GDC0941, MK-2206 and Ku-0063794 for 2h. The proteomics analysis of sensitive and resistant MCF7 cells in basal conditions and phosphoproteomics analysis of the G2 cells in the presence of inhibitors of the PI3K pathway were run in a nano flow ultrahigh pressure liquid chromatography (UPLC, nano Acquity, Waters) coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific). The phosphoproteomics study of sensitive and resistant MCF7 cells in basal conditions was run in a Dionex UltiMate 3000 RSLCnano coupled to an Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific).

INSTRUMENT(S): LTQ Orbitrap, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Pedro Casado-Izquierdo

LAB HEAD: Pedro R. Cutillas

PROVIDER: PXD003594 | Pride | 2018-04-09

REPOSITORIES: Pride

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Publications

Oxidative stress downstream of mTORC1 but not AKT causes a proliferative defect in cancer cells resistant to PI3K inhibition.

Dermit M M Casado P P Rajeeve V V Wilkes E H EH Foxler D E DE Campbell H H Critchlow S S Sharp T V TV Gribben J G JG Unwin R R Cutillas P R PR

Oncogene 20161219 19

Compounds targeting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling are being investigated in multiple clinical settings, but drug resistance may reduce their benefit. Compound rechallenge after drug holidays can overcome such resistance, yet little is known about the impact of drug holidays on cell biochemistry. We found that PI3K inhibitor (PI3Ki)-resistant cells cultured in the absence of PI3Ki developed a proliferative defect, increased oxygen consumption an ...[more]

PMID: 27991931

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Project description:Vitamin D is the strongest known natural anti-proliferative. A large number of studies in a wide spectrum of cancers, including epidemiological, in vitro and animal models, demonstrate that the active form of Vitamin D has anti-cancer benefits, affecting both progression and metastasis. Alike the role in calcium Vitamin D regulation, its anti-proliferative effect is thought to function through the Vitamin D receptor (VDR), although convincing evidence is lacking. Notwithstanding, separation of the calcemic and the anti-proliferative activity of Vitamin D analogues has been a major obstacle in developing new drugs for the treatment of cancer. The work presented attempts to unveil the molecular mechanism behind the anti-proliferative action of Vitamin D using genomic tools. For that purpose four independently developed Vitamin D sensitive/resistant MCF7 cell line pairs were collected. These unique biological replicates enabled us, both to increase the power of our study and to omit the use of Vitamin D. We deem this omission crucial since in the presence of Vitamin D only downstream genes involved in proliferation and cell cycle would be identified rather than causal resistance genes. The use of a variety of genomic techniques including expression, NMD and oligo CGH arrays reveal in the resistant cell lines the 11q13-14 as a region of DNA copy number loss and an altered expression of EGFR signaling pathway genes. Surprisingly, no genes known from calcium Vitamin D regulation were identified, nor did the VDR silencing by RNAi induce resistance to the sensitive cell lines. Keywords: comparative genomic hybridization, cell type comparision Several MCF7 breast tumor cell lines independently derived from the human breast cancer cell line, both resistant and sensitive to Vitamin D, were used. The pairs of cell lines (parental-resistant) were developed in different laboratories and using different methodologies; while some cell lines acquired resistance by long exposure to the physiological concentration of Vitamin D (100nM), others were induced to resistant by being exposed to increasing amounts of Vitamin D. A total of 4 microarray expression experiments were carried out. In all microarray experiments DNA from the Vitamin D resistant cell line was hybrizided agains DNA from the respsective sensitive/parental cell line.

Project description:Tamoxifen, an antagonist to estrogen receptor (ER), is a first line drug used in breast cancer treatment. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying the resistance to tamoxifen, we established a tamoxifen-resistant cell line by treating the MCF7 breast cancer cell line with tamoxifen for over 6 months. We showed that this cell line exhibited resistance to tamoxifen both in vitro and in vivo. In order to quantify the phosphorylation alterations associated with tamoxifen resistance, we performed SILAC-based quantitative phosphoproteomic profiling on the resistant and vehicle-treated sensitive cell lines where we identified >5,600 unique phosphopeptides. We found phosphorylation levels of 1,529 peptides were increased (>2 fold) and 409 peptides were decreased (<0.5-fold) in tamoxifen resistant cells compared to tamoxifen sensitive cells. Gene set enrichment analysis revealed that focal adhesion pathway was the top enriched signaling pathway activated in tamoxifen resistant cells. We observed hyperphosphorylation of the focal adhesion kinases FAK1 and FAK2 in the tamoxifen resistant cells. Of note, FAK2 was not only hyperphosphorylated but also transcriptionally upregulated in tamoxifen resistant cells. Suppression of FAK2 by specific siRNA knockdown could sensitize the resistant cells to the treatment of tamoxifen. We further showed that inhibiting FAK activity using the small molecule inhibitor PF562271 repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 significantly associated with short metastasis-free survival of ER-positive breast cancer patients treated with tamoxifen-based hormone therapy. Our studies suggest that FAK2 is a great potential target for the development of therapy for the treatment of hormone refractory breast cancers.

Dataset Information

An increase in oxidative stress downstream of mTORC1 but not AKT causes a proliferative defect in cancer cells with acquired resistance to PI3K/mTOR inhibitors

Publications

Oxidative stress downstream of mTORC1 but not AKT causes a proliferative defect in cancer cells resistant to PI3K inhibition.

Similar Datasets

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