Normal epithelial cells require matrix attachment for survival and the ability of tumour cells to survive outside their natural extracellular matrix (ECM) niches is dependent on acquisition of anchorage independence1. While apoptosis is the most rapid mechanism for eliminating cells lacking appropriate ECM attachment2, recent reports suggest that non-apoptotic death processes prevent survival when apoptosis is inhibited in matrix-deprived cells3 , 4. Here we demonstrate that detachment of mammary epithelial cells from ECM causes an ATP deficiency due to loss of glucose transport. Overexpression of ErbB2 rescues the ATP deficiency by restoring glucose uptake through stabilization of EGFR and PI(3)K activation and this rescue is dependent on glucose-stimulated flux through the antioxidant-generating pentose phosphate pathway (PPP). Interestingly, we found that the ATP deficiency could be rescued by antioxidant treatment without rescue of glucose uptake. This rescue was found to be dependent on stimulation of fatty acid oxidation (FAO), which is inhibited by detachment-induced reactive oxygen species (ROS). The significance of these findings was supported by evidence of an elevation in ROS in matrix-deprived cells in the luminal space of mammary acini and that antioxidants facilitate the survival of these cells and enhance anchorageindependent colony formation. These results reveal both the importance of matrix attachment in regulating metabolic activity and an unanticipated mechanism for cell survival in altered matrix environments through antioxidant restoration of ATP generation.Epithelial cells are dependent on interactions with specific extracellular matrix (ECM) components for survival, proliferation, and differentiation functions 5 . Loss of matrix attachment of cultured epithelial cells activates a caspase-mediated apoptotic program known as anoikis2. In glandular cancers, like breast cancer, tumour cells are displaced from their * To whom correspondence should be addressed: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, Phone: 617-432-3974, Fax: 617-432-3969, joan_brugge@hms.harvard.edu. 4 Present address: Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 † These authors contributed equally to this work.Supplementary information is linked to the online version of the paper at www.nature.com/nature. A figure summarising the main result of this paper ( Supplementary Fig. 1) is included in the supplementary information.Author Contributions Z.T.S. and J.S.B. were responsible for the overall study design. Z.T.S., A.R.G., H.Y.I., and S.G. conducted experiments. L.S. and Z.J. conducted the experiments measuring native fluorescence of NAD(P)H in 3D cell culture. Z.G.-H. and P.P. designed the fatty acid oxidation studies and Z.T.S. and Z.G.-H. conducted the fatty acid oxidation assays. Z.T.S. and J.S.B. drafted the manuscript and all other authors made revisions. Author InformationReprints and permissions information is available...

Summary 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.

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