Mice were housed and handled by following the local, state, and federal regulations. after their transplantation, significantly increased their growthin vivo. The increase in tumor growth was completely rescued by rapamycin treatment, suggesting a major contribution from mTORC1 hyperactivation. Interestingly, glucose starvation-induced autophagy, but not amino acid starvation-induced autophagy, was increased significantly in Tsc1-null tumor cells. Further analysis of these cells also showed an increased Akt activation but no significant changes in Erk signaling. Together, these results provide insights into the mechanism by which hyperactivation of mTORC1 promotes breast cancer progression through increasing autophagy and Akt activationin vivo. == Introduction == Tuberous sclerosis complex (Tsc)3is an autosomal dominant disease that is caused by mutations in either theTsc1orTsc2gene and is characterized by the formation of benign tumors in multiple systems such as skin, brain, lung, liver, heart, and kidney (13). Tsc1 and Tsc2 proteins form a complex that negatively regulates cell growth/proliferation through the inhibition of mammalian target of rapamycin (mTOR). mTOR is a large protein kinase associated with different protein partners to form two independently regulated hetero-oligomeric complexes, the rapamycin-sensitive and rapamycin-insensitive mTOR complexes (mTORC) 1 and 2, respectively (4,5). mTORC1 contains the catalytic subunit mTOR and its binding partners mLST8/GL, PRAS40, and Raptor, which have been well documented to promote the growth and proliferation of a variety BAY 73-6691 of cells through the phosphorylation of two main regulators of mRNA translation and ribosome biogenesis, ribosomal S6 kinase (S6K) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) (4,6), although a multitude of other targets have also been suggested in recent studies (7). mTORC1 has been shown to play a major role in the regulation of autophagy by phosphorylating components of the autophagy induction machinery (8). Autophagy is an evolutionarily conserved process involved in the degradation of bulk cytoplasmic materials via sequestering them in the double-membraned structures called autophagosomes, followed by delivery to lysosomes for degradation (9,10). In addition to its BAY 73-6691 essential role in BAY 73-6691 a variety of physiological processes, autophagy dysfunction has been linked to many diseases, including cancer, although the underlying molecular mechanisms are not very clear at present (9,1113). Although abnormalities in Tsc/mTOR signaling are best illustrated in the development of benign tumors in many organs, recent studies have also suggested potential functions of this key pathway in the development and progression of several malignant cancers. For example, knockout of raptor inhibited mTORC1 activation and leukemia propagation (14). Liver-specific knockout of Tsc1 led to increased mTORC1 signaling and development of hepatocellular carcinoma in mice (15). Mice with conditional knockout of Tsc1 in prostate epithelial cells developed prostate cancer at an old age (16). However, a potential role and mechanisms of Tsc/mTOR signaling have not been examined directly in breast cancerin vivo, although a previous study showed that overexpression of Tsc1 in a murine breast cancer cell line, 4T1, reduced their growth in nude mice by inhibiting mTORC1-mediated VEGF expression and tumor angiogenesis (17). In this work, we created a novel system that allows one to delete Tsc1 in primary mammary tumor cells bothin vitroandin vivoin an inducible manner and demonstrated TNFSF13B directly that deletion of Tsc1 and consequent activation of mTORC1 promoted mammary tumor growth and metastasis. Moreover, we showed that Tsc1 deletion increased glucose starvation-induced autophagy as well as Akt activation, which could promote tumor cell survival and contribute to the increased tumor growthin vivo. == EXPERIMENTAL PROCEDURES == == == == == == Analysis of Human Breast Cancer Gene Expression Datasets == Oncomine was used to examineTsc1expression in several human cancer datasets, including Radvanyi Breast,GSE1477(18); Richardson Breast 2,GSE3744(19); The Cancer Genome Atlas,http://tcga-data.nci.nih.gov/tcga/; and Curtis (20). == Mice and Genotyping == MMTV-PyMT transgenic mice in the FVB/n background were as described previously BAY 73-6691 (21).Tsc1f/fmice (C57/B6 background) were BAY 73-6691 provided by Dr. David Kwiatkowski (22) and were used to cross with MMTV-PyMT mice to produceTsc1f/f;MMTV-PyMT mice with mixed FVB and C57/B6 backgrounds. Genotyping for theTsc1andPyMTalleles was performed as described previously (21,22). Mice were housed and handled by following the local, state, and federal regulations. The guidelines of the Institutional Animal Care and Use Committee at the University of Michigan were used in all experiments with mice. == Generation of Primary Mammary Tumor Cells Capable of Inducible Deletion of Tsc1 == Primary mammary tumor cells were isolated from femaleTsc1f/f;MMTV-PyMT mice as described previously (21). The.