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Loss of TSC1 function, a crucial negative regulator of mTOR signaling, is a common alteration in bladder cancer. Mutations in other members of the PI3K pathway, leading to mTOR activation, are also found in bladder cancer. This provides rationale for targeting mTOR for treatment of bladder cancer characterized by TSC1 mutations and/or mTOR activation. In this study, we asked whether combination treatment with rapamycin and resveratrol could be effective in concurrently inhibiting mTOR and PI3K signaling and inducing cell death in bladder cancer cells. In combination with rapamycin, resveratrol was able to block rapamycin-induced Akt activation, while maintaining mTOR pathway inhibition. In addition, combination treatment with rapamycin and resveratrol induced cell death specifically in TSC1-/- MEF cells, and not in wild-type MEFs. Similarly, resveratrol alone or in combination with rapamycin induced cell death in human bladder cancer cell lines. These data indicate that administration of resveratrol together with rapamycin may be a promising therapeutic option for treatment of bladder cancer. J. Cell. Physiol. 232: 436-446, 2017. © 2016 Wiley Periodicals, Inc.

Breast cancer is the leading cause of cancer-related deaths among women. Approximately 75% of breast cancers are estrogen receptor α (ERα) positive, underscoring the dependence of cancer cells on estrogen for growth and survival. Patients treated with endocrine therapy often develop resistance, either de novo or acquired, which in some cases is caused by aberrations within the growth factor signaling pathways. The mechanistic target of rapamycin complex 1 (mTORC1) has emerged as a critical node in estrogenic signaling. We have previously shown that mTORC1 can phosphorylate and activate ERα on S167 via its effector the 40S ribosomal S6 kinase 1 (S6K1). Presently, we have uncovered a direct link between mTORC1 and ERα. We found that ERα binds to regulatory-associated protein of mTOR (Raptor) and causes it to translocate to the nucleus upon estrogen stimulation. Additionally, we identified mTOR as the kinase that phosphorylates ERα on S104/106 and activates transcription of ER target genes. Our findings show a direct link between mTORC1 and ERα, which further implicates mTORC1 signaling in the pathogenesis of ER-positive breast cancer and provides rationale for FDA-approved use of mTORC1 inhibitors in combination with endocrine agents for treatment of this disease.

Class I PI 3-kinases signal by producing the signaling lipid phosphatidylinositol(3,4,5) trisphosphate, which in turn acts by recruiting downstream effectors that contain specific lipid-binding domains. The class I PI 3-kinases comprise four distinct catalytic subunits linked to one of seven different regulatory subunits. All the class I PI 3-kinases produce the same signaling lipid, PIP3, and the different isoforms have overlapping expression patterns and are coupled to overlapping sets of upstream activators. Nonetheless, studies in cultured cells and in animals have demonstrated that the different isoforms are coupled to distinct ranges of downstream responses. This review focuses on the mechanisms by which the production of a common product, PIP3, can produce isoform-specific signaling by PI 3-kinases.

Homologous recombination (HR) is a conserved process that maintains genome stability and cell survival by repairing DNA double-strand breaks (DSBs). The RAD51-related family of proteins is involved in repair of DSBs; consequently, deregulation of RAD51 causes chromosomal rearrangements and stimulates tumorigenesis. RAD51C has been identified as a potential tumor suppressor and a breast and ovarian cancer susceptibility gene. Recent studies have also implicated estrogen as a DNA-damaging agent that causes DSBs. We found that in ERα-positive breast cancer cells, estrogen transcriptionally regulates RAD51C expression in ERα-dependent mechanism. Moreover, estrogen induces RAD51C assembly into nuclear foci at DSBs, which is a precursor to RAD51 complex recruitment to the nucleus. Additionally, disruption of ERα signaling by either anti-estrogens or siRNA prevented estrogen induced upregulation of RAD51C. We have also found an association of a worse clinical outcome between RAD51C expression and ERα status of tumors. These findings provide insight into the mechanism of genomic instability in ERα-positive breast cancer and suggest that individuals with mutations in RAD51C that are exposed to estrogen would be more susceptible to accumulation of DNA damage, leading to cancer progression.

Loss of TSC1 function, a crucial negative regulator of mTOR signaling, is a common alteration in bladder cancer. Mutations in other members of the PI3K pathway, leading to mTOR activation, are also found in bladder cancer. This provides rationale for targeting mTOR for treatment of bladder cancer characterized by TSC1 mutations and/or mTOR activation. In this study, we asked whether combination treatment with rapamycin and resveratrol could be effective in concurrently inhibiting mTOR and PI3K signaling and inducing cell death in bladder cancer cells. In combination with rapamycin, resveratrol was able to block rapamycin-induced Akt activation, while maintaining mTOR pathway inhibition. In addition, combination treatment with rapamycin and resveratrol induced cell death specifically in TSC1

The K + channel KCa3.1 is required for Ca 2+ influx and the subsequent activation of CD4 T cells. The class II phosphatidylinositol 3 kinase C2β (PI3KC2β) is activated by the T-cell receptor (TCR) and is critical for KCa3.1 channel activation. Tripartite motif containing protein 27 (TRIM27) is a member of a large family of proteins that function as Really Interesting New Gene (RING) E3 ubiquitin ligases. We now show that TRIM27 functions as an E3 ligase and mediates lysine 48 polyubiquitination of PI3KC2β, leading to a decrease in PI3K enzyme activity. By inhibiting PI3KC2β, TRIM27 also functions to negatively regulate CD4 T cells by inhibiting KCa3.1 channel activity and TCR-stimulated Ca 2+ influx and cytokine production in Jurkat, primary human CD4 T cells, and Th0, Th1, and Th2 CD4 T cells generated from TRIM27 −/− mice. These findings provide a unique mechanism for regulating class II PI3Ks, and identify TRIM27 as a previously undescribed negative regulator of CD4 T cells.

Lymphangioleiomyomatosis (LAM) is a rare neoplastic metastatic disease affecting women of childbearing age. LAM is caused by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) as a consequence of tuberous sclerosis complex (TSC) 1/2 inactivation. Clinically, LAM results in cystic lung destruction. mTORC1 inhibition using rapamycin analogs (rapalogs) is partially effective in reducing disease progression and improving lung function. However, cessation of treatment results in continued progression of the disease. In the present study, we investigated the effectiveness of the combination of rapamycin treatment with resveratrol, an autophagy inhibitor, in the TSC2-null xenograft tumor model. We determined that this combination inhibits phosphatidylinositol-4,5-bisphosphate 3-kinase PI3K/Akt/mTORC1 signaling and activates apoptosis. Therefore, the combination of rapamycin and resveratrol may be an effective clinical strategy for treatment of LAM and other diseases with mTORC1 hyperactivation.

Isoform-specific signaling by Class IA PI 3-kinases depends in part on the interactions between distinct catalytic subunits and upstream regulatory proteins. From among the class IA catalytic subunits (p110α, p110β, and p110δ), p110β has unique properties. Unlike the other family members, p110β directly binds to Gβγ subunits, downstream from activated G-protein coupled receptors, and to activated Rab5. Furthermore, the Ras-binding domain (RBD) of p110β binds to Rac and Cdc42 but not to Ras. Defining mutations that specifically disrupt these regulatory interactions is critical for defining their role in p110β signaling. This chapter describes the approach that was used to identify the Rab5 binding site in p110β, and discusses methods for the analysis of p110β-Rab5 interactions.

Synergistic activation by heterotrimeric guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110beta from other class IA phosphoinositide 3 kinases (PI3Ks). Activation of p110beta is specifically implicated in various physiological and pathophysiological processes such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110beta dependent functions we identified the site in p110beta that binds to the Gbetagamma subunit of G proteins. Mutation of this site eliminated Gbetagamma dependent activation of PI3Kbeta (a dimer of p110beta and the p85 regulatory subunit) in vitro and in cells without affecting basal activity or phosphotyrosine peptide mediated activation. Disrupting the p110beta Gbetagamma interaction by mutation or with a cell permeable peptide inhibitor blocked the transforming capacity of PI3Kbeta in fibroblasts and reduced the proliferation chemotaxis and invasiveness of PTEN null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kbeta could provide a therapeutic approach for tumors that depend on p110beta for growth and metastasis.

Approximately two thirds of all breast cancer cases are estrogen receptor (ER) positive, which underscores the dependence of cancer cells on estrogen for growth and survival. However, resistance to endocrine therapy develops in most cases. mTORC1 (the mechanistic target of rapamycin complex 1) is a critical node of estrogen signaling in the cell, however the mechanism of estrogen activation of mTORC1 is not well understood. We have previously demonstrated that mTORC1 promotes growth factor-mediated ER activation by phosphorylating ERα on Ser167. This phosphorylation, which is mediated by the mTORC1 effector kinase S6K1, is important for ERα dimerization, DNA binding and transcriptional activity, is associated with endocrine resistance and correlates with therapy response. Moreover, ERα promotes expression of S6K1, generating a feed-forward positive activation loop. Our current efforts are focused on fully understanding the nature and mechanism of the relationship between mTORC1 and estrogen pathways. Our data indicate that mTOR, raptor and ER form a complex whereby ER directly interacts with raptor. Moreover, this interaction is stimulated by estrogen as seen by co-immunoprecipitation analysis. We additionally found that ER and raptor co-localize to the nucleus upon estrogen stimulation, where they interact as detected by both immunofluorescence and cellular fractionation experiments. Our current efforts are focused on understanding the role of this interaction and the mechanism of estrogen mediated localization of raptor to the nucleus. We are also investigating the role of raptor and estrogen receptor interaction in endocrine therapy treatment and resistance. Citation Format: Anya Alayev, Rachel S. Salamon, Marina K. Holz. Crosstalk between mTORC1 and estrogen receptor in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A11.