Sheng-Li Cai

A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.

Cytoplasmic Sequestration of p27 via AKT Phosphorylation in Renal Cell Carcinoma

Purpose: p27 localization and expression has prognostic and predictive value in cancer. Little is known regarding expression patterns of p27 in renal cell carcinoma (RCC) or how p27 participates in disease progression or response to therapy. Experimental Design: RCC-derived cell lines, primary tumors, and normal renal epithelial cells were analyzed for p27 expression, phosphorylation (T157 of the NLS), and subcellular localization. RCC-derived cell lines were treated with phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors and effects on p27 localization were assessed. The potential contribution of cytoplasmic p27 to resistance to apoptosis was also evaluated. Results: p27 was elevated in tumors compared with matched controls, and cytoplasmic mislocalization of p27 was associated with increasing tumor grade. Cytoplasmic localization of p27 correlated with phosphorylation at T157, an AKT phosphorylation site in the p27 NLS. In RCC cell lines, activated PI3K/AKT signaling was accompanied by mislocalization of p27. AKT activation and phosphorylation of p27 was associated with resistance to apoptosis, and small interfering RNA knockdown of p27 or relocalization to the nucleus increased apoptosis in RCC cells. Treatment with the PI3K inhibitors LY294002 or wortmannin resulted in nuclear relocalization of p27, whereas mTOR inhibition by rapamycin did not. Conclusions: In RCC, p27 is phosphorylated at T157 of the NLS, with increasing tumor grade associated with cytoplasmic p27. PI3K inhibition (which reduces AKT activity) reduces T157 phosphorylation and induces nuclear relocalization of p27, whereas mTOR inhibition does not. Clinical testing of these findings may provide a rational approach for use of mTOR and PI3K/AKT pathway inhibitors in patients with RCC.

Polycystic Kidney Disease as a Result of Loss of the Tuberous Sclerosis 2 Tumor Suppressor Gene During Development

Somatic loss of function of the tuberous sclerosis 2 (TSC2) tumor suppressor gene leads to the development of benign and malignant lesions of the kidney, brain, uterus, spleen, and liver and germline loss of function of this tumor suppressor gene is embryonic lethal. In addition, the gene product of TSC2, tuberin, is necessary for normal function of the polycystic kidney disease 1 (PKD1) gene product, polycystin-1, which is required for normal cell-cell and cell-matrix interactions. We report here the development of severe polycystic kidney disease in three cases of young Eker rats carrying a germline inactivation of one allele of the Tsc2 gene. Extrarenal tumors were also noted in the spleen and uterus of these animals, which was remarkable given their young age and in the case of the spleen, diffuse involvement of the affected organ. A cell line (EKT2) was established from an affected kidney of one of these animals and used in conjunction with tissues from affected animals to elucidate the defect responsible for the development of these lesions. Affected cells were determined to have lost the wild-type Tsc2 allele while retaining two copies of chromosome 10 containing the mutant Tsc2 allele along with two normal copies of the Pkd1 gene. The genetic data, bilateral nature of the observed kidney disease, and extent of involvement of the spleen and kidney indicate that, in affected animals, loss of the wild-type Tsc2 allele occurred during embryogenesis, probably as a result of chromosome nondisjunction, with affected animals being mosaics for loss of Tsc2 gene function.

Abstract 4831: ATM signals to TSC2 in the cytoplasm to regulate mTORC1 and autophagy in response to ROS

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC ATM is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. ATM deficiency is also associated with increased oxidative stress, and ATM has been implicated in metabolic regulation. The mechanisms responsible for crosstalk between oxidative stress and metabolic pathways are not fully understood, but are important since tumors often exhibit elevated ROS, and redox pathways may be therapeutic targets. We report a novel cytoplasmic pathway whereby ATM activates the TSC2 tumor suppressor, via LKB1 and AMPK in response to ROS, distinct from that of nuclear ATM in the DNA damage response. Knockout MEFs and siRNA approaches illustrated that mTORC1 suppression by ROS was dependent upon Tsc2 and ATM, and independent of p53. Reconstitution of LKB1-deficient HeLa S3 cells with wild-type LKB1 restored mTORC1 repression by ROS, while reconstitution of a mutant LKB1 lacking the ATM phosphorylation site did not. An in vivo cell-based functional assay revealed that a Tsc2 mutant lacking AMPK phosphorylation sites was deficient in repressing mTORC1 activity in response to H22, directly implicating AMPK as the mediator of ATM signaling to Tsc2. We also demonstrated that ATM activation of the LKB1-AMPK pathway was responsible for TSC2-mediated mTORC1 repression exclusively in the cytoplasm with subcellular fractionation and leptomycin B. Importantly, elevated ROS and dysregulation of mTORC1 in both ATM-deficient and Tsc2-deficient cells was inhibited with rapamycin. We also show for that rapamycin rescued lymphomagenesis in Atm-deficient mice, resulting in enhanced survival. mTORC1 negatively regulates autophagy, a cellular process which degrades organelles and long-lived proteins as a survival mechanism, but can also cause cell death. We tested the hypothesis that repression of mTORC1 by ROS results in induction of autophagy. Electron microscopy analysis demonstrated enhanced formation of autophagosomes in response to ROS. In MCF7 and SKOV-3 cells stably expressing GFP-LC3, a marker of autophagosomes, we observed an increase in punctate GFP-labeled autophagosomes, consistent with autophagy induction. A necessary step during autophagosome formation is lipidation of the cytosolic form of LC3 (LC3 I) to LC3 II, which can be detected as a mobility shift by western analysis. Western analysis confirmed that LC3 II was increased, and concomitantly, the autophagic substrate p62 was decreased, in response to ROS-mediated mTORC1 repression. Together, our results identify a pathway for ATM activation of TSC2 to regulate mTORC1 signaling and autophagy in response to ROS, identifying a new integration node for the cellular damage response with key pathways involved in metabolism, protein synthesis, and cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4831.

Abstract P4-07-02: Targeted therapies for TNBC: Exploiting vulnerabilities that arise from DNA damage repair pathway dependencies

We examined the synergistic effects of DNA damage, Chk1 inhibition and poly(ADP-ribose) polymerase (PARP) inhibition in TNBC. This combinatorial targeting allows us to exploit vulnerabilities in two pathways that are often deregulated in TNBCs: DNA damage checkpoint defects due to TP53 deficiency and DNA repair defects due to alterations in homologous recombination repair (HRR). TP53 maintains genome integrity by inhibiting cells that are experiencing genotoxic stress from progressing through the cell cycle, or by inducing apoptosis or senescence. In response to DNA damage, p53 activates gene expression to arrest cells in the G1 phase of the cell cycle and to reinforce the S- and G2-checkpoints. Thus, p53-deficient cells lack a G1 checkpoint and are impaired in their ability to sustain S- and G2-checkpoints. This makes p53-deficient tumors particularly sensitive to agents that abrogate these checkpoints. Because Chk1 inhibitors abrogate both S- and G2-checkpoints, combining Chk1 inhibitors with agents that induce genotoxic stress provides a rational therapeutic strategy for killing p53-deficient TNBC. Loss of HRR increases dependence of cells on a class of enzymes called PARPs, and Chk1 has also been shown to be important for efficient HRR. Thus, by interfering with HRR, Chk1 inhibitors are predicted to sensitize TNBC cells to PARP inhibitors. We tested the hypotheses that by impairing HRR, Chk1 inhibitors will sensitize TNBCs to PARP inhibition, and that therapies that combine Chk1 inhibitors with PARP inhibitors will be effective at killing TNBCs because they will simultaneously induce checkpoint bypass and block DNA repair. We generated a set of isogenic TNBC cell lines that are p53-proficient (p53WT) or p53-deficient (p53KD), and evaluated their sensitivity to Chk1 inhibitors (LY2606368) and DNA damaging agents (cisplatin). Loss of p53 conferred a dramatic increase in sensitivity to treatment with cisplatin + LY2606368. Surprisingly, inhibition of PARP1 (BMN673) did not increase sensitivity to Chk1 inhibitor ± cisplatin. To determine why Chk1 inhibition did not sensitize cells to PARP inhibition, we evaluated the effect of Chk1 inhibition on the ability of cells to recruit HRR proteins to sites of DNA damage. In line with CHK1 regulating HRR, Chk1 inhibition was associated with an inability of Rad51 to localize to sites of DNA double strand breaks. Interestingly, we also found that upstream of Rad51, there was a significant alteration in the formation of phopho-RPA2 foci in cells treated with the Chk1 inhibitor. On-going studies are evaluating whether there are changes in the kinetics of formation and/or resolution of Rad51 and phospho-RPA2 foci in response to Chk1 inhibition. Citation Format: Redwood AB, Cai S, Piwnica-Worms H. Targeted therapies for TNBC: Exploiting vulnerabilities that arise from DNA damage repair pathway dependencies. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-07-02.