Andrey Parkhitko

The mTORC1 Pathway Stimulates Glutamine Metabolism and Cell Proliferation by Repressing SIRT4

Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mammalian target of rapamycin complex 1 (mTORC1) activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP-responsive element binding 2 (CREB2). Thus, a relationship between mTORC1, SIRT4, and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation, and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach.

Tumorigenesis in tuberous sclerosis complex is autophagy and p62/sequestosome 1 (SQSTM1)-dependent

Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome characterized by benign tumors in multiple organs, including the brain and kidney. TSC-associated tumors exhibit hyperactivation of mammalian target of rapamycin complex 1 (mTORC1), a direct inhibitor of autophagy. Autophagy can either promote or inhibit tumorigenesis, depending on the cellular context. The role of autophagy in the pathogenesis and treatment of the multisystem manifestations of TSC is unknown. We found that the combination of mTORC1 and autophagy inhibition was more effective than either treatment alone in inhibiting the survival of tuberin (TSC2)-null cells, growth of TSC2-null xenograft tumors, and development of spontaneous renal tumors in Tsc2+/− mice. Down-regulation of Atg5 induced extensive central necrosis in TSC2-null xenograft tumors, and loss of one allele of Beclin1 almost completely blocked macroscopic renal tumor formation in Tsc2+/− mice. Surprisingly, given the finding that lowering autophagy blocks TSC tumorigenesis, genetic down-regulation of p62/sequestosome 1 (SQSTM1), the autophagy substrate that accumulates in TSC tumors as a consequence of low autophagy levels, strongly inhibited the growth of TSC2-null xenograft tumors. These data demonstrate that autophagy is a critical component of TSC tumorigenesis, suggest that mTORC1 inhibitors may have autophagy-dependent prosurvival effects in TSC, and reveal two distinct therapeutic targets for TSC: autophagy and the autophagy target p62/SQSTM1.

Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices

Significance KRAS is one of the most prevalent and vicious oncogenic proteins, yet no drugs are available to inhibit its pathologic activity in patients. We report that KRAS-targeting stapled peptides, modeled after the native son of sevenless 1 (SOS1) helical domain, engage wild-type and clinically relevant KRAS mutant proteins with nanomolar affinity. To our knowledge, these compounds represent the highest affinity and broadest spectrum binders of KRAS mutants reported to date. The stapled peptides disrupt the SOS1/KRAS protein interaction and directly inhibit nucleotide association to wild-type and mutant KRAS proteins. We correlate functional binding activity with SAH-SOS1 cytotoxicity across a 13-member panel of KRAS-driven cancer cells and demonstrate sequence- and dose-dependent inhibition of the ERK-MAP kinase phosphosignaling cascade downstream of KRAS in vitro and in vivo. Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequence-specific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85DV12 activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.

Estradiol and mTORC2 cooperate to enhance prostaglandin biosynthesis and tumorigenesis in TSC2-deficient LAM cells

Estradiol enhances COX-2 expression and prostaglandin biosynthesis in TSC2-deficient cells via a rapamycin-insensitive, mTORC2-dependent mechanism.

Mammalian Target of Rapamycin Signaling and Autophagy: Roles in Lymphangioleiomyomatosis Therapy

The pace of progress in lymphangioleiomyomatosis (LAM) is remarkable. In the year 2000, TSC2 gene mutations were found in LAM cells; in 2001 the tuberous sclerosis complex (TSC) genes were discovered to regulate cell size in Drosophila via the kinase TOR (target of rapamycin); and in 2008 the results were published of a clinical trial of rapamycin, a specific inhibitor of TOR, in patients with TSC and LAM with renal angiomyolipomas. This interval of just 8 years between a genetic discovery for which the relevant signaling pathway was as yet unknown, to the initiation, completion, and publication of a clinical trial, is an almost unparalleled accomplishment in modern biomedical research. This robust foundation of basic, translational, and clinical research in TOR, TSC, and LAM is now poised to optimize and validate effective therapeutic strategies for LAM. An immediate challenge is to deduce the mechanisms underlying the partial response of renal angiomyolipomas to rapamycin, and thereby guide the design of combinatorial approaches. TOR complex 1 (TORC1), which is known to be active in LAM cells, is a key inhibitor of autophagy. One hypothesis, which will be explored here, is that low levels of autophagy in TSC2-null LAM cells limits their survival under conditions of bioenergetic stress. A corollary of this hypothesis is that rapamycin, by inducing autophagy, promotes the survival of LAM cells, while simultaneously arresting their growth. If this hypothesis proves to be correct, then combining TORC1 inhibition with autophagy inhibition may represent an effective clinical strategy for LAM.

Autophagy-Dependent Metabolic Reprogramming Sensitizes TSC2-Deficient Cells to the Antimetabolite 6-Aminonicotinamide

The mammalian target of rapamycin complex 1 (mTORC1) is hyperactive in many human cancers and in tuberous sclerosis complex (TSC). Autophagy, a key mTORC1-targeted process, is a critical determinant of metabolic homeostasis. Metabolomic profiling was performed to elucidate the cellular consequences of autophagy dysregulation under conditions of hyperactive mTORC1. It was discovered that TSC2-null cells have distinctive autophagy-dependent pentose phosphate pathway (PPP) alterations. This was accompanied by enhanced glucose uptake and utilization, decreased mitochondrial oxygen consumption, and increased mitochondrial reactive oxygen species (ROS) production. Importantly, these findings revealed that the PPP is a key autophagy-dependent compensatory metabolic mechanism. Furthermore, PPP inhibition with 6-aminonicotinamide (6-AN) in combination with autophagy inhibition suppressed proliferation and prompted the activation of NF-κB and CASP1 in TSC2-deficient, but not TSC2-proficient cells. These data demonstrate that TSC2-deficient cells can be therapeutically targeted, without mTORC1 inhibitors, by focusing on their metabolic vulnerabilities. Implications: This study provides proof-of-concept that therapeutic targeting of diseases with hyperactive mTORC1 can be achieved without the application of mTORC1 inhibitors. Mol Cancer Res; 12(1); 48–57. ©2013 AACR.

Autophagy: An ‘Achilles’ heel of tumorigenesis in TSC and LAM

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is activated in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), is a master regulator of cell growth, cellular metabolism, and autophagy. Treatment of TSC and LAM patients with mTORC1 inhibitors partially decreases the size of brain and kidney tumors, and stabilizes pulmonary function. However, the tumors regrow and lung function continues to decline when treatment is discontinued. We hypothesized that dysregulation of autophagy plays a critical role in the pathogenesis of tumors with mTORC1 hyperactivation and in their response to mTORC1-targeted therapy. We found that cells lacking TSC2 have low levels of autophagy under basal and cellular stress conditions. Using genetic and pharmacological approaches, we discovered that the survival of Tsc2-deficient tumor cells is dependent on autophagy induction. Thus, autophagy inhibitors may have therapeutic potential in TSC and LAM, either as single agent therapy or in combination with mTORC1 inhibitors.Mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is activated in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), is a master regulator of cell growth, cellular metabolism, and autophagy. Treatment of TSC and LAM patients with mTORC1 inhibitors partially decreases the size of brain and kidney tumors, and stabilizes pulmonary function. However, the tumors regrow and lung function continues to decline when treatment is discontinued. We hypothesized that dysregulation of autophagy plays a critical role in the pathogenesis of tumors with mTORC1 hyperactivation and in their response to mTORC1-targeted therapy. We found that cells lacking TSC2 have low levels of autophagy under basal and cellular stress conditions. Using genetic and pharmacological approaches, we discovered that the survival of Tsc2-deficient tumor cells is dependent on autophagy induction. Thus, autophagy inhibitors may have therapeutic potential in TSC and LAM, either as single agent therapy or in combination with mTORC1 inhibitors.

Folliculin regulates cell-cell adhesion, AMPK, and mTORC1 in a cell-type-specific manner in lung-derived cells.

Germline loss‐of‐function BHD mutations cause cystic lung disease and hereditary pneumothorax, yet little is known about the impact of BHD mutations in the lung. Folliculin (FLCN), the product of the Birt–Hogg–Dube (BHD) gene, has been linked to altered cell–cell adhesion and to the AMPK and mTORC1 signaling pathways. We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho‐S6, a marker of mTORC1 activation, highlighting the cell type–dependent functions of FLCN. Cell–cell adhesion forces were significantly increased in FLCN‐deficient HBE cells, consistent with prior findings in FLCN‐deficient human kidney‐derived cells. To determine how these altered cell–cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd+/− mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg. Our results indicate that FLCN regulates the AMPK and mTORC1 pathways and cell–cell adhesion in a cell type–dependent manner. FLCN deficiency may impact the physiologic response to inflation‐induced mechanical stress, but further investigation is required. We hypothesize that FLCN‐dependent effects on signaling and cellular adhesion contribute to the pathogenesis of cystic lung disease in BHD patients.

Faslodex Inhibits Estradiol-Induced Extracellular Matrix Dynamics and Lung Metastasis in a Model of Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)-2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)-2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM.

Kinase mTOR: Regulation and Role in Maintenance of Cellular Homeostasis, Tumor Development, and Aging

Serine/threonine protein kinase mTOR regulates the maintenance of cellular homeostasis by coordinating transcription, translation, metabolism, and autophagy with availability of amino acids, growth factors, ATP, and oxygen. The mTOR kinase is a component of two protein complexes, mTORC1 and mTORC2, which are different in their composition and regulate different cellular processes. An uncontrolled activation of the mTOR kinase is observed in cells of the majority of tumors, as well as in diabetes and neurodegenerative and some other diseases. At present, inhibitors of the kinase complex mTORC1 are undergoing clinical trials. This review focuses on different aspects of the regulation of the mTORC1 and mTORC2 complexes, on their role in the regulation of protein synthesis, metabolism, and autophagy, as well as on using mTOR inhibitors for treatment of tumors and slowing of aging.