Erik Zhang

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.

Synthetic lethality of combined glutaminase and Hsp90 inhibition in mTORC1-driven tumor cells

Significance The mammalian target of rapamycin complex 1 (mTORC1)-mediated signaling regulates protein translation, cell size/growth, cell survival, and metabolism. This signaling is commonly deregulated in cancer as well as genetic disorders such as tuberous sclerosis complex and sporadic lymphangioleiomyomatosis. Recent studies have shown that the mTORC1 inhibitor rapamycin and its analogs generally decrease proliferation rather than inducing cell death. In this study, we found a strategy that rapidly triggers death of cells with activated mTORC1-mediated signaling by using the combination of aminohydrolase enyzme glutaminase and chaperone protein heat shock protein 90 inhibitors. We believe this combination strategy may have potential to be developed into therapeutic use for the treatment of mTORC1-driven tumors. The mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals from growth factors, nutrients, and cellular energy status to control a wide range of metabolic processes, including mRNA biogenesis; protein, nucleotide, and lipid synthesis; and autophagy. Deregulation of the mTORC1 pathway is found in cancer as well as genetic disorders such as tuberous sclerosis complex (TSC) and sporadic lymphangioleiomyomatosis. Recent studies have shown that the mTORC1 inhibitor rapamycin and its analogs generally suppress proliferation rather than induce apoptosis. Therefore, it is critical to use alternative strategies to induce death of cells with activated mTORC1. In this study, a small-molecule screen has revealed that the combination of glutaminase (GLS) and heat shock protein 90 (Hsp90) inhibitors selectively triggers death of TSC2-deficient cells. At a mechanistic level, high mTORC1-driven translation rates in TSC1/2-deficient cells, unlike wild-type cells, sensitizes these cells to endoplasmic reticulum (ER) stress. Thus, Hsp90 inhibition drives accumulation of unfolded protein and ER stress. When combining proteotoxic stress with oxidative stress by depletion of the intracellular antioxidant glutathione by GLS inhibition, acute cell death is observed in cells with activated mTORC1 signaling. This study suggests that this combination strategy may have the potential to be developed into a therapeutic use for the treatment of mTORC1-driven tumors.

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.

Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells.

Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease that can lead to respiratory failure. LAM cells typically have inactivating TSC2 mutations, leading to mTORC1 activation. The gender specificity of LAM suggests that estradiol contributes to disease development, yet the underlying pathogenic mechanisms are not completely understood. Using metabolomic profiling, we identified an estradiol-enhanced pentose phosphate pathway signature in Tsc2-deficient cells. Estradiol increased levels of cellular NADPH, decreased levels of reactive oxygen species, and enhanced cell survival under oxidative stress. Mechanistically, estradiol reactivated Akt in TSC2-deficient cells in vitro and in vivo, induced membrane translocation of glucose transporters (GLUT1 or GLUT4), and increased glucose uptake in an Akt-dependent manner. 18F-FDG-PET imaging demonstrated enhanced glucose uptake in xenograft tumors of Tsc2-deficient cells from estradiol-treated mice. Expression array study identified estradiol-enhanced transcript levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. Consistent with this, G6PD was abundant in xenograft tumors and lung metastatic lesions of Tsc2-deficient cells from estradiol-treated mice. Molecular depletion of G6PD attenuated estradiol-enhanced survival in vitro, and treatment with 6-aminonicotinamide, a competitive inhibitor of G6PD, reduced lung colonization of Tsc2-deficient cells. Collectively, these data indicate that estradiol promotes glucose metabolism in mTORC1 hyperactive cells through the pentose phosphate pathway via Akt reactivation and G6PD upregulation, thereby enhancing cell survival under oxidative stress. Interestingly, a strong correlation between estrogen exposure and G6PD was also found in breast cancer cells. Targeting the pentose phosphate pathway may have therapeutic benefit for LAM and possibly other hormonally dependent neoplasms.

Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

mTORC1-driven tumor cells are highly sensitive to therapeutic targeting by antagonists of oxidative stress

mTORC1 is a central signaling node in controlling cell growth, proliferation, and metabolism that is aberrantly activated in cancers and certain cancer-associated genetic disorders, such as tuberous sclerosis complex (TSC) and sporadic lymphangioleiomyomatosis. However, while mTORC1-inhibitory compounds (rapamycin and rapalogs) attracted interest as candidate therapeutics, clinical trials have not replicated the promising findings in preclinical models, perhaps because these compounds tend to limit cell proliferation without inducing cell death. In seeking to address this issue, we performed a high-throughput screen for small molecules that could heighten the cytotoxicity of mTORC1 inhibitors. Here we report the discovery that combining inhibitors of mTORC1 and glutamate cysteine ligase (GCLC) can selectively and efficiently trigger apoptosis in Tsc2-deficient cells but not wild-type cells. Mechanistic investigations revealed that coinhibition of mTORC1 and GCLC decreased the level of the intracellular thiol antioxidant glutathione (GSH), thereby increasing levels of reactive oxygen species, which we determined to mediate cell death in Tsc2-deficient cells. Our findings offer preclinical proof of concept for a strategy to selectively increase the cytotoxicity of mTORC1 inhibitors as a therapy to eradicate tumor cells marked by high mTORC1 signaling, based on cotargeting a GSH-controlled oxidative stress pathway. Cancer Res; 76(16); 4816-27. ©2016 AACR.

Rapamycin-Insensitive Up-Regulation of Adipocyte Phospholipase A2 in Tuberous Sclerosis and Lymphangioleiomyomatosis

Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR), and are also seen in LAM cells in sporadic LAM. We recently reported that prostaglandin biosynthesis and cyclooxygenase-2 were deregulated in TSC and LAM. Phospholipase A2 (PLA2) is the rate-limiting enzyme that catalyzes the conversion of plasma membrane phospholipids into prostaglandins. In this study, we identified upregulation of adipocyte AdPLA2 (PLA2G16) in LAM nodule cells using publicly available expression data. We showed that the levels of AdPLA2 transcript and protein were higher in LAM lungs compared with control lungs. We then showed that TSC2 negatively regulates the expression of AdPLA2, and loss of TSC2 is associated with elevated production of prostaglandin E2 (PGE2) and prostacyclin (PGI2) in cell culture models. Mouse model studies also showed increased expression of AdPLA2 in xenograft tumors, estrogen-induced lung metastatic lesions of Tsc2 null leiomyoma-derived cells, and spontaneous renal cystadenomas from Tsc2+/− mice. Importantly, rapamycin treatment did not affect the expression of AdPLA2 and the production of PGE2 by TSC2-deficient mouse embryonic fibroblast (Tsc2 −/−MEFs), rat uterine leiomyoma-derived ELT3 cells, and LAM patient-associated renal angiomyolipoma-derived “mesenchymal” cells. Furthermore, methyl arachidonyl fluorophosphate (MAFP), a potent irreversible PLA2 inhibitor, selectively suppressed the growth and induced apoptosis of TSC2-deficient LAM patient-derived cells relative to TSC2-addback cells. Our findings suggest that AdPLA2 plays an important role in promoting tumorigenesis and disease progression by modulating the production of prostaglandins and may serve as a potential therapeutic target in TSC and LAM.

Rapamycin-Resistant Poly (ADP-Ribose) Polymerase-1 Overexpression Is a Potential Therapeutic Target in Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a female-predominant cystic lung disease that can lead to respiratory failure. LAM cells typically have inactivating tuberous sclerosis complex 2 (TSC2) mutations and mammalian target of rapamycin (mTOR) complex (mTORC) 1 activation. Clinical response to the mTORC1 inhibitors has been limited, prompting a search for additional therapy for LAM. In this study, we investigated the impact of TSC2 on the expression of poly (ADP-ribose) polymerase (PARP)-1 that initiates the DNA repair pathway, and tested the efficacy of PARP1 inhibitors in the survival of TSC2-deficient (TSC2(-)) cells. We analyzed publicly available expression arrays of TSC2(-) cells and validated the findings using real-time RT-PCR, immunoblotting, and immunohistochemistry. We examined the impact of rapamycin and Torin 1 on PARP1 expression. We also tested the effect of PARP1 inhibitors, 8-hydroxy-2-methylquinazoline-4-one and 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline, on the survival of TSC2(-) cells. We identified the up-regulation of PARP1 in TSC2(-) cells relative to cells in which wild-type TSC2 has been reintroduced (TSC2-addback [TSC2(+)] cells). The transcript levels of PARP1 in TSC2(-) cells were not affected by rapamycin. PARP1 levels were increased in TSC2(-) cells, xenograft tumors of rat-derived TSC2(-) cells, renal cystadenomas from Tsc2(+/-) mice, and human LAM nodules. RNA interference of mTOR failed to reduce PARP1 levels. Proliferation and survival of TSC2(-) cells was reduced in response to PARP1 inhibitor treatment, more so than TSC2(+) cells. TSC2(-) cells exhibit higher levels of PARP1 relative to TSC2(+) cells in an mTOR-insensitive manner. PARP1 inhibitors selectively suppress the growth and induce apoptosis of TSC2(-) cells from patients with LAM. Targeting PARP1 may be beneficial in the treatment of LAM and other neoplasm with mTORC1 activation.

Insulin growth factor binding protein 2 mediates the progression of lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease that almost exclusively affects women. LAM cells migrate to the lungs, where they cause cystic destruction of lung parenchyma. Mutations in TSC1 or TSC2 lead to the activation of the mammalian target of rapamycin complex-1, a kinase that regulates growth factor-dependent protein translation, cell growth, and metabolism. Insulin-like growth factor binding protein 2 (IGFBP2) binds insulin, IGF1 and IGF2 in circulation, thereby modulating cell survival, migration, and invasion in neoplasms. In this study, we identified that IGFBP2 primarily localized in the nucleus of TSC2-null LAM patient-derived cells in vitro and in vivo. We also showed that nuclear accumulation of IGFBP2 is closely associated with estrogen receptor alpha (ERa) expression. Furthermore, estrogen treatment induced IGFBP2 nuclear translocation in TSC2-null LAM patient-derived cells. Importantly, depletion of IGFBP2 by siRNA reduced cell proliferation, enhanced apoptosis, and decreased migration and invasion of TSC2-null LAM patient-derived cells. More interestingly, depletion of IGFBP2 markedly decreased the phosphorylation of MAPK in LAM patient-derived TSC2-null cells. Collectively, these results suggest that IGFBP2 plays an important role in promoting tumorigenesis, through estrogen and ERalpha signaling pathway. Thus, targeting IGFBP2 may serve as a potential therapeutic strategy for women with LAM and other female gender specific neoplasms.