Nicholas C. Wolff

Regulation of TFEB and V-ATPases by mTORC1

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is an important, highly conserved, regulator of cell growth. Ancient among the signals that regulate mTORC1 are nutrients. Amino acids direct mTORC1 to the surface of the late endosome/lysosome, where mTORC1 becomes receptive to other inputs. However, the interplay between endosomes and mTORC1 is poorly understood. Here, we report the discovery of a network that links mTORC1 to a critical component of the late endosome/lysosome, the V‐ATPase. In an unbiased screen, we found that mTORC1 regulated the expression of, among other lysosomal genes, the V‐ATPases. mTORC1 regulates V‐ATPase expression both in cells and in mice. V‐ATPase regulation by mTORC1 involves a transcription factor translocated in renal cancer, TFEB. TFEB is required for the expression of a large subset of mTORC1 responsive genes. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization and in a manner dependent on both TFEB and V‐ATPases, mTORC1 promotes endocytosis. These data uncover a regulatory network linking an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis.

A Validated Tumorgraft Model Reveals Activity of Dovitinib Against Renal Cell Carcinoma

Extensively validated tumorgraft model shows activity of investigational agent dovitinib against renal cell carcinoma. Grafting a Better Cancer Model When it comes to predicting drug responsiveness in cancer patients, the standard mouse models (xenografts) get low marks: Drugs that work in these mice are frequently ineffective in humans. Xenograft models are created by injecting human tumor cell lines—which often acquire new mutations in culture—into immunocompromised mice. The resulting tumors are generally different from the original tumor. Tumorgrafts, instead created by implanting fragments of human tumors directly into mice, are generating new excitement among some researchers. Sivanand et al. now describe and validate a tumorgraft model of renal cell carcinoma (RCC) that shows promise for preclinical drug studies. The scientists implanted small fragments of RCC tumor from 94 patients into mice, placing the fragments under the fibrous capsule that surrounds the kidney. Sixteen stable tumor lines—which could be serially passaged to new mice—were ultimately established. Examination by a clinical pathologist revealed that the tumorgrafts were quite similar histologically to the original tumors; gene expression patterns, DNA copy number changes, and most mutations in protein-coding regions were also preserved. In addition, mice bearing tumorgrafts from patients that developed elevated concentrations of serum calcium similarly developed tumor-induced hypercalcemia. Moreover, tumorgraft growth was inhibited by two drugs (sunitinib, approved for treating RCC, and sirolimus, the active metabolite of an approved RCC drug), but did not respond to a lung cancer drug that is inactive against RCC. Finally, dovitinib, an inhibitor of several growth factor receptors that is being studied in clinical trials, suppressed the tumorgrafts more potently than sunitinib or sirolimus, suggesting that dovitinib would be active against RCC in humans. These results apply more broadly: The tumorgrafts should be useful for testing other targeted drugs preclinically—an important need, given that most anticancer drugs that enter clinical trials fail to gain approval. Most anticancer drugs entering clinical trials fail to achieve approval from the U.S. Food and Drug Administration. Drug development is hampered by the lack of preclinical models with therapeutic predictive value. Herein, we report the development and validation of a tumorgraft model of renal cell carcinoma (RCC) and its application to the evaluation of an experimental drug. Tumor samples from 94 patients were implanted in the kidneys of mice without additives or disaggregation. Tumors from 35 of these patients formed tumorgrafts, and 16 stable lines were established. Samples from metastatic sites engrafted at higher frequency than those from primary tumors, and stable engraftment of primary tumors in mice correlated with decreased patient survival. Tumorgrafts retained the histology, gene expression, DNA copy number alterations, and more than 90% of the protein-coding gene mutations of the corresponding tumors. As determined by the induction of hypercalcemia in tumorgraft-bearing mice, tumorgrafts retained the ability to induce paraneoplastic syndromes. In studies simulating drug exposures in patients, RCC tumorgraft growth was inhibited by sunitinib and sirolimus (the active metabolite of temsirolimus in humans), but not by erlotinib, which was used as a control. Dovitinib, a drug in clinical development, showed greater activity than sunitinib and sirolimus. The routine incorporation of models recapitulating the molecular genetics and drug sensitivities of human tumors into preclinical programs has the potential to improve oncology drug development.

Interplay Between pVHL and mTORC1 Pathways in Clear-Cell Renal Cell Carcinoma

mTOR complex 1 (mTORC1) is implicated in cell growth control and is extensively regulated. We previously reported that in response to hypoxia, mTORC1 is inhibited by the protein regulated in development and DNA damage response 1 (REDD1). REDD1 is upregulated by hypoxia-inducible factor (HIF)-1, and forced REDD1 expression is sufficient to inhibit mTORC1. REDD1-induced mTORC1 inhibition is dependent on a protein complex formed by the tuberous sclerosis complex (TSC)1 and 2 (TSC2) proteins. In clear-cell renal cell carcinoma (ccRCC), the von Hippel-Lindau (VHL) gene is frequently inactivated leading to constitutive activation of HIF-2 and/or HIF-1, which may be expected to upregulate REDD1 and inhibit mTORC1. However, mTORC1 is frequently activated in ccRCC, and mTORC1 inhibitors are effective against this tumor type; a paradox herein examined. REDD1 was upregulated in VHL-deficient ccRCC by in silico microarray analyses, as well as by quantitative real-time PCR, Western blot, and immunohistochemistry. Vhl disruption in a mouse model was sufficient to induce Redd1. Using ccRCC-derived cell lines, we show that REDD1 upregulation in tumors is VHL dependent and that both HIF-1 and HIF-2 are, in a cell-type-dependent manner, recruited to, and essential for, REDD1 induction. Interestingly, whereas mTORC1 is responsive to REDD1 in some tumors, strategies have evolved in others, such as mutations disrupting TSC1, to subvert mTORC1 inhibition by REDD1. Sequencing analyses of 77 ccRCCs for mutations in TSC1, TSC2, and REDD1, using PTEN as a reference, implicate the TSC1 gene, and possibly REDD1, as tumor suppressors in sporadic ccRCC. Understanding how ccRCCs become refractory to REDD1-induced mTORC1 inhibition should shed light into the development of ccRCC and may aid in patient selection for molecular-targeted therapies. Mol Cancer Res; 9(9); 1255–65. ©2011 AACR.

Bap1 is essential for kidney function and cooperates with Vhl in renal tumorigenesis

Significance Despite the discovery of the von Hippel–Lindau (VHL) gene in 1993, and that inactivating germ-line mutations of VHL cause multiple kidney lesions, including clear-cell renal cell carcinoma (ccRCC), Vhl inactivation in the mouse does not lead to ccRCC and a mouse model has been lacking. We discovered that the BRCA1-associated protein-1 (BAP1) two-hit tumor suppressor gene is mutated in ccRCC, and one BAP1 allele is frequently somatically codeleted with VHL in tumors. In the mouse, Vhl and Bap1 are on different chromosomes. We show that SIX homeobox 2 (Six2)-Cre;VhlF/F;Bap1F/+ mice develop premalignant lesions and malignant ccRCC resembling VHL syndrome. More broadly, differences in tumor predisposition across species may result from differences in the location of two-hit tumor suppressor genes across the genome. Why different species are predisposed to different tumor spectra is not well understood. In particular, whether the physical location of tumor suppressor genes relative to one another influences tumor predisposition is unknown. Renal cancer presents a unique opportunity to explore this question. Renal cell carcinoma (RCC) of clear-cell type (ccRCC), the most common type, begins with an intragenic mutation in the von Hippel–Lindau (VHL) gene and loss of 3p (where VHL is located). Chromosome 3p harbors several additional tumor suppressor genes, including BRCA1-associated protein-1 (BAP1). In the mouse, Vhl is on a different chromosome than Bap1. Thus, whereas loss of 3p in humans simultaneously deletes one copy of BAP1, loss of heterozygosity in the corresponding Vhl region in the mouse would not affect Bap1. To test the role of BAP1 in ccRCC development, we generated mice deficient for either Vhl or Vhl together with one allele of Bap1 in nephron progenitor cells. Six2-Cre;VhlF/F;Bap1F/+ mice developed ccRCC, but Six2-Cre;VhlF/F mice did not. Kidneys from Six2-Cre;VhlF/F;Bap1F/+ mice resembled kidneys from humans with VHL syndrome, containing multiple lesions spanning from benign cysts to cystic and solid RCC. Although the tumors were small, they showed nuclear atypia and exhibited features of human ccRCC. These results provide an explanation for why VHL heterozygous humans, but not mice, develop ccRCC. They also explain why a mouse model of ccRCC has been lacking. More broadly, our data suggest that differences in tumor predisposition across species may be explained, at least in part, by differences in the location of two-hit tumor suppressor genes across the genome.

Imatinib Mesylate Efficiently Achieves Therapeutic Intratumor Concentrations in Vivo but Has Limited Activity in a Xenograft Model of Small Cell Lung Cancer

Purpose: Despite recent advances in cancer therapy, long-term survival in small cell lung cancer (SCLC) remains uncommon, underscoring the need for novel therapeutic approaches. Previous studies have identified constitutive expression of the receptor tyrosine kinase, c-Kit, and its ligand, stem cell factor, in a substantial proportion of SCLC specimens. The purpose of this study was to determine whether imatinib mesylate, an inhibitor of c-Kit, could achieve therapeutic concentrations in tumors and in brain (a frequent site of SCLC metastasis) and interfere with SCLC tumor growth in vivo. Experimental Design: Human SCLC tumor cell lines with constitutive c-kit expression and tyrosine phosphorylation (NCI-H209, NCI-H526, and NCI-H1607) were used to establish SCLC tumor xenografts in NCr nude (nu/nu)-immunodeficient mice. SCLC tumor-bearing mice were randomly assigned to imatinib or control (water) administered twice a day by oral gavage. Imatinib concentrations in plasma, brain, and tumor were quantitated and correlated with tumor response. Results: Therapeutic concentrations of imatinib were achieved in plasma and tumor xenografts but not in the brain. Imatinib blocked the constitutive activation of c-kit in SCLC tumor cell lines in vitro but had a negligible effect on SCLC xenograft growth in vivo. Conclusions: Orally administered imatinib rapidly reaches therapeutic concentrations in SCLC xenografts, suggesting the feasibility of combining imatinib with other novel or traditional chemotherapeutic agents in SCLC or other solid tumors. The c-Kit signaling pathway does not appear to play a critical role in SCLC proliferation and viability in vivo, however, suggesting that imatinib is unlikely to be effective as monotherapy for SCLC.

Sirolimus and Temsirolimus for Epithelioid Angiomyolipoma

A 24-year-old man with familial tuberous sclerosis complex (TSC) presented to University of Texas Southwestern (Dallas, TX) in February 2009 with abdominal pain, vomiting, and generalized weakness. Five months previously, at another institution, he had undergone a right radical nephrectomy of a 24-cm epithelioid angiomyolipoma (EAML) that ruptured during surgery. Pathologic examination of the tumor at our institution showed sheets of epithelioid cells surrounding haphazardly shaped vessels embedded in a hyalinized stroma with focal myxoid change (Fig 1A; H&E, hematoxylin and eosin). The neoplastic cells were large, round or oval, with eccentric round nuclei, prominent nucleoli, and eosinophilic cytoplasm. They resembled ganglion cells and rare mitoses were appreciated (one in ten high-power fields). Immunohistochemical studies showed that the neoplastic cells expressed, as is characteristic of this tumor type,

Cell-Type-Dependent Regulation of mTORC1 by REDD1 and the Tumor Suppressors TSC1/TSC2 and LKB1 in Response to Hypoxia

ABSTRACT mTORC1 is a critical regulator of cell growth that integrates multiple signals and is deregulated in cancer. We previously reported that mTORC1 regulation by hypoxia involves Redd1 and the Tsc1/Tsc2 complex. Here we show that Redd1 induction by hypoxia is tissue dependent and that hypoxia signals are relayed to mTORC1 through different pathways in a tissue-specific manner. In the liver, Redd1 induction is restricted to the centrilobular area, and in primary hepatocytes, mTORC1 inhibition by hypoxia is independent of Redd1. Furthermore, Tsc1/Tsc2 and Arnt (Hif-1β) are similarly dispensable. Hypoxia signaling in hepatocytes involves Lkb1, AMP-activated protein kinase (AMPK), and raptor. Differences in signal relay extend beyond hypoxia and involve AMPK signaling. AMPK activation (using 5-aminoimidazole-4-carboxamide riboside [AICAR]) induces raptor phosphorylation and inhibits mTORC1 in both mouse embryo fibroblasts (MEFs) and hepatocytes, but whereas mTORC1 inhibition is Tsc1/Tsc2 dependent in MEFs, it is independent in hepatocytes. In liver cells, raptor phosphorylation is essential for both AMPK and hypoxia signaling. Thus, context-specific signals are required for raptor phosphorylation-induced mTORC1 inhibition. Our data illustrate a heretofore unappreciated topological complexity in mTORC1 regulation. Interestingly, topological differences in mTORC1 regulation by the tumor suppressor proteins Lkb1 and Tsc1/Tsc2 may underlie their tissue specificity of tumor suppressor action.

Interference with the constitutive activation of ERK1 and ERK2 impairs EWS/FLI-1-dependent transformation.

The chimeric gene EWS/FLI-1, the hallmark of the Ewings sarcoma and primitive neuroectodermal tumor family, encodes a fusion protein with enhanced transcriptional activation properties and preserved recognition of canonical ETS binding sites. Although EWS/FLI-1 alters the expression of various genes, the precise mechanism by which EWS/FLI-1 acts as an oncogene remains to be defined. In this study we report that members of the mitogen-activated protein kinase (MAPK) signaling pathway, ERK1 and ERK2, are constitutively activated in NIH 3T3 cells expressing EWS/FLI-1. Interference with ERK activation by either highly specific inhibitors of MEK1 or a dominant negative ras mutant profoundly impaired the ability of EWS/FLI-1 to transform NIH3T3 cells to growth in semi-solid medium. An EWS/FLI-1 mutant defective in DNA-binding and transcriptional activation failed to activate ERK and was also defective in 3T3 cell transformation. Constitutive ERK activation was also evident in several human Ewings sarcoma tumor-derived cell lines. Interestingly, cells expressing the type II EWS/FLI-1 fusion, recently demonstrated more potent in transcriptional activation, showed even greater MAPK activation than cells expressing the more common type I fusion. These results implicate ERK activation in EWS/FLI-1 transformation and suggest that this signaling pathway may be important in the pathogenesis of Ewings sarcoma.

Modeling Renal Cell Carcinoma in Mice: Bap1 and Pbrm1 Inactivation Drive Tumor Grade

Clear cell renal cell carcinoma (ccRCC) is characterized by BAP1 and PBRM1 mutations, which are associated with tumors of different grade and prognosis. However, whether BAP1 and PBRM1 loss causes ccRCC and determines tumor grade is unclear. We conditionally targeted Bap1 and Pbrm1 (with Vhl) in the mouse using several Cre drivers. Sglt2 and Villin proximal convoluted tubule drivers failed to cause tumorigenesis, challenging the conventional notion of ccRCC origins. In contrast, targeting with PAX8, a transcription factor frequently overexpressed in ccRCC, led to ccRCC of different grades. Bap1-deficient tumors were of high grade and showed greater mTORC1 activation than Pbrm1-deficient tumors, which exhibited longer latency. Disrupting one allele of the mTORC1 negative regulator, Tsc1, in Pbrm1-deficient kidneys triggered higher grade ccRCC. This study establishes Bap1 and Pbrm1 as lineage-specific drivers of ccRCC and histologic grade, implicates mTORC1 as a tumor grade rheostat, and suggests that ccRCCs arise from Bowman capsule cells.Significance: Determinants of tumor grade and aggressiveness across cancer types are poorly understood. Using ccRCC as a model, we show that Bap1 and Pbrm1 loss drives tumor grade. Furthermore, we show that the conversion from low grade to high grade can be promoted by activation of mTORC1. Cancer Discov; 7(8); 900-17. ©2017 AACR.See related commentary by Leung and Kim, p. 802This article is highlighted in the In This Issue feature, p. 783.

REDD1/DDIT4-independent mTORC1 inhibition and apoptosis by glucocorticoids in thymocytes.

Glucocorticoids induce apoptosis in lymphocytes and are commonly used to treat hematologic malignancies. However, they are also associated with significant adverse effects and their molecular mechanism of action is not fully understood. Glucocorticoid treatment induces expression of the mTORC1 inhibitor Regulated in Development and DNA Damage Response 1 (REDD1), also known as DNA-Damage Inducible Transcript 4 (DDIT4), and mTORC1 inhibition may distinguish glucocorticoid-sensitive from glucocorticoid-resistant acute lymphoblastic leukemia (ALL). Interestingly, REDD1 induction was impaired in glucocorticoid-resistant ALL cells and inhibition of mTORC1 using rapamycin restored glucocorticoid sensitivity. These data suggest that REDD1 may be essential for the response of ALL cells to glucocorticoids. To further investigate the role of REDD1, we evaluated the effects of glucocorticoids on primary thymocytes from wild-type and REDD1-deficient mice. Glucocorticoid-mediated apoptosis was blocked by a glucocorticoid receptor antagonist and by an inhibitor of transcription, which interfered with REDD1 induction and mTORC1 inhibition. However, REDD1 ablation had no effect on glucocorticoid-induced mTORC1 inhibition and apoptosis in thymocytes ex vivo. Overall, these data not only demonstrate the contextual differences of downstream signaling following glucocorticoid treatment but also provide a better mechanistic understanding of the role of REDD1. Implications: These molecular findings underlying glucocorticoid action and the role of REDD1 are fundamental for the design of novel, more efficacious, and less toxic analogs. Mol Cancer Res; 12(6); 867–77. ©2014 AACR.