Tuberous Sclerosis Complex Tumor Research Articles

Abstract 2501: Rapamycin inhibits epidermal cell proliferation in a xenograft model of tuberous sclerosis skin tumors

Abstract Patients with tuberous sclerosis complex (TSC) develop hamartomatous tumors of the skin that have increased dermal fibroblast-like cells and a thickened epidermis. Our previous studies indicate that dermal cells are neoplastic, showing loss of TSC1/TSC2 function and activation of signaling through the mTORC1 pathway, and that overlying epidermal keratinocytes are reactive. The goals of this study were to determine whether TSC skin tumor cells induce epidermal changes and to assess the potential utility and mechanism of action of rapamycin, an mTORC1 inhibitor. TSC2-null skin tumor cells or fibroblasts from patient normal-appearing skin were incorporated into collagen gels overlaid with normal human neonatal keratinocytes, and these constructs were grafted to the backs of nude mice. Mice grafted with TSC tumor cells (n=27) or TSC normal fibroblasts (n=23) received rapamycin (2 mg/kg) (n=24) or an equal volume of vehicle (n=26) by intraperitoneal injection on alternate days for 12 weeks beginning 5 weeks after grafting. Mice were sacrificed 24 hours after the last injection for analysis of the grafts by immunohistochemistry. Grafts containing TSC tumor cells showed greater activation of the mTORC1 signaling pathway than grafts with normal fibroblasts, as shown by greater numbers of dermal cells staining for phosphorylated ribosomal protein S6 (p-S6). Xenografted epidermis overlying TSC tumor cells also showed stronger p-S6 staining than epidermis overlying TSC fibroblasts, replicating what we had observed in TSC skin tumors. Rapamycin treatment of tumor xenografts dramatically inhibited p-S6 staining in both epidermal and dermal cells. Tumor xenografts showed greater epidermal cell proliferation (as determined by staining with anti-Ki-67 specific antibody) than normal xenografts (p=0.009). Rapamycin treatment decreased the number of Ki-67 positive epidermal cells in grafts containing TSC tumor cells (p=0.046) but not in grafts with TSC normal fibroblasts. These results indicate that TSC tumor cells stimulate mTORC1 signaling and proliferation of overlying epidermal cells, and suggest that one of rapamycin effects on TSC skin tumors is through decreasing epidermal cell proliferation. This work was supported by an Idea Development Award (TS080064) from the Congressionally Directed Medical Research Program to Dr. Darling. Dr. Moss was supported by the Intramural Research Program, NIH, NHLBI. 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 2501.

Signaling Events Downstream of Mammalian Target of Rapamycin Complex 2 Are Attenuated in Cells and Tumors Deficient for the Tuberous Sclerosis Complex Tumor Suppressors

Mutations in the TSC1 and TSC2 tumor suppressor genes give rise to the neoplastic disorders tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis. Their gene products form a complex that is a critical negative regulator of mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and cell growth. We recently found that the TSC1-TSC2 complex promotes the activity of mTOR complex 2 (mTORC2), an upstream activator of Akt, and this occurs independent of its inhibitory effects on mTORC1. Loss of mTORC2 activity in cells lacking the TSC1-TSC2 complex, coupled with mTORC1-mediated feedback mechanisms, leads to strong attenuation of the growth factor-stimulated phosphorylation of Akt on S473. In this study, we show that both phosphatidylinositol 3-kinase-dependent and phosphatidylinositol 3-kinase-independent mTORC2 substrates are affected by loss of the TSC1-TSC2 complex in cell culture models and kidney tumors from both Tsc2(+/-) mice (adenoma) and TSC patients (angiomyolipoma). These mTORC2 targets are all members of the AGC kinase family and include Akt, protein kinase Calpha, and serum and glucocorticoid-induced protein kinase 1. We also show that the TSC1-TSC2 complex can directly stimulate the in vitro kinase activity of mTORC2. The interaction between these two complexes is mediated primarily through regions on TSC2 and a core component of mTORC2 called Rictor. Hence, loss of the TSC tumor suppressors results in elevated mTORC1 signaling and attenuated mTORC2 signaling. These findings suggest that the TSC1-TSC2 complex plays opposing roles in tumor progression, both blocking and promoting specific oncogenic pathways through its effects on mTORC1 inhibition and mTORC2 activation, respectively.

Changing Trends in Presentation, Diagnosis and Management of Renal Angiomyolipoma: Comparison of Sporadic and Tuberous Sclerosis Complex-associated Forms

To evaluate the changing management of sporadic renal angiomyolipoma and renal angiomyolipoma associated with the tuberous sclerosis complex (TSC) during the past 16 years. We retrospectively reviewed the charts of 60 patients with angiomyolipoma seen at our institutions. The median age at presentation was 45 years (range 7-78). The presentation was pain in 30 patients and hematuria in 13; it was incidentally discovered in 17 patients. Of the 60 patients, 43 were females. TSC was present in 14 patients. The median tumor size was 4 cm (range 0.3-40, mean 6.5 +/- 1.1). Of the 60 patients, 31 were followed up expectantly. Surgery or intervention was needed for 29 patients to control hemorrhage or relieve pain or because of the suspicion of malignancy. Of these 29 patients, 12 underwent nephrectomy, 11 partial nephrectomy, and 6 embolization. The patients treated for hemorrhage had a median tumor diameter of 11 cm (range 2-21). Patients were followed up for a mean of 39.3 +/- 5.4 months. The lesions grew an average of 4.7 +/- 3.4 cm for TSC tumors and 0.6 +/- 0.2 cm for sporadic angiomyolipoma tumors. None of the patients developed renal impairment. Patients with TSC presented at a younger age, had larger and bilateral lesions, and were more symptomatic during follow-up. In the past 6 years, a significant trend was seen toward finding tumors in asymptomatic patients and toward the use of conservative or interventional (embolization) treatment. Renal angiomyolipoma has a slow growth rate. The preservation of renal function was noted in all our patients. A recent shift was noted toward finding smaller tumors in asymptomatic patients and the use of conservative and interventional treatment.

Unfolding Possible Cancer Target

Work from Ozcan et al . weaves the unfolded protein response (UPR) [which is initiated in the endoplasmic reticulum (ER) when the capacity of the ER to produce properly folded proteins is exceeded] into a regulatory network regulating insulin signaling and apoptosis. The paper focuses on signaling through the mTORC1 complex, which regulates protein synthesis by controlling the activity of ribosomal protein S6 kinases and eukaryotic initiation factor 4E. Deletion or loss of function of the tuberous sclerosis complex genes (TSC1 and TSC2) leads to sustained activation of mTORC1. In mouse embryo fibroblasts lacking TSC1 and TSC2 or in tumor cells from Tsc2 +/– mice, the UPR response was activated. This UPR-dependent activation of the mitogen-activated protein kinase JNK and serine/threonine phosphorylation of insulin receptor substrate 1 provided a mechanism by which insulin receptor signaling was inhibited. Treatments that alleviated ER stress (such as the addition of a chemical chaperone that promoted proper protein folding) alleviated the defects in insulin signaling in Tsc1 –/– or Tsc2 –/– cells. When ER stress gets really out of hand, cells undergo apoptosis, and the authors showed that TSC-deficient cells had increased vulnerability to apoptosis when treated with chemical agents that enhance ER stress. The authors and Reiling and Sabatini in associated commentary discuss the appealing logic of having the UPR feed back onto the mTORC1 pathway, which responds to nutrients and regulates growth and proliferation, thus coordinating pathways regulating metabolism and survival. They further point out the possibility that the UPR may provide a target to promote elimination of tumor cells, specifically those cells lacking TSC proteins, or more generally in tumor cells whose rapid growth and high rates of protein synthesis might make them more vulnerable than normal cells. U. Ozcan, L. Ozcan, E. Yilmaz, K. Düvel, M. Sahin, B. D. Manning, G. S. Hotamisligil, Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis. Mol. Cell 29 , 541-551 (2008). [PubMed] J. H. Reiling, D. M. Sabatini, Increased mTORC1 signaling UPRegulates stress. Mol. Cell 29 , 533-535 (2008). [PubMed]

Mammalian target of rapamycin complex 1 (mTORC1); tuberous sclerosis complex tumor suppressors 1 and 2 (TSC1 and TSC2); unfolded protein response (UPR)

Mammalian target of rapamycin complex 1 (mTORC1); tuberous sclerosis complex tumor suppressors 1 and 2 (TSC1 and TSC2); unfolded protein response (UPR)

Loss of the Tuberous Sclerosis Complex Tumor Suppressors Triggers the Unfolded Protein Response to Regulate Insulin Signaling and Apoptosis

Mammalian target of rapamycin, mTOR, is a major sensor of nutrient and energy availability in the cell and regulates a variety of cellular processes, including growth, proliferation, and metabolism. Loss of the tuberous sclerosis complex genes (TSC1 or TSC2) leads to constitutive activation of mTOR and downstream signaling elements, resulting in the development of tumors, neurological disorders, and at the cellular level, severe insulin/IGF-1 resistance. Here, we show that loss of TSC1 or TSC2 in cell lines and mouse or human tumors causes endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). The resulting ER stress plays a significant role in the mTOR-mediated negative-feedback inhibition of insulin action and increases the vulnerability to apoptosis. These results demonstrate ER stress as a critical component of the pathologies associated with dysregulated mTOR activity and offer the possibility to exploit this mechanism for new therapeutic opportunities.

PDGFRs are critical for PI3K/Akt activation and negatively regulated by mTOR

The receptor tyrosine kinase/PI3K/Akt/mammalian target of rapamycin (RTK/PI3K/Akt/mTOR) pathway is frequently altered in tumors. Inactivating mutations of either the TSC1 or the TSC2 tumor-suppressor genes cause tuberous sclerosis complex (TSC), a benign tumor syndrome in which there is both hyperactivation of mTOR and inhibition of RTK/PI3K/Akt signaling, partially due to reduced PDGFR expression. We report here that activation of PI3K or Akt, or deletion of phosphatase and tensin homolog (PTEN) in mouse embryonic fibroblasts (MEFs) also suppresses PDGFR expression. This was a direct effect of mTOR activation, since rapamycin restored PDGFR expression and PDGF-sensitive Akt activation in Tsc1-/- and Tsc2-/- cells. Akt activation in response to EGF in Tsc2-/- cells was also reduced. Furthermore, Akt activation in response to each of EGF, IGF, and PMA was reduced in cells lacking both PDGFRalpha and PDGFRbeta, implying a role for PDGFR in transmission of growth signals downstream of these stimuli. Consistent with the reduction in PI3K/Akt signaling, in a nude mouse model both Tsc1-/- and Tsc2-/- cells had reduced tumorigenic potential in comparison to control cells, which was enhanced by expression of either active Akt or PDGFRbeta. In conclusion, PDGFR is a major target of negative feedback regulation in cells with activated mTOR, which limits the growth potential of TSC tumors.

The tuberous sclerosis genes and regulation of the cyclin-dependent kinase inhibitor p27

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor syndrome that affects approximately 1 in 6000 individuals. It is characterized by the development of tumors, named hamartomas, in the kidneys, heart, skin and brain. The latter often cause seizures, mental retardation, and a variety of developmental disorders, including autism. This disease is caused by mutations within the tumor suppressor gene TSC1 on chromosome 9q34 encoding hamartin or within TSC2 on chromosome 16p13.3 encoding tuberin. TSC patients carry a mutant TSC1 or TSC2 gene in each of their somatic cells, and loss of heterozygosity has been documented in a wide variety of TSC tumors. Recent data suggest that functional inactivation of TSC proteins might also be involved in the development of other diseases not associated with TSC, such as sporadic bladder cancer, breast cancer, ovarian carcinoma, gall bladder carcinoma, non-small-cell carcinoma of the lung, and Alzheimer's disease. Tuberin and hamartin form a heterodimer, suggesting they might affect the same processes. Tuberin is assumed to be the functional component of the complex and has been implicated in the regulation of different cellular functions. The TSC proteins regulate cell size control due to their involvement in the insulin signalling pathway. Furthermore, they are potent positive regulators of the cyclin-dependent kinase inhibitor p27, a major regulator of the mammalian cell cycle. Here we review the current knowledge on how mutations within the TSC genes could trigger deregulation of stability and localization of the tumor suppressor p27.

Hamartomas and tubers from defects in hamartin-tuberin

her brain as well as in numerous other organs. 1 In time, additional patients with this inherited disease were studied and found to have benign tumors in virtually every organ, including the brain (cortical tubers, subependymal nodules, and giant cell astrocytomas), eyes (retinal hamartomas), kidneys (angiomyolipomas, cysts), heart (cardiac rhabdomyomas), lungs (lymphangioleiomyomatosis), and skin. Recent molecular investigations have revealed that this potpourri of seemingly unrelated findings, called tuberous sclerosis complex (TSC),results from a dysregulated signaling pathway. These discoveries have profound clinical implications. The skin is paradigmatic for TSC. TSC presents a variable clinical picture, and the skin lesions are no less variable. TSC skin lesions range from subtle to disfiguring. A patient may have one or several types of skin lesions, and each type may be single or multiple. Many of the skin and internal tumors in TSC are hamartomas that are highly vascular. Both skin tumors and internal tumors occur in characteristic locations with typical ages of onset. Proclivity of skin tumors for certain regions of the body is indicated by the names of lesions, such as facial angiofibromas, periungual fibromas, and forehead plaques. In contrast, hypopigmented macules seemingly occur at random locations, and exhibit differences in size and shape (ash leaf vs confetti-like), location (hypopigmented macule vs poliosis), and extent. Hypomelanotic macules are typically present at birth or appear during early infancy. Angiofibromas usually develop at about 3 to 5 years, while periungual fibromas emerge in the teen years or later. 2 Skin lesions may be a presenting feature and can form the basis for the diagnosis of TSC. Among the diagnostic criteria, mucocutaneous lesions comprise 4 major features (angiofibromas or forehead plaque, subungual or periungual fibromas, hypomelanotic macules, and the shagreen patch) and 3 minor features (confetti-like hypopigmentation, gingival fibromas, and multiple dental pits). 3 These 1998 criteria were modified from earlier versions to accommodate recent observations. Multiple facial angiofibromas are no longer considered pathognomonic for TSC, as they are also observed in multiple endocrine neoplasia type 1. More than 3 hypomelanotic macules must be observed to qualify as a major feature of TSC, because solitary lesions are commonly observed in the general population. Finally, consideration is given to the fact that single ungual fibromas may occur in the general popu

Tuberous sclerosis complex tumor suppressor-mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent.

The evolution of mitogenic pathways has led to the parallel requirement for negative control mechanisms, which prevent aberrant growth and the development of cancer. Principally, such negative control mechanisms are represented by tumor suppressor genes, which normally act to constrain cell proliferation (Macleod, K. 2000. Curr. Opin. Genet. Dev. 10:81–93). Tuberous sclerosis complex (TSC) is an autosomal-dominant genetic disorder, characterized by mutations in either TSC1 or TSC2, whose gene products hamartin (TSC1) and tuberin (TSC2) constitute a putative tumor suppressor complex (TSC1-2; van Slegtenhorst, M., M. Nellist, B. Nagelkerken, J. Cheadle, R. Snell, A. van den Ouweland, A. Reuser, J. Sampson, D. Halley, and P. van der Sluijs. 1998. Hum. Mol. Genet. 7:1053–1057). Little is known with regard to the oncogenic target of TSC1-2, however recent genetic studies in Drosophila have shown that S6 kinase (S6K) is epistatically dominant to TSC1-2 (Tapon, N., N. Ito, B.J. Dickson, J.E. Treisman, and I.K. Hariharan. 2001. Cell. 105:345–355; Potter, C.J., H. Huang, and T. Xu. 2001. Cell. 105:357–368). Here we show that loss of TSC2 function in mammalian cells leads to constitutive S6K1 activation, whereas ectopic expression of TSC1-2 blocks this response. Although activation of wild-type S6K1 and cell proliferation in TSC2-deficient cells is dependent on the mammalian target of rapamycin (mTOR), by using an S6K1 variant (GST-ΔC-S6K1), which is uncoupled from mTOR signaling, we demonstrate that TSC1-2 does not inhibit S6K1 via mTOR. Instead, we show by using wortmannin and dominant interfering alleles of phosphatidylinositide-3-OH kinase (PI3K) that increased S6K1 activation is contingent upon the suppression of TSC2 function by PI3K in normal cells and is PI3K independent in TSC2-deficient cells.

Lethality of Drosophila lacking TSC tumor suppressor function rescued by reducing dS6K signaling.

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in one of two tumor suppressor genes, TSC1 and TSC2. Here, we show that absence of Drosophila Tsc1/2 leads to constitutive dS6K activation and inhibition of dPKB, the latter effect being relieved by loss of dS6K. In contrast, the dPTEN tumor suppressor, a negative effector of PI3K, has little effect on dS6K, but negatively regulates dPKB. More importantly, we demonstrate that reducing dS6K signaling rescues early larval lethality associated with loss of dTsc1/2 function, arguing that the S6K pathway is a promising target for the treatment of TSC.

Octreotide Acetate Uptake In Tuberous Sclerosis Complex With Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare progressive lung disease of reproductive age women. It occurs as an isolated disorder (sporadic LAM) or in an association with tuberous sclerosis complex (TSC). Coexisting renal angiomyolipomas occur in TSC and LAM, and there is an association between TSC and neuroendocrine tumors. Commonly used therapies aimed at decreasing endogenous estrogen in LAM have not been verified by randomized trial. CASE REPORT An octreoScan was performed to detect tumors/tissue containing somatostatin receptors (such as neuroendocrine tumors) in a 29 year-old woman with TSC, end-stage renal disease secondary to angiomyolipomas (figure 1), LAM lung (figure 2), and “potato-like” cerebral lesions. Whole body images were obtained 24 hours post IV injection of In-111 pentatreotide 5.5 mCi demonstrating increased uptake of the radio pharmaceutical diffusely in the lung and kidney (figure 3). Figure 1 Figure 1: Triphasic Computerized Tomogram of kidneys showing multiple renal angiomyolipomas, a pseudoaneurysm (pointed by an arrow) and multiple renal cysts. Figure 2 Figure 2: Computerized Tomogram of chest showing bilateral, multiple thin walled cysts suggesting of LAM lung. Octreotide Acetate Uptake In Tuberous Sclerosis Complex With Lymphangioleiomyomatosis 2 of 4 Figure 3 Figure 3: Whole body OctreoScan demonstrating diffusely increased uptake of 5.5mCi Indium 111 Octreotide in lung and kidney. Octreotide Acetate Uptake In Tuberous Sclerosis Complex With Lymphangioleiomyomatosis 3 of 4 DISCUSSION This result suggests somatostatin receptor-bearing tissue in TSC/LAM. This observation suggests a novel approach to the detection and monitoring of this disease and possible treatment with octreotide acetate, a cyclic octapeptide with down-regulating properties similar to the natural hormone somatostatin. Further studies addressing the importance of somatostatin receptor in these disorders is warranted. CORRESPONDENCE TO Daya Upadhyay, MD Division of Pulmonary and Critical Care Medicine 14-707 Tarry Building, 300 East Superior St, Chicago, Illinois 60611. Tel: (312) 908-8163, Fax: (312) 908-4650, E-mail: upadhyayd@pol.net

Parallels between tuberous sclerosis complex and neurofibromatosis 1: Common threads in the same tapestry

Neurofibromatosis type 1 (NF1) and tuberous sclerosis complex (TSC) represent two neurocutaneous disorders in which affected individuals develop tumors at an increased frequency. Although the clinical manifestations of these disorders are distinctive, the identification of the genes responsible for these disorders has demonstrated remarkable similarities on a molecular level between the NF1 and TSC tumor suppressor gene products. The NF1 and TSC2 gene products are hypothesized to function as growth regulators by modulating the activities of small GTPase molecules. The overlap between the functions of these tumor suppressor genes has yielded important insights into the molecular pathogenesis underlying each of these disorders and suggested possible pharmacological therapies specifically targeted for affected individuals.