TSC2 Expression Research Articles

Prognostic implication of TSC1 and mTOR expression in gastric carcinoma

Gastric adenocarcinoma is the sixth most common and third most lethal cancer in the world. Except for HER2-targeted therapy, targeted agents against specific molecules participating in gastric carcinogenesis, including those in the mechanistic target of rapamycin (serine/threonine kinase) (mTOR) pathway have not been proved to be effective. However, some studies have suggested that dysfunction of TSC1 may augment mTOR inhibitor activity. We studied p-mTOR and TSC1 status by immunohistochemical analysis of gastric carcinoma samples using a tissue microarray method and expression values adopted from The Cancer Genome Atlas. High p-mTOR and low TSC1 expression status is associated with adverse clinicopathologic parameters. Patients with high p-mTOR levels showed poor survival. Patients with low TSC1 levels showed unfavorable survival status in the overall patients group. The combination of p-mTOR status and TSC1 status provided more strong survival information than using each parameter alone. In gastric cancer, high p-mTOR expression level is a statistically significant parameter in multivariate and Kaplan-Meier analyses (log-rank test). In addition to p-mTOR, TSC1 expression provided additional information to predict survival. We therefore suggest that evaluation of both p-mTOR and TSC1 status may be helpful in clinical trials related to mTOR inhibitors.

Tsc1-Tp53 loss induces mesothelioma in mice, and evidence for this mechanism in human mesothelioma

Mesothelioma is diagnosed in approximately 2,500 patients in the United States every year, most often arising in the pleural space, but also occurring as primary peritoneal mesothelioma. The vast majority of patients with mesothelioma die from their disease within 3 years. We developed a new mouse model of mesothelioma by bladder or intra-peritoneal injection of adenovirus Cre into mice with conditional alleles of each of Tp53 and Tsc1. Such mice began to develop malignant ascites about 6 months after injection, which was due to peritoneal mesothelioma, based on tumor morphology and immunohistochemical staining. Mesothelioma cell lines were established which showed loss of both Tsc1 and Tp53, with mTORC1 activation. Treatment of mice with malignant ascites due to mesothelioma with rapamycin led to a marked reduction in ascites, extended survival, and a 95–99% reduction in mesothelioma tumor volume, in comparison to vehicle-treated mice. To see if TSC1/TSC2 loss was a common genetic event in human mesothelioma, we examined 9 human mesothelioma cell lines, and found that 4 of 9 showed persistent activation of mTORC1 though none had loss of TSC1 or TSC2. A tissue microarray analysis of 198 human mesothelioma specimens showed that 33% of cases had reduced TSC2 expression and 60% showed activation of mTOR, indicating that mTOR activation is common in human mesothelioma and suggesting that it is a potential therapeutic target.

Abstract 1800: Expression analysis of PI3K pathway genes in head and neck cancer.

Abstract Head and neck cancer (HNC) is a heterogenous group of malignant tumors which includes neoplasias of the oral cavity, the pharynx, the larynx and paranasal sinuses. Squamous cell carcinomas comprise more than 95% of all head and neck cancers. The vast majority of HNC is related to exposure to carcinogens, including tobacco, alcohol, betel nut, and sexually transmitted viral pathogens such as human papilloma viruses (HPV). However, only a small fraction of tobacco and alcohol users develop the disease suggesting that genetic factors may play a key role in HNC. Increasing evidence indicate that mTOR is a central regulator of cell growth, proliferation, survival and dysregulation of the PI3K/Akt/mTOR signaling pathway that occurs in many tumors including head and neck cancer. The TSC complex (TSC1/TSC2) negatively regulates mTOR by inhibiting a small GTP binding protein, Rheb, considered to be the direct activator of mTOR. On the other hand, the Ras protein upregulates PI3K signaling which is an upstream effector of mTOR. When PI3K activates Akt, the dual function phosphatase PTEN negatively regulates it. Human cancers frequently harbor activating mutations in the p110 catalytic subunit of PI3K or genomic amplification of Akt. Activated Akt also promotes survival under conditions of cellular stress. In this study we investigated the expression of the critical genes (mTOR, K-ras, PI3KCA, TSC1, RHEB, PTEN and AKT1) acting in this pathway. For this purpose, we analyzed expression levels of these genes in 49 matched HNC tumor and non-cancerous tissue samples by quantitative RT-PCR. We observed a significant downregulation of the mTOR (p=0.005), TSC1 (p=0.001), and PTEN (p=0.000) mRNA expressions. On the other hand, a slight decrease was also observed in K-ras, Akt1 and PI3KCA mRNA levels in tumor tissues compared to the non-cancerous ones. However, the difference in the expression levels of these genes between the two groups were not statistically significant. Whereas we didn't observe any difference in Rheb mRNA expression level in tumor tissue when compared to the non-cancerous tissue samples. mTOR, TSC1, and PTEN mRNA expression levels were decreased 0.48, 0.44 and 0.32 % in HNC tumor tissue samples compared to non-cancerous tissue samples, respectively . Our result indicate that, the PI3K pathway has an important function in HNC progression with the contribution of more than one gene of this pathway. Citation Format: Nur Buyru, Seda Ekizoglu, Emin Karaman, Ozgun Enver. Expression analysis of PI3K pathway genes in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1800. doi:10.1158/1538-7445.AM2013-1800 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5510: Tranilast inhibits growth of skin tumor cells in a xenograft mouse model of tuberous sclerosis complex.

Abstract Pharmacological approaches for treating tumors in patients with tuberous sclerosis complex (TSC) are inadequate. Although mTORC1 inhibitors such as rapamycin represent a major new avenue for treatment, we and others have shown that rapamycin improves but does not eradicate TSC skin tumors. We tested the effects of tranilast, an antiallergic and antifibrotic drug with antiproliferative effects, on fibroblasts grown from angiofibromas, forehead plaques, periungual fibromas, and normal-appearing skin from 5 patients with TSC. Tumor cells showed loss of TSC2 expression and hyperactivation of the mTORC1 signaling pathway. Tranilast (5, 10, 20, 40 μM) inhibited the viability of TSC skin tumor cells from five patients in an MTT assay. These effects on cell viability appeared to be independent of mTORC1 activity, since tranilast did not inhibit phosphorylation of ribosomal protein S6. Four paired samples of RNA from tumor and normal cells, treated with or without tranilast, were studied using gene expression arrays, revealing that tranilast significantly altered the expression of at least 36 genes, of which 12 are involved in transcription. In a xenograft model in which dermal-epidermal constructs comprising normal human keratinocytes with either TSC angiofibroma cells or TSC normal fibroblasts from a TSC patient were grafted to nude mice (6-8 mice per group), tranilast treatment (300 mg/kg/day) reduced the number of skin tumor cells compared to vehicle control group (P< 0.05). Tranilast treatment did not block angiogenesis or lymphangiogenesis in the xenograft model. These results indicate that tranilast has antiproliferative effects independent of mTORC1 and suggest that tranilast may be useful as an adjunctive agent for the treatment of TSC skin tumors. Citation Format: Shaowei Li, Ji-an Wang, Rajesh L. Thangapazham, Peter Klover, Joel Moss, Thomas N. Darling. Tranilast inhibits growth of skin tumor cells in a xenograft mouse model of tuberous sclerosis complex. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5510. doi:10.1158/1538-7445.AM2013-5510

Abstract 1576: Mesenchymal disruption of Tsc2 in mice results in highly vascular hamartomas.

Abstract Patients with tuberous sclerosis complex (TSC) suffer from the formation of multi-organbenign tumors. Skin tumors from TSC patients are characterized by increased vascularity, fibrosis, and overactivation of mTORC1 signaling due to inactivation of either of the tumor suppressor genes TSC1 or TSC2 in fibroblasts. The role of the TSC2 gene in cells of mesenchymal origin with respect to tumor formation still remains unclear. To investigate the roles of TSC2 in these cells, we used the cre/loxP system to conditionally disrupt mouse Tsc2 in tissues derived from mesenchymal cells. Mice with floxed Tsc2 alleles were mated with mice expressing the cre recombinase transgene under the control of a Prrx1 regulatory element (Prrx1-cre), which is selectively expressed in craniofacial and limb bud mesenchyme. Cells with homozygous Tsc2 floxed alleles expressing the Prrx1-cre transgene (termed here “Tsc2cKO mice”) efficiently eliminated Tsc2 expression from fibroblasts isolated from limb and ventral skin. An EYFP reporter transgene was also present to track cells expressing cre recombinase. Fibroblasts grown from the skin of Tsc2cKO mice expressed EYFP, lacked Tsc2 protein and had elevated levels of phosphorylated ribosomal protein S6, a marker of mTORC1 signaling. Tsc2cKO mice had shortened lifespan with a median survival between 5 and 6 months of age. Tumors were observed in spleen and forepaws of nearly all mice, and tumors involving the liver, skin, skeletal muscle, mediastinum, and kidney also occurred. These results were unexpected due to the lineage restriction of the Prrx1-cre transgene. Tumor formation began to appear on forepaws near the heel of the paw pad as early as 3 weeks of age. By 6 weeks of age the lesions generally showed a reddish appearance. In mice serially imaged by MRI, splenic tumors were apparent by 10-12 weeks of age and renal tumors were apparent by 7-8 weeks. Histologically, the forepaw, splenic, and hepatic tumors were benign fibrovascular proliferations with perivascular cells that stained with smooth muscle actin, most prominently in the spleen. Using ex vivo fluorescent imaging, EYFP fluorescence was not detected in internal organs of control mice containing Prrx1-cre, but strong fluorescence was observed in the splenic, hepatic, and renal tumors of Tsc2cKO mice. In summary, we have developed model system to study the role of Tsc2-deficient mesenchymal cells in hamartoma formation. Citation Format: Peter Klover, Rajesh Thangapazham, Shaowei Li, Jiro Kato, Stasia A. Anderson, Victoria Hoffman, Ji-an Wang, Joshua Bernstock, Elizabeth McCart, Joel Moss, Thomas Darling. Mesenchymal disruption of Tsc2 in mice results in highly vascular hamartomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1576. doi:10.1158/1538-7445.AM2013-1576

The acute effects of strength, endurance and concurrent exercises on the Akt/mTOR/p70S6K1 and AMPK signaling pathway responses in rat skeletal muscle

The activation of competing intracellular pathways has been proposed to explain the reduced training adaptations after concurrent strength and endurance exercises (CE). The present study investigated the acute effects of CE, strength exercises (SE), and endurance exercises (EE) on phosphorylated/total ratios of selected AMPK and Akt/mTOR/p70S6K1 pathway proteins in rats. Six animals per exercise group were killed immediately (0 h) and 2 h after each exercise mode. In addition, 6 animals in a non-exercised condition (NE) were killed on the same day and under the same conditions. The levels of AMPK, phospho-Thr172AMPK (p-AMPK), Akt, phospho-Ser473Akt (p-Akt), p70S6K1, phospho-Thr389-p70S6K1 (p-p70S6K1), mTOR, phospho-Ser2448mTOR (p-mTOR), and phospho-Thr1462-TSC2 (p-TSC2) expression were evaluated by immunoblotting in total plantaris muscle extracts. The only significant difference detected was an increase (i.e., 87%) in Akt phosphorylated/total ratio in the CE group 2 h after exercise compared to the NE group (P = 0.002). There were no changes in AMPK, TSC2, mTOR, or p70S6K1 ratios when the exercise modes were compared to the NE condition (P ≥ 0.05). In conclusion, our data suggest that low-intensity and low-volume CE might not blunt the training-induced adaptations, since it did not activate competing intracellular pathways in an acute bout of strength and endurance exercises in rat skeletal muscle.

Tsc1 (hamartin) confers neuroprotection against ischemia by inducing autophagy

Previous attempts to identify neuroprotective targets by studying the ischemic cascade and devising ways to suppress it have failed to translate to efficacious therapies for acute ischemic stroke1. We hypothesized that studying the molecular determinants of endogenous neuroprotection in two well-established paradigms, the resistance of CA3 hippocampal neurons to global ischemia2 and the tolerance conferred by ischemic preconditioning (IPC)3, would reveal new neuroprotective targets. We found that the product of the tuberous sclerosis complex 1 gene (TSC1), hamartin, is selectively induced by ischemia in hippocampal CA3 neurons. In CA1 neurons, hamartin was unaffected by ischemia but was upregulated by IPC preceding ischemia, which protects the otherwise vulnerable CA1 cells. Suppression of hamartin expression with TSC1 shRNA viral vectors both in vitro and in vivo increased the vulnerability of neurons to cell death following oxygen glucose deprivation (OGD) and ischemia. In vivo suppression of TSC1 expression increased locomotor activity and decreased habituation in a hippocampal-dependent task. Overexpression of hamartin increased resistance to OGD by inducing productive autophagy through an mTORC1-dependent mechanism.

XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons

Truncating or missense mutation of cullin 4B (CUL4B) is one of the most prevalent causes underlying X-linked intellectual disability (XLID). CUL4B-RING E3 ubiquitin ligase promotes ubiquitination and degradation of various proteins. Consistent with previous studies, overexpression of wild-type CUL4B in 293 cells enhanced ubiquitylation and degradation of TSC2 or cyclin E. The present study shows that XLID mutant (R388X), (R572C) or (V745A) CULB failed to promote ubiquitination and degradation of TSC2 or cyclin E. Adenoviruses-mediated expression of wild-type CUL4B decreased protein level of TSC2 or cyclin E in cultured neocortical neurons of frontal lobe. Furthermore, shRNA-mediated CUL4B knockdown caused an upregulation of TSC2 or cyclin E. XLID mutant (R388X), (R572C) or (V745A) CUL4B did not downregulate protein expression of TSC2 or cyclin E in neocortical neurons. By promoting TSC2 degradation, CUL4B could positively regulate mTOR activity in neocortical neurons of frontal cortex. Consistent with this hypothesis, CUL4B knockdown-induced upregulation of TSC2 in neocortical neurons resulted in a decreased protein level of active phospho-mTORSer2448 and a reduced expression of active phospho-p70S6KThr389 and phospho-4E-BP1Thr37/46, two main substrates of mTOR-mediated phosphorylation. Wild-type CUL4B also increased protein level of active phospho-mTORSer2448, phospho-p70S6KThr389 or phospho-4E-BP1Thr37/46. XLID CUL4B mutants did not affect protein level of active phospho-mTORSer2448, phospho-p70S6KThr389 or phospho-4E-BP1Thr37/46. Our results suggest that XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons.

Timing of mTOR activation affects tuberous sclerosis complex neuropathology in mouse models

SUMMARYTuberous sclerosis complex (TSC) is a dominantly inherited disease with high penetrance and morbidity, and is caused by mutations in either of two genes, TSC1 or TSC2. Most affected individuals display severe neurological manifestations – such as intractable epilepsy, mental retardation and autism – that are intimately associated with peculiar CNS lesions known as cortical tubers (CTs). The existence of a significant genotype-phenotype correlation in individuals bearing mutations in either TSC1 or TSC2 is highly controversial. Similar to observations in humans, mouse modeling has suggested that a more severe phenotype is associated with mutation in Tsc2 rather than in Tsc1. However, in these mutant mice, deletion of either gene was achieved in differentiated astrocytes. Here, we report that loss of Tsc1 expression in undifferentiated radial glia cells (RGCs) early during development yields the same phenotype detected upon deletion of Tsc2 in the same cells. Indeed, the same aberrations in cortical cytoarchitecture, hippocampal disturbances and spontaneous epilepsy that have been detected in RGC-targeted Tsc2 mutants were observed in RGC-targeted Tsc1 mutant mice. Remarkably, thorough characterization of RGC-targeted Tsc1 mutants also highlighted subventricular zone (SVZ) disturbances as well as STAT3-dependent and -independent developmental-stage-specific defects in the differentiation potential of ex-vivo-derived embryonic and postnatal neural stem cells (NSCs). As such, deletion of either Tsc1 or Tsc2 induces mostly overlapping phenotypic neuropathological features when performed early during neurogenesis, thus suggesting that the timing of mTOR activation is a key event in proper neural development.

Profiling mTOR pathway in neuroendocrine tumors

The serine/threonine kinase mammalian target of rapamycin (mTOR) plays a central role in regulating critical cellular processes such as growth, proliferation, and protein synthesis. The study of cancer predisposing syndromes within which neuroendocrine tumors (NETs) may arise has furnished clues on the involvement of mTOR pathway in sporadic diseases so far. Recent comprehensive analyses have definitely shown activation of mTOR pathway in both experimental and human sporadic NETs. Upstream regulators of mTOR (PTEN and TSC2) have been found mutated in sporadic pNETs. Activation of mTOR pathways in NETs is already demonstrated by expression profiles analysis that revealed downregulation of TSC2 gene and alterations of TSC2 and PTEN protein expression in the vast majority of well-differentiated tumors. Moreover, a global microRNA expression analysis revealed the overexpression, in highly aggressive tumors, of a microRNA (miR-21) that targets PTEN reducing its expression and therefore leading to mTOR activation as well. Overall, these clues have furnished the rationale for the use of mTOR inhibitors the treatment of pNETs. With the recent approval of Everolimus (mTOR-targeted drug) for the treatment of advanced pNETs, this paradigm has been effectively translated into the clinical setting. In this review, we discuss mTOR pathway involvement in NETs, the clinical evidence supporting the use of mTOR inhibitors in cancer treatment, and the current clinical issues that remain to be elucidated to improve patient management.

Uterine-Specific Knockout of TSC2: A Mouse Model for Leiomyoma and LAM.

Lymphangioleiomyomatosis (LAM) is a rare disease characterized by proliferation of abnormal smooth muscle (SM) cells in the lungs. With time, LAM cells proliferate and lead to progressive loss of lung function. No effective treatment for this life-threatening disease is available, and patients often require lung transplantation. LAM has two unique characteristics: 1) LAM demonstrates strong gender specificity, affecting almost exclusively reproductive-age women; and 2) LAM cells contain mutations in genes (TSC1 or TSC2) that encode the tuberous sclerosis complex, a signaling module that limits mTOR activity and thereby cell growth. The origin of LAM cells is not known; however, data support a metastatic model of LAM pathogenesis. Interestingly, LAM shares many features with uterine leiomyomas, the leading reason for hysterectomy among women. Like LAM, leiomyomas are characterized by abnormal SM cell proliferation and are most prevalent during reproductive years. Furthermore, both LAM and leiomyomas express steroid hormone receptors and respond to estrogen and possibly progesterone. We propose that mutations in TSC2, in addition to estrogen and/or progesterone stimuli, trigger myometrial cells to proliferate and metastasize to the lungs similar to LAM cells. To test our hypothesis, we generated a mouse strain with a uterine-specific loss of TSC2. We show that TSC2 expression is low and mTOR signaling elevated in the uteri of post-pubertal uterine-specific TSC2 knockout (KO) mice. Notably, uteri from TSC2 KO mice develop leiomyomas at 5-6 months of age, and these tumors are prevented by treatment with the mTOR inhibitor rapamycin. Furthermore, these myometrial tumors express high levels of estrogen- and progesterone receptors, and uterine growth can be prevented by eliminating ovarian steroid production through ovariectomy. Importantly, multiple lung lesions were seen in a 34-week old TSC2 KO female. These lung tumors express progesterone receptors and are negative for the lung marker, Nkx2.1, indicating that they might be LAM cells. Together, our studies suggest that LAM may indeed originate from uterine myometrial cells. We hope that our model will improve our understanding of both LAM and leiomyomas, and may lead to the development of novel innovative therapeutic strategies for both tumors.

Graded loss of tuberin in an allelic series of brain models of TSC correlates with survival, and biochemical, histological and behavioral features

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with prominent brain manifestations due to mutations in either TSC1 or TSC2. Here, we describe novel mouse brain models of TSC generated using conditional hypomorphic and null alleles of Tsc2 combined with the neuron-specific synapsin I cre (SynIcre) allele. This allelic series of homozygous conditional hypomorphic alleles (Tsc2(c-del3/c-del3)SynICre(+)) and heterozygote null/conditional hypomorphic alleles (Tsc2(k/c-del3)SynICre(+)) achieves a graded reduction in expression of Tsc2 in neurons in vivo. The mice demonstrate a progressive neurologic phenotype including hunchback, hind limb clasp, reduced survival and brain and cortical neuron enlargement that correlates with a graded reduction in expression of Tsc2 in the two sets of mice. Both models also showed behavioral abnormalities in anxiety, social interaction and learning assays, which correlated with Tsc2 protein levels as well. The observations demonstrate that there are graded biochemical, cellular and clinical/behavioral effects that are proportional to the extent of reduction in Tsc2 expression in neurons. Further, they suggest that some patients with milder manifestations of TSC may be due to persistent low-level expression of functional protein from their mutant allele. In addition, they point to the potential clinical benefit of strategies to raise TSC2 protein expression from the wild-type allele by even modest amounts.

Expression of PI3K pathway proteins in refractory epilepsy associated with cortical malformation development

To investigate the expression of TSC1, TSC2, p-mTOR, p-4E-BP1, p-p70S6K and p-S6 in refractory epilepsy associated malformation of cortical development (MCD) tissues. A total of 43 cases of refractory epilepsy were involved in the study, and all the patients were treated in Xuanwu Hospital during 2005 - 2008, including focal cortical dysplasia type IIa (11 cases) and type IIb (11 cases), tuberous sclerosis complex (10 cases) and ganalioglioma (11 cases), and other 12 cases were used as control. These cases were divided into 7 study groups and immunohistochemical EnVision method was used. To detect the location and intensity of TSC1, TSC2, p-mTOR, p-4E-BP1, p-p70S6K and p-S6 expression in every group. Then the Image-Pro Plus 6.0 image processing and analysis software were used to measure the number, area, integrating absorbance (IA) of positive cells in every samples. The statistical software SPSS 16.0 was used to analyze the data. The immunolocalization of TSC1 and TSC2 was similar. It could be observed the expression of various levels in the cytoplasm of dysmorphic neurons, balloon cells, giant cells, ganglioglioma cells and normal neurons. TSC1 staining in normal neurons was more notably than others but TSC2 staining in giant cells was weaker than other samples. p-mTOR mainly presented in giant cells, which could also be observed in astrocyte. P-4E-BP1 presented in the cytoplasm and nuclear membrane of balloon cells, giant cells and ganglioglioma cells, the staining of giant cells was stronger than balloon cells, but their staining were weaker than ganglioglioma cells. P-p70S6K mainly expressed in giant cells and less commonly presented in balloon cells. P-S6 typically presented in all abnormal glioneuronal cells and it nearly did not present in the normal neurons of N-CTX group. PI3K pathway, at least in part, involves in the occurrence of MCD, and may play an important role in the pathogenesis.

Hepatitis C Virus Activates the mTOR/S6K1 Signaling Pathway in Inhibiting IRS-1 Function for Insulin Resistance

Hepatitis C virus (HCV) infection significantly increases the prevalence of type 2 diabetes mellitus (T2DM). Insulin receptor substrate 1 (IRS-1) plays a key role in insulin signaling, thus enabling metabolic regulation in mammalian cells. We have previously shown that HCV infection modulates phosphorylation of Akt, a downstream target of IRS-1. In this study, we further examined the status of total IRS-1 and the downstream regulation of the Akt pathway in understanding mTOR/S6K1 signaling using HCV genotype 2a (clone JFH1)-infected hepatocytes. Inhibition of IRS-1 expression was observed in HCV-infected hepatocytes compared to that in a mock-infected control. The status of the tuberous sclerosis complex (TSC-1/TSC-2) was significantly decreased after HCV infection of human hepatocytes, showing a modulation of the downstream Akt pathway. Subsequent study indicated an increased level of Rheb and mTOR expression in HCV-infected hepatocytes. Interestingly, the phosphoS6K1 level was higher in HCV-infected hepatocytes, suggesting a novel mechanism for IRS-1 inhibition. Ectopic expression of TSC-1/TSC-2 significantly recovered the IRS-1 protein expression level in HCV-infected hepatocytes. Further analyses indicated that HCV core protein plays a significant role in modulating the mTOR/S6K1 signaling pathway. Proteasome inhibitor MG 132 recovered IRS-1 and TSC1/2 expression, suggesting that degradation occurred via the ubiquitin proteasome pathway. A functional consequence of IRS-1 inhibition was reflected in a decrease in GLUT4 protein expression and upregulation of the gluconeogenic enzyme PCK2 in HCV-infected hepatocytes. Together, these observations suggested that HCV infection activates the mTOR/S6K1 pathway in inhibiting IRS-1 function and perturbs glucose metabolism via downregulation of GLUT4 and upregulation of PCK2 for insulin resistance.

Regulation of PI 3-K, PTEN, p53, and mTOR in Malignant and Benign Tumors Deficient in Tuberin.

The tuberous sclerosis complex (TSC) is caused by mutation in either of 2 tumor suppressor genes, TSC-1 (encodes hamartin) and TSC-2 (encodes tuberin). In humans, deficiency in TSC1/2 is associated with benign tumors in many organs, including renal angiomyolipoma (AML) but rarely renal cell carcinoma (RCC). In contrast, deficiency of TSC function in the Eker rat is associated with RCC. Here, we have investigated the activity of PI 3-K and the expression of PTEN, p53, tuberin, p-mTOR, and p-p70S6K in both Eker rat RCC and human renal AML. Compared to normal tissue, increased PI 3-K activity was detected in RCC of Eker rats but not in human AML tissue. In contrast, PTEN was highly expressed in AML but significantly reduced in the renal tumors of Eker rats. Phosphorylation on Ser(2448) of mTOR and Thr(389) of p70S6K were significantly increased in both RCC and AML compared to matching control tissue. Total tuberin was significantly decreased in AML while completely lost in RCC of Eker rats. Our data also show that while p53 protein expression is lost in rat RCC, it was highly elevated in AML. These novel data provide evidence that loss of TSC-2, PTEN, and p53 as well as activation of PI 3-K and mTOR is associated with kidney cancer in the Eker rat, while sustained expression of TSC-2, PTEN, and p53 may prevent progression of kidney cancer in TSC patients.

RGS19 stimulates cell proliferation by deregulating cell cycle control and enhancing Akt signaling

RGS19 is a regulator of G protein signaling which is upregulated in ovarian cancers and its overexpression promotes cell proliferation in several mammalian cell types. Here we showed that cyclin D1/3 and Cdk6 were upregulated in HEK293 cells overexpressing RGS19, while INK4A and INK4B were reduced. Moreover, RGS19 augmented serum-stimulated PTEN/PDK/Akt and Rb phosphorylations in 293/RGS19 and Caco2/RGS19 cells. These changes were reversed upon the knockdown of RGS19. Consistent with an elevated Akt activity, increased levels of phosphorylated Bad and c-Raf and a diminished expression of TSC2 were detected, thus demonstrating that RGS19 can deregulate cell proliferation via multiple pathways.

Inhibition of mTOR blocks the anti-inflammatory effects of glucocorticoids in myeloid immune cells

AbstractA central role for the mammalian target of rapamycin (mTOR) in innate immunity has been recently defined by its ability to limit proinflammatory mediators. Although glucocorticoids (GCs) exert potent anti-inflammatory effects in innate immune cells, it is currently unknown whether the mTOR pathway interferes with GC signaling. Here we show that inhibition of mTOR with rapamycin or Torin1 prevented the anti-inflammatory potency of GC both in human monocytes and myeloid dendritic cells. GCs could not suppress nuclear factor-κB and JNK activation, the expression of proinflammatory cytokines, and the promotion of Th1 responses when mTOR was inhibited. Interestingly, long-term activation of monocytes with lipopolysaccharide enhanced the expression of TSC2, the principle negative regulator of mTOR, whereas dexamethasone blocked TSC2 expression and reestablished mTOR activation. Renal transplant patients receiving rapamycin but not those receiving calcineurin inhibitors displayed a state of innate immune cell hyper-responsiveness despite the concurrent use of GC. Finally, mTOR inhibition was able to override the healing phenotype of dexamethasone in a murine lipopolysaccharide shock model. Collectively, these data identify a novel link between the glucocorticoid receptor and mTOR in innate immune cells, which is of considerable clinical importance in a variety of disorders, including allogeneic transplantation, autoimmune diseases, and cancer.

A matter of energy stress: p38β meets mTORC1

A matter of energy stress: p38β meets mTORC1

Evidence for population variation in TSC1 and TSC2 gene expression

BackgroundTuberous sclerosis complex (TSC) is an autosomal dominant neurogenetic disorder caused by mutations in one of two genes, TSC1 or TSC2, which encode the proteins hamartin and tuberin, respectively [1-3]. Common features of TSC include intractable epilepsy, mental retardation, and autistic features. TSC is associated with specific brain lesions, including cortical tubers, subependymal nodules and subependymal giant cell astrocytomas. In addition, this disease frequently produces characteristic tumors, termed hamartomas, in the kidneys, heart, skin, retina, and lungs. Disease severity in TSC can be quite variable and is not determined by the primary mutation alone. In fact, there is often considerable variability in phenotype within single families, where all affected individuals carry the same mutation. Factors suspected to influence phenotype in TSC include the specific primary mutation, random occurrence of second-hit somatic mutations, mosaicism, "modifying genes", and environmental factors. In addition to these factors, we hypothesize that differences in mRNA expression from the non-mutated TSC allele, or possibly from the mutated allele, play a part in modifying disease severity. Common genetic variants that regulate mRNA expression have previously been shown to play important roles in human phenotypic variability, including disease susceptibility. A prediction based on this idea is that common regulatory variants that influence disease severity in TSC should be detectable in non-affected individuals.MethodsA PCR/primer extension assay was used to measure allele specific expression of TSC1 and TSC2 mRNAs in leukocytes isolated from normal volunteers. This assay can be used to measure "allelic expression imbalance" (AEI) in individuals by making use of heterozygous "marker" single nucleotide polymorphisms (SNPs) located within their mRNA.ResultsIn this study we show for the first time that TSC1 and TSC2 genes exhibit allele-specific differences in mRNA expression in blood leukocytes isolated from normal individuals.ConclusionsThese results support the possibility that allele-specific variation in TSC mRNA expression contributes to the variable severity of symptoms in TSC patients.

Smooth muscle protein-22-mediated deletion of Tsc1 results in cardiac hypertrophy that is mTORC1-mediated and reversed by rapamycin

Constitutive activation of mammalian target of rapamycin complex 1 (mTORC1), a key kinase complex that regulates cell size and growth, is observed with inactivating mutations of either of the tuberous sclerosis complex (TSC) genes, Tsc1 and Tsc2. Tsc1 and Tsc2 are highly expressed in cardiovascular tissue but their functional role there is unknown. We generated a tissue-specific knock-out of Tsc1, using a conditional allele of Tsc1 and a cre recombinase allele regulated by the smooth muscle protein-22 (SM22) promoter (Tsc1c/cSM22cre+/-) to constitutively activate mTOR in cardiovascular tissue. Significant gene recombination (∼80%) occurred in the heart by embryonic day (E) 15, and reduction in Tsc1 expression with increased levels of phosphorylated S6 kinase (S6K) and S6 was observed, consistent with constitutive activation of mTORC1. Cardiac hypertrophy was evident by E15 with post-natal progression to heart weights of 142 ± 24 mg in Tsc1c/cSM22cre+/- mice versus 65 ± 14 mg in controls (P < 0.01). Median survival of Tsc1c/cSM22cre+/- mice was 24 days, with none surviving beyond 6 weeks. Pathologic and echocardiographic analysis revealed severe biventricular hypertrophy without evidence of fibrosis or myocyte disarray, and significant reduction in the left ventricular end-diastolic diameter (P < 0.001) and fractional index (P < 0.001). Inhibition of mTORC1 by rapamycin resulted in prolonged survival of Tsc1c/cSM22cre+/- mice, with regression of ventricular hypertrophy. These data support a critical role for the Tsc1/Tsc2-mTORC1-S6K axis in the normal development of cardiovascular tissue and also suggest possible therapeutic potential of rapamycin in cardiac disorders where pathologic mTORC1 activation occurs.