Recent Advances in Renal Tumors with TSC/mTOR Pathway Abnormalities in Patients with Tuberous Sclerosis Complex and in the Sporadic Setting.

A spectrum of renal tumors associated with frequent TSC/mTOR (tuberous sclerosis complex/mechanistic target of rapamycin) pathway gene alterations (in both the germline and sporadic settings) have recently been described. These include renal cell carcinoma with fibromyomatous stroma (RCC FMS), eosinophilic solid and cystic renal cell carcinoma (ESC RCC), eosinophilic vacuolated tumor (EVT), and low-grade oncocytic tumor (LOT). Most of these entities have characteristic morphologic and immunohistochemical features that enable their recognition without the need for molecular studies. In this report, we summarize recent advances and discuss their evolving complexity.

Tuberin levels during cellular differentiation in brain development.

Tuberin is a member of a large protein complex, Tuberous Sclerosis Complex (TSC), and acts as a sensor for nutrient status regulating protein synthesis and cell cycle progression. Mutations in the Tuberin gene, TSC2, permits the formation of tumors that can lead to developmental defects in many organ systems, including the central nervous system. Tuberin is expressed in the brain throughout development and levels of Tuberin have been found to decrease during neuronal differentiation in cell lines in vitro. Our current work investigates the levels of Tuberin at two stages of embryonic development in vivo, and we study the mRNA and protein levels during a time course using immortalized cell lines in vitro. Our results show that total Tuberin levels are tightly regulated through developmental stages in the embryonic brain. At a cell biology level, we show that Tuberin levels are higher when cells are cultured as neurospheres, and knockdown of Tuberin results in a reduction in the number of neurospheres. This functional data supports the hypothesis that Tuberin is an important regulator of stemness and the reduction of Tuberin levels might support functional differentiation in the central nervous system. Understanding how Tuberin expression is regulated throughout neural development is essential to fully comprehend the role of this protein in several developmental and neural pathologies.

Angiotensin II receptor type 1 blockade regulates Klotho expression to induce TSC2-deficient cell death.

Lymphangioleiomyomatosis (LAM) is a multisystem disease occurring in women of child-bearing age manifested by uncontrolled proliferation of smooth muscle-like "LAM" cells in the lungs. LAM cells bear loss-of-function mutations in tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2, causing hyperactivation of the proliferation promoting mammalian/mechanistic target of Rapamycin complex 1 pathway. Additionally, LAM-specific active renin-angiotensin system (RAS) has been identified in LAM nodules, suggesting this system potentially contributes to neoplastic properties of LAM cells; however, the role of this renin-angiotensin signaling is unclear. Here, we report that TSC2-deficient cells are sensitive to the blockade of angiotensin II receptor type 1 (Agtr1). We show that treatment of these cells with the AGTR1 inhibitor losartan or silencing of the Agtr1 gene leads to increased cell death in vitro and attenuates tumor progression in vivo. Notably, we found the effect of Agtr1 blockade is specific to TSC2-deficient cells. Mechanistically, we demonstrate that cell death induced by Agtr1 inhibition is mediated by an increased expression of Klotho. In TSC2-deficient cells, we showed overexpression of Klotho or treatment with recombinant (soluble) Klotho mirrored the cytocidal effect of angiotensin blockade. Furthermore, Klotho treatment decreased the phosphorylation of AKT, potentially leading to this cytocidal effect. Conversely, silencing of Klotho rescued TSC2-deficient cells from cell death induced by Agtr1 inhibition. Therefore, we conclude that Agtr1 and Klotho are important for TSC2-deficient cell survival. These findings further illuminate the role of the RAS in LAM and the potential of targeting Agtr1 inhibition in TSC2-deficient cells.

Emerging Link between Tsc1 and FNIP Co-Chaperones of Hsp90 and Cancer.

Heat shock protein-90 (Hsp90) is an ATP-dependent molecular chaperone that is tightly regulated by a group of proteins termed co-chaperones. This chaperone system is essential for the stabilization and activation of many key signaling proteins. Recent identification of the co-chaperones FNIP1, FNIP2, and Tsc1 has broadened the spectrum of Hsp90 regulators. These new co-chaperones mediate the stability of critical tumor suppressors FLCN and Tsc2 as well as the various classes of Hsp90 kinase and non-kinase clients. Many early observations of the roles of FNIP1, FNIP2, and Tsc1 suggested functions independent of FLCN and Tsc2 but have not been fully delineated. Given the broad cellular impact of Hsp90-dependent signaling, it is possible to explain the cellular activities of these new co-chaperones by their influence on Hsp90 function. Here, we review the literature on FNIP1, FNIP2, and Tsc1 as co-chaperones and discuss the potential downstream impact of this regulation on normal cellular function and in human diseases.

LAM Cells as Potential Drivers of Senescence in Lymphangioleiomyomatosis Microenvironment.

Senescence is a stress-response process characterized by the irreversible inhibition of cell proliferation, associated to the acquisition of a senescence-associated secretory phenotype (SASP), that may drive pathological conditions. Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells, featuring the hyperactivation of the mammalian Target of Rapamycin Complex 1 (mTORC1) for the absence of tuberin expression, cause the disruption of the lung parenchyma. Considering that LAM cells secrete SASP factors and that mTOR is also a driver of senescence, we deepened the contribution of senescence in LAM cell phenotype. We firstly demonstrated that human primary tuberin-deficient LAM cells (LAM/TSC cells) have senescent features depending on mTOR hyperactivation, since their high positivity to SA-ß galactosidase and to phospho-histone H2A.X are reduced by inducing tuberin expression and by inhibiting mTOR with rapamycin. Then, we demonstrated the capability of LAM/TSC cells to induce senescence. Indeed, primary lung fibroblasts (PLFs) grown in LAM/TSC conditioned medium increased the positivity to SA-ß galactosidase and to phospho-histone H2A.X, as well as p21WAF1/CIP1 expression, and enhanced the mRNA expression and the secretion of the SASP component IL-8. Taken together, these data make senescence a novel field of study to understand LAM development and progression.

STAT3 suppresses the AMPKα/ULK1-dependent induction of autophagy in glioblastoma cells.

Despite advances in molecular characterization, glioblastoma (GBM) remains the most common and lethal brain tumour with high mortality rates in both paediatric and adult patients. The signal transducer and activator of transcription 3 (STAT3) is an important oncogenic driver of GBM. Although STAT3 reportedly plays a role in autophagy of some cells, its role in cancer cell autophagy remains unclear. In this study, we found Serine-727 and Tyrosine-705 phosphorylation of STAT3 was constitutive in GBM cell lines. Tyrosine phosphorylation of STAT3 in GBM cells suppresses autophagy, whereas knockout (KO) of STAT3 increases ULK1 gene expression, increases TSC2-AMPKα-ULK1 signalling, and increases lysosomal Cathepsin D processing, leading to the stimulation of autophagy. Rescue of STAT3-KO cells by the enforced expression of wild-type (WT) STAT3 reverses these pathways and inhibits autophagy. Conversely, expression of Y705F- and S727A-STAT3 phosphorylation deficient mutants in STAT3-KO cells did not suppress autophagy. Inhibition of ULK1 activity (by treatment with MRT68921) or its expression (by siRNA knockdown) in STAT3-KO cells inhibits autophagy and sensitizes cells to apoptosis. Taken together, our findings suggest that serine and tyrosine phosphorylation of STAT3 play critical roles in STAT3-dependent autophagy in GBM, and thus are potential targets to treat GBM.

The non-essential TSC complex component TBC1D7 restricts tissue mTORC1 signaling and brain and neuron growth.

The tuberous sclerosis complex (TSC) 1 and 2 proteins associate with TBC1D7 to form the TSC complex, which is an essential suppressor of mTOR complex 1 (mTORC1), a ubiquitous driver of cell and tissue growth. Loss-of-function mutations in TSC1 or TSC2, but not TBC1D7, give rise to TSC, a pleiotropic disorder with aberrant activation of mTORC1 in various tissues. Here, we characterize mice with genetic deletion of Tbc1d7, which are viable with normal growth and development. Consistent with partial loss of function of the TSC complex, Tbc1d7 knockout (KO) mice display variable increases in tissue mTORC1 signaling with increased muscle fiber size but with strength and motor defects. Their most pronounced phenotype is brain overgrowth due to thickening of the cerebral cortex, with enhanced neuron-intrinsic mTORC1 signaling and growth. Thus, TBC1D7 is required for full TSC complex function in tissues, and the brain is particularly sensitive to its growth-suppressing activities.

Reappraising Schwannoma-Hemangioma Composite Tumors as Synchronous Tumorigenic Entities With Conjoined Histomorphology: A Case Report.

The most common peripheral nerve sheath and vascular tumors are schwannomas and hemangiomas, respectively. These tumors can affect any organ system and usually occur as separate morphologic and diagnostic entities. Herein, we describe the case of a 24-year-old woman with a tumor demonstrating composite differentiation to both cavernous hemangioma and schwannoma in the scalp. The patient had a slow-growing subcutaneous scalp tumor in the occipital region with an insidious onset and progression. The patient underwent wide local excision for treatment and based on follow-up evaluations has remained asymptomatic with no signs of recurrence.

Reviewing Schwannoma-Hemangioma Composite Tumors With Their Tumorigenetic Molecular Pathways and Associated Syndromic Manifestations.

Schwannomas are common peripheral nerve sheath tumors. Cavernous hemangiomas are vascular tumors that can affect any organ system. The coexistence of cavernous hemangioma with peripheral nervous system neoplasms is a rare occurrence. So far, 37 cases have been documented, and they have been divided into two categories: conjoined association (neoplasms discovered within the tumor tissue) and discrete association (neoplasms discovered outside the tumor tissue, thus placing neoplasms and tumors in close proximity but in different locations). Schwannomas and neurofibromas are the most prevalent tumors linked to cavernous hemangiomas that have been documented. The author provides a comprehensive review of all such cases published in the past with an emphasis on the implications of their tumorigenetic molecular pathways and syndromic manifestations.

A novel role of snail in regulating tuberin/AMPK pathways to promote renal fibrosis in the new mouse model of type II diabetes.

Epithelial-mesenchymal transition (EMT) plays an important role in tissue fibrosis following chronic exposure to hyperglycemia. This study investigates the role of chronic diabetes in regulating tuberin/snail/AMPK to enhance EMT and increase renal fibrosis. A new mouse model of db/db/TSC2 +/- was generated by backcrossing db/db mice and TSC2 +/- mice. Wild type (WT), db/db, TSC2 +/- and dbdb/TSC2 +/- mice were sacrificed at ages 6 and 8 months old. Tuberin protein level was significantly decreased in kidneys from diabetic compared to WT mice at both ages. In addition, tuberin and E-cadherin protein levels were significantly decreased in dbdb/TSC2 +/- compared to TSC2 +/- and db/db mice. In contrast, p-PS6K, NFkB, snail, vimentin, fibronectin, and α-SMA protein levels were significantly increased in dbdb/TSC2 +/- compared to db/db and TSC2 +/- mice at ages 6 and 8 months. Both downregulation of AMPK by DN-AMPK and downregulation of tuberin by siRNA resulted in increased NFkB, snail, and fibronectin protein expression and decreased E-cadherin protein expression in mouse primary renal proximal tubular cells. Interestingly, downregulation of snail by siRNA increased tuberin expression via feedback through activation of AMPK and reversed the expression of epithelial proteins such as E-cadherin as well as mesenchymal proteins such as fibronectin, NF-KB, vimentin, and α-SMA in mouse primary renal proximal tubular cells isolated from kidneys of four mice genotypes. The data show that chronic diabetes significantly decreases tuberin expression and that provides strong evidence that tuberin is a major key protein involved in regulating EMT. These data also demonstrated a novel role for snail in regulating of AMPK/tuberin to enhance EMT and renal cell fibrosis in diabetes.

Beyond Protein Synthesis; The Multifaceted Roles of Tuberin in Cell Cycle Regulation.

The ability of cells to sense diverse environmental signals, including nutrient availability and conditions of stress, is critical for both prokaryotes and eukaryotes to mount an appropriate physiological response. While there is a great deal known about the different biochemical pathways that can detect and relay information from the environment, how these signals are integrated to control progression through the cell cycle is still an expanding area of research. Over the past three decades the proteins Tuberin, Hamartin and TBC1D7 have emerged as a large protein complex called the Tuberous Sclerosis Complex. This complex can integrate a wide variety of environmental signals to control a host of cell biology events including protein synthesis, cell cycle, protein transport, cell adhesion, autophagy, and cell growth. Worldwide efforts have revealed many molecular pathways which alter Tuberin post-translationally to convey messages to these important pathways, with most of the focus being on the regulation over protein synthesis. Herein we review the literature supporting that the Tuberous Sclerosis Complex plays a critical role in integrating environmental signals with the core cell cycle machinery.

The Role of Heat Shock Protein-90 in the Pathogenesis of Birt-Hogg-Dubé and Tuberous Sclerosis Complex Syndromes.

Birt-Hogg-Dubé (BHD) and tuberous sclerosis (TS) syndromes share many clinical features. These two diseases display distinct histologic subtypes of renal tumors: chromophobe renal cell carcinoma and renal angiomyolipoma, respectively. Early work suggested a role for mTOR dysregulation in the pathogenesis of these two diseases, however their detailed molecular link remains elusive. Interestingly, a growing number of case reports describe renal angiomyolipoma in BHD patients, suggesting a common molecular origin. The BHD-associated proteins FNIP1/2 and the TS protein Tsc1 were recently identified as regulators of the molecular chaperone Hsp90. Dysregulation of Hsp90 activity has previously been reported to support tumorigenesis, providing a potential explanation for the overlapping phenotypic manifestations in these two hereditary syndromes.

Architecture of the Tuberous Sclerosis Protein Complex.

The Tuberous Sclerosis Complex (TSC) protein complex (TSCC), comprising TSC1, TSC2, and TBC1D7, is widely recognised as a key integration hub for cell growth and intracellular stress signals upstream of the mammalian target of rapamycin complex 1 (mTORC1). The TSCC negatively regulates mTORC1 by acting as a GTPase-activating protein (GAP) towards the small GTPase Rheb. Both human TSC1 and TSC2 are important tumour suppressors, and mutations in them underlie the disease tuberous sclerosis. We used single-particle cryo-EM to reveal the organisation and architecture of the complete human TSCC. We show that TSCC forms an elongated scorpion-like structure, consisting of a central "body", with a "pincer" and a "tail" at the respective ends. The "body" is composed of a flexible TSC2 HEAT repeat dimer, along the surface of which runs the TSC1 coiled-coil backbone, breaking the symmetry of the dimer. Each end of the body is structurally distinct, representing the N- and C-termini of TSC1; a "pincer" is formed by the highly flexible N-terminal TSC1 core domains and a barbed "tail" makes up the TSC1 coiled-coil-TBC1D7 junction. The TSC2 GAP domain is found abutting the centre of the body on each side of the dimerisation interface, poised to bind a pair of Rheb molecules at a similar separation to the pair in activated mTORC1. Our architectural dissection reveals the mode of association and topology of the complex, casts light on the recruitment of Rheb to the TSCC, and also hints at functional higher order oligomerisation, which has previously been predicted to be important for Rheb-signalling suppression.

An update on the central nervous system manifestations of tuberous sclerosis complex.

The autosomal dominant disorder tuberous sclerosis complex (TSC) is characterized by an array of manifestations both within and outside of the central nervous system (CNS), including hamartomas and other malformations. TSC is caused by mutations in the TSC1 or TSC2 gene resulting in activation of the mechanistic target of rapamycin (mTOR) signaling pathway. Study of TSC has shed light on the critical role of the mTOR pathway in neurodevelopment. This update reviews the genetic basis of TSC, its cardinal phenotypic CNS features, and recent developments in the field of TSC and other mTOR-altered disorders.

Mutation of the co-chaperone Tsc1 in bladder cancer diminishes Hsp90 acetylation and reduces drug sensitivity and selectivity.

The molecular chaperone Heat shock protein 90 (Hsp90) is essential for the folding, stability, and activity of several drivers of oncogenesis. Hsp90 inhibitors are currently under clinical evaluation for cancer treatment, however their efficacy is limited by lack of biomarkers to optimize patient selection. We have recently identified the tumor suppressor tuberous sclerosis complex 1 (Tsc1) as a new co-chaperone of Hsp90 that affects Hsp90 binding to its inhibitors. Highly variable mutations of TSC1 have been previously identified in bladder cancer and correlate with sensitivity to the Hsp90 inhibitors. Here we showed loss of TSC1 leads to hypoacetylation of Hsp90-K407/K419 and subsequent decreased binding to the Hsp90 inhibitor ganetespib. Pharmacologic inhibition of histone deacetylases (HDACs) restores acetylation of Hsp90 and sensitizes Tsc1-mutant bladder cancer cells to ganetespib, resulting in apoptosis. Our findings suggest that TSC1 status may predict response to Hsp90 inhibitors in patients with bladder cancer, and co-targeting HDACs can sensitize tumors with Tsc1 mutations to Hsp90 inhibitors.

Autopsy case of right ventricular rhabdomyoma in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a genetic multisystem disorder characterized by widespread hamartomas in several organs, including the brain, heart, skin, eyes, kidney, lung, and liver. Rhabdomyoma is the most common cardiac tumor diagnosed in fetuses, neonates and infants, and is closely linked to TSC. Here we describe an autopsy case of right ventricular rhabdomyoma in TSC. The deceased was a 3-month-old male infant, and TSC with a cardiac tumor had been diagnosed before his death. Since the cardiac tumor had not been physically blocking the blood flow, he had not undergone surgical intervention. At autopsy, the patient's height was 62 cm and his body weight was 6 kg. The heart weighed 37.3 g and the right ventricle was filled with the tumor. The tumor measured 2.1 cm × 1.6 cm, being a fusion of multiple tumors with several attachment sites to the myocardium. Histologically, the tumor was diagnosed as a rhabdomyoma, and was positive for mammalian target of rapamycin (mTOR). The brain weighed 795.0 g, without hydrocephalus. The cut surface of the brain revealed multiple cortical tubers and subependymal nodules. Through screening for the TSC1 (hamartin) and TSC2 (tuberin) genes, a nonsense mutation, c.1108C>T:p.Gln370∗, was detected in the TSC2 gene. Immediate cause of death was determined to be ventricular obstruction by a cardiac rhabdomyoma with insidious growth. This case highlights the need for forensic pathologists to perform a complete autopsy to determine the cause of sudden death with cardiac tumor, including genetic examination.

Importance of tuberin in carcinogenesis.

The cell cycle is a dynamic process with multiple phases regulating cell growth. The proper regulation is essential for avoiding errors and activation of cell death. Tumour suppressor proteins, including tuberin, are crucial in coordinating adequate cell growth and properly timed cell division. So, the present review article is focused on the latest aspects of the tuberin in the process of carcinogenesis. The PubMed was the main database used for the collection of latest data relating to multiple aspects of tuberin especially in context of cancer. Most of the recent studies revealed that mutation, truncation, and deregulation of the tuberin protein could definitely lead to cancer. Recent studies are also devoted to explore implications towards better understanding the progression of disease involving mis-regulated tuberin.

Moving to the Rhythm with Clock (Circadian) Genes, Autophagy, mTOR, and SIRT1 in Degenerative Disease and Cancer.

BACKGROUND: The mammalian circadian clock and its associated clock genes are increasingly been recognized as critical components for a number of physiological and disease processes that extend beyond hormone release, thermal regulation, and sleep-wake cycles. New evidence suggests that clinical behavior disruptions that involve prolonged shift work and even space travel may negatively impact circadian rhythm and lead to multi-system disease. METHODS: In light of the significant role circadian rhythm can hold over the body's normal physiology as well as disease processes, we examined and discussed the impact circadian rhythm and clock genes hold over lifespan, neurodegenerative disorders, and tumorigenesis. RESULTS: In experimental models, lifespan is significantly reduced with the introduction of arrhythmic mutants and leads to an increase in oxidative stress exposure. Interestingly, patients with Alzheimer's disease and Parkinson's disease may suffer disease onset or progression as a result of alterations in the DNA methylation of clock genes as well as prolonged pharmacological treatment for these disorders that may lead to impairment of circadian rhythm function. Tumorigenesis also can occur with the loss of a maintained circadian rhythm and lead to an increased risk for nasopharyngeal carcinoma, breast cancer, and metastatic colorectal cancer. Interestingly, the circadian clock system relies upon the regulation of the critical pathways of autophagy, the mechanistic target of rapamycin (mTOR), AMP activated protein kinase (AMPK), and silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1) as well as proliferative mechanisms that involve the wingless pathway of Wnt/ß-catenin pathway to foster cell survival during injury and block tumor cell growth. CONCLUSION: Future targeting of the pathways of autophagy, mTOR, SIRT1, and Wnt that control mammalian circadian rhythm may hold the key for the development of novel and effective therapies against aging- related disorders, neurodegenerative disease, and tumorigenesis.

Warming Up to New Possibilities with the Capsaicin Receptor TRPV1: mTOR, AMPK, and Erythropoietin.

BACKGROUND: Transient receptor potential (TRP) channels are a superfamily of ion channels termed after the trp gene in Drosophila that are diverse in structure and control a wide range of biological functions including cell development and growth, thermal regulation, and vascular physiology. Of significant interest is the transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor, also known as the capsaicin receptor and the vanilloid receptor 1, that is a non-selective cation channel sensitive to a host of external stimuli including capsaicin and camphor, venoms, acid/basic pH changes, and temperature. METHODS: Given the multiple modalities that TRPV1 receptors impact in the body, we examined and discussed the role of these receptors in vasomotor control, metabolic disorders, cellular injury, oxidative stress, apoptosis, autophagy, and neurodegenerative disorders and their overlap with other signal transduction pathways that impact trophic factors. RESULTS: Surprisingly, TRPV1 receptors do not rely entirely upon calcium signaling to affect cellular biology, but also have a close relationship with the mechanistic target of rapamycin (mTOR), AMP activated protein kinase (AMPK), and protein kinase B (Akt) that have roles in pain sensitivity, stem cell development, cellular survival, and cellular metabolism. These pathways with TRPV1 converge in the signaling of growth factors with recent work highlighting a relationship with erythropoietin (EPO). Angiogenesis and endothelial tube formation controlled by EPO requires, in part, the activation of TRPV1 receptors in conjunction with Akt and AMPK pathways. CONCLUSION: TRPV1 receptors could prove to become vital to target disorders of vascular origin and neurodegeneration. Broader and currently unrealized implementations for both EPO and TRPV1 receptors can be envisioned for for the development of novel therapeutic strategies in multiple systems of the body.

Autophagy activated by tuberin/mTOR/p70S6K suppression is a protective mechanism against local anaesthetics neurotoxicity.

The local anaesthetics (LAs) are widely used for peripheral nerve blocks, epidural anaesthesia, spinal anaesthesia and pain management. However, exposure to LAs for long duration or at high dosage can provoke potential neuronal damages. Autophagy is an intracellular bulk degradation process for proteins and organelles. However, both the effects of LAs on autophagy in neuronal cells and the effects of autophagy on LAs neurotoxicity are not clear. To answer these questions, both lipid LAs (procaine and tetracaine) and amide LAs (bupivacaine, lidocaine and ropivacaine) were administrated to human neuroblastoma SH-SY5Y cells. Neurotoxicity was evaluated by MTT assay, morphological alterations and median death dosage. Autophagic flux was estimated by autolysosome formation (dual fluorescence LC3 assay), LC3-II generation and p62 protein degradation (immunoblotting). Signalling alterations were examined by immunoblotting analysis. Inhibition of autophagy was achieved by transfection with beclin-1 siRNA. We observed that LAs decreased cell viability in a dose-dependent manner. The neurotoxicity of LAs was tetracaine > bupivacaine > ropivacaine > procaine > lidocaine. LAs increased autophagic flux, as reflected by increases in autolysosome formation and LC3-II generation, and decrease in p62 levels. Moreover, LAs inhibited tuberin/mTOR/p70S6K signalling, a negative regulator of autophagy activation. Most importantly, autophagy inhibition by beclin-1 knockdown exacerbated the LAs-provoked cell damage. Our data suggest that autophagic flux was up-regulated by LAs through inhibition of tuberin/mTOR/p70S6K signalling, and autophagy activation served as a protective mechanism against LAs neurotoxicity. Therefore, autophagy manipulation could be an alternative therapeutic intervention to prevent LAs-induced neuronal damage.