TSC2 Expression Research Articles

Abstract CT161: Clinical study of PD-1 inhibitor with mTOR inhibitor for relapsed and refractory brain tumors

Abstract In this phase I/II study the combination regimen is used to treat recurrent or refractory malignant glioma patients. The inclusion criteria: 1) Histologically confirmed WHO IV. Participants will be eligible if the original histology was low-grade glioma and a subsequent histological diagnosis of glioblastoma. 2) Patients must be at first or second relapse following initial radiation-chemotherapy therapy. 3) Must provide tumor tissue. 4) Molecular diagnosis includes the mutation of PIK3CA, KRAS, TSC1 and PTEN expression decreased, meet the above 2 and above. High expression of PD-L1 in tumor and tumor infiltrating lymphocytes consider as drug specifically targeting T-cell checkpoint pathways. 5) KPS>/= 60. 6) Measurable contrast-enhancing tumor consists of a miaxmal volume of 3*3 cm. 7) Stable doses of steroids for 5 days, no more than 2 mg dexamethasone total per day. 8) Absolute neutrophil count (ANC) >/=1,500 /mcL; platelets >/=100,000/mcL; hemoglobin >/= 9 g/dL; serum creatinine /= 60 mL/min for subject with creatinine levels > 1.5 × institutional ULN. 9) Both sexual and age >/= 18 years on day of signing informed consent, 5 to 18 years of age signing informed consent by parents. Exclusion criteria: 1) Treated previously with bevacizumab. 2) Has tumor localized primarily to the brainstem or spinal cord. 3) Has received prior interstitial brachytherapy, implanted chemotherapy, or CAR-T cell therapy. 4) Currently participating in or has participated in a study of an investigational agent or using an investigational device 4 weeks since last dose of agent administration. 5) Has a diagnosis of immunodeficiency or any other form of immunosuppressive therapy within 7 days prior to the first dose of trial. 6) Has evidence of interstitial lung disease or active, non-infectious pneumonitis. 7) Has an active infection requiring systemic therapy. 8) Pregnant or breastfeeding. 9) Has received prior therapy with an anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CD137 antibody. 10) History of HIV, HBV, HCV. 11) Has received a live vaccine within 30 days prior to the first dose of trial treatment. According to the results of tumor molecular evaluation, patients would be divided into three intervention groups. The interventions are EVE group (Everolimus 10mg PO QD), PD-1 inhibitor monotherapy group (PD-1 inhibitor 2mg/kg ivgtt Q3W) and combination group (Everolimus 10mg PO QD + PD-1 inhibitor 2mg/kg ivgtt Q3W). Primary outcome measure: 6 month progression-free survival rate, progression will be determined by review of MRI exams every month, assessed using iRANO criteria. Secondary outcome measures: 1) Overall survival (OS) and progression-free survival (PFS). 2) Incidence of grade 3+ toxicities. 3) Helper and effecter T cells active percent, T reg and MDSC percent in peripheral blood will be detected before and after treatment Day 1, 3, 7 and 3 weeks in every cycle. This clinical trial has supported by IRB of Renji hospital and registered on ICTRP identifier: ChiCTR-OON-16008330. Citation Format: zhihua Chen, Yongming Qiu. Clinical study of PD-1 inhibitor with mTOR inhibitor for relapsed and refractory brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT161.

TIS21/BTG2 inhibits breast cancer growth and progression by differential regulation of mTORc1 and mTORc2-AKT1-NFAT1-PHLPP2 signaling axis.

It has been reported that PI3K/AKT pathway is altered in various cancers and AKT isoforms specifically regulate cell growth and metastasis of cancer cells; AKT1, but not AKT2, reduces invasion of cancer cells but maintains cancer growth. We propose here a novel mechanism of the tumor suppresser, TIS21/BTG2, that inhibits both growth and invasion of triple negative breast cancer cells via AKT1 activation by differential regulation of mTORc1 and mTORc2 activity. Transduction of adenovirus carrying TIS21/BTG2 gene and transfection of short interfering RNAs were employed to regulate TIS21/BTG2 gene expression in various cell lines. Treatment of mTOR inhibitors and mTOR kinase assays can evaluate the role of mTORc in the regulation of AKT phosphorylation at S473 residue by TIS21/BTG2 in breast cancer cells. Open data and immunohistochemical analysis were performed to confirm the role of TIS21/BTG2 expression in various human breast cancer tissues. We observed that TIS21/BTG2 inhibited mTORc1 activity by reducing Raptor-mTOR interaction along with upregulation of tsc1 expression, which lead to significant reduction of p70S6K activation as opposed to AKT1S473, but not AKT2, phosphorylation via downregulating PHLPP2 (AKT1-specific phosphatase) in breast cancers. TIS21/BTG2-induced pAKTS473 required Rictor-bound mTOR kinase, indicating activation of mTORc2 by TIS21/BTG2 gene. Additionally, the TIS21/BTG2-induced pAKTS473 could reduce expression of NFAT1 (nuclear factor of activated T cells) and its target genes, which regulate cancer microenvironment. TIS21/BTG2 significantly lost in the infiltrating ductal carcinoma, but it can inhibit cancer growth via the TIS21/BTG2-tsc1/2-mTORc1-p70S6K axis and downregulate cancer progression via the TIS21/BTG2-mTORc2-AKT1-NFAT1-PHLPP2 pathway.

Insight on ALPPS – Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy – mechanisms: activation of mTOR pathway

BackgroundALPPS procedure has been introduced to increase the volume of future liver remnant. The mechanisms underlying the accelerated regeneration observed with ALPPS are unknown. It was hypothesized that AMPK/mTOR is activated as an integrating pathway for metabolic signals leading to proliferation and cell growth. Our aim was to analyze increase in liver volume, proliferation parameters and expression of AMPK/mTOR pathway-related molecules in patients undergoing ALPPS. MethodsA single center prospective study of patients undergoing ALPPS was performed from 2013 to 2015. Liver and serum samples, clinical laboratory results and CT-scan data were obtained. ELISA, Ki-67 immunostaining and qRT-PCR were performed in deportalized and remnant liver tissue in both stages of the procedure. Results11 patients were enrolled. Remnant liver volume increased 112 ± 63% (p < 0.05) in 9.1 ± 1.6 days. Proliferation-related cytokines IL-6, TNF-α, HGF and EGF significantly increased, while higher Ki-67 immunostaining and cyclin D expression were observed in remnant livers after ALPPS. mTOR, S6K1, 4E-BP1, TSC1 and TSC2 expression were significantly increased in remnant livers at second stage, while AMPK and Akt increased only in deportalized liver samples. ConclusionRapid liver regeneration with ALPPS might be associated with hepatocyte proliferation induced by mTOR pathway activation.

Electroacupuncture Reduces Weight in Diet-Induced Obese Rats via Hypothalamic Tsc1 Promoter Demethylation and Inhibition of the Activity of mTORC1 Signaling Pathway.

Subject The study aimed to investigate the mechanism of electroacupuncture reducing weight via tuberous sclerosis complex 1 (Tsc1) promoter methylation, inhibiting the mammalian target of rapamycin complex 1 (mTORC1) pathway. Materials and Methods Male Sprague-Dawley rats were divided into chow-fed group (chow group) or high-fat diet group (HF group) for 14 weeks. The obesity rats in HF group were randomly divided into electroacupuncture group (EA group) and diet-induced obesity (DIO) group, which received EA stimulation on bilateral ST25, RN12, SP6, and ST36 for 4 weeks or no further treatment, respectively. Methylation of the Tsc1 gene promoter and expression of agouti-related protein (AgRP), neuropeptide Y (NPY), and proopiomelanocortin (PoMC) were detected at the 18th week. Results At week 18, weight, body fat, and the body fat rate in DIO group were significantly higher than those of the chow and EA group. Compared with the chow group, the DIO group had increased methylation of the Tsc1 gene promoter and expression of mTORC1, AgRP, and NPY gene and decreased PoMC in the hypothalamus; after EA, methylation of the Tsc1 gene promoter, mRNA, and protein of the mTORC1 and expression of AgRP and NPY gene decreased and PoMC increased significantly. Conclusions Our study could shed light on the potential pathway where EA exerts effects on the mechanism of EA treatment for obesity through the hypothalamic Tsc1 promoter demethylation and inhibition of the activity of mTORC1 signaling pathway.

Tsc1-dependent transcriptional programming of dendritic cell homeostasis and function

Dendritic cells (DCs) are pivotal to initiating adaptive immune response. Emerging evidence highlights important roles of tuberous sclerosis complex 1 (Tsc1) in DC development and activation. Our previous study also showed that Tsc1 expression in DCs was required to promote T-cell homeostasis and response partially through inhibiting mammalian target of rapamycin complex1 (mTORC1). However, the molecular mechanism of transcriptional regulation by which Tsc1 control DC homeostasis and function remains largely unknown. Here we globally identified the Tsc1-regulated genes by comparing the transcriptional profiling of Tsc1-deficient DCs with wild-type DCs. It showed that Tsc1 specifically regulated the expression of groups of gene sets critically involved in DC survival, proliferation, metabolism and antigen presentation. The impacts of Tsc1 on DC gene expression were partially dependent on inhibition of mTORC1 signal. Our study thus provides a comprehensive molecular basis for understanding how Tsc1 programs the homeostasis and function of DCs through transcriptional regulation.

TSC1 Mutations in Keratoconus Patients With or Without Tuberous Sclerosis.

PurposeTo test candidate genes TSC1 and TSC2 in a family affected by tuberous sclerosis complex (TSC) where proband was also diagnosed with bilateral keratoconus (KC) and to test the hypothesis that defects in the same gene may lead to a nonsyndromic KC.MethodsNext-generation sequencing of TSC1 and TSC2 genes was performed in a proband affected by TSC and KC. Identified mutation was confirmed by Sanger DNA sequencing. Whole exome sequencing (WES) was performed in patients with nonsyndromic KC. Sanger DNA sequencing was used to confirm WES results and to screen additional patients. RT-PCR was used to investigate TSC1 expression in seven normal human corneas and eight corneas from patients with KC. Various in silico tools were employed to model functional consequences of identified mutations.ResultsA heterozygous nonsense TSC1 mutation g.132902703C>T (c.2293C>T, p.Gln765Ter) was identified in a patient with TSC and KC. Two heterozygous missense TSC1 variants g.132896322A>T (c.3408A>T, p.Asp1136Glu) and g.132896452G>A (c.3278G>A, p.Arg1093Gln) were identified in three patients with nonsyndromic KC. Two mutations were not present in The Genome Aggregation (GnomAD), The Exome Aggregation (ExAC), and 1000 Genomes (1000G) databases, while the third one was present in GnomAD and 1000G with minor allele frequencies (MAF) of 0.00001 and 0.0002, respectively. We found TSC1 expressed in normal corneas and KC corneas, albeit with various levels.ConclusionsHere for the first time we found TSC1 gene to be involved in bilateral KC and TSC as well as with nonsyndromic KC, supporting the hypothesis that diverse germline mutations of the same gene can cause genetic disorders with overlapping clinical features.

IRF-1 SNPs influence the risk for childhood allergic asthma: A critical role for pro-inflammatory immune regulation.

Allergic and non-allergic childhood asthma has been characterized by distinct immune mechanisms. While interferon regulating factor 1 (IRF-1) polymorphisms (SNPs) influence atopy risk, the effect of SNPs on asthma phenotype-specific immune mechanisms is unclear. We assessed whether IRF-1 SNPs modify distinct immune-regulatory pathways in allergic and non-allergic childhood asthma (AA/NA). In the CLARA study, asthma was characterized by doctor's diagnosis and AA vs NA by positive or negative specific IgE. Children were genotyped for four tagging SNPs within IRF-1 (n=172). mRNA expression was measured with qRT-PCR. Gene expression was analyzed depending on genetic variants within IRF-1 and phenotype including haplotype estimation and an allelic risk score. Carrying the risk alleles of IRF-1 in rs10035166, rs2706384, or rs2070721 was associated with increased risk for AA. Carrying the non-risk allele in rs17622656 was associated with lower risk for AA but not NA. In AA carrying the risk alleles, an increased pro-inflammatory expression of ICAM3, IRF-8, XBP-1, IFN-γ, RGS13, RORC, and TSC2 was observed. NOD2 expression was decreased in AA with risk alleles in rs2706384 and rs10035166 and with risk haplotype. Further, AA with risk haplotype showed increased IL-13 secretion. NA with risk allele in rs2070721 compared to non-risk allele in rs17622656 showed significantly upregulated calcium, innate, mTOR, neutrophil, and inflammatory-associated genes. IRF-1 polymorphisms influence the risk for childhood allergic asthma being associated with increased pro-inflammatory gene regulation. Thus, it is critical to implement IRF-1 genetics in immune assessment for childhood asthma phenotypes.

ID:2027 Deregulated expression of key components of phosphoinositide 3 kinase pathway in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is sixth common cancer in males globally. Deregulation of phosphatidylinositol-3-kinase (PI3K) pathway leads to various intracellular responses such as proliferation, survival, and inhibition of apoptosis. We evaluated the expression of key components of PI3K pathway in OSCC patients. RT-PCR and qRT-PCR was used asses the expression of different AKT isoforms, PTEN, TSC1 and TSC2 in tumor and normal tissues. The expression of various components of PI3K pathway e.g.Ser473pAKT, pan-AKT, PTEN and Ser2448pmTOR and mTOR was evaluated by western blot assay. Effect of selected PI3K inhibitors on OSCC cells (SCC-4, SCC-9 and SCC-25) was also studied. Approximately 1.4-fold higher expression of AKT1 and downregulation of AKT2 and AKT3 mRNA was observed in tumor tissue sections of patients as compared to controls. PTEN, TSC1 and TSC2 mRNA was found to be marginally decreases in tumor than the normal area. Significantly strong immunostaining of ser473p-AKT in comparison to AKT1 was documented in all paraffin fixed oral cancer tissues. Additionally, a strong positive correlation between the immuno-histochemical expression of AKT-1 and ser473p-AKT in the paraffin sections of oral cancer tissues was observed (r= 0.7504; p≤0.0001). Aberrant expression of key components of PI3K pathway was also found in OSCC cells that were reversed with the treatment of its inhibitors. Overall, our study suggests that PI3K pathway is deregulated OSCC patients and OSCC cell lines, with AKT1 being the predominantly expressed isoform. PI3K inhibitors restored such aberrations in OSCC cell lines.

MiR-130a upregulates mTOR pathway by targeting TSC1 and is transactivated by NF-κB in high-grade serous ovarian carcinoma.

Activation of mammalian target of rapamycin (mTOR) signaling pathway is associated with poor prognosis of epithelial ovarian cancer. The TSC1-TSC2 complex is a critical negative regulator of mTOR signaling. Here, we demonstrated that TSC1 was frequently downregulated in high-grade serous ovarian carcinoma (HGSOC) and low TSC1 expression level is associated with advanced tumor stage. We next identified miR-130a to be a negative regulator of TSC1 by targeting its 3'UTR. miR-130a was overexpressed in HGSOC and could drive proliferation and invasion/metastasis of ovarian cancer cells. miR-130a could also attenuate rapamycin/starvation-induced autophagy. Ectopic TSC1 expression could block the effects of miR-130a on cell proliferation, migration and autophagy. Finally, we found that miR-130a expression could be upregulated by inflammatory factors and was transactivated by NF-κB. Therefore, our findings establish a crosstalk between inflammation and mTOR signaling that is mediated by miR-130a, which might have a pivotal role in the initiation and progression of HGSOC.

Effect of Redd1 loss on proliferation and metastasis of pancreatic cancer cells with KrasG12D-LOH by inhibiting glycolysis.

e15741 Background: Aerobic glycolysis, regulated by mammalian target of rapamycin (mTOR) pathway, plays an important role in pancreatic carcinogenesis. Regulated in development and DNA damage response (Redd1) constitutes an important regulator of mTOR signaling. Previously, we observed that the loss of heterozygosity (LOH) status of Kras mutations (e.g. KrasG12D) is associated with an increased Redd1 expression. Here, we investigated the functional relevance of Redd1 in the context of KrasG12D-LOH. Methods: Murine KrasG12D-LOH/KrasG12Dpancreatic cancer cells isolated from genetically engineered mice were used in this study. The glycolysis dependence was detected by CCK8 assays after treated by glycolysis inhibitor 2-Deoxy-D-glucose (2-DG). Redd1 expression was down-regulated using shRNA transfection. Cell proliferation was determined by CCK8 and colony formation assays. Cell invasion and migration was measured by transwell and wound-healing assays. The levels of lactic acid and ATP were tested by ELISA kit. Genes related to glycolysis and mTOR signal were evaluated by western-blot and quantitative RT-PCR. Results: Compared with KRASG12D pancreatic cancer cells, the viability of KRASG12D-LOH cells decreased significantly in the presence of 2-DG. After Redd1 suppression, the proliferative and invasive potentials of KRASG12D-LOH cells decreased significantly when compared with blank group and negative group. The levels of lactic acid and ATP were also reduced by Redd1 down-regulation. Furthermore, the signal activity of mTOR pathway and Pkm2 and Hk2 expression were reduced dramatically, while Tsc1 and Tsc2 expression were unaffected. Conclusions: The LOH status of KRASG12D renders the pancreatic cancer cells additive to glycolysis, which further affect the proliferative and invasive potentials via the function of Redd1/mTOR. These data underscore the importance of KRASG12D-LOH in regulating cancer glucose metabolism.

TCS2 Increases Olaquindox-Induced Apoptosis by Upregulation of ROS Production and Downregulation of Autophagy in HEK293 Cells.

Olaquindox, a feed additive, has drawn public attention due to its potential mutagenicity, genotoxicity, hepatoxicity and nephrotoxicity. The purpose of this study was to investigate the role of tuberous sclerosis complex (TSC2) pathways in olaquindox-induced autophagy in human embryonic kidney 293 (HEK293) cells. The results revealed that olaquindox treatment reduced the cell viability of HEK293 cells and downregulated the expression of TSC2 in a dose- and time-dependent manner. Meanwhile, olaquindox treatment markedly induced the production of reactive oxygen species (ROS), cascaded to autophagy, oxidative stress, and apoptotic cell death, which was effectively eliminated by the antioxidant N-acetylcysteine (NAC). Furthermore, overexpression of TSC2 attenuated olaquindox-induced autophagy in contrast to inducing the production of ROS, oxidative stress and apoptosis. Consistently, knockdown of TSC2 upregulated autophagy, and decreased olaquindox-induced cell apoptosis. In conclusion, our findings indicate that TSC2 partly participates in olaquindox-induced autophagy, oxidative stress and apoptosis, and demonstrate that TSC2 has a negative regulation role in olaquindox-induced autophagy in HEK293 cells.

Tsc1 expression by dendritic cells is required to preserve T-cell homeostasis and response

Dendritic cells (DCs) are pivotal to the induction of adaptive T-cell immune responses. Recent evidence highlights a critical role of tuberous sclerosis complex 1 (Tsc1), a primarily upstream negative regulator of mammalian target of rapamycin (mTOR), in DC development, but whether and how Tsc1 directly regulate mature DC function in vivo remains elusive. Here we show that selective disruption of Tsc1 in DCs results in a lymphoproliferative disorder with the spontaneous activation of T cells. Tsc1 deficiency results in the activation of mTORC1-PPARγ pathway, which leads to the upregulation of neuropilin-1 (Nrp1) expression on DCs to stimulate naive T-cell proliferation. However, Tsc1-deficient DCs have defects in the ability to induce antigen-specific T-cell responses in vitro and in vivo owing to impaired survival during antigen transportation and presentation. Indeed, Tsc1 promotes DC survival through restraining independent mTORC1 and ROS-Bim pathways. Our study identifies Tsc1 as a crucial signaling checkpoint in DCs essential for preserving T-cell homeostasis and response.

Cryptotanshinone activates AMPK-TSC2 axis leading to inhibition of mTORC1 signaling in cancer cells

BackgroundCryptotanshinone (CPT), a fat-soluble phenanthraquinone from Salvia miltiorrhiza Bunge, has been demonstrated to inhibit phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a couple of direct downstream effectors of the mammalian target of rapamycin complex 1 (mTORC1), resulting in cancer cell arrested in G0 phase and subsequent inhibition of proliferation. However, its concrete molecular mechanism about how CPT inhibits mTORC1 signaling pathway is unclear.Methodsone solution was used to check cell viability and western blotting for determining expression of the indicated proteins. Molecular docking was performed to assess the binding of CPT with mTOR. The co-immunoprecipitation assay was to analyze whether CPT could disrupt the mTORC1 and TSC1/TSC2 complex. Recombinant adenoviral dominant-negative AMPKα was used to downregulate expression of AMPKα and lentiviral AMPK and TSC2 to silence the AMPK and TSC2 in Rh30 cells.ResultsPrimarily, Rh30 cells expressing rapamycin-resistant mutant mTOR are also sensitive to CPT, while the molecular docking result for CPT binding to mTOR is negative, suggesting that CPT inhibition of mTORC1 is different from rapamycin. Then the related proteins of PTEN-PI3K pathway was proved not to be affected, but the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was activated by a concentration- and time- dependent manner, meaning that it may be associated with AMPK. Further results indicated that compound C, inhibitor of AMPK, could clearly reversed CPT inhibitory effect on Rh30 cells, and dominant-negative AMPK in cancer cells conferred resistance to CPT inhibition of 4E-BP1 and phosphorylation of S6K1, as well as sh-AMPK. Furthermore, compared with AMPK-positive MEF cells, AMPK-negative MEF cells are less sensitive to CPT by the findings that 4E-BP1 and phosphorylation of S6K1 express comparatively more. Additionally, phosphorylation of tuberous sclerosis complex 2 (TSC2) was activated under the treatment of CPT, and down-expression of TSC2 by shRNA slightly recovered expression of 4E-BP1 and phosphorylation of S6K1, while co-immunoprecipitation of TSC2 did not alter expression of TSC1 by CPT.ConclusionCPT inhibiting mTORC1 pathway was mostly due to activation of AMPK-TSC2 axis rather than specific binding to mTORC1. CPT is a potent anticancer agent targeting AMPK.

Kavalactone yangonin induces autophagy and sensitizes bladder cancer cells to flavokawain A and docetaxel via inhibition of the mTOR pathway.

Consumption of kava (Piper methysticum Forst) has been linked to reduced cancer risk in the South Pacific Islands. Kavalactones are major bioactive components in kava root extracts, which have recently demonstrated anti-cancer activities. However, molecular mechanisms of kavalactones' anti-cancer action remain largely unknown. We have identified two kavalactones, yangonin and 5′ 6'-dehydrokawain, as potent inducers of autophagic cell death in bladder cancer cells. The effect of yangonin inducing autophagy is associated with increased expression of beclin and ATG5. In addition, yangonin increases the expression of LKB1 and decreases the phosphorylation of Akt, PRAS40, rpS6, p70S6K and 4E-BP1, leading to increased binding of 4E-BP1 to m7 GTP. The growth inhibitory effects of yangonin were attenuated inTSC1 or LKB1 knockout mouse embryonic fibroblasts, suggesting that TSC1 and LKB1 expression may contribute to optimal growth inhibition by yangonin. Furthermore, yangonin reduces the viability of bladder cancer cell lines derived from different stages of human bladder cancer, and acts synergistically with apoptosis-inducing agents such as docetaxel and flavokawain A. Our results support a novel anti-bladder cancer mechanism by yangonin and further studies are needed to assess the potential use of yangonin for bladder cancer prevention and treatment

Repression of TSC1/TSC2 mediated by MeCP2 regulates human embryo lung fibroblast cell differentiation and proliferation

Pulmonary fibrosis (PF) is a severe inflammatory disease with limited effective treatments. It is known that the transdifferentiation of human embryo lung fibroblast (HELF) cells from pulmonary fibroblasts into myofibroblasts, contributes to the progression of pulmonary fibrogenesis. The tuberous sclerosis proteins TSC1 and TSC2 are two key signaling factors which can suppress cell growth and proliferation. However, the roles of TSC1 and TSC2 in lung fibroblast are unclear. Here, we developed a PF model with bleomycin (BLM) in mice and conducted several simulation experiments in HELF cells. Our study shows that the expression of TSC1 and TSC2 in fibrotic mice lung was reduced and stimulation of HELF cells with TGF-β1 resulted in a down-regulation of TSC1 and TSC2. In addition, overexpression of TSC1 or TSC2 decreased cell proliferation and differentiation. Furthermore, we found that reduced expression of TSC1 and TSC2 caused by TGF-β1 is associated with the promoter methylation status of TSC1 and TSC2. MeCP2, controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. We found that expression of TSC1 and TSC2 can be repressed by MeCP2, which regulates HELF cell differentiation and proliferation as myofibroblasts and lead to PF ultimately.

Tuberous sclerosis complex inactivation disrupts melanogenesis via mTORC1 activation.

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor-suppressor gene syndrome caused by inactivating mutations in either TSC1 or TSC2, and the TSC protein complex is an essential regulator of mTOR complex 1 (mTORC1). Patients with TSC develop hypomelanotic macules (white spots), but the molecular mechanisms underlying their formation are not fully characterized. Using human primary melanocytes and a highly pigmented melanoma cell line, we demonstrate that reduced expression of either TSC1 or TSC2 causes reduced pigmentation through mTORC1 activation, which results in hyperactivation of glycogen synthase kinase 3β (GSK3β), followed by phosphorylation of and loss of β-catenin from the nucleus, thereby reducing expression of microphthalmia-associated transcription factor (MITF), and subsequent reductions in tyrosinase and other genes required for melanogenesis. Genetic suppression or pharmacological inhibition of this signaling cascade at multiple levels restored pigmentation. Importantly, primary melanocytes isolated from hypomelanotic macules from 6 patients with TSC all exhibited reduced TSC2 protein expression, and 1 culture showed biallelic mutation in TSC2, one of which was germline and the second acquired in the melanocytes of the hypomelanotic macule. These findings indicate that the TSC/mTORC1/AKT/GSK3β/β-catenin/MITF axis plays a central role in regulating melanogenesis. Interventions that enhance or diminish mTORC1 activity or other nodes in this pathway in melanocytes could potentially modulate pigment production.

Tuberous sclerosis complex protein 1 expression is affected by VHL Gene alterations and HIF-1α production in sporadic clear-cell renal cell carcinoma

Alterations in von Hippel-Lindau gene (VHL) do not determine deregulation of hypoxia-inducible factors (HIFs) in clear-cell renal carcinoma (ccRCC). Their effects on tuberous sclerosis proteins (TSC1/2) and heat shock protein 90 (Hsp90) expressions in sporadic ccRCC are unknown. Therefore, we analyze the impact of VHL alterations and HIF-α production on the expression of TSC proteins and Hsp90 in these tumors.Alterations in VHL gene region exhibited 37/47 (78.7%) tumors. Monoallelic inactivation (intragenic mutation or LOH) was found in 10 (21.3%) and biallelic inactivation (intragenic mutation plus LOH) in 27 (57.4%) ccRCCs. Tumorous expression of HIF-α mRNAs, HIF-α, Hsp90 and TSC2 were VHL independent; TSC2 was underexpressed in all tumors by immunostaining (P<0.001). Immunoblotting revealed that TSC1 production was lower in tumors with monoallelic VHL inactivation than in control (P=0.01) and tissues with biallelic VHL inactivation (P=0.019), while tumors lacking HIF-1α (16/47) concurrently overexpressed HIF-2α and underexpressed TSC1 in comparison to controls (P=0.01 for both) and HIF-1α positive tumors (P=0.015 and P=0.050). Significant portion of variability (56.4%) in tumor diameter was explained by oscillations in nuclear grade, and TSC1 and HIF-2α expression in VHL altered tumors.In conclusion, while TSC2 is broadly downregulated in sporadic ccRCC, TSC1 expression is reduced in two subsets of these tumors, those with monoallelic VHL gene inactivation and those with concurrent low HIF-1α and high HIF-2α expression. Hence, the involvement of nuclear grade, TSC1 and HIF-2α in the progression of VHL altered tumors, implies the interplay between pVHL and TSC1.

Fatty acid extract from CLA-enriched egg yolks can mediate transcriptome reprogramming of MCF-7 cancer cells to prevent their growth and proliferation.

BackgroundOur previous study showed that fatty acids extract obtained from CLA-enriched egg yolks (EFA-CLA) suppressed the viability of MCF-7 cancer cell line more effectively than extract from non-enriched egg yolks (EFA). In this study, we analysed the effect of EFA-CLA and EFA on transcriptome profile of MCF-7 cells by applying the whole Human Genome Microarray technology.ResultsWe found that EFA-CLA and EFA treated cells differentially regulated genes involved in cancer development and progression. EFA-CLA, compared to EFA, positively increased the mRNA expression of TSC2 and PTEN tumor suppressors as well as decreased the expression of NOTCH1, AGPS, GNA12, STAT3, UCP2, HIGD2A, HIF1A, PPKAR1A oncogenes.ConclusionsWe show for the first time that EFA-CLA can regulate genes engaged in AKT/mTOR pathway and inhibiting cell cycle progression. The observed results are most likely achieved by the combined effect of both: incorporated CLA isomers and other fatty acids in eggs organically modified through hens’ diet. Our results suggest that CLA-enriched eggs could be easily available food products with a potential of a cancer chemopreventive agent.Electronic supplementary materialThe online version of this article (doi:10.1186/s12263-016-0537-z) contains supplementary material, which is available to authorized users.

Oncogenic effects of urotensin-II in cells lacking tuberous sclerosis complex-2.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease that can arise sporadically or in adults suffering from the tumor syndrome tuberous sclerosis complex (TSC). Microscopic tumors (‘LAM nodules’) in the lung interstitium arise from lymphatic invasion and metastasis. These consist of smooth muscle-like cells (LAM cells) that exhibit markers of neural crest differentiation and loss of the tumor suppressor protein ‘tuberous sclerosis complex-2’ (TSC2). Consistent with a neural phenotype, expression of the neuropeptide urotensin-II and its receptor was detected in LAM nodules. We hypothesized that loss of TSC2 sensitizes cells to the oncogenic effects of urotensin-II. TSC2-deficient Eker rat uterine leiomyoma ELT3 cells were stably transfected with empty vector or plasmid for the expression of TSC2. Urotensin-II increased cell viability and proliferation in TSC2-deficient cells, but not in TSC2-reconstituted cells. When exposed to urotensin-II, TSC2-deficient cells exhibited greater migration, anchorage-independent cell growth, and matrix invasion. The effects of urotensin-II on TSC2-deficient cells were blocked by the urotensin receptor antagonist SB657510, and accompanied by activation of Erk mitogen-activated protein kinase and focal adhesion kinase. Urotensin-II-induced proliferation and migration were reproduced in TSC2-deficient human angiomyolipoma cells, but not in those stably expressing TSC2. In a mouse xenograft model, SB657510 blocked the growth of established ELT3 tumors, reduced the number of circulating tumor cells, and attenuated the production of VEGF-D, a clinical biomarker of LAM. Urotensin receptor antagonists may be selective therapeutic agents for the treatment of LAM or other neural crest-derived neoplasms featuring loss of TSC2 or increased expression of the urotensin receptor.

Abstract 58: Metformin impacts IGF-1R/ Akt signaling and energy metabolism in lung adenocarcinoma

Abstract Introduction: Clinical outcomes for type II diabetes patients diagnosed with NSCLC are superior for those receiving metformin relative to other diabetic medications, though the mechanism for this effect remains incompletely elucidated. We hypothesized that metformin induces changes in Akt-based signaling via AMPK that increases glycolytic flux, and adversely affects survival in cell-line models of lung adenocarcinoma. The objective for these studies is to investigate therapeutic strategies involving metformin that have the potential to synergize with strategies targeting IGF-1R/Akt signaling. Methods: The lung adenocarcinoma cell lines, A549 and H358, were treated with 0, 1, 5, 10 mM metformin for 24, 48, and 72 hours and evaluated for changes in expression and activity of IGF-1R/Akt signaling, as well as the expression of 17 enzymes involved in glycolytic and oxidative energy metabolism, all using Luminex® immunobead assays. In parallel, MTT assays were performed for up to 5 days to document changes in cellular proliferation/ death rates. Extracellular acidification was also monitored as a measure of glycolytic flux. All findings were collated and statistically evaluated using SPSS v19 (Chicago, IL) using independent t-tests or one-way ANOVA. Results: We observed that metformin decreased IGF-1R/Akt signaling while increasing the expression of the protein levels of the signaling intermediates. Treatment with metformin increased the expression of Akt, IRS-1, PTEN, GSK3alpha and TSC2 (p&amp;lt;0.05 for all). Further, metformin treatment decreased the mean ratio of phospho-/total pyruvate dehydrogenase and increased the expression of ATP Synthase (both p&amp;lt;0.05), and were associated (p&amp;lt;0.05) with a dose-dependent increase in extracellular acidification. Lower doses (0.1 mM) of metformin did not have a significant impact on cell proliferation at 5 days, but a dose-dependent decrease in cell numbers was observed, with 37% and 29% reduction in cell numbers documented (p&amp;lt;0.05) at 5 mM for the H358 and A549 cells, respectively. Conclusion: This study links changes with Akt signaling with changes in glycolytic flux and oxidative metabolism. This study suggests metformin may make an attractive adjunct for advanced NSCLC patients receiving therapy that mechanistically involves targeting Akt signaling and metabolic control, such as IGF1R inhibition. Citation Format: Gabriela C. Lobato, Steve Li, Imad Tarhoni, Wen-Rong Lie, Jeffrey A. Borgia. Metformin impacts IGF-1R/ Akt signaling and energy metabolism in lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 58.