Activation of an oncogenic TBC1D7 (TBC1 domain family,member 7) protein in pulmonary carcinogenesis

To develop novel biomarkers and therapeutic agents for lung cancers, we screened molecules that were highly expressed in lung cancers by means of cDNA microarray analysis and found an elevated expression of TBC1 domain family, member 7 (TBC1D7) in the majority of lung cancers. Northern‐blot analysis using mRNAs from 16 normal tissues detected its expression only in testis. Immunohistochemical staining using tumor tissue microarrays consisting of 261 archived non‐small cell lung cancer (NSCLC) specimens suggested an association of TBC1D7 expression with poor prognosis for NSCLC patients (P = 0.0063). Treatment of lung cancer cells using siRNA against TBC1D7, suppressed its expression and resulted in inhibition of the cell growth. Furthermore, the induction of exogenous expression of TBC1D7 conferred growth‐promoting activity at in vitro and in vivo conditions. We also identified TBC1D7 to interact with TSC1 protein in lung cancer cells. TSC1 introduction into cells increased the level of TBC1D7 protein, whereas knockdown of TSC1 expression decreased the level of TBC1D7 protein, suggesting that TBC1D7 is stabilized probably through interaction with TSC1. In addition, inhibition of the binding between TBC1D7 and TSC1 by a TBC1D7‐derived 20‐amino acid cell‐permeable peptide (11R‐TBC1D7_152‐171), which corresponded to the binding domain to TSC1, effectively suppressed growth of lung cancer cells. Selective suppression of TBC1D7 and/or inhibition of the TBC1D7‐TSC1 complex formation could be promising therapeutic strategies for lung cancer therapy. © 2010 Wiley‐Liss, Inc.     

Biallelic loss‐of‐function variants in TBC1D2B cause a neurodevelopmental disorder with seizures and gingival overgrowth

The family of Tre2‐Bub2‐Cdc16 (TBC)‐domain containing GTPase activating proteins (RABGAPs) is not only known as key regulatorof RAB GTPase activity but also has GAP‐independent functions. Rab GTPases are implicated in membrane trafficking pathways, such as vesicular trafficking. We report biallelic loss‐of‐function variants in TBC1D2B, encoding a member of the TBC/RABGAP family with yet unknown function, as the underlying cause of cognitive impairment, seizures, and/or gingival overgrowth in three individuals from unrelated families. TBC1D2B messenger RNA amount was drastically reduced, and the protein was absent in fibroblasts of two patients. In immunofluorescence analysis, ectopically expressed TBC1D2B colocalized with vesicles positive for RAB5, a small GTPase orchestrating early endocytic vesicle trafficking. In two independent TBC1D2B CRISPR/Cas9 knockout HeLa cell lines that serve as cellular model of TBC1D2B deficiency, epidermal growth factor internalization was significantly reduced compared with the parental HeLa cell line suggesting a role of TBC1D2B in early endocytosis. Serum deprivation of TBC1D2B‐deficient HeLa cell lines caused a decrease in cell viability and an increase in apoptosis. Our data reveal that loss of TBC1D2B causes a neurodevelopmental disorder with gingival overgrowth, possibly by deficits in vesicle trafficking and/or cell survival.     

Bcat1 is controlled by Tsc2/mTORC1 pathway at expression levels and its deficiency together with Bcat2 inactivation suppresses the growth of a Tsc2−/− tumor cell line

The tuberous sclerosis complex (TSC) gene products (TSC1/TSC2) negatively regulate mTORC1. Although mTORC1 inhibitors are used for the treatment of TSC, incomplete tumor elimination and the adverse effects from long-term administration are problems that need to be solved. Branched-chain amino acid (BCAA) metabolism is involved in the growth of many tumor cells via the mTORC1 pathway. However, it remains unclear how BCAA metabolism affects the growth of mTORC1-dysregulated tumors. We show here that the expression of branched-chain amino transferase1 (Bcat1) was suppressed in Tsc2-deficient murine renal tumor cells either by treatment with rapamycin or restoration of Tsc2 expression suggesting that Bcat1 is located downstream of Tsc2-mTORC1 pathway. We also found that gabapentin, a Bcat1 inhibitor suppressed the growth of Tsc2-deficient tumor cells and increased efficacy when combined with rapamycin. We investigate the functional importance of Bcat1 and the mitochondrial isoform Bcat2 by inhibiting each enzyme separately or both together by genome editing and shRNA in Tsc2-deficient cells. We found that deficiency of both enzymes, but not either alone, inhibited cell growth, indicating that BCAA-metabolic reactions support Tsc2-deficient cell proliferation. Our results indicate that inhibition of Bcat1 and Bcat2 by specific drugs should be a useful method for TSC treatment.     

A Dominant Mutation in the Stereocilia‐Expressing Gene TBC1D24 is a Probable Cause for Nonsyndromic Hearing Impairment

Mutations in TBC1D24 have been linked to a variety of epileptic syndromes and recently to syndromic hearing impairment DOORS syndrome and nonsyndromic hearing impairment DFNB86. All TBC1D24 mutations reported so far were inherited in the recessive mode. In a dominant family segregated with late‐onset, progressive, nonsyndromic hearing impairment, linkage analysis revealed a 2.07 Mb candidate region on chromosome 16p13.3 that contains TBC1D24. Whole‐exome sequencing identified a heterozygous p.Ser178Leu variant of TBC1D24 as the only candidate mutation segregating with the hearing loss within the family. In perinatal mouse cochlea, we detected a restricted expression of Tbc1d24 in the stereocilia of the hair cells as well as in the spiral ganglion neurons. Our study suggested that the p.Ser178Leu mutation of TBC1D24 is a probable cause for dominant, nonsyndromic hearing impairment. Identification of TBC1D24 as the stereocilia‐expressing gene may shed new light on its specific function in the inner ear.     

Mammalian target of rapamycin (mTOR) complex 2 regulates filamin A‐dependent focal adhesion dynamics and cell migration

The serine/threonine kinase mTOR forms two distinct complexes, mTORC1 and mTORC2, and controls a number of biological processes, including proliferation, survival and autophagy. Although the function of mTORC1 has been extensively studied, the mTORC2 signaling pathway largely remains to be elucidated. Here, we have shown that mTORC2 phosphorylates filamin A, an actin cross‐linking protein, at serine 2152 (S2152) both in vivo and in living cells. Treatment of HeLa cells with Torin1 (an mTORC1/mTORC2 inhibitor), but not rapamycin (an mTORC1 inhibitor), suppressed the phosphorylation of filamin A, which decreased the binding of filamin A with β7‐integrin cytoplasmic tail. Torin1 also inhibited focal adhesion formation and cell migration in A7 filamin A‐replete melanoma cells but not in M2 filamin A‐deficient cells, suggesting a pivotal role for mTORC2 in filamin A function. Finally, reduced focal adhesion formation in M2 cells was significantly rescued by expressing wild type but not S2152A nonphosphorylatable mutant of filamin A. Taken together, our results indicate that mTORC2 regulates filamin A‐dependent focal adhesions and cell migration.     

TBC1D24 Mutation Causes Autosomal‐Dominant Nonsyndromic Hearing Loss

Hereditary hearing loss is extremely heterogeneous. Over 70 genes have been identified to date, and with the advent of massively parallel sequencing, the pace of novel gene discovery has accelerated. In a family segregating progressive autosomal‐dominant nonsyndromic hearing loss (NSHL), we used OtoSCOPE® to exclude mutations in known deafness genes and then performed segregation mapping and whole‐exome sequencing to identify a unique variant, p.Ser178Leu, in TBC1D24 that segregates with the hearing loss phenotype. TBC1D24 encodes a GTPase‐activating protein expressed in the cochlea. Ser178 is highly conserved across vertebrates and its change is predicted to be damaging. Other variants in TBC1D24 have been associated with a panoply of clinical symptoms including autosomal recessive NSHL, syndromic hearing impairment associated with onychodystrophy, osteodystrophy, mental retardation, and seizures (DOORS syndrome), and a wide range of epileptic disorders.     

TSC1 involvement in bladder cancer: diverse effects and therapeutic implications

TSC1 is often mutated in bladder cancer. However the importance of this event in disease pathogenesis and its implications for therapy are uncertain. We used genomic sequencing to examine the involvement of TSC1 in bladder cancer, and signalling pathway analysis and small‐molecule screening to identify targeted therapeutic strategies in TSC1 mutant bladder cancer cell lines. TSC1 loss of heterozygosity was seen in 54% of bladder cancers. Two (4.9%) of these 41 bladder cancers had TSC1 mutations by exon‐based sequencing. Analysis of 27 bladder cancer cell lines demonstrated inactivating TSC1 mutations in three: RT‐4, HCV29, 97–1. Interestingly, only RT‐4 showed classic feedback inhibition of AKT, and was highly sensitive to treatment with mTOR inhibitors rapamycin and Torin1. 97–1 cells showed constitutive EGFR activation, and were highly sensitive to combined treatment with the mTOR inhibitor Torin1 and EGFR inhibitors Lapatinib or Afatinib. A BRAF missense mutation G469V was found in HCV29 cells, and AKT activation was dependent on BRAF, but independent of ERK. A kinase inhibitor screen of HCV29 cells showed strong growth inhibition by the Hsp90 inhibitor NVP‐AUY922, and we then found synergistic inhibitory effects of NVP‐AUY922 combined with either Torin1 or rapamycin on cell survival for both HCV29 and 97–1 cells. In aggregate, these findings indicate that there are highly variable mutation profiles and signalling pathway activation in TSC1‐mutant bladder cancer. Furthermore, combined Hsp90/mTOR inhibition is a promising therapeutic approach for TSC1 mutant bladder cancer. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Shedding new light on neurodegenerative diseases through the mammalian target of rapamycin

Neurodegenerative disorders affect a significant portion of the world's population leading to either disability or death for almost 30 million individuals worldwide. One novel therapeutic target that may offer promise for multiple disease entities that involve Alzheimer's disease, Parkinson's disease, epilepsy, trauma, stroke, and tumors of the nervous system is the mammalian target of rapamycin (mTOR). mTOR signaling is dependent upon the mTORC1 and mTORC2 complexes that are composed of mTOR and several regulatory proteins including the tuberous sclerosis complex (TSC1, hamartin/TSC2, tuberin). Through a number of integrated cell signaling pathways that involve those of mTORC1 and mTORC2 as well as more novel signaling tied to cytokines, Wnt, and forkhead, mTOR can foster stem cellular proliferation, tissue repair and longevity, and synaptic growth by modulating mechanisms that foster both apoptosis and autophagy. Yet, mTOR through its proliferative capacity may sometimes be detrimental to central nervous system recovery and even promote tumorigenesis. Further knowledge of mTOR and the critical pathways governed by this serine/threonine protein kinase can bring new light for neurodegeneration and other related diseases that currently require new and robust treatments.     

RagA,an mTORC1 activator,interacts with a hedgehog signaling protein,WDR35/IFT121

Small Ras‐like GTPases act as molecular switches for various signal transduction pathways. RagA, RagB/RagC and RagD are small Ras‐like GTPases that play regulatory roles in mTORC1. Lack of proper activation of mTORC1 can lead to diseases, such as cancer and diabetes. In this study, we found an interaction between RagA and WDR35. Mutations of WDR35 may cause genetic diseases including Sensenbrenner syndrome. WDR35 seems to be a hedgehog signaling protein with a possible ciliary function and a possible upstream regulator of RagA. RagB is a homologue of RagA and is also associated with WDR35. WDR35 is present in the endoplasmic reticulum, but usually not in lysosomes, where Rag family proteins act as an mTORC1 switch. Over‐expression of WDR35 results in decreased phosphorylation of ribosome S6 protein in a RagA‐, RagB‐ and RagC‐dependent manner. Thus, WDR35 is associated with RagA, RagB and RagC and might negatively influence mTORC1 activity.     

Novel Compound Heterozygous Mutations in TBC1D24 Cause Familial Malignant Migrating Partial Seizures of Infancy

Early‐onset epileptic encephalopathies (EOEEs) are a group of rare devastating epileptic syndromes of infancy characterized by severe drug‐resistant seizures and electroencephalographic abnormalities. The current study aims to determine the genetic etiology of a familial form of EOEE fulfilling the diagnosis criteria for malignant migrating partial seizures of infancy (MMPSI). We identified two inherited novel mutations in TBC1D24 in two affected siblings. Mutations severely impaired TBC1D24 expression and function, which is critical for maturation of neuronal circuits. The screening of TBC1D24 in an additional set of eight MMPSI patients was negative. TBC1D24 loss of function has been associated to idiopathic infantile myoclonic epilepsy, as well as to drug‐resistant early‐onset epilepsy with intellectual disability. Here, we describe a familial form of MMPSI due to mutation in TBC1D24, revealing a devastating epileptic phenotype associated with TBC1D24 dysfunction.     

Suppressed Expression of Autophagosomal Protein LC3 in Cortical Tubers of Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is characterized by benign tumors and hamartomas, including cortical tubers. Hamartin and tuberin, encoded by the TSC 1 and 2 genes, respectively, constitute a functional complex that negatively regulates the mammalian target of rapamycin (mTOR) signaling pathway, eventually promoting the induction of autophagy. In the present study, we assessed the induction of autophagy in cortical tubers surgically removed from seven patients with TSC in comparison with five controls of cortical tissue taken from non‐TSC patients with epilepsy. Immunoblotting demonstrated a marked reduction of LC3B‐I and LC3B‐II in tubers relative to the controls. In tubers, strong, diffuse and dot‐like immunoreactivity (IR) for LC3B was observed in dysmorphic neurons and balloon cells, but LC3B‐IR in other neurons with normal morphology was significantly weaker than that in neurons in the controls. Immunoelectron microscopy revealed diffuse distribution of LC3B‐IR within the cytoplasm of balloon cells. The dot‐like pattern may correspond to abnormal aggregation bodies involving LC3. In an autopsy patient with TSC, we observed that LC3B‐IR in neurons located outside of the tubers was preserved. Thus, autophagy is suppressed in tubers presumably through the mTOR pathway, and possibly a pathological autophagy reaction occurs in the dysmorphic neurons and balloon cells.     

Ciliary Genes TBC1D32/C6orf170 and SCLT1 are Mutated in Patients with OFD Type IX

Clinical syndromes caused by defects in the primary cilium are heterogeneous but there are recurrent phenotypic manifestations that define them as a collective group known as ciliopathies. Dozens of genes have been linked to various ciliopathies but large patient cohorts have clearly revealed the existence of additional genetic heterogeneity, which is yet to be fully appreciated. In our search for novel ciliopathy‐linked genes through the study of unmapped ciliopathy phenotypes, we have identified two simplex cases with a severe ciliopathy phenotype consistent with oro‐facio‐digital syndrome type IX featuring midline cleft, microcephaly, and colobomatous microphathalmia/anophthalmia. In addition, there was variable presence of polydactyly, absent pituitary, and congenital heart disease. The autozygome of each index harbored a single novel truncating variant as revealed by exome sequencing, and the affected genes (SCLT1 and TBC1D32/C6orf170) have established roles in centrosomal biology and ciliogenesis. Our findings suggest a previously unrecognized role of SCLT1 and TBC1D32 in the pathogenesis of ciliopathy in humans.     

Diagnosis of TBC1D32-associated conditions: Expanding the phenotypic spectrum of a complex ciliopathy

Exome sequencing is a powerful tool in prenatal and postnatal genetics and can help identify novel candidate genes critical to human development. We describe seven unpublished probands with rare likely pathogenic variants or variants of uncertain significance that segregate with recessive disease in TBC1D32, including four fetal probands in three unrelated pedigrees and three pediatric probands in unrelated pedigrees. We also report clinical comparisons with seven previously published patients. Index probands were identified through an ongoing prenatal exome sequencing study and through an online data sharing platform (Gene Matcher™). A literature review was also completed. TBC1D32 is involved in the development and function of cilia and is expressed in the developing hypothalamus and pituitary gland. We provide additional data to expand the phenotype correlated with TBC1D32 variants, including a severe prenatal phenotype associated with life-limiting congenital anomalies.     

Sclerosing TSC1 mutated renal cell carcinoma: An unusual pattern mimicking MITF family translocation renal cell carcinoma

The tuberous sclerosis genes and MTOR are increasingly being found to have important roles in novel subtypes of renal cancer, particularly emerging entities eosinophilic solid and cystic renal cell carcinoma (RCC) and high‐grade oncocytic renal tumor (HOT)/RCC with eosinophilic and vacuolated cytoplasm. We report a unique renal neoplasm in a 66‐year‐old woman that initially mimicked MITF family translocation RCC due to mixed clear and eosinophilic cells, extensive stromal hyalinization, and psammoma bodies, yet which was negative for TFE3 and TFEB fluorescence in situ hybridization and a next generation sequencing (NGS) gene fusion assay. Cytoplasmic stippling triggered consideration of TSC‐associated neoplasms, and a targeted NGS assay revealed a variant in exon 21 of TSC1 resulting in c.2626G>T p.(Glu876*) truncating mutation. This report adds to the morphologic spectrum of TSC‐related renal neoplasms, including prominent stromal hyalinization as a potentially deceptive pattern. Due to the overlap in cytoplasmic stippling between eosinophilic solid and cystic RCC and HOT/RCC with eosinophilic and vacuolated cytoplasm, it is debatable which category this example would best fit. Further understanding of these entities and other renal neoplasms with alterations in the TSC genes will elucidate whether they should be considered a family of tumors.     

DAPK1 (death associated protein kinase 1) mediates mTORC1 activation and antiviral activities in CD8+ T cells

Mechanistic target of rapamycin complex 1 (mTORC1) regulates CD8⁺ T-cell differentiation and function. Despite the links between PI3K-AKT and mTORC1 activation in CD8⁺ T cells, the molecular mechanism underlying mTORC1 activation remains unclear. Here, we show that both the kinase activity and the death domain of DAPK1 are required for maximal mTOR activation and CD8⁺ T-cell function. We found that TCR-induced activation of calcineurin activates DAPK1, which subsequently interacts with TSC2 via its death domain and phosphorylates TSC2 to mediate mTORC1 activation. Furthermore, both the kinase domain and death domain of DAPK1 are required for CD8⁺ T-cell antiviral responses in an LCMV infection model. Together, our data reveal a novel mechanism of mTORC1 activation that mediates optimal CD8⁺ T-cell function and antiviral activity.     

A Role for the Chromatin‐Remodeling Factor BAZ1A in Neurodevelopment

Chromatin‐remodeling factors are required for a wide range of cellular and biological processes including development and cognition, mainly by regulating gene expression. As these functions would predict, deregulation of chromatin‐remodeling factors causes various disease syndromes, including neurodevelopmental disorders. Recent reports have linked mutations in several genes coding for chromatin‐remodeling factors to intellectual disability (ID). Here, we used exome sequencing and identified a nonsynonymous de novo mutation in BAZ1A (NM_182648.2:c.4043T > G, p.Phe1348Cys), encoding the ATP‐utilizing chromatin assembly and remodeling factor 1 (ACF1), in a patient with unexplained ID. ACF1 has been previously reported to bind to the promoter of the vitamin D receptor (VDR)‐regulated genes and suppress their expression. Our results show that the patient displays decreased binding of ACF1 to the promoter of the VDR‐regulated gene CYP24A1. Using RNA sequencing, we find that the mutation affects the expression of genes involved in several pathways including vitamin D metabolism, Wnt signaling and synaptic formation. RNA sequencing of BAZ1A knockdown cells and Baz1a knockout mice revealed that BAZ1A carry out distinctive functions in different tissues. We also demonstrate that BAZ1A depletion influence the expression of genes important for nervous system development and function. Our data point to an important role for BAZ1A in neurodevelopment, and highlight a possible link for BAZ1A to ID.     

Variant ATRX Syndrome with Dysfunction of ATRX and MAGT1 Genes

A 0.8kb intronic duplication in MAGT1 and a single base pair deletion in the last exon of ATRX were identified using a chromosome X‐specific microarray and exome sequencing in a family with five males demonstrating intellectual disability (ID) and unusual skin findings (e.g., generalized pruritus). MAGT1 is an Mg²⁺ transporter previously associated with primary immunodeficiency and ID, whereas mutations in ATRX cause ATRX‐ID syndrome. In patient cells, the function of ATRX was demonstrated to be abnormal based on altered RNA/protein expression, hypomethylation of rDNA, and abnormal cytokinesis. Dysfunction of MAGT1 was reflected in reduced RNA/protein expression and Mg²⁺ influx. The mutation in ATRX most likely explains the ID, whereas MAGT1 disruption could be linked to abnormal skin findings, as normal magnesium homeostasis is necessary for skin health. This work supports observations that multiple mutations collectively contribute to the phenotypic variability of syndromic ID, and emphasizes the importance of correlating clinical phenotype with genomic and cell function analyses.     

Autophagy is involved in regulating the immune response of dendritic cells to influenza A (H1N1) pdm09 infection

Autophagy can mediate antiviral immunity. However, it remains unknown whether autophagy regulates the immune response of dendritic cells (DCs) to influenza A (H1N1) pdm09 infection. In this study, we found that infection with the H1N1 virus induced DC autophagy in an endocytosis‐dependent manner. Compared with autophagy‐deficient Beclin‐1^+/? mice, we found that bone‐marrow‐derived DCs from wild‐type mice (WT BMDCs) presented a more mature phenotype on H1N1 infection. Wild‐type BMDCs secreted higher levels of interleukin‐6 (IL‐6), tumour necrosis factor‐ α (TNF‐α), interferon‐β (IFN‐β), IL‐12p70 and IFN‐γ than did Beclin‐1^+/? BMDCs. In contrast to Beclin‐1^+/? BMDCs, H1N1‐infected WT BMDCs exhibited increased activation of extracellular signal‐regulated kinase, Jun N‐terminal kinase, p38, and nuclear factor‐κB as well as IFN regulatory factor 7 nuclear translocation. Blockade of autophagosomal and lysosomal fusion by bafilomycin A1 decreased the co‐localization of H1N1 viruses, autophagosomes and lysosomes as well as the secretion of IL‐6, TNF‐α and IFN‐β in H1N1‐infected BMDCs. In contrast to Beclin‐1^+/? BMDCs, H1N1‐infected WT BMDCs were more efficient in inducing allogeneic CD4⁺ T‐cell proliferation and driving T helper type 1, 2 and 17 cell differentiation while inhibiting CD4⁺ Foxp3⁺ regulatory T‐cell differentiation. Moreover, WT BMDCs were more efficient at cross‐presenting the ovalbumin antigen to CD8⁺ T cells. We consistently found that Beclin‐1^+/? BMDCs were inferior in their inhibition of H1N1 virus replication and their induction of H1N1‐specific CD4⁺ and CD8⁺ T‐cell responses, which produced lower levels of IL‐6, TNF‐α and IFN‐β in vivo. Our data indicate that autophagy is important in the regulation of the DC immune response to H1N1 infection, thereby extending our understanding of host immune responses to the virus.     

Micro and Martsolf syndromes in 34 new patients: Refining the phenotypic spectrum and further molecular insights

Micro and Martsolf syndromes are rare clinically and genetically overlapping disorders caused by mutations in RAB3GAP1, RAB3GAP2, RAB18 and TBC1D20 genes. We describe 34 new patients, 27 with Micro and seven with Martsolf. Patients presented with the characteristic clinical manifestations of the two syndromes, including postnatal microcephaly, congenital cataracts, microphthalmia, optic atrophy, spasticity and intellectual disability. Brain imaging showed in the majority of cases polymicrogyria, thin corpus callosum, cortical atrophy, and white matter dysmyelination. Unusual additional findings were pectus excavatum (four patients), pectus carinatum (three patients), congenital heart disease (three patients) and bilateral calcification in basal ganglia (one patient). Mutational analysis of RAB3GAP1 and RAB3GAP2 revealed 21 mutations, including 14 novel variants. RAB3GAP1 mutations were identified in 22 patients with Micro, including a deletion of the entire gene in one patient. On the other hand, RAB3GAP2 mutations were identified in two patients with Micro and all Martsolf patients. Moreover, exome sequencing unraveled a TBC1D20 mutation in an additional family with Micro syndrome. Our results expand the phenotypic and mutational spectrum associated with Micro and Martsolf syndromes. Due to the overlapped severities and genetic basis of both syndromes, we suggest to be comprehended as one entity “Micro/Martsolf spectrum” or “RAB18 deficiency.”