bims-tubesc 2022-10-30 papers

bims-tubesc

Biomed News

on Molecular mechanisms in tuberous sclerosis

Issue of 2022–10–30
eightteen papers selected by

Management of Renal Angiomyolipomas in Tuberous Sclerosis Complex: A Case Report and Literature Review.
Tsc2 shapes olfactory bulb granule cell molecular and morphological characteristics.
Macrodactyly as a rare manifestation of tuberous sclerosis.
Expanding Our Knowledge of Menstrual Irregularities Reported by Females With Tuberous Sclerosis Complex.
Tuberous Sclerosis: A Rare Disease with an Orphan Complex.
Satisfaction and seizure outcomes of epilepsy surgery in tuberous sclerosis: A Swedish population-based long-term follow-up study.
Downregulated formyl peptide receptor 2 expression in the epileptogenic foci of patients with focal cortical dysplasia type IIb and tuberous sclerosis complex.
Analysis of current data on the use of topical mTOR inhibitors in the treatment of facial angiofibromas in Tuberous Sclerosis Complex - An Update.
mTORC1 beyond anabolic metabolism: Regulation of cell death.
Insulin-induced mTOR signaling and gluconeogenesis in renal proximal tubules: A mini-review of current evidence and therapeutic potential.
GATOR2-dependent mTORC1 activity is a therapeutic vulnerability in FOXO1 fusion positive rhabdomyosarcoma.
Extracellular vesicles derived from endometrial epithelial cells deliver exogenous miR-92b-3p to affect the function of embryonic trophoblast cells via targeting TSC1 and DKK3.
mTOR as a Potential Target for the Treatment of Microbial Infections, Inflammatory Bowel Diseases, and Colorectal Cancer.
mTOR Contributes to the Proteome Diversity through Transcriptome-Wide Alternative Splicing.
A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling.
The NRF2 antagonist ML385 inhibits PI3K-mTOR signaling and growth of lung squamous cell carcinoma cells.
Dual roles of mTORC1-dependent activation of the ubiquitin-proteasome system in muscle proteostasis.
Expression of AKT1 Related with Clinicopathological Parameters in Clear Cell Renal Cell Carcinoma.

J Clin Med. 2022 Oct 15. pii: 6084. [Epub ahead of print]11(20):

Management of Renal Angiomyolipomas in Tuberous Sclerosis Complex: A Case Report and Literature Review.

Mitchell Hunter-Dickson, Patrick Wu, Akshay Athavale, Amanda Ying Wang.

  We report a case of misdiagnosed tuberous sclerosis complex (TSC) in a patient without TSC gene variant presenting with bilateral renal angiomyolipomas and seizures in the context of strong family history of polycystic kidney disease. Clinical diagnosis of tuberous sclerosis complex was made and treatment with everolimus reduced size of renal angiomyolipomas. In this case, report we discuss the association between tuberous sclerosis complex and polycystic kidney disease and novel treatment for TSC.

Keywords:  angiomyolipoma; cystic kidney disease; everolimus; genetics; tuberous sclerosis

DOI:  https://doi.org/10.3390/jcm11206084
Front Mol Neurosci. 2022 ;15 970357

Tsc2 shapes olfactory bulb granule cell molecular and morphological characteristics.

Victoria A Riley, Jennie C Holmberg, Aidan M Sokolov, David M Feliciano.

  Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations that inactivate TSC1 or TSC2. Hamartin and tuberin are encoded by TSC1 and TSC2 which form a GTPase activating protein heteromer that inhibits the Rheb GTPase from activating a growth promoting protein kinase called mammalian target of rapamycin (mTOR). Growths and lesions occur in the ventricular-subventricular zone (V-SVZ), cortex, olfactory tract, and olfactory bulbs (OB) in TSC. A leading hypothesis is that mutations in inhibitory neural progenitor cells cause brain growths in TSC. OB granule cells (GCs) are GABAergic inhibitory neurons that are generated through infancy by inhibitory progenitor cells along the V-SVZ. Removal of Tsc1 from mouse OB GCs creates cellular phenotypes seen in TSC lesions. However, the role of Tsc2 in OB GC maturation requires clarification. Here, it is demonstrated that conditional loss of Tsc2 alters GC development. A mosaic model of TSC was created by performing neonatal CRE recombinase electroporation into inhibitory V-SVZ progenitors yielded clusters of ectopic cytomegalic neurons with hyperactive mTOR complex 1 (mTORC1) in homozygous Tsc2 mutant but not heterozygous or wild type mice. Similarly, homozygous Tsc2 mutant GC morphology was altered at postnatal days 30 and 60. Tsc2 mutant GCs had hypertrophic dendritic arbors that were established by postnatal day 30. In contrast, loss of Tsc2 from mature GCs had negligible effects on mTORC1, soma size, and dendrite arborization. OB transcriptome profiling revealed a network of significantly differentially expressed genes following loss of Tsc2 during development that altered neural circuitry. These results demonstrate that Tsc2 has a critical role in regulating neural development and shapes inhibitory GC molecular and morphological characteristics.

Keywords:  granule cell; mammalian target of rapamycin (mTOR); olfactory bulb (OB); tuberous sclerosis complex (TSC); tuberous sclerosis complex 1 (TSC1); tuberous sclerosis complex 2 (TSC2)

DOI:  https://doi.org/10.3389/fnmol.2022.970357
Pediatr Neonatol. 2022 Oct 13. pii: S1875-9572(22)00216-9. [Epub ahead of print]

Macrodactyly as a rare manifestation of tuberous sclerosis.

Eduardo Bandeira.

DOI: https://doi.org/10.1016/j.pedneo.2022.01.011
Front Reprod Health. 2022 ;4 798983

Expanding Our Knowledge of Menstrual Irregularities Reported by Females With Tuberous Sclerosis Complex.

Kate Mowrey, Hope Northrup, Syed Shahrukh Hashmi, David Rodriguez-Buritica.

   Purpose: The purpose of our study is to expand the knowledge regarding intrinsic reproductive dysfunction in females with TSC and to explore the impact of mTOR inhibitors (mTORi) on menstrual irregularity in the Tuberous Sclerosis Complex (TSC) community.
Methods: An electronic survey composed of author-designed questions set out to evaluate reproductive history, presence of menstrual irregularities, mTORi use, as well as maternal reproductive history among females with TSC.
Results: Of the 68 responses from females with TSC regarding age of menarche, the average age was 12.3 years. 56.5% (n = 48) of respondents reported irregular menstrual cycles and noted a total of 102 menstrual irregularities. There was a cohort of 35 women with a reported history of mTORi use. Of these women, 68.6% (n = 24) reported irregular menstrual cycles after taking mTORi. In comparison, among the females with no history of mTORi use (n = 50) only 48% reported irregular menstrual cycles (n = 24).
Conclusions: Our data expands the knowledge regarding intrinsic menstrual dysregulation present in women with TSC, demonstrates a rate of menstrual irregularities among females taking mTORi, and identifies a tendency toward early menarche that may be a previously unrecognized feature of TSC.

Keywords:  mTOR dysregulation; mTOR inhibitors; menstrual irregularity; reproductive health; tuberous sclerosis complex

DOI:  https://doi.org/10.3389/frph.2022.798983
Ir Med J. 2022 08 18. 115(7): 635

Tuberous Sclerosis: A Rare Disease with an Orphan Complex.

C Behan, R Davis, M Vasseghi, P Moloney, S Amin, N Delanty, C P Doherty.

Introduction In the Republic of Ireland, there are no tuberous sclerosis complex (TSC) specialist clinics. Methods A clinical audit was carried out to assess the care received by patients attending two specialist adult epilepsy specialist centres, measuring their care against the UK guidelines. Results Although many baseline investigations are carried out, only one-third of patients had diagnostic genetic testing results available. Neuropsychiatry is largely neglected, and the completion of neuropsychiatric assessments checklists is inadequate. Discussions concerning SUDEP are not happening and access to treatment is limited. Reporting of radiological findings in TSC is inconsistent and the number of adults with TSC accessing specialist epilepsy services appear to be low. Discussion TSC care in the Republic of Ireland is fragmented, difficult to navigate and wasteful of resources due to the complex nature of the disease and no formal clinical setting to manage it. The service gaps echo the demand for an improved care system including consistent radiological reporting of TSC pathology. The absence of a specialist TSC clinic compounds the complexity of navigating care for individuals with TSC, families and healthcare professionals. Extending this audit nationally will give a more complete picture and highlight the resources required to bring care of these patients in line with recommended guidelines.

Keywords: Tuberous Sclerosis
Seizure. 2022 Oct 10. pii: S1059-1311(22)00237-0. [Epub ahead of print]103 39-45

Satisfaction and seizure outcomes of epilepsy surgery in tuberous sclerosis: A Swedish population-based long-term follow-up study.

Kevin Pearsson, Maria Compagno-Strandberg, Erik A Eklund, Olof Rask, Kristina Källén.

   PURPOSE: We conducted a cross-sectional study to evaluate long-term outcomes of epilepsy surgery in tuberous sclerosis complex (TSC) in a Swedish population.
METHODS: Demographic and seizure data was retrieved from the Swedish National Epilepsy Surgery Registry and medical records. Patient reported outcome measurements (PROM) were determined by telephonic interviews at long term follow-up.
RESULTS: Median follow-up was 6 y 8 m (range, 3-15 y 1 m) for tuberectomies (n = 15) and 3 y 6 m (range 2-10 y) for callosotomies (n = 7). Eight of the 15 tuberectomy participants were seizure-free. Four out of seven callosotomies were free from drop attacks. PROMs were provided by caregivers of 18/20 participants (data missing for two callosotomies). In the tuberectomy group, 6/8 patients were seizure-free and 3/7 had continued seizures; surgery was considered satisfactory and beneficial. Overall, satisfaction was high, even among patients who did not achieve remission; 13/15 tuberectomy responders recommended surgery to others with TSC and refractory epilepsy. None of the patients considered the surgery harmful. In the callosotomy group, satisfaction was low and congruent with the seizure outcome. All patients with continued drop attacks were unsatisfied; one considered surgery to be harmful. One participant, who would not recommend surgery to others, still perceived the surgery to be beneficial.
CONCLUSIONS: This study confirmed that both tuberectomy and callosotomy are effective treatment options for TSC. Factors other than seizure outcomes seemed to have a major influence on satisfaction and perception of the benefit of surgery.

Keywords:  Cross-sectional studies; Drug resistant epilepsy; Epilepsy; Epilepsy surgery; Patient reported outcome measures; Tuberous sclerosis

DOI:  https://doi.org/10.1016/j.seizure.2022.10.011
Immun Inflamm Dis. 2022 Nov;10(11): e706

Downregulated formyl peptide receptor 2 expression in the epileptogenic foci of patients with focal cortical dysplasia type IIb and tuberous sclerosis complex.

Kaixuan Huang, Zhongke Wang, Zeng He, Yang Li, Shujing Li, Kaifeng Shen, Gang Zhu, Zhonghong Liu, Shengqing Lv, Chunqing Zhang, Hui Yang, Xiaolin Yang, Shiyong Liu.

   BACKGROUND: Focal cortical dysplasia type IIb (FCDIIb) and tuberous sclerosis complex (TSC) show persistent neuroinflammation, which promotes epileptogenesis and epilepsy progression, suggesting that endogenous resolution of inflammation is inadequate to relieve neuronal network hyperexcitability. To explore the potential roles of formyl peptide receptor 2 (FPR2), which is a key regulator of inflammation resolution, in epilepsy caused by FCDIIb and TSC, we examined the expression and cellular distribution of FPR2.
METHOD: The expression of FPR2 and nuclear factor-κB (NF-κB) signaling pathway was examined by real-time PCR, western blots, and analyzed via one-way analysis of variance. The distribution of FPR2 was detected using immunostaining. The expression of resolvin D1 (RvD1, the endogenous ligand of FPR2) was observed via enzyme-linked immunosorbent assay. Pearson's correlation test was used to evaluate the correlation between the expression levels of FPR2 and RvD1 and the clinical variants.
RESULTS: The expression of FPR2 was significantly lower in FCDIIb (p = .0146) and TSC (p = .0006) cortical lesions than in controls, as was the expression of RvD1 (FCDIIb: p = .00431; TSC: p = .0439). Weak FPR2 immunoreactivity was observed in dysmorphic neurons (DNs), balloon cells (BCs), and giant cells (GCs) in FCDIIb and TSC tissues. Moreover, FPR2 was mainly distributed in dysplastic neurons; it was sparse in microglia and nearly absent in astrocytes. The NF-κB pathway was significantly activated in patients with FCDIIb and TSC, and the protein level of NF-κB was negatively correlated with the protein level of FPR2 (FCDIIb: p = .00395; TSC: p = .0399). In addition, the protein level of FPR2 was negatively correlated with seizure frequency in FCDIIb (p = .0434) and TSC (p = .0351) patients.
CONCLUSION: In summary, these results showed that the expression and specific distribution of FPR2 may be involved in epilepsy caused by FCDIIb and TSC, indicating that downregulation of FPR2 mediated the dysfunction of neuroinflammation resolution in FCDIIb and TSC.

Keywords:  focal cortical dysplasia type IIb; formyl peptide receptor 2; inflammation resolution; resolvin D1; tuberous sclerosis complex

DOI:  https://doi.org/10.1002/iid3.706
J Eur Acad Dermatol Venereol. 2022 Oct 27.

Analysis of current data on the use of topical mTOR inhibitors in the treatment of facial angiofibromas in Tuberous Sclerosis Complex - An Update.

R Balestri, L Rizzoli, A Pedrolli, Sam Urru, G Rech, I Neri, C R Girardelli, M Magnano.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome causing hamartomatous growths in multiple organs. Facial angiofibromas occur in up to 80% of patients and can be highly disfiguring. Treatment for these lesions is challenging. Recently, topical rapamycin has been proposed as an effective option to treat angiofibromas but a commercially available compound has not yet been developed in Europe. We conducted a retrospective review with the aim to update the current data on the use of topical rapamycin in the treatment of angiofibromas in TSC, focusing on the optimal concentration and trying to establish which vehicle should be preferred. Thirty-nine reports describing the use of topical rapamycin in the treatment of angiofibromas in TSC were considered, involving a total of 483 patients. An improvement of the lesions has been shown in over 90% of subjects, particularly if the treatment was started at early stages. Several different formulations (ointment, gel, solution and cream) with a wide range of concentrations (0.003%-1%) were proposed, of which a pharmacological analysis has also been performed. Topical rapamycin can be considered an effective and safe option for the treatment and the prevention of facial angiofibromas in younger patients, but the best formulation has yet to be established. Our review demonstrates that ointment and gel should be preferred, but it is not clear which concentration is optimal. However, according to this study, the 0.1% concentration represents the first choice. Long-term and comparative studies between topical rapamycin formulations are required in order to establish which treatment has a better outcome and lower recurrence rate.

DOI: https://doi.org/10.1111/jdv.18693
J Cell Biol. 2022 Dec 05. pii: e202208103. [Epub ahead of print]221(12):

mTORC1 beyond anabolic metabolism: Regulation of cell death.

Jiajun Zhu, Hua Wang, Xuejun Jiang.

The mechanistic target of rapamycin complex 1 (mTORC1), a multi-subunit protein kinase complex, interrogates growth factor signaling with cellular nutrient and energy status to control metabolic homeostasis. Activation of mTORC1 promotes biosynthesis of macromolecules, including proteins, lipids, and nucleic acids, and simultaneously suppresses catabolic processes such as lysosomal degradation of self-constituents and extracellular components. Metabolic regulation has emerged as a critical determinant of various cellular death programs, including apoptosis, pyroptosis, and ferroptosis. In this article, we review the expanding knowledge on how mTORC1 coordinates metabolic pathways to impinge on cell death regulation. We focus on the current understanding on how nutrient status and cellular signaling pathways connect mTORC1 activity with ferroptosis, an iron-dependent cell death program that has been implicated in a plethora of human diseases. In-depth understanding of the principles governing the interaction between mTORC1 and cell death pathways can ultimately guide the development of novel therapies for the treatment of relevant pathological conditions.

DOI: https://doi.org/10.1083/jcb.202208103
Front Pharmacol. 2022 ;13 1015204

Insulin-induced mTOR signaling and gluconeogenesis in renal proximal tubules: A mini-review of current evidence and therapeutic potential.

Motonobu Nakamura, Nobuhiko Satoh, Shoko Horita, Masaomi Nangaku.

  Energy is continuously expended in the body, and gluconeogenesis maintains glucose homeostasis during starvation. Gluconeogenesis occurs in the liver and kidneys. The proximal tubule is the primary location for renal gluconeogenesis, accounting for up to 25% and 60% of endogenous glucose production during fasting and after a meal, respectively. The mechanistic target of rapamycin (mTOR), which exists downstream of the insulin pathway, plays an important role in regulating proximal tubular gluconeogenesis. mTOR is an atypical serine/threonine kinase present in two complexes. mTORC1 phosphorylates substrates that enhance anabolic processes such as mRNA translation and lipid synthesis and catabolic processes such as autophagy. mTORC2 regulates cytoskeletal dynamics and controls ion transport and proliferation via phosphorylation of SGK1. Therefore, mTOR signaling defects have been implicated in various pathological conditions, including cancer, cardiovascular disease, and diabetes. However, concrete elucidations of the associated mechanisms are still unclear. This review provides an overview of mTOR and describes the relationship between mTOR and renal.

Keywords:  diabetes; gluconeogenesis; insulin resistance; mTOR; proximal tubules

DOI:  https://doi.org/10.3389/fphar.2022.1015204
JCI Insight. 2022 Oct 25. pii: e162207. [Epub ahead of print]

GATOR2-dependent mTORC1 activity is a therapeutic vulnerability in FOXO1 fusion positive rhabdomyosarcoma.

Jacqueline Morales, David V Allegakoen, José A Garcia, Kristen Kwong, Pushpendra K Sahu, Drew A Fajardo, Yue Pan, Max A Horlbeck, Jonathan S Weissman, W Clay Gustafson, Trever G Bivona, Amit J Sabnis.

  Oncogenic FOXO1 gene fusions drive a subset of rhabdomyosarcoma (RMS) with poor survival and to date these cancer drivers are therapeutically intractable. To identify new therapies for this disease, we undertook an isogenic CRISPR-interference screen to define PAX3-FOXO1 specific genetic dependencies and identified genes in the GATOR2 complex. GATOR2 loss in RMS abrogated amino acid-induced lysosomal localization of mTORC1 and consequent downstream signaling, slowing G1-S cell cycle transition. In vivo suppression of GATOR2 impaired the growth of tumor xenografts and favored the outgrowth of cells lacking PAX3-FOXO1. Loss of a subset of GATOR2 members can be compensated by direct genetic activation of mTORC1. RAS mutations are also sufficient to decouple mTORC1 activation from GATOR2, and indeed fusion negative RMS harboring such mutations exhibit amino acid-independent mTORC1 activity. A bi-steric, mTORC1-selective small molecule induced tumor regressions in fusion positive patient-derived tumor xenografts. These findings highlight a vulnerability in FOXO1 fusion positive RMS and provide rationale for the clinical evaluation of bi-steric mTORC1 inhibitors, currently in phase 1 testing, to treat this disease. Isogenic genetic screens can thus identify potentially exploitable vulnerabilities in fusion driven pediatric cancers which otherwise remain mostly undruggable.

Keywords:  Cancer; Drug therapy; Genetics; Oncology; Signal transduction

DOI:  https://doi.org/10.1172/jci.insight.162207
Reprod Biol Endocrinol. 2022 Oct 25. 20(1): 152

Extracellular vesicles derived from endometrial epithelial cells deliver exogenous miR-92b-3p to affect the function of embryonic trophoblast cells via targeting TSC1 and DKK3.

Renwu Hua, Qiaorui Liu, Weisi Lian, Ting Ting Kang, Dengying Gao, Cheng Huang, Yueying Wang, Minggang Lei.

   BACKGROUND: Extracellular vesicles (EVs) could mediate embryo-maternal communication to affect embryo implantation by delivering biology information, including microRNA (miRNA), protein, lipid. Our previous research shows that miR-92b-3p was differentially expressed in EVs of uterine flushing fluids during the embryo implantation period. However, the role of miR-92b-3p from EVs in embryo implantation remains elusive.
MATERIALS AND METHODS: EVs were isolated from porcine endometrial epithelial cells (EECs) by ultracentrifugation. MiR-92b-3p mimics and EVs were used to regulate the expression of miR-92b-3p in porcine trophoblast cells (PTr2 cells). Cell proliferation, migration and adhesion analyses were used to observe the phenotype. RT-qPCR, western blot and dual-luciferase reporter assay were used to assess the targets of miR-92b-3p.
RESULTS: In this study, EVs derived from porcine EECs were identified and could be taken up by PTr2 cells. We found that the EVs derived from EECs transfected with miR-92b-3p mimic (EVs-miR-92b-3p) significantly promoted the proliferation, migration and adhesion of PTr2 cells. We verified that Tuberous sclerosis complex subunit (TSC1) and Dickkopf 3 (DKK3) were the target genes of miR-92b-3p. Moreover, our study showed that miR-92b-3p plays a vital role in PTr2 cells via targeting TSC1 and DKK3. Furthermore, the 3'UTR vectors of TSC1 and DKK3 can rescue the effect of miR-92b-3p on PTr2 cells.
CONCLUSIONS: Taken together, this study reveals a novel mechanism that EVs derived from porcine EECs treated with miR-92b-3p crosstalk with trophoblasts by targeting TSC1 and DKK3, leading to an enhanced ability for implantation.

Keywords:  DKK3; Embryo implantation; Extracellular Vesicles; TSC1; miR-92b-3p

DOI:  https://doi.org/10.1186/s12958-022-01023-z
Int J Mol Sci. 2022 Oct 18. pii: 12470. [Epub ahead of print]23(20):

mTOR as a Potential Target for the Treatment of Microbial Infections, Inflammatory Bowel Diseases, and Colorectal Cancer.

Obaid Afzal, Abdulmalik S A Altamimi, Bismillah Mubeen, Sami I Alzarea, Waleed Hassan Almalki, Salwa D Al-Qahtani, Eman M Atiya, Fahad A Al-Abbasi, Fatima Ali, Inam Ullah, Muhammad Shahid Nadeem, Imran Kazmi.

  The mammalian target of rapamycin (mTOR) is the major controller of a number of important cellular activities, including protein synthesis, cell expansion, multiplication, autophagy, lysosomal function, and cellular metabolism. When mTOR interacts with specific adaptor proteins, it forms two complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The mTOR signaling system regulates gene transcription and protein manufacturing to control proliferation of cell, differentiation of immune cell, and tumor metabolism. Due to its vital role in case of microbial infections, inflammations and cancer development and progression, mTOR has been considered as a key therapeutic target for the development of targeted medication. As autophagy dysfunction is linked to changes in both innate and adaptive immune responses, bacterial clearance defects, and goblet and Paneth cell malfunction, all of these changes are linked to inflammatory bowel diseases (IBD) and colorectal cancer (CRC) pathogenesis. Preclinical and clinical data have shown that the inhibition and induction of autophagy have significant potential to be translated into the clinical applications. In IBD and several CRC models, mTORC1 inhibitors have been found effective. In the recent years, a number of novel mTOR inhibitors have been investigated in clinical trials, and a number of drugs have shown considerably enhanced efficacy when combined with mTOR inhibitors. The future developments in the mTOR targeting medications can benefit patients in individualized therapy. Advanced and innovative medicines that are more effective and have lower drug resistance are still in high demand. New findings could be relevant in medicine development, pharmacological modification, or future mTOR inhibitor research. Therefore, the goal of this review is to present a comprehensive account of current developments on the mTOR pathway and its inhibitors, with an emphasis on the management of microbial infections, the treatment of inflammatory bowel disease, and the management of colon cancer.

Keywords:  colorectal cancer (CRC); inflammatory bowel diseases (IBD); mammalian target of rapamycin (mTOR)

DOI:  https://doi.org/10.3390/ijms232012470
Int J Mol Sci. 2022 Oct 17. pii: 12416. [Epub ahead of print]23(20):

mTOR Contributes to the Proteome Diversity through Transcriptome-Wide Alternative Splicing.

Sze Cheng, Naima Ahmed Fahmi, Meeyeon Park, Jiao Sun, Kaitlyn Thao, Hsin-Sung Yeh, Wei Zhang, Jeongsik Yong.

  The mammalian target of rapamycin (mTOR) pathway is crucial in energy metabolism and cell proliferation. Previously, we reported transcriptome-wide 3'-untranslated region (UTR) shortening by alternative polyadenylation upon mTOR activation and its impact on the proteome. Here, we further interrogated the mTOR-activated transcriptome and found that hyperactivation of mTOR promotes transcriptome-wide exon skipping/exclusion, producing short isoform transcripts from genes. This widespread exon skipping confers multifarious regulations in the mTOR-controlled functional proteomics: AS in coding regions widely affects the protein length and functional domains. They also alter the half-life of proteins and affect the regulatory post-translational modifications. Among the RNA processing factors differentially regulated by mTOR signaling, we found that SRSF3 mechanistically facilitates exon skipping in the mTOR-activated transcriptome. This study reveals a role of mTOR in AS regulation and demonstrates that widespread AS is a multifaceted modulator of the mTOR-regulated functional proteome.

Keywords:  alternative splicing; functional proteome; mTOR signaling; post-transcriptional gene regulation

DOI:  https://doi.org/10.3390/ijms232012416
J Biol Chem. 2022 Oct 20. pii: S0021-9258(22)01072-9. [Epub ahead of print] 102629

A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling.

Johanna B Brüggenthies, Alessandra Fiore, Marion Russier, Christina Bitsina, Julian Brötzmann, Susanne Kordes, Sascha Menninger, Alexander Wolf, Elena Conti, Jan E Eickhoff, Peter J Murray.

  mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.

Keywords:  GCN2; amino acid starvation; integrated stress response; mTORC1; torins

DOI:  https://doi.org/10.1016/j.jbc.2022.102629
Cancer Med. 2022 Oct 28.

The NRF2 antagonist ML385 inhibits PI3K-mTOR signaling and growth of lung squamous cell carcinoma cells.

Lili Ji, Nadeem Moghal, Xinru Zou, Yixuan Fang, Shuning Hu, Yuhui Wang, Ming Sound Tsao.

   BACKGROUND: Lung squamous cell carcinoma (LUSC) currently has limited therapeutic options because of the relatively few validated targets and the lack of clinical drugs for some of these targets. Although NRF2/NFE2L2 pathway activation commonly occurs in LUSC, NRF2 has predominantly been studied in other cancer models. Here, we investigated the function of NRF2 in LUSC, including in organoid models, and we explored the activity of a small molecule NRF2 inhibitor ML385, which has not previously been investigated in LUSC.
METHODS: We first explored the role of NRF2 signaling in LUSC cancer cell line and organoid proliferation through NRF2 knockdown or ML385 treatment, both in vivo and in vitro. Next, we performed Western blot and immunofluorescence assays to determine the effect of NRF2 inhibition on PI3K-mTOR signaling. Finally, we used cell viability and clonogenic assays to explore whether ML385 could sensitize LUSC cancer cells to PI3K inhibitors.
RESULTS: We find that downregulation of NRF2 signaling inhibited proliferation of LUSC cancer cell lines and organoids, both in vivo and in vitro. We also demonstrate that inhibition of NRF2 reduces PI3K-mTOR signaling, with two potential mechanisms being involved. Although NRF2 promotes AKT phosphorylation, it also acts downstream of AKT to increase RagD protein expression and recruitment of mTOR to lysosomes after amino acid stimulation. We also find that ML385 potentiates LUSC growth inhibition by a pan-PI3K inhibitor, which correlates with stronger inhibition of PI3K-mTOR signaling.
CONCLUSIONS: Our data provide additional support for NRF2 promoting LUSC growth through PI3K-mTOR activation and support development of NRF2 inhibitors for the treatment of LUSC.

Keywords:  NRF2; lung squamous cell carcinoma; mTOR; organoid; tumor growth

DOI:  https://doi.org/10.1002/cam4.5311
Commun Biol. 2022 Oct 27. 5(1): 1141

Dual roles of mTORC1-dependent activation of the ubiquitin-proteasome system in muscle proteostasis.

Marco S Kaiser, Giulia Milan, Daniel J Ham, Shuo Lin, Filippo Oliveri, Kathrin Chojnowska, Lionel A Tintignac, Nitish Mittal, Christian E Zimmerli, David J Glass, Mihaela Zavolan, Markus A Rüegg.

Muscle size is controlled by the PI3K-PKB/Akt-mTORC1-FoxO pathway, which integrates signals from growth factors, energy and amino acids to activate protein synthesis and inhibit protein breakdown. While mTORC1 activity is necessary for PKB/Akt-induced muscle hypertrophy, its constant activation alone induces muscle atrophy. Here we show that this paradox is based on mTORC1 activity promoting protein breakdown through the ubiquitin-proteasome system (UPS) by simultaneously inducing ubiquitin E3 ligase expression via feedback inhibition of PKB/Akt and proteasome biogenesis via Nuclear Factor Erythroid 2-Like 1 (Nrf1). Muscle growth was restored by reactivation of PKB/Akt, but not by Nrf1 knockdown, implicating ubiquitination as the limiting step. However, both PKB/Akt activation and proteasome depletion by Nrf1 knockdown led to an immediate disruption of proteome integrity with rapid accumulation of damaged material. These data highlight the physiological importance of mTORC1-mediated PKB/Akt inhibition and point to juxtaposed roles of the UPS in atrophy and proteome integrity.

DOI: https://doi.org/10.1038/s42003-022-04097-y
Curr Issues Mol Biol. 2022 Oct 15. 44(10): 4921-4929

Expression of AKT1 Related with Clinicopathological Parameters in Clear Cell Renal Cell Carcinoma.

Taesoo Choi, Koo Han Yoo, Man S Kim.

  Pathways such as VEGF, EGF and mTOR are known to be one of the major mechanisms of tumorigenesis including kidney cancer. To identify potential signaling pathway proteins, we performed differential/correlation analyses of mTOR-associated genes from three public datasets. AKT1 protein, one of the PI3K/AKT/mTOR pathways, turned out to be the potential by showing a consistent discrepancy between ccRCC-associated conditions as well as strong correlation with other mTOR-associated genes across the datasets. Then, we analyzed how AKT1 alteration affects clear cell renal cell carcinoma. The pathology of 58 kidney cancer patients was constructed to analyze the relationship between the expression level of AKT1 through immunohistochemical staining and their clinicopathological data. Gender, age and TNM stage did not show significant results. AKT1 is a known oncogene. However, in this study, high expression of AKT1 showed a slight correlation with lower WHO/ISUP grade, longer recurrence-free and progression-free survival rates.

Keywords:  AKT1 protein; carcinoma; renal cell; survival analysis

DOI:  https://doi.org/10.3390/cimb44100334