Proteome and Phosphoproteome Analysis of Brown Adipocytes Reveals That RICTOR Loss Dampens Global Insulin/AKT Signaling

Entwisle, Samuel W.; Calejman, Camila Martínez; Valente, Anthony S.; Lawrence, Robert; Hung, Chien-Min; Guertin, David A.; Villén, Judit

doi:10.1074/mcp.ra120.001946

Cited by 9 publications

(4 citation statements)

References 39 publications

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“…The connection between increase in mitochondrial functions and resistance to BRAFi is supported by our experimental evidence, as both NAMPT and ETC inhibition reverses the BRAFi tolerance of RICTOR-depleted cells. Our findings are supported by previous works that show NDUFS1 modulation by posttranslational modifications, and by poorly-characterized associations between RICTOR downregulation and gain of mitochondrial functions [18,19,48].…”

Section: Discussionsupporting

confidence: 91%

“…Notably, the metabolic phenotype found in RICTORknockout keratinocytes, consisting of a switch from a glycolytic-to an OXPHOS-based energetic metabolism fueled by glutamine consumption, is reminiscent of that of targeted therapy-resistant melanoma cells. Consistently, RICTOR deficiency was previously associated with the induction of genes and/or cellular processes that favor mitochondrial ATP synthesis [18][19][20][21]. Based on these notions, we have hypothesized that RICTOR/ mTORC2 deficiency in BRAF V600E melanoma cells may favor BRAF/MEKi resistance.…”

Section: Introductionsupporting

confidence: 58%

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RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition

Ponzone,

Audrito,

Landi

et al. 2024

Mol Cancer

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Background The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. Methods After analyzing The Cancer Genome Atlas MM patients’ database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. Results Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. Conclusions Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

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Section: Discussionsupporting

confidence: 91%

Section: Introductionsupporting

confidence: 58%

RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition

Ponzone,

Audrito,

Landi

et al. 2024

Mol Cancer

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“…Most pronounced programming effect across all tissues at the level of individual upstream regulators was inhibition of RICTOR, a regulatory subunit of the mammalian target of rapamycin complex 2. Loss of RICTOR leads to the global dampening of insulin/AKT signaling ( Entwisle et al, 2020 ). Although this observation did not translate to change of global glucose tolerance in the programmed rat offspring, subtler change in insulin resistance of peripheral tissues cannot be excluded, as suggested by the 3-fold (2-fold in SHR- Zbtb16 ) reduction of Glut4 expression in white adipose tissue of SHR male offspring and, in a network perspective, the glucose metabolism disorder activated node.…”

Section: Discussionmentioning

confidence: 99%

Maternal High-Sucrose Diet Affects Phenotype Outcome in Adult Male Offspring: Role of Zbtb16

et al. 2020

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Overnutrition in pregnancy and lactation affects fetal and early postnatal development, which can result in metabolic disorders in adulthood. We tested a hypothesis that variation of the Zbtb16 gene, a significant energy metabolism regulator, modulates the effect of maternal high-sucrose diet (HSD) on metabolic and transcriptomic profiles of the offspring. We used the spontaneously hypertensive rat (SHR) strain and a minimal congenic rat strain SHR- Zbtb16 , carrying the Zbtb16 gene allele originating from the PD/Cub rat, a metabolic syndrome model. Sixteen-week-old SHR and SHR- Zbtb16 rat dams were fed either standard diet (control groups) or a high-sucrose diet (HSD, 70% calories as sucrose) during pregnancy and 4 weeks of lactation. In dams of both strains, we observed an HSD-induced increase of cholesterol and triacylglycerol concentrations in VLDL particles and a decrease of cholesterol and triacylglycerols content in medium to very small LDL particles. In male offspring, exposure to maternal HSD substantially increased brown fat weight in both strains, decreased triglycerides in LDL particles, and impaired glucose tolerance exclusively in SHR. The transcriptome assessment revealed networks of transcripts reflecting the shifts induced by maternal HSD with major nodes including mir-126, Hsd11b1 in the brown adipose tissue, Pcsk9, Nr0b2 in the liver and Hsd11b1, Slc2a4 in white adipose tissue. In summary, maternal HSD feeding during pregnancy and lactation affected brown fat deposition and lipid metabolism in adult male offspring and induced major transcriptome shifts in liver, white, and brown adipose tissues. The Zbtb16 variation present in the SHR- Zbtb16 led to several strain-specific effects of the maternal HSD, particularly the transcriptomic profile shifts of the adult male offspring.

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“…Recent advances in mass spectrometry (MS)-based phosphoproteomics have enabled widespread quantification of phosphorylation dynamics in diverse biological contexts and organisms [15][16][17][18] . Global studies in skeletal muscle, adipose tissue, and liver of rodents and humans have revealed that insulin regulates thousands of phosphosites on hundreds of proteins 3,4,[19][20][21][22][23] . A major challenge now lies in identifying the connectivity and functionally annotating these signalling networks.…”

Section: Introductionmentioning

confidence: 99%

Personalized phosphoproteomics of skeletal muscle insulin resistance and exercise links MINDY1 to insulin action

Needham,

Hingst,

Onslev

et al. 2024

Preprint

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Type 2 diabetes is preceded by a defective insulin response, yet our knowledge of the precise mechanisms is incomplete. Here, we investigate how insulin resistance alters signalling responses in skeletal muscle and how this is modified by exercise. We measured parallel phenotypes and phosphoproteomes of insulin resistant and insulin sensitive individuals as they responded to exercise and insulin (n=19, 114 biopsies), quantifying over 12,000 phosphopeptides in each biopsy. Our personalized phosphoproteomics approach revealed that insulin resistant individuals have selective and time-dependent signalling alterations. Insulin resistant subjects have reduced insulin-stimulated mTORC1 responses and alterations to non-canonical rather than canonical insulin signalling. Prior exercise promotes insulin sensitivity even in insulin resistant individuals by priming a portion of insulin signalling prior to insulin infusion. This includes MINDY1 S441, which is elevated in insulin-sensitive subjects and primed by prior exercise. MINDY1 contains a missense variant that is protective for type 2 diabetes but its role in disease risk is unknown. We show that MINDY1 S441 phosphorylation is downstream of AKT, and MINDY1 knockdown enhances insulin-stimulated glucose uptake in rat myotubes. This work delineates the signalling alterations in insulin resistant skeletal muscle and how exercise partially counteracts these and identifies MINDY1 as a regulator of insulin action.

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Proteome and Phosphoproteome Analysis of Brown Adipocytes Reveals That RICTOR Loss Dampens Global Insulin/AKT Signaling

Cited by 9 publications

References 39 publications

RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition

RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition

Maternal High-Sucrose Diet Affects Phenotype Outcome in Adult Male Offspring: Role of Zbtb16

Personalized phosphoproteomics of skeletal muscle insulin resistance and exercise links MINDY1 to insulin action

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