The energetic demands of proliferating cells during tumorigenesis require close coordination between the cell cycle and metabolism. While CDK4 is known for its role in cell proliferation, its metabolic function in cancer, particularly in triple-negative breast cancer (TNBC), remains unclear. Our study, using genetic and pharmacological approaches, reveals that CDK4 inactivation only modestly impacts TNBC cell proliferation and tumor formation. Notably, CDK4 depletion or long-term CDK4/6 inhibition confers resistance to apoptosis in TNBC cells. Mechanistically, CDK4 enhances mitochondria-endoplasmic reticulum contact (MERCs) formation, promoting mitochondrial fission and ER-mitochondrial calcium signaling, which are crucial for TNBC metabolic flexibility. Phosphoproteomic analysis identified CDK4’s role in regulating PKA activity at MERCs. In this work, we highlight CDK4’s role in mitochondrial apoptosis inhibition and suggest that targeting MERCs-associated metabolic shifts could enhance TNBC therapy.

Background: Obesity is characterized by adipose tissue dysregulation and predisposes individuals to insulin resistance and type 2 diabetes. At the molecular level, adipocyte dysfunction has been linked to obesity-triggered oxidative stress and protein carbonylation, considering protein carbonylation as a link between oxidative stress and metabolic dysfunction. The identification of specific carbonylated proteins in adipose tissue could provide novel biomarkers of oxidative damage related to metabolic status (i.e prediabetes). Thus, we aimed at characterizing the subcutaneous and omental human adipose tissue carbonylome in obesity-associated insulin resistance. Methods: 2D-PAGE was used to identify carbonylated proteins, and clinical correlations studies and molecular biology approaches including intracellular trafficking, reactive oxygen species assay, and iron content were performed using in vitro models of insulin resistance. Results: The carbonylome of human adipose tissue included common (serotransferrin, vimentin, actin, and annexin A2) and depot-specific (carbonic anhydrase and α-crystallin B in the subcutaneous depot; and α-1-antitrypsin and tubulin in the omental depot) differences that point out the complexity of oxidative stress at the metabolic level, highlighting changes in carbonylated transferrin expression. Posterior studies using in vitro prediabetic model evidence alteration in transferrin receptor translocation, linked to the prediabetic environment. Finally, ligand-receptor molecular docking studies showed a reduced affinity for carbonylated transferrin binding to its receptor compared to wild-type transferrin, emphasizing the role of transferrin carbonylation in the link between oxidative stress and metabolic dysfunction. Conclusions: The adipose tissue carbonylome contributes to understanding the molecular mechanism driving adipocyte dysfunction and identifies possible adipose tissue carbonylated targets in obesity-associated insulin resistance.

Adipocyte dysfunction in obesity is commonly associated with impaired insulin signalling in adipocytes and insulin resistance. Insulin signalling has been associated with caveolae, which are coated by large complexes of caveolin and cavin proteins, along with proteins with membrane‐binding and remodelling properties. Here, we analysed the regulation and function of a component of caveolae involved in growth factor signalling in neuroendocrine cells, neuroendocrine long coiled‐coil protein‐2 (NECC2), in adipocytes. Studies in 3T3‐L1 cells showed that NECC2 expression increased during adipogenesis. Furthermore, NECC2 co‐immunoprecipitated with caveolin‐1 (CAV1) and exhibited a distribution pattern similar to that of the components of adipocyte caveolae, CAV1, Cavin1, the insulin receptor and cortical actin. Interestingly, NECC2 overexpression enhanced insulin‐activated Akt phosphorylation, whereas NECC2 downregulation impaired insulin‐induced phosphorylation of Akt and ERK2. Finally, an up‐regulation of NECC2 in subcutaneous and omental adipose tissue was found in association with human obesity and insulin resistance. This effect was also observed in 3T3‐L1 adipocytes exposed to hyperglycaemia/hyperinsulinemia. Overall, the present study identifies NECC2 as a component of adipocyte caveolae that is regulated in response to obesity and associated metabolic complications, and supports the contribution of this protein as a molecular scaffold modulating insulin signal transduction at these membrane microdomains.

The adipose tissue stores excess energy in the form of neutral lipids within adipocyte lipid droplets (LDs). The correct function of LDs requires the interaction with other organelles, such as the endoplasmic reticulum (ER) as well as with LD coat-associated proteins, including Rab18, a mediator of intracellular lipid trafficking and ER–LD interaction. Although perturbations of the inter-organelle contact sites have been linked to several diseases, such as cancer, no information regarding ER–LD contact sites in dysfunctional adipocytes from the obese adipose tissue has been published to date. Herein, the ER–LD connection and Rab18 distribution at ER–LD contact sites are examined in adipocytes challenged with fibrosis and inflammatory conditions, which represent known hallmarks of the adipose tissue in obesity. Our results show that adipocytes differentiated in fibrotic conditions caused ER fragmentation, the expansion of ER–LD contact sites, and modified Rab18 dynamics. Likewise, adipocytes exposed to inflammatory conditions favored ER–LD contact, Rab18 accumulation in the ER, and Rab18 redistribution to large LDs. Finally, our studies in human adipocytes supported the suggestion that Rab18 transitions to the LD coat from the ER. Taken together, our results suggest that obesity-related pathogenic processes alter the maintenance of ER–LD interactions and interfere with Rab18 trafficking through these contact sites.

Bariatric surgery (BS) is the most effective treatment for obesity and its related comorbidities, resulting in significant adipose tissue (AT) loss and functional recovery. However, the molecular mechanisms in the AT that drive these health benefits remain poorly understood, and predictive factors for adequate weight loss are currently needed. This study aimed to identify the proteomic signature of subcutaneous AT (SAT) linked to BS-induced weight loss and metabolic improvement, and to determine potential predictors of individual response. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics was performed on paired abdominal SAT samples from individuals with obesity before and after BS. Bioinformatic and correlation analyses were conducted between proteomic data and changes in anthropometric and clinical parameters. In addition, the effect of sera from BS participants on subcutaneous adipocytes was assessed in vitro. The SAT response to BS was characterised by an up-regulation of mitochondrial metabolism and a down-regulation of inflammation, which correlated with different anthropometric and circulating markers. Notably, obesity duration was associated with BS-induced weight loss. Individuals with short-standing obesity (SSO) exhibited greater proteomic remodelling, weight reduction, and clinical improvement post-BS than those with long-standing obesity (LSO). Furthermore, a regulatory axis involving miR-223-3p, glucose metabolism, and cytoskeleton reorganisation in adipocytes might be connected to the positive effects of BS in LSO. Overall, our results indicate that obesity duration is related to BS-induced weight loss through SAT proteomic remodelling. Addressing this relationship could help develop new strategies to enhance weight management and metabolic health in people living with obesity.