bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2020‒05‒24
thirty papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Heliyon. 2020 May;6(5): e03910
      A fundamental feature of tumor progression is reprogramming of metabolic pathways. ATP citrate lyase (ACLY) is a key metabolic enzyme that catalyzes the generation of Acetyl-CoA and is upregulated in cancer cells and required for their growth. The phosphoinositide 3-kinase (PI3K) and Src-family kinase (SFK) Lyn are constitutively activate in many cancers. We show here, for the first time, that both the substrate and product of PI3K, phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), respectively, bind to ACLY in Acute Myeloid Leukemia (AML) patient-derived, but not normal donor-derived cells. We demonstrate the binding of PIP2 to the CoA-binding domain of ACLY and identify the six tyrosine residues of ACLY that are phosphorylated by Lyn. Three of them (Y682, Y252, Y227) can be also phosphorylated by Src and they are located in catalytic, citrate binding and ATP binding domains, respectively. PI3K and Lyn inhibitors reduce the ACLY enzyme activity, ACLY-mediated Acetyl-CoA synthesis, phospholipid synthesis, histone acetylation and cell growth. Thus, PIP2/PIP3 binding and Src tyrosine kinases-mediated stimulation of ACLY links oncogenic pathways to Acetyl-CoA-dependent pro-growth and survival metabolic pathways in cancer cells. These results indicate a novel function for Lyn, as a regulator of Acetyl-CoA-mediated metabolic pathways.
    Keywords:  ACLY; Acetyl-CoA; Biochemistry; Biological sciences; Cancer; Cancer research; Health sciences; Lyn; Metabolism; PI3K; Src
  2. Sci Rep. 2020 May 22. 10(1): 8509
      Acute aerobic exercise (AE) increases skeletal muscle insulin sensitivity for several hours, caused by acute activation of AMP-activated protein kinase (AMPK). Acute resistance exercise (RE) also activates AMPK, possibly improving insulin-stimulated glucose uptake. However, RE-induced rapamycin-sensitive mechanistic target of rapamycin complex 1 (mTORC1) activation is higher and has a longer duration than after AE. In molecular studies, mTORC1 was shown to be upstream of insulin receptor substrate 1 (IRS-1) Ser phosphorylation residue, inducing insulin resistance. Therefore, we hypothesised that although RE increases insulin sensitivity through AMPK activation, prolonged mTORC1 activation after RE reduces RE-induced insulin sensitising effect. In this study, we used an electrical stimulation-induced RE model in rats, with rapamycin as an inhibitor of mTORC1 activation. Our results showed that RE increased insulin-stimulated glucose uptake following AMPK signal activation. However, mTORC1 activation and IRS-1 Ser632/635 and Ser612 phosphorylation were elevated 6 h after RE, with concomitant impairment of insulin-stimulated Akt signal activation. By contrast, rapamycin inhibited these prior exercise responses. Furthermore, increases in insulin-stimulated skeletal muscle glucose uptake 6 h after RE were higher in rats with rapamycin treatment than with placebo treatment. Our data suggest that mTORC1/IRS-1 signaling inhibition enhances skeletal muscle insulin-sensitising effect of RE.
  3. Mol Metab. 2020 May 18. pii: S2212-8778(20)30094-6. [Epub ahead of print] 101020
      OBJECTIVE: Insulin signaling via phosphoinositide 3-kinase (PI3K) requires PIK3R1-encoded regulatory subunits. C-terminal PIK3R1 mutations cause SHORT syndrome, including lipodystrophy and insulin resistance (IR), surprisingly without fatty liver or metabolic dyslipidemia. We sought to investigate this discordance.METHODS: The human pathogenic Pik3r1 Y657* mutation was knocked into mice by homologous recombination. Growth, body composition, bioenergetic and metabolic profiles were investigated on chow and high fat diet. Adipose and liver histology was examined, and liver responses to fasting and refeeding assessed transcriptomically.
    RESULTS: Like humans with SHORT syndrome, Pik3r1Y657*/WT mice were small with severe IR, and adipose expansion on high fat diet was markedly reduced. Also like humans, plasma lipid concentrations were low, and insulin-stimulated hepatic lipogenesis was not increased despite hyperinsulinemia. At odds with lipodystrophy, however, no adipocyte hypertrophy nor adipose inflammation was found. Liver lipogenic gene expression was not significantly altered, and unbiased transcriptomics showed only minor changes, including evidence of reduced endoplasmic reticulum stress in the fed state and diminished Rictor-dependent transcription on fasting. Increased energy expenditure, which was not explained by hyperglycemia nor intestinal malabsorption, provided an alternative explanation for uncoupling of IR from dyslipidaemia.
    CONCLUSIONS: Pik3r1 dysfunction in mice phenocopies the IR and reduced adiposity without lipotoxicity of human SHORT syndrome. Decreased adiposity may not reflect bona fide lipodystrophy, but rather increased energy expenditure, and we suggest that study of brown adipose tissue in both humans and mice is warranted.
    Keywords:  Insulin resistance; PI 3-Kinase; Pik3r1; diabetes; lipids; lipotoxicity; p85
  4. Mol Cancer Ther. 2020 May 19. pii: molcanther.1036.2019. [Epub ahead of print]
      Cetuximab, a monoclonal antibody targeting EGFR, is a standard of care for the treatment for locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC). However, despite overexpression of EGFR in over 90% of HNSCC lesions, most HNSCC patients fail to respond to cetuximab treatment. In addition, there are no available biomarkers to predict sensitivity or resistance to cetuximab in the clinic. Here, we sought to advance precision medicine approaches for HNSCC by identifying PI3K-mTOR signaling-network-specific cetuximab resistance mechanisms. We first analyzed the frequency of genomic alterations in genes involved in the PI3K-mTOR signaling circuitry in the HNSCC TCGA dataset. Experimentally, we took advantage of CRISPR/Cas9 genome editing approaches to systematically explore the contribution of genomic alterations in each tumor suppressor gene (TSG) controlling the PI3K-mTOR pathway to cetuximab resistance in HNSCC cases that do not exhibit PIK3CA mutations. Remarkably, we found that many HNSCC cases exhibit pathway-specific gene copy number loss of multiple TSGs that normally restrain PI3K-mTOR signaling. Among them, we found that both engineered and endogenous PTEN gene deletions can mediate resistance to cetuximab. Our findings suggest that PTEN gene copy number loss, which is highly prevalent in HNSCC, may result in sustained PI3K/mTOR signaling independent of EGFR, thereby representing a promising mechanistic biomarker predictive of cetuximab resistance in this cancer type. Further prospective studies are needed to investigate the impact of PTEN loss on cetuximab efficacy in the clinic.
  5. Diabetes. 2020 May 21. pii: db191074. [Epub ahead of print]
      Insulin resistance due to overnutrition places a burden on energy-producing pathways in skeletal muscle (SkM). Nevertheless, energy state is not compromised. The hypothesis that the energy sensor AMP-activated protein kinase (AMPK) is necessary to offset the metabolic burden of overnutrition was tested using chow-fed and high fat (HF)-fed SkM-specific AMPKα1α2 knockout (mdKO) mice and AMPKα1α2lox/lox littermates (WT). Lean mdKO and WT mice were phenotypically similar. HF-fed mice were equally obese and maintained lean mass regardless of genotype. Results did not support the hypothesis that AMPK is protective during overnutrition. Paradoxically, mdKO mice were more insulin sensitive. Insulin-stimulated SkM glucose uptake was ∼two-fold greater in mdKO mice in vivo. Furthermore, insulin signaling, SkM GLUT4 translocation, hexokinase activity, and glycolysis were increased. AMPK and insulin signaling intersect at mTOR, a critical node for cell proliferation and survival. Basal mTOR activation was reduced by 50% in HF-fed mdKO mice, but was normalized by insulin-stimulation. Mitochondrial function was impaired in mdKO mice, but energy charge was preserved by AMP deamination. Results show a surprising reciprocity between SkM AMPK signaling and insulin action that manifests with diet-induced obesity, as insulin action is preserved to protect fundamental energetic processes in the muscle.
  6. J Cell Biol. 2020 Jul 06. pii: e201912098. [Epub ahead of print]219(7):
      Autophagy degrades cytoplasmic cargo by its delivery to lysosomes within double membrane autophagosomes. Synthesis of the phosphoinositide PI(3)P by the autophagic class III phosphatidylinositol-3 kinase complex I (PI3KC3-C1) and conjugation of ATG8/LC3 proteins to phagophore membranes by the ATG12-ATG5-ATG16L1 (E3) complex are two critical steps in autophagosome biogenesis, connected by WIPI2. Here, we present a complete reconstitution of these events. On giant unilamellar vesicles (GUVs), LC3 lipidation is strictly dependent on the recruitment of WIPI2 that in turn depends on PI(3)P. Ectopically targeting E3 to membranes in the absence of WIPI2 is insufficient to support LC3 lipidation, demonstrating that WIPI2 allosterically activates the E3 complex. PI3KC3-C1 and WIPI2 mutually promote the recruitment of each other in a positive feedback loop. When both PI 3-kinase and LC3 lipidation reactions were performed simultaneously, positive feedback between PI3KC3-C1 and WIPI2 led to rapid LC3 lipidation with kinetics similar to that seen in cellular autophagosome formation.
  7. Nat Genet. 2020 May 18.
      Cas9 is commonly introduced into cell lines to enable CRISPR-Cas9-mediated genome editing. Here, we studied the genetic and transcriptional consequences of Cas9 expression itself. Gene expression profiling of 165 pairs of human cancer cell lines and their Cas9-expressing derivatives revealed upregulation of the p53 pathway upon introduction of Cas9, specifically in wild-type TP53 (TP53-WT) cell lines. This was confirmed at the messenger RNA and protein levels. Moreover, elevated levels of DNA repair were observed in Cas9-expressing cell lines. Genetic characterization of 42 cell line pairs showed that introduction of Cas9 can lead to the emergence and expansion of p53-inactivating mutations. This was confirmed by competition experiments in isogenic TP53-WT and TP53-null (TP53-/-) cell lines. Lastly, Cas9 was less active in TP53-WT than in TP53-mutant cell lines, and Cas9-induced p53 pathway activation affected cellular sensitivity to both genetic and chemical perturbations. These findings may have broad implications for the proper use of CRISPR-Cas9-mediated genome editing.
  8. J Clin Invest. 2020 May 19. pii: 138577. [Epub ahead of print]
      Breast cancer stem cells (BCSCs) play a critical role in cancer recurrence and metastasis. Chemotherapy induces BCSC specification through increased expression of pluripotency factors, but how their expression is regulated is not fully understood. Here, we delineate a hypoxia-inducible factor 1 (HIF-1)-controlled pathway that epigenetically activates pluripotency factor gene transcription in response to chemotherapy. Paclitaxel induces HIF-1-dependent expression of S100A10, which forms a complex with ANXA2 that interacts with histone chaperone SPT6 and histone demethylase KDM6A. S100A10, ANXA2, SPT6, and KDM6A are recruited to OCT4 binding sites and KDM6A erases H3K27me3 chromatin marks, facilitating transcription of genes encoding the pluripotency factors NANOG, SOX2, and KLF4, which along with OCT4 are responsible for BCSC specification. Silencing of S100A10, ANXA2, SPT6, or KDM6A expression blocks chemotherapy-induced enrichment of BCSCs, impairs tumor initiation, and increases time to tumor recurrence after chemotherapy is discontinued. Pharmacological inhibition of KDM6A also impairs chemotherapy-induced BCSC enrichment. These results suggest that targeting HIF-1/S100A10-dependent and KDM6A-mediated epigenetic activation of pluripotency factor gene expression in combination with chemotherapy may block BCSC enrichment and improve clinical outcome.
    Keywords:  Breast cancer; Oncology; Transcription; hypoxia
  9. Nat Commun. 2020 May 21. 11(1): 2549
      Mitochondria undergo dynamic fusion/fission, biogenesis and mitophagy in response to stimuli or stresses. Disruption of mitochondrial homeostasis could lead to cell senescence, although the underlying mechanism remains unclear. We show that deletion of mitochondrial phosphatase PGAM5 leads to accelerated retinal pigment epithelial (RPE) senescence in vitro and in vivo. Mechanistically, PGAM5 is required for mitochondrial fission through dephosphorylating DRP1. PGAM5 deletion leads to increased mitochondrial fusion and decreased mitochondrial turnover. As results, cellular ATP and reactive oxygen species (ROS) levels are elevated, mTOR and IRF/IFN-β signaling pathways are enhanced, leading to cellular senescence. Overexpression of Drp1 K38A or S637A mutant phenocopies or rescues mTOR activation and senescence in PGAM5-/- cells, respectively. Young but not aging Pgam5-/- mice are resistant to sodium iodate-induced RPE cell death. Our studies establish a link between defective mitochondrial fission, cellular senescence and age-dependent oxidative stress response, which have implications in age-related diseases.
  10. Ann Oncol. 2020 May 13. pii: S0923-7534(20)39798-2. [Epub ahead of print]
      BACKGROUND: Alpelisib (α-selective PI3K inhibitor) plus fulvestrant is approved in multiple countries for men and post-menopausal women with PIK3CA-mutated, hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer following progression on or after endocrine therapy. A detailed understanding of alpelisib's safety profile should inform adverse event (AE) management and enhance patient care.PATIENTS AND METHODS: AEs in the phase 3 SOLAR-1 trial were assessed in patients with and without PIK3CA mutations. The impact of protocol-specified AE-management recommendations was evaluated, including an amendment to optimize hyperglycemia and rash management.
    RESULTS: Patients were randomized to fulvestrant plus alpelisib (n=284) or placebo (n=287). The most common grade 3/4 AEs with alpelisib were hyperglycemia (grade 3, 32.7%; grade 4, 3.9%), rash (grade 3, 9.9%), and diarrhea (grade 3, 6.7%). Median time to onset of grade ≥3 toxicity was 15 days (hyperglycemia, based on fasting plasma glucose), 13 days (rash), and 139 days (diarrhea). Metformin alone or in combination with other anti-diabetic agents was used by most patients (87.1%) with hyperglycemia. Preventive anti-rash medication resulted in lower incidence (any grade, 26.7% vs 64.1%) and severity of rash (grade 3, 11.6% vs 22.7%) vs no preventative medication. Discontinuations due to grade ≥3 AEs were lower following more-detailed AE management guidelines (7.9% vs 18.1% previously). Patients with PIK3CA mutations had a median alpelisib dose intensity of 248 mg/day. Median progression-free survival (PFS) with alpelisib was 12.5 and 9.6 months for alpelisib dose intensities ≥248 mg/day and <248 mg/day, respectively, compared with 5.8 months with placebo.
    CONCLUSIONS: Hyperglycemia and rash occurred early during alpelisib treatment, while diarrhea occurred at a later timepoint. Early identification, prevention, and intervention, including concomitant medications and alpelisib dose modifications, resulted in less severe toxicities. Reductions in treatment discontinuations and improved PFS at higher alpelisib dose intensities support the need for optimal AE management.
    Keywords:  alpelisib; breast cancer; diarrhea; hyperglycemia; rash
  11. Am J Hum Genet. 2020 May 19. pii: S0002-9297(20)30123-3. [Epub ahead of print]
      Germline variation in PTEN results in variable clinical presentations, including benign and malignant neoplasia and neurodevelopmental disorders. Despite decades of research, it remains unclear how the PTEN genotype is related to clinical outcomes. In this study, we combined two recent deep mutational scanning (DMS) datasets probing the effects of single amino acid variation on enzyme activity and steady-state cellular abundance with a large, well-curated clinical cohort of PTEN-variant carriers. We sought to connect variant-specific molecular phenotypes to the clinical outcomes of individuals with PTEN variants. We found that DMS data partially explain quantitative clinical traits, including head circumference and Cleveland Clinic (CC) score, which is a semiquantitative surrogate of disease burden. We built logistic regression models that use DMS and CADD scores to separate clinical PTEN variation from gnomAD control-only variation with high accuracy. By using a survival-like analysis, we identified molecular phenotype groups with differential risk of early cancer onset as well as lifetime risk of cancer. Finally, we identified classes of DMS-defined variants with significantly different risk levels for classical hamartoma-related features (odds ratio [OR] range of 4.1-102.9). In stark contrast, the risk for developing autism or developmental delay does not significantly change across variant classes (OR range of 5.4-12.4). Together, these findings highlight the potential impact of combining DMS datasets with rich clinical data and provide new insights that might guide personalized clinical decisions for PTEN-variant carriers.
    Keywords:  ASD; PHTS; PTEN; PTEN hamartoma tumor syndrome; autism; autism spectrum disorder; cancer; deep mutational scanning; genotype-phenotype; multiplex assay for variant effect
  12. Proc Natl Acad Sci U S A. 2020 May 18. pii: 201919528. [Epub ahead of print]
      Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum-mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum-mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells.
    Keywords:  ER–mitochondria contacts; proximity labeling; split-TurboID
  13. Nat Genet. 2020 May 18.
      During aging, progenitor cells acquire mutations, which may generate clones that colonize the surrounding tissue. By middle age, normal human tissues, including the esophageal epithelium (EE), become a patchwork of mutant clones. Despite their relevance for understanding aging and cancer, the processes that underpin mutational selection in normal tissues remain poorly understood. Here, we investigated this issue in the esophageal epithelium of mutagen-treated mice. Deep sequencing identified numerous mutant clones with multiple genes under positive selection, including Notch1, Notch2 and Trp53, which are also selected in human esophageal epithelium. Transgenic lineage tracing revealed strong clonal competition that evolved over time. Clone dynamics were consistent with a simple model in which the proliferative advantage conferred by positively selected mutations depends on the nature of the neighboring cells. When clones with similar competitive fitness collide, mutant cell fate reverts towards homeostasis, a constraint that explains how selection operates in normal-appearing epithelium.
  14. Nat Commun. 2020 May 21. 11(1): 2538
      Compromised β-cell identity is emerging as an important contributor to β-cell failure in diabetes; however, the precise mechanism independent of hyperglycemia is under investigation. We have previously reported that mTORC1/Raptor regulates functional maturation in β-cells. In the present study, we find that diabetic β-cell specific Raptor-deficient mice (βRapKOGFP) show reduced β-cell mass, loss of β-cell identity and acquisition of α-cell features; which are not reversible upon glucose normalization. Deletion of Raptor directly impairs β-cell identity, mitochondrial metabolic coupling and protein synthetic activity, leading to β-cell failure. Moreover, loss of Raptor activates α-cell transcription factor MafB (via modulating C/EBPβ isoform ratio) and several α-cell enriched genes i.e. Etv1 and Tspan12, thus initiates β- to α-cell reprograming. The present findings highlight mTORC1 as a metabolic rheostat for stabilizing β-cell identity and repressing α-cell program at normoglycemic level, which might present therapeutic opportunities for treatment of diabetes.
  15. Cell Rep. 2020 May 19. pii: S2211-1247(20)30608-2. [Epub ahead of print]31(7): 107655
      Transcription factors (TFs) play a pivotal role in determining cell states, yet our understanding of the causative relationship between TFs and cell states is limited. Here, we systematically examine the state changes of human pluripotent embryonic stem cells (hESCs) by the large-scale manipulation of single TFs. We establish 2,135 hESC lines, representing three clones each of 714 doxycycline (Dox)-inducible genes including 481 TFs, and obtain 26,998 microscopic cell images and 2,174 transcriptome datasets-RNA sequencing (RNA-seq) or microarrays-48 h after the presence or absence of Dox. Interestingly, the expression of essentially all the genes, including genes located in heterochromatin regions, are perturbed by these TFs. TFs are also characterized by their ability to induce differentiation of hESCs into specific cell lineages. These analyses help to provide a way of classifying TFs and identifying specific sets of TFs for directing hESC differentiation into desired cell types.
    Keywords:  cell differentiation; conditional induction; human embryonic stem cells; transcription factors; transcriptome
  16. Br J Cancer. 2020 May 22.
      BACKGROUND: AKT, a critical effector of the phosphoinositide 3-kinase (PI3K) signalling cascade, is an intensely pursued therapeutic target in oncology. Two distinct classes of AKT inhibitors have been in clinical development, ATP-competitive and allosteric. Class-specific differences in drug activity are likely the result of differential structural and conformational requirements governing efficient target binding, which ultimately determine isoform-specific potency, selectivity profiles and activity against clinically relevant AKT mutant variants.METHODS: We have carried out a systematic evaluation of clinical AKT inhibitors using in vitro pharmacology, molecular profiling and biochemical assays together with structural modelling to better understand the context of drug-specific and drug-class-specific cell-killing activity.
    RESULTS: Our data demonstrate clear differences between ATP-competitive and allosteric AKT inhibitors, including differential effects on non-catalytic activity as measured by a novel functional readout. Surprisingly, we found that some mutations can cause drug resistance in an isoform-selective manner despite high structural conservation across AKT isoforms. Finally, we have derived drug-class-specific phosphoproteomic signatures and used them to identify effective drug combinations.
    CONCLUSIONS: These findings illustrate the utility of individual AKT inhibitors, both as drugs and as chemical probes, and the benefit of AKT inhibitor pharmacological diversity in providing a repertoire of context-specific therapeutic options.
  17. Cell Syst. 2020 May 20. pii: S2405-4712(20)30150-2. [Epub ahead of print]10(5): 424-432.e6
      Selecting appropriate cancer models is a key prerequisite for maximizing translational potential and clinical relevance of in vitro oncology studies. We developed CELLector: an R package and R Shiny application allowing researchers to select the most relevant cancer cell lines in a patient-genomic-guided fashion. CELLector leverages tumor genomics to identify recurrent subtypes with associated genomic signatures. It then evaluates these signatures in cancer cell lines to prioritize their selection. This enables users to choose appropriate in vitro models for inclusion or exclusion in retrospective analyses and future studies. Moreover, this allows bridging outcomes from cancer cell line screens to precisely defined sub-cohorts of primary tumors. Here, we demonstrate the usefulness and applicability of CELLector, showing how it can aid prioritization of in vitro models for future development and unveil patient-derived multivariate prognostic and therapeutic markers. CELLector is freely available at (code at and
    Keywords:  cancer models; cell lines; genomics; in silico prescriptions; in vitro study; molecular subtyping; new algorithm; pharmacogenomics
  18. Cancers (Basel). 2020 May 20. pii: E1290. [Epub ahead of print]12(5):
      Obesity is a global, intractable issue, altering inflammatory and stress response pathways, and promoting tissue adiposity and tumorigenesis. Visceral fat accumulation is correlated with primary tumor recurrence, poor prognosis and chemotherapeutic resistance. Accumulating evidence highlights a close association between obesity and an increased incidence of hepatocellular carcinoma (HCC). Obesity drives HCC, and obesity-associated tumorigenesis develops via nonalcoholic fatty liver (NAFL), progressing to nonalcoholic steatohepatitis (NASH) and ultimately to HCC. The better molecular elucidation and proteogenomic characterization of obesity-associated HCC might eventually open up potential therapeutic avenues. The mechanisms relating obesity and HCC are correlated with adipose tissue remodeling, alteration in the gut microbiome, genetic factors, ER stress, oxidative stress and epigenetic changes. During obesity-related hepatocarcinogenesis, adipokine secretion is dysregulated and the nuclear factor erythroid 2 related factor 1 (Nrf-1), nuclear factor kappa B (NF-κB), mammalian target of rapamycin (mTOR), phosphatidylinositol-3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/Akt, and Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways are activated. This review captures the present trends allied with the molecular mechanisms involved in obesity-associated hepatic tumorigenesis, showcasing next generation molecular therapeutic strategies and their mechanisms for the successful treatment of HCC.
    Keywords:  HCC; NASH; epigenetic changes; genetic factors; obesity; therapeutics
  19. Cell Rep. 2020 May 19. pii: S2211-1247(20)30617-3. [Epub ahead of print]31(7): 107664
      Cullin-RING ligases (CRLs) control key cellular processes by promoting ubiquitylation of a multitude of soluble cytosolic and nuclear proteins. Subsets of CRL complexes are recruited and activated locally at cellular membranes; however, few CRL functions and substrates at these distinct cellular compartments are known. Here, we use a proteomic screen to identify proteins that are ubiquitylated at cellular membranes and found that Lunapark, an endoplasmic reticulum (ER)-shaping protein localized to ER three-way junctions, is ubiquitylated by the CRL3KLHL12 ubiquitin ligase. We demonstrate that Lunapark interacts with mechanistic target of rapamycin complex-1 (mTORC1), a central cellular regulator that coordinates growth and metabolism with environmental conditions. We show that mTORC1 binds Lunapark specifically at three-way junctions, and lysosomes, where mTORC1 is activated, make contact with three-way junctions where Lunapark resides. Inhibition of Lunapark ubiquitylation results in neurodevelopmental defects indicating that KLHL12-dependent ubiquitylation of Lunapark is required for normal growth and development.
    Keywords:  ER three-way junction; Lunapark; cullin-RING ligases; endoplasmic reticulum; lysosome; mTORC1; ubiquitin
  20. Trends Endocrinol Metab. 2020 May 13. pii: S1043-2760(20)30082-5. [Epub ahead of print]
      Disruption of endocrine hormonal balance (i.e., increased levels of insulin, and reduced levels of growth hormone, GH) often occurs in pre-obesity and obesity. Using distinct intracellular signaling pathways to control cell and body metabolism, GH and insulin also regulate each other's secretion to maintain overall metabolic homeostasis. Therefore, a comprehensive understanding of insulin and GH balance is essential for understanding endocrine hormonal contributions to energy storage and utilization. In this review we summarize the actions of, and interactions between, insulin and GH at the cellular level, and highlight the association between the insulin/GH ratio and energy metabolism, as well as fat accumulation. Use of the [insulin]:[GH] ratio as a biomarker for predicting the development of obesity is proposed.
    Keywords:  energy metabolism; glucose metabolism; growth hormone; insulin; lipid metabolism; obesity
  21. Nat Commun. 2020 May 19. 11(1): 2512
      Mutations do not accumulate uniformly across the genome. Human germline and tumor mutation density correlate poorly, and each is associated with different genomic features. Here, we use non-human great ape (NHGA) germlines to determine human germline- and tumor-specific deviations from an ancestral-like great ape genome-wide mutational landscape. Strikingly, we find that the distribution of mutation densities in tumors presents a stronger correlation with NHGA than with human germlines. This effect is driven by human-specific differences in the distribution of mutations at non-CpG sites. We propose that ancestral human demographic events, together with the human-specific mutation slowdown, disrupted the human genome-wide distribution of mutation densities. Tumors partially recover this distribution by accumulating preneoplastic-like somatic mutations. Our results highlight the potential utility of using NHGA population data, rather than human controls, to establish the expected mutational background of healthy somatic cells.
  22. Commun Biol. 2020 May 22. 3(1): 253
      The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig-like domain 20 of FLNc, is doubly phosphorylated. The protein kinases responsible for this dual-site phosphorylation are Akt and PKCα. Proximity proteomics and interaction analysis identified filamin A-interacting protein 1 (FILIP1) as direct FLNc binding partner. FILIP1 binding induces filamin degradation, thereby negatively regulating its function. Here, dual-site phosphorylation of FLNc not only reduces FILIP1 binding, providing a mechanism to shield FLNc from FILIP1-mediated degradation, but also enables fast dynamics of FLNc necessary for its function as signaling adaptor in cross-striated muscle cells.
  23. Am J Physiol Endocrinol Metab. 2020 May 18.
      Fibroblast growth factor 21 (FGF21) is a peptide hormone that acts to enhance insulin sensitivity and reverse many of the metabolic defects associated with consumption of a high-fat diet. Recent studies show that the liver is the primary source of FGF21 in the blood, and that hepatic FGF21 expression is upregulated by glucagon. Interestingly, glucagon acts to upregulate FGF21 production by primary cultures of rat hepatocytes and H4IIE and HepG2 hepatocarcinoma cells independent of changes in FGF21 mRNA abundance, suggesting that FGF21 protein expression is regulated post-transcriptionally. Based on these observations, the goal of the present study was to assess whether or not FGF21 mRNA is translationally regulated. The results show that FGF21 mRNA translation and secretion of the hormone are significantly upregulated in H4IIE cells exposed to 25 nM glucagon, independent of changes in FGF21 mRNA abundance. Furthermore, the glucagon-induced upregulation of FGF21 mRNA translation is associated with suppressed activity of the mechanistic target of rapamycin in complex 1 (mTORC1). Similarly, the results show that rapamycin-induced suppression of mTORC1 leads to upregulation of FGF21 mRNA translation with no change in FGF21 mRNA abundance. In contrast, activation of mTORC1 by refreshing the culture medium leads to downregulation of FGF21 mRNA translation. Notably, re-feeding fasted rats also leads to downregulation of FGF21 mRNA translation concomitantly with activation of mTORC1 in the liver. Overall, the findings support a model in which glucagon acts to upregulate FGF21 production by hepatocytes through suppression of mTORC1 and subsequent upregulation of FGF21 mRNA translation.
    Keywords:  FGF21; glucagon; mRNA translation; mTORC1
  24. Nat Commun. 2020 May 21. 11(1): 2551
      Forkhead box O (FoxO) proteins and thyroid hormone (TH) have well established roles in cardiovascular morphogenesis and remodeling. However, specific role(s) of individual FoxO family members in stress-induced growth and remodeling of cardiomyocytes remains unknown. Here, we report that FoxO1, but not FoxO3, activity is essential for reciprocal regulation of types II and III iodothyronine deiodinases (Dio2 and Dio3, respectively), key enzymes involved in intracellular TH metabolism. We further show that Dio2 is a direct transcriptional target of FoxO1, and the FoxO1-Dio2 axis governs TH-induced hypertrophic growth of neonatal cardiomyocytes in vitro and in vivo. Utilizing transverse aortic constriction as a model of hemodynamic stress in wild-type and cardiomyocyte-restricted FoxO1 knockout mice, we unveil an essential role for the FoxO1-Dio2 axis in afterload-induced pathological cardiac remodeling and activation of TRα1. These findings demonstrate a previously unrecognized FoxO1-Dio2 signaling axis in stress-induced cardiomyocyte growth and remodeling and intracellular TH homeostasis.
  25. Nat Commun. 2020 May 19. 11(1): 2498
      Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5' Untranslated Regions (5'UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and "fate-switching" toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-cell-like phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.
  26. Front Oncol. 2020 ;10 452
      Breast cancer stem cells (BCSCs) are the minor population of breast cancer (BC) cells that exhibit several phenotypes such as migration, invasion, self-renewal, and chemotherapy as well as radiotherapy resistance. Recently, BCSCs have been more considerable due to their capacity for recurrence of tumors after treatment. Recognition of signaling pathways and molecular mechanisms involved in stemness phenotypes of BCSCs could be effective for discovering novel treatment strategies to target BCSCs. This review introduces BCSC markers, their roles in stemness phenotypes, and the dysregulated signaling pathways involved in BCSCs such as mitogen-activated protein (MAP) kinase, PI3K/Akt/nuclear factor kappa B (NFκB), TGF-β, hedgehog (Hh), Notch, Wnt/β-catenin, and Hippo pathway. In addition, this review presents recently discovered molecular mechanisms implicated in chemotherapy and radiotherapy resistance, migration, metastasis, and angiogenesis of BCSCs. Finally, we reviewed the role of microRNAs (miRNAs) in BCSCs as well as several other therapeutic strategies such as herbal medicine, biological agents, anti-inflammatory drugs, monoclonal antibodies, nanoparticles, and microRNAs, which have been more considerable in the last decades.
    Keywords:  angiogenesis; breast cancer stem cell; chemotherapy and radiotherapy resistance; invasion; metastasis
  27. Cancer Cell. 2020 May 15. pii: S1535-6108(20)30205-1. [Epub ahead of print]
      Functional genomics describes a field of biology that uses a range of approaches for assessing gene function with high-throughput molecular, genetic, and cellular technologies. The near limitless potential for applying these concepts to study the activities of all genetic loci has completely upended how today's cancer biologists tackle drug target discovery. We provide an overview of contemporary functional genomics platforms, highlighting areas of distinction and complementarity across technologies, so as to aid in the development or interpretation of cancer-focused screening efforts.
    Keywords:  CRISPR/Cas9; RNAi; cancer; functional genomics; genetic screen; genome editing; oncology; transposon
  28. Nat Commun. 2020 May 20. 11(1): 2517
      Alterations in non-driver genes represent an emerging class of potential therapeutic targets in cancer. Hundreds to thousands of non-driver genes undergo loss of heterozygosity (LOH) events per tumor, generating discrete differences between tumor and normal cells. Here we interrogate LOH of polymorphisms in essential genes as a novel class of therapeutic targets. We hypothesized that monoallelic inactivation of the allele retained in tumors can selectively kill cancer cells but not somatic cells, which retain both alleles. We identified 5664 variants in 1278 essential genes that undergo LOH in cancer and evaluated the potential for each to be targeted using allele-specific gene-editing, RNAi, or small-molecule approaches. We further show that allele-specific inactivation of either of two essential genes (PRIM1 and EXOSC8) reduces growth of cells harboring that allele, while cells harboring the non-targeted allele remain intact. We conclude that LOH of essential genes represents a rich class of non-driver cancer vulnerabilities.
  29. CNS Neurol Disord Drug Targets. 2020 05 17.
      Primary central nervous system lymphoma (PCNSL) is a rare invasive extranodal non-Hodgkin lymphoma, the vast majority of which is diffuse large B-cell lymphoma (DLBCL). Although high-dose methotrexate-based immunochemotherapy achieves a high remission rate, the risk of relapse and related death remains a crucial obstruction to long-term survival. Novel agents for the treatment of lymphatic malignancies have greatly broadened the horizons of therapeutic options for PCNSL. The PI3K/AKT/mTOR signaling pathway is one of the most important pathways for B-cell malignancy growth and survival. Novel therapies that target key components of this pathway have shown antitumor effects in many B-cell malignancies, including DLBCL. This review will discuss the aberrant status of the PI3K/AKT/mTOR signaling pathways in PCNSL and the application prospects of inhibitors in hopes of providing alternative clinical therapy strategies and improving prognosis.
    Keywords:  AKT; PI3K; inhibitors; mTOR; primary central nervous system lymphoma; targeted therapy
  30. Cell Syst. 2020 May 20. pii: S2405-4712(20)30148-4. [Epub ahead of print]10(5): 384-396.e9
      Complex networks of regulatory relationships between protein kinases comprise a major component of intracellular signaling. Although many kinase-kinase regulatory relationships have been described in detail, these tend to be limited to well-studied kinases whereas the majority of possible relationships remains unexplored. Here, we implement a data-driven, supervised machine learning method to predict human kinase-kinase regulatory relationships and whether they have activating or inhibiting effects. We incorporate high-throughput data, kinase specificity profiles, and structural information to produce our predictions. The results successfully recapitulate previously annotated regulatory relationships and can reconstruct known signaling pathways from the ground up. The full network of predictions is relatively sparse, with the vast majority of relationships assigned low probabilities. However, it nevertheless suggests denser modes of inter-kinase regulation than normally considered in intracellular signaling research. A record of this paper's transparent peer review process is included in the Supplemental Information.
    Keywords:  intracellular signaling; machine learning; phosphorylation; protein kinase; signaling networks