bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2019‒08‒18
forty-three papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Immunol Rev. 2019 Sep;291(1): 154-173
      Phosphatidylinositol 3 kinases (PI3K) are a family of lipid kinases that are activated by a variety of cell-surface receptors, and regulate a wide range of downstream readouts affecting cellular metabolism, growth, survival, differentiation, adhesion, and migration. The importance of these lipid kinases in lymphocyte signaling has recently been highlighted by genetic analyses, including the recognition that both activating and inactivating mutations of the catalytic subunit of PI3Kδ, p110δ, lead to human primary immunodeficiencies. In this article, we discuss how studies on the human genetic disorder "Activated PI3K-delta syndrome" and mouse models of this disease (Pik3cdE1020K/+ mice) have provided fundamental insight into pathways regulated by PI3Kδ in T and B cells and their contribution to lymphocyte function and disease, including responses to commensal bacteria and the development of autoimmunity and tumors. We highlight critical roles of PI3Kδ in T follicular helper cells and the orchestration of the germinal center reaction, as well as in CD8+ T-cell function. We further  present data demonstrating the ability of the AKT-resistant FOXO1AAA mutant to rescue IgG1 class switching defects in Pik3cdE1020K/+ B cells, as well as data supporting a role for PI3Kδ in promoting multiple T-helper effector cell lineages.
    Keywords:  APDS/PASLI; T and B lymphocytes; autoimmunity; immunodeficiency; phosphatidylinositol 3 kinase
  2. J Cell Sci. 2019 Aug 16. pii: jcs231639. [Epub ahead of print]132(16):
      Macropinocytosis is an actin-dependent but clathrin-independent endocytic process by which cells nonselectively take up large aliquots of extracellular material. Macropinocytosis is used for immune surveillance by dendritic cells, as a route of infection by viruses and protozoa, and as a nutrient uptake pathway in tumor cells. In this study, we explore the role of class I phosphoinositide 3-kinases (PI3Ks) during ligand-stimulated macropinocytosis. We find that macropinocytosis in response to receptor tyrosine kinase activation is strikingly dependent on a single class I PI3K isoform, namely PI3Kβ (containing the p110β catalytic subunit encoded by PIK3CB). Loss of PI3Kβ expression or activity blocks macropinocytosis at early steps, before the formation of circular dorsal ruffles, but also plays a role in later steps, downstream from Rac1 activation. PI3Kβ is also required for the elevated levels of constitutive macropinocytosis found in tumor cells that are defective for the PTEN tumor suppressor. Our data shed new light on PI3K signaling during macropinocytosis, and suggest new therapeutic uses for pharmacological inhibitors of PI3Kβ.
    Keywords:  Circular dorsal ruffles; HGF; Macropinocytosis; PDGF; PIK3CB; PTEN; Phosphoinositide 3-kinase; Rac; Tumor cells
  3. Nat Commun. 2019 Aug 16. 10(1): 3716
      The Anaphase Promoting Complex (APC) coactivator Cdh1 drives proper cell cycle progression and is implicated in the suppression of tumorigenesis. However, it remains elusive how Cdh1 restrains cancer progression and how tumor cells escape the inhibition of Cdh1. Here we report that Cdh1 suppresses the kinase activity of c-Src in an APC-independent manner. Depleting Cdh1 accelerates breast cancer cell proliferation and cooperates with PTEN loss to promote breast tumor progression in mice. Hyperactive c-Src, on the other hand, reciprocally inhibits the ubiquitin E3 ligase activity of APCCdh1 through direct phosphorylation of Cdh1 at its N-terminus, which disrupts the interaction between Cdh1 and the APC core complex. Furthermore, pharmacological inhibition of c-Src restores APCCdh1 tumor suppressor function to repress a panel of APCCdh1 oncogenic substrates. Our findings reveal a reciprocal feedback circuit of Cdh1 and c-Src in the crosstalk between the cell cycle machinery and the c-Src signaling pathway.
  4. Immunol Rev. 2019 Sep;291(1): 134-153
      T cells play important roles in autoimmune diseases and cancer. Following the cloning of the T cell receptor (TCR), the race was on to map signaling proteins that contributed to T cell activation downstream of the TCR as well as co-stimulatory molecules such as CD28. We term this "canonical TCR signaling" here. More recently, it has been appreciated that T cells need to accommodate increased metabolic needs that stem from T cell activation in order to function properly. A central role herein has emerged for mechanistic/mammalian target of rapamycin (mTOR). In this review we briefly cover canonical TCR signaling to set the stage for discussion on mTOR signaling, mRNA translation, and metabolic adaptation in T cells. We also discuss the role of mTOR in follicular helper T cells, regulatory T cells, and other T cell subsets. Our lab recently uncovered that "tonic signals", which pass through proximal TCR signaling components, are robustly and selectively transduced to mTOR to promote baseline translation of various mRNA targets. We discuss insights on (tonic) mTOR signaling in the context of T cell function in autoimmune diseases such as lupus as well as in cancer immunotherapy through CAR-T cell or checkpoint blockade approaches.
    Keywords:  T cell; autoimmune; cancer; mammalian target of rapamycin; signaling; tonic
  5. Exp Ther Med. 2019 Sep;18(3): 2278-2284
      The aim of the present study was to evaluate the mutation and amplification status of the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) gene, as well as the association with clinicopathological characteristics and prognosis, in Japanese patients with cervical cancer. Fluorescence in situ hybridization and polymerase chain reaction were performed to assess PIK3CA gene amplification and mutation. The inhibitors temsirolimus and NVP-BEZ235 were used to inactivate the phosphatidylinositide 3-kinase (PI3K)/AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin kinase (mTOR) pathway to clarify the roles of PI3K/AKT activation in cervical carcinoma cells harboring associated mutations. Four somatic point mutations (4/71, 5.6%) were found in exon 20 in cervical squamous cell carcinoma samples, whereas three (3/53, 5.7%) were found in exon 9 in cervical adeno/adenosquamous cell carcinoma samples. Amplification of PIK3CA was also observed in this study and amplification was more commonly found in adeno/adenosquamous carcinomas than in cervical squamous cell carcinomas (20.7 vs. 1.4%, respectively, P=0.0003). No significant correlation was obesrved between PIK3CA amplification and progression free survival (P=0.7576) or overall survival (P=0.8859). Moreover, no association between PIK3CA mutation and sensitivity to PI3K/AKT/mTOR inhibitors was observed in cervical carcinoma cells. These results suggest that in Japanese patients with cervical cancer, PIK3CA mutation and amplification cannot act as biomarkers for individualized molecular targeted therapy.
    Keywords:  PIK3CA; adeno/adenosquamous cell carcinoma; cervical cancer; gene amplification; gene mutation; squamous cell carcinoma
  6. J Allergy Clin Immunol. 2019 Aug 08. pii: S0091-6749(19)31029-2. [Epub ahead of print]
      BACKGROUND: Perturbation of epidermal barrier formation will profoundly compromise overall skin function, leading to a dry and scaly, ichthyosis-like skin phenotype, which is the hallmark of a broad range of skin diseases, including ichthyosis, atopic dermatitis, and a multitude of clinical eczema variants. An overarching molecular mechanism that orchestrates the multitude of factors controlling epidermal barrier formation and homeostasis remains to be elucidated.OBJECTIVE: Here we highlight a specific role of mammalian target of rapamycin complex 2 (mTORC2) signaling in epidermal barrier formation.
    METHODS: Epidermal mTORC2 signaling was specifically disrupted by deleting Rictor, encoding an essential subunit of mTORC2 in mouse epidermis (RicEKO). Epidermal structure and barrier function were investigated by a combination of gene expression, biochemical, morphological and functional analysis in RicEKO and control mice.
    RESULTS: RicEKO newborns displayed an ichthyosis-like phenotype characterized by dysregulated epidermal de novo lipid synthesis, altered lipid lamellae structure, and aberrant filaggrin processing. Despite a compensatory transcriptional epidermal repair response, the protective epidermal function was impaired in RicEKO mice as revealed by increased transepidermal water loss, enhanced corneocyte fragility, decreased dendritic epidermal T cells, and an exaggerated percutaneous immune response. Restoration of Akt-Ser473 phosphorylation in mTORC2-deficient keratinocytes by expression of constitutive Akt rescued filaggrin processing.
    CONCLUSION: Our findings reveal a critical metabolic signaling relay of barrier formation where epidermal mTORC2 activity controls filaggrin processing and de novo epidermal lipid synthesis during cornification. Our findings provide novel mechanistic insights into epidermal barrier formation and could open up new therapeutic opportunities to restore defective epidermal barrier conditions.
    Keywords:  Epidermal barrier; epidermal lipid synthesis; filaggrin; ichthyosis; mTORC2; rictor
  7. Oncogene. 2019 Aug 16.
      Mutational activation of the epidermal growth factor receptor (EGFR) is a major player in the pathogenesis of non-small cell lung cancer (NSCLC). NSCLC patients with constitutively active EGFR mutations eventually develop drug resistance against EGFR tyrosine-kinase inhibitors; therefore, better understandings of key components of mutant EGFR (mtEGFR) signaling are required. Here, we initially observed aberrantly high expression of protein kinase Cα (PKCα) in lung adenocarcinomas, especially those with EGFR mutations, and proceeded to examine the role of PKCα in the regulation of the signaling pathways downstream of mtEGFR. The results showed that NSCLC cell lines with constitutively active EGFR mutations tend to have very or moderately high PKCα levels. Furthermore, PKCα was constitutively activated in HCC827 and H4006 cells which have an EGFR deletion mutation in exon 19. Interestingly, mtEGFR was not required for the induction of PKCα at protein and message levels suggesting that the increased levels of PKCα are due to independent selection. On the other hand, mtEGFR activity was required for robust activation of PKCα. Loss of functions studies revealed that the NSCLC cells rely heavily on PKCα for the activation of the mTORC1 signaling pathway. Unexpectedly, the results demonstrated that PKCα was required for activation of Akt upstream of mTOR but only in cells with the mtEGFR and with the increased expression of PKCα. Functionally, inhibition of PKCα in HCC827 led to caspase-3-dependent apoptosis and a significant decrease in cell survival in response to cellular stress induced by serum starvation. In summary, the results identified important roles of PKCα in regulating mTORC1 activity in lung cancer cells, whereby a primary switching occurs from PKCα-independent to PKCα-dependent signaling in the presence of EGFR mutations. The results present PKCα as a potential synergistic target of personalized treatment for NSCLC with constitutively active mutant forms of EGFR and constitutively active PKCα.
  8. Semin Cancer Biol. 2019 Aug 10. pii: S1044-579X(18)30135-4. [Epub ahead of print]
      Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that gets inputs from the amino acids, nutrients, growth factor, and environmental cues to regulate varieties of fundamental cellular processes which include protein synthesis, growth, metabolism, aging, regeneration, autophagy, etc. The mTOR is frequently deregulated in human cancer and activating somatic mutations of mTOR were recently identified in several types of human cancer and hence mTOR is therapeutically targeted. mTOR inhibitors were commonly used as immunosuppressors and currently, it is approved for the treatment of human malignancies. This review briefly focuses on the structure and biological functions of mTOR. It extensively discusses the genetic deregulation of mTOR including amplifications and somatic mutations, mTOR-mediated cell growth promoting signaling, therapeutic targeting of mTOR and the mechanisms of resistance, the role of mTOR in precision medicine and other recent advances in further understanding the role of mTOR in cancer.
    Keywords:  cancer; inhibitors; mTOR; metastasis; molecular target; mutation; oncogene; precision medicine; resistance
  9. J Cachexia Sarcopenia Muscle. 2019 Aug 11.
      BACKGROUND: Skeletal muscle wasting is often associated with insulin resistance. A major regulator of muscle mass is the transforming growth factor β (TGF-β) superfamily, including activin A, which causes atrophy. TGF-β superfamily ligands also negatively regulate insulin-sensitive proteins, but whether this pathway contributes to insulin action remains to be determined.METHODS: To elucidate if TGF-β superfamily ligands regulate insulin action, we used an adeno-associated virus gene editing approach to overexpress an activin A inhibitor, follistatin (Fst288), in mouse muscle of lean and diet-induced obese mice. We determined basal and insulin-stimulated 2-deoxy-glucose uptake using isotopic tracers in vivo. Furthermore, to evaluate whether circulating Fst and activin A concentrations are associated with obesity, insulin resistance, and weight loss in humans, we analysed serum from morbidly obese subjects before, 1 week, and 1 year after Roux-en-Y gastric bypass (RYGB).
    RESULTS: Fst288 muscle overexpression markedly increased in vivo insulin-stimulated (but not basal) glucose uptake (+75%, P < 0.05) and increased protein expression and intracellular insulin signalling of AKT, TBC1D4, PAK1, pyruvate dehydrogenase-E1α, and p70S6K, while decreasing TBC1D1 signaling (P < 0.05). Fst288 increased both basal and insulin-stimulated protein synthesis, but no correlation was observed between the Fst288-driven hypertrophy and the increase in insulin-stimulated glucose uptake. Importantly, Fst288 completely normalized muscle glucose uptake in insulin-resistant diet-induced obese mice. RYGB surgery doubled circulating Fst and reduced activin A (-24%, P < 0.05) concentration 1 week after surgery before any significant weight loss in morbidly obese normoglycemic patients, while major weight loss after 1 year did not further change the concentrations.
    CONCLUSIONS: We here present evidence that Fst is a potent regulator of insulin action in muscle, and in addition to AKT and p70S6K, we identify TBC1D1, TBC1D4, pyruvate dehydrogenase-E1α, and PAK1 as Fst targets. Circulating Fst more than doubled post-RYGB surgery, a treatment that markedly improved insulin sensitivity, suggesting a role for Fst in regulating glycaemic control. These findings demonstrate the therapeutic potential of inhibiting TGF-β superfamily ligands to improve insulin action and Fst's relevance to muscle wasting-associated insulin-resistant conditions in mice and humans.
    Keywords:  Follistatin; Glucose uptake; Glycaemic control; Insulin resistance; Muscle wasting; TGF-β
  10. Cancer Res. 2019 Aug 15. pii: canres.0325.2019. [Epub ahead of print]
      Phosphoinositide-(3)-kinase (PI3K)-targeting therapy represents one of the most sought-after therapies for glioblastoma (GBM). Several small molecule inhibitors have been evaluated in clinical trials; however, the emergence of resistance limits treatment potential. Here, we generated a patient-derived glioma sphere-forming cell (GSC) xenograft model resistant to the PI3K specific inhibitor BKM-120. Integrated RNA-Seq and high-throughput drug screening revealed that the Aurora A kinase (Aurora A)/Polo-like kinase 1 (PLK1)/Cyclin-dependent kinase 1 (CDK1) signaling pathway was the main driver of PI3K inhibitor resistance in the resistant xenografts. Aurora kinase was upregulated and CDK1 was downregulated in resistant tumors from both xenografts and tumor tissues from patients treated with the PI3K inhibitor. Mechanistically, the tyrosine kinase receptor Tie2 physically interacted with FGFR1 promoting STAT3 phosphorylation and binding to the AURKA promoter, which increased Aurora A expression in resistant GSCs. Concurrent inhibition of Aurora A and PI3K signaling overcame PI3K inhibitor-induced resistance. This study offers a proof of concept to target PI3K and the collateral-activated pathway to improve GBM therapy.
  11. Semin Cancer Biol. 2019 Aug 10. pii: S1044-579X(19)30039-2. [Epub ahead of print]
      Breast cancer is the major cause of deaths in women worldwide. Detection and treatment of breast cancer at earlier stages of the disease has shown encouraging results. Modern genomic technologies facilitated several therapeutic options however the diagnosis of the disease at an advanced stage claim more deaths. Therefore more research directed towards genomics and proteomics into this area may lead to novel biomarkers thereby enhancing the survival rates in breast cancer patients. Phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway was shown to be hyperactivated in most of the breast carcinomas resulting in excessive growth, proliferation, and tumor development. Development of nanotechnology has provided many interesting avenues to target the PI3K/Akt/mTOR pathway both at the pre-clinical and clinical stages. Therefore, the current review summarizes the underlying mechanism and the importance of targeting PI3K/Akt/mTOR pathway, novel biomarkers and use of nanotechnological interventions in breast cancer.
    Keywords:  PI3K/Akt/mTOR pathway; breast Cancer; breast cancer biomarkers; deregulated genes; nanotechnological interventions
  12. Lancet Oncol. 2019 Aug 08. pii: S1470-2045(19)30334-1. [Epub ahead of print]
      BACKGROUND: Endocrine therapy-based neoadjuvant treatment for luminal breast cancer allows efficient testing of new combinations before surgery. The activation of the phosphatidylinositol-3-kinase (PI3K) pathway is a known mechanism of resistance to endocrine therapy. Taselisib is an oral, selective PI3K inhibitor with enhanced activity against PIK3CA-mutant cancer cells. The LORELEI trial tested whether taselisib in combination with letrozole would result in an increased proportion of objective responses and pathological complete responses.METHODS: In this multicentre, randomised, double-blind, parallel-cohort, placebo-controlled phase 2, study, we enrolled postmenopausal women (aged ≥18 years) with histologically confirmed, oestrogen receptor (ER)-positive, HER2-negative, stage I-III, operable breast cancer, from 85 hospitals in 22 countries worldwide. To be eligible, patients had have an Eastern Cooperative Oncology Group (ECOG) performance status 0-1, adequate organ function, and had to have evaluable tumour tissue for PIK3CA genotyping. Patients were randomly assigned (1:1) by means of a permuted block algorithm (block size of four) via an interactive voice or web-based response system, to receive letrozole (2·5 mg/day orally, continuously) with either 4 mg of oral taselisib or placebo (on a 5 days-on, 2 days-off schedule) for 16 weeks, followed by surgery. Randomisation was stratified by tumour size and nodal status. Site staff, patients, and the sponsor were masked to treatment assignment. Coprimary endpoints were the proportion of patients who achieved an objective response by centrally assessed breast MRI and a locally assessed pathological complete response in the breast and axilla (ypT0/Tis, ypN0) at surgery in all randomly assigned patients and in patients with PIK3CA-mutant tumours. Analyses were done in the intention-to-treat population. This trial is registered with, number NCT02273973, and is closed to accrual.
    FINDINGS: Between Nov 12, 2014, and Aug 12, 2016, 334 participants were enrolled and randomly assigned to receive letrozole and placebo (n=168) or letrozole and taselisib (n=166). Median follow-up was 4·9 months (IQR 4·7-5·1). The study met one of its primary endpoints: the addition of taselisib to letrozole was associated with a higher proportion of patients achieving an objective response in all randomly assigned patients (66 [39%] of 168 patients in the placebo group vs 83 [50%] of 166 in the taselisib group; odds ratio [OR] 1·55, 95% CI 1·00-2·38; p=0·049) and in the PIK3CA-mutant subset (30 [38%] of 79 vs 41 [56%] of 73; OR 2·03, 95% CI 1·06-3·88; p=0·033). No significant differences were observed in pathological complete response between the two groups, either in the overall population (three [2%] of 166 in the taselisib group vs one [1%] of 168 in the placebo group; OR 3·07 [95% CI 0·32-29·85], p=0·37) or in the PIK3CA-mutant cohort (one patient [1%) vs none [0%]; OR not estimable, p=0·48). The most common grade 3-4 adverse events in the taselisib group were gastrointestinal (13 [8%] of 167 patients), infections (eight [5%]), and skin-subcutaneous tissue disorders (eight [5%]). In the placebo group, four (2%) of 167 patients had grade 3 or worse vascular disorders, two (1%) had gastrointestinal disorders, and two (1%) patients had grade 3 or worse infections and infestations. There was no grade 4 hyperglycaemia and grade 3 cases were asymptomatic. Serious adverse events were more common in the taselisib group (eight [5%] patients with infections and seven [4%] with gastrointestinal effects) than in the placebo group (one [1%] patient each with grade 3 postoperative wound and haematoma infection, grade 4 hypertensive encephalopathy, grade 3 acute cardiac failure, and grade 3 breast pain). One death occurred in the taselisib group, which was not considered to be treatment-related.
    INTERPRETATION: The increase in the proportion of patients who achieved an objective response from the addition of taselisib to endocrine therapy in a neoadjuvant setting is consistent with the clinical benefit observed in hormone receptor-positive, HER2-negative, metastatic breast cancer.
    FUNDING: Genentech and F Hoffmann-La Roche.
  13. Sci Rep. 2019 Aug 15. 9(1): 11928
      Human embryonal carcinoma (EC) cells comprise the pluripotent stem cells of malignant non-seminomatous germ cell tumors (GCTs) and represent the malignant counterpart of embryonic stem cells (ESCs). WNT/β-catenin signaling has been implicated in regulating adult and embryonic stem cells although its role in EC cells is less investigated. Here, we studied WNT signaling in a panel of representative pluripotent and nullipotent human EC cell lines. We found that EC cell lines show distinct levels of intrinsic WNT signaling and respond differently to ectopic WNT activation. Short-term activation of WNT signaling induced a differentiation-response in the pluripotent EC cells (NT2 and NCCIT) whereas the nullipotent EC cells (TERA1 and 2102Ep) were refractory and maintained high levels of OCT4 and SSEA4 expression. Long-term activation of WNT signaling in NCCIT and, to a lesser extent, TERA1 cells led to (re)gain of OCT4 expression and a switch from SSEA4 to SSEA1 surface antigens ultimately resulting in OCT4+/SSEA4-/SSEA1+ profile. Cisplatin treatment indicated that the OCT4+/SSEA4-/SSEA1+ NCCIT cells became more resistant to chemotherapy treatment. Our findings are of particular interest for the GCT and ES cell biology and shed light on the role of WNT signaling in human EC cells.
  14. PLoS Genet. 2019 Aug;15(8): e1008318
      Elevated uric acid (UA) is a key risk factor for many disorders, including metabolic syndrome, gout and kidney stones. Despite frequent occurrence of these disorders, the genetic pathways influencing UA metabolism and the association with disease remain poorly understood. In humans, elevated UA levels resulted from the loss of the of the urate oxidase (Uro) gene around 15 million years ago. Therefore, we established a Drosophila melanogaster model with reduced expression of the orthologous Uro gene to study the pathogenesis arising from elevated UA. Reduced Uro expression in Drosophila resulted in elevated UA levels, accumulation of concretions in the excretory system, and shortening of lifespan when reared on diets containing high levels of yeast extract. Furthermore, high levels of dietary purines, but not protein or sugar, were sufficient to produce the same effects of shortened lifespan and concretion formation in the Drosophila model. The insulin-like signaling (ILS) pathway has been shown to respond to changes in nutrient status in several species. We observed that genetic suppression of ILS genes reduced both UA levels and concretion load in flies fed high levels of yeast extract. Further support for the role of the ILS pathway in modulating UA metabolism stems from a human candidate gene study identifying SNPs in the ILS genes AKT2 and FOXO3 being associated with serum UA levels or gout. Additionally, inhibition of the NADPH oxidase (NOX) gene rescued the reduced lifespan and concretion phenotypes in Uro knockdown flies. Thus, components of the ILS pathway and the downstream protein NOX represent potential therapeutic targets for treating UA associated pathologies, including gout and kidney stones, as well as extending human healthspan.
  15. Hum Mol Genet. 2019 Aug 14. pii: ddz194. [Epub ahead of print]
      Single germline or somatic activating mutations of mTOR pathway genes are emerging as a major cause of Type II Focal Cortical Dysplasia (FCD), hemimegalencephaly (HME), and Tuberous Sclerosis Complex (TSC). A double hit mechanism, based on a primary germline mutation in one allele and a secondary somatic hit affecting the other allele of the same gene in a small number of cells, has been documented in some patients with TSC or FCD. In a patient with HME, severe intellectual disability, intractable seizures, and hypochromic skin patches, we identified the RPS6 p.R232H variant, present as somatic mosaicism at ~ 15.1% in dysplastic brain tissue and ~ 11% in blood, and the MTOR p.S2215F variant, detected as ~ 8.8% mosaicism in brain tissue, but not in blood. Overexpressing the two variants independently in animal models, we demonstrated that MTOR p.S2215F caused neuronal migration delay and cytomegaly, while RPS6 p.R232H prompted increased cell proliferation. Double mutants exhibited a more severe phenotype, with increased proliferation and migration defects at embryonic stage and, at postnatal stage, cytomegalic cells exhibiting eccentric nuclei and binucleation, which are typical features of balloon cells. These findings suggest a synergistic effect of the two variants. This study indicates that, in addition to single activating mutations and double-hit inactivating mutations in mTOR pathway genes, severe forms of cortical dysplasia can also result from activating mutations affecting different genes in this pathway. RPS6 is a potential novel disease-related gene.
  16. Elife. 2019 Aug 16. pii: e47990. [Epub ahead of print]8
      White adipose tissue (WAT) inflammation contributes to the development of insulin resistance in obesity. While the role of adipose tissue macrophage (ATM) pro-inflammatory signalling in the development of insulin resistance has been established, it is less clear how WAT inflammation is initiated. Here, we show that ATMs isolated from obese mice and humans exhibit markers of increased rate of de novo phosphatidylcholine (PC) biosynthesis. Macrophage-specific knockout of phosphocholine cytidylyltransferase A (CCTa), the rate-limiting enzyme of de novo PC biosynthesis pathway, alleviated obesity-induced WAT inflammation and insulin resistance. Mechanistically, CCTa-deficient macrophages showed reduced ER stress and inflammation in response to palmitate. Surprisingly, this was not due to lower exogenous palmitate incorporation into cellular PCs. Instead, CCTa-null macrophages had lower membrane PC turnover, leading to elevated membrane polyunsaturated fatty acid levels that negated the pro-inflammatory effects of palmitate. Our results reveal a causal link between obesity-associated increase in de novo PC synthesis, accelerated PC turnover and pro-inflammatory activation of ATMs.
    Keywords:  cell biology; human; human biology; medicine; mouse
  17. Front Oncol. 2019 ;9 653
      Breast cancer represents the most common malignancy in women worldwide and the ErbB/PI3K pathway has been found to play a crucial role in regulation of the cancer cell growth. MicroRNAs have been implicated in regulating diverse cellular pathways and therefore, understanding the link between the regulatory microRNAs and the ErbB/PI3K signaling pathway could potentially be helpful for breast cancer prevention and treatment. The aim of this study is to examine the regulatory effect of miR-326 on ErbB/PI3K signaling pathway in breast cancer development and progression. The results of qRT-PCR, RNA seq, and array data indicated that miR-326 was remarkably down-regulated in breast tumor tissues and correlated with poor survival outcome. Importantly, very low levels of miR-326 expression were found in aggressive breast cells compared to less-aggressive cell types. Mechanistically, a gene network including EGFR, ErbB2, ErbB3, AKT1, AKT2, and AKT3 targeted by miR-326, thereby providing suppression of ErbB/PI3K pathway, detected by RT-qPCR, and dual luciferase assay. In addition, Western blot analysis revealed that miR-326 upregulation decreased PI3K signaling activity by decreasing total AKT and p-AKT protein level in SKBR3 cell lines. Interestingly, up regulation of ErbB2 rescued the effect of miR-326 on miR-326 target genes. Further functional assays demonstrated that up regulation of miR-326 significantly suppressed cell growth as evidenced by cell cycle, cell cycle associated genes expression, colony formation and MTT assays and induced apoptosis, detected by Annexin V-PI. In addition, EMT markers RT-qPCR, scratch, and Transwell assays showed inhibited cellular migration and invasion following miR-326 upregulation. Altogether, our results revealed that miR-326 play a tumor-suppressive role in breast cancer through inhibiting ErbB/PI3K pathway and miR-326 may serve as a potential therapeutic target for the treatment of patients with breast cancer.
    Keywords:  ErbB/PI3K signaling pathway; breast cancer; miR-326; microRNA; tumor suppressor
  18. Gerontology. 2019 Aug 14. 1-8
      In this manuscript, we summarize published results showing that obesity and aging are inflammatory conditions associated with serious health problems, increased risk for disease and death. We show that fat mass increases with age and represents a major contributor to insulin resistance and the metabolic syndrome. We summarize the effects of age on the adipose tissue (AT), related to the abundance, distribution, cellular composition, endocrine signaling and function of the tissue. The AT is an immunological tissue, with several hallmarks of innate and adaptive immune responses. We show that in both mice and humans, the AT is heavily infiltrated by immune cells that have receptors for pro-inflammatory cytokines and chemokines secreted by the adipocytes and also by the immune cells that have infiltrated the AT. We also show that the AT provides an environment for the secretion of IgG antibodies with anti-self (autoimmune) reactivity. As we have previously shown, this is due to the release of self antigens following cell death due to hypoxia, as well as to the expression of activation-induced cytidine deaminase, the enzyme of class switch recombination, and the transcription factor T-bet by the resident B cells, which also express the membrane marker CD11c, both involved in the production of autoimmune IgG antibodies. We show data in support of the AT as a tertiary lymphoid organ (TLO), showing the examples of TLOs that develop within the AT, such as fat-associated lymphoid clusters and milky spots, as well as artery TLOs that develop in the adventitia areas of the aorta.
    Keywords:  Adipose tissue; Aging; Immunosenescence; Inflammaging
  19. Cancers (Basel). 2019 Aug 14. pii: E1169. [Epub ahead of print]11(8):
      Soft tissue sarcoma (STS) is a rare malignancy of mesenchymal origin classified into more than 50 different subtypes with distinct clinical and pathologic features. Despite the poor prognosis in the majority of patients, only modest improvements in treatment strategies have been achieved, largely due to the rarity and heterogeneity of these tumors. Therefore, the discovery of new prognostic and predictive biomarkers, together with new therapeutic targets, is of enormous interest. Phosphatase and tensin homolog (PTEN) is a well-known tumor suppressor that commonly loses its function via mutation, deletion, transcriptional silencing, or protein instability, and is frequently downregulated in distinct sarcoma subtypes. The loss of PTEN function has consequent alterations in important pathways implicated in cell proliferation, survival, migration, and genomic stability. PTEN can also interact with other tumor suppressors and oncogenic signaling pathways that have important implications for the pathogenesis in certain STSs. The aim of the present review is to summarize the biological significance of PTEN in STS and its potential role in the development of new therapeutic strategies.
    Keywords:  PTEN; Soft tissue sarcoma; epithelioid sarcoma; gastrointestinal stromal tumor; leiomyosarcoma; liposarcoma; malignant peripheral nerve sheath tumor; myxofibrosarcoma; synovial sarcoma; undifferentiated pleomorphic sarcoma
  20. Oncogene. 2019 Aug 16.
      miR-29b has been identified as a rapamycin-induced microRNA (miRNA) in Tsc2-deficient, mTORC1-hyperactive cells. The biological significance of this induction of miR-29b is unknown. We have found that miR-29b acts as an oncogenic miRNA in Tsc2-deficient cells: inhibition of miR-29b suppressed cell proliferation, anchorage-independent cell growth, cell migration, invasion, and the growth of Tsc2-deficient tumors in vivo. Importantly, the combination of miR-29b inhibition with rapamycin treatment further inhibited these tumor-associated cellular processes. To gain insight into the molecular mechanisms by which miR-29b promotes tumorigenesis, we used RNA sequencing to identify the tumor suppressor retinoid receptor beta (RARβ) as a target gene of miR-29b. We found that miR-29b directly targeted the 3'UTR of RARβ. Forced expression of RARβ reversed the effects of miR-29b overexpression in proliferation, migration, and invasion, indicating that it is a critical target. miR-29b expression correlated with low RARβ expression in renal clear cell carcinomas and bladder urothelial carcinomas, tumors associated with TSC gene mutations. We further identified growth family member 4 (ING4) as a novel interacting partner of RARβ. Overexpression of ING4 inhibited the migration and invasion of Tsc2-deficient cells while silencing of ING4 reversed the RARβ-mediated suppression of cell migration and invasion. Taken together, our findings reveal a novel miR-29b/RARβ/ING4 pathway that regulates tumorigenic properties of Tsc2-deficient cells, and that may serve as a potential therapeutic target for TSC, lymphangioleiomyomatosis (LAM), and other mTORC1-hyperactive tumors.
  21. J Mol Endocrinol. 2019 Aug 01. pii: JME-18-0274.R2. [Epub ahead of print]
      Gestational diabetes mellitus (GDM) imposes serious short- and long-term health problems for mother and baby. An effective therapeutic that can reduce the incidence of GDM and improve long-term maternal and fetal outcomes is a major research priority, crucially important for public health. A lack of knowledge about the underlying pathophysiology of GDM has hampered the development of such therapeutics. What we do know, however, is that maternal insulin resistance, low-grade inflammation and endothelial cell dysfunction are three central features of pregnancies complicated by GDM. Indeed, data generated over the past decade have implicated a number of candidate regulators of insulin resistance, inflammation and endothelial cell dysfunction in placenta, maternal adipose tissue and skeletal muscle. These include nuclear factor-κB (NF-κB), peroxisome proliferator activated receptors (PPARs), sirtuins (SIRTs), 5' AMP-activated protein kinase (AMPK), glycogen synthase kinase 3 (GSK3), PI3K/mTOR, inflammasome and endoplasmic reticulum (ER) stress. In this review, the identification of these as key modulators of GDM will be discussed. The biochemical pathways involved in the formation of these may represent potential sites for intervention that may translate to therapeutic interventions to prevent the development of GDM.
  22. FASEB J. 2019 Aug 10. fj201900841R
      AMPK is a central regulator of energy homeostasis. AMPK not only elicits acute metabolic responses but also promotes metabolic reprogramming and adaptations in the long-term through regulation of specific transcription factors and coactivators. We performed a whole-genome transcriptome profiling in wild-type (WT) and AMPK-deficient mouse embryonic fibroblasts (MEFs) and primary hepatocytes that had been treated with 2 distinct classes of small-molecule AMPK activators. We identified unique compound-dependent gene expression signatures and several AMPK-regulated genes, including folliculin (Flcn), which encodes the tumor suppressor FLCN. Bioinformatics analysis highlighted the lysosomal pathway and the associated transcription factor EB (TFEB) as a key transcriptional mediator responsible for AMPK responses. AMPK-induced Flcn expression was abolished in MEFs lacking TFEB and transcription factor E3, 2 transcription factors with partially redundant function; additionally, the promoter activity of Flcn was profoundly reduced when its putative TFEB-binding site was mutated. The AMPK-TFEB-FLCN axis is conserved across species; swimming exercise in WT zebrafish induced Flcn expression in muscle, which was significantly reduced in AMPK-deficient zebrafish. Mechanistically, we have found that AMPK promotes dephosphorylation and nuclear localization of TFEB independently of mammalian target of rapamycin activity. Collectively, we identified the novel AMPK-TFEB-FLCN axis, which may function as a key cascade for cellular and metabolic adaptations.-Collodet, C., Foretz, M., Deak, M., Bultot, L., Metairon, S., Viollet, B., Lefebvre, G., Raymond, F., Parisi, A., Civiletto, G., Gut, P., Descombes, P., Sakamoto, K. AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR.
    Keywords:  991; AICAR; Birt-Hogg-Dubé syndrome; folliculin; folliculin interacting protein
  23. Stem Cell Res Ther. 2019 Aug 14. 10(1): 255
      BACKGROUND: Obesity impairs a variety of cell types including adipose-derived mesenchymal stem cells (ASCs). ASCs are indispensable for tissue homeostasis/repair, immunomodulation, and cell renewal. It has been demonstrated that obese ASCs are defective in differentiation, motility, immunomodulation, and replication. We have recently reported that some of these defects are linked to impaired primary cilia, which are unable to properly convey and coordinate a variety of signaling pathways. We hypothesized that the rescue of the primary cilium in obese ASCs would restore their functional properties.METHODS: Obese ASCs derived from subcutaneous and visceral adipose tissues were treated with a specific inhibitor against Aurora A or with an inhibitor against extracellular signal-regulated kinase 1/2 (Erk1/2). Multiple molecular and cellular assays were performed to analyze the altered functionalities and their involved pathways.
    RESULTS: The treatment with low doses of these inhibitors extended the length of the primary cilium, restored the invasion and migration potential, and improved the differentiation capacity of obese ASCs. Associated with enhanced differentiation ability, the cells displayed an increased expression of self-renewal/stemness-related genes like SOX2, OCT4, and NANOG, mediated by reduced active glycogen synthase kinase 3 β (GSK3β).
    CONCLUSION: This work describes a novel phenomenon whereby the primary cilium of obese ASCs is rescuable by the low-dose inhibition of Aurora A or Erk1/2, restoring functional ASCs with increased stemness. These cells might be able to improve tissue homeostasis in obese patients and thereby ameliorate obesity-associated diseases. Additionally, these functionally restored obese ASCs could be useful for novel autologous mesenchymal stem cell-based therapies.
    Keywords:  Adipose-derived mesenchymal stem cells; Aurora A; Extracellular signal-regulated kinase 1/2; Glycogen synthase kinase 3 beta; Obesity; Primary cilium
  24. Elife. 2019 Aug 14. pii: e49158. [Epub ahead of print]8
      Target of rapamycin complex 1 (TORC1) and AMP-activated protein kinase (AMPK) antagonistically modulate metabolism and aging. However, how they coordinate to determine longevity and if they act via separable mechanisms is unclear. Here, we show that neuronal AMPK is essential for lifespan extension from TORC1 inhibition, and that TORC1 suppression increases lifespan cell non autonomously via distinct mechanisms from global AMPK activation. Lifespan extension by null mutations in genes encoding raga-1 (RagA) or rsks-1 (S6K) is fully suppressed by neuronal-specific rescues. Loss of RAGA-1 increases lifespan via maintaining mitochondrial fusion. Neuronal RAGA-1 abrogation of raga-1 mutant longevity requires UNC-64/syntaxin, and promotes mitochondrial fission cell nonautonomously. Finally, deleting the mitochondrial fission factor DRP-1 renders the animal refractory to the pro-aging effects of neuronal RAGA-1. Our results highlight a new role for neuronal TORC1 in cell nonautonomous regulation of longevity, and suggest TORC1 in the central nervous system might be targeted to promote healthy aging.
    Keywords:  C. elegans; genetics; genomics
  25. Immunol Rev. 2019 Sep;291(1): 75-90
      To efficiently initiate activation responses against rare ligands in the microenvironment, lymphocytes employ sophisticated mechanisms involving signaling amplification. Recently, a signaling amplification mechanism initiated from phosphatidylinositol (PI) 4, 5-biphosphate [PI(4,5)P2] hydrolysis and synthesis for sustained B cell activation has been reported. Antigen and B cell receptor (BCR) recognition triggered the prompt reduction of PI(4,5)P2 density within the BCR microclusters, which led to the positive feedback for the synthesis of PI(4,5)P2 outside of the BCR microclusters. At single molecule level, the diffusion of PI(4,5)P2 was slow, allowing for the maintenance of a PI(4,5)P2 density gradient between the inside and outside of the BCR microclusters and the persistent supply of PI(4,5)P2 from outside to inside of the BCR microclusters. Here, we review studies that have contributed to uncovering the molecular mechanisms of PI(4,5)P2-derived signaling amplification model. Based on these studies, we proposed a "gasoline engine model" in which the activation of B cell signaling inside the microclusters is similar to the working principle of burning gasoline within the engine chamber of a gasoline engine. We also discuss the evidences showing the potential universality of this model and future prospects.
    Keywords:  B cell receptor; PI(4,5)P2; immunological synapse; lymphocyte signaling; microcluster
  26. Transplantation. 2019 Aug 12.
      BACKGROUND: Renal allograft rejection is more frequent under belatacept-based, compared to tacrolimus-based, immunosuppression. We studied kidney transplant recipients experiencing rejection under belatacept-based early corticosteroid withdrawal following T cell depleting induction in a recent randomized trial (BEST Trial, #NCT01729494) to determine mechanisms of rejection and treatment.METHODS: Peripheral mononuclear cells, serum creatinine levels, and renal biopsies were collected from 8 patients undergoing belatacept-refractory rejection. We used flow cytometry, histology and immunofluorescence to characterize CD8 effector memory T cell (TEM) populations in the periphery and graft before and after mammalian target of rapamycin (mTOR) inhibition.
    RESULTS: Here, we found that patients with belatacept-refractory rejection (BRR) did not respond to standard antirejection therapy and had a substantial increase in alloreactive CD8 T cells with a CD28/DR/CD38/CD45RO TEM. These cells had increased activation of the mTOR pathway, as assessed by phosphorylated ribosomal protein S6 (p-RPS6) expression. Notably, everolimus (an mTOR inhibitor) treatment of patients with BRR halted the in vivo proliferation of TEM cells, their ex vivo alloreactivity, and resulted in their significant reduction in the peripheral blood. The frequency of circulating FoxP3 regulatory T cells was not altered. Importantly, everolimus led to rapid resolution of rejection as confirmed by histology.
    CONCLUSIONS: Thus, while prior work has shown that concomitant belatacept + mTOR inhibitor therapy is effective for maintenance immunosuppression, our preliminary data suggest that everolimus may provide an available means for effecting "rescue" therapy for rejections occurring under belatacept that are refractory to traditional antirejection therapy with corticosteroids and polyclonal antilymphocyte globulin.
  27. Front Physiol. 2019 ;10 965
      Embryogenesis is a metabolically intensive process carried out under tightly controlled conditions. The insulin signaling pathway regulates glucose homeostasis and is essential for reproduction in metazoan model species. Three key targets are part of this signaling pathway: protein kinase B (PKB, or AKT), glycogen synthase kinase 3 (GSK-3), and target of rapamycin (TOR). While the role of AKT and GSK-3 has been investigated during tick embryonic development, the role of TOR remains unknown. In this study, TOR and two other downstream effectors, namely S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), were investigated in in vitro studies using the tick embryonic cell line BME26. First, we show that exogenous insulin can stimulate TOR transcription. Second, TOR chemical inhibition led to a decrease in BME26 cell viability, loss of membrane integrity, and downregulation of S6K and 4E-BP1 transcription. Conversely, treating BME26 cells with chemical inhibitors of AKT or GSK-3 did not affect S6K and 4E-BP1 transcription, showing that TOR is specifically required to activate its downstream targets. To address the role of TOR in tick reproduction, in vivo studies were performed. Analysis of relative transcription during different stages of tick embryonic development showed different levels of transcription for TOR, and a maternal deposition of S6K and 4E-BP1 transcripts. Injection of TOR double-stranded RNA (dsRNA) into partially fed females led to a slight delay in oviposition, an atypical egg external morphology, decreased vitellin content in eggs, and decreased larval hatching. Taken together, our data show that the TOR signaling pathway is important for tick reproduction, that TOR acts as a regulatory target in Rhipicephalus microplus embryogenesis and represents a promising target for the development of compounds for tick control.
    Keywords:  BME26; Rhipicephalus microplus; embryogenesis; target of rapamycin; tick embryonic cells
  28. Genome Biol. 2019 Aug 15. 20(1): 167
      The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing by even a single base pair. Target sites might carry genetic variations that are not distinguishable by sgRNA designing tools based on one reference genome. AlleleAnalyzer is an open-source software that incorporates single-nucleotide variants and short insertions and deletions to design sgRNAs for precisely editing 1 or multiple haplotypes of a sequenced genome, currently supporting 11 Cas proteins. It also leverages patterns of shared genetic variation to optimize sgRNA design for different human populations. AlleleAnalyzer is available at .
    Keywords:  CRISPR; Computational biology; Genome editing; Genome surgery; Genomics; sgRNA design
  29. Oncogene. 2019 Aug 12.
      Tumor mitochondria have heightened protein folding quality control, but the regulators of this process and how they impact cancer traits are not completely understood. Here we show that the ATP-directed mitochondrial protease, LonP1 is upregulated by stress conditions, including hypoxia, in tumor, but not normal cells. In mitochondria, LonP1 is phosphorylated by Akt on Ser173 and Ser181, enhancing its protease activity. Interference with this pathway induces accumulation of misfolded subunits of electron transport chain complex II and complex V, resulting in impaired oxidative bioenergetics and heightened ROS production. Functionally, this suppresses mitochondrial trafficking to the cortical cytoskeleton, shuts off tumor cell migration and invasion, and inhibits primary and metastatic tumor growth, in vivo. These data identify LonP1 as a key effector of mitochondrial reprogramming in cancer and potential therapeutic target.
  30. Nat Protoc. 2019 Aug 16.
      Lineage tracing is a powerful tool that can be used to uncover cell fates. Here, we describe a novel method for the quantitative analysis of clonal dynamics in grafted cancer tissues. The protocol involves the preparation and validation of cells for lineage tracing, establishment of grafts and label induction, analysis of clone-size distribution and fitting of the experimental data to a mathematical tumor growth model. In contrast to other lineage-tracing strategies, the method described here assesses stem cell functionality and infers tumor expansion dynamics independently of molecular markers such as putative cancer stem cell (CSC)-specific genes. The experimental system and analytical framework presented can be used to quantify clonal advantages that specific mutations provide, in both the absence and presence of (targeted) therapeutic agents. This protocol typically takes ~20 weeks to complete from cell line selection to inference of growth dynamics, depending on the grafted cancer growth rate.
  31. Cell Death Dis. 2019 Aug 13. 10(8): 607
      Rapamycin (Sirolimus) slows aging, extends life span, and prevents age-related diseases, including diabetic complications such as retinopathy. Puzzlingly, rapamycin can induce insulin sensitivity, but may also induce insulin resistance or glucose intolerance without insulin resistance. This mirrors the effect of fasting and very low calorie diets, which improve insulin sensitivity and reverse type 2 diabetes, but also can cause a form of glucose intolerance known as benevolent pseudo-diabetes. There is no indication that starvation (benevolent) pseudo-diabetes is detrimental. By contrast, it is associated with better health and life extension. In transplant patients, a weak association between rapamycin/everolimus use and hyperglycemia is mostly due to a drug interaction with calcineurin inhibitors. When it occurs in cancer patients, the hyperglycemia is mild and reversible. No hyperglycemic effects of rapamycin/everolimus have been detected in healthy people. For antiaging purposes, rapamycin/everolimus can be administrated intermittently (e.g., once a week) in combination with intermittent carbohydrate restriction, physical exercise, and metformin.
  32. J Clin Oncol. 2019 Aug 13. JCO1802424
      PURPOSE: Genetically engineered T-cell therapy is an emerging treatment of hematologic cancers with potential utility in epithelial cancers. We investigated T-cell therapy for the treatment of metastatic human papillomavirus (HPV)-associated epithelial cancers.METHODS: This phase I/II, single-center trial enrolled patients with metastatic HPV16-positive cancer from any primary tumor site who had received prior platinum-based therapy. Treatment consisted of autologous genetically engineered T cells expressing a T-cell receptor directed against HPV16 E6 (E6 T-cell receptor T cells), a conditioning regimen, and systemic aldesleukin.
    RESULTS: Twelve patients were treated in the study. No dose-limiting toxicities were observed in the phase I portion. Two patients, both in the highest-dose cohort, experienced objective tumor responses. A patient with three lung metastases experienced complete regression of one tumor and partial regression of two tumors, which were subsequently resected; she has no evidence of disease 3 years after treatment. All patients demonstrated high levels of peripheral blood engraftment with E6 T-cell receptor T cells 1 month after treatment (median, 30%; range, 4% to 53%). One patient's resistant tumor demonstrated a frameshift deletion in interferon gamma receptor 1, which mediates response to interferon gamma, an essential molecule for T-cell-mediated antitumor activity. Another patient's resistant tumor demonstrated loss of HLA-A*02:01, the antigen presentation molecule required for this therapy. A tumor from a patient who responded to treatment did not demonstrate genetic defects in interferon gamma response or antigen presentation.
    CONCLUSION: Engineered T cells can induce regression of epithelial cancer. Tumor resistance was observed in the context of T-cell programmed death-1 expression and defects in interferon gamma and antigen presentation pathway components. These findings have important implications for development of cellular therapy in epithelial cancers.
  33. Biochem Biophys Res Commun. 2019 Aug 13. pii: S0006-291X(19)31540-2. [Epub ahead of print]
      Acidic fibroblast growth factors (FGF1s) are heparin binding proteins that regulate a wide array of key cellular processes and are also candidates for promising biomedical applications. FGF1-based therapeutic applications are currently limited due to their inherent thermal instability and susceptibility to proteases. Using a wide range of biophysical and biochemical techniques, we demonstrate that reversal of charge on a well-conserved positively charged amino acid, R136, in the heparin binding pocket drastically increases the resistance to proteases, thermal stability, and cell proliferation activity of the human acidic fibroblast growth factor (hFGF1). Two-dimensional NMR data suggest that the single point mutations at position-136 (R136G, R136L, R136Q, R136K, and R136E) did not perturb the backbone folding of hFGF1. Results of the differential scanning calorimetry experiments show that of all the designed R136 mutations only the charge reversal mutation, R136E, significantly increases (ΔTm = 7 °C) the thermal stability of the protein. Limited trypsin and thrombin digestion results reveal that the R136E mutation drastically increases the resistance of hFGF1 to the action of the serine proteases. Isothermal titration calorimetry data show that the R136E mutation markedly decreases the heparin binding affinity of hFGF1. Interestingly, despite lower heparin binding affinity, the cell proliferation activity of the R136E variant is more than double of that exhibited by either the wild type or the other R136 variants. The R136E variant due to its increased thermal stability, resistance to proteases, and enhanced cell proliferation activity are expected to provide valuable clues for the development of hFGF1- based therapeutics for the management of chronic diabetic wounds.
    Keywords:  Binding affinity; Cell proliferation; Cell signaling; Fibroblast growth factor; Heparin; Stability
  34. Nat Commun. 2019 Aug 13. 10(1): 3646
      Entry into cells is necessary for many nanomaterial applications, and a common solution is to functionalize nanoparticles (NPs) with cell-penetrating ligands. Despite intensive studies on these functionalized NPs, little is known about their effect on cellular activities to engulf other cargo from the nearby environment. Here, we use NPs functionalized with TAT (transactivator of transcription) peptide (T-NPs) as an example to investigate their impact on cellular uptake of bystander cargo. We find that T-NP internalization enables cellular uptake of bystander NPs, but not common fluid markers, through a receptor-dependent macropinocytosis pathway. Moreover, the activity of this bystander uptake is stimulated by cysteine presence in the surrounding solution. The cargo selectivity and cysteine regulation are further demonstrated ex vivo and in vivo. These findings reveal another mechanism for NP entry into cells and open up an avenue of studying the interplay among endocytosis, amino acids, and nanomaterial delivery.
  35. Oncol Lett. 2019 Sep;18(3): 2427-2433
      Breast cancer is one of the major causes of female morbidity and mortality, accounting for ~25% of the total cancer cases in women. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic α subunit (PIK3CA) mutations serve a major role in downstream signaling of receptor tyrosine kinases. The present study aimed to elucidate the frequency of exon 9 and 20 mutations of PIK3CA and their role in disease progression. A total of 118 tumor samples from confirmed breast cancer patients were collected from the histopathology laboratory at King Fahd Hospital of the University (Al-Khobar, Saudi Arabia). Sanger sequencing was performed on extracted DNA to identify the mutations on exons 9 and 20 of PIK3CA. The results were further validated by competitive allele-specific TaqMan polymerase chain reaction. Three mutations, namely E542K and E545K within exon 9, and H1047R within exon 20, were observed in 25 patients (21.2%). Among these, 18 patients carried the H1047R mutation of the kinase domain, while the remaining 7 patients carried mutations in the helical domain. PIK3CA mutations were associated with the estrogen receptor-positive/progesterone receptor-positive (ER+/PR+) group of tumors in contrast to the ER-/PR- group (P=0.021). Furthermore, it was observed that the PIK3CA mutation was associated with a poor disease prognosis. Taken together, the current study emphasized the potential of PIK3CA mutations as an important biomarker for breast cancer classification and the possible use of PIK3CA inhibitor as targeted therapy for breast cancer.
    Keywords:  hormone receptor; prognosis; sequencing; somatic mutation; target therapy
  36. J Clin Invest. 2019 Aug 13. pii: 126091. [Epub ahead of print]130
      BACKGROUND: Idiopathic multicentric Castleman disease (iMCD) is a hematologic illness involving cytokine-induced lymphoproliferation, systemic inflammation, cytopenias, and life-threatening multi-organ dysfunction. The molecular underpinnings of interleukin-6(IL-6)-blockade refractory patients remain unknown; no targeted therapies exist. In this study, we searched for therapeutic targets in IL-6-blockade refractory iMCD patients with the thrombocytopenia, anasarca, fever/elevated C-reactive protein, reticulin myelofibrosis, renal dysfunction, organomegaly (TAFRO) clinical subtype.METHODS: We analyzed tissues and blood samples from three IL-6-blockade refractory iMCD-TAFRO patients. Cytokine panels, quantitative serum proteomics, flow cytometry of PBMCs, and pathway analyses were employed to identify novel therapeutic targets. To confirm elevated mTOR signaling, a candidate therapeutic target from the above assays, immunohistochemistry was performed for phosphorylated S6, a read-out of mTOR activation, in three iMCD lymph node tissue samples and controls. Proteomic, immunophenotypic, and clinical response assessments were performed to quantify the effects of administration of the mTOR inhibitor, sirolimus.
    RESULTS: Studies of three IL-6-blockade refractory iMCD cases revealed increased CD8+ T cell activation, VEGF-A, and PI3K/Akt/mTOR pathway activity. Administration of sirolimus significantly attenuated CD8+ T cell activation and decreased VEGF-A levels. Sirolimus induced clinical benefit responses in all three patients with durable and ongoing remissions of 66, 19, and 19 months.
    CONCLUSION: This precision medicine approach identifies PI3K/Akt/mTOR signaling as the first pharmacologically-targetable pathogenic process in IL-6-blockade refractory iMCD. Prospective evaluation of sirolimus in treatment-refractory iMCD is planned (NCT03933904).
    FUNDING: Castleman's Awareness & Research Effort/Castleman Disease Collaborative Network, Penn Center for Precision Medicine, University Research Foundation, Intramural NIH funding, and National Heart Lung and Blood Institute.
    Keywords:  Cytokines; Hematology; Immunology; Lymphomas
  37. Cell Rep. 2019 Aug 13. pii: S2211-1247(19)30924-6. [Epub ahead of print]28(7): 1860-1878.e9
      Squamous cell carcinoma (SCC), a malignancy arising across multiple anatomical sites, is responsible for significant cancer mortality due to insufficient therapeutic options. Here, we identify exceptional glucose reliance among SCCs dictated by hyperactive GLUT1-mediated glucose influx. Mechanistically, squamous lineage transcription factors p63 and SOX2 transactivate the intronic enhancer cluster of SLC2A1. Elevated glucose influx fuels generation of NADPH and GSH, thereby heightening the anti-oxidative capacity in SCC tumors. Systemic glucose restriction by ketogenic diet and inhibiting renal glucose reabsorption with SGLT2 inhibitor precipitate intratumoral oxidative stress and tumor growth inhibition. Furthermore, reduction of blood glucose lowers blood insulin levels, which suppresses PI3K/AKT signaling in SCC cells. Clinically, we demonstrate a robust correlation between blood glucose concentration and worse survival among SCC patients. Collectively, this study identifies the exceptional glucose reliance of SCC and suggests its candidacy as a highly vulnerable cancer type to be targeted by systemic glucose restriction.
    Keywords:  GLUT1; SGLT2; SOX2; glucose restriction; ketogenic diet; p63; squamous cell carcinoma
  38. Cancers (Basel). 2019 Aug 09. pii: E1141. [Epub ahead of print]11(8):
      Lung cancer is the most common malignancy and cause of cancer deaths worldwide, owing to the dismal prognosis for most affected patients. Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) acts as a powerful tumor suppressor gene and even partial reduction of its levels increases cancer susceptibility. While the most validated anti-oncogenic duty of PTEN is the negative regulation of the PI3K/mTOR/Akt oncogenic signaling pathway, further tumor suppressor functions, such as chromosomal integrity and DNA repair have been reported. PTEN protein loss is a frequent event in lung cancer, but genetic alterations are not equally detected. It has been demonstrated that its expression is regulated at multiple genetic and epigenetic levels and deeper delineation of these mechanisms might provide fertile ground for upgrading lung cancer therapeutics. Today, PTEN expression is usually determined by immunohistochemistry and low protein levels have been associated with decreased survival in lung cancer. Moreover, available data involve PTEN mutations and loss of activity with resistance to targeted treatments and immunotherapy. This review discusses the current knowledge about PTEN status in lung cancer, highlighting the prevalence of its alterations in the disease, the regulatory mechanisms and the implications of PTEN on available treatment options.
    Keywords:  NSCLC; PTEN; epigenetic; genetic; lung cancer; survival; treatment resistance
  39. Nat Chem Biol. 2019 Aug 12.
      The CRISPR-Cpf1 endonuclease has recently been demonstrated as a powerful tool to manipulate targeted gene sequences. Here, we performed an extensive screening of split Cpf1 fragments and identified a pair that, combined with inducible dimerization domains, enables chemical- and light-inducible genome editing in human cells. We also identified another split Cpf1 pair that is spontaneously activated. The newly generated amino and carboxyl termini of the spontaneously activated split Cpf1 can be repurposed as de novo fusion sites of artificial effector domains. Based on this finding, we generated an improved split dCpf1 activator, which has the potential to activate endogenous genes more efficiently than a previously established dCas9 activator. Finally, we showed that the split dCpf1 activator can efficiently activate target genes in mice. These results demonstrate that the present split Cpf1 provides an efficient and sophisticated genome manipulation in the fields of basic research and biotechnological applications.
  40. Oncogene. 2019 Aug 12.
      Tumor-repopulating cells (TRCs) are cancer stem cell (CSC)-like cells with highly tumorigenic and self-renewing abilities, which were selected from tumor cells in soft three-dimensional (3D) fibrin gels with unidentified mechanisms. Here we evaluated the transcriptome alteration during TRCs generation in 3D culture and revealed that a variety of molecules related with integrin/membrane and stemness were continuously altered by mechanical environment. Some key regulators such as MYC/STAT3/hsa-miR-199a-5p, were changed in the TRCs generation. They regulated membrane genes and the downstream mechanotransduction pathways such as Hippo/WNT/TGF-β/PI3K-AKT pathways, thus further affecting the expression of downstream cancer-related genes. By integrating networks for membrane proteins, the WNT pathway and cancer-related genes, we identified key molecules in the selection of TRCs, such as ATF4, SLC3A2, CCT3, and hsa-miR-199a-5p. Silencing ATF4 or CCT3 inhibited the selection and growth of TRCs whereas reduction of SLC3A2 or hsa-miR-199a-5p promoted TRCs growth. Further studies showed that CCT3 promoted cell proliferation and stemness in vitro, while its suppression inhibited TRCs-induced tumor formation. We also contemplated CCT3 as a stemness-related gene. Our findings provide insights in the mechanism of TRCs selection through transcriptome analysis.
  41. Front Genet. 2019 ;10 693
      Type 2 diabetes mellitus (T2DM) is characterized by persistent hyperglycemia and is influenced by genetic and environmental factors. Optimum T2DM management involves early diagnosis and effective glucose-lowering therapies. Further research is warranted to improve our understanding of T2DM pathophysiology and reveal potential roles of genetic predisposition. We have previously developed an obesity-induced diabetic zebrafish model that shares common pathological pathways with humans and may be used to identify putative pharmacological targets of diabetes. Additionally, we have previously identified several candidate genes with altered expression in T2DM zebrafish. Here, we performed a small-scale zebrafish screening for these genes and discovered a new therapeutic target, centromere protein X (CENPX), which was further validated in a T2DM mouse model. In zebrafish, cenpx knockdown by morpholino or knockout by CRISPR/Cas9 system ameliorated overfeeding-induced hyperglycemia and upregulated insulin level. In T2DM mice, small-interfering RNA-mediated Cenpx knockdown decreased hyperglycemia and upregulated insulin synthesis in the pancreas. Gene expression analysis revealed insulin, mechanistic target of rapamycin, leptin, and insulin-like growth factor 1 pathway activation following Cenpx silencing in pancreas tissues. Thus, CENPX inhibition exerted antidiabetic effects via increased insulin expression and related pathways. Therefore, T2DM zebrafish may serve as a powerful tool in the discovery of new therapeutic gene targets.
    Keywords:  CRISPR/Cas9; centromere protein X; gene silencing; insulin; therapeutic gene target; type 2 diabetes mellitus; zebrafish model
  42. Cancer Res. 2019 Aug 15. pii: canres.2086.2018. [Epub ahead of print]
      Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adenocarcinoma and squamous subtypes of non-small cell lung cancer. The consequence of these mutations is stabilized Nrf2 and chronic induction of several Nrf2 target genes. Here, downregulation of Nrf2 resulted in modest growth inhibition of cells growing in 2D; this was more pronounced in cell lines expressing mutant KEAP1. In contrast, downregulation of Nrf2 caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wildtype (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments, and was not prevented by excess extracellular glutathione. Using CRISPR screening we identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1 mutant cells compared to WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2, and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors.
  43. Nat Methods. 2019 Aug 12.
      The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.