bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2020–06–07
twelve papers selected by
Ralitsa Radostinova Madsen, University College London
  1. FAK activates AKT-mTOR signaling to promote the growth and progression of MMTV-Wnt1-driven basal-like mammary tumors.
  2. Novel Miniaturized Drug Conjugate Leverages HSP90 Driven Tumor Accumulation to Overcome PI3K Inhibitor Delivery Challenges to Solid Tumors.
  3. Rab35-regulated lipid turnover by myotubularins represses mTORC1 activity and controls myelin growth.
  4. Acetyl-CoA Derived from Hepatic Peroxisomal β-Oxidation Inhibits Autophagy and Promotes Steatosis via mTORC1 Activation.
  5. Inhibition of decidual IGF-1 signaling in response to hypoxia and leucine deprivation is mediated by mTOR and AAR pathways and increased IGFBP-1 phosphorylation.
  6. Epithelial-Mesenchymal Transition Programs and Cancer Stem Cell Phenotypes: Mediators of Breast Cancer Therapy Resistance.
  7. Structure of the TSC2 GAP Domain: Mechanistic Insight into Catalysis and Pathogenic Mutations.
  8. Inhibition of PI3K by copanlisib exerts potent antitumor effects on Merkel cell carcinoma cell lines and mouse xenografts.
  9. The Long and the Short of PTEN in the Regulation of Mitophagy.
  10. Endocytosis and the internalization of pathogenic organisms: focus on phosphoinositides.
  11. Cre/loxP approach-mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells.
  12. Lysosome activity is modulated by multiple longevity pathways and is important for lifespan extension in C. elegans.
  1. Breast Cancer Res. 2020 Jun 03. 22(1): 59
    FAK activates AKT-mTOR signaling to promote the growth and progression of MMTV-Wnt1-driven basal-like mammary tumors.
    Ritama Paul, Ming Luo, Xueying Mo, Jason Lu, Syn Kok Yeo, Jun-Lin Guan.
       BACKGROUND: Breast cancer is a heterogeneous disease. Hence, stratification of patients based on the subtype of breast cancer is key to its successful treatment. Among all the breast cancer subtypes, basal-like breast cancer is the most aggressive subtype with limited treatment options. Interestingly, we found focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is highly overexpressed and activated in basal-like breast cancer.
    METHODS: To understand the role of FAK in this subtype, we generated mice with conditional deletion of FAK and a knock-in mutation in its kinase domain in MMTV-Wnt1-driven basal-like mammary tumors. Tumor initiation, growth, and metastasis were characterized for these mice cohorts. Immunohistochemical and transcriptomic analysis of Wnt1-driven tumors were also performed to elucidate the mechanisms underlying FAK-dependent phenotypes. Pharmacological inhibition of FAK and mTOR in human basal-like breast cancer cell lines was also tested.
    RESULTS: We found that in the absence of FAK or its kinase function, growth and metastasis of the tumors were significantly suppressed. Furthermore, immunohistochemical analyses of cleaved caspase 3 revealed that loss of FAK results in increased tumor cell apoptosis. To further investigate the mechanism by which FAK regulates survival of the Wnt1-driven tumor cells, we prepared an isogenic pair of mammary tumor cells with and without FAK and found that FAK ablation increased their sensitivity to ER stress-induced cell death, as well as reduced tumor cell migration and tumor sphere formation. Comparative transcriptomic profiling of the pair of tumor cells and gene set enrichment analysis suggested mTOR pathway to be downregulated upon loss of FAK. Immunoblot analyses further confirmed that absence of FAK results in reduction of AKT and downstream mTOR pathways. We also found that inhibition of FAK and mTOR pathways both induces apoptosis, indicating the importance of these pathways in regulating cell survival.
    CONCLUSIONS: In summary, our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells and can serve as a potential therapeutic target.
    Keywords:  Basal-like breast cancer; FAK; WNT1; mTOR
    DOI:  https://doi.org/10.1186/s13058-020-01298-3
  2. Mol Cancer Ther. 2020 Jun 04. pii: molcanther.0964.2019. [Epub ahead of print]
    Novel Miniaturized Drug Conjugate Leverages HSP90 Driven Tumor Accumulation to Overcome PI3K Inhibitor Delivery Challenges to Solid Tumors.
    Samantha Perino, Benoit Moreau, Jessica Freda, Amanda Cirello, Brian H White, James M Quinn, Kristina Kriksciukaite, Ashwajith Someshwar, Janel Romagnoli, Megan Robinson, Sara Movassaghian, Tyler Cipriani, Richard Wooster, Mark T Bilodeau, Kerry A Whalen.
      The Phosphatidylinositol 3 kinase (PI3K) pathway is considered a master regulator for cancer due to its frequent activation, making it an attractive target for pharmacologic intervention. While substantial efforts have been made to develop drugs targeting PI3K signaling, few drugs have been able to achieve the inhibition necessary for effective tumor control at tolerated doses. HSP90 is a chaperone protein that is overexpressed and activated in many tumors and as a consequence, small molecule ligands of HSP90 are preferentially retained in tumors up to 20 times longer than in normal tissue. We hypothesize that the generation of conjugates that use a HSP90 targeting ligand and a payload such as Copanlisib, may open the narrow therapeutic window of this and other PI3K inhibitors. In support of this hypothesis, we have generated a HSP90-PI3K drug conjugate, T-2143 and utilizing xenograft models, demonstrate rapid and sustained tumor accumulation of the conjugate, deep pathway inhibition, and superior efficacy than the PI3K inhibitor on its own. Selective delivery of T-2143 and the masking of the inhibitor active site was also able to mitigate a potentially dose limiting side effect of Copanlisib, hyperglycemia. These data demonstrate that by leveraging the preferential accumulation of HSP90 targeting ligands in tumors, we can selectively deliver a PI3K inhibitor leading to efficacy in multiple tumor models without hyperglycemia in mice. These data highlight a novel drug delivery strategy that allows for the potential opening of a narrow therapeutic window through specific tumor delivery of anticancer payloads and reduction of toxicity.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-19-0964
  3. Nat Commun. 2020 Jun 05. 11(1): 2835
    Rab35-regulated lipid turnover by myotubularins represses mTORC1 activity and controls myelin growth.
    Linda Sawade, Federica Grandi, Marianna Mignanelli, Genaro Patiño-López, Kerstin Klinkert, Francina Langa-Vives, Roberta Di Guardo, Arnaud Echard, Alessandra Bolino, Volker Haucke.
      Inherited peripheral neuropathies (IPNs) represent a broad group of disorders including Charcot-Marie-Tooth (CMT) neuropathies characterized by defects primarily arising in myelin, axons, or both. The molecular mechanisms by which mutations in nearly 100 identified IPN/CMT genes lead to neuropathies are poorly understood. Here we show that the Ras-related GTPase Rab35 controls myelin growth via complex formation with the myotubularin-related phosphatidylinositol (PI) 3-phosphatases MTMR13 and MTMR2, encoded by genes responsible for CMT-types 4B2 and B1 in humans, and found that it downregulates lipid-mediated mTORC1 activation, a pathway known to crucially regulate myelin biogenesis. Targeted disruption of Rab35 leads to hyperactivation of mTORC1 signaling caused by elevated levels of PI 3-phosphates and to focal hypermyelination in vivo. Pharmacological inhibition of phosphatidylinositol 3,5-bisphosphate synthesis or mTORC1 signaling ameliorates this phenotype. These findings reveal a crucial role for Rab35-regulated lipid turnover by myotubularins to repress mTORC1 activity and to control myelin growth.
    DOI:  https://doi.org/10.1038/s41467-020-16696-6
  4. Mol Cell. 2020 May 20. pii: S1097-2765(20)30308-7. [Epub ahead of print]
    Acetyl-CoA Derived from Hepatic Peroxisomal β-Oxidation Inhibits Autophagy and Promotes Steatosis via mTORC1 Activation.
    Anyuan He, Xiaowen Chen, Min Tan, Yali Chen, Dongliang Lu, Xiangyu Zhang, John M Dean, Babak Razani, Irfan J Lodhi.
      Autophagy is activated by prolonged fasting but cannot overcome the ensuing hepatic lipid overload, resulting in fatty liver. Here, we describe a peroxisome-lysosome metabolic link that restricts autophagic degradation of lipids. Acyl-CoA oxidase 1 (Acox1), the enzyme that catalyzes the first step in peroxisomal β-oxidation, is enriched in liver and further increases with fasting or high-fat diet (HFD). Liver-specific Acox1 knockout (Acox1-LKO) protected mice against hepatic steatosis caused by starvation or HFD due to induction of autophagic degradation of lipid droplets. Hepatic Acox1 deficiency markedly lowered total cytosolic acetyl-CoA levels, which led to decreased Raptor acetylation and reduced lysosomal localization of mTOR, resulting in impaired activation of mTORC1, a central regulator of autophagy. Dichloroacetic acid treatment elevated acetyl-CoA levels, restored mTORC1 activation, inhibited autophagy, and increased hepatic triglycerides in Acox1-LKO mice. These results identify peroxisome-derived acetyl-CoA as a key metabolic regulator of autophagy that controls hepatic lipid homeostasis.
    Keywords:  Acox1; Autophagy; Lipid metabolism; NAFLD; Raptor; fatty acid oxidation; lipophagy; mTOR; peroxisomes
    DOI:  https://doi.org/10.1016/j.molcel.2020.05.007
  5. Mol Cell Endocrinol. 2020 Jun 02. pii: S0303-7207(20)30165-9. [Epub ahead of print]512 110865
    Inhibition of decidual IGF-1 signaling in response to hypoxia and leucine deprivation is mediated by mTOR and AAR pathways and increased IGFBP-1 phosphorylation.
    Majida Abu Shehab, Kyle Biggar, Jenica H Kakadia, Manthan Dhruv, Bhawani Jain, Pinki Nandi, Karen Nygard, Thomas Jansson, Madhulika B Gupta.
      Decidual mechanistic target of rapamycin (mTOR) is inhibited, amino acid response (AAR) and protein kinase CK2 are activated, and IGF (insulin-like growth factor) binding protein (IGFBP)-1 is hyperphosphorylated in human intrauterine growth restriction (IUGR). Using decidualized human immortalized endometrial stromal cells (HIESC), we hypothesized that hypoxia and leucine deprivation causing inhibition of decidual IGF-1 signaling is mediated by mTOR, AAR, CK2 and IGFBP-1 phosphorylation. Mass spectrometry demonstrated that hypoxia (1% O2) or rapamycin increased IGFBP-1 phosphorylation singly at Ser101/119/169 (confirmed using immunoblotting) and dually at pSer169 + 174. Hypoxia resulted in mTOR inhibition, AAR and CK2 activation, and decreased IGF-1 bioactivity, with no additional changes with rapamycin + hypoxia. Rapamycin and/or hypoxia promoted colocalization of IGFBP-1 and CK2 (dual-immunofluorescence and proximity ligation assay). Leucine deprivation showed similar outcomes. Changes in IGFBP-1 phosphorylation regulated by mTOR/AAR signaling and CK2 may represent a novel mechanism linking oxygen and nutrient availability to IGF-1 signaling in the decidua.
    Keywords:  Biological availability; Humans; Intrauterine growth restriction; Maternal-fetal exchange; Pregnancy
    DOI:  https://doi.org/10.1016/j.mce.2020.110865
  6. Mol Cancer Res. 2020 Jun 05. pii: molcanres.0067.2020. [Epub ahead of print]
    Epithelial-Mesenchymal Transition Programs and Cancer Stem Cell Phenotypes: Mediators of Breast Cancer Therapy Resistance.
    Alex J Gooding, William P Schiemann.
      Epithelial-mesenchymal transition (EMT) programs play essential functions in normal morphogenesis and organogenesis, including that occurring during mammary gland development and glandular regeneration. Historically, EMT programs were believed to reflect a loss of epithelial gene expression signatures and morphologies that give way to those associated with mesenchymal cells and their enhanced migratory and invasive behaviors. However, accumulating evidence now paints EMT programs as representing a spectrum of phenotypic behaviors that also serve to enhance cell survival, immune tolerance, and perhaps even metastatic dormancy. Equally important, the activation of EMT programs in transformed mammary epithelial cells not only enhances their acquisition of invasive and metastatic behaviors, but also expands their generation of chemoresistant breast cancer stem cells (BCSCs). Importantly, the net-effect of these events results in the appearance of recurrent metastatic lesions that remain refractory to the armamentarium of chemotherapies and targeted therapeutic agents deployed against advanced-stage breast cancers. Here we review the molecular and cellular mechanisms that contribute to the pathophysiology of EMT programs in human breast cancers and how these events impact their "stemness" and acquisition of chemoresistant phenotypes.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0067
  7. Structure. 2020 Jun 04. pii: S0969-2126(20)30177-5. [Epub ahead of print]
    Structure of the TSC2 GAP Domain: Mechanistic Insight into Catalysis and Pathogenic Mutations.
    Patrick Hansmann, Anne Brückner, Stephan Kiontke, Bianca Berkenfeld, Guiscard Seebohm, Pascal Brouillard, Miikka Vikkula, Floor E Jansen, Mark Nellist, Andrea Oeckinghaus, Daniel Kümmel.
      The TSC complex is the cognate GTPase-activating protein (GAP) for the small GTPase Rheb and a crucial regulator of the mechanistic target of rapamycin complex 1 (mTORC1). Mutations in the TSC1 and TSC2 subunits of the complex cause tuberous sclerosis complex (TSC). We present the crystal structure of the catalytic asparagine-thumb GAP domain of TSC2. A model of the TSC2-Rheb complex and molecular dynamics simulations suggest that TSC2 Asn1643 and Rheb Tyr35 are key active site residues, while Rheb Arg15 and Asp65, previously proposed as catalytic residues, contribute to the TSC2-Rheb interface and indirectly aid catalysis. The TSC2 GAP domain is further stabilized by interactions with other TSC2 domains. We characterize TSC2 variants that partially affect TSC2 functionality and are associated with atypical symptoms in patients, suggesting that mutations in TSC1 and TSC2 might predispose to neurological and vascular disorders without fulfilling the clinical criteria for TSC.
    Keywords:  GTPase-activating protein; Rheb; TSC; mTORC1
    DOI:  https://doi.org/10.1016/j.str.2020.05.008
  8. Sci Rep. 2020 Jun 01. 10(1): 8867
    Inhibition of PI3K by copanlisib exerts potent antitumor effects on Merkel cell carcinoma cell lines and mouse xenografts.
    Bin Fang, Aarthi Kannan, Stephanie Zhao, Quy H Nguyen, Samuel Ejadi, Maki Yamamoto, J Camilo Barreto, Haibo Zhao, Ling Gao.
      Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer with steadily increasing incidence and poor prognosis. Despite recent success with immunotherapy, 50% of patients still succumb to their diseases. To date, there is no Food and Drug Administration-approved targeted therapy for advanced MCC. Aberrant activation of phosphatidylinositide-3-kinase (PI3K)/AKT/mTOR pathway is frequently detected in MCC, making it an attractive therapeutic target. We previously found PI3K pathway activation in human MCC cell lines and tumors and demonstrated complete clinical response in a Stage IV MCC patient treated with PI3K inhibitor idelalisib. Here, we found that both PI3K-α and -δ isoforms are abundantly expressed in our MCC cell lines and clinical samples; we therefore examined antitumor efficacy across a panel of five PI3K inhibitors with distinctive isoform-specificities, including idelalisib (PI3K-δ), copanlisib (PI3K-α/δ), duvelisib (PI3K-γ/δ), alpelisib (PI3K-α), and AZD8186 (PI3K-β/δ). Of these, copanlisib exerts the most potent antitumor effects, markedly inhibiting cell proliferation, survival, and tumor growth by suppressing PI3K/mTOR/Akt activities in mouse models generated from MCC cell xenografts and patient-derived tumor xenografts. These results provide compelling preclinical evidence for application of copanlisib in advanced MCC with aberrant PI3K activation for which immunotherapy is insufficient, or patients who are unsuitable for immunotherapy.
    DOI:  https://doi.org/10.1038/s41598-020-65637-2
  9. Front Cell Dev Biol. 2020 ;8 299
    The Long and the Short of PTEN in the Regulation of Mitophagy.
    Liming Wang, Guang Lu, Han-Ming Shen.
      Mitophagy is a key mitochondrial quality control mechanism for effective and selective elimination of damaged mitochondria through the autophagy-lysosome machinery. Defective mitophagy is associated with pathogenesis of important human diseases including neurodegenerative diseases, heart failure, innate immunity, and cancer. In the past two decades, the mechanistic studies of mitophagy have made many breakthroughs with the discoveries of phosphatase and tensin homolog (PTEN)-induced kinase protein 1 (PINK1)-parkin-mediated ubiquitin (Ub)-driven pathway and BCL2/adenovirus E1B 19 kDa protein-interacting proteins 3 (BNIP3)/NIX or FUN14 domain containing 1 (FUNDC1) mitochondrial receptor-mediated pathways. Recently, several isoforms of dual phosphatase PTEN, such as PTEN-long (PTEN-L), have been identified, and some of them are implicated in the mitophagy process via their protein phosphatase activity. In this review, we aim to discuss the regulatory roles of PTEN isoforms in mitophagy. These discoveries may provide new opportunities for development of novel therapeutic strategies for mitophagy-related diseases such as neurodegenerative disorders via targeting PTEN isoforms and mitophagy.
    Keywords:  BNIP3; PINK1; PTEN; PTEN-L; Parkin; mitophagy
    DOI:  https://doi.org/10.3389/fcell.2020.00299
  10. F1000Res. 2020 ;pii: F1000 Faculty Rev-368. [Epub ahead of print]9
    Endocytosis and the internalization of pathogenic organisms: focus on phosphoinositides.
    Glenn F W Walpole, Sergio Grinstein.
      Despite their comparatively low abundance in biological membranes, phosphoinositides are key to the regulation of a diverse array of signaling pathways and direct membrane traffic. The role of phosphoinositides in the initiation and progression of endocytic pathways has been studied in considerable depth. Recent advances have revealed that distinct phosphoinositide species feature prominently in clathrin-dependent and -independent endocytosis as well as in phagocytosis and macropinocytosis. Moreover, a variety of intracellular and cell-associated pathogens have developed strategies to commandeer host cell phosphoinositide metabolism to gain entry and/or metabolic advantage, thereby promoting their survival and proliferation. Here, we briefly survey the current knowledge on the involvement of phosphoinositides in endocytosis, phagocytosis, and macropinocytosis and highlight several examples of molecular mimicry employed by pathogens to either "hitch a ride" on endocytic pathways endogenous to the host or create an entry path of their own.
    Keywords:  endocytosis; inositides; macropinocytosis; pathogen; phagocytosis; phosphoinositides; signaling; traffic
    DOI:  https://doi.org/10.12688/f1000research.22393.1
  11. J Cell Physiol. 2020 May 31.
    Cre/loxP approach-mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells.
    Ting Liu, Jialing Yuan, Caihong Dai, Jinxian Xu, Shude Li, Benjamin D Humphreys, Daniel T Kleven, Jian-Kang Chen.
      Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreERT2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreERT2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1-S6K1-rpS6 signaling pathway.
    Keywords:  class III phosphatidylinositol-3 kinase (Pik3c3); compensatory nephron hypertrophy (CNH); mammalian target of rapamycin complex 1 (mTORC1); renal proximal tubule cells (RPTC); ribosomal protein S6 (rpS6) phosphorylation; unilateral nephrectomy (UNX)
    DOI:  https://doi.org/10.1002/jcp.29811
  12. Elife. 2020 Jun 02. pii: e55745. [Epub ahead of print]9
    Lysosome activity is modulated by multiple longevity pathways and is important for lifespan extension in C. elegans.
    Yanan Sun, Meijiao Li, Dongfeng Zhao, Xin Li, Chonglin Yang, Xiaochen Wang.
      Lysosomes play important roles in cellular degradation to maintain cell homeostasis. In order to understand whether and how lysosomes alter with age and contribute to lifespan regulation, we characterized multiple properties of lysosomes during the aging process in C. elegans. We uncovered age-dependent alterations in lysosomal morphology, motility, acidity and degradation activity, all of which indicate a decline in lysosome function with age. The age-associated lysosomal changes are suppressed in the long-lived mutants daf-2, eat-2 and isp-1, which extend lifespan by inhibiting insulin/IGF-1 signaling, reducing food intake and impairing mitochondrial function, respectively. We found that 43 lysosome genes exhibit reduced expression with age, including genes encoding subunits of the proton pump V-ATPase and cathepsin proteases. The expression of lysosome genes is upregulated in the long-lived mutants, and this upregulation requires the functions of DAF-16/FOXO and SKN-1/NRF2 transcription factors. Impairing lysosome function affects clearance of aggregate-prone proteins and disrupts lifespan extension in daf-2, eat-2 and isp-1 worms. Our data indicate that lysosome function is modulated by multiple longevity pathways and is important for lifespan extension.
    Keywords:  C. elegans; DAF-16/FOXO; SKN-1/NRF2; aging; cell biology; insuin/igf-1 signaling; longevity pathways; lysosome
    DOI:  https://doi.org/10.7554/eLife.55745
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