bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021‒02‒07
twenty-nine papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. J Biol Chem. 2020 Jan 03. pii: S0021-9258(17)49565-2. [Epub ahead of print]295(1): 263-274
    Frias MA, Mukhopadhyay S, Lehman E, Walasek A, Utter M, Menon D, Foster DA.
      Mammalian target of rapamycin complex 1 (mTORC1) promotes cell growth and proliferation in response to nutrients and growth factors. Amino acids induce lysosomal translocation of mTORC1 via the Rag GTPases. Growth factors activate Ras homolog enriched in brain (Rheb), which in turn activates mTORC1 at the lysosome. Amino acids and growth factors also induce the phospholipase D (PLD)-phosphatidic acid (PA) pathway, required for mTORC1 signaling through mechanisms that are not fully understood. Here, using human and murine cell lines, along with immunofluorescence, confocal microscopy, endocytosis, PLD activity, and cell viability assays, we show that exogenously supplied PA vesicles deliver mTORC1 to the lysosome in the absence of amino acids, Rag GTPases, growth factors, and Rheb. Of note, pharmacological or genetic inhibition of endogenous PLD prevented mTORC1 lysosomal translocation. We observed that precancerous cells with constitutive Rheb activation through loss of tuberous sclerosis complex subunit 2 (TSC2) exploit the PLD-PA pathway and thereby sustain mTORC1 activation at the lysosome in the absence of amino acids. Our findings indicate that sequential inputs from amino acids and growth factors trigger PA production required for mTORC1 translocation and activation at the lysosome.
    Keywords:  amino acid; cancer biology; cancer therapy; growth factor; lysosome; mTOR complex (mTORC); phosphatidic acid; phospholipase D; phospholipid vesicle
  2. FEBS Lett. 2021 Feb 04.
    Yan L, Tsujita K, Fujita Y, Itoh T.
      The balance between phosphoinositides distributed at specific sites in the plasma membrane causes polarized actin polymerization. Oncogenic transformations affect this balance by regulating phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog deleted on chromosome ten (PTEN), causing metastatic behavior in cancer cells. Here, we show that the PTEN tumor suppressor gene is required for epithelial cancer cell invasion. Loss of PTEN in Ras-transformed MDCK cells suppressed their migratory phenotype in collagen gel and invasion through Matrigel. Rescue experiments showed a requirement for the C2 domain-mediated membrane recruitment of PTEN, which is typically observed at the rear side of invading cancer cells. These findings support the role of PTEN in suppression of unwanted leading edges necessary for efficient migration of epithelial cancer cells.
    Keywords:  PI3K; PTEN; Ras; cancer cell invasion
  3. J Biol Chem. 2020 Jan 03. pii: S0021-9258(17)49552-4. [Epub ahead of print]295(1): 99-110
    Krycer JR, Elkington SD, Diaz-Vegas A, Cooke KC, Burchfield JG, Fisher-Wellman KH, Cooney GJ, Fazakerley DJ, James DE.
      Insulin action in adipose tissue is crucial for whole-body glucose homeostasis, with insulin resistance being a major risk factor for metabolic diseases such as type 2 diabetes. Recent studies have proposed mitochondrial oxidants as a unifying driver of adipose insulin resistance, serving as a signal of nutrient excess. However, neither the substrates for nor sites of oxidant production are known. Because insulin stimulates glucose utilization, we hypothesized that glucose oxidation would fuel respiration, in turn generating mitochondrial oxidants. This would impair insulin action, limiting further glucose uptake in a negative feedback loop of "glucose-dependent" insulin resistance. Using primary rat adipocytes and cultured 3T3-L1 adipocytes, we observed that insulin increased respiration, but notably this occurred independently of glucose supply. In contrast, glucose was required for insulin to increase mitochondrial oxidants. Despite rising to similar levels as when treated with other agents that cause insulin resistance, glucose-dependent mitochondrial oxidants failed to cause insulin resistance. Subsequent studies revealed a temporal relationship whereby mitochondrial oxidants needed to increase before the insulin stimulus to induce insulin resistance. Together, these data reveal that (a) adipocyte respiration is principally fueled from nonglucose sources; (b) there is a disconnect between respiration and oxidative stress, whereby mitochondrial oxidant levels do not rise with increased respiration unless glucose is present; and (c) mitochondrial oxidative stress must precede the insulin stimulus to cause insulin resistance, explaining why short-term, insulin-dependent glucose utilization does not promote insulin resistance. These data provide additional clues to mechanistically link nutrient excess to adipose insulin resistance.
    Keywords:  adipocyte; glucose; insulin; insulin resistance; mitochondria; oxidative stress; respiration
  4. J Cell Sci. 2021 Feb 01. pii: jcs.248294. [Epub ahead of print]
    Gjelaj E, Hamel PA.
      The most common PIK3CA mutation, producing the H1047R mutant of p110α, arises in myriad malignancies and is typically observed in low grade breast tumours. In contrast, amplification is observed for wildtype PIK3CB, encoding p110β, and occurs at low frequency but in aggressive, high-grade, metastatic tumours. We hypothesized that mutant p110αH1047R and wildtype p110β give rise to distinct transformed phenotypes. We show that p110αH1047R and wildtype p110β, but not wildtype p110α, transform MCF-10A cells and constitutively stimulate PI3K/AKT signalling. However, their resultant morphological transformed phenotypes are distinct. p110αH1047R induced an EMT commensurate with SNAIL induction and loss of E-cadherin. p110β, however, maintained E-cadherin expression despite cells readily delaminating from epithelial sheets. Distinct from the prominent filopodia in p110αH1047R-expressing cells, p110β induced formation of lamellipodia, cells migrating with significantly greater velocity and decreased directionality. p110β-induced phenotypic alterations were accompanied by hyperactivation of RAC1, the dependency of transformation of p110β-binding to Rac1 revealed using a Rac1-binding mutant of p110β. Thus, PIK3CB amplification induces a transformed phenotype that is dependent upon a p110β-Rac1 signalling loop and is distinct from the transformed phenotype induced by p110αH1047R.
    Keywords:  Epithelial-to-mesenchymal transiition; Mammary epithelial cells; Migration; PI3K; PIK3CB; Rac1
  5. J Biol Chem. 2020 Jan 03. pii: S0021-9258(17)49551-2. [Epub ahead of print]295(1): 83-98
    Krycer JR, Quek LE, Francis D, Fazakerley DJ, Elkington SD, Diaz-Vegas A, Cooke KC, Weiss FC, Duan X, Kurdyukov S, Zhou PX, Tambar UK, Hirayama A, Ikeda S, Kamei Y, Soga T, Cooney GJ, James DE.
      Adipose tissue is essential for whole-body glucose homeostasis, with a primary role in lipid storage. It has been previously observed that lactate production is also an important metabolic feature of adipocytes, but its relationship to adipose and whole-body glucose disposal remains unclear. Therefore, using a combination of metabolic labeling techniques, here we closely examined lactate production of cultured and primary mammalian adipocytes. Insulin treatment increased glucose uptake and conversion to lactate, with the latter responding more to insulin than did other metabolic fates of glucose. However, lactate production did not just serve as a mechanism to dispose of excess glucose, because we also observed that lactate production in adipocytes did not solely depend on glucose availability and even occurred independently of glucose metabolism. This suggests that lactate production is prioritized in adipocytes. Furthermore, knocking down lactate dehydrogenase specifically in the fat body of Drosophila flies lowered circulating lactate and improved whole-body glucose disposal. These results emphasize that lactate production is an additional metabolic role of adipose tissue beyond lipid storage and release.
    Keywords:  Drosophila; adipocyte; cell metabolism; fat tissue; glucose disposal; insulin; insulin resistance; lactate; metabolic regulation; whole-body glucose homeostasis
  6. Biochem Soc Trans. 2021 Feb 05. pii: BST20190730. [Epub ahead of print]
    Heberle AM, Rehbein U, Rodríguez Peiris M, Thedieck K.
      Cells have evolved highly intertwined kinase networks to finely tune cellular homeostasis to the environment. The network converging on the mechanistic target of rapamycin (MTOR) kinase constitutes a central hub that integrates metabolic signals and adapts cellular metabolism and functions to nutritional changes and stress. Feedforward and feedback loops, crosstalks and a plethora of modulators finely balance MTOR-driven anabolic and catabolic processes. This complexity renders it difficult - if not impossible - to intuitively decipher signaling dynamics and network topology. Over the last two decades, systems approaches have emerged as powerful tools to simulate signaling network dynamics and responses. In this review, we discuss the contribution of systems studies to the discovery of novel edges and modulators in the MTOR network in healthy cells and in disease.
    Keywords:  amino acids; computational models; mechanistic target of rapamycin; protein kinase B; signaling; systems biology
  7. J Clin Invest. 2021 Feb 02. pii: 143557. [Epub ahead of print]
    Karthikeyan S, Waters IG, Dennison L, Chu D, Donaldson J, Shin DH, Rosen DM, Gonzalez-Ericsson PI, Sanchez V, Sanders ME, Pantone MV, Bergman RE, Davidson BA, Reed SC, Zabransky DJ, Cravero K, Kyker-Snowman K, Button B, Wong HY, Hurley PJ, Croessmann S, Park B.
      Intratumor heterogeneity is an important mediator of poor outcomes in many cancers, including breast cancer. Genetic subclones frequently contribute to this heterogeneity, however their growth dynamics and interactions remain poorly understood. PIK3CA and HER2 alterations are known to co-exist in breast and other cancers. Herein, we present data that describe the ability of oncogenic PIK3CA mutant cells to induce the proliferation of quiescent HER2 mutant cells through a cell-contact mediated mechanism. Interestingly, the HER2 cells proliferated to become the major subclone over PIK3CA counterparts both in vitro and in vivo. Furthermore, this phenotype was observed in both hormone receptor positive and negative cell lines, and was dependent on the expression of fibronectin from mutant PIK3CA cells. Analysis of human tumors demonstrated similar HER2:PIK3CA clonal dynamics and fibronectin expression. Our study provides insights into non-random subclonal architecture of heterogenous tumors, which may aid understanding of tumor evolution and future strategies for personalized medicine.
    Keywords:  Breast cancer; Cell Biology; Molecular biology; Molecular genetics; Oncology
  8. Nature. 2021 Feb 03.
    Alonso-Curbelo D, Ho YJ, Burdziak C, Maag JLV, Morris JP, Chandwani R, Chen HA, Tsanov KM, Barriga FM, Luan W, Tasdemir N, Livshits G, Azizi E, Chun J, Wilkinson JE, Mazutis L, Leach SD, Koche R, Pe'er D, Lowe SW.
      Tissue damage increases the risk of cancer through poorly understood mechanisms1. In mouse models of pancreatic cancer, pancreatitis associated with tissue injury collaborates with activating mutations in the Kras oncogene to markedly accelerate the formation of early neoplastic lesions and, ultimately, adenocarcinoma2,3. Here, by integrating genomics, single-cell chromatin assays and spatiotemporally controlled functional perturbations in autochthonous mouse models, we show that the combination of Kras mutation and tissue damage promotes a unique chromatin state in the pancreatic epithelium that distinguishes neoplastic transformation from normal regeneration and is selected for throughout malignant evolution. This cancer-associated epigenetic state emerges within 48 hours of pancreatic injury, and involves an 'acinar-to-neoplasia' chromatin switch that contributes to the early dysregulation of genes that define human pancreatic cancer. Among the factors that are most rapidly activated after tissue damage in the pre-malignant pancreatic epithelium is the alarmin cytokine interleukin 33, which recapitulates the effects of injury in cooperating with mutant Kras to unleash the epigenetic remodelling program of early neoplasia and neoplastic transformation. Collectively, our study demonstrates how gene-environment interactions can rapidly produce gene-regulatory programs that dictate early neoplastic commitment, and provides a molecular framework for understanding the interplay between genetic and environmental cues in the initiation of cancer.
  9. J Cell Sci. 2021 Feb 04. pii: jcs.246090. [Epub ahead of print]
    Mazloumi Gavgani F, Karlsson T, Tangen IL, Morovicz AP, Arnesen VS, Turcu DC, Ninzima S, Spang K, Krakstad C, Guillermet-Guibert J, Lewis AE.
      The class I phosphoinositide 3-kinase (PI3K) catalytic subunits p110α and p110β are ubiquitously expressed but differently targeted in tumours. In cancer, PIK3CB (encoding p110β) is seldom mutated compared to PIK3CA (encoding p110α) but can contribute to tumorigenesis in certain PTEN-deficient tumours. The underlying molecular mechanisms are however unclear. We have previously reported that p110β is highly expressed in endometrial cancer (EC) cell lines and at the mRNA levels in primary patient tumours. Here, we show that p110β protein levels are high in both the cytoplasmic and nuclear compartments in EC cells. Moreover, high nuclear/cytoplasmic staining ratio were detected in high grade primary tumours. High levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3) were measured in the nucleus of EC cells and pharmacological and genetic approaches showed that its production was partly dependent upon p110β activity. Using immunofluorescence staining, p110β and PtdIns(3,4,5)P 3 were localised in the nucleolus, which correlated with high levels of 47S pre-rRNA. p110β inhibition led to a decrease in 47S rRNA levels and cell proliferation. In conclusion, these results present a nucleolar role for p110β that may contribute to tumorigenesis in EC.
    Keywords:  Endometrial cancer; Nucleolus; PIK3CB; Phosphoinositide 3-kinase; RDNA transcription; p110β
  10. Mol Biol Cell. 2021 Feb 03. mbcE20120784
    Way GP, Kost-Alimova M, Shibue T, Harrington WF, Gill S, Piccioni F, Becker T, Shafqat-Abbasi H, Hahn WC, Carpenter AE, Vazquez F, Singh S.
      Genetic and chemical perturbations impact diverse cellular phenotypes, including multiple indicators of cell health. These readouts reveal toxicity and antitumorigenic effects relevant to drug discovery and personalized medicine. We developed two customized microscopy assays, one using four targeted reagents and the other three targeted reagents, to collectively measure 70 specific cell health phenotypes including proliferation, apoptosis, reactive oxygen species (ROS), DNA damage, and cell cycle stage. We then tested an approach to predict multiple cell health phenotypes using Cell Painting, an inexpensive and scalable image-based morphology assay. In matched CRISPR perturbations of three cancer cell lines, we collected both Cell Painting and cell health data. We found that simple machine learning algorithms can predict many cell health readouts directly from Cell Painting images, at less than half the cost. We hypothesized that these models can be applied to accurately predict cell health assay outcomes for any future or existing Cell Painting dataset. For Cell Painting images from a set of 1,500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts. We provide a web app to browse predictions Our approach can be used to add cell health annotations to Cell Painting datasets.
  11. Cancer Sci. 2021 Feb 04.
    Zhao WJ, Zhu LL, Yang WQ, Xu SJ, Chen J, Ding XF, Liang Y, Chen G.
      Lysophosphatidic acid receptor 5 (LPAR5) is involved in mediating thyroid cancer progression, but the underlying mechanism needs to be further revealed. In this study, we confirmed that LPAR5 is upregulated in papillary thyroid carcinoma (PTC) especially in BRAF-like PTC by analyzing TCGA database and performing immunohistochemistry assay in human thyroid cancer tissues. LPAR5 specific antagonist TC LPA5 4 treatment inhibited CGTH-W3, TPC-1, B-CPAP and BHT-101 cells proliferation, CGTH-W3 and TPC-1 cells migration significantly. In vivo, TC LPA5 4 treatment could delay CGTH-W3 xenograft growth in nude mice. We also found that LPAR5 specific antagonists TC LPA5 4, PI3K inhibitor Wortmannin or mTOR inhibitor Rapamycin pretreatment abrogated phosphorylation of Akt and p70S6K1 stimulated by LPA in CGTH-W3 and TPC-1 cells. Stimulating CGTH-W3 cells transfected with pEGFPC1-Grp1-PH fusion protein with LPA resulted in the generation of phosphatidylinositol (3,4,5)-triphosphate, which indicates that PI3K was activated by LPA directly. The p110β-siRNA instead of p110α-siRNA transfection abrogated the increase of levels of phosphorylated Akt and S6K1 stimulated by LPA. Furthermore, immunoprecipitation assay confirmed an interaction between LPAR5 and p110β. Overall, we provide new insights that the downregulation of LPAR5 decreased the proliferation and migration phenotype via the PI3K/Akt pathway. Inhibition of LPAR5 or PI3K/Akt signal may be the novel therapeutic strategy for treating thyroid cancer.
    Keywords:  GPCR; LPAR5; PI3K; p110β; thyroid cancer
  12. Nature. 2021 Jan 27.
    Ansarullah , Jain C, Far FF, Homberg S, Wißmiller K, von Hahn FG, Raducanu A, Schirge S, Sterr M, Bilekova S, Siehler J, Wiener J, Oppenländer L, Morshedi A, Bastidas-Ponce A, Collden G, Irmler M, Beckers J, Feuchtinger A, Grzybek M, Ahlbrecht C, Feederle R, Plettenburg O, Müller TD, Meier M, Tschöp MH, Coskun Ü, Lickert H.
      Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic β-cells causes overt diabetes in mice; thus, therapies that sensitize β-cells to insulin may protect patients with diabetes against β-cell failure1-3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse β-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir-/-) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir-/- mice showed an increase in the activation of INSR-IGF1R in Iir-/- pancreatic tissue, resulting in an increase in the proliferation and mass of β-cells. Similarly, inducible β-cell-specific Iir-/- knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR-IGF1R and increased proliferation of β-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR-IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR-IGF1R in β-cells. Together, our findings show that inceptor shields insulin-producing β-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR-IGF1R sensitization and diabetes therapy.
  13. J Mol Biol. 2021 Feb 01. pii: S0022-2836(21)00032-2. [Epub ahead of print] 166838
    Martinez NG, Thieker DF, Carey LM, Rasquinha JA, Kistler SK, Kuhlman BA, Campbell SL.
      Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate to generate a key lipid second messenger, phosphatidylinositol 3,4,5-bisphosphate. PI3Kα and PI3Kγ require activation by RAS proteins to stimulate signaling pathways that control cellular growth, differentiation, motility and survival. Intriguingly, RAS binding to PI3K isoforms likely differ, as RAS mutations have been identified that discriminate between PI3Kα and PI3Kγ, consistent with low sequence homology (23%) between their RAS binding domains (RBDs). As disruption of the RAS/PI3Kα interaction reduces tumor growth in mice with RAS- and epidermal growth factor receptor driven skin and lung cancers, compounds that interfere with this key interaction may prove useful as anti-cancer agents. However, a structure of PI3Kα bound to RAS is lacking, limiting drug discovery efforts. Expression of full-length PI3K isoforms in insect cells has resulted in low yield and variable activity, limiting biophysical and structural studies of RAS/PI3K interactions. This led us to generate the first RBDs from PI3Kα and PI3Kγ that can be expressed at high yield in bacteria and bind to RAS with similar affinity to full-length PI3K. We also solved a 2.31 Å X-ray crystal structure of the PI3Kα-RBD, which aligns well to full-length PI3Kα. Structural differences between the PI3Kα and PI3Kγ RBDs are consistent with differences in thermal stability and may underly differential RAS recognition and RAS-mediated PI3K activation. These high expression, functional PI3K RBDs will aid in interrogating RAS interactions and could aid in identifying inhibitors of this key interaction.
    Keywords:  Phosphoinositide 3-kinase (PI3K); RAS; cancer; cell signaling; protein/protein interactions
  14. Nat Commun. 2021 01 27. 12(1): 615
    Li T, Chen X, Qian Y, Shao J, Li X, Liu S, Zhu L, Zhao Y, Ye H, Yang Y.
      Pulsing cellular dynamics in genetic circuits have been shown to provide critical capabilities to cells in stress response, signaling and development. Despite the fascinating discoveries made in the past few years, the mechanisms and functional capabilities of most pulsing systems remain unclear, and one of the critical challenges is the lack of a technology that allows pulsatile regulation of transgene expression both in vitro and in vivo. Here, we describe the development of a synthetic BRET-based transgene expression (LuminON) system based on a luminescent transcription factor, termed luminGAVPO, by fusing NanoLuc luciferase to the light-switchable transcription factor GAVPO. luminGAVPO allows pulsatile and quantitative activation of transgene expression via both chemogenetic and optogenetic approaches in mammalian cells and mice. Both the pulse amplitude and duration of transgene expression are highly tunable via adjustment of the amount of furimazine. We further demonstrated LuminON-mediated blood-glucose homeostasis in type 1 diabetic mice. We believe that the BRET-based LuminON system with the pulsatile dynamics of transgene expression provides a highly sensitive tool for precise manipulation in biological systems that has strong potential for application in diverse basic biological studies and gene- and cell-based precision therapies in the future.
  15. Nat Rev Cancer. 2021 Feb 05.
    Lambert AW, Weinberg RA.
      Epithelial stem cells serve critical physiological functions in the generation, maintenance and repair of diverse tissues through their ability to self-renew and spawn more specialized, differentiated cell types. In an analogous fashion, cancer stem cells have been proposed to fuel the growth, progression and recurrence of many carcinomas. Activation of an epithelial-mesenchymal transition (EMT), a latent cell-biological programme involved in development and wound healing, has been linked to the formation of both normal and neoplastic stem cells, but the mechanistic basis underlying this connection remains unclear. In this Perspective, we outline the instances where aspects of an EMT have been implicated in normal and neoplastic epithelial stem cells and consider the involvement of this programme during tissue regeneration and repair. We also discuss emerging concepts and evidence related to the heterogeneous and plastic cell states generated by EMT programmes and how these bear on our understanding of cancer stem cell biology and cancer metastasis. A more comprehensive accounting of the still-elusive links between EMT programmes and the stem cell state will surely advance our understanding of both normal stem cell biology and cancer pathogenesis.
  16. Sci Adv. 2021 Feb;pii: eabd7819. [Epub ahead of print]7(6):
    Chen J, Sivan U, Tan SL, Lippo L, De Angelis J, Labella R, Singh A, Chatzis A, Cheuk S, Medhghalchi M, Gil J, Hollander G, Marsden BD, Williams R, Ramasamy SK, Kusumbe AP.
      Blood vessels provide supportive microenvironments for maintaining tissue functions. Age-associated vascular changes and their relation to tissue aging and pathology are poorly understood. Here, we perform 3D imaging of young and aging vascular beds. Multiple organs in mice and humans demonstrate an age-dependent decline in vessel density and pericyte numbers, while highly remodeling tissues such as skin preserve the vasculature. Vascular attrition precedes the appearance of cellular hallmarks of aging such as senescence. Endothelial VEGFR2 loss-of-function mice demonstrate that vascular perturbations are sufficient to stimulate cellular changes coupled with aging. Age-associated tissue-specific molecular changes in the endothelium drive vascular loss and dictate pericyte to fibroblast differentiation. Lineage tracing of perivascular cells with inducible PDGFRβ and NG2 Cre mouse lines demonstrated that increased pericyte to fibroblast differentiation distinguishes injury-induced organ fibrosis and zymosan-induced arthritis. To spur further discoveries, we provide a freely available resource with 3D vascular and tissue maps.
  17. Mol Cancer Ther. 2021 Feb 03. pii: molcanther.0566.2020. [Epub ahead of print]
    Herrick WG, Kilpatrick CL, Hollingshead MG, Esposito D, O'Sullivan Coyne G, Gross AM, Johnson BC, Chen AP, Widemann BC, Doroshow JH, Parchment RE, Srivastava AK.
      Ras/Raf/MEK/ERK (MAPK) and PI3K/AKT signaling pathways influence several cell functions involved in oncogenesis, making them attractive drug targets. We describe a novel multiplex immunoassay to quantitate isoform-specific phosphorylation of proteins in the PI3K/AKT and MAPK pathways as a tool to assess pharmacodynamic changes. Isoform-specific assays measuring total protein and site-specific phosphorylation levels of ERK1/2, MEK1/2, AKT1/2/3, and rpS6 were developed on the Luminex platform with validated antibody reagents. The multiplex assay demonstrated satisfactory analytical performance. Fit-for-purpose validation was performed with xenograft models treated with selected agents. In PC3 and HCC70 xenograft tumors, the PI3Kβ inhibitor AZD8186 suppressed phosphorylation of AKT1, AKT2, and rpS6 for 4 to 7 hours post-single dose, but levels returned to baseline by 24 hours. AKT3 phosphorylation was suppressed in PC3 xenografts at all doses tested, but only at the highest dose in HCC70. The AKT inhibitor MK-2206 reduced AKT1/2/3 phosphorylation in SW620 xenograft tumors 2 to 4 hours post-dose, and the MEK inhibitor selumetinib reduced MEK1/2 and ERK1/2 phosphorylation by up to 50% and >90%, respectively. Clinical utility was demonstrated by analyzing biopsies from untreated patients with plexiform neurofibromas enrolled in a clinical trial of selumetinib (NCT02407405). These biopsies showed MEK and ERK phosphorylation levels sufficient for measuring up to 90% inhibition, and low AKT and rpS6 phosphorylation. This validated multiplex immunoassay demonstrates the degree and duration of phosphorylation modulation for three distinct classes of drugs targeting the PI3K/AKT and MAPK pathways.
  18. Leukemia. 2021 Feb 02.
    Ge Z, Song C, Ding Y, Tan BH, Desai D, Sharma A, Gowda R, Yue F, Huang S, Spiegelman V, Payne JL, Reeves ME, Iyer S, Dhanyamraju PK, Imamura Y, Bogush D, Bamme Y, Yang Y, Soliman M, Kane S, Dovat E, Schramm J, Hu T, McGrath M, Chroneos ZC, Payne KJ, Gowda C, Dovat S.
      Children of Hispanic/Latino ancestry have increased incidence of high-risk B-cell acute lymphoblastic leukemia (HR B-ALL) with poor prognosis. This leukemia is characterized by a single-copy deletion of the IKZF1 (IKAROS) tumor suppressor and increased activation of the PI3K/AKT/mTOR pathway. This identifies mTOR as an attractive therapeutic target in HR B-ALL. Here, we report that IKAROS represses MTOR transcription and IKAROS' ability to repress MTOR in leukemia is impaired by oncogenic CK2 kinase. Treatment with the CK2 inhibitor, CX-4945, enhances IKAROS activity as a repressor of MTOR, resulting in reduced expression of MTOR in HR B-ALL. Thus, we designed a novel therapeutic approach that implements dual targeting of mTOR: direct inhibition of the mTOR protein (with rapamycin), in combination with IKAROS-mediated transcriptional repression of the MTOR gene (using the CK2 inhibitor, CX-4945). Combination treatment with rapamycin and CX-4945 shows synergistic therapeutic effects in vitro and in patient-derived xenografts from Hispanic/Latino children with HR B-ALL. These data suggest that such therapy has the potential to reduce the health disparity in HR B-ALL among Hispanic/Latino children. The dual targeting of oncogene transcription, combined with inhibition of the corresponding oncoprotein provides a paradigm for a novel precision medicine approach for treating hematological malignancies.
  19. Sci Rep. 2021 Feb 03. 11(1): 2923
    Takenaga K, Akimoto M, Koshikawa N, Nagase H.
      The antidiabetic adiponectin receptor agonist AdipoRon has been shown to suppress the tumour growth of human pancreatic cancer cells. Because obesity and diabetes affect pancreatic cancer progression and chemoresistance, we investigated the effect of AdipoRon on orthotopic tumour growth of Panc02 pancreatic cancer cells in DIO (diet-induced obese) prediabetic mice. Administration of AdipoRon into DIO mice fed high-fat diets, in which prediabetic conditions were alleviated to some extent, did not reduce either body weight or tumour growth. However, when the DIO mice were fed low-fat diets, body weight and the blood leptin level gradually decreased, and importantly, AdipoRon became effective in suppressing tumour growth, which was accompanied by increases in necrotic areas and decreases in Ki67-positive cells and tumour microvessels. AdipoRon inhibited cell growth and induced necrotic cell death of Panc02 cells and suppressed angiogenesis of endothelial MSS31 cells. Insulin and IGF-1 only slightly reversed the AdipoRon-induced suppression of Panc02 cell survival but had no effect on the AdipoRon-induced suppression of MSS31 cell angiogenesis. Leptin significantly ameliorated AdipoRon-induced suppression of angiogenesis through inhibition of ERK1/2 activation. These results suggest that obesity-associated factors weaken the anticancer effect of AdipoRon, which indicates the importance of weight loss in combating pancreatic cancer.
  20. Science. 2021 Feb 05. pii: eabb4776. [Epub ahead of print]371(6529):
    Zalc A, Sinha R, Gulati GS, Wesche DJ, Daszczuk P, Swigut T, Weissman IL, Wysocka J.
      During development, cells progress from a pluripotent state to a more restricted fate within a particular germ layer. However, cranial neural crest cells (CNCCs), a transient cell population that generates most of the craniofacial skeleton, have much broader differentiation potential than their ectodermal lineage of origin. Here, we identify a neuroepithelial precursor population characterized by expression of canonical pluripotency transcription factors that gives rise to CNCCs and is essential for craniofacial development. Pluripotency factor Oct4 is transiently reactivated in CNCCs and is required for the subsequent formation of ectomesenchyme. Furthermore, open chromatin landscapes of Oct4+ CNCC precursors resemble those of epiblast stem cells, with additional features suggestive of priming for mesenchymal programs. We propose that CNCCs expand their developmental potential through a transient reacquisition of molecular signatures of pluripotency.
  21. Nat Commun. 2021 Feb 05. 12(1): 836
    Schumacher MA, Hsieh JJ, Liu CY, Appel KL, Waddell A, Almohazey D, Katada K, Bernard JK, Bucar EB, Gadeock S, Maselli KM, Washington MK, Grikscheit TC, Warburton D, Rosen MJ, Frey MR.
      Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3β inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.
  22. Cell Rep. 2021 Feb 02. pii: S2211-1247(21)00020-6. [Epub ahead of print]34(5): 108707
    Carrot-Zhang J, Yao X, Devarakonda S, Deshpande A, Damrauer JS, Silva TC, Wong CK, Choi HY, Felau I, Robertson AG, Castro MAA, Bao L, Rheinbay E, Liu EM, Trieu T, Haan D, Yau C, Hinoue T, Liu Y, Shapira O, Kumar K, Mungall KL, Zhang H, Lee JJ, Berger A, Gao GF, Zhitomirsky B, Liang WW, Zhou M, Moorthi S, Berger AH, Collisson EA, Zody MC, Ding L, Cherniack AD, Getz G, Elemento O, Benz CC, Stuart J, Zenklusen JC, Beroukhim R, Chang JC, Campbell JD, Hayes DN, Yang L, Laird PW, Weinstein JN, Kwiatkowski DJ, Tsao MS, Travis WD, Khurana E, Berman BP, Hoadley KA, Robine N, , Meyerson M, Govindan R, Imielinski M.
      RTK/RAS/RAF pathway alterations (RPAs) are a hallmark of lung adenocarcinoma (LUAD). In this study, we use whole-genome sequencing (WGS) of 85 cases found to be RPA(-) by previous studies from The Cancer Genome Atlas (TCGA) to characterize the minority of LUADs lacking apparent alterations in this pathway. We show that WGS analysis uncovers RPA(+) in 28 (33%) of the 85 samples. Among the remaining 57 cases, we observe focal deletions targeting the promoter or transcription start site of STK11 (n = 7) or KEAP1 (n = 3), and promoter mutations associated with the increased expression of ILF2 (n = 6). We also identify complex structural variations associated with high-level copy number amplifications. Moreover, an enrichment of focal deletions is found in TP53 mutant cases. Our results indicate that RPA(-) cases demonstrate tumor suppressor deletions and genome instability, but lack unique or recurrent genetic lesions compensating for the lack of RPAs. Larger WGS studies of RPA(-) cases are required to understand this important LUAD subset.
    Keywords:  TCGA; driver; genome analysis; lung adenocarcinoma; noncoding; oncogene; precision oncology; structural variation; tumor suppressor; whole genome sequencing
  23. Elife. 2021 Feb 01. pii: e65026. [Epub ahead of print]10
    Kinser HE, Mosley MC, Plutzer IB, Pincus Z.
      Across species, lifespan is highly variable among individuals within a population. Even genetically identical Caenorhabditis elegans reared in homogeneous environments are as variable in lifespan as outbred human populations. We hypothesized that persistent inter-individual differences in expression of key regulatory genes drives this lifespan variability. As a test, we examined the relationship between future lifespan and the expression of 22 microRNA promoter::GFP constructs. Surprisingly, expression of nearly half of these reporters, well before death, could effectively predict lifespan. This indicates that prospectively long- vs. short-lived individuals have highly divergent patterns of transgene expression and transcriptional regulation. The gene-regulatory processes reported on by two of the most lifespan-predictive transgenes do not require DAF-16, the FOXO transcription factor that is a principal effector of insulin/insulin-like growth factor (IGF-1) signaling. Last, we demonstrate a hierarchy of redundancy in lifespan-predictive ability among three transgenes expressed in distinct tissues, suggesting that they collectively report on an organism-wide, cell non-autonomous process that acts to set each individual's lifespan.
    Keywords:  C. elegans; aging biomarker; chromosomes; computational biology; gene expression; individual variability; lifespan; systems biology
  24. Trends Cancer. 2021 Feb 01. pii: S2405-8033(20)30334-4. [Epub ahead of print]
    Huang S.
      Post-treatment progression of tumors is commonly explained by somatic Darwinian evolution (i.e., selection of cells carrying genetic mutations that create more aggressive cell traits). But cancer genome and transcriptome analyses now paint a picture far more complex, prompting us to see beyond the Darwinian scheme: non-genetic cell phenotype plasticity explained by alternative stable gene expression states ('attractors'), may also produce aggressive phenotypes that can be selected for, without mutations. Worse, treatment may even induce cell state transitions into more malignant attractors. We review recent evidence for non-genetic mechanisms of progression, explain the theoretical foundation of attractor transitions behind treatment-induced increase of aggressiveness, and provide a framework for unifying genetic and non-genetic dynamics in tumor progression.
    Keywords:  attractor states; non-genetic plasticity; treatment resistance; treatment-induced progression
  25. Sci Adv. 2021 Jan;pii: eabc2100. [Epub ahead of print]7(1):
    Cheng K, Nair NU, Lee JS, Ruppin E.
      Various characteristics of cancers exhibit tissue specificity, including lifetime cancer risk, onset age, and cancer driver genes. Previously, the large variation in cancer risk across human tissues was found to strongly correlate with the number of stem cell divisions and abnormal DNA methylation levels. Here, we study the role of synthetic lethality in cancer risk. Analyzing normal tissue transcriptomics data in the Genotype-Tissue Expression project, we quantify the extent of co-inactivation of cancer synthetic lethal (cSL) gene pairs and find that normal tissues with more down-regulated cSL gene pairs have lower and delayed cancer risk. Consistently, more cSL gene pairs become up-regulated in cells treated by carcinogens and throughout premalignant stages in vivo. We also show that the tissue specificity of numerous tumor suppressor genes is associated with the expression of their cSL partner genes across normal tissues. Overall, our findings support the possible role of synthetic lethality in tumorigenesis.
  26. Sci Adv. 2021 Jan;pii: eabb1703. [Epub ahead of print]7(2):
    Cheah PS, Prabhakar S, Yellen D, Beauchamp RL, Zhang X, Kasamatsu S, Bronson RT, Thiele EA, Kwiatkowski DJ, Stemmer-Rachamimov A, György B, Ling KH, Kaneki M, Tannous BA, Ramesh V, Maguire CA, Breakefield XO.
      Tuberous sclerosis complex (TSC) results from loss of a tumor suppressor gene - TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins formed a complex to inhibit mTORC1-mediated cell growth and proliferation. Loss of either protein leads to overgrowth lesions in many vital organs. Gene therapy was evaluated in a mouse model of TSC2 using an adeno-associated virus (AAV) vector carrying the complementary for a "condensed" form of human tuberin (cTuberin). Functionality of cTuberin was verified in culture. A mouse model of TSC2 was generated by AAV-Cre recombinase disruption of Tsc2-floxed alleles at birth, leading to a shortened lifespan (mean 58 days) and brain pathology consistent with TSC. When these mice were injected intravenously on day 21 with AAV9-cTuberin, the mean survival was extended to 462 days with reduction in brain pathology. This demonstrates the potential of treating life-threatening TSC2 lesions with a single intravenous injection of AAV9-cTuberin.
  27. Nature. 2021 Jan 27.
    Wie J, Liu Z, Song H, Tropea TF, Yang L, Wang H, Liang Y, Cang C, Aranda K, Lohmann J, Yang J, Lu B, Chen-Plotkin AS, Luk KC, Ren D.
      Lysosomes have fundamental physiological roles and have previously been implicated in Parkinson's disease1-5. However, how extracellular growth factors communicate with intracellular organelles to control lysosomal function is not well understood. Here we report a lysosomal K+ channel complex that is activated by growth factors and gated by protein kinase B (AKT) that we term lysoKGF. LysoKGF consists of a pore-forming protein TMEM175 and AKT: TMEM175 is opened by conformational changes in, but not the catalytic activity of, AKT. The minor allele at rs34311866, a common variant in TMEM175, is associated with an increased risk of developing Parkinson's disease and reduces channel currents. Reduction in lysoKGF function predisposes neurons to stress-induced damage and accelerates the accumulation of pathological α-synuclein. By contrast, the minor allele at rs3488217-another common variant of TMEM175, which is associated with a decreased risk of developing Parkinson's disease-produces a gain-of-function in lysoKGF during cell starvation, and enables neuronal resistance to damage. Deficiency in TMEM175 leads to a loss of dopaminergic neurons and impairment in motor function in mice, and a TMEM175 loss-of-function variant is nominally associated with accelerated rates of cognitive and motor decline in humans with Parkinson's disease. Together, our studies uncover a pathway by which extracellular growth factors regulate intracellular organelle function, and establish a targetable mechanism by which common variants of TMEM175 confer risk for Parkinson's disease.
  28. Genome Biol. 2021 Feb 02. 22(1): 55
    Ietswaart R, Gyori BM, Bachman JA, Sorger PK, Churchman LS.
      A bottleneck in high-throughput functional genomics experiments is identifying the most important genes and their relevant functions from a list of gene hits. Gene Ontology (GO) enrichment methods provide insight at the gene set level. Here, we introduce GeneWalk ( ) that identifies individual genes and their relevant functions critical for the experimental setting under examination. After the automatic assembly of an experiment-specific gene regulatory network, GeneWalk uses representation learning to quantify the similarity between vector representations of each gene and its GO annotations, yielding annotation significance scores that reflect the experimental context. By performing gene- and condition-specific functional analysis, GeneWalk converts a list of genes into data-driven hypotheses.
    Keywords:  Differential expression; Functional analysis; GO enrichment; Gene set enrichment analysis; GeneWalk; INDRA (Integrated Network and Dynamical Reasoning Assembler); Machine learning; NET-seq; Network representation learning; Next-generation sequencing; Pathway Commons; RNA-seq
  29. Cancers (Basel). 2021 Jan 26. pii: 468. [Epub ahead of print]13(3):
    Hsu CF, Chen PC, Seenan V, Ding DC, Chu TY.
      Background: High-grade serous carcinoma (HGSC) is mainly derived from the stepwise accumulation of driver mutations in the fallopian tube epithelium (FTE), and it subsequently metastasizes to the ovary and peritoneum that develops into a clinically evident ovarian carcinoma. The developmental process involves cell proliferation/clonal expansion, cell migration, anoikis resistance, anchorage-independent growth (AIG), peritoneum attachment, and cell invasion. Previously, we discovered FTE could be transformed by follicular fluid (FF) released from ovulation, the most crucial risk factor of ovarian cancer, and IGF axis proteins in FF confers stemness activation and clonal expansion via IGF-1R/AKT pathway. However, whether other phenotypes in advanced cancer development are involved is unknown. Methods: A panel of FTE and ovarian HGSC cell lines with different severity of transformation were treated with FF with or without IGF-1R and AKT inhibitors and analyzed for the transformation phenotypes in vitro, ex vivo, and in vivo. Results: FF largely promotes (by order of magnitude) cell migration, AIG, cell invasion, peritoneum attachment, anoikis resistance, and cell proliferation. Most of these activities worked in the full panel of cell lines. The AIG activity largely depends on IGF-1R/AKT phosphorylation, and the proliferation activity depends on an AKT phosphorylation not mediated by IGF-1R. In contrast, both AKT- and non-AKT-mediated signals are responsible for the other transformation activities. Conclusions: Our data demonstrate an extensive transformation activity of FF in the full journey of carcinogenesis, and endorsed ovulation-inhibition for the prevention and AKT-inhibition for the treatment of ovarian HGSC.
    Keywords:  Fallopian tube; follicular fluid; high-grade serous carcinoma; peritoneal metastasis; transformation