bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2019‒09‒01
sixteen papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Nat Cell Biol. 2019 Aug 26.
      Mechanistic target of rapamycin (mTOR) kinase functions in two multiprotein complexes: lysosomal mTOR complex 1 (mTORC1) and mTORC2 at the plasma membrane. mTORC1 modulates the cell response to growth factors and nutrients by increasing protein synthesis and cell growth, and repressing the autophagy-lysosomal pathway1-4; however, dysfunction in mTORC1 is implicated in various diseases3,5,6. mTORC1 activity is regulated by phosphoinositide lipids7-10. Class I phosphatidylinositol-3-kinase (PI3K)-mediated production of phosphatidylinositol-3,4,5-trisphosphate6,11 at the plasma membrane stimulates mTORC1 signalling, while local synthesis of phosphatidylinositol-3,4-bisphosphate by starvation-induced recruitment of class II PI3K-β (PI3KC2-β) to lysosomes represses mTORC1 activity12. How the localization and activity of PI3KC2-β are regulated by mitogens is unknown. We demonstrate that protein kinase N (PKN) facilitates mTORC1 signalling by repressing PI3KC2-β-mediated phosphatidylinositol-3,4-bisphosphate synthesis downstream of mTORC2. Active PKN2 phosphorylates PI3KC2-β to trigger PI3KC2-β complex formation with inhibitory 14-3-3 proteins. Conversely, loss of PKN2 or inactivation of its target phosphorylation site in PI3KC2-β represses nutrient signalling via mTORC1. These results uncover a mechanism that couples mTORC2-dependent activation of PKN2 to the regulation of mTORC1-mediated nutrient signalling by local lipid signals.
  2. ACS Chem Biol. 2019 Aug 28.
      SGK3 is a PX domain containing protein kinase activated at endosomes downstream of Class 1 and 3 PI3K family members by growth factors and oncogenic mutations. SGK3 plays a key role in mediating resistance of breast cancer cells to Class 1 PI3K or Akt inhibitors, by substituting for loss of Akt activity and restoring proliferative pathways such as mTORC1 signaling. It is therefore critical to develop tools to potently target SGK3, and obstruct its role in inhibitor resistance. Here we describe the development of SGK3-PROTAC1, a PROTAC conjugate of the 308-R SGK inhibitor with the VH032 VHL binding ligand, targeting SGK3 for degradation. 0.3 µM SGK3-PROTAC1 induced 50% degradation of endogenous SGK3 within 2 hours, with maximal 80% degradation observed within 8 hours, accompanied by a loss of phosphorylation of NDRG1, an SGK3 substrate. SGK3-PROTAC1 did not degrade closely related SGK1 and SGK2 isoforms that are nevertheless engaged and inhibited by 308-R. Proteomic analysis revealed that SGK3 was the only cellular protein whose cellular levels were significantly reduced following treatment with SGK3-PROTAC1. Low doses of SGK3-PROTAC1 (0.1-0.3 µM) restored sensitivity of SGK3 dependent ZR-75-1 and CAMA-1 breast cancer cells to Akt (AZD5363) and PI3K (GDC0941) inhibitors, whereas the cis epimer analogue incapable of binding to the VHL E3 ligase had no impact. SGK3-PROTAC1 suppressed proliferation of ZR-75-1 and CAMA-1 cancer cell lines treated with a PI3K inhibitor (GDC0941) more effectively than could be achieved by a conventional SGK isoform inhibitor (14H). This work underscores the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors. SGK3-PROTAC1 will be an important reagent to explore the roles of the SGK3 pathway.
  3. Adv Exp Med Biol. 2019 ;1152 283-292
      Based on the insights gleaned from decades of research, it seems clear that mechanistic target of rapamycin (mTOR) is an essential signaling node that integrates environmental clues for regulation of cell survival, metabolism and proliferation of the cells. However, overwhelmingly increasing scientific evidence has added a new layer of intricacy to already complicated and versatile signaling pathway of mTOR. Deregulation of spatio-temporally controlled mTOR-driven pathway played contributory role in breast cancer development and progression. Pharmacologists and molecular biologists have specifically emphasized on the identification and development of mTOR-pathway inhibitors. In this chapter we have attempted to provide an overview of the most recent findings related to therapeutic targeting of mTOR-associated mTORC1 and mTORC2 in breast cancer. We have also comprehensively summarized regulation of mTOR and its partners by microRNAs in breast cancer.
    Keywords:  Apoptosis; Signaling; Therapy; mTOR
  4. Cell Rep. 2019 Aug 27. pii: S2211-1247(19)30967-2. [Epub ahead of print]28(9): 2317-2330.e8
      Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor and bi-functional lipid and protein phosphatase. We report that the metabolic regulator pyruvate dehydrogenase kinase1 (PDHK1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The PTEN protein phosphatase dephosphorylates nuclear factor κB (NF-κB)-activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NF-κB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to induce aerobic glycolysis and PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, a biomarker of decreased patient survival. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers.
    Keywords:  NF-κB; NKAP; PDHK1; PTEN; cancer; metabolism; protein phosphatase; signaling; synthetic lethality
  5. Cold Spring Harb Perspect Med. 2019 Aug 26. pii: a036228. [Epub ahead of print]
      Phosphatase and tensin homolog (PTEN) is a tumor suppressor that directly regulates a diverse array of cellular phenotypes, including growth, migration, morphology, and genome stability. How a single protein impacts so many important cellular processes remains a fascinating question. This question has been partially resolved by the characterization of a slew of missense variants that alter or eliminate PTEN's various molecular functions, including its enzymatic activity, subcellular localization, and posttranslational modifications. Here, we review what is known about how PTEN variants impact molecular function and, consequently, cellular phenotype. In particular, we highlight eight informative "sentinel variants" that abrogate distinct molecular functions of PTEN. We consider two published massively parallel assays of variant effect that measured the effect of thousands of PTEN variants on protein abundance and enzymatic activity. Finally, we discuss how characterization of clinically ascertained variants, establishment of clinical sequencing databases, and massively parallel assays of variant effect yield complementary datasets for dissecting PTEN's role in disease.
  6. Cancers (Basel). 2019 Aug 26. pii: E1247. [Epub ahead of print]11(9):
      Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene frequently found to be inactivated in over 30% of human cancers. PTEN encodes a 54-kDa lipid phosphatase that serves as a gatekeeper of the phosphoinositide 3-kinase pathway involved in the promotion of multiple pro-tumorigenic phenotypes. Although the PTEN protein plays a pivotal role in carcinogenesis, cumulative evidence has implicated it as a key signaling molecule in several other diseases as well, such as diabetes, Alzheimer's disease, and autism spectrum disorders. This finding suggests that diverse cell types, especially differentiated cells, express PTEN. At the cellular level, PTEN is widely distributed in all subcellular compartments and organelles. Surprisingly, the cytoplasmic compartment, not the plasma membrane, is the predominant subcellular location of PTEN. More recently, the finding of a secreted 'long' isoform of PTEN and the presence of PTEN in the cell nucleus further revealed unexpected biological functions of this multifaceted molecule. At the regulatory level, PTEN activity, stability, and subcellular distribution are modulated by a fascinating array of post-translational modification events, including phosphorylation, ubiquitination, and sumoylation. Dysregulation of these regulatory mechanisms has been observed in various human diseases. In this review, we provide an up-to-date overview of the knowledge gained in the last decade on how different functional domains of PTEN regulate its biological functions, with special emphasis on its subcellular distribution. This review also highlights the findings of published studies that have reported how mutational alterations in specific PTEN domains can lead to pathogenesis in humans.
    Keywords:  PI3K; PTEN; phosphatase; regulation
  7. Leukemia. 2019 Aug 28.
      Fcγ receptor (FcγR) signalling in monocyte derived macrophages from chronic lymphocytic leukaemia (CLL) patients is poorly understood. This signalling pathway is the key determinant of the ability of the macrophages to respond to therapeutic antibodies in current clinical use for CLL. Muted FcγR signalling activity accompanies disease progression and results in resistance to therapeutic antibodies. The molecular mechanisms controlling FcγR signalling and resistance are unknown. Here, we demonstrate that the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110δ is essential for CLL-derived macrophages to respond to therapeutic antibodies. Inhibition of p110δ in the macrophages reduces FcγR-mediated antibody immune responses. Surprisingly, our studies indicated that FcγR downstream signalling is independent of SYK and BTK activity. Thus, we show that FcγR antibody responses occur via a previously unidentified p110δ-dependent pathway, which is independent of the previously described SYK/BTK activation pathway. These data provide novel insights into the effectors of antibody responses. Our data also provide mechanistic insights into therapy resistance in CLL.
  8. EMBO Rep. 2019 Aug 30. e48058
      Cyclin-dependent kinase 12 (CDK12) has emerged as an effective therapeutic target due to its ability to regulate DNA damage repair in human cancers, but little is known about the role of CDK12 in driving tumorigenesis. Here, we demonstrate that CDK12 promotes tumor initiation as a novel regulator of cancer stem cells (CSCs) and induces anti-HER2 therapy resistance in human breast cancer. High CDK12 expression caused by concurrent amplification of CDK12 and HER2 in breast cancer patients is associated with disease recurrence and poor survival. CDK12 induces self-renewal of breast CSCs and in vivo tumor-initiating ability, and also reduces susceptibility to trastuzumab. Furthermore, CDK12 kinase activity inhibition facilitates anticancer efficacy of trastuzumab in HER2+ tumors, and mice bearing trastuzumab-resistant HER2+ tumor show sensitivity to an inhibitor of CDK12. Mechanistically, the catalytic activity of CDK12 is required for the expression of genes involved in the activation of ErbB-PI3K-AKT or WNT-signaling cascades. These results suggest that CDK12 is a major oncogenic driver and an actionable target for HER2+ breast cancer to replace or augment current anti-HER2 therapies.
    Keywords:  CDK12; HER2; breast cancer; cancer stem cells; trastuzumab
  9. J Biol Chem. 2019 Aug 29. pii: jbc.RA119.010130. [Epub ahead of print]
      Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play a critical role in transmitting signals from cell surface molecules to intracellular protein effectors. Key PI3Ks include PI3Kα, PI3Kβ and PI3Kδ, which are regulated by receptors. The signaling pathway comprising the PI3Ks, along with a Ser/Thr kinase: AKT, a proto-oncogene product: mouse double minute (MDM)2, and tumor suppressor protein: p53, plays an essential role in experimental proliferative vitreoretinopathy (PVR), which is a fibrotic blinding eye disorder. However, which PI3K isoforms are involved in PVR is unknown. A major characteristic of PVR is the formation of epi (or sub) -retinal membranes that consist of extracellular matrix and cells including retinal pigment epithelium (RPE) cells, glial cells and macrophages. RPE cells are considered key players in PVR pathogenesis. Using immunoblotting and immunofluorescence analyses, we herein provide the evidence that PI3Kδ is highly expressed in human RPEs when it is primarily expressed in leukocytes. We also found that PI3Kδ inactivation through two approaches, CRISPR/Cas9-mediated depletion and a PI3Kδ specific inhibitor (idelalisib) not only blocks vitreous-induced activation of AKT and MDM2 but also abrogates a vitreous-stimulated decrease in p53. Furthermore, we demonstrate that PI3Kδ inactivation prevents vitreous-induced proliferation, migration and contraction of human RPEs. These results suggest that PI3Kδ may represent a potential therapeutic target for RPE-related eye diseases including PVR.
    Keywords:  Akt PKB; migration; mouse double minute 2 homolog (MDM2); p53; phosphatidylinositide 3-kinase (PI 3-kinase)
  10. Nat Rev Cancer. 2019 Aug 27.
      Genetic intratumoural heterogeneity is a natural consequence of imperfect DNA replication. Any two randomly selected cells, whether normal or cancerous, are therefore genetically different. Here, we review the different forms of genetic heterogeneity in cancer and re-analyse the extent of genetic heterogeneity within seven types of untreated epithelial cancers, with particular regard to its clinical relevance. We find that the homogeneity of predicted functional mutations in driver genes is the rule rather than the exception. In primary tumours with multiple samples, 97% of driver-gene mutations in 38 patients were homogeneous. Moreover, among metastases from the same primary tumour, 100% of the driver mutations in 17 patients were homogeneous. With a single biopsy of a primary tumour in 14 patients, the likelihood of missing a functional driver-gene mutation that was present in all metastases was 2.6%. Furthermore, all functional driver-gene mutations detected in these 14 primary tumours were present among all their metastases. Finally, we found that individual metastatic lesions responded concordantly to targeted therapies in 91% of 44 patients. These analyses indicate that the cells within the primary tumours that gave rise to metastases are genetically homogeneous with respect to functional driver-gene mutations, and we suggest that future efforts to develop combination therapies have the potential to be curative.
  11. Elife. 2019 Aug 27. pii: e43668. [Epub ahead of print]8
      Human lung adenocarcinoma exhibits a propensity for de-differentiation, complicating diagnosis and treatment, and predicting poorer patient survival. In genetically engineered mouse models of lung cancer, expression of the BRAFV600E oncoprotein kinase initiates the growth of benign tumors retaining characteristics of their cell of origin, AT2 pneumocytes. Cooperating alterations that activate PI3'-lipid signaling promote progression of BRAFV600E-driven benign tumors to malignant adenocarcinoma. However, the mechanism(s) by which this cooperation occurs remains unclear. To address this, we generated mice carrying a conditional BrafCAT allele in which CRE-mediated recombination leads to co-expression of BRAFV600E and tdTomato. We demonstrate that co-expression of BRAFV600E and PIK3CAH1047R in AT2 pneumocytes leads to rapid cell de-differentiation, without decreased expression of the transcription factors NKX2-1, FOXA1, or FOXA2. Instead, we propose a novel role for PGC1α in maintaining AT2 pneumocyte identity. These findings provide insight into how these pathways may cooperate in the pathogenesis of human lung adenocarcinoma.
    Keywords:  BRAF; PGC1a; PI3K; cancer biology; de-differentiation; lung adenocarcinoma; mouse
  12. Genome Res. 2019 Aug 29.
      Obesity is an increasing pathophysiological problem in developed societies. Despite all major progress in understanding molecular mechanisms of obesity, currently available anti-obesity drugs have shown limited efficacy with severe side effects. CRISPR interference (CRISPRi) mechanism based on catalytically dead Cas9 (dCas9) and single guide RNA (sgRNA) was combined with a targeted nonviral gene delivery system to treat obesity and obesity-induced type 2 diabetes. A fusion peptide targeting a vascular and cellular marker of adipose tissue, prohibitin, was developed by conjugation of adipocyte targeting sequence (CKGGRAKDC) to 9-mer arginine (ATS-9R). (dCas9/sgFabp4) + ATS-9R oligoplexes showed effective condensation and selective delivery into mature adipocytes. Targeted delivery of the CRISPRi system against Fabp4 to white adipocytes by ATS-9R induced effective silencing of Fabp4, resulting in reduction of body weight and inflammation and restoration of hepatic steatosis in obese mice. This RNA-guided DNA recognition platform provides a simple and safe approach to regress and treat obesity and obesity-induced metabolic syndromes.
  13. Commun Biol. 2019 ;2 318
      Knockout of phosphatase and tensin homolog (PTEN-/-) is neuroprotective and promotes axon regeneration in mature neurons. Elevation of mTOR activity in injured neurons has been proposed as the primary underlying mechanism. Here we demonstrate that PTEN-/- also abrogates the inhibitory activity of GSK3 on collapsin response mediator protein 2 (CRMP2) in retinal ganglion cell (RGC) axons. Moreover, maintenance of GSK3 activity in Gsk3 S/A knockin mice significantly compromised PTEN-/--mediated optic nerve regeneration as well as the activity of CRMP2, and to a lesser extent, mTOR. These GSK3S/A mediated negative effects on regeneration were rescued by viral expression of constitutively active CRMP2T/A, despite decreased mTOR activation. Gsk3 S/A knockin or CRMP2 inhibition also decreased PTEN-/- mediated neurite growth of RGCs in culture and disinhibition towards CNS myelin. Thus, the GSK3/CRMP2 pathway is essential for PTEN-/- mediated axon regeneration. These new mechanistic insights may help to find novel strategies to promote axon regeneration.
    Keywords:  Brain injuries; Cellular neuroscience; Regeneration and repair in the nervous system
  14. Oncotarget. 2019 Aug 13. 10(48): 4951-4959
      Gene transcription is a highly complex and strictly regulated process. RNA polymerase II (Pol II) C-terminal domain (CTD) undergoes massive cycles of phosphorylation and dephosphorylation during the process of gene transcription. These post-translational modifications of CTD provide an interactive platform for various factors required for transcription initiation, elongation, termination, and co-transcriptional RNA processing. Pol II CTD kinases and phosphatases are key regulators and any deviation may cause genome-wide transcriptional dysregulation leading to various pathological conditions including cancer. PTEN, a well known tumor suppressor, is one of the most commonly somatically altered in diverse malignancies. When mutated in the germline, PTEN causes cancer predisposition. Numerous studies have demonstrated that PTEN regulates the expression of hundreds of genes, however, no mechanism is known so far. PTEN is a dual specificity phosphatase, using both lipid and protein as substrates. In the present study, we demonstrate that PTEN interacts with the RNA Pol II and that PTEN expression is inversely correlated with global phosphorylation of Pol II CTD. Furthermore, PTEN dephosphorylates Pol II CTD in vitro with a significant specificity for Ser5p. Interestingly, ChIP-seq data analysis revealed that PTEN globally binds to promoter proximal regions, and PTEN loss increases genome-wide Pol II Ser5p occupancy, suggest that PTEN is a Pol II CTD phosphatase. Our observations demonstrate an unexplored function of PTEN with the potential of global transcriptional regulation, adding a new dimension to somatic carcinogenesis and germline cancer predisposition.
    Keywords:  PTEN hamartoma tumor syndrome; Pol II CTD dephosphorylation; RNA polymerase II; cowden syndrome; phosphatase and tensin homolog (PTEN)
  15. Cell Metab. 2019 Aug 19. pii: S1550-4131(19)30385-7. [Epub ahead of print]
      Lipid metabolism is frequently perturbed in cancers, but the underlying mechanism is unclear. We present comprehensive evidence that oncogene MYC, in collaboration with transcription factor sterol-regulated element-binding protein (SREBP1), regulates lipogenesis to promote tumorigenesis. We used human and mouse tumor-derived cell lines, tumor xenografts, and four conditional transgenic mouse models of MYC-induced tumors to show that MYC regulates lipogenesis genes, enzymes, and metabolites. We found that MYC induces SREBP1, and they collaborate to activate fatty acid (FA) synthesis and drive FA chain elongation from glucose and glutamine. Further, by employing desorption electrospray ionization mass spectrometry imaging (DESI-MSI), we observed in vivo lipidomic changes upon MYC induction across different cancers, for example, a global increase in glycerophosphoglycerols. After inhibition of FA synthesis, tumorigenesis was blocked, and tumors regressed in both xenograft and primary transgenic mouse models, revealing the vulnerability of MYC-induced tumors to the inhibition of lipogenesis.
    Keywords:  ChIP; MYC; MYC conditional transgenic mouse models; RNA-seq; SREBP1; acetyl-CoA carboxylase A inhibition; carbon tracing; fatty acid synthesis; glycerophosphoglycerols; mass spectrometry imaging; nuclear run-on
  16. Aging (Albany NY). 2019 Aug 27.
    Keywords:  aging; mTORC1; oxidative stress; rapamycin; skeletal muscle