bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021‒04‒04
eighteen papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Biophys J. 2021 Mar 25. pii: S0006-3495(21)00248-4. [Epub ahead of print]
      Akt plays a key role in the Ras/PI3K/Akt/mTOR signaling pathway. In breast cancer, Akt translocation to the plasma membrane is enabled by the interaction of its pleckstrin homology domain (PHD) with calmodulin (CaM). At the membrane, the conformational change promoted by PIP3 releases CaM and facilitates Thr308 and Ser473 phosphorylation and activation. Here, using modelling and molecular dynamics (MD) simulations we aim to figure out how CaM interacts with Akt's PHD at the atomic level. Our simulations show that the CaM-PHD interaction is thermodynamically stable and involves a β-strand, rather than an α-helix, in agreement with NMR data, and that electrostatic and hydrophobic interactions are critical. PHD interacts with CaM lobes; however, multiple modes are possible. IP4, the polar head of PIP3, weakens the CaM-PHD interaction, implicating the release mechanism at the plasma membrane. Recently, we unraveled the mechanism of PI3Kα activation at the atomistic level and the structural basis for Ras role in the activation. Here, our atomistic structural data clarify the mechanism of how CaM interacts, delivers, and releases Akt - the next node in the Ras/PI3K pathway - at the plasma membrane.
    Keywords:  Akt activation; CaM; MD Simulation; PH domain; PI3K; Phosphorylation; Ras; plasma membrane; signaling pathway
  2. Pharmacol Res. 2021 Mar 24. pii: S1043-6618(21)00163-8. [Epub ahead of print] 105579
      The discovery of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway was a major advance in understanding eukaryotic signal transduction. The high frequency of PI 3-kinase pathway mutations in many cancers stimulated the development of drugs targeting these oncogenic mutants. The PI 3-kinases are divided into three classes and Class I PI 3-kinases, which catalyze the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3), are the main subject of this review. The class I PI 3-kinases are made up of p110α, p110β, p110δ, and p110γ catalytic subunits. These catalytic subunits are constitutively bound to regulatory subunits (p85α, p85β, p55γ, p101, and p87 proteins). The p85/p55 regulatory subunits heterodimerize with p110α or p110δ thereby forming complexes that are regulated chiefly by receptor protein-tyrosine kinases. The p101 and p87 subunits heterodimerize with p110γ to form complexes that are regulated mainly by G protein-coupled receptors (GPCRs). Complexes containing the p110β subunit are activated by receptor protein-tyrosine kinases as well as GPCRs. Following the generation of PIP3, the AKT and mTOR protein-serine/threonine kinases are activated leading to cell growth, proliferation, and survival. Like protein kinases, the PI 3-kinase domains consist of a bilobed structure connected by a hinge-linker segment. ATP and most PI 3-kinase and protein kinase inhibitors form hydrogen bonds with hinge residues. The small and large lobes of PI 3-kinases and protein kinases have a very similar three-dimensional structure called the protein kinase fold. Both PI 3-kinases and eukaryotic protein kinases possess an activation segment that begins with a DFG triad (aspartate-phenylalanine-glycine). The protein kinase catalytic loop has an HRD (histidine-arginine-aspartate) signature while that of the PI 3-kinases reverses the order with a DRH triad. Alpelisib is an orally effective FDA-approved PI 3-kinase-α inhibitor used for the treatment of breast cancer. Copanlisib, duvelisib, idelalisib, and umbralisib are PI 3-kinase-δ inhibitors that are approved for the third-line treatment of follicular lymphomas and other hematological disorders. Copanlisib is also a potent inhibitor of PI 3-kinase-α. Of the five approved drugs, all are orally bioavailable except copanlisib. Idelalisib interacts with the active conformation of PI 3-kinase-δ and is classified as a type I inhibitor. Alpelisib and copanlisib interact with inactive PI 3-kinase-α and PI 3-kinase-γ, respectively, and are classified as a type I½ antagonists. Except for umbralisib with a molecular weight of 571.5, all five drugs conform to the Lipinski rule of five for oral effectiveness. Copanlisib, however, must be given intravenously. Alpelisib and copanlisib inhibit PI 3-kinase-α, which is involved in insulin signaling, and both drugs promote insulin-resistance and produce hyperglycemia. The five FDA-approved PI 3-kinase inhibitors produce significant on-target toxicities, more so than many approved protein kinase antagonists. The development of PI 3-kinase inhibitors with fewer toxicities is an important long-term therapeutic goal.
    Keywords:  Acalabrutinib (PubChem CID: 71226662); Alpelisib (PubChem CID: 56649450); Breast cancer, Chronic lymphocytic leukemia; Copanlisib (PubChem CID: 135565596); Duvelisib (PubChem CID: 50905713); Follicular lymphoma; Fulvestrant (PubChem CID: 104741); Ibrutinib (PubChem CID: 24821094); Idelalisib (PubChem CID: 11625818); Insulin (PubChem CID: 16131098); Marginal zone lymphoma; PI 3-kinase structure; Phosphatidylinositol-3,4,5-trisphosphate (PubChem CID: 53477782); Small lymphocytic lymphoma; Umbralisib (PubChem CID: 72950888)
  3. Int J Mol Sci. 2021 Mar 30. pii: 3567. [Epub ahead of print]22(7):
      Cutaneous squamous cell carcinomas (cSCCs) account for about 20% of keratinocyte carcinomas, the most common cancer in the UK. Therapeutic options for cSCC patients who develop metastasis are limited and a better understanding of the biochemical pathways involved in cSCC development/progression is crucial to identify novel therapeutic targets. Evidence indicates that the phosphoinositide 3-kinases (PI3Ks)/Akt pathway plays an important role, in particular in advanced cSCC. Questions remain of whether all four PI3K isoforms able to activate Akt are involved and whether selective inhibition of specific isoform(s) might represent a more targeted strategy. Here we determined the sensitivity of four patient-derived cSCC cell lines to isoform-specific PI3K inhibitors to start investigating their potential therapeutic value in cSCC. Parallel experiments were performed in immortalized keratinocyte cell lines. We observed that pan PI3Ks inhibition reduced the growth/viability of all tested cell lines, confirming the crucial role of this pathway. Selective inhibition of the PI3K isoform p110α reduced growth/viability of keratinocytes and of two cSCC cell lines while affecting the other two only slightly. Importantly, p110α inhibition reduced Akt phosphorylation in all cSCC cell lines. These data indicate that growth and viability of the investigated cSCC cells display differential sensitivity to isoform-specific PI3K inhibitors.
    Keywords:  BYL719; cutaneous squamous cell carcinoma; isoform-specific PI3K inhibitors; mTOR; phosphoinositide 3-kinases
  4. Nat Commun. 2021 Mar 31. 12(1): 1998
      The heterogeneity of breast cancer plays a major role in drug response and resistance and has been extensively characterized at the genomic level. Here, a single-cell breast cancer mass cytometry (BCMC) panel is optimized to identify cell phenotypes and their oncogenic signalling states in a biobank of patient-derived tumour xenograft (PDTX) models representing the diversity of human breast cancer. The BCMC panel identifies 13 cellular phenotypes (11 human and 2 murine), associated with both breast cancer subtypes and specific genomic features. Pre-treatment cellular phenotypic composition is a determinant of response to anticancer therapies. Single-cell profiling also reveals drug-induced cellular phenotypic dynamics, unravelling previously unnoticed intra-tumour response diversity. The comprehensive view of the landscapes of cellular phenotypic heterogeneity in PDTXs uncovered by the BCMC panel, which is mirrored in primary human tumours, has profound implications for understanding and predicting therapy response and resistance.
  5. Lancet Oncol. 2021 Apr;pii: S1470-2045(21)00034-6. [Epub ahead of print]22(4): 489-498
      BACKGROUND: Alpelisib, a PI3Kα-selective inhibitor and degrader, plus fulvestrant showed efficacy in hormone receptor-positive, HER2-negative, PIK3CA-mutated advanced breast cancer in SOLAR-1; limited data are available in the post-cyclin-dependent kinase 4/6 inhibitor setting. BYLieve aimed to assess alpelisib plus endocrine therapy in this setting in three cohorts defined by immediate previous treatment; here, we report results from cohort A.METHODS: This ongoing, phase 2, multicentre, open-label, non-comparative study enrolled patients with hormone receptor-positive, HER2-negative, advanced breast cancer with tumour PIK3CA mutation, following progression on or after previous therapy, including CDK4/6 inhibitors, from 114 study locations (cancer centres, medical centres, university hospitals, and hospitals) in 18 countries worldwide. Participants aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 2 or less, with no more than two previous anticancer treatments and no more than one previous chemotherapy regimen, were enrolled in three cohorts. In cohort A, patients must have had progression on or after a CDK4/6 inhibitor plus an aromatase inhibitor as the immediate previous treatment. Patients received oral alpelisib 300 mg/day (continuously) plus fulvestrant 500 mg intramuscularly on day 1 of each 28-day cycle and on day 15 of cycle 1. The primary endpoint was the proportion of patients alive without disease progression at 6 months per local assessment using Response Evaluation Criteria in Solid Tumors, version 1.1, in patients with a centrally confirmed PIK3CA mutation. This trial is registered with, NCT03056755.
    FINDINGS: Between Aug 14, 2017, and Dec 17, 2019 (data cutoff), 127 patients with at least 6 months' follow-up were enrolled into cohort A. 121 patients had a centrally confirmed PIK3CA mutation. At data cutoff, median follow-up was 11·7 months (IQR 8·5-15·9). 61 (50·4%; 95% CI 41·2-59·6) of 121 patients were alive without disease progression at 6 months. The most frequent grade 3 or worse adverse events were hyperglycaemia (36 [28%] of 127 patients), rash (12 [9%]), and rash maculopapular (12 [9%]). Serious adverse events occurred in 33 (26%) of 127 patients. No treatment-related deaths were reported.
    INTERPRETATION: BYLieve showed activity of alpelisib plus fulvestrant with manageable toxicity in patients with PIK3CA-mutated, hormone receptor-positive, HER2-negative advanced breast cancer, after progression on a CDK4/6 inhibitor plus an aromatase inhibitor.
    FUNDING: Novartis Pharmaceuticals.
  6. Cancers (Basel). 2021 Mar 26. pii: 1538. [Epub ahead of print]13(7):
      The phosphatidylinositol-3-kinase (PI3K) pathway plays a central role in the regulation of several signalling cascades which regulate biological processes such as cellular growth, survival, proliferation, motility and angiogenesis. The hyperactivation of this pathway is linked to tumour progression and is one of the most common events in human cancers. Additionally, aberrant activation of the PI3K pathway has been demonstrated to limit the effectiveness of a number of anti-tumour agents paving the way for the development and implementation of PI3K inhibitors in the clinic. However, the overall effectiveness of these compounds has been greatly limited by inadequate target engagement due to reactivation of the pathway by compensatory mechanisms. Herein, we review the common adaptive responses that lead to reactivation of the PI3K pathway, therapy resistance and potential strategies to overcome these mechanisms of resistance. Furthermore, we highlight the potential role in changes in cellular plasticity and PI3K inhibitor resistance.
    Keywords:  PI3K pathway; PI3K pathway inhibitors; mechanisms of resistance
  7. Cancers (Basel). 2021 Mar 13. pii: 1280. [Epub ahead of print]13(6):
      Chronic lymphocytic leukemia (CLL) shows constitutive phosphatidylinositol 3-kinase (PI3K) activation resulting from aberrant regulation of the B-cell receptor (BCR) signaling. PI3K inhibitors have been evaluated in CLL therapy, bringing a new treatment opportunity for patients with this disease. Despite the proven therapeutic efficacy, the use of approved PI3K inhibitors is limited by severe immune-mediated toxicities and given the availability of other more tolerable agents. This article reviews the relevance of PI3K signaling and pharmacologic inhibition in CLL. Data on efficacy and toxicity of PI3K inhibitors are also presented, as well as strategies for overcoming barriers for their clinical use in CLL treatment.
    Keywords:  PI3K inhibitors; chronic lymphocytic leukemia; targeted therapy
  8. FASEB J. 2021 May;35(5): e21465
      N6 -methyladenosine (m6A) methylation is the most prevalent RNA modification, and it emerges as an important regulatory mechanism of gene expression involved in many cellular and biological processes. However, the role of m6 A methylation in vascular development is not clear. The m6 A RNA methylation is regulated by dynamic interplay among methyltransferases, binding proteins, and demethylases. Mettl3 is a member of the mettl3-mettl14 methyltransferase complex, referred to as writers that catalyze m6A RNA methylation. Here, we used CRISPR-Cas9 genome editing to develop two lines of knockout (KO) zebrafish for mettl3. Heterozygous mettl3+/- KO embryos show defective vascular development, which is directly visible in fli-EGFP and flk-EGFP zebrafish. Alkaline phosphatase staining and whole mount in situ hybridization with cdh5, and flk markers demonstrated defective development of intersegmental vessels (ISVs), subintestinal vessels (SIVs), interconnecting vessels (ICVs) and dorsal longitudinal anastomotic vessels (DLAV) in both heterozygous mettl3+/- and homozygous mettl3-/- KO zebrafish embryos. Similar phenotypes were observed in zebrafish embryos with morpholino knockdown (KD) of mettl3; however, the vascular defects were rescued fully by overexpression of constitutively active AKT1. KD of METTL3 in human endothelial cells inhibited cell proliferation, migration, and capillary tube formation. Mechanistically, mettl3 KO and KD significantly reduced the levels of m6 A RNA methylation, and AKT phosphorylation (S473) by an increase in the expression of phosphatase enzyme PHLPP2 and reduction in the phosphorylation of mTOR (S2481), a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases. These data suggest that m6 A RNA methylation regulates vascular development via PHLPP2/mTOR-AKT signaling.
    Keywords:   mettl3 ; PHLPP2; m6A methylation; mTOR-AKT; vascular development; zebrafish
  9. Nat Biomed Eng. 2021 Mar 29.
      Cancer recurrence can arise owing to rare circulating cancer stem cells (CSCs) that are resistant to chemotherapies and radiotherapies. Here, we show that a double-network hydrogel can rapidly reprogramme differentiated cancer cells into CSCs. Spheroids expressing elevated levels of the stemness genes Sox2, Oct3/4 and Nanog formed within 24 h of seeding the gel with cells from any of six human cancer cell lines or with brain cancer cells resected from patients with glioblastoma. Human brain cancer cells cultured on the double-network hydrogel and intracranially injected in immunodeficient mice led to higher tumorigenicity than brain cancer cells cultured on single-network gels. We also show that the double-network gel induced the phosphorylation of tyrosine kinases, that gel-induced CSCs from primary brain cancer cells were eradicated by an inhibitor of the platelet-derived growth factor receptor, and that calcium channel receptors and the protein osteopontin were essential for the regulation of gel-mediated induction of stemness in brain cancer cells.
  10. PLoS One. 2021 ;16(3): e0245871
      CD44 is a transmembrane glycoprotein, the phosphorylation of which can directly trigger intracellular signaling, particularly Akt protein, for supporting cell growth, motility and invasion. This study examined the role of CD44 on the progression of Cholangiocarcinoma (CCA) using metabolic profiling to investigate the molecular mechanisms involved in the Akt signaling pathway. Our results show that the silencing of CD44 decreases Akt and mTOR phosphorylation resulting in p21 and Bax accumulation and Bcl-2 suppression that reduces cell proliferation. Moreover, an inhibition of cell migration and invasion regulated by CD44. Similarly, the silencing of CD44 showed an alteration in the epithelial-mesenchymal transition (EMT), e.g. an upregulation of E-cadherin and a downregulation of vimentin, and the reduction of the matrix metalloproteinase (MMP)-9 signal. Interestingly, a depletion of CD44 leads to metabolic pathway changes resulting in redox status modification and Trolox (anti-oxidant) led to the recovery of the cancer cell functions. Based on our findings, the regulation of CCA progression and metastasis via the redox status-related Akt signaling pathway depends on the alteration of metabolic profiling synchronized by CD44.
  11. Nat Cell Biol. 2021 Apr 01.
      Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.
  12. Dev Biol. 2021 Mar 24. pii: S0012-1606(21)00079-8. [Epub ahead of print]
      Newly emerging transformed cells are often eliminated from the epithelium via cell competition with the surrounding normal cells. A number of recent studies using mammalian cell competition systems have demonstrated that cells with various types of oncogenic insults are extruded from the tissue in a cell death-dependent or -independent manner. Cell competition-mediated elimination of transformed cells, called EDAC (epithelial defense against cancer), represents an intrinsic anti-tumor activity within the epithelial cell society to reduce the risk of oncogenesis. Here we delineate roles and molecular mechanisms of this homeostatic process, especially focusing on mammalian models.
  13. Sci Rep. 2021 Mar 31. 11(1): 7259
      Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established.
  14. Cell. 2021 Apr 01. pii: S0092-8674(21)00285-3. [Epub ahead of print]184(7): 1661-1670
      When it comes to precision oncology, proteogenomics may provide better prospects to the clinical characterization of tumors, help make a more accurate diagnosis of cancer, and improve treatment for patients with cancer. This perspective describes the significant contributions of The Cancer Genome Atlas and the Clinical Proteomic Tumor Analysis Consortium to precision oncology and makes the case that proteogenomics needs to be fully integrated into clinical trials and patient care in order for precision oncology to deliver the right cancer treatment to the right patient at the right dose and at the right time.
    Keywords:  cancer diagnosis; cancer treatment; clinical trial; collaboration; data sharing; genomics; molecular medicine; open access; precision medicine; precision oncology; proteogenomics; proteomics; targeted therapy
  15. Nat Cell Biol. 2021 Apr 01.
      Endothelial cells (ECs) adapt their metabolism to enable the growth of new blood vessels, but little is known how ECs regulate metabolism to adopt a quiescent state. Here, we show that the metabolite S-2-hydroxyglutarate (S-2HG) plays a crucial role in the regulation of endothelial quiescence. We find that S-2HG is produced in ECs after activation of the transcription factor forkhead box O1 (FOXO1), where it limits cell cycle progression, metabolic activity and vascular expansion. FOXO1 stimulates S-2HG production by inhibiting the mitochondrial enzyme 2-oxoglutarate dehydrogenase. This inhibition relies on branched-chain amino acid catabolites such as 3-methyl-2-oxovalerate, which increase in ECs with activated FOXO1. Treatment of ECs with 3-methyl-2-oxovalerate elicits S-2HG production and suppresses proliferation, causing vascular rarefaction in mice. Our findings identify a metabolic programme that promotes the acquisition of a quiescent endothelial state and highlight the role of metabolites as signalling molecules in the endothelium.
  16. Nat Commun. 2021 Apr 01. 12(1): 2029
      Mucosal-associated invariant T (MAIT) cells have important functions in immune responses against pathogens and in diseases, but mechanisms controlling MAIT cell development and effector lineage differentiation remain unclear. Here, we report that IL-2/IL-15 receptor β chain and inducible costimulatory (ICOS) not only serve as lineage-specific markers for IFN-γ-producing MAIT1 and IL-17A-producing MAIT17 cells, but are also important for their differentiation, respectively. Both IL-2 and IL-15 induce mTOR activation, T-bet upregulation, and subsequent MAIT cell, especially MAIT1 cell, expansion. By contrast, IL-1β induces more MAIT17 than MAIT1 cells, while IL-23 alone promotes MAIT17 cell proliferation and survival, but synergizes with IL-1β to induce strong MAIT17 cell expansion in an mTOR-dependent manner. Moreover, mTOR is dispensable for early MAIT cell development, yet pivotal for MAIT cell effector differentiation. Our results thus show that mTORC2 integrates signals from ICOS and IL-1βR/IL-23R to exert a crucial role for MAIT17 differentiation, while the IL-2/IL-15R-mTORC1-T-bet axis ensures MAIT1 differentiation.
  17. Mol Cell. 2021 Apr 01. pii: S1097-2765(21)00179-9. [Epub ahead of print]81(7): 1365-1367
      Cieśla et al. (2021) uncover intricate circuits of post-transcriptional regulation induced by the Myc oncogene, including alternative splicing and translational control, which are relevant for breast cancer prognosis and contribute to metabolic reprogramming and stem cell-like features of cancer cells.
  18. Curr Opin Cell Biol. 2021 Mar 28. pii: S0955-0674(21)00033-8. [Epub ahead of print]