bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021‒12‒12
twenty papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Int J Mol Sci. 2021 Nov 26. pii: 12813. [Epub ahead of print]22(23):
      Attribution of specific roles to the two ubiquitously expressed PI 3-kinase (PI3K) isoforms p110α and p110β in biological functions they have been implicated, such as in insulin signalling, has been challenging. While p110α has been demonstrated to be the principal isoform activated downstream of the insulin receptor, several studies have provided evidence for a role of p110β. Here we have used isoform-selective inhibitors to estimate the relative contribution of each of these isoforms in insulin signalling in adipocytes, which are a cell type with essential roles in regulation of metabolism at the systemic level. Consistent with previous genetic and pharmacological studies, we found that p110α is the principal isoform activated downstream of the insulin receptor under physiological conditions. p110α interaction with Ras enhanced the strength of p110α activation by insulin. However, this interaction did not account for the selectivity for p110α over p110β in insulin signalling. We also demonstrate that p110α is the principal isoform activated downstream of the β-adrenergic receptor (β-AR), another important signalling pathway in metabolic regulation, through a mechanism involving activation of the cAMP effector molecule EPAC1. This study offers further insights in the role of PI3K isoforms in the regulation of energy metabolism with implications for the therapeutic application of selective inhibitors of these isoforms.
    Keywords:  adrenergic signalling; insulin receptor; insulin resistance; insulin signalling; obesity; phosphoinositide 3-kinase; type 2 diabetes; β-adrenergic receptor
  2. FASEB J. 2022 Jan;36(1): e22080
      Angiogenesis is required in embryonic development and tissue repair in the adult. Vascular endothelial growth factor (VEGF) initiates angiogenesis, and VEGF or its receptor is targeted therapeutically to block pathological angiogenesis. Additional pro-angiogenic cues, such as CXCL12 acting via the CXCR4 receptor, co-operate with VEGF/VEGFR2 to cue vascular patterning. We studied the role of FGD5, an endothelial Rho GTP/GDP exchange factor (RhoGEF), to regulate CXCR4-dependent signals in the endothelial cell (EC). Patient-derived renal cell carcinomas produce a complex milieu of growth factors that stimulated sprouting angiogenesis and endothelial tip cell differentiation ex vivo that was blocked by EC FGD5 loss. In a simplified model, CXCL12 augmented sprouting and tip gene expression under conditions where VEGF was limiting. CXCL12-stimulated tip cell differentiation was dependent on PI3 kinase (PI3K)-β activity. Knockdown of EC FGD5 abolished CXCR4 signaling to PI3K-β and Akt. Further, inhibition of Rac1, a Rho GTPase required for PI3K-β activity, recapitulated the signaling defects of FGD5 deficiency, suggesting that FGD5 may regulate PI3K-β activity through Rac1. Overexpression of a RhoGEF deficient, Dbl domain-deleted FGD5 mutant reduced CXCL12-stimulated Akt phosphorylation and failed to rescue PI3K signaling in native FGD5-deficient EC, indicating that FGD5 RhoGEF activity is required for FDG5 function. Endothelial expression of mutant PI3K-β with an inactivated Rho binding domain confirmed that CXCL12-stimulated PI3K activity in EC requires Rac1-GTP co-regulation. Together, this data identify the role of FGD5 to generate Rac1-GTP to regulate pro-angiogenic CXCR4-dependent PI3K-β signaling in EC. Inhibition of FGD5 activity may complement current angiogenesis inhibitor drugs.
    Keywords:  CXCL12; Rac1 GTP-binding protein; endothelial cell; neovascularization; phosphatidylinositide 3-kinase; signal transduction
  3. Cold Spring Harb Mol Case Stud. 2021 Dec;pii: a006121. [Epub ahead of print]7(6):
      We report a patient with a germline RIT1 and a mosaic PIK3CA variant. The diagnosis of the RASopathy was confirmed by targeted sequencing following the identification of transient cardiomyopathy in a patient with PIK3CA-related overgrowth spectrum (PROS). Our observation confirms that the PIK3CA gain-of-function (GoF) variant effects dominate those of the RASopathy, and the resulting blended phenotype mostly resembles megalencephaly-capillary malformation syndrome (MCAP PROS). There appears to be interaction between RIT1 and PI3K-AKT because the latter pathway is needed for the growth-promoting activity of the first, at least in adenocarcinomas, but the details of this interaction are not known. If so, the PIK3CA somatic variant may not be just a chance event. It could also be of etiological relevance that Rit activation mediates resistance to cellular stress-that is, promotes cell survival. This anti-apoptotic effect could also make it more likely that a cell that spontaneously acquires a PIK3CA GoF variant will survive and proliferate. We aim to encourage clinicians to investigate atypical findings in individuals with PROS. If further similar cases are reported, this would suggest that the establishment of PROS mosaicism is facilitated by the background of a RASopathy.
    Keywords:  facial midline hemangioma; large placenta; macrocephaly at birth; macrocephaly due to hydrocephalus; polyhydramnios; question mark ear
  4. Cold Spring Harb Mol Case Stud. 2021 Dec;pii: a006147. [Epub ahead of print]7(6):
      Disorganized morphogenesis of arteries, veins, capillaries, and lymphatic vessels results in vascular malformations. Most individuals with isolated vascular malformations have postzygotic (mosaic), activating pathogenic variants in a handful of oncogenes within the PI3K-RAS-MAPK pathway (Padia et al., Laryngoscope Investig Otolaryngol 4: 170-173 [2019]). Activating pathogenic variants in the gene PIK3CA, which encodes for the catalytic subunit of phosphatidylinositol 3-kinase, are present in both lymphatic and venous malformations as well as arteriovenous malformations in other complex disorders such as CLOVES syndrome (congenital, lipomatous, overgrowth, vascular malformations, epidermal anevi, scoliosis) (Luks et al., Pediatr Dev Pathol 16: 51 [2013]; Luks et al., J Pediatr 166: 1048-1054.e1-5 [2015]; Al-Olabi et al., J Clin Invest 128: 1496-1508 [2018]). These vascular malformations are part of the PIK3CA-related overgrowth spectrum, a spectrum of entities that have regionalized disordered growth due to the presence of tissue-restricted postzygotic PIK3CA pathogenic variants (Keppler-Noreuil et al., Am J Med Genet A 167A: 287-295 [2015]). Cerebrofacial vascular metameric syndrome (CVMS; also described as cerebrofacial arteriovenous metameric syndrome, Bonnet-Dechaume-Blanc syndrome, and Wyburn-Mason syndrome) is the association of retinal, facial, and cerebral vascular malformations (Bhattacharya et al., Interv Neuroradiol 7: 5-17 [2001]; Krings et al., Neuroimaging Clin N Am 17: 245-258 [2007]). The segmental distribution, the presence of tissue overgrowth, and the absence of familial recurrence are all consistent with CVMS being caused by a postzygotic mutation, which has been hypothesized by previous authors (Brinjiki et al., Am J Neuroradiol 39: 2103-2107 [2018]). However, the genetic cause of CVMS has not yet been described. Here, we present three individuals with CVMS and mosaic activating pathogenic variants within the gene PIK3CA We propose that CVMS be recognized as part of the PIK3CA-related overgrowth spectrum, providing justification for future trials using pharmacologic PIK3CA inhibitors (e.g., alpelisib) for these difficult-to-treat patients.
    Keywords:  frontal venous angioma; hemorrhage of the eye; intracranial hemorrhage; lymphangioma; ocular pain
  5. Nat Commun. 2021 Dec 07. 12(1): 7113
      Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmark the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in vitro in HeLa cells and in vivo in mouse tissues. Finally, we investigate the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through .
  6. Cell. 2021 Dec 09. pii: S0092-8674(21)01332-5. [Epub ahead of print]184(25): 6119-6137.e26
      Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.
    Keywords:  liver metastases; pancreatic cancer; patient-derived organoid models; plasticity; single-cell RNA-sequencing; transcriptional states; tumor heterogeneity; tumor microenvironment
  7. Anal Chem. 2021 Dec 04.
      Peptide bioreporters were developed to perform multiplexed measurements of the activation of epidermal growth factor receptor kinase (EGFR), Akt kinase (Akt/protein kinase B), and proteases/peptidases in single cells. The performance characteristics of the three reporters were assessed by measuring the reporter's proteolytic stability, kinetic constants for EGFR and Akt, and dephosphorylation rate. The reporter displaying optimal performance was composed of 6-carboxyfluorescein (6-FAM) on the peptide N-terminus, an Akt substrate sequence employing a threonine phosphorylation site for Akt, followed by a tri-D arginine linker, and finally an EGFR substrate sequence bearing a phosphatase-resistant 7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (L-htc) residue as the EGFR phosphorylation site. Importantly, use of a single electrophoretic condition separated the mono- and diphosphorylated products as well as proteolytic forms permitting the quantitation of multiple enzyme activities simultaneously using a single reporter. Because the Akt and EGFR substrates were linked, a known ratio (EGFR/Akt) of the reporter was loaded into cells. A photoactivatable version of the reporter was synthesized by adding two 4,5-dimethoxy-2-nitrobenzyl (DMNB) moieties to mask the EGFR and Akt phosphorylation sites. The DMNB moieties were readily photocleaved following exposure to 360 nm light, unmasking the phosphorylation sites on the reporter. The new photoactivatable reporter permitted multiplexed measurements of kinase signaling and proteolytic degradation in single cells in a temporally controlled manner. This work will facilitate the development of a new generation of multiplexed activity-based reporters capable of light-initiated measurement of enzymatic activity in single cells.
  8. PLoS Genet. 2021 Dec;17(12): e1009941
      The retinoblastoma (RB) tumor suppressor is functionally inactivated in a wide range of human tumors where this inactivation promotes tumorigenesis in part by allowing uncontrolled proliferation. RB has been extensively studied, but its mechanisms of action in normal and cancer cells remain only partly understood. Here, we describe a new mouse model to investigate the consequences of RB depletion and its re-activation in vivo. In these mice, induction of shRNA molecules targeting RB for knock-down results in the development of phenotypes similar to Rb knock-out mice, including the development of pituitary and thyroid tumors. Re-expression of RB leads to cell cycle arrest in cancer cells and repression of transcriptional programs driven by E2F activity. Thus, continuous RB loss is required for the maintenance of tumor phenotypes initiated by loss of RB, and this new mouse model will provide a new platform to investigate RB function in vivo.
  9. Nat Biotechnol. 2021 Dec 09.
      The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.
  10. Science. 2021 Dec 10. 374(6573): 1311
      [Figure: see text].
  11. Aging Cancer. 2021 Sep;2(3): 82-97
      BACKGROUND: To shed light on the earliest events in oncogenesis, there is growing interest in understanding the mutational landscapes of normal tissues across ages. In the last decade, next-generation sequencing of human tissues has revealed a surprising abundance of cells with what would be considered oncogenic mutations.AIMS: We performed meta-analysis on previously published sequencing data on normal tissues to categorize mutations based on their presence in cancer and showcase the quantity of cells with cancer-associated mutations in cancer-free individuals.
    METHODS AND RESULTS: We analyzed sequencing data from these studies of normal tissues to determine the prevalence of cells with mutations in three different categories across multiple age groups: 1) mutations in genes designated as drivers, 2) mutations that are in the Cancer Gene Census (CGC), and 3) mutations in the CGC that are considered pathogenic. As we age, the percentage of cells in all three levels increase significantly, reaching over 50% of cells having oncogenic mutations for multiple tissues in the older age groups. The clear enrichment for these mutations, particularly at older ages, likely indicates strong selection for the resulting phenotypes. Combined with an estimation of the number of cells in tissues, we calculate that most older, cancer-free individuals possess at least a 100 billion cells that harbor at least one oncogenic mutation, presumably emanating from a fitness advantage conferred by these mutations that promotes clonal expansion.
    CONCLUSIONS: These studies of normal tissues have highlighted the specific drivers of clonal expansion and how frequently they appear in us. Their high prevalence throughout cancer-free individuals necessitates reconsideration of the oncogenicity of these mutations, which could shape methods of detection, prevention and treatment of cancer, as well as of the potential impact of these mutations on tissue function and our health.
    Keywords:  cancer evolution; life history theory; mutational landscape; somatic evolution
  12. Elife. 2021 Dec 09. pii: e74912. [Epub ahead of print]10
      Environmental cues, not oncogene-induced senescence, may stop melanocytes with an activating mutation in the BRAF gene from turning into melanoma.
    Keywords:  cancer biology; genetics; genomics; human; melanocytes; melanoma; microRNA; mutation; nevi
  13. ChemMedChem. 2021 Dec 10.
      Many intracellular signaling events remain poorly characterized due to a general lack of tools to interfere with "undruggable" targets. Antibodies have the potential to elucidate intracellular mechanisms via targeted disruption of cell signaling cascades because of their ability to bind to a target with high specificity and affinity. However, due to their size and chemical composition, antibodies cannot innately cross the cell membrane, and thus access to the cytosol with these macromolecules has been limited. Here, we describe strategies for accessing the intracellular space with recombinant antibodies mediated by cationic lipid nanoparticles to selectively disrupt intracellular signaling events. Together, our results demonstrate the use of recombinantly produced antibodies, delivered at concentrations of 10 nM, to selectively interfere with signaling driven by a single posttranslational modification. Efficient intracellular delivery of engineered antibodies opens up possibilities for modulation of previously "undruggable" targets, including for potential therapeutic applications.
    Keywords:  Antibodies, intracellular delivery, cell signaling
  14. Stem Cell Res. 2021 Nov 24. pii: S1873-5061(21)00457-8. [Epub ahead of print]57 102610
      Prime editing uses the Cas9 nickase fused to a reverse transcriptase to copy a DNA sequence into a specific locus from a 'prime editing' guide RNA (pegRNA), eliminating the need for double-stranded DNA breaks and donor DNA templates. To facilitate prime editing in human induced pluripotent stem cells (iPSCs), we integrated a doxycycline-inducible Prime Editor protein (PE2) into the AAVS1 genomic safe harbor locus. Prime editing of iPSCs resulted in precise insertion of three nucleotides in HEK3 locus with high efficiency, demonstrating the utility of this approach. This engineered cell line can be used to edit a single or multiple genomic loci by introducing a target-specific pegRNA for precise and effective genome editing to facilitate disease modeling and functional genetics studies.
  15. Brief Bioinform. 2021 Dec 08. pii: bbab510. [Epub ahead of print]
      Large-scale phosphoproteome profiling using mass spectrometry (MS) provides functional insight that is crucial for disease biology and drug discovery. However, extracting biological understanding from these data is an arduous task requiring multiple analysis platforms that are not adapted for automated high-dimensional data analysis. Here, we introduce an integrated pipeline that combines several R packages to extract high-level biological understanding from large-scale phosphoproteomic data by seamless integration with existing databases and knowledge resources. In a single run, PhosPiR provides data clean-up, fast data overview, multiple statistical testing, differential expression analysis, phosphosite annotation and translation across species, multilevel enrichment analyses, proteome-wide kinase activity and substrate mapping and network hub analysis. Data output includes graphical formats such as heatmap, box-, volcano- and circos-plots. This resource is designed to assist proteome-wide data mining of pathophysiological mechanism without a need for programming knowledge.
    Keywords:  bioinformatics; data visualization; phosphoproteomics; pipeline; proteomics; statistics
  16. Elife. 2021 12 07. pii: e73430. [Epub ahead of print]10
      As part of the Reproducibility Project: Cancer Biology, we published Registered Reports that described how we intended to replicate selected experiments from 29 high-impact preclinical cancer biology papers published between 2010 and 2012. Replication experiments were completed and Replication Studies reporting the results were submitted for 18 papers, of which 17 were accepted and published by eLife with the rejected paper posted as a preprint. Here, we report the status and outcomes obtained for the remaining 11 papers. Four papers initiated experimental work but were stopped without any experimental outcomes. Two papers resulted in incomplete outcomes due to unanticipated challenges when conducting the experiments. For the remaining five papers only some of the experiments were completed with the other experiments incomplete due to mundane technical or unanticipated methodological challenges. The experiments from these papers, along with the other experiments attempted as part of the Reproducibility Project: Cancer Biology, provides evidence about the challenges of repeating preclinical cancer biology experiments and the replicability of the completed experiments.
    Keywords:  cancer biology; human; methodology; mouse; null results; open science; replication; reproducibility; transparency
  17. Nature. 2021 Dec 08.
      All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.
  18. Proc Natl Acad Sci U S A. 2021 Dec 07. pii: e2106623118. [Epub ahead of print]118(49):
      Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via β-arrestin-2 (β-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a β-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)-rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates β-arr2-dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal-regulated kinase 1/2 (ERK1/2) activation is also dependent on β-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-β-arr2-mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, β-arr2-driven signaling pathways in caveolae.
    Keywords:  GPCR; biased signaling; cytoprotection; endothelial dysfunction
  19. Nucleic Acids Res. 2021 Dec 06. pii: gkab1157. [Epub ahead of print]
      Phosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the cellular and organismal functions of RPS6 phosphorylation are known, the molecular consequences of RPS6 phosphorylation on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. We test whether RPS6 phosphorylation differentially affects mRNA translation based on CDS length by genetic removal of RPS6 phosphorylation. We find that RPS6 phosphorylation promotes translation of mRNAs with short CDSs more strongly than mRNAs with long CDSs. Interestingly, RPS6 phosphorylation does not promote translation of mRNAs with 5' TOP motifs despite their short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs and the functional consequence on translation.