bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021‒05‒09
fourteen papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Cell Syst. 2021 Apr 27. pii: S2405-4712(21)00111-3. [Epub ahead of print]
      One goal of precision medicine is to tailor effective treatments to patients' specific molecular markers of disease. Here, we used mass cytometry to characterize the single-cell signaling landscapes of 62 breast cancer cell lines and five lines from healthy tissue. We quantified 34 markers in each cell line upon stimulation by the growth factor EGF in the presence or absence of five kinase inhibitors. These data-on more than 80 million single cells from 4,000 conditions-were used to fit mechanistic signaling network models that provide insight into how cancer cells process information. Our dynamic single-cell-based models accurately predicted drug sensitivity and identified genomic features associated with drug sensitivity, including a missense mutation in DDIT3 predictive of PI3K-inhibition sensitivity. We observed similar trends in genotype-drug sensitivity associations in patient-derived xenograft mouse models. This work provides proof of principle that patient-specific single-cell measurements and modeling could inform effective precision medicine strategies.
    Keywords:  EGF-MAP kinase pathway; breast cancer; cell lines; cellular signaling; drug sensitivity prediction; mechanistic modeling; proteomics; single-cell signaling
  2. Front Immunol. 2021 ;12 671756
      Neutrophils, the most abundant circulating leukocytes in humans have key roles in host defense and in the inflammatory response. Agonist-activated phosphoinositide 3-kinases (PI3Ks) are important regulators of many facets of neutrophil biology. PIP3 is subject to dephosphorylation by several 5' phosphatases, including SHIP family phosphatases, which convert the PI3K product and lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) into PI(3,4)P2, a lipid second messenger in its own right. In addition to the leukocyte restricted SHIP1, neutrophils express the ubiquitous SHIP2. This study analyzed mice and isolated neutrophils carrying a catalytically inactive SHIP2, identifying an important regulatory function in neutrophil chemotaxis and directionality in vitro and in neutrophil recruitment to sites of sterile inflammation in vivo, in the absence of major defects of any other neutrophil functions analyzed, including, phagocytosis and the formation of reactive oxygen species. Mechanistically, this is explained by a subtle effect on global 3-phosphorylated phosphoinositide species. This work identifies a non-redundant role for the hitherto overlooked SHIP2 in the regulation of neutrophils, and specifically, neutrophil chemotaxis/trafficking. It completes an emerging wider understanding of the complexity of PI3K signaling in the neutrophil, and the roles played by individual kinases and phosphatases within.
    Keywords:  PI3K; SHIP1; SHIP2; chemotaxis; lipid second messenger; neutrophil; recruitment
  3. EMBO Rep. 2021 May 05. e52122
      Metabolic regulation is critical for the maintenance of pluripotency and the survival of embryonic stem cells (ESCs). The transcription factor Tfcp2l1 has emerged as a key factor for the naïve pluripotency of ESCs. Here, we report an unexpected role of Tfcp2l1 in metabolic regulation in ESCs-promoting the survival of ESCs through regulating fatty acid oxidation (FAO) under metabolic stress. Tfcp2l1 directly activates many metabolic genes in ESCs. Deletion of Tfcp2l1 leads to an FAO defect associated with upregulation of glucose uptake, the TCA cycle, and glutamine catabolism. Mechanistically, Tfcp2l1 activates FAO by inducing Cpt1a, a rate-limiting enzyme transporting free fatty acids into the mitochondria. ESCs with defective FAO are sensitive to cell death induced by glycolysis inhibition and glutamine deprivation. Moreover, the Tfcp2l1-Cpt1a-FAO axis promotes the survival of quiescent ESCs and diapause-like blastocysts induced by mTOR inhibition. Thus, our results reveal how ESCs orchestrate pluripotent and metabolic programs to ensure their survival in response to metabolic stress.
    Keywords:  Tfcp2l1; diapause; embryonic stem cell; fatty acid oxidation; metabolism
  4. Clin Cancer Res. 2021 May 04. pii: clincanres.0047.2021. [Epub ahead of print]
      PURPOSE: Activating mutations in PIK3CA promote resistance to HER2-targeted therapy in breast cancer, however inhibition of PI3K alone leads to escape via feedback upregulation of HER3. Combined inhibition of HER2, HER3 and PI3K overcomes this mechanism preclinically.EXPERIMENTAL DESIGN: This phase I study investigated the maximum tolerated dose (MTD) of alpelisib given in combination with trastuzumab and LJM716 (a HER3-targeted antibody) in patients with PIK3CA-mutant HER2-positive metastatic breast cancer (MBC) using the continual reassessment method. Secondary analyses included efficacy and exploratory correlative studies.
    RESULTS: Ten patients were treated initially with daily alpelisib (Arm A). Grade {greater than or equal to}3 adverse events seen in {greater than or equal to}2 patients included diarrhea (n=6), hypokalemia (n=3), abnormal liver enzymes (n=3), hyperglycemia (n=2), mucositis (n=2), and elevated lipase (n=2). The MTD of alpelisib in Arm A was 250mg daily. This prompted the opening of Arm B in which 11 patients received intermittently dosed alpelisib. Grade {greater than or equal to}3 adverse events seen in {greater than or equal to}2 patients included diarrhea (n=5), hypokalemia (n=3), and hypomagnesemia (n=2). The MTD of alpelisib in Arm B was 350mg given 4 days on, 3 days off. Among 17 patients assessed, 1 had a partial response, 14 had stable disease, and 2 had disease progression at best response. Five patients had stable disease for >30 weeks. mRNA profiling of pre-and on-treatment tissue demonstrated PIK3CA target engagement by alpelisib via induction of downstream signaling and feedback pathways.
    CONCLUSIONS: Combination treatment with alpelisib, trastuzumab and LJM716 was limited by gastrointestinal toxicity. Further efforts are warranted to target the PI3K pathway in HER2+ MBC.
  5. Nat Biotechnol. 2021 May 03.
      Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of <5 × 10-7 mutants per base pair (bp). We demonstrate that it can evaluate mutations in a single amplicon or simultaneously in multiple amplicons, assess limited quantities of cell-free DNA with high recovery of both strands and reduce the error rate of existing PCR-based molecular barcoding approaches by >100-fold.
  6. Dev Biol. 2021 Apr 30. pii: S0012-1606(21)00109-3. [Epub ahead of print]
      During tumorigenesis, tumor cells interact intimately with their surrounding cells (microenvironment) for their growth and progression. However, the roles of tumor microenvironment in tumor development and progression are not fully understood. Here, using an established benign tumor model in adult Drosophila intestines, we find that non-cell autonomous autophagy (NAA) is induced in tumor surrounding neighbor cells. Tumor growth can be significantly suppressed by genetic ablation of autophagy induction in tumor neighboring cells, indicating that tumor neighboring cells act as tumor microenvironment to promote tumor growth. Autophagy in tumor neighboring cells is induced downstream of elevated ROS and activated JNK signaling in tumor cells. Interestingly, we find that active transport of nutrients, such as amino acids, from tumor neighboring cells sustains tumor growth, and increasing nutrient availability could significantly restore tumor growth. Together, these data demonstrate that tumor cells take advantage of their surrounding normal neighbor cells as nutrient sources through NAA to meet their high metabolic demand for growth and progression. Thus we provide insights into our understanding of the mechanisms underlying the interaction between tumor cells and their microenvironment in tumor development.
    Keywords:  Autophagy; Drosophila; Microenvironment; ROS; Raf; Tumorigenesis
  7. Cell Syst. 2021 May 06. pii: S2405-4712(21)00134-4. [Epub ahead of print]
      Predicting the metastasis risk in patients with a primary breast cancer tumor is of fundamental importance to decide the best therapeutic strategy in the framework of personalized medicine. Here, we present ARIADNE, a general algorithmic strategy to assess the risk of metastasis from transcriptomic data of patients with triple-negative breast cancer, a subtype of breast cancer with poorer prognosis with respect to the other subtypes. ARIADNE identifies hybrid epithelial/mesenchymal phenotypes by mapping gene expression data into the states of a Boolean network model of the epithelial-mesenchymal pathway. Using this mapping, it is possible to stratify patients according to their prognosis, as we show by validating the strategy with three independent cohorts of triple-negative breast cancer patients. Our strategy provides a prognostic tool that could be applied to other biologically relevant pathways, in order to estimate the metastatic risk for other breast cancer subtypes or other tumor types. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  Boolean network; Triple-negative breast cancer; epithelial-mesenchymal transition; metastasis; personalized medicine; tumor aggressiveness
  8. Cell Rep. 2021 May 04. pii: S2211-1247(21)00400-9. [Epub ahead of print]35(5): 109069
      mTOR, the sensor of nutrients and growth factors, has important roles in tissue homeostasis and tumorigenesis. However, how mTOR controls gastric epithelial cell turnover and gastric cancer development, a leading malignancy, remains poorly understood. Here, we provide genetic evidence that mTOR activation promotes proliferation and inhibits differentiation of Lgr5+ gastric epithelial progenitors (GEPs) in gastric homeostasis and tumorigenesis. mTOR signaling increases MEK1 and Smad1 expression and enhances activation of MEK1-ERKs and BMP-Smad1 pathways, respectively, in GEPs and gastric tumors. Mek1 deletion or inhibition rescues hyperproliferation, whereas Bmpr1a ablation or inhibition rescues differentiation defects of Tsc1-/- GEPs. Tsc1 deficiency in Lgr5+ GEPs accelerates gastric tumor initiation and development, which require MEK1-ERKs for hyperplasia and BMP-Smad1 for differentiation suppression. These findings reveal how mTOR signaling controls Lgr5+ GEP homeostasis and cancerization and suggest that ERKs and Smad1 signaling can be safely targeted to substitute mTOR inhibitors in gastric cancer therapy.
    Keywords:  Lgr5; MEK1; Smad1; TSC; differentiation; gastric cancer; gastric epithelial progenitor; homeostasis; mTOR; tumorigenesis
  9. Dev Cell. 2021 Apr 29. pii: S1534-5807(21)00321-X. [Epub ahead of print]
      Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. We show that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity.
    Keywords:  Drosophila; PI3-kinase; Src; Toll-like receptor; actomyosin contractility; convergent extension; epithelial morphogenesis; planar polarity
  10. Sci Signal. 2021 May 04. pii: eabc4078. [Epub ahead of print]14(681):
      The dual-specificity phosphatase PTEN functions as a tumor suppressor by hydrolyzing PI(3,4,5)P3 to PI(4,5)P2 to inhibit PI3K-AKT signaling and cellular proliferation. P-Rex2 is a guanine nucleotide exchange factor for Rho GTPases and can be activated by Gβγ subunits downstream of G protein-coupled receptor signaling and by PI(3,4,5)P3 downstream of receptor tyrosine kinases. The PTEN:P-Rex2 complex is a commonly mutated signaling node in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cellular proliferation. Here, using cross-linking mass spectrometry and functional studies, we gained mechanistic insights into PTEN:P-Rex2 complex assembly and coinhibition. We found that PTEN was anchored to P-Rex2 by interactions between the PDZ-interacting motif in the PTEN C-terminal tail and the second PDZ domain of P-Rex2. This interaction bridged PTEN across the P-Rex2 surface, preventing PI(3,4,5)P3 hydrolysis. Conversely, PTEN both allosterically promoted an autoinhibited conformation of P-Rex2 and blocked its binding to Gβγ. In addition, we observed that the PTEN-deactivating mutations and P-Rex2 truncations combined to drive Rac1 activation to a greater extent than did either single variant alone. These insights enabled us to propose a class of gain-of-function, cancer-associated mutations within the PTEN:P-Rex2 interface that uncouple PTEN from the inhibition of Rac1 signaling.
  11. Proc Natl Acad Sci U S A. 2021 May 11. pii: e2017645118. [Epub ahead of print]118(19):
      Invadopodia are integrin-mediated adhesions with abundant PI(3,4)P2 However, the functional role of PI(3,4)P2 in adhesion signaling remains unclear. Here, we find that the PI(3,4)P2 biogenesis regulates the integrin endocytosis at invadopodia. PI(3,4)P2 is locally produced by PIK3CA and SHIP2 and is concentrated at the trailing edge of the invadopodium arc. The PI(3,4)P2-rich compartment locally forms small puncta (membrane buds) in a SNX9-dependent manner, recruits dynein activator Hook1 through AKTIP, and rearranges into micrometer-long tubular invaginations (membrane tubes). The uncurving membrane tube extends rapidly, follows the retrograde movement of dynein along microtubule tracks, and disconnects from the plasma membrane. Activated integrin-beta3 is locally internalized through the pathway of PI(3,4)P2-mediated membrane invagination and is then actively recycled. Blockages of PI3K, SHIP2, and SNX9 suppress integrin-beta3 endocytosis, delay adhesion turnover, and impede transwell invasion of MEF-Src and MDA-MB-231 cells. Thus, the production of PI(3,4)P2 promotes invasive cell migration by stimulating the trafficking of integrin receptor at the invadopodium.
    Keywords:  integrin trafficking; invadopodia; microtubule; phosphoinositide lipids
  12. Exp Cell Res. 2021 May 03. pii: S0014-4827(21)00133-6. [Epub ahead of print] 112601
      Adhesion of cells to each other and to the extracellular matrix (ECM) are both required for cellular functions. Cell-to-cell adhesion is mediated by cadherins and their engagement triggers the activation of Stat3, which offers a potent survival signal. Adhesion to the ECM on the other hand, activates FAK which attracts and activates Src, as well as receptor tyrosine kinases (RTKs), the PI3k/Akt and Ras/Erk pathways. However, the effect of cell density upon FAK and Akt activity has not been examined. We now demonstrate that, interestingly, despite being potent Stat3 activators, Src and RTKs are unable to activate Stat3 in sparsely growing (i.e., without cadherin engagement), non-neoplastic cells attached to the ECM. In contrast, cell aggregation (i.e., cadherin engagement in the absence of adhesion to a solid substratum) was found to activate both Stat3 and Akt. Pharmacologic or genetic reduction of FAK activity abolished Akt activity at low densities, indicating that FAK is an important activator of Akt in this setting. Notably, FAK knockout increased cellular sensitivity to the Stat3 inhibitor CPA7, while FAK reintroduction restored resistance to this drug. These findings suggest a complementary role of integrin/FAK/Akt and cadherin/Stat3-mediated pro-survival pathways, which may be of significance during neoplastic transformation and metastasis.
  13. Commun Biol. 2021 May 05. 4(1): 527
      The ovarian surface epithelium (OSE) is a monolayer of epithelial cells surrounding the ovary that ruptures during each ovulation to allow release of the oocyte. This wound is quickly repaired, but mechanisms promoting repair are poorly understood. The contribution of tissue-resident stem cells in the homeostasis of several epithelial tissues is widely accepted, but their involvement in OSE is unclear. We show that traits associated with stem cells can be increased following exposure to the cytokine TGFB1, overexpression of the transcription factor Snai1, or deletion of Brca1. We find that stemness is often linked to mesenchymal-associated gene expression and higher activation of ERK signalling, but is not consistently dependent on their activation. Expression profiles of these populations are extremely context specific, suggesting that stemness may not be associated with a single, distinct population, but rather is a heterogeneous cell state that may emerge from diverse environmental cues. These findings support that the OSE may not require distinct stem cells for long-term maintenance, and may instead achieve this through transient dedifferentiation into a stem-like state.
  14. Proc Natl Acad Sci U S A. 2021 May 11. pii: e2102050118. [Epub ahead of print]118(19):
      The epithelial-to-mesenchymal transition (EMT) plays a critical role during normal development and in cancer progression. EMT is induced by various signaling pathways, including TGF-β, BMP, Wnt-β-catenin, NOTCH, Shh, and receptor tyrosine kinases. In this study, we performed single-cell RNA sequencing on MCF10A cells undergoing EMT by TGF-β1 stimulation. Our comprehensive analysis revealed that cells progress through EMT at different paces. Using pseudotime clustering reconstruction of gene-expression profiles during EMT, we found sequential and parallel activation of EMT signaling pathways. We also observed various transitional cellular states during EMT. We identified regulatory signaling nodes that drive EMT with the expression of important microRNAs and transcription factors. Using a random circuit perturbation methodology, we demonstrate that the NOTCH signaling pathway acts as a key driver of TGF-β-induced EMT. Furthermore, we demonstrate that the gene signatures of pseudotime clusters corresponding to the intermediate hybrid EMT state are associated with poor patient outcome. Overall, this study provides insight into context-specific drivers of cancer progression and highlights the complexities of the EMT process.
    Keywords:  EMT; NOTCH; RACIPE; scRNA-seq; signaling cascade