bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2022‒09‒25
thirteen papers selected by
Lucas B. Zeiger
Beatson Institute for Cancer Research


  1. Sci Rep. 2022 Sep 22. 12(1): 15810
      Oncogenic RAS proteins are important for driving tumour formation, and for maintenance of the transformed phenotype, and thus their relevance as a cancer therapeutic target is undeniable. We focused here on obtaining peptidomimetics, which have good pharmacological properties, to block Ras-effector interaction. Computational analysis was used to identify hot spots of RAS relevant for these interactions and to screen a library of peptidomimetics. Nine compounds were synthesized and assayed for their activity as RAS inhibitors in cultured cells. Most of them induced a reduction in ERK and AKT activation by EGF, a marker of RAS activity. The most potent inhibitor disrupted Raf and PI3K interaction with oncogenic KRAS, corroborating its mechanism of action as an inhibitor of protein-protein interactions, and thus validating our computational methodology. Most interestingly, improvement of one of the compounds allowed us to obtain a peptidomimetic that decreased the survival of pancreatic cancer cell lines harbouring oncogenic KRAS.
    DOI:  https://doi.org/10.1038/s41598-022-19703-6
  2. Nat Commun. 2022 Sep 17. 13(1): 5469
      Oncogenic RAS mutations are common in multiple myeloma (MM), an incurable malignancy of plasma cells. However, the mechanisms of pathogenic RAS signaling in this disease remain enigmatic and difficult to inhibit therapeutically. We employ an unbiased proteogenomic approach to dissect RAS signaling in MM. We discover that mutant isoforms of RAS organize a signaling complex with the amino acid transporter, SLC3A2, and MTOR on endolysosomes, which directly activates mTORC1 by co-opting amino acid sensing pathways. MM tumors with high expression of mTORC1-dependent genes are more aggressive and enriched in RAS mutations, and we detect interactions between RAS and MTOR in MM patient tumors harboring mutant RAS isoforms. Inhibition of RAS-dependent mTORC1 activity synergizes with MEK and ERK inhibitors to quench pathogenic RAS signaling in MM cells. This study redefines the RAS pathway in MM and provides a mechanistic and rational basis to target this mode of RAS signaling.
    DOI:  https://doi.org/10.1038/s41467-022-33142-x
  3. Biochem J. 2022 Sep 30. 479(18): 1917-1940
      As first demonstrated in budding yeast (Saccharomyces cerevisiae), all eukaryotic cells contain two, distinct multi-component protein kinase complexes that each harbor the TOR (Target Of Rapamycin) polypeptide as the catalytic subunit. These ensembles, dubbed TORC1 and TORC2, function as universal, centrally important sensors, integrators, and controllers of eukaryotic cell growth and homeostasis. TORC1, activated on the cytosolic surface of the lysosome (or, in yeast, on the cytosolic surface of the vacuole), has emerged as a primary nutrient sensor that promotes cellular biosynthesis and suppresses autophagy. TORC2, located primarily at the plasma membrane, plays a major role in maintaining the proper levels and bilayer distribution of all plasma membrane components (sphingolipids, glycerophospholipids, sterols, and integral membrane proteins). This article surveys what we have learned about signaling via the TORC2 complex, largely through studies conducted in S. cerevisiae. In this yeast, conditions that challenge plasma membrane integrity can, depending on the nature of the stress, stimulate or inhibit TORC2, resulting in, respectively, up-regulation or down-regulation of the phosphorylation and thus the activity of its essential downstream effector the AGC family protein kinase Ypk1. Through the ensuing effect on the efficiency with which Ypk1 phosphorylates multiple substrates that control diverse processes, membrane homeostasis is maintained. Thus, the major focus here is on TORC2, Ypk1, and the multifarious targets of Ypk1 and how the functions of these substrates are regulated by their Ypk1-mediated phosphorylation, with emphasis on recent advances in our understanding of these processes.
    Keywords:  contact sites; lipids; membrane proteins; phosphorylation; plasma membrane; protein kinases
    DOI:  https://doi.org/10.1042/BCJ20220388
  4. Nat Commun. 2022 Sep 19. 13(1): 5478
      Most colorectal (CRC) tumors are dependent on EGFR/KRAS/BRAF/MAPK signaling activation. ARID1A is an epigenetic regulator mutated in approximately 5% of non-hypermutated CRC tumors. Here we show that anti-EGFR but not anti-VEGF treatment enriches for emerging ARID1A mutations in CRC patients. In addition, we find that patients with ARID1A mutations, at baseline, are associated with worse outcome when treated with cetuximab- but not bevacizumab-containing therapies; thus, this suggests that ARID1A mutations may provide both an acquired and intrinsic mechanism of resistance to anti-EGFR therapies. We find that, ARID1A and EGFR-pathway genetic alterations are mutually exclusive across lung and colorectal cancers, further supporting a functional connection between these pathways. Our results not only suggest that ARID1A could be potentially used as a predictive biomarker for cetuximab treatment decisions but also provide a rationale for exploring therapeutic MAPK inhibition in an unexpected but genetically defined segment of CRC patients.
    DOI:  https://doi.org/10.1038/s41467-022-33172-5
  5. J Cell Sci. 2022 Sep 20. pii: jcs.260082. [Epub ahead of print]
      The forkhead box transcription factor FOXQ1 contributes to the pathogenesis of carcinomas. In colorectal cancers, FOXQ1 promotes tumour metastasis by inducing epithelial-to-mesenchymal transition (EMT) of cancer cells. FOXQ1 may exacerbate cancer by activating the oncogenic Wnt/-catenin signalling pathway. However, the role of FOXQ1 in the Wnt pathway remains to be resolved. Here, we report that FOXQ1 is an activator of Wnt-induced transcription and regulator of b-catenin target gene expression. Upon Wnt pathway activation, FOXQ1 synergises with the b-catenin nuclear complex to boost the expression of major Wnt targets. In parallel, we find that FOXQ1 controls the differential expression of various Wnt target genes in a b-catenin-independent manner. Using RNA sequencing of colorectal cancer cell lines, we show that Wnt signalling and FOXQ1 converge on a transcriptional program linked to EMT and cell migration. Additionally, we demonstrate that FOXQ1 occupies Wnt-responsive elements in b-catenin target gene promoters and recruits a similar set of co-factors as the b-catenin-associated transcription factor Tcf7l1. Taken together, our results indicate a multifaceted role of FOXQ1 in Wnt/b-catenin signalling, which may drive the metastasis of colorectal cancers.
    Keywords:  Colorectal cancer; Forkhead box; Gene expression; Proteomics; Wnt signalling
    DOI:  https://doi.org/10.1242/jcs.260082
  6. Exp Mol Med. 2022 Sep 18.
      The small intestine is among the fastest self-renewing tissues in adult mammals. This rapid turnover is fueled by the intestinal stem cells residing in the intestinal crypt. Wnt signaling plays a pivotal role in regulating intestinal stem cell renewal and differentiation, and the dysregulation of this pathway leads to cancer formation. Several studies demonstrate that intestinal stem cells follow neutral drift dynamics, as they divide symmetrically to generate other equipotent stem cells. Competition for niche space and extrinsic signals in the intestinal crypt is the governing mechanism that regulates stemness versus cell differentiation, but the underlying molecular mechanisms are still poorly understood, and it is not yet clear how this process changes during disease. In this review, we highlight the mechanisms that regulate stem cell homeostasis in the small intestine, focusing on Wnt signaling and its regulation by RNF43 and ZNRF3, key inhibitors of the Wnt pathway. Furthermore, we summarize the evidence supporting the current model of intestinal stem cell regulation, highlighting the principles of neutral drift at the basis of intestinal stem cell homeostasis. Finally, we discuss recent studies showing how cancer cells bypass this mechanism to gain a competitive advantage against neighboring normal cells.
    DOI:  https://doi.org/10.1038/s12276-022-00854-5
  7. J Am Chem Soc. 2022 Sep 19.
      The small GTPase Ras is a critical regulator of cell growth and proliferation. Its activity is frequently dysregulated in cancers, prompting decades of work to pharmacologically target Ras. Understanding Ras biology and developing effective Ras therapeutics both require probing Ras activity in its native context, yet tools to measure its activities in cellulo are limited. Here, we developed a ratiometric Ras activity reporter (RasAR) that provides quantitative measurement of Ras activity in living cells with high spatiotemporal resolution. We demonstrated that RasAR can probe live-cell activities of all the primary isoforms of Ras. Given that the functional roles of different isoforms of Ras are intimately linked to their subcellular distribution and regulation, we interrogated the spatiotemporal regulation of Ras utilizing subcellularly targeted RasAR and uncovered the role of Src kinase as an upstream regulator to inhibit HRas. Furthermore, we showed that RasAR enables capture of KRasG12C inhibition dynamics in living cells upon treatment with KRasG12C covalent inhibitors, including ARS1620, Sotorasib, and Adagrasib. We found in living cells a residual Ras activity lingers for hours in the presence of these inhibitors. Together, RasAR represents a powerful molecular tool to enable live-cell interrogation of Ras activity and facilitate the development of Ras inhibitors.
    DOI:  https://doi.org/10.1021/jacs.2c05203
  8. Br J Cancer. 2022 Sep 17.
      BACKGROUND: Cellular metabolism is an integral component of cellular adaptation to stress, playing a pivotal role in the resistance of cancer cells to various treatment modalities, including radiotherapy. In response to radiotherapy, cancer cells engage antioxidant and DNA repair mechanisms which mitigate and remove DNA damage, facilitating cancer cell survival. Given the reliance of these resistance mechanisms on amino acid metabolism, we hypothesised that controlling the exogenous availability of the non-essential amino acids serine and glycine would radiosensitise cancer cells.METHODS: We exposed colorectal, breast and pancreatic cancer cell lines/organoids to radiation in vitro and in vivo in the presence and absence of exogenous serine and glycine. We performed phenotypic assays for DNA damage, cell cycle, ROS levels and cell death, combined with a high-resolution untargeted LCMS metabolomics and RNA-Seq.
    RESULTS: Serine and glycine restriction sensitised a range of cancer cell lines, patient-derived organoids and syngeneic mouse tumour models to radiotherapy. Comprehensive metabolomic and transcriptomic analysis of central carbon metabolism revealed that amino acid restriction impacted not only antioxidant response and nucleotide synthesis but had a marked inhibitory effect on the TCA cycle.
    CONCLUSION: Dietary restriction of serine and glycine is a viable radio-sensitisation strategy in cancer.
    DOI:  https://doi.org/10.1038/s41416-022-01965-6
  9. Sci Rep. 2022 Sep 20. 12(1): 15715
      The serine/threonine protein kinase AKT plays a pivotal role within the PI3K pathway in regulating cellular proliferation and apoptotic cellular functions, and AKT hyper-activation via gene amplification and/or mutation has been implicated in multiple human malignancies. There are 3 AKT isoenzymes (AKT1-3) which mediate critical, non-redundant functions. We present the discovery and development of ALM301, a novel, allosteric, sub-type selective inhibitor of AKT1/2. ALM301 binds in an allosteric pocket created by the combined movement of the PH domain and the catalytic domain, resulting in a DFG out conformation. ALM301 was shown to be highly selective against a panel of over 450 kinases and potently inhibited cellular proliferation. These effects were particularly pronounced in MCF-7 cells containing a PI3KCA mutation. Subsequent cellular downstream pathway analysis in this sensitive cell line revealed potent inhibition of pAKT signalling up to 48 h post dosing. ALM301 treatment was well tolerated in an MCF-7 xenograft model and led to a dose-dependent reduction in tumour growth. Enhanced efficacy was observed in combination with tamoxifen. In summary, ALM301 is a highly specific AKT 1/2 inhibitor with an excellent pharmacological profile suitable for further clinical development.
    DOI:  https://doi.org/10.1038/s41598-022-20208-5
  10. Int J Cancer. 2022 Sep 19.
      Patient derived organoids closely resemble the biology of tissues and tumors. They are enabling ex vivo modeling of human diseases and dissecting key features of tumor biology like anatomical diversity or inter- and intra-tumoral heterogeneity. In the last years, organoids were established as models for drug discovery and explored to guide clinical decision making. In this review, we discuss the recent developments in organoid based research, elaborating on the developments in colorectal cancer as a prime example. We focus our review on the role of organoids to decode cancer cell dynamics and tumor microenvironmental complexity with the underlying bi-directional crosstalk. Additionally, advancements in the development of living biobanks, screening approaches, organoid based precision medicine and challenges of co-clinical trials are highlighted. We discuss ongoing efforts to overcome challenges that the field faces and indicate potential future directions. This article is protected by copyright. All rights reserved.
    Keywords:  Organoid; drug screening; heterogeneity; microenvironment; precision medicine
    DOI:  https://doi.org/10.1002/ijc.34297
  11. Sci Rep. 2022 Sep 17. 12(1): 15628
      Previously, our group has demonstrated establishment of Cancer Stem Cell (CSC) models from stem cells in the presence of conditioned medium of cancer cell lines. In this study, we tried to identify the factors responsible for the induction of CSCs. Since we found the lipid composition could be traced to arachidonic acid cascade in the CSC model, we assessed prostaglandin E2 (PGE2) as a candidate for the ability to induce CSCs from induced pluripotent stem cells (iPSCs). Mouse iPSCs acquired the characteristics of CSCs in the presence of 10 ng/mL of PGE2 after 4 weeks. Since constitutive Akt activation and pik3cg overexpression were found in the resultant CSCs, of which growth was found independent of PGE2, chronic stimulation of the receptors EP-2/4 by PGE2 was supposed to induce CSCs from iPSCs through epigenetic effect. The bioinformatics analysis of the next generation sequence data of the obtained CSCs proposed not only receptor tyrosine kinase activation by growth factors but also extracellular matrix and focal adhesion enhanced PI3K pathway. Collectively, chronic stimulation of stem cells with PGE2 was implied responsible for cancer initiation enhancing PI3K/Akt axis.
    DOI:  https://doi.org/10.1038/s41598-022-19265-7
  12. Sci Rep. 2022 Sep 23. 12(1): 15870
      Leucine (Leu) regulates protein synthesis and degradation via activation of mammalian target of rapamycin complex 1 (mTORC1). Glutamine (Gln) synergistically promotes mTORC1 activation with Leu via glutaminolysis and Leu absorption via an antiporter. However, Gln has also been shown to inhibit mTORC1 activity. To resolve this paradox, we aimed to elucidate the effects of Gln on Leu-mediated mTORC1 activation. We administered Leu, Gln, tryptophan, Leu + Gln, or Leu + tryptophan to mice after 24-h fasting. The mice were then administered puromycin to evaluate protein synthesis and the gastrocnemius muscle was harvested 30 min later. Phosphorylated eukaryotic initiation factor 4E-binding protein 1, 70-kDa ribosomal protein S6 kinase 1, and Unc-51 like kinase 1 levels were the highest in the Leu + Gln group and significantly increased compared with those in the control group; however, Gln alone did not increase the levels of phosphorylated proteins. No difference in glutamate dehydrogenase activity was observed between the groups. Leu concentrations in the gastrocnemius muscle were similar in the Leu-intake groups. Our study highlights a novel mechanism underlying the promotive effect of Gln on Leu-mediated mTORC1 activation, providing insights into the pathway through which amino acids regulate muscle protein metabolism.
    DOI:  https://doi.org/10.1038/s41598-022-20251-2
  13. Nature. 2022 Sep 21.
      Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.
    DOI:  https://doi.org/10.1038/s41586-022-05235-6