bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2023–07–09
twelve papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research



  1. J Cell Biol. 2023 Sep 04. pii: e202208150. [Epub ahead of print]222(9):
      PTEN is a crucial negative regulator of the INS/PI3K/AKT pathway and is one of the most commonly mutated tumor suppressors in cancer. Global overexpression (OE) of PTEN in mice shifts metabolism to favor oxidative phosphorylation over glycolysis, reduces fat mass, and extends the lifespan of both sexes. We demonstrate that PTEN regulates chaperone-mediated autophagy (CMA). Using cultured cells and mouse models, we show that PTEN OE enhances CMA, dependent upon PTEN's lipid phosphatase activity and AKT inactivation. Reciprocally, PTEN knockdown reduces CMA, which can be rescued by inhibiting class I PI3K or AKT. Both PTEN and CMA are negative regulators of glycolysis and lipid droplet formation. We show that suppression of glycolysis and lipid droplet formation downstream of PTEN OE depends on CMA activity. Finally, we show that PTEN protein levels are sensitive to CMA and that PTEN accumulates in lysosomes with elevated CMA. Collectively, these data suggest that CMA is both an effector and a regulator of PTEN.
    DOI:  https://doi.org/10.1083/jcb.202208150
  2. Proc Natl Acad Sci U S A. 2023 07 11. 120(28): e2302485120
      The G12D mutation is among the most common KRAS mutations associated with cancer, in particular, pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over the G12D mutation in HRAS and NRAS. Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and α3 helix, and captured this pocket in the most widely open form reported to date. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of KRAS Asp12, a feature that closely resembles that of a small-molecule inhibitor, MTRX1133. The monobody also directly interacted with H95, a residue not conserved in RAS isoforms. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a monobody generated hundreds of functional and nonfunctional single-point mutants, which identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results further illustrate the plasticity of the S-II pocket, which may be exploited for the design of next-generation KRAS(G12D)-selective inhibitors.
    Keywords:  conformational plasticity; drug discovery; intracellular biologics; protein engineering; protein-protein interaction
    DOI:  https://doi.org/10.1073/pnas.2302485120
  3. Bioessays. 2023 Jul 04. e2300088
      RAS GTPases play essential roles in normal development and are direct drivers of human cancers. Three decades of study have failed to wholly characterize pathways stimulated by activated RAS, driven by engagement with 'effector' proteins that have RAS binding domains (RBDs). Bone fide effectors must bind directly to RAS GTPases in a nucleotide-dependent manner, and this interaction must impart a clear change in effector activity. Despite this, for most proteins currently deemed effectors there is little mechanistic understanding of how binding to the GTPase alters protein function. There has also been limited effort to comprehensively resolve the specificity of effector binding to the full array of RAS superfamily GTPase proteins. This review will summarize what is known about RAS-driven activation for an array of potential effector proteins, focusing on structural and mechanistic effects and highlighting how little is still known regarding this key paradigm of cellular signal transduction.
    Keywords:  AFDN; PI3K; RAF; RAS GTPases; RBD domain; SCRIB; adherens junctions
    DOI:  https://doi.org/10.1002/bies.202300088
  4. Oncotarget. 2023 07 01. 14 672-687
      Ras proteins are small GTPases that regulate cell growth and division. Mutations in Ras genes are associated with many types of cancer, making them attractive targets for cancer therapy. Despite extensive efforts, targeting Ras proteins with small molecules has been extremely challenging due to Ras's mostly flat surface and lack of small molecule-binding cavities. These challenges were recently overcome by the development of the first covalent small-molecule anti-Ras drug, sotorasib, highlighting the efficacy of Ras inhibition as a therapeutic strategy. However, this drug exclusively inhibits the Ras G12C mutant, which is not a prevalent mutation in most cancer types. Unlike the G12C variant, other Ras oncogenic mutants lack reactive cysteines, rendering them unsuitable for targeting via the same strategy. Protein engineering has emerged as a promising method to target Ras, as engineered proteins have the ability to recognize various surfaces with high affinity and specificity. Over the past few years, scientists have engineered antibodies, natural Ras effectors, and novel binding domains to bind to Ras and counteract its carcinogenic activities via a variety of strategies. These include inhibiting Ras-effector interactions, disrupting Ras dimerization, interrupting Ras nucleotide exchange, stimulating Ras interaction with tumor suppressor genes, and promoting Ras degradation. In parallel, significant advancements have been made in intracellular protein delivery, enabling the delivery of the engineered anti-Ras agents into the cellular cytoplasm. These advances offer a promising path for targeting Ras proteins and other challenging drug targets, opening up new opportunities for drug discovery and development.
    Keywords:  Ras oncogene; Ras targeting; anti-Ras therapeutics; protein design; protein engineering
    DOI:  https://doi.org/10.18632/oncotarget.28469
  5. Mol Cell. 2023 Jun 26. pii: S1097-2765(23)00429-X. [Epub ahead of print]
      K-Ras frequently acquires gain-of-function mutations (K-RasG12D being the most common) that trigger significant transcriptomic and proteomic changes to drive tumorigenesis. Nevertheless, oncogenic K-Ras-induced dysregulation of post-transcriptional regulators such as microRNAs (miRNAs) during oncogenesis is poorly understood. Here, we report that K-RasG12D promotes global suppression of miRNA activity, resulting in the upregulation of hundreds of targets. We constructed a comprehensive profile of physiological miRNA targets in mouse colonic epithelium and tumors expressing K-RasG12D using Halo-enhanced Argonaute pull-down. Combining this with parallel datasets of chromatin accessibility, transcriptome, and proteome, we uncovered that K-RasG12D suppressed the expression of Csnk1a1 and Csnk2a1, subsequently decreasing Ago2 phosphorylation at Ser825/829/832/835. Hypo-phosphorylated Ago2 increased binding to mRNAs while reducing its activity to repress miRNA targets. Our findings connect a potent regulatory mechanism of global miRNA activity to K-Ras in a pathophysiological context and provide a mechanistic link between oncogenic K-Ras and the post-transcriptional upregulation of miRNA targets.
    Keywords:  Ago2; CLIP-seq; K-Ras; casein kinase; colon; colorectal cancer; miRNA
    DOI:  https://doi.org/10.1016/j.molcel.2023.06.008
  6. bioRxiv. 2023 Jun 13. pii: 2023.06.12.544386. [Epub ahead of print]
      The Ras/PI3K/ERK signaling network is frequently mutated in various human cancers including cervical cancer and pancreatic cancer. Previous studies showed that the Ras/PI3K/ERK signaling network displays features of excitable systems including propagation of activity waves, all-or-none responses, and refractoriness. Oncogenic mutations lead to enhanced excitability of the network. A positive feedback loop between Ras, PI3K, the cytoskeleton, and FAK was identified as a driver of excitability. In this study, we investigated the effectiveness of targeting signaling excitability by inhibiting both FAK and PI3K in cervical and pancreatic cancer cells. We found that the combination of FAK and PI3K inhibitors synergistically suppressed the growth of select cervical and pancreatic cancer cell lines through increased apoptosis and decreased mitosis. In particular, FAK inhibition caused downregulation of PI3K and ERK signaling in cervical cancer but not pancreatic cancer cells. Interestingly, PI3K inhibitors activated multiple receptor tyrosine kinases (RTKs), including insulin receptor and IGF-1R in cervical cancer cells, as well as EGFR, Her2, Her3, Axl, and EphA2 in pancreatic cancer cells. Our results highlight the potential of combining FAK and PI3K inhibition for treating cervical and pancreatic cancer, although appropriate biomarkers for drug sensitivity are needed, and concurrent targeting of RTKs may be required for resistant cells.
    DOI:  https://doi.org/10.1101/2023.06.12.544386
  7. Biochim Biophys Acta Rev Cancer. 2023 Jun 30. pii: S0304-419X(23)00096-3. [Epub ahead of print]1878(5): 188947
      Recent cryo-electron microscopic (cryo-EM) investigations have succeeded in the analysis of various structural conformations and functional states of PI3Kα, a dimer consisting of the catalytic subunit p110α and the regulatory subunit p85α of class IA of phosphoinositide 3-kinase. High resolution structures have been obtained of the unliganded and of BYL-719-bound PI3Kα. The latter provides information on excessively flexible domains of p85α that are then further analyzed with nanobodies and CXMS (chemical cross-linking, digestion and mass spectrometry). Analysis of p110α helical and kinase domain mutations reveals mutant-specific features that can be linked to the gain of function in enzymatic and signaling activities.
    Keywords:  BYL-719; CXMS; Mutants; Nanobody; Phosphoinositide 3-kinase
    DOI:  https://doi.org/10.1016/j.bbcan.2023.188947
  8. Dig Dis Sci. 2023 Jul 05.
      In this installment of the "Paradigm Shifts in Perspective" series, the authors, all scientists who have been involved in colorectal cancer (CRC) research for most or all of their careers, have watched the field develop from early pathological descriptions of tumor formation to the current understanding of tumor pathogenesis that informs personalized therapies. We outline how our understanding of the pathogenetic basis of CRC began with seemingly isolated discoveries-initially with the mutations in RAS and the APC gene, the latter of which was initially found in the context of intestinal polyposis, to the more complex process of multistep carcinogenesis, to the chase for tumor suppressor genes, which led to the unexpected discovery of microsatellite instability (MSI). These discoveries enabled the authors to better understand how the DNA mismatch repair (MMR) system not only recognizes DNA damage but also responds to damage by DNA repair or by triggering apoptosis in the injured cell. This work served, in part, to link the earlier findings on the pathogenesis of CRC to the development of immune checkpoint inhibitors, which has been transformative-and curative-for certain types of CRCs and other cancers as well. These discoveries also highlight the circuitous routes that scientific progress takes, which can include thoughtful hypothesis testing and at other times recognizing the importance of seemingly serendipitous observations that substantially change the flow and direction of the discovery process. What has happened over the past 37 years was not predictable when this journey began, but it does speak to the power of careful scientific experimentation, following the facts, perseverance in the face of opposition, and the willingness to think outside of established paradigms.
    Keywords:  Colorectal cancer; DNA mismatch repair; Lynch syndrome; Microsatellite instability; Multistep carcinogenesis
    DOI:  https://doi.org/10.1007/s10620-023-08006-z
  9. Oncol Res. 2023 ;31(4): 495-503
      Pancreatic cancer is one of the most aggressive cancers with a median survival time of less than 5 months, and conventional chemotherapeutics are the main treatment strategy. Poly(ADP-ribose) polymerase (PARP) inhibitors have been recently approved for BRCA1/2-mutant pancreatic cancer, opening a new era for targeted therapy for this disease. However, most pancreatic cancer patients carry wild-type BRCA1/2 with resistance to PARP inhibitors. Here, we reported that mammalian target of rapamycin complex 2 (mTORC2) kinase is overexpressed in pancreatic cancer tissues and promotes pancreatic cancer cell growth and invasion. Moreover, we found that knockdown of the mTORC2 obligate subunit Rictor sensitized pancreatic cancer cells to the PARP inhibitor olaparib. Mechanistically, we showed that mTORC2 positively regulates homologous recombination (HR) repair by modulating BRCA1 recruitment to DNA double-strand breaks (DSBs). In addition, we confirmed that combination treatment with the mTORC2 inhibitor PP242 and the PARP inhibitor olaparib synergistically inhibited pancreatic cancer growth in vivo. Thus, this study provides a novel target and strategy for optimizing PARP inhibitor efficiency in pancreatic cancers.
    Keywords:  DNA damage; HR repair; PARP inhibitors; Pancreatic cancer; mTORC2
    DOI:  https://doi.org/10.32604/or.2023.029309
  10. Biomed Pharmacother. 2023 Jul 01. pii: S0753-3322(23)00814-4. [Epub ahead of print]165 115024
      The pleckstrin homology [PH] domain is a structural fold found in more than 250 proteins making it the 11th most common domain in the human proteome. 25% of family members have more than one PH domain and some PH domains are split by one, or several other, protein domains although still folding to give functioning PH domains. We review mechanisms of PH domain activity, the role PH domain mutation plays in human disease including cancer, hyperproliferation, neurodegeneration, inflammation, and infection, and discuss pharmacotherapeutic approaches to regulate PH domain activity for the treatment of human disease. Almost half PH domain family members bind phosphatidylinositols [PIs] that attach the host protein to cell membranes where they interact with other membrane proteins to give signaling complexes or cytoskeleton scaffold platforms. A PH domain in its native state may fold over other protein domains thereby preventing substrate access to a catalytic site or binding with other proteins. The resulting autoinhibition can be released by PI binding to the PH domain, or by protein phosphorylation thus providing fine tuning of the cellular control of PH domain protein activity. For many years the PH domain was thought to be undruggable until high-resolution structures of human PH domains allowed structure-based design of novel inhibitors that selectively bind the PH domain. Allosteric inhibitors of the Akt1 PH domain have already been tested in cancer patients and for proteus syndrome, with several other PH domain inhibitors in preclinical development for treatment of other human diseases.
    Keywords:  Disease; Drugs; Mechanism; PH domain; Phosphatidylinositol; Pleckstrin
    DOI:  https://doi.org/10.1016/j.biopha.2023.115024
  11. Cell Rep. 2023 Jul 04. pii: S2211-1247(23)00735-0. [Epub ahead of print]42(7): 112724
      The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.
    Keywords:  ATF4; CHAC1; CP: Cell biology; GSH; NRF2; SLC7A11; integrated stress response; melanoma
    DOI:  https://doi.org/10.1016/j.celrep.2023.112724