bims-pimaco 2023-03-05 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2023–03–05
eight papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Ras protein abundance correlates with Ras isoform mutation patterns in cancer.
MTORC2 is a physiological hydrophobic motif kinase of S6 Kinase 1.
Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.
Blebs promote cell survival by assembling oncogenic signalling hubs.
Codon-specific KRAS mutations predict survival benefit of trifluridine/tipiracil in metastatic colorectal cancer.
RAS and PP2A activities converge on epigenetic gene regulation.
FAK-mediated phosphorylation at Y464 regulates p85β nuclear translocation to promote tumorigenesis of ccRCC by repressing RB1 expression.
Mechanosensitive mTORC2 independently coordinates leading and trailing edge polarity programs during neutrophil migration.

Oncogene. 2023 Mar 02.

Ras protein abundance correlates with Ras isoform mutation patterns in cancer.

Fiona E Hood, Yasmina M Sahraoui, Rosalind E Jenkins, Ian A Prior.

Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. We observe consistent patterns of KRAS > NRAS»HRAS protein expression in cells that correlate with the rank order of Ras mutation frequencies in cancer. Our data provide support for the model of a sweet-spot of Ras dosage mediating isoform-specific contributions to cancer and development. We suggest that in most cases, being the most abundant Ras isoform correlates with occupying the sweet-spot and that HRAS and NRAS expression is usually insufficient to promote oncogenesis when mutated. However, our results challenge the notion that rare codons mechanistically underpin the predominance of KRAS mutant cancers. Finally, direct measurement of mutant versus wildtype KRAS protein abundance revealed a frequent imbalance that may suggest additional non-gene duplication mechanisms for optimizing oncogenic Ras dosage.

DOI: https://doi.org/10.1038/s41388-023-02638-1
Biochim Biophys Acta Mol Cell Res. 2023 Feb 28. pii: S0167-4889(23)00020-4. [Epub ahead of print]1870(4): 119449

MTORC2 is a physiological hydrophobic motif kinase of S6 Kinase 1.

Sheikh Tahir Majeed, Rabiya Majeed, Aijaz A Malik, Khurshid Iqbal Andrabi.

  Ribosomal protein S6 kinase 1 (S6K1), a major downstream effector molecule of mTORC1, regulates cell growth and proliferation by modulating protein translation and ribosome biogenesis. We have recently identified eIF4E as an intermediate in transducing signals from mTORC1 to S6K1 and further demonstrated that the role of mTORC1 is restricted to inducing eIF4E phosphorylation and interaction with S6K1. This interaction relieves S6K1 auto-inhibition and facilitates its hydrophobic motif (HM) phosphorylation and activation as a consequence. These observations underscore a possible involvement of mTORC1 independent kinase in mediating HM phosphorylation. Here, we report mTORC2 as an in-vivo/physiological HM kinase of S6K1. We show that rapamycin-resistant S6K1 truncation mutant ∆NH∆CT continues to display HM phosphorylation with selective sensitivity toward Torin-1. We also show that HM phosphorylation of wildtype S6K1and ∆NH∆CT depends on the presence of mTORC2 regulatory subunit-rictor. Furthermore, truncation mutagenesis and molecular docking analysis reveal the involvement of a conserved 19 amino acid stretch of S6K1 in mediating interaction with rictor. We finally show that deletion of the 19 amino acid region from wildtype S6K1 results in loss of interaction with rictor, with a resultant loss of HM phosphorylation regardless of the presence of functional TOS motif. Our data demonstrate that mTORC2 acts as a physiological HM kinase that can activate S6K1 after its auto-inhibition is overcome by mTORC1. We, therefore, propose a novel mechanism for S6K1 regulation where mTOR complexes 1 and 2 act in tandem to activate the enzyme.

Keywords:  Hydrophobic motif; Kinase; Rapamycin; S6 Kinase 1; Torin; eIF4E; mTORC1; mTORC2

DOI:  https://doi.org/10.1016/j.bbamcr.2023.119449
Cell Rep. 2023 Feb 25. pii: S2211-1247(23)00183-3. [Epub ahead of print]42(3): 112172

Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.

Manoj K Rathinaswamy, Meredith L Jenkins, Benjamin R Duewell, Xuxiao Zhang, Noah J Harris, John T Evans, Jordan T B Stariha, Udit Dalwadi, Kaelin D Fleming, Harish Ranga-Prasad, Calvin K Yip, Roger L Williams, Scott D Hansen, John E Burke.

  Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gβγ subunits, p110γ-p84 is weakly recruited to membranes by Gβγ subunits alone and requires recruitment by Ras to allow for Gβγ activation. We mapped two distinct Gβγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gβγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.

Keywords:  CP: Cell biology; GPCR; HDX-MS; PI3K; PIK3CG; PIK3R5; PIK3R6; TIRF; p101; p84; phosphoinositide 3-kinase

DOI:  https://doi.org/10.1016/j.celrep.2023.112172
Nature. 2023 Mar 01.

Blebs promote cell survival by assembling oncogenic signalling hubs.

Andrew D Weems, Erik S Welf, Meghan K Driscoll, Felix Y Zhou, Hanieh Mazloom-Farsibaf, Bo-Jui Chang, Vasanth S Murali, Gabriel M Gihana, Byron G Weiss, Joseph Chi, Divya Rajendran, Kevin M Dean, Reto Fiolka, Gaudenz Danuser.

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4-11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

DOI: https://doi.org/10.1038/s41586-023-05758-6
Nat Med. 2023 Mar 02.

Codon-specific KRAS mutations predict survival benefit of trifluridine/tipiracil in metastatic colorectal cancer.

Joris van de Haar, Xuhui Ma, Salo N Ooft, Pim W van der Helm, Louisa R Hoes, Sara Mainardi, David J Pinato, Kristi Sun, Lisa Salvatore, Giampaolo Tortora, Ina Valeria Zurlo, Silvana Leo, Riccardo Giampieri, Rossana Berardi, Fabio Gelsomino, Valeria Merz, Federica Mazzuca, Lorenzo Antonuzzo, Gerardo Rosati, Chara Stavraka, Paul Ross, Maria Grazia Rodriquenz, Michele Pavarana, Carlo Messina, Timothy Iveson, Federica Zoratto, Anne Thomas, Elisabetta Fenocchio, Margherita Ratti, Ilaria Depetris, Massimiliano Cergnul, Cristina Morelli, Michela Libertini, Alessandro Parisi, Michele De Tursi, Nicoletta Zanaletti, Ornella Garrone, Janet Graham, Raffaella Longarini, Stefania Maria Gobba, Angelica Petrillo, Emiliano Tamburini, Nicla La Verde, Fausto Petrelli, Vincenzo Ricci, Lodewyk F A Wessels, Michele Ghidini, Alessio Cortellini, Emile E Voest, Nicola Valeri.

Genomics has greatly improved how patients with cancer are being treated; however, clinical-grade genomic biomarkers for chemotherapies are currently lacking. Using whole-genome analysis of 37 patients with metastatic colorectal cancer (mCRC) treated with the chemotherapy trifluridine/tipiracil (FTD/TPI), we identified KRAS codon G12 (KRASG12) mutations as a potential biomarker of resistance. Next, we collected real-world data of 960 patients with mCRC receiving FTD/TPI and validated that KRASG12 mutations were significantly associated with poor survival, also in analyses restricted to the RAS/RAF mutant subgroup. We next analyzed the data of the global, double-blind, placebo-controlled, phase 3 RECOURSE trial (n = 800 patients) and found that KRASG12 mutations (n = 279) were predictive biomarkers for reduced overall survival (OS) benefit of FTD/TPI versus placebo (unadjusted interaction P = 0.0031, adjusted interaction P = 0.015). For patients with KRASG12 mutations in the RECOURSE trial, OS was not prolonged with FTD/TPI versus placebo (n = 279; hazard ratio (HR) = 0.97; 95% confidence interval (CI) = 0.73-1.20; P = 0.85). In contrast, patients with KRASG13 mutant tumors showed significantly improved OS with FTD/TPI versus placebo (n = 60; HR = 0.29; 95% CI = 0.15-0.55; P < 0.001). In isogenic cell lines and patient-derived organoids, KRASG12 mutations were associated with increased resistance to FTD-based genotoxicity. In conclusion, these data show that KRASG12 mutations are biomarkers for reduced OS benefit of FTD/TPI treatment, with potential implications for approximately 28% of patients with mCRC under consideration for treatment with FTD/TPI. Furthermore, our data suggest that genomics-based precision medicine may be possible for a subset of chemotherapies.

DOI: https://doi.org/10.1038/s41591-023-02240-8
Life Sci Alliance. 2023 May;pii: e202301928. [Epub ahead of print]6(5):

RAS and PP2A activities converge on epigenetic gene regulation.

Anna Aakula, Mukund Sharma, Francesco Tabaro, Reetta Nätkin, Jesse Kamila, Henrik Honkanen, Matthieu Schapira, Cheryl Arrowsmith, Matti Nykter, Jukka Westermarck.

RAS-mediated human cell transformation requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A). However, the phosphoprotein targets and cellular processes in which RAS and PP2A activities converge in human cancers have not been systematically analyzed. Here, we discover that phosphosites co-regulated by RAS and PP2A are enriched on proteins involved in epigenetic gene regulation. As examples, RAS and PP2A co-regulate the same phosphorylation sites on HDAC1/2, KDM1A, MTA1/2, RNF168, and TP53BP1. We validate RAS- and PP2A-elicited regulation of HDAC1/2 chromatin recruitment, of RNF168-TP53BP1 interaction, and of gene expression. Consistent with their known synergistic effects in cancer, RAS activation and PP2A inhibition resulted in epigenetic reporter derepression and activation of oncogenic transcription. Transcriptional derepression by PP2A inhibition was associated with an increase in euchromatin and a decrease in global DNA methylation. Collectively, the results indicate that epigenetic protein complexes constitute a significant point of convergence for RAS hyperactivity and PP2A inhibition in cancer. Furthermore, the work provides an important resource for future studies focusing on phosphoregulation of epigenetic gene regulation in cancer and in other RAS/PP2A-regulated cellular processes.

DOI: https://doi.org/10.26508/lsa.202301928
Cell Rep. 2023 Feb 28. pii: S2211-1247(23)00199-7. [Epub ahead of print]42(3): 112188

FAK-mediated phosphorylation at Y464 regulates p85β nuclear translocation to promote tumorigenesis of ccRCC by repressing RB1 expression.

Yanhua Zhang, Baoyu He, Dong Zhang, Yifan Zhang, Chengkun Chen, Wenye Zhang, Shiyi Yang, Meilian Yao, Gaoping Cui, Jun Gu, Ting Wang, Zhang Lin, Youben Fan, Zuquan Xiong, Yujun Hao.

  PI3K regulatory subunit p85s normally stabilizes and regulates catalytic subunit p110s in the cytoplasm. Recent studies show that p110-free p85s in the nucleus plays important roles in biological processes. However, the mechanisms by which p85s translocate into the nucleus remain elusive. Here, we describe the mechanism by which p85β translocates into the nucleus to promote ccRCC tumorigenesis. Phosphorylation of p85β at the Y464 by FAK facilitates its nuclear translocation in the kidney through enhancing the binding of p85β to KPNA1. PIK3R2/p85β is highly expressed in ccRCC samples and associated with overall survival of ccRCC patients. Nuclear but not cytoplasmic p85β performs oncogenic functions by repressing RB1 expression and regulating the G1/S cell cycle transition. Nuclear p85β represses RB1 expression by stabilizing histone methyltransferase EZH1/EZH2 proteins. Last, the FAK inhibitor defactinib significantly suppresses the tumor growth of ccRCC with high p85β Y464 levels.

Keywords:  CP: Cancer; FAK; RB1; ccRCC; nuclear translocation; p85β; tyrosine phosphorylation

DOI:  https://doi.org/10.1016/j.celrep.2023.112188
Mol Biol Cell. 2023 Mar 01. mbcE22050191

Mechanosensitive mTORC2 independently coordinates leading and trailing edge polarity programs during neutrophil migration.

Suvrajit Saha, Jason P Town, Orion D Weiner.

By acting both upstream and downstream of biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanosensitive mTORC2 programs in neutrophil polarity and motility. We find that the tension-based inhibition of leading edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the regulation of RhoA and Myosin II-based contractility at the trailing edge depend on mTORC2 kinase activity. mTORC2 is essential for spatial and temporal coordination of the front and back polarity programs for persistent migration under confinement. This mechanosensory pathway integrates multiple upstream signals, and we find that membrane stretch synergizes with biochemical co-input PIP3 to robustly amplify mTORC2 activation. Our results suggest that different signalling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs for efficient coordination of neutrophil shape and movement. [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E22-05-0191