bims-pimaco 2023-02-05 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2023–02–05
ten papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Characterisation of a cyclic peptide that binds to the RAS binding domain of phosphoinositide 3-kinase p110α.
Arf5 mediated regulation of mTORC1 at the plasma membrane.
Whole-cell energy modeling reveals quantitative changes of predicted energy flows in RAS mutant cancer cell lines.
AMPK-dependent phosphorylation of the GATOR2 component WDR24 suppresses glucose-mediated mTORC1 activation.
Claspin is Required for Growth Recovery from Serum Starvation through Regulating the PI3K-PDK1-mTOR Pathway in Mammalian Cells.
Regulation of mTOR by phosphatidic acid.
MmCMS: mouse models' consensus molecular subtypes of colorectal cancer.
Slow TCA flux and ATP production in primary solid tumours but not metastases.
Paneth cells as the origin of intestinal cancer in the context of inflammation.
TSC2 regulates tumor susceptibility to TRAIL-mediated T-cell killing by orchestrating mTOR signaling.

Sci Rep. 2023 Feb 02. 13(1): 1889

Characterisation of a cyclic peptide that binds to the RAS binding domain of phosphoinositide 3-kinase p110α.

Mohamed Ismail, Stephen R Martin, Roger George, Francesca Houghton, Geoff Kelly, Raphaël A G Chaleil, Panayiotis Anastasiou, Xinyue Wang, Nicola O'Reilly, Stefania Federico, Dhira Joshi, Hemavathi Nagaraj, Rachel Cooley, Ning Sze Hui, Miriam Molina-Arcas, David C Hancock, Ali Tavassoli, Julian Downward.

P110α is a member of the phosphoinositide 3-kinase (PI3K) enzyme family that functions downstream of RAS. RAS proteins contribute to the activation of p110α by interacting directly with its RAS binding domain (RBD), resulting in the promotion of many cellular functions such as cell growth, proliferation and survival. Previous work from our lab has highlighted the importance of the p110α/RAS interaction in tumour initiation and growth. Here we report the discovery and characterisation of a cyclic peptide inhibitor (cyclo-CRVLIR) that interacts with the p110α-RBD and blocks its interaction with KRAS. cyclo-CRVLIR was discovered by screening a "split-intein cyclisation of peptides and proteins" (SICLOPPS) cyclic peptide library. The primary cyclic peptide hit from the screen initially showed a weak affinity for the p110α-RBD (Kd about 360 µM). However, two rounds of amino acid substitution led to cyclo-CRVLIR, with an improved affinity for p110α-RBD in the low µM (Kd 3 µM). We show that cyclo-CRVLIR binds selectively to the p110α-RBD but not to KRAS or the structurally-related RAF-RBD. Further, using biophysical, biochemical and cellular assays, we show that cyclo-CRVLIR effectively blocks the p110α/KRAS interaction in a dose dependent manner and reduces phospho-AKT levels in several oncogenic KRAS cell lines.

DOI: https://doi.org/10.1038/s41598-023-28756-0
Mol Biol Cell. 2023 Feb 03. mbcE22070302

Arf5 mediated regulation of mTORC1 at the plasma membrane.

Christian Makhoul, Fiona J Houghton, Elizabeth Hinde, Paul A Gleeson.

The mechanistic target of rapamycin (mTOR) kinase regulates a major signalling pathway in eukaryotic cells. In addition to regulation of mTORC1 at lysosomes, mTORC1 is also localised at other locations. However, little is known about the recruitment and activation of mTORC1 at non-lysosomal sites. To identify regulators of mTORC1 recruitment to non-lysosomal compartments, novel interacting partners with the mTORC1 subunit, Raptor, were identified using immunoprecipitation and mass spectrometry. We show that one of the interacting partners, Arf5, is a novel regulator of mTORC1 signalling at plasma membrane ruffles. Arf5-GFP localizes with endogenous mTOR at PI3,4P2 enriched membrane ruffles together the GTPase required for mTORC1 activation, Rheb. Knockdown of Arf5 reduced the recruitment of mTOR to membrane ruffles. The activation of mTORC1 at membrane ruffles was directly demonstrated using a plasma membrane-targeted mTORC1 biosensor and Arf5 shown to enhance the phosphorylation of the mTORC1 biosensor substrate. In addition, endogenous Arf5 was shown to be required for rapid activation of mTORC1-mediated S6 phosphorylation following nutrient starvation and re-feeding. Our findings reveal a novel Arf5-dependent pathway for recruitment and activation of mTORC1 at plasma membrane ruffles, a process relevant for spatial and temporal regulation of mTORC1 by receptor and nutrient stimuli.

DOI: https://doi.org/10.1091/mbc.E22-07-0302
iScience. 2023 Feb 17. 26(2): 105931

Whole-cell energy modeling reveals quantitative changes of predicted energy flows in RAS mutant cancer cell lines.

Thomas Sevrin, Lisa Strasser, Camille Ternet, Philipp Junk, Miriam Caffarini, Stella Prins, Cian D'Arcy, Simona Catozzi, Giorgio Oliviero, Kieran Wynne, Christina Kiel, Philip J Luthert.

  Cellular utilization of available energy flows to drive a multitude of forms of cellular "work" is a major biological constraint. Cells steer metabolism to address changing phenotypic states but little is known as to how bioenergetics couples to the richness of processes in a cell as a whole. Here, we outline a whole-cell energy framework that is informed by proteomic analysis and an energetics-based gene ontology. We separate analysis of metabolic supply and the capacity to generate high-energy phosphates from a representation of demand that is built on the relative abundance of ATPases and GTPases that deliver cellular work. We employed mouse embryonic fibroblast cell lines that express wild-type KRAS or oncogenic mutations and with distinct phenotypes. We observe shifts between energy-requiring processes. Calibrating against Seahorse analysis, we have created a whole-cell energy budget with apparent predictive power, for instance in relation to protein synthesis.

Keywords:  Cellular physiology; Protein

DOI:  https://doi.org/10.1016/j.isci.2023.105931
Nat Metab. 2023 Feb 02.

AMPK-dependent phosphorylation of the GATOR2 component WDR24 suppresses glucose-mediated mTORC1 activation.

Xiaoming Dai, Cong Jiang, Qiwei Jiang, Lan Fang, Haihong Yu, Jinhe Guo, Peiqiang Yan, Fangtao Chi, Tao Zhang, Hiroyuki Inuzuka, John M Asara, Ping Wang, Jianping Guo, Wenyi Wei.

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth in response to amino acid and glucose levels. However, how mTORC1 senses glucose availability to regulate various downstream signalling pathways remains largely elusive. Here we report that AMP-activated protein kinase (AMPK)-mediated phosphorylation of WDR24, a core component of the GATOR2 complex, has a role in the glucose-sensing capability of mTORC1. Mechanistically, glucose deprivation activates AMPK, which directly phosphorylates WDR24 on S155, subsequently disrupting the integrity of the GATOR2 complex to suppress mTORC1 activation. Phosphomimetic Wdr24S155D knock-in mice exhibit early embryonic lethality and reduced mTORC1 activity. On the other hand, compared to wild-type littermates, phospho-deficient Wdr24S155A knock-in mice are more resistant to fasting and display elevated mTORC1 activity. Our findings reveal that AMPK-mediated phosphorylation of WDR24 modulates glucose-induced mTORC1 activation, thereby providing a rationale for targeting AMPK-WDR24 signalling to fine-tune mTORC1 activation as a potential therapeutic means to combat human diseases with aberrant activation of mTORC1 signalling including cancer.

DOI: https://doi.org/10.1038/s42255-022-00732-4
Mol Cell Biol. 2023 Jan;43(1): 1-21

Claspin is Required for Growth Recovery from Serum Starvation through Regulating the PI3K-PDK1-mTOR Pathway in Mammalian Cells.

Chi-Chun Yang, Hisao Masai.

  Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation, which requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. In the absence of Claspin, cells do not proceed into S phase and eventually die partially in a ROS- and p53-dependent manner. Claspin directly interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4EBP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 physically interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. Thus, Claspin plays a novel role as a key regulator for nutrition-induced proliferation/survival signaling by activating the mTOR pathway. The results also suggest a possibility that Claspin may serve as a common mediator that receives signals from different PI3K-related kinases and transmit them to specific downstream kinases.

Keywords:  Claspin; PDK1; PI3 kinase; mTOR; serum stimulation

DOI:  https://doi.org/10.1080/10985549.2022.2160598
Trends Endocrinol Metab. 2023 Jan 31. pii: S1043-2760(23)00015-2. [Epub ahead of print]

Regulation of mTOR by phosphatidic acid.

Maria A Frias, Ahmet Hatipoglu, David A Foster.

  mTORC1, the mammalian target of rapamycin complex 1, is a key regulator of cellular physiology. The lipid metabolite phosphatidic acid (PA) binds to and activates mTORC1 in response to nutrients and growth factors. We review structural findings and propose a model for PA activation of mTORC1. PA binds to a highly conserved sequence in the α4 helix of the FK506 binding protein 12 (FKBP12)/rapamycin-binding (FRB) domain of mTOR. It is proposed that PA binding to two adjacent positively charged amino acids breaks and shortens the C-terminal region of helix α4. This has profound consequences for both substrate binding and the catalytic activity of mTORC1.

Keywords:  FRB; Rheb; mTOR; phosphatidic acid; phospholipase D; structure

DOI:  https://doi.org/10.1016/j.tem.2023.01.004
Br J Cancer. 2023 Jan 30.

MmCMS: mouse models' consensus molecular subtypes of colorectal cancer.

Raheleh Amirkhah, Kathryn Gilroy, Sudhir B Malla, Tamsin R M Lannagan, Ryan M Byrne, Natalie C Fisher, Shania M Corry, Noha-Ehssan Mohamed, Hojjat Naderi-Meshkin, Megan L Mills, Andrew D Campbell, Rachel A Ridgway, Baharak Ahmaderaghi, Richard Murray, Antoni Berenguer Llergo, Rebeca Sanz-Pamplona, Alberto Villanueva, Eduard Batlle, Ramon Salazar, Mark Lawler, Owen J Sansom, Philip D Dunne.

BACKGROUND: Colorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1-4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue.
METHODS: Using transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n = 577) and mouse (n = 57 across n = 8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers.
RESULTS: We developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours.
CONCLUSIONS: When applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package enables researchers to select suitable mouse models of human CRC subtype for their experimental testing.

DOI: https://doi.org/10.1038/s41416-023-02157-6
Nature. 2023 Feb 01.

Slow TCA flux and ATP production in primary solid tumours but not metastases.

Caroline R Bartman, Daniel R Weilandt, Yihui Shen, Won Dong Lee, Yujiao Han, Tara TeSlaa, Connor S R Jankowski, Laith Samarah, Noel R Park, Victoria da Silva-Diz, Maya Aleksandrova, Yetis Gultekin, Argit Marishta, Lin Wang, Lifeng Yang, Asael Roichman, Vrushank Bhatt, Taijin Lan, Zhixian Hu, Xi Xing, Wenyun Lu, Shawn Davidson, Martin Wühr, Matthew G Vander Heiden, Daniel Herranz, Jessie Yanxiang Guo, Yibin Kang, Joshua D Rabinowitz.

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.

DOI: https://doi.org/10.1038/s41586-022-05661-6
Res Sq. 2023 Jan 19. pii: rs.3.rs-2458794. [Epub ahead of print]

Paneth cells as the origin of intestinal cancer in the context of inflammation.

Riccardo Fodde, Mathijs Verhagen, Rosalie Joosten, Mark Schmitt, Andrea Sacchetti, Jiahn Choi, Niko Välimäki, Lauri Aaltonen, Leonard Augenlicht.

Paneth cells (PCs), responsible for the secretion of antimicrobial peptides in the small intestine and for niche support to Lgr5+ crypt-base columnar stem cells (CBCs), have been shown to respond to inflammation by dedifferentiating into stem-like cells in order to sustain a regenerative response 1,2 . Therefore, PCs may represent the cells-of-origin of intestinal cancer in the context of inflammation. To test this hypothesis, we targeted Apc, Kras, and Tp53 mutations in Paneth cells by Cre-Lox technology and modelled inflammation by dextran sodium sulfate (DSS) administration. PC-specific loss of Apc resulted in multiple small intestinal tumors, whereas Kras or Tp53 mutations did not. Compound Apc and Kras mutations in PCs resulted in a striking increase in tumor multiplicity even in the absence of the inflammatory insult. By combining scRNAseq with lineage tracing to capture the conversion of PCs into bona fide tumor cells, we show that they progress through a "revival stem cell" (RSC) state characterized by high Clusterin (Clu) expression and Yap1 signaling, reminiscent of what has been previously observed upon irradiation of the mouse digestive tract 3 . Accordingly, comparison of PC- and Lgr5-derived murine intestinal tumors revealed differences related to Wnt signaling and inflammatory pathways which match the dichotomy of CBCs and injury-induced RSCs 4 between human sporadic colon cancers and those arising in the context of inflammatory bowel diseases. Last, we show that western-style dietary habits, known to trigger a low-grade inflammation throughout the intestinal tract, underlie the analogous de-differentiation of Paneth cells and their acquisition of stem-like features. Taken together, our results show that intestinal cancer arises in the context of inflammation through the dedifferentiation of committed secretory lineages such as Paneth cells and the activation of the revival stem cell state. As such, a true quiescent stem cell identity may be hidden in fully committed and post-mitotic lineages which, upon inflammation, support the regenerative response by re-entering the cell cycle and dedifferentiating into RSCs. The chronic nature of the tissue insult in inflammatory bowel diseases and even in the context of western-style dietary habits is likely to result in the expansion of cell targets for tumor initiation and progression.

DOI: https://doi.org/10.21203/rs.3.rs-2458794/v1
EMBO J. 2023 Jan 30. e111614

TSC2 regulates tumor susceptibility to TRAIL-mediated T-cell killing by orchestrating mTOR signaling.

Chun-Pu Lin, Joleen J H Traets, David W Vredevoogd, Nils L Visser, Daniel S Peeper.

  Resistance to cancer immunotherapy continues to impair common clinical benefit. Here, we use whole-genome CRISPR-Cas9 knockout data to uncover an important role for Tuberous Sclerosis Complex 2 (TSC2) in determining tumor susceptibility to cytotoxic T lymphocyte (CTL) killing in human melanoma cells. TSC2-depleted tumor cells had disrupted mTOR regulation following CTL attack, which was associated with enhanced cell death. Wild-type tumor cells adapted to CTL attack by shifting their mTOR signaling balance toward increased mTORC2 activity, circumventing apoptosis, and necroptosis. TSC2 ablation strongly augmented tumor cell sensitivity to CTL attack in vitro and in vivo, suggesting one of its functions is to critically protect tumor cells. Mechanistically, TSC2 inactivation caused elevation of TRAIL receptor expression, cooperating with mTORC1-S6 signaling to induce tumor cell death. Clinically, we found a negative correlation between TSC2 expression and TRAIL signaling in TCGA patient cohorts. Moreover, a lower TSC2 immune response signature was observed in melanomas from patients responding to immune checkpoint blockade. Our study uncovers a pivotal role for TSC2 in the cancer immune response by governing crosstalk between TSC2-mTOR and TRAIL signaling, aiding future therapeutic exploration of this pathway in immuno-oncology.

Keywords:  T-cell sensitivity; TRAIL; TSC2; mTOR; tumor cells

DOI:  https://doi.org/10.15252/embj.2022111614