bims-pimaco 2023-02-19 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2023–02–19
eight papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Germline variation in RASAL2 may predict survival in patients with RAS-activated colorectal cancer.
Amino acid transporter SLC7A5 regulates Paneth cell function to affect the intestinal inflammatory response.
Efficacious Combination Drug Treatment for Colorectal Cancer that Overcomes Resistance to KRAS G12C Inhibitors.
Activation of AKT induces EZH2-mediated β-catenin trimethylation in colorectal cancer.
The spatial distribution of GPCR and Gβγ activity across a cell dictates PIP3 dynamics.
Positive feedback in Ras activation through modulation of SOS activation probability.
Phosphoproteomic analysis of metformin signaling in colorectal cancer cells elucidates mechanism of action and potential therapeutic opportunities.
Survival improvement for patients with metastatic colorectal cancer over twenty years.

Genes Chromosomes Cancer. 2023 Feb 15.

Germline variation in RASAL2 may predict survival in patients with RAS-activated colorectal cancer.

Christopher Wills, Katie Watts, Timothy S Maughan, David Fisher, Nada A Al-Tassan, Richard S Houlston, Valentina Escott-Price, Jeremy P Cheadle.

   BACKGROUND: Therapeutic agents that specifically target patients with RAS mutant colorectal cancer (CRC) are needed. We sought potential drug targets by relating genome-wide association study and survival data in patients with advanced CRC profiled for mitogen-activated protein kinase kinase (MAPK) pathway mutations.
METHODS: 694 patients from the clinical trials COIN and COIN-B had MAPK-activated CRCs (assigned as KRAS, NRAS or BRAF mutant). Genome-wide SNP, gene and gene-set analyses were performed to identify determinants of survival. For rs12028023 in RAS Protein Activator Like 2 (RASAL2), we studied its effect by MAPK pathway activation status (by comparing to 760 patients without MAPK-activated CRCs), MAPK gene mutation status, surface area of the primary tumour (as a marker of proliferation) and expression on RASAL2.
RESULTS: In MAGMA genome-wide analyses, RASAL2 was the most significant gene associated with survival (P=2.0x10-5 ). Patients carrying the minor (A) allele in the lead SNP, rs12028023 in intron 1 of RASAL2, had a median increase in survival of 167 days as compared to those carrying the major allele. rs12028023 was predictive for survival by MAPK-activation status (PZ-test =2.1x10-3 ). Furthermore, rs12028023 improved survival in patients with RAS mutant (Hazard Ratio [HR]=0.62, 95% Confidence Intervals [CI]=0.5-0.8, P=3.4x10-5 ) but not BRAF mutant (P=0.87) CRCs. The rs12028023 A-allele was associated with reduced surface area of the primary tumour (Beta= -0.037, Standard Error [SE]= 0.017, P=3.2x10-2 ) and reduced RASAL2 expression in cultured fibroblasts (P=1.6x10-11 ).
CONCLUSION: Our data demonstrates a prognostic role for RASAL2 in patients with MAPK-activated CRCs, with potential as a therapeutic target. This article is protected by copyright. All rights reserved.

Keywords:  Colorectal cancer; MAPK-activation; RAS; RASAL2; survival

DOI:  https://doi.org/10.1002/gcc.23133
bioRxiv. 2023 Jan 24. pii: 2023.01.24.524966. [Epub ahead of print]

Amino acid transporter SLC7A5 regulates Paneth cell function to affect the intestinal inflammatory response.

Lingyu Bao, Liezhen Fu, Yijun Su, Zuojia Chen, Zhaoyi Peng, Lulu Sun, Frank J Gonzalez, Chuan Wu, Hongen Zhang, Bingyin Shi, Yun-Bo Shi.

The intestine is critical for not only processing and resorbing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell-specific knockout ( ΔIEC ) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5 ΔIEC reduces mTORC1 signaling. Surprisingly, Slc7a5 ΔIEC mice have increased cell proliferation but reduced secretory cells, particularly mature Paneth cells. scRNA-seq and electron microscopic analyses revealed dedifferentiation of Paneth cells in Slc7a5 ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. We further show that Slc7a5 ΔIEC mice are prone to experimental colitis. Thus, SLC7A5 regulates secretory cell differentiation to affect stem cell niche and/or inflammatory response to regulate cell proliferation.

DOI: https://doi.org/10.1101/2023.01.24.524966
Mol Cancer Ther. 2023 Feb 10. pii: MCT-22-0411. [Epub ahead of print]

Efficacious Combination Drug Treatment for Colorectal Cancer that Overcomes Resistance to KRAS G12C Inhibitors.

Hiroyuki Matsubara, Hiroyuki Miyoshi, Fumihiko Kakizaki, Tomonori Morimoto, Kenji Kawada, Takehito Yamamoto, Kazutaka Obama, Yoshiharu Sakai, Makoto Mark Taketo.

Recent advances in combinatorial chemistry led to the discovery of inhibitors targeting the KRAS G12C mutant protein. However, efficacy of its monotherapy on colorectal cancer (CRC) is limited. Thus, effective combination drugs should be explored for applicable CRC patients to fully benefit from the KRAS G12C inhibitor treatment. Here we employed a patient-derived CRC stem cell (PD-CRC-SC) spheroid culture and demonstrated that three-drug combination of inhibitors against KRAS G12C, EGFR, and FGFR synergistically suppressed the growth of CRC cells carrying the KRAS G12C mutation. Likewise, combination of KRAS G12C and SHP2 inhibitors was also effective. Importantly, activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in heregulin-responsive CRC cells canceled out the effect of KRAS G12C inhibition, which was largely overcome by PI3K inhibitors. These results reveal that evaluating efficacy of combination therapies with PD-CRC-SC spheroids can be a promising strategy to find the best regimen for individual CRC patients.

DOI: https://doi.org/10.1158/1535-7163.MCT-22-0411
bioRxiv. 2023 Feb 04. pii: 2023.01.31.526429. [Epub ahead of print]

Activation of AKT induces EZH2-mediated β-catenin trimethylation in colorectal cancer.

Ahmed H Ghobashi, Truc T Vuong, Jane W Kimani, Heather M O'Hagan.

Colorectal cancer (CRC) develops in part through the deregulation of different signaling pathways, including activation of the WNT/β-catenin and PI3K/AKT pathways. Enhancer of zeste homolog 2 (EZH2) is a lysine methyltransferase that is involved in regulating stem cell development and differentiation and is overexpressed in CRC. However, depending on the study EZH2 has been found to be both positively and negatively correlated with the survival of CRC patients suggesting that EZH2's role in CRC may be context specific. In this study, we explored how PI3K/AKT activation alters EZH2's role in CRC. We found that activation of AKT by PTEN knockdown or by hydrogen peroxide treatment induced EZH2 phosphorylation at serine 21. Phosphorylation of EZH2 resulted in EZH2-mediated methylation of β-catenin and an associated increased interaction between β-catenin, TCF1, and RNA polymerase II. AKT activation increased β-catenin's enrichment across the genome and EZH2 inhibition reduced this enrichment by reducing the methylation of β-catenin. Furthermore, PTEN knockdown increased the expression of epithelial-mesenchymal transition (EMT)-related genes, and somewhat unexpectedly EZH2 inhibition further increased the expression of these genes. Consistent with these findings, EZH2 inhibition enhanced the migratory phenotype of PTEN knockdown cells. Overall, we demonstrated that EZH2 modulates AKT-induced changes in gene expression through the AKT/EZH2/ β-catenin axis in CRC with active PI3K/AKT signaling. Therefore, it is important to consider the use of EZH2 inhibitors in CRC with caution as these inhibitors will inhibit EZH2-mediated methylation of histone and non-histone targets such as β-catenin, which can have tumor-promoting effects.

DOI: https://doi.org/10.1101/2023.01.31.526429
Sci Rep. 2023 Feb 16. 13(1): 2771

The spatial distribution of GPCR and Gβγ activity across a cell dictates PIP3 dynamics.

Dhanushan Wijayaratna, Kasun Ratnayake, Sithurandi Ubeysinghe, Dinesh Kankanamge, Mithila Tennakoon, Ajith Karunarathne.

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a plasma membrane-bound signaling phospholipid involved in many cellular signaling pathways that control crucial cellular processes and behaviors, including cytoskeleton remodeling, metabolism, chemotaxis, and apoptosis. Therefore, defective PIP3 signaling is implicated in various diseases, including cancer, diabetes, obesity, and cardiovascular diseases. Upon activation by G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), phosphoinositide-3-kinases (PI3Ks) phosphorylate phosphatidylinositol (4,5) bisphosphate (PIP2), generating PIP3. Though the mechanisms are unclear, PIP3 produced upon GPCR activation attenuates within minutes, indicating a tight temporal regulation. Our data show that subcellular redistributions of G proteins govern this PIP3 attenuation when GPCRs are activated globally, while localized GPCR activation induces sustained subcellular PIP3. Interestingly the observed PIP3 attenuation was Gγ subtype-dependent. Considering distinct cell-tissue-specific Gγ expression profiles, our findings not only demonstrate how the GPCR-induced PIP3 response is regulated depending on the GPCR activity gradient across a cell, but also show how diversely cells respond to spatial and temporal variability of external stimuli.

DOI: https://doi.org/10.1038/s41598-023-29639-0
Biophys J. 2023 Feb 10. pii: S0006-3495(22)01397-2. [Epub ahead of print]122(3S1): 50a

Positive feedback in Ras activation through modulation of SOS activation probability.

He Ren, Albert Lee, Lap Jung Nugent Lew, Joseph B DeGrandchamp, Jay T Groves.

DOI: https://doi.org/10.1016/j.bpj.2022.11.481
Clin Transl Med. 2023 Feb;13(2): e1179

Phosphoproteomic analysis of metformin signaling in colorectal cancer cells elucidates mechanism of action and potential therapeutic opportunities.

Barbora Salovska, Erli Gao, Sophia Müller-Dott, Wenxue Li, Carlos Chacon Cordon, Shisheng Wang, Aurelien Dugourd, George Rosenberger, Julio Saez-Rodriguez, Yansheng Liu.

   BACKGROUND: The biguanide drug metformin is a safe and widely prescribed drug for type 2 diabetes. Interestingly, hundreds of clinical trials have been set to evaluate the potential role of metformin in the prevention and treatment of cancer including colorectal cancer (CRC). However, the "metformin signaling" remains controversial.
AIMS AND METHODS: To interrogate cell signaling induced by metformin in CRC and explore the druggability of the metformin-rewired phosphorylation network, we performed integrative analysis of phosphoproteomics, bioinformatics, and cell proliferation assays on a panel of 12 molecularly heterogeneous CRC cell lines. Using the high-resolute data-independent analysis mass spectrometry (DIA-MS), we monitored a total of 10,142 proteins and 56,080 phosphosites (P-sites) in CRC cells upon a short- and a long-term metformin treatment.
RESULTS AND CONCLUSIONS: We found that metformin tended to primarily remodel cell signaling in the long-term and only minimally regulated the total proteome expression levels. Strikingly, the phosphorylation signaling response to metformin was highly heterogeneous in the CRC panel, based on a network analysis inferring kinase/phosphatase activities and cell signaling reconstruction. A "MetScore" was determined to assign the metformin relevance of each P-site, revealing new and robust phosphorylation nodes and pathways in metformin signaling. Finally, we leveraged the metformin P-site signature to identify pharmacodynamic interactions and confirmed a number of candidate metformin-interacting drugs, including navitoclax, a BCL-2/BCL-xL inhibitor. Together, we provide a comprehensive phosphoproteomic resource to explore the metformin-induced cell signaling for potential cancer therapeutics. This resource can be accessed at https://yslproteomics.shinyapps.io/Metformin/.

Keywords:  DIA mass spectrometry; colorectal cancer; drug synergy; kinase activity analysis; metformin; network analysis; phosphoproteomics; phosphorylation signature; proteomics

DOI:  https://doi.org/10.1002/ctm2.1179
NPJ Precis Oncol. 2023 Feb 13. 7(1): 16

Survival improvement for patients with metastatic colorectal cancer over twenty years.

Fadl A Zeineddine, Mohammad A Zeineddine, Abdelrahman Yousef, Yue Gu, Saikat Chowdhury, Arvind Dasari, Ryan W Huey, Benny Johnson, Bryan Kee, Michael S Lee, Maria Pia Morelli, Van K Morris, Michael J Overman, Christine Parseghian, Kanwal Raghav, Jason Willis, Robert A Wolff, Yoshikuni Kawaguchi, Jean-Nicolas Vauthey, Ryan Sun, Scott Kopetz, John Paul Shen.

Over the past two decades of successive clinical trials in metastatic colorectal cancer (CRC), the median overall survival of both control and experimental arms has steadily improved. However, the incremental change in survival for metastatic CRC patients not treated on trial has not yet been quantified. We performed a retrospective review of 1420 patients with de novo metastatic CRC who received their primary treatment at the University of Texas M.D. Anderson Cancer Center (UTMDACC) from 2004 through 2019. Median OS was roughly stable for patients diagnosed between 2004 and 2012 (22.6 months) but since has steadily improved for those diagnosed in 2013 to 2015 (28.8 months), and 2016 to 2019 (32.4 months). Likewise, 5-year survival rate has increased from 15.7% for patients diagnosed from 2004 to 2006 to 26% for those diagnosed from 2013 to 2015. Notably, survival improved for patients with BRAFV600E mutant as well as microsatellite unstable (MSI-H) tumors. Multivariate regression analysis identified surgical resection of liver metastasis (HR = 0.26, 95% CI, 0.19-0.37), use of immunotherapy (HR = 0.44, 95% CI, 0.29-0.67) and use of third line chemotherapy (regorafenib or trifluridine/tipiracil, HR = 0.74, 95% CI, 0.58-0.95), but not year of diagnosis (HR = 0.99, 95% CI, 0.98-1), as associated with better survival, suggesting that increased use of these therapies are the drivers of the observed improvement in survival.

DOI: https://doi.org/10.1038/s41698-023-00353-4