bims-pimaco 2021-06-20 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2021–06–20
eight papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Rictor/mTORC2 signaling protects colonocytes from apoptosis and prevents epithelial barrier breakdown.
mTOR-Rictor-EGFR axis in oncogenesis and diagnosis of glioblastoma multiforme.
AIMTOR, a BRET Biosensor for Live Recording of mTOR Activity in Cell Populations and Single Cells.
KRAS-dependent cancer cells promote survival by producing exosomes enriched in Survivin.
PI3K inhibitors are finally coming of age.
GLS1 depletion inhibited colorectal cancer proliferation and migration via redox/Nrf2/autophagy-dependent pathway.
Inhibition of K-Ras4B-plasma membrane association with a membrane microdomain-targeting peptide.
Critical requirement of SOS1 RAS-GEF function for mitochondrial dynamics, metabolism, and redox homeostasis.

Am J Pathol. 2021 Jun 14. pii: S0002-9440(21)00259-5. [Epub ahead of print]

Rictor/mTORC2 signaling protects colonocytes from apoptosis and prevents epithelial barrier breakdown.

Felipe Castro-Martinez, Aurora Candelario-Martinez, Maria Del Rocio Encarnacion-Garcia, Zayda Piedra-Quintero, Raul Bonilla-Moreno, Abigail Betanzos, Rocio Perez-Orozco, Maria de Los Angeles Hernandez-Cueto, Jose Esteban Muñoz-Medina, Genaro Patiño-Lopez, Michael Schnoor, Nicolas Villegas-Sepulveda, Porfirio Nava.

  Epithelial barrier impairment is a hallmark of several pathological processes in the gut, including inflammatory bowel diseases. Several intracellular signals prevent apoptosis in intestinal epithelial cells. Here we shown that in colonocytes, rictor/mTORC2 signaling is a pro-survival stimulus. Mechanistically, mTORC2 activates Akt which in turns inhibits apoptosis by phosphorylating Bad and preventing caspase-3 activation. Nevertheless, during inflammation, rictor/mTORC2 signaling declines and Akt activity is reduced. Consequently, active caspase-3 increases in surface colonocytes undergoing apoptosis/anoikis and causes epithelial barrier breakdown. Likewise, Rictor ablation in intestinal epithelial cells interrupts mTORC2/Akt signaling and increases apoptosis/anoikis of surface colonocytes without affecting the crypt architecture. The increase in epithelial permeability induced by Rictor ablation produces a mild inflammatory response in the colonic mucosa, but minimally affects the development/establishment of colitis. The data identify a previously unknown mechanism by which rictor/mTORC2 signaling regulates apoptosis/anoikis in intestinal epithelial cells during colitis and clarifies its role in the maintenance of the intestinal epithelial barrier.

Keywords:  Akt/PKB; PI3K; apoptosis; inflammation; intestinal epithelial cells; mTORC2; rictor

DOI:  https://doi.org/10.1016/j.ajpath.2021.06.004
Mol Biol Rep. 2021 Jun 16.

mTOR-Rictor-EGFR axis in oncogenesis and diagnosis of glioblastoma multiforme.

M Janaki Ramaiah, K Rohil Kumar.

  Glioblastoma multiforme (GBM) is one of the aggressive brain cancers with patients having less survival period upto 12-15 months. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase, belongs to the phosphatidylinositol 3-kinases (PI3K) pathway and is involved in various cellular processes of cancer cells. Cancer metabolism is regulated by mTOR and its components. mTOR forms two complexes as mTORC1 and mTORC2. Studies have identified the key component of the mTORC2 complex, Rapamycin-insensitive companion of mammalian target of rapamycin (Rictor) plays a prominent role in the regulation of cancer cell proliferation and metabolism. Apart, growth factor receptor signaling such as epidermal growth factor signaling mediated by epidermal growth factor receptor (EGFR) regulates cancer-related processes. In EGFR signaling various other signaling cascades such as phosphatidyl-inositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR pathway) and Ras/Raf/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) -dependent signaling cross-talk each other. From various studies about GBM, it is very well established that Rictor and EGFR mediated signaling pathways majorly playing a pivotal role in chemoresistance and tumor aggressiveness. Recent studies have shown that non-coding RNAs such as microRNAs (miRs) and long non-coding RNAs (lncRNAs) regulate the EGFR and Rictor and sensitize the cells towards chemotherapeutic agents. Thus, understanding of microRNA mediated regulation of EGFR and Rictor will help in cancer prevention and management as well as a future therapy.

Keywords:  EGFR; Glioblastoma multiforme (GBM); HDAC; Rictor; Temozolomide (TMZ); mTOR

DOI:  https://doi.org/10.1007/s11033-021-06462-2
Bio Protoc. 2021 Apr 20. 11(8): e3989

AIMTOR, a BRET Biosensor for Live Recording of mTOR Activity in Cell Populations and Single Cells.

Nathalie Bouquier, Enora Moutin, Julie Perroy, Vincent Ollendorff.

  Mammalian target of rapamycin (mTOR) controls many crucial cellular functions, including protein synthesis, cell size, energy metabolism, lysosome and mitochondria biogenesis, and autophagy. Consequently, deregulation of mTOR signaling plays a role in numerous pathological conditions such as cancer, metabolic disorders and neurological diseases. Developing new tools to monitor mTOR spatiotemporal activation is crucial to better understand its roles in physiological and pathological conditions. However, the most widely used method to report mTOR activity relies on the quantification of specific mTOR-phosphorylated substrates by western blot. This approach requires cellular lysate preparation, which restricts the quantification to a single time point. Here, we present a simple protocol to study mTOR activity in living cells in real time using AIMTOR, an intramolecular BRET-based (bioluminescence resonance energy transfer) biosensor that we recently designed ( Bouquier et al., 2020 ). We describe transfection of AIMTOR in the C2C12 cell line and procedures to monitor BRET in a cell population using a plate reader and in single cells by microscopy. Importantly, this protocol is transposable to any cell line and primary cells. In addition, several subcellular compartment-specific versions of AIMTOR have been developed, enabling compartmentalized assessment of mTOR activity. This protocol describes how to use the sensitive AIMTOR biosensor to investigate mTOR signaling dynamics in living cells. Graphic abstract: AIMTOR protocol overview from seeding cells to live BRET recording.

Keywords:  BRET; Kinase activity; Living cells; Real time imaging; mTOR signaling; mTORC1 biosensor

DOI:  https://doi.org/10.21769/BioProtoc.3989
Cancer Lett. 2021 Jun 08. pii: S0304-3835(21)00255-X. [Epub ahead of print]517 66-77

KRAS-dependent cancer cells promote survival by producing exosomes enriched in Survivin.

Wen-Hsuan Chang, Thuy-Tien Thi Nguyen, Chia-Hsin Hsu, Kirsten L Bryant, Hong Jin Kim, Haoqiang Ying, Jon W Erickson, Channing J Der, Richard A Cerione, Marc A Antonyak.

  Mutations in KRAS frequently occur in human cancer and are especially prevalent in pancreatic ductal adenocarcinoma (PDAC), where they have been shown to promote aggressive phenotypes. However, targeting this onco-protein has proven to be challenging, highlighting the need to further identify the various mechanisms used by KRAS to drive cancer progression. Here, we considered the role played by exosomes, a specific class of extracellular vesicles (EVs) derived from the endocytic cellular trafficking machinery, in mediating the ability of KRAS to promote cell survival. We found that exosomes isolated from the serum of PDAC patients, as well as from KRAS-transformed fibroblasts and pancreatic cancer cells, were all highly enriched in the cell survival protein Survivin. Exosomes containing Survivin, upon engaging serum-starved cells, strongly enhanced their survival. Moreover, they significantly compromised the effectiveness of the conventional chemotherapy drug paclitaxel, as well as a novel therapy that combines an ERK inhibitor with chloroquine, which is currently in clinical trials for PDAC. The survival benefits provided by oncogenic KRAS-derived exosomes were markedly reduced when depleted of Survivin using siRNA or upon treatment with the Survivin inhibitor YM155. Taken together, these findings demonstrate how KRAS mutations give rise to exosomes that provide a unique form of intercellular communication to promote cancer cell survival and therapy resistance, as well as raise interesting possibilities regarding their potential for serving as therapeutic targets and diagnostic markers for KRAS-dependent cancers.

Keywords:  Exosome; Extracellular vesicle; KRAS; Pancreatic cancer; Survivin

DOI:  https://doi.org/10.1016/j.canlet.2021.05.031
Nat Rev Drug Discov. 2021 Jun 14.

PI3K inhibitors are finally coming of age.

Bart Vanhaesebroeck, Matthew W D Perry, Jennifer R Brown, Fabrice André, Klaus Okkenhaug.

Overactive phosphoinositide 3-kinase (PI3K) in cancer and immune dysregulation has spurred extensive efforts to develop therapeutic PI3K inhibitors. Although progress has been hampered by issues such as poor drug tolerance and drug resistance, several PI3K inhibitors have now received regulatory approval - the PI3Kα isoform-selective inhibitor alpelisib for the treatment of breast cancer and inhibitors mainly aimed at the leukocyte-enriched PI3Kδ in B cell malignancies. In addition to targeting cancer cell-intrinsic PI3K activity, emerging evidence highlights the potential of PI3K inhibitors in cancer immunotherapy. This Review summarizes key discoveries that aid the clinical translation of PI3Kα and PI3Kδ inhibitors, highlighting lessons learnt and future opportunities.

DOI: https://doi.org/10.1038/s41573-021-00209-1
Arch Biochem Biophys. 2021 Jun 11. pii: S0003-9861(21)00213-7. [Epub ahead of print]708 108964

GLS1 depletion inhibited colorectal cancer proliferation and migration via redox/Nrf2/autophagy-dependent pathway.

Hui-Yun Liu, Hong-Sheng Zhang, Min-Yao Liu, Hong-Ming Li, Xin-Yu Wang, Miao Wang.

  Cancer cells can metabolize glutamine to replenish TCA cycle intermediates for cell survival. Glutaminase (GLS1) is over-expressed in multiple cancers, including colorectal cancer (CRC). However, the role of GLS1 in colorectal cancer development has not yet fully elucidated. In this study, we found that GLS1 levels were significantly increased in CRC cells. Knockdown of GLS1 by shRNAs as well as GLS1 inhibitor BPTES decreased DLD1 and SW480 cell proliferation, colony formation and migration. Knockdown of GLS1 as well as BPTES induced reactive oxygen species (ROS) production, down-regulation of GSH/GSSG ratio, an decrease in Nrf2 protein expression and an increase in cytoplasmic Nrf2 protein expression in DLD1 and SW480 cells. Furthermore, Knockdown of GLS1 as well as BPTES inhibited autophagy pathway, antioxidant NAC and Nrf2 activator could reversed inhibition of GLS1-mediated an decrease in autophagic flux in DLD1 and SW480 cells. Depletion of GLS1-induced inhibition of DLD1 and SW480 CRC cell proliferation, colony formation and migration was reversed by autophagy inducer rapamycin. These results suggest that targeting GLS1 might be a new potential therapeutic target for the treatment of CRC.

Keywords:  Autophagy; Colorectal cancer; GLS1; Redox

DOI:  https://doi.org/10.1016/j.abb.2021.108964
Chem Sci. 2019 Dec 03. 11(3): 826-832

Inhibition of K-Ras4B-plasma membrane association with a membrane microdomain-targeting peptide.

Fang-Yi Li, Zhen-Feng Zhang, Stephanie Voss, Yao-Wen Wu, Yu-Fen Zhao, Yan-Mei Li, Yong-Xiang Chen.

The association of K-Ras4B protein with plasma membrane (PM) is required for its signaling activity. Thus, direct inhibition of K-Ras4B-PM interaction could be a potential anti-Ras therapeutic strategy. However, it remains challenging to modulate such protein-PM interaction. Based on Ras isoform-specific PM microdomain localization patterns, we have developed a potent and isoform-selective peptide inhibitor, Memrasin, for detachment of K-Ras4B from the PM. Memrasin is one of the first direct inhibitors of K-Ras4B-PM interaction, and consists of a membrane ld region-binding sequence derived from the C-terminal region of K-Ras4B and an endosome-escape enhancing motif that can aggregate on membrane. It forms peptide-enriched domains in the ld region, abrogates the tethering of K-Ras4B to the PM and accordingly impairs Ras signaling activity, thereby efficiently decreasing the viability of several human lung cancer cells in a dose-responsive and K-Ras dependent manner. Memrasin provides a useful tool for exploring the biological function of K-Ras4B on or off the PM and a potential starting point for further development into anti-Ras therapeutics.

DOI: https://doi.org/10.1039/c9sc04726c
Oncogene. 2021 Jun 12.

Critical requirement of SOS1 RAS-GEF function for mitochondrial dynamics, metabolism, and redox homeostasis.

Rósula García-Navas, Pilar Liceras-Boillos, Carmela Gómez, Fernando C Baltanás, Nuria Calzada, Cristina Nuevo-Tapioles, José M Cuezva, Eugenio Santos.

SOS1 ablation causes specific defective phenotypes in MEFs including increased levels of intracellular ROS. We showed that the mitochondria-targeted antioxidant MitoTEMPO restores normal endogenous ROS levels, suggesting predominant involvement of mitochondria in generation of this defective SOS1-dependent phenotype. The absence of SOS1 caused specific alterations of mitochondrial shape, mass, and dynamics accompanied by higher percentage of dysfunctional mitochondria and lower rates of electron transport in comparison to WT or SOS2-KO counterparts. SOS1-deficient MEFs also exhibited specific alterations of respiratory complexes and their assembly into mitochondrial supercomplexes and consistently reduced rates of respiration, glycolysis, and ATP production, together with distinctive patterns of substrate preference for oxidative energy metabolism and dependence on glucose for survival. RASless cells showed defective respiratory/metabolic phenotypes reminiscent of those of SOS1-deficient MEFs, suggesting that the mitochondrial defects of these cells are mechanistically linked to the absence of SOS1-GEF activity on cellular RAS targets. Our observations provide a direct mechanistic link between SOS1 and control of cellular oxidative stress and suggest that SOS1-mediated RAS activation is required for correct mitochondrial dynamics and function.

DOI: https://doi.org/10.1038/s41388-021-01886-3