bims-pimaco 2023-03-12 papers

Cancers (Basel). 2023 Feb 22. pii: 1383. [Epub ahead of print]15(5):

The Anti-Tumor Effect of the Newly Developed LAT1 Inhibitor JPH203 in Colorectal Carcinoma, According to a Comprehensive Analysis.

Rina Otani, Hidehiko Takigawa, Ryo Yuge, Daisuke Shimizu, Misa Ariyoshi, Ryo Miyamoto, Hiroki Kadota, Yuichi Hiyama, Ryohei Hayashi, Yuji Urabe, Akira Ishikawa, Naohide Oue, Yasuhiko Kitadai, Shiro Oka, Shinji Tanaka.

A novel large neutral amino acid transporter 1 (LAT1)-specific inhibitor, JPH203, is expected to cause cancer-specific starvation and possess anti-tumor effects; however, its anti-tumor mechanism for colorectal cancer (CRC) remains unclear. We analyzed LAT family gene expressions in public databases using UCSC Xena and evaluated LAT1 protein expression using immunohistochemistry in 154 cases of surgically resected CRC. We also evaluated mRNA expression using polymerase chain reaction in 10 CRC cell lines. Furthermore, JPH203 treatment experiments were conducted in vitro and in vivo using an allogeneic immune-responsive mouse model with abundant stroma created via the orthotopic transplantation of the mouse-derived CRC cell line CT26 and mesenchymal stem cells. The treatment experiments were followed by comprehensive gene expression analyses with RNA sequencing. Database analyses and immunohistochemistry research on clinical specimens revealed that LAT1 expression was cancer-dominant, and its increase was accompanied by tumor progression. In vitro, JPH203 was effective in an LAT1 expression-dependent manner. In vivo, JPH203 treatment considerably reduced tumor size and metastasis, and RNA sequencing-based pathway analysis showed that not only tumor growth and amino acid metabolism pathways, but also stromal activation-related pathways were suppressed. The results of the RNA sequencing were validated in the clinical specimens, as well as both in vitro and in vivo. LAT1 expression in CRC plays an important role in tumor progression. JPH203 may inhibit the progression of CRC and tumor stromal activity.

Keywords: LAT1; chemotherapy; colorectal cancer; comprehensive analysis; tumor microenvironment

DOI: https://doi.org/10.3390/cancers15051383

Life Sci Alliance. 2023 May;pii: e202201670. [Epub ahead of print]6(5):

Analysis of context-specific KRAS-effector (sub)complexes in Caco-2 cells.

Camille Ternet, Philipp Junk, Thomas Sevrin, Simona Catozzi, Erik Wåhlén, Johan Heldin, Giorgio Oliviero, Kieran Wynne, Christina Kiel.

Ras is a key switch controlling cell behavior. In the GTP-bound form, Ras interacts with numerous effectors in a mutually exclusive manner, where individual Ras-effectors are likely part of larger cellular (sub)complexes. The molecular details of these (sub)complexes and their alteration in specific contexts are not understood. Focusing on KRAS, we performed affinity purification (AP)-mass spectrometry (MS) experiments of exogenously expressed FLAG-KRAS WT and three oncogenic mutants ("genetic contexts") in the human Caco-2 cell line, each exposed to 11 different culture media ("culture contexts") that mimic conditions relevant in the colon and colorectal cancer. We identified four effectors present in complex with KRAS in all genetic and growth contexts ("context-general effectors"). Seven effectors are found in KRAS complexes in only some contexts ("context-specific effectors"). Analyzing all interactors in complex with KRAS per condition, we find that the culture contexts had a larger impact on interaction rewiring than genetic contexts. We investigated how changes in the interactome impact functional outcomes and created a Shiny app for interactive visualization. We validated some of the functional differences in metabolism and proliferation. Finally, we used networks to evaluate how KRAS-effectors are involved in the modulation of functions by random walk analyses of effector-mediated (sub)complexes. Altogether, our work shows the impact of environmental contexts on network rewiring, which provides insights into tissue-specific signaling mechanisms. This may also explain why KRAS oncogenic mutants may be causing cancer only in specific tissues despite KRAS being expressed in most cells and tissues.

DOI: https://doi.org/10.26508/lsa.202201670

Comput Biol Chem. 2023 Mar 01. pii: S1476-9271(23)00026-9. [Epub ahead of print]104 107835

GAP positions catalytic H-Ras residue Q61 for GTP hydrolysis in molecular dynamics simulations, complicating chemical rescue of Ras deactivation.

Lara A Patel, Timothy J Waybright, Andrew G Stephen, Chris Neale.

Functional interaction of Ras signaling proteins with upstream, negative regulatory GTPase activating proteins (GAPs) represents a crucial step in cellular decision making related to growth and survival. Key components of the catalytic transition state for Ras deactivation by GAP-accelerated hydrolysis of Ras-bound guanosine triphosphate (GTP) are thought to include an arginine residue from the GAP (the arginine finger), a glutamine residue from Ras (Q61), and a water molecule that is likely coordinated by Q61 to engage in nucleophilic attack on GTP. Here, we use in-vitro fluorescence experiments to show that 0.1-100 mM concentrations of free arginine, imidazole, and other small nitrogenous molecule fail to accelerate GTP hydrolysis, even in the presence of the catalytic domain of a mutant GAP lacking its arginine finger (R1276A NF1). This result is surprising given that imidazole can chemically rescue enzyme activity in arginine-to-alanine mutant protein tyrosine kinases (PTKs) that share many active site components with Ras/GAP complexes. Complementary all-atom molecular dynamics (MD) simulations reveal that an arginine finger GAP mutant still functions to enhance Ras Q61-GTP interaction, though less extensively than wild-type GAP. This increased Q61-GTP proximity may promote more frequent fluctuations into configurations that enable GTP hydrolysis as a component of the mechanism by which GAPs accelerate Ras deactivation in the face of arginine finger mutations. The failure of small molecule analogs of arginine to chemically rescue catalytic deactivation of Ras is consistent with the idea that the influence of the GAP goes beyond the simple provision of its arginine finger. However, the failure of chemical rescue in the presence of R1276A NF1 suggests that the GAPs arginine finger is either unsusceptible to rescue due to exquisite positioning or that it is involved in complex multivalent interactions. Therefore, in the context of oncogenic Ras proteins with mutations at codons 12 or 13 that inhibit arginine finger penetration toward GTP, drug-based chemical rescue of GTP hydrolysis may have bifunctional chemical/geometric requirements that are more difficult to satisfy than those that result from arginine-to-alanine mutations in other enzymes for which chemical rescue has been demonstrated.

Keywords: Cancer; GAP; GTPase; Interface; RAS

DOI: https://doi.org/10.1016/j.compbiolchem.2023.107835

Oncogene. 2023 Mar 07.

CDK13 phosphorylates the translation machinery and promotes tumorigenic protein synthesis.

Chao Wu, Ting Xie, Ying Guo, Donghai Wang, Min Qiu, Ruyi Han, Guoliang Qing, Kaiwei Liang, Hudan Liu.

Cyclin-dependent kinase 13 (CDK13) has been suggested to phosphorylate RNA polymerase II and is involved in transcriptional activation. However, whether CDK13 catalyzes other protein substrates and how CDK13 contributes to tumorigenesis remain largely unclear. We here identify key translation machinery components, 4E-BP1 and eIF4B, as novel CDK13 substrates. CDK13 directly phosphorylates 4E-BP1 at Thr46 and eIF4B at Ser422; genetically or pharmacologically inhibiting CDK13 disrupts mRNA translation. Polysome profiling analysis shows that MYC oncoprotein synthesis strictly depends on CDK13-regulated translation in colorectal cancer (CRC), and CDK13 is required for CRC cell proliferation. As mTORC1 is implicated in 4E-BP1 and eIF4B phosphorylation, inactivation of CDK13 in combination with the mTORC1 inhibitor rapamycin further dephosphorylates 4E-BP1 and eIF4B and blocks protein synthesis. As a result, dual inhibition of CDK13 and mTORC1 induces more profound tumor cell death. These findings clarify the pro-tumorigenic role of CDK13 by direct phosphorylation of translation initiation factors and enhancing protein synthesis. Therefore, therapeutic targeting of CDK13 alone or in combination with rapamycin may pave a new way for cancer treatment.

DOI: https://doi.org/10.1038/s41388-023-02653-2

Cancers (Basel). 2023 Feb 23. pii: 1416. [Epub ahead of print]15(5):

Phosphoinositide 3-Kinase (PI3K) Inhibitors and Breast Cancer: An Overview of Current Achievements.

Alexandre Bertucci, François Bertucci, Anthony Gonçalves.

The phosphatidylinositol 3-kinase (PI3K) pathway is one of the most altered pathways in human cancers, and it plays a central role in cellular growth, survival, metabolism, and cellular mobility, making it a particularly interesting therapeutic target. Recently, pan-inhibitors and then selective p110α subunit inhibitors of PI3K were developed. Breast cancer is the most frequent cancer in women and, despite therapeutic progress in recent years, advanced breast cancers remain incurable and early breast cancers are at risk of relapse. Breast cancer is divided in three molecular subtypes, each with its own molecular biology. However, PI3K mutations are found in all breast cancer subtypes in three main "hotspots". In this review, we report the results of the most recent and main ongoing studies evaluating pan-PI3K inhibitors and selective PI3K inhibitors in each breast cancer subtype. In addition, we discuss the future of their development, the various potential mechanisms of resistance to these inhibitors and the ways to circumvent them.

Keywords: PI3KCA; breast cancer; overview; pan-PI3K inhibitor; resistance mechanism; selective PI3K inhibitor; target therapy

DOI: https://doi.org/10.3390/cancers15051416

Transl Lung Cancer Res. 2023 Feb 28. 12(2): 181-183

Understanding KRAS for better targeting.

Ronak Patel, Jorge Nieva.

Keywords: Adagrasib; KRAS; KRASG12C; SHP2; Sotorasib

DOI: https://doi.org/10.21037/tlcr-23-47

Nat Metab. 2023 Mar 06.

Primary cilia sense glutamine availability and respond via asparagine synthetase.

Maria Elena Steidl, Elisa A Nigro, Anne Kallehauge Nielsen, Roberto Pagliarini, Laura Cassina, Matteo Lampis, Christine Podrini, Marco Chiaravalli, Valeria Mannella, Gianfranco Distefano, Ming Yang, Mariam Aslanyan, Giovanna Musco, Ronald Roepman, Christian Frezza, Alessandra Boletta.

Depriving cells of nutrients triggers an energetic crisis, which is resolved by metabolic rewiring and organelle reorganization. Primary cilia are microtubule-based organelles at the cell surface, capable of integrating multiple metabolic and signalling cues, but their precise sensory function is not fully understood. Here we show that primary cilia respond to nutrient availability and adjust their length via glutamine-mediated anaplerosis facilitated by asparagine synthetase (ASNS). Nutrient deprivation causes cilia elongation, mediated by reduced mitochondrial function, ATP availability and AMPK activation independently of mTORC1. Of note, glutamine removal and replenishment is necessary and sufficient to induce ciliary elongation or retraction, respectively, under nutrient stress conditions both in vivo and in vitro by restoring mitochondrial anaplerosis via ASNS-dependent glutamate generation. Ift88-mutant cells lacking cilia show reduced glutamine-dependent mitochondrial anaplerosis during metabolic stress, due to reduced expression and activity of ASNS at the base of cilia. Our data indicate a role for cilia in responding to, and possibly sensing, cellular glutamine levels via ASNS during metabolic stress.

DOI: https://doi.org/10.1038/s42255-023-00754-6