bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2022–07–17
eightteen papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research
  1. A proteomic approach identifies isoform-specific and nucleotide-dependent RAS interactions.
  2. Structure of the MRAS-SHOC2-PP1C phosphatase complex.
  3. The Multifaceted Role of Nutrient Sensing and mTORC1 Signaling in Physiology and Aging.
  4. Targeting Oncogenic RAS Protein.
  5. Targeting KRAS G12C-Mutated Advanced Colorectal Cancer: Research and Clinical Developments.
  6. Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover.
  7. Structure-function analysis of the SHOC2-MRAS-PP1C holophosphatase complex.
  8. Structure of the nutrient-sensing hub GATOR2.
  9. mTORC1 Crosstalk With Stress Granules in Aging and Age-Related Diseases.
  10. AKT inhibition sensitizes EVI1 expressing colon cancer cells to irinotecan therapy by regulating the Akt/mTOR axis.
  11. Integrated multi-omics characterization of KRAS mutant colorectal cancer.
  12. Analysis of KRAS, NRAS, and BRAF Mutations, Microsatellite Instability, and Relevant Prognosis Effects in Patients With Early Colorectal Cancer: A Cohort Study in East Asia.
  13. Retrograde movements determine effective stem cell numbers in the intestine.
  14. Blocking the Farnesyl Pocket of PDEδ Reduces Rheb-Dependent mTORC1 Activation and Survival of Tsc2-Null Cells.
  15. Acetyl-CoA-Carboxylase 1-mediated de novo fatty acid synthesis sustains Lgr5+ intestinal stem cell function.
  16. A ROS-dependent mechanism promotes CDK2 phosphorylation to drive progression through S phase.
  17. KIFC3 Promotes Proliferation, Migration, and Invasion in Colorectal Cancer via PI3K/AKT/mTOR Signaling Pathway.
  18. Nuclear RAC1 is a modulator of the doxorubicin-induced DNA damage response.
  1. Mol Cell Proteomics. 2022 Jul 12. pii: S1535-9476(22)00076-7. [Epub ahead of print] 100268
    A proteomic approach identifies isoform-specific and nucleotide-dependent RAS interactions.
    Seth P Miller, George Maio, Xiaoyu Zhang, Felix S Badillo Soto, Julia Zhu, Stephen Z Ramirez, Hening Lin.
      Active mutations in the RAS genes are found in ∼30% of human cancers. Although thought to have overlapping functions, RAS isoforms show preferential activation in human tumors, which prompted us to employ a comparative and quantitative proteomics approach to generate isoform-specific and nucleotide-dependent interactomes of the four RAS isoforms, KRAS4A, KRAS4B, HRAS, and NRAS. Many isoform-specific interacting proteins were identified including HRAS-specific CARM1 and CHK1, and KRAS-specific PIP4K2C and IPO7. Comparing the interactomes of WT and constitutively active G12D mutant RAS isoforms identified several potential previously unknown effector proteins of RAS, one of which was recently reported while this manuscript was in preparation, RADIL. These interacting proteins play important roles as knockdown or pharmacological inhibition leads to potent inhibition of cancer cells. The HRAS-specific interacting protein CARM1 plays a role in HRAS-induced senescence, with CARM1 knockdown or inhibition selectively increases senescence in HRAS-transformed cells but not KRAS4B-transformed cells. By revealing new isoform-specific and nucleotide-dependent RAS interactors, the study here provides insights to help understand the overlapping functions of the RAS isoforms.
    DOI:  https://doi.org/10.1016/j.mcpro.2022.100268
  2. Nature. 2022 Jul 13.
    Structure of the MRAS-SHOC2-PP1C phosphatase complex.
    Zachary J Hauseman, Michelle Fodor, Anxhela Dhembi, Jessica Viscomi, David Egli, Melusine Bleu, Stephanie Katz, Eunyoung Park, Dong Man Jang, Kathryn A Porter, Fabian Meili, Hongqiu Guo, Grainne Kerr, Sandra Mollé, Camilo Velez-Vega, Kim S Beyer, Giorgio G Galli, Saveur-Michel Maira, Travis Stams, Kirk Clark, Michael J Eck, Luca Tordella, Claudio R Thoma, Daniel A King.
      RAS-MAPK signaling is fundamental for cell proliferation and altered in most human cancers1-3. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. While studies revealed snapshots for autoinhibited and active RAF-MEK1-14-3-3 complexes4, the intermediate steps leading to RAF activation remain unclear. The MRAS-SHOC2-PP1c holophosphatase de-phosphorylates RAF on Serine 259 resulting in 14-3-3 partial displacement and RAF-RAS association3,5,6. MRAS, SHOC2 and PP1C are mutated in Rasopathies, developmental syndromes caused by aberrant MAPK pathway activation6-14 and SHOC2 itself has emerged as potential target in RTK-RAS driven tumors15-18. Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we reveal a 1.95 Å X-ray crystal structure of the MRAS-SHOC2-PP1C complex. SHOC2 bridges PP1C and MRAS via its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS-GTP molecules recruit RAF-14-3-3 and SHOC2-PP1C to produce downstream pathway activation. Importantly, we find that Rasopathy and cancer mutations reside at protein-protein interfaces within the holophosphatase, resulting in enhancing affinities and function. Collectively our findings shed light on a fundamental mechanism of RAS biology and on mechanisms for clinically observed enhanced RAS-MAPK signaling, thus providing the structural basis for therapeutic interventions.
    DOI:  https://doi.org/10.1038/s41586-022-05086-1
  3. Front Aging. 2021 ;2 707372
    The Multifaceted Role of Nutrient Sensing and mTORC1 Signaling in Physiology and Aging.
    Stephanie A Fernandes, Constantinos Demetriades.
      The mechanistic Target of Rapamycin (mTOR) is a growth-related kinase that, in the context of the mTOR complex 1 (mTORC1), touches upon most fundamental cellular processes. Consequently, its activity is a critical determinant for cellular and organismal physiology, while its dysregulation is commonly linked to human aging and age-related disease. Presumably the most important stimulus that regulates mTORC1 activity is nutrient sufficiency, whereby amino acids play a predominant role. In fact, mTORC1 functions as a molecular sensor for amino acids, linking the cellular demand to the nutritional supply. Notably, dietary restriction (DR), a nutritional regimen that has been shown to extend lifespan and improve healthspan in a broad spectrum of organisms, works via limiting nutrient uptake and changes in mTORC1 activity. Furthermore, pharmacological inhibition of mTORC1, using rapamycin or its analogs (rapalogs), can mimic the pro-longevity effects of DR. Conversely, nutritional amino acid overload has been tightly linked to aging and diseases, such as cancer, type 2 diabetes and obesity. Similar effects can also be recapitulated by mutations in upstream mTORC1 regulators, thus establishing a tight connection between mTORC1 signaling and aging. Although the role of growth factor signaling upstream of mTORC1 in aging has been investigated extensively, the involvement of signaling components participating in the nutrient sensing branch is less well understood. In this review, we provide a comprehensive overview of the molecular and cellular mechanisms that signal nutrient availability to mTORC1, and summarize the role that nutrients, nutrient sensors, and other components of the nutrient sensing machinery play in cellular and organismal aging.
    Keywords:  aging; amino acids; dietary restriction; mTORC1; nutrient sensing
    DOI:  https://doi.org/10.3389/fragi.2021.707372
  4. N Engl J Med. 2022 Jul 14. 387(2): 184-186
    Targeting Oncogenic RAS Protein.
    Dan L Longo, Neal Rosen.
      
    DOI:  https://doi.org/10.1056/NEJMe2206831
  5. Onco Targets Ther. 2022 ;15 747-756
    Targeting KRAS G12C-Mutated Advanced Colorectal Cancer: Research and Clinical Developments.
    Jingran Ji, Chongkai Wang, Marwan Fakih.
      Identifying mutations in the KRAS gene has become increasingly important in the treatment of colorectal cancer with many prognostic and therapeutic implications. However, efforts to develop drugs that target KRAS mutations have not been successful until more recently with the introduction of the KRAS G12C inhibitors, sotorasib (AMG510) and adagrasib (MRTX849). Both agents have demonstrated safety and promising efficacy in preclinical studies and early phase trials, but it appears that not all tumor types harboring the KRAS G12C mutation are sensitive to monotherapy approaches. In particular, patients with colorectal cancer (CRC) derive less benefit compared to those with non-small cell lung cancer (NSCLC), likely due to rapid treatment-induced resistance through increased epidermal growth factor receptor (EGFR) signaling. As a result, combination therapy trials with EGFR inhibitors are currently underway. Here, we will review the available clinical trial data on KRASG12C inhibitors in KRAS G12C-mutated CRC, possible mechanisms of resistance to monotherapy, the research studying why available agents are proving to be less efficacious in CRC compared to NSCLC, and future directions for these promising new drugs.
    Keywords:  KRASG12C; adagrasib; colorectal cancer; sotorasib; targeted therapy
    DOI:  https://doi.org/10.2147/OTT.S340392
  6. Dev Cell. 2022 Jul 05. pii: S1534-5807(22)00451-8. [Epub ahead of print]
    Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover.
    York Posor, Charis Kampyli, Benoit Bilanges, Sushila Ganguli, Philipp A Koch, Alexander Wallroth, Daniele Morelli, Michalina Jenkins, Samira Alliouachene, Elitza Deltcheva, Buzz Baum, Volker Haucke, Bart Vanhaesebroeck.
      Focal adhesions are multifunctional organelles that couple cell-matrix adhesion to cytoskeletal force transmission and signaling and to steer cell migration and collective cell behavior. Whereas proteomic changes at focal adhesions are well understood, little is known about signaling lipids in focal adhesion dynamics. Through the characterization of cells from mice with a kinase-inactivating point mutation in the class II PI3K-C2β, we find that generation of the phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) membrane lipid promotes focal adhesion disassembly in response to changing environmental conditions. We show that reduced growth factor signaling sensed by protein kinase N, an mTORC2 target and effector of RhoA, synergizes with the adhesion disassembly factor DEPDC1B to induce local synthesis of PtdIns(3,4)P2 by PI3K-C2β. PtdIns(3,4)P2 then promotes turnover of RhoA-dependent stress fibers by recruiting the PtdIns(3,4)P2-dependent RhoA-GTPase-activating protein ARAP3. Our findings uncover a pathway by which cessation of growth factor signaling facilitates cell-matrix adhesion disassembly via a phosphoinositide lipid switch.
    Keywords:  ARAP3; DEPDC1B; PKN2; RhoA signaling; cell migration; class II PI3K; focal adhesion; lipid switch; phosphatidylinositol-3,4-bisphosphate; phosphoinositide
    DOI:  https://doi.org/10.1016/j.devcel.2022.06.011
  7. Nature. 2022 Jul 13.
    Structure-function analysis of the SHOC2-MRAS-PP1C holophosphatase complex.
    Jason J Kwon, Behnoush Hajian, Yuemin Bian, Lucy C Young, Alvaro J Amor, James R Fuller, Cara V Fraley, Abbey M Sykes, Jonathan So, Joshua Pan, Laura Baker, Sun Joo Lee, Douglas B Wheeler, David L Mayhew, Nicole S Persky, Xiaoping Yang, David E Root, Anthony M Barsotti, Andrew W Stamford, Charles K Perry, Alex Burgin, Frank McCormick, Christopher T Lemke, William C Hahn, Andrew J Aguirre.
      Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .
    DOI:  https://doi.org/10.1038/s41586-022-04928-2
  8. Nature. 2022 Jul 13.
    Structure of the nutrient-sensing hub GATOR2.
    Max L Valenstein, Kacper B Rogala, Pranav V Lalgudi, Edward J Brignole, Xin Gu, Robert A Saxton, Lynne Chantranupong, Jonas Kolibius, Jan-Philipp Quast, David M Sabatini.
      Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients1,2. Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L and SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors SESN2 and CASTOR1, respectively3-5. Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function and structure are unknown. Here we used cryo-electron microscopy to determine the three-dimensional structure of the human GATOR2 complex. We found that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 β-propellers. The scaffold contains two WDR24, four MIOS and two WDR59 subunits circularized via two distinct types of junction involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through β-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane-coating complexes6. The scaffold orients the WD40 β-propeller dimers, which mediate interactions with SESN2, CASTOR1 and GATOR1. Our work reveals the structure of an essential component of the nutrient-sensing machinery and provides a foundation for understanding the function of GATOR2 within the mTORC1 pathway.
    DOI:  https://doi.org/10.1038/s41586-022-04939-z
  9. Front Aging. 2021 ;2 761333
    mTORC1 Crosstalk With Stress Granules in Aging and Age-Related Diseases.
    Marti Cadena Sandoval, Alexander Martin Heberle, Ulrike Rehbein, Cecilia Barile, José Miguel Ramos Pittol, Kathrin Thedieck.
      The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a master regulator of metabolism and aging. A complex signaling network converges on mTORC1 and integrates growth factor, nutrient and stress signals. Aging is a dynamic process characterized by declining cellular survival, renewal, and fertility. Stressors elicited by aging hallmarks such as mitochondrial malfunction, loss of proteostasis, genomic instability and telomere shortening impinge on mTORC1 thereby contributing to age-related processes. Stress granules (SGs) constitute a cytoplasmic non-membranous compartment formed by RNA-protein aggregates, which control RNA metabolism, signaling, and survival under stress. Increasing evidence reveals complex crosstalk between the mTORC1 network and SGs. In this review, we cover stressors elicited by aging hallmarks that impinge on mTORC1 and SGs. We discuss their interplay, and we highlight possible links in the context of aging and age-related diseases.
    Keywords:  MTOR; aging hallmarks; amino acids; autophagy; cellular signaling; insulin; stress; stress granules (SGs)
    DOI:  https://doi.org/10.3389/fragi.2021.761333
  10. Cell Oncol (Dordr). 2022 Jul 14.
    AKT inhibition sensitizes EVI1 expressing colon cancer cells to irinotecan therapy by regulating the Akt/mTOR axis.
    Pradeepa, Voddu Suresh, Shantibhusan Senapati, Soumen Chakraborty.
       PURPOSE: Ecotropic viral integration site 1 (EVI1) is an oncogenic transcription factor that has been attributed to chemotherapy resistance in different cancers. As yet, however, its role in colon cancer drug resistance is not completely understood. Here, we set out to investigate the functional and therapeutic relevance of EVI1 in colon cancer drug resistance.
    METHODS: The EVI1 gene was knocked down in colon cancer cells that were subsequently tested for susceptibility to irinotecan using in vitro assays and in vivo subcutaneous mouse colon cancer models. The effect of EVI1 knockdown on the AKT-mTOR signaling pathway was assessed using cell line models, immunohistochemistry and bioinformatics tools. The anti-proliferative activity of AKT inhibitor GSK690693 and its combination with irinotecan was tested in colon cancer cell line models (2D and 3D). Finally, the therapeutic efficacy of GSK690693 and its combination with irinotecan was evaluated in xenografted EVI1 expressing colon cancer mouse models.
    RESULTS: We found that EVI1 knockdown decreased cancer stem cell-like properties and improved irinotecan responses in both cell line and subcutaneous mouse models. In addition, we found that EVI1 downregulation resulted in inhibition of AKT/mTOR signaling and RICTOR expression. Knocking down RICTOR expression increased the cytotoxic effects of irinotecan in EVI1 downregulated colon cancer cells. Co-treatment with irinotecan and ATP-competitive AKT inhibitor GSK690693 significantly reduced colon cancer cell survival and tumor progression rates.
    CONCLUSION: Inhibition of the AKT signaling cascade by GSK690693 may serve as an alternative to improve the irinotecan response in EVI1-expressing colon cancer cells.
    Keywords:  AKT; Colon cancer; Drug resistance; EVI1; Irinotecan; RICTOR
    DOI:  https://doi.org/10.1007/s13402-022-00690-9
  11. Theranostics. 2022 ;12(11): 5138-5154
    Integrated multi-omics characterization of KRAS mutant colorectal cancer.
    Wei Chong, Xingyu Zhu, Huicheng Ren, Chunshui Ye, Kang Xu, Zhe Wang, Shengtao Jia, Liang Shang, Leping Li, Hao Chen.
      KRAS mutation is the most frequent oncogenic aberration in colorectal cancer (CRC). The molecular mechanism and clinical implications of KRAS mutation in CRC remain unclear and show high heterogeneity within these tumors. Methods: We harnessed the multi-omics data (genomic, transcriptomic, proteomic, and phosphoproteomic etc.) of KRAS-mutant CRC tumors and performed unsupervised clustering to identify proteomics-based subgroups and molecular characterization. Results: In-depth analysis of the tumor microenvironment by single-cell transcriptomic revealed the cellular landscape of KRAS-mutant CRC tumors and identified the specific cell subsets with KRAS mutation. Integrated multi-omics analyses separated the KRAS-mutant tumors into two distinct molecular subtypes, termed KRAS-M1 (KM1) and KRAS-M2 (KM2). The two subtypes had a similar distribution of mutated residues in KRAS (G12D/V/C etc.) but were characterized by distinct features in terms of prognosis, genetic alterations, microenvironment dysregulation, biological phenotype, and potential therapeutic approaches. Proteogenomic analyses revealed that the EMT, TGF-β and angiogenesis pathways were enriched in the KM2 subtype and that the KM2 subtype was associated with the mesenchymal phenotype-related CMS4 subtype, which indicated stromal invasion and worse prognosis. The KM1 subtype was characterized predominantly by activation of the cell cycle, E2F and RNA transcription and was associated with the chromosomal instability (CIN)-related ProS-E proteomic subtype, which suggested cyclin-dependent features and better survival outcomes. Moreover, drug sensitivity analyses based on three compound databases revealed subgroup-specific agents for KM1 and KM2 tumors. Conclusions: This study clarifies the molecular heterogeneity of KRAS-mutant CRC and reveals new biological subtypes and therapeutic possibilities for these tumors.
    Keywords:  Colorectal cancer; KRAS mutation; Molecular subtype; Prognosis; Proteogenomics
    DOI:  https://doi.org/10.7150/thno.73089
  12. Front Oncol. 2022 ;12 897548
    Analysis of KRAS, NRAS, and BRAF Mutations, Microsatellite Instability, and Relevant Prognosis Effects in Patients With Early Colorectal Cancer: A Cohort Study in East Asia.
    Yang Li, Jun Xiao, Tiancheng Zhang, Yanying Zheng, Hailin Jin.
       Background: Early colorectal cancer (ECRC) refers to any size of colorectal cancer (CRC) whose depth of invasion is limited to the mucosa and submucosa. About 10% of patients with ECRC die from cancer after surgery. KRAS, NRAS, and BRAF mutations and microsatellite instability (MSI) are considered diagnostic and prognostic markers in CRC. However, their characteristics in ECRC and whether postoperative chemotherapy based on them will benefit ECRC patients or not remain unknown.
    Patients and Methods: Patients with ECRC and 298 patients with advanced colorectal cancer (ACRC) were collected in our hospital from January 2013 to December 2015. The Amplification Refractory Mutation System (ARMS)-PCR was used to perform the KRAS, NRAS, and BRAF mutant tests.
    Results: In ECRC patients, 43 cases of KRAS mutation were found, accounting for 69.35%. Interestingly, among KRAS mutations, there were 10 KRAS multi-site mutation patients (16.13% in 62 ECRC patients). Moreover, the NRAS mutation rate was 3.23% but no BRAF mutation was found and only 1 case of MSI-High was detected. KRAS mutation was only related to the depth of tumor invasion whereas KRAS multi-site mutations were related to mucus components and tumor size. As far as NRAS is concerned, mutations were associated with elevated CEA, mucus components, and the depth of tumor invasion. Notably, compared with 2.35% KRAS multi-site mutation in ACRC, the rate of KRAS multi-site mutation in ECRC was much higher. Furthermore, Cox regression analysis revealed that KRAS mutation could be an independent prognostic factor of ECRC in patients who have undergone endoscopic resection or surgery.
    Conclusion: Patients with ECRC might benefit from KRAS mutation testing but not from postoperative chemotherapy.
    Keywords:  BRAF; KRAS; NRAS; early colorectal cancer; gene mutation; microsatellite instability
    DOI:  https://doi.org/10.3389/fonc.2022.897548
  13. Nature. 2022 Jul 13.
    Retrograde movements determine effective stem cell numbers in the intestine.
    Maria Azkanaz, Bernat Corominas-Murtra, Saskia I J Ellenbroek, Lotte Bruens, Anna T Webb, Dimitrios Laskaris, Koen C Oost, Simona J A Lafirenze, Karl Annusver, Hendrik A Messal, Sharif Iqbal, Dustin J Flanagan, David J Huels, Felipe Rojas-Rodríguez, Miguel Vizoso, Maria Kasper, Owen J Sansom, Hugo J Snippert, Prisca Liberali, Benjamin D Simons, Pekka Katajisto, Edouard Hannezo, Jacco van Rheenen.
      The morphology and functionality of the epithelial lining differ along the intestinal tract, but tissue renewal at all sites is driven by stem cells at the base of crypts1-3. Whether stem cell numbers and behaviour vary at different sites is unknown. Here we show using intravital microscopy that, despite similarities in the number and distribution of proliferative cells with an Lgr5 signature in mice, small intestinal crypts contain twice as many effective stem cells as large intestinal crypts. We find that, although passively displaced by a conveyor-belt-like upward movement, small intestinal cells positioned away from the crypt base can function as long-term effective stem cells owing to Wnt-dependent retrograde cellular movement. By contrast, the near absence of retrograde movement in the large intestine restricts cell repositioning, leading to a reduction in effective stem cell number. Moreover, after suppression of the retrograde movement in the small intestine, the number of effective stem cells is reduced, and the rate of monoclonal conversion of crypts is accelerated. Together, these results show that the number of effective stem cells is determined by active retrograde movement, revealing a new channel of stem cell regulation that can be experimentally and pharmacologically manipulated.
    DOI:  https://doi.org/10.1038/s41586-022-04962-0
  14. Front Pharmacol. 2022 ;13 912688
    Blocking the Farnesyl Pocket of PDEδ Reduces Rheb-Dependent mTORC1 Activation and Survival of Tsc2-Null Cells.
    Marisol Estrella Armijo, Emilia Escalona, Daniela Peña, Alejandro Farias, Violeta Morin, Matthias Baumann, Bert Matthias Klebl, Roxana Pincheira, Ariel Fernando Castro.
      Rheb is a small GTPase member of the Ras superfamily and an activator of mTORC1, a protein complex master regulator of cell metabolism, growth, and proliferation. Rheb/mTORC1 pathway is hyperactivated in proliferative diseases, such as Tuberous Sclerosis Complex syndrome and cancer. Therefore, targeting Rheb-dependent signaling is a rational strategy for developing new drug therapies. Rheb activates mTORC1 in the cytosolic surface of lysosomal membranes. Rheb's farnesylation allows its anchorage on membranes, while its proper localization depends on the prenyl-binding chaperone PDEδ. Recently, the use of PDEδ inhibitors has been proposed as anticancer agents because they interrupted KRas signaling leading to antiproliferative effects in KRas-dependent pancreatic cancer cells. However, the effect of PDEδ inhibition on the Rheb/mTORC1 pathway has been poorly investigated. Here, we evaluated the impact of a new PDEδ inhibitor, called Deltasonamide 1, in Tsc2-null MEFs, a Rheb-dependent overactivated mTORC1 cell line. By using a yeast two-hybrid assay, we first validated that Deltasonamide 1 disrupts Rheb-PDEδ interaction. Accordingly, we found that Deltasonamide 1 reduces mTORC1 targets activation. In addition, our results showed that Deltasonamide 1 has antiproliferative and cytotoxic effects on Tsc2-null MEFs but has less effect on Tsc2-wild type MEFs viability. This work proposes the pharmacological PDEδ inhibition as a new approach to target the abnormal Rheb/mTORC1 activation in Tuberous Sclerosis Complex cells.
    Keywords:  Deltasonamide 1; PDEδ inhibitor; Rheb; Tsc2-null cells; mTORC1 signaling
    DOI:  https://doi.org/10.3389/fphar.2022.912688
  15. Nat Commun. 2022 Jul 09. 13(1): 3998
    Acetyl-CoA-Carboxylase 1-mediated de novo fatty acid synthesis sustains Lgr5+ intestinal stem cell function.
    Shuting Li, Chia-Wen Lu, Elia C Diem, Wang Li, Melanie Guderian, Marc Lindenberg, Friederike Kruse, Manuela Buettner, Stefan Floess, Markus R Winny, Robert Geffers, Hans-Hermann Richnow, Wolf-Rainer Abraham, Guntram A Grassl, Matthias Lochner.
      Basic processes of the fatty acid metabolism have an important impact on the function of intestinal epithelial cells (IEC). However, while the role of cellular fatty acid oxidation is well appreciated, it is not clear how de novo fatty acid synthesis (FAS) influences the biology of IECs. We report here that interfering with de novo FAS by deletion of the enzyme Acetyl-CoA-Carboxylase (ACC)1 in IECs results in the loss of epithelial crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells (ISC). Mechanistically, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/β-catenin in ISCs. The dependency of ISCs on cellular de novo FAS is tuned by the availability of environmental lipids, as an excess delivery of external fatty acids is sufficient to rescue the defect in crypt formation. Finally, inhibition of ACC1 reduces the formation of tumors in colitis-associated colon cancer, together highlighting the importance of cellular lipogenesis for sustaining ISC function and providing a potential perspective to colon cancer therapy.
    DOI:  https://doi.org/10.1038/s41467-022-31725-2
  16. Dev Cell. 2022 Jul 05. pii: S1534-5807(22)00448-8. [Epub ahead of print]
    A ROS-dependent mechanism promotes CDK2 phosphorylation to drive progression through S phase.
    Dilyana Georgieva Kirova, Kristyna Judasova, Julia Vorhauser, Thomas Zerjatke, Jacky Kieran Leung, Ingmar Glauche, Jörg Mansfeld.
      Reactive oxygen species (ROS) at the right concentration promote cell proliferation in cell culture, stem cells, and model organisms. However, the mystery of how ROS signaling is coordinated with cell cycle progression and integrated into the cell cycle control machinery on the molecular level remains unsolved. Here, we report increasing levels of mitochondrial ROS during the cell cycle in human cell lines that target cyclin-dependent kinase 2 (CDK2). Chemical and metabolic interferences with ROS production decrease T-loop phosphorylation on CDK2 and so impede its full activation and thus its efficient DNA replication. ROS regulate CDK2 activity through the oxidation of a conserved cysteine residue near the T-loop, which prevents the binding of the T-loop phosphatase KAP. Together, our data reveal how mitochondrial metabolism is coupled with DNA replication and cell cycle progression via ROS, thereby demonstrating how KAP activity toward CDKs can be cell cycle regulated.
    Keywords:  CDK2; DNA replication; KAP; T-loop phosphorylation; cell cycle; cyclin-dependent kinase; metabolism; mitochondria; proliferation; reactive oxygen species
    DOI:  https://doi.org/10.1016/j.devcel.2022.06.008
  17. Front Genet. 2022 ;13 848926
    KIFC3 Promotes Proliferation, Migration, and Invasion in Colorectal Cancer via PI3K/AKT/mTOR Signaling Pathway.
    Huiling Liao, Lan Zhang, Shimin Lu, Wei Li, Weiguo Dong.
      Background: KIFC3, belongs to kinesin superfamily proteins (KIFs), is well known for its role in intracellular cargo movement. KIFC3 has been identified as a docetaxel resistance gene in breast cancer cells, however, the role of KIFC3 and its potential mechanism in colorectal cancer (CRC) remains elusive. Objectives: We aims to investigate the effects of KIFC3 in proliferation, migration, and invasion in CRC as well as the potential mechanism inside. Methods: We investigated the expression of KIFC3 in the Oncomine, Gene Expression Profiling Interactive Analysis databases. The KIFC3 protein expression and mRNA level in CRC cells were evaluated by western blot and qRT-PCR. Cell proliferation ability was detected by CCK-8, EdU, colony formation assay and xenograft tumor in nude mice. Flow cytometry was used to detect the cell cycle. The effect of KIFC3 on the epithelial-to-mesenchymal transition (EMT) was investigated by transwell and wound healing assay. The association of KIFC3 with EMT and PI3K/AKT/mTOR signaling pathway were measured by western blot and immunofluorescence staining. Results: The expression of KIFC3 was higher in CRC tissues than normal colorectal tissue, and was negatively correlated with the overall survival of patients with CRC. KIFC3 silencing inhibited the proliferation, migration and invasion of CRC cells. Meanwhile, it could decrease the number of cells in S phase. KIFC3 silencing inhibited the expression of proliferating cell nuclear antigen, Cyclin A2, Cyclin E1, and CDK2 and increased the expression of p21 and p53. KIFC3 overexpression promoted the G1/S phase transition. KIFC3 silencing inhibited the EMT process, which decreased the level of N-cadherin, Vimentin, SNAIL 1, TWIST, MMP-2, MMP-9 and increased E-cadherin, while KIFC3 overexpression show the opposite results. Furthermore, the knockdown of KIFC3 suppressed the EMT process by modulating the PI3K/AKT/mTOR signaling pathway. KIFC3 silencing decreased the expression of phosphorylated PI3K, AKT, mTOR, but total PI3K, AKT, mTOR have no change. Inversely, the upregulation of KIFC3 increased the expression of phosphorylated PI3K, AKT and mTOR, total PI3K, AKT, mTOR have no change. In a xenograft mouse model, the depletion of KIFC3 suppressed tumor growth. the increased expression levels of KIFC3 could enhance the proliferation, migration and invasion of CRC cells, and enhance the EMT process through the PI3K/AKT/mTOR pathway. Conclusion: Our study substantiates that KIFC3 can participate in the regulation of CRC progression by which regulates EMT via the PI3K/AKT/mTOR axis.
    Keywords:  KIFC3; PI3K/Akt/mTOR signal pathway; epithelial-to-mesenchymal transition; invasion; migration; proliferation
    DOI:  https://doi.org/10.3389/fgene.2022.848926
  18. Biochim Biophys Acta Mol Cell Res. 2022 Jul 08. pii: S0167-4889(22)00112-4. [Epub ahead of print] 119320
    Nuclear RAC1 is a modulator of the doxorubicin-induced DNA damage response.
    Rebekka Kitzinger, Gerhard Fritz, Christian Henninger.
      Rho GTPases like RAC1 are localized on the inner side of the outer cell membrane where they act as molecular switches that can trigger signal transduction pathways in response to various extracellular stimuli. Nuclear functions of RAC1 were identified that are related to mitosis, cell cycle arrest and apoptosis. Previously, we showed that RAC1 plays a role in the doxorubicin (Dox)-induced DNA damage response (DDR). In this context it is still unknown whether cytosolic RAC1 modulates the Dox-induced DDR or if a nuclear fraction of RAC1 is involved. Here, we silenced RAC1 in mouse embryonic fibroblasts (MEF) pharmacologically with EHT1864 or by using siRNA against Rac1. Additionally, we transfected MEF with RAC1 mutants (wild-type, dominant-negative, constitutively active) containing a nuclear localization sequence (NLS). Afterwards, we analysed the Dox-induced DDR by evaluation of fluorescent nuclear γH2AX and 53BP1 foci formation, as well as by detection of activated proteins of the DDR by western blot to elucidate the role of nuclear RAC1 in the DDR. Treatment with EHT1864 as well as Rac1 knock-down reduced the Dox-induced DSB-formation to a similar extent. Enhanced nuclear localization of dominant-negative as well as constitutively active RAC1 mimicked these effects. Expression of the RAC1 mutants altered the Dox-induced amount of pP53 and pKAP1 protein. The observed effects were independent of S1981 ATM phosphorylation. We conclude that RAC1 is required for a substantial activation of the Dox-induced DDR and balanced levels of active/inactive RAC1 inside the nucleus are a prerequisite for this response.
    Keywords:  DNA double-strand breaks; Doxorubicin; Genotoxicity; Nucleus; RAC1; Small GTPases
    DOI:  https://doi.org/10.1016/j.bbamcr.2022.119320
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