bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2022‒03‒06
eight papers selected by
Lucas B. Zeiger
Beatson Institute for Cancer Research
  1. Same-Cell Co-Occurrence of RAS Hotspot and BRAF V600E Mutations in Treatment-Naive Colorectal Cancer.
  2. Reconstruction and analysis of a large-scale binary Ras-effector signaling network.
  3. Understanding and drugging RAS: 40 years to break the tip of the iceberg.
  4. Silent mutations reveal therapeutic vulnerability in RAS Q61 cancers.
  5. Generation of Highly Expandable Intestinal Spheroids Composed of Stem Cells.
  6. The MYC oncoprotein directly interacts with its chromatin cofactor PNUTS to recruit PP1 phosphatase.
  7. Metformin suppresses the growth of colorectal cancer by targeting INHBA to inhibit TGF-β/PI3K/AKT signaling transduction.
  8. BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states.
  1. JCO Precis Oncol. 2022 Mar;6 e2100365
    Same-Cell Co-Occurrence of RAS Hotspot and BRAF V600E Mutations in Treatment-Naive Colorectal Cancer.
    Rodrigo Gularte-Mérida, Shaleigh Smith, Anita S Bowman, Arnaud da Cruz Paula, Walid Chatila, Craig M Bielski, Monika Vyas, Laetitia Borsu, Ahmet Zehir, Luciano G Martelotto, Jinru Shia, Rona Yaeger, Fang Fang, Rui Gardner, Ruibang Luo, Michael C Schatz, Ronglai Shen, Britta Weigelt, Francisco Sánchez-Vega, Jorge S Reis-Filho, Jaclyn F Hechtman.
      PURPOSE: Mitogen-activated protein kinase pathway-activating mutations occur in the majority of colorectal cancer (CRC) cases and show mutual exclusivity. We identified 47 epidermal growth factor receptor/BRAF inhibitor-naive CRC patients with dual RAS hotspot/BRAF V600E mutations (CRC-DD) from a cohort of 4,561 CRC patients with clinical next-generation sequencing results. We aimed to define the molecular phenotypes of the CRC-DD and to test if the dual RAS hotspot/BRAF V600E mutations coexist within the same cell.MATERIALS AND METHODS: We developed a single-cell genotyping method with a mutation detection rate of 96.3% and a genotype prediction accuracy of 92.1%. Mutations in the CRC-DD cohort were analyzed for clonality, allelic imbalance, copy number, and overall survival.
    RESULTS: Application of single-cell genotyping to four CRC-DD revealed the co-occurrence of both mutations in the following percentages of cells per case: NRAS G13D/KRAS G12C, 95%; KRAS G12D/NRAS G12V, 48%; BRAF V600E/KRAS G12D, 44%; and KRAS G12D/NRAS G13V, 14%, respectively. Allelic imbalance favoring the oncogenic allele was less frequent in CRC-DD (24 of 76, 31.5%, somatic mutations) compared with a curated cohort of CRC with a single-driver mutation (CRC-SD; 119 of 232 mutations, 51.3%; P = .013). Microsatellite instability-high status was enriched in CRC-DD compared with CRC-SD (23% v 11.4%, P = .028). Of the seven CRC-DD cases with multiregional sequencing, five retained both driver mutations throughout all sequenced tumor sites. Both CRC-DD cases with discordant multiregional sequencing were microsatellite instability-high.
    CONCLUSION: Our findings indicate that dual-driver mutations occur in a rare subset of CRC, often within the same tumor cells and across multiple tumor sites. Their presence and a lower rate of allelic imbalance may be related to dose-dependent signaling within the mitogen-activated protein kinase pathway.
    DOI:  https://doi.org/10.1200/PO.21.00365
  2. Cell Commun Signal. 2022 Mar 04. 20(1): 24
    Reconstruction and analysis of a large-scale binary Ras-effector signaling network.
    Simona Catozzi, Camille Ternet, Alize Gourrege, Kieran Wynne, Giorgio Oliviero, Christina Kiel.
      BACKGROUND: Ras is a key cellular signaling hub that controls numerous cell fates via multiple downstream effector pathways. While pathways downstream of effectors such as Raf, PI3K and RalGDS are extensively described in the literature, how other effectors signal downstream of Ras is often still enigmatic.METHODS: A comprehensive and unbiased Ras-effector network was reconstructed downstream of 43 effector proteins (converging onto 12 effector classes) using public pathway and protein-protein interaction (PPI) databases. The output is an oriented graph of pairwise interactions defining a 3-layer signaling network downstream of Ras. The 2290 proteins comprising the network were studied for their implication in signaling crosstalk and feedbacks, their subcellular localizations, and their cellular functions.
    RESULTS: The final Ras-effector network consists of 2290 proteins that are connected via 19,080 binary PPIs, increasingly distributed across the downstream layers, with 441 PPIs in layer 1, 1660 in layer 2, and 16,979 in layer 3. We identified a high level of crosstalk among proteins of the 12 effector classes. A class-specific Ras sub-network was generated in CellDesigner (.xml file) and a functional enrichment analysis thereof shows that 58% of the processes have previously been associated to a respective effector pathway, with the remaining providing insights into novel and unexplored functions of specific effector pathways.
    CONCLUSIONS: Our large-scale and cell general Ras-effector network is a crucial steppingstone towards defining the network boundaries. It constitutes a 'reference interactome' and can be contextualized for specific conditions, e.g. different cell types or biopsy material obtained from cancer patients. Further, it can serve as a basis for elucidating systems properties, such as input-output relationships, crosstalk, and pathway redundancy. Video Abstract.
    Keywords:  Crosstalk; Effectors; Feedbacks; Network hubs; Pathway reconstruction; Ras; Signaling pathways
    DOI:  https://doi.org/10.1186/s12964-022-00823-5
  3. Dis Model Mech. 2022 Feb 01. pii: dmm049519. [Epub ahead of print]15(2):
    Understanding and drugging RAS: 40 years to break the tip of the iceberg.
    Donita C Brady, Julija Hmeljak, Arvin C Dar.
      Several cancers and rare genetic diseases are caused by dysregulation in the RAS signaling pathway. RAS proteins serve as molecular switches that regulate pathways involved in cellular growth, differentiation and survival. These pathways have been an intense area of investigation for four decades, since the initial identification of somatic RAS mutations linked to human cancers. In the past few years, inhibitors against several RAS effectors, as well as direct inhibitors of the K-RAS mutant G12C, have been developed. This Special Issue in DMM includes original Research articles on RAS-driven cancers and RASopathies. The articles provide insights into mechanisms and biomarkers, and evaluate therapeutic targets. Several articles also present new disease models, whereas others describe technologies or approaches to evaluate the function of RAS in vivo. The collection also includes a series of Review articles on RAS biology and translational aspects of defining and treating RAS-driven diseases. In this Editorial, we summarize this collection and discuss the potential impact of the articles within this evolving area of research. We also identify areas of growth and possible future developments.
    Keywords:  Cancer; Developmental disorders; RAS inhibitor; RAS pathway
    DOI:  https://doi.org/10.1242/dmm.049519
  4. Nature. 2022 Mar 02.
    Silent mutations reveal therapeutic vulnerability in RAS Q61 cancers.
    Yoshihisa Kobayashi, Chhayheng Chhoeu, Jiaqi Li, Kristin S Price, Lesli A Kiedrowski, Jamie L Hutchins, Aaron I Hardin, Zihan Wei, Fangxin Hong, Magda Bahcall, Prafulla C Gokhale, Pasi A Jänne.
      RAS family members are the most frequently mutated oncogenes in human cancers. Although KRAS(G12C)-specific inhibitors show clinical activity in patients with cancer1-3, there are no direct inhibitors of NRAS, HRAS or non-G12C KRAS variants. Here we uncover the requirement of the silent KRASG60G mutation for cells to produce a functional KRAS(Q61K). In the absence of this G60G mutation in KRASQ61K, a cryptic splice donor site is formed, promoting alternative splicing and premature protein termination. A G60G silent mutation eliminates the splice donor site, yielding a functional KRAS(Q61K) variant. We detected a concordance of KRASQ61K and a G60G/A59A silent mutation in three independent pan-cancer cohorts. The region around RAS Q61 is enriched in exonic splicing enhancer (ESE) motifs and we designed mutant-specific oligonucleotides to interfere with ESE-mediated splicing, rendering the RAS(Q61) protein non-functional in a mutant-selective manner. The induction of aberrant splicing by antisense oligonucleotides demonstrated therapeutic effects in vitro and in vivo. By studying the splicing necessary for a functional KRAS(Q61K), we uncover a mutant-selective treatment strategy for RASQ61 cancer and expose a mutant-specific vulnerability, which could potentially be exploited for therapy in other genetic contexts.
    DOI:  https://doi.org/10.1038/s41586-022-04451-4
  5. Int J Stem Cells. 2022 Feb 28. 15(1): 104-111
    Generation of Highly Expandable Intestinal Spheroids Composed of Stem Cells.
    Ohman Kwon, Won Dong Yu, Ye Seul Son, Kwang Bo Jung, Hana Lee, Mi-Young Son.
      Many of early findings regarding intestinal stem cells (ISCs) and their niche in the human intestine have relied on colorectal cancer cell lines and labor-intensive and time-consuming mouse models. However, these models cannot accurately recapitulate the physiologically relevant aspects of human ISCs. In this study, we demonstrate a reliable and robust culture method for 3D expanding intestinal spheroids (InSexp) mainly comprising ISCs and progenitors, which can be derived from 3D human intestinal organoids (HIOs). We did functional chararcterization of InSexp derived from 3D HIOs, differentiated from human pluripotent stem cells, and optimization culture methods. Our results indicate that InSexp can be rapidly expanded and easily passaged, and show enhanced growth rates via WNT pathway activation. InSexp are capable of exponential cell expansion and cryopreservation. Furthermore, in vitro-matured HIO-derived InSexp proliferate faster than immature HIO-derived InSexp with preservation of the parental HIO characteristics. These findings may facilitate the development of scalable culture systems for the long-term maintenance of human ISCs and provide an alternative platform for studying ISC biology.
    Keywords:  Expandable Intestinal spheroid (InSexp); Human intestinal organoid (HIO); Intestinal stem cell; Maturity memory; WNT pathway
    DOI:  https://doi.org/10.15283/ijsc21209
  6. Nucleic Acids Res. 2022 Mar 04. pii: gkac138. [Epub ahead of print]
    The MYC oncoprotein directly interacts with its chromatin cofactor PNUTS to recruit PP1 phosphatase.
    Yong Wei, Cornelia Redel, Alexandra Ahlner, Alexander Lemak, Isak Johansson-Åkhe, Scott Houliston, Tristan M G Kenney, Aaliya Tamachi, Vivian Morad, Shili Duan, David W Andrews, Björn Wallner, Maria Sunnerhagen, Cheryl H Arrowsmith, Linda Z Penn.
      Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remain an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation, chromatin occupancy, and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. By NMR we identified a distinct peptide motif within MB0 that interacts with PNUTS residues 1-148, a functional unit, here termed PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its MYC-binding patch. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo, leading to elevated MYC phosphorylation. These data demonstrate that the MB0 region of MYC directly interacts with the PAD of PNUTS, which provides new insight into the control mechanisms of MYC as a regulator of gene transcription and a pervasive cancer driver.
    DOI:  https://doi.org/10.1093/nar/gkac138
  7. Cell Death Dis. 2022 Mar 02. 13(3): 202
    Metformin suppresses the growth of colorectal cancer by targeting INHBA to inhibit TGF-β/PI3K/AKT signaling transduction.
    Qing Xiao, Jiani Xiao, Jiaqi Liu, Jiaxin Liu, Guang Shu, Gang Yin.
      Multiple evidence shows that metformin serves as a potential agent for Colorectal Cancer (CRC) treatment, while its molecular mechanisms still require detailed investigation. Here, we revealed that metformin specifically suppressed the proliferation of CRC cells by causing G1/S arrest, and INHBA is a potential target for metformin to play an anti-proliferation effect in CRC. We verified the oncogene role of INHBA by knocking down and overexpressing INHBA in CRC cells. Silencing INHBA abrogated the cell growth, while overexpression INHBA promotes the proliferation of CRC cells. As an oncogene, INHBA was aberrant overexpression in CRC tissues and closely related to the poor prognosis of CRC patients. In mechanism, INHBA is an important ligand of TGF-β signaling and metformin blocked the activation of TGF-β signaling by targeting INHBA, and then down-regulated the activity of PI3K/Akt pathway, leading to the reduction of cyclinD1 and cell cycle arrest. Together, these findings indicate that metformin down-regulates the expression of INHBA, then attenuating TGF-β/PI3K/Akt signaling transduction, thus inhibiting the proliferation of CRC. Our study elucidated a novel molecular mechanism for the anti-proliferation effect of metformin, providing a theoretical basis for the application of metformin in CRC therapy.
    DOI:  https://doi.org/10.1038/s41419-022-04649-4
  8. Cell Rep. 2022 Mar 01. pii: S2211-1247(22)00165-6. [Epub ahead of print]38(9): 110438
    BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states.
    Joep Beumer, Jens Puschhof, Fjodor Yousef Yengej, Lianzheng Zhao, Adriana Martinez-Silgado, Marloes Blotenburg, Harry Begthel, Charelle Boot, Alexander van Oudenaarden, Ye-Guang Chen, Hans Clevers.
      Intestinal epithelial cells derive from stem cells at the crypt base and travel along the crypt-villus axis to die at the villus tip. The two dominant villus epithelial cell types, absorptive enterocytes and mucous-secreting goblet cells, are mature when they exit crypts. Murine enterocytes switch functional cell states during migration along the villus. Here, we ask whether this zonation is driven by the bone morphogenetic protein (BMP) gradient, which increases toward the villus. Using human intestinal organoids, we show that BMP signaling controls the expression of zonated genes in enterocytes. We find that goblet cells display similar zonation involving antimicrobial genes. Using an inducible Bmpr1a knockout mouse model, we confirm that BMP controls these zonated genes in vivo. Our findings imply that local manipulation of BMP signal strength may be used to reset the enterocyte "rheostat" of carbohydrate versus lipid uptake and to control the antimicrobial response through goblet cells.
    Keywords:  BMP signaling; CRISPR-Cas9; enterocytes; intestinal differentiation; organoids; single-cell RNA sequencing
    DOI:  https://doi.org/10.1016/j.celrep.2022.110438
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