bims-instec Biomed News
on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration
Issue of 2022–01–23
eleven papers selected by
Maria-Virginia Giolito, IRFAC/UMR-S1113 INSERM
  1. Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer.
  2. CDX2 controls genes involved in the metabolism of 5-fluorouracil and is associated with reduced efficacy of chemotherapy in colorectal cancer.
  3. An in vitro chronic damage model impairs inflammatory and regenerative responses in human colonoid monolayers.
  4. Suppressive GLI2 fragment enhances liver metastasis in colorectal cancer.
  5. Inhibition of p38 MAPK increases the sensitivity of 5-fluorouracil-resistant SW480 human colon cancer cells to noscapine.
  6. MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma.
  7. Oncogenic potential of BEST4 in colorectal cancer via activation of PI3K/Akt signaling.
  8. Epigenetic modifier balances Mapk and Wnt signalling in differentiation of goblet and Paneth cells.
  9. Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.
  10. Normal and tumor-derived organoids as a drug screening platform for tumor-specific drug vulnerabilities.
  11. Loss of circadian gene Timeless induces EMT and tumor progression in colorectal cancer via Zeb1-dependent mechanism.
  1. J Pathol. 2022 Jan 17.
    Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer.
    Daisuke Yamamoto, Hiroko Oshima, Dong Wang, Haruna Takeda, Kenji Kita, Xuelian Lei, Mizuho Nakayama, Kazuhiro Murakami, Takashi Ohama, Hirofumi Takemura, Mutsumi Toyota, Hiromu Suzuki, Noriyuki Inaki, Masanobu Oshima.
      Loss-of-function mutations in RNF43 induce activation of Wnt ligand-dependent Wnt/β-catenin signaling through stabilization of the Frizzled receptor, which is often found in microsatellite instability (MSI)-type colorectal cancer (CRC) that develops from sessile serrated adenomas. However, the mechanism underlying how RNF43 mutations promote tumorigenesis remains poorly understood. In this study, we established nine human CRC-derived organoids and found that three organoid lines carried RNF43 frameshift mutations associated with MSI-high and BRAFV600E mutations, suggesting that these CRCs developed through the serrated pathway. RNF43 frameshift mutant organoids required both Wnt ligands and R-spondin for proliferation, indicating that suppression of ZNRF3 and retained RNF43 function by R-spondin are required to achieve an indispensable level of Wnt activation for tumorigenesis. However, active β-catenin levels in RNF43 mutant organoids were lower than those in APC two-hit mutant CRC, suggesting a lower threshold for Wnt activation in CRC that developed through the serrated pathway. Interestingly, transplantation of RNF43 mutant organoids with intestinal myofibroblasts accelerated the β-catenin nuclear accumulation and proliferation of xenograft tumors, indicating a key role of stromal cells in the promotion of the malignant phenotype of RNF43 mutant CRC cells. Sequencing of subcloned organoid cell-expressed transcripts revealed that two organoid lines carried monoallelic RNF43 cis-mutations, with two RNF43 frameshift mutations introduced in the same allele and the wild-type RNF43 allele remaining, while the other organoid line carried two-hit biallelic RNF43 trans-mutations. These results suggest that heterozygous RNF43 frameshift mutations contribute to CRC development via the serrated pathway; however, a second-hit RNF43 mutation may be advantageous in tumorigenesis compared with a single-hit mutation through further activation of Wnt signaling. Finally, treatment with the PORCN inhibitor significantly suppressed RNF43 mutant cell-derived PDX tumor development. These results suggest a novel mechanism underlying RNF43 mutation-associated CRC development and the therapeutic potential of Wnt ligand inhibition against RNF43 mutant CRC. This article is protected by copyright. All rights reserved.
    Keywords:  POCRN inhibitor; RNF43; Wnt ligand; colorectal cancer; organoid; serrated pathway
    DOI:  https://doi.org/10.1002/path.5868
  2. Biomed Pharmacother. 2022 Jan 17. pii: S0753-3322(22)00018-X. [Epub ahead of print]147 112630
    CDX2 controls genes involved in the metabolism of 5-fluorouracil and is associated with reduced efficacy of chemotherapy in colorectal cancer.
    Jean-Baptiste Delhorme, Emilie Bersuder, Chloé Terciolo, Ourania Vlami, Marie-Pierrette Chenard, Elisabeth Martin, Serge Rohr, Cécile Brigand, Isabelle Duluc, Jean-Noël Freund, Isabelle Gross.
      Most patients affected with colorectal cancers (CRC) are treated with 5-fluorouracil (5-FU)-based chemotherapy but its efficacy is often hampered by resistance mechanisms linked to tumor heterogeneity. A better understanding of the molecular determinants involved in chemoresistance is critical for precision medicine and therapeutic progress. Caudal type homeobox 2 (CDX2) is a master regulator of intestinal identity and acts as tumor suppressor in the colon. Here, using a translational approach, we examined the role of CDX2 in CRC chemoresistance. Unexpectedly, we discovered that the prognosis value of CDX2 for disease-free survival of patients affected with CRC is lost upon chemotherapy and that CDX2 expression enhances resistance of colon cancer cells towards 5-FU. At the molecular level, we found that CDX2 expression correlates with higher levels of genes regulating the bioavailability of 5-FU through efflux (ABCC11) and catabolism (DPYD) in patients affected with CRC and CRC cell lines. We further showed that CDX2 directly regulates the expression of ABCC11 and that the inhibition of ABCC11 improves 5-FU-sensitivity of CDX2-expressing colon cancer cells. Thus, this study illustrates how biological functions are hijacked in CRC cells and reveals the therapeutic interest of CDX2/ABCC11/DPYD to improve systemic chemotherapy in CRC.
    Keywords:  ABCC11; Biomarker; Chemoresistance; DPD; Heterogeneity
    DOI:  https://doi.org/10.1016/j.biopha.2022.112630
  3. Cell Rep. 2022 Jan 18. pii: S2211-1247(21)01798-8. [Epub ahead of print]38(3): 110283
    An in vitro chronic damage model impairs inflammatory and regenerative responses in human colonoid monolayers.
    William D Rees, Nikita Telkar, David T S Lin, May Q Wong, Chad Poloni, Ayda Fathi, Michael Kobor, Nicholas C Zachos, Theodore S Steiner.
      Acute damage to the intestinal epithelium can be repaired via de-differentiation of mature intestinal epithelial cells (IECs) to a stem-like state, but there is a lack of knowledge on how intestinal stem cells function after chronic injury, such as in inflammatory bowel disease (IBD). We developed a chronic-injury model in human colonoid monolayers by repeated rounds of air-liquid interface and submerged culture. We use this model to understand how chronic intestinal damage affects the ability of IECs to (1) respond to microbial stimulation, using the Toll-like receptor 5 (TLR5) agonist FliC and (2) regenerate and protect the epithelium from further damage. Repeated rounds of damage impair the ability of IECs to regrow and respond to TLR stimulation. We also identify mRNA expression and DNA methylation changes in genes associated with IBD and colon cancer. This methodology results in a human model of recurrent IEC injury like that which occurs in IBD.
    Keywords:  ER stress; air-liquid interface; chronic damage; epigenetics; flagellin; inflammatory bowel diseases; intestinal stem cells; organoids; regeneration
    DOI:  https://doi.org/10.1016/j.celrep.2021.110283
  4. Oncotarget. 2022 ;13 122-135
    Suppressive GLI2 fragment enhances liver metastasis in colorectal cancer.
    Ruiko Ogata, Shiori Mori, Hitoshi Ohmori, Shingo Kishi, Rina Fujiwara-Tani, Takamitsu Sasaki, Yukiko Nishiguchi, Chie Nakashima, Kei Goto, Isao Kawahara, Yi Luo, Hiroki Kuniyasu.
      Linoleic acid (LA) has been shown to cause inflammation and promote development of colorectal cancer (CRC). Moreover, many literatures show that LA is associated with cancer metastasis. Metastatic cancer cells have high stemness, suggesting that LA might affect the stemness of cancer cells. In this study, we examined the effect of LA on the hedgehog system, which affects cancer stemness. In CT26 cells, LA treatment induced the expression of sonic hedgehog (Shh); the signal transduction factor, and glioma-associated oncogene homolog (Gli) 2, whereas the expression of SRY-box transcription factor (Sox) 17 was suppressed. Furthermore, LA reduced GLI2 ubiquitination, resulting in an increase in the N-terminal fragment of GLI2, known as suppressive GLI2, produced by cleavage of GLI2. LA-induced cleaved GLI2 was also detected in Colo320 and HT29 human CRC cells. Knocking down Gli2 abrogated the LA-mediated suppression of Sox17 expression. These results suggest that LA promotes tumor cell stemness by increasing of suppressive GLI2 fragments via GLI2 modification. In mouse liver metastasis models, LA enhanced metastasis with production of the suppressive GLI2 fragments in CT26 and HT29 cells, whereas knockdown of GLI2 abrogated LA-induced metastatic activity. In human CRCs, the cases with liver metastasis showed the suppressive GLI2 fragments. This study provides mechanistic insights into LA-induced stemness in colon cancer cells. This finding suggests that dietary intake of LA might increase the stemness of cancer cells and enhance metastatic activity of the cancer.
    Keywords:  GLI2; colorectal cancer; linoleic acid; stemness; ubiquitination
    DOI:  https://doi.org/10.18632/oncotarget.28170
  5. Oncol Lett. 2022 Feb;23(2): 52
    Inhibition of p38 MAPK increases the sensitivity of 5-fluorouracil-resistant SW480 human colon cancer cells to noscapine.
    Zheng Han, Liu Meng, Xiaodong Huang, Jie Tan, Weijie Liu, Wei Chen, Yanli Zou, Yishan Cai, Shasha Huang, Aifang Chen, Ting Zhan, Min Huang, Xiaoli Chen, Xia Tian, Qingxi Zhu.
      A major cause of treatment failure in advanced colon cancer is resistance to chemotherapy. p38 mitogen-activated protein kinase (MAPK) has been associated with cellular apoptosis and plays an important role in multidrug resistance (MDR) in cancer cells. In the present study the effect of p38 MAPK on the sensitivity of 5-fluorouracil (5-FU)-resistant SW480 (SW480/5-FU) human colon cancer cells to noscapine was investigated. Following p38 MAPK interference, the inhibitory effect of noscapine on cell viability and proliferation was increased in the SW480/5-FU cells and there was also a decrease in the expression level of minichromosome maintenance proteins, recombinant Ki-67 and proliferating cell nuclear antigen. Inhibition of p38 MAPK also enhanced noscapine-induced G1-phase cell cycle arrest in the SW480/5-FU cells and there was also a decrease in the protein and mRNA expression level of cyclin D, cyclin E and cyclin-dependent kinase 2, and an increase in the expression level of P57. Furthermore, p38 MAPK interference increased noscapine-induced apoptosis of the SW480/5-FU cells and there was an increase in the protein and mRNA expression level of caspases-3 and 8 and Bax, and decreased Bcl-2 expression level. The sensitivity of the SW480/5-FU cells to noscapine was also increased following p38 MAPK interference, as demonstrated by MDR inhibition via decreased Akt activity and reduced protein expression level of the MDR proteins P-glycoprotein, multidrug resistance protein 1 and ATP-binding cassette G2. These observations indicated that inhibition of p38 MAPK increased the sensitivity of the SW480/5-FU cells to noscapine by suppressing proliferation, induction of cell cycle arrest and apoptosis, and reversal of MDR in the SW480/5-FU cells.
    Keywords:  apoptosis; colon cancer; drug resistance; noscapine; p38 MAPK
    DOI:  https://doi.org/10.3892/ol.2021.13170
  6. Cell Death Dis. 2022 Jan 18. 13(1): 63
    MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma.
    Dechen Fu, Luke Pfannenstiel, Abeba Demelash, Yee Peng Phoon, Cameron Mayell, Claudia Cabrera, Caini Liu, Junjie Zhao, Josephine Dermawan, Deepa Patil, Jennifer DeVecchio, Matthew Kalady, Andrew J Souers, Darren C Phillips, Xiaoxia Li, Brian Gastman.
      Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein-protein interactions are critical in MCL1's nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53-/- CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53-/- CRC via exploitation of this unique MCL1-based chemoresistance mechanism.
    DOI:  https://doi.org/10.1038/s41419-021-04334-y
  7. Oncogene. 2022 Jan 21.
    Oncogenic potential of BEST4 in colorectal cancer via activation of PI3K/Akt signaling.
    Xiao-Shun He, Wen-Long Ye, Yu-Juan Zhang, Xiao-Qin Yang, Feng Liu, Jing-Ru Wang, Xiao-Lu Ding, Yun Yang, Ruo-Nan Zhang, Yuan-Yuan Zhao, Hai-Xia Bi, Ling-Chuan Guo, Wen-Juan Gan, Hua Wu.
      BEST4 is a member of the bestrophin protein family that plays a critical role in human intestinal epithelial cells. However, its role and mechanism in colorectal cancer (CRC) remain largely elusive. Here, we investigated the role and clinical significance of BEST4 in CRC. Our results demonstrate that BEST4 expression is upregulated in clinical CRC samples and its high-level expression correlates with advanced TNM (tumor, lymph nodes, distant metastasis) stage, LNM (lymph node metastasis), and poor survival. Functional studies revealed that ectopic expression of BEST4 promoted CRC cell proliferation and metastasis, whereas the depletion of BEST4 had the opposite effect both in vitro and in vivo. Mechanistically, BEST4 binds to the p85α regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and promotes p110 kinase activity; this leads to activation of Akt signaling and expression of MYC and CCND1, which are critical regulators of cell proliferation and metastasis. In clinical samples, the expression of BEST4 is positively associated with the expression of phosphorylated Akt, MYC and CCND1. Pharmacological inhibition of Akt activity markedly repressed BEST4-mediated Akt signaling and proliferation and metastasis of CRC cells. Importantly, the interaction between BEST4 and p85α was also enhanced by epidermal growth factor (EGF) in CRC cells. Therapeutically, BEST4 suppression effectively sensitized CRC cells to gefitinib treatment in vivo. Taken together, our findings indicate the oncogenic potential of BEST4 in colorectal carcinogenesis and metastasis by modulating BEST4/PI3K/Akt signaling, highlighting a potential strategy for CRC therapy.
    DOI:  https://doi.org/10.1038/s41388-021-02160-2
  8. Life Sci Alliance. 2022 Apr;pii: e202101187. [Epub ahead of print]5(4):
    Epigenetic modifier balances Mapk and Wnt signalling in differentiation of goblet and Paneth cells.
    Johanna Grinat, Frauke Kosel, Neha Goveas, Andrea Kranz, Dimitra Alexopoulou, Klaus Rajewsky, Michael Sigal, A Francis Stewart, Julian Heuberger.
      Differentiation and lineage specification are controlled by cooperation of growth factor signalling. The involvement of epigenetic regulators in lineage specification remains largely elusive. Here, we show that the histone methyltransferase Mll1 prevents intestinal progenitor cells from differentiation, whereas it is also involved in secretory lineage specification of Paneth and goblet cells. Using conditional mutagenesis in mice and intestinal organoids, we demonstrate that loss of Mll1 renders intestinal progenitor cells permissive for Wnt-driven secretory differentiation. However, Mll1-deficient crypt cells fail to segregate Paneth and goblet cell fates. Mll1 deficiency causes Paneth cell-determined crypt progenitors to exhibit goblet cell features by unleashing Mapk signalling, resulting in increased numbers of mixed Paneth/goblet cells. We show that loss of Mll1 abolishes the pro-proliferative effect of Mapk signalling in intestinal progenitor cells and promotes Mapk-induced goblet cell differentiation. Our data uncover Mll1 and its downstream targets Gata4/6 as a regulatory hub of Wnt and Mapk signalling in the control of lineage specification of intestinal secretory Paneth and goblet cells.
    DOI:  https://doi.org/10.26508/lsa.202101187
  9. Int J Oncol. 2022 Mar;pii: 22. [Epub ahead of print]60(3):
    Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.
    Maha Abdulla, Thamer Bin Traiki, Mansoor-Ali Vaali-Mohammed, Mohammad S El-Wetidy, Noura Alhassan, Khayal Al-Khayal, Ahmed Zubaidi, Omar Al-Obeed, Rehan Ahmad.
      With >1.85 million cases and 850,000 deaths annually, colorectal cancer (CRC) is the third most common cancer detected globally. CRC is an aggressive malignancy with metastasis and, in spite of advances in improved treatment regimen, distant disease failure rates remain disappointingly high. Mucin‑like 1 (MUCL1) is a small glycoprotein highly expressed mainly in breast cancer. The involvement of the MUCL1 protein in CRC progression and the underlying mechanism have been largely unknown. The aim of the present study was to investigate the MUCL1 expression profile and its functional significance in CRC. The Cancer Genome Atlas dataset revealed that MUCL1 expression was higher in colorectal tumor compared with normal tissues. MUCL1 was also revealed to be expressed in human CRC cell lines. The results demonstrated that MUCL1 promoted cell proliferation and colony formation, confirming its oncogenic potential. Silencing MUCL1 with short interfering RNA inhibited the protein expression of Bcl2 family proteins, such as Bcl2 and BclxL. Targeting MUCL1 resulted in significant inhibition in cell invasive and migratory behavior of HT‑29 and SW620 cells. In addition, the expression of E‑cadherin increased whereas the expression of vimentin decreased in MUCL1‑silenced cells, confirming inhibition of epithelial‑mesenchymal transition (EMT) process. Thus, it was revealed that MUCL1 plays a notable role in cell invasion and migration by inhibiting EMT in CRC. Mechanistically, MUCL1 drives β‑catenin activation by Ser‑552 phosphorylation, nuclear accumulation and transcriptional activation. Targeting MUCL1 increases the drug sensitivity of CRC cells towards irinotecan. These findings thus demonstrated that MUCL1 acts as a modifier of other pathways that play an important role in CRC progression and MUCL1 was identified as a potential target for CRC therapeutics.
    Keywords:  colorectal cancer; epithelial‑mesenchymal transition; invasion; irinotecan; migration; mucin‑like 1
    DOI:  https://doi.org/10.3892/ijo.2022.5312
  10. STAR Protoc. 2022 Mar 18. 3(1): 101079
    Normal and tumor-derived organoids as a drug screening platform for tumor-specific drug vulnerabilities.
    Camilla Calandrini, Jarno Drost.
      Patient-derived tumor organoids can be predictive of patient's treatment responses, and normal tissue-derived organoids allow for drug toxicity testing. Combining both types of organoids therefore enables screening for tumor-specific drug vulnerabilities. Here, we provide a detailed protocol for organoid drug screening using, as proof-of-principle, patient-derived malignant rhabdoid tumor organoids. The protocol can be adapted for drug testing on any tumor and/or normal tissue-derived organoid culture. For complete details on the use and execution of this protocol, please refer to Calandrini et al. (2021).
    Keywords:  Cancer; Cell Biology; Health Sciences; High Throughput Screening; Organoids; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2021.101079
  11. Cell Death Differ. 2022 Jan 15.
    Loss of circadian gene Timeless induces EMT and tumor progression in colorectal cancer via Zeb1-dependent mechanism.
    Tommaso Colangelo, Annalucia Carbone, Francesco Mazzarelli, Roberto Cuttano, Elisa Dama, Teresa Nittoli, Jacopo Albanesi, Giovannina Barisciano, Nicola Forte, Orazio Palumbo, Paolo Graziano, Alessandra di Masi, Vittorio Colantuoni, Lina Sabatino, Fabrizio Bianchi, Gianluigi Mazzoccoli.
      The circadian gene Timeless (TIM) provides a molecular bridge between circadian and cell cycle/DNA replication regulatory systems and has been recently involved in human cancer development and progression. However, its functional role in colorectal cancer (CRC), the third leading cause of cancer-related deaths worldwide, has not been fully clarified yet. Here, the analysis of two independent CRC patient cohorts (total 1159 samples) reveals that loss of TIM expression is an unfavorable prognostic factor significantly correlated with advanced tumor stage, metastatic spreading, and microsatellite stability status. Genome-wide expression profiling, in vitro and in vivo experiments, revealed that TIM knockdown induces the activation of the epithelial-to-mesenchymal transition (EMT) program. Accordingly, the analysis of a large set of human samples showed that TIM expression inversely correlated with a previously established gene signature of canonical EMT markers (EMT score), and its ectopic silencing promotes migration, invasion, and acquisition of stem-like phenotype in CRC cells. Mechanistically, we found that loss of TIM expression unleashes ZEB1 expression that in turn drives the EMT program and enhances the aggressive behavior of CRC cells. Besides, the deranged TIM-ZEB1 axis sets off the accumulation of DNA damage and delays DNA damage recovery. Furthermore, we show that the aggressive and genetically unstable 'CMS4 colorectal cancer molecular subtype' is characterized by a lower expression of TIM and that patients with the combination of low-TIM/high-ZEB1 expression have a poorer outcome. In conclusion, our results as a whole suggest the engagement of an unedited TIM-ZEB1 axis in key pathological processes driving malignant phenotype acquisition in colorectal carcinogenesis. Thus, TIM-ZEB1 expression profiling could provide a robust prognostic biomarker in CRC patients, supporting targeted therapeutic strategies with better treatment selection and patients' outcomes.
    DOI:  https://doi.org/10.1038/s41418-022-00935-y
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