bims-instec 2023-08-27 papers

on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration

Issue of 2023‒08‒27
thirteen papers selected by
Maria-Virginia Giolito, Université Catholique de Louvain, Free University of Brussels, IRFAC/UMR-S1113 INSERM

Matrix stiffness triggers lipid metabolic crosstalk between tumor and stromal cells to mediate bevacizumab resistance in colorectal cancer liver metastases.
Compartment specific responses to contractility in the small intestinal epithelium.
OTULIN protects the intestinal epithelium from apoptosis during inflammation and infection.
Adrenergic nerves regulate intestinal regeneration through IL-22 signaling from type 3 innate lymphoid cells.
Lasting response by vertical inhibition with cetuximab and trametinib in KRAS-mutated colorectal cancer patient-derived xenografts.
Deciphering colorectal cancer progression features and prognostic signature by single-cell RNA sequencing pseudotime trajectory analysis.
Protein post-translational modifications: A key factor in colorectal cancer resistance mechanisms.
Mutant PIK3CA as a negative predictive biomarker for treatment with a highly selective PIM1 inhibitor in human colon cancer.
Hypoxia-induced cancer cell reprogramming: a review on how cancer stem cells arise.
SIRT1 controls enteroendocrine progenitor cell proliferation in high-fat diet-fed mice.
Paired microbiome and metabolome analyses associate bile acid changes with colorectal cancer progression.
Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression.
CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.

Cancer Res. 2023 Aug 23. pii: CAN-23-0025. [Epub ahead of print]

Matrix stiffness triggers lipid metabolic crosstalk between tumor and stromal cells to mediate bevacizumab resistance in colorectal cancer liver metastases.

Yannan Zheng, Rui Zhou, Jianan Cai, Nanyan Yang, Zhaowei Wen, Zhihua Zhang, Huiying Sun, Genjie Huang, Yijin Guan, Na Huang, Min Shi, Yulin Liao, Jianping Bin, Wangjun Liao.

Bevacizumab is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody that plays an important role in the combination treatment of advanced colorectal cancer (CRC). However, resistance remains a major hurdle limiting bevacizumab efficacy, highlighting the importance of identifying mechanism of anti-angiogenic therapy resistance. Here, we investigated biophysical properties of the extracellular matrix (ECM) related to metabolic processes and acquired resistance to bevacizumab. Evaluation of paired pre- and post-treatment samples of liver metastases from 20 CRC patients treated with combination bevacizumab therapy, including 10 responders and 10 non-responders, indicated that ECM deposition in liver metastases and a highly activated fatty acid oxidation (FAO) pathway were elevated in non-responders after anti-angiogenic therapy compared to responders. In mouse models of liver metastatic CRC, anti-VEGF increased ECM deposition and FAO in CRC cells, and treatment with the FAO inhibitor etomoxir enhanced the efficacy of antiangiogenic therapy. Hepatic stellate cells (HSCs) were essential for matrix stiffness-mediated FAO in colon cancer cells. Matrix stiffness activated lipolysis in HSCs via the focal adhesion kinase (FAK)/yes-associated protein (YAP) pathway, and free fatty acids secreted by HSCs were absorbed as metabolic substrates and activated FAO in colon cancer cells. Suppressing HSC lipolysis using FAK and YAP inhibition enhanced the efficacy of anti-VEGF therapy. Together, these results indicate that bevacizumab-induced ECM remodeling triggers lipid metabolic crosstalk between colon cancer cells and HSCs. This metabolic mechanism of bevacizumab resistance mediated by the physical tumor microenvironment represents a potential therapeutic target for reversing drug resistance.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-0025
bioRxiv. 2023 Aug 07. pii: 2023.08.07.552224. [Epub ahead of print]

Compartment specific responses to contractility in the small intestinal epithelium.

Taylor Hinnant, Wenxiu Ning, Terry Lechler.

Tissues are subject to multiple mechanical inputs at the cellular level that influence their overall shape and function. In the small intestine, actomyosin contractility can be induced by many physiological and pathological inputs. However, we have little understanding of how contractility impacts the intestinal epithelium on a cellular and tissue level. In this study, we probed the cell and tissue-level effects of contractility by using mouse models to genetically increase the level of myosin activity in the two distinct morphologic compartments of the intestinal epithelium, the crypts and villi. We found that increased contractility in the villar compartment caused shape changes in the cells that expressed the transgene and their immediate neighbors. While there were no discernable effects on villar architecture, even low levels of transgene induction in the villi caused non-cell autonomous hyperproliferation of the transit amplifying cells in the crypt, driving increased cell flux through the crypt-villar axis. In contrast, induction of increased contractility in the proliferating cells of the crypts resulted in nuclear deformations, DNA damage, and apoptosis. This study reveals the complex and diverse responses of different intestinal epithelial cells to contractility and provides important insight into mechanical regulation of intestinal physiology.Author Summary: The small intestine epithelium is comprised of two main compartments: the villi which contain differentiated cells that function in nutrient absorption, and the crypts which are made up of undifferentiated cells which serve to replenish cells of the villi. Because of their physical location within the tissue, villi and crypts are subjected to different types of insults and mechanical forces. We sought to directly test how villi and crypts respond to mechanical changes in the epithelia by genetically inducing actomyosin contraction. Increasing contractility in villar cells resulted in cell shape changes without affecting their overall polarity or organization. However, it led to a non-autonomous increase in proliferation of the undifferentiated cells of the intestine. In contrast, increased contractility in the proliferative cells of the crypt resulted in nuclear shape changes, DNA damage and ultimately a rapid cell death. Thus, our work demonstrates that the crypt and villi epithelia respond differently to mechanical changes and highlights long-range regulation between villi and crypt compartments.

DOI: https://doi.org/10.1101/2023.08.07.552224
Cell Death Dis. 2023 08 19. 14(8): 534

OTULIN protects the intestinal epithelium from apoptosis during inflammation and infection.

Lien Verboom, Christopher J Anderson, Maude Jans, Ioanna Petta, Gillian Blancke, Arne Martens, Mozes Sze, Tino Hochepied, Kodi S Ravichandran, Lars Vereecke, Geert van Loo.

The intestinal epithelium is a single cell layer that is constantly renewed and acts as a physical barrier that separates intestinal microbiota from underlying tissues. In inflammatory bowel disease (IBD) in humans, as well as in experimental mouse models of IBD, this barrier is impaired, causing microbial infiltration and inflammation. Deficiency in OTU deubiquitinase with linear linkage specificity (OTULIN) causes OTULIN-related autoinflammatory syndrome (ORAS), a severe inflammatory pathology affecting multiple organs including the intestine. We show that mice with intestinal epithelial cell (IEC)-specific OTULIN deficiency exhibit increased susceptibility to experimental colitis and are highly sensitive to TNF toxicity, due to excessive apoptosis of OTULIN deficient IECs. OTULIN deficiency also increases intestinal pathology in mice genetically engineered to secrete excess TNF, confirming that chronic exposure to TNF promotes epithelial cell death and inflammation in OTULIN deficient mice. Mechanistically we demonstrate that upon TNF stimulation, OTULIN deficiency impairs TNF receptor complex I formation and LUBAC recruitment, and promotes the formation of the cytosolic complex II inducing epithelial cell death. Finally, we show that OTULIN deficiency in IECs increases susceptibility to Salmonella infection, further confirming the importance of OTULIN for intestinal barrier integrity. Together, these results identify OTULIN as a major anti-apoptotic protein in the intestinal epithelium and provide mechanistic insights into how OTULIN deficiency drives gastrointestinal inflammation in ORAS patients.

DOI: https://doi.org/10.1038/s41419-023-06058-7
Cell Stem Cell. 2023 Aug 13. pii: S1934-5909(23)00258-8. [Epub ahead of print]

Adrenergic nerves regulate intestinal regeneration through IL-22 signaling from type 3 innate lymphoid cells.

Putianqi Wang, Noelyn Kljavin, Thi Thu Thao Nguyen, Elaine E Storm, Bryan Marsh, Jian Jiang, William Lin, Hari Menon, Robert Piskol, Frederic J de Sauvage.

  The intestinal epithelium has high intrinsic turnover rate, and the precise renewal of the epithelium is dependent on the microenvironment. The intestine is innervated by a dense network of peripheral nerves that controls various aspects of intestinal physiology. However, the role of neurons in regulating epithelial cell regeneration remains largely unknown. Here, we investigated the effects of gut-innervating adrenergic nerves on epithelial cell repair following irradiation (IR)-induced injury. We observed that adrenergic nerve density in the small intestine increased post IR, while chemical adrenergic denervation impaired epithelial regeneration. Single-cell RNA sequencing experiments revealed a decrease in IL-22 signaling post IR in denervated animals. Combining pharmacologic and genetic tools, we demonstrate that β-adrenergic receptor signaling drives IL-22 production from type 3 innate lymphoid cells (ILC3s) post IR, which in turn promotes epithelial regeneration. These results define an adrenergic-ILC3 axis important for intestinal regeneration.

Keywords:  IL-22; adrenergic nerves; innate lymphoid cells; interleukin-22; intestinal regeneration; irradiation; microenvironment; neuroimmune interaction; single-cell RNA sequencing; β-adrenergic signaling

DOI:  https://doi.org/10.1016/j.stem.2023.07.013
Mol Oncol. 2023 Aug 21.

Lasting response by vertical inhibition with cetuximab and trametinib in KRAS-mutated colorectal cancer patient-derived xenografts.

Timm M Reissig, Swetlana Ladigan-Badura, Anja Steinberg, Abdelouahid Maghnouj, Ting Li, Berlinda Verdoodt, Sven T Liffers, Michael Pohl, Heiner Wolters, Christian Teschendorf, Richard Viebahn, Jakob Admard, Nicolas Casadei, Andrea Tannapfel, Wolff Schmiegel, Stephan A Hahn, Deepak B Vangala.

  Although approximately half of all metastatic colorectal cancers (mCRCs) harbor mutations in KRAS or NRAS, hardly any progress has been made regarding targeted treatment for this group over the last few years. Here, we investigated the efficacy of vertical inhibition of the RAS-pathway by targeting epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase kinase (MEK) in patient-derived xenograft (PDX) tumors with primary KRAS mutation. In total, 19 different PDX models comprising 127 tumors were tested. Responses were evaluated according to baseline tumor changes and graded as partial response (PR; ≤-30%), stable disease (SD; between -30% and +20%) or progressive disease (PD; ≥+20%). Vertical inhibition with trametinib and cetuximab induced SD or PR in 74% of analyzed models, compared to 24% by monotherapy with trametinib. In cases of PR by vertical inhibition (47%), responses were lasting (as long as day 137), with a low incidence of secondary resistance. Molecular analyses revealed that primary and secondary resistance was driven by transcriptional reprogramming activating the RAS pathway in a substantial fraction of tumors. Together, these preclinical data strongly support the translation of this combination therapy into clinical trials for CRC patients.

Keywords:  CRC; EGFR; MEK; PDX; Targeted therapy; resistance

DOI:  https://doi.org/10.1002/1878-0261.13510
Biochem Biophys Rep. 2023 Sep;35 101491

Deciphering colorectal cancer progression features and prognostic signature by single-cell RNA sequencing pseudotime trajectory analysis.

Mohammad Javad Bazyari, Zakie Saadat, Ali Ahmadizad Firouzjaei, Seyed Hamid Aghaee-Bakhtiari.

  Colorectal cancer is the third most common cancer and second cancer with the highest mortality rate in the world. Progression, which leads to metastasis, is one of the biggest challenges in cancer treatment, and despite improvement in screening and treatment techniques, 5 years of survival of colorectal cancer patients drop from 91% in stage I to 12% in stage IV. Single-cell RNA sequencing is one of the most powerful tools to study complex diseases such as cancer, and despite its recent emergence, it's rapidly growing. In contrast to bulk RNA sequencing, which averages out expression of thousands of cells, single-cell RNA sequencing can capture intra-tumor heterogeneity. Moreover, cellular dynamic events like progression can be studied by pseudotime trajectory analysis of single-cell RNA sequencing data. Herein we used Samsung Medical Center (SMC) colorectal cancer single-cell RNA sequencing dataset to find important tumor epithelial cells subtypes. Subsequently, we've found important genes with a dynamic pattern along cancer progression by using pseudo-time trajectory analysis. Also, we found TGFB1 and IL1B as effective ligands and several transcription factors which may regulate the expression of pseudo-time related genes. In the end, we've constructed a LASSO cox regression using 20 psudotime genes, which can predict 3-year survival of colorectal cancer patients with AUC >0.7.

Keywords:  Colorectal cancer; Prognostic signature; Progression features; Single-cell RNA sequencing

DOI:  https://doi.org/10.1016/j.bbrep.2023.101491
Biochim Biophys Acta Gene Regul Mech. 2023 Aug 23. pii: S1874-9399(23)00072-X. [Epub ahead of print] 194977

Protein post-translational modifications: A key factor in colorectal cancer resistance mechanisms.

Bo Bi, Miaojuan Qiu, Peng Liu, Qiang Wang, Yingfei Wen, You Li, Binbin Li, Yongshu Li, Yulong He, Jing Zhao.

  Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Despite advances in treatment, drug resistance remains a critical impediment. Post-translational modifications (PTMs) regulate protein stability, localization, and activity, impacting vital cellular processes. Recent research has highlighted the essential role of PTMs in the development of CRC resistance. This review summarizes recent advancements in understanding PTMs' roles in CRC resistance, focusing on the latest discoveries. We discuss the functional impact of PTMs on signaling pathways and molecules involved in CRC resistance, progress in drug development, and potential therapeutic targets. We also summarize the primary enrichment methods for PTMs. Finally, we discuss current challenges and future directions, including the need for more comprehensive PTM analysis methods and PTM-targeted therapies. This review identifies potential therapeutic interventions for addressing medication resistance in CRC, proposes prospective therapeutic options, and gives an overview of the function of PTMs in CRC resistance.

Keywords:  Colorectal cancer; Drug resistance; PTMs; Post-translational modifications; Protein enrichment

DOI:  https://doi.org/10.1016/j.bbagrm.2023.194977
Cancer Biol Ther. 2023 Dec 31. 24(1): 2246208

Mutant PIK3CA as a negative predictive biomarker for treatment with a highly selective PIM1 inhibitor in human colon cancer.

Yoon Sun Park, Joseph Kim, Yea Seong Ryu, Jai-Hee Moon, Yu Jin Shin, Jeong Hee Kim, Seung-Woo Hong, Soo-A Jung, Seul Lee, Seung-Mi Kim, Dae Hee Lee, Do Yeon Kim, Hyeseon Yun, Ji-Eun You, Dong Il Yoon, Chul Hee Kim, Dong-In Koh, Dong-Hoon Jin.

  Significant improvement in targeted therapy for colorectal cancer (CRC) has occurred over the past few decades since the approval of the EGFR inhibitor cetuximab. However, cetuximab is used only for patients possessing the wild-type oncogene KRAS, NRAS, and BRAF, and even most of these eventually acquire therapeutic resistance, via activation of parallel oncogenic pathways such as RAS-MAPK or PI3K/Akt/mTOR. The two aforementioned pathways also contribute to the development of therapeutic resistance in CRC patients, due to compensatory and feedback mechanisms. Therefore, combination drug therapies (versus monotherapy) targeting these multiple pathways may be necessary for further efficacy against CRC. In this study, we identified PIK3CA mutant (PIK3CA MT) as a determinant of resistance to SMI-4a, a highly selective PIM1 kinase inhibitor, in CRC cell lines. In CRC cell lines, SMI-4a showed its effect only in PIK3CA wild type (PIK3CA WT) cell lines, while PIK3CA MT cells did not respond to SMI-4a in cell death assays. In vivo xenograft and PDX experiments confirmed that PIK3CA MT is responsible for the resistance to SMI-4a. Inhibition of PIK3CA MT by PI3K inhibitors restored SMI-4a sensitivity in PIK3CA MT CRC cell lines. Taken together, these results demonstrate that sensitivity to SMI-4a is determined by the PIK3CA genotype and that co-targeting of PI3K and PIM1 in PIK3CA MT CRC patients could be a promising and novel therapeutic approach for refractory CRC patients.

Keywords:  PIK3CA; PIM1; colorectal cancer; mutant KRAS; predictive marker

DOI:  https://doi.org/10.1080/15384047.2023.2246208
Front Oncol. 2023 ;13 1227884

Hypoxia-induced cancer cell reprogramming: a review on how cancer stem cells arise.

Genevieve M Abd, Madison C Laird, Jennifer C Ku, Yong Li.

  Cancer stem cells are a subset of cells within the tumor that possess the ability to self-renew as well as differentiate into different cancer cell lineages. The exact mechanisms by which cancer stem cells arise is still not completely understood. However, current research suggests that cancer stem cells may originate from normal stem cells that have undergone genetic mutations or epigenetic changes. A more recent discovery is the dedifferentiation of cancer cells to stem-like cells. These stem-like cells have been found to express and even upregulate induced pluripotent stem cell markers known as Yamanaka factors. Here we discuss developments in how cancer stem cells arise and consider how environmental factors, such as hypoxia, plays a key role in promoting the progression of cancer stem cells and metastasis. Understanding the mechanisms that give rise to these cells could have important implications for the development of new strategies in cancer treatments and therapies.

Keywords:  Yamanaka factors; cancer stem cells; hypoxia; metastasis; reprogramming

DOI:  https://doi.org/10.3389/fonc.2023.1227884
Cell Mol Gastroenterol Hepatol. 2023 Aug 18. pii: S2352-345X(23)00151-0. [Epub ahead of print]

SIRT1 controls enteroendocrine progenitor cell proliferation in high-fat diet-fed mice.

Masaomi Miura, Masaki Igarashi, Ryosuke Isotani, Yoshiko Nakagawa-Nagahama, Satoshi Kuranami, Kyoko Naruse, Takashi Kadowaki, Toshimasa Yamauchi.

  BACKGROUND & AIMS: We aimed to investigate how SIRT1, a conserved mammalian NAD+-dependent protein deacetylase, regulates the number of enteroendocrine cells (EECs). EECs benefit metabolism, and their increase could potentially treat type 2 diabetes and obesity.METHODS: We used mice with specific Sirt1 disruption in the intestinal epithelium (VilKO, villin-Cre+, and Sirt1flox/flox mice) or enteroendocrine progenitor cells (EEPCs) (NgnKO, neurogenin3(ngns)-Cre+, Sirt1flox/flox mice) and mice with increased SIRT1 activity due to overexpression (Sir2d mice) or 24 h-fasting. Mice were fed a high-fat diet (HFD), and blood glucagon-like peptide 1 (GLP-1) and glucose levels were measured. Intestinal tissues, EECs, and formed organoids were analyzed using quantitative PCR, immunoblotting, and immunohistochemistry.
RESULTS: In HFD-fed VilKO and NgnKO mice, an increase in EECs (42.3% and 37.2%), GLP-1 or -2-producing L cells (93.0% and 61.4%), and GLP-1 (85.7% and 109.6%) was observed after glucose loading, explaining the improved metabolic phenotype of HFD-VilKO mice. These increases were associated with upregulated expression of Ngn3 (EEPC marker) in crypts of HFD-VilKO and HFD-NgnKO mice, respectively. Conversely, Sir2d or 24 h-fasted mice exhibited a decrease in EECs (21.6%), L cells (41.6%), and proliferative progenitor cells. SIRT1 overexpression- or knockdown-mediated change in progenitor cell proliferation was associated with Wnt/β-catenin activity changes. Notably, Wnt/β-catenin inhibitor completely suppressed EEC and L cell increase in HFD-VilKO mice or organoids from HFD-VilKO and HFD-NgnKO mice.
CONCLUSIONS: Intestinal SIRT1 in EECs modulates the EEPC cycle by regulating β-catenin activity and can control the number of EECs in HFD-fed mice, which is a previously unknown role.

Keywords:  GLP-1; cell cycle; type 2 diabetes

DOI:  https://doi.org/10.1016/j.jcmgh.2023.08.006
Cell Rep. 2023 Aug 18. pii: S2211-1247(23)01008-2. [Epub ahead of print] 112997

Paired microbiome and metabolome analyses associate bile acid changes with colorectal cancer progression.

Ting Fu, Tao Huan, Gibraan Rahman, Hui Zhi, Zhenjiang Xu, Tae Gyu Oh, Jian Guo, Sally Coulter, Anupriya Tripathi, Cameron Martino, Justin L McCarville, Qiyun Zhu, Fritz Cayabyab, Brian Low, Mingxiao He, Shipei Xing, Fernando Vargas, Ruth T Yu, Annette Atkins, Christopher Liddle, Janelle Ayres, Manuela Raffatellu, Pieter C Dorrestein, Michael Downes, Rob Knight, Ronald M Evans.

  Colorectal cancer (CRC) is driven by genomic alterations in concert with dietary influences, with the gut microbiome implicated as an effector in disease development and progression. While meta-analyses have provided mechanistic insight into patients with CRC, study heterogeneity has limited causal associations. Using multi-omics studies on genetically controlled cohorts of mice, we identify diet as the major driver of microbial and metabolomic differences, with reductions in α diversity and widespread changes in cecal metabolites seen in high-fat diet (HFD)-fed mice. In addition, non-classic amino acid conjugation of the bile acid cholic acid (AA-CA) increased with HFD. We show that AA-CAs impact intestinal stem cell growth and demonstrate that Ileibacterium valens and Ruminococcus gnavus are able to synthesize these AA-CAs. This multi-omics dataset implicates diet-induced shifts in the microbiome and the metabolome in disease progression and has potential utility in future diagnostic and therapeutic developments.

Keywords:  CP: Cancer; CP: Microbiology; bile acids; colorectal cancer; conjugated bile acids; high-fat diet; metabolome; microbiome

DOI:  https://doi.org/10.1016/j.celrep.2023.112997
Proc Natl Acad Sci U S A. 2023 Aug 29. 120(35): e2208117120

Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression.

Sarah J Kierans, Raphael R Fagundes, Mykyta I Malkov, Ríona Sparkes, Eugène T Dillon, Albert Smolenski, Klaas Nico Faber, Cormac T Taylor.

  The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic adenosine triphosphate (ATP) production, an event with consequences for cellular bioenergetics and cell fate. This response is regulated at the transcriptional level by the hypoxia-inducible factor-1(HIF-1)-dependent transcriptional upregulation of glycolytic enzymes (GEs) and glucose transporters. However, this transcriptional upregulation alone is unlikely to account fully for the levels of glycolytic ATP produced during hypoxia. Here, we investigated additional mechanisms regulating glycolysis in hypoxia. We observed that intestinal epithelial cells treated with inhibitors of transcription or translation and human platelets (which lack nuclei and the capacity for canonical transcriptional activity) maintained the capacity for hypoxia-induced glycolysis, a finding which suggests the involvement of a nontranscriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. In our investigations into potential nontranscriptional mechanisms for glycolytic induction, we identified a hypoxia-sensitive formation of complexes comprising GEs and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of such glycolytic complexes occurs independent of HIF-1-driven transcription. Finally, we provide evidence for the presence of HIF-1α in cytosolic fractions of hypoxic cells which physically interacts with the glucose transporter GLUT1 and the GEs in a hypoxia-sensitive manner. In conclusion, we provide insights into the nontranscriptional regulation of hypoxia-induced glycolysis in intestinal epithelial cells.

Keywords:  HIF; glycolysis; hypoxia; metabolism

DOI:  https://doi.org/10.1073/pnas.2208117120
Clin Epigenetics. 2023 Aug 24. 15(1): 133

CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.

Juan Ramón Tejedor, Alfonso Peñarroya, Javier Gancedo-Verdejo, Pablo Santamarina-Ojeda, Raúl F Pérez, Sara López-Tamargo, Ana Díez-Borge, Juan J Alba-Linares, Nerea González-Del-Rey, Rocío G Urdinguio, Cristina Mangas, Annalisa Roberti, Virginia López, Teresa Morales-Ruiz, Rafael R Ariza, Teresa Roldán-Arjona, Mónica Meijón, Luis Valledor, María Jesús Cañal, Daniel Fernández-Martínez, María Fernández-Hevia, Paula Jiménez-Fonseca, Luis J García-Flórez, Agustín F Fernández, Mario F Fraga.

  BACKGROUND: Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated.RESULTS: In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence.
CONCLUSIONS: These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.

Keywords:  CRISPR screen; Colorectal cancer; DNA methylation; Epigenetics; Gene expression; Tumour suppressor gene

DOI:  https://doi.org/10.1186/s13148-023-01546-1