Wnt/GSK-3β 介导 FLYWCH1 翻译后修饰,调节结肠肠上皮功能和肿瘤发生。

IF 20.1 1区 医学 Q1 ONCOLOGY
Sheema Almozyan, Roya Babaei-Jadidi, Abrar Aljohani, Sepideh Youssefi, William Dalleywater, Prerna Kadam, Bradley Spencer-Dene, Emad Rakha, Mohammad Ilyas, Abdolrahman Shams Nateri
{"title":"Wnt/GSK-3β 介导 FLYWCH1 翻译后修饰,调节结肠肠上皮功能和肿瘤发生。","authors":"Sheema Almozyan,&nbsp;Roya Babaei-Jadidi,&nbsp;Abrar Aljohani,&nbsp;Sepideh Youssefi,&nbsp;William Dalleywater,&nbsp;Prerna Kadam,&nbsp;Bradley Spencer-Dene,&nbsp;Emad Rakha,&nbsp;Mohammad Ilyas,&nbsp;Abdolrahman Shams Nateri","doi":"10.1002/cac2.12625","DOIUrl":null,"url":null,"abstract":"<p>The intestinal epithelium undergoes rapid renewal, with the entire epithelial layer replaced within five days. Intestinal stem cells (ISCs), located in the intestinal crypts, generate all differentiated cell types necessary for intestinal function. Key signalling pathways involved in stem cell maintenance include Wnt, Notch, Hedgehog, and BMP. Wnt signalling, primarily driven by crypt cells, creates a signalling gradient to maintain homeostasis [<span>1</span>]. However, nuclear β-catenin, the key regulator of Wnt signalling, correlates positively with tumorigenesis. While crypt base cells also exhibit high levels of nuclear β-catenin, the regulatory mechanism in normal tissue versus tumor remains unclear [<span>1</span>]. FLYWCH-Type Zinc Finger 1 (FLYWCH1), an uncharacterised transcription factor, binds unphosphorylated-β-catenin [<span>2</span>], is associated with H3K9me3 in (peri)centromeric chromatin [<span>3</span>], and colocalizes with γ-H2AX foci [<span>4</span>]. While its deletion is embryonically lethal in mice [<span>5</span>], the specific role and regulation of FLYWCH1 in tissue homeostasis and tumorigenesis remain unclear.</p><p>This study investigates the role of FLYWCH1 in intestinal stem cell regulation and its impact on colorectal cancer. We hypothesize that FLYWCH1 directly influences ISC function by modulating critical signalling pathways, thereby playing a significant role in the initiation and progression of colorectal cancer (CRC).</p><p>To assess the significance of FLYWCH1 expression in intestinal tissue homeostasis, we first examined its expression in murine tissues. Data from BioGPS (http://biogps.org) and the mouse gene expression database indicate varying tissue expression of <i>Flywch1</i>, with the highest level observed in the brain (Supplementary Figure S1). To confirm this, we conducted in-situ hybridisation (ISH) analysis to identify distinct cell type-specific expression patterns in the brain and intestinal tissues. ISH was performed using a Digoxigenin-labelled antisense-RNA probe for <i>Flywch1</i> mRNA on representative brain, liver and intestinal sections from 16-week-old wild-type mice (Supplementary Figure S2A-G). We observed high expression of <i>Flywch1</i> in cells located alongside the ISC marker Olfactomedin-4 (<i>Olmf4</i>)-positive cells, while <i>Flywch1</i> was not detectable in the differentiated epithelial cells of the intestinal villi (Supplementary Figure S2D-E). In addition, we examined the differential expression of FLYWCH1 during carcinogenesis, initially in the intestine of <i>Apc</i><sup>Min+/−</sup> mice, which harbour tumors and adjacent non-tumor regions. <i>Flywch1</i> expression was substantially downregulated in intestinal neoplastic crypts compared to normal crypts (Supplementary Figure S2F-G). This is consistent with FLYWCH1 expression in human CRC tissues (Figure 1A-B, Supplementary Table S1). Collectively, these studies suggest a potential role for FLYWCH1 in ISC and the development of CRC.</p><p>To investigate the specific role of FLYWCH1 in ISCs and CRC, we performed loss-of-function experiments by expressing <i>Flywch1</i>-gRNA in Cas9-expressing intestinal organoids (Supplementary Figure S3A). This was followed by Southern blot analysis to evaluate the establishment of <i>Flywch1</i>-knockout (<i>Flywch1</i><sup>K/O</sup>) organoid lines (Supplementary Figure S3B). The results indicated that <i>Flywch1</i><sup>K/O</sup> organoids, displayed larger size and increased crypt budding compared to the normal control (Supplementary Figure S4A-E). Additionally, the expression of cycling stem cell marker (<i>Lgr5</i>, <i>Olmf4</i>) and enteroendocrine cell mediators (<i>Ngn3</i>, <i>Pax4</i>) expressions was increased in <i>Flywch1</i><sup>K/O</sup> organoids, while the expression of quiescent markers (<i>Tert</i>, <i>Lrig1</i>) was reduced. <i>Atoh1</i> (a Paneth-cell mediators) remained unchanged (Supplementary Figure S4F-H). In contrast, overexpression of FLYWCH1 (FLYWCH1<i><sup>OE</sup></i>) significantly repressed CRC patient-derived tumor organoids (PDOs) growth (Supplementary Figure S4I-K), and cancer stem-like cell markers (<i>CD44</i>, <i>LGR5</i>) (Supplementary Figure S4L). These observations suggest that FLYWCH1 play a key role in intestinal homeostasis and tumorigenesis.</p><p>To investigate the relationship between FLYWCH1 expression and Wnt signalling, we evaluated the impact of FLYWCH1 on the expression of Wnt target genes in CRC cells. We used qRT-PCR to analyse both <i>FLYWCH1</i>-knockout (<i>FLYWCH1</i><sup>K/O</sup>) (Supplementary Figure S5A-C) and FLYWCH1-overexpressing (FLYWCH1<sup>OE</sup>) SW620 cells (Supplementary Figure S5D-G). We assessed the expression of a subset of Wnt-associated gene, including frizzled genes (FZD5, FZD6, FZD7), WNT1, WNT6, lipoprotein receptor-related protein genes (LRP5, LRP6), homeobox protein CDX2, fibroblast growth factor 4 (FGF4), B-cell CLL/lymphoma 9 (BCL9), and the cell surface adhesion receptor CD44 (Supplementary Figure S5H-I). In line with our previous report [<span>2</span>], differential expression of FLYWCH1 influenced the expression of genes associated with Wnt pathway.</p><p>Additionally, we analysed FLYWCH1 expression in various CRC and normal epithelial cell lines (TIG119, CCD841-CoN). Immunofluorescence (IF) staining revealed that the nuclear foci formed by FLYWCH1 were more pronounced in TIG119 and CCD841-CoN cells compared to the diffuse nuclear expression observed in CRC cells (Figure 1C, Top panels, Supplementary Figure S6A). After 24 hours treatment with Wnt-3A and R-spondin1 (Wnt3A/Rspo), which activate Wnt receptors, we observed the accumulation of unexpected FLYWCH1 foci in the cytoplasm of all tested cell lines (Figure 1C, Bottom panels, Supplementary Figure S6B), along with a decrease in the total level of nuclear FLYWCH1 protein (Figure 1D, Supplementary Figure S6C). However, treatment with FH535, an inhibitor of β-catenin and PPAR that acts at the level of transcription of target genes, notably increased FLYWCH1, inhibited glycogen synthase kinase-3β (GSK-3β) and β-catenin levels (Supplementary Figure S6D-E), while treatment with the GSK-3β-specific β-catenin phosphorylation sites inhibitor (BIO) inhibited FLYWCH1 expression (Supplementary Figure S6F). These results suggest an antagonistic relationship between FLYWCH1 and GSK-3β/β-catenin signalling, where aberrant Wnt pathway activity may induce a negative feedback loop to downregulate FLYWCH1 activity.</p><p>Previously, we reported that FLYWCH1 interacts with β-catenin via its N-terminal domain of β-catenin, where GSK-3β phosphorylates β-catenin [<span>2</span>]. Here, we further investigated the potential crosstalk between FLYWCH1 and GSK-3β. Our bioinformatics databases and molecular docking simulations confirmed potential binding sites and interaction orientations between FLYWCH1 and GSK-3β (Figure 1E, Supplementary Video S1, Supplementary Figure S7A, and Supplementary Tables S2-S4). We performed a comparative binding enzyme-linked immunosorbent assay (ELISA) (Figure 1F, Supplementary Figure S7B) and co-immunoprecipitation (Co-IP) assay, which were attenuated upon Wnt3A/Rspo treatment (Supplementary Figure S7C). Our analysis revealed various post-translational modifications (PTMs), including ubiquitination and GSK-3β consensus sequence phosphorylation sites within FLYWCH1 amino acid residues (Supplementary Figure S8A). A cycloheximide (CHX) chase assay determined the time course of FLYWCH1 protein stability (Supplementary Figure S8B) and demonstrated ubiquitination-mediated degradation (Supplementary Figure S8C). FLYWCH1-IP-based assays and BIO treatment further showed that GSK-3β maintained nuclear ubiquitinated FLYWCH1, while Wnt activation and GSK-3β phosphorylation of FLYWCH1 led to its ubiquitination and subsequent degradation (Supplementary Figure S8D). These data suggest that the crosstalk between nuclear FLYWCH1 and Wnt/GSK-3β increases FLYWCH1 phosphorylation, which could be crucial for proteasome-mediated FLYWCH1 destabilisation.</p><p>To investigate the therapeutic significance of FLYWCH1 and GSK-3β crosstalk in CRC, we screened PDOs for growth with six different Wnt small molecule inhibitors and activators, including the GSK-3β phosphorylation blocker BIO, QS11, WAY-316606, WIK14, FH535, and a GSK-3β inhibitor drug. After 72 hours, the WST-1 proliferation assay showed that BIO induced the most growth, while FH535 resulted in lowest growth compared to control organoids and other drugs (Figure 1G, Supplementary Figure S9A).</p><p>The clinical significance of FLYWCH1/GSK-3β crosstalk was further examined using a CRC tissue microarray (TMA) via immunohistochemistry (IHC) and H-score analysis (Supplementary Figure S9B, Supplementary Table S5). H-score analysis showed that while the nuclear GSK-3β expression was not significantly different between normal and tumor samples, nuclear GSK-3β significantly increased with tumor stage (Supplementary Figure S9B), contrasting with FLYWCH1 (Figure 1B, left panel). However, cytoplasmic GSK-3β staining intensity decreased with tumor stage (Supplementary Figure S9C), similar to FLYWCH1 (Figure 1B, right panel) in tumor samples. Furthermore, patients with low cytoplasmic FLYWCH1 and high cytoplasmic GSK-3β had the shortest overall survival (OS) (Supplementary Figure S10A-B and Supplementary Table S6), while no significant differences were observed for nuclear FLYWCH1 or nuclear GSK-3β (Supplementary Figure S10C-D and Supplementary Table S6).</p><p>In conclusion, we propose a model (Figure 1H) in which FLYWCH1 acts as a negative regulator of Wnt/β-catenin signalling, maintaining ISC homeostasis. Aberrant Wnt activation mediates the cytoplasmic translocation of FLYWCH1 protein, and further dysregulation of FLYWCH1 by GSK-3β disrupts this complex, leading to uncontrolled β-catenin activity during CRC development and progression. Our data, along with previous findings [<span>6</span>], suggest that FH535 might inhibit tumor growth by GSK-3β/β-catenin inhibition, inducing FLYWCH1 expression. Altogether, this highlights FLYWCH1 as a potential therapeutic target in Wnt/β-catenin-driven tumorigenesis. Recent studies have identified associations between FLYWCH1 and the Wnt/β-catenin pathway, particularly with chromatin in DNA damage response and radioresistance [<span>3, 4, 7</span>-<span>10</span>]. A more detailed analysis of FLYWCH1's role in ISC regulation and CRC, including its therapeutic potential and differential subcellular localization, is discussed in the “Extra discussion text” section of the Supplementary Materials. Therefore, future investigations targeting FLYWCH1 for clinical anti-cancer treatment strategies and monitoring treatment efficiency are warranted.</p><p>The authors declare no potential conflicts of interest.</p><p>This research was supported by the Medical Research Council (MRC) (grant number G0700763) and by the University of Nottingham, Nottingham, UK.</p><p>For patient-derived organoids, CRC specimens were collected from the Nottingham Health Sciences Biobank (NHSB) at Queens Medical Centre, University of Nottingham (NHSB approval number: ACP000098 A Nateri CRC). Ethical approval and research and development approval for generating the TMA were obtained from the Local Research Ethics Committee and the Trust Research and Development office in Nottingham (Ethical approval reference number: 05/Q1605/66), respectively. Mice for organoid isolations were kept in a specific pathogen-free condition (PPL number: P375A76FE). All experiments were conducted at the University of Nottingham in accordance with institutional biomedical service unit guidelines.</p>","PeriodicalId":9495,"journal":{"name":"Cancer Communications","volume":"45 1","pages":"9-14"},"PeriodicalIF":20.1000,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11758259/pdf/","citationCount":"0","resultStr":"{\"title\":\"Wnt/GSK-3β mediates posttranslational modifications of FLYWCH1 to regulate intestinal epithelial function and tumorigenesis in the colon\",\"authors\":\"Sheema Almozyan,&nbsp;Roya Babaei-Jadidi,&nbsp;Abrar Aljohani,&nbsp;Sepideh Youssefi,&nbsp;William Dalleywater,&nbsp;Prerna Kadam,&nbsp;Bradley Spencer-Dene,&nbsp;Emad Rakha,&nbsp;Mohammad Ilyas,&nbsp;Abdolrahman Shams Nateri\",\"doi\":\"10.1002/cac2.12625\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>The intestinal epithelium undergoes rapid renewal, with the entire epithelial layer replaced within five days. Intestinal stem cells (ISCs), located in the intestinal crypts, generate all differentiated cell types necessary for intestinal function. Key signalling pathways involved in stem cell maintenance include Wnt, Notch, Hedgehog, and BMP. Wnt signalling, primarily driven by crypt cells, creates a signalling gradient to maintain homeostasis [<span>1</span>]. However, nuclear β-catenin, the key regulator of Wnt signalling, correlates positively with tumorigenesis. While crypt base cells also exhibit high levels of nuclear β-catenin, the regulatory mechanism in normal tissue versus tumor remains unclear [<span>1</span>]. FLYWCH-Type Zinc Finger 1 (FLYWCH1), an uncharacterised transcription factor, binds unphosphorylated-β-catenin [<span>2</span>], is associated with H3K9me3 in (peri)centromeric chromatin [<span>3</span>], and colocalizes with γ-H2AX foci [<span>4</span>]. While its deletion is embryonically lethal in mice [<span>5</span>], the specific role and regulation of FLYWCH1 in tissue homeostasis and tumorigenesis remain unclear.</p><p>This study investigates the role of FLYWCH1 in intestinal stem cell regulation and its impact on colorectal cancer. We hypothesize that FLYWCH1 directly influences ISC function by modulating critical signalling pathways, thereby playing a significant role in the initiation and progression of colorectal cancer (CRC).</p><p>To assess the significance of FLYWCH1 expression in intestinal tissue homeostasis, we first examined its expression in murine tissues. Data from BioGPS (http://biogps.org) and the mouse gene expression database indicate varying tissue expression of <i>Flywch1</i>, with the highest level observed in the brain (Supplementary Figure S1). To confirm this, we conducted in-situ hybridisation (ISH) analysis to identify distinct cell type-specific expression patterns in the brain and intestinal tissues. ISH was performed using a Digoxigenin-labelled antisense-RNA probe for <i>Flywch1</i> mRNA on representative brain, liver and intestinal sections from 16-week-old wild-type mice (Supplementary Figure S2A-G). We observed high expression of <i>Flywch1</i> in cells located alongside the ISC marker Olfactomedin-4 (<i>Olmf4</i>)-positive cells, while <i>Flywch1</i> was not detectable in the differentiated epithelial cells of the intestinal villi (Supplementary Figure S2D-E). In addition, we examined the differential expression of FLYWCH1 during carcinogenesis, initially in the intestine of <i>Apc</i><sup>Min+/−</sup> mice, which harbour tumors and adjacent non-tumor regions. <i>Flywch1</i> expression was substantially downregulated in intestinal neoplastic crypts compared to normal crypts (Supplementary Figure S2F-G). This is consistent with FLYWCH1 expression in human CRC tissues (Figure 1A-B, Supplementary Table S1). Collectively, these studies suggest a potential role for FLYWCH1 in ISC and the development of CRC.</p><p>To investigate the specific role of FLYWCH1 in ISCs and CRC, we performed loss-of-function experiments by expressing <i>Flywch1</i>-gRNA in Cas9-expressing intestinal organoids (Supplementary Figure S3A). This was followed by Southern blot analysis to evaluate the establishment of <i>Flywch1</i>-knockout (<i>Flywch1</i><sup>K/O</sup>) organoid lines (Supplementary Figure S3B). The results indicated that <i>Flywch1</i><sup>K/O</sup> organoids, displayed larger size and increased crypt budding compared to the normal control (Supplementary Figure S4A-E). Additionally, the expression of cycling stem cell marker (<i>Lgr5</i>, <i>Olmf4</i>) and enteroendocrine cell mediators (<i>Ngn3</i>, <i>Pax4</i>) expressions was increased in <i>Flywch1</i><sup>K/O</sup> organoids, while the expression of quiescent markers (<i>Tert</i>, <i>Lrig1</i>) was reduced. <i>Atoh1</i> (a Paneth-cell mediators) remained unchanged (Supplementary Figure S4F-H). In contrast, overexpression of FLYWCH1 (FLYWCH1<i><sup>OE</sup></i>) significantly repressed CRC patient-derived tumor organoids (PDOs) growth (Supplementary Figure S4I-K), and cancer stem-like cell markers (<i>CD44</i>, <i>LGR5</i>) (Supplementary Figure S4L). These observations suggest that FLYWCH1 play a key role in intestinal homeostasis and tumorigenesis.</p><p>To investigate the relationship between FLYWCH1 expression and Wnt signalling, we evaluated the impact of FLYWCH1 on the expression of Wnt target genes in CRC cells. We used qRT-PCR to analyse both <i>FLYWCH1</i>-knockout (<i>FLYWCH1</i><sup>K/O</sup>) (Supplementary Figure S5A-C) and FLYWCH1-overexpressing (FLYWCH1<sup>OE</sup>) SW620 cells (Supplementary Figure S5D-G). We assessed the expression of a subset of Wnt-associated gene, including frizzled genes (FZD5, FZD6, FZD7), WNT1, WNT6, lipoprotein receptor-related protein genes (LRP5, LRP6), homeobox protein CDX2, fibroblast growth factor 4 (FGF4), B-cell CLL/lymphoma 9 (BCL9), and the cell surface adhesion receptor CD44 (Supplementary Figure S5H-I). In line with our previous report [<span>2</span>], differential expression of FLYWCH1 influenced the expression of genes associated with Wnt pathway.</p><p>Additionally, we analysed FLYWCH1 expression in various CRC and normal epithelial cell lines (TIG119, CCD841-CoN). Immunofluorescence (IF) staining revealed that the nuclear foci formed by FLYWCH1 were more pronounced in TIG119 and CCD841-CoN cells compared to the diffuse nuclear expression observed in CRC cells (Figure 1C, Top panels, Supplementary Figure S6A). After 24 hours treatment with Wnt-3A and R-spondin1 (Wnt3A/Rspo), which activate Wnt receptors, we observed the accumulation of unexpected FLYWCH1 foci in the cytoplasm of all tested cell lines (Figure 1C, Bottom panels, Supplementary Figure S6B), along with a decrease in the total level of nuclear FLYWCH1 protein (Figure 1D, Supplementary Figure S6C). However, treatment with FH535, an inhibitor of β-catenin and PPAR that acts at the level of transcription of target genes, notably increased FLYWCH1, inhibited glycogen synthase kinase-3β (GSK-3β) and β-catenin levels (Supplementary Figure S6D-E), while treatment with the GSK-3β-specific β-catenin phosphorylation sites inhibitor (BIO) inhibited FLYWCH1 expression (Supplementary Figure S6F). These results suggest an antagonistic relationship between FLYWCH1 and GSK-3β/β-catenin signalling, where aberrant Wnt pathway activity may induce a negative feedback loop to downregulate FLYWCH1 activity.</p><p>Previously, we reported that FLYWCH1 interacts with β-catenin via its N-terminal domain of β-catenin, where GSK-3β phosphorylates β-catenin [<span>2</span>]. Here, we further investigated the potential crosstalk between FLYWCH1 and GSK-3β. Our bioinformatics databases and molecular docking simulations confirmed potential binding sites and interaction orientations between FLYWCH1 and GSK-3β (Figure 1E, Supplementary Video S1, Supplementary Figure S7A, and Supplementary Tables S2-S4). We performed a comparative binding enzyme-linked immunosorbent assay (ELISA) (Figure 1F, Supplementary Figure S7B) and co-immunoprecipitation (Co-IP) assay, which were attenuated upon Wnt3A/Rspo treatment (Supplementary Figure S7C). Our analysis revealed various post-translational modifications (PTMs), including ubiquitination and GSK-3β consensus sequence phosphorylation sites within FLYWCH1 amino acid residues (Supplementary Figure S8A). A cycloheximide (CHX) chase assay determined the time course of FLYWCH1 protein stability (Supplementary Figure S8B) and demonstrated ubiquitination-mediated degradation (Supplementary Figure S8C). FLYWCH1-IP-based assays and BIO treatment further showed that GSK-3β maintained nuclear ubiquitinated FLYWCH1, while Wnt activation and GSK-3β phosphorylation of FLYWCH1 led to its ubiquitination and subsequent degradation (Supplementary Figure S8D). These data suggest that the crosstalk between nuclear FLYWCH1 and Wnt/GSK-3β increases FLYWCH1 phosphorylation, which could be crucial for proteasome-mediated FLYWCH1 destabilisation.</p><p>To investigate the therapeutic significance of FLYWCH1 and GSK-3β crosstalk in CRC, we screened PDOs for growth with six different Wnt small molecule inhibitors and activators, including the GSK-3β phosphorylation blocker BIO, QS11, WAY-316606, WIK14, FH535, and a GSK-3β inhibitor drug. After 72 hours, the WST-1 proliferation assay showed that BIO induced the most growth, while FH535 resulted in lowest growth compared to control organoids and other drugs (Figure 1G, Supplementary Figure S9A).</p><p>The clinical significance of FLYWCH1/GSK-3β crosstalk was further examined using a CRC tissue microarray (TMA) via immunohistochemistry (IHC) and H-score analysis (Supplementary Figure S9B, Supplementary Table S5). H-score analysis showed that while the nuclear GSK-3β expression was not significantly different between normal and tumor samples, nuclear GSK-3β significantly increased with tumor stage (Supplementary Figure S9B), contrasting with FLYWCH1 (Figure 1B, left panel). However, cytoplasmic GSK-3β staining intensity decreased with tumor stage (Supplementary Figure S9C), similar to FLYWCH1 (Figure 1B, right panel) in tumor samples. Furthermore, patients with low cytoplasmic FLYWCH1 and high cytoplasmic GSK-3β had the shortest overall survival (OS) (Supplementary Figure S10A-B and Supplementary Table S6), while no significant differences were observed for nuclear FLYWCH1 or nuclear GSK-3β (Supplementary Figure S10C-D and Supplementary Table S6).</p><p>In conclusion, we propose a model (Figure 1H) in which FLYWCH1 acts as a negative regulator of Wnt/β-catenin signalling, maintaining ISC homeostasis. Aberrant Wnt activation mediates the cytoplasmic translocation of FLYWCH1 protein, and further dysregulation of FLYWCH1 by GSK-3β disrupts this complex, leading to uncontrolled β-catenin activity during CRC development and progression. Our data, along with previous findings [<span>6</span>], suggest that FH535 might inhibit tumor growth by GSK-3β/β-catenin inhibition, inducing FLYWCH1 expression. Altogether, this highlights FLYWCH1 as a potential therapeutic target in Wnt/β-catenin-driven tumorigenesis. Recent studies have identified associations between FLYWCH1 and the Wnt/β-catenin pathway, particularly with chromatin in DNA damage response and radioresistance [<span>3, 4, 7</span>-<span>10</span>]. A more detailed analysis of FLYWCH1's role in ISC regulation and CRC, including its therapeutic potential and differential subcellular localization, is discussed in the “Extra discussion text” section of the Supplementary Materials. Therefore, future investigations targeting FLYWCH1 for clinical anti-cancer treatment strategies and monitoring treatment efficiency are warranted.</p><p>The authors declare no potential conflicts of interest.</p><p>This research was supported by the Medical Research Council (MRC) (grant number G0700763) and by the University of Nottingham, Nottingham, UK.</p><p>For patient-derived organoids, CRC specimens were collected from the Nottingham Health Sciences Biobank (NHSB) at Queens Medical Centre, University of Nottingham (NHSB approval number: ACP000098 A Nateri CRC). Ethical approval and research and development approval for generating the TMA were obtained from the Local Research Ethics Committee and the Trust Research and Development office in Nottingham (Ethical approval reference number: 05/Q1605/66), respectively. Mice for organoid isolations were kept in a specific pathogen-free condition (PPL number: P375A76FE). All experiments were conducted at the University of Nottingham in accordance with institutional biomedical service unit guidelines.</p>\",\"PeriodicalId\":9495,\"journal\":{\"name\":\"Cancer Communications\",\"volume\":\"45 1\",\"pages\":\"9-14\"},\"PeriodicalIF\":20.1000,\"publicationDate\":\"2024-10-30\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11758259/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cancer Communications\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12625\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"ONCOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cancer Communications","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12625","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ONCOLOGY","Score":null,"Total":0}
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摘要

肠上皮经历快速更新,整个上皮层在5天内被替换。肠干细胞(ISCs)位于肠隐窝中,产生肠功能所需的所有分化细胞类型。参与干细胞维持的关键信号通路包括Wnt、Notch、Hedgehog和BMP。Wnt信号主要由隐窝细胞驱动,产生信号梯度以维持体内平衡[1]。然而,核β-catenin, Wnt信号的关键调节因子,与肿瘤发生呈正相关。虽然隐窝基底细胞也表现出高水平的核β-连环蛋白,但正常组织对肿瘤的调节机制仍不清楚。flywch型锌指1 (FLYWCH1)是一种未表征的转录因子,结合未磷酸化的-β-catenin[2],与(周围)着丝染色质[3]中的H3K9me3相关,并与γ-H2AX焦点[4]共定位。虽然其缺失在小鼠[5]中具有胚胎致死性,但FLYWCH1在组织稳态和肿瘤发生中的具体作用和调控尚不清楚。本研究探讨FLYWCH1在肠道干细胞调控中的作用及其对结直肠癌的影响。我们假设FLYWCH1通过调节关键信号通路直接影响ISC功能,从而在结直肠癌(CRC)的发生和进展中发挥重要作用。为了评估FLYWCH1表达在肠道组织稳态中的意义,我们首先检测了其在小鼠组织中的表达。来自BioGPS (http://biogps.org)和小鼠基因表达数据库的数据表明,Flywch1在不同的组织中表达不同,在大脑中表达水平最高(补充图S1)。为了证实这一点,我们进行了原位杂交(ISH)分析,以鉴定大脑和肠道组织中不同的细胞类型特异性表达模式。使用地高辛标记的反义rna探针在16周龄野生型小鼠的代表性脑、肝和肠切片上检测Flywch1 mRNA进行ISH(补充图S2A-G)。我们观察到Flywch1在ISC标记Olfactomedin-4 (olf4)阳性细胞旁的细胞中高表达,而在肠绒毛分化的上皮细胞中未检测到Flywch1 (Supplementary Figure S2D-E)。此外,我们检测了FLYWCH1在癌变过程中的差异表达,最初是在ApcMin+/−小鼠的肠道中,其中包含肿瘤和邻近的非肿瘤区域。与正常隐窝相比,肠道肿瘤隐窝中的Flywch1表达明显下调(补充图S2F-G)。这与FLYWCH1在人类结直肠癌组织中的表达一致(图1A-B, Supplementary Table S1)。总的来说,这些研究表明FLYWCH1在ISC和CRC的发展中具有潜在的作用。为了研究FLYWCH1在ISCs和CRC中的具体作用,我们通过在表达cas9的肠道类器官中表达FLYWCH1 - grna进行了功能缺失实验(Supplementary Figure S3A)。随后进行Southern blot分析,评估flywch1敲除(Flywch1K/O)类器官系的建立情况(Supplementary Figure S3B)。结果显示,与正常对照相比,Flywch1K/O类器官的体积更大,隐窝出芽增加(Supplementary Figure S4A-E)。此外,循环干细胞标志物(Lgr5、olf4)和肠内分泌细胞介质(Ngn3、Pax4)在Flywch1K/O类器官中的表达增加,而静止标志物(Tert、Lrig1)的表达减少。Atoh1(一种泛细胞介质)保持不变(补充图S4F-H)。相反,FLYWCH1 (FLYWCH1OE)的过表达显著抑制结直肠癌患者衍生的肿瘤类器官(PDOs)的生长(Supplementary Figure S4I-K)和癌症干细胞样细胞标志物(CD44, LGR5) (Supplementary Figure S4L)。这些观察结果表明,FLYWCH1在肠道内稳态和肿瘤发生中起关键作用。为了研究FLYWCH1表达与Wnt信号传导之间的关系,我们评估了FLYWCH1对CRC细胞中Wnt靶基因表达的影响。我们使用qRT-PCR分析了flywch1敲除(FLYWCH1K/O)(补充图S5A-C)和flywch1过表达(FLYWCH1OE)的SW620细胞(补充图S5D-G)。我们评估了wnt相关基因的一个子集的表达,包括卷曲基因(FZD5、FZD6、FZD7)、WNT1、WNT6、脂蛋白受体相关蛋白基因(LRP5、LRP6)、同源盒蛋白CDX2、成纤维细胞生长因子4 (FGF4)、b细胞CLL/淋巴瘤9 (BCL9)和细胞表面粘附受体CD44 (Supplementary Figure S5H-I)。与我们之前的报道[2]一致,FLYWCH1的差异表达影响了Wnt通路相关基因的表达。此外,我们分析了FLYWCH1在各种结直肠癌和正常上皮细胞系(TIG119, CCD841-CoN)中的表达。 免疫荧光(IF)染色显示,与CRC细胞中弥漫性核表达相比,FLYWCH1形成的核灶在TIG119和CCD841-CoN细胞中更为明显(图1C,顶部面板,补充图S6A)。用激活Wnt受体的Wnt- 3a和R-spondin1 (Wnt3A/Rspo)处理24小时后,我们观察到所有被测试细胞系的细胞质中都积累了意想不到的FLYWCH1灶(图1C,底部面板,补充图S6B),同时核FLYWCH1蛋白总水平降低(图1D,补充图S6C)。然而,用FH535(一种作用于靶基因转录水平的β-catenin和PPAR抑制剂)处理后,FLYWCH1显著增加,抑制糖原合成酶激酶3β (GSK-3β)和β-catenin水平(补充图S6D-E),而用GSK-3β特异性β-catenin磷酸化位点抑制剂(BIO)处理后,FLYWCH1表达受到抑制(补充图S6F)。这些结果表明FLYWCH1与GSK-3β/β-catenin信号通路之间存在拮抗关系,其中Wnt通路活性异常可能诱导一个负反馈回路下调FLYWCH1活性。在此之前,我们报道了FLYWCH1通过β-catenin的n端结构域与β-catenin相互作用,GSK-3β在其中磷酸化β-catenin[2]。在这里,我们进一步研究了FLYWCH1和GSK-3β之间的潜在串扰。我们的生物信息学数据库和分子对接模拟证实了FLYWCH1和GSK-3β之间潜在的结合位点和相互作用方向(图1E,补充视频S1,补充图S7A和补充表S2-S4)。我们进行了比较结合酶联免疫吸附试验(ELISA)(图1F,补充图S7B)和共免疫沉淀(Co-IP)试验,这些试验在Wnt3A/Rspo处理后减弱(补充图S7C)。我们的分析揭示了FLYWCH1氨基酸残基中的各种翻译后修饰(PTMs),包括泛素化和GSK-3β一致序列磷酸化位点(补充图S8A)。环己亚胺(CHX)追踪实验确定了FLYWCH1蛋白稳定性的时间过程(补充图S8B),并证明了泛素化介导的降解(补充图S8C)。基于FLYWCH1- ip的实验和BIO处理进一步表明,GSK-3β维持了FLYWCH1的核泛素化,而Wnt激活和GSK-3β磷酸化FLYWCH1导致其泛素化和随后的降解(补充图S8D)。这些数据表明,核FLYWCH1和Wnt/GSK-3β之间的串扰增加了FLYWCH1的磷酸化,这可能是蛋白酶体介导的FLYWCH1不稳定的关键。为了研究FLYWCH1和GSK-3β串扰在结直肠癌中的治疗意义,我们用6种不同的Wnt小分子抑制剂和激活剂筛选PDOs,包括GSK-3β磷酸化阻断剂BIO、QS11、WAY-316606、WIK14、FH535和GSK-3β抑制剂药物。72h后,WST-1增殖实验显示,与对照类器官和其他药物相比,BIO诱导的生长最多,而FH535的生长最低(图1G,补充图S9A)。使用结直肠癌组织芯片(TMA)通过免疫组化(IHC)和h评分分析进一步检测FLYWCH1/GSK-3β串扰的临床意义(补充图S9B,补充表S5)。H-score分析显示,虽然细胞核GSK-3β的表达在正常和肿瘤样本之间没有显著差异,但细胞核GSK-3β随肿瘤分期显著升高(Supplementary图S9B),与FLYWCH1(图1B,左面板)形成对比。然而,细胞质GSK-3β染色强度随着肿瘤分期而降低(补充图S9C),与肿瘤样品中的FLYWCH1(图1B,右图)相似。此外,低细胞质FLYWCH1和高细胞质GSK-3β患者的总生存期(OS)最短(补充图S10A-B和补充表S6),而核FLYWCH1和核GSK-3β患者的总生存期(OS)无显著差异(补充图S10C-D和补充表S6)。总之,我们提出了一个模型(图1H),其中FLYWCH1作为Wnt/β-catenin信号传导的负调节因子,维持ISC稳态。异常的Wnt激活介导了FLYWCH1蛋白的细胞质易位,GSK-3β对FLYWCH1的进一步失调破坏了该复合物,导致结直肠癌发生和进展过程中β-catenin活性失控。我们的数据以及之前的研究结果表明,FH535可能通过抑制GSK-3β/β-catenin来抑制肿瘤生长,诱导FLYWCH1的表达。总之,这突出了FLYWCH1作为Wnt/β-连环蛋白驱动的肿瘤发生的潜在治疗靶点。 最近的研究发现FLYWCH1与Wnt/β-catenin通路之间存在关联,特别是与DNA损伤反应和辐射抗性中的染色质有关[3,4,7 -10]。关于FLYWCH1在ISC调控和CRC中的作用的更详细分析,包括其治疗潜力和差异亚细胞定位,在补充材料的“额外讨论文本”部分进行了讨论。因此,未来研究以FLYWCH1为靶点的临床抗癌治疗策略和监测治疗效果是有必要的。作者声明没有潜在的利益冲突。这项研究得到了医学研究委员会(MRC)(授权号G0700763)和英国诺丁汉大学的支持。对于患者来源的类器官,CRC标本来自诺丁汉大学皇后医学中心的诺丁汉健康科学生物银行(NHSB) (NHSB批准号:ACP000098 A Nateri CRC)。产生TMA的伦理批准和研发批准分别获得了当地研究伦理委员会和诺丁汉信托研究与发展办公室的批准(伦理批准参考号:05/Q1605/66)。类器官分离小鼠保持在特定的无病原体状态(PPL编号:P375A76FE)。所有实验均在诺丁汉大学按照机构生物医学服务单位的指导方针进行。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Wnt/GSK-3β mediates posttranslational modifications of FLYWCH1 to regulate intestinal epithelial function and tumorigenesis in the colon

Wnt/GSK-3β mediates posttranslational modifications of FLYWCH1 to regulate intestinal epithelial function and tumorigenesis in the colon

The intestinal epithelium undergoes rapid renewal, with the entire epithelial layer replaced within five days. Intestinal stem cells (ISCs), located in the intestinal crypts, generate all differentiated cell types necessary for intestinal function. Key signalling pathways involved in stem cell maintenance include Wnt, Notch, Hedgehog, and BMP. Wnt signalling, primarily driven by crypt cells, creates a signalling gradient to maintain homeostasis [1]. However, nuclear β-catenin, the key regulator of Wnt signalling, correlates positively with tumorigenesis. While crypt base cells also exhibit high levels of nuclear β-catenin, the regulatory mechanism in normal tissue versus tumor remains unclear [1]. FLYWCH-Type Zinc Finger 1 (FLYWCH1), an uncharacterised transcription factor, binds unphosphorylated-β-catenin [2], is associated with H3K9me3 in (peri)centromeric chromatin [3], and colocalizes with γ-H2AX foci [4]. While its deletion is embryonically lethal in mice [5], the specific role and regulation of FLYWCH1 in tissue homeostasis and tumorigenesis remain unclear.

This study investigates the role of FLYWCH1 in intestinal stem cell regulation and its impact on colorectal cancer. We hypothesize that FLYWCH1 directly influences ISC function by modulating critical signalling pathways, thereby playing a significant role in the initiation and progression of colorectal cancer (CRC).

To assess the significance of FLYWCH1 expression in intestinal tissue homeostasis, we first examined its expression in murine tissues. Data from BioGPS (http://biogps.org) and the mouse gene expression database indicate varying tissue expression of Flywch1, with the highest level observed in the brain (Supplementary Figure S1). To confirm this, we conducted in-situ hybridisation (ISH) analysis to identify distinct cell type-specific expression patterns in the brain and intestinal tissues. ISH was performed using a Digoxigenin-labelled antisense-RNA probe for Flywch1 mRNA on representative brain, liver and intestinal sections from 16-week-old wild-type mice (Supplementary Figure S2A-G). We observed high expression of Flywch1 in cells located alongside the ISC marker Olfactomedin-4 (Olmf4)-positive cells, while Flywch1 was not detectable in the differentiated epithelial cells of the intestinal villi (Supplementary Figure S2D-E). In addition, we examined the differential expression of FLYWCH1 during carcinogenesis, initially in the intestine of ApcMin+/− mice, which harbour tumors and adjacent non-tumor regions. Flywch1 expression was substantially downregulated in intestinal neoplastic crypts compared to normal crypts (Supplementary Figure S2F-G). This is consistent with FLYWCH1 expression in human CRC tissues (Figure 1A-B, Supplementary Table S1). Collectively, these studies suggest a potential role for FLYWCH1 in ISC and the development of CRC.

To investigate the specific role of FLYWCH1 in ISCs and CRC, we performed loss-of-function experiments by expressing Flywch1-gRNA in Cas9-expressing intestinal organoids (Supplementary Figure S3A). This was followed by Southern blot analysis to evaluate the establishment of Flywch1-knockout (Flywch1K/O) organoid lines (Supplementary Figure S3B). The results indicated that Flywch1K/O organoids, displayed larger size and increased crypt budding compared to the normal control (Supplementary Figure S4A-E). Additionally, the expression of cycling stem cell marker (Lgr5, Olmf4) and enteroendocrine cell mediators (Ngn3, Pax4) expressions was increased in Flywch1K/O organoids, while the expression of quiescent markers (Tert, Lrig1) was reduced. Atoh1 (a Paneth-cell mediators) remained unchanged (Supplementary Figure S4F-H). In contrast, overexpression of FLYWCH1 (FLYWCH1OE) significantly repressed CRC patient-derived tumor organoids (PDOs) growth (Supplementary Figure S4I-K), and cancer stem-like cell markers (CD44, LGR5) (Supplementary Figure S4L). These observations suggest that FLYWCH1 play a key role in intestinal homeostasis and tumorigenesis.

To investigate the relationship between FLYWCH1 expression and Wnt signalling, we evaluated the impact of FLYWCH1 on the expression of Wnt target genes in CRC cells. We used qRT-PCR to analyse both FLYWCH1-knockout (FLYWCH1K/O) (Supplementary Figure S5A-C) and FLYWCH1-overexpressing (FLYWCH1OE) SW620 cells (Supplementary Figure S5D-G). We assessed the expression of a subset of Wnt-associated gene, including frizzled genes (FZD5, FZD6, FZD7), WNT1, WNT6, lipoprotein receptor-related protein genes (LRP5, LRP6), homeobox protein CDX2, fibroblast growth factor 4 (FGF4), B-cell CLL/lymphoma 9 (BCL9), and the cell surface adhesion receptor CD44 (Supplementary Figure S5H-I). In line with our previous report [2], differential expression of FLYWCH1 influenced the expression of genes associated with Wnt pathway.

Additionally, we analysed FLYWCH1 expression in various CRC and normal epithelial cell lines (TIG119, CCD841-CoN). Immunofluorescence (IF) staining revealed that the nuclear foci formed by FLYWCH1 were more pronounced in TIG119 and CCD841-CoN cells compared to the diffuse nuclear expression observed in CRC cells (Figure 1C, Top panels, Supplementary Figure S6A). After 24 hours treatment with Wnt-3A and R-spondin1 (Wnt3A/Rspo), which activate Wnt receptors, we observed the accumulation of unexpected FLYWCH1 foci in the cytoplasm of all tested cell lines (Figure 1C, Bottom panels, Supplementary Figure S6B), along with a decrease in the total level of nuclear FLYWCH1 protein (Figure 1D, Supplementary Figure S6C). However, treatment with FH535, an inhibitor of β-catenin and PPAR that acts at the level of transcription of target genes, notably increased FLYWCH1, inhibited glycogen synthase kinase-3β (GSK-3β) and β-catenin levels (Supplementary Figure S6D-E), while treatment with the GSK-3β-specific β-catenin phosphorylation sites inhibitor (BIO) inhibited FLYWCH1 expression (Supplementary Figure S6F). These results suggest an antagonistic relationship between FLYWCH1 and GSK-3β/β-catenin signalling, where aberrant Wnt pathway activity may induce a negative feedback loop to downregulate FLYWCH1 activity.

Previously, we reported that FLYWCH1 interacts with β-catenin via its N-terminal domain of β-catenin, where GSK-3β phosphorylates β-catenin [2]. Here, we further investigated the potential crosstalk between FLYWCH1 and GSK-3β. Our bioinformatics databases and molecular docking simulations confirmed potential binding sites and interaction orientations between FLYWCH1 and GSK-3β (Figure 1E, Supplementary Video S1, Supplementary Figure S7A, and Supplementary Tables S2-S4). We performed a comparative binding enzyme-linked immunosorbent assay (ELISA) (Figure 1F, Supplementary Figure S7B) and co-immunoprecipitation (Co-IP) assay, which were attenuated upon Wnt3A/Rspo treatment (Supplementary Figure S7C). Our analysis revealed various post-translational modifications (PTMs), including ubiquitination and GSK-3β consensus sequence phosphorylation sites within FLYWCH1 amino acid residues (Supplementary Figure S8A). A cycloheximide (CHX) chase assay determined the time course of FLYWCH1 protein stability (Supplementary Figure S8B) and demonstrated ubiquitination-mediated degradation (Supplementary Figure S8C). FLYWCH1-IP-based assays and BIO treatment further showed that GSK-3β maintained nuclear ubiquitinated FLYWCH1, while Wnt activation and GSK-3β phosphorylation of FLYWCH1 led to its ubiquitination and subsequent degradation (Supplementary Figure S8D). These data suggest that the crosstalk between nuclear FLYWCH1 and Wnt/GSK-3β increases FLYWCH1 phosphorylation, which could be crucial for proteasome-mediated FLYWCH1 destabilisation.

To investigate the therapeutic significance of FLYWCH1 and GSK-3β crosstalk in CRC, we screened PDOs for growth with six different Wnt small molecule inhibitors and activators, including the GSK-3β phosphorylation blocker BIO, QS11, WAY-316606, WIK14, FH535, and a GSK-3β inhibitor drug. After 72 hours, the WST-1 proliferation assay showed that BIO induced the most growth, while FH535 resulted in lowest growth compared to control organoids and other drugs (Figure 1G, Supplementary Figure S9A).

The clinical significance of FLYWCH1/GSK-3β crosstalk was further examined using a CRC tissue microarray (TMA) via immunohistochemistry (IHC) and H-score analysis (Supplementary Figure S9B, Supplementary Table S5). H-score analysis showed that while the nuclear GSK-3β expression was not significantly different between normal and tumor samples, nuclear GSK-3β significantly increased with tumor stage (Supplementary Figure S9B), contrasting with FLYWCH1 (Figure 1B, left panel). However, cytoplasmic GSK-3β staining intensity decreased with tumor stage (Supplementary Figure S9C), similar to FLYWCH1 (Figure 1B, right panel) in tumor samples. Furthermore, patients with low cytoplasmic FLYWCH1 and high cytoplasmic GSK-3β had the shortest overall survival (OS) (Supplementary Figure S10A-B and Supplementary Table S6), while no significant differences were observed for nuclear FLYWCH1 or nuclear GSK-3β (Supplementary Figure S10C-D and Supplementary Table S6).

In conclusion, we propose a model (Figure 1H) in which FLYWCH1 acts as a negative regulator of Wnt/β-catenin signalling, maintaining ISC homeostasis. Aberrant Wnt activation mediates the cytoplasmic translocation of FLYWCH1 protein, and further dysregulation of FLYWCH1 by GSK-3β disrupts this complex, leading to uncontrolled β-catenin activity during CRC development and progression. Our data, along with previous findings [6], suggest that FH535 might inhibit tumor growth by GSK-3β/β-catenin inhibition, inducing FLYWCH1 expression. Altogether, this highlights FLYWCH1 as a potential therapeutic target in Wnt/β-catenin-driven tumorigenesis. Recent studies have identified associations between FLYWCH1 and the Wnt/β-catenin pathway, particularly with chromatin in DNA damage response and radioresistance [3, 4, 7-10]. A more detailed analysis of FLYWCH1's role in ISC regulation and CRC, including its therapeutic potential and differential subcellular localization, is discussed in the “Extra discussion text” section of the Supplementary Materials. Therefore, future investigations targeting FLYWCH1 for clinical anti-cancer treatment strategies and monitoring treatment efficiency are warranted.

The authors declare no potential conflicts of interest.

This research was supported by the Medical Research Council (MRC) (grant number G0700763) and by the University of Nottingham, Nottingham, UK.

For patient-derived organoids, CRC specimens were collected from the Nottingham Health Sciences Biobank (NHSB) at Queens Medical Centre, University of Nottingham (NHSB approval number: ACP000098 A Nateri CRC). Ethical approval and research and development approval for generating the TMA were obtained from the Local Research Ethics Committee and the Trust Research and Development office in Nottingham (Ethical approval reference number: 05/Q1605/66), respectively. Mice for organoid isolations were kept in a specific pathogen-free condition (PPL number: P375A76FE). All experiments were conducted at the University of Nottingham in accordance with institutional biomedical service unit guidelines.

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来源期刊
Cancer Communications
Cancer Communications Biochemistry, Genetics and Molecular Biology-Cancer Research
CiteScore
25.50
自引率
4.30%
发文量
153
审稿时长
4 weeks
期刊介绍: Cancer Communications is an open access, peer-reviewed online journal that encompasses basic, clinical, and translational cancer research. The journal welcomes submissions concerning clinical trials, epidemiology, molecular and cellular biology, and genetics.
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