Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin.

Colorectal cancer (CRC) poses a significant public health challenge, ranking third in incidence and second in mortality among all cancers in 2020. While genetic factors contribute to a portion of CRC risk, the majority is influenced by environmental factors, with the gut microbiota playing a significant role. Infections by certain pathogens like pathogenic E. coli, Salmonella enterica, toxigenic Bacteroides fragilis, Fusobacterium nucleatum (Fn), Peptostreptococcus anaerobius, and Helicobacter pylori have been linked to CRC risk. Among these, F. nucleatum, a gram-negative anaerobic bacillus, has garnered attention for its association with cancer. Recently studies show an increased presence of Fn in human CRC compared to healthy tissues, and higher levels of Fn in CRC tumors are linked to poorer survival rates. Animal and cellular models support the cancer-promoting role of Fn, with FadA, a protein it produces, shown to bind to E-cadherin, activating β-catenin signaling and influencing inflammatory and oncogenic responses. The overexpression of the fadA gene in colon tissue from CRC patients further supports its involvement in cancer development. However, the precise mechanisms driving the progression of cancer initiated by this bacterium are not yet fully understood. In this study, we aimed to determine the prevalence of CRC-associated bacteria F. nucleatum in the biopsied tissues of patients with CRC using quantitative real-time PCR (qPCR) and the fold change Fn abundance in colorectal cancer of Kаzаkhstаni раtiеnts. Furthemore, we determined the association between the epidemiological characteristics of CRC and the presence of F. nucleatum. In our investigation were recruited a total of 80 patients with histologically confirmed colorectal adenocarcinoma (males 38 and females 42, age range 26-86 years) undergoing surgical resections at the National Research Oncology Center, Astana, Kazakhstan between Oktober 2022 and March 2024. Demographic and clinical factors were included such as age, gender, high consumption of red meat, obesity, smoking, and alcohol intake. Prior to the study, informed consent was obtained from all patients in accordance with the guidelines set forth by the RSE Ethics Commission, "National Center for Biotechnology" under the Ministry of Health of the Republic of Kazakhstan. Biopsied tissues from each patient were obtained from colorectal carcinoma tissues, adjacent tissues, and distal normal tissues. In total, 240 number of biopsies were collected. Q-PCR was applied to detect F. nucleatum in CRC and normal tissues. The amount of Fusobacterium between two groups was assessed using the Student’s t-test and Wilcoxon rank sum test. The median abundance of F. nucleatum determined by 2-ΔΔCT in CRC tissues 19.4 (1.9-354.3) was significantly higher than that in normal controls 4.13 (0.98-23.57) (P < 0.001). F. nucleatum was over-represented in 67/80 (83.8%) CRC samples. The location of CRC and tumor size were significantly associated with abundance of Fn (P < 0.051, P < 0.023, respectively). No significant association of F. nucleatum with other clinico-pathological variables was observed (P > 0.05). To conclude, F. nucleatum was enriched in CRC tissues and associated with CRC development. These results imply that assessing F. nucleatum levels could aid in predicting clinical outcomes for colorectal cancer patients.

Colorectal cancer (CRC) is both preventable and curable if diagnosed at early stage, and yet it is the second most common cause of cancer deaths in US. Recent emphasis on the role of human microbiome in cancer led us to investigate new microbial diagnostic marker for CRC. CRC originates in the colon or rectum as a polyp that may invade into surrounding tissue and metastasize to secondary sites through epithelial to mesenchymal transition (EMT) of the cancer cells. Fusobacterium nucleatum, an abundant opportunistic bacterium, is often found in the gut and oral cavity, and implicated in promoting colorectal carcinogenesis. F. nucleatum is known to bind E‐Cadherin, an epithelial cell surface adherence protein, through amyloid‐like adhesin fadA, which initiates EMT. FadA mediates the attachment and invasion of F. nucleatum and activates β‐catenin signaling, causing inflammatory and oncogenic responses. This study sought to assess F. nucleatum and EMT biomarkers in CRC. De‐identified formalin fixed paraffin embedded tissue samples of different stages in CRC, classified as normal, precancerous, and cancerous, were immunostained for E‐Cadherin and Vimentin as markers for epithelial and mesenchymal cells respectively. We used amyloid‐like adhesin fadA immunostaining as a marker for F. nucleatum. As expected, normal tissue specimens expressed E‐Cadherin exclusively in the mucosal epithelial cells while mesenchymal cells in the stroma specifically stained for Vimentin expression. However, E‐Cadherin was found to be downregulated in cancer tissue specimens, signifying a partial EMT. Further, some precancerous and cancerous specimens showed Vimentin‐positive foci within epithelial cells that lacked E‐Cadherin expression implicating EMT. We also observed the presence of F. nucleatum secreted amyloid‐like fadA in cancer tissue specimen. Amyloid‐like fadA plays a key role in transformation of F. nucleatum from commensal to pathogen, hence, its detection in the colon may aid in early detection of CRC and provide an opportunity for targeted intervention. Its role in EMT, however, requires detailed molecular analyses. Supplementing the routine histopathological evaluation with these specific biomarkers would provide more predictive and detailed analysis of cancer initiation and progression in tissue samples to aid in personalized treatment plan with the goal of improved disease outcome.

Circular RNAs (circRNAs) play crucial roles in multiple cancers, including colorectal cancer (CRC). Here, we explored the role of circRNA ArfGAP with coiled-coil, ankyrin repeat and PH domains 2 (circ-ACAP2) in the progression and radioresistance of CRC. Quantitative real-time polymerase chain reaction (qPCR) and Western blot assay were used to detect RNA and protein expression, respectively. The proliferation, apoptosis, migration, invasion and radioresistance of CRC cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, transwell migration assay, transwell invasion assay and colony formation assay. The target interaction between microRNA-143-3p (miR-143-3p) and circ-ACAP2 or frizzled class receptor 4 (FZD4) was verified by dual-luciferase reporter assay. Murine xenograft model was established to explore the role of circ-ACAP2 in vivo. The expression of circ-ACAP2 was prominently enhanced in CRC tissues and cell lines. Circ-ACAP2 facilitated the proliferation, migration, invasion and radioresistance whereas inhibited the apoptosis of CRC cells. MiR-143-3p was a direct target of circ-ACAP2 in CRC cells. Circ-ACAP2 promoted the progression and radioresistance of CRC partly by sponging miR-143-3p. MiR-143-3p interacted with the 3' untranslated region (3'UTR) of FZD4 in CRC cells, and FZD4 overexpression partly reversed miR-143-3p-mediated effects in CRC cells. Wnt/β-catenin signalling was modulated by circ-ACAP2/miR-143-3p/FZD4 axis in CRC cells. Circ-ACAP2 contributed to the development and radioresistance of CRC partly through targeting miR-143-3p/FZD4 axis, which provided novel potential diagnostic and therapeutic targets for CRC.

The aim of the present study was to investigate the effect of the actin‑binding protein Girdin on the proliferation, invasion and migration of colorectal cancer (CRC) cells. Cultured CRC cells (LoVo cell line) were transfected by Girdin‑specific and control shRNA constructs and analyzed for proliferation, invasion and migration by the MTT, Transwell and wound‑healing assays, respectively. The activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway and expression of proinflammatory cytokines was examined by western blotting and ELISA assay, respectively. The effect of Girdin silencing on CRC growth was also evaluated in a xenograft model using nude mice, which were subcutaneously injected with Girdin‑deficient and negative control LoVo cells and analyzed for tumor volume and weight. Transfection of LoVo cells with Girdin‑specific shRNA inhibited Girdin mRNA expression to 27.5% and protein expression to 36.7% when compared with expression levels in the control cells (P<0.001) and significantly demonstrated suppression of LoVo cell proliferation (P<0.05), invasion (P<0.01) and migration (P<0.01). Furthermore, Girdin silencing downregulated the phosphorylation of the signaling proteins JAK (by 42%, P<0.001) and STAT3 (by 34%, P<0.01) and the content of IFN (by 28%, P<0.001) and IL‑6 (by 44%, P<0.001) compared to the control. Notably, inhibition of Girdin expression effectively suppressed tumorigenicity of LoVo cells invivo as evidenced by the reduced volume (P<0.05) and weight (P<0.05) of the tumors derived from Girdin shRNA‑transfected LoVo cells compared to those from the control cells. In conclusion, the silencing of Girdin expression inhibited the malignant behavior of CRC cells via the downregulation of the JAK/STAT signaling pathway, indicating Girdin as a potential therapeutic target in CRC. In the present study, we revealed, for the first time, that the malignant behavior of CRC cells depended on the expression of an actin‑binding protein, Girdin. Silencing of Girdin expression by specific shRNA suppressed the proliferation, invasion, and migration of CRC cells through the decrease in proinflammatory cytokines IFN and IL‑6 and the downregulation of the JAK/STAT signaling pathway. Our findings indicated that Girdin expression may be a potential novel therapeutic target in CRC.

PurposeThe lncRNA IGFL2‐AS1 is a known cancer‐promoting factor in colorectal cancer (CRC); nonetheless, the mechanism of its carcinogenic effects has not yet been elucidated. This study elaborated on the role and underlying molecular mechanism of IGFL2‐AS1 in promoting CRC cell functions.MethodsIGLF2‐AS1 expression levels in CRC tissue/normal tissue and CRC cell line/normal colon epithelial cell line were detected by quantitative real‐time polymerase chain reaction. Cell counting kit‐8, colony formation assay, and EdU assay were performed to assess the effect of IGFL2‐AS1 knockdown or overexpression on the proliferative capacity of CRC cells. The migration and invasion abilities of LoVo cells were measured using transwell assay. The expression relationship between IGFL2‐AS1 and carbonic anhydrase 9 (CA9) and the CA9 expression level in CRC tissues and cells was verified by transcriptome sequencing, western blotting, and immunohistochemical staining. Treatment with MG132 and cycloheximide was utilized to explore the mechanism by which IGFL2‐AS1 affects the hypoxia‐inducible factor‐1α (HIF‐1α)/CA9 pathway. A nude mouse xenograft model was constructed to evaluate the effect of IGFL2‐AS1 on CRC growth in vivo.ResultsWe discovered that IGFL2‐AS1 was highly upregulated in CRC tumor tissues and cells. IGFL2‐AS1 can functionally promote CRC cell proliferation, migration, and invasion in vitro and accelerate CRC occurrence in vivo. Mechanistic studies demonstrated that IGFL2‐AS1 upregulated the CA9 level by affecting the degradation pathway of HIF‐1α, which elucidates its pro‐proliferative effect in CRC. The lncRNA IGFL2‐AS1 mediated the inhibition of HIF‐1α degradation in CRC and increased CA9 expression, thereby promoting CRC progression.ConclusionOur findings suggested that IGFL2‐AS1 is expected to be a promising new diagnostic marker and therapeutic target for CRC.

Aberrant expression of numerous microRNAs (miRNAs/miRs) in colorectal cancer (CRC) significantly affects disease progression. Recently, miR‑629‑5p (miR‑629) was identified as a tumor‑promoting miRNA in the malignant processes of a number of human cancers. However, few studies have been conducted regarding expression profiles and detailed roles of miR‑629 in CRC. In the present study, reverse transcription‑quantitative polymerase chain reaction was used to assess miR‑629 expression in CRC tissues and cell lines. Cell Counting Kit‑8 assay, flow cytometry and Transwell assays were performed to determine the in vitro effects of miR‑629 on CRC cell proliferation, apoptosis, and metastasis, respectively. Xenograft models were employed to determine the in vivo effects of miR‑629 on tumor growth in nude mice. Molecular mechanisms underlying the activity of miR‑629 in CRC cells were explored. miR‑629 expression decreased in CRC tissues and cell lines. The decreased aberrant miR‑629 expression was significantly associated with tumor size, lymphatic metastasis and tumor‑node‑metastasis stage of CRC, and was a predictor of poor prognosis. Restoring miR‑629 expression attenuated CRC cell proliferation, migration and invasion; promoted cell apoptosis in vitro; and inhibited tumor growth in vivo. Low‑density lipoprotein receptor‑related protein 6 (LRP6) was a direct target gene of miR‑629 in CRC cells. Furthermore, the effect of LRP6 knockdown was similar to that of miR‑629 overexpression in CRC cells. Restoration of LRP6 expression neutralized the effects of miR‑629 in CRC cells. miR‑629 suppressed the activation of the Wnt/β‑catenin pathway through LRP6 regulation both in vitro and in vivo. In conclusion, miR‑629 suppressed the development and progression of CRC by directly targeting LRP6 and inhibiting the Wnt/β‑catenin pathway both in vitro and in vivo. Therefore, miR‑629 may be a novel prognostic biomarker and therapeutic target in CRC.

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, and many oncogenes and tumor suppressor genes are involved in CRC. MicroRNAs (miRNAs) are small non-coding RNAs that can negatively regulate gene expression. Previous studies have revealed that miRNAs regulate the development and progression of many cancers. In this study, we investigated the role of microRNA-30a-5p (miR-30a) in CRC and its unknown mechanisms. qRT-PCR was used to detect miR-30a and TM4SF1 mRNA expression in CRC specimens and cell lines. CRC cell migration and invasion were assessed after transfection with miR-30a or TM4SF1 using wound healing and trans-well migration and invasion assays. Transmembrane-4-L-six-family protein (TM4SF1) was validated as a target of miR-30a in CRC through luciferase reporter assay and bioinformatics algorithms. Moreover, two EMT regulators, E-cadherin and VEGF, were also identified using Western blotting and immunohistochemistry. We found that miR-30a was down-regulated in CRC tumor tissues and cell lines, and miR-30a was inversely associated with advanced stage and lymph node metastatic status compared with normal tissues. miR-30a decreased migration and invasion in CRC cell lines, and miR-30a overexpression not only down-regulated TM4SF1 mRNA and protein expression, but also inhibited the expression of VEGF and enhanced expression of E-cadherin. We also showed that TM4SF1 was up-regulated in CRC tumor specimens compared with adjacent normal tissues, and TM4SF1 expression was significantly associated with advanced stage and lymph node status compared with adjacent normal tissues. These results suggest that miR-30a is an important regulator of TM4SF1, VEGF, and E-cadherin for CRC lymph node metastasis, a potential new therapeutic target in CRC.

BackgroundAlthough constitutive activating mutations in the Wnt/β-catenin signalling pathway are important for colorectal cancer development, canonical signalling through Wnt ligands is essential for β-catenin activation. Here, we investigated the role of (pro)renin receptor ((P)RR), a component of the Wnt receptor complex, in the pathogenesis of colorectal cancer.Methods(P)RR silencing was performed in human colorectal cancer cells containing constitutive activating mutations in the Wnt/β-catenin pathway. (P)RR overexpression was induced in normal colon epithelial cells. Protein and mRNA levels of pathway components were detected, and Wnt signalling activity was measured using a β-catenin reporter. Cell proliferative activity and apoptosis were evaluated using WST-1 assay and flow cytometry. Xenografts were induced in nude mice.Results(P)RR expression was greater in colorectal cancer tissues and cells than in normal colorectal samples. Patients with strong (P)RR expression took more proportion in groups with poorly-differentiated, advanced and rapidly-progressing cancers. (P)RR silencing attenuated the pathway in colorectal cancer cells, impaired their proliferation in vitro and vivo. (P)RR overexpression enhanced the pathway and proliferation of normal colon epithelial cells.ConclusionsAberrant (P)RR expression promotes colorectal cancer through the Wnt/β-catenin signalling pathway despite constitutive pathway-activating mutations. (P)RR is a potential diagnostic and therapeutic target for colorectal cancer.

Circular RNAs (circRNAs) are widely expressed in cancer tissues and participate in modulating the progression of malignant tumors, playing a pro- or anti-cancer role. This work is conducted to probe the precise role of circ_0000467 in colorectal cancer (CRC) and its regulatory mechanism. The differentially expressed circRNAs in CRC tissues and paracancerous tissues were screened by bioinformatics analysis. The expression levels of circ_0000467, miR-651-5p and DNA methyltransferases 3B (DNMT3B) mRNA in CRC tissues and cells were detected by qRT-PCR. circ_0000467 knockdown cell model was constructed to investigate the effects of circ_0000467 on CRC cell growth, migration and invasion by CCK-8 and Transwell experiments. Western blot was performed to examine DNMT3B protein expression in CRC cells. Dual-luciferase reporter gene experiment was executed to validate the targeting relationship between circ_0000467 and miR-651-5p, miR-651-5p and DNMT3B. Circ_0000467 expression and DNMT3B mRNA expression were increased and miR-651-5p expression was down-regulated in CRC tissues and cell lines. Knockdown of circ_0000467 repressed CRC cell growth, migration and invasion. Dual-luciferase reporter gene experiments validated that miR-651-5p was a direct target of circ_0000467 and miR-651-5p could specifically bind with DNMT3B 3’UTR. Functional compensation experiments showed that the regulatory effect of circ_0000467 on CRC cells’ behaviors could be partially counteracted by miR-651-5p. Circ_0000467 may enhance the growth and metastasis of CRC cells by targeting miR-651-5p and up-regulating DNMT3B expression. Circ_0000467 may be a potential diagnostic biomarker and therapeutic target for CRC.

Purpose: Dysregulated expression of miRNAs has emerged as a hallmark feature in human cancers. Exportin-5 (XPO5), a karyopherin family member, is a key protein responsible for transporting precursor miRNAs from the nucleus to the cytoplasm. Although XPO5 is one of the key regulators of miRNA biogenesis, its functional role and potential clinical significance in colorectal cancer remains unclear.Experimental Design: The expression levels of XPO5 were initially assessed in three genomic datasets, followed by determination and validation of the relationship between XPO5 expression and clinicopathologic features in two independent colorectal cancer patient cohorts. A functional characterization of XPO5 in colorectal cancer was examined by targeted gene silencing in colorectal cancer cell lines and a xenograft animal model.Results: XPO5 is upregulated, both at mRNA and protein levels, in colorectal cancers compared with normal tissues. High XPO5 expression is associated with worse clinicopathologic features and poor survival in colorectal cancer patient cohorts. The siRNA knockdown of XPO5 resulted in reduced cellular proliferation, attenuated invasion, induction of G1-S cell-cycle arrest, and downregulation of key oncogenic miRNAs in colorectal cancer cells. These findings were confirmed in a xenograft animal model, wherein silencing of XPO5 resulted in the attenuation of tumor growth.Conclusions: XPO5 acts like an oncogene in colorectal cancer by regulating the expression of miRNAs and may be a potential therapeutic target in colorectal cancer. Clin Cancer Res; 23(5); 1312-22. ©2016 AACR.

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Dysregulated expression of miRNAs has emerged as a hallmark feature in human cancers. Exportin-5 (XPO5), a karyopherin family member, is a key protein responsible for transporting precursor miRNAs from the nucleus to the cytoplasm. Although XPO5 is one of the key regulators of miRNA biogenesis, its functional role and potential clinical significance in colorectal cancer remains unclear.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; The expression levels of XPO5 were initially assessed in three genomic datasets, followed by determination and validation of the relationship between XPO5 expression and clinicopathologic features in two independent colorectal cancer patient cohorts. A functional characterization of XPO5 in colorectal cancer was examined by targeted gene silencing in colorectal cancer cell lines and a xenograft animal model.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; XPO5 is upregulated, both at mRNA and protein levels, in colorectal cancers compared with normal tissues. High XPO5 expression is associated with worse clinicopathologic features and poor survival in colorectal cancer patient cohorts. The siRNA knockdown of XPO5 resulted in reduced cellular proliferation, attenuated invasion, induction of G&lt;sub&gt;1&lt;/sub&gt;–S cell-cycle arrest, and downregulation of key oncogenic miRNAs in colorectal cancer cells. These findings were confirmed in a xenograft animal model, wherein silencing of XPO5 resulted in the attenuation of tumor growth.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; XPO5 acts like an oncogene in colorectal cancer by regulating the expression of miRNAs and may be a potential therapeutic target in colorectal cancer. &lt;i&gt;Clin Cancer Res; 23(5); 1312–22. ©2016 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Dysregulated expression of miRNAs has emerged as a hallmark feature in human cancers. Exportin-5 (XPO5), a karyopherin family member, is a key protein responsible for transporting precursor miRNAs from the nucleus to the cytoplasm. Although XPO5 is one of the key regulators of miRNA biogenesis, its functional role and potential clinical significance in colorectal cancer remains unclear.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; The expression levels of XPO5 were initially assessed in three genomic datasets, followed by determination and validation of the relationship between XPO5 expression and clinicopathologic features in two independent colorectal cancer patient cohorts. A functional characterization of XPO5 in colorectal cancer was examined by targeted gene silencing in colorectal cancer cell lines and a xenograft animal model.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; XPO5 is upregulated, both at mRNA and protein levels, in colorectal cancers compared with normal tissues. High XPO5 expression is associated with worse clinicopathologic features and poor survival in colorectal cancer patient cohorts. The siRNA knockdown of XPO5 resulted in reduced cellular proliferation, attenuated invasion, induction of G&lt;sub&gt;1&lt;/sub&gt;–S cell-cycle arrest, and downregulation of key oncogenic miRNAs in colorectal cancer cells. These findings were confirmed in a xenograft animal model, wherein silencing of XPO5 resulted in the attenuation of tumor growth.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; XPO5 acts like an oncogene in colorectal cancer by regulating the expression of miRNAs and may be a potential therapeutic target in colorectal cancer. &lt;i&gt;Clin Cancer Res; 23(5); 1312–22. ©2016 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Zinc-finger proteins play different roles in cancer; however, the function of zinc-finger protein ZNF575 in cancer remains unclear. In the present study, we aimed to determine the function and expression of ZNF575 in colorectal cancer. Proliferation assay, colony formation assay, and tumor model in mice were used to investigate the function of ZNF575 after ectopic expression of ZNF575 in colorectal cancer (CRC) cells. RNA sequencing, ChIP, and luciferase assays were used to investigate the mechanism behind ZNF575 regulation of CRC cell growth. The expression of ZNF575 was determined by IHC staining in 150 pairs of malignant CRC tissues, followed by prognosis analysis. We indicated that ectopic expression of ZNF575 inhibited CRC cell proliferation, colony formation and promoted cell apoptosis in vitro. Tumor growth in CRC was also impaired by ZNF575 in mice. RNA sequencing, follow-up western blotting, and qPCR results demonstrated the increase of p53, BAK, and PUMA in ZNF575-expressing CRC cells. Further results indicated that ZNF575 directly targeted the p53 promoter and promoted the transcription of p53. Downregulation of ZNF575 was confirmed in malignant tissues, and ZNF575 expression was positively correlated with the prognosis of CRC patients. The present study demonstrated the function, underlying mechanism, expression, and the prognosis-predicting role of ZNF575 in CRC, which indicated that ZNF575 would be a potential prognostic predictor and therapeutic target for CRC and other cancers.

PAD4-dependent citrullination of nuclear translocation of GSK3β promotes colorectal cancer progression via the degradation of nuclear CDKN1A

Properties and Clinical Relevance of Speckle-Type POZ Protein in Human Colorectal Cancer