Expression In Colorectal Cancer Cells Research Articles

SNHG4-mediated PTEN destabilization confers oxaliplatin resistance in colorectal cancer cells by inhibiting ferroptosis.

Oxaliplatin resistance poses a significant challenge in colorectal cancer (CRC) therapy, necessitating further investigation into the underlying molecular mechanisms. This study aimed to elucidate the regulatory role of SNHG4 in oxaliplatin resistance and ferroptosis in CRC. Our findings revealed that treatment with oxaliplatin led to downregulation of SNHG4 expression in CRC cells, while resistant CRC cells exhibited higher levels of SNHG4 compared to parental cells. Silencing SNHG4 attenuated oxaliplatin resistance and reduced the expression of resistance-related proteins MRD1 and MPR1. Furthermore, induction of ferroptosis effectively diminished oxaliplatin resistance in both parental and resistant CRC cells. Notably, ferroptosis induction resulted in decreased SNHG4 expression, whereas SNHG4 overexpression suppressed ferroptosis. Through FISH, RIP, and RNA pull-down assays, we identified the cytoplasmic localization of both SNHG4 and PTEN, establishing that SNHG4 directly targets PTEN, thereby reducing mRNA stability in CRC cells. Silencing PTEN abrogated the impact of SNHG4 on oxaliplatin resistance and ferroptosis in CRC cells. In vivo experiments further validated the influence of SNHG4 on oxaliplatin resistance and ferroptosis in CRC cells through PTEN regulation. In conclusion, SNHG4 promotes resistance to oxaliplatin in CRC cells by suppressing ferroptosis through instability of PTEN, thus serves as a target for patients with oxaliplatin-base chemoresistance.

Downregulation of ABCC3 activates MAPK signaling through accumulation of deoxycholic acid in colorectal cancer cells.

ABCC3 (also known as MRP3) is an ATP binding cassette transporter for bile acids, whose expression is downregulated in colorectal cancer through the Wnt/β-catenin signaling pathway. However, it remained unclear how downregulation of ABCC3 expression contributes to colorectal carcinogenesis. We explored the role of ABCC3 in the progression of colorectal cancer-in particular, focusing on the regulation of bile acid export. Gene expression analysis of colorectal adenoma isolated from familial adenomatous polyposis patients revealed that genes related to bile acid secretion including ABCC3 were downregulated as early as at the stage of adenoma formation. Knockdown or overexpression of ABCC3 increased or decreased intracellular concentration of deoxycholic acid, a secondary bile acid, respectively, in colorectal cancer cells. Forced expression of ABCC3 suppressed deoxycholic acid-induced activation of MAPK signaling. Finally, we found that nonsteroidal anti-inflammatory drugs increased ABCC3 expression in colorectal cancer cells, suggesting that ABCC3 could be one of the targets for therapeutic intervention of familial adenomatous polyposis. Our data thus suggest that downregulation of ABCC3 expression contributes to colorectal carcinogenesis through the regulation of intracellular accumulation of bile acids and activity of MAPK signaling.

Urolithin A affects cellular migration and modulates matrix metalloproteinase expression in colorectal cancer cells.

Colorectal cancer (CRC) is the world's second most common gastrointestinal malignancy. Preventing tumor cell proliferation and dissemination is critical for patient survival. Polyphenols have a variety of health advantages and can help prevent cancer. The current study examined different cellular activities of the gut-microbiota metabolite urolithin A (UA) on several colon cancer cell lines. The results revealed that UA suppressed cell growth in a dose- and time-dependent manner. In the current investigation, UA substantially affected cell migration in the wound-healing experiment and greatly decreased the number of colonies generated in each CRC cell culture. UA decreased cellular migration in CRC cells 48 h after treatment, which was significant (p < .001) compared to the migration rate in untreated cells. When compared to untreated cells, UA slowed the process of colony formation by reducing the number of colonies or altering their morphological shape. The western blot analysis investigation revealed that UA inhibits cellular metastasis by lowering the expression levels of matrix metalloproteinases 1 and 2 (MMP1 and MMP2) by more than 43% and 41% (p < .001) in HT29, 28% and 149% (p < .001) in SW480, and 90% and 74% (p < .001) in SW620, respectively, at a 100 µM dosage of UA compared to the control. Surprisingly, at a 100 µM dosage of UA, the expression levels of the tissue inhibitor of metalloproteinases 1 (TIMP1) were elevated in HT29, SW480, and SW620 cells treated with 100 µM of UA by more than 89%, 57%, and 29%, respectively. Our findings imply that UA has anticancer properties and might be used therapeutically to treat CRC. The findings provided the first indication of the influence of UA on cellular migration and metastasis in colon cancer cells. All of these data showed that UA might be used as an adjuvant therapy in the treatment of various forms of CRC.

Sulforaphane Inhibits IL-1β-Induced IL-6 by Suppressing ROS Production, AP-1, and STAT3 in Colorectal Cancer HT-29 Cells

Colorectal cancer (CRC) stands as a major cause of cancer-related mortality globally, accounting for approximately 881,000 deaths each year. Traditional approaches such as chemotherapy and surgery have been the primary treatment modalities, yet the outcomes for patients with metastatic CRC are often unsatisfactory. Recent research has focused on targeting the pathways involved in oxidative stress, inflammation, and metastasis to enhance the survival of CRC patients. Within this context, sulforaphane (SFN), a notable phytochemical found predominantly in cruciferous vegetables, has been recognized as a potential anticancer agent. However, the specific mechanisms through which SFN may exert its chemopreventive effects in CRC remain unclear. This study explores the impact of SFN on IL-1β-induced IL-6 activation and MAPK and AP-1 signaling in HT-29 cells. Our findings reveal that SFN treatment not only diminishes IL-1β-stimulated IL-6 expression but also reduces oxidative stress by curtailing reactive oxygen species (ROS) production. Furthermore, it hinders the proliferation and invasiveness of HT-29 cells through the modulation of MAPK/AP-1 and STAT3 signaling pathways. These results indicate that SFN mitigates IL-1β-induced IL-6 expression in CRC cells by attenuating ROS production and disrupting MAPK/AP-1 signaling. This suggests that SFN holds significant potential as a chemotherapeutic agent for both treating and preventing CRC.

Abstract 1479: ABCC3 (MRP3)-mediated export of deoxycholic acid regulates MAPK signaling in colorectal adenoma and adenocarcinoma

Abstract ABCC3 (also known as MRP3) is an ATP binding cassette transporter and it works as a bile acids transporter. We previously elucidated that ABCC3 expression is downregulated in colorectal cancer through the Wnt/β-catenin signaling pathway. However, it remained unclear how downregulation of ABCC3 expression contributes to colorectal carcinogenesis. We here explored the role of ABCC3 in the progression of colorectal cancer-in particular, focusing on the regulation of bile acid export. First, we performed experiments with clinical samples of familial adenomatous polyposis (FAP) patients to confirm whether ABCC3 expression is already downregulated in colorectal adenomas. Next, we investigated the effect of ABCC3 expression on intracellular bile acid concentrations using ABCC3 knockdown and overexpression colorectal cancer cell lines (HT-29, SW620). The intracellular concentration of deoxycholic acid, which is known to have carcinogenic effects, was verified with fluorescently labeled deoxycholic acid. Then, we tested whether activation of oncogenic signaling by deoxycholic acid is affected by ABCC3 expression changes. Finally, we examined the effect of nonsteroidal anti-inflammatory drugs (NSAIDs), which have been suggested to inhibit the carcinogenesis of FAP, on the expression of ABCC3 in experiments with colorectal cancer cell lines. Gene and protein expression analysis of colorectal adenomas isolated from FAP patients revealed that ABCC3 were downregulated as early as at the stage of adenoma formation, and many other genes related to bile acid efflux were also downregulated. Knockdown or overexpression of ABCC3 increased or decreased intracellular concentration of deoxycholic acid in colorectal cancer cells, respectively. Forced expression of ABCC3 suppressed deoxycholic acid-induced activation of MAPK signaling. Finally, we found that several NSAIDs increased ABCC3 expression in colorectal cancer cells, suggesting that ABCC3 could be one of the targets for therapeutic intervention of FAP. Our data thus suggest that downregulation of ABCC3 expression contributes to colorectal carcinogenesis through the regulation of intracellular accumulation of bile acids and activity of MAPK signaling. Citation Format: Yukihiro Sato, Minoru Kobayashi, Masahiro Ohira, Ryo Funayama, Masamitsu Maekawa, Hideaki Karasawa, Shinobu Ohnuma, Michiaki Unno, Keiko Nakayama. ABCC3 (MRP3)-mediated export of deoxycholic acid regulates MAPK signaling in colorectal adenoma and adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1479.

Abstract 376: Targeting the convergence on HIF-1α of CDK4/6 and MAPK pathway: Implications for enhanced anticancer strategies

Abstract Hypoxia-inducible factor 1-alpha (HIF-1α) plays a pivotal role in orchestrating cellular responses to hypoxia, influencing cancer cell survival and progression. Our previous work identified a non-canonical mechanism wherein Smurf2 mediates HIF-1α degradation under CDK4/6 inhibition. Through proteomic analysis, we discovered that serine 451 phosphorylation occurs on HIF-1α but not in palbociclib-treated samples. Point mutations at this site, substituting serine with alanine, resulted in decreased HIF-1α levels and enhanced interaction with Smurf2. Intriguingly, under palbociclib treatment, we observed phosphorylation at the serine 643 site. This site has been previously associated with MAPK-dependent regulation of HIF-1α localization and activity (Ilias Mylonis, et al., 2006), particularly relevant given the reported MAPK reliance in acquired CDK4/6 inhibitor-resistant scenarios (Renée de Leeuw, et al., 2018). To explore therapeutic implications, we investigated the impact of combined CDK4/6 (palbociclib) and MEK1/2 inhibition (trametinib, selumetinib, PD98059, U0126). Dual inhibition robustly reduced HIF-1α expression in colorectal cancer cells (HCT116, SW480) and suppressed HIF-1α activity in luciferase reporter assays. This effect extended to synergistic inhibition of cell viability under both normoxia and hypoxia in HCT116 and SW480 cells. Such effect is also applicable to other cancer types and cell lines (e.g. U251). In summary, our findings unveil a phosphorylation site on HIF-1α associated with CDK4/6 activity, influencing its protein stabilization. This discovery supports the rationale for combining CDK4/6 and MEK1/2 inhibition as a promising strategy in the treatment of solid tumors. Citation Format: Shuai Zhao, Lanlan Zhou, Shengliang Zhang, Wafik S. El-Deiry. Targeting the convergence on HIF-1α of CDK4/6 and MAPK pathway: Implications for enhanced anticancer strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 376.

Abstract 4125: Exploring the efficacy of alpelisib and combined PI3Kα-S6K inhibition in LIN28B-mediated colorectal cancer metastasis

Abstract Introduction: Colorectal cancer (CRC) is the third most diagnosed cancer worldwide. LIN28B, an RNA-binding protein that regulates mRNA translation, has garnered significant interest in recent cancer research. Our lab has established that LIN28B is an oncogene and promotes CRC metastasis. However, the exact mechanisms through which LIN28B exerts these effects remain elusive. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is altered in 50-70% of CRCs and is characterized by PIK3CA gene mutations in 20-30% of CRC cases. PIK3CA encodes the catalytic subunit p110α of the PI3Kα enzyme, which is critical for PI3K/AKT signaling. Activation of this pathway is associated with worse CRC prognosis. Among the components of this signaling cascade, the ribosomal protein S6 kinase (S6K) is a crucial downstream effector, playing a pivotal role in protein synthesis and cell growth. Despite the frequent occurrence of PIK3CA mutations in CRC, targeted therapies for PIK3CA-mutant CRC are lacking. Alpelisib, a PI3Kα-specific inhibitor approved by the FDA for a subset of PIK3CA-mutant breast cancers, remains largely unassessed in CRC. We hypothesize that LIN28B-mediated CRC metastasis is facilitated by the activation of the PI3K/AKT pathway, representing a potential target for Alpelisib or other related inhibitors. Results: Our data indicate that LIN28B expression in CRC cells promotes liver metastasis in mouse models of metastatic CRC (mCRC). Furthermore, LIN28B expression activates the PI3K/AKT pathway, as measured by an AKT phosphorylation array. Treatment of CRC cells with SC79, a pan-AKT activator, enhanced cell migration and invasiveness. Similarly, VillinCre;R26Pik3ca transgenic mice, expressing a constantly active mutant form of PI3Kα in the intestinal epithelium, demonstrated crypt hyperplasia and an amplified propensity for tumorigenesis, mirroring the effects of LIN28B expression in CRC. Importantly, Alpelisib effectively hinders LIN28B-driven CRC progression in cell lines, transgenic mice-derived organoids, and mCRC mouse models, as well as the growth rate of CRC patient-derived organoids (PDOs). The effects observed with Alpelisib treatment were replicated with inhibition of S6K using either LY25284702 or PF4708671. Moreover, a combination of low-dose Alpelisib with low-dose LY25284702 or PF4708671 synergistically restricted the growth of PDOs. Conclusion: Our data provide compelling evidence that the PI3K/AKT pathway regulates LIN28B-mediated CRC metastasis, and its inhibition by Alpelisib is feasible. This underscores the potential for precision medicine in CRC, offering targeted therapies to PIK3CA-mutant CRC patients who lack specific treatment options. Furthermore, our study highlights the clinical advantages of combined inhibition strategies for improved anti-metastatic outcomes, a field in dire need of interventions in mCRC. Citation Format: Alice E. Shin, Kensuke Sugiura, Secunda W. Kariuki, David A. Cohen, Christopher J. Lengner, Peter A. Sims, Joel T. Gabre, Anil K. Rustgi. Exploring the efficacy of alpelisib and combined PI3Kα-S6K inhibition in LIN28B-mediated colorectal cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4125.

Abstract 5880: Induction of the p-glycoprotein with atorvastatin using colorectal cancer cells

Abstract Chemotherapy resistance is responsible for a majority of the cancer-associated mortalities. Efflux of the P-glycoprotein (P-gp) transporter is one of the many causes for chemotherapy resistance. The increased activity of the P-gp on the surface of the tumor cells leads to the removal chemotherapy drugs and allows a cancer to survive. The purpose of this study was to evaluate how atorvastatin impacts the P-gp expression in colorectal cancer cells. The colorectal cells, CACO2 cell line, were separated into drug treatment groups. The experimental groups were treated with the statin drug, atorvastatin, at different concentrations and doxorubicin as the positive control, for 3-4 months. Atorvastatin was selected for the study since it is a P-gp substrate and a potential inducer. Exon-spanning primers for the ABCB1 gene were used to monitor changes in mRNA expression of the P-gp. Once overexpression of the ABCB1 gene was achieved, the cells were tested for protein expression through western blots and flow cytometry. The P-pg-induced cells, which consisted of the atorvastatin-treated and doxorubicin-treated cells, and the untreated cells were then tested for chemotherapy-resistance using the PrestoBlue proliferation assay. The atorvastatin treatments were repeated for reproducibility by using two other colorectal cancer cell lines. The P-pg-induced cells, which consisted of the atorvastatin-treated and doxorubicin-treated cells, and the untreated cells were also studied for stability by removing the treatments of the atorvastatin for three months and monitoring for changes in expression of the ABCB1 gene with rt-qPCR. Drug pathways of the treated cells were also studied for expression changes using RNA Sequencing (RNA-Seq). For the results, the overexpression of the ABCB1 gene occurred in the 4th month of the study of the bi-weekly atorvastatin and doxorubicin treatments using the CACO2 cells. Protein assays were able to confirm the P-gp overexpression in the atorvastatin-treated and doxorubicin-treated cells. The chemotherapy-resistant assay revealed that the atorvastatin-treated cells were just as drug resistant to the chemotherapy as the doxorubicin-treated cells. Through the reproducibility experiment, the overexpression of the ABCB1 gene was observed in two other cell lines after long-term treatment of the drug therapy. Removal of the atorvastatin for three months revealed that the P-gp overexpression was not reversible. The RNA-Seq analysis identified other possible drug resistant markers in the atorvastatin pathway. This study implicates that late-stage cancer patients, who are treated for hypercholesterolemia, may be at higher risk for developing chemotherapy-resistant tumor cells. With the identification of chemotherapy-resistant tumors in chemotherapy-naïve cancer patients, doctors will be able to provide more precise approaches to treating patients through effective drug therapies. Citation Format: Christopher L. Farrell, Amy Messersmith, Wei Lei, Brianna Dyer. Induction of the p-glycoprotein with atorvastatin using colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5880.

Abstract 3169: The role and mechanism of m6A methylation modification of LncRNA-ATB in regulating the invasion and metastasis of colorectal cancer

Abstract Background: The epigenetic modification of LncRNA N6-methyladenosine (m6A) methylation plays a wide role in the invasion and metastasis of various malignant tumors. However, the role of LncRNA-ATB m6A methylation modification in colorectal cancer (CRC) has not been systematically elucidated. Methods: Detect the regulatory effect of ALKBH5 on LncRNA-ATB by Luciferin reporter gene. LncRNA-ATB expression was detected by IHC and qPCR in CRC tissues and cells. Transwell and scratch tests were used to detect the effect of ALKBH5 overexpression after LncRNA-ATB knockdown and replacement on the invasion and migration ability of CRC cells. Western blotting was used to detect the expression changes of MMPs proteins. Results: ALKBH5 could regulate the methylation level of the LncRNA-ATB 3’ UTR region (p&amp;lt;0.05). ALKBH5 overexpression in CRC cells led to a significant decrease in the expression level of LncRNA-ATB (p&amp;lt;0.05). LncRNA-ATB exhibited elevated expression in CRC cells and demonstrated significantly higher levels in CRC tissues compared to paracancerous tissues (p&amp;lt;0.05), indicating the potential tumor-promoting effect of LncRNA-ATB in CRC, with the mechanism possibly regulated by ALKBH5-mediated m6A demethylation. ALKBH5 overexpression inhibited the proliferation and migration of CRC cells after LncRNA-ATB knockdown. However, this inhibitory effect was attenuated upon LncRNA-ATB rescue, accompanied by an increase in MMP9 and a decrease in TIMP1. Conclusion: LncRNA-ATB plays a crucial role in ALKBH5-mediated m6A demethylation, regulating the invasion and metastasis of CRC. The ALKBH5-LncRNA-ATB m6A demethylation-MMPs signaling axis has been identified as a key regulatory pathway promoting CRC migration. m6A modification of lncRNA-ATB modulated by ALKBH5 provides a theoretical basis for novel therapeutic strategies for patients with metastatic CRC. FUNDING: This study was supported by the National Natural Science Foundation of China (Grant No. 81702414), Natural Science Foundation of Fujian Province of China (Grant No. 2020J05306) and Xiamen Science and Technology Planning Project (Grant No. 3502Z20194003). Citation Format: Yu Lang#, Jia-Lin Lin#, Jia-Peng Kang, Lu Yang, Dan Zhou, Can-Bin Fang*, Feng Ye*, Wei-wei Tang*. The role and mechanism of m6A methylation modification of LncRNA-ATB in regulating the invasion and metastasis of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3169.

Abstract 5599: Role of HOX gene expression in differentiation of colon cancer stem cells

Abstract HOX genes encode a highly conserved set of transcription factors critical for stem cell (SC) function and embryonic development. Several HOX genes have also been implicated in a myriad of cancers, including colorectal cancer (CRC). However, how HOX genes play a role in SC regulation and contributes to cancer development is unclear. HOX expression is regulated by WNT, FGF, and retinoic acid (RA) signaling - pathways which also become dysregulated in cancer. Goal: To determine how regulation of HOX genes specifies differentiation of colonic SCs into specialized cell lineages and how dysregulation of HOX genes contributes to CRC development. Hypotheses: i) Each of the different cell types within human colonic epithelium has a distinct HOX gene expression signature; ii) All-trans retinoic acid (ATRA) induced differentiation of CRC cells changes HOX expression that correlates with specific HOX expression signatures in normal colon and CRC cells. Results: Immunostaining shows that specific HOX proteins (e.g., HOXA9 &amp; HOXC9) are selectively expressed in ALDH-positive SCs and expression of these HOX genes is increased in CRC tissues compared to normal epithelium. Bioinformatics analysis predicted that these HOX genes (plus HOXA4, HOXA10, HOXC8) are regulated by RA signaling. Accordingly, we studied how ATRA influences HOX expression in CRC cells (HT29 &amp; SW480). We found ATRA treatment: 1) Decreases proliferation and increases neuroendocrine cell (NEC) differentiation; and 2). Leads to specific changes in HOX gene expression patterns. Using NanoString profiling, we also discovered that LGR5+ SCs, ALDH+ SCs and GLP2R+ NECs have unique HOX signatures. By defining how these HOX gene expression changes in response to ATRA treatment in comparison to the HOX gene signatures of different colonic cell lineages will help us understand the role that HOX genes play in SC differentiation and how dysregulation of HOX gene expression contributes to CRC development. Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn O. Opdenaker, Chi Zhang, Bruce M. Boman. Role of HOX gene expression in differentiation of colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5599.

Understanding the Potential of mRNA as Biomarker to Revolutionize Diagnosis of Colorectal Cancer.

MicroRNA as potential biomarker for early diagnosis, differentiating various stages, interpreting the success of postoperative curative surgery and predicting early relapse of Colorectal cancer.In the realm of medical research, the quest to find effective biomarkers for various diseases has always been a top priority. Colorectal cancer (CRC), one of the leading causes of cancer-related deaths worldwide, is no exception. The emergence of microRNA (mRNA) as a potential biomarker for CRC has sparked immense interest among scientists and clinicians alike. mRNA, a molecule responsible for translating genetic information into functional proteins, presents a promising avenue for early detection and personalized treatment of this deadly disease. By analyzing the specific patterns and levels of mRNA expression in CRC cells, researchers have the ability to identify signatures that can aid in accurate diagnosis, predict patient prognosis, and even guide targeted therapies. This breakthrough in molecular biology not only enhances our understanding of CRC but also holds the potential to revolutionize the field of cancer diagnostics and treatment. In this article, we will delve deeper into the potential of mRNA as a biomarker for CRC, exploring its benefits and challenges in the field of cancer research.

Mechanism of miR-7 mediating TLR4/TRAF6/NF-κB inflammatory pathway in colorectal cancer

This study is aimed at investigating the roles of Toll-like receptor 4 (TLR4) and microRNA-7 (miR-7) in colorectal cancer (CRC) development and progression. We assessed TLR4 and miR-7 expression in CRC cells and tissues using reverse transcription-quantitative polymerase chain reaction. The relationship between miR-7 and TLR4 was analyzed through dual luciferase reporter assays. MTT, wound healing, and cell invasion assays were conducted to examine the effects of TLR4 and miR-7 on CRC cell proliferation, migration, and invasion. Western blotting was used to explore the involvement of the TRAF6/NF-κB signaling pathway. miR-7 was underexpressed in CRC, while TLR4 levels were increased. miR-7 negatively regulated TLR4 expression and its knockdown enhanced CRC cell proliferation, migration, and invasion. TLR4 knockdown had the opposite effects. The TRAF6/NF-κB pathway was linked to TLR4’s role in tumor progression. miR-7 might inhibit TRAF6/NF-κB target a signaling pathway of TLR4 and promote CRC occurrence. miR-7 may therefore be used as a sensitive biomarker in CRC patients.

Lysine acetyltransferase KAT2A modulates ferroptosis during colorectal cancer development

Background Histone modifications, especially the lysine acetylation, have drawn increasing attention in cancer research area. The aim of this research is to explore the molecular mechanisms underlying the regulation of lysine acetyltransferase 2 A (KAT2A) on colorectal cancer (CRC). Methods Clinical samples were collected from patients with CRC. The expression and correlation between KAT2A and ferroptosis suppressor SLC7A11 and glutathione peroxidase 4 (GPX4) were measured by qPCR and Pearson correlation analysis. NCP cells were transfected with KAT2A overexpression vectors or siRNAs. The proliferation of cells was measured by CCK-8 and colony formation assay. Cell migration and invasion was analyzed by Transwell. The accumulation of lipid peroxidation, ferrous iron, and malondialdehyde (MDA) were analyzed to determine cell ferroptosis. The expression of cell metastasis biomarkers was measured by western blotting assay. Interaction between KAT2A with GPX4 gene was measured by chromatin immunoprecipitation (ChIP). Results The KAT2A, GPX4, and SLC7A11 expression was notably elevated in tumor tissues compared with the paired non-tumor tissues from CRC patients. The expression of KAT2A showed positive correlation with GPX4 and SLC7A11. Overexpression of KAT2A recovered the cell proliferation, migration, and invasion of CRC cells that suppressed by ferroptosis inducer erastin, along with deceased levels of ROS, iron, Fe2+, and MDA. Overexpression of KAT2A suppressed E-cadherin level and increased N-cadherin, Snail, and Vimentin expression in CRC cells. KAT2A interacted with GPX4 promoter region. Conclusions In conclusion, our findings demonstrated that KAT2A modulates the histone acetylation of GPX4 to regulate proliferation, metastasis, and ferroptosis of CRC cells.

Thymol-loaded liposomes effectively induced apoptosis and decreased EGFR expression in colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common and deadly cancers worldwide. Different factors, such as environmental and genetic factors and lifestyle, affect it. Owing to the presence of phenolic, alkaloid, antioxidant, and terpenoid compounds, herbal compounds can be effective in the treatment of various cancers. Thymol is a natural monoterpene phenol that is abundant in some plants and exerts several biological effects. The aim of this study was to investigate the apoptotic, anti-proliferative effect and EGFR gene expression under the influence of thymol-loaded nanoliposome in SW84 and SW111 cell lines derived from colorectal cancer. The lipid thin-film hydration method was used to synthesize thymol-loaded liposomes, and their characterization was performed using TEM, DLS, and HPLC analyses. SW84 and SW1111 cells were treated with thymol- and thymol-loaded liposomes at different doses, the inhibition of cell proliferation was evaluated using an MTT assay, the rate of apoptosis induction was assessed using flow cytometry, and EGFR gene expression was measured using real-time PCR. The nanoparticles produced were spherical, uniform, and 200 ± 10nm in size. HPLC analysis showed that approximately 98% thymol was loaded into the nanoliposome. The results of the MTT assay showed that thymol and thymol-nanoliposomes decreased the proliferation of SW84 and SW1111 cells in a concentration-dependent manner. The IC50 of thymol and thymol-nanoliposomes were 18 and 14.2µg/ml for the SW48 cell line (P = 0.04) and 10.5 and 6.4µg/ml for the SW1116 cell line (P = 0.001). Thymol-nanoliposomes significantly inhibited the proliferation of cancer cells compared to free thymol. Flow cytometry showed an increase in the percentage of apoptotic cells, especially in the thymol-nanoliposome group in the treated cells. Real-time PCR results also showed that thymol and thymol-nanoliposome both caused a decrease in the expression of EGFR genes in both cell lines, but this effect of decreasing gene expression was significantly higher in the thymol-nanoliposome group. Our results showed that thymol-nanoliposomes reduced proliferation, increased apoptosis, and decreased EGFR expression in colorectal cancer-derived cell lines.

5-methylcytosine-mediated upregulation of circular RNA 0102913 augments malignant properties of colorectal cancer cells through a microRNA-571/Rac family small GTPase 2 axis

Circular RNAs (circRNAs) are a class of stable non-coding RNAs that have emerged as key regulators in human diseases including cancer. This study investigates the role of circRNA_0102913 (circ_0102913) in malignant behavior of colorectal cancer (CRC) cells and the underpinning mechanisms. By analyzing CRC-related GSE197991, GSE159669, and GSE223001 datasets, we obtained circ_0102913 as an aberrantly upregulated circRNA in CRC. Increased circ_0102913 expression was detected in CRC tissues and cells. By querying multiple bioinformatics systems (circBank, Circular RNA Interactome, TargetScan, miRDIP, miRwalk, and miRDB), we identified microRNA-571 (miR-571) as a target of circ_0102913 and Rac family small GTPase 2 (RAC2) mRNA as a target of miR-571. Biotinylated-RNA pull-down and/or luciferase assays showed that circ_0102913 bound to miR-571 to restore the expression of RAC2 mRNA. Circ_0102913 silencing or miR-571 overexpression repressed proliferation, migration and invasion, and in vivo tumorigenesis abilities of CRC cells. However, the malignant properties of cells were restored by RAC2 overexpression. The increased circ_0102913 expression in CRC cells was attributed to increased 5-methylcytosine (m5C) modification levels. Silencing of NOP2/Sun RNA methyltransferase 5 reduced the m5C level and therefore reduced stability and expression of circ_0102913 expression in CRC cells. In conclusion, this study demonstrates that m5C-mediated upregulation of circ_0102913 augments malignant properties of CRC cells through a miR-571/RAC2 axis.

Lysine demethylase 5C inhibits transcription of prefoldin subunit 5 to activate c-Myc signal transduction and colorectal cancer progression

BackgroundLysine demethylase 5C (KDM5C) has been implicated in the development of several human cancers. This study aims to investigate the role of KDM5C in the progression of colorectal cancer (CRC) and explore the associated molecular mechanism.MethodsBioinformatics tools were employed to predict the target genes of KDM5C in CRC. The expression levels of KDM5C and prefoldin subunit 5 (PFDN5) in CRC cells were determined by RT-qPCR and western blot assays. The interaction between KDM5C, H3K4me3, and PFDN5 was validated by chromatin immunoprecipitation. Expression and prognostic values of KDM5C and PFDN5 in CRC were analyzed in a cohort of 72 patients. The function of KDM5C/PFDN5 in c-Myc signal transduction was analyzed by luciferase assay. Silencing of KDM5C and PFDN5 was induced in CRC cell lines to analyze the cell malignant phenotype in vitro and tumorigenic activity in nude mice.ResultsKDM5C exhibited high expression, while PFDN5 displayed low expression in CRC cells and clinical CRC samples. High KDM5C levels correlated with poor survival and unfavorable clinical presentation, whereas elevated PFDN5 correlated with improved patient outcomes. KDM5C mediated demethylation of H3K4me3 on the PFDN5 promoter, suppressing its transcription and thereby enhancing the transcriptional activity of c-Myc. KDM5C knockdown in CRC cells suppressed cell proliferation, migration and invasion, epithelial-mesenchymal transition, and tumorigenic activity while increasing autophagy and apoptosis rates. However, the malignant behavior of cells was restored by the further silencing of PFDN5.ConclusionThis study demonstrates that KDM5C inhibits PFDN5 transcription, thereby activating c-Myc signal transduction and promoting CRC progression.

Downregulation of osteoprotegerin in colorectal cancer cells promotes liver metastasis via activating tumor-associated macrophage

Osteoprotegerin (OPG) is a secreted cytokine that functions as a decoy receptor for receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL). Anti-RANKL treatment for bone metastasis has been widely accepted for solid tumors. However, the mechanism of OPG-RANKL-RANK signaling in systemic colorectal cancer (CRC) metastasis remains unclear. In this study, we investigated the relevance and function of OPG expression in CRC liver metastasis. First, we performed in silico analysis using The Cancer Genome Atlas public database and found that lower OPG expression in CRC was associated with poor overall survival. Immunohistochemistry analyses using resected specimen from patients with CRC in our institute confirmed the result. Patient-matched primary CRC and liver metastases showed a significant downregulation of OPG expression in metastatic lesions. In CRC cell lines, OPG expression did not suppress cell proliferation and migration. However, OPG expression inhibited macrophage migration by suppressing the RANKL-RANK pathway. Moreover, in vivo mouse liver metastasis models showed that OPG expression in CRC cells suppressed liver metastases. In addition, treatment with an anti-RANKL neutralizing antibody also suppressed liver metastases. These results showed that downregulation of OPG expression in CRC cells promotes liver metastasis by activating tumor-associated macrophage, which can become a candidate for targeted therapy with anti-RANKL neutralizing antibody for CRC liver metastasis.

Serine and arginine rich splicing factor1‑regulated microtubule interacting and trafficking domain containing1 affects colorectal cancer progression and ferroptosis.

As the third most common type of cancer globally, colorectal cancer (CC) is a prevalent digestive malignancy, with the second highest mortality rate among all types of cancer. It has been reported that microtubule interacting and trafficking domain containing 1 (MITD1) serves a pivotal role in the initiation and progression of diverse types of tumors. Nevertheless, the underlying mechanism of MITD1 in CC has not been previously investigated. The ENCORI and GEPIA databases were used to investigate the expression levels of MITD1 in patients with CC. Immunohistochemistry was used to detect the expression of MITD1 in cancer tissues obtained from patients with CC, while its mRNA and protein expression levels in CC cell lines were determined by reverse transcription-quantitative PCR and western blot analysis, respectively. Subsequently, MITD1 was knocked down in CC cells using an interference plasmid and Cell Counting Kit 8, colony formation, as well as EdU assays were performed to assess cell proliferation. Concurrently, wound healing and Transwell assays were performed to evaluate the migration and invasion abilities of CC cells. Lipid reactive oxygen species (ROS) levels were determined by BODIPY 581/591 C11 staining. In addition, the levels of oxidative stress markers and those of total iron were measured using the corresponding kits. Furthermore, the association between serine and arginine rich splicing factor 1 (SRSF1) and MITD1 was verified by RNA immunoprecipitation and actinomycin D experiments. Finally, to further uncover the mechanism of MITD1, SRSF1 was overexpressed and MITD1 was silenced in CC cells. The results demonstrated that the expression of MITD1 was abnormally elevated in CC tissues and CC cell lines. MITD1 silencing distinctly diminished CC cell viability, increased CC cell ferroptosis and attenuated their invasion and migration abilities. In addition, MITD1 knockdown significantly increased the expression of lipid ROS and total iron levels in CC cells. Additionally, the results showed that SRSF1 could stabilize MITD1 mRNA expression in CC cells. Finally, it was revealed that SRSF1 could regulate MITD1 and affect the progression of CC and ferroptosis via p53/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling. Overall, the results of the current study indicated that SRSF1-regulated MITD1 could affect CC progression and ferroptosis, probably via the p53/SLC7A11/GPX4 signaling pathway.

Hsa_circ_0009092/miR-665/NLK signaling axis suppresses colorectal cancer progression via recruiting TAMs in the tumor microenvironment

BackgroundIt has been demonstrated that circularRNA (circRNAs) plays a critical role in various cancers. While the potential molecular mechanism of circRNAs in the progression of colorectal cancer (CRC) remains uncertain.MethodsDifferentially expressed circRNAs were identified by RNA sequencing. RT-qPCR detected the expression of circ_0009092, miR-665, and NLK in CRC tissues and cells. Functions of circ_0009092 on tumor cell proliferation, migration, and invasion were investigated by a series of in vitro assays. The underlying mechanism of circ_0009092 was explored by bioinformatics analysis, RNA immunoprecipitation (RIP) and luciferase assays. A co-culture assay in vitro was performed to detect the affection of circ_0009092 on macrophage recruitment in the tumor microenvironment (TME). A xenograft mouse model was used to explore the effect of circ_0009092 on tumor growth.ResultsCirc_0009092 was downregulated in CRCand predicted a good prognosis. Overexpression of circ_0009092 reduced tumor cell EMT, proliferation, migration, and invasion in vitro and in vivo. Mechanistically, circ_0009092 elevated the NLK expression via sponging miR-665 and suppressed the Wnt/β-catenin signaling pathway. EIF4EA3 induced circ_0009092 expression in CRC cells. In addition, NLK regulates phosphorylation and O-GlcNAcylation of STAT3 by binding to STAT3, thereby inhibiting CCL2 expression, in which it inhibits macrophage recruitment in the tumor microenvironment (TME).ConclusionEIF4A3 suppressed circ_0009092 biogenesis, whichinhibits CRC progression by sponging miR-665 to downregulate NLK. Circ_0009092/miR-665/NLK suppressed tumor EMT, proliferation, migration, and invasion by acting on the Wnt/β-catenin signaling pathway. NLK directly interacted with STAT3 and decreased the CCL2 expression, inhibiting the recruitment of tumor-associated macrophages (TAMs) in the TME. Our study provided novel insights into the roles of circ_0009092 as a novel promising prognostic and therapeutic target in CRC.

RPF2 mediates the CARM1‑MYCN axis to promote chemotherapy resistance in colorectal cancer cells.

Ribosome production factor 2 homolog (RPF2) plays an important role in the life processes of ribosomal biogenesis; however, the function and mechanism of RPF2 in tumors are unclear. The present study demonstrated that RPF2 expression is involved in chemoresistance in colorectal cancer (CRC) cells. The current study demonstrated that upregulation of RPF2 expression in CRC promoted resistance to chemotherapeutic agents in CRC cells, whereas knockdown of RPF2 leads to increased sensitivity of CRC to chemotherapy. In addition, it was found that overexpression of RPF2 led to an increase in ATP‑binding cassette (ABC)B1 expression in CRC cells; accordingly, inhibition of RPF2 reduced the level of ABCB1 in CRC cells, thus suggesting that ABCB1 may be a downstream factor of RPF2 in the promotion of chemotherapy resistance to CRC. The results also suggested that the expression of N‑myc proto‑oncogene protein (MYCN), an upstream regulator of ABCB1, was affected by RPF2 in CRC cells. In addition, it was also found that the downstream protein coactivator‑associated arginine methyltransferase 1 (CARM1) of RPF2 existed in direct binding to MYCN and this interaction was regulated by RPF2. The above results suggested that RPF2 is probably regulated ABCB1 expression in CRC through the CARM1‑MYCN pathway, thereby promoting CRC drug resistance.