Colorectal Cancer Cell Proliferation Research Articles

Overexpressed FKBP5 mediates colorectal cancer progression and sensitivity to FK506 treatment via the NF-κB signaling pathway.

Colorectal cancer (CRC) is a common and deadly tumor. FK506-binding protein 5 (FKBP5) is associated with some cancers, but the role of FKBP5 in CRC is not clear. The present study aimed to reveal the relationship between FKBP5 and CRC and to uncover the roles of FK506 in CRC. In total, 96 CRC patients were recruited. Survival analysis was conducted using the Kaplan-Meier method and COX regression analyses. Bioinformatics analyses were performed to explore the functions of FKBP5. The mechanisms of FKBP5 and the roles of FK506 in CRC progression were clarified by immunohistochemistry, MTS, scratch assay, transwell and flow cytometric analyses via in vitro and in vivo experiments. FKBP5 was overexpressed in 77 cancer tissues compared to that in matched normal tissues, and the overall survival rate of these patients was relatively shorter. Bioinformatics analyses showed that FKBP5 regulates proliferation, invasion, migration, epithelial-mesenchymal transition and nuclear factor-kappa B (NF-κB) signaling. The upregulation or downregulation of FKBP5 dramatically increases or decreases the proliferation, invasion and migration abilities of CRC cells. The expression of NF-κB, inhibitor B kinase α, matrix metalloproteinase-2 and metalloproteinase-9 positively correlated with FKBP5. FK506 inhibits the progression of CRC via the FKBP5/NF-κB signaling pathway. Our study identified a regulatory role for FKBP5 in CRC progression. Therefore, targeting FKBP5 may provide a novel treatment approach for CRC. FK506 can inhibit the progression of CRC by restraining the FKBP5/NF-κB signaling pathway and is expected to become a new drug for the treatment of CRC.

CBC-1 as a Cynanbungeigenin C derivative inhibits the growth of colorectal cancer through targeting Hedgehog pathway component GLI 1

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers that results in death in worldwide. The Hedgehog (HH) signalling pathway regulates the initiation and progression of CRC. Inhibiting the HH pathway has been presented as a potential treatment strategy in recent years. Cynanbungeigenin C (CBC) is a new type of C21 steroid that has been previously reported for the treatment of medulloblastoma. However, its further investigation was limited by its poor water solubility. In this study, six new CBC derivatives were synthesized through the structural modification of CBC, and four of them showed better water solubility than CBC. Moreover, their antiproliferative activities on CRC were evaluated. It was found that CBC-1 presented the best inhibitory effect on three types of CRC cell lines, and this effect was superior to that of CBC. Mechanistically, CBC-1 inhibited the proliferation of CRC cells through regulation of mRNA and proteins of the HH pathway according to qRT–PCR and Western blotting analysis. Furthermore, Cellular Thermal Shift Assay results indicated that CBC-1 regulated this signalling pathway by targeting glioma‑associated oncogene (GLI 1).In addition, cell apoptosis was induced increasingly by transfection with GLI 1 siRNA or treatment with CBC-1 to downregulate GLI 1. Last, the in vivo results demonstrated that CBC-1 significantly reduced tumour size and downregulated GLI 1 in CRC. Therefore, this study suggests that CBC-1, a new GLI 1 inhibitor derived from natural products, may be developed as a potential antitumour candidate for CRC treatment.

LPCAT2 inhibits colorectal cancer progression via the PRMT1/SLC7A11 axis

Colorectal cancer (CRC) has a high degree of heterogeneity and identifying the genetic information of individual tumor cells could help enhance our understanding of tumor biology and uncover potential therapeutic targets for CRC. In this study, we identified LPCAT2+ tumor cell populations with less malignancy than LPCAT2- tumor cells in human and mouse CRC tissues using scRNA-seq. Combining in vitro and in vivo experiments, we found that LPCAT2 could inhibit the proliferation of CRC cells by inducing ferroptosis. Mechanistically, LPCAT2 arrested PRMT1 in cytoplasm of CRC cells via regulating acetylation of PRMT1 at the K145 site. In turn, PRMT1 enhanced SLC7A11 promoter activity. Thus, LPCAT2 attenuated the positive regulatory effect of PRMT1 on SLC7A11 promoter. Notably, SLC7A11 acts as a ferroptosis regulator. Furthermore, in LPCAT2 knockout mice (LPCAT2−/−) colon cancer model, we found that LPCAT2−/− mice exhibited more severe lesions, while PRMT1 or SLC7A11 inhibitors delayed the progression. Altogether, we elucidated that LPCAT2 suppresses SLC7A11 expression by inhibiting PRMT1 nuclear translocation, thereby inducing ferroptosis in CRC cells. Moreover, inhibitors of the PRMT1/SLC7A11 axis could delay tumor progression in CRC with low LPCAT2 expression, making it a potentially effective treatment for CRC.

A novel four‑gene biomarker for tobacco smoking -induced colorectal cancer progression.

Cigarette smoking greatly promotes the progression and poor prognosis of colorectal cancer (CRC) patients, with the molecular mechanism still not fully clear. In this study, CRC cells were exposed to tobacco specific nitrosamine 4‑(methylnitrosamino)‑1‑(3‑pyridyl) 1‑butanone (NNK), and the differentially expressed smoking-related genes were identified based on both NNK-induced CRC cells and a total of 763 CRC tissues from TCGA cohort. Cox regression analysis, ROC curve and Kaplan-Meier plot were used to establish the risk score model for CRC prognosis. Moreover, qRT-PCR, western blotting, colony formation, migration and invasion assays were performed to verify the core differentially expressed smoking-related gene and its molecular function in NNK-induced CRC progression. Results indicated NNK significantly enhanced CRC cell proliferation, migration and invasion. Moreover, a four-gene signature containing AKR1B10, CALB2, PLAC1, GNA15 was established as CRC prognosis marker. Among these four genes, AKR1B10 was further validated as the core gene, and its expression was significantly inhibited after NNK exposure in CRC cells. Results of gene enrichment analysis and western blotting suggested AKR1B10 might reduce the malignant progression of NNK-induced CRC cells through inhibiting Wnt signaling pathway by promoting E-Cadherin expression and inhibiting the expression of N-Cadherin, β-Catenin, Vimentin and Snail. In conclusion, a new four smoking-related genes can be jointly used as prognostic markers for CRC. AKR1B10 served as a tumor suppressor, can be used as a potential target to inhibit NNK-induced CRC malignant progression through regulating Wnt signaling pathway. This study demonstrates tobacco-derived NNK dependence would promote the malignant progression of colorectal cancer through regulating the expressions of AKR1B10/Wnt signaling pathway. And a novel four-gene signature is established for the prognosis prediction of smoking CRC patients. These findings have important translational implications given the continued use of tobacco and the difficulty in smoking cessation worldwide, which can be applied to alleviate the adverse effects induced by tobacco dependence on colorectal cancer patients.

Combination of bazedoxifene with chemotherapy and SMAC-mimetics for the treatment of colorectal cancer

Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Compound 225# inhibits the proliferation of human colorectal cancer cells by promoting cell cycle arrest and apoptosis induction.

Colorectal cancer (CRC) ranks as the second leading cause of cancer‑related death worldwide due to its aggressive nature. After surgical resection, >50% of patients with CRC require adjuvant therapy. As a result, eradicating cancer cells with medications is a promising method to treat patients with CRC. In the present study, a novel compound was synthesized, which was termed compound 225#. The inhibitory activity of compound 225# against CRC was determined by MTT assay, EdU fluorescence labeling and colony formation assay; the effects of compound 225# on the cell cycle progression and apoptosis of CRC cells were detected by flow cytometry and western blotting; and the changes in autophagic flux after the administration of compound 225# were detected using the double fluorescence fusion protein mCherry‑GFP‑LC3B and western blotting. The results demonstrated that compound 225# exhibited antiproliferative properties, inhibiting the proliferation and expansion of CRC cell lines in a time‑ and dose‑dependent manner. Furthermore, compound 225# triggered G2/M cell cycle arrest by influencing the expression of cell cycle regulators, such as CDK1, cyclin A1 and cyclin B1, which is also closely related to the activation of DNA damage pathways. The cleavage of PARP and increased protein expression levels of PUMA suggested that apoptosis was triggered after treatment with compound 225#. Moreover, the increase in LC3‑II expression and stimulation of autophagic flux indicated the activation of an autophagy pathway. Notably, compound 225# induced autophagy, which was associated with endoplasmic reticulum (ER) stress. In accordance with the in vitro findings, the in vivo results demonstrated that compound 225# effectively inhibited the growth of HCT116 tumors in mice without causing any changes in their body weight. Collectively, the present results demonstrated that compound 225# not only inhibited proliferation and promoted G2/M‑phase cell cycle arrest and apoptosis, but also initiated cytoprotective autophagy in CRC cells by activating ER stress pathways. Taken together, these findings provide an experimental basis for the evaluation of compound 225# as a novel potential medication for CRC treatment.

Long-Noncoding RNA (lncRNA) EGOT Prevents the Malignant Process of Colorectal Carcinoma by Regulating BTG3

This study investigates the role of the long non-coding RNA EGOT in colorectal cancer (CRC) by examining its expression in 40 pairs of CRC and adjacent normal tissues and assessing its impact on clinical outcomes. EGOT was found to be downregulated in CRC tissues, and low EGOT levels were associated with a higher likelihood of lymphatic and distant metastasis, as well as poorer overall and progression-free survival in CRC patients. Functional experiments revealed that overexpression of EGOT in SW480 cells reduced cell viability, migration, and wound closure, while knockdown of EGOT in LoVo cells had the opposite effect. In vivo experiments with nude mice confirmed that EGOT downregulation accelerated CRC growth, whereas its overexpression slowed tumor growth. The study identified BTG3 as the target gene of EGOT, and they exhibited a negative correlation in CRC tissues. Rescue experiments demonstrated that BTG3 could reverse the effects of EGOT on CRC cell phenotypes. In conclusion, EGOT is a downregulated molecule in CRC, closely associated with metastasis and patient prognosis. It exerts a suppressive influence on CRC cell proliferation, migration, and tumorigenesis by negatively regulating BTG3.

Cancer-associated fibroblasts drive colorectal cancer cell progression through exosomal miR-20a-5p-mediated targeting of PTEN and stimulating interleukin-6 production.

This study evaluated the clinical relevance of a set of five serum-derived circulating microRNAs (miRNAs) in colorectal cancer (CRC). Additionally, we investigated the role of miR-20a-5p released by exosomes derived from cancer-associated fibroblasts (CAFs) in the context of CRC. The expression levels of five circulating serum-derived miRNAs (miR-20a-5p, miR-122-5p, miR-139-3p, miR-143-5p, and miR-193a-5p) were quantified by real-time quantitative PCR (RT-qPCR), and their associations with clinicopathological characteristics in CRC patients were assessed. The diagnostic accuracy of these miRNAs was determined through Receiver Operating Characteristic (ROC) curve analysis. CAFs and normal fibroblasts (NFs) were isolated from tissue samples, and subsequently, exosomes derived from these cells were isolated and meticulously characterized using electron microscopy and Western blotting. The cellular internalization of fluorescent-labeled exosomes was visualized by confocal microscopy. Gain- and loss-of-function experiments were conducted to elucidate the oncogenic role of miR-20a-5p transferred by exosomes derived from CAFs in CRC progression. The underlying mechanisms were uncovered through luciferase reporter assay, Western blotting, enzyme-linked immunosorbent assays, as well as proliferation and migration assays. The expression levels of serum-derived circulating miR-20a-5p and miR-122-5p were significantly higher in CRC and were positively correlated with advanced stages of tumorigenesis and lymph node metastasis (LNM). In contrast, circulating miR-139-3p, miR-143-5p, and miR-193a-5p were down-regulated in CRC and associated with early tumorigenesis. Except for miR-139-3p, they showed a negative correlation with LNM status. Among the candidate miRNAs, significantly elevated levels of miR-20a-5p were observed in both cellular and exosomal fractions of CAFs. Our findings indicated that miR-20a-5p induces the expression of EMT markers, partly by targeting PTEN. Exosomal miR-20a secreted by CAFs emerged as a key factor enhancing the proliferation and migration of CRC cells. The inhibition of miR-20a impaired the proliferative and migratory potential of CAF-derived exosomes in SW480 CRC cells, suggesting that the oncogenic effects of CAF-derived exosomes are mediated through the exosomal transfer of miR-20a. Furthermore, exosomes originating from CAFs induced increased nuclear translocation of the NF-kB p65 transcription factor in SW480 CRC cells, leading to increased interleukin-6 (IL-6) production. We established a set of five circulating miRNAs as a non-invasive biomarker for CRC diagnosis. Additionally, our findings shed light on the intricate mechanisms underpinning the oncogenic impacts of CAF-derived exosomes and underscore the pivotal role of miR-20a-5p in CRC progression.

Urocortin-1 promotes colorectal cancer cell migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway

PurposeTo investigate the effect of urocortin-1 (UCN-1) on growth, migration, and apoptosis in colorectal cancer (CRC) in vivo and vitro and the mechanism by which UCN-1 modulates CRC cells in vitro.MethodsThe correlation between UCN-1 and CRC was evaluated using The Cancer Genome Atlas (TCGA) database and a tissue microarray. The expression of UCN-1 in CRC cells was assessed using quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting. In vitro, the influence of UCN-1 on the proliferation, apoptosis, and migration of HT-29, HCT-116, and RKO cells was explored using the celigo cell counting assay or cell counting kit-8 (CCK8), flow cytometry, and wound healing or Transwell assays, respectively. In vivo, the effect of UCN-1 on CRC growth and progression was evaluated in nude mice. The downstream pathway underlying UCN-1-mediated regulation of CRC was determined using the phospho-kinase profiler array in RKO cells. Lentiviruses were used to knockdown or upregulate UCN-1 expression in cells.ResultsBoth the TCGA and tissue microarray results showed that UCN-1 was strongly expressed in the tissues of patients with CRC. Furthermore, the tissue microarray results showed that the expression of UCN-1 was higher in male than in female patients, and high expression of UCN-1 was associated with higher risk of lymphatic metastasis and later pathological stage. UCN-1 knockdown caused a reduction in CRC cell proliferation, migration, and colony formation, as well as an increase in apoptosis. In xenograft experiments, tumors generated from RKO cells with UCN-1 knockdown exhibited reduced volumes and weights. A reduction in the expression of Ki-67 in xenograft tumors indicated that UCN-1 knockdown curbed tumor growth. The human phospho-kinase array showed that the p53 signaling pathway participated in UCN-1-mediated CRC development. The suppression in migration and proliferation caused by UCN-1 knockdown was reversed by inhibitors of p53 signal pathway, while the increase in cell apoptosis was suppressed. On the other hand, overexpression of UCN-1 promoted proliferation and migration and inhibited apoptosis in CRC cells. Overexpression of p53 reversed the effect of UCN-1 overexpression on CRC development.ConclusionUCN-1 promotes migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway.

Cynaroside Induces G1 Cell Cycle Arrest by Downregulating Cell Division Cycle 25A in Colorectal Cancer.

Natural chemicals derived from herbal plants have recently been recognized as potentially useful treatment alternatives owing to their ability to target a wide range of important biological molecules. Cynaroside is one of these natural compounds with promising anticancer activity for numerous tumor types. Nevertheless, the anticancer effects and molecular mechanisms of action of cynaroside on colorectal cancer (CRC) remain unclear. In this study, cynaroside was found to markedly inhibit CRC cell proliferation and colony formation in vitro. Cynaroside also inhibited cell proliferation in vivo and decreased the expression of KI67, a cell nuclear antigen. RNA sequencing revealed 144 differentially expressed genes (DEGs) in HCT116 cells and 493 DEGs in RKO cells that were enriched in the cell cycle signaling pathway. Cell division cycle 25A (CDC25A), a DEG widely enriched in the cell cycle signaling pathway, is considered a key target of cynaroside in CRC cells. Cynaroside also inhibited DNA replication and arrested cells in the G1/S phase in vitro. The expression levels of CDC25A and related G1-phase proteins were significantly elevated after CDC25A overexpression in CRC cells, which partially reversed the inhibitory effect of cynaroside on CRC cell proliferation and G1/S-phase arrest. In summary, cynaroside may be used to treat CRC as it inhibits CDC25A expression.

Exosomal miR-1470 is a diagnostic biomarker and promotes cell proliferation and metastasis in colorectal cancer.

In recent years,the lack of specific markers for the diagnosis of colorectal cancer has led to an upward trend in both morbidity and mortality from this condition. There is an urgent need to identify molecular biomarkers that contribute to early cancer detection. This study aimed to identify specific exosomal microRNAs that hold potential as diagnostic biomarkers for CRC. We screened for differentially expressed miRNAs using the CRC exosome dataset GSE39833. To validate the results in the public database, we collected serum from 168 CRC patients and 168 healthy volunteers. The expression levels of exosomal miR-1470 in healthy volunteers and CRC patients were analyzed using qRT-PCR. To evaluate the diagnostic potential of the selected miR-1470 in distinguishing CRC patients from healthy controls, we analyzed its receiver operating characteristic curve. To explore the biological functions of miR-1470 in CRC cell lines, we detected the miR-1470's ability to regulate the growth and metastasis of CRC cells by CCK8, transwell and other assays after transfection of miR-1470 in SW480, HCT-116 cells. Exosomal miR-1470 exhibited significant up-regulation in CRC patients compared to healthy volunteers. The ROC curve analysis revealed an area under the curve (AUC) of 0.74 (95% confidence interval: 0.6876-0.7920) for exosomal miR-1470, indicating its potential as a diagnostic biomarker. Furthermore, the expression level of miR-1470 in CRC patients showed correlations with age, metastasis, and HDL content. We overexpressed miR-1470 in CRC cell lines. CCK8 proliferation assay showed that miR-1470 promoted the proliferation ability of SW480 and HCT-116 cells. Transwell assay showed that miR-1470 promoted the migration and invasion ability of SW480 and HCT-116 cells. This suggested that non-invasive diagnosis of CRC is possible by detecting the level of miR-1470 in exosomes, which has important implications for early detection and treatment of this disease.

YWHAG promotes colorectal cancer progression by regulating the CTTN-Wnt/β-catenin signaling axis.

Colorectal cancer (CRC) ranks as the third most prevalent cancer type globally. Nevertheless, the fundamental mechanisms driving CRC progression remain ambiguous, and the prognosis for the majority of patients diagnosed at an advanced stage is dismal. YWHA/14-3-3 proteins serve as central nodes in several signaling pathways and are closely related to tumorigenesis and progression. However, their exact roles in CRC are still poorly elucidated. In this study, we revealed that YWHAG was the most significantly upregulated member of the YWHA/14-3-3 family in CRC tissues and was associated with a poor prognosis. Subsequent phenotypic experiments showed that YWHAG promoted the proliferation, migration, and invasion of CRC cells. Mechanistically, RNA-seq data showed that multiple signaling pathways, including Wnt and epithelial-mesenchymal transition, were potentially regulated by YWHAG. CTTN was identified as a YWHAG-associated protein, and mediated its tumor-promoting functions by activating the Wnt/β-catenin signaling in CRC cells. In summary, our data indicate that YWHAG facilitates the proliferation, migration, and invasion of CRC cells by modulating the CTTN-Wnt/β-catenin signaling pathway, which offers a novel perspective for the treatment of CRC.

A transcribed ultraconserved noncoding RNA, uc.285+, promotes colorectal cancer proliferation through dual targeting of CDC42 by directly binding mRNA and protein

The transcribed ultraconserved region (T-UCR) belongs to a new type of lncRNAs that are conserved in homologous regions of the rat, mouse and human genomes. A lot of research has reported that differential expression of T-UCRs can influence the development of various cancers, revealing the ability of T-UCRs as new therapeutic targets or potential cancer biomarkers. Most studies on the molecular mechanisms of T-UCRs in cancer have focused on ceRNA regulatory networks and interactions with target proteins, but the present study reveals an innovative dual-targeted regulatory approach in which T-UCRs bind directly to mRNAs and directly to proteins. We screened T-UCRs that may be related to colorectal cancer (CRC) by performing a whole-genome T-UCR gene microarray and further studied the functional mechanism of T-UCR uc.285+ in the development of CRC. Modulation of uc.285+ affected the proliferation of CRC cell lines and influenced the expression of the CDC42 gene. We also found that uc.285+ promoted the proliferation of CRC cells by directly binding to CDC42 mRNA and enhancing its stability while directly binding to CDC42 protein and affecting its stability. In short, our research on the characteristics of cell proliferation found that uc.285+ has a biological function in promoting CRC proliferation. uc.285+ may have considerable potential as a new diagnostic biomarker for CRC.

Depletion of CPNE7 sensitizes colorectal cancer to 5-fluorouracil by downregulating ATG9B expression.

We aimed to explore the biological function of CPNE7 and determine the impact of CPNE7 on chemotherapy resistance in colorectal cancer (CRC) patients. According to the Gene Expression Profiling Interactive Analysis database and previously published data, CPNE7 was identified as a potential oncogene in CRC. RT-qPCR and Western blotting were performed to verify the expression of CPNE7. Chi-square test was used to evaluate the associations between CPNE7 and clinical features. Cell proliferation, colony formation, cell migration and invasion, cell cycle and apoptosis were assessed to determine the effects of CPNE7. Transcriptome sequencing was used to identify potential downstream regulatory genes, and gene set enrichment analysis was performed to investigate downstream pathways. The effect of CPNE7 on 5-fluorouracil chemosensitivity was verified by half maximal inhibitory concentration (IC50). Subcutaneous tumorigenesis assay was used to examine the role of CPNE7 in sensitivity of CRC to chemotherapy invivo. Transmission electron microscopy was used to detect autophagosomes. CPNE7 was highly expressed in CRC tissues, and its expression was correlated with T stage and tumour site. Knockdown of CPNE7 inhibited the proliferation and colony formation of CRC cells and promoted apoptosis. Knockdown of CPNE7 suppressed the expression of ATG9B and enhanced the sensitivity of CRC cells to 5-fluorouracil invitro and invivo. Knockdown of CPNE7 reversed the induction of the autophagy pathway by rapamycin and reduced the number of autophagosomes. Depletion of CPNE7 attenuated the malignant proliferation of CRC cells and enhanced the chemosensitivity of CRC cells to 5-fluorouracil.

Abstract 1819: Esculetin modulates SLC31A1 and ATP7B to influence copper homeostasis and trigger cell death in colorectal cancer

Abstract Background: Esculetin is a naturally occurring dihydroxycoumarin with anti-inflammatory, antioxidant, and anti-tumor activities. Copper ion transport proteins, notably SLC31A1 and ATP7B, are essential in maintaining intracellular copper homeostasis, averting disruptions that might lead to copper-induced cell death under normal physiological conditions. However, prior research has not explored Esculetin's role in inducing copper-induced cell death. Hence, this study aims to investigate the biological process of Esculetin-induced cell death and the molecular mechanism underlying dysregulation of intracellular copper ion homeostasis. Methods: Initially, the CCK-8 assay was used to determine the IC50 values of Esculetin on colorectal cancer cell lines (HCT116, HT29, SW480) and normal colonic epithelial cell lines (NCM460). In terms of biological behaviors, the effects of Esculetin on the proliferation of colorectal cancer cell lines were assessed using CCK-8, colony formation assay, and EdU staining. Flow cytometry was used to examine the impact of Esculetin on apoptosis and cell cycle distribution in colorectal cancer cells. A subcutaneous xenograft tumor model in nude mice was employed to evaluate the in vivo anti-tumor effects of Esculetin, while IHC staining was conducted to detect Ki-67 levels, and Western blotting was performed to assess EMT-related protein expression. To investigate copper ion homeostasis, qPCR was used to measure the expression levels of copper ion transporters SLC31A1 and ATP7B before and after Esculetin treatment. Results: The CCK-8 results revealed that the IC50 of Esculetin for normal colonic epithelial cell lines was significantly higher than that for colorectal cancer cell lines. Meanwhile, a notable inhibitory effect of Esculetin on the proliferation of colorectal cancer cells was observed. Additionally, Esculetin treatment suppressed colony formation and reduced the proportion of EdU-positive cells in colorectal cancer cells. Flow cytometry demonstrated an increase in apoptosis and S-phase arrest following Esculetin treatment. The subcutaneous xenograft tumor model showed significant inhibition of tumor growth by Esculetin, as evidenced by reduced Ki-67-positive cells in the Esculetin-treated group. Western blot analysis revealed a significant decrease in the expression levels of Vimentin and N-Cadherin and an increase in E-Cadherin expression. Furthermore, qPCR analysis post-Esculetin treatment showed an elevation in the inward copper transporter SLC31A1 levels, while the outward transporter ATP7B expression decreased. Conclusion: Esculetin induces dysregulation of intracellular copper ion homeostasis by upregulating SLC31A1 expression and downregulating ATP7B expression, leading to intracellular copper accumulation and subsequent disruption of copper homeostasis, thereby inducing copper-induced cell death. Citation Format: Fangyue Guo, Mengying Liu, Kaihe Zhu, Jiapeng Ru, Pei Li, Shanfeng Zhang. Esculetin modulates SLC31A1 and ATP7B to influence copper homeostasis and trigger cell death in 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 1819.

Abstract 1478: Chronic perfluorooctanesulfonic acid exposure promotes proliferation of colorectal cancer cells

Abstract Background: Chronic exposure to long-term exposures to per- and polyfluoroalkyl substances (PFAS), widely known as “forever chemicals”, has been associated with multiple negative health outcomes including increased risk of cancer. Perfluorooctanesulfonic acid (PFOS), a “long-chain” subtype of PFAS, has high bioaccumulation potential with a long elimination half-life. PFOS is frequently detected in drinking water leading to its high absorption through the gastrointestinal tract. Recent studies demonstrate that PFAS exposures promote intestinal inflammation and gut barrier dysfunction. However, how a long-term PFOS exposure affects colorectal cancer (CRC) progression is not known. Therefore, the purpose of this study is to delineate the effect of PFOS exposure on CRC cell proliferation and test the potential mitigation strategy for the harmful effects of PFOS. Methods: SW480 and HCT116 cells have been treated with 1 ug/mL PFOS and proliferation was measured at 1, 2, and 3 months using the Presto Blue Cell Viability Reagent fluorescence assay. Proliferation markers were assessed by qPCR and Western blot. Control and PFOS exposed cells were treated with sulforaphane, a nuclear factor erythroid 2-related factor 2 (Nrf-2) inducer, at concentration 5, 10 and 20 uM for 72 hours. Results: We show that chronic, low-dose PFOS exposure promotes proliferation of SW480 and HCT116 cell lines starting at 3 months since the first exposure. The increase in proliferation is associated with upregulation of Cyclin D, pAkt and FASN. We also show that sulforaphane significantly decreases proliferation of HCT116 and SW480 cells, and its efficacy is significantly higher in PFOS exposed CRC cells. The current studies show no effect on sulforaphane on normal colon epithelium cells. Conclusion: Our studies suggest that chronic PFOS exposure promotes proliferation of CRC cells by increasing pro-carcinogenic gene expression and signaling. Furthermore, our findings suggest that supplementation of diet sulforaphane can potentially mitigate the harmful effects from PFOS exposure. Our studies warrant further investigation of the mechanisms behind the effect of PFOS exposure on cancer progression that would guide development of effective intervention strategies to mitigate the harmful effects of the exposure to “forever chemicals”. Citation Format: Jerika Durham, Josiane Weber Tessmann, Bernhard Hennig, Yekaterina Zaytseva. Chronic perfluorooctanesulfonic acid exposure promotes proliferation of 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 1478.

Abstract 5639: TIP60-independent mechanism of BRD8 is involved in colorectal tumorigenesis

Abstract Modifications of histone tails control chromatin structure, and the acetylation is generally associated with euchromatin and transcriptional activation. Acetylated lysine residues are recognized by bromodomains, which serve as acetyl-lysine binding modules and participate in transcriptional regulation. Bromodomain-containing protein 8 (BRD8) is a subunit of the NuA4/TIP60-histone acetyltransferase complex. Although BRD8 has been considered to act as a co-activator of the complex, its biological role remains to be unclear. In this study, we revealed that BRD8 accumulates in colorectal cancer cells through the inhibition of ubiquitin-dependent protein degradation by the interaction with MRG domain binding protein (MRGBP). RNA-seq analysis coupled with genome-wide mapping of BRD8-binding sites disclosed that BRD8 transactivates a set of genes independently of TIP60, a catalytic subunit of NuA4 complex, and that BRD8 regulates the expression of multiple subunits of the pre-replicative complex (pre-RC) in concert with the activator protein-1 (AP-1) transcription factor. Depletion of BRD8 induced cell-cycle arrest at the G1 phase and suppressed proliferation of colorectal cancer cells. We have also shown that the bromodomain of BRD8 is indispensable for not only the interaction with histone H4 or transcriptional regulation but also its own protein stability. These findings provide a new insight into the roles played by BRD8 in human colorectal carcinogenesis and will contribute to the development of new chemotherapeutic agents targeting the bromodomain of BRD8. Citation Format: Kiyoshi Yamaguchi, Saya Nakagawa, Rui Yamaguchi, Paul Sheridan, Kiyoko Takane, Tsuneo Ikenoue, Satoru Nagatoishi, Hiroko Kozuka-Hata, Masaaki Oyama, Susumu Aikou, Yuka Ahiko, Dai Shida, Kouhei Tsumoto, Satoru Miyano, Seiya Imoto, Yoichi Furukawa. TIP60-independent mechanism of BRD8 is involved in colorectal tumorigenesis [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 5639.

Abstract 3176: Otopetrin 3 promotes cell proliferation and regulates glycolysis via interaction with MYC in colorectal cancer

Abstract Otopetrin 3 (OTOP3) stands out as a pivotal regulator of intracellular pH through its control over proton transportation into cells, affecting fundamental cellular processes. Previous studies have shown that OTOP3 is associated with the prognosis of colorectal cancer (CRC) patients. However, the mechanism underlying OTOP3 regulation remains unclear in cancer. Our investigation reveals OTOP3 as a potent oncogene in CRC, with higher OTOP3 levels correlating with diminished overall survival rates in CRC patients. Comparative analysis between cancerous and normal colon cells highlights a pronounced overexpression of OTOP3 in cancer cells. Suppression of OTOP3 expression significantly curtailed CRC cell proliferation, colony formation, migration, and invasion, concurrently inducing apoptosis and cell cycle arrest, ultimately leading to cancer cell death. Moreover, through CHX, FBS inactivation, and MG132 treatment experiments, it was confirmed that OTOP3 regulates c-Myc expression and stability through ubiquitination and has a critical impact on CRC cell proliferation and growth. Additionally, OTOP3's modulation of HK2, PKM2, and LDHA, underscores its role in impacting cancer cell behavior via glycolytic pathways. In summary, the overexpression of OTOP3 in CRC cells drives cell proliferation and growth through the regulation of c-Myc, impacting crucial cellular processes. Additionally, it promotes these processes by influencing glycolysis. These findings provide valuable insights into potential therapeutic targets for CRC treatment, possibly involving OTOP3 and its associated pathways. Citation Format: Hyeung-Jin Jang, Wona Jee, So-Mi Park, Ye-rin Park, Seok Woo Kim, Ji Hoon Jung. Otopetrin 3 promotes cell proliferation and regulates glycolysis via interaction with MYC in 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 3176.

RFC3 drives the proliferation, migration, invasion and angiogenesis of colorectal cancer cells by binding KIF14.

Colorectal cancer (CRC) is a deadly and aggressive type of cancer that has a high fatality rate. The expression levels of replication factor C subunit 3 (RFC3) and kinesin family member 14 (KIF14) have been reported to be increased in CRC. The current study aimed to explore the effects of RFC3 on the malignant behaviors of CRC cells and its possible underlying mechanism involving KIF14. RFC3 and KIF14 expression levels in CRC tissues were analyzed using TNMplot database and Gene Expression Profiling Interactive Analysis database bioinformatics tools. RFC3 and KIF14 levels in CRC cells were examined using reverse transcription-quantitative PCR and western blotting. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays were performed to assess cell proliferation. Cell apoptosis was determined using flow cytometric analysis. Wound healing and Transwell assays were adopted for the evaluation of cell migration and invasion. Tube formation assay in human umbilical vein endothelial cells was used to measure angiogenesis. Western blotting analysis was performed to determine the expression of apoptosis-, migration- and angiogenesis-associated proteins. Additionally, bioinformatics tools predicted the co-expression and interaction of RFC3 and KIF14, which was verified by a co-immunoprecipitation assay. RFC3 displayed elevated expression in CRC tissues and cells, and depletion of RFC3 halted the proliferation, migration, invasion and angiogenesis, while increasing the apoptosis of CRC cells; this was accompanied by changes in the expression levels of related proteins. In addition, RFC3 bound to KIF14 and interference with RFC3 reduced KIF14 expression. Moreover, KIF14 upregulation reversed the effects of RFC3 depletion on the aggressive cellular behaviors in CRC. In conclusion, RFC3 might interact with KIF14 to function as a contributor to the malignant development of CRC.

Abstract 4635: RNF208: A novel diagnostic and prognostic biomarker for colorectal cancer and key regulator of malignant behavior

Abstract Background: RNF208, a member of the RING finger protein family, has been implicated in ubiquitin-mediated degradation of Vimentin, regulating invasion in breast cancer, and serves as a prognostic factor in uveal melanoma. However, its diagnostic and prognostic value in colorectal cancer (CRC), as well as its role in regulating malignant behavior in CRC, remains unclear. This study aims to reveal the potential of RNF208 as a diagnostic and prognostic marker for CRC and its function in regulating malignant behavior in CRC through bioinformatics analysis and experimental validation. Methods: In this study, based on the ExoRbase2.0 human plasma exosome database (CRC=35; Healthy=118) and TCGA-COAD dataset (CRC=478; Healthy=41), we conducted bioinformatics analysis of the transcriptomic differences in plasma exosomes between CRC patients and normal individuals using ExoRbase2.0. Survival data from the TCGA-COAD dataset were integrated to select key molecules for CRC diagnosis and prognosis. Plasma samples from 39 CRC patients and 16 normal individuals were collected from hospital, and exosomes were isolated and purified using a reagent kit. qPCR was employed to validate the levels of RNF208 in plasma exosomes, confirming the results of bioinformatics analysis. Biological behavior was assessed through CCK-8 proliferation assays, flow cytometry experiments, and Transwell migration assays to investigate the relationship between RNF208 and malignant behavior in the HCT116 colorectal cancer cell line. Results: Bioinformatics analysis revealed a significant correlation between elevated RNF208 expression in plasma exosomes and the incidence of CRC, as well as poor prognosis. The qPCR results from clinical samples further confirmed this correlation, and ROC curves demonstrated high sensitivity and accuracy, suggesting RNF208's potential as a diagnostic and independent prognostic factor for CRC. CCK-8 experiments showed that RNF208 knockdown suppressed proliferation activity in HCT116 cells, flow cytometry indicated that RNF208 knockdown promoted apoptosis in HCT116 cells, and Transwell migration assays demonstrated that RNF208 knockdown inhibited the migration of HCT116 cells. Conclusion: RNF208 serves as a novel diagnostic and prognostic biomarker for colorectal cancer, promoting the proliferation and migration of colorectal cancer cells and contributing to the further deterioration of the disease. Citation Format: Fangyue Guo, Shanfeng Zhang, Pei Li. RNF208: A novel diagnostic and prognostic biomarker for colorectal cancer and key regulator of malignant behavior [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 4635.