Colorectal Cancer Cancer Cell Research Articles

Inhibiting circ_0000673 blocks the progression of colorectal cancer through downregulating CPSF6 via targeting miR-548b-3p.

Colorectal cancer (CRC) is one of the most common cancers, and its progression is regulated by several factors, including circular RNA (circRNA). The objective of this study was to determine the role, or roles, of circ_0000673 in CRC. We used quantitative real-time polymerase chain reaction (qPCR) to detect the expression of circ_0000673, miR-548b-3p and cleavage and polyadenylation specific factor 6 (CPSF6) in DLD-1 and RKO cells. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to determine circ_0000673 roles in proliferation. Wound healing and transwell assays were used to detect cell migration and invasion abilities. Expression of CPSF6 protein and stem cell-associated proteins were examined using western blot. The putative relationship between miR-548b-3p and circ_0000673 or CPSF6 was verified with dual-luciferase reporter assay. The role of circ_0000673 in CRC was also investigated in a tumor xenograft assay in nude mice. Circ_0000673 expression was increased in CRC tissues and cancer cells. Silencing circ_0000673 reduced tumor cell proliferation, migration and invasion, while also decreasing cell stemness. MiR-548b-3p was found to be a target of circ_0000673, while CPSF6 was a downstream target of miR-548b-3p. The tumor-regulatory effects of si-circ_0000673, anti-miR-548b-3p and oe-CPSF6 were partially reversed by anti-miR-548b-3p, si-CPSF6 and si-circ_0000673, respectively, in rescue assays. Downregulation of circ_0000673 reduced solid tumor growth in vivo. Circ_0000673 inhibition reduced CPSF6 expression by targeting miR-548b-3p, thereby blocking proliferation, migration and invasion of CRC tumor cells.

Dexmedetomidine promotes colorectal cancer progression via Piwil2 signaling.

α2-adrenoceptor agonist dexmedetomidine (DEX) has been reported to promote tumorigenesis. Stem-cell protein Piwil2 is associated with cancer progression. Whether Piwil2 plays a role in tumor-promoting effects of DEX is unknown. We examined the expression of Piwil2 in human colorectal cancer (CRC) cell lines with/without DEX treatment. We also studied the roles of Piwil2 in proliferation, invasion, migration, as well as expressions of epithelial-mesenchymal transition (EMT)-related proteins in DEX-treated in vitro and in vivo CRC models. And the experiments with genetic and pharmacological treatments were conducted to investigate the underlying molecular mechanism. RNA-sequencing (RNA-seq) analysis found Piwil2 is one of most upregulated genes upon DEX treatment in CRC cells. Furthermore, Piwil2 protein levels significantly increased in DEX-treated CRC cancer cells, which promoted proliferation, invasion, and migration in both CRC cell lines and human tumor xenografts model. Mechanistically, DEX increased nuclear factor E2-related factor 2 (Nrf2) expression, which enhanced Piwil2 transcription via binding to its promoter. Furthermore, in vitro experiments with Piwil2 knockdown or Siah2 inhibition indicated that DEX promoted EMT process and tumorigenesis through Siah2/PHD3/HIF1α pathway. The experiments with another α2-adrenoceptor agonist Brimonidine and antagonists yohimbine and atipamezole also suggested the role of Piwil2 signaling in tumor-promoting effects via an α2 adrenoceptor-dependent manner. DEX promotes CRC progression may via activating α2 adrenoceptor-dependent Nrf2/Piwil2/Siah2 pathway and thus EMT process. Our work provides a novel insight into the mechanism underlying tumor-promoting effects of α2-adrenoceptor agonists.

Abstract 447: Anti-tumor efficacy of ompenaclid, a creatine transporter inhibitor, in KRAS mutant tumors, including NSCLC

Abstract Background: Pre-clinical studies identified creatine metabolism to be activated in KRAS mutant metastatic colorectal cancer (CRC) as a mechanism to fuel the increased energy demands of RAS mutant tumors. Metastatic colon cancer cells overexpress and subsequently release creatine kinase-B (CKB) into the extracellular space, where it phosphorylates creatine to generate the high energy metabolite phosphocreatine. Ompenaclid is a small molecule inhibitor of the creatine transporter SLC6A8. Inhibition of SLC6A8 by ompenaclid prevents import of phosphocreatine/creatine, resulting in depletion of intracellular creatine, phosphocreatine and ATP levels. This results in tumor apoptosis and robust anti-tumor efficacy in CRC models with a wide range of KRAS mutant alleles. Ompenaclid is currently in a Phase 2 trial, in combination with FOLFIRI and bevacizumab, in patients with RAS-mutated second-line advanced/metastatic colorectal cancer. Here, we assessed efficacy of ompenaclid in tumors beyond CRC, including KRAS WT and KRAS mutant NSCLC. Methods: Athymic mice were inoculated with CRC, pancreatic, gastric or NSCLC cancer cell lines and treated with ompenaclid once tumors reached ~150 mm3, until termination. Plasma metabolites were quantified by LC-MS/MS and tumoral expression of CKB and SLC6A8 in NSCLC and CRC tumors was measured by qPCR after treatment with a control or ompenaclid-supplemented diet for ~10 days. Combination treatment with MRTX849, a KRAS G12C inhibitor, was conducted in a pancreatic MiaPaca xenograft model. Results: Our studies demonstrate ompenaclid anti-tumor efficacy in NSCLC and pancreatic xenografts. Notably, and consistent with findings in CRC, efficacy in NSCLC was more robust in RAS mutant cancer models, relative to those bearing KRAS WT tumors. Metabolite analysis of the plasma of tumor-bearing mice at baseline revealed that mice harboring RAS mutant tumors had higher levels of p-creatine and lower levels of ATP, indicating that RAS mutant tumors utilize higher amounts of ATP, which may be converted to phosphocreatine by tumoral CKB, as confirmed by qPCR analysis. This finding is consistent with metastatic RAS mutant CRC tumors utilizing extracellular ATP to generate phosphocreatine and thus exhibiting greater dependency on creatine metabolism relative to RAS WT tumors. Treatment with ompenaclid also demonstrated additive efficacy in combination with KRAS G12C inhibitors. In summary, these data support development of ompenaclid in indications beyond CRC, including NSCLC, as well as in combination with KRAS G12C inhibitors. Citation Format: PuiChi Lo, Victor Sanz Chavez, Shugaku Takeda, Masoud Tavazoie, Isabel Kurth. Anti-tumor efficacy of ompenaclid, a creatine transporter inhibitor, in KRAS mutant tumors, including NSCLC [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 447.

Abstract 1995: DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant colorectal cancer

Abstract The 5-year survival rate for metastatic colorectal cancer (CRC) is less than 14%. Hyperactivation of the ERK/MAPK pathway occurs at a high frequency in CRC, due to mutations (RAS, BRAF) or overexpression/amplification (EGFR) of its signalling components. Aberrant activation of this pathway drives tumour growth and survival and several inhibitors of this pathway are now clinically utilized. The BRAF V600E activating mutation occurs in ~10% of CRC patients and is correlated with an extremely poor prognosis. Use of the small molecule BRAF inhibitor encorafenib in combination with the EGFR inhibitor cetuximab is now approved for BRAF V600E mutant patients, however, the objective response rate is only ~20% and the treatment is rarely curative. Therefore, there is an urgent need to identify predictive biomarkers of response to encorafenib+cetuximab treatment. Dual-specificity phosphatase 4 (DUSP4) is rapidly induced upon activation of ERK/MAPK signaling upon which it acts as a critical negative regulator of signaling output by directly de-phosphorylating ERK. We have observed that DUSP4 is highly expressed in BRAF-mutant CRC cell lines and primary tumours, although there is a range of expression. Whether DUSP4 expression status impacts on response to encorafenib+cetuximab therapy is unknown. The aim of this study therefore was to determine the role of DUSP4 on sensitivity of BRAF-mutant CRC cells to encorafenib+cetuximab treatment in vitro. To examine this, we determined the impact of DUSP4 knock-down, knock-out and re-expression on sensitivity of BRAF-mutant CRC cancer cell lines to encorafenib+cetuximab treatment. Screening a panel of BRAF mutant CRC cell lines for sensitivity to encorafenib+cetuximab revealed a subset of resistant cell lines had either low or undetectable DUSP4 protein expression, while all sensitive cell lines had high DUSP4 expression. siRNA-mediated knockdown of DUSP4 in DUSP4 expressing BRAF-mutant CRC cell lines increased pERK and pFRA1, suggesting activation of ERK/MAPK signalling. DUSP4 knockdown also reduced sensitivity of BRAF mutant CRC cell lines to encorafenib+cetuximab compared to control. These findings were further validated using DUSP4 knock-out with CRISPR-Cas9. Conversely, DUSP4 re-expression in BRAF-mutant CRC cell lines lacking DUSP4 expression, decreased pERK and increased sensitivity to encorafenib+cetuximab treatment. These results demonstrate that DUSP4 regulates ERK/MAPK signalling in BRAF mutant CRC cell lines and that loss of DUSP4 promotes resistance to encorafenib plus cetuximab treatment. These findings warrant investigation of DUSP4 expression status as a predictive biomarker of encorafenib+cetuximab response in patient samples. Citation Format: Kristen Needham, Fiona Chionh, Stan Kaczmarczyk, Connor Kearney, Laura Jenkins, Ian Luk, John M. Mariadason. DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant 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 1995.

High Expression Level of TRIP6 is Correlated with Poor Prognosis in Colorectal Cancer and Promotes Tumor Cell Proliferation and Migration.

Globally, colorectal cancer (CRC) is one of the leading causes of health problems. More reliable molecular biomarkers for early diagnosis in CRC patients are needed. A crucial role for thyroid hormone receptor interacting protein 6 (TRIP6) is played in tumorigenesis and tumor growth. Our study aims to determine the diagnostic and prognostic roles of TRIP6 at CRC. TRIP6 gene expression levels were analyzed in this study from public databases. The relationship between TRIP6 expression and clinicopathological characteristics was explored by logistic regression analysis. Based on Kaplan-Meier (K-M) survival curves and receiver operating characteristic curves (ROC) analysis, the prognostic and diagnostic values of TRIP6 were determined. Protein-protein interaction (PPI) networks analysis were performed using the STRING database. A Spearman's correlation analysis applied for examining the correlation between TRIP6 expression, immune cell infiltration, and immune checkpoint genes. Moreover, colony formation assay and transwell assay were used to investigate the functions of TRIP6. TRIP6 was highly expressed in CRC cancer tissues and cells. K-M survival analysis indicated that a high expression of TRIP6 was associated with poor prognosis. TRIP6 expression was obviously associated with immune cell infiltration and immune checkpoint gene expression. For validation, the results of collected clinical CRC samples show that TRIP6 levels in CRC tumor tissue were higher than those of paired adjacent colorectal tissues. Additionally, in vitro experiments suggested that TRIP6 knockdown suppressed proliferation and migration in CRC cell line RKO. TRIP6 overexpression promoted the proliferation and migration of normal colon cell line NCM460. High TRIP6 expression is associated with poor prognosis in colorectal cancer and promotes tumor cell proliferation and migration which may be a potential diagnostic and prognostic biomarker and a promising therapeutic target for CRC, providing new insights into its role in CRC.

Investigations of the novel checkpoint kinase 1 inhibitor SRA737 in non-small cell lung cancer and colorectal cancer cells of differing tumour protein 53 gene status

Aim: In response to DNA damage the serine/threonine-specific protein kinase checkpoint kinase 1 (CHK1) is activated allowing cells to enter S phase (S) and G2 phase (G2) cell-cycle arrest. CHK1 inhibitors are expected to prevent cells from entering such arrest, thereby enhancing DNA damage-induced cytotoxicity. In contrast, normal cells with intact ataxia–telangiectasia mutated (ATM), CHK2 and tumour suppressor protein 53 (P53) signalling are still able to enter cell-cycle arrest using the functioning G1/S checkpoint, thereby being rescued from enhanced cytotoxicity. The main objective of this work is to investigate the in vitro effects of the novel CHK1 inhibitor SRA737 on pairs of non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines, all with genetic aberrations rendering them susceptible to replication stress but of differing tumour protein 53 (TP53) gene status, focusing on DNA damage induction and the subsequent effects on cell proliferation and viability. Methods: NSCLC cell lines H23 [TP53 mutant (MUT)] and A549 [TP53 wild-type (WT)] and CRC cell lines HT29 (TP53 MUT) and HCT116 (TP53 WT) were incubated with differing micromolar concentrations of SRA737 for 24 h and then analysed using alkaline comet and phosphorylated H2A.X variant histone (γH2AX)-foci assays to assess mostly DNA single strand break and double strand break damage, respectively. Cell-counting/trypan blue staining was also performed to assess cell proliferation/viability. Results: Clear concentration-dependent increases in comet formation and γH2AX-foci/cell were noted for the TP53 MUT cells with no or lower increases being noted in the corresponding TP53 WT cells. Also, greater anti-proliferative and cell killing effects were noted in the TP53 MUT cells than in the TP53 WT cells. Conclusions: This study’s data suggests that P53 status/functioning is a key factor in determining the sensitivity of NSCLC and CRC cancer cells towards CHK1 inhibition, even in circumstances conducive to high replicative stress.

Network pharmacology and metabolomics elucidate the underlying mechanisms of Venenum Bufonis in the treatment of colorectal cancer

Ethnopharmacological relevanceThe present study aims to evaluate the efficacy of Venenum Bufonis (VBF), a traditional Chinese medicine derived from the dried secretions of the Chinese toad, in treating colorectal cancer (CRC). The comprehensive roles of VBF in CRC through systems biology and metabolomics approaches have been rarely investigated. Aims of the studyThe study sought to uncover the potential underlying mechanisms of VBF's anti-cancer effects by investigating the impact of VBF on cellular metabolic balance. Materials and methodsAn integrative approach combining biological network analysis, molecular docking and multi-dose metabolomics was used to predict the effects and mechanisms of VBF in CRC treatment. The prediction was verified by cell viability assay, EdU assay and flow cytometry. ResultsThe results of the study indicate that VBF presents anti-CRC effects and impacts cellular metabolic balance through its impact on cell cycle-regulating proteins, such as MTOR, CDK1, and TOP2A. The results of the multi-dose metabolomics analysis suggest a dose-dependent reduction of metabolites related to DNA synthesis after VBF treatment, while the EdU and flow cytometry results indicate that VBF inhibits cell proliferation and arrests the cell cycle at the S and G2/M phases. ConclusionsThese findings suggest that VBF disrupts purine and pyrimidine pathways in CRC cancer cells, leading to cell cycle arrest. This proposed workflow integrating molecular docking, multi-dose metabolomics, and biological validation, which contented EdU assay, cell cycle assay, provides a valuable framework for future similar studies.

Combination of 5-FU plus KRAS G12D inhibitor MRTX1133 against human colorectal and pancreatic cancer cells and the affects on inhibition of pERK and immune-stimulatory cytokine patterns in in KRAS G12D and KRAS G12V tumor cells.

e16301 Background: Colorectal cancer (CRC) & pancreatic cancer are among deadliest cancers with great disease burden. The most commonly mutated oncogenic alteration in human cancers is KRAS, which is prevalent in pancreatic, colorectal malignancies. KRAS is considered an especially difficult, if not "undruggable" target. The KRAS G12D is present in more than 40% of PDAC & 34.2% of CRC cases. KRAS mutation subtypes are associated with different downstream pathways and varied clinical outcomes in cancer. MRTX1133 has been identified as a noncovalent, potent, and selective inhibitor of KRASG12D which has been shown to suppress KRASG12D signaling in cells and in vivo. We hypothesized that combination of MRTX1133 with 5-FU will be more effective at slowing or stopping tumor growth, and prolonging overall survival time for patients while allowing lower dose 5-FU with less side effects. Methods: We investigated synergistic effects on pERK inhibition and changes in immune tumor killing with 5-FU plus KRAS G12D inhibitor MRTX1133 in pancreatic and colon cancer cell lines. Human CRC (N=6) and pancreatic (N=4) cancer cell lines with different KRAS mutations were used in experiments evaluating drug effects on pERK at 6 and 24 hours. We evaluated changes at 48 hours in cytokine profiles in treated cells using a custom panel of 62 cytokines, chemokines, and growth factors associated with tumor growth, immune stimulation or immune suppression. Results: Colorectal and pancreatic cancer cell lines had IC50 sensitivities ranging from 7 to 12 microM 5-FU, and >100 nM to >5 microM for MRTX1133 (G12D N=4: LS513, 120 nM; HPAF-II, 1.8 microM; SNUC2B, 5.7 microM; PANC-1, 2.8 microM). For non-G12D, the range of IC50 for MRTX1133 was in the range of 2 to 9 microM for CRC lines with G12V, G13D, or wild- type KRAS (N=7). Synergies between 5-FU and MRTX1133 were observed regardless of presence of KRAS G12D mutation with combination indices of <0.5 indicating strong synergy. pERK was suppressed by MRTX1133 treatment of tumor cells regardless of KRAS G12D mutation. IL8/CXCL8, MICA, Angiopoietin 2, VEGF and TNF-alpha were reduced while IL-18/IL-1F4 increased. Conclusions: Our studies reveal strong synergy between MRTX1133 & 5-FU in human pancreatic & colon cancer models at much lower than IC50 dosage which is important for avoiding side effects. This is the first description that effect of KRAS G12D inhibitor MRTX1133 is active on KRAS G12V. The surprising synergy in KRAS G12V samples with combination therapy and the important synergistic change in cytokine patterns suggests potential strong immune stimulatory anti-cancer effects of MRTX1133 & 5FU against mCRC and pancreatic cancer cells regardless of KRAS G12D mutation which should be considered when including patients with respective mutations in clinical trials.

BZD9L1 benzimidazole analogue hampers colorectal tumor progression by impeding angiogenesis.

The development of new vasculatures (angiogenesis) is indispensable in supplying oxygen and nutrients to fuel tumor growth. Epigenetic dysregulation in the tumor vasculature is critical to colorectal cancer (CRC) progression. Sirtuin (SIRT) enzymes are highly expressed in blood vessels. BZD9L1 benzimidazole analogue is a SIRT 1 and 2 inhibitor with reported anticancer activities in CRC. However, its role has yet to be explored in CRC tumor angiogenesis. To investigate the anti-angiogenic potential of BZD9L1 on endothelial cells (EC) in vitro, ex vivo and in HCT116 CRC xenograft in vivo models. EA.hy926 EC were treated with half inhibitory concentration (IC50) (2.5 μM), IC50 (5.0 μM), and double IC50 (10.0 μM) of BZD9L1 and assessed for cell proliferation, adhesion and SIRT 1 and 2 protein expression. Next, 2.5 μM and 5.0 μM of BZD9L1 were employed in downstream in vitro assays, including cell cycle, cell death and sprouting in EC. The effect of BZD9L1 on cell adhesion molecules and SIRT 1 and 2 were assessed via real-time quantitative polymerase chain reaction (qPCR). The growth factors secreted by EC post-treatment were evaluated using the Quantibody Human Angiogenesis Array. Indirect co-culture with HCT116 CRC cells was performed to investigate the impact of growth factors modulated by BZD9L1-treated EC on CRC. The effect of BZD9L1 on sprouting impediment and vessel regression was determined using mouse choroids. HCT116 cells were also injected subcutaneously into nude mice and analyzed for the outcome of BZD9L1 on tumor necrosis, Ki67 protein expression indicative of proliferation, cluster of differentiation 31 (CD31) and CD34 EC markers, and SIRT 1 and 2 genes via hematoxylin and eosin, immunohistochemistry and qPCR, respectively. BZD9L1 impeded EC proliferation, adhesion, and spheroid sprouting through the downregulation of intercellular adhesion molecule 1, vascular endothelial cadherin, integrin-alpha V, SIRT1 and SIRT2 genes. The compound also arrested the cells at G1 phase and induced apoptosis in the EC. In mouse choroids, BZD9L1 inhibited sprouting and regressed sprouting vessels compared to the negative control. Compared to the negative control, the compound also reduced the protein levels of angiogenin, basic fibroblast growth factor, platelet-derived growth factor and placental growth factor, which then inhibited HCT116 CRC spheroid invasion in co-culture. In addition, a significant reduction in CRC tumor growth was noted alongside the downregulation of human SIRT1 (hSIRT1), hSIRT2, CD31, and CD34 EC markers and murine SIRT2 gene, while the murine SIRT1 gene remained unaffected, compared to vehicle control. Histology analyses revealed that BZD9L1 at low (50 mg/kg) and high (250 mg/kg) doses reduced Ki-67 protein expression, while BZD9L1 at the high dose diminished tumor necrosis compared to vehicle control. These results highlighted the anti-angiogenic potential of BZD9L1 to reduce CRC tumor progression. Furthermore, together with previous anticancer findings, this study provides valuable insights into the potential of BZD9L1 to co-target CRC tumor vasculatures and cancer cells via SIRT1 and/or SIRT2 down-regulation to improve the therapeutic outcome.

LIM domain-containing protein Ajuba inhibits chemotherapy-induced apoptosis by negatively regulating p53 stability in colorectal cancer cells.

LIM protein-domain containing protein Ajuba (encoded by AJUBA) functions as a scaffold protein to regulate protein-protein interactions, signalling transduction and genes transcription. AJUBA expression is higher in colorectal cancer (CRC) tissues than normal tissues, but its specific molecular function in CRC progression is still not very clear. Here, we found that, in CRC cancer cell lines, overexpression of AJUBA decreased p53 levels, whereas knock-down of AJUBA significantly increased p53 levels. Although the presence of Ajuba did not influence p53 transcription, it formed a complex with p53 and MDM2 to promote the degradation of p53. AJUBA overexpression reduced the sensitivity of cancer cells to chemotherapeutic drugs and vice versa. In addition, chemotherapeutic drugs significantly induced AJUBA expression, which was largely dependent on the presence of p53. Therefore, Ajuba formed a negative feedback loop to regulate p53 expression and activity. In conclusion, as a novel p53-negative regulator, Ajuba inhibits the apoptosis of CRC cells induced by chemotherapeutic drugs and it may be a new therapeutic target for CRC treatment.

Upregulation of APOC1 Promotes Colorectal Cancer Progression and Serves as a Potential Therapeutic Target Based on Bioinformatics Analysis.

Approximately 10% of cancer patients worldwide have colorectal cancer (CRC), a prevalent gastrointestinal malignancy with substantial mortality and morbidity. The purpose of this work was to investigate the APOC1 gene's expression patterns in the CRC tumor microenvironment and, using the findings from bioinformatics, to assess the biological function of APOC1 in the development of CRC. The TCGA portal was employed in this investigation to find APOC1 expression in CRC. Its correlation with other genes and clinicopathological data was examined using the UALCAN database. To validate APOC1's cellular location, the Human Protein was employed. In order to forecast the relationship between APOC1 expression and prognosis in CRC patients, the Kaplan-Meier plotter database was used. TISIDB was also employed to evaluate the connection between immune responses and APOC1 expression in CRC. The interactions of APOC1 with other proteins were predicted using STRING. In order to understand the factors that contribute to liver metastasis from CRC, single-cell RNA sequencing (scRNA-seq) was done on one patient who had the disease. This procedure included sampling preoperative blood and the main colorectal cancer tissues, surrounding colorectal cancer normal tissues, liver metastatic cancer tissues, and normal liver tissues. Finally, an in vitro knockdown method was used to assess how APOC1 expression in tumor-associated macrophages (TAMs) affected CRC cancer cell growth and migration. When compared to paracancerous tissues, APOC1 expression was considerably higher in CRC tissues. The clinicopathological stage and the prognosis of CRC patients had a positive correlation with APOC1 upregulation and a negative correlation, respectively. APOC1 proteins are mostly found in cell cytosols where they may interact with APOE, RAB42, and TREM2. APOC1 was also discovered to have a substantial relationship with immunoinhibitors (CD274, IDO1, and IL10) and immunostimulators (PVR, CD86, and ICOS). According to the results of scRNA-seq, we found that TAMs of CRC tissues had considerably more APOC1 than other cell groups. The proliferation and migration of CRC cells were impeded in vitro by APOC1 knockdown in TAMs. Based on scRNA-seq research, the current study shows that APOC1 was overexpressed in TAMs from CRC tissues. By inhibiting APOC1 in TAMs, CRC progression was reduced in vitro, offering a new tactic and giving CRC patients fresh hope.

Studies on 1,4-Quinone Derivatives Exhibiting Anti-Leukemic Activity along with Anti-Colorectal and Anti-Breast Cancer Effects

Colorectal cancer (CRC), breast cancer, and chronic myeloid leukemia (CML) are life-threatening malignancies worldwide. Although potent therapeutic and screening strategies have been developed so far, these cancer types are still major public health problems. Therefore, the exploration of more potent and selective new agents is urgently required for the treatment of these cancers. Quinones represent one of the most important structures in anticancer drug discovery. We have previously identified a series of quinone-based compounds (ABQ-1-17) as anti-CML agents. In the current work, ABQ-3 was taken to the National Cancer Institute (NCI) for screening to determine its in vitro antiproliferative effects against a large panel of human tumor cell lines at five doses. ABQ-3 revealed significant growth inhibition against HCT-116 CRC and MCF-7 breast cancer cells with 2.00 µM and 2.35 µM GI50 values, respectively. The MTT test also showed that ABQ-3 possessed anticancer effects towards HCT-116 and MCF-7 cells with IC50 values of 5.22 ± 2.41 μM and 7.46 ± 2.76 μM, respectively. Further experiments indicated that ABQ-3 induced apoptosis in both cell lines, and molecular docking studies explicitly suggested that ABQ-3 exhibited DNA binding in a similar fashion to previously reported compounds. Based on in silico pharmacokinetic prediction, ABQ-3 might display drug-like features enabling this compound to become a lead molecule for future studies.

In Vitro Cytotoxicity Evaluation of Plastoquinone Analogues against Colorectal and Breast Cancers along with In Silico Insights.

Colorectal cancer (CRC) and breast cancer are leading causes of death globally, due to significant challenges in detection and management. The late-stage diagnosis and treatment failures require the discovery of potential anticancer agents to achieve a satisfactory therapeutic effect. We have previously reported a series of plastoquinone analogues to understand their cytotoxic profile. Among these derivatives, three of them (AQ-11, AQ-12, and AQ-15) were selected by the National Cancer Institute (NCI) to evaluate their in vitro antiproliferative activity against a panel of 60 human tumor cell lines. AQ-12 exhibited significant antiproliferative activity against HCT-116 CRC and MCF-7 breast cancer cells at a single dose and further five doses. MTT assay was also performed for AQ-12 at different concentrations against these two cells, implying that AQ-12 exerted notable cytotoxicity toward HCT-116 (IC50 = 5.11 ± 2.14 μM) and MCF-7 (IC50 = 6.06 ± 3.09 μM) cells in comparison with cisplatin (IC50 = 23.68 ± 6.81 μM and 19.67 ± 5.94 μM, respectively). This compound also augmented apoptosis in HCT-116 (62.30%) and MCF-7 (64.60%) cells comparable to cisplatin (67.30% and 78.80%, respectively). Molecular docking studies showed that AQ-12 bound to DNA, forming hydrogen bonding through the quinone scaffold. In silico pharmacokinetic determinants indicated that AQ-12 demonstrated drug-likeness with a remarkable pharmacokinetic profile for future mechanistic anti-CRC and anti-breast cancer activity studies.

CircPTK2 accelerates tumorigenesis of colorectal cancer by upregulating AKT2 expression via miR-506-3p.

With the rapid increase in its incidence in the last decade, colorectal cancer (CRC) is becoming one of the most life-threatening cancers. Circular RNA PTK2 (circPTK2) has multiple functions in oncogenesis, including in CRC. However, it remains elusive if circPTK2 also plays an important role in CRC malignancy. The levels of circPTK2, miR-506-3p, and AKT serine/threonine kinase 2 (AKT2) were measured by qPCR. The protein level of AKT2 was evaluated by western blotting assay. The proliferation, migration, and invasion of CRC cancer cells were evaluated by MTT, colony formation, wound-healing, and transwell assays. The interaction between circPTK2 and miR-506-3p and between miR-506-3p and AKT2 mRNA were verified by dual-luciferase reporter assay. The expressions of circPTK2 and AKT2 were elevated in CRC cells, with a concomitant reduction of miR-506-3p. The knockdown of circPTK2 suppressed the proliferation, migration, and invasion of CRC cells. CircPTK2 targeted miR-506-3p and negatively regulated its expression. Furthermore, miR-506-3p overexpression suppressed the CRC progression by downregulating the AKT2 expression. AKT2 overexpression or miR-506-3p inhibition restored the suppression of growth and invasiveness of CRC cancer cells caused by circPTK2 silencing. The circPTK2/miR-506-3p/AKT2 axis plays a novel and essential role in promoting CRC progression, providing potential targets for CRC therapeutic modality.

Synthesis and biological evaluation of a novel c-Myc inhibitor against colorectal cancer via blocking c-Myc/Max heterodimerization and disturbing its DNA binding

c-Myc is a transcription factor that is aberrantly expressed in the majority of human cancers. Recent studies unveiled that abnormal expression of c-Myc protein is involved in the development of colorectal cancer (CRC). Previously, we reported a novel phenoxy-N-phenylaniline derivative A-42 that can inhibit c-Myc protein and the growth of different CRC cancer cells potently. To look for a better candidate, the structure-activity relationship (SAR) of A ring, D ring and the linker between A and B rings of A-42 was investigated, and a series of compounds were synthesized. Among them, compound B13 was identified as the most active c-Myc inhibitor with cytotoxicity activity against HT29 and HCT116 cells at IC50 0.29 μM and IC50 0.64 μM, respectively, which is superior to that of A-42. According to the bioassays, compound B13 not only can suppress CRC cells proliferation and migration, but also inhibit the binding of c-Myc/Max dimer to DNA, which further interfere with the expression of the relevant proteins of apoptosis pathway. Furthermore, B13 could inhibit HT29 tumor growth in xenograft mouse models potently with tumor growth inhibitions (TGIs) up to 65.49% at dose of 40 mg/kg, which is superior to A-42 (55.82%, 40 mg/kg). Overall, B13 may potentially serve as an effective CRC therapy via blocking c-Myc/Max binding with DNA.

Celastrol upregulated ATG7 triggers autophagy via targeting Nur77 in colorectal cancer

BackgroundCelastrol is a biologically active ingredient extracted from Tripterygium wilfordii that has exerted properties of anti-cancer. We explored the anti-tumor activities of celastrol against colorectal cancer (CRC) and the potential signaling pathways involved in its mechanism in this study. PurposeThe main purpose was to investigate the anti-CRC effects of celastrol and its novel potential mechanisms. Study designHCT-116 and SW480 cell lines were used for in vitro studies, the mouse xenograft model of CRC tumor was performed for in vivo studies. MethodsThe effects of celastrol on colorectal cancer cells in vitro and underlying mechanisms were examined by using western blot analysis, cell proliferation assays, PI and Annexin-V staining assays, immunofluorescence and qRT-PCR assay. CRC xenografts model and IHC-staining were mainly used to evaluate the effects of celastrol in vivo. ResultsThe results demonstrated that celastrol induced apoptosis and inhibited proliferation in CRC cells. The expression of Nur77 influenced the anti-CRC effects of celastrol, and inhibitory effect of celastrol on CRC cells could be reversed by overexpressing Nur77. Celastrol induced autophagy and the autophagy inhibition enhanced the anti-CRC effects. The ATG7 was up-regulated obviously after celastrol treatment for Nur77 overexpressing CRC cancer cells. Treating mice implanted with CRC cells with celastrol showed that it effectively inhibited tumor growth, which was associated with the down-regulation of Nur77. Levels of Nur77 and ATG7 were correlated with survival in human colorectal cancer. ConclusionCelastrol induced apoptosis and autophagy played an important role in human colorectal cancer, Nur77 was involved in the anti-CRC effect of celastrol and decreased expression of Nur77 induced high expression of ATG7. Celastrol exerted anti-CRC effects by inhibiting Nur77 to induce high expression of ATG7 signaling and Nur77/ATG7 signaling may be a potential pathway for colorectal cancer treatment.

Transcriptome analysis of clock disrupted cancer cells reveals differential alternative splicing of cancer hallmarks genes

Emerging evidence points towards a regulatory role of the circadian clock in alternative splicing (AS). Whether alterations in core-clock components may contribute to differential AS events is largely unknown. To address this, we carried out a computational analysis on recently generated time-series RNA-seq datasets from three core-clock knockout (KO) genes (ARNTL, NR1D1, PER2) and WT of a colorectal cancer (CRC) cell line, and time-series RNA-seq datasets for additional CRC and Hodgkin’s lymphoma (HL) cells, murine WT, Arntl KO, and Nr1d1/2 KO, and murine SCN WT tissue. The deletion of individual core-clock genes resulted in the loss of circadian expression in crucial spliceosome components such as SF3A1 (in ARNTLKO), SNW1 (in NR1D1KO), and HNRNPC (in PER2KO), which led to a differential pattern of KO-specific AS events. All HCT116KO cells showed a rhythmicity loss of a crucial spliceosome gene U2AF1, which was also not rhythmic in higher progression stage CRC and HL cancer cells. AS analysis revealed an increase in alternative first exon events specific to PER2 and NR1D1 KO in HCT116 cells, and a KO-specific change in expression and rhythmicity pattern of AS transcripts related to cancer hallmarks genes including FGFR2 in HCT116_ARNTLKO, CD44 in HCT116_NR1D1KO, and MET in HCT116_PER2KO. KO-specific changes in rhythmic properties of known spliced variants of these genes (e.g. FGFR2 IIIb/FGFR2 IIIc) correlated with epithelial-mesenchymal-transition signalling. Altogether, our bioinformatic analysis highlights a role for the circadian clock in the regulation of AS, and reveals a potential impact of clock disruption in aberrant splicing in cancer hallmark genes.

M6A methylated EphA2 and VEGFA through IGF2BP2/3 regulation promotes vasculogenic mimicry in colorectal cancer via PI3K/AKT and ERK1/2 signaling

Exploring the epigenetic regulation mechanism of colorectal cancer (CRC) from the perspective of N6-methyladenosine (m6A) modification may provide a new target for tumor therapy. Analysis using high-throughput RNA-seq profile from TCGA found that the gene expression of Methyltransferase-like 3 (METTL3) was significantly upregulated among 20 m6A binding proteins in CRC, which was also validated in CRC cancer tissues and cell lines. Moreover, transcriptome sequencing in METTL3 knockdown cells using CRISPR/Cas9 editing suggested that EphA2 and VEGFA were differential expression, which were enriched in the vasculature development, PI3K/AKT and ERK1/2 signal pathway through the functional enrichment analysis. The results in vitro revealed that METTL3 as the m6A “writers” participates the methylation of EphA2 and VEGFA, which were recognized by the m6A “readers”, insulin-like growth factor 2 mRNA binding protein 2/3 (IGF2BP2/3), to prevent their mRNA degradation. In addition, EphA2 and VEGFA targeted by METTL3 via different IGF2BP-dependent mechanisms were found to promote vasculogenic mimicry (VM) formation via PI3K/AKT/mTOR and ERK1/2 signaling in CRC. The study suggests that intervention with m6A-binding proteins (METTL3 and IGF2BP2/3) may provide a potential diagnostic or prognostic target of VM-based anti-metastasis drugs for CRC.

Overexpression of OTU domain-containing ubiquitin aldehyde-binding protein 1 exacerbates colorectal cancer malignancy by inhibiting protein degradation of β-Catenin via Ubiquitin-proteasome pathway

ABSTRACT Although major advances were achieved in colorectal cancer (CRC) therapy, major concerns still remain on proper control of cancer metastasis and chemo-resistance in order to achieve satisfactory general treatment response. Previous studies suggested that OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1) serves as regulator of gene ubiquitination and participates in the pathogenesis of multiple malignancies. Therefore, to discover its molecular mechanism in CRC tumor growth and metastasis will contribute in CRC treatment strategy development. Clinical tissues and CRC cancer cell lines were utilized to evaluate OTUB1 expression pattern. Functional tests including cellular proliferation, migration and invasion, as well as chemo-resistance, etc., were evaluated to investigate the role of OTUB1/β-catenin regulatory pathway on CRC malignant biological behaviors. Both CRC tumor tissues and CRC cell lines exhibited promoted OTUB1 expression level. Subsequent experiments further suggested that OTUB1 promoted CRC malignancy by enhancing protein stability of β-catenin, via inhibition of its protein degradation by UPP pathway, which indicated its crucial role in enhancement of CRC tumor cellular proliferative and chemo-resistant capabilities. This study reported that OTUB1 exhibited novel pro-survival and pro-metastatic function by interaction of β-Catenin via Ubiquitin-proteasome pathway. Our research indicated that OTUB1/β-Catenin regulatory axis might be potential druggable target for CRC cancer patients’ treatment.

The Anticancer Effect of a Conjugated Antimicrobial Peptide Against Colorectal Cancer (CRC) Cells.

Although antimicrobial peptides (AMPs) were initially known as compounds of the innate immune system to fight microbial pathogens, it has been recently proposed that differences in normal and cancer cell membranes cause the anticancer effect of these peptides. The aim of this study was to evaluate the anticancer effect of MELITININ+BMAP27-conjugated peptide against colorectal cancer (CRC) cells. The MELITININ+BMAP27-conjugated peptides were designed and the β-naphthylalanine residues were added to the termini to improve the anticancer effect. CRC cancer cell lines including HT29, SW742, HCT-116, and WiDr were used. After preparing concentrations of 5, 10, 25, 50, 100, 150, 200, and 400μg/mL of peptide solution, the rate of cell death after 12, 24, and 48h was assessed using MTT test. After confirmation of the 30µg/mL efficacy and nontoxic concentration, the cells were exposed to this concentration, and the total RNA was extracted. The quantitative real-time PCR (RT-qPCR) technique was performed for the amplification of Bax, caspase3, atg5, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase as the internal control) genes. The cytotoxicity of peptide against normal cells exhibited that the IC50 at 24 and 4h included 80 and 100µg/mL, respectively. After 24-72h of treatment, a significant difference in the mean percentage of CRC living cells was observed at concentrations of 50-400μg/mL of conjugated peptide (p < 0.05). The IC50 of the peptide at 24, 48, and 72h of exposure was measured as 30, 20, and 10μg/mL, respectively. The peptide resulted in a significant increase of 2.35-fold in the mean expression of Bax gene in CRC cells (p < 0.001). It also caused a significant increase of 1.75 times (p = 0.0112) of caspase 3 gene and 1.2 times (p = 0.0217) of atg5 gene. There was no significant difference among cell lines regarding the expression of each gene. The conjugated peptide caused the death of CRC lines via induction of the apoptosis and necrosis mechanisms. More studies are needed in this regard.