Human CRC Cell Lines Research Articles

P53 genotype-independent anticancer effects of olibanum extract and 11-keto-beta-boswellic acid on human colorectal cancer cells

BackgroundColorectal cancer (CRC) is the third most common cancer, and CRC-related mortality is increasing annually. A key regulator of the cell cycle and cell death, the p53 gene, is frequently found in patients with CRC as mutants. Since tumors with abnormal p53 sometimes show resistance to anticancer drugs, it is important to develop anticancer drugs according to genetic characteristics. Although evidence shows that boswellic acids (BAs) might be potential anticancer agents, the anticancer effects and mechanisms involved in their activities in CRC are unclear. Methods and ResultsHere, we investigated whether olibanum (Boswellia serrata Roxb.) extract and 11-keto-β-boswellic acid exerted cytotoxic effects against p53 wildtype, p53 mutated-, and p53-deleted human CRC cell lines. Results show that 11-keto-β-boswellic acid considerably reduced cell viability and induced cell cycle arrest in HCT116, HT29, and SW1417 cells. And increase of apoptotic cell population and morphological changes in apoptotic cells induced by 11-keto-β-boswellic acid were observed. The 11-keto-β-boswellic acid mediated cell cycle arrest and apoptosis were accompanied by changes in the expression of factors linked to apoptosis, regardless of p53 genotype. In addition, 11-keto-β-boswellic acid-treated cells dissociated from their spheroidal structure and appeared in an irregular form. ConclusionsAlthough further investigations are required to fully understand the underlying mechanisms, these results may provide insight into the potential of using olibanum extract and 11-keto-β-boswellic acid as therapeutic agents, with no difference in sensitivity depending on the p53 genotype.

Abstract 2086: Effect of XPO1 inhibition on colorectal cancer tumorigenesis

Abstract Colorectal Cancer (CRC) is the second leading cause of cancer-related death in the U.S. A subset of individuals faces a notably higher likelihood of developing CRC within their lifetime. Hence, there is a compelling need for innovative chemopreventive treatments aimed at minimizing CRC tumorigenesis. Exportin 1 (XPO1; also referred to as CRM1) plays a pivotal role in transporting proteins from the nucleus to the cytoplasm. The overexpression of XPO1 has been identified across several cancers, including CRC. XPO1 interacts with a diverse spectrum of more than 1000 proteins. The overexpression of XPO1 leads to the excessive removal of tumor suppressors from the nucleus, rendering these proteins functionally inert. To counteract the overexpression of XPO1 in tumors, a novel class of drugs known as Selective Inhibitors of Nuclear Export (SINE) has been developed. Among these, Eltanexor (KPT-8602) has emerged as a promising therapeutic agent, demonstrating fewer side effects compared to its precursors, and is currently under evaluation in Phase 1/2 clinical trials. The treatment effects of SINE compounds on CRC remain largely unknown. We have assessed Eltanexor's ability to limit CRC tumorigenesis using a genetic CRC model in Apcmin/+ mice. Our findings indicate that Eltanexor substantially reduces tumor burden by approximately 3-fold and induces a significant reduction of tumors exceeding 1mm in size. Furthermore, a xenograft experiment using the human CRC cell line, HCT116, reveals nearly a 3-fold reduction in tumor size upon Eltanexor treatment. In both CRC models, Eltanexor exhibits noteworthy tolerability. Additionally, XPO1 protein levels are consistently diminished in both models, aligning with our in vitro observations. To further ascertain Eltanexor's specificity for CRC tumors versus normal tissue, we conducted drug sensitivity assays using organoids derived from Apcmin/+ tumors and wild-type mice small intestine tissue. We found that Apcmin/+ tumoroids have heightened sensitivity to Eltanexor-treatments, when compared to wild-type organoids. In an endeavor to elucidate Eltanexor's potent in vivo tumorigenesis inhibitory effects, we conducted multiple assays which revealed a reduction in COX-2 RNA and protein expression levels in Eltanexor-treated cells and reduced COX-2 protein in the tumors of Eltanexor-treated Apcmin/+ mice. In CRC, COX-2 is implicated in CRC development and is currently the target of multiple CRC chemopreventive agents. Our observations reveal that Eltanexor-treated CRC cells exhibit reduced COX-2 gene expression through both inhibiting COX-2’s promoter and its mRNA post-transcriptional stability. Collectively, our findings underscore XPO1 as a potent target for inhibiting CRC tumorigenesis. Future studies will aim to further assess how Eltanexor is impacting COX-2’s promoter, while additionally testing Eltanexor’s tumorigenesis-inhibiting potential in a colitis-induced CRC mouse model. Citation Format: Andrew E. Evans, Dan A. Dixon. Effect of XPO1 inhibition on colorectal cancer 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 2086.

Abstract 1566: Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties 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 1566.

A54 SHP2 FUNCTION IN NORMAL AND APC-MUTANT INTESTINAL EPITHELIAL CELLS

Abstract Background Somatic SHP2 gain-of-function mutations were found in some colorectal tumours. However, its function in colorectal epithelium remains to be determined. SHP2 is a tyrosine phosphatase necessary for RAS/MAPK pathway activation by most, if not all, tyrosine kinase receptors, as well as by GPCRs and cytokine receptors. SHP2 can also regulate the PI3K, Jak/Stat, RhoA and Hippo pathways in different cell settings. As a result, SHP2 can control cellular processes including growth, migration and differentiation. Previously, we observed that SHP2 levels are enhanced in human colorectal tumours, notably in late adenomas. Likewise, SHP2 silencing inhibited ERK activation and tumour properties of established human CRC cell lines. Hence, these findings support a model in which SHP2 activation promotes tumorigenesis in the intestinal epithelium. But how? Aims To analyze the exact cellular and molecular mechanisms underlying SHP2 pro-oncogenic function in intestinal epithelial cells. Methods The impact of SHP2 pharmaco-inhibition (RMC-4550 and/or SHP099) on intracellular signaling, cell cycle, proliferation and survival was evaluated in three complementary models: 1) HIEC-6 cells, normal human fetal intestinal crypt-like cells; 2) organoids derived from normal mouse small intestine or ApcMin/+ tumors; 3) Caco-2/15 cells, colorectal cancer cells with APC mutation. Results 1- Pharmaco-inhibition of SHP2 blocks proliferation of HIEC-6 cells (loss of pRb phosphorylation, decreased EdU staining) and induces cell enlargement and darkening of the outside of perinuclear area. This phenotype was reminiscent of cellular senescence, and we indeed observed increased senescence-associated β-galactosidase activity (SA-β-Gal) and INK4A gene expression. 2- As expected, SHP2 inhibition in normal mouse organoids resulted in a reduction in the number of protrusions and EdU positive cells. RNA sequencing was performed, and gene set enriched revealed significant down regulation of several Ras signaling-dependant genes including cell cycle regulated genes (E2F targets, G2/M checkpoint) and Myc targets 48h after SHP2 inhibition when compared to DMSO treated organoids. Genes associated with PI3K signaling were also diminished in SHP099 treated organoids but no significant modulation of genes associated with Wnt/b-catenin signaling was observed. 3- Notably, SHP2 pharmaco-inhibition in Caco-2/15 cells as well as APC-mutant organoids rapidly induced cell death associated with increased expression of stem cell markers. Conclusions These results suggest that SHP2 is required for proliferation of normal IEC and protects against senescence. Notably, its inhibition restrains the proliferative and tumorigenic potential conferred by APC inactivation. Overall, our data suggest that SHP2 could represent a potential target to counter colorectal tumour initiation. Funding Agencies CIHRFRQS

Abstract C111: A novel human monoclonal antibody targeting active ADAM10 demonstrates anti-tumor effects in colorectal cancer

Abstract Colorectal cancer (CRC) is the third most diagnosed cancer in the USA and accounts for more than 600,000 deaths annually worldwide, primarily due to relapse with highly aggressive, chemo-resistant disease characterized by poorly differentiated cancer cells with stem cell-like properties. A common signature of these chemo-resistant tumors is dysregulation of Notch receptor signaling, as well as upregulation of its metalloprotease activator, ADAM10. Although small molecule inhibition of either Notch or ADAM10 has been shown to produce potent anti-tumor effects, these therapeutic strategies have failed in clinical trials primarily due to systemic toxicities, especially cytotoxic effects on the gastrointestinal tract and musculoskeletal system, highlighting the need for development of more targeted approaches. We have previously demonstrated that ADAM10 predominantly exists in an inhibited state in normal tissues but is activated in tumor cells through a conformational change in the extracellular domain, thus providing a potential target for tumor-specific modulation of ADAM10 activity. Here, we look to characterize a novel human monoclonal antibody agent (1H5) that selectively targets an exposed extracellular region of activated ADAM10 on tumor cells. We previously demonstrated that treatment with a murine version of the antibody (8C7), specific for the activated form of both mouse and human ADAM10, conferred a significant reduction in tumor burden against human CRC cell lines in cell culture and transplants in xenograft models, and relapse was prevented when 8C7 was combined with chemotherapy. Here, we demonstrate that the 1H5 monoclonal antibody effectively reduces cell viability in numerous colorectal cancer cell lines in vitro and induces marked tumor regression in established xenograft models through inhibition of Notch signaling. Through the use of additional genetic tools combined with in vitro and in vivo models, our preclinical study aims to fully evaluate 1H5 as novel therapeutic agent to treat aggressive colorectal cancer. We aim to propel this novel agent toward clinical-stage development. Citation Format: Pargol Mashati, Dan Sun, Eduardo Garcia Reino, Jessica A. Blandino, Ben Mason, Nayanendu Saha, Dimitar B. Nikolov, Prem Premsrirut. A novel human monoclonal antibody targeting active ADAM10 demonstrates anti-tumor effects in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C111.

Abstract C043: Discovery of new drug combinations for metastatic colorectal cancer

Abstract Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide. Despite a range of therapeutic options, drug resistance remains a significant challenge in CRC treatment. Drug combination therapy offers an opportunity to enhance therapeutic responses. There are several combination drug options that have been used for CRC patients such as FOLFOX (5-FU + Oxaliplatin) and FOLFIRI (5-FU + Irinotecan). This study aimed to discover novel drug combinations for CRC by conducting high-throughput screening of 800 pharmacologically active compounds in combination with the three main chemotherapy backbones used in CRC treatment: 5-FU, SN-38, and Oxaliplatin. Initially, we performed a primary drug screen using 11 human CRC cell lines, resulting in the identification of 72 candidate drug combinations (24 candidates x 3 backbones) exhibiting synergistic effects. Subsequently, we validated these candidates in a subset of 5 cell lines. Among them, 24 drug combinations (8 candidates x 3 backbones) were selected and further evaluated in an expanded panel of 44 CRC cell lines to determine the spectrum of drug responses and identify potential mutation, gene and protein expression biomarkers, that could predict the efficacy of the drug combinations. We investigated the molecular mechanisms underlying the synergistic effects of final 4 candidates combined with chemotherapeutic agents. Chk1 inhibitor MK-8776 showed significantly higher synergy scores in combination with SN-38 in TP53 mutated cell lines, which was further validated by in vitro apoptosis analysis and in vivo xenograft models using TP53 wildtype and knockout isogenic CRC cell lines. MK-8776 abrogated SN-38-mediated G2 cell cycle arrest, resulting in an increased cytotoxicity. These findings highlight the potential of the combination treatment involving SN-38 and MK-8776 as a promising strategy for CRC patients with TP53 mutations. Citation Format: Eun-Jung In, Tao Tan, Dmitri Mouradov, Anuratha Sakthianandeswaren, Amanda Au, Kym Lowes, Oliver Sieber. Discovery of new drug combinations for metastatic colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C043.

PEGylated Fluorescent Anti-carcinoembryonic Antigen Antibody Labels Colorectal Cancer Tumors in Orthotopic Mouse Models

IntroductionColorectal cancer (CRC) patients often develop liver metastasis. However, curative resection of liver metastasis is not always possible due to poor visualization of tumor margins. The present study reports the characterization of a humanized anti-carcinoembryonic antigen monoclonal antibody conjugated to a PEGylated near-infrared dye, that targets and brightly labels human CRC tumors in metastatic orthotopic mouse models. MethodsThe hT84.66-M5A (M5A) monoclonal antibody was conjugated with a polyethylene glycol (PEG) chain that incorporated a near infrared (NIR) IR800 dye to establish M5A-IR800 Sidewinder (M5A-IR800-SW). Nude mice with CRC orthotopic primary tumors and liver metastasis both developed from a human CRC cell line, were injected with M5A-IR800-SW and imaged with the Pearl Trilogy Imaging System. ResultsM5A-IR800-SW targeted and brightly labeled CRC tumors, both in primary-tumor and liver-metastasis models. M5A-IR800-SW at 75 μg exhibited highly-specific tumor labeling in a primary-tumor orthotopic model with a median tumor-to-background ratio of 9.77 and in a liver-metastasis orthotopic model with a median tumor-to-background ratio of 7.23 at 96 h. The precise labeling of the liver metastasis was due to lack of hepatic accumulation of M5A-IR800-SW in the liver. ConclusionsM5A-IR800-SW provided bright and targeted NIR images of human CRC in orthotopic primary-tumor and liver-metastasis mouse models. The results of the present study suggest the clinical potential of M5A-IR800-SW for fluorescence-guided surgery including metastasectomies for CRC. The lack of hepatic NIR signal is of critical importance to allow for precise labeling of liver tumors.

Blastocystis sp. reduces the efficacy of 5-fluorouracil as a colorectal cancer chemotherapeutic treatment

Blastocystis is an enteric protozoan parasite with extensive genetic variation and unclear pathogenicity. It is commonly associated with gastrointestinal symptoms such as nausea, diarrhea, vomiting and abdominal pain in immunocompromised individuals. In this study, we explored the in vitro and in vivo effects of Blastocystis on the activity of a commonly used CRC chemotherapeutic agent, 5-FU. The cellular and molecular effects of solubilized antigen of Blastocystis in the presence of 5-FU were investigated using HCT116, human CRC cell line and CCD 18-Co, normal human colon fibroblast cells. For the in vivo study, 30 male Wistar rats were divided into six groups, as follows; Control Group: oral administration of 0.3 ml Jones' medium, Group A: rats injected with azoxymethane (AOM), Group A-30FU: Rats injected with AOM and administered 30 mg/kg 5-FU, Group B-A-30FU: rats inoculated with Blastocystis cysts, injected with AOM and administered 30 mg/kg 5-FU, Group A-60FU: rats injected with AOM and administered 60 mg/kg 5-FU and Group B-A-60FU: rats inoculated with Blastocystis cysts, injected with AOM and administered 60 mg/kg 5-FU. The in vitro study revealed that the inhibitory potency of 5-FU at 8 μM and 10 μM was reduced from 57.7% to 31.6% (p < 0.001) and 69.0% to 36.7% (p < 0.001) respectively when co-incubated with Blastocystis antigen for 24 h. However, the inhibitory potency of 5-FU in CCD-18Co cells was not significantly affected in the presence of Blastocystis antigen. The reduced inhibitory potency of 5-FU against cancer cell proliferation due to the presence of Blastocystis is consistent with the upregulation of expression of type 2 cytokines, transforming growth factor (TGF-β) and nuclear factor E2-related factor 2 (Nrf2) gene expression. Increased inflammation and abnormal histopathological findings along with a significant cancer multiplicity and adenoma incidence were evident in the intestine of the B-A-30FU and B-A-60FU groups when compared with the A-30FU and A-60FU groups respectively. Our in vitro and in vivo findings indicate that Blastocystis infection could potentially interfere with chemotherapy regimens such as 5-FU in CRC patients undergoing chemotherapy.

SPATA2 and CYLD inhibit T cell infiltration into colorectal cancer via regulation of IFN-γ/STAT1 axis.

Colorectal cancer (CRC) is largely refractory to currently available immunotherapies such as blockade of programmed cell death protein-1 (PD-1). In this study, we identified SPATA2 and its protein partner CYLD as novel regulators of CXC-ligand 10 (CXCL10), a T-cell-attractant chemokine, in CRC. By specifically deleting SPATA2 and CYLD in human and mouse CRC cell lines, we showed that these two proteins inhibit STAT1 accumulation and activation and subsequently CXCL10 expression in tumor cells. At steady-state, STAT1 is highly ubiquitinated in a SPATA2/CYLD-dependent manner. Finally, we demonstrated that tumor-specific deletion of SPATA2 and CYLD enhances anti-PD-1 response in vivo. Our data suggest that SPATA2 and CYLD represent two potential novel targets for treatment of immune-excluded, PD-1-resistant tumors.

Lactate promotes metastasis of normoxic colorectal cancer stem cells through PGC-1α-mediated oxidative phosphorylation

Uneven oxygen supply in solid tumors leads to hypoxic and normoxic regions. Hypoxic cells exhibit increased secretion of lactate, which creates an acidic tumor microenvironment (TME). This acidic TME is positively associated with tumor metastasis. Despite the increased metastatic capacity of hypoxic cells, they are located relatively further away from the blood vessels and have limited access to the circulatory system. Studies have shown that cancer stem cells (CSCs) are enriched for tumor metastasis-initiating cells and generally undergo aerobic respiration, which could be enhanced by lactate. We therefore hypothesized that TME-derived lactate may promote the metastasis of normoxic CSCs. In the present study, the abundance of hypoxic and normoxic CSCs was analyzed in primary CRC tumors. It was found that the proportion of normoxic CSCs was positively associated with tumor stage. Using two human CRC cell lines, LoVo and SW480, and a patient-derived xenograft (XhCRC), it was found that treatment with lactate promoted normoxic CSC metastasis. Metabolism analysis indicated that, upon treatment with lactate, oxidative phosphorylation (OXPHOS) activity in normoxic CSCs was enhanced, whereas hypoxic CSCs were rarely altered. At the molecular level, the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of lactate oxidation, was found to be elevated in normoxic CSCs. Furthermore, PGC-1α knockdown markedly reduced the metastatic potential of normoxic CSCs. Notably, both the PGC-1α-mediated OXPHOS activity and metastatic potential were impaired when hypoxia-inducible factor-1α (HIF-1α) was activated in normoxic CSCs. Together, these findings provide a therapeutic strategy against tumor metastasis through the targeting of PGC-1α and, thus, the suppression of lactate-feeding OXPHOS in normoxic CSCs may improve the therapeutic benefit of patients with cancer, particularly CRC.

TACH101, a first-in-class pan-inhibitor of KDM4 for treatment of gastrointestinal cancers.

132 Background: TACH101 is a novel, potent small molecule inhibitor of KDM4, a novel epigenetic target for cancer therapy. KDM4 is a family of histone lysine demethylases that, when overexpressed, drives key processes linked to cancer. Validation of KDM4 as a driver gene was confirmed across gastrointestinal tumor types including esophageal, colon and gastric cancers, and is associated with formation of aggressive tumors. Methods: TACH101 was evaluated in in vitro and in vivo studies including cell inhibition assays, patient-derived xenograft (PDX) and organoid models, and bioinformatics analyses studies. Results: In vitro, TACH101 treatment potently inhibited cell proliferation in cell lines and organoid models representing esophageal, CRC, and gastric cancers. TACH101 induced apoptosis in human CRC (HT-29) and esophageal (KYSE-150) cancer cell lines (EC50s 0.033 - 0.092 µM). Further evaluation using a panel of &gt; 300 cell lines from different tumor types showed potent activity of TACH101 against gastric cancer and CRC. In gastric cancer, 2D cell viability inhibition assays conducted on a panel of 11 gastric cancer cell lines showed 9/11 (82%) were sensitive to TACH101 treatment (IC50 0.004 - 0.072 µM); in PDX models, 4/5 (80%) were sensitive to TACH101 treatment (IC50 0.007 - 0.039 µM). In CRC, bioinformatics analysis indicated increased TACH101 sensitivity in cell lines with MSI-H status (IC50 1-150 nM). Sensitivity of MSI-H CRC to TACH101 was further confirmed in a panel of 14 CRC PDX models and 7 CRC organoid models in culture-based viability inhibition assays. In PDX models, 5/5 (100%) characterized as MSI were sensitive to TACH101 treatment (IC50 0.001 - 0.014 µM), whereas 4/8 (50%) characterized as MSS were sensitive to TACH101 (IC50 0.003 - 0.270 µM). In patient derived CRC organoid models, 3/3 (100%) characterized as MSI were sensitive to TACH101 treatment (IC50 0.022 - 0.149 µM) whereas 0/3 (0%) characterized as MSS were sensitive (IC50 &gt; 10 µM). In vivo, TACH101 triggered effective tumor control (≥70%) in xenograft models of CRC (SU60), esophageal (KYSE-150) and gastric (GXA-3036) cancers. Pharmacologic studies showed TACH101 demonstrated favorable cell permeability, good oral bioavailability, and high metabolic stability. Conclusions: Preclinical work on TACH101 KDM4 inhibitor demonstrates compelling data and applicability as a potential therapy for gastrointestinal cancers. Preparations to advance TACH101 into clinical trials are underway.

Effects of dihydroartemisinin, a metabolite of artemisinin, on colon cancer chemoprevention and adaptive immune regulation.

Artemisinin (ART) is an anti-malaria natural compound with a moderate anticancer action. As a metabolite of ART, dihydroartemisinin (DHA) may have stronger anti-colorectal cancer (CRC) bioactivities. However, the effects of DHA and ART on CRC chemoprevention, including adaptive immune regulation, have not been systematically evaluated and compared. Coupled with a newly-established HPLC analytical method, enteric microbiome biotransformation was conducted to identify if the DHA is a gut microbial metabolite of ART. The anti-CRC potential of these compounds was compared using two different human CRC cell lines for cell cycle arrest, apoptotic induction, and anti-inflammation activities. Naive CD4+T cells were also obtained for testing the compounds on the differentiation of Treg, Th1 and Th17. Using compound extraction and analytical methods, we observed for the first time that ART completely converted into its metabolites by gut microbiome within 24h, but no DHA was detected. Although ART did not obviously influence cancer cell growth in the concentration tested, DHA very significantly inhibited the cancer cell growth at relatively low concentrations. DHA included G2/M cell cycle arrest via upregulation of cyclin A and apoptosis. Both ART and DHA downregulated the pro-inflammatory cytokine expression. The DHA significantly promoted Treg cell proliferation, while both ART and DHA inhibited Th1 and Th17 cell differentiation. As a metabolite of ART, DHA possessed stronger anti-CRC activities. The DHA significantly inhibited cell growth via cell cycle arrest, apoptosis induction and anti-inflammation actions. The adaptive immune regulation is a related mechanism of actions for the observed effects.

A Chemerin Peptide Analog Stimulates Tumor Growth in Two Xenograft Mouse Models of Human Colorectal Carcinoma.

Simple SummaryThe chemoattractant adipokine chemerin has been found to be elevated in several types of cancer, including colorectal carcinoma. The functional role of chemerin in colorectal carcinoma, however, has not been elucidated to date. This study analyses the impact of the chemerin analog CG34 on proliferation, colony formation, and migration in the human colorectal cancer cell lines HCT116, HT29 and SW620. In addition, the effect of systemic CG34 treatment is investigated in two xenograft mouse models of colorectal cancer (HCT116-luc and HT29-luc). The results of this study suggest there is a stimulatory role of chemerin receptor activation on the growth of colorectal carcinoma.Background: Chemerin plasma concentration has been reported to be positively correlated with the risk of colorectal cancer. However, the potential regulation of CRC tumorigenesis and progression has not yet been investigated in an experimental setting. This study addresses this hypothesis by investigating proliferation, colony formation, and migration of CRC cell lines in vitro as well as in animal models. Methods: In vitro, microscopic assays to study proliferation, as well as a scratch-wound assay for migration monitoring, were applied using the human CRC cell lines HCT116, HT29, and SW620 under the influence of the chemerin analog CG34. The animal study investigated HCT116-luc and HT29-luc subcutaneous tumor size and bioluminescence during treatment with CG34 versus control, followed by an ex-vivo analysis of vessel density and mitotic activity. Results: While the proliferation of the three CRC cell lines in monolayers was not clearly stimulated by CG34, the chemerin analog promoted colony formation in three-dimensional aggregates. An effect on cell migration was not observed. In the treatment study, CG34 significantly stimulated both growth and bioluminescence signals of HCT116-luc and HT29-luc xenografts. Conclusions: The results of this study represent the first indication of a tumor growth-stimulating effect of chemerin signaling in CRC.

Epigenetic Effects of Blackberry Extract on Human Colorectal Cancer Cells

Fruit-derived polyphenolic compounds have been shown to exert anticancer effects via epigenetic mechanisms. In this study, we investigated the effect of blackberry extract on the expression of DNMTs (Dnmt1, Dnmt3a, and Dnmt3b) and HDACs (HDAC1-4 and SIRT1) and its influence on the cellular differentiation and promoter DNA methylation of tumor-related genes using a panel of six human CRC cell lines. Treatment with IC20 and IC50 concentrations of blackberry extract for 72 h significantly reduced Dnmt1 and Dnmt3b transcript levels in HCT116, SW480, HT29/219, SW742, and LS180 cells in a dose-dependent manner. Blackberry also induced promoter DNA demethylation of SFRP2 and p16 genes in four tested CRC cell lines. Berry treatment, however, upregulated Dnmt3a genes in SW480, SW742, and HT29/219 cell lines. A dose-dependent and cell-type-specific reduction of HDAC1, HDAC2, and HDAC4 expressions were observed in CRC-treated cells. Treatment with berry extract induced the expression of SIRT1 gene in HCT116 and HT29/219 cells and increased the expression of two colonic epithelial cell differentiation markers, carcinoembryonic antigen (CEA) and alkaline phosphatase in LS180 cells in a time-dependent manner. This study is the first to report the epigenetic effects of blackberry in cancer cells.

Nuclear partitioning of Prohibitin 1 inhibits Wnt/β-catenin-dependent intestinal tumorigenesis.

The Wnt/β-catenin signaling pathway is aberrantly activated in the majority of colorectal cancer cases due to somatic mutations in the adenomatous polyposis coli (APC) gene. Prohibitin 1 (PHB1) serves pleiotropic cellular functions with dynamic subcellular trafficking facilitating signaling crosstalk between organelles. Nuclear-localized PHB1 is an important regulator of gene transcription. Using mice with inducible intestinal epithelial cell (IEC)-specific deletion of Phb1 (Phb1iΔIEC) and mice with IEC-specific overexpression of Phb1 (Phb1Tg), we demonstrate that IEC-specific PHB1 combats intestinal tumorigenesis in the ApcMin/+ mouse model by inhibiting Wnt/β-catenin signaling. Forced nuclear accumulation of PHB1 in human RKO or SW48 CRC cell lines increased AXIN1 expression and decreased cell viability. PHB1 deficiency in CRC cells decreased AXIN1 expression and increased β-catenin activation that was abolished by XAV939, a pharmacological AXIN stabilizer. These results define a role of PHB1 in inhibiting the Wnt/β-catenin pathway to influence the development of intestinal tumorigenesis. Induction of nuclear PHB1 trafficking provides a novel therapeutic option to influence AXIN1 expression and the β-catenin destruction complex in Wnt-driven intestinal tumorigenesis.

Cyclooxygenase activity mediates colorectal cancer cell resistance to the omega-3 polyunsaturated fatty acid eicosapentaenoic acid

PurposeThe naturally-occurring omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) is safe, well-tolerated and inexpensive, making it an attractive anti-cancer intervention. However, EPA has only modest anti-colorectal cancer (CRC) activity, when used alone. Both cyclooxygenase (COX) isoforms metabolise EPA and are over-expressed in CRC cells. We investigated whether COX inhibition increases the sensitivity of CRC cells to growth inhibition by EPA.MethodsA panel of 18 human and mouse CRC cell lines was used to characterize the differential sensitivity of CRC cells to the growth inhibitory effects of EPA. The effect of CRISPR-Cas9 genetic deletion and pharmacological inhibition of COX-1 and COX-2 on the anti-cancer activity of EPA was determined using in vitro and in vivo models.ResultsGenetic ablation of both COX isoforms increased sensitivity of CT26 mouse CRC cells to growth inhibition by EPA in vitro and in vivo. The non-selective COX inhibitor aspirin and the selective COX-2 inhibitor celecoxib increased sensitivity of several human and mouse CRC cell lines to EPA in vitro. However, in a MC38 mouse CRC cell tumour model, with dosing that mirrored low-dose aspirin use in humans, thereby producing significant platelet COX-1 inhibition, there was ineffective intra-tumoral COX-2 inhibition by aspirin and no effect on EPA sensitivity of MC38 cell tumours.ConclusionCyclooxygenase inhibition by non-steroidal anti-inflammatory drugs represents a therapeutic opportunity to augment the modest anti-CRC activity of EPA. However, intra-tumoral COX inhibition is likely to be critical for this drug-nutrient interaction and careful tissue pharmacodynamic profiling is required in subsequent pre-clinical and human studies.

Effect of the isoflavone corylin from cullen corylifolium on colorectal cancer growth, by targeting the STAT3 signaling pathway

BackgroundColorectal cancer (CRC) is one of the most common cancers worldwide. Corylin is an isoflavone extracted from Cullen corylifolium (L.) Medik., which is widely used anti-inflammatory and anticancer in Asian countries. Signal transducer and activator of transcription 3 (STAT3) plays an important role in the occurrence and development of CRC. PurposeTo analyze the antitumor activity of corylin in CRC and to elucidate its molecular mechanisms of action. MethodsThe human CRC cell lines HCT116, RKO, and SW480 and immunodeficient mice were used as models to study the antitumor effect of corylin. The potent anti-proliferative, anti-migration and proapoptotic effects of corylin were observed by cell viability, colony formation assays, wound-healing migration assay, and cell apoptosis assay. Immunostaining analysis and western blot analysis revealed inhibition of the STAT3 signaling axis. ResultsWe found that corylin could significantly reduce the viability and stimulate apoptosis in human CRC cells in a dose-dependent manner. Corylin decreased the expression levels of P-STAT3 and STAT3 target proteins, such as myeloid cell leukemia-1(MCL-1), Survivin, VEGF and B-cell lymphoma 2 (BCL-2). It also upregulated the expression levels of the proapoptotic proteins BCL-2-associated X protein (BAX) and Cl-caspase 3. Moreover, corylin reduced the nuclear localization of STAT3. Furthermore, corylin inhibited the growth of the tumor in CRC mouse models. ConclusionsOur findings provide convincing results that could support the role of corylin in the treatment of CRC through inhibiting the STAT3 pathway. It is conceivable that corylin should be further explored as a unique STAT3 inhibitor in antitumor therapy.

FUT9-Driven Programming of Colon Cancer Cells towards a Stem Cell-Like State

Simple SummaryAberrant glycosylation, for instance heightened expression of fucosylated structures, is a frequent feature observed in tumor cells. Our paper outlines the role of aberrant fucosylation by the Fucosyltransferase 9 (FUT9) as a potent reprogramming factor marking the acquisition of a stem-like state both by murine and human colon cancer cells. Importantly, our study reinforces the implication of aberrant fucosylation in promoting tumor growth and resistance to chemotherapy in the context of colon cancer.Cancer stem cells (CSCs) are located in dedicated niches, where they remain inert to chemotherapeutic drugs and drive metastasis. Although plasticity in the CSC pool is well appreciated, the molecular mechanisms implicated in the regulation of cancer stemness are still elusive. Here, we define a fucosylation-dependent reprogramming of colon cancer cells towards a stem cell-like phenotype and function. De novo transcriptional activation of Fut9 in the murine colon adenocarcinoma cell line, MC38, followed by RNA seq-based regulon analysis, revealed major gene regulatory networks related to stemness. Lewisx, Sox2, ALDH and CD44 expression, tumorsphere formation, resistance to 5-FU treatment and in vivo tumor growth were increased in FUT9-expressing MC38 cells compared to the control cells. Likewise, human CRC cell lines highly expressing FUT9 displayed phenotypic features of CSCs, which were significantly impaired upon FUT9 knock-out. Finally, in primary CRC FUT9+ tumor cells pathways related to cancer stemness were enriched, providing a clinically meaningful annotation of the complicity of FUT9 in stemness regulation and may open new avenues for therapeutic intervention.

AXL is a predictor of poor survival and of resistance to anti-EGFR therapy in RAS wild-type metastatic colorectal cancer

BackgroundRAS mutations are the only validated biomarkers in metastatic colorectal cancer (mCRC) for anti-epidermal growth factor receptor (EGFR) therapy. Limited clinical information is available on AXL expression, marker of epithelial to mesenchymal transition, in mCRC. MethodsAXL was retrospectively assessed by immunohistochemistry in 307 patients. RAS wild-type (WT) patients (N = 136) received first-line anti-EGFR–based therapy; RAS mutant patients (N = 171) received anti-angiogenic–based regimens.Preclinical experiments were performed using human RAS WT CRC cell lines and xenograft models. AXL RNA levels were assessed in a cohort of patients with available samples at baseline and at progression to anti-EGFR treatment and in the GSE5851 dataset. ResultsAXL was expressed in 55/307 tumour tissues, correlating with worse survival in the overall population (AXL-positive, 23.7 months; AXL-negative, 30.8 months; HR, 1.455, P = 0.032) and in RAS WT patients (AXL-positive, 23.0 months; AXL-negative, 35.8 months; HR,1.780, P = 0.032). Progression-free survival (PFS) in the RAS WT cohort was shorter in the AXL-positive cohort (6.2 months versus 12.1 months; HR, 1.796, P = 0.013). Three-dimensional cultures obtained from a patient following anti-EGFR therapy resulted AXL-positive, showing resistance to anti-EGFR drugs and sensitivity to AXL inhibition. AXL transfection in CRC cell lines induced AXL overexpression and resistance to the EGFR blockade. At progression to cetuximab, 2/10 SW48-tumour xenograft mice showed AXL expression. Consistently, AXL RNA levels increased in 5/7 patients following anti-EGFR therapy. Moreover, in the GSE5851 dataset higher AXL RNA levels correlated with worse PFS with cetuximab in KRAS-exon2 WT chemorefractory patients. ConclusionsAXL is a marker of poor prognosis in mCRC with consistent clinical and preclinical evidences of involvement in primary and acquired resistance to anti-EGFR drugs in RAS WT patients.

Novel benzoate-lipophilic cations selectively induce cell death in human colorectal cancer cell lines

IntroductionColorectal cancer (CRC) is a critical health issue worldwide. The high rate of liver and lung metastasis associated with CRC creates a significant barrier to effective and efficient therapy. Tumour cells, including CRC cells, have metabolic alterations, such as high levels of glycolytic activity, increased cell proliferation and invasiveness, and chemo- and radio-resistance. However, the abnormally elevated mitochondrial transmembrane potential of these cells also provides an opportunity to develop drugs that selectively target the mitochondrial functions of tumour cells. MethodsIn this work, we used a new batch of benzoic acid esters with cytotoxic activities attached to the triphenylphosphonium group as a vehicle to target tumour mitochondria and improve their activity. We evaluated the cytotoxicity, selectivity, and mechanism of action of these derivatives, including the effects on energy stress-induced apoptosis and metabolic behaviour in the human CRC cell lines HCT-15 and COLO-205. ResultsThe benzoic acid derivatives selectively targeted the tumour cells with high potency and efficacy. The derivatives induced the uncoupling of the oxidative phosphorylation system, decreased the transmembrane potential, and reduced ATP levels while increasing AMPK activation, thereby triggering tumour cell apoptosis in both tumour cell lines tested. ConclusionThe benzoic acid derivatives studied here are promising candidates for assessing in vivo models of CRC, despite the diverse metabolic characteristics of these tumour cells.