Colorectal Tumor Cells Research Articles

Novel 1,3,5-Triazinyl Aminobenzenesulfonamides Incorporating Aminoalcohol, Aminochalcone and Aminostilbene Structural Motifs as Potent Anti-VRE Agents, and Carbonic Anhydrases I, II, VII, IX, and XII Inhibitors.

A series of 1,3,5-triazinyl aminobenzenesulfonamides substituted by aminoalcohol, aminostilbene, and aminochalcone structural motifs was synthesized as potential human carbonic anhydrase (hCA) inhibitors. The compounds were evaluated on their inhibition of tumor-associated hCA IX and hCA XII, hCA VII isoenzyme present in the brain, and physiologically important hCA I and hCA II. While the test compounds had only a negligible effect on physiologically important isoenzymes, many of the studied compounds significantly affected the hCA IX isoenzyme. Several compounds showed activity against hCA XII; (E)-4-{2-[(4-[(2,3-dihydroxypropyl)amino]-6-[(4-styrylphenyl)amino]-1,3,5-triazin-2-yl)amino]ethyl}benzenesulfonamide (31) and (E)-4-{2-[(4-[(4-hydroxyphenyl)amino]-6-[(4-styrylphenyl)amino]-1,3,5-triazin-2-yl)amino]ethyl}benzenesulfonamide (32) were the most effective inhibitors with KIs = 4.4 and 5.9 nM, respectively. In addition, the compounds were tested against vancomycin-resistant Enterococcus faecalis (VRE) isolates. (E)-4-[2-({4-[(4-cinnamoylphenyl)amino]-6-[(4-hydroxyphenyl)amino]-1,3,5-triazin-2-yl}amino)ethyl]benzenesulfonamide (21) (MIC = 26.33 µM) and derivative 32 (MIC range 13.80–55.20 µM) demonstrated the highest activity against all tested strains. The most active compounds were evaluated for their cytotoxicity against the Human Colorectal Tumor Cell Line (HCT116 p53 +/+). Only 4,4’-[(6-chloro-1,3,5-triazin-2,4-diyl)bis(iminomethylene)]dibenzenesulfonamide (7) and compound 32 demonstrated an IC50 of ca. 6.5 μM; otherwise, the other selected derivatives did not show toxicity at concentrations up to 50 µM. The molecular modeling and docking of active compounds into various hCA isoenzymes, including bacterial carbonic anhydrase, specifically α-CA present in VRE, was performed to try to outline a possible mechanism of selective anti-VRE activity.

SPERT gene silencing inhibits the growth of human colon cancer xenograft tumor in nude mice via p38MAPK/HSP27 signaling pathway.

Colorectal cancer is one of the most common gastrointestinal malignancies and is also a disease of genetic heterogeneity. Our previous studies have shown that SPERT (sprermatid-associated protein) gene may be an underlying oncogene that is associated with the progression of the disease in colorectal cancer patients, and SPERT gene silencing can inhibit the proliferation of colorectal tumor cells and promote cell apoptosis. Here, we use the stably transfected human colorectal cancer cell line RKO to construct an animal xenograft model and study the effect of SPERT gene silencing on animal xenografts. The results showed that SPERT gene silencing can inhibit tumor growth in animals. In addition, through signaling pathway analysis, we found that the p38MAPK/HSP27 signaling pathway may be the molecular mechanism by which SPERT gene silencing inhibits the growth of xenograft tumors in nude mice. Combined with previous data, SPERT gene silencing has the same inhibitory effect on tumor growth in vitro and in vivo. These data suggest that SPERT gene may be a potential target for the treatment of colorectal cancer in clinic.

Colorectal Cancer Progression Is Potently Reduced by a Glucose-Free, High-Protein Diet: Comparison to Anti-EGFR Therapy.

Simple SummaryTo study the interplay between nutrition and intestinal metabolism in the context of colitis-driven colorectal carcinoma (CRC), we here investigated a nutritional therapy strategy in the presence or absence of EGFR-directed antibody therapy in mice to treat established colitis-driven CRCs in vivo. After CRC development, mice were fed a control diet or an isoenergetic glucose-free high-protein (GFHP) diet in the presence or absence of EGFR-directed antibody therapy. The GFHP diet was accompanied by a metabolic shift of the mice towards lower glycolysis activity. Both, GFHP diet or anti-EGFR antibody treatment, improved tumor differentiation and anti-tumor immune response, resulting in an efficient reduction of colonic tumor burden.To enable rapid proliferation, colorectal tumor cells up-regulate epidermal growth factor receptor (EGFR) signaling and aerobic glycolysis, resulting in substantial lactate release into the tumor microenvironment and impaired anti-tumor immune responses. We hypothesized that a nutritional intervention designed to reduce aerobic glycolysis may boost the EGFR-directed antibody (Ab)-based therapy of pre-existing colitis-driven colorectal carcinoma (CRC). CRC development was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) administration to C57BL/6 mice. AOM/DSS-treated mice were fed a glucose-free, high-protein diet (GFHPD) or an isoenergetic control diet (CD) in the presence or absence of an i.p. injection of an anti-EGFR mIgG2a or respective controls. AOM/DSS-treated mice on a GFHPD displayed a reduced systemic glucose metabolism associated with reduced oxidative phosphorylation (OXPHOS) complex IV expression and diminished tumor loads. Comparable but not additive to an anti-EGFR-Ab therapy, the GFHPD was accompanied by enhanced tumoral goblet cell differentiation and decreased colonic PD-L1 and splenic CD3ε, as well as PD-1 immune checkpoint expression. In vitro, glucose-free, high-amino acid culture conditions reduced proliferation but improved goblet cell differentiation of murine and human CRC cell lines MC-38 and HT29-MTX in combination with down-regulation of PD-L1 expression. We here found GFHPD to systemically dampen glycolysis activity, thereby reducing CRC progression with a similar efficacy to EGFR-directed antibody therapy.

Musarin, a novel protein with tyrosine kinase inhibitory activity from Trametes versicolor, inhibits colorectal cancer stem cell growth

Colorectal cancer is the second deadly cancer in the world. Trametes versicolor is a traditional Chinese medicinal mushroom with a long history of being used to regulate immunity and prevent cancer. Trametes versicolor mushroom extract demonstrates strongly cell growth inhibitory activity on human colorectal tumor cells. In this study, we characterized a novel 12-kDa protein that named musarin, which was purified from Trametes versicolor mushroom extract and showed significant growth inhibition on multiple human colorectal cancer cell lines in vitro. The protein sequence of musarin was determined through enzyme digestion and MS/MS analysis. Furthermore, Musarin, in particular, strongly inhibits aggressive human colorectal cancer stem cell-like CD24+CD44+ HT29 proliferation in vitro and in a NOD/SCID murine xenograft model. Through whole transcription profile and gene enrichment analysis of musarin-treated CSCs-like cells, major signaling pathways and network modulated by musarin have been enriched, including the bioprocess of the Epithelial-Mesenchymal Transition, the EGFR-Ras signaling pathway and enzyme inhibitor activity. Musarin demonstrated tyrosine kinase inhibitory activity in vitro. Musarin strongly attenuated EGFR expression and down-regulated phosphorylation level, thereby slowing cancer cells proliferation. In addition, oral ingestion of musarin significantly inhibited CD24+CD44+ HT29 generated tumor development in SCID/NOD mice with less side effects in microgram doses. Targeting self-renewal aggressive stem-cell like cancer cell proliferation, with higher water solubility and lower cytotoxicity, musarin has shown strong potence to be developed as a promising novel therapeutic drug candidate against colorectal cancers, especially those that acquire chemo-resistance.

Multifunctional Magnesium Anastomosis Staples for Wound Closure and Inhibition of Tumor Recurrence and Metastasis.

Biodegradable magnesium (Mg) implants spontaneously releasing therapeutic agents against tumors are an intriguing therapeutic approach for both tissue repair and tumor treatment. Anastomotic staples are extensively used for wound closure after surgical resection in patients with colorectal tumors. However, the safety of Mg anastomosis implants for intestinal closure and the effect of tumor suppression remain elusive. Here, we used a high-purity Mg staple to study these issues. Based on the results, we found that it has the potential to heal wounds produced after colorectal tumor resection while inhibiting relapse of residual tumor cells in vitro and in vivo. After implantation of Mg staples for 7 weeks in rabbits, the intestinal wound gradually healed with no adverse effects such as leakage or inflammation. Furthermore, the implanted Mg staples inhibit the growth of colorectal tumor cells and block migration to normal organs because of the increased concentration of Mg ions and released hydrogen. Such an antitumor effect is further confirmed by the in vitro cell experiments. Mg significantly induces apoptosis of tumor cells as well as inhibits cell growth and migration. Our work presents a feasible therapeutic opinion to design Mg anastomotic staples to perform wound healing and simultaneously release tumor suppressor elements in vivo to decrease the risk of tumor recurrence and metastasis.

FBXW11 contributes to stem-cell-like features and liver metastasis through regulating HIC1-mediated SIRT1 transcription in colorectal cancer

Colorectal tumorigenesis is a heterogeneous disease driven by multiple genetic and epigenetic alterations. F-box and WD repeat domain containing 11 (FBXW11) is a member of the F-box protein family that regulates the ubiquitination of key factors associated with tumor growth and aggressiveness. Our study aimed to explore the role of FBXW11 in the development and metastasis of colorectal cancer (CRC). FBXW11 was overexpressed in colorectal tumor tissues and its overexpression was associated with a poor prognosis of CRC patients. The upregulation of FBXW11 not only promoted cell proliferation, invasion, and migration, but also contributed to maintaining stem-cell features in colorectal tumor cells. Further analysis revealed that FBXW11 targeted hypermethylated in cancer 1 (HIC1) and reduced its stability in CRC cells through ubiquitination. Moreover, the expression of sirtuin 1 (SIRT1), a deacetylase in tumor cells was upregulated by FBXW11 via regulating HIC1 expression. The mouse xenograft models of CRC confirmed that FBXW11 knockdown impeded colorectal tumor growth and liver metastasis in vivo. In summary, our study identified FBXW11 as an oncogenic factor that contributed to stem-cell-like properties and liver metastasis in CRC via regulating HIC1-mediated SIRT1 expression. These results provide a rationale for the development of FBXW11-targeting drugs for CRC patients.

Di-2-pyridylketone 4, 4-dimethyl-3-thiosemicarbazone effectively induces human colorectal carcinoma cell apoptosis via mTOR pathway

Background: To investigate the anticancer mechanisms of di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) in human colon cancer cells. Human colorectal carcinoma (HCC) is one of the most commonly diagnosed cancers in both males and females. Current studies have found that iron chelators can be used as novel anticancer drugs; however, the anticancer activity of iron chelators and their target genes in HCC has been rarely reported. Methods: Dp44mT was used to treat two colorectal tumor cell lines, SW480 and HT-29. The proapoptotic effects of different concentrations of Dp44mt were measured using flow cytometry and Hoechst 33258 staining. Ferric ammonium citrate (FAC) was used as an additional iron donor to inhibit the effects of Dp44mT. Apoptosis and DNA damage-related proteins were examined by Western blot analysis. Results: In this study, we found that the iron chelators Dp44mT could induce the apoptosis in two colorectal tumor cell lines SW480 and HT-29, upregulate the expression level of p-histone H2A.X, and inhibit the phosphorylation level of mTOR in a dose-dependent way. Those effects could be reversed by the additional iron donor FAC. Conclusion: These data indicate that iron depletion and/or the presence of iron can modulate the HCC apoptosis progression in vitro, which may be a potential target for future HCC therapy.

β-Caryophyllene Induces Apoptosis and Inhibits Angiogenesis in Colorectal Cancer Models.

Beta-Caryophyllene (BCP), a naturally occurring sesquiterpene abundantly found in cloves, hops, and cannabis, is the active candidate of a relatively new group of vascular-inhibiting compounds that aim to block existing tumor blood vessels. Previously, we have reported the anti-cancer properties of BCP by utilizing a series of in-vitro anti-tumor-related assays using human colorectal carcinoma cells. The present study aimed to investigate the effects of BCP on in-vitro, ex-vivo, and in-vivo models of anti-angiogenic assays and evaluate its anti-cancer activity in xenograft tumor (both ectopic and orthotopic) mice models of human colorectal cancer. Computational structural analysis and an apoptosis antibody array were also performed to understand the molecular players underlying this effect. BCP exhibited strong anti-angiogenic activity by blocking the migration of endothelial cells, tube-like network formation, suppression of vascular endothelial growth factor (VEGF) secretion from human umbilical vein endothelial cells and sprouting of rat aorta microvessels. BCP has a probable binding at Site#0 on the surface of VEGFR2. Moreover, BCP significantly deformed the vascularization architecture compared to the negative control in a chick embryo chorioallantoic membrane assay. BCP showed a remarkable reduction in tumor size and fluorescence molecular tomography signal intensity in all the mice treated with BCP, in a dose-dependent relationship, in ectopic and orthotopic tumor xenograft models, respectively. The histological analysis of the tumor from BCP-treated mice revealed a clear reduction of the density of vascularization. In addition, BCP induced apoptosis through downregulation of HSP60, HTRA, survivin, and XIAP, along with the upregulation of p21 expressions. These results suggest that BCP acts at multiple stages of angiogenesis and could be used as a promising therapeutic candidate to halt the growth of colorectal tumor cells.

A high-throughput customized cytokinome screen of colon cancer cell responses to small-molecule oncology drugs.

Inflammatory cytokines, chemokines, and growth factors are molecular messengers that circulate and have the capability to modify the tumor microenvironment and impact therapeutic response. The characterization of soluble mediators as biomarkers for diagnosis and prognosis is of interest in oncology. We utilize the cytokinome to characterize the response of colorectal tumor cell lines to selected small-molecules in oncology as a proof-of-concept dataset with immunomodulatory analyte heat map rankings for drug and cell line combinations. We observed overall trends in drug class effects with MEK-, BRAF-, PARP-inhibitors, and Imipridones in cytokine, chemokine, and growth factor responses that may help guide therapy selection. MEK-inhibitor treatment downregulated analytes VEGF, CXCL9/MIG, and IL-8/CXCL8 and upregulated CXCL14/BRAK, Prolactin, and CCL5/RANTES. BRAF-inhibitor treatment downregulated VEGF and IL-8/CXCL8, while increasing soluble TRAIL-R2. Treatment with PARP-inhibitors decreased CXCL9/MIG, IL-8/CXCL8, CCL3/MIP-1 alpha, VEGF, and CXCL14/BRAK, while treatment increased soluble TRAIL-R2 and prolactin. Treatment with Imipridones decreased CCL3/MIP-1 alpha, VEGF, CXCL14/BRAK, IL-8/CXCL8, and Prolactin and increased CXCL5/ENA-78. We also observed differential responses to therapeutics depending on the mutational profile of the cell line. In the future, a similar but larger dataset may be utilized in the clinic to aid in the prediction of patient response to immunomodulatory therapies based on tumor genotype.

EGFR targeting of [177Lu] gold nanoparticles to colorectal and breast tumour cells: Affinity, duration of binding and growth inhibition of Cetuximab-resistant cells

ObjectiveRadioimmunotherapy (RIT) is a systemic therapy currently used in the treatment of patients with lymphoma. RIT complexes consist of a targeting molecule, commonly an antibody, radionuclide chelates and a linker which can be a nanoparticle platform. Nanoparticles facilitate the attachment of multiple radionuclides and targeting groups to a single complex. Here the target affinity, duration of target association and inhibition of colony formation of Cetuximab-resistant tumour cells with Cetuximab-targeted [177Lu]-AuNPs were investigated. Dose distribution in xenografts derived from EGFR-overexpressing cells was also determined. MethodsCetuximab-targeted [177Lu]-AuNPs were generated by functionalising 15nm AuNPs with the chelator DOTA and Cetuximab and radiolabelling with 177LuCl3. KDis, a measure of affinity, was determined by competitive binding to EGFR expressing cells. Radio-sensitivity was determined in EGFR expressing tumour cells including the Cetuximab resistant cell line HCT116 using a colony formation assay. Dose distribution was measured in sections from xenografts grown in nude mice using autoradiography. ResultsKDis for the complex binding to EGFR on MDA-MB-468 cells was 20 nM. Loss of cell associated [177Lu] activity was biphasic with loss of about 50% of activity in about 4 h. Remaining activity dissociated over a period of about 4 days. HCT8 and MDA-MB-468, but not HCT116 cells were sensitive to the growth inhibitory effect of Cetuximab. However, treatment with Cetuximab-targeted [177Lu]-AuNPs inhibited colony formation in all 3 cell lines. Dose distribution across sections from xenografts was found to demonstrate a co-efficient of variation of 15%. ConclusionCetuximab-targeted [177Lu]-AuNPs demonstrate high affinity for EGFR and could be an effective treatment for Cetuximab-resistant colorectal cancer cells. A strategy involving pre-treatment with receptor targeted[177Lu] to improve RIT therapeutic ratios has the potential to enhance clinical outcomes.

MiR-582-5p inhibits migration and chemo-resistant capabilities of colorectal cancer cells by targeting TNKS2.

Metastasis and chemo-resistance are still important factors that limit the overall efficacy of colorectal cancer treatment. Understanding the detailed molecular mechanism and identifying potential biomarkers are of great value in prognosis prediction and risk stratification. We investigated the role of miR-582-5p in colorectal cancer pathogenesis, progression and chemo-resistance. Furthermore, we explored the underlying molecular mechanism of miR-582-5p in modulation of malignant behaviors of colorectal cancer cells. Clinical samples and colorectal cancer cell lines were applied to explore miR-582-5p expression level and its significance on tumor cell metastasis and chemo-resistance. Transwell study and cellular survivability study were performed to explore the influences of miR-582-5p expression modulation on tumor cell chemo-resistance and invasion/migration. Dual-luciferase reporter gene assay was conducted to explore the influences of miR-582-5p on its target gene TNKS2. Colorectal cancer patients with lymph node or distal organ metastatic diseases exhibited significantly lower level of miR-582-5p. In vitro studies have indicated that miR-582-5p inhibition significantly increased migration and chemo-resistant capabilities of tumor cells. And dual-luciferase reporter gene assay demonstrated that miR-582-5p exhibited its influences on the biological behavior of tumor cells by targeting TNKS2. Our study demonstrated for the first time that miR-582-5p played an important role for colorectal tumor cell metastasis and chemo-resistance. Our research also indicated that miR-582-5p and its target gene TNKS2 could be novel biomarkers for metastatic disease prediction, overall prognosis evaluation, as well as potential therapeutic target for colorectal cancer patients.

Supramolecular Chemotherapy: Noncovalent Bond Synergy of Cucurbit[7]uril against Human Colorectal Tumor Cells.

Supramolecular chemotherapy has drawn increasing interest due to its ability to improve the efficiency of antitumor drugs and fewer associated toxic side effects. In this study, the smart supramolecular cargo, the doxorubicin-ZnO-cucurbit[7]uril (CDZ) nanocomplex, was constructed through ion-dipole interactions between cucurbit[7]uril {CB[7]} and doxorubicin-ZnO (dox-ZnO). The binding affinity of CB[7] and dox-ZnO was determined to be 104 M-1 by isothermal titration calorimetry. Importantly, spermine had a stronger binding affinity (106 M-1) with CB[7] than dox-ZnO through host-guest interactions. In the tumor microenvironment, spermine disassembled the CDZ nanocomplex, and dox was released from the nanocomplex by XRD, UV-visible spectra, and contact angle analysis. Compared to the single drug dox, the CDZ nanocomplex was demonstrated to possess higher activity of killing colorectal tumor cells by confocal laser scanning microscopy and cytotoxicity, which could be attributed to spermine concentration, spermine synthase, free radical damage, and G1 cell cycle arrest. Overall, the supramolecular delivery of dox can enhance the inhibition of human colorectal tumor cell proliferation and reduce cytotoxicity in human myocardial cells through the noncovalent bond synergy of {CB[7]}.

Relationship between miR-204 and ANGPTL2 expression and diagnosis, pathological stage, and prognosis in patients with colon cancer.

BackgroundAngiopoietin-like protein 2 (ANGPTL2) is linked to various tumors. MicroRNA-204 (miR-204) is associated with colorectal cancer (CRC). Bioinformatic analysis has demonstrated a targeting relationship between miR-204 and ANGPTL2. The present study aimed to investigate the role of miR-204 in the proliferation and apoptosis of colorectal tumor cells.MethodsColorectal tumor tissues were collected. Normal colon mucosa was used as a control. The relationship between miR-204 and ANGPTL2 expression and tumor stage and prognosis was analyzed. The dual-luciferase reporter assay confirmed targeted regulation between miR-204 and ANGPTL2. SW480 cells were allocated to the miR-NC group and the miR-204 mimic group, followed by apoptotic analysis using flow cytometry and cellular proliferation analysis using EdU staining.ResultsCompared with normal colonic mucosa, miR-204 expression was decreased in colorectal tumor tissues and ANGPTL2 expression was increased, which correlated with TNM staging. The prognosis of patients with low miR-204 expression and high ANGPTL2 expression was worse than for patients with high miR-204 expression and low ANGPTL2 expression. The dual-luciferase reporter assay confirmed a targeting regulation relationship between miR-204 and ANGPTL2. Transfection of miR-204 mimic significantly inhibited the expression of ANGPTL2 and cell proliferation in SW480 cells and promoted apoptosis.ConclusionsDownregulating miR-204 expression plays a vital role in upregulating ANGPTL2 expression and promoting the pathogenesis of CRC. MiR-204 is able to hinder the proliferation of colorectal tumor cells and encourage apoptosis by targeting the inhibition of ANGPTL2 expression.

ZNF326 promotes colorectal cancer epithelial–mesenchymal transition

Zinc-finger protein 326 (ZNF326) activity has been reported in different tumors, but its expression and possible mechanism of action in colorectal cancer are not known. In this study, we applied immunohistochemistry to detect the expression of ZNF326 in colorectal tissues. Next, we used a ZNF326 expression plasmid and small interfering (si) RNA-ZNF326 (siZNF326) to transfect colorectal cancer cell lines in order to determine the effect of ZNF326 on cell migration and as well as its potential role in promoting epithelial–mesenchymal transition (EMT). A higher ZNF326 expression in the nuclei of colorectal tumor cells compared to normal mucosa was observed (70.3%, 109/155 specimens vs. 23.2%, 36/155 specimens). A high ZNF326 expression level was positively correlated with tumor differentiation, tumor-node-metastasis (TNM) staging, and lymph node metastasis. Transfection of cancer cell lines (SW480 and SW620) with a ZNF326-overexpression vector promoted colorectal cancer cell invasion and altered the expression of EMT-related proteins. Vimentin, N-cadherin, Snail, and Slug were upregulated, whereas E-cadherin and zonula occludens-1 (ZO-1) were downregulated. In contrast, downregulation of ZNF326 expression using siRNA-ZNF326 in cancer cell lines (CL187 and RKO) resulted in the opposite findings. ZNF326 overexpression also upregulated the expression of latent transforming growth factor beta binding protein 4 (LTBP4) and p-Smad2/3. In conclusion, ZNF326 promoted the EMT and invasiveness of colorectal cancer cells. These findings are likely due to LTBP4 and p-Smad2/3 upregulation and, in turn, transforming growth factor beta (TGF-β) signaling activation.

Antitumor Activity of Fucus vesiculosus-Derived Phlorotannins through Activation of Apoptotic Signals in Gastric and Colorectal Tumor Cell Lines.

Seaweeds are one of the largest producers of biomass in the marine environment and a source of multiple bioactive metabolites with valuable health benefits. Among these, phlorotannins have been widely recognized for their promising bioactive properties. The potential antitumor capacity of Fucus vesiculosus-derived phlorotannins remains, however, poorly explored, especially in gastrointestinal tract-related tumors. Therefore, this work aimed to evaluate the cytotoxic properties and possible mechanisms by which F. vesiculosus crude extract (CRD), phlorotannin-rich extract (EtOAc), and further phlorotannin-purified fractions (F1–F9) trigger cell death on different tumor cell lines of the gastrointestinal tract, using flow cytometry. The results indicate that F. vesiculosus samples exert specific cytotoxicity against tumor cell lines without affecting the viability of normal cells. Moreover, it was found that, among the nine different phlorotannin fractions tested, F5 was the most active against both Caco-2 colorectal and MKN-28 gastric cancer cells, inducing death via activation of both apoptosis and necrosis. The UHPLC-MS analysis of this fraction revealed, among others, the presence of a compound tentatively identified as eckstolonol and another as fucofurodiphlorethol, which could be mainly responsible for the promising cytotoxic effects observed in this sample. Overall, the results herein reported contribute to a better understanding of the mechanisms behind the antitumor properties of F. vesiculosus phlorotannin-rich extracts.

ORAI3 contributes to hypoxia-inducible factor 1/2α-sensitive colon cell migration.

Hypoxia is a pivotal initiator of tumor angiogenesis and growth through the stabilization of hypoxia-inducible factors (HIFs). This study set out to examine the involvement of HIF-1α and HIF-2α in colon cancer and ascertained whether ORAI3 was involved in the pathway. Patients and murine models as well as human colorectal adenocarcinoma tumor (CW2) cells were included to examine the levels of ORAI1/3 and HIF-1/2α levels. Calcium imaging was utilized to ascertain the activity of calcium channel. Scratch assay was used to assess the migration capacity of the cells. Tumors from murine colon cancer xenograft models and patients with colon cancer displayed high ORAI1/3 and HIF-1/2α levels. Hypoxia treatment, mimicking the tumor microenvironment in vitro, increased ORAI1/3 and HIF-1/2α expression as well as store-operated Ca2+ entry (SOCE). Of note is that HIF-1/2α silencing decreased SOCE, and HIF-1/2α overexpression facilitated SOCE. Furthermore, ORAI3 rather than ORAI1 expression was inhibited by HIF-1/2α silencing while increased by ML228. Luciferase assay also confirmed that ORAI3 was elevated in the presence of ML228, indicating the linkage between HIF-1/2α and ORAI3. Additionally, colony-forming potential and cell migration capacity were decreased in siHIF-1α and siHIF-2α as well as siORAI3 cells, and the facilitating effect of ML228 on cell migration and colony-forming potential was also decreased in siORAI3 CW-2 cells, which points out the importance of ORAI3 in HIF1/2α pathway. Our findings allow to conclude that both HIF-1α and HIF-2α facilitate ORAI3 expression, hence enhancing colon cancer progression.

Substrate Rigidity Dictates Colorectal Tumorigenic Cell Stemness and Metastasis via KIAA1211 Dependent Mechanotransduction

Recurrence and metastasis rank among the most prominent clinical problems in colorectal cancer (CRC) treatment. Colorectal tumorigenic cell was identified to explore the process of CRC stemness maintenance and progression by simulating tumor mechanical properties. Here, we show that F-actin regulator KIAA1211 negatively correlates with CRC progression, stemness, and metastasis. Mechanistically, decreased KIAA1211 in soft substrates induces YAP retention in the cytoplasm, restores the repression effect on stemness markers NANOG and OCT4, thereby promotes CRC stemness and metastasis. Furthermore, KIAA1211 deficiency promotes colorectal tumor cell softening and regulates EMT states, contributing to its metastasis potential. Clinically, KIAA1211 expression is correlated with malignant degrees and metastasis in CRC patients. To sum up, our work uncovers an essential role of actin regulator KIAA1211 in anti-cancer and mechanical signal transduction of extracellular matrix in CRC.

Abstract 2478: Elevating SOX2 inhibits the proliferation of tumor cells independently of DNA binding

Abstract Purpose: The purpose of these studies was to understand how elevated SOX2 affects tumor cell proliferation. The stem cell transcription factor SOX2 has been implicated in ~20 different tumor types. Importantly, several studies have demonstrated that the effects of SOX2 during development and malignancy are dependent on its dosage. High levels of SOX2 in both contexts are associated with a quiescent, stem-like phenotype. SOX2high quiescent tumor cells present a significant clinical challenge as these cells are largely resistant to conventional chemotherapies and, thus, provide a reservoir to drive disease recurrence. Therefore, understanding the mechanisms through which elevated SOX2 inhibits tumor cell proliferation will provide insights into the biology of drug-resistant, quiescent tumor cells. Methods: Tumor cell lines representative of medulloblastoma (MB) and colorectal (CRC) tumor cells were engineered for the doxycycline (Dox)-inducible expression of mutant forms of SOX2 that are unable to bind to DNA. One SOX2 mutant was created by substituting a proline for a glycine in the DNA binding domain of SOX2 [SOX2(G76P)]. A second SOX2 mutant replaces the DNA binding domain of SOX2 with the DNA binding domain of the yeast transcription factor Gal4 (Gal4-SOX2). The effects of elevating mutant forms of SOX2 on cell proliferation and the cell cycle distribution of the cells were compared to the effects of elevating wild-type SOX2. Results: Our studies demonstrate that elevating SOX2, SOX2(G76P) or Gal4-SOX2 decreases the proliferation of MB and CRC cells to similar extents. In contrast, inducible expression of the DNA binding domain of Gal4 had no effect on cell proliferation. We previously reported that while SOX2 elevation inhibits the proliferation of a diverse set of tumor cell lines, it does not significantly alter their cell cycle distribution. Similarly, elevating SOX2(G76P) or Gal4-SOX2 did not significantly alter cell cycle distribution. To extend these findings, we are engineering additional SOX2 mutants to map the location of the domain of SOX2 required for growth inhibition. We recently engineered MB and CRC cells to express a mutant form of SOX2 that lacks the c-terminal R1 transactivation domain [Gal4-SOX2(ΔR1)]. Significantly, removing the R1 transactivation domain eliminated the growth inhibitory effects of Gal4-SOX2 when elevated. Conclusion: Our findings demonstrate that elevating two mutant forms of SOX2 that cannot bind to DNA inhibit the proliferation of tumor cell lines that represent two different forms of cancer, similar to the effects of wild-type SOX2. Equally important, our studies indicate that the c-terminal R1 transactivation domain of SOX2 is necessary, but no sufficient, to inhibit proliferation when SOX2 levels are elevated. Collectively, these findings and our previously published work indicate that elevated levels of SOX2 inhibit cell proliferation independently of DNA binding. Citation Format: Ethan Patrick Metz, Phillip J. Wilder, Angie Rizzino. Elevating SOX2 inhibits the proliferation of tumor cells independently of DNA binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2478.

Abstract 998: Mechanism of action of RXC004, a Wnt pathway inhibitor, in genetically-defined models of cancer

Abstract Background: RXC004 is a porcupine inhibitor, and thus a potent and selective inhibitor of Wnt ligand-dependent signalling. RXC004 is currently being investigated in a safety and tolerability study in cancer patients with solid tumours (NCT03447470). Certain Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, result in higher levels of the Wnt receptor Fzd on the cell surface increasing Wnt-ligand dependent signalling. These alterations are implicated in colorectal and pancreatic cancer. We present pre-clinical data on the effects of RXC004 in genetically defined models of cancer. Methods: RXC004 in vitro effects on the metabolic activity of both sensitive (genetically selected RNF43 mutant or RSPO fusion models) and insensitive (beta-catenin mutant model) colorectal and pancreatic tumour cells lines were assessed. To test if these effects translated in vivo, RXC004 was also evaluated in an in vivo FDG-PET study in an RSPO fusion tumour cell line. In addition, as we had previously noted that RXC004 caused cell differentiation in an RSPO fusion model, we studied if pre-treatment with RXC004 would affect subsequent tumour growth post RXC004 induced cell differentiation in vivo. Results: In tumour cell lines in vitro, RXC004 significantly reduced glucose uptake only in genetically selected tumour cell lines. In vivo FDG-PET studies also showed a reduction in FDG-PET signals post 7 days treatment of 5mg/kg QD RXC004. Imaging demonstrated that MaxSUV (maximum standardised uptake value) decreased by 31% in RXC004 treated versus vehicle treated tumours, furthermore biodistribution of PET ligand was significantly inhibited in tumours post 7 days RXC004 treatment. Pre-treatment of RXC004 in an in vivo mouse xenograft model for 7 days resulted in a significant reduction in tumour growth when tumour fragments from these pre-treated mice were re-implanted into naïve mice (P<0.05). Conclusion: These data demonstrate that RXC004 monotherapy results in tumour cell differentiation accompanied by reduced metabolic activity in genetically selected tumours. RXC004-induced cell differentiation may therefore continue to provide patient benefit even post dosing. Moreover, FDG-PET imaging in clinical studies may allow early detection of the positive effects of RXC004 in a non-invasive manner, and prior to any changes in tumour volume by conventional RECIST measurements. Citation Format: Caroline Phillips, Simon Woodcock, Catherine Eagle, Andrew Saunders, Craig Tilston, Inder Bhamra, Richard Armer. Mechanism of action of RXC004, a Wnt pathway inhibitor, in genetically-defined models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 998.

Abstract 2676: GSK3β inhibition by small molecule 9-ING-41 decreases VEGF and other cytokines, and boosts NK and T cell-mediated killing of colorectal tumor cells

Abstract The GSK3β/beta-catenin axis activates vascular endothelial growth factor (VEGF) signaling in endothelial cells to promote angiogenesis. VEGF has been noted as immunosuppressive, it dampens the immune cell response by promoting recruitment of tumor associated macrophages (TAMs), and has been proposed as a target to modulate antitumor immunity. A panel of human colorectal cell lines was chosen to provide a varied mutational background (TP53, KRAS, BRAF, TRK, APC, PIK3CA). Cells were treated with 9-ING-41, a selective and potent small molecule inhibitor of GSK3β, at doses up to 50 µM for 72 hours to determine IC10, IC30, IC50, and IC70. Cytokine, chemokine, and growth factor levels were analyzed in the tumor cell culture supernatants after treatment at IC10, IC30, IC50, and IC70 for 48 hours using a Luminex 200 multiplexing instrument. Co-culture experiments were conducted with GFP+ SW480 colorectal cancer cells and either NK-92 natural killer cells or TALL-104 T cells at various effector/target ratios in a 48-well plate, in the presence or absence of 9-ING-41. Overall cytokine levels showed a decreasing trend in response to increasing doses of 9-ING-41. Among the most prominently decreased growth factors in the profile was VEGF. In an immune cell-killing co-culture assay we observed a significant increase in natural killer (NK-92) cell and T cell (TALL-104) killing of the colorectal cancer cells in response to treatment with 9-ING-41 as compared to controls without drug treatment. Follow-up experiments compared the effect of pre-treating either the effector or the target cell population with 9-ING-41 before the co-culture experiment was started. Pre-treatment of the target tumor cells, but not the effector immune cells, bolstered cell-killing, implying that the drug is sensitizing the tumor cells to killing by the immune cells. Moreover, we saw an increase in the expression of chemokine CXCL14 (BRAK) in the tumor cell culture supernatant with increasing doses of 9-ING-41. BRAK is known to stimulate activated NK cell migration and could have a beneficial therapeutic effect by increasing NK cell migration into the tumor microenvironment. Furthermore, we saw a decrease in macrophage colony-stimulating factor (M-CSF) which, along with VEGF, has been associated with recruitment of TAMs. We hypothesize that a 9-ING-41-mediated decrease of VEGF in conjunction with a 9-ING-41-mediated increase of BRAK secreted by the tumor cells may increase the capacity of NK- and T cell-mediated killing of the tumor cells. Utilizing a compound such as 9-ING-41 could be a way to increase the host's anti-tumor immune response to decrease tumor burden in conjunction with other therapeutic agents. Citation Format: Kelsey E. Huntington, Shengliang Zhang, Benedito A. Carneiro, Wafik S. El-Deiry. GSK3β inhibition by small molecule 9-ING-41 decreases VEGF and other cytokines, and boosts NK and T cell-mediated killing of colorectal tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2676.