Treatment Of CRC Research Articles

Understanding the regulation of β-catenin expression and activity in colorectal cancer carcinogenesis: beyond destruction complex.

Aberrant Wnt/β-catenin signaling is central to colorectal cancer carcinogenesis. The well-known potential of targeting the canonical Wnt signaling pathway for the treatment of CRC is largely attributed to the ability of this pathway to regulate various cellular processes such as cell proliferation, metastasis, drug resistance, immune response, apoptosis, and cellular metabolism. However, with the current approach of targeting this pathway, none of the Wnt-targeted agents have been successfully implicated in clinical practice. Instead of using classical approaches to target this pathway, there is a growing need to find new and modified approaches to achieve the same. For this, a better understanding of the regulation of β-catenin, a major effector of the canonical Wnt pathway is a must. The present review addresses the importance of understanding the regulation of β-catenin beyond the destruction complex. Few recently discovered β-catenin regulators such as ZNF281, TTPAL, AGR2, ARHGAP25, TREM2, and TIPE1 showed significant potential in regulating the development of CRC through modulation of the Wnt/β-catenin signaling pathway in both in vitro and in vivo studies. Although the expression and activity of β-catenin is influenced by many protein regulators, the abovementioned proteins not only influence its expression and activation but are also directly involved in the development of CRC and various other solid tumors. Therefore, we hypothesise that focusing the current research on finding the detailed mechanism of action of these regulators may assist in providing with a better treatment approach or improve the current therapeutic regimens.

CircCDYL2, Overexpressed in Highly Migratory Colorectal Cancer Cells, Promotes Migration by Binding to Ezrin.

BackgroundColorectal cancer (CRC) is one of the most common malignancies with high mortality worldwide, particularly due to metastasis. However, there are no clinically available strategies for treating CRC metastasis. Exploring the mechanisms underlying CRC metastasis is the key to improve the treatment of CRC with metastasis.MethodsIn this study, we generated the highly migratory CRC cell subline H-RKO using a repeated transwell migration assay to identify circRNAs involved in CRC migration by high-throughput RNA sequencing. Upregulated circRNAs were validated by RT-qPCR to identify the most elevated circRNA. The expression of this circRNA (circCDYL2) was evaluated in 40 pairs of CRC tissues and four CRC cell lines by RT-qPCR. Transwell migration and wound healing assays were performed to verify the function of circCDYL2 in cell migration. The cellular distribution of circCDYL2 was confirmed using PCR. RNA pulldown and RNA immunoprecipitation were used to confirm the interaction between circCDYL2 and Ezrin. Western blotting, immunohistochemistry, and rescue experiments were used to determine the role of circCDYL2 in regulating Ezrin protein expression and AKT phosphorylation.ResultsAmong the candidate circRNAs, circCDYL2 was the highest overexpressed circRNA in H-RKO compared to parental N-RKO cells. Furthermore, circCDYL2 expression was elevated in CRC tissues and cell lines. Gain- and loss-of-function assays indicated that circCDYL2 enhanced the migration of CRC cells. circCDYL2 was located in the cytoplasm of CRC cells and interacted with Ezrin to upregulate its protein levels, resulting in AKT phosphorylation. Ezrin knockdown abrogated the CRC cell migration induced by circCDYL2 overexpression.ConclusionsOur study demonstrated for the first time that circCDYL2 promotes CRC migration by binding Ezrin and activating the AKT pathway. CircCDYL2 represents a potential therapeutic target for preventing CRC metastasis.

TMEM100 Modulates TGF-β Signaling Pathway to Inhibit Colorectal Cancer Progression.

Objectives This study investigated the functional mechanism of transmembrane protein 100 (TMEM100) as a tumor inhibitor gene in CRC cells and offered a reference for the treatment of CRC. Methods The mRNA expression data of CRC were acquired from the TCGA database to mine differentially expressed mRNAs. The role of TMEM100 in the progression of CRC cells was evaluated by MTT, colony formation, scratch healing, and Transwell assays. The influence of TMEM100 on the TGF-β signaling pathway was detected by western blot. Results TMEM100 was markedly lowly expressed in CRC. CRC cell growth was significantly suppressed by overexpressing TMEM100 but noticeably facilitated by silencing TMEM100. Overexpression of TMEM100 inhibited the activation of the TGF-β signaling pathway, thus inhibiting malignant progression of CRC. Conclusion TMEM100 is lowly expressed in CRC, which can suppress CRC cell growth by regulating the TGF-β signaling pathway.

The combined effect of dichloroacetate and 3-bromopyruvate on glucose metabolism in colorectal cancer cell line, HT-29; the mitochondrial pathway apoptosis

Background5-Fluorouracil (5-FU) is regarded as the first line treatment for colorectal cancer; however, its effectiveness is limited by drug resistance. The ultimate goal of cancer therapy is induction of cancer cell death to achieve an effective outcome with minimal side effects. The present work aimed to assess the anti-cancer activities of mitocans which can be considered as an effective anticancer drug due to high specificity in targeting cancer cells.MethodsMTT (3–4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay was performed to determine the effects of our mitocans on cell viability and cell death. Apoptosis and necrosis, caspase 3 activity, mitochondrial membrane potential and ROS production in HT29 cell lines were analyzed by ApopNexin™ FITC/PI Kit, Caspase- 3 Assay Kit, MitoTracker Green and DCFH-DA, respectively. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression level of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) genes in HT29 cell lines.ResultsTreatment with mitocans (3Br-P + DCA) inhibited the growth of HT29. Moreover, 3Br-P + DCA significantly induced apoptosis and necrosis, activation of caspase 3 activity, depolarize the mitochondrial membrane potential, and ROS production. At a molecular level, 3Br-P + DCA treatment remarkably down-regulated the expression of Bcl-2, while up-regulated the expression of Bax.ConclusionMitocans, in particular the combined drug, 3Br-P + DCA, could be regarded and more evaluated as a safe and effective compound for CRC treatment. Targeting hexokinase and pyruvate dehydrogenase kinase enzymes may be an option to overcome 5-FU -mediated chemo-resistant in colorectal cancer.

20 (S)-ginsenoside Rh2 inhibits colorectal cancer cell growth by suppressing the Axl signaling pathway in vitro and in vivo

BackgroundColorectal cancer (CRC) has a high morbidity and mortality worldwide. 20 (S)-ginsenoside Rh2 (G-Rh2) is a natural compound extracted from ginseng, which exhibits anticancer effects in many cancer types. In this study, we demonstrated the effect and underlying molecular mechanism of G-Rh2 in CRC cells in vitro and in vivo. MethodsCell proliferation, migration, invasion, apoptosis, cell cycle, and western blot assays were performed to evaluate the effect of G-Rh2 on CRC cells. In vitro pull-down assay was used to verify the interaction between G-Rh2 and Axl. Transfection and infection experiments were used to explore the function of Axl in CRC cells. CRC xenograft models were used to further investigate the effect of Axl knockdown and G-Rh2 on tumor growth in vivo. ResultsG-Rh2 significantly inhibited proliferation, migration, and invasion, and induced apoptosis and G0/G1 phase cell cycle arrest in CRC cell lines. G-Rh2 directly binds to Axl and inhibits the Axl signaling pathway in CRC cells. Knockdown of Axl suppressed the growth, migration and invasion ability of CRC cells in vitro and xenograft tumor growth in vivo, whereas overexpression of Axl promoted the growth, migration, and invasion ability of CRC cells. Moreover, G-Rh2 significantly suppressed CRC xenograft tumor growth by inhibiting Axl signaling with no obvious toxicity to nude mice. ConclusionOur results indicate that G-Rh2 exerts anticancer activity in vitro and in vivo by suppressing the Axl signaling pathway. G-Rh2 is a promising candidate for CRC prevention and treatment.

Abstract 977: Mcl-1 levels critically determines the sensitivities of human colon cancer cells to APG1244, a novel Bcl-2 and Bcl-XLinhibitor, that induces Bax- and caspase-8- dependent apoptosis

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide due to its poor prognosis. Most of the patients receive surgical resection when diagnosed as CRC, whereas the recurrence rate is very high. Therefore, additional treatment options, such as targeted therapy, are still urgently needed. The novel BH3 mimetic, APG1244 (a prodrug for APG1252), is a highly potent Bcl-2 and Bcl-XL dual inhibitor and being tested in clinical trials as a potential cancer therapeutic agent. The current study focuses on demonstrating the therapeutic efficacies of APG1244 against colorectal cancer and understanding the underlying mechanisms. A panel of CRC cell lines with different levels of Bcl-2, Bcl-XL and Mcl-1 possessed different sensitivities to APG1244. APG1244 effectively decreased the survival of CRC cell lines, particularly those expressing relative low levels of Mcl-1, with induction of apoptosis. High levels of Mcl-1 in these cell lines were significantly correlated to decreased sensitivity to APG1244. APG1244, when combined with a Mcl-1 inhibitor, synergistically decreased the survival and induced apoptosis of APG1244-insensitive cell lines with high levels of Mcl-1. This combination could further decreased the survival and enhanced apoptosis even in the cell lines with relatively low levels of Mcl-1, whereas enforced expression of ectopic Mcl-1 in these cells abrogated APG1244's effects on decreasing cell survival and inducing apoptosis, which can be reversed by Mcl-1 inhibition. In our study, it appears that APG1244 quickly activated caspase-8 and caspase-3 and induced PARP cleavage with induction of cytochrome C and Smac release from mitochondria. Deficiency of Bax, Smac, Bim or PUMA, but not FADD, in CRC cells protected (BAX-KO) or partially protected cells from APG1244-induced apoptosis, indicating that APG1244 induces Bax-dependent apoptosis. Furthermore, blockage of caspase-8 with a caspase-8 specific inhibitor abolished cleavage of caspase-3 and PARP including attenuation of cytochrome C and Smac release induced by APG1244, suggesting a caspase-8-dependent mechanism. The current study thus demonstrates the potential of APG1244 as a monotherapy in the treatment of CRC, particularly those with low Mcl-1 expression, or in combination with a Mcl-1 inhibitor for CRC treatment, warranting further evaluation in vivo and in the clinic. Citation Format: Weilong Yao, Shi-Yong Sun. Mcl-1 levels critically determines the sensitivities of human colon cancer cells to APG1244, a novel Bcl-2 and Bcl-XLinhibitor, that induces Bax- and caspase-8- dependent apoptosis [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 977.

Abstract 2926: CT26.WT-luc orthotopic syngeneic tumor model of colon cancer: development and immunotherapy response

Abstract Colorectal cancer (CRC) is a major contributor to the whole of cancer as a disease, representing 10% of all cases of cancer. Recent clinical studies and FDA approvals demonstrate strong possibilities for the future of immunotherapy in the treatment of CRC. Highly translational preclinical models of the disease are required to assess the effectiveness of novel immuno-oncology agents. The CT26.WT murine colon carcinoma model is a widely used syngeneic tumor model to evaluate the preclinical efficacy of immuno-oncology agents. Subcutaneous tumor models are limited in translatability compared to the orthotopic implant site including differential stromal interactions, immune cell infiltrate, and response to immunotherapy. We present the development of a robust murine orthotopic colon tumor model (CT26.WT-luc) assessed by bioluminescence imaging (BLI) and assessed for response to common checkpoint targets. All animal work was performed in an AAALAC accredited facility, in alignment with applicable animal welfare regulations and with predetermined humane euthanasia criteria on all studies. Subcutaneous CT26.WT-luc tumors were dissected into fragments and grafted onto the cecum of naïve Balb/c mice. Mice were subjected to BLI twice weekly for four weeks for longitudinal monitoring of tumor growth. The tumor engraftment rate was 95% based on BLI and necropsy, no spontaneous regressions were observed. The BLI derived tumor volume doubling time was 1.5 days and the median time on study was 21 days. Common clinical observations associated with disease progression were abdominal distension due to tumor growth and bodyweight gain. Necropsy revealed large primary masses attached to the cecum with large vascular involvement, and small nodules on the liver and abdominal wall. To assess the model response to immunotherapy, Balb/c mice bearing orthotopic CT26.WT-luc tumors were treated by intraperitoneal administration of 10mg/kg isotype control, anti-PD-L1, anti-CTLA-4 or anti-PD-1 twice weekly for two weeks (Bio X Cell). Administration of anti-PD-L1 resulted in one complete regression. Anti-CTLA-4 treatment resulted in a day 23 ΔT/ΔC of 0.1% and five partial regressions with no visibly detectable disease at necropsy. Administration of anti-PD-1 did not result in regressions or tumor free survivors. Administration of either anti-PD-L1 or anti-CTLA-4 provided an increase of life span over isotype control of 13 and 24 days, respectively. Terminal necropsy revealed no evidence of tumors or other lesions in 1 out of 8 animals administered anti-PD-L1, and 5 out of 8 animals administered anti-CTLA-4. This model represents a powerful tool with translational potential for assessing the efficacy of novel immune modulating agents with the possibility of combination therapy. Assessment of tumor response, cell and molecular differences between subcutaneous and orthotopic CT26.WT-luc tumors is ongoing. Citation Format: Patrick Allison, Derrik Germain, Meredith Baugher, Maryland Franklin. CT26.WT-luc orthotopic syngeneic tumor model of colon cancer: development and immunotherapy response [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 2926.

Abstract 2842: 6-gingerol attenuates metastasis in aged mice model of colorectal cancer through modulation of epithelial-mesenchymal transition mediators genes and e-cadherin upregulation

Abstract Colorectal cancer (CRC) is the third most common cancer in America, and the third cause of cancer related death. Metastasis is the major cause of death in patient with CRC. Many CRC are likely to spread from primary site to the other parts of the body before and after detection of malignant tumor. 6-gingerol, the bioactive component of fresh ginger have been reported to have myriad of biological activities including anti-inflammatory and antioxidant properties. We have previously reported the anti-CRC effect of 6-gingerol. However there is dearth of information on the anti-metastatic effect 6-gingerol in CRC. Thus, the effect of 6-gingerol on epithelial-mesenchymal transition mediators' genes and E-Cadherin repression in aged mice model of CRC was assessed in this study. Following institutional ethical approval. Aged male BALB/c mice (10 months) were divided into four groups of 10 mice each. Mice were fed with AIN-93 diet and allowed access to water ad libitum. Group 1 mice serve as control, group 2 mice received 6-gingerol 10mg/kg/day by oral gavage for 10 weeks. Group 3 mice received a single dose of azoxymethane (AOM) 10mg/kg (iP) and on the seventh day they received three cycles of dextran sulfate sodium (DSS) 2% (W/V) in drinking water ( each cycle is define as one week of DSS followed by two weeks of plain drinking water) Group 4 mice received 6-gingerol 10mg/kg/day by oral gavage in combination with single dose of azoxymethane 10mg/kg (iP) and on the seventh day they received three cycles of dextran sulfate sodium salt 2% (W/V) in drinking water. Mice were humanely sacrificed under anesthesia and colon tissues were processed for RT-PCR and multiplex immunohistochemistry analysis. Mice that received both 6-gingerol and AOM and DSS showed a significant decreased expression of Lymphocyte function-associated antigen-1, N-cadherin, SNAIL 1, ZEB 1, AP4 and HMGA1 with concomitant Upregulation miR-34, miR-15a, miR-218 and miR-302 expression as compared with the AOM and DSS only group. The result obtained from this study showed 6-gingerol inhibited metastasis through modulation of epithelial-mesenchymal transition mediators' genes and E-Cadherin upregulation, and as such 6-gingerol could be a promising anti-metastatic agent in CRC treatment. Citation Format: Babajide Oluwaseun Ajayi, Ayomiposi Adenigba, Anuoluwapo Adeshina. 6-gingerol attenuates metastasis in aged mice model of colorectal cancer through modulation of epithelial-mesenchymal transition mediators genes and e-cadherin upregulation [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 2842.

The Promising Effect of Peucedanum chenur Chloroformic Extract on Prevention of Human Colorectal Cancer Progression by Modulating miR-135b, miR-21, and APC Genes.

The therapeutic use of herbal medicines for the diseases, including cancer, is increasing due to their lower side effects. The present research evaluated the effect of Peucedanum chenur chloroformic extract (PCCE) on cell proliferation against HCT-116 human colorectal cancer cell line. The cytotoxic effect of PCCE was evaluated by MTT assay. The activity of the Wnt/B-catenin pathway was assayed through measuring the expression of miR-135b, miR-21, and APC genes by real-time PCR. The flow cytometry and scratch tests were used to study the cell cycle and cell migration, respectively. Also, the antioxidant activity of PCCE was measured by DPPH and iron-chelating tests. The results showed the downregulation of miR-135b and miR-21 and overexpression of the APC gene. Furthermore, PCCE decreased the free radicals, cell migration, and cell proliferation. The antioxidant activity of PCCE was confirmed by standard tests. Altogether, our findings suggest that purified compounds of PCCE could be developed as a potent chemo-preventive drug for the treatment of CRC.

Abstract 1071: Studying the ability of retinoids to inhibit growth of CRC cells based on their retinoid pathway genotype

Abstract Colorectal cancer (CRC) tumorigenesis arises through acquisition of mutations in a colonic stem cell (SC) that transforms it into a cancer stem cell (CSC). There are no curative treatments for advanced CRC because CSCs appear to be resistant to chemotherapy and/or radiation. Because all-trans retinoic acid (ATRA) is a highly effective and often curative treatment for acute promyelocytic leukemia patients, we conjecture that retinoids may be an efficacious treatment for CRC. Retinoic acid (RA) is a metabolite of vitamin A which is known to induce differentiation of SCs and inhibit cell proliferation. Our goal is to discover how to therapeutically target human CRC CSCs using the differentiation-inducing effects of retinoid agents. Our previous study on the RA metabolizing enzyme aldehyde dehydrogenase (ALDH) shows that ALDH marks normal and malignant colonic SCs and that RA signaling mainly occurs through colonic ALDH+ SCs. Hypothesis: The ability of retinoid agents, ATRA, 9-cis retinoic acid, 13-cis retinoic acid and Liarozole, to induce differentiation of CSCs depends on the RA pathway genotype according to mutations in RA signaling components in CRC cells. Indeed, our bioinformatics analysis shows that many RA pathway genes are overexpressed and often mutated in CRC. We have also evaluated the ability of the different RA ligands to inhibit cell growth and induce differentiation using a panel of CRC cell lines that have specific RA signaling mutations. Further investigation of RA signaling mechanisms that regulate colon SCs and how dysregulation contributes to the SC overpopulation that drives CRC growth should provide insight into strategies for designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the ability of retinoids to inhibit growth of CRC cells based on their retinoid pathway genotype [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 1071.

An Original Ferroptosis-Related Gene Signature Effectively Predicts the Prognosis and Clinical Status for Colorectal Cancer Patients.

BackgroundColorectal cancer (CRC) is one of the most common malignant tumors in the world. Ferroptosis is a newly defined form of cell death, distinguished by different morphology, biochemistry, and genetics, and involved in CRC progression and treatment. This study aims to establish a predictive model to elucidate the relationship between ferroptosis and prognosis of CRC patients, to explore the potential value of ferroptosis in therapeutic options.MethodsThe ferroptosis-related genes were obtained from the GeneCards and FerrDb websites. The limma R package was used to screen the differential ferroptosis-related genes (DEGs) in CRC from The Cancer Genome Atlas (TCGA) dataset. The least absolute shrinkage and selection operator (LASSO) and multivariate Cox regressions were to establish the 10-gene prognostic signature. The survival and receiver operating characteristic (ROC) curves were illustrated to evaluate the predictive effect of the signature. Besides, independent prognostic factors, downstream functional enrichment, drug sensitivity, somatic mutation status, and immune feature were analyzed. Moreover, all these conclusions were verified by using multiple datasets in International Cancer Genome Consortium (ICGC) and Gene Expression Omnibus (GEO).ResultsTen ferroptosis-related gene signature (TFAP2C, SLC39A8, NOS2, HAMP, GDF15, FDFT1, CDKN2A, ALOX12, AKR1C1, ATP6V1G2) was established to predict the prognosis of CRC patients by Lasso cox analysis, demonstrating a good performance on Receiver operating characteristic (ROC) and Kaplan–Meier (K–M) analyses. The CRC patients in the high- or low-risk group showed significantly different fractions of immune cells, such as macrophage cells and CD8+ T cells. Drug sensitivity and somatic mutation status like TP53 were also closely associated with the risk scores.ConclusionsIn this study, we identified a novel ferroptosis-related 10-gene signature, which could effectively predict the prognosis and survival time of CRC patients, and provide meaningful clinical implications for targeted therapy or immunotherapy. Targeting ferroptosis is a good therapeutic option for CRC patients. Further studies are needed to reveal the underlying mechanisms of ferroptosis in CRC.

An analysis of the rates of discontinuation and non-publication of colorectal cancer clinical trials.

Non-publication and premature discontinuation for clinical trials pose an ethical dilemma for trial participants, patients, clinicians, and researchers, as well as the general public as these studies receive significant public funding that may be further contributing to research waste. Here, we investigate the rate of trial discontinuation and non-publication among CRC trials using ClinicalTrials.gov. We performed an advanced search on ClinicalTrials.gov pertaining to the treatment of CRC using the keyword colorectal cancer. For each clinical trial, links to the publication provided by ClinicalTrials.gov were searched and verified to be correct. If a publication was unable to be found using the methods above, we attempted to contact the lead investigator via email for the reason for non-publication. Of the 123 (123/428, 28.7%) discontinued trials, a reason for discontinuation was provided for 57 (57/123, 46.3%) trials. Of the 305 (305/428, 71.3%) completed trials, 244 (244/305, 80.0%) had a verifiable publication, while 61 (61/305, 20.0%) did not publish their findings or were unable to be located. We found that more than one-quarter of trials were prematurely ended, and almost one-third of completed trials did not publish their findings. Subjecting trial participants to potentially harmful treatments and interventions that fail to complete or publish study findings have the potential to undermine the patient-provider relationship, as well as public confidence in government-sponsored clinical trials.

PCN5 EARLY-Onset Colorectal Cancer in the United States: A Narrative Review of the Evidence

The incidence and mortality of early-onset (<50 years of age) colorectal cancer (EOCRC) in comparison to colorectal cancer in patients greater than 50 years of age is steadily rising. Due to a delay in screening, younger populations are susceptible to later staged diagnoses, more aggressive treatment strategies, adverse histologic features, and increased mortality. This study summarizes the evidence on early-onset CRC screening, diagnosis, treatment, and survival in the United States MEDLINE, the Cochrane Library, and Google Scholar were searched for English language publications describing studies on early-onset CRC. A study was eligible for inclusion if it reported information on CRC or EOCRC incidence, screening, survival, risk factors, pathophysiology, clinical characteristics, genetic features, diagnostic criteria, treatment strategy or evaluation, racial, geographic, and socioeconomic disparities, and was published in English between January 2000 and October 2020. Patients diagnosed with EOCRC tend to have a distinct histological type including poorly differentiated tumors and signet cells, making diagnosis and treatment more difficult. Genetic features such as hereditary syndromes can contribute to EOCRC, but do not account for the majority of cases. Over-treatment patterns of young adults with EOCRC were observed. EOCRC patients that were over-treated were not shown to have greater survival rates. Ethnic minorities and low socioeconomic classes, especially African-Americans (AA), had greater mortality rates and under-treatment patterns. EOCRC incidence and mortality rates are rising rapidly, and many young adults are not being treated appropriately. Young patients have a greater likelihood of being over-treated and/or misdiagnosed, leading to long-term toxicity effects and decreased quality of life. Special attention needs to be addressed to young AA adults, to counter the observed trend, as they have the highest incidence of CRC among young population groups by race/ethnicity.

Vesicle transporter GOLT1B mediates the cell membrane localization of DVL2 and PD-L2 and promotes colorectal cancer metastasis

BackgroundColorectal cancer (CRC) is the third most diagnosed and second leading cause of cancer death worldwide. Hallmark proteins processing is usually dysregulated in cancers. Finding key regulatory molecules is of great importance for CRC metastasis intervention. GOLT1B is a vesicle transport protein which is involved in cytosolic proteins trafficking. However, its role in cancer has never been addressed.MethodsCRC cell lines and subcutaneous xenograft animal model were utilized to investigate the biological function of GOLT1B. Patients samples were used to validate the correlation between GOLT1B and clinical outcome. In vivo targeted delivery of GOLT1B-siRNA was investigated in PDX (Patient derived tumor xenograft) model.ResultsWe found that GOLT1B was highly expressed in CRC, and was an independent prognostic marker of overall survival (OS) and progression free survival (PFS). GOLT1B could promote CRC metastasis in vitro and in vivo. GOLT1B overexpression could increase DVL2 level and enhance its plasma membrane translocation, which subsequently activated downstream Wnt/β-catenin pathway and increase the nuclear β-catenin level, hence induce epithelial-mesenchymal transition (EMT). In addition, GOLT1B could also interact with PD-L2 and increase its membrane level. Co-culture of GOLT1B-overexpresed CRC cells with Jurkat cells significantly induced T cells apoptosis, which might further promote cancer cell the migration and invasion. Further, targeted delivery of GOLT1B siRNA could significantly inhibit tumor progression in GOLT1B highly expressed PDX model.ConclusionTaken together, our findings suggest that the vesicle transporter GOLT1B could promote CRC metastasis not only by assisting DVL2 translocation and activating Wnt/β-catenin pathway, but also facilitating PD-L2 membrane localization to induce immune suppression. Targeted inhibition of GOLT1B could be a potential therapeutic strategy for CRC treatment.

Combination of Anti-EGFR and Anti-VEGF Drugs for the Treatment of Previously Treated Metastatic Colorectal Cancer: A Case Report and Literature Review.

The standard third-line treatment of metastatic colorectal cancer (mCRC) includes the small-molecule anti-vascular drugs (Regofenib and Fruquintinib) and the chemotherapy drug trifluridine and tipiracil hydrochloride (TAS-102). There is no standard treatment for mCRC if the third-line treatment failed. Therefore, it is a pressing need to develop new therapeutic approaches to improve the survival of patients who developed drug resistance to the third-line treatment. In this study, we report a case of mCRC with RAS/BRAF wild-type, who was successfully treated using cetuximab in combination with fruquintinib after resistance to chemotherapy, bevacizumab, cetuximab and regorafenib. This patient responded to this combination regimen. Then, we discuss the mechanisms of action of this combination. Furthermore, we introduce the clinical trials on the combination regimens of anti-EGFR with anti-vascular monoclonal antibodies. Finally, we discuss the clinical explorations of using combination of anti-EGFR with small-molecule anti-VEGF drugs and their potential benefits. The clinical effects of small-molecule anti-vascular drugs in combination with anti-EGFR in the treatment of CRC warrant further explored.

Long noncoding RNA TUG1 regulates the progression of colorectal cancer through miR-542-3p/TRIB2 axis and Wnt/\u03b2-catenin pathway

BackgroundColorectal cancer (CRC) is one of the third normal malignancy worldwide. Taurine-upregulated gene 1 (TUG1), a member of long noncoding RNAs (lncRNAs), has been reported to be involved in various cancers. However, the mechanism underlying TUG1 in the progression of CRC remains unclear.MethodsThe expression of TUG1, microRNA-542-3p (miR-542-3p), and tribbles homolog 2 (TRIB2) in CRC tissues and cells (LoVo and HCT116) were detected by quantitative real-time PCR (qRT-PCR). Methyl thiazolyl tetrazolium (MTT), transwell and flow cytometry assays were employed to evaluate the effects of TUG1 in CRC cells. The interaction between miR-542-3p and TUG1 or TRIB2 were verified by dual-luciferase reporter assay. A xenograft tumor model in nude mice was established to investigate the biological role of TUG1 in CRC in vivo.ResultsTUG1 was increased in CRC tissues and cells (LoVo and HCT116) in contrast with adjacent normal tissues and normal intestinal mucous cells (CCC-HIE-2). Downregulation of TUG1 or TRIB2 suppressed the proliferation, migration, invasion, and induced apoptosis in CRC cells. And knockdown of TUG1 repressed tumor growth in vivo. Besides, overexpression of TRIB2 reversed the effects of TUG1 depletion on the progression of CRC. Meanwhile, TUG1 interacted with miR-542-3p and TRIB2 was a target of miR-542-3p. Furthermore, miR-542-3p knockdown or TRIB2 overexpression partly reversed the suppression effect of TUG1 depletion on the Wnt/β-catenin pathway.ConclusionsTUG1 served as a tumor promoter, impeded the progression of CRC by miR-542-3p/TRIB2 axis to inactivate of Wnt/β-catenin pathway, which providing a novel target for CRC treatment.

Variation in the use of genomic testing in patients with metastatic colorectal cancer.

e15557 Background: Oncology has greatly benefited from the use of clinical genomics to aid diagnostic and treatment decisions. To inform targeted therapy, standard practice dictates molecular testing of metastatic colorectal cancer (mCRC) tumors through genomic testing (GT), including molecular biomarker, single gene, or next-generation sequencing (NGS). While persisting disparities in CRC screening, treatment, and outcomes have been well described, little is known about differences in GT for mCRC. Using all-payer electronic health record-derived de-identified data from a real-world database generated from routine clinical care across the U.S., we identified combinations of demographic and clinical characteristics, rather than individual factors, associated with GT. Methods: This study used the nationwide Flatiron Health electronic health record (EHR)-derived de-identified database. During the study period, the de-identified data originated from approximately 280 US cancer clinics (̃800 sites of care). Our study population included 26,524 patients with mCRC during the years 2013-2020. We evaluated documentation of receipt of GT (individual biomarker or NGS) measured within one year of diagnosis of metastatic disease, by demographic (age, sex, and race/ethnicity), payer type, de novo metastatic cancer (vs. progression to metastatic disease), tumor site (colon vs. rectum), Eastern Cooperative Oncology Group (ECOG) performance status, and year of diagnosis. We identified Elixhauser comorbid conditions from ICD codes, and grouped them by count (0-4, 5+). We conducted Classification and Regression Tree (CART) analysis, a machine learning approach to identify combinations of demographic and clinical characteristics associated with receipt of GT. Results: Nearly 83% had documented GT, 90% of whom did so within 6 months of diagnosis. The largest group of patients with GT at 90% consisted of individuals with colon cancer, 64 years of age or younger, 0-4 comorbidities, and an ECOG score of 0 or 1 (n = 2,004). Conversely, the lowest rates of GT ( &lt; 60%) were observed among women 75 years of age or older, despite having 0-4 comorbidities (n = 1,314). In this group of patients, GT was higher among those with ECOG score of 1 than among those with high ECOG scores (66% and 54%, respectively). On the other hand, 71% of patients 75 years of age or older with 5+ comorbidities and high ECOG scores received GT. Conclusions: Considerable variations exist in GT across subgroups of the population. Additional analysis is warranted to characterize young and healthy patients who did not receive GT, and those who did get tested despite their older age and compromised health status. Future analyses will investigate whether documentation of receipt of GT is associated with treatment decisions and outcomes.

Cryopreservation of Whole Tumor Biopsies from Rectal Cancer Patients Enable Phenotypic and In Vitro Functional Evaluation of Tumor-Infiltrating T Cells.

Simple SummaryColorectal cancer (CRC) remains the third most common malignancy. Tumor-infiltrating lymphocytes (TILs) have emerged as correlates to CRC patient outcome after treatment. The pro- or anti-tumor responses of TILs are usually assessed in cell suspensions of fresh tumors that were surgically removed a few hours earlier. We propose a platform for concurrent enumeration and in vitro functional evaluation of TILs in cryopreserved tumor biopsies, offering the benefit of postponing tumor processing and analyses of TILs in cell suspensions until clinical post-treatment responses are established. Our platform is practical considering the inconsistent time when patient samples become available for research purposes and can be readily utilized by other laboratories. With a fresh portion of tumor biopsies as benchmark, we validated the recovery of viable TILs capable of interferon (IFN)-γ responses in the cryopreserved portion of same biopsies. Ultimately, this platform could provide sufficient information on TILs, to also predict patient outcome after CRC treatments.TILs comprise functionally distinct conventional and unconventional T cell subsets and their role in responses to CRC treatments is poorly understood. We explored recovery of viable TILs from cryopreserved tumor biopsies of (chemo)-radiated patients with rectal cancer to establish a platform for retrospective TIL analyses of frozen tumors from pre-selected study cohorts. Frequencies of TIL subsets and their capacity to mount IFN-γ responses in cell suspensions of fresh vs. cryopreserved portions of the same tumor biopsies were determined for platform validation. The percentages and proportions of CD4+ TILs and CD8+ cytotoxic T lymphocytes (CTLs) among total TILs were not affected by cryopreservation. While recovery of unconventional γδ T cells and mucosal-associated invariant T cells (MAIT cells) was stable after cryopreservation, the regulatory T cells (Tregs) were reduced, but in sufficient yields for quantification. IFN-γ production by in vitro-stimulated CD4+ TILs, CTLs, γδ T cells, and MAIT cells were proportionally similar in fresh and cryopreserved tumor portions, albeit the latter displayed lower levels. Thus, the proposed platform intended for TIL analyses on cryopreserved tumor biobank biopsies holds promises for studies linking the quantity and quality of TIL subsets with specific clinical outcome after CRC treatment.

Reovirus enhances cytotoxicity of natural killer cells against colorectal cancer via TLR3 pathway

BackgroundCetuximab has been approved for use for first-line treatment of patients with wild-type KRAS metastatic colorectal cancer (CRC). However, treatment with cetuximab has shown limited efficacy as a CRC monotherapy. In addition, natural killer (NK) cell function is known to be severely attenuated in cancer patients. The goal of this study was to develop a new strategy to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells, in combination with cetuximab against CRC cells.MethodsEx vivo expanded NK cells were stimulated with reovirus, and reovirus-activated NK cells mediated ADCC assay were performed on CRC cells in combination with cetuximab. The synergistic antitumor effects of reovirus-activated NK cells and cetuximab were tested on DLD-1 tumor-bearing mice. Finally, Toll-like receptor 3 (TLR3) knockdown in NK cells, along with chemical blockade of TLR3/dsRNA complex, and inhibition of the TLR3 downstream signaling pathway, were performed to explore the mechanisms by which reovirus enhances NK cell cytotoxicity.ResultsWe first confirmed that exposure of NK cells to reovirus enhanced their cytotoxicity in a dose-dependent manner.We then investigated whether reovirus-activated NK cells exposed to cetuximab-bound CRC cells exhibited greater anti-tumor efficacy than either monotherapy. Co-culture of CRC cell lines with reovirus-activated NK cells indicated that NK cytotoxicity was significantly higher in combination with cetuximab, regardless of KRAS mutation status or EGFR expression level. We also found that reovirus activation of NK cells, in conjunction with cetuximab, resulted in significantly stronger anti-tumor efficacy.Finally, TLR3 knockdown, inhibition of TLR3/dsRNA complex or TBK1/IKKε demonstrated that activation of NK cells by reovirus was dependent on TLR3 and its downstream signaling pathway.ConclusionsThis study demonstrated that combination treatment of reovirus-activated NK cells with cetuximab synergistically enhances their anti-tumor cytotoxicity, suggesting a strong candidate strategy for clinical treatment of CRC.

Recombinant Bacteroides fragilis enterotoxin-1 (rBFT-1) promotes proliferation of colorectal cancer via CCL3-related molecular pathways.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers worldwide and stands among the leading causes of cancer-related deaths. Although deregulation of the microbiota in the gastrointestinal tract has been frequently described in CRC, very little is known about the precise molecular mechanisms by which bacteria and their toxins modulate the process of tumorigenesis and behavior of cancer cells. In this study, we produced recombinant Bacteroides fragilis enterotoxin-1 (rBFT1) and demonstrate that rBFT1 could promote cell proliferation in colorectal cancer cells and accelerate tumor growth in vivo. To identify the mechanisms, we further investigated CCL3/CCR5 and NF-κB pathway. We found that CCL3, CCR5, NF-κB, and TRAF-6 were dramatically upregulated after rBFT1 treatment, thus suggesting that the role of rBFT1 in CRC progression may be associated with CCL3/CCR5 and NF-κB pathways. Collectively, our results indicate that rBFT1 serves as a tumor promoter and plays a crucial role in inducing the proliferation of CRC via accelerating CCL3-related molecular pathway, thus giving insights into mechanistic underpinnings for the prevention and treatment of CRC.