KCTD10 promoting PD-L1 expression in colorectal cancer enhanced the anti-tumor effect of PD-1 antibody.

Platostoma palustre jelly is a traditional food. Platostoma palustre has been used as folk medicine and is effective against heat-shock, hypertension and diabetes. Therefore, the aim of this study was to determine the effects of ethanolic extracts and commercial herbal tea of Platostoma palustre in inhibiting colorectal cancer cell viability. The ethanolic extracts of Platostoma palustre by using 90% ethanol for extraction. In this study, 2-fold serial dilution of 100 mg/mL Platostoma palustre extracts were applied. On other hand, the same dilution fold was also performed for 100% commercial herbal tea with Platostoma palustre. Additionally, CT-26 and HT-29 colorectal cancer cell lines were also used in this study. After co-culturing for 24 hours, the cell viability of CT-26 and HT-29 colorectal cancer cell lines were performed by using 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. According to these data, the 1.56-100 mg/mL Platostoma palustre extracts possessed the significant inhibition effects of CT-26 colorectal cancer cell viability. The 3.13-100% commercial herbal tea with Platostoma palustre possessed the significant inhibition effects of CT-26 colorectal cancer cell viability. The 6.25-100 mg/mL Platostoma palustre extracts possessed the significant inhibition effects of HT-29 colorectal cancer cell viability. The 25-100% commercial herbal tea with Platostoma palustre possessed the significant inhibition effects of HT-29 colorectal cancer cell viability. However, the 0.39-3.13 mg/mL Platostoma palustre extracts possessed the significant promoting effects of HT-29 colorectal cancer cell viability. The 0.39-12.5% commercial herbal tea with Platostoma palustre also possessed the significant promoting effects of HT-29 colorectal cancer cell viability. Comparison of CT-26 and HT-29 cell lines was on the cell viability after Platostoma palustre ethanolic extracts and commercial herbal tea treatments, CT-26 cell line was better sensitive than HT-29 cell line on the inhibition of cell viability after treatment of Platostoma palustre ethanolic extracts and the commercial herbal tea. Taken these results together, Platostoma palustre ethanolic extracts and commercial herbal tea may have a potential for inhibiting the growth of colorectal cancer cells.

Colorectal cancer is still unmanageable despite advances in target therapy. However, extracellular vesicles (EVs) have shown potential in nanomedicine as drug delivery systems, especially for modulating the immune cells in the tumor microenvironment (TME). In this study, M1 Macrophage EVs (M1EVs) were used as nanocarriers of oxaliplatin (M1EV1) associated with retinoic acid (M1EV2) and Libidibia ferrea (M1EV3), alone or in combination (M1EV4) to evaluate their antiproliferative and immunomodulatory potential on CT-26 and MC-38 colorectal cancer cell lines and prevent metastasis in mice of allograft and peritoneal colorectal cancer models. Tumors were evaluated by qRT-PCR and immunohistochemistry. The cell death profile and epithelial-mesenchymal transition process (EMT) were analyzed in vitro in colorectal cancer cell lines. Polarization of murine macrophages (RAW264.7 cells) was also carried out. M1EV2 and M1EV3 used alone or particularly M1EV4 downregulated the tumor progression by TME immunomodulation, leading to a decrease in primary tumor size and metastasis in the peritoneum, liver, and lungs. STAT3, NF-kB, and AKT were the major genes downregulated by of M1EV systems. Tumor-associated macrophages (TAMs) shifted from an M2 phenotype (CD163) to an M1 phenotype (CD68) reducing levels of IL-10, TGF-β and CCL22. Furthermore, malignant cells showed overexpression of FADD, APAF-1, caspase-3, and E-cadherin, and decreased expression of MDR1, survivin, vimentin, and PD-L1 after treatment with systems of M1EVs. The study shows that EVs from M1 antitumor macrophages can transport drugs and enhance their immunomodulatory and antitumor activity by modulating pathways associated with cell proliferation, migration, survival, and drug resistance.

Lauric acid (LAA) is a 12-carbon medium-chain fatty acid that reportedly has antitumor and muscle-protecting effects. However, the details of these antitumor effects remain unclear. Therefore, in this study, we investigated the mechanism underlying the antitumor effects of LAA in CT26 and HT29 colorectal cancer (CRC) cell lines. Our in vitro findings demonstrated that LAA suppressed CRC cell proliferation, induced mitochondrial oxidative stress (reactive oxygen species (ROS)), inhibited oxidative phosphorylation (OXPHOS), and induced apoptosis. Moreover, in vivo analysis of LAA showed a more pronounced antitumor effect in CT26 cells in a syngeneic mouse tumor model than in vitro; therefore, we further investigated its impact on host antitumor immunity. We observed that LAA increased the number of effector T cells in mouse tumors, while in vitro LAA activated mouse splenocytes (SplC) and promoted OXPHOS. In two-dimensional co-culture of SplC and CT26 cells, LAA induced cell death in cancer cells. In three-dimensional co-culture, LAA promoted SplC infiltration and suppressed the formation of tumor spheres. Thus, LAA may exert antitumor effects through increased ROS production in cancer cells and effector T cell activation via increased energy metabolism. These results suggest that LAA, when used in combination with existing anti-cancer drugs, is likely to exhibit sensitizing effects in terms of both antitumor and antitumor immune effects, and future clinical studies are anticipated.

This study aimed to evaluate the combination effect of gefitinib (GEF) and regorafenib (REG) against HCT116, CT26 and SW948 colorectal cancer cell lines. Results showed synergistic effects on HCT116 and CT26 cells, while the additive effect was observed on SW948 cells. Combination of GEF and REG induced sub-G1 peak as the apoptotic population on HCT116 cells, through flow cytometry histogram. Downregulation of AKT1 and TGFB2 and upregulation of CASP3 were observed in the combination of GEF and REG in HCT116 cells, using quantitative real-time PCR analysis. HSA binding properties exhibit that the first drug increased binding affinity between the second drug and HSA; as a result, HSA could transport both drugs. Thus, we hope this study creates a promising strategy to treat colorectal cancer.

Loss of the Ras Regulator RASAL1: Another Route to Ras Activation in Colorectal Cancer

Background: Ferula assa-foetida L has several therapeutic effects such as anticancer traits. The aim of this study was investigation of anticancer effect of Ferula assa-foetida gum alcoholic extract against CT-26, HT-29, SW742 and WiDr colorectal cancer cell lines. Methods: The hot extract was obtained by soxhlet using 70% ethanol solvent. Various cell lines were cultured in DMEM medium and in vitro anticancer effects using MTT assay was performed. A dilution (5, 10, 25, 50 and 100µg/mL) of the gum extract were subjected to cell lines in triplicate. and incubated for 24, 48 and 72hrs. The main phytochemicals within the gum extract were detected through Gas and mass chromatography. Results: cytotoxicity rates were observed at 50µg/mL and >100µg/mL, respectively for all cell lines. And morphological change was noticed after 72 hr. application of the gum extract on SW742 cell line. The compounds found in the Ferula assa-foetida L mostly included oxygen-containing sesquiterpenes (45.25%) and sesquiterpenes (30.45%). Conclusion: the viability decreasing was observed for all examined colorectal adenocarcinoma cell lines including SW742, CT-26, HT-29 and WiDr lines depend on time and concentration of the applied extract of F. assa-foetida gum. The potent cytotoxicity was observed in 50-100 µg/Ml. Furthermore, a morphological change was detected after 72 hr. on SW742. A high percentages of anticancer compounds which were detected within the gum extract supports the idea of purifying these components and considering them in cancer drugs, phytochemistry.

Background: N1-methyladenosine (m1A) within tRNAs, an essential and highly conserved RNA modification, has recently emerged as a significant epigenetic modification that plays a pivotal role in tumor progression in some cancers. However, the role of m1A methylation in colorectal cancer (CRC) remains largely unexplored. TRMT6 is the most critical methyltransferase that catalyzes N1-methyladenine modification within tRNA, and early evidence suggests that this TRMT6-mediated m1A methylation is also required for the self-renewal of cancer stem cells (CSCs). Herein, we systematically interrogated the role of TRMT6 in mediating stemness and pathogenesis of CRC. Methods: We first analyzed the expression levels of all m1A regulators in paired tumor and adjacent-normal samples from TCGA and GEO databases. Subsequently, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting (WB) assays were performed to validate the dysregulation of TRMT6 in CRC tissues and cell lines. m1A dot-blot assays were undertaken to detect methylation levels in CRC tissues and RKO and SW620 CRC cell lines. Cell viability, colony formation, spheroid formation, wound healing, and invasion assays were conducted to investigate the biological functions of TRMT6 in CRC. Results: Higher m1A methylation levels were detected in CRC than in normal tissues. By analyzing the expression of ten m1A regulators in public datasets, and subsequent qRT-PCR validation in a CRC clinical cohort, we identified that high TRMT6 expression was significantly associated with advanced tumor stages (III&IV vs. I&II, p<0.05) and poor prognosis (5-year relapse-free survival rates in high vs. low expression groups: 50.9% vs. 65.1%, p<0.05). TRMT6 silencing in CRC cell lines inhibited cell growth, colony formation, and metastatic efficiency (p<0.01-0.001). Furthermore, we discovered that the TRMT6 could enhance stem cell-like properties required for CSC self-renewal. Transcriptomic profiling identified that the epidermal growth factor receptor (EGFR) pathway is one of the critical signaling regulators of TRMT6 in CRC. The TRMT6 knockdown resulted in significant inhibition of p-EGFR and p-ERK, but not p-AKT in CRC cell lines (all p<0.05), which suggested that m1A modification exerts its oncogenic effects in CRC by activating EGFR downstream pathway. Conclusion: Our study elucidates for the first time the oncogenic effects of TRMT6 on catalyzing m1A modification through the EGFR/ERK pathway, which could offer a promising therapeutic strategy in CRC. Citation Format: SILEI SUI, CAIMING XU, Ying Qing, Hideo Baba, Ajay Goel. TRMT6-mediated N1-methyladenosine methylation promotes tumorigenesis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1713.

The unsatisfactory efficacy of immunotherapy for colorectal cancer (CRC) remains a major challenge for clinicians and patients. The tumor microenvironment may promote CRC progression by upregulating the expression of hypoxia-inducing factor (HIF) and PD-L1. Therefore, this study explored the expression and correlation of HIF-1α and PD-L1 in the CRC microenvironment. The expression and correlation of HIF-1α and PD-L1 in CRC were analyzed using bioinformatics and Western blotting (WB). The hypoxia and inflammation of the CRC microenvironment were established in the CT26 cell line. CT26 cells were stimulated with two hypoxia mimics, CoCl2 and DFO, which were used to induce the hypoxic environment. Western blotting was used to assess the expression and correlation of HIF-1α and PD-L1 in the hypoxic environment.LPS stimulated CT26 cells to induce the inflammatory environment. WB and bioinformatics were used to assess the expression and correlation of TLR4, HIF-1α, and PD-L1 in the inflammatory environment. Furthermore, the impact of curcumin on the inflammatory environment established by LPS-stimulated CT26 cells was demonstrated through MTT, Transwell, molecular docking, network pharmacology and Western blotting assays. In this study, we found that the HIF-1α/PD-L1 pathway was activated in the hypoxic and inflammatory environment and promoted immune escape in CRC. Meanwhile, curcumin suppressed tumor immune escape by inhibiting the TLR4/HIF-1α/PD-L1 pathway in the inflammatory environment of CRC. These results suggest that combination therapy based on the HIF-1α/PD-L1 pathway can be a promising therapeutic option and that curcumin can be used as a potent immunomodulatory agent in clinical practice.

Purpose Tumor RNA vaccines can activate dendritic cells to generate systemic anti-tumor immune response. However, due to easily degraded of RNA, direct RNA vaccine is less effective. In this study, we optimized the method for preparing PEGylated liposom-polycationic DNA complex (LPD) nanoliposomes, increased encapsulate amount of total RNA derived from CT-26 colorectal cancer cells. Tumor RNA LPD nanoliposomes vaccines improved anti-tumor immune response ability of tumor RNA and can effectively promote anti-tumor therapeutic effect of oxaliplatin. Methods Total tumor-derived RNA was extracted from colorectal cancer cells (CT-26 cells), and loaded to our optimized the LPD complex, resulting in the LPD nanoliposomes. We evaluated the characteristics (size, zeta potential, and stability), cytotoxicity, transfection ability, and tumor-growth inhibitory efficacy of LPD nanoliposomes. Results The improved LPD nanoliposomes exhibited a spherical shape, RNA loading efficiency of 9.07%, the average size of 120.37 ± 2.949 nm and zeta potential was 3.34 ± 0.056 mV. Also, the improved LPD nanoliposomes showed high stability at 4 °C, with a low toxicity and high cell transfection efficacy toward CT-26 colorectal cancer cells. Notably, the improved LPD nanoliposomes showed tumor growth inhibition by activating anti-tumor immune response in CT-26 colorectal cancer bearing mice, with mini side effects toward the normal organs of mice. Furthermore, the effect of the improved LPD nanoliposomes in combination with oxaliplatin can be better than that of oxaliplatin alone. Conclusion The improved LPD nanoliposomes may serve as an effective vaccine to induce antitumor immunity, presenting a new treatment option for colorectal cancer.

BackgroundColorectal cancer is a common malignancy occurring in the digestive system and ranks second in cancer mortality worldwide. In colorectal cancer, hydrogen sulfide (H2S) is selectively upregulated, resulting in the further exacerbation of the disease. Therefore, the clearance of H2S and the regulation of the enzymes on the H2S pathways are of great significance for colorectal cancer therapy.MethodsHere, we investigated the H2S content in various clinical tumor tissues from patients and confirmed that overproduced concentration of H2S in colorectal cancer. Accordingly, we developed an H2S-responsive nanoplatform based on zinc oxide coated virus-like silica nanoparticles (VZnO) for the therapy of colorectal cancer.ResultsOwing to its excellent H2S scavenging ability, VZnO could effectively reduce H2S content in colorectal cancer to prohibit the growth of CT26 and HCT116 colorectal cancer cells. Moreover, the removal of H2S in colorectal cancer also leads to tumor inhibition through activating ferroptosis, a non-apoptotic form of cell death. The biosafety-related toxicological and pathological analysis confirmed the low toxicity and high safety of VZnO in colorectal cancer treatment. Furthermore, as an H2S-responsible nanosystem, VZnO appears to have no therapeutic effect on other non H2S rich cancers, such as the 4T1 breast cancer model.ConclusionsWe anticipate that the H2S-depletion-induced ferroptosis strategy using zinc oxide-based nanomaterials would provide insights in designing nanomedicines for colorectal cancer-target theranostics and may offer clinical promise.Graphic abstract

Colorectal cancer (CRC) is one of the most common cancers and the fourth leading cause of cancer-related deaths worldwide. We aimed to determine the role of miR-650 in CRC pathogenesis. In this study, we examined the expression of miR-650 and KISS1 in 80 CRC patients who either received or did not receive chemo agents. For this aim, we assessed the miR-650 and KISS1 expression levels in 80 CRC tissues, 30 of which had no history of chemotherapy. The effect of miR-650 and 5-FU on KISS1 expression was measured using qPCR and Western blotting. Also, the 5- FU effect on miR-650 expression in the CRC cell lines was measured by qRT-PCR. Next, MTT assay and Flowcytometry assays were conducted to determine the role of miR-650 in cell viability and apoptosis. The results showed that miR-650 was down-regulated in CRC tissues. However, patients who received 5-FU before surgery showed increased expression of miR-650. The results for KISS1 were insignificant while administering 5-FU to patients preoperatively increased its expression. In-vitro studies showed that 5-FU led to the up-regulation of miR-650 in the SW480 CRC cell line. Furthermore, the administration of miR-650 and 5-FU downregulated KISS1, especially when combined. Moreover, miR-650 with 5-FU significantly reduced cell viability in CRC cell lines by inducing apoptosis. These results indicate that miR-650 has a tumor suppressive function, overcoming 5-FU chemoresistance in CRC, and induces apoptosis probably by alleviating KISS1. These results suggest that miR-650 is a potential contributor to CRC pathogenesis.

In the Western world, colorectal cancer (CRC) is the third most common cancer with poor prognosis. To identify the proteins and to elucidate the possible mechanisms involved in colorectal carcinogenesis, 2-DE coupled with MS/MS analysis were employed to compare the global protein profile between CRC and individual matched normal tissues from 8 CRC patients. Of 36 proteins identified, carbonic anhydrase II (CA II) was one of most significantly altered and its downregulation in CRC tissues was verified by RT-PCR, western blotting and immunohistochemistry methods, suggesting that CA II may serve as a potential biomarker for CRC diagnosis. To investigate the function and mechanisms of CA II in CRC, a stable SW480 colorectal cancer cell line overexpressing CA II was established. It was shown that overexpression of CA II remarkably suppressed tumor cell growth both in vitro and in vivo, which was in part interpreted by cell cycle arrest at G0/G1 and G2 phase. Further mechanism analysis revealed that the sensitivity of colorectal cancer cells to chemotherapy drugs could be increased by CA II overexpression. Taken together, these data suggest that CA II may be a potential biomarker for early diagnosis of CRC and the results may contribute to a better understanding of the molecular mechanism of CRC and colorectal cancer treatment.

Introduction: Colorectal cancer (CRC) is one of the most common types of cancers worldwide and it is the third leading cause of cancer-related death. Therefore, several efforts have been made to identify not only new diagnostic, prognostic and predictive biomarkers, but also therapeutic targets. One of the promising approaches is the characterization of the tumors using microRNAs (miRNAs). Patients and Methods: We have analyzed expression profiles of 667 miRNAs in 8 patients diagnosed for CRC and 8 paired adjacent non-tumoral tissues using TaqMan Low Density miRNA arrays. We have found miR-215 to be one of the most deregulated miRNAs, therefore its expression was further validated on independent cohort of 250 paired samples and correlated with clinicopathological features of the patients. Consequently, involvement of this miRNA in CRC pathogenesis was investigated using numerous in vitro and in vivo assays. Results: By use of miRNA expression profiling we have identified miR-215 to be highly down-regulated in tumor tissue of CRC patients (P < 0,0001). Subsequent validation on independent cohort of 250 paired samples of tumor tissues and adjacent non-tumor tissues confirmed this result, moreover, correlation between low expression of this miRNA and clinical stage (P < 0.0001) and lymph node metastasis (P < 0.0001) has been observed. In addition, significantly reduced levels of miR-215 have been detected in metastatic tissue (P < 0.0001) compared to primary tumor tissue and low expression of miR-215 was associated with shorter disease free survival (P = 0.0403). Subsequently, in vitro analyses have been performed using stable colorectal cancer cell lines in which the level of miR-215 was increased using transient or stable transfection. Higher levels of miR-215 lead to the cell cycle arrest in G1 phase of HCT-116+/+ cells (P = 0.01) and in G2/M phase of DLD-1 cells (P = 0.05), HCT-116-/- cells (P < 0.001) and HT-29 cells (P = 0.01), increased apoptosis of HCT-116+/+ cells (P < 0.001), reduced migration of DLD-1 cells (P < 0.001) and HCT-116+/+ cells (P < 0.001) and decreased proliferation activity of all analyzed cell lines (P < 0.001). Using qRT-PCR we have validated several predicted targets of miR-215 including XIAP, CD164, ALCAM or HOXB9. Finally, we have established human tumor xenografts in immunodeficient mice. We have observed that HCT-116+/+ transduced with vector containing precursor of miR-215 were associated with the slower growth of the tumors in vivo (N = 5; P = 0.04). Conclusions: The reduced expression of miR-215 has been observed in several cancers, therefore we presume that this miRNA functions as an important tumor suppressor. The results of our study indicate that miR-215 could serve as a new diagnostic and prognostic biomarker as well as potential therapeutic target for the treatment of CRC patients. The work has been supported by IGA MZCR NT13549-4/2012, NT13860-4/2012, NT13547-4/2012 and NT13514-4/2012. Citation Format: Petra Vychytilova-Faltejskova, Jana Merhautova, Jitka Mlcochova, Lenka Radova, Robert Iliev, Marek Svoboda, Rostislav Vyzula, Ondrej Slaby. MiR-215 is a tumor suppressor in colorectal cancer in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3116. doi:10.1158/1538-7445.AM2015-3116

Ribosomal protein S15A (RPS15A) has been implicated in tumorigenesis, but its role in colorectal cancer (CRC) is not fully studied. The objective of this study was to investigate the role of RPS15A in CRC carcinogenesis. RBSP15A expression was detected in 120 colorectal adenocarcinoma biopsies by immunohistological staining, and we examined the association of RSP15A expression with clinicopathological outcomes. We generated RPS15A stable knockdown CRC cell lines using shRNAs and assessed cell proliferation by MTT assays, clonogenicity by colony formation assays, and apoptosis and cell cycle arrest by flow cytometric analyses. A mouse tumor xenograft model was used to confirm the influence of RPS15A expression on CRC in vivo. RPS15A expression was predictive for poor disease-free survival. Knockdown of RPS15A expression significantly inhibited cell proliferation and colony formation and augmented apoptosis in both the RKO and SW620 CRC cell lines. Moreover, RPS15A knockdown arrested RKO cells at the G2/M phase and SW620 cells at the G0/G1 phase. KEGG pathway analysis of 785 genes differentially expressed between wild-type and shRPS15A RKO cells showed enrichment for the pathway in cancer and MAPK signaling pathway KEGG terms. RPS15A knockdown induced apoptosis via regulation of BIRC3, p38 MAPK, and Chk1. Consistently, RPS15A knockdown significantly impaired the growth of subcutaneous CRC xenografts in nude mice. These results indicate that RPS15A is a novel, potentially oncogenic gene involved in colorectal carcinogenesis. RPS15A knockdown may be an attractive strategy for treating CRC with gene therapy.

590 Background: PD-1/PD-L1 blockade showed therapeutic efficacy in only microsatellite (MSI) colorectal carcinomas (CRC), however, the profile of PD-L1 and PD-1 expression in CRC is only partially described. Methods: We thus analyzed on FFPE whole-tissue sections of 80 CRC, the expression profile of PD-L1 by tumor and/or immune cells by immunohistochemistry (clone E1L3N) depending on the MSI status and the histological subtype, and correlated to the density of PD-1+ and Tbet+ (able to secrete IFNg known to induce PD-L1) tumor-infiltrating lymphocytes (TIL). Results: 78% of MSI CRC (32/41) overexpressed PD-L1 either by tumor or immune cells versus 46% of MSS CRC (18/39) (p 0.005). This overexpression was heterogeneous within the same tumor in most of cases. Among MSI CRC, PD-L1 was preferentially overexpressed in medullary carcinomas (MC, 19/21, 90%) compared with 65% (13/20) in non-medullary adenocarcinomas (p 0.06). PD-L1 expression by tumor cells was only observed in MSI CRC (19/41, 46% with PD-L1 expression in more than 5% of tumor cells – score 1), and preferentially in MC (57% vs 5% in no medullary adenocarcinomas, with PD-L1 expression in more than 50% of tumor cells – score 3, p 0.0005). Conversely, PD-L1 expression by immune cells was observed in MSI CRC (23/41, 56% with PD-L1 expression by more than 5 sheets of 50 positive cells) but also in MSS CRC (18/39, 43%) (p 0.5). The density of PD-1+ cells was significantly correlated to the PD-L1 expression, as well as the density of Tbet+ TIL. Conclusions: PD-L1 expression is 1) heterogeneous in CRC, among CRC but also within the same tumor, 2) preferentially observed in MSI CRC (78%), especially in MC (90%), where PD-L1 is expressed by tumor cells, 3) correlated with the density of PD-1+ or T-bet+ TIL, and 4) observed in a significant proportion of MSS CRC (46%) by immune cells only. From a clinical point of view, PD-L1 expression has to be determined at best in full tissue section and besides its preferential expression in MSI CRC, its significant frequency and expression profile (only by immune cells) in MSS CRC should be taken into account in the future clinical trials testing the efficacy of anti-PD-1/PD-L1 antibodies.