circEXOC6B interacting with RRAGB, an mTORC1 activator, inhibits the progression of colorectal cancer by antagonizing the HIF1A-RRAGB-mTORC1 positive feedback loop.

BACKGROUND: In recent years, an increasing number of studies have indicated that circular RNA plays crucial roles in regulating tumor development and chemoresistance. Using two high-throughput RNA sequence datasets, we previously found that circEXOC6B was downregulated in colon cancer. However, its role and mechanism in colorectal cancer (CRC) remained unknown. METHODS: Real-time quantitative PCR was used to examine the expression of circEXOC6B in CRC tissues. In vivo and in vitro functional experiments were performed to determine the suppressor role of circEXOC6B in CRC progression. RNA pull-down, mass spectrometry, RNA-binding protein immunoprecipitation, co-immunoprecipitation, fluorescence in situ hybridization, and immunofluorescence were applied to investigate the possible mechanisms connecting circEXOC6B to CRC growth and 5-fluorouracil-induced apoptosis. Chromatin immunoprecipitation, dual-luciferase assay, western blot, and immunohistochemistry were used to explore the mechanisms underlying the HIF1A regulation of RRAGB transcription. RESULTS: circEXOC6B was downregulated in CRC tissues, and its lower expression was associated with poor prognosis of patients. Functional experiments showed that circEXOC6B inhibited growth and increased the 5-fluorouracil-induced apoptosis of CRC cells in vitro and in vivo. Mechanistically, circEXOC6B inhibited the heterodimer formation of RRAGB by binding to it, thereby suppressing the mTORC1 pathway and HIF1A level. In addition, HIF1A upregulated the transcription of RRAGB by binding to its promoter region. Altogether, the results demonstrated that a HIF1A-RRAGB-mTORC1 positive feedback loop drives tumor progression in CRC, which could be interrupted by circEXOC6B. CONCLUSIONS: circEXOC6B inhibits the progression of CRC and enhances the chemosensitivity of CRC cells to 5-fluorouracil by antagonizing the HIF1A-RRAGB-mTORC1 positive feedback loop. circEXOC6B is a possible therapeutic target for CRC treatment.

CXCL10 mediates CD8+ T cells to facilitate vessel normalization and improve the efficacy of cetuximab combined with PD-1 checkpoint inhibitors in colorectal cancer.

The immunotherapy and anti-EGFR targeted treatment occupying a pivotal position in colorectal cancer (CRC), is still limited to a group of patients who display specific molecular alterations and inevitably escape from resistance, further studies are still needed in colorectal cancer. We found that chemokine ligand 10 (CXCL10) expression correlates with intratumoral CD8+ T cell infiltration and reprograms tumor vasculatures in colorectal cancer. CXCL10 overexpression not only suppressed tumor growth but also increased CD8+ T cell infiltration and induced tumor vascular normalization in vivo. Additionally, the growth inhibition and tumor vascular normalization induced by CXCL10 can be reversed by the depletion of CD8+ T cells in vivo. Mechanically, CXCL10 interacts with VCAN to mediate tumor vascular normalization. The VCAN expression correlated inversely with the expression of CXCL10 and the infiltration of CD8+ T cells in CRC. Elevated CXCL10 expression sensitized colorectal cancer cells to cetuximab/anti-PD1 combination therapy compared with cetuximab or anti-PD1 alone. We propose that CXCL10 could be used to increase the anti-EGFR therapy and immunotherapy effect, targeting both tumor vessels and immune cells in colorectal cancer.

HRK inhibits colorectal cancer cells proliferation by suppressing the PI3K/AKT/mTOR pathway.

Background: As one of the most common malignant tumor, colorectal cancer (CRC) continues to have a high incidence and mortality rate. HRK belongs to the BCL-2 protein family, which has been shown to have antitumor effects in prostate cancer. However, its role in colorectal cancer is not yet known. Methods: In this study, we verified the expression levels of HRK in colorectal cancer tissues by public database search as well as immunohistochemistry. Next, we analyzed HRK expression levels in CRC tissues,adjacent non-cancerous tissues, cell lines and normal intestinal epithelial cells by qPCR and Western blotting. CCK-8 proliferation assays, transwell assays, wound healing assays, colony assays and flow cytometry were performed to clarified the effect of HRK on CRC cells. Western blotting and rescue experiments were used to determine the role of HRK in regulating PI3K/AKT/mTOR signaling pathway. Results: HRK expression was lower in CRC tissues and cell lines. Gain and loss of function experiments showed that HRK decreased proliferation, invasion and migration of CRC cells. Low expression of HRK inhibited CRC cell apoptosis as well as activated the PI3K/AKT/mTOR signaling pathway. In addition, rapamycin inhibits the activation of PI3K/AKT/mTOR signaling pathway and reverses HRK-induced alterations in cell biological functions. Conclusion: Our study demonstrates that HRK is lowly expressed in colorectal cancer tissues. And for the first time, HRK was shown to promote apoptosis and inhibit proliferation of colorectal cancer cells by inhibiting PI3K/AKT/mTOR signaling pathway. HRK represents a potential target for the treatment of CRC.

circCDYL2, Overexpressed in Highly Migratory Colorectal Cancer Cells, Promotes Migration by Binding to Ezrin.

BACKGROUND: Colorectal 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. METHODS: In 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. RESULTS: Among 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. CONCLUSIONS: Our 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.