Regulator of G protein signaling 16 restrains apoptosis in colorectal cancer through disrupting TRAF6-TAB2-TAK1-JNK/p38 MAPK signaling.

Colorectal cancer (CRC) remains a major global cause of cancer-related mortality, lacking effective biomarkers and therapeutic targets. Revealing the critical pathogenic factors of CRC and the underlying mechanisms would offer potential therapeutic strategies for clinical application. G protein signaling (RGS) protein family modulators play essential role within regulating downstream signaling of GPCR receptors, with function in cancers unclear. Our study focused on the expression patterns of RGS proteins in CRC, identifying Regulator of G protein signaling 16 (RGS16) as a prospective diagnostic and therapeutic target. Analyzing 899 CRC tissues revealed elevated RGS16 levels, correlating with clinicopathological features and CRC prognosis by immunohistochemistry (IHC) combined with microarray. We confirmed the elevated RGS16 protein level in CRC, and found that patients with RGS16-high tumors exhibited decreased disease-specific survival (DSS) and disease-free survival (DFS) compared to those with low RGS16 expression. Functional assays demonstrated that RGS16 promoted the CRC progression, knockdown of RGS16 led to significantly increased apoptosis rates of CRC in vitro and in vivo. Notably, we also confirmed these phenotypes of RGS16 in organoids originated from resected primary human CRC tissues. Mechanistically, RGS16 restrained JNK/P38-mediated apoptosis in CRC cells through disrupting the recruitment of TAB2/TAK1 to TRAF6. This study provides insights into addressing the challenges posed by CRC, offering avenues for clinical translation.

USP37 promotes angiogenesis and metastasis in colorectal cancer by facilitating ß-catenin stability.

Ubiquitin-specific peptidase 37 (USP37) is a novel deubiquitinating enzyme, which has been found to be involved in the progression of multiple tumors. However, its function in colorectal cancer (CRC) remains unclear. Here, we firstly proved that USP37 was up-regulated in CRC cases, and high USP37 expression predicted poor survival of CRC cases. USP37 up-regulation promoted the proliferation, cell cycle progression, apoptosis inhibition, migration, invasion, epithelial mesenchymal transition (EMT) and stemness of CRC cells; moreover, USP37 facilitated the angiogenesis of human umbilical vein endothelial cells (HUVECs). However, USP37 silencing showed the opposite function. In vivo experiment suggested that USP37 silencing suppressed the growth and lung metastasis of CRC in nude mice. Interestingly, we found that CTNNB1 (gene coding for ß-catenin) level was positively correlated with USP37 level in CRC and USP37 silencing suppressed the expression of ß-catenin in CRC cells and xenograft tumor tissues. Further mechanistic studies showed that USP37 could enhance the stability of ß-catenin by inhibiting its ubiquitination. Taken together, USP37 acts as an oncogene in CRC, which promotes angiogenesis, metastasis and stemness by enhancing ß-catenin stability via inhibiting its ubiquitination. USP37 may be a usefully target in CRC clinical treatment.

CNN2 silencing inhibits colorectal cancer development through promoting ubiquitination of EGR1.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignant tumors of the digestive tract. H2-calponin (CNN2), an actin cytoskeleton-binding protein, is an isoform of the calponin protein family whose role in CRC is still unknown. Research based on clinical samples showed the up-regulation of CNN2 in CRC and its association with tumor development, metastasis, and poor prognosis of patients. Both in vitro loss-of-function and gain-of-function experiments showed that CNN2 participates in CRC development through influencing malignant cell phenotypes. In vivo, xenografts formed by CNN2 knockdown cells also showed a slower growth rate and smaller final tumors. Furthermore, EGR1 was identified as a downstream of CNN2, forming a complex with CNN2 and YAP1 and playing an essential role in the CNN2-induced regulation of CRC development. Mechanistically, CNN2 knockdown down-regulated EGR1 expression through enhancing its ubiquitination, thus decreasing its protein stability in a YAP1-dependent manner. In summary, CNN2 plays an EGR1-dependent promotion role in the development and progression of CRC, which may be a promising therapeutic target for CRC treatment.

ZNF516 suppresses stem cell-like characteristics by regulating the transcription of Sox2 in colorectal cancer.

This study aimed to explore the biological function and the molecular mechanism of the action of zinc-finger protein 516 (ZNF516) in suppressing stem cell-like characteristics and tumor progression in colorectal cancer (CRC). The expression profiles of ZNF516 in clinical samples and from The Cancer Genome Atlas (TCGA) CRC database were analyzed. Cell transfection was used to overexpress and knockdown ZNF516 in CRC cells. Cell counting kit-8 (CCK8) assays, transwell assays and flow cytometry were used to study cell proliferation, invasion and stem cell-like characteristics, respectively. Cycloheximide (CHX) was used to examine the effect of ZNF516 expression on Sox2 degradation. Finally, the effects of ZNF516 on tumor growth and metastasis were tested on xenograft tumor models and lung metastasis models in immunocompromised mice. We found that the expression level of ZNF516 was lower in TCGA CRC tissue and clinical CRC samples compared with that in normal colorectal mucosal cells. Overexpression of ZNF516 in CRC cells inhibited cell proliferation, colony formation, migration and invasion, whereas ZNF516 knockdown showed the opposite effects. In addition, ZNF516 overexpression inhibited the sphere-forming ability of CRC cells and suppressed the expression of CD133, CD44 and Oct4 in CRC cells. ZNF516 decreased the stability of Sox2 through a mechanism mediated by EGFR. By in vivo experiments using mouse tumor models, we further confirmed that ZNF516 attenuated tumor growth and alleviated lung metastasis in mice. In conclusion, ZNF516 functions as a tumor suppressor by regulating the transcription of Sox2 to inhibit cell proliferation, invasion, and the development of stem cell-like characteristics in CRC cells.

LncRNA CASC21 induces HGH1 to mediate colorectal cancer cell proliferation, migration, EMT and stemness.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) have been increasingly reported to serve vital parts in malignancies including CRC. Although cancer susceptibility 21 (CASC21) has been uncovered to play a part in CRC, its mechanism still needs further explanation. Thus, our study aimed to further explore the influence and mechanism of CASC21 in CRC progression. Quantitative real-time RT-PCR and western blot were performed to detect gene expression; a series of functional assays were performed to investigate the effect of CASC21 on CRC cells; in vivo tumour growth was evaluated via the nude mice xenograft model. The results revealed that CASC21 facilitated CRC cell proliferation, migration, epithelial-mesenchymal transition (EMT) and stemness. In addition, CASC21 was co-expressed with and bound to transcription factor POU5F1B (POU class 5 homeobox 1B). CASC21 recruited POU5F1B to HGH1 promoter to activate the transcription of HGH1 homolog. Also, CASC21 served as a competitive endogenous RNA (ceRNA) to up-regulate HGH1 via endogenously sponging miR-485-5p. Moreover, HGH1 overexpression counteracted the suppression of CASC21 deficiency on CRC tumour growth. In summary, our study indicated that CASC21 enhanced the expression of HGH1 to promote the malignancy of CRC by recruiting POU5F1B and sponging miR-485-5p, suggesting a key role of CASC21 in CRC progression.

Prognostic and Predictive Value of BGN in Colon Cancer Outcomes and Response to Immunotherapy.

BACKGROUND: Colon cancer is one of the most frequent malignancies and causes high mortality worldwide. Exploring the tumor-immune interactions in the tumor microenvironment and identifying new prognostic and therapeutic biomarkers will assist in decoding the novel mechanism of tumor immunotherapy. BGN is a typical extracellular matrix protein that was previously validated as a signaling molecule regulating multiple processes of tumorigenesis. However, its role in tumor immunity requires further investigation. METHODS: The differentially expressed genes in three GEO datasets were analyzed, and BGN was identified as the target gene by intersection analysis of PPIs. The relevance between clinical outcomes and BGN expression levels was evaluated using data from the GEO database, TCGA and tissue microarray of colon cancer samples. Univariable and multivariable Cox regression models were conducted for identifying the risk factors correlated with clinical prognosis of colon cancer patients. Next, the association between BGN expression levels and the infiltration of immune cells as well as the process of the immune response was analyzed. Finally, we predicted the immunotherapeutic response rates in the subgroups of low and high BGN expression by TIS score, ImmuCellAI and TIDE algorithms. RESULTS: BGN expression demonstrated a statistically significant upregulation in colon cancer tissues than in normal tissues. Elevated BGN was associated with shorter overall survival as well as unfavorable clinicopathological features, including tumor size, serosa invasion and length of hospitalization. Mechanistically, pathway enrichment and functional analysis demonstrated that BGN was positively correlated with immune and stromal scores in the TME and primarily involved in the regulation of immune response. Further investigation revealed that BGN was strongly expressed in the immunosuppressive phenotype and tightly associated with the infiltration of multiple immune cells in colon cancer, especially M2 macrophages and induced Tregs. Finally, we demonstrated that high BGN expression presented a better immunotherapeutic response in colon cancer patients. CONCLUSION: BGN is an encouraging predictor of diagnosis, prognosis and immunotherapeutic response in patients with colon cancer. Assessment of BGN expression represents a novel approach with great promise for identifying patients who may potentially benefit from immunotherapy.

MicroRNA-95-3p inhibits cell proliferation and metastasis in colorectal carcinoma by HDGF.

BACKGROUND: MicroRNAs (miRNAs) play an important regulatory role in carcinogenesis and cancer progression. MiR-95-3p has been reported to be an oncogene in hepatocellular carcinoma. However, the role of miR-95-3p in colorectal carcinoma (CRC) remains unclear. METHODS: miR-95-3p was validated in an independent validation sample cohort of 215 CRC tissues. Functional assays, Cell proliferation (MTT) assay colony formation, wound healing, transwell and animal xenograft assays were used to determine the oppressor role of miR-95-3p in human CRC progression. Furthermore, Bioinformatics analysis, western blotting and dual-luciferase reporter assay were used to determine the mechanism by which miR-95-3p suppresses progression of CRC cells. RESULTS: In this study, we found that miR-95-3p was downregulated in CRC tissues. The low level of miR-95-3p in CRC tumors was correlated with aggressive clinicopathological characteristics, and it predicted poor prognosis in CRC patients. The overexpression of miR-95-3p significantly inhibited CRC cell proliferation, colony formation and metastasis in vitro and in vivo. Bioinformatic analysis further identified hepatoma-derived growth factor (HDGF) as a novel target of miR-95-3p in CRC cells. These findings suggest that miR-95-3p regulates CRC cell survival, partially through the downregulation of HDGF. CONCLUSIONS: Therefore, the miR-95-3p/HDGF axis might serve as a novel therapeutic target in patients with CRC.

Inverse relationship between CD40L expression and cytolytic molecule expression by CD8+CXCR5+ T follicular cytotoxic cells in colorectal cancer.

CXCR5+ CD8 T cells, sometimes termed T follicular cytotoxic (Tfc) cells, are characterized by high proinflammatory cytokine and cytolytic molecule expression and low exhaustion and checkpoint molecule expression. Additionally, Tfc cells could promote B cell responses and support Ig release. It is yet unclear how Tfc cells could help B cells when they have the potential to mediate cytotoxicity at the same time. In this study, we found that Tfc cells expressed significantly higher levels of CD40L than non-Tfc CD8 T cells. Interestingly, Tfc cells from colorectal cancer (CRC) patients presented significantly higher CD40L than Tfc cells from healthy controls in a manner that was associated with CRC stage. Coincubation of Tfc cells and autologous B cells resulted in higher CD40L expression in a time-dependent manner. Interestingly, activated Tfc cells, when incubated with B cells, presented rapid downregulation of perforin and granzyme B. In general, greater than 50% of tumor-infiltrating Tfc cells expressed CD40L. In addition, the level of CD40L in tumor-infiltrating Tfc cells was higher in stage IV CRC patients than in stage II and stage III CRC patients. Interestingly, the levels of perforin and granzyme B expression by tumor-infiltrating Tfc cells were inversely correlated with the level of CD40L expression by tumor-infiltrating Tfc cells. Overall, we demonstrated that an inverse association existed between CD40L and cytotoxic molecule expression in Tfc cells from CRC patients.

Toll-like receptor 4 regulates spontaneous intestinal tumorigenesis by up-regulating IL-6 and GM-CSF.

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll-like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%-90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The ApcMin/+ (multiple intestinal neoplasia) model mouse is a well-utilized model of FAP, an inherited form of intestinal cancer. In this study, ApcMin/+ intestinal adenoma mice were generated on TLR4-sufficient and TLR4-deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of ApcMin/+ WT and ApcMin/+ TLR4-/- mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high-throughput RNA-Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine-cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM-CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of ApcMin/+ TLR4-/- mice compared with ApcMin/+ WT mice. In the functional study of core down-regulation factors, it was found that IL6, GM-CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.

CD8+CXCR5+ T cells in tumor-draining lymph nodes are highly activated and predict better prognosis in colorectal cancer.

Tumor-draining lymph nodes (TDLNs) are the primary sites of tumor antigen presentation, as well as the origin of metastasis in most cases. Hence, the type and function of immune cells in TDLNs are critical to the microenvironment and potentially affect the clinical outcome of the malignancy. CD8+CXCR5+ T cells are recently described to present high effector functions in infectious diseases, but their role in colorectal cancer (CRC) remains unclear. In forty-four Stage III CRC patients, we examined the CD8+CXCR5+ T cells in blood, tumor, and TDLN. CD8+CXCR5+ T cells represented lass than 2% of CD3+ T cells in blood, but a much larger population in tumor. In TDLN, the CD8+CXCR5+ T cells represented the vast majority of CD8+ T cells and between 9.3% and 32.9% of CD3+ T cells. The prevalence of CD8+CXCR5+ T cells in tumor was not associated with their frequency in peripheral blood, but was positively correlated with their frequency in TDLN. The transcription of effector genes, including IFNG, TNF, IL2, PRF1, and GZMB, and exhaustion markers, including PD1, TIM3, 2B4, and LAG3, were examined in CD8+CXCR5+ T cells and CD8+CXCR5- T cells. With a few exceptions, CD8+CXCR5+ T cell presented significantly higher effector gene expression, and significantly lower exhaustion marker expression than their CXCR5- counterparts. In addition, the prognosis of CRC patients was positively associated with the frequency of TDLN CD8+CXCR5+ T cells, and with the expression of IFNG, PRF1, and GZMB expression by tumor and TDLN CD8+CXCR5+ T cells. Together, these results demonstrated that CD8+CXCR5+ T cells were significant participants of CRC-associated immunity and could potentially serve as therapeutic options.

miR-320b suppresses cell proliferation by targeting c-Myc in human colorectal cancer cells.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs that potentially play a critical role in tumorigenesis. Mounting evidence indicates that one specific miRNA: miR-320b is down regulated in numerous human cancers, including colorectal cancer (CRC); making the hypothesis that miR-320b may play a key role in tumorigenesis plausible. However, its role in carcinogenesis remains poorly defined. The goal of this study is to better clarify the role of miR-320b in tumor growth of CRC. METHODS: Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was conducted to detect the expression of miR-320b in CRC tissues and 5 CRC cell lines. The effect of miR-320b on cell proliferation was analyzed in vitro and in vivo. Furthermore, a luciferase reporter assay was performed to measure the target effects of miR-320b. Lastly, the messenger RNA (mRNA) and protein levels of the gene c-MYC were measured in CRC cell lines and tissues by qRT-PCR, and confirmed via Western blot and Immunohistochemical (IHC) staining. RESULTS: The results presented here showed that miR-320b expression was down regulated in both CRC tissues and cells. Overexpression of miR-320b in CRC cells was statistically correlated with a decrease of cell growth in vitro and in vivo, while c-MYC was identified as a target gene of miR-320b in CRC. Furthermore, it was found that up-regulation of c-Myc can attenuate the effects induced by miR-320b. CONCLUSIONS: Our identification of c-MYC as a target gene of miR-320b provides new insights into the pathophysiology of CRC proliferation, and identifies miR-320b as a novel therapeutic target for the treatment of CRC.

Combine MEK inhibition with PI3K/mTOR inhibition exert inhibitory tumor growth effect on KRAS and PIK3CA mutation CRC xenografts due to reduced expression of VEGF and matrix metallopeptidase-9.

Although epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) cetuximab are used widely to treat KRAS wild-type metastatic colorectal cancer (mCRC), patients become resistant by various mechanisms, including KRAS, BRAF, and PIK3CA mutations, thereafter relapsing. AZD6244 is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we investigated the mechanisms of AZD6244 alone or with BEZ235, an orally available potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), in a KRAS and PIK3CA mutation CRC xenograft model. HCT116 (KRAS (G13D) , PIK3CA (H1047R) mutant) cells were subcutaneously injected into the nude mice. Mice were randomly assigned to treatment with vehicle, cetuximab, AZD6244, BEZ235, or AZD6244 plus BEZ235, for up to 3 weeks; then, all mice were sacrificed, and tumor tissues were subjected to Western blot analysis and immunohistochemical staining. AZD6244 or BEZ235 slightly inhibit tumor growth of HCT116 xenografts, and the combination treatment markedly enhanced their antitumor effects. However, cetuximab had no effect on tumor growth. Western blot analysis and immunohistochemical staining revealed that treatment with AZD6244 or BEZ235 could significantly reduce the phosphorylation level of ERK1/2 or AKT in HCT116 tumor tissues. More interesting, the antiangiogenic effects were substantially enhanced when the agents were combined which may due to the reduced expression of VEGF and matrix metallopeptidase-9 (MMP-9) in tumor tissues. These results suggest that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in CRC harboring with KRAS and PIK3CA mutations. The mechanisms of synergistic antitumor effects may be due to antiangiogenesis.

[Analysis on misdiagnosis of 271 patients with rectal cancer].

OBJECTIVE: To investigate the common reasons for the misdiagnosis of rectal cancer. METHODS: A retrospective study was performed in 568 cases of rectal cancer in the Changhai Hospital from January 2007 to December 2008. Age at diagnosis, gender distribution, symptom, delay in diagnosis, TNM stage, and grade of differentiation were recorded and analyzed. The importance of digital examination and colonoscopy were addressed. RESULTS: Two hundred and seventy-one(47.7%) out of 568 patients were misdiagnosed for iatrogenic reasons. Rectal cancer patients who presented hematochezia were more likely to be misdiagnosed. There were 110 cases of stage III(40.6%) and 68 cases of stage IV(12.5%) in patients who were misdiagnosed, which was significantly higher than those who were diagnosed correctly(P<0.05). Patients under 40 years old were more likely to be misdiagnosed, and their correct diagnosis was often delayed longer and the tumors were in more advanced stage as compared to the older groups(P<0.05). CONCLUSIONS: The misdiagnosis rate of rectal cancer is high. Tumor stage of patients misdiagnosed is significantly more advanced than those who are correctly diagnosed. Digital examination and colonoscopy should be emphasized, especially for patients under the age of 40.