Metastasis Of Colorectal Cancer Cells Research Articles

Elevated Sodium Pump α3 Subunit Expression Promotes Colorectal Liver Metastasis via the p53-PTEN/IGFBP3-AKT-mTOR Axis.

The sodium pump α3 subunit is associated with colorectal liver metastasis. However, the underlying mechanism involved in this effect is not yet known. In this study, we found that the expression levels of the sodium pump α3 subunit were positively associated with metastasis in colorectal cancer (CRC). Knockdown of the α3 subunit or inhibition of the sodium pump could significantly inhibit the migration of colorectal cancer cells, whereas overexpression of the α3 subunit promoted colorectal cancer cell migration. Mechanistically, the α3 subunit decreased p53 expression, which subsequently downregulated PTEN/IGFBP3 and activated mTOR, leading to the promotion of colorectal cancer cell metastasis. Reciprocally, knockdown of the α3 subunit or inhibition of the sodium pump dramatically blocked this effect in vitro and in vivo via the downregulation of mTOR activity. Furthermore, a positive correlation between α3 subunit expression and mTOR activity was observed in an aggressive CRC subtype. Conclusions: Elevated expression of the sodium pump α3 subunit promotes CRC liver metastasis via the PTEN/IGFBP3-mediated mTOR pathway, suggesting that sodium pump α3 could represent a critical prognostic marker and/or therapeutic target for this disease.

Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells.

Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.

MVP Expression Facilitates Tumor Cell Proliferation and Migration Supporting the Metastasis of Colorectal Cancer Cells.

Cancer cells show significant dysregulation of genes expression, which may favor their survival in the tumor environment. In this study, the cellular vault’s components MVP (major vault protein), TEP1 (telomerase-associated protein 1) and vPARP (vault poly(ADP-ribose) polymerase) were transiently or completely inhibited in U2OS cells (human bone osteosarcoma epithelial cells) to evaluate their impact on the cell proliferative and migratory capacity as well as on the development of their resistance to the drug vinorelbine. Comparative analysis of MVP protein expression level in normal colon tissue, primary colorectal tumor, and metastasis showed that the expression of this protein does not increase significantly in the primary tumor, but its expression increases in metastatic cells. Further comparative molecular analysis using the whole transcriptome microarrays for MVP-positive and MVP-negative cells showed that MVP is involved in regulating proliferation and migration of cancer cells. MVP may facilitate metastasis of colon cancer due to its impact on cell migration. Moreover, two vault proteins, MVP and TEP1, contribute the resistance to vinorelbine, while vPARP does not.

ASPM facilitates colorectal cancer cells migration and invasion by enhancing β-catenin expression and nuclear translocation.

Increased abnormal spindle-like microcephaly (ASPM) expression has been linked to clinical stage and poor prognosis in cancers, but the molecular mechanisms by which ASPM promotes cell metastasis in colorectal cancer (CRC) has not been identified. This study showed that the abilities of cell migration, invasion, and epithelial-mesenchymal transition (EMT) were attenuated in ASPM-deficient CRC cell lines. Furthermore, we reported that attenuation of ASPM expression inhibited CRC cell metastasis in vivo. Additionally, the expression of ASPM was positively correlated with β-catenin level in CRC tissues. Mechanistically, ASPM can upregulate β-catenin transcription by stimulating the β-catenin promoter and enhancing the nuclear translocation of β-catenin in CRC cells, which leads to the activation of the Wnt/β-catenin pathway. Finally, we showed that ASPM effectively induced CRC cell migration and invasion in a β-catenin-dependent manner.

PHF5A promotes colorectal cancer progression by alternative splicing of TEAD2

Dysregulated alternative splicing (AS) plays critical roles in driving cancer progression, and the underlying mechanisms remain largely unknown. Here, we demonstrated that PHF5A, a component of U2 small nuclear ribonucleoproteins, was frequently upregulated in colorectal cancer (CRC) samples and associated with poor prognosis. PHF5A promoted proliferation and metastasis of CRC cells in vitro and in vivo. Transcriptomic analysis identified PHF5A-regulated AS targets and pathways. Particularly, PHF5A induced TEAD2 exon 2 inclusion to activate YAP signaling, and interference of TEAD2-L partially reversed the PHF5A-mediated tumor progression. Pharmacological inhibition of PHF5A using pladienolide B had potent antitumor activity. Collectively, these data revealed the oncogenic role of PHF5A in CRC through regulating AS and established PHF5A as potential therapeutic target.

ERp29 forms a feedback regulation loop with microRNA-135a-5p and promotes progression of colorectal cancer

Expression of endoplasmic reticulum (ER) stress-associated genes is often dysregulated in cancer progression. ER protein 29 (ERp29) is abnormally expressed in many neoplasms and plays an important role in tumorigenesis. Here, we showed ERp29 is a novel target for microRNA-135a-5p (miR-135a-5p) to inhibit the progression of colorectal cancer (CRC); correspondingly, ERp29 acts as an oncoprotein in CRC by promoting proliferation and metastasis of CRC cells, and suppressing apoptosis of the cells. More importantly, we found that miR-135a-5p expression is reversely upregulated by ERp29 through suppressing IL-1β-elicited methylation of miR-135a-5p promoter region, a process for enterocyte to maintain a balance between miR-135a-5p and ERp29 but dysregulated in CRC. Our study reveals a novel feedback regulation loop between miR-135a-5p and ERp29 that is critical for maintaining appropriate level of each of them, but partially imbalanced in CRC, resulting in abnormal expression of miR-135a-5p and ERp29, which further accelerates CRC progression. We provide supporting evidence for ERp29 and miR-135a-5p as potential biomarkers for diagnosis and treatment of CRC.

AHA1 regulates cell migration and invasion via the EMT pathway in colorectal adenocarcinomas

The progression of colorectal cancer (CRC) has been well studied and understood with the development of molecular and genetic techniques. However, specific marker(s) that could be used to predict lymph node (LN) involvement, which is the most important prognostic factor for CRC, have not been identified so far. Our previous study, in which network analysis of LN(+) and LN(−) CRC gene expression was carried out with data obtained from the Cancer Genome Atlas, led to the identification of AHA1. AHA1 is a co-chaperone activator of the Hsp90 ATPase activity. However, the role of AHA1 expression in cancer cells is still unclear. To investigate how AHA1 expression regulates the cancer cell progression and/or metastasis of human CRC, the expression levels of AHA1 and Hsp90 were examined in 105 CRC tissue samples and compared with those in paired normal tissue. The RNA expression levels of AHA1 and Hsp90aa1, but not Hsp90ab, were significantly higher in cancer tissues than in adjacent paired normal tissues (p = 0.032 and p = 0.0002, respectively). In particular, AHA1, but not Hsp90aa1 and Hsp90ab, was closely associated with the TNM stage, LN stage, and tumor metastasis (p = 0.035, p = 0.012, and p = 0.0003, respectively). Moreover, the expression of AHA1 was not only higher in the CRC cell lines than in the normal colon fibroblast cell line but was also associated with the progression of these CRC cell lines. Overexpression of AHA1 in SW480 cells increased, whereas suppression of AHA1 expression in HCT116 cells reduced cell migration and invasion through the regulation of Snail, E-cadherin, pSRC, and pAKT, which are associated with EMT signaling. Taken together, our study suggests that AHA1 contributes to the metastatic advantage of human CRC.

FOXO4 Inhibits the Migration and Metastasis of Colorectal Cancer by Regulating the APC2/β-Catenin Axis.

Objective: Adenomatous polyposis coli 2 (APC2) is a colorectal cancer (CRC) tumor-suppressor gene. The progression of several kinds of cancer is closely associated with Forkhead box O4 (FOXO4). However, the function of FOXO4 in CRC is unclear. This study focused on the role of FOXO4 and the relationship between FOXO4 and APC2 in CRC migration and metastasis.Methods: The expressions of FOXO4, APC2, and p(S37)-β-catenin were detected in CRC tissues by immunohistochemistry, and their correlation was analyzed using the Spearman coefficient. Chromatin immunoprecipitation was used to test whether FOXO4 binds and regulates APC2 as a transcription factor. Either FOXO4 overexpression or APC2 knockdown was performed in CRC cell lines. The roles of FOXO4 and APC2 were investigated in CRC migration and metastasis.Results: FOXO4 was downregulated in CRC tissues compared with normal tissues and positively correlated with APC2 and p(S37)-β-catenin. FOXO4 could combine the promoter region of APC2 to upregulate its expression and increase the phosphorylated degradation of β-catenin. Stemness genes (CD133, ABCG1, and SOX2) were inhibited by FOXO4 overexpression in SW620 and HCT116 cell lines. Overexpressed FOXO4 suppressed epithelial–mesenchymal transition and the migration of CRC cell lines and metastasis of HCT116 in both the spleen and liver of nude mice, which was reversed by APC2 knockdown.Conclusion: This research demonstrates that overexpressed FOXO4 inhibits the migration and metastasis of CRC cells by enhancing the APC2/β-catenin axis, suggesting that FOXO4 is a potential therapeutic target of CRC.

WDR74 promotes proliferation and metastasis in colorectal cancer cells through regulating the Wnt/β-catenin signaling pathway.

Colon cancer (CRC) is a common type of cancer and has a high incidence worldwide. Protein 74 (WDR74), which consists of the WD repetition sequence, has been previously associated with tumor tumorigenesis. However, its mechanism of action in CRC remains unclear. Here, we found that WDR74 expression was upregulated in CRC tissues and cells. Downregulation of WDR74 repressed the proliferation and cell cycles in CRC cells. In addition, WDR74 knockdown induced cell apoptosis and suppressed both cell metastasis and invasion. Mechanistically, WDR74 decreased the phosphorylation of β-catenin and induced nuclear β-catenin accumulation, activating the Wnt/β-catenin signaling pathway in CRC cells. Further investigation showed that blocking the Wnt/β-catenin signaling pathway by XAV-939 reversed the effects of WDR74 on cell proliferation, migration, and invasion in HCT116 cells. Overall, WDR74 induced β-catenin translocation to the nucleus and activated the Wnt/β-Catenin, thus facilitated CRC cell proliferation and metastasis. In summary, WDR74 could be a potential target for the intervention of CRC.

RBP-J promotes cell growth and metastasis through regulating miR-182-5p-mediated Tiam1/Rac1/p38 MAPK axis in colorectal cancer

BackgroundRBP-J is involved in number of cellular processes. However, the potential mechanisms of RBP-J on colorectal cancer (CRC) development have not been clearly defined. In this study, we aimed to investigate the role and molecular mechanism of RBP-J in CRC. MethodsThe expression levels of RBP-J and Tiam1 in CRC tissues and cells were evaluated by RT-qPCR or western blot. RBP-J was knocked down with sh-RBP-J or overexpressed by pcDNA3.1-RBP-J in CRC cells. Cell proliferation, migration and invasion abilities were analyzed by MTT, wound healing, and transwell assay, respectively. CHIP-qPCR, RIP and dual luciferase reporter assays were performed to confirm the interaction between miR-182-5p and RBP-J or Tiam1. Expression levels of p-p38 MAPK, p38 MAPK, Slug-1, Twist1 and MMP-9 were analyzed by western blot. G-LISA test was used to detect Rac1 activity. ResultsOur results showed that the expression of RBP-J and Tiam1 was significantly up-regulated in CRC tissues and cells. RBP-J overexpression promoted proliferation, migration and invasion of CRC cells. Moreover, RBP-J was found to directly target miR-182-5p promoter and positively regulate the Tiam1/Rac1/p38 MAPK signaling pathway in CRC cells. It was also proved that miR-182-5p can bind Tiam1 directly. Furthermore, experiments revealed that RBP-J could promote CRC cell proliferation, migration and invasion via miR-182-5p-mediated Tiam1/Rac1/p38 MAPK axis. In addition, knockdown of RBP-J reduced tumor growth and metastasis in CRC mice. ConclusionRBP-J regulates CRC cell growth and metastasis through miR-182-5p mediated Tiam1/Rac1/p38 MAPK signaling pathway, implying potential novel therapeutic targets for CRC patients.

SphK1-driven autophagy potentiates focal adhesion paxillin-mediated metastasis in colorectal cancer.

Invasion and metastasis are the main causes of colorectal cancer (CRC)‐related death. Accumulating evidence suggested that sphingosine kinase 1 (SphK1) promoted the metastasis of CRC and autophagy played an important role in SphK1 promoting the metastasis of malignancy. However, the mechanism by which SphK1‐driven autophagy promotes invasion and metastasis in CRC remains to be clarified. In the present study, immunohistochemical detection showed the expression of SphK1 and paxillin was higher in human CRC tissues than those of normal colorectal mucosal tissues, they were both associated with TNM staging, lymphatic, and distance metastasis. In addition, study of in situ tumor transplantation model in nude mice showed that the suppression of SphK1 inhibited the growth of colonic orthotopic implantation tumors and the expression of paxillin, p‐paxillin, LC3 in the tumor. So, SphK1 may promote CRC metastasis via inducing the expression of paxillin expression and its phosphorylation, in vivo. Furthermore, results of CCK8 assay, transwell and wound healing assays showed that SphK1 promoted the viability, invasion, and metastasis of CRC cells. Transmission electron microscopy detection showed that SphK1 is the key factor in autophagy induction in CRC cells. Moreover, western blot examination indicated that the expression of LC3Ⅱ/Ⅰ, paxillin, p‐paxillin, MMP‐2, and vimentin was enhanced in SphK1‐overexpressed CRC cells and suppressed in SphK1 knockdown CRC cells, meanwhile, the expression of E‐cadherin was suppressed in SphK1‐overexpressed CRC cells and enhanced in SphK1 knockdown CRC cells. Suppression of autophagy by 3MA reversed the expression of paxillin and its phosphorylation in SphK1‐overexpressed CRC cells, indicated that SphK1‐driven autophagy induced the expression of paxillin and its phosphorylation in CRC cells. Together, these findings reveal that SphK1‐driven autophagy may promote the invasion and metastasis of CRC via promoting the expression of focal adhesion paxillin and its phosphorylation.

Nerve growth factor orchestrates NGAL and matrix metalloproteinases activity to promote colorectal cancer metastasis.

Colorectal cancer (CRC) is one most cancer type of high incidence and high mortality rate. Metastasis play an important role in survival rate and life quality of colorectal cancer patients. Nerve growth factor (NGF) has been shown to be involved in the metastasis and deterioration in many cancers, but the detail mechanisms in promoting the metastasis of colorectal cancer remain unknown. In this study, we aimed to explore the mechanism of NGF promoting colorectal cancer metastasis to provide new insights for developing NGF anti-colorectal cancer drugs. We examined the expression of NGF in human colorectal cancer by immunohistochemical staining, and Western blot to evaluate the relationship between NGF and colorectal cancer metastasis. Using biochemical experiments including wound healing assay, transwell migration and invasion assay, RT-PCR, Western blot and ELISA to explore the relative mechanism of NGF promoting colorectal cancer cells metastasis in vivo. Our results found that the high expression of NGF was related with high incidence of metastasis. The binding of NGF to TrkA phosphorylated TrkA, which activated MAPK/Erk signaling pathway increasing the expression NGAL to enhance the activity of MMP2 and MMP9, promoted colorectal cancer metastasis. Our finding demonstrated that NGF increased NGAL expression to enhance MMPs activity to promoted colorectal cancer cell metastasis by TrkA-MAPK/Erk axis.

NOP14 regulates the growth, migration, and invasion of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway.

Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide. Recently, nucleolar complex protein 14 (NOP14) has been discovered to play a critical role in cancer development and progression, but the mechanisms of action of NOP14 in colorectal cancer remain to be elucidated. In this study, we used collected colorectal cancer tissues and cultured colorectal cancer cell lines (SW480, HT29, HCT116, DLD1, Lovo), and measured the mRNA and protein expression levels of NOP14 in colorectal cancer cells using qPCR and Western blotting. GFP-NOP14 was constructed and siRNA fragments against NOP14 were synthesized to investigate the importance of NOP14 for the development of colorectal cells. Transwell migration assays were used to measure cell invasion and migration, CCK-8 kits were used to measure cell activity, and flow cytometry was applied to the observation of apoptosis. We found that both the mRNA and protein levels of NOP14 were significantly upregulated in CRC tissues and cell lines. Overexpression of GFP-NOP14 markedly promoted the growth, migration, and invasion of the CRC cells HT19 and SW480, while genetic knockdown of NOP14 inhibited these behaviors. Overexpression of NOP14 promoted the expression of NRIP1 and phosphorylated inactivation of GSK-3β, leading to the upregulation of β-catenin. Genetic knockdown of NOP14 had the opposite effect on NRIP1/GSK-3/β-catenin signals. NOP14 therefore appears to be overexpressed in clinical samples and cell lines of colorectal cancer, and promotes the proliferation, growth, and metastasis of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway.

Abstract 2388: p73 - lncRNA Fer1l4 axis plays a critical role in suppression of cancer cell migration, invasion and metastasis in a p73-dependent manner via inhibition of miR-1273g-3p

Abstract p73 is a member of the p53 tumor suppressor family and exerts its tumor suppressor functions by suppressing metastasis. It is increasingly evident that long noncoding RNAs (lncRNAs) play a significant role in tumor suppression. The present study is aimed to identify novel lncRNAs that play a role in p73-mediated suppression of metastasis in colorectal cancer cells. Transcriptome analysis was performed to detect the differentially expressed lncRNAs in presence and absence of p73. Out of these, FER1L4 lncRNA was found to be significantly induced in a p73-dependent manner. p73 binding to FER1L4 promoter was confirmed through bioinformatic analysis, luciferase reporter, ChIP and site-directed mutagenesis assays. Knockdown of FER1L4 and p73 significantly increased the invasion and migration rate of colorectal cancer cells as confirmed by wound-healing assay. Knockdown of FER1L4 decreased the expression of E-cadherin and increased the expression of prominent EMT markers such as N-cadherin, Snail, Vimentin and Fibronectin. FER1L4 causes a G2/M cell cycle arrest in a p73-dependent manner in HCT116p53-/-p73+/+ cells and upon FER1L4kd, normal cell cycle progression was observed. Annexin V/PI and TUNEL apoptosis assays revealed that FER1L4 induced apoptosis in HCT116p53-/-p73+/+ cells with increase in time-dependent treatment of etoposide and FER1L4kd inhibited apoptosis even in the presence of p73. The protein expression level of pro-apoptotic genes such as Bad, Bax, Bik, Bim, BID, Bak and PUMA decreased upon FER1L4kd and p73kd, confirming that FER1L4 plays a role in p73 mediated apoptosis and cell cycle arrest. FER1L4 also sponges the expression of miR-1273g-3p, thus inhibiting its oncogenic role. RNA-In situ hybridization (RNA-ISH) confirmed the decreased expression of p73 and FER1L4 mRNA in 30 human metastatic CRC tissues as compared to 30 human non-metastatic CRC tissues. Taken together, we provide conclusive proof that p73 exerts its anti-metastatic function by inducing the expression of lncRNA FER1L4 in response to genotoxic stress. Citation Format: Apoorva Uboveja, Yatendra Kumar Satija, Fouzia Siraj, Chanchal Bareja, Daman Saluja. p73 - lncRNA Fer1l4 axis plays a critical role in suppression of cancer cell migration, invasion and metastasis in a p73-dependent manner via inhibition of miR-1273g-3p [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 2388.

Snail enhances arginine synthesis by inhibiting ubiquitination‐mediated degradation of ASS1

Snail is a dedicated transcriptional repressor and acts as a master inducer of EMT and metastasis, yet the underlying signaling cascades triggered by Snail still remain elusive. Here, we report that Snail promotes colorectal cancer (CRC) migration by preventing non-coding RNA LOC113230-mediated degradation of argininosuccinate synthase 1 (ASS1). LOC113230 is a novel Snail target gene, and Snail binds to the functional E-boxes within its proximal promoter to repress its expression in response to TGF-β induction. Ectopic expression of LOC113230 potently suppresses CRC cell growth, migration, and lung metastasis in xenograft experiments. Mechanistically, LOC113230 acts as a scaffold to facilitate recruiting LRPPRC and the TRAF2 E3 ubiquitin ligase to ASS1, resulting in enhanced ubiquitination and degradation of ASS1 and decreased arginine synthesis. Moreover, elevated ASS1 expression is essential for CRC growth and migration. Collectively, these findings suggest that TGF-β and Snail promote arginine synthesis via inhibiting LOC113230-mediated LRPPRC/TRAF2/ASS1 complex assembly and this complex can serve as potential target for the development of new therapeutic approaches to treat CRC.

Circular RNA circFAT1(e2) Promotes Colorectal Cancer Tumorigenesis via the miR-30e-5p/ITGA6 Axis.

circRNAs (circular RNAs) are a family of noncoding RNAs and have diverse physiological and pathological functions. However, the functions and mechanisms of circRNAs in the development and progression of colorectal cancer (CRC) remain largely unknown. Here, we aimed to explore the functions and roles of circFAT1(e2) in CRC. qRT-PCR revealed that circFAT1(e2) in CRC tumor tissues was upregulated compared with that in adjacent normal tissues and was also upregulated in CRC cell lines. Small interfering RNAs (siRNAs) against circFAT1(e2) were used to decrease the expression of circFAT1(e2) in HCT116 and RKO cells in vitro. The roles of circFAT1(e2) in CRC cell metastasis and proliferation were then determined by transwell and CCK-8 assays. The results showed that circFAT1(e2) silencing markedly suppressed CRC growth. Moreover, we identified circFAT1(e2) as a promoter of CRC metastasis. Knockdown of circFAT1(e2) evidently reduced HCT116 and RKO cell migration and invasion. Furthermore, the regulatory relationship between circFAT1(e2) and its target miRNAs was verified by a luciferase reporter assay. We demonstrated that circFAT1(e2) could sponge miR-30e-5p, which regulated the expression level of integrin α6 (ITGA6), the downstream target gene of miR-30e-5p. Rescue assays demonstrated that knockdown of miR-30e-5p enhanced CRC proliferation and migration via ITGA6. Taken together, our results reveal the novel oncogenic roles of circFAT1(e2) in CRC through the miR-30e-5p/ITGA6 axis.

TSHZ3 functions as a tumor suppressor by DNA methylation in colorectal cancer

ObjectiveTeashirt zinc finger homeobox 3 (TSHZ3) is currently reported to be aberrantly expressed in several tumors, but the detailed functions and epigenetic mechanisms of TSHZ3 in colorectal cancer (CRC) remain unclear. Materials and methodsIn this study, the TSHZ3 expression in 118 CRC and normal adjacent tissues (NATs) was evaluated, and the methylation status of the TSZH3 promoter region in CRC tissues and cell lines was also analyzed. ResultsThe results of PCR analysis showed that TSHZ3 was significantly down-regulated in CRC tissues, and patients with low TSHZ3 levels had a poorer 5-year overall survival (OS) rate. Analyzing the promoter sequence (−1000∼0) by MethPrimer, TSHZ3 promoter was found to harbor abundant of CpG islands. The methylation specific PCR (MSP) analysis presented a relatively hypermethylated status of THSZ3 promoter in CRC samples. The data of MSP and bisulfite sequencing PCR (BSP) also confirmed that CpG sites of TSHZ3 promoter were methylated in CRC cells, and the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-Aza) could effectively restored the TSHZ3 expression in vitro. Functionally, the proliferation, apoptosis and metastasis of CRC cells were regulated by TSZH3 over-expression, and the suppressing effects of TSHZ3 in CRC were also confirmed in a xenograft mouse model. ConlusionsOur results indicated that promoter methylation was one of the mechanisms contributing to the down-regulation of TSHZ3 in CRC, and TSZH3 might served as a potential tumor suppressor gene in the development and progression of CRC.

Cyr61 from adipose-derived stem cells promotes colorectal cancer metastasis and vasculogenic mimicry formation via integrin αV β5.

Adipose‐derived stem cells (ADSCs) play a vital role in colorectal cancer (CRC) progression, but the mechanism remains largely unknown. Herein, we found that ADSCs isolated from CRC patients produced more cysteine‐rich 61 (Cyr61) than those from healthy donors, and the elevated serum Cyr61 levels were associated with advanced TNM stages. Moreover, serum Cyr61 displayed a better diagnostic value for CRC compared to carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19‐9). Mechanistically, integrin αVβ5 was identified as the functional receptor by which Cyr61 promotes CRC cell metastasis in vitro and in vivo by activating the αVβ5/FAK/NF‐κB signaling pathway. In addition, Cyr61 promotes vasculogenic mimicry (VM) formation, thereby promoting tumor growth and metastasis through a αVβ5/FAK/HIF‐1α/STAT3/MMP2 signaling cascade. Histologically, xenografts and clinical samples of CRC both exhibited VM, which was correlated with HIF‐1α and MMP2 activation. Notably, we demonstrated the synergistic effect of combined anti‐VM therapy (integrin αVβ5 inhibitor) and anti‐VEGF therapy (bevacizumab) in patient‐derived xenograft models. Further investigation showed that CRC cell‐derived exosomal STAT3 promoted Cyr61 transcription in ADSCs. These findings indicate that Cyr61 derived from ADSCs plays a critical role in promoting CRC progression via integrin αVβ5 and provides a novel antitumor strategy by targeting Cyr61/αVβ5.

Circ_0084615 is an oncogenic circular RNA in colorectal cancer and promotes DNMT3A expression via repressing miR-599

BackgroundCircular RNAs (circRNAs) are implicated in modulating cancer progression, exerting a pro- or anti-cancer effect. This work is aimed to probe the biological function of circ_0084615 in colorectal cancer (CRC) and its underlying mechanism. MethodsCirc_0084615 was selected from two circRNA microarray datasets (GSE138589 and GSE142837). Circ_0084615, microRNA (miR)-599 and DNA methyltransferases 3A (DNMT3A) mRNA expression in CRC tissues and cell lines were examined by qRT-PCR. The relationship between circ_0084615 expression level and clinical features were analyzed with chi-square test. Circ_0084615 knockdown model was constructed by siRNA in two CRC cell lines. The biological functions of circ_0084615 in CRC cells were evaluated by CCK-8 and Transwell experiments. The effect of circ_0084615 on CRC cell metastasis in vivo was examined with lung metastasis model of nude mice. Dual luciferase reporter gene assay was used to determine whether circ_0084615 and miR-599, and miR-599 and DNMT3A interacted with each other. Western blot was employed to examine the regulatory effects of circ_0084615 and miR-599 on DNMT3A protein expression in CRC cells. ResultsCirc_0084615 was up-regulated in CRC and was correlated with poor overall survival rate and advanced clinical stage of CRC patients. Functional assays validated that depletion of circ_0084615 impeded CRC cell proliferation, migration and invasion. Circ_0084615 acted as a molecular sponge for miR-599 to repress its expression. DNMT3A was a downstream target of miR-599. Functional compensation experiments showed that miR-599 inhibitors partially counteracted the the biological effects of silencing circ_0084615 on CRC cells. ConclusionsCirc_0084615 is a tumor-promoting circRNA in CRC that functions as a competing endogenous RNA to regulate DNMT3A expression via sponging miR-599. Our research provides a potential therapeutic target for CRC patients.

HSP90-dependent PUS7 overexpression facilitates the metastasis of colorectal cancer cells by regulating LASP1 abundance

BackgroundPseudouridine synthase (PUS) 7 is a member of the PUS family that catalyses pseudouridine formation. It has been shown to be involved in intellectual development and haematological malignancies. Nevertheless, the role and the underlying molecular mechanisms of PUS7 in solid tumours, such as colorectal cancer (CRC), remain unexplored. This study elucidated, for the first time, the role of PUS7 in CRC cell metastasis and the underlying mechanisms.MethodsWe conducted immunohistochemistry, qPCR, and western blotting to quantify the expression of PUS7 in CRC tissues as well as cell lines. Besides, diverse in vivo and in vitro functional tests were employed to establish the function of PUS7 in CRC. RNA-seq and proteome profiling analysis were also applied to identify the targets of PUS7. PUS7-interacting proteins were further uncovered using immunoprecipitation and mass spectrometry.ResultsOverexpression of PUS7 was observed in CRC tissues and was linked to advanced clinical stages and shorter overall survival. PUS7 silencing effectively repressed CRC cell metastasis, while its upregulation promoted metastasis, independently of the PUS7 catalytic activity. LASP1 was identified as a downstream effector of PUS7. Forced LASP1 expression abolished the metastasis suppression triggered by PUS7 silencing. Furthermore, HSP90 was identified as a client protein of PUS7, associated with the increased PUS7 abundance in CRC. NMS-E973, a specific HSP90 inhibitor, also showed higher anti-metastatic activity when combined with PUS7 repression. Importantly, in line with these results, in human CRC tissues, the expression of PUS7 was positively linked to the expression of HSP90 and LASP1, and patients co-expressing HSP90/PUS7/LASP1 showed a worse prognosis.ConclusionsThe HSP90-dependent PUS7 upregulation promotes CRC cell metastasis via the regulation of LASP1. Thus, targeting the HSP90/PUS7/LASP1 axis may be a novel approach for the treatment of CRC.