Mechanism of colorectal cancer cell exosome-derived circ_0044366 in regulating release of MMP2 in fibroblasts and promoting metastasis of colorectal cancer

Breast cancer is the second most common fatal malignancy of women in the United States. Matrix metalloproteinases (MMPs) have been implicated in virtually all stages of breast cancer. MMP2 was identified as part of a subset of genes upregulated in breast to lung metastases (Minn). These studies focused on epithelial derived MMP2, however, a host of other sources have emphasized the importance of stromal cell derived MMP2 in breast cancer growth. We have directed our studies to investigate the role of MMP2 from stromal cells in tumor outgrowth at the secondary site. We hypothesize that stromal MMP2 at the secondary site contributes to metastatic progression. Spontaneous metastasis models have shown that MMP2−/− mice demonstrate reduced lung metastatic lesions compared to wildtype (WT) animals. In an experimental metastasis model using luciferase labeled tumor cells, we have found that MMP2−/− mice have a significant reduction in lung tumor outgrowth compared to WT animals as measured by bioluminescent imaging. The reduction in growth only manifests approximately seven days post inoculation, suggesting that decreased tumor growth is due to defects in tumor outgrowth and not seeding ability or survival. Immunohistochemical staining of Ki67 revealed that MMP2 selectively facilitates the proliferation of tumor cells in the lung. MMP2 in our model is primarily localized to stromal cells surrounding lung tumor nodules. We determined by dual labeling for MMP2 and vimentin or αSMA positive areas that fibroblasts were the major cellular source of MMP2. Hematopoietic cells (CD45 positive areas) and endothelial cells (CD31 positive areas) showed minimal expression of MMP2 and were ruled out as a significant source. To determine if there is a direct role of MMP2 in tumor cell proliferation, we took an in vitro approach and analyzed tumor cell proliferation after treatment with recombinant protein or fibroblast conditioned media. There was no difference in two-dimensional (2D) proliferation after either treatment, suggesting that MMP2 plays an indirect role in the proliferation of tumor cells. In our present studies, we investigate the role of MMP2 in fibroblasts and the effect this has on the outgrowth of tumor cells in the lung. A knock down approach was used to assess the impact of MMP2 on 2D fibroblast proliferation by cell counting. Our results demonstrated no difference in 2D proliferation between control and MMP2 knockdown fibroblasts. We have developed a 3D co-culture system to evaluate the role of MMP2 in fibroblast promotion of tumor spheroid proliferation. Our present data suggests that MMP2 mediated recruitment and functionality of tumor associated fibroblasts promotes tumor outgrowth via proliferation. Results from these experiments will lead to new insights on the role of MMP2 in fibroblasts and how these cells then enable a permissive environment for tumor outgrowth at secondary sites. Minn, AJ et al. Nature. 2005 Jul 28;436(7050):518-24 Citation Format: Andreia Bates, Barbara Fingleton. The role of fibroblast MMP2 in breast to lung metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5062. doi:10.1158/1538-7445.AM2013-5062

To investigate the role of miR-26b in the invasion and metastasis of colorectal cancer. Data of public chip databases were extracted to analyze the relationship between miR-26b expression and lymph node metastasis. Two types of colorectal cancer cell lines, Caco2 and DLD1, were selected, and the miR-26b-high colorectal cancer cell line was constructed using the method of lentivirus infection. The effects of up-regulating miR-26b expression on the invasion and metastasis of colorectal cancer cells were analyzed by Transwell migration and invasion experiment and wound healing assay. The effect of up-regulating miR-26b expression on stem cell phenotype of colorectal cancer cells was analyzed by sphere-formation assay. The microarray detection results showed that the expression of miR-26b in tumor tissues of patients with lymph node metastasis was significantly higher than those without lymph node metastasis[(12.04±0.20) vs. (11.31±0.19), t=2.646, P = 0.010]. In the in vitro experiment section, the Transwell experiment results showed that the number of invasive cells [(16.40±1.36) vs. (3.80±0.86), t=7.814, P=0.000] and migrating cells [(33.40±2.93) vs. (8.80±2.40), t=6.505, P=0.000] in miR-26b-high colorectal cancer cells was significantly higher as compared to miR-26b-low cells(all P<0.05). Would healing assay also confirmed that the migration speed of miR-26b-high colorectal cancer cells was significantly accelerated. Both the rate and the density of sphere formation were higher in miR-26b-high colorectal cancer cells than those in miR-26b-low colorectal cancer cells [Caco2:(168.3±11.7) vs. (54.2±10.8), t=7.185,P=0.002; DLD1:(4 076.0±409.8) vs.(1 613.0±210.1), t=5.349, P=0.006]. miR-26b may promote the invasion and metastasis of colorectal cancer by accelerating the migration and invasion of colorectal cancer cells and enhancing the stem cell phenotype of tumor cells.

Recent studies highlight long non-coding RNAs (lncRNAs) as key regulators of cancer biology that contribute to carcinogenesis. The lncRNA HOXA transcript at the distal tip (HOTTIP) is involved in the development of several cancers. Previous studies demonstrated that HOTTIP could promote colorectal cancer (CRC) cell proliferation via silencing of p21 expression. However, the potential role of HOTTIP in CRC metastasis has not yet been discussed. Here, we found that HOTTIP level was significantly higher in CRC than in corresponding adjacent normal tissues, and patients with a larger tumor size, advanced pathological stage, or distant metastasis had higher HOTTIP expression. Moreover, silencing HOTTIP expression by siRNA or shRNA could inhibit CRC cell migration and invasion in vitro and in vivo, whereas HOTTIP overexpression promoted cell metastasis, as documented in the SW480 cell lines. Mechanistic analyses indicated that HOTTIP regulates CRC cell metastasis partly through the downregulation of tumor suppressor DKK1 expression. Collectively, our results suggest that tumor expression of lncRNA HOTTIP plays an important role in CRC metastasis. HOTTIP may serve as a candidate biomarker in this disease.

The single nucleotide polymorphism (SNP), -397G > T (rs13278062) polymorphism, in the promoter of Death Receptor 4 (DR4) had been reported to be associated with a significantly increased risk for bladder cancer. However, the association of this SNP with the risk of colorectal cancer has not been reported. In this study, we performed a case-control study in 1,078 colorectal cancer patients and 1,175 matched healthy controls to evaluate the association of the potential functional genetic variants in DR4 with risk and survival of colorectal cancer. PCR-TaqMan were used to genotype the rs13278062, rs1000294 and rs2235126 polymorphisms. We found that subjects carrying the rs13278062 GT/TT genotypes had a significantly lower risk and increased survival time when compared to the GG genotype. We also constructed the rs13278062 GT/TT genotype in SW480 and SW620 cells (rs13278062 is GG in both cell lines) with the CRISPR/Cas9 system. Flow cytometry experiments showed that the rs13278062 TT genotype promoted apoptosis in colorectal cancer cells. In vitro and in vivo experiments established that the rs13278062 G to T mutation inhibited carcinogenesis and metastasis of colorectal cancer. Chromatin immunoprecipitation (ChIP) assays revealed that the rs13278062 G > T polymorphism altered the binding affinity of the transcription factors Sp1/NF1 to the rs13278062 mutation region. Immunohistochemistry, western blot, and qPCR corroborated that the rs13278062 GT/TT genotypes increased the expression of DR4 protein in colorectal cancer tissues and cells. In conclusion, these findings indicate that DR4 mediated progression, invasion, metastasis and survival of colorectal cancer via the Sp1/NF1 switch axis on genomics locus.

Colorectal cancer is a common malignancy and a common cause of tumor-related death. Long non-coding RNAs (lncRNAs) have become an important regulatory factor and tissue specific biomarker for a variety of cancers, including colorectal cancer. Recent evidence indicates that the novel lncRNA SLC30A10 plays an important role in tumor progression and metastasis. However, its role and molecular mechanisms in colorectal cancer are unclear. SLC30A10 expression was detected in 12 colorectal cancer and adjacent normal tissues by quantitative reverse transcription PCR. Insights into the underlying mechanisms of competitive endogenous RNAs (ceRNAs) were determined by transwell assay, CCK8 assay, and luciferase assay. SLC30A10 was down-regulated in colorectal cancer tissues and cell lines, and its low expression was positively correlated with colorectal cancer progression and metastasis. Functionally, SLC30A10 depletion promotes cell proliferation and migration in colorectal cancer cells, while SLC30A10 overexpression has the opposite effect. Bioinformatics prediction and luciferase assay indicated that miR-21c is a direct target of SLC30A10, which plays the role of ceRNA in regulating colorectal cancer metastasis. In addition, miR-21c specifically targets APC gene. Our findings suggest that reduced expression of SLC30A10 is associated with aggressive tumor phenotypes and poor patient outcomes in colorectal cancer. SLC30A10 inhibits colorectal cancer progression and metastasis by acting as a ceRNA for miR-21c to regulate APC expression, suggesting that SLC30A10 may serve as a potential prognostic biomarker and anti-metastatic therapeutic target for colorectal cancer.

Background: Colorectal Cancer (CRC) is the second highest cause of cancer related deaths in United States. Metastatic dissemination of CRC cells to distant organs (primarily to the liver and lungs) makes the disease more lethal and incurable. Inappropriate cell survival signaling mechanisms contribute to successful colonization of cells at metastatic sites. Akt is a major cell survival-signaling node, which undergoes aberrant activation in various types of cancer. The Akt family has three members: Akt1, Akt2 and Akt3 which share structural homology and have similar mechanism of activation; however, differences in the phenotype of Akt1, Akt2 and Akt3 knock out mice suggest that they have non-redundant functional characteristics. Recently Akt2 has been identified as a significant contributor to colon cancer malignancy in addition to Akt1. Earlier reports have shown that knockdown of Akt2 results in reduced liver metastasis of CRC cells, however there are no studies related to the mechanism responsible for the reduction in metastasis. We observed a significant increase in the expression of Akt2 (microarray analysis) in liver metastatic samples formed by highly metastatic IGF1R-dependent CRC cells in comparison to the primary tumor. This was further confirmed by western blot and immunohistochemistry (IHC) analysis. Additionally, correlative studies using human primary colon carcinoma and corresponding liver metastatic samples showed a significant (p&amp;lt;0.001) increase in the expression of Akt2. These preliminary data led us to hypothesize that Akt2 may be playing a role in regulating cell survival and metastasis of CRC cells. Materials and Methods: IGF1R-dependent CRC cells (GEO and CBS) were used for the study. Two doxycycline inducible shRNAs for Akt2 were introduced by lentiviral transfection. Non-Targeting shRNA was used as control. Results: shRNA-mediated knockdown of Akt2 led to reduction in the expression of XIAP and survivin. We and others have shown that these IAPs (XIAP and survivin) are implicated to play an important role in cancer cell survival and metastasis. Furthermore, loss of Akt2 resulted in loss of pBad (S136), which is an essential cell survival marker. Loss of pBad results in increased cell death by PARP activation. Akt2 knockdown also affects cell survival by increasing p21 and decreasing the expression of cyclin E. PCR array analysis for metastatic genes revealed that a metastatic suppressor gene MTSS1 was 9 fold increased on knockdown of Akt2. It has been shown that MTSS1 inhibits metastasis by affecting cell junction assembly and stability. Conclusion: We have shown that loss of Akt2 promotes cell death and inhibits cell proliferation. This is the first study, which suggests the mechanism of Akt2 mediated cell death by XIAP and survivin downregulation. These results suggest that Akt2 inhibits metastasis by increasing the expression of MTSS1. Citation Format: Ekta Agarwal. Role of Akt2 in cell survival and metastasis of colorectal cancer (CRC) cells. [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 4139. doi:10.1158/1538-7445.AM2015-4139

Background: Colorectal cancer (CRC) is one of the most prevalent cancers. In case of CRC with distant metastasis, it is initially treated by chemotherapy, and oxaliplatin is often used in first-line regimen. However, chemoresistance often becomes an issue and it remains unclear. We previously reported that RNA editing enzyme ADAR1 causes stemness (JCI Insight, 2018) and induces the poor prognosis in the liver metastasis of CRC (Scientific Reports, 2023). In this study, we cleared the role of RNA editing in chemoresistance of CRC. Methods: We performed immunohistochemistry (IHC) of ADAR1 in 151 cases of CRC with distant metastasis. In vitro, we analyzed ADAR1 expression and RNA editing levels in both cancer cells and macrophages with the CRC cell lines and differentiated THP-1(dTHP-1). RNA-sequence was performed to compare wild type and ADAR1 knockdown dTHP-1 co-cultured with CRC cells respectively. In vivo, xenograft mouse models were made by mixing CRC cells and macrophages, and treatment experiment was conducted using oxaliplatin. Results: IHC scoring showed that high expression of ADAR1 in CRC cancer cells relates the poor prognosis (p&amp;lt;0.01). Moreover, high expression of ADAR1 in the stromal mononuclear cells relates the poor prognosis (p=0.032) and resistance to oxaliplatin (p&amp;lt;0.01). Additionally, there was a positive correlation between the expression levels of ADAR1 in macrophages in tumor tissue and cancer cells (ρ=0.74, p &amp;lt; 0.001). In vitro, macrophages increased the intensity of ADAR1 expression when indirectly co-cultured with CRC cells (p&amp;lt;0.01). These results suggested that ADAR1 might be supplied from CRC cells to macrophages through some liquid factors. We focused on extracellular vesicles (EVs). Western blotting revealed that EVs from CRC cells contained ADAR1. When these EVs were added to macrophages, ADAR1 expression increased in the macrophages. ADAR1 increased RNA editing levels in macrophages, including AZIN1 and GLI1. Next, RNA-sequence analysis showed an enrichment of pathways associated with distant tumor metastasis in macrophages with high ADAR1 expression (p&amp;lt;0.05). Additionally, an increase in SPP1 gene expression, which is involved in tumor immune suppression, was observed. In vivo, xenograft mouse models were made, knockdown of ADAR1 in macrophages enhanced the effect of oxaliplatin to inhibit tumor growth (p&amp;lt;0.01). Treatment model mouse was made using oxaliplatin in combination with filgotinib, JAK inhibitor that reduces ADAR1 expression. The results showed a significant inhibition of tumor growth in the oxaliplatin plus JAK inhibitor group (p=0.0197). Conclusions: We showed that ADAR1-high macrophages are induced by stimulation from CRC cells via EVs and it may cause chemoresistance. Cancer cells and macrophages dual-targeted RNA editing suppression therapy was considered a promising treatment option. Citation Format: Kazuya Moriwake, Kunitoshi Shigeyasu, Hibiki Umeda, Toshiaki Takahashi, Masashi Kayano, Kaori Nitta, Shunsuke Nakamura, Masafumi Takahashi, Yuya Sakurai, Yuki Matsumi, Yoshitaka Kondo, Fuminori Teraishi, Hiroshi Tazawa, Toshiyoshi Fujiwara. ADAR1-high tumor-associated macrophages induce drug resistance and are therapeutic targets in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4105.

ABSTRACTOur previous study has demonstrated that knockdown of Grainyhead-like 2(GRHL2) in colorectal cancer (CRC) cells inhibited cell proliferation by targeting ZEB1. This study aimed at researching whether knockdown of GRHL2 promoted CRC progression and metastasis via inducing epithelial-mesenchymal transition (EMT). GRHL2-upregulated SW-620/GRHL2+ and GRHL2-knockdown HCT116/GRHL2-KD, HT29/GRHL2-KD cells and their control cells were generated. The morphological changes after overexpression and knockdown GRHL2 were observed. qRT-PCR, Western blotting, and Immunofluorescence were used to detect EMT markers: E-cadherin, Vimentin, p-catein, ZO-1 and ZEB1 expression. Then, sh-ZEB1 was transfected to GRHL2 knockdown cells to research the relationship between GRHL2 and ZEB1. Transwell and wound healing assays were further performed to detect the impact of GRHL2 on invasion and migration in vitro. CRC cells were injected into mice tail vein to verify the impact of GRHL2 on CRC metastasis. Morphological change of mesenchymal-epithelial transition (MET) could be observed in SW620/GRHL2+ cell. The expression of epithelial markers: E-cadherin, β-catenin, ZO-1 were up-regulated, while mesenchymal markers: Vimentin was decreased. Meanwhile, opposite EMT morphological change could be observed in HCT116/GRHL2-KD cell, accompanied by reverse change of E-cadherin, β-catenin, ZO-1, and Vimentin. The expression level of GRHL2 and ZEB1 was found negative in both SW620/GRHL2+ and HCT116/GRHL2-KD cells. Knockdown of ZEB1 by siRNA in HCT116/GRHL2-KD and HT29/GRHL2-KD could upregulate expression of E-cadherin and GRHL2. GRHL2 knockdown also promoted migration, invasion in vitro and CRC metastasis in mice model. In conclusion, GRHL2/ZEB1 axis inhibits CRC progression and metastasis via oppressing EMT.

Survival signaling is critical for the metastatic program of cancer cells. The current study investigated the role of Akt survival proteins in colorectal cancer (CRC) metastasis and explored potential mechanisms of Akt-mediated metastasis regulation. Using an orthotopic implantation model in mice, which uniquely recapitulates the entire multistep process of CRC metastasis, combined with an inducible system of short hairpin RNA-mediated Akt isoform knockdown in human CRC cells, our studies confirm a role of Akt2 in CRC cell dissemination to distant organs in vivo. Akt2 deficiency profoundly inhibited the development of liver lesions in mice, whereas Akt1 had no effect under the experimental conditions used in the study. Array analysis of human metastatic genes identified the scaffolding protein metastasis suppressor 1 (MTSS1) as a novel Akt2-regulated gene. Inducible loss of Akt2 in CRC cells robustly upregulated MTSS1 at the messenger RNA and protein level, and the accumulated protein was functionally active as shown by its ability to engage an MTSS1-Src-cortactin inhibitory axis. MTSS1 expression led to a marked reduction in levels of functional cortacin (pcortactin Y421), an actin nucleation-promoting factor that has a crucial role in cancer cell invasion and metastasis. MTSS1 was also shown to mediate suppressive effects of Akt2 deficiency on CRC cell viability, survival, migration and actin polymerization in vitro. The relevance of these findings to human CRC is supported by analysis of The Cancer Genome Atlas (TCGA) and NCBI GEO data sets, which demonstrated inverse changes in expression of Akt2 and MTSS1 during CRC progression. Taken together, the data identify MTSS1 as a new Akt2-regulated gene, and point to suppression of MTSS1 as a key step in the metastasis-promoting effects of Akt2 in CRC cells.

Deranged metabolism is a hallmark of cancer, playing a significant role in driving the disease process. One such example is the induction of carcinogenesis by the oncometabolite D-2 hydroxyglutarate (D-2HG), which is produced by the mutated enzyme isocitrate dehydrogenase (IDH) occurring in subsets of leukaemias and brain tumours. The oncogenic property of D-2HG appears to stem from its ability to interfere with the activities of α-ketoglutarate-dependent dioxygenases, including the Jumonji family histone demethylases. Here, we find in colorectal cancer cells that even in the absence of IDH mutation, the levels of D-2HG and its enantiomer L-2HG were elevated through glutamine anaplerosis. D-2HG, but not L-2HG, increased the trimethylation of histone H3 lysine 4 of the promoter region of ZEB1, a master regulator of epithelial-mesenchymal transition (EMT), and increased the expression of the ZEB1 gene to directly induce EMT in colorectal cancer cells. EMT promotes the ability of cancer cells to invade the local tissue and enter into the bloodstream, leading to distant organ metastasis. D-2HG levels were elevated in colorectal cancer specimens, particularly in those associated with distant metastasis, supporting the observations in vitro and implicating the contribution of D-2HG in metastasis, the major cause of death in this disease.

Introduction: Metastatic disease is responsible for 90% of colorectal cancer (CRC) deaths. Studies suggest that metastasis is closely associated with the presence of CRC tumor-initiating cells (Co-TIC) and their interaction with the lymph node (LN) stromal microenvironment. Prior to developing extra-nodal metastasis, these cells acquire a chemotherapy-resistant phenotype developing genetic alterations making them resistant to conventional treatments. In addition to cell-cell contact and secreted molecules, a recently discovered means of intercellular signaling is the exchange of extra-cellular vesicles. These microvesicles (MVs) carry complex biological information, including mRNA, miRNA, as well as soluble and transmembrane proteins that can affect the behavior of target cells. MVs have been detected in patient specimens with diverse malignancies and may play a role in communication between the LN stromal microenvironment and Co-TIC. We hypothesize that MVs are involved in intracellular trafficking between LN stromal cells and CRC cells promoting tumor formation and distant organ metastasis. Methods: MVs released by human mesenteric LN stromal cells (LNSC) derived from surgical specimens and the established LN stromal cell line (HK cell) were isolated using differential centrifugation and gradient purification. The MVs were visualized using GFP-HK cell and RFP-HT-29 cell (CRC cell line) and florescence microscopy. The functional properties of LN stromal MVs and their effect on CRC proliferation and metastasis was analyzed using established in vitro co-culture models and a humanized orthotopic intra-rectal (IR) injection mouse model, tracked by bioluminescent imaging (BLI). Results: A 100,000 g pellet containing MVs derived from LNSC and HK cells have a similar size profile when analyzed by NanoSight. Budding CD63-RFP tagged MVs were released by LNSC and HK cells and uptake by GFP tagged CRC cells was confirmed through time-lapse experiments using deconvoluting microscopy. When HK cell or LN stromal cell-derived MVs were co-cultured with HT-29 cells in vitro, they supported HT-29 cell growth at a similar level as that of HK cell or LN stromal cell conditioned media, respectively. By adding LNSC- or HK-derived MVs to HT-29-Luc cells or patient derived CRC cells (CRC-Pt-Luc cells) in our IR model, we demonstrated that MVs enhanced CRC tumor growth as well as distant organ metastasis in vivo. Conclusion: MVs isolated from LNSCs traffic between the stromal cells and CRC cells. These MVs promote tumor formation and distant organ metastasis in vivo suggesting that they play a crucial role in the communication between the LN stromal microenvironment and CRC cells. Further analyzing the functional properties of effector MV RNAs may help identify novel targetable candidates for therapeutic strategies that target CRC metastasis using our unique patient derived orthotopic mouse model. Citation Format: Xin Zhang, Ryan Sullivan, Nathan Hite, Grace Maresh, Linh Hellmers, Zhen Lin, Erik Flemington, Carlos Salomon, Heather Green, David Margolin, Li Li. The critical role of lymph node stromal cell-derived microvesicles in colorectal cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1558.

This study aimed to investigate the expression of PEBP4 protein in colorectal carcinoma tissues and its correlation with the clinical pathology of colorectal cancer and to investigate the relationship between PEBP4 expression and the invasion and metastasis of colorectal cancer cells, which could provide an experimental basis for future biological treatments of human colorectal cancer. RT-PCR and western blot methods were applied to detect the mRNA and protein expressions, respectively, of PEBP4 in colorectal cancer tissues and normal pericarcinoma tissues, and their correlations with the tumorigenesis and development of colorectal cancer, as well as its clinical pathology, were analyzed. Using the RNA interference technology, the expression of PEBP4 was knocked down in the human colorectal cancer cell HCT116, and the changes of the invasion capability of HCT116 were monitored. The positive mRNA expression rate of PEBP4 in colorectal cancer tissue was significantly higher than that in the normal pericarcinoma tissue (p < 0.05). Also, the positive expression rate in the cancer tissues from patients with positive lymph node and distant metastasis was significantly higher than that from the patients negative for lymph node and distant metastasis (p < 0.05). The positive expression rate of PEBP4 in the cancer tissues from the patients in early stages (I, II) was significantly lower than the expression rate in patients in advanced stages (III, IV) (p < 0.05). A lower degree of differentiation in colorectal cancer corresponded to a higher positive mRNA expression rate of PEBP4 (p < 0.05). However, this was independent of the patient's gender, age, and tumor size (p > 0.05). In colorectal cancer tissue, the expression of PEBP4 protein was consistent with its mRNA. Namely, PEBP4 protein expression in colorectal cancer tissues was significantly higher than that in the normal pericarcinoma tissues (p < 0.05), the expression in the cancer tissues from the patients with positive lymph node and distant metastasis was significantly higher than that from the patients who were negative for these metastases (p < 0.05), and a lower degree of differentiation in colorectal cancer corresponded to a higher TNM staging along with a higher PEBP4 protein expression (p < 0.05). After HCT116 cells transfected with PEBP4 siRNA, they showed a significantly lower expression level of PEBP4 protein (p < 0.05), and the number of cells that passed through the Transwell chamber was significantly lower compared to the non-transfected or the transfected controls (p < 0.05). The over-expression of PEBP4 protein may be related to the tumorigenesis, development, metastasis, and invasion of colorectal cancer.

Multiregion whole-exome sequencing of matched primary and metastatic tumors revealed genomic heterogeneity and suggested polyclonal seeding in colorectal cancer metastasis

ABSTRACTWe aimed at the effects of long non-coding RNA (lncRNA) SNHG5 on proliferation, metastasis and migration of colorectal cancer (CRC) cells. We also investigated regulatory relationships among miR-132-3p, SNHG5 and CREB5 and their roles in CRC. 25 pairs of samples containing CRC tissues and matched para-tumor tissues were obtained to examine SNHG5, miR-132-3p and CREB5 expression by qRT-PCR or Western blot. The targeted relationship between miR-132-3p and SNHG5 or CREB5 was confirmed by dual luciferase report assay as well as RNA pull down assay. The expression of SNHG5, miR-132-3p and CREB5 in CRC cells were regulated by cell transfection. CRC cellular proliferation was assayed by CCK-8 and meanwhile flow cytometry was adopted to observe apoptosis. Metastasis and migration of CRC cells were determined respectively by means of Transwell assay and scratch test. The effects of SNHG5 on CRC were researched in vivo, too. SNHG5 or CREB5 was up-regulated in CRC tissues and cells, whereas miR-132-3p was down-regulated. Overexpression of SNHG5 and CREB5 resulted in the enhancement of proliferation, metastasis, migration and the inhibition of apoptosis in CRC cells, while miR-132-3p led to the opposite result. LncRNA SNHG5 promoted proliferation, migration and metastasis of CRC cells but inhibited apoptosis by modulating miR-132-3p/CERB5.

The aim was to determine the diagnostic value of epithelial-mesenchymal transition typing of circulating tumour cells (CTCs) in colorectal cancer (CRC). Peripheral blood samples were collected from 51 CRC patients before anti-tumour treatment from April 2016 to June 2018 at the Peking University Shenzhen Hospital. The blood samples were analysed using the CanPatrol CTC typing technique (SurExam, Guangzhou, China), which combines nanomembrane enrichment with mRNA in situ hybridization. Based on the marker expression, the CTCs were classified into epithelial, epithelial mesenchymal and mesenchymal (M-CTC) types. The correlation between the CTC counts and clinicopathological characteristics such as gender, age, TNM stage, lymph node metastasis and distant metastasis were analysed by univariate and multivariate Cox regression models. The overall survival and progression-free survival (PFS) of patients demarcated by CTC typing were analysed using the Kaplan-Meier method and log-rank tests. M-CTCs were detected more frequently in patients with lymph node metastasis (N2 81.8%; N1 72.7%; N0 38.9%) as well as distant metastasis (M0 50%; M1a 81.25%; M1b 85.7%) compared to those without metastasis. In addition, the presence of M-CTCs was significantly correlated with distant metastasis (P<0.01). Univariate analysis showed that lymph node metastasis (P=0.035), distant metastasis (P<0.001) and total CTC count ≥4 (P=0.007) and M-CTC count ≥1 (P<0.001) were significantly associated with unfavourable PFS, and lymph node metastasis (P=0.04), distant metastasis (P=0.01) and M-CTC count ≥1 (P<0.001) were significantly associated with unfavourable overall survival. Multivariate analysis showed that the presence of M-CTCs was the only independent prognostic factor for poor PFS, and patients with M-CTCs had significantly shorter PFS than those without (P=0.011). M-CTCs are significantly associated with CRC severity and metastasis, and M-CTC count is an independent predictor of prognosis.