Angiogenesis and hemostasis in colorectal cancer

Tumor angiogenesis is an important factor which precipitates recurrence and metastasis of colorectal cancer (CRC). Angiogenesis is also a significant feature which accompanies invasion and metastasis of CRC. Tumor hypoxia activates hypoxia inducible factor (HIF), which promotes angiogenesis in CRC. HIF significantly promotes cell proliferation and angiogenesis in CRC, facilitating invasion and metastasis. Tanshinone IIA (Tan IIA) has been revealed to effectively inhibit angiogenesis in CRC, although the underlying mechanism remains to be determined. The aim of the present study was to determine the effects of HIF-1α on hypoxia induced angiogenesis in CRC cells, the effects of Tan IIA on the expression of pro-angiogenic factors in CRC cells, and on human umbilical vein endothelial cell (HUVEC) tube formation in normal and hypoxic conditions. The results of the present study revealed that Tan IIA not only decreased HIF-1α expression and inhibited the secretion level of vascular endothelial growth factor and basic fibroblast growth factor, but also efficiently decreased proliferation, tube formation and metastasis of HUVECs. The results highlight the potential of Tan IIA-mediated targeting of HIF-1α as a potential therapeutic option for treatment of patients with CRC.

Angiogenesis and lymphangiogenesis are involved in the dissemination of tumor cells from solid tumors to regional lymph nodes and various distant sites. KAI1 COOH-terminal interacting tetraspanin (KITENIN) contributes to tumor progression and poor clinical outcomes in various cancers including colorectal cancer. The aim of the present study was to evaluate whether KITENIN affects tumor angiogenesis and lymphangiogenesis in colorectal cancer. A KITENIN small interfering RNA vector was used to silence KITENIN expression in colorectal cancer cell lines including DLD1 and SW480 cells. To evaluate the ability of KITENIN to induce angiogenesis and lymphangiogenesis in human umbilical vein endothelial cells (HUVECs) and lymphatic endothelial cells (HLECs), we performed Matrigel invasion and tube formation assays. Immunohistochemistry was used to determine the expression of KITENIN in colorectal cancer tissues. Angiogenesis and lymphangiogenesis were evaluated by immunostaining with CD34 and D2-40 antibodies. KITENIN silencing inhibited both HUVEC invasion and tube formation in the DLD1 and SW480 cells. KITENIN silencing led to decreased expression of the angiogenic inducers vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor-1α and increased expression of the angiogenic inhibitor angiostatin. KITENIN silencing did not inhibit either HLEC invasion or tube formation in all tested cells, but it resulted in decreased expression of the lymphangiogenic inducer VEGF-C. KITENIN expression was significantly associated with tumor stage, depth of invasion, lymph node and distant metastases and poor survival. The mean microvessel density was significantly higher in the KITENIN-positive tumors than that in the KITENIN-negative tumors. However, the mean lymphatic vessel density of KITENIN-positive tumors was not significantly higher than that of the KITENIN-negative tumors. These results suggest that KITENIN promotes tumor progression by enhancing angiogenesis in colorectal cancer.

Tumour angiogenesis is a critical step in the growth, metastatic spread and regrowth of colorectal cancer. Angiogenesis specific to tumour is a complicated process, the mechanisms of which remain unclear. Metastasis of colorectal cancer may result from passive entry into the circulation secondary to the effect of angiogenic factors. The survival and growth of colorectal tumour and thus their metastases are dependent on the balance of endogenous angiogenic and anti-angiogenic factors such that the outcome favours increased angiogenesis. Angiogenesis has become an attractive target for anticancer drug development, based on its important roles in tumour growth, invasion and metastasis. Several growth factors have been identified that regulate angiogenesis in colorectal cancer; the most important of these are vascular endothelial growth factors (VEGF), and of the several angiogenic factors, VEGF expression at the deepest invasive site of tumour is the most statistically significant prognostic indicator in advanced colorectal carcinoma. In this review article, we provide an overview on angiogenic factors and their receptors, and discuss the role of newly identified tumour endothelial markers (TEMs) that are involved in tumour-associated angiogenesis in colorectal cancer.

Recent studies proved that an inactivation of RUNX3 (runt-related transcription factor-3) expression is highly associated with lymph node metastasis and poor prognosis in various cancer types. However, the mechanism of RUNX3-mediated suppression of tumor metastasis remains unclear. Herein, we aimed to clarify the effect of RUNX3 on metastasis and angiogenesis in colorectal cancer (CRC). First, we found that the reduction of expression of RUNX3 in CRC tissues when compared with tumor adjacent normal colon tissues, as indicated by reduced RUNX3 staining, was significantly correlated with TNM stage. Second, we demonstrated that RUNX3 overexpression inhibited CRC cell migration and invasion resulting from the elevated upregulation of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) expression. In contrast, the knockdown of RUNX3 reduced the inhibition of migration and invasion of CRC cells. Last, Vascular endothelial growth factor(VEGF) is an inducer of angiogenesis and lymphangiogenesis. We found that restoration of RUNX3 decreased vascular endothelial growth factor (VEGF) secretion and suppressed endothelial cell growth and tube formation in CRC cells. Taken together, our data demonstrated that RUNX3 may provide insights into the development of RUNX3 for CRC metastasis diagnostics and therapeutics. Citation Format: Bo Ram Kim, Jung Lim Kim, Yoo Jin Na, Seong Hye Park, Sun Il Lee, Sanghee Kang, Sung Yup Joung, Suk Young Lee, Hong-Jun Kim, Dae-Hee Lee, Byung-Wook Min, Sang Cheul Oh. RUNX3 inhibits metastasis and angiogenesis in colorectal cancer. [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 1617.

Increasing evidence indicates that angiogenesis plays a pivotal role in tumor progression. Formin‐like 2 (FMNL2) is well‐known for promoting metastasis; however, the molecular mechanisms by which FMNL2 promotes angiogenesis in colorectal cancer (CRC) remain unclear. Here, we found that FMNL2 promotes angiogenesis and metastasis of CRC in vitro and in vivo. The GDB/FH3 domain of FMNL2 directly interacts with epidermal growth factor‐like protein 6 (EGFL6). Formin‐like 2 promotes EGFL6 paracrine signaling by exosomes to regulate angiogenesis in CRC. Cytoskeleton associated protein 4 (CKAP4) is a downstream target of EGFL6 and is involved in CRC angiogenesis. Epidermal growth factor‐like protein 6 binds to the N‐terminus of CKAP4 to promote the migration of HUVECs by activating the ERK/MMP pathway. These findings suggest that FMNL2 promotes the migration of HUVECs and enhances angiogenesis and tumorigenesis in CRC by regulating the EGFL6/CKAP4/ERK axis. Therefore, the EGFL6/CKAP4/ERK axis could be a candidate therapeutic target for CRC treatment.

Recent studies have shown that nuclear factor-kappa B/RelA (NF-kappa B/RelA) is involved in tumor angiogenesis. This study examined whether NF-kappa B/RelA expression is associated with vascular endothelial growth factor (VEGF) expression and microvessel density in human colorectal cancer. Ten specimens from normal colorectal mucosa and 52 colorectal adenocarcinomas were obtained by surgery or endoscopy. Immunohistochemical expression of NF-kappa B/RelA, VEGF, and CD34 was detected on paraffin-embedded tissue sections. NF-kappa B/RelA and VEGF were significantly overexpressed and associated with microvessel density in colorectal cancer. A significant association was found between NF-kappa B/RelA and VEGF expression. Clinicopathological features were not correlated with NF-kappa B/RelA, VEGF expression, or microvessel density. Our results suggest that increased expression of NF-kappa B/RelA contributes to tumor angiogenesis in colorectal cancer. VEGF may play an important role in mediating the NF-kappa B/RelA angiogenic pathway.

ATPase family AAA domain-containing protein 2 (ATAD2) is involved in various types of cancers, including colorectal cancer. This study aimed to determine the role of ATAD2 in angiogenesis in colorectal cancer. Here, we downregulated ATAD2 expression in HCT116 and SW480 cells, and collected the conditioned medium (CM) from control and ATAD2-silenced cells. The effect of CM on human umbilical vein endothelial cells (HUVEC) was evaluated by using CCK-8, wound healing, tube formation, Western blot, and dual-luciferase reporter assays. Our results showed that the proliferation, migration, and tube formation of HUVEC were reduced in presence of ATAD2-silenced CM, and the levels of phosphorylated vascular endothelial growth factor receptor 2 (P-VEGFR2), CD31, and CD34 were downregulated. Mechanism studies showed that ATAD2 silencing regulated the expression of vascular endothelial growth factor A (VEGFA) and miR-520a. Moreover, we found that miR-520a could bind to ATAD2, and its inhibitor partly reversed the alterations in HUVEC induced by CM from ATAD2-silenced cells. In addition, we demonstrated that miR-520a directly bound to 3'-UTR of VEGFA and inhibited its expression. Collectively, our results indicate that ATAD2 inhibition suppresses VEGFA secretion by increasing miR-520a levels. Our study suggests ATAD2 as a potential therapeutic target for angiogenesis in colorectal cancer.

Up regulation of fatty acid synthase (FASN), a key enzyme of de novo lipogenesis, is associated with metastasis in colorectal cancer (CRC). However, the mechanisms of regulation of metastasis by FASN are elusive. The induction of angiogenesis is crucial for metastasis. Therefore, the purpose of the present study was (i) to determine the role of FASN in regulation of secretion of angiogenic factors by CRC cells, (ii) to evaluate the effect of tumor-specific expression of FASN on endothelial cells, and (iii) to assess the role of FASN in regulation of tumor vasculature. METHODS: The effect of FASN inhibition on secretion of angiogenic factors was analyzed using the Human Angiogenesis Antibody Array 1 (RayBiotech). Co-culture of CRC and HMVEC-L cells was used to assess the effect of FASN expression on growth, migration, and tube formation of endothelial cells. To evaluate the effect of FASN on vasculature, we utilized murine colonoscopy, the orthotopic colon cancer model, the chick chorioallantoic membrane (CAM) model, and the Matrigel plug assay. RESULTS: We found that inhibition of FASN is associated with an evident change in the profile of secreted angiogenic factors by CRC cells and this change is associated with a significant decrease in microvessel density as determined by analysis of CD31 staining (Aperio Image Scope) in orthotopic colon tumors. A decrease in expression of FASN is also associated with “normalization” of tumor vasculature in the CAM model. The mechanisms of inhibition of angiogenesis by knockdown of FASN included down regulation of VEGF189, an isoform associated with aggressive CRC, up regulation of anti-angiogenic isoform VEGF165b, and a decrease in expression and activity of MMP-9. Furthermore, medium conditioned on FASN-knockdown CRC cells inhibited proliferation, migration, and tube formation of endothelial cells via inhibition of VEGFR-2 activation and its downstream signaling as compared to control medium. CONCLUSIONS: This study suggests that FASN promotes angiogenesis via alteration of the profile of secreted angiogenic factors by CRC cell and stimulation of endothelial cell proliferation. Furthermore, the regulation of angiogenesis by FASN, at least in part, is mediated by up-regulation of VEGF189, a VEGF-A isoform that tightly correlates with poor prognosis in CRC, and by an increase in activity of MMP-9. Together, these findings suggest that inhibition of FASN upstream of VEGF-A and other angiogenic pathways can be a novel therapeutic strategy to prevent or inhibit metastasis in CRC. Citation Format: Yekaterina Y. Zaytseva, Victoria Elliott, Piotr Rychahou, William Mustain, Ji Tae Kim, Joseph Valentino, Tianyan Gao, Kathleen O'Connor, Janna Neltner, Heidi Weiss, B. Mark Evers. Tumor-specific expression of fatty acid synthase promotes angiogenesis in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1009. doi:10.1158/1538-7445.AM2014-1009

LncRNA OGFRP1 promotes angiogenesis and epithelial-mesenchymal transition in colorectal cancer cells through miR-423-5p/CTCF axis

In order to better understand the trends and hot spots in angiogenesis research in metastatic colorectal cancer, this study uses bibliometric analysis to conduct a systematic analysis. Publications were retrieved from Web of Science Core Collection from 2000 to 2023. All studies were output for further analysis in R and CiteSpace software. A total of 5875 studies were recruited via the Web of Science Core Collection from 2000 to 2023 regarding the field of angiogenesis in metastatic colorectal cancer. China and USA were the leading country with top country publications. Among all contributed authors, the main three subjects were determined as cell biology, oncology, and peripheral vascular disease. Furthermore, network analysis of all recruited studies identified 16 research subjects. Next, the top 25 countries, authors references, institutions, and keywords with the strongest citation bursts were identified. To further delineate the growth and change of two types of keywords, Keywords Plus® and author keywords, a heatmap analysis was employed. Last, the most influential references relating to this field were identified using the strongest citation bursts. This study explored the research hot spots and trends of angiogenesis in metastatic colorectal cancer through bibliometric analysis. It would help researchers better understand the current status and future development hotspots of global research to promote further development in this field.

Up-regulation of cyclooxygenase 2 gene expression correlates with tumor angiogenesis in human colorectal cancer

microRNA (miRNA) levels are dysregulated in many cancers, suggesting that miRNA-based therapy may be effective. The molecular pathways of colorectal cancer (CRC) development are unknown. Understanding miRNAs implicated in CRC formation may reveal new diagnostic and therapeutic targets. Angiogenesis is a key mechanism in tumor growth. CRC treatment may involve inhibiting angiogenesis, but existing drugs can cause negative effects. Tranexamic acid, an FDA-approved medication, may reduce the adverse effects of angiogenesis inhibitors. This work examined miRNAs implicated in CRC angiogenesis and how miR-16 and tranexamic acid may synergistically decrease CRC cell migration and angiogenesis. We identified miRNAs targeting CRC angiogenesis genes using bioinformatic databases. Proteins were docked with tranexamic acid utilizing the PyRx software. Quantitative Real-time PCR was used to analyze the effects of overexpressed miRNA and tranexamic acid on the expression of target genes. Scratch, transwell migration, and Chicken Chorioallantoic Membrane (CAM) assays were used to evaluate the effect of selected miRNA and tranexamic acid on the invasion and angiogenesis of CRC cells. in silico studies identified hsa-miR-16-5p, -101-3p, and 34a-5p as possible CRC angiogenesis modulators. The study found that miR-16 and tranexamic acid influence the expression of VEGFA, ANGPT2, MMP9, and HIF1A. miR-16 and tranexamic acid influenced CRC cell movement in scratch tests and transwell migration assays. Furthermore, the CAM assay results demonstrated that miR-16 and tranexamic acid can alter angiogenesis in CRC. These findings highlight the potential of miR-16 and tranexamic acid as combination therapeutic agents for CRC, with the ability to simultaneously target tumorigenesis and angiogenesis.

Background. Of all intestinal microbiome-derived metabolites, trimethylamine N-oxide (TMAO) has received increasing attention because of its potent role in colorectal cancer development. Accumulating evidence suggests that TMAO generated by the gut microbiota is a new and important player in the etiological process of colorectal cancer. Nevertheless, the carcinogenic mechanism of TMAO in colorectal cancer remains unclear. In this study, TMAO induced colorectal cancer cell proliferation and produced higher vascular endothelial growth factor A (VEGFA) levels in vitro. In vivo, after long-term choline feeding in tumor-bearing mice, circulating TMAO levels, tumor volume, new blood vessel formation, and VEGFA and CD31 amounts were increased significantly. This study revealed that TMAO exerts oncogenic effects by promoting cell proliferation and angiogenesis in colorectal cancer.

Introduction: Angiogenesis plays an important role in the growth and metastasis of colorectal carcinoma, which is currently one of the targets of cancer therapy. It has been reported that the Endoglin (CD105) involved in angiogenesis and is a powerful marker for angiogenesis in colorectal carcinoma. Level Quantitative angiogenesis in peritumor and an intratumor area is important to know because it is closely related to the micro-environmental factors that influence the occurrence of cancer angiogenesis. The goal of this study to analyze the distribution pattern of angiogenesis in colorectal cancer by comparing the distribution of angiogenesis in peritumor and intratumor areas between well, moderate and poorly differentiated colorectal carcinoma, and between metastasis and non-metastatic colorectal cancer. Methods: This study analyzed fifty samples of resected adenocarcinoma colorectal. Angiogenesis was assessed by the immunohistochemical method using a primary monoclonal antibody Endoglin (CD105). Positive expression of CD105 was assessed through the CD105 protein expression in neovascular endothelial cells, while the distribution pat- tern of angiogenesis assessed by counting the positive expression of CD105 protein in hot spots by using the MVD (microvessel density) in the peritumor and intratumor areas and then performed statistical analysis. Results: There is a significant difference between the quantitative level of an- giogenesis in peritumor and intratumor areas of well (P<0.01), moderate (P<0.01) and poorly (<0.05) differentiated adenocarcinoma. The significant difference between the quantitative levels of angio- genesis in peritumor and intratumor areas of non-metastatic colorectal cancer (P<0.01) and lymph node metastases (<0.05) was found, but not in colorectal cancer with liver metastasis. Conclu- sion: Angiogenesis pattern is more concentrated in peritumor compared to intratumor areas. This showed the role of stromal cells in-angiogenesis. There is significant expression between angio- genesis in peritumor and intratumor areas.

Background and Aims: Angiogenesis is a hallmark of cancer development that has been considered an attractive therapeutic target. In this study, we aimed to unravel the molecular mechanism of tumor angiogenesis in colorectal cancer (CRC). Materials and Methods: We isolated endothelial and epithelial cells from surgically resected 14 human CRC tissues by using antibodies against endothelial (CD146) and epithelial markers (EpCAM). RNA sequencing was carried out using 3 pairs of normal and tumor endothelial cells. Expression of the genes was validated using quantitative RT-PCR, immunohistochemistry. Functions of a selected gene were carried analyzed by tumor conditioned medium (TCM) experiments, tube formation assay, gene expression microarray and cell cycle analysis. Results: Through RNA-seq analysis, we identified 18 genes, which were upregulated in the endothelial cells isolated from CRC tissues. We further validated the results by performing quantitative RT-PCR and immunohistochemical analysis in a larger series of clinical samples, and identified gene A as a novel candidate of the tumor endothelium-related gene. Expression of gene A was also upregulated in human umbilical vein endothelial cells (HUVECs) treated with TCM obtained from CRC cell lines. Knockdown of gene A in HUVECs suppressed in vitro tube formation and induced G1 cell cycle arrest. Microarray analysis revealed that gene A knockdown induced expression changes of approximately 300 genes in HUVECs, and gene ontology analysis showed that genes related to cell cycle or cell division were significantly enriched in the affected genes. To confirm our findings in vivo, we co-transplanted CRC cells with HUVECs into nude mice. We found that gene A knockdown in HUVECs resulted in reduced micro vessel formations in the xenograft tissues. Conclusion: We identified elevated expression of gene A in tumor endothelial cells of primary colorectal cancer tissues. Our results suggest that gene A may play an important role in the angiogenesis in colorectal cancer, and that it could be a potential therapeutic target. Citation Format: Akira Yorozu, Eiichiro Yamamoto, Reo Maruyama, Masahiro Kai, Toshihiko Nishidate, Tomohisa Furuhata, Tamotsu Sugai, Hiromu Suzuki. Identification of tumor endothelium-related genes in colorectal cancer. [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 3385.