Lymphangiogenesis In Colorectal Cancer Research Articles

Identification of a novel lymphangiogenesis signature associated with immune cell infiltration in colorectal cancer based on bioinformatics analysis

BackgroundLymphangiogenesis plays an important role in tumor progression and is significantly associated with tumor immune infiltration. However, the role and mechanisms of lymphangiogenesis in colorectal cancer (CRC) are still unknown. Thus, the objective is to identify the lymphangiogenesis-related genes associated with immune infiltration and investigation of their prognosis value.MethodsmRNA expression profiles and corresponding clinical information of CRC samples were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The lymphangiogenesis-related genes (LymRGs) were collected from the Molecular Signatures database (MSigDB). Lymphangiogenesis score (LymScore) and immune cell infiltrating levels were quantified using ssGSEA. LymScore) and immune cell infiltrating levels-related hub genes were identified using weighted gene co-expression network analysis (WGCNA). Univariate Cox and LASSO regression analyses were performed to identify the prognostic gene signature and construct a risk model. Furthermore, a predictive nomogram was constructed based on the independent risk factor generated from a multivariate Cox model.ResultsA total of 1076 LymScore and immune cell infiltrating levels-related hub genes from three key modules were identified by WGCNA. Lymscore is positively associated with natural killer cells as well as regulator T cells infiltrating. These modular genes were enriched in extracellular matrix and structure, collagen fibril organization, cell-substrate adhesion, etc. NUMBL, TSPAN11, PHF21A, PDGFRA, ZNF385A, and RIMKLB were eventually identified as the prognostic gene signature in CRC. And patients were divided into high-risk and low-risk groups based on the median risk score, the patients in the high-risk group indicated poor survival and were predisposed to metastasis and advanced stages. NUMBL and PHF21A were upregulated but PDGFRA was downregulated in tumor samples compared with normal samples in the Human Protein Atlas (HPA) database.ConclusionOur finding highlights the critical role of lymphangiogenesis in CRC progression and metastasis and provides a novel gene signature for CRC and novel therapeutic strategies for anti-lymphangiogenic therapies in CRC.

Expression of an oxysterol-metabolizing enzyme in colorectal cancer and its relation to tumor cell behavior and prognosis

Oxysterols and oxysterol-metabolizing enzymes have been implicated in the pathogenesis of various cancers. However, the distinct function of the oxysterol-metabolizing enzyme cytochrome P450 family 39 Subfamily A Member 1 (CYP39A1) in colorectal cancer (CRC) remains unclear. The aims of the current study were to evaluate whether CYP39A1 affects the oncogenic behaviors of CRC cells and to investigate the prognostic value of its expression in CRC. A CYP39A1 small-interfering RNA was used to block CYP39A1 gene expression in DLD1 and SW480 cells. The expression of CYP39A1 in CRC tissues was investigated by immunohistochemistry. Tumor angiogenesis and lymphangiogenesis were assessed by CD34 and D2–40 immunohistochemical staining, respectively. CYP39A1 knockdown inhibited tumor cell migration and invasion in DLD1 and SW480 cells. Angiogenesis was also inhibited through the decreased expression of vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor (HIF)−1α, and angiostatin and endostatin expression increased. In addition, CYP39A1 knockdown inhibited the lymphangiogenesis by decreasing the expression of VEGF-C. CYP39A1 expression was increased in CRC tissues compared with normal colorectal mucosa. CYP39A1 expression was associated with tumor stage, depth of invasion, lymph node metastasis, distant metastasis, and poor survival. The microvessel and lymphatic vessel density values of CYP39A1-positive tumors were significantly higher than those of CYP39A1-negative tumors. These results indicate that CYP39A1 is associated with tumor progression by influencing tumor cell angiogenesis and lymphangiogenesis in CRC.

TRAF6 regulates the signaling pathway influencing colorectal cancer function through ubiquitination mechanisms.

Tumor necrosis factor receptor‐associated factor‐6 (TRAF6) is a ubiquitin E3 ligase. TRAF6 plays an important role in tumor invasion and metastasis. However, the specific mechanism by which TRAF6 promotes colorectal cancer (CRC) metastasis is incompletely understood. This study aimed to determine whether TRAF6 affects the LPS‐NF‐κB‐VEGF‐C signaling pathway through ubiquitination, which plays a role in colorectal cancer metastasis. Here, our results showed that TRAF6 affected lymphangiogenesis through the LPS‐NF‐κB‐VEGF‐C signaling pathway. Using ubiquitination experiments, we found that TRAF6 was mainly ubiquitinated with the K63‐linked chains, and LPS promoted ubiquitination of TRAF6 and K63‐linked chains. More importantly, TRAF6 124mut is the main ubiquitination site of TRAF6 interacting with K63‐linked chains. TRAF6 affected the migration, invasion, and lymphatic metastasis of colorectal cancer through its ubiquitination. In subcutaneous xenograft models, TRAF6 124mut inhibited tumor growth. In conclusion, our results provide new insight for studying the mechanism of lymphangiogenesis in colorectal cancer to promote cancer metastasis, which may provide new ideas for tumor immunotherapy.

Qingjie Fuzheng Granule suppresses lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway

ScopeTo investigate the effect of Qingjie Fuzheng Granule (QFG) on lymphangiogenesis and lymphatic metastasis in colorectal cancer. MethodsThe effects of QFG on the expression and secretion of vascular endothelial growth factor-C (VEGF-C) in HCT-116 cells were investigated both in vitro and in vivo. HCT-116 cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. The VEGF-C expression level was determined using RT-qPCR and western blotting, and the VEGF-C concentration in supernatant was measured by ELISA. Tumor xenograft models of HCT-116 cells were generated using BALB/c nude mice, and the mice were randomly divided into a control group (gavaged with normal saline) and QFG group (gavaged with 2 g/kg QFG). The effect of QFG on tumor growth was evaluated by comparing the volume and weight of tumors between two groups. Immunohistochemistry (IHC) and RT-qPCR were performed to detect the expression levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1). ELISA was performed to measure the concentration of serum VEGF-C. TMT proteomics technology and Reactome pathway analysis were used to explore the mechanism of QFG inhibiting lymphangiogenesis in tumor. The VEGF-C (5 ng/mL)-stimulated human lymphatic endothelial cell (HLEC) model was conducted to evaluate the effect of QFG on lymphangiogenesis in vitro. The model cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. Cell viability was then determined using an MTT assay. The cell migration, invasion, and tube-formation ability were analyzed using transwell migration, matrigel invasion and tube formation assays, respectively. The underlying mechanism was uncovered, the levels of VEGFR-3, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), p-PI3K/PI3K, p-AKT/AKT and p-mTOR/ mTOR were detected using western blotting. ResultsQFG significantly reduced VEGF-C expression and secretion in HCT-116 cells. QFG evidently suppressed in vivo tumor growth and the expression of VEGF-C, VEGFR-3, and LYVE-1. The serum VEGF-C level was also reduced by QFG. Moreover, TMT proteomics technology and Reactome pathway analysis identified 95 differentially expressed protein and multiple enriched pathway about matrix metalloproteinase and extracellular matrix, which is direct associate with lymphangiogenesis. In vitro experiment, QFG inhibited the viability, migration, invasion and tube formation of HLECs. Additionally, QFG reduced the VEGFR-3, MMP-2, MMP-9 expression levels, and the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/ mTOR ratios. ConclusionQFG can exert its effect on both tumor cells and HLECs, exhibiting ani- lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway pathway.

Detection and prognostic value of intratumoral and peritumoral lymphangiogenesis in colorectal cancer.

BackgroundColorectal cancer (CRC) with lymphatic invasion is one of the critical prognostic factors in lymph node metastasis. Lymphangiogenesis has a significant effect on lymphatic metastasis and tumor progression. However, the significance of intratumoral and peritumoral lymphangiogenesis has been controversial in CRC. The aim of this study is to investigate the different role of introtumoral and peritumoral lymphangiogenesis in CRC progression and prognosis.MethodsLymphangiogenesis of 120 CRC specimens, as measured by lymphatic vessel density (LVD), was examined by immunostaining for podoplanin, a lymphatic vessel-specific marker. The mean number of lymphatic vessels of three hotspots was measured in intratumoral and peritumoral areas as intratumoral LVD (LVDit) and peritumoral LVD (LVDpt), respectively. The association of LVDit and LVDpt with the clinicopathological findings and prognosis was investigated.ResultsCompared to the peritumoral lymphatics, the intratumoral lymphatics were small, collapsed and irregular. The mean LVDpt was higher than the mean LVDit (P<0.001). LVDit was positively correlated with tumor size (P=0.009), tumor histologic grade (P=0.023), and overall survival (P=0.036). LVDpt was correlated with lymph node metastasis (P<0.001), tumor stage (P=0.004), and overall survival (P=0.016).ConclusionsLVDpt plays a prominent role in lymph node metastasis, whereas LVDit is more closely correlated with tumor growth and histopathological differentiation. Both LVDpt and LVDit contribute to CRC progression and prognosis.

CCBE1 promotes tumor lymphangiogenesis and is negatively regulated by TGFβ signaling in colorectal cancer.

Collagen and calcium-binding EGF domain-1 (CCBE1) is essential for lymphatic vascular development as it promotes vascular endothelial growth factor C (VEGFC) proteolysis. A recent study reported that CCBE1 was overexpressed in epithelial colorectal cancer (CRC) cells; however, the role of CCBE1 in tumor lymphangiogenesis and the mechanism underlying dysregulated CCBE1 expression in CRC remain undefined.Methods: The role of CCBE1 in tumor lymphangiogenesis and lymphatic metastasis was investigated using human lymphatic endothelial cells (HLECs) model in vitro, and a hindfoot lymphatic metastasis model in vivo. Immunochemistry analysis was performed to assess CCBE1 expression, prognostic value and correlation with clinicopathological characteristics in CRC. The biochemical function and transcriptional regulatory mechanism of CCBE1 were explored by western blot, qPCR, and chromatin immunoprecipitation.Results: Cancer cell-derived CCBE1 enhances VEGFC proteolysis in vitro, facilitates tube formation and migration of HLECs in vitro, and promotes tumor lymphangiogenesis and lymphatic metastasis in vivo. In addition to CRC cells, tumor stroma within CRC tissue shows high CCBE1 expression, which is associated with high lymphatic vessel density, increased lymph node metastasis and poor prognosis. Cancer-associated fibroblasts (CAFs) express and secret CCBE1, thereby contributing to VEGFC maturation and tumor lymphangiogenesis in CRC. Transforming growth factor beta (TGF-β) downregulates the transcription and lymphangiogenic function of CCBE1 in CAFs and CRC cells through direct binding of SMADs to CCBE1 gene locus. Inactivation of the TGF-β pathway correlates with increased CCBE1 expression in CRC.Conclusion: Our results demonstrate the protumorigenic role of CCBE1 in promoting lymphangiogenesis and lymphatic metastasis in CRC, revealing a new mechanism by which loss of TGF-β signaling promotes CRC metastasis.

Pazopanib Inhibits Tumor Growth, Lymph-node Metastasis and Lymphangiogenesis of an Orthotopic Mouse of Colorectal Cancer.

Pazopanib (PAZ) can inhibit tumor progression, but whether PAZ inhibits lymph node metastasis and lymphangiogenesis in colorectal cancer is still unknown. The aim of the present study was to determine the efficacy of PAZ on tumor growth, lymph node metastasis and lymphangiogenesis in an orthotopic nude mouse model in colorectal cancer. CT-26-green fluorescence protein (GFP)-expressing mouse colon cancer cells were injected into nude mice to establish a subcutaneous colorectal cancer model and were treated with saline and PAZ. Additionals subcutaneous tumors were harvested and cut into 5 mm3 fragments, then tumor fragments were implanted orthotopically in the cecum to establish an orthotopic colorectal-cancer nude mouse model. Orthotopic mice were randomized into two groups for the treatment with saline and PAZ, respectively. Tumor width, length and mouse body weight was measured twice a week. The Fluor Vivo imaging system was used to image the GFP. Hematoxylin & eosin staining and immunohistochemical staining was used for histological analysis. PAZ inhibited the growth of subcutaneous colorectal cancer, as wells as orthotopic transplanted colorectal cancer tumors. PAZ suppressed lymph node metastasis and lymphangiogenesis in the orthotopic colon cancer model. No significant changes were observed in the body weight between the control and the mice treated with PAZ. PAZ can inhibit the growth of colorectal cancer and inhibit lymph node metastasis and lymphangiogenesis in orthotopic colon cancer nude mouse models.

Hedyotis diffusa Willd. inhibits VEGF‑C‑mediated lymphangiogenesis in colorectal cancer via multiple signaling pathways.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies worldwide. For patients diagnosed with the presence of metastatic disease, surgery is not suitable for the majority of them. Lymphangiogenesis is a key factor during cancer metastasis and is regulated by vascular endothelial growth factor C (VEGF‑C). Hedyotis diffusa Willd. (HDW) is a Chinese herb of the Rubiaceae family that reportedly inhibits tumor metastasis. However, its underlying anticancer mechanisms have not yet been elucidated. In the present study, we investigated the effects of an ethanol extract of HDW (EEHDW) on the migration capacity by wound healing and Transwell assays, and the effect on the VEGF‑C expression in different CRC cell lines by western blot analysis and ELISA assays. A model of VEGF‑C‑stimulated human lymphatic endothelial cells (HLECs) was constructed. It was found that EEHDW suppressed lymphangiogenesis via the mediation of multiple pathways, which attenuated the migration of cells and their tube formation abilities. Multiple signaling pathways were found to be involved in the VEGF‑C‑mediated lymphangiogenesis. After EEHDW treatment in VEGF‑C‑stimulated HLECs, EEHDW was found to downregulate the expression levels of multiple signaling pathways. Taken together, these results indicate that EEHDW possesses significant anti‑metastatic activities. Moreover, the suppressive effect of EEHDW on lymphangiogenesis, particularly via downregulation of VEGF‑C, partly explains the potential molecular mechanism underlying the inhibitory effect of EEHDW on CRC metastasis.

Lymphangiogenesis and colorectal cancer.

Lymphatic metastasis is an important event in the progress of metastasis in colorectal cancer (CRC). The purpose of this article is to assess the role of lymphangiogenesis on CRC. In peritumoral areas of CRC, the lymphatic microvessel density (LMVD) is higher than those in normal colorectal tissues. Morever, the high LMVD is correlated with DFS and local recurrence in CRC. The VEGF-C/VEGF-D/VEGFR-3 pathway, sonic hedgehog (Shh) signaling pathway and extracellular matrix (ECM) are involved in the regulation of lymphangiogenesis in CRC. Inhibition of the VEGF-C/VEGF-D/VEGFR-3 pathway by specific antibodies has been reported to efficiently inhibit experimental tumor lymphangiogenesis and metastasis in animal experiments. Although lymphangiogenesis has been reported to play an important role in the occurrence of colon cancer and to be associated with prognosis, it remains unclear whether it is a valid therapeutic target molecule. Further study of the potential of targeting this process for anti-lymphatic therapies is worthwhile.

Pien Tze Huang suppresses VEGF-C-mediated lymphangiogenesis in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. The majority of patients are not suitable for surgery due to the presence of metastatic disease at the time of diagnosis, which has led to a high mortality rate for patients with CRC. Lymphangiogenesis, formation of new lymphatic vessels, plays an critical role in cancer progression particularly in cancer metastasis. Vascular endothelial growth factor-C (VEGF-C) has been previously demonstrated to play a pivotal role in cancer metastasis and therefore has become an attractive target for anticancer treatments. Pien Tze Huang (PZH) is a well-known traditional Chinese formula, which has exhibited significant therapeutic effects against CRC. However, the molecular mechanisms underlying its anticancer effects, particularly in regards to antimetastasis activity, still require further elucidation. In the present study, we evaluated the effects of PZH on cell migration and VEGF-C expression using various human CRC cell lines. Moreover, using a VEGF‑C-stimulated human lymphatic endothelial cell (HLEC) model, we demonstrated that PZH suppresses lymphangiogenesis by attenuating cell migration and tube formation. This indicates that PZH possesses significant antimetastatic activity. Moreover, suppression of lymphangiogenesis by PZH via the downregulation of VEGF-C may be a potential molecular mechanism by which PZH inhibits metastasis in CRC.

BRG1 targeting STAT3/VEGFC signaling regulates lymphangiogenesis in colorectal cancer.

Tumor lymphangiogenesis is an important early event in tumorigenesis, one that promotes lymphatic metastasis. BRG1 (also known as SMARCA4) is a central component of the SWI/SNF chromatin-remodeling complex. In a previous work, we have reported that decreased BRG1 could promote colon cancer cell migration and invasion, and that the BRG1 expression level is negatively correlated with lymphatic metastasis. In the current study, we provide a comprehensive analysis of the role of BRG1 during lymphangiogenesis in colorectal cancer. Lymphatic vessels are more abundant in BRG1 low-expression tumors than in BRG1 high-expression tumors. We investigate the process by which BRG1 can promote VEGFC transcription and induce lymphangiogenesis in vivo and in vitro. We show that BRG1 controls lymphangiogenesis by binding to STAT3 and regulating STAT3 activation. We also prove the mechanisms through clinical samples. In summary, our demonstration of the important roles of the BRG1/STAT3/VEGFC in tumor-associated lymphangiogenesis might lead to the discovery of novel therapeutic targets in the treatment of cancers with BRG1 loss of function.

Impact of KITENIN on tumor angiogenesis and lymphangiogenesis in colorectal cancer.

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.

Smad4 acts as tumor suppressor by antagonizing lymphangiogenesis in colorectal cancer

Vascular endothelial growth factor-C (VEGF-C) is considered a vital promoter to tumor lymphangiogenesis. Smad4 is an important downstream mediator of transforming growth factor beta (TGF-β) signaling pathway. Smad4 is regarded as a tumor suppressor in colon cancer, but its role in lymphangiogenesis in colorectal cancer is still unclear. In the present study, we investigated the potential inhibiting effect of Smad4 in lymphangiogenesis, and its correlation with VEGF-C secretion in colon cancer. We investigated endogenous Smad4 expression in 7 human colorectal cancer cell lines, and found that six of colon cancer cell lines existed with loss of Smad4. Expression of Smad4 in SW480 cell line is extremely low. SW480 cells were used to establish a Smad4 over-expression model. We investigated the relationship between Smad4 and VEGF-C expression in SW480 cells and found that Smad4 can inhibit the expression of VEGF-C. In an in vitro assay, we found that overexpression of Smad4 can reduce migration and invasion ability in SW480 cells, but had no effect on cell proliferation. Interestingly, Smad4 definitely inhibited the growth in an in vivo assay, and the volumes of transplanted tumors which originated from Smad4 overexpressing SW480 cells in nude mice were smaller than those of normal SW480 cells. In addition, we also demonstrated that Smad4 can inhibit lymphangiogenesis in vivo by immunohistochemistry. In summary, our findings provide molecular evidence that Smad4 may reduce lymphangiogenesis of colon cancer cell by attenuating VEGF-C secretion and act as tumor suppressor by inhibiting migration, invasion and tumorigenicity.

Mo1666 Nicotine Suppresses Colonic Tumorigenesis Associated With Chronic Colitis Through the Inhibition of IL-6/STAT3 Pathway in Mice

Background: Chronic intestinal inflammation occurring in Inflammatory Bowel Disease (IBD) predisposes to colitis-associated colorectal cancer (CAC). Tumor-induced lymphangiogenesis is one of the processes taking place in cancer, but no mechanistic data is available on the molecules modulating lymphangiogenesis in CAC. VEGFC is a key player in lymphangiogenesis that signals through its cognate receptor VEGFR3 and its blockade has been used in several models of metastatic cancer. However, little is known about the role played by adherens' junction proteins, such as Vascular Endothelial (VE)-cadherin on the lymphatic endothelium during the metastatic process. We hypothesized that lymphatics-targeted therapymay be effective in CAC formation and would abate metastasis dissemination bymaintaining lymphatic vessel junctions' integrity. Methods: The AOM/DSS model of CAC was used. In addition, colitic mice were orthotopically injected with CT26 colon cancer cells. Modulation of lymphangiogenesis was obtained by systemic inhibition of VEGFR3 or adenoviral overexpression of VEGFC. Tumor density and size were measured at the end of the AOM/ DSS protocol by visual, endoscopic and histological inspection. In the xenotransplantation model, liver, lung and draining lymphnodes were processed for metastasis quantification by FACS analysis.Whole mounts of colonswere stained to analyze area density and dimension of lymphatic vessels (LVs) within peritumoral and tumoral regions. Moreover, VE-cadherin distribution and expression were studied in vivo on LVs and in vitro on Human intestinal lymphatic endothelial cells (HILEC) stimulated with VEGFC or with a VEGFR3 inhibitor. Results: In both the AOM/DSS and the xenotransplantation models, systemic inhibition of VEGFR3 inhibited lymphangiogenesis in peritumoral and tumoral regions, reducing both area density and LVs dimension. In addition, it reduced tumor density and size, together with an abatement of metastatic dissemination, when compared to untreated animals. In contrast, tumor density and growth, includingmetastasis formation was enhanced by VEGFC, which in turn increased lymphangiogenesis within the same regions. Whole mount staining showed that VEGFC altered VE-cadherin expression both in vivo and in vitro, whereas VEGFR3 inhibitor kept endothelial junction integrity, thus linking the VEGFC/VEGFR3 pathway to VE-cadherin-dependent metastasis dissemination. Conclusion: Our findings demonstrate that VEGFC/VEGFR3-dependent lymphangiogenesis plays a key role both in CAC growth and metastasis dissemination. Our study reveals a novel mechanism of control of lymphatic junction integrity which may be a promising target for the treatment of CAC and the associated metastatic process.

Mo1665 The VEGFC/VEGFR3 Pathway Controls Colitis Associated Cancer Formation and Progression Through Lymphangiogenesis and Lymphatic Endothelial VECadherin

Background: Chronic intestinal inflammation occurring in Inflammatory Bowel Disease (IBD) predisposes to colitis-associated colorectal cancer (CAC). Tumor-induced lymphangiogenesis is one of the processes taking place in cancer, but no mechanistic data is available on the molecules modulating lymphangiogenesis in CAC. VEGFC is a key player in lymphangiogenesis that signals through its cognate receptor VEGFR3 and its blockade has been used in several models of metastatic cancer. However, little is known about the role played by adherens' junction proteins, such as Vascular Endothelial (VE)-cadherin on the lymphatic endothelium during the metastatic process. We hypothesized that lymphatics-targeted therapymay be effective in CAC formation and would abate metastasis dissemination bymaintaining lymphatic vessel junctions' integrity. Methods: The AOM/DSS model of CAC was used. In addition, colitic mice were orthotopically injected with CT26 colon cancer cells. Modulation of lymphangiogenesis was obtained by systemic inhibition of VEGFR3 or adenoviral overexpression of VEGFC. Tumor density and size were measured at the end of the AOM/ DSS protocol by visual, endoscopic and histological inspection. In the xenotransplantation model, liver, lung and draining lymphnodes were processed for metastasis quantification by FACS analysis.Whole mounts of colonswere stained to analyze area density and dimension of lymphatic vessels (LVs) within peritumoral and tumoral regions. Moreover, VE-cadherin distribution and expression were studied in vivo on LVs and in vitro on Human intestinal lymphatic endothelial cells (HILEC) stimulated with VEGFC or with a VEGFR3 inhibitor. Results: In both the AOM/DSS and the xenotransplantation models, systemic inhibition of VEGFR3 inhibited lymphangiogenesis in peritumoral and tumoral regions, reducing both area density and LVs dimension. In addition, it reduced tumor density and size, together with an abatement of metastatic dissemination, when compared to untreated animals. In contrast, tumor density and growth, includingmetastasis formation was enhanced by VEGFC, which in turn increased lymphangiogenesis within the same regions. Whole mount staining showed that VEGFC altered VE-cadherin expression both in vivo and in vitro, whereas VEGFR3 inhibitor kept endothelial junction integrity, thus linking the VEGFC/VEGFR3 pathway to VE-cadherin-dependent metastasis dissemination. Conclusion: Our findings demonstrate that VEGFC/VEGFR3-dependent lymphangiogenesis plays a key role both in CAC growth and metastasis dissemination. Our study reveals a novel mechanism of control of lymphatic junction integrity which may be a promising target for the treatment of CAC and the associated metastatic process.

Association between c-Met and lymphangiogenic factors in patients with colorectal cancer.

e22199 Background: Lymphangiogenesis plays an important role in cancer metastasis. Although animal models show a strong relationship between lymphangiogenesis and lymph node metastasis and survival, the clinical significance of lymphangiogenesis in colorectal cancer (CRC) remains uncertain. The goal of this study was to evaluate the association between c-Met and lymphangiogenic factors and to elucidate their prognostic significance for patients with CRC. Methods: A total of 379 tissue samples were obtained from surgically resected specimens from patients with CRC in Soonchunhyang University Cheonan Hospital between January 2002 and December 2010. The expressions of c-Met, vascular endothelial growth factor (VEGF)-C, VEGF-D, VEGF receptor (VEGFR)-3, and podoplanin were examined by immunohistochemistry. The expression of each marker and clinical factors were analyzed. Results: Three hundred and one of 379 (79.4%) tissues had c-Met expression. High expression of c-Met in tumor cells was significantly associated with high expression of VEGF-C (P &lt; .001) and VEGFR-3 (P = .001). But, there was no statistically significant association with podoplanin (P = .587) and VEGF-D (P = .096). Of the 103 evaluable patients, expression of c-Met in tumor cells was significantly associated with advanced clinical stage (P = .020), positive lymph node status (P = .038), and high expression of VEGF-C (P = .020). But, there was no statistically significant association with podoplanin (P = .518), VEGFR-3 (P = .085), VEGF-D (P = .203), and overall survival (P = .360). Conclusions: Our results provide indirect evidence for an association and possible regulatory link of c-Met with the lymphangiogenic factors. But, c-Met expression in patients with CRC are not prognostic indicator for overall survival in this retrospective study.

Metastasis-associated protein 1 induces VEGF-C and facilitates lymphangiogenesis in colorectal cancer

To study the correlation between high metastasis-associated protein 1 (MTA1) expression and lymphangiogenesis in colorectal cancer (CRC) and its role in production of vascular endothelial growth factor-C(VEGF-C). Impact of high MTA1 and VEGF-C expression levels on disease progression and lymphovascular density (LVD, D2-40-immunolabeled) in 81 cases of human CRC was evaluated by immunohistochemistry. VEGF-C mRNA and protein expressions in human LoVo and HCT116 cell lines were detected by real-time polymerase chain reaction and Western blotting, respectively, with a stable expression vector or siRNA. The elevated MTA1 and VEGF-C expression levels were correlated with lymph node metastasis and Dukes stages (P < 0.05). Additionally, high MTA1 expression level was correlated with a large tumor size (P < 0.05). A significant correlation was found between MTA1 and VEGF-C protein expressions in tumor cells (r = 0.371, P < 0.05). Similar to the VEGF-C expression level, high MTA1 expression level was correlated with high LVD in CRC (P < 0.05). Furthermore, over-expression of MTA1 significantly enhanced the VEGF-C mRNA and protein expression levels, whereas siRNAs - knocked down MTA1 decreased the VEGF-C expression level. MTA1, as a regulator of tumor-associated lymphangiogenesis, promotes lymphangiogenesis in CRC by mediating the VEGF-C expression.

Clinical and biological significance of angiogenesis and lymphangiogenesis in colorectal cancer

Angiogenesis and lymphangiogenesis are essential for tumour development and progression. However, in colorectal cancer (CRC), the relationship between angiogenesis and clinical outcome is controversial, and the prognostic significance of lymphangiogenesis is not well examined because of the lack of specific a marker for lymphatic vessels. To evaluate blood microvessel density (MVD) following the proposed standard method for MVD assessment given by the first international consensus and lymphatic vessel density (LVD), and investigate their clinicopathologic and biologic significance in CRC. MVD and LVD in primary tumours (n=210), along with their corresponding adjacent normal mucosa (n=105) and distant normal mucosa (n=27) specimens, were immunohistochemically examined by using CD31 and D2-40 antibodies. Both MVD and LVD were higher in tumour compared with the corresponding normal mucosa. In tumours, MVD was positively related to particular interesting new cysteine-histidine-rich protein (PINCH) expression (P=0.006), but not with clinicopathologic variables. LVD, in both intratumoural and peritumoural areas of tumours, was reversely related to Dukes' stage. There was no association between MVD or LVD and patients' survival (P>0.05). Angiogenesis and lymphangiogenesis occurred in CRC development, but were not related to CRC patient prognosis. PINCH may play a potential role in tumour angiogenesis.

Lymphangiogenesis in colorectal cancer—Prognostic and therapeutic aspects

Colorectal cancer (CRC) represents the second most common cause of cancer mortality in the western world. The tumors frequently show metastatic spread which affects different organs such as lymph nodes, liver and lungs. Although the pattern of spread may vary, the initial step usually involves dissemination to regional lymph nodes. At present it is clear that neovessel formation, including lymphangiogenesis, represents key events in tumor progression. However, to what extent lymphangiogenesis contributes in the progression of CRC is unclear. This work focuses on recent progress within the field of tumor lymphangiogenesis with special reference to CRC, and on novel therapeutic strategies for anti-lymphangiogenic therapies. Inhibition of metastatic spread may be achieved by restriction of lymphatic vessel growth by using targeted therapeutic strategies towards molecules involved in lymphangiogenic signalling. Such adjuvant therapeutic approaches in addition to existing therapeutic strategies may represent a favourable treatment for CRCs with higher than average risk of disease recurrence and progression.

Vascular endothelial growth factor-C expression and lymphangiogenesis in colorectal cancer

BACKGROUND: Colorectal cancer is a common cause of death throughout the world. Approximately 145,290 new cases of large bowel cancer are diagnosed each year in the United States, of which 104,950 are colon and the remainder rectal cancers. In 2005, more than 56,000 Americans will die of CRC, accounting for approximately 10 percent of all cancer deaths. In the United States, CRC ranks second to lung cancer as a cause of cancer death, and it is third both in frequency and cause of cancer death, in men and women separately. In 2004 in Europe, there were an estimated 2 886 800 incident cases of cancer diagnosed and 1 711 000 cancer deaths. The most common incident form of cancer was lung cancer (13.3 % of all incident cases), followed by colorectal cancer (13.2 %) and breast cancer (13 %). Lung cancer was also the most common cause of cancer death (341 800 deaths), followed by colorectal (203 700), stomach (137 900) and breast (129 900). According to the ASO-ASSO (Austrian Society of Surgical Oncology) in Austria CRC is the second most common cancer and the incidence rate is increasing. As in many types of cancer, the lymphatic system is the primary pathway of metastasis, representing the first step of generalisation. However, lymphangiogenesis and the mechanism of lymphatic metastasis in colorectal cancer remain unclear. Aim of this study was to investigate the prognostic role of lymphangiogenesis (LMVD), lymphovascular invasion (LVI) and the lymphangiogenic growth factor VEGF-C in a cohort of colorectal cancers. METHODS: Forty-seven cases of colorectal cancer were investigated. VEGF-C-expression, LMVD, LVI and lymphatic vessel size (LVS) were evaluated semiquantitatively by immunohistochemistry. RESULTS: A significant association was seen between LMVD and LVI (p ≤ 0.005, Mann-Whitney test). LVI correlated significantly with the lymph-node status (p = 0.04, Spearman's coefficient of correlation) and LVS (p = 0.018, Mann-Whitney test). In a mean observation time of 145 months univariate survival analysis showed a significant difference in disease-free survival (DFS) and a clear trend in overall survival (OS) between patients with and without LVI (p = 0.0148 and p = 0.14, respectively, log-rank test). Further VEGF-C-expression in tumor-cells had a significant influence on the OS in univariate survival analysis (p = 0.0391, log-rank test). CONCLUSIONS: These data provide further evidence for the important role of lymphangiogenesis and lymphovascular invasion in the prognosis of colorectal cancer implying new potential anti-tumor targets.