The Role Of Endothelial DLL4 In Renal Cell Carcinoma Growth, Invasion, Angiogenesis And Hematogenous Metastasis

Objective:Renal cell carcinoma (RCC) is a vascular-rich neoplasm, whose tumorigenesis and progression largely depend on angiogenesis. Previous studied indicated that majority of RCC harbors VHL (Von-Hippel-Lindau) gene mutation. The mutant VHL protein loses the function of degrading HIF (Hypoxia Inducible Factor) subunit, which induces RCC cancer cell secreting a lot of VEGF (Vascular Endothelial Growth Factor). The major function of VEGF is to induce angiogenesis and ultimately promotes RCC growth and metastasis. Basing on these theories, first-line target therapies are anti-VEGF drugs. However, the overall response rates of target therapies are about 30-50%, which mean the rest of them are anti-VEGF therapies refractory. Furthermore, the sensitive tumors gradually become resistant ones after a period of anti-VEGF therapy. These phenomenon indicate that there are other factors or signaling pathways controlling RCC angiogenesis and tumor progression. Endothelial DLL4 was previously reported to play an important role in angiogenesis in VEGF-dependent or independent manner. However, the VEGF-independent mechanism of regulating DLL4 was rarely documented. In addition, whether endothelial DLL4 could directly influence the functions of RCC cancer cells such as proliferation and metastasis, has not yet been elucidated. Therefore, we hypothesize that DLL4 may play dual roles both in angiogenesis and proliferation and invasion of RCC cancer cells, which determine the hematogenous metastasis of RCC. This study was to ①figure out a VEGF-independent mechanism controlling DLL4 in angiogenesis; ②figure out the impact of DLL4 in proliferation and invasion of RCC cancer cells and uncover its downstream signaling pathway;③ figure out the role of DLL4 in RCC hematogenous metastasis and its clinical significance.Materials and Methods: ①The mRNA and protein levers of DLL4 and relative genes in 120 cases of RCC specimen and 28 cases of adjacent normal tissue were detected using real-time PCR, western blot and immunochemistry, respectively. Then, the expressions of these genes were associated with some important clinical and pathological factors using univariate and multivariate analyses; ②We used flow cytometer and MTS assay to detect the proliferation ability of endothelial cell HUVEC and RCC cancer cell lines 769-P,786-O and caki-1 after modulation of DLL4 in endothelial cells. Furthermore, we also applied transwell assay to detect the invasive ability of HUVEC and RCC cancer cell mentioned above after modulation of DLL4. These assays were to determine influence of DLL4 in proliferation and invasiveness of RCC cancer cells and tumor angiogenesis. ③Bioinformatical analysis were performed to explore miRNAs targeting DLL4. Then, luciferase assay, western blot and functional assay were used to validate whether the candidate miRNA could regulate the expression and functions of DLL4 in endothelial cells. After RCC cells were cocultured with cells expressing high level DLL4 or adding recombinant DLL4 protein (rDLL4), the downstream genes/signaling pathway inside the cancer cells were detected. To examine whether these connections were clinical significant, the expressions candidate genes were also associated with DLL4 expression in RCC samples. ④We followed up the patients included to see whether DLL4 expression was associated with RCC hematogenous metastasis and effectiveness of anti-VEGF target therapies.Results:①The expression of DLL4 in RCC samples was higher than that in adjacent normal tissue and localized in the vascular endothelial cells; DLL4 expression, microvessel density (MVD) and DLL4 density (reference to endothelial cell number) increased from the non-metastatic and lymphatic metastasis groups to the hematogenous metastasis group, which showed statistical significance (p<0.05); DLL4 expression showed difference among the RCC subtypes and was upregulated in clear cell RCC (ccRCC) by increased MVD rather than increased DLL4 density relative to papillary RCC (pRCC) and chromophobe RCC (chRCC); Multivariate analysis method called logistic regression revealed that hematogenous metastasis of RCC were not only correlated with the tumor size and MVD, but also with DLL4 density (p<0.05).②Overexpression of DLL4 increased the proliferation and invasive capacities of endothelial cell HUVEC; After RCC cells were cocultured with cells expressing high level DLL4 or adding rDLL4, the proliferation and invasiveness of these RCC cells were dramatically promoted; DLL4 promoted RCC cells proliferation probably through regulating the cell cycle.③Bioinformatical prediction showed that miR-30a targeted DLL4. Luciferase and functional assays validated that miR-30a down-regulated DLL4 through binding to its 3’UTR, which influenced the functions of DLL4 in endothelial cells; Addition of DLL4 by coculture with cells expressing high level DLL4 or adding rDLL4 activated the Notch signaling pathway in RCC cells, which upregulated the MMP9 through Notch signal downstream genes Heyl. Upregulated MMP9 promoted invasiveness of RCC cells. ④After 4-year follow-up, we found that miR-30a and DLL4 expression density predicted hematogenous metastasis of RCC. Furthermore, tumors with high level DLL4 density were more sensitive to anti-VEGF target therapies.Conclusions:①Endothelial DLL4 promoted RCC hematogenous metastasis through increasing angiogenesis, as well as growth and invasiveness of RCC cells. ②Mechanism dissection showed that DLL4 activated Notch/Hey 1/MMP9 signal to promote RCC progression. ③Down-Regulated miR-30a in ccRCC promoted angiogenesis by upregulating DLL4. ④DLL4 expression was associated with hematogenous metastasis of RCC and effectiveness of target therapies. Targeting the DLL4/Notch/Heyl/MMP9 signal pathway may be a new approach to battle RCC and a combined therapy using an-VEGF and anti-DLL4 drugs may be better to suppress RCC progression.