Mutual Enhancement Of Heparanase And VEGF: A Novel Mechanism For Melanoma Progression

Malignant melanoma (MM), arising from melanocytes, is the most invasive and deadly form of skin cancer at present. Metastatic melanoma has a very poor prognosis and 5-year survival rate of less than 5%, because of being largely refractory to existing therapies. However, mechanisms responsible for melanoma progression to highly aggressive disease are not fully understood.Heparanase (HPSE-1), a mammalian endo-β-D-glucuronidase, plays an important role in invasion, metastatic potential, prognosis and poor survival of a variety of tumors. HPSE-1 participates in degradation and remodeling of the extracellular matrix (ECM) due to its degrading carbohydrate chains of heparan sulfate proteoglycans (HSPGs), the main polysaccharide component of the basement membrane (BM) and ECM. HPSE-1 overexpression has been documented in a range of human tumors and correlates closely with tumor progression. Conversely, downregulation of HPSE expression may serve as an efficient therapeutic strategy for human malignancies. Although much progress has been made in studying the role of HPSE, the mechanism for HPSE promoting tumor progression is largely unknown.Much evidence has shown that HPSE-1 is closely related to growth factors in the role of promoting tumor progression. HPSE-1 regulates the expression and activity of growth factors, mainly by its extracellular enzymatic function and participating in intracellular signal transduction of tumor cells. In these growth factors, vascular endothelial growth factor (VEGF), acting as a key factor in angiogenesis, is necessary for tumor growth and metastasis. On one hand, degradation of HSPGs by HPSE-1 liberates ECM-resident VEGF, leading to the performance of its biologic functions. On the other hand, HPSE-1 can also regulate VEGF expression by participating in intracellular signal transduction in several human non-melanoma cell lines. The release and expression of VEGF are capable of initiate relative signaling pathways, after binding to its receptors. The activation of these pathways results in upregulation of transcriptional factors related with cell proliferation, survival, invasion, and metastasis. It should be noted that in these transcription factors, some of them can bind to the HPSE-1 promoter and regulate its transcription. In general, it is possible that VEGF has an unknown function of regulating HPSE-1 expression in tumors.According to the evidence given above, we hypothesized a novel mechanism that mutual enhancement of HPSE-1 and VEGF plays a crucial role in tumor progression. Thus, the objective of this study is to testify the hypothetical mutual enhancement between HPSE-1 and VEGF and elucidate its effect on melanoma progression.1. The mutual enhancement between HPSE-1 and VEGF in MM cellsMethods and results: ①Construction and identification of HPSE-1 siRNA vectorAfter identification by restriction enzyme digestion and sequencing, the HPSE-1 siRNA vector was successfully constructed.②Transfection of vectors and the expression of transfected genesBy G418 pressure screening, HPSE-1 and VEGF cDNA were stably transfected into A2058 cells, and the expression of HPSE-1 and VEGF genes were both upregulated at mRNA and protein levels, respectively. While HPSE-1 and VEGF SiRNA were transfected transiently into A2058 cells, resulting in the obvious inhibition of HPSE-1 and VEGF expression.③The expression regulation of VEGF by HPSE-1By use of real-time quantitative RT-PCR, ELISA and Western blot, the level of VEGF expression increased due to HPSE-1 upregulation, and decreased by HPSE-1 downregulation in MM cells.④The expression regulation of HPSE-1 by VEGFAfter transfection of VEGF expression vector, addition of exogenous VEGF and RNA interference, the HPSE-1 expression also changed correspondingly whatever at mRNA and protein levels.⑤The effect of PD98059 on the mutual enhancement between HPSE-1and VEGFPD98059, as a specific inhibitor of MEK/ERK pathways, was used in our study. Due to the addition of PD98059 to HPSE-1 and VEGF stable expression cell lines respectively, the mutual enhancement between HPSE-1and VEGF was downregulated incompletely. Meanwhile, the activation of MEK/ERK pathway was also inhibited.Conclusion:For the first time, we testified in vitro that there exists a mutual enhancement between HPSE-1 and VEGF in MM cells, which could be blocked incompletely by PD98059, a specific inhibitor of MEK/ERK pathways.2. The role of the mutual enhancement between HPSE-1 and VEGF in malignant biological behaviors of MM cellsMethods and results:①Analysis of cell proliferation by methyl thiazolyl tetrazolium (MTT) assay Analysis of MTT results showed that the cell proliferation was accelerated in transfected A2058 cell lines stably expressing HPSE-1 and VEGF, and PD98059 also inhibited the acceleration of proliferation partly.②Analysis of Matrigel invasion assayThe results of Matrigel invasion assay were consistent with the cell proliferation assay by MTT. After staining, we found that both the HPSE1-transfected and the VEGF-transfected cells demonstrated an enhanced transmigration capacity, compared to the control group, as evidenced by increase of migrated cell numbers. Similarly, PD98059 also had an imcomplete inhibition.③TumorigenicityAfter 30 days of injections, the nude mice were sacrificed and tumor weight was measured. The results told us that the HPSE1-transfected and the VEGF-transfected cells showed a reduced tumor weight, after administration of Avastin, a neutralizing monoclonal antibody of VEGF.④Morphological observation of tumor tissueUsing HE staining of tumor tissue from nude mice, we found that the HPSE1-transfected cell group without Avastin revealed not only a good growth status, but also much microvasculature formation. But the same cell group with Avastin appeared much patchy necrosis. The results suggested that these morphological feasures should be contributed to by neutralization of Avastin to VEGF, which prevented tumor angiogenesis.⑤Measurement of HPSE-1mRNA level from tumor tissueIn the VEGF-transfected cell group, HPSE-1 mRNA level with Avastin from tumor tissue of nude mice was lower than the group without Avastin. It suggested that VEGF could regulate HPSE-1 expression in vivo.Conclusion:The results whatever in vitro and in vivo, showed that the mutual enhancement between HPSE-1 and VEGF endows MM cells with increased proliferation, invasion and growth. In addition, the block of their relationship by the administration of PD98059 in vitro or Avastin in vivo, could reduce and inhibit the malignant effect from the mutual enhancement between them.Generally speaking, for the first time, we proposed and testified that HPSE-1 and VEGF have a mutual enhancement in MM cells, which seems to be the souce power of promote MM cells to indefinite proliferation, resistance to apoptosis, increased ability to metastasis and angiogenesis. Conversely, blocking the relationship between them could reverse and inhibit the malignant effects on MM cells. Thus, the clarification on the mutual enhancement between HPSE-1 and VEGF will provide us with new ideas about revealing pathogenesis of tumors and define a range of novel and exciting therapeutic approaches.