Impact Of Overexpressing EGFR In A Human Melanoma Brain Metastasis Cell Line H1

Background and ObjectiveMelanoma is a highly invasive and metastatic cancer which can originate in skin, mucosa, uvea and leptomeninges. Although only 4% of skin cancers are melanomas, but which leads to nearly 80% of skin cancer-related deaths. Over the last 60 years the incidence of melanoma increases approximate 700% and mortality has also increased by 165%. Now the preferred treatment is still tumor removal by surgery, but the cure rate largely depends on early detection. Melanoma metastasizes quickly, and because of poor prognosis and low response rate to standard chemotherapy the median overall survival of metastatic melanoma is less than one year. Unfortunately, the under-lying biological mechanisms manipulating melanoma development and metastasis have not been clarified clearly. The epidermal growth factor receptor (EGFR) which is a 170-kDa transmembrane tyrosine kinase receptor belong to the ErbB family of receptor tyrosinase kinases (TKIs), including ErbB2 (HER2 orNeu), ErbB3 (HER3) and ErbB4 (HER4). Both epidermal growth factor (EGF) and transforming growth factor a (TGF-a) are its ligands. EGFR is known to be correlated with tumor progression and poor outcome in many human neoplasms. [6] EGFR is also involved in progression and metastasis of melanomas. Therefore the contribution of EGFR makes EGFR become a promising target for melanomas therapy. Our research is to clarify the contributions of EGFR to the metastasis mechanism in a human melanoma brain metastasis cell line (H1), thus provide a theoretical basis for targeted therapy of melanoma.Material and MethodsA human melanoma brain metastasis cell line (H1) which had already established in our laboratory was chosen. Cells were transfected with EGFR wild-type to get EGFR overexpression cell line (HIEGFRwt), at the same time cells transfected with green fluorescent protein GFP (H1GFP) were as control. Observing the results of wound healing assay with different concentrations of epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) stimulated. Colony formation assay was used to investigate the effect of EGFR on the cell independent viability and colony forming ability. Western blot was used to explore the signaling pathways activated by EGFR under different concentrations of EGF or TGF-a stimulated. Furthermore in vivo we observed the tumor development and metastasis with MRI after H1EGFRwt and HI GFP cells intracardially implanted into nodscid mice. ResultsWound healing experiment showed that EGFR increased the migration ability of H1 cell line when stimulated by EGF and TGF-a, compared with the control group the healing rate of H1EGFRwt cells was significantly improved (P<0.05). Colony formation assay confirmed that the independent survival ability and colony-forming ability were significantly improved by overexpressing EGFR in H1 cells compared with control. Western blot showed EGFR can activate all these downstream signals including Phospho-Phosphoinositide-specific phospholipase C (pPLC y), Phospho-signal transducer and activation of transcription 5 (pStat 5),Phospho-signal transducer and activation of transcription 3(pStat 3),Phospho-Akt (Ser473)(pAkt) and Phospho-p44/42 MAPK(pMAPK) when stimulated by EGF. However, when treated with the same concentration of TGF-a, none of these signals were activated. MRI results showed that after HIEGFRwt cell implantation, tumor signals can only be detected in brain, compared with the control group no meaningful discrepancy was found.ConclusionOverexpressing EGFR improved the independent survival ability and colony forming ability of Hlcells in metastasis mechanism. When stimulated with EGF and TGF-a, overexpressing EGFR can significantly improve the migration ability of HIEGFRwt cells. Further, all the downstream signals including pPLC y, pSTAT 5, pSTAT 3, pAKT, and pMAPK can be activated by EGF but can’t be activated by the same concentration of TGF-a, the reason of which is not clear. Similarly, for the data of vivo experiment, we believe that more animal experiments including more mice and PET-CT which can dynamically monitored the growth and metastasis of melanoma need to be done to confirm the results. In conclusion, overexpressing EGFR might play a role in promoting melanoma metastasis, but more vitro and vivo studies still need for further study, besides EGFR may become a promising target for melanoma metastasis therapy, and our study also provided some theoretical basis.