The Effect Of Hypoxia-inducible Factor-1α Regulating SCF/c-kit Pathway In The Pathogenesis Of Acral Melanoma

Cutaneous malignant melanoma (CMM) is a highly malignant skin tumors with remarkable mortality rate. Acral melanoma is a major clinical subtype of CMM in Asians. Studies revealed that c-KIT gene point mutations, mostly at L576P and K642E, can be found in acral melanoma, suggesting mutation may be associated with tumor formation. On the other hand, acral CMMs are located in the extremities, which suggest that hypoxic microenvironment in extremities may associate with tumor occurrence and progression, considering tissue hypoxia is one of the basic characteristics of the tumor microenvironment.Part1. Study on c-KIT gene expression and mutation in cutaneous acral malignant melanoma of Chinese populationImmunohistochemical staining, PCR and gene sequencing methods were operated in this part. Genomic DNA was extracted from paraffin-embedded tissue from Chinese patients with melanoma in recent years in our hospital, and exon11,13and17sequences of c-KIT were amplified and sequenced. Four patients were detected with c-KIT gene mutation out of93acral CMM patients, and the mutation rate was4.3%. Immunohistochemical analysis showed that, in93cases of acral CMM, the c-kit protein expression was found in84cases (90.3%), and, in22cases of non-acral CMM, the c-kit protein expression was found in18cases (81.8%); when in situ, invasive and proximal metastatic acral CMM were compared, c-kit expression showed no significant correlation with clinicopathological features of tumors (all P>0.05).Part2. Hypoxia-inducible factor-la expression in acral malignant melanoma and its correlation with the SCF/c-kit pathwaysImmunohistochemical staining was applied to detect the expression of HIF-la protein in acral CMM, non-acral CMM, acral melanocytic nevi, and normal acral skin (as a control), and to analyze HIF-la expression and its correlation with clinicopathological features of the tumors. The results showed that HIF-1α was highly expressed in acral CMM, and was positively correlated to tumoral stages, progression and invasiveness. On the other hand, c-kit protein was expressed in acral CMM, as well, and was analyzed its relationship with HIF-1α, both of which were coordinatively expressed in the same tissue, suggesting that HIF-la and SCF/c-kit pathway may be both involved in the pathogenesis of acral CMM. Part3. Construction of stable over-expression of wild type and mutant c-KIT gene melanoma cell linesLentiviral vectors were constructed to contain the wild type c-KIT gene and the mutant type c-KIT gene, L576P and K642E, respectively. A375human melanoma cells were infected with constructed lentiviral vectors, and three groups of melanoma cell lines as A375-WT (with wild type c-KIT gene), A375-L576P and A375-K642E (including c-KIT mutant gene) were all successfully established. These constructed cell lines could provide good experimental model in vitro.ConclusionThe results showed that c-KIT gene mutations in acral CMMs were found with low frequencies in Chinese population we had studied. And the most two common mutation points were located at L576P and K642E. HIF-la protein was highly expressed in the acral CMMs, which showed coordinated expression with the c-kit protein expression in the same tissues. Above results revealed that HIF-la and SCF/c-kit pathway may be both involved in the pathogenesis of acral CMMs. Finally, stable over-expression of c-KIT wild type and mutant genes of melanoma cell lines were successfully established which could provide good experimental models in vitro to help further study of c-KIT gene mutations, hypoxia and its relationship with the SCF/c-kit pathway in melanoma.