Recent advantages in DNA sequencing rises new approaches of analyzing cancer genomes. Instead of looking at a cancer as a result of a series of point mutations in oncogenes or tumor suppressor genes researches assume tumors as completely different type of cells . Cancer cells pass through lots of cycles of natural selection accompanied by genome reorganization and DNA repair system modulation [25, 26]. Most of mutations in cancer genome are not driver ones but passenger. They do not improve tumor surviving ability and therefore may experience a negative selection . In recent works most laboratories all over the world concentrated at finding driver mutations in target genes to defeat the growth and/or malignancy of tumors. The most common way of searching–comparing genomes of cancer samples and adjusting healthy tissues and identification of genes significantly mutated in cancer .
The approach that was proposed by Pyatnitskiy et al. revealed 91 gene . It was suggested that these genes could be essential for melanoma surviving. Our knockdown experiments in melanoma cell lines could prove their theory.
According to Fantom5 data only less than a half of the genes (44 gene) were expressed in melanoma cell lines. Such discrepancy could be explained by a difference between patient`s samples on which the conclusions were made and cell lines presented in Fantom5: it is well known that immortal cell lines have high difference with patient samples. We also tried to exclude genes which are associated with any disease. To do it we used the most complete and numerous study of human genomes is the deCODE genetics research project.
In a list of 18 genes obtained after there were a part that confirm our hypothesis and a part that was not in a line with it. For instance, knockdown of UNC45A lead to decreased proliferation of cultured myogenic and ovarian cancer cells ; PTK2B inhibition by shRNA vectors led to reduction of multiple myeloma tumor growth in vivo as well as decreased cell proliferation, cell-cycle progression, and adhesion ability in vitro . On the other hand, there are examples of driver mutation genes: MYCT1 was down-regulated in the majority of gastric carcinoma tissues and when overexpressed, could promote apoptosis of gastric carcinoma cell lines ; TGM5 being overexpressed induces cell death, mutations in this gene are associated with acral peeling skin syndrome, knockdown effects are unknown .
For our work we selected 7 genes. For some of these genes it has been shown that knockdown can lead to decrease of viability of some cancer cell types but not for melanoma. Also, for some genes it has been shown the role in cell cycle but not in cancer cells viability. With these different genes that were interesting for our purpose we performed expression analysis in available melanoma cell lines: sk-mel-1, sk-mel-5, G361 and A375. In our experiments with knockdown we selected 4 genes (UNC45A, STK11IP, RHPN2 and ZNFX1), which have the highest expression in suitable cell line A375.
The results on knockdown experiments show that the selected genes are not essential for melanoma cells surviving, but they have influence at distinct processes in the cells. Particularly, knockdown of all of the tested genes increased the speed of cell migrating. Moreover, the viability of cells assessed by MTT test was not changed despite the Wound-healing results.
It indicates that although the genes play some role in melanoma cells life they, at least individually, are not as essential as we expected according to bioinformatical analysis. Perhaps the data shows that the contribution of the proteins of these genes to survivability is not crucial. They can make a significant contribution to the progression of turmeric processes when they are initiated by other mutations in the driver genes.
Anyway, this work of Pyatnitskiy et al. is the first try in such novel direction. For more accurate results we need to accumulate more data for every and each cancer type. For now, it seems to reveal a lot of false positive results. In this work, we used different methods to evaluate any sign of essentiality of the genes for melanoma cells surviving, but obtained controversial results that do not prove the theory. If the idea had had an extension, development and a good implementation, it could have led to appearance of novel approaches for cancer treatment.