Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets.
BACKGROUND
Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alphavbeta3-integrin and low levels of RHOC.


METHODS
Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified.


RESULTS
We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library.


CONCLUSIONS
This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.
Sousa
Josane F
JF
Department of Cellular and Molecular Biology and Pathogenic Bioagents of Faculty of Medicine of Ribeirão Preto - University of São Paulo, Ribeirão Preto, SP, Brazil. jdfsousa@gmail.com
Espreafico
Enilza M
EM
D045744:Cell Line, Tumor; D002999:Clone Cells; D018076:DNA, Complementary / Q000032:analysis; D018450:Disease Progression; D015972:Gene Expression Regulation, Neoplastic; D005819:Genetic Markers; D006684:HLA-DR Antigens / Q000378:metabolism; D059805:HLA-DR alpha-Chains; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D059648:Kisspeptins; D008545:Melanoma / Q000235:genetics* / Q000473:pathology / Q000556:secondary*; D009367:Neoplasm Staging; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D000075024:rhoC GTP-Binding Protein
D018076:DNA, Complementary; D005819:Genetic Markers; D006684:HLA-DR Antigens; D059805:HLA-DR alpha-Chains; D039302:Integrin alphaVbeta3; C106628:KISS1 protein, human; D059648:Kisspeptins; D025521:Tumor Suppressor Proteins; C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D000075024:rhoC GTP-Binding Protein