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. 2008 Jan 22:8:19.
doi: 10.1186/1471-2407-8-19.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential _targets

Josane F Sousa  1 , Enilza M Espreafico
Affiliations

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential _targets

Josane F Sousa et al. BMC Cancer. .

Abstract

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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
KISS1 and RHOC mRNA expression in a panel of RGP, VGP and metastatic melanoma cell lines. Comparison of the expression levels of the KISS1 metastasis suppressor gene (A) and the small GTPase RHOC (B) among melanoma cell lines of different stages of tumor progression supported the selection of WM1552C cell line as the RGP representative for suppression subtractive hybridization against a pool of metastatic cell lines. Samples of 20 μg of total RNA from different melanoma cell lines were submitted to electrophoresis in 1% agarose-formaldehyde gel and transferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech) by standard methods. Fragments of the indicated genes were radiolabeled with [α-32P]-dCTP by random-priming (Rad-prime kit, Invitrogen) and used as probes for Northern blot hybridization. In order to correct for loading differences, after stripping, the blots were probed with a ACTB (β-actin) cDNA fragment.
Figure 2
Figure 2
Subtracted cDNA profiles of the RGP and metastatic (Met) cells. PCR-1 represents the PCR products generated using a single primer directed towards both adaptors, after 27 amplification cycles from two duplicate samples of subtracted (S1 and S2) or non-subtracted (NS) cDNA of the RGP (WM1552C) and the metastatic (a pool of WM9, WM852, 1205Lu and WM1617) cell lines. PCR-2 represents the PCR product generated after 10 amplification cycles by nested-PCR using a specific primer for each adaptor. Note the difference between the subtracted and non-subtracted profiles.
Figure 3
Figure 3
Validation by Northern blot and RT-PCR of the expression pattern of seven genes identified in the SSH libraries. Frames depict the names of cell lines used in the construction of the libraries. The inserts of cDNA clones corresponding to the genes DCN (decorin) (A), ALS2CR7 (B) and MBOAT1 (C) of the RGP library; YWHAZ (14-3-3 ξ) (D) identified in both libraries; and MITF (E) and PLP1 (F) from the Met library were isolated and used as probes for hybridization in Northern blots containing total RNA from the melanoma cell lines indicated above the panels – Blank lanes mean that the corresponding cell line was not included in the Northern blot, and were introduced to allow alignment among panels. Northern blots were prepared as described in Figure 1. HLA-DRA (G) identified in the Met library was validated by RT-PCR. For RT-PCR, total RNA samples (2 μg) from the indicated cell lines were, after DNase treatment, submitted to reverse transcription with Superscript II (Invitrogen) using oligo dT as primer and the cDNA was used as template for PCR amplification with HLA-DRA primers. After 25, 28, 30 and 32 amplification cycles, 5 μl aliquots were collected for agarose gel electrophoresis. As endogenous control, a pair of primers for the ACTB (β-actin) mRNA was used. C: Control RT-PCR amplification using as template RNA (DNase treated) without prior reverse transcription.
Figure 4
Figure 4
Genes from distinct chromosome locations are differentially enriched between the RGP and Met libraries. Chromosome locations of all genes/ESTs were obtained from GenBank accession number reports or through BLAT alignment. (A) Represents the total number of genes per human chromosome for each library; (B and C) Represent the chromosome locations for all genes identified in the RGP (B) and Met (C) libraries, along the length (bp) of all human chromosomes. The absence of genes mapping to Y chromosome in the RGP library is not explained by lack of this chromosome since the RGP cell line WM1552C was obtained from a male patient.
Figure 5
Figure 5
Genes identified in the SSH library distinguish non-neoplastic from neoplastic tissues and primary from metastatic melanomas in a microarray study on melanoma progression. Expression data from the microarray analysis by Haqq et al [16] were collected for the genes identified in the RGP and Met libraries. The expression data for each gene group were submitted to SAM (FDR = 0) in a two-class analysis for detection of genes differentially expressed between primary and metastatic tumors and between non-neoplastic (skin and melanocytic nevi) and neoplastic (primary and metastatic melanomas) samples. The results from SAM analysis were extracted using SAMTERS and visualized by CLUSTER 3.0 and Java TreeView – Red and green squares represent genes up-regulated and down-regulated, respectively. (A) Expression profiles from primary and metastatic tumors for genes from the RGP library. (B – C) Expression profiles from non-neoplastic and neoplastic samples for genes from the RGP (B) and Met (C) libraries. Vertical blue lines on the left side indicate: (B) Genes from the RGP library that showed up-regulation in non-neoplastic samples; and (C) Genes from the Met library up-regulated in neoplastic tissues.
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