The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis

Abstract

Background: The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples.

Methods: We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes.

Results: We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including SPRR1A/B, KRT16/17, CD24, LOR, GATA3, MUC15, and TMPRSS4, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as MAGE, GPR19, BCL2A1, MMP14, SOX5, BUB1, RGS20, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (SPP-1, MITF, CITED-1, GDF-15, c-Met, HOX loci) and suppressor genes (PITX-1, CST-6, PDGFRL, DSC-3, POU2F3, CLCA2, ST7L), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype.

Conclusion: The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.

Figure 1

Clinical primary and metastatic melanoma samples procured and cryopreserved at the time of surgery. A: Intraoperative illustrations of the spectrum of PCM and MM samples procured. The PCM represents varying tumor thicknesses, measured utilizing Breslow's depth of invasion. All procured lymph node metastases were macroscopically involved, often completely replacing the entire lymph node parenchyma. Distant metastatic (subcutaneous and solid organ) melanoma often exhibited varying degrees of pigmentation, however, surrounding stroma was avoided in procurement of melanoma samples. B: A distinct change in the gene expression patterns is apparent within the comparative groups of thin/I.M. to thick PCM samples. Gene over-expression (upper graph) is evident at the I.M. thickness sample set, with an average Breslow's tumor thickness of 2.1 mm and 19 mm for thick melanomas. Contrary, there is a decrease in gene expression (lower graph) of the same set of genes, with a comparative difference in gene down-regulation evident at the same interphase of I.M. to thick PCM. Proceeding from left to right: normal skin, BCC, SCC, MIS, I.M., thick primary, metastatic melanoma (subcutaneous, lymph node and distant) and melanoma cell lines derived from patients with stage IV melanoma.

Figure 2

Comparative gene expression analysis of putative oncogenes and tumor suppressor genes. A: Comparative gene expression analysis utilizing qPCR data of putative tumor suppressor genes (CST6, DSC3, PITX1, POU2F3) and oncogenes (GDF15, SPP-1) in PCM (n = 7) and MM (n = 13) samples. Relative quantitation of target gene expression for each sample was determined using the equation 2^-ΔΔCt, where GAPDH was used as the internal reference and normal skin as the calibrator. Values were Log base 10 transformed (y-axis) so that all values below zero represent a down-regulation in gene expression and values above zero represent an up-regulation in gene expression, compared to normal skin. B: Correlative microarray analysis of gene expression levels in primary and metastatic melanoma samples compared with normal skin. The statistical differences of gene expression between primary (PCM) and metastatic melanoma (MM) samples were analyzed by Wilcoxon's signed rank test; two-tailed significance level was set at α = 0.05. Compared to PCM samples (n = 7), the expression levels of 4 putative tumor suppressor genes (CST6, p < 0.0001; DSC3, p < 0.0001; PITX1, p = 0.0043, POU2F3, p < 0.0001) were significantly decreased in MM samples (n = 40), while the expression of putative oncogenes (GDF15, p = 0.0027; SPP1, p < 0.0001) were significantly increased in MM samples.

Figure 3

Semi-quantitative RT-PCR analysis of oncogene and tumor suppressor genes. A: A panel of 12 putative oncogenes and 6 TSG were analyzed, with the relative levels of mRNA expression as follows: negative band (-), faintly visible band (+/-), visible band (+), strongly visible band (++), N = Not Done. β-actin served as the internal comparative control. The grey values of PCR products of each gene are analyzed by the AlphaEase 3 software and standardized according to β-actin in every sample. B: a, b, Daughter melanoma cell lines secrete SPP1 (osteopontin). The melanoma cell lysates and conditioned cell-free media was resolved by SDS-PAGE and transferred to a PVDF membrane. The blot was probed with anti-SPP1 antibody. c-f, Antibodies for DSC-3 (c), CLCA2 (d), PDGFRL (e) and α-tubulin (f) as an internal control. Lanes 1–3 are PCM and lanes 4–6 are MM cell lines.