M3 and MM3 are two related mammary adenocarcinomas with different lung metastasizing abilities. In the present work we have compared the global gene expression profile of primary cultures of both tumors in order to identify genes with altered expression associated with the metastatic behavior.
To avoid stromal contamination, we performed primary cultures. Initially, we confirmed whether M3 and MM3 primary cultures preserve the metastatic potential of their original tumors by inoculating s.c. in syngeneic Balb/c mice. Animals, inoculated with MM3 primary cultures presented significantly higher incidence (95% vs. 53%, p<0.01) and number (median: 11 (0-62) vs. 1 (0-6) p<0.01) of lung metastasis than M3 ones. Next, gene expression profile was determined using Clontech Atlas Mouse cDNA microarray. A total of 88 from 1,176 genes were differentially expressed in MM3 primary cultures, being most of them (86) upregulated (>2.7-fold). The altered expression of relevant genes was also confirmed by Western blot.
Modulated genes were grouped according to their functions as signal transductors and transcription regulators (36 genes), growth factors and receptors, oncogenes and tumor suppressors genes (20 genes i.e.: TGFβ), adhesion and motility (14 genes), regulation of cell proliferation and cell death (6 genes), invasion and angiogenesis (5 genes i.e.: uPA), others (5 genes). Interestingly, the expression FN, a plasma and extracellular matrix (ECM) glycoprotein which loss has been associated with the acquisition of a malignant phenotype, was 1 of the only 2 significantly downregulated genes in the more metastatic cells (19-fold reduction p<0.01). This interesting result confirmed our previous findings that the silencing of FN gene in MM3 cells occur at transcriptional level involving the 220-bp proximal promoter region and that the re-expression of FN in these same cells reduced experimental and spontaneous metastasis development by a mechanism that does not involve the formation of the ECM. Other gene modulations presented here were also validated by our previous studies reporting that the TGF-β and uPA/uPAR systems were upregulated in the MM3 cells as compared with the M3 tumor, suggesting an effect on cell proliferation as well as in the migratory and invasive capability.
Our results, showing that the reduction of FN expression in the highly metastatic mammary tumor MM3 cells is almost a unique event in an environment of upregulated genes, places FN downregulation in a new context, and may help to understand better its role in tumor progression, supporting the hypothesis of FN as a main metastasis suppressor gene in mammary cancer.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA