Invasion and Metastasis

The two hallmarks of cancer are uncontrolled proliferation and invasion. Invasion and subsequent metastasis create serious problems for the management and treatment of cancer because they have often occurred before diagnosis. Local invasion of the primary tumour into surrounding tissue makes complete surgical removal difficult, or at some sites impossible, while distant metastases may not be detected. Increasing evidence suggests invasion can occur earlier during tumour progression than previously thought. Understanding the molecular biology of the mechanism of cell invasion can help develop cancer diagnostics and therapeutics through targeting the proteins involved. The aim of our research is to define the invasion transcriptome through gene expression profiling.

Differentially expressed genes cooperate to enhance pseudopod elongation and invasion.
AP-1 activity is required by oncogenes that function in the growth factor-ras signal transduction pathway to render the cells morphologically transformed and invasive. Gene expression profiling of v-fos transformed cells demonstrates that AP-1 targets genes involved in invasion; up-regulated genes function as effectors of invasion and down-regulated genes function as suppressors of invasion. We have demonstrated that both up- and down-regulated genes contribute to pseudopod elongation and invasion. The up-regulated genes ezrin and kelch-related protein 1 (Krp1) are necessary for pseudopod elongation. Krp1 binding to lasp-1 and the co-localisation of both proteins with dynamic actin structures at the extending tip of elongating pseudopods is dependent on transformation. Lasp-1, although it is not differentially expressed, is necessary for pseudopod extension and invasion.

Fibronectin (FN) is dramatically down-regulated in v-fos transformed cells. Exposure of the transformed cells to exogenous FN inhibits invasion and results in morphological reversion with the appearance of actin stress fibres and focal adhesions. In FN treated cells Krp1 no longer co-localises with Lasp-1 to pseudopods. FN mediated morphological reversion is dependent upon integrin activation of RhoA and Rho kinase, which are not differentially expressed. The results support the concept of a transcriptionally regulated invasion programme, whereby differential expression of genes alters the cellular context and, in doing so, the activity of proteins that are not differentially expressed, so as to enhance invasion.

Histone Deacetylase activity suppresses the expression of invasion suppressor genes
Histone deacetylase activity is necessary for transformation by epigenetically silencing expression of tumour suppressors. Many components of histone deacetylase complexes including HDACs are up-regulated in v-fos transformed cells. Pharmacological inhibition of HDAC activity by trichostatin A (TSA) or valproic acid (VPA) in transformed cells inhibits invasion and induces morphological reversion with the appearance of actin stress fibres and focal adhesions at concentrations of inhibitor that do not affect proliferation or 2-dimensional cell migration. Both inhibitors also induce the expression of some, but not all, of the down-regulated genes thereby confirming that their suppression is mediated by HDAC activity. Three of the down-regulated genes (RYBP, STAT6 and PDCH2), when expressed individually in v-fos transformed cells, inhibit invasion without affecting cell morphology, the actin cytoskeleton, 2-dimensional migration or proliferation. A fourth gene, fibronectin (FN), is also suppressed by HDAC activity, and exposure of transformed cells to FN reverts their morphological transformation and inhibits invasion. Together these results indicate that the morphological transformation by the v-fos oncogene is maintained by the down-regulation of FN and that its expression is incompatible with the invasive phenotype.

TSC-36 is a bi-functional invasion suppressor
TSC-36 is down-regulated in v-fos and H-ras transformed fibroblasts and in many human tumours. Expression of TSC-36 in human lung cancer derived cell lines suppresses proliferation, whilst expression of TSC-36 in v-fos transformed cells inhibits invasion but not proliferation.

TSC-36 is a member of the SPARC family of follistatin related proteins and is a secreted heparin binding glycoprotein. TSC-36 can be divided into multiple domains; Follistatin and KAZAL domain (FS) and the extracellular calcium binding domain (EC). Recombinant TSC-36 inhibits invasion of v-fos transformed rat and human fibroblasts, H-ras transformed human fibroblasts and human HT1080 cells that are derived form a fibrosarcoma. Recombinant FS inhibits v-fos- and H-ras- transformed fibroblasts, but not HT1080 cells, whilst recombinant EC inhibits v-fos transformed fibroblasts and HT1080 cells, but not H-ras transformed fibroblasts. This demonstrates that TSC-36 is a bi-functional invasion suppressor. Surprisingly, rTSC-36, rFS and rEC inhibit MMP2 protease activity. In the FS domain the KAZAL motif is responsible for MMP2 inhibition. Thus TSC-36 is a potent and at least a bi-functional suppressor of invasion that is down-regulated in a variety of oncogene transformed cells and a wide variety of human tumours. Future work will determine the mechanism of suppression of invasion for each domain.

Gene expression profiling defines the invasion transcriptome
Human fibroblasts are relatively resistant to v-fos or H-ras mediated transformation. We expressed v-fos and H-ras in human fibroblasts to determine if the AP-1 multigenic invasion programme was activated in these cells, even though they were not fully transformed. Human fibroblasts expressing v-fos (Tif-Fos) or H-Ras (Tif-Ras) are rendered highly invasive without becoming fully transformed and their invasion is independent of the tumour suppressors p53 and p16^INK4a. These results indicate that the invasion programme can be activated in the context of normal fibroblasts and demonstrate that invasion could occur early during tumour progression. Gene expression profiling demonstrates that v-fos activates a multigenic invasion programme in human fibroblasts. This is highlighted by 10 fold up-regulation of expression of the EGF receptor that is necessary for the reduction in actin stress fibres and invasion of Tif-Fos cells, but is not required for their proliferation. Gene expression profiling indicates that the pattern of gene expression is more complex in the Tif-Ras cells compared to Tif-Fos cells in that genes involved in proliferation and invasion are targeted in Tif-Ras cells. Strikingly, there is little overlap in the genes that are up-regulated in Tif-Ras cells and Tif-Fos cells, but there is an extensive overlap in the down-regulated genes. Future research is directed towards validating the genes as members of the invasion programme.

Fig. 1: Up- and Down-regulated Genes enhance pseudopod elongation and invasion In a program the differentially expressed proteins interact and alter the activity of proteinsthat are not differentially expressed to enhance invasion