Therapeutic Potential Of Hai1 And Hai2 As Anticancer Agents

The last decade has witnessed the rapid increase of knowledge available on the role of HGF and c-Met in human cancer. HGF stimulates, through c-Met coupling, the metastatic spread and angiogenesis of tumours. Therefore, the blockade of HGF signalling has become a strategy to inhibit tumour invasion and metastasis, as indicated in recent articles (55, 81, 165). An increasing number of reports support this theory, as shown by recent NK4 studies.

Figure 6. (A) Ribozyme transgenes were used to inhibit HAI-1 and HAI-2 expression in MDA-MB-231 breast cancer cells (Wild type = control; HAI-1 suppressed = HAI-1 KO; HAI-2 inhibited = HAI-2 KO). HAI-1 KO and HAI-2 KO breast cancer cells revealed a dramatically more aggressive nature compared to the control group. (B) Crystal Violet Staining of Invaded Breast Cancer Cells. (i) Wild type breast cancer cell control group following 72 hour incubation. (ii) The elimination HAI-1 resulted in a significantly higher degree of breast cancer cell invasion. (iii) Suppression of HAI-2 expression dramatically influenced the nature of these cells, resulting in enhanced tumour cell invasion. (C) Wound Closure Migration Assay. The knockdown of HAI-1 or HAI-2 expression significantly increased the migratory nature of the breast cancer cells. (D) Recombinant HAI's were used to potently reduce, MRC5 fibroblast-induced, breast cancer cell invasion. (E) Retroviral Expression of HAIs within human MRC5 fibroblasts inhibited the ability of these fibroblasts to induce breast cancer cell invasion. (Adapted from Parr et al, 2006. Int J Cancer; 119: 1176-1183).

Figure 6. (A) Ribozyme transgenes were used to inhibit HAI-1 and HAI-2 expression in MDA-MB-231 breast cancer cells (Wild type = control; HAI-1 suppressed = HAI-1 KO; HAI-2 inhibited = HAI-2 KO). HAI-1 KO and HAI-2 KO breast cancer cells revealed a dramatically more aggressive nature compared to the control group. (B) Crystal Violet Staining of Invaded Breast Cancer Cells. (i) Wild type breast cancer cell control group following 72 hour incubation. (ii) The elimination HAI-1 resulted in a significantly higher degree of breast cancer cell invasion. (iii) Suppression of HAI-2 expression dramatically influenced the nature of these cells, resulting in enhanced tumour cell invasion. (C) Wound Closure Migration Assay. The knockdown of HAI-1 or HAI-2 expression significantly increased the migratory nature of the breast cancer cells. (D) Recombinant HAI's were used to potently reduce, MRC5 fibroblast-induced, breast cancer cell invasion. (E) Retroviral Expression of HAIs within human MRC5 fibroblasts inhibited the ability of these fibroblasts to induce breast cancer cell invasion. (Adapted from Parr et al, 2006. Int J Cancer; 119: 1176-1183).

NK4 is a variant of HGF that competitively blocks HGF binding to the c-Met receptor, thereby reducing HGF-related pro-metastatic effects. NK4 acts as an antagonist of HGF-Met coupling, and has demonstrated significant potential as a novel anti-cancer agent (166-174). Similarly, small molecule inhibitors to the HGF receptor have also been recently reported, e.g., PHA-665752 and SU5416 (175, 176).

The HAI inhibitors address the issue of HGF suppression from a different angle to NK4, in that HAI inhibitors prevent pro-HGF being converted into the active form in the first instance. Pro-HGF is ineffective as a pro-metastatic factor prior to its interaction and subsequent activation via the HGFA or matriptase proteases (94, 105, 177, 178). The conversion of pro-HGF to the biologically active HGF is the critical limiting step in the HGF regulatory system and will play a key role in the control of metastatic events. If no active HGF is available in the tumour micro-environment, tumour cells, or tumour-infiltrated endothelial cells will not receive pro-invasive signals even if their c-Met levels ensure high ligand sensitivity.

The importance of suppressing pro-HGF processing was recently highlighted in an interesting study that described, through a single amino acid substitution in the proteolytic site, how an uncleavable form of pro-HGF suppressed tumour growth and dissemination in a mouse model (179). HAI-1 and HAI-2 are two novel Kunitz-type serine protease inhibitors that inhibit the influence of a range of proteases, most notably HGFA and matriptase (91, 92, 125). Our most recent studies have generated recombinant HAI-1 and HAI-2 proteins to further assess the function and anti-cancer properties of these protease inhibitors. Crucially, these HAI studies further implicated the potential value of the HAI-1 and HAI-2 as anti-cancer agents (160). Addition of either rHAI-1 or rHAI-2 to cultured fibroblasts significantly reduced the production of biologically active HGF (Figure 6). These HAI-1 and HAI-2 proteins also dramatically reduced fibroblast-mediated breast cancer cell invasion and migration (160). These exciting results may be due to the ability of the HAI's to interact and inhibit the pro-invasive function of matriptase, HGFA and hepsin. Therefore, the natural ability of fibroblasts to facilitate cancer cell invasion had been suppressed by the addition of recombinant HAI proteins.

Suppression of HGF activity is due to a shift in the balance between the HGF-converting proteases and the HAI inhibitors. This shift between HGF activation and HGF activation suppression is the crucial step controlling the metastatic influence of HGF, and may represent a method of limiting tumour progression. The presence of our HAI-1 or HAI-2 proteins altered the balance to favour suppression of HGF activity. Therefore, the ability of HGFA, matriptase, and hepsin to enhance the potent effects of HGF on tumour cells, has been quenched through the presence of HAI-1 and HAI-2. Importantly, the most effective suppression of breast cancer cell invasion was observed when rHAI-1 and rHAI-2 were used in combination. These inhibitors appear to possess additional and individual inhibitory properties (98, 113, 122, 180, 181), and may therefore benefit from being deployed in tandem. However, it has to be pointed out that the development of the HGF activation inhibitors is in the early stages. The true clinical value of using HAIs as therapeutic modalities requires substantial work.

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