5887

Melanoma is a highly malignant disease occurring in the skin and accounts for 2% of all newly diagnosed cancers each year. Current treatments include radiotherapy, surgery, chemotherapy and the use of biological agents such as interferon. Although selected HOX genes are variably expressed in leukemias and solid malignancies, their relationship with the neoplastic phenotype remains unclear. The dysregulation of specific HOX genes are closely associated with the onset and subsequent progression of melanoma. This study focuses on the role of a subset of the HOX genes and their potential as a target for therapeutic intervention. We have designed a synthetic peptide, HXP4, that disrupts the interaction between HOX and PBX leading to growth inhibition in vitro. An aggressive mouse melanoma, B16, was utilised to determine the efficacy of HXP4 as a therapeutic agent. Using this cell line we tested the efficacy of HXP4 in vitro. Cells were treated with HXP4 for four days and analysed. All results are expressed relative to untreated control cells. Following a 60μM dose of HXP4, no viable cells were detected as determined by trypan blue staining, suggesting that HXP4 was cytotoxic. Following treatment with a lower dose of 6μM of HXP4, and re-suspension in drug-free medium for a further 6 days, cell regrowth was observed, suggesting a cytostatic effect. BrdU analysis is in progress to establish cell cycle status. Quantative RT-PCR studies were undertaken to determine downstream targets of HXP4. B16 cells were treated with 60μM HXP4 and harvested after 1, 2 and 4 hours. HXP4 downregulated N-RAS 9-fold as compared to control cells at 24 hours. Complete loss of MAPK expression was seen 24 hours after HXP4 treatment. CD34 expression was 16-fold less in HXP4 treated cells 24 hours post treatment as compared to control. Subsequent work has focused on the in vivo response of melanoma cells to HXP4. Recipient mice were subcutaneously injected with one million B16 cells. Mice received treatment on day 11 when tumours were palpable. HXP4 was administered intraperitoneally at 15mg/kg three times a day, rested for two days and treatment repeated on day 16 at the same dose and schedule. Mice were euthanised on day 17. Anti-tumour activity was determined by measurement of tumour diameter. Mice receiving HXP4 therapy had approximately 30% less growth as compared to control treated mice. Similar results were also observed using the K1375 in vivo model. These preliminary results suggest that HXP4 is a cytostatic agent at relatively low concentrations, with a reversible antiproliferative effect. Some downstream genes regulated by disrupting the HOX-PBX interaction with HXP4 have been identified by RT-PCR, but microarray analysis will provide a more comprehensive screen for target genes. Further analysis of in vivo data is currently in progress. In conclusion blocking the interaction between HOX and PBX may represent a therapeutic strategy in melanoma treatment.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]