The use of the soluble protein SLIT2 as a potential therapeutic in lung and breast cancer.

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The SLIT2 gene encodes a 200kDa secreted protein, which functions as the endogenous ligand of the Robo1 transmembrane receptor. The SLIT2 protein is cleaved to yield 140kDa and 60kDa fragments. Although best known for its role in the guidance of axonal crossing of the midbrain, SLIT2 has been shown to play a role in the pathogenesis of several types of human malignancy. Genetic studies show LOH at the SLIT2 locus (4p15.2) is a common event in lung and breast cancers, and SLIT2 is subject to tumor specific promoter hypermethylation, correlating with a loss of expression in a range of human cancers (lung (NSCLC 53%, SCLC 36%), breast (43%), colorectal (72%) and glioma (59%)). Furthermore reintroduction of SLIT2 has been shown to lead to a reduction in cell growth in lung and breast cancer cell lines (46-84%). This data coupled with its soluble nature suggests that SLIT2 could be used as a therapeutic in the treatment of lung and breast cancer. Treatment of a panel of lung (11) and breast (2) cancer cell lines with soluble SLIT2 (300ug/ml) (obtained from conditioned media from cos7 cells transfected with SLIT2) led to a reduction in colony formation and cell growth (21-98%). While this confirms that soluble SLIT2 can mediate cell death it also suggested that different cell lines have different sensitivities to treatment. After treatment with different concentrations of SLIT2 protein (0.003-300ug/ml), the cell lines could be clearly classified based on their IC50 values into 3 distinct groups “sensitive” (IC50<0.3ug/ml), “intermediate” (IC50 approx 30ug/ml) or “resistant” (IC50>300ug/ml). Interestingly the HCC38 breast cancer cell line (homozygous deletion of the Robo1 receptor) displayed “intermediate” sensitivity to SLIT2 (IC50 approx 3ug/ml). This result is surprising as loss of its receptor would be expected to lead to resistance to SLIT2 mediated cell death, suggesting that SLIT2 mediates at least part of its effects through Robo1 independent pathways. The normal immortalized human bronchial epithelial cell line (HBEC) was resistant to treatment with SLIT2, suggesting a tumor specific mechanism of action. Affymetrix microarray expression analysis of “sensitive” and “resistant” lung cell lines revealed a gene signature for SLIT2 sensitivity. In summary our data suggest that soluble SLIT2 can act as a potent therapeutic drug in in-vitro assays, with cell lines showing differing sensitivity to treatment. Normal human HBEC appear resistant suggesting a tumor specific function for SLIT2. These data suggest that SLIT2 has potential as a chemotherapeutic agent requiring further study in in-vitro and in-vivo models. The detection of a SLIT2 sensitivity signature could enable us to better discern the mechanisms by which SLIT2 mediates its cytotoxic action, and could be used to predict “sensitivity” or “resistance” to SLIT2 in cell lines and primary tumors.