Abstract B33: PTTG1 expression predicts sensitivity to the Src inhibitor Saracatinib in ovarian cancer cell lines

Abstract

Abstract Background: Increasing numbers of tyrosine kinases have been investigated as potential targets in molecular cancer therapy. Src, a nonreceptor tyrosine kinase, is one such attractive target since it is highly expressed in many malignancies including ovarian cancer, and plays a key role in tumor progression and metastasis. In clinical trials, however, Src kinase inhibitors exhibited limited activity in unselected patients. To understand the detailed mechanisms of Src inhibition, we developed a gene expression profile using a panel of ovarian cancer cell lines with known IC50 values to the Src inhibitor saracatinib to identify genes likely to associate with sensitivity/resistance to saracatinib. Among the genes identified, we sought to find and characterize a gene that could be predictive of response to saracatinib. Methods: We utilized a Src kinase inihibitor, saracatinib (AZD0530), which was kindly provided by AstraZeneca. We performed MTT based cell proliferation assays for 18 ovarian cancer cell lines to determine their sensitivities to saracatinib, and classified them as either sensitive or resistant according to their IC50 data. The mean of log10(IC50) across 18 lines was used as a cutoff value. A gene expression profile associated with the Src inhibitor sensitivity was developed by processing extracted RNA from those cell lines and running Affymetrix Human Gene 1.0 ST arrays. Data normalization and statistical analysis were conducted using Partek Genomics Suite software. To explore the expression changes in protein level for the selected gene before and after treatment with saracatinib, we performed western blots for several drug-sensitive and resistant cell lines. Cells were treated for 24 hours with physiological concentrations of saracatinib. Results: Out of eighteen ovarian cancer cell lines, 11 and 7 cell lines were categorized into sensitive and resistant groups respectively based on the results from cell proliferation assays. The mean of log10(IC50) was 1.51 μM, which was used as cutoff value to define as sensitive or resistant. Statistical analysis for the gene expression profile detected seven differentially expressed genes between the two groups (p < 2.31×10−5, two sample t-test for sensitive group versus resistant group and false discovery rate of < 0.05). Among them, we focused on pituitary tumor transforming gene 1 (PTTG1; securin) since it is involved in a variety of cancer-related processes such as cell transformation, mitotic regulation and angiogenesis. In addition, previous studies have shown higher expressions of PTTG1 gene in many cancers, including ovarian cancer, when compared with normal tissues. We found that PTTG1 gene expression levels were higher in resistant lines (mean resistant/sensitive ratio = 2.0, p-value = 5.74×10−6). Interestingly, our western blot results demonstrated that in sensitive cell lines, the protein level of PTTG1 was significantly reduced by treatment with saracatinib, but no reduction was seen in the resistant lines. These results indicate that enhanced and preserved PTTG1 expression correlates with resistance to Src inhibition. The ability of this gene to work as a predictive biomarker for saracatinib sensitivity should be further investigated in practical tumor specimens. Conclusions: Highly expressed PTTG1 gene correlates with resistance to Src inhibition. PTTG1 could be a predictive biomarker for sensitivity to Src kinase inhibitor.