Gene array and fluorescence in situ hybridization biomarkers of activity of saracatinib (AZD0530), a Src inhibitor, in a preclinical model of colorectal cancer

Abstract

Purpose: To evaluate the efficacy of saracatinib (AZD0530), an oral Src inhibitor, in colorectal cancer (CRC) and to identify biomarkers that predict antitumor activity.

Experimental design: Twenty-three CRC cell lines were exposed to saracatinib, and baseline gene expression profiles of three sensitive and eight resistant cell lines in vitro and in vivo were used to predict saracatinib sensitivity in an independent group of 10 human CRC explant tumors using the gene array K-Top Scoring Pairs (K-TSP) method. In addition, fluorescence in situ hybridization (FISH) and immunoblotting determined both Src gene copy number and activation of Src, respectively.

Results: Two of 10 explant tumors were determined to be sensitive to saracatinib. The K-TSP classifier (TOX>GLIS2, TSPAN7>BCAS4, and PARD6G>NXN) achieved 70% (7 of 10) accuracy on the test set. Evaluation of Src gene copy number by FISH showed a trend toward significance (P = 0.066) with respect to an increase in Src gene copy and resistance to saracatinib. Tumors sensitive to saracatinib showed an increase in the activation of Src and FAK when compared with resistant tumors.

Conclusions: Saracatinib significantly decreased tumor growth in a subset of CRC cell lines and explants. A K-TSP classifier (TOX>GLIS2, TSPAN7>BCAS4, and PARD6G>NXN) was predictive for sensitivity to saracatinib. In addition, increased activation of the Src pathway was associated with sensitivity to saracatinib. These results suggest that FISH, a K-TSP classifier, and activation of the Src pathway have potential in identifying CRC patients that would potentially benefit from treatment with saracatinib.

Fig. 1

The effects of saracatinib on proliferation of CRC cell lines. A, 23 CRC cell lines were treated with saracatinib (0.0625 μmol/L → 2 μmol/L) and assayed by SRB 72 h after exposure to drug. Cell lines (H508, LS180, and LS174T) with an IC₅₀ of <1 μmol/L were considered sensitive to saracatinib. Cell lines are listed in order from the most resistant to the most sensitive; (R), resistant; (S), sensitive. B, two sensitive and two resistant cell lines (identified from the SRB assay) were injected in a xenograft model to confirm the antiproliferative effects of saracatinib in vivo. When tumor volumes reached ~200 mm³, mice were randomized and treated with saracatinib 50 mg/kg/d by oral gavage for 7 to 28 d. Tumor size was evaluated twice per week by caliper measurements using the formula: tumor volume = (length × width²) × 0.52. Treatment with saracatinib resulted in a significant decrease in growth in the LS180 (*, P < 0.05) and LS174T (***, P < 0.001). C, cell cycle was analyzed in two sensitive (H508 and LS174T) and two resistant (SW620 and SW480) cell lines by flow cytometry. Treatment with saracatinib resulted in a G₁ cell cycle arrest (*, P < 0.05) in the sensitive cell lines. There were no effects on cell cycle in the resistant cell lines.

Fig. 2

Evaluation of Src gene copy number by FISH. A, representative graphs of interphase nuclei showing normal copies (~2 genes per cell) of the Src gene (CRC007). B, gain (~5.3 gene copies per cell) in Src gene copy (CRC027).

Fig. 3

Heat map of the K-TSP gene pairs for predicting saracatinib sensitivity. A, relative gene expression profiles in the training set of the three gene pairs in (three sensitive and eight resistant) CRC cell lines in vitro and in vivo. The three gene pairs that predict sensitivity to saracatinib include TOX > GLIS2, TSPAN7 > BCAS4, and PARD6G > NXN. High (red) and low (green) gene expression. B, heat map of expression in the validation set of the three gene pairs. CRC006 and CRC007 were predicted to be sensitive.

Fig. 4

Three gene pairs identified by K-TSP predicts saracatinib sensitivity in an independent test set. A, prospective validation of efficacy in 10 xenografts predicted by the K-TSP classifier. The 10 xenografts predicted for saracatinib sensitivity were treated with saracatinib 50 mg/kg/d by oral gavage for 28 d. Tumor size was evaluated twice per week by caliper measurements using the formula: tumor volume = (length × width²) × 0.52. TGI was calculated by relative tumor growth of treated mice divided by relative tumor growth of control mice × 100. Cases with a TGI of <50% were considered sensitive, and TGI of >50% were considered resistant to saracatinib. Two xenografts (CRC006 and CRC040) were sensitive to saracatinib (TGI ≤ 50%), and eight xenografts were resistant to saracatinib (TGI > 50%). CRC001, CRC006 (predicted sensitive), and CRC040 (predicted resistant) by the K-TSP classifier were misclassified. Columns, mean (n = 8-0 tumors per group); bars, SEM. *, significance (P < 0.05) compared with vehicle-treated tumors. B, representative graph of a sensitive tumor (CRC007) and (C) resistant tumor (CRC027) treated with saracatinib.

Fig. 5

Saracatinib-sensitive CRC cell lines and explant have increased activation of Src and downstream mediators. A, the sensitive cell lines H508 and LS174T had greater activation of Src, FAK, and Stat-3 when compared with the resistant cell lines SW620 and SW480. Treatment with saracatinib (500 nmol/L) for 1 h resulted in a decrease in the phosphorylation of Src, FAK, and Stat-3 (n = 3). B, evaluation of Src, FAK, and Stat-3 activation in CRC explants by immunoblotting showed that CRC007 and CRC040, sensitive tumors, had the highest activation of Src and FAK but not Stat-3. C, representative graph of two tumors (CRC007 and CRC027) treated with saracatinib (50 mg/kg/d) showed a decrease in the activation of Src. D, immunohistochemical staining of p-Src (Y416) shows positive staining in the sensitive tumor (CRC007) and negative staining in the resistant tumor (CRC027).