Common PIK3CA mutants and a novel 3' UTR mutation are associated with increased sensitivity to saracatinib

Abstract

Purpose: Dysregulation of the phosphoinositide 3-kinase (PI3K) and Src signaling pathways commonly occur in colorectal cancer. Mutations in the PIK3CA gene are associated with an increase in severity of disease and worse clinical outcomes. Elevated levels of Src have been identified in premalignant lesions and are suggested to play a central role in tumor progression. Because these pathways appear to enhance tumor growth and metastasis, molecularly targeted agents for both pathways are currently being evaluated in early-phase clinical trials.

Experimental design: We used colorectal cancer cell lines and a patient-derived explant model to investigate the efficacy of saracatinib. Mutations in the PIK3CA were evaluated to examine the association between mutations in the PIK3CA gene and sensitivity to saracatinib.

Results: We have identified a subset of patients with a PIK3CA (exon 9 and 20) mutation with increased sensitivity to saracatinib. A novel 3' untranslated region (UTR) mutation was also shown to be associated with increased sensitivity to saracatinib and have a reduced affinity for miR-520a and miR-525a. Importantly, we show that Src inhibition reduces the interaction between Src and p85, subsequently decreasing Akt-dependent signaling.

Conclusion: These results indicate that a personalized approach in targeting Src in PIK3CA-mutant patients with colorectal cancers may prove effective in a subset of patients with this genetic alteration.

Figure 1

Saracatinib effects on tumor growth in colorectal cancer explants. A, seventeen colorectal cancer explants were treated with saracatinib 50 mg/kg/d by oral gavage for 28 days. Tumor size was evaluated twice per week by caliper measurements by 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 < 50% were considered sensitive, TGI > 50% were considered resistant to saracatinib. Two xenografts (CRC007 and CRC040) were sensitive to saracatinib (TGI ≤ 50%) and 15 xenografts were resistant to saracatinib (TGI > 50%). Columns, mean (n = 8–10 tumors per group); bars, SE; and *, significance (P < 0.05) compared with vehicle-treated tumors. Mutational status of PIK3CA, PTEN, KRAS, APC, CTNNB1, and p53 are shown for each explant. Mutations in the PIK3CA gene were identified in CRC007 [3′ UTR; (c.*19T>C)], CRC040 (E542K), CRC042 [3′ UTR; (c.*19T>C)], and CRC020 (E542G). B, the relationship between PIK3CA mutation and saracatinib effects (TGI) in colorectal cancer explants. A significant association (P = 0.0157) between PIK3CA mutants and sensitivity was identified. CRC, colorectal cancer; WT, wild-type.

Figure 2

PTEN-null cells exhibit sensitivity to saracatinib in a xenograft model. The (A) PTEN-null (−/−) and (B) wild-type (+/+) cell lines were injected in axenograft model to examine the antitumor effects of saracatinib in vivo. When tumor volumes reached approximately 200 mm³, mice were randomized and treated with saracatinib 50 mg/kg/d by oral gavage for 12 days. Tumor size was evaluated twice per week by caliper measurements by the formula: tumor volume = (length × width²) × 0.52. Treatment with saracatinib resulted in a significant decrease in growth in the PTEN-nul cell line (*, P < 0.05). C, evaluation of the activation of Src and Akt in PTEN wild-type and -null tumors. Baseline levels of p-Src and p-Akt were increased in the PTEN-null tumor when compared with wild-type.

Figure 3

Effect of saracatinib on the Src and PI3K signaling pathways. A, treatment with saracatinib (0.5 µmol/L) at 30 minutes, 1, 4, 8, and 24 hours resulted in a decrease in the phosphorylation of Src, FAK, and Stat-3 in the LS180 sensitive cell line (N = 3). Src inhibition also decreased the activation of PDK-1, AKT, and S6 and increased protein levels of p27^kip1. B, co-immunoprecipitation (IP) of Src showed a decrease in binding to p85 at 0.5, 1, and 4 hours after treatment with saracatinib. Conversely, immunoprecipitation of p85 resulted in an increase in binding the p110-α subunit (N = 3). C, the activation of the PI3K pathway (Akt and S6) was decreased with saracatinib treatment in CRC040 (sensitive), CRC020 (resistant), and HT29 (resistant). CRC, colorectal cancer.

Figure 4

Src pathway is enriched in sensitive cell lines and explants. A, pathways enriched in the sensitive (SEN) cell lines and explants, and focal adhesion is one of the pathways with false discovery rate less than 1%. B, heatmap of the core genes of the focal adhesion pathway. Red and green represent over- and underexpressed genes, respectively. RES, resistant.

Figure 5

A novel mutation in the PIK3CA 3′ UTR affects the binding of miR-520a-5p and miR-525-5p. CRC007 (A) and CRC042 (B) had increased sensitivity to saracatinib. Data are presented as an average of treated (T)/control (C) × 100 at 28 days (end of study). A novel PIK3CA mutation in the 3′ UTR was identified in both explants by sequencing the PIK3CA gene in (A) CRC007 tumor and (B)CRC042 normal and tumor. C, Western blot analysis of the p110-α protein. Protein levels of the p110-α subunit were the greatest in CRC007. D, cotransfection of miR-520a or miR-525 mimic and wild-type psi-PIK3CA-T plasmid significantly decreases the luciferase activity from HEK293 cell lysates compared with control group. While cotransfection with the mutant identified in these studies psi-PIK3CA-C was not significantly different from the empty vector control. ***P < 0.001. CRC, colorectal cancer. CRC, colorectal cancer; WT, wild-type.