Evaluation of the efficacy of dasatinib, a Src/Abl inhibitor, in colorectal cancer cell lines and explant mouse model

Abstract

Background: Dysregulation of the Src pathway has been shown to be important at various stages of cancer. Dasatinib is a potent Src/Abl inhibitor and has demonstrated to have anti-proliferative and anti-invasive activity in many preclinical models. The objective of this study was to determine the anti-tumor activity of dasatinib using in vitro and in vivo preclinical colorectal (CRC) models.

Methods: CRC cell lines and patient-derived tumor explant (PDX) models were used to investigate the efficacy of dasatinib. We treated 50 CRC cell lines with dasatinib for 72 hours and proliferation was assayed by a sulforhodamine B (SRB) assay; an IC50 ≤ 0.08 μmol/L was considered sensitive. We treated 17 patient-derived CRC explants with dasatinib (50 mg/kg/day, administered once-daily) for 28 days to determine in vivo efficacy. Tumor growth inhibition (TGI) ≥ 50% was considered sensitive.

Results: We found that 8 out of 50 CRC cell lines reached an IC50 ≤ 0.08 μmol/L with dasatinib treatment. In addition, of 17 CRC explants grown in the xenograft mouse model, 2 showed sensitivity to dasatinib. The anti-tumor effects observed in this study were a result of G1 cell cycle arrest as the dasatinib sensitive CRC cell lines exhibited G1 inhibition. Moreover, those CRC cell lines that were responsive (0.08 μmol/L) to treatment demonstrated a significant baseline increase in Src and FAK gene expression.

Conclusion: Dasatinib demonstrated significant anti-proliferative activity in a subset of CRC cell lines in vitro, especially in those with increased Src expression at baseline, but only showed modest efficacy in CRC explants. Dasatinib is currently being studied in combination with chemotherapy in patients with advanced CRC, as its use as a single agent appears limited.

Fig 1. The effects of dasatinib on CRC cell lines in vitro.

Fifty CRC cell lines were treated with dasatinib (dose 0.08–5 μmol/L) and proliferation was determined by an SRB assay. An IC₅₀ ≤ 0.08 μM were deemed sensitive to dasatinib. The CRC cell lines KM20, LS174T, SNU-977, WiDR, HCT-116, SW1417, SNU-796, and SKCO1 demonstrated sensitivity to dasatinib.

Fig 2. Efficacy of dasatinib in CRC PDX models.

A) Seventeen colorectal cancer explants were treated with dasatinib and tumor size was evaluated twice per week. A TGI was calculated by relative tumor growth of treated mice divided by relative tumor growth of control mice x 100. CRC explants that exhibited a TGI < 50% were considered sensitive and TGI > 50% were considered resistant to dasatinib. B-C) Two explants (CRC036 and CRC047) demonstrated sensitivity to dasatinib. Columns, mean (n = 10 tumors per group); bars, SEM; and *, significance (P < 0.05) compared with control.

Fig 3. Cell cycle analysis of sensitive and resistant CRC cell lines.

Sensitive CRC cell lines (HCT116 and LS174T) demonstrated an increase in G1 cell cycle arrest compared to resistant cell lines (HT15 and SW620) after treatment with dasatinib.

Fig 4. Pharmacodynamic effects of dasatinib on the Src pathway.

A) Treatment with dasatinib (0.8 μmol/L) at 0.5h, 1h, 2h, 4h, and 8h significantly reduced the activation of Src, FAK and paxillin at all time points examined in the HCT116 sensitive CRC cell line when compared to control. B) A decrease in Src activity was seen in 1 out of 3 CRC explants treated with dasatinib in the sensitive (CRC036) and resistant CRC explant (CRC027) measured at end of study (day 28). However, in both cases FAK activity appeared to be increased.

Fig 5. Evaluation of baseline pathway gene expression between sensitive and resistant CRC cell lines.

A) Pathway analysis of sensitive and resistant gene expression and KEGG pathway diagram shows that adherens junction is a pathway enriched in sensitive CRC cell lines (red shows the genes that are increased in the sensitive cell lines and include Src and FAK. B) Src and C) FAK gene expression are significantly elevated in sensitive when compared to resistant CRC cell lines.