Acquired resistance to 5-fluorouracil via HSP90/Src-mediated increase in thymidylate synthase expression in colon cancer

Abstract

5-fluorouracil (5-FU), one of the first-line chemotherapeutic agents for the treatment of gastrointestinal malignancies, has shown limited efficacy. The expression of thymidylate synthase (TYMS) has been reported to be associated with the resistance to 5-FU. Here, we demonstrate that the enhanced HSP90 function and subsequent activation of Src induce expression of TYMS and acquired resistance to 5-FU in colon cancer. We show that the persistent 5-FU treatment granted 5-FU-sensitive HCT116 colon cancer cells morphologic, molecular, and behavioral characteristic of the epithelial-mesenchymal transition (EMT), contributing to emergence of acquired resistance to 5-FU. HCT116/R, a HCT116 colon cancer cell subline carrying acquired resistance to 5-FU, showed increased expression and activation of HSP90's client proteins and transcriptional up-regulation of TYMS. Forced overexpression of HSP90 or constitutive active Src in HCT116 cells increased TYMS expression. Conversely, pharmacological blockade of HSP90 or Src in HCT116/R cells effectively suppressed the changes involved in 5-FU resistance in vitro and xenograft tumor growth, hematogenous spread, and metastatic tumor development in vivo. This study suggests a novel function of HSP90-Src pathway in regulation of TYMS expression and acquisition of 5-FU resistance. Thus, therapeutics targeting this pathway may be an effective clinical strategy to overcome 5-FU resistance in colon cancer.

Keywords: 5-fluorouracil; HSP90; Src; colon cancer.

Figure 1. Generation and characterization of 5-FU resistant cells

A. Colon cancer cells were treated with increasing concentrations of 5-FU for 3 days. Cell viability was determined by the MTT assay. B–E. Basal phenotype alterations including anchorage-dependent (B) and -independent colony formation (C), migration (D), and invasion (E) of 5-FU-resistant HCT116 cells (HCT116/R) compared with parental cells (HCT116/P) were determined. **P < 0.01; ***P < 0.001.

Figure 2. Acquisition of EMT phenotype in 5-FU resistant cells

A. Morphological changes were examined using a phase-contrast microscope. B. immunofluorescence staining of each cells using anti-E-cadherin (Scale bar: 20 μm, 400× magnifications). C. E-cadherin, β-catenin, and TGF-β1 expression in each cells was determined by Western blot. D. The expression of E-cadherin, β-catenin, and TGF-β1 mRNA in each cells was analyzed by RT-PCR.

Figure 3. Effects of 5-FU on HCT116/P and HCT116/R cells

A. Cell viability was determined by the MTT assay after 3 days of 5-FU treatment. B, C. The effect of 5-FU on anchorage-dependent (B) and -independent (C) colony formation in HCT116/R cells compared with HCT116/P cells was determined. D. Migration and invasion of HCT116/P and HCT116/R cells with 5-FU treatment was compared with untreated cells. E, F. Resistance to 5-FU-induced cell death in HCT116/R cells. HCT116/P and HCT116/R cells were treated with 5-FU (50 μM) for 2 days. (E) Increases in apoptotic cell populations were determined by flow cytometry after staining unfixed cells with annexin V and PI. (F) PARP cleavage modulation upon 5-FU treatment was determined by Western blot. **P < 0.01; ***P < 0.001.

Figure 4. Activation of the HSP90-mediated Src signaling, contributing to increase in TYMS expression

A. Western blot and RT-PCR analysis for TYMS level in HCT116/P and HCT116/R cells. B. Changes in various signaling pathways in HCT116/R cells compared with HCT116/P cells were analyzed by Western blot. C. Cells were exposed to cycloheximide (CHX, 100 μg/ml) for indicated times, and protein lysates were harvested. EGFR, IGF-1R, and Src protein expression was determined by Western blot. Numbers below the blot indicate densitometric quantification relative to 0 h treatment. D. HCT116/P cells were transfected with HSP90 (left) or constitutively-active Src {Src(Y527F); right} and expression of TYMS were detected by RT-PCR. E. HCT116/R cells were treated with 17-AAG (100 nM; left) or dasatinib (50 nM; right) for 48 h and expression of TYMS, E-cadherin, and Src phosphorylation were detected by RT-PCR or Western blot. (E, right) Src expression was reduced using siRNA in HCT116/R cells, and expression of TYMS was determined by RT-PCR or Western blot.

Figure 5. HSP90 or Src blockade suppresses growth, migration, and invasion of 5-FU resistant cells

A. Anchorage-independent colony formation of HCT116/P and HCT116/R cells under dasatinib or 17-AAG treatment. B. HCT116/R cells were treated with 5-FU alone or in combination with dasatinib (100 nM) or 17-AAG (200 nM), and cell viability was determined by the MTT assay. Relative cell viability was presented as percentage of untreated cells. The effect of each inhibitor on the regulation of its target signaling was determined by Western blot. C, D. The effects of 5-FU combined with dasatinib or 17-AAG on HCT116/R cell migration (C) and invasion (D) **P < 0.01; ***P < 0.001. E. Cleaved PARP under dasatinib or 17-AAG in HCT116/R cells was detected by Western blot.

Figure 6. HSP90 or Src inhibition decreases primary tumor growth, the number of circulating tumor cells, and lung metastasis

A–E. Effects of HSP90 or Src inhibition in renal capsule implantation model of HCT116/R cells. The volume (A) and weight (B) of primary tumors derived from cells implanted under the renal capsule were examined after 3 weeks of drug treatment; dasatinib (70 mg/kg, oral gavage) or 17-AAG (70 mg/kg, intraperitoneal injection) were administered to mice 5 days per week. (C) The dissemination of implanted cells in the circulation in each treatment group was determined by observation of smeared blood under a fluorescence microscope and the enumeration of circulating tumor cells is shown. CTC = Circulating Tumor Cell. (D) Quantification of metastatic lung tumor nodule formation in each treatment group compared with the vehicle-treated control group. **P < 0.01; ***P < 0.001. (E) Lung metastases were decreased upon HSP90 or Src inhibition as determined by observation under a fluorescence microscope. Lung tissue specimens stained with hematoxylin and eosin were observed under an inverted microscope to confirm metastatic tumor formation (Scale bar: 50 μm, 100 × magnifications). N = 5 to 7 per group. F. Schematic illustration of the relationships among HSP90, Src, and TYMS in 5-FU resistance. Upon chronic exposure to 5-FU, enhanced HSP90 function results in Src activation followed increase in TYMS expression, and this leads 5-FU resistance by activating cancer cell proliferation, survival, migration, and invasion.