Cancer Res Treat.  2018 Jan;50(1):239-254. 10.4143/crt.2016.580.

FOXO1 Suppression is a Determinant of Acquired Lapatinib-Resistance in HER2-Positive Gastric Cancer Cells Through MET Upregulation

• Park J
• Choi Y
• Ko YS
• Kim Y
• Pyo JS
• Jang BG
• Kim MA
• Lee JS
• Chang MS
• Park JW
• Lee BL

• KMID: 2403493
• DOI: http://doi.org/10.4143/crt.2016.580

Abstract

PURPOSE
Lapatinib is a candidate drug for treatment of trastuzumab-resistant, human epidermal growth factor receptor 2 (HER2)-positive gastric cancer (GC). Unfortunately, lapatinib resistance renders this drug ineffective. The present study investigated the implication of forkhead box O1 (FOXO1) signaling in the acquired lapatinib resistance in HER2-positive GC cells.
MATERIALS AND METHODS
Lapatinib-resistant GC cell lines (SNU-216 LR2-8) were generated in vitro by chronic exposure of lapatinib-sensitive, HER2-positive SNU-216 cells to lapatinib. SNU-216 LR cells with FOXO1 overexpression were generated by stable transfection of a constitutively active FOXO1 mutant (FOXO1A3). HER2 and MET in SNU-216 LR cells were downregulated using RNA interference. The sensitivity of GC cells to lapatinib and/or cisplatin was determined by crystal violet assay. In addition, Western blot analysis, luciferase reporter assay and reverse transcription-polymerase chain reaction were performed.
RESULTS
SNU-216 LR cells showed upregulations of HER2 and MET, but downregulation of FOXO1 compared to parental SNU-216 cells. FOXO1 overexpression in SNU-216 LR cells significantly suppressed resistance to lapatinib and/or cisplatin. In addition, FOXO1 negatively controlled HER2 and MET at the transcriptional level and was negatively controlled by these molecules at the post-transcriptional level. A positive crosstalk was shown between HER2 and MET, each of which increased resistance to lapatinib and/or cisplatin.
CONCLUSION
FOXO1 serves as an important linker between HER2 and MET signaling pathways through negative crosstalks and is a key regulator of the acquired lapatinib resistance in HER2-positive GC cells. These findings provide a rationale for establishing a novel treatment strategy to overcome lapatinib resistance in a subtype of GC patients.

Keyword

Stomach neoplasms; ErbB-2 receptor; Drug resistance; Lapatinib; Human FOXO1 protein; Human MET protein

MeSH Terms

Blotting, Western
Cell Line
Cisplatin
Down-Regulation
Drug Resistance
Gentian Violet
Humans
In Vitro Techniques
Luciferases
Parents
Receptor, Epidermal Growth Factor
Receptor, ErbB-2
RNA Interference
Stomach Neoplasms*
Transfection
Up-Regulation*
Cisplatin
Gentian Violet
Luciferases
Receptor, Epidermal Growth Factor
Receptor, ErbB-2

Figure

• 

  Fig. 1. Effect of chronic lapatinib treatment on SNU-216 cells. (A) Lapatinib-resistant (LR), human epidermal growth factor receptor 2 (HER2)–positive gastric cancer (GC) cell lines (SNU-216 LR2-8) were generated from a lapatinib-sensitive, HER2-positive SNU-216 GC cell line by chronic exposure to lapatinib over a period of 8 months. Twenty-four hours after plating, parental and lapatinib-resistant (LR) SNU-216 cells were treated with the indicated concentrations of lapatinib for 3 days, and cell viability was determined using crystal violet assay. The percentage of viable cells is shown relative to untreated cells (considered as 100%). (B) Comparative analysis of total and phosphorylated HER2, MET, and AKT as well as total forkhead box O1 (FOXO1) by Western blot analysis (WB). mRNA expressions of HER2, MET, and FOXO1 were determined by reverse transcription–polymerase chain reaction (RT-PCR). β-Actin protein and mRNA were served as loading controls. (C) FOXO1 transcriptional activity was determined by the luciferase reporter assay and was normalized by β-galactosidase activity. Luciferase activity in parental SNU-216 cells was arbitrarily set to 1. Each bar represents the mean±standard deviation. a)p < 0.05 vs. parental SNU-216 cells.

• 

  Fig. 2. Effect of forkhead box O1 (FOXO1) overexpression on lapatinib and/or cisplatin sensitivity in lapatinib-resistant (LR) cell lines. SNU-216 LR3 and LR7 cells were transfected with empty pcDNA3 vector (pcDNA3) or FOXO1A3 mutant vector (FOXO1A3). Cell viability was measured by crystal violet assay. (A) FOXO1 overexpression was confirmed by Western blot analysis. (B) FOXO1 transcriptional activity was analyzed by the luciferase reporter assay. (C) Cells were treated with the indicated concentrations of lapatinib, and cell viability was measured after 3 days. The percentage of viable cells is shown relative to untreated cells (considered as 100%). (D) Cells were treated with the indicated concentrations of cisplatin, and cell viability was measured after 3 days. The percentage of viable cells is shown relative to untreated cells (considered as 100%). (E) Cells were treated with the 1 μmol/L lapatinib alone or combined with 10 μg/mL cisplatin (CDDP), and cell viability was measured after 3 days. The percentage of viable cells is shown relative to untreated cells (considered as 100%). Each bar represents the mean±standard deviation. ns, not significant. a)p < 0.05 vs. pcDNA3 cells, b)p < 0.05 vs. lapatinib-treated pcDNA3 cells, c)p < 0.05 vs. cisplatin-treated pcDNA3 cells, d)p < 0.05 vs. pcDNA3 cells, e)p < 0.05 vs. lapatinib-treated FOXO1A3 cells.

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  Fig. 3. Association between forkhead box O1 (FOXO1) and human epidermal growth factor receptor 2 (HER2)/MET in lapatinib-resistant (LR) cell lines. SNU-216 LR3 and LR7 cells were transfected with empty pcDNA3 vector (pcDNA3) or FOXO1A3 mutant vector (FOXO1A3). (A) The protein expressions of total and phosphorylated HER2, MET, and AKT were determined by Western blot analysis (WB). (B) The mRNA expressions of HER2 and MET were evaluated by reverse transcription-polymerase chain reaction (RT-PCR).

• 

  Fig. 4. Effect of human epidermal growth factor receptor 2 (HER2) downregulation on lapatinib/cisplatin resistance in lapatinib-resistant (LR) cell lines. SNU-216 LR3 and LR7 cells were infected with a lentivirus containing either control shRNA (shCtrl) or HER2 shRNA (shHER2). Cell viability was measured by crystal violet assay. (A) The protein expressions of HER2, pAKT, and AKT were determined by Western blot analysis. (B) Twenty-four hours after plating, cells were cultured for 3 days and cell growth was determined at the indicated times. (C) Cells were treated with the indicated concentrations of lapatinib, and cell viability was measured after 3 days. The percentage of viable cells is shown relative to untreated cells (considered as 100%). (D) Cells were treated with the indicated concentrations of cisplatin, and cell viability was measured after 3 days. The percentage of viable cells is shown relative to untreated cells (considered as 100%). (E) Cells were treated with the 1 μmol/L lapatinib alone or combined with 10 μg/mL cisplatin (CDDP), and cell viability was measured after 3 days. The percentage of viable cells is shown relative to untreated cells (considered as 100%). Each bar represents the mean±standard deviation. ns, not significant. a)p < 0.05 vs. shCtrl cells, b)p < 0.05 vs. lapatinib-treated shCtrl cells, c)p < 0.05 vs. cisplatin-treated shCtrl cells, d)p < 0.05 vs. shCtrl cells, e)p < 0.05 vs. lapatinib-treated shHER2 cells.

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  Fig. 5. Effect of MET downregulation on lapatinib/cisplatin resistance in lapatinib-resistant cell lines. SNU-216 LR3 and LR7 cells were transfected with pGFP-v-RS vectors containing either control scrambled shRNA (shCtrl) or MET shRNA (shMET). Cell viability was measured by crystal violet assay. (A) The protein expressions of MET, pAKT, and AKT were termined by Western blot analysis. (B) Cells were cultured for 3 days, and cell growth was determined at the indicated times. (C) Cells were treated with the indicated concentrations of lapatinib and cell viability was measured after 3 days. The percentage of viable cells is shown relative to untreated cells (considered as 100%). (D) Cells were treated with the indicated concentrations of cisplatin, and cell viability was measured after 3 days. The percentage of viable cells is shown relative to untreated cells (considered as 100%). (E) Cells were treated with the 1 μmol/L lapatinib alone or combined with 10 μg/mL cisplatin (CDDP), and cell viability was measured after 3 days. The percentage of viable cells is shown relative to untreated cells (considered as 100%). Each bar represents the mean±standard deviation. ns, not significant. a)p < 0.05 vs. shCtrl cells, b)p < 0.05 vs. lapatinib-treated shCtrl cells, c)p < 0.05 vs. cisplatin-treated shCtrl cells, d)p < 0.05 vs. shCtrl cells, e)p < 0.05 vs. lapatinib-treated shMET cells.

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  Fig. 6. The relationships between human epidermal growth factor receptor 2 (HER2), MET, and forkhead box O1 (FOXO1) in lapatinib-resistant cells. (A-C) Cells were infected with a lentivirus containing either control shRNA (shCtrl) or HER2 shRNA (shHER2). (A) The expression and pMET, MET, and FOXO1 protein expression were determined by Western blot analysis. (B) FOXO1 transcriptional activity was determined by the luciferase reporter assay. (C) The mRNA expression of FOXO1 was evaluated by reverse transcription–polymerase chain reaction (RT-PCR). (D-F) Cells were transfected with pGFPv-RS vectors containing either control scrambled shRNA (shCtrl) or MET shRNA (shMET). (D) The expression and pHER2, HER2, and FOXO1 protein expression were determined by Western blot analysis. (E) FOXO1 transcriptional activity was determined by the luciferase reporter assay. (F) mRNA expression of FOXO1 was evaluated by RT-PCR. Each bar represents the mean±standard deviation. a)p < 0.05 vs. shCtrl cells, b)p < 0.05 vs. shCtrl cells.

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  Fig. 7. Model for forkhead box O1 (FOXO1)–dependent acquired lapatinib resistance and the crosstalk among FOXO1, human epidermal growth factor receptor 2 (HER2) and MET in lapatinib-resistant, HER2-positive gastric cancer (GC) cells. Downregulation of FOXO1 leads to coactivation of HER2 and MET, which are essential to lapatinib resistance. Reintroduction of FOXO1 is necessary to reduce the lapatinib resistance in a subpopulation of HER2-positive GC patients showing lapatinib resistance.

Reference

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