Breast cancers that overexpress HER2 are generally considered to be poor survival subtypes. Recent advances in anti-HER2 drugs have significantly improved the outcomes of patients with HER2 + breast cancer. However, the curative effects of these targeting drugs alone are limited. Therefore, they are typically administered in combination with chemotherapeutics to maximize the therapeutic effect. 5-FU is commonly used in combination with anti-HER2-targeting therapies for breast cancers. However, some patients with breast cancer treated with 5-FU-based chemotherapy experience recurrence. Vulsteke et al. reported that 15.3% of patients with breast cancer experience disease relapse following treatment with 5-FU-based chemotherapy. This recurrence is predominantly due to the development of 5-FU resistance during treatment. About the mechanisms of 5-FU resistance, some studies have highlighted a few, such as overexpression of TS, ABCG2 and dihydropyrimidine dehydrogenase[3, 4]. However, much was still incompletely understood about this process.
In this study, we established two 5FUresistant breast cancer cell lines, SK-BR-3/FU and MDA-MB-453/FU. In vitro, inhibitory effect of 5FU on SK-BR-3/FU cells and MAD-MB-453/FU cells was slight, the IC50 values for 5-FU in these cell lines were significantly higher than those in the parental cell lines. In addition, in vivo xenograft experiment confirmed that 5-FU did not suppress SK-BR-3/FU-tumors. These results indicated that SK-BR-3/FU cells and MAD-MB-453/FU cells were highly resistant to 5FU compared to the parental cells. Further We investigated the relationship between 5-FU resistance and TS or ABCG2.
TS is an enzyme that targets 5FU. Our results showed that the mRNA and protein levels of TS were higher in SK-BR-3/FU and MDA-MB-453/FU cells than in the corresponding parental cell lines. Several reports also have shown TS overexpression in some types of 5-FU-resistant cancer cells[21,22]. We used TS siRNAs to knock down TS in SK-BR-3/FU cells and MDA-MB-453/FU cells, the TS mRNA and protein expression levels were found to be significantly lower in TS-knockdown SK-BR-3/FU and MDA-MB-453/FU cells than in the control cells. Moreover, the inhibition rate of 5-FU was also found to be remarkably higher in TS siRNAs-transfected cells than in control siRNAs-transfected cells. Similarly, Kadota et al. also reported that a short hairpin RNA (shRNA) targeting TS effectively downregulated TS expression in 5-FU-resistant tumor cells and the sensitivity to 5-FU were restored in these cells. Therefore, these findings suggested that high TS expression is a mechanism of 5FU resistance.
Recent studies have showed that ATP-binding cassette (ABC) transport proteins could pumped cytotoxic drugs out of cells, and was a major mechanism of multidrug resistance (MDR). Breast cancer resistance protein (BCRP), which is also called ABCG2, is a ABC transport protein, its overexpression has been observed in some tumors[4, 25]. In addition, it has been reported that there was an effect-dose relationship between resistance to 5-FU and BCRP expression in the cells, and 5-FU resistance could be reversed by knocking down BCRP through siRNA. Our results showed that the intracellular concentration of 5-FU was remarkably lower and the mRNA and protein expression levels of ABCG2 were noticeably higher in SK-BR-3/FU and MDA-MB-453/FU cells than in the parental cell lines. We used ABCG2 siRNAs to knock down ABCG2 in SK-BR-3/FU cells and MDA-MB-453/FU cells, the ABCG2 mRNA and protein expression levels were found to be significantly lower in ABCG2-knockdown SK-BR-3/FU and MDA-MB-453/FU cells than in the control cells. Moreover, the inhibition rate of cell growth and intracellular concentration of 5-FU were also found to be remarkably higher in ABCG2 siRNAs-transfected cells than in control siRNAs-transfected cells. But TS knockdown had no effect on the intracellular accumulation of 5-FU in SK-BR-3/FU and MDA-MB-453/FU cells. The 5-FU resistance of breast cancer cells was reversed by knockdown of ABCG2. These findings indicated that 5-FU is a substrate of BCRP in breast cancer cells and is pumped out by BCRP. In addition, a combination of TS and ABCG2 knockdown made the cells more sensitive to 5-FU than TS or ABCG2 knockdown alone, which suggested that TS and ABCG2 were simultaneously involved in 5-FU-resistance in breast cancer cells.
Some studies showed that a number of targets can restore 5FUresistant breast cancer cells to 5FU[21,22], for example, Minegaki et al. reported that valproic acid and suberanilohydroxamic acid, two histone deacetylase inhibitors, re-sensitized 5FUresistant breast cancer cells to 5FU. In this study, our results revealed that pyrotinib not only increased the sensitivity to 5-FU in 5-FU-sensitive breast cancer cells, but also re-sensitized 5-FU-resistant breast cancer cells to 5-FU in vitro and in vivo. In addition, mice that received the combination treatment did not show lower body weights than the mice in the groups treated with 5-FU or pyrotinib alone. Thus, our data show that pyrotinib restored sensitivity to 5-FU, without an increase in toxicity.
We further investigated the mechanisms of pyrotinib re-sensitizing 5-FU-resistant breast cancer cells to 5-FU. Our results showed that pyrotinib markedly decreased TS mRNA and protein expression in both 5FUresistant cell lines as well as the corresponding parental cell lines, thus allowing 5-FU to better suppress the remaining TS activity. Because the apoptosis induced by 5-FU is related to the extent of enzymatic inhibition13. Being consistent with our result, Chefrour et al. reported that lapatinib, a reversible inhibitor of HER1 and HER2, decreased TS activity and increased the cytotoxic effect of 5′-dFUR in HER2 + breast cancer cell lines. However, the molecular mechanism by which pyrotinib and lapatinib downregulates TS is unknown.
Moreover, pyrotinib also decreased ABCG2 mRNA and protein expression in both SK-BR-3/FU and MDA-MB-453/FU cells. The intracellular concentration of 5-FU in 5-FU-resistant cells was increased in pyrotinib-treated cells almost to the level of that in the parental cells, and the sensitivity to 5-FU was restored. However, pyrotinib had no effect on ABCG2 mRNA and protein expression in SK-BR-3 and MDA-MB-453 cells. This might be due to the little ABCG2 expression levels in these cells.
Here, we performed a further investigation of the mechanisms by which ABCG2 expression was inhibited by pyrotinib. Our results showed that the levels of p-AKT, pHer-2, and pHer-4 were higher in SK-BR-3/FU and MDA-MB-453/FU cells than in the parental cell lines, and pyrotinib significantly decreased p-AKT, pHer-2, and pHer-4 levels in SK-BR-3/FU and MDA-MB-453/FU cells, without affecting total AKT, Her-2, or Her-4 levels in virto. pyrotinib-treated SK-BR-3/FU xenograft tissues also had lower pAKT, pHER2 and pHER4 expression, which indicated that BCRP is suppressed by pyrotinib via inhibition of the PI3K/Akt signaling pathway. Similarly, Chefrour et al. reported that lapatinib significantly downregulated pHer-2 and p-AKT levels and increased the cytotoxic effect of 5′-dFUR in HER2 + breast cancer cell lines.
Some previous studies showed that HER2 overexpression activated the PI3K/Akt signal transduction pathway and led to increased BCRP expression in breast cancer cells[27, 28]. PI3K inhibition also has been reported to be particularly effective in sensitizing breast cancer to cytotoxic agents by decreasing ACCG2 expression as a consequence of inhibiting p-AKT activity[29, 30]. The present data strongly suggest that the downregulation of ABCG2 by pyrotinib, via inhibition of p-Akt signaling and the pHER2/pHER4 pathway, could be a mechanism of the restoration of sensitivity to 5-FU in 5FUresistant breast cancer cell lines.