PIK3CA mutation confers resistance to chemotherapy in triple-negative breast cancer by inhibiting apoptosis and activating the PI3K/AKT/mTOR signaling pathway

Abstract

Background: Triple-negative breast cancer (TNBC) is a malignant subtype of breast cancer, the main treatments for which are chemotherapy and surgery. PIK3CA is an oncogene that encodes the p110α subunit of class IA PI3K to regulate cell proliferation and apoptosis. Some reports have observed neoadjuvant chemotherapy (NAC) to have poor pathological complete response (pCR) rates in TNBC with PIK3CA mutation. This study aimed to explore the mechanism of how mutant PIK3CA alters chemotherapeutic susceptibility in TNBC.

Methods: TNBC cell lines (MDA-MB-231 and MDA-MB-468) with PIK3CA gene mutations (E545K and H1047R regions) and overexpression were established by transfection. NOD/SCID mice were used for in vivo experiments. Epirubicin was used as the chemotherapeutic agent. Cell viability, cell cycle, apoptosis, and Transwell assays were conducted for phenotype analysis. Western blot, quantitative reverse transcription-polymerase chain reaction, and immunohistochemistry were used to detect gene and protein expression levels. A clinical analysis of 50 patients with TNBC was also performed.

Results: Cell viability and Transwell assays showed that PIK3CA mutation promoted TNBC cell growth and conferred an enhanced migratory phenotype. Cell cycle and apoptosis assays showed that PIK3CA mutation moderately improved the proliferation ability of TNBC cells and remarkably inhibited their apoptosis. After epirubicin therapy, the proportion of early apoptotic cells decreased among cells with PIK3CA mutation. Further, xenograft tumors grew faster in NOD/SCID mice injected with mutated cell lines than in control group, suggesting that PIK3CA mutation caused chemotherapy resistance. Importantly, western blot and immunohistochemical analysis showed that cells and mouse tumors in the PIK3CA mutation groups exhibited different expression levels of apoptosis-related markers (Xiap, Bcl-2, and Caspase 3) and proteins associated with the PI3K/AKT/mTOR pathway (p110α, AKT, p-AKT, mTOR, p-mTOR, p-4E-BP1, p-p70S6K, and Pten). Moreover, prognostic analysis of 50 patients with TNBC indicated that PIK3CA mutation might be linked with relapse and death.

Conclusions: PIK3CA mutation confers resistance to chemotherapy in TNBC by inhibiting apoptosis and activating the PI3K/AKT/mTOR signaling pathway.

Keywords: PI3K/AKT/mTOR pathway; PIK3CA mutation; Triple-negative breast cancer (TNBC); apoptosis; chemotherapy resistance.

Figure 1

PIK3CA mutation promotes growth, inhibits apoptosis, and confers enhanced migratory phenotype in TNBC cells. (A) The cell proliferation rates of TNBC cells (MDA-MB-231 and MDA-MB-468) were measured by CCK-8 assays. (B,C) Cell cycle analysis was conducted using the propidium iodide staining method. Columns represented cell proportions in the S phase. (D,E) Apoptosis assays were performed by staining cells with APC-Annexin V and 7-AAD and performing flow cytometric analysis. The histogram shows the proportion of early apoptotic cells. (F,G) The migratory properties of 2 TNBC cell lines were analyzed using Transwell assays by staining cells with crystal violet. Scale bar =100 μm. Quantitation of results is shown as columns. All experiments were performed in triplicate, and the representative data are shown. Data represents mean ± SD. Different conditions were compared using one-way ANOVA and Tukey’s multiple comparisons test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns means no significance for PIK3CA^Oe, PIK3CA^E545K, PIK3CA^H1047R vs. PIK3CA^ctrl.

Figure 2

PIK3CA mutation induces resistance to chemotherapy, mainly through inhibiting apoptosis in TNBC cells. (A,B) TNBC cells (MDA-MB-231 and MDA-MB-468) were treated with epirubicin at a concentration close to their IC₅₀ value (1.5 μmol/L for MDA-MB-231 cells; 0.5 μmol/L for MDA-MB-468 cells) followed by staining with APC-Annexin V and 7-AAD for flow cytometric analysis. The columns show the proportions of early apoptotic cells. The IC₅₀ value obtained through CCK-8 assays is shown in Figure S1D and Table S2. (C) The expression levels of apoptosis related proteins (Xiap and Bcl-2) in MDA-MB-231 cells treated with epirubicin were detected by western blot. (D) The growth trend of tumor volume in mice treated with epirubicin (10 mg/kg, i.p. once a week, 3 times in total) in different groups. The black arrow represents the first treatment and the red arrow represents tumor extraction. (E) The indicated proteins of tumor tissues of mice were detected by immunohistochemistry. Bar, 50 μm. Data shown are representative of 3 independent experiments. Data represented mean ± SD. Different conditions were compared using one-way ANOVA and Tukey’s multiple comparisons test. *, P<0.05; **, P<0.01; ***, P<0.001; ****P<0.0001; ns means no significance for PIK3CA^Oe, PIK3CA^E545K, PIK3CA^H1047R vs. PIK3CA^ctrl.

Figure 3

PIK3CA mutation activates PI3K/AKT/mTOR signaling pathway to resist chemotherapy in TNBC cells. (A) Western blot analysis was used to detect the expression levels of AKT, p-AKT, mTOR, and p-mTOR protein in MDA-MB-231 cell lines treated with or without epirubicin. (B) Western blot analysis was used to detect the expression levels of p110α, p-AKT, Pten, mTOR, p-p70S6 kinase, and p-4E-BP1 protein in tumors from mice in each group treated with epirubicin. (C) Quantitative analysis of messenger RNA (p110α, AKT, mTOR, Pten, and 4E-BP1) was conducted by RT-qPCR. n=3. Data shown as mean ± SD. Different conditions were compared using one-way ANOVA and Tukey’s multiple comparisons test. **, P<0.01; ***, P<0.001; ****, P<0.0001; ns means no significance for PIK3CA^Oe, PIK3CA^E545K, PIK3CA^H1047R vs. PIK3CA^ctrl. (D) Immunohistochemistry was used to detect AKT, mTOR, and Pten proteins in tumor tissues from mice. Bar, 50 μm. (E) Immunohistochemistry was used to detect apoptosis-related proteins and proteins pertaining to the PI3K/AKT/mTOR pathway in patients with TNBC. Bar, 50 μm.