PIK3CA mutations confer resistance to first-line chemotherapy in colorectal cancer

Abstract

Chemotherapy represents an important treatment option for colorectal cancer (CRC), but only half of the patients benefit from these regimens. We explored the potential predicting value and mechanism of PIK3CA mutation in CRC chemotherapy. CRC specimens from 440 patients were retrospectively collected and examined with a fluorescence PCR-based method. The correlation of first-line chemotherapy response and PIK3CA mutation was evaluated according to follow-up and medical records. The underlying mechanism of PIK3CA mutation in chemotherapy resistance was assessed with CRC tumors and primary cells. The mutation frequency of the PIK3CA gene in CRC patients was 9.55%, which was correlated with late TNM staging and lower histological grade. The CRC patients with PIK3A mutation showed worse response to first-line chemotherapy than those without PIK3CA mutation. PIK3A mutation tumor cells showed poor sensitivity to first-line chemotherapy in vitro and in vivo. PIK3CA mutation induced PI3K/Akt signaling activation to increase LGR5⁺ CRC stem cells survival and proliferation, from which lead to chemotherapy resistance. Furthermore, PIK3CA ^mutation/LGR5⁺ expression was an independent detrimental factor for CRC patients. Our findings indicated that PIK3CA mutation induced PI3K/Akt activation contributed to CRC stem cells survival and proliferation, from which cells further resistance to chemotherapy. PIK3CA ^mutation/LGR5⁺ expression was a potential biomarker for monitoring chemotherapy resistance in CRC.

Fig. 1. The correlation between chemotherapy response and PIK3CA mutation status in CRC patients.

a The percentage of PIK3CA mutation in the patients effective and non-effective to first-line chemotherapy. *p < 0.05. b Chemotherapy rsponses among patients with wild-type, PIK3CA mutant, exon 9 mutant and exon 20 mutant tumors

Fig. 2. PIK3CA mutation correlates with first-line chemotherapy resistance.

a Western blot analysis of pAkt, Akt and GAPDH (loading control) in primary CRC cells. b CC-1 and CC-2 cells were incubated with 5-Fu (25 µM) for 72 h. Cell viability was examined by the MTT assay. Results represent the mean ± S.E.M. of the values relative to vehicle controls; *P < 0.05. c CC-1 cells were incubated combined perifosine or LY294002 with 5-FU. Cell viability was examined by the MTT assay. The data are shown as means ± S.E.M. d Immunoblots showing caspase-3 and GAPDH (loading control) expression levels in CC-1 cells treated with perifosine or LY294002 combined with 5-Fu (1 μM) for 24 h, and in CC-2 cells. e The colorectal cancer cells (CC-1) were treated with perifosine, LY294002 or 5-Fu for 48 h and the cells were collected for apoptosis analysis by flow cytometry (n = 3). f In vivo tumorigenicity of CRC xenograft tumors treated with or without perifosine. The tumor growth was monitored every week until 6 weeks after injection. Data are shown as means ± S.E.M. *P < 0.05

Fig. 3. PIK3CA mutation related PI3K/Akt signaling increased CRC stem cell survival.

a Quantitative analysis of primary and secondary sphere formation by CC-1 and CC-2 cells. b Quantitative analysis of sphere formation by CRC cells treated with perifosine or LY294002. c Flow cytometry analysis of LGR5 expression in CC-1 and CC-2 cells. d qRT-PCR analysis of stem cell markers, CD133, C-MYC, LGR5, ALDH1A1, NANOG and CXCR4 genes expression in CC-1 and CC-2. e Immunoblots showing p-Akt, Akt, LGR5, C-myc and GAPDH (loading control) expression levels in PIK3CA mutant tumor tissue comparing with that without mutation. f Immunoblots showing p-Akt, Akt, LGR5, C-myc and GAPDH (loading control) expression levels in CC-1 cells treated with perifosine, LY294002 or BpV (phen) comparing with vehicle

Fig. 4. The prognostic value of PIK3CA mutation and LGR5⁺ in CRC patients.

a, b Kaplan–Meier analysis of the correlation between PIK3CA mutation and disease-free survival of 440 CRC patients from Jinan Military General Hospital. c Kaplan–Meier analysis of the correlation between PIK3CA mutation and overall survival of 440 CRC patients