Impact of inducible nitric oxide synthase (iNOS) expression on triple negative breast cancer outcome and activation of EGFR and ERK signaling pathways

Abstract

Inflammation is implicated in triple negative breast cancer (TNBC) progression. TNBC carries a worse prognosis than other breast cancer subtypes, and with the clinical and molecular heterogeneity of TNBC, there is a lack of effective therapeutic targets available. Identification of molecular targets for TNBC subtypes is crucial towards personalized patient stratification. Inducible nitric oxide synthase (iNOS) has been shown to induce p53 mutation accumulation, basal-like gene signature enrichment and transactivation of the epidermal growth factor receptor (EGFR) via s-nitrosylation. Herein we report that iNOS is associated with disease recurrence, distant metastasis and decreased breast cancer specific survival in 209 cases of TNBC. Employing TNBC cell lines representing normal basal breast, and basal-like 1 and basal-like 2 tumors, we demonstrate that nitric oxide (NO) induces EGFR-dependent ERK phosphorylation in basal-like TNBC cell lines. Moreover NO mediated cell migration and cell invasion was found to be dependent on EGFR and ERK activation particularly in basal-like 2 TBNC cells. This occurred in conjunction with NF-κB activation and increased secretion of pro-inflammatory cytokines IL-8, IL-1β and TNF-α. This provides substantial evidence for EGFR as a therapeutic target to be taken into consideration in the treatment of a specific subset of basal-like TNBC overexpressing iNOS.

Keywords: EGFR; inflammation; metastasis; nitric oxide; triple negative breast cancer.

Conflict of interest statement

CONFLICTS OF INTEREST Authors declare no conflicts of interest.

Figures

Figure 1. iNOS expression immunohistochemistry in TNBC

Representative images of TNBC tumor with (A) negative, (B) low, (C) moderate, and (D) strong iNOS tumor epithelial staining.

Figure 2. iNOS expression is associated with decreased recurrence free survival, distant metastasis free survival and breast cancer survival in TNBC

(A) Kaplan-Meier cumulative recurrence free survival curves for TNBC patients by iNOS status (n = 199). Patients with weak iNOS expression (n = 79) or moderate iNOS expression (n = 59) were significantly associated with decreased recurrence free survival compared to patients with negative iNOS expression (n = 27) (P=0.036 & P=0.007, log-rank test). Patients with strong iNOS expression (n = 34) showed a trend toward worse survival compared to patients with negative iNOS expression (n = 27), but did not reach significance (P=0.078, log-rank test). (B) Kaplan-Meier cumulative distant metastasis free survival curves for TNBC patients by iNOS status (n = 199). Patients with weak iNOS expression (n = 79) or moderate levels of iNOS expression (n = 59) were significantly associated with increased risk of distant metastasis compared to patients with negative iNOS expression (n = 27) (P=0.017 & P=0.002, log-rank test). Patients with strong iNOS expression (n = 34) showed a trend toward increased risk of distant metastasis compared to patients with negative iNOS expression (n = 27), but did not reach significance (P=0.052, log-rank test). (C) Kaplan-Meier cumulative breast cancer–specific survival curves for TNBC patients by iNOS status (n = 200). Patients with weak iNOS expression (n = 78) or moderate levels of iNOS expression (n = 59) were significantly associated with increased risk of death due to breast cancer compared to patients with negative iNOS expression (n = 28) (P=0.012 & P=0.007, log-rank test). Patients with strong iNOS expression (n = 35) showed a trend toward increased risk of distant metastasis compared to patients with negative iNOS expression (n = 28), but did not reach significance (P=0.093, log-rank test).

Figure 3. NO induces increased EGFR and ERK phosphorylation in TNBC cell lines

Phosphorylation status of EGFR (A) in the MCF-10A, MDA-MB-468 and HCC1806 cell lines after 24 hours exposure to increasing doses of DETA/NO alone or in combination with 100nM of PD153035 (EGFR inhibitor). (B) Phosphorylation status of the MAP kinases ERK1 and ERK2 after 24 hours exposure to 0.5mM of DETA/NO alone or in combination with 100nM of PD153035 or 200nM PD198306 (MEK inhibitor).

Figure 4. Densitometry analysis of EGFR phosphorylation in response to DETA/NO

Quantification of western blots (Figure 3A) examining EGFR phosphorylation at Y1068, Y1173 and Y1045 and total EGFR expression in the MCF-10A, MDA-MB-468 and HCC1806 cell lines after 24 hours exposure to increasing doses of DETA/NO alone or in combination with 100nM of PD153035 (EGFR inhibitor).

Figure 5. Densitometry analysis of ERK phosphorylation in response to DETA/NO

Quantification of western blots (Figure 3B) examining ERK phosphorylation at Y204 and total ERK expression in the MCF-10A, MDA-MB-468 and HCC1806 cell lines after 24 hours exposure to increasing doses of DETA/NO alone or in combination with 100nM of PD153035 (EGFR inhibitor) or 200nM PD198306 (MEK inhibitor).

Figure 6. Induction of COX-2 by NO in HCC1806 is suppressed by EGFR or MEK inhibitiors

COX-2 protein levels in MCF-10A, MDA-MB-468 and HCC1806 after 24 hours exposure to 0.5mM of DETA/NO alone or in combination with 100nM of PD153035 or 200nM PD198306.

Figure 7. NO induction of IL-1β and IL-8 secretion is perturbed by EGFR inhibition in HCC1806

Cytokine profiling of released IL-6 (A) IL-1β (B) and Il-8 (C) in MCF-10A, MDA-MB-468 and HCC1806 after 24 hours exposure to vehicle or 0.5mM of DETA/NO alone or in combination with 100nM of PD153035. * p<0.05 vs vehicle; # p<0.05 vs DETA/NO plus PD153035

Figure 8. NO induces increased TNFα secretion and activation of NF-κB in HCC1806

TNFα released (A) in the three cell lines after 24 hours exposure to vehicle or 0.5mM of DETA/NO alone or in combination with 100nM of PD153035. (B) Luciferase activity of NF-κB reporter after 24 hours exposure to vehicle or 0.5mM of DETA/NO alone or in combination with 50μg/ml of Infliximab (TNFα blocking antibody), 10ng/ml of recombinant TNFα were used as a positive control. * p<0.05 vs vehicle; # p<0.05 vs DETA/NO plus PD153035.

Figure 9. NO induces increased MMP expression and activity

(A) qPCR showing the mRNA expression levels of MMP1, MMP2 and MMP9 after 24 hours exposure to vehicle or 0.5mM of DETA/NO alone or in combination with 100nM of PD153035. (B) Zymography showing the gelatinase activity of MMP9 and MMP2 after 24 hours exposure to vehicle or 0.5mM of DETA/NO alone or in combination with 100nM of PD153035.

Figure 10. Impact of EGFR inhibition on NO induction of cell migration and invasion

(A) Cell migration, or invasion through (B) gelatin and (C) collagen after 24 hours exposure to vehicle or 0.5mM of DETA/NO alone or in combination with 100nM of PD153035 or 200nM PD198306.* p<0.05 vs vehicle; # p<0.05 vs DETA/NO plus PD153035.

Figure 11. Representative images of NO effects on migration and invasion

Representative images of migratory and invasion potential (A) and invasive potential of the cell lines to invade through a 0.2% gelatin (B) or 1mg/ml collagen (C) of the cell lines after 24 hours exposure to vehicle or 0.5mM of DETA/NO alone or in combination with 100nM of PD153035 or 200nM PD198306.