Abstract
PurposeEstrogen plays a critical role in the invasiveness and metastasis of non-small cell lung cancer (NSCLC) through estrogen receptor β (ERβ). However, the antimetastatic effect of the ERβ antagonist fulvestrant was still limited in NSCLC patients. Recently, Toll-like receptor 4 (TLR4) signaling was implicated in NSCLC metastasis. Our present study aimed to evaluate the synergistic antimetastatic effect of a combination of fulvestrant and the TLR4-specific inhibitor CLI-095 (TAK-242) on human NSCLC cells.MethodsThe expression levels of ERβ and TLR4 were detected by immunohistochemical (IHC) analysis of 180 primary NSCLC and 30 corresponding metastatic lymph node samples. The association between ERβ and TLR4 expression was analyzed. The aggressiveness of NSCLC cells treated with fulvestrant, CLI-095 or the drug combination and formation status of their invadopodia, invasion-associated structures, were investigated. The protein levels in NSCLC cells in different groups were determined by Western blot and immunofluorescence analyses.ResultsHere, a positive correlation between ERβ and TLR4 expression was observed in both primary NSCLC tissue (Spearman’s Rho correlation coefficient = 0.411, p < 0.001) and metastatic lymph node tissue (Spearman’s Rho correlation coefficient = 0.374, p = 0.009). The protein levels of ERβ in NSCLC cell lines were decreased by fulvestrant, and this suppressive effect was significantly enhanced when fulvestrant was combined with CLI-095 (p < 0.05). Both the migration and invasion of NSCLC cells were suppressed by fulvestrant or CLI-095 alone, and the combination of fulvestrant + CLI-095 showed the strongest inhibitory effect (p < 0.05). In addition, the results demonstrated that CLI-095 also helped fulvestrant restrict the formation and function of invadopodia in NSCLC cells (p < 0.05).ConclusionsCollectively, our study results suggested that CLI-095 enhances the antimetastatic effect of fulvestrant on NSCLC and provided support for further investigation of the antitumor activity of combined therapy with antiestrogen and anti-TLR4 agents in the clinic.
Highlights
Abbreviations non-small cell lung cancer (NSCLC) Non-small cell lung cancer estrogen receptor β (ERβ) Estrogen receptor beta TLR4 Toll-like receptor 4 matrix metalloprotease 2 (MMP2) Matrix Metalloproteases 2 IHC Immunohistochemical
To determine whether there is an association between ERβ and TLR4, we first analyzed a range of standard clinicopathological parameters and protein expression levels by immunohistochemistry (Fig. 1a)
The total positive rate of ERβ expression in the primary NSCLC samples was 73.33%, and TLR4 expression appeared in 80.00%
Summary
Abbreviations NSCLC Non-small cell lung cancer ERβ Estrogen receptor beta TLR4 Toll-like receptor 4 MMP2 Matrix Metalloproteases 2 IHC Immunohistochemical. Our previous studies first detected a significantly higher expression level of ERβ in metastatic lymph nodes of non-small cell lung cancer (NSCLC) patients than in primary tumor tissue. The results further indicated that treatment with estrogen or ERβ activation significantly promotes lung cancer cell metastasis by upregulating invasiveness-associated matrix metalloprotease 2 (MMP2) both in vitro and in vivo [13]. Recent studies have provided evidence that TLR4 activation augments NSCLC cell adhesion to murine hepatic sinusoids and the formation of hepatic metastases in vivo, and an upregulation of MMP2 expression in response to LPS induction has been identified [21]. Novel inhibition of NSCLC cell proliferation induced by knocking down TLR4 expression has been detected in vitro [26], the antimetastatic effect of specific TLR4 signaling inhibition is still unclear
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