There is increasing evidence for steroid hormone effects in lung cancer. We report here for the first time that cytoplasmic ERβ (specifically ERβ-1) is an independent negative prognostic factor for lung cancer and confirm a previous report that PR is an independent positive prognostic factor. We further show that the combined analysis of these two receptors significantly increases calculated risk of progression and death from lung cancer. Nuclear ERβ or nuclear and cytoplasmic ERβ considered together showed no significant effect on survival in our analysis, suggesting that cytoplasmic ERβ signaling is more important for patient survival than its nuclear signaling. In the case of PR, impact on survival was only significant when considering the sum of cytoplasmic and nuclear PR staining, suggesting both cellular compartments contribute to PR effects in lung cancer. We observed few sex differences in the impact of cytoplasmic ERβ and PR on survival when the markers were considered together. It is possible that ERβ cytoplasmic expression has more impact on survival in men than women when considered as a single variable, but much less so when considered together with PR. Further, there were no significant interactions between sex and any of the biomarker(s) of interest when controlling for other variables. It does not appear that differences in these steroid hormone receptors can account for the large survival difference observed in general between men and women with lung cancer. We further found in both men and women that ERβ is upregulated in lung tumor tissues compared to normal lung tissue, while PR is downregulated in lung tumors compared to normal lung. We observed that these pathways are dysfunctional to the same extent in male and female lung cancer.
The reported expression frequency and localization of hormone receptors in lung cancer has been variable. These differences could be due to lack of standardization in multiple factors including 1) interpretation of the staining 2) antibody and dilution used 3) variability in the scoring assessment or 4) differences in patient cohort characteristics. These discrepancies probably contribute to variable results regarding prognostic significance. There are reports of nuclear ERβ as a positive prognostic factor for lung cancer (8
) but in a recent report, survival effect of nuclear ERβ was limited to patients with EGFR mutations. Recently an association between high ER expression and EGFR mutant lung tumors was found (12
), and strong ERβ expression predicted a good clinical response and longer progression-free survival after EGFR TKI treatment for lung adenocarcinoma patients (29
). In our cohort, EGFR mutation status was not available on all patients; EGFR mutations occur mainly in never smokers (30
) and our cohort consisted of only 13 never smokers. We found 4 EGFR mutant tumors in these 13 patients. No statistical differences were observed in any of the biomarker expression levels between the 4 EGFR mutant tumors and the 9 EGFR wild-type tumors. Several EGFR mutations may have been missed, but are unlikely to contribute to survival effects we observed. We found no difference in ERα or ERβ cytoplasmic or nuclear expression between never smokers and all other patients, and no prognostic significance of ERβ when the never smokers were examined separately. PR expression however was significantly higher in never smokers, and could be an important survival variable for patients without tobacco exposure. Determining a selective effect of tobacco history or EGFR mutation on the variables we examined would require a much larger population of never smokers.
The significance of ERβ cytoplasmic staining as an independent negative prognostic factor for lung cancer has not been reported previously. In part, this could be because cytoplasmic ERβ staining has been largely ignored during staining interpretation or alternatively could be due to which antibody was used. We used an antibody that specifically detects the ERβ-1 isoform. Other commercial antibodies directed towards the amino-terminus of ERβ, such as H-150, may also recognize other isoforms of ERβ which may confound the results. A recent report shows that ERβ-1, but not ERβ-2 is linked to worse prognosis in Stage I lung adenocarcinoma in women (31
). We have previously shown that full-length ERβ protein was detected by Western blot in both the nuclear and cytosolic fractions of NSCLC cells, demonstrating that cytoplasmic staining for ERβ is biologically meaningful (7
). Hormone receptors do mediate effects through non-genomic pathways, which usually occur in the cytoplasm and involve bidirectional cross-talk with growth factor receptor pathways. We and others have reported rapid activation of MAPK by β-estradiol in NSCLC cells, which is dependent on immediate release of EGFR ligands (21
). Our survival results suggest that non-genomic ERβ signaling in the lung has important clinical implications. We also observed that ERβ cytoplasmic and nuclear expression values are moderately correlated with each other but in our study nuclear expression did not predict survival. It is unknown how cellular compartmentalization of ERβ is controlled. We were unable to analyze the combined effect of high ERβ cyto/low ERβ nuclear versus the opposite due to the small number of patients whose tumors exhibited this combination. However, there may be sub-groups in which amount of nuclear ERβ protein is prognostic. Supplemental Table 1
summarizes the results of our study and others on the prognostic significance of ERβ. Either an antibody that specifically recognizes ERβ-1 or an antibody to the N-terminus which recognizes all ERβ isoforms was used. Our study lies in the middle of the extremes of the staging reported in other studies and most likely does not contribute to differences in ERβ prognostic value observed. The main difference in these studies is in the scoring and interpretation. Whether or not nuclear staining only or nuclear and cytoplasmic staining were scored separately or together is a major variable as well as the cut-off points used for statistical survival analyses. Our study compared patients with the highest ERβ expression versus all others, unlike all other studies which compared negative versus positive or negative/weak versus strong.
We could not demonstrate a predictive value for extent of ERα expression, but did demonstrate that expression is dependent upon which antibody is used. Antibody reactivity is a function of presence or absence of the epitope against which the antibody is raised. Results from mRNA analysis of NSCLC cell lines suggest that a series of ERα mRNA splice variants are present with little or no full-length mRNA in the lung cancer cell lines that we examined. By IHC in lung tumors, an antibody to the ERα C-terminus, which is predicted to be present in proteins translated from the splice variants we detected, was the most reactive. Raso et al.
recently tested a panel of antibodies for ERα IHC expression and reported varying expression results based on the antibody (15
). Either ERα expression was not observed at all or high cytoplasmic expression was observed, in line with our findings. Whether or not ERα variants have any functional role is not known. We have previously shown that an ERα selective agonist does not activate transcription from an estrogen response element in NSCLC cells, does not cooperate with EGF to promote lung tumor growth, and does not induce lung tumor xenograft growth (7
), while ERβ selective agonists are active in these assays.
We found no general association between aromatase and survival in our cohort whereas Mah et al.
have shown that aromatase was predictive, but only in women age 65 and older with early stage disease (19
). In our patient population, only 41 women met these criteria, compared to 103 in the study by Mah et al.
There was no difference in OS or TTP among high versus low aromatase for these 41 patients. Small sample size most likely limits our ability to observe this effect.
Our results confirm a previous report that PR is a strong protective factor for lung cancer (16
), similar to what is observed in breast cancer (32
). Recent results from the Women’s Health Initiative showed that HRT (estrogen plus progestin) increased the risk of lung cancer death (6
). Furthermore, the Vitamins and Lifestyle (VITAL) study demonstrated that women who took combined estrogen and progestin had an increased risk of lung cancer while estrogen-only used showed no effect (33
). Progesterone exerts its tumorigenic effects by increasing angiogenesis (34
), however progesterone is normally only produced during the menstrual cycle and pregnancy. Progesterone levels found in post-menopausal women and men are below the 1ng/ml or higher serum range required to stimulate the PR (35
). In breast cancer, PR is known to signal through ligand-independent mechanisms due to phosphorylation by kinases (36
). One molecular mechanism for loss of PR expression in breast tumors is down-regulation of PR by increased growth factor signaling which portends a more aggressive biology (37
). It is unknown how the PR signals in NSCLC, however ligand-independent mechanisms may be involved since the populations most at risk are older men and women, groups that do not have a source of progesterone synthesis. Additionally, the antibody used in this study does not distinguish between the PR-A and PR-B isoforms, which could exert different functions.
It will be important to determine the role of multiple sex hormone-related proteins in predicting lung cancer survival and understanding how interactions between hormones and growth factors are involved in lung cancer. A recent report shows that nuclear ERβ and aromatase are often expressed together (14
). ERβ and aromatase were only weakly correlated in our study. The significant markers in our study, ERβ cyto, PR total, and EGFR were only weakly correlated suggesting that results with more than one marker are not artifacts of using markers that are highly correlated. In regards to survival, the combined contribution of ERβ and PR held up in a multivariate regression model after adjusting for other variables such as sex and stage. The HRs found for combined cytoplasmic ERβ and total PR suggested a 2.6 to 6.0-fold increase in poor outcome for at-risk patients and the increased risk for the combination was significantly different from that of ERβ cyto high alone. Combining other markers singly with either ERβ or PR was not significant.
Expression of EGFR by IHC has been reported in a wide range (35–83%) of lung tumors in clinical studies (38
). A meta-analysis of 2972 NSCLC patients concluded that EGFR expression considered as a single variable has no effect on survival (38
). While EGFR expression was only marginally significant as a single variable, adding EGFR expression to combined ERβ and PR expression showed enhanced survival effects for OS, but not for TTP. Whether this is a true selective effect or due to sample size constraints is not known. Because of cross-talk between EGFR and ER in lung cancer, degree of EGFR expression might modify ER survival contributions. EGFR gene copy number or mutations are known to contribute to survival and response to therapy. EGFR copy number or mutation might have additional predictive value in a model including expression of EGFR, PR, and ERβ.
Based on our findings and those previously reported, a standardized approach (same antibody epitope, dilutions, epitope retrieval method, scoring system and interpretation) should be developed and validated for screening of lung tumor ERα, ERβ-1, PR, EGFR and aromatase expression in patients. A larger cohort will be needed to examine and validate the predictive value of these markers on lung cancer survival. EGFR and aromatase expression contributed to survival effects only in context with steroid hormone receptors. Following validation, systematic examination of these markers could potentially be prognostic and may be useful to identify patients of both sexes who might respond to anti-estrogen therapy, growth factor TKI therapy, or a combination of both.