A mounting body of evidence from cell culture and mouse models has shown that estrogen and activation of the estrogen receptor pathways are important not only in lung embryogeneis [26
] but also in lung cancer pathogenesis [9
] Aromatase mediated conversion of androstenedione to estradiol [30
] may also be important for lung cancer progression [29
]. Evidence suggests that the majority of intratumoral estradiol is produced locally by aromatase in the lung tumors themselves, possibly from circulating androgens [11
]. The exact functions of estrogens in lung cancers however are not clear and gender-based differences also need to be further elucidated. In NSCLC cells, estradiol (E2) was shown to increase cell proliferation in both in vitro
and in vivo
]. In contrast, agents which blocked estrogen synthesis (such as the aromatase inhibitors anastrazole, exemestane) or interfered with receptor function (such as the pure antiestrogen fulvestrant) inhibited tumor xenograft proliferation [9
]. Effects of estrogen in lung cancers may be mediated directly by receptor binding followed by nuclear localization and activation of transcription or indirectly by extranuclear pathways that engage kinase signaling to modulate transcription and tumor progression. In addition certain oxidative metabolites of estrogen such as catechol estrogen-3,4-quinones can react with DNA to form depurinating adducts, possibly leading to mutations that promote cancer initiation [33
Gender related differences in lung cancer have been well documented and suggest a role for hormonal influences. Of non-smokers with lung cancer, a higher percentage are women [34
]. Women have increased susceptibility to lung cancers from tobacco exposure but have overall better prognoses in some studies but not others [35
]. In an analysis of the SEER database, a survival advantage for older (55-59 years) versus younger (40-49 years) women was seen for squamous cell carcinoma and bronchioloalveolar carcinoma suggesting postmenopausal status might be advantageous at least for these histologic subtypes [16
]. Also recent studies from the Women's Health Initiative (WHI) and the Vitamins and Lifestyle studies showed a possible increase in incidence and mortality from NSCLC in post-menopausal women treated with combined estrogen and progesterone hormone replacement therapy [39
]. A somewhat analogous finding was reduced lung cancer mortality in breast cancer patients who had been treated with anti-estrogens[42
We have set out to profile key elements of the estrogen / ER signaling pathway in individuals with lung cancer. Previous proteomics results showed that aromatase expression levels were a strong predictor of survival in women over 65 years of age with NSCLC. Lower levels of aromatase predicted a significantly longer survival. Here, we have continued to assess the estrogen signaling pathway by examining the expression levels and localization of ERα and ERβ. While ERβ in contrast to ERα displayed enhanced expression with increasing grade, neither ERα nor ERβ alone was predictive for survival in individuals with NSCLC nor any patient subgroup examined (gender, histology, stage, or smoking status). However, when we combined ERβ expression with aromatase expression, we found that higher levels of both proteins together predicted a significantly poorer survival outcome. While this observation held true for all individuals with NSCLC examined together, it was somewhat stronger for women of age 65 years and older and weakest in men under 65 years of age.
ER Signaling Pathways
Of note, cytoplasmic levels of ERβ were a stronger predictor of outcome than nuclear expression. Extranuclear estrogen receptor signaling may have considerable importance through interactions with tyrosine kinase receptors (EGFR and IGF1-R), MAP kinase and/or PI3/AKT kinase signaling [16
]. In breast cancer cells, recent data suggest proline-glutamic acid-leucine-rich protein-1 (PELP1) couples ERs to signaling pathways such as Src-MAPK, PI3K-Akt and EGFR-Stat3 [45
ERs have also been found to cross-communicate with EGFR signaling in NSCLC cells such that combined targeting of ER and EGFR enhances antitumor effects [17
]. EGFR mutations are well documented in a subset of individuals with lung cancer; such mutations are more prevalent in women in East Asian populations, never-smokers and individuals with adenocarcinoma [48
]. Recently, Nose et al. observed that in individuals with EGFR mutations, higher ERβ expression significantly correlated with lung cancer-free survival [49
]. While we do not currently have the EGFR mutation status in the cohort we examined, the interplay between ER and EGFR is certainly an aspect that we are actively examining.
The observation that cytoplasmic levels of ERβ were more strongly predictive of survival in the presence of high aromatase than nuclear ERβ levels could suggest that extranuclear signaling, likely in concert with kinase signaling pathways, may have important factors in lung cancer progression. Indeed, it was recently reported that extranuclear ER forms are critical in regulating invasion and metastatic progression in breast cancer [45
]. It will be important to determine if similar functions are mediated by extranuclear ERs in lung cancer.
Niikawa et al.
recently reported measurements of aromatase and estradiol within lung tumors [11
]. They found that estradiol levels correlated with aromatase expression and that median estradiol concentrations were significantly higher in NSCLCs than non-neoplastic lung tissues. In addition, they also observed that when tumors were dichotomized based on the median level of estradiol, the group with both elevated estradiol and ER positivity, tended to have a worse prognosis. Although these earlier results did not quite reach statistical significance, they indicated a trend consistent with our current report.
Published data on both the presence and effects of ERα and ERβ on survival in lung cancer is somewhat varied [11
]. This might be due to a lack of reproducibility in IHC with different anti-ER antibodies and diverse preparative methods [7
] and/or to the relatively lower levels of ER proteins in lung tissue as compared to breast tissue. Of note, there is currently no standardized IHC assay for the measure of ERs in lung cancer nor for breast cancer [54
]. In addition, as is seen for EGFR, ethnic and regional variations in the several patient populations may account for different effects of estrogen signaling. Nevertheless, general trends are starting to emerge with overall higher expression levels of ERβ in lung cancer compared to ERα and a trend towards the former playing a role in lung cancer pathobiology and reflecting disease aggressiveness [8
]. As one example, Hershberger et al. reported that ERβ agonists are highly effective in promoting proliferation of lung tumor cells [16
]. This contrasts somewhat with what is observed in breast cancers where ERα tends to predominate over ERβ expression [11
The results presented here are consistent with our overall hypothesis that NSCLC cells hijack the estrogen signaling pathway. We predict that the mechanism of progression for most if not all NSCLCs involves dysfunction at one or more nodes in this pathway. It is interesting to note that while aromatase was primarily predictive in women 65 years or older, the combination of aromatase plus ERβ levels was a strong indicator of outcome in both men and women. This observation could reflect hormonal differences as a function of age. Estrogen levels in women decline after menopause while levels remain relatively constant in men throughout life. In postmenopausal women, the ovaries respond to higher gonodotropin levels and therefore contribute significantly to the circulating pool of testosterone and androgens. This appears to be maintained for at least ten years past the onset of menopause [56
]. Levels of androgens fall in women during the reproductive years, then may continue to fall [57
] or level off after approximately 65 years of age [58
]. Effects of other interacting proteins such as sex hormone-binding globulin (SHBG) may also play a role in their bioavailability. Androgens are the substrates used by aromatase for estrogen synthesis. As we continue to map branches of the ER signaling pathway with regard to expression level and activation in men and women with NSCLC, we predict that different nodes will be preferentially enhanced in different substrata of individuals. Such characterization may provide useful clinical tools to determine disease aggressiveness as well as potential targets of therapeutic attack. We continue to explore both of these possibilities.