Results from the present study demonstrate for the first time that a panel of estrogen metabolism genes is expressed in cultured human head and neck cells. Detection of transcripts for these genes in both premalignant lesions and HNSCCs suggests that these enzymes contribute to cellular metabolism throughout tumorigenesis. While the importance of E2
signaling and ERβ in HNSCC has been suggested previously (23
), the contribution of the estrogen pathway to the premalignant stage of head and neck tumorigenesis has not been evaluated. Furthermore, even though the expression of individual xenobiotic/hormone metabolism genes has been evaluated in head and neck cells (26
), the potential role of estrogen in head and neck carcinogenesis has not been investigated in a comprehensive manner.
We demonstrate here for the first time that CYP1B1
is upregulated in MSK-Leuk1 but not in HNSCC cells following E2
exposure. The mechanistic basis for this differential upregulation of CYP1B1 remains unclear. However, it has been shown for lung cancer that the timing of hormone exposure relative to a diagnosis of lung cancer may make a difference with respect to whether the hormonal effect is protective or adverse (29
). CYP1B1 metabolizes hormones, including E2
, and xenobiotics, including tobacco-associated carcinogens, to species that can cause DNA damage (11
). Enhanced expression of CYP1B1
, in the absence of an elevation in the expression of the conjugation gene COMT
, could potentially promote the accumulation of mutagenic DNA damage and contribute to the formation of HNSCCs. Indeed, the CYP1B1
*3 allele, which confers increased catalytic activity, has been associated with increased susceptibility for HNSCC (31
In order to assess the functional role of CYP1B1 in premalignant head and neck cells, the migratory potential of MSK-Leuk1 cells deficient in CYP1B1 was compared to that of the same cell line carrying control vector. The novel finding that CYP1B1 deficiency leads to the decreased migration of oral leukoplakia cells is consistent with a prior report of an association between CYP1B1 depletion and the decreased invasiveness of cultured endometrial cancer cells (33
). The invasive properties of MSK-Leuk1 cells were previously shown to be enhanced by tobacco smoke via induced expression of the urokinase-type plasminogen activator (34
). Tobacco smoke also induces CYP1B1 (14
); however, the role of this pathway in the invasiveness of MSK-Leuk1 cells was not investigated. In the present study, the observed decrease in cell migration was not attributed to decreased proliferation (). However, when a longer exposure time was investigated (72 hours, ), cell proliferation was decreased in CYP1B1-deficient cells, as compared to vector-expressing cells. The ability of CYP1B1, independent of E2
, to promote the migration and proliferation of oral premalignant cells suggests that it may play a role in the clonal spread of leukoplakic lesions within the oral mucosa and facilitate cancer progression within the head and neck.
Exposure to E2
failed to alter the rate of cell proliferation in MSK-Leuk1 cells, irrespective of CYP1B1 levels. In contrast, E2
inhibited apoptosis in both control and CYP1B1-deficient MSK-Leuk1 cells. A previous report indicates that E2
exposure increased the proliferation of cultured HNSCC cells derived from locally advanced tumors marginally; however, the effect of E2
on apoptosis was not analyzed (23
). The observed ability of E2
to decrease apoptosis in premalignant cultured cells suggests that estrogens may be involved in the progression of premalignant lesions to HNSCCs. The ability of the pure antiestrogen fulvestrant to antagonize E2
-mediated inhibition of apoptosis, suggests that this effect is ER-mediated and that antiestrogens may be beneficial as chemopreventive agents for HNSCC.
Using TMAs of surgical specimens from 128 patients, we have demonstrated that CYP1B1 protein is present at detectable levels in normal, dysplastic and tumor tissues of the head and neck. Previously, CYP1B1
mRNA and/or protein have been detected in HNSCC cell lines (35
) and MSK-Leuk1 cells (37
); however, human head and neck tissues have not been analyzed for CYP1B1 protein previously. The present results further demonstrate that CYP1B1 is overexpressed in HNSCCs, as compared to the normal epithelium of the head and neck. CYP1B1 overexpression in tumors relative to normal tissue has been demonstrated for a number of organs, including breast, uterus, skin, lung and esophagus, but not for head and neck (39
). Our observation of enhanced expression of CYP1B1 in HNSCCs suggests that CYP1B1 may be a marker of head and neck tumorigenesis.
MSK-Leuk-1 cells, as well as cultured HNSCC cells, stained positive for ERβ expression. Consistent with these data, ERβ was detected in human HNSCCs and dysplastic tissues as well as in the normal epithelium. ER isoforms have been detected previously in human HNSCC tissues and cell lines (23
), but not in premalignant cells, with ERβ being the predominant transcriptionally active form (23
). The detection of ERβ in both dysplastic and HNSCC cells suggests the potential contribution of estrogen signaling to the development of HNSCCs at both the premalignant and malignant stages. The absence of a gender difference in the intensity of immunohistochemical staining for CYP1B1, ERβ or E2
indicates that the estrogen pathway may contribute to head and neck carcinogenesis in both males and females.
In summary, we have shown that a number of estrogen metabolism genes are expressed in cells derived from both early- and late-stage lesions of the head and neck. This study is the first to report the detection of estrogen within human head and neck tissue, and demonstrate that both estrogen and CYP1B1 may contribute to the progression of HNSCCs. These data suggest that CYP1B1 may be an important biomarker of tumorigenesis in the head and neck and may represent a novel target for chemopreventive intervention in patients with premalignant lesions.