In lung cancer GRPR and its ligand are involved in autocrine growth stimulation [6
]. Previous results showed that lung tumor tissues have elevated GRPR
expression, and we have previously described elevated GRPR
mRNA levels in histologically normal mucosal tissues adjacent to HNSCC compared to oral mucosal tissues from cancer-free control subjects [6
mRNA expression has also been detected in prostate tumors and tissues adjacent to prostate cancers [15
]. Our new findings reported here indicate that in a prospectively collected lung cancer case-control population, GRPR
expression in at-risk upper aerodigestive mucosa was significantly associated with lung cancer. Importantly, even after controlling for the effects of possible confounding by age, sex, and tobacco use, GRPR
expression in non-cancerous mucosal tissues was significantly associated with lung cancer among never and former smokers and appeared to confer similar risk to both sexes.
Our finding here of no difference in bronchial epithelial cell GRPR
expression between men and women did not replicate our previous result [2
]. In our previous bronchial cell study, which had a smaller sample size of 78 patients, the presence of cancer was not separately evaluated, and only one never smoker with lung cancer was male [2
]. In retrospect, it is likely that the associations between GRPR
expression, female sex, and smoking observed in our previous study were actually surrogates for the underlying association between bronchial epithelial GRPR
expression and lung cancer, which appears to be most significant in never smokers. Therefore, this study presents important revisions to our previous understanding of the role of GRPR
in lung cancers arising in females and males; this study supports a similar role for bronchial GRPR
mRNA expression and lung cancer risk for both females and males.
Although in the current study we also found no association between GRPR
expression and pack-years of smoking, we did observe associations between smoking status (active versus former smoker versus never smoker) and GPRR
expression. We observed these associations for both cases and controls separately, but the relationships were different. The proportion of GRPR
positive actively smoking cancer-free control subjects was similar to the proportion of actively smoking lung cancer cases. These findings were consistent with our previously reported findings that GRPR
expression in bronchial epithelium was activated with tobacco use [2
] and with findings that bombesin-like peptide receptors play a role in wound healing following airway injury [16
]. Similar to our previous findings of persistent bronchial GRPR
expression after tobacco cessation [5
], we detected bronchial GRPR
expression in the majority of former smoking lung cancer cases. In contrast, in the current study bronchial GRPR
expression was detected in only a minority of cancer-free controls who were former smokers. The inclusion of more cancer-free control subjects in this study compared to our 1997 study has allowed us to evaluate lung cancer patients and cancer-free controls separately and has revealed new insights regarding the relationship between bronchial GRPR
expression and tobacco use in cancer-free controls.
Although the number of active smoking surgical controls in our current study was small, the data suggest that bronchial GRPR expression may be induced by tobacco use in subjects without lung cancer, but that the increase in GRPR expression in bronchial mucosa likely subsides following the cessation of smoking in most subjects without lung cancer. Among former smoking lung cancer cases, bronchial GRPR expression may be aberrantly maintained following cessation of smoking, or similar to never smoking lung cancer cases, bronchial GRPR expression may reflect risk that is independent of tobacco use. Our 1997 report indicated that of the 4 cancer-free subjects with defined bronchial GRPR expression and smoking status, 1 active smoker was GRPR positive while 3 subjects who were former or never smokers were negative for GRPR expression. Therefore, although the numbers are small, among cancer-free control subjects, the relationship between smoking status and bronchial GRPR expression in the 1997 study is consistent with our current study results.
Of special interest was the finding of frequent bronchial GRPR expression among never smoking lung cancer cases. While GRPR expression was detected in only a minority of never smoking cancer-free controls, GRPR expression was detected in almost 90% of never smoking lung cancer cases. Though the specific cause of GRPR expression in never smoking lung cancer cases is unknown, we posit that bronchial GRPR expression may reflect an inherent or conferred risk factor that can be best observed in the absence of the more potent risk factor of tobacco use. Though bronchial GRPR expression was more common among cancer-free controls with a diagnosis of granuloma, suggesting a possible inflammatory component to bronchial GRPR expression among cancer-free controls, bronchial GRPR expression was not increased in lung cancer cases or controls with more severe pulmonary obstruction, which also has an inflammatory component. Therefore, the role of inflammation in elevated bronchial GRPR expression remains undefined.
GRPR expression in normal bronchial tissues was not correlated with clinical disease stage. Therefore, our data suggest that GRPR expression in surrogate tissues did not reflect tumor burden and, perhaps, was not a direct consequence of the prevalent cancer. Our finding that detectable GRPR expression in normal upper aerodigestive tissues was not an indication of poor overall survival for lung cancer cases indicates that elevated GRPR bronchial cell expression was not associated with disease progression and is, instead, likely to be a marker of risk exposure or a marker of host susceptibility.
Lung cancer cases positive for GRPR
bronchial expression were significantly younger than cases negative for GRPR
expression, which supports the role of GRPR
bronchial expression as conferring lung cancer risk. Though a prospective cohort study will be required to fully understand the relationship between GRPR
expression levels in surrogate tissues and the development of lung cancer, GRPR
expression in normal bronchial tissues has potential value as a marker for elevated risk, especially in those with little or no tobacco exposure. Though GRPR
is overexpressed in many solid tumors, only one other group has evaluated GRPR
and/or their gene product levels in surrogate tissues of cancer patients to date. Uchida et al. reported that serum levels of proGRP, as measured by enzyme-linked immunosorbent assay (ELISA), correlated with tumor GRP
gene expression levels in small cell lung cancer (SCLC) patients [17
expression levels in tumors were not evaluated in our study, as material for analysis was not available.
The increased risk due to elevated GRPR
expression may be most apparent in never and former smokers because the contribution of GRPR
expression to risk is obscured in active smokers by factors such as genetic abnormalities and inflammatory processes that confer substantial risk from tobacco use. Elevated GRPR
expression in the lung may independently contribute to increased cancer risk by promoting proliferation. GRPR
is expressed at early embryonic stages in the nervous, urogenital, respiratory, and gastrointestinal systems and expression in these tissues is generally down-regulated before birth [18
]. The GRPR ligand, GRP, a bombesin-like peptide (BLP) growth factor, is expressed by pulmonary neuroendocrine cells and has been shown to stimulate lung development in utero and to increase growth and maturation of human fetal lung organ cultures [20
]. In non-cancerous tissues, BLPs stimulate growth of bronchial, gastrointestinal and pancreatic epithelial cells and lead to ligand-dependent hyperplasia [5
]. GRPR and GRP are involved in an autocrine stimulation loop in lung cancer and HNSCC [6
], and GRPR
expression has been shown to be positively regulated by GRP [20
]. Increased GRPR
expression in the lung may, therefore, reflect a state that is more nascent and proliferative in nature than epithelium with low or undetected GRPR
We acknowledge our case-control study population limitations. This case-control study required hospital surgical controls, and this limited recruitment with the result that our lung cancer cases are older than our controls and include fewer men. In addition, the exhaustion of samples made quantitative measurements of GRPR mRNA expression impossible. We have confined our analysis to GRPR mRNA because of antibody reagent limitations at the time the samples were evaluated. This leaves the question of whether GRPR protein levels also differ unanswered. Despite these limitations, a high degree of association between detectable GRPR expression in normal bronchial tissue and lung cancer was demonstrated in the case-control population even after adjusting for sex and age.
Though we did not assess the epidermal growth factor receptor (EGFR
) mRNA or protein expression in bronchial epithelial cultures, we speculate that increased GRPR
expression contributes to lung cancer through EGFR-dependent and/or -independent mechanisms. The EGFR pathway has been reported to be activated in lung tumors from never smokers with EGFR mutations, and it is possible that lung tumors developing in never smokers have multiple mechanisms for EGFR activation. The GRPR pathway is known to interact with the EGFR pathway in lung cancer cells by increasing the release of EGFR ligands such as amphiregulin [8
], which could act to further promote cancer in never smokers who develop EGFR mutations. Alternatively, activation of the GRPR pathway may increase EGFR bronchial cell signaling in the absence of EGFR mutation, providing another route to lung cancer development in never smokers.