We evaluated the association of VDR expression with OS in a small cohort of patients with NSCLC who had received no prior therapy. We found that high nuclear VDR expression was independently associated with better OS in the overall study population. No such association was observed for cytoplasmic VDR expression.
Our results support our hypothesis that VDR expression is associated with improved survival in NSCLC. The role of VDR signaling in mediating improved survival and preventing metastatic growth has been shown in various malignancies in preclinical studies [34
] and lend support to our findings. In vivo
studies have demonstrated that in a defined animal model, acting through VDR, 1,25(OH)2
D inhibits metastatic growth of lung carcinoma cell lines [34
], while in vitro
studies in breast cancer have shown that ablation of VDR shortens survival in MMTV-neu mice [36
]. Additionally, a pilot study of human carcinomas (including lung) that measured VDR concentration using an immunoradiometric assay showed that an alteration in VDR number occurs when a cell undergoes malignant transformation and suggested a role for VDR measurement as a marker of prognosis [37
There have been few studies of VDR expression in lung cancer [22
]. Although these studies performed immunohistochemistry using the same monoclonal antibody to VDR as our study, they each used a different approach to quantify the immunohistochemical expression of VDR. Kaiser et al [22
], detected only nuclear VDR expression in their 37 NSCLC samples, and described the percentages of cells displaying nuclear receptor expression across various histologies. In our study, we detected both nuclear and cytoplasmic VDR expression among the various histological subtypes of NSCLC. Menezes et al [23
] performed a descriptive study of VDR immunohistochemical expression across the lung carcinogenesis spectrum in 35 patients with lung cancer and 78 premalignant bronchoscopic biopsy specimens. They assessed the intensity of staining and percentage of positive staining separately for nuclear and cytoplasmic VDR expression, and found that VDR expression was similar among squamous cell carcinoma and adenocarcinoma histologies with more nuclear than cytoplasmic expression, and noted that cytoplasmic VDR was generally lacking in their tumor samples compared to non-tumor samples. We found high nuclear VDR expression among adenocarcinomas and squamous cell carcinomas. A higher proportion of adenocarcinomas in our study also retained high cytoplasmic expression compared to other histologies. Both Menezes et al and Kaiser et al did not evaluate the relation of VDR expression with outcome. Therefore, we were unable to perform a direct comparison of our other study findings.
Our findings suggest that both VDR expression characteristics, intensity of staining and percentage of stained cells, may influence the mortality end-point for nuclear (but not cytoplasmic) VDR expression.
Upon ligand binding, VDR transits from the cytoplasm to the nucleus, where it regulates gene expression. In colon cancer cells, it has been shown that a cytosolic pool of VDR can also rapidly activate an intracellular signaling cascade (in response to 1,25(OH)2
D treatment) that ultimately enhances transcriptional regulation by nuclear VDR [38
]. To evaluate the potential importance of this non-genomic signaling pathway, we separately quantified VDR expression in the nucleus and cytoplasm of tumor cells. We observed high cytoplasmic VDR expression in women, and subjects with adenocarcinoma histology. The biological mechanism for this differential VDR expression among certain individual characteristics is unclear. In a recent study of 619 patients with colorectal cancer, cytoplasmic VDR overexpression was independently associated with K-ras and PI3KCA mutations [18
]. Additionally, studies conducted in NSCLC cell lines in our laboratory (unpublished data, Hershberger PA) reveal that cell lines with K-ras mutations tend to have lower nuclear VDR expression compared to cell lines without K-ras mutations. K-ras mutations occur predominantly in lung adenocarcinomas, and most of these mutations are smoking-related G-T transversions [39
]. Since the majority of patients in our study were current or former smokers, it is possible that they may harbor K-ras mutations that may be responsible for the higher cytoplasmic (and lower nuclear VDR) expression among these patients.
The differences in VDR expression between males and females in our study cannot simply be attributed to differences in estrogen exposure for two reasons. First, the mean age of patients analyzed in our study was 69. Thus, a majority of the females we analyzed were likely to be post-menopausal. Secondly, recent studies have shown that a majority (>80%) of primary lung tumors from both males and females express aromatase, the enzyme that synthesizes estradiol from androgens [40
]. Because lung tumors in both males and females have the capacity to locally synthesize and respond to estradiol, any effect of estrogen on VDR expression could potentially occur in both sexes.
VDR activity may also be affected by other members of the Vitamin D signaling pathway [3
]. Additionally, polymorphisms in the VDR gene may influence VDR expression in lung cancer tissue and need to be investigated in future studies.
Zhou et al and Heist et al studied the effect of Vitamin D status in early and advanced stage NSCLC respectively [12
]. In a study of 456 patients with early stage NSCLC, Zhou et al found that improved survival was associated with seasonality (surgery during summer) and high recent vitamin D intake [12
]. In the same cohort of early stage patients, circulating 25(OH)D levels were also associated with improved survival, and Cdx-2 polymorphisms in the VDR gene were associated with improved survival in patients with squamous cell carcinoma [13
]. However, there was no association of survival with circulating 25(OH)D levels in late stage NSCLC [41
]. In our early stage patients, high nuclear (but not cytoplasmic) VDR expression was associated with better survival, and a similar trend was seen in our late stage patients although it was statistically non-significant. Larger studies are warranted for definitive conclusions, and if our findings are confirmed, indicate that vitamin D based therapies may also be beneficial in late stage NSCLC patients with high nuclear VDR expression. In fact, the protective effects of a sufficient vitamin D status on various diseases, including cancer, has already led to recommendations for clinical practice [42
Our study has many limitations. Our conclusions are limited by the small sample size. However, despite this limitation, nuclear VDR expression was independently associated with improved overall survival in NSCLC after controlling for some of the important determinants of survival suggesting that the effect size was large enough to be significant. A major limitation is the absence of serum 25(OH)D levels in our NSCLC patients. Hence, we were unable to evaluate the joint effects of VDR expression and serum 25(OH)D levels. Our smoking status information was based only on data collected at the time of recruitment, and is subject to recall bias. For the survival analysis, we assessed OS only as we were unable to distinguish deaths due to lung cancer from deaths due to other causes. Additionally, other factors that influence survival in lung cancer such as performance status, adjuvant therapy after surgical resection were not assessed. Other unadjusted factors in our study include solar UV-B exposure, season of surgery, dietary intake, and body mass index, all of which influence Vitamin D levels. A majority of our study population was Caucasian. This may influence vitamin D status as dark-skinned individuals require more exposure to UV-B radiation than light-skinned individuals for producing an equivalent amount of vitamin D [1
]. Another limitation is generalizability of findings to other populations as our study is based on a convenient sample of patients who were recruited based on certain eligibility criteria and underwent surgical resection of their primary lung cancer at a tertiary care hospital (UPMC). Despite the limitations, there is biological plausibility to support our study results, and even the upper limit of our estimate indicates a 21% improvement in overall survival for patients with high nuclear VDR expression after adjusting for some other important predictors of survival in NSCLC. These findings suggest a role for VDR as a biological marker of prognosis in NSCLC. The patients in our study received no prior neoadjuvant chemotherapy or radiation therapy, thereby eliminating any bias associated with the predictive value of VDR on outcome that may be related to prior therapy. There was minimal missing data in our study.