To the best of our knowledge, this study represents the largest study on susceptibility to SCCHN risk associated with potentially functional IGFBP5 polymorphisms. In this study of 1,082 SCCHN patients and 1,120 cancer-free controls of non-Hispanic whites, we did not find evidence of a main effect of IGFBP5 -1195T>C and -709G>C on SCCHN risk in the case-control analysis. However, the -1195C variant genotypes were associated with late-stage SCCHN at HPV-unrelated sites. Further case-only analysis revealed that -1195CC and CT+CC genotypes were significantly associated with the late-stage tumors (stage III and IV) at HPV-unrelated sites. In addition, we found that the change of -1195T allele to C allele resulted in the decreased promoter activity of IGFBP5, which may be regulated by the AP-1 transcription factor.
Very few association studies have been conducted to evaluate the roles of IGFBP5
polymorphisms in cancer risk. In the only published population-based case-control study of 460 African-American women with breast cancer and 279 matched controls as well as 461 Nigerian women with breast cancer and 163 matched controls, a 50-kb DNA sequence covering three exons in the 3′ end of IGFBP2
and three exons in the 3′ end of IGFBP5
was significantly associated with breast cancer risk (20
), but this study did not include any functional SNPs, nor the promoter polymorphisms that were studied in our current study of non-Hispanic whites. Although these two studies did not provide relevant data for the comparisons, our larger sample size provided adequate statistical power for detecting a meaningful main effect of IGFBP5
variant genotypes on SCCHN risk and tumor progression in non-Hispanic whites.
In our study, the genotype distribution of -709G>C in controls did not follow HWE. This could have been caused by genotyping error of 1% in our study, but this is unlikely to play a major role; selection bias could not be fully ruled out, because our control subjects were recruited from the cancer-free visitors to MD Anderson Cancer Center, who may not represent the general population; another explanation is that the adverse effect of the rare homozygous CC may be lethal during the embryonic development, because only 22 out of 2202 study subjects had the CC genotype.
IGFBP-5 has been reported to be involved in tumorigenesis and metastasis of various cancers, including SCCHN. Although we did not find any main effect on overall risk of SCCHN, we did find that the -1195C allele was significantly associated with late TNM stages of SCCHN at HPV-unrelated sites. It is possible that the very limited sample size of patients with early-stage I and II oropharyngeal cancer (9 stage I and 38 stage II) did not provide enough power to detect such an association in this HPV-related site. Because a direct association between HPV-infection and the -1195 SNP was not established here, larger studies with more early-stage I and II oropharyngeal cancer patients are needed to confirm this observed association.
Nevertheless, our findings are biologically plausible, because several studies have shown that IGFBP-5 regulate cell migration, invasion and angiogenesis through interactions with ECM proteins. For example, IGFBP-5 has been shown to function as a tumor suppressor by inhibiting tumor vascularity of human ovarian cancer in a xenograft model (21
). An important ECM protein, matrix metalloproteinase 7 (MMP-7) has been shown to cleave IGFBP-5 and other IGFBPs, hence to affect the bioavailability of IGFs and to regulate tumor cell growth during invasion and metastasis (22
). Another study showed that IGFBPs, including IGFBP-5, bound to vitronectin and enhanced keratinocyte cell migration (23
). Indeed, the expression levels of IGFBP-5 in multiple cancers, including breast cancer, ovarian cancer, cervical cancer, mesothelioma, and SCCHN, were associated with advanced TNM stages (15
). In SCCHN, it has been shown that the down-regulation of IGFBP-5 was associated with a higher tendency for nodal metastasis (15
). Therefore, our findings may provide a useful biomarker for the association between IGFBP5
polymorphisms and SCCHN risk, particularly for late-stage tumors, if validated by others.
To provide additional mechanistic insight into our findings, we also showed that the transcription factor AP-1 differentially bound to the IGFBP5
promoter region containing either T or C allele, which may lead to different expression levels of IGFBP-5. AP-1 is a dimeric transcription factor that consists of members of Fos (c-Fos, FosB, Fra1, and Fra2) and Jun (c-Jun, JunB, and JunD) gene families, and Fos/Jun heterodimers are the predominant forms of AP-1 in most tissues (27
). A number of extracellular signals, including growth factors, cytokines and ECM proteins can activate AP-1. It is reported that AP-1 may play critical roles in tumor invasion and metastasis through regulating the expression of genes, MMPs
, involved in cell migration and invasion (28
In an early study, multiple AP-1 and AP-2 transcription factor binding sites were found to be present in the rat IGFBP5
promoter region (29
), suggesting that human IGFBP5
may also be regulated by AP-1 at the highly conserved promoter region. Although a direct interaction between AP-1 and IGFBP-5 has not been fully studied, the activation of the mitogen activated protein kinase (MAPK) pathway can increase the expression of IGFBP-5. Indeed, IGFBP-5 induce lung fibroblast and mononuclear cell migration through the activation of the MAPK signaling pathway, as blocking MEK1/2, the key mediator of the MAPK pathway, inhibited the IGFBP-5 induced cell migration (30
). IGF-I has been reported to regulate the expression of IGFBP-5 (31
), and the MAPK pathway played critical roles in IGF-I induced expression of IGFBP-5 in intestinal smooth muscle cells (32
). More recently, it was shown that inhibition of MEK1/2 abolished IGFBP-5 mediated cell survival in pancreatic cancer cells (33
It is well known that AP-1 can be activated through the induction of the MAPK pathway by growth factors, including IGF-I (34
). Therefore, it is possible that IGF-I or other growth factors may activate the MAPK pathway and then induce AP-1, and the activated AP-1 then binds to the IGFBP5
promoter and regulates its expression. Our data suggested that IGFBP-5 with the C allele at the -1195T>C site may have a weaker binding to AP-1, compared with that with the T allele. Consequently, lower expression of IGFBP-5 in tissues of C allele carriers may explain the higher TNM stage. These results are consistent with the previous work showing that decreased expression of IGFBP-5 was related to the higher propensity for metastasis in SCCHN (15
). However, more detailed molecular studies are needed to provide direct evidences that the differential binding of AP-1 to the IGFBP5
promoter alters the expression level of IGFBP-5.
In summary, our study showed that carriers of the -1195C allele were likely to have late-stage or invasive SCCHN and that T and C alleles at -1195T>C resulted in different promoter activities, which may be regulated by the AP-1 transcription factor. These results provide evidence of a possibly novel mechanism by which the IGFBP-5 regulates SCCHN metastasis. Although our large study provides adequate statistical power and reliable association estimates, an uncontrolled selection bias may exist due to the nature of a hospital-based case-control study. Larger, population-based, preferably prospective studies should be performed to further validate our findings.