Background
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer-related death in the world [
1]. It accounts for over 80% of all human liver cancer, and is responsible for between 500,000 and 1 million worldwide deaths annually [
2]. Predisposing factors for HCC include chronic hepatitis B and C virus infections (HBV and HCV, respectively), exposure to aflatoxin B1, chronic alcohol consumption, or any hepatic disease associated with cirrhosis. Nevertheless, the molecular pathogenesis of HCC remains largely unknown. Recognized abnormalities in HCC include aberrant signaling through the mitogen-activated protein kinase, PI3K/Akt and mTOR pathways, and overactivation of several growth factor receptors (although research has focused mainly on the epidermal growth factor receptor) [
3]. Recurrent allelic losses or gains have also been detected on 14 chromosome arms in more than 30% of all HCCs analyzed [
4]. Despite the large number of scientific and clinical studies performed to date, overall survival of patients with HCC has not improved in the last two decades.
There are three IQGAP proteins in humans, termed IQGAP1, IQGAP2 and IQGAP3 [
5]. IQGAP1 is the best-characterized member of the IQGAP family. Unlike IQGAP2, which is expressed primarily in the liver and platelets [
6,
7], and IQGAP3, where expression is limited to the brain [
8], IQGAP1 is expressed ubiquitously [
5]. IQGAP1 binds F-actin through calponin homology domains [
9], interacts with multiple calmodulin molecules (in a Ca
2+-regulated fashion) through repetitive IQ motifs (IQxxxRGxxR), and binds the Rho GTPases Cdc42 and Rac1 by means of a C-terminal RasGAP-related domain [
5]. In addition to the established binding partners listed above, IQGAP1 associates with the ERK and MEK kinases [
10,
11], β-catenin [
12,
13], E-cadherin [
14,
15], adenomatous polyposis coli (APC) [
16], mTOR [
17], and Sec 3 and 8 (which are involved in exocytosis and invasion) [
18]. IQGAP1 has been shown to regulate cell proliferation and migration
in vitro [
19‐
21], and is overexpressed in aggressive cancers [
22]. In order to elucidate the physiological functions of IQGAP2 (one of the less well studied IQGAP1 homologs), a conventional
Iqgap2 knockout mouse was generated in our laboratory [
6]. We showed that IQGAP2 deficiency results in an 86% incidence of HCC. Of equal importance, mice deficient in both
Iqgap1 and
Iqgap2 (
Iqgap1
-/-
/Iqgap2
-/-
) display relative protection against HCC, and have improved long-term survival. These data suggest that, at least in mice, changes in IQGAP expression contribute to the pathogenesis of HCC.
In humans,
Iqgap2 silencing, by hypermethylation, contributes to the pathogenesis of certain forms of gastrointestinal cancer [
23]. For example,
Iqgap2 methylation was detected in 47% of gastrointestinal tumors, but not in normal mucosa. Additionally, IQGAP2 protein was absent from all samples in which the
Iqgap2 promoter was hypermethylated, and a significant correlation was noted between
Iqgap2 methylation and cancer aggressiveness. These data, viewed in conjunction with the data from our mouse model, prompted us to hypothesize that decreased IQGAP2 expression, as a result of hypermethylation of the
Iqgap2 promoter, may contribute to the pathogenesis of human HCC. In the present study, our aim was to examine IQGAP1 and IQGAP2 expression in human HCC, their sensitivity and specificity as biomarkers of this type of tumor, and the methylation profile of the
Iqgap2 promoter.
Discussion
We recently showed that IQGAP1 and IQGAP2 have opposing roles in a murine model of hepatic carcinogenesis [
6]. In that study,
Iqgap2
-/-
mice developed age-dependent HCC, whereas mice deficient in both
Iqgap1 and
Iqgap2 displayed relative protection against HCC and showed significantly improved long-term survival. These data suggest that, in HCC, IQGAP2 may represent a tumor suppressor and IQGAP1 an oncogene. In the current study, we evaluated the expression of IQGAP1 and IQGAP2 in human HCC. We showed that a reciprocal relationship existed between IQGAP1 and IQGAP2 expression in human liver cancer cell lines. Furthermore, IQGAP2 was downregulated in 78.0% of HCC specimens, and IQGAP1 protein was overexpressed in 84.1% of tumors. Finally, we demonstrated that IQGAP2 mRNA is decreased in HCC compared to normal livers (although we did not detect any significant change in the IQGAP1 transcript), and showed that the
Iqgap2 promoter is not hypermethylated in HCC. Viewed collectively, these data strongly suggest that IQGAP1 and IQGAP2 contribute to the pathogenesis of HCC, and that these proteins are highly sensitive and specific biomarkers of this type of tumor.
Our data indicate that Sk-Hep-1, SNU475 and SNU387 cells have high levels of IQGAP1 and low levels of IQGAP2. Interestingly, these cells are mesenchymal and lack E-cadherin, while the other cell lines we studied have E-cadherin and are epithelial [
29]. Loss of E-cadherin results in metastasis and a poor prognosis in many tumors [
30]. Congruent with this, microarray analysis revealed that genes involved in invasion and metastasis are overexpressed in Sk-Hep-1, SNU475 and SNU387 cells, but not in HepG2, Hep3B or Huh7 cells [
31] (which have low IQGAP1 and high IQGAP2 expression). Viewed collectively, these data suggest that increased IQGAP1 and/or decreased IQGAP2 expression may be a characteristic of a more invasive and metastatic HCC phenotype. Nevertheless, as we did not observe a difference in IQGAP1 positivity or IQGAP2 negativity between different HCC grades, it is also possible that these observations are a consequence of cell line immortalization. Future studies are necessary to reconcile these discrepant data.
Differentiation of HCC from benign hepatocellular tumors, such as hepatic adenomas, macroregenerative nodules, and high grade dysplastic nodules, can be difficult morphologically [
32,
33]. Several immunohistochemical markers, such as CD10, polyclonal CEA, and Hep Par1 may be used to help determine a hepatocellular origin of a particular lesion, but are not helpful in differentiating benign from malignant tumors [
34,
35]. Moreover, Glypican-3, a heparin-sulfate proteoglycan recently reported to show a high degree of specificity for HCC versus benign hepatocellular proliferations, is limited by its relatively low sensitivity [
33,
36]. In the current study, we demonstrated a high degree of sensitivity and specificity for IQGAP1 positivity, and IQGAP2 negativity, in HCC. Indeed, 84.1% of HCCs were positive for IQGAP1, whereas all hepatic adenomas, cirrhosis cases, and most (82.1%) normal livers were IQGAP1 negative. Similarly, IQGAP2 was negative in 78.0% of HCCs, but was positive in 100%, 100% and 78.6% of hepatic adenomas, cirrhosis cases and normal livers, respectively. Based on these data, we propose that IQGAP1 and IQGAP2, either alone or in combination, are highly sensitive and specific biomarkers of HCC. As a result, their use may be valuable in routine diagnostic pathology.
As mentioned above, our qRT-PCR results showed that IQGAP2 transcript expression in human HCC was significantly lower than normal tissue. To our knowledge, we are the first group to report this finding in human HCC. The only other report of reduced IQGAP2 mRNA in HCC comes from our previous study in mice [
6]. Consistent with our current findings, IHC revealed that IQGAP2 protein, which was abundant in normal livers, was undetectable in most (78.0%) HCC specimens studied. Our qRT-PCR results also indicated that IQGAP1 mRNA expression did not differ significantly between normal livers and HCC. These findings are different from both our current IHC data, and mRNA data observed by other investigators [
37]. Several factors may account for these differences, including the source of the original samples. For instance, our qRT-PCR experiments were performed on a commercial cDNA array for which no information regarding the etiology of HCC was available. Furthermore, it has been demonstrated that the expression of certain genes in HCC may differ depending on a patient's HBV and HCV status [
38,
39]. Moreover, although we could not detect IQGAP1 protein in normal hepatocytes, it was expressed in Kupffer cells. Thus, if the cDNA in the array specimens was crudely extracted from homogenized liver, rather than from hepatocytes exclusively, possible changes in IQGAP1 mRNA expression may have been masked by mRNA from other cell types. Recent examination of microarray datasets of human HCC revealed that the
Iqgap1 gene is significantly upregulated in human HCC specimens compared to normal livers [
37]. Conversely, in agreement with our results, Liao and colleagues reported no significant difference in
Iqgap1 expression between normal livers and HCC [
40]. A more quantitative assessment of
Iqgap1 gene expression in a larger cohort of HCC specimens is necessary to reconcile these discrepancies.
Silencing of tumor suppressor genes by promoter hypermethylation is the most extensively studied epigenetic mechanism in tumorigenesis [
41‐
43]. In our current study, we hypothesized that downregulation of IQGAP2 expression in HCC specimens may be due to aberrant methylation of the
Iqgap2 promoter. To test this theory, we utilized pyrosequencing of bisulfite-treated DNA, and evaluated the
Iqgap2 promoter methylation profile of our HCC specimens. Pyrosequencing is a quantitative technique which allows for precise determination of methylation of individual CpG sites within a specific CpG island. Additionally, it has been shown to be effective for methylation analysis of FFPE tissues [
44]. Other commonly used methylation analysis methods, such as methylation specific PCR, do not allow resolution of single-CpG sites (which is especially important for the analysis of genes with heterogeneous methylation patterns). A methylation level of ≤ 5% is considered background noise, and thus has questionable significance [
45]. The overall
Iqgap2 methylation levels detected in our current study were less than 5% in both FFPE HCC specimens and in normal livers. In contrast,
Iqgap2 hypermethylation was detected in Kato III cells, suggesting that our pyrosequencing assay was technically sound. We confirmed the results of our FFPE HCC specimens and normal livers using an independent set of snap-frozen patient tissue. Viewed collectively, these data strongly suggest that methylation of the
Iqgap2 promoter is not the principle mechanism by which IQGAP2 is downregulated in HCC. It has been reported that ectopic expression of specific miRNAs in HCC cells results in silencing of
Iqgap1 and a concomitant decrease in cell proliferation [
46]. Thus, it is possible that IQGAP2 expression in HCC may also be regulated by miRNAs, instead of by promoter methylation.
This study is the first assessment of IQGAP1 and IQGAP2 expression in human HCC. Based on the data presented here, the stochiometry between IQGAP1 and IQGAP2 is central to hepatocellular carcinogenesis. The precise mechanism by which IQGAPs contribute to neoplastic transformation and tumor progression is still poorly understood. Numerous IQGAP1 binding partners are known to be involved in tumorigenesis [
22]. In contrast, little is known about the proteins with which IQGAP2 interacts. Future studies will provide insight into the role of IQGAPs in liver cancer.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CDW, HK and RDO performed and interpreted all immunohistochemistry. DVG performed and interpreted all qRT-PCR and methylation analyses. ZL performed and interpreted all Western blotting. DBS provided intellectual input into many experiments. VAS performed and interpreted all methylation analyses and was in overall charge of the study. All authors have read and approved the final manuscript.