IQGAP1 and IQGAP2 are reciprocally altered in hepatocellular carcinoma

Abstract

Background: IQGAP1 and IQGAP2 are homologous members of the IQGAP family of scaffold proteins. Accumulating evidence implicates IQGAPs in tumorigenesis. We recently reported that IQGAP2 deficiency leads to the development of hepatocellular carcinoma (HCC) in mice. In the current study we extend these findings, and investigate IQGAP1 and IQGAP2 expression in human HCC.

Methods: IQGAP1 and IQGAP2 protein expression was assessed by Western blotting and immunohistochemistry. IQGAP mRNA was measured by quantitative RT-PCR. The methylation status of the Iqgap2 promoter was determined by pyrosequencing of bisulfite-treated genomic DNA.

Results: IQGAP1 and IQGAP2 expression was reciprocally altered in 6/6 liver cancer cell lines. Similarly, immunohistochemical staining of 82 HCC samples showed that IQGAP2 protein expression was reduced in 64/82 (78.0%), while IQGAP1 was present in 69/82 (84.1%). No IQGAP1 staining was detected in 23/28 (82.1%) normal livers, 4/4 (100.0%) hepatic adenomas and 23/23 (100.0%) cirrhosis cases, while IQGAP2 was increased in 22/28 (78.6%), 4/4 (100.0%) and 23/23 (100.0%), respectively. Although the Iqgap2 promoter was not hypermethylated in HCC at any of the 25 CpG sites studied (N = 17), IQGAP2 mRNA levels were significantly lower in HCC specimens (N = 23) than normal livers (N = 6).

Conclusions: We conclude that increased IQGAP1 and/or decreased IQGAP2 contribute to the pathogenesis of human HCC. Furthermore, downregulation of IQGAP2 in HCC occurs independently of hypermethylation of the Iqgap2 promoter. Immunostaining of IQGAP1 and IQGAP2 may aid in the diagnosis of HCC, and their pharmacologic modulation may represent a novel therapeutic strategy for the treatment of liver cancer.

Figure 1

IQGAP1 and IQGAP2 are reciprocally altered in human liver cancer cell lines. Equal amounts of cell lysate from the cultured liver cancer cell lines indicated were resolved by SDS-PAGE and processed by Western blotting. Immunoblots were probed with anti-IQGAP1, anti-IQGAP2 and anti-β-Tubulin antibodies. Representative data from four independent experiments are shown.

Figure 2

IQGAP1 and IQGAP2 protein expression is altered in hepatocellular carcinoma. Representative immunohistochemistry images showing IQGAP1 and IQGAP2 protein expression in normal (b-c), adenoma (e-f), cirrhosis (h-i) and carcinoma (k-l). Hematoxylin and eosin (H&E) stained images corresponding to each case are also shown (a, d, g, j). Scale bar represents 10 μm, and the final magnification is 400×.

Figure 3

IQGAP2 RNA transcript expression is altered in hepatocellular carcinoma. IQGAP1 (top panel) and IQGAP2 (bottom panel) mRNA expression in cDNAs from patients with HCC and normal livers was evaluated by qRT-PCR. The boxed area represents 50% of samples (from the 25^th to the 75^th percentile) and the band inside the box represents the median. *, p < 0.05 versus normal livers.

Figure 4

The Iqgap2 promoter is not hypermethylated in hepatocellular carcinoma. The degree of methylation for each CpG site was expressed as a percentage of methylated cytosines over the sum of total cytosines. The data are shown for 25 CpG sites and grouped by tissue type. Genomic DNA from the Kato III human gastric cancer cell line, which is known to have high methylation levels of the Iqgap2 gene promoter [23], was used as a positive control. The boxed area represents 50% of samples (from the 25^th to the 75^th percentile) and the band inside the box represents the median. Error bars (whiskers) indicate the 10th and 90th percentiles (shown only for plots with N = 9 or more). Symbols outside whiskers represent outliers.