Transcript profiling identifies iqgap2(-/-) mouse as a model for advanced human hepatocellular carcinoma

Abstract

It is broadly accepted that genetically engineered animal models do not always recapitulate human pathobiology. Therefore identifying best-fit mouse models of human cancers that truly reflect the corresponding human disease is of vital importance in elucidating molecular mechanisms of tumorigenesis and developing preventive and therapeutic approaches. A new hepatocellular carcinoma (HCC) mouse model lacking a novel putative tumor suppressor IQGAP2 has been generated by our laboratory. The aim of this study was to obtain the molecular signature of Iqgap2(-/-) HCC tumors and establish the relevance of this model to human disease. Here we report a comprehensive transcriptome analysis of Iqgap2(-/-) livers and a cross-species comparison of human and Iqgap2(-/-) HCC tumors using Significance Analysis of Microarray (SAM) and unsupervised hierarchical clustering analysis. We identified the Wnt/β-catenin signaling pathway as the top canonical pathway dysregulated in Iqgap2(-/-) livers. We also demonstrated that Iqgap2(-/-) hepatic tumors shared genetic signatures with HCC tumors from patients with advanced disease as evidenced by a 78% mouse-to-human microarray data set concordance rate with 117 out of 151 identified ortholog genes having similar expression profiles across the two species. Collectively, these results indicate that the Iqgap2 knockout mouse model closely recapitulates human HCC at the molecular level and supports its further application for the study of this disease.

Figure 1. Hepatic RNA microarray analysis of Iqgap2^−/− mice.

A: Schema of a four-way microarray analysis of Iqgap2^−/− knockout (KO) and wild-type (WT) mouse livers at 6 months and 24 months of age. Four-way comparison using SAM identified 554 genes that were differentially expressed among four groups (cutoff fold change ≥3 and FDR≤0.05). B: Unsupervised hierarchical cluster analysis identified 11 subsets of genes (clusters) within 554 genes that change expression in similar pattern across four groups; C: Top canonical biological pathways for genes from the cluster # 9 as identified by Ingenuity Pathway Analysis (IPA).

Figure 2. Q-PCR validation of the Wnt/β-catenin signaling pathway alterations in Iqgap2^−/− livers.

The same liver RNA samples used for microarray (24-month-old WT, N  =  3, and 24-month-old KO, N  =  3) were analyzed using the Mouse Wnt Signaling Pathway RT² Profiler PCR Array. 11 genes belonging to the pathway and found to be differentially expressed in Iqgap2^−/− livers are shown.

Figure 3. Unsupervised hierarchical clustering analysis of the 399 genes subset representing the Iqgap2^−/− HCC transcript signature.

The two-dimension hierarchical clustering procedure was performed based on the 1- Pearson correlation distance and the average linkage method. All data were centered by rows to mean 0 and standard deviation of 1, meanwhile the data range was confined to −3 to 3 for a more comparable scale. The data are presented in a matrix format with columns representing individual samples and rows representing genes, thereby each cell in the matrix represents the expression level of a gene feature in an individual sample. The red and green colors in cells reflect high and low expression levels, respectively, as indicated in the fold-change scale bar. Tumor-free samples have blue font labels and HCC samples have maroon font labels.WT-1, WT-2, WT-3 – wild-type liver samples from 6-month-old mice; KO-1, KO-2, KO-3 - Iqgap2^−/− liver samples from 6-month-old mice; WT-4, WT-5, WT-6 - wild-type liver samples from 24-month-old mice; and KO-4, KO-5, KO-6 - Iqgap2^−/− liver tumor samples from 24-month-old mice. Note that the three Iqgap2^−/− HCC tumor samples from 24-month-old mice show a distinct pattern and form a separate cluster. The rest of the samples have more similar transcript profiles with the highest similarity found between livers from the younger (6-month-old) mice irrespectively of genotype.

Figure 4. Comparison of HCC transcript profiles of Iqgap2^−/− mouse model and human HCC.

Cross-species clustering profiles were obtained using comparative functional genomics approach. The clustering dendrogram was generated based on 1 - Pearson correlation and an average distance linkage. The data scale was also confined to a range of −3 to 3 for a more comparable heat map. Hierarchical clustering analysis was performed on 151 ortholog genes shared between 24-month-old wild-type and the age-matched Iqgap2^−/− mice (N = 3 in each group, see Figure 3 legend), and the human GSE6222 integrated microarray data set. This data set included four T1 HCC tumors (early stage HCC), six T3 HCC tumors (advanced HCC), and 2 normal liver controls. Mouse and human tumor-free samples have blue font labels; human T1 HCC samples have red font labels; and Iqgap2^−/− HCC and human T3 HCC samples have maroon font labels. All three Iqgap2^−/− HCC samples (KO.4 through KO.6) co-clustered with four out of six human T3 HCC samples. Genes differentially expressed between mouse and human livers are enclosed in a box at the top of the heat diagram.