Identification of the possible therapeutic targets in the insulin-like growth factor 1 receptor pathway in a cohort of Egyptian hepatocellular carcinoma complicating chronic hepatitis C type 4

Abstract

Background: Molecular targeted drugs are the first line of treatment of advanced hepatocellular carcinoma (HCC) due to its chemo- and radioresistant nature. HCC has several well-documented etiologic factors that drive hepatocarcinogenesis through different molecular pathways. Currently, hepatitis C virus (HCV) is a leading cause of HCC. Therefore, we included a unified cohort of HCV genotype 4-related HCCs to study the expression levels of genes involved in the insulin-like growth factor 1 receptor (IGF1R) pathway, which is known to be involved in all aspects of cancer growth and progression.

Aim: Determine the gene expression patterns of IGF1R pathway genes in a cohort of Egyptian HCV-related HCCs. Correlate them with different patient/tumor characteristics. Determine the activity status of involved pathways.

Methods: Total ribonucleic acid (RNA) was extracted from 32 formalin-fixed paraffin-embedded tissues of human HCV-related HCCs and 6 healthy liver donors as controls. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) using RT² Profiler PCR Array for Human Insulin Signaling Pathway was done to determine significantly up- and downregulated genes with identification of most frequently coregulated genes, followed by correlation of gene expression with different patient/tumor characteristics. Finally, canonical pathway analysis was performed using the Ingenuity Pathway Analysis software.

Results: Six genes - AEBP1, AKT2, C-FOS, PIK3R1, PRKCI, SHC1 - were significantly overexpressed. Thirteen genes - ADRB3, CEBPA, DUSP14, ERCC1, FRS3, IGF2, INS, IRS1, JUN, MTOR, PIK3R2, PPP1CA, RPS6KA1 - were significantly underexpressed. Several differentially expressed genes were related to different tumor/patient characteristics. Nitric oxide and reactive oxygen species production pathway was significantly activated in the present cohort, while the growth hormone signaling pathway was inactive.

Conclusions: The gene expression patterns identified in this study may serve as possible therapeutic targets in HCV-related HCCs. The most frequently coregulated genes may serve to guide combined molecular targeted therapies. The IGF1R pathway showed evidence of inactivity in the present cohort of HCV-related HCCs, so targeting this pathway in therapy may not be effective.

Keywords: Gene expression; Hepatitis C virus; Hepatocellular carcinoma; Molecular therapeutic targets.

Fig. 1 -

Bar chart representing the 6 significantly upregulated and the 13 significantly downregulated genes in the hepatocellular carcinoma group

Fig. 2 -

Scatter plot representing the normalized expression of each gene on the polymerase chain reaction array between the two groups. Log base 10 of 2^–ΔCT value of each gene in the control group is plotted on the x-axis against the corresponding value in the hepatocellular carcinoma (HCC) group, which is plotted on the y-axis to visualize gene expression changes. The central boundary line indicates unchanged gene expression. The upper left section of the scatter plot (above the fold-change boundary lines) contains genes upregulated in the HCC group as compared to the control group. The lower right section of the scatter plot (below the fold-change boundary lines) contains genes downregulated in the HCC group as compared to the control group. It shows the upregulated genes in red and the downregulated genes in green.

Fig. 3 -

Heat map is a color-coded representation of fold regulation expression data between HCC and control groups overlaid onto the polymerase chain reaction array plate layout. The black color represents the average magnitude of gene expression. The brightest green represents the most downregulated genes, the brightest red represents the most upregulated genes in the HCC cases compared to the normal controls.

Fig. 4 -

Analysis of canonical pathways involved in hepatitis C virus-related hepatocellular carcinoma pathogenesis with the Ingenuity Pathway Analysis (IPA) database using the present dataset (the 84 tested genes). The IGF pathway was found to be inactive. On the other hand, the nitric oxide and reactive oxygen species production pathway were significantly activated. Description: The white bars mean that half the molecules suggest an activation and half suggest inhibition, so an overall z-score of 0 (Ingenuity Systems). The blue bar indicates that there is evidence that the pathway is inhibited and a negative z-score. The orange bar means that there is a positive z-score indicating that there is evidence for activation of this pathway. The horizontal threshold line indicates the threshold for the overrepresentation analysis and was set at p = 0.05. Only bars that are more significant than p < 0.05 are shown. The ratio line represents the ratio of the number of molecules in the present dataset that map into the pathway relative to the whole size of the pathway (Ingenuity Systems). The pathway analysis was generated through the use of IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis