Inositol 1,4,5-trisphosphate receptor type 3 is involved in resistance to apoptosis and maintenance of human hepatocellular carcinoma

Abstract

The expression of the inositol 1,4,5-trisphosphate receptor type 3 (ITRP3) in hepatocytes is a common event in the pathogenesis of hepatocellular carcinoma (HCC), regardless of the type of underlying liver disease. However, it is not known whether ITPR3 expression in hepatocytes is involved in tumor maintenance. The aim of the present study was to determine whether there is an association between ITPR3 expression and clinical and morphological parameters using HCC samples obtained from liver explants from patients (n=53) with different etiologies of underlying chronic liver disease (CLD). ITPR3 expression, mitosis and apoptosis were analyzed in human liver samples by immunohistochemistry. Clinical and event-free survival data were combined to assess the relationship between ITPR3 and liver cancer growth in patients. RNA sequencing analysis was performed to identify apoptotic genes altered by ITPR3 expression in a liver tumor cell line. ITPR3 was highly expressed in HCC tumor cells relative to adjacent CLD tissue and healthy livers. There was an inverse correlation between ITPR3 expression and mitotic and apoptotic indices in HCC, suggesting that ITPR3 contributed to the maintenance of HCC by promoting resistance to apoptosis. This was confirmed by the upregulation of CTSB, CHOP and GADD45, genes involved in the apoptotic pathway in HCC. The expression of ITPR3 in the liver may be a promising prognostic marker of HCC.

Keywords: apoptosis; calcium signaling; chronic diseases; liver cancer; mitosis.

Figure 1.

Representative images of ITPR3 labeling in HCC, underlying cirrhotic parenchyma and non-tumor livers (controls). (A) ITPR3 labeling in cirrhosis in patients diagnosed with HCV (n=31), ALD (n=16), CC (n=6) and healthy (control; n=2). Scale bar=200 µm (magnification, ×5), 50 µm (magnification, × 20) and 25 µm (magnification, ×40). Black arrows indicate ITPR3 perinuclear localization in hepatocytes. Blue arrows indicate ITPR3 cytoplasmatic localization in hepatocytes. (B) ITPR3 labeling in tumor (HCC) region of patients diagnosed with HCV (n=31), ALD (n=16), CC (n=6) and healthy (control; n=3). Black arrows indicate ITPR3 perinuclear localization in hepatocytes. Blue arrows indicate ITPR3 cytoplasmatic localization in hepatocytes. (C) Representative graph of the intensity of ITPR3 marking in HCC regions and cirrhotic parenchyma of patients with HCV compared with non-tumor livers (control; n=31; ****P<0.0001; ***P<0.0001). (D) Representative graph the intensity of ITPR3 marking in HCC regions and cirrhotic parenchyma of patients with ALD compared with non-tumor livers (control; n=16; **P<0.0057; ***P<0.0001). (E) Representative graph the intensity of ITPR3 marking in HCC regions and cirrhotic parenchyma of patients with CC compared with non-tumor livers (control; n=06; ****P<0.0001). (F) Representative graph the ITPR3 labeling intensity in tumor samples and cirrhotic parenchyma comparing the three etiologies (****P<0.0001). ITPR3, inositol 1,4,5-trisphosphate type 3 receptor; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; ALD, alcoholic liver disease; CC, cryptogenic cirrhosis; ALD, alcoholic liver disease.

Figure 2.

Correlation between ITPR3 labeling intensity and mitotic and apoptosis percentages (ssDNA). (A) Low mitotic (<5), high mitotic (≥5); n=35. **P<0.0098 Student's t-test. (B) Representative images of ssDNA labeling by immunohistochemistry, low nuclear labeling for ssDNA (≤50)% and high ssDNA labeling (>50%); Scale bar=25 µm (magnification, × 40). (C) Correlation graph between ITPR3 labeling intensity and apoptosis percentage. Low apoptosis percentage (≤50%), high apoptosis percentage (>50%); n=33. **P<0.0003 Student's t-test. (D) Correlation graph between mitotic index and percentage of apoptosis (n=15; *P<0.0220). ITPR3, inositol 1,4,5-trisphosphate type 3 receptor; ssDNA, single-stranded DNA; HPF, high-power fields.

Figure 3.

Heatmap of the most upregulated and downregulated genes in WT and ITPR3 KO HepG2 cells. Five different samples were analyzed by RNA-seq and several genes were altered when ITPR3 was not expressed. To confirm efficient depletion of ITPR3 in these cells, the ITPR3 gene was highlighted between the downregulated genes. CTSB, CHOP and GADD45 were the genes associated with apoptosis. WT, wild type; KO, knockout; ITPR3, inositol 1,4,5-trisphosphate type 3 receptor.

Figure 4.

Analysis of the EFS among the three etiological groups. Kaplan-Meier curve, with a difference in the comparison of the EFS between the etiological groups (Renyi test=0.015). Blue line representing the EFS of patients with HCV-associated tumor; Green line representing the EFS of patients with CC-associated tumor and yellow line representing the EFS of patients with ALD-associated tumor. EFS, event-free survival; HCV, hepatitis C virus; CC, cryptogenic cirrhosis; ALD, alcoholic liver disease.