Decreased PCSK9 expression in human hepatocellular carcinoma

Abstract

Background: The management of hepatocellular carcinoma (HCC) is limited by the lack of adequate screening biomarkers and chemotherapy. In response, there has been much interest in tumor metabolism as a therapeutic target. PCSK9 stimulates internalization of the LDL-receptor, decreases cholesterol uptake into hepatocytes and affects liver regeneration. Thus, we investigated whether PCSK9 expression is altered in HCC, influencing its ability to harness cholesterol metabolism.

Methods: Thirty-nine patients undergoing partial hepatectomy or liver transplantation for HCC were consented for use of HCC tissue to construct a tissue microarray (TMA). The TMA was immunostained for PCSK9. Imagescope software was used to objectively determine staining, and assess for pathological and clinical correlations. PCSK9 and LDL receptor mRNA levels in flash-frozen HCC and adjacent liver tissue were determined by quantitative RT-PCR. Serum PCSK9 levels were determined by ELISA.

Results: By immunohistochemistry, there was significantly lower expression of PCSK9 in HCC as compared to adjacent cirrhosis (p-value < 0.0001, wilcoxon signed-rank test). Significantly greater staining of PCSK9 was present in cirrhosis compared to HCC (p value <0.0001), and positivity (percentage of positive cells) was significantly greater in cirrhosis compared to HCC (p-value < 0.0001). Conversely, significantly higher expression of LDL-R was present in HCC as compared to the adjacent cirrhosis (p-value < 0.0001). There was no significant correlation of PCSK9 staining with grade of tumor, but there were significant correlations between PCSK9 staining and stage of fibrosis, according to spearman correlation test. PCSK9 mRNA levels were relatively less abundant within HCC compared to adjacent liver tissue (p-value =0.08) and normal control tissue (p-value =0.02). In contrast, serum PCSK9 levels were significantly increased among patients with HCC compared to those with chronic liver disease without HCC (p-value =0.029). LDL receptor mRNA was consistantly greater in HCC when compared to normal control tissue (p-value = 0.06) and, in general, was significantly greater in HCC when compared to adjacent liver (p-value = 0.04).

Conclusions: The decreased expression of PCSK9 and conversely increased LDL-R expression in HCC suggests that HCC modulates its local microenvironment to enable a constant energy supply. Larger-scale studies should be conducted to determine whether PCSK9 could be a therapeutic target for HCC.

Fig. 1

Comparison of PCSK9 immunohistochemical staining in hepatocellular carcinoma and adjacent liver tissue. a Total intensity of positives of 30413022.89 ± 2916711.84 pixel intensity versus 47844692.78 ± 3501425.25 pixel intensity (p-value < 0.0001); (b) Total intensity of strong positives of 244849.22 ± 30352.09 versus 591716 ± 95497.21 pixel intensity (p-value < 0.0001); (c) Positivity of 0.72 ± 0.03 versus 0.89 ± 0.009 (p-value < 0.0001); (d) proportion of strong positives of 0.005 ± 0.0003 versus 0.011 ± 0.001 (p-value < 0.0001); (e) Representative core of hepatocellular carcinoma adjacent to cirrhosis, stained with PCSK9 antibody; (f) Representative core of cirrhosis strongly staining with PCSK9 antibody

Fig. 2

a No significant correlation of PCSK9 expression by immunohistochemistry with grade of tumor. b Significant correlation of PCSK9 expression by immunohistochemistry with stage of fibrosis

Fig. 3

Comparison of LDL-receptor immunohistochemical staining in hepatocellular carcinoma and adjacent liver tissue (a) Total intensity of positives of 184722732 ± 15287852.05 pixel intensity versus 91021737 ± 8108288.90 pixel intensity (p-value < 0.0001); (b) Total intensity of strong positives of 4329282 ± 887724.04 versus 1066570 ± 235470.9 pixel intensity (p-value < 0.0001); (c) Positivity of 0.992 ± 0.001 versus 0.986 ± 0.002 (p-value = 0.013); (d) Proportion of strong positives of 0.022 ± 0.004 versus 0.009 ± 0.002 (p-value < 0.0001). (e) Representative core of hepatocellular carcinoma, strongly staining with LDL-R antibody; (f) Representative core of cirrhosis staining with LDL-R antibody

Fig. 4

a PCSK9 is less expressed in tumor as compared to adjacent liver parenchyma and control tissue, whereas b LDL-receptor mRNA is more highly expressed in tumor tissue as compared to adjacent parenchyma and control tissue

Fig. 5

Comparison of serum PCSK9 levels among patients with hepatocellular carcinoma versus those with cirrhosis or chronic liver disease without cirrhosis, demonstrating: (a) Logistic fit of HCC by PCSK9 model, with serum levels of PCSK9 in HCC patients being significantly higher than those in patients with cirrhosis or chronic liver disease (p-value =0.029); (b) Logistic fit of PCSK9 by HCC tumor diameter model, showing no correlation between these two parameters; (c) Logistic fit of Cirrhosis by PCSK9 model, with serum levels of PCSK9 in patients with cirrhosis being higher than those in patients without cirrhosis (p-value =0.048)