Cancer Res Treat.  2020 Jan;52(1):10-23. 10.4143/crt.2019.145.

Cathepsin C Interacts with TNF-α/p38 MAPK Signaling Pathway to Promote Proliferation and Metastasis in Hepatocellular Carcinoma

• Zhang GP
• Yue X
• Li SQ

• KMID: 2501197
• DOI: http://doi.org/10.4143/crt.2019.145

Abstract

Purpose
Although cathepsin C (CTSC) has been reported to maintain malignant biological properties in various cancers, its functions in hepatocellular carcinoma (HCC) remain obscure. We aimed to investigate the potential role of CTSC in HCC.
Materials and Methods
HCC tissue microarrays (n=122) were employed to analyze the correlation between CTSC expression and clinicopathological characteristics through immunohistochemistry staining. Quantitative real-time polymerase chain reaction, western blot assay, Cell Counting Kit-8 assay, colony formation, cell migration, and invasion assays, xenograft mice model were adopted to validate what had been indicated by the bioinformatic web tools.
Results
By bioinformatic tools and tissue microarrays, CTSC was found upregulated in HCC compared with normal liver tissues, and its higher expression was correlated with poor prognosis of HCC patients (hazard ratio, 2.402; 95% confidence interval, 1.493 to 3.865; p < 0.001). By gain/loss-of-function assays, we implicated that CTSC functioned as an oncogene to promote the proliferation and metastasis of HCC cells. Mechanistically, we revealed that CTSC was involved in several cancer-related signaling pathways by Gene Set Enrichment Analysis, among which tumor necrosis factor α (TNF-α)/p38 pathway was verified to be activated by CTSC. Furthermore, we found that TNF-α could activate CTSC expression in a concentration- dependent manner. Ralimetinib, an oral p38 mitogen-activated protein kinase (MAPK) inhibitor could inhibit CTSC expression. These indicated a potential positive feedback loop between CTSC and TNF-α/MAPK (p38) signaling.
Conclusion
Taken together, CTSC plays an important role in the growth and metastasis of HCC and may be a promising therapeutic target upon HCC.

Keyword

Cathepsin C; Hepatocellular carcinoma; Cell proliferation; Metastasis; TNF-α/MAPK (p38) pathway; Ralimetinib

Figure

• 

  Fig. 1. Cathepsin C (CTSC) is frequently upregulated in hepatocellular carcinoma (HCC) and correlated with poor prognosis of HCC patients. (A) The mRNA level of CTSC in HCC specimens analyzed by GEPIA web tool. (B) Western blot assay detected CTSC expression in both HCC and normal tissues. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (C) The correlation between CTSC expression and overall survival (OS) analyzed by GEPIA. (D) The differential expression in HCC specimens upon tissue microarrays. T, tumor; N, adjacent normal liver. (E) Kaplan-Meier survival analysis of OS in 122 HCC patients upon tissue microarrays.

• 

  Fig. 2. The expression of cathepsin C (CTSC) in hepatocellular carcinoma (HCC) cell lines. (A) The mRNA level in HCC cell lines analyzed by Cancer Cell Line Encyclopedia. (B) Western blot assay verified CTSC expression in HCC cell lines. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (C) The confirmation of CTSC overexpression in SH-HEP-1 and SMMC-7721 cells by quantitative real-time polymerase chain reaction. (D) Knockdown of CTSC in SK-HEP-1 and PLC/PRF/5 cells which was validated by western blot assay. ***p < 0.001.

• 

  Fig. 3. Cathepsin C (C TSC) promotes proliferation, migration, an d in vasion of hepatocellular carcinoma (HCC) cell lines. (A) The proliferation of SH-HEP-1 and SMMC-7721 cells infected with lentiviral LV-C TSC vector, or SK-HEP-1 and PLC/PRF/5 cells transfected with CTSC siRNA were assessed by Cell Counting Kit-8 assay. (B) The effects of CTSC on viability was assessed by colony formation assays. (C) Effects of overexpressing CTSC on tu morigenesis in vivo. (D) Trans-well chamber assays indicated the migratory ability of HCC cell lines. (E) Boyden chamber assays indicated thein vasiveness of HCC cell lines. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Fig. 4. Tumor necrosis factor α (TNF-α)/mitogen-activated protein kinase (MAPK, p38) pathway is involved in cathepsin C (CTSC) mediated hepatocellular carcinoma progression. (A) The signaling pathway acquired by Gene Set Enrichment Analysis. (B) The activation of TNF-α/MAPK (p38) signaling pathway after overexpressing CTSC in SK-HEP-1 and SMMC-7721 cells, demonstrated by western blot assay. (C) The expression of matrix metalloproteinase (MMP) 3, MMP-9, vimentin after CTSC overexpression in SK-HEP-1 and SMMC-7721 cells detected by western blot assay. (D) The expression of TNF-α after overexpressing CTSC in SK-HEP-1 and SMMC-7721 cells detected by quantitative real-time polymerase chain reaction. (E) The correlation between CTSC and MMP-3, MMP-9, Vimentin, and TNF-α in clinical specimens analyzed by the GEPIA. (F) The western blot assay was used to investigate the effect of TNF-α on CTSC expression. (G, H) The western blot assay was used to investigate the effect of Ralimetinib on CTSC expression. PI3K, phosphoinositide 3-kinase; NF-κB, nuclear factor κB; TNF-α, tumor necrosis factor α; HIF-1, hypoxia-inducible factor 1; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. **p < 0.01, ***p < 0.001.

Reference

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