Elevated FAM134B expression induces radiation-sensitive in hepatocellular carcinoma

Abstract

Background: Previous studies have shown that Family with sequence similarity 134 member B (FAM134B) was involved in the occurrence and development of malignancy, however, the function and molecular mechanism of FAM134B in Hepatocellular Carcinoma (HCC) radiotherapy resistance remain unclear. Therefore, it may clinical effective to clarify the molecular mechanism and identify novel biomarker to overcome radiotherapy resistance in HCC.

Methods: The protein and mRNA expression of FAM134B were determined using Real-time PCR and Western blot, respectively. IHC assay was performed to investigate the association between FAM134B expression and the clinicopathological characteristics of 132 HCC patients. Functional assays, such as in situ model, colon formation, FACS, and Tunel assay were used to determine the oncogenic role of FAM134B in human HCC progression. Furthermore, western blotting and luciferase assay were used to determine the mechanism of FAM134B promotes radiation-sensitive in HCC cells.

Results: We noted that FAM134B was downregulated in HCC, which was correlated with the radiation resistance in patients with HCC. Overexpression of FAM134B contribute to radiation sensitive in HCC; however, inhibition of FAM134B confers HCC cell lines to radiation resistance both in vitro and in vivo. Moreover, we found that FAM134B interacts with FMS related receptor tyrosine kinase 3 (FLT3) and downregulation of FAM134B activated JAK/Stat3 signaling pathway. Importantly, pharmacological inhibition of JAK/Stat3 signaling pathway significantly counteracted downregulation of FAM134B-induced radiation resistance and enhanced radiation therapeutic efficacy in HCC.

Conclusions: Our findings suggest that FAM134B may be a potential therapeutic biomarker for the treatment of HCC patients with radiotherapy tolerance.

Keywords: FAM134B; FLT3; HCC; JAK/Stat3 signaling pathway; Radiotherapy-resistance.

Fig. 1

FAM134B is downregulation and correlates with radiation sensitive in HCC. A. Downregulated FAM134B in tumor tissue compared with normal tissue (n = 421, TCGA). B. FAM134B was downregulated in HCC who revised radiotherapy and exhibit stable disease (SD) or progressive disease (PD), compared with patient’s exhibit (PD) complete response (CR) or partial response (PR) (n = 7, TCGA). C GSEA plot analyze the correlation between the mRNA levels of FAM134B and post-radiation gene signatures in published datasets. D Real-time PCR analysis showed FAM134B expression was decrease in 8 HCC tissues. E Western blotting analysis showed FAM134B expression was decrease in 8 HCC tissues. β-actin was used as a loading control

Fig. 2

FAM134B downregulation correlates with progression and poor prognosis in HCC patients A Survival analysis of FAM134B expression in HCC patients. B GSEA plot analyze the correlation between the mRNA levels of FAM134B and the liver cancer survival in published datasets. C IHC staining analyze the FAM134B protein expression in HCC (left); The Kaplan-Meier survival curves compare HCC patients with low and high FAM134B expression levels (right), P < 0.001

Fig. 3

FAM134B contribute to radiation sensitive in HCC in vitro A. Real-time PCR analysis of FAM134B expression in the indicated cells. B. Western blotting analysis of FAM134B expression in the indicated cells. β-actin was used as a loading control. C. Annexin V-FITC and PI staining of the indicated cells. D. Representative micrographs (left) and quantification (right) of colonies in the colony formatiom assay. E. Representative micrographs (left) and quantification (right) of Tunel positive signaling in the indicated assay. Each bar represents the mean ± SD of three independent experiments. * P < 0.05

Fig. 4

FAM134B contribute to radiation sensitive in HCC in vivo A. The luminescence of the tumor xenografts in the indicated groups. B. The Kaplan-Meier survival curves compare mice with low and high FAM134B expression levels. C. Concentration of AST and ALT in the indicated groups. * P < 0.05

Fig. 5

Downregulation of FAM134B activates the JAK/Stat3 signaling pathway in HCC. A. Coomassie brilliant blue analysis of proteins interacting with FAM134B. B. MS sequencing showing that FAM134B interacted with FLT3. C. Co-IP assay showing that FAM134B interacted with FLT3. D. GSEA plot analyze the correlation between the mRNA levels of FAM134B expression in HCC published datasets. E. Analysis of luciferase reporter activity in the indicated cells. F. Western blotting analysis of the expression levels of indicate proteins in the indicated cells. β-actin was used as a loading control. Each bar represents the mean ± SD of three independent experiments. * P < 0.05

Fig. 6

JAK/Stat3 signaling pathway is required for FAM134B induced radiation sensitive A. Annexin V-FITC and PI staining of the indicated cells. B. Colony formation analyze in the indicated cells. C. Correlation analysis of FAM134B expression and p-Stat3 expression in 8 freshly collected human HCC tissue samples (T); β-actin was used as a loading control. Each bar represents the mean ± SD of three independent experiments. * P < 0.05