Regulatory Factor X5 Promotes Hepatocellular Carcinoma Progression by Transactivating Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Theta and Suppressing Apoptosis
Hepatocellular carcinoma (HCC) is a major public health challenge worldwide, accounting for a significant portion of cancer-related deaths. Despite advances in medical science, the 5-year survival rate for HCC remains unsatisfactory, primarily due to the lack of effective treatment options. Understanding the molecular mechanisms underlying HCC carcinogenesis and progression is crucial for developing novel therapeutic strategies. This study focuses on the role of regulatory factor X5 (RFX5) in HCC, revealing its biological significance and the underlying mechanisms that drive HCC development.
RFX5 is a classical transcription regulator of major histocompatibility complex class II (MHCII) genes. Previous studies have shown that RFX5 is overexpressed in HCC tumors, but its role in HCC carcinogenesis and progression was not well understood. This study aimed to elucidate the biological significance of RFX5 in HCC and uncover the molecular mechanisms by which it promotes tumor development.
The study began by analyzing the expression and copy number variation (CNV) of RFX5 in HCC tumors and cell lines using data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The results showed that RFX5 was frequently amplified and overexpressed in HCC tumor tissues compared to corresponding non-tumor tissues. Specifically, RFX5 was amplified in 24.8% of HCC patients, with a 1.5-fold increase in tumor versus normal tissue. The mRNA expression level of RFX5 was significantly higher in HCC tumor tissues, and this overexpression was strongly correlated with its DNA copy number (r = 0.4, P < 0.001). Immunohistochemistry (IHC) analysis of a tissue microarray (TMA) containing 128 primary HCC specimens further confirmed that RFX5 protein was overexpressed in 71.1% of HCC cases, predominantly located in the nucleus of tumor cells.
To investigate the biological significance of RFX5 in HCC, functional studies were conducted using HCC cell lines. RFX5 was silenced using lentiviral short hairpin RNA (shRNA) and CRISPR/Cas9 systems, and its impact on colony formation and subcutaneous tumor growth was monitored. The results demonstrated that RFX5 knockdown significantly reduced the clonogenic forming ability of HCC cells in vitro and suppressed subcutaneous tumor growth in vivo. For example, in MHCC-97H cells, RFX5 knockdown led to a dramatic decrease in tumor volume (94.00 ± 45.81 mm³ vs. 226.8 ± 26.86 mm³, P = 0.0387) and tumor weight (0.041 ± 0.017 g vs. 0.115 ± 0.053 g, P = 0.0358). These findings indicated that RFX5 is essential for the growth and survival of HCC cells.
The study then sought to identify the downstream target genes of RFX5 in HCC cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays revealed that tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (YWHAQ), also known as 14-3-3 tau, is a key transcriptional target of RFX5. RFX5 directly binds to the promoter region of YWHAQ and enhances its transcription. Overexpression of RFX5 significantly upregulated the mRNA and protein levels of YWHAQ in HCC cells, while RFX5 knockdown had the opposite effect. For instance, in HepG2 cells, RFX5 knockdown reduced YWHAQ mRNA levels (P < 0.05), and overexpression of RFX5 increased YWHAQ protein levels.
YWHAQ belongs to the 14-3-3 family of proteins, which are known to play roles in various cellular processes, including cell cycle regulation, apoptosis, and signal transduction. The study found that YWHAQ is overexpressed in HCC tumor tissues and is associated with poor prognosis. Patients with higher YWHAQ mRNA expression levels had shorter recurrence-free survival times (P = 0.0496). IHC analysis of a TMA containing 51 primary HCC specimens showed that YWHAQ overexpression was detected in 70.59% of HCC cases.
To determine the functional significance of the RFX5-YWHAQ pathway in HCC, rescue experiments were performed. Overexpression of YWHAQ in RFX5 knockdown HCC cells largely restored the clonogenic growth and tumorigenicity that were suppressed by RFX5 depletion. For example, in SK-HEP-1 cells, YWHAQ overexpression rescued tumor growth, with tumor volumes increasing from 365.3 ± 29.42 mm³ to 807.0 ± 156.90 mm³ (P < 0.05) and tumor weights increasing from 0.059 ± 0.001 g to 0.199 ± 0.051 g (P < 0.05). These results demonstrated that YWHAQ is a downstream effector of RFX5 and plays a crucial role in HCC progression.
The study further investigated the mechanism by which the RFX5-YWHAQ pathway promotes HCC progression. Apoptosis assays revealed that RFX5 and YWHAQ overexpression significantly inhibited apoptosis in HCC cells. For instance, in HepG2 and SK-HEP-1 cells treated with Dactinomycin D (ActD), overexpression of RFX5 or YWHAQ reduced apoptosis levels, while RFX5 knockdown increased apoptosis. Western blotting analysis showed that RFX5 and YWHAQ overexpression downregulated the protein levels of p53 and Bax, key regulators of apoptosis. Conversely, RFX5 knockdown upregulated p53 and Bax levels, and this effect was reversed by YWHAQ overexpression. These findings indicated that the RFX5-YWHAQ pathway protects HCC cells from apoptosis by suppressing the p53-Bax signaling pathway.
In conclusion, this study revealed that RFX5 is a putative driver gene in HCC, playing a critical role in tumor development and progression. RFX5 is frequently amplified and overexpressed in HCC, and its overexpression is associated with increased YWHAQ expression. The RFX5-YWHAQ pathway promotes HCC progression by suppressing apoptosis through the downregulation of p53 and Bax. These findings provide new insights into the molecular mechanisms of HCC and highlight the potential of targeting the RFX5-YWHAQ pathway as a novel therapeutic strategy for HCC treatment.
doi.org/10.1097/CM9.0000000000000296
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