RFX5 promotes the progression of hepatocellular carcinoma through transcriptional activation of KDM4A.

Regulatory factor X-5 (RFX5) represents a key transcription regulator of MHCII gene expression in the immune system. This study aims to explore the molecular mechanisms and biological significance of RFX5. Firstly, by analyzing ENCODE chromatin immunoprecipitation (ChIP)-seq in HepG2 and TCGA RNA-seq data, we discovered lysine-specific demethylase 4A (KDM4A), also named JMJD2A, to be a major downstream target gene of RFX5. Moreover, RFX5 was verified to bind directly to the KDM4A's promoter region and sequentially promoted its transcription determined by the ChIP-PCR assay and luciferase assay. In addition, RFX5-dependent regulation of KDM4A was demonstrated in HCC. Compared with adjacent non-tumor tissues, the expression levels of KDM4A were significantly raised in HCC tumor tissues. Notably, elevated levels of KDM4A were strongly correlated with HCC patient prognosis. Functionally, KDM4A overexpression largely rescued the growth inhibitory effects of RFX5 deletion, highlighting KDM4A as a downstream effector of RFX5. Mechanistically, the RFX5-KDM4A pathway promoted the progression of the cell cycle from G0/G1 to S phase and was protective against cell apoptosis through regulation of p53 and its downstream genes in HCC. In conclusion, RFX5 could promote HCC progression via transcriptionally activating KDM4A expression.

Genome-scale CRISPR activation screening identifies a role of ELAVL2-CDKN1A axis in paclitaxel resistance in esophageal squamous cell carcinoma.

Neoadjuvant chemotherapy (NAC) may provide survival benefits for patients with advanced esophageal squamous cell carcinoma. However, tumor cells can display primary or secondary resistance to paclitaxel (PTX), a primary component of induction chemotherapy regimen. To identify genes capable of conveying PTX resistance, we performed a genome-wide CRISPR transcriptional activation library in human KYSE-180 cells. High throughput next generation sequencing was further applied to establish the phenotype-to-genotype relationship. Our highest-ranking hits are CDKN1A, TSPAN4, ELAVL2, JUNB and PAAF1. We generated evidence that esophageal tumors with high CDKN1A, ELAVL2 and TSPAN4 levels, quantified using qRT-PCR and Western blot assays, showed poorer chemotherapy response. Higher expression levels of TSPAN4 and ELAVL2 protein are independent risk factors for poor chemotherapy response in ESCC patients. We then found that overexpression of CDKN1A, ELAVL2 or TSPAN4 in ESCC cell lines significantly promoted the resistance to PTX by inhibiting cell apoptosis. Interestingly, ESCC cells overexpressed CDKN1A, ELAVL2 or TSPAN4 also acquired resistance to cisplatin (DDP). This phenomenon may be explained by cross-resistance of chemotherapy. We additionally found an association between ELAVL2 and CDKN1A, which may be regarded as the upstream and downstream factors that synergistically involved in the regulation of chemo-resistance in ESCC. Therefore, our study demonstrated that the genome-wide CRISPR activation library is a powerful strategy for the discovery of chemo-resistant genes critical for ESCC and we reported the first evidence that the ELAVL2-CDKN1A axis may be an important mechanism involved in chemo-resistance in ESCC.

Regulatory factor X5 promotes hepatocellular carcinoma progression by transactivating tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta and suppressing apoptosis.

BACKGROUND: Our previous studies have shown that regulatory factor X5 (RFX5), a classical transcription regulator of MHCII genes, was obviously overexpressed in hepatocellular carcinoma (HCC) tumors. However, the role of RFX5 in the carcinogenesis and progress of HCC remains unknown. This study aimed to reveal its biological significance and the underlying mechanism in HCC. METHODS: RFX5 mRNA expression level and copy number variation in HCC tumors and cell lines were determined by analyzing deposited data sets in the Cancer Genome Atlas and Gene Expression Omnibus database. The biological significance of RFX5 in HCC was investigated by monitoring the colony formation and subcutaneous tumor growth capacity when RFX5 was silenced with lentiviral short hairpin RNA and CRISPR/Cas9 system in HCC cell lines. The downstream gene transcriptionally activated by RFX5 in HCC cells was determined by chromatin immunoprecipitation and luciferase reporter assay. The involvement of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (YWHAQ) in HCC development was further determined by performing colony formation rescue assay and subcutaneous tumor growth rescue experiment. The association of YWHAQ with recurrence-free survival of patients with HCC was assessed by Kaplan-Meier analysis. Moreover, apoptosis level and the protein level of p53 pathway were determined to reveal the mechanism of RFX5 in driving HCC development. RESULTS: RFX5 was amplified and highly overexpressed in HCC tumor tissues compared with the corresponding non-tumor tissues. The mRNA expression level of RFX5 was significantly correlated with its DNA copy number (r = 0.4, P < 0.001). Functional study demonstrated that RFX5 was required for both clonogenic forming in vitro and subcutaneous tumor growth in vivo of HCC cells. Further study identified YWHAQ, namely 14-3-3 tau, as a key downstream transcriptional target gene of RFX5, which was tightly regulated by RFX5 in HCC. Moreover, overexpression of YWHAQ largely rescued the clonogenic growth of HCC cells that was suppressed by RFX5 knockdown. In addition, overexpression of YWHAQ in primary tumor was linked to poor prognosis of patients with HCC. These results demonstrated that YWHAQ was a downstream effector of RFX5 in HCC. Notably, RFX5-YWHAQ pathway could protect cells from apoptosis by suppressing the p53 and Bax in HCC. CONCLUSION: RFX5 is a putative HCC driver gene that plays an important role in the development and progression of HCC by transactivating YWHAQ and suppressing apoptosis.