Regulatory factor X1 is a new tumor suppressive transcription factor that acts via direct downregulation of CD44 in glioblastoma

Background: The biological functions of regulatory factor (RF)X1, a transcription factor, are not known. Since the RFX1 gene is often epigenetically silenced and clusters of differentiation (CD)44 proteins that regulate cancer cell biology are increased in human glioblastomas, we designed this study to determine whether RFX1 could regulate CD44 expression in glioblastoma.

Methods: Regulatory factor X1 was overexpressed in 4 human glioblastoma cell lines. CD44 expression and cell proliferation, apoptosis, and invasion were assayed under in vitro conditions. In vivo growth of human glioblastoma xenografts was determined in mice. The expression of RFX1 and CD44 in human glioblastoma tissues was quantified.

Results: A putative RFX1 binding sequence existed in the first exon of the human CD44 gene. The transcription activity of the DNA fragment containing this putative sequence was decreased in cells overexpressing RFX1. Regulatory factor X1 bound to the CD44 gene in glioblastoma cells. It reduced CD44 expression and activated Akt and extracellular signal-regulated kinase, signaling molecules downstream of CD44 to regulate cell proliferation and survival. Overexpression of RFX1 inhibited the survival, proliferation, and transwell invasion of glioblastoma cells and in vivo growth of human glioblastoma xenografts. CD44 overexpression reversed RFX1 effects on cell proliferation. Finally, CD44 protein levels were inversely correlated with RFX1 protein levels in human glioblastoma tissues.

Conclusions: These results suggest that RFX1 directly regulates CD44 expression. This mechanism may contribute to RFX1's effects on proliferation, survival, and invasion of glioblastoma cells. Our results provide initial evidence that RFX1 may be an important target/regulator of the malignancy of glioblastoma.