Three Epigenetic Drugs Up-Regulate Homeobox GeneRhox5in Cancer Cells through Overlapping and Distinct Molecular Mechanisms

Abstract

Epigenetic therapy of cancer using inhibitors of DNA methyltransferases (DNMT) or/and histone deacetylases (HDACs) has shown promising results in preclinical models and is being investigated in clinical trials. Homeodomain proteins play important roles in normal development and carcinogenesis. In this study, we demonstrated for the first time that an epigenetic drug could up-regulate homeobox genes in the reproductive homeobox genes on chromosome X (Rhox) family, including murine Rhox5, Rhox6, and Rhox9 and human RhoxF1 and RhoxF2 in breast, colon, and other types of cancer cells. We examined the molecular mechanisms underlining selective induction of Rhox5 in cancer cells by three epigenetic drugs: 5-aza-2Ј-deoxycytidine (DAC; decitabine), arsenic trioxide (ATO), and dine-3-ylmethoxy-carbonyl)aminomethyl]benzamide]. DAC induced Rhox5 mRNA expression from both distal promoter (Pd) and proximal promoter, whereas MS-275 and ATO induced gene expression from the Pd only. DAC and ATO inhibited both DNMT1 and DNMT3B protein expression, whereas MS-275 significantly reduced DNMT3B protein. In contrast to DAC, neither MS-275 nor ATO induced DNA demethylation on the Pd region. All three drugs led to enhanced acetylation of histones H3 and H4 at the promoter region. The occupancy of the activating histone mark dimethylated lysine 4 of H3 at Pd was enhanced by DAC and MS-275 but not ATO. Because they modulate gene expression with different potencies through shared and distinct epigenetic mechanisms, these epigenetic drugs may possess great potential in different applications for epigenetic therapy of cancer and other diseases.

The Rhox genes

“…This is probably also the mechanism responsible for the dramatic induction of Rhox5 transcripts in 10T 1 ⁄ 2 mesenchymal stem cells reported earlier (Sasaki et al 1991). However, 5AzaC treatment also increased the levels of transcription-promoting histone modifications at the Pd, complicating the interpretation of its mechanism of action (Li et al 2009). In the future, it will be important to untangle the independent contributions of DNA methylation and histone modifications, a challenging task given that these two events are intimately linked at many levels (Berger 2007).…”

“…The selective expression pattern of Rhox genes in reproductive organs is conserved, as the rat Rhox genes are also selectively expressed in the ovary, testis, epididymis, and placenta , Maiti et al 1996a, MacLean et al 2005b, Morris et al 2006; JA MacLean, unpublished observations), and the two best-characterized human RHOX genes appear to be specifically expressed in the human testis (Geserick et al 2002, Wayne et al 2002a. While Rhox genes normally exhibit highly specific and regulated expression patterns, some Rhox family members are ubiquitously expressed in tumors, including mouse Rhox5, Rhox6, and Rhox9, as well as human RHOXF1 (Wilkinson et al 1990, Rao et al 2002a, Wayne et al 2002a, Li et al 2009). …”

“…Further insights into the regulation of Pd transcription have come from a recent study by Li et al (2009) who used 'epigenetic' inhibitors to study the role of DNA methylation and histone modifications in regulating the Pd. The drugs used by the investigators were the DNMT inhibitor 5AzaC, the histone deacetylase inhibitor MS-275, and the chemotherapeutic drug arsenic trioxide (ATO), all of which trigger both DNA demethylation and histone modifications favorable for transcription.…”

Identification of RHOXF2 (PEPP2) as a cancer-promoting gene by expression cloning

“…In addition, RHOXF2 expression was found in a variety of cancer tissues. Recently, several studies have reported that expression of RHOXF2 is regulated by some epigenetic mechanisms, especially in cancer tissues (16). These findings suggest that RHOXF2 plays a role in tumorigenesis and that it can be controlled epigenetically.…”

“…Homeobox proteins, share a DNA-binding homeodomain motif of 60 amino acids, and are transcription factors that regulate development. The Rhox genes belong to a recently discovered homeobox family whose members are clustered on the X chromosome (14)(15)(16) Rhox family consists of >30 members (14)(15)(16) (19). A murine pro-B cell line Ba/F3 was maintained in RPMI-1640 with 10% FCS and 1 ng/ml mouse IL-3 (mIL-3), and a murine myelomonocytic cell line HF6 was maintained in RPMI-1640 with 20% FCS and 10 ng/ml mIL-3 (11).…”