The reproductive homeobox on X-chromosome (Rhox ) gene cluster in mouse contains 33 known genes , and three members of this gene family (Rhox2, Rhox4b and Rhox5 ) are crucial for self-renewal and differentiation of embryonic stem (ES) cells [2–4]. The founding member of Rhox gene cluster, Rhox5 (formerly pem), is expressed in early embryos and ES cells [5–7], embryonic carcinoma (EC) cells, and primordial and pre-muscle stem cells . Intriguingly, Rhox5 is predominantly expressed in female blastocysts from the paternally inherited X chromosome , yet the paternal copy is silent in placenta cells . In adult mice, Rhox5 expression is restricted to germline tissues in both male and female and is silenced in most somatic tissues [1, 10, 11]. Rhox5 is expressed from its two promoters, a distal promoter (Pd) and a proximal promoter (Pp), that give rise to transcripts with different 5'-ends encoding the same protein. The transcription from Pp depends on both androgen receptor and androgen . Rhox5 plays an essential role in self-renewal and differentiation of ES cells. It has been shown that Rhox5 over-expression is able to maintain murine ES cells in a pluripotent state in a leukemia inhibitory factor-independent manner , and can also block ES cell differentiation [3, 11]. It promotes differentiation and survival of germ cells in germline tissues . Targeted disruption of Rhox5 increases male germ cell apoptosis and reduces sperm production, sperm motility, and fertility .
Rhox5 is expressed not only in established cancer cell lines [13–15], but also in cancers in vivo, e.g., adenomas and carcinomas in the APCMin/+ mice and large intestine tumors of Msh2-deficient mice conditionally expressing K-ras (V12) [16, 17]. The Pd promoter was regarded as the promoter directing the aberrant expression in tumor cells .
Rhox5 may exert important functions in cancer based on the following evidence. First, partners for Rhox5 include: menin, a tumor suppressor , prosaposin, a multifunctional protein , and the cell division cycle 37 (Cdc37) homolog protein . Second, Rhox5 also mediates transcriptional repression of the netrin-1 receptor gene Unc5c, a tumor suppressor in colorectal cancer . Third, Rhox5 gene Pd activity in tumor cells requires Ras signaling . Fourth, in a colon adenoma model induced by conditional activation of K-rasV12 in Msh2 knockout mice, Rhox5 is one of three genes significantly up-regulated . Finally, Rhox5 renders tumor cells resistant to apoptotic cell death induced by anticancer therapies . In addition, it may play a role in cancer initiating cells (or cancer stem [CS] cells) . CS cells are cancer cells that possess characteristics associated with normal stem cells. They have the ability to give rise to all cell types found in a particular tumor. It is possible that ES and CS cells share some key regulatory genes that are tightly regulated by similar epigenetic mechanisms.
While there are a total of 33 known Rhox genes clustered in the X chromosome in mouse , only two RHOX genes have been characterized in humans: RHOXF1 (originally called OTEX and hPEPP1) and RHOXF2A (originally hPEPP2) [25, 26]. While there is no human homolog of mouse Rhox5, human RHOXF1 is closest to murine Rhox5 in terms of chromosomal location of the gene, tissue expression profiles, and potential functions. RHOXF1 is expressed at relatively high levels in human ES cells and adult germline stem cells . It is expressed in human colorectal cancer and testicular seminoma in vivo[28, 29], as well as in some cancer cell lines [15, 26]. Therefore, it is possible that Rhox5 and RhoxF1 may have comparable functions despite low sequence conservation and therefore they may be considered orthologues.
DNA methylation regulates gene expression in normal mammalian development [30, 31]. In cancer, aberrant promoter hypermethylation plays a major role in transcriptional silencing of critical growth regulators such as tumor suppressor genes [32, 33], while aberrant promoter hypomethylation upregulates germline genes (such as Rhox5 ) that are normally expressed in embryo stages and stem cells yet silenced in all or most somatic tissues [34, 35]. Histone modifications together with DNA methylation in the chromatin regulate many regulatory genes [36, 37]. All known acetylations of histones are correlated with transcriptional activation . Histone methylations at lysine and arginine residues are another class of epigenetic marks [39, 40]. A recent high-resolution profiling study in the human genome indicated that H3K4 trimethylation and the monomethylations of H3K9, H3K27, H3K79, H4K20 and H2BK5 are linked to gene activation, whereas trimethylations of H3K27, H3K9 and H3K79 are linked to repression . In addition, a "bivalent domain" (repressive mark H3K27me3 and permissive mark H3K4me2/me3) marks key developmental genes in ES cells [41, 42]. This chromatin bivalent domain in stem/progenitor cells predisposes tumor suppressor genes to DNA hypermethylation and heritable silencing [43–45].
RHOX5 may be regulated by epigenetic mechanisms. First, DNA methylation regulates long-range silencing of Rhox gene cluster including Rhox5 during the post-implantation development of mice . Second, Rhox5 could be upregulated in ES cells and embryonic fibroblast cells by inactivation of DNA methyltransferase genes [46, 47], or in ES cells null for linker histone H1 . While this paper was under revision, Wilkinson, MacLean, and coworkers showed that the Rhox gene cluster is imprinted and regulated by histone H1 and DNA methylation in ES cells . Third, Rhox5 is one of the X-linked cancer-germline (CG) genes, many of which are regulated by DNA methylation [14, 35, 49]. Finally, we have demonstrated that epigenetic drugs could upregulate Rhox5 in cancer cells through enrichment of active histone marks in the promoter region preferentially with DNA demethylation .
We and our collaborators have previously investigated epigenetic regulation of genes in normal development and cancer [15, 35, 50–52]. In this study, we have confirmed that Rhox5 is expressed in ES cells, EC cells, and cancer cells. We found that Rhox5 is expressed in side population (SP) cells that enrich for cancer stem/progenitor cells. We have examined the epigenetic marks in the promoter region, including both DNA methylation and histone acetylation (H3ac, H4ac and H3K9ac) and methylation (H3K4me2, H3K9me2 and H3K27me3), and related them to levels of expression in various cells types. We showed that epigenetic drugs could induce differentiation of F9 teratocarcinoma cells, but not SP cells, with Rhox5 upregulation and concurrent epigenetic changes. Finally, we demonstrated that Rhox5 gene knockdown by small hairpin RNA (shRNA) in CT26 colon cancer cells resulted in reduced tumor cell migration and cell proliferation in vitro and attenuated tumor growth in vivo .