Current Medicinal Chemistry, 2011, 18, ????-???? 1

0929-8673/11 $58.00+.00 © 2011 Bentham Science Publishers

The HOX Genes Network in Uro-Genital Cancers: Mechanisms and Potential

Therapeutic Implications

M. Cantile1, R. Franco

1, G. Schiavo

2, A. Procino

2, L. Cindolo

3, G. Botti

1 and C. Cillo*

,2

1Surgical Pathology Department, National Cancer Institute "G.Pascale", Naples, Italy;

2Department of Clinical & Experimental

Medicine, Federico II University Medical School, Via S. Pansini 5, 80131, Naples, Italy; 3Urology Unit S.Pio da Pietralcina

Hospital, Vasto, Italy

Abstract: Genito-urinary malignancies (prostate, bladder, renal and testicular cancers) rank high among human tumors with an incidence

that varies with age and organ involvement. Prostate cancer is the most commonly detected male cancer followed by bladder and kidney

cancers, less frequent in women. Testicular cancer, although rare, is the most frequent cancer in males under 35. The majority of

oncogenic and tumor suppressor signaling pathways involved with urogenital cancers converge on sets of transcription factors that

ultimately control gene expression resulting in tumor formation and metastatic progression. The activity of these transcription factors is

modulated by multiple mechanisms spanning from transcriptional regulation, deregulation of the splicing, maturation, export and location

of mRNAs, protein synthesis and post-translational modifications. The recent involvement of the epigenitic mechanisms in the generation

and the evolution of cancer has produced a great deal of interest. This is related to the possibility that revealing these mechanisms able to

regulate the cell memory program (the gene systems polycomb, trithorax and HOX) may generate important biological and therapeutic

achievements. The HOX gene network is the only physically and functionally identifiable transcription factor network located in the

human genome controlling crucial cellular processes. Here we describe the implication of the HOX genes in the urogenital embryonic

development and cancers. We further highlight the mechanisms uncovered along these processes and involving the HOX genes. Finally,

we foresee the specific targeting of HOX genes and in general the cell memory gene program in the therapeutic setting of urogenital

malignancies due to their upstream location in these stepwise cell processes and their early deregulation in cancer evolution.

Keywords: Epigenetics and cancer, HOX genes, HOX genes and uro-genital cancers, HOX genes and epithelial-mesenchymal interaction, HOXC6 and prostate cancer, HOXC4, HOXC5 and HOXC6 and bladder cancer, HOXC11 and kidney cancer, HOX genes and cell identity, HOX genes and miRNA, HOX genes and ncRNA, HOTAIR, HOTTIP, Epigenetic therapies, Anti-Epigenetic Drugs, HOX/PBX and therapy.

HOMEOBOX AND HOX GENES

The Cell Memory Gene Program

Aberrant epigenetic modifications involving the cell memory gene program play a major role in the generation and evolution of neoplasia [1], making it possiblee to conceive the therapeutic potential of drugs acting on the cell memory between the potential treatments against cancer. The fate of each cell of our body is determined by the cell memory gene program (Fig. 1) which includes the whole set of gene functions capable of transferring the complete genome from mother to daughter cell through cellular reproduction [1]. The memory program contains much information crucial to the cell life cycle: where the daughter cell will be located; what phenotype identity will aquire; when both these properties will be express; the number of cell division the daughter cell will be able to perform and when, if ever, will go through apoptosis [2]. The cell memory is regulated by three gene families: the Polycomb genes (H3K27m3), able to block DNA-chromatin interaction leading to Hox gene silencing [3]; the Trithorax genes (H3K4m3), able to induce mRNA transcription through an open configuration of DNA-chromatin interaction and leading to Hox gene activation [4]; finally the Hox genes involved in the orchestration of phenotype specific gene program mostly through the fine regulation of mRNA transcription.

Homeobox and Hox Genes

Homeobox genes are a super-family of originally identified transcription factors mostly involved with the determination of the developmental identity of animal body plan [5]. They are subdivided in several classes. Class I homeobox genes (Hox in mouse, HOX in human) are 39 transcriptional regulators of embryonic development containing a 183 nucleotide sequence (homeobox) encoding for a 61 amino acid domain (hoemodomain)

*Address correspondence to this author at the Dept. Clinical & Experimental Medicine, Federico II University Medical School, Via S. Pansini 5, 80131 Naples – Italy;

Tel/Fax: 0039 081 5454790; E-mail: clecillo@unina.it

included in the corresponding protein (homeoprotein) [6]. The Hox genes are organized into four chromosomal clusters or loci

[7]

(HOX A at 7p15.3, HOX B at 17q21.3, HOX C at 12q13.3 and HOX D at 2q31

[8]) each containing from 9 to 11 genes (Fig. 2). On

the basis of sequence similarity and position in the locus, corresponding genes of the four clusters can be aligned with each other into 13 paralogous groups

[9]. The Hox gene network

organization has evolutionarily evolved through subsequent replication and transposition events from a unique ancestral (proto-Hox) gene [10]. In contast to Polycomb and Trithorax genes, dispersed inside the genome, Hox genes generate the only physically and functionally identifiable network of higher vertebrate genomes [11]. During mammalian development, Hox gene expression controls the identity of various regions along the body axis according to the rules of spatio-temporal co-linearity, with 3’ Hox genes (retinoic acid responsive) expressed early in development and controlling anterior regions, followed by progressively more 5’ genes (FGF responsive) expressed later and controlling more posterior regions [12]. The HOX gene network, the most repeat-poor regions of the human genome [11], is also active in normal adult organs[13]. Homeobox and Hox genes appear to regulate normal development, phenotype cell identity and cell differentiation, and control primary cellular processes [13] as proven by the description of congenital [14], somatic [15], metabolic [16] and neoplastic [17] alterations involving these genes. New crucial functions have recently been ascribed to HOX genes and homeoproteins mostly related to their interaction with miRNAs [18] and ncRNAs [19].

SPATIO-TEMPORAL HOX REGULATION DURING

EMBRYONIC DEVELOPMENT AND ADULTHOOD

Hox and Developmental Localization

The Hox gene network is progressively active, during vertebrate embryonic development, along the antero-posterior axis starting from the rhombencephalon, through the thoracic region and ending with the caudal body structures (Fig. 2) [6]. The anterior brain does non express Hox genes during embryonic development.

2 Current Medicinal Chemistry, 2011 Vol. 18, No. 2 Cantile et al.

The same colinear antero-posterior organization is followed, during the generation of limb and genitourinary structures, for the expression of caudal Hox genes (paralogous 9-13) [20]. During embryonic development Hox genes of paralogous group 1-4 are involved in the generation of the branchial archs, the hearth, the circulatory system and the lung [21]. Thoracic paralogous Hox genes (5-8) control, during development, axial extension and moto-neural migration [22] and act as species-specific markers. Knock-out experiments have identified the phenotypes generated by loss of function of single Hox gene, pairs of genes, a complete paralogous group and two paralogous groups Hox genes (to a maximum of six genes) [23]. Resulting phenotypes display homeiotic transformation concerning the axial skeleton and organs and/or body structure agenesis [24].

Fig. (2). Schematic representation of the HOX gene network (see text for

details).

Hox Role in Adulthood

HOX genes are active in adult human tissues and organs to regulate homeostasis and to preserve the spatio-temporal

coordinates established during embryonic development [25]. Each adult organ displays a characteristic combination of HOX gene expression, a sort of molecular fingerprint, resulting from the patterns of the different cell phenotypes included [26]. Extended organs such as gut or the adipose tissue display a combination of HOX expression patterns both organ specific and related to the antero-posterior localization of the body structure [27]. Organs sharing a common embryonic origin (such as the organs derived from primitive intestine) share common characteristics on their HOX expression patterns.

In the nervous system HOX genes are more frequently active and at higher level in neuronal cells [28]. Evolutionarily, the reason for this peculiarity is probably related to the origin of the Hox network as major determinant of neuro-ectodermal tissues [29]. At cellular level, locus A and B HOX genes play a crucial role in haematopoietic differentiation, the self-renewal and the repopulation ability of haematopoietic stem cells and represent a key molecular system to characterize the molecular bases of leukemogenesis [30]. The expression of the locus C HOX gene discriminates epithelial cells with respect to their origin and location and identifies significant alteration of HOX gene expression in connection to the neoplastic progression of several organs [31]. The expression of the HOX genes in human fybroblasts, in vitro as well as in vivo, represents the most prominent gene expression patterns connected to their spatio-temporal memory related to the organ-tissue origin and the antero-posterior body location [32]. Thus, the expression of the HOX network in adult eukariotic cells represents the fingerprint of each cell and characterizes its location in the organism. It is fair to say that spatio-temporal architecture during embryonic development and adulthood is controlled at cell, tissue and organ level by the HOX gene network.

Aim of the Review

In this review we will describe the involvement of HOX genes with uro-genital cancers. We will further present a unified

Fig. (1). The cell memory gene program (see text for details).

HOX Genes and Uro-Genital Cancer Current Medicinal Chemistry, 2011 Vol. 18, No. 2 3

molecular mechanism accounting for the HOX gene deregulation in uro-genital malignancies. We will finish by discussing the possibility for identifying new molecular markers useful for detecting early stages of disease as well as for defining new therapeutic strategies for targeting the cell memory gene program with anti-epigenetic drugs in uro-genital malignancies.

PROSTATE CANCER

Molecular Mechanisms of Prostate Cancer

In spite of the significant progress made during recent years, prostate cancer still represents the second leading cause of cancer death in men. Prostate development requires the reciprocal interactions between the urogenital sinus mesenchyme and epithelium under the stimulation of testicular androgen [33]. Posterior developmental signaling pathways and morphogens include Wnt [34] (canonical and non canonical Wnt5a [35]), FGF and Hedgehog are key players in mediating epithelial-mesenchymal interaction during prostate organogenesis [36, 37]. Both prostate development and cancer are strictly dependent of the interaction with androgen receptor (AR) [38]. Crucial molecular determinants of prostate cancer include the down regulation of the homeobox related gene NKX3.1 and MYC over-expression as initiation events [39, 40], the generation of the TMPRSS2-ERG fusion gene [41], the negative PTEN and positive ERK/MAPK activation as progression steps [42,43], finally the over-expression of the polycomb gene EZH2 as determinant of the castration-resistance epithelial-mesechymal transition [44]. The precise identification of the molecular pathways involved with castration resistance and neuroendocrine differentiation of prostate cancer could be useful to identify new therapeutic approaches. The molecular mechanisms of the bone tropism for metastatic prostate cancer remain to be elucidated.

Hox and Prostate Development

Hox genes have originally been involved with prostate development: HoxD13 plays a role in prostate ductal morphogenesis since the deficient mouse display diminished ductal branching of the ventral and dorsal prostate [45]. HoxC11 is active during development of the posterior urogenital sinus that gives rice to urethra, vagina and prostate [46]. HoxB13 plays a specific role in the differentiation pathway that determine ventral prostate epithelium identity, as well as a more general role in the ventral prostate morphogenesis in redundancy with other Hox13 paralogous genes [47].

Hox and Prostate Cancer

Concerning the function of Hox genes in prostate cancer, a transcriptome analysis has identified a 5 gene expression model able to predict patient recurrence with 90% accuracy following prostatectomy, and one of these genes is HOXC6 [48]. The other genes are chromogranin A, platelet-derived growth factor receptor

(PDGFR ), inositol triphosphate receptor 3 (IPTR3) and sialyltransferase. These data support the notion that the clinical behaviour of prostate cancer is linked to underlying differences in gene expression, detectable at the time of diagnosis. In support of this finding, the aberrant HOXC expression has been also detected in prostate cancer cells and in lymph node metastases [49]. The deregulation mostly concerns the close related genes HOXC4, HOXC5 and HOXC6 (belonging to the same transcription unit) and HOXC8 able to suppress transactivation by androgen receptors in LNCaP cells [49]. HOXC8 deregulation inhibits androgen receptor mediated gene induction by blocking i) the AR-dependent recruitment of the steroid receptor coactivator-3 (SRC-3); ii) the binding of CREB to the enhancer of the androgen regulated gene PSA; iii) the histone acetylation of androgen regulated genes [50].

HOXB13 is another key determinant of prostate cancer androgen response due to its molecular interaction with the androgen receptor capable of blocking the transcription of androgen response element containing genes [51]. On the other hand, the interaction androgen receptor/HOXB13 confers androgen responsiveness to promoters containing a specific HOXB13-responsive element. HOXB13 and androgen receptor synergize to enhance the transcription of genes containing the androgen responsive element and the homeobox sequence juxtaposed [51].

Polycomb and Tritorax Genes in Prostate Cancer

Epigenetic regulation plays an increasing role in cancer. The deregulated mRNA and protein expression of the polycomb gene EZH2, component of the PRC2 histone methyltrasferase complex required for the silencing of HOX gene expression during embryonic development (see the cell memory gene program above), is involved in the progression of prostate cancer. In addition clinically localized prostate cancers expressing higher concentrations of EZH2 show a poorer prognosis [52].

The comparison of transcriptome and Chip-Chip analyses between prostate cancers and prostate primary cells reveals that epigenetic modifications such as loss and/or gain of H3K4me3 and/or H3K27me3 are strongly associated with differential gene expression [53]. Promoter patterns of H3K4me3 and H3K27me3 strongly correspond with coordinated expression changes of HOX genes, miRNA genes and gene encoding cell junction proteins [54]. Several epigenetically regulated oncogenes and tumor suppressor genes are up/down regulated accordingly in prostate cancer cells.

Hox and Physio-Pathology of Prostate Cancer

The expression pattern of the complete HOX gene network in human prostate cell phenotype representing different stages of prostate physiology and prostate cancer progression make it possible to discriminate between different human prostate cell phenotypes and to identify HOX genes mostly involved with prostate cancer organogenesis and cancerogenesis [55]. Exposure of epithelial cell phenotypes to cAMP alters the expression of lumbo-dacral HOX D genes located on the chromosome region 2q31-33 where the cAMP effector genes (CREB1, CREB2 and cAMP-GEFII) are located. Interestingly, this same chromosomal area harbors: (i) a global cis-regulatory DNA control region able to coordinate the expression of HOXD and contiguous phylogenetically unrelated genes [56]; (ii) a prostate specific miRNA gene associated with high-rosk prostate cancer (PCGEM1) [57]; (iii) a series of neurogenic-related genes involved with epithelial-neuronal cell conversion. The in vivo expression of one of them, Neuro D1, in human advanced prostate cancers correlate with the state of tumor differentiation as measured by Glison score [58]. Neuro D1, a gene of the atonal family involved in the generation of sensory organs, is a transcription factor originally identified in -pancreatic cells which acts as a neuronal differentiation factor, regulates digestive endocrine differentiation, interacts with Rb to activate pro-opiomelanocortin expressing cells (POMC) and is a marker of the neuroendocrine differentiation of prostate cancer [58, 59].

The expression of HOXD13 homeoprotein in 79 different tumor types has recently highlighted significant differences between specific normal tissues and corresponding tumor types with the majority of cancers showing an increase of HOXD13 expression the exception being pancreatic and stomach cancer subtypes which display an opposite trend [60]. In prostate cancers an increase of HOXD13 expression is detectable in untreated prostate adenocar-cinoma versus normal prostate without significant variations for the hormone-refractory prostate adenocarcinoma. In contrast detection in pancreas-tissue microarrays showed that HOXD13 homeoprotein expression decreases in pancreatic cancer versus normal tissue and

4 Current Medicinal Chemistry, 2011 Vol. 18, No. 2 Cantile et al.

display a significant and adverse effect on the prognosis of patients with pancreatic cancers, independent of the T or N stage at the time of diagnosis [60]. Recently another homeobox genes PDX-1, crucially involved with pancreas embryonic dvelopment has been described as over-expressed in prostate cancer versus normal prostate [61]. The expression of PDX-1 decreases with prostate cancer progression (Increasing Geason score). Thus, prostate, pancreas and other endocrine related cancer phenotypes such as breast seem to share molecular identity mechanisms.

Molecular Determinant of Bone Tropism in Prostate Cancer

According to our view of the HOX network as a complex genetic system displaying crucial cellular functions, we postulate here a hypothesis concerning the role of the HOX network as molecular determinant of prostate cancer cell phenotype identity along tumor progression. The locus C HOX genes, located on chromosome 12q13.15 appear to be a determinant of epithelial differentiation [62] and of lymphoid cells [63], in addition to the role the whole C cluster plays in regulating neuronal migration [64]. The complete activation of locus C HOX genes characterizes the neoplastic transformation of the dental lamina epithelium (ameloblastoma) [31]; HOXC4 activates the promoter of the AID cytidine deaminase gene capable of inducing class switch recombination and somatic hypermutation in B lymphocytes [65]; the transcription unit HOXC4, HOXC5 and HOXC6 characterize the neoplastic transformation of epithelial bladder cells [66]; HOXC appears to be the HOX locus mostly involved with prostate cancer. Inside the locus HOXC is located the ncRNA HOTAIR [19, 67] that can be activated by paralogous group 13 HOX genes, involved in prostate development. HOTAIR activation entails the cis-regulation of the whole HOXC locus as well as the tras-regulation of the lumbo-sacral part of the HOXD locus on chromosome 2q31-33 [19]. This chromosomal area appears to be extremely well preserved during evolution, contains a series of neurogenic (brain related) and neuroendocrine genes deregulated in the neuroendocrine differentiation of prostate and pancreatic cancer cells (NeuroD1, HOXD13) [58, 60], a prostate specific ncRNA (PCGEM1) associated with high risk prostate cancer [57], a global controlling region (GCR) able to transcriptionally regulate the lumbo-sacral HOXD locus [56] and a large series of upstream genes including SATB2, the gene encoding for the nuclear matrix attachment protein [68]. SATB2 has been reported to be able to interact with the anterior part of the HOXA locus on chromosome 7p13-15 blocking the expression of HOXA2, the most anterior expressed homeoprotein involved with branchial archs generation [69]. The block of HOXA2 expression induces, through the interaction with the major osteogenic determinants such as Runx2 and Sox9 the realization of the osteogenic program [69]. The molecular program described here going from locus C to posterior locus D and anterior locus A of the HOX network could account, in our view, for the osteogenic tropism observed along prostate cancer progression.

In conclusion, HOXC appears to be the HOX locus mostly involved with prostate cancer. Lumbo-sacral HOX genes of paralogous groups 13 and 11 play a crucial role in normal prostate development and adult neoplastic prostate evolution.

BLADDER CANCER

Molecular Mechanisms of Bladder Cancer

Bladder cancer is the second most frequently diagnosed urogenital neoplasia after prostate cancer. The majority of bladder cancers are of epithelial origin and correspond to transitional cell carcinomas (TCC) [70]. An initial event on chromosome 9 causes the highest mutation index detectable in TCC. Subsequent gene events involve mutations of oncosuppressor such as p53 and Rb as

well as oncogenes as RAS, growth factor receptors (FGFR3) and signaling pathways (PI3K) [71]. Other chromosomal deletions (ch.5,3,10,6,11,18) are involved in TCC as well as the amplification of the chromosomal region 12q13-15 [72]. Finally a PAX homeobox gene (PAX-5), linked to embryonic development, seems to play a role in TCC when inappropriately expressed [73].

Hox Genes and Bladder Cancer

Lumbo-sacral genes of the HOX network are involved during the embryonic development of uro-genital organs. More specifically paralogous group 11 and 13 HOX genes are active during posterior urogenital sinus generation and regulate epitleilal-mesenchymal interaction, respectively [46,47].

The comparison of the whole HOX network expression in pairs normal/TCC biopsies highlights an unusual locus C HOX gene expression even in normal bladder tissues [66]. Unlike the expression of network genes located on the A, B and D HOX loci, where constitutive active HOX genes are equal (locus B) or more numerous than (locus A and D) silent genes, the constitutive inactive HOXC genes are more numerous than the unique active HOXC8 gene. This observation already suggests a special role for the locus C HOX genes in bladder epithelium. In TCC, where the expression of the HOX network tends to increase for the whole network, a dramatic variation is detected in the expression of the HOXC locus, as four of the six genes (HOXC4, HOXC5, HOXC6 and HOXC11), constitutive silent in normal bladder, became active in the majority of the bladder cancer analysed. Furthermore, paralogous groups 11 (HOXA11, HOXC11 and HOXD11) and 13(HOXA13, HOXB13, HOXC13 and HOXD13) genes, already involved during urogenital organs development, appear to be deregulated in the comparison between normal urothelium and TCC [66, 74]. Finally HOXB13 protein expression enables discrimination between invasive and non-invasive TCCs (Cantile M., Personal Communication).

Cytokeratins and Bladder Cancer

Cytokeratins (CKs) are intermediate filament proteins regulating cell shape and cell-cell communication [75]. In bladder cancer, alteration of the expression of a series of CKs normally active in the urothelium, such as CK8, CK18 and CK19 has been described, making it possible to suggest a distinction between TCCs through cytokeratin expression patterns [78]. Genes coding for the cytokeratins CK8 and CK18 are located on the chromosome 12q13 along with the cytokeratin genes CK1, CK3, CK5, CK6A and CK6B [79] in physical contiguity to the 3’ end HOX C locus, whose huge alteration is detectable in TCCs. In addition, the genes for CK7 and the basic hair keratin 1 and 6 are located at the 5’ end of the HOX C locus and in the same physical contiguity, and are transcriptionally regulated by HOXC13 [80]. Another group of cytokeratin genes is located on chromosome 17p21.3 in physical contiguity with the HOXB locus [81]. In biology, the concept of common functions performed by physically and/or evolutionarily contiguous genes is gaining more and more support [56]. Based on this we postulate an interaction between locus C HOX genes and keratins in the regulation of gene expression through the recently reported involvement of keratins in mRNAs transport [76] and protein synthesis [77]. We hypothesize that the interaction between HOX genes and keratins acts as a potential epigenetic resetting of epithelial cells. These mechanisms are functionally integrated in the epithelial tooth compartment and deregulated along the neoplastic evolution of tooth epithelium as we have recently reported in human ameloblastomas (Schiavo et al. 2011). Thus, the physical and functional interaction of locus C HOX and keratin genes and its deregulation occurring along tumor progression suggest this mechanism as potentially involved with the neoplastic

transformation of urogenital epithelial tissues.

HOX Genes and Uro-Genital Cancer Current Medicinal Chemistry, 2011 Vol. 18, No. 2 5

In conclusion, as with prostate cancers, HOXC appears to be the HOX locus mostly involved with bladder cancer. Furthermore, lumbo-sacral HOX genes of paralogous groups 13 and 11 are mostly involved with normal bladder development and bladder cancer progression. The interaction between HOX genes and keratins in the epigenetic resetting of epithelial cell identity deserves a deeper understanding.

KIDNEY CANCER

Molecular Mechanisms of Kidney Organogenesis

Homeobox and Hox genes are involved in different stages of kidney organogenesis, from early events in intermediate mesoderm to terminal differentiation of glomerular and tubular epithelia. The molecular mechanisms involved in early kidney organogenesis require expression in the matanephric blastema of the genes Hox A11 and Hox D11 [82] to induce the outgrowth of ureteric bud from the Wolffian duct through the expression of several transcription factors (Wt1, Pax 2, Sall1, Fox C1 and Eya1) [83-87], of the nuclear protein formin [88], the growth factors GDNF [89], its tyrosine kinase receptor Ret [90] and the co-receptor Gfra1 [91]. During mouse development, the targeted disruption of paralogous group 11 Hox genes (HoxA11, HoxC11 e HoxD11) results in the bilateral kidney agenesis due to alterations in the epithelial-mesenchymal interaction, further preventing ureteric bud outgrowth [24, 92].

Hox Genes and Kidney Cancers

Comparing the expression of the whole HOX gene network in late kidney organogenesis with normal adult kidneys and clear cell kidney cancers (RCCs) highlight the involvement, in kidney cancerogenesis, of lumbo-sacral HOX genes crucial in early and late kidney organogenesis [93]. Paralogous group 9, 11 and 13 and locus D HOX genes manifest a substantial deregulation in RCCs versus normal kidneys. HOXC11 and paralogous group 13 HOX genes tend to increase their expression in RCCs whereas paralogous groups 9, HOXA11 and HOXD11 manifest a decreased expression in RCCs versus normal kidneys. HOX A11, whose effector is 8 integrin, is again active at the 15

th week of development, HOX C11

is expressed from the 18th

week, HOX D11 starts again to be active from the 23

rd week until birth and is always active in normal adult

human kidneys. The expression of HOXD11 and HOXD9 parallels the acquisition of kidney functions as the kidney starts functioning from the 23

rd week of development [94]. HOX D9 and HOX D11

are constitutive expressed in normal adult human kidneys and are the only lumbo-sacral HOX D genes active in primary epithelial tubular kidney cells to prove that the function of these homeoproteins is performed in tubular epithelial kidney cells [93]. Finally HOX D9 and HOX D11 are inactive in the majority of RCCs. It has been suggested that, during tumour evolution, the gene profiles responsible for identifying specific cell phenotypes undergo a de-differentiation programme towards early developmental stages [95].

The expression of lumbo-sacral HOXD genes marks a molecular de-differentiation process towards embryonic life as consequence of the overlapping patterns of foetal kidneys and RCCs. This observation supports the alteration of a kidney function in relation to HOXD homeoproteins and is connected to the role played by lumbo-sacral HOX D genes in controlling epithelial cell differentiation and epithelial-mesenchymal transition through the interaction of a putative mesenchymal enhancer (acting from HOXD12 to HOXD9) and a ureteric bud enhancer (acting from HOXD9 to HOXD1). HOXD9 is the only HOXD gene responsive to both enhancers [96]. That is why the expression of HOXD11 and HOXD9 parallels the acquisition of kidney functions. The same chromosomal area 12q13-15 where HOXC11 is located harbours

the gene AQP2 coding for the water channel aquaporin-2 involved in water excretion, as part of the vasopressin hormone system, in renal collecting duct cells. The proteomic profiling of renal inner medullary collecting duct cells has recently identified HOX genes among the transcription factors expressed by duct cells [97]. The key involvement of paralogous group 11 and posterior HOXD genes in kidney cancers suggest the possibility to target these homeoproteins for diagnostic and therapeutic purposes in the clinical management of clear cell kidney cancers.

In conclusion, lumbo-sacral HOX genes of paralogous groups 9, 11 and 13 are mostly involved with normal kidney development and kidney cancer progression.

TESTICULAR CANCER

Testicular Cancer Generalities

Testicular germ cell tumors (TGCTs) are the most common malignancies in young male [98]. They are histologically classified as seminomas and non-seminomas. The high chemiosensitivity allows this type of cancer to be considered as a model of curative disease, although a small percentage escape therapy and metastasize. Is thus crucial to understand the role of genetic and epigenetic factors in the etiology of germ cell tumors [98].

Homeobox and Hox Genes in Testicular Cancers

Allelic variants of paralogous group 13 HOX genes (mostly HOXA13 and HOXD13) are involved in the pathogenesis of cryptorchidism, a congenital anomaly associated with increased risk for infertility and testicular cancer [99]. The homeobox gene Oct-4 (POU5f1), normally active in germ cells and required to maintain their stemness, is able to induce TGC tumorigenesis when inappropriately expressed. The TGCT growth is further related to the level of Oct-4 expression and turns off by turning off the inducible Oct-4 gene [100].

The inactivation of gene expression through promoter hypermethylation results crucial in germ cell tumorigenesis. Overall, the non-seminomas are significantly more frequently methylated than seminomas. The most frequent methylated genes among candidate genes are SGCB3A1, RASSF1A and HOXA9; CDH13 and HOXB5 are methylated at low frequencies and EMX2, MSX1, RUNX3, SORBS1 and XPA are only rarely methylated [101]. The study of methylation detection in testicular tumors took advantage of the observation that the normally methylated 5’ end of the XIST gene, a ncRNA involved in the process of X inactivation that maps on the X chromosome, is hypomethylated in testicular tumor cells in the presence of normally methylated 5’ XIST gene in the somatic cells of the same subject [102]. The presence of the hypomethylated gene was observed in 16 out of 25 (64%) paired plasma samples from patients whose testicular tumours had hypomethylated XIST. The unmethylated product was absent in 24 plasma samples from patients with renal and bladder cancer. Thus XIST hypomethylation seems to be more specific for existing serum tumor markers of testicular cancer [102].

Pluripotent embryonic carcinomas (EC) are the malignant counterparts to embryonic stem cells and are considered the stem cells of TGCTs. Human EC cells are highly sensitive to 5-aza-deoxycytidine (5-ara-CdR) in contrast to somatic solid tumor cells, display a decreased proliferation and survival associated with ATM activation, H2AX phosphorylation, increased p21 expression, and the induction of genes methylated in TGCTs (MGMT, RASSF1A and HOXA9) [103]. 5-aza-CdR hypersensitivity is associated with increased expression of the pluripotency-associated DNA methyltrasferase 3B (DNMT3B) compared with somatic tumor cells. Thus, high expression of DNMT3B sensitizes TGCT-derived EC cells to low-dose 5-aza-CdR treatment [103]. The RASSF1A

6 Current Medicinal Chemistry, 2011 Vol. 18, No. 2 Cantile et al.

tumor suppressor gene is epigenetically silenced in a variety of cancers. The epigenetic silencing of RASSF1A requires the homeprotein HOXB3 that binds to the DNA methyltrandferase DNMT3B gene and increases its expression [104]. DNMT3B, in turn, is recruited to the RASSF1A promoter resulting in the hypermethylation and silencing of RASSF1A expression. DNMT3B recruitment is facilitated through interactions with Polycomb repressor complex 2 and MYC, which is bound to the RASSF1A promoter. The oncogeneic activity of HOXB3 is, at least in part, due to epigenetic silencing of RASSF1A. The RASSF1A epigenetic silencing mechanism described here may be common to diverse cancer types [104].

In conclusion, in spite of the involvement of lumbo-sacral HOX genes in testicular cancer, this malignancy further manifests the implication of anterior HOXB genes (already involved in pancreatic cancer) and deserves a deeper analysis of the expression pattern of the whole HOX network.

MOLECULAR MECHANISMS INSIDE THE HOX NET-

WORK

Hox Genes Evolution

The Hox network acts as a complex genetic system where, behind the properties of the single gene components, complex biological functions are realized by the network as a whole. Phylogenetic evidences have initially supported these observations [105]. During evolution the vertebrate genome has gone through a remarkable expansion when compared to the invertebrate one. While the idea that genome expansion occurred durino early vertebrate evolution is accepted, the molecular processes involved are not so evident [106]. The duplication of the single invertebrate Hox locus to generate the four loci of vertebrate Hox network is often considered as paradigmatic of the genome expansion. The one to four ratio is thus at the basis of the double whole genome duplication events, a concept called the two rounds (2R) hypothesis, occurred in early vertebrates and responsible for the tethraploidization of the modern vertebrate genome [107].

Hox Genes, miRNAs and ncRNAs

Recent studies have identified small RNAs (single strended, 21-23 nuclotides in lenght) inside the genome displaying a regulative role (miRNAs) as well as long non coding RNA (ncRNAs) ranging from 300 nucletides to over 10 kb that are spliced, polyadenylated, and as diverse as protein-coding RNAs [108, 109].

At least 30 of the 39 mammalian Hox 3’UTR have one or more conserved matches to vertebrate miRNAs, several of which have been supported experimentally [110]. Five genes encoding miRNAs have been identified as located inside the Hox network. Three genes encoding miRNA 196 (mir-196b, mir-196a-1, mir-196a-2) are located between paralogous Hox gene group 9-10 and two genes encoding for miRNA 10a and 10b between paralogous group 4 and 5 of the Hox network. Thus, mir-196 and mir-10 miRNAs display the same paralogous organization of the Hox genes in the network [110]. The Drosophila homologue of miRNA 196, named iab-4, generates the homeotic dominant transformation of alter to wings [111]. Functional overlapping is thus shared between Hox genes and miRNAs.

ncRNAs may perform diverse role in gene regulation, especially in epigenetic control of chromatin [112]. The most important example of ncRNA is the silencing of the inactive X chromosome by the ncRNA XIST through the interaction with Polycomb gene products (see cell memory program) [113]. Recently, 231 ncRNAs have been identified inside the four human HOX loci [19]. Between them there is a 2.2 kilobase ncRNA residing in the HOXC locus on chromosome 12q13-15, termed

HOTAIR, which is able to repress transcription in trans across 40 kilobases of the HOXD locus on chromosome 2q31-33, interacting with the Polycomb Responsive Element (PRE) [114]. Another ncRNA, HOTAIRM1, has been identified at the 3’ end of the HOXA locus, between HOXA1 and HOXA2. HOTAIRM1 play a major role modulating gene expression of the anterior HOX A locus during myelopoiesis [115]. Thus, transcription of ncRNAs may act on chromosomal domains of gene silencing from a distance. This discovery has broad implications for gene regulation in development, diseases and regenerative medicine.

Global Controlling Region of Posterior HOXD Cluster

During limb development the coordinate expression of several HoxD genes is required to generate presuntive digits. Searching for the underlying upstream controlling sequences, Denis Duboule and coworkers have identified Lunapark (Lpk) a gene involved with limb and CNS expression specificity with both HoxD and Evx, another gene located nearby [56]. All these genes are transcriptionally regulated by the same regulative region, named global controlling region (GCR), located 100 Kb upstream the HoxD cluster [56]. GCR is 50 Kb in length and is able, as the locus controlling region (LCR) of the globin gene, to generate a transcriptional wave able to regulate, in cis transcription of unrelated genes included between CGR, Lpk and the HoxD locus. On the chromosome 2q31-33, between the GCR and HoxD and under GCR regulation, is the gene SATB2, already known to induce, when mutated, cleft palate[68]. SATB2 encodes a nuclear matrix protein and is able to repress HoxA2 expression [69]. HoxA2 is located on the locus Hox A on the chromosome 7q13, regulate the segmentation of the branchial archs and inhibits osteogenesis[69]. Repression of Hox A2 by SATB2 activate Runx2 to promote osteoblast differentiaton and bone formation[69]. These observation demonstrate a molecular interaction between the HoxD locus (chromosome 2q32) and the locus HoxA (chromosome 7q13) during ostreogenesis and branchial arch patterning (see prostate cancer section).

Molecular Interactions Beween HOX Clusters

The HOXA13 gene is required to maintain the distal specific transcriptional program in adult skin fibroblasts including the expression of WNT5A, a WNT non canonical gene required for distal development [116]. Through Chip-Chip analysis the promoters of seven genes most occupied by HOXA13 have recently been identified suggesting their positive transcriptional activation by HOXA13. The same analysis has also identified the genes negatively regulated by HOXA13 because of their anti-correlation to the expression level of HOXA13. Interestingly the most highly anti-regulated genes of HOXA13 include HOXB2, HOXB4, HOXB5, HOXB6 and HOXB7 suggesting a molecular interaction between two HOX loci, namely HOXA and HOXB, located on separate chromosomes [116].

Very recently a lincRNA, called HOTTIP, transcribed from the 5’ end of the HOXA locus that coordinates the activation of several 5’ HOXA genes in vivo has been identified [117]. HOTTIP RNA targets trithorax genes, driving histone H3 lysine 4 trimethylation and gene transcription along the HOXA locus and organizing chromatin domains to coordinate long range gene activation. Consistent with its genomic location 5’ to HOXA13, HOTTIP is expressed from development to adulthood in lumbo-sacral anatomical locations. Depletion of HOTTIP RNA in mice induces defects resembling HoxA11 and HoxA13 inactvation, suggesting the in vivo control of lumbo-sacral Hox genes by HOTTIP RNA [117].

In conclusion, the molecular interactions described between Hox genes inside the network support the concept of molecular software able to regulate cell identity and cell-cell communication.

HOX Genes and Uro-Genital Cancer Current Medicinal Chemistry, 2011 Vol. 18, No. 2 7

A UNIFIED MECHANISM POTENTIALLY INVOLVED

WITH URO-GENITAL CANCERS

Based on these observations we propose a unified molecular mechanism potentially involved with uro-genital malignancies (Fig. 3). We speculate that activation on the chromosome 7p15 of HOTTIP, ncRNA able to induce the regulation of the closest located 5’ HOXA genes through interaction with the trithorax gene WDR5, is perturbed along uro-genital cancer evolution. The consequence is the deregulation of 5’ HOXA genes (HOXA13, HOXA11) detectable in these malignancies.

On the other hand, HOXA13 is able to activate a sub-network including the HOTAIR ncRNA located on the chromosome 12q13. HOTAIR has already been shown to act as prognostic factor in human cancers acting on cell proliferation and micrometastases generation. The HOXA13 sub-network is also able to induce, through its overexpression, the down-regulation of 3’ end HOXB genes (chromosome 17p13-15), already reported to be involved in cancerogenesis. Once activated, HOTAIR is capable i) of regulating, in cis, adjacent locus C HOX genes (deregulate in kidney, bladder and prostate cancers) and ii) of repressing, in trans, through its interaction with the D11.12 Polycomb Responsive Element (PRE), the expression of 5’ HOXD genes on chromosome 2q31-33. Repression of 5’ HOX D locus genes induces (possibly through the activation of the Global Controlling Region (GCR) upstream of the HOXD locus) cell phenotype reprogramming with epithelial-mesenchymal transition, mesenchymal condensation and neuro-endocrine differentiation. Neuro-endocrine differentiation occurs in prostate cancer progression with the involvement of the prostate specific miRNA PCGEM and the atonal Neuro D1 genes, present in the same chromosomal location. HOTAIR repression acts also on the near located gene SATB2, which encodes an A-T rich binding protein of the nuclear matrix. SATB2 activated osteogenesis interacting with HOXA2, as reported in the prostate cancer section. SATB2 also interacts with its homologue SATB1, to the Xist ncRNA in the process of X chromosome inactivation. SATB2 induces the deregulation of miR-31 detectable in several cancer types including prostate. SATB2-miR31 interaction account for the export of tumorigenicity to the surrounding stroma with consequent cell migration and metastatization.

We postulate that the interaction between the transcripts WDR5-HOTTIP-HOXA13-HOTAIR-PRED11.12-SATB2 is part of the mechanism of epigenetic regulation related to posteriorization function and realised, for the caudal organs, through the gene systems CDX/HOX/WNT, during embryonic

development. This mechanism appears deregulated in uro-genital cancers.

Supplementary functions of Hox proteins

Hox Genes as Morphogens

We have described the Hox genes as transcriptional regulators of gene program involved with embryonic development, the regulation of body structure homeostasis and the neoplastic evolution. The potential use of this complex robust gene system in cell physiology and pharmacology requires thar we mention other functions ascribable to it. It has been reported that homeoproeins can be transferred from cell to cell translocating across biological membranes and internalized through the action of the homeodomain third helix [118]. This has led to the discovery of the messenger protein concept and the identification of the first transduction peptide (PTD) or cell permeability peptide (CPP) called Penetratin [119]. Target cells will be instructed to a different cell fate independent of their own memory program [119]. This intercellular transfer has important biotechnological consequences, but the real interest come from the physiological implication of this new mode of signal transdaction. This function confers to the homeoproteins the role of real morphogens able to instruct surrounding cells inside a tissue [120].

Hox Genes and Post-Transcriptional Regulation, the RNA

Operon

Post-transcriptional regulation of gene expression is crucial in several cellular processes from stress response, to immuno response, oxidative metabolism, circadian rhythms and cancerogenesis. A molecular mechanism for mRNAs co-regulation, at post-transcriptional level, by sequence-specific RNA-binding proteins that orchestrate the splicing, export, stability, localization and translation has recently been proposed [121]. According to this model mRNAs encoding functionally related proteins are coordinately regulated during cell growth and differentiation as post-transcriptional “RNA operons”, through a ribonucleoprotein-driven mechanism [121]. mRNAs functionally connected are linked to RNA-binding proteins and ncRNA through USER (untraslated sequence element for regulation) sequences located at their 5’ UTR. The USER code will determine the combinatorial association events between RBPs and mRNAs [121]. Thus, RNA binding factors coordinately regulate in this manner the fate of several transcripts functionally related.

Fig. (3). A unified mechanism potentially involved with uro-genital cancers (see text for details).

8 Current Medicinal Chemistry, 2011 Vol. 18, No. 2 Cantile et al.

The eukaryotic initiation factor 4E and the AU (Adenine-Uridine) binding proteins HuR are key node on their respective “RNA operons” [122,123]. eIF4E regulates mRNAs containing 4E sensitive elements at their 5’ UTR through the modification of their nuclear export and translation. HuR proteins modulate stability, translation and nuclear export of mRNAs containing AU rich regions (ARE) at their 3’UTR [124]. Recent results reveal that eIF4E and HuR are functionally linked [125]. Inside the cell nucleus, the eIF4E nuclear bodies are inhibited by their interaction with the hematopoietic homeobox (Hhex) [126] and promyelocytic leukaemia (PML) [127] proteins but modulated through their interaction with the HOX homeoproteins resulting in a lack of nuclear export (Hhex/PML) or in the export (HOX) of specific transcripts (ODC, Cyclin D1, c-myc, VEGF, FGF-2) [128] connected to crucial activities of the cell (cell-cycle, angiogenesis).

The Hox Network as a Privileged Site of Eukariotic Cell

On the basis of these observations the regulation of gene expression can not be limited to the transcriptional process. Post-trasnscriptional events related to mRNAs combinatorial coupling, their nuclear export and the cytoplasmic localization to get translated represent crucial events in order to originate a protein from a nucleotide sequence. The possibility for the homeoproteins to be involved with each one of these events makes the Hox network as a privileged site of the eukariotic cell. The spatio-temporal organization of the Hox network could account for the subcellular mRNA localization as well as for the coordinate mRNA regulation-timing. Thus the idea that Hox genes could drive transcriptional and post-transcriptional regulation of downstream targets is definitely fascinating.

HOX THERAPEUTIC TARGETING

Oligo Antisense, RNA Interference and RNA Activator

The identification of HOX gene deregulation in genito-urinary and other cancer phenotype suggests that HOX genes may be targeted for cancer therapy. This is the consequence of the increased understanding of the HOX gene network in the epigenetic regulation of the DNA-chromatin interaction and the transcriptional control of the gene programs involved in the determination of the cell memory and the spatio- temporal cell identity.

To inhibit the different steps of tumor progression requires the repair of several gene deregulation, of entire gene programs and of several signaling proteins to make the use of a single protein or a few proteins impracticable. Acting directly on specific transcription factors seems more feasible because their number is much lower than the number of oncogenes and oncosuppressor genes. Furthermore transcription factors connected to epigenetic programming are usually deregulated long time before the neoplastic pathology, rendering targeted therapy against epigenetically related transcription factors a possibility to inhibit cancer formation. Finally we propose that selective transcription be inhibited, rather than general transcription as currently occurs with anthracycline used in clinical practice [129]. Acting on specific targets implies some toxicity, but the advantage of blocking the growth of an invasive tumour outweighs the risk.

A limited number of pharmacological agents could be able to interact with crucial HOX genes in the network that are responsible for playing crucial roles in several tumour phenotypes, at present considered as different biological entities, but sharing common molecular characteristics. In theory, antisense oligonucleotide (ASO) and small interfering RNA (siRNA) molecules are ideal tools to serve the purpose of targeted therapy. These two major approaches could also be used in the targeting of HOX transcription factors in cancer. Antisense oligonucleotides against the majority of known oncogenes (c – myc, myb, mdm2) or anti-apoptotic genes

have been experimentally tested in a series of human tumours [130]. Treatment by oligo antisense against oncogenes inhibits the transformed phenotypes, while against anti-apoptotic genes it increases apoptosis.

Oligonucleotide antisenses have been extensively used to inhibit HOX gene expression of tumour cells. The inhibition, by antisense oligonucleotides, of retinoic acid induced activation of 3’ human HOX B genes (HOX B1 – HOX B3) in embryonic carcinoma cells, affects sequential activation of genes located upstream (with a 3’ to 5’ polarity) in the four loci of the HOX network [131]. The targeted downregulation of MLL – AF9 gene (MLL gene is the corresponding gene in vertebrates of the Drosophila gene Trithorax) by antisense oligonucleotide reduces the expression of HOXA7 and HOXA10 genes and induces apoptosis in human leukemic cells [132]. Hox B3 transfection in fetal lung cells has been shown to increase retinol – induced gene expression of Clara cell specific secretory protein, while reducing the expression of surfectant-associated protein C. These alterations are attenuated by the transfection with Hox B3 antisense nucleotide.

However, satisfactory results in cancer treatment have not yet been obtained with the in vivo use of antisense oligonucleotides due to their fragility and toxicity, although there have been many successful in vitro examples, such as the ones we have described. To circumvent these problems, RNA interference using small double stranded siRNA (small interference RNA) has been applied.

RNA interference (RNAi) has the potential to investigate molecular physiology by means of specific and effective gene silencing. Short interfering RNAs are short double stranded RNA molecules with 2-nucleotide 3’overhangs derived from the cytoplasmic processing of long dsRNA by the enzyme Dicer. siRNAs are similar to non coding RNA molecules naturally used by cells to regulate gene expression. They bind to the RISC complex, which promotes the cleavage of the RNA containing a particular sequence. A further way to generate siRNAs duplex is by synthetic short hairpin RNA (shRNA). The production of this synthetic shRNA is obtained from gene therapy vectors (either viral or nonviral) and it is an efficient means of experimentally eliciting RNAi in vivo. This mechanism leads to the virtual abolition of the expression of the target gene with almost absolute specificity. The selectivity conveyed by the gene sequence, which could potentially be as stringent as a single nucleotide difference between two genes, promises to aid in therapeutic approaches by providing much better target selectivity [133]. However significant drawbacks, such as incomplete suppression of target genes, non-specific immune response, in vivo delivery and the so-called off-target effects, either mediated by siRNA acting as a microRNA, or by the non-specific stress response of the cell need to be overcome before this technology can be successfully translated into clinical therapeutic practice. The fact that siRNAs are specifically and rapidly incorporated into RISC decreases the possibility of non-specific protein binding. A series of control experiments are required in order to minimize these risks. The lowest effective concentration of siRNA should be used to avoid unspecific gene silencing.

Using siRNA to inhibit genes in vitro and in vivo has improved studies on the mechanism of action for many disease genes, including those involved in the angiogenic process. The ability to use siRNA in vivo to validate angiogenesis factors as drug targets is uniquely important, because its pathological impact can only be characterized accurately in animal disease models. siRNAs protect mice from fulminant hepatitis, viral infection, sepsis and tumour growth. With the emergence of clinically viable delivery vehicles, anti-angiogenesis RNAi agents appear to have a promising and unprecedented role for the treatment of many serious human diseases that result from excessive angiogenesis. Using this systemic delivery of siRNA targeting VEGF pathway factors at sites of neovascularization, anti-angiogenesis efficacies has been

HOX Genes and Uro-Genital Cancer Current Medicinal Chemistry, 2011 Vol. 18, No. 2 9

achieved in a neuroblastoma tumor model [134]. Furthermore siRNAs targeting human VEGF have been found to inhibit the secretion of VEGF in the human prostate cancer cell line PC-3 [135]. The siRNA approach for gene silencing holds great therapeutic promise, as siRNAs, like microRNAs, are naturally used by cells to regulate gene expression and are therefore non toxic and highly effective. HOX genes and siRNA interference have only recently been coupled following the discovery of siRNA functionality in mammalian cells just a few years ago. Deregulation of HOX A9 by RNA interference decreases cell migration and tube formation of endothelial human cells suggesting that HOX A9 plays a role in endothelial cell migration and may exert its function by regulating the expression of EphB4 [136]. The oncogene Bmi1 stimulates H2A ubiquitylation for Hox gene silencing and normal cell growth. The role of Bmi1 in H2A ubiquitylation and Hox C5 gene expression has been analyzed in vivo by means of RNA interference experiments. Knockdown of Bmi1 causes a global and loci-specific loss of H2A ubiquitylation, up-regulation of the Hox C5 gene, and slower cell growth [137].

A new role has further been proposed for RNAs: instead of turning off genes (RNAi), synthetic RNAs are able to activate target genes (RNAa) [138]. Recent experiments indicate that RNAa-mediated over-expression of the transcription factor KLF4 inhibits prostate cancer cell growth and migration altering the expression of cell-cycle related genes [139]. This finding reveals an increasing role for small RNA molecules in the regulation of gene expression compared to what has so far been recognized and could be very powerful, in terms of potential anticancer therapeutic applications.

HOX/PBX

A Peptide to Target HOX/PBX Interaction

The redundancy of the Hox function is based in part upon the binding of similar DNA sequences, whereas the specificity of this binding is acquired through the interaction of the Hox protein with a common set of cofactors. Pre B cell leukemia transcription factors (PBX) are a family of genes belonging to the TALE super-family of homeobox genes [140]. PBX homeoproteins are able to interact with a subset of HOX proteins influencing the regulation of transcription and are required for many aspect of the Hox function. In addition to the interaction with Hox, PBX were subsequently identified as involved in the regulation and assembly of the cytoskeleton. More recently the role of PBX has been revaluated as a system to integrate transduction signals to regulate gene expression during embryonic development [141].

The identification of a small, cell-permeable peptide (HXR9) able to antagonize the HOX/PBX interaction has recently been described. HOX/PBX dimers have an increasing binding affinity and specificity for target DNA sequences when compared to the HOX monomer alone [142]. HXR9 triggers apoptosis and necrotic cell death in kidney, ovary and melanoma cells in vitro and in vivo, while sparing normal adult somatic cells and hematopoietic progenitors [143]. HXR9 further retards the in vivo growth of ovarian cancers [144]. The differences in sensitivity to HXR9 probably involve other regulator of the genes targeted by the peptide such as c-Fos. Targeting HOX/PBX interaction may represent a potential alternative or addition to current cancer therapies when drug resistance develops.

eIF4E

Hox and Protein Synthesis

The involvement of the mRNA cap-binding protein eIF4E in tumor evolution has been demonstrated in a serie of malignancies including prostate and bladder cancers in relation to tumor transformation and progression [128]. Although eIF4E globally

regulates cytoplasmic cap-dependent protein synthesis through the recruitment of mRNA to ribosomes, a substantial fraction (up to 68%) of eIF4E is found in the nucleus where generates nuclear bodies [124]. eIF4E reachs the nucleus through the nuclear pore where selects the mRNAs that will be trasferred from nucleus to cytoplasm [124]. m7G cap mRNAs compete with the promyelocytic leukemia protein (PML), a protein expressed in all eukaryotic cell types, for the binding to eIF4E leading to the inhibition of eIF4E nuclear functions [126]. A tissue specific regulation of cyclin D1 mRNA transport was demonstrated by the tissue specific partner of PML called proline-rich homeodomain (PRH/Hhex) by the direct interaction with eIF4E [126]. In contrast the HOXA9 protein stimulates the eIF4E-dependent export of cyclin D1 mRNA by competing with the factors that repress eIF4E activity. The eIF4E binding domain contains the motif YXXXXL , where is a hydrophobic amino acid residue [124]. The statistical occurrence of this motif in 803 homeoproteins found in the Swissprot database showed the presence of YXXXXL motif in 199 proteins [126]. Of the sexteen lumbo-sacral HOX genes here described as involved in uro-genital malignancies nine contain the YXXXXL motif.

Pre-clinical and clinical studies indicated that ribavirin, a broad-spectrum anti-viral drug that physically mimic the m7Gcap thus blocking eIF4E activity, has a positive clinical response in sevral cancer type (leukemia, prostate, breast, liver) [145]. eIF4E over-expressing M4/M5 AML leukemic cells depend on eIF4E for growth and survival displaying an oncogenic addition to eIF4E [145]. Ribavirin in vitro leukemic cell exposure generate a re-localization of eIF4E from nucleus to cytoplasm that correlates with reduced eIF4E dependent export of target transcripts (NBS1, cyclin D1) [146]. Micromolar plasma concentrations of ribavirin are achievable in humans with minimal toxicity. Further studies are needed on the role of the target related to the interaction between eIF4E and other lumbo-sacral homeoprotein, differing from HOXA9, and on ribavirin and analog as a therapy. The ribavirin treatment (either alone or in combination) could be clinically beneficial in the 30% of cancers characterized by elevated eIF4E levels. The post-transcriptional regulation of mRNA by homeoproteins in different tissues and organs may generate an excellent therapeutic target against cancer. In contrast to eIFs, the ribosomes have been considered to display a constitutive role in mRNA translation. However, recent evidence suggest a rather regulatory role for the ribosome in the control of gene expression during embryonic development: the ribosomal protein L38 alters the translation of specific HOX mRNAs generating tissue and structure specific loss-of-function [147]. Thus, the ribosome regulates gene expression during embryonic development by promoting the translation of Hox mRNAs.

ANTI-EPIGENETIC DRUGS

Acetylases and Methylases

Aberrant epigenetic modifications are involved in cancer generation and progression through deregulation of transcription and modifications of gene expression. Thus, epigenetic mechanisms represent a new powerful target for cancer therapy. A potential approach for anti-epigenetic therapeutic intervention involves the use of drugs that target histone acetylation and methylation. Histone acetylation is associated with transcriptional activation and the use of histone deacetylase (HDAC) enzymes as inhibitors allowing histone acetylation to be reversed in vitro and in vivo [148]. Importantly, the effects of HDAC inhibitors is mediated by specific HDAC isoforms (HDAC1 and HDAC2) suggesting a clear therapeutic role for these enzimes [148]. This family of drugs including suberoylanilide hydroxamic acid (SAHA), sodium butyrate and trichostatin A (TSA), generates histone hyper-adenilation and inhibits specific HDAC enzyme isoforms. These

10 Current Medicinal Chemistry, 2011 Vol. 18, No. 2 Cantile et al.

drugs are already used or are in clinial trias to be inserted in the treatment of urogenital cancers [149].

Histone methylase and demethylase are a different type of epigenetic modification potentially useful in cancer therapy, instead of the incompletely defined role they perform as transcriptional repressor or activator. In reality, demethylation at H3K9 appears to act as repressor whereas at H3K4 (such as Mll) as activator of transcription. Zebularine and 5-aza-deoxycytidine are nucleoside analogs able to inhibit DNA methylation activity, although both require replication to incorporate into DNA. Zebularine targets bladder, prostate and other cancer cell lines, exhibits low toxicity in vivo and oral bioavalability [148]. In contrast with other aza-nucleoside analogs, effective in treating various cancers, zebularine does not form irreversible covalent bonds with DNA causing long-term side effects including mutagenesis during DNA replication [148]. A different DNMT inhibitor, RG108 intercts directly with DNMT active site circumventing the need for DNA replication [150]. These drugs are potentially useful in the future therapeutic setting against cancer.

CONCLUSIONS

The expression of the HOX gene network in genito-urinary malignancies identifies lumbo-sacral HOX genes, already involved in posterior embryonic development, as determinant of urogenital cancers.The mechanisms involved support the role of the HOX gene network as complex gene system regulating cell identity and spatio-temporal determination. In addition to the evolutionarily related function of the Hox network as major determinant of neuro-ectodermal tissues, the available data allow locus C HOX genes to be connected with epithelial differentiation, locus A and D genes with epithelial-mesenchymal transition and mesenchymal condensation (locus D) and locus B and D genes to be connected with neurogenic differentiation. At molecular level these functions are performed through the control of mRNA transcription, editing, nuclear export, localization and translation with miRNA, ncRNA and nuclear matrix attachment proteins acting as major interactors of the HOX gene network. Targeting of these mechanisms (RNAi, HOX/PBX, eIF4E, anti-epigenetic drugs) represent a potential implementation in the cancer therapy resources. The implication of the HOX gene network in the regulation of these basic cellular processes allows postulation that the homeoproteins would possibly be good target for therapy, both for their involvement at the very onset of cancer evolution as well as for the possibility to target different and yet unrelated tumor phenotypes with an unique epigenetic approach.

ACKNOWLEDGEMENTS

We are grateful to Dr Barbara Cullen, University of Basel CH, for editing the manuscript.

POTENTIAL CONFLICT OF INTEREST

Nothing to declare

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