Clemente Cillo

Homeobox genes in normal and malignant cells

Homeobox genes are transcription factors primarily involved in embryonic development. Several homeobox gene families have so far been identified: Hox, EMX, PAX, MSX as well as many isolated divergent homeobox genes. Among these, Hox genes are most intriguing for having a regulatory network structure organization. Recent indications suggest the involvement of homeobox genes in (i) crucial adult eukariotic cell functions and (ii) human diseases, spanning from diabetes to cancer. In this review we will discuss the mechanisms through which homeobox genes act, and will propose a model for the function of the Hox gene network as decoding system for achieving specific genetic programs. New technologies for whole‐genome RNA expression will be crucial to evaluate the clinical relevance of homeobox genes in structural and metabolic diseases.

Expression of homeobox-containing genes in primary and metastatic colorectal cancer

Homeobox genes are a network of genes encoding nuclear proteins functioning as transcriptional regulators. Human and murine homeobox genes of the HOX family are organised in four clusters on different chromosomes. Gene order within each cluster is highly conserved, perhaps in direct relation to their expression. Homeobox genes have recently been involved in normal development and oncogenesis. We have analysed HOX gene expression in normal human colon and in primary and metastatic colorectal carcinomas. The majority of HOX genes are active in normal adult colon and their overall expression pattern is characteristic of this organ. Furthermore, the expression of some HOX genes is identical in normal and neoplastic colon indicating that these genes may exert an organ-specific function. In contrast, other HOX genes exhibit altered expression in primary colon cancers and their hepatic metastases which may suggest an association with colon cancer progression.

HOX gene network is involved in the transcriptional regulation of in vivo human adipogenesis

Adipogenesis is regulated by the sequential activation of a series of transcription factors: the C/EBP proteins of type β and δ trigger the process while PPARγ and C/EBPα induce the differentiation from pre‐adipocyte to adipocyte, followed by adipo‐specific gene expression. A number of observations suggest the involvement of genes controlling embryonal development in adipogenesis. In human thyroid follicular carcinoma, it has been recently identified an oncogenetic fusion protein resulting from the interaction between the isoform PPARγ1 of PPARγ and the homeoprotein encoded by the PAX‐8 gene. Recent observations have pointed out that gene expression associated with adipocyte differentiation in vivo and in vitro, although partially overlapping, is actually different. HOX genes make up a network of transcription factors (homeoproteins) controlling embryonal development as well as crucial functions of adult eukaryotic cells. The molecular organization of this network of 39 genes appears to be unique in the genome and probably acts regulating phenotypic cell identity. In the present study we have analyzed the expression of the complete HOX gene network, in vivo, in different deposits of human white adipose tissue and in embryonal brown adipose tissues. Most of the genes in the HOX network are active in white as well as brown adipose tissue. Furthermore HOX genes display a deposit‐specific expression in white adipose tissue. Moreover, expression of the paralogous group 4 genes (HOX A4, HOX B4, HOX C4, and HOX D4), together with that of isolated genes in the network, appears to discriminate between white and brown adipose tissue. This data allows us to postulate the involvement of the HOX network in transcriptional regulation of human adipogenesis and to hypothesize on the molecular mechanisms that could be implicated.

Adult human neural crest-derived cells for articular cartilage repair.

HOX-negative, differentiated neural crest–derived adult cells from the nasal septum display self-renewal capacity and environmental plasticity and are compatible for articular cartilage repair. Cells from Nose Repair Tissue in Joint Cartilage repair remains a yet unmet clinical need, with few viable cell therapy options available. Taking cells from the knee or ankle to repair worn cartilage requires additional surgery and, in turn, pain and healing for the patient. As such, a new, accessible cell source would greatly benefit these patients. Here, Pelttari and colleagues looked up the nose for cells that may have the capacity to regenerate cartilage. Nasal septum cells arise from the neuroectoderm—the tissue that gives rise to the nervous system—and are better at repairing tissues than their mesoderm counterparts. These regenerative capabilities have been attributed to a lack of homeobox (HOX) gene expression. The authors therefore investigated whether nasal chondrocytes (HOX-negative, neuroectoderm origin) were compatible with an articular cartilage environment, like the knee joint (HOX-positive, mesoderm origin). The authors discovered that adult human nasal chondrocytes were able to self-renew and also, to their surprise, adopt a HOX-positive profile upon implantation into a mesoderm environment; in goats, this led to repair of experimental articular cartilage defects. In an ongoing clinical trial, human nasal chondrocytes have been shown to be safe once transplanted, suggesting translation of this new, easy-to-access cell source for repairing damaged joints. In embryonic models and stem cell systems, mesenchymal cells derived from the neuroectoderm can be distinguished from mesoderm-derived cells by their Hox-negative profile—a phenotype associated with enhanced capacity of tissue regeneration. We investigated whether developmental origin and Hox negativity correlated with self-renewal and environmental plasticity also in differentiated cells from adults. Using hyaline cartilage as a model, we showed that adult human neuroectoderm-derived nasal chondrocytes (NCs) can be constitutively distinguished from mesoderm-derived articular chondrocytes (ACs) by lack of expression of specific HOX genes, including HOXC4 and HOXD8. In contrast to ACs, serially cloned NCs could be continuously reverted from differentiated to dedifferentiated states, conserving the ability to form cartilage tissue in vitro and in vivo. NCs could also be reprogrammed to stably express Hox genes typical of ACs upon implantation into goat articular cartilage defects, directly contributing to cartilage repair. Our findings identify previously unrecognized regenerative properties of HOX-negative differentiated neuroectoderm cells in adults, implying a role for NCs in the unmet clinical challenge of articular cartilage repair. An ongoing phase 1 clinical trial preliminarily indicated the safety and feasibility of autologous NC–based engineered tissues for the treatment of traumatic articular cartilage lesions.

The HOX gene network in hepatocellular carcinoma

Liver organogenesis and cancerogenesis share common mechanisms. HOX genes control normal development, primary cellular processes and are characterized by a unique genomic network organization. Less is known about the involvement of HOX genes with liver cancerogenesis. The comparison of the HOX gene network expression between nontumorous livers and hepatocellular carcinomas (HCCs) highlights significant differences in the locus A HOX genes, located on chromosome 7, with a consistent overexpression of HOXA13 mRNA thus validating this gene deregulation as a feature of HCC. HOXA13 is a determinant of gut primordia and posterior body structures. Transcriptome analysis of HCC/nontumorous liver mRNAs, selected on the basis of HOXA13 overexpression, recognizes a set of deregulated genes. The matching of these genes with previously reported HCC transcriptome analysis identifies cell‐cycle and nuclear pore‐related HCC phenotype displaying poor prognosis. HOXA13 and HOXA7 homeoproteins share a consensus sequence that physically links eIF4E nuclear bodies acting on the export of specific mRNAs (c‐myc, FGF‐2, vascular endothelial growth factor (VEGF), ornithine decarboxylase (ODC) and cyclin D1). We report the protein–protein interaction between HOXA13 and eIF4E in liver cancer cells and the deregulation of eIF4E mRNA and protein in cell cycle/nuclear pore HCC group phenotype and in T4 stage HCCs, respectively. Thus, transcriptional and post‐transcriptional HOXA13 deregulation is involved in HCC possibly through the mRNA nuclear export of eIF4E‐dependent transcripts.

Differential patterns of HOX gene expression are associated with specific integrin and ICAM profiles in clonal populations isolated from a single human melanoma metastasis

Homeobox‐containing genes comprise a gene family coding for transcription factors involved in normal development. Class I human homeobox (HOX) genes display a peculiar chromosomal organization, perhaps directly related to their function. Aberrant expression of homeobox genes has been associated with both morphological abnormalities and oncogenesis. We have reported that HOX gene expression is (i) specific for normal adult human organs (kidney, colon, lung) and (ii) altered in cancer specimens according to their histological type and stage of tumor progression. Here, we have investigated whether patterns of HOX gene expression are associated with tumor heterogeneity by analyzing the expression of the entire panel of 38 HOX genes in clones isolated from a single human metastatic melanoma cell line (Me 665/2). The differential expression of a block of genes located at the 5′ end of the HOX C locus allows melanoma clones to be classified into 2 major groups. The 2 patterns of HOX gene expression are inversely associated with 2 distinct surface phenotypes for integrins (VLA‐2, VLA‐5 and VLA‐6) and the adhesion molecule ICAM‐I. The genes of the HOX C locus are silent in the clones with high levels of integrins VLA‐2, VLA‐5 and VLA‐6 and of the adhesion molecule ICAM‐I but actively expressed in the clones with low levels of ICAM‐I and lacking VLA‐2, VLA‐5 and VLA‐6. Our results indicate that HOX gene expression reflects the intra‐tumor heterogeneity of melanoma clones and suggest that the expression of surface molecules involved in cell‐cell and cell‐matrix interactions may be related to the patterns of HOX gene expression.

Hyperexpression of locus C genes in the HOX network is strongly associated in vivo with human bladder transitional cell carcinomas

Bladder carcinogenesis remains unclear despite the identification of chemical, environmental and genetic factors. It has recently been reported that the chromosomal region 12q13–q15, containing crucial cancer genes such as MDM2, CDK4 and GLI, is amplified in bladder cancer. In the same region are also located the genes of the locus HOX C, flanked by keratin genes whose protein product may be a prognostic marker of bladder cancer. The HOX genes constitute a network of transcription factors controlling embryonal development and play an important role in crucial adult eukaryotic cell functions. The molecular organization of this 39-gene network is unique in the genome and probably acts by regulating phenotypical cell identity. We have analysed the expression of the whole HOX gene network in pairs of normal–tumour bladder and in tumoral biopsies. Comparison between normal urothelium and bladder tumour has identified dramatic variations of expression in a block of three genes (HOX C4, HOX C5 and HOX C6) localized in the HOX C locus on the chromosome 12q13 and in the paralogous group 11 HOX genes, involved during normal development in the formation of the urogenital system. These data suggest a key involvement of the HOX gene network, and especially the locus C, in bladder cancer.

HOX D13 expression across 79 tumor tissue types.

HOX genes control normal development, primary cellular processes and are characterized by a unique genomic network organization. Locus D HOX genes play an important role in limb generation and mesenchymal condensation. Dysregulated HOXD13 expression has been detected in breast cancer, melanoma, cervical cancer and astrocytomas. We have investigated the epidemiology of HOXD13 expression in human tissues and its potential deregulation in the carcinogenesis of specific tumors. HOXD13 homeoprotein expression has been detected using microarray technology comprising more than 4,000 normal and neoplastic tissue samples including 79 different tumor categories. Validation of HOXD13 expression has been performed, at mRNA level, for selected tumor types. Significant differences are detectable between specific normal tissues and corresponding tumor types with the majority of cancers showing an increase in HOXD13 expression (16.1% normal vs. 57.7% cancers). In contrast, pancreas and stomach tumor subtypes display the opposite trend. Interestingly, detection of the HOXD13 homeoprotein in pancreas‐tissue microarrays shows that its negative expression has a significant and adverse effect on the prognosis of patients with pancreatic cancer independent of the T or N stage at the time of diagnosis. Our study provides, for the first time, an overview of a HOX protein expression in a large series of normal and neoplastic tissue types, identifies pancreatic cancer as one of the most affected by the HOXD13 hoemoprotein and underlines the way homeoproteins can be associated to human cancerogenesis.

Kinetics of Erythroid and Myeloid Stem Cells in Post-Hypoxia Polycythaemia

Summary. The number of erythroid burst‐ (BFU‐E) and colony‐forming units (CFU‐E), as well as of myeloid‐macrophage colony‐forming units (CFU‐C), has been evaluated in tibial marrow and spleen of ex‐hypoxic polycythaemic mice, at sequential time intervals after the end of hypoxia.

cAMP induced modifications of HOX D gene expression in prostate cells allow the identification of a chromosomal area involved in vivo with neuroendocrine differentiation of human advanced prostate cancers

The acquisition of epithelial‐neuroendocrine differentiation (ND) is a peculiarity of human advanced, androgen‐independent, prostate cancers. The HOX genes are a network of transcription factors controlling embryonal development and playing an important role in crucial adult eukaryotic cell functions. The molecular organization of this 39‐gene network is unique in the genome and probably acts by regulating phenotype cell identity. The expression patterns of the HOX gene network in human prostate cell phenotypes, representing different stages of prostate physiology and prostate cancer progression, make it possible to discriminate between different human prostate cell lines and to identify loci and paralogous groups harboring the HOX genes mostly involved in prostate organogenesis and cancerogenesis. Exposure of prostate epithelial phenotypes to cAMP alters the expression of lumbo‐sacral HOX D genes located on the chromosomal region 2q31‐33 where the cAMP effector genes CREB1, CREB2, and cAMP‐GEFII are present. Interestingly, this same chromosomal area harbors: (i) a global cis‐regulatory DNA control region able to coordinate the expression of HOX D and contiguous phylogenetically unrelated genes; (ii) a prostate specific ncRNA gene associated with high‐risk prostate cancer (PCGEM1); (iii) a series of neurogenic‐related genes involved with epithelial‐neuronal cell conversion. We report the expression of neurexin 1, Neuro D1, dlx1, and dlx2 in untreated and cAMP treated epithelial prostate cells. The in vivo expression of Neuro D1 in human advanced prostate cancers correlate with the state of tumor differentiation as measured by Gleason score. Thus, we suggest that the chromosomal area 2q 31‐33 might be involved in the epithelial‐ND characteristic of human advanced prostate cancers.