Hox-Mediated Spatial and Temporal Coding of Stem Cells in Homeostasis and Neoplasia

Abstract

Hox genes are fundamental components of embryonic patterning and morphogenesis with expression persisting into adulthood. They are also implicated in the development of diseases, particularly neoplastic transformations. The tight spatio-temporal regulation of Hox genes in concordance with embryonic patterning is an outstanding feature of these genes. In this review we have systematically analyzed Hox functions within the stem/progenitor cell compartments and asked whether their temporo-spatial topography is retained within the stem cell domain throughout development and adulthood. In brief, evidence support involvement of Hox genes at several levels along the stem cell hierarchy, including positional identity, stem cell self-renewal, and differentiation. There is also strong evidence to suggest a role for Hox genes during neoplasia. Although fundamental questions are yet to be addressed through more targeted and high- throughput approaches, existing evidence suggests a central role for Hox genes within a continuum along the developmental axes persisting into adult homeostasis and disease.

Introduction

Hox genes are evolutionarily conserved master regulators of embryonic patterning and morphogenesis [1,2]. In humans, the 39 Hox genes are organized within four paralogous clusters (HOXA to D) located on chromosomes 7, 17, 12, and 2, respectively (Fig. 1A) [3 –5]. These genes encode homeobox-containing transcription factors with a DNA-binding domain located in exon2 (Fig. 1B). Hox proteins can act as activators or repressors of target genes, in configurations of homo or heterodimers, or in heterotrimeric complexes with other members of the homeodomain protein family [6]. Moreover, intergenic splicing events have been reported in Hox genes, where a certain sequence within one Hox transcript acts as a splice acceptor for a different Hox. For example, sequences within the second exon of Hoxd12 act as an internal splicing sequence to produce a read-through in-frame transcript that connects the coding region of Hoxd12 and Hoxd11 and swaps the homeobox region of Hoxd11 into Hoxd12 [7].

FIG. 1.

Hox clusters and their gene structure: (A) The human Hox cluster organized in four paralog groups on separate chromosomes. Note the alignment of genes along the antero-posterior developmental axis. (B) Typical structure of Hox genes, with the second exon containing the conserved homeodomain.

Hox genes are accredited as integral components of temporospatial patterning during embryonic and limb development, although a full list of their downstream target genes is yet to be defined [8]. It is, however, well established that Hox gene regulation and function follows three basic principles: First, “spatial co-linearity,” where the 3′ to 5′ position of Hox genes along the clusters corresponds to their positional expression along the antero-posterior axis; usually, 3′ Hox genes are expressed more anteriorly in the embryo with 5′ expression seen more posteriorly (Fig. 1A) [9,10]. Second, “posterior prevalence” where Hox genes located more 5′ in the cluster have a dominant effect on more 3′ Hox genes. And finally, Hox gene expression follows “temporal co-linearity,” where the timing of each Hox expression corresponds to its location along the 3′ to 5′ axis, with 3′ Hoxes expressed earlier in development [8,9]. Given their tight spatial and temporal regulation during early axial patterning and their crucial role in development and differentiation of distinct tissues in the nascent embryos, it is of particular interest to know whether Hox genes play a role in the stem/progenitor compartments of developing and adult tissues and whether part or all of their temporal and/or spatial patterns of expression is preserved within these domains in homeostasis and disease.

Hox Code Encrypts Positional and Tissue-Specific Identities in Adult Stem Cells

Hox genes are determinants of segmental identity in the developing embryos. It is therefore valid to ask whether a similar Hox or segmental memory persists in downstream tissue-resident stem cells with different temporo-spatial topography. The majority of evidence for such an anatomical code focuses on a population of adult stem cells referred to as mesenchymal stem cells (MSC). MSCs were initially isolated from bone marrow and characterized by their ability to adhere to plastic and to form differentiated progeny of mesenchymal origin including fat, bone, and cartilage [11]. Since then, MSCs have been isolated from almost all organs and despite the similarities, phenotypic and transcriptional differences are reported based on their tissue of origin. Studies suggest an inherent positional (or organ-specific) identity for MSCs that is coded as early as formation of postsegmentation mesoderm. In case of early hematopoiesis, for example, the Hoxb6⁺ lateral plate mesoderm is suggested as an important, and to some degree, exclusive source of all adult hemogenic progenitor cells [12,13]. Different MSC populations have individual Hox codes that act as a fingerprint for their developmental origin [14]. Moreover, MSCs derived from various organs showed heterogeneous but yet characteristic Hox profiles in concordance with their anatomical origin. Interestingly, the Hox signature persisted throughout the differentiation process, suggesting an intrinsic link to basic MSC characteristics [15]. Studies in adult mouse aorta suggested enriched expression of Hox6-10 paralog groups in the thoracic aorta compared to the low level expressions in the aortic arch. These distinct Hox profiles may further translate into higher resistance to atherosclerosis in the smooth muscle cells (SMC) lining the thoracic aorta, and lower activity of proinflammatory and proatherogenic nuclear factor-κB along with its target genes. The resistance to the atherogenesis is attributed to inhibitory effects of HoxA9 and is similarly observed in human embryonic stem (ES) cell-derived SMC [16]. Based on these and other studies, Hox codes are suggested as fingerprints to differentiate between functionally distinct stem cell subpopulations. In the cord blood for example, there are two distinct stem cell populations: the cord blood-derived MSCs that resemble bone marrow MSCs and the unrestricted somatic stem cells (USSC) that show a differentiation potential into all three germ layers. Similar to the ES cells, no expression of Hox genes were detected in USSCs with the exception of low levels of HOXD8 expressed in one of the USSC replicates. On the other hand, expression of four Hox genes A9, B7, C10, and D8 was observed in the cord blood MSCs, suggesting discriminatory Hox codes between two types of cord blood stem cell populations [17]. In a similar approach, HOXC10 was suggested as a distinct marker between amnion and decidua-derived MSCs. In fact, high expression of HOXC10 was readily detectable in all 10 tested amnion-derived MSCs, whereas only two decidua-derived MSCs showed detectable expression levels of this Hox gene [18]. In a separate study, the regenerative capacities of cranial neural crest and mesoderm-derived skeletal progenitors in healing mandibular and tibial bones were compared. Expression of Hoxa11 in mesodermal progenitors, contributed to these cells being selective in healing tibial but not mandibular bone, suggesting the exclusive ability of these cells to heal bone tissue with mesodermal embryonic origin. The Hoxa11-negative neural crest progenitors, however, performed more plastic, regenerating both mandibular (neural crest-derived) and tibial (mesoderm-derived) bones [19]. Hox genes are also negatively selected during early development of the facial skeleton. Mis-expression of several anterior Hox genes including Hoxa3 and Hoxb4 in the Hox-negative section of the anterior neural folds resulted in severe perturbations in facial morphogenesis [20]. In summary, specific spatial and temporal imprinting of Hox codes in tissue-resident progenitors assigns inherent functional properties to these cellular compartments.

Hox Genes Play Critical Roles in Lineage-Specific Differentiation of Stem Cells

Undifferentiated ES cells manifest a range of Hox expression levels and profiles, from rare levels of posterior Hox transcripts in human ES cells [21], to relatively outstanding levels in murine ES cells [22]. Once the differentiation process begins, there is however, a tendency for Hox profiles to become more prominent and refined [22,23]. In fact, well-tuned in vitro differentiation of ES cells in response to factors such as retinoic acid [24 –26], or successive treatments with fibroblast growth factor (FGF), Wnt/β-Catenin, and growth differentiation factor [27] could induce collinear Hox gene expression similar to murine embryos.

In the course of differentiation, Hox genes appear to function in three major phases: lineage specification, early differentiation, and tissue maturation (Fig. 2). In cranial neural crest cells for example, suppression of Hoxa2 is essential for osteo/chondrogenic patterning. In fact, expression of Hoxa2 inhibited Sox9 and hence impeded the ability of neural crest cells to develop into ectoMSCs involved in osteochondrogenesis [20,23]. In the hematopoietic system, different Hox clusters show lineage-restricted patterns of expression with HOXA genes predominantly expressed in myeloid cells, HOXB genes in erythroid cells, and HOXC genes in lymphoid cells [28]. In accordance, gain of function studies suggest a lineage-instructive role for Hox genes. In human CD34⁺ umbilical cord blood cells, overexpression of HOXA9 or HOXA10 suppressed erythropoiesis in favor of hematopoietic stem cells (HSC) and downstream progenitors [29]. In a different study, overexpression of Hoxa9 or Hoxa10 in murine or human bone marrow expanded the monocyte/granulocyte lineage at the expense of other hematopoietic lineages [30]. In mouse models, Hoxb1 plays an essential role during early patterning of the rhombomeres and in assigning a hindbrain identity to neural stem cells [31]. In addition, ectopic expression of Hoxb1 and Hoxa2 could sufficiently induce motor neuron differentiation [32] again suggesting a role in lineage specification.

FIG. 2.

Role of Hox genes during stem/progenitor cell differentiation: Hox genes are involved at different stages of the differentiation process from lineage commitment, to early differentiation and tissue-specific maturation. Note the distinct Hox signatures during each stage.

Once committed, cells will progress through differentiation and tissue-specific maturation events. Hox effects during early differentiation have been reported in a number of systems. During chondrogenic differentiation, expression of HOXC8 negatively affected the progression of chondrocytes and their differentiation process by causing accumulation of proliferating precursors [33,34]. In vascular wall-resident multipotent stem cells, differentiation into SMCs is dependent on the expression of HOXB7, C6, and C8. Silencing of these Hox genes resulted in significantly reduced sprouting capacity and expression of SMC markers such as TAGLN and Calponin. Interestingly, these Hox transcripts were not expressed in mature aortic SMC suggesting a specific role during early SMC differentiation [21]. In adult endothelial cells and in vitro-differentiated embryonic bodies, a prominent role was suggested for three anterior HOX genes A3, D3, and B3 in activating an angiogenic phenotype [35 –38]. Likewise, a global reduction in the expression of Hoxa, b and c in mice lacking mixed lineage leukemia (MLL) gene, an upstream regulator of Hox genes, significantly impaired the production of hematopoietic colonies despite unperturbed development of early hematopoietic progenitors [39,40] suggesting an effect downstream of hematopoietic lineage commitment.

The final domain of Hox function is the late stage of differentiation or tissue maturation. In adult endothelial cells, vessel maturation and maintenance of a quiescent endothelial state relies on the expression of HOXA5 and HOXD10 [36,38]. Mice lacking Hoxa11 and d11 also showed impaired mesenchymal condensation with no differentiation into mature hypertrophic chondrocytes [41]. Likewise, during the development of facial structures, maturation and maintenance of facial nerves is dependent on Hoxa2 and Hoxb1 [42,43]. Although the above studies suggest a positive role for Hox genes during tissue maturation, studies have also suggested an essential role for timed withdrawal of Hox proteins for correct progression through final differentiation stages. For example, during lumbar motor neuron development, timed repression of Hoxb8 mediated by microRNA miR-196 was shown to be essential for the exit of motor neurons from the cell cycle and subsequent axonal projection [44] (Fig. 3A). Likewise, in hematopoietic system, Hox gene expression is almost lacking in CD34⁻ cells that are considered as differentiated bone marrow cells [28]. Overall, studies confirm the strict requirement for temporal and spatial tuning of Hox gene expression at the stem/progenitor cell compartment during tissue specification, differentiation, and maturation.

FIG. 3.

Hox genes along the stem/progenitor cell hierarchy: (A) Expression of Hoxb8 mRNA in lumbar neural tube of chick embryos (Left: HH16, Right: HH22). Note the dynamics of Hoxb8 expression in the motor neuron domain. MN, motor neuron; nc, notochord. (B) Different domains of Hox gene activity throughout stem cell hierarchy.

Hox Genes Are Functional Components of Stem Cell Self-Renewal and Expansion in Homeostasis and Neoplasia

Adult and cancer stem cells are often quiescent but exhibit rare events of cell division referred to as self-renewal to replenish or maintain the existing stem cell pool. Stem cell proliferation and self-renewal processes are enhanced in the events of tissue injury response or, in case of cancer stem cells, during tumorigenesis and/or metastasis. The role of Hox genes in stem cell self-renewal and expansion has been particularly elaborated in the hematopoietic system. Expression of HOX paralog groups A, B, and C members is reported in various human hematopoietic cell lines and in CD34⁺ hematopoietic progenitors [45] with certain Hox genes such as HOXB3 and HOXB4 specifically enriched in progenitor cell populations [46]. Retroviral overexpression of Hoxb4 in murine bone marrow cells conferred a proliferative and bone marrow repopulating advantage to these cells with little effect on their lymphoid or myeloid differentiation [47]. In a similar study, Hoxb4-transduced murine HSC were also assessed using in vitro colony formation and pluripotency as readouts for the HSC compartment within the bone marrow isolates. As previously reported, Hoxb4- transduced HSCs demonstrated a selective advantage in growth, and colony formation, and proved to be more competitive in repopulating irradiated bone marrow compared to control cells. Interestingly, the in vivo expansion occurred at a polyclonal level, suggesting a role for Hoxb4 in preservation of multiple HSC clones during the repopulation process [48]. Although in these and other studies a minimal effect on lympho-myeloid differentiation of transduced cells was reported, a similar approach to overexpress HOXB4 in CD34⁺ cells of human cord blood could replicate the effects seen at the level of competitive growth advantages, but at the expense of differentiation potential to lymphoid and myeloid lineages [49]. The differences in these observations are probably due to higher levels of HOXB4 expression in the latter approach, suggesting an essential dosage threshold to maintain the balance between differentiation and self-renewal. Studies suggest a considerable level of functional redundancy between members of “group4” Hox paralogs [50]. Similar effects of HOXB4 and HOXC4 proteins were observed where CD34⁺ hematopoietic progenitor cells were transduced with either of these Hox proteins [51]. In this approach, CD34⁺ cells cocultured with HOXB4/C4-producing stromal cells were assessed for in vivo expansion and repopulation where in the case of both proteins a significant effect was observed. Furthermore, comparative transcriptome analysis between HSCs transduced with either of the Hox proteins showed no prominent difference, suggesting that they act through similar pathways. Interestingly, among the target transcripts that were upregulated in response to either Hox proteins, factors involved in hypoxia and myc-mediated apoptosis, and cell cycle checkpoint mediators were enriched. In a separate study, Hoxa4-transduced HSCs were readily expanded with little or no effect on the balance between lymphoid and myeloid progenitors. Bone marrow transplantation assays, however, confirmed a tendency for these cells to favor B-cell progenitors over other primitive lineages suggesting an additional role beyond unbiased expansion of the stem cell pool [52]. In fact, Hoxa4 was further shown to expand short-term repopulating HSCs to higher degrees compared to Hoxb4, suggesting positive effects for this gene during engraftment [52]. Despite the inductive effects of the group 4 paralog genes in the HSC compartment, mice lacking Hoxb4 were surprisingly viable with normal HSC numbers [53]. Moreover, cKit⁺ fetal liver cells from Hoxb4^−/− mice were similarly competitive in repopulation of irradiated bone marrow as their wild-type counterparts. While this could arise from the inherent redundancy in paralog Hox genes, expression of Hoxa4, c4, and d4 genes in cKit⁺ fetal liver cells of Hoxb4^−/− animals were either unaltered or showed a slight decrease suggesting a more complex cross-regulatory network among Hox proteins. Similar to Hox4 paralog genes, Hoxa9 overexpression in HSCs conferred enhanced growth advantages to chimeric bone marrows, evident by several folds increase in transplantable lympho-myeloid long-term repopulating cells [54]. Studies as well suggest a growth stimulatory role for Hoxa10 in the HSC compartment, where its overexpression leads to significantly enhanced repopulating capacity for these cells [55]. Moreover, the inductive effects of Hoxa10 were dose-dependent, suggesting a requirement for tightly controlled regulation of the gene. As discussed above, functional redundancy and high degree of cross-regulation between Hox genes does not allow to understand the role of individual genes in hematopoiesis through single gene knockout approaches. A study of complete Hoxa locus ablation however, demonstrated a significant role for the Hoxa cluster genes in long-term repopulating HSCs [56]. Mice heterozygous for the Hoxa locus (Hoxa^+/−), showed a general increase in primitive hematopoietic progenitors, a dramatic decrease in side population, exhaustion of B-cell progenitors with age, and accelerated maturation of myeloid progenitors, all indicating that Hoxa locus plays a significant role in definitive hematopoiesis.

Given the functional significance of Hox proteins during stem cell proliferation and expansion, it is well anticipated for any misregulation in the Hox network to potentially lead to neoplasia. In fact, aberrant expression of Hox proteins has been reported in a variety of cancers with functional roles during various stages of tumorigenesis and progression. Extensive studies have dissected the role of Hox genes in hematopoietic abnormalities, including leukemias and lymphomas [28,30,57]. There is also evidence for involvement of Hox genes in other types of cancer including renal carcinoma [58], esophageal adenocarcinoma [59], hepatocellular carcinoma [60], gastric carcinoma [61,62], and lung adenocarcinoma [60], to name a few. Evidence for changes in Hox profiles during carcinogenesis have been comprehensively reviewed before [63,64]. Based on the existing evidence however, we wanted to specifically look for domains of continuity in stem cell-specific Hox activity during homeostasis and neoplastic transformations and the downstream functional pathways involved.

Although limited, evidence of such Hox-mediated molecular bridges have been identified in a number of tissues. Gene expression analysis of resident colonic crypt stem cells and colon carcinomas, for example, confirmed HOXA4 and HOXD10 to be commonly expressed between the two compartments with an enhanced expression in colon carcinomas, suggesting a functional continuum between tissue-resident stem cell self-renewal/maintenance and cancer progression [65]. An MLL-induced HOXA10 expression within the glioblastoma stem cell domain has as well elaborated a stem cell-specific function, involving additional Hox genes downstream, that affected the tumorigenic properties of glioblastoma stem cells [66].

The exact functional network downstream of Hox proteins is yet to be determined. Studies, however, suggest that Hox genes directly communicate with the cell cycle and/or the metastatic machinery to induce tumor progression. In colorectal cancer, overexpression of HOXB7 upregulated the G1 phase CyclinD1 in the expense of cyclin-dependant kinase inhibitor P21^Kip1, hence accelerating growth and proliferation [67]. Same Hox gene was shown to induce markers of epithelial to mesenchymal transition, a central process for metastatic transformation of tumors, in human lung adenocarcinoma [68]. Studies focusing on hepatocellular carcinoma also suggest a second Cyclin/Cdk complex, CyclinE1/Cdk2, as a downstream effector of HOXA7, again resulting in tumor proliferation and enhanced clonogenicity [60]. The tumorigenic effects of Hox genes could as well be mediated through signaling pathways that globally affect cell proliferation. In gastric cancer, HOXB7 affected the PI3/Akt signaling pathway, hence causing malignant properties [61]. In a similar cancer model, HOXB5 induced invasion and metastasis through direct binding to β-Catenin, thus activating the canonical Wnt pathway, with clear proliferation-enhancing downstream effects such as induction of CyclinD1 and c-Myc [62]. In breast carcinoma, overexpression of HOXB7 upregulated FGF, and hence enhanced signaling through the FGF and the downstream mitogen-activated protein kinase pathways [69]. Likewise, expression of HOXA9 in acute myeloid leukemia coincided with upregulation of fms-related tyrosine kinase 3 (FLT-3) [70]. Hox effects could as well be mediated through pathways in dialogue with inflammatory response cues. In breast cancer cells, for example, overexpression of HOXB13 enhanced invasiveness through downregulation of estrogen receptor α and upregulation of interleukin 6 [71]. The favorable effects of Hox genes in the maintenance of the stem cell pool could as well be mediated through modulation of cell survival and apoptosis. In MCF10A breast cancer cells, HOXA5 was shown to act downstream of retinoic acid signaling, thus rendering the cells apoptotic when the pathway was active. Furthermore, loss of both HOXA5 and nuclear retinoic receptor β occurred during neoplastic transformation suggesting HOXA5 as an anticancer and chemoprevention target [71].

Overall, there is enough evidence to support a functional compartment for Hox genes within the continuum of stem cells during homeostasis and disease. Studies even suggest for certain Hox profiles to be potentially used as prognostic markers. Examples of such applications include cases of clear cell renal carcinoma [58], lung adenocarcinoma [68], and acute meyoeloid leukemia [72], where HOXC11, HOXB7, and HOXA9 could respectively serve as prognostic markers, expression of all relating to poor prognosis.

Conclusion

Hox genes are regarded as molecular determinants of early embryonic patterning with an extensive body of evidence supporting their role in primary coding of anteroposterior axial identity. In the course of morphogenesis, and in synchrony with formation of secondary and tertiary developmental axes, Hox genes adopt new and more refined functional domains along with new roles (Fig. 3A, B). Positional and tissue-specific identities of adult stem cells are at least partially encrypted by their Hox code suggesting a heritable temporal component that persists from early developmental stages all through adulthood. The Hox code, further affects differentiating compartments at various stages of lineage commitment, early differentiation, and maturation. Interestingly, the Hox signature determining every stage appears to be discrete, with negligible overlap. Further understanding of differential Hox codes, at different stages of differentiation and/or tissue specification, may allow targeted expansion of selected compartments, either as a solo trial with single/multiple Hox genes, or in combination with additional transcription or signaling factors.

Combined roles of Hox genes in stem cell self-renewal, positional identity, and the secondary functions in neoplastic transformations, might as well suggest a role for these genes as tracers of tissue of origin in cancer. Such applications require large-scale systematic analysis however, that is yet to be performed. Overall, expanding evidence supports functional roles for Hox genes at several levels of stem cell hierarchy, part of which is in continuum with their developmental domains of activity that renders them as a reliable resource in generation of functional genetic signatures. Realization of this aim however, requires systematic, high-throughput approaches and well-designed analytical tools to define Hox codes along the stem cell hierarchy during homeostasis and in disease.

Footnotes

Acknowledgments

The authors acknowledge Prof. Michael Kessel and his laboratory at the Max Planck Institute of Biophysical Chemistry, Goettingen-Germany, for kind provision of the Hoxb8 in situ hybridization image used in .

Author Disclosure Statement

No competing financial interests exist.

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