EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma.

PURPOSE: Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity. EXPERIMENTAL DESIGN: Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays. RESULTS: We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis. CONCLUSIONS: Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13. See related commentary by Weiss and Bailey, p. 4360.

The Lung Elastin Matrix Undergoes Rapid Degradation Upon Adult Loss of Hox5 Function.

Hox genes encode transcription factors that are critical for embryonic skeletal patterning and organogenesis. The Hoxa5, Hoxb5, and Hoxc5 paralogs are expressed in the lung mesenchyme and function redundantly during embryonic lung development. Conditional loss-of-function of these genes during postnatal stages leads to severe defects in alveologenesis, specifically in the generation of the elastin network, and animals display bronchopulmonary dysplasia (BPD) or BPD-like phenotype. Here we show the surprising results that mesenchyme-specific loss of Hox5 function at adult stages leads to rapid disruption of the mature elastin matrix, alveolar enlargement, and an emphysema-like phenotype. As the elastin matrix of the lung is considered highly stable, adult disruption of the matrix was not predicted. Just 2 weeks after deletion, adult Hox5 mutant animals show significant increases in alveolar space and changes in pulmonary function, including reduced elastance and increased compliance. Examination of the extracellular matrix (ECM) of adult Tbx4rtTA; TetOCre; Hox5a f a f bbcc lungs demonstrates a disruption of the elastin network although the underlying fibronectin, interstitial collagen and basement membrane appear unaffected. An influx of macrophages and increased matrix metalloproteinase 12 (MMP12) are observed in the distal lung 3 days after Hox5 deletion. In culture, fibroblasts from Hox5 mutant lungs exhibit reduced adhesion. These findings establish a novel role for Hox5 transcription factors as critical regulators of lung fibroblasts at adult homeostasis.

Squamous cell carcinoma subverts adjacent histologically normal epithelium to promote lateral invasion.

Recurrent and new tumors, attributed in part to lateral invasion, are frequent in squamous cell carcinomas and lead to poor survival. We identified a mechanism by which cancer subverts adjacent histologically normal epithelium to enable small clusters of cancer cells to burrow undetected under adjacent histologically normal epithelium. We show that suppression of DMBT1 within cancer promotes aggressive invasion and metastasis in vivo and is associated with metastasis in patients. Cancer cells via TGFß1 and TNFα also suppress DMBT1 in adjacent histologically normal epithelium, thereby subverting it to promote invasion of a small population of tumor cells. The sufficiency of DMBT1 in this process is demonstrated by significantly higher satellite tumor nests in Dmbt1-/- compared with wild-type mice. Moreover, in patients, invasion of small tumor nests under adjacent histologically normal epithelium is associated with increased risk for recurrence and shorter disease-free survival. This study demonstrates a crucial role of adjacent histologically normal epithelium in invasion and its important role in the tumor microenvironment and opens new possibilities for therapeutic strategies that reduce tumor recurrence.

Novel Lineage-Tracing System to Identify Site-Specific Ectopic Bone Precursor Cells.

Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; however, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and inducible lineage-tracing mouse (Hoxa11-CreERT2;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors specifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and adipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion.

Ovarian Cells Have Increased Proliferation in Response to Heparin-Binding Epidermal Growth Factor as Collagen Density Increases.

It is well known that during ovarian cancer progression, the omentum transforms from a thin lacy organ to a thick tougher tissue. However, the mechanisms regulating this transformation and the implications of the altered microenvironment on ovarian cancer progression remain unclear. To address these questions, the global and local concentrations of collagen I were determined for normal and metastatic human omentum. Collagen I was increased 5.3-fold in omenta from ovarian cancer patients and localized to areas of activated fibroblasts rather than regions with a high density of cancer cells. Transforming growth factor beta 1 (TGFß1) was detected in ascites from ovarian cancer patients (4 ng/mL), suggesting a potential role for TGFß1 in the observed increase in collagen. Treatment with TGFß1 induced fibroblast activation, proliferation, and collagen deposition in mouse omental explants and an in vitro model with human omental fibroblasts. Finally, the impact of increased collagen I on ovarian cancer cells was determined by examining proliferation on collagen I gels formulated to mimic normal and cancerous omenta. While collagen density alone had no impact on proliferation, a synergistic effect was observed with collagen density and heparin-binding epidermal growth factor treatment. These results suggest that TGFß1 induces collagen deposition from the resident fibroblasts in the omentum and that this altered microenvironment impacts cancer cell response to growth factors found in ascites. Impact statement Using quantitative analysis of patient samples, in vitro models of the metastatic ovarian cancer microenvironment were designed with pathologically relevant collagen densities and growth factor concentrations. Studies in these models support a mechanism where transforming growth factor ß1 in the ascites fluid induces omental fibroblast proliferation, activation, and deposition of collagen I, which then impacts tumor cell proliferation in response to additional ascites growth factors such as heparin-binding epidermal growth factor. This approach can be used to dissect mechanisms involved in microenvironmental modeling in multiple disease applications.

Differential Contribution of Pancreatic Fibroblast Subsets to the Pancreatic Cancer Stroma.

BACKGROUND & AIMS: Although the healthy pancreas consists mostly of epithelial cells, pancreatic cancer and the precursor lesions known as pancreatic intraepithelial neoplasia, are characterized by an extensive accumulation of fibroinflammatory stroma that includes a substantial and heterogeneous fibroblast population. The cellular origin of fibroblasts within the stroma has not been determined. Here, we show that the Gli1 and Hoxb6 markers label distinct fibroblast populations in the healthy mouse pancreas. We then set out to determine whether these distinct fibroblast populations expanded during carcinogenesis. METHODS: We developed genetically engineered models using a dual-recombinase approach that allowed us to induce pancreatic cancer formation through codon-optimized Flp recombinase-driven epithelial recombination of Kirsten rat sarcoma viral oncogene homolog while labeling Gli1+ or Hoxb6+ fibroblasts in an inducible manner. By using these models, we lineage-traced these 2 fibroblast populations during the process of carcinogenesis. RESULTS: Although in the healthy pancreas Gli1+ fibroblasts and Hoxb6+ fibroblasts are present in similar numbers, they contribute differently to the stroma in carcinogenesis. Namely, Gli1+ fibroblasts expand dramatically, whereas Hoxb6+ cells do not. CONCLUSIONS: Fibroblasts present in the healthy pancreas expand during carcinogenesis, but with a different prevalence for different subtypes. Here, we compared Gli1+ and Hoxb6+ fibroblasts and found only Gli1+ expanded to contribute to the stroma during pancreatic carcinogenesis.

Hox11 expressing regional skeletal stem cells are progenitors for osteoblasts, chondrocytes and adipocytes throughout life.

Multipotent mesenchymal stromal cells (MSCs) are required for skeletal formation, maintenance, and repair throughout life; however, current models posit that postnatally arising long-lived adult MSCs replace transient embryonic progenitor populations. We previously reported exclusive expression and function of the embryonic patterning transcription factor, Hoxa11, in adult skeletal progenitor-enriched MSCs. Here, using a newly generated Hoxa11-CreERT2 lineage-tracing system, we show Hoxa11-lineage marked cells give rise to all skeletal lineages throughout the life of the animal and persist as MSCs. Hoxa11 lineage-positive cells give rise to previously described progenitor-enriched MSC populations marked by LepR-Cre and Osx-CreER, placing them upstream of these populations. Our studies establish that Hox-expressing cells are skeletal stem cells that arise from the earliest stages of skeletal development and self-renew throughout the life of the animal.

Bone morphology is regulated modularly by global and regional genetic programs.

Bone protrusions provide stable anchoring sites for ligaments and tendons and define the unique morphology of each long bone. Despite their importance, the mechanism by which superstructures are patterned is unknown. Here, we identify components of the genetic program that control the patterning of Sox9+/Scx+ superstructure progenitors in mouse and show that this program includes both global and regional regulatory modules. Using light-sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9+/Scx+ progenitors to the formation of bone superstructures. Then, by combining literature-based evidence, comparative transcriptomic analysis and genetic mouse models, we identified Gli3 as a global regulator of superstructure patterning, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in a coordinated manner. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning, which further supports the notion that long bone development is a modular process.This article has an associated 'The people behind the papers' interview.

Anatomic Origin of Osteochondrogenic Progenitors Impacts Sensitivity to EWS-FLI1-Induced Transformation.

Ewing sarcomas predominantly arise in pelvic and stylopod bones (i.e., femur and humerus), likely as a consequence of EWS-FLI1 oncogene-induced transformation of mesenchymal stem/progenitor cells (MSCs). MSCs located in the embryonic superficial zone cells (eSZ) of limbs express anatomically distinct posterior Hox genes. Significantly, high expression of posterior HOXD genes, especially HOXD13, is a hallmark of Ewing sarcoma. These data drove our hypothesis that Hox genes in posterior skeleton MSCs contribute to Ewing sarcoma tumorigenesis. We isolated eSZ cells from stylopod and zeugopod (i.e., tibia/fibula, radius/ulna) bones, from wild-type and Hoxd13 mutant embryos, and tested the impact of EWS-FLI1 transduction on cell proliferation, gene expression, and tumorigenicity. Our data demonstrate that both stylopod and zeugopod eSZ cells tolerate EWS-FLI1 but that stylopod eSZ cells are relatively more susceptible, demonstrating changes in proliferation and gene expression consistent with initiation of malignant transformation. Significantly, loss of Hoxd13 had no impact, showing that it is dispensable for the initiation of EWS-FLI1-induced transformation in mouse MSCs. These findings show that MSCs from anatomically distinct sites are differentially susceptible to EWS-FLI1-induced transformation, supporting the premise that the dominant presentation of Ewing sarcoma in pelvic and stylopod bones is attributable to anatomically-defined differences in MSCs.

Hox genes in the adult skeleton: Novel functions beyond embryonic development.

Hox genes encode evolutionarily conserved transcription factors that control skeletal patterning in the developing embryo. They are expressed in regionally restricted domains and function to regulate the morphology of specific vertebral and long bone elements. Recent work has provided evidence that Hox genes continue to be regionally expressed in adult tissues. Fibroblasts cultured from adult tissues show broadly maintained Hox gene expression patterns. In the adult skeleton, Hox genes are expressed in progenitor-enriched populations of mesenchymal stem/stromal cells (MSCs), and genetic loss-of-function analyses have provided evidence that Hox genes function during the fracture healing process. This review will highlight our current understanding of Hox expression in the adult animal and its function in skeletal regeneration. Developmental Dynamics 246:310-317, 2017. © 2016 Wiley Periodicals, Inc.

Regionally Restricted Hox Function in Adult Bone Marrow Multipotent Mesenchymal Stem/Stromal Cells.

Posterior Hox genes (Hox9-13) are critical for patterning the limb skeleton along the proximodistal axis during embryonic development. Here we show that Hox11 paralogous genes, which developmentally pattern the zeugopod (radius/ulna and tibia/fibula), remain regionally expressed in the adult skeleton. Using Hoxa11EGFP reporter mice, we demonstrate expression exclusively in multipotent mesenchymal stromal cells (MSCs) in the bone marrow of the adult zeugopod. Hox-positive cells express PDGFRα and CD51, are marked by LepR-Cre, and exhibit colony-forming unit fibroblast activity and tri-lineage differentiation in vitro. Loss of Hox11 function leads to fracture repair defects, including reduced cartilage formation and delayed ossification. Hox mutant cells are defective in osteoblastic and chondrogenic differentiation in tri-lineage differentiation experiments, and these defects are zeugopod specific. In the stylopod (humerus and femur) and sternum, bone marrow MSCs express other regionally restricted Hox genes, and femur fractures heal normally in Hox11 mutants. Together, our data support regional Hox expression and function in skeletal MSCs.

In vivo structural and cellular remodeling of engineered bone-ligament-bone constructs used for anterior cruciate ligament reconstruction in sheep.

Anterior cruciate ligament (ACL) ruptures rank among the most prevalent and costly sports-related injuries. Current tendon grafts used for ACL reconstruction are limited by suboptimal biomechanical properties. We have addressed these issues by engineering multiphasic bone-ligament-bone (BLB) constructs that develop structural and mechanical properties similar to native ACL. The purpose of this study was to examine the acute remodeling process that occurs as the BLB grafts advance toward the adult ligament phenotype in vivo. Thus, we implanted BLB constructs fabricated from male cells into female host sheep and allowed 3, 7, 14, or 28 days (n = 4 at each time point) for recovery. To address whether or not graft-derived cells were even necessary, a subset of BLB constructs (n = 3) were acellularized, implanted, and allowed 28 days for recovery. At each recovery time point, the following histological analyses were performed: picrosirius red staining to assess collagen alignment and immunohistochemistry to assess both graft development and host immune response. Polymerase chain reaction (PCR) analysis, performed on every explanted BLB, was used to detect the presence of graft-derived male cells remaining in the constructs and/or migration into surrounding host tissue. The analysis of the PCR and histology samples revealed a rapid migration of host-derived macrophages and neutrophils into the graft at 3 days, followed by increased collagen density and alignment, vascularization, innervation, and near complete repopulation of the graft with host cells within 28 days. This study provides a greater understanding of the processes of ligament regeneration in our BLB constructs as they remodel toward the adult ligament phenotype.

Hox5 interacts with Plzf to restrict Shh expression in the developing forelimb.

To date, only the five most posterior groups of Hox genes, Hox9-Hox13, have demonstrated loss-of-function roles in limb patterning. Individual paralog groups control proximodistal patterning of the limb skeletal elements. Hox9 genes also initiate the onset of Hand2 expression in the posterior forelimb compartment, and collectively, the posterior HoxA/D genes maintain posterior Sonic Hedgehog (Shh) expression. Here we show that an anterior Hox paralog group, Hox5, is required for forelimb anterior patterning. Deletion of all three Hox5 genes (Hoxa5, Hoxb5, and Hoxc5) leads to anterior forelimb defects resulting from derepression of Shh expression. The phenotype requires the loss of all three Hox5 genes, demonstrating the high level of redundancy in this Hox paralogous group. Further analyses reveal that Hox5 interacts with promyelocytic leukemia zinc finger biochemically and genetically to restrict Shh expression. These findings, along with previous reports showing that point mutations in the Shh limb enhancer lead to similar anterior limb defects, highlight the importance of Shh repression for proper patterning of the vertebrate limb.

Sox9 plays multiple roles in the lung epithelium during branching morphogenesis.

Lung branching morphogenesis is a highly orchestrated process that gives rise to the complex network of gas-exchanging units in the adult lung. Intricate regulation of signaling pathways, transcription factors, and epithelial-mesenchymal cross-talk are critical to ensuring branching morphogenesis occurs properly. Here, we describe a role for the transcription factor Sox9 during lung branching morphogenesis. Sox9 is expressed at the distal tips of the branching epithelium in a highly dynamic manner as branching occurs and is down-regulated starting at embryonic day 16.5, concurrent with the onset of terminal differentiation of type 1 and type 2 alveolar cells. Using epithelial-specific genetic loss- and gain-of-function approaches, our results demonstrate that Sox9 controls multiple aspects of lung branching. Fine regulation of Sox9 levels is required to balance proliferation and differentiation of epithelial tip progenitor cells, and loss of Sox9 leads to direct and indirect cellular defects including extracellular matrix defects, cytoskeletal disorganization, and aberrant epithelial movement. Our evidence shows that unlike other endoderm-derived epithelial tissues, such as the intestine, Wnt/ß-catenin signaling does not regulate Sox9 expression in the lung. We conclude that Sox9 collectively promotes proper branching morphogenesis by controlling the balance between proliferation and differentiation and regulating the extracellular matrix.

The role of Pax2 in mouse prostate development.

BACKGROUND: Loss-of-function of Pax2 results in severe defects of the male reproductive system, and Pax2 expression is detected in mouse prostate lobes and human prostatic cancers. However, the role for Pax2 in prostate development remains poorly understood. METHODS: The expression of Pax2 was examined by in situ hybridization at various developmental stages. Urogenital sinuses were dissected out at E18.5 from mouse Pax2 mutants and controls, cultured in vitro or grafted under the renal capsule of CD1 nude mice. The expression of prostate developmental regulatory factors was analyzed by semi-quantitative real-time PCR or immuohistochemistry. RESULTS: Pax2 is expressed in the epithelial cells of prostate buds. Loss-of-function of Pax2 does not affect the initiation of prostatic buds, but in vitro culture assays show that the prostates of Pax2 mutants are hypomorphic and branching is severely disrupted compared to controls. RT-PCR data from Pax2 mutant prostates demonstrate increased expression levels of dorsolateral prostate marker MSMB and ventral prostate marker SBP and dramatically reduced expression levels of anterior prostate marker TGM4. CONCLUSIONS: Pax2 is essential for mouse prostate development and regulates prostatic ductal growth, branching, and lobe-specific identity. These findings are important for understanding the molecular regulatory mechanisms in prostate development.

Axial Hox9 activity establishes the posterior field in the developing forelimb.

Current models hold that the early limb field becomes polarized into anterior and posterior domains by the opposing activities of Hand2 and Gli3. This polarization is essential for the initiation of Shh expression in the posterior margin of the limb bud, but how this polarity is established is not clear. Here we show that initial anteroposterior polarization of the early forelimb field requires the function of all four Hox9 paralogs (Hoxa9, Hoxb9, Hoxc9, and Hoxd9). This is unexpected, given that only HoxA and HoxD AbdB group genes have been shown to play a role in forelimb patterning, regulating the activation and maintenance of Shh expression and subsequent proximal-distal patterning of the forelimb. Our analysis of Hox9 quadruple mutants demonstrates that Hox9 function is required for the expression of Hand2 in the posterior limb field. Subsequently, Gli3 expression is not repressed posteriorly, Shh expression is not initiated, and collinear expression of HoxA/D10-13 is not established, resulting in severely malformed forelimbs lacking all posterior, Shh-regulated elements. This Hox9 mutant phenotype is restricted to the forelimbs; mutant hindlimbs are normal, revealing fundamental differences in the patterning mechanisms governing the establishment of forelimb and hindlimb fields.

Hox11 paralogous genes are required for formation of wrist and ankle joints and articular surface organization.

Limb skeletal elements are connected by distinct synovial joints, but the mechanisms regulating joint formation, diversity, and organization remain unclear. Previous studies showed that Hox11 mouse mutants have severe developmental defects in radius and ulna and tibia and fibula, but wrist and ankle joint formation and characteristics were not examined in detail. We now find that E11.5 and E12.5 triple Hox11aaccdd mutants exhibit a significant reduction in prospective carpal and tarsal mesenchyme. Although the mesenchyme became segmented into individual carpal and tarsal skeletal elements with further development, the elements were ill defined and the more proximal elements (radiale, ulnare, talus, and calcaneous) actually underwent involution and/or fusion. Wild-type carpal and tarsal elements displayed a thick articulating superficial zone at their outer perimeter that expressed genes typical of developing joint interzones and articulating cells, including Gdf5, Erg, Gli3, collagen IIA, and lubricin, and defined each element anatomically. In mutant wrists and ankles, the superficial zone around each element was thin and ill defined, and expression of several of those genes was low and often interrupted. These and other data provide novel and clear evidence that Hox11 paralogous genes regulate wrist and ankle joint organization and are essential for establishing carpal and tarsal element boundary and maintaining their articulating surface tissue.

Hox10 and Hox11 genes are required to globally pattern the mammalian skeleton.

Mice in which all members of the Hox10 or Hox11 paralogous group are disrupted provide evidence that these Hox genes are involved in global patterning of the axial and appendicular skeleton. In the absence of Hox10 function, no lumbar vertebrae are formed. Instead, ribs project from all posterior vertebrae, extending caudally from the last thoracic vertebrae to beyond the sacral region. In the absence of Hox11 function, sacral vertebrae are not formed and instead these vertebrae assume a lumbar identity. The redundancy among these paralogous family members is so great that this global aspect of Hox patterning is not apparent in mice that are mutant for five of the six paralogous alleles.

Hox11 paralogous genes are essential for metanephric kidney induction.

The mammalian Hox complex is divided into four linkage groups containing 13 sets of paralogous genes. These paralogous genes have retained functional redundancy during evolution. For this reason, loss of only one or two Hox genes within a paralogous group often results in incompletely penetrant phenotypes which are difficult to interpret by molecular analysis. For example, mice individually mutant for Hoxa11 or Hoxd11 show no discernible kidney abnormalities. Hoxa11/Hoxd11 double mutants, however, demonstrate hypoplasia of the kidneys. As described in this study, removal of the last Hox11 paralogous member, Hoxc11, results in the complete loss of metanephric kidney induction. In these triple mutants, the metanephric blastema condenses, and expression of early patterning genes, Pax2 and Wt1, is unperturbed. Eya1 expression is also intact. Six2 expression, however, is absent, as is expression of the inducing growth factor, Gdnf. In the absence of Gdnf, ureteric bud formation is not initiated. Molecular analysis of this phenotype demonstrates that Hox11 control of early metanephric induction is accomplished by the interaction of Hox11 genes with the pax-eya-six regulatory cascade, a pathway that may be used by Hox genes more generally for the induction of multiple structures along the anteroposterior axis.