Jeffrey S. Rubin

Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast.

The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-β and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives.

Secreted Frizzled-related Protein-1 Binds Directly to Wingless and Is a Biphasic Modulator of Wnt Signaling

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields ∼3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.

Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium.

Epithelial-mesenchymal interactions mediate aspects of normal lung growth and development and are important in the restoration of normal alveolar architecture after lung injury. To determine if fibroblasts are a source of soluble growth factors for alveolar type II cells, we investigated the effect of fibroblast-conditioned medium (CM) on alveolar type II cell DNA synthesis. Serum-free CM from confluent adult human lung fibroblasts was concentrated fivefold by lyophilization. Type II cells were isolated from adult rats by elastase dissociation and incubated with [3H]thymidine and varying dilutions of concentrated CM and serum from day 1 to 3 of culture. Stimulation of type II cell DNA synthesis by fibroblast-CM was maximal after 48 h of conditioning and required the presence of serum. The activity of the CM was eliminated by boiling and by treatment with trypsin, pepsin, or dithiothreitol and was additive with saturating concentrations of acidic fibroblast growth factor, epidermal growth factor, and insulin. The growth factor activity bound to heparin-Sepharose and was eluted with 0.6 and 1.0 M NaCl. Neutralizing antibody studies demonstrated that the primary mitogens isolated in the 0.6 and 1.0 M NaCl fractions were keratinocyte growth factor (KGF, fibroblast growth factor 7) and hepatocyte growth factor/scatter factor (HGF/SF), respectively. HGF/SF was demonstrated in the crude CM and KGF was detected in the 0.6 M NaCl eluent by immunoblotting. Northern blot analysis confirmed that the lung fibroblasts expressed both KGF and HGF/SF transcripts. Human recombinant KGF and HGF/SF induced a concentration- and serum-dependent increase in rat alveolar type II cell DNA synthesis. We conclude that adult human lung fibroblasts produce at least two soluble heparin-binding growth factors, KGF and HGF/SF, which promote DNA synthesis and proliferation of rat alveolar type II cells in primary culture. KGF and HGF/SF may be important stimuli for alveolar type II cell proliferation during lung growth and after lung injury.

Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats.

Alveolar type II cell proliferation occurs after many forms of lung injury and is thought to play a critical role in alveolar epithelial repair. Keratinocyte growth factor/fibroblast growth factor 7 (KGF) has been shown to promote alveolar type II cell growth in primary culture and alveolar epithelial hyperplasia in vivo. In this study, we used immunohistochemical analysis to determine the intrapulmonary distribution and cellular localization of recombinant human KGF (rhKGF) instilled into the trachea of rats. 6 h after administration, immunoreactive KGF was observed within the lung parenchyma and along alveolar epithelial cell membranes. By 18-24 h, KGF was detected intracellularly in alveolar epithelial cells and intraalveolar macrophages. Immunoreactive KGF was not demonstrable 48 h after delivery or in lung sections from PBS-treated animals. Intratracheal instillation of 5 mg/kg rhKGF stimulated a marked, time-dependent increase in the alveolar type II cell specific labeling index to a maximum level of 33 +/- 3% 48 h after rhKGF administration compared with 1.3 +/- 0.3% after PBS instillation. In addition, this increase in type II cell proliferation in vivo was documented by flow cytometric analysis of isolated type II cells which revealed a nearly fivefold increase in the proportion of cells traversing through the S and G2/M phases of the cell cycle. To test the hypothesis that KGFs effects on type II cells in vivo might affect the response to lung injury, rats were treated with rhKGF and exposed to hyperoxia. Animals that received 1 or 5 mg/kg rhKGF exhibited dramatically reduced mortality (P < 0.001, for both doses). Survival for animals treated with 0.1 mg/kg rhKGF was not significantly different from either untreated rats or animals treated with heat-denatured rhKGF. The lungs of rhKGF-treated animals that survived hyperoxia exposure had minimal hemorrhage and no exudate within the intraalveolar space. These experiments established that intratracheal administration of rhKGF stimulated alveolar type II cell proliferation in vivo and reduced hyperoxia-induced lung injury in rats. Directed delivery of KGF to the lungs may provide a therapeutic strategy to preserve or restore the alveolar epithelium during exposure to hyperoxia or other injurious agents.

Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea.

In the mammalian cochlea, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are unidirectionally oriented. Development of this planar polarity is necessary for normal hearing as stereociliary bundles are only sensitive to vibrations in a single plane; however, the mechanisms governing their orientation are unknown. We report that Wnt signaling regulates the development of unidirectional stereociliary bundle orientation. In vitro application of Wnt7a protein or inhibitors of Wnt signaling, secreted Frizzled-related protein 1 or Wnt inhibitory factor 1, disrupts bundle orientation. Moreover, Wnt7a is expressed in a pattern consistent with a role in the polarization of the developing stereociliary bundles. We propose that Wnt signaling across the region of developing outer hair cells gives rise to planar polarity in the mammalian cochlea.

Wnt signaling in B-cell neoplasia

Wnts comprise a family of secreted proteins that interact with receptors consisting of a Frizzled (Fz) family member alone or complexed with LDL receptor-related proteins (LRP5/6). Wnt signaling plays a crucial role in both development and differentiation, and activation of a ‘canonical’ Wnt pathway resulting in β-catenin stabilization is associated with several types of human cancers. To date, little is known about potential Wnt signaling in mature lymphocytes or lymphoid neoplasia. Herein, we have analysed Wnt signaling in mature B cells (lymphomas) and plasma cells (multiple myeloma). Both Fz and LRP5/6 mRNAs were expressed in myeloma lines, but LRP5/6 were not observed in lymphomas. In myelomas, a canonical Wnt signaling pathway was activated following treatment with Wnt-3a as assessed by accumulation of β-catenin, but β-catenin levels actually decreased in lymphoma cells. Wnt-3a treatment further led to striking morphological changes in myeloma cells accompanied by rearrangement of the actin cytoskeleton. Morphological changes were associated with a second Wnt pathway dependent on Rho activation. These results suggest that Wnt responsiveness is a stage-specific phenomenon in B-cell development and that the morphological changes associated with Wnt signaling may play a role in the motility and metastatic potential of myeloma cells.

Identification of SFRP1 as a Candidate Mediator of Stromal-to-Epithelial Signaling in Prostate Cancer

Genetic changes in epithelial cells initiate the development of prostatic adenocarcinomas. As nascent tumors grow and undergo progression, epithelial tumor cells are intimately associated with stromal cells. Stromal cells within the tumor microenvironment acquire new properties, including the capacity to promote phenotypic and genetic progression in adjacent epithelial cells. Affymetrix microarrays were used to identify 119 genes differentially expressed between normal-derived and carcinoma-derived prostatic stromal cells. These included 31 genes encoding extracellular proteins that may act as stromal-to-epithelial paracrine signals. Further investigation of one of these genes, secreted frizzled related protein 1 (SFRP1), revealed that its expression parallels prostatic growth with high expression during prostatic development, low expression in the adult prostate, and elevated expression in prostatic tumor stroma. In addition, as prostatic epithelial cells progressed to a tumorigenic state under the influence of tumor stroma, SFRP1 became overexpressed in the progressed epithelial cells. To further understand the roles of SFRP1 in the prostate, we tested the affects of increased SFRP1 levels on prostatic tissues and cells. Treatment of developing prostates with SFRP1 in culture led to increased organ growth. Treatment of a human prostatic epithelial cell line with SFRP1 led to increased proliferation, decreased apoptosis, and decreased signaling through the Wnt/beta-catenin pathway in vitro and increased proliferation in vivo. These data suggest that overexpression of SFRP1 by prostatic tumor stroma may account for the previously reported capacity of prostatic tumor stroma to provide a pro-proliferative paracrine signal to adjacent epithelial cells.

Metalloproteinase inhibitor TIMP‐1 affects hepatocyte cell cycle via HGF activation in murine liver regeneration

Liver regeneration depends on timely restoration of cellular mass while orchestrating structural matrix remodeling. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the extracellular matrix (ECM) turnover and, more recently, the processing of growth factors and cytokines. We have previously demonstrated that TIMP‐1 inhibits preneoplastic hepatocyte proliferation by attenuating growth factor bioavailability. In the present study, we examined the role of TIMP‐1 in de novo hepatocyte cell division during liver regeneration. Comprehensive real‐time reverse‐transcriptase polymerase chain reaction analyses of regenerating livers revealed significant inductions in the messenger RNA of TIMP‐1, TIMP‐3, TIMP‐4, MMP‐2, MMP‐9, MMP‐13, MMP‐14, and MMP‐24, while MMP‐15 expression was significantly reduced. Induction of TIMP‐1 occurred during the peak of hepatocyte DNA synthesis. Studies using genetically altered mice revealed that TIMP‐1 loss of function accelerated hepatocyte cell cycle progression. This finding was demonstrated by earlier expression of cyclin D1, proliferating cell nuclear antigen, and phosphorylated histone H3, which mark the G1‐S, S, and M phase, respectively. Conversely, TIMP‐1 gain of function delayed cell cycle progression. MMP activity was increased in the absence of Timp‐1. Examination of hepatocyte growth factor (HGF), and its receptor Met, both of which provide a mitogenic signal for hepatocyte division, showed increased HGF activity in Timp‐1−/−–regenerating livers. HGF is released from the ECM and is proteolytically processed to its active form. Active HGF was elevated in Timp‐1−/− mice, leading to increased immunostaining of phosphorylated Met as well as activation of a downstream effector, p38. In conclusion, TIMP‐1 is a novel negative regulator of HGF activity during liver regeneration. (HEPATOLOGY 2005.)

GLI1 Is a Direct Transcriptional Target of EWS-FLI1 Oncoprotein

Ewing sarcoma family of tumors (ESFT) is an undifferentiated neoplasm of the bone and soft tissue. ESFT is characterized by a specific chromosomal translocation occurring between chromosome 22 and (in most cases) chromosome 11, which generates an aberrant transcription factor, EWS-FLI1. The function of EWS-FLI1 is essential for the maintenance of ESFT cell survival and tumorigenesis. The Hedgehog pathway is activated in several cancers. Oncogenic potential of the Hedgehog pathway is mediated by increasing the activity of the GLI family of transcription factors. Recent evidence suggests that EWS-FLI1 increases expression of GLI1 by an unknown mechanism. Our data from chromatin immunoprecipitation and promoter reporter studies indicated GLI1 as a direct transcriptional target of EWS-FLI1. Expression of EWS-FLI1 in non-ESFT cells increased GLI1 expression and GLI-dependent transcription. We also detected high levels of GLI1 protein in ESFT cell lines. Pharmacological inhibition of GLI1 protein function decreased proliferation and soft agar colony formation of ESFT cells. Our results establish GLI1 as a direct transcriptional target of EWS-FLI1 and suggest a potential role for GLI1 in ESFT tumorigenesis.

Secreted Frizzled-related proteins can regulate metanephric development.

Wnt-4 signaling plays a critical role in kidney development and is associated with the epithelial conversion of the metanephric mesenchyme. Furthermore, secreted Frizzled-related proteins (sFRPs) that can bind Wnts are normally expressed in the developing metanephros, and function in other systems as modulators of Wnt signaling. sfrp-1 is distributed throughout the medullary and cortical stroma in the metanephros, but is absent from condensed mesenchyme and primitive tubular epithelia of the developing nephron where wnt-4 is highly expressed. In contrast, sfrp-2 is expressed in primitive tubules. To determine their role in kidney development, recombinant sFRP-1, sFRP-2 or combinations of both were applied to cultures of 13-dpc rat metanephroi. Both tubule formation and bud branching were markedly inhibited by sFRP-1, but concurrent sFRP-2 treatment restored some tubular differentiation and bud branching. sFRP-2 itself showed no effect on cultures of metanephroi. In cultures of isolated, induced rat metanephric mesenchymes, sFRP-1 blocked events associated with epithelial conversion (tubulogenesis and expression of lim-1, sfrp-2 and E-cadherin); however, it had no demonstrable effect on early events (compaction of mesenchyme and expression of wt1). As shown herein, sFRP-1 binds Wnt-4 with considerable avidity and inhibits the DNA-binding activity of TCF, an effector of Wnt signaling, while sFRP-2 had no effect on TCF activation. These observations suggest that sFRP-1 and sFRP-2 compete locally to regulate Wnt signaling during renal organogenesis. The antagonistic effect of sFRP-1 may be important either in preventing inappropriate development within differentiated areas of the medulla or in maintaining a population of cortical blastemal cells to facilitate further renal expansion. On the other hand, sFRP-2 might promote tubule formation by permitting Wnt-4 signaling in the presence of sFRP-1.