The DEK oncogene promotes cellular proliferation through paracrine Wnt signaling in Ron receptor-positive breast cancers.

Disease progression and recurrence are major barriers to survival for breast cancer patients. Understanding the etiology of recurrent or metastatic breast cancer and underlying mechanisms is critical for the development of new treatments and improved survival. Here, we report that two commonly overexpressed breast cancer oncogenes, Ron (Recepteur d'Origine Nantaise) and DEK, cooperate to promote advanced disease through multipronged effects on ß-catenin signaling. The Ron receptor is commonly activated in breast cancers, and Ron overexpression in human disease stimulates ß-catenin nuclear translocation and is an independent predictor of metastatic dissemination. Dek is a chromatin-associated oncogene whose expression has been linked to cancer through multiple mechanisms, including ß-catenin activity. We demonstrate here that Dek is a downstream target of Ron receptor activation in murine and human models. The absence of Dek in the MMTV-Ron mouse model led to a significant delay in tumor development, characterized by decreased cell proliferation, diminished metastasis and fewer cells expressing mammary cancer stem cell markers. Dek complementation of cell lines established from this model was sufficient to promote cellular growth and invasion. Mechanistically, Dek expression stimulated the production and secretion of Wnt ligands to sustain an autocrine/paracrine canonical ß-catenin signaling loop. Finally, we show that Dek overexpression promotes tumorigenic phenotypes in immortalized human mammary epithelial MCF10A cells and, in the context of Ron receptor activation, correlates with disease recurrence and metastasis in patients. Overall, our studies demonstrate that DEK overexpression, due in part to Ron receptor activation, drives breast cancer progression through the induction of Wnt/ß-catenin signaling.

The Ron receptor tyrosine kinase activates c-Abl to promote cell proliferation through tyrosine phosphorylation of PCNA in breast cancer.

Multiple growth pathways lead to enhanced proliferation in malignant cells. However, how the core machinery of DNA replication is regulated by growth signaling remains largely unclear. The sliding clamp proliferating cell nuclear antigen (PCNA) is an indispensable component of the DNA machinery responsible for replicating the genome and maintaining genomic integrity. We previously reported that epidermal growth factor receptor (EGFR) triggered tyrosine 211 (Y211) phosphorylation of PCNA, which in turn stabilized PCNA on chromatin to promote cell proliferation. Here we show that the phosphorylation can also be catalyzed by the non-receptor tyrosine kinase c-Abl. We further demonstrate that, in the absence of EGFR, signaling to PCNA can be attained through the activation of the Ron receptor tyrosine kinase and the downstream non-receptor tyrosine kinase c-Abl. We show that Ron and c-Abl form a complex, and that activation of Ron by its ligand, hepatocyte growth factor-like protein (HGFL), stimulates c-Abl kinase activity, which in turn directly phosphorylates PCNA at Y211 and leads to an increased level of chromatin-associated PCNA. Correspondingly, HGFL-induced Ron activation resulted in Y211 phosphorylation of PCNA while silencing of c-Abl blocked this effect. We show that c-Abl and Y211 phosphorylation of PCNA is an important axis downstream of Ron, which is required for cell proliferation. Treatment with a specific peptide that inhibits Y211 phosphorylation of PCNA or with the c-Abl pharmacological inhibitor imatinib suppressed HGFL-induced cell proliferation. Our findings identify the pathway of Ron-c-Abl-PCNA as a mechanism of oncogene-induced cell proliferation, with potentially important implications for development of combination therapy of breast cancer.

The Ron receptor promotes prostate tumor growth in the TRAMP mouse model.

The Ron receptor tyrosine kinase (TK) is overexpressed in many cancers, including prostate cancer. To examine the significance of Ron in prostate cancer in vivo, we utilized a genetically engineered mouse model, referred to as TRAMP mice, that is predisposed to develop prostate tumors. In this model, we show that prostate tumors from 30-week-old TRAMP mice have increased Ron expression compared with age-matched wild-type prostates. Based on the upregulation of Ron in human prostate cancers and in this murine model of prostate tumorigenesis, we hypothesized that this receptor has a functional role in the development of prostate tumors. To test this hypothesis, we crossed TRAMP mice with mice that are deficient in Ron signaling (TK-/-). Interestingly, TK-/- TRAMP+ mice show a significant decrease in prostate tumor mass relative to TRAMP mice containing functional Ron. Moreover, TK-/- TRAMP+ prostate tumors exhibited decreased tumor vascularization relative to TK+/+ TRAMP+ prostate tumors, which correlated with reduced levels of the angiogenic molecules vascular endothelial growth factor and CXCL2. Although Ron loss did not alter tumor cell proliferation, a significant decrease in cell survival was observed. Similarly, murine prostate cancer cell lines containing a Ron deficiency exhibited decreased levels of active nuclear factor-κB, suggesting that Ron may be important in regulating prostate cell survival at least partly through this pathway. In total, our data show for the first time that Ron promotes prostate tumor growth, prostate tumor angiogenesis and prostate cancer cell survival in vivo.

ß-Catenin is required for Ron receptor-induced mammary tumorigenesis.

Our previous studies demonstrated that selective overexpression of the Ron receptor tyrosine kinase in the murine mammary epithelium leads to mammary tumor formation. Biochemical analysis of mammary tumor lysates showed that Ron overexpression was associated with increases in ß-catenin expression and tyrosine phosphorylation. ß-Catenin has also been shown to be regulated through tyrosine phosphorylation by the receptor tyrosine kinases Met, Fer and Fyn. However, the molecular and physiological roles of ß-catenin and ß-catenin tyrosine phosphorylation downstream of Ron are not known. To investigate this association, we show that Ron and ß-catenin are coordinately elevated in human breast cancers. Our data also demonstrate that activation of Ron, through ligand binding by hepatocyte growth factor-like protein (HGFL), induces the tyrosine phosphorylation of ß-catenin, primarily on tyrosine residues Tyr 654 and Tyr 670. In addition, HGFL-mediated Ron activation induces both ß-catenin nuclear localization and transcriptional activity, with Tyr 654 and Tyr 670 residues of ß-catenin being critical for these processes. We also demonstrate that a knockdown of Ron in breast cancer cell lines leads to a loss of HGFL-induced ß-catenin-dependent transcriptional activation and cell growth, which can be rescued by activation of canonical Wnt/ß-catenin signaling. Moreover, we show that HGFL-dependent Ron activation mediates upregulation of the ß-catenin target genes cyclin D1 and c-myc, and that expression of these target genes in breast cancer cells is decreased following inhibition of Ron and/or ß-catenin. Finally, we show that genetic ablation of ß-catenin in Ron-expressing breast cancer cells decreases cellular proliferation in vitro, as well as mammary tumor growth and metastasis, following orthotopic transplantation into the mammary fat pad. Together, our data suggest that ß-catenin is a crucial downstream regulator of Ron receptor activation and is an important mediator of mammary tumorigenesis.

The human DEK oncogene stimulates ß-catenin signaling, invasion and mammosphere formation in breast cancer.

Breast cancer is a major cause of cancer-related deaths in American women; therefore, the identification of novel breast cancer-related molecules for the discovery of new markers and drug targets remains essential. The human DEK gene, which encodes a chromatin-binding protein and DNA topology regulator, is upregulated in many types of cancer. DEK has been implicated as an oncogene in breast cancer based on mRNA expression studies, but its functional significance in breast cancer growth and progression has not yet been tested directly. We demonstrate that DEK is highly expressed in breast cancer cells compared with normal tissue, and functionally important for cellular growth, invasion and mammosphere formation. DEK overexpression in non-tumorigenic MCF10A cells resulted in increased growth and motility, with a concomitant downregulation of E-cadherin. Conversely, DEK knockdown in MCF7 and MDA-MB-468 breast cancer cells resulted in decreased growth and motility with upregulation of E-cadherin. The use of DEK-proficient and -deficient breast cancer cells in orthotopic xenografts provided further in vivo evidence that DEK contributes to tumor growth. Activation of the ß-catenin signaling pathway is important for normal and cancer stem cell character, growth and metastasis. We show that DEK expression stimulated, and DEK knockdown repressed ß-catenin nuclear translocation and activity. Importantly, the expression of constitutively active ß-catenin rescued breast cancer invasion defects of DEK knockdown cells. Together, our data indicate that DEK expression stimulates the growth, stem cell character and motility of breast cancer cells, and that DEK-dependent cellular invasion occurs at least in part via ß-catenin activation.

The Ron receptor tyrosine kinase positively regulates angiogenic chemokine production in prostate cancer cells.

Overexpression of the Ron receptor tyrosine kinase has recently been shown in a wide variety of human cancers. However, no studies have examined Ron receptor expression or function during prostate tumorigenesis. In this study we report that Ron is highly expressed in human prostate adenocarcinoma and metastatic lymph nodes when compared with normal prostate or benign prostate hyperplasia. Furthermore, we show that Ron is overexpressed in PC-3 and DU145 prostate cancer cell lines, and that the levels of angiogenic chemokines produced by prostate cancer cells positively correlate with Ron expression. The knockdown of Ron in PC-3 or DU145 cells results in a significant decrease in angiogenic chemokine production and is associated with a decreased activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Moreover, exogenous overexpression of Ron in LNCaP cells is sufficient to induce a significant increase in angiogenic chemokines that can be abrogated by inhibition of NF-kappaB signaling. Given that the function of angiogenic chemokines is important in the development of new blood vessels, we also examined the ability of Ron to modulate endothelial cell migration. Our data show that knockdown of Ron in prostate cancer cells results in significantly less endothelial cell chemotaxis when compared with Ron-expressing cells in vitro as well as in reduced tumor growth and decreased microvessel density after orthotopic transplantation into the prostate in vivo. In total, our data suggest that the Ron receptor is important in modulating prostate tumor growth by modulating angiogenic chemokine production and subsequent endothelial cell recruitment.

Point mutations and overexpression of Ron induce transformation, tumor formation, and metastasis.

The receptor tyrosine kinase Ron is a member of the receptor family that includes the proto-oncogene Met and the avian oncogene Sea. The interaction of Ron with its ligand, known as hepatocyte growth factor-like protein (HGFL) or macrophage stimulating protein (MSP), induces crucial cellular responses including invasive growth, proliferation, cell scattering, and branching morphogenesis. Based on the homology and functional similarities between Met and Ron it was hypothesized that Ron may be important in tumor formation and metastasis. To test this hypothesis, wild-type mouse Ron and three mutant forms of Ron containing mutations similar to those found in the Met gene in human hereditary papillary renal carcinoma (HPRC), were expressed in NIH3T3 cells. A transformed phenotype was produced in cell lines expressing either wild-type Ron or the mutated Ron proteins. Further, these cell lines displayed oncogenic potential by exhibiting increased proliferation and constitutive phosphorylation of Ron. These cell lines were also tested for the ability to form solid tumors. Cells expressing wild-type Ron and the three proteins with single amino acid substitutions were highly tumorigenic in vivo. In a model of experimental metastasis, two of the cell lines with altered Ron protein formed highly aggressive tumors in the lungs. These results suggest that Ron may be an aggressive oncogene when either overexpressed or when activated by mutation.

Ron-mediated cytoplasmic signaling is dispensable for viability but is required to limit inflammatory responses.

Ron receptor activation induces numerous cellular responses in vitro, including proliferation, dissociation, and migration. Ron is thought to be involved in blood cell development in vivo, as well as in many aspects of the immune response including macrophage activation, antigen presentation, and nitric oxide regulation. In previous studies to determine the function of Ron in vivo, mice were generated with a targeted deletion of the extracellular and transmembrane regions of this gene. Mice homologous for this deletion appear to die early during embryonic development. To ascertain the in vivo function of Ron in more detail, we have generated mice with a germline ablation of the tyrosine kinase domain. Strikingly, our studies indicate that this domain of Ron, and therefore Ron cytoplasmic signaling, is not essential for embryonic development. While mice deficient in this domain are overtly normal, mice lacking Ron signaling have an altered ability to regulate nitric oxide levels and, in addition, have enhanced tissue damage following acute and cell-mediated inflammatory responses.

The Ron/STK receptor tyrosine kinase is essential for peri-implantation development in the mouse.

The Ron/STK receptor tyrosine kinase is a member of the c-Met family of receptors and is activated by hepatocyte growth factor-like protein (HGFL). Ron activation results in a variety of cellular responses in vitro, such as activation of macrophages, proliferation, migration, and invasion, suggesting a broad biologic role in vivo. Nevertheless, HGFL-deficient mice grow to adulthood with few appreciable phenotypic abnormalities. We report here that in striking contrast to the loss of its only known ligand, complete loss of Ron leads to early embryonic death. Embryos that are devoid of Ron (Ron-/-) are viable through the blastocyst stage of development but fail to survive past the peri-implantation period. In situ hybridization analysis demonstrates that Ron is expressed in the trophectoderm at embryonic day (E) 3.5 and is maintained in extraembryonic tissue through E7.5, compatible with an essential function at this stage of development. Hemizygous mice (Ron+/-) grow to adulthood; however, these mice are highly susceptible to endotoxic shock and appear to be compromised in their ability to downregulate nitric oxide production. These results demonstrate a novel role for Ron in early mouse development and suggest that Ron plays a limiting role in the inflammatory response.

Expression of hepatocyte growth factor-like protein is repressed by retinoic acid and enhanced by cyclic adenosine 3',5'-monophosphate response element-binding protein (CREB)-binding protein (CBP).

In an effort to understand the molecular mechanisms involved in the regulation of expression of the gene encoding hepatocyte growth factor-like protein (HGFL), it was found that all-trans-retinoic acid dramatically represses expression of the endogenous HGFL gene in HepG2 cells, a human hepatocyte-derived cell line. This repression requires the sequence between nucleotides -135 and -105 in the 5'-flanking sequence of the HGFL gene, a site that has previously been shown to bind the transcription factor hepatocyte nuclear factor-4 (HNF-4). Electrophoretic mobility shift analysis suggests that the retinoic acid receptor does not bind to this site, and that retinoic acid does not alter binding of HNF-4 to this DNA site. However, the transcriptional coactivator, CREB-binding protein (CBP) coactivates expression of this gene through an indirect interaction with the HNF-4-binding site, and overexpression of CBP in HepG2 cells eliminates retinoic acid repression of reporter gene expression driven by the HGFL promoter. Overexpression of CBP also protects the endogenous HGFL gene from down-regulation by retinoic acid. These results suggest that HGFL gene expression requires CBP, and competition for limiting amounts of CBP by retinoic acid receptor may be a means of modifying the activity of HNF-4 at the HGFL gene promoter.

Multiple initiations in the c-myc replication origin independent of chromosomal location.

At supramolecular resolution, DNA synthesis begins at preferred replication origins in the chromosomes of metazoan cells. To characterize one of these origins in detail, the initiation of replication was examined in the HeLa c-myc origin. Polymerase chain reaction (PCR) amplification of size-fractionated nascent chromosomal DNAs revealed multiple replication initiation sites over a 12-kb region spanning the c-myc origin, including the transcribed region and the 5' and 3' flanking DNA of the gene. Two of the start sites for chromosomal replication occurred inside a 2.4-kb region of the origin that exhibits autonomously replicating sequence (ARS) activity. When a plasmid containing the 2.4-kb ARS region was transfected into HeLa cells, PCR mapping of nascent plasmid DNA confirmed that the plasmid replicated semiconservatively and autonomously and that replication did not initiate at random sites but rather began at multiple sites in a limited zone overlapping the c-myc DNA insert. Within the resolution of the PCR assay, the same sites that were used in the chromosomal c-myc origin were used in the 2.4-kb ARS fragment. The locations of replication start sites determined by PCR are considered in the context of other functional and structural elements of the c-myc origin.

Characterization of the mouse Ron/Stk receptor tyrosine kinase gene.

In an effort to understand the mechanisms governing the regulation of the mouse Ron receptor gene, a mouse genomic library was screened and overlapping clones coding for the Ron gene and flanking DNA were identified. Continuous DNA sequence was obtained for approximately 16.4 kilobases. The gene, from the initiator methionine to the polyadenylation site, is contained within 13 244 basepairs and contains 19 exons. Primer extension analyses were performed to determine the transcription start site of the mouse Ron transcript. Multiple transcription start sites were found which also appear to be used in transfected reporter constructs containing Ron 5' flanking DNA. To determine the location of sites which may be critical for the function of the Ron gene promoter, a series of chimeric genes containing serial deletions of the Ron gene promoter fused to the coding sequences for the chloramphenicol acetyl-transferase gene were constructed. Transient transfection analyses of these hybrid genes into various cell lines demonstrated that two regions of the Ron gene promoter, encompassing nucleotides -585 to -465 and from -465 to -285, are important for expression of this transcript in CMT-93 cells. Further analysis of the Ron promoter utilizing gel mobility shift analyses suggests that regions encompassing nucleotides -585 to - 508 and nucleotides -375 to -285 appear to bind specific proteins which may be involved in the negative and positive regulation, respectively, of the mouse Ron gene.

Structure of the human D1F15S1A locus: a chromosome 1 locus with 97% identity to the chromosome 3 gene coding for hepatocyte growth factor-like protein.

The human chromosome 3 locus coding for hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL/MSP) is homologous to two sets of amplified loci on human chromosome 1 at 1p36. One copy of one of the amplified loci (D1F15S1A) has been further characterized by restriction enzyme and DNA sequence analysis. A total of 8331 bp of continuous sequence was determined for this locus. The first 6878 bp of sequence is 96.1% identical to the HGFL/MSP gene, while there is no homology between the two genes following nucleotide 6878. Based on the presence of a 5 bp deletion in putative exon 2 and several downstream stop codons it is very likely that this gene is a pseudogene. Screening of a human liver cDNA library with a chromosome 1-specific probe indicates that at least several other members of the chromosome 1 loci are transcribed.

Functional characterization of domains contained in hepatocyte growth factor-like protein.

To delineate the functional protein domains necessary for the biological activity of hepatocyte growth factor-like protein (HGFL), we created various site-directed and deletion mutated cDNAs coding for this protein. Wild-type and mutated versions of HGFL were produced after transfection of the corresponding cDNAs into tissue culture cells. The biological importance of the domains within HGFL was then examined by addition of recombinant wild-type or mutant forms of HGFL to assays aimed at elucidating regions involved in the stimulation of DNA synthesis, the induction of shape changes in macrophages, and the ability to stimulate cell scattering. Mutant proteins lacking the serine protease-like domain (light chain) were not biologically active in any of the assays tested and could not compete with wild-type HGFL in cell scattering experiments. These data, in addition to direct enzyme-linked immunosorbent assay analyses, suggest that the light chain may play an important role in the interaction of HGFL with its receptor, Ron. Elimination of the proposed protease cleavage site between the heavy and light chains (by mutation of Arg-483 to Glu) produced a protein with activity comparable to wild-type HGFL. Further studies with this mutated protein uncovered an additional proteolytic cleavage site that produces biologically active protein. Deletion of the various kringle domains or the amino-terminal hairpin loop had various effects in the multiple assays. These data suggest that the heavy chain may play a pivotal role in determining the functional aspects of HGFL.

DNA replication initiates non-randomly at multiple sites near the c-myc gene in HeLa cells.

The origin of replication of the c-myc gene in HeLa cells was previously identified at low resolution within 3.5 kb 5' to the P1 promoter, based on replication fork polarity and the location of DNA nascent strands. To define the initiation events in the c-myc origin at higher resolution the template bias of nascent DNAs in a 12 kb c-myc domain has been analyzed by hybridization to strand specific probes. Strong switches in the asymmetry of nascent strand template preference confirm that replication initiates non-randomly at multiple sites within 2.4 kb 5' to the c-myc P1 promoter, and at other sites over a region of 12 kb or more. The strongest template biases occur in the 2.4 kb region 5' of the P1 promoter, shown earlier to contain sequences which allow the autonomous semiconservative replication of c-myc plasmids. An asymmetric pyrimidine heptanucleotide consensus sequence has been identified which occurs 12 times in the c-myc origin zone, and whose polarity exactly correlates with the polarity of nascent strand synthesis.

Hepatocyte nuclear factor-4 is responsible for the liver-specific expression of the gene coding for hepatocyte growth factor-like protein.

In an attempt to understand the molecular mechanism regulating the expression of the gene coding for human hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL), our laboratory has isolated and characterized approximately 4200 bp of the 5'-flanking region of the HGFL gene. To determine the location of sites which may be critical for the function of the HGFL gene promoter, we constructed a series of hybrid genes containing serial deletions of this region attached to the coding sequences for chloramphenicol acetyltransferase. Expression of these chimeric plasmids was examined by transient transfection of HepG2 and 293 cells. Our results suggest that the transcriptional activity of the HGFL promoter is modulated in HepG2 cells by one positive element at position -135 to -105 (-135/-105). In contrast, only background levels of chloramphenicol acetyltransferase expression have been detected in 293 cells. The -135/-105 region appears to bind a liver-specific transcription factor essential for expression of this gene. Gel mobility shift experiments with antibodies against hepatocyte nuclear factor-4 (HNF-4) and transactivation of the HGFL promoter by a HNF-4 cDNA expression vector suggest that HNF-4 binds to the -135/-105 region and is responsible for the liver-specific expression of HGFL.

Cis-acting effects of sequences within 2.4-kb upstream of the human c-myc gene on autonomous plasmid replication in HeLa cells.

We have used density shift analysis to monitor the autonomous replicating sequence (ARS) activity of plasmids containing various DNA fragments from the 5'-flanking region of the human c-myc gene. The ARS activity of certain of these plasmids implied that structures in the c-myc DNA could be recognized for the initiation of replication in the absence of chromosomal integration. The plasmid pNeo.Myc-2.4 contains 2.4 contains 2.4 kb of c-myc 5'-flanking DNA, and replicated semiconservatively as a circular extrachromosomal element. Deletion derivatives of pNeo.Myc-2.4 containing either of two nonoverlapping regions of c-myc DNA semiconservatively incorporated bromodeoxyuridine into discrete populations of heavy-light supercoiled molecules to roughly the same extent as the chromosomal DNA in the same cultures. Some constructs displayed lower ARS activity, implying that distinct cis-acting sequences in the c-myc 5'-flanking DNA may independently affect DNA replication. The ARS activity of two separate c-myc sequences suggests that replication initiation signals are redundant in the c-myc origin. The smallest c-myc insert that displayed substantial ARS activity was 930 bp long and contained three 10/11 matches to the yeast ARS consensus and several additional features found in eukaryotic replication origins.