Overexpression of ERBB4 JM-a CYT-1 and CYT-2 isoforms in transgenic mice reveals isoform-specific roles in mammary gland development and carcinogenesis.

INTRODUCTION: Human Epidermal Growth Factor Receptor (ERBB4/HER4) belongs to the Epidermal Growth Factor receptor/ERBB family of receptor tyrosine kinases. While ERBB1, ERBB2 and ERBB3 are often overexpressed or activated in breast cancer, and are oncogenic, the role of ERBB4 in breast cancer is uncertain. Some studies suggest a tumor suppressor role of ERBB4, while other reports suggest an oncogenic potential. Alternative splicing of ERBB4 yields four major protein products, these spliced isoforms differ in the extracellular juxtamembrane domain (JM-a versus JM-b) and cytoplasmic domain (CYT-1 versus CYT-2). Two of these isoforms, JM-a CYT-1 and JM-a CYT-2, are expressed in the mammary gland. Failure to account for isoform-specific functions in previous studies may account for conflicting reports on the role of ERBB4 in breast cancer. METHODS: We have produced mouse mammary tumour virus (MMTV) -ERBB4 transgenic mice to evaluate potential developmental and carcinogenic changes associated with full length (FL) JM-a ERBB4 CYT-1 versus ERBB4 CYT-2. Mammary tissue was isolated from transgenic mice and sibling controls at various developmental stages for whole mount analysis, RNA extraction, and immunohistochemistry. To maintain maximal ERBB4 expression, transgenic mice were bred continuously for a year after which mammary glands were isolated and analyzed. RESULTS: Overexpressing FL CYT-1 isoform resulted in suppression of mammary ductal morphogenesis which was accompanied by decreased number of mammary terminal end buds (TEBs) and Ki-67 positive cells within TEBs, while FL CYT-2 isoform had no effect on ductal growth in pubescent mice. The suppressive ductal phenotype in CYT-1 mice disappeared after mid-pregnancy, and subsequent developmental stages showed no abnormality in mammary gland morphology or function in CYT-1 or CYT-2 transgenic mice. However, sustained expression of FL CYT-1 isoform resulted in formation of neoplastic mammary lesions, suggesting a potential oncogenic function for this isoform. CONCLUSIONS: Together, we present isoform-specific roles of ERBB4 during puberty and early pregnancy, and reveal a novel oncogenic property of CYT-1 ERBB4. The results may be exploited to develop better therapeutic strategies in breast cancer.

Convergent and divergent cellular responses by ErbB4 isoforms in mammary epithelial cells.

UNLABELLED: Associations of ErbB4 (ERBB4/HER4), the fourth member of the EGFR family, with cancer are variable, possibly as a result of structural diversity of this receptor. There are multiple structural isoforms of ERBB4 arising by alternative mRNA splicing, and a subset undergo proteolysis that releases membrane-anchored and soluble isoforms that associate with transcription factors and coregulators to modulate transcription. To compare the differential and common signaling activities of full-length (FL) and soluble intracellular isoforms of ERBB4, four JM-a isoforms (FL and soluble intracellular domain (ICD) CYT-1 and CYT-2) were expressed in isogenic MCF10A cells and their biologic activities were analyzed. Both FL and ICD CYT-2 promoted cell proliferation and invasion, and CYT-1 suppressed cell growth. Transcriptional profiling revealed several new and underexplored ERBB4-regulated transcripts, including: proteases/protease inhibitors (MMP3 and SERPINE2), the YAP/Hippo pathway (CTGF, CYR61, and SPARC), the mevalonate/cholesterol pathway (HMGCR, HMGCS1, LDLR, and DHCR7), and cytokines (IL8, CCL20, and CXCL1). Many of these transcripts were subsequently validated in a luminal breast cancer cell line that normally expresses ERBB4. Furthermore, ChIP-seq experiments identified ADAP1, APOE, SPARC, STMN1, and MXD1 as novel molecular targets of ERBB4. These findings clarify the diverse biologic activities of ERBB4 isoforms, and reveal new and divergent functions. IMPLICATIONS: ErbB4 as a regulator of Hippo and mevalonate pathways provides new insight into milk production and anabolic processes in normal mammary epithelia and cancer.

Interactions of ErbB4 and Kap1 connect the growth factor and DNA damage response pathways.

ErbB4 is unusual among receptor tyrosine kinases because some isoforms can be efficiently cleaved at the plasma membrane to release a soluble intracellular domain. The cleavage product has high kinase activity and homes to the nucleus. A screen for proteins that associate with the ErbB4 intracellular domain identified candidate interactors including ITCH, WWP2, Nucleolin, and Krab-associated protein 1 (Kap1). Kap1 binds to multiple isoforms of ErbB4 but does not require ErbB4 kinase activity for binding, nor is it an ErbB4 substrate. Kap1 reduces ERBB4 transcription and either directly or indirectly modulates the expression of genes that are themselves regulated by ErbB4. Upregulation of ErbB4 and suppression of MDM2 jointly enhance and accelerate the accumulation of p21(CIP1) in response to DNA damage. Overall, these findings further substantiate the role of ErbB4 in conjoint regulation of growth factor signaling and DNA damage responses.

The multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer.

Although glyceraldehyde-3-phosphate dehydrogenase's (GAPDH) predilection for AU-rich elements has long been known, the expected connection between GAPDH and control of mRNA stability has never been made. Recently, we described GAPDH binding the AU-rich terminal 144 nt of the colony-stimulating factor-1 (CSF-1) 3' untranslated region (UTR), which we showed to be an mRNA decay element in ovarian cancer cells. CSF-1 is strongly correlated with the poor prognosis of patients with ovarian cancer. We investigated the functional significance of GAPDH's association with CSF-1 mRNA and found that GAPDH small interfering RNA reduces both CSF-1 mRNA and protein levels by destabilizing CSF-1 mRNA. CSF-1 mRNA half-lives were decreased by 50% in the presence of GAPDH small interfering RNA. RNA footprinting analysis of the 144 nt CSF-1 sequence revealed that GAPDH associates with a large AU-rich-containing region. The effects of binding of GAPDH protein or ovarian extracts to mutations of the AU-rich regions within the footprint were consistent with this finding. In a tissue array containing 256 ovarian and fallopian tube cancer specimens, we found that GAPDH was regulated in these cancers, with almost 50% of specimens having no GAPDH staining. Furthermore, we found that low GAPDH staining was associated with a low CSF-1 score (P = 0.008). In summary, GAPDH, a multifunctional protein, now adds regulation of mRNA stability to its repertoire. We are the first to evaluate the clinical role of GAPDH protein in cancer. In ovarian cancers, we show that GAPDH expression is regulated, and we now recognize that one of the many functions of GAPDH is to promote mRNA stability of CSF-1, an important cytokine in tumor progression.

Glyceraldehyde-3-phosphate dehydrogenase binds to the AU-Rich 3' untranslated region of colony-stimulating factor-1 (CSF-1) messenger RNA in human ovarian cancer cells: possible role in CSF-1 posttranscriptional regulation and tumor phenotype.

The overexpression of the colony-stimulating factor-1(CSF-1) by epithelial ovarian cancer cells enhances invasiveness and metastatic properties, contributing to the poor prognosis of the patients. It has been suggested that CSF-1 3' untranslated region containing AU-rich elements (ARE) could regulate CSF-1 posttranscriptional expression and be responsible for its aberrant abundance in such cancer cells. In this study, normal (NOSE.1) and malignant (Hey) ovarian epithelial cells were used to examine CSF-1 expression and regulation. CSF-1 overexpression in Hey cells was found to associate with increased invasiveness, motility, urokinase activity, and virulence of tumorigenicity, compared with NOSE.1 cells, which expressed little CSF-1. CSF-1 ARE was further found to serve as an mRNA decay element that correlates with down-regulation of protein translation. Moreover, such down-regulation was found more prominent in NOSE.1 than in Hey cells, suggesting differences in posttranscriptional regulation. As a variety of trans-acting factors [AU-binding protein (AUBP)] are known to modulate messenger stability through binding to such elements, we examined the protein content of both cell lines for their ability to bind the CSF-1 ARE. Our results strongly suggested the abundance of such AUBP activity in Hey cells. We isolated a 37-kDa AUBP, which was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To summarize, our study identified GAPDH as an AUBP abundant in Hey cells, where it binds to CSF-1 ARE that imparts mRNA decay. These data suggest that GAPDH binding to CSF-1 ARE sequence prevents CSF-1 mRNA decay and subsequent down-regulation of CSF-1 protein translation, leading to CSF-1 overexpression and increased metastatic properties seen in ovarian cancer.

cDNA microarray analysis of invasive and tumorigenic phenotypes in a breast cancer model.

The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets.

SGK1, a potential regulator of c-fms related breast cancer aggressiveness.

The aggressive behavior of breast cancer cells can at times be modulated by hormonal mechanisms. Exposure to glucocorticoids (GC) has been shown to stimulate the invasiveness, motility and adhesiveness of breast cancer cells containing the glucocorticoid receptor. This is largely explained by GC-associated overexpression of the c-fms proto-oncogene, which encodes the receptor for the colony stimulating factor-1 (CSF-1). Our objective is to investigate additional GC-associated genetic alterations that could modulate c-fms related malignant behavior in breast cancer cells. A microarray technique using an oligonucleotide array representing 16,700 known expressed human genes was used to analyze the gene expression profile of breast cancer cells exposed to dexamethasone (Dex) or vehicle. Results were confirmed by western blot analysis. Six genes were found to be consistently differentially overexpressed in the Dex-exposed cells compared to control. We focused on serum-glucose kinase 1 (SGK1), a serine-threonine kinase known to be involved in intracellular signal transduction pathways and induced by GC and serum. An adhesion assay was performed on extracellular matrix after exposing the breast cancer cells to Dex, CSF-1 or to Dex or CSF-1 plus LY294002, a functional inhibitor of SGK1 action. Exposure to LY294002 significantly decreased both CSF-1 and Dex-induced adhesiveness to the level of control cells. SGK1 may act as a downstream intracellular regulator of c-fms, particularly of c-fms-induced adhesiveness of breast cancer cells after exposure to GC or CSF-1. This finding may have implications for potential therapeutic interventions aimed at decreasing the aggressiveness of breast cancer cells.