The accumulation of mutations in mammary epithelium is thought to be a contributing factor in the initiation of premalignant mammary lesions and their progression to malignancy and metastasis. We have used a mouse model in which the carcinogen is the mouse mammary tumor virus (MMTV) which induces clonal premalignant mammary lesions and malignant mammary tumors by insertional mutagenesis. Identification of the genes and signaling pathways affected by MMTV-induced mouse mammary lesions provides a rationale for determining whether genetic alteration of the human orthologues of these genes/pathways may contribute to human breast carcinogenesis. A high-throughput platform for inverse PCR to identify MMTV-host junction fragments and to determine their nucleotide sequences in a large panel of MMTV-induced lesions was developed. Common integration site (CIS) means that the gene was altered by an MMTV proviral insertion in at least two independent lesions arising in different hosts. Three of the new genes identified as CIS for MMTV were assayed for their capability to confer on HC11 mouse mammary epithelial cells the ability for invasion, anchorage independent growth and tumor development in nude mice. In addition, we have interrogated the published transcriptome and nucleotide sequence analysis of primary human breast tumors and determined that the expression of the human orthologues of many of these new MMTV CIS genes is deregulated or mutated in human breast cancer. Analysis of MMTV induced mammary premalignant hyperplastic outgrowth (HOG) lines and mammary tumors led to the identification of CIS restricted to 35 loci. We determined that within these loci members of the Wnt, Fgf and Rspo gene families plus two linked genes (Npm3 and Ddn) and three unlinked genes (Notch4, Sfmbt2 and Pdgfr-alpha) were frequently activated in tumors induced by MMTV irrespective of the mouse strain or the strain of virus. A second group of 13 CIS occur at a low frequency (2-5 observations) in mammary HOGs or tumors and are mouse or virus strain specific. In this latter group the expression of either Phf19 or Sdc2 was shown to increase HC11 cells invasion capability. Foxl1 expression conferred on HC11 cells the capability for anchorage-independent colony formation in soft agar and tumor development in nude mice. Twenty of the human orthologues of MMTV CIS associated genes are deregulated and/or mutated in human breast tumors. These results have been published in Callahan et al., 2012. Among the genes whose expression is altered by MMTV integration in the same transcriptional orientation within the gene (Notch4, Usp31 and Cadm2) we have shown previously that "activated" Notch4 intracellular domain (Int3) can induce mammary tumors development as a consequence of an Rbpj independent component of the Notch signaling pathway. My current aim is to investigate the Rbpj independent pathway and to determine whether the activation of NF-kappaB, in the context of Int3 signaling, contributes to malignant growth. Microarray analysis of HC11-Int3 RNAs revealed high steady-state levels of the NF-kappaB target genes;IL1, IL6, IL-8, CXCL1 and CXCL2, as compared to control HC11 cells. Blocking of NF-kappaB signaling with the IKK-alpha inhibitor (IMD-0345) resulted in a complete regression of Wap-Int3 mammary tumors. In addition we demonstrated that Int3 activates the expression of NFkappaB P50 independent of Rbpjk. Investigation of the levels of activation/phosphorylation of NF-kappaB targets in normal mammary tissue from FVB/N females and Wap-Int3 mammary glands showed no difference in the phosphorylation of Ikk-alpha and P65 however the phosphorylation of P50 was significantly higher in the Wap-Int3 mammary glands than in the FVB/N glands. Total and phospho-P50 levels are not affected by the presence or absence of Rbpjk in the Wap-Int3 mammary tumors. However blocking of Int3 expression resulted in significant reduction of the phospho-P50 levels. The incidence of mammary tumorigenesis in Wap-Int3 transgenic mice dropped from 85% after the second pregnancy to 0% in the Wap-Int3/ NF-kappaB -P50-/- mice after five pregnancies. Taken together, these data are consistent with NF-kappaB-P50 being a component of Notch signaling that contributes to mammary tumorigenesis. A manuscript describing this work is being prepared. Wap-Int3 mice develop tumors with 100% penetrance. The treatment of Wap-Int3 tumor-bearing mice with Imatinib mesylate (Gleevec) is associated with the complete regression of the tumor, suggesting that Gleevec is an inhibitor of Notch signaling. To elucidate the mechanism by which Gleevec inhibits Notch, we treated HC11 mouse mammary epithelial cells expressing Int3 with Gleevec. We have found that there is a loss of Int3 protein but no change in mRNA levels. We hypothesized that Gleevec affects the stability of Int3 by promoting the degradation of Int3. The addition of MG132, a proteasome inhibitor, shows increased ubiquitination in HC11-Int3 cells in the presence of Gleevec. By studying the c-Kit and PDGFR pathway, we began to investigate substrate kinases ILK, CDK8, and GSK3-beta to ascertain a possible role in the stability of Int3. We have found that inhibiting GSK3-beta by drug treatment (SB216763) or GSK3-beta siRNA in the presence of Gleevec Int3 expression is preserved. Utilization of HC11-Int3 deletion mutants demonstrate differentially expressed phosphorylated GSK3-beta. Currently we are attempting to identify which E3 ligases are activated by GSK3-beta for ubiquitination of Int3 in response to Gleevec treatment. In addition we are mutating the serine/threonine residues at the C-terminus of Int3 to assess the phosphorylation site(s) of GSK3-beta and its role in the degradation of Int3 by Gleevec. The R-spondin (Rspo) protein family consists of four homologous members that are dynamically expressed during embryogenesis and have important roles in development. Rspo2 and Rspo3 are CIS for MMTV preneoplastic mammary hyperplastic alveolar nodules and mammary tumors, respectively. Currently we are developing transgenic mice expressing from MMTV LTR Rspo2 in the mammmary gland to study the biology of its involvement the initiation of mammary tumor mammary tumor development. Our previous studies demonstrated that synergy in Wnt and Rspo2 signaling on conferring on cells the capability for anchorage independent growth and invasion was independent of beta- catenin signaling. Currently we are exploring the Wnt/beta-catenin independent signaling pathways which confer these growth properties on cells.
|Li, Sheng-Jian; Yen, Ten-Yang; Endo, Yoshimi et al. (2009) Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function. Cell Signal 21:916-25|
|Raafat, A; Lawson, S; Bargo, S et al. (2009) Rbpj conditional knockout reveals distinct functions of Notch4/Int3 in mammary gland development and tumorigenesis. Oncogene 28:219-30|