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) that induces clonal premalignant mammary lesions and malignant mammary tumors by insertional mutagenesis. The novel aspect of this model is that the mouse strain lacks genetically transmitted MMTV genomes so that the viral genome(s) detected in tumor DNA represents an acquired viral genome(s). 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. 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. Notch4, Usp31 and Cadm2 are among the genes whose expression is altered by MMTV integration within the gene and in these cases the viral genome is in the same transcriptional orientation as the gene. We have shown previously that MMTV """"""""activated"""""""" Notch4 intracellular domain (Int3) represents a gain of function mutation and when expressed in the mammary gland can block mammary alveolar/lobular development as well as induce mammary tumors. Currently we are developing vectors that express Usp31 (Ubiquitin specific peptidase 31) and Cadm2 (Cell adhesion molecule 2) to determine whether the truncated gene products resulting from the integration of MMTV in the gene represents a gain of function or loss of function mutation. Previously we have shown that blockage of mammary alveolar/lobular development is a consequence of an Rbpj dependent component of the Notch signaling pathway. Mammary tumor development seems to be independent of Notch4/Rbpj signaling. My current aim is to investigate the Rbpj independent pathway and to determine whether the activation of NFkappaB, 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 as compared to control HC11 cells. Wap-Int3 mice develop mammary tumors with 100% penetrance. Blocking of NFkappaB signaling with the IKK-alpha inhibitor (IMD-0345) resulted in a complete regression of Wap-Int3 mammary tumors. In addition, 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. 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. Previously we showed that 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. We have examined effect of Gleevec on mammary gland development in Wap-Int3 females. When the drug is administered as soon as the female is plugged alveolar lobular development occurs during the first pregnancy and the mouse lactates. A similar result occurs if the drug is applied after plugging at the second pregnancy. Interestingly at subsequent pregnancies and in the absence of the drug the females retain the ability to lactate and do not develop mammary tumors. This suggests that there is a progression of Notch sensitive progenitor cells to Notch insensitive cells that are capable of providing alveolar lobular development in subsequent pregnancies and lactation with the development of no mammary tumors. 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 (SB216763) treatment or GSK3-beta siRNA in the presence of Gleevec Int3 expression is preserved. Currently we are attempting to identify which E3 ligases are activated by GSK3-beta for ubiquitination of Int3 in response to Gleevec treatment. We have expanded our studies of Gleevec to HC11 cells stably expressing Myc, Fgf3, ErbB2 and Hras. Each of these cell lines exhibits the capacity for anchorage independent growth in soft agar, but only HC11-Myc and HC11-Hras cell growth is inhibited by Gleevec. Similarly, Gleevec treatment of HC11-Myc and HC11-Hras tumor bearing nude mice also leads to the regression of the tumor. We have begun to look at the mechanism by which Gleevec inhibits Myc and Ras signaling. In the case of Myc it may be similar to Int3 in that Gleevec has no effect on Myc RNA expression but is associated with absence of Myc protein. At the present time this is a focus of our studies. Our results suggest that Gleevec treatment may relevant for the 30% of breast cancer patients bearing tumors having an amplification of cMyc. 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. We have developed four founder lines of transgenic mice in which Rspo2 expression in the mammary gland is driven by the MMTV LTR to study the biology of its involvement the initiation of 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.
Showing the most recent 10 out of 11 publications
