Genetic Pathways Altered by MMTV in Mouse Mammary Tumors

Division Of Basic Sciences - Nci

Investigators

Abstract

The panels of genomic DNAs analyzed by inverse PCR were from 96 mammary tumors each from CzechII, Balb/cfCZ, and Balb/cfSP mice along with 48 independent CzechII mammary preneoplastic hyperplastic outgrowth (HOG) lines and 48 HOG-derived mammary tumors. The nucleotide sequence of 48 recombinant DNA clones of the inverse PCR products was determined per tumor. We found that 69-82% of the tumor DNAs contained at least one retroviral integration site (RIS) per tumor. This corresponds to an average of 5.2 RIS per tumor. A similar copy number of 2-3 acquired MMTV genomes were detected by a Southern blot analysis of these same tumors DNAs. In our tumor panels, of those tumors that had at least 1 RIS on average 58% had one or more candidate common integration sites (CIS) (Table1). I define a CIS as a locus in which a MMTV genome has integrated in the gene or near the target gene in two or more independent mammary tumors, the integration event has been shown to alter the target genes expression relative to other tumors having no viral integrations at that site or in normal mammary gland tissue, and the integration either confers on normal mammary epithelial cells a transformed phenotype or in vivo contributes to mammary tumorigenesis. The most frequent CIS target genes were members of the Wnt, Fgf, and RSpo gene families.We have verified the expression of these genes by quantitative RT-PCR of RNA from tumors having a viral integration near the particular target gene. With the possible exceptions of a higher frequency of Wnt activation in Balb/cfC3H compared to CzechII mammary tumors or the higher frequency of RSpo activation in our tumor panels compared to Balb/cfC3H mammary tumors, the overall similarity in the frequency with which the Wnt, Fgf, and RSpo gene families are activated in the four tumor panels suggests to me that they represent the "Core" MMTV CIS. Furthermore, activation of these Core CIS by MMTV appears to be an early event since 14 out of 31 HOGs having a MMTV RIS contained an activated member of one of these gene families. This suggests that the Core CIS are necessary but not sufficient for the malignant transformation of the mammary gland. Interestingly, in those cases where a Wnt gene was activated it was always Wnt1. Similarly, in those HOGs where a RSpo gene was activated it was always RSpo2. This suggests that selection for MMTV activation of particular members of these gene families may depend on the presence of other potentially collaborating CIS or somatic mutations, particular properties of the activated gene product, or the cellular context in which the gene is activated. In addition to Wnt1, Fgf 3/4, and RSpo2, MMTV was integrated into eIF3e/Int6 in 4 independent CzechII HOGs. In each case the virus had integrated into a different intron of the gene and in the opposite transcriptional orientation of the gene. MMTV integration into eIF3e has only been detected in CzechII mammary HOGs and tumors, consistent with specificity for the CzechII genetic background. Notch4/Int3 is a frequent CIS in CzechII and M.m jkg mammary tumors, but is a low frequency CIS in BR6 and Balb/cfC3H mammary tumors. Interestingly, Notch4/Int3 and Rspo3 have never been found to be MMTV CIS in HOGs, suggesting that their activation by MMTV represent later events during mammary tumor progression. In addition to the Core MMTV CIS, we have identified 70 low frequency (n=2-3 tumors) candidate CIS. Their distribution seems to be specific to particular strains of MMTV or mouse strains. Alternatively, the strain specificity (mouse and MMTV) may be more apparent, because of low numbers, than real and simply reflect the complexity of mutations that are capable of contributing to mammary tumorigenesis. Although most of these candidate target genes are uncharacterized, the function of several are known, such as, PDGFR?, HDAC6, FoxD3, Sox9, c-Myc, Sfmbt2 and Fgfr2.