Our Science – Callahan Website
Robert Callahan, Ph.D.
The aim of my research has been to define and characterize the mutations that are required to deregulate normal mammary gland developmental controls and contribute to the evolution of malignant progression, terminating in metastasis. I developed a novel mouse mammary tumor virus (MMTV)-induced mammary tumor model system in a feral strain of mice (CzechII) that lacks genetically transmitted MMTV but is congenitally infected with MMTV through the milk. In this approach I have used the viral genome as a molecular tag to identify genes which are rearranged by MMTV integration or whose expression is affected by MMTV integration adjacent to the target gene. These studies led to the identification of two novel genes that are activated/inactivated by MMTV in mouse mammary tumors: Int3 and Int6. In each case MMTV integration in the target gene led to expression of a truncated target gene product. Int3 is a member (Notch4) of the Notch gene family that encodes a transmembrane receptor protein involved in cell fate decisions during development. Int6 has been shown to encode the p48 subunit of the eucaryotic translation initiation factor-3 complex (Eif3e).
More recently we have expanded these studies to other mouse strains infected with MMTV (CzechII) and MMTV (Spret) using the reverse polymerase chain reaction methodology to obtain recombinant clones of host-viral junction restriction fragments from MMTV induced mammary tumor DNA. These studies include certain transgenic mouse strains where we hope to identify viral induced mutations that collaborate with the transgene in malignant progression. The preliminary results suggest that, using this approach we have found at least 28 new common integration sites for MMTV in mouse mammary tumors. One of these is Rspo2, a member of the R-spondin gene family. Members of this family encode proteins that are 'non-conventional' ligands for the Wnt co-receptors (LRP5/6 and Fz) and synergizes with Wnt in the canonical signaling pathway.
To begin to understand the mechanism and consequences of Notch4/int3 signaling on mammary gland development and tumorigenesis we have developed several transgenic mouse strains. The MMTV LTR -Int3 and Wap-Int3 transgenic mice exhibit two phenotypes with 100% penetrance: lack of mammary alveolar/lobular development during pregnancy and mammary tumor development. We have begun to dissect and identify the different components of Notch4/In3 signaling that are responsible for these phenotypes. To determine whether these phenotypes result from the canonical Notch4/Int3-Rbpj signaling pathway, we conditionally deleted exons 6 and 7 of the Rbpj gene in the presence of the mammary gland specific Wap-Cre transgene. Loss of the Rbpj gene in Wap-Int3/Wap-Cre mice is associated with normal mammary gland development, but 100% of the females still develop mammary tumors. At the present time we are attempting to identify the component(s) of Notch4/Int3 signaling that is responsible for mammary tumor development.
It seems that Notch4/Int3 mediates tumorigenesis of the mammary gland by affecting the proliferation of stem cells at different stages of mammary gland development. To begin to understand the molecular consequences of activated Notch4/Int-3 on the expression of genes that are downstream in this signaling pathway, we have taken advantage of the transgenic mouse strains that we developed. In these experiments gene expression is being measured by microarray analysis of RNA prepared from mammary tissue at different stages of mammary gland development and/or from mammary tumors arising in these mice.
Another high priority for my lab has been to determine the role of Rspo2 in mammary tumorigenesis. Mammary preneoplastic lesions and tumors containing an MMTV activated Rspo2/Int7 were also found to have two or three additional viral insertions that occurred flanking a member of the Wnt (Wnt3a, Wnt10) and/or FGF (Fgf3, Fgf4) gene families. This suggests a possible collaboration between Rspo2 and Wnt/Fgf genes during mammary tumorigenesis. Currently we are focused on elucidating the cellular transforming abilities of Rspo2, its mechanism of action and possible cooperation with other signaling pathways.
This page was last updated on 3/31/2014.