The overall goal of Dr. Crawford's research is to determine how germline variation modulates tumor progression and metastasis, focusing primarily on prostate and breast cancers. Although tumorigenesis is initiated by somatic mutation, and populations of cells in the primary tumor gain the capacity to metastasize through these same mutational mechanisms, it is clear that hereditary variation substantially modifies metastasis risk. Dr. Crawford's group uses approaches that strictly control conditions in the laboratory mouse - most notably the two variables that confound human association studies: genetic variation and environmental exposure.
In his prostate studies, Dr. Crawford concentrates on identifying novel germline variants associated with tumor progression and metastasis. His approach leverages both the tremendous variation captured in inbred mouse strains and modern genomics tools to determine the effects of these variants. His group is analyzing the effects of germline variation on susceptibility to aggressive forms of prostate cancer in different mouse models of tumorigenesis to determine variants that inhibit or promote metastasis. In other studies, Dr. Crawford utilizes the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) model of aggressive neuroendocrine (NE) prostate cancer to identify germline susceptibility genes associated with aggressive disease. Although pure NE prostate cancer is rare, adenocarcinomas - the most common histological subtype of prostate cancer - that display neuroendocrine differentiation (NED) are associated with a poorer prognosis.
Dr. Crawford's group has bred the TRAMP mouse to a variety of inbred and wild-derived mouse strains to investigate genetic modifiers influencing NED and aggressive disease development. The resulting transgene-positive F1 males display a wide spectrum of tumor growth and metastasis burden, proving that germline polymorphism does indeed modulate prostate cancer progression. This earlier work has facilitated the definition of tumor progression and metastasis quantitative trait loci (QTLs) by using an F2 intercross approach as well as breeding TRAMP mice to Diversity Outbred stock - a genetically diverse mouse resource used for high-resolution genetic mapping. These approaches have revealed multiple QTLs for aggressive disease progression associated with NE prostate tumorigenesis. Candidate genes have been identified using a number of fine mapping strategies, most notably expression QTL (eQTL) mapping. Ongoing research involves characterization of the roles of these genes in aggressive forms of prostate cancer. These studies will encompass both in vitro and in vivo functional validation of individual candidate genes, as well as candidate gene validation in a human prostate cancer dataset, including epidemiological association studies in human cohorts and human prostate cancer tumor gene expression datasets.
In addition to his work in prostate cancer, Dr. Crawford's group is continuing to advance its studies in breast cancer. As is the case with prostate cancer, mouse models of mammary tumorigenesis have proven a powerful tool in the identification of germline-encoded progression and metastasis susceptibility genes in breast cancer. Breast cancer metastasis susceptibility genes include RRP1B and NDN, which are currently under investigation in the Crawford lab to further understand the role of germline polymorphisms in both transcriptional regulation and metastasis. Both genes were identified as metastasis modifiers by introducing germline polymorphism through breeding into the FVB/N-Tg(MMTVPyVT)634Mul/J mouse model of mammary tumorigenesis. Dr. Crawford demonstrated that activation of either RRP1B or NDN induces a gene expression signature that accurately predicts survival in human breast cancer. Additionally, RRP1B contains a non-synonymous coding polymorphism (G1421A; P436L), the variant allele of which is associated with improved survival in multiple human breast cancer cohorts.
The majority of work performed thus far has focused on the functional characterization of RRP1B. Dr. Crawford's earlier studies suggested that RRP1B regulates transcription through multiple mechanisms, including mRNA splicing regulation and by interacting with chromatin. Using RNA-seq, Dr. Crawford's group demonstrated that RRP1B dysregulation induces global changes in splicing patterns by physically interacting with spliceosomal regulators such as SRSF1 and CROP, identifying the first example of a direct link between a metastasis suppressor and alternative mRNA splicing. Additionally, ChIP-seq analysis revealed that RRP1B regulates metastasis-associated gene expression by binding to chromatin and inducing global histone methylation. Specifically, RRP1B physically interacts with several heterochromatin-associated proteins to induce transcriptional repression via trimethylation of the histone H3 at the lysine-9 position in several prominent metastasis-associated genes. Collectively, these observations provide insight into how RRP1B regulates transcription at the genome-wide level, and suggest a possible mechanism by which it suppresses metastasis. Ongoing work with RRP1B is focusing on the functional significance of the survival-associated P436L SNP, and its role in the RNA binding properties of RRP1B.
Metastasis Genetics Section Members
Jonathan Andreas, M.S., Biologist
Minnkyong Lee, Ph.D., Postdoctoral Fellow
Amanda Wardlaw, B.S., Biologist
Kendra Williams, Ph.D., Postdoctoral Fellow
Posted: January 5, 2015