We are interested in mechanisms that transform normal cells into cancer cells. Currently, a good portion of the lab is focused on furthering our understanding of the genetics of inherited cancers. We use, among other techniques, the tools of next generation sequencing of the germlines of patients with a family history of cancer to understand the molecular basis of their genetic inheritance. The goal is to identify new genes and cellular pathways involved in the abnormal biology of cancer cells. We’ve recently sequenced a cohort of 278 patients from the clinic and identified candidate genes for further analysis (Foley et al, 2015, EBiomedicine). We are also investigating why some patients with BRCA1 mutations get cancer while others don't by looking for genes that modify the genetic penetrance of BRCA1 mutations. These studies dovetail with our laboratory’s bench work that seeks a more precise understanding of the regulation of BRCA1 expression and the role of BRCA1 in normal physiology in humans and mice (Soyombo et al., 2013, Stem Cell Reports).
Cover art depicting skin biopsies from patients with BRCA1 mutations. These biopsy samples provided fibroblasts used for the generation and study of BRCA1 mutant induced pluripotent stem cells.Click to enlarge
For more than a decade, another part of the lab has focused on the Huntingtin Interacting Protein 1 (HIP1) family. HIP1 is linked to neurology by virtue of its interaction with Huntingtin, the protein mutated in Huntington's disease. It was originally linked to neoplasia by our discovery of the oncogenic HIP1/PDGFβ Receptor (HIP1/PDGFβR) fusion protein that resulted from a t(5;7) chromosomal translocation in a patient with chronic myelomonocytic leukemia. Subsequently others have discovered that the HIP1 gene is also a partner for the ALK tyrosine kinase gene in chromosomal translocations that cause a type of non-small cell lung cancer - a lung cancer type that is sensitive to ALK inhibitors (e.g. crizotinib). The protein structures of HIP1 and HIP1-related (HIP1r) (the only HIP1 relative) suggest that they link the actin cytoskeleton, clathrin trafficking and phosphatidylinositol turnover. One part of the lab has focused on understanding the transforming biology of HIP1/PDGFβR and other oncogenic tyrosine kinase fusion proteins such as BCR/ABL. We have generated and analyzed conditional HIP1/PDGFβR (Oravecz-Wilson et al, 2009, Cancer Cell) and BCR/ABL knock-in alleles (Foley et al., 2013, Cell Reports) and discovered that the biology resulting from these mutations in mice is quite distinct from what has been observed in the past when standard retroviral models of oncogene expression were used. This difference is most likely due to the fact that knockin alleles are more physiologic as expression is more likely in the right cell, at the right time and in the right amount compared to the wrong cell, time and amount that occurs as a result of standard retroviral models.
Peripheral blood smear showing severe myeloid leukocytosis resulting from targeted "knock-in" expression of the HIP1/PDGFβR and the t(8;21)-associated AML1-ETO oncogenic fusion proteins in mouse bone marrow. Click to enlarge.
The lab is also works on the roles of HIP1 and HIP1r in tumorigenesis, hematopoiesis, and clathrin trafficking. We have found that, although HIP1 is expressed in some normal epithelial tissues, its expression is restricted to the colon and prostate neoplastic epithelia and not expressed in normal colon and prostate epithelia. We have evaluated the clinical implications of this in prostate cancer patients and found that HIP1 expression is a strong marker of tumor progression (Rao et al., 2002,, J Clin Invest). The mechanism of how HIP1 may promote tumorigenesis and progression is not known. However, we have shown that its expression is necessary in some cells to survive, that it directly transforms NIH/3T3 cells and that the transformed cells have altered levels of multiple growth factor receptors (GFRs) (Rao et al., 2003, Cancer Cell).
Confocal immunofluorescent analysis of HIP1 and clathrin heavy chain localization in normal 3T3 cells and 3T3 over-expressing HIP1. HIP1 causes redistribution of clathrin, decreased endocytosis, and increased growth factor receptor signaling. Click to enlarge
To test if HIP1 and HIP1r directly affect the trafficking of GFRs we have evaluated the effects of transiently altered levels of HIP1 and HIP1r on activated GFRs. These proteins, but not their dominant negative counterparts, directly stabilize endosome pools of activated GFRs. In addition HIP1 knockout mice have a general "cell loss" phenotype as evidenced by hematopoietic defects, testicular degeneration, spinal defects and cataracts. These findings suggest that HIP1 and HIP1r stabilization of GFRs is necessary for survival or proliferation of cells from diverse tissues.
Expression of HIP1 is also found in multiple cell types that show no overt phenotypes as a result of the HIP1's deletion. We therefore suspect that some lost HIP1 functions can be compensated for by expression of HIP1r, which is also expressed in multiple tissue types. The lab has generated compound HIP1r/HIP1 deficient mice to test the latter hypothesis and found that although the HIP1r deficient mice alone are normal, loss of HIPr drastically accelerates the HIP1 deficient phenotype and the mice die by 4 months of age (Bradley, et al. 2007, Hum. Mol. Genetics). Although HIP1 and HIP1r clearly function in the same cellular pathway we have found that, in contrast to HIP1, HIP1r expression is not altered in primary cancers.
The putative regulation of clathrin mediated trafficking and indirectly GFR activation by HIP1 and HIP1r could explain our observations. I.e. alteration of the levels and activation of GFRs may be the reason for the altered growth characteristics during over-expression or knockout of HIP1 and HIP1r.
The spinal defects and diminished weight of "double knockout" mice deficient in both HIP1 and HIP1-related protein are rescued by transgenic expression of the human HIP1 protein. Click to enlarge
In sum, although much of the lab's past focus was to understand the role of the clathrin trafficking in the biology of malignancy and to assess if modulation of this pathway in tumor cells is therapeutic, we've expanded and are now working to merge studies of patients from our cancer genetics clinic with laboratory discovery of new genes and pathways that contribute to tumorigenesis.