Predoctoral Trainees

Nancy Castro
Lange lab
Phone: 624-1971
E-mail: castr043@umn.edu

Protein tyrosine kinases (PTKs) play a major role in the regulation of normal cell growth and differentiation and contribute to cancer progression. The non-receptor PTK Brk (breast tumor kinase) was originally cloned from a human metastatic breast tumor and is highly expressed in 65% of human breast tumors and breast tumor cell lines, but is not found in normal breast tissue. Brk overexpression may account for high PTK activity in breast cancer cells and contribute to increased growth or survival signals in breast tumors. In the mouse, the PTK src-related intestinal kinase (Sik) is 80% identical to human Brk. Sik mRNA expression is restricted to differentiating cell layers located just above the proliferative cell zone of rapidly renewing intestinal and skin epithelia, indicating a highly tissue specific role in signal transduction within differentiating epithelial tissues. Like Sik, Brk is also present in human normal epithelial cells of the gastrointestinal tract and skin; Brk expression is increased in numerous cancers, including melanomas. Nancy's research is focused on understanding the function of Brk in normal cells (using HaCaT cells as a skin cell model) and how inappropriate expression of Brk may contribute to breast cancer. Nancy is using ligands for the MET family of receptors, which include hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), as potential signaling pathways relevant to both wound healing in skin cells and cancer cell migration during metastasis. Both ligands play a role in the migration of keratinocytes and have been implicated in cancer metastatsis. Nancy hypothesizes that Brk mediates cell migration in response to stimulation of MET receptor signaling. Understanding the normal function of Brk during keratinocyte migration may reveal its role(s) in breast cancer progression and metastasis. A better understanding of how intracellular signaling and cytoskeletal cross talk occurs in breast cancer cells compared to normal contexts may lead to the development of better treatment strategies for advanced invasive breast cancer.

Justin Haworth
Bielinsky lab
Phone: 624-0460
E-mail: hawo0007@umn.edu

In the US, cancer accounts for one in every four deaths, stressing the need for a better understanding of the basic processes important for the proliferation of cancer cells, such as DNA replication. A major focus of the Bielinsky laboratory is to fully understand the role of minichromosome maintenance (Mcm) 10 in DNA replication. Critical for Mcm10's function during replication is its DNA-binding activity. The crystal structure of X. laevis Mcm10 was recently solved. Based on this structural data, residues in Mcm10 were identified that, when mutated, significantly decrease the DNA-binding activity of X. laevis Mcm10 in vitro. Justin has shown that the corresponding mutations in S. cerevisiae Mcm10 lead to defects in DNA replication, providing evidence that DNA binding is important in vivo. In addition to its DNA-binding activity, Mcm10 regulates DNA polymerase-a (pol-a), the only enzyme capable of de novo DNA synthesis in yeast and human cells. In the absence of Mcm10, the catalytic subunit of pol-a, Cdc17 in yeast, is rapidly degraded. Degradation of Cdc17 is proteasome-dependent and, more specifically, requires the ubiquitin conjugating (E2) enzyme Ubc4 and the ubiquitin ligase (E3) Not4. The regulation of pol-a by Mcm10 is conserved in human cells, suggesting that this is a functionally important pathway in the cell. Justin is currently testing the functional relevance of this pathway by deregulating Ubc4- and Not4-dependent pol-a turnover in the presence of Mcm10 in yeast cells.

Eric Rahrmann
Largaespada lab
Phone: 612-66-6971
E-mail: rahr0003@umn.edu

Prostate cancer is responsible for 27,000 deaths each year in the United States alone. Due to the genetic heterogeneity of the disease, knowledge of the genetic pathways involved in prostate cancer initiation and progression is incomplete. Existing mouse models are limited in that they typically utilize a single genetic defect that induces either robust tumor formation without metastasis or develop precursor prostate cancer lesions that never progress to full blown tumors. To better mimic human prostate cancer initiation and progression in mice, a new model that generates heterogeneous tumors is necessary. Eric is developing a new prostate cancer mouse model that can identify novel genes and genetic pathways involved in prostate cancer. This work involves a forward genetic screen using the Sleeping Beauty transposon mediated mutagenesis system to scan the genome for novel oncogenes and tumor suppressors. Previous work from Dr. David Largaespada's lab has demonstrated that the sleeping beauty system can induce solid tumor formation in mice. This screen successfully identified cooperating genes in sarcoma formation. In order to develop a mouse model specific to prostate cancer, Eric has created a transgenic mouse that expresses the Sleeping Beauty transposase specifically in the adult prostate epithelium using the androgen regulated probasin promoter. These mice will be crossed to the oncogenic transposon transgenic mice developed in the Largaespada lab. Erci hypothesizes that multiple transposition events will occur in the prostate, generating heterogeneous tumors similar to human prostate cancer development. This work will circumvent the limitations of existing mouse models by creating a genetically heterogeneous prostate disease rather than a single genetic defect and provide models of transgenic mice at various stages of prostate cancer progression. These mouse models will provide a unique and important tool to screen for new genetic pathways involved in prostate cancer initiation and progression, and they will be useful for pre-clinical evaluation of new candidate prostate cancer therapies.

Mark Stenglein
Harris lab
Phone: 624-0459
E-mail: sten0171@umn.edu

In humans, the APOBEC3s form a seven-member family of polynucleotide cytosine deaminases. These enzymes catalyze the conversion of cytosines to uracils in DNA. APOBEC3s defend cells against potentially dangerous genetic pathogens by actively impeding the replication of a variety of retroviruses (most notably HIV) and retrotransposons. These mobile genetic elements pose a serious threat to the genomic stability of cells. Thus, the APOBEC3 paradox is that they are DNA-mutating enzymes that safeguard the genetic integrity of cells. This paradox means that APOBEC3s play a unique role in cancer biology in that they have both the potential to prevent and to cause cancer. Mark's thesis research explores both sides of this APOBEC3 paradox. First, Mark has demonstrated that APOBEC3 proteins limit the replication of LINE-1 retrotransposons. LINE-1s are the only transposable element currently active in humans, and de novo LINE-1 insertions have been shown to cause somatic cancers. Mark has also focused on the way that APOBEC3s—especially deregulated APOBEC3s—might cause DNA mutations that lead to cancer. The mutations in certain types of cancer are predominantly C/G to T/A transition mutations, exactly the type of mutation that APOBEC3s trigger. Mark is using APOBEC3 and deregulated APOBEC3 mutants to demonstrate that, in the absence of normal regulatory mechanisms, APOBEC3s indeed mutate the genomic DNA of cells and could contribute to the development or progression of cancer. These studies show how APOBEC3 proteins represent a Faustian bargain for the cell: they provide powerful intrinsic immunity against mutagenic mobile elements but are themselves potentially dangerous oncoproteins.

Postdoctoral Trainees

Christine Clouser, Ph.D.
Mansky Lab
Phone: 612-624-5172
E-mail: cclouser@umn.edu

The introduction of combination therapy to treat HIV significantly reduced the prevalence of AIDS-related cancers such as Non-Hodgkin's lymphoma, Hodgkin's lymphoma, Kaposi's sarcoma, cervical cancer and lung cancer. However, the emergence and transmission of drug resistant HIV has limited the efficacy of current anti-HIV drugs. The prevalence of drug resistant HIV threatens the advances made in the prevention of AIDS-related cancers as well as the in the treatment of HIV. Thus, novel drugs are needed to better treat HIV thereby reducing mortality from AIDS-related cancers. The high mutation rate of HIV enables the virus to evade the immune system and develop resistance to drug therapy. However, the high mutation rate comes at a cost to the virus. It is thought that the majority of mutations that arise during reverse transcription are deleterious to virus survival and that an intentional increase in mutation rate could render the virus unable to replicate with enough fidelity to remain infectious. This method of eliminating virus is known as lethal mutagenesis and has been proposed as a rationale approach to treating HIV. Lethal mutagenesis would offer a novel therapy that exploits a new HIV target—the mutation rate. Ribonucleotide reductase inhibitors and nucleoside analogs have each been shown to increase the mutation rate of HIV. Christine has identified two FDA approved drugs from these drug classes that potently inhibit HIV infectivity without significant cytotoxicity. The loss of infectivity correlates with a 3.4 fold increase in mutant frequency, suggesting that these drugs work in combination by targeting the mutation rate of HIV. The current and future work is focused on examining this drug combination in a murine AIDS model. Additionally, Christine will use the approach of lethal mutagenesis on other RNA viruses such as Hepatitis B and HTLV1, both of which contribute to oncogenesis.

Christy Hagan
Lange lab
Phone: 624-1971
E-mail: hagan018@umn.edu

Hormone-dependent activation of the progesterone receptor (PR) is a critical step in normal breast epithelial cell proliferation and mammary gland development. Dr. Lange’s laboratory has recently discovered that PRs induce rapid signaling events through members of the MAP kinase (MAPK) family that lead to the regulation of genes required for breast cancer cell proliferation. Christy proposes that hormonal and growth factor signals converge on the progesterone receptor. Inappropriate PR signaling, in response to activation of MAPK pathways (i.e. under sub-physiological steroid hormone concentrations) may lead to hyperplastic breast cell growth.  Understanding the normal role of cross-talk between the PR and MAPK pathways may decipher how this interplay is dysregulated in breast cancer. Christy’s research is focusing on identifying functional domains within PR that are necessary for direct interaction with MAPK signaling molecules, and determining how these subsequent interactions propagate the signal to mediate downstream effects on PR post-translational modifications (i.e phosphorylation events) and transcriptional activity at selected promoters.  Kinases are essential regulators of steroid hormone receptor action, and understanding their interactions with PR as model receptors may provide useful avenues for breast cancer prevention and chemotherapeutic targeting.

Jason Mitchell, Ph.D.
Shimizu lab
Phone: 612-626-6713
E-mail: mitch490@umn.edu

The regulation of integrin mediated adhesion is critical for immunosurveillance against the development of cancer cells. The induction of T cell adhesion mechanisms allows for sustained contacts during cancer antigen recognition, leading to productive initiation of the immune response. T cell adhesion regulation is accomplished through signaling molecules binding to integrin cytoplasmic domains, which regulate cytoskeletal mediated integrin clustering and the induction of high affinity integrin conformations. The focus of Jason's research is to determine the molecular mechanisms regulating integrin activation in T cells in response to antigen mimetic signals. One such mechanism involves T cell receptor-mediated activation of the small GTPase Rap1. To determine the importance of Rap1 activation in integrin activation, Jason is using an adenovirus infection model to express mutant signaling proteins in T cell lines and mouse primary cells with genetic knockouts. These cells are then used in adhesion and soluble ligand binding assays to assess which molecular interactions are important to activate Rap1 and scaffold to integrin cytoplasimc domains to regulate T cell receptor-directed integrin activation.

Marion Pepper Pew, Ph.D.
Jenkins lab
Phone: 612-626-1188
E-mail: peppe033@umn.edu

Marion's research will determine how endogenous CD4+ monoclonal populations are expanded, contracted or maintained during an ongoing immune response and examine the influence of repertoire composition upon the maintenance of a polyclonal pool.  Using a novel technique for making MHC Class II multimers, Marion can enrich specific monoclonal CD4+ T cell populations.  Peptide-MHC Class II multimers are currently being generated against several immunogenic antigens expressed during extracellular or intracellular stages of infection.  These multimers will then be used to isolate, characterize and compare the in vivo kinetics of multiple monoclonal populations enabling the analysis of the endogenous TCR repertoire diversity over time.  It is hypothesized that the pressure to maintain CD4+ TCR heterogeneity through intraclonal competition determines the composition of the memory population that persists rather than the need to maintain the overall frequency of any one specific clonal population.  The proposed experiments will test this hypothesis as the resulting repertoire of the endogenous memory pool can be examined.  Furthermore, the effects of prior immunization either with specific proteins or live attenuated bacteria on clonal composition can also be assessed.  Generation of reagents with peptides derived from tumor antigens will enable similar studies in a cancer model.