Kay-Uwe Wagner - US grants

Our long-term objective is to determine the mechanisms that regulate cellular growth and neoplastic transformation in breast tissue. Our current studies focus on how the tumor susceptibility gene 101 (TSG101) prevents tumorigenesis in mammary epithelial cells. TSG101 has been recently identified as an important factor for growth restriction and maintenance of genome stability in murine cell lines. The protein encoded by this gene has a very high similarity between human and rodents. Although chromosomal deletions of TSG 101 are rare events, aberrant transcripts are observed quite frequently in human cancers. Despite its important role for the maintenance of genome integrity in cultured cells, the biological function of TSG101 in vivo is largely unknown. The specific aims of this proposal are to determine the role of TSG101 during normal development and tumorigenesis in vivo. We use genetically engineered mice that carry somatic mutation for TSG101 in mammary tissue. We will investigate whether TSG101 has characteristics of a tumor suppressor gene or mammary tissue. The critical domains within TSG101 that mediate its role during tumorigenesis will be determined by replacing the endogenous TSG1021 protein with shorter with shorter TSG101 mutants that lack specific parts of the protein. Finally, we will investigate the suggested role of TSG101 as a co-factor for the transcriptional repression of targeted by DNA methyltransferase 1. TSG101 might function in various different pathways that are important for growth, differentiation, and cancer progression. Thus, investigating the regulation of TSG101 expression and its biological function will provide us with new insights that will allow us to intercept oncogenic pathways, that require adequate levels of this protein to function normally.

Our long-term objective is to elucidate how growth-regulatory signaling networks regulate normal growth and neoplastic transformation of mammary epithelial cells. Our studies focus on Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). JAKs and STATs are important intermediaries in "the lines of fire" of various growth factor receptors that are implicated in breast cancer. A primary objective of our current research is to examine how JAK/STAT signaling is altered in breast cancer models that initiate tumorigenesis through hyperstimulation of growth factor receptors (i.e.prolactin receptor and ErbB2) that utilize Jak2 and/or StatS and Stat3 as signal transducers. We developed a unique model system that enables us to genetically modify JAK/STAT signaling both prior to growth factor-mediated neoplastic transformation and during particular stages of the progressing disease. We hypothesize that inhibiting the growth factor- mediated activation of STATs through inactivation of Jak2 will reduce the onset of neoplastic transformation in these tumor models. This will suggest that targeting Jak2 is a relevant strategy for breast cancer prevention in individuals with hyperprolactinemia or patients that are at risk of developing pregnancy-associated breast cancers that are frequently ErbB2-positive. In contrast, the ablation of Jak2/Stat5/3 signaling in neoplastic cells (therapeutic intervention) might result in a different outcome depending on the type of growth factor- initiated transformation, and,more importantly, the stage of the progressing lesion. The specific aims of this proposal are to determine a hierarchy of diverse signaling transducers (aim 1) that serve as biomarkers for the analysis of prolactin and ErbB2 overexpressing cancer cells. Furthermore, the proposed studies will address mechanistic aspects about the autocrine role of prolactin in breast cancer (aim2) as well as the suggested Jak2-mediated receptor crosstalk between the prolactin receptor and ErbB2 (aim 3). The results of these analyses might, discriminate subtypes of breast cancer, in which targeting Jak2 is therapeutically relevant. Furthermore, they might reveal whether a combinatorial therapy of pan-ErbB tyrosine kinase inhibitors and Jak2 inhibitors would be beneficial for the treatment of ErbB2-positive breast cancers.

DESCRIPTION (provided by applicant): Our long-term objective is to elucidate how growth-regulatory signaling networks regulate the multiplication and survival of normal and neoplastic mammary epithelial cells. Our current studies focus on biological and biochemical functions of the oncoprotein encoded by the Tumor Susceptibility Gene 101 (Tsg101). Tsg101 is a key member of the endosomal sorting complex required for transport (ESCRT). This pathway is crucial for the transport, sorting, and lysosomal degradation of ubiquitinated cell surface receptors, in particular such as the EGFR and ErbB2 (Her2/neu) that are important receptor tyrosine kinases implicated in breast tumorigenesis and other human malignancies. A primary objective of our current research is to examine how Tsg101 engages with and modifies active ErbB-signaling complexes. In particular, we are interested in the function of Tsg101's intrinsic PTAP amino acid motif to regulate the activity of Tsg101 and its effect on the downregulation of oncogenic Her2/neu in normal and neoplastic mammary epithelial cells. Our central hypothesis is that altering the expression of Tsg101 or modifying its activity through inhibition of its self-regulatory PTAP domain will affect the onset and progression of Her2/neu-mediated mammary tumorigenesis. In the first specific aim of this proposal, we will determine how altering the expression level of wildtype Tsg101 modifies the growth properties of mouse and human mammary cancer cells in culture as well as tumor progression in vivo. We will also examine whether Tsg101 deficiency affects the survival of Trastuzumab-resistant human breast cancer cells. The second specific aim focuses on the mechanisms of the activation of Tsg101 that mediate an accelerated ubiquitination and degradation of the Tsg101 protein and its cargo (i.e. ErbB2). We will study the interaction of constitutively active Tsg101 with the ubiquitin ligase Tal as well as known members of the ESCRT complex. Additionally, we will use a proteomics approach to identify novel regulators for the activation of Tsg101. In the third specific aim, we will address whether activating Tsg101 will result in a more efficient downregulation of oncogenic ErbB2 in mammary cancer cells in vitro and in vivo. We propose the development of advanced genetically engineered models to predict the outcome of a possible targeted therapy against Her2/neu-mediated breast cancer by modifying the activity of Tsg101. Using these tools we might be able to herald the efficacy of this therapeutic strategy before pharmaceutical agents become available. PUBLIC HEALTH RELEVANCE: A significant subset of invasive breast cancers exhibit Her2/neu gene amplification with an unfavorable prognosis. The objective of this proposal is to elucidate how altering the expression of Tsg101 or modifying its activity will affect the onset and progression of Her2/neu-mediated breast cancer. The genetic and molecular studies outlined in this application will help to predict the potential value of Tsg101 as a therapeutic target before a drug will be developed and tested in preclinical trials.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. One goal of this COBRE proposal is to establish the triangle UNMC-Creighton-Boys Town NRH as a recognized Center of Research Excellence in Neurosensory Development. With the commitment of all three institutions to human health care, the mouse will be the paramount model organism for future biomedical research on this topic. The presence and availability of a Mouse Core will, therefore, not only benefit the initial COBRE Investigators through the on-site establishment of the pertinent mouse models, but will serve as an essential recruiting tool once the initial COBRE Investigators will rotate off and new Investigators will be brought aboard. We anticipate that such recruits will also focus on the mouse, use genetically manipulated mice to advance their research. In particular, junior Investigators with limited experience with mice would greatly benefit from Mouse Core support services. Furthermore, we expect that the expression profiling experiments of several projects of this proposal will yield results that merit the generation of new transgenic and knockout mouse models for the respective genes in order to study their function in neurosensory development. We are therefore convinced that the availability of a Mouse Core will significantly enhance the competitiveness of new applications for extramural funding that are fostered by this COBRE proposal.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Histology Core has the mission to serve the investigators of the COBRE Program with their needs in specialized morphological and histological analysis of neurosensory development and relevant mousephenotypes. A Core Facility for histology offers several advantages to the COBRE investigators and the COBRE Program: 1) Enrichment of Resources: The establishment of the Core Facility with expert personnel will provide the individual investigators with a multitude of services, equipment resources and technical expertise that is not readily available within their individual laboratories. This will significantly broaden the depth of the individual research projects and support the development of state-of-the-art and technologically comprehensive research programs within the individual junior investigators'laboratories. 2) Efficiency of Personnel and Cost Management: The concentration of such resources within the Core allows for better efficiency and standardization in the usage of typically costly reagents, such as for example, specialized antibodies. Furthermore, the involvement of the highly trained and experienced Director of Phenotyping Services at UNMC, Ms. M. Anita Jennings, will reduce the need for each PI to allocate their personnel to methodologies that require extensive training and time in order to be proficiently carried out. In addition, the use of common reagents will better allow investigators from the different laboratories to compare and interpret results. 3) Quality Assurance, Mentoring and Information Flow: Scientifically, the Core will enable a standardization of procedures, of flow of information, and of frameworks for interpretation that will greatly enhance the infrastructure for success of individual projects through common quality assurance, data generation and interpretation standards. In this way, investigators benefit from the combined expertise generated through all of the projects. Most importantly, information flow will be improved through the use of a common Website for access to the Histology Core that integrates the efforts of individual investigators along common themes. 4) Enhancement of Infrastructure: The long-term objective of the COBRE, namely, to establish a critical mass of investigators on one area through the Center for Neurosensory Development in Nebraska, will also be served by the Core Facility. The availability of a specialized Histology Core Facility will increase the competitiveness of COBRE investigators for extramural funding, serve in retention of funded investigators, and attract and recruit new investigators into the Center for Neurosensory Development.

DESCRIPTION (provided by applicant): The long-term objective in our laboratory is to elucidate how genetic alterations contribute to malignant transformation of normal epithelial cells. Our current studies focus more closely on the continuous biological relevance of cancer initiating mutations during subsequent stages of malignant progression of pancreatic ductal adenocarcinoma (PDAC), which accounts for the majority of pancreatic malignancies. In addition, we aim to identify cellular subtypes that are primary targets for neoplastic transformation or for the maintenance of pancreatic cancers. Previous studies have demonstrated that not all genetic alternations that initiate neoplastic transformation are equally important for the survival of cancer cells. Consequently, the selection of appropriate molecular targets that are crucial for the growth of cancer cells is of the utmost importance. In preliminary studies, we found that the majority of human ductal precursor lesions (PanINs) highly express c-Myc, and this oncogene is also upregulated in a subset of primary and metastatic PDACs. To assess the role of c-Myc and other oncogenes during tumor initiation and progression, we have developed a novel mouse model that allows for a strong, ligand-controlled expression of genes in a temporally controlled manner in pancreatic ductal cells of adult animals. Using this unique mouse model, we can demonstrate that the upregulation of the c-Myc oncogene is sufficient to cause rapid transformation and the appearance of ductal neoplasia after a short latency period. These neoplastic lesions progress quickly into primary PDACs with sporadic metastases to the liver. The treatment of tumor-bearing mice with a ligand (doxycycline) results in the suppression of c-Myc expression, which subsequently leads to apoptosis of neoplastic cells within PanIN lesions and solid pancreatic tumors. The primary goal of the proposed studies is to further examine whether c-Myc is equally required for the survival of all or a subset of metastatic carcinoma cells. Based on our preliminary data, we hypothesize that ablating the expression of the c-Myc oncogene in invasive pancreatic adenocarcinoma cells will cause the regression of metastatic lesions in tumor-bearing mice. We anticipate that the vast majority of cancer cells will undergo cell death, but a few cells will remain dormant that might serve as precursors for disease recurrence. To experimentally address the hypothesis in an exploratory-type (R21) study, we will determine in the first specific aim the importance of the c-Myc oncogene during maintenance and metastatic progression of PDAC and how its significance in advanced cancers is affected by secondary genetic events. In the second aim, we will employ our unique cancer model to identify and characterize residual cells that survive the ablation of c- Myc expression, and we will assess whether these cells are responsible for disease recurrence. Collectively, the results of both specific aims will yield novel data about the importance of cancer-initiating genetic events during the final stages of PDAC progression as well the differential effects of the ablation of an oncogene on cancer cell subtypes that may facilitate disease recurrence.

The majority of PDAC cases in humans carry mutations in the KRAS gene. While oncogenic KRAS is sufficient to initiate the formation of pre-cancerous lesions in the pancreas, the development of invasive PDAC requires an accumulation of additional genetic and epigenetic alterations in other tumor susceptibility loci as well as changes in cytokine signaling. Inflammatory cytokines produced by the stroma and, more importantly, by the cancer cells themselves are important drivers for pancreatic cancer progression. Interleukin-6 (IL-6)-class cytokines are considered master regulators of cancer-associated inflammation and they all signal through specific ligand-receptor complexes that share the glycoprotein 130 (gp130) signal transducing subunit, which activates Janus kinases (JAKs) and their downstream Signal Transducers and Activators of Transcription (STATs). IL-6-class cytokines are responsible for the persistent activation of STAT3 in cancer cells, which is a prerequisite for the progression of KRAS-driven pancreatic neoplasms. Using a novel genetically engineered mouse model, we can demonstrate that JAK1, and not JAK2 as commonly believed, is crucial for the activation of STAT3. Given the importance of IL-6 and STAT3 in malignant transformation, our findings provide a sound rationale for elucidating a potentially pivotal role for JAK1 in pancreatic carcinogenesis. The immediate objectives of this proposal are to mechanistically define how JAK1/STAT3 signaling contributes to the genesis and progression of PDAC. To accomplish this goal, we will use human pancreatic cancer cells and a state-of- the-art genetically engineered mouse model of pancreatic cancer to determine the biological significance of JAK1 in pancreatic cancer progression (aim 1). After establishing that targeting JAK1 is sufficient to block the activation of STAT3 and prevent pancreatic cancer metastasis, we will perform genome-wide transcriptome analyses to ascertain and validate new targets of JAK1/STAT3 signaling in PDAC. To assist the development of pharmacological agents that specifically target JAK1, we will further define sets of downstream target genes that can be used as biomarkers for a successful inhibition of this particular signaling pathway in human pancreatic cancer cells (aim 2). In the final aim of this project, we will examine the biological significance of two JAK1 downstream targets, RUNX1 and c-FOS, in pancreatic cancer progression with particular emphasis on their suggested role in the regulation of factors for extra cellular matrix remodeling. In an effort towards clinical translation, we will test a recently developed JAK1 inhibitor to assess whether this agent is able to selectively block the activation of STAT3 and expression of validated downstream target genes in pancreatic cancer cells. In summary, this will be the first comprehensive study that addresses a specific role for JAK1 in adenocarcinoma, and the collective results of this project are expected to provide new and detailed insight into the molecular and biologically relevant functions of this important member of the Janus kinase family in pancreatic carcinogenesis.

PROJECT SUMMARY Aberrant signaling through receptor tyrosine kinases of ERBB family members plays a key role in the etiology of breast cancer. Approximately 20-25% of invasive breast cancers exhibit an amplification of the ERBB2 locus or a constitutive activation of the receptor tyrosine kinase encoded by this gene. There is a substantial body of experimental and clinical data that shows that ERBB2-overexpressing breast cancer cells have an elevated metastatic potential. Despite initial response to targeted therapies against ERBB2 using trastuzumab and pertuzumab, the majority of patients eventually relapse and succumb to metastatic disease. Therefore, novel therapeutic strategies are needed to prevent and treat this aggressive breast cancer subtype. As a component of the endocytic machinery, the protein encoded by the Tumor Susceptibility Gene 101 (TSG101) is crucial for the intra-cellular trafficking, sorting, and lysosomal degradation of ubiquitinated cargo proteins including ERBB2 and other receptor tyrosine kinases. TSG101 is a central node in trafficking as it is able to simultaneously bind cargo proteins as well as PTAP amino acid motif-containing regulatory proteins and ubiquitin ligases that modify the sorting and trafficking of cargo complexes. TSG101 itself possesses an intrinsic PTAP motif near its C-terminal end, and we gathered preliminary evidence that this domain can associate with the PTAP binding groove at the N-terminus of TSG101. Moreover, we can demonstrate that mutating the intrinsic PTAP motif into an ATAA amino acid sequence and thereby blocking the intramolecular binding modality leads to a very significant decrease in the steady-state level of TSG101 and a simultaneous decline in the expression of its cargo proteins ERBB2, EGFR, and IGF-R1. The immediate objectives of this exploratory project are to extend the preliminary findings from in vitro studies and to develop a TSG101-ATAA knockin mouse model using the CRISPR/Cas9-based gene editing approach to firmly establish that disrupting the intramolecular association of the intrinsic PTAP domain with its binding grove increases the turnover and reduces the expression of TSG101 in vivo. We will then apply this mutant TSG101-ATAA knockin model to assess whether lowering the steady-state level of TSG101 will be sufficient to co-downregulate oncogenic ERBB2 and prevent the onset and progression of ERBB2-indcued mammary cancer in transgenic mice. Accelerating the turnover of TSG101 and its cargos represents a novel approach to prevent and treat ERBB2- positive breast cancer, and the collective results from this exploratory project will provide strong in vivo evidence for the development of a new class of therapeutics that target specific intramolecular associations within TSG101.