Lisa Coussens - US grants

DESCRIPTION: (provided by applicant) Transgenic mouse models of human cancers present unique opportunities to elucidate important cellular and genetic pathways of cancer development, through which normal cells progressively are converted into aberrant cancers. By studying a transgenic mouse model of squamous cell carcinoma development, we have identified an important paracrine modifier of epithelial carcinogenesis, e.g., gelatinase B/matrix metalloproteinase-9 (MMP9), whose role previously was believed to facilitate basement membrane degradation. During neoplastic development, MMP-9 regulates epithelial cell proliferation, differentiation, and overall malignancy of emergent cancers, all previously unappreciated regulatory capabilities for this extracellular proteinase. These realizations imply that MMP-9 exerts a more profound role during early events in tumor evolution. The overall goal of this proposal is to identify the target epithelial cells regulated by MMP-9, identify molecules that interact with MMP9 mediating proliferative responses and, identify intracellular signal transduction pathways activated as a consequence of MMP-9-induced proliferation. We will address the characteristics and functional significance of these parameters/mechanisms by combined in vivo and in vitro approaches. The specific aims of this project are: Aim 1. To determine mechanisms regulating neoplastic epithelkil cell proliferation by MMP-9. We will determine the identity of the target epithelial cells responsive to MMP-9 and assess candidate protein substrates of MMP-9 mediating proliferative responses. Aim 2. To determine the mechanism(s) of activation responsible for MMP-9 -induced proliferation. Using in vitro cell culture systems that phenocopy proliferative responses to MMP-9 observed in vivo, we will determine the identify of critical protein targets and intracellular signal transduction pathways specifically activated by MMP-9. Aim 3. Determine the impact of MMP-9 on the initiation, promotion, and malignant conversion steps of multistage epithelial carcinogenesis. The functional importance of MMP-9 at discrete steps of multi-stage epithelial carcinogenesis will be ascertained using the classical 'two-stage' model of chemical carcinogenesis. These experiments will reveal if MMP-9 plays a more significant modifier role at the initiation, promotion, or malignant conversion stages of cancer development. The immediate ramifications of this work are in its applications to use of MMP-Inhibitors (MMPIs) as anticancer agents. Only a thorough understanding of the actions and effects of MMP-9 will effectively guide use of MMPIs in the treatment of cancer.

[unreadable] DESCRIPTION (provided by applicant): Transgenic mouse models of human cancers present unique opportunities to elucidate important cellular and genetic pathways of cancer development, through which normal cells progressively are converted into aberrant cancers. The overall goal of this proposal is to identify cellular signaling pathways impacting tumor development regulated by collagenolytic matrix metalloproteinases (MMPs) and their major stromal substrate, type I collagen, during squamous neoplastic progression in the skin. We will address the characteristics and functional significance of epithelial-stromal interactions regulated by collagenolytic MMPs and type I collagen by combined in vivo and in vitro approaches. The specific aims of this proposal are: Aim 1. Determine the mechanism(s) of type I collagen remodeling during tumor progression. We will determine the expression, localization, and activity of collagenolytic enzymes, and expression, synthesis, and degradation characteristics of type I collagen by molecular, biochemical, morphologic, and histochemical criteria during the development and metastasis of epithelial cancer in a transgenic mouse model of squamous carcinoma development. Aim 2. Determine the consequences of altered type I collagen degradation in vivo: Altered substrate susceptibility. We will determine the impact of collagenaseresistant type I collagen on neoplastic progression, tumorigenesis, tumor stroma development, and the local metastasis of cancers in HPV16 transgenic mice. Aim 3. Determine the consequences of type I collagen remodeling on cell behavior. We will determine the functional consequences of type I collagen remodeling on epithelial and endothelial cell behavior by combined in vivo and in vitro analyses. These studies will reveal the molecular and cellular mechanisms engaged by different populations of cells resulting from type I collagen remodeling during multi-stage neoplastic progression. The immediate ramifications of this work are in its applications to use of MMP-Inhibitors (MPIs) as anticancer agents and will provide proof-of-principle that eliminating productive access to a critical MMP substrate is an efficacious intervention for neoplastic progression. Only a thorough understanding of the actions and effects of MMPs and their major substrates will effectively guide use of MPIs in the treatment of cancer. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): It is well established that chronic inflammation contributes to cancer development. Many studies have demonstrated that inflammatory leukocytes promote epithelial cancer by providing soluble growth and survival factors to initiated cells and contribute to tissue remodeling and angiogenesis via synthesis of extracellular proteases; thus, physiological processes necessary for tumor development (enhanced cell survival, tissue remodeling and angiogenesis) are regulated, in part, by leukocytes and the soluble mediators they deliver. However, molecular mechanisms mediating the dialogue between infiltrating immune cells with initiated epithelia are poorly characterized. Moreover, the degree to which these interactions alter stem cell niches in neoplastic environments have not been explored. We hypothesize that infiltrating immune cells regulate niche autonomy of putative lung cancer stem cells through activation of Wnt and Sonic hedgehog (Shh) signaling cascades in initiated lung epithelia; thus, the goals of this project are to define the lineages of functionally significant immune cells that potentiate cancer development in lung, determine which of these regulate Wnt and Shh signaling in lung epithelia, and determine if in so doing, they confer stem cell niche autonomy to initiated epithelial cells and therefore enhance tumorigenic potential. To assess our hypothesis, we propose to, 1) define the profile of immune cells associated with human and mouse lung carcinogenesis and determine how these correlate with presence of CD133+ cells and activation of Wnt and Shh signaling cascades, 2) define functional significance of recruited immune cells as regulators of Wnt and Shh signaling during lung carcinogenesis, and 3) Define the functional significance of immune cells as regulators of Wnt and Shh signaling and their combined effects on putative lung cancer stem cells. [unreadable] [unreadable] [unreadable]

DESCRIPTION: Breast cancer is the most common cancer and second leading cause of death in women. Neoadjuvant chemotherapy is increasingly used to "shrink" tumors prior to surgery and enable breast conservative approaches;however, long-term survival remains poor, in part due to factors that limit delivery of cytotoxic drugs to tumor tissue. Abnormal tumor blood vessels and altered transendothelial permeability and increased interstitial fluid pressures (IFP), conspire to limit delivery of macromolecular cytotoxic drugs. Thus, approaches that aim to alter tumor vessel hemodynamics and vascular permeability would effectively increase tissue accumulation of chemotherapeutic agents by overcoming high IFP and promoting convection driven uptake of large macromolecular agents into tissues. To this end, we have discovered a novel, endogenous pathway regulating vascular permeability that remains functional in tumor vessels. Whereas transforming growth factor beta 1 (TGF?1) restricts normal vascular permeability, inhibition of the type I TGF??receptor Alk5 expressed in vascular cells, enhances vascular permeability in normal as well as tumor vasculature (Sounni et al. manuscript submitted). Thus, we propose to administer an Alk5 inhibitor, in combination with macromolecular chemotherapeutic agents to improve breast tumor perfusion and accumulation of conventional chemotherapeutic agents. Paradoxically, while increases in tumor perfusion and improved penetration of cytotoxic agents have also been achieved by 'normalization'of tumor vasculature by blocking vascular endothelial growth factor (VEGF) to reduce vascular permeability, these effects are transient and largely limited to immature vessels more frequently associated with early stage tumors. As tumors progress, tumor vessels mature and become refractory to VEGF blockade. Importantly, we have also observed that TGFb1-mediated vascular stabilization remains functional in more mature tumor vessels, and further predict that Alk5 blockade will improve cytotoxic drug penetration in both early as well as late stage tumors. We will compare accumulation of Doxil in mammary tumor-bearing mice treated with Alk5 inhibitor as opposed to those treated with anti-VEGF antibody (DC101), and assess vascular permeability and accumulation of Doxil in both early and late stage tumors in MMTV-PymT mice. Moreover, while previous studies have used MR imaging data to asses breast cancer responses after cytotoxic drug administration, we will demonstrate that MR imaging of macromolecular contrast media (MMCM) in the presence or absence of Alk5 blockade correlates with Doxil accumulation and thus can be used to predict macromolecular drug distribution and to identify tumors most likely to benefit from cytotoxics combined with agents that improve vascular permeability. In addition, we will assess the anti-tumor impact of improved Doxil accumulation following Alk5 blockade by monitoring histopathologic characteristics of mammary adenocarcinomas, as well as tumor burden, tumor latency (to endpoint), and frequency of pulmonary metastasis, and further demonstrate that MR imaging based predictions of drug accumulation also correlates with therapeutic responses. We will also examine responses of orthotopically implanted human breast tumor cells in mice treated with Alk5 inhibitor plus Doxil versus Doxil as monotherapy, to demonstrate enhanced efficacy of Doxil in human tumor cell killing. Together these studies will establish the effectiveness of 1) improved cytotoxic drug accumulations in the presence and absence of ALK5 inhibitors at different tumor stages, 2) MRI predictions of tumor microvascular permeability and cytotoxic drug accumulation, and 3) enhanced tumor responses and reduced cytotoxicity from improved chemotherapeutic drug accumulation in tumors. Thus, by utilizing new predictive MRI correlations and exploiting a novel endogenous pathway regulating vascular permeability that remains functional in breast cancer, the delivery of chemotherapeutic agents and subsequent response of both early and late stage breast tumors will be radically improved.

DESCRIPTION (provided by applicant): Mouse studies offer tremendous insight into the development of normal tissues and of diseased tissues including cancer. The ability to image mice repeatedly and non-invasively is necessary for understanding the course of therapeutic interventions and to minimize the use of mice in experimentation. Ultrasound imaging is the accepted standard for high resolution imaging, offering the ability for repeated, inexpensive, investigator-operated, real-time monitoring of mice. The many NIH-funded investigators using mice at the University of California San Francisco Parnassus have relied on a Vevo 770 ultrasound small animal imager that will be moved to the newly opened research campus at Mission Bay. This planned move will leave the investigators without the ability for noninvasive imaging. Because the mice are behind a barrier, longitudinal studies by other imaging modalities such as MRI, CT or PET scanning are not possible due to their locations outside the barrier facility. Thus, these investigators have joined to apply for a new ultrasound and, in the process, have decided to take advantage of the higher resolution, greater depth of field and capabilities for vascular analysis of the new Vevo 2100. The multiple major users on this application are diverse, but are united by the desire to carry out longitudinal, non-invasive studies in mice. The high resolution imaging will enhance the ability of researchers to diagnose tumor development de novo, localize tissues for accurate injection, and confirm early implantation and monitor embryonic development in models of lung development. The increased depth of focus will enable visualization of organs throughout the abdomen, needed for detecting and monitoring pancreatic tumors and mesothelioma. The Doppler components (pulsed wave and power) will permit visualization of the vasculature of a tissue and its change over time. The 3D capability will rapidly and accurately measure the volume of a defined structure, such as a tumor, and will display the heterogeneity of tissues so that different regions can be analyzed separately. Finally, the contrast enhancement features expand the capabilities of the ultrasound: with non-targeted synthetic bubbles, the Vevo 2100 can quantify relative blood flow to an organ; with synthetic bubbles targeted to various endothelial targets such as VEGF receptor, the Vevo 2100 can identify relative expression of the targets characteristic of tumor angiogenesis. A Vevo 2100 Small Animal Imager would immediately address the needs of the NIH- funded investigators included here as major users and would enhance opportunities for all mouse users of the barrier facility. With the added support of the Simmons Mesothelioma Foundation to provide long-term maintenance, this state-of-the-art small animal imager will enhance research for the diverse community of those studying mice at UCSF. Relevance to Public Health: Mouse models of human disease are essential for understanding the underlying biology of disease and for developing effective therapies. The translational value of studies will be greatly enhanced by the ability to image living mice non-invasively and repetitively over time. A small animal ultrasound imaging system will allow investigators to monitor mice used in their research for the development of conditions and the response to therapy, thereby increasing efficiency, reducing the number of mice needed, and speeding the development of potential treatments for patients.