Douglas Hanahan - US grants

The goal of this proposal is to study the effects of a series of oncogenes when expressed in a specific cell type of transgenic mice, namely the insulin producing beta cells of the endocrine pancreas. These are epithelial cells which are central to the maintenance of proper carbohydrate balance. It is expected that this analysis will contribute to our knowledge of the properties of oncogenes, which in large part has resulted from assaying their effects on cells cultured in vitro. The specific experimental design is to target oncogene expression to the beta cells using the insulin gene regulatory region. We have already established that these sequences are capable of directing expression of a linked oncogene (the early region of SV40) exclusively to the beta cells of the pancreas. Among the new hybrid oncogenes to be examined are Hras, E1A, v-myc, cmyc, p53, v-src, and v-erbB. The consequences of oncogene expression will be evaluated from several perspectives: 1) does targeted expression of individual oncogenes produce tumors, and at what rate, and in what number per animal, 2) what are the effects when pairs of hybrid oncogenes are combined by mating individual transgenic mice in a genetic complementation assay; 3) do individual oncogenes or specific pairs of oncogenes cause metastasis of the beta cell tumors, and 4) do certain insulin promoted oncogenes cause diabetes rather than cancer, as a direct or indirect result of their expression in beta cells. The tumors will be characterized with respect to cellular and chromosomal anatomy, alterations in endogenous gene expression, and the possibility that secondary mutations occurred during oncogenesis. In addition, the immunological and physiological consequences of oncogene expression in the islets of Langethans will be evaluated.

Bovine papillomavirus type 1 induces skin tumors when it is transmitted through the germ line of transgenic mice. The two frequent skin pathologies which arise -- abnormal skin and protuberant tumors -- will be analyzed in order to assess several notable characteristics of tumorigenesis: the slow appearance of abnormalities, at sites prone to irritation and wounding; the inactivity of the BPV genome in normal skin tissue and its activation in all abnormal conditions; and the apparent progression in the transformed phenotype of affected dermal fibroblasts. Using tissue biopsies and primary cultures of dermal fibroblasts from normal transgenic skin, abnormal skin, and protuberant tumors, we will characterize: 1) the four apparent states of dermal fibroblasts, so as to establish criteria with which to distinguish them on molecular and cellular levels; 2) the nature of the activation mechanism for BPV in normal skin, as to whether its silence results from cis or trans effects; 3) the possible role of the immune system in slow oncogenesis; and 4) tumor progression, both as it occurs naturally in these mice, as well as in response to attempts to accelerate it. Two mutant BPV genomes will be tested in new lines of transgenic mice, to assess the necessity of extrachromosomal replication and the 31% "late" region in tumorigenesis. The BPV E5, E6, and E2 ORFs will be widely expressed as hybrid genes in transgenic mice, in order to assess the tissue specificity of their activities. The genomes of human papilloma viruses type 5 and type 18 will be established in lines of transgenic mice, in order to develop mouse models for the human cancers associated with these tumor viruses. We will characterize the ability of HPV-5 and HPV-18 to heritably induce tumor formation in transgenic mice, and analyze the types of abnormalities which arise, with respect to the expression of the HPV oncogenes, the histological character of the affected cells, and their cellular phenotypes. Additional experiments will seek to induce and/or accelerate tumorigenesis with specific agents, which will include carcinogens, tumor promoters, radiation and retroviruses expressing oncogenes such as Ha-ras and c-myc. The E6/E7 and E5 regions will be expressed under broad specificity promoters in transgenic mice, so as to address the penetrance (of specificity) of these oncogenes.

This Program Project intends to collectively investigate in the individual characteristics, interactions, developmental interrelationships, and regulation of the four pancreatic islet cell types, which synthesize and secrete either glucagon (alpha cells), insulin (beta cells) , somatostatin (delta cells) , or pancreatic polypeptide (PP cells). Project 1 (Hanahan) will establish transgenic mice developing specific tumors of all four islet cell types, derive cell lines of each and study the expression of the four hormone genes in each cell type. The development of the islet cells will be studied with the goal of identifying the putative islet stem cell. CDNA libraries to each cell type will be produced and genes isolated that correlate with the common (unique) features of the islet cells and/or their development. Project 2 (Baekkeskov) will produce a 2D protein gel database for the four islet cell types, and compare them by various criteria. This section further intends t identify, isolate and clone the genes for beta-cell specific genes, and to clone cDNAs for two proteins which correlate with the maturation of the islets. Projects 2 and 3 will investigate cell adhesion and its involvement of cell organization within the islets an will seek to identify and characterize the cell adhesion molecules which are involved. Project 3 (Kelly) will study hormone secretion in the four islet cell types, with particular emphasis on the beta-cell. Two types of secretory dysfunctions will be studied those that arise in tumor cells, and those that effect diabetes in transgenic mice overexpressing MHC molecules on their beta-cells. Cell polarization will be examined in all four islet cells, as will development of their secretory capability during embryogenesis. Project 4 (Rutter) will examine the expression of known growth factor receptors an nuclear transcription factors in each cell type, and will seek to isolate the protein and clone the genes for the glucagon and somatostatin receptors, and to examine the mechanisms by which the different islet cells communicate so as to regulate each other's hormone gene expression. This project also intends (with Project 1) to isolate cDNAs of novel islet cell receptors and transcription factors. This Program Project will substantially increase our knowledge of the cell an molecular biology of the four islet cell types, and especially, the distinctive proper ties of the insulin-producing beta-cell, which is so remarkably susceptible to specific destruction and the consequent onset of insulin-dependent diabetes.

neoplastic process; Langerhans'cell; growth factor receptors; viral carcinogenesis; insulinlike growth factor; diabetes mellitus genetics; pancreatic islet neoplasm; metastasis; telomerase; gene expression; cell growth regulation; tumor suppressor genes; loss of heterozygosity; cell adhesion molecules; cell population study; apoptosis; gene targeting; cell line; genetically modified animals; laboratory mouse; polymerase chain reaction; simian virus 40;

Bovine papillomavirus type 1 induces skin tumors when it is transmitted through the germ line of transgenic mice. The two frequent skin pathologies which arise -- abnormal skin and protuberant tumors -- will be analyzed in order to assess several notable characteristics of tumorigenesis: the slow appearance of abnormalities, at sites prone to irritation and wounding; the inactivity of the BPV genome in normal skin tissue and its activation in all abnormal conditions; and the apparent progression in the transformed phenotype of affected dermal fibroblasts. Using tissue biopsies and primary cultures of dermal fibroblasts from normal transgenic skin, abnormal skin, and protuberant tumors, we will characterize: 1) the four apparent states of dermal fibroblasts, so as to establish criteria with which to distinguish them on molecular and cellular levels; 2) the nature of the activation mechanism for BPV in normal skin, as to whether its silence results from cis or trans effects; 3) the possible role of the immune system in slow oncogenesis; and 4) tumor progression, both as it occurs naturally in these mice, as well as in response to attempts to accelerate it. Two mutant BPV genomes will be tested in new lines of transgenic mice, to assess the necessity of extrachromosomal replication and the 31% "late" region in tumorigenesis. The BPV E5, E6, and E2 ORFs will be widely expressed as hybrid genes in transgenic mice, in order to assess the tissue specificity of their activities. The genomes of human papilloma viruses type 5 and type 18 will be established in lines of transgenic mice, in order to develop mouse models for the human cancers associated with these tumor viruses. We will characterize the ability of HPV-5 and HPV-18 to heritably induce tumor formation in transgenic mice, and analyze the types of abnormalities which arise, with respect to the expression of the HPV oncogenes, the histological character of the affected cells, and their cellular phenotypes. Additional experiments will seek to induce and/or accelerate tumorigenesis with specific agents, which will include carcinogens, tumor promoters, radiation and retroviruses expressing oncogenes such as Ha-ras and c-myc. The E6/E7 and E5 regions will be expressed under broad specificity promoters in transgenic mice, so as to address the penetrance (of specificity) of these oncogenes.

This program project continues its investigation of the cell and molecular biology of the four neuroendocrine cell types comprising the pancreatic islets, so as to clarify their inter-relationships and distinguishing features. Of particular relevance to the mechanism of type I diabetes are the unique qualities of the insulin producing beta cells, which are selectively destroyed, leaving the alpha, delta, and PP cells intact. An exciting candidate is the 65kd isoform of glutamic acid decarboxylase, which was discovered by Baekkeskov et al. in the context of this program project to be the '64kd' beta cell autoantigen in type I diabetes. GAD65 has been and will continue to be a major focus of this interactive program, with all three projects planning studies on the biology of GAD65. Our plans are: Project 1 (Hanahan) will produce mice carrying gene knockouts of GAD65 and betacellulin, a TGF-alpha family member expressed in beta cells, to assess the roles of these genes in embryogenesis and in islet development and function. The oncogene SV40 Tag will be integrated into the genes for somatostatin and pancreatic polypeptide, and gene-targeted mice produced, from which delta and PP cell tumors and tumor cell lines will be derived, to complete the set of representative islet cell lines. A cell type (mPAC) associated with juvenile islets will be investigated both for its potential as an islet progenitor, and as a target for pancreatic ductal adenocarcinoma. Project 2 (Baekkeskov) will characterize the biosynthetic pathway of GAD65, which is membrane anchored, and identify the sequences that elicit its subcellular localization to synaptic like micro-vesicles. Tansgenic mice over-expressing human GAD65 will be investigated to learn more about the normal functions of GAD in beta cells, and its ability to disrupt beta cell function through improper expression. Similarly, the GAD65 knockout mice will be studied to ascertain the physiological consequences of a failure to produce this enzyme. The capability of GAD to act as an autoantigen in beta cells of transgenic mice will be investigated, which should contribute to our understanding of its roles in the human autoimmune disease. Project 3 (German/Rutter) will investigate transcriptional regulation of genes implicated in islet cell function and disease, including the islet hormones, glucokinase, islet amyloid, and GAD65, using cell lines representative of differentiated and 'progenitor' islet cell types. The goal is to define the key transcriptional regulatory proteins that elicit cell type specificity among the closely inter-related alpha, beta, delta, and PP cells, with the expectation that this information will help explain the unique susceptibility of b cells to immune destruction. Having identified candidates for key islet regulatory factors, gene knockout mice will be produced to assess the importance of these factors for embryogenesis in general, and islet cell development and function in particular.

The central goal of this project is to develop relevant transgenic mouse models of carcinogenesis elicited by human papillomavirus oncogenes, and to use those models to characterize and define the mechanisms underlying each stage in the development of HPV associated cancer. A set of transgenic mice has been produced in which expression of the human papillomavirus type 16 (HPV16) oncogenes is directed to the basal cells of the epidermis, resulting in the predictable development of progressive dysplasias in the external epidermis, and anal papillomas. These neoplastic lesions are analogous to those seen in infected humans, of which only a small fraction go on to develop cancer. A major focus of this project is to define the necessary genetic or epigenetic events for malignant conversion to carcinoma. It is our conviction that these events will prove of relevance to the mechanisms of PHV associated cancers in humans, and hence their identification and characterization are crucial both to understand the biology of the tumorigenesis process, and to set a foundation for design of rational therapeutic interventions. The specific aims of this proposal are to: characterize the various stages of HPV16 associated pathology in terms of abnormal cell proliferation, abrogation of orderly keratinocyte differentiation, and differential expression of the HPV16 oncoproteins; similarly analyze the anal papillomas and their preneoplastic stages; identify transgenic mouse lines showing HPV oncogene expression in cervix, and assess the pathological consequences and susceptibility to cervical carcinogenesis; derive keratinocyte cell cultures representing the stages in skin and anal/genital neoplastic development, and compare their cellular and biochemical properties; define the requirements for progression to carcinoma, assessing general genetic background, two specific candidate genes (TGFalpha and p53), environmental carcinogens, and immunodeficiency; elucidate the functions of the individual HPV oncogenes in neoplastic progression; and begin an investigation of cellular factors that contribute to carcinogenesis.

This project has sought to exploit genetic manipulation of the mouse genome in order to study the development and characteristics of the cells comprising the pancreatic islets. Our proposition has been that such information will contribute to basic knowledge of the pancreas and its diseases (not only diabetes but also cancer), and perhaps to the eventual design of strategies to replenish the beta cells through the control of beta cell growth and differentiation. A major new focus of this project is applying to diabetes research the technology of homologous integration into embryonic stems cells so as to produce mice with targeted gene disruptions (or knockouts). A second new focus is on an exciting cell type associated with pancreatic islets that may represent a multipotential 'stem-like' cell for islet neogenesis and pancreatic cancer. Our specific aims are to: 1. Produce mice carrying a targeted knockout of the gene encoding the autoantigen glutamic acid decarboxylase (GAD65), discovered by Project 2 during the last funding period, and assess by its absence the functional contributions of GAD65 to normal development and postnatal activities of the pancreatic islets. 2. Produce mice carrying a knockout of betacellulin, a TGF/alpha family member implicated in beta cell tumorigenesis, so as to investigate its involvement in islet cell development and islet morphogenesis, as well as physiological function in the adult. 3. Homologously integrate the oncogene SV40 T-antigen into the somatostatin and pancreatic polypeptide genes in embryonic stem cells, and derive mice that carry the modified SMS/Tag and PP/Tag alleles with consequent d and PP cell tumorigenesis, from which representative tumor cell lines will be established. The knockout mice will also be bred to homozygosity, to assess the effects that absence of somatostatin and pancreatic polypeptide have on embryogenesis, as well as islet development, morphogenesis, and function. 4. Introduction of polyomavirus mT oncogene into juvenile islets resulted in establishment of epitheloid cell lines (mPAC) that heterogeneously co-express a pancreatic duct cell marker, cytokeratin, and a neuroendocrine marker, A2B5, in addition to somatostatin and pancreatic polypeptide mRNAs. Upon transplantation, mPAC cells produce classic ductal adenocarcinomas analogous to the major human pancreatic cancer. The cell type from which mPAC originate within juvenile islets, the cellular characteristics of mPAC, and the signaling pathway used in their transformation will be studied to clarify the possibility that mPAC cells represent on the one hand a duct cell progenitor of the islets, and on the other a target cell for pancreatic cancer.

pancreatic islet function; pancreatic islets; biomedical facility; physiology; histology; nucleic acid sequence; genetically modified animals; laboratory mouse;

Transgenic mouse models of cancer present a spectrum of experimental opportunities, including elucidation of pathways of tumor development and progression. As in human cancers, genetic changes during mouse tumorigenesis have the potential to be instructive about mechanisms underlying pathways to cancer. Over the last decade the RIP-Tag mouse model of islet cell carcinoma has proved a valuable prototype for investigating parameters of multistage tumorigenesis. Specific changes include characteristic losses of heterozygosity/DNA copy number on chromosomes 9 (designated LOH9) and 16 (LOH16), acquisition of resistance to apoptosis, and induction of angiogenesis. These observations have lead to the hypothesis that LOH9 encodes an apoptosis regulatory gene and LOH16 encodes an angiogenesis suppressor gene. This project brings together complementary talents of the Hanahan lab, which has expertise and experience in transgenic mouse models and their characterization, and the Gray lab, which has expertise in cancer genetics and in technologies for characterizing cancer cell genomes. Together these labs shall precisely determine the minimal extents of LOH9 and LOH16, test the hypothesis that apoptosis and angiogenesis are partly controlled by tumor suppressor genes in these regions and identify the involved genes, designated loh9 and loh16; respectively. In so doing, this project will shed light on the mechanism of tumorigenesis in this model, and serve to develop and refine technological strategies that should prove broadly applicable to mouse models of cancer. Specifically, this project will: Develop genome screening technologies to detect and fine structure map these tumor suppressor loci utilizing multiplex LOH and array-based CGH, and rigorously compare these techniques during the analysis of a bank of approximately 450 islet carcinomas; Assess the hypotheses that LOH9 encodes an apoptosis regulatory gene and that LOH16 encodes a gene that suppresses angiogenesis, using in vitro and in vivo bioassays in conjunction with genomic analysis, functional selection, and genetic complementation via BAC DNA transfer; Isolate loh9 and loh16 using functional complementation and/or positional cloning techniques, and begin to analyze their expression and roles in the islet carcinoma pathway, and in human cancers.

DESCRIPTION: (adapted from the investigator's abstract) Transgenic mouse models of human cancers present the opportunity to elucidate pathways of cancer development, through which a normal cell in its natural microenvironment is progressively converted into an aberrant cancer, acquiring characteristics that contribute to the resultant cancer phenotype. This laboratory has developed transgenic mice that express the human papillomavirus type 16 (HPV-16) oncogenes in basal keratinocytes; these 'K14-HPV16' mice develop squamous cell carcinomas of the epidermis, and, in concert with chronic estrogen treatment, cervical and vaginal squamous cancers. Both epidermal and cervical pathways to carcinoma are characterized by progression through histologically distinct stages. In humans, the oncogenes of HPV16 and related HPV subtypes are found in a majority of cervical carcinomas, and in premalignant lesions thought to precede those cancers. The overall goal of this proposal is to characterize and assess the function contributions of cellular parameters that appear during tumor progression in this mouse model of squamous carcinoma, parameters hypothesized to be influencing the developing cancers in distinct and complementary ways to those directly effected by the human viral oncogenes; these parameters are: 1. Selective upregulation of a fibroblast growth factor receptor in aggressive, metastatic cancers. 2. The acquired resistance to induction of apoptosis, the process of programmed cell suicide, which is implicated as a growth-limiting mechanism that must be controlled by successful cancers. 3. The upregulation of telomerase, an enzyme that protects the ends of chromosomes during cell proliferation and thereby sustains tumor growth potential, in aggressive epidermal carcinomas. 4. The apparent capability of CD8+ T cells to restrict the appearance of invasive squamous cancers. 5. Dermal infiltration by mast cells in epidermal dysplasias, and their role in malignant progression. The long term goal is to define the pathways to epidermal and cervical cancer in mice expressing oncogenes implicated in a relevant human cancer, identifying the critical cellular and molecular parameters governing progression, and eventually utilizing the knowledge of mechanism to design therapeutic and preventative interventions that can be evaluated in this preclinical model of human carcinogenesis.

DESCRIPTION (provided by applicant): This project will use a genetically engineered mouse model of cancer to study the efficacy of drugs that inhibit the function of receptor tyrosine kinases expressed in endothelial or peri-endothelial support cells of the angiogenic vasculature that is induced during tumorigenesis. The model involves pancreatic islet carcinogenesis in RIP1-Tag2 transgenic mice, wherein a pathway unfolds through the sequential appearance of distinguishable premalignant and malignant stages. The RIP-Tag model has been used to develop a set of therapeutic trial designs that are initiated at different stages of islet carcinogenesis; the result is that certain angiogenesis inhibitors show stage-specific efficacy, with some most effective at early stages and others at late stages of disease progression. Provocatively, trials with two kinase inhibitors suggest the hypothesis that both tumor endothelial cells and tumor pericytes can be separately targeted, and that by functionally inhibiting receptor kinases on each cell type, combinatorial efficacy with broader stage specificity can be achieved. Based on exciting preliminary results, this project will test the proposition that combinatorial targeting of kinases expressed in these vascular cell types, with and without chemotherapy, can produce objective and significant responses against both premalignant (prevention) and malignant disease. The aims are to: 1. Evaluate stage specificity and relative benefits of agents that variously interfere with VEGF receptor signaling in tumor endothelial cells. 2. Investigate the hypothesis that inhibitors of PDGF receptor signaling target pericytes and thereby disrupt the tumor vasculature, impairing angiogenesis, vascular integrity, and tumor growth. 3. Evaluate combinatorial strategies involving targeting of multiple cell types with receptor tyrosine kinase inhibitors to broaden stage specificity and improve efficacy. 4. Assess efficacy and target cell types of 'metronomic' chemotherapy in the stages of islet carcinogenesis, and determine the benefits of combining traditional vs. metronomic chemotherapy with RTK inhibitors. 5. Identify biological response markers of the distinctive targeting strategies using microarray expression profiling technology that might be used in translational studies with these drugs in other models and in human clinical trials. The results will give new perspective into the potential of targeting two component cell types of the tumor vasculature with kinase inhibitors so as to broaden activity and improve efficacy in distinct stages of progression, and thereby may significantly impact the clinical applications of such agents in treating different stages and kinds of human cancer.

DESCRIPTION (provided by applicant): This MMHC consortium proposal will address the immunobiology of carcinogenesis, in particular three critical issues related to mechanisms by which organ specific cancers develop, and to strategies aimed at treating or preventing cancer, as revealed by and to be studied in several genetically engineered mouse models of human cancer. The goals of the consortium team center upon: i) elucidating the cell and molecular mechanisms of immune enhancement, whereby cells of the innate and acquired immune system are recruited to infiltrate neoplastic lesions, wherein they have been shown to be capable of functionally enhancing neoplastic progression, in part by activating angiogenesis and amplifying tumor cell growth; ii) identifying the molecular basis of implicit barriers to T cell inflammation and killing of solid tumors that are evident in certain mouse models and further assessing its generality, and iii) refining strategies for induction of efficacious T cell immunotherapies in fully immunocompetent models of organ-specific carcinogenesis, both assessing and seeking to circumvent the potential interference with efficacy by immune enhancement and inflammatory barriers. The consortium team will investigate the regulation and manifestation of these interrelated facets of tumor immunobiology in a select group of organ-specific cancers, of breast, cervix, and pancreatic islet. The group will ask whether immune enhancement and inflammatory barriers are factors for premalignant and nascent tumor stages of carcinogenesis in these organs, and/or for the continuing growth and progression of well established solid tumors. The experimental designs will reveal whether immune enhancement and inflammatory barriers vary as a function of organ site and stage of progression, and provide insight into the possibility that these parameters can impact upon the success of tumor immunotherapy. The prospects for improving the efficacy of T cell immunity against either premalignant or malignant lesions in breast, cervix, and pancreas by combining mechanism-based disruptions in immune enhancement or inflammatory barriers will be assessed in preclinical trials. A remarkable group of tumor biologists and immunologists has been assembled to pursue these strategic goals with their collectively enabling and complementary expertise.

DESCRIPTION (provided by applicant): This project will address a hypothesis, based on a provocative set of preliminary results, that Prefoldin-4, a component of the Prefoldin pre-chaperonin complex, acts as a dominant tumor progression factor when upregulated by gene copy number increases in tumors. The new results, both from a mouse model of human cancer, and from bioinformatic assessment of PFDN-4 expression profiles in human tumors, implicates PFDN-4 as a gene whose upregulation functionally contributes to tumor progression. The hypothesis presents a rationale for the recurrent chromosomal amplification of human chromosome 20q13 detected in a significant fraction of breast, ovarian, and other human tumors, and for gains of the syntenic chromosomal locus in mouse models of pancreatic neuroendocrine and breast cancer. The specific aims are to: 1. Elucidate the mechanistic basis for the oncogenic activity associated with upregulated Prefoldin-4, assessing in particular the possibility that its transforming action is related to its normal function as a component of the Prefoldin prechaperonin complex that modulates actin and tubulin biosynthesis. 2. Assess the causality and roles of Prefoldin-4 upregulation in mouse models of multistage carcinogenesis, of pancreatic islets and breast. 3. Investigate the patterns and association of PFDN-4 upregulation in lesional stages of human tumors prone to DNA copy number increases of the Chr_20q13 locus, assessing in particular the implication that increased copy and expression of PFDN-4 will correlate with regions of increased malignancy, in support of the hypothesis that Prefoldin-4 is a tumor progression factor whose upregulation underlies the recurrent amplification of the 20q13 locus in human cancers. If the data forthcoming substantiate the hypothesis, and clarify when and how PFDN-4 exerts its oncogenic effects in the course of multi-step tumorigenesis, Prefoldin activity and/or the Prefoldin-4 subunit may well become the target for development of pharmacological inhibitors seeking to down-modulate activity, in particular in tumors cancers where Chr_20q13 is amplified and PFDN-4 upregulated, much as for Herceptin targeting of Her2/Neu in those breast cancers where it is upregulated.

DESCRIPTION (provided by applicant): The strategic goal of this project has for almost 20 years been to define the ontogeny of neuroendocrine carcinomas of the pancreas that develop via a series of stepwise transitions, inferred to reflect the acquisition of necessary capabilities to manifest a tumor, the so-called 'hallmarks of cancer'. One can foresee it will be possible to identify the regulatory determinants and functional effectors that dictate and underlay each of the stepwise transitions from normality to lethal cancer for this prototypical pathway, establishing a conceptual framework from which to consider pathways to cancer in other mouse models and in human cancers. The general approach involves a transgenic mouse model, RIP-Tag2, which presents the opportunity to follow the genesis of tumors from their earliest beginnings in the pancreatic islets up through their lethal culmination as invasive cancers. The pathway can be readily probed and perturbed with specifically altered genes or pharmacological inhibitors, to identify critical functional determinants and clarify mechanistic principles. The specific focus of this renewal is to investigate regulatory determinants of the 'invasive switch' that is activated during pancreatic tumorigenesis. Functional tests will assess the hypothesis that the invasive switch is mediated in significant part by an intracellular signaling circuit emanating from the IGF-1 receptor tyrosine kinase, as well as the ancillary possibility that members of the EGF receptor family are contributing to this or other capabilities that determine pancreatic tumorigenesis. Critical nodal points in these signaling pathways will be perturbed both pharmacologically and genetically, aiming to prevent progression to the invasive growth phenotype, or revert it, thereby clarifying their importance as regulators of invasion as well as other hallmark capabilities that enable tumorigenesis, potentially guiding therapeutic applications. The specific aims are to Aim 1. Assess the causality of IGF-1 receptor signaling for the invasive switch during islet tumorigenesis, and begin to define the critical downstream effectors of its pro-invasive signaling. Aim 2. Interfere with signaling by candidate EGF ligands and their multiple receptors to address the hypothesis that members of the EGF ligand/receptor family are functionally contributing to the regulation of islet tumorigenesis, including the invasive growth capability. Aim 3. Assess roles of IFG1R and EGFR-family signaling of the invasive switch in pancreatic ductal adenocarcinoma using a mouse model that recapitulates the human disease's lesional progression and oncogenic events.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This project will develop targeted nano-probes for molecular imaging to enable non-invasive early detection of incipient cancer, affording substantive improvements in sensitivity and selectivity. It brings together three research groups with complementary expertise in angiogenesis and mouse models of cancer (Hanahan, UCSF), in vascular profiling (Ruoslahti, Burnham Institute), and in clinical and experimental molecular imaging (Franc, UCSF). Peptides have been discovered that specifically home through the circulatory system to the angiogenic blood or lymphatic neo-vasculature of high-grade neoplasias and/or invasive carcinomas. These vascular signatures can distinguish cancerous lesions from their cognate normal tissue, as well as from blood/lymphatic vessels in other organs and neoplastic conditions. The aims are: [unreadable] 1. Develop imaging nanoprobes for detecting the blood and lymphatic neo-vasculature in two mouse models of cancer that undergo stepwise progression to carcinoma, using validated signature-finding peptides as modular specificity elements linked to agents appropriate for imaging with SPECT, PET, or MRI. [unreadable] 2. Discover and characterize a repertoire of new signature-finding peptides for blood and lymphatic vasculature of cervical and pancreatic ductal neoplasias and cancer, and determine which identify analogous lesions in the cognate human diseases. [unreadable] 3. Competitively evaluate mouse/human signature-finding peptides (and mixtures thereof) from Aim 2 to identify the best at delivering imaging reporters to the aberrant blood and lymphatic vasculatures in the mouse models of cervical (as a prototype) and pancreatic ductal cancer (for its an unmet clinical need). [unreadable] 4. In partnership with Centers of Excellence in Cancer Nanotechnology, test nano-probes in the mouse models consisting of the best human/mouse signature-finding peptides linked to new imaging nanostructures being developed by those centers, to identify optimal candidate nanoprobes for clinical evaluation. [unreadable] [unreadable] The modular imaging nanoprobes to be developed, by delivering imaging agents to organ sites of tumor angiogenesis and lymphangiogenesis, hold promise to enable early detection of human cancer. [unreadable] [unreadable]

Project 3 will use mouse models of multistage pancreatic carcinogenesis previously developed and concurrently analyzed by Project 1 to investigate the neoplastic microenvironment and its constituent cell types as functions of losses in the INK4a/Arf, p53, and/or SMAD4 tumor suppressors, and of activity of the PI3 Kinase pathway, all in the context of mutationally activated KRAS signaling. Microenvironmental parameters to be assessed include: angiogenesis and the character of the blood vasculature;the association of pericytes with the tumor vasculature;lymphangiogenesis and the morphology of the lymphatic vasculature;the abundance and types of infiltrating (tumor-enhancing) leucocytes and expression/activity of the matrix-degrading enzymes they produce;and the characteristics of the fibroblastic stromal cells. A central goal is to test the hypothesis that the angiogenic phenotype and other features of the neoplastic microenvironment of PanIN and PDAC are differentially regulated by the loss of particular tumor suppressor genes that are signatures of this disease. An ancillary goal, to be pursued in collaboration with Project 2, is to determine the importance of KRAS signaling via the PIS kinase network in the cancer cells for induction of the aberrant tumor microenvironment. Analysis of human tumor biopsies with the Experimental Pathology Core will assess the correlation of genetic and phenotypic parameters identified in the mouse. Neoplastic stage-specific preclinical trial designs will be established and used to test innovative chemotherapeutic regimens, targeted antiangiogenic therapies, and combinations that might guide future human clinical trials. Joint studies with the Imaging Core will develop probes that non-invasively visualize parameters of the microenvironment, both to monitor lesional progression, and responses to therapy. Biomarkers of the cell-of-origin/pancreatic cancer stem cell identified by Project 4 will be used to assess specific responses to therapy and roles in relapse/progression. . These studies, in engineered mouse models of de novo pancreatic cancer, will characterize in unprecedented detail the aberrant lesional microenvironment and its regulation by genetic mutations during multistage progression, and begin knowledge-based pre-clinical therapeutic trials with the potential to reveal strategies that could be translated into improved treatments for the human disease.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This project will develop targeted nano-probes for molecular imaging to enable non-invasive early detection of incipient cancer, affording substantive improvements in sensitivity and selectivity. It brings together three research groups with complementary expertise in angiogenesis and mouse models of cancer (Hanahan, UCSF), in vascular profiling (Ruoslahti, Burnham Institute), and in clinical and experimental molecular imaging (Franc, UCSF). Peptides have been discovered that specifically home through the circulatory system to the angiogenic blood or lymphatic neo-vasculature of high-grade neoplasias and/or invasive carcinomas. These vascular signatures can distinguish cancerous lesions from their cognate normal tissue, as well as from blood/lymphatic vessels in other organs and neoplastic conditions. The aims are: [unreadable] 1. Develop imaging nanoprobes for detecting the blood and lymphatic neo-vasculature in two mouse models of cancer that undergo stepwise progression to carcinoma, using validated signature-finding peptides as modular specificity elements linked to agents appropriate for imaging with SPECT, PET, or MRI. [unreadable] 2. Discover and characterize a repertoire of new signature-finding peptides for blood and lymphatic vasculature of cervical and pancreatic ductal neoplasias and cancer, and determine which identify analogous lesions in the cognate human diseases. [unreadable] 3. Competitively evaluate mouse/human signature-finding peptides (and mixtures thereof) from Aim 2 to identify the best at delivering imaging reporters to the aberrant blood and lymphatic vasculatures in the mouse models of cervical (as a prototype) and pancreatic ductal cancer (for its an unmet clinical need). [unreadable] 4. In partnership with Centers of Excellence in Cancer Nanotechnology, test nano-probes in the mouse models consisting of the best human/mouse signature-finding peptides linked to new imaging nanostructures being developed by those centers, to identify optimal candidate nanoprobes for clinical evaluation. [unreadable] [unreadable] The modular imaging nanoprobes to be developed, by delivering imaging agents to organ sites of tumor angiogenesis and lymphangiogenesis, hold promise to enable early detection of human cancer. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This project will develop targeted nano-probes for molecular imaging to enable non-invasive early detection of incipient cancer, affording substantive improvements in sensitivity and selectivity. It brings together three research groups with complementary expertise in angiogenesis and mouse models of cancer (Hanahan, UCSF), in vascular profiling (Ruoslahti, Burnham Institute), and in clinical and experimental molecular imaging (Franc, UCSF). Peptides have been discovered that specifically home through the circulatory system to the angiogenic blood or lymphatic neo-vasculature of high-grade neoplasias and/or invasive carcinomas. These vascular signatures can distinguish cancerous lesions from their cognate normal tissue, as well as from blood/lymphatic vessels in other organs and neoplastic conditions. The aims are: [unreadable] 1. Develop imaging nanoprobes for detecting the blood and lymphatic neo-vasculature in two mouse models of cancer that undergo stepwise progression to carcinoma, using validated signature-finding peptides as modular specificity elements linked to agents appropriate for imaging with SPECT, PET, or MRI. [unreadable] 2. Discover and characterize a repertoire of new signature-finding peptides for blood and lymphatic vasculature of cervical and pancreatic ductal neoplasias and cancer, and determine which identify analogous lesions in the cognate human diseases. [unreadable] 3. Competitively evaluate mouse/human signature-finding peptides (and mixtures thereof) from Aim 2 to identify the best at delivering imaging reporters to the aberrant blood and lymphatic vasculatures in the mouse models of cervical (as a prototype) and pancreatic ductal cancer (for its an unmet clinical need). [unreadable] 4. In partnership with Centers of Excellence in Cancer Nanotechnology, test nano-probes in the mouse models consisting of the best human/mouse signature-finding peptides linked to new imaging nanostructures being developed by those centers, to identify optimal candidate nanoprobes for clinical evaluation. [unreadable] [unreadable] The modular imaging nanoprobes to be developed, by delivering imaging agents to organ sites of tumor angiogenesis and lymphangiogenesis, hold promise to enable early detection of human cancer. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The strategic goal of this revised application is to develop mechanism-based experimental therapeutic targeting strategies aimed at circumventing 'evasive resistance'to anti-angiogenic therapy, a phenomenon discovered in the course of the current term of this project, whereby tumors whose angiogenic vasculature is disrupted by inhibitors of VEGF signaling acquire means to relapse and recommence growth after a transitory period of beneficial therapeutic response. The general approach involves using mouse models of cancer, principally neuroendocrine pancreatic tumors and glioblastoma, whose histopathology and angiogenic phenotypes mirror human tumors, along with pharmacological agents (monoclonal antibodies, hybrid antibody-ligand traps, and small molecules) targeting regulatory circuits governing tumor angiogenesis and vascular homeostasis. The specific aims of this renewal intend to characterize and identify therapeutic targets for three distinctive mechanisms of evasive resistance to inhibitors of VEGF signaling: Aim 1. Investigate the induction of additional and alternative pro-angiogenic signaling circuits as an evasion mechanism to anti-VEGFR therapy;Aim 2. Evaluate the capability of recruited bone marrow-derived cells (BMD-C) to help circumvent anti-angiogenesis drugs. Aim 3. Elucidate the parameters and assess mechanism-based therapeutic targeting of increased tumor invasiveness in response to angiogenesis inhibition. While much heralded, the arrival of angiogenesis inhibitors into the clinic, in particular ones targeting the VEGF signaling pathway, is not in general resulting in enduring responses, but rather transitory periods of improved quality of life (tumor shrinkage or stabilization;months of survival advantage) followed by renewed tumor growth and progression. The postulates forthcoming from this project to be pursued in its renewal are that 1) a component to such relapse is the induction of evasive resistance to VEGF inhibition, and 2) therapeutic strategies targeting evasive resistance mechanisms hold promise to produce more enduring anti-angiogenic therapies for human cancers. PUBLIC HEALTH RELEVANCE: While much heralded, the arrival of angiogenesis inhibitors into the clinic, in particular ones targeting the VEGF signaling pathway, is not in general resulting in enduring responses, but rather transitory periods of improved quality of life (modest tumor shrinkage or stabilization;months of survival advantage) followed by renewed tumor growth and progression. This project has discovered a basis for such transitory efficacy, involving the induction of three 'evasive resistance'mechanisms: revascularization mediated by alternative angiogenic signals, recruitment of vascular progenitor cells from the bone marrow, and increased invasiveness, parameters to be elucidated in this resubmitted competitive renewal application. Targeting such resistance mechanisms holds promise to produce more enduring anti-angiogenic therapies for human cancers.