Hallgeir Rui - US grants

Prolactin (PRL) receptors regulate vital physiological processes such as reproduction, immune function, morphogenesis, cell proliferation and differentiation. Human PRL receptors are activated by PRL as well as placental lactogen and growth hormone. Overstimulation of PRL receptors has been associated with breast and prostate tumor promotion. the long-term objective of this proposal is to understand the molecular basis for how prolactin receptors activate multiple parallel signal transduction pathways in target cells. As our first major objective in pursuit of this goal, we proposed on the basis of work that will presented under Preliminary Studies to test the central hypothesis that prolactin receptors signal via multiple parallel transduction pathways that are cell-dependent and selectively modulated by external stimuli. This proposal has three major, intimately linked goals, each maintaining a primary focus processes. These goals are: 1) To determine how PRL receptors interact with multiple JAK tyrosine kinases (JAK1, JAK2 and TYk2) in a cell dependent manner, 2) to identify PRL receptor-mediated activation of Src-tyrosine kinases in mammary epithelial cells and hematopoietic cells, and investigate the importance o cytoplasmic receptor regions for cell-dependent recruitment of specific Src kinases and the activation of the SHC/RAS/MAPK pathway, 3) to determine whether activation of multiple parallel signaling pathways by PRL in target cells can be selectively regulated by external physiological and pharmacological modulators, and specifically explore the impact of maintained JAK2/STAT5a/b signals and uncoupling of STAT1/3SHC- RAS-MAPK signals on PRL inducted proliferation and gene regulation in Nb2 lymphoctyes. The best models of direct studies of these initial biochemical reactions remain simple, homogenous cell populations cultured in vitro. Rat Nb2 pre-T lymphocytes and several well-differentiated human mammary epithelial cells lines will be used for these studies. PRL signaling will also be reconstituted in naive myeloid cells and b the introduction of novel PRL receptor variants into mammary epithelial cells. Our expectations are that, at the conclusion of the proposed period of support, we will have identified how PRL receptors can activate multiple JAKs, and defined regions of the human PRL receptor specifically responsible for triggering SHC/RAS/MAPK and Src tyrosine kinases in relevant human mammary cells. We also expect to shed new light on external modulation of individual PRL receptor signaling pathways. With our extensive background in signal transduction, we expect to converge to regulate vital physiological processes such as cellular growth and differentiation.

DESCRIPTION: Prostate carcinomas resist chemotherapy due to a low fraction of dividing cells, and new strategies are sought that also will induce apoptosis in non-dividing cells. This goal might be achieved through inhibition of signaling pathways used by the most promising autocrine growth and survival factors for prostate cancer, interleukin-6, EGF and prolactin. Signaling by these cytokines converge on Jak tyrosine kinases and Stat transcription factors. To date there are no published studies on Jak-Stat signaling in normal or malignant prostate. Based on compelling new data and novel preliminary observations, we propose to test the central hypothesis that Jak2 tyrosine kinase and Stat5 transcription factors mediate anti-apoptotic cytokine signals in normal and malignant prostate epithelial cells. Genetic (knockout mice), molecular/epigenetic (adenoviral gene delivery), and pharmacological approaches will be continued. Specific Aim 1: Employ adenoviral transfer of dominant-negative mutants to assess the apoptotic and growth-inhibitory effect of specific suppression of Jak2 tyrosine kinase and Stat5 transcription factors in human prostate cancer cells and tumor tissue explants in vitro and in human prostate tumor models in vivo. Specific Aim 2: Test whether Stat5 deficiency in mice enhances castration-induced apoptosis, and perform a morphological and functional characterization of specific epithelial defects we have discovered in prostates of Stat5-deficient mice. Specific Aim 3: Test whether pharmacologic inhibitors of the Jak2-Stat5 pathway will induce apoptosis in organ cultures of normal and malignant prostate and in a panel of prostate cancer cell lines under various growth conditions. Specific Aim 4: Identify the molecular structure of a 55 kDa short form of Stat5 that is uniquely activated inhuman prostate cancer cell lines and in the transplantable human CWR22 prostate cancer model, and functionally characterize its transcription regulatory effects. We are uniquely qualified due to our experience with Jak-Stat signal transduction, background in prostate biology and cancer, and unique research tools. We expect to determine the efficacy of suppression of Jak-Stat signals on survival and growth of normal and malignant prostate cells. The research is significant and important because it will provide novel insight into the roles of Jak2-Stat5 pathways in prostate function. More effective therapeutic strategies could be a result.

DESCRIPTION (provided by applicant): Each year, more than one million new cases of breast cancer are diagnosed worldwide, and an estimated 370,000 women die from breast cancer. The vast majority of fatal breast cancer cases involve metastatic spread of the primary tumor, but the metastatic process remains a complex and poorly understood process. Our long-range goal is to identify the molecular mechanisms of breast cancer progression from solitary tumor to metastasis. Based on our extensive molecular analysis of clinical human breast cancer specimens and a series of experimental breast cancer models, we now propose to test the following central hypothesis: Loss of activation of transcription factor Stat5 is a cancer progression event that favors epithelial-to-mesenchymal dedifferentiation, invasiveness, and increased metastatic potential of breast cancer cells. To accomplish the objectives of this application, we will pursue three specific aims: Aim #1: Analyze 1,300 human breast cancer specimens to establish the relationship between levels of active Stat5 and measures of tumor cell invasiveness. Aim #2: Establish whether Stat5 stimulates human breast cancer cell differentiation and adhesion, and suppresses tumor cell invasion, in vitro and in vivo. Aim #3: Determine the effect of Stat5 activation on invasion and metastasis of mouse breast cancer models in vivo. Our expectations are that by the end of the proposed project period, we will have established whether Stat5 activation status in breast cancer is a useful clinical predictor of disease progression and clinical outcome in lymph node-negative breast cancer. This is important because active Stat5 may serve as a simple immunohistochemical marker to identify node-negative breast cancer patients with excellent prognosis, permitting more individualized treatment, including selection of antiestrogen therapy. The results of this work could lead to new preventative and therapeutic strategies for primary and metastatic breast cancer.

[unreadable] DESCRIPTION (provided by applicant): The 2008 Prolactin and Growth Hormone Gordon Research Conference will provide a forum for the presentation and active discussion of the most cutting-edge information and ideas on Prolactin, Growth Hormone and related peptides. The Gordon Research Conference format will bring together investigators that are international leaders in the structural, biochemical, cell signaling, physiological, developmental and clinicalpathological aspects of these hormone ligands and their receptors. We expect that this conference will provide a thorough and in-depth coverage of prolactin and growth hormone biology, thus continuing the tradition of this being the premier meeting in this field. The peptide ligands and receptors for PRL and GH have been shown to mediate critical biological actions related to reproduction and growth, respectively. However, PRL has now been documented to also affect many nonreproductive targets, such as modulating behavior, promoting neurogenesis, inducing tumorigenesis, regulating angiogenesis and bone formation, and impacting the immune system. Similarly, GH biology extends beyond its classical IGF-1-stimulatory and growth-promoting effects. Indeed, the GH/IGF-1 system has been implicated in carcinogenesis, cellular maintenance and repair, and longevity/aging pathways. While these recent advances have provided important insights into PRL and GH actions, nevertheless the precise mechanisms by which these two hormones mediate their effects remain unanswered. We encourage investigators at all career stages interested in these areas to attend the PRL and Growth Hormone GRC. [unreadable] [unreadable] [unreadable]

Project Summary Despite enormous expenditures and efforts by academic, government, and pharmaceutical institutions, most drugs that show promise against human breast cancer in preclinical testing in mice, fail to cure breast cancer in the clinic. There is a great need for improved drug response-predictive testing of human breast cancer in the preclinical setting to identify the best drug candidates. Based on novel insight gained in our laboratories, as a key first step we propose to correct the mouse endocrine environment to more adequately mimic the endocrine environment of breast cancer patients. Novel and independent insight from the two collaborating laboratories suggest that prolactin acts as a modulator of drug sensitivity in human breast cancer. Prolactin receptors are expressed, often at elevated levels, in a majority of human breast cancers. However, current mouse models are inadequate since mouse prolactin prevents activation of human prolactin receptors. Thus, current predictive testing of drugs against human breast cancer is performed on human breast cancer lines selected for growth under prolactin-free conditions. We will now test the role of prolactin as a modulator of breast cancer drug sensitivity and biology. For this study, a mouse model that expresses physiological levels of human prolactin has been genetically engineered for more accurate predictive testing of drugs on human breast cancer xenotransplants. The central hypothesis is that PRL receptor signaling, through its effects on mammary cell survival, growth, and differentiation, modulates sensitivity of breast cancer cells to anti-tumor agents. Consistent with this, we further hypothesize that mice, in which endogenous mouse prolactin has been replaced with physiological levels of human prolactin, will restore prolactin receptor signaling in human breast cancer xenotransplants and provide a more relevant endocrine environment for improved prediction of clinical responsiveness of breast cancer to therapeutic agents. Finally, we hypothesize that the hPRL expressing mice will allow us to establish new and transplantable breast cancer lines that more closely resemble primary breast human cancer than existing metastasis-derived tumor cell lines. If successful, the new mouse model would be available for testing of a broad number of human breast cancer drug candidates, with the potential for more reliable prediction of clinical efficacy. In addition, successful transplantation of primary breast cancer tissue with epithelial and stromal components will pave the road to a new personalized medicine approach to treatment of breast cancer patients. Project Narrative Most drugs that work when tested on human breast cancer in mice, fail to cure breast cancer in humans. There is a great need to improve the ability to predict whether candidate breast cancer drugs will work in patients. To this end, we have developed a new mouse model with a hormone environment that more closely resembles that of breast cancer patients. We will test the hypothesis that the new mouse represents an improved preclinical model for more accurate predictive testing of breast cancer drug candidates.

ENDOCRINE MECHANISMS AND HORMONE ACTION IN CANCER PROGRAM The Endocrine Mechanisms and Hormone Action in Cancer (EMHAC) Program is a new cancer center program, initiated in 2006. The overall goal of the program is to discover molecular alterations and signatures of hormone-related cancer and translate these findings into new strategies for effective detection, diagnosis, and cure. The program maintains a focus on translational. research on tumors arising from hormone-responsive tissues, including breast and prostate. Three program areas of focus include: 1) detection and diagnosis, 2) prognosis and progression, and 3) targets and therapy. The main specific Scientific Goals of the program are to: Develop innovative strategies and technologies for early detection of primary cancer and monitoring of therapy response and disease recurrence. Improve diagnostic tumor classification, prognostic and predictive marker analyses, and design of pre-clinical models for drug response prediction to facilitate improved tailored treatment. Exploit mechanisms of hormone action for molecular target identification, improved therapy, and prevention of hormone responsive cancer. Several collaborative initiatives are ongoing between the 15 members from 6 departments, supported by peer-reviewed funding of $4.2 million ($2.7 NCI) per year. An P01 program project grant proposal on breast cancer has been submitted from the program. Although this is a new program, several program members have worked collaboratively over many years, with more than 100 joint publications. Since the program's inception in 2006, there have been more than 20 intra-programmatic publications.

DESCRIPTION (provided by applicant): Despite enormous expenditures and efforts by academic, government, and pharmaceutical institutions over many years, most breast cancer drugs that show promise in mice, fail to cure breast cancer in patients. Such inefficiency is enormously costly and hampers identification of new and effective breast cancer drugs. This discrepancy suggests that mice are not a reliable model for correct prediction of drug responsiveness of human breast cancer. Nonetheless, before candidate drugs against breast cancer are allowed into clinical trials, FDA requires promising effects on human breast cancer grown in immunodeficient mice. We have discovered and corrected a key hormonal deficiency of mice that make regular mice suboptimal for modeling and drug testing of estrogen receptor-positive human breast cancer. We have created genetically engineered mice that restore the missing hormone component, pituitary human prolactin, and have determined that the prolactin-humanized mice are excellent recipients for estrogen receptor-positive patient-derived breast cancer tissues. Intriguingly, two of the first estrogen receptor-positive patient derived lines tha we have established spontaneously metastasize to lungs from orthotopic mammary implantation sites. These models will for the first time allow us to explore new therapeutic strategies to improve the efficacy of anti-estrogens in the metastatic setting. This is important because adjuvant treatment for breast cancer is given after surgical resection of primary tumors, and breast cancer patients die from metastases and not from the primary tumor. The objectives will be achieved by exploring combination treatment of antiestrogens with prolactin-pathway suppressive drugs as well as parallel molecular profiling of lung metastases and primary tumors to identify alternative candidate drug targets. The novel concepts of this exploratory R21 project are supported by solid scientific rationale and previously unavailable experimental models. The potential impact is strong, with the possibility of uncovering improved therapeutic strategies to metastatic ER-positive breast cancer that could benefit patients within a very short time frame. This project is significant because the majority of patients who die from breast cancer were initially diagnosed with ER-positive disease. Finally, the new hormonally improved mouse model could benefit individual patients immediately by serving as a culture vessel to determine the sensitivity of a patient's tumor against a panel of existing drugs. Life-saving, tailored therapy fr metastatic breast cancer could be a result.

? DESCRIPTION (provided by applicant): Estrogen receptor-a positive (ER?+) breast cancer (BC) represents 70-80% of newly diagnosed cases. While potentially responsive to anti-estrogens, progression of ER?+ BC to anti-estrogen refractory disease in the metastatic setting is a common occurrence. Endocrine resistance mechanisms may differ between subtypes of ER?+ BC and involve both ER?-independent and ER?-dependent forms that remain poorly understood. Prolactin (PRL) interacts with estrogens to stimulate growth of certain subtypes of ER?+ BC. At the same time, the PRL-Jak-Stat5 pathway promotes differentiation and inhibits invasive features of BC, and loss of Stat5a signaling in a subgroup of ER?+ BC is associated with anti-estrogen therapy failure. The involvement of PRL pathways in growth and progression of anti-estrogen refractory BC subtypes remains to be determined. There is a lack of preclinical human ER?+ BC models that recapitulate progression from localized mammary gland growth to distant metastasis. Xenografts of patient-derived ER?+ BC exhibit poor take rate in mice. We have discovered that murine PRL is a poor agonist and a potent antagonist for human PRL receptor (PRLr). Bovine PRL is also a poor agonist with antagonist activity for human PRLr. Laboratory human BC lines therefore have been selected for PRL-independent growth and may only represent subtype(s) of ER?+ BC. To address this problem, we generated hPRL knock-in mice in the immunodeficient Nod-Scid-IL2R? strain that express physiological levels of circulating hPRL. Remarkably, PRL-humanized mice display greatly increased take rate of patient-derived xenografts of ER?+ BC. We established a novel panel of serially transplantable ER?+ Luminal B BC lines, several of which spontaneously metastasize to lungs and liver. The distant metastases become anti-estrogen refractory despite continued expression of ER?+, but show PRL-dependence. Our long-range goal is to determine mechanisms of anti-estrogen refractoriness of BC to improve clinical management. Aim 1 is focused on a recently identified Luminobasal subtype of ER?+ BC (ER?+/CK5+). Aim 1 explores a distinct ER?-independent mechanism of anti-estrogen refractory BC in pre-existing ER?+ laboratory cell lines. We hypothesize that in Luminobasal BC, loss of PRL-Stat5 signaling promotes loss of ER? and subsequent anti-estrogen resistance due to defective Stat5a-driven differentiation. Aim 2 is focused on the Luminal B BC subtype (ER?+/CK5-/Ki67high) and is centered on our new PRL-dependent patient-derived xenograft lines. Aim 2 will test the hypothesis that in Luminal B BC, unlike in Luminobasal BC, PRL facilitates growth and survival of metastases and that PRLr-pathway targeting cooperates with anti-estrogens to eliminate distant metastases.