Bryan R. Haugen - US grants

The anterior pituitary gland contains at least five terminally differentiated cell types, which includes somatotropes, lactotropes, thyrotropes, corticotropes, and gonadotropes. Each of these cells has highly specialized endocrine functions to produce hormones that have diverse effects on mammalian physiology. Sparingly little information is currently known about the developmental requirements for pituitary-specific gene expression. To date, only one pituitary-specific gene transcription factor has been discovered, called Pit-1/GHF1. RNA transcripts encoding Pit-1 are found in all pituitary cells, but protein expression is found only in somatotropes, lactotropes and thyrotropes. Despite its ability to transactivate both growth hormone and prolactin promoters, the role of Pit- 1 in TSHbeta gene expression in thyrotropes in unclear. Specifically, it is unable to stimulate TSHbeta subunit promoter activity in both homologous thyrotrope cells and in cells of non-pituitary origin. I have now discovered another pituitary-specific transcription factor whose mRNA and protein expression are restricted to cells of thyrotropic origin,. This factor is a variant isoform of Pit-1 and specifically transactivates the thyrotrope-specific TSHbeta subunit gene. I have called this new factor Pit-1T, to emphasize its structural similarity to Pit-1 and its restricted expression in thyrotrope cells. The overall goal of this grant proposal is to gain specific information on the role of Pit-1 and Pit-1T in thyrotrope cells. To achieve these goals, I propose three specific aims. First, specific protein-DNA interactions between Pit-1T and the TSHbeta promoter will be studied. I will use DNase I protection analysis, mutational experiments with the TSHbeta promoter, and gel mobility shift analysis to better understand these interactions. Secondly, I will take advantage of the unique Pit-1T transactivation domain to further analyze the protein-protein interactions that mediate the TSHbeta promoter specific effect of Pit-1T. I will use Far Western blot analysis and subsequent Far Western library screening to identify these interactions. Finally, I will use the information generated from the first two specific aims to reconstitute TSHbeta promoter activity and gene expression in a thyrotrope-derived cell line which has lost the ability to express the TSHbeta gene. I will use stable transfections of Pit-1, Pit-1T, and other identified factors into the thyrotrope-derived alphaTSH cell line to achieve this goal. Acquisition of new knowledge gained from these studies will advance our understanding of TSHbeta gene expression in thyrotropes, and provide insight into the complex molecular mechanisms governing cell-specific regulation of highly restricted genes.

A single center study of patients with thyroid disease studied before and after treatment, as well as 10 normal controls. Metabolic energy expenditure, exercise capacity and cardiovascular function will be evaluated in overt hyperthyroidism, subclinical hyperthyroidism, overt primary hypothyroidism, subclinical hypothyroidism and normal controls. Tissue specific effects will be evaluated in the treatment of patients with overt hypothyroidism using levothyroxine (LT4) to normalize TSH and 3,5,3' - trithyroacetic acid (Triac) to normalize TSH.

There is currently no effective chemotherapy for anaplastic carcinoma of the thyroid, which has a 50% survival of 6 months after diagnosis. Cell culture and mouse studies have shown that placlitaxel (taxol) is effective against anaplastic thyroid carcinoma. The current study is a multi-center phase i/ii looking at the effect of a 96 hour continuous infusion of taxol in patients with anaplastic thyroid carcinoma. Total accrual is expected to B 20-24 patients with 2-3 patients enrolled at each site.

DESCRIPTION: (Adapted from the applicant's abstract) Vitamin A has a well recognized, but poorly understood effect on the pituitary-thyroid axis. Vitamin A excess causes central hypothyroidism with suppressed levels of serum TSH and T4. The effects of vitamin A are mediated through two nuclear hormone receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Many synthetic derivatives of vitamin A, or retinoids, have been developed, and these retinoids exhibit unique chemotherapeutic and chemopreventive properties in many different cancers. An RXR-selective retinoid (Targretin, LG 1069) caused a reversible central hypothyroidism in patients, suggesting that the vitamin A effect on this axis is mediated, at least in part, through an RXR-liganded mechanism. The principal investigator's laboratory has utilized molecular techniques to show that retinoids directly suppress activity of thyrotrope-specific TSHbeta gene promoter. This mechanism appears to require the RXRgamma isoform, which has expression limited to the thyrotropes within the anterior pituitary gland. The goals of this proposal are to define the specific mechanisms governing the effect of retinoids on TSH regulation, to use the TSHbeta promotor as a model of negative gene regulation by retinoids, and to further define the role of RXR and the unique role of the RXR isoform.

DESCRIPTION (provided by applicant): Thyroid cancer is the most common endocrine malignancy with an annual incidence of 21,000 in the U.S. 5- 10% of patients have advanced thyroid cancer that is unresponsive to standard therapy, and 1,500 patients die each year from this disease. There are Currently no effective therapies for patients with advanced thyroid cancer. Retinoid therapy (derivatives of vitamin A) is effective in some patients with leukemia, head & neck cancer, lung cancer and breast cancer. This therapy has shown some promise for patients with advanced thyroid cancer, but only 20-40% of patients responsd to retinoid treatment. We hypothesized that patients with advanced thyroid cancer would respond to retinoid therapy based on retinoic acid receptor (RAR a, b, gamma) and retinoid X receptor (RXR a, b, gamma) expression. While testing this hypothesis, we identified a unique pattern of expression of two receptors (RARb and RXRgamma) in thyroid cancer cell lines and tumor tissue. The expression of these receptors is associated with growth inhibition by synthetic retinoids, and this heterodimer (RARb /RXRgamma) may be critical in mediating the retinoid response. We have also shown that expression of another nuclear hormone receptor (PPARgamma) is associated with growth inhibition by thiazolidinediones (TZD), and may form a unique heterodimer with RXRgamma to mediate this response. Based on these preliminary results, we hypothesize that two unique heterodimer pathways are necessary and sufficient for response to retinoid and TZD treatment: RARb/RXRgamma, which mediates growth suppression and differentiation, and PPARgamma/RXRgamma, which mediates growth suppression and apoptosis. The goals of this proposal are to define the roles of the RXRgamma, RARb and PPARgamma nuclear hormone receptors in mediating response to retinoid and TZD therapy for advanced thyroid cancer through in vitro cell line models and in vivo mouse thyroid carcinoma models. We will also explore the role of leukemia inhibitory factor (identified by microarray analysis) as a direct mechanism through which the effects of retinoids and TZD mediate growth arrest in advanced thyroid carcinoma. Successful completion of these aims will provide a fundamental understanding of how retinoids and TZD affect cancer cell growth and differentiation, as well as identify molecular tools to design clinical studies that will be the basis of individualized therapy for patients with cancer.

Arteries; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; Blood Tests; Blood Vessels; CRISP; Cardiac Diseases; Cardiac Disorders; Cholest-5-en-3-ol (3beta)-; Cholesterol; Clinical; Computer Retrieval of Information on Scientific Projects Database; Development; Disease; Disorder; Dysfunction; Functional disorder; Funding; Grant; Head and Neck, Thyroid; Health; Heart Diseases; Hematologic Tests; Hematological Tests; Hematology Testing; Hypothyroidism; Institution; Investigators; NIH; National Institutes of Health; National Institutes of Health (U.S.); Patients; Physiopathology; Process; Research; Research Personnel; Research Resources; Researchers; Resources; Risk Factors; Source; Thick; Thickness; Thyroid; Thyroid Function Tests; Thyroid Gland; Thyroid Gland Function Tests; United States National Institutes of Health; atheromatosis; atherosclerotic vascular disease; cardiac disease risk; cardiac disorder risk; disease/disorder; heart disease risk; heart disorder; heart disorder risk; improved; interest; pathophysiology; thyroid function; vascular

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area (15) Translational Science, 15-CA-103: Thyroid Cancer Cell Line Project which is in direct response to our recent discovery showing that 17 out of 40 "thyroid" cancer cell lines are either redundant or misidentified (i.e. of a different tumor lineage including melanoma and colon cancer). The remaining 23 lines are unique and are likely of thyroid origin based on the expression of a limited number of thyroid-specific genes. These cell lines, however, have not been genetically linked to tumors of origin or carefully characterized at a genetic or molecular level. This problem for the research community was reviewed at a recent NCI conference in which the Principal Investigators participated. The need for well- validated thyroid cancer cell lines to study mechanisms of cancer development and progression as well as discovery of novel therapeutic targets was emphasized. In this proposal, we will use three complementary strategies to provide a large panel of comprehensively characterized thyroid cancer cell lines representing primary and metastatic tumors of different histologic types and mutational subtypes (BRAF, RET/PTC, RAS, PIK3CA, AKT1, CTNNB1). We will focus on developing cell lines from poorly differentiated thyroid cancer as well as metastatic lesions that are more likely to harbor mutations that are not represented in the current panel of thyroid cancer cell lines. To establish new cell lines, we will use a parallel in vivo and in vitro approach to maximize our success rate. The new cell lines will be carefully characterized and compared to the corresponding tumor tissue of origin by short tandem repeat (STR) profiling to confirm their derivation. STR profiling and expression of thyroid-specific genes will be used investigate the origins of an additional 36 thyroid cell lines reported in the literature in order to assemble a complete panel of thyroid cancer cell lines. We will use expression arrays of authentic thyroid cancer cell lines to identify a signature that can be used to validate cell lines that have lost expression of thyroid-specific markers or from which patient DNA is unavailable. Finally, we will apply global molecular and genomic approaches with novel computational analyses to the new and existing thyroid cancer cell lines to uncover pathways important in thyroid cancer development and progression. All cell lines, as well as the genomic and gene expression data, will be made available to the research community through a central repository with the assistance of the NCI, and data will be accessible through the NCBI GEO database and UCSC genome browser. Feasibility and Impact: The generation of new cell lines from solid tumors is a challenging task. We propose to generate at least 10 new cell lines in a 12-15 month time period using parallel in vivo and in vitro approaches. There is significant expertise at the University of Colorado Cancer Center (UCCC) with a proven record of success in generating cell lines from solid cancers. We believe that this ambitious proposal is feasible within the two-year time frame because the unique collaboration between the UC Denver and MSKCC has all of these advanced technologies currently in place (new procedures to develop cell lines, gene expression platforms, mass spectrometry genotyping, CGH array analysis, mutational screen of key oncogenes and tumor suppressor genes and high throughput pathway activation analysis by Western blotting). A majority of the sample preparation and studies for the arrays and mutation screening will be performed by experienced personnel in well-equipped cores with rapid turn-around times. Furthermore, we are collaborating with nationally recognized bioinformatics and computational experts to apply novel computational biology strategies to identify important signaling pathways that will better define disease pathogenesis. Successful completion of these aims will provide approximately 40 well-characterized thyroid cancer cell lines for use by the research community to uncover mechanisms of thyroid cancer development, progression, metastatic potential and to serve as a platform for identification of novel therapeutic targets for advanced disease. Lay Summary: Thyroid cancer is the most common endocrine malignancy with an annual incidence of ~34,000. At least 300,000 people are living with a diagnosis of thyroid cancer in the United States, and more than 1,500 of them die each year from this disease. Permanent thyroid cancer cell lines derived from patient's cancers are critical to help us understand the causes of thyroid cancer, how the tumor grows and spreads, and how we can find new ways to treat advanced disease for which there is currently no cure. Recent studies by our research groups have shown that nearly half of the "thyroid" cancer cell lines in current use are not unique and many did not even originate from a thyroid cancer. In order to provide well characterized thyroid cancer cell lines for study by the research community, we will generate new thyroid cancer cell lines that can be linked back to the original tumor tissue. We will also develop approaches by which we can determine whether some of the existing thyroid cancer cell lines are authentic or not. We will develop genetic profiles of all the cancer cell lines, from which we will be able to characterize the abnormal signaling networks that may explain how these cancers develop. Once we know what makes these cancers grow, we will have a better understanding of how to treat them. With the help of the National Cancer Institute, we will make these cell lines widely available to the research community. PUBLIC HEALTH RELEVANCE: The generation of a well-characterized panel of thyroid cancer cell lines has significant relevance for public health as validated tools for the broader research community. Successful completion of the proposed aims will provide approximately 40 well-characterized thyroid cancer cell lines in a central repository for use by the research community to uncover mechanisms of thyroid cancer development, progression, metastatic potential and to serve as a platform for identification of novel therapeutic targets for many types of advanced cancer.

DESCRIPTION (provided by applicant): Thyroid cancer is the most common endocrine malignancy and the incidence is rising. Approximately 1,700 patients with thyroid cancer die each year in the U.S. and many others suffer from progressive, symptomatic disease. There are currently no effective, approved systemic therapies for patients with advanced, radioiodine- resistant disease. Novel targeted therapies and markers of disease activity are desperately needed for patients with advanced thyroid cancer. Many solid tumors share activation of the nuclear factor kappa B (NFkB) pathway including the endocrine-related cancers (thyroid, parathyroid, breast, prostate and endometrial). NFkB signaling regulates diverse processes in cancer including epithelial to mesenchymal transition (EMT), invasion, angiogenesis and metastases. Furthermore, current therapies for advanced cancer, including chemotherapy and radiation, activate the NFkB pathway, which can diminish therapeutic efficacy. A better understanding of NFkB signaling in advanced thyroid cancer will lead to selective diagnostic markers and inhibitors which will be therapeutically useful as single agents or in combination with other therapies. The current field is concentrating on long-term inhibition of this complex pathway as a potential 'targeted' cancer therapy, which will likely have many untoward side-effects since NFkB signaling is important in normal physiology, particularly immune function. This proposed application will shift the current translational research paradigm in two ways: (1) investigate rational combination therapy with intermittent blockade of the NFkB pathway in novel preclinical orthotopic and metastatic mouse models of advanced thyroid cancer that recapitulates tumor invasion as well as lymph node and distant metastases, and (2) employ a novel proteomic approach to identify NFkB-dependent secreted mediators ('secretome') of tumor angiogenesis and invasion, which will lead to more specific targeted therapies and disease markers. To accomplish these goals, the following approaches will be used: (1) Genetic and pharmacologic inhibition of the NFkB pathway alone and in combination with taxane-based chemotherapy in murine models of human thyroid cancer, (2) Comparative proteomic analysis of thyroid cancer cells with and without a genetically inhibited NFkB pathway to more broadly define the NFkB-dependent secretome in advanced thyroid cancer, (3) Genetic and functional manipulation of interleukin 8, identified as an important NFkB-dependent secreted protein in thyroid cancer, and other identified NFkB targets using in vitro and in vivo models, and (4) Use of a unique, large thyroid tumor tissue/plasma bank and preclinical mouse models to determine if IL8 and other NFkB-dependent targets may be useful molecular markers or therapeutic targets in advanced thyroid cancer. Successful completion of the proposed aims will provide novel tools to study thyroid cancer development, progression, metastases and resistance to conventional therapies. These studies will likely lead to new tumor markers and therapeutic targets to improve treatment and monitoring of patients with advanced thyroid cancer.

DESCRIPTION (provided by applicant): Thyroid cancer is the most common endocrine malignancy. A majority of patients have differentiated thyroid cancer (DTC) and are managed successfully with a combination of surgery, radioiodine and thyroid hormone replacement therapy. Anaplastic thyroid cancer (ATC), on the other hand, is one of the most aggressive human malignancies with an average survival of only 4 to 6 months. There are currently no effective therapies for ATC indicating that we need better tools to understand and treat this aggressive disease. TXNIP is a potent tumor suppressor, plays an important role in oxidative stress and is a major regulator of glucose uptake in cells. This protein has not been studied in normal thyrocytes or thyroid cancer. We have, for the first time, shown that TXNIP is expressed in normal thyroid cells and differentiated thyroid cancer, but is undetectable or expressed at low levels in poorly differentiated and undifferentiated (ATC) thyroid cancer and cells lines. We believe that this tumor suppressor and regulator of glucose uptake is responsible for the indolent behavior of many well-differentiated thyroid cancers and may explain why some patients with PET negative (low glucose uptake) metastatic disease do well, while most patients with PET positive (high glucose uptake) metastatic disease have a much worse prognosis. In this proposal, we will genetically manipulate TXNIP levels in normal thyroid cells as well as differentiated and undifferentiated thyroid cancer cells to assess the role of this potential tumor suppressor in cancer progression. We have also treated a panel of ATC cell lines with pharmacologic inhibitors of important signaling pathways in thyroid cancer and made the novel observation that inhibitors of the PI3K-mTOR pathway induce re-expression of TXNIP in these ATC cells. We will test these genetic and pharmacologic approaches to TXNIP regulation in vivo using a clinically-relevant orthotopic tumor model where thyroid cancer cells are injected directly into the thyroid glands of mice. We will also utilize a metastatic model of direct intracardiac injection of cancer cells, which leads to lung, bone and brain metastases. This model was developed by our research group and is completely new to the thyroid cancer field. Finally we will develop novel transgenic mouse lines which will genetically delete or overexpress TXNIP in the thyroid cells. Successful completion of the aims in this proposal will advance our understanding of the role of TXNIP in normal thyrocyte development and function, susceptibility to development of thyroid cancer, as well as advanced thyroid cancer tumor progression, dedifferentiation, metastases and metabolism. Ultimately, these studies will provide new tools to understand thyroid physiology and cancer pathophysiology, and will lead to new tumor markers and therapeutic targets to improve treatment and monitoring of patients with advanced thyroid cancer.

Project Summary Anaplastic thyroid cancer (ATC) is one of the deadliest human malignancies with median survival of < 6 months despite treatment with a combination of surgery, external beam radiation and cytotoxic chemotherapy. Paclitaxel and docetaxel are cytotoxic drugs that work by stabilizing mitotic spindles and increasing the rate of chromosomal segregation errors during cell division. Taxanes have shown a modest benefit in patients with ATC, but even with treatment, patient survival remains very poor. Recently, the combination of dabrafenib and trametinib have shown efficacy in patients with BRAFV600E mutated tumors, and this combination has been approved by the FDA for use in patients with this specific mutation. We recently published mutation status of the largest group of patients with ATC (Pozdeyev N, Clin Cancer Res, 2018). Of 196 patients, 41% had a BRAF mutation, leaving 59% of patients not eligible for the dabrafenib/trametinib therapy. More effective drugs and rational synergistic drug combinations are urgently needed for these patients. We have used a novel functional genomics discovery approach to identify the spindle assembly checkpoint (SAC) pathway as a key synergistic lethal vulnerability with taxanes in ATC. We have also shown that ATC and poorly differentiated thyroid cancer (PDTC) tumors have high levels of critical SAC components, MPS1 and BUBR1. The primary objective of this application is to study the combination of taxanes with the drugs blocking the SAC pathway for the treatment of ATC. Our central hypothesis is that the inhibition of the SAC pathway, responsible for the prevention of chromosomal segregation errors in ATC and PDTC cells, which already have high baseline chromosomal instability (CIN), will sensitize a vulnerable subset of tumor cells to taxanes and cause massive aneuploidy incompatible with cell survival. We further predict that the aneuploidy generated by this therapy will activate the immunosurveillance system, which we will test in an immunocompetent preclinical model. We will test these hypotheses using in vitro human ATC/PDTC cells lines to elucidate mechanisms of synergy sensitivity and resistance, validate these findings in orthoptopic xenograft models, and extend these studies into syngeneic orthotopic models to determine the contribution of immunosurveillance on effectiveness of therapy. Successful completion of these Aims will determine the preclinical efficacy of combination taxane therapy and SAC pathway inhibition in ATC and PDTC. These studies will also provide mechanistic insights that could lead to other novel combination therapies and critical biomarkers for focused clinical trials. If successful, this work should rapidly lead to clinical trials that will improve survival in these patients who desperately need better therapies. These studies will likely also inform treatments in other advanced cancers. `