Judy L. Meinkoth - US grants

The ability to respond to environmental signals is absolutely essential for proper development and cell survival. Extracellular signals in the form of growth factors affect many aspects of cell development including proliferation, differentiation and survival. The coordination of proliferation and differentiation affects not only embryonic development, but tissue replenishment and repair in the adult. The decision to divide or differentiate is influenced by numerous growth factors acting both individually and in concert to either stimulate or inhibit proliferation. One way in which growth factors act is through changes in gene expression. The elucidation of the molecular components of the pathways through which growth factors alter patterns of gene expression will provide potential targets for the augmentation or inhibition of these signalling pathways. The ability to manipulate these pathways has widespread applications in tissue maintenance and repair as well as in chemotherapeutic rationales. Moreover, the identification of the mediators of signal transduction pathways from the cell surface to the nucleus will provide a better understanding of the potential interactions between growth factors and insight into some of the cellular mechanisms of growth control. Much of our knowledge regarding signal transduction has resulted from the characterization of two predominant pathways active in fibroblasts, one including tyrosine kinase receptors and the other G-protein coupled receptors which activate phospholipases. Yet, elevations in cAMP are mitogenic in a variety of endocrine and other cell types. Moreover, activating mutations in the stimulatory G protein of adenylate cyclase have been isolated from a variety of tumors in which cAMP induces proliferation. We wish to elucidate the molecules which function in cAMP and ras mediated mitogenic pathways since activating mutations in both pathways have been found in human tumors. Thyroid follicular cells provide an ideal model system for this analysis since their growth can be stimulated by multiple signalling pathways including a cAMP mediated pathway stimulated by thyrotropin (TSH). Moreover, activating mutations in Gs and in all three cellular ras genes have been identified in thyroid adenomas, follicular and papillary carcinomas. Using needle microinjection and other molecular approaches, we will perform a molecular dissection of mitogenic pathways which include the ras protooncogene and cAMP as a second messenger. Microinjection allows for the introduction of putative signalling molecules or inhibitors directly into living cells in high concentrations. Unlike other techniques, biological effects in response to injected molecules can be measured in very short times. Since activating mutations in Gs and ras are found in a variety of human tumors, the identification of the molecular components of cAMP and ras mediated proliferation should contribute significantly to our understanding of growth control and transformation.

Cyclic AMP, the first discovered second messenger of hormone action, elicits cell type-specific effects on proliferation. Proliferation in many cells is inhibited by cAMP, effects mediated in part through interference with Ras-mediated signaling. In endocrine and other cells, cAMP is a mitogen, and in the appropriate cellular context, an oncogene. Mutations leading to constitutive activation of cAMP-mediated signaling have been identified in human pituitary and thyroid tumors. Despite this, relatively little is known regarding how cAMP stimulates proliferation. Our work in thyroid epithelial cells has revealed that cAMP-stimulated proliferation proceeds through divergent pathways, only some of which require PKA activity. The recent discover of cAMP-regulated guanine nucleotide exchange factors for Rap1, a small GTP-binding protein closely related to Ras, establishes that the effects of cAMP are far more divergent than currently appreciated. Crosstalk between cAMP and Ras, and potentially between Ras and Rap, is an important feature of cAMP- mediated mitogenesis. cAMP influences Ras-mediated signaling at points both upstream and downstream from Ras. We are one of very few laboratories that have developed and characterized cellular models in which to investigate cAMP-regulated growth control. The long term goals of our studies are to identify the molecules which participate in cAMP-stimulated cell cycle progression, and to elucidate how cAMP- and Ras-mediated signals are integrated in the control of epithelial cell proliferation. Our immediate goals are to define the contributions of PKA and Ras in the regulation of PI3K activity; to identify the mechanisms through which cAMP elevating agents activate both Ras and Rap, and to elucidate how Ras and PKA cooperatively regulate Rap1 activity. The identification of sites of crosstalk between cAMP and Ras may reveal novel drug targets for future therapeutic intervention. Given the high frequency (ca 100 percent) of thyroid tumors observed at autopsy, identification of growth regulatory circuits targeted by cAMP is an important and exciting area for future investigation.

DESCRIPTION (Taken from the applicant's abstract) Because cells respond to a wide array of signals received at the cell surface, their ability to integrate and respond appropriately to these signals is essential. One of the best studied examples of signaling crosstalk is the interplay between Ras- and cAMP-mediated pathways. Cyclic AMP inhibits proliferation in many cells, in part through uncoupling Ras from one of its downstream effectors, Raf-1. Elevations in cAMP, however, are not universally growth inhibitory. In thyrocytes, pituitary somatotrophs and other cells, cAMP stimulates proliferation. We discovered that thyrotropin (TSH) stimulates proliferation through a novel pathway involving both Ras and the cAMP-dependent protein kinase, but not the well described downstream effectors of Ras, Raf-1 and the mitogen-activated protein kinase cascade. The effectors used by Ras in the presence of TSH and elevated cAMP levels remain to be elucidated, but may include phosphatidylinositol 3-kinase, the isoform of protein kinase C, and other small G proteins. It is the goal of these studies to elucidate those molecules which transduce TSH-stimulated mitogenic signals, including the identification of novel Ras effectors, and the sites of interaction between Ras- and cAMP-mediated pathways. Using microinjection of purified signaling molecules and highly specific inhibitors, the biological activity of a panel of Ras mutants defective in various effector interactions will be assessed. Ras-overexpressing thyroid cells will be screened for novel proteins that bind to GTP-bound Ras. Other molecules proposed to function as Ras effectors will be assessed for growth-stimulating activity in the thyrocyte. Together these approaches will provide insight into novel Ras-mediated signaling pathways active in thyroid cells. Once these molecules are identified, we will test their activity in growth stimulation in other epithelial cells, including pituitary somatotrophs and mammary epithelial cells. Ras gene activation and the constitutive activation of cAMP-mediated signaling pathways have been identified in human thyroid and pituitary cancer. The elucidation of the molecular components of, and interactions between these pathways may identify novel targets for therapeutic intervention. My long term career goal is to identify the molecular components of growth-signaling pathways in a variety of epithelial cell types in a challenging academic environment. The University of Pennsylvania provides an exciting and rich research environment in which to pursue these studies.

Immune-mediated cell destruction is one of the central pathologic events in the development of autoimmune disease. In Hashimoto's thyroiditis (HT), a specific immune response to thyroid antigens leads to lymphocytic infiltration, follicular cell destruction and clinical hypothyroidism. It is now evident that apoptosis is one of the major pathologic mechanisms underlying HT and other forms of autoimmune disease. Proinflammatory cytokines are thought to play an initiating role in autoimmunity and apoptosis. Interferon-gamma (IFN-gamma) and interleukin 1-beta have been implicated in the pathogenesis of HT and other autoimmune disorders. Recently, these cytokines were shown to induce Fas receptor expression on normal human thyrocytes, cells that express FasL. Cross-linking of Fas resulted in massive apoptosis, leading to the speculation that following inflammation and cytokine release, thyroid cells are pruned to die by fratricidal mechanisms. Thyrotropin (TSH) regulates thyroid function, proliferation and possibly, survival. TSH stimulates the expression of thyroid-specific genes, effects that are opposed by (IFN-gamma). In turn, TSH has been reported to decrease IFN-gamma effects on Fas expression and apoptosis. Our aims are to elucidate the effects of proinflammatory cytokines in a continuous line of rat thyroid cells. We wish to explore the mechanisms of cytokine-induced cell death, and of survival promoted by TSH using a combination of biochemistry, cell biology and microinjection. Crosstalk between cytokines and hormones, given their opposing effects on Fas expression, may significantly alter the susceptibility of endocrine cells to apoptosis, a factor with profound implications for autoimmune diseases including thyroiditis, diabetes and rheumatoid arthritis. Ras activation, a frequent event in thyroid cancer, sensitizes many cells to cytokine-induced apoptosis, and numerous studies implicate a close linkage between signals activated by Ras and Fas. TSH appears to alter the balance in Ras-mediated signals from proliferation to apoptosis, a finding that may explain the infrequent occurrence of mutations in Ras and in Gs or the TSH receptor, mutations leading to the constitutive activity cAMP- mediated signaling pathways, in thyroid tumors. We will examine the acute effects of Ras on apoptosis, and the signaling pathways responsible for these effects. We will identify cellular factors that contribute to the ability of Ras-transformed cells to escape apoptosis. Taken together, these studies will provide new insight into the regulation of thyroid cell survival and transformation applicable to other endocrine cells.

DESCRIPTION (provided by applicant): Ras mutations are a hallmark of human tumors, including those of the thyroid gland. Ras mutations are found at high frequency in follicular adenomas and carcinomas. Although Ras mutations are infrequent in papillary carcinomas, ret mutations occur at high frequency in these tumors and RET has been shown to signal through Ras. Despite years of active investigation, the contribution of Ras to neoplastic transformation is not well understood. Ras signals through complex signaling networks that are utilized in a cell type-dependent manner. The phenotypic consequences that follow Ras activation depend upon the level and duration of Ras activity, the effectors activated by Ras and importantly, cell context. The effects of activated Ras in primary thyroid cells are unusual. Unlike primary fibroblasts where activated Ras induces growth arrest, Ras stimulates sustained proliferation in primary human thyroid cells. We have shown that acute expression of activated Ras stimulates apoptosis in rat thyroid cells. In these cells as in human thyrocytes, apoptosis is preceded by cell proliferation. However, cell cycle progression in response to acute Ras expression is highly aberrant. Ras-expressing cells progress through G1, are delayed in S phase and perish by apoptosis. Moreover, the effects of Ras on the cell cycle machinery are strikingly different from those of thyroid cell mitogens. Following its acute expression, Ras decreased cyclin D1 and p27 protein levels, and increased p21 expression. Intriguingly, Ras elicited a marked increase in cyclin-dependent kinase-2 (cdk-2) activity predominantly in apoptotic cells. This was accompanied by the cleavage of cyclin A and p27 selectively in apoptotic cells. As observed in thyroid tumors, cyclin D1 was upregulated in thyroid cells selected to survive constitutive expression of activated Ras. It is our hypothesis that apoptosis induced by Ras is a direct consequence of unrestrained cdk-2 activity initiated by Ras effects on the cyclin-dependent kinase inhibitors, p27 and p21. The specific goals of this application are to determine whether cdk-2 activity is both necessary and sufficient for apoptosis stimulated by Ras, to elucidate the molecular mechanisms that contribute to unrestrained cdk-2 activity and apoptosis following acute expression of activated Ras, and to identify the secondary changes that transpire to allow for the survival of thyroid cells harboring Ras.

DESCRIPTION (provided by applicant): Thyroid tumors are the most common endocrine malignancy. It has been estimated that up to 90% of the autopsies performed in this country reveal the presence of slow-growing thyroid tumors. While the prognosis for patients with well-differentiated follicular and papillary thyroid tumors is good, anaplastic thyroid tumors are rapidly fatal. Activating Ras mutations are particularly prevalent in human thyroid tumors. Ras mutations are found in benign adenomas and at a higher frequency in follicular and anaplastic carcinomas. Mutations in B-Raf, a downstream Ras effector, are the most frequent mutational event in papillary thyroid tumors. These observations support roles for Ras in the initiation and progression of thyroid tumors. A large proportion of papillary thyroid tumors exhibit amplification and rearrangement of the PKCepsilon gene, leading to the expression of an N-terminal fragment of PKCepsilon structurally similar to the V1 domain, a peptide that selectively inhibits PKCepsilon translocation. Interestingly, most papillary thyroid tumors exhibit decreased expression of PKCepsilon. Moreover, expression of the RET/PTC oncogene induced the selective translocation, followed by downregulation of PKCepsilon. PKCalpha expression is increased in follicular thyroid tumors, tumors that also harbor Ras mutations. It is our hypothesis that individual PKC isozymes play essential roles in the initiation and maintenance of thyroid cell transformation by Ras. Our preliminary data demonstrate that PKCdelta selectively reproduces the acute effects of oncogenic Ras on aberrant cell cycle progression and apoptosis;that PKCepsilon is required for Ras-induced morphological changes;that PKCs mimic the inhibitory effects of Ras on thyroid differentiation;and that Ras-transformed thyroid cells exhibit alterations in PKC expression and activity. The proposed studies investigate the roles of individual PKC isozymes in the initiation and maintenance of Ras transformation in rat thyroid cells. This will be accomplished using highly specific molecular reagents including adenoviruses for PKC isozymes, selective PKC peptide activators and inhibitors and RNA interference. This analysis will provide novel insight into the molecular mechanisms through which Ras dysregulates thyroid cell proliferation, differentiation and survival, and may give rise to the development of new strategies to selectively impair tumor cell proliferation and/or reactivate differentiated gene expression.

DESCRIPTION (provided by applicant): One of the most critical steps in the progression to malignancy is the acquisition by tumor cells of the ability to metastasize. The hypothesis to be tested in this proposal is that downregulation of Rap1GAP contributes to thyroid tumor progression. This notion is based on data showing that Rap1GAP is highly expressed in normal human thyroid cells, and that its expression is dramatically reduced in the majority of invasive papillary thyroid carcinomas. Studies in human thyroid carcinoma cell lines revealed a striking correlation between loss of Rap1GAP and loss of epithelial structure. Rap1GAP-deficient tumor cells lacked E-cadherin and acquired the expression of vimentin, indicative of epithelial-to-mesenchymal transition. These cells exhibited enhanced migratory and invasive properties compared to tumor cells that retained Rap1GAP. Restoring Rap1GAP to Rap1GAP-deficient cells inhibited cell migration, invasion and anchorage-independent proliferation. Silencing Rap1GAP expression in thyroid carcinoma cells that retain an epithelial morphology caused the cells to disperse, enhanced the dissociation of cell aggregates, and dysregulated E-cadherin, suggestive of defects in cell/cell adhesion. As disruption of cell/cell junctions is associated with serious pathological consequences, we propose that downregulation of Rap1GAP contributes to the pathogenesis of thyroid tumors by attenuating cell/cell adhesion. In vitro studies will explore the molecular mechanism through which decreased Rap1GAP expression attenuates cell/cell adhesion and assess whether eliminating Rap1GAP endows tumor cells with altered migratory properties and invasive potential. As TSH regulation of Rap1GAP is lost from the human thyroid tumor cell lines, but is likely to be retained in primary thyroid tumors, complementary studies will be performed in differentiated rat thyroid cells. These studies will be conducted in cells in three-dimensional environments, conditions that more closely reproduce the matrix compliance of human tissues and where cell/cell contacts are maximized. The in vitro studies are complemented with studies in human tumor specimens that will identify the subtypes of thyroid tumors in which Rap1GAP expression is decreased and probe the clinical significance of loss of Rap1GAP. In summary, this proposal presents a highly cohesive plan to investigate the contribution and clinical significance of Rap1GAP depletion to the progression of human thyroid tumors.