Pituitary-Specific Oncogene Signaling Mechanisms

Gutierrez-Hartmann, Arthur

Abstract

Despite significant efforts, our understanding of clinically relevant mechanisms underlying human pituitary tumorigenesis remains ashamedly meager. My lab has focused on defining the precise molecular mechanisms by which ubiquitous components of growth factor/Ras/MAPK signaling pathways result in pituitary lactotrope cell-specific transcription responses. We discovered an elegant tri-partite molecular code, involving the protooncoprotein c-Ets-1, the POU-homeodomain protein Pit-1, and a composite DNA element containing an Ets binding site adjacent to a Pit-1 site. Pit-1, Ets-1 and this composite site were shown to be required not only for the oncogenic V12Ras response of the rat (r) prolactin (PRL) promoter, but also to reconstitute lactotropespecific rPRL promoter activity in HeLa nonpituitary cells. During the past funding cycle we made important advances, showing that Ets factors, particularly GABP, are critical for basal and growth factor-induced endogenous rPRL gene expression in GH4 cells and lactotrope ontogeny in transgenic mice;in dissecting the TAD subdomains of Ets-1 and Pit-1;and in identifying co-activators and co-repressors governing basal and Ras-regulated PRL gene expression. In this application, we propose to move our Ras work to a more translational level, and address the role of Ras/MAPK in pituitary prolactinoma tumor formation, and whether PTTG-1 and Menin act as downstream effectors in this pathway. Thus, the main goal of this proposal is to develop pre-clinical GH4 cell line and transgenic mouse models that target V12Ras to pituitary lactotropes, to determine the functionally relevant role of persistently-activated pMAPK and downstream effectors (eg, Menin, PTTG-1 and Mediator components) in regulating lactotrope gene expression, cell proliferation and tumorigenesis. Our unifying hypothesis is that persistent MAPK activation in lactotropes regulates specific Mediator components leading to the stimulation of prl and pttg-1 gene expression, and inhibition of menin gene expression, which leads to increased cell cycle progression, cell proliferation and tumorigenesis.
Specific Aims : (1) To use Dox-inducible V12Ras to determine the role of distinct levels of activated MAPK in mediating cell proliferation and differentiation in GH4 somatolactotropes. (2) To use ChIP analysis to define the MAPKdependent chromatin alterations on the PRL, GH, PTTG-1 and Menin promoters and on recruitment of specific Mediator components to these genes. (3) To use shRNA and over-expression to identify the key MAPK downstream effectors required for MAPK-regulated promoter activity and GH4 cell proliferation. (4) To target a V12Ras-IRES-Luc transgene to pituitary lactotropes and determine whether persistent MAPK activation results in tumorigenesis and if drug induced lactotrope hyperplasia enhances tumor formation. Knowledge gained from these studies will provide critical insights into the mechanisms by which pMAPK links to the Mediator complex, and how this link regulates prl, pttg-1 and menin gene expression, thus providing a mechanistic framework for how the MAPK pathway intersects with clinically relevant proteins to contribute to lactotrope tumorigenesis

Public Health Relevance

Pituitary tumors, such a prolactinomas, are very common intracranial tumors, with the vast majority of prolactinomas remaining as adenomas and rarely progressing to metastatic carcinomas. To date, we neither understand the biological cause for these tumors, nor do we understand why they fail to progress to a frank carcinoma. Here, I plan to establish clever cell line and transgenic mouse models that will directly interrogate the role of the MAPK pathway in prolactinoma formation, and the role of cell cycle inhibitors as ?brakes? that impede adenoma progression to cancer