Activation of Mek Alters Morphology and Neuroendocrine Phenotype in Medullary Thyroid Cancer Cell

Abstract

SynopsisActivation of the MEK pathway in MTC cells elicited similar effects of raf-1 pathway activation, resulting in cell growth suppression. Therefore, MEK may serve as a molecular target and a therapeutic strategy to treat patients with MTC. IntroductionMedullary thyroid cancer (MTC) is a neuroendocrine (NE) tumor that arises from the parafollicular cells (C-cells) of the thyroid gland [1]. MTC accounts for 5-10% of all thyroid malignancies and responds poorly to chemotherapy [2]. Thus, the only curative option for MTC is surgical resection [1]. MTC is the end result of an oncogenic activation of protein cascades in the NE cells of the thyroid gland [3]. Hence, understanding the molecular pathways that control C-cell proliferation is vital for developing novel therapies for patients with advanced MTC [4].MTC cells produce several hormones and NE markers such as calcitonin, Chromogranin A (CgA) [5], and Achaete Scute Complex-Like 1 (ASCL1), also known as human ASH1 (Achaete Scute Homolog-1) [6,7]. ASCL1 is involved in C-cell expansion and is critical for neuroendocrine cell differentiation and MTC tumor growth. The importance of ASCL1 for C-cell growth was observed in transgenic mice with an ASCL1 knockout gene, which failed to develop thyroid C-cells [1]. Moreover, ASCL1 has been found to be involved in the development of other NE cells such as pulmonary endocrine cells and chromaffin cells [1]. AbstractBackground: Medullary thyroid cancer (MTC) is a neuroendocrine (NE) tumor of the thyroid C cells. Surgery is the only curative therapy. Activation of raf-1 in MTC cells resulted in growth suppression and NE marker reduction. However, the exact mediator of this effect is not clearly understood. We hypothesize that MEK, a key downstream target of raf-1 pathway, may be involved in the effect seen with raf-1 activation in MTC. To determine the role of MEK, we established a doxycyclineinducible MEK in MTC TT cells and assessed the effects on morphology and NE phenotype.Methods: Doxycycline-inducible TT-MEK cells were created by stable transfection of a pRevTRE-MEK plasmid in TT cells expressing a Tet responsive protein. TT-MEK cells were treated with 0-1.0μg/ml of doxycycline for four days. The level of total and active MEK expression was determined by western blot analysis using MEK and phosphorylated ERK1/2 antibodies. In addition, the lysates were analyzed for levels of the NE markers achaete-scute complex like 1 (ASCL1) and Chromogranin A (CgA). Morphology of the treated and control cells was observed under a light microscope.Results: Treatment of TT-MEK cells with doxycycline led to an induction of MEK protein, which is associated with activation of ERK1/2 in a dose-dependent manner. Importantly, the levels of NE markers were reduced by increasing MEK activation. Similar to RAF-1 activation, there was a striking morphological change with activation of MEK1/2, with a decrease in horizontal spread of each cell resulting in rounder, larger cells. Conclusion:We demonstrate, for the first time, that the over exp...