mTOR and AMPK uncoupling in Follicular Thyroid Cancer
Specific aims
Mechanistic target for rapamycin (mTOR) has recently emerged as central regulator of cell metabolism with key role in cell proliferation and cancer development. Our investigation of human follicular thyroid cancer (FTC) tissue samples demonstrated activation of mTOR along with p-CREB. Our transgenic mouse model of human FTC by protein kinase A (PKA) activation (due to knockdown of the regulatory subunit PRKAR1A gene implicated as tumor suppressor in human FTC), faithfully replicated the human FTC results with activation of mTOR and p-CREB. Examination of signaling pathways in our mouse model, we interestingly found activation of AMPK (Thr-172) which is not only an energy sensor but also termed as negative regulator of mTOR pathway. However, AMPK is also essential to prevent cellular apoptosis in cells subjected to various kinds of stress. We therefore hypothesize that activation of both AMPK and mTOR pathways are essential, balancing the cellular metabolic pathways leading to FTC and both the pathways are uncoupled from each other in FTC allowing growth of the tumor.
To test this hypothesis, we are going to
Characterize HOW? the two pathways are uncoupled from each other in our transgenic mouse models of FTC.
a) RSK-1 has been described in some contexts as inhibitor of TSC-2. We will examine its role in inhibiting TSC-2 activation by gene manipulation and pharmacological means.
b) Examine the post-translational modification of mTOR allowing it to be active in spite of negative regulation by AMPK. Using enzymatic treatment of mTOR and raptor proteins, we will assess the role of usual post-translational modifications such as glycosylation, acetylation, phosphorylation, ubiquitination etc. We will also subject human and mouse FTC samples to proteomics approach such as mass-spectrometry for detailed examination of possible modifications.
Identifying the mechanism of WHY? two pathways are uncoupled from each other in FTC tumors.
a) Examine the role of AMPK and mTOR in cellular metabolism and its impact on tumor growth. We will manipulate the tumor microenvironment by subjecting tumor cells in vivo and in vitro with nutrient-rich or deficient conditions and study its impact on cellular apoptosis and proliferation, coupled with manipulation of AMPK and mTOR pathways.
b) Role of oxidative stress and its influence on AMPK and mTOR pathways. We will manipulate the oxidative stress of tumor cells in vitro and in vivo coupled with AMPK and mTOR pathways and assess influence on cellular apoptosis and proliferation.
Background and Significance
Thyroid cancer, although effects about one percent of population, has rising incidence in recent years. Apart from that, it is the most common endocrine malignancy. Of the thyroid cancers, majority are caused by a subtype called papillary thyroid cancer (PTC) followed by follicular thyroid cancer (FTC). Unlike PTC, FTC is more aggressive and has a metastatic potential. FTC interestingly has been found in association with Carney complex and Cowden syndrome, two tumor predisposition syndromes. Carney complex is caused by inactivating mutations in PRKAR1A, encoding for the type 1a regulatory subunit of cAMP dependent protein kinase, or PKA. Cowden syndrome on the other hand has inactivating mutations of PTEN, which regulates the PI3K/AKT pathway. In order to have a mechanistic understanding of FTC, and with an eye on therapeutic targets, our lab has generated a PRKAR1A thyroid specific KO mice. These mice develop FTCs by 1 yr of age in around 40% cases. We also have generated thyroid specific double knockouts of PRKAR1A and PTEN (DKO) which leads to more aggressive and metastatic FTCs within 6 months of age, mimicking the human FTC cases.
cAMP regulates number of cellular processes such as cell growth, differentiation etc. PKA also phosphorylates a number of physiological substrates modifying their actions. Perturbations in amp/atp ratios is in the center of cellular metabolism, and is tightly regulated. AMPK in turn shuts down energy consumption and enhances energy production trying to restore ATP levels to improve the AMP/ATP ratio. AMPK which is a metabolic stress-sensing enzyme is now believed to be the master regulator of energy metabolism. AMPK has been shown to be activated during metabolic stress where there is an increase in AMP/ATP ratio. In addition to AMP/ATP ratio, recent studies have shown that ROS is another upstream activator of AMPK. MTOR is another important regulator of cellular metabolism required for enhanced cellular growth. In normal cells, AMPK and mTOR are acting inversely where AMPK regulates and shuts down mTOR pathway for increased catabolism for cell survival, whereas mTOR is needed for cellular growth and proliferation. In many tumors, there is an activation of mTOR pathway which is needed for proliferation and cellular growth needed for cancer development. Since AMPK is the negative regulator of mTOR pathway, it was termed as a tumor suppressor. Recent literature however demonstrates that AMPK may also be acting as a tumor promoter, promoting cellular survival under stressful conditions. In certain cases, both catabolism to enhance glucose levels and anabolism to improve the cellular stocks of fatty acids may be beneficial. Indeed in case of some intracellular pathogens which require both increased glucose levels and continued fatty acid synthesis, both AMPK and mTOR pathways are activated. In some cases such as that of hcmv, a viral protein helps uncouple the AMPK and mTOR pathways.
Coming to thyroid, AMPK is involved in regulation of glucose metabolism. TSH has also been shown to inhibit AMPK phosphorylation as it counters the TSH action of increased uptake of iodide. It has been shown that AMPK is upregulated in some instances of differentiated thyroid cancers such as PTC. Interestingly mTOR pathway has also been shown to be active in some dtcs. Dual activation of mTOR and AMPK in thyroid cancers has many unexplained questions, most importantly in their role in cell survival, tumor growth, progression, invasion and impact on TSH signaling.
Preliminary Investigation
In human FTC thyroid samples, we found localization of p70S6K (which is a marker for mTOR activation) and p-CREB (which is a marker for PKA activation). Assessment of same in our transgenic mice, we found similar activation in PRKAR1A/PTEN DKO mice and little in PRKAR1A KO mice. To confirm co-localization of the two proteins, we performed immunofluorescene on tumor sections and found co-localization in PRKAR1A/PTEN KO mice compared to little co-localization in PRKAR1A KO mice and none in wt and PTEN KO mice (Fig. 1). To assess the mTOR activation in our FTC transgenic mouse model, we performed western blot of thyroid tumor samples from our various mice and found mTOR (total and pmTOR) and raptor migrating to a higher molecular mass in PRKAR1A and DKO mice compared to that of PTEN and wt (Fig. 2). The shift observed was around 8-10 kda. To assess the downstream targets of mTOR activation, we blotted for phospho-eIF4b and phospho-p70S6K1 protein in tumor lysate and found them to be active in PRKAR1A KO, DKO, PTEN but not in wt mice (Fig. 3). Examination of AMPK activation revealed interesting findings where phospho-T172 AMPK was observed in all PRKAR1A and DKO tumor samples tested, whereas for PTEN tumors, 1 out of 3 thyroids (Fig. 3). Since AMPK is considered to be the negative master regulator of mTOR pathway, we next blotted for phospho-TSC-2 which is the target of AMPK to turn off mTOR. Interestingly, phospho-TSC-2 levels were similar in all the lanes suggesting lack of activation. We are presently assessing other targets of AMPK activation and its influence on cellular metabolism and on mTOR pathway.
Experimental Design and Methods
Characterize HOW? the two pathways are uncoupled from each other in our transgenic mouse models of FTC.
a) RSK-1 has been described in some contexts as inhibitor of TSC-2. We will examine its role in inhibiting TSC-2 activation by gene manipulation and pharmacological means.
b) Examine the post-translational modification of mTOR allowing it to be active in spite of negative regulation by AMPK. Using enzymatic treatment of mTOR and raptor proteins, we will assess the role of usual post-translational modifications such as glycosylation, acetylation, phosphorylation, ubiquitination etc. We will also subject human and mouse FTC samples to proteomics approach such as mass-spectrometry for detailed examination of possible modifications.
Identifying the mechanism of WHY? two pathways are uncoupled from each other in FTC tumors.
a) Examine the role of AMPK and mTOR in cellular metabolism and its impact on tumor growth. We will manipulate the tumor microenvironment by subjecting tumor cells in vivo and in vitro with nutrient-rich or deficient conditions and study its impact on cellular apoptosis and proliferation, coupled with manipulation of AMPK and mTOR pathways.
b) Role of oxidative stress and its influence on AMPK and mTOR pathways. We will manipulate the oxidative stress of tumor cells in vitro and in vivo coupled with AMPK and mTOR pathways and assess influence on cellular apoptosis and proliferation.
Comments
Post a Comment