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Annals of Clinical & Laboratory Science 40:211-217 (2010)
© 2010 Association of Clinical Scientists

Morphoproteomics Demonstrates Activation of mTOR Pathway in Anaplastic Thyroid Carcinoma: A Preliminary Observation

Jing Liu and Robert E. Brown
Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston Medical School, Houston, Texas

Address correspondence to Jing Liu, M.D., Ph.D., Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston Medical School, 6431 Fannin Street, MSB 2.260A, Houston, Texas, USA; tel 713 500 5327; fax 713 500 0695; e-mail jing.liu.1{at}uth.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
The mammalian target of rapamycin (mTOR) signaling pathway was studied using immunohistochemical stains on paraffin-embedded tumor tissue from two patients with anaplastic thyroid carcinoma (ATC) and on paraffin-embedded normal thyroid tissue from 23 control patients. Immunoreactivities of p-mTOR, p-Akt, p-p70S6K, and PLD1 were observed in both of the ATCs, with nuclear translocation of p-mTOR, p-Akt, and p-p70S6K. Increased expression of Ki-67, Skp2, and cyclin D1, decreased expression of p27kip1, and increased mitotic index (MI) were noted in the ATCs in comparison with those of normal thyroid tissue. The results provide evidence of (a) constitutive activation of the mTOR pathway, (b) mTORC2 activation, suggested by the nuclear translocation of p-mTOR, and (c) enhanced cell cycle progression in ATCs. These preliminary findings warrant future studies in a large series of patients with ATC to evaluate a possible molecular basis for treating chemoradioresistant ATC.

Keywords: morphoproteomics, anaplastic thyroid carcinoma, mTOR pathway


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Anaplastic thyroid carcinoma (ATC), one of the most aggressive human malignancies, has a very poor prognosis. The overall median survival is limited to months [1]. Surgery, radiotherapy, and standard chemotherapy do not meaningfully improve survival [2]. Consequently, there is a need for new therapeutic approaches. Individualized targeted therapy incorporating small molecule inhibitors into tumor cells might be an effective strategy against ATC. However, the signaling pathways involved in the tumorigenesis of ATC have not been well defined.

Among the cancer signaling pathways, the mammalian target of rapamycin (mTOR) pathway has been increasingly studied and its activation has been reported in a variety of malignant neoplasms. It is estimated that mTOR is upregulated in 70% of all tumors [3]. Only a few studies of the mTOR pathway in ATC using cultured cell lines have been published. These studies were limited to a few genes and their products [4–7]. It is desirable to define the role of mTOR pathway in ATC because mTOR inhibitors (rapamycin, its various analogues, and metformin) have been identified as potential pharmaceutical agents and utilized in clinical or preclinical studies of a number of malignancies including glioblastoma multiforme, renal cell carcinoma (RCC), and prostate cancer [8–10].

The mTOR is a highly conserved protein kinase. Its activation promotes cell cycle progression and suppresses apoptosis in a variety of human cancers [11]. The mTOR is found in two structurally and functionally distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). They have different components– most significantly raptor for mTORC1 and rictor for mTORC2. Rapamycin is an mTOR inhibitor that prevents mTOR-dependent downstream signaling transduction. mTORC1 is rapamycin sensitive while mTORC2 is relatively rapamycin insensitive. Since activation of mTORC2 likely confers resistance to rapamycin-based targeted therapy, it is crucial to identify mTORC2 activation in tumors.

The activation of mTORC1 by p-Akt has been well established (ie, by phosphorylating mTORC1 predominantly on Ser2448 [12]), but the mechanism that activates mTORC2 is unclear. Phospholipase D (PLD) has recently been shown to be another mTOR activator and phosphatidic acid (PA), a catalytic product of PLD, has been reported to impact mTORC1 and mTORC2 by facilitating the assembly of mTOR complexes [13–17].

mTORC1 and mTORC2 signal via different sets of effector pathways. p70S6K is one of the downstream effectors of mTORC1 [18]. mTORC1 phosphorylatively activates p70S6K (threonine 389) [19–21]. The activation status is indicated by nuclear translocation of p-p70S6K [22]. Akt has recently been demonstrated to be the downstream effector of mTORC2 [3,23–25].

Investigation of the mTOR signaling pathway in ATC is still at an initial stage, although this pathway has been extensively studied in other neoplasms. In particular, studies of m-TORC2 and PLD involvement in activating the mTOR pathway in ATC have not been reported.

Morphoproteomics utilizes phosphospecific probes and cellular compartmentalization to assess the state of activation of protein analytes in tissues [19]. In this study, we investigated the mTOR signaling pathway in ATC using morphoproteomics in an effort to elucidate the signaling transduction circuitry and potential molecular targets for individualized therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Acknowledgements
 References
 
Archival thyroidectomy specimens were obtained from two patients with ATC and from 23 control patients with morphologically normal thyroid tissue. This study protocol was approved by the Institutional Review Board of the University of Texas Health Science Center at Houston.

Immunohistochemial staining was performed on formalin-fixed, paraffin-embedded unstained sections of 4 µm thickness. Eight primary antibodies were utilized to detect (a) three phosphorylated (p) antigens, including p-Akt (Ser473) (Cell Signaling Technology, Beverly, MA), p-mTOR (Ser2448) (Cell Signaling Technology), and p-p70S6K (Thr389) (Cell Signaling Technology); (b) phospholipase D1 (PLD1) (Santa Cruz Biotechnology, Santa Cruz, CA); and (c) four cell cycle-associated proteins, including p27kip1 (Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK), S phase kinase-associated protein (Skp2) (Santa Cruz Biotechnology), Ki-67 (Dako Corp., Carpinteria, CA), and cyclin D1 (Biocare Medical, Concord, CA). We used a semiautomatic method and monoclonal antibodies against the three phosphorylated antigens (p-Akt, p-mTOR, and p-p70S6K) and an automatic stainer for PLD1, Skp2, p27kip1, Ki-67, and cyclin D1.

Chromogenic signals and subcellular expression patterns of the immunohistochemical stains were assessed by brightfield microscopy. Both staining intensity and extensiveness (percentage of tumor cells staining) were evaluated for p-Akt (Ser473), p-mTOR (Ser2448), p-p70S6K (Thr389), and PLD1. Staining intensity was graded as negative (0), weak (1+), moderate (2+), and strong (3+). Staining extensiveness was the percentage of tumor cells positively stained, ranging from 0% to 100%. The immunostained sections were semiquantitatively assessed by the authors. A combined score was calculated using the staining intensity (1+, 2+, or 3+) multiplied by the percentage of positive staining. The overall score of staining was recorded in each group, with a range from 0 to 300. The subcellular expression pattern was evaluated and characterized as nuclear, cytoplasmic, or plasmalemmal. Only unequivocal nuclear staining was considered in assessing expressions of p27kip1, Skp-2, Ki-67, and cyclin D1 antigens. The mitotic index (MI) was calculated as the number of mitoses/10 high power fields (HPF).

The level of immunoreactivity was graded according to the combined scores: weak expression (1 to 100), moderate expression (>100 to 200), and strong expression (>200). Unpaired t-tests were used to compare the mean combined scores of ATC versus those of normal thyroid tissue for p-Akt, p-mTOR, p-p70S6K, and PLD1.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Acknowledgements
 References
 
Moderate or strong expressions of p-mTOR, p-Akt, PLD1, and p-p70S6K were observed in the ATCs (n = 2). Strong immunoreactivity of p-mTOR was seen in both nuclear and cytoplasmic locations in ATC (Fig. 1AGo, page 214). In contrast, 22 of the 23 normal thyroid tissue sections did not show any nuclear expression of p-mTOR and one section showed focal and weak expression. Although the normal thyrocytes demonstrated cytoplasmic and plasmalemmal immunoreactivity of p-mTOR, the level of expression in cytoplasm was significantly lower than that in ATC (p <0.05) and the expression in the plasmalemmal location was not significantly greater (p = 0.17) (Fig. 1BGo).


Figure 1
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  		Fig. 1. Anaplastic thyroid carcinoma shows moderate to strong expression of phosphorylated mammalian target of rapamycin (p-mTOR) (Ser2448) (panel A), while normal thyroid tissue does not express p-mTOR in the nuclear location and has variable or focal and generally less intensity of expression in the cytoplasm with focal immunoreactivity in the plasmalemma (panel B). Expression of p-Akt is present in the cytoplasm and nucleus in ATC (panel C) and in normal thyroid tissue (panel D), with stronger expression in ATC than in normal thyroid tissue. (Immunohistochemical stains, original magnification x400.)

 
Expression of p-Akt was noted in the cytoplasm and nuclei of ATC (Fig. 1CGo) and normal thyroid tissue (Fig. 1DGo). However, the level of expression of p-Akt in cytoplasm was significantly higher in ATC than in normal thyroid tissue. The levels of nuclear expression of p-Akt in ATC and normal thyroid were not significantly different (p=0.11).

The expression of PLD1 was moderate and located in the cytoplasm in both of the ATCs and in the plasmalemma in one ATC (Fig. 2AGo, page 215), while it was not detected in any of the 23 normal thyroid sections (p <0.05) (Fig. 2BGo).


Figure 2
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  		Fig. 2. Anaplastic thyroid carcinoma shows moderate expression of phospholipase D1 (PLD1) in the cytoplasm and plasmalemma (panel A), while normal thyroid tissue does not express PLD1 (panel B). Note the expression of nuclear p-p70S6K (Thr 389) in ATC (panel C) versus normal thyroid tissue(panel D). (Immunohistochemical stains, original magnification x400.)

 
The p-p70S6K was observed in nuclei in all ATCs (Fig. 2CGo) and normal thyroid tissue sections (Fig. 2DGo). The expression seemed more extensive in ATC than in normal thyroid tissue, although the difference was not statistically significant (p = 0.11). The overall combined scores for each group are shown in Table 1Go.


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  		Table 1. Comparison of overall combined immunostaining scores for p-Akt, p-mTOR, p-p70S6K, and PLD1 in anaplastic thyroid carcinoma (ATC) and normal thyroid tissue (NTT).
 
Of the cell cycle markers, the mean expressions of Ki-67, Skp2, cyclin D1, and the mitotic index (MI) in ATC were 32%, 51%, 36%, and 8/10 HPFs, respectively. In contrast, no immunoreactivities for these biomarkers and 0 mitoses/10 HPFs were detected in normal thyroid tissue. Expression of p27skp1 was not detected in either case of ATC, but it was seen in all of the normal thyroid sections with an average extensiveness of 56% (range: 10 – 90%). Expressions of cell cycle markers are listed in Table 2Go.


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  		Table 2. Mean expression of cell cycle progression markers in anaplastic thyroid carcinoma (ATC) and normal thyroid tissue (NTT).
 
The ATCs in this study demonstrate: (a) constitutive activation of mTOR signal transduction pathway with mTORC2 activation, indicated by the nuclear translocation of p-mTOR (Ser 2448); (b) activation of the upstream regulators of mTORCs including Akt and PLD1; (c) activation of p70S6K and Akt, the downstream effectors of mTORC1 and mTORC2, respectively; and (d) significantly enhanced cell cycle progression.

Previous studies have shown that mTORC1 is expressed in cytoplasm and/or plasmalemma in its activated state, as demonstrated in RCC, gastric adenocarcinoma, and other tumors [20,26,27]. Moreover, Rosner and Hengstschlager [28] found that mTORC1 assembly is predominantly in the cytoplasm whereas mTORC2 is abundant in both cytoplasm and nucleus of non-transformed, non-immortalized human diploid fibroblasts. These findings imply that the expression of mTOR in the nuclear location reflects the presence of mTORC2. Our study demonstrates strong expression of p-mTOR in both cytoplasmic and nuclear locations and correlative nuclear expressions of p-p70S6K and p-Akt, the downstream effectors of both mTORCs, suggesting the activation of both mTORC1 and mTORC2 in the ATCs. The activated state of the mTORCs is further supported by the phosphorylation of these proteins [29].

There is a substantial difference in the stability of the two mTOR complexes, with mTORC2 being far more stable than mTORC1. This differential stability explains the relative resistance of mTORC2 to rapamycin; it takes higher concentrations of rapamycin for a longer time to interfere with the more stable mTORC2, which rarely dissociates [30]. mTORC2 is the more critical target for strategies to retard or kill cancer cells. Thus, it is important to identify the activation of mTORC2 in a particular tumor.

In this study, the expression of p-Akt in ATC cases was only moderate and was not as strong as expected, which appears to be slightly discordant with the strong nuclear expression of p-mTOR (mTORC2). This discordance may be related to the interplay between Akt, mTORC1, and mTORC2, in which the Akt is both the upstream regulator of mTORC1 and the downstream effector of mTORC2. A few previous studies have suggested that after Akt activates mTORC1, the latter elicits a negative feedback loop to inhibit Akt activity [31–33]. Therefore, the Akt is placed under positive and negative control mediated by the mTORCs. Thus, the relatively low immunoreactivity of Akt in this study may be due to a negative feedback loop elicited by m-TORC1.

Moreover, the moderate expression of p-Akt as an upstream regulator appears not to coincide with the strong expression of p-mTOR. To explain the expression discrepancy between p-Akt and p-mTOR, there may possibly exist alternate activation pathways for the mTORCs.

The present study demonstrates moderate expression of PLD1 in the majority of tumoral cells in both ATC cases, but no expression in normal thyroid tissue. This finding suggests that PLD1 may involve the activation of mTOR pathway in ATC. This contention accords with previous reports that rapamycin competes with PA for binding mTORCs by blocking the association between mTOR and raptor, and between mTOR and rictor in cultured cells [24,34].

The end result of the activation of the mTOR pathway is the promotion of cell growth and proliferation. The enhancement of cell cycle progression is evidenced by increased expression of cell cycle analytes including S phase kinase-associated protein2 (Skp2), cyclin D1, Ki-67, and the mitotic index, and decreased expression of p27 in ATCs, as shown in this study. Skp2 is a cyclin A-CDK2 complex binding protein that promotes entry into S phase of both normal and transformed cells [35]. p27, a cyclin-dependent kinase inhibitor, helps to regulate the transition from the G1 to S phase of the cell cycle [36,37]. It has been demonstrated that the expression of p27 protein is controlled at the post-transcriptional level and by inhibitory regulators, such as Skp2 [38,39]. There is an inverse relationship between the levels of p27 and Skp2 proteins, and p27 is frequently down regulated in cultured thyroid tumor cells [38,40]. Cyclin D1 is a regulatory subunit of certain protein kinases thought to advance the G1 phase of the cell cycle, while the Ki-67 antigen is a nuclear protein that is expressed in proliferating cells during all active phases of the cell cycle (G1, S, G2, and M-phases), but is absent in quiescent cells (G0- phase) [41]. In this context, our results suggest that the majority of ATC cells are progressing from G1 to S phase and, therefore, ATCs may be candidates for an S phase active chemotherapeutic agent.

In summary, this is the first study in tumor tissue that indicates the constitutive activation of mTORCs and related pathways in ATC, as well as cell cycle progression from G1 to S and M phases. The concomitant expression of p-Akt, p-mTOR, p-p70S6K, and the upregulated cell cycle analytes are consistent with activation of Akt/m-TORC1/p70S6K pathway and its end effect–increased cell cycle progression. The nuclear expression of p-mTOR (Ser 2448) is consistent with activation of mTORC2, according to the literature. Concomitant activation of PLD1 is also detected in ATC. Together these findings suggest that activation of PLD1/mTORC2/Akt pathway may be involved in the tumorigenesis and drug resistance of ATC. These observations suggest a molecular basis for the chemoradioresistance of ATC. The constitutive activation of mTORC2 may serve as a therapeutic target for this tumor. The results of this preliminary study warrant future studies in large series of ATC cases.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
References
 
We thank Pamela K. Johnston, HT (ASCP), Miryam Naranjo, HT (ASCP), and Richard A. Breckenridge, HT (ASCP), for technical assistance, Bheravi Patel for secretarial and graphic assistance, and Dr. Ronny Zhang for statistical assistance.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 

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