Abstract
Pituitary ademonas are benign tumours with different biological behaviour, especially with regard to tumour size, invasion, endocrine function, intratumour cystic lesion and apoplexy. There is little understanding of the growth and the control of progression of pituitary tumours. In the present study, we investigated the expression of mammalian target of rapamycin (mTOR) pathway regulators, in clinical pituitary adenomas. Pituitary adenomas from 95 patients were included in the study. Fresh pituitary tumours were obtained immediately after surgery and processed for histological, immunohistological and molecular based analyses. Histolopathological and clinical information including tumour stage, invasion characteristic and endocrine status were analysed against the gene transcript expression of mTOR, RAPTOR and RICTOR. There was a stepwise and significantly increased relation-ship between RICTOR expression and tumour size, namely p=0.0012 and p=0.0055 for tumours 1-2 cm and tumours >3 cm compared with tumours <1 cm respectively. Significantly higher levels of mTOR were seen in tumours with cystic lesions (p=0.044). There was no significant correlation between mTOR, RAPTOR and RICTOR and tumour apoplexy, nor a correlation between mTOR, RAPTOR and RICTOR with suprasephanous spread and sella floor destruction. However, pituitary tumours with cavernous sinus invasion, namely Knosp stage 3-4 had significantly lower levels of RAPTOR than those of Knosp stage 1-2 (p=0.01). A similar but statistically insignificant trend was seen with RICTOR. Using modified Hardy's staging, it was found that there was a significant correlation between tumour stage and RAPTOR and RICTOR expression. mTOR and RAPTOR levels differed in tumours with different endocrine functions, although no statistical difference was observed. However, Growth Hormone (GH) -, Follicle-Stimulating Hormone (FSH)-, Thyroid Stimulating Hormone (TSH)-secreting tumours had significantly lower levels of RICTOR compared with nonfunctional tumours. Finally, levels of mTOR were found to be significantly correlated with levels of both RAPTOR and RICTOR. It is noteworthy that RAPTOR and RICTOR levels were also significantly correlated. In conclusion, mTOR pathway regulators, mTOR, RAPTOR and RICTOR are significantly correlated with the invasion, staging, and tumour growth of pituitary adenomas and thus have an important predictive and prognostic value in patients with pituitary adenoma.
Mammalian target of rapamycin (mTOR) (also known as the mechanistic target of rapamycin) is a serine/threonine kinase that belongs to the Phosphatidylinositide 3-kinases (PI 3-kinases or PI3Ks) family (1). It is a key intracellular signalling regulator that connects the extracellular events to the intracellular process. A protein of about 289 kDa, mTOR forms two major protein complexes with other protein partners, namely mTORC1 and mTORC2, which differ in the protein components and in their sensitivity to rapamycin. mTORC1 is formed by mTOR, Regulatory-associated protein of mTOR (RAPTOR), Proline-roch Akt Substrate 40 KDa (PRAS40), and DEP domain containing mammalian target of rapamycin (mTOR) (DEPTOR). mTORC2 is formed by mTOR, Rapamycin Independent Companion of mTOR (RICTOR), DEPTOR, stress-activated protein kinase-interacting protein 1 (SIN1) and Protor-I (2-4). A major difference between the two complexes is the mTOR regulating proteins RAPTOR and RICTOR. Interactions between mTOR and RAPTOR and RICTOR are mutually exclusive. RICTOR, is essential in recruiting other mTORC2 proteins to the protein complex. A similar role for RAPTOR (also called KIAA1303) is known for the mTORC1 complex (5-7).
For the mTORC1 complex, extracellular signals including cytokines, inflammatory intermidators, oxygen and energy levels send signals to AKT and the Tuberous Sclerosis Complex (TSC)1/2 proteins, negative regulators of mTOR, which deactivate small Ras-related GTPase (8-10). This results in activation of mTORC1 and its downstream pathways including S6 Kinase-1 (S6K1), PRAS40 and Lipin1, and regulate protein synthesis, suppression of autophagy, gene expression, mitochondrial metabolism and adipogenesis metabolisms. On the other hand, activation of mTORC2 will either go on to activate the mTORC1 pathway, or directly regulate the PKC, ion transportation and cell growth via serum- and glucocorticoid-regulated kinase (SGK) pathways (11,12). There is compelling evidence which show that the mTOR pathways are aberrant in cancer, whether via over expression of the mTOR pathway molecules, loss of mutation of mTOR negators such as TSC proteins, or excessive extracellular signals such as cytokines. Collectively, this has made mTOR pathways viable targets in cancer treatment.
Pituitary adenomas are benign tumours with monoclonal origin. Pituitary adenomas have overall prevalence of 16.7% in the general population, 14.4% in autopsy studies and 22.5% in radiological studies (13). Although most tumours are slow growing, they do however have aggressive clinical course, including invasion of surrounding tissues, namely sella bones etc.
The mTOR pathway takes part in the process of tumour development and may have a role in the development and progression of pituitary tumours. Dai et al. affirmed that down regulation of PI3K/AKT/mTOR pathway improved the chemotherapy result in pituitary adenoma cell lines in vitro and in xenografted pituitary adenoma in nude mice (14). Some of the tumours showed a rapid rate of growth, and invaded the suprasella region, sphenoid sinus and cavernous sinus. Clinically, it is very difficult for neurosurgeons to totally remove pituitary tumours, which can reoccur rapidly after initial surgery. Invasive pituitary adenomas are obviously different from the classical ones. There may be a relationship between mTOR pathways and unrestrained growth of invasive pituitary adenoma. Although there was no difference found in phosphorylated mTOR in pituitary adenomas and control tissues, there are indications that its regulators may be activated and that there may be possible changes in message expression of mTOR (15). In pituitary adenomas which secrete growth hormone and in non-functional pituitary adenomas, mTOR has been found to be active (16, 17). In non-human pituitary adenoma cells, PI3K/mTOR inhibitor, XL765, was shown to influence the growth and apoptosis of tumour cells (14). Inhibiting mTOR pathway also reduced the growth of pituitary adenoma cells (18, 19). RAPTOR and RICTOR are mutually exclusive proteins when forming the mTOR complex, mTORC1 and mTORC2. The two mTOR complexes have related but also different functions in cells. It is unclear if mTOR, RAPTOR and RICTOR have a related expression pattern and if they play a distinct or orchestrated role (s) in cancer. In a recent report, it was shown that these three molecules were aberrantly expressed in human breast cancer (20). In the present study, we investigated the expression of mTOR, RAPTOR and RICTOR in human pituitary adenomas and aimed to establish a possible link between the expression pattern, and their relationship with the pathological and clinical features of pituitary adenomas.
Clinical and pathological information of the patients.
Materials and Methods
Clinical and pathological demographics of the patients. Patient's clinical history, diagnostic images, endocrine tests were routinely recorded. This study included a total of 95 patients with pituitary adenomas who underwent transsphenoidal or craniotomy surgical resection from Jan 2012 to Dec 2012 at the Department of Neurosurgery of Beijing TianTan Hospital, PRC. Age, gender, hormonal function were also reviewed and recorded. The diagnosis was confirmed by postoperative pathology. Immunohistochemical examination was used to determine the endocrine type. Preoperative magnetic resonance imaging (MRI) was performed to determine image characteristic: tumour size, cystic lesion, intratumour haemorrhage, invasion type. The greatest diameter of tumour obtained on gadolinium (Gd)-enhanced T1WI MRI was measured as the tumour size. Intratumoral haemorrhage and cystic lesion were defined by preoperative MRI and were confirmed intraoperatively. The invasion type was determined based on the invasion site of the tumour and recorded as cavernous sinus invasion, sphenoid sinus invasion, and suprasellar invasion. The criteria for suprasellar invasion was tumour growth toward the suprasellar region with invasion in the third ventricle and/or lateral ventricle. The criterion for sphenoid sinus invasion was tumour growth downward to the sphenoid sinus cavity or tumour growth into the clivas. Cavernous sinus invasion was defined as tumour extending lateral to the lateral tangent of the intra- and supracavernous internal carotid artery (ICA) (grade 0-2) or beyond that (grade 3 or 4), with reference to the classification proposed by Knosp et al. (21). Hardy's classification includes four tumour stages, in which tumour size and invasion were both taken into account, these were used in the present study (22). Full details of the clinicopathological information are given in Table I.
Primers used in the study.
Expression level of Mammalian Target Of Rapamycin (mTOR), Regulatory associated protein of mTOR (RAPTOR) and Rapamycin Independent Companion of mTOR (RICTOR) according to clinicopathological characteristices of patient.
Levels of Mammalian Target Of Rapamycin (mTOR), Regulatory associated protein of mTOR (RAPTOR) and Rapamycin Independent Companion of mTOR (RICTOR) are correlated with the tumour size in pituitary adenomas. *The level of the RICTOR transcripts for tumours 1-2 cm and tumours >3 cm are significantly higher than that of tumours <1 cm.
Levels of Mammalian Target Of Rapamycin (mTOR), Regulatory associated protein of mTOR (RAPTOR) and Rapamycin Independent Companion of mTOR (RICTOR) differ with the appearance of cystic lesion in pituitary tumours. mTOR levels were significantly higher in tumours with cystic lesion. RAPTOR and RICTOR levels were marginally lower in tumours with cystic lesion, although those differences were not significant.
Levels of Mammalian Target Of Rapamycin (mTOR), Regulatory associated protein of mTOR (RAPTOR) and Rapamycin Independent Companion of mTOR (RICTOR) are correlated with the cavernous sinus invasion (Knosp stage). *The level of the RAPTOR transcripts for Knosp stage 3/4 was significantly lower than stage 1-2. A similar trend was seen with RICTOR and an opposite trend was seen with mTOR.
Levels of Mammalian Target Of Rapamycin (mTOR). Regulatory associated protein of mTOR (RAPTOR) and Rapamycin Independent Companion of mTOR (RICTOR) are correlated with the Hardy's tumour stage in pituitary adenomas. *The levels of the respective transcripts in high stage groups were significantly different from these of low tumour stage.
Collection of pituitary adenomas. Pituitary adenoma samples were freshly-collected immediately after microsurgical resection at the Department of Neurosurgery of Beijing TianTan Hospital, supported by an ethics approval by the Local Research Ethics Committee with patient's consent. A total of 95 patients entered the current study. The tissues were immediately frozen and stored in liquid nitrogen until use.
Tissue processing and generation of genetic materials for genetic based analyses. Frozen tumour tissues were frozen-sectioned at 10 μm. A small number of sections were used for histological evaluation and the rest were combined and then homogenised in an RNA extraction solution. Total RNA was extracted based on the manufacturer's instructions (Sigama-Aldrich Inc, St Louis, MO, USA) and quantified using a spectrophotometer. Equal amounts of total RNA was used to generate complementary DNA, cDNA, using a reverse transcription kit from Sigma (Sigma-Aldrich Inc, St Louis, MO, USA). The quality of cDNA samples were verified using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a house keeping control.
Quantitative gene transcript analyses. This was based on the Amplifluor™ Technology in quantitative gene transcript analysis. Briefly, pairs of specific primers were designed to amplify a region of mTOR, RAPTOR and RICTOR. To one primer of the primer sets was added a Z sequence which is complementary to the Amplifluor probe (FAM tagged, synthesised by Biosearch Technologies, Inc., Novato, CA, USA). Each reaction comprised of a forward primer, a reserve Z primer at one tenth of the strength, the Amplifluor probe, cDNA from tumours and custom master mix (Life Technologies, Paisley, UK) (Table II). An internal standard was included as control. Quantitative Polymerase Chain Reaction (PCR) analysis was conducted using a SteponePlus instrument (ABI, Paisley, Scotland, UK). GAPDH transcript, was also simultaneously quantified and was used as a house keeping control.
Statistical analysis. The statistical package used was SigmaPlot (Version 11). Student t-test and ANOVA were used for normalised data and Mann-Whitney U-test and Kruskall–Walis test for non-normalised data.
Results
mTOR, RAPTOR and RICTOR expression and tumour size. There was a stepwise increase in the levels of mTOR and RAPTOR with increasing tumour size (Figure 1), although the differences were not significant. It is interesting to note that there was a similar but significant positive correlation between RICTOR and tumour size, namely p=0.0012 and p=0.0055 for tumours 1-2 cm and tumours >3 cm compared with tumours <1 cm (Figure 1 and Table III).
Expression of mTOR, RAPTOR and RICTOR was not significantly associated with sex and age (Table III). Significantly higher levels of mTOR were seen in tumours with cystic lesions (p=0.044 vs. tumours without cysts). Interestingly, RAPTOR and RICTOR levels were marginally lower in tumours with cystic lesions, although these differences were not significant (Figure 2). There was no correlation between mTOR, RAPTOR or RICTOR and intratumour haemorrhage (Table III).
MRI images of typical cases of Hardy's stage I-IV (A-D, respectively), with stepwise increasing levels of Mammalian Target Of Rapamycin (mTOR). Regulatory associated protein of mTOR (RAPTOR) and Rapamycin Independent Companion of mTOR (RICTOR). White arrowhead indicates the tumour.
mTOR, RAPTOR and RICTOR and invasion. We did not find any correlation between mTOR, RAPTOR or RICTOR with suprasephanous spread and invasion (Table III). There were also no significant difference between tumours with sella floor destruction and without destruction for all three molecules (Table III). Advanced pituitary tumours, namely Knosp stage 3/4, had significantly lower levels of RAPTOR than those of Knosp stage 1/2 (p=0.010). A similar trend was seen for RICTOR although this was not significant (p=0.09). An opposite but insignificant trend was seen for mTOR (p=0.53) (Figure 3).
mTOR, RAPTOR and RICTOR and tumour staging. A modified Hardy's staging showed a clear trend of increased levels of all three molecules with increasing tumour stage (Figure 4). It is interesting to note that stage-I tumours had very little expression of RAPTOR, significantly lower than stage II-IV tumours (p<0.01 stage-I vs. other stages) (Figure 4 middle). Likewise, the level was also low in stage-I tumours: Stage-II, III and IV tumours had significantly higher levels of RICTOR transcript than stage-I tumours (p=0.004, 0.014 and 0.006 respectively) (Figure 4 bottom). Transcript levels of mTOR had a similar trend to that of RAPTOR and RICTOR, although this was not statistically significant (p=0.065, stage-I vs. other stages). Figure 5 shows some representative MRI images from patients with differing levels of mTOR, RAPTOR, RICTOR and different tumour staging.
mTOR, RAPTOR and RICTOR and endocrine functions. mTOR and RAPTOR levels differed in tumours with different endocrine functions, although no statistical difference was observed. However, GH-, FSH-, TSH-secreting tumours had significantly lower levels of RICTOR compared with non-functional tumours (Figure 6).
mTOR, RAPTOR and RICTOR are intercorrelated. As shown in Table IV and Figure 7, mTOR was found to be significantly correlated with both RAPTOR and RICTOR. It is noteworthy that RAPTOR and RICTOR were found to be highly significantly correlated.
Expression of Mammalian Target Of Rapamycin (mTOR). Regulatory associated protein of mTOR (RAPTOR) and Rapamycin Independent Companion of mTOR (RICTOR) in relation to the endocrine function of pituitary tumours. Growth Hormone (GH)-, Follicle-Stimulating Hormone (FSH)-, Thyroid Stimulating Hormone (TSH)-secreting tumours had significantly low levels of RICTOR compared with non-functional tumours. PRL: Prolactin; ACTH: Adreno-Cortico-Tropic-Hormone; LH: Luteinizing Hormone.
Discussion
The mTOR pathway plays a pivotal role in the regulation of cell growth and death, energy, lipid and protein metabolism and cell migration. Its expression is aberrant in a number of human solid tumour and has been shown to be a viable target in cancer treatment (23-25). Here, we provide fresh evidence that both mTOR and its protein complex facilitators, RAPTOR and RICTOR which form different mTOR complexes, are also aberrantly expressed in human pituitary adenomas.
Regulatory associated protein of mTOR (RAPTOR) and Rapamycin Independent Companion of mTOR (RICTOR) were highly significantly correlated.
Spearman's rank correlations of Mammalian Target Of Rapamycin (mTOR), Regulatory associated protein of mTOR (RAPTOR) and Rapamycin Independent Companion of mTOR (RICTOR).
The most interesting finding of the present study is the link between mTOR, RAPTOR and in particular RICTOR with tumour staging. With increasing tumour stage, there were stepwise increases in the transcript levels of mTOR, RAPTOR and RICTOR. Hardy's classification divides the tumours into four stages by taking into consideration tumour size and invasion. Thus, Hardy staging and the Knosp staging methods are quite different, with the latter being a practical classification for cavernous sinus invasion.
The tumour size is closely related to the clinical symptoms. With an increase in tumour size, patients may first lose visual acuity. With subsequent invasion into the thalamus, the tumour can cause diabetes insipidus. Blocking the interventricular foramen can lead to hydrocephalus and invasion into the carvernous sinus will cause cranial nerve palsy. Thus, there is a certain relationship between the changes of clinical symptoms in patients with changes in mTOR pathway. The mTOR pathway can promote the proliferation of tumour cells. Strong proliferation of tumour cells is precisely the material basis of pituitary adenoma recurrence and invasive growth. The data presented here, together with the limited reports in the literature, strongly argue that mTOR, RAPTOR and RICTOR have an important predictive and prognostic value in patients with pituitary tumours (25-27).
The finding that RAPTOR and RICTOR expression are both increased together with mTOR is also interesting. RAPTOR and RICTOR form mutually exclusive protein complexes with the mTOR protein. In the protein complexes mTORC1 and mTORC2, there are different components largely as a result of the association between mTOR with either RAPTOR or RICTOR (2-4, 23). It has been established that the two protein complexes have different regulatory roles in the cells, namely mTORC1 is more dominant than mTORC2 in regulating lipid and protein metabolism, cell growth and gene expression. In contrast, mTORC2 is more dominant in regulating cytoskeletal organisation and thus cell migration, as well as activation of mTORC1. The two protein complexes differ in their protein components and in some aspects of cell functions, but also communicate with each other. Thus, the findings that both expression of RAPTOR and RICTOR were highly raised in aggressive pituitary tumours may suggest that both play a key role in pituitary adenoma.
The other intriguing finding is that mTOR expression is significantly correlated with that of both RAPTOR and RICTOR. It is also interesting to observe that RAPTOR and RICTOR are also highly correlated. Presently, the mechanism(s) by which the aberrant expression occurs is not clear. Those three genes are located on different chromosomes. RAPTOR is located on 17q25.3, RICTOR on chromosome 5, and mTOR on 1p36.22 (28-30). The regulation of gene transcription of all three genes is yet to be fully established. Given that the raised levels of all the molecules are seen at message level, a possibility exists that there may be transcriptional aberration in pituitary adenomas. Further investigation is necessary to establish whether this is the case.
mTOR inhibitor has been indicated in both endocrine-active and -inactive pituitary tumours (24-26). The present study did not show a consistent pattern of the expression of these molecules and the endocrine functions of the tumours. From the above data, we argue that mTOR, RAPTOR and RICTOR have no clear link with the endocrine functions of the tumours, however, a large cohort is required to firmly establish this.
In conclusion, the current study adds further weight the ongoing development and indicates that pituitary adenomas, endocrine-active or -inactive, have aberrant expression of mTOR and its regulator, and appear to be an ideal tumour type for mTOR therapies.
Acknowledgements
The Authors wish to thank Cancer Research Wales and the Albert Hung Foundation for supporting their work.
- Received May 26, 2013.
- Revision received June 16, 2013.
- Accepted June 18, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
