Abstract
Background/Aim: Mammalian target of rapamycin (mTOR) plays a critical role in the regulation of tumor cell motility, invasion and cancer cell metastasis. mTOR consists of two separate multi-protein complexes, mTOR complex (mTORC) 1 and mTORC2. Materials and Methods: We investigated the expression levels of mTORC1 and mTORC2 immunohistochemically in oral squamous cell carcinoma (OSCC). Results: mTORC1 and mTORC2 were more highly expressed in tumors than in normal oral mucosa. mTORC1 expression was correlated with T classification, N classification, and survival rate (p<0.05), whereas mTORC2 expression was only correlated with T classification (p<0.05). Histologically, the expression levels of mTORC1 and mTORC2 correlated with cancer cell invasion and the expression of proliferating cell nuclear antigen (p<0.05), respectively. Expression levels of vascular endothelial growth factors and hypoxia-inducible factor 1 in the mTORC1 (−)/ mTORC2 (+) group were significantly lower than those in other groups. Conclusion: These findings suggested that mTORC1 and mTORC2 could be promising anti-tumor targets in OSCC, and mTORC1 (−)/mTORC2 (+) may have a correlation with the malignant potential of OSCC.
Cancer cell migration and invasion play fundamental roles in cancer metastasis. Mammalian target of rapamycin (mTOR) is a 289-kDa serine/threonine kinase, belonging to the phosphoinositide 3-kinase (PI3K)-related kinase family, that regulates cell growth, proliferation, and progression of the cell cycle (1). Recent studies have shown that mTOR also plays a critical role in the regulation of tumor cell motility and invasion and cancer cell metastasis (2, 3).
mTOR is activated by the phosphorylation of Ser2448 through the PI3K/Akt signaling pathway, which then completes its functions by activating p70 ribosomal S6 kinase and phosphorylating the eukaryotic initiation factor 4E binding protein 1 (4, 5). The mTOR pathway and mTOR genes were discovered while investigating the mechanism of action of their inhibitor rapamycin and the causes of resistance (6). Rapamycin, a macrolide antibiotic product, was first discovered in 1975 from samples collected on Easter Island in the South Pacific (6). A few years later, rapamycin demonstrated significant immunosuppressive activity in preventing the development of allergic encephalomyelitis and adjuvant arthritis in rats. The basis of its immunosuppressive properties was established by many later studies and led to the establishment of rapamycin as a major immunosuppressant against transplant rejection (6).
mTOR consists of two separate multi-protein complexes, mTOR complex (mTORC) 1 and mTORC2, which are both activated by growth factor stimulation (6, 7). mTORC1 phosphorylates p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), and regulates cell growth, proliferation, survival, and motility (8). mTORC2, which phosphorylates Akt, protein kinase C, and focal adhesion proteins, controls the activities of small GTPases, and regulates cell survival and the actin cytoskeleton (8). Rapalogs are first-generation mTOR inhibitors and they allosterically inhibit mTORC1 but not mTORC2 (9, 10). Activated mTOR has been associated with poor prognosis in various cancers, including oral squamous cell carcinoma (OSCC) (11-14). However, the anti-tumor effects of mTOR inhibitors in OSCC remain unclear. To ascertain the effects of mTOR inhibitors in OSCC, it is important to investigate the expression levels of mTORC1 and mTORC2 in OSCC.
In the present study, we investigated immunohistochemically the expression levels of mTORC1 and mTORC2 in OSCC and examined their relationships with clinical and pathological factors.
Materials and Methods
Paraffin-embedded sections were obtained from biopsy specimens from 72 patients with OSCC who underwent radical surgery in our department between January 2000 and December 2007. The tumor stage was classified according to the TNM classification of the International Union Against Cancer (15). The histological differentiation of tumors was defined according to the WHO classification (16) and the invasive grade was assessed by the Yamamoto-Kohama (YK) mode of invasion (17).
Deparaffinized sections in xylene were soaked in 10 mmol/l citrate buffer (pH 6.0) and placed in an autoclave at 121°C for 5 min for antigen retrieval. Endogenous peroxidase was blocked by incubation with 0.3% H2O2 in methanol for 30 min. Immunohistochemical staining was performed using an Envision system (ENVISION+; DAKO, Glostrup, Denmark). The primary antibodies used were against mTORC1, mTORC2, proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF)-A, VEGF-C, and hypoxia-inducible factor 1α (HIF-1α) (DAKO, Glostrup, Denmark). The sections were then washed in Dulbecco's phosphate buffered saline (PBS), followed by incubation with the primary antibodies at 4°C over night. The reaction products were visualized by immersing the sections in diaminobenzidine (DAB) solution, and the samples were counterstained with Mayer's hematoxylin and mounted.
Results were evaluated by calculating the total immunostaining score as the product of the proportional score and intensity score. The proportional scores described the estimated percentage of positively stained tumor cells (0, none; 1, <10%; 2, 10-50%; 3, 50-80%; 4, >80%). The intensity score represented the estimated staining intensity (0, no staining; 1, weak; 2, moderate; 3, strong). Total scores ranged from 0 to 12. Immunohistochemical overexpression was defined as a total score of 4 or greater, because immunohistochemical expression levels in samples showed a bimodal distribution with the discriminating nadir at a total score value of 3 to 4. mTOR (−) was defined as a total mTOR immunostaining score ranging from 0 to 3 and mTOR (+) was defined as total mTOR immunostaining score of 4 or greater.
The relationships between the sample expression of target molecules and clinicopathological features were assessed using Fischer's exact test. Survival analysis was calculated using the Kaplan–Meier method and compared using the log-rank test. A multiple regression study was performed using Cox's proportional hazard analysis and chi-squared test. p-Values <0.05 were considered to be significant.
Results
mTORC1 and mTORC2 were expressed mainly in the cytoplasm of the tumor cells (Figure 1). In OSCC specimens, mTORC1 and mTORC2 were detected in 37 tumors and 50 tumors, respectively. mTORC1 and mTORC2 protein expression was absent or minimal in the cytoplasm of epithelial cells in normal oral tissue. mTORC1 expression correlated with T classification, N classification, and survival rate (p<0.05), whereas mTORC2 expression was only correlated with T classification (p<0.05) (Table I). Histologically, the expression levels of mTORC1 and mTORC2 correlated with cancer cell invasion by Y-K classification (Table II) and the expression of PCNA (p<0.05) (Table III), respectively. Concerning the survival rate, there was significant difference between mTORC1(+) and mTORC1(−), but there was no significant difference between mTORC2(+) and mTORC2(−) (Figure 2).
The numbers of case with mTORC1 (−)/mTORC2 (−), mTORC1 (−)/mTORC2 (+), mTORC1 (+)/mTORC2 (−), and mTORC1 (+)/mTORC2 (+) were 16, 17, 6, and 33, respectively. In OSCC samples, VEGF-A, VEGF-C and HIF-1α protein expressions were detected 50, 51 and 50 samples, respectively. VEGF-A, VEGF-C and HIF-1α were expressed in cytoplasm of OSCC (Figure 1). The expression levels of VEGFs and HIF-1α in the mTORC1 (−) mTORC2 (+) group were significantly lower than those in the other groups (Table IV).
Discussion
The anticancerous activity of rapamycin was first demonstrated in human glioma tumor xenografts in mice (6, 18). In the last few years, significant advances have been made in understanding the role of mTOR in cancer development and progression (3, 19). Increased mTOR signaling in cancers often occurs as a result of mutations in the pathways closely related to mTOR. Recently, activating mutations of mTOR itself have been identified through mining of human cancer genome databases (20). Activation of the PI3K/Akt/mTOR pathway through such mechanisms has been shown to correlate with tumor progression and reduced survival in patients across a variety of tumor types (21, 22).
To investigate the role in initiation and development of cancer, molecular approaches have been used to study the specific components of the mTOR pathway (3). Some researchers have demonstrated that molecular inhibition of mTOR, Rictor, or Raptor leads to a significant decrease in proliferation of cancer cells and attenuates cell cycle progression (21, 23, 24).
mTOR also plays a key role in advanced and metastatic disease. Although the molecular mechanisms of the regulation of cell motility and metastasis by mTOR are not fully understood, there are some reports concerning metastasis in breast cancer, gliomas and head and neck cancer (25-27). In colorectal cancer, Gulhati et al. demonstrated that mTORC1 and mTORC2 are intimately involved in epithelial-mesenchymal transition, motility, and metastasis (26). In head and neck squamous cell carcinoma, Patel et al. (28) developed an orthotopic model of head and neck squamous cell carcinoma (HNSCC) consisting of the implantation of HNSCC cells into the tongue of immunocompromised mice, and they found that inhibition of mTOR with rapamycin and the rapalog RAD001 diminished lymphangiogenesis in the primary tumors and prevented the dissemination of head and neck cancer cells to the cervical lymph nodes. In addition, knockdown of mTORC1 and mTORC2 was reported to sensitize colorectal cancer cell lines to apoptosis induced by oxaliplatin (26).
Immunohistochemically determined expression levels of mTORC1, mTORC2, VEGF-A and VEGF-C. Immunohistochemically-positive cells can be seen in the cancer cell nest. A; mTORC1, B; mTORC2. C; VEGF-A, D; VEGF-C.
Kaplan–Meier survival curve. Significant differences in survival are present between the mTORC1 positive and negative groups. There is no significant difference between the mTORC2-positive and -negative groups.
Correlation between mTORC1 and mTORC2 expression and clinicopathological features.
Correlation between mTORC1 and mTORC2 expressions and YK mode of invasion of oral SCC.
Correlation between mTORC1 and mTORC2 expressions and PCNA-LI.
mTORC1 is comprised of mTOR, regulatory-associated protein of mTOR (Raptor), mLST8/GβL, Deptor, and proline-rich AKt substrate 40 (29). mTORC1 influences cell growth and proliferation by promoting the biosynthesis of proteins, lipids, and organelles, and by limiting catabolic processes (8). In addition, activation of mTORC1 is sufficient for tumorigenesis, such as the promotion of glycolysis, increased flux through the oxidative branch of the pentose pathway, and enhanced de novo lipogenesis (30). mTORC2 consists of mTOR, rapamycin-insensitive companion of mTOR (Rictor), mLST8/GβL, Protor, Deptor, and mammalian stress-activated protein kinase interacting protein (mSIN1) (8). Compared with mTORC1, the regulation and functions of mTORC2 are less well understood. mTORC2 was first reported to be involved in the control of actin cytoskeleton organization (31). Subsequent studies have suggested that mTORC2 also plays a part in the regulation of proliferation, survival, and nutrient uptake in cancer cells (32-34). In the present study, both mTORC1 and mTORC2 expression levels correlated with T stage and PCNA expression. Hence, we suggested that mTORC1 and mTORC2 play roles in the cell proliferation of OSCC. In addition, in present study, we demonstrated that cases of mTORC1(−)/mTORC2(+) had significantly lower expression levels of VEGFs and HIF1-α compared with the other expression pattern groups.
Correlation between VEGF and HIF-α expression and mTORC1/mTORC2 expressions.
VEGF-A overexpression has been reported in most types of cancer, including oral cancer, and it is thought to be a prognostic factor for survival (2). VEGF-C has been detected in several different types of cancer, and its level in some studies seemed to correlate with nodal metastasis and patient survival (2). The HIF-1α transcription factor plays an essential role in oxygen homeostasis, and high expression of HIF-1α protein has been found to be associated with both tumor aggressiveness and an unfavorable prognosis in various types of cancer (2). Hence, we suggest mTORC1(−)/mTORC2(+) may have a low potential for malignancy in OSCC, and furthermore have correlation with the effectiveness of anti-mTOR drugs.
Conclusion
mTORC1 and mTORC2 were found to be overexpressed in OSCC, and they were significantly correlated with clinicopathological factors. In a previous study, we concluded that expression levels of mTORC1-HIF1-VEGF had a significant correlation with the effects of anti-mTOR drugs. In the present study, the mTORC1 (−)/mTORC2 (+) group showed significantly lower VEGF and HIF1 expression levels. Hence, it was suggested that mTORC1 and mTORC2 could be promising targets for anti-tumor effects in OSCC, and mTORC1 (−)/mTORC2 (+) may have a correlation with the effectiveness of anti-mTOR drugs.
- Received November 13, 2017.
- Revision received December 18, 2017.
- Accepted December 20, 2017.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.