Torin 2

Therapeutic implication of mTORC2 in oral squamous cell carcinoma
Tomofumi Naruse a,⇑, Souichi Yanamoto a, Kohei Okuyama a, Kentaro Yamashita a, Keisuke Omori a,
Yuji Nakao b, Shin-ichi Yamada b, Masahiro Umeda a
a Department of Clinical Oral Oncology, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan
b Department of Dentistry and Oral Surgery, Shinshu University School of Medicine, Matsumoto, Nagano 390-8621, Japan


Article history:
Received 26 July 2016
Received in revised form 9 November 2016 Accepted 14 December 2016

Rictor SGK1
Torin-1 Cetuximab


The aim of the present study was to clarify the association of mTORC2 expression with the cancer pro- gression and the anti-tumor effects of Torin-1 alone and combined treatment with Cetuximab in OSCC cells. The expressions of Rictor and SGK1 were immunohistochemically evaluated and the relationships between the expressions of molecular markers and clinicopathological factors were determined. Moreover, OSCC cells were treated with Torin-1, Cetuximab or combined agents, and anti-tumor effects of OSCC cells were examined in vitro and in vivo. Rictor and SGK1 expressions were significantly associ- ated with tumor stage and pattern of invasion in OSCC sections (P < 0.05 and P < 0.01, respectively). Treatment of OSCC cell lines with Torin-1 resulted in dose and time-dependent inhibition of proliferation with decrease of phosphorylation on downstream molecules. Combined treatment with Torin-1 and Cetuximab resulted in enhanced anti-tumor effects in vitro compared with either agent alone. Furthermore, treatment of mice bearing OSCC xenografts with Torin-1 and Cetuximab also demonstrated a remarked growth inhibition of tumor volumes. The results suggested that new regimens of systemic therapy combined with Cetuximab and Torin-1 may be useful for very advanced OSCC patients. © 2016 Elsevier Ltd. All rights reserved. Introduction Oral squamous cell carcinoma (OSCC) represents 2–3% of all human cancers and is the 6th most frequent cancer in the world [1,2]. OSCC has continually shown a poor prognosis and remains a lethal disease for >50% of cases diagnosed annually [3]. The cur- rent management and treatment of OSCC for most patients is sur- gery, and postoperative concurrent chemoradiotherapy is a widely accepted standard of care for high-risk OSCC [4]. Recently, Cetux- imab, an epidermal growth factor receptor (EGFR) inhibitor, was approved for the treatment of locally advanced (LA) and recur- rent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) in December 2012 in Japan. National Comprehensive Can- cer Network (NCCN) Clinical Practice Guideline has provided that systemic therapy including Cetuximab is the standard therapy with strong evidence for very advanced HNSCC [4]. Despite new treatment options developed in the last some years, the prognosis for patients with unresectable LA or R/M OSCC remains poor. We previously reported that Cetuximab therapy was shown to result in improved 1year OS, but not improved 2-year OS using a histor- icalcontrol study [5]. Although Cetuximab alone has been reported

⇑ Corresponding author.
E-mail address: [email protected] (T. Naruse).

active and generally well tolerated in the treatment of R/M HNSCC [6], the modest benefit of EGFR blockade when Cetuximab alone is administered could be attributed to compensation by other signal- ing pathways that are independent of EGFR [7].
The mammalian target of rapamycin (mTOR) is a 289-KDa ser- ine/threonine kinase belonging to the phosphoinositide 3-kinase (PI3 K)-related kinase family that regulates cell growth, prolifera- tion, and progression of the cell cycle, and therefore a potential tar- get for anti-cancer therapy [8–10]. The mTOR complex is comprised of two complexes, mTOR complex1 (mTORC1) and mTOR complex2 (mTORC2). mTORC1 is activated by the phospho- rylation of Ser2448 through the PI3K/AKT signaling pathway, and completes these functions by activating p70 ribosomal S6 kinase (p70S6K) and phosphorylating the eukaryotic initiation factor 4E binding protein 1 (4E-BP1). One of the common features of this pathway include activated p70S6K phosphorylates Rictor and/or Insulin receptor substrate (IRS)-1, consequently mTORC2 and PI3K/AKT signaling were inhibited; these signaling formed mTORC1 dependent negative feedback [8–10]. On the other hand, mTORC2 contains exclusively rictor (rapamycin-insensitive com- panion of mTOR) (Avo3 in S.cerevisiae), mSin1 (Avo1 in S.cere- visiae), and PRR5/protor. mTORC2 plays an important role in cell survival, metabolism, proliferation and cytoskeleton organization, as it phosphorylates Protein Kinase Ca (PKCa), SGK1, as well as 1368-8375/© 2016 Elsevier Ltd. All rights reserved.

Akt, allowing for its complete activation [8–10]. Particularly, forced expression of Rictor promotes mTORC2 activation, and activated mTORC2-SGK1 axis has been associated with drug resistance, lead- ing to the poor prognosis in human tongue cancer [11].
Recently, in addition to p70S6K-IRS-1 axis, mTORC1 phosphory- lates Growth Factor Receptor Bound Protein 10 (Grb10), leading to accumulation of Grb10 and negative feedback inhibition of PI3K and Microtubule-associated protein kinase/Extracellular signaling regulated kinase (MAPK/ERK) pathway [12]. This means that dis- ruption of mTORC1 dependent negative feedback results in more upregulation of PI3K-Akt pathway, leading to the pro-survival rather than anti-tumor effect of the mTORC1 rapalog. Therefore, a new generation of mTOR inhibitors, which compete with ATP in the catalytic site of mTOR and inhibit both mTORC1 and mTORC2 with high degree of selectivity, has been expected [13].
With regard to the dual mTORC1/mTORC2 inhibitor, recent studies demonstrated the efficacy of the Torin 1, PP242, WYE354, KU63794 and OSI-027 in some solid tumor cell lines [14–18]. Among them, Torin-1 is the most powerful and selective inhibitor, and is possible to inhibit the tumorigenic potentials of stem-like cells in metastatic colorectal cancer [13,17]. However, anti-tumor effects of Torin-1 against OSCC have not yet investigated. The aim of the present study is to determine the clinicopathological significance of mTORC2-SGK1 axis in OSCC. We also examined the anti-tumor effects of Torin-1 alone or in combination with Cetuximab in OSCC preclinical models (Fig. 1).

Materials and methods


This study was approved by the independent ethics committee of our hospital (Approval No. 15061128). We retrospectively reviewed the records of 70 patients who underwent radical surgery with or without adjuvant RT/CCRT for OSCC between January 2001 and December 2007, and tumor outcome data is available. Patients who underwent preoperative chemotherapy or radiation therapy or who had insufficient preoperative records were excluded. The tumor stage was classified according to the TNM classification of

the International Union Against Cancer [19]. Tumor histologic dif- ferentiation was defined according to the WHO classification. Pat- tern of invasion (POI) was classified according to Bryne’s classification [20].

Immunohistochemical staining and evaluation

Biopsy specimens were taken from the patient with OSCC, formalin-fixed, and paraffin-embedded after surgery. The speci- mens of normal oral mucosa from 10 healthy individuals were used as controls. Sections deparaffinized 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 the EnVision method (EnVision+; Dako, Glostrup, Denmark). The fol- lowing antibodies were used as primary antibodies: polyclonal antibody for Rictor (ab70374; Abcam; dilution 1:100, Cambridge, UK) and SGK1 (ab59337; Abcam; dilution 1:100, Cambridge, UK) derived from rabbit. Anti-PCNA monoclonal mouse antibody (PC10; Dako, dilution 1:100, M0879, Denmark) were used as prolif- eration marker. The sections were then washed in Dulbecco’s phos- phate buffered saline (PBS), followed by incubation with the
primary antibodies at 4 °C overnight. The reaction products were visualized by immersing the sections in diaminobenzidine (DAB)
solution, and the samples were counterstained with Meyer’s hema- toxylin and mounted.
Rictor and SGK1 protein expressions were evaluated by calcu- lating the total immunostaining score as the product of the propor- tional score and the intensity score in the invasion front of the tumor in each of the 2 visual fields. The proportional scores are based on the estimated fraction of positively stained tumor cells (0, none; 1, <10%; 2, 10–50%; 3, 50–80%; 4, >80%). The intensity
score represents the estimated staining intensity (0, no staining; 1, weak; 2, moderate; 3, strong). The total immunostaining score therefore ranges from 0 to 12. Positive cases were defined as those with a total score >4, as the patient samples showed a bimodal dis- tribution of immunohistochemical expression, where in the dis- criminating nadir was a total score value of 3–4. The PCNA

Fig. 1. Mechanisms of mTOR pathway in cancer cells.

labeling index (LI) of tumor cells was defined as the percentage of PCNA-positive cells in each of the 2 visual fields was counted, and mean values was calculated. All immunohistochemical assess- ments were performed by two examiners in a blinded fashion, and based on the results of these assessments, we compared the responses and characterized the tumors by stage and progression.

Cell lines and reagents

All 5 human OSCC cell lines, SAS, HSC-2, HSC-3, SCC25, and Ca9-22, used in this study were cultured in a 1:1 mixture of Ham’s F-12/DMEM supplemented with 10% FBS (Trace Scientific,
Melbourne, Australia). All cells were maintained under humidified 5% CO2 and 19% O2 incubation at 37 °C. Torin-1, a novel molecular targeted drug, was purchased from Tocris Bioscience (Bristol,
BS119QD, UK). It was dissolved in DMSO and adjusted to the final concentration with culture medium. Cetuximab (C225, ErbituxTM) was provided by Merck (Darmstadt, Germany). Working solutions were freshly prepared from the stock solution by dilution in cell culture medium on the day of the experiment.

Cell proliferation assay

Cells were seeded in 96-well plates at a concentration of
1.5 103 per well and incubated for 24 h. Cells were exposed to Torin-1 and cetuximab doses ranging from 10 nmol/L to 1000 nmol/L. At the end of the treatment for 72 h, cells were incubated with 0.5 mg/ml 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT; Sigma-Aldrich). Four hours later, the medium was replaced with 100 ll dimethylsulfoxide (DMSO; Sigma-Aldrich) and vortexed for 10 min. Absorbance was then recorded at 570 nm using a microplate auto reader (Multiskan FC, Thermo Fisher Scientific Inc). Cell viability (%) was calculated as a percentage of the absence of agents. The 50% of cell growth inhi- bition (IC50) values were appropriately derived from the results obtained with the MTT assay.

Western blot analysis

Cells were harvested by trypsinization, washed, and precipi- tated by centrifugation. The Mammalian Cell Extraction Kit (Biovi- sion Research Products, Mountain View, CA) was used to extract proteins. All subsequent manipulations were performed on ice. The cells were incubated in Extraction Buffer Mix. Lysed cells were centrifuged at 15,000 rpm for 5 min, and the resultant supernatant was used. The protein concentration of each sample was measured with micro-BCA protein assay reagent (Pierce Chemica. Co., Rock- ford, IL). After the samples were denatured in SDS sample buffer,
they were heated at 70 °C for 10mins and then loaded onto a 4– 12% NuPAGE NOVEX bis-Tris polyacrylamide gel or 3–8% NuPAGE
NOVEX Tris-Acetate polyacrylamide gel. After electrophoresis, the separated proteins were transferred to iBlot polyvinylidene difluo- ride membranes using the iBlot Dry Blotting System and signals were detected by the Western Breeze Immunodetection Kit (life technologies, Tokyo, Japan). Antibodies against Rictor, SGK (Abcam, Cambridge, UK), p-Rictor Thr1135, p-SGK1 Ser78, mTOR, p-mTOR Ser2448, p70S6K and p-p70S6K Tyr389 (Cell Signaling Technology, Danvers, MA) were used at 1:1000 dilution. Anti-b-actin (Santa Cruz Biotech, CA) was used as a blotting control.

Cell migration and invasion assay

A Biocoat Matrigel invasion chamber containing an internal chamber with an 8-lm porous membrane bottom coated with Matrigel (Becton Dickinson, Bedford, MA) was used for the inva- sion assay. Twelve cell culture inserts and 24-well multiwall com-

panion plate were used for the experiment. The membranes were rehydrated with warm serum-free medium for 2 h. Cells were col- lected by tripsinization, followed by sedding in the internal cham- ber at 1.25 105 cells in serum-free medium. The lower chamber was filled with medium containing 10% FBS as a chemoattractant. Cells were incubated for 72 h, non-invading cells were removed from the top of the wells with a cotton swab, and cells that were transferred to the inverse surface of the membrane were subjected to Diff-Quick staining. Cells were counted under a microscope at
100 magnification. Cells that passed through a control chamber without Matrigel were calculated for the migration index. The per- centage of the cell count that passed through the Matrigel chamber to the control cell count was calculated as the invasion index. All experiments were performed in triplicate, and cell numbers were counted in at least 2 fields/well.

Xenograft model and tumor growth inhibition assay

All animal experiments were performed in compliance with the Guildlines for Animal Experiments at Nagasaki University Gradu- ate School of Biomedical Sciences. SAS cells (1 × 107) were sus-
pended in 100 ll of Dulbecco’s PBS and were injected
subcutaneously into the back of 6-week old BALB/c-nu nude mice using 21-gauge needle. After growing to 10–15 mm in diameter, the tumor was extirpated, cut into 1 mm3 pieces, and transplanted subcutaneously into the back of the different nude mice.
SAS tumor was transplanted in 12 mice and divided into 4 groups (Control, Cetuximab, Torin-1 and combination group, respectively). After growing to 5–10 mm in diameter, the tumor- bearing mice were treated for 4 weeks with Cetuximab (20 mg/kg, three times/week) and/or Torin-1 (10 mg/kg, three times/week). Cetuximab was diluted 1:4 in saline. Torin-1 was dissolved by 20% N-methyl-2-pyrrolidone and adjusted by 40% PEG400 and 40% distilled water (DW). Control group received 50% PEG400 and 50% DW only [21]. These agents were delivered by i.p. injec- tion, and tumor size were measured three times a week by the ver-
nier caliper at the same time. Tumor volume was evaluated using the formula 4/3p (width/2)2 (length/2)2 because the tumor growth after transplantation was spherical. Humane endpoint was set to 20 mm in greatest dimension according to the Guildlines of our university. Under the same condition as previously described, after the mice were treated for 4 weeks, xenografted tumors were excised, fixed in buffered formalin and embedded in paraffin for histological and immunohistochemical examination used by p-EGFR Tyr1068 and p-mTOR Ser2448 antibodies (Cell Signaling Technology, Danvers, MA).

Statistical analysis

The associations between the expression of targeted molecules and clinicopathological features were analysed by Fisher’s exact test and Spearman rank-correlation test. Continuous data were given as means ± standard deviation. Survival analyses were calcu- lated by the Kaplan-Meier method and compared using the log- rank test. Differences between two groups were compared with the t-test. Multiple comparison test among the four groups was used by sheffe’s method. P values of < 0.05 were considered significant. Results Expression of Rictor and SGK1 in OSCC Among the 70 patients with OSCC, immunohistochemical staining were positive for Rictor in 68.5% and positive for SGK1 in 60.0%. Rictor was expressed primarily in the cytoplasm and nucleus of the tumor cells, ranging from low to strong intensity. The distributions of both Rictor and SGK1 were observed in tumor nests and at the invasive front, with especially strong expression observed at the invasive front. In the normal oral epithelium, expression of these molecular markers were absent (Fig. 2A and B). Association of Rictor and SGK1 expression with clinicopathological factors and survival Rictor and SGK1 expression levels in OSCC were examined as a function of clinicopathological factors. Rictor expression was significantly associated with T classification and pattern of invasion (POI) (P < 0.05, respectively). SGK1 expression was also Fig. 2. Representative immunohistochemical staining for Rictor and SGK1. (A) Negative staining for Rictor is seen in Normal epithelium. OSCC with Bryne’s score 3 demonstrates strong Rictor cytoplasmic and nuclear expression (staining index of 12, ×100 and ×400, respectively). (B) Negative staining for SGK1 is seen in the normal epithelium. OSCC with Bryne’s score 3 demonstrates strong SGK1 cytoplasmic and nuclear expression (staining index of 12, original magnification ×100 and ×400, respectively). significantly associated with T classification and POI (P < 0.01, respectively; Table 1). Both Rictor and SGK1 double positive were observed in 38 of 70 cases, and significantly correlation between Rictor and SGK1 was found (rs = 0.5779, P < 0.001). Immunohistochemical PCNA expression levels were examined in the cancer cells to determine the interaction between tumor cell proliferation and the function of the Rictor and SGK1. PCNA expres- sion was detected immunohistochemically in the nucleus of the tumor cells. The average PCNA labeling index (LI) was 43.615% and 43.374% in Rictor and SGK1 positive cases respectively. In con- trast, the average PCNA LI were 26.682% and 30.071% in Rictor and SGK1 negative cases respectively. This difference was significant both Rictor and SGK1 between positive cases and negative case (Table 2). The 5-year DSS rates according to Rictor and SGK1 expressions were determined. Univariate analysis using the log-rank test and the Kaplan-Meier method showed that both Rictor and SGK1 were likely to be associated with 5-year DSS, but no significant differ- ence was found (Fig. 3A and B, P = 0.23, 0.06, respectively). In addi- tion, a subgroup analysis regarding in conjuction with T stage revealed that high Rictor and SGK1 expressions were likely to be associated with poor prognosis, although not significantly. (Fig. 3C and D, P = 0.11, 0.07, respectively). In the summary of immunohistochemical analysis, Rictor and SGK1 expressions were observed in OSCC specifically, and were associated with cell prolif- erations and invasion. Inhibitions of Rictor and SGK1 by Torin-1 suppressed cell growth in OSCC cell lines At the protein levels, Rictor and SGK1 expressions were evalu- ated by western blotting in OSCC cell lines. Since SAS and HSC-3 cell lines were shown the high expressions, we selected these cell lines in next experiments (Fig. 4). In the MTT assay, Torin-1 signif- icantly inhibited cell proliferation in a dose-dependent manner in both cell lines tested (Fig. 5A and B, p < 0.05). Moreover, the expression levels of the intrinsic and phosphorylated forms of Rictor, SGK1, mTOR and p70S6K were analysed by western blotting. The results showed that Torin-1 decreased the phosphorylations of p-Rictor, p-SGK1, p-mTOR and p- p70S6K at the IC50 concentrations compared to controls (Fig. 5C). Effects of agent alone and combined treatment with Torin-1 and Cetuximab on the migration and invasion potential We analysed migration and invasion potentials on both cell lines to examine the effects of either agent alone and combined treatment with Torin-1 and Cetuximab. The evaluation of cell migration revealed a significant decrease in both either agent alone and combined agents compared with controls (Fig. 6A–C, SAS, P < 0.01; HSC-3, P < 0.01). No significant differences between either agent alone and combined agents were observed, but migration potential in combined agents was likely to decrease compared with Table 1 Associations between the expression of Rictor and SGK1, and clinicopathological factors. Characteristic n Rictor P-value SGK1 P-value — + — + Normal epithelium 10 10 0 <0.0001 10 0 <0.001 SCC 70 22 48 28 42 Sex Male 41 14 27 0.606 21 20 0.463 Female 29 8 21 12 17 Age = 67 26 7 19 0.791 7 19 0.133 <67 44 15 29 21 23 T classification T1 + T2 51 20 31 <0.05 25 26 <0.01 T3 + T4 19 2 17 3 16 N classification N0 55 20 35 0.123 25 30 0.085 N1 + N2 15 2 13 3 12 Stage classification Stage Ⅰ + Ⅱ 48 18 30 0.165 23 25 0.066 Stage III + IV 22 4 18 5 17 Differentiation Well 64 21 43 0.658 26 38 1.000 Moderate. poor. 6 1 5 2 4 Pattern of invasion Grade 1/2 51 20 31 <0.05 25 26 <0.01 Grade 3/4 19 2 17 3 16 Table 2 Associations between the expression of Rictor and SGK1, and the PCNA labeling index. n PCNA LI (%) P-value Rictor SGK1 — 22 + 48 — 28 26.7 ± 24.8 43.6 ± 24.2 30.1 ± 25.3 P < 0.001 P < 0.001 + 42 43.4 ± 24.3 Values are means ± SD. Fig. 3. Kaplan-Meier survival curve of the 5-year disease-specific survival (DSS) rate. No significant correlation between Rictor positive patients and negative patients, and SGK1 positive patients and negative patients were observed in the 5- year DSS rate (P = 0.23 and 0.06, respectively). A subgroup analysis regarding in conjuction with T stage revealed that high Rictor and SGK1 expressions were likely to be associated with poor prognosis (P = 0.11, 0.07, respectively). Differences between the two groups were evaluated with the log-rank test. either agent alone. The evaluation of cell invasion revealed a signif- icant decrease in both cetuximab alone and combined agents com- pared with controls (Fig. 6D–F, SAS, P < 0.01; HSC-3, P < 0.05). As Fig. 4. Representative Western blotting analysis for Rictor and SGK1 in OSCC cell lines (SAS, HSC-2, HSC-3, SCC25, Ca9-22). well as migration assay, no significant differences between either agent alone and combined agents were observed in invasion assay, but invasion potential in combined agents was also likely to decrease compared with either agent alone. In the summary of cell biological analyses, Torin-1 as a single agent had anti-tumor effect against OSCC cell lines and combined treatment with Torin-1 and Cetuximab was likely to enhance the anti-tumor effects. Effects of agent alone and combined treatment with Torin-1 and Cetuximab in vivo Since given the anti-tumor effect on both mTORC1/C2 and EGFR in vitro, we next assessed the in vivo effects. Control tumors reached the maximum allowed tumor size on day 25. During administration, Cetuximab treated tumors significantly grew slower than control tumors (Fig. 7A, P < 0.01). Combined treated tumors also significantly grew slower than control tumors. No sig- nificant differences between Torin-1 treated tumors and control tumors were observed. Torin-1 treated tumors reached the maxi- mum allowed tumor size on day 30. Moreover, a comparison of Cetuximab treated tumors and combined treated tumors after the end of treatment demonstrated that Cetuximab treated tumors re-grew rapidly two weeks later, whereas combined treated tumors greatly re-grew slowly. These results suggested that anti- tumor effects of combined treatment are long-lasting compared with single agent treatment, and additional benefit was observed. At the protein levels of the excised tumors, both p-EGFR and p- mTOR expressions were evaluated by immunohistochemistry. Both p-EGFR and p-mTOR expressions were observed in control tumors, whereas only p-EGFR expression was observed in Torin-1 treated tumors. In the cetuximab treated tumors and combined treated tumors, both p-EGFR and p-mTOR expressions were not observed (Fig. 7B). In the summary of xenografts model analyses, Torin-1 as a single agent had minimal anti-tumor effect, but com- bined treatment with Torin-1 and Cetuximab was significantly enhance the anti-tumor effects against the OSCC xenograft models. Discussion Although NCCN Clinical Practice Guideline has provided that systemic therapy including Cetuximab is the standard therapy for very advanced HNSCC, administration of Cetuximab alone have been associated with only modest clinical response to date [6], and hence, better understanding of the underlying mechanisms is need to improve the current regimens of systemic therapy. mTORC1 rapalog (Temsirolimus and Everolimus) has been approved by FDA for the treatment of advanced some cancer, and we previously reported the effectiveness of Everolimus against the OSCC [22]. However, many negative feedback loop exists in Fig. 5. Effect of Torin-1 on cell proliferation and mTOR signaling in OSCC cell lines. (A) SAS and HSC-3 cell lines were exposed to doses of Torin-1 ranging from 10 nM to 1000 nM, (B) and were exposed to time of Torin-1 ranging from 24 h to 72 h. The percentage cell viability (%) and dose of the drug that inhibited cell growth by 50% (IC50) were calculated. (C) SAS and HSC-3 cell lines were exposed to Torin-1 at the IC50 concentrations and subsequently assessed for protein expression and phos- phorylation by western blotting. the PI3K-Akt-mTOR pathway, which makes it difficult to treatment with rapalogs [12]. Therefore, mTORC2 located upstream of AKT may play a crucial role. This study aimed to apply and develop an OSCC treatment strategy by using mTOR inhibitors, conse- quently are more effective and provide the additional benefit in OSCC. As the clinicopathological evidences of our hypothesis, we found that both Rictor configure the mTORC2 and SGK1 expres- sions are associated with T classification and POI (P < 0.05, P < 0.01, respectively). We also found that high expressions of both Rictor and SGK1 were associated with an increased PCNA LI, but were not associated with 5-year DDS. These results suggest that mTORC2/SGK1 axis affects the progression of OSCC. To our best knowledge, all previous retrospective studies with regard to the Rictor and SGK1 expressions analysed by immunohistochemistry suggested that Rictor or SGK1 was associated with the high degree of malignancy in some solid tumors [23–28]. With regard to the 5- year DSS, our results indicated that high expressions of Rictor or SGK1 is likely to be associated with poor survival. Many studies has reported that Rictor is positively associated with poor survival in tongue cancer [11], gastric cancer [23], hepatocellular carcinoma [24], endometrial carcinoma [26] and breast cancer [27]. SGK1 has also been reported the association of poor survival in lung squamous cell carcinoma [28]. It has been reported that SGK1 was indicated the main mTORC2 effector [8–10,12,13] and our results suggest that mTORC2/SGK1 axis is implicated in the pro- gression of OSCC and might be used as a novel biomarker for prog- nosis given the previous reports in some solid tumors. As the cell biological evidences of our hypothesis, we found that OSCC cell lines were sensitive to the anti-proliferative effects of Torin-1. In addition, growth inhibition was accompanied by decreased phosphorylation of downstream markers of mTORC1 and mTORC2 in a dose-dependent and time-dependent manners. This results is in agreement with the previous study [13,17,29], showing effectiveness of Torin-1 in OSCC. We also hypothesized that Cetuximab and Torin-1 might show anti-cancer effect of each other additively in vitro and in vivo through selective blocking of the extracellular ligand-binding domain of EGFR by Cetuximab, while Torin-1 compete with intracellular catalytic site of mTOR. More recently, some preclinical study with regard to combined treatment with mTORC1/C2 inhibitor and Cetuximab in HNSCC models has been reported [16,30,31]. But, OSCC cell lines and pre- clinical model were not included in these studes. Extreme trial has been described [32], since cetuximab sensitivity is different between OSCC and other HNSCC, it is necessary to study specializ- ing in OSCC. With regard to the tumor migration and invasion potentials in the mTORC2 pathway, previous studies have reported that the inhibition of mTORC1 and/or mTORC2 decreased these potentials in some solid tumors [33–35]. In the present study, as expected, both cetuximab and Torin-1 were inhibited the invasion and migration potentials, and combined treatment with Torin-1 and Cetuximab results in the additive inhibition of OSCC migration and invasion potentials compared with the treatment with agent alone. The immunohistochemical staining of Rictor and SGK1 in the present study also revealed strong positivity in the invasive front of tumors and were associated with POI, which was consis- tent with these findings. Moreover, combined treatment with Torin-1 and Cetuximab significantly inhibit the progression of OSCC compared with the treatment with agent alone in vivo. Particularly, significant corre- lation between Cetuximab alone treatment and combined treat- ment is observed, suggesting that combined treatment with Torin-1 and Cetuximab is effectiveness in OSCC. In the clinical practice, we have often experienced the discontinuation of Cetux- imab monotherapy by the complete response (CR), age or perfor- mance status even if the disease progressed or unacceptable toxic effects were not occurred. To our knowledge, two cases have been reported that tumor progression was observed in both a few months after discontinuation of cetuximab monotherapy since obtaining the CR in OSCC [36,37]. Therefore, in the present study, we continued to observe the tumor size after the mice were treated for 4 weeks. Interestingly, remark differences in the speed of re-growth between the treatment with Cetuximab alone and combined treatment with Torin-1 and Cetuximab were observed. Fig. 6. Effect of Torin-1 alone, Cetuximab alone and combined treatment on migration and invasion potentials in the OSCC cell lines. Migration in SAS (A) and HSC-3 (B) cells and the percentage of migrated cells were determined, as described in the Material and Methods. (C) The graph shows significant decreases in the migration index of SAS and HSC-3 cells. Invasion in SAS (D) and HSC-3 (E) cells and the percentage of invaded cells were also determined. (F) The graph shows significant decreases in the invasion index of SAS and HSC-3 cells. These results suggested that combined treatment with Torin-1 and Cetuximab lasted the anti-tumor effect longer than the treatment with agent alone. In the present study, higher Torin-1 concentrations could increase anti-tumor response in vitro. Previous studies have reported that 20 mg/kg Torin-1 was administrated in nude mice, and it was sufficient to inhibit the tumor expansion [17,21]. We also first started that the dose of 20 mg /kg Torin-1 was adminis- trated to nude mice. However, adverse events such as cyanosis and weight loss were observed, and it reduced the dose to 10 mg/kg Torin-1. Therefore, in the present study, anti-tumor effect of Torin-1 treated tumors was insufficient in vivo compared with cetuximab alone. To our knowledge, one study of biotherapy with Torin-1 has not been reported so far, detailed adverse events against patients remains unclear. Therefore, further investigations of this pathway should be performed to determine whether these agents might be useful in the clinic for treatment of patients with OSCC. Potential weakness of immunohistochemical study is a retro- spective study from single institution and the small number of Fig. 7. (A) The graph shows the SAS tumor volumes for control and treated mice over time. 10 mg/kg Torin-1, 20 mg/ Cetuximab, combined Torin-1 and Cetuximab, and vehicle were delivered by i.p injection three times a week for 4 weeks after the tumor reached the size of 5–10 mm in diameter. (Statistical analysis at the end of administration among the four groups was used by sheffe’s method. ⁄ P < 0.01) (B) Images of paraffin-embedded sections of control, Cetuximab treated, Torin-1 treated and combined treated SAS tumors stained with H-E, p-EGFR Tyr1068 and mTOR Ser2448. cases. Moreover, there were some biases that were inherent in these studies, including the data recorded by the authors. Despite uniforming the patient characteristics and treatment strategy in OSCC to reduce biases associated with retrospective study design, statistical power of our study may be low. Therefore, an intergroup study with a further number of cases is needed. In conclusion, this study suggested that mTORC2 axis plays a crucial role in cancer progression, and combined treatment with Torin-1 and Cetuximab may provide some additional benefit com- pared with agent alone in OSCC. 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