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Morphoproteomic and Pharmacoproteomic Rationale for mTOR Effectors as Therapeutic Targets in Head and Neck Squamous Cell Carcinoma^*

Address correspondence to Robert E. Brown, M.D., at his current address: Department of Pathology, University of Texas Houston Medical School, 6431 Fannin Street, Room 2.286, Houston, TX 77030, USA; tel 713 500 5332; fax 713 500 0732; e-mail robert.brown{at}uth.tmc.edu,

	Abstract

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Head and neck squamous cell carcinoma (HNSCC) has a relatively high mortality rate and poor prognosis. Recently, we showed that overexpression of phosphorylated (p) nuclear factor-kappaB (NF-B) in squamous cell carcinoma of the tonsil (SCCT) and high grade dysplasia is associated with a poor prognosis. Because the mammalian target of the rapamycin (mTOR) pathway contributes to the activation of NF-B through immunophilin/mTOR signaling, we investigated: (a) the immunohistochemical expression and state of activation and potential clinical significance of components of the mTOR signal transduction pathway in SCCT patients (morphoproteomics); and (b) the inhibitory effects of rapamycin on the growth and state of activation of mTOR in 2 HNSCC cell lines (pharmacoproteomics). Archival biopsy materials from 39 patients with SCCT were studied by immunohistochemistry for the expression of p-mTOR (Ser 2448), and p-p70S6K (Thr 389), and/or cyclin D1. Results for SCCT were compared with adjacent non-neoplastic epithelium, when present, and with normal tonsillar epithelium from approximately age-matched controls; clinical outcomes were also assessed. SCCT showed mTOR (Ser 2448) expression in 93% (30/32 cases) with 2+ or 3+ plasmalemmal and/or cytoplasmic intensity in 84% vs 42% in surface epithelium from normal tonsils (p <0.001). The mean combined expression score (signal intensity x percentage of positive cells) for p-p70S6K was significantly greater in the SCCT group vs adjacent non-neoplastic squamous epithelium and normal tonsillar epithelium of the control group (p <0.05). A relationship existed between higher p-p70S6K expression levels in the non-neoplastic squamous epithelium adjacent to the SCCT and increased risk of death from disease (hazard ratio = 7.9; 95% confidence interval (CI) = 2.1 to 29.9; p = 0.002). There was also a relationship between nuclear expression of cyclin D1 in SCCT and shortened recurrence-free survival (p = 0.015). Two human HNSCC cell lines, SCC-15 and FaDu, were incubated with and without rapamycin to assess its impact on growth and on the expression of p-mTOR. Rapamycin in a dose-dependent fashion inhibited growth more in SCC-15, which correlated with a greater reduction in constitutively activated p-mTOR (Ser 2448) as shown by Western blotting. In conclusion, these morphoproteomic and pharmacoproteomic data collectively provide a rationale for selecting mTOR effectors as therapeutic targets in HNSCC.

Keywords: mTOR, squamous cell carcinoma, rapamycin, p70S6K, cyclin D1

Abbreviations: PDGFR: platelet-derived growth factor receptor; EGFR: epidermal growth factor receptor; VEGFR: vascular endothelial growth factor receptor; VEGF: vascular endothelial growth factor; PI3-K: phosphatidylinositol 3-kinase; ERK: extracellular signal-regulated kinase; IKK: inhibitor kappa B kinase; I-B; inhibitor-kappaB; NF-B: nuclear factor-kappaB; HIF: hypoxia-inducible factor; mRNA: messenger ribonucleic acid

	Introduction

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Identification of signal transduction pathways and appreciation of their potential cross-talk and interplay are essential to an understanding of the mechanisms involved in proliferation, resistance to apoptosis, chemoresistance, radioresistance, and migratory potential of HNSCC. The protein circuitry that comprises these pathways can be delineated by various techniques including two-dimensional gel electrophoresis/mass spectrometry, Western blotting, and immunohistochemistry (morphoproteomics). Delineating such circuitry uncovers potential targets amenable to specific molecular interventions and enables the construction of therapeutic templates that take into account heterogeneity among tumoral phenotypes and to some extent, individual biological variation [1].

One approach would be to focus on points of convergence in signaling so that the effects of cross-talk and redundancy could be minimized and fewer small molecule inhibitors would be required to retard tumoral growth and reverse chemoresistance and/or radioresistance. Because we previously found that activated (phosphorylated) NF-B is associated with a poor clinical outcome in patients with tonsillar squamous cell carcinoma and / or high grade dysplasia [2] and because phosphorylated (p)-Akt and one of its downstream effectors in the mTOR pathway (ie, immunophilin) activates NF-B [3,4], we considered the role of the mTOR pathway in HNSCC, both in terms of tumorigenicity and as a potential therapeutic target.

The purpose of this report is twofold: first, to assess the expression, state of activation, and potential clinical significance of components of the mTOR signal transduction pathway, including downstream cyclin D1, in patients with SCCT, and second, to determine the inhibitory effects of rapamycin on the growth and state of activation of mTOR in 2 HNSCC cell lines.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patients with tonsillar squamous cell carcinoma and controls.  With prior Institutional Review Board (IRB) approval, case histories and archival paraffin-embedded tissues from 39 patients with SCCT were studied. In addition, similar materials were available on 35 approximately age-matched control patients who underwent tonsillectomy for nontumoral indications. Twenty-five of the archival tissues of SCCT cases had accompanying non-neoplastic squamous epithelium. Representative sections of each case were reacted in immunohistochemical procedures for detection of activated (phosphorylated) mTOR and p70S6K antigens (32 cases with adequate material) and for cyclin D1 (39 cases). The primary antibodies were purchased from Cell Signaling Technology, Beverly, MA, and from DAKO Corp., Carpintiria, CA, and were directed against (a) mTOR phosphorylated at serine 2448, (b) p70S6K at threonine 389, and (c) cyclin D1. All reactions were performed according to the manufacturer’s specifications; the results were graded on a scale of 0–3+ and compartmentalized according to their plasmalemmal (cell membranous), cytoplasmic, and nuclear subcellular localizations using bright-field microscopy.

Human HNSCC cell lines.  The FaDu and SCC-15 cancer cell lines were obtained from American Type Culture Collection (Manassas, VA). They had been established from HNSCC of the pharynx and tongue, respectively, that had been previously collected from patients. These cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM [Gibco-BRL, Gaithersburg, MD]) supplemented with 10% bovine serum and cultured in 95% air and 5% CO₂.

Inhibition studies.  The 2 HNSCC cell lines were placed into 96 well plates and allowed to attach to the wells for 3 days. Rapamycin (also known as sirolimus [Rapamune]) was used as a potential pharmaceutical inhibitor in the cell cultures. It was purchased from Calbiochem (San Diego, CA). Rapamycin was initially dissolved in dimethyl sulfoxide (DMSO, Fisher Scientific) and then diluted in culture medium and added to each of the cell lines to give the final desired concentrations in nM. (Separate experiments were carried out to determine the impact of the vehicle alone on each of the cell lines, and no effects were identified). After 4 days of incubation with or without increasing concentrations of rapamycin, the viable cells in each well were determined colorimetrically (CellTiter 96 Aqueous ONE Solution Proliferation Assay, Promega, Madison, WI). Inhibition rates were calculated as the cell numbers in control groups minus those in treated groups, divided by control [ie, (control – treated) / control].

Western blotting.  Control and rapamycin-treated HNSCC cells were harvested and sonicated. Whole cell homogenates (30 µg total protein/lane) were separated on 6% or 12% SDS-PAGE and transferred onto PVDF membranes. Primary antibodies used in the immunostaining procedure included those against phosphorylated (p)-mammalian target of rapamycin p-mTOR [Ser 2448]; Cell Signaling Technology, Beverly, MA; p-p70S6K [Thr 389], Cell Signaling; and p-Akt [Ser 473], Cell Signaling. Anti-actin antibody was purchased from Santa Cruz Biotechnology, Santa Cruz, CA. The secondary antibody was horseradish-peroxidase-linked. Immunoreactive proteins were visualized by an enhanced chemiluminescence-Western blotting system (Amersham).

Statistics.  Clinical data from the patients were collected through a review of their Geisinger medical records and linked to the respective immunohistochemical data, As previously noted [2], the clinical data comprised age, gender, history of smoked/smokeless tobacco, TNM status, stage (I–IV), and recurrence and survival. This study focused on the latter. A comparison of the incidence of moderate (2+) and strong (3+) expression intensities for p-mTOR (Ser 2448) in the cases of SCCT vis-à-vis normal surface epithelium from tonsillar controls was assessed using Fisher’s exact test. Combined scores for nuclear p-p70S6K (Thr 389) expression were calculated using staining intensity score multiplied by percentage of positively staining cells. A one-way ANOVA was used to detect overall differences between means of each group. The scores of the p-p70S6K (Thr 389) in SCCT and in the adjacent non-neoplastic epithelium were tested for correlation with survival and recurrence using Cox regression. Prior to regression analysis, the combined scores (which fell in the range of 0–300) were transformed into 0, 1+, 2+ or 3+. Similarly, the presence of cyclin D1 in the nuclei of SCCT was tested for correlation with survival and recurrence using Cox regression. Results of in vitro inhibitory rate were expressed as mean ± SE. The unpaired Student t test was used to compare mean inhibitory rates in the two cell lines. Statistical analyses and the data manipulations were conducted using SAS software (version 8.1, Statistical Analysis Systems, Cary, NC). All p-values were two-sided and those <0.05 were considered statistically significant.

	Results

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Expression of p-mTOR (Ser 2448) was evident in 93% (30/32 cases) with plasmalemmal and/or cytoplasmic intensity scores at 2+ or 3+ in 84% (27/32 cases) vs 42% (14/35 cases) in the surface epithelium from normal tonsillar controls (p <0.001; Fisher’s exact test). Among the cases of SCCT, the percentage of tumor cells showing 2+ or 3+ expressions ranged from ~1% to ~80%. Parenthetically, it should be noted that the normal crypt epithelium generally exhibited a strong chromogenic signal for p-mTOR (Ser 2448) antigen, contrasting with the companionate surface epithelium. These findings are illustrated in Fig. 1. Similarly, a more generalized expression of the downstream effector of p-mTOR (Ser 2448), namely p-p70S6K (Thr 389), was evident in the tumoral nuclei in some cases of SCCT as compared with that in the normal tonsillar surface epithelium of controls (Fig. 1). Correlative expression of both p-mTOR (Ser 2448) and p-p70S6K (Thr 389) was noted in invasive SCCT and in tumor emboli lying in vascular spaces (Fig. 2). Quantification of (primarily) nuclear, p-p70S6K (Thr 389) expression gave the following combined (intensity x percentage) scores: 151.7 ± 19.4, SCCT (n = 32); 91.2 ± 12.1, non-neoplastic squamous epithelium adjacent to SCCT (n = 25); and 85.6 ± 13.8, tonsillar surface squamous epithelium in control subjects (n = 32). This represents a statistically significant increase in the mean ± SE in the SCCT group versus the latter 2 groups, with p values of 0.0163 and 0.0072, respectively. Analysis of individual p-p70S6K (Thr 389) marker scores vs 2 clinical parameters (death from disease or recurrence) revealed no significant relationships in SCCT patients for either parameter (p = 0.20 and p = 0.13, respectively). Although there was no significant relationship between p-p70S6K (Thr 389) expression and recurrence (p = 0.36), there was a significant relationship between higher p-p70S6K (Thr 389) expression in adjacent non-neoplastic squamous epithelium from SCCT subjects and increased risk of death from disease (hazard ratio = 7.9; 95% CI = 2.1 to 29.9; p = 0.002).

View larger version (150K): [in this window] [in a new window]   Fig. 1. Immunohistochemical probes for mTOR (phosphorylated on serine 2448) and for p70S6K (phosphorylated on threonine 389) showing: (a) mild to moderate brown signal for p-mTOR in the cytoplasm of surface squamous epithelial cells of normal tonsil (upper left frame) contrasting with strong chromogenic signal for p-mTOR in normal crypt epithelium (see inset); (b) moderate to strong expression of p-mTOR in the cytoplasm of tonsillar squamous cell carcinoma (SCCT) with occasional plasmalemmal translocation (upper right frame); and (c) primary nuclear expression of p-p70S6K, depicted in lower one-half of normal tonsillar epithelium (lower left frame), contrasting with more generalized expression of p-p70S6K in nuclei of SCCT (lower right frame) [3-3' diaminobenzidine (DAB) chromogen; original magnification x200 upper left frame and x600 for inset and remaining frames).

View larger version (147K): [in this window] [in a new window]   Fig. 2. Correlative expression of p-mTOR (Ser 2448) and its downstream effector, p-p70S6K (Thr 389) in invasive SCCT (upper left and right hand frames) and in tumor emboli of SCCT, lying in a vascular space (lower left and right hand frames). [DAB chromogen; original magnification x600.]

View larger version (32K): [in this window] [in a new window]   Fig. 3. Recurrence-free survival curves (Kaplan-Meier) illustrating the shortened recurrence-free survival (p = 0.015) in association with cyclin D1 expression in tumoral nuclei (inset: DAB chromogen; original magnification x600]).

View larger version (23K): [in this window] [in a new window]   Fig. 4. Dose response pattern of growth inhibition in two HNSCC cell lines, SCC-15 and FaDu cells, after incubation with and without rapamycin for 4 days, illustrating a greater inhibitory effect in SCC-15 cells (upper frame). Corresponding Western blotting analysis (lower frame) shows a correlative reduction in p-mTOR (Ser 2448) in whole cell lysates from both cell lines but with a greater reduction in the SCC-15 cell line (75.9 vs 33.7% by densitometry) following rapamycin exposure. Protein level of p-p70S6K (Thr 389)/p-p85S6K was reduced in both cell lines by rapamycin but p-Akt (Ser 473) remained unchanged, as expected (lower frame).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The prognostic implication of p-p70S6K (Thr 389) scores in non-neoplastic squamous epithelium adjacent to their SCCT, as noted in this study, coincides with a related observation by Nathan et al [12]. They showed that immunohistochemical detection of elevated eukaryotic initiation-factor 4E (eIF-4E) in the surgical margins is an independent predictor of significantly increased risk of recurrence in head and neck cancer. Such an observation could reflect the action of p-mTOR in effecting phosphorylative inactivation of 4E-binding protein (4E-BP)1 [13], thereby permitting the release and promoting the translational activity of eIF-4E in HNSCC.

Finally, with respect to the activation and correlative expression of the specific protein analytes in our study, namely, p-mTOR (Ser 2448) and p-p70S6K (Thr 389), there are morphoproteomic and molecular aspects of note. The demonstration of cytoplasmic and plasmalemmal expression of p-mTOR (Ser 2448) and nuclear translocation of p-p70S6K (Thr 389) strengthens the thesis that the functional state of these molecules is also reflected in the cellular compartments in which they are situated (ie, membrane and nuclear compartments, respectively [14–19]). Moreover, the phosphorylation of mTOR on serine 2448 coincides with downstream activation by p-Akt [10,20], and the phosphorylation of p70S6K on threonine 389, accords with the downstream activation by p-mTOR [10,21–25].

Pharmacoproteomic validation of an activated and functional mTOR pathway is offered by our in vitro studies on 2 HNSCC cell lines in which exposure to rapamycin resulted in reduced constitutive expression of p-mTOR (Ser 2448) by Western blotting and in a dose-response fashion, of growth inhibition. Collectively, these observations provide a rationale for targeting the mTOR pathway in patients with HNSCC with rapamycin or rapamycin analogs [1]. Additional support for such a therapeutic approach was recently provided by Amornphimoltham and co-workers [26] who noted aberrant accumulation of the phosphorylated active form of S6, the most downstream target of the Akt-mTOR-p70-S6 kinase pathway, in clinical specimens from patients with HNSCC and their derived cell lines. Most importantly, they were able to reduce the level of the phosphorylated active form of S6 in HNSCC cell lines and xenograft models at clinically relevant doses of rapamycin and to produce tumor regression.

The potential role of downstream signaling by activated mTOR effectors in promoting tumorigenicity is probably best considered in the context of the molecular concomitants in HNSCC. The latter include p-NF-Bp65 (Ser 536) [2], nuclear cyclin D1 [27], hypoxia-inducible factor (HIF)-alpha, and vascular endothelial growth factor (VEGF). To expand on these, an activated immunophilin mTOR pathway has been reported to activate inhibitor-kappaB kinase (IKK) [4], which leads to proteasomal degradation and release of p-NF-Bp65 (Ser 536) for translocation to the nucleus. Phosphorylated and translocated NF-B promotes both tumor cell growth, in part through cyclin D1 production; it also enhances migratory potential and stimulates the production of anti-apoptotic and angiogenic proteins (eg, bcl-2, bcl-xL, and bfl-1 and VEGF, respectively) through transcriptional activation [28–32]. Similarly, phosphorylative activation of p70S6K leads to increased G1 cell cycle progression with tumor cell proliferation [6,7], and to phosphorylative activation of S6 ribosomal protein, which stimulates the translation of messenger ribonucleic acids [26]. Phosphorylative inactivation of 4E-BP1 by the mTOR pathway releases eIF4-E to effect translational protein synthesis, including anti-apoptotic proteins [33–35]. High nuclear expression of cyclin D1, which is an independent predictor for increased local recurrence, inferior disease-free survival, and inferior overall survival at 5 yr in oropharyngeal squamous cell carcinoma [27], and of a shortened recurrence-free survival in this study could act in concert with p-p70S6K (Thr 389) in advancing G1 cell cycle progression [5–7] and proliferation of tumor cells. Overexpression of HIF-1 alpha and HIF-2 alpha has been associated with high microvessel density and with VEGF expression in squamous cell head-and-neck cancer, with incomplete response to chemoradiation, with poor local relapse-free survival, and with poor overall survival [36]. The mTOR pathway participates in the activation of HIF-1 alpha signaling [37,38]and VEGF production [39].

Finally, the therapeutic debate over whether or not to focus on upstream signal transducers or pathways of convergence in HNSCC is relevant and deserves mention. A number of potential upstream tyrosine kinase targets have been identified in HNSCC, including epidermal growth factor receptor (EGFR [2,40,41]), platelet-derived growth factor receptor (PDGFR [41]), c-kit [41], and vascular endothelial growth factor receptor (VEGFR [42]). Although overexpression of EGFR protein has been reported to occur in HNSCC, including our previous study of SCCT [2], and has been correlated with a high mitotic index and advanced stage tumors in HNSCC patients [40]; the results of a phase II therapeutic trial using gefitinib (Iressa, ZD1839), a selective EGFR-tyrosine kinase inhibitor, in patients with recurrent or metastatic HNSCC produced a response rate of only 10.6% [43]. This apparent discordance might be explained on the basis of the following: (a) heterodimerization with other tyrosine kinase molecules such as PDGFR, leading to transactivation of EGFR [1,44,45]; and (b) co-expression of other activated and independent tyrosine kinase signaling pathways, including PDGFR and VEGFR-2 [1,41,42]. Adaptive signaling mechanisms are also possible. For example, imatinib mesylate, an inhibitor of PDGFR signaling, induced EGFR activation and downstream signaling through the mitogen-activated protein kinase (MAPK) pathway in head and neck squamous cell carcinoma cells [46]. In this regard, it is noteworthy that each of the aforementioned upstream tyrosine kinases signal through both the PI3'-K/Akt/mTOR and Raf kinase/MAPK/extracellular signal-regulated kinase (ERK) pathway[1,47,48]. Recently, we demonstrated the expression of constitutively activated p-ERK 1/2 (Thr 202/Tyr 204) in this same series of SCCT cases and we regard it as another pathway of convergence in HNSCC [1,49], capable of contributing to the activation of the mTOR [1,26] and NF-B pathways [1,48] in such patients. This complex protein circuitry in HNSCC is illustrated in Fig. 5. Focusing on these pathways of convergence in HNSCC, one could consider rapamycin (Rapamune) to block mTOR signaling [1,26], or Nexavar (sorafenib, BAY43-9006) as an inhibitor of the Raf kinase/MAPK/ERK pathway [54], and bortezomib (Velcade), a proteasome inhibitor to block NF-B [1] (see Fig. 5) in singular or combinatorial fashion. Van Waes and co-workers [31] have employed the latter approach, combining bortezomib with irradiation in patients with HNSCC, and have achieved tumor reduction in some cases.

View larger version (103K): [in this window] [in a new window]   Fig. 5. A composite of the complex protein circuitries identified by the authors and others utilizing immunohistochemistry in cases of HNSCC (analytes with*) and depicting the interrelationships with the mTOR pathway. Specifically, downstream signaling by the tyrosine kinases, PDGFR, EGFR, and VEGFR and their ligands proceeds through the PI3-K/Akt and Raf kinase/ERK 1/2 pathways. The former leads to phosphorylative activation of mTOR and its effector p70S6K and the latter (p-ERK 1/2) converges on p70S6K to assist in G1 cell cycle progression, and both pathways lead to the translational synthesis of protein through the activation, and/or release of eIF-4E and the phosphorylation by activated p70S6K of ribosomal protein S6 [50–53]. Nuclear cyclin D1, when present, could also promote G1 cell cycle progression. Notably, HIF-1 induced by mTOR signaling is relevant to VEGF production and autocrine stimulation of VEGFR-2 signaling in HNSCC. Bipathway signal transduction also converges on phosphorylative activation of NF-kappaBp65 with nuclear translocation, thereby contributing to tumoral proliferation, chemoradioresistance, anti-apoptosis, and angiogenesis. Opportunities for therapeutic intervention include: rapamycin or a rapamycin analog; Nexavar, a VEGFR and Raf kinase inhibitor; and Velcade, a proteasome inhibitor.

	Acknowledgments

 
Acknowledgments

The authors thank Glen Kauwell and Laurie Kneller-Walter for technical assistance and Sharon Coup-Stroh for secretarial support and assistance with the graphics.

	Footnotes

 
^* The results of this investigation were presented in part at the Annual Meeting of the United States and Canadian Academy of Pathology in Atlanta, GA, on 11–17 February 2006.

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