Persistent Activation of the Akt Pathway in Head and Neck Squamous Cell Carcinoma

Human Tissues.

Formalin-fixed, paraffin-embedded tissue specimens from head and neck cancer patients were retrieved from the archives of Department of Diagnostic Science and Pathology, Dental School, University of Maryland, with the approval of the Institutional Review Board. All tissues were stained H&E for pathological diagnosis. The tissues were divided, according to their histology, into four distinct groups: normal/metaplastic epithelium, which included normal epithelium adjacent to more advanced lesions; dysplasias; carcinomas in situ; and invasive SCCs.

Mouse Tissues.

Samples of normal mouse skin, hyperplasia, papilloma and SCC were obtained from SENCARB mice using the two-stage skin carcinogenesis model by treated with 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate as previously described (10) in accordance with approved animal protocols. Tissues were snap-frozen or fixed in buffered formalin and embedded in paraffin. Five-μm sections were obtained and stained with H&E for diagnostic purposes or left unstained for immunocytochemistry.

Antibodies.

Rabbit polyclonal phospho-Akt (Ser⁴⁷³) immunohistochemistry (IHC)-specific antibody (Cell Signaling Technology, Beverly, MA; dilution 1:50) was used for IHC; rabbit polyclonal phospho-Akt (Ser^472/473/474; BD PharMingen), rabbit polyclonal Akt antibody (BD PharMingen), mouse monoclonal glycogen synthase kinase-3β (GSK-3β; BD PharMingen), rabbit polyclonal phospho-mTOR (Ser²⁴⁴⁸; Cell Signaling Technology), rabbit polyclonal mTOR antibody (Cell Signaling Technology), and rabbit polyclonal phospho-GSK-3 α/β (Ser^21/9; Cell Signaling Technology) were used for Western blotting at a dilution 1:1000.

IHC.

The tissues slides were dewaxed in xylene, hydrated through graded alcohols and distilled water, and washed with PBS. Antigen retrieval was performed using a commercial unmasking solution (Vector Laboratories, Burlingame, CA) in a microwave for 20 min (2 min at 100% power and 18 min at 10% power). Slides were allowed to cool down for 30 min at room temperature, rinsed twice with PBS, and incubated in 3% hydrogen peroxide in PBS for 30 min to quench the endogenous peroxidase. The sections were then washed in distilled water and PBS and incubated in blocking solution (5% horse serum) for 1 h at room temperature. Excess solution was discarded, and the sections incubated with the primary antibody diluted in blocking solution at 4°C overnight. After washing with PBS, the slides were sequentially incubated with the biotinylated secondary antibody (1:300; Vector Laboratories) for 1 h, followed by the avidin-biotin complex method (Vector Stain Elite, ABC kit; Vector Laboratories) for 30 min at room temperature. The slides were washed and developed in 3,3′-diaminobenzidine (Sigma FASTDAB tablet; Sigma Chemical, St. Louis, MO) under microscopic control. The reaction was stopped in tap water, and the tissues were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. To capture the entire areas of interest, several partially overlapping high-definition images were taken from each case with a total magnification of ×160, using a real-time Retiga 1300 digital camera with the QImaging Pro software (MVIA, Inc., Monaca, PA). The images were then stitched together using the PanaVue Image Assembler Software (PanaVue Co., Quebec City, Quebec, Canada). The resulting image was a high-quality panoramic picture of the lesion that allowed us to analyze simultaneously a high number of positively and negatively stained cells, frequently including within the same section, nonneoplastic reactive epithelium, as well as different degrees of tumor progression, which were analyzed under the same software parameters. Images were quantified using the Northern Eclipse Image Analysis Software (Empix Imaging, North Tonawanda, NY) and expressed as the percentage of phospho-Akt (p-Akt)-positive cells with respect to the total number of cells in each histologically defined area.

Immunocytochemistry.

Cells were grown on the coverslips until 50% confluent, serum-starved, subjected to different experimental conditions, and washed twice in cold PBS. Coverslips were immersed in 100% methanol at −20°C for 10 min and washed twice in cold PBS.

Blocking step, antibody incubation, and staining conditions were all performed as described in IHC.

Cells Cultures.

HNSCC cell lines HN4, HN6, HN8, HN12, HN13, HN17, HN19, HN22, HN26, HN30, and HN31 and HaCaT cells were maintained in DMEM with 10% FCS, penicillin, and streptomycin in 5% CO₂ at 37°C as described previously (11) .

Pharmacological Treatment.

UCN-01 was provided by Kyowa Hakko Kogyo Co. Ltd., Japan, to the Development Therapeutics Program, National Cancer Institute. The compound was reconstituted in DMSO as a 10 mm stock solution, which was additionally diluted to the working concentration in culture medium (1–1000 nm), at a final concentration of ≤0.1%. Epidermal growth factor (EGF) was purchased from Sigma Chemical and used as indicated (100 ng/ml for 10 min). Wortmannin was purchased from Calbiochem (San Diego, CA) and used at a concentration of 50 nm.

Western Blotting.

Cells were rinsed twice in PBS, lysed with protein lysis buffer [62.5 mm Tris-HCl (pH 6.8), 2% v/v SDS, and 50 mm DTT], scraped immediately, and transferred to microcentrifuge tubes. Lysate was sonicated for 20 s. The protein yield was quantified using the DC protein assay kit (Bio-Rad, Hercules, CA). Equivalent amounts of protein (50 μg) were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Equivalent loading was confirmed by staining membranes with Ponceau-S. The membranes were blocked for 1 h in blocking buffer (1× Tris-buffered saline, 5% nonfat dry milk, and 0.1% Tween 20), which was then replaced by the primary antibody at the concentration described above, in blocking buffer, overnight at 4°C. After incubation, the membranes were washed three times in washing buffer (1× Tris-buffered saline and 0.1% Tween 20). Primary antibody was detected using horseradish peroxidase-linked goat antimouse (Santa Cruz Biotechnology, Santa Cruz, CA) or goat antirabbit IgG antibody (Santa Cruz Biotechnology) and visualized with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). The bands were scanned and quantified using NIH image software.

Akt Kinase Assay.

To assay for Akt kinase activity, cells were serum-starved, subjected to the different experimental conditions, washed twice in cold PBS, and lysed on ice with 500 μl of lysis buffer containing 20 mm Tris (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm Na PP_I, 1 mm β-glycerophosphate, 1 mm Na₃VO₄, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. Equal amounts of lysates (300 μg of protein) were precleared by centrifugation and preabsorbed with protein A-protein G (1:1) agarose slurry. Immunoprecipitation was carried out for 18 h using an immobilized anti-Akt1G1 monoclonal antibody cross-linked to agarose. Immunoprecipitates were washed three times with lysis buffer and twice with Akt kinase buffer (25 mm Tris (pH 7.5), 5 mm β-glycerophosphate, 2 mm DTT, 0.1 mm Na₃VO₄, and 10 mm MgCl₂). Kinase assays were performed for 30 min at 30°C under continuous agitation in kinase buffer containing 200 μm ATP and 1 μg of GSK-3 fusion protein as a substrate, according to the manufacturer’s instructions (Cell Signaling Technology). The incorporation of phosphate into GSK-3 was assessed by Western blot using an antiphospho-specific GSK-3 α/β (Ser^21/9) antibody and horseradish peroxidase-conjugated antirabbit antibody. To assess the level of expression of Akt, parallel total cell lysates were analyzed by Western blot, using a polyclonal goat antibody against human Akt.

Cell Proliferation.

[³H]Thymidine (ICN Pharmaceuticals, Inc., Costa Mesa, CA) uptake was performed as described previously (12) . Briefly, HNSCC and HaCaT cells (1–2 × 10⁴/well) were allowed to attach overnight in 24-well plates, and the medium was replaced with serum-free medium 24 h for the vehicle control wells. After treatment (20 h), the cells were pulsed with [³H]thymidine (1 μCi/well) for 4 h, precipitated DNA (5% trichloroacetic acid), and solubilized (0.5 m NaOH) before scintillation counting. Experiments were performed in triplicate.

Statistical Analysis.

ANOVA followed by the Turkey t test was used to analyze the differences between groups after immunohistochemical staining of human HNSCC for p-Akt protein. Data analysis was performed with using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, CA;5 P values < 0.05 were considered statistically significant.

Detection of Activated Akt in Mouse Tissues.

The recent development of new immunological tools detecting the phosphorylated form of Akt, which represents its activated state (13) , provides a unique opportunity to monitor the activation of Akt in tissue sections, thereby facilitating the analysis of its status in defined cell populations and even subcellular locations within a tumor. Fig. 1⇓ shows the expression of p-Akt detected by IHC in normal or pathological tissue from SENCARB mice under the two-step chemically induced carcinogenesis treatment (10) . Expression of the activated form of Akt correlated with SCC progression, paralleling our prior enzymatic analysis of Akt activity (9) . Normal skin exhibited only isolated nuclear staining in basal cells of skin and hair follicles (Fig. 1A)⇓ . The intensity and number of positive cells for p-Akt increased dramatically in skin papillomas, where cytoplasmic staining was observed in every sample (Fig. 1B)⇓ . At the carcinoma stage, almost all cells showed cytoplasmic immunoreactivity for p-Akt, which was even stronger in invasive areas (Fig. 1C)⇓ .

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Fig. 1.

A, phospho-Akt (p-Akt) staining in normal mouse skin. Isolated positive cells can be seen scattered in the epidermis (×200). Inset: moderate cytoplasmic or nuclear staining in normal epithelial cells (×400). B, papilloma; p-Akt-positive staining is evident in the lower third of the hyperplastic squamous epithelium (×200). Inset: strong cytoplasmic staining of ∼100% of the cells in this area (×400). C, infiltrating SCC; most neoplastic malignant keratinocytes are positive for p-Akt (×200). Inset: staining distribution is cytoplasmic in all malignant cells (×400).

Accumulation of Phospho-Akt in Human HNSCC.

Because the immunodetection of p-Akt in mouse SCC correlated well with the enhanced Akt activity in these tissues (9) , these findings prompted us to evaluate the status of Akt activation in human HNSCC using the IHC approach. Indeed, we found that human HNSCC exhibited a similar pattern of expression of p-Akt, which correlated with disease progression (Figs. 2⇓ and 3⇓ ). In normal tissues, the staining was limited to few cells (Figs. 2⇓ and 3⇓ ) within the basal or parabasal layers and almost always confined to the cell nucleus. The image was similar in metaplastic or inflammatory areas, although the percentage of stained cells was marginally higher. Focal cytoplasmic staining could be seen in normal squamous epithelium adjacent to dysplastic or malignant lesions. In dysplastic areas, a high fraction of the cells presented with strong nuclear staining (Figs. 2⇓ and 3⇓ ). Carcinoma in situ also presented with strong nuclear staining, as well as p-Akt staining in the cytoplasm. Only in a few cases was nuclear staining observed in invasive lesions; in most instances, p-Akt staining was predominantly cytoplasmic (Fig. 2, C–E)⇓ , a staining pattern similar to that observed in mouse tissues. The percentage of positively stained cells among the different groups showed significant differences between normal/metaplasia to dysplasia (P < 0.001), carcinoma in situ (P < 0.01), or SCC (P < 0.05).

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Fig. 2.

A, normal oral mucosa exhibits only mild phospho-Akt (p-Akt) staining in isolated cells in the basal epithelial layer. B, predominant nuclear staining for p-Akt in the lower half of the squamous epithelium in a mild dysplasia of the oral mucosa. Note positive immunoreactivity in isolated fibroblastic-like stromal cells and endothelial cells. C and D, two squamous carcinomas of the head and neck showing different p-Akt immunoreactivity distribution patterns; staining in C is almost exclusively cytoplasmic, whereas D presents extensive cytoplasmic as well as nuclear positive staining. E, p-Akt staining in squamous carcinoma of the head and neck, showing positive staining in the cytoplasm of the invasive front of squamous cell carcinoma, whereas only very mild staining for p-Akt can be observed in the normal adjacent cells (arrowheads).

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Fig. 3.

Quantitative analysis of immunohistochemistry: data are representing the percentage of positively staining cells observed in normal and metaplasia, dysplasia, carcinoma in situ (CIS), and invasive squamous carcinoma (SCC), compared with total cells present in each histologically defined area. Number of cases analyzed is indicated in parenthesis.

Constitutive Active Akt in HNSCC.

To further control the experimental conditions used to examine active Akt in tissues, we used the same IHC conditions for the immunocytochemical analysis of p-Akt in a more defined cellular system in vitro. Indeed, using HaCaT cells, an immortalized human epidermal cell line, we observed that there was limited p-Akt staining under basal conditions but that treating with EGF (100 ng/ml for 10 min) dramatically enhanced p-Akt immunoreactivity (Fig. 4A)⇓ . On the contrary, treatment with wortmannin (50 nm 30 min), a potent PI3K inhibitor (14) , can abolish basal p-Akt staining and diminished the p-Akt response to EGF (Fig. 4A)⇓ . These observations were additionally confirmed by the parallel Western blot analysis of cell lysates using a distinct p-Akt antibody that recognizes the phosphorylation of Ser^472/473/474 of all Akt isoforms. As shown in Fig. 4B⇓ , there was a remarkably high level of p-Akt when cells were treated with EGF, which was inhibited by the treatment with wortmannin without affecting the total level of Akt protein. Enhanced p-Akt levels correlated with increased Akt kinase activity, as judged by immunocomplex Akt kinase reactions using GSK3α/β as a biologically relevant substrate in vitro (Fig. 4B)⇓ .

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Fig. 4.

Activated Akt is frequently found in squamous carcinoma of the head and neck (HNSCC) cell lines. A and B, immunostaining for phospho-Akt (p-Akt) correlates with the level of phosphorylated Akt and its in vitro kinase activity. A, serum-starved HaCaT cells were treated with epidermal growth factor (EGF; 100 ng/ml for 10 min) with or without pretreatment with wortmannin (WM; 50 nm, 30 min). Immunocytochemical analysis was performed as described in “Materials and Methods.” B, cell lysates from cells treated as in A were processed for Western blotting (WB) with anti-Akt (Akt), or with phosphorylation-specific anti-Akt antibodies (p-Akt). In vitro immunocomplex Akt kinase reactions were performed and the phosphorylated product glycogen synthase kinase-3 (p-GSK-3) revealed with a phospho-specific GSK-3α/β antibody. C, expression pattern of p-Akt in HNSCC cells. Cell lysates were harvested from serum-deprived HNSCC cells. Immunoblotting for total Akt was performed in the same filter. D, immunocomplex Akt kinase assays were performed in HNSCC cell lines as described in B. A representative group is shown.

We then investigated whether the increased Akt activity observed in human HNSCC was reflected in Akt activation in HNSCC-derived cells lines. For this analysis, we selected a panel of 11 HNSCC cell lines derived from primary and secondary cancer lesions of contrasting clinical staging (11) . These cells have been comprehensively explored for alterations of major tumor suppressor genes (15 , 16) . For example, aberrant forms of p53 and p16^INK4A were presented in all of the cell lines tested with the exception of one, HN30, which was found to express wild-type p53, thus displaying normal p53 function (16) . Accordingly, we performed Western blot analysis using a p-Akt antibody in lysates from this panel of HNSCC cell lines (Fig. 4C)⇓ . The majority of the HNSCC cells exhibited increased levels of active Akt, albeit to different extents. HN6 cells demonstrated the highest levels of constitutively active Akt among all of the cells examined, followed by HN12, HN13, and HN26, whereas HN17, HN19, and HN 31 displayed a moderate level of activation. By comparison, HN4, HN8, HN22, and HN30 cells had low level of constitutively active Akt (Fig. 4C)⇓ . Total Akt levels were similar in all cells. The status of phosphorylation of Akt in these cells correlated tightly with their in vitro Akt enzymatic activity (Fig. 4D⇓ and data not shown).

UCN-01 Inhibits the PI3K-Akt Signaling Pathway in HNSCC.

UCN-01 (7-hydroxystaurosporine) is a derivative of staurosporine, which was originally developed as an inhibitor of the protein kinase C family of serine-threonine kinases (17) . This compound exhibits potent antiproliferative effects on a variety of tumor cells such as those derived from breast, lung, and colon in both in vitro, as well as in vivo experimental models (18, 19, 20, 21) . Furthermore, in a recent study, we have shown that UCN-01 effectively inhibits the growth of HNSCC cells and HNSCC tumor xenografts (12) . Indeed, when we extended these original observations to the full panel of HNSCC cells used in this study, we observed that UCN-01 inhibits the incorporation of [³H]thymidine into DNA in all HNSCC cells, with an EC₅₀ ranging from 6 to 30 nm (data not shown). However, recent studies have revealed that the antiproliferative and proapoptotic activities of UCN-01 do not correlate with the blockade of protein kinase C (22) . Instead, UCN-01 has been shown to inhibit other kinases important in cell cycle regulation, including Chk1 (23) and, indirectly, the cyclin-dependent kinases, the latter occurring through increases in the level of expression of the endogenous cyclin-dependent kinase inhibitors p21^WAF1 and p27^KIP1 (12) .

Of interest, in a very recent study it was shown that UCN-01 can also block PDK1, a kinase that acts directly upstream of Akt (24) . On the basis of the antiproliferative effect of UCN-01 in HNSCC cells and the fact that these cells exhibit hyperactive Akt, we next examined whether UCN-01 could inhibit the activity of Akt in these cells at concentrations that are consistent with the growth inhibitory effect of this compound at concentrations compatible with those achieved in tissues during recent clinical trials (25) . As shown in Fig. 5A⇓ , UCN-01 potently decreased the levels of active Akt in a dose-dependent manner in HNSCC cells. Using as an example HN6, the EC₅₀ for Akt inhibition was ∼19 nm (range, 8–45 nm; n = 4), which corresponded well with the antiproliferative property of UCN-01 in these cells when assessed by [³H]thymidine incorporation (EC₅₀ ∼7 nm). Furthermore, using a concentration of UCN-01 of 100 nm (EC₉₀), we observed that the activity of Akt in cells exhibiting high levels of constitutively active Akt decreased rapidly in a time-dependent manner, in most cases at least by 50–90% in ∼30 min (Fig. 5B)⇓ . In contrast, UCN-01 did not decrease but instead increased the activity of a distinct signaling route that leads to activation of extracellular signal-regulated kinases, as previously reported (22 , 26) , thus supporting the specificity of this effect on the Akt pathway.

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Fig. 5.

Constitutively active Akt in squamous carcinoma of the head and neck (HNSCC) cells is inhibited by UCN-01. A, UCN-01 potently decreased the levels of activated phospho-Akt (p-Akt) in a dose-dependent manner in HNSCC cells. Lysates were prepared from serum-starved HNSCC cell lines treated with UCN-01 at the indicated concentration for 20 min. p-Akt levels in HN6 were assessed as indicated in “Materials and Methods” and plotted against UCN-01. Akt inhibition by UCN-01 (EC₅₀ ∼19 nm) correlated with the antiproliferative property of this antineoplastic agent in these cells when assessed by [³H]thymidine incorporation (EC₅₀ ∼7 nm). In contrast, UCN-01 leads to activation of extracellular signal-regulated kinases (ERKs) as previously shown (26) . B, inhibition of Akt activation by UCN-01 is an early event. HNSCC cells were treated with UCN-01 (100 nm) for the indicated time, and p-Akt and total Akt levels determined in cellular lysates by Western blotting (WB).

We next evaluated whether the inhibition of the Akt pathway by UCN-01 was reflected by the decreased phosphorylation of endogenous Akt substrates. We focused our attention on GSK-3, which was initially identified as an enzyme that regulates glycogen synthesis, and was recently shown to be a critical downstream element of PI3K/Akt cell survival pathway (27) . Using HN6 as an example, we observed that UCN-01 (100 nm) inhibited the phosphorylation of GSK-3 within 5–30 min (Fig. 6)⇓ , similar to that caused by wortmannin, used as a control (albeit the response to wortmannin was faster). The mammalian target of rapamycin (mTOR, also known as FK506-binding protein 12-rapamycin associated protein [FRAP] or rapamycin and FKBP12 target-1 protein [RAFT]) is also known to play a critical role in the transduction of proliferative signals mediated through the PI3K/Akt signaling pathway, and thus represents a prime target for anti-cancer therapy development (28 , 29) . Indeed, treatment of HN6 cells with UCN-01 or wortmannin inhibited mTOR phosphorylation (Fig. 6)⇓ . Both results support the conclusion that inhibition of the PI3K/Akt pathway by UCN-01 results in decreased phosphorylation of its downstream targets.

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Fig. 6.

Inhibition of downstream targets of Akt by UCN-01 in squamous carcinoma of the head and neck. HN6 cells were used as a representative squamous carcinoma of the head and neck cell line and treated with UCN-01 (100 nm) or wortmannin (50 nm) as a control for the indicated time. Levels of total and phosphorylated Akt (p-Akt) in cell lysates, as well as Akt in vitro kinase activity, were assessed as in Fig. 4⇓ . The phosphorylation status of endogenous glycogen synthase kinase-3 (p-GSK-3) and mTOR (p-mTOR) was determined by Western blotting (WB) using phospho-specific antibodies. Total levels of these proteins were also determined.