Everolimus downregulates STAT3/HIF-1α/VEGF pathway to inhibit angiogenesis and lymphangiogenesis in TP53 mutant head and neck squamous cell carcinoma (HNSCC).

TP53 mutant head and neck squamous cell carcinoma (HNSCC) patients exhibit poor clinical outcomes with 50-60% recurrence rates in advanced stage patients. In a recent phase II clinical trial, adjuvant therapy with everolimus (mTOR inhibitor) significantly increased 2-year progression-free survival in p53 mutated patients. TP53-driven mTOR activation in solid malignancies causes upregulation of HIF-1α and its target, downstream effector VEGF, by activating STAT3 cell signaling pathway. Here, we investigated the effects of everolimus on the STAT3/HIF-1α/VEGF pathway in TP53 mutant cell lines and xenograft models. Treatment with everolimus significantly inhibited cell growth in vitro and effectively reduced the growth of TP53 mutant xenografts in a minimal residual disease (MRD) model in nude mice. Everolimus treatment was associated with significant downregulation of STAT3/HIF-1α/VEGF pathway in both models. Further, treatment with everolimus was associated with attenuation in tumor angiogenesis and lymphangiogenesis as indicated by decreased microvessel density of vascular and lymphatic vessels in HN31 and FaDu xenografts. Everolimus downregulated the STAT3/HIF-1α/VEGF pathway to inhibit growth and in vitro tube formation of HMEC-1 (endothelial) and HMEC-1A (lymphatic endothelial) cell lines. Our studies demonstrated that everolimus inhibits the growth of TP53 mutant tumors by inhibiting angiogenesis and lymphangiogenesis through the downregulation of STAT3/HIF-1α/VEGF signaling.

Rapalogs induce non-apoptotic, autophagy-dependent cell death in HPV-negative TP53 mutant head and neck squamous cell carcinoma.

TP53 is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis, necessitating additional agents for treatment. Since mutant p53 causes sustained activation of the PI3K/AKT/mTOR signaling pathway, we investigated the effect of rapalogs RAD001 and CCI-779 on HPV-negative mutTP53 HNSCC cell lines and xenografts. Rapalogs significantly reduced cell viability and colony formation. Interestingly, rapalogs-induced autophagy with no effect on apoptosis. Pretreatment with autophagy inhibitors, 3-methyladenine (3-MA) and ULK-101 rescued the cell viability by inhibiting rapalog-induced autophagy, suggesting that both RAD001 and CCI-779 induce non-apoptotic autophagy-dependent cell death (ADCD). Moreover, rapalogs upregulated the levels of ULK1 and pULK1 S555 with concomitant downregulation of the mTORC1 pathway. However, pretreatment of cells with rapalogs prevented the ULK-101-mediated inhibition of ULK1 to sustained autophagy, suggesting that rapalogs induce ADCD through the activation of ULK1. To further translate our in vitro studies, we investigated the effect of RAD001 in HPV-negative mutTP53 (HN31 and FaDu) tumor cell xenograft model in nude mice. Mice treated with RAD001 exhibited a significant tumor volume reduction without induction of apoptosis, and with a concomitant increase in autophagy. Further, treatment with RAD001 was associated with a considerable increase in pULK1 S555 and ULK1 levels through the inhibition of mTORC1. 3-MA reversed the effect of RAD001 on FaDu tumor growth suggesting that RAD001 promotes ACDC in HPV-negative mutTP53 xenograft. This is the first report demonstrating that rapalogs promote non-apoptotic ADCD in HPV-negative mutTP53 HNSCC via the ULK1 pathway. Further studies are required to establish the promising role of rapalogs in preventing the regrowth of HPV-negative mutTP53 HNSCC.

4NQO enhances differential activation of DNA repair proteins in HPV positive and HPV negative HNSCC cells.

Tobacco exposure and human papillomavirus (HPV) infection are among the main risk factors for the development of head and neck squamous cell carcinoma (HNSCC). Interestingly, recent studies show that tumors from HPV positive (HPV+) smokers and non-smokers have similar mutational profiles, which suggests that HPV could prevent mutation induction or accumulation in the intermediate risk group composed of HPV+ smokers. Hence, we tested this observation by analyzing the effects of 4-Nitroquinoline N-oxide (4NQO), a mutagen and smoking mimetic, in NOK (normal oral keratinocytes), NOKE6.E7 (NOK cells transfected with E6.E7 oncogenes of HPV), HPV+ and HPV negative (HPV-) HNSCC cells. Oxidative DNA damage, γH2AX foci formation, DNA repair protein activation, cell cycle phase analysis, apoptotic cell death, cell viability and clonogenic cell survival were analyzed after 4NQO treatment in NOK, NOKE6.E7, HPV+ and HPV- HNSCC cells. 4NQO increased oxidative base damage and γH2AX foci formation in NOKE6.E7, HPV+ and HPV- HNSCC cells. Phosphorylation of homologous recombination (HR) repair proteins was higher in NOKE6.E7 and HPV+ HNSCC cells compared to NOK and HPV- HNSCC cells respectively. HPV+ and HPV- HNSCC cells showed differential activation of cell cycle regulatory proteins, increased apoptosis, and decreased cell viability upon 4NQO-induced DNA damage. Taken together, 4NQO (a smoking mimetic), induced higher activation of HR repair in HPV+ HNSCC cells compared to HPV- HNSCC cells. This may allow for increased mutational resistance and help explain why HPV+ smokers have a worse prognosis than HPV+ non-smokers.

Rapamycin targets Interleukin 6 (IL-6) expression and suppresses endothelial cell invasion stimulated by tumor cells.

mTOR inhibitors have potent antiangiogenic and anti-lymphangiogenic effects in addition to their growth inhibitory effects in head and neck squamous cell carcinoma (HNSCC). Lymphatogenous spread is much more predominant in HNSCC than hematogenous spread and significantly decreases survival. In this study we evaluated the effects of rapamycin on targeting tumor-stroma crosstalk in HNSCC. HNSCC tumor cells (FaDu) and human lymphatic endothelial cells (HMEC-1A) were co-cultured in various combinations using transwell cell culture inserts to study tumor-stroma crosstalk and the effects of mTOR inhibitor rapamycin. Levels of growth factors and cytokines in cell culture media were measured using Milliplex bead immunoassay (EMD Millipore) and ELISA assay (R&D Systems). We found that conditioned media collected from tumor cells or co-culture with tumor cells significantly increased the invasiveness of lymphatic and blood vascular endothelial cells (P<0.05), while there was no effect of conditioned media collected from endothelial cell cultures or co-culture with endothelial cells on tumor cell invasiveness. There was a significant effect of rapamycin on both baseline and tumor cell stimulated invasiveness of endothelial cells (P<0.05). Importantly the level of IL-6 secreted in media increased significantly in tumor-endothelial cell co-culture compared to monocultures. Rapamycin significantly suppressed secretion of IL-6 by tumor cells (P<0.05). Thus, HNSCC cells produce chemotactic stimuli that promote endothelial cell invasion toward tumor cells that can stimulate lymphangiogenesis. Rapamycin effectively reverted the stimulatory effect of IL-6 secreted by tumor cells on endothelial cell invasiveness.

Photopreventive Effect and Mechanism of AZD4547 and Curcumin C3 Complex on UVB-Induced Epidermal Hyperplasia.

Aggressive cutaneous squamous cell carcinoma (cSCC) of the skin is the second most common type of skin cancer in the United States due to high exposure to ultraviolet B (UVB) radiation. In our previous studies, Curcumin C3 complex (C3), a standardized preparation of three curcumonoids, delayed UVB-induced tumor incidence and inhibited multiplicity. Exposure to UVB activates mTOR and FGFR signaling that play a key role in skin tumorigenesis. The purpose of this study was to investigate the efficacy of C3 complex to afford protection against acute UVB-induced hyperproliferation by targeting the mTOR and FGFR signaling pathways. Pretreatment with C3 complex significantly inhibited UVB-induced FGF-2 induction, FGF-2-induced cell proliferation, progression and colony formation, mTORC1 and mTORC2 activation, and FGFR2 phosphorylation in the promotion-sensitive JB6 cells epithelial cells. Further, FGFR was critical for UVB-induced mTOR activation, suggesting an important role of FGFR2 in UVB-induced mTOR signaling. SKH-1 mice pretreated with C3 (15 mg/kg/b.w.) for 2 weeks followed by a single exposure to UVB (180 mj/cm(2)) significantly attenuated UVB-induced mTORC1, mTORC2, and FGFR2 activation. To further assess the role of FGFR in UVB-induced hyperproliferation, SKH-1 mice were pretreated with AZD4547 (5 mg/kg/b.w.); a selective pan-FGFR kinase inhibitor followed by single exposure to UVB (180 mj/cm(2)). AZD4547 significantly inhibited UVB-induced mTORC1 and mTORC2 activation, epidermal hyperplasia and hyperproliferation. Our studies underscore the importance of FGFR signaling in UVB-induced acute skin changes and the role of FGFR/mTOR signaling in mediating the effects of C3 complex in the pathogenesis of skin cancer.

EBV and not HPV sensitizes tobacco-associated head and neck cancer cell line FaDu to radiotherapy.

Conclusion EBV radiosensitized the p53 mutant tobacco associated head and neck cell line, FaDu. Objectives In the head and neck, HPV is a major risk factor associated with tonsil and base of tongue cancers, while a majority of undifferentiated nasopharyngeal cancers are positive for EBV. Clinically, head and neck tumors positive for HPV or EBV are more radiosensitive than tumors associated with tobacco and alcohol. This study aimed to evaluate whether viral infections can sensitize tobacco-associated head and neck squamous cell carcinoma cell line that harbors multiple mutations, especially TP53, to radiotherapy. Method Four FaDu cell lines (vector control - FaDu-DN; FaDu expressing HPV16 E6/E7 - FaDu-HPV; FaDu infected with EBV - FaDu-EBV; and FaDu-HPV infected with EBV - FaDu-HE) were evaluated for their radiation sensitivity using clonogenic assay. Cell cycle, protein expression, apoptosis, and cellular senescence were analyzed. Results FaDu-EBV and FaDu-HE exhibited significantly increased radiosensitivity in comparison with the control cell line. Radiation-induced cell cycle arrest was altered in all cell lines expressing viral genes. The observed distribution of cells at G1 and S phases was associated with a significant increase in expression of p21 protein along with decreased levels of pAKT/AKT and pERK/ERK ratio (p < 0.05) and increased cellular senescence (p < 0.05).

Enhanced Systemic Bioavailability of Curcumin Through Transmucosal Administration of a Novel Microgranular Formulation.

BACKGROUND/AIM: Curcumin is a promising nutraceutical for chemoprevention of head and neck squamous cell carcinoma (HNSCC). Capsular formulations of curcumin demonstrate low systemic bioavailability. We aimed to determine if curcumin levels were higher in healthy volunteers and cancer patients with microgranular curcumin that allows for transmucosal absorption and identify a consistent biomarker. PATIENTS AND METHODS: Eight healthy volunteers and 15 HNSCC patients completed the trials. Serum levels of curcumin were measured by HPLC. Biological activity of curcumin was assessed with Multiplex Immunoassay and immunohistochemistry. RESULTS: We achieved higher serum levels of curcumin compared to trials using capsular formulation. In cancer patients a significant decrease in expression of fibroblast growth factor-2 (FGF-2) in post-biopsy samples and decreased serum levels of FGF-2, granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-17 (IL-17) (p<0.05) was observed. CONCLUSION: Transmucosal administration of microgranular curcumin leads to enhanced curcumin bioavailability that is associated with significant biological effects.

Association between human papilloma virus/Epstein-Barr virus coinfection and oral carcinogenesis.

BACKGROUND: The recent epidemic of head and neck squamous cell carcinomas associated with human papilloma virus (HPV) has not addressed its association with lymphoid tissue in the oropharynx or the potential role of Epstein-Barr virus (EBV)/HPV coinfection. METHODS: The prevalence of HPV and EBV infection/coinfection and CD21 mRNA expression were determined in normal and cancerous tissues from the oropharynx using in situ hybridization (ISH), p16, and quantitative reverse transcriptase PCR (qRT-PCR). The effects of coinfection on tumorigenicity were evaluated using proliferation and invasion assays. RESULTS: Normal oropharynx, tonsil, non-cancer base of tongue (BOT), and BOT from sleep apnea patients demonstrated EBV positivity ranging from 7% to 36% depending on the site and methods of detection used (qRT-PCR or ISH). Among non-malignant BOT samples, HPV positivity was noted only in 20%. The percent of tonsil and BOT cancers positive for HPV (up to 63% and 80%, respectively) or coinfected with HPV/EBV (up to 25% and 70%, respectively) were both significantly associated with cancer status. Notably, HPV/EBV coinfection was observed only in malignant tissue originating in lymphoid-rich oropharynx sites (tonsil, BOT). CD21 mRNA (the major EBV attachment receptor) was detected in tonsil and BOT epithelium, but not in soft-palate epithelium. Coinfected cell lines showed a significant increase in invasiveness (P < 0.01). CONCLUSIONS: There is a high prevalence of HPV/EBV infection and coinfection in BOT and tonsil cancers, possibly reflecting their origins in lymphoid-rich tissue. In vitro, cells modeling coinfection have an increased invasive potential.

Anti-lymphangiogenic properties of mTOR inhibitors in head and neck squamous cell carcinoma experimental models.

BACKGROUND: Tumor dissemination to cervical lymph nodes via lymphatics represents the first step in the metastasis of head and neck squamous cell carcinoma (HNSCC) and is the most significant predictor of tumor recurrence decreasing survival by 50%. The lymphatic suppressing properties of mTOR inhibitors are not yet well understood. METHODS: Lymphatic inhibiting effects of rapamycin were evaluated in vitro using two lymphatic endothelial cell (LEC) lines. An orthotopic mouse model of HNSCC (OSC-19 cells) was used to evaluate anti-lymphangiogenic effects of rapamycin in vivo. The incidence of cervical lymph node metastases, numbers of tumor-free lymphatic vessels and those invaded by tumor cells in mouse lingual tissue, and expression of pro-lymphangiogenic markers were assessed. RESULTS: Rapamycin significantly decreased lymphatic vascular density (p = 0.027), reduced the fraction of lymphatic vessels invaded by tumor cells in tongue tissue (p = 0.013) and decreased metastasis-positive lymph nodes (p = 0.04). Rapamycin also significantly attenuated the extent of metastatic tumor cell spread within lymph nodes (p < 0.0001). We found that rapamycin significantly reduced LEC proliferation and was correlated with decreased VEGFR-3 expression in both LEC, and in some HNSCC cell lines. CONCLUSIONS: The results of this study demonstrate anti-lymphangiogenic properties of mTOR inhibitors in HNSCC. mTOR inhibitors suppress autocrine and paracrine growth stimulation of tumor and lymphatic endothelial cells by impairing VEGF-C/VEGFR-3 axis and release of soluble VEGFR-2. In a murine HNSCC orthotopic model rapamycin significantly suppressed lymphovascular invasion, decreased cervical lymph node metastasis and delayed the spread of metastatic tumor cells within the lymph nodes.

Topical curcumin-based cream is equivalent to dietary curcumin in a skin cancer model.

Skin squamous cell carcinoma (SCC), the most common cancer in the USA, is a growing problem with the use of tanning booths causing sun-damaged skin. Antiproliferative effects of curcumin were demonstrated in an aggressive skin cancer cell line SRB12-p9 (P < 0.05 compared to control). Topical formulation was as effective as oral curcumin at suppressing tumor growth in a mouse skin cancer model. Curcumin at 15 mg administered by oral, topical, or combined formulation significantly reduced tumor growth compared to control (P = 0.004). Inhibition of pAKT, pS6, p-4EBP1, pSTAT3, and pERK1/2 was noted in SRB12-p9 cells post-curcumin treatment compared to control (P < 0.05). Inhibition of pSTAT3 and pERK1/2 was also noted in curcumin-treated groups in vivo. IHC analysis revealed human tumor specimens that expressed significantly more activated pERK (P = 0.006) and pS6 (P < 0.0001) than normal skin samples. This is the first study to compare topical curcumin to oral curcumin. Our data supports the use of curcumin as a chemopreventive for skin SCC where condemned skin is a significant problem. Prevention strategies offer the best hope of future health care costs in a disease that is increasing in incidence due to increased sun exposure.

Curcumin inhibits skin squamous cell carcinoma tumor growth in vivo.

OBJECTIVE: Squamous cell carcinoma (SCCa) has increased from 4% to 10% over 4 decades, stimulating interest in developing novel agents that slow sun-damaged skin progression. This is the first study evaluating the naturally occurring bioactive food compound curcumin on skin cancer xenografts. Low bioavailability of curcumin has slowed its transition to clinical trials. It is hypothesized that curcumin has growth-inhibitory effects through the TOR pathway and chemopreventive potential in skin SCCa where local application could bypass bioavailability problems. STUDY DESIGN: A randomized experimental animal and laboratory study. SETTING: Louisiana State University Health Sciences Center, Shreveport, Louisiana. SUBJECTS AND METHODS: SCID mice were pretreated with 0, 5, or 15 mg of curcumin (n = 8 per group), 3 days prior to injecting 106 SRB12-p9 skin SCCa cells in each flank, and were gavaged daily thereafter. Tumor volumes were measured and tumors were harvested on day 24 when mice were sacrificed. Immunohistochemical analysis of pS6 expression (n = 3 per group) and tumor volumes in the 3 groups were compared using 1-way analysis of variance and pairwise comparisons were determined with the Tukey t test if overall comparisons were significant. RESULTS: Tumor volume increased 2.3 times faster in control mice compared with the group receiving 15 mg of curcumin (P = .0003). A significant difference in average tumor volumes was seen (P = .0012), especially with treatment of 15 mg of curcumin compared with control P = .0003). Curcumin inhibited S6 phosphorylation (P = .0027), suggest-ing inhibition of the MTOR pathway. CONCLUSION: Curcumin appears to inhibit skin SCCa growth and blocks tumor progression by inhibiting pS6 even when gavage is used to deliver curcumin, indicating even more significant effects in future experiments with local application.

Efficacy and comparative effectiveness of sirolimus as an anticancer drug.

OBJECTIVES/HYPOTHESIS: To evaluate antitumor efficacy of the generic mammalian target of rapamycin (mTOR) inhibitor sirolimus in preclinical animal models of head and neck squamous cell carcinoma (HNSCC) and compare its effects with those of the patented analogue temsirolimus. STUDY DESIGN: In vivo study. METHODS: To develop xenograft established tumor model (ETM) of HNSCC, FaDu cells were injected subcutaneously into nude mice. When tumors reached 50 to 60 mm(3), mice were randomized into five groups and treated daily intraperitoneally with sirolimus at various doses for 5 days per week for 3 weeks. Tumor volumes were measured. The results were compared with historical data on temsirolimus effects. In the minimal residual disease (MRD) model, surgical wounds were created and FaDu cells implanted. After 72 hours, animals were randomized into two groups and were injected intraperitoneally with 0 or 5 mg/kg sirolimus for 5 days per week for 30 days. RESULTS: In the ETM, sirolimus significantly inhibited tumor growth (P < .01), although there was no overall significant difference in tumor growth inhibition between sirolimus and temsirolimus. In the MRD model, sirolimus significantly suppressed growth of tumors (P < .001) and improved survival compared with controls (P < .01). There was a significant decrease in pS6 expression, indicating mTOR inhibition. CONCLUSIONS: In this study, we demonstrate that the generic mTOR inhibitor sirolimus shows potent antitumor activity in HNSCC and produces comparable effects to the patent drug temsirolimus. Sirolimus has the potential of serving as an economic and comparative targeted agent to temsirolimus in the treatment of HNSCC.

Curcumin inhibits carcinogen and nicotine-induced Mammalian target of rapamycin pathway activation in head and neck squamous cell carcinoma.

Curcumin appears to be a safe, bioactive food compound that is a potential chemopreventive for patients at a high risk for head and neck squamous cell carcinoma (HNSCC). Identification and validation of intermediate endpoints is an important step in evaluating chemopreventive agents. AKT/MTOR pathway biomarkers are intrinsic to the carcinogenic process as well as the mechanism of intervention with curcumin. Antiproliferative effects of curcumin were assayed in 9 HNSCC and a keratinocyte cell line. Nicotine, a genotoxic alkaloid involved in tobacco addiction, forms DNA adducts and has been implicated in upper aerodigestive tract cancer promotion. The antiproliferative effects of curcumin were associated with inhibition of the AKT/MTOR pathway in presence and absence of nicotine, which also induced this pathway. Curcumin was highly effective at suppressing growth of SCC40 xenografts and its activity is associated with modulation of MTOR's downstream target pS6. Curcumin at 15 mg significantly increased survival (286 ± 37 vs. 350 days) in the 4NQO carcinogenic model survival study. A major cause of lethal progression of HNSCC is local regional migration and invasion of malignant cells, and curcumin significantly inhibited cancer cell migration and invasion in vitro and in vivo where downregulation of pS6 was associated with a significant decrease in MMP-9. This is the first study to demonstrate that curcumin inhibits the adverse effects of nicotine by blocking nicotine-induced activation of the AKT/MTOR pathway in HNSCC, which retards cell migration. These studies indicate that inhibiting the AKT/MTOR pathway with curcumin may be useful as an oral chemopreventive agent.

Teasing out the best molecular marker in the AKT/mTOR pathway in head and neck squamous cell cancer patients.

OBJECTIVES/HYPOTHESIS: No reliable molecular biomarker is currently available for clinical application in the management of head and neck cancer patients. The AKT/mTOR pathway is activated in 90% to 100% of head and neck squamous cell cancer (HNSCC) and could be promising biomarkers closely linked to cancer incidence. STUDY DESIGN: Retrospective study of HNSCC and non-cancer patients. METHODS: Oral mucosa from noncancer patients were compared to HNSCC tumors and junctional zone mucosa. The candidate biomarkers mTOR, AKT, 4EBP1, and S6 kinase, signaling components upstream and downstream of mTOR that appear dysregulated in HNSCC, were evaluated using immunohistochemistry (IHC) and Western blot analysis. RESULTS: Expression of phosphorylated AKT and phosphorylated mTOR were significantly higher in cancer patient tumors compared to noncancer oral mucosa samples (P = .004 and P = .026, respectively) by Western blot analysis. Expression of p-mTOR and p-4EBP1 were higher in patient junctional zones compared to tumors (p = 0.017 and p = 0.022, respectively) and no difference in p-AKT or p-S6 expression in HNSCC patients' junctional zone compared to tumors. IHC-demonstrated p-mTOR expression was 81.9% sensitive and 100% specific in differentiating cancer from noncancer mucosa, whereas p-4EBP1 expression by IHC was only 50.0% sensitive and 95.5% specific in differentiating normal mucosa from HNSCC (P < .01). CONCLUSIONS: Phosphorylated mTOR appears to be a reliable biomarker by both Western blot analysis (P = .026) and IHC in human head and neck cancer (P < .001). Moreover, phosphorylated AKT, which is immediately upstream of mTOR, is a potential biomarker that should be further studied. Clinical trials with mTOR inhibitors are being evaluated for HNSCC, and selecting patients that are likely to respond to these inhibitors requires identifying and validating predictive biomarkers of response.

Pharmacodynamic evaluation of temsirolimus in patients with newly diagnosed advanced-stage head and neck squamous cell carcinoma.

BACKGROUND: Activation of the mammalian target of rapamycin (mTOR) pathway in surgical margins of head and neck squamous cell carcinoma (HNSCC) is a predictor of recurrence and patients with minimal residual disease may benefit from adjuvant therapy with temsirolimus, an mTOR inhibitor. METHODS: The effects of 3 weekly doses of 25 mg of temsirolimus on Akt/mTOR pathway biomarkers were evaluated in tumor and peripheral blood mononuclear cells (PBMCs) of patients with HNSCC. Adverse events were assessed. RESULTS: Temsirolimus significantly decreased pS6 and p4E-BP1 in tumors, and pS6 and pAkt in PBMCs (p < .05). There was no significant upregulation of pAkt(Ser(473)) in tumor tissue. Side effects were minimal and reversible. CONCLUSION: Significant inhibition of the mTOR pathway was noted in both tumors and PBMCs of HNSCC with minimal side effects. The mTOR inhibitors can potentially be used as adjuvant therapy for patients with minimal residual disease and PBMCs are potential surrogate markers in this setting.

Comparison of radiosensitizing effects of the mammalian target of rapamycin inhibitor CCI-779 to cisplatin in experimental models of head and neck squamous cell carcinoma.

To determine if the mammalian target of rapamycin (mTOR) inhibitor CCI-779 can sensitize head and neck squamous cell carcinoma (HNSCC) to radiotherapy (XRT) and compare the radiosensitizing effects to cisplatin with its known considerable toxicity. Radiosensitizing effects of CCI-779 were assayed on HNSCC cell lines in vitro. CCI-779 (5 mg/kg), cisplatin (1 mg/kg), and XRT (2 Gy) alone and in combination were evaluated for antitumor activity in mice bearing FaDu and SCC40 xenografts. Effects of CCI-779 on radiation-induced activation of the Akt/mTOR pathway were analyzed. Although CCI-779 did not sensitize HNSCC cells to ionizing radiation in vitro, combination of CCI-779 and XRT significantly augmented the in vivo tumor growth-inhibitory effects of XRT and CCI-779 (P < 0.05). In addition, CCI-779 + XRT suppressed tumor growth more effectively than cisplatin + XRT (P < 0.05). CCI-779 + XRT significantly improved survival compared with XRT alone in both cisplatin-sensitive FaDu (P < 0.01) and cisplatin-resistant SCC40 (P < 0.05) xenograft mice. There were no additional benefits of adding cisplatin to CCI-779 + XRT. CCI-779 significantly attenuated irradiation-induced up-regulation of the mTOR pathway, increased apoptosis and displayed potent antiangiogenic activity in FaDu xenografts that was further enhanced by its combination with XRT (P < 0.05), which may explain the mechanism of its selective radiosensitizing effects in vivo and not in vitro. Antitumor activity of XRT was enhanced when combined with CCI-779 in HNSCC xenograft model. CCI-779 + XRT showed antitumor activity superior to conventional chemoradiotherapy with cisplatin. These results pave the way for clinical trials using molecular targeted therapy with CCI-779 in combination with XRT for HNSCC treatment.

Mammalian target of rapamycin inhibitors as possible adjuvant therapy for microscopic residual disease in head and neck squamous cell cancer.

Molecular therapeutics identifies an aberration in tumors to select patients that benefit from molecular targeted therapy. Overexpression of eIF4E in histologically "tumor-free" surgical margins of head and neck squamous cell cancer (HNSCC) patients is an independent predictor of recurrence and is functionally activated through the Akt/mammalian target of rapamycin (mTOR) pathway. Although mTOR inhibitors are cytostatic agents, best used in combination therapy, we hypothesize that they can be used as long-term single agents in an HNSCC model of minimal residual disease (MRD). CCI-779, an mTOR inhibitor, arrested growth of a phosphatase and tensin homologue deleted on chromosome 10 (PTEN) abnormal HNSCC cell line FaDu, inhibiting phosphorylation of 4E-binding protein 1, resulting in increased association with eIF4E and inhibition of basic fibroblast growth factor and vascular endothelial growth factor. Fluorescence in situ hybridization detected PTEN abnormalities in 68% of patient tumors and 35% of tumor-free margins. CCI-779 inhibited growth of established tumors in nude mice. However, in the MRD model, there were significant differences in the tumor-free rate between the control (4%) and the treatment group (50%), and the median tumor-free time was 7 versus 18 days, respectively (P < 0.0001). In those animals that formed tumors, CCI-779 caused a significant decrease in the tumor volume. The Kaplan-Meier curve showed that CCI-779 significantly increased survival (P < 0.0001). The mTOR pathway was inhibited in peripheral blood mononuclear cells potential surrogate markers of response to therapy. Stable transfection of FaDu with luciferase allowed us to monitor the effects of CCI-779 with bioluminescence imaging in the MRD model. These results pave the way for a clinical trial using targeted molecular therapy with CCI-779 as a single agent for mTOR-activated residual cells.

Overexpressed eIF4E is functionally active in surgical margins of head and neck cancer patients via activation of the Akt/mammalian target of rapamycin pathway.

PURPOSE: Overexpression of eIF4E in surgical margins of head and neck cancer patients is an independent risk factor for recurrence. We hypothesize that overexpressed eIF4E is functionally active in tumor margins through activation of the Akt/mammalian target of rapamycin (mTOR) pathway EXPERIMENTAL DESIGN: Western blots and/or immunohistochemistry were performed to determine whether phosphorylation of mTOR and activation of its downstream molecules eIF4E-binding protein-1 (4E-BP1) and p70 S6 kinase and the upstream modulator of mTOR, Akt, were expressed in margins overexpressing eIF4E. RESULTS: There was a significant association between phospho-4E-BP1 and eIF4E expression of a margin or a significant difference in phospho-4E-BP1 expression between the eIF4E-positive and -negative margins (P < 0.01). A significant association between eIF4E and phospho-p70 S6 kinase as well as eIF4E and phospho-mTOR was also noted (P < 0.05). Western blot analysis indicated a highly significant difference in the phosphorylation status of 4E-BP1 between tumors and resection margins. A total of 89% of the 4E-BP1-expressing margins expressed more of the phosphorylated (beta, gamma, and delta) isoforms, whereas 81% of the 4E-BP1-expressing tumors expressed more of the unphosphorylated alpha isoform. A similar difference in Akt activation was noted between eIF4E-positive margins and tumors (P < 0.05). CONCLUSIONS: Overexpression of eIF4E is functionally active in tumor margins through activation of the Akt/mTOR signaling pathway. The greater degree of expression of downstream targets and upstream regulators of mTOR in margins compared with the tumors indicates preferential activation of the Akt/mTOR signaling pathway in margins overexpressing eIF4E. Rapamycin analogs can potentially be used as adjuvant therapy for patients with eIF4E-positive margins.

NS 398 radiosensitizes an HNSCC cell line by possibly inhibiting radiation-induced expression of COX-2.

PURPOSE: Cyclooxygenase-2 (COX-2) protein is frequently elevated in squamous cell carcinoma of the head and neck (HNSCC). The aim of this study was to determine if COX-2 inhibitors have radiosensitizing effects in HNSCC and understand the mechanism by which this occurs. MATERIALS AND METHODS: The radiosensitizing effects of a selective COX-2 inhibitor, NS398, on a HNSCC cell line HEp3, were determined using clonogenic survival assay. Cells were pretreated with the dose of NS398 at which 50% growth inhibition occurred (IC(50)) and then irradiated. COX-2 protein and mRNA were then determined in the presence and absence of NS398. RESULTS: NS398 significantly decreased (p < 0.0001) the calculated survival fraction (SF) for all radiation doses (0.79 to 0.41 at 2 Gy). A significant increase in COX-2 protein of 2.8 fold for 2 Gy and 3.5 fold for 6 Gy was noted 48 h after radiation. Interestingly, the upregulation of COX-2 protein with radiation was suppressed when cells were pretreated with NS398. Quantitative reverse transcriptase polymerase chain reaction showed no significant corresponding increase in COX-2 mRNA at 48 h with ionizing radiation. CONCLUSIONS: The radiosensitizing effect of NS398 could be due to inhibition of radiation-induced COX-2 upregulation by this drug. NS398, known as an inhibitor of COX-2 enzyme activity, down-regulated COX-2 protein expression, which may indicate that NS398 can act upstream of COX-2, and this change appears to be post-transcriptional.