Research ArticleExperimental Studies

Targeting mTOR and AREG with Everolimus, Sunitinib and Sorafenib in HPV-positive and -negative SCC

CHRISTOPH ADERHOLD, ANNE FABER, CLAUDIA UMBREIT, RICHARD BIRK, CHRISTEL WEISS, JÖRG ULRICH SOMMER, KARL HÖRMANN and JOHANNES DAVID SCHULTZ
Anticancer Research April 2015, 35 (4) 1951-1959;

Abstract

Background/Aim: Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. It is the most common neoplasm appearing in the upper aerodigestive tract and the sixth most common cancer worldwide. The five-year survival rate remains poor despite advances in surgery, radiation and chemotherapy. Furthermore, the incidence of human papillomavirus (HPV)-associated oropharyngeal cancer is rising. Thus, innovative therapy approaches are imperative in order to improve the situation. Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR) and sorafenib and sunitinib, multityrosine kinase inhibitors, have been notably effective in the therapy of different tumor entities. The modest side-effects and oral application of the drugs might improve patient compliance. Expression levels of mTOR and Amphiregulin (AREG) in p16-positive and -negative SCC (squamous cell carcinoma) and the effect of everolimus, sorafenib or sunitinib on the expression levels of these target proteins were assessed. As far as we are aware of, this is one of the first in vitro studies to evaluate the effect of these small-molecule drugs with regard to the p16 status of SCC cells. Materials and Methods: p16-negative HNSCC 11A and 14C cells and p16-positive CERV196 cells were exposed to different concentrations of everolimus, sorafenib and sunitinib for 2-8 days. Expression levels of mTOR and AREG were determined by enzyme-linked immunosorbent assay (ELISA) and compared against a chemonaïve control. Results: AREG and mTOR were expressed in all tested cell lines. CERV196 displayed a remarkable increase of mTOR expression compared to p16-negative HNSCC. On the contrary, AREG levels were reduced by 50% in CERV196. Everolimus, sorafenib and sunitinib significantly reduced mTOR expres sion. Everolimus significantly decreased AREG expression independently of the HPV status. Sunitinib and sorafenib increased AREG expression in HNSCC 11A and 14C but not in CERV196. Conclusion: The applied drugs showed remarkable suppression of mTOR expression, which might delay tumor progression. Interestingly, sorafenib and sunitinib increased AREG in HNSCC 11A and 14C, which could be a possible evasive mechanism following incubation with these drugs. On the contrary, p16-positive CERV196 showed increased susceptibility to sorafenib and sunitinib concerning suppression of AREG expression. Further studies are required to evaluate the HPV-dependent differences of therapy response and the possible consequences for treatment options.

Head and neck squamous cell carcinoma (HNSCC) is the most common epithelial malignancy in the upper aerodigetive tract. Every year, this tumor entity accounts for about 686,000 new cases and approximately 375,000 deaths. It is the sixth most common cancer in the world, characterized by variable tumor aggressiveness and response to treatment (1). The overall 5-year survival rate is less than 50% and has not improved despite innovative diagnostics, surgical procedures, radiation and chemotherapy (2). The most important risk factors for HNSCC are smoking and alcohol abuse (3, 4). The recent decrease in HNSCC incidence could be explained by the falling prevalence of tobacco abuse (5).

In contrast, the incidence of oropharyngeal cancer has increased in patients aged 20-44 (6). These oropharyngeal carcinomas are associated with human papillomavirus (HPV) and their rising incidence underlines the importance of the viral etiology (7-9). A systematic review reported an overall HPV-prevalence of about 26% (10). HPV is significantly more common in oropharyngeal HNSCC (35.6%) compared to oral (23.5%) or laryngeal locations (24%) (11). It is remarkable that approximately 95% of HPV-positive HNSCC contain DNA (deoxyribonucleic acid) of the high-risk subtype 16 (12). The circular, double-stranded DNA genome of HPV encodes three oncoproteins and two structural capsid proteins (13). The oncoprotein E6 degrades tumor suppressor protein p53 (14). At present, over 100 subtypes are known and can be classified as low- or high-risk sub-populations based on their cancerogenic potency (15).

Amphiregulin (AREG) is a ligand of epidermal growth factor receptor (EGFR) and is related to epidermal growth factor (EGF) and transforming growth factor α (TGF-α). Activation of EGFR affects several cellular signal cascades. EGFR can induce tumor angiogenesis and inhibit apoptosis via the PI3K (phosphatidylinositol 3-kinase)/Akt signaling pathway (16). Cell cycle progression can be realized via activation of the Ras/Raf cascade (17). Over-expression of EGFR can be found in 42-80% of HNSCC and EGFR gene amplification occurs in up to 30% (18-21). This is associated with poorer prognosis (22). The relevance of EGFR and its ligands is illustrated by the fact that an EGFR-targeted therapy with cetuximab is part of the guideline therapy for recurrent or advanced-stage HNSCC with distant metastasis (23). Currently, cetuximab is the only Food and Drug Administration (FDA)-approved targeted-therapy for HNSCC. Mutations of EGFR cause resistance to cetuximab and could be a possible evasive mechanism of the tumor cell during a targeted therapy (24).

The mammalian target of rapamycin (mTOR) is a phosphatidylinositol 3-kinase-related kinase. It integrates the upstream signaling from several messengers including EGFR (25). mTOR detects nutrient, oxygen and energy levels in the cell, as well as oxidative stress. It is part of two functional protein complexes: mTORC1 and mTORC2. mTOR and the PI3K/Akt signaling cascade are closely related. mTORC1, for instance, can be activated via the PI3K/Akt signaling pathway. mTORC2, on the other hand, is able to activate Akt (26-28). mTOR alters DNA transcription and protein synthesis of vascular endothelial growth factor (VEGF), and therefore induces angiogenesis (29, 30). Additional functions of mTOR are regulation of proliferation, cell motility, cell survival and protein synthesis.

Given the poor prognosis of HNSCC, new pharmacological approaches are necessary. Tumor heterogeneity gives hope for a personalized anticancer therapy but the only FDA-approved targeted therapy for HNSCC is cetuximab. Personalized therapy approaches are still missing for HNSCC.

The mTOR inhibitor everolimus is used as an immunosuppressant to prevent rejection after organ transplantation (31). It is also part of chemotherapeutic regimens against various types of human cancers, including HNSCC (32-34). Solid tumors that developed resistance to VEGF or vascular endothelial growth factor receptor (VEGFR)-targeted therapy with bevacizumab showed sensitivity towards mTOR inhibitors (32). mTOR is an integration center for several intra cellular signaling pathways frequently dysregulated in cancer (26, 33). Synergistic effects of mTOR inhibitors with other targeted therapy drugs and radiochemotherapy illustrate the integrative function of mTOR (35-37). Lymph node metastases of HNSCC often show activated mTOR signaling. Its inhibition with everolimus reduces lymphangiogenesis at the primary tumor site and prevents lymphatic dissemination (38).

Sorafenib and sunitinib are multi-tyrosine kinase inhibitors. Both drugs target VEGFR and platelet-derived growth factor receptor (PDGFR). Furthermore, sorafenib inhibits Raf kinases, while sunitinib inhibits c-KIT, RET and colony stimulating factor (CSF). Sorafenib was applied in the therapy of hepatocellular, thyroid and renal cell carcinoma, lung cancer, melanoma and HNSCC (39-42). The combination of sorafenib with other targeted anticancer drugs, chemotherapy and radiation led to promising synergistic effects (43, 44).

Sunitinib was established in treatment regimens for gastrointestinal stromal tumors (GIST), renal cell carcinoma, pancreatic neuroendocrine tumors, breast, lung, thyroid and colorectal cancers (45, 46). Recently, it was evaluated for HNSCC therapy in clinical trials (47-49).

The application of everolimus, sorafenib and sunitinib in HNSCC therapy has been evaluated in several clinical trials (36, 37, 47-52); however, none of these studies took into account the HPV status of SCC (squamous cell carcinoma) cells. Our study evaluated the protein levels of mTOR and AREG in HPV-positive (CERV196) and HPV-negative (HNSCC 11A and 14C) SCC and their alteration following incubation with everolimus, sorafenib and sunitinib. As far as we are aware of, this is one of the first in vitro studies to investigate the impact of these small-molecule drugs on SCC, particularly with regard to HPV-dependent differences.

Materials and Methods

Cell lines. The human cell lines HNSCC 11A and 14C were received from Dr. T.E. Carey (University of Michigan, Ann Arbor, MI, USA) and originated from a SCC of the oropharynx and larynx. CERV196 cells were obtained from a p16-positive SCC of the uterine cervix (CLS, Eppelheim, Germany). Dulbecco's modified essential medium (DMEM) (Fisher Scientific and Co., Pittsburgh, PA, USA) supplemented with 10% fetal calf serum (FCS) and antibiotics (Life Technologies Inc., Gaithersburg, MD, USA) was used as medium for HNSCC 11A and 14C. CERV196 cells were kept in Eagle's minimum essential medium with 2 mM L-glutamine, 10% FCS and Earle's balanced salt solution (BSS) with 1.5 g/l sodium bicarbonate, 0.1 mM amino acids and 1.0 mM sodium pyruvate. Cell cultures were carried out at 37°C in a fully humidified atmosphere with 5% CO2. Everolimus (Novartis, Basel, Switzer land), sorafenib (Bayer, Leverkusen, Germany) and sunitinib (Pfizer, New York, NY, USA) were made up in dimethylsulfoxide (DMSO) at the time of use. The cells were exposed to drug concentrations ranging from 1.0-25.0 μmol/ml for 2 to 8 days. These drug concentrations were chosen after the alamarBlue (AbD Serotec, Oxford, UK) cell proliferation assay was performed, which measured the proliferation of HNSCC tumor cell lines quantitatively and indicated the relative cytotoxicity of the studied drugs.

Figure 1.
Figure 1.

mTOR expression in HNSCC 11A, 14C and CERV196 after incubation with everolimus, sunitinib or sorafenib compared to the negative control. Data are mean values. Standard deviation is indicated.

Enzyme-linked immunosorbent assay (ELISA) for total mTOR and AREG. Following incubation, cells were washed with phosphate buffered saline (PBS). Then, 350 μl of lysis buffer were added to each well. The lysed cells were stirred up with a vortex at 2-8°C for 30 min and microcentrifuged with 5,000×g for 5 min. The supernatant was pipetted into a clean tube.

Protein concentrations were determined with the ELISA technique (R&D Systems, Wiesbaden, Germany): DuoSet IC Human phosphorylated mTOR (DYC1665) and DuoSet Human Amphiregulin (AREG) (DY262).

The sandwich ELISA system used a solid-phase capture antibody specific for either human phosphorylated mTOR or AREG and a specific detection antibody using a standard streptavidin-horseradish peroxidase (HRP) format specific for phosphorylated mTOR or AREG. The capture antibody was diluted to its working con centration (2 μg/ml for both mTOR and AREG). Then, 100 μl of the capture antibody were added to each well and incubated overnight. The contents of each well were aspirated and the wells were washed three times with 400 μl of Tween buffer. Subsequently, 300 μl of block buffer were added to each well and incubated for 1-2 h followed by another Tween buffer wash as described above. The detection antibody was diluted to its working concentration (1 μg/ml for mTOR and 2 μg/ml for AREG) and 100 μl of the detection antibody were added to each well for 2 h. The wells were washed again as described above and 100 μl of streptavidin-HRP (diluted according to the manufacturer's instructions) were added to each well followed by 20 min of incubation at room temperature. Afterwards, the wells were washed as described above and 100 μl of substrate solution were added to each well for 20 min followed by 50 μl of stop solution. According to the manufacturer's directions, each ELISA was performed with 100 μl of supernatant. All analyses and calibrations were carried out three times. The calibrations on each microtiter plate included recombinant human mTOR and AREG standards that were provided in the kits. Optical density was measured using a microplate reader at a wavelength of 450 nm. Wavelength correction was set to 540 nm, and concentrations were reported as pg/ml. The range of detection for mTOR was 156-10,000 pg/ml and 15.6-1,000 pg/ml for AREG. The interassay coefficient of variation reported by the manufacturer was below 10%.

Measuring total protein. Total protein was measured with the DC Protein Assay (BioRad, Hercules, CA, USA). Cells were incubated, lysed and centrifuged as previously described. Dilutions of protein standard were prepared according to the manufacturer's instructions. Measurement was performed on 100 μl of protein standard or cell supernatant with a spectrophotometer set to 750 nm; concentrations were reported as μg/ml.

View this table:
Table I.

ELISA of mTOR expression in HNSCC 11A, 14C and CERV196 after incubation with everolimus, sunitinib or sorafenib compared to the negative control (statistical significance is shown in bold).

Statistical analysis. Statistical analysis was performed in cooperation with PD Dr. C. Weiss, Institute of Biomathematics, Faculty of Medicine, Mannheim, Germany. A p-value ≤0.05 was considered statistically significant. The two-coefficient variance test (SAS Statistics, Cary, NC, USA) and Dunnett's test were used.

Results

Total protein assay. Total protein levels were evaluated in order to differentiate a decline of AREG and mTOR protein expression from cytotoxicity of the applied drugs. The total protein level of the cell lysate was compared to the protein level of the target values. We detected a slight increase of the protein quotient (expression of the target value/total protein level) for HNSCC 11A and 14C after incubation with sunitinib and sorafenib in a time-dependent manner, which could be evidence of increased apoptosis. A similar effect was not observed in p16-positive CERV196. Everolimus did not increase protein quotient (data not shown).

ELISA for mTOR expression in HNSCC 14C, 11A and CERV 196 cells. mTOR was expressed in all three cell lines. Expression levels of mTOR in CERV196 were twice as high compared to p16-negative HNSCC 11A and 14C. In CERV 196, sunitinib and sorafenib significantly decreased mTOR expression independent of the incubation time. The p-values for sunitinib varied from <0.001-0.002 and for sorafenib from 0.006-0.024. In HNSCC 11A, sunitinib suppressed mTOR expression significantly after 3, 5 and 8 days of incubation (p=0.003-0.025). Similar results were observed for sorafenib in HNSCC 11A with a significant decrease of mTOR expression after 2, 3 and 8 days of incubation (p=0.015-0.072). In HNSCC 14C, neither sunitinib nor sorafenib led to a statistically significant reduction of mTOR expression with the exception of sunitinib after 2 days of incubation (p=0.003). Although everolimus considerably decreased mTOR expression in CERV196 and HNSCC 11A in an incubation-time dependent manner, the reduction was not statistically significant. On the contrary, we detected significant suppression of mTOR expression by everolimus in HNSCC 14C independent of the incubation time (p=0.009-0.039). Increasing drug concentrations had no statistically significant effect on mTOR expression. For simplification, only the data for 25 μmol/ml are show in Table I and Figure 1.

ELISA of AREG expression in HNSCC 14C, 11A and CERV196. Expression of AREG was detected in all three cell lines. In CERV196, expression levels were considerably lower (reduced by almost 50%) compared to HNSCC 11A and 14C. Both sunitinib and sorafenib significantly suppressed AREG in CERV196. Sunitinib almost completely abolished the expression of AREG in CERV196 compared to the chemonaïve control with p-values between 0.001 and 0.005. Sorafenib displayed a statistically significant impact on AREG expression in CERV196 (p<0.001). Everolimus significantly suppressed AREG in CERV196 (p=0.014-0.026), with one exception after 120 h of incubation (p=0.229). The impact of everolimus on HNSCC 11A and 14C was much smaller. Significant suppression of AREG could be detected after 72 h of incubation in HNSCC 11A only (p=0.019). Due to technical difficulties, the sample for the negative control after 192 h of incubation could not be analyzed. Consequently, we were unable to determine a p-value for this sample. In HNSCC 14C, we detected a significant alteration of AREG levels under incubation with everolimus after 48 h and 72 h (p<0.001 and 0.048, respectively). Interestingly, both multi-tyrosine kinase inhibitors sunitinib and sorafenib significantly increased the expression of AREG in HNSCC 14C and 11A independent of the incubation time (p≤0.008). Drug concentrations of 10 μmol/ml or higher were significantly more effective at altering protein expression. For simplification, only the data for 25 μmol/ml are show in Table II and Figure 2.

Figure 2.
Figure 2.

AREG expression in HNSCC 11A, 14C and CERV196 after incubation with everolimus, sunitinib or sorafenib compared to the negative control. Data are mean values. Standard deviation is indicated.

Discussion

The formation of a vasculature is a crucial step for sustained tumor growth, progression, invasion and migration of tumor cells, which eventually leads to lymph node or distant metastasis. Tumor cells express a set of various angiogenic factors that facilitate the formation of new blood vessels. Therefore, it is reasonable to target pro-angiogenic factors in anticancer therapy in order to prevent or decelerate the aforementioned process. Riedel et al. demonstrated a correlation between the microvessel density in a tumor and the recurrence or metastasis of a tumor (53, 54). A set of angiogenic factors either released by the tumor or the stroma regulate the process of vascularization and neoangiogenesis (55, 56).

This is one of the first in vitro studies to investigate the impact of everolimus, sunitinib and sorafenib on the expression of AREG and mTOR in p16-positive and - negative SCC. Our results showed that both AREG and mTOR are expressed in all three cell lines. We found that the expression of AREG in CERV196 is considerably lower (reduced by almost 50%) when compared to HNSCC 11A and 14C. Tinhofer et al. reported that HNSCC patients with high expression levels of EGFR and AREG are less likely to benefit from a combination therapy with cetuximab and docetaxel (57). Thus, our findings support the hypothesis that HPV positivity in HNSCC is a rather favorable factor concerning prognosis and outcome for the patient. On the contrary, expression levels for mTOR were considerably higher in CERV196 when compared to HNSCC 11A and 14C, which implies increased activity of mTOR signaling path ways in CERV196.

Sunitinib and sorafenib suppressed AREG expression in CERV196 independently of the incubation time, which could lead to an anti-angiogenic effect and delay tumor progression and dissemination. Interestingly, sunitinib almost completely abolished AREG expression in an incubation time-dependent manner. In HNSCC 11A and 14C, only a slight reduction of AREG expression could be detected after incubation with everolimus. Surprisingly, we detected increased expression levels of AREG in HNSCC 11A and 14C after incubation with sunitinib and sorafenib irrespective of the incubation time. Sunitinib and sorafenib are inhibitors of VEGFR and PDGFR but not EGFR, the receptor of AREG. As previously mentioned, EGFR is over-expressed in 42-80% of HNSCC (18-21). An up-regulation of AREG expression following exposure to sunitinib and sorafenib could be explained as an evasive mechanism for VEGFR and PDGFR inhibition by an autocrine pro-angiogenic stimulus via AREG and EGFR. This is distinct from a mechanism of drug resistance because inhibition of the targets VEGFR and PDGFR is still possible but the anti-angiogenic potency of the multi-tyrosine kinase inhibitors is compromised. It is also a possible explanation why a monotherapy with sunitinib or sorafenib is not sufficient for HNSCC therapy and should be used in a combination therapy (47-51). Interestingly, p16-positive CERV196 did not increase AREG expression under sunitinib and sorafenib. Furthermore, expression levels of AREG in the chemonaïve control of CERV196 were remarkably lower when compared to HNSCC 11A and 14C. This could be evidence that p16-positive CERV196 has higher susceptibility towards a targeted anti-angiogenic therapy with sunitinib and sorafenib as already reported for PDGFR (58).

View this table:
Table II.

ELISA of AREG expression in HNSCC 11A, 14C and CERV196 after incubation with everolimus, sunitinib or sorafenib compared to the negative control (statistical significance is shown in bold).

As previously described, mTOR integrates the signaling cascades of several messengers, including EGFR, and induces angiogenesis via increased protein synthesis of, for instance, VEGF (25, 29, 30). mTOR inhibition has been shown to increase cetuximab sensitivity in EGFR-resistant HNSCC (59). Hirashima and colleagues reported that the mTOR pathway is aberrantly activated in many SCCs (60). We demonstrated that sunitinib and sorafenib significantly decrease mTOR expression apart from their main mechanism of action. Consequently, we have to state a pleiotropic effect of these two drugs. Sunitinib appeared to be more effective compared to sorafenib; however, this difference was not significant. Again, p16-positive CERV196 was more susceptible towards a reduction of mTOR compared to HNSCC 11A and 14C. Everolimus reduced mTOR expression in all cell lines. Surprisingly, the effect of everolimus in HNSCC 11A and CERV196 did not reach significance. Reduced expression of mTOR would lead to an impaired PI3K/Akt signaling pathway and increase the expression of the tumor suppressor proteins PTEN (phosphatase and tensin homolog deleted on chromosome 10) and p53 (27, 61). Patel and colleagues reported a decreased formation of lymphatic metastasis under mTOR inhibition in HNSCC (38).

As previously mentioned, the protein quotient (expression of the target value/total protein level) increased under incubation with sunitinib and sorafenib in an incubation time-dependent manner in HNSCC 11A and 14C but not in CERV196. Saito et al. reported that sunitinib induces apoptosis via a VEGFR2/Akt/mTOR pathway (62). The fact that CERV196 cells showed higher expression levels of mTOR even under incubation with sunitinib and sorafenib when compared to the chemonaïve control of HNSCC 11A and 14C might explain why a similar apoptotic effect of sunitinib and sorafenib is missing in CERV196. Interestingly, we found no signs of increased apoptosis after incubation with everolimus.

Our results confirm that a targeted monotherapy may lead to evasive mechanisms within cellular signaling. CERV196 was characterized by an increased susceptibility towards AREG suppression under everolimus, sorafenib and sunitinib. Further studies are mandatory to investigate HPV-dependent differences in therapy response and possible consequences for treatment regimens in the future.

Acknowledgements

The Authors would like to thank Petra Prohaska for her outstanding technical assistance and PD Dr. C. Weiss for her distinguished advice in statistical analysis.

  • Received December 14, 2014.
  • Revision received February 7, 2015.
  • Accepted February 10, 2015.

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Targeting mTOR and AREG with Everolimus, Sunitinib and Sorafenib in HPV-positive and -negative SCC
CHRISTOPH ADERHOLD, ANNE FABER, CLAUDIA UMBREIT, RICHARD BIRK, CHRISTEL WEISS, JÖRG ULRICH SOMMER, KARL HÖRMANN, JOHANNES DAVID SCHULTZ
Anticancer Research Apr 2015, 35 (4) 1951-1959;
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