Research ArticleExperimental Studies

Impact of Small Molecules on β-Catenin and E-Cadherin Expression in HPV16-positive and -negative Squamous Cell Carcinomas

BENEDIKT KRAMER, CLEMENS HOCK, JOHANNES DAVID SCHULTZ, ANNE LAMMERT, BEATRICE KUHLIN, RICHARD BIRK, KARL HÖRMANN and CHRISTOPH ADERHOLD
Anticancer Research June 2017, 37 (6) 2845-2852;

Abstract

Background: The validation of potential molecular targets in head and neck squamous cell carcinoma (SCC) is mandatory. β-Catenin and E-cadherin are crucial for cancer progression through epithelial–mesenchymal transition. We analyzed the effect of the tyrosine kinase inhibitors nilotinib, dasatinib, erlotinib and gefitinib on β-catenin and E-cadherin expression in SCC with respect to human papillomavirus (HPV) status. Materials and Methods: Expression of β-catenin and E-cadherin in cell lines UMSCC 11A, UMSCC 14C and CERV196 under the influence of tyrosine kinase inhibitors were analyzed by enzyme-linked immunosorbent assay. Results: All agents reduced β-catenin and E-cadherin expression of HPV16-negative cells. Increased E-cadherin expression was observed after treatment with gefitinib and dasatinib in HPV16-positive cells. Conclusion: All substances, nilotinib, dasatinib, erlotinib and gefitinib have a significant impact on β-catenin and E-cadherin expression in both HPV16-positive and HPV16-negative cells in vitro. Alterations of β-catenin and E-cadherin could provide novel insights for future targeted therapies of head and neck SCC.

Squamous cell carcinoma (SCC) of the head and neck (HNSCC) is the sixth most common cancer type and the seventh most frequent cause of cancer-associated death worldwide, with annual incidence of more than 680,000 cases and mortality rate of 375,000 (1). Despite ongoing improvement of diagnostics and therapy within the past decades, the prognosis for patients remains poor, with an overall 5-year survival of approximately 50% (2). Especially for advanced-stage HNSCC, therapeutic options supplementary to surgery and radiation, as well as chemotherapy, are limited (3).

Two different types of different HNSCC can be distinguished, based on the status of human papillomavirus (HPV) infection. Diverse risk profiles, molecular causes and survival prognoses have been described (4). Over 150 different types of HPV have been discovered, but more than 82% of HNSCCs and 58% of cervical uterine cancer tested positively for HPV were infected with high-risk type 16 (5, 6). The overexpression of viral oncogenes E6 and E7 results from an increase of viral deoxyribonucleic acid (DNA) integration into the host's genome, with subsequent stimulation of cell proliferation (7). In contrast to the decreasing incidence of HPV-negative HNSCC of the larynx, the prevalence of HPV positive HNSCC of the oropharynx is rising (8). The clinical appearance of HPV-related HNSCC is grave as it initially presents with a high rate of lymph nodal metastases at an early T-stage (9). However, HPV16-positive HNSCC is associated with a better response to platinum-based chemotherapy and radiation. HPV-related HNSCC is also associated with a better overall prognosis (4, 8).

A crucial factor in carcinogenesis is the loss of cell adhesion, with subsequent increased cell motility. This so called epithelial–mesenchymal transition (EMT) enables cancer cells to invade tissues and metastasize (10). A loss of function of E-cadherin is characteristic of EMT and results in the dissociation of the stabilizing E-cadherin–β-catenin–α-catenin-complex (11). Furthermore, cleavage products of E-cadherin and β-catenin influence several signaling pathways, such as the epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) pathways, which are involved in the regulation of the cell cycle (11, 12). Intracellular β-catenin connects E-cadherin indirectly to the actin cytoskeleton (13). Thus, it is of essential importance between intracellular and extracellular communication. Moreover, β-catenin is an important factor of the canonical Wingless-related integration site (WNT) pathway (14). If the WNT pathway is inactive, β-catenin is bound by a complex consisting of adenomatous polyposis coli (APC), axis inhibition protein (axin) and glycogen synthase kinase 3. This complex induces phosphorylation, ubiquitination and degradation of β-catenin. The WNT pathway is activated by WNT ligands, a poorly characterized family of pleiotrophic, cysteine-rich, and lipid-modified glycoproteins, binding to the so called frizzled-receptor. Subsequently, the degradation complex is blocked by dishevelled, which binds axin. β-Catenin then accumulates and is translocated into the cell nucleus. After conjugation with the DNA-binding proteins lymphoid enhancer factor/T-cell factor, it induces DNA transcription (14, 15). Consequently, target genes such as oncoprotein c-myelocytomatosis (c-MYC) and cyclin D1 gene (CCND1) stimulate cell proliferation (15). Deregulation of the dual function of β-catenin leads to enhanced cell proliferation and attenuated cell adhesion. This mechanism is a critical factor for cancer development. Inactivating mutations of APC have been reported in up to 85% of sporadic colorectal carcinomas (16).

E-Cadherin is an extracellular calcium-dependent glycoprotein that facilitates the extracellular connection between squamous cells. Through its transmembrane domain it connects indirectly with β-catenin and several components of the cytoskeleton (17). Loss of E-cadherin-mediated cell adhesion correlates with an accumulation of unbound β-catenin and enhanced β-catenin-dependent transcription of several proliferation factors (18). Down-regulation of E-cadherin expression is achieved by activated catenin (19). Soluble E-cadherin is a (pro-)oncogene and activates receptors of the EGFR family in SCC. Activation of EGFR-induced signaling pathways such as the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway or the phosphatidylinositol-3-kinase/protein kinase B pathway (PI3K/AKT) can induce cell proliferation and inhibit apoptosis (20-24). The activation of EGFR additionally enhances the activity of matrix metalloproteases, which subsequently can cleave E-cadherin, resulting in a positive feedback loop with a continuous stimulation of cell proliferation and invasion (18).

The activity of tyrosine kinases is essential for intracellular signaling and plays an important role in cancer development. The cause of chronic myeloid leukemia (CML) is a chromosomal translocation between chromosomes 9 and 22, called the Philadelphia chromosome, which leads to activation of a specific tyrosine kinase, enhanced cell proliferation and inhibition of apoptosis (25). Therapeutic approaches to inhibit this tyrosine kinase activity have been established in the treatment of CML. Small molecules can act as selective tyrosine kinase inhibitors by competitive binding of the ATP-binding site, which results in inactivation of enzymatic activity (26). Small-molecule inhibition of tyrosine kinases is approved for effective targeted therapy in several cancer entities other than HNSCC (27). Dasatinib and nilotinib inhibit multiple tyrosine kinases, such as those active or inactive in CML, platelet-derived growth factor β receptor, ephrin receptor kinases and mast/stem cell growth factor receptor. Dasatinib also inhibits sarcoma tyrosine kinase (SRC) family kinases and was the first orally available alternative to imatinib in the therapy of CML and acute lymphoid leukemia (25, 28, 29). Interactions of SRC and WNT-dependent signaling have been described and consequently, the expression levels of β-catenin and E-cadherin may possibly be altered by these signaling pathways (30).

Erlotinib and gefitinib are reversible tyrosine kinase inhibitors of intracellular EGFR autophosphorylation and are approved for the therapy of non-small cell lung cancer, erlotinib can also be used in therapy of metastatic pancreatic cancer (31, 32). Additionally, transforming growth factor-α, vascular endothelial growth factor, basic fibroblast growth factor and interleukin 8 stimulate the migration, proliferation, and functional differentiation of endothelial cells and can be inhibited by gefitinib and erlotinib (33). This study was focused on the interaction of EGFR to WNT and on the interaction between soluble E-cadherin and EGFR under the influence of small molecule inhibition (17, 34).

Regarding the molecular mechanisms of these four tyrosine kinase inhibitors and their effective results for other tumor entities, a promising impact on HNSCC might be possible (34). To our knowledge, this is the first study to evaluate alteration of expression levels of the cell adhesion factors β-catenin and E-cadherin under treatment of tyrosine kinase inhibitors nilotinib, dasatinib, erlotinib and gefitinib in HPV16-positive and -negative SCC cells in vitro.

Materials and Methods

Cell lines, drugs and study design. HPV-negative University of Michigan Squamous Cell Carcinoma (UMSCC) cell lines were kindly provided by T.E. Carey, Ph.D., University of Michigan, Ann Arbor, USA: UMSCC-11A originated from a primary SCC of human epiglottis; UMSCC 14C originated from a human skin metastasis of a floor of mouth SCC after surgery, radiation and chemotherapy. HPV16-positive CERV196 cell line was acquired from Cell Lines Service GmbH (Eppelheim, Germany) and derived from SCC cells of the uterine cervix.

For UMSCC cell culture, Eagle's minimum essential medium (Gibco, Life Technologies, Carlsbad, CA, USA) was used, with addition of 2 mM of L-glutamine and 10% fetal calf serum (Gibco, Life Technologies). Antibiotics and antimycotics were supplemented according to the manufacturer's instructions (Pen-Strep; Gibco, Life Technologies). CERV196 tumor cells were cultured in Eagle's minimum essential medium (Gibco, Life Technologies), supplemented with 2 mM L-glutamine, 1.0 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 g/l sodium pyruvate and 10% of fetal bovine serum (Gibco, Life Technologies). The cells were incubated under standardized conditions at 37°C, 5% CO2 and 95% humidity. For further use, subcultures of the cells were generated. Therefore, we used 0.05% trypsin/0.02% EDTA solution (Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes at 37°C.

Nilotinib, dasatinib, gefitinib and erlotinib were kindly provided by Professor Dr. Hofheinz, Oncological Department, University Medical Centre Mannheim, Medical Faculty Mannheim, University of Heidelberg, Germany. The drugs were stored at room temperature and dissolved in dimethylsulfoxide for further use. 96-Well microtiter plates were used for the cell proliferation assay. The four agents were added to the cell cultures at 5, 10 and 20 μmol/l. Cell cultures were incubated for 24, 48, 72 and 96 h and compared to the untreated cells (control). Each experiment was independently repeated at least three times (n=3).

Enzyme-linked immunosorbent assay (ELISA) for E-cadherin and β-catenin. The determination of protein concentrations was accomplished using the ELISA technique. Each ELISA was performed according to the manufacturer's directions. Sandwich ELISA was used for a quantitative measurement of E-cadherin and β-catenin. DuoSet ELISA development kits (R&D Systems, Wiesbaden, Germany) for both of the target proteins were used (DYC1329 for β-catenin and DY648 for E-cadherin. MRX Microplate Reader (DYNEX Technologies, Chantilly, VA, USA) at a wavelength of 450 nm with a wavelength correction of 540 nm was used to measure the optical density. The concentrations are reported as pg/ml. The detection range was 187.00-12000 pg/ml for E-cadherin and 312.00-20,000 pg/ml for β-catenin. Interassay coefficient of variation reported by the manufacturer was below 10%.

Statistical analysis. The statistical analysis was performed using the means of each experiment. Multiple-coefficient variance test with the following determining factors was used: time of incubation, cell line, applied drug and concentration of applied drug (including the control). The concentration of the applied drug had no statistical relevance in this initial analysis. Therefore, a drug concentration of 20 μM was used to perform all subsequent experiments. The resulting p-values were adjusted with Dunnett's test in order to evaluate statistical significance (Version 9.3 SAS/STAT of SAS Institute, Inc., Cary, NC, USA). For all analyses, p≤0.05 was considered to be statistically significant. Statistical analysis was performed in cooperation with Professor Dr. C. Weiss, Institute of Biomathematics, Faculty of Medicine Mannheim, University of Heidelberg, Germany.

Results

β-Catenin expression. β-Catenin was detected in all three tested cell lines. Expression of β-catenin increased time-dependently in the untreated UMSCC 11A cells. In contrast, after exposure to any of the tested drugs, the level of β-catenin decreased statistically significantly (p<0.029), with one exception, for gefitinib after 48 h. The expression of β-catenin in UMSCC 14C cells was comparable to those of UMSCC 11A. β-Catenin expression increased time-dependently in untreated cells, with an exception after 48 h. β-Catenin expression in UMSCC 14C was suppressed by all of the agents tested. Interestingly, the expression of β-catenin increased time-dependently after exposure to nilotinib and dasatinib up to 72 h in contest to the untreated control UMSCC 14C cells. However, after 96 h of incubation with these agent, the level of protein expression of β-catenin in UMSCC 14C cells was lower compared to that after 72 h. Only gefitinib obtained a statistically significant decrease of β-catenin for all the tested time periods (p<0.048). β-Catenin was significantly reduced by erlotinib after 48 (p=0.048), 72 (p<0.001) and 96 (p<0.001) h. Dasatinib significantly reduced β-catenin after 72 (p=0.032) and 96 (p<0.001) h. For nilotinib, a statistically significant reduction of β-catenin protein expression was observed after 24 (p<0.001) and 96 (p=0.019) h.

In control HPV16-positive CERV196 cells, the level of β-catenin expression was higher than in corresponding HPV16-negative UMSCC tumor cells for all of the tested time periods. The level of β-catenin expression increased time dependently under the influence of dasatinib and nilotinib, whereas such an expression pattern was not observed in erlotinib- or gefitinib-treated cells. We recorded a statistically relevant suppression of β-catenin expression in CERV196 cells by all of the tested drugs. Nilotinib and erlotinib significantly reduced β-catenin after 48-96 h (p<0.001). Under the influence of dasatinib and gefitinib, β-catenin expression levels significantly decreased after all tested time periods (p<0.006). For simplification, only the data for a drug concentration of 20 μmol/l are shown in Table I.

E-Cadherin expression. E-Cadherin expression was observed in all three cell lines tested. The levels of expression were the lowest in HPV16-negative UMSCC 11A cells. In UMSCC 14C cells and HPV16-positive CERV196 cells, elevated E-cadherin expression was observed. Expression of E-cadherin increased time dependently for the control for all of the tested cell lines, whereas a time-dependent fluctuation of protein expression was seen under the influence of the tested tyrosine kinase inhibitors. A statistically significant decrease of E-cadherin protein expression was detected for all of the agents for all three cell lines.

In UMSCC 11A cells, nilotinib, dasatinib and erlotinib reduced E-cadherin expression statistically significantly only after 72 h (p<0.001 for erlotinib and nilotinib, p=0.001 for dasatinib). For gefitinib, a statistically relevant suppression of E-cadherin was seen after 24 (p=0.048) and 72 (p=0.002) hours. E-Cadherin protein levels were significantly reduced in UMSCC 14C cells by nilotinib, dasatinib, erlotinib and gefitinib after all of the tested time periods (p<0.012).

The expression patterns of E-cadherin in CERV196 cells fluctuated under the influence the tyrosine kinase inhibitors. After exposure to nilotinib, a statistically significant reduction of E-cadherin was observed after 48-96 h (p<0.001). Dasatinib significantly reduced E-cadherin expression only after 24 (p=0.017) h. Interestingly, a statistically significant increase of E-cadherin expression was detected in CERV196 cells after treatment with dasatinib after 48 (p=0.002) hours of incubation. An increase of E-cadherin expression was also seen after 72 h of dasatinib exposure, although this was not statistically significant. Erlotinib significantly reduced the expression of E-cadherin in CERV196 cells after 48 (p<0.001) and 96 (p<0.001) h. Surprisingly, an increase of E-cadherin protein was observed after 24 and 72 h of exposure to erlotinib, however, without statistical significance. A statistically significant decrease of E-cadherin expression in gefitinib-treated CERV196 cells was observed after 24 (p=0.016) and 96 (p<0.001) h of incubation. As we reported above for erlotinib in CERV196 cells, the level of E-cadherin expression was also statistically significantly increased after 48 h of gefitinib treatment (p=0.002). An increase of gefitinib-mediated E-cadherin expression was also observed after 72 h, although not statistically significant. For simplification, only the data for drug concentration of 20 μmol/l are shown in Table II.

Discussion

The present study was designed to determine the expression patterns of β-catenin and E-cadherin in HPV16-positive and negative SCC under the influence of small molecule tyrosine kinase inhibitors nilotinib, dasatinib, erlotinib and gefitinib. E-Cadherin and β-catenin are crucial factors in carcinogenesis by regulating cell–cell communication (35). The impact of targeted anticancer therapy which is well-established in other tumor entities could provide important information for further therapeutic approaches in HNSCC. The progression of cancer cells in solid tumors is highly associated with EMT (36, 37). Intracellular β-catenin stabilizes cell–cell communication as a significant part of the canonical WNT pathway (14). Dysregulated β-catenin triggers tumorigenesis (15). During EMT in cancer cells, the interaction of β-catenin and E-cadherin is impaired (19).

Expression of β-catenin was observed for all three cell lines tested and enhanced in HPV16-postive CERV196 cells. This observation is interesting as it indicates an increased activity of β-catenin-dependent signaling in HPV16-positive tumor cells. The effect of the tested small-molecule inhibitors on β-catenin expression was statistically relevant for all tested cell lines. Interestingly, reduction of β-catenin expression was considerably more effective in UMSCC 11A than UMSCC 14C although both types of cells originate from HPV16- negative HNSCC. One possible explanation for this could be that UMSCC 11A originated from a primary tumor of the epiglottis and might therefore have a better response to selective tyrosine kinase inhibition than UMSCC 14C which was derived from metastatic tissue.

None of the tested agents act as direct inhibitors of β-catenin but the complex process of cancer development involves the interaction of several signaling pathways as has been shown for WNT and EGFR signaling (32). Erlotinib and gefitinib act through direct inhibition of EGFR (26). The association of EGFR expression and HPV16-positive tumor cells is well described for HNSCC (38, 39). Both WNT and EGFR signaling are strongly related to tumorigenesis as EGFR-mediated PI3K/AKT activation promotes β-catenin transactivation and tumor cell invasion (34). Our results confirm the findings of Umbreit and colleagues, who showed distinct up-regulation of β-catenin after EGF/transforming growth factor β1 co-stimulation in SCC, and gefitinib-mediated inhibition of β-catenin (40, 41). Consequently, all EGFR-inhibiting agents tested here might reduce β-catenin activity and promote the degradation of inactive β-catenin. Dasatinib-induced down-regulation of EGFR expression has been described in various tumor entities, including HNSCC (28, 32). Phosphorylation of β-catenin can be achieved by SRC with a subsequent disruption of β-catenin–cadherin binding (42). This mechanism results in a loss of cadherin mediated cell–cell adhesion and an increase in the level of cytoplasmic β-catenin (43). Thus, the inhibition of SRC family kinases is a possible mechanism for reduced β-catenin expression following dasatinib treatment.

As mentioned above, the HPV16-positive CERV196 cells had significantly higher β-catenin protein levels than did the HPV16-negative cell lines tested. The expression of β-catenin in CERV196 was approximately 2.2-fold higher than in UMSCC 11A and UMSCC 14C cells. A crucial mechanism for HPV16-related malignant cell transformation is the up-regulation of oncoproteins E6 and E7, which can inactivate tumor-suppressor proteins such as p53 and retinoblastoma (4). Growing evidence suggests that various cell signaling pathways can be modulated by these oncoproteins and therefore contribute to carcinogenesis (9). Both E6 and E7 expression were confirmed to up-regulate β-catenin expression in HNSCC, while the exact molecular mechanisms are yet unknown (44). Interestingly, HPV-associated oncoproteins E6 and E7 have been shown to be regulated by the level of nuclear-translocated β-catenin (45). EGFR and other cell signaling-modulating protein kinases are involved in the translocation of membrane-bound β-catenin to the nucleus (46, 47). One possible explanation for increased β-catenin protein levels in HPV16-positive SCC cells could therefore be EGFR-mediated regulation of nuclear translocation of β-catenin by HPV-related oncogenes (48). The results of our study, with increased β-catenin expression in HPV16-positive tumor cells support this hypothesis but further research is necessary to determine the exact mechanism of HPV16-mediated regulation of various transcription factors, including β-catenin.

View this table:
Table I.

β-Catenin expression as determined by enzyme-linked immunosorbent assay in HPV16-negative UMSCC 11A and 14C squamous cell carcinoma (SCC) of the head and neck, and HPV16-positive CERV196 SCC cells after incubation with nilotinib, dasatinib, erlotinib or gefitinib compared to untreated control cells. Data are mean values±standard deviation (pg/ml). Statistically significant differences (p<0.05) are shown in bold.

View this table:
Table II.

E-Cadherin expression as determined by enzyme-linked immunosorbent assay in HPV16-negative UMSCC 11A and 14C squamous cell carcinoma (SCC) of the head and neck, and HPV16-positive CERV196 SCC cells after incubation with nilotinib, dasatinib, erlotinib or gefitinib compared to untreated control cells. Data are mean values±standard deviation (pg/ml). Statistically significant differences (p<0.05) are shown in bold.

In non-small cell lung cancer cells, β-catenin overexpression has also been associated with increased chemoresistance to gefitinib by affecting the activation of EGFR and downstream intracellular signaling (49). In our study, we found the highest β-catenin concentration under gefitinib treatment in HPV16-positive CERV196 cells with an overall time-dependent increase after 96 hours but β-catenin expression was still significantly lower than that of the control cells. The up-regulation of β-catenin by EGFR-inhibiting tyrosine kinase proteins could also be interpreted as a possible evasive mechanism from EGFR inhibition in order to protect the cell from apoptosis. This effect can be induced through an up-regulation of WNT/β-catenin signaling by viral oncoproteins (50). Increased β-catenin expression could increase the integrity of the cell and therefore reduce its susceptibility for extrinsic influences such as selective EGFR inhibition.

In summary, β-catenin-associated signaling is involved in SCC carcinogenesis and all the tyrosine kinase inhibitors tested here were found to have a negative impact on its expression in both HPV16-positive and -negative SCC cells. Intervention in β-catenin-associated signaling might stabilize cell–cell adhesion and contribute to reduced tumor progression and formation of metastases. For future therapeutic approaches in SCC, additional in vitro and in vivo research will be mandatory.

E-Cadherin expression. Intercellular adhesion between squamous cells is also mediated through various transmembrane proteins such as E-cadherin (15). Down-regulation of E-cadherin expression leads to a separation of cells from the primary cell mass (16). Loss of E-cadherin is associated with a worse prognosis in patients with cancer, through enhanced invasiveness and enhanced metastatic spread through EMT (11, 17, 51). As mentioned above, β-catenin is anchored by E-cadherin to the cell membrane as part of a transmembrane complex which prevents β-catenin from entering the nucleus and inducing the expression of EMT-related transcription factors (15). Consequently, loss of E-cadherin promotes WNT-dependent signaling (18). However, not all invasive tumors are characterized by reduced E-cadherin expression (52, 53). This paradox might be explained by the fact that higher E-cadherin levels enable further production of oncogenically-active fragments (e.g. soluble E-cadherin) (17).

The level of E-cadherin expression was significantly reduced by all the tested tyrosine kinase inhibitors in both, HPV16-positive and -negative cell lines in comparison with control cells, dependent on the time of incubation. Of note, we observed markedly higher levels of E-cadherin expression in CERV196 cells at certain points of time under the influence of all of the small molecules tested. None of the applied agents acts a direct stimulator or inhibitor of E-cadherin. However, interaction of cell signaling pathways aIso affects E-cadherin via tyrosine kinase inhibition. EGFR inhibition in particular has an impact on E-cadherin level (54). An inverse correlation between EGFR activation and loss of cell–cell adhesion with a subsequent decrease of E-cadherin expression has been described (55). The interaction is presumed to be mediated by the Rho family of small-GTPases, RhoA and by merlin protein (54, 55). Hence, the reduction of E-cadherin expression under the influence of EGFR inhibitors erlotinib and gefitinib could be explained by this mechanism. Lower E-cadherin level can be explained by a reduced response to EGFR inhibition (56). This might be a possible mechanism for an increase of the E-cadherin concentration in a time-dependent course in erlotinib- or gefitinib-treated cells. Moreover, a positive feedback loop with subsequent EGFR activation through soluble E-cadherin and following E-cadherin cleavage has been reported (18). A possible mechanism of selective EGFR inhibition could therefore result in an increase of E-cadherin expression by inhibiting the degradation of E-cadherin. Another signaling inter-communication with E-cadherin has been reported for SRC. Activation of SRC kinases induces a switch from cadherin to integrin expression, resulting in reduced cell–cell adhesion (54). E-Cadherin concentration can therefore possibly be altered through SRC-mediated signaling by dasatinib.

Possible interactions between HPV and E-cadherin have been described (57). HPV16-related oncoprotein E7 can reduce E-cadherin expression through epigenetic repression (39-41). In our study, we found markedly higher concentrations of E-cadherin in HPV16-positive cells compared to HPV16-negative cells. Nilotinib-treated SCC cells had a markedly lower expression of E-cadherin compared to their controls. This observation contrasts with the findings of Laurson and colleagues, who found reduced levels of E-cadherin in HPV-infected cells (41). Lefevre and co-workers indicate that EMT is not necessarily associated with the status of HPV infection in oral SCC and that β-catenin and E-cadherin are not reliable prognostic markers regarding the outcome of HPV16-related SCC (58). This hypothesis contrasts with our observations which indicate that EMT-regulating proteins, such as β-catenin and E-cadherin, can be altered depending on the status of HPV infection and therefore possibly influence the outcome of HPV-related SCC. A possible explanation for this discrepancy could be due to difficulty in comparability because of i) methodical differences regarding the inclusion criteria for cancer types, and ii) non homogeneous methods of approaching outcome measures (59).

In conclusion, we observed significant alterations of β-catenin and E-cadherin expression under the influence of the small-molecule tyrosine kinase inhibitors erlotinib, gefitinib, dasatinib and nilotinib in both HPV16-positive and -negative SCC. β-Catenin and E-cadherin concentrations were significantly reduced by all the tested agents. Regarding EMT as a crucial factor for tumor progression and the formation of metastases, such targeted therapy could possibly contribute to improving the outcome of patients with SCC.

Acknowledgements

The Authors thank Petra Prohaska, Mannheim, Germany, for technical support and Professor Dr. C. Weiss, Institute of Biomathematics, Faculty of Medicine, Mannheim, Germany, for advice on statistical analysis.

Footnotes

  • This article is freely accessible online.

  • Received April 18, 2017.
  • Revision received May 4, 2017.
  • Accepted May 9, 2017.

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Impact of Small Molecules on β-Catenin and E-Cadherin Expression in HPV16-positive and -negative Squamous Cell Carcinomas
BENEDIKT KRAMER, CLEMENS HOCK, JOHANNES DAVID SCHULTZ, ANNE LAMMERT, BEATRICE KUHLIN, RICHARD BIRK, KARL HÖRMANN, CHRISTOPH ADERHOLD
Anticancer Research Jun 2017, 37 (6) 2845-2852;
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