The human HECA interacts with cyclins and CDKs to antagonize Wnt-mediated proliferation and chemoresistance of head and neck cancer cells.

There is a growing evidence that the human homologue of the Drosophila headcase (HECA) plays an important role in human carcinogenesis. So far specific protein interaction partners and affected signaling pathways of HECA are still elusive. In a recent study we showed that HECA overexpression in oral squamous-cell carcinoma (OSCC) keratinocytes has tumor suppressive effects resulting in a recuperation of cell cycle control concerning the entry and progression of S-phase, G2- and M-phase. Currently, quantitative RT-PCR and immunohistochemical analysis of primary tumor tissue from OSCC patients demonstrate that HECA expression is markedly decreased compared to normal control patients with abundant HECA expression. Additionally, there is nearly no HECA expression in OSCC metastases. Here, we show that HECA expression is negatively controlled by the Wnt-pathway and TCF4, a Wnt related transcription factor, binds to the HECA promoter. Furthermore, immunocytochemistry reveals colocalization of HECA with the cyclin dependent kinase CDK9. Immunoprecipitation experiments and proximity ligation assays further reveal an interaction of HECA with CDK2, CDK9, Cyclin A and Cyclin K, a direct transcriptional target of the p53 tumor suppressor. Silencing HECA in OSCC cell lines leads to a significant increase of cell division and a markedly increased resistance against the chemotherapeutic cisplatin. On the contrary, HECA overexpressing OSCC cell lines show decreased resistance of OSCC cells against cisplatin. Therefore, HECA could be considered as future therapeutic agent against Wnt-dependent tumor progression.

Slit-2 facilitates interaction of P-cadherin with Robo-3 and inhibits cell migration in an oral squamous cell carcinoma cell line.

Slits are a group of secreted glycoproteins that act as molecular guidance cues in cellular migration. Recently, several studies demonstrated that Slit-2 can operate as candidate tumour suppressor protein in various tissues. In this study, we show Slit-2 expression in basal cell layers of normal oral mucosa colocalized with P-cadherin expression. In contrast, there is a loss of Slit-2 and P-cadherin expression in mucosa of oral squamous cell carcinoma (OSCC). Our in vitro investigations reveal a correlation of P-cadherin and Slit-2 expression: OSCC cells with induced P-cadherin expression (PCI52_PC) display an increased Slit-2 expression. However, abrogating P-cadherin function with a function-blocking antibody decreases Slit-2 secretion confirming a direct link between P-cadherin and Slit-2. Moreover, experiments with OSCC cells show that Slit-2 interferes with a Wnt related signalling pathway, which in turn affects Slit-2 expression in a feedback loop. Functionally, transwell migration assays demonstrate a Slit-2 dose-dependent decrease of PCI52_PC cell migration. However, there is no influence on migration in mock control cells. Responsible for this migration block might be an interaction of P-cadherin with Roundabout (Robo)-3, a high affinity receptor of Slit-2. Indeed, proximity ligation assays exhibit P-cadherin/Robo-3 interactions on PCI52_PC cells. Additionally, we detect a modulation of this interaction by addition of recombinant Slit-2. Down-regulation of Robo-3 expression via small interfering RNA neutralizes Slit-2 induced migration block in PCI52_PC cells. In summary, our experiments show antitumorigenic effects of Slit-2 on P-cadherin expressing OSCC cells supposedly via modulation of Robo-3 interaction.

Induction of type XVI collagen expression facilitates proliferation of oral cancer cells.

Type XVI collagen belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). Recently, high affinity to integrin alpha1beta1 has been shown allowing cells expressing those integrins to attach and spread on recombinant type XVI collagen. Here, we show that type XVI collagen is overexpressed in dysplastic areas of mucosal epithelium from oral squamous cell carcinoma (OSCC) patients. Induction of its expression in OSCC cell lines (COLXVI cells) leads to an increased expression of Kindlin-1. Moreover, we demonstrate a significantly increased Kindlin-1/beta1-integrin interaction. Additionally, we detected a higher number of activated beta1-integrins in COLXVI cells and found a neo-expression of alpha1 integrin subunit on these cells. FACS analysis revealed a significantly higher amount of COLXVI cells in S-phase and G2/M-phase 6h after synchronisation leading to a markedly higher proliferation activity. Blocking beta1-integrins with a specific antibody resulted in reduced proliferation of COLXVI cells. In summary, we demonstrate that overexpression of type XVI collagen in aberrant oral keratinocytes leads to Kindlin-1 induction, increased Kindlin-1/beta1-integrin interaction, integrin activation and subsequently to a proliferative cellular phenotype.

Impact of MAGE-A antigens on taxane response in oral squamous cell carcinoma.

MAGE-A antigens are a subgroup of cancer/testis antigens that are exclusively expressed in malignant cells. Only scarce information on the function of MAGE-A antigens is available. There is some evidence that they may influence the response to chemotherapeutic drugs. This study aimed to evaluate the impact of the MAGE-A antigen subgroups MAGE-A2, -A3, -A4 and -A6 on oral squamous cell carcinoma cell lines treated with docetaxel and paclitaxel. Five oral squamous cell carcinoma cell lines were characterized for their quantitative expression of MAGE-A2, -A3, -A4 and -A6. The cell lines were treated with concentrations ranging from 0.025 to 0.8 µM of docetaxel and paclitaxel. The amount of viable cells after 24 and 48 h was measured. The measurements were statistically correlated with MAGE-A expression. All cell lines responded to docetaxel and paclitaxel. One cell line showed a statistically significant weaker response to the taxane treatment. This cell line was the only one that expressed MAGE-A4. MAGE-A4 has a statistically significant impact on the tumour response to docetaxel and paclitaxel in oral squamous cell carcinoma. This may influence treatment options and the course of the disease. Therefore, patients should be evaluated for MAGE-A4 expression before treatment with taxanes.

MAGE-A antigens in patients with primary oral squamous cell carcinoma.

MAGE-A antigens are only expressed on tumor cells. The aim of this study was to identify their expression in patients with oral squamous cell carcinoma (OSCC). Forty-seven patients with primary OSCC was selected retrospectively. Histo-pathological sections were stained immunohistochemically with MAGE-A antibody 57B. The results were evaluated regarding tumor size (T), lymph-node metastasis (N), blood vessel infiltration (V), lymph vessel infiltration (L), grading (G), and sex. MAGE-A antigens were expressed in 55% of all patients. Expression increased with tumor size (T1 = 56%; T2 = 44%; T3 = 67%; T4 = 71%). Lymph-node metastasis had no influence (N0 and N1 about 50%). Tumors with blood and lymph vessel infiltration had higher expression (V0 = 50%; V1 = 100%; L0 = 46%; L1 = 71%). Less-differentiated tumors showed higher rates (G1 = 50%; G2 = 45%; G3 = 83%). OSCC in men were positive in 62% and in women in 38%. MAGE-A antigens are frequently expressed in OSCC. Their expression seems to increase with tumor dedifferentiation.

The human homolog of the Drosophila headcase protein slows down cell division of head and neck cancer cells.

The human homolog of the Drosophila headcase (HECA) belongs to a new class of cell differentiation regulators. In Drosophila, the HECA protein regulates the proliferation and differentiation of cells during adult morphogenesis. There is growing evidence that HECA plays an important role in human carcinogenesis. In different tumor entities, an altered HECA expression was found (colorectal, pancreatic and renal cancer). Colorectal cancer studies also suggested HECA as a marker for early disease stages. Therefore, we speculated whether human HECA affects cell cycle progression and proliferation in head and neck cancer cells. In vivo, we found a distinct HECA protein expression in basal and superficial cells of a healthy oral epithelium via immunohistochemistry, whereas in tissues of oral squamous cell carcinoma (OSCC), a weaker staining was observed, particularly in basal cells. In vitro, mRNA and protein expression analyses of OSCC cell lines exhibited that HECA expression correlates with the state of cellular differentiation. In further investigations, we overexpressed HECA in the OSCC cell line PCI 13 and performed functional assays. HECA-overexpressing OSCC cells revealed a significant extended doubling time (up to 45%, 17 h) and yielded a lower number of proliferating cells (up to 30%) than controls. Flow cytometry analyses have shown that HECA-overexpressing OSCC cells forced to hold in the G(2)/M-Phase. In summary, our results show that human HECA slows down cell division of OSCC cells and may therefore act as a tumor suppressor in head and neck cancer.

P-cadherin induces an epithelial-like phenotype in oral squamous cell carcinoma by GSK-3beta-mediated Snail phosphorylation.

Cadherins belong to a family of Ca(2+)-dependent homophilic cell-cell adhesion proteins that are important for correct cellular localization and tissue integrity. They play a major role in the development and homeostasis of epithelial architecture. Recently, it has become more and more evident that P-cadherin contributes to the oncogenesis of many tumors. To analyze the role of P-cadherin in oral squamous cell carcinoma (OSCC), we used a cell line that was deficient of the classical cadherins, P-cadherin, E-cadherin and N-cadherin. This cell line was transfected with full-length P-cadherin (PCI52_PC). After overexpression of P-cadherin, PCI52_PC gained an epithelial-like brickstone morphology in contrast to the mock-transfected cells with a spindle-shaped mesenchymal morphology. Immunohistochemical analysis revealed a strong nuclear Snail staining in mock-transfected cells compared with a significantly reduced nuclear staining and translocation to the cytoplasm in P-cadherin-overexpressing cells. Interestingly, the effects triggered by P-cadherin overexpression could be reversed by transfecting the cells with an antisense P-cadherin plasmid construct. Additional investigations showed a reexpression of E-cadherin in all P-cadherin-transfected cell clones in contrast to the mock controls. Analyzing the signaling mechanism behind it, we found glycogen-synthase-kinase-3beta (GSK-3beta) bound to Snail in all cell clones. Furthermore, P-cadherin-overexpressing cell lines showed activated GSK-3beta that phosphorylated Snail leading to its cytoplasmic translocation. In summary, our results reveal P-cadherin as one major component in reconfiguring mesenchymal cells with epithelial features by triggering GSK-3beta-mediated inactivation and cytoplasmatic translocation of Snail in OSCC.

Analysis of expression profiles of MAGE-A antigens in oral squamous cell carcinoma cell lines.

BACKGROUND: The immunological response to solid tumours is insufficient. Therefore, tumour specific antigens have been explored to facilitate the activation of the immune system. The cancer/testis antigen class of MAGE-A antigens is a possible target for vaccination. Their differential expression profiles also modulate the course of the cancer disease and its response to antineoplastic drugs. METHODS: The expression profiles of MAGE-A2, -A3, -A4, -A6 and -A10 in five own oral squamous cell carcinoma cell lines were characterised by rt-PCR, qrt-PCR and immunocytochemistry with a global MAGE-A antibody (57B) and compared with those of an adult keratinocyte cell line (NHEK). RESULTS: All tumour cell lines expressed MAGE-A antigens. The antigens were expressed in groups with different preferences. The predominant antigens expressed were MAGE-A2, -A3 and -A6. MAGE-A10 was not expressed in the cell lines tested. The MAGE-A gene products detected in the adult keratinocyte cell line NHEK were used as a reference. CONCLUSION: MAGE-A antigens are expressed in oral squamous cell carcinomas. The expression profiles measured facilitate distinct examinations in forthcoming studies on responses to antineoplastic drugs or radiation therapy. MAGE-A antigens are still an interesting aim for immunotherapy.

Truncated P-cadherin is produced in oral squamous cell carcinoma.

Cadherins belong to a family of homophilic cell-cell adhesion proteins that are responsible for the establishment of a precise cell architecture and tissue integrity. Moreover, experimental data suggest that loss of intercellular adhesion is inversely correlated with cellular differentiation. Furthermore, dedifferentiation is closely linked to tumor progression. Recently, we have shown that a secreted 50 kDa N-terminal fragment of P-cadherin plays a role in the progression of malignant melanoma. In this study, we have detected both the full-length and the truncated versions of P-cadherin in cell lysates of differentiated head and neck oral squamous cell carcinoma cell lines, whereas in cell lysates of dedifferentiated cell lines, we detected only the truncated 50 kDa version of P-cadherin. Treatment of the cell lines with a recombinantly expressed biotinylated, soluble 50 kDa form of the N-terminal part of P-cadherin revealed a major effect on cell aggregation and migration of oral squamous cell carcinoma cells. However, the 50 kDa N-terminal fragment of P-cadherin did not show any influence on cell proliferation in 2D and 3D cell culture. These results suggest that generation of truncated P-cadherin during the progression of oral squamous carcinoma attenuates tissue integrity, facilitates cellular separation, and leads to the acquisition of a more migratory phenotype.

Different expression of MAGE-A-antigens in foetal and adult keratinocyte cell lines.

MAGE-A-antigens are an immunologic marker for many cancers. The goal of this study was to compare the expression profiles of MAGE-A2, -A3, -A4, -A6 and -A10 in foetal and adult keratinocytes with an oral squamous cell carcinoma (OSCC) cell line. Expression of MAGE-A2, -A3, -A4, -A6 and -A10-antigens were detected with PCR in foetal and adult keratinocyte cell lines and in an OSCC cell line (pT4N1M0). Quantitative expression of the single MAGE-A-antigens was measured with rtq-PCR. The results were compared to the reference value of the adult keratinocyte cell line. MAGE-A-antigens were detected in all cell lines. Expression profiles of adult and foetal keratinocyte cell lines differed significantly. Expression profiles of foetal and carcinoma cell lines differed significantly also. MAGE-A-antigens were detected in foetal keratinocyte cell line and oral squamous cell carcinoma cell line but differ in their expression profiles. Up to now MAGE-A-antigens were not detected in foetal keratinocytes. Their role is still unknown.