Selectively high efficacy of eribulin against high-grade invasive recurrent and/or metastatic squamous cell carcinoma of the head and neck.

Patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) have a poor prognosis. Over the past decade, a major development in the first-line treatment of R/M SCCHN was the introduction of cetuximab in combination with platinum plus 5-fluorouracil chemotherapy. Currently, a promising novel treatment option in R/M SCCHN has emerged, termed immune checkpoint inhibitors. However, only a few patients presenting with R/M SCCHN have exhibited meaningful tumor regression with these agents. Therefore, novel agents are required to order improve the overall survival of patients with R/M SCCHN. Recently, we demonstrated that R/M SCCHN cells are highly sensitive to eribulin. In the present study, the effects of eribulin, paclitaxel and vinblastine were investigated in R/M SCCHN (OLC-01 and OSC-19) and locally advanced SCCHN (OSC-20) cells. Tumour-inhibitory activities of eribulin against R/M SCCHN were evaluated in orthotopic xenograft models. The data revealed that eribulin has sub-nM growth inhibitory activities in vitro against OLC-01 cells, and that it is more potent than paclitaxel and vinblastine. The reduced expression of Tubulin Beta 3 Class III (TUBB3) following treatment was correlated with a high sensitivity to eribulin. Histological analysis of OLC-01 cells in NOD-SCID mice demonstrated that they had a higher invasiveness in the tissue around the alveolar cancer when compared with the histology of OSC-19 cells, which has been reported in our previous study. Treatment with eribulin revealed marked inhibitory activities in vivo at 0.125 mg/kg against OLC-01 cells orthotopic xenografts. In conclusion, the results highlight the existence of invasive-type heterogeneity in R/M SCCHN with respect to eribulin sensitivity. Eribulin is already an approved clinical agent; therefore, the continued investigation of its preclinical antitumor attributes may contribute significantly to the future process of identifying novel uses of eribulin against R/M SCCHN.

Clinicopathological Significance of the ET Axis in Human Oral Squamous Cell Carcinoma.

The interaction between cancer cells and the surrounding microenvironment in malignant tumor tissue is known to be closely associated with cancer cell invasion and proliferation. Endothelin (ET) present in the microenvironment surrounding tumors has been reported to play a role in cancer cell invasion and proliferation by binding to receptors on the cell membrane of cancer cells. Here, we immunohistologically detected the expression of ET-1 and its receptor ETAR in oral squamous cell carcinoma (OSCC) and evaluated the association between the expression of each as well as their co-expression (ET-axis expression) and clinicopathological factors. A significant difference was observed between the invasion pattern as a parameter of cancer cell malignancy and the expressions of ET-1 and ETAR. The survival rates were significantly lower among the patients who were strongly positive for ET-1 and the ETAR-positive patients compared to negative patients. There was also a significant difference between ET-axis expression and the degree of histological differentiation and mode of invasion, and the survival rate of the positive cases was significantly lower than that of the negative cases. Our findings suggested that ET-axis assessments are important for assessing the malignancy of cancer cells and predicting the prognoses of OSCC patients.

Regulation of programmed-death ligand in the human head and neck squamous cell carcinoma microenvironment is mediated through matrix metalloproteinase-mediated proteolytic cleavage.

Recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. According to recent clinical studies, tumour growth can be effectively reduced and survival can be improved by blocking the programmed death receptor-1 (PD-1)/programmed death-ligand 1 (PD­L1) pathway. PD-L1 expression has been proposed as a potential causative mechanism, as HNSCC is highly immunosuppressive. However, anti-PD-1 treatment is beneficial only for certain patients. Therefore, the mechanisms controlling PD-L1 expression warrant further investigation in order to provide a better understanding of the predicting efficacy of and optimising anti-PD-1 therapy, alone or in combination. In this study, PD-L1 protein extracted from the cell membrane was found to be downregulated in OSC-20 cells compared with OSC-19 cells, despite a higher PD-L1 expression in the total cell lysate of the OSC-20 compared with the OSC-19 cells. Several matrix metalloproteinases (MMPs) were found to be upregulated in HNSCC; in particular, MMP-7 and -13 were upregulated in the OSC-20 compared with the OSC-19 cells. Purified PD-L1 was degraded by recombinant MMP-13 and -7. The expression of PD-L1 was significantly restored by a specific inhibitor of MMP-13 (CL82198), which suggested the involvement of MMP-13 in the shedding/cleavage of PD-L1 in the OSC-20 cells. Among the anticancer drugs conventionally used in the treatment of patients with HNSCC, paclitaxel increased MMP-13 expression in R/M HNSCC cells (HOC313 cells) co-cultured without/with dendritic cells (DCs). These results suggest that the shedding/cleavage of PD-L1 by MMP-13 is one of the mechanisms behind the protective effect against invasion and metastasis. Thus, MMP-13 has potential value as a marker predictive of the decreased efficacy of anti-PD-1 therapy. In addition, paclitaxel is a particularly promising candidate for combination therapy in R/M HNSCC with anti-PD-1 therapy.

Regulation of PD-L1 expression in a high-grade invasive human oral squamous cell carcinoma microenvironment.

Blockade of the programmed-death 1 receptor (PD-1)/programmed-death ligand (PD-L1) pathway efficiently reduces tumour growth and improves survival. Durable tumour regression with blockade of the PD-1/PD-L1 checkpoint has been demonstrated in recent clinical studies. Oral squamous cell carcinoma (OSCC) is highly immunosuppressive, and PD-L1 expression has been proposed as a potential mechanism responsible for this phenotype. Despite the fact that anti-PD-1 treatment can produce durable responses, such therapy appears to benefit only a subset of patients. Thus, it is important to understand the mechanisms underlying regulation of PD-L1 expression in the OSCC microenvironment. In this study, we showed that PD-L1 expression in high-grade invasive OSCC cell lines was lower than that in a low-grade invasive OSCC line and found a close correlation between PD-L1 expression and the epithelial-mesenchymal transition (EMT). PD-L1 expression was upregulated in macrophages and dendritic cells (DCs) in high-grade invasive human OSCC tissues or co-cultured with mesenchymal-phenotype OSCC cells in vitro. TLR4-inhibitory peptide successfully suppressed PD-L1 upregulation on macrophages and DCs co-cultured with mesenchymal-phenotype OSCC cells, suggesting that some EMT-induced tumour antigen is critical for PD-L1 induction on tumour-associated macrophages and DCs. Further studies are necessary to explore the impact of EMT on the tumour immune microenvironment and to identify potential biomarkers for selecting patients who might preferentially benefit from PD-1/PD-L1 blockade or immunotherapies more broadly.

Eribulin sensitizes oral squamous cell carcinoma cells to cetuximab via induction of mesenchymal-to-epithelial transition.

Inhibition of epidermal growth factor receptor (EGFR) signalling has emerged as a new treatment strategy for oral squamous cell carcinoma (OSCC). Previously, we found that loss of EGFR expression in OSCC was associated with epithelial-mesenchymal transition (EMT), and may have functional implications with regard to resistance to cetuximab, a monoclonal anti-EGFR antibody. Eribulin (a microtubule inhibitor) reportedly renders breast cancer less aggressive, and less likely to metastasise, by triggering mesenchymal­to­epithelial (MET) transition. In the present study we evaluated whether eribulin-induced MET was associated with re-sensitization of resistant OSCC cell lines to cetuximab. In vitro antiproliferative activities were determined in three human OSCC lines (OSC-20, OSC-19 and HOC313) treated with eribulin. These three human OSCC represented different EMT/MET states. Interestingly, HOC313 cells (mesenchymal phenotype) were highly sensitive to eribulin in comparison with other cell lines, and significantly enhanced the anti-proliferative effect of cetuximab in response to the drug. Eribulin also underwent a MET-associated gene switch that resulted in morphological changes and high EGFR expression in HOC313 cells, and abrogated a TGF-ß-induced EMT gene expression signature. Eribulin-dependent sensitization of OSCC to cetuximab is likely due to induction of MET. Combination therapies based on eribulin and cetuximab have potential as a novel treatment regimen in OSCC.

Loss of epidermal growth factor receptor expression in oral squamous cell carcinoma is associated with invasiveness and epithelial-mesenchymal transition.

Inhibition of epidermal growth factor receptor (EGFR) signaling has emerged as a novel therapeutic strategy for the treatment of oral squamous cell carcinoma (OSCC). The EGFR-directed inhibitor cetuximab is currently the only approved targeted therapy for the treatment of OSCC. EGFR status may affect the patient response to cetuximab treatment. In the present study, via analysis of the immunomarker for EGFR, it was revealed that 58.3% of the total cases investigated stained positively for EGFR expression, and furthermore, that invasiveness was inversely correlated with EGFR expression. Expression levels of EGFR were quantified, and the correlation between EGFR expression and cetuximab sensitivity was investigated using three varying grades of invasive human OSCC line. EGFR expression in high-grade invasive cells was significantly downregulated compared with that of low-grade invasive cells. There was no significant antiproliferative effect in the high-grade invasive cells treated with various concentrations of cetuximab. The EMT-associated genes, N-cadherin, vimentin and Snail, were upregulated in the high-grade invasive cells. The low-grade invasive cells exhibited characteristics of typical epithelial cells, including the expression of E-cadherin and absence of the expression of N-cadherin, vimentin and Snail. Transforming growth factor-ß induced low-grade invasive cells to undergo an epithelial-mesenchymal transition (EMT)-associated gene switch, which resulted in low levels of EGFR expression. The results of the present study suggested that loss of EGFR expression in OSCC was associated with EMT, and may have functional implications with regard to tumor invasiveness and the resistance to cetuximab treatment.

Prognostic value of vascular endothelial growth factors A and C in oral squamous cell carcinoma.

BACKGROUND: Vascular endothelial growth factor (VEGF) family members play a major role in angiogenesis and vascularization. VEGF-A promotes tumor angiogenesis by stimulating the growth of tumor vascular endothelial cells. In addition, VEGF-C has been identified as a potent inducer of lymphangiogenesis in tumor and lymph node metastasis. Previous studies have investigated the association between clinicopathological factors and the expression of VEGF-A and VEGF-C in oral squamous cell carcinoma cancer (OSCC), but the results are contradictory. In this study, we investigated the relationship between VEGF-A and VEGF-C expression and OSCC clinicopathological factors and prognosis. METHODS: Expression of VEGF-A and VEGF-C was evaluated in surgical specimens from 61 patients with OSCC and three human oral cancer cell lines (OSC-19, OSC-20 and HOC313) by immunohistochemical staining and enzyme-linked immunosorbent assay, respectively. We also determined the relationship between the 5-year survival rate and clinicopathological factors, such as TNM classification (Union for International Cancer Control, UICC), lymph node metastasis, recurrence, histological differentiation, location, and mode of invasion. RESULTS: VEGF-A expression correlated significantly with lymph node metastasis. VEGF-C expression was associated with lymph node metastasis, recurrence, and a poorer 5-year survival rate. A multivariate analysis demonstrated that VEGF-C is an independent prognostic factor for patients with OSCC. VEGF-C expression was significantly up-regulated in HOC313 cells compared to OSC-19 and OSC-20 cells. CONCLUSIONS: These results indicate that VEGF-C may be a predictive factor for OSCC outcome, lymph node metastasis, and recurrence. Moreover, VEGF-C may be an important factor in the development of new therapies for OSCC patients.

Loss of claudin-7 is a negative prognostic factor for invasion and metastasis in oral squamous cell carcinoma.

Claudin-7 belongs to the claudin family, which consists of 24 subtypes of essential tight junction (TJ) integral membrane proteins with molecular weights of 20-27 kDa. We investigated the interrelationship between clinicopathological findings and claudin-7 expression in oral squamous cell carcinoma (OSCC). Using immunohistochemical techniques to examine the expression levels of claudin-7 in 67 cases of OSCC, claudin-7 expression was detected in 35 (52.2%) of the 67 cases. We also compared the clinicopathological features of the OSCC cases with claudin-7 expression levels. Moreover, six cell lines with various invasive properties were investigated in vitro to compare mRNA and protein levels of claudin-7 using reverse transcription-polymerase chain reaction (RT-PCR) and the western blotting method. Decreased claudin-7 expression correlated significantly with T-category (p<0.05), lymph node metastasis (p<0.01), and mode of invasion (p<0.001). Patients with positive claudin-7 expression had a significantly better prognosis (p<0.05). Claudin-7 protein and mRNA levels were lower in the HOC313 and TSU cells, which have higher invasive potentials compared with other cell lines. These results suggest that loss of claudin-7 expression is associated closely with invasion and lymph metastasis and is an unfavorable prognostic factor in patients with OSCC.

Expression of urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor and maspin in oral squamous cell carcinoma: Association with mode of invasion and clinicopathological factors.

It is well documented that the binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR), which has been implicated in cancer invasion and metastasis, is regulated by several inhibitors such as maspin. In this study, we investigated the interrelationship between clinicopathologic findings and expression of uPA, uPAR and maspin in oral squamous cell carcinoma (OSCC) to elucidate the participation of maspin in the uPA/uPAR system in the malignant behavior of OSCC. Using immunohistochemical techniques to examine the expression levels of uPA, uPAR and maspin in 54 cases of OSCC, we also compared the clinicopathologic features of OSCC with the expression levels of each. Moreover, we examined the expression of uPA, uPAR and maspin in six cell lines derived from OSCC using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. uPA and uPAR showed a positive correlation with the mode of cancer invasion; conversely maspin showed a negative correlation with the mode of invasion. Multivariate analysis revealed that only two factors (N-category and uPA+/uPAR+/maspin- expression pattern) were significant and independent variables with relative risks of 3.84 and 2.52, respectively. In particular, tumors exhibiting an expression pattern of uPA+/uPAR+/maspin- were highly malignant and were associated with the worst survival rate (5-year overall survival rate, 29.4%), while tumors with an expression pattern, uPA-/uPAR-/Μaspin+, showed the most favorable survival rate (5-year overall survival rate, 77.8%). In vitro, lower expression of maspin was also noted in the cell lines derived from grade 4D OSCC, which exhibited a stronger invasive potential than the cells lines derived from the other grades of OSCC, while uPA and uPAR demonstrated an expression trend opposite to maspin. These results indicate that uPA, uPAR and maspin expression patterns may be useful markers for evaluating the clinical course or prognosis of OSCC patients.

Expression form of p53 and PCNA at the invasive front in oral squamous cell carcinoma: correlation with clinicopathological features and prognosis.

BACKGROUND: Abnormalities in cell-cycle-controlling genes are important in the malignant transformation and proliferation of tumors. Among these genes, the tumor suppressor gene p53 is the most notable, and its mutations provide an indicator of tumor progression and prognosis. Proliferating cell nuclear antigen (PCNA) is a highly conserved nuclear protein that is expressed during cell replication and DNA repair. This study examined the expression of p53 and PCNA at the invasive front of oral squamous cell carcinomas (OSCC) by immunohistochemical staining, and investigated the relationship of these proteins to clinicopathological findings and prognosis. METHODS: Fifty-nine biopsy cases of OSCC were examined by immunohistochemical staining. Clinicopathological data were gathered and patient survival was analyzed. RESULTS: The p53 labeling index (p53-LI) and PCNA labeling index (PCNA-LI) were examined at the invasive front of the tumors. A high p53-LI (p53+) was observed in 17 of the 59 cases (28.8%) and a high PCNA-LI (PCNA+) was observed in 28 of the 59 cases (47.5%). Among the modes of cancer invasion, many of the p53+/PCNA+ cases could be confirmed as highly invasive cancer (P < 0.05). In addition, the p53+/PCNA+ cases showed a high risk of tumor recurrence compared with the other expression forms, and patients with p53+/PCNA+ had a worse prognosis than those with the other expression forms. High labeling indices of p53 and PCNA are associated with poor prognosis in patients with OSCC. CONCLUSION: We suggest that it is important to investigate the expression of p53 and PCNA at the invasive front of OSCC.

Loss of maspin is a negative prognostic factor for invasion and metastasis in oral squamous cell carcinoma.

OBJECTIVE: Maspin, a 42-kDa protein, belongs to the serpin family of protease inhibitors and is known to have tumor-suppressor function. In this study, we investigated the interrelationship between clinicopathologic findings and maspin expression in oral squamous cell carcinoma (OSCC). METHODS: Using immunohistochemical techniques to examine the expression levels of maspin in OSCC, maspin expression in OSCC was detected in 46 (64.8%) of 71 cases. We also compared the clonicopathologic features of OSCC cases with maspin expression levels. Moreover, we examined expression of maspin in eight cell lines derived from OSCC using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: There was a significant correlation between decreased maspin expression and T-category (P < 0.01), lymph metastasis (P < 0.0001), and mode of invasion (P < 0.0001). Patients with positive maspin expression had a significantly better prognosis (P < 0.001). Lower expression of maspin was also seen in cell lines derived from grade 4D, which shows stronger invasive potential than other grades of OSCC. CONCLUSION: Maspin may be a useful marker to identify the potential for progression in OSCC.

Integrin expression levels correlate with invasion, metastasis and prognosis of oral squamous cell carcinoma.

The present study evaluated the relationship between alpha 3, alpha 6A, and beta 1 integrin expression in cancer cells at the invasive front of oral squamous cell carcinoma (OSCC) and survival rates, as well as the clinical and pathological characteristics. Sections of 100 specimens of primary OSCC were immunostained to assess alpha 3, alpha 6A, and beta 1 integrin expression in cancer cells at the invasive front. OSCC patients with higher expression levels of alpha 3, alpha 6A, and beta 1 integrin had significantly better prognosis than those with lower expression levels (median survival at low vs. high expression levels: alpha 3, 37.1 months vs. 55.7 months; alpha 6A , 38.3 months vs. 47.9 months; and beta 1, 26.1 months vs. 46.1 months) (P < 0.05). In addition, beta 1 integrin expression showed the highest correlation with clinical and pathological characteristics. This study concludes that alpha 3, alpha 6A, and beta 1 integrin expression in cancer cells at the invasive front are related to the mode of invasion and prognosis in OSCC.

Apoptosis-associated markers and clinical outcome in human oral squamous cell carcinomas.

BACKGROUND: Apoptosis is a genetically regulated cell death involved in the deletion of cells in normal or malignant tissues. Proteins of the Bcl-2 family play a key role in the control of apoptosis and carry out both pro-apoptotic and anti-apoptotic functions. The present study evaluated the prognostic value of Bcl-2 and Bax expression at the invasive front of oral squamous cell carcinoma (OSCC), taking clinicopathological findings into account. METHODS: Fifty-six specimens of OSCC were randomly selected, and Bcl-2 and Bax expression was evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded pre-treated specimens at the invasive front of OSCC. Clinicopathological data were gathered and patient survival was analysed. RESULTS: No significant relationship was found between Bcl-2 or Bax expression and clinical variables. Patients with Bcl-2 expression had a worse prognosis than those without Bcl-2 expression, but the difference did not reach statistical significance. Patients with Bax expression had a significantly better prognosis than those without Bax expression (P < 0.05). In univariate analyses, T category, mode of cancer invasion and Bax expression showed significant correlations. Multivariate analysis revealed that the mode of cancer invasion and Bax expression were significant and independent variables. Bax expression was found to be the strongest independent prognostic parameter. Patients with negative Bcl-2 expression and positive Bax expression had a significantly better prognosis (P < 0.005). CONCLUSION: We suggest that Bax expression at the invasive front of OSCC is a significant predictor of prognosis and that it is therefore important to investigate the expression of Bcl-2 and Bax in this disease.

Copper efflux transporter (ATP7B) contributes to the acquisition of cisplatin-resistance in human oral squamous cell lines.

Acquired resistance to cisplatin (CDDP) is an issue in cancer chemotherapy. This resistance has been reported to be correlated with the expression of the Cu influx copper transporter 1 (CTR1) and two copper efflux transporters (ATP7A, ATP7B). We investigated the correlation between the expression of these transporters and the sensitivity to CDDP using three pairs of parent cell lines and resistant cell lines derived from various types of invasive oral squamous cell carcinoma (OSCC). Using multiple steps, each of the CDDP-resistant cell lines, HSC-4-R, OSC-19-R, HOC313-R, was selected from HSC-4 cells derived from a cancer with medium invasiveness, OSC-19 cells derived from a cancer with high invasiveness and HOC313 cells derived from a cancer with the highest invasiveness. Resistant cell lines had a stronger expression of ATP7B in conjunction with the acquisition of CDDP-resistance than parent cell lines. Furthermore, OSC-19-R cells transfected with the ATP7B siRNA had a 10.6-fold higher sensitivity to CDDP compared to OSC-19-R cells transfected with a nonsense siRNA. These results suggest that each of the resistant cell lines had acquired resistance to CDDP due to the overexpression of ATP7B. On the other hand, the expression of CTR1 was the same between sensitive cell lines and resistant cell lines and ATP7A mRNA expression was barely noted. We conclude that ATP7B is correlated with the acquisition of CDDP resistance more closely than either CTR1 or ATP7A. ATP7B may be a key determinant in the acquired resistance to CDDP in OSCC.