Hiroko Kitahara

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.

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.

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.

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.

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-β 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.

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 mesenchymal-to-epithelial (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-β-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.

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 (PD-L1) 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.

Expression of Copper Efflux Transporter (ATP7B) in the Transport of Cisplatin in Cell Lines Derived From Invasive Oral Squamous Cell Carcinoma

Abstract Intrinsic or acquired resistance to cisplatin (CDDP) is a problem for its use in cancer chemotherapy. This resistance has been reported to correlate with expression of the human copper transporter 1 and two copper export pumps, ATP7A and ATP7B. In the current study, we investigated the correlation between the expression of these transporters and sensitivity to CDDP using four cell lines derived from each of high invasive oral squamous cell carcinoma (OSCC) and low invasive OSCC. We found that the amount of CDDP accumulated in high invasive OSCC cell lines (Yamamoto-Kohama criteria : grade 4C and 4D) with strong intrinsic tolerance was lower than in low invasive OSCC cell lines (grade 3) with weak intrinsic tolerance. Additionally, overexpression of ATP7B mRNA in cell lines derived from high invasive OSCC conferred low sensitivity to CDDP. Furthermore, the accumulation and sensitivity of CDDP was higher in HOC313 cells transfected with the ATP7B siRNA than in cells transfected with the nonsense siRNA. These results suggest that the overexpression of ATP7B results in the export of and, therefore, resistance to CDDP. Furthermore, ATP7B may be a key determinant of the intrinsic resistance to CDDP.