Tumor-suppressive roles of ΔNp63ß-miR-205 axis in epithelial-mesenchymal transition of oral squamous cell carcinoma via targeting ZEB1 and ZEB2.

We previously revealed that epithelial-to-mesenchymal transition (EMT) was mediated by ΔNp63ß, a splicing variant of ΔNp63, in oral squamous cell carcinoma (OSCC). Recent studies have highlighted the involvement of microRNA (miRNA) in EMT of cancer cells, though the mechanism remains unclear. To identify miRNAs responsible for ΔNp63ß-mediated EMT, miRNA microarray analyses were performed by ΔNp63ß-overexpression in OSCC cells; SQUU-B, which lacks ΔNp63 expression and displays EMT phenotypes. miRNAs microarray analyses revealed miR-205 was the most up-regulated following ΔNp63ß-overexpression. In OSCC cells, miR-205 expression was positively associated with ΔNp63 and negatively with zinc-finger E-box binding homeobox (ZEB) 1 and ZEB2, potential targets of miR-205. miR-205 overexpression by miR-205 mimic transfection into SQUU-B cells led to decreasing ZEB1, ZEB2, and mesenchymal markers, increasing epithelial markers, and reducing cell motilities, suggesting inhibition of EMT phenotype. Interestingly, the results opposite to this phenomenon were obtained by transfection of miR-205 inhibitor into OSCC cells, which express ΔNp63 and miR-205. Furthermore, target protector analyses revealed direct regulation by miR-205 of ZEB1 and ZEB2 expression. These results showed tumor-suppressive roles of ΔNp63ß and miR-205 by inhibiting EMT thorough modulating ZEB1 and ZEB2 expression in OSCC.

Differential roles of kallikrein-related peptidase 6 in malignant transformation and ΔNp63ß-mediated epithelial-mesenchymal transition of oral squamous cell carcinoma.

We previously reported that epithelial-to-mesenchymal transition (EMT) was mediated by ΔNp63ß in oral squamous cell carcinoma (OSCC). In this study, DNA microarray analyses were performed using ΔNp63ß-overexpressing OSCC cells to identify genes associated with ΔNp63ß-mediated EMT. Thereby, we focused on kallikrein-related peptidase (KLK) 6, most up-regulated following ΔNp63ß-overexpression, that activates protease-activated receptors (PARs). In RT-PCR analyses, ΔNp63 was positively associated with KLK6 and PAR2 and negatively with PAR1 in OSCC cells. By ΔNp63 knockdown, KLK6 and PAR2 expression was decreased and PAR1 was increased. Furthermore, KLK6 knockdown led to enhancing migration and invasion, and inhibiting proliferation, suggesting EMT-phenotypes. Although, in the KLK6 or PAR2 knockdown cells, phosphorylation of ERK was reduced, it was restored in the KLK6 knockdown OSCC cells treated with recombinant KLK6 proteins. Immunohistochemistry showed ΔNp63, KLK6, and PAR2 were more strongly expressed in the epithelial dysplasia and central region of OSCC than normal oral epithelium, whereas PAR1 expression was undetectable. Interestingly, at the invasive front of OSCC, ΔNp63, KLK6, and PAR2 were reduced, but PAR1 was elevated. In addition, the OSCC patients with decreasing KLK6 expression at the invasive front had more unfavourable prognosis. These results suggested differential roles of KLK6 in malignant transformation and EMT; high ΔNp63ß expression up-regulates KLK6-PAR2 and down-regulates PAR1, inducing malignant transformation in oral epithelium with stimulating proliferation through ERK signal activation. Moreover, KLK6-PAR2 expression is down-regulated and PAR1 is up-regulated when ΔNp63ß expression is decreased, leading to EMT with enhancing migration and invasion through ERK signal reduction at the invasive front.

Cytokeratin 17 mRNA as a prognostic marker of oral squamous cell carcinoma.

Despite diagnostic and therapeutic advances, the 5-year survival rate of oral squamous cell carcinoma (OSCC) remains between 70-80% due to recurrences and secondary metastases to cervical lymph nodes. It is difficult to find these recurrences and metastases postoperatively, thus, careful follow-up is recommended. Cytokeratins (CKs) are intermediate filaments of the cytoskeleton and candidate prognostic biomarkers for OSCC, as they are overexpressed in OSCC compared with normal mucosa. The aim of the present study was to determine the relative levels of occurrence of 3 CK mRNA (CK17, CK19, CK20) transcripts in peripheral blood mononuclear cells (PBMC) using reverse transcription-quantitative polymerase chain reaction. The study comprised pre- and post-operative PBMC samples from 19 OSCC patients. In the good-prognosis group, 10 of 13 patients demonstrated reduced CK17 mRNA expression post-operatively, compared with pre-operative samples, conversely, only 3 of 6 patients in the poor-prognosis group had reduced post-operative CK17 mRNA expression. This difference was statistically significant (P<0.01). The disease-free survival rate of the group with reduced post-operative CK17 mRNA expression was significantly increased compared with the elevated CK17 mRNA group (P<0.01); however, the overall survival rates of the two groups were not significantly different. Neither CK19 mRNA nor CK20 mRNA were significantly expressed in the PBMC of OSCC patients. Overall, CK17 mRNA expression may be a useful prognostic biomarker for OSCC.

Critical roles of Wnt5a-Ror2 signaling in aggressiveness of tongue squamous cell carcinoma and production of matrix metalloproteinase-2 via ΔNp63ß-mediated epithelial-mesenchymal transition.

OBJECTIVES: We previously showed that ΔNp63ß, a splicing variant of ΔNp63, mediated EMT and affected cell motility. DNA microarray was thus performed to elucidate the mechanism that ΔNp63ß affects cell motility. As the results, Wnt5a was significantly down-regulated by ΔNp63ß overexpression in tongue SCC cell line (SQUU-B) with EMT phenotype. MATERIALS AND METHODS: Seven OSCC cell lines were used. Expression of ΔNp63, Wnt5a, its receptor Ror2, and matrix metalloproteinases (MMPs) were analyzed by RT-PCR, real-time PCR, and western blotting, and gelatin zymography. Furthermore, we examined the effects of siRNA for Wnt5a or Ror2 and recombinant human Wnt5a (rhWnt5a) on motility of tongue SCC cells. Biopsy specimens from tongue SCC patients were used for immunohistochemical staining of Wnt5a and Ror2. RESULTS: Wnt5a and Ror2 were expressed only in SQUU-B cells without ΔNp63 expression, and negatively associated with ΔNp63 expression in other cells. ΔNp63ß overexpression in SQUU-B cells decreased Wnt5a and Ror2 expression. By Wnt5a or Ror2 knockdown, cell motility was remarkably inhibited, but EMT markers expression was unaffected. MMP-2 expression and the activities inversely correlated with ΔNp63 expression, and were inhibited by Wnt5a or Ror2 knockdown. Cell motility and MMP-2 activities were recovered by adding rhWnt5a in the cells with Wnt5a knockdown, but not in those with Ror2 knockdown. Moreover, immunohistochemical analyses in tongue SCC specimens found that high expression of Wnt5a or Ror2 was associated with poorer prognosis. CONCLUSION: Wnt5a-Ror2 signaling enhanced tongue SCC cell aggressiveness and promoted production of MMP-2 following ΔNp63ß-mediated EMT.

CD163+CD204+ tumor-associated macrophages contribute to T cell regulation via interleukin-10 and PD-L1 production in oral squamous cell carcinoma.

Tumor-associated macrophages (TAMs) promote cancer cell proliferation, invasion, and metastasis by producing various mediators. Although preclinical studies demonstrated that TAMs preferentially express CD163 and CD204, the TAM subsets in oral squamous cell carcinoma (OSCC) remain unknown. In this study, we examined the expression and role of TAM subsets in OSCC. Forty-six patients with OSCC were analyzed for expression of TAMs in biopsy samples by immunohistochemistry. We examined TAM subsets and their production of immune suppressive molecules (IL-10 and PD-L1) in peripheral blood mononuclear cells from three OSCC patients by flow cytometry. CD163 was detected around the tumor or connective tissue, while CD204 was detected in/around the tumors. Flow cytometric analysis revealed that CD163+CD204+ TAMs strongly produced IL-10 and PD-L1 in comparison with CD163+CD204- and CD163-CD204+ TAMs. Furthermore, the number of activated CD3+ T cells after co-culture with CD163+CD204+ TAMs was significantly lower than that after co-culture with other TAM subsets. In clinical findings, the number of CD163+CD204+ TAMs was negatively correlated with that of CD25+ cells and 5-year progression-free survival. These results suggest that CD163+CD204+ TAMs possibly play a key role in the invasion and metastasis of OSCC by T-cell regulation via IL-10 and PD-L1 production.

Tongue squamous cell carcinoma producing both parathyroid hormone-related protein and granulocyte colony-stimulating factor: a case report and literature review.

BACKGROUND: Paraneoplastic syndrome generally results from tumor-derived hormones or peptides that cause metabolic derangements. Common metabolic conditions include hyponatremia, hypercalcemia, hypoglycemia, and Cushing's syndrome. Herein, we report a very rare case of tongue carcinoma presenting with leukocytosis and hypercalcemia. CASE PRESENTATION: A 57-year-old man was admitted to our hospital with tongue squamous cell carcinoma (cT4aN0M0, stage IV). He underwent radical resection following preoperative chemoradiotherapy, but locoregional recurrence was detected 2 months after surgery. He presented with marked leukocytosis and hypercalcemia with elevated serum levels of granulocyte colony-stimulating factor (G-CSF) and parathyroid hormone-related protein (PTHrP). He was therefore managed with intravenous fluids, furosemide, prednisolone, elcatonin, and pamidronate. However, the patient died 1 month later of carcinomatous pleuritis following distant metastasis to the lung. Immunohistochemical analyses of the resected specimens revealed positive staining for PTHrP and G-CSF in the cancer cells. CONCLUSIONS: In this case, it was considered that tumor-derived G-CSF and PTHrP caused leukocytosis and hypercalcemia.

Clinicopathological evaluation of pre-operative chemoradiotherapy with S-1 as a treatment for locally advanced oral squamous cell carcinoma.

The administration of pre-operative chemotherapy with S-1 and concurrent radiotherapy at a total dose of 30 Gy was clinicopathologically evaluated as a treatment for locally advanced oral squamous cell carcinoma (OSCC) in the present study. The participants comprised 81 patients with OSCC, consisting of 29 patients with stage II disease, 12 patients with stage III disease and 40 patients with stage IV disease. All patients received a total radiation dose of 30 Gy in daily fractions of 2 Gy, 5 times a week, for 3 weeks, and the patients were concurrently administered S-1 at a dose of 80-120 mg, twice daily, over 4 consecutive weeks. Radical surgery was performed in all cases at 2-6 weeks subsequent to the end of pre-operative chemoradiotherapy. The most common adverse event was oropharyngeal mucositis, but this was transient in all patients. No severe hematological or non-hematological toxicities were observed. The clinical and histopathological response rates were 70.4 and 75.3%, respectively. Post-operatively, local failure developed in 6 patients (7.4%) and neck failure developed in 2 patients (2.5%). Distant metastases were found in 7 patients (8.6%). The overall survival rate, disease-specific survival rate and locoregional control rate at 5 years were 87.7, 89.9 and 90.6%, respectively. Locoregional recurrence occurred more frequently in patients that demonstrated a poor histopathological response compared with patients that demonstrated a good response (P<0.01). These results indicate that pre-operative S-1 chemotherapy with radiotherapy at a total dose of 30 Gy is feasible and effective for patients with locally advanced OSCC, and that little or no histopathological response may be a risk factor for locoregional recurrence in this treatment.

Increased expression of interleukin-6 predicts poor response to chemoradiotherapy and unfavorable prognosis in oral squamous cell carcinoma.

Recent studies have revealed that cancer cells are exacerbated by chronic inflammation. The present study examined the immunohistochemical expression for interleukin-6 (IL-6), a pleiotropic inflammatory cytokine, in oral squamous cell carcinoma (OSCC) to elucidate the association of IL-6 expression with tumor progression, chemoresistance and prognosis. Seventy-eight patients with primary OSCC were analyzed by immunohistochemical staining for IL-6. These labeling indexes (LIs) were calculated and evaluated in association with the clinicopathologic characteristics and prognosis in the OSCC patients. The patients were divided into three groups as follows: negative group = LI <5%; low IL-6 group = 5% ≤ LI <30%; high IL-6 group = LI ≥30%. The patient numbers of the negative, low and high expression groups were 24, 22 and 32, respectively. In the high IL-6 expression group, IL-6 receptor (IL-6R), phosphor-signal tranducer and activator of transcription 3 (p-STAT3) were also detected in almost all the cancer cells. The prevalence of the cervical lymph node or the distant metastasis in the high expression group was significantly higher than those in the negative and low expression groups. Furthermore, the high expression group had a significantly poorer tumor response to the preoperative chemoradiotherapy and a more unfavourable prognosis than the negative and the low expression groups. Interestingly, IL-6, IL-6R and p-STAT3 were expressed in the residual cancer cells of all the patients in the high expression group with poor response to chemoradiotherapy. These results suggested that IL-6 signaling possibly is involved in the progression and treatment-resistance of OSCC and IL-6 expression in cancer cells could be a useful predictive factor of poor response to chemoradiotherapy and unfavorable prognosis.

Possible involvement of ΔNp63 downregulation in the invasion and metastasis of oral squamous cell carcinoma via induction of a mesenchymal phenotype.

Epithelial-to-mesenchymal transition (EMT), an essential developmental program, is involved in tumor progression. ΔNp63, a homolog of p53, is associated with the EMT program, but the detailed mechanism remains to be elucidated. In this study, we investigated the role of ΔNp63 in EMT during progression of oral squamous cell carcinoma (OSCC). Five OSCC cell lines and specimens from 78 patients with OSCC were used. The expressions of ΔNp63, p63α, p63ß and epithelial markers (cytokeratins 5 and 14) was detected in the OSCC cells, but not in SQUU-B cells (high metastatic potential). E-cadherin was expressed in all OSCC cells. Mesenchymal markers were strongly expressed in the SQUU-B cells. Knockdown of endogenous ΔNp63 in HSC-2 cells induced morphological changes to the spindle shape, decreased the expression of epithelial markers, increased the expression of mesenchymal markers, increased migration and reduced proliferation. By contrast, SQUU-B cells overexpressing ΔNp63ß showed changed their morphology from stromal cell-like to epithelial cells. However, E-cadherin expression was not affected by ΔNp63 knockdown or overexpression. Immunohistochemical staining revealed that cancer cells expressing vimentin were found at the invasive front in the OSCC specimens. The intensity of ΔNp63 expression was also decreased in these cells. Interestingly, the vimentin positivity or decreased intensity of ΔNp63 was positively associated with metastases and poor prognosis in the OSCC patients. These results indicated that ΔNp63 downregulation in cancer cells induces a mesenchymal phenotype that is related to tumor progression of OSCC.

Immunohistochemical location of the p75 neurotrophin receptor (p75NTR) in oral leukoplakia and oral squamous cell carcinoma.

BACKGROUND: Recent studies have demonstrated that the p75 neurotrophin receptor (p75NTR) is a useful marker of keratinocyte stem cells. Although the stem cell markers of original normal tissue have been used to identify cancer stem cells in a variety of cancers, the expression and function of p75NTR have been poorly understood in oral squamous cell carcinoma (OSCC). The objective of this study is, thus, to examine p75NTR expression immunohistochemically in oral leukoplakia (OL), the most frequent precancerous lesion, and OSCC, and to reveal the usefulness of p75NTR as a marker for undifferentiated cancer cells and a novel prognostic factor for OSCC patients. METHODS: In this study immunohistochemical expression of p75NTR, Ki-67, cytokeratin (CK) 5, and CK14 was examined in 112 cases of OL and 81 of OSCC. The labeling indices (LIs) of p75NTR and Ki-67 were calculated, and the association of these LIs with histopathologic characteristics was then evaluated. RESULTS: In the normal oral epithelium and OL, p75NTR was expressed only in the basal layer, and its LI was invariant, irrespective of the extent of epithelial dysplasia. In OSCC, however, p75NTR-LI was significantly increased in association with upgrading of histologic grade and mode of tumor invasion. Furthermore, the prognosis of the high p75NTR-LI group (LI ≥ 53.1%) was poorer than that of the low p75NTR-LI group (LI < 53.1%). CONCLUSIONS: These results suggest that p75NTR is expressed in undifferentiated cell populations in OL and OSCC. Furthermore, p75NTR is possibly involved in invasion and poor prognosis in OSCC.

Clinical significance of combined assessment of the maximum standardized uptake value of F-18 FDG PET with nodal size in the diagnosis of cervical lymph node metastasis of oral squamous cell carcinoma.

RATIONALE AND OBJECTIVES: This study aimed to elucidate the diagnostic accuracy of F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) for nodal involvement in oral squamous cell carcinoma (OSCC), and to reveal clinically useful factors to distinguish between true-positive (TP) and false-positive (FP) nodes. MATERIALS AND METHODS: Thirty-eight patients with primary OSCC who underwent neck dissection were assessed. The diagnostic accuracy of F-18 FDG PET/CT was evaluated, and then compared with that of CT/ultrasonography (US). Furthermore, the association of the maximum standardized uptake value (SUVmax) and nodal size with the histopathologic findings was examined. RESULTS: Sensitivity and specificity using F-18 FDG PET/CT were 77.1% and 97.3%, and those using CT/US were 72.9% and 98.9%, respectively. The SUVmax of TP nodes was significantly higher than that of FP nodes. Nodes with SUVmax >4.5 were pathologically confirmed as metastasis. Nodes with SUVmax ≤4.5 were further discriminated between TP and FP nodes by using the long axis diameters or the ratios of long to short axis diameter as clinical parameters. Positive correlation between the SUVmax and the short-axis diameter was found in TP nodes. The AUC obtained from the ROC curves of the SUVmax alone (AUC, 0.804) was improved by combination with the long-axis diameter (AUC, 0.867) or the short-axis diameter (AUC, 0.846), although no significant difference was found. CONCLUSIONS: These results indicated that F-18 FDG PET/CT was potentially useful in diagnosing preoperative nodal state. Furthermore, combined assessment of SUVmax with nodal size could be significant in the identification of metastatic lymph nodes in OSCC patients.

Increased ΔNp63 expression is predictive of malignant transformation in oral epithelial dysplasia and poor prognosis in oral squamous cell carcinoma.

This study examined immunohistochemical expression of ΔNp63, a keratinocyte stem cell marker, in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and then to elucidate usefulness of ΔNp63 as a marker for diagnosis and prognosis. One-hundred and twelve cases of OL and 81 cases of OSCC were analyzed by immunohistochemical staining for ΔNp63, Ki-67, and cytokeratin 14. These labeling indices (LIs) were calculated, and the association of these LIs with clinicopathologic characteristics in the OL and OSCC was evaluated. In the OL, these LIs increased significantly according to the severity of epithelial dysplasia (p<0.0001). ΔNp63-LI in the OL with malignant transformation was significantly higher than that in the OL without (49.3 vs. 34.2%; p<0.01). In the OSCC, the LIs increased significantly in association with the histologic grade (p<0.0001). A significant difference between the high and low ΔNp63-LI groups was found in the incidence of cervical lymph node and distant metastasis (p<0.05). The prognosis of the high ΔNp63-LI (mean value >73.8%) group is poorer than that of the low ΔNp63-LI (mean value ≤73.8%) group (p<0.05). These results suggested that increased ΔNp63 expression is involved in malignant transformation in epithelial dysplasia and poor prognosis in OSCC.