Ryoji Kitamura

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), phospho-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, p63β 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 ΔNp63β 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.

Critical roles of Wnt5a–Ror2 signaling in aggressiveness of tongue squamous cell carcinoma and production of matrix metalloproteinase-2 via ΔNp63β-mediated epithelial–mesenchymal transition

OBJECTIVES We previously showed that ΔNp63β, a splicing variant of ΔNp63, mediated EMT and affected cell motility. DNA microarray was thus performed to elucidate the mechanism that ΔNp63β affects cell motility. As the results, Wnt5a was significantly down-regulated by ΔNp63β 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. ΔNp63β 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 ΔNp63β-mediated EMT.

Significant association of increased PD-L1 and PD-1 expression with nodal metastasis and a poor prognosis in oral squamous cell carcinoma

Programmed cell death ligand 1 (PD-L1) and its receptor PD-1 are immune checkpoint molecules that attenuate the immune response. Blockade of PD-L1 enhances the immune response in a variety of tumours and thus serves as an effective anti-cancer treatment. However, the biological and prognostic roles of PD-L1/PD-1 signalling in oral squamous cell carcinoma (OSCC) remain to be elucidated. The purpose of this study was to examine the correlation of PD-L1/PD-1 signalling with the prognosis of OSCC patients to assess its potential therapeutic relevance. The expression of PD-L1 and of PD-1 was determined immunohistochemically in 97 patients with OSCC and the association of this expression with clinicopathological characteristics was examined. Increased expression of PD-L1 was found in 64.9% of OSCC cases and increased expression of PD-1 was found in 61.9%. Univariate and multivariate analysis revealed that increased expression of PD-L1 and PD-1 positively correlated with cervical lymph node metastasis. The expression of CD25, an activated T-cell marker, was negatively correlated with the labelling index of PD-L1 and PD-1. Moreover, the patient group with PD-L1-positive and PD-1-positive expression showed a more unfavourable prognosis than the group with PD-L1-negative and PD-1-negative expression. These data suggest that increased PD-L1 and PD-1 expression is predictive of nodal metastasis and a poor prognosis and is possibly involved in cancer progression via attenuating the immune response.

In vitro induction of specific CD8+ T lymphocytes by tumor-associated antigenic peptides in patients with oral squamous cell carcinoma

The aim of this study was to clarify candidate peptides for peptide-based specific immunotherapy of patients with oral squamous cell carcinoma (SCC). Thirteen peptides were examined for in vitro induction of peptide-specific CD8(+) T lymphocyte (CD8(+)TL) activity in peripheral blood mononuclear cells from 35 patients with oral SCC. A correlation between the induction ability of CD8(+)TL and in vivo immune response of host was carried out immunohistochemically in 23 patients. Peptide-specific activities of CD8(+)TL for at least one peptide were detectable in 21/35 patients (60.0%). The potent peptides were SART-1(690) in 9/35 (25.7%), SART-2(93), and ART4(75) in 7/35 (20.0%), respectively. In the 9 patients with SART-1(690)-specific activity, the whole of activities was significantly inducible for more number of other peptides compared to that in 26 patients without the activity (P=0.035). Cellular responses in 7 patients with SART-1(690)-specific activity were significantly stronger than those in 16 patients without the activity (P=0.027). Furthermore, the number of CD3(+) T cells around the SCC was also significantly different between the 2 groups of patients (P=0.041). In conclusion, SART-1(690), SART-2(93), and ART4(75) could be applicable as peptide-based specific immunotherapies for the majority of patients with oral SCC.

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.

Apoptotic function of tumor-associated antigen RCAS1 in oral squamous cell carcinoma

BackgroundReceptor-binding cancer antigen expressed on SiSo cell (RCAS1) is derived from uterine adenocarcinoma and can induce apoptosis in lymphocytes, allowing tumor cells to escape from immune surveillance. RCAS1 is reportedly expressed in a membranous pattern on tumor cell or soluble one in serum of patients. The aim of this study was to investigate expression patterns of RCAS1 and the effect on apoptosis in oral squamous cell carcinoma (OSCC) cell lines.MethodsIn four kinds of OSCC cell lines (HSC-2, HSC-3, SQUU-A, and SQUU-B), RCAS1 mRNAs and proteins were determined by RT-PCR and immunocytochemistry. Membranous RCAS1 was determined by flow cytometry. Culture supernatants were analyzed for detection of soluble RCAS1 by dot blotting and enzyme-linked immunosorbent assay. Apoptotic ability of RCAS1 on the erythroid leukemia cell line K562 with the putative receptor was evaluated by flow cytometry in co-culture with highly metastatic SQUU-B, with knocked-down RCAS1 cells or in a no-cell contact condition.ResultsRCAS1 mRNA and proteins were expressed in all of OSCC cell lines. Membranous pattern were expressed in all cell lines, while soluble pattern was detected in all supernatants. RCAS1 mRNA, membranous and soluble RCAS1 were significantly seen in SQUU-B more than the other 3 cell lines (P < 0.05). K562 apoptosis was induced in co-culture with each of all cell lines, particularly with SQUU-B. Apoptosis was markedly reduced in co-culture with RCAS1 knockdown cells, but was induced in co-culture without cell contract of SQUU-B.ConlusionsOur study suggests that RCAS1 has an apoptotic function via membranous/soluble expression pattern in OSCC cells. RCAS1 may thus affect tumor escape from immune surveillance in OSCC by inducing apoptosis.