Norihiko Narita

Cancer Cells Expressing Toll-like Receptors and the Tumor Microenvironment.

Toll-like receptors (TLRs) play a crucial role in the innate immune response and the subsequent induction of adaptive immune responses against microbial infection or tissue injury. Recent findings show that functional TLRs are expressed not only on immune cells but also on cancer cells. TLRs play an active role in carcinogenesis and tumor progression during chronic inflammation that involves the tumor microenvironment. Damage-associated molecular patterns (DAMPs) derived from injured normal epithelial cells and necrotic cancer cells appear to be present at significant levels in the tumor microenvironment, and their stimulation of specific TLRs can foster chronic inflammation. This review discusses how carcinogenesis, cancer progression, and site-specific metastasis are related to interactions between cancer cells, immune cells, and DAMPs through TLR activation in the tumor microenvironment.

Activation of toll-like receptors 2, 3, and 4 on human melanoma cells induces inflammatory factors

Toll-like receptors (TLR) have been shown to be expressed on various types of cancers; however, their functional activity is not known. We examined TLR profiles of human melanoma cells and showed that TLR2, TLR3, and TLR4 were found to be highly expressed. By PCR array analysis, specific stimulation of TLR2, TLR3, and TLR4 on melanoma cells showed significant activation of the adaptor protein MyD88, as well as downstream signal transduction factors nuclear factor-κB and inflammatory response–related factors. Specific ligand activation of TLR2, TLR3, and TLR4 was shown to induce cell migration. Peripheral blood lymphocytes and melanoma purified RNA was shown to activate TLR3 on melanoma cells. These studies show expression and functional activity of specific TLRs on melanoma cells and as potential therapeutic targets to control tumor progression. [Mol Cancer Ther 2008;7(11):3642–53]

Functional RET G691S Polymorphism in Cutaneous Malignant Melanoma

RET proto-oncogene encodes a receptor tyrosine kinase whose ligand is glial cell line-derived neurotrophic factor (GDNF), and its polymorphism at G691S juxtamembrane region (RETp) is a germline polymorphism. Cutaneous melanomas, particularly the desmoplastic subtype, are highly neurotropic; thus we sought to determine the frequency of RETp in cutaneous melanoma and its functional responsiveness to GDNF. RETp was assessed in 71 non-desmoplastic cutaneous melanomas (non-DMs) and 70 desmoplastic melanomas (DMs). Melanoma cell lines with RETp, RET wild type (RETwt), BRAF V600E mutation (BRAFmt) or BRAF wild type (BRAFwt) were assessed for functional activity. RETp frequency was significantly higher in DMs (61%) than in non-DMs (31%, P<0.001). BRAFmt was detected in only 11% of DMs. GDNF stimulation significantly amplified cell proliferation, migration and invasion in RETp, but not in RETwt melanoma cells. GDNF stimulation of RETp cell lines enhanced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt of the RET-RAS-RAF-ERK and RET-phosphatidylinositol 3-kinase (PI3K)-Akt pathways, respectively. GDNF response of RETp cells in signal transduction and other functional studies were not affected by BRAFmt. The study demonstrates that RETp is frequently found in cutaneous melanoma, particularly desmoplastic subtypes, and responds to GDNF inducing events favorable for tumor progression.

B7-h3 ligand expression by primary breast cancer and associated with regional nodal metastasis.

Objective: B7 ligand family members have been shown to be important immunoregulatory factors in host tumor immune responses. We hypothesized that B7–H3, a coinhibitory factor, is expressed by primary breast cancer cells and associated with metastasis to regional tumor-draining lymph nodes. Experimental Design: American Joint Committee on Cancer stage I to III primary breast cancers (n = 82) and normal breast specimens (n = 17) were assessed for B7–H3 expression using paraffin-embedded archival tissues. B7–H3 expression by breast cancer cells was assessed by a quantitative real-time reverse transcription-polymerase chain reaction, and B7–H3 protein expression was evaluated using immunohistochemistry. Results: B7–H3 mRNA expression was detected in 32 of 82 (39%) primary breast tumors but not in normal breast tissues (P = 0.0029). B7–H3 expression in primary tumors significantly correlated with increasing tumor size, American Joint Committee on Cancer stage, and lymphovascular invasion (P < 0.0001, P < 0.0001, P = 0.0071). B7–H3 expression was highly correlated to sentinel lymph node and overall number of lymph nodes with metastasis P = 0.003, and P = 0.004, respectively). In a multivariate analysis, B7–H3 mRNA expression of the primary tumor significantly predicted metastasis to regional lymph nodes (P = 0.021, and P = 0.023, respectively). Antibody staining analysis of paraffin-embedded archival tissue breast tumors and flow cytometry of breast cancer cell lines demonstrated B7–H3 protein expression. Conclusions: B7–H3 protein expressed by primary breast cancer cells is a tumor progression factor and is associated with extent of regional nodal metastasis.

Efficacy of mometasone furoate nasal spray for nasal symptoms, quality of life, rhinitis-disturbed sleep, and nasal nitric oxide in patients with perennial allergic rhinitis.

Intranasal corticosteroid therapy has exhibited effectiveness for improving nasal symptoms and quality of life (QOL) scores associated with seasonal allergic rhinitis. We prospectively investigated the efficacy of mometasone furoate nasal spray (MFNS) for improving the total nasal symptom score, QOL score, and sleep quality in subjects with perennial allergic rhinitis (PAR). Nasal airway conditions were also objectively assessed by measuring nasal nitric oxide (NO). Fifty-seven patients with PAR were randomized to MFNS or placebo for a 14-day, double-blind, crossover study. The subjects recorded their symptoms on nasal symptom forms and a visual analog scale. QOL and sleep quality were surveyed in accordance with the Japanese version of the Rhinoconjunctivitis Quality of Life Questionnaire (JRQLQ) and the Japanese version of the Epworth Sleepiness Scale. Nasal NO was measured during a single exhalation using a chemiluminescence analyzer. MFNS treatment achieved significant reductions versus placebo for total nasal symptoms (p < 0.001). There were significant decreases of the usual daily activity domain (p < 0.005), outdoor activities (p < 0.01), social function (p < 0.05), and the overall QOL score (p < 0.05) of JRQLQ with MFNS therapy versus placebo. A significant reduction of the sleepiness scale was also observed in the MFNS group with high sleep disturbance (p < 0.01). A significant decrease of nasal NO was found in the MFNS group (p < 0.01), especially among patients with severe nasal symptoms (p < 0.005). This prospective study indicated that MFNS therapy significantly improves nasal symptoms, QOL, sleep quality, and upper airway condition in Japanese subjects with PAR.

Gene Expression Analysis of Human Middle Ear Cholesteatoma Using Complementary DNA Arrays

Objective To identify genes regulated in human cholesteatoma compared with normal skin tissue using complementary DNA arrays.

Prognostic Molecular Biomarkers for Cutaneous Malignant Melanoma

Molecular signatures of melanoma have propelled new approaches to early diagnosis, monitoring of treatment response, and targeted therapy. This review discusses messenger RNA (mRNA), genomic, and epigenomic melanoma biomarkers in blood and tissue specimens. The major focus is on tissue‐based molecular assays to upstage sentinel lymph nodes (SLNs), and blood‐based assays to detect melanoma progression by monitoring levels of circulating tumor cells (CTC) and circulating DNA. J. Surg. Oncol. 2011; 104:438–446.

Poly(I:C) induces BLyS-expression of airway fibroblasts through phosphatidylinositol 3-kinase

B lymphocyte stimulator (BLyS), B cell activating factor (BAFF), a member of the tumor necrosis factor ligand superfamily has potent co-stimulatory activity on B cells, and BLyS-production in the airway mucosa is of potential importance as it triggers innate and adaptive immune responses. To investigate whether airway fibroblast could express BLyS, we examined BLyS-expression in human nasal airway fibroblasts and compared to its expression in tonsillar and skin fibroblasts as well as the effect of the Toll-like receptor (TLR) ligands on that in human nasal airway fibroblasts. The expression of BLyS by nasal fibroblasts in the presence of polyinocinic-polycytidykic acid (poly(I:C)) was markedly induced, to a level of more than 100 times higher than that observed in the absence of poly(I:C). In order to demonstrate the intracellular pathways involved in poly(I:C)-induced BLyS-expression, we used specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), spleen tyrosine kinase (Syk), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and extracellular-signal related kinase (ERK)-signaling in these events. Pre-incubation with the PI3-kinase inhibitor LY294002 or Wortmanin reversed the poly(I:C)-induced production and expression of BLyS. Syk kinase inhibitor Piceatannol partially reduced its production and expression. Thus, we were able to show that PI3-kinase signaling is directly involved in poly(I:C)-induced BLyS-expression in nasal airway fibroblasts. These results indicate that human nasal airway fibroblasts strongly induce BLyS-expression and production by poly(I:C) through PI3-K signaling during airway immune responses.

Inhibition of histone deacetylase 3 stimulates apoptosis induced by heat shock under acidic conditions in human maxillary cancer.

To elucidate the molecular mechanisms for the enhancement of heat-induced apoptosis on exposure to acidic conditions, human maxillary carcinoma IMC-3 cells were heat-shocked at 44°C for 30 min at either pH 7.4 or 6.7. Analyses with cDNA arrays, the reverse transcription–polymerase chain reaction (RT–PCR), and Western blotting were performed. We found that histone deacetylase 3 (HDAC3) was specifically induced after hyperthermia at 44°C for 30 min at pH 6.7. Although the cytotoxicity of heating at 44°C for 30 min was enhanced by decreasing the pH from 7.4 to 6.7, it was enhanced even more by antisense RNA oligonucleotides for HDAC3. The induction of G2/M arrest after heating occurred earlier at pH 6.7 than at pH 7.4. The inhibition of HDAC3 by the antisense RNA oligonucleotides suppressed partially the induction of G2/M arrest, resulting in an enhancement of the apoptosis caused by the heating under acidic conditions. Antisense RNA oligonucleotides for HDAC3 enhanced apoptosis 48 h after hyperthermia at 43°C for 30 min in vivo. Analyses of p65 activity suggested that NF-κB is involved in this enhancement of hyperthermia. HDAC3 may be a novel target enhancing hyperthermia and combined treatment with hyperthermia and HDAC inhibitors is a possible modality for cancer therapy.

Development of Sporadic Microsatellite Instability in Colorectal Tumors Involves Hypermethylation at Methylated-In-Tumor Loci in Adenoma

Microsatellite instability (MSI) and genomic hypermethylation of methylated-in-tumor (MINT) loci are both strong prognostic indicators in a subgroup of patients with sporadic colorectal cancer (CRC). The present study was designed to determine whether the methylation of MINT loci during the progression of adenoma to CRC is related to MSI in CRC cases. Methylation index (MI) was measured by absolute quantitative assessment of methylated alleles at seven MINT loci in primary CRC with contiguous adenomatous and normal tissues of 79 patients. Results were then validated in primary CRC tissues from an independent group of 54 patients. Increased MI of both MINT loci 1 and 31 was significantly associated with MSI in CRC and was specific for adenoma. Total MI and the number of methylated loci were threefold (P=0.02) and fivefold (P=0.004) higher, respectively, in adenomas associated with microsatellite-stable CRC versus microsatellite-unstable CRC. MINT MI was found to be correlated with mismatch repair protein expression, MSI, BRAF (V600E) mutation status, mut-L homologue 1 methylation status, and disease-specific survival in the second independent validation group of patients. MI of specific MINT loci may be prognostic indicators of colorectal adenomas that will develop into sporadic microsatellite-unstable CRCs. Increased MINT locus methylation appears to precede MSI and may have utility in defining clinical pathology in the absence of features of malignant invasive tumors.