Miho Harada

Long non-coding RNA TSIX is upregulated in scleroderma dermal fibroblasts and controls collagen mRNA stabilization

Long non‐coding RNAs (lncRNAs) are thought to have various functions other than RNA silencing. We tried to evaluate the expression of lncRNAs in patients with systemic sclerosis (SSc) and determined whether lncRNAs controls collagen expression in dermal fibroblasts. lncRNA expression was determined by real‐time PCR and in situ hybridization. Protein and mRNA levels of collagen were analysed using immunoblotting and real‐time PCR. We found TSIX, one of the lncRNAs, was overexpressed in SSc dermal fibroblasts both in vivo and in vitro, which was inhibited by the transfection of transforming growth factor (TGF)‐β1 siRNA. TSIX siRNA reduced the mRNA expression of type I collagen in normal and SSc dermal fibroblasts, but not the levels of major disease‐related cytokines. In addition, TSIX siRNA significantly reduced type I collagen mRNA stability, but not protein half‐lives. Furthermore, we first investigated serum lncRNA levels in patients with SSc, and serum TSIX levels were significantly increased in SSc patients. TSIX is a new regulator of collagen expression which stabilizes the collagen mRNA. The upregulation of TSIX seen in SSc fibroblasts may result from activated endogenous TGF‐β signalling and may play a role in the constitutive upregulation of collagen in these cells. Further studies on the regulatory mechanism of tissue fibrosis by lncRNAs in SSc skin lead to a better understanding of the pathogenesis, new diagnostic methods by their serum levels and new therapeutic approaches using siRNAs.

Evaluation of sentinel node biopsy for cutaneous squamous cell carcinoma.

Sentinel lymph node biopsy (SLNB) is a standard care for cutaneous melanoma but its role in cutaneous squamous cell carcinoma (SCC) has not been established. Clinical data was obtained from 54 patients with SCC who received SLNB with the usage of blue dye and radioisotope colloid methods. The positive rate of SLNB in SCC was 7.4%. If the cases were limited to more than T2, the positive rate was 12.9%. Three of 41 patients who was estimated negative LN metastasis by the preoperative tests had micrometastasis (7.3%). Among 13 patients who were suggested to have metastasis in the preoperative tests, only one patient had histological metastasis. One patient with SCC located in the lower lip showed negative SLNB and subsequently developed node recurrence. In conclusion, the efficacy of SLNB in SCC is comparable to that of melanoma in the positive rate. There are two kinds of benefit, avoidance of unnecessary complete lymph node dissection and early detection of metastasis.

miR-205 down-regulation promotes proliferation of dermatofibrosarcoma protuberans tumor cells by regulating LRP-1 and ERK phosphorylation

Dermatofibrosarcoma protuberans (DFSP) is an intermediate malignancy of the skin. Although COL1A1/PDGFB fusion gene was identified in the tumor cells recently, not all of the cases were positive for the fusion gene, and further researches are still needed to clarify the pathogenesis of DFSP. In this study, we investigated the role of microRNAs in the tumor. microRNA PCR array showed several microRNAs increased or decreased in DFSP in vivo compared with dermatofibroma (DF) and normal skin. Among them, the expression of miR-205 was down-regulated in DFSP compared with DF and normal skin. In situ hybridization showed that miR-205 expression was evident in dermal fibroblasts of normal skin although hardly detected in tumor cells of DF or DFSP. miR-205 inhibitor increased cell proliferation and the luciferase activity of 3′UTR of low-density lipoprotein receptor-related protein-1 (LRP-1) in cultured normal dermal fibroblasts. Immunohistochemistry showed the expression of LRP-1 was increased in DFSP tissue. Knockdown of LRP-1 suppressed cell growth and down-regulated extracellular signal-regulated kinase (ERK) phosphorylation without affecting MEK phosphorylation in cultured DFSP cells. Taken together, LRP-1 overexpression caused by the miR-205 down-regulation may play a role in the abnormal proliferation of DFSP cells via directly regulating ERK phosphorylation.

NUP160-SLC43A3 Is a Novel Recurrent Fusion Oncogene in Angiosarcoma

Angiosarcoma is a malignant vascular tumor originating from endothelial cells of blood vessels or lymphatic vessels. The specific driver mutations in angiosarcoma remain unknown. In this study, we investigated this issue by transcriptome sequencing of patient-derived angiosarcoma cells (ISO-HAS), identifying a novel fusion gene NUP160-SLC43A3 found to be expressed in 9 of 25 human angiosarcoma specimens that were examined. In tumors harboring the fusion gene, the duration between the onset of symptoms and the first hospital visit was significantly shorter, suggesting more rapid tumor progression. Stable expression of the fusion gene in nontransformed human dermal microvascular endothelial cells elicited a gene-expression pattern mimicking ISO-HAS cells and increased cell proliferation, an effect traced in part to NUP160 truncation. Conversely, RNAi-mediated attenuation of NUP160 in ISO-HAS cells decreased cell number. Confirming the oncogenic effects of the fusion protein, subcutaneous implantation of NUP160-SLC43A3-expressing fibroblasts induced tumors resembling human angiosarcoma. Collectively, our findings advance knowledge concerning the genetic causes of angiosarcoma, with potential implications for new diagnostic and therapeutic approaches.

Altered expression of CD63 and exosomes in scleroderma dermal fibroblasts

BACKGROUND Exosomes are small vesicles shed from various cells. They contain proteins, lipids, and nucleic acids, and are regarded as a tool of cell-cell communication. OBJECTIVES To reveal the putative role of exosomes in systemic sclerosis (SSc), and to elucidate the effect of exosomes on wound healing. METHODS The expression of common markers for exosomes (CD63, CD9, and CD81) and type I collagen were examined with real-time PCR, immunohistochemical analysis, ELISA, immunoblotting, and flow cytometry. The effect of serum-derived exosomes on wound healing was tested on full-thickness wounds in the mid-dorsal skin of BALB/c mice. RESULTS The expression levels of CD63 as well as CD9 and CD81 tended to be increased in SSc dermal fibroblasts compared to normal fibroblasts. Increased exosomes in a cultured media of SSc fibroblasts stimulated the expression levels of type I collagen in normal fibroblasts. As the mechanism, collagen-related microRNA levels in SSc fibroblast-derived exosomes were dysregulated, indicating that both the amount and the content of exosomes were altered in SSc. On the other hand, SSc sera showed significantly decreased exosome levels compared to normal sera. The frequencies of vascular involvements, including skin ulcers or pitting scars, were significantly increased in patients with decreased serum exosome levels. The healing of mice wounds was accelerated by treatment with serum-derived exosomes. CONCLUSIONS Vascular abnormalities in SSc may account for the decreased serum exosome levels by the disturbed transfer of exosomes from the skin tissue to the blood stream. Our study suggests the possibility that SSc patients with vascular involvements have decreased serum exosome levels, which causes the delay of wound healing due to down-regulation of collagen, resulting in higher susceptibility to pitting scars and/or ulcers. Exosome research will lead to a detailed understanding of SSc pathogenesis and new therapeutic approaches.

The expression of miR-124 increases in aged skin to cause cell senescence and it decreases in squamous cell carcinoma.

Skin senescence is induced by various factors including intrinsic aging and extrinsic aging. The current study compared the expression of microRNAs in young facial skin and senescent facial skin, and this study identified skin aging-related microRNAs. According to the results from a microRNA PCR Array, miR-124 was the microRNA that increased the most in senescent skin compared to young skin. Real-time PCR with a greater number of samples indicated that the increase in miR-124 levels in senescent facial skin was statistically significant. In situ hybridization was performed, and results indicated that the signal for miR-124 was evident in keratinocytes of senescent skin but not in those of young skin. The morphology of cultured normal human epidermal keratinocytes (NHEKs) transfected with a miR-124 mimic changed to an enlarged and irregular shape. In addition, the number of NHEKs positive for senescence-associated β-galactosidase (SA-β-gal) increased significantly as a result of the overexpression of the miR-124 mimic. The expression of miR-124 increased in UVB-irradiated NHEKs compared to controls in a dose-dependent manner. Expression of miR-124 in A431, a human cutaneous squamous cell carcinoma (SCC) cell line, decreased significantly compared to that in NHEKs. Forced overexpression of miR-124 as a result of the transfection of a miR-124 mimic in A431 resulted in the significant suppression of the proportion of cancer cells. The current results indicated that miR-124 increases as a result of cell senescence and that it decreases during tumorigenesis. The effect of supplementation of miR-124 in an SCC cell line suggests that senescence induction therapy with microRNA may be a new therapeutic approach for treatment of SCC.

The role of miR-210, E2F3 and ephrin A3 in angiosarcoma cell proliferation

BackgroundAlthough angiosarcoma exhibits aggressive progression and is associated with unfavourable prognosis, its pathogenesis is poorly understood.ObjectivesIn the present study, we investigated the possibility that microRNAs play a role in the pathogenesis of angiosarcoma.Materials & methodsmicroRNA expression was evaluated by array analysis and real-time PCR, and protein expression was determined by immunohistochemistry and immunoblotting.ResultsmiR-210 expression was decreased in angiosarcoma cells both in vivo and in vitro. E2F3 and ephrin A3 are putative targets of miR-210, and their protein expressionwas up-regulated in the tumour cells. Knockdown of E2F3 or ephrin A3 resulted in a significant decrease in the number of angiosarcoma cells.ConclusionFurther investigations into the regulatory mechanisms of oncogenesis associated with miR-210/E2F3/ephrin A3 signalling may lead to a new therapeutic approach against angiosarcoma.

Cell division cycle-associated protein 1 as a new melanoma-associated antigen.

Immune checkpoint inhibitors have increased the median survival of melanoma patients. To improve their effects, antigen‐specific therapies utilizing melanoma‐associated antigens should be developed. Cell division cycle‐associated protein 1 (CDCA1), which has a specific function at the kinetochores for stabilizing microtubule attachment, is overexpressed in various cancers. CDCA1, which is a member of cancer–testis antigens, does not show detectable expression levels in normal tissues. Quantitative reverse transcription polymerase chain reaction and immunoblotting analyses revealed that CDCA1 was expressed in all of the tested melanoma cell lines, 74% of primary melanomas, 64% of metastatic melanomas and 25% of nevi. An immunohistochemical analysis and a Cox proportional hazards model showed that CDCA1 could be a prognostic marker in malignant melanoma (MM) patients. CDCA1‐specific siRNA inhibited the cell proliferation of SKMEL2 and WM115 cells, but did not reduce the migration or invasion activity. These results suggest that CDCA1 may be a new therapeutic target of melanoma.

Cytokine expression profiles in the sera of cutaneous squamous cell carcinoma patients

We focused on the interaction of cytokines in squamous cell carcinoma (SCC), and determined the expression profile of multiple cytokines in the serum of each patient with SCC in the present study. Serum samples were obtained from 12 SCC patients and 7 normal subjects. Four cytokines (IFN-γ, IL-6, GM-CSF, and TGF-β) were selected because they are reported to be involved in keratinocyte proliferation and SCC progression. Serum levels were measured using ELISA. We found a statistically significant increase of serum IFN-γ levels in SCC patients compared to those in normal subjects, and areas under the curve (AUC) of 0.82 for the serum levels of IFN-γ were higher than those for other cytokine levels according to ROC curve analysis. Patients with increased IFN-γ levels had a significantly more severe cancer stage. Furthermore, the combination of IFN-γ levels and TGF-β levels could improve the AUC to 0.84. We also found there was a significant correlation between IFN-γ levels and GM-CSF levels or between GM-CSF levels and TGF-β levels only in SCC patients. Our results suggest that the combination of IFN-γ levels and TGF-β levels is more effective to diagnose SCC, while serum levels of IFN-γ alone are useful to evaluate tumor progression. Furthermore, expression of these cytokines was not independent, but may be regulated by common upstream factors (e.g. cytokines or methylation) in SCC patients, and such factors may play some roles in the pathogenesis of SCC.

Hidradenocarcinoma showing prominent mucinous and squamous differentiation and associated pagetoid cells.

Herein, we report a 63‐year‐old man presenting with hidradenocarcinoma showing prominent mucinous and squamous differentiation on his back. The tumor was dermal‐based, solid and cystic. Tumor cells with squamous differentiation and with keratin pearl formation were identified predominantly in the superficial dermis, and mucinous cells were identified principally in the cystic lesion in the deep dermis. Interestingly, the additional feature of pagetoid cells was identified in the overlying epidermis. Both the mucinous cells in hidradenocarcinoma and pagetoid cells had intracytoplasmic mucin; however, they had different histopathologic findings and immunophenotypes. Mucinous cells in hidradenocarcinoma had small nuclei and abundant intracytoplasmic mucin presenting goblet cells with low rate of positive immunostaining for p53 and Ki67. In contrast, pagetoid cells had larger nuclei with less intracytoplasmic mucin. Both p53‐ and Ki67‐positive cells were increased in pagetoid cells. Additionally, mucinous cells in hidradenocarcinoma were MUC1(+)/MUC2(−)/MUC5AC(+)/MUC6(+), but pagetoid cells were MUC1(+; focal)/MUC2(−)/MUC5AC(−)/MUC6(+; focal). The derivation of pagetoid cells is unclear; however, the localized small region of pagetoid cells over the hidradenocarcinoma in the present case may suggest a common histogenesis of these two malignant neoplasms.