Inhibition of Endoglin Exerts Antitumor Effects through the Regulation of Non-Smad TGF-ß Signaling in Angiosarcoma.

Angiosarcoma is a rare malignant tumor derived from endothelial cells, and its prognosis is poor because advanced angiosarcoma is often resistant to taxane therapy. Endoglin (CD105) acts as a coreceptor for TGF-ß signaling and is overexpressed in tumor-associated endothelial cells and enhances tumor angiogenesis. Numerous clinical trials are testing the effectiveness of anti-endoglin antibodies in various types of malignancies. Here, we investigated the role of endoglin in the pathogenesis of angiosarcoma and whether endoglin inhibition results in antitumor activity. Endoglin was overexpressed in angiosarcoma, and its inhibition was effective in promoting apoptosis and the suppression of migration, invasion, tube formation, and Warburg effect in angiosarcoma cells. Knockdown of endoglin activated caspase 3/7 that is essential for apoptosis, reduced survivin levels, and decreased paxillin and vascular endothelial cadherin phosphorylation and matrix metalloproteinase 2 and matrix metalloproteinase 9 activities in angiosarcoma cells. Although endoglin is a coreceptor that regulates TGF-ß signaling, the antitumor effect of endoglin in angiosarcoma was not based on Smad signaling regulation but on non-Smad TGF-ß signaling. Taken together, these results indicated that endoglin could be a novel therapeutic target for angiosarcoma.

A potential significance of circ_0024169 down regulation in angiosarcoma tissue.

Circular RNAs (circRNAs) are recently characterized non-coding RNAs that have a closed continuous loop. CircRNAs might play important roles in the oncogenesis of several cancers. However, little is known about association between circRNAs and skin tumors. In this study, we tried to demonstrate the expression change of circ_0024169 in angiosarcoma, and to elucidate correlations between circ_0024169 expression in angiosarcoma tissues and clinical manifestation. RNA expression was evaluated by quantitative real-time PCR with TaqMan systems for circ_0024169 and linear isoform CUL5. Both relative circRNA levels (corrected for EEF1A1 levels) and circRNA levels/linear RNA expression ratio were evaluated. We found that both relative circ_0024169 levels and circ_0024169/CUL5 ratio was decreased in normal human dermal microvascular endothelial cells (HDMEC) and angiosarcoma cell line in vitro, compared to squamous cell carcinoma line. circ_0024169/ CUL5 ratio was significantly reduced in angiosarcoma and pyogenic granuloma than other tumors in vivo, which were more evident than decreased relative circ_0024169 levels. On the other hand, relative circ_0024169 levels showed mild inverse correlation with the follow-up periods (duration between the first hospital visit and the last hospital visit/the date of death) of angiosarcoma patients. Taken together, circ_0024169/CUL5 ratio are likely to be useful as a diagnostic biomarker for vascular tumors, whereas circ_0024169 levels may have more potential as a prognostic marker of angiosarcoma. The future studies of the function of circRNAs may lead to the clarification of detailed mechanism of oncogenesis of angiosarcoma.

Hypoxia accelerates the progression of angiosarcoma through the regulation of angiosarcoma cells and tumor microenvironment.

BACKGROUND: Angiosarcoma is a rare malignant tumor with a poor prognosis. It is known that hypoxic condition activates tumor progression in several cancers. Additionally, hypoxic tumor microenvironment accelerates immune escape. However, the presence and significance of hypoxia in angiosarcoma has not been adequately investigated. OBJECTIVE: To study the role of hypoxia in the progression of angiosarcoma. METHODS: The protein level of hypoxia inducible factor-1α (HIF-1α) in angiosarcoma was examined using immunohistochemistry and immunoblotting. To study the effect of hypoxia on tumor progression, cell proliferation, migration, invasion, and tube formation assays were performed in angiosarcoma cells. The influence of tumor cell supernatant in hypoxia from angiosarcoma cells on immune escape and angiogenesis was analysed to investigate the modulatory effect of hypoxia on tumor microenvironment of angiosarcoma. The molecular mechanism related to these results was investigated using immunoblotting and real time RT-PCR. RESULTS: HIF-1α protein was over-expressed in angiosarcoma tissues and cell lines under hypoxic conditions, and there was heterogeneity of oxygen supply in angiosarcoma. Hypoxia enhanced the proliferation, migration, and invasion abilities and inhibited tube formation in angiosarcoma cells. Tumor cell supernatant in hypoxia from angiosarcoma cells activated the monocyte invasion ability, facilitated its differentiation into M2-like macrophages, and suppressed cell-adhesion. These in vitro results were compatible to the pathological findings of angiosarcoma patients. CONCLUSION: Hypoxia plays a major role in progression of angiosarcoma cells by enhancing cell proliferation, migration, and invasion and by modulating the tumor microenvironment.

Oral lichenoid reaction showing multiple ulcers associated with anti-programmed death cell receptor-1 treatment: A report of two cases and published work review.

Anti-programmed cell death receptor-1 (PD-1) antibodies represent an effective treatment opinion for advanced melanoma and non-small-cell lung cancer, as well as other cancerous entities. Immune checkpoint inhibitors such as anti-PD-1 antibody result in a unique side-effect profile, commonly described as immune-related adverse events (irAE). These irAE affect the skin, gastrointestinal tract, liver, endocrine system and other organ systems. We report two cases of oral lichenoid reaction showing multiple ulcers associated with nivolumab treatment. Both patients presented with multiple ulcers covered with fibrinous plaque over the entire oral mucosa, lips and tongue. Histopathological examination of ulceration showed epithelial necrosis and subepidermal clefts with dense band-like layers of lymphohistiocytic infiltrate within the upper dermis. Nivolumab was interrupted in both cases. Case 1 responded well to topical corticosteroids. Case 2 required oral corticosteroids, however, nivolumab could be restarted without recurrence of oral ulcers. We provide a comprehensive review of reported cases of lichenoid reaction showing multiple oral ulcers associated with anti-PD-1 therapy to date. Early recognition and management may improve treatment, avoid discontinuation of life-saving therapy and maintain quality of life in these patients.

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

BACKGROUND: Although angiosarcoma exhibits aggressive progression and is associated with unfavourable prognosis, its pathogenesis is poorly understood. OBJECTIVES: In the present study, we investigated the possibility that microRNAs play a role in the pathogenesis of angiosarcoma. MATERIALS & METHODS: microRNA expression was evaluated by array analysis and real-time PCR, and protein expression was determined by immunohistochemistry and immunoblotting. RESULTS: miR-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 expression was up-regulated in the tumour cells. Knockdown of E2F3 or ephrin A3 resulted in a significant decrease in the number of angiosarcoma cells. CONCLUSION: Further investigations into the regulatory mechanisms of oncogenesis associated with miR-210/E2F3/ephrin A3 signalling may lead to a new therapeutic approach against angiosarcoma.

Infiltration of PD-1-positive cells in combination with tumor site PD-L1 expression is a positive prognostic factor in cutaneous angiosarcoma.

Cutaneous angiosarcoma (CAS) is a malignant sarcoma with poor prognosis. Programmed cell death-1 (PD-1)/programmed cell death-1 ligand-1 (PD-L1) expression reflects antitumor immunity, and is associated with patient prognosis in various cancers. The purpose of this study is to investigate the relationship between PD-1/PD-L1 expression and CAS prognosis. CAS cases (n = 106) were immunohistochemically studied for PD-L1 and PD-1 expression, and the correlation with patient prognosis was analyzed. PD-L1 expression was assessed by flow cytometry on three CAS cell lines with or without IFNγ stimulation. A total of 30.2% of patients' samples were positive for PD-L1, and 17.9% showed a high infiltration of PD-1-positive cells. Univariate analysis showed a significant relationship between a high infiltration of PD-1-positive cells with tumor site PD-L1 expression and favorable survival in stage 1 patients (p = 0.014, log-rank test). Multivariable Cox-proportional hazard regression analysis also showed that patients with a high infiltration of PD-1-positive cells with tumor site PD-L1 expression were more likely to have favorable survival, after adjustment with possible confounders (hazard ratio (HR) = 0.38, p = 0.021, 95% confidence interval (CI) 0.16-0.86). Immunofluorescence staining of CAS samples revealed that PD-L1-positive cells were adjacent to PD-1-positive cells and/or tumor stroma with high IFNγ expression. In vitro stimulation with IFNγ increased PD-L1 expression in two out of three established CAS cell lines. Our results suggest that PD-1/PD-L1 expression is related to CAS progression, and the treatment with anti-PD-1 antibodies could be a new therapeutic option for CAS.

Immune Milk Suppresses Herpes Simplex Virus Type 1.

Immune milk has been developed as a substitute for colostrum and contains a high concentration of IgG antibodies specific to the immunized pathogens. Meanwhile, bovine herpesvirus type-1 (BHV-1) naturally infects cattle worldwide, and its antibody is found in milk. Moreover, BHV-1 glycoprotein K, the major antigen, exhibits substantial homology with human herpes virus simplex 1 (HSV-1) glycoprotein K. On the basis of this evidence, we hypothesized BHV-1 antibody exists in immune milk and suppresses HSV-1 activity. This study investigated whether immune milk IgG recognizes HSV-1 and suppresses HSV-1 activity. IgG in immune milk was purified by affinity Protein A columns, and HSV-1-reactive IgG in immune milk IgG was detected and quantified by ELISA. The efficacy of the IgG against HSV-1 was analyzed using a reduction assay based on the cytopathic effect due to HSV-1 in the presence of macrophages. We detected a high concentration of HSV-1-reactive IgG in immune milk. Furthermore, IgG suppressed HSV-1 pathogenicity in the presence of macrophages. These results indicate immune milk has protective activity against HSV-1 by opsonic activity owing to its high concentration of HSV-1-reactive IgG, which is likely the BHV-1 antibody. HSV-1 is currently a refractory infection with a worldwide distribution. Primary infection occurs via the oral cavity, but there is no effective precaution at this time. However, the present results suggest that taking oral immune milk may be an effective measure to prevent primary HSV-1 infection in the oral cavity.

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.

[Gemcitabine Monotherapy for Advanced Mycosis Fungoides--Two Case Reports and a Literature Review].

Gemcitabine, a pyrimidine nucleoside analogue, is gaining recognition as a potential therapeutic agent for advanced-stage and refractory cutaneous T-cell lymphoma (CTCL). We report of 2 patients whose advanced-stage mycosis fungoides was not sufficiently controlled by prior CHOP therapy. Both patients showed great improvement in the skin lesions with weekly gemcitabine therapy (1,000-1,200 mg/m2). The patients received four and 8 cycles of gemcitabine monotherapy, respectively, and no grade 3-4 hematological or hepatic adverse events occurred. This is the first report of the efficacy of gemcitabine for CTCL in Japan. Gemcitabine is well tolerated and is an effective monotherapy for CTCL.

Association of D2-40 and MMP-1 expression with cyst formation in lung metastatic lesions of cutaneous angiosarcoma on the scalp: immunohistochemical analysis of 23 autopsy cases.

Cutaneous angiosarcoma of the scalp can rapidly develop into pulmonary metastasis. The pulmonary metastatic lesions display a unique appearance, so-called thin-walled cysts, which cause a fatal relapsed pneumothorax by rupturing. We analyzed 23 autopsy cases of angiosarcoma with pulmonary metastasis to elucidate the mechanism of the thin-walled cyst development. Of the 23 cases of cutaneous angiosarcoma of the scalp with pulmonary metastasis, radiological examination revealed pulmonary metastatic lesions as thin-walled cysts (39%), nodules (39%), mixed cysts and nodules (13%), and ground-glass opacity (9%). All the cases but one with cystic metastases were complicated by pneumothorax. The cystic lesions were accompanied by podoplanin (D2-40)-positive tumor cells in the luminal surface of the cysts. In both primary cutaneous lesions and pulmonary metastatic lesions, the D2-40 expression was positive for angiosarcoma cells in 100% and 92% of the cases, respectively. While the estrogen-regulated gene (ERG) expression was also positive for most of the primary and metastatic pulmonary angiosarcomas, D2-40 was a more useful marker to differentiate tumor cells from the background than was the ERG expression of the vascular endothelium. Matrix metalloproteinase-1 (MMP-1) expression was also predominant in primary lesions (95%) and pulmonary metastatic lesions (82.6%). Proteinases, like MMP-1, might be associated with a developing thin-walled cyst, although there were no differences in the MMP-1 expression in either the cystic or nodular metastasis. Two extremely aggressive cases showed cystic metastasis with central necrosis that was not observed in other cases. These results suggest a pathogenesis of thin-walled cysts in some progressive cases.

Establishment and characterization of a novel lymphangiosarcoma cell line (MO-LAS) compared with the hemangiosarcoma cell line (ISO-HAS).

The concept of "lymphangiosarcoma" remains obscure. Therefore, we reported a patient with lymphangiosarcoma, resistant to immunotherapy. The patient presented with impressive and discriminative features: clinically an ill-defined edematous lesion with lymphorrhea and pathologically atypical vascular channel formation without extravasation of blood, clearly distinguished from common angiosarcoma with hemorrhage. From this case, a lymphangiosarcoma cell line, MO-LAS, was established and its characteristics were compared with the hemangiosarcoma cell line, ISO-HAS. Flow cytometric analysis revealed that MO-LAS was negative for factor VIII-related antigen, but positive for CD31, D2-40, NZ-1, and vascular endothelial growth factor receptor-3 (VEGFR-3), similar to ISO-HAS. However, MO-LAS expressed a much higher level of homeobox gene PROX1, indicating a lymphatic phenotype, compared with ISO-HAS. Furthermore, MO-LAS showed a much lesser expression of oncogenes and much lower sensitivity against lymphokine-activated killer (LAK) cells. Lymphangiosarcoma may be difficult to recognize by the immune system. Conclusively, the establishment of MO-LAS, a novel angiosarcoma cell line bearing lymphatic characters, strongly suggests the entity of lymphangiosarcoma.

Attenuation of Mycobacterium tuberculosis functionally disrupted in a fatty acyl-coenzyme A synthetase gene fadD5.

One key adaptation that Mycobacterium tuberculosis established to survive long term in vivo is a reliance on lipids as an energy source. M. tuberculosis H37Rv has 36 fadD genes annotated as putative fatty acyl-coenzyme A (CoA) synthetase genes, which encode enzymes that activate fatty acids for metabolism. One such gene, fadD5 (Rv0166), is located within the mce1 operon, a cluster of genes associated with M. tuberculosis persistence. We disrupted the putative fatty acid-binding site of fadD5 in H37Rv M. tuberculosis. No significant differences were found in the growth of the mutant and wild-type strains in vitro in nutrient-rich broth or in activated RAW264.7 cells. However, the fadD5 mutant was diminished in growth in minimal medium containing mycolic acid but not other long-chain fatty acids. C57BL/6 mice infected with the fadD5 mutant survived significantly longer than those infected with the wild type, and the mutant never attained the plateau phase of infection in mouse lungs. Infection in the steady-state phase was maintained for up to 168 days at a level that was 1-2 logs less than that noted in the wild type. These observations raise the rather intriguing possibility that FadD5 may serve to recycle mycolic acids for the long-term survival of the tubercle bacilli.