BRAF inhibition promotes ER stress-mediated cell death in uveal melanoma.

Melanoma with a BRAF mutation is more common to develop into a fatal disease. BRAF mutation inhibitor-induced autophagy affects the drug efficacy in many cancer types. The role of autophagy during BRAF inhibition in uveal melanoma (UM) remains unclear. In this study, we examined the autophagic flux and compared the number of autophagic vacuoles during the BRAF inhibition in UM. The PKR-like endoplasmic reticulum (ER) kinase (PERK) arm was studied to test whether the ER stress was involved. The effects of downregulation of ER stress by targeting the PERK arm (pharmacologically and genetically) were also assessed. We found a dose-dependent increase of autophagic flux in OCM1A cells during the BRAF inhibition. This phenomenon was further verified by an enhanced number of GFP-LC3 puncta and was finally confirmed by raised autophagic index examined by transmission electron microscopy. Pathway analysis revealed that the vemurafenib (the BRAF inhibitor)-induced autophagy was independent of the MAPK signaling pathway. Instead, it was possibly regulated via the enhanced ER stress response. We further found that the inhibition of ER stress response rescued cell death. Therefore, our results suggest BRAF inhibition promotes ER stress response-induced autophagy in UM. Targeting ER stress response can partially revert autophagy and rescue cell death, which may impair the anti-tumor effect of BRAF inhibitor in UM.

MiR-423-5p activated by E2F1 promotes neovascularization in diabetic retinopathy by targeting HIPK2.

BACKGROUND: Diabetic retinopathy (DR) is a diabetic complication and the primary cause of blindness in the world. However, the treatments of DR are challenging given its complicated pathogenesis. Here, we investigated the molecular mechanisms of DR by focusing on the function of E2F1/miR-423-5p/HIPK2/HIF1α/VEGF axis. METHODS: Cultured retinal endothelial cells (hRMECs, hRECs) were treated with 25 mM glucose to mimic the high glucose-induced DR in vitro. Streptozotocin (STZ) was injected into mice to induce DR in mice. qRT-PCR, western blotting, immunohistochemistry, and ELISA were employed to measure levels of E2F1, miR-423-5p, HIPK2, HIF1α, and VEGF. H&E staining was utilized to examine retinal neovascularization. CCK-8 assay, transwell assay, and vascular tube formation assay were used to assess the cell viability, migration, and angiogenesis. Dual luciferase assay was performed to validate interactions between E2F1 and miR-423-5p, miR-423-5p and HIPK2. RESULTS: HG treatment increased the cell viability, migration, and angiogenesis accompanied by upregulation of E2F1, miR-423-5p, HIF1α, and VEGF levels, but reduction in HIPK2 expression. Knockdown of E2F1 or miR-423-5p suppressed the HG-induced increases in cell viability, migration, and angiogenesis. E2F1 transcriptionally activated miR-423-5p expression and miR-423-5p mimics blocked the effects of E2F1 knockdown on angiogenesis. Moreover, miR-423-5p directly targeted HIPK2 to disinhibit HIF1α/VEGF signaling. Knockdown of HIPK2 reversed the effects of miR-423-5p inhibitor on cell viability, migration, and angiogenesis. Knockdown of E2F1 suppressed neovascularization during DR in vivo. CONCLUSIONS: E2F1 activates miR-423-5p transcription during DR to promote angiogenesis via suppressing HIPK2 expression to disinhibit HIF1α/VEGF signaling. Strategies targeting E2F1/miR-423-5p/HIPK2 axis could be potentially used for DR treatment.

Identification of Versican as an Independent Prognostic Factor in Uveal Melanoma.

OBJECTIVE: To assess the role of versican (VCAN) in uveal melanoma (UVM) from its expression, prognostic value and biological function. METHODS: The general profile of VCAN mRNA and protein expression levels were obtained using bioinformatic approaches. Then, UALCAN database was adopted to examine the association of VCAN mRNA expression and clinical factors in UVM. The prognostic value of VCAN was assessed by UALCAN, GEPIA and TISIDB databases. Besides, Cox regression analysis was performed to predict the independent prognostic factors for UVM. Further, functional enrichment analysis was conducted to reveal the biological functions of VCAN involved in UVM through DAVID, Cytoscape and GSEA analyses. RESULTS: VCAN showed a relative low expression level in normal eye but was highly expressed in UVM cell lines. Tumor histology and stage in UVM were significantly related to VCAN mRNA expression (all P <0.05). Besides, high VCAN mRNA expression led to unfavorable prognosis of UVM patients, especially in female patients and those aged <60 years (all P <0.05). Cox regression analysis indicated that VCAN mRNA expression was an independent prognostic factor for overall survival in UVM. Enrichment analysis suggested that VCAN was mainly involved in cytokine-cytokine receptor interaction, chemokine signaling pathway and T cell receptor signaling pathway (all P <0.05). Meanwhile, hyaluronic acid was revealed to be a potential drug for the UVM treatment. CONCLUSION: VCAN served as an independent prognostic factor for UVM. Further analysis found that VCAN was positively correlated with metastasis-related pathway, which might imply the metastasis risk of UVM. Our study initially revealed the vital role of VCAN in the process of UVM and provided a therapeutic target for UVM treatment.

A Comparison of the Seventh and Eighth Editions of the AJCC Staging Systems to Predict Recurrence in Papillary Thyroid Microcarcinoma.

BACKGROUND: The incidence of papillary thyroid microcarcinoma has been constantly rising in recent decades. The tumor, node, metastasis staging system is designed to predict prognosis in patients with papillary thyroid carcinoma. Recent studies have shown that the American Joint Committee on Cancer (AJCC) 8th edition is superior to the 7th edition for predicting tumor recurrence in PTC patients. To date, whether the 8th edition is also better able to predict recurrence in papillary thyroid microcarcinoma (PTMC) remains unclear. METHOD: We enrolled 1007 cases from our thyroid cancer database in the First Affiliated Hospital, Zhejiang University School of Medicine, from 1997 to 2011. Univariable and multivariate Cox hazard regression analyses were used to identify the association between variables and recurrence. Disease-free survival was calculated using the Kaplan-Meier method and compared using the log-rank test. RESULTS: A total of 1007 PTMC patients were enrolled, with a median follow-up of 67 months. Of 93 (9.2%) patients downstaged by the changes in versions, 49 (52.7%) were downstaged because the age-at-diagnosis cut-off used for staging increased from 45 to 55 years, while 35 (37.6%) were downstaged due to the weakening of the effects of lymph node metastasis. The recurrence rate of PTMC was 4.17%. Univariate Cox hazards regression analyses showed that TNM stage according to the AJCC 8th edition was significantly associated with recurrence, while the recurrence survival curves showed that TNM stage (stage I vs. stage II-IV) according to the AJCC 8th edition, but not the 7th edition, was significantly associated with disease-free survival (p < 0.05). CONCLUSIONS: The AJCC 8th edition has better ability to predict recurrence in PTMC patients than the 7th edition.

NFE2/miR-423-5p/TFF1 axis regulates high glucose-induced apoptosis in retinal pigment epithelial cells.

BACKGROUND: A study has shown that miR-423-5p is highly expressed in proliferative diabetic retinopathy. However, the exact biological functions and mechanisms of miR-423-5p in diabetic retinopathy (DR) progression are currently unclear. This study aimed to investigate the role of miR-423-5p in DR and the underlying mechanism. RESULTS: Our data demonstrate that the expression of miR-423-5p is significantly increased in HG-induced RPE cells and DR patient plasma. Moreover, the overexpression of miR-423-5p exacerbates HG-induced apoptosis. Mechanistically, our results provide evidence that miR-423-5p directly targets TFF1. MiR-423-5p exerts its effect on HG-induced apoptosis in RPE cells through TFF1, and the NF-κB pathway is involved in the regulatory mechanism. Further analysis revealed that the transcription factor NFE2 regulates miR-423-5p promoter activity. In addition, NFE2 regulates the levels of TFF1 and NF-κB pathway-associated proteins by regulating the expression of miR-423-5p. CONCLUSION: The NFE2-miR-423-5p-TFF1 axis is a novel molecular mechanism and provides a new direction for the study and treatment of DR.

Targeting Autophagy Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib.

Context: The RAF inhibitor vemurafenib has provided a major advance for the treatment of patients with BRAF-mutant metastatic melanoma. However, BRAF-mutant thyroid cancer is relatively resistant to vemurafenib, and the reason for this disparity remains unclear. Anticancer therapy-induced autophagy can trigger adaptive drug resistance in a variety of cancer types and treatments. To date, role of autophagy during BRAF inhibition in thyroid cancer remains unknown. Objective: In this study, we investigate if autophagy is activated in vemurafenib-treated BRAF-mutant thyroid cancer cells, and whether autophagy inhibition improves or impairs the treatment efficacy of vemurafenib. Design: Autophagy level was determined by western blot assay and transmission electron microscopy. The combined effects of autophagy inhibitor and vemurafenib were assessed in terms of cell viability in vitro and tumor growth rate in vivo. Whether the endoplasmic reticulum (ER) stress was in response to vemurafenib-induced autophagy was also analyzed. Results: Vemurafenib induced a high level of autophagy in BRAF-mutant thyroid cancer cells. Inhibition of autophagy by either a pharmacological inhibitor or interfering RNA knockdown of essential autophagy genes augmented vemurafenib-induced cell death. Vemurafenib-induced autophagy was independent of MAPK signaling pathway and was mediated through the ER stress response. Finally, administration of vemurafenib with the autophagy inhibitor hydroxychloroquine promoted more pronounced tumor suppression in vivo. Conclusions: Our data demonstrate that vemurafenib induces ER stress response-mediated autophagy in thyroid cancer and autophagy inhibition may be a beneficial strategy to sensitize BRAF-mutant thyroid cancer to vemurafenib.

BRAF V600E-dependent role of autophagy in uveal melanoma.

BACKGROUND: Autophagy can function in a dual role in cancer development and progression: It can be cytoprotective or contribute to cell death. Therefore, determining the contextual role of autophagy between these two opposing effects is important. So far, little is known about the role of autophagy in uveal melanoma. In the present study, we looked to investigate the autophagic process, as well as its effect on cell survival in uveal melanoma cell lines under stressed conditions (starvation). The possible role of autophagy during BRAF inhibition in uveal melanoma was also sought. METHODS: Two human uveal melanoma cell lines, OCM1A, which harbors the BRAF mutation V600E and Mel 290, which is BRAF wild type, were studied. Autophagy levels were determined by Western blot assay with/without the addition of autophagic flux inhibitor (bafilomycin A1). Cell proliferation was assessed by an MTT assay. RESULTS: Starvation triggered autophagy in BRAF V600E-mutant OCM1A cells but not in BRAF wild-type Mel 290 cells. Enhanced autophagy helped the OCM1A cells survive under stressed conditions. The BRAF inhibitor vemurafenib upregulated autophagy through suppression of the PI3K/Akt/mTOR/p70S6 K pathway in BRAF V600E-mutant uveal melanoma cells. Autophagy inhibition impaired the treatment efficacy of vemurafenib in BRAF V600E-mutant uveal melanoma cells. CONCLUSIONS: Our data demonstrate that starvation-trigged autophagy, which is BRAF V600E dependent, promotes cancer cell survival in uveal melanoma. Vemurafenib induces autophagic cell death rather than adaptive cell survival in BRAF V600E-mutant melanoma.

Comparison of the clinicopathologic features and prognosis of bilateral versus unilateral multifocal papillary thyroid cancer: An updated study with more than 2000 consecutive patients.

BACKGROUND: Bilaterality is common in papillary thyroid cancer (PTC), but its clinical and prognostic implications are still controversial, and it remains unclear whether its behavior is more aggressive than multifocality. METHODS: The clinicopathologic features of 2211 consecutive patients with PTC who underwent surgical treatment at the authors' institute between 1997 and 2011 were reviewed. Among these surgical patients, 425 (19.2%) had bilateral PTCs, and 1786 had unilateral PTCs. The patients who had unilateral PTCs were subdivided into a group with unilateral-multifocal PTCs (210 patients) and a group with solitary PTCs (1576 patients). The 10-year disease-free survival (DFS) rates were calculated to compare the prognosis between groups. B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutation status was examined by direct DNA sequencing. RESULTS: Patients who had bilateral PTCs were likely to have larger tumors, higher rates of extrathyroid extension and lymph node metastasis, and more advanced tumor stage than those who had unilateral-multifocal PTCs. Multivariate analysis identified only lymph node metastasis as an independent risk factor for PTC recurrence (P < .001). The 10-year DFS rate for patients with bilateral PTCs was much lower than that for those with unilateral-multifocal and solitary PTCs (78.8% vs 85.7% and 89.3%, respectively; P = .005). It is noteworthy that patients who had bilateral PTCs with lymph node metastasis had the worst prognosis in terms of DFS. Incidence of the BRAF V600E mutation (valine to glutamic acid mutation at position 600) was higher in the bilateral PTC group than that in the unilateral and unilateral-multifocal PTC groups. CONCLUSIONS: The current results provide initial evidence that bilateral PTCs are more aggressive than unilateral-multifocal PTCs, and patients who have bilateral disease have more advanced stage and shorter DFS. The poorer outcome of patients with bilateral PTCs may be caused in part by their high incidence of lymph node metastasis. Cancer 2016;122:198-206. © 2015 American Cancer Society.