SHROOM2 inhibits tumor metastasis through RhoA-ROCK pathway-dependent and -independent mechanisms in nasopharyngeal carcinoma.

SHROOM2 is a key mediator of RhoA-ROCK pathway that regulates cell motility and actin cytoskeleton organization. However, the functions of SHROOM2 beyond RhoA/ROCK signaling remain poorly understood. Here, we report that SHROOM2 not only participates in RhoA-ROCK-induced stress fiber formation and focal adhesion, but also had an unanticipated role in suppressing epithelial-to-mesenchymal transition (EMT) and tumor metastasis. Depletion of SHROOM2 in nasopharyngeal carcinoma (NPC) cells enhances mesenchymal characteristics and reduces epithelial markers, concomitant with increased motility, enabling the development of invasion and tumor metastasis, which are largely ROCK-independent, as ROCK inhibitor Y-27632 did not cause EMT phenotype; furthermore, combination of ROCK inhibition and SHROOM2 depletion resulted in the most robust increases in cell migration and invasion, indicating that SHROOM2 and ROCK work synergistically rather than epistatic. Analysis of clinical samples suggested that SHROOM2 is downregulated in NPC and the expression of SHROOM2 in metastatic NPC was even lower than in the primary tumors. Our findings uncover a non-canonical role of SHROOM2 as a potent antagonist for EMT and NPC metastasis.

BET bromodomain inhibitor JQ1 preferentially suppresses EBV-positive nasopharyngeal carcinoma cells partially through repressing c-Myc.

The management of advanced nasopharyngeal carcinoma (NPC) remains a challenge. The ubiquitous nature of Epstein-Barr virus (EBV) infection in nonkeratinizing NPC has forced us to investigate novel drugs for NPC in the presence of EBV. In this study, we performed a small-scale screening of a library of compounds that target epigenetic regulators in paired EBV-positive and EBV-negative NPC cell lines. We found that bromodomain and extra-terminal (BET) inhibitor JQ1 preferentially inhibits the growth of EBV-positive NPC cells. JQ1 induces apoptosis, decreases cell proliferation and enhances the radiosensitivity in NPC cells, especially EBV-positive cells. Significantly, JQ1-induced cell death is c-Myc-dependent. Notably, RNA-seq analysis demonstrated that JQ1 represses TP63, TP53 and their targets. JQ1 also lessens the expression of PD-L1 in NPC. Moreover, the high potency of JQ1 in NPC cells was further confirmed in vivo in CNE2-EBV+ tumor-bearing mice. These findings indicate that JQ1 is a promising therapeutic candidate for advanced NPC.

Epstein-Barr virus activates F-box protein FBXO2 to limit viral infectivity by targeting glycoprotein B for degradation.

Epstein-Barr virus (EBV) is a human cancer-related virus closely associated with lymphoid and epithelial malignancies, and EBV glycoprotein B (gB) plays an essential role in viral entry into both B cells and epithelial cells by promoting cell-cell fusion. EBV gB is exclusively modified with high-mannose-linked N-glycans and primarily localizes to the endoplasmic reticulum (ER) with low levels on the plasma membrane (PM). However, the mechanism through which gB is regulated within host cells is largely unknown. Here, we report the identification of F-box only protein 2 (FBXO2), an SCF ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans and attenuates EBV infectivity by targeting N-glycosylated gB for degradation. gB possesses seven N-glycosylation sites, and FBXO2 directly binds to these high-mannose moieties through its sugar-binding domain. The interaction promotes the degradation of glycosylated gB via the ubiquitin-proteasome pathway. Depletion of FBXO2 not only stabilizes gB but also promotes its transport from the ER to the PM, resulting in enhanced membrane fusion and viral entry. FBXO2 is expressed in epithelial cells but not B cells, and EBV infection up-regulates FBXO2 levels. In summary, our findings highlight the significance of high-mannose modification of gB and reveal a novel host defense mechanism involving glycoprotein homeostasis regulation.

TNFRSF19 Inhibits TGFß Signaling through Interaction with TGFß Receptor Type I to Promote Tumorigenesis.

Genetic susceptibility underlies the pathogenesis of cancer. We and others have previously identified a novel susceptibility gene TNFRSF19, which encodes an orphan member of the TNF receptor superfamily known to be associated with nasopharyngeal carcinoma (NPC) and lung cancer risk. Here, we show that TNFRSF19 is highly expressed in NPC and is required for cell proliferation and NPC development. However, unlike most of the TNF receptors, TNFRSF19 was not involved in NFκB activation or associated with TRAF proteins. We identified TGFß receptor type I (TßRI) as a specific binding partner for TNFRSF19. TNFRSF19 bound the kinase domain of TßRI in the cytoplasm, thereby blocking Smad2/3 association with TßRI and subsequent signal transduction. Ectopic expression of TNFRSF19 in normal epithelial cells conferred resistance to the cell-cycle block induced by TGFß, whereas knockout of TNFRSF19 in NPC cells unleashed a potent TGFß response characterized by upregulation of Smad2/3 phosphorylation and TGFß target gene transcription. Furthermore, elevated TNFRSF19 expression correlated with reduced TGFß activity and poor prognosis in patients with NPC. Our data reveal that gain of function of TNFRSF19 in NPC represents a mechanism by which tumor cells evade the growth-inhibitory action of TGFß.Significance:TNFRSF19, a susceptibility gene for nasopharyngeal carcinoma and other cancers, functions as a potent inhibitor of the TGFß signaling pathway.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3469/F1.large.jpg Cancer Res; 78(13); 3469-83. ©2018 AACR.

OVOL2 links stemness and metastasis via fine-tuning epithelial-mesenchymal transition in nasopharyngeal carcinoma.

Rationale: Metastasis is the leading cause of disease-related death among patients with nasopharyngeal carcinoma (NPC). Mounting evidence suggest that epithelial-mesenchymal transition (EMT) is crucial for cancer cells to acquire metastatic ability. In this study, we aim to clarify the extent to which EMT is involved in various cancer properties and identify novel markers for predicting the prognosis of NPC patients. Methods: Two cellular models derived from the same NPC cell line with distinct metastasis ability were used for microarray analysis to identify key transcriptional factors that drive metastasis. Cell migration and invasion were analyzed by wound healing and Transwell analysis. Lung metatasis was determined by tail vein injection assay. Cancer stemness was analyzed using colony formation and xenograft assay. The EMT extent was evaluated using immunoblotting, RT-qPCR and immunofluorescence of EMT markers. The value of OVOL2 in prognosis was determined by immunohistochemistry in NPC biopsies. Results: OVOL2 was the most significantly down-regulated EMT transcription factor (EMT-TF) in cellular models of NPC metatasis. Low levels of OVOL2 were associated with poor overall survival of NPC patients and the reduced expression is partly due to promoter methylation and epithelial dedifferentiation. Knockout of OVOL2 in epithelial-like NPC cells partially activates EMT program and significantly promotes cancer stemness and metastatic phenotypes. Conversely, ectopically expression of OVOL2 in mesenchymal-like cells leads to a partial transition to an epithelial phenotype and reduced malignancy. Reversing EMT by depleting ZEB1, a major target of OVOL2, does not eliminate the stemness advantage of OVOL2-deficient cells but does reduce their invasion capacity. A comparison of subpopulations at different stages of EMT revealed that the extent of EMT is positively correlated with metastasis and drug resistance; however, only the intermediate EMT state is associated with cancer stemness. Conclusion: Distinct from other canonical EMT-TFs, OVOL2 only exhibits modest effect on EMT but has a strong impact on both metastasis and tumorigenesis. Therefore, OVOL2 could serve as a prognostic indicator for cancer patients.

A novel Smac mimetic APG-1387 demonstrates potent antitumor activity in nasopharyngeal carcinoma cells by inducing apoptosis.

Despite advances in the development of radiation against nasopharyngeal carcinoma (NPC), the management of advanced NPC remains a challenge. Smac mimetics are designed to neutralize inhibitor of apoptosis (IAP) proteins, thus reactivating the apoptotic program in cancer cells. In this study, we investigated the effect of a novel bivalent Smac mimetic APG-1387 in NPC. In vitro, APG-1387 in combination with TNF-α potently decreased NPC cell viability by inducing apoptosis in majority of NPC cell lines. The in vitro antitumor effect was RIPK1-dependent, whereas it was independent on IAPs, USP11, or EBV. Of note, the inhibition of NF-κB or AKT pathway rendered resistant NPC cells responsive to the treatment of APG-1387/TNF-α. In vivo, APG-1387 displayed antitumor activity as a single agent at well-tolerated doses, even in an in vitro resistant cell line. In summary, our results demonstrate that APG-1387 exerts a potent antitumor effect on NPC. These findings support clinical evaluation of APG-1387 as a potential treatment for advanced NPC.