Shengnan Chen

Oxidored-nitro domain containing protein 1 (NOR1) expression suppresses slug/vimentin but not snail in nasopharyngeal carcinoma: Inhibition of EMT in vitro and in vivo in mice

Oxidored-nitro domain containing protein 1 (NOR1) is a putative tumor suppressor gene. In this study, NOR1 expression was detected in NPC tissues and non-cancerous nasopharyngeal epithelium. The data showed that NOR1 protein was decreased in NPC tissues. Lost expression NOR1 protein was associated with poor overall and event-free survival of NPC patients. Overexpression of NOR1 in NPC cells resulted in a significant morphological change and decreased expression of epithelial-to-mesenchymal transition (EMT) mediators (e.g., slug and vimentin), but induced cytokeratin 13 expression. A nude mouse metastasis assay revealed that overexpression of NOR1 decreased NPC tumor cells metastasis capacity in vivo. Knockdown of NOR1 expression in HeLa cells was sufficient to abrogate epithelial traits and to enhance cell migration and invasion. Concomitant inhibition of slug or vimentin alleviated induction of EMT, migration or invasion by NOR1 siRNA in HeLa cells in vitro. In conclusion, the data from the current study suggest, for the first time, that NOR1 plays an important role in NPC in ex vivo, in vitro, and in vivo.

Significance of the NOR1-FOXA1/HDAC2-Slug regulatory network in epithelial-mesenchymal transition of tumor cells.

The epithelial-mesenchymal transition (EMT) process is believed to play a crucial role in nasopharyngeal carcinoma (NPC) progression, a squamous cell carcinoma of the head and neck with the tendency to metastasize early. At present, much attention has been given to the inducer of EMT involved in NPC progression, while antagonists have been less intensively characterized. In this study, unbiased analysis of EMT-associated gene expression patterns was performed using data mining of global gene expression profiles derived from NPC samples, leading to the successful identification of NOR1, FOXA1, and Slug, all of which showed aberrant expression during NPC progression. The effect of tumor suppressor NOR1 on Slug-induced NPC cells during the EMT process was investigated by use of ectopic expression and RNA interference methods. The molecular mechanisms underlying the tumor-suppressing effect of NOR1 on Slug-induced EMT were thought to be dependent on the cooperation of NOR1 with the FOXA1-HDAC2 complex. We also showed that FOXA1 and HDAC2 bind the slug promoter and directly repress its transcription. Our data revealed a previously unrecognized role of the NOR1-FOXA1/HDAC2-Slug network in the regulation of the EMT process and aggressiveness of NPC.

NOR1 Suppresses Cancer Stem-Like Cells Properties of Tumor Cells via the Inhibition of the AKT-GSK-3β-Wnt/β-Catenin-ALDH1A1 Signal Circuit.

Cancer stem cells (CSCs) play a key role in tumor radiotherapy and chemotherapy resistance, relapse, and metastasis, and are primarily maintained in a resting state in vivo. The failure of conventional therapies to target CSCs is the main cause of treatment failure. The discovery of CSCs in nasopharyngeal carcinoma (NPC) tumors is becoming more prevalent; however, the understanding of the mechanisms underlying the maintenance of tumor stemness is still limited. We previously cloned NOR1, a tumor suppressor gene downregulated in NPC cell lines and tissues. In this study, we demonstrate that Wnt/β‐catenin and ALDH1A1 form a signal circuit and that NOR1 antagonizes the tumor stem cell‐like phenotype in NPC cell lines: the ectopic overexpression of NOR1 reduced β‐catenin and ALDH1A1 expression; β‐catenin/TCF4 targeted the regulation of ALDH1A1 transcription in NPC cells; silencing ALDH1A1 reduced AKT (total and phosphorylated) and GSK‐3β (phosphorylated) expression; and eventually feedback decreased β‐catenin expression levels. We also found that NOR1 expression decreased cancer stem‐like cell properties of NPC cells, reduced their ability to form tumor spheroids in vitro, reduced tumorigenicity in nude mice in vivo, and increased sensitivity to chemotherapy agents. Taken together, our findings illustrated a new function of NOR1 that suppresses cancer stem‐like cell properties in tumor cells by inhibiting the AKT‐GSK‐3β‐Wnt/β‐catenin‐ALDH1A1 signal circuit. The study suggests that NOR1 deletion expression in NPC cells may be a potential molecular target for cancer stem cell therapy. J. Cell. Physiol. 232: 2829–2840, 2017.

Vimentin is a crucial target for anti-metastasis therapy of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck cancer, with tendency to spread to regional lymph nodes and distant organs at early stage. Vimentin, a major cytoskeletal protein constituent of the intermediate filament, plays a critical role in the epithelial–mesenchymal transition. Overexpression of vimentin is considered to be a critical prerequisite for metastasis in numerous human cancers. Therefore, targeting vimentin for cancer therapy has gained a lot of interest. In the present study, we detected vimentin expression in NPC tissues and found that overexpression of vimentin is associated with poor prognosis of NPC patients. Silencing of vimentin in NPC CNE2 cells by RNAi suppresses cells migration and invasion in vitro. However, blocking vimentin did not affect cell proliferation of CNE2 cells. In addition, the in vivo metastatic potential of CNE2 cells transfected with Vimentin shRNA was suppressed in a nude mouse model of pulmonary metastasis. Silencing of Vimentin in CNE2 cells leads to a decrease of microvessel density and VEGF, CD31, MMP2, and MMP9 expressions in pulmonary metastatic tumors. Importantly, we found that it is easier for the tumor cells from the high vimentin-expressing pulmonary metastatic tumors to reinvade the microvessel and to form stable tumor plaques attached to the endothelial cells, which resemble the resource of circulating tumor cells and are very hard to eliminate. However, depletion of vimentin inhibits the formation of vascular tumor plaques. Our findings suggest that RNAi-based vimentin silencing may be a potential and promising anti-metastatic therapeutic strategy for NPC.

Abberent expression of NOR1 protein in tumor associated macrophages contributes to the development of DEN-induced hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver and the sixth most common lethal cancer worldwide. Recent evidences demonstrated that oxidored nitro domain containing protein 1 (NOR1), a putative tumor suppressor gene, is overexpressed in human HCC tissues. However, the role of NOR1 in HCC development remains unclear. Here, we described that NOR1 protein level is elevated in HCC and is associated with poorer clinical outcome. However, ecotopic overexpression of NOR1 protein in human HCC cell line HepG2 cells had no effect on cells proliferation, migration, and clonality. Immunofluoresence assay indicates NOR1 protein is mainly expressed at CD163 positive M2 tumor associated macrophages (TAMs). To explore the role of NOR1 in the development of HCC, we interrogated the susceptibility of mice lacking the NOR1 gene to DEN‐induced hepatocarcinogenesis. NOR1 deficient mice displayed resistance to DEN‐induced HCC. We also demonstrate that mNOR1 protein is enriched in F4/80 positive Kupffer cells (KCs) infiltrated in DEN induced murine HCC tissues. Loss of NOR1 led to increase of iNOS whereas decrease of Arg1, Ym1 expression in KCs. Overexpression of NOR1 in THP‐1 macrophages led to decrease of iNOS but increase of Arg1. Mechanistic investigations showed that inflammatory cytokines IL‐6, TNF‐α production, and NF‐κB activation were also decreased in NOR1 knockout mice exposed to DEN treatment. Our data suggested that NOR1 is overexpressed in HCC associated TAMs and promotes M2 alternative polarization. Genetic deletion of NOR1 in mice leads to impairment of IL‐6 production and NF‐κB activation, which in turn attenuates DEN‐induced HCC development.

Rediscovery of NF-κB signaling in nasopharyngeal carcinoma: How genetic defects of NF-κB pathway interplay with EBV in driving oncogenesis?

Nasopharyngeal carcinoma (NPC) is a unique EBV‐associated subtype of head and neck cancer, which has the highest incidence in Southern China and eastern South Asia. The interaction between genetic risk factors and environmental challenge, have been considered to contribute to the development of nasopharyngeal carcinogenesis. Constitutive activation of NF‐κB signaling has been seen in NPC tissues and is associated with unfavorable prognosis. Recently, several whole exome sequencing study consistently revealed that high frequency mutations of NF‐κB pathway negative regulators is common in nasopharyngeal carcinoma, which reinforce the importance of NF‐κB driving oncogenesis. This review focuses on the current state of research in role of NF‐κB in NPC carcinogenesis. We summarized the newly identified loss of function (LOF) mutations on NF‐κB negative regulators leading to its activation bypass LMP‐1 stimulation. We discussed the critical role of NF‐κB activation in immortalization and transformation of nasopharygeal epithelium. We also depicted how NF‐κB signaling mediated chronic inflammation contribute to persistent EBV infection, immune evasion of EBV infected cells, metabolic reprogramming, and cancer stem cells (CSCs) formation in NPC. Lastly, we discussed the clinical resonance of targeting NF‐κB for NPC precise therapy.

Emerging role of lipid metabolism alterations in Cancer stem cells

BackgroundCancer stem cells (CSCs) or tumor-initiating cells (TICs) represent a small population of cancer cells with self-renewal and tumor-initiating properties. Unlike the bulk of tumor cells, CSCs or TICs are refractory to traditional therapy and are responsible for relapse or disease recurrence in cancer patients. Stem cells have distinct metabolic properties compared to differentiated cells, and metabolic rewiring contributes to self-renewal and stemness maintenance in CSCs.Main bodyRecent advances in metabolomic detection, particularly in hyperspectral-stimulated raman scattering microscopy, have expanded our knowledge of the contribution of lipid metabolism to the generation and maintenance of CSCs. Alterations in lipid uptake, de novo lipogenesis, lipid droplets, lipid desaturation, and fatty acid oxidation are all clearly implicated in CSCs regulation. Alterations on lipid metabolism not only satisfies the energy demands and biomass production of CSCs, but also contributes to the activation of several important oncogenic signaling pathways, including Wnt/β-catenin and Hippo/YAP signaling. In this review, we summarize the current progress in this attractive field and describe some recent therapeutic agents specifically targeting CSCs based on their modulation of lipid metabolism.ConclusionIncreased reliance on lipid metabolism makes it a promising therapeutic strategy to eliminate CSCs. Targeting key players of fatty acids metabolism shows promising to anti-CSCs and tumor prevention effects.

Dual-functionality of RASSF1A overexpression in A375 cells is mediated by activation of IL-6/STAT3 regulatory loop

The RASSF1A, a microtubule associated protein, is a well-known tumor suppressor silenced in various cancer via promoter hypermethylation. RASSF1A is implicated in the regulation of cellular proliferation and apoptosis. However, its role in melanoma A375 cells invasion and metastasis remain unclear. Here, we report an unusual dual function role of ectopic RASSF1A in A375 cells. RASSF1A suppressed A375 cells proliferation but enhanced cells migration, invasiveness and metastatic potential in vivo. We demonstrated RASSF1A simultaneously up-regulated p21 and vimentin expression in A375 cells. Increase of vimentin expression contributes to RASSF1A mediated enhancement of cells mobility and invasion. Transcriptome assay unclosed that RASSF1A promoted IL-6 expression in A375 cells, which in turn activate JAK2/STAT3 signaling. Treatment with recombinant IL-6 enhanced both p21 and vimentin protein level in the empty vector transfected A375 cells to similar level as RASSF1A expressing cells. In contrast, knockdown IL-6 expression by siRNAs decreased p21 and vimentin level in RASSF1A expressing cells. Blockade of JAK2/STAT3 signaling by use of JAK2 inhibitor WP1066 led to decrease of IL-6, p21 and vimentin protein in RASSF1A expressing cells. Our findings unclosed a unusual dual functionality of ectopic RASSF1A overexpression in A375 cells by regulating IL-6/STAT3 regulatory loop, suggesting it should be cautious about the safety of RASSF1A-based gene therapy.

Correction to: Emerging role of lipid metabolism alterations in Cancer stem cells

In the publication of this article [1], there is an error in the Figure caption of Figs. 2, 3 and 4. This has now been included in this correction. The authors declare that these corrections do not change the results or conclusions of this paper.

FOXA1 Reprograms the TGF-β-Stimulated Transcriptional Program from a Metastasis Promoter to a Tumor Suppressor in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck squamous carcinoma that is notorious for its high metastatic potential. In this study, we reported that FOXA1 protein was decreased in NPC cells. Loss of FOXA1 is associated with lymph node metastasis and poor prognosis. Silencing FOXA1 in NP69 and C666-1 NPC cells accelerated cell proliferation and migration, while re-expression of FOXA1 has opposite effects. Microarray and RNA-seq analysis revealed that re-expression of FOXA1 in NPC cells reprogrammed the TGF-β-stimulated transcription program, which is characterized by promotion of TGF-β-inducible tumor-suppressive targets but repression of TGF-β-inducible oncogenes expression in NPC cells, leading to restoration of NPC cell sensitivity to TGF-βs growth-inhibitory effect. BAMBI, a TGF-β responsive tumor suppressor, was induced by FOXA1 in NPC cells. FOXA1 binding on the BAMBI gene facilitated SMAD2/3 binding to the BAMBI promoter via increasing BAMBI associated H3K4me1 and H3K27ac modification. Enforced expression of BAMBI in NPC cells suppressed cell proliferation and invasiveness. Our data suggested that FOXA1 is a master factor in controlling the TGF-β-stimulated transcriptome and a regulator of TGF-β biological functions in NPC oncogenesis.