Artemisinin suppressed tumour growth and induced vascular normalisation in oral squamous cell carcinoma via inhibition of macrophage migration inhibitory factor.

BACKGROUND: Tumour vascular normalisation therapy advocates a balance between pro-angiogenic factors and anti-angiogenic factors in tumours. Artemisinin (ART), which is derived from traditional Chinese medicine, has been shown to inhibit tumour growth; however, the relationship between ART and tumour vascular normalisation in oral squamous cell carcinoma (OSCC) has not been previously reported. METHODS: Different concentrations(0 mg/kg, 25 mg/kg, 50 mg/kg, 100 mg/kg)of ART were used to treat the xenograft nude mice model of OSCC. The effects of ART on migration and proliferation of OSCC and human umbilical vein endothelial cells (HUVEC) cells were detected by scratch assay and CCK-8 assay. OSCC cells with macrophage migration inhibitory factor (MIF) silenced were constructed to explore the effect of MIF. RESULTS: Treatment with ART inhibited the growth and angiogenesis of OSCC xenografts in nude mice and downregulated vascular endothelial growth factor (VEGF), IL-8, and MIF expression levels. ART reduced the proliferation, migration, and tube formation of HUVEC, as well as the expression of VEGFR1 and VEGFR2. When the dose of ART was 50 mg/kg, vascular normalisation of OSCC xenografts was induced. Moreover, VEGF and IL-8 were needed in rhMIF restoring tumour growth and inhibit vascular normalisation after the addition of rhMIF to ART-treated cells. CONCLUSION: Artemisinin might induce vascular normalisation and inhibit tumour growth in OSCC through the MIF-signalling pathway.

Fatty acid synthase contributes to epithelial-mesenchymal transition and invasion of salivary adenoid cystic carcinoma through PRRX1/Wnt/ß-catenin pathway.

Fatty acid synthase (FASN) has been shown to be selectively up-regulated in cancer cells to drive the development of cancer. However, the role and associated mechanism of FASN in regulating the malignant progression of salivary adenoid cystic carcinoma (SACC) still remains unclear. In this study, we demonstrated that FASN inhibition attenuated invasion, metastasis and EMT of SACC cells as well as the expression ofPRRX1, ZEB1, Twist, Slug and Snail, among which the level of PRRX1 changed the most obviously. Overexpression of PRRX1 restored migration and invasion in FASN knockdown cells, indicating that PRRX1 is an important downstream target of FASN signalling. Levels of cyclin D1 and c-Myc, targets of Wnt/ß-catenin pathway, were significantly decreased by FASN silencing and restored by PRRX1 overexpression. In addition, FASN expression was positively associated with metastasis and poor prognosis of SACC patients as well as with the expression of PRRX1, cyclin D1 and c-Myc in SACC tissues. Our findings revealed that FASN in SACC progression may induce EMT in a PRRX1/Wnt/ß-catenin dependent manner.

MIF promotes perineural invasion through EMT in salivary adenoid cystic carcinoma.

Macrophage migration inhibitory factor (MIF) is a prominent orchestrator during the onset and progression of cancer. Recently, MIF was detected in salivary adenoid cystic carcinoma (SACC). However, its functional effect in perineural invasion (PNI) of SACC remained unknown. To illuminate the effect of MIF in genesis of PNI in SACC, we examined the expression of MIF, epithelial-to-mesenchymal transition (EMT)-related markers, and Schwann cell markers by immunohistochemical analysis in 158 cases of SACC samples. Meanwhile, the correlation between MIF and PNI of SACC species was analyzed. Our data indicated that MIF expression was associated with PNI of SACC significantly. In vitro, the silence and overexpression experiments of MIF were performed in SACC cell lines. The ability of migration, invasion and PNI could be inhibited significantly by siRNA-mediated MIF silence, and the occurrence of EMT and Schwann-like cell differentiation was also inhibited by MIF silence in SACC-LM cells. Overexpression of MIF in SACC-83 cells using expressive plasmid showed the opposite effects. Our findings identified that an association between PNI and MIF expression existed. MIF may promote PNI of SACC by participating in cytoskeletal reorganization and pseudo foot formation induced by EMT and the Schwann-like cell differentiation of SACC cells.

Macrophage migration inhibitory factor promotes the invasion and metastasis of oral squamous cell carcinoma through matrix metalloprotein-2/9.

Macrophage migration inhibitory factor (MIF) has been shown to closely associate with the malignant progression of a variety of human carcinomas. However, the role and its underlying molecular mechanisms of MIF in the invasion and metastasis of oral squamous cell carcinoma (OSCC) still remains unclear. Here, we found that MIF silencing reduced the cell proliferation, migration, and invasion, as well as matrix metalloprotein-2 (MMP-2) and MMP-9 in OSCC cells. Overexpression of MMP-2 or MMP-9 restored the migration and invasion of MIF-knockdown cells, indicating that MMP-2 and MMP-9 are downstream targets of MIF. In the xenograft model, MIF silencing inhibited tumor growth and in lymph metastasis model, MIF silencing reduced tumor metastasis. More importantly, immunohistochemistry staining in a tissue microarray (TMA) demonstrated that MIF expression was positively correlated with clinic stage, recurrence, metastasis, and poor prognosis of patients with OSCC as well as with the levels of MMP-2 or MMP-9 in TMA. Therefore, our findings suggest that MIF may promote the invasion and metastasis of OSCC through the activation of MMP-2 and MMP-9 and prompt further investigation into the therapeutic value of MIF for OSCC treatment.

NR2F1 contributes to cancer cell dormancy, invasion and metastasis of salivary adenoid cystic carcinoma by activating CXCL12/CXCR4 pathway.

BACKGROUND: Salivary adenoid cystic carcinoma (SACC) can recur after removal of the primary tumor and treatment, where they can keep no clinical symptoms and dormant state for 10-15 years. NR2F1 has been demonstrated to regulate the tumor cell dormancy in various malignant tumors and has a potential impact on recurrence and metastasis of carcinoma. However, the role and significance of NR2F1 in SACC dormancy still remain unknown. METHODS: A total number of 59 patients with a diagnosis of SACC were included to detected expression of NR2F1, Ki-67 by immunohistochemical (IHC) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL). Fisher's exact test was used to examine the NR2F1 expression and clinicopathologic parameters of SACC. In vitro, SACC cell lines were transfected NR2F1 and knockdown NR2F1 respectively. CCK-8, flow cytometry, wound healing assay and transwell invasion determined SACC cell proliferation, apoptosis, cell cycle, migration and invasion respectively. Chromatin immunoprecipitation (ChIP) assays were utilized to demonstrate the potential role of NR2F1 in SACC invasion via CXCL12/CXCR4 axis. In vivo, xenografts of nude mice via subcutaneous injection or tail vein injection were used to testify the results in vitro. RESULTS: Among the 59 patients with SACC, 23.73% (14/59) were positive to NR2F1 expression, a lower rate of expression compared with 60% (6/10) in normal salivary gland samples. NR2F1 was correlated with metastasis, relapse and dormancy of SACC. SACC cells with transfected NR2F1 remained dormant, as well as enhanced invasion and metastasis. Knockdown of NR2F1 via siRNA after NR2F1 overexpression restored the proliferation and the cell number in G2/M phases, and reduced the abilities of migration and invasion. In addition, NR2F1 promoted the expression of CXCL12 and CXCR4, and overexpression of CXCL12 at least partly rescued the proliferation, migration, and invasion activities induced by NR2F1 silencing. CONCLUSIONS: NR2F1 may be an underlying mechanism of SACC recurrence and metastasis via regulating tumor cell dormancy through CXCL12/CXCR4 pathway.

Corrigendum: Overexpression Cathepsin D Contributes to Perineural Invasion of Salivary Adenoid Cystic Carcinoma.

[This corrects the article DOI: 10.3389/fonc.2018.00492.].

PRRX1-induced epithelial-to-mesenchymal transition in salivary adenoid cystic carcinoma activates the metabolic reprogramming of free fatty acids to promote invasion and metastasis.

OBJECTIVES: Increasing evidences demonstrate a close correlation between epithelial-to-mesenchymal transition (EMT) induction and cancer lipid metabolism. However, the molecular mechanisms have not been clarified. MATERIALS AND METHODS: In our study, the relative expression level of PRRX1 was detected, its relationship with free fatty acid (FFA) and PPARG2 was analysed in 85 SACC tissues and 15 salivary glands from the benign salivary tumours. We also compared the FFAs composition and levels in these SACC cells. PPARG2 was detected in PRRX1-induced FFAs treatment as well as Src and MMP-9 were detected in FFAs treatment-induced invasion and migration of SACC cells, and ChIP test was performed to identify the target interactions. RESULTS: Our data showed that overexpression of PRRX1 induced EMT and facilitated the invasion and migration of SACC cells, and PRRX1 expression was closely associated with high FFAs level and poor prognosis of SACC patients. Furthermore, PRRX1 silence led to the increase of PPARG2 and the reduction of FFAs level and the migration and invasion of SACC cells. And inhibition of PPARG2 rescued FFAs level and migration and invasion capabilities of SACC cells. Free fatty acids treatment induced an increase of Stat5-DNA binding activity via Src- and MMP-9-dependent pathway. CONCLUSIONS: Collectively, our findings showed that the PRRX1/PPARG2/FFAs signalling in SACC was important for accelerating tumour metastasis through the induction of EMT and the metabolic reprogramming of FFAs.

Interplay between cancer cells and M2 macrophages is necessary for miR-550a-3-5p down-regulation-mediated HPV-positive OSCC progression.

BACKGROUND: Human papillomavirus (HPV)-positive oral squamous cell carcinoma (OSCC) is increasing worldwide with typically higher grade and stage, while better prognosis. microRNAs (miRNAs) has been shown to play a critical role in cancer, however, their role in HPV-positive OSCC progression remains unclear. METHODS: miRNA microarray was performed to identify differentially expressed miRNAs. qRT-PCR and FISH were performed to determine the relative expression of miR-550a-3-5p. CCK-8, Flow cytometry, Wound healing, Cell invasion assays and xenograft experiments were conducted to analyze the biological roles of miR-550a-3-5p. Tumor-associated macrophages (TAMs) generation, co-culturing of cancer cells with TAMs, Western blot, Dual-luciferase reporter gene assay, Immunohistochemistry and animal studies were performed to explore the mechanisms underlying the functions of miR-550a-3-5p. RESULTS: We identified 19 miRNAs differentially expressed in HPV-positive OSCC specimens and miR-550a-3-5p was down-regulated. The low expression of miR-550a-3-5p correlated with higher tumor size and nodal metastasis of HPV-positive OSCC patients. Then, we found that miR-550a-3-5p suppressed the migration, invasion and EMT of HPV-positive OSCC cells dependent on decreasing M2 macrophages polarization. Moreover, miR-550a-3-5p, down-regulated by E6 oncoprotein, inhibited M2 macrophages polarization by YAP/CCL2 signaling, which in turn abrogating EMT program in HPV-positive OSCC cells. In addition, in both xenografts and clinical HPV-positive OSCC samples, miR-550a-3-5p levels were inversely associated with YAP, CCL2 expressions and the number of M2 macrophages. CONCLUSIONS: E6/miR-550a-3-5p/YAP/CCL2 signaling induces M2 macrophages polarization to enhance EMT and progression, revealing a novel crosstalk between cancer cells and immune cells in HPV-positive OSCC microenvironment.

Myeloid derived suppressor cells contribute to the malignant progression of oral squamous cell carcinoma.

PURPOSE: The tumor-related myeloid derived suppressor cells (MDSCs), important immunosuppressive cells in tumor microenvironment, play an important role in the cancer progression. This study is aimed to investigate the crosstalk between MDSCs and oral squamous cell carcinoma (OSCC) cells and their role in the malignant progression of OSCC. METHODS: Immunochemistry (IHC) was used to investigate the expression of CD33 in 200 OSCC, 36 premalignant. CD33+ MDSCs were sorted and enriched via magnetic-activated cell sorting (MACS) from OSCC patients or health donor, and their phenotypes were identified by flow cytometry. With a co-culture system of MDSCs and OSCC, the effects of MDSCs on OSCC proliferation, apoptosis, migration invasion, epithelial-mesenchymal transition (EMT), and vasculogenic mimicry formation (VM) formation were assessed, respectively. Besides, peripheral blood mononuclear cells (PBMCs) from health donor were cultured with OSCC supernatant, the level of MDSCs and expressions of Arginase (Arg-1) and inducible nitric oxide synthase (iNOS) were measured. RESULTS: The number of MDSCs was increased in tumor tissues of OSCC patients, and was positively related to the T stage, pathological grade, lymph node metastasis and poor prognosis. Tumor-related MDSCs of the co-culture system promoted OSCC progression by contributing to cell proliferation, migration and invasion as well as inducing EMT and VM. In turn, OSCC cells had potential to induce MDSCs differentiation from PBMCs and increase the expression of Arg-1 and iNOS. CONCLUSION: These indicated that the crosstalk between MDSCs and tumor cells facilitated the malignant progression of OSCC cells and the immune suppressive properties of MDSCs, which may provide new insights into tumor treatment on targeting tumor-associated immunosuppressive cells.

Non-coding RNAs derailed: The many influences on the fatty acid reprogramming of cancer.

Non-coding RNAs (NcRNAs), a family of functional RNA molecules that cannot translate into proteins but control specific gene expression programs, have been shown to be implicated in various biological processes, including fatty acid metabolism. Fast-growing tumor cells rewire their fatty acid metabolic circuitry in order to meet the needs of energy storage, membrane proliferation, and the generation of signaling molecules, which is achieved by regulating a variety of key enzymes along with related signaling pathways in fatty acid metabolism. This review presents an update of our knowledge about the regulatory network of ncRNAs-specifically, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs)-in this metabolic shift and discusses the possibility of ncRNA-based therapeutics being applied to the restoration of cancer-related fatty acid metabolism.

Cathepsin B defines leader cells during the collective invasion of salivary adenoid cystic carcinoma.

Cathepsin B (CTSB) has been reported to be involved in cancer metastasis by altering extracellular matrix (ECM) remodeling and facilitating invasion. However, the contribution of CTSB to collective cell invasion in salivary adenoid cystic carcinoma (SACC) and the underlying mechanisms remain unclear. The present study demonstrated that collective cell invasion is commonly observed in SACC without a complete epithelial­mesenchymal transition signature. CTSB was found to be overexpressed in the invasive front of SACC compared to the tumor center, and was associated with a poor prognosis of patients with SACC. Subsequently, a 3D spheroid invasion assay was established in order to recapitulate the collective cell invasion of SACC and the results revealed that CTSB was only expressed in leader cells. The knockdown of CTSB by siRNA inhibited the migration and invasion of SACC­83 cells and impaired the formation of leader cells. CTSB knockdown also disrupted cytoskeletal organization, altered cell morphology and inhibited ECM remodeling by downregulating matrix metalloproteinase­9, focal adhesion kinase and Rho/ROCK function. Therefore, the present study provides evidence that CTSB may define leader cells in SACC and is required for collective cell invasion as a potential key regulator of ECM remodeling.

PRRX1 Regulates Cellular Phenotype Plasticity and Dormancy of Head and Neck Squamous Cell Carcinoma Through miR-642b-3p.

BACKGROUND: Dormancy is one characteristic of cancer cells to make patients remain asymptomatic before metastasis and relapse, which is closely related to the survival rate of cancer patients, including head and neck squamous cell carcinoma (HNSCC). PRRX1 has previously been implicated in the invasion and metastasis of the epithelial-mesenchymal transition (EMT) process in different types of human carcinoma. However, whether PRRX1 can regulate cancer dormancy and its reactivation, leading to the migration and invasion of HNSCC cells, remains elusive. The aim of this study was to determine the role of PRRX1 in cellular phenotype plasticity and cancer dormancy of HNSCC cells and its association with miRNAs in HNSCC. METHODS: The expression of PRRX1 was detected by immunohistochemical staining in primary HNSCC samples and the metastatic lymph nodes. Meanwhile, the role of PRRX1 and its relationship with miR-642b-3p and EMT in cellular phenotype plasticity and cancer dormancy of HNSCC were investigated in vitro and in vivo. RESULTS: PRRX1 was significantly higher at the invasive front of HNSCC samples compared with the metastatic lymph nodes, and such switch process was accompanied by the cellular phenotype plasticity and cell dormancy activation. In HNSCC cell lines, PRRX1 positively promoted the expression of known EMT inducers and cooperated with activated TGF-ß1 to contribute to EMT and migration and invasion of HNSCC cells. Then, we found that overexpression of miR-642b-3p, one of the most significantly downregulated miRNAs in PRRX1-overexpressed cells, significantly reduced the migration and invasion, and increased cell proliferation and apoptosis. And miR-642b-3p restoration reversed PRRX1-induced cell dormancy and EMT of HNSCC cells through TGF-ß2 and p38. Finally, we demonstrated that overexpressed PRRX1 was closely correlated with miR-642b-3p downregulation and the upregulation of TGF-ß2 and p38 in a xenograft model of HNSCC. CONCLUSIONS: Our findings showed that PRRX1 may be one of the main driving forces for the cellular phenotype plasticity and tumor dormancy of HNSCC. Therefore, we can raise the possibility that EMT may help to keep cancer cell in dormant state and mesenchymal-epithelial transition may resurge dormancy in HNSCC.

Hypoxia promotes vasculogenic mimicry formation by vascular endothelial growth factor A mediating epithelial-mesenchymal transition in salivary adenoid cystic carcinoma.

OBJECTIVES: To investigate the role of hypoxia in vasculogenic mimicry (VM) of salivary adenoid cystic carcinoma (SACC) and the underlying mechanism involved. MATERIALS AND METHODS: Firstly, wound healing, transwell invasion, immunofluorescence and tube formation assays were performed to measure the effect of hypoxia on migration, invasion, EMT and VM of SACC cells, respectively. Then, immunofluorescence and RT-PCR were used to detect the effect of hypoxia on VE-cadherin and VEGFA expression. And pro-vasculogenic mimicry effect of VEGFA was investigated by confocal laser scanning microscopy and Western blot. Moreover, the levels of E-cadherin, N-cadherin, Vimentin, CD44 and ALDH1 were determined by Western blot and immunofluorescence in SACC cells treated by exogenous VEGFA or bevacizumab. Finally, CD31/ PAS staining was performed to observe VM and immunohistochemistry was used to determine the levels of VEGFA and HIF-1α in 95 SACC patients. The relationships between VM and clinicopathological variables, VEGFA or HIF-1α level were analysed. RESULTS: Hypoxia promoted cell migration, invasion, EMT and VM formation, and enhanced VE-cadherin and VEGFA expression in SACC cells. Further, exogenous VEGFA markedly increased the levels of N-cadherin, Vimentin, CD44 and ALDH1, and inhibited the expression of E-cadherin, while the VEGFA inhibitor reversed these changes. In addition, VM channels existed in 25 of 95 SACC samples, and there was a strong positive correlation between VM and clinic stage, distant metastases, VEGFA and HIF-1α expression. CONCLUSIONS: VEGFA played an important role in hypoxia-induced VM through regulating EMT and stemness, which may eventually fuel the migration and invasion of SACC.

Targeting VEGF pathway to normalize the vasculature: an emerging insight in cancer therapy.

Vascular normalization is a new concept of targeting angiogenesis to restore vessel structure and function and to increase blood perfusion and delivery of drugs. It has been confirmed that vascular normalization can decrease relapse and benefit other cancer therapy, including chemotherapy, radiotherapy, and immune cell therapy. The key point of this therapy is to inhibit pro-angiogenic factors and make it be balanced with anti-angiogenic factors, resulting in a mature and normal vessel characteristic. Vascular endothelial growth factor (VEGF) is a key player in the process of tumor angiogenesis, and inhibiting VEGF is a primary approach to tumor vessel normalization. Herein, we review newly uncovered mechanisms governing angiogenesis and vascular normalization of cancer and place emphasis on targeting VEGF pathway to normalize the vasculature. Also, important methods to depress VEGF pathway and make tumor vascular are discussed.

Porphyromonas gingivalis Promotes 4-Nitroquinoline-1-Oxide-Induced Oral Carcinogenesis With an Alteration of Fatty Acid Metabolism.

Microbiota has been widely considered to play a critical role in human carcinogenesis. Human papilloma virus, hepatitis B and C virus, and Helicobacter pylori are implicated in the pathogenesis of cancer of uterine cervix, liver, and stomach, respectively. However, whether Porphyromonas gingivalis (P. gingivalis), a common Gram negative oral bacteria, is associated with oral carcinogenesis still remains unclear and its underlying mechanism needs to be addressed. Here, we established a combined experimental system of 4NQO-induced oral carcinoma model and chronic periodontitis model and investigated the effects of P. gingivalis infection on oral carcinogenesis and fatty acid metabolism during oral carcinogenesis. The data showed that in this animal model, P. gingivalis infection induced mice periodontitis, increased the tongue lesion size and multiplicity of each mouse and promoted oral cancer development. P. gingivalis treatment significantly increased the level of free fatty acids and altered the fatty acid profile in tongue tissues and the serum of mice. And P. gingivalis induced the formation of fatty liver of the mice. Besides, immunohistochemical analysis and qRT-PCR showed that the expression of fatty-acid synthase and acetyl-CoA carboxylase 1 were increased in the tongue and liver tissues of 4NQO-treated mice infected with P. gingivalis. These results showed that P. gingivalis promoted oral carcinogenesis and aggravated disturbance of fatty acid metabolism, indicating a close association among P. gingivalis, lipid metabolic and oral carcinogenesis.

Autophagy is positively associated with the accumulation of myeloid­derived suppressor cells in 4­nitroquinoline­1­oxide­induced oral cancer.

It has previously been demonstrated that autophagy and inflammation act synergistically to promote carcinogenesis. However, the precise roles of autophagy in multistep oral carcinogenesis are still unclear, particularly regarding its association with tumor inflammation. The present study established a 4NQO­induced oral cancer mouse model and investigated autophagy status in the multistep process of oral carcinogenesis using immunohistochemistry, western blotting and immunofluorescence staining. Furthermore, the number of Gr­1+CD11b+ myeloid derived suppressor cells (MDSCs) and CD4+ Foxp3+ regulatory T cells (Tregs) during oral carcinogenesis and the association with autophagy status was also examined. The results revealed that the expression of autophagy biomarkers, including dihydrosphingosine 1-phosphate phosphatase LCB3 (LC3B), p62/SQSTM1 (p62) and Beclin 1 increased during 4NQO­induced carcinogenesis and in human oral cancer. The number of MDSCs and Tregs also increased during oral carcinogenesis. Furthermore, the expression of LC3B and p62 significantly correlated with the accumulation of MDSCs and the expression of Beclin 1 correlated with the increase of Tregs. These data indicated that autophagy may be activated by the tumor inflammation microenvironment during oral carcinogenesis.

Overexpression Cathepsin D Contributes to Perineural Invasion of Salivary Adenoid Cystic Carcinoma.

Objective: Cathepsin D (CTSD) is a pivotal orchestrator in the occurrence and development of tumors. Recently, CTSD was detected in salivary adenoid cystic carcinoma (SACC). However, its functional role in perineural invasion (PNI) of SACC remained elusive. We conducted the present study to detect the expression of CTSD in SACC, analyze the correlation between CTSD expression and prognosis of SACC patients and elucidate the role of CTSD in occurrence of PNI in SACC to lay the foundation for further studies. Methods: Immunohistochemical analysis was conducted to assess CTSD and Ki67 expression in 158 SACC samples and 20 normal salivary gland samples adjacent to carcinoma. Meanwhile, the correlation between CTSD and PNI of SACC specimens was analyzed using Wilcoxon test. QRT-PCR, immunofluorescence and western blot analysis were used to examine the levels of CTSD mRNA and protein in SACC-LM cell line. SiRNA-mediated CTSD silence was performed. Scratch wound healing assay, transwell invasion assay and DRG co-culture assay of PNI was used to detect the ability of migration, invasion and PNI. FITC-phalloidin was used to detect cytoskeletal organization. Results: Our data demonstrated that the positive expression of CTSD was observed in 74.1% (117/158) of SACC cases, and the expression of CTSD was significantly correlated with the PNI (p < 0.05). The ability of migration, invasion, and PNI could be inhibited significantly by siRNA-mediated CTSD silence (p < 0.01). Furthermore, siRNA-mediated CTSD silence inhibited cytoskeletal organization and pseudo foot formation in SACC-LM cells. Conclusion: Our results suggested that an association between PNI and expression of CTSD existed. CTSD may promote PNI of SACC accompanied by cytoskeletal organization and pseudo foot formation.