Fibroblast Programmed Cell Death Ligand 1 Promotes Osteoclastogenesis in Odontogenic Keratocysts.

Local aggressive growth of odontogenic keratocysts (OKCs) can cause serious bone destruction, even resulting in pathologic fractures of the mandible. The mechanism of osteoclastogenesis in OKCs was explored by investigating the role of programmed cell death ligand 1 (PD-L1), a key immune checkpoint, in OKCs and its relationship with the M2 isoform of pyruvate kinase (PKM2), a key enzyme of glycolysis. The data from immunohistochemistry, real-time quantitative PCR, Western blot, and flow cytometry indicated that the expression level of PD-L1 was significantly increased in the stroma and fibroblasts of OKCs (OKC-Fs) when compared with oral mucosa. Double-labeling staining demonstrated that osteoclasts in OKCs spatially interacted with PD-L1-positive OKC-Fs. Exogenous expression of PD-L1 in OKC-Fs promoted osteoclastogenesis when OKC-Fs were co-cultured with osteoclast precursors (RAW264.7 cells). Because OKC-Fs exhibit energy dependency and acquire energy from PKM2-mediated glycolysis, this study generated stable PKM2 knockdown OKC-Fs using shRNAs against PKM2, and found that PD-L1 expression level was decreased by PKM2 knockdown. Furthermore, Spearman rank correlation analysis showed that there was a positive correlation between the immunostaining of PKM2 and PD-L1 in OKC samples. In addition, double-labeling immunofluorescence showed colocalizations between PKM2 and PD-L1 in the fibrous tissue walls of OKCs. In conclusion, PD-L1 in fibroblasts promotes osteoclastogenesis in OKCs, which is regulated by PKM2.

Endoplasmic reticulum stress is attenuated by glycolysis in lymphatic malformations.

BACKGROUND: This study aims to investigate the role of endoplasmic reticulum stress (ER stress) in human dermal lymphatic endothelial cells (HDLECs) and lymphatic malformations (LMs) and its relationship with aerobic glycolysis and inflammation. METHODS: The proliferation and apoptosis of HDLECs were examined with lipopolysaccharide (LPS) treatment. ER stress-associated proteins and glycolysis-related markers were detected by western blot. Glycolysis indexes were detected by seahorse analysis and lactic acid production assay kits. Immunohistochemistry was used to reveal the ER stress state of lymphatic endothelial cells (LECs) in LMs. RESULTS: LPS induced ER stress in HDLECs but did not trigger detectable apoptosis. Intriguingly, LPS-treated HDLECs also showed increased glycolysis flux. Knockdown of Hexokinase 2, a key enzyme for aerobic glycolysis, significantly inhibited the ability of HDLECs to resist ER stress-induced apoptosis. Moreover, compared to normal skin, glucose-regulated protein 78 (GRP78/BIP), and phosphorylation protein kinase R-like kinase (p-PERK), two key ER stress-associated markers, were upregulated in LECs of LMs, which was correlated with the inflected state. In addition, excessively activated ER stress inhibited the progression of LMs in rat models. CONCLUSIONS: These data indicate that glycolysis could rescue activated ER stress in HDLECs, which is required for the accelerated development of LMs. IMPACT: Inflammation enhances both ER stress and glycolysis in LECs while glycolysis is required to attenuate the pro-apoptotic effect of ER stress. Endoplasmic reticulum (ER) stress is activated in lymphatic endothelial cells (LECs) of LMs, especially in inflammatory condition. The expression of ER stress-related proteins is increased in LMs and correlated with Hexokinase 2 expression. Pharmacological activation of ER stress suppresses the formation of LM lesions in the rat model. ER stress may be a promising and effective therapeutic target for the treatment of LMs.

NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma.

The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome plays cell- and tissue-specific roles in cancer, meaning that its activation in different tumors or cells may play different roles in tumor progression. We have previously described the tumor-promoting function of tumor-intrinsic NLRP3/IL-1ß signaling in head and neck squamous cell carcinoma (HNSCC), but its role in immune cells remains unclear. In this study, we found that NLRP3 was highly expressed in tumor-associated macrophages (TAMs) in both mouse and human HNSCC, and the expression of NLRP3 was positively correlated with the density of TAMs according to immunohistochemistry, immunofluorescence, and flow cytometry analyses. Importantly, the number of NLRP3high TAMs was related to worse overall survival in HNSCC patients. Knocking out NLRP3 inhibited M2-like macrophage differentiation in vitro. Moreover, the carcinogenic effect induced by 4-nitroquinoline-1-oxide was decreased in Nlrp3-deficient mice, which had smaller tumor sizes. Genetic depletion of NLRP3 reduced the expression of protumoral cytokines, such as IL-1ß, IL-6, IL-10, and CCL2, and suppressed the accumulation of TAMs and myeloid-derived suppressor cells (MDSCs) in mouse HNSCC. Thus, activation of NLRP3 in TAMs may contribute to tumor progression and have prognostic significance in HNSCC.

Blockage of the NLRP3 inflammasome by MCC950 improves anti-tumor immune responses in head and neck squamous cell carcinoma.

The NLRP3 inflammasome is a critical innate immune pathway responsible for producing active interleukin (IL)-1ß, which is associated with tumor development and immunity. However, the mechanisms regulating the inflammatory microenvironment, tumorigenesis and tumor immunity are unclear. Herein, we show that the NLRP3 inflammasome was over-expressed in human HNSCC tissues and that the IL-1ß concentration was increased in the peripheral blood of HNSCC patients. Additionally, elevated NLRP3 inflammasome levels were detected in tumor tissues of Tgfbr1/Pten 2cKO HNSCC mice, and elevated IL-1ß levels were detected in the peripheral blood serum, spleen, draining lymph nodes and tumor tissues. Blocking NLRP3 inflammasome activation using MCC950 remarkably reduced IL-1ß production in an HNSCC mouse model and reduced the numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). Moreover, inhibiting NLRP3 inflammasome activation increased the numbers of CD4+ and CD8+ T cells in HNSCC mice. Notably, the numbers of exhausted PD-1+ and Tim3+ T cells were significantly reduced. A human HNSCC tissue microarray showed that NLRP3 inflammasome expression was correlated with the expression of CD8 and CD4, the Treg marker Foxp3, the MDSC markers CD11b and CD33, and the TAM markers CD68 and CD163, PD-1 and Tim3. Overall, our results demonstrate that the NLRP3 inflammasome/IL-1ß pathway promotes tumorigenesis in HNSCC and inactivation of this pathway delays tumor growth, accompanied by decreased immunosuppressive cell accumulation and an increased number of effector T cells. Thus, inhibition of the tumor microenvironment through the NLRP3 inflammasome/IL-1ß pathway may provide a novel approach for HNSCC therapy.

TRAF6 regulates tumour metastasis through EMT and CSC phenotypes in head and neck squamous cell carcinoma.

Epithelial-mesenchymal transition (EMT) is associated with metastasis formation, generation and maintenance of cancer stem cells (CSCs). However, the regulatory mechanisms of CSCs have not been clarified. This study aims to investigate the role of TNF receptor-associated factor 6 (TRAF6) on EMT and CSC regulation in squamous cell carcinoma of head and neck (SCCHN). We found TRAF6 was overexpressed in human SCCHN tissues, and high TRAF6 expression was associated with lymphatic metastasis and resulted in poor prognosis in patients with SCCHN. In addition, elevated TRAF6 expression was observed in several HNSCC cell lines, and wound healing and transwell assay results showed that TRAF6 knockdown inhibited the migration and invasion ability of the SCCHN cells. Moreover, the expression of Vimentin, Slug and N-cadherin was down-regulated and that of E-cadherin was elevated after TRAF6 knockdown but decreased by transforming growth factor beta 1 (TGF-ß1) and CAL27 similar to mesenchymal cells formed after TGF-ß1 induction. In addition, the expression levels of CD44, ALDH1, KLF4 and SOX2 were inhibited after TRAF6 knockdown, and the anchor-dependent colony formation number and sphere number were remarkably reduced. Flow cytometry showed TRAF6 knockdown reduced ALDH1-positive cancer stem cells. We also demonstrated that TRAF6 is closely associated with EMT process and cancer stem cells using a Tgfbr1/Pten 2cKO mice SCCHN model and human SCCHN tissue microarray. Our findings indicate that TRAF6 plays a role in EMT phenotypes, the generation and maintenance of CSCs in SCCHN, suggesting that TRAF6 is a potential therapeutic target for SCCHN.

Expression of VISTA correlated with immunosuppression and synergized with CD8 to predict survival in human oral squamous cell carcinoma.

V-domain Ig suppressor of T cell activation (VISTA), a novel immune checkpoint regulatory molecule, suppresses T cell mediated immune responses. The aim of the present study was to profile the immunological expression, clinical significance and correlation of VISTA in human oral squamous cell carcinoma (OSCC). Human tissue microarrays, containing 165 primary OSCCs, 48 oral epithelial dysplasias and 43 normal oral mucosae, were applied to investigate the expression levels of VISTA, CD8, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed death ligand 1 (PD-L1), PI3Kα p110, IL13Rα2, phospho-STAT3 at tyrosine 705 (p-STAT3) and myeloid-derived suppressor cell (MDSC) markers (CD11b and CD33) by immunohistochemistry and digital pathology analysis. The results demonstrated that the protein level of VISTA was significantly higher in human OSCC specimens, and that VISTA expression in primary OSCCs was correlated with lymph node status. VISTA expression did not serve as an independent predictor for poor prognosis, while patient subgroup with VISTA high and CD8 low expression (22/165) had significantly poorer overall survival compared with other subgroups based on the multivariate and Cox hazard analyses among the primary OSCC patients in the present cohort. Additionally, the expression of VISTA was significantly correlated with PD-L1, CTLA-4, IL13Rα2, PI3K, p-STAT3, CD11b and CD33 according to Pearson's correlation coefficient test. Taken together, the results indicated that the VISTA high and CD8 low group, as an immunosuppressive subgroup, might be associated with a poor prognosis in primary OSCC. These findings indicated that VISTA might be a potential immunotherapeutic target in OSCC treatment.

Correlation of ALDH1, CD44, OCT4 and SOX2 in tongue squamous cell carcinoma and their association with disease progression and prognosis.

BACKGROUND: Recently, studies indicated that cancer stem cell plays a key role in cancer development and progression. However, the role of cancer stem cell has not been well elucidated in tongue squamous cell carcinoma (TSCC). The objective of this study was to investigate the relationships between the expressions of stem cell markers and the prognosis of TSCC. MATERIALS AND METHODS: Immunohistochemistry was employed to analyse the protein expression levels of ALDH1, CD44, OCT4 and SOX2 in 66 TSCC tissue samples. The results were then evaluated semiquantitatively and compared with other clinicopathological variables. RESULTS: Immunohistochemistry revealed that the ALDH1, CD44, OCT4 and SOX2 proteins were overexpressed in the 66 TSCC specimens used in this study. Spearman's correlation analysis showed that the expressions of ALDH1 and CD44 were significantly correlated with SOX2 except other proteins (P < 0.05) and that OCT4 and SOX2 were significantly related (P < 0.01). Kaplan-Meier analysis revealed that T category, node metastasis, TNM stage, differentiation and distant metastasis were associated with poor patient survival (P < 0.05). Multivariate Cox regression analysis demonstrated that SOX2, recurrence and distant metastasis were independent prognostic factors of overall survival in patients with TSCC. CONCLUSION: Taken together, these data suggest that the stem cell markers ALDH1, CD44, OCT4 and SOX2 are closely related in TSCC, and the expression of SOX2 can be used as a prognostic indicator of TSCC.

Tumor-host colluding through erythroid progenitor cells: Mechanisms and opportunities.

Immunotherapy, particularly immune checkpoint blockade (ICB), has shown great promise in the treatment of cancer and emerged as a beacon of hope for patients who have exhausted traditional therapeutic options. Despite ICB's approval for the treatment of advanced tumors, its efficacy remains limited to a small subset of patients. As a systemic disease, cancer can induce changes in the composition and function of the systemic immune system, and ICB resistance often involves a dialog between the tumor microenvironment (TME) and the systemic immune macroenvironment. While investigations into tumor progression and ICB resistance have largely focused on the TME itself, the alterations in the systemic immune system and immune macroenvironment are still poorly understood. Given the spleen's role as the largest secondary lymphoid organ, its examination and discussion may provide valuable insights into the systemic immune status and TME components. Recent studies have highlighted the importance of the spleen in tumor progression and immunotherapy, particularly in the context of erythroid progenitor cells (EPCs), a significant cell subpopulation. In this review, we discuss the mechanisms and role of splenic extramedullary hematopoiesis (EMH) as an intermediary in tumor-host interactions and explore the mechanism of EPC-TME collusions. We further summarize the progress in EPC-targeting strategies and emphasize the potential for further research into the role and mechanisms of EPCs in tumor progression and treatment, which could have far-reaching implications.

CSRP2 promotes cell stemness in head and neck squamous cell carcinoma.

BACKGROUND: Cysteine-rich protein 2 (CSRP2) is discovered as oncogene. The study aims to investigate the clinical significance and potential mechanism of CSRP2 in head and neck squamous cell carcinoma (HNSCC). METHODS: CSRP2 expression was explored by immunohistochemistry tissue microarrays and Western blotting in HNSCC. The effect of CSRP2 on the cancer stemness and epithelial-to-mesenchymal transition (EMT) of HNSCC cells was investigated by sphere formation, wound healing, and transwell assays. The vitro and vivo experiments revealed that CSRP2 modulated cancer stemness and EMT phenotypes in HNSCC. RESULTS: CSRP2 was overexpressed in HNSCC patients and presented poor prognosis. CSRP2 knockdown inhibited the migration and invasion ability of the HNSCC cells. And CSRP2 expression was closely associated with CSCs markers, EMT-transcription factor, new oncoprotein, and immune checkpoint. CONCLUSION: The overexpression of CSRP2 indicates poor prognosis and plays a key role in maintaining the cancer cell stemness and EMT.

Sunitinib attenuates reactive MDSCs enhancing anti-tumor immunity in HNSCC.

Enhancer of zeste homolog 2 (EZH2) is implicated in promoting HNSCC malignant progression. However, EZH2 inhibitors, when used alone, increase the number of myeloid-derived suppressor cells (MDSCs), which are responsible for enhancing tumor stemness and promoting tumor immune escape. We aimed to determine whether combining tazemetostat (an EZH2 inhibitor) and sunitinib (a MDSC inhibitor) can improve the response rate to an immune-checkpoint-blocking (ICB) therapy. We evaluated the efficacy of the above treatment strategies by bioinformatics analysis and animal experiments. EZH2 overexpression and abundant MDSCs in patients with HNSCC are associated with tumor progression. Tazemetostat treatment alone had limited inhibitory effect on HNSCC progression in the mouse models, accompanied by a surge in the number of MDSCs in the tumor microenvironment. Conversely, the combined use of tazemetostat and sunitinib reduced the number of MDSCs and regulatory T cell populations, promoting intratumoral infiltration of T cells and inhibiting of T cell exhausting, regulating of wnt/ß-catenin signaling pathway and tumor stemness, promoting the intratumoral PD-L1 expression and improved the response rate to anti-PD-1 therapy. The combined use of EZH2 and MDSC inhibitors effectively reverses HNSCC-specific immunotherapeutic resistance and is a promising strategy for overcoming resistance to ICB therapy.

Association of increased ligand cyclophilin A and receptor CD147 with hypoxia, angiogenesis, metastasis and prognosis of tongue squamous cell carcinoma.

AIMS: We evaluated the association of ligand cyclophilin A (CypA) and receptor CD147 with hypoxia, angiogenesis, lymph node metastasis and prognosis of patients with tongue squamous cell carcinoma (TSCC). METHODS AND RESULTS: We studied the expression of CypA, CD147, hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor A and C (VEGF-A and VEGF-C) protein by immunohistochemistry in 80 specimens of TSCC. CypA, CD147, HIF-1α, VEGF-A and VEGF-C were overexpressed in TSCCs, and were significantly higher than those in normal oral mucosa tissues (P<0.01). Increased ligand CypA and receptor CD147 correlated significantly with expression of HIF-1α, VEGF-A and VEGF-C. A significant relationship between VEGF-A and VEGF-C was also detected (P<0.01). Patients with overexpression of CypA, CD147, HIF-1α and VEGF-C had significantly worse overall survival (P<0.05) using Kaplan-Meier analysis. Multivariate Cox regression analysis showed that HIF-1α, recurrence and distant metastasis were independent prognostic factors on overall survival in TSCC patients. CONCLUSIONS: The association of expression of ligand CypA and receptor CD147 with carcinogenesis, hypoxia, angiogenesis, metastasis and prognosis of TSCC suggests that ligand CypA and receptor CD147 may have prognostic value and could be regarded as potential therapeutic targets in TSCC.

Bleomycin restricts the glycolysis of lymphatic endothelial cells by inhibiting dimeric PKM2 formation: A novel mechanism for lymphatic malformation treatment.

Glycolysis is activated in lymphatic endothelial cells and contributes to the development of lymphatic malformations (LMs). Bleomycin (BLM) is the most wildly used sclerosant for LMs, but its mechanisms are unclear. Here, our data showed that BLM suppressed the glycolysis of human dermal lymphatic endothelial cells (HDLECs) via inhibiting the expression and nucleus translocation of pyruvate kinase M2 isoform (PKM2) and inhibited dimeric PKM2 formation. Furthermore, the proliferation of LM lesions was inhibited by BLM through the down-regulation of nuclear PKM2 in the rat model. Additionally, PKM2, especially the nuclear PKM2 along with Ki-67, was inhibited in the lymphatic vessels of BLM-treated LMs. Our findings provide a new molecular mechanism of BLM in LM sclerotherapy treatment.

Mammalian target of rapamycin pathway promotes tumor-induced angiogenesis in adenoid cystic carcinoma: its suppression by isoliquiritigenin through dual activation of c-Jun NH2-terminal kinase and inhibition of extracellular signal-regulated kinase.

Tumor-induced angiogenesis is essential for invasive growth and hematogenous metastasis of adenoid cystic carcinoma (ACC), a highly aggressive neoplasm mostly occurring in salivary glands. Previous studies have indicated that strategies directed against angiogenesis will help develop new therapeutic agents for ACC. The Chinese folk medicine licorice has been used for years as a natural remedy for angiogenesis-related diseases. In this study, we examined the effects of isoliquiritigenin (ISL), a flavonoid isolated from licorice, on the growth and viability of ACC cells and observed a concentration-dependent (0-20 microM) inhibition of cell growth without cell death at 24 h. In a further mimic coculture study, ISL effectively suppressed the ability of ACC cells to induce in vitro proliferation, migration, and tube formation of human endothelial hybridoma (EAhy926) cells as well as ex vivo and in vivo angiogenesis, whereas it exerted no effect on EAhy926 cells when added directly or in the presence of vascular endothelial growth factor (VEGF). The data also showed that the specific suppression of tumor angiogenesis by ISL was caused by down-regulation of mammalian target of rapamycin (mTOR) pathway-dependent VEGF production by ACC cells, correlating with concurrent activation of c-Jun NH(2)-terminal kinase (JNK) and inhibition of extracellular signal-regulated kinase (ERK). Most importantly, ISL also significantly decreased microvessel density within xenograft tumors, associating with the reduction of VEGF production and suppression of the mTOR pathway coregulated by JNK and ERK, as revealed by immunohistochemical studies and clustering analysis. Taken together, our results highlight the fact that ISL is a novel inhibitor of tumor angiogenesis and possesses great therapeutic potential for ACC.

NLRP3 inflammasome activation promotes inflammation-induced carcinogenesis in head and neck squamous cell carcinoma.

BACKGROUND: NLRP3 inflammasome acts as a danger signal sensor that triggers and coordinates the inflammatory response. However, the roles of NLRP3 inflammasome in the tumorigenesis and development of cancer stem cells (CSCs) of squamous cell carcinoma of the head and neck (SCCHN) remain ambiguous. METHODS: In our study, tissue microarrays, ELISA, sphere-forming assay, colony formation assay and Western blot analysis were performed to evaluate the effect of NLRP3 inflammasome on the development of CSCs in human SCCHN tissue specimen, cell lines, and transgenic mouse SCCHN model. RESULTS: The components of NLRP3 inflammasome, namely, NLRP3, ASC, Caspase-1, and IL-18 were correlated with CSCs markers BMI1, ALDH1 and CD44 in human SCCHN specimens. Moreover, NLRP3, Caspase-1, IL-1ß, and IL-18 were highly expressed in SCCHN cell lines. NLRP3 inflammasome activated by LPS and ATP promoted sphere-forming and colony formation capacities along with an upregulation of BMI1, ALDH1 and CD44. In addition, NLRP3 inflammasome blockade by NLRP3 inhibitor MCC950 reduced sphere and colony number, also decreased the expression of BMI1, ALDH1 and CD44 in SCCHN cell lines. Expression of NLRP3, ASC, Caspase-1, IL-1ß, IL-18, BMI1, ALDH1 and CD44 was upregulated in Tgfbr1/Pten 2cKO mouse SCCHN model, and NLRP3 inflammasome expression was closely related to those CSCs makers in mice SCCHN. However, MCC950 treatment reduced the expression of NLRP3 inflammasome, CSCs markers BMI1, ALDH1 and CD44 in Tgfbr1/Pten 2cKO mice SCCHN. In addition, blockade of NLRP3 inflammasome can also delayed the tumor-burdened speed in SCCHN mice. CONCLUSIONS: Our study demonstrates that NLRP3 inflammasome was upregulated and associated with the carcinogenesis and CSCs self-renewal activation in SCCHN. NLRP3 inflammasome can be a potential target in the development of novel approaches for head and neck squamous cell carcinoma therapy.

T-cell immunoglobulin mucin 3 blockade drives an antitumor immune response in head and neck cancer.

T-cell immunoglobulin mucin 3 (TIM3) contributes to immune suppression during progression of many cancers, but the precise role of TIM3 in head and neck squamous cell carcinoma (HNSCC) is not clearly understood. In this study, we report that TIM3 expression was significantly up-regulated in patients with HNSCC and associated with lymph node metastasis. Additionally, TIM3 expression was increased in patients with recurrent HNSCC and patients with preradiotherapy or prechemotherapy. We also characterized CD8+ T cells and CD11b+ CD33+ myeloid-derived suppressor cells (MDSCs) in human HNSCC, and found that their expression was positively correlated with TIM3 expression. To determine the underlying mechanism of TIM3 in immune response during HNSCC progression, we utilized the Tgfbr1/Pten 2cKO HNSCC mouse model with TIM3 overexpression. Treatment with anti-TIM3 monoclonal antibody effectively suppressed tumor growth through restoring effector T-cell function by targeting CD4+ TIM3+ cells and CD8+ TIM3+ cells and decreasing MDSCs. Our findings demonstrate TIM3 expression in patients with HNSCC and suggest anti-TIM3 immunotherapy as a novel therapeutic approach for effective treatment of HNSCC.

Expression of LC3, LAMP2, KEAP1 and NRF2 in Salivary Adenoid Cystic Carcinoma.

Salivary Adenoid Cystic Carcinoma (SACC) is a tumor characterized by inevitable local progression and terminal hematogenous metastasis. This study aimed to investigate the expression of LC3, LAMP2, KEAP1 and NRF2 in SACC. Human salivary gland tissue microarray which contains 74 SACC, 12 pleomorphic adenoma and 18 normal salivary gland specimens. High expression of LC3, LAMP2, KEAP1 and NRF2 were found in SACC patients, and LC3, LAMP2, KEAP1 and NRF2 expression were significantly higher in SACC than as compared with pleomorphic adenoma and (or) normal salivary gland. The expression of NRF2 was correlated with pathological type of human SACC (P < 0.05). Moreover, the high-expression of KEAP1 had significant correlations with LC3 (P < 0.001, R = 0.3195), and LAMP2 (P < 0.001, R = 0.3346) and NRF2 (P < 0.05, R = 0.2246) by using the Pearson correlation coefficient test. Our findings demonstrated that up-regulation of LC3, LAMP2, KEAP1 and NRF2 were associated with carcinogenesis and progression of SACC patients, suggesting that they may be useful molecular targets in salivary adenoid cystic carcinoma.

Hypoxia induces TFE3 expression in head and neck squamous cell carcinoma.

To assess the role of transcription factor µE3 (TFE3) in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC), human HNSCC tissue arrays were investigated for TFE3 expression. Human HNSCC tissues with neoadjuvant inductive chemotherapey (docetaxel, cisplatin and fluorouracil, TPF) and mice HNSCC tissues from transgenic mice model were evaluated for TFE3 expression and the hypoxia pathway. The roles of EGF/EGFR mediated hypoxia in TFE3 nuclear expression were analyzed in vitro and in vivo. TFE3 expression was higher in human HNSCC tissues compared with that in normal oral mucosa. Moreover, high TFE3 expression was related to HIF-1α, PAI-1, and EGFR, which demonstrated the activation of the hypoxia pathway in HNSCC tissues. Furthermore, elevated TFE3 expression was observed in HNSCC after cisplatin-based chemotherapy, and high TFE3 expression may indicate poor response to TPF inductive chemotherapy. Furthermore, similar changes with increased TFE3 were observed in HNSCC of the transgenic mouse HNSCC model. Hypoxic culture in the human HNSCC cell line increased TFE3 expression, which promoted cell survival under hypoxia. EGFR inhibiton by cetuximab could attenuate hypoxia-induced TFE3 in the HNSCC cell line and transgenic mouse HNSCC model. These findings indicated that TFE3 was an important hypoxia-induced transcriptional factor in HNSCC. TFE3 could be regarded as a durgable therapeutic oncotarget by EGFR inhibition.

NOTCH1 inhibition enhances the efficacy of conventional chemotherapeutic agents by targeting head neck cancer stem cell.

Cancer stem cells (CSCs) are considered responsible for tumor initiation and chemoresistance. This study was aimed to investigate the possibility of targeting head neck squamous cell carcinoma (HNSCC) by NOTCH1 pathway inhibition and explore the synergistic effect of combining NOTCH inhibition with conventional chemotherapy. NOTCH1/HES1 elevation was found in human HNSCC, especially in tissue post chemotherapy and lymph node metastasis, which is correlated with CSCs markers. NOTCH1 inhibitor DAPT (GSI-IX) significantly reduces CSCs population and tumor self-renewal ability in vitro and in vivo. Flow cytometry analysis showed that NOTCH1 inhibition reduces CSCs frequency either alone or in combination with chemotherapeutic agents, namely, cisplatin, docetaxel, and 5-fluorouracil. The combined strategy of NOTCH1 blockade and chemotherapy synergistically attenuated chemotherapy-enriched CSC population, promising a potential therapeutic exploitation in future clinical trial.

Anterior gradient protein 2 expression in high grade head and neck squamous cell carcinoma correlated with cancer stem cell and epithelial mesenchymal transition.

Anterior gradient protein 2 (AGR2) is a novel biomarker with potential oncogenic role. We sought to investigate the diagnostic and prognostic role of AGR2 on head and neck squamous cell carcinoma (HNSCC) with an emphasis on its correlation of cancer stemloid cells (CSC) and epithelial mesenchymal transition (EMT). We found that AGR2 protein levels were higher in HNSCC than in normal oral mucosa. High levels of AGR2 were associated with the T category, pathological grade and lymph node metastasis of HNSCC. Expression of AGR2 increased in recurring HNSCC after radiotherapy and in post cisplatin-based chemotherapeutic tissues. In HNSCC cell lines, knock-down of AGR2 induced apoptosis, reduced sphere formation, and down-regulated Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog and Oct4. In addition, over-expressed AGR2 in transgenic mice with spontaneous HNSCC was associated with lost function of Tgfbr1 and/ or lost function of Pten. In vitro knockdown TGFBR1 in HNSCC cell lines increased AGR2 expression. These results suggest that AGR2 is involved in EMT and self-renewal of CSC and may present a potential therapeutic target (oncotarget) for HNSCC.

C4.4A as a biomarker of head and neck squamous cell carcinoma and correlated with epithelial mesenchymal transition.

C4.4A, a member of the Ly6/uPAR family of membrane proteins, has been identified as a metastasis-associated molecule, but little is known about its actual expression and possible function in head and neck squamous cell carcinoma (HNSCC). To explore diagnostic and prognostic roles of C4.4A in HNSCC, we investigated the expression of C4.4A in human HNSCC tissue array which contains 43 HNSCC, 6 epithelial dysplasia and 16 normal oral mucosa. Expression of C4.4A was significantly increased in epithelial dysplasia and HNSCC when compared with normal oral mucosa. Moreover, high C4.4A expression indicated a rather poor prognosis of HNSCC patients. To better understand the function of C4.4A in HNSCC progression, we investigated epithelial to mesenchymal transition (EMT) associated proteins including transforming growth factor (TGF-ß1), Slug and CD147 in HNSCC. The expression of TGF-ß1, Slug, and CD147 was significantly increased in HNSCC when compared with normal oral mucosa. Meanwhile, the expression of C4.4A was significantly correlated with TGF-ß1, Slug, and CD147 in HNSCC tissue array. Furthermore, knockdown of C4.4A decreased the cell invasion and migration in CAL27 cell line and suppressed the EMT with increased E-cadherin and decreased N-cadherin and Slug. Our study demonstrated that C4.4A was a potential marker for prognosis of HNSCC, and C4.4A participated in EMT program in HNSCC progression.