PDK1 promotes metastasis by inducing epithelial-mesenchymal transition in hypopharyngeal carcinoma via the Notch1 signaling pathway.

Hypopharyngeal squamous cell carcinoma (HSCC) is a relatively rare malignancy and has the worst prognosis among head and neck cancer. Metastasis is the major cause of poor prognosis in HSCC patients. In this study, we found that 3-phosphoinositide-dependent protein kinase 1 (PDK1 or PDPK1) was overexpressed in HSCC. The overexpression was positively correlated lymph node metastasis, clinical stage, and distant metastasis and indicated poor outcome. Loss and gain-of-function revealed that PDK1 increased cell proliferation, migration and invasion in vitro as well as tumor growth and metastasis in vivo. Mechanically, PDK1 induced epithelial-mesenchymal transition and promoted metastasis by activating the Notch1 signaling pathway. We further illustrated that PDK1 bound with the Notch1 intracellular domain, thereby inhibiting its ubiquitin-mediated degradation in a protein kinase B (Akt-) independent manner. In summary, PDK1/Notch1 axis played an important role in HSCC metastasis, and this investigation provided a new perspective on potential therapeutic targets for HSCC.

MicroR-140-5p suppresses tumor cell migration and invasion by targeting ADAM10-mediated Notch1 signaling pathway in hypopharyngeal squamous cell carcinoma.

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides that negatively regulate gene expression at the post-transcriptional level. Downexpression of miR-140-5p was reported in some human cancers, and combined with a reduction of cell migration and invasion, suggesting that miR-140-5p functions as a tumor suppressor. However, little is known about the expression and function of miR-140-5p in hypopharyngeal squamous cell carcinoma (HSCC). In this research, we found that miR-140-5p was significantly downregulated in HSCC tissues and correlated to tumor classification and lymph node metastasis. Restoration of miR-140-5p suppressed the migration and invasion of FaDu cells, and decreased the protein expression levels of ADAM10. Furthermore, the luciferase reporter assay revealed that miR-140-5p was directly bound to ADAM10 mRNA and knockdown of ADAM10 could inhibit FaDu cell migration and invasion and reduced the protein expression levels of and Notch1 intracellular domain (NICD1). Of note, knockdown of Notch1 could inhibit the migration and invasion of FaDu cells and rescued the effect of miR-140-5p inhibitor in FaDu cells. Taken together, our study demonstrates that miR-140-5p suppresses tumor migration and invasion by inhibiting ADAM10-mediated Notch1 signaling pathway and suggests that miR-140-5p could have potential therapeutic applications in HSCC.

MTDH in macrophages promotes the vasculogenic mimicry via VEGFA-165/Flt-1 signaling pathway in head and neck squamous cell carcinoma.

Vasculogenic mimicry (VM) refers to vessel-like structures formed by aggressive tumor cells and is closely associated with cancer invasion and metastasis. Here, we investigated the effect of macrophage-derived MTDH on VM formation in head and neck squamous cell carcinoma (HNSCC) and its underlying mechanism. Macrophages with MTDH overexpression (Mac-MTDH) promoted cancer cell VM formation, migration, and invasion in vitro. Moreover, MTDH overexpression triggered macrophage polarization into M2 type tumor-associated macrophages. Analysis of HNSCC clinical samples revealed that MTDH+ macrophages were predominantly located in the tumor-stromal region in proximity to VM and correlated with lymph node metastasis. Mechanistically, Mac-MTDH enhanced the expression and secretion of VEGFA-165 rather than other VEGFA isoforms via ß-catenin. The VEGFA-165/Flt-1 axis was responsible for Mac-MTDH's effects in cancer cells through p-STAT3/Twist1/VE-cadherin pathway. Using mouse model, we further confirmed that Mac-MTDH increased VM formation and cancer metastasis in vivo. Furthermore, in subcutaneous xenograft mouse model, HN6 + Mac-MTDH tumor exhibited elevated expression of p-STAT3 and Twist1 than HN6 + Mac-NC tumors. This study revealed that Mac-MTDH promoted VM formation, cancer cell migration and invasion, and cancer metastasis through VEGFA-165/Flt-1 axis, and that macrophage-derived MTDH could be a potential therapeutic target in HNSCC.

PHF20 inhibition promotes apoptosis and cisplatin chemosensitivity via the OCT4­p­STAT3­MCL1 signaling pathway in hypopharyngeal squamous cell carcinoma.

Cisplatin is a widely used platinum­based chemotherapeutic agent for hypopharyngeal squamous cell carcinoma (HSCC). However, resistance to cisplatin limits its use for the treatment of HSCC, and the underlying molecular mechanism requires further investigation. The present study performed functional assays to determine whether the expression of plant homeodomain finger protein 20 (PHF20) may be involved in the apoptosis and cisplatin resistance of HSCC. The expression levels of PHF20 were higher in cisplatin­resistant HSCC cells compared with those in cisplatin­sensitive cells. The inhibition of PHF20 suppressed cell viability but did not affect the migratory and invasive abilities of HSCC cells compared with those of negative control­transfected cells. Furthermore, PHF20 inhibition reduced cell viability by enhancing apoptosis compared with those in the control cells in vitro. Notably, the inhibition of PHF20 sensitized HSCC cells to cisplatin, thus increasing apoptosis via the signal transducer and activator of transcription 3 (STAT3)­myeloid cell leukemia­1 (MCL1) pathway. Octamer­binding transcription factor 4 (OCT4) overexpression restored phosphorylated STAT3­MCL1­mediated apoptosis induced by PHF20 inhibition. In vivo experiments confirmed that PHF20 silencing induced tumor growth and increased apoptosis in HSCC cells compared with those in the control cells. Thus, PHF20 inhibition may promote apoptosis and improve cisplatin chemosensitivity via the OCT4­p­STAT3­MCL1 signaling pathway in HSCC.

Prognostic Significance of PD-L1 Expression and Its Tumor-Intrinsic Functions in Hypopharyngeal Squamous Cell Carcinoma.

PURPOSE: The expression of programmed death-ligand 1 (PD-L1) is common in various solid human cancers and it is an important therapeutic target. However, the expression pattern, clinical significance and potential mechanism of PD-L1 in hypopharyngeal squamous cell carcinoma (HSCC) are still lacking. METHODS: PD-L1 expression in HSCC tumor tissues and paired adjacent hypopharyngeal mucosal tissues was detected using immunohistochemistry assay, and the clinical significance of PD-L1 in HSCC was characterized. In vitro assays including cell viability assays, migration assays, invasion assays as well as Western blot assays were performed to illuminate the biological functions and underlying molecular mechanisms of PD-L1 in HSCC development. RESULTS: PD-L1 expression was detected in HSCC samples but we found no positive expression in matched normal hypopharyngeal mucosal tissues. The levels of PD-L1 expression were significantly correlated with advanced clinical progression and poor patient survival. Multivariable analysis of Cox model showed that PD-L1 expression was an independent predictor for the prognosis of HSCC patients. Functional experiments showed that the ectopic expression of PD-L1 markedly influenced the proliferation, migration and invasion of FaDu cells in vitro. Mechanistically, investigations demonstrated that PD-L1 could promote the epithelial-mesenchymal transition of FaDu cells. Meanwhile, PD-L1 knockdown inhibited, while PD-L1 overexpression activated the Akt-mTOR signaling pathway in FaDu cells. The EMT induced by PD-L1 overexpression could be reversed by the Akt inhibitor. CONCLUSION: In summary, the expression of PD-L1 can act as a significant biomarker for the adverse clinicopathological features and poor prognosis of patients with HSCC. PD-L1 can promote the proliferation, migration and invasion of FaDu cells and consequently enhance the aggressiveness. Moreover, PD-L1 induces EMT through AKT-mTOR signaling pathway. These suggest that PD-L1 has important tumor-intrinsic functions independent of its immunopathogenic effects.

Notch1 serves as a prognostic factor and regulates metastasis via regulating EGFR expression in hypopharyngeal squamous cell carcinoma.

OBJECTIVE: Hypopharyngeal squamous cell carcinoma (HSCC) remains one of the most lethal malignancies in head and neck. Notch1 has been validated to play prominent roles in the occurrence and development of various types of cancer. The aim of this study was to explore the function and underlying mechanism of Notch1 in HSCC. PATIENTS AND METHODS: Seventy-one cancer tissue samples and adjacent noncancerous formalin-fixed paraffin embedded tissue specimens were analyzed by immunohistochemistry. As Notch1 is overexpressed in HSCC, we further questioned whether there was a relationship between Notch1 and the clinicopathological characteristics. After confirming the successful knockdown of Notch1 by siRNA, the migration and invasion after gene knockdown were investigated by Transwell chambers. We then tried to identify YBX1 and EGFR expression using real-time PCR (RT-PCR) and Western blot analyses. To further determine whether the downexpression of EGFR was caused by YBX1 and the overexpression of YBX1 was caused by gene amplification, the expression of EGFR was detected by RT-PCR and Western blot assays. RESULTS: We found that the expression of Notch1 and EGFR in HSCC tissues was upregulated compared with those in the adjacent noncancerous tissues. Further clinicopathological characteristics analysis revealed that the expression of Notch1 was positively correlated with distant metastasis (P=0.003) and tumor differentiation (P=0.031). The high expression of Notch1 is an independent prognostic factor for a poor overall survival in patients with HSCC (P=0.015, χ 2=10.403). Knocking down of Notch1 significantly inhibits the migration and invasion of FaDu cells in vitro. Mechanistic investigation reveals that Notch1 knockdown is found suppressing the expression of EGFR at transcriptional level. Interestingly, we further found that Notch1 knockdown also decreased the expression of YBX1, which is a transcription factor of EGFR. Moreover, the upregulation of YBX1 reverses the suppression of Notch1 on EGFR. Furthermore, forced overexpression of YBX1 induced the invasion of FaDu cells. CONCLUSION: Taken together, we found a positively cross-linked role of Notch1 signaling in the outcome of HSCC, providing a novel valuable prognostic marker and potential therapeutic target for the treatment of HSCC patients. Notch1 is a core signaling molecule for regulating migration and invasion via interplaying with EGFR in HSCC cells.

[miR-140-5p affects the migration and invasion of hypopharyngeal carcinoma cells by downregulating ADAM10 expression].

OBJECTIVE: To investigate the expression of miR-140-5p and ADAM10 in hypopharyngeal carcinoma tissues and their effects on the migration and invasion of FaDu cells and underlying mechanism. METHODS: The miR-140-5p and ADAM10 mRNA levels in 33 cases of hypopharyngeal carcinoma tissues and adjacent normal tissues were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Transwell migration assay and transwell invasion assay were used to test the metastasis ability of FaDu cells after upregulation or downregulation of miR-140-5p and downregulation of ADAM10. The protein expression levels of ADAM10 in hypopharyngeal carcinoma tissues and the FaDu cells after transfection were determined by Western blot assays. RESULTS: The expression level of miR-140-5p was significantly downregulated in hypopharyngeal carcinoma tissues compared with adjacent tissues (t=-4.016, P<0.01), which was significantly correlated with tumor classification and lymph node metastasis (P<0.05). Conversely, mRNA and protein expressions of ADAM10 were significantly upregulated in tumor tissues (t=3.960, P<0.01; t=12.089, P<0.01), and were significantly downregulated in the FaDu cells after tranfected with si-ADAM10 (t=8.653, P<0.05; t=5.191, P<0.05). Transwell assay showed that compare with control group, the migration and invasive cells decreased significantly in hsa-mir-140-5p group (t=3.255, P<0.05; t=2.942, P<0.05), while increased significantly in anti-hsa-mir-140-5p group, (t=-13.521, P<0.05; t=-6.223, P<0.05). The migration and invasive cells in si-ADAM10 group were less than those in control group (t=4.759, P<0.05; t=3.663, P<0.05). The downregulation of ADAM10 attenuated the effect of anti-mir-140-5p in FaDu cells. Western blot assay showed that ADAM10 expression was apparently decreased in hsa-mir-140-5p group and increased in anti-mir-140-5p group compared with control group. CONCLUSIONS: The expression of miR-140-5p was significantly downregulated in hypopharyngeal carcinoma tissues and correlated with tumor classification and lymph node metastasis. ADAM10 was upregulated in tumor tissues. MiR-140-5p suppresses the migration and invasion abilities of FaDu cells, possibly through downregulation of ADAM10.

Afatinib down-regulates MCL-1 expression through the PERK-eIF2α-ATF4 axis and leads to apoptosis in head and neck squamous cell carcinoma.

Afatinib is the second generation of irreversible inhibitor of EGFR, HER2 and HER4, which has shown encouraging phase II and III clinical outcomes in the treatment of head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanism of afatinib-induced apoptosis in HNSCC is poorly understood. In the present investigation, we discovered that down-regulation of MCL-1, an anti-apoptotic member of BCL-2 family, was responsible for afatinib-triggered apoptosis. And the inactivation of AKT-mTOR signaling caused by afatinib lead to translational inhibition of MCL-1 expression. As a crucial branch of ER stress, PERK-eIF2α-ATF4 axis was also stimulated in HNSCC cells after afatinib incubation. Silencing either eIF2α or ATF4 by siRNA transfection relieved afatinib-caused suppression of AKT-mTOR activity, attenuating MCL-1 down-regulation as well as subsequent apoptosis. Collectively, the results show that afatinib hampers AKT-mTOR activation by stimulating PERK-eIF2α-ATF4 signaling pathway, giving rise to MCL-1 down-regulation mediated apoptosis in HNSCC cells. Therefore, our findings reveal the elaborate molecular network of afatinib-induced apoptosis in HNSCC, which would provide substantial theoretical underpinnings for afatinib clinical application and highlight its promising prospect in HNSCC treatment.