Lactate dehydrogenase B as a metabolism-related marker for immunotherapy in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies. Lactate dehydrogenase family genes (LDHs) play a critical role in tumor metabolism, but their functions in HNSCC have not been investigated thoroughly. Thus, we aimed to explore the value of LDHs in HNSCC. METHODS: The association between LDHs expression and mutations, methylation, copy number variations (CNVs), alternative splicing (AS) and competing endogenous RNA (ceRNA) was investigated in The Cancer Genome Atlas (TCGA). The expression level of LDHs in OSCC tissues and adjacent normal tissues was verified by qPCR. Algorithms, such as ssGSEA, ESTIMATE, xCell and TIDE were utilized to analyze the characteristics of immune infiltration. Pathway alternations were enriched by GO, GSEA and KEGG analysis. The Mantel test was employed to elucidate the correlation between metabolism and the tumor microenvironment (TME). Subsequently, MTT and colony formation assays were utilized to assess the impact of LDHB knockdown on cellular proliferation. Additionally, ATP and lactate assays were performed to examine metabolic alterations. Co-culture experiments further investigated the effect of LDHB knockdown on T cell differentiation. RESULTS: LDHs were completely analyzed in multiple databases, among which LDHB was differentially expressed in HNSCC and significantly associated with prognosis. Low LDHB expression had better clinicopathological characteristics. Downregulated LDHB expression was associated with enhanced immune cell infiltration and could influence tumor metabolism. Despite having worse cytotoxic T lymphocyte dysfunction, the LDHBlow group was predicted to respond more favorably to immune checkpoint inhibitors (ICIs) therapy. Moreover, the correlation between metabolism and TME was depicted. In vitro, LDHB knockdown resulted in inhibited cell proliferation, increased lactate levels and decreased ATP levels, while promoted the Th1 differentiation of T cells. CONCLUSIONS: Our study provided a comprehensive analysis of the LDHs and illustrated low LDHB expression could inhibit tumor cell proliferation and ATP production by influencing metabolism, with improved immune cell infiltration and better response to immunotherapy.

KRAS mutations upregulate Runx1 to promote occurrence of head and neck squamous cell carcinoma.

Gene mutations play an important role in head and neck squamous cell carcinoma (HNSCC) by not only promoting the occurrence and progression of HNSCC but also affecting sensitivity to treatment and prognosis. KRAS is one of the most frequently mutated oncogenes, which has been reported to have a mutation rate from 1.7% to 12.7% and may lead to poor prognosis in HNSCC, but its role remains unclear. Here, we found that the KRAS mutation can promote HNSCC generation through synergism with 4-Nitroquinoline-1-Oxide(4NQO). Mechanistically, KRAS mutations can significantly upregulate Runx1 to promote oral epithelial cell proliferation and migration and inhibit apoptosis. Runx1 inhibitor Ro 5-3335 can effectively inhibit KRAS-mutated HNSCC progression both in vitro and in vivo. These findings suggest that the KRAS mutation plays an important role in HNSCC and that Runx1 may be a novel therapeutic target for KRAS-mutated HNSCC.

Exosomal miR-146b-5p derived from cancer-associated fibroblasts promotes progression of oral squamous cell carcinoma by downregulating HIPK3.

OBJECTIVES: Cancer-associated fibroblasts (CAFs) are vital constituents of the tumor microenvironment (TME) and play a predominant role in oral squamous cell carcinoma (OSCC) progression. We aimed to investigate the effect and mechanism of exosomal miR-146b-5p derived from CAFs on the malignant biological behavior of OSCC. MATERIALS AND METHODS: Illumina small RNA (sRNA) sequencing was conducted to determine the differential expression patterns of microRNAs (miRNAs) in exosomes derived from CAFs and normal fibroblasts (NFs). Transwell and cell counting kit-8 (CCK-8) assays and xenograft tumor models in nude mice were used to investigate the effect of CAF exosomes and miR-146b-p on the malignant biological behavior of OSCC. Reverse transcription quantitative real-time PCR (qRT-PCR), luciferase reporter, western blotting (WB) and immunohistochemistry assays were employed to investigate the underlying mechanisms involved in CAF exosomes that promote OSCC progression. RESULTS: We demonstrated that CAF-derived exosomes were taken up by OSCC cells and enhanced the proliferation, migration, and invasion ability of OSCC. Compared with NFs, the expression of miR-146b-5p was increased in exosomes and their parent CAFs. Further studies showed that the decreased expression of miR-146b-5p inhibited the proliferation, migration and invasion ability of OSCC cells in vitro and the growth of OSCC cells in vivo. Mechanistically, miR-146b-5p overexpression led to the suppression of HIKP3 by directly targeting the 3'-UTR of HIPK3, as confirmed by luciferase assay. Reciprocally, HIPK3 knockdown partially reversed the inhibitory effect of the miR-146b-5p inhibitor on the proliferation, migration, and invasion ability of OSCC cells and restored their malignant phenotype. CONCLUSIONS: Our results revealed that CAF-derived exosomes contained higher levels of miR-146b-5p than NFs, and miR-146b-5p overexpression in exosomes promoted the malignant phenotype of OSCC by targeting HIPK3. Therefore, inhibiting exosomal miR-146b-5p secretion may be a promising therapeutic modality for OSCC.

Upregulated miR­411­5p levels promote lymph node metastasis by targeting RYBP in head and neck squamous cell carcinoma.

Metastasis is the primary cause of the high mortality rates in head and neck squamous cell carcinoma (HNSCC). MicroRNA (miR)­411­5p has been discovered to serve an important role in cancer metastases. However, to the best of our knowledge, the association between miR­411­5p expression levels and HNSCC metastasis has not been thoroughly investigated. The present study aimed to research the function of miR­411­5p in HNSCC metastasis. The results of the present study revealed that miR­411­5p expression levels were upregulated in patients with HNSCC with lymph node metastasis and the upregulated expression levels of miR­411­5p were positively associated with the metastatic potential of HNSCC. Moreover, miR­411­5p promoted HNSCC cell migration, invasion and epithelial­mesenchymal transition (EMT). The results of the dual­luciferase reporter assays identified RING1 and YY1 binding protein (RYBP) as a functional downstream target gene for miR­411­5p. Therefore, whether miR­411­5p downregulated the expression levels of RYBP in HNSCC cells was subsequently investigated. Notably, the silencing of RYBP expression restored the stimulatory effects of miR­411­5p on HNSCC cell migration, invasion and EMT. In addition, the mRNA expression levels of miR­411­5p and RYBP were found to be inversely correlated in HNSCC samples. In conclusion, the results of the present study indicated that the miR­411­5p­mediated downregulation of RYBP expression levels may exert an important role in HNSCC metastasis and may provide a novel target for the treatment of HNSCC.

Metformin Combined with 4SC-202 Inhibited the Migration and Invasion of OSCC via STAT3/TWIST1.

BACKGROUND: Oral squamous cell carcinoma (OSCC), the most common epithelial malignant neoplasm in the head and neck, characterizes with local infiltration and metastasis of lymph nodes. The five-year survival rate of OSCC remains low despite the advances in clinical methods. Thus, it is necessary to develop a new effective therapeutic scheme for OSCC. Our previous results showed that metformin and 4SC-202 synergistically promoted the intrinsic apoptosis of OSCC in vitro and in vivo, but the effects on invasion and migration remained unclear. METHODS: Human OSCC cell lines HSC6 and CAL33 were cultured with metformin (16 mM) or/and 4SC-202 (0.4 µM) for 72 h. STAT3 inhibitor S31-201 was applied at concentration of 60 µM for 48 h. Wound-healing assays and transwell assays were used to determine the invasion and migration ability of OSCC. qRT-PCR and Western blot were performed to detect mRNA levels and protein levels. RESULTS: Metformin or/and 4SC-202 suppressed the migration and invasion of OSCC cells. Importantly, the expression of TWIST1 was suppressed by metformin and 4SC-202, while the invasion and migration inhibitory effects of metformin and 4SC-202 were countered by the overexpression of TWIST1. In addition, the phosphorylation level of STAT3 decreased after the administration of metformin or/and 4SC-202. Furthermore, inhibition of STAT3 by S31-201 suppressed the expression of TWIST1 and led to a decline in migration and invasion of OSCC, while overexpression of TWIST1 attenuated these effects. CONCLUSION: Metformin and 4SC-202 suppressed the invasion and migration of OSCC through inhibition of STAT3/TWIST1, and this scheme can serve as a novel therapeutic strategy for OSCC.

Targeting methyltransferase PRMT5 retards the carcinogenesis and metastasis of HNSCC via epigenetically inhibiting Twist1 transcription.

Protein arginine methyltransferase 5 (PRMT5) is an important type II arginine methyltransferase that can play roles in cancers in a highly tissue-specific manner, but its role in the carcinogenesis and metastasis of head and neck squamous cell carcinoma (HNSCC) remains unclear. Here, we detected PRMT5 expression in HNSCC tissues and performed series of in vivo and in vitro assays to investigate the function and mechanism of PRMT5 in HNSCC. We found that PRMT5 was overexpressed in dysplastic and cancer tissues, and associated with lymph node metastasis and worse patient survival. PRMT5 knockdown repressed the malignant phenotype of HNSCC cells in vitro and in vivo. PRMT5 specific inhibitor blocked the formation of precancerous lesion and HNSCC in 4NQO-induced tongue carcinogenesis model, prevented lymph node metastasis in tongue orthotopic xenograft model and inhibited cancer development in subcutaneous xenograft model and Patient-Derived tumor Xenograft (PDX) model. Mechanistically, PRMT5-catalyzed H3R2me2s promotes the enrichment of H3K4me3 in the Twist1 promoter region by recruiting WDR5, and subsequently activates the transcription of Twist1. The rescue experiments indicated that overexpressed Twist1 abrogated the inhibition of cell invasion induced by PRMT5 inhibitor. In summary, this study elucidates that PRMT5 inhibition could reduce H3K4me3-mediated Twist1 transcription and retard the carcinogenesis and metastasis of HNSCC.

Salivary exosomal miR-24-3p serves as a potential detective biomarker for oral squamous cell carcinoma screening.

OBJECTIVES: miRNAs in salivary exosomes are used as novel non-invasive biomarkers for detection strategies of human disease. Here, we aimed to investigate the diagnostic potential of salivary exosomal miRNAs as biomarkers for screening oral squamous cell carcinoma (OSCC) and to explore the underlying mechanisms of OSCC pathogenesis. MATERIALS AND METHODS: Differentially expressed miRNAs were obtained from salivary exosomes of four healthy controls and four OSCC patients using miRNA microarray analysis. The expression of miR-24-3p in the salivary exosomes was then verified by qRT-PCR. The diagnostic power was assessed by receiver operating characteristic (ROC) analysis. Cell proliferation was measured using CCK-8 cell viability assay and colony formation assay. The target gene of miR-24-3p was confirmed by dual luciferase reporter assay. RESULTS: A total of 109 miRNAs were found to be more than 2-fold altered in the salivary of patients and healthy individuals by miRNA microarray. qRT-PCR analysis further confirmed a significant increase of miR-24-3p in the salivary exosomes from 45 preoperative OSCC patients compared to 10 normal controls. ROC analysis showed that miR-24-3p has excellent diagnostic accuracy for OSCC (area under the ROC curve [AUC] = 0.738; P = 0.02). Similarly, we found that miR-24-3p expressed a higher level in OSCC neoplastic tissues, suggesting that circulating miR-24-3p may originate from tumor cells. Furthermore, exogenous exosomal miR-24-3p increased proliferation of recipient malignant cells. Additionally, overexpression of miR-24-3p promoted the proliferation of OSCC cells and regulated the expression of cell cycle-related genes. Dual luciferase reporter assay indicated that miR-24-3p can interact with PER1 directly. CONCLUSIONS: Salivary exosomal miR-24-3p is a potential novel diagnostic biomarker for OSCC, and miR-24-3p can maintain the proliferation of OSCC cells through targeting PER1.

Metformin and 4SC-202 synergistically promote intrinsic cell apoptosis by accelerating ΔNp63 ubiquitination and degradation in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common and aggressive epithelial tumor in the head and neck region with a rising incidence. Despite the advances in basic science and clinical research, the overall survival rate of OSCC remains low. Thus finding novel effective therapeutic agents for OSCC is necessary. In this study, we investigated the effects and mechanisms of combined metformin and 4SC-202 in OSCC. Our results showed that metformin and 4SC-202 synergistically suppressed the proliferation and promoted the intrinsic apoptosis of OSCC cells in vitro and in vivo. Importantly, the proteasome inhibitor MG132 impeded the ΔNp63-decreasing effects after metformin and 4SC-202 treatment, indicating that metformin and 4SC-202 could promote the degradation of ΔNp63 protein. Moreover, ubiquitination level of ΔNp63 increased after metformin or/and 4SC-202 administration. Furthermore, we revealed that ΔNp63 mediated anticancer effects of metformin and 4SC-202, as overexpression or suppression of ΔNp63 could attenuate or facilitate the apoptosis rate of OSCC under metformin or/and 4SC-202 treatment. Collectively, metformin and 4SC-202 synergistically promote intrinsic apoptosis through accelerating ubiquitin-mediated degradation of ΔNp63 in OSCC, and this co-treatment can serve as a potential therapeutic scheme for OSCC.

Combined class I histone deacetylase and mTORC1/C2 inhibition suppresses the initiation and recurrence of oral squamous cell carcinomas by repressing SOX2.

Treatment of oral squamous cell carcinoma (OSCC) remains a challenge because of the lack of effective early treatment strategies and high incidence of relapse. Here, we showed that combined 4SC-202 (a novel selective class I HDAC inhibitor) and INK128 (a selective mTORC1/C2 inhibitor) treatment exhibited synergistic effects on inhibiting cell growth, sphere-forming ability, subcutaneous tumor formation and ALDH1+ cancer stem cells (CSCs) in OSCC. The initiation of OSCC was significantly inhibited by combined treatment in 4NQO-induced rat model. In addition, upregulated SOX2 was associated with advanced and metastatic tumors in OSCC patients and was responsible for the drug-resistance property of OSCC cells. The inhibitory effect of combined treatment on cell viability and ALDH1+ CSCs were attenuated by SOX2 verexpression. Furthermore, combined treatment can effectively overcome chemoresistance and inhibit the growth of recurrent OSCC in vitro and in vivo. Mechanistically, 4SC-202 and INK128 repressed SOX2 expression through miR-429/miR-1181-mediated mRNA degradation and preventing cap-dependent mRNA translation, respectively. These results suggest that combined class I histone deacetylase and mTORC1/C2 inhibition suppresses the carcinogenesis and recurrence of OSCC by repressing SOX2.

Chemokine (CC motif) ligand 18 upregulates Slug expression to promote stem-cell like features by activating the mammalian target of rapamycin pathway in oral squamous cell carcinoma.

Chemokine (CC motif) ligand 18 (CCL18) is involved in remodeling of the tumor microenvironment and plays critical roles in oncogenesis, invasiveness, and metastasis. We previously investigated the overexpression of CCL18 in primary oral squamous cell carcinoma (OSCC) tissues and its association with advanced clinical stage in OSCC patients. However, the underlying mechanisms of this CCL18-derived activity remains unidentified. This study showed exogenous CCL18 increased cell migration and invasion and induced cell epithelial-mesenchymal transition (EMT), and that E-cadherin, an epithelial marker, decreased and N-cadherin, a mesenchymal marker, increased, compared to negative control in OSCC cells. Furthermore, we detected that CCL18 induced the acquisition of cancer stem(-like) cell characteristics in oral cancer cells, but also found a significantly positive correlation between the expression of CCL18 and Bmi-1 (P < 0.001) in OSCC surgical specimens by immunohistochemistry analysis. The expression of octamer-binding transcription factor 4 and Bmi-1 were significantly upregulated, and proportions of aldehyde dehydrogenasehigh+ cells and CD133+ cells were markedly increased in CCL18-treated cells compared to untreated cells. Sphere formation ability was observably enhanced when cells were continually exposed to high levels of CCL18. Moreover, CCL18 upregulated Slug expression by stimulating the mammalian target of rapamycin (mTOR) signaling pathway in OSCC cell lines. Inhibition of the mTOR pathway by INK128, or Slug knockdown by RNA interference, reversed CCL18-induced EMT and the stemness response at both molecular and functional levels. In conclusion, our data suggested that CCL18 upregulated Slug expression to promote EMT and stem cell-like features by activating the mTOR pathway in oral cancer. These findings provide new potential targets for the early diagnosis and treatment of OSCC.

Prognostic Value of Cancer Stem Cell Markers in Head and Neck Squamous Cell Carcinoma: a Meta-analysis.

Bmi-1, CD133, Nanog and Oct-4 have been reported as cancer stem cell (CSC) markers in head and neck squamous cell carcinoma (HNSCC). However, the prognostic value of them in HNSCC remains controversial. Hence, this meta-analysis was conducted to access the association between the four CSC markers and survival outcome of HNSCC patients. A total of 22 articles with 27 studies met the inclusion criteria and the combined hazard ratio (HR) and 95% confidence intervals (95% CI) were calculated. Data analysis showed that high expression of CSC markers was associated with poor overall survival (OS) (HR = 1.93; 95% CI: 1.46-2.55, P < 0.001) and disease free survival (DFS) (HR = 4.78; 95% CI: 2.95-7.75, P < 0.001) but not disease specific survival (DSS) (HR = 1.17; 95% CI: 0.74-1.84, P = 0.50) of HNSCC patients. Subgroup analysis indicted that high expression of CD133 (HR = 2.33, 95%CI: 1.42-3.83, P < 0.001), Oct-4(HR = 2.10, 95%CI: 1.36-3.22, P = 0.007) and Nanog (HR = 2.49, 95%CI: 1.66-3.72, P < 0.001) could predict poor OS in HNSCC patients respectively whereas overexpression of Bmi-1 was not related to the reduced OS in HNSCC patients (HR = 1.32, 95%CI: 0.66-2.65, P = 0.43). Therefore, we concluded that CSC markers, especially CD133, Nanog and Oct-4, might be predictive factors in HNSCC patients.