Human Oral Squamous Cell Carcinoma Cell Research Articles

Possible Role of miR-375-3p in Cervical Lymph Node Metastasis of Oral Squamous Cell Carcinoma.

No clinically useful predictors of latent cervical lymph node metastasis (LNM) in early oral squamous cell carcinoma (OSCC) are available. In this study, we focused on the microRNAs (miRNAs) involved in the expression of numerous genes and explored those associated with latent cervical LNM in early OSCC (eOSCC). First, microarray and RT-PCR analyses revealed a significant downregulation of miR-375-3p expression in primary eOSCC tissues with latent cervical LNM. Next, we examined the effects of miR-375-3p mimics on the growth and migration of four human OSCC cell lines that do not express miR-375-3p. The overexpression of miR-375-3p significantly suppressed the cell proliferation and migration of human OSCC cells in vitro. Furthermore, miR-375-3p mimics markedly inhibited the subcutaneously xenografted human OSCC tumors. Finally, we found the genes involved in the PI3K-AKT pathway and cell migration as target gene candidates of miR-375-3p in human OSCC cells. These findings suggest that miR-375-3p functions as a tumor suppressive-miRNA in OSCC and may serve as a potential biomarker for the prediction of latent cervical LNM in eOSCC and a useful therapeutic target to suppress OSCC progression.

Abstract 3795: Possible role of miR-375-3p in cervical lymph node metastasis of oral squamous cell carcinoma

Abstract Latent cervical lymph node metastasis (LNM) is the most important prognostic factor in early oral squamous cell carcinoma (OSCC). However, no clinically useful predictors of LNM in early OSCC are available. In this study, we focused on the microRNAs (miRNAs) involved in the expression of numerous genes and explored those associated with latent cervical LNM in OSCC. Microarray analysis of miRNA expression showed that the expression levels of miR-375-3p were significantly reduced in primary OSCC tissues with latent cervical LNM. Subsequently, we quantified the expression of miR-375-3p by RT-qPCR and digital PCR. Similar to the results of miRNA microarray analysis, miR-375-3p was significantly downregulated in primary OSCC tissues with latent cervical LNM. The results of digital PCR showed a more significant difference than those of RT-qPCR. When the reference value for predicting latent cervical LNM was set to less than 57.85 copies based on the receiver operating characteristic curve, the sensitivity, specificity, accuracy, and area under the curve were 80%, 100%, 90%, and 0.942, respectively. Next, we examined the effects of miR-375-3p mimics on the growth and migration of four human OSCC cell lines (SAS-L1, HSC2, HSC3, and Ca9-22) that do not express miR-375-3p. When an miR-375-3p mimic was introduced into these cells, cell proliferation and migration were significantly suppressed. Total RNA was extracted from these cells reverse-transfected with miR-375-3p mimic for 48 hours. Microarray analysis and Ingenuity Pathway Analysis (IPA) miRNA target filter showed that miR-375-3p overexpression reduced the expression of 266 genes, of which 37 genes had the target sequences of miR-375-3p in their 3’-untranslated region. Among these target genes, CEPT1 and TIMM8A knockdown significantly inhibited the migration of all human OSCC cells, similar to miR-375-3p mimics. Furthermore, IPA core analysis revealed a decrease in expression of genes involved in the activation of PI3K-AKT pathways by miR-375-3p overexpression. These results indicated that downregulating miR-375-3p expression could promote cervical LNM by enhancing cell proliferation and migration in OSCC. We also found that CEPT1 and TIMM8A as novel target genes of miR-375-3p regulated the migration of human OSCC cells. The expression levels of miR-375-3p in primary OSCC tissues appear to be a useful biomarker for predicting latent cervical LNM. Citation Format: Masato Saika, Koh-ichi Nakashiro, Norihiko Tokuzen, Hiroyuki Shirai, Daisuke Uchida. Possible role of miR-375-3p in cervical lymph node metastasis of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3795.

PHA-665752's Antigrowth and Proapoptotic Effects on HSC-3 Human Oral Cancer Cells.

c-Met is a tyrosine-kinase receptor, and its aberrant activation plays critical roles in tumorigenesis, invasion, and metastatic spread in many human tumors. PHA-665752 (PHA) is an inhibitor of c-Met and has antitumor effects on many hematological malignancies and solid cancers. However, the activation and expression of c-Met and its role and the antitumor effect of PHA on human oral squamous cell carcinoma (OSCC) cells remain unclear. Here, we investigated the activation and expression of c-Met and the effects of PHA on the growth of a highly tumorigenic HSC-3 human OSCC cell line with high c-Met phosphorylation and expression. Of note, c-Met was highly expressed and phosphorylated on Y1234/1235 in HSC-3 cells, and PHA treatment significantly suppressed the growth and induced apoptosis of these cells. Moreover, PHA that inhibited the phosphorylation (activation) of c-Met further caused the reduced phosphorylation and expression levels of Src, protein kinase B (PKB), mammalian target of rapamycin (mTtor), and myeloid cell leukemia-1 (Mcl-1) in HSC-3 cells. In addition, the antiangiogenic property of PHA in HSC-3 cells was shown, as evidenced by the drug's suppressive effect on the expression of hypoxia-inducible factor-1α (HIF-1α), a critical tumor angiogenic transcription factor. Importantly, genetic ablation of c-Met caused the reduced growth of HSC-3 cells and decreased Src phosphorylation and HIF-1α expression. Together, these results demonstrate that c-Met is highly activated in HSC-3 human oral cancer cells, and PHA exhibits strong antigrowth, proapoptotic, and antiangiogenic effects on these cells, which are mediated through regulation of the phosphorylation and expression of multiple targets, including c-Met, Src, PKB, mTOR, Mcl-1, and HIF-1α.

Tetraspanin CD82 Correlates with and May Regulate S100A7 Expression in Oral Cancer.

Many metastatic cancers with poor prognoses correlate to downregulated CD82, but exceptions exist. Understanding the context of this correlation is essential to CD82 as a prognostic biomarker and therapeutic target. Oral squamous cell carcinoma (OSCC) constitutes over 90% of oral cancer. We aimed to uncover the function and mechanism of CD82 in OSCC. We investigated CD82 in human OSCC cell lines, tissues, and healthy controls using the CRISPR-Cas9 gene knockout, transcriptomics, proteomics, etc. CD82 expression is elevated in CAL 27 cells. Knockout CD82 altered over 300 genes and proteins and inhibited cell migration. Furthermore, CD82 expression correlates with S100 proteins in CAL 27, CD82KO, SCC-25, and S-G cells and some OSCC tissues. The 37-50 kDa CD82 protein in CAL 27 cells is upregulated, glycosylated, and truncated. CD82 correlates with S100 proteins and may regulate their expression and cell migration. The truncated CD82 explains the invasive metastasis and poor outcome of the CAL 27 donor. OSCC with upregulated truncated CD82 and S100A7 may represent a distinct subtype with a poor prognosis. Differing alternatives from wild-type CD82 may elucidate the contradictory functions and pave the way for CD82 as a prognostic biomarker and therapeutic target.

Semilicoisoflavone B induces oral cancer cell apoptosis by targeting claspin and ATR-Chk1 signaling pathways.

The prevalence of oral squamous cell carcinoma (OSCC) is increasing worldwide mainly due to poor oral hygiene and unrestricted lifestyle. Advanced-stage OSCC is associated with poor prognosis and a 5-year survival rate of only 30%-50%. The present study was designed to investigate the anticancer effect and mode of action of Glycyrrhiza-derived semilicoisoflavone B (SFB) in 5-fluorourasil (5FU)-resistant human OSCC cell lines. The study findings revealed that SFB significantly reduces OSCC cell viability and colony formation ability by arresting cell cycle at the G2/M and S phases and reducing the expressions of key cell cycle regulators including cyclin A, cyclin B, CDC2, and CDK2. The compound caused a significant induction in the percentage of nuclear condensation and apoptotic cells in OSCC. Regarding pro-apoptotic mode of action, SFB was found to increase Fas-associated death domain and death receptor 5 expressions and reduce decoy receptor 2 expression, indicating involvement of extrinsic pathway. Moreover, SFB was found to increase pro-apoptotic Bim expression and reduce anti-apoptotic Bcl-2 and Bcl-xL expressions, indicating involvement of intrinsic pathway. Moreover, SFB-mediated induction in cleaved caspases 3, 8, and 9 and cleaved poly(ADP-ribose) polymerase confirmed the induction of caspase-mediated apoptotic pathways. Regarding upstream signaling pathway, SFB was found to reduce extracellular signal regulated kinase 1/2 (ERK) phosphorylation to execute its pro-apoptotic activity. The Human Apoptotic Array findings revealed that SFB suppresses claspin expression, which in turn caused reduced phosphorylation of ATR, checkpoint kinase 1 (Chk1), Wee1, and CDC25C, indicating disruption of ATR-Chk1 signaling pathway by SFB. Taken together, these findings indicate that SFB acts as a potent anticancer compound against 5FU-resistant OSCC by modulating mitogen-activated protein kinase (MAPK) and ATR-Chk1 signaling pathways.

TLR9 activation induces immunosuppression and tumorigenesis via PARP1/PD-L1 signaling pathway in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer, and metastasis and immunosuppression are responsible for the poor prognosis of OSCC. Previous studies have shown that poly(ADP-ribose) polymerase (PARP)1 plays a key role in the pathogenesis of OSCC. Therefore, PARP1 may serve as an important research target for the potential treatment of OSCC. Here, we aimed to investigate the role of PARP1 in the tumorigenesis of OSCC and elucidate the key molecular mechanisms of its upstream and downstream regulation in vivo and in vitro. In human OSCC tissues and cells, Toll-like receptor (TLR)9 and PD-L1 were highly expressed and PARP1 was lowly expressed. Suppression of TLR9 remarkably repressed CAL27 and SCC9 cell proliferation, migration, and invasion. After coculture, we found that low expression of TLR9 inhibited PD-L1 expression and immune escape. In addition, TLR9 regulated PD-L1 expression through the PARP1/STAT3 pathway. PARP1 mediated the effects of TLR9 on OSCC cell proliferation, migration, and invasion and immune escape. Additionally, in vivo experiments further verified that TLR9 promoted tumor growth and immune escape by inhibiting PARP1. Collectively, TLR9 activation induced immunosuppression and tumorigenesis via PARP1/PD-L1 signaling pathway in OSCC, providing important insights for subsequent in-depth exploration of the mechanism of OSCC.NEW & NOTEWORTHY In this research, we took PARP1 as the key target to explore its regulatory effect on oral squamous cell carcinoma (OSCC). The key molecular mechanisms involved in its upstream and downstream regulation were elucidated in OSCC cell lines in vitro and tumor-bearing mice in vivo, combined with clinical OSCC tissues.

Astaxanthin Synergizes with Ionizing Radiation (IR) in Oral Squamous Cell Carcinoma (OSCC).

Astaxanthin (ATX) is known for its antioxidant and anti-inflammation functions yet its role in cancers requires more research. This study is aimed to reveal the potential synergetic effect of ATX with ionizing radiation (IR) in OSCC. Cell survival was measured after human OSCC cells including CAL27 and SCC9, and normal human oral keratinocytes (NHOKs) were treated with different concentrations of ATX for 24h. Colony formation assays were performed after OSCC cells were treated with IR, ATX (20μM), or combined and survival fraction was analyzed. Malondialdehyde (MDA), glutathione (GSH), and intercellular iron levels were measured. Western blot method was used to measure the ferroptosis-related proteins, GPX4, SLC7A11, and ACSL4. In xenograft mice model, we evaluated the tumor volumes, tumor growth, and examined the GPX4/ACSL4 proteins in tumor tissues using Immunohistochemistry (IHC). ATX inhibited viability of OSCC cells but not NHOK. In OSCC cells, ATX further enhanced the cell death induced by IR. In addition, ATX promoted the MDA content, Iron levels but inhibited the GSH regulated by IR in cells. ATX could synergize with IR, further inhibiting GPX4, SLC7A11 and promoting ACSL4 in OSCC cells. In vivo, ATX and IR treatment inhibited OSCC tumor growth and the group with combined treatment showed the most inhibitory effect. GPX4 was inhibited by IR and further inhibited in the combined group while ACSL4 was promoted by IR and enhanced more significantly in the combined group. ATX might synergize with IR treatment in OSCC partly via ferroptosis.

Apoptotic activity of genipin in human oral squamous cell carcinoma in vitro by regulating STAT3 signaling.

Genipin, a natural compound derived from the fruit of Gardenia jasminoides Ellis, was reported to have activity against various cancer types. In this study, we determined the underlying mechanism for genipin-induced cell death in human oral squamous cell carcinoma (OSCC). The growth-inhibitory effects of genipin in human OSCC cells was examined by the Cell Counting Kit-8 and soft agar assays. The effects of genipin on apoptosis were assessed by nuclear morphological changes by 4',6-diamidino-2-phenylindole staining, measurement of the sub-G1 population, and Annexin V-fluorescein isothiocyanate/propidium iodide double staining. The underlying mechanism of genipin activity was analyzed by western blot analysis, subcellular fractionation of the nucleus and cytoplasm, immunocytochemistry, and quantitative real-time polymerase chain reaction. Genipin inhibited the growth of OSCC cells and induced apoptosis, which was mediated by a caspase-dependent pathway. Genipin reduced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr705 and its nuclear localization. Furthermore, inhibition of p-STAT3Tyr705 levels following genipin treatment was required for the reduction of survivin and myeloid cell leukemia-1 (Mcl-1) expression, leading to apoptotic cell death. The genipin-mediated reduction in survivin and Mcl-1 expression was caused by transcriptional and/or posttranslational regulatory mechanisms. The results provide insight into the regulatory mechanism by which genipin induces apoptotic cell death through the abrogation of nuclear STAT3 phosphorylation and suggest that genipin may represent a potential therapeutic option for the treatment of human OSCC.

Podophyllotoxin reduces the aggressiveness of human oral squamous cell carcinoma through myeloid cell leukemia‑1.

Podophyllotoxin (PPT), which is derived from the podophyllum plant, exhibits marked cytotoxic effects against cancer cells; however, the precise molecular mechanism underlying its activity against human oral squamous cell carcinoma (OSCC) has not been elucidated. In the present study, the mechanism by which PPT induced cytotoxicity in two OSCC cell lines, HSC3 and HSC4, was determined. The effects of PPT on cytotoxicity in HSC3 and HSC4 cells were analyzed using Annexin V/PI double staining, Sub‑G1 analysis, soft agar assays, western blotting, and quantitative PCR. The changes in the mitochondrial membrane potential were assessed using a JC‑1 assay and cytosolic and mitochondrial fractionation. A myeloid cell leukemia‑1 (Mcl‑1) overexpression cell lines were also established to study the role of Mcl‑1 on apoptosis. The results showed that PPT inhibited the growth of the two human OSCC cell lines and induced apoptosis, which was accompanied by mitochondrial membrane depolarization. Compared with the control, PPT reduced the expression of Mcl‑1 in both cell lines through a proteasome‑dependent protein degradation process. Overall, these results suggested that targeting of Mcl‑1 protein by PPT induced apoptosis, providing a foundation for further pre‑clinical and clinical study of its value in the management of OSCC.

Transcutaneous carbon dioxide application suppresses the expression of cancer-associated fibroblasts markers in oral squamous cell carcinoma xenograft mouse model.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer. Cancer-associated fibroblasts (CAFs) are the main stromal cells in the tumor microenvironment (TME). As CAFs promote tumor progression and hypoxia in the TME, regulating the conversion of normal fibroblasts (NFs) into CAFs is essential for improving the prognosis of patients with OSCC. We have previously reported the antitumor effects of transcutaneous carbon dioxide (CO2) application in OSCC. However, the effects of reducing hypoxia in the TME remain unclear. In this study, we investigated whether CO2 administration improves the TME by evaluating CAFs marker expression. Human OSCC cells (HSC-3) and normal human dermal fibroblasts (NHDF) were coinjected subcutaneously into the dorsal region of mice. CO2 gas was applied twice a week for 3 weeks. The tumors were harvested six times after transcutaneous CO2 application. The expression of CAFs markers (α-SMA, FAP, PDPN, and TGF-β) were evaluated by using real-time polymerase chain reaction and immunohistochemical staining. The expression of α-SMA, FAP, PDPN, and TGF-β was significantly increased over time after co-injection. In the CO2-treated group, tumor growth was significantly suppressed after treatment initiation. In addition, the mRNA expression of these markers was significantly inhibited. Furthermore, immunohistochemical staining revealed a significant decrease in the protein expression of all CAFs markers in the CO2-treated group. We confirmed that transcutaneous CO2 application suppressed CAFs marker expression and tumor growth in OSCC xenograft mouse model.

MicroRNA-1289 Functions as a Novel Tumor Suppressor in Oral Squamous Cell Carcinoma.

Recently, numerous tumor-suppressive microRNAs (TS-miRs) have been identified in human malignancies. Here, we attempted to identify novel TS-miRs in oral squamous cell carcinoma (OSCC). First, we transfected human OSCC cells individually with 968 synthetic miRs mimicking human mature miRs individually, and the growth of these cells was evaluated using the WST-8 assay. Five miR mimics significantly reduced the cell growth rate by less than 30%, and the miR-1289 mimic had the most potent growth inhibitory effect among these miRs. Subsequently, we assessed the in vivo growth-inhibitory effects of miR-1289 using a mouse model. The administration of the miR-1289 mimic-atelocollagen complex significantly reduced the size of subcutaneously xenografted human OSCC tumors. Next, we investigated the expression of miR-1289 in OSCC tissues using reverse transcription-quantitative PCR. The expression level of miR-1289 was significantly lower in OSCC tissues than in the adjacent normal oral mucosa. Furthermore, 15 genes were identified as target genes of miR-1289 via microarray and Ingenuity Pathway Analysis (IPA) microRNA target filtering. Among these genes, the knockdown of magnesium transporter 1 (MAGT1) resulted in the most remarkable cell growth inhibition in human OSCC cells. These results suggested that miR-1289 functions as a novel TS-miR in OSCC and may be a useful therapeutic tool for patients with OSCC.

Dual roles of cellular communication network factor 6 (CCN6) in the invasion and metastasis of oral cancer cells to bone via binding to BMP2 and RANKL.

The acquisition of motility via epithelial-mesenchymal transition (EMT) and osteoclast induction are essential for the invasion and metastasis of oral squamous cell carcinoma (OSCC) to bone. However, the molecule suppressing both EMT and osteoclastogenesis is still unknown. In this study, we found that cellular communication network factor 6 (CCN6) was less produced in a human OSCC cell line, HSC-3 with mesenchymal phenotype, than in HSC-2 cells without it. Notably, CCN6 interacted with bone morphogenetic protein 2 (BMP2) and suppressed the cell migration of HSC-3 cells stimulated by BMP2. Moreover, knockdown of CCN6 in HSC-2 cells led to the promotion of EMT and enhanced the effect of transforming growth factor-β (TGF-β) on the promotion of EMT. Furthermore, CCN6 combined with BMP2 suppressed EMT. These results suggest that CCN6 strongly suppresses EMT in cooperation with BMP2 and TGF-β. Interestingly, CCN6 combined with BMP2 increased the gene expression of receptor activator of nuclear factor-κB ligand (RANKL) in HSC-2 and HSC-3 cells. Additionally, CCN6 interacted with RANKL, and CCN6 combined with RANKL suppressed RANKL-induced osteoclast formation. In metastatic lesions, increasing BMP2 due to the bone destruction led to interference with binding of CCN6 to RANKL, which results in the promotion of bone metastasis of OSCC cells due to continuous osteoclastogenesis. These findings suggest that CCN6 plays dual roles in the suppression of EMT and in the promotion of bone destruction of OSCC in primary and metastatic lesions, respectively, through cooperation with BMP2 and interference with RANKL.

Role of Stromal Fibroblast–Induced WNT7A Associated with Cancer Cell Migration Through the AKT/CLDN1 Signaling Axis in Oral Squamous Cell Carcinoma

Wnt signaling plays a crucial role in the progression of various cancers, including oral squamous cell carcinoma (OSCC). However, the tumor microenvironment (TME) regulating Wnt signaling has not yet been fully elucidated. In this study, we investigated whether cancer-associated fibroblasts (CAFs), the primary components of the TME, activate Wnt signaling and promote tumor progression in OSCC. We conducted a Transwell coculture assay using human OSCC cell lines and normal human dermal fibroblasts (NHDFs). NHDFs stimulated WNT7A expression in several OSCC cell lines, especially HO-1-N-1 and HSC-5. An immunohistochemical study using 122 human OSCC samples indicated that high WNT7A expression in tumor cells was significantly associated with invasion depth and poor prognosis. Moreover, WNT7A expression in OSCC cells was positively correlated with α–smooth muscle actin expression in CAFs. WNT7A knockdown in OSCC cells demonstrated that OSCC cells cocultured with NHDFs significantly promoted tumor cell migration and invasion, which was dependent on WNT7A expression in OSCC cells. We also isolated HSC-5 cells from the coculture and conducted microarray analysis to investigate the factors that promote tumor progression induced by WNT7A. Among the various differentially expressed genes, we identified a downregulated gene encoding CLDN1 and confirmed that WNT7A negatively regulated CLDN1 expression in OSCC cells and CLDN1 knockdown in OSCC cells promoted their migration. Phosphokinase array analysis showed that WNT7A activates protein kinase B (AKT) phosphorylation. Activating AKT signaling using the SC79 agonist induced CLDN1 downregulation in OSCC cells. In the coculture assay, the AKT inhibitor MK2206 significantly recovered CLDN1 expression downregulated by WNT7A, resulting in OSCC cell migration suppression. These results suggest that CAFs stimulate OSCC cells to produce WNT7A, following CLDN1 expression downregulation by activating AKT signaling, promoting cancer cell migration. These findings highlight the importance of molecular therapies targeting the TME in OSCC.

Abstract 6049: Exogenous serine promotes cancer stem cells in oral squamous cell carcinoma

Abstract Cancer development and progression is associated with metabolic reprogramming of tumor cells required to meet their proliferative, bioenergetic and survival challenges. Some metabolites, including a non-essential amino acid, serine, have been shown to play a role in tumorigenesis by promoting aggressive cell states, such as cancer stem cells (CSCs). Previous studies from our laboratory showed that the nuclear branch of the Wnt/β-catenin signaling pathway, the β-catenin/CBP/MLL1 axis, enhanced CSC states during oral squamous cell carcinoma (OSCC) evolution through H3K4 trimethylation (H3K4me3) and epigenetic remodeling of the chromatin landscape. Given that metabolism has also been shown to modulate cell plasticity via epigenomic modifications, we have aimed to decode the relationship between serine synthesis and cell identity in OSCC. We have shown that under serine starvation conditions, OSCC cells upregulate steady-state mRNA and protein levels of serine synthesis pathway enzymes. The latter is associated with an increase in the by-product, alpha-ketoglutarate (αKG), a co-substrate for nuclear αKG-dependent dioxygenases, which demethylate histone marker H3K27me3 and de-repress differentiation-associated genes. Furthermore, increased production of αKG is associated with downregulation of the β-catenin/CBP/MLl1 complex and CSC-associated H3K4me3. In this study, we used human OSCC cell lines CAL27 and HSC3, derived from a primary tongue tumor and a metastatic site, respectively. Each cell line was cultured in either complete medium or serine starvation medium. Serine starvation conditions led to a downregulation of H3K27me3 along with H3K4me3, promoting a switch from cancer stem cell to differentiation-enhancing chromatin landscape. The observed changes in cell plasticity were further investigated through tumorsphere formation, a surrogate assay for cancer stem cells. We found that OSCC CAL27 and HSC3 cells readily formed tumorspheres replete with stem cell markers BMI1, KRT14 and SOX2, under non-adhesive conditions when grown in complete media, supporting our findings that utilization of exogenous serine enables OSCC cells to maintain stemness. Our findings suggest that a switch from exogenous serine uptake to the endogenous serine biosynthesis promotes OSCC cell differentiation concomitant with the loss of CSC identity. Citation Format: Stacy Ann Jankowski, Nina C. Hardy, Maria A. Kukuruzinska. Exogenous serine promotes cancer stem cells in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6049.

Abstract 1327: Mechanism of tumor microenvironment remodeling and progression based on FAM20C-mediated extracellular kinome network in oral squamous cell carcinoma

Abstract Tumorigenesis and tumor growth are accomplished through the crosstalk between intra- and extracellular molecules, which is often mediated by phosphorylation, and research on intracellular signal transduction has made great progress. Despite the significance of the tumor microenvironment (TME) for tumor progression, relatively few kinome studies have been conducted on phosphorylation cascade in the TME of oral squamous cell carcinoma (OSCC). OSCC is an aggressive cancer with a high recurrence rate of 40-60%, but there are uncertain molecular classification and no identifiable driver mutation genes to be a druggable target. As the presence of extranodal extension (ENE) is currently the most significant risk factor for locoregional recurrence or distant metastasis, differences in molecular profiles between patients with and without ENE may provide insight into regulatory mechanisms that are critical for the recurrence and progression of OSCC. Using LC-MS-based secretome analysis, we examined total and phosphoproteins from ten patients' plasma with or without ENE. Global proteome analysis revealed that the ENE group had higher levels of protein phosphorylation, the ERK 1/2 cascade, signal transduction, phagocytosis, and the integrin-mediated signaling pathway than the without ENE group. Furthermore, 319 phosphosites originating from 165 proteins were discovered, with the majority of phosphorylated proteins having lipid binding, complement system, extracellular matrix, and calcium binding. Interestingly, we discovered 82 proteins with Ser-x-Glu/pSer sites among the phosphorylated proteins, indicating that they could be substrates of the unique extracellular secreted serine kinase, family with sequence similarity 20C (FAM20C). Proteins associated to tumor metastasis, such as insulin-like growth factor-binding proteins (IGFBPs), osteopontin (OPN), and serine protease inhibitors (Serpins), are known as substrates for the FAM20C. In accordance with the proteome results in plasma, gene ontology (GO) term analysis using the NanoString oncology panel revealed that extracellular structure organization-related genes were up-regulated and enzyme inhibitor, kinase regulator, and protein tyrosine kinase activity-related genes were down-regulated in human OSCC cell line, Cal-27, with knockout of FAM20C. Furthermore, we functionally demonstrated that FAM20C depletion inhibited invasion by lowering protein production of Vimentin, TGF-β, and ERK phosphorylation. Collectively, we discovered that the secreted kinase FAM20C controls metastasis by cross-talking with proteins in the TME and propose that this may be the evidence for OSCC prognosis. (This research was supported by National Cancer Center, Korea (No. 2210980) and National Research Foundation of Korea grant funded by the Korea government (No. 2020M3A9A5036362)). Citation Format: Mi Rim Lee, Yu-Sun Lee, Sumin Kang, Hye Won Shon, Jeong Eun Gong, Hye Joo Park, Jung-Ah Hwang, Kyung-Hee Kim, Sung Weon Choi, Yun-Hee Kim. Mechanism of tumor microenvironment remodeling and progression based on FAM20C-mediated extracellular kinome network in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1327.

Proteasome inhibitor MG132 enhances the sensitivity of human OSCC cells to cisplatin via a ROS/DNA damage/p53 axis.

Cis-diamine-dichloroplatinum II (cisplatin, CDDP) is a key chemotherapeutic regimen in the treatment of oral squamous cell carcinoma (OSCC). However, the therapeutic efficacy of cisplatin in OSCC may be hampered by chemoresistance. Therefore, the development of novel combination therapy strategies to overcome the limitations of CDDP is of great importance. The proteasome inhibitor MG132 exhibits anti-cancer properties against various types of cancer. However, our knowledge of its anti-cancer effects in combination with CDDP in OSCC cells remains limited. In the current study, the synergetic effects of MG132 and CDDP were evaluated in the human CAL27 OSCC cell line. CAL27 cells were treated with CDDP alone or in combination with MG132. The results showed that MG132 significantly reduced cell viability in a dose-dependent manner. Additionally, cell viability was significantly reduced in CAL27 cells treated with 0.2 µM MG132 and 2 µM CDDP compared with cells treated with MG132 or CDDP alone. In addition, MG132 significantly enhanced the CDDP-induced generation of intracellular reactive oxygen species and DNA damage in OSCC cells. Furthermore, treatment with CDDP or MG132 alone notably inhibited colony formation and proliferation of OSCC cells. However, co-treatment of OSCC cells with MG132 and CDDP further hampered colony formation and proliferation compared with cells treated with either MG132 or CDDP alone. Finally, in cells co-treated with MG132 and CDDP, the expression of p53 was markedly elevated and the p53-mediated apoptotic pathway was further activated compared with cells treated with MG132 or CDDP alone, as shown by the enhanced cell apoptosis, Bax upregulation, and Bcl-2 downregulation. Overall, the results of the current study support the synergistic anti-cancer effects of a combination of MG132 and CDDP against OSCC, thus suggesting that the combination of MG132 and CDDP may be a promising therapeutic strategy for the management of OSCC.

Machilin D Promotes Apoptosis and Autophagy, and Inhibits Necroptosis in Human Oral Squamous Cell Carcinoma Cells.

Oral squamous cell carcinoma (OSCC) accounts for about 90% of all head and neck cancers, the prognosis is very poor, and there are no effective targeted therapies. Herein, we isolated Machilin D (Mach), a lignin, from the roots of Saururus chinensis (S. chinensis) and assessed its inhibitory effects on OSCC. Herein, Mach had significant cytotoxicity against human OSCC cells and showed inhibitory effects against cell adhesion, migration, and invasion by inhibiting adhesion molecules, including the FAK/Src pathway. Mach suppressed the PI3K/AKT/mTOR/p70S6K pathway and MAPKs, leading to apoptotic cell death. We investigated other modes of programmed cell death in these cells and found that Mach increased LC3I/II and Beclin1 and decreased p62, leading to autophagosomes, and suppressed the necroptosis-regulatory proteins RIP1 and MLKL. Our findings provide evidence that the inhibitory effects of Mach against human YD-10B OSCC cells are related to the promotion of apoptosis and autophagy and inhibition of necroptosis and are mediated via focal adhesion molecules.

Is CCL2 an Important Mediator of Mast Cell-Tumor Cell Interactions in Oral Squamous Cell Carcinoma?

In this study, we aimed to evaluate the influence of interactions between mast cells (MCs) and oral squamous cell carcinoma (OSCC) tumor cells on tumor proliferation and invasion rates and identify soluble factors mediating this crosstalk. To this end, MC/OSCC interactions were characterized using the human MC cell line LUVA and the human OSCC cell line PCI-13. The influence of an MC-conditioned (MCM) medium and MC/OSCC co-cultures on the proliferative and invasive properties of the tumor cells was investigated, and the most interesting soluble factors were identified by multiplex ELISA analysis. LUVA/PCI-13 co-cultures increased tumor cell proliferation significantly (p = 0.0164). MCM reduced PCI-13 cell invasion significantly (p = 0.0010). CC chemokine ligand 2 (CCL2) secretion could be detected in PCI-13 monocultures and be significantly (p = 0.0161) increased by LUVA/PCI-13 co-cultures. In summary, the MC/OSCC interaction influences tumor cell characteristics, and CCL2 could be identified as a possible mediator.

NCBP2 and TFRC are novel prognostic biomarkers in oral squamous cell carcinoma

There are few prognostic biomarkers and targeted therapeutics currently in use for the clinical management of oral squamous cell carcinoma (OSCC) and patient outcomes remain poor in this disease. A majority of mutations in OSCC are loss-of-function events in tumour suppressor genes that are refractory to conventional modes of targeting. Interestingly, the chromosomal segment 3q22-3q29 is amplified in many epithelial cancers, including OSCC. We hypothesized that some of the 468 genes located on 3q22-3q29 might be drivers of oral carcinogenesis and could be exploited as potential prognostic biomarkers and therapeutic targets. Our integrative analysis of copy number variation (CNV), gene expression and clinical data from The Cancer Genome Atlas (TCGA), identified two candidate genes: NCBP2, TFRC, whose expression positively correlates with worse overall survival (OS) in HPV-negative OSCC patients. Expression of NCBP2 and TFRC is significantly higher in tumour cells compared to most normal human tissues. High NCBP2 and TFRC protein abundance is associated with worse overall, disease-specific survival, and progression-free interval in an in-house cohort of HPV-negative OSCC patients. Finally, due to a lack of evidence for the role of NCBP2 in carcinogenesis, we tested if modulating NCBP2 levels in human OSCC cell lines affected their carcinogenic behaviour. We found that NCBP2 depletion reduced OSCC cell proliferation, migration, and invasion. Differential expression analysis revealed the upregulation of several tumour-promoting genes in patients with high NCBP2 expression. We thus propose both NCBP2 and TFRC as novel prognostic and potentially therapeutic biomarkers for HPV-negative OSCC.

Effects of Sangju Honey on Oral Squamous Carcinoma Cells.

Since ancient times, honey has been used in traditional medicine owing to its pharmacological effects. It possesses anticancer properties. However, the therapeutic implications of Sangju honey in cancer remains unknown. Therefore, we aimed to demonstrate the potential anticancer effects of Sangju honey on human oral squamous cell carcinoma (OSCC), particularly focusing on epithelial-mesenchymal transition (EMT) and apoptotic and mitogen-activated protein kinase (MAPK) signaling pathways. Ca9-22 and YD-10B human OSCC cells were treated with 0.25% or 0.5% Sangju honey, and the cell viability was examined using the Cell Counting Kit-8 assay. Cell morphology studies were conducted to observe morphological changes, and the wound-healing assay was performed to evaluate the proliferation of honey-treated OSCC cells. Western blot analysis was conducted to investigate protein expression related to EMT and apoptotic and MAPK signaling pathways. Sangju honey reduced cell viability, induced morphological changes, and significantly suppressed the proliferation and migration of Ca9-22 and YD-10B cells. The expression of E-cadherin and N-cadherin was increased and decreased, respectively, in both OSCC cell lines. Moreover, Sangju honey stimulated apoptosis by increasing the expression of p21, p53, cleaved caspase 3, and caspase 9. Furthermore, it downregulated the expression of phospho (p)-extracellular signal-regulated kinases 1 and 2, p-c-Jun amino-terminal kinase, and p-p38 in Ca9-22 and YD-10B cells. Sangju honey inhibits Ca9-22 and YD-10B cell proliferation by regulating EMT, inducing apoptosis, and suppressing the MAPK signaling pathway. Thus, it is a potential anticancer agent for human OSCC.