SCC Cell Lines Research Articles

Influence of tumor cell-derived TGF-β on macrophage phenotype and macrophage-mediated tumor cell invasion.

In oral squamous cell carcinoma (OSCC), macrophages are the most abundant immune cell type in the tumor microenvironment (TME). Macrophage infiltration is inversely proportional to prognosis and disease survival, particularly when these tumor-associated macrophages (TAM) assume an M2-like phenotype. This phenotype is determined by cues from the microenvironment, especially tumor cell-secreted molecules, and is associated with increased production of extracellular-matrix-degrading enzymes, angiogenic molecules and immunosuppressing cytokines. This study investigates, in vitro and in vivo, the relative contribution of OSCC cell-secreted transforming growth factor beta (TGF-β) on the phenotype of macrophages and on macrophage-facilitated tumor invasion. TCGA database shows a positive correlation between high expression of TGFB1 and macrophage infiltrate in Head and neck squamous cell carcinoma (HNSCC). THP-1 derived-macrophages were exposed to the secretome of two OSCC cell lines using two strategies to block the effects of neoplastic cell-secreted TGF-β: pre-treatment with a TGF-β receptor type I kinase inhibitor (LY364947) and antibody-mediated depletion. RT-qPCR, ELISA and flow cytometry determined macrophage phenotype after exposure to conditioned medium (CM) from H-314 (TGF-βhigh) or SCC-9 (TGF-βlow) cell lines. The influence of TGF-β on macrophage-mediated tumor cell invasion (myogel and CAM assays) and chemotaxis (Boyden chamber) was assessed using co-cultures of macrophages and OSCC cells in which macrophages were pre-conditioned with the secretome of OSCC cells in the presence and absence of LY364947. Blocking the effects of TGF-β skewed macrophages to the M1 end of the phenotype by differential effects depending on the strategy for inhibiting the influence of TGF-β and on the neoplastic cell secretome. In vitro and in vivo invasion of H-314 cell line was reduced by inhibiting TGFBR1 signaling in macrophages, whereas SCC-9 cell invasion was not affected. SCC-9/macrophage reciprocal chemotaxis were enhanced by inhibiting TGFBR1 signaling in macrophages, whereas only macrophage chemotaxis to H314 products was inhibited by inhibiting TGFBR1. In summary, blocking the effects of OSCC cell-secreted TGF-β in macrophages attenuates M2-like phenotypical traits of macrophages and can impact invasion and chemotaxis of tumor cells differentially.

Abstract A021: Development of a novel MEK inhibitor, NFX-179, as a chemoprevention agent for squamous cell carcinoma

Abstract Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer comprising at least 20% of all non-melanoma skin cancers. While cSCC contributes to significant morbidity and mortality in high-risk individuals, deployment of otherwise effective chemoprevention of cSCC is limited by toxicities. To help address this, we conducted a systematic computational drug repositioning screen which predicted that selumetinib, an FDA-approved MEK inhibitor (MEKi), would reverse transcriptional signatures associated with cSCC development. This is consistent with our genomic analysis implicating ETS2, a transcription factor in the canonical RAS/RAF/MEK/ERK Mitogen-Activated Protein Kinase (MAPK) pathway, as an upstream regulator of cSCC development. Therefore, as a key regulator of the MAPK pathway, we reasoned MEK would be a viable chemopreventive target. Although systemic MEK inhibition suppresses the formation of cSCC in mice, systemic MEKi administration causes significant adverse effects, including diarrhea, peripheral edema, cardiomyopathy and retinal toxicity. Here, we report the development of a topically formulated, metabolically labile, novel MEKi, NFX-179, designed to potently and selectively suppress the MAPK pathway in the skin prior to rapid metabolism in the systemic circulation. NFX-179 was identified from a targeted drug discovery effort based on its biochemical and cellular potency, selectivity, and rapid metabolism upon systemic absorption. In our UV-induced cSCC mouse model, topical application of NFX- 179 gel reduced the formation of new cSCCs by an average of 60% at doses of 0.1% and greater at 28 days. No systemic or skin toxicities were observed in this model. Furthermore, we conducted a second split-mouse randomized controlled study in which NFX-179 0.5% gel was applied to one half of the back and vehicle was applied to the opposite half of each of the UV-irradiated mice. Near complete suppression of cSCC was observed only in the drug-treated area, demonstrating the targeted and dermal effect of the intervention. NFX-179 inhibits the growth of human SCC cell lines in a dose-dependent manner and topical NFX-179 application penetrates human skin and inhibits MAPK signaling in human cSCC explants. Together our data provide compelling rationale for using topical MEK inhibition through application of NFX-179 gel as an effective strategy for cSCC chemoprevention. Citation Format: Kavita Y. Sarin, John Kincaid, Brittney Sell, Jahanbanoo Shahryari, Matthew A. J. Duncton, Elaine Morefield, Wenchao Sun, Omar Chavez-Chiang, Scott R. Plotkin, Gerd G. Kochendoerfer, Peter Fenn, Christopher Powala, Kenneth Y. Tsai. Development of a novel MEK inhibitor, NFX-179, as a chemoprevention agent for squamous cell carcinoma [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A021.

Synthesis of First Coumarin Fluorescent Dye for Actin Visualization.

Selective fluorescence imaging of actin protein hugely depends on the fluorescently labeled actin-binding domain (ABD). Thus, it is always a challenging task to image the actin protein (in vivo or in vitro) directly with an ABD-free system. To overcome the limitations of actin imaging without an ABD, we have designed a facile and cost-effective red fluorescent coumarin dye 7-hydroxy-4-methyl-8-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl-2H-chromen-2-one (CTC) for actin binding. The selective binding of the dye was investigated using the gut and eye of the model organism Drosophila melanogaster and C2C12 and SCC-9 cell lines. Our result suggests two major advantages of CTC over the dyes presently used for imaging actin proteins. First, the dye can bind to actin efficiently without any secondary intermediate. Second, it is much more stable at room temperature and exhibits excellent photostability. To the best of our knowledge, this is the first fluorescent dye that can bind to the actin protein without employing any secondary intermediate/actin-binding domain. These findings could pave the way for many biologists and physicists to successfully employ the CTC dye for imaging and tracking actin proteins by fluorescence microscopy in various in vivo and in vitro systems.

Selinexor assists vorinostat in inhibiting HDAC activity via promoting the accumulation of maspin in the nucleus of oral tongue squamous cell carcinoma cells.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cancer with a low overall survival rate, necessitating effective treatments. This study reports the anti-OTSCC effect of vorinostat and selinexor. OTSCC cell lines SCC-4 and SCC-25 were cultured to determine the effects of vorinostat and/or selinexor on cell survival, invasion, migration, and apoptosis. The transplanted tumor model of SCC-25 in nude mice was established to observe the therapeutic effects of vorinostat and/or selinexor. Western blotting was used to determine protein expressions in tumor cells. The results showed that histone deacetylase 1 (HDAC1) and exportin 1 (XPO1) were highly expressed, while nuclear maspin was expressed at a low rate in SCC-4 and SCC-25 compared to the normal tongue tissue. In vitro, both vorinostat and selinexor effectively inhibited cell viability, invasion, and migration, promoted cell apoptosis, down-regulated HDAC1, Matrix Metalloproteinase 2 (MMP2), and B cell leukemia/lymphoma 2 (Bcl-2), and up-regulated nuclear maspin and cleaved caspase 3. In vivo, both vorinostat and selinexor inhibited the growth of SCC-25-bearing tumors, down-regulated the expression of Ki67, HDAC1, MMP2, and Bcl-2, and promoted the expression of nuclear maspin and cleaved caspase 3. The combination of these two drugs exhibited synergistic effects both in vivo and in vitro. Our evidence shows that vorinostat combined with selinexor is an effective treatment for OTSCC. The mechanism may be that selinexor promotes the accumulation of maspin in the nucleus, an endogenous HDAC1 inhibitory protein to inhibit the HDAC1 activity of vorinostat and exert a synergistic anti-OTSCC effect.

Understanding of black salve toxicity by multi-compound cytotoxicity assays

BackgroundBlack salve is a controversial complementary and alternative medicine (CAM) associated with skin toxicity and skin cancer treatment failures. Black salve formulations vary between manufacturers and contain a number of botanical and synthetic constituents. The skin cancer cytotoxicity of a number of these constituents has not been assessed to date. The alkaloids from the rhizomes of Sanguinaria canadensis, a key black salve ingredient, have had their single compound cytotoxicity assessed; however, whether they possess synergistic cytotoxicity with other compounds has not been studied and is of direct clinical relevance. This research aimed to improve our understanding of the skin cancer cytotoxicity of black salve constituents.MethodsThe cytotoxicity of individual and combination black salve constituents were assessed against the A375 melanoma and A431 squamous cell carcinoma cell lines. Cytotoxicity was determined using the Resazurin assay with fluorescence measured using a Tecan Infinite 200 Pro Microplate reader, compound cytotoxicity being compared to that of the topical cancer therapeutic agent, 5- fluouracil. Docetaxal was used as a positive control. Dunnetts p value was used to determine whether significant synergistic cytotoxicity was present.ResultsSanguinarine was the most cytotoxic compound tested with a 24-hour IC50 of 2.1 μM against the A375 Melanoma cell line and 3.14 μM against the A431 SCC cell line. All black salve constituents showed greater cytotoxicity against the two skin cancer cell lines tested than the skin cancer therapeutic 5-Fluouracil with 24 hours of compound exposure. Chelerythrine and minor Quaternary Benzophenanthridine Alkaloids (QBAs) present in black salve, at concentrations not having a cytotoxic effect by themselves, boosted the cytotoxic effects of sanguinarine. This could be a synergistic rather than additive cytotoxic effect although the synergistic effect was cell line and concentration dependent.ConclusionsBlack salve contains several cytotoxic compounds, a number of which have been found to possess synergistic cytotoxicity for the first time against skin cancer cell lines. In addition, these compounds together increase the overall cytotoxic effect. Assessing multi-compound cytotoxicity in herbal medicine can provide additional information about both their therapeutic and toxicity potential. As black salve is currently being used by patients, further cytotoxicity work should be undertaken to assess whether synergistic cytotoxicity exists when tested in normal skin cells.

Duguetia pycnastera Sandwith (Annonaceae) Leaf Essential Oil Inhibits HepG2 Cell Growth In Vitro and In Vivo.

Duguetia pycnastera Sandwith (Annonaceae) is a tropical tree that can be found in the Guyanas, Bolivia, Venezuela, and Brazil. In Brazil, it is popularly known as “ata”, “envira”, “envira-preta”, and “envira-surucucu”. In the present work, we investigated the in vitro and in vivo HepG2 cell growth inhibition capacity of D. pycnastera leaf essential oil (EO). The chemical composition of the EO was determined by GC–MS and GC–FID analyses. The alamar blue assay was used to examine the in vitro cytotoxicity of EO in cancer cell lines and non-cancerous cells. In EO-treated HepG2 cells, DNA fragmentation was measured by flow cytometry. The in vivo antitumor activity of the EO was assessed in C.B-17 SCID mice xenografted with HepG2 cells treated with the EO at a dosage of 40 mg/kg. Chemical composition analysis displayed the sesquiterpenes α-gurjunene (26.83%), bicyclogermacrene (24.90%), germacrene D (15.35%), and spathulenol (12.97%) as the main EO constituents. The EO exhibited cytotoxicity, with IC50 values ranging from 3.28 to 39.39 μg/mL in the cancer cell lines SCC4 and CAL27, respectively. The cytotoxic activity of the EO in non-cancerous cells revealed IC50 values of 16.57, 21.28, and >50 μg/mL for MRC-5, PBMC, and BJ cells, respectively. An increase of the fragmented DNA content was observed in EO-treated HepG2 cells. In vivo, EO displayed tumor mass inhibition activity by 47.76%. These findings imply that D. pycnastera leaf EO may have anti-liver cancer properties.

Eugenol Inhibits the Biological Activities of an Oral Squamous Cell Carcinoma Cell Line SCC9 via Targeting MIF.

Oral squamous cell carcinoma (OSCC) is a rampant cancer type in head and neck cancers with a poor prognosis and a high recurrence rate. Eugenol shows an anticancer effect in a variety of cancers, but it has been rarely studied in oral squamous cell carcinoma (OSCC). The purpose of this study was to explore the role of Eugenol in OSCC and the underlying mechanism. After different concentrations of Eugenol (0, 200, 400, and 800 μM) treatment, the viability, proliferation, migration, and invasion of OSCC cell line SCC9 were measured by CCK-8, colony formation, wound-healing, and transwell assays, respectively. TUNEL staining was employed to detect apoptosis. Western blotting was used to evaluate gene expression at the protein level. Molecular docking was used to identify the target of Eugenol. Eugenol decreased the proliferation and reduced the abilities of invasion and migration along with the expression of matrix metalloproteinases (MMP) 2 and MMP9 in SCC9 cells. On the contrary, the ratio of apoptotic cells was increased by Eugenol. In addition, Eugenol down-regulated B cell lymphoma-2 (Bcl-2) expression, but up-regulated BCL-2 associated X (Bax), cleaved caspase 3, and cleaved poly-ADP ribose polymerase (PARP) expression. Meanwhile, Eugenol exerted its effect on SCC9 cells in a concentration-dependent manner. Eugenol could bind to macrophage migration inhibitory factor (MIF), the expression of which was down-regulated after Eugenol treatment. Besides, overexpression of MIF reversed all the effects of Eugenol on OSCC cells. In summary, Eugenol suppressed the malignant processes of OSCC cells by targeting MIF, which could guide the clinical application of Eugenol in OSCC.

Pingyangmycin Activates Oral Carcinoma Cell Autophagy via the Phosphorylation of the PI3K/AKT/mTOR Axis to Achieve the Purpose of Treating Oral Carcinoma.

Objective The aim of the study is to investigate the role of pingyangmycin (PYM) in oral carcinoma (OC) cell autophagy via the PI3K/AKT/mTOR axis. Methods 200 μL PYM culture solution with a concentration of 100 μg/ml (low PYM (L-PYM) group), 300 μg/ml (middle PYM (M-PYM) group), 500 μg/ml (high PYM (H-PYM) group), and the same amount of conventional medium (normal control (NC)) were added to the purchased OC cell line SCC-25, respectively, and the PI3K/AKT/mTOR pathway expression, autophagy protein levels, cell activity, and apoptosis rate were determined. Subsequently, we selected OC cells co-cultured with PYM with the concentration of the most significant intervention effect and 740Y-P, a specific activator of the PI3K/AKT/mTOR axis, and those treated with 740Y-P alone for the aforementioned detection. Results L-PYM, M-PYM, and H-PYM groups all showed decreased PI3K, AKT, mTOR, and phosphorylated protein levels (P < 0.05). Beclin1 and LC3-II protein levels and apoptosis rate of PYM-intervened OC cells increased, but the activity decreased (P < 0.05). Under 740Y-P intervention, the PI3K/AKT/mTOR pathway was activated, cell activity was increased, and the apoptosis rate and autophagy were decreased (P < 0.05). Simultaneous use of PYM and 740Y-P led to no difference in cell condition compared with NC (P > 0.05P>0.05). Conclusion PYM can activate OC cell autophagy by inhibiting the phosphorylation of the PI3K/AKT/mTOR axis, and thus, achieving the goal of killing tumor cells.

Effect of low-level light therapy before radiotherapy in oral squamous cell carcinoma: An in vitro study.

Radiation therapy for head and neck squamous cell carcinoma (HNSCC) is associated with several complications. Although photobiomodulation (PBM) has radioprotective effects in normal tissue, it could also enhance the growth of neoplastic cells. Thus, the present study aimed to investigate the cellular response of oral squamous cell carcinoma with pre-exposure to low-level phototherapy before radiotherapy. SCC9, Cal-27, A431, and HaCaT cell lines were subjected to low-level light therapy and radiotherapy. The cells were treated with a single energy density (300 J/cm2) of a light-emitting diode (660 nm) prior to ionizing radiation at different doses (0, 2, 4, and 6 Gy). After 24 h, wound scratch, proliferation, clonogenic cell survival, cell death, and reactive oxygen species (ROS) analyses were performed to evaluate cell response. The cell lines pre-exposed to PBM at the analyzed dosage were radiosensitive. The treatment significantly reduced cell proliferation and clonogenic cell survival. Migration and cell death assays also revealed positive results, with the treatment group showing lower rate of migration and higher cell death than did the control group. Moreover, PBM effectively increased the intracellular levels of ROS. PBM at 300 J/cm2 is a promising radiosensitizing modality to reduce the radiation dose and avoid the intolerable side effects of radiotherapy for HNSCC, thus increasing the probability of successful treatment. However, further studies are needed to support and confirm the results.

INVERSE EXPRESSION BETWEEN TGF-β1 AND TWIST IN ORAL SQUAMOUS CELL CARCINOMA CELL LINE AFTER CHOLESTEROL DEPLETION

Objectives To evaluate the transforming growht factor-β1 and twist protein expression in oral squamous cell carcinoma (OSCC) cell line SCC-9 after cholesterol depletion with methyl-β-cyclodextrin (MβCD). Study Design SCC-9 cells were treated with MβCD at 7.5, 10, and 15 mM concentrations over 1 hour. After 24 hours of depletion, the gene expression of TGF-β1 and TWIST was accessed by TaqMan RT-qPCR. Results The TGF-β1 gene expression in SCC-9 treated by different concentrations of MβCD was upregulated upon cholesterol depletion at 7.5 mM compared to untreated SCC-9 (P = .06, 1-way analysis of variance [ANOVA]). Conversely, TWIST gene expression was uniform through the different concentrations of MβCD (P = .9, 1-way ANOVA). A significant difference between expressions of TGF-β1 treated with 7.5 mM (mean value = 1.9) and TWIST treated with 7.5 mM (mean value = 0.9) and 10 mM (mean value = 1.0) was noted (P = .01, 1-way ANOVA). No correlation was observed in the expression of TGF-β1 and TWIST in each concentration of MβCD (Pearson's correlation analysis). Conclusions Cholesterol depletion may affect TGF-β1 and TWIST expression. This finding should be associated with others related to epithelial-mesenchymal transition inducers, due to their potential in promoting carcinogenesis, in order to better understand OSCC biology. To evaluate the transforming growht factor-β1 and twist protein expression in oral squamous cell carcinoma (OSCC) cell line SCC-9 after cholesterol depletion with methyl-β-cyclodextrin (MβCD). SCC-9 cells were treated with MβCD at 7.5, 10, and 15 mM concentrations over 1 hour. After 24 hours of depletion, the gene expression of TGF-β1 and TWIST was accessed by TaqMan RT-qPCR. The TGF-β1 gene expression in SCC-9 treated by different concentrations of MβCD was upregulated upon cholesterol depletion at 7.5 mM compared to untreated SCC-9 (P = .06, 1-way analysis of variance [ANOVA]). Conversely, TWIST gene expression was uniform through the different concentrations of MβCD (P = .9, 1-way ANOVA). A significant difference between expressions of TGF-β1 treated with 7.5 mM (mean value = 1.9) and TWIST treated with 7.5 mM (mean value = 0.9) and 10 mM (mean value = 1.0) was noted (P = .01, 1-way ANOVA). No correlation was observed in the expression of TGF-β1 and TWIST in each concentration of MβCD (Pearson's correlation analysis). Cholesterol depletion may affect TGF-β1 and TWIST expression. This finding should be associated with others related to epithelial-mesenchymal transition inducers, due to their potential in promoting carcinogenesis, in order to better understand OSCC biology.

ANTI-CANCER PROPERTIES OF CEPHAELINE IN ORAL SQUAMOUS CELL CARCINOMA CELL LINE

Objectives To evaluate the anticancer properties of cephaeline in an oral squamous cell carcinoma (OSCC) cell line, including in cancer stem cells (CSC) regulation. Study Design The SCC-9 cell line was used as an in vitro OSCC model. Briefly, cell viability was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and cell migration by wound healing scratch assay. Gistone acetylation (H3K9ac) status was determined by immunofluorescence staining. CSC was evaluated by clonogenic assay. Results A single-dose administration of cephaeline reduced viability of OSCC cells and delayed cell migration. Moreover, cephaeline halted CSC colony formation. Additionally, cephaeline affected histone acetylation status. Conclusions Our findings suggest that cephaeline has anti-cancer properties in OSCC. Further analysis with other cell lines and in vivo studies are needed to validate these findings. To evaluate the anticancer properties of cephaeline in an oral squamous cell carcinoma (OSCC) cell line, including in cancer stem cells (CSC) regulation. The SCC-9 cell line was used as an in vitro OSCC model. Briefly, cell viability was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and cell migration by wound healing scratch assay. Gistone acetylation (H3K9ac) status was determined by immunofluorescence staining. CSC was evaluated by clonogenic assay. A single-dose administration of cephaeline reduced viability of OSCC cells and delayed cell migration. Moreover, cephaeline halted CSC colony formation. Additionally, cephaeline affected histone acetylation status. Our findings suggest that cephaeline has anti-cancer properties in OSCC. Further analysis with other cell lines and in vivo studies are needed to validate these findings.

ROLE OF PHARMACOLOGICAL NFkB INHIBITION IN CANCER STEM CELLS POPULATION OF HEAD AND NECK SQUAMOUS CELL CARCINOMA

<h3>Objectives</h3> To evaluate the effect of pharmacological inhibition of nuclear factor-kappa beta (NFkB) using emetine and CBL0137 in cancer stem cells (CSCs) of head and neck squamous cell carcinoma cell lines resistant to cisplatin (CisR). <h3>Study Design</h3> CAL27 and SCC9 cell lines were used. NFkB expression was investigated by quantitative polymerase chain reaction and Western blot. MTS and spheres formation assay were assessed to determine the inhibition doses of emetine and CBL0137. Flow cytometry was performed to quantify the CSC population. <h3>Results</h3> Both CisR cell lines presented NFkB accumulation. CAL27 CisR IC50 dose was 1.8 μM for emetine and 1.5 μM for CBL0137. Emetine IC50 dose for SCC9 CisR was 1.7 μM and 1.5 μM for CBL0137. Emetine was able to eliminate CAL27 CisR CSC at 0.5 μM and CBL0137 at 0.75 μM. For both drugs, 0.75 μM eliminated CSC of the SCC9 CisR cell line. In combination with cisplatin, emetine was more effective in reducing CSC of CAL27 CisR and CBL0137 showed better response for SCC9 CisR. Interestingly, cisplatin alone did not have any effect on CisR cell lines and emetine or CBL0137 alone was more effective in reducing the CSC population than in combination with cisplatin. <h3>Conclusions</h3> Pharmacological inhibition of NFkB is efficient in reducing the CSC population, decreasing cisplatin chemoresistance.

UNRAVELING PATHWAYS THAT DRIVE CISPLATIN CHEMORESISTANCE IN ORAL CARCINOMA CELL LINES

<h3>Objectives</h3> Investigate the mechanisms that contribute to cancer stem cells (CSCs) accumulation and chemoresistance phenotype in cisplatin chemoresistant cell lines. <h3>Study Design</h3> CAL27 and SCC9 cell lines were treated with cisplatin until the establishment of the chemoresistance (CisR). RNA-seq and gene ontology (GO) analysis were performed to identify genes and biologic processes modified by chemoresistance. CSC accumulation was assessed by flow cytometry and sphere formation. Aggressiveness was investigated by clonogenic assay, MTS assay, Ki-67 immunostaining, wound healing assay, invasion, and tumor growth in a xenograft model. Epithelial mesenchymal transition (EMT) was detected by changes in E-cadherin and vimentin. <h3>Results</h3> The transcriptome of chemoresistant cell lines revealed upregulation of genes involved with proliferation, cell migration, and stemness, including TGFβ-2 and TGFβR-2; an increase in inflammatory processes; and an association with activation of EMT. RNA-seq also revealed downregulation of genes related to adhesion and drug metabolism. Chemoresistant cells presented accumulation of CSCs as well as increased proliferation, migration, and invasion. Increases in vimentin and decreases in E-cadherin were observed in CisR cell lines, suggesting activation of EMT. For CAL27 CisR we also observed a high in vivo tumorigenic potential. <h3>Conclusions</h3> A reprogramming in the expression of genes signaling stemness and tumor aggressiveness drives chemoresistance induced by cisplatin.

ANTICANCER EFFECTS OF DIFFERENT FATTY ACID SYNTHASE INHIBITORS ON ORAL CANCER

<h3>Objectives</h3> Overexpression of fatty acid synthase (FASN) is associated with aggressiveness, poor prognosis, and risk of metastasis in several human cancers, including oral squamous cell carcinomas (OSCC). Here, we compared the in vitro effects of 3 FASN inhibitors on malignant phenotypes of the OSCC cell line SCC-9. <h3>Study Design</h3> SCC-9 cells were treated with C75, triclosan (TCS), and orlistat (ORL) in their respective IC12.5, IC25, and IC50. We analyzed proliferation using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, cell cycle, and apoptosis by flow cytometry. Cell migration and invasion were evaluated by transwell inserts and adhesion using type I collagen or fibronectin. <h3>Results</h3> C75 showed the lowest IC50, whereas ORL presented the highest IC50. Each drug promoted distinct morphologic changes in SCC-9 cells and was able to significantly reduce FASN activity. All drugs promoted cell cycle arrest in G0/G1 and induced apoptosis at IC50. C75 reduced cell migration and invasion and increased the adhesion of SCC-9 cells. TCS reduced adhesion, migration, and invasion of SCC-9 cells. ORL reduced cell migration but did not significantly modify the adhesion and invasion of SCC-9 cells. <h3>Conclusions</h3> FASN inhibitors affected significant steps of tumor progression, and TCS showed better anticancer effects on SCC-9 cells.

CHOLESTEROL DEPLETION SUPPORT A STEM CELL–LIKE AND PROLIFERATIVE PROFILE IN OSCC CELL LINE

Objectives Oral squamous cell carcinoma (OSCC) is able to invade and develop metastasis. Cancer stem cells have been associated with tumor development, progression, and recurrence of OSCC. Tumor cells have high cholesterol rates in their membranes, and cholesterol depletion affects these cells and interferes in carcinogenesis. The objective was to evaluate the effect of cholesterol depletion with methyl-β-cyclodextrin (MβCD) on the expression of stem cell markers CD44 and ESA in OSCC cell line SCC-9. Study Design SCC-9 cells were depleted with MβCD at concentrations 0, 7.5, 10, and 15 mM and evaluated by flow cytometry for CD44 and ESA markers. Results An increase in the CD44-high/ESA-high population was observed after cholesterol depletion at all evaluated concentrations. This increase was accompanied by a decrease in the CD44-low population, indicating that depletion favors the selection of stem cell—like cells. There was no difference in the percentage of CD44-high/ESA-low cells in the treated groups compared to the control group. Conclusions Cholesterol depletion seems to induce a stem cell–like profile with greater proliferative capacity in OSCC cells. Oral squamous cell carcinoma (OSCC) is able to invade and develop metastasis. Cancer stem cells have been associated with tumor development, progression, and recurrence of OSCC. Tumor cells have high cholesterol rates in their membranes, and cholesterol depletion affects these cells and interferes in carcinogenesis. The objective was to evaluate the effect of cholesterol depletion with methyl-β-cyclodextrin (MβCD) on the expression of stem cell markers CD44 and ESA in OSCC cell line SCC-9. SCC-9 cells were depleted with MβCD at concentrations 0, 7.5, 10, and 15 mM and evaluated by flow cytometry for CD44 and ESA markers. An increase in the CD44-high/ESA-high population was observed after cholesterol depletion at all evaluated concentrations. This increase was accompanied by a decrease in the CD44-low population, indicating that depletion favors the selection of stem cell—like cells. There was no difference in the percentage of CD44-high/ESA-low cells in the treated groups compared to the control group. Cholesterol depletion seems to induce a stem cell–like profile with greater proliferative capacity in OSCC cells.

Heterotypic neutrophil-in-tumor structure: A novel pathological feature first discovered in the tissues of OPSCC

ObjectiveTo reveal a novel pathological feature: heterotypic neutrophil-in-tumor structure (hNiT) first discovered in patients with oropharyngeal squamous cell carcinoma (OPSCC), to analyze the prognostic role of hNiT in OPSCC patients and to explore the role of p16 in the formation of hNiT structures.MethodsClinically, 197 patients were enrolled. Clinicopathological information was extracted and analyzed. All pathologic sections made from primary tumors were re-evaluated by immunohistochemistry and immunostaining. In vitro, we cocultured OPSCC cell line SCC-15 with neutrophils to form hNiT structures, which were then subject to fluorescence staining. By RNAi and overexpression techniques, we investigated the role of CDKN2A in the formation of hNiTs. We validated the two techniques by qPCR and Western Blot.ResultsThe hNiT as a novel pathological feature was first discovered in the tissues of OPSCC. The FNiT was significantly associated with tumor stage, disease stage, p16 and tumor grade. A total of 119 patients died of the disease, and the 5-year disease-specific survival (DSS) rate was 36%. The median survival time was 52.6 months. In patients with an FNiT<0.5%, the 5-year DSS rate was 40%; in patients with an FNiT>=0.5%, the 5-year DSS was 28%, and the difference was significant (p=0.001). Cox model analysis showed that FNiT along with disease stage, p16 and tumor grade was an independent prognostic factor for DSS. Immunostaining results of p16 expression showed hNiT formation was negatively correlated to p16 in OPSCC as well as in the hNiT formation assays in vitro indicated by fluorescent staining. Function assays of CDKN2A implied that reduce CDKN2A promoted the formation of hNiT while elevated CDKN2A impeded the hNiT formation.ConclusionThe hNiT as a novel pathological feature is associated with the adverse prognosis of OPSCC patients with p16 inhibiting the formation of hNiT structures.

EZH2 regulates a SETDB1/ΔNp63α axis via RUNX3 to drive a cancer stem cell phenotype in squamous cell carcinoma.

Enhancer of zeste homolog 2 (EZH2) and SET domain bifurcated 1 (SETDB1, also known as ESET) are oncogenic methyltransferases implicated in a number of human cancers. These enzymes typically function as epigenetic repressors of target genes by methylating histone H3 K27 and H3-K9 residues, respectively. Here, we show that EZH2 and SETDB1 are essential to proliferation in 3 SCC cell lines, HSC-5, FaDu, and Cal33. Additionally, we find both of these proteins highly expressed in an aggressive stem-like SCC sub-population. Depletion of either EZH2 or SETDB1 disrupts these stem-like cells and their associated phenotypes of spheroid formation, invasion, and tumor growth. We show that SETDB1 regulates this SCC stem cell phenotype through cooperation with ΔNp63α, an oncogenic isoform of the p53-related transcription factor p63. Furthermore, EZH2 is upstream of both SETDB1 and ΔNp63α, activating these targets via repression of the tumor suppressor RUNX3. We show that targeting this pathway with inhibitors of EZH2 results in activation of RUNX3 and repression of both SETDB1 and ΔNp63α, antagonizing the SCC cancer stem cell phenotype. This work highlights a novel pathway that drives an aggressive cancer stem cell phenotype and demonstrates a means of pharmacological intervention.

Abstract 5734: The effect of PRAME on retinoid response and cell proliferation in cutaneous and head and neck squamous cell carcinoma

Abstract As cells undergo terminal differentiation, they adopt their tissue-specific functions and permanently exit the cell cycle. Inducing differentiation of premalignant cells and malignant cells are proposed strategies for cancer prevention and treatment, respectively. Retinoids, compounds related to retinol, drive terminal differentiation of numerous cell types. These compounds exhibit efficacy for the prevention of cutaneous squamous cell carcinomas (cSCC) and head and neck squamous cell carcinomas (HNSCCs), and their incorporation into treatment plans for these cancers is supported by laboratory and clinical studies. Preferentially Expressed Antigen in Melanoma (PRAME) is a cancer-testis antigen that represses retinoid signaling, and is associated with adverse outcomes in a plethora of malignancies. Although PRAME is known to be expressed in subsets of cutaneous SCC (cSCC) and head and neck SCC (HNSCC) tumors, its functions, prognostic and therapeutic significance have never been investigated in these cancers. We hypothesize that PRAME expression in SCC cells confers resistance to the anti-neoplastic effects of retinoids and supports cell proliferation. PRAME expression was evaluated in human cSCC tumors, and in cSCC and HNSCC cell lines by immunoblotting and qRT-PCR. PRAME-overexpressing immortalized keratinocyte, cSCC and HNSCC cell lines were generated. shRNA-mediated knockdown of PRAME was performed in a cSCC and a HNSCC cell line. Cells were treated with all-trans retinoic acid (ATRA) for 24, 48 or 72 hours. Expression of differentiation markers was assessed by immunoblotting and qRT-PCR of markers of differentiation. Cell counting assays, immunoblot analysis of cell cycle genes and Ki67 immunofluorescence staining were used to assess proliferation. PRAME expression is detected in subsets of cSCC tumors and in select SCC cell lines. Overexpression of PRAME in HNSCC cells enhanced cell proliferation compared to control cells. Treatment with ATRA did not promote differentiation of PRAME-expressing cells. Furthermore, PRAME overexpression attenuated the anti-proliferative effect of ATRA in HNSCC cells. We conclude that PRAME enhances proliferation of malignant keratinocytes in vitro and may confer resistance to retinoid-induced differentiation and proliferation arrest. Investigations to assess the prognostic and therapeutic significance of PRAME expression in SCCs are warranted. Citation Format: Brandon Liam Ramchatesingh, Ivan Litvinov. The effect of PRAME on retinoid response and cell proliferation in cutaneous and head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5734.

Abstract 3834: Assessing the role of extracellular matrix-integrins in metastatic squamous cell carcinoma

Abstract Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) have a dismal survival rate. The effects of the extracellular matrix (ECM) on cancer progression have been long studied, but the roles of specific integrins in the process of HNSCC metastasis remain to be dissected. This study aims to determine how HNSCC cells affect the production of laminin-binding integrins, and how these integrins participate in the ECM interactions necessary for a metastatic phenotype. Experimental design: Our laboratory has produced syngeneic mouse SCC cell lines, P029 and A223, derived from Keratin15+ stem cells with Smad4 loss and KrasG12D mutation. In syngeneic recipients, SCCs derived from P029 cells transplanted to the flank mouse skin produced spontaneous metastases to the lung while SCCs derived from A223 cells did not form metastases. Having the same genotype, these cell lines serve as models to examine cancer cell interactions with the ECM and resulting effects on invasion and metastasis. Bulk RNA sequencing (RNAseq) was performed to compare cultured metastatic P029 cells versus non-metastatic A223 cells and identify differentially expressed genes that regulate SCC cells and ECM interactions. Immunoassays and functional invasion assays were performed to evaluate ECM-cancer cell signaling and influence on cancer cell invasion in these two cell lines. Results: RNAseq analysis revealed that, relative to A223 cells, P029 cells have increased levels of integrins, the main mechanoreceptors for numerous ECM ligands and matrix proteins. Laminin-binding integrins, including integrins α4 and β6, were greater at the RNA and protein levels. Additionally, P029 cells expressed higher levels of fibronectin and laminin coding genes compared to non-metastatic A223 cells. Correlatively, high expression of these matrix proteins is associated with worse patient survival. Additionally, P029 SCC cells displayed differential gene expression of tight and apical junctions, PI3K signaling components, and regulators of cytoskeletal remodeling. Furthermore, ELISA and western blot analysis revealed that P029 has aberrant TGFβ-Smad signaling as indicated by the elevated release of TGFβ-1 protein and higher levels of phosphorylated Smad2 and Smad3 relative to non-metastatic A223. Treating P029 cells with TGFβ-1 significantly increased their motility and invasion. Conversely, migration and motility of P029 cells were radically reduced by the TGFβ inhibitor galunisertib. Additionally, the mouse cytokine array revealed that mouse plasma bearing P029 tumors had a greater circulation of CXCL16, MMP-9, proliferin, and serpin E1. These proteins are associated with ECM remodeling and metastasis in HNSCC. Conclusions: SCC cells with metastatic properties upregulate integrins which bind to non-collagen matrix proteins and have elevated invasive and migratory capacity contributed from activated TGFβ signaling. Citation Format: John Aleman, Khoa A. Nguyen, Yao Ke, Christian D. Young, Xiao-Jing Wang. Assessing the role of extracellular matrix-integrins in metastatic squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3834.

Fibroblast Growth Factor 3 Is Associated with Tongue Squamous Cell Carcinoma: A Controlled Study.

Objective To explore the effects of fibroblast growth factor 3 (FGF3) on the proliferation, cell cycle, and apoptosis of the tongue squamous cell carcinoma SCC-9 cell line (SCC-9). Methods We measured the proliferation of SCC-9 cells in a control group, an FGF3 intervention group, and a fibroblast growth factor (FGFR) inhibitor intervention group in cholecystokinin octapeptide (CCK-8) experiments. We studied effects of FGF3 on the cell cycle and apoptosis of tongue cancer cells using flow cytometry. We further explored the IRS1/PI3K/AKT signaling pathway by measuring BCL-2 and Bcl-2 Associated X-protein (BAX) mRNA and protein levels with RT-PCR and western blot, respectively. Results Results from the CCK-8 experiment showed that survival rates of cells in the control group, FGF3 intervention group, and FGFR inhibitor intervention group were 100.000% ± 4.026%, 136.330% ± 9.779%, and 83.199% ± 4.954%, respectively; survival rates of SCC-9 cells in all three groups were statistically significant (P < 0.05). Compared with that in the control group, the ratio of cells in G0/G1 phase in the FGFR inhibitor intervention group was higher (P < 0.05) and that in G2/M phase was lower, while the FGF3 intervention group showed opposite results (P < 0.05). The apoptosis rate of tongue cancer cells differed significantly between the FGFR inhibitor intervention and the control groups (P < 0.05). The mRNA and protein expression levels of IRS1, PI3K, and BCL-2 were all increased in the FGF3 intervention group (P < 0.05), while BAX mRNA and protein expression levels were decreased (P < 0.05). The mRNA expression levels of protein kinase B (AKT) showed no differences between groups. The p-AKT protein was overexpressed, while the total amount of AKT protein remained stable (P < 0.05). Conclusion FGF3 contributes to the proliferation of SCC-9 cells by increasing the proportion of cells in G2/M phase. Therefore, appropriately timed inhibition of FGF3 can potentially promote tumor apoptosis through the IRS1/PI3K/AKT signaling pathway. Our results demonstrate the role of FGF3 in the tumor microenvironment in tongue squamous cell carcinoma SCC-9 cells and suggest new therapeutic targets.