Human Squamous Cell Research Articles

Effect of HMGB1 knockdown by RNA interference on cell proliferation and migration of esophageal squamous cell carcinoma after X-ray radiation

Objective To evaluate the effect of X-ray radiation on cell proliferation, migration, survival ability and cell cycle of human esophageal squamous cell carcinoma after RNA interference-mediated down-regulation of HMGB1 gene expression. Methods The expression of HMGB1 at mRNA and protein levels in the human esophageal squamous cell carcinoma cell lines ECA109 and KYSE30 was determined using RT-PCR and Western blot assays. MTS and Transwell assays were employed to examine the proliferation and migration of ECA109 and KYSE30 cell lines. The cellular survival ability in vitro was assessed by clone formation assay. The cell cycle after X-ray radiation in different groups was detected by flow cytometry. Results The expression of HMGB1 at mRNA and protein levels in ECA109 and KYSE30 cells were markedly higher in a dose-dependent and time-dependent manner in the radiation group than that in the control group (all P<0.05). MTS results demonstrated that the proliferation of ECA109 and KYSE30 cells was obviously lower at each time point after radiation than that in the group without radiation (all P<0.01). The expression of HMGB1 at mRNA and protein levels was significantly inhibited in the HMGB1 siRNA group than those in the control and NC groups (both P<0.01). The data from the clone formation assay revealed that the radiosensitivity was significantly increased after down-regulation of HMGB1 expression (P<0.01). Transwell migration assay revealed that the number of migrating cells at the fourth hour after X-ray irradiation in the HMGB1 siRNA group was significantly lower than those in the control and negative groups (both P<0.01). In the HMGB1 siRNA group, the percentage of cells at G0/G1 phase was obviously higher, whereas the percentage of S phase was significantly lower than those in the control and NC groups, and the trend was even more significant after X-ray radiation (all P<0.01). Conclusion Inhibition of HMGB1 expression by siRNA can suppress the proliferation and migration of ECA109 and KYSE30 cells and enhance the radiosensitivity by increasing the cell cycle arrest at G0/G1 stage after X-ray irradiation in vitro. Key words: HMGB1 gene; Cell proliferation and migration; Cell cycle; Radiosensitivity

Modulation of EPS8 functions by phosphorylation

Objectives Epidermal growth factor receptor pathway substrate 8 (EPS8) is a scaffolding protein involved in regulating cell proliferation, actin dynamics, and receptor trafficking in human cells. EPS8 expression is increased in a range of human cancers, including head and neck squamous cell carcinoma (HNSCC). Previous studies have indicated that overexpression of EPS8 enhances mitogenesis and migration of tumor cells and is sufficient to convert nontumorigenic cells to a tumorigenic phenotype. The nonreceptor tyrosine kinase Src is reported to phosphorylate EPS8 at 4 tyrosine residues, although the impact of this on EPS8 function is unknown. The purpose of this study was to investigate the role of tyrosine phosphorylation of EPS8 at Src target sites in modulating biochemical functions, cell growth, and motility in HNSCC. Study Design Expression plasmids encoding EPS8 with amino acid substitutions to phenylalanine (F) at the 4 putative Src phosphorylation sites (Y485 F, Y525 F, Y602 F, Y774 F), and all 4 combined (4 F), were prepared by site-directed mutagenesis. To evaluate the effect of unphosphorylated EPS8 on downstream signals and biologic behavior, plasmids were transduced into a model cell line expressing a normal endogenous level of EPS8. Additionally, cells were treated with dasatinib, a Src inhibitor, to block phosphorylation of Src substrates. Expression of downstream targets was evaluated by Western blotting. Wound closure and proliferation assays were used to investigate the impact of these mutations on cell motility and growth. Results FOXM1, AURKA, and AURKB levels were decreased in cells expressing the nonphosphorylatable 4 F and Y602 F EPS8 mutants, whereas cells harboring the Y485 F, Y525 F, and Y774 F EPS8 mutants showed no differences in the expression of these proteins compared with controls. Moreover, dasatinib treatment resulted in a significant decrease in the expression of EPS8 downstream targets. In addition, both Y602 F and 4 F mutants elicited a significant reduction in tumor cell proliferation and migration. Conclusions The nonphosphorylatable Y602 F-EPS8 and 4 F-EPS8 mutants decreased the expression of EPS8 downstream targets, as well as reducing tumor cell growth and motility, implying a crucial role for phosphorylation of EPS8, principally at Y602, in mediating protumorigenic signal transduction.

Abstract B034: IFIT1 and IFIT3 function as Hsp90 co-chaperones to modulate the drug response in human oral squamous cell carcinoma

Abstract Oral squamous cell carcinoma (OSCC) is one of the most common malignant cancers with poor clinical outcome in the patients treated with chemotherapeutics. To improve the potential treatment approaches for OSCC patients, it is an urgent need to understand the molecular signaling mechanism involved in OSCC drug resistance. The failure of chemotherapy or targeted therapy is due to the development of drug resistance to anti-cancer drugs by various intrinsic cellular mechanisms. In the tumor microenvironment, cancer cells are exposed to many cellular components such as growth factors, extracellular matrix, and cytokines, etc. Interferons are pleiotropic cytokines promote cancer cells to acquire malignant phenotype and drug resistance through transcriptional induction of interferon-stimulated genes (ISGs). The human interferon-induced protein with tetratricopeptide repeats (IFIT) genes: IFIT1, IFIT2, IFIT3, and IFIT5 are well-known ISGs with multiple tetratricopeptide motifs that mediate protein-protein interactions. We have previously shown that high IFIT1 or IFIT3 expression promoted OSCC invasion and metastasis by EGFR signaling via enhancing EGFR endocytic recycling. However, the clinical significance of IFIT1 and IFIT3 in OSCC drug resistance remains unexplored. In the present study, we found that overexpression of IFIT1 or IFIT3 proteins in OSCC cells notably increased the resistance to DNA damaging agents including cisplatin, carboplatin, and oxaliplatin. In addition, xenograft tumors derived from IFIT1 or IFIT3-overexpressed cells showed resistance to cisplatin compared to controls. Intriguingly, our protein-protein interaction studies have revealed that IFIT1 and IFIT3 interacted with molecular chaperone Hsp90. Furthermore, ectopic expression of IFIT1 or IFIT3 induced C-terminal phosphorylation of Hsp90-α and subsequently activated of its client proteins, such as EGFR, AKT, p38, and SAPK/JNK in OSCC cells. Also, IFIT1 or IFIT3-overexpressed cells showed resistance to Hsp90 inhibitors such as geldanamycin and ganetespib. Suppression of Hsp90 activation by ganetespib resulted in decreased activation of downstream client proteins in IFIT1- or IFIT3-overexpressing cells. Moreover, IFIT1- or IFIT3-overexpressing cells showed enhanced expression of cancer stem cell marker c-Met compared to vector control cells. These results suggested a novel role of IFIT1 and IFIT3 in activating Hsp90 and several downstream signaling regulators which are critical for drug resistance in OSCC cells. Collectively, our results indicate that IFIT1 and IFIT3 possibly act as co-chaperones and serve as potentially important therapeutic targets for OSCC treatment in the future. Citation Format: Vijaya Pidugu, Hima Pidugu, Yu-Hsiang Teng, Te-Chang Lee. IFIT1 and IFIT3 function as Hsp90 co-chaperones to modulate the drug response in human oral squamous cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B034. doi:10.1158/1535-7163.TARG-19-B034

Curcumin enhances cisplatin-induced human laryngeal squamous cancer cell death through activation of TRPM2 channel and mitochondrial oxidative stress

In this study, laryngeal tumor cells were killed through the production of excessive reactive oxygen species (ROS) and Ca2+ influx by cisplatin (CISP). Nevertheless, a resistance was determined against CISP treatment in the tumor cells. We have investigated the stimulating role of curcumin (CURC) on CISP-induced human laryngeal squamous cancer (Hep2) cell death through TRPM2 channel activation, and its protective role against the adverse effects of CISP in normal kidney (MPK) cells. Hep2 and MPK cells were divided into four groups as control group, CURC group (10μM for 24 hrs), CISP group (25 μM for 24 hrs), and CURC + CISP combination group. CISP-induced decrease of cell viability, cell count, glutathione peroxidase and glutathione level in Hep2 cells were further increased by CURC treatment, but the CISP-induced normal MPK cell death was reduced by the treatment. CISP-induced increase of apoptosis, Ca2+ fluorescence intensity, TRPM2 expression and current densities through the increase of lipid peroxidation, intracellular and mitochondrial oxidative stress were stimulated by CURC treatment. In conclusion, CISP-induced increases in mitochondrial ROS and cell death levels in Hep2 cells were further enhanced through the increase of TRPM2 activation with the effect of CURC treatment. CISP-induced drug resistance in Hep2 cells might be reduced by CURC treatment.

Effects of Polygonatum sibiricum Polysaccharides (PSP) on Human Esophageal Squamous Cell Carcinoma (ESCC) via NF-κB Signaling Pathway

Objective. To explore the effects of different concentrations of Polygonatum sibiricum polysaccharides (PSP) on human esophageal squamous cell carcinoma (ESCC) cell line Eca109 and explore the new approach for the treatment of ESCC. Methods. Eca109 cells were divided into 5 groups, including one control group and 4 experimental groups where the concentrations of PSP used were 50, 100, 200, and 400 μg/mL. The proliferation rate of Eca109 cells in each group was measured with the CCK8 assay, and the apoptosis rate in each group was analyzed by flow cytometry; the in vitro scratch assay was used to determine the migration ability of Eca109 cells after PSP treatment; the expression levels of IL-1, IL-6, IL-10, TNF-α, and TGF-β were measured by RT-PCR, and the expression levels of TLR4 and proteins that are related to NF-κB signaling pathways were determined by Western blot. Results. PSP significantly inhibited the proliferation of Eca109 cells (p&lt;0.05) on a time- and dose-dependent manner; the apoptosis rates of Eca109 cells in experimental groups were significantly increased after 48 h of culture (p&lt;0.05); PSP significantly reduced the migration and invasion ability of Eca109 cells (p&lt;0.05); RT-PCR results showed that the expression of IL-10 in Eca109 cells increased significantly after treatment with PSP (p&lt;0.05), while the expression of IL-1, IL-6, TNF-α, and TGF-β decreased significantly (p&lt;0.05). Compared with the control group, the expression level of TLR4, NF-κB/p50, and NF-κB/p65 protein in each experimental group was significantly lower than that in the control group (p&lt;0.05). Conclusions. PSP significantly inhibited the proliferation, invasion, and migration of Eca109 cells and promoted cell apoptosis. These observed effects were probably due to the PSP’s inhibition on the NF-κB signaling pathway in Eca109 cells via the regulation of the TLR4 expression.

Expression of neuritin in human lung squamous cell carcinoma

Background: This study aimed to investigate the expression of neuritin in four common cancers, and to explore the association between neuritin expression and the occurrence and development of cancer. Methods: We initially examined neuritin expression in human cervical, endometrial, oesophageal, and lung cancer tissues by immunohistochemistry (IHC). Based on these results, we further examined its expression in lung squamous cell carcinoma (LUSC) tissues by IHC and reverse transcription-polymerase chain reaction (RT-PCR). Results: Neuritin expression levels were higher in all cancer tissues compared with normal control tissues. Neuritin protein and gene expression levels were significantly higher in LUSC tissues compared with normal lung tissues (P<0.001), according to IHC and RT-PCR, respectively. Neuritin expression levels decreased significantly with increased clinical TNM stage (I-IV) and distant metastasis (P<0.05). Conclusion: Neuritin may have clinical value as a novel diagnostic and prognostic marker in patients with LUSC.

Role of SATB1 in invasion and metastasis of human laryngeal squamous cell carcinoma cell line Hep-2 and its mechanisms

Objective To investigate the role of special AT-rich sequence-binding protein 1 (SATB1) in invasion and metastasis of human laryngeal squamous cell carcinoma cell line Hep-2 and its mechanism. Methods Hep-2 cells were cultured in vitro. The cell lines were divided into two groups. The cells in the experimental group was transfected with Lipofectamine 2000 liposome for SATB1 small interfering RNA (SATB1-siRNA), and the cells in the control group was transfected with Lipofectamine 2000 liposome for NC-siRNA. The before and after transfection SATB1, E-cadherin and Snail genes relative expression levels between the two groups were detected and compared using RT-PCR. Transwell cell invasion experiments were applied to evaluate the cells invasive ability of the two groups. The scratch experiments were applied to evaluate the 48 h cell migration ability of the two groups. Results The differences in the relative expression level of SATB1, E-cadherin and Snail genes in pre-transfected laryngeal squamous cell line Hep-2 between the two groups were not statistically significant (all P>0.05). Compared with the control group, the relative expression levels of the mRNA of SATB1 and Snail genes decreased in the experimental group after transfection, while the relative expression levels of the mRNA of E-cadherin gene increased, and the differences were statistically significant (all P<0.05). After the transfection, the number of transwell membrane cells and cell migration distances of the two groups of cells decreased. The values of cell number and migration distance in the experimental group were lower than those in the control group, and the differences were statistically significant (all P<0.05). Conclusions SATB1 can promote the invasion and metastasis of Hep-2 cells, and its mechanism may be related to the down-regulation of E-cadherin and the up-regulation of Snail gene expression to induce epithelial-mesenchymal transition. Key words: Laryngeal neoplasms; Carcinoma, squamous cell; Neoplasm invasiveness; Neoplasm metastasis; Special AT-rich sequence-binding protein 1

Mechanism of YAP1-JUN promoting proliferation and EMT transformation of human laryngeal squamous cell carcinoma cells

Objective To investigate the role of Yes-associated protein 1 (YAP1)-JUN in promoting the proliferation and epithelial mesenchymal transitions (EMT) transformation of laryngeal squamous cell carcinoma. Methods The expression and subcellular localization of YAP1 were detected by tissue immunofluorescence assay. The effects of YAP1 on the proliferation of laryngeal squamous cell were examined by cell clone formation experiment. Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the effect of YAP1 on the expression and transcriptional level of EMT-related molecular markers. The interaction between YAP1 and JUN was detected by immunocoprecipitation (CoIP) and Western blot assay, which regulated the expression of downstream genes to control the EMT process. Results The expression of YAP1 in laryngeal squamous cell carcinoma tissue increased and transferred from cytoplasm to nucleus. The number of clones increased significantly after YAP1 up regulation (P<0.01). The expression level of key gene E-cadherin in epithelial cells was significantly inhibited after YAP1 up (P<0.01), while the expression level of the key genes of interstitial cells, β-catenin, vimentin and N-cadherin was significantly up (P<0.01). YAP1 was interacted with nuclear transcription factor JUN, and the proliferation ability of YAP1 decreased significantly (P<0.01) after the inhibition of JUN expression (P<0.01), and the expression of EMT related molecular markers decreased significantly (P<0.01). Conclusions YAP1 combined with JUN gene promotes the proliferation and EMT transformation of human laryngeal squamous cell carcinoma cells. Key words: Laryngeal neoplasms; Carcinoma, squamous cell; Yes-associated protein 1; Cell proliferation; Epithelial-mesenchymal transition

(Cyto)genomic and epigenetic characterization of BICR 10 cell line and three new established primary human head and neck squamous cell carcinoma cultures.

Head and neck squamous cell carcinoma cell lines are useful preclinical models to understand the molecular processes underlying the development of such tumors, and to establish targeted therapies. We performed a comprehensive (cyto)genomic and epigenetic characterization of three new established primary human head and neck squamous cell carcinoma cultures and an established, yet undercharacterized cell line: BICR 10. Karyotyping, multiplex fluorescence in situ hybridization, array comparative genomic hybridization and methylation-specific multiplex ligation-dependent probe amplification were applied. The three primary cultures turned out to be a near-triploid and BICR 10 near-diploid. Banding and molecular cytogenetic analysis revealed non-random numerical and structural aberrations. The most common rearrangements identified in BICR 10 cell line were non-complex derivatives of reciprocal translocations, in which the breakpoints often appeared in centromeric/near-centromeric regions. In the 3 primary cell cultures the most common rearrangements observed were iso- and derivatives chromosomes derived from translocations. Overall, gains of 7p, 8q and losses at 3p, 8p, 9p, 18q and Xp were present in all four studied samples. Among the analyzed genes, BICR 10 cell line exhibited enhanced methylation of gene promoter; however, in all studied samples PAX5, WT1 and GATA5 were methylated. The here reported comprehensive characterization of BICR 10 cell line and the new established cultures enriches the resources available for head and neck cancer research, especially for testing therapeutic agents.

LKB1, a Key Driver Gene of Human Lung Squamous Cell Carcinoma

Identification of driver genes not only is a challenging work for cancer study, including lung cancer, but also is a foundation of precision medicine [1].

Abstract 2098: IFIT1 and IFIT3 modulate the drug response in human oral squamous cell carcinoma through interaction and activation of Hsp90

Abstract Interferon-induced protein with tetratricopeptide repeats (IFITs) family, well-known interferon-stimulated genes, have multiple tetratricopeptide repeats with helix-turn-helix structural motifs that mediate a variety of protein-protein interactions. Four IFIT genes have been identified in humans: IFIT1, IFIT2, IFIT3, and IFIT5. While the importance of IFIT1 and IFIT3 in the prognosis of cancer has been reported, the molecular basis of IFIT1 and IFIT3 in cancer progression remains unexplored. We have previously shown that high IFIT1 and IFIT3 expression associated with advanced T-stage, lymph node metastasis, perineural invasion, lymphovascular invasion, extranodal extension, and poor overall survival rate in oral squamous cell carcinoma (OSCC) patients. Also, we have demonstrated that high IFIT1 or IFIT3 expression promoted OSCC invasion and metastasis by EGFR signaling via enhancing EGFR endocytic recycling. Intriguingly, our recent studies have revealed the involvement of IFIT1 and IFIT3 in the resistance to chemotherapeutic agents in OSCC cells. The failure of chemotherapy or targeted therapy was often associated with the intrinsic or acquired drug resistance in cancer cells. In the present study, we intended to explore the biological role of IFIT1 and IFIT3 on drug resistance in OSCC cells. Our results showed that ectopic expression of IFIT1 or IFIT3 proteins in OSCC cells significantly increased the resistance to various therapeutic drugs such as cisplatin, carboplatin, 5-FU, and doxorubicin. Whereas, silencing of IFIT1 and IFIT3 by shRNA enhanced the sensitivity to these drugs. We further demonstrated that enhanced expression of IFIT1 and IFIT3 significantly increased the levels of C-terminal phosphorylation of Hsp90 and activation of its client proteins, such as EGFR, AKT, p38, and SAPK/JNK. Using mass spectrophotometric, immunoprecipitation and immunofluorescence analyses, we identified the interactions of IFIT1 and IFIT3 with Hsp90. Suppression of Hsp90 by a specific inhibitor resulted in decreased activation of its downstream targets in IFIT1- or IFIT3-overexpressing cells. These results suggested a novel mechanism of IFIT1 and IFIT3 in activating Hsp90 and several downstream signaling regulators which are crucial for OSCC tumor progression and drug resistance. Thus, IFIT1 and IFIT3 may act as co-chaperones and serve as potentially important prognostic biomarkers for OSCC progression. Citation Format: Vijaya Kumar Pidugu, Meei-Maan wu, Hima Bindu Pidugu, Te-Chang Lee. IFIT1 and IFIT3 modulate the drug response in human oral squamous cell carcinoma through interaction and activation of Hsp90 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2098.

Abstract 3553: Targeting miR-361-3p suppresses the growth of human oral squamous cell carcinoma cells in vitro &amp; in vivo

Abstract MicroRNAs (miRNAs) are small non-coding RNAs with size of 20-25 nucleotides, and inhibit protein translation by binding the 3’-untranslated region (3’-UTR) of target mRNA. Each miRNA can regulate multiple mRNAs and each mRNA can be targeted by a number of miRNAs. In cancer, miRNAs can act as not only tumor suppressor genes but also oncogenes. Addiction to oncogenic miRNAs (OncomiRs) may provide therapeutic opportunities in human cancers such as oncogene addiction. In this study, we have attempted to identify OncomiRs in human oral squamous cell carcinoma (OSCC) cells and considered whether targeting miRNAs can be possible for cancer therapy. Functional screening for OncomiRs in human OSCC cells was performed with the use of miRCURY LNATM microRNA Knockdown Library-Human v12.0 (EXIQON). We transfected 918 locked nucleic acid (LNA)/DNA antisense oligonucleotides for specific human mature miRNAs into human OSCC cells (GFP-SAS). After 80 hours, each cell growth was evaluated by WST-8 assay. We identified LNA/DNA antisense oligonucleotide against miR-361-3p (LNA-miR-361-3p) which showed remarkable growth inhibition in GFP-SAS cells. Subsequently, we confirmed the target specificity of LNA-miR-361-3p by quantitative RT-PCR (qRT-PCR). LNA-miR-361-3p significantly reduced the expression of miR-361-3p. Co-transfection of synthetic mature miR-361-3p abrogated the growth inhibitory effect of LNA-miR-361-3p in GFP-SAS cells. Furthermore, transfection of synthetic mature miR-361-3p resulted in 20% increase of cell growth in GFP-SAS cells. Next, we identified odd-skipped related 2 (OSR2) as a target gene of miR-361-3p by the use of microarray, GeneSpring GX, and Ingenuity Pathway Analysis (IPA). The expression of OSR2 after transfection with LNA-miR-361-3p was significantly induced, and co-transfection of the OSR2 3’-UTR luciferase reporter plasmid and LNA-miR-361-3p into GFP-SAS cells produced higher luciferase activity than cells in co-transfected with LNA-non target (LNA-NT). Finally, we assessed the effect of LNA-miR-361-3p on the in vivo growth of GFP-SAS cells. We administered LNA-miR-361-3p into the xenograft tumors every 3 days. We found that LNA-miR-361-3p significantly reduced the size of subcutaneously xenografted GFP-SAS tumors, compared to the control group treated with LNA-NT. The expression of miR-361-3p in excised tumors was examined by qRT-PCR. LNA-miR-361-3p suppressed the expression levels of miR-361-3p compared with the control tumors. These results suggest that miR-361-3p support the growth of human OSCC cells and that targeting miR-361-3p may be a useful therapeutic approach for patients with OSCC. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Himiko Ogawa, Hiroyuki Goda. Targeting miR-361-3p suppresses the growth of human oral squamous cell carcinoma cells in vitro &amp; in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3553.

Abstract 3828: Differential transcriptional response of MYC to bromodomain inhibition in HPV positive head and neck squamous cell carcinoma

Abstract Differential transcriptional response of MYC to bromodomain inhibition in HPV positive head and neck squamous cell carcinoma Targeting of bromodomain (BRD) and extra-terminal domain (BET) family protein activity has the potential to reduce proliferation in many cancers. We hypothesize that targeting BRDs using its inhibitors will lead to reduced proliferation in human papilloma virus (HPV) infected head and neck cancer squamous cell carcinoma (HNSCC). HPV infected HNSCC is predicted to surpass the incidence of cervical cancer by 2020. Amongst various HPV serotypes, HPV16 is classified as oncogenic for a various number of cancer sites including head and neck squamous cell carcinoma (HNSCC). Integration of HPV into host genomes is not part of its life cycle; however, the exception is HNSCC wherein E6 and E7 viral proteins predominantly drive its tumor progression. HPV integrated HNSCC cells will be utilized to unravel the fundamental question of epigenetic regulation of viral-host interaction using the HPV-positive HNSCC model. Results from our lab indicate that despite high nanomolar IC50 in cell proliferation assays, downregulation of MYC expression was not observed using BRD inhibitor in all MYC expressing HNSCC cell lines. Further interrogation revealed differential phosphorylation of tumor suppressors - p53 and pRb might influence the sensitivity to BRD inhibition, which in turn could explain the contrasting regulation of MYC RNA levels. Intriguingly, combining radiation with bromodomain inhibitors led to increased radio-sensitization suggesting that BRDs have a role to play in DNA damage repair pathway and altered cell cycle regulation. Taken together, we will present MYC dependent and independent mechanism following BRD inhibition in HPV positive HNSCC. Note: This abstract was not presented at the meeting. Citation Format: Hassler Rengifo, Albert R. Wang, Mohammad Titi, Kwang Nickel, Randy Kimple, Gopal Iyer. Differential transcriptional response of MYC to bromodomain inhibition in HPV positive head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3828.

Abstract 1261: Targeting Wee1 in laryngeal squamous cell carcinoma

Abstract WEE1 is a tyrosine kinase that regulates G2/M cell cycle checkpoint and frequently overexpressed in various tumors. However, the expression and clinical significance of WEE1 in human laryngeal squamous cell carcinoma (LSCC) are still unknown. In this study, we found that WEE1 was highly expressed in LSCC tissues compared with adjacent normal tissues. Importantly, overexpression of WEE1 was correlated with T stages, lymph node metastasis, clinical stages and poor prognosis of LSCC patients. Furthermore, inhibition of WEE1 by MK-1775 induced cell growth inhibition, G2/M cell cycle arrest and apoptosis with the increased intracellular reactive oxygen species (ROS) levels in LSCC cells. Pretreatment with ROS scavenger N-acetyl-L-cysteine could reverse MK-1775-induced ROS accumulation and cell apoptosis in LSCC cells. MK-1775 also inhibited the growth of LSCC xenografts in nude mice. Altogether, these findings suggest that WEE1 is a potential therapeutic target in LSCC, and inhibition of WEE1 is the prospective strategy for LSCC therapy. Note: This abstract was not presented at the meeting. Citation Format: Meng-Ling Yuan, Qi-Wei Jiang, Yang Yang, Jia-Rong Huang, Zhi Shi. Targeting Wee1 in laryngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1261.

Abstract 2638: Galectin-3 activates the receptor tyrosine kinase Tyro3 and stimulates ERK signaling, cell survival and migration in human head and neck cancer cells

Abstract The beta-galactoside-binding protein galectin-3 (Gal-3) has been shown to influence the tumorigenic behaviour of cancer cells, and elevated serum levels of Gal-3 have been linked to tumor metastasis in several different cancers. Previously, Gal-3 was shown to be a ligand MerTK, a member of the TAM (Tyro3, Axl, MerTK) subfamily of receptor tyrosine kinases (RTKs) - these RTKs are implicated in tumorigenesis and/or enabling tumor chemoresistance in many different cancers. However, it is not known whether Gal-3 is also a ligand for the other two TAM RTKs, nor what signaling pathways it activates to regulate cancer cell functions. This study is aimed at determining the ligand properties of Gal-3 against Tyro3 and Axl RTKs in human cancer cells and the oncogenic signal pathways linked to them. The cultured human head and neck squamous cell carcinoma (HNSCC) cell line SCC-25 was determined by western blot and qPCR to express both Axl and Tyro3, but not MerTK. Recombinant Gal-3 and the TAM ligands Gas6 and ProS1 were added to serum-starved cells over a time-period of minutes and cells lysed to determine activation/phosphorylation of RTKs and intracellular signal mediators by western blotting. Longer incubations were performed to measure cell viability (MTS assay) and cell migration rate (scratch assay). All experiments were carried out a minimum three times. The known TAM ligands Gas6 and ProS1 stimulated TAM RTKs differentially in SCC-25 cells: Gas6 activated Axl strongly within minutes, with ProS1 having a weak effect. In contrast, ProS1 was the strongest ligand for Tyro3, activating it by fourfold, followed by Gal-3 at threefold and Gas6 at twofold. All three exogenous proteins also rapidly stimulated ERK1/2 phosphorylation by two-fold, whereas only Gas6 and Gal-3 stimulated Akt phosphorylation. All three proteins significantly maintained cell survival during acute induction of apoptosis by staurosporine. However, only Gas6 conferred a survival effect over long-term serum starvation-induced cell death. In addition, Gal-3 significantly stimulated cell migration during Axl blockade with the small molecule Axl inhibitor BGB324, further suggesting a role for Tyro3 in this effect. These results have shown that Gal-3 is a ligand for Tyro3 RTK, and that Gal-3 promotes cell survival and migration in HNSCC cells, coupled with activation of both the related signal pathways ERK and Akt. The data therefore reveal a new mechanism of Tyro3 activation; however, it remains to be determined whether Gal-3 interacts with the glycosyl chain on the RTK or else via other interfaces. Finally, cancer therapeutic strategies targeting Gal-3 action and/or cell surface glycosylation may be viable approaches in further research. Citation Format: Sassan Hafizi, Nour Al Kafri. Galectin-3 activates the receptor tyrosine kinase Tyro3 and stimulates ERK signaling, cell survival and migration in human head and neck cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2638.

Abstract 2750: Physical and functional landscape of deubiquitinating enzymes (DUBs) in KRAS mutant lung cancer

Abstract DUBs are involved in tumorigenesis and are of high interest as potential targets for cancer therapy. However, target discovery is problematic due to the absence of clear DUB activity within subsets of human cancers. Our goal was to profile DUBs and their activity in KRAS-mutated lung cancer cell lines using chemical biology strategies. Approximately 25% of patients with Lung adenocarcinoma have tumor associated KRAS mutations in non-small lung cancers (NSCLC), yet specific RAS inhibitors against KRAS-mutated lung cancer have not yet been successfully developed. We used activity-based protein profiling (ABPP) to profile DUBs in 25 distinct human lung cancer cell lines harboring KRAS mutations. ABPP uses chemical probes that are active-site directed and covalently bind to a class of enzymes in complex proteome. Whole cell lysates were incubated with HA-UB-VME and HA-UB-PA probes and ABPP pull-down was performed. Enriched proteins were trypsin digested and peptides were analyzed using liquid chromatography and tandem mass spectrometry (LC-MS/MS). MAXQUANT software was used to quantify DUBs. We related activity of observed DUBs to effects on cell viability by examining publicly available RNAi (Project DRIVE) and CRISPR (PICKLES) databases. A total of 50 DUBs were identified in our ABPP screen. Each cell line expressed at least 32 different DUBs and 22 DUBs were represented in all cell lines. The identified DUBs include 48% USP, 36% OTU, 8% JAMM and 8% UCH &amp; MJD family. USP5, USP7, USP14, USP15, UCHL5, UCHL3, OTUB1, PRP8, PSMD7 and PSMD14 are the highly expressed DUBs. Functional enrichment approach and protein-protein interaction network reveal that 70% of the active DUBs are involved in cell cycle regulation. DDR, RNA splicing, TGF-beta receptor, NF-kB and Wnt signaling are some other enriched pathways. Of the 35 cell cycle DUBs, CRISPR screens identify 14 DUBs and DRIVE data identified 11 DUBS as having effects on cell viability. Combining the ABPP data, shRNA and CRISPR results, OTUD5, BRCC3, UCHL5, UFD1L, USP5, YY1, USP3, USP39, USP37, OTUB1, USP9X and COPS5 have activity in KRAS mutant lung cancer cell lines and demonstrate reduced cell viability with loss of function through CRISPR or RNAi. We have identified and prioritized DUB targets in KRAS mutant lung cancer. Future studies will further validate DUB as targets and identify mechanisms of activity in KRAS mutant lung cancers, as well as examine DUB activity using ABPP in human lung cancer tumor tissues, including adenocarcinoma (KRAS mutant and wild-type) and squamous cell lung cancer. Citation Format: Emma Adhikari, Shikha Mahajan, Harshani Lawrence, Yan Yang, Bin Fang, Eric Haura. Physical and functional landscape of deubiquitinating enzymes (DUBs) in KRAS mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2750.

Identifying adverse molecular features of HPV+ head and neck cancers using patient-derived models.

6057 Background: Advancing therapy for human papilloma virus-related (HPV+) head and neck squamous cell carcinoma (HNSCC) is hindered by inadequate preclinical models and risk stratification tools. This study addresses both barriers by characterizing a panel of patient-derived xenografts (PDXs) that includes nine new models of HPV+ disease. Methods: Exome-sequenced genetic features of the PDXs were compared to their growth properties and the outcomes of their patients of origin. Genetic traits with potential prognostic utility were validated in multiple retrospective patient cohorts. Results: The HPV+ PDXs avoided known artifacts in HPV+ cell lines, including 3q amplifications and loss of PIK3CA mutants, while enriching for alterations in H3K4 methyltransferases and Notch pathway genes. A positive association emerged between PDX tumor mutational burden (TMB) and their growth efficiency both in vivo and as organoids. This finding led to identification in The Cancer Genome Atlas (TCGA) of an association between high TMB and worse survival of early-stage HPV-negative cancers but not HPV+ ones. Insight into aggressive HPV+ disease came from a PDX established from a patient before lethal relapse. The reduced levels of viral E7 and p16INK4A present in this model were also detected in early lethal HPV+ cases in TCGA as well as the recurrences in a second HPV+ HNSCC cohort (JHU). This observation suggested a diminished contribution of viral oncogenes to cell cycle dysregulation in aggressive cases. To evaluate this possibility, hierarchical clustering of both cohorts was performed based on expression of E2F target genes. This analysis discovered a distinct cell cycle-related transcriptional pattern in the clusters of cases containing the recurrences and early lethal events. Furthermore, a subset of these transcripts proved to be stage-independent predictors of survival for the HPV+ HNSCCs in both TCGA and JHU cohorts. Conclusions: Characterizing the most HPV+ patient-derived models described to date revealed novel prognostic utility for E2F target expression in HPV+ HNSCCs and TMB in HPV-negative HNSCCs. These features have potential for application to risk stratification, biomarker development, and trial design.

Preparation of Graphene Nano Drug-Loading System and Its Killing Effect on Human Oral Squamous Cell Carcinoma Cells

Preparation of Graphene Nano Drug-Loading System and Its Killing Effect on Human Oral Squamous Cell Carcinoma Cells

H19 Facilitates Tongue Squamous Cell Carcinoma Migration and Invasion via Sponging miR-let-7

The long noncoding RNA (lncRNA) H19 has been described to participate in the metastasis of various tumors. Nevertheless, whether H19 promotes or impedes tongue squamous cell carcinoma (TSCC) cell migration and invasion remains controversial. Here we found that the expression of H19 was elevated in TSCC tissues compared with adjacent normal tissues. Moreover, we demonstrated that the expression of H19 was higher in metastasized tumors compared with unmetastasized tumors. Consistently, TSCC cells express higher levels of H19 than human squamous cells. Subsequently, depletion of H19 impaired the migration and invasion abilities of TSCC cells. Mechanistically, we demonstrated that H19 functions as a competing endogenous RNA (ceRNA) to sponge miRNA let-7a, leading to an increase in a let-7a target, the key regulator of tumor metastasis HMGA2, which is enriched in TSCC tissues and cell lines. Intriguingly, inhibition of let-7a significantly rescued the short hairpin H19 (shH19)-induced decrease in TSCC migration and invasion. These findings revealed that the H19/let-7a/HMGA2/EMT axis plays a critical role in the regulation of TSCC migration and invasion, which may provide a new therapeutic target for TSCC.

Effect of 5-fluorouracil on cellular response to proton beam in esophageal cancer cell lines according to the position of spread-out Bragg peak

Introduction: To investigate enhancement by 5-fluorouracil (5-FU) of the sensitivity of cancer cells to proton beam irradiation and clarify the differences in the responses of the 5-FU-treated cells to proton beam irradiation according to the position of the cells on the spread-out Bragg peak (SOBP).Methods: OE21 human esophageal squamous cells were irradiated with a 235-MeV proton beam at four different positions on the SOBP. The effects of the irradiation plus 5-FU treatment on the cell survival were assessed by clonogenic assays and determination of the sensitizer enhancement ratio (SER). In addition, DNA double-strand breaks were estimated by measuring phospho-histone H2AX (γH2AX) foci formation in the cells at 0.5 and 24 h after irradiation.Results: The relative biological effectiveness (RBE) of proton beam irradiation against vehicle-control cells tended to increase with an increase in the depth of the cells on the SOBP. On the other hand, the degree of enhancement of the cellular sensitivity to proton beam irradiation by 5-FU was similar across all the positions on the SOBP. Furthermore, a marked increase in the number of residual γH2AX foci at 24 h post-irradiation was observed in the cells at the distal end of the SOBP.Conclusions: Our data indicated that the degree of enhancement by 5-FU of the sensitivity of OE21 cells to 235-MeV proton beam irradiation did not differ significantly depending on the position of the cells on the SOBP. Furthermore, the degree of increase in the number of γH2AX foci at 24 h after proton beam irradiation with or without 5-FU exposure did not differ significantly according to the position on the SOBP. The effect of 5-FU in enhancing the effect of proton beam irradiation on cancer cells may be constant for all positions on the SOBP.