Human Squamous Cell Research Articles

Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer.

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ Tcell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.

Roles of IGFBP-3 in cell migration and growth in an endophytic tongue squamous cell carcinoma cell line

Insulin-like growth factor binding protein-3 (IGFBP-3) is a member of the IGFBP family that has high affinity for IGFs and functions as either an oncogene or tumor suppressor in various types of cancer. We previously found that IGFBP3 mRNA levels are higher in endophytic-type human tongue squamous cell carcinoma (TSCC) that is more invasive and more prone to metastasis than exophytic and superficial types. This finding prompted us to investigate the roles of IGFBP-3 in TSCC using SAS cells, which were originally derived from endophytic-type TSCC. Specifically, we used SAS cells that express a fluorescent ubiquitination-based cell-cycle indicator (Fucci). RNA-sequencing analysis indicated that IGFBP-3 is associated with cell migration and cell growth. In fact, IGFBP-3 knockdown downregulates cell migration and causes cells to arrest in G1. This migratory potential appears to be cell cycle–independent. IGFBP-3 knockdown also reduced levels of secreted IGFBP-3; however, decreased migratory potential was not rescued by exogenous recombinant human IGFBP-3. Furthermore, ERK activity was downregulated by IGFBP-3 depletion, which suggests that MEK/ERK signaling may be involved in IGFBP-3-mediated cell migration. We therefore conclude that intracellular IGFBP-3 enhances cell migration independently of the cell cycle in TSCC with a higher metastatic potential.

Abstract 2229: Targeted locus amplification based sequencing for mapping viral integration sites in human papillomavirus positive head and neck squamous cell carcinomas

Abstract Background: Human papillomavirus (HPV) infections are the principal cause of cervical cancers, subsets of anogenital and head and neck cancers (HNC). During persistent infection, viral DNA integration into the host genome may occur, which is suggested to affect carcinogenesis. One of the most critical limitations of currently used HPV integration detection techniques (PCR-based or NGS-based) is their application to FFPE material, because of DNA fragmentation. The aim of this study was to assess HPV integration in HPV-positive HNSCC cell lines and FFPE tissue comparing the new Targeted Locus Amplification (TLA) technology with previously used PCR technology (APOT/DIPS). Methods: Seven HPV-positive cell lines and FFPE material of 10 HPV-positive HNSCC were used for HPV integration detection by TLA, a proximity ligation-based next-generation sequencing technique. Crosslinked DNA is digested with restriction enzymes, and re-ligated into chimeric DNA molecules. For cell lines, a PCR based HPV16 target enrichment is performed, and for FFPE material a capture-based target enrichment is performed for HPV16 and HPV18 sequences, both followed by Illumina sequencing. Results: TLA was able to sequence up to 100 kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with APOT/DIPS PCR data and the literature. In the FFPE tissue samples, TLA identified integrated HPV in 6 out of 10 tumors, with simple and complex integration patterns. In general, TLA confirmed PCR data and detected additional integration sites. Conclusion: TLA provides the opportunity for reliable and robust detection of HPV integration in HNSCC cell lines and FFPE tissue. This new sequencing technology could be a useful tool for further research on HPV integration in disease and patient outcome and eventually in clinical diagnostics. Citation Format: Imke Demers, Harini Balaji, Harma Feitsma, Irinia Sergeeva, Joost Swennenhuis, Nora Wuerdemann, Steffen Wagner, Bernd Kremer, Christian Huebbers, Jens Peter Klussmann, Ernst-Jan Speel. Targeted locus amplification based sequencing for mapping viral integration sites in human papillomavirus positive head and neck squamous cell carcinomas [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 2229.

Abstract 5808: Diclofenac inhibits esophageal cancer cell growth by depleting mitochondrial functions

Abstract Esophageal cancer is one of the most aggressive forms of human malignancy with a 5-yearsurvival rate of less than 20%. Mitochondria are dynamic organelles that play essential roles in various cellular processes, including energy metabolism, redox homeostasis, and apoptotic cell death. Alterations in mitochondrial biology are associated with esophageal carcinogenesis and esophageal cancer cell response to therapy. As the anti-inflammatory drug diclofenac (DCF)induces mitochondrial dysfunction, we hypothesized that DCF may inhibit esophageal carcinogenesis by affecting mitochondrial processes. In the human esophageal squamous cell carcinoma (ESCC) cell line TE11, we performed MTT assay and Annexin-V/Propidium Iodide (PI) to evaluate the impact of DCF on proliferation and cell death, respectively. RNA sequencing and Ingenuity Pathway Analysis (IPA) identified differentially expressed genes (DEGs) and altered canonical pathways in DCF-treated TE11 cells. To assess metabolic function, we measured the levels of pyruvate, lactate, and ATP. Mitochondrial membrane potential and ROS production were assessed by flow cytometry for MitoTracker red/green and MitoSox red, respectively. Mice bearing subcutaneous syngeneic ESCC tumors were treated with DCF.DCF inhibited proliferation of TE11 in dose dependent manner with an IC50 concentration of 76.7µM. By contrast, the IC50 concentration of DCF in normal esophageal keratinocytes was 876.2µM, supporting selectivity of DCF for esophageal cancer cells. In TE11, 200 µM DCF induced apoptosis in 60% of cells. RNA-sequencing identified that DCF significantly altered expression of3287 genes (FC ≥ 1.50) in TE11, including the top cancer-associated genes: PLK1, MCM2, MCM3,MCM7, MCM10 and SKP2. IPA analysis revealed that DCF activated p53 signaling while inhibited Gluconeogenesis I, Glycolysis I and Oxidative Phosphorylation. DCF significantly inhibited glycolysis as demonstrated by reduction in concentrations of pyruvate (1.76-fold) and lactate (7.30-fold) and downregulation of the glycolysis- associated genes PKM2, PFKM, LDHA, and PKM1. A decreased production of ATP (5-fold) was also noted in DCF-treated TE11 cells. In addition, DCF depleted mitochondrial membrane potential (2.5-fold) and increased mitochondrial ROS production (5-fold). A reduction in ROS by mitochondrial antioxidant MitoTempo increased viability of the DCF-treated TE11 cells, indicating that ROS contributes to the anticancer activity of DCF. Consistent with antitumor activity of DCF in TE11, the drug significantly decreased the tumor volume in syngeneic ESCC tumors in vivo. Our preclinical findings indicate that DCF may limit esophageal cancer cell growth through the inhibition of mitochondrial functions, identifying a novel experimental therapeutic for ESCC. Future studies will define the precise molecular mechanisms through which DCF promotes cell death in vitro and in vivo. Citation Format: Mohammad Faujul Kabir, Adam Karami, Anbin Mu, Don-Gerard Conde, Kelly A. Whelan. Diclofenac inhibits esophageal cancer cell growth by depleting mitochondrial functions [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 5808.

Abstract 1390: Immunotherapy boosts B cell responses in lung adenocarcinoma

Abstract Introduction: B cells constitute a major part of the lung tumour microenvironment where they are organised in tertiary lymphoid structures (TLS) that resemble antibody-producing germinal centres. The presence of TLS has been associated with improved patient outcome and response to immunotherapy, though the mechanisms remain unclear. Similarly, the contribution of B cells and TLS to anti-tumour immunity in non-small cell lung cancer (NSCLC) remains largely unexplored. Methods: We evaluated TRACERx and TCGA NSCLC cohorts to compare B cell and TLS signatures as well as BCR sequences from human adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Using a novel immunogenic mouse model of LUAD, we applied lineage tracing, 3D IF, and single-cell sequencing to characterise the evolution of lung germinal centre and antibody responses over tumour progression and immunotherapy treatment. We validated immunotherapy observations in a cohort of melanoma patients with pre- and post-treatment samples. Results: We found that B cell and TLS gene set scores were significantly higher in LUAD compared to LUSC. We examined B cell signals in specific mutational backgrounds, and observed an increase in BCR CDR3 diversity and IgG class switch in the context of clonal TP53 mutation. We therefore investigated the mechanistic basis of B cells in LUAD using an immunogenic mouse model on a KRAS/TP53 background. We found that TLS organisation was triggered by CXCL13 and that exogenous CXCL13 treatment increased B cell infiltration and overall survival. Using serum transfer experiments, we demonstrated that tumour-binding class-switched antibodies played an anti-tumour role and increased survival. We found that immunotherapy increased germinal centre B cell responses and antibody quantity and quality. Using single-cell BCR sequencing of immunotherapy-treated mice, we showed clonal expansion of B cells reactive to re-expressed endogenous retroviral antigens. Furthermore, we found that KRAS G12C but not MEK inhibition boosted B cell and antibody responses. Finally, combined treatment with CXCL13 and checkpoint blockade improved survival, suggesting that boosting local B cell responses can improve immunotherapy outcome. In checkpoint blockade-treated melanoma patients, we found that BCR diversity and the number of unique antigen specificities was elevated in responders post-therapy. Non-responders showed a loss of expanded clones, suggesting that both maintenance of expanded clones and recruitment of new clones associate with successful response. Conclusions: Our work describes a novel role for B cells in the lung tumour microenvironment and establish that TLS directly contribute to anti-tumour immunity by producing class-switched protective antibodies. We demonstrate that selective boosting of anti-tumour B cells shows therapeutic potential in combination with targeted and immunotherapy. Citation Format: Katey Enfield, Kevin Ng, Jesse Boumelha, Kevin Litchfield, Oriol Pich, TRACERx Consortium, Charles Swanton, George Kassiotis, Julian Downward. Immunotherapy boosts B cell responses in lung adenocarcinoma [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 1390.

Abstract 4222: Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway

Abstract Oral cancer is the sixth most prevalent malignancy in the world and oral squamous cell carcinoma accounts for majority of all oral malignancies. Upregulated NLRP3 inflammasome innate immune pathway is of importance to tumor development. Current efforts are being focused on identifying small molecules that exhibit anti-cancer activity as inflammasome pathway inhibitors. Our previously published work on phenstatin based indole linked chalcone scaffold 9a with 1-methyl, 2- and 3-methoxy substituents in the aromatic ring revealed 9a as an anti-oral cancer compound. 9a was found to act through inhibiting tubulin polymerization at protein level, using in vitro models oral cancer cell line/spheroid cells and in vivo animal oral cancer xenograft model. 9a had also shown significant reduction in radiolabeled-glucose uptake in xenograft mice model. Current study was undertaken to evaluate if small molecule inhibitor 9a acts through regulating the NLRP3 pathway. Using computation approach, we predicted the binding of 9a with NLRP3NACHT domain, which revealed stable interaction as similar to that exhibited by NLRP3 inhibitors MCC950 and ADP. Further, we checked immune mechanistic activity of 9a on NLRP3 pathway intermediates in oral cancer cells. AW13516 cell line which was human tongue squamous tumor-derived cell line; indigenously developed at our department previously, was activated for NLRP3 inflammasome pathway using LPS and activator Nigericin in presence of 9a. MCC950 treated cells and only LPS or LPS/Nigericin treated cells served as controls. NLRP3, caspase-1 and mitochondrial protein expression was analyzed in these cells by immunofluorescence (IF) and found to be increased upon LPS/NIG activation and reduced significantly upon MCC950 and 9a treatment. Activation led to puncta formation which was found diffused after MCC950/9a treatment. Similarly treated AW13516 cells were also validated using western blotting experiments. Expression of 118kDa NLRP3 protein was found increased upon inflammasome activation that was significantly reduced in 9a treated cells and reduction was dose dependent. 9a had shown significant reduction in oral cancer xenograft of AW13516 in NOD-SCID mice model. We tested formalin-fixed paraffin sections of these tumors by immunohistochemistry. Tumor areas were assessed for expression of NLRP3 pathway markers and there was significant reduction in NLRP3, Caspase-1, GSDMD and IL-1β in 9a treated tumors compared to control tumors. This reduction was at par with that shown by Adriamycin. Summarizingly, 9a has been found to be regulating inflammatory immune mechanisms and can be developed further as immunomodulatory anti-cancer agent. Since macrophages are major resource immune cells of NLRP3, further studies are underway to test 9a on macrophages from nlrp3-/- and caspase-/- mice in comparison with normal mice. Citation Format: Jyoti Kode, Jitendra Maharana, K. Nirmal Kumar, Trupti Pradhan, Arvind Ingle, Madan Barkume, Meena Patkar, Namitha Thampi, Ankita Patil, Anand Vaibhaw, Jeshma Kovvuri, Ahmed Kamal. Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway [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 4222.

Assessing Gene Expression Related to Cisplatin Resistance in Human Oral Squamous Cell Carcinoma Cell Lines

Cisplatin-based chemotherapy has been effectively used to treat oral cancer, but treatment often fails owing to the development of drug resistance. However, the important gene expression alterations associated with these resistances remain unclear. In this study, we aimed to identify the gene expressions related to cisplatin resistance in oral squamous cell carcinoma (OSCC) cell lines. RNA samples were obtained from three cisplatin-resistant (YD-8/CIS, YD-9/CIS, and YD-38/CIS) and -sensitive (YD-8, YD-9, and YD-38) cell lines. Global gene expression was analyzed using RNA sequencing (RNA-Seq). Differentially expressed genes were determined. Based on the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, functional enrichment and signaling pathways analyses were performed. Candidate genes selected from RNA-Seq analysis were validated by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The YD-8/CIS and YD-9/CIS samples had very similar expression patterns. qRT-PCR analysis was performed on selected genes commonly expressed between the two samples. The expression levels of 11 genes were changed in cisplatin-resistant samples compared with their parental samples; several of these genes were related to cell adhesion molecules and proteoglycans in cancer pathways. Our data provide candidate genes associated with cisplatin resistance in OSCC, but further study is required to determine which genes have an important role. Nevertheless, these results may provide new ideas to improve the clinical therapeutic outcomes of OSCC.

MiR-214 Modulates the Growth and Migration of Oral Cancer before and after Chemotherapy through Mediating ULK1.

The role of miRNAs as crucial components in carcinogenesis has been well documented. However, whether and how miR-214 influences oral cancer cells' drug resistance remains to be elucidated, and its downstream targets are still under investigation. Hence, this research is aimed at determining miR-214 and ULK1 expression in oral cancer before and after chemotherapy and their correlations with cancer cell growth. Human oral normal epithelial cells and human tongue squamous cell carcinoma CAL-27 cells were cultured to detect miR-214 and ULK1 levels. It was found that before chemotherapy, miR-214 was higher, while ULK1 was underexpressed in CAL-27 cells, versus normal epithelial cells. After chemotherapy, miR-214 decreased obviously in CAL-27 cells, while ULK1 level increased significantly. In addition, autophagy-related genes (Beclin 1, mTOR, and P53) in CAL-27 cells were found to be significantly inhibited before chemotherapy and were obviously increased after chemotherapy. Moreover, to further determine the impacts of miR-214 and ULK1 on oral cancer cell growth after chemotherapy, the two were overexpressed or silenced in CAL-27 cells after transfection. We found that ULK1 could effectively decrease the activity and invasion of CAL-27 cells and increase their apoptosis level, while miR-214 could antagonize its antitumor effect. Therefore, miR-214 can be used as an early prognostic biomarker for oral cancer, and ULK1 is a new candidate therapeutic target.

Non-Robustness of Ang's Risk Classification in Human Papillomavirus-Related Oropharyngeal Squamous Cell Carcinoma in Japanese Patients.

Simple SummaryHuman papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) has a favorable prognosis in comparison to HPV-unrelated OPSCC. Risk classification by Ang is known to predict prognosis of HPV-related OPSCC, but it remains to be studied whether it is useful in predicting prognosis in operatively treated Japanese patients. HPV-related OPSCC treated from 2010 to 2018 in a single Japanese institution by various methods were analyzed. We found that Ang’s risk classification lost its statistical significance in predicting prognosis in the multivariate analysis and it turned out to not be a robust prognostic factor for Japanese patients with HPV-related OPSCC treated by various methods. Additionally, Ang’s risk classification was not prognostically significant in the patients treated by definitive radiation in this series. Smoking appeared to have a larger impact on prognosis in the operatively treated patients than in the radiotherapeutically treated patients.Background: Validity of the risk classification by Ang for human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) remains to be studied in the patients treated by modalities other than chemoradiotherapy and in Japanese patients. Materials and Methods: Between 2010 and 2018, 122 patients with HPV-related OPSCC in stages III and IV by the TNM classification 7th edition (TNM-7) were treated curatively at a single institution in Japan. The median age was 62.7 years. Over 50% of the patients underwent surgery with or without adjuvant therapy. The influence of multiple factors on survival was analyzed. Results: The amount of smoking dichotomized at 10 pack-year, which was used in Ang’s risk classification, was not predictive of prognosis, and Ang’s risk classification was not significantly influential on prognosis in multivariate analysis. In the patients treated with definitive radiation therapy, Ang’s risk classification was not predictive of the prognosis in univariate analysis. The impact of smoking was significant only in the patients undergoing the definitive operation. Conclusions: Ang’s risk classification was not robust in predicting the prognosis of general Japanese HPV-related OPSCC patients. The amount of smoking might have different prognostic influences depending on the therapeutic method.

Palbociclib-based high-throughput combination drug screening identifies synergistic therapeutic options in HPV-negative head and neck squamous cell carcinoma

BackgroundDeregulation of cell-cycle pathway is ubiquitously observed in human papillomavirus negative (HPVneg) head and neck squamous cell carcinoma (HNSCC). Despite being an attractive target, CDK4/6 inhibition using palbociclib showed modest or conflicting results as monotherapy or in combination with platinum-based chemotherapy or cetuximab in HPVneg HNSCC. Thus, innovative agents to augment the efficacy of palbociclib in HPVneg HNSCC would be welcomed.MethodsA collection of 162 FDA-approved and investigational agents was screened in combinatorial matrix format, and top combinations were validated in a broader panel of HPVneg HNSCC cell lines. Transcriptional profiling was conducted to explore the molecular mechanisms of drug synergy. Finally, the most potent palbociclib-based drug combination was evaluated and compared with palbociclib plus cetuximab or cisplatin in a panel of genetically diverse HPVneg HNSCC cell lines and patient-derived xenograft models.ResultsPalbociclib displayed limited efficacy in HPVneg HNSCC as monotherapy. The high-throughput combination drug screening provided a comprehensive palbociclib-based drug-drug interaction dataset, whereas significant synergistic effects were observed when palbociclib was combined with multiple agents, including inhibitors of the PI3K, EGFR, and MEK pathways. PI3K pathway inhibitors significantly reduced cell proliferation and induced cell-cycle arrest in HPVneg HNSCC cell lines when combined with palbociclib, and alpelisib (a PI3Kα inhibitor) was demonstrated to show the most potent synergy with particularly higher efficacy in HNSCCs bearing PIK3CA alterations. Notably, when compared with cisplatin and cetuximab, alpelisib exerted stronger synergism in a broader panel of cell lines. Mechanistically, RRM2-dependent epithelial mesenchymal transition (EMT) induced by palbociclib, was attenuated by alpelisib and cetuximab rather than cisplatin. Subsequently, PDX models with distinct genetic background further validated that palbociclib plus alpelisib had significant synergistic effects in models harboring PIK3CA amplification.ConclusionsThis study provides insights into the systematic combinatory effect associated with CDK4/6 inhibition and supports further initiation of clinical trials using the palbociclib plus alpelisib combination in HPVneg HNSCC with PIK3CA alterations.

CGAS Regulates the Radioresistance of Human Head and Neck Squamous Cell Carcinoma Cells.

Cyclic GMP-AMP synthase (cGAS) plays an important role in biological responses to pathogens. The activation of the cGAS pathway in immune cells is known to induce antitumor effects, but the role of cGAS in cancer cells remains poorly understood. In silico analysis using public databases suggested that high cGAS expression in head and neck squamous cell carcinoma (HNSCC) is indicative of a poor prognosis for HNSCC patients. We therefore investigated the role of cGAS in malignancies and the cellular radiation response of human HNSCC cells (SAS and Ca9-22) in vitro, because radiotherapy is one of the treatments most commonly used for HNSCC. Although cGAS knockdown failed to suppress the proliferation of non-irradiated HNSCC cells, it enhanced the radiosensitivity of HNSCC cells. The administration of the cGAS agonist increased the radioresistance of HNSCC cells. cGAS knockdown increased radiation-induced mitotic catastrophe, apoptosis, or cellular senescence, depending on the cell line, and this cell line-dependent response might be due to different responses of p21 after irradiation. Collectively, our findings indicate that the cGAS pathway regulates the radioresistance of HNSCC cells.

COX-2 strengthens the effects of acid and bile salts on human esophageal cells and Barrett esophageal cells

AimsInvestigate the effect and mechanism of COX-2 on viability, intestinal metaplasia, and atypia in human esophageal squamous and Barrett esophageal cell lines.MethodsHuman esophageal squamous and Barrett esophageal cell lines were transfected with a COX-2 expression vector and a COX-2 siRNA, and then were treated with acid, bile salts, and a mixture of both. Cell viability, the expression of COX-2, NF-κB(p65), CDX-2, MUC2, c-myb, and BMP-4, and the morphology and microstructure of cells were then observed.ResultsThe viability of COX-2 overexpressed cells was significantly higher than that of control cells, while the viability of COX-2 siRNA-treated cells was significantly lower than that of control cells. Intestinal metaplasia and atypia were observed in cells overexpressing COX-2. Acid, bile salts, and their mixture inhibited the viability of these two cell lines, but the inhibitory effect of the mixture was stronger than a single treatment in either. SiRNA mediated knockdown of COX-2 strengthened the antiproliferative effects of the mixture on HET-1A and BAR-T cells. The expression of p-p65, CDX-2, and BMP-4 was positively correlated with COX-2 expression, while the expression levels of p65, MUC2, and c-myb remained unchanged.ConclusionCOX-2 may influence the viability, atypia, and intestinal metaplasia of human esophageal cells and Barrett esophageal cells. Activation of the p-p65, CDX-2, and BMP-4 signaling pathways by COX-2 may be part of this mechanism.

Porphyromonas gingivalis W83 Membrane Components Induce Distinct Profiles of Metabolic Genes in Oral Squamous Carcinoma Cells.

Periodontitis, a chronic inflammatory disease is caused by a bacterial biofilm, affecting all periodontal tissues and structures. This chronic disease seems to be associated with cancer since, in general, inflammation intensifies the risk for carcinoma development and progression. Interactions between periodontal pathogens and the host immune response induce the onset of periodontitis and are responsible for its progression, among them Porphyromonas gingivalis (P. gingivalis), a Gram-negative anaerobic rod, capable of expressing a variety of virulence factors that is considered a keystone pathogen in periodontal biofilms. The aim of this study was to investigate the genome-wide impact of P. gingivalis W83 membranes on RNA expression of oral squamous carcinoma cells by transcriptome analysis. Human squamous cell carcinoma cells (SCC-25) were infected for 4 and 24 h with extracts from P. gingivalis W83 membrane, harvested, and RNA was extracted. RNA sequencing was performed, and differential gene expression and enrichment were analyzed using GO, KEGG, and REACTOME. The results of transcriptome analysis were validated using quantitative real-time PCR with selected genes. Differential gene expression analysis resulted in the upregulation of 15 genes and downregulation of 1 gene after 4 h. After 24 h, 61 genes were upregulated and 278 downregulated. GO, KEGG, and REACTONE enrichment analysis revealed a strong metabolic transcriptomic response signature, demonstrating altered gene expressions after 4 h and 24 h that mainly belong to cell metabolic pathways and replication. Real-time PCR of selected genes belonging to immune response, signaling, and metabolism revealed upregulated expression of CCL20, CXCL8, NFkBIA, TNFAIP3, TRAF5, CYP1A1, and NOD2. This work sheds light on the RNA transcriptome of human oral squamous carcinoma cells following stimulation with P. gingivalis membranes and identifies a strong metabolic gene expression response to this periodontal pathogen. The data provide a base for future studies of molecular and cellular interactions between P. gingivalis and oral epithelium to elucidate the basic mechanisms of periodontitis and the development of cancer.

Increased PPARD Expression May Play a Protective Role in Human Lung Adenocarcinoma and Squamous Cell Carcinoma.

Peroxisome proliferator-activated receptor-δ, encoded by gene PPARD, is overexpressed in a majority of human lung cancer subtypes, but its role in the tumor progression remains poorly understood. We have analyzed the expression of PPARD in lung adenocarcinoma (LA) and squamous cell carcinoma (LSCC) datasets. The potential roles of PPARD in the pathological development of LA and LSCC were explored through literature-based pathway analysis and pathway enrichment analysis. In all LA datasets (N = 11) and in seven out of nine LSCC studies, the levels of PPARD were increased as compared to control tissues (log-fold changes were 0.37 ± 0.20 and 0.10 ± 0.37 for LA and LSCC, respectively). On average, the expression levels of PPARD in LA were higher than those in LSCC (p = 0.036). Pathway analysis showed that the overexpression of PPARD might play both positive and negative roles in the development of both LA and LSCC. Specifically, PPARD inhibits seven LSCC promoters and seven LA promoters and activates one LSCC inhibitor and another LA inhibitor. However, PPARD also activates six and one promoters of LA and LSCC, respectively, which would facilitate the development of LA/LSCC. Our results suggested a mixed role of PPARD in LA/LSCC, which may add new insights into the understanding of the PPARD-lung cancer relationship.

Anti-Cancer Effects of Disulfiram in Cervical Cancer Cell Lines Are Mediated by Both Autophagy and Apoptosis.

Disulfiram (DSF), which is used to treat alcohol dependence, has been reported to have anticancer effects in various malignant tumors. We studied the anti-cancer effects and mechanism of action of DSF on cervical cancer cell lines HeLa and SiHa. The anti-cancer effects of DSF were confirmed in vivo using a xenograft tumor model. The anti-cancer effects of DSF in human head and neck squamous cell carcinoma were found to be copper (Cu)-dependent. The anti-tumor effects of DSF/Cu were time- and dose-dependent and were mediated by both autophagy and apoptosis. DSF/Cu shows stronger cytotoxicity to adenocarcinoma cell lines with higher malignant behavior, and valosin-containing protein (VCP) is its potential target. The cytotoxic effect of DSF/Cu against cervical cancer cell lines in vitro was mediated by apoptosis and autophagy simultaneously. Analysis of the clinical relevance of DSF/Cu on a xenograft animal model showed that DSF markedly stimulated tumor necrosis. DSF may contribute to improved survival of patients with cervical cancer. The antitumor characteristic of DSF can be used in the development of a new drug for advanced and refractory patients with cervical cancer.

ESCCAL-1 promotes cell-cycle progression by interacting with and stabilizing galectin-1 in esophageal squamous cell carcinoma

Long non-coding RNAs (LncRNAs) play important roles in the development of human esophageal squamous cell carcinoma (ESCC). Our previous studies have shown that knockdown of LncRNA ESCCAL-1 expression inhibits the growth of ESCC cells, but the mechanisms remain largely unknown. In this study, we show that over-expression of ESCCAL-1 promotes ESCC cell proliferation and cell-cycle progression by blocking ubiquitin-mediated degradation of an oncoprotein galectin-1 (Gal-1). Multiple LncRNA expression datasets as well as our own data together reveal that ESCCAL-1 is evidently up-regulated in ESCC tissues and exhibits promising diagnostic value. Over-expression of ESCCAL-1 augmented ESCC cell proliferation and cell-cycle progression, whereas down-regulation of ESCCAL-1 resulted in the opposite effects. Mechanistically, LncRNA ESCCAL-1 directly binds to Gal-1 and positively regulates its protein level without affecting its mRNA level. Up-regulation of Gal-1 facilitated ESCC cell proliferation and cell-cycle progress. Knockdown of Gal-1 mitigated the effects of ESCCAL-1-mediated high cellular proliferation, NF-κB signaling activation and tumorigenicity of ESCC cells. Thus, our findings provide novel insight into the mechanism by which ESCCAL-1 facilitates ESCC tumorigenesis and cell-cycle progression by interacting with and stabilizing Gal-1 protein, suggesting a potential therapeutic target for ESCC.

Tumor radioresistance caused by radiation-induced changes of stem-like cell content and sub-lethal damage repair capability

Cancer stem-like cells (CSCs) within solid tumors exhibit radioresistance, leading to recurrence and distant metastasis after radiotherapy. To experimentally study the characteristics of CSCs, radioresistant cell lines were successfully established using fractionated X-ray irradiation. The fundamental characteristics of CSCs in vitro have been previously reported; however, the relationship between CSC and acquired radioresistance remains uncertain. To efficiently study this relationship, we performed both in vitro experiments and theoretical analysis using a cell-killing model. Four types of human oral squamous carcinoma cell lines, non-radioresistant cell lines (SAS and HSC2), and radioresistant cell lines (SAS-R and HSC2-R), were used to measure the surviving fraction after single-dose irradiation, split-dose irradiation, and multi-fractionated irradiation. The SAS-R and HSC2-R cell lines were more positive for one of the CSC marker aldehyde dehydrogenase activity than the corresponding non-radioresistant cell lines. The theoretical model analysis showed that changes in both the experimental-based ALDH (+) fractions and DNA repair efficiency of ALDH (−) fractions (i.e., sub-lethal damage repair) are required to reproduce the measured cell survival data of non-radioresistant and radioresistant cell lines. These results suggest that the enhanced cell recovery in SAS-R and HSC2-R is important when predicting tumor control probability in radiotherapy to require a long dose-delivery time; in other words, intensity-modulated radiation therapy is ideal. This work provides a precise understanding of the mechanism of radioresistance, which is induced after irradiation of cancer cells.

Repositioning Fenofibrate to Reactivate p53 and Reprogram the Tumor-Immune Microenvironment in HPV+ Head and Neck Squamous Cell Carcinoma.

Simple SummaryA critical need for optimal management of human papillomavirus-associated head and neck squamous cell carcinoma (HPV+ HNSCC) patients is the development of therapeutic strategies to exploit the inherent vulnerabilities of this unique disease. We identified fenofibrate, an FDA-approved drug, as a potent anti-cancer agent for HPV+ HNSCC. Fenofibrate induced the accumulation of the p53 tumor suppressor and re-programmed the tumor microenvironment to drive immune cell infiltration. We provide compelling evidence to reposition fenofibrate as a single agent or in combination with standard therapies for the HPV+ HNSCC setting.Human papillomavirus-associated head and neck squamous cell carcinoma (HPV+ HNSCC) is recognized as a distinct disease with unique etiology and clinical features. Current standard of care therapeutic modalities are identical for HPV+ and HPV− HNSCC and thus, there remains an opportunity to develop innovative pharmacologic approaches to exploit the inherent vulnerabilities of HPV+ HNSCC. In this study, using an inducible HPVE6E7 knockdown system, we found that HPV+ HNSCC cells are addicted to HPVE6E7, such that loss of these viral oncogenes impaired tumorigenicity in vitro and in vivo. A number of druggable pathways, including PPAR and Wnt, were modulated in response to HPVE6E7 loss. Fenofibrate showed significant anti-proliferative effects in a panel of HPV+ cancer cell lines. Additionally, fenofibrate impaired tumor growth as monotherapy and potentiated the activity of cisplatin in a pre-clinical HPV+ animal model. Systemic fenofibrate treatment induced p53 protein accumulation, and surprisingly, re-programmed the tumor-immune microenvironment to drive immune cell infiltration. Since fenofibrate is FDA-approved with a favorable long-term safety record, repositioning of this drug, as a single agent or in combination with cisplatin or checkpoint blockade, for the HPV+ HNSCC setting should be prioritized.

Mannose enhances the radio-sensitivity of esophageal squamous cell carcinoma with low MPI expression by suppressing glycolysis

BackgroundTo investigate the effect of mannose on radio-sensitivity of human esophageal squamous cell carcinoma (ESCC) cell line and its possible mechanism.MethodsThe expression of mannose phosphate isomerase (MPI) in human esophageal cancer cell lines were detected by Western blot. The inhibitory effect of mannose on human esophageal cancer cell lines were observed by MTT assay. Plate clone formation assay was performed to investigate the efficacy of mannose on radio-sensitivity of human esophageal cancer cells. The apoptosis rates of tumor cells treated with mannose and/or radiation therapy was calculated by flow cytometry. Furthermore, we analyzed intracellular metabolites using liquid chromatography mass spectrometry to identify selective sugar metabolites.ResultsMPI expression was various in human esophageal cancer cells. KYSE70 cells was associated with the highest MPI expression whereas KYSE450 cells had the lowest MPI expression level. When administrated with 11.1 mM/L mannose, the same inhibitory effect was observed in both KYSE70 and KYSE450 cell lines. Moreover, the inhibitory effect was significant on KYSE450 cell lines with an increased mannose concentration. The application of 11.1 mM/L mannose could significantly enhance the radio-sensitivity of KYSE450 cell line; and tumor cell apoptosis rate was also increased. However, there was limited efficacy of mannose on the radio-sensitivity and apoptosis rate of KYSE70 cell line. Additionally, intracellular metabolites analyzation revealed that glycolysis could be disturbed by mannose when combined with radiation therapy in esophageal cancer cells.ConclusionIn esophageal cancer cell lines with low MPI expression, the administration of mannose was associated with enhanced radio-sensitivity.

Synthesis of 3-(2-(subsituted-(trifluoromethyl)phenylamino)acetyl)-2H-chromen-2-one derivatives as new anticancer agents

Under solvent free conditions and in presence of a base 3-(2-(subsituted-(trifluoromethyl)phenylamino)acetyl)-2H-chromen-2-one derivatives were synthesized by grinding technique. Structural investigations were carried out with IR studies, HRMS, 1HNMR and 13CNMR. The compounds were checked for their in vitro anticancer activities against three different human cancer cell lines viz human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa) and human oral squamous cell carcinoma (SCC-40) using SRB method. All the title compounds showed low toxicity towards non-malignant PBMC cells indicating their tumour selectivity. The compounds exhibited good in vitro anti-proliferative potency at lower concentrations against HeLa and MCF-7 cell lines and remain moderately active against SCC-40.