Common Gene and Immune Cell Dynamics Across Oral Carcinogenesis: A Multi-Stage Transcriptomic Analysis.

VEGF expression from human dysplastic or malignant oral epithelium may be related to mast cell density and the subsequent angiogenetic phenomena

Previous studies have shown that asthma is a risk factor for lung cancer, while the mechanisms involved remain unclear. We attempted to further explore the association between asthma and non-small cell lung cancer (NSCLC) via bioinformatics analysis. We obtained GSE143303 and GSE18842 from the GEO database. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) groups were downloaded from the TCGA database. Based on the results of differentially expressed genes (DEGs) between asthma and NSCLC, we determined common DEGs by constructing a Venn diagram. Enrichment analysis was used to explore the common pathways of asthma and NSCLC. A protein-protein interaction (PPI) network was constructed to screen hub genes. KM survival analysis was performed to screen prognostic genes in the LUAD and LUSC groups. A Cox model was constructed based on hub genes and validated internally and externally. Tumor Immune Estimation Resource (TIMER) was used to evaluate the association of prognostic gene models with the tumor microenvironment (TME) and immune cell infiltration. Nomogram model was constructed by combining prognostic genes and clinical features. 114 common DEGs were obtained based on asthma and NSCLC data, and enrichment analysis showed that significant enrichment pathways mainly focused on inflammatory pathways. Screening of 5 hub genes as a key prognostic gene model for asthma progression to LUAD, and internal and external validation led to consistent conclusions. In addition, the risk score of the 5 hub genes could be used as a tool to assess the TME and immune cell infiltration. The nomogram model constructed by combining the 5 hub genes with clinical features was accurate for LUAD. Five-hub genes enrich our understanding of the potential mechanisms by which asthma contributes to the increased risk of lung cancer.

A few studies indicate that human papillomavirus (HPV) induces aberrant expression of microRNAs (miRNAs) and correlate this with p16INK4a in oral dysplasia (OD) and oral squamous cell carcinoma (OSCC). Therefore, this study aimed to evaluate the expression of miRNA-21, miRNA-22, and miRNA-224 by q-PCR and the p16 < sup > INK4a < /sup > by immunohistochemical (IHC) as markers for HPV-positive OSCC and OD in comparison to controls as miRNA expression can be altered by the HPV oncogenes and hence can be used as a biomarker for HPV positive cases. Fifty-two specimens were collected from archived paraffin blocks for patients aged between 19 and 88 (31 males and 21 females) from various oral sites. They were examined by IHC using p16 < sup > INK4a < /sup > , by RT-PCR for the detection of HPV (6, 11, 16, 18, 31, 33, 35, 42, 43, 44, 45, 52, 53, 56), and by q-PCR for the expression of miRNA-21, miRNA-22, and miRNA-224 in positive specimens. Out of the 15 OD, three were positive by both techniques. Meanwhile, 17 out of all OSCC specimens showed intense nuclear and cytoplasmic staining by p16 < sup > INK4a < /sup > , and only 16 were also positive by RT-PCR. However, all control specimens were negative. MiRNA-21, miRNA-22, and miRNA-224 were overexpressed in 3 specimens of OD and 16 of OSCC. MiRNA-21, miRNA-22, and miRNA-224, besides p16 < sup > INK4a < /sup > , could be used as indicators for HPV-associated OD and OSCC as their expression is attributed to the HPV oncoprotein. Further studies using follow-up data should be done to correlate it with miRNA overexpression.

Inflammatory Bowel Diseases (IBD), which encompass ulcerative colitis (UC) and Crohn's disease (CD), are characterized by chronic inflammation and tissue damage of the gastrointestinal tract. This study aimed to uncover novel disease-gene signatures, dysregulated pathways, and the immune cell infiltration landscape of inflamed tissues. Eight publicly available transcriptomic datasets, including inflamed and non-inflamed tissues from CD and UC patients were analyzed. Common differentially expressed genes (DEGs) were identified through meta-analysis, revealing 180 DEGs. DEGs were implicated in leukocyte transendothelial migration, PI3K-Akt, chemokine, NOD-like receptors, TNF signaling pathways, and pathways in cancer. Protein-protein interaction network and cluster analysis identified 14 central IBD players, which were validated using eight external datasets. Disease module construction using the NeDRex platform identified nine out of 14 disease-associated genes (CYBB, RAC2, GNAI2, ITGA4, CYBA, NCF4, CPT1A, NCF2, and PCK1). Immune infiltration profile assessment revealed a significantly higher degree of infiltration of neutrophils, activated dendritic cells, plasma cells, mast cells (resting/activated), B cells (memory/naïve), regulatory T cells, and M0 and M1 macrophages in inflamed IBD tissue. Collectively, this study identified the immune infiltration profile and nine disease-associated genes as potential modulators of IBD pathogenesis, offering insights into disease molecular mechanisms, and highlighting potential disease modulators and immune cell dynamics.

A novel inhibitor of apoptosis, survivin, plays a role in oncogenesis. To determine the potential involvement of survivin in oral carcinogenesis, we investigated the distribution of survivin protein expression in oral squamous cell carcinomas (OSCCs) and oral pre-malignant lesions. The mRNA expression level and methylation status of the gene also were evaluated in OSCCs and OSCC-derived cell lines. In immunohistochemistry, 58% of tumors and 37% of pre-malignant lesions examined were positive for survivin, while no immunoreaction was observed in corresponding normal tissues. The reverse-transcription/polymerase chain-reaction revealed similar changes in survivin gene expression levels. Furthermore, of the 9 normal oral tissues with no survivin gene expression, 4 showed methylation of the gene, while no methylation was detected in the corresponding tumorous tissues. The results suggest that survivin plays an important role during oral carcinogenesis, and that the gene expression may be regulated by an epigenetic mechanism.

Carcinogenesis occurs when the cell cycle is compromised. Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X are expressed in cells in G1 phase, S/G2 /M phases, and apoptosis, respectively, and these three markers may be useful for histological evaluation of malignant lesions. Here, we aimed to identify cell cycle phases and apoptosis using immunohistochemistry in oral epithelial precursor lesions and oral squamous cell carcinoma. Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X expression levels were immunohistochemically examined in tissue specimens from 55 patients with oral epithelial precursor lesions and 50 patients with oral squamous cell carcinoma. Associations of clinicopathological variables with marker expression were assessed. Chromatin licensing and DNA replication factor 1 was expressed in the prickle cell layer of oral epithelial precursor lesions and many carcinoma cells of oral squamous cell carcinoma. Geminin reactivity was widely distributed in high-grade dysplasia and oral squamous cell carcinoma rather than low-grade or no dysplastic cases. γ-H2A histone family member X was expressed in the superficial layer of oral epithelial precursor lesions and scattered carcinoma cells of oral squamous cell carcinoma. In oral squamous cell carcinoma, lower geminin expression was observed in recurrent cases. Geminin and γ-H2A histone family member X were associated with the degree of differentiation and mode of invasion. Chromatin licensing and DNA replication factor 1, geminin, and γ-H2A histone family member X expression levels were correlated with oral carcinogenesis; these markers were associated with clinicopathological behaviors in oral squamous cell carcinoma.

Recent experiments have showed that it is possible to use pharmacological agents that inactivate oncogenes to treat at least some types of human malignancies. However, the development of molecular targeted therapy in oral squamous cell carcinoma (OSCC) is lagging behind compare to other cancers. In this study, we attempted to identify an appropriate target molecule and to determine whether targeting such a molecule is plausible therapeutic approach for the treatment of patients with OSCC. We determined the gene expression profiles of 9 human OSCC cell lines and non-neoplastic keratinocyte cell line by microarray analysis, and then identified Aurora kinase A (AURKA) as a cancer-related gene. AURKA has been shown to be related to the progression, survival, histological differentiation, and metastasis in various tumors. We revealed the overexpression of AURKA mRNA and protein in human OSCC cell lines and tissues. To clarify the function of AURKA in the cell proliferation of OSCC cells, we investigated the effect of small interfering RNA (siRNA) specific for AURKA (siAURKA) and MLN8237, an AURKA selective inhibitor, on the growth of human OSCC cells in vitro and in vivo. All siAURKAs almost completely suppressed the expression of AURKA protein, and significantly inhibited the growth of these cells compared to non-targeting siRNA. MLN8237 also markedly reduced the growth rate of human OSCC cells. Next, we assessed the growth inhibitory effect of siAURKA and MLN8237 using mouse model. Synthetic siAURKA/atelocollagen complexes (40 μM) were administrated into mouse tail vain every 3 days, and MLN8237 (20 mg/kg) was received orally for 14 consecutive days. We found both siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSCC tumors. To confirm the usefulness of targeting AURKA in OSCC, we attempted to culture the resected tumor tissues from 3 patients with OSCC and obtained primary cultured cells. Knockdown of AURKA expression and MLN8237 induced the growth inhibition of OSCC primary cultured cells. Furthermore, the expression of AURKA mRNA in OSCC tissues resected from patients was examined. In 37 of 50 primary OSCC tissues, the expression levels of AURKA mRNA were more than 2-fold increase compared to normal oral mucosa tissues, and notably we found significant association between AURKA mRNA expression levels and histological differentiation and lymph node metastasis. These results suggest that AURKA plays a critical role in the growth of human OSCC cells, and targeting AURKA appears to be a potentially useful therapeutic approach for patients with OSCC. Citation Format: Hiroshi Tanaka, Koh-ichi Nakashiro, Kazuki Iwamoto, Norihiko Tokuzen, Yohei Fujita, Rikimaru Shirakawa, Ryota Oka, Hiroyuki Goda, Hiroyuki Hamakawa. Aurora kinase A as a novel therapeutic target in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3003. doi:10.1158/1538-7445.AM2013-3003

Interleukin-1 beta in oral submucous fibrosis, verrucous hyperplasia and squamous cell carcinoma tissues.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Up to 20% of oral dysplasia cases have been suggested to undergo malignant transformation to OSCC; however, there are no methods to predict OSCC development. In this study, to identify the genes associated with oral dysplasia progression, we performed genomic copy number analyses of genomic DNA samples isolated from primary oral dysplasia and OSCC via the microdissection method and found elevated expression of transferrin receptor C (TfR1/TFRC) with genomic amplification in oral dysplasia and OSCC. The expression rate of TFRC in OSCC was significantly higher than that in dysplasia, suggesting that OSCC disease progression might be related to TFRC expression. Additionally, we investigated the in vitro and in vivo impacts of a newly established anti-human TFRC monoclonal antibody, which was isolated from a human cDNA library using the phage-display method, on cell proliferation and survival. The anti-TFRC antibody blocked the interaction between transferrin and TFRC and consequently inhibited iron uptake, leading to the iron deprivation-mediated suppression of cell growth and induction of apoptosis. Moreover, we demonstrated that the anti-TFRC antibody efficiently inhibited tumor growth in a murine xenograft OSCC model. Therefore, we suggest our developed complete human anti-human TFRC antibody as a useful, novel treatment for oral dysplasia and OSCC.

To evaluate the expression of voltage-gated potassium channel Kv3.4 and to determine its significance in oral squamous cell carcinoma (OSCC) and precancerous lesions. Immunohistochemical SP methods were performed to detect the expression of Kv3.4 at tissue level on 57 paraffin-embedded samples collected from Pathology Department of Anhui Province Stomatological Hospital during January 2013 to June 2014. The relationships between the expression of Kv3.4 and precancerous lesion and clinical pathologic factors of oral squamous cell carcinoma, such as pathologic classification, clinical stage and lymph node metastasis, were also analyzed. Totally 6 samples of normal oral mucosa tissue, 23 samples of OSCC and 13 samples of precancerous lesions were collected from the Department of Oral and Maxillofacial Surgery of The First Affiliated Hospital of Anhui Medical University during May 2014 to March 2015. Real-time quantitative PCR and Western blotting were used to detect the expression of Kv3.4 in these 42 samples at molecular and protein levels. Real-time quantitative PCR showed the relative expression quantity of Kv3.4 in normal oral mucosa, precancerous lesions, and OSCC tissues were 0.85±0.48, 3.50±2.51 and 18.48±7.70, respectively. The relative expression quantity of Kv3.4 in OSCC and precancerous lesions were higher than that in normal group, the differences were both statistically significant (P=0.002, P=0.029). The relative expression quantities of Kv3.4 protein in precancerous and OSCC tissues were 0.87±0.14 and 0.35±0.03 respectively by Western blotting, and both were higher than that in normal tissues (0.18±0.10). The differences were statistically significant (P=0.002). In 57 paraffin-embedded samples, the positive expression rates of Kv3.4 in normal, precancerous and OSCC tissues were 2/6, 13/18 and 95% (37/39), respectively. The differences were statistically significant(P<0.05). However, the expression of Kv3.4 did not show obvious correlation with patients' genders, presence of lymph node metastasis and clinical stages (P>0.05). The expression of Kv3.4 is positively correlated to OSCC's occurrence and development. Detection of the expression of Kv3.4 may be used for early diagnosis and prognostic judgment of oral squamous cell carcinoma and precancerous lesions.

Amplifications of TAOS1 and EMS1 genes in oral carcinogenesis: association with clinicopathological features

&lt;div&gt;AbstractPurpose:&lt;p&gt;Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/β-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis.&lt;/p&gt;Experimental Design:&lt;p&gt;We performed a model of tobacco-induced oral cancer &lt;i&gt;in vitro&lt;/i&gt;, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an &lt;i&gt;in vivo&lt;/i&gt; model of oral carcinogenesis and &lt;i&gt;ex vivo&lt;/i&gt; samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59.&lt;/p&gt;Results:&lt;p&gt;Both &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;ex vivo&lt;/i&gt; oral carcinogenesis approaches revealed decreased levels of nuclear β-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 &lt;i&gt;in vitro&lt;/i&gt; resulted in diminished cell migration, viability, and invasion. Finally, by using an &lt;i&gt;in vivo&lt;/i&gt; model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions.&lt;/p&gt;Conclusions:&lt;p&gt;The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.&lt;/p&gt;&lt;/div&gt;

In this study, the molecular mechanisms underlying malignant transformation from oral lichen planus (OLP) to oral squamous cell carcinoma (OSCC) were examined. High-throughput sequencing of long noncoding RNAs (lncRNAs) and mRNAs of normal subjects and patients with OLP and OSCC was conducted. RNA-seq reads were mapped, lncRNA and mRNA transcripts were assembled, and expression levels were estimated. The targets of lncRNAs were predicted. Finally, Gene Ontology (GO) and pathway enrichment analyses of differentially expressed genes (DEGs) and lncRNA targets were performed. High-quality sequence data were generated and the mapping ratios for OSCC, normal, and OLP samples were high. In total, 820, 656, and 582 DEGs were obtained from OPL vs. normal, OSCC vs. normal, and OSCC vs. OPL, respectively. A total of 1721 known lncRNAs and 133 predicted lncRNAs and targets were obtained. Keratinization was significantly enriched by OSCC-related DEGs, but not OPL-related DEGs. The pathway of olfactory transduction was enriched by OPL- and OSCC-related DEGs. Defense response to virus and viral carcinogenesis were enriched by DEGs and lncRNA targets in all comparisons. GO term related to the metabolic process was enriched by lncRNA targets in the OPL vs normal comparison, and antigen processing and presentation via MHC class I was significantly enriched by lncRNA targets in the other 2 comparisons. Keratinization and MHC class I antigen processing and presentation were activated during the malignant transformation from OLP to OSCC. Additionally, the olfactory transduction pathway may be important for OSCC.

Five-year survival of patients with oral squamous cell carcinoma (OSCC) has not changed apparently for the past 30 years. Although cylindromatosis (CYLD) is thought as a potent tumor suppressor, its biological significances in malignancies are largely unknown. The aim of this study was to clarify the role of CYLD in OSCC progression. We investigated expression of CYLD in OSCC including intraepithelial neoplasia (IEN; n = 48) and invasive carcinomas (n = 133) tissues and normal oral mucosal tissues (n = 35) by immunohistochemistry. In addition, effects of CYLD knockdown by siRNA or its overexpression on OSCC progression using OSCC cell lines. Our immunohistochemical analyses revealed that CYLD expression was significantly reduced at invasive lesions in OSCC tissues whereas it was conserved in normal epithelium and IEN. In addition, lower CYLD expression was associated with poor overall survival in invasive OSCC. Accordingly, CYLD knockdown led to acquisition of mesenchymal state and increased migratory activity in OSCC cells. Interestingly, loss of CYLD enhanced TGFβR1 (ALK5) protein expression by promoting ALK5 stability while overexpression of wild-type CYLD downregulated ALK5. Furthermore, EMT-like changes induced by CYLD knockdown were completely blocked by a TGFβR1 kinase inhibitor in OSCC cells. These findings suggest that downregulation of CYLD promotes invasion through EMT-like changes via ALK5 stabilization in OSCC cells. Further studies for these mechanisms probably provide new insights into the TGFβ signal transduction in cancer and development of a novel therapy in OSCC. Citation Format: Satoru Shinriki, Hirofumi Jono, Jian-Dong Li, Takuya Nakamura, Mitsuhiro Hayashi, Jianying Guo, Masanori Shinohara, Yukio Ando. Loss of CYLD promotes cell migration through stabilized ALK5 in human oral squamous cell carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4314. doi:10.1158/1538-7445.AM2013-4314

The 5-year survival of patients with oral squamous cell carcinoma (OSCC) has not changed apparently for the past 30 years. Although cylindromatosis (CYLD) is thought as a potent tumor suppressor, its biological significances in malignancies are largely unknown. The aim of this study was to clarify the role of CYLD in OSCC progression. We investigated expression of CYLD in OSCC including intraepithelial neoplasia (IEN; n = 48) and invasive carcinomas (n = 133) tissues and normal oral mucosal tissues (n = 35) by immunohistochemistry. In addition, effects of CYLD knockdown by siRNA transfection on OSCC progression using 5 OSCC cell lines (SAS, Tu4, HSC3, Ca9-22, and SCC-NA) and HaCaT keratinocytes. Our immunohistochemical analyses revealed that CYLD expression was significantly reduced at invasive lesions in OSCC tissues whereas it was conserved in normal epithelium and IEN. In addition, lower CYLD expression was associated with the correlation with the increased tumor size, advanced clinical stage, and poor overall survival in invasive OSCC. Accordingly, CYLD knockdown led to acquisition of mesenchymal state and increased migratory activity in all the OSCC cell lines as well as HaCaT keratinocytes. Notably, such EMT-like changes were completely blocked by a TGFβR1 inhibitor in all the OSCC cell lines, but not in HaCaT keratinocytes. Furthermore, treatment with excess amount of anti-TGFβ antibody (1D11) did not inhibit the EMT-like changes induced by CYLD repression. These findings suggest that downregulation of CYLD promotes invasion through EMT-like changes via ligand-independent TGFβR1 activation in OSCC cells in mechanisms distinct from normal epithelium. Further studies for these mechanisms probably provide new insights into the biology including “TGFβ switch” and development of a novel therapy in OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 16. doi:1538-7445.AM2012-16