Promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions are associated with tumor differentiation, nodal involvement and survival.

Differentially methylated regions (DMRs) can be used as head and neck squamous cell carcinoma (HNSCC) diagnostic, prognostic and therapeutic targets in precision medicine workflows. DNA from 21 HNSCC and 10 healthy oral tissue samples was hybridized to a genome-wide tiling array to identify DMRs in a discovery cohort. Downstream analyses identified differences in promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions associated with tumor differentiation, nodal involvement and survival. Genome-wide DMR analysis showed 2,565 DMRs common to the three subsites. A total of 738 DMRs were unique to laryngeal cancer (n=7), 889 DMRs were unique to oral cavity cancer (n=10) and 363 DMRs were unique to pharyngeal cancer (n=6). Based on the genome-wide analysis and a Gene Ontology analysis, 10 candidate genes were selected to test for prognostic value and association with clinicopathological features. TIMP3 was associated with tumor differentiation in oral cavity cancer (P=0.039), DAPK1 was associated with nodal involvement in pharyngeal cancer (P=0.017) and PAX1 was associated with tumor differentiation in laryngeal cancer (P=0.040). A total of five candidate genes were selected, DAPK1, CDH1, PAX1, CALCA and TIMP3, for a prevalence study in a larger validation cohort: Oral cavity cancer samples (n=42), pharyngeal cancer tissues (n=25) and laryngeal cancer samples (n=52). PAX1 hypermethylation differed across HNSCC anatomic subsites (P=0.029), and was predominantly detected in laryngeal cancer. Kaplan-Meier survival analysis (P=0.043) and Cox regression analysis of overall survival (P=0.001) showed that DAPK1 methylation is associated with better prognosis in HNSCC. The findings of the present study showed that the HNSCC subsites oral cavity, pharynx and larynx display substantial differences in aberrant DNA methylation patterns, which may serve as prognostic biomarkers and therapeutic targets.

Molecular Characterization of Esophageal Squamous Cell Carcinoma Using Quantitative Proteomics.

Esophageal squamous cell carcinoma (ESCC) is a heterogeneous cancer associated with a poor prognosis in advanced stages. In India, it is the sixth most common cause of cancer-related mortality. In this study, we employed high-resolution mass spectrometry-based quantitative proteomics to characterize the differential protein expression pattern associated with ESCC. We identified several differentially expressed proteins including PDPN, TOP2A, POSTN and MMP2 that were overexpressed in ESCC. In addition, we identified downregulation of esophagus tissue-enriched proteins such as SLURP1, PADI1, CSTA, small proline-rich proteins such as SPRR3, SPRR2A, SPRR1A, KRT4, and KRT13, involved in squamous cell differentiation. We identified several overexpressed proteins mapped to the 3q24-29 chromosomal region, aligning with CNV alterations in this region reported in several published studies. Among these, we identified overexpression of SOX2, TP63, IGF2BP2 and RNF13 that are encoded by genes in the 3q26 region. Functional enrichment analysis revealed proteins involved in cell cycle pathways, DNA replication, spliceosome, and DNA repair pathways. We identified the overexpression of multiple proteins that play a major role in alleviating ER stress, including SYVN1 and SEL1L. The SYVN1/SEL1L complex is an essential part of the ER quality control machinery clearing misfolded proteins from the ER. SYVN1 is an E3 ubiquitin ligase that ubiquitinates ER-resident proteins. Interestingly, there are also other non-canonical substrates of SYVN1 which are known to play a crucial role in tumor progression. Thus, SYVN1 could be a potential therapeutic target in ESCC.

A Novel Class of Ribosome Modulating Agents Exploits Cancer Ribosome Heterogeneity to Selectively Target the CMS2 Subtype of Colorectal Cancer.

Ribosomes in cancer cells accumulate numerous patient-specific structural and functional modifications that facilitate tumor progression by modifying protein translation. We have taken a unique synthetic chemistry approach to generate novel macrolides, Ribosome modulating agents (RMA), that are proposed to act distal to catalytic sites and exploit cancer ribosome heterogeneity. The RMA ZKN-157 shows two levels of selectivity: (i) selective translation inhibition of a subset of proteins enriched for components of the ribosome and protein translation machinery that are upregulated by MYC; and (ii) selective inhibition of proliferation of a subset of colorectal cancer cell lines. Mechanistically, the selective ribosome targeting in sensitive cells triggered cell-cycle arrest and apoptosis. Consequently, in colorectal cancer, sensitivity to ZKN-157 in cell lines and patient-derived organoids was restricted to the consensus molecular subtype 2 (CMS2) subtype that is distinguished by high MYC and WNT pathway activity. ZKN-157 showed efficacy as single agent and, the potency and efficacy of ZKN-157 synergized with clinically approved DNA-intercalating agents which have previously been shown to inhibit ribogenesis as well. ZKN-157 thus represents a new class of ribosome modulators that display cancer selectivity through specific ribosome inhibition in the CMS2 subtype of colorectal cancer potentially targeting MYC-driven addiction to high protein translation. Significance: This study demonstrates that ribosome heterogeneity in cancer can be exploited to develop selective ribogenesis inhibitors. The colorectal cancer CMS2 subtype, with a high unmet need for therapeutics, shows vulnerability to our novel selective ribosome modulator. The mechanism suggests that other cancer subtypes with high MYC activation could also be targeted.

Clinical and Prognostic Significance of the Eighth Edition Oral Cancer Staging System.

Objectives: The most notable changes in the eighth edition of the AJCC Cancer Staging System include incorporating the depth of invasion (DOI) into T staging and extranodal extension (ENE) into N staging. In this study, we retrospectively assessed the prognostic and clinical implications of the eighth TNM staging system. Materials and Methods: Patients with Oral Squamous Cell Carcinoma (OSCC) who were treated surgically between 2010 and 2017 were retrospectively reviewed. Tumors were first staged according to the seventh edition and restaged using the eighth edition. The prognostic value of the resultant upstaging was evaluated. Results: Integrating the DOI into the T classification resulted in the upstaging of 65 patients, whereas incorporating ENE into the N staging resulted in the upstaging of 18 patients (p < 0.001). Upstaging due to DOI integration had no significant effect on OS or DSS (p > 0.05). Conclusion: Our results demonstrate the importance of incorporating ENE into nodal staging and considering adjuvant therapy when ENE is present.

Tyrosine Phosphorylation Profiling Revealed the Signaling Network Characteristics of CAMKK2 in Gastric Adenocarcinoma.

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions via the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.

Precision health diagnostic and surveillance network uses S gene target failure (SGTF) combined with sequencing technologies to track emerging SARS-CoV-2 variants.

INTRODUCTION: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic revealed a worldwide lack of effective molecular surveillance networks at local, state, and national levels, which are essential to identify, monitor, and limit viral community spread. SARS-CoV-2 variants of concern (VOCs) such as Alpha and Omicron, which show increased transmissibility and immune evasion, rapidly became dominant VOCs worldwide. Our objective was to develop an evidenced-based genomic surveillance algorithm, combining reverse transcription polymerase chain reaction (RT-PCR) and sequencing technologies to quickly identify highly contagious VOCs, before cases accumulate exponentially. METHODS: Deidentified data were obtained from 508,969 patients tested for coronavirus disease 2019 (COVID-19) with the TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) in four CLIA-certified clinical laboratories in Puerto Rico (n = 86,639) and in three CLIA-certified clinical laboratories in the United States (n = 422,330). RESULTS: TaqPath data revealed a frequency of S Gene Target Failure (SGTF) > 47% for the last week of March 2021 in both, Puerto Rico and US laboratories. The monthly frequency of SGTF in Puerto Rico steadily increased exponentially from 4% in November 2020 to 47% in March 2021. The weekly SGTF rate in US samples was high (>8%) from late December to early January and then also increased exponentially through April (48%). The exponential increase in SGFT prevalence in Puerto Rico was concurrent with a sharp increase in VOCs among all SARS-CoV-2 sequences from Puerto Rico uploaded to Global Influenza Surveillance and Response System (GISAID) (n = 461). Alpha variant frequency increased from <1% in the last week of January 2021 to 51.5% of viral sequences from Puerto Rico collected in the last week of March 2021. CONCLUSIONS: According to the proposed evidence-based algorithm, approximately 50% of all SGTF patients should be managed with VOCs self-quarantine and contact tracing protocols, while WGS confirms their lineage in genomic surveillance laboratories. Our results suggest this workflow is useful for tracking VOCs with SGTF.

A robust and interpretable gene signature for predicting the lymph node status of primary T1/T2 oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC) affects more than 30 000 individuals in the United States annually, with smoking and alcohol consumption being the main risk factors. Management of early-stage tumors usually includes surgical resection followed by postoperative radiotherapy in certain cases. The cervical lymph nodes (LNs) are the most common site for local metastasis, and elective neck dissection is usually performed if the primary tumor thickness is greater than 3.5 mm. However, postoperative histological examination often reveals that many patients with early-stage disease are negative for neck nodal metastasis, posing a pressing need for improved risk stratification to either avoid overtreatment or prevent the disease progression. To this end, we aimed to identify a primary tumor gene signature that can accurately predict cervical LN metastasis in patients with early-stage OSCC. Using gene expression profiles from 189 samples, we trained K-top scoring pairs models and identified six gene pairs that can distinguish primary tumors with nodal metastasis from those without metastasis. The signature was further validated on an independent cohort of 35 patients using real-time polymerase chain reaction (PCR) in which it achieved an area under the receiver operating characteristic (ROC) curve and accuracy of 90% and 91%, respectively. These results indicate that such signature holds promise as a quick and cost effective method for detecting patients at high risk of developing cervical LN metastasis, and may be potentially used to guide the neck treatment regimen in early-stage OSCC.

Correction: Targeting focal adhesion kinase overcomes erlotinib resistance in smoke induced lung cancer by altering phosphorylation of epidermal growth factor receptor.

[This corrects the article DOI: 10.18632/oncoscience.395.].

Doublecortin-Like Kinase 1 (DCLK1) Is a Novel NOTCH Pathway Signaling Regulator in Head and Neck Squamous Cell Carcinoma.

Despite recent advancements, the 5 year survival of head and neck squamous cell carcinoma (HNSCC) hovers at 60%. DCLK1 has been shown to regulate epithelial-to-mesenchymal transition as well as serving as a cancer stem cell marker in colon, pancreatic and renal cancer. Although it was reported that DCLK1 is associated with poor prognosis in oropharyngeal cancers, very little is known about the molecular characterization of DCLK1 in HNSCC. In this study, we performed a comprehensive transcriptome-based computational analysis on hundreds of HNSCC patients from TCGA and GEO databases, and found that DCLK1 expression positively correlates with NOTCH signaling pathway activation. Since NOTCH signaling has a recognized role in HNSCC tumorigenesis, we next performed a series of in vitro experiments in a collection of HNSCC cell lines to investigate the role of DCLK1 in NOTCH pathway regulation. Our analyses revealed that DCLK1 inhibition, using either a pharmacological inhibitor or siRNA, resulted in substantially decreased proliferation, invasion, migration, and colony formation. Furthermore, these effects paralleled downregulation of active NOTCH1, and its downstream effectors, HEY1, HES1 and HES5, whereas overexpression of DCLK1 in normal keratinocytes, lead to an upregulation of NOTCH signaling associated with increased proliferation. Analysis of 233 primary and 40 recurrent HNSCC cancer biopsies revealed that high DCLK1 expression was associated with poor prognosis and showed a trend towards higher active NOTCH1 expression in tumors with elevated DCLK1. Our results demonstrate the novel role of DCLK1 as a regulator of NOTCH signaling network and suggest its potential as a therapeutic target in HNSCC.

Hyperactivation of MEK/ERK pathway by Ca2+ /calmodulin-dependent protein kinase kinase 2 promotes cellular proliferation by activating cyclin-dependent kinases and minichromosome maintenance protein in gastric cancer cells.

Although CAMKK2 is overexpressed in several cancers, its role and relevant downstream signaling pathways in gastric cancer (GC) are poorly understood. Treatment of AGS GC cells with a CAMKK2 inhibitor, STO-609, resulted in decreased cell proliferation, cell migration, invasion, colony-forming ability, and G1/S-phase arrest. Quantitative phosphoproteomics in AGS cells with the CAMKK2 inhibitor led to the identification of 9603 unique phosphosites mapping to 3120 proteins. We observed decreased phosphorylation of 1101 phosphopeptides (1.5-fold) corresponding to 752 proteins upon CAMKK2 inhibition. Bioinformatics analysis of hypo-phosphorylated proteins revealed enrichment of MAPK1/MAPK3 signaling. Kinase enrichment analysis of hypo-phosphorylated proteins using the X2K Web tool identified ERK1, cyclin-dependant kinase 1 (CDK1), and CDK2 as downstream substrates of CAMKK2. Moreover, inhibition of CAMKK2 and MEK1 resulted in decreased phosphorylation of ERK1, CDK1, MCM2, and MCM3. Immunofluorescence results were in concordance with our mass spectroscopy data and Western blot analysis results. Taken together, our data reveal the essential role of CAMKK2 in the pathobiology of GC through the activation of the MEK/ERK1 signaling cascade.

How to Achieve Therapeutic Response in Erlotinib-Resistant Head and Neck Squamous Cell Carcinoma? New Insights from Stable Isotope Labeling with Amino Acids in Cell Culture-Based Quantitative Tyrosine Phosphoproteomics.

Resistance to cancer chemotherapy is a major global health burden. Epidermal growth factor receptor (EGFR) is a proven therapeutic target for multiple cancers of epithelial origin. Despite its overexpression in >90% of head and neck squamous cell carcinoma (HNSCC) patients, tyrosine kinase inhibitors such as erlotinib have shown a modest response in clinical trials. Cellular heterogeneity is thought to play an important role in HNSCC therapeutic resistance. Genomic alterations alone cannot explain all resistance mechanisms at play in a heterogeneous system. It is thus important to understand the biochemical mechanisms associated with drug resistance to determine potential strategies to achieve clinical response. We investigated tyrosine kinase signaling networks in erlotinib-resistant cells using quantitative tyrosine phosphoproteomics approach. We observed altered phosphorylation of proteins involved in cell adhesion and motility in erlotinib-resistant cells. Bioinformatics analysis revealed enrichment of pathways related to regulation of the actin cytoskeleton, extracellular matrix (ECM)-receptor interaction, and endothelial migration. Of importance, enrichment of the focal adhesion kinase (PTK2) signaling pathway downstream of EGFR was also observed in erlotinib-resistant cells. To the best of our knowledge, we present the first report of tyrosine phosphoproteome profiling in erlotinib-resistant HNSCC, with an eye to inform new ways to achieve clinical response. Our findings suggest that common signaling networks are at play in driving resistance to EGFR-targeted therapies in HNSCC and other cancers. Most notably, our data suggest that the PTK2 pathway genes may potentially play a significant role in determining clinical response to erlotinib in HNSCC tumors.

Whole-Exome Sequencing Analysis of Oral Squamous Cell Carcinoma Delineated by Tobacco Usage Habits.

Oral squamous cell carcinoma (OSCC) is a common cancer of the oral cavity in India. Cigarette smoking and chewing tobacco are known risk factors associated with OSCC. However, genomic alterations in OSCC with varied tobacco consumption history are not well-characterized. In this study, we carried out whole-exome sequencing to characterize the mutational landscape of OSCC tumors from subjects with different tobacco consumption habits. We identified several frequently mutated genes, including TP53, NOTCH1, CASP8, RYR2, LRP2, CDKN2A, and ATM. TP53 and HRAS exhibited mutually exclusive mutation patterns. We identified recurrent amplifications in the 1q31, 7q35, 14q11, 22q11, and 22q13 regions and observed amplification of EGFR in 25% of samples with tobacco consumption history. We observed genomic alterations in several genes associated with PTK6 signaling. We observed alterations in clinically actionable targets including ERBB4, HRAS, EGFR, NOTCH1, NOTCH4, and NOTCH3. We observed enrichment of signature 29 in 40% of OSCC samples from tobacco chewers. Signature 15 associated with defective DNA mismatch repair was enriched in 80% of OSCC samples. NOTCH1 was mutated in 36% of samples and harbored truncating as well as missense variants. We observed copy number alterations in 67% of OSCC samples. Several genes associated with non-receptor tyrosine kinase signaling were affected in OSCC. These molecules can serve as potential candidates for therapeutic targeting in OSCC.

Molecular alterations in oral cancer between tobacco chewers and smokers using serum proteomics.

BACKGROUND: Tobacco exposure (through smoking or chewing) is one of the predominant risk factors associated with the development of oral squamous cell carcinoma (OSCC). Despite the growing number of patients diagnosed with OSCC, there are few circulating biomarkers for identifying individuals at a higher risk of developing the disease. Successful identification of candidate molecular markers for risk assessment could aid in the early detection of oral lesions and potentially be used for community screening of high-risk populations. OBJECTIVE: Identification of differentially expressed proteins in the serum of oral cancer patients which can serve as biomarkers for the diagnosis of the onset of oral cancer among tobacco users. METHODS: We employed a tandem mass tag (TMT)-based quantitative proteomics approach to study alterations in the serum proteomes of OSCC patients based on their tobacco exposure habits (chewing and smoking) compared to healthy individuals with no history of using any form of tobacco or any symptoms of the disease. RESULTS: Mass spectrometry-based analysis resulted in the identification of distinct signatures in the serum of OSCC patients who either chewed or smoked tobacco. Pathway analysis revealed opposing effects of dysregulated proteins enriched in the complement-coagulation signaling cascades with a high expression of the Serpin family of proteins observed in OSCC patients who chewed tobacco compared to healthy individuals whereas these proteins showed decreased levels in OSCC patients who smoked. ELISA-based validation further confirmed our findings revealing higher expression of SERPINA6 and SERPINF1 across serum of OSCC patients who chewed tobacco compared to healthy individuals. CONCLUSIONS: This study serves as a benchmark for the identification of serum-based protein markers that may aid in the identification of high-risk patients who either chew tobacco or smoke tobacco.

Proteomic and phosphoproteomic profiling of shammah induced signaling in oral keratinocytes.

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. Exposure to shammah has been linked with dental diseases and oral squamous cell carcinoma. There is limited literature on the prevalence of shammah and its role in pathobiology of oral cancer. In this study, we developed a cellular model to understand the effect of chronic shammah exposure on oral keratinocytes. Chronic exposure to shammah resulted in increased proliferation and invasiveness of non-transformed oral keratinocytes. Quantitative proteomics of shammah treated cells compared to untreated cells led to quantification of 4712 proteins of which 402 were found to be significantly altered. In addition, phosphoproteomics analysis of shammah treated cells compared to untreated revealed hyperphosphorylation of 36 proteins and hypophosphorylation of 83 proteins (twofold, p-value ≤ 0.05). Bioinformatics analysis of significantly altered proteins showed enrichment of proteins involved in extracellular matrix interactions, necroptosis and peroxisome mediated fatty acid oxidation. Kinase-Substrate Enrichment Analysis showed significant increase in activity of kinases such as ROCK1, RAF1, PRKCE and HIPK2 in shammah treated cells. These results provide better understanding of how shammah transforms non-neoplastic cells and warrants additional studies that may assist in improved early diagnosis and treatment of shammah induced oral cancer.

Molecular Profiling Associated with Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2)-Mediated Carcinogenesis in Gastric Cancer.

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related death worldwide. We showed previously that calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2), a serine-threonine kinase, is highly expressed in gastric cancer and leads to progression. In the present study, we identified the molecular networks involved in CAMKK2-mediated progression of gastric adenocarcinoma. Treatment of gastric cancer cell lines with a CAMKK2 inhibitor, STO-609, resulted in decreased cell migration, invasion, and colony-forming ability and a G1/S-phase arrest. In addition, tandem mass tag (TMT)-based quantitative proteomic analysis resulted in the identification of 7609 proteins, of which 219 proteins were found to be overexpressed and 718 downregulated (1.5-fold). Our data identified several key downregulated proteins involved in cell division and cell proliferation, which included DNA replication licensing factors, replication factor C, origin recognition complex, replication protein A and GINS, and mesenchymal markers, upon CAMKK2 inhibition. Immunoblotting and immunofluorescence results showed concordance with our mass spectroscopy data. Taken together, our study supports CAMKK2 as a novel therapeutic target in gastric cancer.

Proteomic Alterations Associated with Oral Cancer Patients with Tobacco Using Habits.

Tobacco abuse is a major risk factor associated with the development of oral squamous cell carcinoma. Differences in molecular aberrations induced by tobacco exposure by chewing or smoking form are not well studied in case of oral cancer. We used tandem mass tag-based quantitative proteomic approach to delineate proteomic alterations in oral cancer patients based on their history of tobacco using habits (patients who chewed tobacco, patients who smoked tobacco, and those with no history of tobacco consumption). Our data identified distinct dysregulation of biological processes and pathways in each patient cohort. Bioinformatics analysis of dysregulated proteins identified in our proteomic study revealed dysregulation of collagen formation and antigen processing/presentation pathway in oral cancer patients who smoked tobacco, whereas proteins associated with the process of keratinization showed enrichment in patients who chewed tobacco. In addition, we identified overexpression of proteins involved in immune pathways and downregulation of muscle contraction-mediated signaling events in all three cohorts, irrespective of tobacco using habits. This study lays the groundwork for identification of protein markers that may aid in identification of high-risk patients for cancer development based on the history of tobacco exposure habits.

Molecular alterations in oral cancer using high-throughput proteomic analysis of formalin-fixed paraffin-embedded tissue.

Loss of cell differentiation is a hallmark for the progression of oral squamous cell carcinoma (OSCC). Archival Formalin-Fixed Paraffin-Embedded (FFPE) tissues constitute a valuable resource for studying the differentiation of OSCC and can offer valuable insights into the process of tumor progression. In the current study, we performed LC-MS/MS-based quantitative proteomics of FFPE specimens from pathologically-confirmed well-differentiated, moderately-differentiated, and poorly-differentiated OSCC cases. The data were analyzed in four technical replicates, resulting in the identification of 2376 proteins. Of these, 141 and 109 were differentially expressed in moderately-differentiated and poorly differentiated OSCC cases, respectively, compared to well-differentiated OSCC. The data revealed significant metabolic reprogramming with respect to lipid metabolism and glycolysis with proteins belonging to both these processes downregulated in moderately-differentiated OSCC when compared to well-differentiated OSCC. Signaling pathway analysis indicated the alteration of extracellular matrix organization, muscle contraction, and glucose metabolism pathways across tumor grades. The extracellular matrix organization pathway was upregulated in moderately-differentiated OSCC and downregulated in poorly differentiated OSCC, compared to well-differentiated OSCC. PADI4, an epigenetic enzyme transcriptional regulator, and its transcriptional target HIST1H1B were both found to be upregulated in moderately differentiated and poorly differentiated OSCC, indicating epigenetic events underlying tumor differentiation. In conclusion, the findings support the advantage of using high-resolution mass spectrometry-based FFPE archival blocks for clinical and translational research. The candidate signaling pathways identified in the study could be used to develop potential therapeutic targets for OSCC.

Signaling alterations in oral keratinocytes in response to shisha and crude tobacco extract.

BACKGROUND: Tobacco consumption in smoking and non-smoking forms has been consequential in the rise of oral cancer cases. Among different forms, epidemiological studies from Middle Eastern countries and rural parts of northern India have reported increasing association of oral cancer with waterpipe (hookah) smoking. However, molecular mechanisms and role played by waterpipe smoking in the onset of oral carcinogenesis remains unexplored. METHODS: In this study, immortalized normal human oral keratinocytes were chronically treated with extracts of two varieties of waterpipe tobacco-crude tobacco and processed shisha. Phenotypic changes and molecular aberrations were examined using cell culture-based assays and mass spectrometry-based quantitative proteomic analysis, respectively. Bioinformatics analysis was utilized to analyze proteomics data and identify dysregulated pathways. RESULTS: Our data indicate that chronic treatment with waterpipe tobacco extracts increased proliferation, invasion, migration, and significant dysregulation of protein expression in oral keratinocytes. Altered expression of proteins involved in interferon signaling pathway were observed with both varieties of tobacco. Overexpression of cholesterol metabolism and vesicle-mediated transport proteins were identified exclusively in cells treated with crude tobacco extract. Bioinformatics analyses revealed different oncogenic response in oral cells based on the type of waterpipe tobacco used. CONCLUSIONS: This study may serve as a useful resource in understanding the early onset of oral cancer attributed to waterpipe smoking.

Multi-Omics Analysis to Characterize Cigarette Smoke Induced Molecular Alterations in Esophageal Cells.

Though smoking remains one of the established risk factors of esophageal squamous cell carcinoma, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. To investigate molecular alterations associated with chronic exposure to cigarette smoke, non-neoplastic human esophageal epithelial cells were treated with cigarette smoke condensate (CSC) for up to 8 months. Chronic treatment with CSC increased cell proliferation and invasive ability of non-neoplastic esophageal cells. Whole exome sequence analysis of CSC treated cells revealed several mutations and copy number variations. This included loss of high mobility group nucleosomal binding domain 2 (HMGN2) and a missense variant in mediator complex subunit 1 (MED1). Both these genes play an important role in DNA repair. Global proteomic and phosphoproteomic profiling of CSC treated cells lead to the identification of 38 differentially expressed and 171 differentially phosphorylated proteins. Bioinformatics analysis of differentially expressed proteins and phosphoproteins revealed that most of these proteins are associated with DNA damage response pathway. Proteomics data revealed decreased expression of HMGN2 and hypophosphorylation of MED1. Exogenous expression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of smoke exposed cells. Immunohistochemical labeling of HMGN2 in primary ESCC tumor tissue sections (from smokers) showed no detectable expression while strong to moderate staining of HMGN2 was observed in normal esophageal tissues. Our data suggests that cigarette smoke perturbs expression of proteins associated with DNA damage response pathways which might play a vital role in development of ESCC.