Event In Carcinogenesis Research Articles

Cell of Origin Influences Pancreatic Cancer Subtype.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a 5-year survival rate of approximately 9%. An improved understanding of PDAC initiation and progression is paramount for discovering strategies to better detect and combat this disease. Although transcriptomic analyses have uncovered distinct molecular subtypes of human PDAC, the factors that influence subtype development remain unclear. Here, we interrogate the impact of cell of origin and different Trp53 alleles on tumor evolution, using a panel of tractable genetically engineered mouse models. Oncogenic KRAS expression, coupled with Trp53 deletion or point mutation, drives PDAC from both acinar and ductal cells. Gene-expression analysis reveals further that ductal cell-derived and acinar cell-derived tumor signatures are enriched in basal-like and classical subtypes of human PDAC, respectively. These findings highlight cell of origin as one factor that influences PDAC molecular subtypes and provide insight into the fundamental impact that the very earliest events in carcinogenesis can have on cancer evolution. SIGNIFICANCE: Although human PDAC has been classified into different molecular subtypes, the etiology of these distinct subtypes remains unclear. Using mouse genetics, we reveal that cell of origin is an important determinant of PDAC molecular subtype. Deciphering the biology underlying pancreatic cancer subtypes may reveal meaningful distinctions that could improve clinical intervention.This article is highlighted in the In This Issue feature, p. 521.

Genotoxicity, oxidative stress, and inflammatory response induced by crack-cocaine: relevance to carcinogenesis.

Crack-cocaine is a cocaine by-product widely consumed by general population in developing countries. The drug is low cost and is associated with more intense effects when compared to other illicit drugs. Genotoxicity, oxidative stress, and inflammatory response are considered crucial events in carcinogenesis, since they actively participate in the multistep process. The purpose of this paper was to provide a mini review regarding the relationship between carcinogenesis and genotoxicity, oxidative stress, and inflammation induced by crack-cocaine. The present study was conducted on search of the scientific literature from the published studies available in PubMed, MEDLINE, Scopus, and Google Scholar for all kind of articles (all publications to November 2020) using the following key words: crack-cocaine, DNA damage, genotoxicity, cellular death, cytotoxicity, mutation, oxidative stress, inflammation, and mutagenicity. The results showed that published papers available were almost all in vivo test system being conducted in humans or rodents. Crack-cocaine was able to induce genotoxicity and oxidative stress in mammalian cells. However, the role of inflammatory response after exposure to crack-cocaine was not conclusive so far. In summary, this study is consistent with the notion that crack-cocaine is a chemical carcinogen as a result of genotoxicity and oxidative stress induced in mammalian and non-mammalian cells.

Mechanisms of DNA Abnormal Methylation in the Development of Lung Cancer in Rats

In order to study the mechanism of abnormal deoxyribonucleic acid methylation in the process of lung cancer in rats, Wistar rats are used as the research object. The lung cancer model of rats is established by bronchial perfusion of carcinogens. Hematoxylin-eosin staining is used to observe the pathological changes of lung tissue in rats. The overall methylation level of genome is analyzed by immunohistochemistry, and the methylation status is detected by methylation specific polymerase chain reaction. The results show that the nuclei of normal bronchial epithelial cells are brown and yellow uder optical microscope. With the development of carcinogenesis, the staining gradually fades to light yellow in the infiltrating nuclei of cancer cells. Compared with normal bronchial epithelial cells, the optical density and integrated optical density values of 5-mC immunohistochemistry decrease. The methylation frequency of p16 gene in atypical hyperplasia, carcinoma in situ and invasive cancer tissues is significantly higher than that in normal and proliferative tissues. The methylation frequency of p57 gene is significantly higher in carcinoma in situ and invasive tissues than in normal and proliferative tissues. Abnormal methylation-mediated down-regulation of p16 and p57 gene transcription may be an important molecular biological event in carcinogenesis of lung cancer rats induced by carcinogens. There are still some shortcomings in the research process, but the results of the study still provide some reference and guidance for exploring more precise mechanism in the future. Therefore, this study is a significant research topic.

Promoter CpG island hypermethylation and down regulation of XRCC1 gene can augment in the gastric carcinogenesis events.

Gastric cancer (GC) is a multistep process characterized by a gradual accumulation of genetic and epigenetic alterations in genes at various stages of progression. Epigenetic alterations like DNA methylation play an important role in cancer and may serve as a biomarker for cancer. The present study was aimed to investigate the promoter hypermethylation, expression profile, and Arg399Gln gene polymorphism of DNA repair gene XRCC1 (X-ray repair cross complimentary group I) in GC patients. A total of 60 histopathologically confirmed GC subjects were recruited in the study. Information on various dietary, lifestyle and environmental factors was obtained in face-to-face interviews using a structured questionnaire from each subject. Tissue samples were taken along with adjacent non-cancerous tissues for analysis. Promoter methylation status and expression of XRCC1 gene was evaluated using MS-PCR and western blotting respectively; while as Arg399Gln polymorphism was analyzed by PCR-RFLP. We found that the XRCC1 gene promoter of 38.3% cancerous tissues were methylated compared to 13.3% of adjacent normal tissues. The promoter hypermethylation status of the gene was found to be significantly associated with the loss of protein expression (P < 0.0001, OR = 14.63; 95% CI 4.01-53.43). However, we did not find any significant association of polymorphism of XRCC1 Arg399Gln with promoter methylation or protein expression. Further, comparison of methylation status and protein expression with clinical parameters like age, smoking status, etc. was also not significant (P > 0.05). The present study indicates that XRCC1 undergoes aberrant promoter hypermethylation with subsequent loss of protein expression in gastric cancer.

Integrated differential DNA methylation and gene expression of formalin-fixed paraffin-embedded uveal melanoma specimens identifies genes associated with early metastasis and poor prognosis

PurposeUveal melanoma (UM) is an aggressive malignancy, in which nearly 50% of the patients die from metastatic disease. Aberrant DNA methylation is recognized as an important epigenomic event in carcinogenesis. Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable source of tumor tissue, and recent technology has enabled the use of these samples in genome-wide DNA methylation analyses. Our aim was to investigate differential DNA methylation in relation to histopathological classification and survival data. In addition we sought to identify aberrant DNA methylation of genes that could be associated with metastatic disease and poor survival. MethodsFFPE samples from UM patients (n = 23) who underwent enucleation of the eye in the period 1976–1989 were included. DNA methylation was assessed using the Illumina Infinium HumanMethylation450 array and coupled to histopathological data, Cancer Registry of Norway- (registered UM metastasis) and Norwegian Cause of Death Registry- (time and cause of death) data. Differential DNA methylation patterns contrasting histological classification, survival data and clustering properties were investigated. Survival groups were defined as “Early metastasis” (metastases and death within 2–5 years after enucleation, n = 8), “Late metastasis” (metastases and death within 9–21 years after enucleation, n = 7) and “No metastasis” (no detected metastases ≥18 years after enucleation, n = 8). A subset of samples were selected based on preliminary multi-dimensional scaling (MDS) plots, histopathological classification, chromosome 3 status, survival status and clustering properties; “Subset Early metastasis” (n = 4) vs “Subset No metastasis” (n = 4). Bioinformatics analyses were conducted in the R statistical software. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) in various comparisons were assessed. Gene expression of relevant subgroups was determined by microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). ResultsDNA methylation analyses identified 2 clusters that separated the samples according to chromosome 3 status. Cluster 1 consisted of samples (n = 5) with chromosome 3 disomy (D3), while Cluster 2 was comprised of samples (n = 15) with chromosome 3 monosomy (M3). 1212 DMRs and 9386 DMPs were identified in M3 vs D3. No clear clusters were formed based on our predefined survival groups (“Early”, “Late”, “No”) nor histopathological classification (Epithelioid, Mixed, Spindle). We identified significant changes in DNA methylation (beta FC ≥ 0.2, adjusted p < 0.05) between two sample subsets (n = 8). “Subset Early metastasis” (n = 4) vs “Subset No metastasis” (n = 4) identified 348 DMPs and 36 DMRs, and their differential gene expression by microarray showed that 14 DMPs and 2 DMRs corresponded to changes in gene expression (FC ≥ 1.5, p < 0.05). RNF13, ZNF217 and HYAL1 were hypermethylated and downregulated in “Subset Early metastasis” vs “Subset No metastasis” and could be potential tumor suppressors. TMEM200C, RGS10, ADAM12 and PAM were hypomethylated and upregulated in “Subset Early metastasis vs “Subset No metastasis” and could be potential oncogenes and thus markers of early metastasis and poor prognosis in UM. ConclusionsDNA methylation profiling showed differential clustering of samples according to chromosome 3 status: Cluster 1 (D3) and Cluster 2 (M3). Integrated differential DNA methylation and gene expression of two subsets of samples identified genes associated with early metastasis and poor prognosis. RNF13, ZNF217 and HYAL1 are hypermethylated and candidate tumor suppressors, while TMEM200C, RGS10, ADAM12 and PAM are hypomethylated and candidate oncogenes linked to early metastasis. UM FFPE samples represent a valuable source for methylome studies and enable long-time follow-up.

Immunohistochemical expression of Tazarotene-induced Gene 3 in oral squamous cell carcinoma.

The prognosis of hyperproliferative skin lesions, such as psoriasis, basal cell carcinoma, and non-melanoma skin cancers, is significantly benefited from the levels of tazarotene-induced gene-1 (TIG3) expression and subsequent treatment with tazarotene. Such observations suggest that TIG3 could be used as a biomarker for apoptosis, differentiation, and proliferation. The current study aimed to evaluate the expression of TIG3 in normal oral mucosa (NOM) and oral squamous cell carcinoma (OSCC) compared with normal skin (NS) and skin squamous cell carcinoma (SSCC) using immunohistochemistry. Seventeen cases each of SSCC, OSCC, NOM, and NS were evaluated. Each section was immunohistochemically stained with a rabbit polyclonal TIG3 antibody. The entire procedure was blinded and evaluated by 5 observers. Statistical analysis was performed using the chi-square test. There was a significant decrease in TIG3 protein expression in OSCC and SSCC compared with that in NOM and NS (P=0.008). The progressive loss of expression was observed as the grade of both malignancies increased. However, there was no significant difference in the expression among the normal tissue groups and within SCC groups of similar grades. The present study suggests that the loss of TIG3 is an important event in carcinogenesis. TIG3 acts as a regulator of keratinocyte proliferation and terminal differentiation. Therefore, TIG3 could be a potential biomarker to differentiate aggressive and non-aggressive neoplasms.

Rapid and ultrasensitive detection of circulating human papillomavirus E7 cell-free DNA as a cervical cancer biomarker.

Circulating cell-free DNA (cfDNA) has attracted attention as a non-invasive biomarker for diagnosing and monitoring various cancers. Given that human papillomavirus (HPV) DNA integration and overexpression of E6/E7 oncogenes are pivotal events for carcinogenesis, we sought to determine if HPV E7 cfDNA could serve as a specific biomarker for cervical cancer detection. We applied droplet digital PCR (ddPCR) to quantify HPV16/18 E7 cfDNA from the serum of patients with cervical cancer, cervical intraepithelial neoplasia, and controls. HPV16/18 E7 cfDNA was highly specific for cervical cancer, displaying 30.77% sensitivity, 100% specificity, and an area under the curve of 0.65. Furthermore, we developed a sensitive isothermal detection of HPV16/18 E7 and the PIK3CA WT reference gene based on recombinase polymerase amplification combined with a lateral flow strip (RPA-LF). The assay took less than 30 min and the detection limit was 5-10 copies. RPA-LF exhibited 100% sensitivity and 88.24% specificity towards HPV16/18 E7 cfDNA in clinical samples. The agreement between RPA-LF and ddPCR was 83.33% (κ = 0.67) for HPV16 E7 and 100% (κ = 1.0) for HPV18 E7, indicating a good correlation between both tests. Therefore, we conclude that HPV E7 cfDNA represents a potential tumor marker with excellent specificity and moderate sensitivity for minimally invasive cervical cancer monitoring. Moreover, the RPA-LF assay provides an affordable, rapid, and ultrasensitive tool for detecting HPV cfDNA in resource-limited settings.

Epigenetic regulation of cancer stem cell formation and maintenance.

Cancerous tumours contain a rare subset of cells with stem‐like properties that are termed cancer stem cells (CSCs). CSCs are defined by their ability to divide both symmetrically and asymmetrically, to initiate new tumour growth and to tolerate the foreign niches required for metastatic dissemination. Accumulating evidence suggests that tumours arise from cells with stem‐like properties, the generation of CSCs is therefore likely to be an initiatory event in carcinogenesis. Furthermore, CSCs in established tumours exist in a dynamic and plastic state, with nonstem tumour cells thought to be capable of de‐differentiation to CSCs. The regulation of the CSC state both during tumour initiation and within established tumours is a desirable therapeutic target and is mediated by epigenetic factors. In this review, we will explore the epigenetic parallels between induced pluripotency and the generation of CSCs, and discuss how the epigenetic regulation of CSCs opens up novel opportunities for therapeutic intervention.

Mitotic Spindle Apparatus Abnormalities in Chronic Obstructive Pulmonary Disease Cells: A Potential Pathway to Lung Cancer.

Chronic obstructive pulmonary disease (COPD) is a long-term lung disease characterized by irreversible lung damage resulting in airflow limitation, abnormal permanent air-space enlargement, and emphysema. Cigarette smoking is the major cause of COPD with 15% to 30% of smokers developing either disease. About 50% to 80% of patients with lung cancer have preexisting COPD and smokers who have COPD are at an increased risk for developing lung cancer. Therefore, COPD is considered an independent risk for lung cancer, even after adjusting for smoking. A crucial early event in carcinogenesis is the induction of the genomic instability through alterations in the mitotic spindle apparatus. To date, the underlying mechanism by which COPD contributes to lung cancer risk is unclear. We hypothesized that tobacco smoke carcinogens induce mitotic spindle apparatus abnormalities and alter expression of crucial genes leading to increased genomic instability and ultimately tumorigenesis. To test our hypothesis, we assessed the genotoxic effects of a potent tobacco-smoke carcinogen [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK)] on bronchial epithelial cells from patients with COPD and normal bronchial epithelial cells and identified genes associated with mitotic spindle defects and chromosome missegregation that also overlap with lung cancer. Our results indicate that exposure to NNK leads to a significantly altered spindle orientation, centrosome amplification, and chromosome misalignment in COPD cells as compared with normal epithelial cells. In addition, we identified several genes (such as AURKA, AURKB, and MAD2L2) that were upregulated and overlap with lung cancer suggesting a potential common pathway in the transition from COPD to lung cancer.

Effects of Gut Microbiome on Carcinogenic DNA Damage.

The human intestine is host to a vast microbial community: the gut microbiome (GM). The GM has been considered as a key modulator of human health in the past decade. In particular, several studies have supported that altered GM is associated with cancer, such as colorectal cancer, adenocarcinoma, and pancreatic cancer. The formation of a DNA adduct is one of the key events in carcinogenesis, and whether GM can influence DNA adducts has yet to be examined. This study analyzed 10 DNA adducts (N2-Me-dG, N6-Me-dA, N2-Et-dG, OH-Me-dG, OH-Me-dA, N2-EtD-dG, O6-Me-dG, 1,N2-ε-dG, 8-oxo-dG, and 5-Cl-dC), attributed to various endogenous processes and physiological stressors, using highly sensitive LC-MS/MS in germ-free (GF) and conventionally raised (CONV-R) mice. Our results showed that significant differences in specific DNA adducts appeared in liver, colon, and small intestine samples between GF and CONV-R mice. The differences in adduct levels may indicate that GM can locally or systemically regulate endogenous processes including neutrophil bactericidal activity (represented by 5-Cl-dC), lipid peroxidation (1,N2-ε-dG), oxidative stress generation (8-oxo-dG), and endogenous aldehyde metabolism (OH-Me-dA). Further studies are warranted to elucidate how the GM influences endogenous process, DNA damage, and the risks of developing cancer.

EMMPRIN/BASIGIN as a biological modulator of oral cancer and COVID-19 interaction: Novel propositions

Extracellular matrix metalloproteinase inducer (EMMPRIN), which is also called BASIGIN/CD147, is a cell surface glycoprotein that belongs to the immunoglobulin superfamily and plays a significant role in intercellular recognition in immunology, cellular differentiation and development. Apart from ACE-2, recently EMMPRIN, has been regarded as a target for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attachment and entry into the host cell. Since one of the routes of entry for the virus is the oral cavity, it becomes imperative to percept oral comorbidities such oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs) in terms of EMMPRIN as a target for SARS-CoV-2. In the present paper, it is proposed that OSCC, by the virtue of upregulation of EMMPRIN expression, increases the susceptibility to coronavirus disease (COVID-19). In turn, COVID-19 in OSCC patients causes exhaustion of EMMPRIN receptor due to binding with ‘S’ receptor leading to a downregulation of related carcinogenesis events. We proposed that in the ACE-2 depleted situation in OSCC, EMMPRIN receptor might get high jacked by the COVID-19 virus for the entry into the host cells. Apart from the anti-monoclonal antibody, it is recommended to explore the use of grape seed and skin containing mouthwash as an adjunct, which could also have anti EMMPRIN effects in patients with OSCC and OPMDs.

Abstract B29: Ovarian hormones regulate C/EBPD induced EMT/MET transition in the human fallopian tube epithelia

Abstract Introduction: The histologically normal BRCA1 mutation carrier fallopian tube epithelia (FTE), compared to controls, showed that CEBPD was upregulated in the luteal phase of the ovulatory cycle. CEBPD is involved with the maintenance of genomic stability, promoting cellular differentiation, and regulating the cell cycle in response to cytotoxic stressors. In breast epithelial cells, CEBPD protein expression correlated with estrogen receptor (ER) and progesterone receptor (PR) and was found to be associated with increased progression-free survival in breast cancer patients. In fallopian tube epithelia (FTE), CEBPD was also found to modulate the epithelial-to-mesenchymal (EMT)/mesenchymal-to-epithelial transition (MET) by modulating target genes of this pathway. Given the hormonal response of this gene and its function in modulating an EMT/MET, the objective of this study was to determine whether sex hormones influence CEBPD regulation of EMT/MET in the fallopian tube and thus ovarian cancer. Methods: Fresh fallopian tube (FTE) tissues were obtained from patients approved for collection by IRB. Immunohistochemical profiling on normal fallopian tube tissue and HGSC was performed using CEBPD, ER, and PR protein markers. FTE cell lines with a p53 mutation (R175H) were subjected to estradiol (50nM and 100nM) and 4-hydroxytamoxifen (10nM) and assayed using qRT-PCR and PCR. ANOVA and t-tests were conducted in GraphPad Prism software with significance set at p&amp;lt;0.05. Results: E-cadherin in normal fallopian tube tissue was highly expressed in both the luteal and follicular phase whereas vimentin was highly expressed in the follicular phase but showed a range of expression (low expression to high expression) in the luteal phase. CEBPD overexpression increased SNAIL expression (p&amp;lt;0.0001), consistent with previous findings; treatment with 50nM estradiol (E2) resulted in increased SNAIL and SLUG mRNA expression in FTE cell lines overexpressing CEBPD (p&amp;lt;0.0001) relative to controls and decreased ZEB1 and ZEB2 mRNA expression. Addition of tamoxifen to cells overexpressing CEBPD increased SNAIL mRNA expression compared to cells without tamoxifen (p&amp;lt;0.0001); however, a combination of both tamoxifen and estradiol added to these cells decreased SNAIL expression relative to controls. IL6 mRNA expression level was increased in CEBPD overexpressing cells compared to controls (p&amp;lt;0.0001), which was further increased by E2 (p&amp;lt;0.0001). Conclusion: Together these results demonstrate a role for CEBPD in modulating EMT/MET in normal FTE in a hormonally regulated manner, which during cancer formation and spread is critical for anoikis and metastasis. Furthermore, these data will facilitate an understanding of the early events of carcinogenesis in fallopian tube epithelia. Citation Format: Ramlogan Sowamber, Leah V. Dodds, Patricia Shaw, Sophia H.L. George. Ovarian hormones regulate C/EBPD induced EMT/MET transition in the human fallopian tube epithelia [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B29.

Tumor DNA hypomethylation of LINE-1 is associated with low tumor grade of breast cancer in Tunisian patients.

DNA hypomethylation of long interspersed repetitive DNA retrotransposon (LINE-1) and Alu repeats elements of short interspersed elements family (SINEs) is an early event in carcinogenesis that causes transcriptional activation and leads to chromosomal instability. In the current study, DNA methylation levels of LINE-1 and Alu repeats were analyzed in tumoral tissues of invasive breast cancer in a Tunisian cohort and its association with the clinicopathological features of patients was defined. DNA methylation of LINE-1 and Alu repeats were analyzed using pyrosequencing in 61 invasive breast cancers. Median values observed for DNA methylation of LINE-1 and Alu repeats were considered as the cut-off (59.81 and 18.49%, respectively). The results of the current study demonstrated a positive correlation between DNA methylation levels of LINE-1 and Alu repeats (rho=0.284; P<0.03). DNA hypomethylation of LINE-1 was also indicated to be associated with low grade (P=0.023). To the best of our knowledge, the current study is the first study regarding DNA methylation of LINE-1 and Alu repeats element in breast cancer of the Tunisian population. The results of the current study suggest that, since hypomethylation of LINE-1 is associated with low grade, it could be used as a biomarker for prognosis for patients with breast cancer.

Immunohistochemical Expression of BubR1 and Telomerase in Oral Squamous Cell Carcinoma

Objective: Aberrant BubR1 and Telomerase expressions are considered as important markers in tumor progression and clinical outcome. This study purposed to describe, compare, and correlate the immunohistochemical expression of BubR1 and Telomerase in oral squamous cell carcinoma (OSCC).&#x0D; Methods: Immunohistochemical staining was performed for 28 OSCC samples. The samples graded according to Bryne’s grading system. The reaction positivity, intracellular localization, and intensity were recorded. The expression distribution and mean-ranks of these markers were related to OSCC grades by Fisher’s exact and Kruskal Wallis tests. Finally, the correlation between them was achieved by Spearman’s rho test.&#x0D; Results: BubR1 was detected in 89.3% of OSCCs, equally within scores 2 and 3 (35.7%), presented with 50% faint intensity, and 64.3% cytoplasmic localization. Grade III had a significantly higher mean-rank of BubR1 scoring. Whereas Telomerase observed in 75% of OSCCs, mainly found at scores 2 and 3 (60.7%), and 42.9% was within the cytoplasm. Half of OSCCs had faint intensity. No significant differences reported in the mean-rank and stain intensity among the grades. There was no significant correlation between BubR1 and Telomerase expression.&#x0D; Conclusions: OSCCs had high cytoplasmic BubR1 and telomerase expression that cannot ensure proper function. Mean-rank of BubR1 expression related to OSCC grading and being more in high grade. It determines the loss of differentiation and aggressiveness of OSCC. While the mean-rank of Telomerase did not relate to histopathological grading and considered an early event in carcinogenesis, it can use for the diagnostic approach of oral carcinogenesis. These markers have an independent role in cancer progression.

Continuum Models of Membrane Fusion: Evolution of the Theory.

Starting from fertilization, through tissue growth, hormone secretion, synaptic transmission, and sometimes morbid events of carcinogenesis and viral infections, membrane fusion regulates the whole life of high organisms. Despite that, a lot of fusion processes still lack well-established models and even a list of main actors. A merger of membranes requires their topological rearrangements controlled by elastic properties of a lipid bilayer. That is why continuum models based on theories of membrane elasticity are actively applied for the construction of physical models of membrane fusion. Started from the view on the membrane as a structureless film with postulated geometry of fusion intermediates, they developed along with experimental and computational techniques to a powerful tool for prediction of the whole process with molecular accuracy. In the present review, focusing on fusion processes occurring in eukaryotic cells, we scrutinize the history of these models, their evolution and complication, as well as open questions and remaining theoretical problems. We show that modern approaches in this field allow continuum models of membrane fusion to stand shoulder to shoulder with molecular dynamics simulations, and provide the deepest understanding of this process in multiple biological systems.

Methylated DNA markers for plasma detection of ovarian cancer: Discovery, validation, and clinical feasibility.

6072 Background: Effective screening tests for ovarian cancer (OC) are lacking; most cases present at advanced stage and portend poor prognosis. DNA methylation is an early event in carcinogenesis and can be detected in blood plasma samples from cancer patients. In DNA extracted from tissues, we first discovered, then validated discriminant methylated DNA marker (MDM) candidates for OC and subsequently tested independent plasma from women with and without OC. Methods: For discovery, DNA from 67 frozen tissues (18 high grade serous (HGS), 18 endometrioid, 15 clear cell (CC), 6 mucinous OCs; 10 benign fallopian tube epithelium (FT); and 19 buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify MDMs associated with OC. Candidate MDM selection was based on receiver operating characteristic (ROC) discrimination, methylation fold change, and low background methylation among controls. Blinded biological validation was performed using methylated specific PCR on DNA extracted from independent FFPE tissues from OCs (36 HGS, 22 endometrioid, 21 CC, and 14 mucinous) and 29 FT. Top performing MDMs in tissue were tested using long-probe quantitative amplified signal assays in independent pre-treatment plasma samples from women newly-diagnosed with OC and population-sampled healthy women. A random forest modeling analysis was performed to generate predictive probability of disease; results were 500-fold in silico cross-validated. Results: After RRBS discovery and biological validation, 33 MDMs showed marked methylation fold changes (10 to &gt; 1000) across all OC histologies vs FT. The top 11 MDMs ( GPRIN1, CDO1, SRC, SIM2, AGRN, FAIM2, CELF2, DSCR6, GYPC, CAPN2, BCAT1) were tested on plasma from 91 women with OC (76 (84%) HGS) and 91 without OC; the cross-validated 11-MDM panel highly discriminated OC from controls (96% (95%CI 89-99%) specificity; 79% (69-87%) sensitivity, and AUC 0.91 (0.86 - 0.96)). Among HGS, the panel correctly identified 83%, including 5/6 stage I/II, and the majority of other subtypes (Table). Conclusions: Whole methylome sequencing, stringent filtering criteria, and biological validation yielded outstanding candidate MDMs for OC that performed with promisingly high sensitivity and specificity in plasma. Larger plasma-based OC MDM testing studies, with larger numbers of non-HGS histologies are warranted. [Table: see text]

An organoid-based carcinogenesis model induced by in vitro chemical treatment.

Animal carcinogenesis models induced by environmental chemicals have been widely used for basic and applied cancer research. However, establishment of in vitro or ex vivo models is essential for molecular mechanistic elucidation of early events in carcinogenesis, leading to clarification of the total mode of action. In the present study, to establish an organoid-based chemical carcinogenesis model, mouse organoids were treated in vitro with 4 genotoxic chemicals, e.g. ethyl methanesulfonate (EMS), acrylamide (AA), diethylnitrosamine (DEN) and 7,12-dimethylbenz[a]anthracene (DMBA) to examine their tumorigenicity after injection to nude mice. The four chemicals were reported to induce lung, liver or mammary carcinomas in mouse models. DMBA-treated mammary tissue-derived organoids with Trp53 heterozygous knockout exhibited tumorigenicity, but not those with wild-type Trp53, reflecting previous reports of corresponding animal models. Treatment of lung organoids with or without Trp53 knockout with EMS or AA resulted in carcinogenic histopathological characteristics, and the activation of oncogenic kinases was demonstrated in the nodules from the nude mouse subcutis. DEN-treated liver (biliary tract) organoids also had an increased number of similar changes. In conclusion, an ex vivo model for chemical carcinogenesis was established using normal mouse tissue-derived organoids. This model will be applied to detect early molecular events, leading to clarification of the mode of action of chemical carcinogenesis.

Association of the TP53 Arg72Pro Polymorphism with Oral Squamous Cell Carcinoma: A Meta-Analysis

Background: The loss of function in TP53 gene is an early event of carcinogenesis and is now considered as a full etiological factor in oral squamous cell carcinoma (OSCC). The most common polymorphism in this gene that is associated to OSCC and has been extensively studied as a potential risk factor for the development of malignancies is the single nucleotide polymorphism (SNP) encoding either proline or arginine at residue 72. Today, despite the multiplicity of publications and approaches used, the contradiction of the results have not remove the ambiguity of the possible impact of this polymorphism in OSCC. So we aimed this meta-analysis to investigate the distribution of TP53 codon 72 genotypes and alleles in patients with OSCC and healthy matched controls, by using an ethnicity subgroups analysis. Method: A literature search was conducted to identify studies concerning TP53 codon 72 polymorphism and OSCC risk. Retrieved publications from 2000 to 2014 were classified by ethnicity, according to the sampling collection continent. Allelic frequencies were estimated by using genotypic data and Hardy-Weinberg Equilibrium and distributions were checked with Chi-2 test. Statistical tests for contrast models of association were performed with Proline allele as reference stratum. Results: Arg allele distribution was almost similar in both cases and controls for African and Caucasian populations whereas Arg frequency was significantly greater in cases than in controls for Asians (p=0.011). After stratified statistical analyses, we’ve found again a significant association between Arginine allele and OSCC risk in the Asian subgroup (OR=1.31; 95% CI=1.09-1.58; P=0.004). Conclusion: This current study revealed that allelic distribution of TP53 Arg72Pro polymorphism may depend on ethnicity and latitude, and highlighted that Arginine carrier in Asian populations may be considered to be predisposed to OSCC.

P16 AND MGMT GENE METHYLATION IN CHRONIC MECHANICAL IRRITATION OF THE ORAL MUCOSA

Objective Chronic mechanical irritation (CMI) has been proposed as a risk factor for oral cancer. Epigenetic alterations, particularly methylation, have been mentioned as early events in carcinogenesis, and it has been proposed that CMI could induce them. Thus, the aim of this study is to describe p16 and MGMT gene methylation in a specific CMI lesion: chronic traumatic ulcer (CTU). Study Design A split-mouth design was used, and 2 samples per individual were taken using cytobrush: the CTU lesion and clinically normal mucosa of a contralateral site. After extracting DNA, p16 protein and MGMT gene methylation status were assessed using quantitative polymerase chain reaction (PCR). The statistical analysis was performed by McNemar test. Results Twenty-seven patients were studied with a mean age of 59.1 years. CTU evolution time showed an average of 18.4 months. On CTU, methylation of both p16 and MGMT presented a significant difference (P Conclusions CMI-affected sites showed consistently more methylation of p16 and MGMT than mucosa devoid of CMI. Since in each case both control and study samples were from the same individual, effects of confounders were reduced. This study suggests that CMI could foster carcinogenesis through epigenetic alterations.

Haploinsufficient tumor suppressor Tip60 negatively regulates oncogenic Aurora B kinase

The Aurora kinases represent a group of serine/threonine kinases which are crucial regulators of mitosis. Dysregulated Aurora kinase B (AurkB) expression, stemming from genomic amplification, increased gene transcription or overexpression of its allosteric activators, is capable of initiating and sustaining malignant phenotypes. Although AurkB level in cells is well-orchestrated, studies that relate to its stability or activity, independent of mitosis, are lacking. We report that AurkB undergoes acetylation in vitro by lysine acetyltransferases (KATs) belonging to different families, namely by p300 and Tip60. The haploinsufficient tumor suppressor Tip60 acetylates two highly conserved lysine residues within the kinase domain of AurkB which not only impinges the protein stability but also its kinase activity. These results signify a probable outcome on the increase in "overall activity" of AurkB upon Tip60 downregulation, as observed under cancerous conditions. The present work, therefore, uncovers an important functional interplay between AurkB and Tip60, frailty of which may be an initial event in carcinogenesis.