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Tumor heterogeneity is a major challenge for cancer treatment, especially due to the presence of various subpopulations with stem cell or progenitor cell properties. In mouse melanomas, both CD34+p75− (CD34+) and CD34−p75− (CD34−) tumor subpopulations were characterized as melanoma-propagating cells (MPC) that exhibit some of those key features. However, these two subpopulations differ from each other in tumorigenic potential, ability to recapitulate heterogeneity, and chemoresistance. In this study, we demonstrate that CD34+ and CD34− subpopulations carrying the BRAFV600E mutation confer differential sensitivity to targeted BRAF inhibition. Through elevated KDM5B expression, melanoma cells shift toward a more drug-tolerant, CD34− state upon exposure to BRAF inhibitor or combined BRAF inhibitor and MEK inhibitor treatment. KDM5B loss or inhibition shifts melanoma cells to the more BRAF inhibitor–sensitive CD34+ state. These results support that KDM5B is a critical epigenetic regulator that governs the transition of key MPC subpopulations with distinct drug sensitivity. This study also emphasizes the importance of continuing to advance our understanding of intratumor heterogeneity and ultimately develop novel therapeutics by altering the heterogeneous characteristics of melanoma.

Figure S1. Responses of different YUMM cell lines to BRAFi treatment; Figure S2. Inhibition of KDM5 family members by shRNA knockdown and pan-KDM5 inhibitors; Figure S3. Subpopulation composition in tumors of different volumes; Figure S4. In vivo BRAFi/MEKi treatment of grafted YUMM1.7 tumors induced Kdm5b expression and increased CD34- subpopulation; Table S1. List of RT-qPCR primers; Table S2. Subpopulation composition of different YUMM cell lines.

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium–specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer.Significance:This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.

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Table S1, S2 and S3

Tumours use various strategies to evade immune surveillance1,2. Immunotherapies targeting tumour immune evasion such as immune checkpoint blockade have shown considerable efficacy on multiple cancers3,4 but are ineffective for most patients due to primary or acquired resistance5–7. Recent studies showed that some epigenetic regulators suppress anti-tumour immunity2,8–12, suggesting that epigenetic therapies could boost anti-tumour immune responses and overcome resistance to current immunotherapies. Here we show that, in mouse melanoma models, depletion of KDM5B—an H3K4 demethylase that is critical for melanoma maintenance and drug resistance13–15—induces robust adaptive immune responses and enhances responses to immune checkpoint blockade. Mechanistically, KDM5B recruits the H3K9 methyltransferase SETDB1 to repress endogenous retroelements such as MMVL30 in a demethylase-independent manner. Derepression of these retroelements activates cytosolic RNA-sensing and DNA-sensing pathways and the subsequent type-I interferon response, leading to tumour rejection and induction of immune memory. Our results demonstrate that KDM5B suppresses anti-tumour immunity by epigenetic silencing of retroelements. We therefore reveal roles of KDM5B in heterochromatin regulation and immune evasion in melanoma, opening new paths for the development of KDM5B-targeting and SETDB1-targeting therapies to enhance tumour immunogenicity and overcome immunotherapy resistance. KDM5B recruits SETDB1 to repress endogenous retroelements such as MMVL30, suppressing anti-tumour immunity, and the depletion of KDM5B induces a robust adaptive immune response and enhances the response to immune checkpoint blockade.

Tissue engineered vascular grafts (TEVGs) represent a promising therapeutic option for emergency vascular intervention. Although the application of small-diameter TEVGs using patient-specific primary endothelial cells (ECs) to prevent thrombosis and occlusion prior to implantation could be hindered by the long time course required for in vitro endothelialization, human induced pluripotent stem cells (hiPSCs) provide a robust source to derive immunocompatible ECs (hiPSC-ECs) for immediate TEVG endothelialization. To achieve clinical application, hiPSC-ECs should be derived under culture conditions without the use of animal-derived reagents (xenogeneic-free conditions), to avoid unwanted host immune responses from xenogeneic reagents. However, a completely xenogeneic-free method of hiPSC-EC generation has not previously been established. Herein, we substituted animal-derived reagents used in a standard method of xenogeneic hiPSC-EC differentiation with functional counterparts of human origin. As a result, we generated xenogeneic-free hiPSC-ECs (XF-hiPSC-ECs) with similar marker expression and function to those of human primary ECs. Furthermore, XF-hiPSC-ECs functionally responded to shear stress with typical cell alignment and gene expression. Finally, we successfully endothelialized decellularized human vessels with XF-hiPSC-ECs in a dynamic bioreactor system. In conclusion, we developed xenogeneic-free conditions for generating functional hiPSC-ECs suitable for vascular tissue engineering, which will further move TEVG therapy toward clinical application.

Induced pluripotent stem cell (iPSC) technology offers a practically infinite and ethically acceptable source to obtain a variety of somatic cells. Coupled with the biotechnologies of cell therapy or tissue engineering, iPSC technology will enormously contribute to human regenerative medicine. Before clinical application, such human iPSC (hiPSC)-based therapies should be assessed using large animal models that more closely match biological or biomechanical properties of human patients. Therefore, it is critical to generate large animal iPSCs, obtain their iPSC-derived somatic cells, and preclinically evaluate their therapeutic efficacy and safety in large animals. During the past decade, the establishment of iPSC lines of a series of large animal species has been documented, and the acquisition and preclinical evaluation of iPSC-derived somatic cells has also been reported. Despite this progress, significant obstacles, such as obtaining or preserving the bona fide pluripotency of large animal iPSCs, have been encountered. Simultaneously, studies of large animal iPSCs have been overlooked in comparison with those of mouse and hiPSCs, and this field deserves more attention and support due to its important preclinical relevance. Herein, this review will focus on the large animal models of pigs, dogs, horses, and sheep/goats, and summarize current progress, challenges, and potential future directions of research on large animal iPSCs.

Tumor heterogeneity is a major challenge for cancer treatment, especially due to the presence of various subpopulations with stem cell or progenitor cell properties. In mouse melanomas, both CD34+p75− (CD34+) and CD34−p75− (CD34−) tumor subpopulations were characterized as melanoma-propagating cells (MPC) that exhibit some of those key features. However, these two subpopulations differ from each other in tumorigenic potential, ability to recapitulate heterogeneity, and chemoresistance. In this study, we demonstrate that CD34+ and CD34− subpopulations carrying the BRAFV600E mutation confer differential sensitivity to targeted BRAF inhibition. Through elevated KDM5B expression, melanoma cells shift toward a more drug-tolerant, CD34− state upon exposure to BRAF inhibitor or combined BRAF inhibitor and MEK inhibitor treatment. KDM5B loss or inhibition shifts melanoma cells to the more BRAF inhibitor–sensitive CD34+ state. These results support that KDM5B is a critical epigenetic regulator that governs the transition of key MPC subpopulations with distinct drug sensitivity. This study also emphasizes the importance of continuing to advance our understanding of intratumor heterogeneity and ultimately develop novel therapeutics by altering the heterogeneous characteristics of melanoma.

Tissue engineered vascular grafts (TEVGs) generated from human primary cells represent a promising vascular interventional therapy. However, generation and application of these TEVGs may be significantly hindered by the limited accessibility, finite expandability, donor-donor functional variation and immune-incompatibility of primary seed cells from donors. Alternatively, human induced pluripotent stem cells (hiPSCs) offer an infinite source to obtain functional vascular cells in large quantity and comparable quality for TEVG construction. To date, TEVGs (hiPSC-TEVGs) with significant mechanical strength and implantability have been generated using hiPSC-derived seed cells. Despite being in its incipient stage, this emerging field of hiPSC-TEVG research has achieved significant progress and presented promising future potential. Meanwhile, a series of challenges pertaining hiPSC differentiation, vascular tissue engineering technologies and future production and application await to be addressed. Herein, we have composed this review to introduce progress in TEVG generation using hiPSCs, summarize the current major challenges, and encapsulate the future directions of research on hiPSC-based TEVGs. Graphical abstract.

Epigenetic therapies are emerging strategies to prime host immune system to immunotherapies. Here we show that depletion of H3K4 demethylase KDM5B in melanoma cells induces anti-tumor T cell immunity through up-regulation of retroelements, which activates cytosolic nucleic acid sensing pathways and subsequent type I interferon responses. Mechanistically, KDM5B recruits H3K9 methyltransferase SETDB1 to the chromatin to repress these retroelements. Ablation of KDM5B enhances responses of poorly immunogenic melanoma tumors to anti PD-1 treatment. Our studies suggest that therapies targeting KDM5B are a novel approach to enhance tumor immunogenicity and overcome immunotherapy resistance.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs., Graphical Abstract, Highlights • Developed monkey CRISPR library to screen pathogenic coronaviruses in Vero-E6 cells • Screens identified genes that are SARS-CoV-2, MERS-CoV, and pan-coronavirus specific • Therapeutic targets, including SMARCA4, identified for SARS-CoV-2 infection • HMGB1 is novel regulator of ACE2 expression and critical for viral entry, To identify potential therapeutic targets for SARS-CoV-2 and related pathogenic coronaviruses, Wei et al. conduct genome-wide CRISPR screens in Vero-E6 cells using SARS-CoV-2, MERS-CoV, and pseudoviruses presenting SARS-CoV-1 or SARS-CoV-2 spike proteins. They identify pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively, and demonstrate that HMGB1 is critical for SARS lineage viral entry because it has a critical role in ACE2 expression.

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium–specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer. Significance: This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.

Previously, we showed that oral application of the environmental pollutant dibenzo[a,l]pyrene (DB[a,l]P) induces oral tumors in mice. Thus, in the present investigation we examined the effect of alcohol on DB[a,l]P-induced DNA damage and immune regulation; we showed that alcohol (6.4% v/v in the diet, 35% of Calories) significantly enhanced the levels of (−)-anti-trans-DB[a,l]P-dA while decreased the levels of GSH in the mouse oral tissues. Analysis of RNA expression revealed that DB[a,l]P alone upregulates inflammatory genes while alcohol suppresses several markers of immune surveillance. Collectively, these results suggest that alcohol may enhance oral carcinogenesis induced by DB[a,l]P.

Worldwide, cancers of the oral cavity and pharynx comprise the sixth most common malignancies. Histologically, more than 90% of oral cancers are squamous cell carcinoma (SCC). Epidemiologic data strongly support the role of exogenous factors such as tobacco, alcohol, and human papilloma virus infection as major causative agents. Avoidance of risk factors has only been partially successful, and survival rates have not improved despite advances in therapeutic approaches. Therefore, new or improved approaches to prevention and/or early detection are critical. Better understanding of the mechanisms of oral carcinogenesis can assist in the development of novel biomarkers for early detection and strategies for disease prevention. Toward this goal, several animal models for carcinogenesis in the oral cavity have been developed. Among these are xenograft, and transgenic animal models, and others employing the synthetic carcinogens such as 7,12-dimethylbenz[a]anthracene in hamster cheek pouch and 4-nitroquinoline-N-oxide in rats and mice. Additional animal models employing environmental carcinogens such as benzo[a]pyrene and N'-nitrosonornicotine have been reported. Each model has certain advantages and disadvantages. Models that (1) utilize environmental carcinogens, (2) reflect tumor heterogeneity, and (3) accurately represent the cellular and molecular changes involved in the initiation and progression of oral cancer in humans could provide a realistic platform. To achieve this goal, we introduced a novel nonsurgical mouse model to study oral carcinogenesis induced by dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and tobacco smoke constituent, and its diol epoxide metabolite (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene [(±)-anti-DB[a,l]PDE]. On the basis of a detailed comparison of oral cancer induced by DB[a,l]P with that induced by the other above-mentioned oral carcinogens with respect to dose, duration, species and strain, cellular and molecular targets, and relative carcinogenic potency, our animal model may offer a more realistic platform to study oral carcinogenesis. In this perspective, we also discuss our preclinical studies to demonstrate the potential of black raspberry extracts on the prevention of OSCC. Specifically, we were the first to demonstrate that black raspberry inhibited DB[a,l]P-DNA binding and of particular importance its capacity to enhance the repair of DB[a,l]P-induced bulky lesions in DNA. We believe that the information presented in this perspective will stimulate further research on the impact of environmental carcinogens in the development of oral cancer and may lead to novel strategies toward the control and prevention of this disease.

In pace with the advancement of tissue engineering during recent decades, tissue-engineered blood vessels (TEBVs) have been generated using primary seed cells, and their impressive success in clinical trials have demonstrated the great potential of these TEBVs as implantable vascular grafts in human regenerative medicine. However, the production, therapeutic efficacy, and readiness in emergencies of current TEBVs could be hindered by the accessibility, expandability, and donor-donor variation of patient-specific primary seed cells. Alternatively, using human induced pluripotent stem cells (hiPSCs) to derive seed vascular cells for vascular tissue engineering could fundamentally address this current dilemma in TEBV production. As an emerging research field with a promising future, the generation of hiPSC-based TEBVs has been reported recently with significant progress. Simultaneously, to further promote hiPSC-based TEBVs into vascular grafts for clinical use, several challenges related to the safety, readiness, and structural integrity of vascular tissue need to be addressed. Herein, this review will focus on the evolution and role of hiPSCs in vascular tissue engineering technology and summarize the current progress, challenges, and future directions of research on hiPSC-based TEBVs.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.

Multi-scale transforms are considered to be the effective methods in image fusion. However, infrared intensity and polarization images fusion based on these methods frequently arise the issues such as limited contrast ratio improvement and marginal area distortion. The embedded multi-scale decomposition has been proposed to integrate the advantages of different methods in this paper, where the low-frequency image continues to be decomposed by another multi-scale transform. The detailed procedure is shown as the following. Firstly, the low-frequency components and support value image sequences of infrared intensity and polarization images are obtained respectively with support value transform (SVT). Secondly, multi-scale bright and dim information are extracted from the last layer of the low-frequency images using the multi-scale top-hat decomposition, respectively. Thirdly, the resulted images are respectively fused and enhanced, and then fused with the two low-frequency images to get the fused low-frequency image. After that, the image is reversely transformed with the support value image sequences fused by extracting maximum gray to get the final image. Compared with the simple SVT and the multi-scale top-hat decomposition, the method suggested successfully improves the contrast ratio, the distortion of marginal area and the local coarseness. The validity of the method proposed is proven.

Summary Vascular smooth muscle cells (VSMCs) can be derived in large numbers from human induced pluripotent stem cells (hiPSCs) for producing tissue-engineered vascular grafts (TEVGs). However, hiPSC-derived TEVGs are hampered by low mechanical strength and significant radial dilation after implantation. Here, we report generation of hiPSC-derived TEVGs with mechanical strength comparable to native vessels used in arterial bypass grafts by utilizing biodegradable scaffolds, incremental pulsatile stretching, and optimal culture conditions. Following implantation into a rat aortic model, hiPSC-derived TEVGs show excellent patency without luminal dilation and effectively maintain mechanical and contractile function. This study provides a foundation for future production of non-immunogenic, cellularized hiPSC-derived TEVGs composed of allogenic vascular cells, potentially serving needs to a considerable number of patients whose dysfunctional vascular cells preclude TEVG generation via other methods.

We previously reported that dibenzo[a,l]pyrene (DB[a,l]P), the most potent known environmental carcinogen among polycyclic aromatic hydrocarbons (PAH) congeners, is carcinogenic in the oral tissues of mice. We have now developed a new mouse model which employs the oral application of the fjord region diol epoxide, (±)-anti−11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE), a metabolite of the tobacco smoke constituent DB[a,l]P, and we show its specific induction of oral squamous cell carcinoma (OSCC) in both tongue and other oral tissues. Groups of B6C3F1 mice (20/group) received 6 or 3 nmol of (±)-anti-DB[a,l]PDE administered into the oral cavity; 3 times per week for 38 weeks. Additional groups received the vehicle alone or were left untreated. Mice were sacrificed 42 weeks after the first carcinogen administration. The high dose induced 74 and 100% OSCC in the tongue and other oral tissues, respectively; the corresponding values at the lower dose were 45 and 89%. Using immunohistochemistry, we showed that DB[a,l]PDE resulted in overexpression of p53 and COX-2 proteins in malignant tissues when compared to normal oral tissues and tongues. Consistent with the carcinogenicity, we demonstrated powerful mutagenicity in cII gene in B6C3F1 (Big Blue) mouse tongue. The mutational profile in lacI reporter gene is similar to those detected in human head and neck cancer, and p53 mutations were observed in mouse oral tumor tissues. Taken together, we conclude that the formation of diol epoxides plays a major role among the mechanisms by which DB[a,l]P exerts its oral mutagenicity and tumorigenicity.

We were the first to demonstrate that direct application of the environmental pollutant and tobacco smoke constituent dibenzo[a,l]pyrene (DB[a,l]P) into the oral cavity of mice induced squamous cell carcinoma (SCC) in oral tissues but not in the tongue; however, the mechanisms that can account for the varied carcinogenicity remain to be determined. Furthermore, we also showed that not only dA adducts, but also dG adducts can account for the mutagenic activity of DB[a,l]P in the oral tissues in vivo. In this study, we initially focused on DB[a,l]P-induced genotoxic effects in both oral and tongue tissues. Therefore, to fully assess the contribution of these DNA adducts in the initiation stage of carcinogenesis induced by DB[a,l]P, an LC-MS/MS method to simultaneously detect and quantify DB[a,l]PDE-dG and -dA adducts was developed. Mice were orally administered with DB[a,l]P (24 nmole, 3 times per week for 5 weeks) or its fjord region diol epoxide, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE, 12 nmole, single application); animals were sacrificed at 2, 7, 14, and 28 days after the last dose of carcinogen administration. Oral and tongue tissues were obtained and DNA were isolated followed by enzymatic hydrolysis. Following the development of an isotope dilution LC-MS/MS method, we successfully detected (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(2)-dG, as well as (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(6)-dA in oral and tongue tissues of mice treated with DB[a,l]P. Levels of (-)-anti-trans-DB[a,l]PDE-N(6)-dA were ≥2 folds higher than (-)-anti-cis-DB[a,l]PDE-N(6)-dA adduct and those of dG adducts in the oral tissues and tongue at all time points selected after the cessation of DB[a,l]P treatment. Levels of dG adducts were comparable in both tissues. Collectively, our results support that DB[a,l]P is predominantly metabolized to (-)-anti-DB[a,l]PDE, and the levels and persistence of (-)-anti-trans-DB[a,l]PDE-N(6)-dA may, in part, explain the carcinogenicity of DB[a,l]P in the oral tissues but not in the tongue.

The CRISPR/Cas9 system is a powerful genome editing tool and has been widely used for biomedical research. However, many challenges, such as off-target effects and lack of easy solutions for multiplex targeting, are still limiting its applications. To overcome these challenges, we first developed a highly efficient doxycycline-inducible Cas9-EGFP vector. This vector allowed us to track the cells for uniform temporal control and efficient gene disruption, even in a polyclonal setting. Furthermore, the inducible CRISPR/Cas9 system dramatically decreased off-target effects with a pulse exposure of the genome to the Cas9/sgRNA complex. To target multiple genes simultaneously, we established simple one-step cloning approaches for expression of multiple sgRNAs with improved vectors. By combining our inducible and multiplex genome editing approaches, we were able to simultaneously delete Lysine Demethylase (KDM) 5A, 5B and 5C efficiently in vitro and in vivo. This user friendly and highly efficient toolbox provides a solution for easy genome editing with tight temporal control, minimal off-target effects and multiplex targeting.

Tobacco smoking is an etiological factor of ovarian cacner; however, the mechanisms remain largely undefined. Therefore, as an initial investigation, we examined the carcinogenicity and DNA adducts formation in the ovary of mice treated with DB[a,l]P, a tobacco smoke constituent and environmental pollutant. Ovarian tumors in B6C3F1 mice were induced by direct application of DB[a,l]P (24, 12, 6, and 3 nmol/mouse, three times a week for 38 weeks) into the oral cavity of mice. At 6 nmol, DB[a,l]P induced the highest total ovarian tumor incidence (79%), but the incidence of malignancy was only 15%. However, at the dose of 12 nmol, the total ovarian tumor incidence was 75%, and the incidence of malignancy was 65%. In addition to ovarian tumors, at the dose of 24 nmol, DB[a,l]P induced lesions in sites distal from the ovaries including the skin, mammary, lung, and oral tissues, which were rare at doses lower than 24 nmol. Another bioassay was conducted to detect and quantify DNA adducts induced by DB[a,l]P (24 nmol, three times a week for 5 weeks) in the ovary at 48 h and 1, 2, and 4 weeks after the last administration of DB[a,l]P. DNA was isolated, and the dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE)-DNA adducts were analyzed by a LC-MS/MS method. DB[a,l]P resulted in the formation of (-)-anti-cis-DB[a,l]PDE-dA and (-)-anti-trans-DB[a,l]PDE-dA adducts, which were 0.8 and 1.6 fmol/10(6) dA, respectively, in ovaries of mice within 48 h, and the level of adducts decreased over a week. Our results indicated that DB[a,l]P can be metabolized to form (-)-anti-DB[a,l]PDE; the latter may, in part, account for DB[a,l]P-induced ovarian cancer. This animal model should assist to better understand the mechanisms, account for the induction of ovarian cancer by tobacco carcinogens, and facilitate the development of chemopreventive agents against ovarian cancer.

Tobacco smoking is one of the leading causes for oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke constituent, is the most carcinogenic polycyclic aromatic hydrocarbon (PAH) tested to date in several animal models (target organs: skin, lung, ovary, and mammary tissues). We have recently demonstrated that DB[a,l]P is also capable of inducing oral cancer in mice; however, its metabolic activation to the ultimate genotoxic metabolite dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) in mouse oral cavity has not been examined. Here we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify (±)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [(15)N(5)]-(±)-anti-DB[a,l]PDE-N(6)-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR, and CD analysis. The detection limit of the method is 8 fmol with 100 μg of digested DNA as the matrix. Two adducts were detected and identified as (-)-anti-cis and (-)-anti-trans-DB[a,l]PDE-dA in the oral tissues of mice following the direct application of DB[a,l]P (240 nmol per day, for 2 days) into the oral cavity, indicating that DB[a,l]P is predominantly metabolized into (-)-anti-DB[a,l]PDE in this target organ. We also compared the formation and removal of adducts as a function of time, following the direct application of DB[a,l]P (24 nmol, 3 times per week for 5 weeks) into the oral cavity of mice. Adducts were quantified at 48 h, 1, 2, and 4 weeks after the last dose. Maximal levels of adducts occurred at 48 h, followed by a gradual decrease. The levels (fmol/μg DNA) of (-)-anti-trans adducts (4.03 ± 0.27 to 1.77 ± 0.25) are significantly higher than (-)-anti-cis-DB[a,l]PDE-dA adduct (1.63 ± 0.42 to 0.72 ± 0.04) at each time point (p0.005). The results presented here indicate that the formation and persistence of (-)-anti-DB[a,l]PDE-dA adducts may, in part, contribute to the initiation of DB[a,l]P-induced oral carcinogenesis.

Cancer of the oral cavity is a serious disease, affecting about 30,000 individuals in US annually. There are several animal models of oral cancer, but each has certain disadvantages. As a new model, we investigated whether topical application of the tobacco smoke carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) is mutagenic and carcinogenic in the oral cavity of the B6C3F1 lacI and B6C3F1 mouse, respectively. B6C3F1 lacI mice received DB[a,l]P (0, 3, 6, 12 nmol) 3× per week. B6C3F1 mice received the same doses and also 24 nmol. At 38 weeks mutagenesis was measured in oral tissues in lacI mice. For the high dose group, the mutant fraction (MF) in upper mucosa and tongue increased about twofold relative to that in vehicle-alone. The increases were statistically significant. The mutational profile in the DB[a,l]P-induced mutants was compared with that induced by benzo[a]pyrene (BaP) in oral tissue. BaP is mutagenic in many tissues when administered by gavage. The mutational profile for DB[a,l]P was more similar to that reported for p53 mutations in head and neck cancers than was that of BaP. At 47 weeks, oral squamous cell carcinomas (OSCC) were found in 31% of the high-dose B6C3F1 group. Elevations of p53 and COX-2 protein were observed in tumor and dysplastic tissue. As DB[a,l]P induces mutations and tumors in the oral cavity, and has a mutational profile in oral tissue similar to that found in p53 in human OSCC, the treatment protocol described here may represent a new and relevant model for cancer of the oral cavity.

BRB powder and extracts have been shown to inhibit tumorigenesis in oral and other alimentary-tract sites in animal models; and have promising effects on markers of carcinogenesis in several human trials. The purpose of this study was to compare the inhibitory effects of BRB and two related compounds (protocatechuic acid and kaempferol) on mutagenesis and DNA adduct formation induced by the tobacco carcinogen dibenzo[a,l]pyrene (DBP) and its metabolites, DBP-diol and DBP-diol epoxide (DBPDE), in a rat oral epithelial cell line. Mutagenesis and DNA adduct formation induced by DBP, DBP-diol and DBPDE were monitored in rat oral epithelial and fibroblast cells, containing a lacI mutagenesis reporter gene; and the order of potency in both adduct formation and mutagenesis was DBPDE>>DBP-diol>>DBP in both cell types. In the fibroblasts for mutagenesis, the potencies (in units of mutants/10E5 pfu /uM) were: DBPDE, 16,320; DBP-diol, 25; DBP1.7, (above background); background was 2.9., Similar results were obtained for epithelial cells. Analysis of DNA adducts levels is still in progress and results thus far (in units of cis + trans isomers of anti-DBPDE-dA/10E6 A, are: DBPDE, 2096, DBP-diol ≈1.8, DBP < 1.0. The above results are consistent with mutagenesis being dependent on DNA adducts and indicate this cell culture model reflects initiation of carcinogenesis. The background level of mutagenesis was greater in the epithelial cells, so the fibroblasts were then used for subsequent mutagenesis experiments. As initiation by DBP and DBP-diol requires biotransformation to an ultimate mutagen/carcinogen (in this case DBPDE) we initially assayed inhibition of DBP-diol induced mutagenesis, so that effects of BRB on processes involved in activation, as well as effects on steps after formation of DBPDE would be detected. The parent compound, DBP, was not employed because it was very weakly mutagenic, and studies with DBPDE are still in progress. Treatment of cells with one µM DBP-diol led to an increase in mutant fraction from 2.9 +/- 0.8 to 25 +/- 2.3 mutants/10E5 pfu. When pretreated with BRB and its related compounds 4 hours before the addition of DBP-diol, significant inhibition of mutagenesis was observed. With 3 uM protocatechuic acid mutagenesis was reduced to 15.6 +/- 3.7 and with 3uM kaempferol to 13.3 +/- 4.1 mutants/10E5 pfu. Pretreatment of the cells with 25 ug/ml of an anthocyanin-enriched BRB extract (a concentration previously used in colon cancer cells), reduced mutagenesis to 9.8 +/- 0.35 mutants/10E5 pfu. These results suggest that BRB and specific related components will be protective against carcinogenesis induced by certain tobacco carcinogens. Supported by NIH grant #CA173465. Citation Format: Joseph B. Guttenplan, Wieslawa Kosinska, Tianzhen Han, Kun-Ming Chen, Shangmin Zhang, Krishnegowda Gowdahalli, Amin Shantu, Gary Stoner, Karam El-Bayoumy. Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by the tobacco carcinogen, dibenzo(a,l)pyrene and its metabolites in cultured rat oral fibroblasts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2014-252

Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck region; in the USA, over 45,000 cases and about 11,000 deaths from the disease occur annually. Progress in the prevention and control of OSCC has been hampered by the lack of appropriate animal models that would reflect human exposure. Tobacco smoking is considered a major etiological factor in the development of oral cancer. Previous studies have demonstrated the ability of diets containing 5-10% freeze-dried black raspberry (BRB) powder to inhibit the development of chemically-induced cancers in multiple organ sites in rodents including 7,12-dimethylbenz(a)anthracene (DMBA) induced squamous cell carcinomas (SCC) in the hamster cheek pouch. However, DMBA is not present in the environment and the hamster cheek pouch model may not be applicable to humans. We had previously reported the carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, in mouse oral cavity. Recently, we demonstrated that administration of BRB at 5% in the diet can significantly reduce DNA adducts resulted from the administration of DB[a,l]P. It has also been shown that anthocyanins are the most abundant compounds in BRBs that can account for much of their antioxidant activity. In the current study, under identical conditions we compare the inhibitory effects of an anthocyanin enriched fraction extracted from black raspberry (BRBE), protocatechuic acid (PA, a major metabolite of anthocyanins) or ferulic acid (FA, a major phenolic acid in raspberries shown to reduce oral carcinogenesis) with BRB on DNA adducts induced by DB[a,l]P in mouse oral cavity. Levels of DB[a,l]PDE-DNA adducts were quantified by a LC-MS/MS method. We demonstrated that the administration of BRBE (1.6 %), protocatechuic acid (0.2%) or ferulic acid (0.05%) in the diet, starting 2 weeks before DB[a,l]P (24 nmol, 3 times a week for 5 weeks) significantly resulted in 29.6%, 23.0% or 25.3% reduction of the level of (-)-anti-trans-DB[a,l]PDE-dA in murine oral tissues, respectively, compared to a 19.2% reduction of BRB. Our results clearly showed that the anthocyanin components of BRB and its metabolite, PA, are responsible for the inhibitory effects of BRB on the DB[a,l]P- induced DNA adducts formation; in addition, BRB and its related components can inhibit the metabolic activation of DB[a,l]P, and may prevent the subsequent mutagenesis and carcinogenesis resulting from exposure to DB[a,l]P. Supported by NIH grant #CA173465. Citation Format: Kun-Ming Chen, Shangmin Zhang, Yuan-Wan Sun, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Gary Stoner, Karam El-Bayoumy. The inhibitory effects of an anthocyanin enriched fraction of black raspberry (BRB), protocatechuic acid and ferulic acid on DB[a,l]P-induced DNA adduct formation in mouse oral tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 230. doi:10.1158/1538-7445.AM2014-230

Alcohol acts synergistically with tobacco smoke to greatly increase the risk of oral squamous cell carcinoma (OSCC) up to 20-fold; the mechanisms of this synergism remain unknown. Research in this area has been hampered by the lack of appropriate animal models that would mimic chronic simultaneous human exposure to alcohol and representative carcinogens in tobacco or tobacco smoke as a whole. Recently, we developed a more relevant animal model (Int. J. Cancer, 2012, 2783-90) and showed that dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, induces SCC in oral tissues of mice. We also showed that its diol epoxide metabolite, (±)-anti-DB[a,l]PDE, is a potent and specific carcinogen in tongue and other oral tissues in mice. We hypothesize that chronic alcohol consumption will increase the carcinogenic potency of DB[a,l]P in the mouse oral cavity by multiple mechanisms including the depletion of glutathione (GSH) and increasing levels of covalent DB[a,l]PDE-DNA adducts. As an initial study to test our hypothesis, mice were fed Isocaloric Lieber-DeCarli-type liquid diets formulated by BioServ (Frenchtown, N.J.) with or without ethanol (35% of total calories, increased gradually from 0, 11, 22 to 35%). After 3 weeks, mice were treated with 2 daily doses of DB[a,l]P (240 nmol each) and/or tobacco smoke particulates (TSP, 2R4F, 2.5 mg/mouse). Mice were sacrificed 24 hrs later and DB[a,l]PDE-DNA adduct levels were quantified by our recently developed LC-MS/MS method (Chem. Res. Toxicol. 2011, 1297-303). We showed that alcohol consumption significantly increased the levels of (-)-anti-trans-DB[a,l]P-dA in the absence of TSP (25%) or in the presence of TSP (75%). We also demonstrated that GSH levels in buccal mucosa were decreased (38%) in mice fed with liquid diet (35% calories from ethanol) compared with mice fed with control diet (0.89±0.13 μmol/g vs 1.23±0.17 μmol/g). Collectively, our preliminary results support our hypothesis that alcohol consumption will increase the carcinogenic potency of DB[a,l]P by altering the metabolic detoxification via depleting GSH and increasing DNA damage in oral tissues of mice. Citation Format: Shang-Min Zhang, Kun-Ming Chen, John R. Richie, Ana Calcagnotto, Cesar Aliaga, Arthur Berg, Yuan-Wan Sun, Arun K. Sharma, Shantu Amin, Karam El-Bayoumy. Effect of alcohol on DB[a,l]P, an environmental pollutant and a tobacco smoke constituent, on DNA adduct formation in mouse oral tissues. [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 3602. doi:10.1158/1538-7445.AM2013-3602

Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck region; in the USA, over 45,000 cases and about 11,000 deaths from the disease occur annually. Progress in the prevention and control of OSCC has been hampered by the lack of appropriate animal models that would reflect human exposure. Tobacco smoking is considered a major etiological factor in the development of oral cancer. Previous studies have demonstrated the ability of diets containing 5-10% freeze-dried black raspberry (BRB) powder to inhibit the development of chemically-induced cancers in multiple organ sites in rodents including 7,12-dimethylbenz(a)anthracene (DMBA) induced squamous cell carcinomas (SCC) in the hamster cheek pouch. DMBA, however, is not present in the environment and the hamster cheek pouch model may not be applicable to humans. Recently, we developed a relevant animal model (Int. J. Cancer, 2012, 2783-90) and showed that dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, induces SCC in oral tissues of mice. In the current study, we evaluated the effects of BRB and related components including kaempferol and resveratrol on DNA adducts induced by DB[a,l]P in mouse oral cavity as an initial step to explore the potential preventive activity of BRB and its related components on DB[a,l]P-induced oral cancer. The DB[a,l]PDE-DNA adduct levels were quantified by our recently developed LC-MS/MS method (Chem. Res. Toxicol. 2011, 1297-303). We demonstrated that the administration of BRB (5%), kaempferol (0.025%) or resveratrol (0.002%) in the diet, starting 2 weeks before DB[a,l]P (24 nmol, 3 times a week for 5 weeks) resulted in 27%, 36% or 65% reduction of the level of (-)-anti-trans-DB[a,l]PDE-dA in murine oral tissues, respectively. In conclusion, our results support that dietary consumption of BRB and its related components can inhibit the metabolic activation of DB[a,l]P, and may prevent the subsequent mutagenesis and carcinogenesis resulting from exposure to DB[a,l]P. Citation Format: Kun-Ming Chen, Shang-Min Zhang, Cesar Aliaga, Yuan-Wan Sun, Arthur Berg, Arun Sharma, Shantu Amin, Gary Stoner, Karam El-Bayoumy. Effects of black raspberry and related components on DB[a,l]P-induced DNA adduct formation in mouse oral tissues. [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 3708. doi:10.1158/1538-7445.AM2013-3708

Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Recently we developed a novel mouse model of oral cancer induced by DB[a, l]P; we also showed the remarkable carcinogenicity and specificity of the fjord region diol epoxide metabolite, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a, l]pyrene (DB[a, l]PDE). Using immunohistochemistry (IHC), we have shown over-expression of p53 protein in mice oral squamous cell carcinoma and dysplastic tissues induced by DB[a, l]P and DB[a, l]PDE. Current understanding of molecular pathogenesis of oral cancer in humans suggests that both genetic and epigenetic alterations are crucial and complement each other. Our hypothesis is that both genetic and epigenetic alterations induced by DB[a, l]P can contribute to the development of oral cancer. P53 protein overexpression detected by IHC may result from p53 gene mutation or exposure to genotoxic stress. To determine whether p53 over-expression is in part due to p53 mutations, Exons 5 to 8 of p53 from 5 tumor tissues were analyzed by polymerase chain reaction single-strand conformation polymorphisms (PCR-SSCP) and direct sequencing. G to T transversion was detected in Exon 5, leading to mutation of codon 155 Arg to Leu; A to T transversion was detected in Exon 7, resulting in mutation of codon 232 Lys to stop codon. To test the epigenetic effect of DB[a, l]P, methylation specific PCR and bisulfate sequencing were used to detect methylation alteration of p16 and RAR-β promoters. Promoter hypermethylation of both p16 and RAR-β were detected in tumors induced by DB[a, l]PDE; also in oral tissues of mice treated with DB[a, l]P (24nmol, 3 times per week, for 5 weeks, sacrificed at 48 h, 1, 2 and 4 weeks after last dose). These results suggested that genotoxic DNA adducts derived from DB[a, l]P can induce mutations in critical genes, such as tumor suppressor gene p53; this environmental carcinogen also can induce p16 and RAR-β promoter hypermethylation at early stage, which can contribute to the promotion and progression of oral carcinogenesis. 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 5470. doi:1538-7445.AM2012-5470

Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Our laboratory had developed a mouse model of oral cancer induced by DB[a, l]P. Our working hypothesis is that the stereochemical course of DB[a, l]P metabolism, the conformations of the DNA adducts formed and their removal by mammalian DNA repair enzymes, and their mutagenic properties if not removed in an error-free manner, all play critical roles during the induction of oral carcinogenesis. As an initial study to test our hypothesis, we have previously developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify DB[a, l]PDE-N6-dA adducts in oral tissues of mice treated with DB[a, l]P. We have shown that (-)-anti-cis- and (-)-anti-trans-DB[a, l]PDE-N6-dA adducts were detected from oral tissues of mice treated with DB[a, l]P. In the present study, to further test our hypothesis, we report on the development of a LC-MS/MS method to detect DB[a, l]PDE-N2-dG adducts in vivo. (±)-anti-[15N5]-DB[a, l]PDE-N2-dG adducts were synthesized as internal standards. The stereochemistry of adducts were characterized. Following the addition of internal standards, DNA isolated from oral tissues of mice treated with DB[a, l]P or DB[a, l]PDE was enzymatically hydrolyzed to 2′-deoxyribonucleosides and partially purified by solid-phase extraction. The LC-MS/MS analysis was carried out by monitoring transitions m/z 620 [M+H]+→ m/z 504 [(M+H)+−2′-deoxyribose] for DB[a, l]PDE-N2-dG adducts and m/z 625α m/z 509 for the internal standards. We have detected two N2-dG adducts from oral tissues of mice treated with DB[a, l]P, and four N2-dG adducts from oral tissues of mice treated with (±)-anti-DB[a, l]PDE. Collectively, the in vivo detection of dA and dG adducts indicated that DB[a, l]P is predominantly metabolized to (-)-anti-DB[a, l]PDE in oral tissues of mice. This sensitive LC-MS/MS method, is capable of simultaneously detecting both dA and dG adducts derived from anti-DB[a, l]PDE. Our results indicated that levels of dA adducts are significantly higher than dG adducts in vivo, which are consistent with those reported in literature in organs other than oral tissues, demonstrating that fjord region diol epoxide of DB[a, l]P predominantly form dA adducts than dG adducts. 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 5469. doi:1538-7445.AM2012-5469

Oral cancer is the major form of head and neck squamous cell carcinoma, which is the sixth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Our laboratory had developed a novel mouse model of oral cancer induced by DB[a,l]P. The metabolic activation of DB[a,l]P and genotoxic effect in rodent oral cavity have not been examined before. Our working hypothesis is that the stereochemical course of DB[a,l]P metabolism, the conformations of the DNA adducts formed, their removal by mammalian DNA repair enzymes, and their mutagenic properties if not removed in an error-free manner, all play critical roles during the induction of oral carcinogenesis. As an initial study to test our hypothesis, here we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify DB[a,l]PDE-N6-dA adducts in oral tissues of mice treated with DB[a,l]P. (±)-anti-[15N5]-DB[a,l]PDE-N6-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR and CD analysis. After addition of internal standards, DNA was enzymatically hydrolyzed to 2’-deoxyribonucleosides and partially purified by solid-phase extraction. The LC-MS/MS analysis was carried out by monitoring transitions m/z 604 [M+H]+ to m/z 335 [(M+H) +-dA-H2O] for DB[a,l]PDE-N6-dA adducts and m/z 609 to m/z 335 for the internal standards. The detection limit for DB[a,l]PDE-dA adduct is approximately 20 fmol in 100 ug DNA. We have compared the formation and removal of adducts as a function of time following direct application of DB[a,l]P into the oral cavity of B6C3F1 mice (24 nmol, 3 times per week, for 5 weeks). Adducts were measured at 48h, 1, 2 and 4 weeks after the last dose. Only (−)-anti-cis-and (−)-anti-trans- DB[a,l]PDE- N6-dA adducts were detected from oral tissues of DB[a,l]P treated mice, ranging from 1.9± 0.3 to 0.7± 0.1 and from 4.8 ± 0.2 to 2.3 ± 0.3 fmol/ug DNA, respectively. The levels of both adducts decreased in a time-dependent manner; approximately 50% of (−)-anti-cis- and (−)-anti-trans- DB[a,l]PDE- N6-dA adducts were removed within 4 weeks. The results suggest that DB[a,l]P is predominantly metabolized to (−)-anti-DB[a,l]PDE in oral tissues of mice, which is consistent with studies in other preclinical model systems. In this study we developed for the first time a sensitive LC-MS/MS method for the detection and quantification of DNA adducts derived from DB[a,l]P. The persistent (−)-anti-DB[a,l]PDE- N6-dA adducts may account for the initiation of carcinogenesis in the oral tissues of mice treated with DB[a,l]P. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5550. doi:10.1158/1538-7445.AM2011-5550

Ovarian cancer ranks second among gynecologic cancers, and it is the fifth leading cause of cancer deaths among women; 13,850 deaths are expected in 2010. There is usually no obvious symptoms of early stage ovarian cancer; therefore only 15% of ovarian cancers are diagnosed in the localized stage, and the five-year survival rate for ovarian cancer patients is 46%. A positive relation of mucinous ovarian tumors to tobacco smoking had been reported; furthermore, an association was shown between mucinous ovarian cancer with ex-smokers as well. The mechanisms that can account for the role of tobacco smoking on this disease remain unknown. Currently, there is no preclinical animal model that can accurately reflect the ovarian cancer resulting from chronic exposure to chemical carcinogens present in cigarette smoke. A previous report showed that dibenzo[a,l]pyrene (DB[a,l]P) can induce ovarian cancer in mice. Therefore, in this study we examined the carcinogenicity and DNA adducts formation in the ovary of mice treated with DB[a,l]P. In carcinogenesis study, ovarian tumors in B6C3F1 mice were induced by administering DB[a,l]P into the oral cavity of mice. Several groups of B6C3F1 mice (20/group) received various doses of DB[a,l]P (24, 12, 6, and 3 nmol/mouse), and additional groups received DMSO 3 times a week for 38 weeks or were left untreated as controls. Mice were sacrificed 4 weeks after the last carcinogen administration. A short-term bioassay was also conducted to detect and quantify DNA-adducts induced by DB[a,l]P as a function of time in the ovary; mice were administered orally (24 nmol) 3 times a week for 5 weeks. Ovaries were removed from mice 48 h, 1, 2 and 4 weeks after the last dose. DNA were isolated and hydrolyzed enzymatically, and the DB[a,l]PDE-DNA adducts were analyzed by our newly developed LC-MS/MS method. At the dose of 6 nmol in carcinogenesis study, we observed the highest total ovarian tumor incidence (79%), but the incidence of malignancy was only 15%. However, at the dose of 12 nmol, the total tumor incidence was 75%, and the incidence of malignant granulosa cell tumor was 65%. In addition to ovarian tumors, we also observed lesions in sites distal from the ovaries including the skin, mammary, lung, and oral tissues at the dose of 24 nmol, which were rare in other dosing groups. Treatment of DB[a,l]P in mice resulted in the formation of (−)-anti-cis-DB[a,l]PDE-dA and (−)-anti-trans-DB[a,l]PDE-dA adducts, which were 1.6 and 3.1 fmol/µg DNA respectively in ovaries of mice within 48 hours, and the level of adducts decreased dramatically over a week. Our results indicated that DB[a,l]P can be activated to form (−)-anti-DB[a,l]PDE; the latter may account for DB[a,l]P-induced ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1333. doi:10.1158/1538-7445.AM2011-1333

The annual incidence of cancer of the oral cavity and pharynx in the US is about 30,000 cases, similar to that for leukemia and pancreas, and resulting in a 5 yr survival rate of about 50%. Current models utilizing 4-nitroquinoline-N-oxide in rats and mice or 7,12-dimethylbenzanthracene in the hamster cheek pouch, suffer from relevancy of these synthetic carcinogens and/or the target site. One 2-year feeding study reported that dietary administration of the tobacco smoke carcinogen, benzo(a)pyrene (BaP) leads to tumors at several sites including tongue, but only at very high doses, and mainly in forestomach (Culp, S.J., et al., Carcinogenesis, 9, 117-124, 1998). Here as a potential new model, we investigated whether the more powerful tobacco smoke carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) is mutagenic and carcinogenic in the oral cavity of the B6C3F1 lacI and B6C3F1 mouse resp. upon topical application. Four groups of B6C3F1 lacI mice (6/group) received DB[a,l]P (12, 6, and 3 nmol) or vehicle 3x per week. B6C3F1 mice (20/group) received the same treatment, with an additional 24 nmol group. At 38 weeks the lacI mice were euthanized and DNA was isolated from tongue and upper oral mucosa. For the 12 nmol dose group, the mutant fraction (MF) in the cII gene in upper mucosa and tongue increased about 2-fold relative to that in vehicle-alone (5.2 +/- 0.8 and 4.6 +/- 1.2 vs 2.7 +/- 1.3 and 2.6 +/- 1.2) resp. in units of mutants/105 pfu. The increases were statistically significant (ρ = 0.023 and 0.016) for upper mucosa and tongue resp. The MF's at lower doses were non-significantly elevated. The mutational profile in the DB[a,l]P-induced mutants was compared with that induced by BaP in upper mucosa. BaP was previously shown to be mutagenic in many tissues including oral tissues when administered by gavage (Guttenplan, J.B, et al., Mutation Res., 559,199-210, 2004). Only 4% of BaP-induced mutations were at AT base pairs vs. 31% for DB[a,l]P; consistent with a report of the comparative mutational profile of BaP and DB[a,l]P in lung (Leavitt, S.A., et al., Mutagenesis 23, 445-450, 2008). The fraction of mutations at AT base pairs for DB[a,l]P is very similar to that observed in p53 mutations in the human oral cavity. One yr after the start of treatments, B6C3F1 mice were euthanized and oral squamous cell carcinomas (OSCC) were found in 31% of the high-dose group. Tumors were located in several sites in the oral cavity, although not in tongue. Immunohistochemical analyses revealed that p53 protein was overexpressed in malignant tissues, but not controls. The facts that DB[a,l]P induces mutations and tumors in the oral cavity, and has a mutational profile in oral tissue similar to that found in p53 in human OSCC, indicate DB[a,l]P administration to the mouse oral cavity will likely provide the basis for a new and more relevant model for cancer of the oral cavity than existing models. Supported by NCI grant no. R01 CA 100924 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1968.