Anticancer Natural Compounds as Epigenetic Modulators of Gene Expression.

Accumulating evidence shows that hallmarks of cancer include: "genetic and epigenetic alterations leading to inactivation of cancer suppressors, overexpression of oncogenes, deregulation of intracellular signaling cascades, alterations of cancer cell metabolism, failure to undergo cancer cell death, induction of epithelial to mesenchymal transition, invasiveness, metastasis, deregulation of immune response and changes in cancer microenvironment, which underpin cancer development". Natural compounds as bioactive ingredients isolated from natural sources (plants, fungi, marine life forms) have revolutionized the field of anticancer therapeutics and rapid developments in preclinical studies are encouraging. Natural compounds could affect the epigenetic molecular mechanisms that modulate gene expression, as well as DNA damage and repair mechanisms. The current review will describe the latest achievements in using naturally produced compounds targeting epigenetic regulators and modulators of gene transcription in vitro and in vivo to generate novel anticancer therapeutics.

Natural Compounds As Modulators of Non-apoptotic Cell Death in Cancer Cells.

Cell death is an innate capability of cells to be removed from microenvironment, if and when they are damaged by multiple stresses. Cell death is often regulated by multiple molecular pathways and mechanism, including apoptosis, autophagy, and necroptosis. The molecular network underlying these processes is often intertwined and one pathway can dynamically shift to another one acquiring certain protein components, in particular upon treatment with various drugs. The strategy to treat human cancer ultimately relies on the ability of anticancer therapeutics to induce tumor-specific cell death, while leaving normal adjacent cells undamaged. However, tumor cells often develop the resistance to the drug-induced cell death, thus representing a great challenge for the anticancer approaches. Numerous compounds originated from the natural sources and biopharmaceutical industries are applied today in clinics showing advantageous results. However, some exhibit serious toxic side effects. Thus, novel effective therapeutic approaches in treating cancers are continued to be developed. Natural compounds with anticancer activity have gained a great interest among researchers and clinicians alike since they have shown more favorable safety and efficacy then the synthetic marketed drugs. Numerous studies in vitro and in vivo have found that several natural compounds display promising anticancer potentials. This review underlines certain information regarding the role of natural compounds from plants, microorganisms and sea life forms, which are able to induce non-apoptotic cell death in tumor cells, namely autophagy and necroptosis.

Tumor Protein (TP)-p53 Members as Regulators of Autophagy in Tumor Cells upon Marine Drug Exposure.

Targeting autophagic pathways might play a critical role in designing novel chemotherapeutic approaches in the treatment of human cancers, and the prevention of tumor-derived chemoresistance. Marine compounds were found to decrease tumor cell growth in vitro and in vivo. Some of them were shown to induce autophagic flux in tumor cells. In this study, we observed that the selected marine life-derived compounds (Chromomycin A2, Psammaplin A, and Ilimaquinone) induce expression of several autophagic signaling intermediates in human squamous cell carcinoma, glioblastoma, and colorectal carcinoma cells in vitro through a transcriptional regulation by tumor protein (TP)-p53 family members. These conclusions were supported by specific qPCR expression analysis, luciferase reporter promoter assay, and chromatin immunoprecipitation of promoter sequences bound to the TP53 family proteins, and silencing of the TP53 members in tumor cells.

Disruptive environmental chemicals and cellular mechanisms that confer resistance to cell death.

Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.

p63 and p73: teammates or adversaries?

The status and interrelationship of p53 family members are critical elements in tumor progression. An intriguing paper in this issue of Cancer Cell (Rocco et al., 2006) reveals a new twist in the interactions between p63 and p73 following DNA damage, underscoring a role for p73 in the proapoptotic regulation of Puma, Noxa, and Bcl-2 in head and neck squamous cell carcinomas (HNSCC). These data define a pathway in which deltaNp63alpha promotes survival in squamous epithelial malignancy by repressing a p73-dependent proapoptotic transcriptional program, suggesting that p63 levels and p73 status may be key determinants of tumor response in patients with HNSCC.

Tumor Protein p63/microRNA Network in Epithelial Cancer Cells.

Non-coding microRNAs are involved in multiple regulatory mechanisms underlying response of cancer cells to stress leading to apoptosis, cell cycle arrest and autophagy. Many molecular layers are implicated in such cellular response including epigenetic regulation of transcription, RNA processing, metabolism, signaling. The molecular interrelationship between tumor protein (TP)-p53 family members and specific microRNAs is a key functional network supporting tumor cell response to chemotherapy and potentially playing a decisive role in chemoresistance of human epithelial cancers. TP63 was shown to modulate the expression of numerous microRNAs involved in regulation of epithelial cell proliferation, differentiation, senescence, "stemness" and skin maintenance, epithelial/ mesenchymal transition, and tumorigenesis in several types of epithelial cancers (e.g. squamous cell carcinoma, ovarian carcinoma, prostate carcinoma, gastric cancer, bladder cancer, and breast tumors), as well as in chemoresistance of cancer cells. TP63/microRNA network was shown to be involved in cell cycle arrest, apoptosis, autophagy, metabolism and epigenetic transcriptional regulation, thereby providing the groundwork for novel chemotherapeutic venues.

Tumor protein p63/nuclear factor κB feedback loop in regulation of cell death.

Tumor protein (TP)-p53 family members often play proapoptotic roles, whereas nuclear factor κB (NF-κB) functions as a proapoptotic and antiapoptotic regulator depending on the cellular environment. We previously showed that the NF-κB activation leads to the reduction of the TP63 isoform, ΔNp63α, thereby rendering the cells susceptible to cell death upon DNA damage. However, the functional relationship between TP63 isotypes and NF-κB is poorly understood. Here, we report that the TAp63 regulates NF-κB transcription and protein stability subsequently leading to the cell death phenotype. We found that TAp63α induced the expression of the p65 subunit of NF-κB (RELA) and target genes involved in cell cycle arrest or apoptosis, thereby triggering cell death pathways in MCF10A cells. RELA was shown to concomitantly modulate specific cell survival pathways, making it indispensable for the TAp63α-dependent regulation of cell death. We showed that TAp63α and RELA formed protein complexes resulted in their mutual stabilization and inhibition of the RELA ubiquitination. Finally, we showed that TAp63α directly induced RelA transcription by binding to and activating of its promoter and, in turn, leading to activation of the NF-κB-dependent cell death genes. Overall, our data defined the regulatory feedback loop between TAp63α and NF-κB involved in the activation of cell death process of cancer cells.

DeltaNp63 induces beta-catenin nuclear accumulation and signaling.

The P53 homolog p63 encodes multiple proteins with transactivating, apoptosis-inducing, and oncogenic activities. We showed that p63 is amplified and that DeltaNp63 isotypes are overexpressed in squamous cell carcinoma (SCC) and enhance oncogenic growth in vitro and in vivo. Moreover, p53 associated with DeltaNp63alpha and mediated its degradation. Here, we report that DeltaNp63 associates with the B56alpha regulatory subunit of protein phosphatase 2A (PP2A) and glycogen synthase kinase 3beta (GSK3beta), leading to a dramatic inhibition of PP2A-mediated GSK3beta reactivation. The inhibitory effect of DeltaNp63 on GSK3beta mediates a decrease in phosphorylation levels of beta-catenin, which induces intranuclear accumulation of beta-catenin and activates beta-catenin-dependent transcription. Our results suggest that DeltaNp63 isotypes act as positive regulators of the beta-catenin signaling pathway, providing a basis for their oncogenic properties.

Phosphorylated TP63 induces transcription of RPN13, leading to NOS2 protein degradation.

Head and neck squamous cell carcinoma cells exposed to cisplatin display ATM-dependent phosphorylation of the most predominant TP63 isoform (ΔNp63α), leading to its activation as a transcription factor. Here, we found that the phospho-ΔNp63α protein binds to the genomic promoter of RPN13 through the TP63-responsive element. We further found that the phospho-ΔNp63α protein associates with other transcription factors (DDIT3 (also known as CHOP), NF-Y, and NF-κB), activating RPN13 gene transcription. Furthermore, cisplatin-induced and phospho-ΔNp63α-dependent RPN13 gene transcription leads to NOS2 degradation. Finally, we show that RPN13 knockdown by siRNA essentially rescues NOS2 from cisplatin-dependent inactivation. These data provide a novel mechanism for the phospho-ΔNp63α-dependent regulation of NOS2 function in cells upon cisplatin treatment, contributing to the cell death pathway of tumor cells.

Cigarette smoke induces promoter methylation of single-stranded DNA-binding protein 2 in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the sixth most frequent cause of cancer death in the world, and cigarette smoke is a key factor in esophageal carcinogenesis. To identify molecular changes during cigarette smoke-induced ESCC, we examined the methylation status of 13 gene promoters in the human immortalized, nontumorigenic esophageal epithelial cell line (Het-1A) that were exposed to mainstream (MSE) or sidestream cigarette smoke extract (SSE) for 6 months in culture. The promoter of sequence-specific single-stranded DNA-binding protein 2 (SSBP2) was methylated in the Het-1A cells exposed to MSE (MSE-Het-1A). Promoter methylation (86%, 56/70) and downregulation of SSBP2 expression were frequently detected in tumor tissues from ESCC patients. In addition, reintroduction of SSBP2 in an ESCC cell line (TE1) that does not express SSBP2 and in the MSE-Het-1A cells inhibited expression of LRP6 and Dvl3, which are mediators of the Wnt signaling pathway. SSBP2 expression markedly decreased the colony-forming ability of ESCC cell lines and significantly inhibited cell growth of the MSE-Het-1A cells. Our results indicate that cigarette smoking is a cause of SSBP2 promoter methylation and that SSBP2 harbors a tumor suppressive role in ESCC through inhibition of the Wnt signaling pathway.

ΔNp63α/IRF6 interplay activates NOS2 transcription and induces autophagy upon tobacco exposure.

Tobacco-induced oxidative stress leads to chronic inflammation and is implicated in the development of many human epithelial cancers, including head and neck cancer. Cigarette smoke exposure was shown to induce the expression of the ΔNp63α and nitric oxide synthase (NOS)-2 in head and neck squamous cell carcinoma cells and immortalized oral keratinocytes. The NOS2 promoter was found to contain various cognate sequences for several transcription factors including interferon regulatory factor (IRF)-6 and p63, which were shown in vivo binding to the NOS2 promoter in response to smoke exposure. Small interfering (si)-RNAs against both ΔNp63α and IRF6 decreased the induction of NOS2 promoter-driven reporter luciferase activity and were shown to inhibit NOS2 activity. Furthermore, both mainstream (MSE) and sidestream (SSE) smoking extracts induced changes in expression of autophagic marker, LC3B, while siRNA against ΔNp63α, IRF6 and NOS2 modulated these autophagic changes. Overall, these data support the notion that ΔNp63α/IRF6 interplay regulates NOS2 transcription, thereby underlying the autophagic-related cancer cell response to tobacco exposure.

CDC91L1 (PIG-U) is a newly discovered oncogene in human bladder cancer.

Genomic amplification at 20q11-13 is a common event in human cancers. We isolated a germline translocation breakpoint at 20q11 from a bladder cancer patient. We identified CDC91L1, the gene encoding CDC91L1 (also called phosphatidylinositol glycan class U (PIG-U), a transamidase complex unit in the glycosylphosphatidylinositol (GPI) anchoring pathway), as the only gene whose expression was affected by the translocation. CDC91L1 was amplified and overexpressed in about one-third of bladder cancer cell lines and primary tumors, as well as in oncogenic uroepithelial cells transformed with human papillomavirus (HPV) E7. Forced overexpression of CDC91L1 malignantly transformed NIH3T3 cells in vitro and in vivo. Overexpression of CDC91L1 also resulted in upregulation of the urokinase receptor (uPAR), a GPI-anchored protein, and in turn increased STAT-3 phosphorylation in bladder cancer cells. Our findings suggest that CDC91L1 is an oncogene in bladder cancer, and implicate the GPI anchoring system as a potential oncogenic pathway and therapeutic target in human cancers.

Cellular transformation by cigarette smoke extract involves alteration of glycolysis and mitochondrial function in esophageal epithelial cells.

Cigarette-smoking increases the risk of developing various types of human cancers including esophageal cancers. To test the effects of chronic cigarette smoke exposure directly on esophageal epithelium, cellular resistance to mainstream extract (MSE), or sidestream smoke extract (SSE) was developed in chronically exposed nonmalignant Het-1A cells. Anchorage-independent growth, in vitro invasion capacity and proliferation of the resistant cells increased compared with the unexposed, sensitive cells. An epithelial marker E-cadherin was down-regulated and mesenchymal markers N-cadherin and vimentin were up-regulated in the resistant cells. Het-1A cells resistant to MSE or SSE consumed more glucose, and produced more lactate than the sensitive cells. The increased anchorage-independent cell growth of the resistant cells was suppressed by a glycolysis inhibitor, 2-deoxy-D-glucose, indicating that these cells are highly dependent on the glycolytic pathway for survival. Decreased mitochondrial membrane potential and ATP production in the resistant cells indicate the presence of mitochondrial dysfunction induced by chronic exposure of cigarette smoke extract. Increased expression of nuclear genes in the glycolytic pathway and decreased levels of mitochondrial genes in the resistant cells support the notion that cigarette smoking significantly contributes to the transformation of nonmalignant esophageal epithelial cells into a tumorigenic phenotype.

International Research Symposium on Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate (AEC) syndrome.

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome (Hay-Wells syndrome, MIM #106220) is a rare autosomal dominant ectodermal dysplasia syndrome. It is due to mutations in the TP63 gene, known to be a regulatory gene with many downstream gene targets. TP63 is important in the differentiation and proliferation of the epidermis, as well as many other processes including limb and facial development. It is also known that mutations in TP63 lead to skin erosions. These erosions, especially on the scalp, are defining features of AEC syndrome and cause significant morbidity and mortality in these patients. It was this fact that led to the 2003 AEC Skin Erosion Workshop. That conference laid the groundwork for the International Research Symposium for AEC Syndrome held at Texas Children's Hospital in 2006. The conference brought together the largest cohort of individuals with AEC syndrome, along with a multitude of physicians and scientists. The overarching goals were to define the clinical and pathologic findings for improved diagnostic criteria, to obtain tissue samples for further study and to define future research directions. The symposium was successful in accomplishing these aims as detailed in this conference report. Following our report, we also present 11 manuscripts within this special section that outline the collective clinical, pathologic, and mutational data from 18 individuals enrolled in the concurrent Baylor College of Medicine IRB-approved protocol: Characterization of AEC syndrome. These collaborative findings will hopefully provide a stepping-stone to future translational projects of TP63 and TP63-related syndromes.

Midkine promotes tetraspanin-integrin interaction and induces FAK-Stat1alpha pathway contributing to migration/invasiveness of human head and neck squamous cell carcinoma cells.

The heparin-binding growth factor, MK, promoting tumorigenesis and survival was found to associate with alpha6beta1 integrins. We showed for the first time that MK interacted with TSPAN1 and facilitated the association between TSPAN1 and integrin alpha6beta1 proteins in head and neck squamous cell carcinoma (HNSCC) cells. We found that MK mediated an integrin-dependent tyrosine phosphorylation of FAK and activation of paxillin and Stat1alpha pathways. As result, downstream target genes implicated in cell migration and invasiveness (e.g. MMP-2 and MMP-26) were overexpressed. We observed that RNAi silencing of the critical signaling intermediates led to decrease of MK-induced migration/invasiveness of HNSCC cells. The major finding of this study is a novel MK-triggered signaling mechanism implicated in migration and invasiveness of HNSCC cells.

Methylation of the DFNA5 increases risk of lymph node metastasis in human breast cancer.

The pathogenesis of breast cancer involves multiple genetic and epigenetic events. In this study, we report an epigenetic alteration of DFNA5 in human breast cancer. DFNA5 gene was silenced in breast cancer cell lines that were methylated in the DFNA5 promoter, and restored by treatment with the demethylating agent, 5-aza-dC, and gene knock-down of DFNA5 increased cellular invasiveness in vitro. The mRNA expression of DFNA5 in breast cancer tissues was down-regulated as compared to normal tissues. Moreover, the DFNA5 promoter was found to be methylated in primary tumor tissues with high frequency (53%, 18/34). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary breast cancer tissues from normal breast tissues (15.3%, 2/13). Moreover, methylation status of DFNA5 was correlated with lymph node metastasis in breast cancer patients. Our data implicate DFNA5 promoter methylation as a novel molecular biomarker in human breast cancer.

Membrane trafficking of AQP5 and cAMP dependent phosphorylation in bronchial epithelium.

Phosphorylation pathway has been identified as an important step in membrane trafficking for AQP5. We generated stably transfected BEAS-2B human bronchial epithelial cells with various over-expression constructs on permeable support. In stable cells with wild-type AQP5 and S156A (AQP5 mutant targeting PKA consensus sequence), AQP5 expression was predominantly polarized to the apical membrane, whereas stable cells with N185D (AQP5 mutant targeting second NPA motif), mainly localized to the cytoplasm. Treatment with H89 and/or chlorophenylthio-cAMP (cpt-cAMP) did not affect membrane expression of AQP5 in any of three stable cells. In cells with wild-type AQP5 and N185D, AQP5s were phosphorylated by PKA, while phosphorylation of AQP5 was not detected in cells with S156A. These results indicate that, in AQP5, serine156 may be phosphorylated by PKA, but membrane expression of AQP5 may not be regulated by PKA phosphorylation. We conclude that AQP5 membrane targeting can include more than one mechanism besides cAMP dependent phosphorylation.