Loss of Canonical Smad4 Signaling Promotes KRAS Driven Malignant Transformation of Human Pancreatic Duct Epithelial Cells and Metastasis

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRAS ^G12V expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells.     

Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

The recent development of mutant-selective inhibitors for the oncogenic KRAS^G12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS^G12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pK_a of the C12 thiol is depressed (pK_a ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRAS^Y137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS^G12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, K_d = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pK_a of the C12 thiol, we found that reversible and irreversible oxidation of KRAS^G12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS^G12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRAS^G12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS^G12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.     

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients

Background

Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation.

Methods

Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene.

Results

The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38–76 %, depending on the gene. The highest MI value was found for RASSF1A (52 %) and the lowest for NPRL2/G21 (5 %). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71 % tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = −0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75–92 % NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found.

Conclusions

The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn’t seem to be a critical determinate of its promoter hypermethylation.     

Remifentanil up‐regulates HIF1α expression to ameliorate hepatic ischaemia/reperfusion injury via the ZEB1/LIF axis

Ischaemia/reperfusion (I/R)‐induced hepatic injury is regarded as a main reason of hepatic failure after transplantation or lobectomy. The current study aimed to investigate how the opioid analgesic remifentanil treatment affects I/R‐induced hepatic injury and explore the possible mechanisms related to HIF1α. Initially, an I/R‐induced hepatic injury animal model was established in C57BL/6 mice, and an in vitro hypoxia‐reoxygenation model was constructed in NCTC‐1469 cells, followed by remifentanil treatment and HIF1α silencing treatment. The levels of blood glucose, lipids, alanine transaminase (ALT) and aspartate transaminase (AST) in mouse serum were measured using automatic chemistry analyser, while the viability and apoptosis of cells were detected using CCK8 assay and flow cytometry. Our results revealed that mice with I/R‐induced hepatic injury showed higher serum levels of blood glucose, lipids, ALT and AST and leukaemia inhibitory factor (LIF) expression, and lower HIF1α and ZEB1 expression (P < .05), which were reversed after remifentanil treatment (P < .05). Besides, HIF1α silencing increased the serum levels of blood glucose, lipids, ALT and AST (P < .05). Furthermore, hypoxia‐induced NCTC‐1469 cells exhibited decreased HIF1α and ZEB1 expression, reduced cell viability, as well as increased LIF expression and cell apoptosis (P < .05), which were reversed by remifentanil treatment (P < .05). Moreover, HIF1α silencing down‐regulated ZEB1 expression, decreased cell viability, and increased cell apoptosis (P < .05). ZEB1 was identified to bind to the promoter region of LIF and inhibit its expression. In summary, remifentanil protects against hepatic I/R injury through HIF1α and downstream effectors.     

An AP Endonuclease Functions in Active DNA Demethylation and Gene Imprinting in Arabidopsis

Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l^+/−zdp^−/− mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.     

The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferentially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA polymerase beta in long patch base excision repair

Growing evidence suggests that the Rad9-Rad1-Hus1 complex (the 9-1-1 complex), besides its functions in DNA damage sensing and signaling pathways, plays also a direct role in various DNA repair processes. Recent studies have demonstrated that the 9-1-1 complex physically and functionally interacts with several components of the base excision repair (BER) machinery namely DNA polymerase β (Pol β), flap endonuclease 1 (Fen 1), DNA ligase I (Lig I) and the MutY homologue of Schizosaccharomyces pombe. In this work, we found for the first time that the 9-1-1 complex interacts in vitro and in vivo with the apurinic/apyrimidinic endonuclease 1 (APE 1), an early component of BER, and can stimulate its AP-endonuclease activity. Moreover, we show that the 9-1-1 complex possesses a stimulatory effect on long patch base excision repair (LP-BER) reconstituted in vitro. The enhancement of LP-BER activity is due to the specific stimulation of the two early components of the repair machinery, namely APE 1 and Pol β, suggesting a hierarchy of interactions between the 9-1-1 complex and the BER proteins acting in the repairosome. Overall, our results indicate that the 9-1-1 complex is directly involved in LP-BER, thus providing a possible link between DNA damage checkpoints and BER.     

RNA-cleaving properties of human apurinic/apyrimidinic endonuclease 1 (APE1)

We have recently identified apurinic/apyrimidinic endonuclease 1 (APE1) as an endoribonuclease that cleaves c-myc mRNA in vitro and regulates c-myc mRNA levels and half-life in cells. This study was undertaken to further unravel the RNA-cleaving properties of APE1. Here, we show that APE1 cleaves RNA in the absence of divalent metal ions and, at 2 mM, Zn²⁺, Ni²⁺, Cu²⁺, or Co²⁺ inhibited the endoribonuclease activity of APE1. APE1 is able to cleave CD44 mRNA, microRNAs (miR-21, miR-10b), and three RNA components of SARS-corona virus (orf1b, orf3, spike) suggesting that, when challenged, it can cleave any RNAs in vitro. APE1 does not cleave strong doublestranded regions of RNA and it has a strong preference for 3’ of pyrimidine, especially towards UA, CA, and UG sites at single-stranded or weakly paired regions. It also cleaves RNA weakly at UC, CU, AC, and AU sites in single-stranded or weakly paired regions. Finally, we found that APE1 can reduce the ability of the Dicer enzyme to process premiRNAs in vitro. Overall, this study has revealed some previously unknown biochemical properties of APE1 which has implications for its role in vivo.     

Aptamer Selection Based on G4-Forming Promoter Region

We developed a method for aptamer identification without in vitro selection. We have previously obtained several aptamers, which may fold into the G-quadruplex (G4) structure, against target proteins; therefore, we hypothesized that the G4 structure would be an excellent scaffold for aptamers to recognize the target protein. Moreover, the G4-forming sequence contained in the promoter region of insulin can reportedly bind to insulin. We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products. We designated this aptamer identification method as “G4 promoter-derived aptamer selection (G4PAS).” Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers. Surface plasmon resonance (SPR) analysis revealed that the dissociation constant (K _d) values of VEGF165, PDGF-AA, and RB1 DNA aptamers were 1.7 × 10⁻⁷ M, 6.3 × 10⁻⁹ M, and 4.4 × 10⁻⁷ M, respectively. G4PAS is a simple and rapid method of aptamer identification because it involves only binding analysis of G4 DNAs to the target protein. In the human genome, over 40% of promoters contain one or more potential G4 DNAs. G4PAS could therefore be applied to identify aptamers against target proteins that contain G4 DNAs on their promoters.     

Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta

X-ray analysis of enzyme–DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9–10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (ΔG° ~ −8.7 to −9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1–DNA interactions provide only one order of magnitude (ΔG° ~ −1.1 to −1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.     

Somatic Mutations in Exocrine Pancreatic Tumors: Association with Patient Survival

KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.     

Rational design of small-molecules to recognize G-quadruplexes of c-MYC promoter and telomere and the evaluation of their in vivo antitumor activity against breast cancer

DNA G4-structures from human c-MYC promoter and telomere are considered as important drug targets; however, the developing of small-molecule-based fluorescent binding ligands that are highly selective in targeting these G4-structures over other types of nucleic acids is challenging. We herein report a new approach of designing small molecules based on a non-selective thiazole orange scaffold to provide two-directional and multi-site interactions with flanking residues and loops of the G4-motif for better selectivity. The ligands are designed to establish multi-site interactions in the G4-binding pocket. This structural feature may render the molecules higher selectivity toward c-MYC G4s than other structures. The ligand–G4 interaction studied with ¹H NMR may suggest a stacking interaction with the terminal G-tetrad. Moreover, the intracellular co-localization study with BG4 and cellular competition experiments with BRACO-19 may suggest that the binding targets of the ligands in cells are most probably G4-structures. Furthermore, the ligands that either preferentially bind to c-MYC promoter or telomeric G4s are able to downregulate markedly the c-MYC and hTERT gene expression in MCF-7 cells, and induce senescence and DNA damage to cancer cells. The in vivo antitumor activity of the ligands in MCF-7 tumor-bearing mice is also demonstrated.     

Methylation of the Claudin 1 Promoter Is Associated with Loss of Expression in Estrogen Receptor Positive Breast Cancer

Downregulation of the tight junction protein claudin 1 is a frequent event in breast cancer and is associated with recurrence, metastasis, and reduced survival, suggesting a tumor suppressor role for this protein. Tumor suppressor genes are often epigenetically silenced in cancer. Downregulation of claudin 1 via DNA promoter methylation may thus be an important determinant in breast cancer development and progression. To investigate if silencing of claudin 1 has an epigenetic etiology in breast cancer we compared gene expression and methylation data from 217 breast cancer samples and 40 matched normal samples available through the Cancer Genome Atlas (TCGA). Moreover, we analyzed claudin 1 expression and methylation in 26 breast cancer cell lines. We found that methylation of the claudin 1 promoter CpG island is relatively frequent in estrogen receptor positive (ER+) breast cancer and is associated with low claudin 1 expression. In contrast, the claudin 1 promoter was not methylated in most of the ER-breast cancers samples and some of these tumors overexpress claudin 1. In addition, we observed that the demethylating agents, azacitidine and decitabine can upregulate claudin 1 expression in breast cancer cell lines that have a methylated claudin 1 promoter. Taken together, our results indicate that DNA promoter methylation is causally associated with downregulation of claudin 1 in a subgroup of breast cancer that includes mostly ER+ tumors, and suggest that epigenetic therapy to restore claudin 1 expression might represent a viable therapeutic strategy in this subtype of breast cancer.