Small molecule targeting of transcription-replication conflict for selective chemotherapy.

Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeutic avenue for exploiting this cancer-selective vulnerability.

Novel Peptide Therapeutic Approaches for Cancer Treatment.

Peptides are increasingly being developed for use as therapeutics to treat many ailments, including cancer. Therapeutic peptides have the advantages of target specificity and low toxicity. The anticancer effects of a peptide can be the direct result of the peptide binding its intended target, or the peptide may be conjugated to a chemotherapy drug or radionuclide and used to target the agent to cancer cells. Peptides can be targeted to proteins on the cell surface, where the peptide-protein interaction can initiate internalization of the complex, or the peptide can be designed to directly cross the cell membrane. Peptides can induce cell death by numerous mechanisms including membrane disruption and subsequent necrosis, apoptosis, tumor angiogenesis inhibition, immune regulation, disruption of cell signaling pathways, cell cycle regulation, DNA repair pathways, or cell death pathways. Although using peptides as therapeutics has many advantages, peptides have the disadvantage of being easily degraded by proteases once administered and, depending on the mode of administration, often have difficulty being adsorbed into the blood stream. In this review, we discuss strategies recently developed to overcome these obstacles of peptide delivery and bioavailability. In addition, we present many examples of peptides developed to fight cancer.

A Quantitative Analysis of Surgical Smoke Exposure as an Occupational Hazard.

OBJECTIVE: We hypothesized that OR airborne PM was different in quantity and mutagenic potential than office air and cigarette smoke. SUMMARY OF BACKGROUND DATA: Exposure to surgical smoke has been equated to cigarette smoking and thought to be hazardous to health care workers despite limited data. METHODS: PM was measured during 15 operations in ORs with 24.8 ±â€Š2.0 air changes/h, and in controls (cigarettes, office air with 1.9-2.9 air changes/h). Mutagenic potential was assessed by gamma Histone 2A family member X staining of DNA damage in small airway epithelial cells co-cultured with PM. RESULTS: Average PM concentration during surgery was 0.002 ±â€Š0.002 mg/m3 with maximum values at 1.08 ±â€Š1.30 mg/m3. Greater PM correlated with more diathermy (ρ = 0.69, P = 0.006). Values were most often near zero, resulting in OR average values similar to office air (0.002 ±â€Š0.001 mg/m3) (P = 0.32). Cigarette smoke average PM concentration was significantly higher, 4.8 ±â€Š5.6 mg/m3 (P < 0.001). PM collected from 14 days of OR air caused DNA damage to 1.6% ±â€Š2.7% of cultured cells, significantly less than that from office air (27.7% ±â€Š11.7%, P = 0.02), and cigarette smoke (61.3% ±â€Š14.3%, P < 0.001). CONCLUSIONS: The air we breathe during surgery has negligible quantities of PM and mutagenic potential, likely due to low frequency of diathermy use coupled with high airflow. This suggests that exposure to surgical smoke is associated with minimal occupational risk.

Molecular Targeting of Cancer-Associated PCNA Interactions in Pancreatic Ductal Adenocarcinoma Using a Cell-Penetrating Peptide.

Pancreatic ductal adenocarcinoma is a particularly difficult cancer to treat due to a lack of effective screening or treatment. Pancreatic cancer cells exhibit high proliferating cell nuclear antigen (PCNA) expression, which is associated with poor prognosis. PCNA, an important nuclear DNA replication and repair protein, regulates a myriad of proteins via the interdomain connector loop. Within this region, amino acids 126-133 are critical for PCNA interactions in cancer cells. Here, we investigate the ability of a decoy cell-penetrating peptide, R9-caPeptide, that mimics the interdomain connector loop region of PCNA to disrupt PCNA-protein interactions in pancreatic cancer cells. Our data suggest that R9-caPeptide causes dose-dependent toxicity in a panel of pancreatic cancer cell lines by inhibiting DNA replication fork progression and PCNA-regulated DNA repair, ultimately causing lethal DNA damage. Overall, these studies lay the foundation for novel therapeutic strategies that target PCNA in pancreatic cancer.

The Anticancer Activity of a First-in-class Small-molecule Targeting PCNA.

PURPOSE: Proliferating cell nuclear antigen (PCNA) plays an essential role in regulating DNA synthesis and repair and is indispensable to cancer cell growth and survival. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was ubiquitously expressed in a broad range of cancer cells and tumor tissues, but not significantly in nonmalignant cells. We found the L126-Y133 region of caPCNA is structurally altered and more accessible to protein-protein interaction. A cell-permeable peptide harboring the L126-Y133 sequence blocked PCNA interaction in cancer cells and selectively kills cancer cells and xenograft tumors. On the basis of these findings, we sought small molecules targeting this peptide region as potential broad-spectrum anticancer agents. EXPERIMENTAL DESIGN: By computer modeling and medicinal chemistry targeting a surface pocket partly delineated by the L126-Y133 region of PCNA, we identified a potent PCNA inhibitor (AOH1160) and characterized its therapeutic properties and potential toxicity. RESULTS: AOH1160 selectively kills many types of cancer cells at below micromolar concentrations without causing significant toxicity to a broad range of nonmalignant cells. Mechanistically, AOH1160 interferes with DNA replication, blocks homologous recombination-mediated DNA repair, and causes cell-cycle arrest. It induces apoptosis in cancer cells and sensitizes them to cisplatin treatment. AOH1160 is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it does not cause significant toxicity at 2.5 times of an effective dose. CONCLUSIONS: These results demonstrated the favorable therapeutic properties and the potential of AOH1160 as a broad-spectrum therapeutic agent for cancer treatment.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

The Mechanism by Which MYCN Amplification Confers an Enhanced Sensitivity to a PCNA-Derived Cell Permeable Peptide in Neuroblastoma Cells.

Dysregulated expression of MYC family genes is a hallmark of many malignancies. Unfortunately, these proteins are not amenable to blockade by small molecules or protein-based therapeutic agents. Therefore, we must find alternative approaches to target MYC-driven cancers. Amplification of MYCN, a MYC family member, predicts high-risk neuroblastoma (NB) disease. We have shown that R9-caPep blocks the interaction of PCNA with its binding partners and selectively kills human NB cells, especially those with MYCN amplification, and we now show the mechanism. We found elevated levels of DNA replication stress in MYCN-amplified NB cells. R9-caPep exacerbated DNA replication stress in MYCN-amplified NB cells and NB cells with an augmented level of MYC by interfering with DNA replication fork extension, leading to Chk1 dependence and susceptibility to Chk1 inhibition. We describe how these effects may be exploited for treating NB.

Expression of a novel peptide derived from PCNA damages DNA and reverses cisplatin resistance.

BACKGROUND: An 8 amino acid peptide sequence derived from proliferating cell nuclear antigen (PCNA) has been shown to effectively kill several breast cancer and neuroblastoma cell lines when added exogenously to cell cultures. METHODS: In this study, the expression of the 8 amino acid peptide sequence (caPeptide) was placed under control of a tetracycline responsive promoter in MDA-MB-231 cells. RESULTS: Endogenous expression of the peptide resulted in an increase in genomic DNA damage. CaPeptide induction combined with treatment of sublethal doses of cisplatin resulted in a marked increase in death of the cisplatin-resistant MDA-MB-231 cell line. CaPeptide was found to interact with POLD3, one of the subunits of DNA polymerase delta necessary for binding to PCNA. CONCLUSION: These results suggest an important line of inquiry into the possible role that caPeptide might play in the reversal of cisplatin resistance in breast and other cancers. This is of particular interest in those cancers where cisplatin is the first line of chemotherapy and where the acquisition of resistance is a common malady.

CRM1 mediates nuclear-cytoplasmic shuttling of mature microRNAs.

Drosha-processed microRNAs (miRNAs) have been shown to be exported from the nucleus to the cytoplasm by Exportin 5, where they are processed a second time to generate mature miRNAs. In this work we show that miRNAs also use CRM1 for nuclear-cytoplasmic shuttling. Inhibition of CRM1 by Leptomycin B results in nuclear accumulation of miRNA guide sequences. Nuclear to cytoplasmic transport can be actively competed by synthetic small interfering RNAs, indicating that this pathway is shared by different classes of processed small RNAs. We also find that CRM1 coimmunoprecipitates with Ago-1, Ago-2, Topo2alpha, EzH2, and Mta, consistent with a role of Argonautes and small RNAs in chromatin remodeling.

Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC.

Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs.