Eddy S. Yang

Genomic instability in human cancer: Molecular insights and opportunities for therapeutic attack and prevention through diet and nutrition

Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology.

The poly(ADP-Ribose) polymerase inhibitor ABT-888 reduces radiation-induced nuclear EGFR and augments head and neck tumor response to radiotherapy ☆

BACKGROUND AND PURPOSE Current therapies for head and neck cancer frequently are not curative, necessitating novel therapeutic strategies. Thus, we studied whether inhibition of poly(ADP-Ribose) polymerase (PARP), a key DNA repair enzyme, could improve efficacy of radiotherapy in human head and neck cancer. MATERIALS AND METHODS UM-SCC1, UM-SCC5, UM-SCC6, and FaDu human head and neck cancer cellular susceptibility to the PARP inhibitor (PARPi) ABT-888 and/or radiation (IR) was assessed using colony formation assays. DNA damage was evaluated using the alkaline comet assay and immunostaining for γ-H2AX foci. Non-homologous end-joining (NHEJ) mediated repair was measured using phospho-DNA-Pk foci. Epidermal growth factor receptor (EGFR) location was assessed by immunostaining. Poly ADP-Ribose polymerization (PAR) levels were assessed using immunoblotting. RESULTS Human head and neck cancer cells exhibited enhanced cytotoxicity with IR and ABT-888 compared to either agent alone. This increased susceptibility correlated with reduced nuclear EGFR, attenuation of NHEJ, and persistence of DNA damage following IR. Interestingly, a subset of head and neck cancer cells which had elevated basal PAR levels was susceptible to PARPi alone. CONCLUSIONS Combining radiotherapy and PARP inhibition may improve outcomes and quality of life for head and neck cancer patients treated with radiotherapy. Furthermore, this novel strategy may also be feasible in other tumor types. Moreover, PAR levels should be investigated as a potential biomarker for tumor susceptibility to PARP inhibition.

Beyond DNA Repair: Additional Functions of PARP-1 in Cancer

Poly(ADP-ribose) polymerases (PARPs) are DNA-dependent nuclear enzymes that transfer negatively charged ADP-ribose moieties from cellular nicotinamide-adenine-dinucleotide (NAD+) to a variety of protein substrates, altering protein–protein and protein-DNA interactions. The most studied of these enzymes is poly(ADP-ribose) polymerase-1 (PARP-1), which is an excellent therapeutic target in cancer due to its pivotal role in the DNA damage response. Clinical studies have shown susceptibility to PARP inhibitors in DNA repair defective cancers with only mild adverse side effects. Interestingly, additional studies are emerging which demonstrate a role for this therapy in DNA repair proficient tumors through a variety of mechanisms. In this review, we will discuss additional functions of PARP-1 – including regulation of inflammatory mediators, cellular energetics and death pathways, gene transcription, sex hormone- and ERK-mediated signaling, and mitosis – and the role these PARP-1-mediated processes play in oncogenesis, cancer progression, and the development of therapeutic resistance. As PARP-1 can act in both a pro- and anti-tumor manner depending on the context, it is important to consider the global effects of this protein in determining when, and how, to best use PARP inhibitors in anticancer therapy.

Synthetic lethal interactions between EGFR and PARP inhibition in human triple negative breast cancer cells.

Few therapeutic options exist for the highly aggressive triple negative breast cancers (TNBCs). In this study, we report that a contextual synthetic lethality can be achieved both in vitro and in vivo with combined EGFR and PARP inhibition with lapatinib and ABT-888, respectively. The mechanism involves a transient DNA double strand break repair deficit induced by lapatinib and subsequent activation of the intrinsic pathway of apoptosis. Further dissection of the mechanism reveals that EGFR and BRCA1 can be found in the same protein complex, which is reduced by lapatinib. Interestingly, lapatinib also increases cytosolic BRCA1 and EGFR, away from their nuclear DNA repair substrates. Taken together, these results reveal a novel regulation of homologous recombination repair involving EGFR and BRCA1 interaction and alteration of subcellular localization. Additionally, a contextual synthetic lethality may exist between combined EGFR and PARP inhibitors.

PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation.

Cetuximab augments cytotoxicity with poly (adp-ribose) polymerase inhibition in head and neck cancer.

Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of head and neck cancers and confers increased resistance and inferior survival rates. Despite targeted agents against EGFR, such as cetuximab (C225), almost half of treated patients fail this therapy, necessitating novel therapeutic strategies. Poly (ADP-Ribose) polymerase (PARP) inhibitors (PARPi) have gained recent attention due to their unique selectivity in killing tumors with defective DNA repair. In this study, we demonstrate that C225 enhances cytotoxicity with the PARPi ABT-888 in UM-SCC1, UM-SCC6, and FaDu head and neck cancer cells. The mechanism of increased susceptibility to C225 and PARPi involves C225-mediated reduction of non-homologous end-joining (NHEJ)- and homologous recombination (HR)-mediated DNA double strand break (DSB) repair, the subsequent persistence of DNA damage, and activation of the intrinsic apoptotic pathway. By generating a DSB repair deficiency, C225 can render head and neck tumor cells susceptible to PARP inhibition. The combination of C225 and the PARPi ABT-888 can thus be an innovative treatment strategy to potentially improve outcomes in head and neck cancer patients. Furthermore, this strategy may also be feasible for other EGFR overexpressing tumors, including lung and brain cancers.

The BET bromodomain inhibitor JQ1 suppresses growth of pancreatic ductal adenocarcinoma in patient-derived xenograft models

The primary aim of this study was to evaluate the antitumor efficacy of the bromodomain inhibitor JQ1 in pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (tumorgraft) models. A secondary aim of the study was to evaluate whether JQ1 decreases expression of the oncogene c-Myc in PDAC tumors, as has been reported for other tumor types. We used five PDAC tumorgraft models that retain specific characteristics of tumors of origin to evaluate the antitumor efficacy of JQ1. Tumor-bearing mice were treated with JQ1 (50 mg/kg daily for 21 or 28 days). Expression analyses were performed with tumors harvested from host mice after treatment with JQ1 or vehicle control. An nCounter PanCancer Pathways Panel (NanoString Technologies) of 230 cancer-related genes was used to identify gene products affected by JQ1. Quantitative RT–PCR, immunohistochemistry and immunoblots were carried out to confirm that changes in RNA expression reflected changes in protein expression. JQ1 inhibited the growth of all five tumorgraft models (P<0.05), each of which harbors a KRAS mutation; but induced no consistent change in expression of c-Myc protein. Expression profiling identified CDC25B, a regulator of cell cycle progression, as one of the three RNA species (TIMP3, LMO2 and CDC25B) downregulated by JQ1 (P<0.05). Inhibition of tumor progression was more closely related to decreased expression of nuclear CDC25B than to changes in c-Myc expression. JQ1 and other agents that inhibit the function of proteins with bromodomains merit further investigation for treating PDAC tumors. Work is ongoing in our laboratory to identify effective drug combinations that include JQ1.

Endoglin (CD105) Contributes to Platinum Resistance and Is A Target for Tumor-Specific Therapy in Epithelial Ovarian Cancer

Purpose: Endoglin (CD105) is a membranous protein overexpressed in tumor-associated endothelial cells, chemoresistant populations of ovarian cancer cells, and potentially stem cells. Our objective was to evaluate the effects and mechanisms of targeting endoglin in ovarian cancer. Experimental Design: Global and membranous endoglin expression was evaluated in multiple ovarian cancer lines. In vitro, the effects of siRNA-mediated endoglin knockdown with and without chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation, and quantitative PCR. In an orthotopic mouse model, endoglin was targeted with chitosan-encapsulated siRNA with and without carboplatin. Results: Endoglin expression was surprisingly predominantly cytoplasmic, with a small population of surface-positive cells. Endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Targeting endoglin downregulates expression of numerous DNA repair genes, including BARD1, H2AFX, NBN, NTHL1, and SIRT1. BARD1 was also associated with platinum resistance, and was induced by platinum exposure. In vivo, antiendoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared with control (35%–41% reduction, P < 0.05). Endoglin inhibition with carboplatin was associated with even greater inhibitory effect when compared with control (58%–62% reduction, P < 0.001). Conclusions: Endoglin downregulation promotes apoptosis, induces significant DNA damage through modulation of numerous DNA repair genes, and improves platinum sensitivity both in vivo and in vitro. Antiendoglin therapy would allow dual treatment of both tumor angiogenesis and a subset of aggressive tumor cells expressing endoglin and is being actively pursued as therapy in ovarian cancer. Clin Cancer Res; 19(1); 170–82. ©2012 AACR.

BRCA1-Ku80 Protein Interaction Enhances End-joining Fidelity of Chromosomal Double-strand Breaks in the G1 Phase of the Cell Cycle

Background: Quality control of DNA double-strand break repair is poorly understood. Results: BRCA1 enhances the fidelity of NHEJ repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G1. Conclusion: BRCA1 ensures high fidelity repair of NHEJ and thus prevents deletional end-joining of chromosomal DSBs during G1. Significance: BRCA1 not only promotes homologous recombination in G2/M phase but also facilitates fidelity of NHEJ repair during G1. Quality control of DNA double-strand break (DSB) repair is vital in preventing mutagenesis. Non-homologous end-joining (NHEJ), a repair process predominant in the G1 phase of the cell cycle, rejoins DSBs either accurately or with errors, but the mechanisms controlling its fidelity are poorly understood. Here we show that BRCA1, a tumor suppressor, enhances the fidelity of NHEJ-mediated DSB repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G1. BRCA1 binds and stabilizes Ku80 at DSBs through its N-terminal region, promotes precise DSB rejoining, and increases cellular resistance to radiation-induced DNA damage in a G1 phase-specific manner. These results suggest that BRCA1, as a central player in genome integrity maintenance, ensures high fidelity repair of DSBs by not only promoting homologous recombination repair in G2/M phase but also facilitating fidelity of Ku80-dependent NHEJ repair, thus preventing deletional end-joining of chromosomal DSBs during G1.

Inhibition of STAT-3 results in greater cetuximab sensitivity in head and neck squamous cell carcinoma

OBJECTIVE The inhibition of epidermal growth factor receptor (EGFr) with the monoclonal antibody cetuximab reduces cell proliferation and survival which correlates with increased DNA damage. Since the signal transducer and activator of transcription-3 (STAT-3) is involved in the EGFr-induced signaling pathway, we hypothesized that depletion of STAT-3 may augment cetuximab-induced processes in human head and neck cancer cells. MATERIALS AND METHODS Human head and neck squamous carcinoma cells (UM-SCC-5) were transfected with short hairpin RNA (shRNA) against STAT-3 (STAT3-2.4 and 2.9 cells). A mutated form of this shRNA was transfected for a control (NEG4.17 cells). Radiosensitivity was assessed by a standard colony formation assay. Proliferation was assessed by daily cell counts following treatment and apoptosis was assessed by an annexin V-FITC assay. The alkaline comet assay was used to assess DNA damage. RESULTS The STAT-3 knockdown cells (STAT3-2.4 and STAT3-2.9 cells) demonstrated enhanced radiosensitivity compared to control NEG4.17 cells, which correlated with increased apoptosis. Also, the STAT-3 knockdown cells demonstrated decreased proliferation with cetuximab treatments compared to control cells (NEG4.17). The increased cetuximab sensitivity of the STAT-3 knockdown cells correlated with increased apoptosis and DNA damage compared to control cells (NEG4.17). CONCLUSION These studies revealed that the greater anti-proliferative effects and increased cytotoxicity of cetuximab in the STAT3-2.4 and STAT3-2.9 cells compared to control NEG4.17 cells, may be a result of STAT3-mediated effects on cellular apoptosis and DNA damage.