Focus Formation Assay Research Articles

Abstract 840: ATR inhibition is a promising radiosensitizing strategy for chemotherapy-resistant triple negative breast cancer (TNBC)

Abstract Purpose: TNBC represents an aggressive subtype characterized by earlier relapse and worse survival compared to non-TNBC. Patients with residual TNBC after neoadjuvant chemotherapy (NAC) have a high risk of locoregional recurrence despite aggressive subsequent local therapies, including RT. Thus, new radiosensitizers that address chemoresistant TNBC (i.e residual disease after NAC) are needed. ATR plays a key role in the cellular response to replication stress and DNA damage. TNBC is characterized by G1 checkpoint loss, homologous recombination deficiency (HRD), and other features which increase dependence on ATR pathway signaling. We hypothesized that VX-970, a selective inhibitor of ATR, would effectively radiosensitize TNBC. Methods: Cell lines representing varying subtypes of TNBC (MDA-MB-231, HCC1806, and BT-549) and the normal breast epithelial cell line MCF10a were investigated in clonogenic survival, cell cycle, and DNA damage signaling and repair assays. In addition, Mayo Clinic (MC)TNBC1 and MCTNBC2, patient derived xenograft models (PDXs) generated prospectively from baseline and chemoresistant residual disease surgical specimens after NAC, respectively, of unique patients in the Breast Cancer Genome Guided Therapy Study were analyzed. Tumors were injected into hind-legs of athymic nude mice and animals were randomized to: 1) Vehicle 2) RT 3) VX-970 4) RT + VX-970. RT was delivered in 5 daily fractions of 2 Gy. VX-970 was administered daily 1 hour prior to RT at a dose of 60 mg/kg. Exome sequencing was assessed for germ-line and/or somatic alterations in HR genes and an ex-vivo RAD51 foci formation assay was applied to confirm the functional status of HR in each PDX model. Results: In vitro, VX-970 preferentially inhibited ATR-Chk1-CDC25a signaling, abrogated the RT-induced G2/M checkpoint, delayed resolution of γH2AX and 53BP1 foci and reduced colony formation after RT in MDA-MB-231, HCC1806, and BT-549 relative to MCF10a. In vivo, VX-970 did not exhibit single agent activity at the dose administered but markedly sensitized both MCTNBC1 and MCTNBC2 to RT, suggesting that VX-970 could overcome chemoresistant TNBC biology. For MCTNBC1 and MCTNBC2, the median time to tumor tripling was 26 vs 49 days (p=0.002) and 25 vs 43 days (p=0.006) for the RT and RT + VX-970 groups, respectively. Ex-vivo RAD51 foci formation was robust in MCTNBC1, a PDX without HR gene alterations. In contrast, there was no RT-induced RAD51 foci formation in MCTNBC2, suggesting a BRCA1 mutation in the corresponding human tumor resulted in loss of HR function in that model. Conclusion: VX-970 + RT effectively radiosensitized TNBC PDXs established from both pre-treatment biopsy specimens and chemoresistant residual tumor samples and in both HR deficient and HR proficient settings. ATR inhibition should be investigated in the clinic in combination with RT for patients with residual TNBC after NAC. Citation Format: Robert W. Mutter, Mohamed M. Kahila, Xinyi Tu, Qin Zhou, Jia Yu, Krishna R. Kalari, Liewei Wang, Judy C. Boughey, Matthew P. Goetz, Jann N. Sarkaria, Zhenkun Lou. ATR inhibition is a promising radiosensitizing strategy for chemotherapy-resistant triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 840.

Abstract 334: Detection of BRCAness by a functional biomarker

Abstract Homologous recombination (HR) is an error-free repair pathway to eliminate DNA double-strand breaks and cells with deficiency of HR repair are highly sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Thus, targeting HR-defective cells becomes a new therapeutic approach for cancer patients. HR deficiency can be identified as BRCAness which may be due to mutations or epigenetic silencing in genes involved in HR, such as BRCA1 and BRCA2. Recent studies have developed several approaches to analyze HR status of cancer cells including RAD51 focus formation assay, large-scale mutational signatures analysis, and genome-wide transcriptome profiling. However, these assays provide an indirect estimation of HR status. We wish to directly measure HR rate in cancer cells. We have successfully developed two approaches, plasmid-based and virus-based functional assays, to directly quantify HR activity in cells and serve as a functional biomarker. Our results show that the signal of the functional biomarker specifically depends on the presence of the key enzyme, RAD51 recombinase, in HR. Finally, these functional assays can be utilized in different cancer cell types to determine the HR activity and can be a functional biomarker to evaluate the “BRCAness” status for the treatment of PARP inhibitors. Citation Format: Chih-Ying Lee, Min-Yu Ko, Kai-Hang Lei, Peter Chi. Detection of BRCAness by a functional biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 334.

Hypoxia restrains the expression of complement component 9 in tumor-associated macrophages promoting non-small cell lung cancer progression

The tumor microenvironment, including stroma cells, signaling molecules, and the extracellular matrix, critically regulates the growth and survival of cancer cells. Dissecting the active molecules in tumor microenvironment may uncover the key factors that can impact cancer progression. Human NSCLC tumor tissue-conditioned medium (TCM) and adjacent nontumor tissue-conditioned medium (NCM) were used to treat two NSCLC cells LSC1 and LAC1, respectively. Cell growth and foci formation assays were applied to assess the effects of TCM and NCM on cancer cells. The active factors were identified by protein mass spectrometry. Cell growth and foci formation assays showed that 8 of 26 NCM and none of TCM could effectively lead to tumor cell lysis, which was known as tumoricidal activity. And then protein mass spectrometry analysis and functional verifications confirmed that complement component 9 (C9) played a crucial role in the complement-dependent cytotoxicity (CDC)-mediated tumoricidal activity in vitro. Furthermore, immunofluorescent staining revealed that C9 specifically expressed in most alveolar macrophages (AMs) in adjacent lung tissues and a small fraction of tumor-associated macrophages (TAMs) in NSCLC tissues. Most importantly, the percentage of C9-positive cells in AMs or TAMs was responsible for the tumoricidal activity of NCM and TCM. Herein, we found that high expression of C9 in TAMs was a significant independent prognostic factor (P = 0.029), and associated with beneficial overall survival (P = 0.012) and disease-free survival (P = 0.016) for patients with NSCLC. Finally, we unveiled that hypoxic tumor microenvironment could switch the phenotype of macrophages from M1 to M2 forms, accompanying with the downregulation of C9 in TAMs. Collectively, our findings elucidated a novel role of TAMs expressing C9 in the prognosis of NSCLC patients, which provided a promising strategy in the development of anticancer treatments based on the CDC-mediated tumoricidal activity.

Estrogen receptor modulators genistein, daidzein and ERB-041 inhibit cell migration, invasion, proliferation and sphere formation via modulation of FAK and PI3K/AKT signaling in ovarian cancer

BackgroundOvarian cancer is the most lethal gynaecological malignancy. Chemotherapy is the main stay of treatment for metastatic disease, with modest response rates but significant side effects. Therefore, there is a need for alternative therapies that can control the disease while offering good quality of life. Ovarian cancer cells express both estrogen receptor subtypes (ERα and ERβ). There is growing evidence that ERβ is anti-oncogenic. Genistein and daidzein are phytoestrogens found in soybeans and they display higher affinity to bind ERβ. ERB-041 is a potent selective ERβ agonist. In this study, we aimed to investigate the effects of genistein, daidzein and ERB-041 on ovarian cancer.MethodsOvarian cancer cell lines were treated with genistein, daidzein and ERB-041 in pharmacological doses. Cell migration, invasion, proliferation, cell cycle arrest, apoptosis and sphere formation were assessed by Transwell migration and invasion assays, XTT assay, focus formation, flow cytometry and sphere formation assay, respectively. Immunoblotting analysis was performed to determine the downstream signaling pathways.ResultsWe found that genistein, daidzein and ERB-041 significantly inhibited ovarian cancer cell migration, invasion, proliferation, as well as induced cell cycle arrest and apoptosis. Significantly inhibitory effect on the size and number of sphere formed in genistein, daidzein and ERB-041 treated cells was also demonstrated. Moreover, genistein, daidzein and ERB-041 treatment reduced p-FAK, p-PI3K, p-AKT, p-GSK3β, p21 or cyclin D1 expression in ovarian cancer cells.ConclusionGenistein, daidzein and ERB-041 decreased ovarian cancer cell migration, invasion, proliferation and sphere formation, and induced cell cycle arrest and apoptosis with altered FAK and PI3K/AKT/GSK signaling and p21/cyclin D1 expression, suggesting their roles on ovarian cancer cell metastasis, tumorigenesis and stem-like properties and their potential as alternative therapies for ovarian cancer patients.

The CXCL5/CXCR2 axis contributes to the epithelial-mesenchymal transition of nasopharyngeal carcinoma cells by activating ERK/GSK-3\u03b2/snail signalling

BackgroundDistant metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). Although several biomarkers correlate with metastasis and prognosis, the molecular mechanisms of NPC development and progression remain unclear.MethodsQuantitative RT-PCR (qRT-PCR), western blotting, cell growth, foci formation, migration and invasion assays, and xenograft mouse models were utilized to examine the expression levels and functions of the CXCL5/CXCR2 axis in NPC. A luciferase reporter assay, western blotting, immunofluorescence, and migration and invasion assays were used to identify and verify the ERK/GSK-3β/Snail signalling pathway.ResultsCXCL5 was significantly increased in the sera of NPC patients, and high expression levels of CXCL5/CXCR2 in NPC primary tissues indicated poor survival. CXCL5 and CXCR2 were upregulated in NPC cell lines. Ectopic expression of the CXCL5/CXCR2 axis promoted NPC cell migration and invasion in vitro and the formation of lung metastases in vivo. Mechanistically, the dual overexpression of CXCL5 and CXCR2 promoted cell spreading by inducing the epithelial-mesenchymal transition (EMT) through the activation of the ERK/GSK-3β/Snail signalling pathway.ConclusionThe CXCL5/CXCR2 axis contributes to the EMT of NPC cells by activating ERK/GSK-3β/Snail signalling, and this axis may be a potential diagnostic marker and therapeutic target for patients with NPC.

Neoisoliquiritigenin Inhibits Tumor Progression by Targeting GRP78-β- catenin Signaling in Breast Cancer.

Breast cancer mortality has been stable or decreasing in the world, its incidence and recurrence rates have sharply risen worldwide in the recent years. To investigate the clinicopathological significance and potential function of GRP78 in the development and progression of breast cancer. To explore the effects of neoisoliquiritigenin (NISL) in breast cancer and the underlying mechanism. GRP78 was detected by immunohistochemistry (IHC) using breast cancer tissue microarrays (TMAs), and the association between GRP78 levels and clinicopathological factors and prognosis was analyzed. The functional effects of GRP78 on breast cancer were validated by an MTT assay, foci formation assay, Matrigel invasion assay and mouse xenograft assay. The effects of NISL were tested by an MTT assay, apoptosis assay and mouse xenograft assay. A LigandFit algorithm, ATPase activity assay, western blot and IHC assay were used to discover the underlying mechanism of the effects of NSIL. GRP78 was highly expressed in breast cancer cell lines and tissues. In addition, high expression of GRP78 was correlated to poor outcomes and distant metastasis. Functional experiments showed that GRP78 promoted breast cancer proliferation and invasion in vitro and in vivo. NISL inhibited cell proliferation and induced cell apoptosis in breast cancer by directly binding to GRP78 to regulate the β-catenin pathway. Taken together, these results highlighted the significance of GRP78 in breast cancer development and suggested NISL as a natural candidate to inhibit breast cancer by targeting GRP78 and β-catenin signaling.

Determining the effect of different environmental conditions on Ebola virus viability in clinically relevant specimens

In 2013–2016, West Africa experienced the largest and longest Ebola virus disease outbreak ever documented. The wide geographic spread and magnitude of the outbreak often limited the timely and rapid testing of diagnostic samples from patients with suspected Ebola virus disease, raising questions regarding the optimal storage and shipping conditions of clinically relevant specimens, including EDTA-whole blood, plasma, capillary blood, urine and seminal fluid (associated with sexual transmission of the Ebola virus after recovery from the disease). Therefore, the aim of our study was to identify the extent to which storage temperature and clinical specimen type influence Ebola virus viability. Virus infectivity was determined using a fluorescent focus-forming assay. In our study, we show that Ebola virus was the most stable in EDTA-whole blood and plasma samples, whereas rapid decay of infectivity was observed in simulated capillary blood, urine and semen samples, especially when these samples were stored at higher temperatures. The analysis of variance results demonstrated that both temperature and clinical specimen type have significant effects on virus viability, whereas donor differences were not observed. Repeated freeze and thaw cycles of the samples also had a notable impact on virus viability in EDTA-whole blood and urine. Due to the rapid temperature- and specimen-dependent degradation of the virus observed here, our study highlights the importance of proper clinical sample storage at low temperatures during transportation and laboratory analysis.

A major component of vitamin E, α-tocopherol inhibits the anti-tumor activity of crizotinib against cells transformed by EML4-ALK

Crizotinib is an inhibitor of anaplastic lymphoma kinase (ALK) and is of significant therapeutic benefit to patients with non-small cell lung cancer (NSCLC) harboring the EML4-ALK fusion gene. In the present study, we demonstrated that α-tocopherol, a major component of vitamin E, attenuated the effects of crizotinib independently of its anti-oxidant properties. α-Tocopherol significantly inhibited crizotinib-induced apoptosis in cells transformed by EML4-ALK. It also effectively attenuated the crizotinib-induced inhibition of EML4-ALK and its downstream molecules, STAT3 and ERK, and suppressed the inhibitory effects of crizotinib on EML4-ALK-mediated transformation in the focus formation assay. On the other hand, other members of the vitamin E family, namely, β-tocopherol, γ-tocopherol, δ-tocopherol, and α-tocotrienol, and a water-soluble analog of vitamin E, Trolox had no effects on the anti-tumor activity of crizotinib in cells transformed by EML4-ALK. Collectively, these results revealed the risk of the anti-tumor activity of crizotinib being attenuated when it is administrated in combination with vitamin E supplements containing α-tocopherol as a major component.

Effects of 1800 MHz RF-EMF exposure on DNA damage and cellular functions in primary cultured neurogenic cells

Purpose: To systematically evaluate the effects of 1800 MHz radiofrequency electromagnetic fields (RF-EMF) exposure on DNA damage and cellular functions in primary cultured neurogenic cells.Materials and methods: The primary cultured astrocytes, microglia and cortical neurons were exposed to RF-EMF at a SAR of 4.0 W/kg. The DNA damage was evaluated by γH2AX foci formation assay. The secretions of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in astrocytes and microglia, microglial phagocytic activity and neuronal development were examined by enzyme-linked immunosorbent assay, phagocytosis assay and immunofluorescent staining on microtubule-associated protein tau, microtubule-associated protein 2, postsynaptic density 95 and gephyrin, respectively.Results: RF-EMF exposure did not significantly induce γH2AX foci formation in three primary cultured neurogenic cells. Furthermore, RF-EMF exposure did not significantly affect the secretion of cytokines in astrocytes and microglia, and the morphological indicators of dendrites or synapses of cortical neurons. However, the exposure significantly reduced the phagocytic activity of microglia and inhibited the axon branch length and branch number of cortical neurons.Conclusions: Our data demonstrated that exposure to RF-EMF did not elicit DNA damage but inhibited the phagocytic ability of microglia and the axon branch length and branch number of cortical neurons.

Stable knockdown of ZBTB7A promotes cell proliferation and progression in nasopharyngeal carcinoma

Although high expression of ZBTB7A is positively relative to metastasis in nasopharyngeal carcinoma (NPC) patients, the association between its low expression and metastasis of NPC remains unclear. The present study aimed to definitely identify the association. The level of ZBTB7A was effectively knocked down by stable transfection of short hair RNA plasmid in NPC cell lines CNE2 and 5-8F (shRNA-CNE2 and shRNA-5-8F), compared with the cells that stably transfected empty plasmid (NC-CNE2 and NC-5-8F). The levels of ZBTB7A were assessed by real-time polymerase chain reaction and Western blot in the cell lines. MTT assay, colorimetric focus-formation assay, flow cytometry, wound healing assay, transwell assays, and xenograft model were performed to analyze cell vitality, proliferation, cell cycle, migration, invasion, and tumorigenicity. The levels of ZBTB7A were effectively reduced in shRNA-CNE2 and shRNA-5-8F. Their carcinogenicity was stronger separately than the abilities of NC-CNE2 and NC-5-8F. NC-CNE2 and shRNA-CNE2 were selected to establish the xenograft model because of their stronger tumorigenicity than NC-6-10B and shRNA-5-8F. The assay showed that shRNA-CNE2 had stronger tumorigenicity than NC-CNE2. The results demonstrated the reverse association between the expression of ZBTB7A and the tumorigenicity of NPC. We postulate that some oncogenic pathways, which are suppressed by ZBTB7A, will vicariously promote the proliferation and progression of NPC when ZBTB7A is decreased.

Diverse BRCA1 and BRCA2 Reversion Mutations in Circulating Cell-Free DNA of Therapy-Resistant Breast or Ovarian Cancer.

Purpose: Resistance to platinum-based chemotherapy or PARP inhibition in germline BRCA1 or BRCA2 mutation carriers may occur through somatic reversion mutations or intragenic deletions that restore BRCA1 or BRCA2 function. We assessed whether BRCA1/2 reversion mutations could be identified in circulating cell-free DNA (cfDNA) of patients with ovarian or breast cancer previously treated with platinum and/or PARP inhibitors.Experimental Design: cfDNA from 24 prospectively accrued patients with germline BRCA1 or BRCA2 mutations, including 19 patients with platinum-resistant/refractory ovarian cancer and five patients with platinum and/or PARP inhibitor pretreated metastatic breast cancer, was subjected to massively parallel sequencing targeting all exons of 141 genes and all exons and introns of BRCA1 and BRCA2 Functional studies were performed to assess the impact of the putative BRCA1/2 reversion mutations on BRCA1/2 function.Results: Diverse and often polyclonal putative BRCA1 or BRCA2 reversion mutations were identified in cfDNA from four patients with ovarian cancer (21%) and from two patients with breast cancer (40%). BRCA2 reversion mutations were detected in cfDNA prior to PARP inhibitor treatment in a patient with breast cancer who did not respond to treatment and were enriched in plasma samples after PARP inhibitor therapy. Foci formation and immunoprecipitation assays suggest that a subset of the putative reversion mutations restored BRCA1/2 function.Conclusions: Putative BRCA1/2 reversion mutations can be detected by cfDNA sequencing analysis in patients with ovarian and breast cancer. Our findings warrant further investigation of cfDNA sequencing to identify putative BRCA1/2 reversion mutations and to aid the selection of patients for PARP inhibition therapy. Clin Cancer Res; 23(21); 6708-20. ©2017 AACR.

BRCA2 protects mammalian cells from heat shock

Purpose: Heat shock induces DNA double-strand breaks (DSBs) in mammalian cells. Mammalian cells are capable of repairing DSBs by utilising the homologous recombination (HR) pathway. Breast cancer susceptibility gene 2 (BRCA2) is known to regulate the HR pathway. Here, we investigate the role of BRCA2 in repairing DNA damage induced by heat shock.Materials and methods: Chinese hamster lung fibroblast cell lines and human tongue squamous cell carcinoma SAS cells were used. RAD51 foci formation assay was used as an HR indicator. Heat sensitivity was analysed with colony forming assays. Phosphorylated histone H2AX (γH2AX) intensity, which correlates with the number of DSBs, was analysed with flow cytometry.Results: RAD51 foci appeared with heat shock, and the number of cells with RAD51 foci was maximal at about 4 h after heat shock. Heat-induced RAD51 foci co-localised with γH2AX foci. BRCA2-deficient cells were sensitive to heat when compared to their parental wild-type cells. Heat-induced γH2AX was higher in BRCA2-deficient cells compared to parental cells. In SAS cells, cells transfected with BRCA2-siRNA were more sensitive to heat than cells transfected with negative control siRNA. Apoptotic bodies increased in number more rapidly in BRCA2-siRNA transfected cells than in cells transfected with negative control siRNA when cells were observed at 48 h after a heat treatment. In addition, cells deficient in BRCA2 were incapable of activating heat-induced G2/M arrest.Conclusion: BRCA2 has a protecting role against heat-induced cell death. BRCA2 might be a potential molecular target for hyperthermic cancer therapy.

Inhibition of PI3K-AKT-mTOR pathway sensitizes endometrial cancer cell lines to PARP inhibitors

BackgroundPhosphatase and Tensin homolog (PTEN) is a tumor suppressor gene. Loss of its function is the most frequent genetic alteration in endometrioid endometrial cancers (70–80%) and high grade tumors (90%). We assessed the sensitivity of endometrial cancer cell lines to PARP inhibitors (olaparib and BMN-673) and a PI3K inhibitor (BKM-120), alone or in combination, in the context of their PTEN mutation status. We also highlighted a direct pathway linking PTEN to DNA repair.MethodsUsing endometrial cancer cellular models with known PTEN status, we evaluated their homologous recombination (HR) functionality by RAD51 foci formation assay. The 50% Inhibitory concentration (IC50) of PI3K and PARP inhibitors in these cells was assessed, and western blotting was performed to determine the expression of proteins involved in the PI3K/mTOR pathway. Moreover, we explored the interaction between RAD51 and PI3K/mTOR by immunofluorescence. Next, the combination effect of PI3K and PARP inhibitors on cell proliferation was evaluated by a clonogenic assay.ResultsCells with mutated PTEN showed over-activation of the PI3K/mTOR pathway. These cells were more sensitive to PARP inhibition compared to PTEN wild-type cells. In addition, PI3K inhibitor treatment reduced RAD51 foci formation in PTEN mutated cells, and sensitized these cells to PARP inhibitor.ConclusionTargeting both PARP and PI3K might lead to improved personalized therapeutic approaches in endometrial cancer patients with PTEN mutations. Understanding the complex interaction of PTEN mutations with DNA repair in endometrial cancer will help to better select patients that are likely to respond to some of the new and costly targeted therapies.

MiR-22 suppresses tumorigenesis and improves radiosensitivity of breast cancer cells by targeting Sirt1

BackgroundmiR-22 has been shown to be frequently downregulated and act as a tumor suppressor in multiple cancers including breast cancers. However, the role of miR-22 in regulating the radioresistance of breast cancer cells, as well as its underlying mechanism is still not well understood.MethodsThe expressions of miR-22 and sirt1 at mRNA and protein levels were examined by qRT-PCR and Western Blot. The effects of miR-22 overexpression and sirt1 knockdown on cell viability, apoptosis, radiosensitivity, γ-H2AX foci formation were evaluated by CCK-8 assay, flow cytometry, colony formation assay, and γ-H2AX foci formation assay, respectively. Luciferase reporter assay and qRT-PCR analysis were performed to confirm the interaction between miR-22 and sirt1.ResultsmiR-22 was downregulated and sirt1 was upregulated at both mRNA and protein levels in breast cancer cells. miR-22 overexpression or sirt1 knockdown significantly suppressed viability, induced apoptosis, reduced survival fraction, and increased the number of γ-H2AX foci in breast cancer cells. Sirt1 was identified as a target of miR-22 and miR-22 negatively regulated sirt1 expression. Ectopic expression of sirt1 dramatically reversed the inhibitory effect of miR-22 on cell viability and promotive effect on apoptotic rates and radiosensitivity in breast cancer cells.ConclusionsmiR-22 suppresses tumorigenesis and improves radiosensitivity of breast cancer cells by targeting sirt1, providing a promising therapeutic target for breast cancer.

CYT-Rx20 Inhibits Cervical Cancer Cell Growth and Migration Through Oxidative Stress-Induced DNA Damage, Cell Apoptosis, and Epithelial-to-Mesenchymal Transition Inhibition.

The β-nitrostyrene family has been reported to possess anticancer properties. However, the anticancer activity of β-nitrostyrenes on cervical cancer cells and the underlying mechanisms involved remain unexplored. In this study, a β-nitrostyrene derivative CYT-Rx20 (3'-hydroxy-4'-methoxy-β-methyl-β-nitrostyrene) was synthesized, and its anticancer activity on cervical cancer cells and the mechanisms involved were investigated. The effect of CYT-Rx20 on human cervical cancer cell growth was evaluated using cell viability assay. Reactive oxygen species (ROS) generation and annexin V staining were detected by flow cytometry. The protein expression levels of cleaved caspase-3, cleaved caspase-9, cleaved poly (ADPribose) polymerase, γH2AX, β-catenin, Vimentin, and Twist were measured by Western blotting. DNA double-strand breaks were determined by γ-H2AX foci formation and neutral comet assay. Migration assay was used to determine cancer cell migration. Nude mice xenograft was used to investigate the antitumor effects of CYT-Rx20 in vivo. CYT-Rx20 induced cytotoxicity in cervical cancer cells by promoting cell apoptosis via ROS generation and DNA damage. CYT-Rx20-induced cell apoptosis, ROS generation, and DNA damage were reversed by thiol antioxidants. In addition, CYT-Rx20 inhibited cervical cancer cell migration by regulating the expression of epithelial-to-mesenchymal transition markers. In nude mice, CYT-Rx20 inhibited cervical tumor growth accompanied by increased expression of DNA damage marker γH2AX and decreased expression of mesenchymal markers β-catenin and Twist. CYT-Rx20 inhibits cervical cancer cells in vitro and in vivo and has the potential to be further developed into an anti-cervical cancer drug clinically.

Differences in Zbtb7a expression cause heterogeneous changes in human nasopharyngeal carcinoma CNE3 sublines

The present study aimed to determine the association between changes in Zbtb7a expression levels and heterogeneity of nasopharyngeal carcinoma (NPC) CNE3 sublines. CNE3 sublines were established by screening of serial dilution and continuous passage. Proliferative ability and tumorigenicity of the sublines were analyzed separately by soft-agar colony formation and mouse studies. The NPC tissues from mice were analyzed by histological evaluation and immunohistochemistry. The expression levels of Zbtb7a mRNA and protein were analyzed separately by quantitative reverse transcription polymerase chain reaction and western blotting. According to findings from the soft-agar colony formation and mouse studies, two sublines with increased tumorigenicity compared with other sublines were transfected transiently with Zbtb7a short hairpin RNA (shRNA) recombinant plasmid. The changes in viability, migration and invasion abilities were evaluated separately by MTT, colorimetric focus-formation, Transwell migration and invasion assays. The sublines CNE3-GX6 and CNE3-GX11 were selected for subsequent study due to increased tumorigenicity and increased Zbtb7a expression levels compared with the other sublines. High metastatic potency was not observed in all of the sublines. Zbtb7a expression levels were positively associated with tumorigenic degree of the sublines. The growth, migration and invasion abilities of the sublines transfected with Zbtb7a shRNA plasmid were decreased compared with the cells transfected with empty vector in the negative control group. The findings suggest Zbtb7a expression levels may be associated with heterogeneity of CNE3 sublines. Therefore, Zbtb7a may have an important role in the regulatory mechanism of NPC heterogeneity.

Abstract 5056: The bromodomain inhibitor JQ1 sensitizes homologous recombination proficient ovarian cancer cells to the PARP inhibitor olaparib

Abstract Introduction: Lack of effective treatment options for high-grade serous ovarian cancers (HGSOC) retaining functional homologous recombination (HR) DNA repair pathways is a significant clinical problem. HR-proficient HGSOC tumors, for example those harboring cyclin E amplifications, have poorer clinical outcomes and show relative resistance to DNA-damaging platinum agents and newer poly ADP ribose polymerase inhibitors (PARPi). We and others have shown that using epigenetic drugs to reduce HR efficiency in HR-proficient HGSOC sensitizes these cancer cells to DNA damaging agents. One mechanism by which these drugs reduce HR efficiency is by transcriptional down-regulation of HR pathway components. An emerging class of epigenetic mediators of pro-tumorigenic transcription is the bromodomain (BRD) family of proteins, and BRD inhibitors (BRDi) have shown promising preclinical anti-tumor efficacy. However, it is unknown whether BRDi sensitize HR-proficient HGSOC to DNA damaging agents. Aims: To test the hypothesis that BRDi decrease efficiency of HR DNA repair in HR-proficient ovarian cancer cells, thereby sensitizing them to PARPi. Methods: The HR-proficient ovarian cancer cell lines, OVCAR-3 (cyclin E-amplified) and SKOV3, were treated with 0.01% DMSO vehicle, the PARPi olaparib (Astra Zeneca), the BRDi JQ1 or with the olaparib/JQ1 combination. Sulforhodamine B (SRB) assays assessed cell growth and viability (72 hours treatment). Immunofluorescence (IF) assays assessed markers of DNA damage (pH2AX), apoptosis (cleaved caspase-3), and HR efficiency (RAD51 foci, and GFP expression in cells co-transfected with I-Sce1 endonuclease and DRGFP HR reporter plasmids) (24 hours treatment). Steady state levels of the HR protein BRCA1, pH2AX and cleaved caspase-3 were assessed by western blot (24 hours treatment). Results: The combination of JQ1 and olaparib synergistically reduced cancer cell viability following isobologram analyses of SRB experiments. Compared to either drug alone, the JQ1/olaparib combination also significantly reduced BRCA1 expression and increased protein levels of cleaved caspase-3 and pH2AX in western blots, and also increased the number of cells displaying DNA damage and apoptosis in IF assays. Finally, JQ1 and olaparib combined to significantly reduce HR efficiency in our RAD51 foci formation and DRGFP assays compared to olaparib alone. Conclusions: Our results suggest that BRDi sensitize HR-proficient cells to DNA damaging drugs, in part by reducing efficiency of HR DNA repair. These findings have important implications for expanding the use of PARPi in HR-proficient HGSOC through rational combinations with epigenetic drugs such as BRDi that target the HR pathway. Citation Format: Andrew J. Wilson, Janese Thompson, Abdirahman Osman, Jeanette Saskowski, Dineo Khabele. The bromodomain inhibitor JQ1 sensitizes homologous recombination proficient ovarian cancer cells to the PARP inhibitor olaparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5056. doi:10.1158/1538-7445.AM2017-5056

Abstract 1431: Therapeutic exploitation of mutant BRAF in childhood glioma

Abstract Low grade gliomas (LGG) are the most common tumors of the central nervous system in children, accounting for about 50% of all brain tumors. They represent a heterogeneous group of grade I and II tumors according to the WHO classification. Pediatric LGG, is associated with activation of BRAF through a tandem duplication that results in the KIAA1549-BRAF fusion or through an activating point mutation of BRAF (predominantly V600E). More recent findings suggest that the KIAA1549-BRAF fusion is restricted to Grade 1 tumors (70-90%) whereas BRAF(V600E) occurs more frequently in Grade 2-4 tumors (~23%). Findings for BRAF mutation, similar to other tumors with activated BRAF (e.g. melanoma), and the phase I activity of MEK inhibitor in the PBTC-029 protocol, suggest that activated BRAF may provide a validated drug target. Previous studies from our lab indicate that, in the context of mutant BRAF, inhibition of MEK inhibits TORC1 signaling and may induce a ‘BRCA-like’ phenotype, through depletion of FANCD2. Potentially suppression of TORC1 could have effects on other DNA damage response pathways that could compensate for loss of FANCD2. To understand the consequences of MEK inhibition in the context of BRAF abreation in LGG we surveyed DNA repair genes that may be regulated via the MEK/TORC1 pathway in BRAF mutant cells. The objective of this particular study was to examine the mechanism and significance of MEK inhibition to the repair of DNA damage by the homologous recombination (HR) pathway. For this study, we have used glioma cell lines having BRAF(V600E) mutation; the BT40 cell line was developed from patient-derived astrocytoma xenograft (PDX) model in mice in our lab and AM38c1 and DBTRG-05MG cells were generously provided by T. Nicolades. Annexin binding assay demonstrated that MEKi treatment significantly increased the percentage of apoptosis and necrosis of glioma cells. Human DNA repair PCR Array analysis identified MEKi induced down regulation of genes involved in Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and Double-Strand Break (DSB) repair pathways. DSB repair genes were further validated by real time qPCR analysis. Immunoblot analysis of glioma cell lines indicate that MEKi treatment enhanced the gamma-H2AX levels. To elucidate the mechanism of DSB repair pathway in presence of MEKi, clonogenic assay, nuclear foci formation assay, and GFP reporter assay for homologous recombination (HR) and non-homologous end-joining (NHEJ) are ongoing. Collectively, these findings demonstrate for the first time a previously unknown role for MEKi in treatment of glioma cells that involves inhibition of DNA double-strand break repair pathways. Further, we will examine the mechanism and significance of MEK/ERK and TORC1 signaling axis in regulation of DNA repair genes and its effect on LGG. Understanding how MEK plays a role in DNA repair pathways will be useful in maximizing treatment opportunities for childhood glioma. Citation Format: Sudipa Saha Roy, Terry Shackleford, Peter Houghton. Therapeutic exploitation of mutant BRAF in childhood glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1431. doi:10.1158/1538-7445.AM2017-1431

Abstract NTOC-092: THE BROMODOMAIN INHIBITOR JQ1 SENSITIZES HOMOLOGOUS RECOMBINATION PROFICIENT OVARIAN CANCER CELLS TO THE PARP INHIBITOR OLAPARIB

Abstract INTRODUCTION: Lack of effective treatment options for high-grade serous ovarian cancers (HGSOC) retaining functional homologous recombination (HR) DNA repair pathways is a significant clinical problem. HR-proficient HGSOC tumors, for example those harboring cyclin E amplifications, have poorer clinical outcomes and show relative resistance to DNA-damaging platinum agents and newer poly ADP ribose polymerase inhibitors (PARPi). We and others have shown that using epigenetic drugs to reduce HR efficiency in HR-proficient HGSOC sensitizes these cancer cells to DNA damaging agents. One mechanism by which these drugs reduce HR efficiency is by transcriptional down-regulation of HR pathway components. An emerging class of epigenetic mediators of pro-tumorigenic transcription is the bromodomain (BRD) family of proteins, and BRD inhibitors (BRDi) have shown promising preclinical anti-tumor efficacy. However, it is unknown whether BRDi sensitize HR-proficient HGSOC to DNA damaging agents. AIMS: To test the hypothesis that BRDi decrease efficiency of HR DNA repair in HR-proficient ovarian cancer cells, thereby sensitizing them to PARPi. METHODS: The HR-proficient ovarian cancer cell lines, OVCAR-3 (cyclin E-amplified) and SKOV3, were treated with 0.01% DMSO vehicle, the PARPi olaparib (Astra Zeneca), the BRDi JQ1 or with the olaparib/JQ1 combination. Sulforhodamine B (SRB) assays assessed cell growth and viability (72 hours treatment). Immunofluorescence (IF) assays assessed markers of DNA damage (pH2AX), apoptosis (cleaved caspase-3), and HR efficiency (RAD51 foci, and GFP expression in cells co-transfected with I-Sce1 endonuclease and DRGFP HR reporter plasmids) (24 hours treatment). Steady state levels of the HR protein BRCA1, pH2AX and cleaved caspase-3 were assessed by western blot (24 hours treatment). RESULTS: The combination of JQ1 and olaparib synergistically reduced cancer cell viability following isobologram analyses of SRB experiments. Compared to either drug alone, the JQ1/olaparib combination also significantly reduced BRCA1 expression and increased protein levels of cleaved caspase-3 and pH2AX in western blots, and also increased the number of cells displaying DNA damage and apoptosis in IF assays. Finally, JQ1 and olaparib combined to significantly reduce HR efficiency in our RAD51 foci formation and DRGFP assays compared to olaparib alone. CONCLUSIONS: Our results suggest that BRDi sensitize HR-proficient cells to DNA damaging drugs, in part by reducing efficiency of HR DNA repair. These findings have important implications for expanding the use of PARPi in HR-proficient HGSOC through rational combinations with epigenetic drugs such as BRDi that target the HR pathway. Citation Format: Andrew J. Wilson, Janese Thompson, Abdirahman Osman, Jeanette Saskowski, Dineo Khabele. THE BROMODOMAIN INHIBITOR JQ1 SENSITIZES HOMOLOGOUS RECOMBINATION PROFICIENT OVARIAN CANCER CELLS TO THE PARP INHIBITOR OLAPARIB [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-092.

P53 pathway determines the cellular response to alcohol-induced DNA damage in MCF-7 breast cancer cells.

Alcohol consumption is associated with increased breast cancer risk; however, the underlying mechanisms that contribute to mammary tumor initiation and progression are unclear. Alcohol is known to induce oxidative stress and DNA damage; likewise, p53 is a critical modulator of the DNA repair pathway and ensures genomic integrity. p53 mutations are frequently detected in breast and other tumors. The impact of alcohol on p53 is recognized, yet the role of p53 in alcohol-induced mammary carcinogenesis remains poorly defined. In our study, we measured alcohol-mediated oxidative DNA damage in MCF-7 cells using 8-OHdG and p-H2AX foci formation assays. p53 activity and target gene expression after alcohol exposure were determined using p53 luciferase reporter assay, qPCR, and Western blotting. A mechanistic study delineating the role of p53 in DNA damage response and cell cycle arrest was based on isogenic MCF-7 cells stably transfected with control (MCF-7/Con) or p53-targeting siRNA (MCF-7/sip53), and MCF-7 cells that were pretreated with Nutlin-3 (Mdm2 inhibitor) to stabilize p53. Alcohol treatment resulted in significant DNA damage in MCF-7 cells, as indicated by increased levels of 8-OHdG and p-H2AX foci number. A p53-dependent signaling cascade was stimulated by alcohol-induced DNA damage. Moderate to high concentrations of alcohol (0.1–0.8% v/v) induced p53 activation, as indicated by increased p53 phosphorylation, reporter gene activity, and p21/Bax gene expression, which led to G0/G1 cell cycle arrest. Importantly, compared to MCF-7/Con cells, alcohol-induced DNA damage was significantly enhanced, while alcohol-induced p21/Bax expression and cell cycle arrest were attenuated in MCF-7/sip53 cells. In contrast, inhibition of p53 degradation via Nutlin-3 reinforced G0/G1 cell cycle arrest in MCF-7 control cells. Our study suggests that functional p53 plays a critical role in cellular responses to alcohol-induced DNA damage, which protects the cells from DNA damage associated with breast cancer risk.