bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3519: Paroxetine induces apoptosis in human breast cancer MCF-7 cells through mitochondrial dysfunction and modulation of K+ channels
Authors: Kang Kee R. , Cho Young-Woo , Ryu Ji Hyun , Kim Eun-Jin , Han Jaehee , Kang Dawon
Published: July 2016

Purpose: Selective serotonin reuptake inhibitors (SSRIs) are widely used for the treatment of patients with depression. Approximately 60% of women with breast cancer suffer from depression. This study was performed to investigate the effect of SSRIs on cell viability of human breast cancer MCF-7 cells.

Methods: Cell viability, ROS generation, and Ca2+ increase were quantified using MTT, H2DCFDA, and Fluo-3 AM. Apoptotic signaling pathways were analyzed by immuno-blotting and -staining. Plasma and mitochondrial membrane potentials were determined by DiO PMP and JC-1 dyes, respectively. K+ channel activity was analyzed by whole-cell and single-channel recordings.

Results: Of the antidepressants tested in this study, paroxetine most reduced the viability of MCF-7 cells. Paroxetine (10 and 30 μM) treatment resulted in a significant increase in the number of apoptotic cells, an increase in Bax/Bcl-2 ratio, an activation of caspase-8, 9, and PARP, and a release of cytochrome c from mitochondria. In addition, paroxetine markedly increased ROS generation and intracellular Ca2+ levels. Moreover, paroxetine blocked K+ channels, such as large conductance Ca2+-activated K+ channel and TASK-3 channel, which are present in plasma membrane and mitochondria. The paroxetine-induced apoptosis was reduced by antioxidants, in particular NAC. Combination with SB202190 (a p38 MAPK inhibitor) prevented cells from undergoing apoptosis.

Conclusion: Our findings show that apoptotic effect of paroxetine is mediated by mitochondrial dysfunction and modulation of K+ channels in MCF-7 cells. We suggest that paroxetine could be an agent for anti-tumor therapy in breast cancer.

Citation Format: Kee R. Kang, Young-Woo Cho, Ji Hyun Ryu, Eun-Jin Kim, Jaehee Han, Dawon Kang. Paroxetine induces apoptosis in human breast cancer MCF-7 cells through mitochondrial dysfunction and modulation of K+ channels. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3519.
Abstract 3524: D-glucose exposure induced DNA damage and apoptosis in MCF-7 cells
Authors: Farah Ibrahim O. , Tchounwou Christine K. , Yedjou Clement G. , Tchounwou Paul B.
Published: July 2016

D-glucose is the simple carbohydrate sugar that our bodies rely on to produce ATP energy. It has been shown that sustained high glucose burden is related to the promotion of many long-term health problems to various vital target organs including the kidneys, nervous system, eyes, heart (failure and stroke), erectile dysfunction in men and pregnancy complications in women. Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. Furthermore, the D-glucose paradox as cancer-promoting energy source as well as being toxic to cancer cells is puzzling. Therefore in the present study, we will investigate the mechanisms of glucose-induced toxicity in MCF-7 cells as an in vitro cellular model to simulate diabetic complications in tumor cells using trypan blue exclusion and MTT assays. Mechanisms of DNA damage and apoptosis were tested by alkaline single cell gel electrophoresis (Comet) assay, and flow cytometry analysis using Annexin V FITC/PI and caspase-3 analysis. The MTT assay indicated that low dose (5 mg/mL) of D-glucose slightly increase cell viability upon 2 h of exposure. On the other hand, high doses (10-80 mg/mL) of D-glucose significantly reduced the viability of MCF-7 cells in a dose and time-dependent manner. Similar trends were seen with the trypan blue exclusion test. Data obtained from the comet assay indicated that D-glucose caused DNA damage in MCF-7 cells in a dose-dependent manner. The flow cytometry assessment (Annexin V FITC/PI) showed a strong dose-response relationship between high glucose exposure and Annexin V positive MCF-7 cells undergoing early stage apoptosis. Similarly, a statistically significant and concentration-dependent increase (p <0.05) were recorded for caspase-3 activity in MCF-7 cells undergoing late apoptosis. Based on these in vitro findings, our study provides clear evidence that elevated level of D-glucose induced cytotoxicity to MCF-7 cells via induction of DNA damage, externalization of phosphatidylserine and caspase-3 activation as indicators for the apoptotic mechanisms of D-glucose exposure.

This research is supported by a grant from the National Institutes of Health-NIH (Grant No. NIMHD-G12MD007581).

Citation Format: Ibrahim O. Farah, Christine K. Tchounwou, Clement G. Yedjou, Paul B. Tchounwou. D-glucose exposure induced DNA damage and apoptosis in MCF-7 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3524.
Abstract 3525: The effects of Berberis vulgaris on bacterial inhibition and the p53 gene in cancerous Caenorhabditis elegans
Authors: Bhatt Jesal P.
Published: July 2016

Recently, there has been an interest in the use of natural products to inhibit bacterial growth rather than manufactured antibiotics due to the problem of bacterial resistance. This study aims at finding the most effective natural antibacterial remedy. The disk diffusion method was used to compare the antibacterial properties of manuka honey, barberry, aloe vera, and colloidal silver. After a 48-hour incubation period, the zone of inhibition was measured for the following bacteria: Bacillus megaterium, Serratia marcescens, Pseudomonas fluorescens, Rhodospirillum rubrum, and Escherichia coli. Barberry was the most efficient natural product (p = 0.02). Further tests were performed to determine whether barberry contained strong anticancer properties. Barberry was tested on Caenorhabditis elegans (C.elegans), strain JR1279, exposed to ultraviolet-C radiation and the population was counted daily. Prior to this experiment, barberry had never before been tested on a model organism. The purpose of this experiment would determine if barberry could increase the lifespan of the C.elegans by the addition of this reagent. It was shown that the C.elegans exposed to barberry had a longer lifespan than those that were not exposed to barberry (p = 0.01), indicating that barberry could be a potential anticancer supplement.

Citation Format: Jesal P. Bhatt. The effects of Berberis vulgaris on bacterial inhibition and the p53 gene in cancerous Caenorhabditis elegans. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3525.
Abstract 3527: Inhibition of autophagosome closure promotes iDISC-mediated apoptosis in AML cells
Authors: Tang Zhenyuan , Takahashi Yoshinori , Wang Hong-Gang
Published: July 2016

Macroautophagy (hereafter referred to as autophagy) is a lysosomal degradation process whereby cellular components, such as damaged organelles, are enclosed in double-membrane structures called autophagosomes and delivered to lysosomes for degradation and recycling. Autophagy is known to be essential for cancer pathogenesis and progression through its intimate crosstalk with programed cell death machinery, apoptosis. However, the role of autophagy in apoptosis pathway remains highly controversial and context-dependent, which makes developing autophagy-targeted anti-cancer treatments hard to achieve. We have previously reported that autophagosomal membranes can serve as a platform for caspase-8 activation through the assembly of an intracellular Death-Inducing Signaling Complex (iDISC). In the present study, we demonstrate that inhibition of phagophore closure by depleting Atg2A/B stabilizes iDISCs and induces caspase-8-initiated apoptosis in acute myeloid leukemia (AML) cells. We found that knockout of Atg2A/B, but not Atg7, promotes the oligomerization of caspase-8 on Atg16L- and LC3-positive phagophore membranes upon nutrient starvation. Starvation-induced capase-8 activation and apoptosis induction in Atg2A/B-deficient cells were strongly suppressed by the inhibition of caspase-8. These results suggest that autophagy inhibition at the phagophore closure step can shift cytoprotective autophagy to a pro-death mechanism and thus may be a promising strategy to enhance the efficacy of anticancer therapy.

Citation Format: Zhenyuan Tang, Yoshinori Takahashi, Hong-Gang Wang. Inhibition of autophagosome closure promotes iDISC-mediated apoptosis in AML cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3527.
Abstract 3544: Determining autophagic activity following cannabidiol treatment of melanoma: a potential new and novel treatment
Authors: Simmerman Erika L. , Qin Xu , Motamed Kouros , Yu Jack C. , Baban Babak
Published: July 2016

Introduction:

Autophagy is a catabolic pathway utilized for energy metabolism in nutrient-depleted tumors. Recent studies have reported autophagy to be a survival mechanism found in cancer during stress secondary to cancer progression or treatment. In this study we tested for autophagic activity in malignant melanoma tumors via detection of LC3 antibody following treatment with Cannabidiol (CBD) and cisplatin. The objective of this study was to investigate the effect of treatment with a cannabinoid derivative on autophagic activity in malignant melanoma.

Materials and Methods:

Murine B16F10 melanoma tumors were established subcutaneously in C57BL/6 mice. These mice were then subjected to treatment with local vehicle (PBS) injection (control - group 1, n = 5), systemic Cisplatin injection (group 2; n = 5), and local cannabidiol injection (group 3; n = 5). Tumor cells were subsequently submitted to immunohistochemistry, Brightfield microscopy, and quantitative analysis (Bioquant) for LC3 expression.

Results:

The expression level of LC3 was significantly reduced in groups 2 and 3 when compared to the control group 1. Quantitative measurement of the slides using Bioquant software confirmed our immunohistochemistry and microscopic results.

Conclusions:

If CBD does indeed decrease autophagic activity, it may represent a new and novel therapeutic target in the treatment of malignant melanoma. Additional studies to clarify the specific relationship between CBD and autophagy are in progress and will be reported upon completion.

Citation Format: Erika L. Simmerman, Xu Qin, Kouros Motamed, Jack C. Yu, Babak Baban. Determining autophagic activity following cannabidiol treatment of melanoma: a potential new and novel treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3544.
Abstract 3553: A novel allosteric mechanism for molecular inhibition of anti-apoptotic MCL-1
Authors: Lee Susan , Wales Thomas E. , Gallagher Catherine , Cohen Daniel T. , Escudero Silvia , Luccarelli James , Huhn Annissa J. , Cohen Nicole A. , Bird Gregory H. , Engen John R. , Walensky Loren D.
Published: July 2016

MCL-1 is an anti-apoptotic BCL-2 family protein that has emerged as a major pathogenic factor across a broad range of human cancers. Thus, pharmacologic inhibition of MCL-1 has been the focus of intensive drug development efforts. Like BCL-2, MCL-1 bears a C-terminal surface groove whose function is to bind and sequester the essential BH3 killer domains of pro-apoptotic BCL-2 family proteins, a mechanism harnessed by cancer cells to create formidable apoptotic blockades. Whereas drugging the BH3-binding groove has been successfully achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small molecule covalent modification of an interaction site that is distant from the BH3-binding groove. Biochemical and hydrogen-deuterium exchange mass spectrometry analyses revealed that both the BH3-binding capacity of MCL-1 and its functional suppression of BAX-mediated membrane poration is impaired by molecular engagement, a phenomenon recapitulated by mutagenic mimicry at the novel regulatory region. This allosteric mechanism for disrupting the anti-apoptotic, BH3-binding activity of MCL-1 could inform a new therapeutic strategy for disarming MCL-1 in cancer.

Citation Format: Susan Lee, Thomas E. Wales, Catherine Gallagher, Daniel T. Cohen, Silvia Escudero, James Luccarelli, Annissa J. Huhn, Nicole A. Cohen, Gregory H. Bird, John R. Engen, Loren D. Walensky. A novel allosteric mechanism for molecular inhibition of anti-apoptotic MCL-1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3553.
Abstract 3559: The rare BCL-2 isoform BCL-2β is associated with melanoma survival and the apoptotic response to UV and cisplatin
Authors: Warren Chloe , Vilain Ricardo E. , Ashton Katie A. , Almazi Juhura G. , Braye Stephen , Moscato Pablo , Bowden Nikola A.
Published: July 2016

There are two isoforms of the anti-apoptotic protein BCL-2. BCL-2α (wild-type) is well characterised and is known predominantly for its role in apoptosis, BCL-2β lacks the C-terminal transmembrane (TM) domain and its function has not yet been characterised. The aim of this study was to quantify and determine the biological role of the BCL-2β isoform in melanoma.

BCL-2α and BCL-2β isoforms were quantified at the mRNA and protein level in a cohort of 141 FFPE melanoma tumours using qPCR and multiple reaction monitoring (MRM) tandem mass spectrometry and compared to clinical parameters including age at diagnosis, gender, solar elastosis, BRAF/NRAS mutation, Breslow thickness, disease free survival and overall survival. The role of the BCL-2α and BCL-2β isoforms in apoptosis were investigated by quantifying transcript expression and apoptosis before and up to 72 hours after UVB irradiation (650J/m2) and cisplatin treatment (10ug/mL). siRNA knockdown targeted to each individual isoform transcript was used to verify the apoptotic response.

Expression of the BCL-2β isoform in tumours was significantly associated with increased overall survival (686.4 weeks, 95% CI 462.5-910.3). BCL-2α response to UVB and cisplatin (i.e. downregulation prior to apoptosis) was the same in melanocyte and melanoma cell lines. However, BCL-2β response was varied across the melanoma cell lines, but was similar to that of BCL-2α in melanocytes. siRNA knock-down of BCL-2α resulted in increased apoptosis and cell death in melanoma cell lines, but knock-down of BCL-2β delayed onset of apoptosis, suggesting the BCL-2β is pro-apoptotic.

Our current understanding of the role of BCL-2β is based on the concept that it lack the C-terminal transmembrane domain, and is incapable of localising to target organelles. It is currently thought that the isoform is of null function. However, these observations are based on studies of non-representative synthetic versions of BCL-2β. This is the first time the naturally transcribed version of the rare isoform has been studied. We have demonstrated herein that BCL-2β performs an apoptotic role, and that its regulation in melanoma may be a biomarker of better overall survival.

Citation Format: Chloe Warren, Ricardo E. Vilain, Katie A. Ashton, Juhura G. Almazi, Stephen Braye, Pablo Moscato, Nikola A. Bowden. The rare BCL-2 isoform BCL-2β is associated with melanoma survival and the apoptotic response to UV and cisplatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3559.
Abstract 3563: Ras-mediated regulation of cytochrome c-induced caspase activation is dependent on the status of extracellular matrix attachment
Authors: McCallister Chelsea M. , Rayavarapu Raju , Pagani Nicholas , Heiden Brendan , Shaw Melissa , Shuff Sydeny , Schafer Zachary T.
Published: July 2016

Hyperactivating mutations in Ras are found in a significant percentage of cancers, with a particularly high frequency in pancreatic and colon carcinomas. The hyperactivation of Ras drives a vast number of distinct downstream signaling pathways that play an important role in disease progression. One outcome of overactive oncogenic Ras signaling is the inhibition of anoikis, a form of apoptotic cell death caused by detachment from the extracellular matrix (ECM). It is well-established that one mechanism of anoikis inhibition by Ras involves impaired mitochondrial cytochrome c release. In addition, here, we have found that Ras-mediated anoikis inhibition can also occur downstream of mitochondrial cytochrome c release. Our data suggest that anoikis inhibition following cytochrome c release is not dependent on changes in the abundance of the Apaf-1, pro-caspase-9, or pro-caspase-3. Instead, our data suggest that Ras signaling leads to an inhibition in the ability of caspase-9 to bind Apaf-1 which thereby inhibits proper formation of the apoptosome and caspase activation. Interestingly, in stark contrast to the inhibition of cytochrome c-induced apoptosis in ECM-detached cells, the overexpression of oncogenic Ras in ECM-attached cells results in enhanced sensitivity to exogenous cytochrome c. This sensitization was found to be due to upregulation of apoptosomal proteins (e.g. Apaf-1, pro-caspase-9) in an ERK/MAPK dependent manner. In aggregate, our data suggest that in addition to inhibiting the release of cytochrome c in both attachment and detachment, oncogenic Ras drives additional mechanisms that prevent apoptosome formation and caspase activation in detachment. Furthermore, our data support a model whereby ECM-attached cells containing oncogenic Ras mutations could be selectively eliminated by cytochrome c or agents that mimic its action.

Citation Format: Chelsea M. McCallister, Raju Rayavarapu, Nicholas Pagani, Brendan Heiden, Melissa Shaw, Sydeny Shuff, Zachary T. Schafer. Ras-mediated regulation of cytochrome c-induced caspase activation is dependent on the status of extracellular matrix attachment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3563.
Abstract 3601: Evaluation and quantification of biomarkers of DNA damage in human plucked hair
Authors: Bonavita Aude-Marine , Walker Liam , Boanas Adam , Tonge Nicola , Tudor Gregory , Booth Cath , Reed Ben
Published: July 2016

The hair follicle is a well vascularised structure that contains rapidly dividing epithelial cells. As a result it is a potentially useful surrogate biomarker for less accessible but highly similar tissues, such as rapidly proliferating tumours. We have developed an ex vivo culture method to validate target expression changes following exposure to DNA damage and/or potential therapeutics. Proof of concept studies performed ex vivo can then inform the design of clinical validation studies. Earlier work evaluated the response of targets such as pERK, pAKT and pSMAD2 to various kinase inhibitors, whilst recently we have used the model to measure the DNA damage response (DDR) following exposure to UV radiation (as a model damaging agent) and more clinically relevant chemotherapeutic agents.

Human plucked hair was placed in maintenance media in the presence of known chemotherapeutic agents (such as Gemcitabine, DNA topoisomerase inhibitor I, Irinotecan and Paclitaxel) or exposed to 0.2-0.5J/cm2 UVB radiation. Hairs were then fixed at a range of time points post-exposure and longitudinal sections labelled for various markers including pChk1, gamma-H2AX, p53, Ki67 and thymine dimers (TDM1). Quantitative image analysis to measure the level of labelling was performed using an Aperio® ScanScope®.

Following UVB exposure thymine dimer formation occurred immediately on the hair and was stronger on the side directly facing the source of radiation. It was followed rapidly by phosphorylation of Chk1 localised in the inner root sheath at first (10min to 1h after injury), then gamma-H2AX and p53 induction in the outer root sheath. Further studies are looking at DNA damage response in an UV dose dependent manner.

Chemotherapeutic agents known for their different mechanism of actions were tested at two concentrations with various responses depending on their mechanism of action. For example, Gemcitabine, which is linked to DNA polymerase inhibition, strongly induced p53 after 24h but no gamma-H2AX activity was seen above background level. We have profiled the large panel of responses for each chemotherapeutic agent, over an acute time course.

The ex vivo plucked hair follicle method is a rapid and convenient way of studying the DDR induced by exposure to various DNA damage agents. This model allows for screening of novel chemotherapeutic agents to test their efficacy in preventing or treating DNA damage.

Citation Format: Aude-Marine Bonavita, Liam Walker, Adam Boanas, Nicola Tonge, Gregory Tudor, Cath Booth, Ben Reed. Evaluation and quantification of biomarkers of DNA damage in human plucked hair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3601.
Abstract 361: Single cell detection of protein and RNA: Development of a novel method combining immunocytochemistry and in situ hybridization in breast cancer cells
Authors: Ma Lisa J. , Frank Richard T. , Li Xiaoxiao , Lai Steve , Schmidt Jeanette , Just Peggy , Becker-Catania Sara
Published: July 2016

Immunocytochemistry (ICC) utilizes antibodies to visualize the localization of specific proteins in cells while in situ hybridization (ISH) is a useful research tool for examining the presence and changes in levels of gene expression in cells. The ability to simultaneously visualize the co-localization of protein and RNA species in individual cells is necessary to address important biological questions. Additionally, studying both protein and RNA can be useful in cases where expression levels vary between mRNA and protein and to detect post-translational modifications. However, a combined technique has proved challenging due to the incompatibility of ICC and ISH protocols. Furthermore, traditional ISH methods have lacked sensitivity to detect low abundance RNA species and have limited multiplexing capability. Here, we describe a novel technique combining ICC staining with RNA ISH (based on the ViewRNA Assay), which utilizes branched DNA signal amplification technology allowing for single copy mRNA sensitivity. We demonstrate the utility and versatility of this technique using a variety of antibody and probe combinations relevant for cancer research. We compared expression of protein and mRNA of HER2, estrogen receptor alpha, and progesterone receptor in several breast cancer cell lines (MCF7, SKBR3, MDA-MB-175, MDA-MB-231, and BT474) known to express differential levels of these targets. We also compared expression of several microRNAs (let-7a, miR-17, miR-30d, miR-107, and miR-93) known to play a role in cancer development in combination with staining for proteins involved in proliferation (Ki-67), anti-apoptosis (survivin), and cell structure (tubulin). We show successful detection of low abundance RNA species together with various protein markers. This novel technique is a valuable tool not only for cancer researchers, but also for investigators across numerous fields in biological research.

Citation Format: Lisa J. Ma, Richard T. Frank, Xiaoxiao Li, Steve Lai, Jeanette Schmidt, Peggy Just, Sara Becker-Catania. Single cell detection of protein and RNA: Development of a novel method combining immunocytochemistry and in situ hybridization in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 361.
Abstract 3611: SMRT® sequencing of DNA samples extracted from formalin-fixed and paraffin embedded tissues
Authors: Baybayan Primo , Weiand Michael , Eng Kevin , Durin Guillaume , Kujawa Steve
Published: July 2016

Recent advances in next generation sequencing have led to the increased use of formalin-fixed and paraffin-embedded (FFPE) tissues for medical samples in disease and scientific research. Single Molecule Real-Time (SMRT®) sequencing offers a unique advantage in that it allows direct analysis of FFPE samples without amplification. However, obtaining ample long read information from FFPE samples has been a challenge due to the quality and quantity of the extracted DNA. DNA samples extracted from FFPE often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and amplification. Additionally, the quality and quantity of the recovered DNA also vary depending on the extraction methods used.

We have evaluated the Adaptive Focused Acoustics (AFA™) system by Covaris® as a method for obtaining high molecular weight DNA suitable for SMRTbell template preparation and subsequent single molecule sequencing. Using this method, genomic DNA was extracted from normal kidney FFPE scrolls acquired from Cooperative Human Tissue Network (CHTN), University of Pennsylvania. Damaged sites present in the extracted DNA were repaired using a DNA Damage Repair step, and the treated DNA was constructed into SMRTbell libraries suitable for sequencing on the RSII System. Using the same repaired DNA, we also tested PCR efficiency of target gene regions of up to 5 kb. The resulting amplicons were constructed into SMRTbell templates for full-length sequencing on the RS II System.

We found the Adaptive Focused Acoustics (AFA™) system by Covaris® to be effective and efficient. This system is easy and simple to use, and the resulting DNA is compatible with SMRTbell™ library preparation for targeted and whole genome SMRT sequencing. The data presented here demonstrates single molecule sequencing of DNA samples extracted from tissues embedded in FFPE.

Citation Format: Primo Baybayan, Michael Weiand, Kevin Eng, Guillaume Durin, Steve Kujawa. SMRT® sequencing of DNA samples extracted from formalin-fixed and paraffin embedded tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3611.
Abstract 3653: Mutational analysis of PARK2, a regulator of cyclin D1 stability, in sporadic parathyroid adenoma
Authors: Brewer Kelly , Costa-Guda Jessica , Arnold Andrew
Published: July 2016

The molecular mechanisms underlying the pathogenesis of sporadic parathyroid adenomas are incompletely understood. The oncoprotein cyclin D1 is overexpressed in 20-40% of sporadic parathyroid adenomas; however, known aberrations in the gene that encodes cyclin D1, CCND1, account for less than half of these cases. Genetic alterations of PARK2, which encodes the E3 ubiquitin ligase responsible for targeting cyclin D1 for degradation, have been identified as a frequent finding in a variety of human tumors. Interestingly, genetic abnormalities of PARK2 were mutually exclusive of cyclin D1 gene alterations, suggesting a common pathway in tumorigenesis. We postulate that inactivating mutations in PARK2 lead to increased stability of cyclin D1 and thereby may contribute to the development of sporadic parathyroid adenoma. We therefore sought to identify PARK2 coding region mutations in a series of sporadic parathyroid adenomas by direct DNA sequencing. The entire coding region and intron-exon boundaries of PARK2 were examined in a cohort of eighty typically presenting, sporadic parathyroid adenomas. Six known polymorphisms were identified in several of the adenomas in this series; however, none of the polymorphisms are associated with cancer, and no other cancer-associated mutations were found. The absence of intragenic mutations in PARK2 suggests that such mutations do not commonly contribute to the development of sporadic parathyroid adenoma. However, because such intragenic mutations could be rare and limited to only those samples in which overexpression of cyclin D1 cannot be explained by genetic abnormalities in the CCND1 gene, a significant role for cyclin D1 accumulation in parathyroid tumor formation due intragenic inactivating mutations of PARK2 cannot be ruled out. Further research must be conducted in order to elucidate the mechanism(s) of cyclin D1 accumulation in parathyroid adenomas in which CCND1 aberrations are not present.

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Citation Format: Kelly Brewer, Jessica Costa-Guda, Andrew Arnold. Mutational analysis of PARK2, a regulator of cyclin D1 stability, in sporadic parathyroid adenoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3653.
Abstract 3659: DIRAS1 and DIRAS2 are novel ovarian cancer tumor suppressors that regulate cell growth, motility and autophagy
Authors: Sutton Margie N. , Lu Zhen , Yang Hailing , Huang Gilbert , Wang Yan , Mao Weiqun , Bast Robert C.
Published: July 2016

DIRAS3 (also known as ARHI; Aplasia Ras Homolog Member I) is a potent tumor suppressor that has been well characterized for its role in ovarian cancer and autophagy. DIRAS3 is a maternally imprinted tumor suppressor gene that encodes a 26kDa GTPase which shares 60% homology to Ras and Rap. DIRAS3 is downregulated in many tumor types including ovarian cancer. DIRAS3 is downregulated by multiple mechanisms including loss of heterozygosity, transcriptional regulation by E2F1 and E2F4, hypermethylation of the second allele, and regulation by miRNAs 221 and 222. Re-expression of DIRAS3 at physiologic levels inhibits cancer cell growth, reduces motility, induces autophagy and promotes tumor dormancy. The mechanisms by which DIRAS3 induce autophagic cell death in vitro and tumor dormancy in vivo have been well characterized by previous work in our laboratory demonstrating that DIRAS3 is required for the induction of autophagy, and that upon inhibition of autophagy you can prevent outgrowth of dormant ovarian cancer cells in vivo. Interestingly, mice do not have DIRAS3 as it was lost during evolutionary rearrangement of murine chromosomes 3 and 6 nearly 60 million years ago, yet murine cells can still undergo autophagy. The DIRAS family members, DIRAS1 and DIRAS2 share 50-60% homology with DIRAS3 and are found in the murine genome. These 22kDa GTPases differ from DIRAS3 by the truncation of their N-terminal extension. Although DIRAS1 and DIRAS2 have not previously been characterized in ovarian cancer, TCGA analysis suggests that higher mRNA expression of these genes results in a survival advantage for patients with high grade serous ovarian cancer. In this study we demonstrate that re-expression of DIRAS1 and DIRAS2 inhibit ovarian cancer cell growth in vitro and induce autophagy in both human and murine cells. DIRAS1 and DIRAS2 are required for murine autophagy induced by rapamycin or amino acid starvation. Overexpression of DIRAS1 and DIRAS2 inhibits cancer cell growth and motility, and results in the inhibition of both the PI3K and Ras/MAPK signaling pathways. Thus DIRAS1 and DIRAS2 provide many of the functions of DIRAS3 in normal and malignant murine cells.

Citation Format: Margie N. Sutton, Zhen Lu, Hailing Yang, Gilbert Huang, Yan Wang, Weiqun Mao, Robert C. Bast. DIRAS1 and DIRAS2 are novel ovarian cancer tumor suppressors that regulate cell growth, motility and autophagy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3659.
Abstract 3660: ATF4 is a putative tumor suppressor gene in medullary thyroid cancer
Authors: Bagheri-Yarmand Rozita , Cote Gilbert J. , Grubbs Elizabeth G. , Williams Michelle D. , Sherman Steven I. , Gagel Robert F.
Published: July 2016

Medullary thyroid carcinoma (MTC) is derived from the calcitonin-producing C-cells of the thyroid gland. Oncogenic mutations of the RET proto-oncogene are found in all hereditary MTC and ∼45% of the sporadic cases. However, it is clear that additional genetic lesions contribute to the development of both RET-dependent and RET independent MTC. We analyzed 30 primary medullary thyroid cancer (MTC) tumors by comparative genomic hybridization (CGH) arrays (Agilent 244K) and a subset of those tumors by ultra-high-resolution SNP arrays (Affymetrix SNP6.0). A consistent finding with both arrays was a loss localized to 22q13.1 in 40% of tumors. This region contains the ATF4 gene and loss of this gene was validated by real time PCR.

ATF4 is a central mediator of integrated stress response pathway and activates expression of a cohort of downstream target genes known to be involved in cell survival, apoptosis, autophagy and senescence. We hypothesize that the loss of ATF4 expression contributes to MTC progression through mechanisms involving aberrant proliferation and escape from apoptosis. We present several findings to support this. First, ATF4 protein levels were found downregulated in 50% MTC tumor tissues (n = 43) and low ATF4 expression was associated with poor overall survival in MTC patients (HR = 5.015, 95% CI: 1.38-10.64), P = 0.013). A negative correlation was observed between RET and ATF4 protein expression in MTC tumors (r = -0.89, R square = 0.799, p<0.0001). Second, we showed that targeted heterozygote and homozygous deletion of Atf4 in mice causes C cell hyperplasia, a precancerous lesion preceding MTC. Third, ATF4 overexpression inhibits proliferation and survival of MTC derived cell lines. ATF4 decreases RET protein levels by inducing ubiquitination and degradation of activated RET. In contrast, ATF4 depletion upregulates RET protein levels as well as RET autophosphorylation. Finally, combination treatment of tyrosine kinase inhibitor (sunitinib) and eeyarestatin that stimulates ATF4 transcription, induces apoptosis of the MTC cells in a synergistic manner through activation of ATF4 and its target genes NOXA and PUMA as well. These results suggest ATF4 as a potential tumor suppressor gene in MTC. We postulate that inactivation of ATF4 is a central impediment to induction of apoptosis in MTC tumors. Therefore, strategies to upregulate ATF4 during TKI therapy would render tumor cells exquisitely sensitive to stress induced apoptosis, offering a novel synergistic treatment.

Citation Format: Rozita Bagheri-Yarmand, Gilbert J. Cote, Elizabeth G. Grubbs, Michelle D. Williams, Steven I. Sherman, Robert F. Gagel. ATF4 is a putative tumor suppressor gene in medullary thyroid cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3660.
Abstract 3665: WAC: A candidate tumor suppressor gene in colorectal cancer
Authors: Clark Christopher R. , Conboy Caitlin , Maile Makayla , Janik Callie , Hatler Julia , Cormier Robert , Largaespada David , Starr Timothy K.
Published: July 2016

Our lab recently performed a DNA transposon forward genetic screen in mice that was designed to identify low-frequency mutations that contribute to colorectal cancer (CRC) initiation and progression. Results from this screen identified the WW domain-containing adaptor with coiled-coil (WAC) gene as a top DNA transposon insertion site. WAC has previously been implicated in several cellular processes including amino acid starvation-induced autophagy, golgi biogenesis, and transcription associated histone modification but has never before been linked to tumorigenesis. Transposon mutagenesis screens performed by others (Takeda et al. Nature Genetics 2015) have also identified Wac as a common insertion site, a result that further implicates WAC as a candidate CRC driver gene. Analyses of transposon insertion patterns within Wac predict loss of gene function and a role as a tumor suppressor. Soft agar colony formation assays reveal that shRNA mediated silencing of Wac cooperates with Apc mutations in mouse colorectal cells to promote cellular transformation. Additional colony formation assays using immortalized human colonic epithelial cells and the adenoma derived AAC1 cell line also shows that silencing WAC is protumorigenic. Using a zebrafish model we demonstrated that overexpression of wild type but not cancer-associated mutant forms of WAC induce expression of the cell cycle inhibitor p21, which suggests that loss of WAC may lead to uncontrolled cellular proliferation. Finally, using publicly available mutation data we determined that WAC is somatically mutated in both breast and lung cancers; a finding that indicates WAC may serve a critical tumor suppressive role in several tissues. Currently we are developing a conditional knockout mouse to further investigate the role of WAC in CRC tumor formation.

Citation Format: Christopher R. Clark, Caitlin Conboy, Makayla Maile, Callie Janik, Julia Hatler, Robert Cormier, David Largaespada, Timothy K. Starr. WAC: A candidate tumor suppressor gene in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3665.
Abstract 3667: Potential role of DUSP4 as a tumor suppressor in pancreatic cancer
Authors: Singh Chandra K. , George Jasmine , Nihal Minakshi , Ahmad Nihal
Published: July 2016

Pancreatic cancer remains one of the most lethal of all human malignancies, with 48,960 expected new cases and 40,560 deaths predicted in the USA in the year 2015. In order to develop novel strategies for the management of pancreatic cancer, it is of utmost importance to intensify our efforts to dissect the molecular mechanisms involved in the pathophysiology of this fatal neoplasm. Dual-specificity phosphatase-4 (DUSP4), a family member of dual-specificity phosphatases, is capable of dephosphorylating both phosphotyrosine and phosphoserine/phosphothreonine residues and possesses activity against key signaling components of the MAPK pathway that plays an important role in cancer growth. DUSP4 has been shown to be widely expressed in different tissues and implicated in cancer development, differentiation, apoptosis and inflammation. In a recent study from our laboratory that was aimed at defining the molecular targets of the anti-cancer plant-based alkaloid sanguinarine in pancreatic cancer, we identified DUSP4 as a key protein modulated by sanguinarine. Specifically, based on proteomics data and validation by qRT-PCR and immunoblot analysis, we found that sanguinarine significantly increases DUSP4 in BxPC-3 and MIA PaCa-2 pancreatic cancer cells. Indeed, an association of DUSP4 loss with cancer progression has been shown in certain cancers. However, the role of DUSP4 in pancreatic cancer is not known. This study was designed to determine the role and importance of DUSP4 in pancreatic cancer. We determined the expression profile of DUSP4 in pancreatic cancer using immunohistochemical analysis of a tissue microarray containing a variety of pancreatic cancers and normal pancreatic tissues. The DUSP4-immunostaining was microscopically imaged followed by quantification of DUSP4 protein by multispectral Vectra™ system coupled with inForm software. We found a significant downregulation of DUSP4 in human pancreatic cancer tissues, when compared to normal pancreatic tissues. Further, to understand the importance of DUSP4 in pancreatic cancer, we determined the effect of a force overexpression of DUSP4 in MIA PaCa-2 pancreatic cancer cells. We found that DUSP4 overexpression in MIA PaCa-2 cells resulted in a significant i) decrease in cell growth and proliferation, ii) clonogenic survival, and iii) induction of apoptosis. These preliminary findings suggest a potential tumor suppressive function of DUSP4 in pancreatic cancer. Further detailed studies are ongoing in our laboratory to validate the role and functional significance of DUSP4 in pancreatic cancer.

Citation Format: Chandra K. Singh, Jasmine George, Minakshi Nihal, Nihal Ahmad. Potential role of DUSP4 as a tumor suppressor in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3667.
Abstract 3672: Coordinated roles of CHD1 and SPOP in prostate cancer pathogenesis
Authors: Vasudevan Divya , Blattner Mirjam , Deonarine Lesa D. , Chen Yu , Rubin Mark A. , Barbieri Christopher E.
Published: July 2016

Prostate cancer (PCa) is a clinically heterogeneous disease, consequently presenting challenges while designing efficacious therapeutic strategies across diverse patient cohorts. There is an increasing need to improve our understanding of the disease by classifying PCa into distinct molecular subtypes characterized by specific genomic alterations. To this end, loss of the gene encoding chromodomain helicase DNA binding protein 1 (CHD1) represents one of the largest classes of recurrent gene deletions detected exclusively in PCa, hallmarking up to 20% of characterized tumors. CHD1 is well known to facilitate the assembly of euchromatin during transcription. Therefore, we hypothesized that loss of CHD1 would induce a significant change in overall transcription and alter prostate cell fate, promoting tumorigenesis. To confer a high degree of physiological relevance to our studies, we utilized genetically engineered mouse (GEM) models harboring prostate-specific CHD1 deletions. Since loss of CHD1 has a significant tendency to co-occur with mutations in the SPOP gene, we generated GEM models that also co-express the SPOP F133V transgene. We derived organoid lines from GEM prostates to recapitulate prostate histology in a three dimensional matrix, which allowed us to track changes at the molecular level and overall phenotype as they relate to PCa development. We found that CHD1 loss associated strongly with the presence of an increased luminal cell population compared to wild type controls. Since luminal cells are recognized to be cells of origin in PCa, this observation is consistent with the notion that CHD1 deletion represents a pro-tumorigenic event. Additionally, our data indicates that co-occurrence of CHD1 deletions and SPOP mutations may confer a growth advantage to prostate cells. The overarching goal of this study is to define the coordinated role of CHD1 loss and SPOP mutations in PCa, highlight altered signaling pathways and phenotypes, and nominate potential therapeutic strategies for patients with this PCa subtype.

Citation Format: Divya Vasudevan, Mirjam Blattner, Lesa D. Deonarine, Yu Chen, Mark A. Rubin, Christopher E. Barbieri. Coordinated roles of CHD1 and SPOP in prostate cancer pathogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3672.
Abstract 3674: MIG-6 suppresses epithelial cell proliferation via inhibiting AKT phosphorylation during endometrial tumorigenesis
Authors: Kim Tae Hoon , Yoo Jung-Yoon , Lee Jae Hee , Kim Byung Gak , Broaddus Russell R. , Jeong Jae-Wook
Published: July 2016

Endometrial cancer is the most frequently diagnosed malignancy of the female genital tract. Hysterectomy is typically the first line therapeutic strategy for endometrial cancer. However, there is an increasing demand for nonsurgical approaches for endometrial cancer, especially for women of reproductive age with endometrial cancer who wish to preserve their fertility. Most endometrial cancers are characterized by actively proliferating epithelial cells, increased AKT signaling and association with prolonged, unopposed E2 exposure.

Mitogen-inducible gene 6 (Mig-6) is an important mediator of progesterone receptor action in the murine uterus. As P4 achieves the inhibition of proliferation by coordinating stromal-epithelial cross-talk, we generated a mouse model in which we specifically ablate epithelial endometrial Mig-6 using Sprr2f-cre mice (Sprr2fcre+Mig-6f/f) to understand the role of epithelial Mig-6 in the uterus. Sprr2fcre+Mig-6f/f mice displayed endometrial hyperplasia upon 10 weeks of age and develop endometrial cancer by E2 treatment for 3 months. The levels of epithelial proliferation by Ki67 staining were significantly increased in epithelial cells of Sprr2fcre+Mig-6f/f mice at 10 weeks of age. The levels of phospho-AKT and phospho-S6, downstream of AKT, were remarkably higher in Sprr2fcre+Mig-6f/f mice. Interestingly, the hyperplasia exhibited by Sprr2fcre+Mig-6f/f mice was prevented by P4 treatment for 1 week in morphological and histological analysis. Aberrant activation of proliferation as well as AKT signaling was decreased in the epithelium of Sprr2fcre+Mig-6f/f mice by P4 treatment. Furthermore, we identified that MIG-6 inhibits AKT phosphorylation in human endometrial cancer cells. These data suggest that stromal P4 signaling including Mig-6 is critical in regulation of epithelial cell proliferation via regulating AKT phosphorylation during endometrial cancer development and progression.

(This work was supported by American Cancer Society Research Grant, RSG-12-084-01-TBG to J.W.J.)

Citation Format: Tae Hoon Kim, Jung-Yoon Yoo, Jae Hee Lee, Byung Gak Kim, Russell R. Broaddus, Jae-Wook Jeong. MIG-6 suppresses epithelial cell proliferation via inhibiting AKT phosphorylation during endometrial tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3674.
Abstract 3691: MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation
Authors: Qi Donglai , Cobrinik David E.
Published: July 2016

Retinoblastomas can arise from cone photoreceptor precursors in response to the loss of pRB function. Some aspects of cone precursor-specific circuitry cooperate with pRB loss to initiate this process and subsequently continue to contribute to the malignancy. Intrinsic high level MDM2 expression is a key component of this circuitry and is thought to inactivate p53-mediated tumor surveillance that could otherwise be induced by aberrant cell cycle entry. However, the MDM2-related MDM4 has also been proposed to abrogate p53-mediated tumor surveillance in the absence of detectable MDM2. Here we report that high-level MDM2 but not MDM4 has a consistent, critical role in retinoblastoma cell proliferation. Reduction of MDM2 and MDM4 only weakly induced p53, yet reduction of MDM2 but not MDM4 severely impaired proliferation and survival through a p53-independent mechanism. Specifically, MDM2 up-regulated the translation of another component of the cone circuitry, MYCN, in retinoblastoma cells. These findings indicate that high-level MDM2 expression is needed in order to perform a critical p53-independent function and may obviate the need for genomic alterations to the p53 pathway in retinoblastoma cells.

Citation Format: Donglai Qi, David E. Cobrinik. MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3691.