bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3030: De novo engineering of chromosomal amplifications in human cells
Authors: Lauring Josh
Published: August 2015

Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to recurrent amplification events are lacking. Moreover, some common amplification events, such as that at 8p11-12 in breast cancer, harbor multiple candidate driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our general strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We chose regions recurrently amplified at high frequency in breast cancer, including 8p11-12, 17q12, and 11q13-14. After confirmation of successful gene targeting, the targeted cells are subjected to selective pressure directed at the amplifiable marker. Spontaneous DNA breakage events lead to varying degrees of co-amplification of linked loci near the targeting site. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using either the E. coli inosine monophosphate dehydrogenase enzyme IMPDH or a mutant dihydrofolate reductase gene as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR. Genome-wide array comparative genomic hybridization confirmed that amplification had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including “sawtooth” patterns, distal copy number loss suggesting a breakage-fusion-bridge mechanism, and large continuous regions of copy number gain. Amplification was confirmed by FISH. RT-PCR and immunoblotting demonstrated overexpression of selected amplified transcripts and proteins. In summary, we have used genome engineering techniques to create focal chromosomal amplification events de novo in human cell lines. These amplifications share many of the features of amplifications observed in primary human cancers. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a system to model oncogene cooperativity in amplifications harboring multiple candidate driver genes.

Citation Format: Josh Lauring. De novo engineering of chromosomal amplifications in human cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3030. doi:10.1158/1538-7445.AM2015-3030
Abstract 3034: A high-content screen to identify novel chromosome instability genes
Authors: Thompson Laura L. , Baergen Allison , Lichtensztejn Zelda , McManus Kirk J.
Published: August 2015

Chromosome instability (CIN) is defined as an increase in the rate at which whole chromosomes or large chromosomal fragments are gained or lost. It is a characteristic of virtually all cancer types that is frequently observed in highly aggressive, drug resistant tumors. Despite this, the majority of human CIN genes have yet to be elucidated, highlighting the need for studies aimed at identifying the defective genes that underlie CIN. In this study we developed image-based approaches capable of detecting CIN-associated phenotypes following RNAi-based silencing of candidate CIN genes. The first assay involves quantifying nuclear areas following silencing, where changes in mean nuclear area relative to controls act as a surrogate marker of CIN. The second approach monitors micronucleus (MN) formation where increases in the number of micronuclei are indicative of CIN. These assays were employed in a high-content screen of 164 human candidate CIN genes in two unrelated cell lines, HT1080 and hTERT. In HT1080, the nuclear area and MN enumeration assays identified 43 and 83 putative CIN genes, respectively. In hTERT, the nuclear area and MN assays identified 55 and 48 putative CIN genes, respectively. Preliminary data collected through Western blotting, mitotic spreads and flow cytometry, has provided evidence to support the validation of a subset of these putative CIN genes (e.g. SKP1), as bona fide human CIN genes. Identifying novel CIN genes will provide critical insights into CIN and tumorigenesis, as well as identify potential targets that could be exploited for the development of superior therapeutic strategies.

Citation Format: Laura L. Thompson, Allison Baergen, Zelda Lichtensztejn, Kirk J. McManus. A high-content screen to identify novel chromosome instability genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3034. doi:10.1158/1538-7445.AM2015-3034
Abstract 3036: The SUMO-targeted ubiquitin ligase RNF4 regulates BLM helicase's function in dormant origin firing
Authors: Ellis Nathan A. , Yang Wei-Chih , Yagle Mary , Zhu Jianmei , Huang Jing , Seidman Michael , Matunis Michael J.
Published: August 2015

Regulation of dormant origin firing under conditions of replication stress is poorly understood. The Bloom syndrome DNA helicase BLM functions in maintaining replication fork stability, with sumoylation regulating this function, and BLM deficiency is associated with increased dormant origin firing. We found here that BLM sumoylation levels increase in response to replication stalling by hydroxyurea (HU) and to proteasome inhibition; depletion of the SUMO-targeted ubiquitin ligase RNF4 also causes increased levels of BLM sumoylation. RNF4 directly interacts with BLM and can ubiquitylate sumoylated BLM in vitro. These data indicate that sumoylated BLM is an RNF4 substrate. RNF4 is recruited to DNA repair foci in response to replication stalling. RNF4 depletion causes the accumulation of increased numbers of HU-induced BLM foci, whereas the numbers of RPA, RAD51, and gamma-H2AX foci are unaffected by fork stalling in RNF4-depleted cells. Sister chromatid exchanges are normal in RNF4-depleted cells. These data indicate that RNF4 can regulate BLM retention at stalled forks without affecting repair by homologous recombination. RNF4 depletion reduces cell proliferation and survival of untreated cells, and it confers hypersensitivity to replication stalling by HU. Studies of cell-cycle progression using bromodeoxyuridine incorporation and flow cytometry show that RNF4 depletion causes a delay of re-entry into S phase in HU-treated cells and the replication delay is partially rescued by BLM mutation. The delay is not caused by modification of checkpoint kinase activities. Analysis of replication dynamics using the DNA fiber assay show that RNF4 depletion causes an increase of permanently stalled replication forks and a decrease in activation of dormant origins following recovery from HU-induced replication stress. Co-depletion of RNF4 and BLM partially rescues these defects. Replication dynamics is unaffected by RNF4 depletion in untreated cells. These data indicate that RNF4 regulates BLM's functions in replication fork stability and dormant origin firing, without affecting BLM's roles in homologous recombination. We propose that sumoylation of BLM at stalled replication forks leads to RNF4-mediated ubiquitylation and subsequent proteasome-dependent degradation of BLM, which relieves BLM's active inhibition of dormant origin firing.

Citation Format: Nathan A. Ellis, Wei-Chih Yang, Mary Yagle, Jianmei Zhu, Jing Huang, Michael Seidman, Michael J. Matunis. The SUMO-targeted ubiquitin ligase RNF4 regulates BLM helicase's function in dormant origin firing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3036. doi:10.1158/1538-7445.AM2015-3036
Abstract 304: Formation of uniform and reproducible 3D cancer spheroids in a high throughput plate format
Authors: Neeley Cindy , Revankar Chetana M.
Published: August 2015

Cells grown on flat two dimensional (2D) tissue culture substrates can differ considerably from the three dimensional (3D) physiological environment. There is growing evidence that tumor cell aggregates or cancer spheroids are more representative of tumors in vivo and they exhibit several physiological traits including similar morphology, the formation of cell-cell contacts, decreased proliferation, increased survival rates and a hypoxic core. However, variability in cancer spheroid culture has been a persistent problem for researchers. This variability is linked to medium composition and volume, cell density, duration in culture, and most importantly, the cellular interactions with the culture dish itself. Here, we demonstrate that a hydrophilic polymer coated surface, Thermo Scientific Nunclon Sphera, inhibits cell attachment to the culture dish by blocking binding of the extracellular matrix (ECM). The U bottom wells in the Nunclon Sphera plate provide the ideal size and shape for single spheroid formation in each well, offering consistency and reproducibility to the 3D tumor model system in vitro. The growth of cancer spheroids in the Nunclon Sphera 96U-well plate is superior to that of the non-treated plate with less “satellite colonies” and faster cell aggregation at low seeding density. Cancer cell viability and spheroid health are also evaluated by fluorescence- and colorimetric- based assays in the Nunclon Sphera plates. The Nunclon Sphera 96U-well plate provides an excellent solution for reproducibly generating uniform 3D cancer spheroids that are suitable for many cell-based drug discovery procedures, co-culture studies, and high-throughput drug screening.

Citation Format: Cindy Neeley, Chetana M. Revankar. Formation of uniform and reproducible 3D cancer spheroids in a high throughput plate format. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 304. doi:10.1158/1538-7445.AM2015-304
Abstract 305: An innovative 3D porous scaffold-based perfusion bioreactor system for the in vitro maintenance and expansion of primary breast cancer tissue
Authors: Muraro Manuele G. , Muenst Simone , Spagnoli Giulio C. , Oertli Daniel , Weber Walter P. , Soysal Savas D.
Published: August 2015

Introduction: Development of cancer treatment protocols has traditionally been based on studies in experimental animal models or taking advantage of human established tumor cell lines for in vitro and in vivo assays. Here we present an innovative method to culture breast cancer tissues in porous 3D scaffolds by using a perfusion-based bioreactor system.

Experimental procedures: Freshly excised estrogen-receptor positive breast cancer specimens were fragmented and cultured in a 3D “sandwich-like format” between porous scaffolds under perfusion flow, with “on-line” monitoring of environmental condition. DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium in the presence or absence of estrogens. The ability of tumor cells to survive and expand into the scaffold in perfusion culture as well as their ability to recapitulate features of the original breast cancer specimens was histologically assessed.

Results: With this innovative method we were able to preserve viability and to promote the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Expanding cancer cells were viable after 21 days and recapitulating the initial histology with formation of glands. Administration of estrogen to the culture medium was associated with increased expansion of cancer cells into the scaffold starting at 14 days of culture.

Conclusions: Culture of breast cancer tissue in a 3D scaffold-based bioreactor preserves malignant, interstitial and immunocompetent cells present in surgically excised breast cancer samples, thus allowing direct testing of basic cancer research hypotheses in intact, complex human tumor microenvironments. Furthermore, it might allow a direct evaluation of the effects of various treatments on malignant and non-transformed cells. This engineered in vitro model could be extended beyond the context of our primary scientific interest, namely breast cancer, as a broader platform allowing animal-free testing of innovative approaches for the treatment of human malignancies, possibly in the direction of personalized medicine.

Citation Format: Manuele G. Muraro, Simone Muenst, Giulio C. Spagnoli, Daniel Oertli, Walter P. Weber, Savas D. Soysal. An innovative 3D porous scaffold-based perfusion bioreactor system for the in vitro maintenance and expansion of primary breast cancer tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 305. doi:10.1158/1538-7445.AM2015-305
Abstract 3052: NAMPT inhibition induces mitochondrial dysfunction leading to apoptosis in chronic lymphocytic leukemia cells
Authors: Bouchard Eric DJ , Mejia Edgard M. , Gehrke Iris , Poeppl Armando G. , Hewitt Donna , Johnston James B. , Gibson Spencer B. , Hatch Grant M. , Banerji Versha
Published: August 2015

We have previously shown that the NAD depletion following inhibition of NAMPT by the NA-mimetic FK866 leads to loss of mitochondrial membrane potential, depletion of cellular ATP, cytochrome C release and caspase activation in primary Chronic Lymphocytic Leukemia (CLL) cells in vitro, and that these events are coupled with an increased production of reactive oxygen species uncharacteristic of metabolic inhibition. In the present study we characterize the effect of NAD depletion on CLL mitochondria and the downstream mechanism of cell death.

We profiled the mitochondrial respiration of CLL cells and control B-lymphocytes, by extracellular flux analysis, over the 48 hours following FK866 treatment and found time-dependant inhibition of respiratory capacity consistent with disruption of the electron transport chain (ETC) in CLL cells. This led to suppression of basal respiration concomitant with the rise in ROS and approximately one day before loss of cellular viability. Interestingly, Zap70-positive CLL cells exhibited increased mitochondrial respiration and respiratory capacity over Zap70-negative CLL cells, but responded similarly to NAMPT inhibition. Additionally, mitochondrial respiration was not effected by NAMPT inhibition in control B-lymphocytes.

Increase autophagic flux, caspase-dependant apoptosis and mitochondrio-nuclear translocation of Apoptosis Inducing Factor (AIF) were detected by western blot. However, neither pan-caspase inhibition with Z-VAD-fmk nor inhibition of autophagy with 3-methyladenine were sufficient to prevent FK866-induced CLL cell death. We conclude that NAD depletion following inhibition of NAMPT leads to disruption of the mitochondrial ETC in CLL cells, leading to mitochondrial initiation of the intrinsic apoptosis pathway. As healthy B-lymphocytes are resistant to this NAD depletion, these downstream effects are also selective for malignant cells.

We are currently working to characterize the roles of NAD and Zap70 in the regulation and function of the CLL ETC. This will not only lead to an improved understanding of CLL metabolism, but will also inform new therapeutic strategies that will effectively employ NAMPT inhibition to treat this yet-incurable disease.

Citation Format: Eric DJ Bouchard, Edgard M. Mejia, Iris Gehrke, Armando G. Poeppl, Donna Hewitt, James B. Johnston, Spencer B. Gibson, Grant M. Hatch, Versha Banerji. NAMPT inhibition induces mitochondrial dysfunction leading to apoptosis in chronic lymphocytic leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3052. doi:10.1158/1538-7445.AM2015-3052
Abstract 3056: Mitochondrial DNA copy number and risk of second primary tumors in long-term breast cancer survivors
Authors: Santibanez Miguel , Rodriguez Alma , Hildebrandt Michelle A. , Chang David W. , Wu Xifeng
Published: August 2015

Background. Mitochondria is constantly exposed to reactive oxygen species (ROS), an important factor in carcinogenesis. Altered mitochondrial DNA (mtDNA) copy number in peripheral blood has been associated with increased risks of several cancers, including breast cancer. However, the association of mtDNA copy number with clinical outcomes of breast cancer has not been reported. This study aimed to assess the association of mtDNA copy number with second primary tumors (SPT) in long-term breast cancer survivors.

Methods. We measured mtDNA by quantitative PCR in patients in 68 long-term breast cancer survivors with SPT, 100 long-term breast cancer survivors without SPT and 97 newly diagnosed breast cancer cases. Cases with and without SPT were matched by years since diagnosis, age at time of sample collection and race. Differences between mtDNA copy number among groups were analyzed using the Wilcoxon rank sum test.

Results. MtDNA copy number was significantly higher in long-term breast cancer survivors (either with SPT or not SPT) compared with newly diagnosed breast cancer patients (Mean (SD), 1.03 ± 0.25 vs 0.95 ± 0.27; p = 0.005). We did not observe significant differences of mtDNA copy number between long-term survivors with SPT and without SPT. However, in stratified analyses by epidemiological and clinical variables, we observed lower mtDNA copy number in HER2-positive long-term survivors with SPT than those without SPT (0.84 ± 0.13 vs 1.27 ± 0.44; p = 0.010) and in pre-menopause long-term survivors with SPT than those without SPT (0.95 ± 0.20 vs 1.06 ± 0.22; p = 0.034). A borderline significance of mtDNA copy number between SPT and non-SPT ER-negative long-term survivors was also noted (0.90 ± 0.17 vs 1.18 ± 0.41, p = 0.056).

Conclusion. Higher mtDNA copy number was found in long-term breast cancer survivors compared to newly diagnosed cases. There were significant associations between lower mtDNA copy number and SPT in long-term breast cancer survivors who are HER2+ or are pre-menopause. These results indicate a role of mtDNA copy number in long-term breast cancer survivorship.

Citation Format: Miguel Santibanez, Alma Rodriguez, Michelle A. Hildebrandt, David W. Chang, Xifeng Wu. Mitochondrial DNA copy number and risk of second primary tumors in long-term breast cancer survivors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3056. doi:10.1158/1538-7445.AM2015-3056
Abstract 3057: Potentiation of LPS-induced toxicity in human hepatoma HepG2 cells by aspirin and its protection by N-acetyl cysteine
Authors: Raza Haider , John Annie , shafarin Jasmin
Published: August 2015

Bacterial lipopolysaccharides (LPS) induce cytotoxicity in vitro and inflammation-induced toxic responses in vivo. Use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, has been reported to be beneficial in inflammation-associated diseases like cancer and diabetes. Their precise molecular mechanisms, however, are not clearly understood. Our previous studies on aspirin treated HepG2 cells strongly suggest cell cycle arrest and induction of apoptosis associated with mitochondrial dysfunction. In the present study, we have further demonstrated that HepG2 cells treated with LPS alone or in combination with aspirin induces subcellular toxic responses which are accompanied by increase in reactive oxygen species (ROS) production, oxidative stress, apoptosis and mitochondrial respiratory dysfunction. The LPS/Aspirin induced toxicity was attenuated by pretreatment of cells with N-acetyl cysteine (NAC). Oxidative stress and glutathione-dependent redox-homeostasis in the mitochondria were altered more severely compared to the extramitochondrial compartment of cells. Pre-treatment of HepG2 cells with NAC has demonstrated selective protection in redox homeostasis and mitochondrial dysfunction. Our results suggest that altered glutathione (GSH)-redox metabolism, oxidative stress and mitochondrial function in HepG2 cells play a critical role in LPS/aspirin-induced cytotoxicity. These results may help in better understanding the pharmacological and toxicological properties of NSAIDs in cancer cells exposed to bacterial endotoxins. (Supported by Sheikh Hamdan Medical Research Grant and Terry Fox Cancer Research Funds and a fund from CMHS Research Committee).

Citation Format: Haider Raza, Annie John, Jasmin shafarin. Potentiation of LPS-induced toxicity in human hepatoma HepG2 cells by aspirin and its protection by N-acetyl cysteine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3057. doi:10.1158/1538-7445.AM2015-3057
Abstract 3065: MicroRNA-137 confers chemoresistance by suppressing CASP3 in lung cancer cells
Authors: Shu Te-Jen , Yu Sung-Liang , Chen Jeremy J.W.
Published: August 2015

Lung cancer is the leading cause of cancer-related death. Although molecular targeting therapy (MTT) could benefit patients with EGFR activating mutations, chemotherapy still is the first choice of EGFR wild type patients, MTT failed ones and patients of developing countries without regarding their EGFR status. However most of these chemotherapy-treated patients will develop drug resistance eventually. Here, we identified miR-137 might contribute to chemo-resistance in lung cancer in part. Our previous study identified 5 miRNAs (let-7a, miR-182*, miR-372, miR-137 and miR-221) whose expression can be an independent marker to predict the outcome of non-small cell lung cancer (NSCLC) patients. Among them, miR-137 acts as a risky miRNA. Caspase-3 (CASP3) was predicted as one of miR-137 potential targets. CASP3 is a critical effector caspase that promotes programmed cell-death when activated in the apoptotic cells both by extrinsic and intrinsic pathways that are highly conserved and related to drug resistance. We found miR-137 could regulate CASP3 through binding to its 3′UTR. Endogenous CASP3 can be up-regulated by over-expressing miR 137 in CL1-5, H1437 and A549 cells and down-regulated by silencing miR-137 in H1299, CL1-0 and A549 cells. Suppression of CASP3 by miR-137 providing lung cancer cells with ability of anti-apoptosis under UV-induced cell death. MiR-137 also increase IC50 of CL1-5, H1437, A549 and H1975 cells under cisplatin treatment to at least 2 folds. Restoration of CASP3 could reverse the effect. Our IHC data also shows the CASP3 protein expression is negative correlated with miR-137 level. In summary, manipulation of miR-137 might provide a potential treatment for EGFR wild-type and MTT failed NSCLC.

patients in the future.

Citation Format: Te-Jen Shu, Sung-Liang Yu, Jeremy J.W. Chen. MicroRNA-137 confers chemoresistance by suppressing CASP3 in lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3065. doi:10.1158/1538-7445.AM2015-3065
Abstract 3066: Regulation of oncogenic Zeb1 by microRNA-23b in bladder cancer
Authors: Majid Shahana , Dar Altaf A. , Saini Sharanjot , Yamamura Soichiro , Shahryari Varahram , Mitsui Yozo , Tanaka Yuichiro , Dahiya Rajvir , Deng Guoren
Published: August 2015

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation. In this study we show that miRNA-23b (miR-23b) acts as a tumor suppressor in bladder cancer. Quantitative real-time PCR analysis showed that miR-23b is significantly down-regulated in bladder cancer cell lines and tumor tissues compared to non-malignant cells and normal tissue samples. We also demonstrate that miR-23b expression has a potential to be diagnostic and prognostic biomarker in bladder cancer. High miR-23b expression is positively correlated with higher overall survival of bladder cancer patients as revealed by Kaplan-Meier analysis. ROC analysis showed that miR-23b expression can distinguish between normal and bladder cancer tissues. Further we elucidated the biological significance of miR-23b in bladder cancer. Over-expression of miR-23b in bladder cancer cells inhibited cell proliferation and impaired colony formation. Fluorescence activated cell sorting (FACS) analysis revealed that re-expression of miR-23b in bladder cancer cells induced G0/G1 cell cycle arrest and apoptosis while inhibiting cell migration and invasion. Luciferase reporter assays demonstrated that Zeb1, a crucial regulator of epithelial-to-mesenchymal transition (EMT), is a direct target of miR-23b in bladder cancer. These results show that loss of miR-23b confers a proliferative advantage and promotes bladder cancer cell migration and invasion. Furthermore, re-expression of miR-23b may be a beneficial therapeutic strategy for the treatment of human bladder cancer.

Citation Format: Shahana Majid, Altaf A. Dar, Sharanjot Saini, Soichiro Yamamura, Varahram Shahryari, Yozo Mitsui, Yuichiro Tanaka, Rajvir Dahiya, Guoren Deng. Regulation of oncogenic Zeb1 by microRNA-23b in bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3066. doi:10.1158/1538-7445.AM2015-3066
Abstract 3067: Expression of miR-301a regulates metastasis and act as a lethal marker for prostate cancer
Authors: Das Trinath P. , Suman Suman , PapuJohn Arokya Ms , Ankhem Akhila , Rao Sandhya R. , Messer Jamie C. , Alatassi Houda , Ankhem Murali M. , Damodaran Chendil
Published: August 2015

Prostate Specific Antigen (PSA) screening contributes as a biomarker for diagnosis of prostate cancer; however, PSA screening has been considered an over diagnosis in some cases. This has lead to the identification of novel invasive markers which could eventually differentiate patients with lethal prostate cancer from those with indolent disease. Emerging evidences suggest that a small set of microRNAs (miRNA) are linked to prostate cancer pathogenesis leading to castration resistant prostate cancer (CRPC).

In our studies of genome wide miRNA profiling, we have found that miR-301a is highly expressed in prostate cancer (CaP) cell lines as compared to normal prostate epithelial cells. While analyzing CaP samples, we found higher expression of miR-301a at specific stages. Interestingly, the miR-301a induction varies from 10 to 250 fold in aggressive tumors which clearly correlate with metastases. To confirm these findings, we utilized metastatic lesions from transgenic mice model of Trans Adenocarcinoma of Mouse Prostate (TRAMP) tissues, where higher levels of miR301a expression were observed in prostate tumors in comparison with normal prostate. Further, micro dissecting the tumors (metastasis) from lung showed ∼15 fold induction of miR301a expression compared to the normal lung components. Silencing specifically miR-301a resulted in the inhibition of proliferation, colony forming abilities, invasion, and migration and cell adhesion properties in CaP cell lines. Our molecular studies also suggest that miR-301 regulates Rho GTPases family proteins and transcriptionally activates Epithelial-Mesenchymal-Transition (EMT) markers like slug and β-catenin.

Our ongoing studies may not only reveal whether miR-301 regulates Rho-associated kinases (ROCK) but also the potential role of EMT in CaP. Furthermore, we intend to perform a case control study by screening an additional 100 CaP cases besides indolent controls. As mentioned earlier, besides revealing the functional role in CaP pathogenesis, results from these findings will also suggest whether miR-301 expression distinguishes lethal CaP from indolent disease.

Citation Format: Trinath P. Das, Suman Suman, Arokya Ms PapuJohn, Akhila Ankhem, Sandhya R. Rao, Jamie C. Messer, Houda Alatassi, Murali M. Ankhem, Chendil Damodaran. Expression of miR-301a regulates metastasis and act as a lethal marker for prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3067. doi:10.1158/1538-7445.AM2015-3067
Abstract 3072: miR-551b amplification regulates STAT3 expression and resistance to anoikis of ovarian cancer cells
Authors: Chaluvally Raghavan Pradeep , Pradeep Sunila , Lu Yiling , Sood Anil , Mills Gordon
Published: August 2015

Background and Hypothesis: The 3q26.2 locus is highly amplified in high grade serous ovarian cancer (HGSOC). Recently we identified two microRNAs, miR569 and miR551b are located in the 3q26 amplicon are highly expressed in the HGSOC due to the 3q26.2 amplification. However, the potential role of microRNAs amplified at 3q26 amplicon and their functions in tumorigenesis have not been well studied. In this study we have identified the mechanism how miR551b promote the tumor growth and metastasis of ovarian cancer cells.

Methods and Results: High-resolution SNP-based copy number analysis of 533 high-grade serous ovarian cancer samples demonstrated that miR551b is highly amplified in ∼35% of HGSOC patients’ samples. Quantitative real-time PCR (qRT-PCR) analysis of ovarian cancer samples demonstrated a marked increase in the level of mature miR551b in tumors with the 3q26.2 amplicon, relative to the non-amplified tumor samples as a consequence of the 3q26.2 amplicon. Of note, miR551b is expressed highly in the metastatic tumors than the primary tumors of ovary. Our reverse phase protein array (RPPA) demonstrated that enforced expression of miR551b increased the expression c-KIT, STAT3 and PCNA proteins. Importantly miR551b expression activated the ERK signaling pathway and inhibited the expression of BIM protein. Strikingly, the expression of miR551b induced the survival and proliferation of ovarian surface epithelial cells detached from extracellular matrix. Knock down of STAT3 using target specific siRNA abrogate the miR551b mediated c-KIT and PCNA expression and abolished the resistance to anoikis.

Summary: Our results posit that copy number alterations (CNAs) of miR551b primarily regulate the increased levels of mature miR551b. Mechanistic studies revealed that miR-551b regulate the expression of important oncogenes such as STAT3 and c-KIT, in turn causes resistance to anoikis. Resistance to anoikis is the key mechanism enhances the survival and metastasis of ovarian cancer cells in the peritoneal cavity. Of note our results demonstrates that miR-551b regulate c-Kit expression in STAT3 dependent manner. In conclusion, our results propose that the STAT3 expression induced by miR551b is the key mechanism regulates survival and metastasis of HGSOC cells.

Citation Format: Pradeep Chaluvally Raghavan, Sunila Pradeep, Yiling Lu, Anil Sood, Gordon Mills. miR-551b amplification regulates STAT3 expression and resistance to anoikis of ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2015-3072
Abstract 3075: miRNA-10a* enhances invasiveness via suppression of HDAC5 in lung cancer
Authors: Hsu Pin-Yen , Ho Bing-Ching , Cheng Chiou-Ling , Chen Hsuan-Yu , Yu Sung-Liang , Huang Guan-Tarn
Published: August 2015

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Metastasis, the spread process of cancer cells from a primary tumor to seed secondary tumors in distant sites, is one of the big challenges in cancer treatment today. For most patients, when the cancer is detected, metastasis has already occurred. The growing evidences implied that miRNAs play an important role in developmental lineage and different states of cancers as well as cancer invasion. In this study, we would like to establish a metastasis-based prognostic miRNA signature. First, 16 lung adenocarcinoma cells were divided into the high or low-invasive group. The miRNAs whose expression correlated with invasion were identified. Next, we investigated whether the expressions of identified miRNAs are correlated to patients’ survival. Quantitative RT-PCR results showed that the expression of miR-10a* is significantly correlated to the five-year survival in 98 lung adenocarcinoma patients. The inhibition of miR-10a* attenuated the invasion abilities of the highly invasive lung cancer cells. Moreover, we investigated the underlying regulation of miR-10a*. We found that miR-10a* negatively regulated Histone deacetylase 5 (HDAC5) was assayed by Western Blot, RT-PCR and luciferase reporter assay. These evidences suggested that HDAC5 is a direct target of miR-10a*. Besides, inhibition of HDAC5 promoted invasive ability. RNAs derived from two paired groups (pre-miR-10a* and pre-NC; sh-HDAC5 and sh-LacZ) were assayed by microarray and pathway analysis. The analysis result of MetaCore implied that miR-10a* trigger TGF-beta signalings. RT-PCR and Western Blot were utilized to validate the differentially altered expression of certain genes in miR-10a*-mediated pathways. The rescue effect of HDAC5 expression on miR-10a*-mediated phenotypic changes is also evaluated to elucidate the importance of HDAC5 in miR-10a*-mediated invasive ability. Our findings indicated that miR-10a* may play an oncogenic miRNA role in lung cancer.

Citation Format: Pin-Yen Hsu, Bing-Ching Ho, Chiou-Ling Cheng, Hsuan-Yu Chen, Sung-Liang Yu, Guan-Tarn Huang. miRNA-10a* enhances invasiveness via suppression of HDAC5 in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3075. doi:10.1158/1538-7445.AM2015-3075
Abstract 3082: miR-155-5p expression is associated with response to neoadjuvant paclitaxel treatment of locally advanced breast cancer
Authors: Straume Anne Hege , Birkeland Einar , Knappskog Stian , Lønning Per Eystein
Published: August 2015

Introduction

Resistance to chemotherapy remains a prominent obstacle to cure in cancer. We have previously shown that genetic defects in key pathways involved in processes like apoptosis, cell-cycle arrest and senescence may be involved in resistance to chemotherapy in vivo.

MicroRNAs (miRNAs) are known to be crucial regulators of gene expression, and to potentially affect the response to drugs, including chemotherapy. Here, we aimed to explore the potential role of miRNAs in mechanisms of breast cancer chemoresistance in-vivo, by analyzing miRNA expression in breast cancer patients with recorded responses to chemotherapy.

Patient materials and Methods

Breast tumour biopsies were collected before chemotherapy in a prospective clinical study, where patients were randomized to receive epirubicin or paclitaxel monotherapy (n = 223). Information about the response to therapy, as defined by the UICC criteria, in addition to long-term outcome, was available for all patients. The global miRNA expression levels were analyzed in 50 breast tumour biopsies by deep sequencing. The patients were selected based on their response to first-line chemotherapy. To verify the results obtained by miRNA sequencing, selected miRNAs were quantified in all patients by qPCR.

Results

No global miRNA patterns were observed when performing unsupervised clustering of miRNA sequencing data. Among samples from paclitaxel-treated patients, we identified three differentially expressed miRNAs between response groups; the expression of miR-155 was significantly lower (p = 0.005) in patients with poor response to paclitaxel, and the expression of both miR-181a and miR-181b were significantly higher (p = 0.003 and 0.012, respectively) in patients with poor response to paclitaxel.

qPCR data validated the initial association identified between high miR-155-5p expression levels and response to paclitaxel treatment. Intriguingly though, high levels of miR-155 were also associated with a poor prognosis in the study population as a whole (p = 0.012), as well as with the presence of mutations in TP53 (p < 0.001).

Conclusion

Here, we show that breast cancer patients responding to paclitaxel treatment have higher expression levels of miR-155-5p. Also, patients with elevated levels of miR-155-5p expression have a poor prognosis and a higher frequency of TP53 mutations.

Citation Format: Anne Hege Straume, Einar Birkeland, Stian Knappskog, Per Eystein Lønning. miR-155-5p expression is associated with response to neoadjuvant paclitaxel treatment of locally advanced breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3082. doi:10.1158/1538-7445.AM2015-3082
Abstract 3089: CDKN2A and p53 status predicts response to CDK4/6 inhibition in melanoma
Authors: Martin Claire A. , Kirby Laura , Wong Stephen , McArthur Grant A. , Sheppard Karen E.
Published: August 2015

The p16-CyclinD1-CDK4-RB pathway regulates G1-S cell cycle transition. This pathway is deregulated in 90% of melanomas, through amplification or increased expression of CDK4 or Cyclin D1 (CCND1), or reduced expression of the CDK inhibitor p16INK4A (CDKN2A).

To explore if CDK4 may be a viable target for the treatment of human melanoma we screened a panel of melanoma cell lines with the highly selective CDK4/6 inhibitor PD-0332991 (PD-991). A majority of cell lines were found to be sensitive to PD-991, although there was a distinct population of resistant cell lines. In sensitive melanoma cell lines, treatment with PD-991 resulted in loss of hyperphosphorylated RB and a concomitant G0/G1 cell cycle arrest. Treated cells displayed an enlarged flattened morphology and an increase in SA-β-galactosidase staining compared to vehicle controls, indicative of cellular senescence. RB-null cell lines were resistant to CDK4/6 inhibition, and did not display cell cycle arrest or senescence. Expression and mutational analysis was performed to identify additional genomic predictors of sensitivity and resistance. Loss of CDKN2A/p16 correlated with sensitivity to PD-991 (p<0.02), and this was confirmed at the level of protein expression. Mutation of p53 and the corresponding low expression of p53 targets p21 and MDM2 correlated with resistance (p<0.0001). There was no increase in p53 transcriptional targets or evidence of apoptosis in response to PD-991 treatment of sensitive cells, and senescence occurs in both p53 WT and mutant cells, indicating that this response does not require functional p53. However, in p53 WT cells, combination treatment with PD-991 and the p53 pathway activator Nutlin3a results in increased sensitivity to PD-991 compared to treatment with PD-991 alone. This additive effect is not observed in p53 mutant cells, suggesting there is a functional role for WT p53 in the response to PD-991.

Conclusions: These preliminary data identify RB, p16 and p53 as predictors of response to PD-991, and support further evaluation of CDK4/6 inhibitors in melanoma.

Citation Format: Claire A. Martin, Laura Kirby, Stephen Wong, Grant A. McArthur, Karen E. Sheppard. CDKN2A and p53 status predicts response to CDK4/6 inhibition in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3089. doi:10.1158/1538-7445.AM2015-3089
Abstract 3092: In-situ Kinome wide profiling of Palbociclib treated COLO-205 human tumor xenograft samples
Authors: Sharma Geeta , Vogeti Subha , Grant Wendy , Wu Shuzhen , Dias Christa , Nomanbhoy Tyzoon , Wu Jiangyue , Aban Arwin
Published: August 2015

Palbociclib (PD0332991) is a cyclin dependent kinase 4 and 6 inhibitor being developed for treatment of breast cancer. In order to evaluate the kinome-wide impacts of Palbociclib in an in-vivo setting, nude mice inoculated with COLO-205 (human colon carcinoma) cells were administered Palbociclib at different doses (12.5, 75 and 150mg/kg once daily for 3 days. Tumors were collected at various time points after the final dose (1, 3, 6, 12, 24 & 48 hours), lysed and subjected to in-situ kinase profiling using the KinativTM technology. Tumor lysates were probed with an ATP acylphosphate desthiobiotin probe which covalently modifies lysine residues in the kinase active site. Samples are then denatured, trypsinized, and the probe-labeled peptides captured with streptavidin, eluted, and quantitated by targeted mass spectrometry. The presence of an inhibitor bound to the active site of a kinase prevents the probe binding, resulting in the loss of MS signal for peptides from that kinase. In addition, the upregulation or downregulation of kinase proteins can also result in changes in the MS signals in palbociclib versus vehicle-treated samples. A total of 180 kinases were quantified. As expected, direct targets of palbociclib were observed to be inhibited in this study (i.e., kinases inhibited by Palbociclib in Colo205 lysate), including CDK4 &6 as well as ERK5, JNK, CDK16 &17, PIK3C3 and PIP4K2C. The observed inhibition of these targets was both dose and time dependent. Significantly, the MS signals for additional kinases not observed to directly bind Palbociclib in Colo205 lysate were also strongly downregulated in this study. These include CDC2, AurA, AurB, PLK1 and MASTL; kinases critical for cell cycle progression. We hypothesize that the downregulation of these kinases was due to cell cycle arrest in G1 phase. Consistent with this hypothesis, the downregulation of these kinases corresponded well with the inhibition of phosphorylated (Ser780; pRb) as demonstrated by Western Blots of a part of tumor tissue used for the Kinome profiling. Taken together, these results highlight the utility of kinome wide profiling in tumor samples collected from xenograft mice to better understand both pharmacodynamics of target engagement as well as pathway effects elicited by compound treatment.

Citation Format: Geeta Sharma, Subha Vogeti, Wendy Grant, Shuzhen Wu, Christa Dias, Tyzoon Nomanbhoy, Jiangyue Wu, Arwin Aban. In-situ Kinome wide profiling of Palbociclib treated COLO-205 human tumor xenograft samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3092. doi:10.1158/1538-7445.AM2015-3092
Abstract 31: WEE1 kinase inhibition enhances PI3K inhibitor response in p53 deficient gliomas
Authors: Wu Shaofang , Zheng Siyuan , Wang Shuzhen , Yung W.K.Alfred , Koul Dimpy
Published: August 2015

Glioblastoma multiforme (GBM) is an aggressive brain tumor that is resistant to chemotherapy and radiation, and is almost always fatal. Oncogenic activation of the PI3K signaling pathway plays a pivotal role in the development of GBM and is a major molecular target for therapeutic development. However, clinical results of single-agent PI3K inhibitors have been modest to date. PI3K inhibitors have led to tumor stabilization and some disease responses; however, dramatic tumor regressions are not typical. Multiple recent reports have described mechanisms of resistance to PI3K inhibitors. The goal of the present study was to identify ways to improve the efficacy of PI3K inhibitor-based therapy by overcoming adaptive resistance. In this study we show that PI3K inhibitor BKM induces G2/M cell cycle arrest and inhibits GBM cell proliferation. We identified WEE1 as a putative treatment target of resistance to PI3K inhibition based on its potential function as a mitotic gatekeeper and its specific overexpression in GBM. WEE1 kinase is a key molecule in maintaining G2-cell-cycle checkpoint arrest for premitotic DNA repair. We show that that BKM treatment activates WEE1, which in turn phosphorylates CDC2 at Tyr15, inactivates CDC2 and inhibits CDC2 activity, leading to G2/M arrest in glioma initiating cells (GICs). We show that treatment with WEE1 inhibitor MK1775 inhibited BKM-induced Cdc2 Tyr15 phosphorylation, and forced GICs to enter into the mitotic phase of the cell cycle. Using a panel of GICs, we show that MK1775 synergizes with BKM to inhibit GBM cell proliferation. Further, MK1775 and BKM as a single agent alone partially blocks proliferation, however, combination of MK1775 and BKM induces apoptosis as evaluated by Annexin V positive cells, cleaved PARP and caspase 3, and TUNEL staining. This effect was observed specifically in p53 mutant cells. Finally we show that depletion of WEE1 by shRNAs synergizes with BKM to inhibit GBM cell proliferation and induce apoptosis. These results indicate that WEE1 inhibition potentiated the effectiveness of PI3K targeted inhibition in GBM, and suggest a combinational inhibition of WEE1 and PI3K might allow successful targeted therapy in p53 mutant GBM.

Citation Format: Shaofang Wu, Siyuan Zheng, Shuzhen Wang, W.K.Alfred Yung, Dimpy Koul. WEE1 kinase inhibition enhances PI3K inhibitor response in p53 deficient gliomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 31. doi:10.1158/1538-7445.AM2015-31