bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1638: Imaging the interaction of αv integrin-GFP in osteosarcoma cells with RFP-expressing host stromal cells and tumor scaffold collagen in nude mice.
Authors: Tome Yasunori , Uehara Fuminori , Maehara Hiroki , Miwa Shinji , Tsuchiya Hiroyuki , Tomita Katsuro , Kanaya Fuminori , Bouvet Michael , Hoffman Robert M.
Published: August 2014

Human 143B osteosarcoma cells were stably transfected with an αv integrin-GFP vector. Confocal microscopy demonstrated that αv integrin immunofluorescence staining co-localized with αv integrin-GFP fluorescence. The αv integrin-GFP expressing tumors were established in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary tumors acquired RFP-expressing stroma and were passaged orthotopically in the tibia in non-colored nude mice, which maintained the RFP stroma. The interaction of αv integrin-GFP expression in 143B cells with RFP-expressing host stromal cells was observed by confocal microscopy using the Olympus FV1000. Collagen fibers were imaged simultaneously in reflectance mode. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in the tumors which persisted even 3 weeks after passage. CAFs expressing RFP were aligned between collagen fibers and cancer cells expressing αv integrin-GFP. Six weeks after transplantation, pulmonary metastases expressing αv integrin-GFP could be identified. TAMs expressing RFP accompanied osteosarcoma cells expressing αv integrin-GFP in the lung. The present study demonstrates the importance of αv integrin interaction with stromal elements.

Citation Format: Yasunori Tome, Fuminori Uehara, Hiroki Maehara, Shinji Miwa, Hiroyuki Tsuchiya, Katsuro Tomita, Fuminori Kanaya, Michael Bouvet, Robert M. Hoffman. Imaging the interaction of αv integrin-GFP in osteosarcoma cells with RFP-expressing host stromal cells and tumor scaffold collagen in nude mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1638. doi:10.1158/1538-7445.AM2013-1638
Abstract 1652: A new approach to study intracellular pathways of metastasis in living tumor cells.
Authors: Colbert Leiam , Ward Christopher W. , Onel Kenan
Published: August 2014

Although genes associated with metastasis have been identified, the core intracellular events of the process remain obscure. Recently, it has been shown that cytoskeletal reorganisations, indicated by changes in the tensional force generated by the actin-myosin interaction in the cell, are critical for the development of invasive phenotypes. These changes are mediated in part by the microenvironment through mechanotransduction (6,7). Moreover, reduced cytoskeletal stiffness and increased mutability can distinguish metastatic cells from non[[Unsupported Character - Codename ­]]invasive cancer cells and from normal cells. This suggests that many cancer cells are distinguished by altered physical properties and that biomechanical measurements of isolated cells may have significant diagnostic and prognostic value (2-4, 8). Thus, we hypothesize that by using isogenic cancer cell lines of differing metastatic potential transfected with fluorescently labelled motility reporter constructs, we will be able to elucidate altered mechanotransduction signalling as an important component in tumour formation and progression, which can then be studied mechanistically.

We have recently demonstrated novel cytoskeleton dependent mechanotransduction pathways in heart (5) and skeletal muscle (1) cells in which stretch-dependent signal transduction activates the local production of reactive oxygen species (ROS) to trigger Ca2+ signaling events. These discoveries were made possible by combining new mechano-optical techniques. We intend to apply this approach to further investigate abnormal kinase-mediated sensing of mechanical cues that may result in the development of fundamental abnormalities in the cancer microvasculature. We present preliminary data from different cancer lines that suggest manipulating kinase-mediated tension, using different inhibitors (ROCK inhibitor Y27632, MAPK inhibitor U0126), alone or jointly with one another can reprogram the tumour cells so that they normalize their reorientation response to cyclic strain and their ability to form networks (9).

Citation Format: Leiam Colbert, Christopher W. Ward, Kenan Onel. A new approach to study intracellular pathways of metastasis in living tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1652. doi:10.1158/1538-7445.AM2013-1652
Abstract 1656: Mouse embryonic fibroblasts null for krüppel-like factor 4 show reduced autophagy and elevated mTOR activity.
Authors: Engda Hagos , DeRoo Elise
Published: August 2014

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor named for its homology to the Drosophila protein Krüppel. KLF4 negatively regulates cell-cycle progression and, cell proliferation, and is a known tumor suppressor in certain cancers. Autophagy, or self-eating, is a process through which intracellular aggregates can be degraded and their components recycled. The role of autophagy in cancer appears to be context dependent. In pre-cancerous tissue, autophagy may serve a protective purpose, removing cytotoxic aggregates and promoting a healthy intracellular environment. In cancerous tissue, however, autophagy may allow cancer cell survival. Previous studies examining gene expression in WT (KLF4+/+) and KLF4 null (KLF4-/-) mouse embryonic fibroblasts (MEFs) revealed autophagy related gene expression to be downregulated in KLF4-/- MEFs. In order to evaluate whether KLF4 is important in autophagy, we examined microtubule associated protein-1 light chain 3 protein (LC3, a marker of autophagy) and a downstream target of mTOR (p70S6K, a marker of autophagy inhibition) in KLF4+/+ and KLF4-/- MEFs. Our results show that levels of LC3 are reduced and levels of p70S6K are elevated in both KLF4-/- MEFs and MEFs in which KLF4 has been transiently silenced. Rapamycin treatment, which inhibits mTOR activity, fails to restore autophagy in KLF4 null MEFs. Collectively, these findings suggest that KLF4 may negatively regulate mTOR activity and that KLF4 may also be necessary for induction of autophagy either downstream or entirely independent of mTOR.

Citation Format: Hagos Engda, Elise DeRoo. Mouse embryonic fibroblasts null for krüppel-like factor 4 show reduced autophagy and elevated mTOR activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2013-1656

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1658: Cell death and growth arrest pathways mediating the actions of stilbene 5c in HCT-116 colon cancer cells and B16F10 melanoma cells.
Authors: Alotaibi Moureq R.
Published: August 2014

The stilbene derivative, cis-3, 4’, 5-trimethoxy-3’-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding pocket in microtubules. Earlier studies have shown that stilbene 5c induces cell death in ovarian cancer cells and leukemic cells (Cao TM et al. Am J Hematol. 2008;83:390-7; Durrant D et al. Gynecol Oncol. 2008; 110:110-7).). The present study was designed to investigate the effectiveness of this microtubule poison against the HCT-116 human colon cancer cell line as well as B16F10 melanoma cells and its mechanisms of action. Time course studies demonstrated that stilbene 5c produces a decrease in cell viability in both cell lines. The capacity of the cells to proliferate was not restored upon removal of the drug after 5 days of exposure. Consistent with the results of the time course studies, β-galactosidase staining indicated that treatment with stilbene 5c also promotes senescence. In addition to senescence, HCT-116 cells and B16F10 melanoma cells treated with stilbene 5c displayed formation of autophagic vesicles by acridine orange staining, which was supported by fluorescence-activated cell sorting (FACS). Further evidence of autophagy was derived from treatment of HCT116 cells expressing an RFP-LC3 construct with stilbene 5c, in which LC3 puncta formation increased in a time-dependent manner. DAPI staining, TUNEL, and Annexin 5 staining indicated that apoptosis is also occurring in stilbene 5c-treated HCT-116 cells. Cell cycle analysis of HCT116 cells demonstrated growth arrest at both G1 and G2/M, and an increase in the subG1 population at days 3 and 5, which correspond to senescence and apoptosis respectively. Interestingly, DAPI and Hoechst staining of HCT116 cells revealed morphological changes in the cell nuclei (binucleated and micronucleated cells), which suggest that mitotic catastrophe may also serve as a mode of cell death after treatment with stilbene 5c. Our studies also indicated that stilbene 5c works in a p53-independent manner. p53-null HCT116 cells demonstrated similar sensitivity to stilbene as p53-wild type HCT116 cells. Consistent with previous studies in other experimental cancer models, this work indicates that stilbene 5c could potentially be effective against melanoma and colon cancer through the promotion of multiple modes of cell death.

Citation Format: Moureq R. Alotaibi. Cell death and growth arrest pathways mediating the actions of stilbene 5c in HCT-116 colon cancer cells and B16F10 melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1658. doi:10.1158/1538-7445.AM2013-1658
Abstract 1664: A delicate balance between radiation-induced autophagy and apoptosis in HPV-positive head and neck cancer.
Authors: Fowler Tyler L. , Ludwig Kai D. , Djavadian Reza , Bednarz Bryan P. , Kimple Randall J.
Published: August 2014

Purpose: The molecular mechanisms responsible for improved outcomes seen in patients with human papillomavirus (HPV) positive head and neck cancer (HNC) remain largely unknown. Ionizing radiation can induce autophagy, a genetically programmed, evolutionarily conserved response to stress whereby cellular constituents are engulfed, digested, and recycled to sustain cellular metabolism. We investigated whether radiation-induced cellular autophagy is modulated by HPV oncogenes leading to altered therapeutic responses between HPV+ and HPV- HNCs.

Methods: An immortalized human tonsillar epithelial (HTE) cell line engineered to stably express LXSN vector alone, HPV-16 E6, or HPV-16 E7 was used for all experiments. All constructs received a single radiation dose of 4 Gy. Radiation survival was assessed by clonogenic survival. Activation of autophagy proteins LC3-A and LC3-B were analyzed at 4 and 24 hr time points using western blotting and fluorescence microscopy along with acridine orange staining. Apoptosis was evaluated by caspase activity at the same time points.

Results: HPV-16 E6 resulted in greater sensitivity, while HPV-16 E7 resulted in radiation resistance relative to than control cells (surviving fraction after 4 Gy = 8.4+/-0.5% vs. 35+/-3.8% vs. 20+/-1.1%, respectively, 1way ANOVA p<0.0001). At baseline, expression of HPV-16 E6 had no affect on LC3-B expression while HPV-16 E7 and HPV-16 E6/E7 resulted in a significant increase in autophagy. Four hours following radiation, autophagy was increased in HPV-16 E6 (2.0 fold), E7 (2.6 fold) and E6/E7 (2.2 fold) expressing cells whereas vector and untransfected control cells showed no effect (1.2 and 1.1 fold, respectively). This increase was maximal at 4 hrs and had returned to baseline 24 hrs after radiation. No significant change in apoptosis was seen 4 hrs following radiation, but a gradual increase was seen between 12 and 48 hrs.

Conclusions: The HPV oncoproteins E7 up regulates autophagic pathways at baseline correlating with radiation resistance. Four hours following radiation, both E6 and E7 result in significant upregulation of autophagy. This is followed by upregulation of apoptosis, suggesting a transition from an initial protective response to cellular death.

Citation Format: Tyler L. Fowler, Kai D. Ludwig, Reza Djavadian, Bryan P. Bednarz, Randall J. Kimple. A delicate balance between radiation-induced autophagy and apoptosis in HPV-positive head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1664. doi:10.1158/1538-7445.AM2013-1664
Abstract 1698: Evaluation of pesticide involvement in breast cancer.
Authors: Patterson Kathryn , Hale Julie , Bush Jason
Published: August 2014

There is considerable evidence linking cumulative and sustained exposure to estrogens as a key promoter of breast tumor proliferation. Chemicals with estrogenic activity can bind to the estrogen receptor (ER) to affect downstream signaling of estrogen-responsive genes. Organochlorines are a class of chemical pesticides that can act as xenoestrogens to disrupt normal endocrine function. Mitochondria play a significant role in maintenance of energy regulation and cellular detoxification and mitochondrial DNA is sensitive to levels of estrogen. Some members of the Estrogen Related Receptor (ERR) family are known to interact with hypoxia inducible factors to modulate mitochondrial response through the mTOR pathway which is pivotal to cell homeostasis and cellular detoxification. This study investigates the link between two organochlorine pesticides, Methoxychlor and Toxaphene, and the cellular effects by examining the hypothesis that exposure induces differential molecular pathways. Two breast cancer cell lines, MCF-7 (ER+) and MDA-MB-231 (ER-), and a normal breast epithelial cell line MCF-10A are used as a model to evaluate the response to Methoxychlor and Toxaphene treatment. Cytotoxicity studies established different sensitivities of the two breast cancer cell lines to pesticides. MCF-7 was more sensitive to both pesticides, which supports the premise that organochlorines may be acting as endocrine disruptors. Furthermore, pesticide-resistant clones of MCF-7 and MDA-MB-231 were established and compared to their sensitive counterparts. RNA was collected from cells treated in a hypoxic environment, cells treated with a high dose pesticide, and cells that had a co-treatment of hypoxia and pesticide for evaluation of ERR, hypoxic factor expression, and markers of mTOR pathway. Flow cytometry, RT-PCR and immunoblotting demonstrated that treatment affected mitosis and mitochondrial function and we found that ERR and hypoxic factor expression changed in a similar manner suggesting a common mechanism by pesticide treatment or hypoxic conditions. These results provide a basis for understanding specific pesticide-induced molecular mechanisms and their possible relationship to ER+ and ER- mammary epithelial biology.

Citation Format: Kathryn Patterson, Julie Hale, Jason Bush. Evaluation of pesticide involvement in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1698. doi:10.1158/1538-7445.AM2013-1698
Abstract 1716: Calcitriol and progesterone synergistically inhibit growth in endometrial cancer cells.
Authors: Lee Laura R. , Hood Brian L. , Nguyen Huyen , Kavandi Leyla , Turbov Jane M. , Thaete Larry G. , Hamilton Chad A. , Rodriguez Gustavo C. , Maxwell George L. , Conrads Thomas P. , Syed Viqar
Published: August 2014

Human studies suggest that progesterone and calcitriol may prove beneficial in preventing or inhibiting oncogenesis, but the underlying mechanism is not fully understood. The current study investigates the effects of progesterone, calcitriol and their combination on immortalized human endometrial epithelial cells and endometrial cancer cells and identifies their targets of action. The combined cell treatment enhanced vitamin D receptor expression and synergistically inhibited cell proliferation through caspase-3 activation and induction of G0/G1 cell cycle arrest with associated down-regulation of cyclin D1, D3 and p27 induction. We used mass spectrometry-based proteomics to measure protein abundance differences between calcitriol-, progesterone-, or combination-exposed endometrial cells. A total of 117 proteins showed differential expression amongst these three treatments. Four proteins were then selected for validation studies: histone H1.4 (HIST1H1E), histidine triad nucleotide-binding protein 2 (HINT2), interferon-induced, double-stranded RNA-activated protein kinase (EIF2AK2), and Bcl-2-associated X protein (BAX). Abundance levels of selected candidates were low in endometrial cancer cell lines versus the immortalized endometrial epithelial cell line. All four proteins displayed elevated expression in cancer cells upon exposure to calcitriol, progesterone or the combination. Further BAX analysis through gain or loss of function experiments revealed that upregulation of BAX decreased cell proliferation by changing the BAX:Bcl2 ratio. Knock down of BAX attenuated progesterone- and calcitriol-induced cell growth inhibition. Our results showed that progesterone and calcitriol up-regulate the expression of BAX along with other apoptosis-related proteins, which induced inhibition of endometrial cancer cell growth by apoptosis and cell cycle arrest.

Citation Format: Laura R. Lee, Brian L. Hood, Huyen Nguyen, Leyla Kavandi, Jane M. Turbov, Larry G. Thaete, Chad A. Hamilton, Gustavo C. Rodriguez, George L. Maxwell, Thomas P. Conrads, Viqar Syed. Calcitriol and progesterone synergistically inhibit growth in endometrial cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1716. doi:10.1158/1538-7445.AM2013-1716
Abstract 1719: Differential Mcl-1 processing in evasion of DCA-induced apoptosis of colorectal cancer cells.
Authors: Delaney Leanne M. , Coomber Brenda L.
Published: August 2014

Recent evidence indicates that dichloroacetate (DCA) may be a cancer-specific agent that targets the unique metabolism of cancer cells, avoiding disruption of normal somatic cells. However, our laboratory has shown that some human colorectal cancer cell lines do not show the predicted response to DCA treatment, and the molecular mechanism responsible for this is currently unknown. Of interest is the Bcl-2 protein family, which consists of both pro- and anti-apoptotic members. The expression, regulation, and interactions of the Bcl-2 proteins will mediate the release of cytotoxic molecules such as cytochrome c from the mitochondria into the cytosol, activating apoptosis via a caspase cascade. Differential regulation of the Bcl-2 proteins in response to DCA treatment may indicate whether apoptosis will be induced in cancer cell lines. The human colorectal cancer cell line HCT116 was cultured and exposed to DCA as well as 5-fluorouracil, a known initiator of Bcl-2 protein-mediated apoptosis. Key changes in Bcl-2 protein expression were analyzed using immunoblotting. The expression of Mcl-1, an anti-apoptotic Bcl-2 protein, occurred at 40-kDa in all treatments, and at 28-kDa only in non-DCA treatments. PCR for the known splice variants of Mcl-1 confirmed only one gene product, suggesting the involvement of post-translational modification. The presence of cleaved caspase 3 indicates that an increase in 28-kDa Mcl-1 compared to 40-kDa Mcl-1 occurs with apoptosis in 5-fluorouracil treated cells, but does not occur in DCA-treated cells, which were not apoptotic. This suggests that Mcl-1 modification may be acting to modulate susceptibility to DCA treatment. The interactions involved in or resulting from these responses are currently under investigation. Using modified HCT116 cells without functional p53 or without functional Bax, the role of these two proteins will be emphasized. Co-immunoprecipitation of Mcl-1 will allow changes in its binding partners, such as p53, to be observed in response to DCA treatment. By utilizing mitochondrial and cytosolic fractions for protein analysis, the effect of DCA on mitochondrial localization of proteins such as Bax and the release of cytochrome c from the mitochondria will be observed. Further research may provide insight into the mechanism by which some human cancer cell lines are protected against DCA-induced cell death, and the potential role of a pathway involving Mcl-1 in this cell-specific protection.

Citation Format: Leanne M. Delaney, Brenda L. Coomber. Differential Mcl-1 processing in evasion of DCA-induced apoptosis of colorectal cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1719. doi:10.1158/1538-7445.AM2013-1719
Abstract 1723: The BH4 domain of Bcl-2 is sufficient to induce RNA expression profile changes in human prostate cancer cell lines.
Authors: Cohen Jennifer R. , Williams Simon A.
Published: August 2014

Bcl-2 is the archetypal gene and first characterized member of the family bearing its name. Prostate cancer was the first solid tumor malignancy in which an elevation of cellular Bcl-2 levels was associated with advanced disease. It is thought that the anti-apoptotic activity of Bcl-2 contributes to the uniformly lethal nature of castration-resistant advanced prostate cancer. Bcl-2 is a validated target for cancer therapies. Current small molecule drug inhibitors mimic the BH3 domain of pro-apoptotic Bcl-2 family members, binding to a pocket composed mainly of Bcl-2 homology (BH) domains 1-3, and only partially BH4. Surprisingly, while these agents are effective inducers of apoptosis, they do not inhibit the oncogenic functions of the BH4 domain. The BH4 domain is essential for the antiapoptotic functions of Bcl-2, and appears to mediate interactions with proteins not part of the Bcl-2 family. This explains the small but significant body of evidence supporting an oncogenic signaling role for Bcl-2 driven by the BH4 domain. The full extent of the non-apoptotic roles of Bcl-2, including the contribution of the BH4 domain, has yet to be clarified. Our work aims to address this in prostate cancer cells, developing and utilizing a system that will implicate both the full length gene product and the BH4 domain. Eukaryotic expression constructs were designed to contain the full length Bcl-2 gene built to co-express with GFP, or a fusion gene composed of the Bcl-2 BH4 domain, GFP, and the Bcl-2 C-terminal transmembrane domain. These plasmids were then transfected into the 22Rv1, LNCaP, and LAPC4 human prostate cancer cell lines to generate stable expression clones. The clones were validated for protein expression by western blot prior to a determination of classical functionality by treatment with apoptosis-inducing agents. RNA was then isolated from the clones for expression array analysis. We successfully generated human prostate cancer cells stably overexpressing Bcl-2 and the Bcl-2 BH4 domain. These clones displayed enhanced resistance to apoptosis induction. Bcl-2 overexpression also induced expression profile changes in these clones. This was mimicked by expression of the BH4 domain alone. We have developed a useful system to help enhance our understanding of Bcl-2 by highlighting changes in RNA expression profiles induced by the gene's overexpression. We also show that the BH4 domain drives much of this function. The mechanisms responsible for these Bcl-2/BH4-induced changes warrant further investigation.

Citation Format: Jennifer R. Cohen, PhD, Simon A. Williams, PhD. The BH4 domain of Bcl-2 is sufficient to induce RNA expression profile changes in human prostate cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1723. doi:10.1158/1538-7445.AM2013-1723
Abstract 1724: BRAF inhibition is enhanced by BH3-mimetic through BIM induction.
Authors: Frederick Dennie T. , Salas Fragomeni Roberto A. , Hoff Taylor , Cooper Zachary A. , Mier James W. , Wargo Jennifer A. , Sullivan Ryan J.
Published: August 2014

Vemurafenib (Vem) is a highly selective inhibitor of mutant BRAF, FDA-approved for the treatment of patients with BRAF-positive advanced melanoma. While response rates to Vem are high, the emergence of resistance is common, placing high priority in the development of strategies to enhance Vem efficiency. Navitoclax is a BH3-mimetic that binds and inactivates the anti-apoptotic proteins BCL-2, BCL-xl, and BCL-w and leads to apoptosis in tumors with low expression of another anti-apoptotic protein, MCL-1. Our data demonstrates that treatment of BRAF-mutant melanoma patients with Vem is associated with decreased expression of MCL-1 in addition to an increase in the pro-apoptotic, BH3-only protein BIM in serial biopsy samples. Therefore, we hypothesize that the combination of PLX-4720 (Vem analog) with navitoclax will result in increased anti-tumor activity. To test our hypothesis, we performed cell viability studies, Annexin:PI and ELISA apoptosis assays in addition to protein and mRNA analysis. In-vivo studies were performed in two BRAF mutant xenograft models using navitoclax and PLX-4720. In-vitro, our cell viability studies demonstrated that concurrent treatment with PLX-4720 and navitoclax results in an additive viability reduction in BRAF mutant but not in BRAF WT cell lines. The decrease in cell viability correlated to downmodulation of MCL-1 and BIM induction after PLX-4720 treatment, with further enhancement of BIM expression and PARP cleavage after adding navitoclax. In vivo, the navitoclax enhanced the efficacy of PLX-4720 in both xenograft models. The combination of a selective BRAF inhibitor with a BH3-mimetic may be a promising therapeutic strategy to enhance the clinical efficacy of BRAF inhibitors.

Citation Format: Dennie T. Frederick, Roberto A. Salas Fragomeni, Taylor Hoff, Zachary A. Cooper, James W. Mier, Jennifer A. Wargo, Ryan J. Sullivan. BRAF inhibition is enhanced by BH3-mimetic through BIM induction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2013-1724
Abstract 1735: Obatoclax enhances CDDP and trail-induced apoptosis in epithelial ovarian adenocarcinoma cells.
Authors: Martinez-Paniagua Melisa A. , Romero-Gonzalez Luis A. , Anguiano-Hernandez Yu Mei , Gonzalez-Torres Carolina , Vega Mario I.
Published: August 2014

Epithelial ovarian cancer comprises the majority of malignant ovarian tumors in adult women. Ovarian cancer is the fifth cause of death in women and annually 13,850 deaths are reported in United States. Effective screening strategies are lacking and the majority of cases are diagnosed with advanced stage disease. Chemo is the conventional therapy after surgery but the initial response is between 60 to 80%, however, some patients that initially responded to the therapy suffer of recurrent disease or become resistant to the treatment. As a consequence, the overall 5 year survival is only about 30%. It is important to identify novel therapies for ovarian cancer to treat or sensitize tumor cells to the existence therapy. It has been suggested that the tumor cells become resistance due to an increased expression of antiapoptotic proteins especially from the Bcl2 family (Bcl-2, Bcl-XL, Mcl-1). Obatoclax is a small molecule that mimics the BH3 domain of the BH3 only family members and inhibits the effect of antiapoptotic Bcl-2 members. The objective of this study was to assess the susceptibility of ovarian cancer cell lines to chemo or immuno therapy mediated apoptosis by obatoclax and also to elucidate the mechanism. Four different epithelial ovarian adenocarcinomas cell lines (SKOV3, NIH-OVCAR-3, TOV112D and TOV21G) were treated with different concentrations of Obatoclax (nM-μM), CDDP (5-50μg/ml) or TRAIL (50-1200ng/ml) alone or with a combination of treatment to assess the viability, measured by the XTT kit (Roche). Basal and cell treated total protein expressions were assessed by western. Induction of apoptosis was measured by TUNEL. As a single agent, Obatoclax or CDDP inhibited the viability of the cells in a dose response form; TRAIL did not show an evident response. The combination of the treatment after selecting the IC50 and IC20 response showed an increased inhibition of the viability. TUNEL positive cells were measured and the induction of apoptosis was mediated by a caspase dependent manner and PARP cleavage. Our data suggest that obatoclax in combination with CDDP or TRAIL can enhance the induction of apoptosis in resistance cell lines, suggesting to be an optional treatment of patients with ovarian cancer.

Citation Format: Melisa A. Martinez-Paniagua, Luis A. Romero-Gonzalez, Yu Mei Anguiano-Hernandez, Carolina Gonzalez-Torres, Mario I. Vega. Obatoclax enhances CDDP and trail-induced apoptosis in epithelial ovarian adenocarcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1735. doi:10.1158/1538-7445.AM2013-1735
Abstract 1746: Coordination of overexpressed NIMA-related kinase 2 and TRF1 results in mitotic abnormalities in breast cancer cells.
Authors: Lee Jaehyung
Published: August 2014

NIMA-related kinase 2 (NEK2), a serine-threonine protein kinase, plays a role in features of mitotic progression including timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Kinase death NEK2 mutant expression or NEK2 depleted cells lead to failure of centrosome separation while NEK2 overexpression results in premature centrosome separation. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with NEK2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between NEK2 and TRF1 are far from clear. Here, we show that mitotic aberrations through NEK2 overexpression are likely to require TRF1. Our results demonstrate that NEK2 directly binds and phosphorylates TRF1 in vitro and in vivo through multiple sites on TRF1. NEK2 overexpression in breast cancer cells, MCF 7 and MDA-MB-231, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the occurrence of unaligned chromosomes by NEK2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1 depleted cells demonstrated ≤ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA transfected cells. Therefore, we propose that TRF1 is required for overexpressed NEK2 to trigger abnormal mitosis and chromosomal instability.

Citation Format: Jaehyung Lee. Coordination of overexpressed NIMA-related kinase 2 and TRF1 results in mitotic abnormalities in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1746. doi:10.1158/1538-7445.AM2013-1746
Abstract 1749: Cell cycle requirements shape ovarian cancer progression.
Authors: Taylor-Harding Barbie , Agadjanian Hasmik , Orsulic Sandra , Walsh Christine , Karlan Beth Y. , Wiedemeyer Wolf-Ruprecht
Published: August 2014

Acquired resistance to chemotherapeutic drugs and subsequent recurrence of drug-resistant cancer are serious challenges associated with epithelial ovarian cancer. Most high-grade tumors initially respond well to the combination of platinum and taxane-based agents but recur as platinum-resistant disease, which is incurable. In addition, a subset of primary tumors, including CCNE1-amplified tumors, are inherently less susceptible to standard chemotherapy. Studies by The Cancer Genome Atlas have revealed that CCNE1 (cyclin E) amplification is mutually exclusive with mutations in BRCA1 and BRCA2, which render ovarian cancer cells sensitive to DNA-damaging agents and PARP inhibitors, as a result of synthetic lethality. In order to develop a similar therapeutic concept for CCNE1-amplified cancers, we have studied responses and resistance mechanisms of human ovarian cancer cells to several pharmacological inhibitors of cyclin-dependent kinases (CDK). Functional and biochemical studies have established cyclin E as an activator of CDK, which phosphorylate the RB tumor suppressor. However, despite the functional importance of the RB pathway in cancer, CDK inhibitors (CDKi) have yet to make a significant impact on the management of solid tumors.

Here, we describe oncogenic pathways that are activated in CDKi-resistant cells and play critical roles in tumor progression and drug resistance. Targeting individual CDK or subsets of multiple CDK in vitro and in vivo, we found that cyclin E dependence was the main driving force that shaped tumor progression in different model systems of CCNE1-amplified and recurrent ovarian cancer. We identified robust and specific compensatory mechanisms that sustain cyclin E-associated activity in CDKi-resistant cells via activation of oncogenic signaling upstream of the cyclin E-RB-E2F axis. Specific targeting of cooperating oncogenes increased CDKi efficacy and prevented the outgrowth of CDKi-resistant cancer cells. Collectively, our results suggest that CDKi can be highly effective elements of combination therapy.

Citation Format: Barbie Taylor-Harding, Hasmik Agadjanian, Sandra Orsulic, Christine Walsh, Beth Y. Karlan, Wolf-Ruprecht Wiedemeyer. Cell cycle requirements shape ovarian cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1749. doi:10.1158/1538-7445.AM2013-1749
Abstract 1750: Clinically relevant paclitaxel concentrations cause chromosome missegregation rather than mitotic arrest.
Authors: Zasadil Lauren M. , Burkard Mark , Weaver Beth A.
Published: August 2014

Paclitaxel is a chemotherapeutic, microtubule-stabilizing drug used to treat a variety of cancers, including those of the breast. In the neoadjuvant setting, paclitaxel effects tumor shrinkage in up to half of patients. Identification of a biomarker to predict response to paclitaxel would reduce side effects and improve patient outcomes. It has long been known that micromolar concentrations of paclitaxel cause cells in culture to arrest in mitosis, which can lead to cell death. However, the concentration range found within patient tumors remains unclear, both because it has not been directly measured and because paclitaxel accumulates intracellularly up to 1000 fold. In a pilot clinical trial, we are directly measuring the intratumoral drug concentration in breast tumors following neoadjuvant paclitaxel treatment. Our preliminary data support the hypothesis that treatment of cultured cells with a low nanomolar concentration more accurately models the intratumoral drug levels in breast cancer patients. Unlike treatment with micromolar concentrations, treatment with low nanomolar paclitaxel does not cause a significant mitotic arrest. Rather, cells treated with low nanomolar paclitaxel frequently proceed through mitosis with multipolar mitotic spindles, resulting in chromosome missegregation and the production of two or more daughter cells. This alternative mechanism of action may explain why tumors treated with paclitaxel fail to show an increase in mitotic index. Previous studies have shown that low rates of chromosome missegregation are well tolerated, but high rates of chromosome missegregation cause cell death due to loss of both copies of one or more essential chromosomes. Therefore, the fidelity of chromosome segregation may be of use as a predictor of response to paclitaxel. In chromosomally stable breast cancer cells, exposure to paclitaxel for two days is sufficient to induce aneuploidy, and within three days there is an increase in cell death. However, a subset of cells are able to survive in low nM paclitaxel for over one week, and survival is higher than in cells which exhibit chromosomal instability (CIN), and have a preexisting rate of chromosome missegregation during division. In patients, chromosomal stability is of potential use as a predictive biomarker to select patients most likely to respond to paclitaxel treatment.

Citation Format: Lauren M. Zasadil, Mark Burkard, Beth A. Weaver. Clinically relevant paclitaxel concentrations cause chromosome missegregation rather than mitotic arrest. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1750. doi:10.1158/1538-7445.AM2013-1750
Abstract 1752: Consequences of high Chromosome Instability (CIN) in SGO1(Shugoshin1) CIN model mice.
Authors: Yamada Hiroshi Y. , Zhang Yuting , Dai Wei , Rao Chinthalapally V.
Published: August 2014

Abstract

Mitotic errors have been suspected to be a cause of aneuploidy, rapid accumulation of mutations, and tumorigenesis. In addition, recent cell biological studies demonstrated that mitotic errors can result in DNA damage, which would also aid accumulation of mutations then carcinogenesis. However, no observations in vivo have been provided to support the cell biological observations to date. Previously we developed two strains of high Chromosome Instability (CIN) model mice, which show an increase in mitotic errors due to haploinsufficiency in chromosome cohesion system component Sgo1 or mitotic spindle checkpoint component BubR1. The pathways affected by these mutations are suggested to play a role in aging and carcinogenesis in human. Consistently, compared with wild type, both mice strains showed a significant risk for tumor development in colon with chemical carcinogenesis assays. In this study, we used these CIN mice to test whether (i) spontaneous carcinogenesis occurs in the mice, (ii) spontaneous DNA damage occurs in the mice, (iii) whether associated marker expressions are altered, and (iv) if so, whether it is affected by the age. In 12 month old (middle-age) SGO1 and BUBR1 mice, we observed an increase in spontaneous epithelial cancers, although the carcinogenic effect is not aggressive (p<0.05 in SGO1). In histologically normal-looking liver tissue in the mice, increased signals of DNA damage marker pohspho-gamma-H2AX and p53 were observed (4∼5-fold higher in CIN mice), along with altered expressions of p16INK4A and Bcl2 (2∼6-fold higher in CIN mice), providing the first evidence for DNA damage in vivo with mutations that increase mitotic errors. In 4 month old (younger adult) SGO1 mice, significantly (p<0.05) higher percentage of p-H2AX signal in comparison with wild type was also observed. The results demonstrate that mitotic error and CIN-causing mutation can cause higher DNA damage in the tissue-level, indicating that an additional path to carcinogenesis is at work in vivo. Marker analysis in the CIN model mice would indicate molecular consequences of this type of mutation and would lead to the discovery of novel cancer prevention drug targets.

Citation Format: Hiroshi Y. Yamada, Yuting Zhang, Wei Dai, Chinthalapally V. Rao. Consequences of high Chromosome Instability (CIN) in SGO1(Shugoshin1) CIN model mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1752. doi:10.1158/1538-7445.AM2013-1752
Abstract 1756: Intravital imaging of cancer cell cycle in the bone marrow.
Authors: Yano Shuya , Miwa Shinji , Tome Yasunori , Uehara Fuminari , Digman Michelle , Gratton Enrico , Hoffman Robert M.
Published: August 2014

The cell cycle of cancer cells in the bone marrow was imaged using a fluorescence ubiquitination cell cycle indicator (FUCCI). With FUCCI, quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). FUCCI-expressing cancer cells were injected into the bone marrow of tibia of mice. At the early stage after injection (7 days), approximately 90% of the cells were in G0/G1. In contrast, at late stage after injection (28 days more), in the endosteal lesion, approximately 80% of the cancer cells were in S/G2/M phases and in the central zone, approximately 75% of cancer cells were in G0/G1. Thus, cancer-cell location and time in the bone marrow determines cell cycle position and possibly dormancy. Thus, quiescent (dormant) cancer cells in the bone marrow need to be targeted as well as proliferating cancer cells, which is a significant current challenge.

Citation Format: Shuya Yano, Shinji Miwa, Yasunori Tome, Fuminari Uehara, Michelle Digman, Enrico Gratton, Robert M. Hoffman. Intravital imaging of cancer cell cycle in the bone marrow. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1756. doi:10.1158/1538-7445.AM2013-1756
Abstract 1759: Immunohistochemical detection of human telomerase reverse transcriptase in human cholangiocarcinoma and risk condition.
Authors: Endo Kanenori , Tsujitani Shunichi , Yoshimoto Miwa , Watanabe Jyoji , Tokuyasu Naruo , Sakamoto Teruhisa , Naka Takuji , Hirooka Yasuaki , Ikeguchi Masahide
Published: August 2014

Background/Aims: Telomerase activation plays a critical role in several human carcinogenesis through the maintenance of telomeres. The catalytic subunit of telomerase is human telomerase reverse transcriptase (hTERT), whose expression is the critical determinant of telomerase activity. Studies on hTERT in human cholangiocarcinoma (ChC) are rare, and there are no immunohistochemical studies on the correlations with its expressions and ChC patient's clinicopathological parameters.

Methods : HTERT Immunoreactivity was measured in cancerous parenchyma of 28 archival cases of human cholangiocarcinoma (ChC) compared with hyperplastic small biliary ducts in surrounding non-neoplastic liver. And hTERT immunoreactivity was also assessed in both large and small hyperplastic biliary ducts (HBDs) in 9 archival cases of primary sclerosing cholangitis (PSC), which is risk condition for human cholangiocarcinogenesis. Measurements of intrahepatic biliary duct sizes and quantitative microdensitometry measurements of immunostaining intensities were determined using a color video image analysis system. Micro-optical density values related to a standard optical density curve were generated for each set of measurements, using arbitrary units of immunostaining intensity ranging from 0 (nondetectable) to 255 (highest intensity).

Results : HTERT immunoreactivity was presented in nuclei of ChC and PSC cells. Mean micro-optical density values for hTERT in tumorous cells were almost 8-fold greater than that measured in small HBDs in surrounding non-tumorous liver within the same tissue sections. Although there was no significantly difference of tumor differentiation in hTERT expression, its expression was stronger in tubular type than in papillary type and in tubulopapillary type. In PSC cases, hTERT expression was significantly stronger in large HBDs (>500μm cross-sectional diameter) than in small HBDs (<500μm cross-sectional diameter).

Conclusion : Our findings suggest that hTRET protein in human ChC may emerge at a early stage in the progression of the malignant process. The results also suggest hTERT as potentially important targets relevant to chemoprevention or adjunct therapy of ChC.

Citation Format: Kanenori Endo, Shunichi Tsujitani, Miwa Yoshimoto, Jyoji Watanabe, Naruo Tokuyasu, Teruhisa Sakamoto, Takuji Naka, Yasuaki Hirooka, Masahide Ikeguchi. Immunohistochemical detection of human telomerase reverse transcriptase in human cholangiocarcinoma and risk condition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1759. doi:10.1158/1538-7445.AM2013-1759
Abstract 1763: Frequency of homologous recombination repair defects across breast cancer subtypes.
Authors: Timms Kirsten M. , Abkevich Victor , Neff Chris , Morris Brian , Potter Jennifer , Tran Thanh V. , Chen Jian , Sangale Zaina , Tikishvili Eliso , Zharkikh Andrey , Perry Michael , Gutin Alexander , Lanchbury Jerry
Published: August 2014

Introduction: Defects in homologous recombination (HR) repair have potential therapeutic relevance, and current clinical studies are focused on examining the efficacy of agents that exploit HR deficiency in triple negative breast cancer. A homologous recombination deficiency (HRD) score based on whole genome tumor LOH profiles has been developed that is highly correlated with defects in BRCA1/2, and other HR pathway genes, in ovarian cancer(1), and which predicts response to platinum-based neoadjuvant therapy in triple negative breast cancer(2). This study examines the frequency of BRCA1/2 defects and elevated HRD score across breast cancer subtypes as defined by IHC hormone receptor status.

Methods: A targeted custom hybridization panel was developed targeting BRCA1, BRCA2, and 50,000 selected SNPs across the entire human genome. This panel, in combination with sequencing on the Illumina HiSeq, was used to analyze approximately 50 randomly ascertained tumors from each of 4 breast cancer subtypes (triple negative, ER+/Her2-, ER-/Her2+, ER+/Her2+) for BRCA1/2 somatic and germline mutations, and SNP allele frequencies. HRD scores were calculated using LOH profiles reconstructed from the SNP analysis. A BRCA1 promoter methylation assay was also performed on all samples.

Results: BRCA1/2 somatic and germline mutations were detected in all breast cancer subtypes. BRCA1/2 mutations were observed most frequently in triple negative and ER+/Her2+ tumors. BRCA1 promoter methylation was confined almost exclusively to triple negative tumors. Association between elevated HRD score and BRCA defects was observed regardless of tumor type, and BRCA1/2 intact tumors with elevated HRD scores were observed in all breast cancer subtypes.

Conclusions: Elevated HRD score is significantly associated with BRCA1/2 defects in breast cancer. BRCA1/2 defects and elevated HRD scores were observed in all subtypes of breast cancer, suggesting the presence of HR defects in genes other than BRCA1/2 are present in all breast cancer subtypes. HRD score could potentially be used to facilitate the expansion of platinum or PARP inhibitor therapy beyond triple negative breast cancer into other subtypes.

Citation Format: Kirsten M. Timms, Victor Abkevich, Chris Neff, Brian Morris, Jennifer Potter, Thanh V. Tran, Jian Chen, Zaina Sangale, Eliso Tikishvili, Andrey Zharkikh, Michael Perry, Alexander Gutin, Jerry Lanchbury. Frequency of homologous recombination repair defects across breast cancer subtypes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1763. doi:10.1158/1538-7445.AM2013-1763