bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2784: Mitochondrial genomic backgrounds affect nuclear DNA methylation and gene expression
Authors: Vivian Carolyn J. , Brinker Amanda E. , Gooden Gerald C. , Legendre Christophe , Turpin Samuel , Koestler Devin C. , Salhia Bodour , Welch Danny R.
Published: July 2016

Increasing evidence shows mitochondrial DNA (mtDNA) genetics contribute to complex diseases like cancer, but the mechanisms responsible are mostly unknown. A new genetically engineered mouse model was created that contains nuclear DNA from one mouse strain and mtDNA from different mouse strain(s), designated Mitochondrial Nuclear Exchange (MNX). We have demonstrated that specific mtDNA-nDNA combinations could modify metastasis efficiency. The underlying mechanism is hypothesized to be due to mtDNA-driven changes in nDNA expression, but how mtDNA regulates the coordinated gene expression is not yet known. Since DNA methylation is an important epigenetic modification that occurs in all vertebrate genomes and retrograde and anterograde cross-talk exists between the nuclear and mitochondrial genomes, we hypothesized that the nuclear epigenome, specifically methylation, changes as a result of mitochondrial haplotype. To test this hypothesis, we performed Meth-Seq using the Agilent Mouse SureSelectXT, RNA-Seq using Illumina HiSeq 2500®, and Affymetrix GeneChip® Mouse Transcriptome Array. Initial studies were performed using four male mouse brains (8 wk) that were pooled from different litters and cages of wild-type and MNX mice. Significant and selective differential DNA methylation and gene expression patterns were observed and indicate that there are some global, some subtle, but mostly reproducible, differences between these strains. Pathway analysis show clustering of changes involving cell adhesion, ion channels, cell surface receptors, metabolism and molecules involved in transport. Together these observations provide insights into how mtDNA could be altering epigenetic regulation and thereby contribute to cancer pathogenesis. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res, Steiner Family Fund for Metastasis Research, Kansas Bioscience Authority CA134981, P30-CA168524

Citation Format: Carolyn J. Vivian, Amanda E. Brinker, Gerald C. Gooden, Christophe Legendre, Samuel Turpin, Devin C. Koestler, Bodour Salhia, Danny R. Welch. Mitochondrial genomic backgrounds affect nuclear DNA methylation and gene expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2784.
Abstract 2799: Exosomes from hypoxic pancreatic cancer cells confer resistance to subsequent hypoxia insult
Authors: Patton Mary C. , Zubair Haseeb , Patel Girijesh K. , Singh Seema , Singh Ajay P.
Published: July 2016

Pancreatic cancer (PC) remains one of the leading causes of cancer-associated deaths in the US due to its highly aggressive nature and lack of effective treatments. Pancreatic tumors are characterized by a dense, desmoplastic reaction and poor vasculature leading to reduced oxygen levels within the tumor microenvironment. It is believed that this encounter of pancreatic tumor cells to intratumoral hypoxic condition is a major cause of their persistent survival under a variety of stresses, increased aggressiveness and therapeutic-resistance. However, underlying molecular mechanisms responsible for PC's adaptation to hypoxia (along with enhanced growth and aggressiveness) remain elusive. In this study, we examined the role of exosomes, which are nanoscale biological vesicles with a lipid bilayer membrane, in imparting survival benefit to PC cells. For this, we first examined the effect of conditioned media (CM) from PC cells cultured under hypoxic (0.1% O2, H-CM) or normoxic (21% O2, N-CM) conditions on the growth of fresh PC cells cultured under hypoxic (0.1% O2) conditions. We found that H-CM conferred significant survival benefit to cancer cells under hypoxia as compared to N-CM. Thereafter, we isolated exosomes from N-CM (N-exo) or H-CM (H-exo) and used them to treat pancreatic cancer cells under hypoxic culture conditions. We observed that H-exo imparted a significantly higher survival advantage to cancer cells under hypoxia as compared to N-exo. Interestingly, protein quantification indicated the presence of higher levels of exosomes from cells cultured under hypoxic conditions as compared to those grown in normoxic environment. Western blot analysis with anti-CD9 and anti-CD63 antibodies confirmed enrichment of exosomes. Moreover, Dynamic Light Scattering-based size distribution suggested that H-exo were smaller in size compared to N-exo. Further studies are underway to identify the molecular determinants in exosomes responsible for inducing growth-promoting effects under hypoxia. Overall, our data provides the first evidence that hypoxic PC cells secrete exosomes that enhance survivability under hypoxic conditions.

Citation Format: Mary C. Patton, Haseeb Zubair, Girijesh K. Patel, Seema Singh, Ajay P. Singh. Exosomes from hypoxic pancreatic cancer cells confer resistance to subsequent hypoxia insult. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2799.
Abstract 2802: Exogenous pyruvate supports oxygen-independent tumor cell proliferation by serving as an oxygen surrogate to maintain homeostasis of NAD+/NADH
Authors: Yin Chengqian , He Dan , Chen Shuyang , Sang Nianli
Published: July 2016

Hypoxia is usually associated with solid tumors and ischemic lesions. Because oxygen is the common final electron acceptor in a variety of metabolic reactions, hypoxia leads to cell injury and death in normal tissues but promotes tumor progression and metastasis. Hypoxic cells use pyruvate from glycolysis as electron acceptor to support ATP production; anaerobic glycolysis, in which the glycolytic product pyruvate is converted to lactate, has been well established as an adaptive energy strategy for hypoxic cells. In tumor cells, pyruvate may also be used as a substrate to support an active biosynthesis, as well as to maintain the homeostasis of NAD+/NAD. However, whether exogenous pyruvate promotes hypoxic tumor cell proliferation by serving as an electron acceptor or a biosynthetic substrate remains unclear. By using both hypoxia and ρ0 cell with defective mitochondrial electron transfer chain, we show that exogenous pyruvate sustains the proliferation of tumor cells that cannot use oxygen as the electron acceptor. Pyruvate-derived metabolites including acetyl-coA, α-ketoglutarate, succinate, alanine, and aspartate were not able to substitute pyruvate to support oxygen-independent proliferation. Knockdown of metabolic enzymes including pyruvate carboxylase, pyruvate dehydrogenase and citrate synthetase showed no negative effects on pyruvate-facilitated hypoxic cell proliferation, whereas knockdown of lactate dehydrogenase significantly inhibited proliferation, indicating that the crucial role of exogenous pyruvate is to serve as the electron acceptor other than a substrate for proliferating biosynthesis. Consisting with this notion, we show that exogenous pyruvate significantly increased lactate generation and increased the ratio of NAD+/NADH. We also show that in our model cells, hypoxia did not cause accumulation of reactive oxygen species, suggesting that pyruvate-facilitated proliferation of hypoxic cells is not achieved by alleviating oxidative stress. Finally, we show that well-oxygenated cells release pyruvate, providing an in vivo source of pyruvate exogenous to the hypoxic cells. Taken together, our data support a novel model that well-oxygenated cells release pyruvate, which is utilized by hypoxic cells as an electron acceptor to facilitate proliferation. This model provides a potential new therapeutic target to suppress tumor progression.

Citation Format: Chengqian Yin, Dan He, Shuyang Chen, Nianli Sang. Exogenous pyruvate supports oxygen-independent tumor cell proliferation by serving as an oxygen surrogate to maintain homeostasis of NAD+/NADH. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2802.
Abstract 2809: ROS signaling by NADPH oxidase 5 (Nox5) modulates proliferation of human melanoma UACC-257 cells and prolongs growth in the absence of serum or growth factors
Authors: Antony Smitha , Wu Yongzhong , Haines Diana C. , Jiang Guojian , Meitzler Jennifer L. , Juhasz Agnes , Lu Jiamo , Roy Krishnendu K. , Doroshow James H.
Published: July 2016

Reactive oxygen species (ROS) produced by the NADPH oxidase (Noxs) homologs participate in signaling cascades regulating proliferation. Recently, we have reported substantial overexpression of Nox5 in several human cancers including those of prostate, ovary and melanomas. Despite being upregulated in many human cancers and implicated in promoting cell proliferation, the molecular mechanisms modulated by Nox5 are poorly understood. In this study, the functional significance of Nox5 was assessed in human UACC-257 and WM-852 melanoma cells by generating Nox5-overexpressing and knockdown cells. Stable overexpression of Nox5 in UACC-257 cells resulted in enhanced cell growth, increased BrdU positive cells and decreased protein tyrosine phosphatase activity. Additionally, these cells had increased normoxic Hif-1α expression and decreased p27Kip1 expression. Conversely, knockdown of endogenous Nox5 in UACC-257 cells resulted in decreased cell growth, decreased BrdU positive cells, decreased Hif-1α expression and increased p27Kip1 expression. Likewise, in human WM-852 melanoma cells, transient overexpression of Nox5 decreased p27Kip1 expression and knockdown of endogenous Nox5 resulted in increased p27Kip1 and gamma-H2AX expression with decreased cell growth. Cadherin switch, loss of E-cadherin expression and upregulation of N-cadherin was observed in UACC-257 Nox5 overexpressing cells indicative of an invasive phenotype; conversely, an upregulation of E-cadherin expression in Nox5 knockdown cells was noted. Cell invasion assay through matrigel-coated membranes also demonstrated enhanced invasion by Nox5 overexpressing cells. Additionally, 3D cultures of Nox5 overexpressing UACC-257 cells exhibit an amoeboid morphology compared to that of the Nox5 knockdown cells that were mesenchymal, suggestive of an amoeboid - mesenchymal (AMT) transition. Strikingly, UACC-257 cells that overexpress Nox5 were able to proliferate in serum-free conditions for over a month. In summary, our findings suggest that ROS signaling by Nox5 in human melanoma could modulate multiple signaling networks that regulate Hif-1α and p27Kip1 expression, contributing in part, to cell proliferation and the ability to grow in the absence of serum or growth factors.

Citation Format: Smitha Antony, Yongzhong Wu, Diana C. Haines, Guojian Jiang, Jennifer L. Meitzler, Agnes Juhasz, Jiamo Lu, Krishnendu K. Roy, James H. Doroshow. ROS signaling by NADPH oxidase 5 (Nox5) modulates proliferation of human melanoma UACC-257 cells and prolongs growth in the absence of serum or growth factors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2809.
Abstract 2818: An unbiased tumor cell panel profiling method to identify drug-drug interactions reveals synergy between the CDK4 and CDK 6 inhibitor abemaciclib and the Raf dimer and pan-Raf inhibitor LY3009120 in Ras mutant cancers
Authors: Gong Xueqian , Wu Wenjuan , Chio Li-Chun , Pratt Susan , King Constance , Webster Yue , Lallena Maria Jose , Boehnke Karsten , Torres Raquel , Iversen Philip , Reinhard Christoph , Chen Shih-Hsun , Bechmann Richard , Peng Sheng-Bin , Buchanan Sean
Published: July 2016

Drug sensitivity profiling across genomically characterized panels of tumor cells can identify the molecular determinants of drug response. By testing compound combinations in an unbiased format, the same methodology can be used to identify the genomic context of drug-drug synergy. Based on this principle, we developed an unbiased combination screening protocol to identify synergistic interactions with LY3009120, a novel Raf dimer inhibitor that inhibits all three Raf isoforms (Peng et al. 2015, Cancer Cell 28:384-98). Inhibitors of the Ras-MAPK pathway have proven very effective in the treatment of BRAF-mutant melanoma but are, in general, only partially effective in the treatment of BRAF-mutant colorectal cancer and Ras mutant cancers. LY3009120 combined with various compounds was screened across panels of genomically characterized tumor cells. These screens identified a strong synergy with abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4 and CDK6). Statistical analysis of effects in over 500 cancer cell lines showed that mutations in BRAF or Ras family genes were strongly associated with sensitivity to this combination. Strong synergy was observed in skin, colorectal, lung and pancreatic cancers with Ras/Raf mutations, but was also observed in various cancer cells wild type for Ras pathway genes. This included tumor types sensitive to single agent abemaciclib, such as mantle cell lymphoma, ER+ breast cancers, certain leukemias, squamous non-small cell lung cancer, and/or lung cancer with receptor tyrosine kinase activation. In vitro and in vivo analyses of the effects of the combination treatment on signaling pathways in KRAS mutant cancers led to potential mechanistic explanations for the differing efficacy of the combination, which manifests as regression of tumor xenografts in rodent models.

Citation Format: Xueqian Gong, Wenjuan Wu, Li-Chun Chio, Susan Pratt, Constance King, Yue Webster, Maria Jose Lallena, Karsten Boehnke, Raquel Torres, Philip Iversen, Christoph Reinhard, Shih-Hsun Chen, Richard Bechmann, Sheng-Bin Peng, Sean Buchanan. An unbiased tumor cell panel profiling method to identify drug-drug interactions reveals synergy between the CDK4 and CDK 6 inhibitor abemaciclib and the Raf dimer and pan-Raf inhibitor LY3009120 in Ras mutant cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2818.
Abstract 2825: Identification of CDK4/6-response biomarkers using estrogen receptor-positive breast cancer patient-derived xenografts (PDX)
Authors: Serra Violeta , Palafox Marta , Herrera Maria-Teresa , Rivas Martin A , Guzmán Marta , Rodriguez Olga , Grueso Judit , Bellet Meritxell , Oliveira Mafalda , Saura Cristina , di Tomaso Emmanuelle , Camponigro Giordi , Turner Nicholas C. , Cortés Javier , Baselga José
Published: July 2016

Endocrine resistance is a clinical challenge for the treatment of estrogen receptor positive (ER+) breast cancer (BC). CDK4/6 blockade in combination with endocrine therapy has shown clinical activity in metastatic ER+ BC refractory to anti-hormonal treatment. However, there is a need for biomarkers that can predict the response to this treatment and improve patient stratification. We aimed to address this issue using xenograft models established from samples of ER+ BC patients.

Six ER+ PDXs were treated with continuous doses of a CDK4/6 inhibitor (LEE011, 75mg/kg, 6IW) and a PI3K-alpha inhibitor (BYL719, 35mg/kg, 6IW) as single agents and in combination, and intrinsic sensitivity to these agents was evaluated. The models were then genomically characterized using a capture-based sequencing panel and by digital PCR.

One PDX model was intrinsically sensitive to single-agent CDK4/6 inhibition and experienced tumor regression, but all individual tumors eventually escaped therapy after 50 days of treatment. This particular model harbored an ESR1-mutation and concomitant losses of CDKN2A/B. At relapse, we identified the acquisition of an RB1 frameshift mutation. Interestingly, upfront combined treatment with a PI3K-alpha inhibitor delayed the onset of tumor progression. Two out of the remaining five CDK4/6-resistant PDXs harbored either a frameshift mutation in RB1 (plus loss of heterozygosity) or had low pRb protein expression. Two other resistant models harbored CCND1 and MYC amplifications. The remaining one harbored a TSC1 loss. In all the CDK4/6-resistant PDX, however, the combination of CDK4/6 and PI3K-alpha inhibition resulted in tumor regression.

From our results, we conclude that loss of G1-cell cycle checkpoint control, such as mutation/loss of RB1 and CCND1-amplification, is associated with lack of response to CDK4/6 blockade in ER+ BC PDX. The addition of a PI3K-alpha inhibitor results in improvement of disease control in all experimental models tested.

Citation Format: Violeta Serra, Marta Palafox, Maria-Teresa Herrera, Martin A Rivas, Marta Guzmán, Olga Rodriguez, Judit Grueso, Meritxell Bellet, Mafalda Oliveira, Cristina Saura, Emmanuelle di Tomaso, Giordi Camponigro, Nicholas C. Turner, Javier Cortés, José Baselga. Identification of CDK4/6-response biomarkers using estrogen receptor-positive breast cancer patient-derived xenografts (PDX). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2825.
Abstract 2826: Sustained melanoma regression is achieved with continuous palbociclib and PLX4720 treatment but not with intermittent or sequential dosing
Authors: Sheppard Karen E. , Cullinane Carleen , Martin Claire , Kirby Laura , Haynes Nicole , Waldeck Kelly , Young Richard , VanArsdale Todd , McArthur Grant
Published: July 2016

In melanoma the development of resistance to BRAF inhibitors limits clinical responses. Thus the search for novel single agent and combination therapies as well as sequencing strategies that overcome or delay the emergence of resistance is needed. The p16-CDK4- cyclinD-RB1 pathway (CDK4 pathway) is deregulated in approximately 90% of melanomas and most melanoma cell lines are sensitive to palbociclib a specific CDK4/6 inhibitor. We hypothesized that dual targeting the MAPK/ERK and CDK4 pathways would lead to robust inhibition of the CDK4/Cyclin D complex and consequently induce greater tumor regression than single agent treatment. Proliferation and colony formation assays demonstrated that sequential and intermittent treatment with PLX4720 and palbociclib was not as effective as continuous combinational dosing. Only the combination of PLX4720 and palbociclib overcame the development of resistance leading to sustained inhibition of proliferation, cell death and induction of senescence. In A375 and HT144 human tumor xenograft models, palbociclib and PLX4720 initially induced tumor regression and tumor stasis, respectively, however resistance eventually developed to both agents. In contrast, the combination treatment induced rapid and sustained tumor regression. Biomarker studies indicate resistance to single agent PLX4720 was due to reactivation MAPK/ERK pathway and resistance to palbociclib to partial restoration of phosphorylated RB1. Tumor regression in the combination treatment was associated with the infiltration of leukocytes including activated natural killer cells indicating that these cells were involved in tumor clearance. These data demonstrate that dual targeting CDK4/6 and mutant BRAF evades resistance to single agents and leads to sustained tumor regression

Citation Format: Karen E. Sheppard, Carleen Cullinane, Claire Martin, Laura Kirby, Nicole Haynes, Kelly Waldeck, Richard Young, Todd VanArsdale, Grant McArthur. Sustained melanoma regression is achieved with continuous palbociclib and PLX4720 treatment but not with intermittent or sequential dosing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2826.
Abstract 2837: The CDK4/6 inhibitor has potent activity in combination with mTOR inhibitor in head and neck squamous cell carcinoma
Authors: Ku Bo Mi , Koh Jiae , Bae Yeon-Hee , Sun Jong-Mu , Lee Se-hoon , Ahn Jin Seok , Park Keunchil , Ahn Myung-Ju
Published: July 2016

Head and neck squamous cell carcinoma (HNSCC) is a rare lethal human malignancy with no effective therapy. It is critical to identify new therapeutic strategies for NHSCC. Deregulation of CDKN2A (p16) or CCND1 (cyclin D1) occurs commonly in HNSCC and induces sustained cyclin-dependent kinase (CDK) 4/6 activation. The selective CDK4/6 inhibitor, LY2835219 inhibited CDK4/6-dependent Ser780 phosphorylation in retinoblastoma (RB) and induced cell cycle arrest and growth inhibition. In an animal xenograft model of HNSCC, LY2835219 treatment led to reduced tumor growth. However, because single-agent treatment with LY2835219 showed a limited effect in HNSCC, a combinational strategy is necessary for effective therapy. At the molecular level, we found that LY2835219 inhibited activation of AKT and ERK, but not mTOR. The combination of LY2835219 with mTOR inhibitor was found to be more effective than either drug alone in vitro and in vivo. Taken together, our findings suggest that a combination treatment with LY2835219 and mTOR inhibitor is a promising therapeutic approach for HNSCC.

Citation Format: Bo Mi Ku, Jiae Koh, Yeon-Hee Bae, Jong-Mu Sun, Se-hoon Lee, Jin Seok Ahn, Keunchil Park, Myung-Ju Ahn. The CDK4/6 inhibitor has potent activity in combination with mTOR inhibitor in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2837.
Abstract 2847: Phenyl 2-pyridyl ketoxime induces cellular senescence-like alterations via NO production in human diploid fibroblasts
Authors: Jang Hyun-Jin , Jo Eunbi , Chung Young-Ho , Park JunSoo , Park Sung-Jun , Jang Ik-Soon
Published: July 2016

Phenyl-2-pyridyl ketoxime (PPKO) was found to be one of small molecules enriched in the extracellular matrix of near-senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose and time-dependent manner, and resulted in senescence-associated β-galactosidase (SA-β-gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence-associated proteins, such as phosphorylated ERK1/2, caveolin-1, p53, p16ink4a, and p21waf1, were elevated in PPKO-treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II, and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N-acetylcysteine, 2,2,6,6-tetramethylpiperidinyloxy and L-buthionine-(S,R)-sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L-NG-nitroarginine methyl ester and L-NG-monomethylarginine, PPKO-induced transient NO production and SA-β-gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence-associated proteins.

Citation Format: Hyun-Jin Jang, Eunbi Jo, Young-Ho Chung, JunSoo Park, Sung-Jun Park, Ik-Soon Jang. Phenyl 2-pyridyl ketoxime induces cellular senescence-like alterations via NO production in human diploid fibroblasts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2847.
Abstract 2852: CITED2, an emerging regulator in hypoxia- and anoikis- mediated cancer cell growth
Authors: Kuo Ming-Han , Chan Chia Ee , Chou Yu-Ting
Published: July 2016

In solid tumors, hypoxia and anoikis are two common phenomena occurred in the central of region, inducing a series of genes to switch metabolic pathways, prevent apoptosis and promote continuous growth of cancer cells. Herein, we reported that CITED2 (CBP/p300-interacting transactivators with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain 2) responded to hypoxia and anoikis induction, preventing lung cancer cells from cell cycle arrest. We found that hypoxia induced CITED2 through a HIF2α dependent pathway; moreover, three-dimensional (3D) growth of lung cancer cells induced the expression of CITED2. 3D growth condition elicited anoikis-mediated cell cycle arrest, which was further potentiated by hypoxia stimulation. Knockdown of CITED2 or HIF2α in lung cancer cells enhanced anoikis, causing G1/S cell cycle arrest. Soft agar analysis displayed that both HIF2α and CITED2 are necessary for anchorage-independent cell growth under the hypoxic condition. Spheroid assay indicated that HIF2α-CITED2 signaling axis plays an important role in spheroid formation. Pearson correlation analysis showed that CITED2 was associated with HIF2α expression in lung adenocarcinoma. Kaplan Meier analysis revealed that CITED2 expression was correlated with poor survival outcomes in patients with lung adenocarcinoma. Thus, our findings support the notion that CITED2 plays a critical role in hypoxia- and anokis- mediated cancer cell growth with the potential as a prognostic biomarker for predicting lung cancer progression.

Citation Format: Ming-Han Kuo, Chia Ee Chan, Yu-Ting Chou. CITED2, an emerging regulator in hypoxia- and anoikis- mediated cancer cell growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2852.
Abstract 2853: Apoptosis and proliferation in micropapillary structures of colorectal polyps and carcinomas
Authors: Patankar Madhura , Vayrynen Sara , Tuomisto Anne , Makinen Markus , Karttunen Tuomo
Published: July 2016

Background: Micropapillary structures (MIP) can be defined as focal piles of epithelial cells in columnar epithelium. MIP's are found in many cancers, but they are characteristic for serrated colorectal carcinomas. Suprabasal cells of MIP show no contact with extracellular matrix (unpublished).

Objective: To evaluate whether MIP cells in colorectal polyps and cancers would show evidence for anoikis inhibition we assessed proliferation and apoptosis indexes (PI and AI, respectively) in MIP and non-MIP epithelium.

Materials and methods: We stained human colorectal lesions: non-serrated adenomas (n = 15), serrated polyps (n = 29), conventional adenocarcinomas (n = 32), and serrated adenocarcinomas (n = 30) with antibodies to Ki67 and M30. To obtain PI and AI two independent observers counted proportions of positive cells separately in the cells of MIP and in non-MIP epithelium.

Results: In carcinomas, AI was lower in the cells of MIP (M30; median 0, range 0-40) than in the non-MIP cells (median 2, range 0-40; p<0.001, Wilcoxon). Similarly, PI was lower in the MIP cells (Ki67; MIP: median 17, range 0-93; non-MIP: median 30, range 1-93; p<0.001). Similar patterns were evident in subsets of serrated lesions, in both carcinomas (p<0.001) and polyps (p<0.001), and in non-serrated carcinomas (p<0.004). Apoptosis/proliferation (AI/PI) ratio was lower in the MIP cells than in the non-MIP epithelium in both cancers and their precursor lesions (P<0.01).

Conclusion: Apoptosis rate was lower in MIP's than in other tumor epithelium indicating ability of these cells to survive without matrix contact, i.e. Anoikis is inhibited in them. Lower PI could suggest that cells in the MIP are in some kind of quiescent stage. However, significantly lower AI/PI ratio in the MIP infers that these structures may form a fast growing subpopulation of tumor cells. Further studies are in progress to dissect mechanism of formation of MIP and matrix independent survival of tumor cells in them.

Citation Format: Madhura Patankar, Sara Vayrynen, Anne Tuomisto, Markus Makinen, Tuomo Karttunen. Apoptosis and proliferation in micropapillary structures of colorectal polyps and carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2853.
Abstract 2867: Functional role of FHL2 in the splicing machinery of liver cells
Authors: Cao Ye , Liu Minghua , Yang Yi , Zhang Yinfeng , Tsui Kwok Wing
Published: July 2016

Hepatocellular carcinoma (HCC) is a highly lethal cancer with increasing worldwide incidence. The four-and-a-half LIM domain protein 2 (FHL2) has been identified as an oncogene / tumor suppressor gene in a tissue/cell specific manner by acting as an adaptor protein to form different complexes and involving in various signaling pathways. To clarify the intriguing functions of FHL2, we have identified a batch of interacting partners of FHL2 by immunoprecipitation and liquid chromatography coupling with mass spectrometry. Many of these proteins, including SNRNP70, DAZAP1, SFPQ, TIAL1 and so on, are involved in spliceosome components and mRNA surveillance. The interaction among several proteins and FHL2 has been confirmed by co-immunoprecipitation. To further elucidate the functional role of FHL2 in the splicing process in liver cancer cells, we have studied the transcriptomes of FHL2 overexpression and FHL2 knockdown liver cells, whereas empty vector and scrambled siRNA were used as controls respectively. Followed by bioinformatics analysis, we found significant concordance between the immunoprecipitation and transcriptome datasets. Selected genes were further validated by real-time PCR such as FGFR4. In summary, FHL2 is probably regulating the mRNA splicing by interacting with components of the splicing machinery and modulating splicing related genes at the transcriptional level.

Citation Format: Ye Cao, Minghua Liu, Yi Yang, Yinfeng Zhang, Kwok Wing Tsui. Functional role of FHL2 in the splicing machinery of liver cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2867.
Abstract 2874: Genetic suppression reveals DNA repair-independent antagonism between BRCA1 and COBRA1 in mammary gland development
Authors: Zhang Xiaowen , Nair Sreejith J. , Chiang Huai-Chin , Wang Yao , Jahid Md Jamiul , Ruan Jianhua , Jin Victor X. , Hu Yanfen , Li Rong
Published: July 2016

Breast cancer susceptibility gene BRCA1 is well known for its function in double strand break (DSB) DNA repair. While BRCA1 is also implicated in transcriptional regulation, the physiological significance remains unclear. Cofactor of BRCA1 (COBRA1) is a BRCA1-binding protein and an important regulator of transcription elongation. Here we used mouse genetics to interrogate functional interaction between BRCA1 and COBRA1 during mammary gland development. Tissue-specific deletion of Cobra1 reduced mammary epithelial compartments and blocked ductal morphogenesis, alveologenesis, and lactogenesis, demonstrating a pivotal role of COBRA1 in adult tissue development. Remarkably, these developmental deficiencies due to Cobra1 knockout were largely rescued by additional loss of full-length Brca1. Furthermore, Brca1/Cobra1 double knockout restored developmental transcription at puberty, altered luminal epithelial homeostasis, yet remained deficient in homologous recombination-based DSB repair. Thus our genetic suppression analysis uncovers a previously unappreciated, DNA repair-independent function of BRCA1 in antagonizing COBRA1-dependent transcription program during mammary gland development.

Citation Format: Xiaowen Zhang, Sreejith J. Nair, Huai-Chin Chiang, Yao Wang, Md Jamiul Jahid, Jianhua Ruan, Victor X. Jin, Yanfen Hu, Rong Li. Genetic suppression reveals DNA repair-independent antagonism between BRCA1 and COBRA1 in mammary gland development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2874.
Abstract 2875: Genes associated with neuronal development, including splicing regulatory factors, increase in expression across epithelial-mesenchymal transition
Authors: Khodor Yevgenia L. , Riquelme Daisy , Gertler Frank , Burge Christopher B.
Published: July 2016

The program by which epithelial cells lose their polarity and close contacts with neighboring cells and acquire migratory and invasive properties is known as epithelial-mesenchymal transition (EMT). Initially identified as a critical developmental process, EMT is now understood to occur during cancer, where it has been shown to confer resistance to senescence and apoptosis. Previous work indicates that cancer cells that escape the site of the primary tumor hijack at least a portion of the EMT program to increase their invasive properties and metastasize. Earlier work in the field has focused on cell signaling and transcriptional regulation changes that lead to EMT, but changes in the gene expression and post-transcriptional regulation of the cell have only recently become the focus of study. We characterized gene expression and splicing across EMT in the luminal epithelial MCF7ras SLUG+SOX9 breast cancer cell line by collecting samples at 2-day intervals across its 6-day transition and profiling the RNA by RNA-seq. We found over 4,000 significantly changing genes, and that the majority of gene expression and splicing changes occurred in the first two days of the transition, with few changes noted between days 4 and 6. Genes that changed could be broadly categorized into those whose expression decreased, increased, or peaked in the middle of the transition before decreasing. Intriguingly, genes whose expression increased during EMT were enriched for gene ontology terms related to cell signaling and neuronal development. We specifically observed increased expression of mRNAs encoding splicing factors and other RNA-binding proteins (RBPs) associated with neuronal development, in contrast to the majority of RBPs whose expression decreases in EMT. We are currently exploring various aspects of this program, including effects of increased expression of the neuronal-associated splicing regulatory factor quaking (QKI). These may include downregulation of the Notch pathway via effects on splicing of NUMB, a Notch inhibitor. We are currently validating the role of QKI on this and other RNA processing changes and will report our findings.

Citation Format: Yevgenia L. Khodor, Daisy Riquelme, Frank Gertler, Christopher B. Burge. Genes associated with neuronal development, including splicing regulatory factors, increase in expression across epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2875.
Abstract 288: Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel
Authors: Jarvis Courtney L. , Nelius Thomas , Martinez-Marin Dalia , Cheerla Srirupa , Filleur. Stéphanie
Published: July 2016

Despite recently approved novel agents, taxane-based chemotherapy remains the major therapeutic strategy for metastatic castration-resistant prostate cancer (mCRPC). Still mCRPC continues to be incurable. Furthermore, patients often experience severe side effects, prompting a re-evaluation of conventional regimen. Past studies have demonstrated promise for Low-Dose Metronomic (LDM) chemotherapy; schedule defined as the frequent administration of low doses of chemotherapeutic drugs with no prolonged drug-free breaks. Yet relative activities of LDM taxanes and combinations with known anti-neoplasic agents have to be investigated. Using CRPC cell lines (PC3, Du145 and the LNCaP-derivative CL1), we have shown that cabazitaxel-treated cells have a significantly lower EC50 compared to docetaxel, with Du145 cells presenting the greatest differences. In cell cycle analysis, both low-dose taxanes increased the sub-G1 cells population. However, the sub-G1 increase was significantly greater in cabazitaxel- than in docetaxel-treated Du145 cells, but not in PC3 and CL1. Accordingly, plasma membrane Annexin V elevation occurred in Du145 cells at lower doses of cabazitaxel than docetaxel validating a higher efficacy due to increased apoptosis. As other possible cell death mechanisms, autophagy and necrosis were investigated. Beclin1 expression levels remain unchanged in all three cell lines. In contrast, necrosis was stimulated in all the taxanes-treated cell lines in a dose dependent manner. In vivo, LDM cabazitaxel was significantly more efficient in delaying tumor growth than docetaxel. This effect was markedly increased when cabazitaxel was combined with the angio-inhibitor and anti-tumor Pigment Epithelium-Derived Factor (PEDF). Other results showed that PEDF and LDM chemotherapy combination induces more phagocytosis of CRPC cells when compared to single treatments. In conclusion, our data demonstrate a higher efficacy of cabazitaxel on CRPC both in vitro and in vivo, and suggest that LDM taxane chemotherapy/PEDF combination could be used as a novel therapeutic strategy for CRPC.

Citation Format: Courtney L. Jarvis, Thomas Nelius, Dalia Martinez-Marin, Srirupa Cheerla, Stéphanie Filleur. Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 288.
Abstract 2891: High throughput single cell gene expression profiling by multiplex qPCR
Authors: Okino Steven T. , Kong Michelle , Ma Jason , Fenrich Joshua , Boveri Luca , Jouvenot Yann , Wang Yan
Published: July 2016

Single cell gene expression analysis is a powerful technique that provides a unique and insightful perspective on biological pathways and processes. Here we present a robust workflow that enables fast and accurate analysis of up to 100 genes in isolated single cells. Our workflow is highly sensitive, by assessing RNA reference standards we find that a single RNA transcript is detected with about 80% efficiency. We used this workflow to study differentiation in cultured NTera2 cells (NT2), a human embryonic stem cell model system. We analyzed untreated NT2 cells, and NT2 cells treated with low and high doses of retinoic acid (RA) for 10 days to initiate differentiation to a neuronal lineage. The expression levels of 16 genes were quantified in 164 single cells by multiplex real-time qPCR with two technical replicates. The entire experiment, from cultured cells to results, can be completed in 2-3 days and requires four 384-well qPCR plates for gene expression quantification. We find that control cells and cells treated with a high dose of RA (10 uM) are relatively homogeneous in the expression levels of the targeted genes. However cells treated with a low dose of RA (0.25 uM) exhibit significant heterogeneity with respect to gene expression; about half of the cells are similar to the high-dose RA cells, the other cells exhibit a wide range of partial differentiation. Interestingly, we find that LEFTY2 expression is almost exclusive to the low dose RA cells and strongly correlates with partial differentiation. A time-course study analyzing cell populations reveals that LEFTY2 is only transiently expressed in differentiating NT2 cells with peak expression at 3 days of high dose RA treatment. These findings imply that, in NT2 cells, LEFTY2 is a potential biomarker of early differentiation. In summary, we present an accurate, sensitive and robust single cell analysis procedure that uses standard reagents and platforms. We envision that this workflow will enable researchers to investigate cell heterogeneity in biological pathways in a cost-effective way.

Citation Format: Steven T. Okino, Michelle Kong, Jason Ma, Joshua Fenrich, Luca Boveri, Yann Jouvenot, Yan Wang. High throughput single cell gene expression profiling by multiplex qPCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2891.
Abstract 2903: Nuclear localization of a secretion protein NAG-1/GDF15
Authors: Min Kyung-Won , Silva Gabriel , Baek Seung J.
Published: July 2016

Nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1, also known as GDF15) is a divergent member of the transforming growth factor-beta (TGF-β) superfamily, which exert their biological activity by binding to membrane receptors as a secretion protein. Abnormal regulation of TGF-β signaling can contribute to various diseases such as cancer. Although there is sequence similarity between NAG-1 and other TGF-β superfamily members, NAG-1 does not bind to the TGF-β receptors; therefore the biological consequence of NAG-1 remains to be elucidated. Here, we report that NAG-1 has two putative non-canonical nuclear localization signals (NLSs) that mediate NAG-1 nuclear localization. Nuclear import assay shows that NAG-1 enters the nucleus through nuclear pore complex (NPC) in the presence of ATP, indicating nuclear entry of NAG-1 is active transport. Furthermore, we found the nuclear export signal (NES) recognized by CRM1 at near N-terminal region of NAG-1, that is conserved across various mammalian species. Impeding NES via chemical or genetic approaches promotes nuclear accumulation of NAG-1. We also found that nuclear NAG-1 dampens TGF-β-mediated cell migration and invasion via the inhibition of DNA binding of Smads complex. Overall, our data suggest that nuclear NAG-1 affects TGF-β-induced metastasis activity in cancer cells.

Grant support: This work was supported by grant from the University of Tennessee Center of Excellence in Livestock Diseases and Human Health.

K Min, Current address: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA

Citation Format: Kyung-Won Min, Gabriel Silva, Seung J. Baek. Nuclear localization of a secretion protein NAG-1/GDF15. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2903.
Abstract 2908: The clinical significance of Nrf2 overexpression in esophageal cancer
Authors: Kitano Yuki , Baba Yoshifumi , Yamamura Kensuke , Shigaki Hironobu , Mima Kousuke , Kurashige Junji , Iwatsuki Masaaki , Sakamoto Yasuo , Miyamoto Yuji , Yoshida Naoya , Baba Hideo
Published: July 2016

Background: NF-E2-related factor 2 (Nrf2) is a key transcription regulator for antioxidant and detoxification enzyme and is overexpressed in various cancer cells. In this study, prognostic significance of Nrf2 expression in esophageal cancer was investigated. In addition, we examined the relationship between the expression of Nrf2 and cell proliferation using in vitro assay.

Methods: We immunohistochemically evaluated the protein expression of Nrf2 in the surgically resected specimens in 83 esophageal cancer patients. And ten esophageal cancer cell lines were utilized in vitro assay.

Results: We examined the protein expression of Nrf2 in 10 esophageal cancer cell lines. TE-11 cell had the highest expression of Nrf2, while KYSE30 had the lowest expression of Nrf2. Importantly, there was no correlation between the protein level and mRNA level of Nrf2 in these cell lines. The cell proliferation was inhibited in TE-11 cells, and not inhibited in KYSE30 cells by treatment with Nrf2-siRNA. Moreover, Flow Cytometry Assay revealed that knockdown of Nrf2 by siRNA arrested the cell cycle at G1 phase in TE-11 cells. In immunohistological study, Nrf2 was predominantly identified in the nucleus of esophageal cancer cells and the expression level of Nrf2 was higher in the tumor than normal esophageal epithelium (P<0.001). There was no relationship between Nrf2 protein expression and clinicopathological characteristics. The overall survival of the high-Nrf2 expression group was significantly poorer than that of low-Nrf2 expression group (P = 0.004).

Conclusions: The expression of Nrf2 is up-regulated in esophageal cancer cell lines and resected tumor specimen. Our in vitro assay revealed that Nrf2 appeared to support cancer cell proliferation. Therefore, strategies to pharmacologically manipulate the level and / or activity of Nrf2 may have potential to reduce tumor growth and improve the prognosis of esophageal cancer patients.

Citation Format: Yuki Kitano, Yoshifumi Baba, Kensuke Yamamura, Hironobu Shigaki, Kousuke Mima, Junji Kurashige, Masaaki Iwatsuki, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. The clinical significance of Nrf2 overexpression in esophageal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2908.