bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1224: Identification of novel p53 targets by RNA sequencing in mice liver tissues
Authors: Koguchi Tomoyuki , Tanikawa Chizu , Kojima Yoshiyuki , Nakagawa Hidewaki , Nakamura Yusuke , Matsuda Koichi
Published: August 2015

In response to various type of cellular stress such as X-ray irradiation, ultraviolet exposure, and oxidative stress, the p53 protein is activated and regulates its target genes that induce cell cycle arrest, senescence, DNA repair, and apoptosis. Inactivation of the p53 gene is the most common alterations observed in human cancers. Therefore, identification of p53-target genes would help us to understand molecular carcinogenesis. However, an entire picture of the p53 signaling pathway has not been disclosed yet.

To elucidate the mechanism whereby p53 suppress tumor development, we purified RNA of 19 organs from p53+/+ or p53-/- mice with or without 10 Gy of X-ray irradiation. RNA sequence analysis of 228 tissues provided expression profile of 23813 genes. Then we screened genes which satisfy the following criteria at least one organ; median FPKM value after radiation in p53WT mice is more than two-fold higher than that of those in other groups with p-value of 0.05 by t-test; median FPKM value after radiation in p53WT mice is more than one. Among 119 previously identified p53 targets with functional p53 binding motifs, 70 targets were induced by DNA damage in at least one mice tissues.

Then we selected 16 candidates those were induced in mice liver. Quantitative PCR analysis using HepG2 human hepatocellular carcinoma cells revealed that four genes were induced by adriamycin treatment in a p53-dependent manner. Through these screening, here we identified p53-dependent inositol phosphate converting enzyme (PICE) as a direct target of p53. p53 induced PICE protein and mRNA in both human and mice. In addition we identified p53 binding sequence within its promoter region and exon 7 of the PICE gene. p53 also increased an inositol isomer synthesis in response to DNA damage. Our findings revealed the novel role of p53 in inositol phosphate metabolism.

Citation Format: Tomoyuki Koguchi, Chizu Tanikawa, Yoshiyuki Kojima, Hidewaki Nakagawa, Yusuke Nakamura, Koichi Matsuda. Identification of novel p53 targets by RNA sequencing in mice liver tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1224. doi:10.1158/1538-7445.AM2015-1224
Abstract 1225: Proteome-wide analysis of gain-of-function mutant p53 targets in breast cancer implicates PARP, PCNA and MCM4 as oncogenic drivers
Authors: Polotskaia Alla , Xiao Gu , Reynoso Katherine , Martin Che , Qiu Wei-Gang , Hendrickson Ronald , Bargonetti Jill
Published: August 2015

Gain-of-function mutant p53 (mtp53) changes the cancer cell transcriptome. However no analysis of mtp53-associated proteome diversity has been carried out. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53 driven proteome. Using SILAC followed by tandem mass spectrometry (LC-MS/MS) we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells both up and down-regulated multiple proteins and metabolic pathways. We sequenced 73,154 peptide pairs that corresponded to 3010 proteins detected under reciprocal labeling conditions. Importantly the high impact regulated targets included the previously identified transcriptionally regulated mevalonate pathway proteins but also identified two new levels of mtp53 protein regulation for non-transcriptional targets. Interestingly, mtp53 depletion profoundly influenced poly (ADP-ribose) polymerase 1 (PARP1) localization, with increased cytoplasmic and decreased chromatin-associated PARP1. An enzymatic PARP shift occurred with high mtp53 expression resulting in increased poly-ADP-ribosylated proteins in the nucleus. Mtp53 increased the level of PCNA and MCM4 protein without changing the amount of pcna or mcm4 transcript. Pathway enrichment analysis ranked the DNA replication pathway above the cholesterol biosynthesis pathway as an R273H mtp53 activated proteomic target. Knowledge of the proteome diversity driven by mtp53 suggests DNA replication and repair pathways are major targets of mtp53 and highlights consideration of combination chemotherapeutic strategies targeting cholesterol biosynthesis and PARP inhibition.

Acknowledgements. This work was supported by a grant to JB from the Breast Cancer Research Foundation.

Citation Format: Alla Polotskaia, Gu Xiao, Katherine Reynoso, Che Martin, Wei-Gang Qiu, Ronald Hendrickson, Jill Bargonetti. Proteome-wide analysis of gain-of-function mutant p53 targets in breast cancer implicates PARP, PCNA and MCM4 as oncogenic drivers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1225. doi:10.1158/1538-7445.AM2015-1225
Abstract 1229: E3 ubiquitin ligase TRIM32 negatively regulates tumor suppressor p53 to promote tumorigenesis
Authors: Zhang Cen , Liu Juan , Wang Xiaolong , Young Ken H. , Hu Wenwei , Feng Zhaohui
Published: August 2015

Tumor suppressor p53 plays a key role in maintaining genomic stability and preventing tumorigenesis through its regulation of cellular stress responses, including apoptosis, cell cycle arrest and senescence. To ensure its proper levels and functions in cells, p53 is tightly regulated mainly through post-translational modifications. Ubiquitination is the most important post-translational modification for p53. TRIM32 is a protein of the tripartite motif (TRIM) family. The proteins of TRIM family have been reported to be involved in different biological processes, including cell growth, differentiation, development, muscular physiology, innate immune response and cancer. However, like the majority of TRIM family proteins, the biological function of TRIM32 is not well-understood. Here, we identified TRIM32 as a novel p53 target gene and E3 ubiquitin ligase for p53. In response to stress, including ionizing radiation (IR) and chemotherapeutic agents (e.g. etoposide and 5-fluorouracil), p53 protein binds to the p53 responsive element in the promoter of the TRIM32 gene and transcriptionally induces the expression of TRIM32 in cells. In turn, TRIM32 protein interacts with p53 protein and promotes p53 degradation through ubiquitination to negatively regulate p53-mediated apoptosis, cell cycle arrest and senescence in response to stress. Thus, p53 and TRIM32 form a novel negative feedback loop in cells to regulate p53 protein levels and function. TRIM32 is frequently overexpressed in different types of human tumors, including lung, colorectal and liver cancers. TRIM32 overexpression promotes the oncogenic transformation of primary mouse embryonic fibroblasts in a largely p53-dependent manner. Furthermore, TRIM32 overexpression promotes tumorigenesis in mice through the down-regulation of p53 function. Taken together, our results demonstrated that as a novel p53 target and a novel negative regulator for p53, TRIM32 plays an important role in regulation of p53 and p53-mediated cellular stress responses. Furthermore, our results revealed that impairing p53 function is a novel mechanism for TRIM32 in tumorigenesis.

Citation Format: Cen Zhang, Juan Liu, Xiaolong Wang, Ken H. Young, Wenwei Hu, Zhaohui Feng. E3 ubiquitin ligase TRIM32 negatively regulates tumor suppressor p53 to promote tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1229. doi:10.1158/1538-7445.AM2015-1229
Abstract 123: Mechanism of oncogenic signal activation by the novel fusion kinase FGFR3-BAIAP2L1
Authors: Nakanishi Yoshito , Akiyama Nukinori , Tsukaguchi Toshiyuki , Fujii Toshihiko , Satoh Yasuko , Mizuno Hideaki , Ishii Nobuya , Aoki Masahiro
Published: August 2015

Recent cancer genome profiling studies have identified many novel genetic alterations, including rearrangements of genes encoding fibroblast growth factor receptor (FGFR) family members. However, most fusion genes are not functionally well characterized, and the oncogenicity of some fusions as well as their potential sensitivity to targeted therapy are still unclear. In a previous study, we investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like 1 (BAIAP2L1). The FGFR3-BAIAP2L1 fusion gene was identified in 4 bladder cancer patients and 2 lung cancer patients via screens involving PCR and a break-apart fluorescence in situ hybridization assay. The functional analysis of FGFR3-BAIAP2L1 transfectant in Rat-2 fibroblast cells (Rat-2_F3-B) indicated that FGFR3-BAIAP2L1 forms a dimer via the Bin-Amphiphysin-Rvs (BAR) BAIAP2L1 dimerization domain that constitutively activates its FGFR3 kinase activity. CH5183284/Debio 1347*, a selective FGFR inhibitor, effectively inhibits growth of Rat-2_F3-B both in vitro and in vivo, indicating that the FGFR3 kinase activity is critical for tumorigenic activity of this fusion. These results indicate a potential application of FGFR inhibitors to treat FGFR3-BAIAP2L1fusion gene positive patients.

In this study, we further elucidate the mechanism of tumorigenic potential of FGFR3-BAIAP2L1A by a comprehensive gene expression analysis of 4 cell lines (Rat-2_mock, Rat-2_FGFR3, Rat-2_F3-B, and Rat-2_BAIAP2L1) using RNA sequencing. We identified 143 up-regulated and 67 down-regulated genes specifically engaged by FGFR3-BAIAP2L1. Our gene signature analysis with this gene set revealed that FGFR3-BAIAP2L1 activates growth signals, such as the mitogen-activated protein kinase pathway, and inhibits tumor-suppressive signals, such as the p53, RB1, and CDKN2A pathways. Analysis by Western blot in xenograft tissues confirmed the activation and inactivation status of those pathways. These data suggest that a concurrent regulation of an oncogenic pathway and a tumor-suppressive pathway results in the tumorigenic potential of FGFR3-BAIAP2L1.

*CH5183284/Debio 1347 was discovered by Chugai Pharmaceutical Co., Ltd. and is being developed by Debiopharm International S.A. under an exclusive worldwide license.

Citation Format: Yoshito Nakanishi, Nukinori Akiyama, Toshiyuki Tsukaguchi, Toshihiko Fujii, Yasuko Satoh, Hideaki Mizuno, Nobuya Ishii, Masahiro Aoki. Mechanism of oncogenic signal activation by the novel fusion kinase FGFR3-BAIAP2L1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 123. doi:10.1158/1538-7445.AM2015-123
Abstract 1233: Impairment of TFF3 induces cellular apoptosis in prostate cancer cell lines and overexpression of TFF3 may be a prognostic biomarker in human prostate cancers
Authors: Kim So-Youn , Liu Ji-Ying , Chung Yeun-Jun
Published: August 2015

Background: Human trefoil factor 3(TFF3)/ Intestinal trefoil factor(ITF) is originally known well as an essential regulator for epithelial restitution in gastrointestinal tract and also suggested as a potential cancer marker in serum. Furthermore, the aberrant overexpression of TFF3 has been reported in various human cancers, majorly colorectal cancers, suggesting that TFF3 may play a critical role in tumor growth. However, its molecular signaling pathway remains unclear.

Objective: To clarify whether TFF3 is related to survival/anti-apoptotic cell signaling pathways, and to determine that tff3 could be a molecular marker in prostate cancers.

Design, Setting, and Participants: TFF3 gene was silenced by using siRNA in prostate cancer two kinds of cell lines, LNCap and PC3, following investigation of genes related to apoptosis and cell migration. The expression of TFF3 was analyzed by immunohistochemistry in 109 prostate cancer and 106 normal prostate tissues.

Results and Limitations: TFF3 silenced prostate cancer cell lines exhibited increased annexin V binding and the activation of caspase-9 and caspase-3 which are crucial for programmed cell death, consistent with an increased rate of apoptosis, reduced rate of proliferation and migration. In human prostate cancer tissue microarray, TFF3 was positive in 54.7% (49.1%, 2+; 5.6%, 1+) of cancer cases (59/108) and 2.8% (1.9%, 2+; 0.9%, 1+) of morphologically normal tissue cases (3/106), P<0.0001.

Conclusions: The results suggest that TFF3 expression seems play a role in promoting cell survival and be a potential useful marker in prostate cancers. In these contexts, TFF3 in human prostate cancers deserves further investigation to exploit as a cancer marker and anticancer drug.

Citation Format: So-Youn Kim, Ji-Ying Liu, Yeun-Jun Chung. Impairment of TFF3 induces cellular apoptosis in prostate cancer cell lines and overexpression of TFF3 may be a prognostic biomarker in human prostate cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1233. doi:10.1158/1538-7445.AM2015-1233
Abstract 1240: Boric acid inhibits cell proliferation through RB1 protein in head and neck cancer
Authors: Gunduz Esra , Hatipoglu Omer Faruk , Cigdem Sadık , Erdogan Kubra , Elitok Mustafa Semih , Grenman Reidar , Erdamar Husamettin , Gunduz Mehmet
Published: August 2015

Boric acid is a very common chemical used in a wide variety of applications as well as in medicine. In this study, the usage of boric acid as a potential alternative cancer therapeutic was investigated by examining its effect in head and neck cancer cell line proliferation and tumor suppressor gene expression.

Head and neck cancer cell lines UT-SCC 6A, 6B, 9A, 16A, 16B, 24A, 54C, 74A, and 74B were treated with varying doses of boric acid, ranging from 200 ug/mL to 1800 ug/mL and XTT assay was used to measure the dose response. Subsequently, RNA was isolated from treated cells and was converted to cDNA to evaluate the expression of P53, RB1, ING1 and ING3 via Real-time PCR. Also protein was isolated from the same cell lines for Western Blot analysis.

Boric acid treatment caused a significant decrease in proliferation in all of the head and neck cancer cell lines investigated, including UT-SCC 6A, 6B, 9A, 16A, 16B, 24A, 54C, 74A, and 74B. Results from boric acid treatment and untreated controls were analyzed for statistical significance using Mann Whitney U test. The results approached statistical significance with p value of 0.05. After determining the effective doses, cell lines were treated with boric acid and gene expression of P53, RB, ING1 and ING3 were analyzed. All of the genes’ expression were increased in several of the cell lines examined. We couldn't detect the protein expression of ING1 and ING3. But we’ve detected the higher expression of Phospho (PP)-RB and Bax, lower expression of bcl2 protein.

In short, our results displayed similar results to prostate cancer reported in literature in terms of inhibition of proliferation. We’ve showed that boric acid has reduced levels of cell proliferation in head and neck cancer cell lines for the first time in our study. Also higher expression of PP-RB indicated that boric acid stopped cell cycle at G1. As a pro-apoptotic Bax expression was up-regulated and anti-apoptotic bcl2 was down-regulated in some of the cell lines showing that boric acid may lead the cells to apoptosis. In the light of these results, boric acid appears to be a candidate alternative therapeutic agent for cancer. Of course, further experiments are necessary in order to strengthen this conclusion.

Citation Format: Esra Gunduz, Omer Faruk Hatipoglu, Sadık Cigdem, Kubra Erdogan, Mustafa Semih Elitok, Reidar Grenman, Husamettin Erdamar, Mehmet Gunduz. Boric acid inhibits cell proliferation through RB1 protein in head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1240. doi:10.1158/1538-7445.AM2015-1240
Abstract 1251: Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells
Authors: Antony Smitha , Wu Yongzhong , Jiang Guojian , Meitzler Jennifer L. , Liu Han , Juhasz Agnes , Lu Jiamo , Anver Miriam R. , Roy Krishnendu K. , Doroshow James H.
Published: August 2015

NADPH oxidase 5 (Nox5), one of the seven members of the NADPH oxidase gene family that generate reactive oxygen species (ROS), has been implicated in physiological and pathophysiological processes. Recently, we have reported substantial overexpression of Nox5 in several human cancers including those of prostate, ovary and melanomas; expression in most non-malignant tissues was weak to modest. Despite being upregulated in many human cancers and implicated in promoting cell proliferation, the molecular mechanisms regulated by Nox5 are poorly understood. In this study, the functional significance of Nox5 was assessed in human UACC-257 melanoma cells by generating stable Nox5-overexpressing clones and knockdown clones. Overexpression of Nox5 in UACC-257 cells resulted in enhanced cell growth, increased BrdU positive cells and decreased 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. Cadherin switch, loss of E-cadherin expression and upregulation of N-cadherin was observed in 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 cells exhibit an amoeboid morphology compared to that of the Nox5 knockdown cells that were mesenchymal, suggestive of an amoeboid - mesenchymal (AMT) transition. Strikingly, cells that overexpress Nox5 were able to proliferate in serum-free conditions for over a month. In summary, our findings suggest that Nox5 expression in human melanoma UACC-257 cells could contribute to cell proliferation, invasion, amoeboid morphology, and the ability to grow in the absence of serum or growth factors in part due to the generation of high local concentrations of extracellular ROS that modulate multiple signaling networks that regulate Hif-1α, p27Kip1, and the cadherin switch.

Citation Format: Smitha Antony, Yongzhong Wu, Guojian Jiang, Jennifer L. Meitzler, Han Liu, Agnes Juhasz, Jiamo Lu, Miriam R. Anver, Krishnendu K. Roy, James H. Doroshow. Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1251. doi:10.1158/1538-7445.AM2015-1251
Abstract 1253: Selectivity of regulated IRE1 dependent decay of RNA controls pro-oncogenic signaling in glioblastoma
Authors: Chevet Eric
Published: August 2015

Glioblastoma multiforme (GBM) is the most lethal form of glioma with an overall survival at 5 years nearly null (< 5%). Increasing evidences point towards the RNase activity of IRE1 as a central player in GBM development, particularly in cancer cell invasion and tumor vascularization. Indeed, IRE1 promotes XBP1 mRNA splicing, a well-described cytoprotective transcription factor that is required for cancer cell survival. Moreover, regulated IRE1 dependent decay of RNA (RIDD) leads to opposite effects on cell fate depending the ER stress intensity or duration and on the target RIDD substrates. Recent studies unraveled the presence of somatic mutations on the IRE1 gene in GBM that could play a driver role but without providing any functional information. Here, we have sequenced IRE1 exons in 23 human GBMs and identified a new somatic mutation on the IRE1 gene. Interestingly, using biochemical approaches and an orthotopic tumor graft mouse model we show that this variant displays an altered RIDD selectivity in particular towards the specific RIDD substrates already characterized in GBM (SPARC and PER1) and also miR-17, a previously described RIDD substrate whose expression has also a prognostic value in GBM. IRE1-dependent gene expression pattern alterations lead to major functional consequences in terms of tumor phenotype including (i) a magnified induction of the inflammatory and pro-angiogenic pathways based on transcriptome signatures and (ii) an increase of the aggressiveness/infiltration potential of U87 derived tumors in mice. This study provides the first evidence that RIDD substrates selectivity can be modulated and unravels the first mechanistic example of how a somatic mutation in the IRE gene can provide adaptive advantages to glioblastoma cells.

Note: This abstract was not presented at the meeting.

Citation Format: Eric Chevet. Selectivity of regulated IRE1 dependent decay of RNA controls pro-oncogenic signaling in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1253. doi:10.1158/1538-7445.AM2015-1253
Abstract 1263: Cellular senescence in Hodgkin's lymphoma
Authors: Gopas Jacob , Zurgil Udi , Zeev Estee , Benharoch Daniel , Ezratty Jodi , Shubinsky Giora , Livneh Etta
Published: August 2015

Hodgkin's lymphoma (HL), a B cell originated malignancy of the immune system, is one of the most frequent lymphomas in the Western world. Although the rate of cure is high, about 20-35% of patients relapse, and about half of them eventually die of the disease or treatment-related late toxicities and secondary malignancies. Conceptually novel treatment strategies are thus needed, particularly for this category of patients. The malignant cells, called Hodgkin and Reed-Sternberg (HRS) cells, only make up 1-2% of the total tumor cellularity; the remaining mass comprising a mixed infiltrate population is thought to be recruited to the lymph node by HRS-driven pro-inflammatory signals. The origin of the pro-inflammatory signals remains elusive, and represents a critical gap in our knowledge of the pathogenesis of HL. We propose that a sub-population of HRS cells, which we call herein large RS cells, have characteristics of senescent cells, and, thus produce large amounts of inflammatory mediators (the so-called “senescence-associated secretory phenotype”, or SASP). Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. We show that the senescence markers the cell cycle inhibitors p16INK4a and p21Cip1 are expressed in large RS cells in all HL biopsies examined. Moreover, the large RS cells are negative for Ki-67 staining, demonstrating that these cells have ceased to proliferate. We also show that large, ‘RS-like’ cells in a HL-derived line, L428, stained for the characteristic senescence marker β-galactosidase (β-gal). Oxidative stress and chemotherapy increased the proportion of β-gal positive large RS cells. Furthermore, we show that the large RS cells secrete high levels of cytokines. We suggest that these senescent RS cells may be responsible, at least in part, for creating a pro-inflammatory microenvironment, promoting HL pathogenesis, and mediating chemo-resistance in relapsed disease. Understanding the pathways important for the establishment of senescence in HRS cells, as well gaining insight into targetable mechanisms for the eradication of these cells, will provide new therapeutic approaches for HL patients with recurrent or chemo-resistant disease.

Citation Format: Jacob Gopas, Udi Zurgil, Estee Zeev, Daniel Benharoch, Jodi Ezratty, Giora Shubinsky, Etta Livneh. Cellular senescence in Hodgkin's lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1263. doi:10.1158/1538-7445.AM2015-1263
Abstract 1268: Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration
Authors: Buchheit Cassandra , Angarola Brittany , Steiner Allison , Weigel Kelsey , Schafer Zachary
Published: August 2015

Inflammatory breast cancer (IBC) is a rare and highly invasive type of breast cancer, and patients diagnosed with IBC often face a very poor prognosis. IBC is characterized by the lack of primary tumor formation and the rapid accumulation of cancerous epithelial cells in the dermal lymphatic vessels. Given that normal epithelial cells require attachment to the extracellular matrix (ECM) for survival, a comprehensive examination of the molecular mechanisms underlying IBC cell survival in the lymphatic vessels is of paramount importance to our understanding of IBC pathogenesis. Due to the inherent invasive nature of IBC cells, we hypothesized that IBC cells could evade detachment-induced apoptosis (anoikis), and rely on tightly regulated intracellular signaling pathways to do so. Here we demonstrate that in contrast to normal mammary epithelial cells, IBC cells robustly survive in ECM-detached conditions. ErbB2 and EGFR knockdown in KPL-4 and SUM149 cells, respectively, causes decreased colony growth in soft agar and increased caspase activation following ECM detachment, suggesting overexpression/hyperactivation of these oncogenes is vital for IBC evasion of anoikis. Downstream of ErbB2 and EGFR, ERK/MAPK signaling was found to protect cells from anoikis by phosphorylating Bim-EL at serine 59. This phosphorylation of Bim-EL results in a physical interaction between Bim-EL, LC8, and Beclin-1 and prevents Bim-EL from localizing to the mitochondria and promoting cell death. Mutation of the serine 59 site results in increased cell death and a diminished capacity to form a protein complex with LC8 and Beclin-1. Bim-EL knockdown also rescued ECM-detachment-induced death in IBC cells, suggesting Bim-EL is necessary for the induction of anoikis in IBC cells. Interestingly, Bim-EL levels were found to be elevated in ECM-detached IBC cells and present in a significant percentage of IBC patient samples, despite their evasion of anoikis. Therefore, the sequestration of Bim-EL away the mitochondria provides a compelling rationale for how IBC cells survive in the presence of high Bim-EL levels. Furthermore, this mechanism provides a unique and novel opportunity for chemotherapeutic approaches aimed at freeing the already elevated Bim-EL levels from sequestration to specifically induce cell death in IBC cells.

Citation Format: Cassandra Buchheit, Brittany Angarola, Allison Steiner, Kelsey Weigel, Zachary Schafer. Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1268. doi:10.1158/1538-7445.AM2015-1268
Abstract 1269: Role of ribosomal protein, Rpl22 in regulating leukemic transformation
Authors: Patel Nehal Solanki , Kushnir Noa Greenberg , Rao Shuyun , Peri Suraj , Rhodes Michele , Nagahawatte Panduka , Mullighan Charles , Wiest David L.
Published: August 2015

Inactivation of ribosomal proteins (RP) is known to cause diseases called ribosomopathies, which are often associated with abnormal hematopoiesis and an increased risk for development of leukemia. Our laboratory has recently reported an important link between inactivation of one such ribosomal protein, Rpl22, and poor survival in T cell acute lymphoblastic leukemia (T-ALL) patients. Preliminary bioinformatic analysis shows that RPL22 is lost in ∼10% of pediatric T-ALL patients, who exhibit a more aggressive disease course. Deletion of the RPL22 locus is also enriched in the early T-cell precursor (ETP-ALL) subset of T-ALL patients, which exhibit a more aggressive disease course. Thus, RPL22 loss appears to be a marker for poor outcome in pediatric T-ALL patients. While RP inactivation has previously been linked to increased cancer risk, the mechanistic basis for this linkage remained unclear. We have recently demonstrated that inactivation of Rpl22 promotes leukemic transformation by activating NF-κB and inducing the stem cell gene, LIN28B. Nevertheless, the molecular link between Rpl22 inactivation and the induction of NF-κB activity remained unclear. We have now determined that inactivation of Rpl22 activates NF-κB signaling by exacerbating ER stress responses. Indeed, Rpl22 loss results in hyperactivation of the PERK-EIF2a-ATF4 stress pathway, which is known to activate NF-κB. We have demonstrated that knockdown of PERK using shRNA abrogates the activation of NF-κB in Rpl22 mutant cells and returns Lin28B expression levels to baseline. This reduction in Lin28B eliminates the predisposition to transformation exhibited by Rpl22 mutant cells, as evidenced by decreased formation of colonies in soft agar. Taken together, these data suggest that RPL22 inactivation may serve as a negative prognostic indicator in T-ALL and that pharmacologic targeting of ER stress pathways may represent a novel therapeutic alternative in this molecularly-defined subclass of T-ALL.

Citation Format: Nehal Solanki Patel, Noa Greenberg Kushnir, Shuyun Rao, Suraj Peri, Michele Rhodes, Panduka Nagahawatte, Charles Mullighan, David L. Wiest. Role of ribosomal protein, Rpl22 in regulating leukemic transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1269. doi:10.1158/1538-7445.AM2015-1269
Abstract 1271: MDM2 small molecule inhibitors synergize with chemotherapeutics to attenuate senescence-driven inflammatory secretion
Authors: Schaum Nicholas , Almirah Fatouma , Scott Gary , Benz Christopher , Campisi Judith , Davalos Albert R.
Published: August 2015

An attractive therapeutic approach for treatment of cancers with wild-type (wt) p53 is the emergent class of small molecule MDM2 inhibitors, including the imidazoline nutlin-3 and more potent spiro-oxindole MI-63. Both compounds rapidly increase intracellular wt p53 levels and induce apoptosis or a senescence growth arrest, depending on the target cell. A critical question in the clinical development of MDM2 inhibitors is their potential for normal tissue toxicity owing to their ability to cause p53-dependent apoptosis or cellular senescence. Recent work suggest senescent cells exhibit a senescence-associated secretory phenotype (SASP) that can stimulate cancer progression. We critically examined the effect of MDM2 small molecule inhibitors Nutlin-3a and MI-63 on non-senescent and senescent cells. Both compounds caused normal cells to undergo a transient growth arrest. Continuous incubation with MDM2 small molecule inhibitors caused normal cells to exhibit some hallmarks of senescence (arrested growth, SA-β-Gal expression and secretion of High Mobility Group Box 1 protein), but the cells resumed growth upon removal of the MDM2 inhibitors. Senescent cells cultured with MDM2 inhibitors failed to resume growth upon removal of the compounds, as expected. However, their SASP was markedly dampened, as determined by multiplex ELISA assays. Since NF-κB and IL-1α are major drivers of the SASP, we examined their status in senescent cells cultured with MDMD2 inhibitors. The small molecule MDM2 inhibitors significantly reduced NF-κB activity and IL-1α mRNA expression in senescent cells. Our data suggest using combinatorial therapy of chemotherapeutics or irradiation and MDMD2 small molecule inhibitors may provide a potent therapeutic strategy to promote apoptosis of p53 positive tumor cells after radio- or chemo-therapy and attenuate the potentially deleterious effects of the SASP.

Citation Format: Nicholas Schaum, Fatouma Almirah, Gary Scott, Christopher Benz, Judith Campisi, Albert R. Davalos. MDM2 small molecule inhibitors synergize with chemotherapeutics to attenuate senescence-driven inflammatory secretion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1271. doi:10.1158/1538-7445.AM2015-1271
Abstract 1273: Relation between tumor-associated macrophage (TAM) subsets and CD47 expression on squamous cell carcinoma of the head and neck (SCCHN) in tumor microenvironment
Authors: Sakakura Koichi , Takahashi Hideyuki , Chikamatsu Kazuaki
Published: August 2015

Introduction: In general, TAM is classified into M1 producing Th1 cytokines and M2 secreting IL-10. Numerous reports have demonstrated that M2 infiltration to tumor microenvironment associates with local immunosuppression and proliferation of cancer cells. In this study, we investigated phagocytic potentials of M1 and M2 in SCCHN in relation to the expression of CD47 molecule, which is known as “Don't eat me” signal against macrophage (Mϕ).

Material and Methods: CD68 (pan-Mϕ marker) positive CD163 (M2 marker) negative M1 (derived with GM-CSF and IFN-γ) and CD68+CD163+M2 (derived with M-CSF and IL-10) were induced from positively selected CD14+ cells in PBMC from a healthy donor. Phagocytosis of CD47+/CD47-SCCHN cell lines labeled with CFSE by M1/M2 were assessed using multi-color flow cytometry. Suppressions of the phagocytosis by neutralizing anti-CD47 antibody were also evaluated. Furthermore, we examined CD68, CD163 and CD47 expressions immunohistochemically in 74 cases of oral squamous cell carcinoma, and relation between Mϕ counts or CD47 expression on cancer cells and clinicopathological parameters or prognoses.

Results: M1 derived from PBMC showed higher phagocytic potential comparing to M2. Phagocytosis of M1 was dose-dependently inhibited by anti-CD47 neutralizing antibody, however M2 phagocytosis was not suppressed. In immunohistochemical analysis, while CD47 was expressed in almost half cases of oral cancer, CD47 expression did not correlate to the numbers of total Mϕ nor M2. Patients with high CD47 expression on cancer cells, increasing number of total Mϕ or M2 showed significantly shorter overall survival.

Conclusion: Only M1 showed CD47-dependent phagocytosis of CD47-expressed cancer cells in vitro. While CD47 expression on patient samples did not correlate with number of total Mϕ or M2, each of them independently showed significant association with prognosis. Various mechanisms were presumed between CD47 expression on SCCHN cells and tumor-infiltrating Mϕ subsets.

Citation Format: Koichi Sakakura, Hideyuki Takahashi, Kazuaki Chikamatsu. Relation between tumor-associated macrophage (TAM) subsets and CD47 expression on squamous cell carcinoma of the head and neck (SCCHN) in tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1273. doi:10.1158/1538-7445.AM2015-1273
Abstract 128: Tyrosine 823 in the activation loop of c-Kit regulates the transforming capacity of the oncogenic mutant D816V and its sensitivity to kinase inhibitors
Authors: Agarwal Shruti , Kazi Julhash U. , Mohlin Sofie , Påhlman Sven , Rönnstrand Lars
Published: August 2015

Oncogenic mutations in c-Kit have been shown to lead to ligand-independent receptor activation and contribute to transformation. A substitution of an aspartate for a valine at position 816 (D816V) is one of the most commonly found oncogenic c-Kit mutation and is found in more than 90% of cases of mastocytosis and in a subgroup of germ cell tumors, core-binding factor acute myeloid leukemia and mucosal melanomas. Previous studies have shown that the D816V mutation causes a structural change in the activation loop resulting in weaker binding of the activation loop to the juxtamembrane domain, leading to a release of the negative constraint it poses on kinase activity. Here we have investigated the role of Y823 in the activation loop of c-Kit and its role in oncogenic transformation by c-Kit/D816V. Although dispensable for the kinase activity of c-Kit/D816V, the presence of Y823 was shown to be crucial for cell proliferation and survival. Furthermore, mutation of Y823 selectively down-regulated the Ras/Erk and Akt pathways. It also led to decreased phosphorylation of STAT5 and reduced the transforming capacity of D816V/c-Kit in vitro. The sensitivity to kinase inhibitors, such as dasatinib and nilotinib, was decreased in cells expressing c-Kit/Y823F/D816V compared to cells c-Kit/D816V. Mice injected with cells expressing c-Kit/D816V/Y823F displayed significantly reduced tumor size as well as tumor weight compared to controls. Finally, microarray array analysis, comparing c-Kit/Y823F/D816V cells with cells expressing c-Kit/D816V, demonstrate that mutation of Y823 causes up-regulation of pro-apoptotic genes whereas genes of survival pathways are down-regulated. Thus, phosphorylation of Y823 is not necessary for kinase activation, but essential for the transforming ability of the c-Kit/D816V mutant.

Citation Format: Shruti Agarwal, Julhash U. Kazi, Sofie Mohlin, Sven Påhlman, Lars Rönnstrand. Tyrosine 823 in the activation loop of c-Kit regulates the transforming capacity of the oncogenic mutant D816V and its sensitivity to kinase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 128. doi:10.1158/1538-7445.AM2015-128
Abstract 1283: In situ activity of tumor specific CTL is a prognostic factor in colorectal cancer
Authors: Stamova Slava , Reissfelder Christoph , Gossmann Christina , Braun Marion , Bonertz Andreas , Walliczek Ute , Koch Moritz , Benner Axel , Jäger Dirk , Halama Niels , Khazaie Khashayarsha , Weitz Jürgen , Beckhove Philipp
Published: August 2015

The extent of T cell infiltrates in the primary tumor is one of the most important prognostic factors in colorectal cancer (CRC). Still, a putative causal role of T cell infiltrates for prolonged patient survival remains unclear. To answer this question, we studied the presence and functional activity of tumor infiltrating lymphocytes (TIL) together with their specificity in CRC patients and correlated the obtained results to long term survival.

We have utilized intracytoplasmic cytokine staining, conjointly with human leukocyte antigen (HLA)- multimers loaded with peptides derived from tumor antigen (TA), as well as antigen specific cytokine secretion assays, to demonstrate that tumor necrosis factor alpha (TNFα) expression characterizes a population of TA specific, in situ active cytotoxic T cells. TNFα was increased in CRC tumors only when patients develop spontaneous systemic T cell responses, as elucidated by IFN-γ Elispot, and this increase correlated with the number of TNFα expressing tumor infiltrating T cells. Based on these observations we concluded that intratumoral TNFα can be a direct indicator of the in situ functionality of tumor specific effector T cells in CRC.

In accordance, intratumoral TNFα concentration was an independent long term prognostic factor in a retrospective multivariate analysis of a cohort of 102 CRC patients involving various other immune parameters, such as CD4+, CD8+ T cell infiltration, presence of regulatory T cells or mast cells.

In conclusion, our findings provide evidence for a causal relationship between T cell infiltrates and improved survival in CRC patients as the prognostic impact of T cell infiltrates depends on small subpopulations of in situ active TA specific T cells. Furthermore TNFα, as an indicator for the functionality of TIL, might be better and more accurate prognostic parameter in CRC than mere T cell infiltration.

Citation Format: Slava Stamova, Christoph Reissfelder, Christina Gossmann, Marion Braun, Andreas Bonertz, Ute Walliczek, Moritz Koch, Axel Benner, Dirk Jäger, Niels Halama, Khashayarsha Khazaie, Jürgen Weitz, Philipp Beckhove. In situ activity of tumor specific CTL is a prognostic factor in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1283. doi:10.1158/1538-7445.AM2015-1283
Abstract 1288: Does an increased underlying immune/inflammatory response in healthy females put them at a higher risk for thyroid cancer
Authors: Singh Karnika , Jones Melanie , Moscatello Augustine , Shin Edward , Tiwari Raj , Geliebter Jan
Published: August 2015

The SEER (Surveillance, Epidemiology, and End Results Program) and American Cancer Society data indicate that the incidence of papillary thyroid cancer is ∼ 3 times greater in females, as compared to males. The American Cancer Society estimates that out of a total of ∼62,980 new thyroid cancer cases in 2014, approximately 47,790 of these will be women. There is strong evidence that supports the role of estrogen in the pathogenesis of thyroid cancer. Estrogen also plays a critical role in the inflammatory process. For example, mouse models have demonstrated that in vivo exposure to estrogen promotes an inflammatory response, including the enhanced secretion of Th-1 related cytokines (IFNγ, IL-12, IL-1β) and inflammatory chemokines (MCP-1 and MCP-5). Studies in rats and mouse models have shown that females exhibit stronger humoral and cellular immune responses as compared to males. As the association between inflammation and cancer is widely accepted, we hypothesize that an underlying immune and inflammatory response in the normal female thyroid puts it at a higher risk of developing cancer. The Cancer Genome Atlas (TCGA) provides a means to assess gender differences at the molecular level. Using the SUBIO platform version 1.17 (SUBIO Inc., Amami Island, Japan), we performed bioinformatics and statistical analyses on the Level 3 RNA-Seq data available in the TCGA Data Portal. Differential gene expression analysis of 39 normal thyroid samples (26 females and 13 males) revealed that out of the 20,531 genes analyzed, 73 genes exhibited >2 fold increased expression in females vs. males (p-value, 0.05). Of these 73 genes, 10 genes were involved in immune response and inflammation. We are currently analyzing expression levels of these genes on the RNA and protein levels. Such analysis will help us to elucidate a possible link between the gender disparity in thyroid cancer and inflammation.

Citation Format: Karnika Singh, Melanie Jones, Augustine Moscatello, Edward Shin, Raj Tiwari, Jan Geliebter. Does an increased underlying immune/inflammatory response in healthy females put them at a higher risk for thyroid cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1288. doi:10.1158/1538-7445.AM2015-1288
Abstract 129: RAD17 loss of function is synthetically lethal with the checkpoint kinase inhibitors AZD7762 or MK-1775
Authors: Shen John Paul , Srivas Rohith , Bojorquez-Gomez Ana , Licon Katherine , Sivaganesh Vignesh , Xu Jia L. , Yeerna Huwate , Gross Andrew , Li Jian Feng , Sobol Robert , Ideker Trey
Published: August 2015

Synthetic lethal interactions are a type of genetic interaction in which the simultaneous loss of function of two genes in combination results in cell death. Recently, there has been much interest in the discovery of drugs that are selectively toxic to cancer cells by targeting proteins that form synthetic lethal interactions with tumor specific mutations. He we have identified that RAD17 loss of function is synthetically lethal with AZD7762, an inhibitor of Chk1 and Chk2, and with MK-1775, an inhibitor of Wee1. HeLa or LN428 glioblastoma cells were selectively responsive to AZD7762 or MK-1775 following shRNA-mediated depletion of RAD17. Cells with dual knockdown of CHEK1 and CHEK2 or knockdown of WEE1 were also sensitized to RAD17 knockdown. At baseline, cells with RAD17 knockdown had greater expression of the DNA damage marker H2AX, an effect that was magnified by either chemical inhibition or siRNA-mediated knockdown of CHEK1/2 or WEE1. Accumulation of H2AX was observed primarily in S and G2 phases, RAD17 loss did not have a significant effect on cell cycle progression. The combination of AZD7762 and MK-1775 resulted in synergistic toxicity in RAD17 knockdown but not control cells. Collectively, these results demonstrate that RAD17 loss of function sensitizes cells to inhibition of S and G2/M checkpoint kinases, resulting in greater accumulation of DNA damage and ultimately cell death. We suggest that AZD7762 and MK-1775 may have greater clinical efficacy in tumors with RAD17 mutation or deletion.

Citation Format: John Paul Shen, Rohith Srivas, Ana Bojorquez-Gomez, Katherine Licon, Vignesh Sivaganesh, Jia L. Xu, Huwate Yeerna, Andrew Gross, Jian Feng Li, Robert Sobol, Trey Ideker. RAD17 loss of function is synthetically lethal with the checkpoint kinase inhibitors AZD7762 or MK-1775. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 129. doi:10.1158/1538-7445.AM2015-129