bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5424: Metformin increases PDH and suppresses HIF1A under hypoxic conditions and induces cell death in oral squamous cell carcinoma
Authors: Guimarães Talita , Farias Lucyana , Fraga Carlos , Paula Alfredo De , Santos Sergio , Gomez Ricardo , Guimaraes Andre
Published: July 2017

Background: Metformin is a biguanide, belonging to the oral hypoglycemic agents and is a widely used in the treatment of type 2 diabetes. Evidence indicate that Metformin inhibits cell proliferation in several human cancers and inhibits the Warburg phenomenon in tumor cells.

Objective: The present study aims to explore the effects of Metformin in hypoxic conditions. Specifically, we focused on pyruvate dehydrogenase (PDH), (hypoxia-inducible factor 1α) HIF-1α levels and the oral squamous cell carcinoma (OSCC) cell phenotype. Additionally, we also investigated a theoretical consequence of Metformin treatment.

Methods: PDH levels in patients with OSCC and oral dysplasia were evaluated. Metformin was administered in vitro to test the effect of Metformin under hypoxic conditions. The results were complemented by Bioinformatics analyses.

Results: Low PDH levels were observed in OSCC, and Metformin promotes an increase in PDH levels in hypoxic conditions. Metformin also reduced HIF-1α mRNA and protein levels. Metformin demonstrated antiproliferative effects, inhibited migration, increased the number of apoptotic cells and increased the transcription of caspase 3.

Conclusions: In conclusion, our current findings show that Metformin reduces HIF-1α gene expression and increases PDH expression. Metformin inhibits cell proliferation and migration in the OSCC cell line model. Additionally, Metformin enhances the number of apoptotic cells and caspase 3 levels. Interestingly enough, Metformin did not increase the mutant p53 levels under hypoxic conditions.

Note: This abstract was not presented at the meeting.

Citation Format: Talita Guimarães, Lucyana Farias, Carlos Fraga, Alfredo De Paula, Sergio Santos, Ricardo Gomez, Andre Guimaraes. Metformin increases PDH and suppresses HIF1A under hypoxic conditions and induces cell death in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5424. doi:10.1158/1538-7445.AM2017-5424
Abstract 5430: GAPDH loss in a tumorigenic human glioblastoma cell line
Authors: Gaertner Zachary , Lu Yi , Kim Jinsuh , Mohapatra Gayatry , Mehta Ankit , Lakka Sanjani , Engelhard Herbert
Published: July 2017

Background: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a ubiquitous, multifunctional 37 kDa enzyme best known for catalyzing the sixth step in glycolysis. GAPDH expression is so consistent that it has been granted “housekeeping” status, and is used as a standard (along with β-actin) for normalizing Western blots. While increased GAPDH expression has been described in hypoxia, diabetes, and some cancers, decreased expression is rare. Here we describe a new human glioblastoma cell line that exhibits GAPDH loss, while maintaining a high degree of malignancy.

Methods: Primary cultures of human glioblastoma cells were initiated and maintained in DMEM with 10% FBS. This particular cell line (designated E297) grew readily, and was passaged more than 40 times. E297 cells were confirmed to be negative for HIV, HTLV, hepatitis B and C, CMV, and mycoplasma contamination. Cells in exponential growth were stained for routine markers, and studied by DNA flow cytometry. GAPDH expression was studied by Western blot analysis using polyclonal rabbit anti-human GAPDH IgG (Abcam, Rosemont, IL), with HeLa, U138, U87 and U251 cells (as well as β-actin staining) as positive controls. For intracranial implantation into Wistar-Furth rats (male, 10-11 weeks), cells were suspended at 20 x 106 cells/ml. Using aseptic technique, rats were anesthetized, and placed into a stereotactic frame. A burr hole was drilled 3 mm right of the bregma and 25 µl of suspension injected (5 mm depth) over 10 min. by Hamilton syringe. Rats were studied by MRI and euthanized when symptomatic.

Results: E297 cells have glial/epithelioid morphology, with a doubling time of 24 + 2 hours in logarithmic growth phase, with a single DNA subpopulation with 28% S phase. They stain positively for GFAP, vimentin, bFGF, c-myc and p53, but fail to express GAPDH. 10/10 animals implanted intracerebrally developed tumors, becoming symptomatic and requiring sacrifice at 25 days (mean). Loss of GAPDH expression was confirmed by Western blot analysis.

Conclusions: The E297 human glioblastoma cell line is highly aggressive and proliferates rapidly. It is tumorigenic in nonimmunosuppressed rats but lacks GAPDH protein. We conclude that GAPDH expression is not essential for glioblastoma cell proliferation under routine culture conditions, and care needs to be taken when using GAPDH as a normalization standard for Western blots of cancer cells.

Citation Format: Zachary Gaertner, Yi Lu, Jinsuh Kim, Gayatry Mohapatra, Ankit Mehta, Sanjani Lakka, Herbert Engelhard. GAPDH loss in a tumorigenic human glioblastoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5430. doi:10.1158/1538-7445.AM2017-5430
Abstract 5441: Circulating exosomal microRNA-203 and microRNA-373 as non-invasive biomarkers for predicting prognosis and metastasis in human hepatocellular carcinoma
Authors: Kim Gyeonghwa , Lee Eunhye , Jang Se Young , Tak Won Young , Kweon Young Oh , Park Soo Young , Hur Keun
Published: July 2017

Background and Aim Hepatocellular carcinoma (HCC) is the fifth most common cancer and the most leading cause of cancer-related death worldwide. Although considerable progress has been made in treatment of HCC, early detection is still highly considered the key to improved survival. Thus, for the earlier diagnosis and accurate prediction of HCC, identification of non-invasive molecular biomarkers for HCC patients is an imperative need. Recently, cancer cell-derived extracellular vesicles (EVs) have been known to contain various intracellular biomolecules including microRNAs (miRNAs). The aim of this study was to evaluate whether exosomal miRNAs can serve as a serum-based biomarker in HCC.

Materials and Methods We isolated exosome from serum samples from HCC patients as well as normal healthy controls using ultracentrifugation, and it was confirmed by expression of exosome markers (CD9, CD63, ALIX, and TSG101) based on immunoblotting. Next, the expression of 6 miRNAs (miRNA-24, -130a, -182, -203, -373, and -423) was analyzed in the exosome samples. We also investigated expression status of the 6 miRNAs in matched HCC tissues and corresponding normal liver tissues. MiRNAs expression was determined by quantitative real-time PCR (qRT-PCR), and miRNAs expression was normalized relative to cel-miR-39 and RNU6B expression for serum and tissue samples, respectively.

Results We successfully purified exosome from serum clinical samples and detected miRNAs in the exosome. We observed that a subset of miRNAs from serum exosome, including miRNA-24, -130a, -182, -203, and -373 were enhanced in HCC patients than normal healthy controls. In further comparison between early stage and advanced stage of HCC patients, serum exosomal miRNA-203 (P<0.05) and miRNA-373 (P<0.05) were significantly up-regulated in advanced HCC patients. While no significant changes in the expression of other serum exosomal miRNAs (miRNA-24, -130a, -182, and -423) according to HCC progression. More interestingly, high serum exosomal miRNA-203 and miRNA-373 was associated with HCC progression (P<0.01) as well as prognosis (P<0.05) of HCC patients.

Conclusions We provided the novel evidence for usefulness of serum circulating exosomal miR-203 and miR-373 expressions as strong potential biomarkers for predicting prognosis and metastasis of HCC patients.

Citation Format: Gyeonghwa Kim, Eunhye Lee, Se Young Jang, Won Young Tak, Young Oh Kweon, Soo Young Park, Keun Hur. Circulating exosomal microRNA-203 and microRNA-373 as non-invasive biomarkers for predicting prognosis and metastasis in human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5441. doi:10.1158/1538-7445.AM2017-5441
Abstract 5446: Tumor-derived miR-200c and 141 contribute to high levels of plasma microRNA-200c and 141 through exosomes
Authors: Wang Lizhong , Liu Runhua , Gao Song , Wang Yicun , Wang Meng , Li Zhi
Published: July 2017

Circulating microRNAs (miRs) have potential as cancer biomarkers, but their regulatory mechanisms remain elusive. Here we used a breast cancer model (Foxp3 heterozygous Scurfy mutant female mice, Foxp3sf/+) for identification of circulating miR biomarkers, and the formation and regulation of these miRs were investigated. Aging Foxp3sf/+ female mice developed spontaneous breast cancers and lung metastases. Levels of miR-200c and 141 were lower in the Foxp3sf/+ breast cancer cells than in normal breast epithelial cells, but plasma levels of miR-200c and 141 in the Foxp3sf/+ mice increased during tumor progression, especially during metastasis to the lung. Likewise, 259 participants, including patients with breast cancer or benign breast tumors, members of breast cancer families, and matched normal healthy controls, were assessed for circulating levels of candidate miRs. The results showed that: 1) the levels of miR-200c and 141 were lower in the FOXP3low breast cancer cells relative to those with FOXP3high, especially for late-stage and metastatic cancer cells; 2) the levels of miR-200c and 141 were higher in plasma of patients with metastatic breast cancer than in plasma of those with localized breast cancer, with benign breast tumors, with a family history of breast cancer, or normal healthy controls. Furthermore, in Foxp3sf/+ mice, plasma miR-200c and 141 were released from tumor cells through exosomes. Thus, plasma levels of miR-200c and 141 are potential biomarkers for early detection of metastases. MiR-200c and 141 appear to be released from breast cancer cells, through exosomes, into the circulation.

Citation Format: Lizhong Wang, Runhua Liu, Song Gao, Yicun Wang, Meng Wang, Zhi Li. Tumor-derived miR-200c and 141 contribute to high levels of plasma microRNA-200c and 141 through exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5446. doi:10.1158/1538-7445.AM2017-5446
Abstract 545: Prognostic alternative mRNA splicing signature in non-small cell lung cancer
Authors: Li Yuan , Sun Nan , Lu Zhiliang , Sun Shouguo , Huang Jianbing , Chen Zhaoli , He Jie
Published: July 2017

Alternative splicing provides a major mechanism to generate protein diversity. Increasing evidence suggests a link of dysregulation of splicing associated with cancer. However, alternative splicing in non-small cell lung cancer (NSCLC) is largely unstudied. In this study, seven types of alternative splicing were profiled in 491 lung adenocarcinoma (LUAD) and 471 lung squamous cell carcinoma (LUSC) patients in TCGA using RNA sequencing data. Prognostic predictors for LUAD, LUSC and merged NSCLC patients were built by integrated survival analysis. Gene network and splicing correlation network analyses were also conducted. Overall, we detected 45,062 and 50,732 alternative splicing events in LUAD and LUSC, respectively, almost half of them were exon skipping events. A total of 3,691 and 2,403 alternative splicing events were significantly associated with patient survival in LUAD and LUSC, respectively. Gene network analysis uncovered important hub genes or nodes such as EGFR, CD44, PIK3C3, RRAS2, MAPKAP1 and FGFR2. The area under the curve (AUC) of the receiver-operator characteristic (ROC) curve for prognostic predictors in LUAD and LUSC were 0.960 and 0.940 at 2000 days of overall survival. Most relevant for clinical application, we also built the prognostic predictor for NSCLC patients with high performance. The AUC of ROC curve in NSCLC was 0.817 which were also over 0.8 in LUAD and LUSC, separately. Moreover, expression of 49 and 9 survival associated splicing factors were significantly correlated with 75 survival associated alternative splicing events in LUAD and LUSC, respectively. Interestingly, splicing correlation networks uncovered that “oncogenic“ splicing factors were up-regulated in LUAD while “tumor suppressor” splicing factors were down-regulated in LUSC. Survival associated splice factors might have opposite roles in the regulation of RNA splicing in LUAD and LUSC. In conclusion, we created prognostic predictors based on alternative splicing events with high performances for risk stratification in NSCLC patients and uncovered interesting splicing networks in LUAD and LUSC which could be underlying mechanisms.

Citation Format: Yuan Li, Nan Sun, Zhiliang Lu, Shouguo Sun, Jianbing Huang, Zhaoli Chen, Jie He. Prognostic alternative mRNA splicing signature in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 545. doi:10.1158/1538-7445.AM2017-545
Abstract 5450: Urinary microRNA as diagnostic and prognostic biomarkers for prostate cancer
Authors: Fredsøe Jacob , Rasmussen Anni K. , Thomsen Anni R. , Mouritzen Peter , Høyer Søren , Borre Michael , Ørntoft Torben F. , Sørensen Karina D.
Published: July 2017

Widespread use of Prostate Specific Antigen (PSA) testing for prostate cancer (PC) detection has led to extensive overdiagnosis and overtreatment. Thus, we aimed to train and validate urine-based microRNA (miRNA) biomarkers that may assist in the diagnosis and prognosis of PC. To this end, we profiled the expression levels of 92 miRNAs by RT-qPCR in exosome-enriched cell-free urine samples from 20 patients with benign prostatic hyperplasia (BPH) and 188 patients with clinically localized PC (cohort 1). The diagnostic potential of individual miRNAs and multi-miRNA ratio models were assessed by receiver operating characteristic (ROC) curve analysis. Prognostic potential was evaluated by Kaplan-Meier, uni- and multivariate Cox regression analyses using biochemical recurrence (BCR) after radical prostatectomy (RP) as endpoint. Our findings were validated in an independent cohort of 20 BPH patients and 197 patients with clinically localized PC (cohort 2). We identified and validated several deregulated miRNAs in urine samples from PC patients. In addition, we trained a novel diagnostic 3-miRNA model that distinguished BPH and PC patients with an area under the curve (AUC) of 0.95 in cohort 1, and which was successfully validated in cohort 2 (AUC=0.89). Furthermore, we trained a novel prognostic 3-miRNA model that predicted time to BCR after RP independently of routine clinicopathological parameters in cohort 1, and was successfully validated in cohort 2.

Citation Format: Jacob Fredsøe, Anni K. Rasmussen, Anni R. Thomsen, Peter Mouritzen, Søren Høyer, Michael Borre, Torben F. Ørntoft, Karina D. Sørensen. Urinary microRNA as diagnostic and prognostic biomarkers for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5450. doi:10.1158/1538-7445.AM2017-5450
Abstract 546: Supporting neoantigen identification for personalized cancer vaccines trough analytical validation of an augmented content enhanced (ACE) transcriptome
Authors: Yen Jennifer L. , Boyle Sean , Alla Ravi , Harris Jason , Lefterova Martina , Chen Richard
Published: July 2017

The identification of neoantigens has become a critical step in the development of neoantigen-based personalized cancer vaccines and other immunotherapy applications. Since neoantigens can be generated from tumor specific mutations in any expressed gene, the first step in neoantigen identification typically involves deep sequencing of the tumor exome and transcriptome combined with exome sequencing of the matched normal. As personalized vaccines enter clinical trials, there is a growing need for strong analytical validation of these platforms.

To address this, we have developed the ACE Exome (~200X) and Transcriptome platforms for neoantigen identification which harness an augmented exome approach specifically designed to increase sensitivity for neoantigens in low complexity, difficult to sequence regions. To enable this platform for neoantigen based personalized cancer vaccines, we have performed a validation of both our ACE Exome (tumor and normal) and ACE transcriptome platforms for detecting DNA-based SNVs and Indels, as well as for RNA based small variant and fusion calls, variant types that are especially important for neoantigen identification. In this abstract, we describe the ACE Transcriptome validation.

To validate the ACE Transcriptome, we assessed analytical sensitivity (AS) and positive predictive accuracy (PPA). AS was calculated from a reference set of 894 SNVs and 19 indels across 11 tumor cancer cell lines with matched normals. The reference set was constructed based on previously observed variants in CCLE, COSMIC or had been validated by Sanger sequencing. PPA and limit of detection (LOD) were calculated in a series of tumor-normal dilutions representing allele frequencies ranging from 10% to 100%. For fusions, we further selected an additional 10 cell lines to test the detection of 16 previously characterized fusion events including several clinically actionable fusions (ALK and BCR-ABL1).

We report an analytical sensitivity for SNVs of >99%, and a PPA of >96% for small variants with >=10% minor allele frequency (MAF). For fusion events, we report an analytical sensitivity of >99%, with the detection of all 16 fusion events supported by at least 5 reads.

We demonstrate that our ACE Transcriptome assay and RNA cancer pipeline is a highly sensitive and robust platform for detecting small variants and fusions in the RNA.

Citation Format: Jennifer L. Yen, Sean Boyle, Ravi Alla, Jason Harris, Martina Lefterova, Richard Chen. Supporting neoantigen identification for personalized cancer vaccines trough analytical validation of an augmented content enhanced (ACE) transcriptome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 546. doi:10.1158/1538-7445.AM2017-546
Abstract 5461: Nuclear localization of the caspase 3 cleaved form of p73 in anoikis
Authors: Alsafadi Samar , Tourpin Sophie , Salome-Desnoulez Sophie , Bessoltane Nadia , Andre Fabrice , Ahomadegbe Jean Charles
Published: July 2017

The transcription factor p73 is a homologue of p53 that can be expressed as pro- or anti-apoptotic isoforms. Unlike p53, p73 is rarely mutated or lost in cancers and it is found to replace defective p53 inducing apoptosis. Here, we investigated the p73 involvement in anoikis, a type of apoptosis caused by inadequate cell-matrix interactions. Breast cancer cell lines with different p53 status were treated with doxorubicin or docetaxel and cells detached from the extracellular matrix were analyzed. We demonstrate for the first time that doxorubicin-induced cell detachment is associated with p73 cleavage and caspase activation, independently of the p53 status. However, we did not detect p73 cleavage or caspase activation in detached cells under docetaxel treatment. Overexpressing the apoptotic isoform of p73 led to cell detachment associated with p73 cleavage and caspase activation. Interestingly, p73 cleaved forms localize to the nucleus during the late phase of cell death indicating an increase in the transcriptional activity. Our study suggests that the cleavage of p73 on specific sites may release its pro-apoptotic function and contribute to cell death.

Note: This abstract was not presented at the meeting.

Citation Format: Samar Alsafadi, Sophie Tourpin, Sophie Salome-Desnoulez, Nadia Bessoltane, Fabrice Andre, Jean Charles Ahomadegbe. Nuclear localization of the caspase 3 cleaved form of p73 in anoikis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5461. doi:10.1158/1538-7445.AM2017-5461
Abstract 5462: GTP cyclohydrolase in brain tumor stem cells is implicated in glioblastoma growth
Authors: Tran Anh N. , Walker Kiera , Harisson David , Chen Wei , Mobley James , Hocevar Lauren , Hackney James R. , Sedaka Randee S. , Pollock Jennifer S. , Cooper Sara J. , Gillespie George Y. , Hjelmeland Anita B.
Published: July 2017

Glioblastomas (GBMs) are the most common primary brain tumors in adults and one of the most aggressive cancers with high rates of recurrence and therapeutic resistance. In GBMs, subpopulations of highly tumorigenic cells called brain tumor initiating cells (BTICs) have been characterized by their unique capacity to promote tumor maintenance, therapeutic resistance, and angiogenesis. BTIC maintenance is known to be regulated by reactive oxygen and nitrogen species. GTP cyclohydrolase, or GCH1, is a critical molecule regulating reactive species levels. We found that expression of GCH1 RNA and protein were upregulated in BTICs in comparison to non-BTICs. Overexpression of GCH1 in glioma cells increased cell growth in vitro and increased tumor formation and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 depletion with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal orthotopic models. Furthermore, genetic modulation of GCH1 led to altered ROS levels in GBM xenolines. In silico analyses demonstrate that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in BTICs promotes tumor maintenance and is a key regulator of reactive oxygen species in glioblastoma.

Citation Format: Anh N. Tran, Kiera Walker, David Harisson, Wei Chen, James Mobley, Lauren Hocevar, James R. Hackney, Randee S. Sedaka, Jennifer S. Pollock, Sara J. Cooper, George Y. Gillespie, Anita B. Hjelmeland. GTP cyclohydrolase in brain tumor stem cells is implicated in glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5462. doi:10.1158/1538-7445.AM2017-5462
Abstract 5472: Yeast Bax Inhibitor (BXI1) is involved in redox and calcium homeostasis of the ER in S. cerevisiae
Authors: Eagan David , Alisch Joseph , McDonough Liam , Benko Savannah , Jacob Walter , Ritzer Lukas , Mullin James , O'Reilly John , Austriaco Nicanor
Published: July 2017

In mammals, the Bax inhibitor (TMBIM) family of proteins has cytoprotective properties that are most evident in paradigms of endoplasmic reticulum (ER) stress. The canonical gene of the family called BI-1 has been linked to several human cancers. The crystal structure of a prokaryotic member of the family, BsYetJ, has revealed that the Bax inhibitor proteins are pH sensitive calcium leaks. Our published studies have shown that the yeast Bax Inhibitor, Bxi1p, is localized to the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by endoplasmic reticulum (ER) stress. We now show that cells lacking BXI1 have an altered redox microenvironment in the ER: The ER of Δbxi1 cells exposed to dithiothreitol (DTT) and tunicamycin, two drugs known to induce the unfolded protein response (UPR), become more reduced than their wildtype counterparts. Moreover, we also report that cells lacking BXI1 accumulate more calcium in the ER than wildtype cells. Together, our data suggests that Bxi1p is involved in regulating the ER microenvironment. Other members of the TMBIM family may have the same role in normal and cancer cells. [Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]

Note: This abstract was not presented at the meeting.

Citation Format: David Eagan, Joseph Alisch, Liam McDonough, Savannah Benko, Walter Jacob, Lukas Ritzer, James Mullin, John O'Reilly, Nicanor Austriaco. Yeast Bax Inhibitor (BXI1) is involved in redox and calcium homeostasis of the ER in S. cerevisiae [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5472. doi:10.1158/1538-7445.AM2017-5472
Abstract 5476: CIP2A can be a therapeutic target of rapamycin in radioresistant head and neck cancer with P53 mutation
Authors: Kim Song Hee , Lee Won hyeok , Lee Myung Jin , Chang Hyo Won , Kim Ji Won , Kim Mi Ra , Park Jung Je , Han Myung Woul , Kim Sang Yoon
Published: July 2017

The presence of p53 mutation was associated with poor survival in various tumors. The involvement of p53 in apoptosis and cell-cycle control makes it a plausible biomarker of prognosis . Classifying TP53 mutations in HNSCC described disruptive TP53 mutation in either the L2 or L3 loop of the DNA binding domain, resulting in polarity change within the protein, or stop cordon. Disruptive TP53 mutation in HNSCC tumors predicts for locoregional recurrence, due to increased radioresistance via the inhibition of senescence. Senescence induction contributes to cancer therapy responses and is crucial for p53-mediated tumor suppression. However, whether p53 inactivation actively suppresses senescence induction has been unclear. Here, we found that overexpression CIP2A (cancerous inhibitor of protein phosphatase 2A) positively correlated with presence of p53 mutation in head and neck cancer and mediates radioresistance through suppression of radiation induced senescence. And we demonstrated that rapamycin could induce senescence via CIP2A downregulation and increase radiosensitivity in p53 disruptive mutation cell line. This is the first investigation to analyze the role of CI2A in resistance to the radiotherapy of head and neck cancer. As a consequence, a greater understanding of radioresistance mechanisms through our results in head and neck cancer with p53 mutation will enable the rational design of combination regimens and sequential treatment algorithms to improve clinical outcomes and points to the usefulness of CIP2A as a biomarker to predict clinical response to rapamycin in head and neck cancer.

Citation Format: Song Hee Kim, Won hyeok Lee, Myung Jin Lee, Hyo Won Chang, Ji Won Kim, Mi Ra Kim, Jung Je Park, Myung Woul Han, Sang Yoon Kim. CIP2A can be a therapeutic target of rapamycin in radioresistant head and neck cancer with P53 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5476. doi:10.1158/1538-7445.AM2017-5476
Abstract 5478: The TRPA1 Ca2+-permeable channel mediates a non-canonical redox adaptation in cancer cells
Authors: Takahashi Nobuaki , Chen Hsing-Yu , Harris Isaac , Stover Daniel , Bronson Roderick , Deraedt Thomas , Cichowski Karen , Welm Alana , Mills Gordon , Brugge Joan
Published: July 2017

Generation of reactive oxygen species (ROS), a natural byproduct of oxygen metabolism, occurs in all aerobic organisms at a controlled rate. Cancer cells are subjected to numerous cellular insults, including dysregulated oncogenes and dissociation from their natural extracellular matrix (ECM) niches, leading to the generation of high levels of ROS. Although cancer cells possess enhanced canonical antioxidant programs that neutralize ROS, they nevertheless exhibit prominent ROS levels in response to these insults, suggesting the existence of additional programs that allow cancer cells to tolerate elevated ROS.

In this study, we provide evidence for an unconventional mechanism for redox adaptation involving the ROS-activated, Ca2+-permeable TRPA1 channel, which normally functions as an irritant receptor in sensory neurons but is highly upregulated in breast, lung, malignant neural sheath, and other tumors. We found that TRPA1 overexpression enhances survival and proliferation of MCF-10A cells under conditions of matrix-detachment and this effect was dependent on TRPA1 activation by ROS generated as loss of matrix anchorage. In MCF-10A acini, TRPA1 expression induced Ca2+ entry in ECM-deprived cells in the luminal space and promoted their survival and proliferation. Conversely, TRPA1 knockdown inhibited Ca2+ responses to ROS generated through ECM detachment and induced clearance of cells from the luminal space in breast cancer spheroids. TRPA1 was also activated by ROS-inducing chemotherapies and drove chemoresistance in breast, lung and malignant peripheral nerve sheath tumor cells, and its downregulation suppressed breast xenograft tumor growth and enhanced chemosensitivity. TRPA1 mediated these effects independent of antioxidant responses, but through activation of cellular survival and anti-apoptotic programs involving the RAS-ERK/AKT/mTOR pathways.

Together, our findings describe an unexpected mechanism whereby cancer cells co-opt the neuronal TRPA1 channel in order to adapt to oxidative environments. This TRPA1-induced response is distinct from canonical redox adaptation mechanisms that rely on antioxidant program. As TRPA1 inhibitors are currently in clinical trials for pain and respiratory therapies, these studies raise the possibility of using these inhibitors as therapeutic chemosensitizers in TRPA1-enriched tumors.

Citation Format: Nobuaki Takahashi, Hsing-Yu Chen, Isaac Harris, Daniel Stover, Roderick Bronson, Thomas Deraedt, Karen Cichowski, Alana Welm, Gordon Mills, Joan Brugge. The TRPA1 Ca2+-permeable channel mediates a non-canonical redox adaptation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5478. doi:10.1158/1538-7445.AM2017-5478
Abstract 5482: Constitutive overexpression of nrf2 in esophageal adenocarcinoma protects cancer cells from bile salts-induced DNA damage and favors cancer cell survival
Authors: Peng Dunfa , Hu Tianling , Zhu Shoumin , El-Rifai Wael
Published: July 2017

Background: Esophageal adenocarcinoma (EAC) is the major type of malignant cancer of esophagus in the USA. The incidence rate has increased 4-10% per year among men since 1976 in the USA, more rapidly than for any other type of cancer. EAC is known to originate from premalignant Barrett’s esophagus (BE) through BE-dysplasia-EAC process. Normal cells have intact anti-oxidative mechanisms, among which NFE2-related factor 2 (Nrf2) plays a pivotal role in regulating cellular response to various stimuli. Cancer cells have high levels of reactive oxygen species and oxidative stress due to activation of oncogenes, inflammatory microenvironment, and dysfunction of anti-oxidative mechanisms. However, the role of NRF2 in Barrett’s related esophageal carcinogenesis is barely known.

Methods and Results: We have found that Nrf2 protein expression is significantly upregulated in EAC cell lines as compared to BE cell lines (CPA, BAR10T) and normal esophageal squamous cell lines. We detected a similar overexpression of NRF2 in primary EAC tissue samples. Using luciferase reporter assay, we demonstrated significant induction of NRF2 transcription activity in EAC cells in response to exposure to acidic bile acids, as compared to controls (P<.01). We detected significant increase in the expression of Heme Oxygenase 1 (HO-1) and Glutathione Reductase (GR) in these cells (P<.01). Knockdown of NRF2 by siRNA or crisp/cas9, significantly increased cellular ROS level, oxidative DNA damage (8-oxoguanine), and double strand DNA breaks (p-H2AX). Knockdown of NRF2 sensitized cells to apoptosis following exposure to bile acids and CDDP. Concordant with these results, the use of NRF2 inhibitor, Brusatol, sensitized EAC cells to CDDP treatment with a significant increase in cell death, as compared to single agent treatment (P<.01). Conclusion: These results indicate that constitutive overexpression of NRF2 in EAC cells protects cancer cells from high level of ROS and promotes cancer cell survival. Inhibition of NRF2 through its specific inhibitor may have therapeutic value in EAC.

Citation Format: Dunfa Peng, Tianling Hu, Shoumin Zhu, Wael El-Rifai. Constitutive overexpression of nrf2 in esophageal adenocarcinoma protects cancer cells from bile salts-induced DNA damage and favors cancer cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5482. doi:10.1158/1538-7445.AM2017-5482
Abstract 5499: The reprogramming of the steroid receptor binding landscape at active enhancers is associated with a fast DNA residence time through a mechanism termed dynamic assisted loading in breast cancer cells
Authors: Swinstead Erin , Miranda Tina , Paakinaho Ville , Beak Songjoon , Hager Gordon
Published: July 2017

The transcription factor (TF), FoxA1 has been described as a pioneer factor, implicated in steroid receptor (SR) binding patterns. Classically it has been proposed that the pioneer factor model is a slow and static binding event. However, it has recently been described that the estrogen and glucocorticoid receptors (ER and GR), can regulate the binding patterns of each other in a mouse mammary cell line defined as dynamic assisted loading. Further, this phenomenon has been extending demonstrating that both ER and GR can recruit FoxA1 to specific binding sites associated with an increase in chromatin accessibility and fast DNA residence times. This was also associated with the absence of a DNaseI footprint for the initiating and secondary factor supporting the assisted loading model. To further investigate the molecular mechanism behind the dynamic assisted loading model we have characterized the binding and transcriptional landscape of ER and GR in a number of breast cancer cell line models. Genome wide analysis of ChIP and DNaseI hypersensitivity assays upon single and dual activation show that both SRs can recruit each other to a specific subset of binding sites associated with an increase in chromatin accessibility. Therefore, depending on the chromatin landscape, one factor functions as the initiating factor and the other the secondary factor. Further, RNA polymerase II (RNAP II), p300, and H3K27ac binding patterns are associated with the newly programed ER or GR binding sites. Together this suggests chromatin reorganization upon dual activation of either ER or GR (the initiating factors) and recruitment of the secondary factor (ER or GR) through an assisted loading mechanism. Further, we have demonstrated an association of the transcriptional machinery at these newly acquired binding sites at active enhancers. These results propose that many TFs in a given cell have the potential to affect the binding landscape of other TFs, depending on the chromatin context. In addition, this study has shifted our classical understanding of pioneer factors in breast cancer, demonstrating that not only can SRs alter the response of FoxA1 but also each other though highly dynamic and fast DNA interactions associated with active enhancers and transcription.

Citation Format: Erin Swinstead, Tina Miranda, Ville Paakinaho, Songjoon Beak, Gordon Hager. The reprogramming of the steroid receptor binding landscape at active enhancers is associated with a fast DNA residence time through a mechanism termed dynamic assisted loading in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5499. doi:10.1158/1538-7445.AM2017-5499
Abstract 551: Assessment of computational approaches for quantification of immune cell infiltration from gene expression profiles of complex biological samples
Authors: Hodkinson Brendan P. , Schaffer Michael E. , Gormley Michael
Published: July 2017

Anticancer immunotherapies target immune cells to block immune suppression and/or promote immune activation in order to eliminate cancer cells. Ipilumumab and Nivolumab, targeting CTLA4 and PD1 respectively, have demonstrated dramatic responses in melanoma and lung cancer (Hodi et al. 2010; Risvi et al. 2015). However, poor response rates in other types of cancer underscore the need to better understand immunomodulatory mechanisms (Topalian et al. 2012). Examination of immune cell specific signals, in either the tumor microenvironment or the periphery, has proven to be a useful tool in developing prognostic biomarkers (Chi et al. 2014; Gentles et al. 2015), inferring mechanisms of action (Krejcik et al. 2016), and discerning immune-based predictors of drug response (Tumeh et al. 2014). In this study we evaluate various gene expression-based computational approaches for inferring immune cell identity in complex cellular mixtures in order to rank their relative utility for illuminating the details of the tumor microenvironment and potentially revealing new biomarkers for prognosis and response prediction.

Immune cell identity has been inferred by previous researchers using a variety of mathematical approaches, including least squares (Abbas et al. 2009), quadratic programming (Gong et al. 2011; Zhong et al. 2013), maximum likelihood (Qiao et al. 2012; Liebner et al. 2013), machine learning (Newman et al. 2015), and enrichment type approaches (Angelova et al. 2015). We used publically-available gene expression microarray data from blood, normal tissue, and tumor samples to assess the effectiveness of these methods in sample types relevant to oncology research. The results from comparisons of these different methods demonstrate that certain approaches are significantly more robust to noise, and are therefore more suitable for complex cellular mixtures such as tumor samples. The discerning use of methods to infer immune cell type proportions from gene expression profiles may lead to improved prognosis, predictive biomarkers for immunotherapy to assist in patient stratification, and new immunoncology targets by indicating immunosuppressive mechanisms.

Citation Format: Brendan P. Hodkinson, Michael E. Schaffer, Michael Gormley. Assessment of computational approaches for quantification of immune cell infiltration from gene expression profiles of complex biological samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 551. doi:10.1158/1538-7445.AM2017-551
Abstract 5513: Notch directly represses transcription by recruitment of PRC2 to the ternary complex
Authors: Han Xiaoqing , Ranganathan Prathibha , Capobianco Anthony
Published: July 2017

It is well established that Notch functions as a transcriptional activator through the formation of a ternary complex that comprises Notch, Maml and CSL. This ternary complex then serves to recruit additional transcriptional cofactors that link to higher order transcriptional complexes. The mechanistic details of these events remain unclear. In this study, we report that the Notch ternary complex can also serve to direct the formation of a repressor complex to terminate genes expression of select target genes. Herein we demonstrate that p19Arf and Klf4 are transcriptionally repressed directly by Notch. Data indicate that Notch recruits PRC2 and Lsd1 to these promoters, which leads to changes in the state of the epigenetic landscape and repression of transcription. The demethylase activity of Lsd1 is a pre-requisite for Notch-mediate transcriptional repression. Furthermore, we identified a stable Notch transcriptional repressor complex containing Lsd1, PRC2 and the Notch ternary complex. This study demonstrates proof of concept for a novel function of Notch and provides further insight into the mechanisms of Notch mediated tumorigenesis. This finding provides rationale for the targeting of epigenetic enzymes to inhibit Notch activity or use in combinatorial therapy to provide a more profound therapeutic response.

Note: This abstract was not presented at the meeting.

Citation Format: Xiaoqing Han, Prathibha Ranganathan, Anthony Capobianco. Notch directly represses transcription by recruitment of PRC2 to the ternary complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5513. doi:10.1158/1538-7445.AM2017-5513
Abstract 5515: Dicer suppresses cytoskeleton remodeling and tumorigenesis of colorectal epithelium by miR-324-5p mediated suppression of HMGXB3 and WASF-2
Authors: Sun Lina , Li Jianming
Published: July 2017

Emerging evidence indicates that microRNAs, a class of small and well-conserved noncoding RNAs, participate in many physiological and pathological processes. RNase III endonuclease DICER is one of the key enzymes for microRNA (miRNA) biogenesis. However, the role of Dicer and its downstream miRNAs in colorectal cancer (CRC) remain largely unclear. Here, we found that Dicer was downregulated in tumour samples of CRC patients at both mRNA and protein levels. Importantly, more tumours were developed in mice with intestinal epithelial cells specific deletion of Dicer (Dicerloxp/+&VillinCre mice) after azoxymethane (AOM) and dextran sulfate sodium (DSS) administration. Interestingly, deletion of Dicer in intestinal epithelial cells led to severe chronic inflammation and cytoskeleton remodeling of intestinal epithelium layer in mice with or without DSS administration. Microarray analysis of 3 paired Dicer deletion CRC cell lines (RKO-WT/RKO-DICER-/-, HCT116-WT/ HCT116-DICER-/- and DLD-WT/DLD-DICER-/-) showed that miR-324-5p was one of the most significant decreased miRNAs. In the intestinal epithelium of Dicerloxp/loxp&VillinCre mice, miR-324-5p was also found to be markedly downregulated. Mechanistically, miR-324-5p directly bound to the 3'untranslated regions (3'UTRs) of HMGXB3 and WASF-2, two key proteins participated in cytoskeleton remodeling, to suppress their expression. Intraperitoneal injection of miR-324-5p AgomiR curtailed chronic inflammation and cytoskeleton remodeling of colorectal epithelium and restored intestinal barrier function in Dicerloxp/+&VillinCre mice induced by DSS. Therefore, our study reveals a key role of a DICER/miR-324-5p/HMGXB3/WASF-2 axis in cytoskeleton remodeling and maintaining integrity of intestinal barriers and as such alludes to the possibility of screening for small-molecule compounds that boost Dicer activity as a potential strategy in the treatment of colorectal cancer.

Citation Format: Lina Sun, Jianming Li. Dicer suppresses cytoskeleton remodeling and tumorigenesis of colorectal epithelium by miR-324-5p mediated suppression of HMGXB3 and WASF-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5515. doi:10.1158/1538-7445.AM2017-5515
Abstract 5516: Regulation of TFPI-2 in the progression of ovarian cancer
Authors: Fry Jacqueline , Kato Sumie , Abarzua Lorena , Gonzalez Pamela , Ramirez Carolina , Cumsille Elisa , Bravo Erasmo , Correa Raimundo , Leiva Juan E. , Paredes Cesar , Roa Juan C. , Ibañez Carolina , Cuello Mauricio , Owen Gareth I. , Bravo Maria L.
Published: July 2017

Ovarian cancer metastasis occurs when malignant cells migrate from the primary tumor into the ascitic fluid and form a secondary tumor in the peritoneal cavity. The cells responsible for this process, Metastases Initiating Cells (MICs), suffer a series of changes in protein expression, with increased Epithelial Mesenquimal Transition and stem cell markers having been reported. Tissue Factor Pathway Inibitor-2 (TFPI-2) is a serine proteinase inhibitor and potential tumor suppressor gene, whose reduction is correlated with more metastases and poor prognosis. As the exact stage of TFPI-2 loss is still unknown, the purpose of our study was to determine TFPI-2 during ovarian cancer progression. Protein and RNA levels were assessed in primary tumor and metastatic tissue from papillary serous carcinoma by immunohistochemistry and qPCR, respectively. TFPI-2 RNA and protein levels were analyzed in cancer cell lines and primary cultured cancer cells isolated from ascitic fluid of advanced ovarian cancer patients and in cancer spheres derived from these cultures. Epigenetic changes of 3 CpG were analized by bisulfite and pirosequencing technique in ovarian cancer cell lines and in cancer spheres derived from these cultures. We report that significantly decreased TFPI-2 protein levels in metastatic tissue compared to the primary tumor, a result confirmed in matched primary and metastatic lesions from the same ovarian cancer patients. Furthermore, cancer spheres (representing potential MICs) displayed significantly lower levels of TFPI-2 than corresponding cell line and some primary cultured cancer cells. The cancer spheres showed a modest increase in methylation percentage in three specific CpG sites within the TFPI-2 promoter. Our results suggest that TFPI-2 loss plays a role in the cancer cell escape from the primary tumor. Future studies will determine the mechanism and the biological consequences of the progressive loss of TFPI-2 in cancer. Funding: FONDECYT 11140657, 1160800, 1140970, 3140335, 3150028, CORFO L2 13IDL2-18608, IMII-P09/016-F, CONICYT-FONDAP 15130011, BMRC 13CTI21526-P6

Citation Format: Jacqueline Fry, Sumie Kato, Lorena Abarzua, Pamela Gonzalez, Carolina Ramirez, Elisa Cumsille, Erasmo Bravo, Raimundo Correa, Juan E. Leiva, Cesar Paredes, Juan C. Roa, Carolina Ibañez, Mauricio Cuello, Gareth I. Owen, Maria L. Bravo. Regulation of TFPI-2 in the progression of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5516. doi:10.1158/1538-7445.AM2017-5516