bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4373: AR signaling in concert with PP2A/B regulates YAP1 expression in prostate cancer cells
Authors: Cinar Bekir , Moreno Carlos S.
Published: August 2018

The transcriptional coactivator YAP1 is a key nuclear effector of the Hippo pathway. STK4/MST1 and LATS1/2 are the core kinase components of the Hippo pathway, which restricts organ size and prevents tumorigenesis by attenuating cell proliferation and inducing cell death. Previously, we have reported that STK4/Hippo-YAP1 signaling could play a critical role in prostate cancer progression and therapeutic relapse. Here, we investigated the effects of androgen hormone signaling on YAP1 expression and post-transcriptional modifications in prostate cancer cells. We demonstrated that androgen exposure reduced Ser127 phosphorylation on YAP1 in a time-dependent manner in the castration-sensitive prostate cancer cell line, LNCaP, but without altering the levels of YAP1-Ser127 phosphorylation in the C4-2 cell line, a castration-resistant subline of LNCaP cells. As demonstrated by imaging and cell fractionation methods, androgen exposure promoted the nuclear accumulation of YAP1 in LNCaP; however, regardless of androgen exposure, YAP1 was accumulated in the nucleus of C4-2 cells. In addition, we demonstrated that androgen exposure reduced YAP1-Ser127 phosphorylation that was induced by okadaic acid, a potent activator of Ser/Thr phosphatases PP1 and PP2A. Also, we demonstrated that androgen exposure increased PP2A/B protein expression. Moreover, reduction of phospho-Ser127-YAP1 was correlated with increases in total YAP1 protein, which coincided with androgen receptor (AR) nuclear accumulation by androgen. Consistent with these observation, the genetic (siRNA) or the pharmacologic (enzalutamide) inhibition of AR signaling attenuated the expression of YAP1 protein. Furthermore, our analysis of the publicly available TCGA (The Cancer Genome Atlas) set indicated that YAP1 and AR mRNA expressions were positively correlated in prostate clinical samples. These observations suggest that YAP1 is a direct target of androgen hormone signaling, implicating that the AR-PP2A/B-YAP1 axis is a viable cancer drug target.

Citation Format: Bekir Cinar, Carlos S. Moreno. AR signaling in concert with PP2A/B regulates YAP1 expression in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4373.
Abstract 438: High-quality CNV segments from low-coverage whole genome sequencing from FFPE cancer biopsies based on an evaluation of multiple CNV tools
Authors: Lee HoJoon , Xia Li Charlie , Greer Stephanie , Bell John , Grimes Sue M. , Bouwens Christina Wood , Shin Giwon , Lau Billy TC , Johnson Lucas , Andor Noemi , Day Kenneth , Miller Mickey , Escobar Helaman , Nadauld Lincoln , Ji Hanlee P. , Hummelen Paul Van
Published: August 2018

Changes in DNA copy number, i.e., somatic CNVs, are common genetic aberrations in cancers. The effects of CNV include alteration in gene dosage across large segments of the cancer genome affecting the expression of cancer driver genes by amplifications, or cancer suppressor genes by deletions. In addition, CNVs are markers of underlying rearrangements within or between chromosomes and there is increasing evidence supporting a greater role for CNVs in developing and maintaining neoplastic cell population diversity.

Copy number aberrations can be estimated from next generation sequencing data, with high sensitivity and genomic resolution by sequencing the whole genome (WGS). For this study, we demonstrated that high quality CNV calls can be extracted in a fast and cost-effective way from low-coverage whole genome sequencing. Novaseq S2 flowcells (Illumina Inc) enables to obtain an average coverage of 3-4x per sample after pooling up to 96 samples per flowcell.

We examined three different copy number detection tools (CNVkit, BicSeq, and seqCBS) from paired tumor and normal WGS using microarray data as a reference. Pearson correlations were computed between the reference and CNVs from the WGS in two fashion; i) segment based and ii) gene based. The segment based comparison used sliding window of 100 K bp while gene based comparison used segments at the gene level.

We found high correlations between microarray and WGS segments. The highest correlations were obtained by CNVkit, ranging from 0.964 to 0.985 (SD: 0.973 - 0.007) and BicSeq, ranging from 0.963 to 0.986 (SD: 0.975 - 0.008). These results open the prospect of assessing large cancer cohorts of hundreds of samples at a reasonable cost. We are planning to apply this method to a large cohort of Stage III colon cancer patients and determine the clinical relevance of CNVs for survival.

Citation Format: HoJoon Lee, Li Charlie Xia, Stephanie Greer, John Bell, Sue M. Grimes, Christina Wood Bouwens, Giwon Shin, Billy TC Lau, Lucas Johnson, Noemi Andor, Kenneth Day, Mickey Miller, Helaman Escobar, Lincoln Nadauld, Hanlee P. Ji, Paul Van Hummelen. High-quality CNV segments from low-coverage whole genome sequencing from FFPE cancer biopsies based on an evaluation of multiple CNV tools [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 438.
Abstract 4380: Role of FAM3B/PANDER in inhibition of cell death and tumor growth in breast and prostate cancer
Authors: Caldeira Izabela D.
Published: August 2018

FAM3B/PANDER is a novel cytokine-like protein that induces apoptosis in beta-cells and regulates the effects of insulin in peripheral tissues. Since previous data revealed that FAM3B can be expressed by prostate and breast tumors, we evaluated the role of this cytokine in prostate and breast tumor progression. FAM3B expression was compared by quantitative PCR in LnCAP, PC-3 and DU145 prostate tumor cell lines and MCF-7 and MDA-MB- 231 breast tumor cell lines. After treatment with either recombinant FAM3B protein or secreted FAM3B obtained from conditioned media (CM) derived from FAM3B-overexpressing 293T cells, the cell death and viability of DU145 and MDA-MB- 231 cell lines were evaluated. DU145 and MDA-MB- 231 cells overexpressing FAM3B protein were produced by lentiviral-mediated transduction of full-length FAM3B cDNA. Cell viability and apoptosis were analyzed in DU145-FAM3B and MDA-231-FAM3B cells after treatment with cell death inducers. Anchorage-independent growth and scratch wound assays were used to evaluate in vitro tumorigenicity and cell migration, respectively. In vivo tumorigenicity and invasiveness were evaluated by tumor xenograft growth in nude mice. We observed that FAM3B was highly expressed by hormone responsive cells (LnCAP and MCF-7) and low expressed in unresponsive hormone cells (PC-3, DU145 and MDA-MB- 231). Cell viability and survival of DU145 and MDA-MB- 231 cells increased after exogenous treatment with recombinant FAM3B protein or CM containing FAM3B-secreted protein. Overexpression of FAM3B in DU145 and MDA-MB- 231 cells promoted an inhibition of apoptosis triggered by TNF-alpha and staurosporine. Cell death inhibition was accompanied by increased gene expression of the anti-apoptotic Bcl-2 and Bcl-xL genes, and by slight decrease in expression of pro-apoptotic gene Bax and with a decrease of caspases proteolytic activities. When compared to control, cells overexpressing FAM3B displayed a decreased anchorage independent growth and increased motility in vitro, as well as an increased tumor growth in xenografted nude mice. In agreement Immunohistochemistry analysis from tumor xenografts revealed similar anti-apoptotic phenotype in FAM3B overexpression, with an increase of Bcl-2. The results showed that FAM3B was capable to activates pro-survival mechanisms with an increasing of tumor growth by, activation of Bcl-2 and Bcl-xl pathways, suggesting the role of this cytokine in breast and in prostate tumors progression.

Citation Format: Izabela D. Caldeira. Role of FAM3B/PANDER in inhibition of cell death and tumor growth in breast and prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4380.
Abstract 4382: Complement system mutational landscape reveals C4BPA mutations enhance apoptosis in an immune-independent manner
Authors: Olcina Monica M. , Balanis Nikolas G. , Kim Ryan K. , Thompson Michael J. , Graeber Thomas G. , Giaccia Amato J.
Published: August 2018

The complement system is an important pathway in immunity. When dysregulated in the tumor microenvironment, complement is associated with suppression of antitumor immunity and tumorigenesis promotion. While complement has been reported to have important intracellular functions in immune cells, the role of intracellular complement in cancer has remained poorly understood to date. In this study we investigated the prevalence and significance of pathway-wide complement mutations as well as their role with respect to intracellular complement in cancer. We describe mutations in both individual genes as well as within functional groups that are significantly associated with altered survival outcomes. Analyzing mutations occurring across multiple TCGA cancer types highlighted the potential clinical significance of certain mutations that would otherwise not have surfaced at a single cancer level. As an example, we test the significance of mutations in complement regulator C4BPA in vitro and in vivo in a single cancer type. This approach allowed us to uncover a new immune-independent biologic function of the complement system with potential clinical implications for colorectal cancer patients. Specifically, we find that colorectal cancer cells with specific C4BPA mutations display increased oxaliplatin-induced intracellular C4BPA stabilization. By studying the mechanistic basis of this association we report novel crosstalk between intracellular complement and apoptosis signaling, occurring in an NFκB/RelA-dependent manner. Based on both experimental and patient outcome data, we therefore propose that assessing complement mutation status might facilitate patient stratification. In the case of C4BPA mutations, this would be particularly relevant to improve accuracy of prognosis assessment in stage II colorectal patients where TNM staging alone does not accurately predict outcome in patients who might benefit from adjuvant chemotherapy.

Citation Format: Monica M. Olcina, Nikolas G. Balanis, Ryan K. Kim, Michael J. Thompson, Thomas G. Graeber, Amato J. Giaccia. Complement system mutational landscape reveals C4BPA mutations enhance apoptosis in an immune-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4382.
Abstract 4387: Triplet nucleotide repeat-based siRNAs are highly toxic to cancer cells
Authors: Murmann Andrea E. , Putzbach Will , Gao Quan , Patel Monal , Bartom Elizabeth T. , Law Calvin , Bridgeman Bryan , Chen Siquan , McMahon Kaylin M. , Thaxton Shad C. , Peter Marcus E.
Published: August 2018

Triplet nucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by both protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR can be used to kill cancer cells by targeting genes containing reverse complimentary long TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the 6 members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG-induced cell death occurs in vitro in all tested cancer cell lines and in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anticancer reagents.

Citation Format: Andrea E. Murmann, Will Putzbach, Quan Gao, Monal Patel, Elizabeth T. Bartom, Calvin Law, Bryan Bridgeman, Siquan Chen, Kaylin M. McMahon, Shad C. Thaxton, Marcus E. Peter. Triplet nucleotide repeat-based siRNAs are highly toxic to cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4387.
Abstract 4391: CD95L mRNA is toxic to cancer cells
Authors: Putzbach William , Haluck-Kangas Ashley , Gao Quan , Sarshad Aishe A. , Bartom Elizabeth , Stults Austin , Qadir Abdul S. , Scholtens Denise M. , Hafner Markus , Peter Marcus
Published: August 2018

CD95/Fas ligand (CD95L) is best characterized for its role in activating extrinsic apoptosis through binding to its receptor, CD95. However, we recently reported that the CD95L mRNA is enriched in sequences that when converted to si- or shRNAs kill cancer cells through an RNAi-dependent mechanism. When loaded into the RNA Induced Silencing Complex (RISC), CD95L-derived small RNAs downregulate genes critical for cancer cell survival, eliciting a unique form of cell death we call Death Induced by Survival Gene Elimination (DISE). We now report that expression of full-length CD95L mRNA itself is toxic to cells. Acute expression of either wild-type CD95L mRNA in CD95-deficient cells or a CD95L mRNA with a premature stop codon in CD95 wild-type cells induces cell death. RNA-Seq analysis of small RNA reveals that CD95L mRNA is specifically processed into discrete clusters ~19-25 nucleotides long that map along the CD95L ORF. These clusters of CD95L sequences were bound by AGO proteins in cells about to die, suggesting that the mRNA may be killing cells through an RNAi-dependent mechanism. Generation of AGO-bound CD95L mRNA sequences does not appear to be dependent on the canonical miRNA biogenesis pathway, as clusters of CD95L mRNA were detectable in both Drosha and Dicer deficient HCT116 cells. We propose that CD95L mRNA is toxic to cells through processing and loading of small CD95L-derived RNA sequences into the RISC.

Citation Format: William Putzbach, Ashley Haluck-Kangas, Quan Gao, Aishe A. Sarshad, Elizabeth Bartom, Austin Stults, Abdul S. Qadir, Denise M. Scholtens, Markus Hafner, Marcus Peter. CD95L mRNA is toxic to cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4391.
Abstract 4393: Electrochemotherapy with irreversible electroporation and FOLFIRINOX improves survival in murine pancreatic adenocarcinoma by increasing apoptosis and decreasing tumor cell proliferation
Authors: Bhutiani Neal , Pandit Harshul , Zheng Qianqian , Li Suping , Li Yan , Martin Robert C.
Published: August 2018

Introduction: Combining gemcitabine (gem) with irreversible electroporation (IRE) has been shown to result in increased tumor cell apoptosis in a murine model of pancreatic adenocarcinoma (PDAC). However, it remains unclear whether this effect can be augmented locally and systemically by utilizing more active chemotherapies. The objective of this study was to evaluate the efficacy of IRE+FOLFIRINOX (FOL: 5-FU, leucovorin, irinotecan, and oxaliplatin).

Methods: Athymic nude mice underwent intrapancreatic injection with S2-013 PDAC cells. After 7-10 days, tumors were confirmed with ultrasound and mice were treated with chemotherapy (gem or FOL), IRE, or chemotherapy+IRE. Four hours after last chemotherapy, pancreas was evaluated histologically. Levels of irinotecan and its active metabolite (SN-38) were evaluated in organs of mice treated with FOL and IRE+FOL. Experiments were repeated and survival analyses performed to evaluate long-term efficacy. Pancreas was harvested for histology and evaluation of apoptotic machinery, cell cycle proteins, and tumor cell proliferation.

Results: IRE+FOL (ECT) resulted in increased tumor cells apoptosis compared to gem, gem+IRE, or FOL (apoptotic index (AI) 34.6% vs. 3.8%, 7.3%, 5.8%, respectively, p<0.001). High performance liquid chromatography (HPLC) demonstrated a 1.5-2-fold increase in irinotecan and SN-38 in the pancreas and a 4-fold increase of both compounds in the liver in mice treated with ECT vs. FOL. ECT significantly improved overall survival when compared to mice treated with IRE or FOL (median 7 days ECT vs. 4 days IRE vs. 3 days FOL, p=0.03). Histology demonstrated increased tumor cell apoptosis at time of death (AI = 34.2% ECT vs. 13.2% IRE vs. 4.4% FOL, p=0.026). Western blot demonstrated an increase in active caspase 3 (0.14 ECT vs. 0.00 IRE vs. 0.03 FOL fold change/GAPDH, p=0.02). ECT resulted in lower cell proliferation compared to IRE or FOL (0.2% vs. 0.3% vs. 0.9, p<0.001).

Conclusions: ECT with IRE and FOL causes significant tumor cell apoptosis, decreases tumor cell proliferation, enhances systemic FOL exposure, and improves overall survival. Human Phase 1 trials have been initiated to evaluate safety and efficacy of this therapeutic modality.

Citation Format: Neal Bhutiani, Harshul Pandit, Qianqian Zheng, Suping Li, Yan Li, Robert C. Martin. Electrochemotherapy with irreversible electroporation and FOLFIRINOX improves survival in murine pancreatic adenocarcinoma by increasing apoptosis and decreasing tumor cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4393.
Abstract 4407: Targeting BMP4 and miR-139-5p in EGFR-mutant non-small cell lung cancer cells
Authors: Bach Duc-Hiep , Luu Thi-Thu-Trang , Kim Dong-Hwa , An Yong Jin , Park Hyen Joo , Park Sunghyouk , Lee Sang Kook
Published: August 2018

Non-small cell lung cancer (NSCLC) cells with harboring EGF receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Recent studies suggest that bone morphogenetic proteins (BMPs) and microRNAs (miRNAs) might act as oncogenes or suppressors in tumor microenvironment. We identified the potential roles of BMPs and miRNAs involved in EGFR TKIs resistance by analyzing datasets from a pair of NSCLC-sensitive and NSCLC-resistant cells. BMP4 was found to be significantly over-expressed in EGFR-TKI resistant cells and its underlying mechanism was highly associated with induction of energy metabolism for cancer cells through modulating Acyl-CoA synthetase long-chain family member 4. In addition, miR-139-5p was found to be importantly down-regulated in the resistant NSCLC cells. The combination of miR-139-5p and yuanhuadine, a natural product-derived antitumor agent, synergistically suppressed the BMP4 expression in the resistant cells. We furthermore confirmed that LDN-193189, a small molecule BMP receptor 1 inhibitor inhibited the tumor growth in a xenograft nude mouse model implanted with drug resistant cells. These findings suggest a role of BMP4-mediated tumorigenesis in the progression of acquired drug resistance in EGFR-mutant NSCLC cells.

Key words: microRNAs, bone morphogenetic proteins, drug resistance, metabolism, EGFR-TKIs, NSCLC.

Acknowledgements

This study was funded by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A02062012).

Citation Format: Duc-Hiep Bach, Thi-Thu-Trang Luu, Dong-Hwa Kim, Yong Jin An, Hyen Joo Park, Sunghyouk Park, Sang Kook Lee. Targeting BMP4 and miR-139-5p in EGFR-mutant non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4407.
Abstract 4410: miRNA-34a sensitizes triple-negative breast cancer cells to paclitaxel reducing cancer stem cell population and lung colonization
Authors: Climent Montserrat , Bonetti Paola , Nicassio Francesco
Published: August 2018

Background: Cancer stem cells (CSCs) have inherent or developed drug resistance for which chemotherapy fails to eliminate all tumor cells, a property that is critically contributing to recurrence and metastasis. Here, we combine miR-34a, a well-known tumor suppressor miRNA that controls proliferation, apoptosis and self-renewal, with Paclitaxel (PTX), a commonly used first-line chemotherapy drug. Based on our preliminary observations (Bonetti et al., manuscript under revision), we hypothesized a combinatorial approach, coupling miRNA- and chemo- treatment, aimed at reducing CSCs in vitro and recurrence in vivo using triple-negative breast cancer (TNBC) as a model. Methodology: As a model, we exploited SUM159pt cells (TNBC mesenchymal-like, with CSC features) to mimic aggressive breast cancer, which harbor a vector for pLUC, which is induced by luciferin in vivo, to monitor tumor growth in real time. Furthermore, we used i) pSlik34a, with tunable expression of miR-34a by doxycycline; ii) treatment with PTX to select for resistant cells (RC); and combined the two, by treating RC with doxycycline. CSC content was evaluated by ALDH (Aldehyde Dehydrogenase) activity, sphere-forming efficiency (SFE) and qRT-PCR of known markers. A similar approach was followed for in vivo studies, with NSG female injected through tail vein injection with 500,000 SUM159/pLUC/pSlik34a cells, and separated into three groups: not treated (NT), PTX alone, and PTX+34a. Results: As observed for aggressive breast cancer, treatment of SUM159 with PTX generated resistant cells (RC) with increased CSC content (ALDH activity and spheres forming ability). When miR-34a was induced in the RC, cells changed morphology, slowed proliferation rate, and decreased CSC content (ALDH activity and spheres). Moreover, a second PTX treatment almost completely abolished the ALDH activity and SFE. The combined effect was evaluated in vivo, following by luminescence the growth of metastases after lung colonization, with PTX+34a mice reducing lung colonization than the PTX treatment or miR-34a treatment alone. Conclusions: Overall, these results indicate that miR-34a sensitize PTX-resistant cells reducing cancer stem-cell capacities, and in this preclinical setting limited the aggressive phenotype of breast cancer, including proliferation, drug resistance, relapse and metastasis causing in delay of lung colonization in vivo.

Citation Format: Montserrat Climent, Paola Bonetti, Francesco Nicassio. miRNA-34a sensitizes triple-negative breast cancer cells to paclitaxel reducing cancer stem cell population and lung colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4410.
Abstract 4429: FOXP3 transcriptional regulation of MicroRNAs in human breast cancer
Authors: Yi Baozhu , Li Ming , Liu Ying , Liu Runhua
Published: August 2018

MicroRNA (miR)-155 has been found to be overexpressed in breast cancer cells, but its transcriptional regulation remains elusive. The role of miR-155 is also not determined in breast cancer development and progression. In this study, we identified a FOXP3-BRCA1-miR155 axis in breast cancer cells. MiR-155 is induced by FOXP3 through a transcriptional repression of BRCA1 in breast cancer cells. Likewise, overexpression of miR-155 was found in patients with early stage or triple negative breast cancer, but not in patients with high stage or metastatic breast cancer. Of note, expression of miR-155 in blood cells was higher in patients with localized but not metastatic tumors. In addition, although miR-155 in tumor cells is induced by FOXP3, free or exosomal miR-155 in culture medium was not changed with induction of FOXP3. There is a transcriptional axis of FOXP3-BRCA1-miR-155 in breast cancer cells. MiR-155 is a potential biomarker for early stage breast cancer. Plasma miR-155 appears to be related to that in blood cells but not in tumor cells.

Citation Format: Baozhu Yi, Ming Li, Ying Liu, Runhua Liu. FOXP3 transcriptional regulation of MicroRNAs in human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4429.
Abstract 4434: Contribution of heterogenous ribonucleoprotein A18 in prostate cancer progression
Authors: Chang Elizabeth Tsuying , Choi Eun Yong , Parekh Palak R. , Carrier France
Published: August 2018

Prostate cancer is the third leading cause of cancer deaths among men in the United States, with recurrent metastasis being the predominant cause of mortality. In spite of the significant progress made in the past half century with hormonal therapy for metastatic prostate cancer, a majority eventually progresses to a lethal stage of the disease commonly known as castration-resistant prostate cancer. New treatment strategies are therefore needed. Previous work from our lab has identified the RNA-binding protein heterogenous ribonucleoprotein A18 (hnRNP A18) as a new regulator of protein translation in cancer cells. While its localization is predominantly nuclear, hnRNP A18 translocates out to the cytosol in response to cellular stress like hypoxia. Immunohistochemistry data from tissue microarrays reveals increased expression of hnRNP A18 in prostate hyperplasia, and even greater overexpression in prostate adenocarcinoma as compared to the surrounding normal tissue. Furthermore, a greater percentage of hnRNP A18 is seen to shift from the nucleus to the cytosol as the disease progresses. In the cytosol, hnRNP A18 recognizes and binds to a specific 51 nucleotide RNA motif in the 3' untranslated region of targeted transcripts, stabilizing them and enhancing their translation. Transcripts targeted by hnRNP A18 confer tumor growth advantages. The down-regulation of hnRNP A18 was seen to reduce cancer cell proliferation, migration, and invasion. Moreover, down-regulation of hnRNP A18 significantly reduces prostate cancer growth in a mouse tumor xenograft model. Taken together, our data suggests that hnRNP A18 could be a potentially new target to stop or limit prostate cancer progression.

Citation Format: Elizabeth Tsuying Chang, Eun Yong Choi, Palak R. Parekh, France Carrier. Contribution of heterogenous ribonucleoprotein A18 in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4434.
Abstract 4448: Mutations in the center MATH domain of Speckle Type BTB/POZ Protein lead to enhanced proteinstability in prostate cancer
Authors: Fried Joshua , Khattar Vinayak , Ma Jinlu , Zeng Qinghua , Zhang Wei , Xu Bo
Published: August 2018

The Speckle type Poz Protein (SPOP) is a tumor suppressor that is often mutated in prostate cancer. Mutation of SPOP is linked to genomic instability and it plays a critical role in prostate cancer initiation. SPOP mutations are found to be in the heterozygous state in patients. The relevance of these mutations on its protein expression has not been systematically investigated. Here we report that mutations in the center MATH domain of SPOP lead to enhanced protein stability in prostate cancer. Among the mutations, F102C, S119N, F125V and WI31G showed increased protein stability. Further we show that these mutations in the MATH domain increase half-life of SPOP protein. Interestingly, the mutant SPOPs enhanced the endogenous wild-type SPOP level. We further explored the structural insights of these SPOP mutants. We present a computational

model to predict how SPOP mutations potentially lead to conformational changes within the protein, promoting its stability. Finally, immunohistochemistry in clinical samples of prostate cancer augment our findings. Together, we highlight a subset of clinically relevant SPOP mutations that have impact on endogenous SPOP protein stability, allowing to further understand its role in prostate cancer initiation and evaluating of potentially altered therapeutic response in patients harboring these mutations.

Citation Format: Joshua Fried, Vinayak Khattar, Jinlu Ma, Qinghua Zeng, Wei Zhang, Bo Xu. Mutations in the center MATH domain of Speckle Type BTB/POZ Protein lead to enhanced proteinstability in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4448.
Abstract 4453: Osteopontin-c mediated drug resistance in breast and ovarian carcinoma cells
Authors: Brum Mariana C. , Guimaräes Isabella S. , Ferreira Luciana Bueno , Rangel Letícia B. , Maia Raquel C. , Moraes Gabriela N. de , Gimba Etel R.
Published: August 2018

Osteopontin (OPN) has been described as an important gene product mediating resistance to chemotherapeutic drugs. However, the specific roles of each OPN splice variant in mediating chemoresistance should be further investigated. This work aimed to evaluate OPNc expression patterns and its correlations with resistance to doxorubicin (DOX) and cisplatin (CIS) in breast and ovarian tumor cell lines, respectively. We used one breast cancer cell line resistant to DOX (MCF-7 DoxR), and one ovarian cancer cell line resistant to CIS (ACRP) and their corresponding parental cell lines (MCF-7 and A2780). Knockdown of OPNc expression levels has been achieved by transfecting cell lines with specific anti-OPNc phosphothiotate modified DNA oligomers. Cytotoxicity assays have been carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), crystal violet and clonogenic assays. Gene expression has been assessed by quantitative real time PCR. Our results showed that drug-resistant cells overexpress OPNc and P-glycoprotein (P-gp), when compared to their age matched parental cell lines. After transfecting both MCF-7 DoxR and ACRP resistant cell lines with the anti-OPNc oligomers, we found a significant knockdown of OPNc isoform expression, as well as the expression of P-gp. Moreover, these cells exhibited a significantly increased sensitivity to DOX and CIS, and inhibited cell growth and colony formation (p<0,05). Additionally, the epithelial-plasticity phenotype of both MCF-7 DoxR and ACRP resistant cell lines has been reversed, as well the expression of epithelial-mesenchymal transition (EMT)-related interleukins. Altogether, these data evidence that OPNc overexpression can mediate resistance to these chemotherapeutic drugs, possibly by modulating the expression of P-gp and the EMT phenotype. In summary, our data provide early insights on the role of OPNc isoform as a potential molecular target in cancer. Therapeutic strategies aiming to interfere with this isoform expression are likely to overcome resistance to chemotherapeutic agents.

Citation Format: Mariana C. Brum, Isabella S. Guimaräes, Luciana Bueno Ferreira, Letícia B. Rangel, Raquel C. Maia, Gabriela N. de Moraes, Etel R. Gimba. Osteopontin-c mediated drug resistance in breast and ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4453.
Abstract 4457: Determining the role of novel GLI1 splice variants in breast cancer
Authors: Dixon Maria S. , Chdid Lhoucine , Lamson David , Tarpley Michael , Fleming Jodie M. , Freedman Jennifer A. , Devi Gayathri R. , Williams Kevin P.
Published: August 2018

The purpose of this study is to investigate the biological significance of novel GLI1 splice variants in breast cancer. 15-20% of breast cancers are triple negative/basal-like, are associated with poor clinical outcomes and show disproportionately higher prevalence in younger women of African descent. The Hedgehog (Hh)/GLI1 developmental pathway has emerged as a therapeutic target in many cancers including studies on breast cancer such as those from our lab. Overexpression of the main Hh transcriptional mediator GLI1 correlates with poor patient prognosis and relapse. The human GLI1 transcript undergoes alternative splicing producing two shorter isoforms, an N-terminal deletion variant (GLI1ΔN) and a truncated GLI1 (tGLI1) which have been reported to have different patterns of tissue expression and function. tGLI1 has been identified as being highly expressed in several cancers, including in breast cancer. We undertook an in silico analysis of the NCBI database for evidence of additional human GLI1 transcripts and have identified novel GLI1 splice variants that we have subsequently shown to be highly expressed in breast cancer cell lines. Our objectives were to examine expression differences between GLI1 splice variants in basal-like and luminal breast cancer cell models, AA and White patient samples and to investigate the biological function of GLI1 splice variants in breast cancer. In our studies, we have utilized phenotypic and functional assays to examine the functional impact of these novel isoforms. Our preliminary data suggest that these newly identified GLI1 isoforms are expressed in basal-like breast cancers at substantially higher levels than GLI1, tGLI1 or GLI1ΔN. Previous studies assessing GLI1 expression have not considered these new isoforms and the relative contribution of each to breast cancer aggressiveness and require further investigation.

Citation Format: Maria S. Dixon, Lhoucine Chdid, David Lamson, Michael Tarpley, Jodie M. Fleming, Jennifer A. Freedman, Gayathri R. Devi, Kevin P. Williams. Determining the role of novel GLI1 splice variants in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4457.
Abstract 4460: BRCA1-associated R-loop accumulation at noncoding putative ERα enhancer area regulates expression of adjacent genes
Authors: Chiang Huai-Chin , Zhang Xiaowen , Zhang Chi , Zhao Xiayan , Li Jingwei , Hu Yanfen , Li Rong
Published: August 2018

Most BRCA1-associated breast tumors are basal-like yet originate from luminal progenitor cells. BRCA1 is best known for its functions in double strand break (DSB) repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions of BRCA1 fully explains the cell lineage-specific increase in breast tumor development. By sorting and profiling lineage-specific cells from precancerous human breast tissue, I found that BRCA1 mutation carriers tend to have a luminal cell-specific accumulation of R-loop, a transcriptional byproduct of DNA-RNA hybrid structure important for transcriptional regulation and genomic instability. R-loops accumulate preferentially at enhancers and transcription start sites (TSS) of luminal fate-related genes. Using CRISPR-Cas9 system, I've deleted an ERα enhancer area with BRCA1-associated R-loops accumulation in MCF7 cells. The deletion of this non-coding putative ESR1 enhancer area reduced the gene expression level of the neighboring genes of the deleted region, including ERα, RMND1, and CCDC170. In addition, compare to the reduced expression level of ERα, RMND1, and CCDC170 in BRCA1 knockdown of the parental MCF7, knocking down BRCA1 in these deletion clones showed less reduction of these genes. On the other hand, overexpression RNASEH1, an enzyme that specifically removes R-loop, had little effect in these genes after BRCA1 knockdown. These results indicate BRCA1-associated R-loop accumulation at least partially affect gene transcription at these loci. To investigate whether BRCA1-assocaited R-loop accumulation directly contributes to luminal differentiation blockage, I overexpressed RNASEH1 in breast epithelial cells freshly isolated from BRCA1 mutation carriers, flow cytometry comparing different breast lineage reveal a luminal population shift toward more mature luminal. All in all, the results infer that BRCA1 promotes luminal epithelial differentiation by alleviating R-loop accumulation at enhancer of ERα. Aberration in luminal transcription could contribute to lineage-specific BRCA1-associated tumorigenesis.

Citation Format: Huai-Chin Chiang, Xiaowen Zhang, Chi Zhang, Xiayan Zhao, Jingwei Li, Yanfen Hu, Rong Li. BRCA1-associated R-loop accumulation at noncoding putative ERα enhancer area regulates expression of adjacent genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4460.
Abstract 4465: Variants of uncertain significance of the cancer-predisposing genes in two thousand Japanese whole-genome sequencing data
Authors: Yasuda Jun , Iida Keita , Kumada Kazuki , Ogishima Soichi , Shibuya Yusuke , Tokunaga Hideki , Yaegashi Nobuo
Published: August 2018

Many of the cancer predisposing genes are known to be associated with juvenile cancer syndromes such as Li Fraumeni syndrome. The responsible variants of the juvenile cancer syndromes are usually very deleterious and show strong biological effects on the gene functions. It is not so easy to estimate the clinical significance of the moderately deleterious variants in those genes that may have intermediate effects on the hosts' cancer predisposition and these moderately deleterious (or hypomorphic) variants are now classified as variants of uncertain significance (VUSs). The insight that VUSs may have moderate but clinically significant effects on cancer susceptibility now attracts cancer geneticists. To elucidate the state of VUSs of 135 cancer predisposing genes in general population, we functionally annotate the single nucleotide variants (SNVs) in the genes from a 2049 whole genome sequence high confident dataset (2KJPN-HC) obtained from participants in a genome cohort by Tohoku Medical Megabank Organization. Frequency spectrum of the Combined Annotation-Dependent Depletion (CADD) scores of the 135 genes showed very similar pattern to the whole 2KJPN-HC and a weak negative correlation (r = -0.0579) is observed between allele frequencies and CADD scores for each SNV. Among the variants found in 2KJPN, some of the variants are considered as responsible for hereditary cancer syndromes. We used the CADD and Eigen, other scoring system for estimation of impact of the variants, for the known damaging variants in the BRCA1 and BRCA2 genes to narrow down the candidate VUSs that may have moderate risks for cancer predisposition and found one VUS that is identified as a moderate risk SNP for breast cancer in the other studies. Combination of CADD and Eigen will be useful to narrow down the candidate deleterious SNVs for cancer predisposition.

Citation Format: Jun Yasuda, Keita Iida, Kazuki Kumada, Soichi Ogishima, Yusuke Shibuya, Hideki Tokunaga, Nobuo Yaegashi. Variants of uncertain significance of the cancer-predisposing genes in two thousand Japanese whole-genome sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4465.
Abstract 4473: Tumorigenesis by deregulation of CRAD-controlled cytoskeleton dynamics
Authors: Jung Youn-Sang , Wang Wenqi , Jun Sohee , Zhang Jie , Srivastava Mrianl , Kim Moon Jong , Lien Esther M. , Shang Joan , McCrea Pierre D. , Zhang Songlin , Chen Junjie , Park Jae-Il
Published: August 2018

Epithelial cell behavior and homeostasis are mainly modulated by the cytoskeleton and cell adhesion. However, much concerning epithelial cell deregulation in cancer remains unknown. We identified Cancer-related Regulator of Actin Dynamics (CRAD) as frequently mutated or transcriptionally downregulated in colorectal cancer. We found that CRAD is a capping protein inhibitor and stabilizes the cadherin-catenin-actin (CCA) complex. CRAD inactivation disrupts the CCA complex via inhibiting actin polymerization, liberating β-catenin and aberrantly activating Wnt pathway. In mice, CRAD knockout induces the loss of epithelial cell integrity and hyperactivates Wnt signaling, resulting in intestinal adenoma development. With APC mutation, CRAD knockout induces and accelerates mucinous and invasive adenoma development in the colorectum. These results define CRAD as a new tumor suppressor, of which inactivation deregulates the cytoskeleton and hyperactivates Wnt signaling, initiating mucinous intestinal tumorigenesis. Our study reveals the unexpected roles of an actin cytoskeletal regulator in maintaining epithelial cell integrity.

Citation Format: Youn-Sang Jung, Wenqi Wang, Sohee Jun, Jie Zhang, Mrianl Srivastava, Moon Jong Kim, Esther M. Lien, Joan Shang, Pierre D. McCrea, Songlin Zhang, Junjie Chen, Jae-Il Park. Tumorigenesis by deregulation of CRAD-controlled cytoskeleton dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4473.
Abstract 4476: Role of MAP-Kinase suppressor in IBD
Authors: Khare Sharad , Zhang Qiong
Published: August 2018

BACKGROUND & AIMS: Endogenous suppressor of Mitogen Activated Kinase Pathway (MAPK) are considered cancer suppressors. In this regard , Sprouty (SPRY) proteins that are endogenous suppressor of receptor tyrosine kinase signaling are reported to be tumor suppressors in various cancers, whereas we discovered that SPRY2 augments Epithelial Mesenchymal Transition (EMT) in colorectal cancer (CRC) [Oncogene, 2016 Jun 16; 35(24): 3151-62]. Significance of SPRY proteins in Inflammatory Bowel Disease (IBD) is not clearly understood.

MATERIALS & METHODS: Biopsies from Crohn's Colitis patients were assessed for SPRY, Beta-catenin, Occludin and ZO1 expression. Tight junction profiler was employed to assess expression of integrin proteins in CaCO2 colon cancer cell line. SPRY was suppressed in colon cancer cells by siRNA and CRISPR/Cas9 technology and changes were assessed by immunoblotting, real time PCR, phase-contrast and confocal microscopy. Spry1 and Spry2 floxed mice were utilized to dissect SPRY functions in mesenchymal cells.

RESULTS: SPRY, E-cadherin, Beta-catenin and Occludin mRNA transcripts were significantly different in IBD patients (p<0.05). No significant changes in ZO1 mRNA transcripts were noted. Suppression of SPRY significantly decreased expression of several integrins in CaCO2 cells (p<0.05) . Treatment of CaCO2 cells with Dextran Sodium Sulfate (DSS) significantly altered SPRY, Beta-catenin, Occludin and ZO1 expressions that was reversed by SPRY suppression. Spry1-/- and Spry2-/- double mutant murine embryonic fibroblasts (MEFs) demonstrated restoration of several epithelial markers which protected cells from DSS-induced changes.

CONCLUSIONS: SPRY expression in colonic epithelium regulates IBD. SPRY has prognostic value in IBD.

Citation Format: Sharad Khare, Qiong Zhang. Role of MAP-Kinase suppressor in IBD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4476.
Abstract 4479: Protein arginine methyltransferase PRMT6 regulates cancer stemness through CRAF methylation in hepatocellular carcinoma
Authors: Chan LH , Zhou L , Ng Kai Yu , Wong TL , Lee TK , Ching YP , Yuan YF , Xie D , Richard S , Huen MS , Guan XY , Ma S
Published: August 2018

Arginine methylation is a post-translational modification that plays pivotal roles in signal transduction and gene transcription during cell fate determination. We found protein methyltransferase 6 (PRMT6) to be frequently down-regulated in hepatocellular carcinoma (HCC) cells and its expression to negatively correlate with aggressive cancer features in HCC patients. Silencing of PRMT6 promoted the tumor-initiating, metastasis and therapy resistance potential of HCC cells. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a DEN+CCL4 HCC induced PRMT6-/- mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and that PRMT6 interacted with CRAF, and likely other RAF family members, and their methylation at conserved arginine 100, negatively regulating its activity, and as a consequence resulting in enhanced MEK/ERK signaling. Our work uncovered a critical repressive function for PRMT6 in maintenance of HCC cells by regulating the MEK/ERK pathway via arginine methylation of RAF, providing a new avenue of molecular mechanism by which ERK mediated stemness in HCC cells are developed.

Citation Format: LH Chan, L Zhou, Kai Yu Ng, TL Wong, TK Lee, YP Ching, YF Yuan, D Xie, S Richard, MS Huen, XY Guan, S Ma. Protein arginine methyltransferase PRMT6 regulates cancer stemness through CRAF methylation in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4479.
Abstract 4482: MTAP regulates cell malignancy and is novel prognostic factor for kidney cancer
Authors: Huang Carissa , Xu Jihao , Wu Yichin , Weiss Robert H. , Chen Ching-Hsien
Published: August 2018

Renal cell carcinoma (RCC), the 6th most common cancer in the US, is now considered a metabolic disease, which is increasing in incidence and demonstrates resistance usually within 2 years to all available therapies. Given that patients with metastatic RCC have unusually poor prognosis, it is urgent to discover potential molecules for predicting malignant changes that will lead to RCC. Herein, we have identified S-methyl- 5'-thioadenosine phosphorylase (MTAP) and its substrate methylthioadenosine (MTA) as a possible biomarker for early detection of RCC. In a screen of patients with RCC, we found that low MTAP expression is accompanied by high MTA level in RCC specimens, whereas concomitant high level of MTAP and low MTA abundance are detected in adjacent normal kidney tissues. Datasets for RCC (n=538) from The Cancer Genome Atlas (TCGA) showed that the patients with low MTAP levels have a significantly shorter overall survival as compared to the high MTAP group. Immunohistochemistry staining of normal kidney tissues confirmed an increase of MTAP protein expression compared to RCC tissues, and MTAP gene expression is inversely proportional to tumor grade. Accumulation of the metabolite MTA, a major substrate of MTAP, was observed in high-grade tumors showing high malignant potential. MTAP-knockout RCC cells displayed an elongated, spindle-like morphology with extended pseudopodial branches. Genetic manipulation of MTAP studies demonstrated that MTAP expression inhibits epithelial-mesenchymal transition, invasion and migration of RCC cells. Loss of MTAP resulted in an activation of IGF1R signaling in RCC cells. Taken together, our findings indicate a major contribution of MTAP loss to kidney cancer cell malignancy and provide a viable biomarker for tumor detections.

Citation Format: Carissa Huang, Jihao Xu, Yichin Wu, Robert H. Weiss, Ching-Hsien Chen. MTAP regulates cell malignancy and is novel prognostic factor for kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4482.