bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1786: One in four Hispanic women with early onset breast cancer carry BRCA1, BRCA2, or PALB2 mutations: Results from a population-based study in South America
Authors: Tuazon Anna Marie , Bohorquez Mabel , Ramirez Carolina , Lott Paul , Estrada Ana , Criollo Angel , Wang Cathy , Echeverry Magdalena , Suarez John ,   COLUMBUS Consortium,   LaFamilia Consortium, Carvajal Carmona Luis G.
Published: July 2016

Breast cancer is the leading cause of cancer incidence and mortality among Hispanic women, who are often diagnosed with late stage tumors and are more likely to die after diagnosis when compared to non-Hispanic whites. While strides have been made in understanding Hispanic breast cancer genetics, most studies have been limited to investigating cases from high-risk clinics. In this study, we aimed to assess the prevalence of pathogenic mutations in three most important breast cancer genes (BRCA1, BRCA2 and PALB2) in an unselected Hispanic breast cancer cohort. Initially, six hundred and forty-six unselected Colombian breast cancer cases were screened for four known Hispanic BRCA1/2 mutations by genotyping. Subsequently, cases that remained mutation negative, as well as 186 cancer-free controls, were screened for BRCA1, BRCA2 and PALB2 mutations using targeted next generation sequencing. We identified 67 cases with pathogenic mutations, one case with a likely-pathogenic variant, and 16 carriers of variants of unknown significance. Among the pathogenic mutation carriers, 88.1% harbored a founder mutation (n = 59), which includes the known BRCA1 3450del4 mutation, strikingly found in 32 unrelated cases. Remarkably, we found that 1 in 4 of the cases diagnosed with breast cancer by age 40 years, regardless of family history of cancer, carried a pathogenic mutation. The high frequency of pathogenic mutations in this unselected cohort (10.4%), in particular those with an early age of onset (25.3%), suggests that population-based genetic testing among Hispanic communities can identify most carriers who would otherwise ineligible for testing. Identifying mutation carriers of these genes has implications in clinical management and surveillance for Hispanic women, a population that is vulnerable to breast cancer disparities in the U.S. and Latin America.

Citation Format: Anna Marie Tuazon, Mabel Bohorquez, Carolina Ramirez, Paul Lott, Ana Estrada, Angel Criollo, Cathy Wang, Magdalena Echeverry, John Suarez, COLUMBUS Consortium, LaFamilia Consortium, Luis G. Carvajal Carmona. One in four Hispanic women with early onset breast cancer carry BRCA1, BRCA2, or PALB2 mutations: Results from a population-based study in South America. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1786.
Abstract 1797: Loss of FoxO1 expression is associated with decreased IGF-1R levels and alters IGFBP-1 induction in tamoxifen-resistant breast cancer cells
Authors: Vaziri-Gohar Ali , Houston Kevin D.
Published: July 2016

Breast cancer is one of the leading causes of cancer-related deaths in the world. The majority of breast cancers express estrogen receptor alpha (ERα) and tamoxifen, a selective estrogen receptor modulator, is commonly prescribed as adjuvant therapy for ERα-positive breast cancer patients. The development of chemoresistance is a common occurrence in treated patients, however, the molecular mechanisms associated with acquired tamoxifen resistance are not well defined. Previous work from our lab identified a novel mechanism of tamoxifen action that involved the accumulation of extracellular Insulin-like growth factor binding protein-1 (IGFBP-1) and modulates IGF-1-dependent signaling transduction. Tamoxifen-induced IGFBP-1 induction in this model was mediated by G protein-coupled estrogen receptor (GPER)-dependent CREB activation as shown by phosphorylation on serine 133. To determine IGFBP-1 induction is altered during the development of chemoresistance, this mechanism of action was studied in tamoxifen resistant MCF-7 (TamR) cells. Whereas CREB was constitutively phosphorylated in TamR cells, IGFBP-1 levels were not elevated when compared to parental cells and IGFBP-1 was not induced by tamoxifen treatment in TamR cells. These data suggest that another transcription factor(s) are also necessary for the induction of IGFBP-1 in breast cancer cells. FoxO1 is a known regulator of IGFBP-1 transcription and we tested whether FoxO1 contributes to tamoxifen-induced IGFBP-1 transcription. Our results indicate that FoxO1 expression levels were significantly reduced in TamR cells compared with parental cells. Furthermore, FoxO1 knockdown experiments demonstrated this transcription factor mediates tamoxifen-induced IGFBP-1 induction in MCF-7 cells. Consistent with previously published observations, IGF-1R expression was reduced in TamR cells compared to parental MCF-7 cells. To determine if loss of IGF-1R expression in involved in the loss of FoxO1 expression in TamR cells, IGF-1R was exogenously expressed and FoxO1 expression was measured. IGF-1R expression increases FoxO1 levels and partially restores tamoxifen-induced IGFBP-1 induction in TamR cells. These results suggest that FoxO1-mediated IGFBP-1 accumulation is altered during the development of tamoxifen resistance and is associated with decreased IGF-1R expression.

Citation Format: Ali Vaziri-Gohar, Kevin D. Houston. Loss of FoxO1 expression is associated with decreased IGF-1R levels and alters IGFBP-1 induction in tamoxifen-resistant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1797.
Abstract 1811: ERβ sensitizes NSCLC cells to chemotherapeutic agents by regulating DNA damage response
Authors: Bado Igor , Nikolos Fotis , Gustafsson Jan-Åke , Thomas Christoforos
Published: July 2016

The expression of wild-type estrogen receptor β (ERβ1) correlates with increased survival in patients with Non-Small Cell Lung Cancer (NSCLC). However, the molecular mechanism that accounts for this association is unknown. ERβ1 was previously shown to sensitize breast cancer cells to chemotherapeutic agents. The role of the receptor in regulating sensitivity of NSCLC cells to chemotherapy, a common treatment option for advanced disease, has not been studied. Here we show that upregulation of ERβ1 decreases the survival of NSCLC cells in response to treatment with doxorubicin. This effect was observed in p53-defective but not wild-type p53-expressing cells. ERβ1 enhanced G2/M cell cycle arrest in NSCLC cells by activating the checkpoint kinase 1 (Chk1) and altering downstream signaling. The expression of the p63 target gene cyclin G2 that regulates the G2/M checkpoint was induced by ERβ1 proposing an ERβ1-p63 transcriptional cooperation. Our findings suggest the involvement of ERβ1 in the regulation of DNA damage response in NSCLC cells and support the potential predictive value of the receptor in clinical management of the disease.

Citation Format: Igor Bado, Fotis Nikolos, Jan-Åke Gustafsson, Christoforos Thomas. ERβ sensitizes NSCLC cells to chemotherapeutic agents by regulating DNA damage response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1811.
Abstract 182: Dual inhibition of the MEK5 and PI3K pathways most effectively reduces proliferation and migration in triple negative breast cancer cells
Authors: Wright Thomas , Raybuck Christopher , Wendekier Katheryn , Cavanaugh Jane E.
Published: July 2016

Aberrations in the MAPK/extracellular signal-regulated kinase (MEK/ERK) and phosphoinositide-3-kinase (PI3K) pathways have been linked to increased proliferation and survival in triple negative breast cancer (TNBC) cells. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. Promising combinations of MEK and PI3K inhibition have been evaluated in phase I clinical trials for various cancer types. However, these clinical trials have had limited efficacy and have yet to encompass the MEK5/ERK5 pathway, which has been shown to promote cell survival. The goal of this study was to examine the crosstalk between the MEK1/2, MEK5, and PI3K pathways and determine the most promising combination of the MEK1/2, ERK5, and PI3K inhibitors, U0126, XMD8-92, and LY294002, respectively, in a diverse panel of triple negative breast cancer cell lines: BT549, MDA-MB-231, and MDA-MB-468. Our results indicate that dual inhibition of the MEK5 and PI3K pathways significantly reduced both proliferation (45.53%) and migration (39.37%) in MDA-MB-231 TNBC cells. In contrast, inhibition of ERK1/2 alone or in combination with PI3K or ERK5 inhibition yielded mixed responses. Interestingly, dual inhibition of the MEK5 and PI3K pathways increased the activity of the MEK1/2 pathway. These data suggest that crosstalk between these kinases occurs and dual inhibition of PI3K and ERK5 may be a novel therapeutic approach for treating TNBC.

Citation Format: Thomas Wright, Christopher Raybuck, Katheryn Wendekier, Jane E. Cavanaugh. Dual inhibition of the MEK5 and PI3K pathways most effectively reduces proliferation and migration in triple negative breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 182.
Abstract 1823: Mechanism of the anterograde transport of the androgen receptor
Authors: Li Jianzhuo , Zhang Guanyi , Park Hee-Won , Zhang Haitao
Published: July 2016

Androgen receptor (AR) is a ligand-activated nuclear receptor that plays a critical role in normal prostate physiology, as well as in the development and progression of prostate cancer. In addition to the classical paradigm in which AR exerts its biological effects by activating the transcription of its target genes, there is considerable evidence to support a rapid, non-genomic action mediated by membrane-associated AR, which facilitates the activation of kinase signaling cascades, leading to cell proliferation. While the nuclear translocation of AR is well studied, the molecular events controlling the recruitment of AR to the plasma membrane remain poorly understood. In this study, we set out to elucidate the mechanism underlying AR transport to the membrane. We found that a subpopulation of AR was localized to the membrane fraction shortly after androgen stimulation. Treatment with microtubule-targeting agents, but not actin inhibitors, blocked androgen-induced AR membrane localization, suggesting that AR transport to the membrane is mediated by the microtubule cytoskeleton. By utilizing dominant negative mutants, we identified the KIF5B, a member of the kinesin 1 family of motor proteins, is involved in the anterograde transport of AR. We further demonstrated that AR interacts directly with KIF5B, and such interaction is enhanced by androgens. Through a series of deletion constructs, a region includes the N-terminal and DNA binding domains of AR was determined to be involved in the association with KIF5B. Disruption of AR membrane transport decreased AKT phosphorylation. Together, our data suggest that KIF5B mediates AR membrane transport and regulates the non-genomic function of AR. Disruption of AR membrane translocation may represent a novel strategy to target AR signaling in prostate cancer.

Citation Format: Jianzhuo Li, Guanyi Zhang, Hee-Won Park, Haitao Zhang. Mechanism of the anterograde transport of the androgen receptor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1823.
Abstract 1829: The validation of a pyrosequencing KRAS mutation detection assay
Authors: Poulin Matthew L. , Meyer Ann , Mead E. Andrew , Xu Jessica , Drennan Ryan , Yan Liyin
Published: July 2016

Mutations in the KRAS gene, especially affecting the codon 12 and 13 region of the Kras protein, have implications in the treatment of certain cancer types. Because of the current potential for the FDA to oversee laboratory developed procedures (LPDs), it is imminently important that the validation of such tests be published to ensure that it is shown that these LPDs are properly reviewed and properly tested within the CLIA guidelines, thus keeping LPDs within the CLIA regulatory domain. We have developed a pyrosequencing assay that can detect eleven mutations in the codon 12 and 13 position of the KRAS gene. Our validation consisted of a sensitivity study in which a purified mutant PCR product was introduced into a wildtype DNA background and diluted down to undetectable levels. This sensitivity test was done on six different KRAS codon 12/13 mutants to determine the detection limit of the assay. Intra- and inter-assay precision and assay accuracy was determined by comparing assay results of over 40 total samples, both mutant and wild type, over five days carried out by two technicians. The results will show that this KRAS pyrosequencing assay falls within the acceptance criteria for sensitivity, accuracy and precision.

Citation Format: Matthew L. Poulin, Ann Meyer, E. Andrew Mead, Jessica Xu, Ryan Drennan, Liyin Yan. The validation of a pyrosequencing KRAS mutation detection assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1829.
Abstract 1841: Dissecting gene expression programs that define tumor aggression and patient outcome in pancreatic cancer
Authors: Fassler Danielle J. , Escobar-Hoyos Luisa F. , Shroyer Kenneth R.
Published: July 2016

By the year 2020, pancreatic cancer (PDAC) is projected to be the second leading cause of cancer deaths in the United States. Current systemic therapies offer limited survival advantages in subgroups of patients, suggesting underlying biological differences in this deadly cancer. Our lab has recently identified keratin-17 (K17) gene expression as a prognostic and predictive biomarker in PDAC. Furthermore, we found that K17 acts as an oncoprotein by promoting degradation of tumor suppressor p27KIP1, while knockdown of K17 limits tumor size and sensitizes cancer cells to chemotherapy agents. Together, these findings suggest that targeting K17 expression in PDACs may increase patient survival and response to treatment. The factors that lead to increased K17 gene expression in only a subset of PDAC cases, however, are unknown. K17 is expressed in embryonic ectoderm and aberrantly induced in keratinocytes from psoriasis patients through paracrine signaling from the microenvironment. To determine how K17 gene expression is upregulated in PDAC, we tested the effects of two major contributing factors in PDAC pathogenesis: driver mutations and inflammatory mediators present in tumor microenvironment. Using in vitro and in vivo loss- and gain-of function approaches, we modulated oncogene expression in and performed acute cytokine treatments on cells to test for changes in K17 gene expression. K17 expression was evaluated by qRT-PCR and western blot. We found that forced expression of oncogenes like KRASG12D, Q61K or TP53R248W significantly increase K17 expression, yet these cell-autonomous factors are not sufficient to explain K17 gene expression in PDACs. In addition, we found that pro-inflammatory cytokines, typically released by helper T cells, induce a strong increase in K17 gene expression. Together these findings suggest that K17 gene expression across otherwise clinically identical PDAC cases may be explained by early transformation events and maintained by positive feedback loops associated with patient specific adaptive immune response. Ongoing studies are seeking to test the combined effects of genetic and paracrine events in K17 expression, and understand the signaling pathways and transcription factors downstream of these inducing factors. Ultimately, insight into the mechanisms that mediate K17 overexpression in PDACs could help guide future research to develop K17 as a potential therapeutic target.

Citation Format: Danielle J. Fassler, Luisa F. Escobar-Hoyos, Kenneth R. Shroyer. Dissecting gene expression programs that define tumor aggression and patient outcome in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1841.
Abstract 1842: Overexpressing human endogenous retroviral elements (HERVs) in chemorefractory colorectal adenocarcinoma cells are repressed by antiviral drugs
Authors: Diaz-Carballo David
Published: July 2016

Background: HERVs account for ca. 8% of all transposable elements found in the genome of humans and other animals. They represent a genetic footprint of ancestral germ-cell infections of exoviruses that is transmittable to the progeny by Mendelian segregation. Traces of human endogenous retroviruses are physiologically expressed in ovarial, testicular and placental tissues as well as in stem cells. In addition, a number of these fossil viral elements have also been related to carcinogenesis. However, a relation between endoretroviruses expression and chemoresistance has not been reported yet. Methods: Twenty colorectal carcinoma patient samples were analyzed for HERV-WE1 and HERV-FRD1 endoretroviruses using immunohistochemical approaches. In addition, to examine for differential expression of HERVs in chemotherapy refractory cells, a resistant HCT8 colon carcinoma subline was developed by serial etoposide exposure. Endoretroviral elements were detected by immunocytochemical staining, qPCR and ELISA. IC50-values of antiviral and cytostatic drugs in HCT8 cells were determined by MTT proliferation assay. The antivirals-cytostatics interaction was evaluated by the isobologram method. Results: In this work, we show for the first time that HERV-WE1, HERV-FRD1, HERV-31, and HERV-V1 are a) simultaneously expressed in treatment-naïve colon carcinoma cells from patients and b) upregulated after cytostatic exposure in colon-cancer cell lines, suggesting that these retroviral elements are intimately related to chemotherapy resistance. A number of antiviral drugs showed not only cytotoxic activity but also downregulation of HERV proteins in vitro. We also demonstrate that the use of different antiviral compounds alone or in combination with anticancer agents present synergistic antiproliferative activity and downregulation of different endoretroviral elements in highly chemotherapy-resistant colorectal tumor cells. Conclusions: Enhanced HERV-expression is associated with chemo-resistance in colon carcinoma cells which can be repressed by antiviral drugs alone or in combination with anticancer drugs. Therefore, the introduction of antiviral compounds to antiproliferative chemotherapy regimens potentially improves patient outcomes.

Citation Format: David Diaz-Carballo. Overexpressing human endogenous retroviral elements (HERVs) in chemorefractory colorectal adenocarcinoma cells are repressed by antiviral drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1842.
Abstract 1859: Bioinformatic and experimental evaluation of regulatory variants in breast cancer susceptibility genes
Authors: Sevcik Jan , Burke Leslie , Gambino Gaetana , Brewster Brooke L. , Tudini Emma , Whiley Philip J. , Manoukian Siranoush ,   ENIGMA Consortium, van Overeem Hansen Thomas , Santamariña Pena Marta , Vega Ana , Caligo Maria A. , Radice Paolo , Peterlongo Paolo , Rouleau Etienne , Spurdle Amanda B. , Brown Melissa A.
Published: July 2016

Many germline DNA sequence variants in gene regulatory elements are associated with an increased risk of cancer. These include large genomic deletions of the promoter of the BRCA1 gene, through to single nucleotide variants in the promoter, UTRs and long-range enhancers of multiple other cancer related genes. Next generation sequence analysis of early onset and familial breast cancer cases is currently identifying an escalating number of variants in non-coding regions of the genome, however for most of these the significance in unknown. This project aims to determine the functional and clinical significance of breast cancer associated variants in regulatory regions of BRCA1 and BRCA2, as part of an international collaborative project arising from the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. To date over two hundred variants mapping to promoter, UTR and deep intronic regions have been identified in early onset or familial breast cancer patients that have no identifiable coding or splicing changes in BRCA1 or BRCA2. Through a pipeline of bioinformatics analyses using ENCODE datasets, these variants have been prioritized for experimental evaluation. This has lead to the identification of several variants that significantly alter the regulation of BRCA1 or BRCA2 expression, through mechanisms including altered binding of transcription factors and microRNAs. These studies will lay the groundwork for comprehensive statistical analyses, in which bioinformatics and experimental data is combined with clinical and genetic data to establish multifactorial risk prediction models that can ultimately be used in a clinical setting.

Citation Format: Jan Sevcik, Leslie Burke, Gaetana Gambino, Brooke L. Brewster, Emma Tudini, Philip J. Whiley, Siranoush Manoukian, ENIGMA Consortium, Thomas van Overeem Hansen, Marta Santamariña Pena, Ana Vega, Maria A. Caligo, Paolo Radice, Paolo Peterlongo, Etienne Rouleau, Amanda B. Spurdle, Melissa A. Brown. Bioinformatic and experimental evaluation of regulatory variants in breast cancer susceptibility genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1859.
Abstract 1861: The role and regulation of MTA1-ETS2 axis in prostate cancer
Authors: Kumar Avinash , Dhar Swati , Butt Nasir , Rimando Agnes , Martinez Luis , Levenson Anait
Published: July 2016

Overexpression of the chromatin modifier protein, metastasis associated 1 (MTA1) in prostate cancer contributes to tumor aggressiveness, but the molecular mechanism involved has not been fully elucidated. MTA1 ChIP-Seq analysis in prostate tissues from the prostate-specific Pten heterozygous mice, which exhibit elevated levels of MTA1 revealed MTA1 recruitment onto the ETS2 promoter. Loss of function studies with MTA1 in various prostate cancer cell lines exhibited reduced expression for ETS2 at both mRNA and protein levels. We also observed increased expression of ETS2 at both mRNA and protein levels in prostate tissues from the prostate-specific MTA1 transgenic mice. Meta-analysis of prostate cancer samples in the GEO database exhibited a positive correlation between MTA1 and ETS2 levels. These results suggested that MTA1 functions as a coactivator for regulating ETS2 expression in prostate cancer. We found direct interaction between MTA1 and ETS2 in cells expressing ectopic MTA1 and ETS2. Interestingly, the prostate-specific Pten heterozygous mice, when fed with diet supplemented with pterostilbene, a natural pharmacological inhibitor of MTA1, showed a decrease in ETS2 mRNA and protein levels, perhaps due to decreased recruitment of MTA1 onto the ETS2 promoter. These results suggest that MTA1 functions as a coactivator for regulating ETS2 expression in prostate cancer at the transcriptional level and co-operates with ETS2 to promote prostate cancer progression. At the same time, pterostilbene may become a novel chemopreventive and therapeutic candidate for clinical development in prostate cancer due to its ability to target the MTA1-ETS2 axis.

Citation Format: Avinash Kumar, Swati Dhar, Nasir Butt, Agnes Rimando, Luis Martinez, Anait Levenson. The role and regulation of MTA1-ETS2 axis in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1861.
Abstract 1865: Areca nut-induced JNK/ATF2/Jun axis regulates TGF-beta signaling contributing to pre-cancerous oral submucous fibrosis
Authors: Pant Ila , Kondaiah Paturu
Published: July 2016

Oral Submucous Fibrosis (OSF) is an inflammatory, pre-cancerous condition of the oral submucosa. It affects individuals with prolonged areca nut chewing habit and therefore areca nut is regarded as the etiological agent for OSF manifestation. Transforming Growth Factor beta is an established promoter of cancer and fibrosis. Up-regulation of TGF-β and its target genes has also been reported in OSF. However, the underlying mechanism for the regulation of TGF-β by areca nut has not been elucidated.

The present study demonstrates that TGF-β pathway is activated in epithelial cells after two hour exposure to areca nut extract. Areca nut increases TGF-β transcript as well as protein and thereby the downstream canonical Smad2/3 signaling. Pre-treatment with various pathway inhibitors showed JNK pathway activation as a pre-requisite to TGF-β pathway activation by areca nut. Areca nut activated JNK pathway within half an hour of treatment and this was reversed by atropine treatment suggesting involvement of muscarinic acid receptor signaling. Second messengers upstream to JNK activation and regulated by areca nut were calcium/CAMKII and reactive oxygen species. Further, areca nut treatment resulted in JNK dependent phosphorylation of ATF2 and c-Jun transcription factors. These were found to bind TGF-β promoter, as revealed by chromatin immunoprecipitation experiments. Expectedly, knockdown of these transcription factors abrogated areca nut induced TGF-β pathway activation.

Based on the data obtained a model is proposed to elucidate activation of TGF-β pathway by areca nut extract involving activation of JNK and ATF2, c-Jun resulting in the over expression of TGF-β in epithelial cells which may contribute to the manifestation of OSF. Thus, this study highlights ATF2/c-Jun as the critical nodes for therapeutic intervention to prevent TGF-β over production in OSF.

Funding by DST and CSIR is acknowledged.

Citation Format: Ila Pant, Paturu Kondaiah. Areca nut-induced JNK/ATF2/Jun axis regulates TGF-beta signaling contributing to pre-cancerous oral submucous fibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1865.
Abstract 1872: Development of reference reagents to accelerate research on the RAS pathway
Authors: Esposito Dominic , Mehalko Jennifer , Wall Vanessa , Grose Carissa , Gillette William , Stephens Robert
Published: July 2016

The RAS Reference Reagents program within the NCI RAS Initiative is produces and distributes qualified DNA vectors, cell lines, and viruses to the external community to facilitate research into RAS biology and development of RAS cancer therapeutics. DNA vectors representing a variety of RAS mutations, as well as a complete collection of 180 genes in the RAS pathway are available as Gateway-compatible Entry clones. These clones represent the most commonly expressed transcript forms of the 180 genes, many of which were not previously available in full-length, wildtype forms. The vectors can be used with the FNLCR combinatorial cloning library to easily generate a large number of different types of expression clones with various promoters, fusion tags, and plasmid backbones. Additional reagents under construction include validated plasmid and lentiviral reagents for shRNA and CRISPR delivery, qualified cell lines for use in research into the RAS pathway, and systems for the generation of highly purified and characterized Ras and Ras-related proteins. A highlight of these reagents are materials for the high yield production of properly processed, prenylated KRAS protein using an engineered insect cell expression system. This system could be used to produce other prenylated small GTPases at levels not previously attainable. All of the resources of the RAS Reference Reagents program are readily available to the academic community through simple mechanisms, and can be licensed to industry scientists as well. The program is also interested in identifying the needs of the community for additional RAS-related resources, and is developing methods for vetting requests from the outside to provide additional reagents.

Citation Format: Dominic Esposito, Jennifer Mehalko, Vanessa Wall, Carissa Grose, William Gillette, Robert Stephens. Development of reference reagents to accelerate research on the RAS pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1872.
Abstract 1873: The role of p21-activated kinase 4 (PAK4) in cancer stemness and epithelial-to-mesenchymal transition
Authors: Azmi Asfar S. , Muqbil Irfana , Aboukameel Amro , Senapedis William , Baloglu Erkan , Landesman Yosef , Shacham Sharon , Kauffman Michael , Philip Philip A. , Mohammad Ramzi M.
Published: July 2016

The p21-activated kinase 4 (PAK4) is a key downstream effector of the Rho family GTPases. PAK4 is found to be over-expressed in many oncogenic Ras-driven cancers and cell lines but not in their normal counterparts. Mesenchymal pancreatic ductal adenocarcinoma (PDAC) stem cells (CSCs) that are triple positive for stemness markers (CD133+, CD44+, and EpCam+) as well as Ras and snail transduced human mammary epithelial cells (HMLER-snail) showed enhanced expression of PAK4 along with activation of Rho, Rac1 and CDC42. PAK4 RNA interference resulted in disruption of PDAC CSC spheroids, reduction of mesenchymal markers (i.e. vimentin and snail) and suppression of CSCs potential to form subcutaneous tumors in mice. These findings point to a novel role of PAK4 in promoting oncogenic Ras-driven EMT and stemness. Using PDAC CSCs (MiaPaCa-2 and L3.6pl) and human mammary epithelial cells (HMECs) that are transduced with Ras and the EMT promoter snail (HMLE-Snail and HMLER-Snail), we investigated the impact of PAK4 allosteric modulators [PAMs (KPT-7189, KPT-7523, KPT-9274 and KPT-9307)] on the reversal of EMT and stemness. The toxicity and efficacy of PAMs were evaluated in vitro and in multiple sub-cutaneous xenograft models. PAMs show anti-proliferative activity in vitro against different PDAC CSCs and HMECs while sparing normal cells. Cell growth inhibition was concurrent with apoptosis induction, suppression of colony formation and reversal of mesenchymal morphology to epithelial (MET). PAMs not only reduced PAK4 RNA and protein steady state levels but also inhibited proliferative and anti-apoptotic signals downstream of PAK4. PAMs caused suppression of EMT markers (EpCAM, vimentin and snail) and re-expression of an epithelial promoter E-cadherin. PAM treatment inhibited RhoA, Rac1 and CDC42 activation in GLISA assays. KPT-9274 showed remarkable anti-tumor activity in sub-cutaneous xenograft models of CSCs and residual tumors showed suppression of EMT markers that was concurrent with inhibition of PAK4 signaling. These studies open a novel therapeutic opportunity against EMT and cancer stem cells through PAK4 inhibition.

Citation Format: Asfar S. Azmi, Irfana Muqbil, Amro Aboukameel, William Senapedis, Erkan Baloglu, Yosef Landesman, Sharon Shacham, Michael Kauffman, Philip A. Philip, Ramzi M. Mohammad. The role of p21-activated kinase 4 (PAK4) in cancer stemness and epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1873.
Abstract 1876: Reduced Pak1 activity sensitizes FA/BRCA-proficient breast cancer cells to PARP inhibition
Authors: Villamar-Cruz Olga , Prudnikova Tatiana , Johnson Neil , Chernoff Jonathan , Arias Romero Luis E.
Published: July 2016

Cells that are deficient in homologous recombination, such as those that have mutations in any of the Fanconi Anemia (FA)/BRCA pathway genes, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, FA/BRCA-deficient tumors represent only a small fraction of breast cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. p21-activated kinase 1 (Pak1) is a serine/threonine protein kinase located in the chromosome 11q13 and is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme controls many cellular processes by phosphorylating its downstream substrates; in addition to its role in the cytoplasm, Pak1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that Pak1 activation is a component of the DNA damage response. Here, we show that depletion or inhibition of Pak1 down-regulated the expression of several genes involved in the FA/BRCA pathway and compromised the ability of cells to repair DNA by homologous recombination, induced cell cycle arrest, promoted apoptosis and resulted in reduced colony formation. Combined inhibition of Pak1 and PARP in pak1 amplified breast cancer cells had a synergistic effect, enhanced apoptosis, resulted in reduced colony formation and delayed tumor growth in a xenograft setting. Inhibition of Pak1 did not sensitize non-transformed or Pak1 non-amplified cells to inhibition of PARP. Because reduced Pak1 activity impaired FA/BRCA function and consequently, repair by homologous recombination, inhibition of this kinase in pak1 amplified and/or overexpressing breast cancer cells represents a plausible strategy for expanding the utility of PARP inhibitors to FA/BRCA-proficient cancers.

Citation Format: Olga Villamar-Cruz, Tatiana Prudnikova, Neil Johnson, Jonathan Chernoff, Luis E. Arias Romero. Reduced Pak1 activity sensitizes FA/BRCA-proficient breast cancer cells to PARP inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1876.
Abstract 1878: Inhibition of the Transforming Growth Factor beta pathway in human glioblastoma cell lines induces apoptosis and inhibits anoikis escape
Authors: Gallo-Oller Gabriel , Dotor Javier , Fan Xing , Castresana Javier S.
Published: July 2016

Glioblastoma Multiforme (GBM) is the most prevalent malignant brain tumor accounting for 60-70% of all gliomas. Current achieved median patient survival ranges only 12-15 months, and improvements in survival over the last century can be measured only in weeks. A hallmark of this malignancy is the intrinsic resistance to current therapies. The Transforming Growth Factor beta (TGF-beta) signaling pathway plays a key role in GBM. Several inhibitors of different elements and regulators of the TGF-beta pathway have entered to clinical trials.

Here, we analyzed the potential effect of P144, a TGF-beta pathway inhibitor peptide, over cell proliferation and apoptosis in commercial GBM cell lines (A172 and U-87 MG). We found that treatment with P144 significantly decreased cell proliferation of both cell lines analyzed. In addition, different apoptosis determinations such as ELISA and Acridine Orange/Ethidium Bromide (AO/EB) staining, confirmed a significant increase in apoptosis in both cell lines.

Quantification of SMAD2 phosphorylation status and its nuclear translocation, by western blot, confirmed the inhibition of TGF-beta signaling by P144. These data support the correlation between TGF-beta pathway inhibition and the apoptotic process induction.

Anoikis escape sustains invasiveness and metastatic potential in GBM, and has been established as a clear target against tumor progression. Due to P144 clear effect on apoptosis, we analyzed its influence on anoikis escape. The inhibition of anoikis escape by P144 was confirmed in A172 and U-87 MG cell lines under in vitro anchorage-independent culture conditions. The significant upregulation of BAX, Lamin A and Lamin C produced by P144 confirmed the induction of this apoptotic subtype process in the analyzed cell lines.

We can conclude that P144 increases apoptosis and induces anoikis through inhibition of the TGF-beta signaling pathway. Our results highlight the therapeutic potential of P144 for the treatment against GBM. However, previous to further clinical development, additional studies are required in order to confirm the effect of P144 over GBM in the brain environment, as well as to explore the therapeutic potential of P144 in combination with current and emerging molecular based therapies.

Citation Format: Gabriel Gallo-Oller, Javier Dotor, Xing Fan, Javier S. Castresana. Inhibition of the Transforming Growth Factor beta pathway in human glioblastoma cell lines induces apoptosis and inhibits anoikis escape. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1878.
Abstract 1879: Structural and biochemical characterization of farnesylated and methylated KRAS-membrane interactions
Authors: Van Que , Gillette William K. , Esposito Dominic , Ghirlando Rodolfo , Heinrich Frank , Stephen Andrew G.
Published: July 2016

KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed in several enzymatic steps that results in the addition of a farnesyl and a methyl group to the terminal cysteine residue. Plasma membrane localization of KRAS4b requires this processing along with a polybasic stretch of lysines within the hypervariable region. KRAS4b can bind to RAF-RBD in solution but membrane bound KRAS4b is required for RAF-kinase activation. In order to determine why membrane interaction of KRAS4b is required for MAPK signal transduction we under took a structural and biochemical analysis of KRAS4b-membrane interactions. We have developed methods using an engineered baculovirus for the insect cell production of farnesylated and methylated KRAS4b (KRAS4b-FME) and this material was used in these studies. We have demonstrated that the stable interaction of KRAS4b-FME to lipid Nanodiscs and tethered lipid bilayers is dependent on the presence of anionic phospholipids. Using analytical ultracentrifugation we have evaluated the binding stoichiometry of KRAS4b-FME with Nanodiscs. In addition we have used neutron reflectivity to determine the orientation of KRAS4b-FME on tethered lipid bilayers.

Citation Format: Que Van, William K. Gillette, Dominic Esposito, Rodolfo Ghirlando, Frank Heinrich, Andrew G. Stephen. Structural and biochemical characterization of farnesylated and methylated KRAS-membrane interactions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1879.
Abstract 1883: Treatment with histone deacetylase (HDAC) and BRAF inhibitors upregulates the expression of the plasma membrane Ca2+ pump, PMCA4b and alters intracellular Ca2+ handling in BRAF mutant melanoma cells
Authors: Hegedus Luca , Garay Tamas , Molnar Eszter , Varga Karolina , Padanyi Rita , Paszty Katalin , Hegedus Balazs , Wolf Mathias , Grusch Michael , Kallay Eniko , Enyedi Agnes
Published: July 2016

The aim of our study was to test the effects of histone deacetylase (HDAC) and BRAF inhibitors on the expression and activity of the plasma membrane Ca2+ pump isoform 4b (PMCA4b) in BRAF mutant melanoma cell lines. Remodeling of Ca2+ homeostasis during malignancy is caused by the rearrangement of the Ca2+ signaling machinery, including Ca2+ pumps, Na+/Ca2+ exchangers and channels. Plasma membrane calcium ATPases (ATP2B - or PMCA) maintain the resting low intracellular calcium concentration by pumping out excess calcium from the cytosol. Changes in PMCA expression during malignant transformation have been described previously in colorectal and breast cancer cells, and most recently by our group in melanomas. We found that in BRAF mutant melanoma cells the PMCA 4b protein level was markedly elevated by BRAF inhibitor treatment. Overexpression of PMCA4b suppressed motility and metastatic potential. Previously, it was shown that HDAC inhibitors-induced differentiation up-regulated PMCA4b expression in gastric, colon and breast cancer cells. In the present study we treated BRAF mutant and BRAF wild type melanoma cells with HDAC inhibitors (SAHA (suberanilohydroxamic acid) or valproic acid) and tested the changes in abundance, localization and function of PMCA4b. Expression levels of the PMCA proteins were analyzed by qRT-PCR and Western Blotting. Subcellular localization of the PMCAs and the effects of treatments on cytosolic Ca2+ signaling were analyzed by confocal microscopy. We found that treatment with the HDAC inhibitors increased the level of PMCA4b expression at both mRNA and protein levels in both BRAF wild type and BRAF mutant cell types. The increased PMCA4b level was coupled with enhanced plasma membrane localization and with a faster Ca2+ clearance after stimulation. Our results show that in melanoma cells the expression of PMCA4b is under epigenetic control and HDAC inhibitors efficiently enhanced PMCA4b expression independent of the BRAF status of cells.

Citation Format: Luca Hegedus, Tamas Garay, Eszter Molnar, Karolina Varga, Rita Padanyi, Katalin Paszty, Balazs Hegedus, Mathias Wolf, Michael Grusch, Eniko Kallay, Agnes Enyedi. Treatment with histone deacetylase (HDAC) and BRAF inhibitors upregulates the expression of the plasma membrane Ca2+ pump, PMCA4b and alters intracellular Ca2+ handling in BRAF mutant melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1883.
Abstract 1894: Understanding the role of microRNA-206 in breast cancer
Authors: Adorno-Cruz Valery , Liu Huiping
Published: July 2016

MicroRNAs are short non-coding RNA sequences that that can modify genes in post transcriptional manner and are often de-regulated in cancer. Cancer stem cells, a subset of cancer cells with stem cell properties, are considered the root of tumorigenesis and seeds of metastasis. In this study we are interested in elucidating the role of miR-206 in breast cancer stem cell-mediated tumorigenesis and metastasis. We have observed that microRNA-206 (miR-206) regulate the metastatic traits of breast cancer cells, both in vitro and in vivo. Our studies have elucidated a signaling cascade that culminates in the silencing of cytokine (such as interleukin) and integrin expression. Knockdown of the interleukin and integrin genes by siRNAs mimics the phenotype of miR-206 expression in breast cancer cells. DNA content analysis using propidium iodide staining revealed that knockdown of miR-206 targets as well as over-expression of miR-206 in MDA-MB-231 cells regulates cell cycle arrest. Our results indicate that miR-206 mediates its regulatory effects through inhibiting multiple targets. MiR-206 may serve as a new therapeutic candidate for the future treatment of breast cancer.

Citation Format: Valery Adorno-Cruz, Huiping Liu. Understanding the role of microRNA-206 in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1894.
Abstract 1908: Lung tumorigenesis and metastasis is suppressed by the miR-200-GATA4/6-BMP4 axis
Authors: Kim Jeong Seon , Kurie Jonathan M. , Ahn Young-Ho
Published: July 2016

MicroRNA-200 (miR-200) family members suppress the epithelial-mesenchymal transition of various cancer cells, including lung adenocarcinoma cells. Previously, we found that forced expression of miR-200 decreased bone morphogenetic protein 4 (BMP4). In this study, we explored the mechanism and role of BMP4 depletion by miR-200 in murine lung adenocarcinoma cells. miR-200 down-regulated BMP4 via direct targeting of the GATA4 and GATA6 transcription factors that stimulate Bmp4 transcription. Bmp4 knockdown suppressed cancer cell growth, migration, and invasion and inhibited tumorigenesis and metastasis of lung cancer cells when injected into syngeneic mice. In addition, BMP4 was required for normal sphere formation in Matrigel 3-D culture, which might be mediated by MYH10, a downstream target of BMP4. These findings suggest that BMP4 functions as a pro-tumorigenic factor in a murine lung cancer model, and its transcription is regulated by miR-200 and GATA4/6.

Citation Format: Jeong Seon Kim, Jonathan M. Kurie, Young-Ho Ahn. Lung tumorigenesis and metastasis is suppressed by the miR-200-GATA4/6-BMP4 axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1908.