bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3311: The cholesterol/ 27-hydroxycholesterol axis is a novel therapeutic target in castrate resistant prostate cancer
Authors: Alfaqih Mahmoud A. , Nelson Erik R. , Safi Rachid , Jasper Jeff , Chang Ching-yi , Freedland Stephen J. , McDonnell Donald P.
Published: May 2015

The most recent estimates indicate that men have a seventeen percent chance of developing invasive prostate cancer (PCa) over their lifetime. This makes PCa the most commonly diagnosed malignancy among men in industrialized countries, accounting for the second leading cause of cancer-related deaths. Although PCa almost always develops resistance to androgen-deprivation therapy, both androgen receptor (AR) itself and the processes downstream of the receptor remain active and are necessary for cancer progression and thus continue to be viable targets for therapeutic intervention. In this regard, it is of significance that several independent studies demonstrate a link between elevated intra-prostatic levels of cholesterol and the presence of malignancy. This puts into context our current finding that 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol, regulates pathways and processes that oppose AR action. For example, AR activation results in a net uptake of cholesterol by PCa cells and enhances denovo lipogenesis, while 27HC down-regulates genes involved in cholesterol uptake and activates those involved in its efflux. More importantly, we show that treatment of PCa cell lines with 27HC inhibits their proliferation, an activity that is reversed upon supplementing the cells with an exogenous source of cholesterol or upon over-expression of low density lipoprotein receptor (LDLR), a membrane protein that mediates the uptake of cholesterol. In prostatic tissues, 27HC is synthesized directly from cholesterol by the cytochrome P450 enzyme, CYP27A1. To further highlight the above axis as a novel therapeutic target, using data mining approaches from clinical databases, we show that (1) transcript levels of CYP27A1 are dramatically down-regulated during PCa progression and (2) patients whose tumors express high levels of CYP27A1transcript exhibit longer disease free survival. Most notably, we were able to show that over-expression of CYP27A1 inhibits the proliferation of PCa cell lines in vitro and delays the growth of xenografts in castrated immune-deficient mice. In summary, our data thus far strongly implicates that CYP27A1 is a potential tumor suppressor in PCa and highlights the cholesterol/27HC axis as a target for the development of novel therapeutics for castrate resistant disease.

Citation Format: Mahmoud A. Alfaqih, Erik R. Nelson, Rachid Safi, Jeff Jasper, Ching-yi Chang, Stephen J. Freedland, Donald P. McDonnell. The cholesterol/ 27-hydroxycholesterol axis is a novel therapeutic target in castrate resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3311. doi:10.1158/1538-7445.AM2014-3311
Abstract 3313: 17β-Estradiol-ERβ axis confers proliferation and migration through IGF-1R-dependent expression of TMPRSS2 in castration-resistant PC-3 cells
Authors: Kim Hogyoung , Datta Amrita , Elmageed Zakaria Y. Abd , Abdel-Mageed Asim B.
Published: May 2015

Prostate cancer (PCa) is the most common and the second leading cause of cancer-related death among males in the Western world. TMPRSS2, a gene that encodes serine-protease type II transmembrane domain, has been shown to be frequently prevalent as a TMPRSS2-ERG gene fusion during early event in the genesis of PCa. However, TMPRESS2 function remains largely unknown, especially in castration-resistant PCa cells. While TMPRESS2 has previously been shown to be an androgen-regulated gene, TMPRSS2-ERG expression is regulated by a novel ERβ-dependent mechanism. The objectives of this study are to determine the mechanisms of expression and the functional significance of TMPRSS2 in growth and migration of castration-resistant AR-null PC-3 cells. 17β-Estradiol (E2; 10nM) induced TMPRSS2 expression (4-fold higher) in an ERβ-dependent manner as evidenced by shRNA silencing, but not by ERα-specific shRNA, in PC-3 cells. Intriguingly, the E2-induced TMPRSS2 expression was blocked by AG1024 (IGF-1R inhibitor) or knockdown of IGF-1R by siRNA, but not by AG1478 (EGFR inhibitor), suggesting that IGF-1R mediates expression of TMPRESS2 by the E2-ERβ axis in PC-3 cells. Moreover, TMPRSS2-specific siRNA markedly attenuated E2-induced cell growth and migration of PC-3 cells, suggesting that TMPRSS2 may be pivotal to E2-mediated progression of castration-resistant disease in vivo. The effects of TMPRSS2 silencing seem to be mediated through inhibition of E2-induced activation of NF-κB-signaling in these cells. Taken together, our results unraveled the underlying mechanisms that govern the expression and function of TMPRSS2 in mediating E2-induced growth and migration of castration-resistant PC-3 cells. Delineation of such critical players may culminate in identifying therapeutic targets to selectively circumvent castration-resistant outgrowth in PCa patients.

Citation Format: Hogyoung Kim, Amrita Datta, Zakaria Y. Abd Elmageed, Asim B. Abdel-Mageed. 17β-Estradiol-ERβ axis confers proliferation and migration through IGF-1R-dependent expression of TMPRSS2 in castration-resistant PC-3 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3313. doi:10.1158/1538-7445.AM2014-3313
Abstract 3315: RON-mediated hijacking of AR signaling in androgen-independent prostate cancer
Authors: Batth Izhar Singh , Meng Peng , Bedolla Roble , Reddick Robert E. , Kumar Addanki Pratap
Published: May 2015

Despite significant progress in developing effective strategies for management of localized prostate cancer, the 5-year survival for metastatic castrate-resistant disease is less than 30%. Previous studies from our own and other laboratories showed the anti-tumorigenic potential of 2-Methoxyestradiol (2-ME2) in various tumor models. Although variety of mechanisms including anti-angiogenic, pro-apoptotic, cell cycle deregulation has been reported to contribute to 2-ME2-mediated biological effects, the precise mechanism is unclear. To explore the role of 2-ME2, we carried out gene expression array analysis and discovered Recepteur d’Origine Nantais (RON) as one of the most significantly down regulated gene in response to 2-ME2 treatment. Subsequently, RON was found to be significantly upregulated in advanced-stage PCA cell lines, which do not express androgen receptor (AR); and high-grade human prostate tumors. Stable silencing of RON in androgen independent, PTEN-/- PC3 cells showed noticeable changes in cell morphology, actin filament organization, as well as markers of epithelial-to-mesenchymal transition (EMT). In PTEN wild type DU145 cells, RON knockdown caused an increase in AR expression and promoter activity but decreased the transcriptional activation and expression of AR target genes such as PSA. In contrast, RON overexpression in androgen-responsive LNCAP cells leads to a suppression of the transcriptional activation of both AR and its downstream targets, including PSA. Furthermore, we found that RON silencing decreases expression and promoter activity of the anti-apoptotic protein FLICE-like inhibitory protein (FLIP) that is accompanied by the induction of apoptosis. In addition, intervention with 2-ME2 reduced tumor growth that is accompanied with decreased RON expression in the prostate in transgenic adenocarcinoma of mouse prostate (TRAMP) model. These observations implicate RON as a hijacker of AR signaling that contributes in part to PCA growth. Therefore, restoration of native AR signaling via RON targeting could possibly be exploited in combination with antiandrogenic therapy to prevent recurrence of castrate-resistant prostate cancer. Supported by CA135451 (APK).

Citation Format: Izhar Singh Batth, Peng Meng, Roble Bedolla, Robert E. Reddick, Addanki Pratap Kumar. RON-mediated hijacking of AR signaling in androgen-independent prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3315. doi:10.1158/1538-7445.AM2014-3315
Abstract 3316: Characterizing mechanisms of resistance to androgen deprivation in prostate cancer
Authors: Botta Ginevra , Jane-Valbuena Judit , Wong Terence , Doench John , Garraway Levi A.
Published: May 2015

Despite significant advances in the treatment of prostate cancer (PCa), this malignancy remains a leading cause of cancer death among men. PCa cells require androgen receptor (AR) signaling for their growth and survival. Androgen deprivation therapy (ADT) constitutes the main therapeutic option for patients with advanced PCa. However, the major cause of death in men with metastatic prostate cancer involves progression to castration-resistant prostate cancer (CRPC). Characterizing mechanisms of resistance to ADT could enable the development of more effective therapeutic strategies. To identify genes whose silencing drives resistance to ADT in androgen-sensitive PCa cells, we performed a systematic genome-wide suppressor RNAi screen in the androgen-sensitive LNCaP cells, using a 98,000-shRNA library, targeting approximately 16,000 human genes. After infection, cells were cultured in absence of androgens for several weeks. Cells showing the ability to proliferate upon androgen deprivation were harvested at different time points and shRNA sequences were identified and quantitated through nextgen sequencing. Hit genes were those with at least 2 shRNAs enriched >4 fold, and whose silencing exerted the most robust effects on cell proliferation. To validate the identified top-tier genes in vitro, we infected LNCaP cells with lentiviruses carrying shRNAs (2 shRNAs/candidate gene) targeting the top ∼30 candidate genes, in parallel with 15 negative control shRNAs. We monitored the ability of each shRNA to increase proliferation under androgen-deprived conditions, using cell viability assays. To determine whether any shRNAs drive castration resistance by reactivating the AR-signaling pathway, we analyzed the expression of known AR effector genes in the presence of candidate shRNAs and also tested their effect on cell proliferation in the presence of the direct AR antagonist MDV3100. In future, we will also validate candidate genes in vivo, using a LNCaP xenograft model. This study has identified several genes whose silencing may modulate resistance to ADT in prostate cancer. Additional functional studies should determine whether a subset of these may promote new therapeutic strategies for men with CRPC.

Citation Format: Ginevra Botta, Judit Jane-Valbuena, Terence Wong, John Doench, Levi A. Garraway. Characterizing mechanisms of resistance to androgen deprivation in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3316. doi:10.1158/1538-7445.AM2014-3316
Abstract 3317: The cytosolic domain of a disintegrin and metalloprotease (ADAM)-15 promotes proliferation of non-small cell lung cancer (NSCLC)
Authors: Hou Hsin-Han , Yu Chong-Jen
Published: May 2015

Lung cancer is the leading cause of cancer-related deaths in worldwide, including in Taiwan. Lung cancer can typically be grouped into two large categories: small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which account for 15% and 85% of lung cancers, respectively. Despite the continuing endeavors by scientists and clinicians, the cure rate remains low and there is urgent to develop a novel target for NSCLC therapy. The a disintegrin and metalloprotease (ADAM)-15 is a transmembrane protease mediating a wide variety of activities including proteolysis, adhesion, cell fusion and signaling. The expression of ADAM-15 is significantly up-regulated in NSCLC compared to normal tissue. However, the mechanisms of ADAM-15-modulated tumorigenesis in NSCLC are remained unclear. The external enzymetic activity of ADAM15 doesn’t involve in the common pathways in tumoregenesis of NSCLC, such as HER3-EGFR signaling. ADAM15 isoforms are different in cytoplasmic tails, which may drive different biological behavior through intracellular signaling. Therefore, we hypothesized that the diverse cytosolic domain of ADAM15 isoforms plays a crucial role in tumoregenesis of NSCLC. In this study, using real-time quantitative PCR measuring the mRNA expression of 22 ADAMs in NSCLC, we identified a correlation of higher expression of ADAM15 in NSCLC of non-smokers (p=0.002, compared with ex- and current smokers) and in adenocarcinoma (p=0.038, compared with squamous cell ca). Comparing to non-invasive CL1-0 cells and matched normal tissue, CL1-5 cells and NSCLC tissue expressed more the longest ADAM-15 isoform with the largest amount of Src homology (SH)3-binding domain. The association ADAM-15 with SH3-motif containing protein, growth factor receptor-bound (Grb) 2, promoted the ability of proliferation in NSCLC in vitro and in vivo. In conclusion, we suggested that the cytosolic domain of ADAM-15 promotes proliferation of NSCLC via enhance of Grb2-associated signaling pathways, and the inhibition of ADAM-15 may be a potential therapeutic target for NSCLC.

Citation Format: Hsin-Han Hou, Chong-Jen Yu. The cytosolic domain of a disintegrin and metalloprotease (ADAM)-15 promotes proliferation of non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3317. doi:10.1158/1538-7445.AM2014-3317
Abstract 3320: Functional RANK expression is observed in disaggregated primary human lung tumors
Authors: Rossi John M. , Pan Yang , Dougall William C. , Branstetter Daniel , Jacob Allison , Canon Jude , Loberg Robert D.
Published: May 2015

The receptor activator of nuclear factor-κβ (RANK) and its ligand RANKL play a key role in the regulation of bone remodeling. RANK pathway activation has also been implicated in various aspects of lung tumor biology. In mouse models of NSCLC bone metastasis, RANKL inhibition led to decreased skeletal tumor progression and increased survival. By IHC, RANK expression has been reported to occur in approximately 60-70% of NSCLC cases profiled. Expression and function have been observed using human lung cancer cell line models. However, there is a lack of understanding with respect to the role RANK signaling plays in primary human lung tumor cells. To better understand RANK biology in lung cancer a novel platform was employed to measure RANK expression and pathway response to RANKL stimulation in freshly resected human lung tumors.

RANK expression and function were investigated in viable, non-apoptotic disaggregated primary NSCLC samples by flow cytometry using previously described methodology. Cell surface RANK expression was evaluated using a monoclonal antibody (N2B10, Amgen). Evidence of receptor function was investigated by analysis of relevant intracellular signaling pathways using phospho-specific antibodies following ex vivo RANKL stimulation (2.5 pM - 5 µg/mL, 30 minutes). A cocktail of EGF, HGF and IGF1 was also employed as a positive control for functional pathway activation. Molecular profiling of somatic mutations is ongoing to further elucidate the molecular characteristics of tumors that have functional RANK signaling. Table 1 summarizes RANK expression and RANKL response profiles.

This study demonstrates that RANK is expressed and functional in a subset of primary human lung tumors. Data presented here confirms RANK expression on tumor cells of NSCLC and suggests that RANK pathway utilization is restricted to the sub-set of tumors that express detectable RANK.

Table 1SquamousAdenocarcinomaOther# Samples12217RANK Positive373NFκβ p65 Response1pAKT Response1pERK Response2p38 Responsep65 + pAKT Response1p38 + pERK Response1p65 + p38 + pAKT Response1% Responder (Total*)17%19%14%% Responder (RANK positive*)67%57%33%% Responder (EGF/ HGF/ IGF1)75%95%86%Other: large cell (1), carcinoid (1), adenosquamous (3), neuroendocrine (2)*Within Histology subtype

Citation Format: John M. Rossi, Yang Pan, William C. Dougall, Daniel Branstetter, Allison Jacob, Jude Canon, Robert D. Loberg. Functional RANK expression is observed in disaggregated primary human lung tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3320. doi:10.1158/1538-7445.AM2014-3320
Abstract 3321: Using NLPs to study EGFR structure, activation, and inhibition
Authors: Scharadin Tiffany M. , Saldana Matthew , Schlein Michael , Hoang-Phou Steven , Trans Denise , Chang Dennis , He Wei , Lam Kit , Carraway Kermit L. , Coleman Matthew A. , Henderson Paul T.
Published: May 2015

The mammalian ErbB receptor tyrosine kinase family is critical for the development and maintenance of a variety of tissues. Though not completely understood, the mechanism of ErbB receptor activation involves the binding of ligand to the extracellular domain leading to a conformational change that allows dimerization and phosphorylation to initiate downstream signaling pathways. Mutations, amplification, and aberrant activation of these receptors lead to oncogenesis and tumor progression in several cancer types, including lung, breast, and colon. Current ErbB-targeted therapies include monoclonal antibodies and tyrosine kinase inhibitors. These treatments are initially effective but many tumors develop resistance, necessitating the discovery of a more specific and efficient drug. Studying the ErbB receptors is often difficult because of their large size and poor water solubility. Here we report success in assembling EGFR into nanolipoprotein particles (NLPs) to study activation and inhibition of the correctly-folded, full-length, and active receptor. NLPs are ∼20 nm cell membrane analogs composed of an apolipoprotein surrounding a lipid bilayer. We produced a homogenous population of EGFR-NLPs, by FLAG-purification of EGFR from mammalian cells, which are of the correct size, phosphorylated, and can be quantified. Furthermore, these EGFR-NLPs can be utilized as a novel target in a one-bead-one-compound (OBOC) screen of small molecules and peptides to identify unique therapeutics. Studies of ligand binding, kinase activity, and EGFR structure are ongoing. Future directions are to incorporate disease-relevant EGFR mutations into NLPs. The T790M mutation is of particular interest because it is a treatment-induced mutation observed in half of all non small cell lung cancers and confers resistance to current ErbB-targeted therapies.

Citation Format: Tiffany M. Scharadin, Matthew Saldana, Michael Schlein, Steven Hoang-Phou, Denise Trans, Dennis Chang, Wei He, Kit Lam, Kermit L. Carraway, Matthew A. Coleman, Paul T. Henderson. Using NLPs to study EGFR structure, activation, and inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3321. doi:10.1158/1538-7445.AM2014-3321
Abstract 3323: PRAF2 regulates Rab5-dependent endocytic trafficking of EGFR in neuroblastoma
Authors: Yco Lisette P. , Bachmann Andre S.
Published: May 2015

Receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), mediate extracellular signaling that activates downstream cell proliferation, differentiation, and migration. Through ligand stimulation, clathrin-mediated endocytosis regulates the EGFR signal transduction pathway. Prenylated Rab acceptor 1 domain family, membrane 2 (PRAF2) is a novel 19-kDa protein with a prenylated Rab acceptor 1 (PRA1) motif and four transmembrane-spanning domains. Similar to PRAF1 and PRAF3, PRAF2 belongs to the PRAF family of vesicle transport-associated proteins. Our lab identified PRAF2 as a candidate prognostic marker of neuroblastic tumors. Using RNA interference, we showed that PRAF2 plays an essential role in neuroblastoma tumorigenesis and metastasis. In the present study, we examined the function of PRAF2 in EGFR endocytic trafficking. We generated stable PRAF2-knockdown (SK-shPRAF2) and PRAF2-overexpressing (SK-PRAF2) neuroblastoma SK-N-SH cell lines and stimulated both cell lines with EGF (10 ng/ml) for various time periods in order to determine the role of PRAF2 in EGFR endocytic trafficking. As judged by immunofluorescence microscopy, we detected larger Rab5-positive punctuate structures in PRAF2-overexpressing SK-PRAF2 cells compared to PRAF2-knockdown SK-shPRAF2 cells or control cells, after 15 min EGF stimulation. After EGF treatment, PRAF2 clearly colocalized with Rab5-positive punctae in SK-PRAF2 cells, suggesting that PRAF2 interacts with Rab5, a critical regulator for receptor endocytosis and early endosome fusion. Moreover, using a receptor internalization assay, we observed a delay in the internalization of EGFR in EGF-treated SK-shPRAF2 cells, in contrast to SK-PRAF2 and control cells. This is in support of our immunofluorescence microscopy results in which Rab5-postive punctae distribution is observed mostly along the membrane of the EGF-treated SK-shPRAF2 cells. Notably, the level of phosphorylation of Akt (Ser473) transiently elevated in EGF-treated SK-PRAF2 cells after 15 min and diminished thereafter, suggesting that overexpression of PRAF2 accelerates the EGFR internalization and degradation. Based on our findings, we propose that PRAF2 affects EGFR endocytosis by regulating early endosome trafficking and Akt activation in neuroblastoma.

Citation Format: Lisette P. Yco, Andre S. Bachmann. PRAF2 regulates Rab5-dependent endocytic trafficking of EGFR in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3323. doi:10.1158/1538-7445.AM2014-3323
Abstract 3324: The endocannabinoid system inhibits non-small cell lung cancer tumorigenesis by modulating the EGF/EGFR pathway
Authors: Ravi Janani , Sneh Amita , Shilo Konstantin , Nasser Mohd , Ganju Ramesh
Published: May 2015

Non-small cell lung cancer (NSCLC) is a leading form of cancer with limited treatment regimens and is a major cause of cancer related mortality in the United States. The endocannabinoid system composed of two cannabinoid receptors- CB1 and CB2, their endogenous ligands and the enzymes for their metabolism has been implicated in several pathophysiological conditions. They have been extensively studied in various cancers as inhibitors of tumor cell growth by modulating key cell survival pathways. Anandamide (AEA), a well characterized neurotransmitter is an endogenous cannabinoid and agonist for the cannabinoid receptor CB1. One way to enhance the effect of endocannabinoid signaling is to inhibit the enzyme fatty acid amide hydrolase (FAAH), which metabolizes AEA by using the selective FAAH inhibitor URB597. To understand the physiological role of the endocannabinoid system, the NSCLC cell lines A549 and H460 were treated with either Met-F-AEA, URB597 or in combination and subjected to further analysis. Cells which were incubated in combination with Met-F-AEA and URB597 showed reduced proliferative and chemotactic activities in vitro when compared to AEA alone, which were confirmed by reduced MMP2 secretion and stress fiber formation. Also, we have shown that the combination treatment modulates activation of epidermal growth factor receptor (EGFR) and its downstream targets. In response to EGFR pathway activation, cells treated with Met-F-AEA in combination with URB597 underwent GO/G1 cell cycle arrest, ultimately leading to apoptosis via activation of caspase-9 and PARP. Furthermore, these results were validated in vivo in a xenograft nude mouse model system. Mouse xenografts treated with both Met-F-AEA and URB597 underwent reduction in tumor growth when compared to Met-F-AEA or URB597 alone. In summary, our results presented here show that Met-F-AEA in combination with URB597 inhibits the EGFR pathway, resulting in cell cycle arrest and apoptosis. Our findings suggest a novel perspective for the antitumorigenic activity of the endocannabinoid Met-F-AEA when used in combination with URB597 in NSCLC.

Citation Format: Janani Ravi, Amita Sneh, Konstantin Shilo, Mohd Nasser, Ramesh Ganju. The endocannabinoid system inhibits non-small cell lung cancer tumorigenesis by modulating the EGF/EGFR pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3324. doi:10.1158/1538-7445.AM2014-3324
Abstract 3328: Novel regulation of HER2 degradation and breast cancer cell proliferation by junctional adhesion molecule-a (JAM-A)
Authors: Brennan Kieran , McSherry Elaine A. , Hudson Lance , Kay Elaine W. , Young Leonie S. , Hill Arnold DK , Hopkins Ann M.
Published: May 2015

Background: We have previously shown that the adhesion protein Junctional Adhesion Molecule-A (JAM-A) is over-expressed in a population of aggressive and metastatic breast tumours, and that it regulates the migration of breast cells. JAM-dependent regulation of migration could contribute to the likelihood of metastatic disease, since migration is an early event in metastasis. Our objective was to further interrogate the links between JAM-A signalling and markers of disease progression in breast cancer.

Approach and Results: Immunohistochemical analysis of JAM-A in two breast cancer tissue microarrays (TMA) revealed that high JAM-A expression correlated significantly with HER2 protein expression, ERnegativity, high grade breast cancers and aggressive luminal B, HER2 and basal subtypes of breast cancer. Using LCC1 and MCF7-HER2 cells which co-express high levels of JAM-A and HER2, we silenced the gene expression of each one in order to examine potential consequences for protein expression of the other. Knockdown of HER2 had no effect on JAM-A protein levels, but knockdown of JAM-A significantly reduced the protein expression of HER2 and its downstream pro-survival effector pAkt. Accordingly, JAM-A knockdown increased apoptosis via a pathway involving Akt activation, as evidenced by apoptosis normalisation upon pharmacological inhibition of Akt. The expressional regulation of HER2 and pAkt by JAM-A was sensitive to inhibition by the proteasomal inhibitor MG132, suggesting that JAM-A regulates the proteasomal degradation of HER2. Finally, over-expression of wild-type JAM-A was sufficient to significantly enhance proliferative and migratory behaviour in breast cell lines.

Conclusions: Our results are consistent with a model whereby JAM-A expression inhibits the proteasomal degradation of HER2, facilitating a pro-survival phenotype through increased Akt activity. Therefore JAM-A antagonists may have future therapeutic value in the treatment of breast tumours overexpressing HER2.

Citation Format: Kieran Brennan, Elaine A. McSherry, Lance Hudson, Elaine W. Kay, Leonie S. Young, Arnold DK Hill, Ann M. Hopkins. Novel regulation of HER2 degradation and breast cancer cell proliferation by junctional adhesion molecule-a (JAM-A). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3328. doi:10.1158/1538-7445.AM2014-3328
Abstract 3329: Investigations into the tumor specific regulation of HER2 by Grp94 in breast cancer
Authors: Yan Pengrong , Patel Hardik , Yang Chenghua , Taldone Tony , Chiosis Gabriela
Published: May 2015

Background: Grp94 is the Endoplasmic Reticulum (ER) paralog of the Hsp90 family of chaperones. Overexpression of Grp94 in tumors correlates with advanced stage and poor survival in various cancers and is closely linked to cancer growth and metastasis. Until recently, the major focus of cancer related studies on Grp94 was on the involvement of this protein in the secretion of IGF-II and the regulation of Toll-Like Receptors (TLRs) and integrins.

Objective/Hypothesis: Our group combined compound library screening with computational tools to develop potent and selective inhibitors of Grp94 and of Hsp90, the cytosolic HSP90 paralog. We hypothesize that these small molecules are important pharmacologic tools to investigate, in a tumor-by-tumor manner, Grp94 and Hsp90 associated oncogenic mechanisms.

Approach/Results: Using the Hsp90 paralog specific inhibitors we produce preliminary evidence for an unanticipated role for Grp94 in maintaining the architecture of high density HER2 formations at the plasma membrane, particularly in cancer cells where HER2 is required to channel an amplified signaling through the receptor. Our data indicate that under conditions in which stress is imposed on the cell by proteome alterations (i.e. HER2 overexpression), the chaperoning function of Grp94 is vital for proper HER2 functioning. In these cells Grp94 translocates to the plasma membrane where it functions to maintain an active conformation of HER2 and to stabilize downstream signaling through the receptor. Our data show that inhibition of Grp94 in these cells is sufficient to destabilize membrane HER2, inhibit its signaling properties and target HER2 towards a degradative pathway. These effects are associated with cancer cell viability loss and induction of apoptosis.

Significance: These findings reveal a novel mechanism of HER2-regulation at the plasma membrane, where Grp94 is implicated in HER2 regulation at this location in the specific case of HER2 positive tumors. They also provide a strong rationale for the use of these inhibitors in the investigation of Grp94 associated oncogenic mechanisms. Further, this study proposes the development of Grp94-based cancer therapeutics for HER2 overexpressing breast cancer.

Citation Format: Pengrong Yan, Hardik Patel, Chenghua Yang, Tony Taldone, Gabriela Chiosis. Investigations into the tumor specific regulation of HER2 by Grp94 in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2014-3329
Abstract 333: ZBP-89 downregulates histone deacetylase 3 (HDAC3) through inhibition of I kappa B in hepatocelluar carcinoma (HCC)
Authors: Lai Paul B. , Ye Cai-Guo , Liu Liping , He Ming-Liang , Ho Rocky , Chen George G.
Published: May 2015

Background

Histone deacetylase 3 (HDAC3) is frequently overexpressed in hepatocelluar carcinoma (HCC) and its inhibition enhances the efficacy of anti-HCC chemotherapy. ZBP-89 is known to induce apoptosis via an increase of pro-apoptotic Bak. But the relationship between ZBP-89 and HDAC3 is largely unknown.

Method

Human HCC cell lines PLC/PRF/5 and HepG2 were cultured in Minimum Essential Medium and treated with various agents to modulate NF-κB. These agents included CAY10576 and MG132 (which are inhibitors of I kappa B (IκB) degradation) as well as SN50 (an inhibitor of p65/p50 translocation). A xenograft tumor model was established by subcutaneous injection of HCC PLC/PRF/5 cells into nude mice and this model was used to confirm data obtained in HCC cell culture experiments. The expressions of relevant proteins were measured by Western blot or/and immunohistochemical staining.

Results

Our results showed that ZBP-89 reduced the expression of HDAC3 in HCC cells. ZBP-89 could form a complex with IκB and induce the phosphorylation of IκB, which inhibited IκB. Furthermore, ZBP-89-mediated HDAC3 reduction could be suppressed by IκB degradation inhibitors CAY10576 and MG132 but not NF-κB subunit p65/p50 translocation inhibitor SN50. These findings indicate that IκB decreases rather than increases the activity of NF-κB; and such may be involved in the ZBP-89-mediated HDAC3 reduction. In the HCC xenograft model, it was confirmed that the expression of HDAC3, measured by immunohistochemical staining and Western blot, was reduced in HCC with ZBP-89 overexpression, compared with the control. In contrast to HDAC3, ZBP-89 enhanced the expression of Bak.

Conclusion

ZBP-89 downregulates HDAC3 by decreasing IκB rather than elevating activity of NF-κB. This novel pathway identified helps us understand more the mechanism of ZBP-89-mediated inhibition of HCC.

Citation Format: Paul B. Lai, Cai-Guo Ye, Liping Liu, Ming-Liang He, Rocky Ho, George G. Chen. ZBP-89 downregulates histone deacetylase 3 (HDAC3) through inhibition of I kappa B in hepatocelluar carcinoma (HCC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 333. doi:10.1158/1538-7445.AM2014-333
Abstract 3330: HER2 overexpression induces membrane deformation that increases cell motility
Authors: Chung Inhee , Reichelt Mike , Dowbenko Donald , Mellman Ira , Sliwkowski Mark
Published: May 2015

Approximately 20% of breast cancers (BC) are characterized by the gene amplification and overexpression of HER2, a member of the ErbB/HER receptor family. While targeted therapies against HER2 effectively delay disease progression in this BC subtype, details of how overexpressed HER2s drive these tumors to malignancy are still unclear. To better understand this process, we investigated various cellular responses to HER2 overexpression in individual live cells. We developed novel single receptor diffusion analyses that determine the activation status of HER2 by diffusivity, to estimate average HER2 activity per single cell. Surprisingly, we found HER2 overexpression induces membrane deformation, which depends only on the HER2 density, but not the receptor activation status. Moreover, this membrane deformation lowers the available surface area for formation of focal adhesion sites, resulting in reduced cell adhesion and increased cell motility. These findings suggest there are signaling-independent roles of HER2 overexpression in disease progression of HER2 positive BCs.

Citation Format: Inhee Chung, Mike Reichelt, Donald Dowbenko, Ira Mellman, Mark Sliwkowski. HER2 overexpression induces membrane deformation that increases cell motility. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3330. doi:10.1158/1538-7445.AM2014-3330
Abstract 3331: CD24 promotes HER2 signaling pathways and CD24 inhibition sensitizes anti-HER2 therapy in breast cancer
Authors: Arima Yoshimi , Hosonaga Mari , Saya Hideyuki
Published: May 2015

Human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancers are treated with HER2-targeted agents such as trastuzumab and lapatinib, which suppress HER2-initiated activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) cascades. CD24 is a glycosyl phosphatidylinositol (GPI)-anchored membrane protein and its overexpression is correlated with a poor prognosis in breast cancer patients. It has been reported that CD24 tends to be highly expressed in the HR-HER2+ breast cancer subgroup, which is negative for the hormone receptors (HRs) and positive for HER2. To determine the relationship between CD24 and HER2, we overexpressed HER2 in MDA-MB-231 breast cancer cells, which were negative for CD24 and HER2. We found that the expression of CD24 was increased by the overexpression of HER2. Flow cytometry analysis showed that the expression of CD24 was higher in the HER2-positive fraction than in the HER2-negative fraction. Conversely, in BT474 cells which express both CD24 and HER2, CD24 knockdown reduced the expression of HER2, while the knockdown of HER2 did not affect the expression of CD24. Furthermore, the knockdown of CD24 decreased the phosphorylation of Akt, which is known as a downstream molecule of PI3K and is related to cell survival. Accordingly, CD24 plays an important role in the regulation of the HER2-initiated signaling pathway. Based on these findings, we speculated that the expression of CD24 promotes a resistance to HER2-targeted therapies, leading to tumor progression in HER2-overexpressing cancers. We found that the knockdown of CD24 enhanced the sensitivity to lapatinib treatment. Consequently, we suggest using an anti-CD24 therapy to enhance the HER2-targeted therapy.

Citation Format: Yoshimi Arima, Mari Hosonaga, Hideyuki Saya. CD24 promotes HER2 signaling pathways and CD24 inhibition sensitizes anti-HER2 therapy in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3331. doi:10.1158/1538-7445.AM2014-3331
Abstract 3334: The role of nuclear HER3 in breast cancer resistance
Authors: Tang Patrick Ming Kuen , Khoo Ui Soon , Harris Adrian L. , Kong Anthony
Published: May 2015

HER3 is reported to be overexpressed in 18-29% of human breast cancers and suggested to be a marker of reduced disease-specific survival in 4,406 patients with invasive breast carcinoma. Overexpression and subcellular localization of HER3 may be a negative predictive marker for targeted therapies, and HER3 status may play a role in the development of drug resistance and metastasis. Translocation of ERBB family members EGFR, HER2 and HER4 into nucleus for regulating gene transcription have been reported, but only a few studies about nuclear HER3 have been reported and the biological role of nuclear HER3 is still unknown. HER3 was detected in the nucleus of 57% in Japanese lung cancers, and has also been detected in the nuclei of prostate cancers and other cancer cell lines. A study suggested that nuclear HER3 by IHC is associated with favorable overall survival in 128 uveal melanoma patients. The potential mechanism of HER3 translocation and its role on drug resistance was intensively studied in the work. According to our observation, nuclear HER3 was found to be significantly increased in HER2-positive breast cancer cells SKBr3 during an EGFR-targeted therapy Iressa, and increased sub-band of HER3 was detected by Western blot in the Iressa-resistant SKBr3 cells. The Iressa-induced HER3 nuclear localization in SKBr3 and BT474 cells can be inhibited by the application of ADAM17 and gamma-secretase inhibitors or siRNA, the additional inhibitors can sensitize the cells to Iressa treatment which may be a new target for preventing HER3-mediated drug resistance. If the HER3 subcellular localization is important for cancer development and drug resistance, inhibitor for this pathway will enhance the efficiency of recent therapies on breast cancer.

Citation Format: Patrick Ming Kuen Tang, Ui Soon Khoo, Adrian L. Harris, Anthony Kong. The role of nuclear HER3 in breast cancer resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3334. doi:10.1158/1538-7445.AM2014-3334
Abstract 3337: LRIG1 decreases cell proliferation and motility through downregulation of ErbB2 611-CTF
Authors: Cedano-Prieto Maria E.
Published: May 2015

The receptor ErbB2 is a membrane bound protein that controls important cell functions including cell cycle progression; deregulated expression of this protein is associated with the development of a variety of cancer types. Notably, ErbB2 over-expression is observed in approximately 25% of human breast tumors.

Trastuzumab, an FDA approved therapeutic currently used to treat ErbB2 positive breast cancer patients, is a monoclonal antibody that directly binds the ErbB2 extracellular domain. Unfortunately, only a fraction of patients treated with trastuzumab durably respond to the treatment. Moreover, patients that are treated long term with this antibody develop resistance. One of the proposed mechanisms of trastuzumab resistance is the expression of 611-CTF, a C-terminal fragment of ErbB2 which lacks most of its extracellular domain. Remarkably, 611-CTF is hyperactive and has been associated with increased cell migration, tumor progression and metastasis.

Currently nothing is known regarding mechanisms which lead to 611-CTF down-regulation. This represents a significant knowledge gap in our understanding of 611-CTF and receptor fragments in general. Since 611-CTF is associated with poor clinical outcome and therapeutic resistance, it is essential to clarify the mechanisms by which this ErbB2 fragment is regulated. This knowledge could ultimately lead to the development of new therapeutics which could improve the response rate of 611-CTF-positive breast cancer.

LRIG1 is a tumor suppressor that directly interacts with ErbB2 leading to receptor degradation. In this study we examined whether 611-CTF is susceptible to LRIG1-mediated down-regulation. We find that these proteins interact and that expression of LRIG1 is sufficient to decrease 611-CTF protein expression in three different cell lines. Furthermore, LRIG1 is capable of reducing 611-CTF-driven tumor cell proliferation and migration. Our results are unexpected because LRIG1 is thought to recognize its targets through mutual extracellular domain interactions and 611-CTF lacks a large portion of the ErbB2 extracellular domain.

Citation Format: Maria E. Cedano-Prieto. LRIG1 decreases cell proliferation and motility through downregulation of ErbB2 611-CTF. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3337. doi:10.1158/1538-7445.AM2014-3337
Abstract 3338: IGFBP2 promotes EGFR/STAT3 nuclear crosstalk in glioma
Authors: Chua YingXuan , Liu Yuexin , Hu Limei , Granberg Kirsi , Verploegen Maartje , Zhang Wei
Published: May 2015

Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic secreted protein that is overexpressed in various cancers including glioma. Extracellular IGFBP2 binds and modulates the activity of insulin growth factors I and II (IGFI and IGFII) while cytoplasmic IGFBP2 interacts with signaling pathways essential for cell proliferation and survival. The versatility of IGFBP2 as a secreted or cytoplasmic signaling effector is widely characterized; however, the functions of nuclear IGFBP2 remain largely unknown. Through genomic and functional analyses of glioma, we mechanistically elucidated the role of nuclear IGFBP2 and discovered a functional interaction of IGFBP2 with epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription 3 (STAT3) proteins. Our findings established a signaling hierarchy of IGFBP2-EGFR-STAT3; IGFBP2 not only upregulates EGFR/STAT3 signaling activity, but also facilitates EGFR nuclear translocation and subsequent nuclear EGFR/STAT3 activity. Furthermore, mutation of the nuclear localization signal of IGFBP2 demonstrated that nuclear translocation of IGFBP2 is critical to promote the nuclear activity of EGFR. By identifying the role of IGFBP2 in perpetuating nuclear crosstalk between EGFR/STAT3, our study further reveals the importance of exploiting IGFBP2 as a target for glioma therapy.

Citation Format: YingXuan Chua, Yuexin Liu, Limei Hu, Kirsi Granberg, Maartje Verploegen, Wei Zhang. IGFBP2 promotes EGFR/STAT3 nuclear crosstalk in glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3338. doi:10.1158/1538-7445.AM2014-3338
Abstract 334: Mcl-1 protects prostate cancer cells from chemotherapy-induced DNA damage
Authors: Perez-Stable Carlos , Reiner Teresita , Pozas Alicia de las , Parrondo Ricardo
Published: May 2015

Mcl-1 (myeloid cell leukemia sequence 1 [Bcl2-related]) is predominantly localized in the cytoplasm and protects cells from apoptosis by blocking mitochondrial outer membrane permeabilization. There is evidence that nuclear localization of Mcl-1 has pleiotropic effects independent of apoptosis, including functional roles in DNA damage response. Mcl-1 is highly expressed in castration-resistant prostate cancer (CRPC), resulting in resistance to apoptosis and association with poor prognosis. The role of nuclear Mcl-1 in mediating sensitivity to chemotherapy-induced DNA damage and apoptotic cell death in prostate cancer (PCa) is not known. The novel antimitotic agent ENMD-1198 is a more stable and potent derivative of 2-methoxyestradiol (2ME2). We confirmed that 1198 was a more potent inhibitor of androgen-dependent LNCaP and castration-resistant DU145 and PC3 PCa cell viability compared to 2ME2 and induced apoptotic cell death in docetaxel-resistant PC3 cells that overexpress the multi-drug resistance protein (MDR). The ability of 1198 to increase the proteasomal-mediated degradation of Mcl-1 correlated with enhanced apoptotic cell death and shRNA knockdown of Mcl-1 further sensitized LNCaP and PC3 cells to 1198-mediated cell death. We then showed that the combination of 1198 with betulinic acid (BA, a plant-derived small molecule that can increase apoptosis specifically in cancer but not in normal cells) reduced nuclear Mcl-1, increased a pro-death Mcl-1 isoform, increased gamma histone 2AX (marker of DNA damage), and enhanced apoptotic cell death in human PC3 CRPC cells. In the TRAMP mouse model of PCa, the 1198 (75 mg/kg) + BA (10 mg/kg) combination significantly reduced PCa weights compared to 1198, BA alone, and vehicle controls. However, the reduced PCa weights between the 1198 + BA combination and BA alone was more associated with decreased Mcl-1 and increased gamma-H2AX (DNA damage) immunostaining compared to cleaved caspase-3 (apoptosis), CD31(angiogenesis), or Ki67 (proliferation). Furthermore, our results showed that blocking the DNA damage response signaling pathway with an ATM kinase (KU55933) or DNA-PK (NU7441) inhibitor lowered apoptotic cell death induced by the 1198 + BA combination in PC3 cells. Overall, our results demonstrate that nuclear Mcl-1 has an important role in protecting PCa cells from DNA damage and that agents such as 1198 and BA that reduce total/nuclear Mcl-1 and increase pro-death Mcl-1 isoforms will sensitize PCa cells to antimitotic DNA damaging agents.

Citation Format: Carlos Perez-Stable, Teresita Reiner, Alicia de las Pozas, Ricardo Parrondo. Mcl-1 protects prostate cancer cells from chemotherapy-induced DNA damage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 334. doi:10.1158/1538-7445.AM2014-334