bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4534: In vivo antitumor activity of PM060184 in patient-derived xenografted tumors (avatar)
Authors: Hidalgo Manuel , Guillén M. José , López-Casas Pedro Pablo , Sarno Francesca , Cataluña Oscar , Palomares Mandy , Cuevas Carmen , Aviles Pablo M.
Published: May 2015

Background: PM060184 is a synthetic marine-derived compound originally isolated from the marine sponge Lithoplocamia lithistoides. PM060184 induces disorganization and disruption of the microtubule network as well as aberrant mitotic spindle multipolarization and chromosome missegregation. These effects give rise to prometaphase arrest and formation of multinucleated cells. Then, cells enter to caspase-driven apoptosis or are arrested in a pseudo-senescent state. PM060184 is currently under evaluation in Phase I clinical studies in patients with advanced cancer diseases.

Material and Methods: Athymic female nu/nu mice were subcutaneously implanted with different patient-derived tumors: NSCLC (N=4)), pancreas (N=5), colon (N=4) and gastric (N=2). Tumor (ca. 300 mm3) bearing animals (N=6-10/group) were randomly allocated to receive PM060184 (16 mg/kg) or placebo. Treatments were administered weekly for 5 consecutive weeks. Antitumor effect was calculated using ΔT/ΔC (%), defined as a percentage of the change in tumor size for treated (T) and placebo (C) groups during the placebo-treated survival time (D). Complete tumor regression (CR) was defined when tumor volume < 63 mm3 for 2 or more consecutive measurements.

Results: The treatment with PM060184 produced lowest ΔT/ΔC values summarized as follows:

TumorMinimal ΔT/ΔC (%)on Day NSCLCPulm-0052.232Pulm 00920.414Pulm-0160.045Pulm-02114.738PancreasPanc-21537.335Panc-26512.126Panc-35429.532JH-02450.039ColonCCR-02051.628CCR-02535.639CCR-02933.535CCR-03034.328GastricGastric-00853.331Gastric-0102.538

During the treatment, complete remissions (CR) of tumors were also seen in animals bearing the following xenografts: Pulm-005 (7 CR/7 mice), Pulm-016 (10 CR/10 mice) and Gastric-010 (9 CR/9 mice).

Conclusion: The treatment with PM060184 demonstrated significant in vivo antitumor activity in patient-derived xenografts of human NSCLC, pancreas, colon and gastric tumors.

Citation Format: Manuel Hidalgo, M. José Guillén, Pedro Pablo López-Casas, Francesca Sarno, Oscar Cataluña, Mandy Palomares, Carmen Cuevas, Pablo M. Aviles. In vivo antitumor activity of PM060184 in patient-derived xenografted tumors (avatar). [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 4534. doi:10.1158/1538-7445.AM2014-4534
Abstract 4535: Inhibition of cell proliferation by (R,R')-4′-methoxy-1-naphthylfenoterol in breast cancer cell lines
Authors: Paul Rajib K. , Wnorowski Artur , Bernier Michel , Wainer Irving W.
Published: May 2015

(R,R')-4′-methoxy-1-naphthylfenoterol (MNF) is a bitopic ligand that acts as an agonist of the β2-adrenergic receptor and an antagonist of the orphan G-protein-coupled receptor GPR55. Activation of GPR55 is pro-oncogenic through cannabinoid-related signal transduction and MNF treatment inhibits proliferation of different tumor cell lines, representing cancers of the liver, pancreas and brain. Of significance, MNF exerts antitumor activity in a xenograft glioma model in nude mice. In the current study, the anti-proliferative response mediated by MNF has been examined in human breast cancer cell lines. In this study, MCF-7 and MDA-MB-231 cells were screened for the presence of epidermal growth factor receptor (EGFR) and GPR55 proteins by Western blotting. The EGFR in such cancer cells plays a key role in the control of growth and proliferation. When compared to MCF-7 cells, MDA-MB-231 cells contained high levels of EGFR and GPR55 and showed exquisite sensitivity to MNF as evidenced by the significant reduction in constitutive phosphorylation of the extracellular signal-regulated kinase (ERK1/2), with an IC50 of 0.08 nM. [3H]-Thymidine incorporation assay was performed and revealed that MNF elicited a 2-fold higher growth inhibition in MDA-MB-231 cells as compared to MCF-7 cells (50% vs. 24.9% inhibition at 10 µM of MNF; p<0.001). Evidence for the altered expression and/or signaling pathway activation triggered by EGFR and GPR55 in response to MNF will be presented. We conclude that MDA-MB-231 cells represent a suitable model for the study of possible crosstalk between two classes of pro-oncogenic cell surface receptors and the analysis of MNF as novel therapeutic agent for the management of cancer. This work was supported entirely by the Intramural Research Program of the National Institute on Aging, NIH.

Citation Format: Rajib K. Paul, Artur Wnorowski, Michel Bernier, Irving W. Wainer. Inhibition of cell proliferation by (R,R')-4′-methoxy-1-naphthylfenoterol in breast cancer cell lines. [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 4535. doi:10.1158/1538-7445.AM2014-4535
Abstract 4536: ABT-737 and ABT-199 complement the multikinase inhibitor TG02 to induce apoptosis in acute myeloid leukemia cells
Authors: Abdul-Aziz Amina , Burrows Francis , Yu Ning , Russell Nigel H. , Seedhouse Claire H. , Pallis Monica
Published: May 2015

TG02 is a novel multikinase inhibitor currently in Phase 1 trials for AML and CLL, with a unique spectrum of molecular targets. We have previously shown that TG02 significantly downregulates MCL1 but not BCL2 or BCL2L2 in AML cells at nanomolar concentrations. ABT-737 is a BH3 mimetic which inhibits BCL2, but its effects are impaired in cells which over-express MCL1. Both these agents are documented to target dormant as well as proliferating leukaemia cells and thus have the potential to reduce relapse risk, but cellular responses are heterogeneous. In primary AML samples, BCL-2 expression correlated with that of BCL-XL but not of MCL-1. The response at 48 hours to 100 nM TG02 was measured in primary samples. (This concentration did not inhibit clonogenic growth of haematopoietic stem cells from healthy donors). We show that resistance to TG02 is significantly associated with BCL2 over-expression (P=0.001) but not with MCL1. In CD34+CD38- KG1-a cells, using TG02 with ABT-737 or with the novel and more specific BCL-2 inhibitor ABT-199 at a fixed dose ratio of 1(TG02):20, the combination indices for drug interactions were calculated. For TG02 with ABT-737 and ABT-199 respectively these were IC50 0.59 and 0.26; IC90 0.13 and 0.14, indicating strong synergy, particularly at the IC90. We also show that the OCI-AML-3 cell line, which is resistant to both ABT compounds, was sensitive to TG02 (TG02 IC50 43 nM, IC90 97 nM; ABT-737/199 IC50>4,000nM). All three agents exposed activation-specific epitopes of bax, reduced mitochondrial membrane potential, cleaved caspase 3 and induced phosphatidylserine exposure. 50 nM TG02 + 50 nM ABT199 or ABT-737 was significantly (P<0.05) more toxic than 100 nM of any of the 3 agents individually in a cohort of 12 patient samples treated in vitro for 48 hours. Moreover samples which were sensitive to ABT-199 alone clustered separately from samples sensitive to TG02 alone (p=0.03), indicating that in this heterogeneous disease, the combinatorial approach broadens the range of samples that can be targeted as well as enhancing individual toxicities. We conclude that the cytotoxic actions of TG02 and ABT-737 /ABT-199 are complementary.

Citation Format: Amina Abdul-Aziz, Francis Burrows, Ning Yu, Nigel H. Russell, Claire H. Seedhouse, Monica Pallis. ABT-737 and ABT-199 complement the multikinase inhibitor TG02 to induce apoptosis in acute myeloid leukemia 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 4536. doi:10.1158/1538-7445.AM2014-4536
Abstract 4541: Dual targeting of CDK4 and MEK as a combination treatment strategy for metastatic colorectal cancer
Authors: Ziemke Elizabeth , Dosch Joseph , Shettigar Amrith , Wan Shanshan , Welling Theodore , Hardiman Karin , Sebolt-Leopold Judith
Published: May 2015

Aberrant hyperactivation of KRAS plays a prominent role in tumor initiation and progression of a broad spectrum of human malignancies. The incidence of KRAS mutations is especially high in colorectal cancers, where it occurs at a frequency of roughly 50%. The present study addresses the hypothesis that dual targeting of CDK4 and MEK represents a viable treatment strategy for the subpopulation of colorectal cancers exhibiting hyperactivated KRAS signaling. We have generated both in vitro and in vivo data in highly passaged colorectal xenograft models to provide compelling support for further exploration of this combination treatment strategy. We have now turned to the study of primary human models that are anticipated to be more predictive of ultimate clinical activity in patients. A heterogeneous panel of low passage colorectal patient-derived xenografts (PDX) has been established from biospecimens procured during patient surgeries conducted at our institution (n=12). All xenograft models have been genomically profiled and their histologies mimicked those observed in the original samples. As anticipated, approximately half of these models harbor an oncogenic KRAS mutation and exhibit widely differing histologies, consistent with the heterogeneity encountered in the clinic. Immunoblotting analysis has further revealed that that the majority (>80%) are positive for both phosphorylated RB and constitutive pERK expression and are therefore candidates for treatment regimens combining inhibitors of CDK4 and MEK. Employing the CDK4 inhibitor palbociclib and multiple MEK inhibitors, in vivo efficacy studies are ongoing against our colorectal PDX panel. Preliminary data have shown a higher frequency of regressions in the combination arm compared to either single agent arm at the respective maximum tolerated doses. Our results therefore suggest that combined inhibition of CDK4 and MEK may provide an attractive therapeutic strategy for the treatment of colorectal cancer.

Citation Format: Elizabeth Ziemke, Joseph Dosch, Amrith Shettigar, Shanshan Wan, Theodore Welling, Karin Hardiman, Judith Sebolt-Leopold. Dual targeting of CDK4 and MEK as a combination treatment strategy for metastatic colorectal 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 4541. doi:10.1158/1538-7445.AM2014-4541
Abstract 4545: Anticancer effects and mechanism of VR23, a novel chloroquine derivative
Authors: Vu Hai-Yen Thi , Pundir Sheetal , Solomon Raja V. , Lee Hoyun
Published: May 2015

Chloroquine, a worldwide used drug in treatment of malaria, rheumatoid and other human diseases, has recently been focused as a potential anti-cancer drug as well as a chemo-sensitizer when given in combination with other anti-cancer drugs. In cancer treatment, chloroquine possesses a major potential therapeutic advantage in which it specifically targets cancer cells. Unfortunately, however, its anticancer activity is rather low. To further improve its efficacy and specificity in killing cancer cells, we created and characterized several chloroquine derivatives using a hybrid pharmacophore approach, which led us to identify VR23 as the most promising compound. Our in-vitro model of cancer cells shows that VR23 functions as a novel proteasome inhibitor. It also deregulates activity of cyclin E and other centrosomal proteins, resulting in induction of multiple centrosome amplification, abnormal spindle formation, uneven cytokinesis, irreversible mitotic arrests, and eventually apoptosis. These VR23-induced phenomena occur in cancer-specific manner and cause massive death in a variety of cancer cell types. The treatment of engrafted animals with VR23 (30 mg/kg) twice per week for three weeks effectively inhibits tumor growth. Interestingly, administration of VR23 following 24 hours pre-treatment with paclitaxel (20mg/kg/week) enhances paclitaxel's anti-tumor activity by 70%. Our histological data shows that VR23 causes substantial decrease in mitotic index, inhibition of tumor infiltration into surrounding tissues and remarkable reduction of tumor cell proliferation and angiogenesis. Most importantly, VR23 shows very low toxicity to vital organs (liver, spleen, kidney and lung) of non-tumor-bearing nude mice. In addition, VR23 seems to reduce paclitaxel-induced toxicity when used in combination. Taken together, VR23, our novel chloroquine derivative and newly discovered proteasome inhibitor, appears to be an effective and safe anticancer therapeutic alone or in combination with other anticancer agents.

Citation Format: Hai-Yen Thi Vu, Sheetal Pundir, Raja V. Solomon, Hoyun Lee. Anticancer effects and mechanism of VR23, a novel chloroquine derivative. [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 4545. doi:10.1158/1538-7445.AM2014-4545
Abstract 4554: Downstream effects of rigosertib (ON 01910.Na) in cancer cells involves impairment of protein translation via eIF2 and eIF4
Authors: Merali Salim , Perez-Leal Oscar , Barrero Carlos
Published: May 2015

Purpose: Rigosertib, a novel synthetic styryl benzyl sulfone, is cytotoxic against a variety of human tumor cell lines in vitro and in vivo. Significantly, rigosertib is currently being tested in a randomized phase III clinical trial in patients with higher risk myelodysplastic syndrome (MDS) who have failed treatment with hypomethylating agents. Previous studies have shown that rigosertib interacts with both the phosphoinositide 3-kinase (PI3K) and polo-like kinase (PLK1) pathways. Because these pathways can affect several cellular functions, we used a combination of unbiased global proteomics and interactomics approaches to assemble a rigosertib target profile. Here, we confirm and elaborate the pathways modulated by rigosertib.

Experimental Design: Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics was used to identify the differentially expressed proteins in HeLa cells with and without 8 hours of treatment with 10 μM rigosertib. In addition, rigosertib protein interaction (interactomics) studies were performed to validate the proteomics observations.

Results: Using a combination of SILAC and subcellular fractionation we were able to quantify more than 1500 proteins, among which 125 were significantly altered by rigosertib treatment. Quantified proteins were selected and clustered by biological function. We performed pathway and network analysis using Ingenuity® Systems computational pathway analysis. The pathways that were identified at statistically significant levels (p < 0.05) were eukaryotic initiation factor (eIF2) signaling, eIF4 signaling and nuclear erythroid factor 2 (Nrf2)-mediated oxidative stress response. In total, 19 proteins were down-regulated and 2 proteins were up-regulated in the eIF2 signaling pathway, suggesting that rigosertib represses protein translation in tumor cells. These observations have been noted in other experimental systems using different experimental approaches. To validate these findings and to determine extent of interaction of these proteins with rigosertib, we used drug affinity pull-down followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis to confirm that rigosertib interacts with eIF2 and eIF4 via Raf and p70S6K, respectively.

Conclusions: Collectively, these results demonstrate that a terminal effect of rigosertib in cancer cells is the inhibition of protein translation mediated by eIF2 and eIF4.

Citation Format: Salim Merali, Oscar Perez-Leal, Carlos Barrero. Downstream effects of rigosertib (ON 01910.Na) in cancer cells involves impairment of protein translation via eIF2 and eIF4. [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 4554. doi:10.1158/1538-7445.AM2014-4554
Abstract 4562: Ginkgetin inhibits the growth of cancer cells via the cell cycle arrest at G2-phase
Authors: Kwon Byoung-Mog , Lee Yu-Jin
Published: May 2015

Ginkgetin, isolated from herbal medicines, has been reported to play a role in antitumor activity. However, the relevant pathway integrating cell cycle regulation and signaling pathways involved in growth inhibition in cancer cells remains to be identified. In the present study, ginkgetin treatment of colon cancer cells resulted in significant dose-dependent growth inhibition together with apoptosis, G2-phase cell cycle arrest at a 5 μM (IC50) dose in HCT116colon cancer cells and ginkgetin also inhibits tumor growth in a mouse xenograft model of HCT116 cells. We found that ginkgetin regulated the expression of genes which are critically involved in cell cycle, cell signaling transduction, and cell proliferation using DNA microarray analysis. Furthermore, we provided evidence that ginkgetin inhibited BUB3, cyclin B, CDC2, and b-Myb expression. Especially ginkgetin induced G2-phase arrest by down-regulates expression of b-Myb Our data have suggested that ginkgetin exerts its anticancer effects through induction of cell cycle arrest at G2 phase as well as inhibition cell proliferation.

Note: This abstract was not presented at the meeting.

Citation Format: Byoung-Mog Kwon, Yu-Jin Lee. Ginkgetin inhibits the growth of cancer cells via the cell cycle arrest at G2-phase. [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 4562. doi:10.1158/1538-7445.AM2014-4562
Abstract 487: The Integrated Stress Response kinase GCN2 regulates cell proliferation and survival through combined transcriptional and translational control of p21 (Cip1/Waf1)
Authors: Lehman Stacey L. , Ye Jiangbin , Koumenis Constantinos
Published: May 2015

The tumor microenvironment is often characterized by deficiencies in nutrients, such as oxygen, glucose, and amino acids. Activation of the nutrient-sensing kinase GCN2, which constitutes one arm of the Integrated Stress Response (ISR), is one mechanism by which tumor cells cope with and adapt to nutrient stress. GCN2 phosphorylates the alpha subunit of eukaryotic translation initiation factor eIF2, leading to global downregulation of translation to conserve amino acids and initiation of a transcriptional program through ATF4 to promote recovery from nutrient deprivation. Loss of GCN2 results in decreased tumor cell survival in vitro under conditions of amino acid deprivation and attenuated tumor growth in vivo in a variety of implanted animal tumor models. Our recent studies have shown that amino acid deprivation induces the cell cycle inhibitor p21 in a GCN2-dependent manner. GCN2 regulates p21 at both the transcriptional and translational level. Upregulation of p21 results in cell cycle arrest and promotes survival under amino acid deprivation. In agreement with a pro-survival role, we observe p21 upregulation concomitant with ISR activation in a genetically engineered mouse model of soft tissue sarcoma. These findings point to a novel mechanism of p21 regulation in order to coordinate cell division with nutrient availability.

Research supported by NCI R01CA094214.

Citation Format: Stacey L. Lehman, Jiangbin Ye, Constantinos Koumenis. The Integrated Stress Response kinase GCN2 regulates cell proliferation and survival through combined transcriptional and translational control of p21 (Cip1/Waf1). [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 487. doi:10.1158/1538-7445.AM2014-487
Abstract 5010: Osteoblasts-derived WISP-1 increases prostate cancer metastasis through VCAM-1 upregulation
Authors: Chang An-Chen , Wang Shih-Wei , Tang Chih-Hsin
Published: May 2015

The skeleton is the most common metastatic site for prostate cancer, and increases the risk of intractable bone pain. Therefore, study of the interactions between prostate cancer and the bone microenvironment is very important. Wnt-1 induced secreted protein 1 (WISP-1) belongs to the CCN family (CTGF/CYR61/NOV), which plays an important role in bone formation and many cellular processes. However, the effects of WISP-1 on the prostate cancer metastasis are not clearly understood. First, we found that WISP-1 presents in osteoblast-condition medium (OB-CM), which increases prostate cancer cell migration and invasion. In addition, OBs were infected with lentivirus containing human WISP-1 shRNA which reduced OB-CM-mediated prostate cancer metastasis. We also demonstrated that OB-derived WISP-1 enhanced cell migration via vascular cell adhesion molecule 1(VCAM-1) up-regulation in prostate cancer. Transfection of cells with VCAM-1 siRNA reduced OB-CM and WISP-1-mediated cell migration. The incresing of prostate cancer cell migration by OB-derived WISP-1 is associated with FAK and p38 activation, as well as mir-126 downregulation. Pretreatment of cells with FAK and p38 inhibitors reduced WISP-1-mediated cell migration. In contrast, WISP-1-mediated migration activity was inhibited by mir-126 mimic. Over all, we characterized an important role for OB-derived WISP-1 which regulates cell migration and invasion by increasing VCAM-1 expression in human prostate cancer via the activation of FAK, p38 and downregulation of mir-126 signaling pathway.

Citation Format: An-Chen Chang, Shih-Wei Wang, Chih-Hsin Tang. Osteoblasts-derived WISP-1 increases prostate cancer metastasis through VCAM-1 upregulation. [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 5010. doi:10.1158/1538-7445.AM2014-5010
Abstract 5466: The Mdm2 inhibitor, NVP-CGM097, in combination with the BRAF inhibitor NVP-LGX818 elicits synergistic antitumor effects in melanoma
Authors: Wang Hui Qin , Zubrowski Matthew , Emerson Erling , Pradhan Elina , Jeay Sébastien , Wiesmann Marion , Caponigro Giordano , Wuerthner Jens , Schlegel Robert , Cao Z. Alexander , Huang Alan , Halilovic Ensar
Published: May 2015

RAF kinase inhibitors have shown substantial therapeutic effects in patients with BRAF-mutant melanoma. However, despite impressive initial responses, therapeutic effects are often temporary due to acquired resistance. This resistance highlights the need to identify therapeutic approaches to improve the durability of responses to RAF inhibitors. In this study, we demonstrate that a novel inhibitor of p53-Mdm2 interaction, NVP-CGM097, causes marked reduction of melanoma cell viability. The activation of p53 signaling and consequent reduction in cell viability required wild-type p53, but was independent of BRAF status. Moreover, combined inhibition of Mdm2, using NVP-CGM097, and BRAF using NVP-LGX818, synergistically inhibited the viability of BRAF mutant melanoma cells in vitro and tumor growth in vivo. NVP-CGM097 caused induction of p21 and Bax, while NVP-LGX818 did not. Instead, NVP-LGX818 caused induction of p27 and Bim. Therefore, together they induced a complementary set of anti-proliferative and apoptosis stimulating molecules, which resulted in strong synergistic antitumor effects. In vivo, the combination treatment resulted in sustained tumor regressions and markedly prolonged survival relative to either single agent. Collectively, these data show that activation of p53, through inhibition of Mdm2, leads to antitumor effects in melanoma. Furthermore, the data show that concomitant activation of tumor suppressor p53 and inhibition of BRAF synergistically suppresses melanoma growth. Therefore, combined inhibition of Mdm2 and BRAF in melanoma may provide an effective therapeutic modality capable of overcoming the resistance observed with the BRAF inhibitor monotherapy and thus lead to more durable responses in the clinic.

Citation Format: Hui Qin Wang, Matthew Zubrowski, Erling Emerson, Elina Pradhan, Sébastien Jeay, Marion Wiesmann, Giordano Caponigro, Jens Wuerthner, Robert Schlegel, Z. Alexander Cao, Alan Huang, Ensar Halilovic. The Mdm2 inhibitor, NVP-CGM097, in combination with the BRAF inhibitor NVP-LGX818 elicits synergistic antitumor effects in melanoma. [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 5466. doi:10.1158/1538-7445.AM2014-5466
Abstract 5544: Patient-derived xenograft study reveals the pharmacology and the role of ESR1 gene aberrations in endocrine therapy resistance of ER positive breast cancer
Authors: Sen Triparna , Li Shunqiang , Shao Jieya , Crowder Robert , Kitchens Robert , Ellis Matthew J.
Published: May 2015

Background: The study of estrogen-receptor-positive patient-derived xenografts (PDXs) from patients with disease progression despite endocrine therapy has revealed recurrent ESR1 mutation, gene amplification and ESR1 gene translocations as likely drivers of resistance. We addressed this hypothesis further using pharmacological and genetic approaches in the PDX system.

Methodology: ER positive PDXs that harbored one of four recurrent ligand binding domain (LBD) mutations (Y537S/N/C and D538G) as well as a ESR1/YAP1 translocation were studied in parallel with T47D and MCF7 cells expressing these mutants through lentivirus constructs. Single cell suspensions generated from PDX models were using for in vitro experiments using with a panel of endocrine inhibitors and ESR1 shRNA constructs. In vitro effects were confirmed using in vivo tumor response.

Results: Cell suspensions from ER+ PDX models in short-term culture successfully reproduced the endocrine therapy resistant phenotypes exhibited by the donor patient, thereby facilitating drugs screens designed to identify new endocrine approaches for ESR1 mutant breast cancer using authentic models rather than an exclusive reliance on models derived by transfection of mutant constructs into standard indicator cell lines. We were also able to demonstrate that that lentivirus-driven shRNA mediated knockdown of ESR1-YAP1 caused inhibition of PDX tumor growth in in vivo and reduced cell growth in vitro, confirming that this chimeric protein is a driver of estradiol-independent growth.

Conclusion: ER+ PDX can be manipulated in vitro and in vivo to successfully model ESR1 mutation and gene-rearrangement driven endocrine therapy resistance. PDX studies complement standard ectopic expression systems for the study of ESR1 mutation-driven resistance and, we hypothesize, will provide pharmacological data that will predict the activity of pharmacological agents designed to improve outcomes for patients with ESR1 mutant driven endocrine therapy resistance.

Citation Format: Triparna Sen, Shunqiang Li, Jieya Shao, Robert Crowder, Robert Kitchens, Matthew J. Ellis. Patient-derived xenograft study reveals the pharmacology and the role of ESR1 gene aberrations in endocrine therapy resistance of ER positive 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 5544. doi:10.1158/1538-7445.AM2014-5544
Abstract 556: Interleukin 32 is an independent prognostic biomarker of human gastric cancer correlated with tumor progression and metastasis
Authors: Wang Chia-Siu , Tsai Chung-Ying , Lin Kuan-Huei
Published: May 2015

Background: The pro-inflammatory cytokine, interleukin 32 (IL-32), is a novel tumor marker highly expressed in various human carcinomas including gastric cancer (GC).

Materials and Methods: A total of 120 patients with gastric cancer undergoing gastrectomy were enrolled into this study. Real-time quantitative RT-PCR, western blot analysis and immunohistochemistry (IHC) on resected specimens were used to confirm the IL-32 overexpression in surgical patients. The clinicopathological correlation and survival outcome of IL-32 were analysed for clinical significance in these patient. In vitro study, using ectopic overexpression and knockdown of IL-32 in GC cell lines, was established to investigate the invasiveness and the signaling pathway of IL-32 in GC.

Results: IL-32 was significantly upregulated in GC and positively correlated with aggressiveness of cancer and poor prognosis. Stable suppression or overexpression of IL-32 revealed marked influence of cell migration and invasion by modulating phosphorylation of AKT and glycogen synthase kinase 3β (GSK3β), leading to activation of β-catenin. Expression of IL-32 was accompanied by the induction of interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP2) and 9 (MMP9) in vitro and in vivo.Conclusion: Our results clearly suggest that IL-32 is an important mediator for GC metastasis and an independent prognostic predictor of GC.

Citation Format: Chia-Siu Wang, Chung-Ying Tsai, Kuan-Huei Lin. Interleukin 32 is an independent prognostic biomarker of human gastric cancer correlated with tumor progression and metastasis. [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 556. doi:10.1158/1538-7445.AM2014-556
Abstract 5578: Breast cancer cell growth regulatory mechanisms of tamoxifen metabolites
Authors: Thomas T. , Hsu Huichen , Yang PingAr , Hyvonen Mervi T. , Keinanen Tuomo A.
Published: May 2015

The proliferation of estrogen receptor alpha (ERalpha)-positive breast cancer cells is stimulated by estradiol. Tamoxifen [(Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine] is the most commonly used endocrine treatment for ERalpha-positive breast cancer in pre- and post-menopausal women. Tamoxifen is metabolized to 4-hydroxytamoxifen and endoxifen. ICI 182780 (Faslodex or Fulvestrant) is a pure antiestrogen targeted to the ERalpha. We investigated the effects of endoxifen and 4-hydroxytamoxifen on ERalpha-positive MCF-7 breast cancer cells using cell growth assay, oncogene expression and polyamine regulatory pathways. Polyamines are ubiquitous cellular components and are essential for cell growth and differentiation. Treatment of cells for 1-5 days with 4 nM estradiol increased cells number, whereas the addition of endoxifen, 4-hydroxytamoxifen or ICI 182780 in combination with estradiol suppressed cell growth in a concentration- and time-dependent manner. Real time quantitative polymerase chain reaction (RT-qPCR) experiments showed that the addition of 4 nM estradiol to MCF-7 cells increased the expression of c-myc, c-fos and Tiff1 genes significantly, while the addition endoxifen, 4-hydroxytamoxifen, or ICI 182780 suppressed estradiol-mediated increase in gene expression. Polyamine biosynthetic enzymes were also affected by tamoxifen metabolites. Estradiol increased the activities of the polyamine biosynthetic enzyme, ornithine decarboxlase (ODC) and S-adenosylmethionine decarboxlase (AdoMetDC), whereas the antiestrogens suppressed ODC and AdoMetDC activities in a concentration- and time-dependent manner. However, these agents had little effect on the activity of the polyamine catabolic enzyme, spermidine/spermine N1-acetyl transferase (SSAT). The change in biosynthetic enzyme activities was reflected in polyamine pools, as decreased putrescine and spermidine levels. There was no significant change in spermine level. These results suggest that the mechanism of action of tamoxifen metabolites involves multiple cell growth regulatory pathways. In addition, endoxifen can act as an antiestrogen and suppress cell growth stimulatory effects of estradiol on ERalpha-positive breast cancer cells in a manner similar to that of ICI 182780.

Note: This abstract was not presented at the meeting.

Citation Format: T. Thomas, Huichen Hsu, PingAr Yang, Mervi T. Hyvonen, Tuomo A. Keinanen. Breast cancer cell growth regulatory mechanisms of tamoxifen metabolites. [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 5578. doi:10.1158/1538-7445.AM2014-5578
Abstract 5591: Simultaneous isolation and quantification of circulating exosomes for cancer biomarker discovery
Authors: Kanwar Shailender S. , Dunlay Christopher J. , Simeone Diane M. , Nagrath Sunitha
Published: May 2015

Membrane bound extracellular vesicles, including microvesicles and exosomes, are secreted by both normal and cancerous cells into the extracellular space and in blood circulation. These circulating extracellular vesicles (cirEVs) and exosomes in particular, are recognized as a potential source of disease biomarkers and also carry signature molecules for early stage cancer detection. However, to exploit the use of circulatory exosomes as a new diagnostic tool, a rapid, high-throughput and reproducible method is required for their isolation and to allow their molecular analysis. We have developed a simple and a low cost microfluidic-based platform to analyze cirEVs enriched in exosomes directly from blood serum in a single step allowing simultaneous capture and quantification of exosomes. To capture exosomes with specificity, we employed a microfluidic device “ExoChip” functionalized with antibodies against exosomes to capture cirEVs. Subsequent staining with a fluorescent dye that specifically labels the exosomes, we quantitated exosomes using a standard plate-reader. Ten independent ExoChip experiments performed using serum obtained from five pancreatic cancer patients and five healthy individuals revealed a statistically significant increase (2.34±0.31 fold, p <0.001) in exosomes captured in cancer patients when compared to healthy individuals. Exosomal origin of ExoChip immobilized vesicles were further confirmed using immuno-electron-microscopy and Western blotting. In addition, we demonstrate the ability of ExoChip to recover exosomes with intact RNA enabling profiling of exosomal-microRNAs through openarray analysis, which has potential applications in biomarker discovery. Based on our findings, ExoChip is a well suited platform to be used as an exosome-based diagnostic and research tool for molecular screening of human cancers.

Citation Format: Shailender S. Kanwar, Christopher J. Dunlay, Diane M. Simeone, Sunitha Nagrath. Simultaneous isolation and quantification of circulating exosomes for cancer biomarker discovery. [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 5591. doi:10.1158/1538-7445.AM2014-5591
Abstract 5603: Aryl hydrocarbon receptor, a receptor for environmental pollutants, promotes tumorigenesis via intestine-specific homeobox expression in hepatocellular carcinoma
Authors: Wang Li-Ting , Wang Shen-Nien , Hsu Shih-Hsien
Published: May 2015

Environmental pollutants often cause diseases. Aryl hydrocarbon receptor (AHR), a major transcriptional factor in the xenobiotic response, regulates cellular signaling and metabolism, thereby influencing disease progression. The detailed regulatory mechanisms, however, remain unclear. Here we demonstrate that AHR expression promotes tumorigenesis and is associated with shorter survival time in HCC patients. These phenomena appear to be mediated by activation of a target proto-oncogene, intestine-specific homeobox (ISX), in vitro and in vivo. The strong correlation of expression and cellular transcriptional regulation between AHR and ISX (in tumor and hepatoma cells) underscore the significant role of this duo in HCC tumorigenesis and prognosis. Our experiments with the ablation of ISX expression in AHR-mutant mice and hepatoma cells further support the essential roles of AHR and ISX in liver and tumor development. These results advance the understanding of the cellular function of AHR and of its downstream gene, ISX, in HCC progression. Our data may help to develop a new therapeutic approach to HCC.

Citation Format: Li-Ting Wang, Shen-Nien Wang, Shih-Hsien Hsu. Aryl hydrocarbon receptor, a receptor for environmental pollutants, promotes tumorigenesis via intestine-specific homeobox expression in hepatocellular carcinoma. [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 5603. doi:10.1158/1538-7445.AM2014-5603
Abstract 561: A natural copy number variant in the GAEC1 oncogene is associated with breast cancer susceptibility
Authors: Gopalan Vinod , Smith Robert A. , Pillai Suja , Griffiths Lyn R. , Lam Alfred K-Y
Published: May 2015

The recently discovered GAEC1 oncogene, located at 7q22, has been found to be amplified in esophageal and colorectal cancers and is associated with altered clinical characteristics in those diseases. Amplification studies in these cancers showed variation in copy number in normal tissues, indicating a potential natural copy number variant (CNV) of GAEC1. In this research, the presence of such a CNV and its association with sporadic breast cancer development was tested in a population of 190 Caucasian women with breast cancer and a population of age, sex and ethnicity matched controls. Data produced by qPCR confirmed the presence of a CNV for GAEC1 in both cancer and control populations, with three common copy number types and several uncommon values, representing up to a 64-fold difference in GAEC1 copies. In addition, Chi-square analysis of copy number types indicated that increased GAEC1 copy number is associated with cancer development. This includes analysis using only the common copy number types (p=0.05) as well as considering all genotypes on a High/Low gene dosage basis (p=0.03). Overall risk for breast cancer was found to be slightly increased in high dose individuals (OR 1.552 (95% CI 1.035-2.338)). These results indicate that GAEC1 may be an important gene in the development of breast cancer, and that examination of its copy number in patient DNA may be a useful addition to genetic risk markers in sporadic cases.

Citation Format: Vinod Gopalan, Robert A. Smith, Suja Pillai, Lyn R. Griffiths, Alfred K-Y Lam. A natural copy number variant in the GAEC1 oncogene is associated with breast cancer susceptibility. [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 561. doi:10.1158/1538-7445.AM2014-561
Abstract 575: NKX6.1 functions as a tumor suppressor in hepatocellular carcinoma
Authors: Li Hsin-Jung , Shih Yu-Lueng , Yan Ming-De , Yu Pei-Ning , Lin Ya-Wen
Published: May 2015

In addition to genetic alterations, epigenetic alterations have been increasingly recognized as a key event for carcinogenesis. Methylation of CpG islands in promoter regions is almost associated with transcriptional silencing and is implicated in tumor suppressor gene (TSG) inactivation in cancer cells. Our previous data showed that NKX6.1 promoter is hypermethylated and downregulated in hepatocellular carcinoma (HCC) cell lines. The goal of this study was to elucidate whether NKX6.1 is a tumor suppressor gene in HCC.

All HCC samples with NKX6.1 promoter hypermethylation showed downregulation of NKX6.1 expression using quantitative methylation-specific PCR and quantitative RT-PCR analysis. In addition, the expression of NKX6.1 in HCC cell lines can be restored by treatment with DNA methyltransferase (DNMT) inhibitor, 5-aza-2′-deoxycytidine. These findings indicate that NKX6.1 is frequently silenced in HCC via promoter methylation. We then cloned and determined the putative promoter of NKX6.1 by luciferase reporter assay. The Sp1 site in the NKX6.1 promoter region is important for the promoter activity. When we treated cells with Trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), the NKX6.1 promoter activity increased. These data suggest that promoter methylation and histone modification may take part in the epigenetic silencing of NKX6.1 in HCC. Overexpression of NKX6.1 significantly suppressed transformation in HepG2 and SK-Hep1 cells; however, there is no significant effect on cell viability. Moreover we use a xenograft model to demonstrate the suppressor effects of NKX6.1 on tumor formation in vivo. Taken together, these data suggest that NKX6.1 might be a candidate tumor suppressor in HCC in vitro and in vivo.

Citation Format: Hsin-Jung Li, Yu-Lueng Shih, Ming-De Yan, Pei-Ning Yu, Ya-Wen Lin. NKX6.1 functions as a tumor suppressor in hepatocellular carcinoma. [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 575. doi:10.1158/1538-7445.AM2014-575
Abstract 578: RARRES1 is a tumor suppressor in triple-negative breast cancer cell lines
Authors: Coyle Krysta M. , Vaghar-Kashani Ahmad , Wong Florence , Dean Cheryl , Giacomantonio Carman , Marcato Paola
Published: May 2015

Increasing evidence suggests that retinoic acid (RA) signaling is of great importance in the progression and metastasis of different cancer types. We have previously reported that MDA-MB-231 and MDA-MB-468 triple-negative breast cancer cell lines display different responses to increased retinoic acid: retinoic acid increases MDA-MB-231 xenograft growth and decreases growth MDA-MB-468 xenografts. To investigate what may be responsible for these opposite responses to RA, we have investigated the role of retinoic acid receptor responder protein 1 (RARRES1).

RARRES1 is an RA-inducible gene which has not yet been fully characterized. Despite its unknown function, many independent studies have reported the tumor suppressive function of RARRES1 in multiple cancer types. Interestingly, our previous data indicates that high levels of RARRES1 in clinical breast cancer samples is correlated with poorer outcomes for patients. In particular, high expression of RARRES1 is associated with triple-negative breast cancers as well as with more aggressive cancers (by Ki67 positivity, as well as nuclear, mitotic, and overall grade).

Thus, to determine the role of RARRES1 in breast cancers, we have performed in vitro cell proliferation and in vivo xenograft assays. We report that knockdown of RARRES1 in MDA-MB-231 and MDA-MB-468 cells leads to increased cell proliferation in vitro and in in vivo xenografts.

RARRES1 is proposed to regulate a number of genes, including the hypothesized tumor suppressors discs large homolog 2 and protein phosphatase 2A, the proto-oncogene valosin-containing protein (p97), and ankyrin repeat domain-containing protein 26 which is of unknown function. We are investigating the regulation of these genes by RARRES1 in MDA-MB-231 and MDA-MB-468 triple-negative cell lines. We expect that the RARRES1 tumor suppressor is one of many genes induced by RA which form a complex signaling pathway regulating the response of cells to RA.

We conclude that RARRES1 is a tumor suppressor in MDA-MB-231 and MDA-MB-468 triple-negative breast cancer cell lines; however, we continue to investigate the discrepancy between this finding and our previous clinical data.

Citation Format: Krysta M. Coyle, Ahmad Vaghar-Kashani, Florence Wong, Cheryl Dean, Carman Giacomantonio, Paola Marcato. RARRES1 is a tumor suppressor in triple-negative breast cancer cell lines. [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 578. doi:10.1158/1538-7445.AM2014-578
Abstract 579: HPP1's tumor suppressive effects are mediated by ectodomain shedding but not by juxtacrine signaling
Authors: Elahi Abul , Ajidahun Abidemi , Clark Whalen , Hernandez Jonathan , Humphries Leigh Ann , Shibata David
Published: May 2015

Introduction: The tumor suppressor gene; HPP1 is downregulated in over 80% of colorectal cancers (CRC) and has potential as a serum and stool-based biomarker. HPP1 is a secreted transmembrane protein that contains an Epidermal Growth Factor (EGF) - like domain, two follistatin modules, and a cytosolic tail with a potential G-protein activating motif. The soluble ectodomain of HPP1 gene may undergo cleavage and functions as an EGF-like ligand which may bind to erbB family receptors. We sought to investigate the importance of cleavage and the possiblity of a juxtacrine role of the flanking EGF-like domain in the context of HPP1's tumor suppressive function..

Methods: An HPP1 double deletion construct (DD) lacking the juxtamembrane stalk cleavage site and an additional 4 amino acid residues (299-320) that constitutes the critical portion of the EGF-like domain was developed by PCR-based overlap extension mutagenesis. HPP1 non-expressing HCT116 cell lines were transfected with either wild-type HPP1 (WT), empty vector (EV) or double deletion construct (DD). Effects on proliferation (MTT assay) and anchorage-indpendent growth (soft agar assay) were evaluated.

Results: We have previously shown that overexpression of HPP1 results in substantial reduction in proliferation, growth in soft agar, and tumorigenicity. We have also identified that transfection of HPP1 with a deleted stalk sequence cleavage site (amino acid residues 303-320) results in a near-complete attenuation of its tumor suppressive effects. In order to determine whether there is a contributory juxtacrine effect of non-cleaved HPP1, we transfected the double deletion construct with a non-functioning EGF-like domain. Successful transfections of EV, WT, and DD were confirmed by RT-PCR. DD cells had similar proliferative potential and growth in soft agar when compared to EV cells (p = 0.609 and p= 0.263, respectively). When compared to WT, DD cells demonstrated a loss of HPP1's growth suppressive impact on cellular proliferation (optical density; 0.16± 0.03 vs. 0.31±0.04, p= 0.0001) and colony formation in soft agar (14.7 ± 5.5 vs. 278 ± 95.0, p= 0.009).

Conclusion: HPP1's tumor suppressive effects in colorectal cancer require cleavage and ectodomain shedding. However, it is unlikely that HPP1's biologic functions are mediated via an additional juxtacrine mechanism in this setting.

Citation Format: Abul Elahi, Abidemi Ajidahun, Whalen Clark, Jonathan Hernandez, Leigh Ann Humphries, David Shibata. HPP1's tumor suppressive effects are mediated by ectodomain shedding but not by juxtacrine signaling. [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 579. doi:10.1158/1538-7445.AM2014-579