bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1598: Grainyhead-like 2 (GRHL2) regulates the expression of forkhead box transcription factor M1 (FoxM1) in human oral cancer cells and determines tumorigenicity
Authors: Chen Wei , Yi Jin-Kyu , Shin Ki-Hyuk , Kim Reuben , Mehrazarin Shebli , Park No-Hee , Kang Mo K.
Published: May 2015

GRHL2 is a proto-oncogene that regulates epithelial proliferation and differentiation through transcriptional regulation of large number of target genes. The current study was performed to determine its role in oral carcinogenesis through regulation of forkhead box 1 (FoxM1), which is a known oncogene for many solid tumors. GRHL2 expression was highly elevated in oral squamous cell carcinoma (OSCC) tissues compared with normal oral epithelium (NHOM), and its protein expression is localized in the proliferative cells at the outer border of invasive islands of OSCC. In cultured OSCCs, GRHL2 and FoxM1 levels were both highly upregulated compared with normal human oral keratinocytes (NHOK), supporting their roles in oral carcinogenesis. When GRHL2 was knocked down in SCC4 cells by RNA interference, the cells have lost their proliferative capacity and colony formation when plated at low density. GRHL2 knockdown also resulted in marked loss of tumor spheroid formation, which is hallmark of cancer self-renewal. GRHL2 knockdown also led to loss of tumorigenic ability of cells upon xenograft transplantation in immunocompromised mice. GRHL2 appeared to determine the epithelial phenotype in OSCC cells. GRHL2 knockdown led to loss of E-cadherin and miR-200 family genes, which are epithelial markers. These data indicate that GRHL2 is required to maintain the transformed phenotype in OSCC. We also noted that GRHL2 knockdown led to drastic loss of FoxM1 expression, suggesting that GRHL2 may regulate FoxM1. To test this possibility, we performed in vivo binding assay by chromatin immunoprecipitation (ChIP), which showed GRHL2 binding at the proximal promoter of FoxM1. Luciferase reporter plasmid under FoxM1 promoter showed that GRHL2 directly regulates the gene promoter activity. FoxM1 knockdown resulted in loss of OSCC proliferation and tumor spheroid formation, while its overexpression conferred transformed phenotype in non-tumorigenic human oral keratinocytes (HOKs). Therefore, our data indicate that GRHL2 is required for the maintenance of transformed phenotype and that it plays a role in oral carcinogenesis through regulation of FoxM1. This study was supported in part by the grants from NIDCR/NIH (DE18295 and DE18959) and Jack Weichman Endowed Fund.

Note: This abstract was not presented at the meeting.

Citation Format: Wei Chen, Jin-Kyu Yi, Ki-Hyuk Shin, Reuben Kim, Shebli Mehrazarin, No-Hee Park, Mo K. Kang. Grainyhead-like 2 (GRHL2) regulates the expression of forkhead box transcription factor M1 (FoxM1) in human oral cancer cells and determines tumorigenicity. [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 1598. doi:10.1158/1538-7445.AM2014-1598
Abstract 1606: Global mapping of tyrosine kinase signalling in breast cancer by combined use of RNAi and SILAC quantitative proteomics
Authors: Zhang Hua , Stebbing Justin , Xu Yichen , Giamas Georgios
Published: May 2015

Dysregulation of protein tyrosine kinases (TKs) signalling can contribute to tumorigenesis. However, less than half of the 90 TKs described so far have been thoroughly studied and even in these cases a global functional analysis is still lacking. A combined use of RNA interference (RNAi) and stable isotope labelling by amino acids in cell culture (SILAC) was applied in breast cancer MCF7 cells. After knocking down individually each TK, a proteomic map consisting of ∼3,000 regulated proteins per TK was acquired.

Quantitative and statistical analyses resulted in the clustering of the significantly modulated proteins/TK into various biological and molecular categories.

Further use of mathematical modelling tools resulted in a new classification and phylogeny of the TK family members depending on connections between their regulated proteomes. This taxonomy has little overall correlation when compared to their catalytic domain sequence grouping. These data suggest that despite the primary structure homology of kinases, their regulated proteomic landscapes can significantly vary and may depend on other factors, notably their dynamic interactions with other proteins.

Our results and bionformatic analyses will be presented.

Citation Format: Hua Zhang, Justin Stebbing, Yichen Xu, Georgios Giamas. Global mapping of tyrosine kinase signalling in breast cancer by combined use of RNAi and SILAC quantitative proteomics. [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 1606. doi:10.1158/1538-7445.AM2014-1606
Abstract 1607: Quantitative membrane proteome profiling to discover therapeutic targets for adult T-cell leukemia (ATL)
Authors: Ishihara Makoto , Araya Natsumi , Sato Tomoo , Fujii Risa , Tatsuguchi Ayako , Saichi Naomi , Nakagawa Hidewaki , Yamano Yoshihisa , Ueda Koji
Published: May 2015

Human T-cell leukemia virus type 1 (HTLV-1) is one of retroviruses known as the causative agent of adult T-cell leukemia (ATL), which is infected within 20 million people in the world. ATL develops at the prevalence of only 5% among HTLV-1-infected individuals after a long latency period over 30 years. However, the outcomes of conventional treatment for ATL are not satisfactory, exhibiting overall 5-year survival rate of 20%. To discover novel therapeutic targets for ATL, we performed label-free quantitative membrane proteome analysis for CD4+ T-cell subpopulation in which HTLV-1 infected cells are enriched. Tryptic digests of CD4+ T-cells were prepared from 14 normal donors (ND), 21 asymptomatic carriers (AC), and 13 ATL patients. To focus on the membrane derived peptides, glycosylated peptides were enriched by lectin column chromatography (Mol Cell Proteomics. 2010,9(9),1819) since most of membrane proteins are glycosylated. The eluates were analyzed by LC/MS/MS and 8,658 N-glycosylated peptides derived from 1,134 N-glycosylated proteins were identified.

The therapeutic targets were subsequently extracted by a couple of criteria as follows. The all-or-nothing analysis explored peptides detected in none or 1 case of ND+AC group, resulting in identification of 31 peptides derived from 19 proteins. Additionally Welch's t-test (ATL vs. ND+AC) identified 33 peptides derived from 14 proteins (p < 0.05 and fold change > 2.0).

The identified therapeutic target molecules above included cell adhesion molecule 1 (CADM1 or TSLC1) which overexpresses in ATL cells and induces organ infiltration of tumor cells. Programmed cell death 1 ligand 1 (PD1L1) was also found in our therapeutic target list, which was known to be up-regulated in ATL cells and involved in induction of immune tolerance. These facts strongly emphasize the credibility of our quantitative profiling results. We would like to show the results of functional inhibitory experiments for novel therapeutic targets and discuss the uncovered molecular mechanisms of ATL progression.

Note: This abstract was not presented at the meeting.

Citation Format: Makoto Ishihara, Natsumi Araya, Tomoo Sato, Risa Fujii, Ayako Tatsuguchi, Naomi Saichi, Hidewaki Nakagawa, Yoshihisa Yamano, Koji Ueda. Quantitative membrane proteome profiling to discover therapeutic targets for adult T-cell leukemia (ATL). [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 1607. doi:10.1158/1538-7445.AM2014-1607
Abstract 1608: Profiling c-Met and EGFR kinase inhibitor resistance pathways in non-small lung cancer cells
Authors: Bomgarden Ryan D. , Jacobs Ryan , Fong Jason , Moravec David , Botting Gregory M. , Blank Michael , Viner Rosa I. , Rogers John C. , Puri Neelu
Published: May 2015

Tyrosine kinase inhibitors (TKIs) against c-MET and EGFR have positive therapeutic effects in a subset of Non-Small Cell Lung Cancer (NSCLC) patients; however, their clinical efficacies are limited due to the development of TKI resistance. To determine mechanisms of TKI resistance, two NSCLC model cell lines H2170 and H358 were made resistant to the c-Met/EGFR TKIs SU11274, erlotinib or a combination of both by exposing the cells to progressively increasing concentrations of inhibitor. Initial characterization of these cell lines using phospho-specific antibodies showed that phospho-c-Met was found to be downregulated 4- and 1.5- fold in SU11274-resistant (SR) H2170 and SR H358 cells, respectively. Phospho-EGFR was found to be constitutively autophosphorylated (upregulated 19-fold) in erlotinib-resistant (ER) H2170 cells, but downregulated 6-fold in ER H358 cells. Interestingly, c-Met/EGFR downstream signaling proteins p-mTOR, p-p70S6K and p-ERK were found to be 2-4-fold upregulated, 2-fold upregulated and 2-5-fold upregulated, respectively, in ER H2170 and H358 cells. Phospho-p70S6K was also found to be upregulated 20-fold in SR H2170 cells. These results indicate that alternative signaling pathways may confer TKI resistance in these cell lines. To identify which signaling pathways may be responsible for TKI resistance, H358 parental and resistant cell lines were analyzed by mass spectrometry (MS). Differences in protein expression in cell lines treated with and without EGF and/or erlotinib were measured using higher multiplexing Thermo Scientific™ Tandem Mass Tag™ (TMT™) reagents. More than 1500 protein groups were identified and quantified using a Thermo Scientific™ Orbitrap™ Fusion™ platform with HCD-MS2 and MS3 fragmentation methods. Protein expression in ER H358 cells showed a marked increase in expression of DNA replication proteins, transcription factors, apoptosis inhibitors and some Ras-associated signal transduction pathways. To further investigate the mechanism of TKI resistance, phospho-peptide enrichment was also used in combination with TMT quantitation to profile changes in protein phosphorylation state. Overall, the combination of phospho-peptide enrichment coupled with higher multiplexing TMT and high resolution MS analysis enabled deeper global profiling of TKI resistance signaling pathways compared to previous studies without enrichment.

Citation Format: Ryan D. Bomgarden, Ryan Jacobs, Jason Fong, David Moravec, Gregory M. Botting, Michael Blank, Rosa I. Viner, John C. Rogers, Neelu Puri. Profiling c-Met and EGFR kinase inhibitor resistance pathways in non-small lung cancer 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 1608. doi:10.1158/1538-7445.AM2014-1608
Abstract 1609: Proline-directed kinase signaling in human hepatocellular carcinomas developed on non-fibrotic liver
Authors: Taouji Saïd , Leong Kristen , Beausang Lee A. , Arma Daniela , Moreau Violaine , Balabaud Charles , Bioulac-Sage Paulette , Zucman-Rossi Jessica , Latterich Martin , Rosenbaum Jean , Chevet Eric
Published: May 2015

Ten to 40% hepatocellular carcinoma (HCC) arise on non-cirrhotic liver, including 5% on non-fibrotic liver (nfHCC). These tumors provide an interesting model for the analysis of the hepatocarcinogenesis pathways without the confounding factors associated with cirrhosis. Carcinogenesis has been often associated with the deregulation of phosphorylation-dependent signaling pathways. Quantitative phosphoproteomics recently allowed us to unravel the specific activation of proline-directed kinases (PDK) in nfHCC. Our aim was then to use medium throughput analytical tools (Alphascreen® and AMMP®) to evaluate the activation status of PDK dependent signaling pathways in a series of nfHCC samples. We have used a phosphoproteomics set comprising surgical samples from nfHCC (METAVIR F0 or F1) and samples of histologically normal livers. Proteins were extracted and analyzed for the activation status of select signaling pathways such as MAPK using both Alphascreen® and AMMP® technologies. Because MAPK are prototypic PDKs and are deregulated in many cancers, we analyzed their activation in a series of 25 nfHCC as compared to 6 non-tumor samples and found an overall increased activity in nfHCC samples. Moreover the phosphorylation status of Calnexin, a MAPK substrate found in the phosphoproteomics analysis was monitored and compared in nfHCC samples. In conclusion, we have identified phosphopeptide signatures in human samples of nfHCC that led to the identification of a deregulated PDK-dependent network in those tumors. The elucidation of the functional causes and consequences of such deregulation is ongoing.

Citation Format: Saïd Taouji, Kristen Leong, Lee A. Beausang, Daniela Arma, Violaine Moreau, Charles Balabaud, Paulette Bioulac-Sage, Jessica Zucman-Rossi, Martin Latterich, Jean Rosenbaum, Eric Chevet. Proline-directed kinase signaling in human hepatocellular carcinomas developed on non-fibrotic liver. [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 1609. doi:10.1158/1538-7445.AM2014-1609
Abstract 161: Activation of mTOR pathway in myeloid-derived suppressor cells with lysosomal acid lipase deficiency stimulates cancer cell proliferation and metastasis
Authors: Zhao Ting , Du Hong , Ding Xinchun , Walls Katlin , Yan Cong
Published: May 2015

Inflammation plays crucial roles at all stages of tumor development, from tumor initiation to metastatic progression, during which myeloid-derived suppressor cells (MDSCs) are an important participant. Although MDSCs are known to suppress immune surveillance, their roles in directly stimulating cancer cell proliferation and metastasis currently remains unclear. Lysosomal acid lipase (LAL) deficiency causes systemic expansion and infiltration of MDSCs in multiple organs and subsequent inflammation. In the LAL-deficient (lal-/-) mouse model, we found that melanoma metastasized massively in allogeneic lal-/- mice, which was suppressed in allogeneic lal+/+ mice due to immune rejection. Therefore, we hypothesized that MDSCs with LAL deficiency directly stimulate cancer cell proliferation and metastasis. Bone marrow-derived MDSCs from lal-/- mice directly stimulated B16 melanoma cell proliferation in vitro by co-culture analysis and in vivo by co-implantation in the Matrigel plugs. These tumor cell-stimulatory effects were diminished when myeloid-specific human LAL (hLAL) was expressed in myeloid cells in lal-/- mice. In addition, lal-/- MDSCs facilitated B16 melanoma cell metastasis in the lungs of recipient lal+/+ mice via tail vein injection. Furthermore, the mammalian target of rapamycin (mTOR) and its downstream gene products were significantly up-regulated in lal-/- MDSCs. Knockdown of mTOR in lal-/- MDSCs suppressed their stimulation on B16 melanoma cell proliferation, growth and metastasis, indicating the tumor-promoting function of lal-/- MDSCs is mediated, at least in part, through over-activation of the mTOR pathway. Finally, lal-/- MDSCs stimulated proliferation and growth of Lewis lung carcinoma (LLC), and transgenic mouse prostate cancer (TRAMP-C2) cells, and these effects were impaired after mTOR inhibition. Our results indicate that LAL plays a critical role in regulating MDSCs ability to directly stimulate cancer cell proliferation, and overcome immune rejection of cancer metastasis in allogeneic mice through modulation of the mTOR pathway, which provides a mechanistic basis for targeting MDSCs to reduce the risk of cancer metastasis. Therefore, MDSCs possess dual functions to facilitate cancer metastasis: suppress immune surveillance, and stimulate cancer cell proliferation and growth.

Citation Format: Ting Zhao, Hong Du, Xinchun Ding, Katlin Walls, Cong Yan. Activation of mTOR pathway in myeloid-derived suppressor cells with lysosomal acid lipase deficiency stimulates cancer cell proliferation 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 161. doi:10.1158/1538-7445.AM2014-161
Abstract 1622: Design and preclinical evaluation of single agents with tubulin and multiple receptor tyrosine kinase inhibition
Authors: Gangjee Aleem , Pavana Roheeth K. , Ihnat Michael , Hamel Ernest
Published: May 2015

Vascular endothelial growth factor receptor-2 (VEGFR2) inhibitors lead to the reduction of angiogenesis but are cytostatic, not tumoricidal. These inhibitors have shown significant promise in the clinic when used in combination with cytotoxic agents, and several clinical trials with such combinations are in progress. We have previously reported preclinical studies on AG119, a compound that inhibits both VEGFR2 and tubulin, thereby affording antiangiogenic and cytotoxic effects in a single agent. Structural modifications of AG119 afforded AG321, a unique compound with four separate mechanisms of action in a single agent. AG321 inhibits platelet derived growth factor receptor-β (PDGFRβ) and epidermal growth factor receptor (EGFR), in addition to VEGFR2 and tubulin. AG321 is a colchicine site binding agent with potency comparable with that of combretastatin A-4 against bovine brain tubulin assembly. AG321 has potency comparable with that of erlotinib in tumor cell lines overexpressing EGFR and potency comparable with those of erlotinib, PD153035 and DMBI and in cell lines overexpressing PDGFRβ. It also has potency comparable with those of sunitinib and semaxinib in cell lines overexpressing VEGFR2. AG321 reduced tumor size (control: 15.7 ± 2.9 mm3/day; AG321 treated: 5.7 ± 0.7 mm3/day; p < 0.01 by one way ANOVA with Tukey multiple comparisons post test) and the number of lung metastases in the 4T1 triple negative mouse breast cancer allograft model and was superior to docetaxel, without overt systemic toxicity. A range of potential therapies that target EGFR are currently in development or in clinical trials for the treatment of glioblastoma. Targeting both VEGFR2 and PDGFRβ, which are two key mediators of tumor angiogenesis, increases the antiangiogenic effect even in the often intractable late stage of solid tumors. Hence, AG321, a multiple receptor tyrosine kinase (RTK) inhibitor which may also be effective against EGFR-amplified glioblastoma [AG119 reduced tumor size and vascularity in vivo in the U251 glioma model]. In summary, AG321 with antiangiogenic effects via multiple RTK inhibition and cytotoxic effects via tubulin inhibition in a single entity may delay or prevent tumor cell resistance, circumvent pharmacokinetic problems that can occur with multiple agents, avoid drug-drug interactions, be used at lower doses to alleviate toxicity and be and have reduced toxicity.

Citation Format: Aleem Gangjee, Roheeth K. Pavana, Michael Ihnat, Ernest Hamel. Design and preclinical evaluation of single agents with tubulin and multiple receptor tyrosine kinase 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 1622. doi:10.1158/1538-7445.AM2014-1622
Abstract 1627: The novel camptothecin analog and antiapoptotic protein inhibitor FL118 appears to be a great backbone platform for development of personalized anticancer drugs
Authors: Li Fengzhi , Ling Xiang , Westover David , Liu Xiaojun , Jin Chunyang , Wani Mansukh
Published: May 2015

We recently characterized a novel camptothecin analog FL118. Interestingly, different from the two clinically used camptothecin analogs irinotecan and topotecan, which are topoisomerase 1 (Top1) inhibitors, FL118 appears to be a poor Top1 inhibitor at its therapeutic dose range. Instead, FL118 selectively inhibits multiple cancer survival and proliferation-associated antiapoptotic proteins including survivin, Mcl-1, XIAP, and cIAP2. We have demonstrated that genetic knockdown or overexpression of survivin, Mcl-1, XIAP, or cIAP2 modulates FL118's ability to inhibit cancer cell growth and induce apoptosis. FL118 is able to eliminate both small and large human tumor xenografts in animal models. Importantly, FL118 effectively overcomes topotecan treatment-induced resistance of human colon tumor in animal models. Pharmacokinetics (PK) studies indicated that FL118 prefers to be accumulated in tumor and rapidly cleared in blood stream in animal models. Furthermore, our most recent studies demonstrated that maintenance of a free hydroxyl group in the lactone ring of FL118 is critical for its antitumor activity, and the superior antitumor efficacy of FL118 heavily depends on its steric configuration (Zhao et al., Molecular Pharmaceutics). Based on the current observation of FL118 properties, we hypothesize that FL118 is an ideal lead for development of novel analogs that may possess distinct antitumor selectivity to overcome different treatment resistance. In this regard, we tested 146 new camptothecin analogs, which were synthesized based on either the FL118 core structure or the camptothecin core structure, in four cancer cell lines and two normal cell lines. We identified 19 top candidates that showed inhibition of cancer cell growth and induction of apoptosis that are better than or comparable to FL118. Intriguingly, all of the 19 top compounds possess the FL118 core structure. Further investigation of the 19 compounds using cancer cells with distinct treatment resistant backgrounds indicated that these compounds showed distinct antitumor selectivity among different treatment resistant cancer cells. Therefore, while our finding would need further investigation using in vivo human tumor animal models, we propose to develop a series of FL118 analogs for personalized cancer treatment to overcome different treatment resistance.

Citation Format: Fengzhi Li, Xiang Ling, David Westover, Xiaojun Liu, Chunyang Jin, Mansukh Wani. The novel camptothecin analog and antiapoptotic protein inhibitor FL118 appears to be a great backbone platform for development of personalized anticancer drugs. [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 1627. doi:10.1158/1538-7445.AM2014-1627
Abstract 1631: Structural activity relationship studies of aza-podophyllotoxin derivatives on breast cancer cell line
Authors: Santana Michael Concepcion , Kumar Ajay , Kumar Vineet , Alegria Antonio E. , Malhotra Sanjay V.
Published: May 2015

Podophyllotoxin is a natural precursor for the semi-synthesis of novel anticancer drugs and its an anti-tumor and antibacterial ligand derived from Podophyllum peltatum and Podophyllum emodi (Berberidaceae). Etoposide, etopophoside and tenoposide are anti-cancer drugs in pharmaceutical clinics since 1989 derived from a natural lignin podophyllotoxin. Synthesis of podophyllotoxin in a laboratory is one of the tedious and biggest challenges. In our previous work we developed a very simple method to synthesize extensively modified libraries of several novel derivatives, which were previously impossible to synthesize from podophyllotoxin (Kumar and Alegría, 2010). This method also allows us to explore the synthesis and applications of novel aza-podophyllotoxin derivatives which led to several new analogs with anti-cancer potential. Most of the compounds were found very active against Colon cancer, Melanoma, Ovarian, Renal, prostate and breast cancers at NCI upon a panel of 60 types of human cancer cell lines (Kumar, et.al. 2011). Based on structure activity relationship (SAR) of our compounds and their recent unpublished extraordinary activity especially against colon cancer (COLO 205) cell lines, we designed and synthesized novel aza-podophyllotoxin derivatives using microwave synthesis technique. We will be discussing the synthesis, SAR and biological activity of our novel 2,3-didehyropodophyllotoxin derivatives.

Keywords: Pharmacology, Organic Chemistry and Toxicology

Disclosure of Chemical Structure: The complete chemical structures of the compounds of the compounds used will be disclosed at the time of presentation at the meeting.

Funded by INBRE-NIGMS-NIH gran #8 P20 GM 102475 and REU NSF 1262826

Citation Format: Michael Concepcion Santana, Ajay Kumar, Vineet Kumar, Antonio E. Alegria, Sanjay V. Malhotra. Structural activity relationship studies of aza-podophyllotoxin derivatives on breast cancer cell line. [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 1631. doi:10.1158/1538-7445.AM2014-1631
Abstract 1632: Discovery of a novel series of androgen receptor antagonists with potential therapeutic applications in castration-resistant prostate cancer
Authors: Li Huifang , Hassona Mohamed DH , Lack Nathan A. , Axerio-Cilies Peter , Leblanc Eric , Guns Emma T. , Rennie Paul S. , Cherkasov Artem
Published: May 2015

The human androgen receptor (AR) represents a well-established drug target for prostate cancer treatment. All clinically used antiandrogens, such as Bicalutamide and Enzalutamide, possess similar chemical structures and bind to the AR in the androgen binding site (ABS). These AR antagonists are initially effective, but resistance invariably developed in the castration-resistant prostate cancer (CRPC). Even in late-stage patients, the AR activity is still persistent in the progression of the disease. Thus, there is a continuing need for novel chemical classes of AR antagonists that could overcome the drug resistance.

In this study, we performed a virtual screening against the AR ABS, and identified novel AR antagonists with chemical structures completely different from existing antiandrogens. A 10-(4-Hydroxybenzylidene)anthracen-9(10H)-one compound was discovered that not only effectively inhibits AR transcription and strongly displaces androgen in the ABS, but induces AR degradation in prostate cancer cells. Starting from the initial hit compound, a series of 10-benzylidene-10H-anthracen-9-ones were synthesized and evaluated by in vitro assays. A close analogue with enhanced potency was identified as a lead compound, which demonstrated strong androgen displacement potency, effective AR transcriptional inhibition, and a profound ability to cause degradation of AR. Notably, it exhibited significant activity against MDV3100-resistant prostate cancer cells. This lead compound was evaluated in both non-castrated and castration-resistant LNCaP xenograft models, and demonstrated significant effect on inhibiting tumor growth and reducing prostate specific antigen (PSA). This series of compounds were predicted to adopt a different binding mode from current antiandrogens, which may circumvent the resistance rendered by identified gain-of-function mutations. Further development of this series of AR antagonists may have great therapeutic potential in CRPC.

Citation Format: Huifang Li, Mohamed DH Hassona, Nathan A. Lack, Peter Axerio-Cilies, Eric Leblanc, Emma T. Guns, Paul S. Rennie, Artem Cherkasov. Discovery of a novel series of androgen receptor antagonists with potential therapeutic applications in castration-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 1632. doi:10.1158/1538-7445.AM2014-1632
Abstract 1634: A novel small molecule inhibitor of IRE1alpha reverses endocrine resistance in breast cancer cells
Authors: Shajahan-Haq Ayesha N. , Smith Jacqueline , Dakshanamurthy Siva , Wang Difei , Eltayeb Ahreej E. , Brown Milton L. , Clarke Robert
Published: May 2015

Over two-thirds of all breast cancer patients are estrogen receptor alpha positive (ER+). However, almost 50% of all ER+ breast tumors do not respond or respond and later fail on endocrine therapy. Resistance to endocrine therapy remains a significant clinical problem and advanced ER+ breast cancer is largely an incurable disease. The mechanisms of resistance to endocrine therapy remain unclear. We have reported that the unfolded protein response (UPR), a stress-induced cellular adaptive mechanism, acts as a switch controlling the balance between cell death and cell survival in response to these therapies. UPR is activated by three molecular sensors: IRE1alpha, ATF6, PERK. Several components of the UPR, including IRE1alpha, are up-regulated in endocrine resistant cells. We now report a novel small molecule inhibitor of IRE1alpha: N-(4-Phenoxy-phenyl)-2-(5-pyridin-3-yl-2H-[1,2,4]triazol-3-ylsulfanyl)-acetamide (NPPTA). We show that NPPTA interacts with IRE1alpha in vitro and can decrease IRE1alpha protein levels. NPPTA treatment alone inhibits cell proliferation in antiestrogen resistant ER+ breast cancer cells more effectively than in sensitive cells, and in a dose- and time-dependent manner. Normal breast epithelial cells are unaffected by NPPTA. In addition, NPPTA and TAM synergistically inhibit cell proliferation in TAM resistant breast cancer cells. NPPTA's activities are associated with induced G1 cell cycle arrest, increased apoptosis/necrosis, and inhibition of anti-apoptotic signaling. Targeting IRE1alpha may be a new and effective strategy to reverse endocrine resistance in breast cancer.

Citation Format: Ayesha N. Shajahan-Haq, Jacqueline Smith, Siva Dakshanamurthy, Difei Wang, Ahreej E. Eltayeb, Milton L. Brown, Robert Clarke. A novel small molecule inhibitor of IRE1alpha reverses endocrine resistance in breast cancer 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 1634. doi:10.1158/1538-7445.AM2014-1634
Abstract 1636: NMR discovery and molecular-basis of small molecule inhibitors of STAT3
Authors: Namanja Andrew T. , Buettner Ralf , Jove Richard , Chen Yuan
Published: May 2015

Hyperactivation of the signal transducer and activator of transcription 3 (STAT3) transcription factor is a critical molecular anomaly that promotes the constitutive transcription of pro-oncogenic genes in many tumor cells. Therefore, the direct inhibition of STAT3 signaling is a promising strategy to treat cancer. However, targeting transcription factors with small molecule drugs has proved to be challenging. To facilitate the generation of inhibitor leads with good ADME (absorption, distribution, metabolism, and excretion) attributes, we employed fragment-based screening by nuclear magnetic resonance spectroscopy (NMR) in conjunction with electrophoretic mobility shift assay (EMSA) to identify small molecule inhibitors of STAT3. The inhibitors confer specificity for STAT3; inhibit the activation and nuclear accumulation of STAT3; inhibit STAT3 transcription of cell cycle regulatory genes and the proliferation of STAT3-dependent cancer cell lines. We have used two-dimensional methyl NMR of STAT3 to reveal atomic resolution insights on the basis for inhibition. Our results establish an innovative approach to targeting STAT3 that may also be applied to other transcription factors associated with cancer and other diseases.

Citation Format: Andrew T. Namanja, Ralf Buettner, Richard Jove, Yuan Chen. NMR discovery and molecular-basis of small molecule inhibitors of STAT3. [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 1636. doi:10.1158/1538-7445.AM2014-1636
Abstract 1638: Effect of the phytochemical crinumaquine on prostate mesenchymal type cells
Authors: Marvyin Kristo , Liu Run-hui , Jin Huizi , Hua Yaping , Qu Yi , Ke Xisong , Kalland Karl-Henning , Zhang Wei-dong , Oyan Anne Margrete
Published: May 2015

In a prostate carcinogenesis cell culture model benign EP156T prostate epithelial cells underwent epithelial to mesenchymal transition (EMT), and a series of progeny EPT mesenchymal type cells accumulated malignant features in a stepwise fashion. Through the screening of phytochemicals using this model, we have found that crinumaquine inhibited proliferation of mesenchymal type EPT1 and EPT2 cells more efficiently than the compound inhibited epithelial type EP156T cells. Genome-wide gene expression analysis revealed a pattern where crinumaquine induced patterns associated with cell cycle arrest at both G1-S and G2-M phase checkpoints and strong up-regulation of genes involved in DNA repair and CDKN1A and CDKN1C and chromatin rearrangement. Differentially expressed genes were uploaded to the Connectivity Map tools (The Broad Institute, MA, USA) and the highest score was associated with histone deacetylase inhibitors. ChemMap analysis based on its chemical structure indicated that crinumaquine targets ER-β and cyclines. Crinumaquine was also found to induce up-regulation of CDH1 and other genes associated with an incomplete mesenchymal to epithelial transition (MET) of EPT1 and EPT2 cells, and this was associated with increased apoptosis markers in gene expression and Western blot analyses. Due to the selective inhibition of mesenchymal type and more malignant cells of the model, crinumaquine will be further explored concerning its anti-cancer leading compound potential.

Citation Format: Kristo Marvyin, Run-hui Liu, Huizi Jin, Yaping Hua, Yi Qu, Xisong Ke, Karl-Henning Kalland, Wei-dong Zhang, Anne Margrete Oyan. Effect of the phytochemical crinumaquine on prostate mesenchymal type 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 1638. doi:10.1158/1538-7445.AM2014-1638
Abstract 1639: Development of novel thiobarbituric acid derivatives as potential cancer therapeutics
Authors: Ramisetti Srinivasa R. , Lee Sang Y. , Pandey Manoj K. , Amin Shantu G. , Sharma Arun K.
Published: May 2015

Chemotherapy drugs for the treatment of cancer are generally directed to inhibit the reproduction of malignant cells and killing cells, thereby preventing tumor growth or reducing tumor size. Many of these drugs develop resistance to tumor cells by different mechanisms. Therefore a significant clinical need exists for the development of additional chemotherapeutic agents that are toxic to a wide range of tumor cells, in particular tumors that are resistant to current treatments such as radiotherapy and chemotherapy. From recent past, it has been known that barbituric acid derivatives exhibit different biological activities such as anti-depressant, anti-convulsant, anti-viral and anti-cancer activities. Among them some thiobarbituric acid derivatives, such as merocyanine and merbarone have been proposed as possible modulators of apoptosis in several cancer cells including glioblastoma multiforme (GBM), lung, melanoma, leukemia, breast cancer, prostate cancer, and cervical cancer; albeit not particularly for cancers that are resistant to current therapy. We designed and synthesized novel thio- and seleno- barbituric derivatives by varying the substituents at N1, N3 (ethyl, methyl, allyl, and phenyl) and C5 tethered with dienyl and trienyl moieties attached with different substituents like phenyl, 2-furanyl, 2-thiophenyl, 1-naphthyl & 3-pyridyl. These compounds were tested against both chemotherapy sensitive and resistant, melanoma (UACC903 and CHL-1), GBM (U87-MG, T98G, and CCF-STTG1) , and lung cancer (A549, H460, H520and H69) cells. Most of these compounds reduced cell viability in a dose and time dependent manner. Among all the compounds ASR-198, ASR-199, ASR-243 and ASR-244 were most cytotoxic to both chemotherapy resistant and sensitive cells. In addition, these compounds triggered a dose dependent induction of apoptosis by activation of caspase-3 and PARP cleavage in melanoma cells. Overall compound ASR-198, having a 2-thiophen-dienyl substitution and C-5 position, emerged as the most efficacious compound in all three cancer cell lines. Interestingly its isosteric selenium analogs were relatively less cytotoxic. In summary, this structure-activity relationship study clearly indicates that more efficacious compounds, compared to existing thiobarbituric analogs, can be developed based on barbituric acid scaffold, by rational modifications of the chemo type compounds. Detailed results of these investigations will be presented.

Citation Format: Srinivasa R. Ramisetti, Sang Y. Lee, Manoj K. Pandey, Shantu G. Amin, Arun K. Sharma. Development of novel thiobarbituric acid derivatives as potential cancer therapeutics. [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 1639. doi:10.1158/1538-7445.AM2014-1639
Abstract 1651: Profiling immune cells within the tumor microenvironment for optimal model selection for pre-clinical investigations
Authors: Leishman Andrew , Harris Olivia , Ulrichsen Jane Coates , Harper James , Popple Amy , Papaspyridonos Marianna , Williams Geoff , Mullins Stefanie , Valge-Archer Viia , Stewart Ross , Sainson Richard , Morrow Michelle , Wilkinson Robert
Published: May 2015

Infiltrating host immune cells can have a significant impact on the growth and outcome of many tumor types. Several leukocyte populations have been implicated in establishing an immune suppressive microenvironment (e.g. myeloid derived suppressor cells [MDSCs) and regulatory T cells [Treg cells]) and thus making it harder for anti-tumor immune effector cells to function (e.g. CD8 cytotoxic T cells). Immune-Mediated Therapy (IMT) is emerging as a key strategy for the treatment of cancer through by enhancing a patient's anti-tumor immune response. In order to maximise the potential of IMT strategies it is important to understand the immune status within the tumor microenvironment as well as systemically. Therefore, fully immune-competent preclinical models are valuable tools to enable the study of the immune system, both locally within the tumor and systemically, to understand the mechanism of action of IMT monotherapies and combination therapies in vivo. Consequently, we have profiled the immune status of a panel of syngeneic mouse tumor models to assist optimal model selection to aid these investigations. We developed multi-parameter flow cytometry techniques to identify specific immune subsets in both the tumor microenvironment and the periphery. These investigations revealed a number of tumor infiltrating immune cell end-points that can be used to correlate with response to IMT depending on the syngeneic model being used. For example, we observed differences in the MDSC and Treg cell populations across the models investigated, which will influence optimal model selection depending on the mechanism of action of the IMT being tested.

Citation Format: Andrew Leishman, Olivia Harris, Jane Coates Ulrichsen, James Harper, Amy Popple, Marianna Papaspyridonos, Geoff Williams, Stefanie Mullins, Viia Valge-Archer, Ross Stewart, Richard Sainson, Michelle Morrow, Robert Wilkinson. Profiling immune cells within the tumor microenvironment for optimal model selection for pre-clinical investigations. [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 1651. doi:10.1158/1538-7445.AM2014-1651
Abstract 1660: Retroviral replicating vector transduced alloresponsive T lymphocytes retain mobility and cytotoxic functionalities
Authors: Erickson Kate L. , Malone Colin C. , Hickey Michelle J. , Owens Geoffrey C. , Kato Yuki , Prins Robert M. , Kasahara Noriyuki , Kruse Carol A.
Published: May 2015

The radial monolayer cell migration assay was originally developed to measure the infiltrative properties of adherent tumor cells on individual extracellular matrix (ECM) proteins, or on ECM extracted from tumor cell monolayers. For this assay a single cell suspension of tumor cells is seeded using a cell sedimentation manifold (CSM), precisely into the center of Teflon-masked, ECM-coated slide wells. The adherent tumor cells establish a focal, circular monolayer from which horizontal motility can be microscopically-viewed and photographed over time. We adapted use of the CSM to evaluate the in vitro mobility of non-adherent effector T cells on confluent tumor cell monolayers or on ECM extracts from them. Cytotoxic T lymphocytes (CTL) sensitized to the human leukocyte antigens (HLA) on 13-06-MG glioma cells or on brain-tropic breast MDA-MB-231 carcinoma cells were generated by one-way mixed lymphocyte tumor cell reactions. These alloresponsive CTL were then transduced with retroviral replicating vectors (RRV) coding for green fluorescent protein (GFP). Using the CSM, the RRV-GFP transduced CTL were seeded into the center of wells with a confluent monolayer of glioma cells that were partially HLA matched to 13-06-MG. By light and fluorescence microscopy, we visualized not only the a) mobility of individual GFP+ T cells, but b) the cytotoxicity engendered to the underlying adherent glioma cell monolayer by the overlaid nonadherent CTL, in addition to c) transfer of RRV from the CTL to tumor cells. The in vitro cytotoxic functionality of the RRV-transduced CTL was confirmed by real-time xCELLigence impedance cytotoxicity assays. In other experiments, the RRV-GFP transduced CTL were also stained with a vital fluorescent red dye, CMPTX, to distinguish transduced from nontransduced CTL. The CTL were placed into the center of wells coated with ECM harvested from MDA-MB-231BR cells and radial movement of the transduced and nontransduced CTL was also observed over 72 hr time. We conclude that effector T cells transduced with RRV maintain mobility and cytotoxic functionality, and further, are capable of transmitting RRV to tumor cells. Thus, transduced alloresponsive CTL can facilitate immunogene therapy by their capability of gene delivery to infiltrating tumor cells in the brain.

Citation Format: Kate L. Erickson, Colin C. Malone, Michelle J. Hickey, Geoffrey C. Owens, Yuki Kato, Robert M. Prins, Noriyuki Kasahara, Carol A. Kruse. Retroviral replicating vector transduced alloresponsive T lymphocytes retain mobility and cytotoxic functionalities. [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 1660. doi:10.1158/1538-7445.AM2014-1660
Abstract 1661: The pro-inflammatory role of the putative oncogene Bcl-3
Authors: Tang Wanhu , Wang Hongshan , Claudio Estefania , Siebenlist Ulrich
Published: May 2015

Bcl-3 is an oncogene first identified by its translocation into the immunoglobulin alpha-locus in some cases of chronic B cell lymphocytic leukemia. Bcl-3 is an atypical member of the IκB family that exhibits unique properties compared with classic IκBs. It readily enters nuclei, where it interacts with p50/NF-κB1 or p52/NF-κB2 homodimers to transcriptional activity. Depending on the cellular context and target gene, Bcl-3 may promote or inhibit gene expression. Recently it was reported that Bcl-3 could promote tumor growth as well as metastasis in several mouse tumor models. Bcl-3 is also known to play important roles in innate and adaptive immunity. For example, mice lacking Bcl-3 become highly susceptible to various pathogens, including T. gondii and K. pneumoniae, but the underlying mechanisms remain poorly understood. In addition, little is known about potential roles of Bcl-3 in inflammation that may contribute to cancers. Here we report that in a mouse model of colitis, which is dependent on the functions of Th1 cells, Bcl-3 had an intrinsic role to maintain Th1 cells and thus promote inflammation over time. This could also be demonstrated in vitro, where Bcl-3 had no effect on differentiation of Th1 cells per se, but was required for their maintenance. We will show how Bcl-3 promoted the maintenance of specifically Th1, but not Th17 cells. We will also provide evidence that Bcl-3 likely mediated these effects via p52/NF-κB2. Our data demonstrate that the oncoprotein Bcl-3 can contribute to chronic inflammatory conditions that in turn may be associated with cancer development.

Note: This abstract was not presented at the meeting.

Citation Format: Wanhu Tang, Hongshan Wang, Estefania Claudio, Ulrich Siebenlist. The pro-inflammatory role of the putative oncogene Bcl-3. [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 1661. doi:10.1158/1538-7445.AM2014-1661
Abstract 1663: CD40 agonists drive the anti-tumor functions of macrophages and induce systemic immune activation survival the Pan02 orthotopic pancreatic model
Authors: Luheshi Nadia , Harper James , Coates-Ulrichsen Jane , Sainson Richard , Wilkinson Robert W. , Davies Gareth , Legg James William
Published: May 2015

Pancreatic cancer represents an area of high unmet medical need. Antibodies agonising CD40 (αCD40) inhibit tumor growth in preclinical models, and are showing activity in early clinical trials in pancreatic cancer. αCD40 antibodies can directly drive apoptosis and promote antibody-dependent cell cytotoxicity of CD40 positive tumor cells, and may also drive antitumor effects through endothelial cells and B cells. However, data from preclinical models indicate that a major mechanism of action for αCD40 antibodies is through targeting of macrophages and dendritic cells, in some cases promoting long term CD8 T cell-dependent anti-tumor immunity. Pancreatic tumors often become highly infiltrated with immunosuppressive tumor macrophages. Our aim was therefore to test the hypothesis that αCD40 inhibits pancreatic tumor growth and spread by re-educating tumor macrophages to have anti-tumor functions, promoting a Th1 anti-tumor immune response, and gaining direct tumoricidal activity. We found that CD40 activation licenses human macrophage anti-tumor functions in vitro, and that this was dependent on the presence of T cell derived cytokines. In a syngeneic orthotopic model of pancreatic cancer (Pan02), we found that αCD40 significantly improved survival. This correlated with a significant enhancement in macrophage activation and in systemic immune activation (reduction in myeloid derived suppressor cells and increase in CD8 T cells) as determined by flow cytometry analysis. These data add support to the hypothesis that αCD40 inhibits pancreatic tumor growth through promoting anti-tumor functions of tumor macrophages.

Citation Format: Nadia Luheshi, James Harper, Jane Coates-Ulrichsen, Richard Sainson, Robert W. Wilkinson, Gareth Davies, James William Legg. CD40 agonists drive the anti-tumor functions of macrophages and induce systemic immune activation survival the Pan02 orthotopic pancreatic model. [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 1663. doi:10.1158/1538-7445.AM2014-1663