bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 608: Oncogenic role of cullin 4 E3 ubiquitin ligase (CRL4-CDT2) in epithelial ovarian cancer
Authors: Ring Kari E. , Abbas Tarek , Bryant Jennifer , Dutta Anindya , Jazaeri Amir A.
Published: June 2012

Objective: Ubiquitination is a key posttranslational modification involved in the tight regulation of biological processes including cell-cycle regulation and DNA replication and repair. Our objective was to investigate the expression and function of CDT2, a substrate recognition factor for the CUL4 E3 ubiquitin ligase family (CRL4-CDT2) in ovarian cancer.

Methods: CDT2 expression was evaluated using realtime RT-PCR in 26 serous ovarian cancers 11 normal ovaries, and 14 fallopian tube fimbria specimens and confirmed by Western blotting on a subset of the above. Immunohistochemistry was performed using tissue microarrays consisting of over 130 independent ovarian cancer samples of various histologies, as well as, normal fallopian tube samples using antibodies to CDT2 and p21. Each samples was represented at least in triplicate and scored semi-quantitatively. CDT2 knockdown was accomplished using siRNA and overexpression via retroviral transduction. Cell growth was evaluated using MTT assays. Statistical analysis were performed using either parametric (t test) as well as non-parametric (Mann-Whitney and Kruskal Wallis) methods as indicated. Comparison of p21 and CDT2 expression was performed using Spearman's correlation.

Results: CDT2 mRNA expression was approximately 9 fold higher in serous ovarian cancers compared to normal ovary and fallopian tube fimbria samples (P<0.00001). Western blotting confirmed this differential expression at the protein level. Immunohistochemistry validated CDT2 overexpression in ovarian cancers and did not reveal any differences between serous and other histological subtypes. Immunohistochemical staining did not reveal any significant correlation between the expression of CDT2 and p21, a previously identified target of CRL4-CDT2. Knockdown of CDT2 expression resulted in 31% reduction in cell viability in high CDT2 expressing ES2 cells (P<0.0001) but had minimal effect in low CDT2 expressing SKOV3 cells. However, overexpression of CDT2 in SKOV3 cells did not influence growth of these cells. Knockdown of other component of CRL4-CDT2 ubiquitin ligase (Cul4A and DDB1) also reduced growth of ES2 cells. Finally CDT2 protein levels significantly increased upon in vitro transformation of fallopian tube epithelial cells (described elsewhere) and in subsequent mouse xenografts established from these cells.

Conclusions: The results of this ongoing investigation are the first to suggest an oncogenic role for the CRL4-CDT2 ubiquitin ligase in ovarian cancer. The mechanism of action remains unknown but does not appear to involve p21 degradation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 608. doi:10.1158/1538-7445.AM2011-608
Abstract 619: O6-Benzylguanine inhibits tamoxifen-resistant breast cancer cell growth and resensitizes breast cancer cells to anti-estrogen therapy
Authors: Joshua Smith S. , Bobustuc George C. , Isley Beth , Limaye Arati , Ticku Jonathan , Madero-Visbal Rafael , Colon Jimmie , Baker Cheryl H. , Kalkunte Srivenugopal S. , Konduri Santhi D.
Published: June 2012

Endocrine therapies using anti-estrogens are less toxic and very effective for breast cancers however tumor resistance to tamoxifen remains a stumbling block for successful therapy. Based on our recent study on the involvement of the DNA repair protein MGMT in pancreatic cancer (Clin Cancer Res. 15, 6087, 2009), we investigated whether MGMT overexpression mediates tamoxifen resistance. Specifically, we determined whether administration of MGMT inhibitor [O6-benzylguanine (BG)] at a non-toxic dose alone or in combination with the anti-estrogens (tamoxifen/fulvestrant) curtails human tamoxifen resistant breast cancer cell growth. Furthermore, we also determined whether BG sensitizes breast cancer cells to tamoxifen using tamoxifen resistant cells.

MGMT expression was found to be increased in breast cancer cells relative to normal breast epithelial cells. Also, MGMT levels were significantly higher in tamoxifen resistant MCF-7 compared to the parent cells. Silencing of the ER-α expression using a specific siRNA resulted in augmentation of MGMT mRNA and protein levels by two fold. We also observed an inverse correlation between MGMT and p53 levels in breast cancer cell lines; moreover, p53 downregulation was accompanied by increased MGMT expression. Other experiments showed that BG alone or BG in combination with tamoxifen or fulvestrant decreased ER-α expression, whereas tamoxifen alone and fulvestrant alone increased and decreased the same respectively. All these treatments increased the p21cip1 mRNA and protein expression significantly. BG inhibited tamoxifen resistant breast cancer growth in a dose-dependent manner and it also resensitized resistant breast cancer cells to anti-estrogen therapy (TAM/ICI). These combinations also enhanced the cytochrome C release and PARP cleavage, indicative of apoptosis. In breast cancer xenografts, BG alone or a combination of BG with tamoxifen or fulvestrant caused a significant tumor growth delay and immunohistochemistry revealed that BG inhibited the expression of MGMT, ER- α, ki-67 and increased p21cip1 staining. These findings suggest that MGMT inhibition may provide a novel and effective approach for overcoming tamoxifen resistance (supported by a Florida Biomedical grant to SK).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2011-619
Abstract 62: Inhibition of Men1-deficient tumor growth by inactivation of the Rbp2 histone demethylase
Authors: Lin Wenchu , Francis Joshua , Watanabe Hideo , Yan Qin , Kaelin William G. , Meyerson Matthew
Published: June 2012

Abstract: Mutation of the tumor suppressor gene MEN1 causes both human inherited and somatic tumors in multiple endocrine tumors. Dissection of mechanism on MEN1 tumor model will shed light on tumor biology and the targeting therapies on neuroendocrine tumors lacking Men1. Loss of tumor suppressor menin may be tumorigenic because it leads to decreased histone H3 lysine 4 trimethylation and therefore decreased expression of specific genes such as CDKN1B. Reversing the tumorigenesis caused by menin deficiency might therefore be achieved by inhibition of histone H3 lysine 4 demethylation. Rbp2 (also known as KDM5A) is an excellent candidate for such a demethylase because loss of the Rbp2 gene leads to increased expression of CDKN1B. Using a tissue-specific knockout approach, I have disrupted Men1 alone or both Men1 and Rbp2 in mouse pancreatic islets through RIP-Cre transgene (RIP = “rat insulin promoter”) and have observed that loss of Rbp2 dramatically inhibits tumor growth in Men1-deficient islets. Histological assays have found that 100 percent of Men1f/f RIP-Cre mice (13 out of 13) at 10 months old or older harbor tumors of the pancreatic islets, whereas only 7 out of 21 Men1f/f; Rbp2f/f RIP-Cre mice contain tumors or large hyperplastic islets. Furthermore, Inactivation of Rbp2 extends the life span of Men1-deficient mice. The median survival age of Men1f/f RIP-Cre mice in this cohort is 313 days, the median survival age of Men1f/f; Rbp2f/f RIP-Cre (n=72) is 486 days (p<0.0001). In addition, a number of targets shared by both Men1 and Rbp2 have been identified via expression profiling. Studies from this project may provide insight to the cause of endocrine tumors and may develop a molecular target for treatment of endocrine tumors and the MEN-1 syndrome through modulation of histone methylation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 62. doi:10.1158/1538-7445.AM2011-62
Abstract 627: Etp-47187 a novel potent and efficacious dual inhibitor of phosphoinositide-3-kinases and mTOR
Authors: Kurz Guido , Pizcueta Pilar , Rico Rosario , Cebrián David , Aguirre Enara , Soilan David , Ortega Miguel A. , Martinez Sonia , Rabal Obdulia , Saluste C Gustavo , Casas Estela , Villanueva Patricia , Garcia Ana B. , Albarran Maria I. , Alfonso Patricia , Gonzalez Ester , Lorenzo Milagros , Ramos Francisco , Palacios Irene , Cebria Antonio , Rodriguez Antonio , Noya Beatriz , Garcia Ana M. , Rodriguez Sonsoles , Martin José I. , Alvarez Rosa , Hernandez Ana I. , Salgado Antonio , Cerdon Elena , Fominaya Jesus , Granda Teresa G. , Lopez Ana Rodriguez , Pastor Joaquin , Bischoff James R.
Published: June 2012

The phosphoinositide-3-kinase (PI3K) signaling pathway is activated in a variety of solid and non-solid tumors. In many instances this is due to either activating mutations in the catalytic subunit of PI3Kα, p110α, or inactivating mutations or deletions of the tumor suppressor PTEN. In addition, the PI3K pathway is activated by mutations in certain receptor tyrosine kinases as well as by mutation of the oncogene KRAS. All of these lesions lead to enhanced activity of both PI3K and mTOR. Hence there is great interest to discover inhibitors of PI3K and mTOR for the treatment for cancer. Following a rational design strategy, we identified the fused thiadiazole derivative ETP-47187 as a potent dual inhibitor of PI3Kα and mTOR Kis = 0.18 nM and 1.2 nM, respectively. ETP-47187 also inhibits three oncogenc mutants of p110α: p110α E542K Ki = 0.38 nM, p110α E545K Ki = 0.2 nM and p110α H1047R Ki = 0.29 nM as well as PI3Kβ, PI3KΔ and PI3Kγ Kis 2.7, 0.26 and 1.5 nM, respectively. The compound inhibits PI3K signaling in treated tumor cell lines; the EC50 for inhibition of the phosphorylation of Akt was 5 nM. ETP-47187 has a pharmacokinetic profile suitable for oral dosing in mice (%F = 73%, Cl = 0.11 L/hr/kg; Vds = 0.38 L/h/kg). Treatment of tumor bearing mice with the compound causes a dose dependent reduction in P-Akt levels in the tumor. Once a day treatment of mice bearing human tumor xenografts with ETP-47187 results in significant tumor growth delay and is well tolerated. In a mouse model of lung cancer induced by expression of an oncogenic mutant KRAS, treatment with ETP-47187 blocked tumor growth and lead to a significant PET response. These and combination data will be discussed. We believe ETP-47187 and compounds like it are suitable to propose for clinical development in cancer patients with activated PI3K signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 627. doi:10.1158/1538-7445.AM2011-627
Abstract 636: C-Met antibodies with distinctive characteristics including induction of receptor degradation without activation and tumor specificity
Authors: Greenall Sameer , Gherardi Ermanno , Scott Andrew , Johns Terrance G.
Published: June 2012

The hepatocyte growth factor receptor (c-Met) is a cell surface, disulphide linked heterodimeric receptor tyrosine kinase consisting of an extracellular 50 kDa α-chain and an extracellular/intracellular 145 kDa β-chain containing the tyrosine kinase domain. The two chains of the mature c-Met arise from the proteolytic cleavage of a single-chain precursor. The α-chain and N-terminal part of the β-chain associate to form the SEMA domain which contains the binding site for hepatocyte growth factor (HGF). Aberrant signalling due to dysregulation of the c-Met receptor is often associated with more aggressive cancers in a range of tumor types, making it a target for cancer drug development. Mice were immunized with the SEMA domain of c-Met followed by cancer cells expressing c-Met in order to generate antibodies that target this receptor. We created 3 antibodies that exclusively bound the unprocessed form of c-Met. Fine epitope mapping using overlapping linear peptides showed all 3 antibodies had distinct binding sites; one each for the α- and β-chain and one with a conformational epitope. Since this unprocessed form of c-Met is only found on the surface of cancer cells, and not normal cells, these antibodies are tumor specific and may be extremely valuable as targeting agents. We also identified a series of antibodies that bind a defined peptide on the α-chain of c-Met and stimulate c-Met degradation without concurrent activation of the receptor; a problem with previous antibodies that enhance c-Met degradation. Through their ability to down-regulate c-Met, these antibodies inhibited HGF induced migration of SK-OV-3 ovarian cancer cells, inhibited ligand-independent proliferation of A549 lung cancer cells and had anti-tumor activity against U87MG glioma xenografts that possess a HGF/c-Met autocrine loop. The generation of cancer-specific c-Met antibodies and novel antagonist antibodies opens up new strategies for targeting this crucial receptor in range of cancer types.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 636. doi:10.1158/1538-7445.AM2011-636
Abstract 637: MEHD7945A, an EGFR/ErbB3 dual specific antibody, overcomes acquired resistance to EGFR inhibitors in head and neck and lung tumors
Authors: Huang Shyhmin , Li Chunrong , Sliwkowski Mark X. , Harari Paul M.
Published: June 2012

The development of acquired resistance to EGFR inhibitors is emerging as a potential treatment barrier for EGFR targeted therapies. Efforts to better understand underlying mechanisms of acquired resistance to EGFR inhibitors, and strategies to overcome this resistance are highly desired. We have previously identified that acquired resistance to EGFR inhibitors may result from the trans-activation of ErbB3 by EGFR. We therefore examined the capacity of the EGFR/ErbB3 dual specific antibody, MEHD7945A to overcome acquired resistance to EGFR inhibitors.

We first examined the antiproliferative effects of MEHD7945A across a panel of human head and neck (HNSCC) and non-small cell lung (NSCLC) tumor cell lines. MEHD7945A significantly inhibited the proliferation in tumors cells that express EGFR and ErbB3. We then applied MEHD7945A to EGFR inhibitor-resistant clones from HNSCC (SCC-1 and SCC-6) and NSCLC lines (H226) that have been chronically exposed to cetuximab or erlotinib and show EGFR inhibitor-resistant phenotypes. Using identical doses of MEHD7945A and cetuximab, we found that MEHD7945A inhibited the growth of tumor cells that exhibit resistance to cetuximab. Similar results were observed in our erlotinib-resistant clones. MEHD7945A also significantly inhibited EGFR and ErbB3-dependent downstream signaling. Additional studies to confirm the antitumor activity of MEHD7945A in xenograft models using EGFR inhibitor-resistant clones are in progress. In addition, studies to investigate the impact of MEHD7945A on radiation response profiles in a panel of HNSCC, NSCLC and EGFR-inhibitor resistant cell lines are underway. These data not only support an important role of ErbB3 in acquired resistance to EGFR inhibitors but also suggest MEHD7945A may provide a valuable treatment strategy for patients who develop acquired resistance to EGFR targeting agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 637. doi:10.1158/1538-7445.AM2011-637
Abstract 645: Preclinical efficacy of AZD8055, an ATP-competitive mammalian target of rapamycin (mTOR) kinase inhibitor, in vitro in clear cell renal cell carcinoma (RCC)
Authors: Gupta Gopal N. , Lin Kelly Y. , Sourbier Carole , Baba Masaya , Guichard Sylvie , Linehan W. Marston , Srinivasan Ramaprasad
Published: June 2012

Inhibitors of the mTORC1 complex such as the rapamycin analogues temsirolimus and everolimus are approved by the FDA for treatment of advanced renal cell carcinoma. However, objective responses with these agents are infrequent and clinical benefits transient, with the majority of patients eventually developing progressive disease and dying from metastatic disease. Dual mTORC1/2 inhibitors may overcome some of the limitations of rapamycin analogues by suppressing the feedback activation of AKT induced by mTORC1 inhibition. In this study, we investigated the in vitro efficacy of AZD8055, a potent inhibitor of both mTORC1 and mTORC2, in clear cell RCC utilizing 2 von Hippel Lindau (VHL) null clear cell kidney cancer cell lines, UOK139 and UOK140. AZD8055 strongly suppressed mTORC2 signaling in RCC and at concentrations ranging from 50nM-250nM, downregulated pAKTs473 and pmTORs2481 in a concentration-dependent fashion. Phosphorylation of AKTt308 was also diminished in both cell lines at these concentrations. In contrast, rapamycin did not significantly inhibit either mTORC2 signaling or AKTt308 phosphorylation. Both AZD8055 and rapamycin were able to downregulate the mTORC1 substrate pS6; however, only AZD8055 was effective in downregulating p4E-BP1, a critical component of cap-dependent translation that is regulated by mTORC1. The vast majority of clear cell renal tumors are characterized by inactivation of the VHL tumor suppressor gene due to mutation or promoter hypermethylation. One well characterized consequence of VHL inactivation is upregulation of hypoxia inducible factor-2α (HIF-2α), an important mediator of both tumorigenesis and neo-angiogenesis in clear cell RCC. While HIF expression is dependent on mTOR activity, it has been reported that mTORC1 inhibition does not reliably suppress HIF-2α expression; we show that treatment with AZD8055 resulted in significant downregulation of HIF-2α levels in both UOK139 and UOK140, an effect that may be mediated by mTORC2 inhibition. AZD8055 exhibited potent in vitro antitumor activity against clear cell RCC, with an IC50 of 100-150nM in the MTT assay; rapamycin had minimal antitumor activity at these concentrations (IC50 not reached at 10uM). These data demonstrate that AZD8055 is a potent inhibitor of mTORC2 and a more effective inhibitor of downstream mTORC1 targets than rapamycin. Simultaneous mTORC1/2 kinase inhibition may be superior to selective mTORC1 inhibition, and is worthy of clinical evaluation in clear cell RCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 645. doi:10.1158/1538-7445.AM2011-645
Abstract 65: Regulation of KEAP1 expression by promoter methylation in malignant gliomas and association with patient's outcome
Authors: Barbano Raffaela , Muscarella Lucia Anna , D'Angelo Vincenzo , Copetti Massimiliano , Coco Michelina , Balsamo Teresa , la Torre Annamaria , Notarangelo Angelo , Troiano Michele , Parisi Salvatore , Icolaro Nadia , Catapano Domenico , Valori Vanna Maria , Pellegrini Fabio , Merla Giuseppe , Carella Massimo , Fazio Vito Michele , Parrella Paola
Published: June 2012

The Keap1 (Kelch-like ECH-associated protein 1) protein tightly regulates the functions of Nrf2 (nuclear factor-erythroid 2-related factor 2) which plays a pivotal role in the cellular response to oxidative stress. Initial reports uncovered mutations in the KEAP1 gene in non-small cell lung cancer (NSCLC) leading to a permanent nrf2 activation. Mutations were often associated with loss of heterozigosity at the KEAP1 gene locus and promoter hypermethylation suggesting that biallelic inactivation is a common event in NSCLC.

In this study, we sought to determine whether KEAP1 gene is epigenetically regulated in malignant gliomas and if promoter aberrant methylation may impact patient's outcome.

We developed a Quantitative Methylation Specific PCR (QMSP) assay to analyze 86 malignant gliomas and 20 normal brain tissues. The discriminatory power of the assay was assessed by Receiving Operating Curve Characteristics (ROC) analysis. The AUC value of the curve was 0.823 (95%CI: 0.764-0.883) with an optimal cut off value of 0.133 yielding a 74% sensitivity (95%CI: 63%-82%) and an 85% specificity (95%CI: 64%-95%). Bisulfite sequencing analysis confirmed QMSP results and demonstrated a direct correlation between percentage of methylated CpGs and methylation levels (Spearman's Rho 0.929, P=0.003). Remarkably, a strong inverse correlation was observed between methylation levels and KEAP1 mRNA transcript in tumour tissue (Spearman's Rho -0.656 P=0.0001) and in a cell line before and after treatment with 2-deoxy-5 Azacytidine (P=0.003). RECPAM multivariate statistical analysis studying the interaction between MGMT and KEAP1 methylation in subjects treated with radiotherapy and temozolomide (n=70), identified three prognostic classes of glioma patients at different risk to progress. While simultaneous methylation of MGMT and KEAP1 promoters was associated with the lowest risk to progress, patients showing only MGMT methylation were the subgroup at the higher risk (HR 5.54, 95% CI 1.35-22.74). Our results indicate that that KEAP1 expression is frequently methylated in malignant gliomas and may represent a novel predictor of patient's outcome.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 65. doi:10.1158/1538-7445.AM2011-65
Abstract 653: Combination of TORC1/2 and MEK kinase inhibitors in colorectal cancer models
Authors: Garcia-Garcia Celina , Serra Violeta , Scaltriti Maurizio , Ibrahim Yasir , Guzman Marta , Jessen Katti , Liu Yi , Rommel Christian , Tabernero Josep , Baselga Jose
Published: June 2012

MAPK pathway mutations are frequent in colorectal cancer (CRC) and associated with resistance to standard therapies. This provides a rationale for the use of targeted therapies against the MAPK pathway. However, the response of CRC cells to MEK inhibitors varies depending on coexisting mutations in both MAPK and PI3K pathways. As a matter of fact, dual inhibition of MEK and PI3K or AKT in mouse models of CRC has proven effective due to aberrations in both pathways. Inhibition of TORC1, a shared downstream effector of the MAPK and PI3K pathways, was described to be partly responsible for the efficacy of the dual blockade.

In order to explore additional mechanisms involved in the superior antitumor activity of combined MAPK and PI3K pathway suppression we directly targeted TORC1/2 by active-site kinase inhibitor of mTOR, in addition to MEK, in our cell models. We tested the antiproliferative activity of INK128, a selective and potent TORC1/2 inhibitor, in combination with a MEK inhibitor, PD-0325901, in a panel of colon cancer cell lines with diverse alterations of the MAPK and PI3K pathways. PD-0325901 treatment effectively inhibited ERK phosphorylation, which resulted in decreased phosphorylation of Rb and reduced expression of E2F in all the cell lines tested. In CRC cell lines harboring sole mutations in the MAPK pathway, we observed sensitivity to PD-0325901 that was not further enhanced when combining with INK-128. In contrast, CRC cell lines harboring concurrent mutations of both pathways exhibited a striking sensitivity to dual ERK and TORC1/2 blockade associated with an enhanced arrest in cell cycle and, above all, a concomitant increase in apoptosis. Inhibition of TORC1 and TORC2 by INK128 could not be further enhanced by MEK inhibition in any of the cell lines tested. These data suggest that, besides complete suppression of both pathways, additional mechanisms must be at work to explain the observed enhanced cell death following dual suppression of PI3K and MEK. Interestingly, we observed differential regulation of key components of the cell cycle in response to dual blockade. We are currently investigating whether this specific biochemical behaviour plays a causative role in the observed phenotype.

In summary, dual inhibition of TORC1/2 and MEK has potential for effective antitumor activity in CRCs with dysregulation of both signaling cascades. A better understanding of the molecular mechanisms responsible for the enhanced efficacy of this therapeutic combination will help defining the best biomarkers that need to be assessed in phase I combination trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 653. doi:10.1158/1538-7445.AM2011-653
Abstract 655: Combination of MM-111, an ErbB2/ErbB3 bispecific antibody, with endocrine therapies as an effective strategy for treatment of ER+/HER2+ breast cancer
Authors: Zhang Bo , Huhalov Alexandra , Burenkova Olga , Adams Sharlene , Nguyen Stephanie , Overland Ryan , Kudla Arthur J. , Nielsen Ulrik B. , Niyikiza Clet , McDonagh Charlotte F. , MacBeath Gavin
Published: June 2012

Approximately 75% of breast cancers are estrogen receptor (ER) positive. Although endocrine therapies such as tamoxifen, fulvestrant, and letrozole have demonstrated significant efficacy in treating ER+ breast cancer patients, intrinsic or acquired resistance has limited their success. Recent studies suggest that crosstalk between ErbB receptor signaling and ER signaling may contribute to resistance to endocrine therapy. Overexpression of human epidermal growth factor receptor 2 (HER2, synonymous with ErbB2) and upregulation of the ErbB3 ligand heregulin are associated with poor prognosis and reduced overall survival.

MM-111 is a novel bispecific antibody fusion protein that specifically targets the ErbB2/ErbB3 heterodimer and blocks heregulin binding to ErbB3. MM-111 inhibits ligand-induced ErbB3 phosphorylation, tumor cell cycle progression, and tumor growth when ErbB2 is overexpressed. We hypothesized that combination of endocrine therapies with MM-111 may improve anti-tumor efficacy. In an estrogen-stimulated BT474-M3 ER-positive breast cancer cell three-dimensional spheroid assay, MM-111, when used as a single agent, showed growth inhibitory effects similar to the anti-estrogen drugs tamoxifen and fulvestrant. Combination of MM-111 with anti-estrogen therapy showed superior activity to either drug alone. In the presence of heregulin, MM-111 maintained its growth inhibitory activity, whereas the inhibitory effect of tamoxifen and fulvestrant was diminished. This suggests that activation of ErbB3 confers tumor cell resistance to anti-estrogen therapies. When both estrogen and heregulin were present, the combination of MM-111 and the anti-estrogen drugs demonstrated a significantly greater inhibitory effect than either drug alone. Western blot analysis showed that treatment of BT-474-M3 cells with the combination of MM-111 and the anti-estrogen drugs significantly increased apoptosis markers such as cytochrome C and BAX. Furthermore, an in vivo BT474-M3 xenograft model showed resistance to tamoxifen treatment (5 mg/pellet, 60-day release). In this xenograft model MM-111 sensitized tumor response to tamoxifen and the combination treatment dramatically inhibited tumor growth. In conclusion, the combination of MM-111 and endocrine therapies may provide a potent regimen that overcomes acquired resistance to endocrine therapies in ER+, ErbB2-overexpressing breast cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 655. doi:10.1158/1538-7445.AM2011-655
Abstract 658: CBS9106 is a novel low toxic reversible oral CRM1 inhibitor with CRM1 degrading activity
Authors: Sakakibara Keiichi , Saito Naoya , Sato Takuji , Suzuki Atsushi , Hasegawa Yoko , Friedman Jonathan M. , Suganuma Masashi , Kufe Donald W. , VanHoff Daniel D. , Iwami Tadahiko , Kawabe Takumi
Published: June 2012

CRM1 plays an important role in the nuclear export of cargo proteins bearing Nuclear Exporting Signal sequences. Leptomycin B (LMB), a well-known irreversible inhibitor of CRM1, possesses strong anti-tumor properties, however, its toxicity prevents it from being clinically useful. In this study, we show that a novel compound, CBS9106, inhibits CRM1-dependent nuclear export and induces G1-arrest and apoptosis in time- and dose-dependent manner in a broad-spectrum of cancer cells including multiple myeloma cells. IC50s of growth inhibition by CBS9106 (48h) are in the range from 46.7 to 366 nM in multiple myeloma cell lines (MM.1R, MM.1S, ARH-77 and RPMI-8226). In contrast with LMB, CBS9106 acts reversibly and reduces CRM1 protein without affecting the mRNA expression level. CBS9106-dependent reduction of CRM1 is abolished by MG132, a proteasome inhibitor. Pretreatment of cells with LMB suppresses CBS9106-induced CRM1 degradation. Moreover, CBS9106-biotin captured CRM1 in a pull down analysis and the amount of CRM1 captured by CBS9106-biotin was reduced by LMB treatment and vice versa. These results suggest that CBS9106 acts on the domain of CRM1 that includes LMB's covalent binding site at Cys-528, leading to proteasome-dependent CRM1 degradation. Oral administration of CBS9106 significantly suppressed tumor growth and prolonged survival duration in xenograft model mice. It caused prolonged reduction of CRM1 protein in tumor xenografts without showing significant body weight loss. Taken together, the results indicate that CBS9106 is a novel CRM1 inhibitor and a promising clinical candidate.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 658. doi:10.1158/1538-7445.AM2011-658
Abstract 661: Efficiency of the multivalent pseudopeptide nucant 6L in castration- and chemo-resistant prostate cancers
Authors: Destouches Damien , Gostoli Julie , Terry Stéphane , Briand Jean-Paul , Zimmer Robert , Courty José , de la Taille Alexandre , Vacherot Francis
Published: June 2012

Introduction: Targeted therapies on advanced prostate cancer, resistant to castration and chemotherapy, offer significant hope for future treatments. NucAnt 6L (N6L) is a ligand of surface nucleolin. This protein is overexpressed at the cell surface of tumoral and endothelial cells, and is involved in tumor growth and associated angiogenesis. It has been demonstrated that N6L displays, in vitro and in vivo, a double anti-tumoral activity. It inhibits growth of various human tumor cell lines derived from mammary and colorectal carcinoma, melanoma, glioblastoma and lymphoma. In addition, it can compromise associated angiogenesis. In this context, N6L might be of value for therapeutic intervention. Previous studies have demonstrated drug's efficacy of this agent in prostate cancer cell lines. However, its effect on therapeutic resistant lines has yet to be documented. Using in vivo and in vitro models, the aim of this work is to evaluate N6L activity on castration and chemo-resistant prostate cancers.

Methods: When maintained in medium deprived of androgens, androgen-responsive LNCaP cells undergo a “transdifferentiation” process giving rise to neuroendocrine (NE) like LNCaP cells which are resistant to androgen deprivation and chemotherapeutic agents such as docetaxel. Here, the anti-tumoral activity of N6L has been evaluated, in vitro, on human LNCaP-NE cells. Cell viability, as assessed by MTT test, and soft agar assays were performed at 72 hours after treatment with N6L (range of 0.1 to 100 µM). In vivo, anti-tumoral activity was then confirmed using VCaP ectopic xenografts into athymic nude mice, a condition that mimics clinical evolution of advanced prostate cancers in human. Mice bearing tumors of about 600 mm3 were castrated. Mice were treated with either vehicle or N6L at 10 mg/kg by the intraperitoneal route at different times of tumor growth. Tumors volumes were monitored at different time points using the caliper method.

Results: N6L induces death of LNCaP-NE cells with a IC50 of 16.2 µM. These findings were supported by soft agar assays in which LNCaP-NE cells were found to be sensitive to N6L treatment whereas they were resistant to docetaxel. In vivo, N6L inhibits VCaP tumor growth at different stages of tumor progression, the castration-sensitive state and also the castration resistant state.

Conclusions: N6L, which is currently in I/IIb phase of clinical trials, might represent a new targeted approach to treat advanced prostate cancers resistant to castration and chemotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 661. doi:10.1158/1538-7445.AM2011-661
Abstract 677: Effects of the vascular disrupting agent ASA404 on tumor growth of gastric cancer in an experimental model
Authors: Moser Christian , Lang Sven A. , Jung Ernst M. , Pfister Karin , Geissler Edward K. , Schlitt Hans J.
Published: June 2012

Background: A functional vascular system is essential for growth of solid malignancies including gastric cancer. For establishment and maintenance of such a vascular system endothelial cells (ECs) and pericytes (e.g. vascular smooth muscle cells, VSMC) are required. We hypothesized that targeting tumor vasculature with the vascular disrupting agent (VDA) ASA404 reduces tumor growth in a model of gastric cancer. Methods: Gastric cancer (GC) cell lines, ECs and VSMCs were used for experiments. Effects of ASA404 on growth of GC, EC and VSMC were assessed by MTT assays. Impact of ASA404 (20 mg/kg on day 1, 5, 9) in combination with paclitaxel (10 mg/kg on day 1 and 7) on tumor growth was assessed in a subcutaneous tumor model. Treatment was started when tumors reached a size of approximately 200 mm3. Tumors were measured and harvested on day 23 for IHC analyses. Effect of ASA404 on blood perfusion of tumors during therapy was monitored by contrast-enhanced ultrasound (CEUS). Results: In vitro ASA404 impaired growth of ECs and VSMCs upon stimulation with condition media from gastric cancer cells. No direct effect on tumor cells was observed. In vivo, treatment with ASA404 led to marked decrease of tumor perfusion and an increase of necrosis as determined by CEUS. Furthermore, combination of ASA404 with paclitaxel showed significant reduction of tumor growth compared to controls (P<0.05). In addition, tumor vascularisation and tumor cell proliferation were significantly reduced as determined by CD31-positive vessel area and BrdU-positive cells (P<0.05). Conclusion: Combination of the VDA ASA404 with paclitaxel impairs tumor growth and perfusion of gastric cancer in an experimental model. Hence, targeting tumor vasculature with ASA404 appears to be a promising strategy for therapy of gastric cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 677. doi:10.1158/1538-7445.AM2011-677
Abstract 678: Study of 2, 9-disecbutyl-1, 10-phenanthroline as a novel anticancer agent
Authors: Wang Dongsheng , Amin A.R.M. Ruhul , Peng Shifang , Sanghvi Chinar , Shin Dong M. , Eichler Jack F.
Published: June 2012

Development of new, safe, and effective drugs or strategies targeting molecular events and pathways which are responsible for cancer initiation and progression is an urgent need to improve cancer patient care. In this study, we tested the antitumor effect of a novel agent, 2, 9-disecbutyl-1, 10-phenanthroline (dsBPT), a 1, 10-phenanthroline derivative. Both in vitro cell line and in vivo xenograft models were applied to study the antitumor efficacy and underlying mechanism of this compound. IC50s were initially determined using lung cancer cell lines: A549 (0.25 μM) and H1703 (0.1 μM), head and neck cancer cell lines: TU686 (0.08 μM) and TU212 (0.2 μM), and normal bronchial epithelial cells: BEAS-2B (2.0 μM), indicating that the growth inhibitory effect of dsBPT on cancer cell lines was about 10 times greater than that on normal human epithelial cells. Further studies demonstrated that dsBPT at a concentration of 1-2 μM induced autophagy and G1 cell cycle arrest. At higher doses between 4-8 μM, the compound further induced apoptosis (40-80%). Combination of dsBPT with the cytotoxic agent cisplatin synergistically inhibited cancer cell growth (combination index = 0.3) and induced apoptosis (80-90%) at a concentration of 1 μM for each drug. Two experiments were then performed to test dsBPT in vivo. First, xenografted mice implanted with TU212 cells were treated with three drug doses (2mg/kg, 5mg/kg, 10mg/kg). Compound was given by IV injection once per week for 3 weeks. We found that 10 mg/kg treatment significantly reduced tumor growth as compared with the control group (p = 0.004). Under the same treatment schedule, further study showed that the combination of dsBPT (2 mg/kg) with cisplatin (2 mg/kg) significantly reduced the tumor volume as compared with the untreated control (p = 0.0017), while each of the single drugs at the same concentration showed no significant inhibitory effect on tumor growth (p = 0.10 and 0.15 for cisplatin and dsBPT, respectively). Neither significant weight loss nor major organ damage was observed by histology examination in the experimental animals in this study. In summary, our study suggests that dsBPT is a novel, highly potent antitumor drug that warrants further preclinical and clinical development either as a single agent or in combination with known chemotherapy drugs such as cisplatin. (This study is supported by grants from DOD W81XWH-07-1-0306 Project 5, and GCC Distinguished Scholar Award to ZC).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 678. doi:10.1158/1538-7445.AM2011-678
Abstract 702: Drug resistance and invasion in diffuse large B-cell lymphoma are mediated by Akt, 14-3-3ζ, and Vimentin
Authors: Cherry Evan M. , Bayless Kayla J. , Maxwell Steve A.
Published: June 2012

Acquired multi-drug resistance to the anthracycline-based CHOP chemotherapy is a major cause of unsuccessful treatment and mortality in 40% of diffuse large B-cell lymphoma (DLBCL) patients. To investigate molecular mechanisms of multidrug resistance, we generated CHOP-resistant DLBCL cells through repeated selection in the presence of a clinically-relevant CHOP cocktail (cyclophosphamide, doxorubicin, vincristine, prednisone at the ratio of 80:5.5:0.16:11.1). Differential protein expression between CHOP-sensitive and CHOP-resistant DLBCL cells was investigated by two-dimensional differential in-gel (DIGE) analysis, which identified 10 differentially-expressed proteins related to glycolysis (Triosephosphate isomerase-1, Enolase-1), cytoskeletal structure (Ezrin, Vimentin, Tubulin-specific chaperone B), purine biosynthesis (Serine hydroxymethyltransferase), calcium-binding (Sorcin), and apoptosis (p53, 14-3-3zeta, Akt). Upregulation of Akt, 14-3-3ζ, and Vimentin was observed by Western blotting in CHOP-resistant cells compared to CHOP-sensitive cells. These proteins were also heterogenously upregulated in primary DLBCL tissues relative to normal lymphatic tissue. Our previous studies showed that siRNA-mediated knockdown of 14-3-3ζ reversed CHOP resistance in DLBCL cells (Maxwell et al [2009] J. Biol. Chem. 284:22379-89). Our current study showed that chemical Akt inhibition overcame CHOP resistance in DLBCL cells and CHOP-resistant cells exhibited a five-fold greater ability to invade collagen matrices than CHOP-sensitive cells. Moreover, knockdown of Vimentin by siRNA or inhibition by Withaferin A decreased invasion capacity of the CHOP-resistant cells in collagen matrices. The data indicate activation of an Akt-14-3-3ζ-mediated signaling pathway as a contributor to the multi-drug resistant phenotype associated with vimentin-dependent invasive behavior in DLBCL cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 702. doi:10.1158/1538-7445.AM2011-702
Abstract 716: O6-Methylguanine DNA methyl transferase (MGMT) and tamoxifen resistance in patients with breast cancer
Authors: Konduri Santhi D. , Maddipatla Sreeram , Smith Joshua , Isley Beth , Caparas Maria-Lourdes M. , Constantino Susan M. , Shah Nikita , Baidas Said
Published: June 2012

Tamoxifen is an estrogen receptor antagonist in breast tissue. It has been used as a hormonal therapy for early and advanced breast cancer both in premenopausal and postmenopausal patients with estrogen receptor positive breast cancer. Acquired resistance to tamoxifen therapy is a common therapeutic encounter. Different mechanisms for Tamoxifen resistance have been postulated. In the present study we investigated if over expression of MGMT causes tamoxifen resistance in breast cancer patients.

Purpose: We sought to determine MGMT expression levels in various breast cancer cell lines as well as in breast cancer patient's samples before and after treatment with tamoxifen.

Experimental Design: MGMT expression levels were compared in breast cancer tissue samples obtained from patients before starting treatment with Tamoxifen and after tumor progression due to development of Tamoxifen resistance. MGMT expression level was evaluated using western blot and immunohistochemical assays. Several commercial breast cell lines were evaluated for expression of MGMT.

Results: We found that MGMT is over expressed in the majority of breast cancer cell lines compared to normal breast epithelial cells. Compared to pre Tamoxifen treatment, post Tamoxifen treated breast cancer patient samples have significantly higher levels of MGMT expression.

Conclusion: Collectively, our results demonstrate that breast cancer cells showed significantly higher levels of MGMT expression and furthermore, Tamoxifen treatment induces further MGMT over expression in breast cancer patients. According to our results over expression of MGMT is a possible cause for Tamoxifen resistance. Inhibition of MGMT might overcome Tamoxifen resistance and restore breast cancer sensitivity to Tamoxifen treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 716. doi:10.1158/1538-7445.AM2011-716
Abstract 739: Small molecule inhibitor, PZ39C8, promotes BCRP degradation via binding to TM5-loop-TM6
Authors: Qi Jing , Mo Wei , Dong Zizheng , Peng Hui , Zhang Jian-Ting
Published: June 2012

Multidrug resistance (MDR) and existing cancer stem cells (CSCs) are two major problems in successful treatment of cancers. Expression of breast cancer resistance protein (BCRP) is known to associate with both MDR and CSCs, suggesting that BCRP is an interesting and ideal target for development of chemosensitizing agents for better treatment of drug resistant cancers and elimination of CSCs. Recently, we identified a novel BCRP inhibitor PZ39 and its analogue compound PZ39C8 which are capable of inhibiting BCRP function and inducing BCRP degradation in lysosome. In the present study, we (1) investigated the mechanism of inhibitor-induced BCRP degradation using a stable cell line expressing GFP-BCRP in combination with pharmacological agents of endocytosis and trafficking and (2) determined the inhibitor binding site in BCRP using compound-conjugated Sepharose 4B beads and pull-down assay. We found that (1) the lysosomal inhibitor Bafilomycin A but not the proteosome inhibitor MG132 abolished PZ39C8-induced BCRP degradation; (2) dynasore, a potent endocytosis inhibitor, and 3-Methyladenine, a autophagy inhibitor, both efficiently blocked the PZ39C8-induced BCRP degradation from plasma and internal membranes; (3) following co-treatment with PZ-39C8 and endocytosis, trafficking, and lysosome inhibitors, BCRP co-localized with the LAMP-1, LC3II and Clathrin, EEA1, the markers of Lysosome, autophagy and endocytic pathways, respectively; and (4) PZ39C8 directly binds BCRP and the binding site/s appears to locate in the domain including TM5-loop-TM6. Taken together, we conclude that PZ39C8 binds to TM5-ECL3-TM6 of BCRP and induces BCRP degradation in lysosome via endocytic and autophagic dependent pathways. Thus, PZ39C8 is potentially a valuable probe for structure-function studies of BCRP and a lead compound for developing therapeutics targeting BCRP-mediated MDR in combinational cancer chemotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 739. doi:10.1158/1538-7445.AM2011-739
Abstract 74: Genome-wide DNA methylation analysis of pT1a renal cell carcinoma and renal oncocytomas
Authors: Ibragimova Ilsiya , Banumathy Gowrishankar , Al-Saleem Tahseen , Uzzo Robert G. , Cairns Paul
Published: June 2012

Nephron sparing surgery (partial nephrectomy) is currently performed on small renal masses (4cm or less in size) confined to the kidney (pT1a). Up to 25% of small renal masses are benign, the majority being oncocytomas. Benign oncocytomas lack distinct radiographic findings on computerized tomography (CT) and cannot be diagnosed accurately using imaging alone. No difference in tumor size at presentation or tumor growth rate was noted between oncocytoma and RCC in a review of the literature. A needle biopsy or non-invasive urine based differential diagnosis of a benign lesion potentially would allow patients with equivocal renal lesions to avoid unnecessary surgery as well as anxiety if under active surveillance.

The epigenetic alteration of aberrant DNA methylation has potential benefits as a choice of a biomarker because aberrant methylation occurs frequently and early in tumorgenesis and can be detected at extremely sensitive and specific levels by currently available technology. It is known that aberrant hypermethylation of the VHL gene occurs in a subset of clear cell RCC but is absent in oncocytoma. We hypothesized that additonal genes with a differential methylation status between benign oncocytoma and RCC could be identified.

In this study, we performed genome-wide detection of DNA methylation in 25 RCC (pT1a), 25 oncocytomas of 4cm or less in size, and 4 age-matched histologically normal kidney tissues using the Infinium HumanMethylation27 BeadChip. To date, we have evidence that the profile of aberrant gene methylation is different between benign and malignant small renal masses matched for median size. Infinium-identified differentially methylated genes are validated using direct bisulfite sequencing, then a qMSP assay designed. If the panel of differentially methylated genes we have identified are verified in an independent set of small renal masses, we can then determine the predictive value of methylation-based detection of benign oncocytoma versus malignant RCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 74. doi:10.1158/1538-7445.AM2011-74