bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-204: Matrix metalloproteinase-10 is required for lung cancer stem cell maintenance, tumor initiation and metastatic potential
Authors: Justilien Verline , Regala Roderick P. , Tseng I-Chu , Walsh Michael P. , Batra Jyotica , Radisky Evette S. , Murray Nicole R. , Fields Alan P.
Published: August 2012

Matrix metalloproteinase 10 (Mmp10; stromelysin 2) is a member of a large family of structurally related matrix metalloproteinases, many members of which have been implicated in tumor progression, invasion and metastasis. However, emerging evidence suggests a possible role for Mmps in tumor initiation. Here we reveal an unexpected role for Mmp10 in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mouse lung cancer cell cultures enriched in stem-like cells grow as undifferentiated tumor “oncospheres” that express elevated levels of the cancer stem cell markers Aldh1, CD133, Nanog, Notch3, Notch 4, Hey 1 and Hey 2. These cells also express elevated levels of Mmp10 mRNA and secrete elevated levels of Mmp10 protein. Functionally, these lung CSCs exhibit self-renewal, the ability to clonally expand, enhanced transforming potential in vitro, and enhanced tumorigenic properties when injected orthotopically into the lungs of syngeneic mice. RNAi-mediated knockdown of Mmp10 in these cells leads to a loss of stem cell marker gene expression and a dramatic inhibition of oncosphere growth, clonal expansion, transformed growth in vitro, and lung tumor formation and metastasis in vivo. In contrast, oncospheres implanted into syngeneic non-transgenic or Mmp10−/− mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human tumors demonstrates that Mmp10 is elevated in many human tumor types including lung, head and neck, esophageal, colon, breast, melanoma, bladder, cervical, ovarian, prostate and brain. Furthermore, gene set enhancement analysis (GSEA) demonstrates that elevated Mmp10 expression correlates with tumor metastasis and with cancer stem-like genomic signatures in human lung tumors. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-204. doi:1538-7445.AM2012-LB-204
Abstract LB-216: Receptor targeted (CD206) Tc99m tilmanocept localization in sentinel nodes in breast cancer and melanoma is concordant with preoperative imaging and a reduction in missed disease
Authors: Cope Frederick O. , Metz Wendy , Abbruzzese Bonnie , Shuping Jo , Hartman Richard , King Dennis , Christman Lori , Blue Michael
Published: August 2012

Purpose: Tilmanocept is wholly synthetic molecule composed of a dextran-10 backbone with both multiple mannose side moieties (for receptor biding) and DTPA (for chelation of divalent cations). Tilmanocept is designed as a high affinity ligand for the human mannose binding receptor (CD206) of reticuloendothelial cells (RECs), and more specifically, those RECs that reside in tumor-draining lymph nodes. Tilmanocept is designed to provide high sensitivity and specificity for intraoperative lymphatic mapping in melanoma, breast cancer, head/neck squamous cell carcinoma, and other solid tumors. This study was designed to evaluate the correlation of preoperative imaging using Tc99m tilmanocept and intraoperative tumor bed-adnexed node findings, along with metatstatic disease finding for Tc99m tilmaocept with its “truth” comparator, Lymphazurin. Results/Conclusions: The results of combined prospective phase 3 clinical trials in breast cancer and melanoma indicate that there is a near 99% correlation between the preoperative imaging (lymphoscintigraphy) based on the use of the CD206 targeted Tc99m tilmanocept and the intraoperative/surgical findings of tumor-adnexed lymph nodes. Additionally, these findings provide exceptional guidance for nodal extraction in contrast to the “truth” mapping agent Lymphazurin, where Tc99m provides a significantly lower failed detection rate leading to a ∼10 percent improved staging rate for breast cancer and mealnoma patients (less missed disease and appropriate post-surgical treatment program).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-216. doi:1538-7445.AM2012-LB-216
Abstract LB-218: Immune response assessment in a phase II trial of AE37 HER2 peptide vaccine
Authors: Hale Diane F. , Vreeland Timothy J. , Dabney Raetasha S. , Clifton G Travis , Sears Alan K. , Pappou Efi , Anastasopoulou Eleftheria , Ardavanis Alexandros , Ponniah Sathibalan , Papamichail Michael , Perez Sonia , Shumway Nathan , Peoples George E. , Mittendorf Elizabeth
Published: August 2012

Introduction: AE37 is the Ii-Key hybrid of the HER2 derived peptide AE36 (776-790). A phase I trial administering AE37 with the immunoadjuvant GMCSF demonstrated the vaccine to be safe and capable of stimulating CD4+helper T cells with HER2 specific anti-tumor activity. Here we present an analysis of our immunologic testing of our prospective, randomized, single-blinded, phase II trial of the AE37+GMCSF vs. GMCSF alone for the prevention of breast cancer recurrence. Methods: After completion of indicated standard therapy; disease-free, node positive or high risk node negative breast cancer patients were randomized to receive either AE37+GMCSF (VG) or GMCSF (CG) in 6 monthly intradermal inoculations. Patients were enrolled with any level of HER2 expression (IHC 1+ 2+ or 3+). Specific immunologic responses to both AE36 and AE37 were evaluated. In vitro responses were measured using [3H]-thymidine incorporation assay and in vivo responses using delayed-type hypersensitivity (DTH) reactions. T regulatory cells (Tregs) were measured throughout vaccination series. Data was analyzed using Pearson chi-squared tests. Results: To date 217 patients have enrolled (VG=109, CG=108). HER2 over-expression was present in 54 (49.5%) VG and 51 (47.2%) CG, p=0.783. VG had 59 (51.4%) ER positive and CG had 58 (53.7%, p=0.985). The in vitro proliferation responses with a stimulation index (SI) ≥2 were classified as high responders VG 36 (33.0%) vs CG 8 (7.4%, p<0.001), SI 1.5-2 were classified as low responders VG 19 (17.4%) vs CG 16 (14.8%, p=0.600) or SI <1.5 as non responders VG 54(49.5%) vs CG 84(77.8%, p<0.001). The in vivo DTH reactions measured in 149 (VG n=86, CG n=63) were stratified as responders with ≥5mm (VG 74(86.0%) vs CG 17(27.0%) p<0.0001) or non responders with <5mm (VG 12 (14.0%) vs CG 46(73.0%) p<0.001). Tregs responses were measured in 107 patients (VG n=56, CG=51) and categorized according to percent change from pre-vaccination baseline as an increase >110% (5(8.9%) VG vs 10 (19.6%) CG, p=0.112), no change 90-110% (10 (17.9%) VG vs 13 (25.5%) CG, p=0.337), or decrease <90% (41 (73.2%) VG vs 28 (54.9%), p=0.048). Conclusion: VG patients had significant immunologic responses compared to CG. VG had a statistical decrease in Tregs compared to CG. Monitoring immunologic tests and Tregs throughout the vaccination process may stratify patients into responders and non responders and thus assist in identifying patients that will have recurrence.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-218. doi:1538-7445.AM2012-LB-218
Abstract LB-224: A randomized phase 2 study of two doses of itraconazole in men with metastatic castration-resistant prostate cancer (mCRPC)
Authors: Antonarakis Emmanuel S. , Heath Elisabeth I. , Smith David C. , Rathkopf Dana , Blackford Amanda L. , Danila Daniel C. , King Serina , Frost Anja , Ajiboye A. Seun , Zhao Ming , Mendonca Janet , Kachhap Sushant K. , Rudek Michelle A. , Carducci Michael A.
Published: August 2012

BACKGROUND: The antifungal drug itraconazole inhibits angiogenesis and Hedgehog signaling, and delays tumor growth in murine prostate cancer xenograft models. Unlike ketoconazole, it does not suppress androgen synthesis. METHODS: A phase 2 study of oral itraconazole in men with mCRPC was conducted at 4 sites. Men were randomized to low dose (LD) or high dose (HD) itraconazole (200 or 600 mg/day) until disease progression or unacceptable toxicity. The primary endpoint was PSA progression-free survival (PPFS) at 24 wk (PSA progression = 25% PSA rise above baseline/nadir); a 45% success rate in either arm was prespecified as constituting clinical significance. Secondary endpoints were progression-free survival (PFS) at 24 wk (progression = clinical/radiographic progression or death, but not rising PSA); median PFS; median PPFS; and max PSA decline. Exploratory outcomes included CTC enumeration, analysis of adrenal androgens (serum testosterone and DHEA), VEGF analysis, and GLI1 expression in skin biopsy samples. RESULTS: The HD arm enrolled to completion (N=29), but the LD arm closed early (N=17) due to a prespecified futility analysis. Table 1 shows efficacy results. In addition, 3/5 men (60%) in the HD arm and 2/3 men (67%) in the LD arm with unfavorable (≥5) CTCs at baseline converted to favorable (<5) CTC counts with treatment. Itraconazole did not reduce serum testosterone or DHEA levels. It had no effect on circulating VEGF levels. In skin biopsies, downmodulation of GLI1 was seen in 33% and 68% of men in the LD and HD arms, respectively. Median PPFS was superior in men who achieved GLI1 downmodulation compared to those who did not (P=.028). Toxicities with itraconazole included fatigue, nausea, anorexia, rash, and a syndrome of hypokalemia/hypertension/edema. CONCLUSIONS: Only the HD arm met its primary endpoint. Itraconazole 600 mg/day may have activity in men with mCRPC that is not mediated by androgen suppression, and may correlate with GLI1 downmodulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-224. doi:1538-7445.AM2012-LB-224
Abstract LB-23: Ras signaling interrupts TGF-beta-induced KLF5 acetylation to alter the KLF5-Smads transcriptional assembly in epithelial cells
Authors: Guo Peng , Xing Changsheng , Dong Xue-Yuan , Dong Jin-Tang
Published: August 2012

β (TGF-β) suppresses early stage tumorigenesis but promotes late stage tumor progression. While also presenting dual roles in tumorigenesis, KLF5 in the acetylated form is essential for TGF-β to properly regulate gene transcription in the inhibition of cell proliferation. The Ras oncogenic signaling not only converts TGF-β from a tumor suppressor to a tumor promoter but also uses KLF5 as a functional effector. One intriguing question is whether and how Ras signaling uses the KLF5 transcription factor to modulate TGF-β function. In this study, we addressed this question by examining whether and how Ras signaling alters TGF-β-induced KLF5 acetylation and the assembly of the p300-KLF5-Smads transcriptional complex in gene regulation. We found that Ras inhibited TGF-β-induced KLF5 acetylation and interfered with TGF-β in p15 induction and Myc repression. In addition, TGF-β-induced Smad3 phosphorylation at the C-terminal region was necessary for TGF-β to induce KLF5 acetylation, and Ras interrupted this phosphorylation. Ras signaling further interrupted the interactions among p300, KLF5 and Smad4, as well as the binding of the p300-KLF5-Smads complex onto the TGF-β-responsive promoter elements for both p15 and Myc. Without TGF-β, however, Ras signaling increased KLF5 acetylation, enhanced the p300-KLF5 interaction, and promoted the binding of the KLF5 transcriptional complex onto the TGF-β-responsive promoter elements. These findings suggest that KLF5 mediates the crosstalk between TGF-β and Ras signaling, and that suppression of TGF-β-induced KLF5 acetylation by Ras signaling alters TGF-β-induced assembly of p300-KLF5-Smads complex onto gene promoters, thus switching the function of TGF-β in gene regulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-23. doi:1538-7445.AM2012-LB-23
Abstract LB-239: ROCK1 inhibition reduces tumor collagen content in the KPC model for PDAC
Authors: Whatcott Clifford J. , Lobello Janine , Watanabe Aprill , Sepulveda Mario , Von Hoff Daniel D. , Han Haiyong
Published: August 2012

ROCK1 is a multifunctional member of the AGC (protein kinase A/G/C) kinase family that has been implicated in the modulation of stress-fiber assembly, cell contraction, apoptosis, and in the migration and invasion of multiple cancer cell types. It has been reported that ROCK1 plays an important role in the activation and function of pancreatic stellate cells, including in the formation of desmoplasia in pancreatic ductal adenocarcinoma (PDAC)(Masamune, 2003, Br J Pharmacol, 140:1292). Desmoplasia and its effects are thought to contribute to poor drug delivery in PDAC treatment. We therefore hypothesized that ROCK1 inhibition may reduce desmoplasia and enhance drug efficacy in PDAC treatment. We previously reported that ROCK1 is overexpressed in human pancreatic tumor cells and amplified in a small subset of PDAC patient samples. In this study we report that the ROCK inhibitor, fasudil, reduced collagen mRNA levels by 3.9-fold in cultured pancreatic stellate cells derived from PDAC tissues treated at 100µM. We also observed a 3.0-fold reduction in collagen protein expression in co-cultured tumor and stellate cells at the same concentration of fasudil. Similar reductions were observed in immunofluorescence analysis of co-cultured tumor and stellate cells treated with fasudil. We next sought to determine if ROCK1 inhibition would result in a reduction in collagen expression in the the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mouse model for PDAC. KPC mice were treated orally, b.i.d., with 100mg/kg fasudil and by i.p. with gemcitabine (q3dx4) at 80mg/kg. Tumors in KPC mice treated with the combination of fasudil and gemcitabine showed a marked reduction in overall tumor collagen content when compared to vehicle-treated mice as measured by a pentachrome staining. Histochemical analysis of tumors also showed reductions in the overall proliferation of the pancreatic stellate cell compartment. Ongoing efforts are being made to assess whether this treatment regimen may enhance gemcitabine efficacy in the KPC mice. These results indicate that ROCK1 is a potential therapeutic target in pancreatic cancer, and may provide an effective means to enhance current treatment regimens.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-239. doi:1538-7445.AM2012-LB-239
Abstract LB-243: Cetuximab and Artesunate synergistically inhibit the invasion and distant metastasis of non-small cell lung cancer
Authors: Rasheed Suhail Ahmed , Mudduluru Giridhar , Patil Nitin , Leupold Jörg Hendrik , Effert Thomas , Allgayer Heike
Published: August 2012

Cancer is a complex disease and multi step process evolving from malfunctions of molecules and their complex networks which regulate this process. The blockage of the cancer signalling network is crucial in the cancer treatment. In some cases, single drug therapy might not be comprehensive enough to inhibit the activity of secondary signals, feedback regulations and resistance to particular molecules, due to their abundant expression or activity. An understanding of these complex phenomena in cancer therapy for the benefit of patients is essential, especially since it bears the chance to establish novel concepts of combination drug therapy. In the present study, we have tried to find a compound combination to overcome resistance towards Cetuximab, which blocks ligand binding to the epidermal growth factor receptor (EGFR), in non-small cell lung cancer (NSCLC). The anti-malarial drug Artesunate (ART) has been shown to have a profound cytotoxic action against cancer cell lines, and we recently showed its anti-metastatic action in NSCLC. Initial screening experiments in NSCLC cell lines showed that Cetuximab sensitivity is positively correlated with the E-cadherin expression, whereas ART sensitivity was negatively associated with EGFR expression. A549 (high E-cadherin and EGFR, sensitive to Cetuximab alone and not sensitive to ART alone) and H1299 (no E-cadherin and medium EGFR, resistant to Cetuximab alone and sensitive to ART alone) were chosen as model cell lines for this study. Cell proliferation and colony formation assays were performed either with Cetuximab/ART alone or with the combination of both. Interestingly, Cetuximab alone was not able to reduce cell proliferation and colony formation significantly in comparison to ART. In contrast to that combination of both of the drugs able to reduce these phenomena significantly even more than ART alone (p≤0.05). Combination isobologram analysis for ART and Cetuximab showed a significantly reduced proliferation rate in both the tested cell lines. In vivo, chorioallantoic membrane (CAM) metastasis assays revealed that combinatorial treatment of the cells with both drugs significantly reduced primary tumor growth of A549 (p=0.01) and H1299 (p=0.02) cells, in contrast to the treatment by Cetuximab alone. Our findings suggest that ART is resensitizing Cetuximab resistant NSCLC cells for treatment with this antibody. Currently, our study will be completed by experiments to identified major molecules and signalling cascades mediating compound interaction and re-sensitization.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-243. doi:1538-7445.AM2012-LB-243
Abstract LB-249: Characterization of a novel potent and selective CDK4/6 inhibitor and a resistance mechanism
Authors: Dai Kang , Li Cong , Liang Lingming , Rong Minqing , Orf Jessica , Keegan Kathy , Li Zhihong , Fu Jiasheng , Ma Ji , Carlson Tim , McGee Larry , Medina Julio , Kamb Alexander , Wickramasinghe Dineli
Published: August 2012

CDK4 and CDK6 are two highly related cyclin D-dependent cell cycle regulatory kinases. CDK4/6 activity promotes G1 to S phase transition by phosphorylating the retinoblastoma (Rb) tumor suppressor protein. Here, we report the characterization of a potent, selective and orally-available small molecule inhibitor of CDK4/6 for treating Rb-positive cancer. The inhibitor was over 1000-fold more potent in inhibiting CDK4 than CDK1. It also showed Rb-dependent anti-proliferative activity both in cancer cell lines and in xenograft tumor models. A greater degree of variation in sensitivity to the inhibitor was observed in colon cancer cell lines compared to the other cell lines tested. To understand this difference in sensitivity, we isolated CDK4/6 inhibitor-resistant colon cancer cells by growing sensitive cells in the presence of the inhibitor. Gene expression in the parental and the resistant cells was analyzed and compared. Cyclin E1, a CDK2 cyclin, was found to be the most significantly overexpressed gene in the resistant cells. Consistent with this, CDK2-associated cyclin E1 protein and CDK2 kinase activity were also significantly increased in the cells. These results suggest that high expression of cyclin E1 may make tumors less sensitive to CDK4/6 inhibition and could be a potential sensitivity marker for patient selection for treatment with a CDK4/6 inhibitor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-249. doi:1538-7445.AM2012-LB-249
Abstract LB-251: Manipulation of macrophage phenotype enhances the apoptotic response to chemotherapy in mesothelioma
Authors: Broaddus V. Courtney , Battula Sailaja , Barbone Dario , Blakely Collin , Sugarbaker David J. , Bueno Raphael , Coussens Lisa M.
Published: August 2012

Macrophages within the tumor microenvironment (TME) may contribute to tumor chemoresistance. We have found that macrophages constitute a significant percentage of the infiltrating leukocytes in mesothelioma, a highly chemoresistant tumor. We asked whether reprogramming of macrophage phenotype from a Th2 (pro-tumor) to a Th1 (anti-tumor) phenotype would alter the mesothelioma chemoresponsiveness. To address this, we used 3D organotypic models of mesothelioma growth: 1) multicellular spheroids, with mesothelioma cells grown alone or co-cultured with macrophages derived from peripheral blood monocytes, and 2) primary tumor fragment spheroids derived from small fragments of freshly isolated human mesothelioma. Macrophages were incubated with Th1 (LPS & IFNγ) or Th2 (IL-4 &IL-13) cytokines prior to co-culture with mesothelioma spheroids, followed by exposure to standard-of-care chemotherapy, cisplatin plus pemetrexed. Gene expression was evaluated in macrophages to affirm Th1-type (TNF, IL-12, IFNα) versus Th2-type (CD206, IL10) programming. In both spheroid approaches, Th1-type macrophage programming significantly increased the apoptotic response of mesothelioma cells to chemotherapy. Moreover, when CSF1R signaling in macrophages was inhibited by incubation with a small molecular weight kinase antagonist, GW2850, chemoresponsiveness was significantly increased as evidenced by increased presence of apoptotic tumor cells in spheroids. Enhanced chemoresponsiveness of mesothelioma tumor cells was dependent on presence of macrophages because incubation of tumor cells with Th1-type cytokines or GW2850 in the absence of macrophages was without effect. We conclude that manipulating the TME may be a promising therapeutic approach in mesothelioma. This work was supported by a DOD Mesothelioma Program grant PR80717 to Broaddus and Coussens and a T32 Training Grant to Battula and Blakely.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-251. doi:1538-7445.AM2012-LB-251
Abstract LB-252: Enzymology of the mechanism of action for MDX-1203 antibody drug conjugate
Authors: Derwin Daniel W. , Passmore David , Zhang Qian , Sufi Bilal , Pan Chin , Rao Chetana , Huber Mary , Gangwar Sanjeev , Cardarelli Pina , Deshpande Shrikant , Rangan Vangipuram
Published: August 2012

MDX-1203 is an antibody drug conjugate under clinical evaluation by Bristol-Myers Squibb. The antibody portion is MDX-1115, a fully human anti-CD70 IgG. The drug portion is MED-2460, a small molecule which itself is composed of the releasable prodrug MED-2284, a cleavable di-peptide linker, and a maleimide reactive group intended to facilitate protein attachment. The mechanism of action of MDX-1203 involves tumor specific delivery and cellular uptake of ADC, followed by the intervention of two different enzymatic pathways. Release of prodrug is accomplished through proteolysis of a citruline-valine di-peptide linker, presumably by Cathepsin B activity in the acidic environment of the lysosome. Activation of released prodrug, MED-2284, occurs in the endoplasmic reticulum via esterase cleavage in the of the piperazine protecting group by the enzyme human Carboxylesterase 2. Michaelis Menten methodologies are used to probe activity of each enzymatic pathway and their relative role in the activity of the drug. Proteolysis is investigated through the use of Human Liver Cathepsin B protease. A kinetic model is constructed for a cysteine modified analogue MED-2460, and then applied to the more complex system involving antibody conjugated drug. Enzymatic activation of released prodrug MED-2284 is investigated with recombinant Human Carboxylesterase 2. For each pathway, the kinetic parameters Km, kcat, and enzymatic efficiency are measured and reported. Parameters are compared in an effort to define the enzymatic rate limiting step in the activation MDX-1203, and define their respective roles in the overall activity of the drug.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-252. doi:1538-7445.AM2012-LB-252
Abstract LB-257: Ganitumab (AMG 479), an anti IGF-1R antibody, inhibits the proliferation of melanoma cells
Authors: Von Euw Erika M. , Konkankit Veerauo V. , Gong Ke-Wei , Calzone Frank , Finn Richard S. , Chmielowski Bartosz , Slamon Dennis J.
Published: August 2012

It has been shown that the Insulin-like growth factor receptor type I (IGF-1R) signaling pathway is highly active in metastatic melanoma. IGF-1 binding induces autophosphorylation of IGF-1R and initiation of MAPK and Akt pathways signaling which control cellular proliferation and survival. IGF-1 is a critical factor for growth of many types of cancer, including melanomas, and inhibition of its activity can trigger apoptosis by suppressing the PI3K-AKT and MAPK pathways. Our main objective in this study was to evaluate the therapeutic potential of a novel antibody to the IGF-1R (ganitumab) in a large panel of melanoma cancer cells. We have evaluated 41 human melanoma lines with well established genotypes. Among the 41 cell lines, 22 harbored the BRAF mutation, 10 were NRAS mutant and 9 were wild type for BRAF and NRAS mutations. Treatment with ganitumab (100µg/ml) resulted in inhibition of cell proliferation at 120 hrs in 20 out of 41 cell lines. We found that the response to ganitumab was independent of genotype. We chose 6 cell lines, 3 from the responder population and 3 non-responders (2 BRAF, 2 NRAS and 2 wild types) to further investigate the mechanism of action of ganitumab. Results from flow cytometry suggest that treatment of melanoma cells with 100 µg/ml ganitumab caused a 10% increase in apoptosis 72 hrs after treatment in sensitive cell lines compared with the resistant cell lines where we could not detect an increase in apoptotic cells. The effect of ganitumab was also investigated in a 3D colony soft agar formation assay. Melanoma cell lines sensitive to ganitumab showed a significant reduction in the ability to form colonies in soft agar compared with the cells that did not respond to the antibody. Ganitumab treatment led to a decrease in pAKT, pERK and pS6 in sensitive cell lines regardless of their driver oncogenic mutations when compared with non responder cell lines that showed no inhibition of these markers. In conclusion, inhibition of oncogenic MAPK and PI3K signaling by ganitumab resulted in activity in vitro against a large subset of melanoma cell lines. Our findings provide the rationale for further preclinical evaluation of ganitumab as a melanoma therapeutic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-257. doi:1538-7445.AM2012-LB-257
Abstract LB-258: Developing therapeutic siRNA against HER2 in breast cancer cells
Authors: Hu Zhi , Gu Shenda , Rantala Juha , Ngamcherdtrakul Worapol , Yantasee Wassana , Gray Joe W.
Published: August 2012

Over-expression of oncogene HER2 was found in 20-30% primary breast cancers which involved in tumor initiation and progression. Breast cancer patients with high levels of HER2 (HER2 positive) showed poor clinical outcome. Increasing evidence indicated that the majority of patients with HER2 positive did not respond well to anti-HER2 drugs Trastuzumab (Herceptin) and Lapatinib. Even those who showed initial response ultimately became resistant to the treatment. The molecular mechanism of resistance includes the mutation of HER2 gene and the blocking of antigen of HER2 protein. The discovery of small interfering RNA (siRNA) raises the possibility of exploring new approach for cancer treatment. The siRNA against HER2 (siHER2) can reduce HER2 protein level by degrading mRNA expression. In our study, seventy-six siRNA sequences targeting the coding region of human HER2 gene were designed and synthesized. We quantified the RNA levels of HER2 and cell viability after HER2+ breast cancer cells (BT474, SkBr3 HCC1954) were transfected with siHER2 and scramble control siRNAs. Ten siRNA sequences were selected with greater than 50% knockdown efficiency and 40% inhibition of cell viability. We also measured the dose of 50% inhibition of cell growth (GI50) of top 5 siHER2 sequences in 20 HER2+ cell lines. The GI50 data showed that most of cell lines which were resistant to Trastuzumab were still sensitive to siHER2 treatment. Therefore, our data indicated that the siRNA against HER2 can potentially be used to overcome resistance of HER2 drugs, making it an alternative cancer therapeutic for HER2 positive patients. Currently, in vivo delivery of optimized siHER2 using nanoparticle is under evaluation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-258. doi:1538-7445.AM2012-LB-258
Abstract LB-259: Knockdown of slingshot2 serine phosphatase induces caspase3 activation in human carcinoma cell lines with the loss of the Birt-Hogg-Dubé tumor suppressor gene
Authors: Lu Xiaohong , Boora Uncaar , Fenton Janine , Reiman Anne , Seebra Laurence , Maher Eamonn R.
Published: August 2012

Birt-Hogg-Dubé (BHD) Syndrome, is a dominantly familial cancer syndrome associated with susceptibility to renal cancer carcinoma (RCC) caused by inactivating mutations in the folliculin (FLCN) gene. The precise functions of the FLCN gene product are still under investigation but RCC from BHD patients show loss of the wild type allele consistent with a tumour suppressor gene function. In search for potential synthetic-lethal targets for FLCN using a phosphatase siRNA library screen approach, we found that knockdown of SSH2 serine phosphatase (one of the three members of Slingshot family and actively involved in Actin reorganization) specifically induced caspase 3/7 activity in a dose-dependent manner (up to 6 fold increase, 10nM, 72hrs) in two human cell lines (BHD-origin renal cell carcinoma UOK-257 and thyroid carcinoma FTC-133) with loss of FLCN expression, but not in their folliculin expressing isogenic cell lines. SSH2 siRNA-induced knockdown was verified by real-time PCR (up to 60% reduction in SSH2 transcripts, 10nM siRNA, 48hrs), but SSH1 and SSH3 transcripts were increased up to 3 fold (suggesting a compensatory regulatory mechanism among members of SSH family). Further investigation of the underlining mechanism of caspase induction by SSH2 knockdown in FLCN-null cells exhibited that there was a dose-dependent increase in G1 cell population (up to 20% increase, 20nM SSH2 siRNA, 72hrs). Combination treatment of SSH2 + SSH1 siRNAs, but not SSH2 + SSH3 siRNAs potentiated induction of caspase 3 activity and G1 cell cycle arrest. These data indicate that apoptotic cell death in FLCN-null cells can be triggered by SSH2 knockdown through G1 arrest and provide a tool to develop potential therapeutic agents for BHD patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-259. doi:1538-7445.AM2012-LB-259
Abstract LB-260: Identification of DTYMK and CHEK1 as therapeutic targets in LKB1 mutant non-small cell lung cancer
Authors: Liu Yan , Cowley Glenn S. , Cohoon Travis J. , Root David E. , Kwok-kin Wong
Published: August 2012

The LKB1 tumor suppressor encodes a key metabolic sensor that integrates cell growth and metabolism. LKB1 is mutationally inactivated in multiple adult malignancies, including >20% of lung cancers, often simultaneously with activating KRAS mutations. LKB1 mutations are an important predictor of poor outcome and resistance to current therapeutic approaches. We employed an integrative approach to define novel therapeutic targets in Lkb1 mutant lung cancers. Matched cell lines from genetically engineered mouse models of cancer driven by activated Kras alone or in combination with Lkb1 deletion, were employed in high-throughput RNAi, kinase inhibitor, and metabolite screens. These screens identified knockdown of either Dtymk (deoxythymidylate kinase) or Chek1 (checkpoint kinase 1) as synthetically lethal with Lkb1 deficiency in both mouse and human lung cancer cell lines, and revealed that Lkb1 inactivation conferred marked sensitivity to treatment with CHEK1 inhibitors. Lkb1 deficient cells had a distinct metabolic profile, characterized by striking decreases in multiple nucleotide metabolites. Knockdown of DTYMK inhibited dTTP biosynthesis and, consequently, DNA synthesis, and knockdown of CHEK1 caused accumulation of DNA damage. We hypothesize that Lkb1 loss enhances dependence on these enzymes due to broad defects in nucleotide metabolism. Our studies support the development of therapies target DTYMK and CHEK1 in LKB1 mutant non-small cell lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-260. doi:1538-7445.AM2012-LB-260
Abstract LB-262: Targeting T-type calcium channels induces the extrinsic apoptotic pathway and sensitizes glioblastoma cancer cells to radio- and chemotherapy
Authors: Dziegielewski Jaroslaw , Valerie Nicholas C. K. , Dziegielewska Barbara , Hosing Amol S. , Gray Lloyd S. , Larner James M.
Published: August 2012

The purpose of this study is to develop a novel and effective mechanistic target for sensitizing glioblastoma tumors to anti-cancer therapy. Glioblastoma multiforme (GBM) are primary brain tumors with an exceptional resistance to radio- and chemotherapy and, therefore, have extremely poor prognosis. Conventional treatment consists of aggressive surgery, chemotherapy and/or radiotherapy; however in spite of these treatments, the median survival of GBM patients is only 9 to 12 months. T-type channels are a type of voltage-gated calcium channel important for normal cell proliferation and differentiation, but they could be also over-expressed or aberrantly activated in several human cancers, including GBM. In this study we investigate the effects of T-type channel inactivation, either by selective antagonists (mibefradil and TTL1177) or by siRNA-directed knock-down, on GBM cells survival and resistance to therapy in vitro. Our results show that blocking T-type channels not only reversibly arrests cells in G1/G0 phase of the cell cycle but also induces irreversible loss of clonogenic potential and cell death. The execution of cell death is preceded (or coincides) by induction of autophagy, as characterized by LC-3 cleavage and punctuate staining. Furthermore, we also establish that inactivation of T-type channels stimulates the extrinsic pathway of programmed cell death, as evidenced by caspase 8 activation and Bid protein cleavage. Finally, we demonstrate that inactivation of T-type calcium channels sensitizes GBM cells to radio- and chemotherapy. Taken together with the promising outcomes of the in vivo mibefradil plus temozolomide trials, our studies suggest that targeting T-type calcium channels could be a novel and very efficient therapy for glioblastoma brain tumors, either as a single modality or in combination with already established modalities. (This work was financed in part by the University of Virginia Cancer Center Pilot Grant, the Department of Radiation Oncology Pilot Grant and the Department of Chemistry Research Fund)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-262. doi:1538-7445.AM2012-LB-262
Abstract LB-263: Synthetic lethal targeting of PTEN-associated homologous recombination (HR) deficient melanoma cells by human apurinic/apyrimidinic endonuclease (APE1) inhibitors
Authors: Abbotts Rachel M. , Sultana Rebeka , Seedhouse Claire , Patel Poulam M. , Wilson David M. , Madhusudan Srinivasan
Published: August 2012

Background Mutation of the tumour suppressor PTEN is well-characterised in many advanced malignancies, including 30-50% of metastatic melanomas. Emerging evidence suggests a new role for PTEN in DNA repair, through transcriptional downregulation of the critical HR protein RAD51. Tumour cells deficient in HR (such as BRCA-deficient breast and ovarian cancers) have been therapeutically targeted in a synthetic lethality (SL) strategy using PARP inhibitors that block single strand break repair (SSBR), a pathway related to base excision repair (BER). We have previously identified novel inhibitors of the critical BER enzyme APE1, and demonstrated these can induce SL in BRCA-deficient cell lines. In the current study, we have evaluated SL relationships in PTEN-proficient and -deficient melanoma systems for a potential novel treatment strategy. Method We screened a panel of melanoma cell lines (MeWo, MM418, SkMel28, SkMel30, HT144, UACC62) for PTEN, APE1 and RAD51 protein expression. We tested for SL using clonogenic survival assays in PTEN- and RAD51-deficient cell lines using our recently developed APE1 DNA repair domain inhibitors, methoxyamine (indirect APE1 inhibitor) and NU1025 (PARP inhibitor), either alone or in combination. Aldehyde reactive probe assay, neutral comet assay, γH2AX immunofluorescence and FACS analyses were performed to confirm biological activity in cells. Results Of the screened cell lines, SkMel28, HT144 and UACC62 were deficient in PTEN and RAD51 protein expression. APE1 inhibitors were more toxic to PTEN-deficient cells compared to PTEN-proficient cells (p<0.01). Significant sensitivity was also seen in PTEN-deficient cells treated with methoxyamine (p<0.05) and PARP inhibitors (p<0.001), confirming that BER modulation results in SL in PTEN deficiency. This increased cytotoxicity in PTEN deficiency is associated with accumulation of DNA double strand breaks (DSB), as assessed by comet assay (p<0.001) and γH2AX accumulation (p<0.05). DSB accumulation leads to G2/M arrest and apoptosis, as demonstrated by FACS analysis. Conclusion Our data provides the first pre-clinical evidence that targeting APE1 in PTEN-RAD51 deficient melanoma cells is a promising new SL strategy. We are complementing the current small molecule studies by developing PTEN knockout systems using shRNA technology.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-263. doi:1538-7445.AM2012-LB-263
Abstract LB-267: A role for the Sin3 histone deacetylase complex in cell migration
Authors: Smith Karen T. , Martin-Brown Skylar A. , Seidel Chris , Mushegian Arcady , Egidy Rhonda , Florens Laurence , Washburn Michael P. , Workman Jerry L.
Published: August 2012

Histone deacetylases (HDACs) remove acetyl groups from histones and other proteins, leading to alterations in protein activity and gene expression. Drugs that target these enzymes (HDAC inhibitors or HDACis) are currently approved for the treatment of specific cancers and are under study in numerous additional clinical trials. These drugs work by causing cell cycle arrest, differentiation or apoptosis. However, less is known about their potential effects on cancer cell migration and invasion, processes implicated in metastasis. There are eleven human zinc dependent histone deacetylases and many HDACis target several of these structurally related proteins in cells. Our studies are focused on HDACs1 and 2 which reside in large protein complexes, one of which is the Sin3 complex. This complex is important for promoter-mediated histone deacetylation, transcriptional repression and cell cycle control. We previously found that specific HDACis disrupt the function of the Sin3 complex in distinct ways. Here we describe the function of a novel subunit of the Sin3 complex, a previously uncharacterized protein, named “Family with sequence similarity 60, member A” (FAM60A). We find that FAM60A represses expression of genes linked to cancer cell migration and invasion. Moreover, we show that loss of FAM60A, or a core subunit of the Sin3 complex can increase the migration of lung cancer cells. This suggests that not all HDAC-related functions are cancer promoting, and adds to data suggesting that some HDAC-related functions may act in steps in metastasis suppression pathways. This suggests that some HDACis could increase the potential for metastasis. Current studies are focused on delineating the cancer-promoting functions of the complex from those that are tumor or metastasis suppressive and testing the effects of specific HDACis on cancer cell migration and invasion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-267. doi:1538-7445.AM2012-LB-267
Abstract LB-269: A new role for the gain-of-function activity of p53 in breast cancer
Authors: St-Pierre Yves
Published: August 2012

Using tissue microarrays constructed from samples obtained from normal breast tissues and breast carcinomas, we previously found that galectin-7 is specifically expressed in myoepithelial cells but not luminal cells. We also found that abnormally high levels of galectin-7 were restricted to basal-like breast cancers, a heterogeneous group of cancers with a poor prognosis. These results were consistent with the genomic profiling data previously reported by the group of Charles Perou who recently provided a molecular portrait of human breast tumors. Their data revealed that while galectin-7 transcripts were expressed at low levels in normal breast tissues and mammary epithelial cell lines, they were highly expressed in estrogen receptor (ER)-negative breast cancer. Our data were also consistent with microarray studies by Jones and colleagues who identified galectin-7 as a myoepithelial-specific genes. In fact, galectin-7 ranked first on a list of 42 most predictive genes that distinguish luminal and myoepithelial cells. This expression of galectin-7 is not only important to study in terms of biomarkers of aggressive forms of breast cancer, but also because increased galectin-7 in breast cancer cells is known to increase their metastatic behavior. However, the expression of galectin-7 in aggressive forms of breast cancer is somewhat paradoxical if not surprising because galectin-7 has been considered an apoptosis regulator under the control of p53. In a previous report, Galectin-7 (also called p53-induced gene 1) was found to be one of the 14 transcripts that were induced by wild-type p53 in colon cancer cells. Here, we have examined this apparent contradiction by investigating the molecular mechanisms controlling galectin-7 expression in human breast cancer cells. We found that galectin-7 expression in human breast cancer cells is dictated by a gain-of-function activity of mutant p53. This conclusion is based on the following: 1) expression of galectin-7 in cancer cell lines correlated with constitutively high levels of NF-κB activity; 2) transfection of NF-κB subunits or treatment of cells with TNFα, a well-known inducer of NF-κB, increased endogenous level of galectin-7; 3) inhibitors of NF-κB reduced constitutive galectin-7 expression; 4) transfection of a transcriptionally inactive form of p53 (R175H) into a p53-null human breast cancer cell line induced galectin-7 via increased NF-kB activity. These findings identify galectin-7 as a novel player through which mutp53 exerts its gain-of-function activity in breast cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-269. doi:1538-7445.AM2012-LB-269
Abstract LB-272: A novel isoform of Sox5 is expressed in TRAF3-deficient mouse B lymphomas
Authors: Edwards Shanique K.E. , Desai Anand , Moore Carissa R. , Liu Yan , Hart Ronald P. , Xie Ping
Published: August 2012

TRAF3 is a novel tumor suppressor identified in human non-Hodgkin lymphoma and multiple myeloma. We recently reported that TRAF3 deletion causes vastly prolonged survival of mature B cells, which eventually leads to B lymphoma development in mice. The long latency of B lymphoma development observed in B-TRAF3−/− mice suggest that additional oncogenic pathways are required for B lymphomagenesis. To delineate such oncogenic pathways in TRAF3−/− B lymphomas, we performed microarray analyses and identified Sox5 as a gene recurrently up-regulated in B lymphomas spontaneously developed in different individual B-TRAF3−/− mice. We confirmed the striking up-regulation of Sox5 expression in TRAF3−/− B lymphomas at both the mRNA and protein levels by quantitative real time PCR and Western blot analyses, respectively. We further cloned the full-length cDNA of Sox5 from B lymphomas derived from 4 different individual B-TRAF3−/− mice. Surprisingly, we found that the sequence of Sox5 expressed in TRAF3−/− B lymphomas represents a novel isoform of Sox5, which has not been previously reported. This new isoform of Sox5 contains a 35 aa deletion in the N-terminal region in front of the leucine zipper domain. When transduced into human multiple myeloma cells, the cloned Sox5 cDNA was expressed into a protein of 80 kDa, a size identical to that detected in TRAF3−/− B lymphomas. Taken together, our findings identified a novel isoform of Sox5 as a candidate oncogene in B lymphoma. Our ongoing experiments will further elucidate the roles and mechanisms of Sox5 in TRAF3 deficiency-initiated B cell malignant transformation. This study is supported by a seed grant from the New Jersey Commission on Cancer Research (10-1066-CCR-EO, P. Xie), a Faculty Research Grant (P. Xie), and the Arthur Herrmann Endowed Cancer Research Fund (P. Xie); supported in part by a grant from NCI (R. Hart) and an Aresty Research Grant (A. Desai).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-272. doi:1538-7445.AM2012-LB-272