bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C42: p16-enhanced the migration phenotypes in hepatocellular carcinoma cells is Rb-independent.
Authors: CHU HSIAO-CHIEN , Lin Ze-Shiang , Lewis Brian C , Chen Ya-Wen
Published: June 2012

The occurrence of tumor metastasis to the extra-hepatic region contributes to a poor prognosis of hepatocellular carcinoma (HCC). However, the molecular mechanisms of metastasis in HCC remain unclear. Our previous studies showed that concomitant loss of Ink4a/Arf locus accelerated tumor formation and metastasis in mice with liver specific Trp53 deletion, indicating roles of Ink4a/Arf locus in tumorigenesis and metastasis. In this study, we focus on the p16 (Ink4a), a well-known tumor suppressor in regulation of cell cycle. When re-introduction of p16 into Trp53 and Ink4a/Arf-null liver cancer cells, we unexpectedly observed that p16 could enhance cell migration in HCC cell lines, including murine and human orgins by transwell assay and ectopic p16 expression also increased colonization in the lungs via tail vein injection into immune-deficient mice. Interestingly, there was no significant change in expression of epithelial mesenchymal transition (EMT)-related proteins. We further demonstrated that activity of Cdc42 and Rac1 was increased in MM189 cell with p16 over-expression but activity of RhoA was partially decreased. By knockdown of small GTPase, Cdc42 GTPase was required for p16-enhanced cell migration, whereas the related RhoA GTPase inhibited p16-mediated migration. Analysis of p16 mutants indicated that the Cdk4 interacting domain was required for stimulation of HCC cell migration, knockdown of Cdk4 and Cdk6 showed that these proteins are dispensable for this phenomenon. We therefore hypothesized that p16-mediated migration is not involved in pRb-dependent pathways. To validate our hypothesis, knockdown of pRb did not impair p16-enhanced migration in HepG2 cells with ectopic p16 expression. Our study suggested that p16-enhanced migration is not mediated by pRb-dependent pathways and more studies needs to be performed to elucidate this.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C42.
Abstract C45: Validation of thromboxane A2 receptor (TP) as a target to block breast cancer metastasis.
Authors: Zhang Xuejing , Nie Daotai
Published: June 2012

The receptor of thromboxane A2 (TXA2), TP, is a seven transmembrane, G-protein-coupled receptor. Increased expression of TP at RNA level was found to correlate with a poor prognosis in breast cancer patients but it is unknown how TP expression and activities are involved in breast cancer progression. Here we report that TP is expressed in metastatic breast cancer cells at both RNA and protein levels. Treatment of breast cancer MDA-MB-231 cells by U46619, a TP agonist, induced cell body contraction. The U46619-induced contraction was blocked by SQ29548, a high-affinity TP antagonist. Further studies found that small GTPase RhoA was activated by TP activation and that pretreatment of MDA-MB-231 cells with Y27632, a Rho kinase (ROCK) inhibitor, blocked U46619-induced cell contraction. The requirement of RhoA activation in U46619 induced cell contraction was further confirmed by the ability of dominant negative RhoA to block cell contraction induced by U46619. MDA-MB-231 cells with TP knockdown displayed a reduced percentage of contraction under the treatment of U46619 and TXA2 agonist I-BOP as well as attenuated invasion ability. When injected into mice, MDA-MB-231 cells with TP depleted did not present significant changes in primary tumor formation or growth. However, TP depletion reduced the ability of MDA-MB-231 cells to metastasize to the lung and the liver in an experimental metastasis model. We further demonstrated that TP depletion reduced the lung colonization ability of MDA-MB-231 cells by blocking active extravasation. Taken together, TP is expressed in breast cancer and that activation of TP has profound effects on tumor cell cytoskeleton and contraction through activation of RhoA. Inhibition or depletion of TP reduces the metastatic dissemination of breast cancer cells in vivo by blocking the extravasation of tumor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C45.
Abstract C5: Worse prognosis of KRAS c.35 G > A mutant metastatic colorectal cancer (MCRC) patients treated with intensive triplet chemotherapy plus bevacizumab (FIr-B/FOx).
Authors: Bruera Gemma , Cannita Katia , Giacomo Daniela Di , Lamy Aude , Troncone Giancarlo , Dal Mas Antonella , Coletti Gino , Frebourg Thierry , Sabourin Jean Christophe , Tosi Mario , Ficorella Corrado , Ricevuto Enrico
Published: June 2012

Introduction: Bevacizumab (BEV) addition to doublet chemotherapy significantly increases efficacy without differentially affecting prognosis in KRAS wild-type (wt) and mutant (m) MCRC. Present study evaluates clinical outcome of BEV added to triplet chemotherapy, FIr-B/FOx (Bruera G et al, BMC Cancer 2010, 10:567), according to KRAS genotype.

Methods: MCRCpatients (pts) were treated with first line FIr/FOx regimen including BEV and triplet chemotherapy (weekly alternating irinotecan or oxaliplatin associated to fluorouracil). Treatment schedule: weekly 12h-timed-flat-infusion/5-fluorouracil 900 mg/m2, days 1–2, 8–9, 15–16, 22–23; irinotecan 160 mg/m2 plus BEV 5 mg/kg, days 1,15; oxaliplatin, 80 mg/m2, days 8, 22; every 4 weeks. Tumoral samples were simultaneously screened for KRAS codon 12 and 13, and BRAF c.1799 T > A mutations by SNaPshot multiplex assay (Di Fiore F, Br J Cancer 2007;96(8):1166–1169) and/or direct sequencing. Activity and efficacy were evaluated and compared using log-rank test.

Results: Forty-five MCRC pts were evaluated: 25 KRAS wt (56%), 20 KRAS m (44%). After 30 months median follow-up, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were, respectively: KRAS wt 88% (C.I. × 14), 14 months, 38 months; KRAS m 80% (C.I. × 19), 12 months, 21 months. PFS and OS were not significantly different. Among KRAS m pts, ORR, PFS and OS at 19 months median follow-up in c.35 G > A (13 pts) were 69% (C.I. × 26), 9 months, 11 months, respectively. OS of c.35 G > A KRAS m was significantly worse compared to KRAS wt (p = 0.003) and to other than c.35 G > A mutant pts (p = 0.022).

Conclusions: First line FIr-B/FOx intensive regimen adding BEV to triplet chemotherapy can further increase activity and efficacy without significantly affecting different clinical outcome in KRAS wt and m MCRC pts. For the first time in MCRC pts treated with BEV-containing chemotherapy, present study shows a significant interaction between the most prevalent c.35 G > A KRAS mutation and worse prognosis compared to KRAS wt pts and to other than c.35 G > A KRAS exon 2 mutations, depending from different biological aggressiveness and sensitivity to BEV addition to triplet chemotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C5.
Abstract C58: Humanization and characterization of an anti-ErbB-3 murine monoclonal antibody.
Authors: Sala Gianluca , Traini Sara , Risio Annalisa Di , Vianale Giovina , Tinari Nicola , Natali Piergiorgio , Muraro Raffaella , Iacobelli Stefano
Published: June 2012

Background: The signaling network downstream of the human epidermal growth factor receptors ErbB-1 and ErbB-2 have been extensively targeted by cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance which is often associated with reactivation of the ErbB-3/PI3K-Akt axis. This may be overcome by ErbB-3 ligands that abrogates receptor-mediated signaling.

Methods: the murine MP-RM-1 monoclonal antibody that specifically binds ErbB-3 ectodomain was chimerized by replacing the murine constant regions with the human constant regions and humanized by gratfing Complementarity Determining Regions from the murine into a human antibody framework using a computer assisted molecular modelling. Recombinant genes were placed into the pCDNA3.1 expression vector and transfected into Chinese Hamster Ovary-S cells. Antibodies were screened on the basis of their ability to inhibit Neuregulin-1 (NRG-1)-induced activation of the ErbB-3/PI3K-Akt axis in IR-8 human melanoma cells (Table 1). Four antibody variants, i.e., cMP-RM-1 #1, hMP-RM-1 #6, hMP-RM-1 #10, and hMP-RM-1 #20, were selected and further tested for their ability to promote ErbB-3 downregulation and to inhibit in vitro and in vivo growth of tumor cells.

Results: Four chimeric (c) and sixteen humanized (h) antibody variants were obtained. All variants showed to possess antigen binding affinity comparable to that of their parental antibody. Initial screening identified one chimeric (cMP-RM-1 #1) and three humanized (hMP-RM-1 #6, hMP-RM-1 #10, hMP-RM-1 #20) antibodies as the most effective in inhibiting ErbB-3/Akt phosphorylation and promotion of ErbB-3 downregulation. Further testing revealed the antibodies potently inhibited colony formation ability of tumor cells and halted growth of melanoma and ovarian cancer xenografts in nude mice. Treatment with the antibodies was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue.

Conclusions: This successful production of chimeric/humanized antibodies possessing antitumor activity in vitro and in vivo may provide novel candidates for ErbB-3-targeted therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C58.
Abstract C59: Antibody targeting of CD24 efficiently retards growth of experimental carcinomas.
Authors: Salnikov Alexey V. , Bretz Niko , Perne Claudia , Fogel Mina , Keller Sascha , Schlange Thomas , Moldenhauer Gerhard , Altevogt Peter
Published: June 2012

It has been shown that CD24 drives lung metastasis formation of bladder cancer and might be a good candidate for mAb-dependent therapy. We tested whether mAb SWA11 to CD24 would be effective in preventing tumor growth in xenotransplanted mice. In an A549 lung carcinoma model cells were implanted s.c. into SCID/beige mice followed by multiple injections of mAb over 37 days. Therapy with anti-CD24 mAb SWA11 lead to substantially reduced tumor load. The final tumor mass of treated animals was significantly smaller compared to isotype-control mAb treated animals. Similar observations were made in therapy models of CD24-positive BxPC3 pancreatic adenocarcioma cells in NOD/SCID mice and SKOV-3ip ovarian carcinoma cells in CD1 mice. The therapeutic effects were associated with changes in tumor cell proliferation, apoptosis and angiogenesis. Surprisingly, we found that SWA11 mAb treatment significantly altered cytokine tumor microenvironment. Also the mAb treatment lead to the increased infiltration of tumor tissue with immune cells suggesting involvement of ADCC. Addition of SWA11 mAb to the gemcitabine treatment strongly potentiated its anti-cancer efficacy in A549 lung cancer model. Our data demonstrate that targeting of CD24 could be beneficial for the anti-cancer treatment using standard chemotherapy regimes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C59.
Abstract C62: 5A2, a new IL-3Ra (CD123) monoclonal antibody for the treatment of AML.
Authors: Huang Liang , Wang Na , Wang Huaying , Cao Zhixiang , Chen Yiyou , Li Henry
Published: June 2012

Acute myeloblast leukemia (AML) is a classic stem cell disease driven by leukemic stem cells (LSCs). LSCs in general are resistant to the existing therapies and the current treatment prognosis are rather poor. Targeting AML-LSCs has thus been suggested as a new potential approach to improve the current treatment. IL-3 receptor -chain (IL-3R, or CD123) has been shown over-expressed on leukemic blasts, progenitors and LSCs. CD123 signaling contributes to leukemic development. CD123 may thus represent a suitable target for monoclonal antibody based treatment approaches against AML.

We immunized mice with purified IL3RA ECD/Fc fusion protein and the IL3RA expressing plasmid respectively. From the immunized mice we generated 3 and 8 hybridoma cell lines that secreted monoclonal antibodies recognize to IL3RA ECD/Fc and CD123 expressed on TF-1 (a CD123 expressing AML cell line) and 8G9 (a CD123 stable-expressing CHO established by us). In particular, among the 11 positive cell lines, one of the antibodies, 5A2, was confirmed with antagonist property to block the binding of IL-3/IL3RA. We demonstrated that 5A2 dose-dependently suppresses the IL-3 induced proliferation of TF-1 cells. In addition, 5A2 can also efficiently internalize into TF-1 cells. These observations suggest that 5A2 can be a potential candidate for development as an antibody based therapy against AML. At present, we are in process of further characterizing 5A2 (including determination of Kd, epitope, cross-species reactivity, inhibition of ex vivo proliferation of patient leukemic cells, along with in vivo efficacy in a suitable animal model, etc.). We are also initiating humanization of 5A2 to identify clinical candidate. We plan to also report and discuss the progresses on some of these studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C62.
Abstract C67: Investigating PI3K/mTOR inhibition in a mouse model of colorectal cancer mutant for both Apc and Pten.
Authors: Raja Meera , Shaw Paul , Clarke Alan
Published: June 2012

This study evaluates the effect of dual PI3K and mTOR inhibition as a potential novel therapeutic strategy in a preclinical model of colorectal cancer.

Loss of Pten in the context of Apc loss in the murine intestine accelerates tumorigenesis through increased activation of Akt. Tumor bearing AhCreER Apcfl/+ Ptenfl/fl mice were exposed to acute doses of a PI3K/mTOR inhibitor to investigate the immediate biological effects on tumors. To investigate the therapeutic potential of PI3K/mTOR inhibition, mice were treated with the PI3K/mTOR inhibitor twice daily to a survival end point.

Short term exposure to the PI3K/mTOR inhibitor confirmed inhibition of the pathway in tumor tissue through reduction of phospho-AKT, phospho-mTOR and phospho-S6, however this did not induce apoptosis. Chronic exposure resulted in a significant increase in longevity of mice from a median survival of 100 days post induction to beyond 300 days post induction with 20% of mice alive at a year post induction. Tumor burden and grade analysis indicates that chronic PI3K/mTOR inhibition slows down the growth rate of tumors and prevents tumor progression but does not prevent initiation of microadenomas after 80 days of treatment. Resistant tumors which were exposed to a longer period of treatment were found to be larger and on average more aggressive. Currently, we are investigating the mechanisms of resistance to chronic PI3K/mTOR inhibition to identify genes which may be responsible for resistant tumor growth and thus identify new drug targets.

Simultaneous inhibition of the PI3K and mTOR signaling pathways effectively prolongs survival of mice with aggressive adenocarcinoma and prevents tumor progression. Here we provide evidence that dual PI3K and mTOR inhibition may be a rational therapeutic strategy for patients with similar tumor profiles.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C67.
Abstract C7: Analysis of high-risk prostate cancer markers at RNA and protein level.
Authors: Masarik Michal , Gumulec Jaromir , Hlavna Marian , Sztalmachova Marketa , Cernei Natalia , Babula Petr , Adam Vojtech , Krizkova Sona , Hrabec Roman , Rovny Arne , Kizek Rene
Published: June 2012

Background: In many developed countries, prostate cancer is the second leading cause of cancer-related deaths among men. Current diagnostic possibilities do not differentiate aggressive forms of prostate cancer from latent tumors. Therefore, having reliable and easy-to-detect marker is needed to treat this disease successfully. We determined potential novel tumor markers of high-risk prostate cancer (A) at the cell level, represented by cell lines 22Rv1 and PNT1A and (B) in the serum of 82 histologicaly -verified cases and 51 controls.

Material and Methods: 22Rv1 cell line derived from primary prostate tumor and PNT1A cell line derived from normal prostate epithelium were used in our study. The real time PCR, electrophoretical separation technique, immunochemical detection and novel electrochemical analysis were used in tumor markers determination.

Findings and Interpretation: Cell line 22Rv1 represents Gleason sum 9 primary tumor, compared to widely used cell lines PC-3, LNCaP and PD-145, representing metastasis, therefore, is more related to real tumor tissue. We determined RNA and protein level of genes alpha-methyl acyl-CoA racemase (AMACR), caveolin-1, metalothionein, p53, NF-kB, FOS, JUN, Ki-67, ZIP1 and ZnT-1. At the RNA level in cell lines we found significant (p < 0.01) down-regulation of Cav-1 (50-fold), NF-kB (11-fold) and others and significant (p < 0.01) up-regulation of metalothionein (2.4-fold), AMACR (8.4-fold), Ki-67 (2-fold) and zinc transporters ZnT-1 (25-fold) and ZIP1 (45-fold). Conversely, we observed down-regulation of metallothionein at protein level. In the serum of prostate cancer patients we found no significant differences in AMACR and caveolin-1 level and significantly (p < 0.001) higher metallothionein level suggesting metalothionein as a complementary marker to PSA. We found weak correlation of PSA on caveolin-1 (r = 0.41 at p = 0.002) and, interestingly, significant elevation of caveolin-1 in high-grade tumors (tumor stage 4 (2.3-fold elevation), Gleason sum 9, correlation at r = 0,29 at p = 0,028) suggesting caveolin-1 as potential high-risk prostate cancer marker.

This study provides important new information about selected genes connected with prostate cancer and describes new electrochemical approaches for their determination in lower concentrations.

Financial support from IGA MH of Czech republic no. NS10200–3 is greatly acknowledged.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C7.
Abstract C74: Eradicating cancer stem cells by targeting mTOR pathway with metformin.
Authors: Park Heon Joo , Lee Hyemi , Williams Brent , Song Chang Won
Published: June 2012

Cancer stem cells are resistant to conventional radiotherapy and chemotherapy. It is, therefore, important to eradicate cancer stem cells to achieve complete control of cure of cancer. It has been demonstrated that Akt/mTOR pathway plays a critical role for survival and proliferation of cancer cells, particularly cancer stem cells. We investigated the effect of metformin, ionizing radiation and mild heating alone or combined on mTOR activity and survival of cancer stem cells. Metformin inactivated mTOR via activation of AMPK, thereby preferentially killing stem cells of various cancer cells. For example, the % of cancer stem cells in MCF-7 cells before treatment was about 3.4%, and it decreased to 1.2% after 48 h treatment with 5 mM metformin, as determined with FACS using CD44+/CD24- surface markers. Metformin also selectively killed the stem cells of MIAPaCa-2 pancreatic cancer cells expressing CD44+/CD24+. Mammosphere formation study also demonstrated that metformin is effective to kill cancer stem cells. We observed that irradiation and heating also suppress mTOR activity through activation of AMPK. However, irradiation was ineffective to kill cancer stem cells while heating was effective to kill cancer stem cells. Interestingly, irradiation upregulated Akt activity while mild heating downregulated Akt activity. It appeared that the radiation-induced inhibition of mTOR activity is counterbalance by Akt activation. Treatment of MIAPaCa-2 xenografts with combination of metformin and irradiation was markedly more effective than metformin or irradiation alone. It is concluded that metformin in combination with ionizing radiation and mild hyperthermia are potentially effective to eradicate cancer stem cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C74.
Abstract C75: CYP2S1 and CYP2W1 mediate 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (GW610, NSC 721648) sensitivity in breast and colorectal cancer cells.
Authors: Leong Chee-Onn , Tan Boon Shing , Tiong Kai Hung , Muruhadas Ashwin , Randhawa Nirmal , Choo Heng Lungh , Stevens Malcolm FG , Bradshaw Tracey D.
Published: June 2012

Phortress, the lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F203, NSC 703786) is under clinical evaluation. CYP1A1-catalyzed bioactivation of 5F203 is crucial for antitumor activity. 2-(3,4-Dimethoxyphenyl)-5-fluorobenzothiazole (GW610, NSC 721648) is a structurally related experimental antitumor agent. In order to understand the mechanism of action of GW610, we investigated roles of cytochrome P450 enzymes CYP2S1 and CYP2W1 in mediating benzothiazoles activity. Stable isogenic breast (MCF-7, MDA-MB-468) and colorectal (HCC2998, KM12) cancer cell lines depleted for CYP1A1, CYP2S1 or CYP2W1 were generated. The sensitivity of these cells to 5F203 and GW610 was compared with vector control cells.

5F203 exhibited potent activity against breast cancer cells, whereas GW610 was effective against both breast and colorectal carcinoma cells.

CYP1A1 was induced in breast and colon cancer cells, whereas induction of CYP2S1 and CYP2W1 was observed in breast cancer cells only following treatment with 5F203 or GW610. Depletion of CYP1A1 abrogated 5F203 and GW610 activity; in contrast, CYP2S1 depletion enhanced sensitivity of breast and colon cancer cells to 5F203 and GW610. Intriguingly, CYP2W1 knockdown caused marked resistance to GW610 in HCC2998 (>7-fold) and KM12 (>90-fold) colorectal cancer cells only.

The results indicate that CYP2S1 is involved in deactivation of benzothiazoles, but CYP2W1, expressed in colorectal tumors and a putative therapeutic target, is important for bioactivation of GW610 in colorectal carcinoma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C75.
Abstract C77: Sangivamycin-like molecule 6 (SLM6) exhibits potent and selective anti-multiple myeloma activity through inhibition of P-TEFb.
Authors: Dolloff Nathan G. , Talamo Giampaolo , Dicker David T. , El-Deiry Wafik S.
Published: June 2012

We previously reported the anti-tumor and anti-angiogenic activity of a new class of sangivamycin-like molecules (SLMs). In this study we aimed to further the preclinical development of these compounds by identifying tumor types with the highest sensitivity to their cytotoxic effects. Of 35 tumor cell lines from 8 different tissues of origin that we screened, we found that multiple myeloma (MM) cells were approximately 3–10 times more sensitive to SLMs than other tumor types. SLM3 as well as SLM5, SLM6, and SLM7 reduced viability and induced apoptosis of MM cells at sub-micromolar concentrations in vitro. By contrast, SLMs induced negligible amounts of apoptosis in other tumor and normal cell lines. SLM6 exhibited the most potent activity in vivo where it significantly inhibited the growth of xenografted MM tumors. We found that SLM6 inhibited cyclin dependent kinase 9 (CDK9) resulting in hypophosphorylation of RNA polymerase II (Ser2) and transcriptional repression of oncogenes that are known to drive MM progression and resistance to therapy (c-Maf, cyclin D1, c-Myc, L-Myc, and Mcl-1). Furthermore, we found that SLM6 increased the cytotoxic activity of therapies that are currently approved for MM treatment (bortezomib, lenalidomide, dexamethasone, and doxorubicin). These findings suggest that CDK9 inhibition is a particularly effective targeted strategy for MM, and SLM6 is a promising CDK9-targeted agent, both as a single agent and in combination with currently approved therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C77.
Abstract C85: ck2 inhibitors in luminal and basal-like breast cancer cells.
Authors: Patton Michelyn C. , Williams Christopher
Published: June 2012

Protein kinase casein kinase II (CK2) is a heterotetrameric serine/threonine kinase involved in proliferation, apoptosis and embryonic development. It is expressed at elevated levels in proliferating tissue, human cancer cell lines and virtually all forms of solid cancers. Recently it has been shown that CK2 inhibition induces apoptosis in cell models of acquired anti-estrogen resistance. These findings suggest that there may be tumor phenotype specific effects of CK2 in breast cancer and consequently phenotype specific sensitivity to CK2 inhibition. As such we sought to determine if luminal and basal cell types display differential sensitivity to selective CK2 inhibitors. Utilizing crystal violet and alamar blue assays, it was found that CK2 inhibition decreased viability of both luminal and basal breast cancer cell lines, with basal-like ER(−) MDA MB 231 cells showing notable sensitivity. Hypertrophic morphologic changes consistent with cellular senescence were noted upon inhibition of CK2. Additionally, flow cytometric analysis revealed an increase in mean vimentin expression in basal but not luminal breast cancer cells following CK2 inhibition. These studies suggest that CK2 utilizes multiple molecular mechanisms to promote tumorigenesis, and that CK2 inhibition may disrupt cell-type specific signaling pathways to induce breast cancer cell death.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C85.
Abstract C91: Systematically analyzing molecular mechanism of growth inhibition induced by liver × receptor agonists in HT-29 human colon cancer cells using high-throughput micro-Western array and microarray.
Authors: Lin Ching-Yu , Huo Chiech , Lin Hui-Ping , Jiang Shih Sheng , Chiang Chi-Yi , Chuu Chih-Pin
Published: June 2012

Colon cancer is the third leading cause of cancer-related death in the world. Previously, we and other groups reported that agonists for liver × receptors (LXRs) suppressed the proliferation of several human cancer cell lines. LXR and LXR are nuclear receptors and are important regulators of cholesterol, fatty acid, and glucose homeostasis. We recently developed a high-throughput proteomics technology named Micro-western arrays (Ciaccio et al., Nature Methods, 2010), which is an array-based Western blotting assay enable quantitative, sensitive and high-throughput assessment of protein abundance and modifications after electrophoretic separation of micro-arrayed cell lysates. This method allowed us to measure 96 to 384 different phosphosites on multiple proteins of a set of 6 to 15 different samples at the same time at. In order to study the molecular mechanism lying underneath the anticancer activity of LXR agonists (22(R)-hydroxycholesterol, T0901317, and GW3965) on colon cancer cells, we use 96-well proliferation assay, colony formation assay, flow cytometry analysis, invasion/migration assay, gene overexpression and shRNA/siRNA knockdown, qRT-PCR, gene microarray, and Micro-Western Array to systematically investigate the suppressive effect of LXR agonists on HT-29 human colon adenocarcinoma cells. LXR agonists suppress proliferation of HT-29 cells dose-dependently at the range from 100 nM to 10 μM. LXR agonists treatment cause G1 cell cycle arrest and affect several cell cycle regulating proteins. Micro-Western Array and microarray reveals that several signaling pathways regulating survival, stress, proliferation, and cell cycle of cells are being affect by LXR agonists. Our observation suggests that LXR agonist treatment may serve as a potential adjuvant therapy for human colon cancer.

This work was supported by CS-100-PP-12 (National Health Research Institutes, NHRI) and NSC 99–2320-B-400–015-MY3 (National Science Council, NSC) in Taiwan for CPC, as well as DOH100-TD-C-111–014 (Department of Health) for SSJ and CPC. We thank support from Protein Chemistry Core Facility of Core Instrument Center in NHRI and Taiwan Bioinformatics Institute Core Facility of the National Core Facility Program for Biotechnology by NSC (NSC100–2319-B-400–001).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C91.
Abstract PL04-03: Engineering improved cancer vaccines.
Authors: Dranoff Glenn
Published: June 2012

Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in some advanced cancer patients. The detailed analysis of patients achieving sustained clinical benefits from these therapies illustrated the importance of a coordinated cellular and humoral anti-tumor immune response. Indeed, the extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs. Moreover, therapy induced a potent antibody response to several key tumor cell surface and secreted proteins; these antibodies manifest functional activity in vitro, antagonizing tumor cell survival, invasive potential, and angiogenesis.

Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg activity. While blockade of MFG-E8 can enhance therapeutic immunity in the context of cancer vaccines, loss of Treg homeostasis with MFG-E8 deficiency can lead to chronic inflammation. Mice deficient in GM-CSF and IFN-develop at high frequency germinal center derived lymphomas and nonsmall cell lung carcinomas. The pathogenesis of these tumors reflects a combination of tumor-promoting inflammation and a loss of protective tumor immunity. Taken together, these findings highlight dual roles for immunity in cancer pathogenesis and therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL04-03.
Abstract 1176: The expression of RN7SK paralogs in tumors of renal cell carcinoma is associated with patient survival
Authors: Zhang Xiaofan , Hildebrandt Michelle A. , Horikawa Yohei , Pu Xia , Wood Christopher G. , Gu Jian
Published: June 2012

Purpose: Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults, and also the most lethal type of genitourinary cancer. RN7SK RNA, a highly expressed non-coding RNA, was originally found to negatively regulate the transcription elongation factor P-TEFb, which can activate RNA polymerase II and pre-mRNA processing. Previous studies have shown the important role of this machinery in growth control and tumorigenesis. In human genome, eleven homologous non-coding RN7SK RNAs (http://www.noncode.org) have been speculated to possess similar function, but no experimental data have been reported. In this study, we determined the function of these RN7SK paralogs and evaluated their association with RCC development and prognosis.

Experimental Design: Co-immunoprecipitation (Co-IP) was performed to determine the involvement of these RN7SK paralogs in the transcription elongation using antibody of LARP7, a RN7SK specific binding protein. Then, 114 paired tumor tissues and corresponding adjacent normal tissues from histologically confirmed RCC patients were used to measure the expression level of these predicted RN7SK RNAs by real-time PCR. Associations with overall survival, recurrence, and stage were analyzed with adjustment for age, gender, race, smoking status, family hypertension, and family history of cancer.

Results: Co-IP results showed that eight of the eleven RN7SK paralogs were present in RN7SK ribonucleoprotein complex. Each detectable RN7SK RNA showed high expression level in both normal and tumor tissue, with significantly higher expression in tumor than in normal tissue except for n6233. High expression of n283 and n6233 was significantly associated with increased risk of death in RCC patients with hazard rations (HRs) of 3.23 (95% confidence interval [CI], 1.33-7.82; p=0.009) and 2.23 (95% CI; 1.03-4.79; p=0.041), respectively. Four other RN7SK paralogs were also significantly associated with survival, including n206 (p=0.022), n274 (p=0.037), n283 (p=0.007), and n6233 (p=0.014). No significant associations were identified for recurrence risk for any of the eight RN7SKRNAs. N133 (p=0.0005) and n139 (p=0.02) showed significantly different expression level between early (stage I&II) and late stage (stage III&IV) tumors.

CONCLUSIONS: Our results suggest that RN7SK paralogs may be involved in RCC tumorigenesis and the expression of select RN7SK may be valuable biomarkers of RCC prognosis.

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 1176. doi:10.1158/1538-7445.AM2011-1176
Abstract 1348: The discovery of novel inhibitors that downregulate human thymidylate synthase
Authors: Repalli Jayanthi , Chapman James , McInnes Campbell , Berger Sondra H.
Published: June 2012

Inhibitors of the enzyme thymidylate synthase (TS) are typically used as anti-cancer drugs to limit the growth of cancer cells. These inhibitors deplete TTP which is essential for DNA replication and repair. The enzyme in humans is postulated to undergo conformational switching between active and inactive forms. We are testing the hypothesis that stabilization of the inactive conformation will lead to increased turnover of hTS. Stabilization of hTS to degradation is observed after exposure to TS inhibitors in clinical use, which are active site-targeted. X-ray crystallographic studies of 1,3-propanediphosphonic acid (PDPA) bound to hTS led to the prediction that PDPA stabilizes the inactive conformation. Furthermore, PDPA exhibited competitive-noncompetitive behavior in TS catalytic reactions, consistent with allosteric inhibition. A cell-permeable isostere of PDPA inhibited hTS in catalytic assays, inhibited cell proliferation in a thymidine-dependent manner, and induced down regulation of hTS in cultivated cells. This is the first TS inhibitor to decrease the stability of hTS to degradation. To identify lead compounds that bind to the inactive conformation, molecular docking and structure-based virtual screening techniques were applied. Putative interaction sites were obtained from crystal structures of liganded hTS (with PDPA) and docked with 10,000 compounds using Ligand Fit (Drug Discovery Studio). Lead molecules with highest binding scores were selected and their efficacy analyzed in in vitro catalytic assays with hTS. Several compounds showed 30% – 50% inhibition of TS activity at 10 μM concentration. These derivatives were more potent than PDPA and inhibited the growth of cells expressing hTS, with IC50s of 100 µM – 200 µM. Data derived from computational and in vitro experiments are being employed in iterative cycles in the design of more potent, allosteric TS inhibitors.

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 1348. doi:10.1158/1538-7445.AM2011-1348
Abstract 1461: The heparan sulfate mimetic PG545 potently inhibits spontaneous lung metastasis and significantly enhances overall survival in the 4T1 breast carcinoma model
Authors: Dredge Keith , Hammond Edward , Handley Paul , Rowley Jessica , Vincent Christina , Brandt Ralf , Bytheway Ian
Published: June 2012

PG545 is a fully synthetic heparan sulfate (HS) mimetic entering Phase I clinical trials for advanced cancer. PG545 inhibits key processes in tumor progression (a) angiogenesis – by interfering with growth factor binding and (b) metastasis – by inhibition of heparanase activity. The syngeneic 4T1 breast carcinoma murine model is a well known model of spontaneous metastasis and PG545 was assessed for effects on primary tumour growth, metastatic development in lung and overall survival. PG545 and the tyrosine kinase inhibitor (TKI) sorafenib (used as a comparative antiangiogenic agent) were investigated for their effect on primary tumour growth and the number of lung metastases after treatment for two weeks. PG545 significantly inhibited solid tumour growth and potently inhibited the formation of lung metastases when administered on a twice-weekly dosing schedule (2xqw). Although sorafenib inhibited primary tumour growth to a similar extent as PG545, sorafenib significantly increased the number of lung metastases. In a separate study to investigate overall survival, mice had their primary tumours resected (mastectomy) on day 11 post-inoculation and treatment with PG545 (1xqw), sorafenib (1xqd) or cisplatin (1xqw) was administered for 55 days. Interim data on day 30 using satellite animals confirmed the potent antimetastatic activity of PG545 by enumeration of lung metastases. This reduction in metastasis correlated with overall survival in the PG545-treated mice in the main study which was significantly enhanced compared to vehicle control or sorafenib (which had a similar survival profile as control). PG545 also outperformed cisplatin. Mice culled due to clinical signs associated with disease progression correlated with histological assessment of metastatic burden in lung tissue. The antimetastatic effects of PG545 were confirmed by histological assessment at the end of the study on day 55, with very few metastases observed in PG545-treated mice. In conclusion, PG545 has potent anti-metastatic activity that would appear to differentiate it from other antiangiogenic therapies such as TKI's which have also been recently reported by other groups to aggravate metastatic development under certain circumstances.

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 1461. doi:10.1158/1538-7445.AM2011-1461
Abstract 1615: Gene expression profile predicts response to smoothened inhibitors in epithelial cancers
Authors: Galimberti Fabrizio , Busch Alex , Chinyengetere Fadzai , Ma Tian , Sekula David , Memoli Vincent , Dragnev Konstantin , Liu Fang , Andrew Angeline S. , Greninger Patricia , Robbins David , Settleman Jeff , Benes Cyril , Dmitrovsky Ethan
Published: June 2012

The hedgehog (HH) signaling pathway is important for embryonic development as it controls cell fate and proliferation. Deregulation of the HH pathway is linked to the onset and maintenance of multiple cancers. Clinical trials revealed promising effects of targeting the HH pathway with smoothened (Smo) inhibitors, particularly in basal cell carcinomas and medulloblastomas where HH pathway activation occurs. This study sought to uncover a gene signature that would predict response to Smo inhibitors, such as cyclopamine or SANT-1. A panel of 705 human cancer cell lines from diverse epithelial cancers was interrogated for sensitivity to cyclopamine. A subset of cancer cell lines exhibited substantial growth inhibition across a broad array of epithelial cancers. Sensitivity was significantly linked to high Gli1, high cyclin E, and low insulin-like growth factor binding protein 6 (IGFBP6) expression levels (P < 0.05). Cyclin E overexpression and IGFBP6 knockdown conferred increased sensitivity to either cyclopamine or SANT-1 Smo inhibitors in lung cancer cell lines. This functionally validated the importance of this gene signature. To ascertain the biological impact of this profile, cyclin E transgenic mice that spontaneously developed premalignant and malignant lung lesions were studied. These lung cancers expressed the gene signature profile that predicts sensitivity to Smo inhibitors. As expected, cyclopamine treatment of these transgenic mice caused substantial immunohistochemical repression of cyclin D1 and Ki-67 in both premalignant and malignant lung lesions. To confirm and extend these findings, cyclopamine antineoplastic effects were examined using a previously described murine syngeneic and transplantable lung cancer model. Cyclopamine treatment significantly repressed lung cancer formation in these mice (P < 0.01). Notably, a subset of examined human lung cancers was found to express the same gene signature that predicted response to Smo inhibitors. Taken together, these findings reveal a gene expression profile that predicts response of diverse epithelial cancers to Smo inhibitors. Future work should discern whether this same gene signature also predicts response to HH inhibitors in epithelial cancers of 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 1615. doi:10.1158/1538-7445.AM2011-1615
Abstract 1658: STAT5b is expressed in human pancreatic cancer and serves as a target for anti-neoplastic therapy
Authors: Lang Sven Arke , Moser Christian , Schenk Hedwig , Ruemmele Petra , Hackl Christina , Geissler Edward K. , Stoeltzing Oliver , Schlitt Hans J.
Published: June 2012

The transcription factor STAT5b (signal transducer and activator of transcription 5b) is implicated in tumor growth and metastases in various cancer entities including breast and prostate cancer. Activation of STAT5 signaling can be mediated via oncogene mutation (e.g. src kinase), cytokines (e.g. IL-6) and growth factors (e.g. EGF). So far, the role of STAT5b in human pancreatic adenocarcinoma (HPAC) has not been elucidated. Hence, HPAC tissue samples were investigated for nuclear STAT5b expression by immunohistochemical staining. Results show that nuclear expression of STAT5b can be detected in 42/80 HPAC tissue samples. Since specific inhibitors of STAT5b are currently not available, a shRNA approach was chosen for stable knock-down in human pancreatic cancer cells (HPAF-II, BxPC-3, L3.6pl). In vitro, no differences regarding tumor growth were detected by MTT assays and cell counts. However, knock-down of STAT5b led to significant reduction of tumor cell motility as determined in Boyden Chamber assays (P<0.05). Furthermore, upon stimulation with interleukin-6 (IL-6) and epidermal growth factor (EGF) as significant reduction in tumor cell migration was observed (P<0.05). In addition, expression of prometastatic factors Cav-1, MMP-2 and uPAR were impaired upon STAT5b blockade as determined by RT-PCR. In vivo, blockade of STAT5b significantly reduced tumor growth in subcutaneous tumor models (P<0.05). Immunohistochemical analyses of these tumors revealed no differences in tumor cell proliferation (BrdU) whereas vessel area (CD31) was significantly reduced (P<0.05). In conclusion, STAT5b is expressed in almost 50% of HPACs investigated in our study. Since blockade of STAT5b impairs tumor growth and cancer cell motility, this might be an interesting novel target for therapy of human pancreatic adenocarcinoma.

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 1658. doi:10.1158/1538-7445.AM2011-1658