bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 597: Bacteria targeting and tumor vascular destruction by S. typhimurium A1-R depend on the vascularity of the tumor site
Authors: Liu Fang , Zhao Ming , Zhang Lei , Hoffman Robert M.
Published: February 2011

Our laboratory has previously developed a tumor-targeting double-auxotrophic mutant of S. typhimurium termed A1-1 (Cancer Research 66, 7647-7652, 2006; Proc. Natl. Acad. Sci. USA 104, 10170-10174, 2007). The A1-R mutant has high tumor virulence but requires leucine and argentine which disables the bacteria from growing in normal tissue. Our previous studies suggested that the rich vascularity of tumors may contribute to bacterial tumor targeting by A1-R. The goal of the present study was to determine the effect of A1-R on the tumors growing at different sites with different vascularity. In this study, we administered A1-R to Lewis lung carcinoma-bearing mice with tumor growing at three different sites. We observed that tumor location influences the efficacy of targeting of A1-R to the Lewis lung tumor. RFP-expressing Lewis lung cancer cells (1×107 cells/ml) were transplanted subcutaneously in the ear (50 µl), back skin (100 µl), and foot pad (25 µl) of nestin-GFP transgenic nude mice, which selectively express GFP in nascent blood vessels. The tumor-bearing mice were then treated with A1-R bacteria (5×107) via tail-vein injection. Bacteria tumor targeting and vascular damage were evaluated by fluorescence imaging with the Olympus IV100 and OV100 small-animal imaging systems designed for fluorescence imaging. Tumors in the ear had the fastest growth rate and were the most sensitive to bacteria targeting and hemorrhaged severely the day after A1-R injection. Tumors growing in the back were the second most sensitive to bacteria targeting and appeared necrotic 3 days after A1-R injection. Tumors growing in the food pad were the least sensitive to A1-R. Tumor vascularity correlated positively with A1-R targeting with the ear being the most vascular site and the foot pad the least vascular site. The present study suggests that bacteria tumor targeting and hemorrhage of the tumor depend on the vascularity of the tumor site.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 597.
Abstract 6: A triple-helix forming oligonucleotide targeting a genomic locus is capable of sequence specific mutagenesis in human lymphoblastoid TK6 cells
Authors: Reshat Reshat , Priestley Catherine , Gooderham Nigel J.
Published: February 2011

It has been reported that DNA triplex formation induces mutagenesis as determined using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). Triplex mediated mutagenesis has been shown to include point mutations, deletions, small insertions and homologous recombination. To the best of our knowledge, no study has successfully examined the mutagenic potential of a non-conjugated triplex-forming oligonucleotide (TFO) targeting a genomic sequence. In this study, we have designed a TFO that targets the hemizygous hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus, in the human lymphoblastoid TK6 cell line, and assessed mutagenicity through resistance to 6-thioguanine.

Our TFO, TFO27, has been designed to form a triplex with a purine tract in exon 3 of the HPRT gene. Triplex formation at the target motif was shown to occur at nanomolar concentrations, confirmed by Electrophoretic Mobility Shift Assays. A scrambled oligonucleotide, SCR27, failed to form a triplex at the target motif. A range of transfection reagents were evaluated for facilitated delivery of TFO27, and resulted in variable cellular toxicity. Transfection of high concentrations of oligonucleotide, with acceptable levels of cytotoxicity, resulted in HPRT mutation with TFO27 but not SCR27. Similar experiments failed to result in mutation at the non-targeted thymidine kinase (TK) locus, suggesting locus specificity for the mode of action of TFO27. The target specificity and sequence context of these mutagenic events is being determined to establish the mechanism of mutation.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 6.
Abstract 60: Altering tumor cell metabolism may contribute to the antitumor activity of ethyl pyruvate
Authors: Liang Xiaoyan , de Vivar Chavaz Antonio Romo , Schapiro Nicole E. , Thorne Stephen H. , Wang Tao , van Houten Ben , Lotze Michael T. , de Vera Michael
Published: February 2011

Pyruvate, a key metabolite in cellular energy metabolism, is the end-product of glycolysis and the starting substrate for the TCA cycle which generates NADH and ATP during oxidative phosphorylation. According to the Warburg hypothesis, increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors. We have previously shown that ethyl pyruvate (EP), the ethyl ester of pyruvate, significantly inhibits the growth of hepatic tumors. The mechanism of this anti-tumor effect is unclear, and here we hypothesized that EP alters tumor cell metabolism. We analyzed the bioenergetic phenotype of tumor cells by measuring cellular respiration and glycolysis rate using a novel real-time assessment of oxidative phosphorylation and glycolysis.

METHODS: C57BL/6 mice were injected with 2×105 MC38 colorectal and PANCO2 pancreatic tumor cells via the portal vein. In vitro, 4x104 MC38 and PANCO2 cells/well were seeded into XF24 V7 microplates. Cells were pretreated with/without EP (5-10mM) for 60 minutes before measurement. The XF24 Analyzer (Seahorse Bioscience) was used to measure extracellular flux changes of oxygen consumption rate (OCR, measure of oxidative phosphorylation) and the rate of proton excretion into the media (extracellular acidification rate, ECAR, measure of glycolytic rate).

RESULTS: Mice injected with MC38 and PANCO2 cells resulted in the growth of bulky tumors in the liver. EP (80 mg/kg ip) administered 30 minutes prior to infusion of tumor cells and continuing daily for 9 days significantly inhibited tumor growth in a dose-dependent fashion, with 80 mg/kg EP achieving >70% reduction in the number of tumor nodules in comparison to untreated animals. TUNEL staining demonstrated a marked increase in tumor cell apoptosis in EP-treated mice. MC38 and PANCO2 tumor cells had substantially different bioenergetic phenotypes. MC38 cells had lower basal OCR (148 ± 13 vs 434 ± 32 pmoles/min, p< 0.05) and higher basal ECAR than PANCO2 cells (56 ± 6 vs 8 ± 2 mpH/min, respectively, p< 0.05). Use of 2-deoxyglucose (2-DG) to block glycolysis significantly enhanced OCR in PANCO2 from 437 ± 50 to 1486 ± 154 pmoles/min but not in MC38, suggesting that MC38 cells are more dependent on glycolysis. EP enhanced mitochondrial cellular respiration (OCR) in MC38 and decreased basal ECAR significantly without observable changes in PANCO2 cells, suggesting a reversal of the Warburg phenomenon. SUMMARY: Altering tumor cell metabolism by increasing mitochondrial oxidative phosphorylation and decreasing glycolysis may contribute to the antitumor activity of EP.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 60.
Abstract 604: Erucin treatment through induction of thioredoxin reductase may alter tamoxifen resistance in LCC2 cells
Authors: Penney Rosalind B. , Felty Quentin , Slingerland Joyce , Roy Deodutta
Published: February 2011

Tamoxifen-resistant growth is a considerable problem in the treatment of estrogen receptor-positive breast cancer. While the mechanisms of tamoxifen do not rely on an increase in oxidants, reactive oxygen species have been shown to increase with tamoxifen treatment, and tamoxifen-resistance coincides with a high intracellular oxidant state. We postulate that the chronic use of tamoxifen leads to a pro-oxidant environment in cells, leading to tamoxifen resistance. Here we present data that show that increasing a reduced state in the cell through an induction of thioredoxin reductase and subsequent reduction of oxidized thioredoxin, may restore tamoxifen sensitivity to previously resistant LCC2 cells. The isothiocyanate erucin has been found to increase thioredoxin reductase in MCF-7 cells. As such, we tested this in the MCF-7 derivative cell line LCC2, which is sensitive to fulvestrant but resistant to tamoxifen. We found that erucin not only increases expression of thioredoxin reductase, but also increased expression of thioredoxin in LCC2 cells. Erucin treatment led to a decrease in DNA synthesis, as measured by BrdU incorporation. In vitro tumor formation, as measured by colony count with soft agar colony formation assay, was also decreased in tamoxifen-treated cells after erucin treatment. Western blotting shows an increase in p27 with erucin treatment, and probing for p27 in a Jab1 immunoprecipitation shows a decrease in p27/Jab1 interactions after erucin treatment, similar to the effects of fulvestrant in these cells. These data support our hypothesis that altering thioredoxin's redox status can affect the sensitivity of tamoxifen to previously resistant cells, and this alteration may be through the p27/Jab1/thioredoxin pathway.

Supported by BCRP-funded project W81XWH-07-1-0417, BC060125.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 604.
Abstract 606: Heat shock protein 90 inhibitors: New mode of therapy to overcome aromatase inhibitor resistance
Authors: Wong Cynthie , Chen Shiuan
Published: February 2011

Results from several major trials have revealed that aromatase inhibitors (AIs) are superior to tamoxifen with regard to disease progression, incidences of locoregional and distant relapses, and contralateral breast cancers. Although AIs are highly effective drugs (with outstanding specificity and potency), unfortunately, a significant number of AI-treated breast cancer patients will eventually develop disease progression due to acquired resistance. Hormone refractory breast cancers result from complex molecular changes and are challenging to cure. Recently, we examined whether the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) can inhibit the growth of AI-resistant breast cancers and the mechanisms by which 17-DMAG affects proliferation. AI-responsive MCF-7aro and AI-resistant LTEDaro breast epithelial cells were used in this study. We observed that 17-DMAG inhibited proliferation in both MCF-7aro and LTEDaro cells in a dose-dependent manner. 17-DMAG induced apoptosis and G2 cell cycle arrest in both cell lines. Although inhibition of HSP90 decreased the levels of ERα, the ERα transcriptional activity was not affected when cells were treated with 17-DMAG together with estradiol. Moreover, detailed mechanistic studies suggested that 17-DMAG inhibits cell growth via degradation of HSP90 client proteins AKT and HER2. Furthermore, our experiments using AI responsive MCF-7aro cells have revealed that HSP90 inhibitors such as 17-DMAG and AIs such as letrozole can suppress the proliferation of hormone-dependent breast cancer cells in a synergistic manner.

To study de novo AI-resistance, our lab has generated MCF-7 cell models to overexpress both aromatase and either Akt (MCF-7Aktaro) or HER2 (MCF-7aroHER2). We have found that HSP90 inhibitor 17-DMAG is also effective on suppressing the proliferation of these de novo AI resistant cells. Collectively, results from this study provide data to support the theory that HSP90 inhibitors may be an effective therapy to delay and treat AI-resistant breast cancers (This research was supported by the NIH grant CA44735).

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 606.
Abstract 607: Loss of pigment epithelium derived-factor (PEDF) is associated with breast cancer progression and antihormone drug resistance
Authors: Wambi Joan S. , Kim Helen , Jordan V. Craig
Published: February 2011

Despite the benefits of antihormones in treating estrogen receptor (ER)-positive breast cancer, many tumors develop resistance. Pigment epithelium-derived factor (PEDF) is an endogenous antiangiogenic protein that is showing promise as a potential anticancer agent. PEDF is considered a potential tumor suppressor because its expression is very high in normal tissues but is loss or reduced in various types of malignancies. Previously, it was reported that PEDF expression was signficantly reduced in human breast cancer and that a lack of PEDF expression was a potent factor for the enhancement of tumor growth and angiogenesis. At present however, there are no studies which have examined whether PEDF plays a role in antihormone resistance. In this study, we have characterized the role of PEDF in breast cancer progression and assessed whether PEDF, through a possible interaction with the ER, plays a role in antihormone drug resistance. We found that PEDF expression was dramatically reduced in antihormone-resistant breast cancer cells compared to antihormone-sensitive cells and that reexpression of PEDF in these cells resensitized them to antiestrogens. In addition, we found that treatment of antihormone resistant breast cancer cells with recombinant PEDF significantly reduced the growth of these cells by inducing apoptosis and it reduced the ability of these cells to migrate and invade through an artificial basement membrane. Interestingly, we also found that PEDF expression was dramatically reduced in ER-negative versus ER-positive breast cancer cells; however, there was no significant correlation between PEDF expression and ER in our antihormone resistant cells which continued to expressed high levels of ER despite their PEDF-negative status. Overall, these observations support the hypothesis that a loss of PEDF expression plays an important role in antihormone resistance in breast cancer and they suggest that the presence of PEDF might be an important indicator of antihormonal responsiveness.

This work was supported by the NIH Career Development Grant K01CA120051-01A2, the American Cancer Society Grant IRG-92-027-14; and the Hollenbach Family Fund.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 607.
Abstract 608: HOXB7 as a key regulator in the development of tamoxifen resistance
Authors: Jin Kideok , Zhu Tao , Shah Tariq , Bhujwalla Zaver M. , Sukumar Saraswati
Published: February 2011

Multiple factors including long term treatment with tamoxifen are involved in the development of selective estrogen receptor modulator resistance of ERα positive breast cancer. It is well known in the endocrinology field that increased levels of ErbB/HER family members can also directly alter the cellular response to tamoxifen, but the mechanisms underlying increased expression of ErbB/HERs are not clear. In this report we show that HOXB7 is an ERα responsive gene and the downregulation of HOXB7 expression by estradiol is abrogated by tamoxifen in MCF7 cells. HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. EGFR and HER2 expression is upregulated by overexpression of HOXB7, while knockdown of HOXB7 with siRNA in tamoxifen-resistant cell lines causes decrease of EGFR and HER2 expression and loss of tamoxifen resistance. In addition, our work demonstrates that to mediate upregulation of EGFR and HER2, HOXB7 binds to ER-alpha and promotes HER2 transcription activity through direct binding to the HER2 enhancer region by competing with other cofactors. Also, HOXB7 enhances EGFR transcription activity via binding to EGFR promoter region. However, depletion of HOXB7 affects the binding affinity of HOXB7 and the cofactors to the HER2 enhancer element. Higher expression levels of HOXB7 significantly correlated with poorer disease free survival in ERα positive breast cancer patients on adjuvant tamoxifen monotherapy. These findings implicate overexpression of HOXB7 as a key event in the initiation and maintenance of tamoxifen resistance. These studies suggest that HOXB7 acts as a key regulator orchestrating two major groups of target molecules in the oncogene heirarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 608.
Abstract 610: Metformin augments rituximab and chemotherapeutic agents-induced apoptosis via mTOR inhibition in rituximab-resistant B lymphoma cell lines
Authors: Kim Min Kyoung , Yoo Eun Kyung , Kim Nam-Yi , Lee Kyung Hee , Hyun Myung Soo , Kang Min Kyu , Lee ChuHee
Published: February 2011

With elusive mechanisms, a subgroup of NHL patients who treated with rituximab in combination with cytotoxic chemotherapeutic agents becomes unresponsive. Patients who relapsed lymphoma are known to have poorer outcome when treated with rituximab compared with treated without rituximab. Since activation of mTOR pathway has been recently suggested for progression of NHL, especially in salvage setting, we'd like to examine the effect of metformin which inhibits mTOR pathway in rituximab-resistant NHL. Three lymphoma cell lines (Raji, Raji-2R, Raji-4RH) were used, which were kindly given by Dr Czuczman MS from Rosewell Park Cancer Institute (Ref. Czuczmana MS et al. Clin Cancer Res 2008;14:1561, 2008;14:1550). These cells were treated with cisplatin, adriamycin, vincristine and rituximab in the absence or presence of metformin for 24, 48, 72 hrs. Cell growth and proliferation was evaluated by MTT assay and cell cycle progression was determined FACS analysis. To characterize molecular mechanisms involved in metformin-induced inhibition of cell proliferation, the level of BAX, BAK, Bcl-2, Bcl-XL, Mcl-1, AMPK, pAMPK, pACC, mTOR, p70S6K and S6K was determined by western blot analysis.

Compared with rituximab-sensitive cell line (RSCL) which is parental lymphoma cell, Raji, rituximab-resistant cell lines (RRCLs), that are Raji-2R and Raji-4RH, have shown decreased cytotoxicity in response to chemotherapeutic agents including cisplatin, adriamycin and vincristine as well as rituximab. However, cytotoxic effect of these chemotherapeutic agents and rituximab was improved in combination of metformin in both RSCL and RRCLs. To us surprise, a better synergistic inhibition of cell proliferation was found in RRCLs as a result of combination of metformin and chemotherapeutic agents or rituximab, suggesting that intracellular signaling pathway induced by metformin might not be overlapped to that by chemotherapeutic agent and rituximab. Notably, it has known that in RRCLs, the level of proapototic proteins, BAX, BAK, is relatively low compared to in RSCL decreased, but the expression of anti-apoptotic protein and AMPK activity is increased and dysregulation of mTOR pathway is found. Our data showed that metformin restored dysregulation of mTOR pathway in RRCLs, since activation of p70S6K and S6K posphorylation was decreased. At the same time, expression of BAX and BAK was increased and that of anti-apoptotic proteins was decreased.

Metformin enhances rituximab-induced and chemotherapy-induced apoptosis via mTOR inhibition in rituximab-resistant NHL.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 610.
Abstract 614: ErbB-2 Suppresses Notch-1 Signaling by Regulating Jagged-1 Localization in ErbB-2 positive breast cancer
Authors: Mehta Kinnari , Meeke Kathleen , Patel Parul , Miele Lucio , Osipo Clodia
Published: February 2011

ErbB-2 is amplified and overexpressed in 20-30% of breast tumors. Today, ErbB-2 positive breast cancers are treated with trastuzumab plus chemotherapy. In spite of dramatic improvements in survival for women with ErbB-2+ breast tumors, resistance remains a major problem in metastatic disease. We have recently published that ErbB-2 suppresses Notch-1 activity and this is reversed by trastuzumab or a dual EGFR/ErbB-2 tyrosine kinase inhibitor similar to lapatinb. Furthermore, we demonstrated that trastuzumab resistance is reversed with a Notch-1 siRNA or a gamma-secretase inhibitor (GSI). Coincidentally, co-overexpression of Notch-1 and its ligand, Jagged-1, in breast cancer have been associated with the poorest overall survival. We know that activated Notch-1 plays an important role in ErbB-2 positive breast cancer and trastuzumab resistance; however, the precise mechanism by which Notch-1 is activated is not yet known. Thus, we hypothesized that overexpression of ErbB-2 inhibited expression and/or cell surface localization of Jagged-1 to suppress Notch-1 activity. This would be reversed in response to anti-ErbB-2 treatment. The results demonstrated that trastuzumab or lapatinib treatment of SKBr3 cells increased protein expression of Jagged-1 by Flow Cytometry. Notch-1 and Jagged-1 co-localized with early endosome antigen-1 (EEA-1) to submembraneous endosomes in ErbB-2 positive breast cancer cells as measured by confocal immunofluorescence. However upon trastuzumab treatment, Notch-1 and Jagged-1 exited EEA-1 positive vesicles and Notch-1 accumulated in the nucleus while Jagged-1 localized to the cell membrane. Cell surface biotinylation assays showed that Jagged-1 was enriched on the cell surface in response to trastuzumab treatment and this was mimicked by expression of K44A Dynamin. Cell surface accumulation of Jagged-1 was associated with an increase in Notch-1 transcriptional activity as measured by an increase in Deltex1 mRNA by real-time PCR. Conversely, knockdown of Jagged-1 by siRNA in SKbr3 and BT474 cells prevented the trastuzumab-induced increase in Deltex1 mRNA. Co-culture studies confirmed that culturing SKBr3 cells with fibroblast-expressing Jagged-1 or DLL4 increased Notch activity as measured by a CBF-1/Notch reporter assay. Taken together, these results suggest that ErbB-2 suppresses Notch-1 activity by retention of Jagged-1 or possibly other Notch ligands within early endosomes, which may be reversed by trastuzumab or lapatinib treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 614.
Abstract 618: Role of multidrug resistance transporters in the biological response to trastuzumab-cytotoxic drug conjugates
Authors: Guo Jun , Li Guangmin , Phillips Gail D. Lewis
Published: February 2011

Trastuzumab-DM1 (T-DM1, TMAb-mcc-DM1, trastuzumab emtansine) is an antibody-cytotoxic drug conjugate that is composed of the maytansine derivative DM1 directly coupled, via a thioether SMCC linker, to trastuzumab. T-DM1 is currently undergoing clinical evaluation in metastatic HER2-positive breast cancer patients. T-DM1 has been shown to retain all of the mechanisms of action of trastuzumab and to deliver DM1 to HER2-overexpressing tumor cells. In the present study, we investigated potential mechanisms of resistance to T-DM1 and other trastuzumab conjugates, including a different class of anti-microtubule drugs, the auristatins (MMAE and MMAF). Expression of selected multi-drug resistance (MDR) transporters MDR1, MRP1, MRP3 and BCRP was determined by qRT-PCR (Taqman). MDR1 was expressed at moderate levels by only one line, while expression of MRP3 occurred in several HER2-overexpressing cell lines. Cell viability assays with either free drugs (DM1 or MMAE) or the respective trastuzumab conjugates showed that all lines were sensitive to treatment with these agents. The NCI-AdrRes line, which expresses high levels of MDR1, was transfected to overexpress HER2 and used as a positive control. This line was resistant to treatment with DM1 or MMAE, consistent with previous reports that these drugs are substrates for MDR1. Additional studies were performed with the clinical conjugate, T-DM1, on the HER2-amplified breast cancer line SK-BR-3 transfected to express either MDR1, MRP1, MRP3 or BCRP, focusing on the clinical conjugate, T-DM1. In cell proliferation assays, both NCI-AdrRes/HER2 and SK-BR-3/MDR1 were resistant to free DM1, and the addition of XR9051 (a specific MDR1 inhibitor) reversed this resistance. Although the sensitivity to T-DM1 for both cell lines was restored by the addition of XR9051, NCI-AdrRes/HER2 showed more resistance to T-DM1 in the absence of XR9051. SK-BR-3 cells expressing MRP1 or BCRP maintained sensitivity to free and conjugated drugs, while cells expressing MRP3 showed a very modest induction of resistance only at high drug concentrations. The effects of linker stability were also investigated by comparing trastuzumab conjugates comprised of stable vs. cleavable (reducible or protease-sensitive) linkers. MDR1 expressing cells showed relatively more resistance to conjugates with cleavable linkers, presumably due to efflux of free drug upon release after linker cleavage. Together, these data show that, of the MDR transporters studied, only MDR1 confers high level resistance to trastuzumab-cytotoxic drug conjugates comprised of DM1 or auristatins, and that linking the drug through a stable linker may allow evasion of drug efflux through MDR-mediated mechanisms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 618.
Abstract 620: Addition of silibinin to EGFR-TKIs overcomes T790M-mediated drug resistance
Authors: Rho Jin Kyung , Choi Yun Jung , Choi Su Jin , Cheon Gi Jeong , Kim Hye-Ryoun , Kim Cheol Hyeon , Choi Chang-Min , Lee Jae Cheol
Published: February 2011

Although epidermal growth factor receptor EGFR tyrosine kinase inhibitors (EGFR-TKIs) produce an initially exhibit an initial dramatic response in lung cancer patients harboring a mutation in the EGFR gene, development of acquired resistanceis almost inevitable. The secondary mutation of T790M is associated with approximately one- half of the cases of acquired resistance. In this study, we investigated whether addition of silibinin to gefitinib or erlotinib could overcome T790M-mediated drug resistance considering that silibinin has various anti-tumor effects including EGFR modulation. Silibinin selectively reduced the activity of the EGFR family (EGFR, HER2 and ErbB3) in lung cancer cells with EGFR mutations, but not in those harboring wild type. In primary and acquired resistant cells with T790M, addition of silibinin enhanced the ability of EGFR-TKIs to down-regulate EGFR signals and to inhibit cell growth. Similarly, the combination of silibinin and erlotinib effectively suppressed the tumor growth in erlotinib resistance-bearing PC-9 xenografts. The results indicate that In conclusion, the addition of silibinin to EGFR-TKIs is can be a promising strategy to overcome T790M-mediated drug resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 620.
Abstract 621: High-dose gefitinib overcomes the resistance to gefitinib related to hepatocyte growth factor
Authors: Kashihara Hiromi , Ichihara Eiki , Takata Saburo , Ohashi Kadoaki , Kubo Toshio , Takeda Hiromasa , Fujii Masanori , Takigawa Nagio , Tanimoto Mitsune , Matsumoto Kunio , Kiura Katsuyuki
Published: February 2011

Molecular targeted agents are being a mainstay in non-small cell lung cancer. Dramatic responses to the epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitor (TKI) are observed in advanced non-small cell lung cancer (NSCLC), especially with EGFR mutation. Different from cytotoxic agents, it remains unclear whether EGFR-TKI exerts its anti-tumor effect dose-dependently. Gefitinib could not show survival benefit in previously treated unselected patients with NSCLC in a randomized phase III trial, whereas erlotinib did. The difference is considered resulting from the fact that erlotinib was dosed at its maximum-tolerated dose (MTD), while gefitinib was dosed at about one third of its MTD. To investigate the dose-dependency of gefitinib in the cells harboring mutated EGFR, we compared two doses of gefitinib (15 or 50 mg/kg) using PC-9 (adenocarcinoma of lung) xenografts. Both doses of gefitinib reduced tumors. However, the tumors regrew in the low-dose group after several weeks of treatment, whereas did not in the high dose group. This result indicates that gefitinib might have the dose-dependency in the tumors harboring mutated EGFR. We resected tumors at 10 weeks after gefitinib treatment. The removed tumors did not acquired T790M mutation and MET amplification. Interestingly, the cells (PC-9/RX) that were derived from the xenograft tumors treated with low-dose gefitinib remained the sensitivity to gefitinib. Thus, we investigated hepatocyte growth factor (HGF) as the environmental factor. Tumor tissues treated with gefitinib were positively stained with anti-mouse HGF. Mouse HGF mRNA and protein expression in the tumor tissues treated with gefitinib were significantly high when compared to those treated with vehicle alone, and similar results were obtained by ELISA. We confirmed that mouse HGF induced resistance to gefitinib in an ex vivo experiment. To elucidate the role of HGF in vivo, PC-9 xenografts were treated with gefitinib in combination with rabbit anti-HGF antibody. The combination of gefitinib with anti-HGF antibody inhibited tumor growth partially compared with the low-dose gefitinib alone. Taken together, HGF might induce resistance to low-dose of gefitinib treatment in an in vivo xenograft model before the tumor cells themselves acquired the resistant genetic changes.

In conclusion, higher-dose of gefitinib might prevent resistance to gefitinib related to HGF and produce longer response duration.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 621.
Abstract 625: Transcription factor slug conferring resistance To EGFR tyrosine kinase inhibitor gefitinib
Authors: Chang Tzu-Hua , Shih Jin-Yuan , Tsai Meng-Feng , Yang Chih-Hsin , Yang Pan-Chyr
Published: February 2011

Purpose: Somatic EGFR mutations of non-small cell lung cancer (NSCLC) are reported to be associated with clinical responsiveness to tyrosine kinase inhibitor gefitinib. Despite the dramatic responses to gefitinib, most patients ultimately have a relapse. The underlying mechanism of the drug resistance is not well clear. Recently, many studies have shown that epithelial to mesenchymal transition (EMT) is involved in resistance to gefitinib in EGFR. Zinc finger transcription factor slug is one of the EMT regulators. In this study, we will investigate whether the slug plays a role in the gefitinib resistance. Materials and Methods: PC9/IR (gefitinib-resistant cell line) was isolated from PC9 (gefitinib-sensitive cell line) after long-term treatment of gefitinib. To identify the factors involved in gefitinib resistance, we analyzed the expression levels of EMT regulators (Slug, Snail, Twist, Zeb-1) in PC9/IR and PC9 cells using quantitative real time PCR and western blot. To investigate the association of Slug and gefitinib-induced resistance, we also engineered to stably express Slug and short hairpin RNA targeting Slug transcript in PC9 and PC9/IR, respectively. Results: The findings showed that mRNA and protein levels of Slug obviously increased in PC9/IR comparing with PC9 and expression of other EMT regulators Snail, Twist, Zeb-1 were similar between these two cell lines. After gefitinib treatment, PC9/IR expressing shRNA against Slug underwent apoptosis and activation of the intrinsic apoptosis pathway. Ectopic expression of Slug protected PC9 cells from undergoing apoptosis in response to gefitinib treatment. Knockdown the expression of slug reversed gefitinib resistance of PC9/IR cells by restoring Bim expression. Conclusions: These results suggest that expression of slug may contribute to resistance to gefitinib in PC9/IR and may be a useful target for treatment of resistance to EGFR tyrosine kinase inhibitor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 625.
Abstract 626: Therapeutic effect of HGF inhibitors against HGF-induced EGFR-TKI resistance in lung cancer harboring EGFR mutations
Authors: Yano Seiji , Wang Wei , Li Qi , Donev Ivan S. , Takeuchi Shinji , Yamada Tadaaki , Matsumoto Kunio , Nishioka Yasuhiko , Sone Saburo
Published: February 2011

Introduction: Non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-activating mutations (EGFRmu) responds favorably to the EGFR tyrosine kinase inhibitors (EGFR-TKIs), gefitinib or erlotinib. However, the responders invariably acquire resistance to EGFR-TKIs. Two mechanisms, second-site point mutation that substitutes methionine for threonine at position 790 (T790M) in EGFR and amplification of MET proto-oncogene, which contribute to acquired resistance to EGFR-TKIs have been reported. As the third mechanism, we recently demonstrated that hepatocyte growth factor (HGF), specific ligand for MET, induced resistance to EGFR-TKIs. The purpose of this study was to examine whether inhibitor of HGF could overcome resistance to EGFR-TKIs induced by HGF in NSCLC cells with EGFRmu.

Results: HGF induced the resistance to gefitinib and erlotinib in lung cancer cells (PC-9 and HCC827) with EGFRmu, by restoring Akt phosphorylation through MET but not EGFR or ErbB3. These NSCLC cells also became resistant to EGFR-TKIs when co-cultured in vitro with HGF-producing fibroblasts and co-injected into SCID mice. Importantly, combined use of gefitinib plus anti-HGF antibody or the HGF antagonist, NK4 successfully overcame the fibroblast-induced EGFR-TKI resistance both in vitro and in vivo.

Conclusions: These findings indicate that HGF inhibitor, such as anti-HGF antibody and NK4, may be useful for controlling HGF-induced EGFR-TKI resistance in NSCLC harboring EGFRmu in humans.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 626.
Abstract 630: Interleukin-8 as a potential mediator for renal cell carcinoma resistance to antiangiogenic sunitinib therapy
Authors: Huang Dan , Ding Yan , Zhou Ming , Rini Brian , Petillo David , Qian Chao-Nan , Kahnoski Richard , Futreal P. Andrew , Furge Kyle A. , Teh Bin Tean
Published: February 2011

Sunitinib is considered the first line therapy for advanced clear cell renal cell carcinoma (ccRCC), a deadly form of kidney cancer. Unfortunately, most patients exhibit progressive disease after about one year of treatment. The mechanisms of progression are poorly understood. The aim of this study was to evaluate the mechanism of resistance to sunitinib and to identify potential targets to overcome sunitinib resistance. We generated xenograft models that mimicked clinical resistance to sunitinib. Higher microvessel density was found in sunitinib-resistant tumors, which indicated that an escape from anti-angiogenesis occurred in these tumors. This escape coincided with increased tumor secretion of interleukin-8 (IL-8) into the plasma. Co-administration of an IL-8 neutralizing antibody resensitized these tumors to sunitinib treatment, demonstrating the functional contribution of IL-8 to sunitinib resistance. Immunohistochemical staining showed that IL-8 expression was elevated in ccRCC tumors from patients who were refractory to sunitinib treatment, indicating IL-8 levels may predict clinical response to sunitinib. In conclusion, our results demonstrate that IL-8 is an important contributor, among others, to sunitinib resistance in ccRCC. IL-8 may serve as a therapeutic target for treatment of sunitinib resistant ccRCC, as well as a biomarker for both acquired and intrinsic sunitinib resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 630.
Abstract 631: Androgen receptor(AR) splice variants inhibit responses to IGF-IR inhibition in prostate cancer
Authors: Sun Shihua , Haugk Kathleen , Vessella Robert L. , Nguyen Holly , Soriano Kathryn , Plymate Stephen R.
Published: February 2011

Constitutively active splice variants of the androgen receptor (AR) are frequently expressed (59% of metastases) following castration in prostate cancer patients and are associated with earlier tumor recurrence compared to tumors containing only wild-type (wt) AR. In most tumors the variant is expressed along with wt-AR. When wtAR and variant AR are expressed in cells following castration there is a marked decrease in IGF type 1 receptor expression (IGF-IR), an AR target whose expression is increased by wtAR. In order to understand these findings we expressed the AR splice variant in which exons 5, 6, and 7 are skipped during AR splicing resulting in constitutively active ARv567es along with wtAR in a prostate cancer cell line not expressing AR, M12, as well as ARv567es in AR positive LnCaP cells. In both lines ARv567es was found only in the nucleus in the absence of ligand but wtAR had also been transported to the nucleus and absolute levels of AR protein were increased in the absence of ligand in cells expressing the variant. The increase in wtAR protein is due to decreased degradation of wtAR associated with ARv567es. When ligand was added, PSA response was increased 5-fold over cells transfected only with wtAR. Further, whereas transfection with wtAR enhances IGF-IR expression and response to IGF-I ligand, in the presence of ARv567es IGF-IR expression in response to IGF-I was suppressed. When ARv567es was HA tagged and expressed along with wtAR pull-down of ARv567es with HA resulted in concomitant precipitation of wtAR. Subsequent deletion analysis of ARv567es demonstrated direct binding between the two ARs in specific cystine-rich domains. Further, wtAR-induced IGF-IR expression is a non-genomic AR cytoplasmic action through binding to src and MAPK phosphorylation. Although ARv567es binds src, phosphorylation of MAPK does not occur. Finally, when LnCaP cells expressing ARv567es were grown sc with MatrigelTM in immuno-compromised mice there was no response to castration as opposed to slowed growth of LnCaP controls. These data indicate that the presence of the castration-induced ARv567es not only effects progression of prostate cancer by its own constitutive activity but also changes the function of the wt or full-length AR and provides an additional mechanism for AR-induced prostate cancer progression following castration.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 631.
Abstract 637: Filling the gap between preclinical and clinical oncology drug evaluation- Molecular profiling and PD biomarkers on HuPrimeTM tumors models
Authors: Chen Taiping , Song Xiaoming , Chen Dawei , Yu Shijun , Wery Jean-Pierre , Chen Yiyou
Published: February 2011

Most anticancer drug candidates, currently in clinical trials, have clear targets in molecular and cellular systems. However, such molecular targets are not always affected (or not correlated with efficacious endpoints) when the candidate is evaluated in a clinical setting. The gap between preclinical and clinical drug evaluation remains the bottle neck for anticancer drug development. There is an urgent need to develop better biological tools and models to ease the transition between in vitro and in vivo, and between preclinical and clinical settings. In our translational platforms, 170 human primary tumor models (HuPrimeTM) have been established directly from tumor fragments of the patients with many cancer types. In order to pick the best models for evaluation of targeted therapeutics, we have conducted a comprehensive molecular profiling of our models. This profiling includes Affymetrix whole genome expression, Illumina 1400 SNP of cancer related cancer genes for amplification, deletion, and allelic imbalance. Methylation statuses for selected gene sets were also analyzed by SuperArray platform. Genomic amplifications were further confirmed with protein analysis by western blot or immunohistochemistry. The models selected in this way have facilitated pharmacodynamic (PD) biomarker strategies. We have developed a unique platform combining our novel HuPrimeTM xenograft models with molecular profiling and PD biomarker exploration. We have now validated immunohistochemistry (IHC) assays on many molecules covering most signaling pathways that are involved in proliferation, apoptotic, necrotic, and cell cycle regulation, G2M phase arrest, DNA damage response, etc. Additional markers including angiogenesis have also been validated for certain therapeutic compounds. To illustrate the usefulness of our platform, we have applied it to understand the pharmacodynamic changes (at the molecular level) which are associated with the activity of Sunitinib, Sorafenib and Tarceva in several HuPrimeTM models. The PD biomarkers validated in this approach have potential for clinical application and patient stratification.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 637.
Abstract 639: Measuring PI3K and MAPK pathways inhibition, comparison between conventional IHC and AQUA-TMA technology
Authors: Prudkin Ludmila , Serra Violeta , Aura Claudia , Jimenez José , Scaltriti Maurizio , Baselga José
Published: February 2011

The incorporation of molecular biomarkers to assess therapy efficacy is crucial in both preclinical and clinical phases of drug development. New generation targeted therapy requires sensitive and robust analyses that accurately measure up- or down-regulation of specific pathways in response to a given therapeutic strategy. Paraffin embedded tissue is the sample commonly available in the clinic, and immunohistochemistry (IHC) has become the gold standard for biomarker detection. Although the reproducibility of this method has improved over time, IHC remains a semi-quantitative and subjective evaluation (H-score) of both intensity and expression extent of a given protein. These drawbacks can be by-passed by using automated quantitative analysis-tissue microarray (AQUA-TMA) technology. This platform analyzes double immunofluorescence (dIF) images and provides precise and objective staining quantifications using the AQUAnalysis software.

In this work we show a comparison between conventional IHC and AQUA-TMA technology in assessing the efficacy of Hsp90 inhibition, PI3K/mTOR inhibition and Akt inhibition in a HER2-driven breast cancer xenograft model measuring a number of biomarkers.

Both IHC and dIF techniques have been performed on TMAs and several molecular markers have been evaluated. Specifically, we have measured the levels of HER2, AKT and MAPK total proteins as well as phosphorylation of AKT, MAPK, PRAS40, p70-S6K and S6RP in response to the aforementioned therapies. Moreover, since the AQUAnalysis software also recognizes cellular and sub-cellular compartments of fluorescent signals, we investigated FOXO nuclear translocation.

Our results show that the AQUA technology adds more accuracy than IHC to the assessment of the studied biomarkers. TMAs are readily scanned and analyzed by the software, and the quantification values provided by the system are directly used for statistical analysis.

Our work suggests that, although IHC and H-Score is the current method of evaluation, an accurate and unbiased measurement of molecular markers using the AQUA-TMA platform can improve the assessment of target inhibition in both preclinical and clinical settings.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 639.
Abstract 642: Vimentin is a novel anticancer therapeutic target
Authors: Zhu Quansheng , Lahat Guy , Bolshakov Svetlana , Liu Jeffery , Torres Keila , Langley Robert R. , Lazar Alexander J. , Hung Mien Chie , Lev Dina
Published: February 2011

Vimentin is a ubiquitous mesenchymal intermediate filament supporting mechano-structural integrity of quiescent cells while participating in adhesion, migration, survival, and cell signaling processes via dynamic assembly/disassembly in activated cells. Soft tissue sarcomas and some epithelial cancers exhibiting “epithelial to mesenchymal transition” phenotypes express vimentin. Withaferin-A (WFA), a naturally derived bioactive compound, may molecularly target vimentin, so we sought to evaluate its effects on tumor growth in vitro and in vivo thereby elucidating the role of vimentin in drug-induced responses. WFA elicited marked apoptosis and vimentin cleavage in vimentin-expressing tumor cells but significantly less in normal mesenchymal cells. This proapoptotic response was abrogated after vimentin knockdown or by blockade of caspase-induced vimentin degradation via caspase inhibitors or overexpression of mutated caspase-resistant vimentin. Pronounced anti-angiogenic effects of WFA were demonstrated, with only minimal effects seen in non-proliferating endothelial cells. Moreover, WFA significantly blocked soft tissue sarcoma growth, local recurrence, and metastasis in a panel of soft tissue sarcoma xenograft experiments. Apoptosis, decreased angiogenesis, and vimentin degradation were all seen in WFA treated specimens. In light of these findings, evaluation of WFA, its analogs, or other anti-vimentin therapeutic approaches in soft tissue sarcoma and ”epithelial to mesenchymal transition” clinical contexts is warranted.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 642.