bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 796: Novel mechanisms of drug resistance in leukemia cells associated with changes in HoxA9 and microRNA expression
Authors: Ho Tsui-Ting , He Xiaolong , Beck William T.
Published: August 2012

The acquisition of resistance to anticancer drugs is a key obstacle to successful cancer therapy. An increasing number of studies have investigated the roles of microRNAs in drug resistance. We previously reported that the upregulation of miR-135b and miR-196b correlated positively with acquired drug resistance in the human T-cell leukemia cell line, CCRF-CEM (CEM), and that the elevation of expression of these two microRNAs in response to the DNA damaging agent etoposide appears to be histiotype-specific (T-T Ho et al, Proceedings of the 102nd Annual Meeting of the AACR 2011). To develop a mechanistic understanding of why these microRNAs are differentially expressed, we have studied miR-196b, which maps between the HoxA9 and HoxA10 genes on chromosome 7p15.2, suggesting that miR-196b and HoxA genes might be co-activated. Indeed, we found upregulation of HoxA9 mRNA after short-term (48 h) exposure of CEM cells to the topoisomerase II inhibitors etoposide and doxorubicin. Of note, we also found that HoxA9 is constitutively overexpressed in multidrug-resistant CEM/VM-1-5 cells. Of interest, we found that HoxA9 is clearly associated with drug resistance, as knockdown of HoxA9 using esiRNA (a heterogeneous mixture of siRNAs that all target the same mRNA) partially sensitized multidrug-resistant CEM/VM-1-5 cells to etoposide. This indicates that the expression of miR-196b is linked to HoxA gene transcription and to drug responsiveness. We subsequently confirmed that the expression of HoxA9 mRNA follows the same pattern in other leukemia cells. For example, both miR-196b and HoxA9 are upregulated in response to short-term etoposide exposure in HL-60 (acute promyelocytic leukemia), but not in solid tumor-derived MCF7 (breast cancer cell line) and its etoposide-resistant subline MCF7/VP, or Rh30 (rhabdomyosarcoma cell line) and its etoposide-resistant subline, Rh30/V1. We are presently assessing the role of HoxA9 in drug resistance in other leukemia cell lines and investigating the mechanistic basis for this effect. In summary, we report here a novel mechanism of anticancer drug resistance in leukemia cells that is associated with HoxA9 and specific changes in microRNA expression. This is the first report that HoxA9 may impact on anticancer drug resistance. (Supported in part by R01 CA40570 from NCI to WTB, and in part by UIC.)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 796. doi:1538-7445.AM2012-796
Abstract 799: Comprehensive proteomic analysis of cisplatin resistance in ovarian cancer
Authors: Teng Pang-ning , Hood Brian L. , Litzi Tracy , Chappell Nicole P. , Hamilton Chad A. , Maxwell G. Larry , Conrads Thomas P.
Published: August 2012

We have conducted a comprehensive proteomic analysis of an ovarian cancer cell line (A2780) and its cisplatin resistant daughter line (A2780-CP20) to identify key regulators and signaling pathways that confer cisplatin resistance. Quantitative proteomic profiling was conducted of defined biological compartments including that from global lysates, the nuclear fraction, the phosphoproteome and the secretome. Total cell lysates and nuclear fractions were harvested from A2780 and A2780-CP20 cultured in the presence or absence of cisplatin (3 µM) for 72 h. The secretome fractions were collected from A2780 and A2780-CP20 cultured in serum-free, phenol red-free media in the presence or absence of cisplatin (3 µM) for 24 h. Phosphopeptides were enriched from peptide digests of total lysates of A2780 and A2780-CP20 cultured in the presence or absence of cisplatin (3 µM) for 15 min using TiO2. Sample digests were analyzed by high-resolution LC-MS/MS that resulted in the identification of 2703, 2286, 1815, and 1362 proteins from the global, nuclear, secretome, and phosphoproteome fractions, respectively. Spectral counting was utilized to quantify the relative abundance of proteins identified. Nuclear proteins identified at elevated abundance in the cisplatin resistant A2780-CP20 including an array of chromatin remodeling proteins, such as SATB1, SATB2, coilin, AT-rich interactive domain-containing protein 3A (ARID3A), ARID3B, and ARID3Cwere validated by Western blot and qPCR in A2780/A2780-CP20 as well as other gynecologic cancer cell lines. Modulation of the abundance level of these candidates and the phenotypic impact on cisplatin resistance utilizing shRNA knockdown will be described. Differential proteins selected from the secretome analysis for western blot validation include Fras1-related extracellular matrix protein 2 (FREM2), stanniocalcin-1 (STC1), angio-associated migratory cell protein (AAMP), plasminogen activator inhibitor 1 (SERPINE1), high mobility group protein B1 (HMGB1), and chloride intracellular channel protein 2 (CLIC4), stromelysin-2 (MMP10), interstitial collagenase (MMP2), and CD166 antigen. Validated candidate protein targets from the secretome analysis will be tested by IHC on formalin fixed tissue sections as well as ELISA and multiple reaction monitoring (MRM) by mass spectrometry in serum from recurrent and non-recurrent ovarian cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 799. doi:1538-7445.AM2012-799
Abstract 802: A key role for ARF in drug resistance in invasive bladder cancer
Authors: Kobayashi Takashi , Delto Joan C. , Abate-Shen Cory
Published: August 2012

Although most superficial bladder cancers can be removed transurethrally with excellent prognosis, clinical outcome is much more problematic for patients with muscle-invasive disease. Indeed, invasive bladder cancer is a major clinical challenge since it is highly associated with postoperative recurrence and metastasis. Current treatments for those lethal forms of bladder cancer include systemic chemotherapy and molecular targeted therapy; however, survival is poor since most patients develop resistance to the drugs within a short timeframe. Clearly, there is a need to identify novel therapeutic options for invasive bladder cancer as well as a greater understanding of the molecular mechanisms of drug resistance. We have been investigating mechanisms of drug resistance using genetically-engineered mouse models of invasive bladder cancer based on the combinatorial deletion of p53 and Pten in bladder epithelium. p53 and PTEN are frequently inactivated in human bladder cancers, particularly those with poor prognosis. Indeed, allograft tumors established from these mice responded initially to the treatment with cisplatin, docetaxel, or rapamycin but eventually developed resistance. We have observed that these p53; PTEN deficient tumors express robust levels of p19Arf, while targeted deletion of Arf retards the aquisition of resistance following drug treatment. The significance of this expression was further suggested by analysis of a published gene expression profile of human bladder cancers which revealed that high Arf expression is an independent predictor of poor survival of invasive bladder cancer patients. Furthermore, following drug treatment in the mouse model, the Arf-positive, compared to the Arf-negative, tumors were significantly enriched in extracellular matrix/integrin signaling gene signature consistent with activation of downstream PI3K-mTOR pathway, suggesting that a model for drug resistance is via PI3K pathway activation. We have observed similar results in human bladder cancer cell lines either following knock-down of p14Arf in J82 cells that express endogenous p14Arf or following forced expression of p14Arf in ARF-negative UMUC3 human bladder cancer cells, and in both cases it was coincident with deregulated activation of PI3K-mTOR pathway. Thus, we propose that Arf confers drug resistance on bladder cancer by activating PI3K-mTOR pathway, highlighting a potential therapeutic target for advanced invasive bladder cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 802. doi:1538-7445.AM2012-802
Abstract 805: The role of aldo-keto reductases and estrogen signalling in acquired anthracycline resistance
Authors: Chewchuk Simon , Parissenti Amadeo M.
Published: August 2012

Breast cancer is the second leading cause of death from cancer in North American women. The disease can be difficult to treat by adjuvant or neoadjuvant chemotherapy because of intrinsic or acquired resistance to chemotherapy agents. To better understand the mechanisms for acquired resistance to the anthracycline class of chemotherapy agents in breast cancer, a series of MCF-7 breast tumour cell lines have been developed that exhibit progressive resistance to epirubicin (MCF-7EPI cells) or to doxorubicin (MCF-7DOX2 cells). Following mRNA microarray analysis, several genes were identified whose expression was associated with the acquisition of anthracycline resistance. Among these were several Aldo-keto Reductases (AKRs) responsible for estrogen synthesis from estrone and anthracycline detoxification to 13-hydroxymetabolites. AKR 1C3 showed more than a 10-fold increase in expression in anthracycline-resistant breast tumour cells. We hypothesized that the over-expression of AKR 1C3 would lead to both increased drug detoxification and an increase in estrogen synthesis. This would result in increased tumour cell survival and growth in the presence of anthracyclines. Recently, we observed that estrogen levels were, in fact, increased as cells became anthracycline-resistant. In contrast, the expression of estrogen receptor ≤ (ESR1) was reduced by 2- to 5-fold at the gene level and 2- to 20- fold at the protein level, with a concomitant reduction in the estrogen-dependent expression of Bcl-2 and Cyclin D1. Diminished Bcl-2 and cyclin D1 expression would be expected to reduce the growth rate of the cells, a hypothesis we have confirmed in cell culture experiments. Taken together, our findings suggest that selection for anthracycline resistance in breast tumour cells results in alterations in AKR expression, resulting in enhanced detoxification of doxorubicin, the promotion of estrogen biosynthesis, and the reduced estrogen-dependent expression of Bcl-2 and cyclin D1. The reduced expression of Bcl-2 and cyclin D1 blunts the growth rate of anthracycline-resistant cells, which would further increase the ability of these cells to survive in the presence of anthracyclines.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 805. doi:1538-7445.AM2012-805
Abstract 806: Extracellular matrix metalloprotease inducer (EMMPRIN) and CD44 protein complexes are exclusively formed in basal- and normal-like breast cancer cell lines
Authors: Timmermans Annemieke M. , Montazeri Hamed , Trapman-Jansen Anita M. , Martens John W. , Foekens John A. , Umar Arzu
Published: August 2012

Extracellular Matrix Metalloprotease Inducer (EMMPRIN) is a multifunctional glycoprotein that is involved in tumor cell invasion and metastasis, and is a predictor for tamoxifen resistance in breast cancer. EMMPRIN is implicated in development of therapy resistance by forming a functional transporter complex through interaction with CD44 and monocarboxylate transporter 4. Presence of EMMPRIN transporter protein complexes may therefore be a better predictor for therapy resistance and a direct target for drugs. In this study, we aimed to identify EMMPRIN-CD44 protein interactions in a large panel of breast cancer cell lines. EMMPRIN-CD44 protein interaction was investigated in a panel of 41 breast cancer cell lines, which represent the luminal (n=12), ErbB2 (n=13), basal (n=7), and normal-like (n=9) intrinsic subtypes, using in situ Proximity Ligation Assay (isPLA). In addition, protein expression of both EMMPRIN and CD44 in the cell lines was evaluated by immunohistochemistry (IHC). IHC and isPLA read-out was performed by bright field microscopy and 0-1-2-3 scoring was applied to none-weak-moderate-strong staining (IHC) or interaction (isPLA), respectively. Moderate and strong EMMPRIN expression was observed in 50% of luminal, 36% of ErbB2, 86% of basal, and 100% of normal-like cell lines, and moderate to high CD44 expression was detected in 64%, 83%, 86%, and 100% in luminal, ErbB2, basal, and normal-like cell lines, respectively. Interestingly, moderate to strong EMMPRIN-CD44 interaction was exclusively detected in basal (64%) and normal-like (67%) cell lines, whereas luminal and ErbB2 cells lines had no detectible, or only very weak, EMMPRIN-CD44 interaction (≥square = 23.5, P<0.0001). Our results indicate that EMMPRIN-CD44 protein complexes are formed exclusively in basal and normal-like breast cancer cell lines. These breast cancer subtypes are associated with an aggressive phenotype and poor patient survival. We speculate that active EMMPRIN-CD44 transporters are involved in drug resistance, resulting in poor clinical outcome, and will investigate this in more detail in clinical breast cancer specimens.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 806. doi:1538-7445.AM2012-806
Abstract 809: Prenatal and postnatal management of infants at 100%-risk for developing retinoblastoma
Authors: Chan Helen S.L. , Dimaras Helen , Héon Elise , Khetan Vikas , Gallie Brenda L.
Published: August 2012

Purpose: Prenatal diagnosis by amniocentesis of infants at 100%-risk of developing retinoblastoma (RB) is possible when the familial RB1 mutation is known in the parent. Since retinal tumors may develop in the third trimester, we assessed the success of a protocol of early delivery of such infants for early institution of therapy with the aim of optimizing outcome. Methods: Since 1999, we treated ten 100%-risk infants with early delivery in a Special Pregnancy Unit, at 32-37 weeks gestation and when deemed to be safe by their obstetrician. From birth, weekly dilated indirect fundoscopy, and/or contact imaging, and/or optical coherent tomography (OCT) examinations were performed with no sedation, with 3-weekly examinations under anesthesia (EUAs) only started once the first tumor was identified, or by the age of 3 months. The tumors were ablated by focal laser therapy and/or cryotherapy. In selected cases, subtenons topotecan (local chemotherapy that causes little side-effects systemically), was used to reduce the sizes of tumors that were too large, or near the optic nerve, or affecting the macula, for focal therapy ablation. When possible, low-dose or single-agent systemic chemotherapy was avoided to avoid induction of the multidrug resistance (MDR) P-glycoprotein in the tumors. If indicated, short-course but high-dose cyclosporin (CSA) MDR-circumventing high-dose carboplatin-etoposide-vincristine (CEV) chemotherapy was given as soon as the infant's immature renal function normalized. Secondary malignancy-inducing radiation and/or eye enucleation was only used for tumors progressing under this regimen. Results: At median followup of 5.7 years (range 0.7-11.7 years), both eyes were saved in 9 children, seven with 20/20-20/50 vision in at least one eye (two were too young for vision-testing), and only one child required radiation and then enucleation of one eye, but retained 20/30 vision in the remaining radiated eye. Five children were only treated with focal laser therapy and/or cryotherapy, two with added subtenons topotecan, four with 1 to 4-cycle CSA-modulated CEV, and one with 2-cycle non-CSA-modulated/dose-adjusted CEV because of immature renal function. Conclusions: This protocol warrants multicenter trial to confirm the favorable efficacy with low toxicity and excellent vision preservation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 809. doi:1538-7445.AM2012-809
Abstract 81: Role of GRM1 in breast cancer
Authors: Mehta Madhura S. , Dolfi Sonia , Bronfenbrener Roman , Bilal Erhan , Medina Daniel , Haffty Bruce , Toppmeyer Deborah , Ganesan Shridar , Hirshfield Kim M.
Published: August 2012

Despite epidemiologic studies suggesting a link between melanoma and breast cancer, molecular mechanisms leading to these associations have not been elucidated. Overexpression of metabotropic glutamate receptor 1 (GRM1) has been implicated in melanomagenesis. Its primary action occurs through activation of the MAPK signaling pathway and other pathways through intracellular calcium release. A recent study implicated GRM1 as playing a role in angiogenesis in hormone receptor negative breast cancer. However, the role of GRM1 in hormone receptor positive disease is yet to be described. Given the associations between melanoma and breast cancer, we evaluated three SNPs in GRM1 for genotype associations with breast cancer clinicopathologic variables using DNA isolated from peripheral blood of 1,096 breast cancer patients. Our study found that a SNP in GRM1 resulting in a proline to serine substitution correlates with age at diagnosis of breast cancer and reflects the receptor status of the breast cancer, e.g. for progesterone receptor positive (PR+) disease, proline carriers were diagnosed 8 years earlier than serine carriers. Genotype-specific associations with risk of developing specific molecular subtypes of breast cancer were observed, i.e. serine carriers were twice as likely to develop estrogen receptor positive (ER+) and PR+ disease. Furthermore, carrying the proline isoform also correlated with higher risk of recurrence of breast cancer and shorter time to recurrence. Immunohistochemical evaluation of breast microarrays showed that GRM1 expression is higher in breast cancers as compared to normal tissue and is higher in ER+ as compared to ER- breast cancers. This parallels higher expression observed in ER+ breast cancer cells lines in comparison to ER- lines. In vitro studies with breast cancer cell lines were also performed to evaluate the expression of GRM1 as a function of hormone exposure and cell line receptor status. Treatment of ER+ cell lines with estradiol resulted in increased GRM1, while tamoxifen decreased expression. Gene expression data on ER+ breast cancers treated with tamoxifen were reanalyzed for association between GRM1 expression and recurrence. Analysis of breast cancers treated with tamoxifen from an independent cohort demonstrated that high expressors of GRM1 have a worse distant recurrence free survival. The results of these studies implicate GRM1 in hormone receptor positive breast cancer biology. Additional knowledge regarding the interaction between GRM1 and hormone signaling could have significant implications in breast cancer risk, prevention, and treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 81. doi:1538-7445.AM2012-81
Abstract 821: Developing a molecular understanding of non small cell lung cancer (NSCLC) resistance to platin-taxane chemotherapy
Authors: Dalvi Maithili P. , Behrens Carmen , Suraokar Milind , Girard Luc , Xie Yang , Wistuba Ignacio , Minna John D.
Published: August 2012

Purpose: While chemotherapy has improved lung cancer patient survival, relapse rates after neoadjuvant or adjuvant chemotherapy (minimal residual disease settings) are high. This could be due to cancer cells acquiring resistance to therapy or selective survival of pre-existing resistant cells. The molecular basis for primary or acquired resistance to standard platinum doublet chemotherapies are unknown and their elucidation is necessary for developing curative therapy that can be personalized for each patient. Methods: 3 complementary approaches were used to explore mechanisms underlying NSCLC resistance to platin-taxane chemotherapy: A) NSCLC lines were exposed in vitro to cycles of paclitaxel + carboplatin to mimic clinical treatment and resistance validated with cell viability and colony formation assays. Genome wide mRNA expression profiles of resistant and parental lines were compared using Illumina arrays and qRT-PCR. B) A large dataset of clinically and molecularly annotated NSCLC tumor specimens obtained after no chemotherapy or neoadjuvant platin-taxane chemotherapy were subjected to mRNA microarray analysis. C) Lung cancer xenografts were treated with chemotherapy in vivo in mice and profiled for mRNA expression. Biostatistical and bioinformatics approaches were used to identify genes that are differentially expressed in resistant cells in these three settings, and their function studied using over-expression or shRNA mediated knockdown. Results: NSCLC lines H1299 and H1355 were treated in vitro with paclitaxel + carboplatin and showed progressive increases in resistance to chemotherapy (after 5 to 15 cycles) ultimately achieving 30 (H1299) or 60 fold (H1355) differences compared to parental cells. In addition, multiple single cell clones also demonstrated chemotherapy resistance. Resistant NSCLC strains were also resistant to platin+docetaxel and platin+pemetrexed combinations. Expression profiles revealed upregulation of ABCB1/MDR1, down regulation of epithelial-specific genes suggesting EMT, differences in expression of epigenetic modulators as well as cancer testis family of GAGE genes and many of these genes had expression correlated with recurrence after neoadjuvant chemotherapy and with poor survival. Resistance was only partially reversed by verapamil indicating that it can be only partly explained by MDR mechanisms. Mechanistic studies of genes identified by these three approaches provide opportunities for overcoming chemotherapy resistance and developing novel therapies for NSCLC. Grant support: UT-Lung SPORE (5 P50 CA70907-11) and DoD PROSPECT (W81XWH-07-1-0306)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 821. doi:1538-7445.AM2012-821
Abstract 83: Differential regulation of PTEN in ERα positive and negative ovarian cancer cells during cisplatin induced apoptosis
Authors: Singh Mohan , Chaudhry Parvesh , Asselin Eric
Published: August 2012

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study we have investigated the regulation of PTEN levels during cisplatin induced apoptosis in estrogen receptor alpha (ERα) positive (OVCAR-3 and SKOV-3) and negative (A2780 and A270-CP) ovarian cancer cell lines. Cells were treated with 10μM cisplatin for 24hrs. Transcript and protein levels were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting, respectively. The results showed that PTEN levels were significantly decreased in ERα-negative A2780 cells at both the transcript and protein levels; however, there was no change in PTEN levels in ERα-positive cells and cisplatin resistant cells (A2780-CP) in the presence of cisplatin. The decrease in PTEN was accompanied with an increase in the levels of Akt phosphorylation in A2780 cells. Cisplatin treatment induced the activation of caspase-3, -7, -8, -9 and cleavage of Parp in all the cell lines tested in the study except A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-VAD-fmk (caspase inhibitor) and not with MG132 (proteasome inhibitor), suggesting that, in addition to the regulation of PTEN at transcript level, caspases are also involved in the decrease of PTEN protein levels in A2780. The decrease in pro-apoptotic PTEN levels both at the transcriptional and post-translational levels and increase in survival factor pAkt in ERα-negative ovarian cancer cells suggest that cisplatin treatment could further exacerbates drug resistance in ERα-negative ovarian cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 83. doi:1538-7445.AM2012-83
Abstract 834: Inhibition of Gli1 results in altered c-jun activation, inhibition of cisplatin-induced up-regulation of ERCC1, XPD, and XRCC1, and inhibition of platinum-DNA adduct repair
Authors: Gavin Elaine J. , Kudo Kenji , Das Shamik , Amable Lauren , Shevde Lalita A. , Reed Eddie
Published: August 2012

The transcription of ERCC1 and other nucleotide excision repair genes is strongly influenced by c-jun. C-jun is transcriptionally regulated by Gli proteins of the Hedgehog pathway. We therefore studied the possible relationships between Gli1, c-jun, and the upregulation of ERCC1, XPD, and XRCC1 in cisplatin-resistant human ovarian cancer cells. We studied the paired human ovarian cancer cell lines A2780 and A2780-CP70. We used a shRNA construct that specifically degrades Gli1 message; which has been previously published by one of the co-authors (Shevde-Samant et al., 2009). Genes we assessed for mRNA and/or protein levels included: c-jun, ERCC1, XPD, XRCC1, Gli1, Gli2, SHH, IHH, GAPDH, and β-tubulin. Platinum-DNA adduct repair was assessed by atomic absorbance spectrometry with Zeeman background correction. Use of the anti-Gli1 shRNA in cisplatin-resistant cells, resulted in a block of the cells’ ability to up- regulate genes in response to cisplatin treatment, including: c-jun, ERCC1, XPD, and XRCC1. This block in upregulation of c-jun was concurrent with a change in the phosphorylation pattern of the c-jun protein, shifting that pattern from a Ser63/73 dominant pattern, to a Thr91/93 dominant pattern. A2780-CP70 cells were treated at their cisplatin IC50, and DNA repair was assessed after pretreatment with anti-Gli1 shRNA or scrambled shRNA control. Control cells repaired 78% of platinum-DNA adducts at 12 hours; compared to 33% repair in cells pretreated with anti-Gli1 shRNA; a 2.4 fold difference. Pretreatment of A2780-CP70 cells with anti-Gli1 shRNA resulted in supra-additive cell killing with cisplatin; shifting the cisplatin IC50 from 30 µM to 5 µM. Pretreatment of these cells with cyclopamine, did not shift the cisplatin IC50. We conclude that the transcriptional protein Gli1 is important in the upregulation of these three DNA repair genes in human ovarian cancer cells. And that Gli1 thereby strongly influences platinum-DNA adduct repair, and cellular sensitivity to cisplatin. This Gli1 role has c-jun as an intermediate in the pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 834. doi:1538-7445.AM2012-834
Abstract 838: A metabolomic analysis revealed the involvement of deoxycytidine kinase and CTP synthase in the sensitivity of human pancreatic cancer cells to gemcitabine
Authors: Nishi Koji , Fujimura Yurika , Nishimuta Akito , Sugimoto Masahiro , Soga Tomoyoshi , Tanigawara Yusuke
Published: August 2012

Gemcitabine (GEM), a pyrimidine nucleoside analogue, is used in patients with pancreatic cancer as the standard chemotherapeutic agent. However, pancreatic cancer cells often show resistance to GEM in vivo and vitro. In this study, we explored the factors involved in the sensitivity of GEM in human pancreatic cancer cells, Miapaca-2 and AsPC-1, by a metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The sensitivity of Miapaca-2 (IC50: 66 nM) to GEM was higher than that of AxPC-1 (IC50: 16 nM), whereas there were little differences in the growth rates of these cells. This result indicates that the difference in the sensitivities between Miapaca-2 and AsPC-1 is not dependent on their growth rates. At 12 hr after adding GEM, intracellular level of GEM in Miapaca-2 was higher than that in AsPC-1, whereas levels of GEM monophosphate, diphosphate and triphosphate in Miapaca-2 were lower than that in AsPC-1. In addition, expression level of deoxycytidine kinase (dCK), which catalyzes the reaction from GEM to GEM monophosphate, in Miapaca-2 was lower than that in AsPC-1. Expression levels of human equilibrium transporter (hENT1), which is known to major transporter of GEM, were similar in both cells. These results suggest that dCK is rate-limiting enzyme in GEM phosphorylation and one of the factors involved in the sensitivity. In pyrimidine metabolism, GEM was observed to inhibit CTP synthase, which converts UTP to CTP, resulting increase in UTP level, decrease in CTP level and depletion of dCTP in both cells at 12 hr after adding GEM. The degree of increase in UTP and decrease in CTP was lower in Miapaca-2 than that of AsPC-1. Indeed, the ratio of UTP to CTP in Miapaca-2 was about 2.5, whereas that of AsPC-1 was about 11. In addition, the expression level of CTP synthase in Miapaca-2 was higher than that in AsPC-1. Ribonucleotide reductase, which catalyzes the conversion of CDP to dCDP, is known to be inhibited by GEM diphosphate and be involved in the sensitivity of pancreatic cancer cells to GEM. GEM decreased CDP in AsPC-1. This may be caused by inhibiting CTP synthase. Expression level of ribonucleotide reductase in Miapaca-2 was higher than that in AxPC-1. Therefore, ribonucleotide reductase also may be involved in the sensitivity to GEM. Furthermore, we previously found that GEM mainly inhibited CTP synthase in pyrimidine metabolism, resulting in depletion of dCTP. Our current study suggests that dCK, CTP synthase and ribonucleotide reductase are involved in the sensitivity of pancreatic cancer cells to GEM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 838. doi:1538-7445.AM2012-838
Abstract 84: Evidence supporting a role of FKBP51 in melanoma pathogenesis
Authors: Srivastava Sanjeev K. , Arora Sumit , Bhardwaj Arun , Scammell Jonathan G. , Owen Laurie B. , Grizzle William E. , Singh Ajay P. , Singh Seema
Published: August 2012

Melanoma accounts for the majority of skin tumor-related deaths worldwide. The tumors originate from melanocytes and are associated with risk factors such as ultraviolet radiation exposure, fair skin type, and predisposing gene mutations. Although there has been some progress in early melanoma diagnosis, current therapeutic modalities still fail frequently in treating this deadly malignancy once it has metastasized. FKBP51 is a high molecular weight FK506-binding protein involved in the regulation of diverse biological processes. Importantly, it has been shown to be overexpressed in melanoma, activate anti-apoptotic NF-kB signaling, and inhibit apoptosis in irradiated melanoma cells. In this study, we investigated the role of FKBP51 in melanoma pathogenesis in a set of in vitro experiments. FKBP51 was stably repressed in two metastatic melanoma cell lines, A375-SM and FEMX-1, through short-hairpin RNA (shRNA)-mediated silencing and its effect on melanoma phenotype was examined. Silencing of FKBP51 led to a decrease in growth, clonogenicity, motility and invasiveness of both the melanoma cell lines, whereas enhanced cell-cell interaction was observed. Immunoblot analysis demonstrated loss of mesenchymal markers N-cadherin, vimentin, Slug and Snail and gain of epithelial marker, E-cadherin in FKBP51-silenced A375-SM and FEMX-1 cells, indicating a role of FKBP51 in epithelial to mesenchymal transition (EMT) of melanoma cells. Interestingly, in FKBP51-silenced melanoma cells, we also observed a decreased production of interleukin-8 (IL-8/CXCL-8), a chemokine that has earlier been shown to promote melanoma growth, metastasis and angiogenesis. Altogether, these studies provide experimental evidence for a functional role of FKBP51 in altered growth and malignant behavior of the melanoma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 84. doi:1538-7445.AM2012-84
Abstract 842: Proteomics-based comparative analysis of mitochondria isolated from human cisplatin-sensitive and resistant ovarian carcinoma cell lines
Authors: Chappell Nicole P. , Teng Pang-ning , Hood Brian L. , Hamilton Chad A. , Maxwell G. Larry , Conrads Thomas P.
Published: August 2012

Epithelial ovarian cancer (EOC) is the leading cause of death among women with gynecologic malignancies and the fifth leading cause of cancer death in women in the United States. Cisplatin is one of the most effective and active agents in the treatment of advanced EOC, however, development of chemoresistance is a major obstacle. A recurrent theme is emerging that mitochondria and mitochondrial dysfunction are centerpiece in contributing to an aggressive carcinogenic phenotype. Similarly, we hypothesized that changes in the mitochondrial proteome are required to support development of cisplatin resistance in human EOC. We applied an organellar proteomics approach to elucidate quantitative alterations in protein abundance in mitochondria enriched from an isogenic pair of cisplatin sensitive (A2780) and resistant (A2780-CP20) human EOC cells. Protein isolates from mitochondria were analyzed by high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and quantitative determination of changes in the relative abundance level of identified proteins was accomplished by spectral counting. This analysis confirmed that there are substantial alterations in the mitochondrial proteomic composition in cisplatin resistant EOC cells. Differential proteins selected for western blot validation include hypoxia up-regulated protein 1 (HYOU1), activated leukocyte cell adhesion molecule (ALCAM), isoform 1 of dynamin-like 120kDa protein (OPA1), A kinase anchoring protein 12 (AKAP12), and ferredoxin reductase (FDXR). Our proteomic analysis demonstrated overexpression of HYOU1, ALCAM, AKAP12, and OPA1 in the resistant EOC cells. This panel of protein candidates have been implicated in other tumor types and are involved in relevant cell mechanisms to include evasion of apoptosis, increased tumor invasiveness, tumor hypoxia, cellular respiration, and mitochondrial fragmentation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 842. doi:1538-7445.AM2012-842
Abstract 854: Patient stratification strategies for NVP-BGJ398, a selective pan-FGFR inhibitor in phase I clinical trials
Authors: Porta Diana Graus , Guagnano Vito , Stamm Christelle , Woehrle Simon , Kauffmann Audrey , Yao Yao , Brümmendorf Thomas , Bordas Vincent , Douget Mickaël Le , Zhang Yun , Chen Zhi , Ito Moriko , Li Fang , Monahan John , Gaither Alex , Borawski Jason , Wilson Christopher J. , Venkatesan Kavitha , Hofmann Francesco , Sellers William
Published: August 2012

The mammalian fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) family comprises 22 FGF ligands, which exert their action through 4 highly conserved transmembrane tyrosine kinase receptors (FGFR1, FGFR2, FGFR3 and FGFR4). This highly complex signaling network regulates fundamental developmental pathways and physiological processes in the adult organism. A growing body of evidence linking deregulation of the FGF/FGFR system, as a consequence of genetic alterations, to the pathogenesis of cancer makes these receptors attractive targets for therapeutic intervention. To this purpose we have developed NVP-BGJ398, an orally bioavailable, selective pan-FGFR kinase inhibitor which is currently in clinical Phase I dose escalation trial. In order to preclinically identify and validate patient stratification biomarkers that accurately predict for response to NVP-BGJ398 in the clinic, we have utilized the Cancer Cell Line Encyclopedia (CCLE). The CCLE is a collection of almost 1000 cancer cell lines representing multiple tumor types that, in a collaborative effort between The Novartis Institutes for BioMedical Research and the Broad Institute, has been comprehensively annotated in terms of genome-scale mRNA expression, gene copy number alterations and gene mutations (http:/www.broadinstitute.org/ccle). In addition, over half of these cell lines were subjected to high-throughput cell viability assays in the presence NVP-BGJ398, with the aim of generating cell line sensitivity data. Here we show the validation of predictive biomarkers of response in primary human tumor xenografts, revealed through the analysis of the CCLE, as well as the identification of potentially new indications for NVP-BGJ398.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 854. doi:1538-7445.AM2012-854
Abstract 86: Inhibition of oncogenic Pim kinases, including Pim-2, alters cell growth and signaling in pancreatic cancer
Authors: Austin Dana M. , Nandiwada Vijaya B. , Baldino Carmen M. , Caserta Justin , Lee Chee S. , Flanders Yvonne , Dumas Stephane , Baines Antonio T.
Published: August 2012

Pancreatic ductal adenocarcinoma (PDAC) is the most common cancer of the pancreas, comprising over 85% of all cases. PDAC has a relative 1-year survival rate of 20% and a 5-year survival rate of 4%. A better understanding of the molecular mechanisms leading to the development of pancreatic cancer remains a major goal for defining appropriate treatment strategies and earlier detection. Past studies have shown that members of the oncogenic Pim kinase family (Pim-1, Pim-2, and Pim-3) are aberrantly expressed in a variety of solid tumors, including pancreatic cancer. Currently, only Pim-1 and Pim-3 have been shown to be anti-apoptotic and aberrantly expressed in PDAC. Although members of the Pim kinase family have been shown to have a high degree of homology at the amino acid level, past studies have demonstrated functional differences among the members of the Pim family. To better understand the significance of these observations, we have investigated the role of Pims, including Pim-2, in growth transformation of pancreatic cancer. We hypothesize that inhibition of Pim-2 will be effective in antagonizing the aberrant growth of PDAC. Initially, we confirmed by western blot analysis that Pim-2 is aberrantly expressed in PDAC tissues and cell lines. Also, we found that inhibition of Pim-2 expression by shRNA resulted in decreased cell viability of PDAC cells. Additionally, inhibition of PIm-2 expression resulted in decreased anchorage-independent growth. These effects were further demonstrated by inhibiting Pim activity using novel small molecule Pim inhibitors developed by Jasco Pharmaceuticals that inhibit Pim-2 activity in the picomolar range. To begin to determine a mechanism for this growth inhibition, we studied the activity of various downstream substrates of Pim kinases in PDAC cells such as 4EBP-1. Overall our research continues to validate the role of Pim-2 in growth transformation, as well as further support Pim-2 as a viable target for drug therapy in pancreatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 86. doi:1538-7445.AM2012-86
Abstract 861: Selective small-molecule kinase inhibitors of the c-Src:FAK complex
Authors: Moroco Jamie A. , Gray Nathanael S. , Smithgall Thomas E.
Published: August 2012

The c-Src protein-tyrosine kinase is the prototype of a large non-receptor kinase family involved in a variety of signaling pathways in virtually every cell type. In normal cells, c-Src controls cell-cell adhesion, cell-matrix adhesion, and cell motility by interacting with and phosphorylating focal adhesion and extracellular matrix (ECM) proteins. Focal adhesion kinase (FAK), a key component of focal adhesions, activates c-Src by binding to its SH2 and SH3 domains and disrupting their negative regulatory influence on the kinase domain. The c-Src:FAK complex plays a major role in the migration and invasion of both normal and cancer cells. Both c-Src and FAK are over-expressed and constitutively active in many tumor types, enhancing focal adhesion turnover, cell migration and metastatic potential. The goal of our project is to determine whether distinct conformational changes occur in the c-Src kinase domain as a function of binding to FAK that can be selectively targeted with small molecule inhibitors. Using the FRET-based Z’-Lyte in vitro kinase assay, we first identified assay conditions where recombinant, downregulated c-Src activation was dependent on a synthetic phosphopeptide based on the FAK SH3- and SH2-binding motifs for c-Src. We then screened a chemical library of about 600 kinase-biased inhibitors for compounds that preferentially inhibited FAK-peptide-dependent c-Src activation compared to c-Src alone. Remarkably, 13 compounds showed at least five-fold selectivity for the FAK peptide:Src complex in the primary screen. Several of these compounds are of the Type II kinase inhibitor class and may exploit unique conformations of c-Src that are induced by FAK peptide binding to the SH3 and SH2 region of c-Src. These compounds are currently being evaluated for their impact on cell growth, invasion and migration in both fibroblasts and epithelial tumor cell lines. Discovery and development of selective inhibitors for a unique FAK-induced c-Src conformation may provide a new approach to targeted therapy for metastatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 861. doi:1538-7445.AM2012-861
Abstract 868: Oral SHP-1 inhibitor TPI-1a4 increases TILs in melanoma tumors and interacts positively with CTLA4 antibody for melanoma cure in mice
Authors: Yi Taolin , Lindner Daniel , Tuthill Ralph , Loibner Hans , Borden Ernest
Published: August 2012

SHP-1 phosphatase is a promising target for developing inhibitors as novel cancer immune therapeutics given its key role in negative regulation of immune cells. TPI-1a4 is a small molecule SHP-1 inhibitor identified recently and demonstrated activity for melanoma and colon cancer tumors in mice as a tolerated single oral agent. TPI-1a4 has been further evaluated herein for mechanism of action and potential clinical translation. Oral TPI-1a4 increased (up to 8x) in TILs in K1735 melanoma tumors in syngeneic mice in a dose-dependent manner and in correlation with tumor growth inhibition. The growth of 4-day established K1735 tumors (s.c.) in syngeneic C3H/HeJ mice was inhibited 44% (p < 0.05) by TPI-1a4 at 0.4 mg/kg (5 d/wk, gavage) or 90% (p < 0.01) at 0.8 mg/kg (5 d/wk, gavage) during a treatment period of 4 wks. Evaluation of tumor histology (H.E.) revealed increases (2x and 4x for the two treatments) of tumor-infiltrating lymphocytes (TIL). Immunohistochemistry analyses of the tumor samples demonstrate increases (∼3 and 6x) of CD45+ cells (pan-leukocytes), CD3+ cells (T lineage), B220+ cells (B/NK lineages), and Mac3+ cells (myeloid lineages). Oral TPI-1a4 interacted positively with CTLA4 antibody for survival extension and cure in K1735 melanoma tumor mice. TPI-1a4 (1 mg/kg, 5d/wk, gavage) or CTLA4 antibody (9H10, 100 ug x3, ip) as individual agents delayed tumor growth for ∼1-4 wks in C3H/HeJ mice with 4-day established K1735 tumors (s.c.). Their combination delayed tumor growth for 4-14 wks (4/5 mice) or completely blocked tumor growth (1/5 mice) by 20th wk in association with resistance to re-challenging with the melanoma tumor (s.c.). The combination was more effective in C3H/HeJ mice with 1-day established K1735 tumors, blocked tumor growth in most mice (4/5) comparing to mostly delayed tumor growth for several wks by the individual agents. TPI-1a4 in drinking water ad lib with weekly renewal was effective against the melanoma tumors in mice, suggesting chemical stability. Indeed, mass spectrometry verified structural integrity of TPI-1a4 stored as solid compound or in solvent at room temperature for 2 months. These data provide further evidences designate TPI-1a4 as a novel anti-cancer immune agent and a promising candidate for clinical translation. Our studies demonstrate for the first time the potential of TPI-1a4 to improve the clinical efficacy of CTLA4 antibody and other related cancer therapeutics. Future studies of TPI-1a4 analogs and derivatives are warranted and may lead to more refined and effective SHP-1 inhibitors and immune cell activators.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 868. doi:1538-7445.AM2012-868