Insensitive Cells Research Articles

Medullary serotonin neurons are CO2 sensitive in situ

Brainstem central chemoreceptors are critical to the hypercapnic ventilatory response, but their location and identity are poorly understood. When studied in vitro, serotonin-synthesizing (5-HT) neurons within the rat medullary raphé are intrinsically stimulated by CO2/acidosis. The contributions of these neurons to central chemosensitivity in vivo, however, are controversial. Lacking is documentation of CO2-sensitive 5-HT neurons in intact experimental preparations and understanding of their spatial and proportional distribution. Here we test the hypothesis that 5-HT neurons in the rat medullary raphé are sensitive to arterial hypercapnia. We use extracellular recording and hypercapnic challenge of spontaneously active medullary raphé neurons in the unanesthetized in situ perfused decerebrate brainstem preparation to assess chemosensitivity of individual cells. Juxtacellular labeling of a subset of recorded neurons and subsequent immunohistochemistry for the 5-HT-synthesizing enzyme tryptophan hydroxylase (TPH) identify or exclude this neurotransmitter phenotype in electrophysiologically characterized chemosensitive and insensitive cells. We show that the medullary raphé houses a heterogeneous population, including chemosensitive and insensitive 5-HT neurons. Of 124 recorded cells, 16 cells were juxtacellularly filled, visualized, and immunohistochemically identified as 5-HT synthesizing, based on TPH-immunoreactivity. Forty-four percent of 5-HT cells were CO2 stimulated (increased firing rate with hypercapnia), while 56% were unstimulated. Our results demonstrate that medullary raphé neurons are heterogeneous and clearly include a subset of 5-HT neurons that are excited by arterial hypercapnia. Together with data identifying intrinsically CO2-sensitive 5-HT neurons in vitro, these results support a role for such cells as central chemoreceptors in the intact system.

Oncogenic Herpesvirus HHV-8 Promotes Androgen-Independent Prostate Cancer Growth

Mechanisms underlying progression to androgen-independent prostate cancer following radical ablation therapy remain poorly defined. Although intraprostatic infections have been highlighted as potential cofactors, pathogen influences on pathways that support tumor regrowth are not known. To explore this provocative concept, we derived androgen-sensitive and -insensitive prostate epithelial cells persistently infected with human herpesvirus 8 (HHV-8), an oncogenic herpesvirus that has been detected in normal prostate epithelium, prostate adenocarcinoma, and biologic fluids of patients with prostate cancer, to explore its effects on transition to hormone-refractory disease. Strikingly, we found that HHV-8 infection of androgen-sensitive prostate cancer cells conferred the capacity for androgen-independent growth. This effect was associated with altered expression and transcriptional activity of the androgen receptor (AR). However, HHV-8 infection bypassed AR signaling by promoting enhancer of zeste homolog 2 (EZH2)-mediated epigenetic silencing of tumor-suppressor genes, including MSMB and DAB2IP that are often inactivated in advanced disease. Furthermore, we found that HHV-8 triggered epithelial-to-mesenchymal transition. Although HHV-8 has not been linked etiologically to prostate cancer, virologic outcomes revealed by our study provide mechanistic insight into how intraprostatic infections could constitute risk for progression to androgen-independent metastatic disease where EZH2 has been implicated. Taken together, our findings prompt further evaluations of the relationship between HHV-8 infections and risk of advanced prostate cancer.

Strengthening Context-Dependent Anticancer Effects on Non–Small Cell Lung Carcinoma by Inhibition of Both MET and EGFR

The MET and EGFR receptor tyrosine kinases (RTK) are often coexpressed and may cross-talk in driving the development and progression of non-small cell lung carcinoma (NSCLC). In addition, MET amplification is an alternative resistance mechanism for escaping EGFR-targeted therapy. To assess the benefits of combined targeting of MET and EGFR for treating NSCLCs, we investigated the activities of these two RTK pathways in NSCLC cell lines and evaluated their responses to SGX523 and erlotinib, the small-molecule kinase inhibitors of MET and EGFR, respectively. We showed that MET interacts with and cross-activates EGFR in MET-amplified or -overexpressed cells. The inhibition of both MET and EGFR results in maximal suppression of downstream signaling and of cell proliferation when their ligands are present. Furthermore, we showed that SGX523 plus erlotinib strengthens anticancer activity in vivo in a cellular context-dependent manner. The combination led to the regression of H1993 tumors by enhancing the suppression of proliferation and inducing apoptosis, whereas H1373 tumor growth was significantly reduced by the combination via suppression of proliferation without inducing apoptosis. SGX523 alone was sufficient to achieve near-complete regression of EBC-1 tumors; its combination with erlotinib strongly inhibited the viability of a population of insensitive cells emerging from an SGX523-treated EBC-1 tumor recurrence. Our data suggest that inhibition of both MET and EGFR can enhance anticancer effects against NSCLCs in a context-dependent manner and thus provide a strong rationale for combining MET and EGFR inhibitors in treating NSCLCs.

Blocking transforming growth factor-β signaling pathway augments antitumor effect of adoptive NK-92 cell therapy

Natural killer (NK) cells hold great potential for improving the immunotherapy of cancer. However, existing data indicate that tumor cells can effectively escape NK cell-mediated apoptosis through immunosuppressive effect in the tumor microenvironment. Transforming growth factor-β (TGF-β) is a potent immunosuppressant. The present study was intended to develop a treatment strategy through adoptive transfer of TGF-β insensitive NK-92 cells. To block TGF-β signaling pathway, NK-92 cells were genetically modified with dominant negative TGF-β type II receptor (DNTβRII) by optimizing electroporation using the Amaxa Nucleofector system. These genetically modified NK-92 cells were insensitive to TGF-β and able to resist the suppressive effect of TGF-β on Calu-6 lung cancer cells in vitro. To determine the antitumor activity in vivo, recipient mice were challenged with a single subcutaneous injection of Calu-6 cells. Adoptive transfer of TGF-β insensitive NK-92 cells decreased tumor proliferation, reduced lung metastasis, produced more IFN-γ, and increased the survival rate of nude mice bearing established Calu-6 cells. Hence, we have demonstrated that blocking transforming growth factor-β signaling pathway in NK cells provides a novel therapeutic strategy and warrants further investigation.

A gene expression profile indicative of early stage HER2 tyrosine kinase inhibitor response.

e11536 Background: Lapatinib, afatinib and neratinib are tyrosine kinase inhibitors (TKIs) of HER2 and EGFR growth receptors. A panel of breast cancer cell lines was treated with these agents and gefintib (EGFR inhibitor) and the expression pattern of a specific panel of genes investigated as a potential marker of early drug response. Methods: RNA was extracted from breast cancer cell lines (BT474, SKBR3 and MDAMB453) with differing HER2 expression patterns and sensitivity to lapatinib before and 12hrs after treatment with 1 µM of lapatinib, 150nM of afatinib, 150nM of neratinib or 1µM of gefitinib. Gene expression changes were measured by Taqman RT-PCR and RQ values were determined using the comparative cycle threshold (Ct) method. Results: The expression of RB1CC1, ERBB3, FOXO3a, NR3C1 was directly correlated with the degree of sensitivity of the cell line to lapatinib and was observed to “switch” from up-regulated to down-regulated in the HER2 expressing lapatinib insensitive cell line (MDAMD453). The CCND1 gene (functionally linked to the other four genes) demonstrated an inversely proportional response to drug exposure; showing a trend of strong down-regulation in lapatinib-sensitive lines. A similar expression pattern was observed following the treatment with both neratinib and afatinib. In contrast, gefitinib treatment, resulted in a completely different expression pattern change. Conclusions: In these HER2-expressing cell models, lapatinib, neratinib and afatinib treatment generated a common, characteristic and specific gene expression response, proportionate to the sensitivity of the cell lines to the HER2 inhibitor. Characterisation of changes in these genes shortly after drug treatment may therefore give a valuable predictor of the likely extent and specificity of tumour HER2 inhibitor response in patients, potentially guiding more specific use of these agents.

Abstract 5455: Differential regulation of E2A (TCF3) by androgens in prostate cancer cells.

Abstract Introduction: E2A (TCF3) is a multifunctional basic helix loop helix (bHLH) transcription factor. E2A promotes cell differentiation, acts as a negative regulator of cell proliferation in normal cells and cancer cell lines and is required for normal B-cell development. Previous studies from our laboratory has shown that E2A expression is highly increased in prostate cancer as compared to normal prostate and that it acts as a tumor promoter in prostate cancer. Given the diverse biological pathways regulated/ influenced by E2A little is known about its regulation in prostate cancer. Experimental design: E2A expression in androgen sensitive LNCaP and insensitive C81 prostate cancer cell lines was determined by western blot after treatments with androgen receptor (AR) agonist R1881 and antagonist casodex. Putative Androgen Response Elements (ARE) were identified in the first intronic region of the E2A gene using some of the online bioinformatics tools and confirmed by Chromatin Immunoprecipitation (ChIP) with AR antibody and Luciferase reporter assays on the above mentioned cell lines after treatments with R1881 and casodex. Results: E2A expression was found to increase with the increasing aggrasiveness of prostate cancer cell lines as compared to normal prostate epithelial cell line RWPE1. When androgen responsive cell line LNCaP was treated with R1881 there was an increased expression of E2A which decreased upon treatment with antiandrogen casodex whereas the E2A expression remained unaltered upon similar treatments in androgen insensitive cell line C81. The first intronic region of the E2A gene was predicted to contain two putative ARE sites. ChIP after treatment of LNCaP and C81 cells with R1881 and casodex showed that the intronic region was bound by AR in LNCaP cells only in the presence of R1881, whereas C81 cells showed a pulldown with AR in presence as well as absence of R1881. Similar results were observed in luciferase reporter assays indicating that E2A is transactivated by AR in LNCaP cell lines whereas it is independent of androgens in C81 cell line. Furthermore, Luciferase reporter assays also confirmed that only one of the two predicted putative AREs was functionally active and responsible for the androgen mediated regulation of E2A. Conclusion: Our results indicate that E2A is differentially regulated in Prostate cancer cell lines. The increased expression of E2A and its role as a tumor promoter in prostate cancer cell lines may be contributed to its loss of androgen dependence as is evident in its progression from androgen dependent LNCaP to independent C81 cells. Acknowledgements: This study was supported by NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12RR03062. Citation Format: Divya Patel, Jaideep Chaudhary. Differential regulation of E2A (TCF3) by androgens in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5455. doi:10.1158/1538-7445.AM2013-5455

Abstract 965: Establishment of a Sunitinib resistant cell line from ccRCC by long term drug application.

Abstract Introduction: Sunitinib (SUN) acts as a multitargeted tyrosine kinase inhibitor (TKI) in the treatment of advanced renal cell carcinoma. It addresses predominantly the neo-angiogenic pathway by inhibition of proliferation and induction of apoptosis. But in clinical practice patients fail therapy. To elucidate the mechanism behind the failure of SUN we started to investigate the efficacy of SUN in long term cell culture studies using permanent cell lines representing ccRCC. Material and Methods: CAKI-1/2, A-498 and 786-O cell lines, normal fibroblasts and HUVEC were subjected to different concentrations of SUN for up to 5 days. Efficacy of SUN was monitored by quantification of viability (WST-1), apoptosis (CDD+), necrosis (LDH+) and proliferation (BrdU). Under long term SUN-treatment growth behavior was assessed by cell counting spanning a cultivation period of several weeks. miRNA profiling was performed by microarray analysis at different point within this schedule. Results: ccRCC cell lines offer a decrease in viability down to 40-60% at 2-5μM/L SUN dependent on cell-type and cell concentration as a result of f proliferation-inhibition. Endothelial cells and fibroblasts react more sensitive showing a growth inhibition between 80-90% compared to untreated controls. Long term application of SUN to these ccRCC-cells results in SUN-insensitive cell clones offering no inhibition of proliferation at 2-5μM/l drug. Specific miRNA expression changes were identified in insensitive cells. Conclusion: Drug resistance by a long term SUN treatment provides cell lines to study the molecular mechanisms underlying this phenomenon. We have shown that resistance is associated with miRNA alterations. 3D-combination of these cell lines together with endothelial cells and fibroblasts offer a powerful tissue-like model to develop new therapeutic schedules to overcome the failure of actual treatment. Citation Format: Gerhard Unteregger, Simone Krein, Anne Weiland, Michael Stoeckle, Kerstin Junker. Establishment of a Sunitinib resistant cell line from ccRCC by long term drug application. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 965. doi:10.1158/1538-7445.AM2013-965

Abstract 1620: Dovitinib has anti-tumor activity in gastrointestinal stromal tumor (GIST) cell lines.

Abstract Background: Gastrointestinal stromal tumors (GISTs) are the most common gastrointestinal tract sarcoma, with 5000-6000 cases per year diagnosed in the United States. Most GISTs carry activating mutations in KIT (≈ 75% of cases) or PDGFR alpha (10%-15%). Imatinib, a KIT/PDGFR inhibitor, is the frontline treatment for GIST, although secondary mutations commonly lead to acquired resistance. Dovitinib inhibits several kinase targets important in the development and maintenance of GISTs, including KIT, PDGFR, VEGFR, and FGFR. This study was designed to evaluate the single-agent activities of imatinib and dovitinib in a panel of GIST cell lines and an imatinib-sensitive mouse model. Methods: In vitro sensitivity was examined in both imatinib-sensitive (GIST-T1 and GIST882) and -insensitive (GIST430 and GIST48) cell lines. KIT phosphorylation was measured by Western blot and sandwich ELISA. Imatinib and dovitinib monotherapy were evaluated for tumor growth inhibition and delay in a nu/nu mouse xenograft model using GIST-T1 xenografts. Dovitinib was administered by mouth at either 60 mg/kg daily for 14 days or 30 mg/kg daily for 21 days. Imatinib was given at 100 mg/kg by mouth twice daily. Following treatment, dosing was terminated, and tumors were allowed to regrow off treatment. Kaplan-Meier analysis was used to determine tumor growth delay, reported as median time to reach endpoint (tumor volume ≥ 1000 mm3). Results: Imatinib and dovitinib inhibit KIT phosphorylation in GIST-T1, GIST882, GIST430 and GIST48 cells, although imatinib appeared to be more potent. Dovitinib and imatinib both potently inhibited the proliferation, GI50 10-100 nM, of imatinib-sensitive GIST-T1 and GIST882 cell lines; GI50 was > 500 nM in imatinib-insensitive GIST48 and GIST430 cell lines. In vivo, imatinib and dovitinib were well tolerated for 14- and 21-day treatment cycles. In the 14-day treatment model, imatinib and dovitinib led to 52% and 59% tumor regression, respectively, and median time to regrowth was 69 days for both agents compared with 31 days for vehicle. In the 21-day treatment model, imatinib and dovitinib led to 66% and 36% tumor regression, respectively, and median time to regrowth was 81 and 69 days, respectively, compared with 27 days for vehicle. Conclusions: Dovitinib inhibits KIT signaling and has similar growth-inhibition activity to imatinib in imatinib-sensitive GIST cell lines. In xenograft models, both imatinib and dovitinib are well tolerated, induce tumor regression, and delay tumor regrowth. The additional kinase inhibitory activities of dovitinib offer the possibility for enhanced or differentiated activity to imatinib in certain settings. Experiments are ongoing to explore this possibility. These data suggest that dovitinib may have therapeutic benefit in GIST, with efficacy resulting from a combination of the inhibition of KIT and other kinases. Citation Format: Joseph D. Growney, Fang Li, Shumei Qiu, Bella Gorbatcheva, Linda Battalagine, Juergen Mestan, Paul Manley, Matthew Squires, Alex Cao, John E. Monahan. Dovitinib has anti-tumor activity in gastrointestinal stromal tumor (GIST) cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1620. doi:10.1158/1538-7445.AM2013-1620

Abstract 4389: E2F7 regulates sensitivity to the cytotoxic actions of anthracyclins in squamous cell carcinoma.

Abstract Squamous cell carcinoma (SCC) of the skin and head and neck region is a common and potentially fatal malignancy. Current chemotherapy treatments are generally not curative. Therefore, there is considerable need to develop new and targeted therapies to treat SCC. There is abundant data to show that E2Fs play an important role in many KC functions such as differentiation, growth and apoptosis. However, the identification of two new inhibitory forms of E2F, E2F7 and E2F8 has caused a rethink of our previous models. In this study, we examined the effects of E2F1, E2F7 and E2F8 on cytotoxic responses to chemotherapeutic agents. For in vitro experiments, we generated murine KCs from E2F7 fl/fl or E2F8 fl/fl mice that were made deficient in E2F7 and E2F8 following infection with Cre expressing adenoviral constructs. In addition, we had KCs generated from E2F1 KO mice. Our results showed that E2F8 deficient KCs displayed unaltered responses to 3 structurally different classes of chemotherapeutics represented by doxorubicin (anthracycline), cisplatin (platinum) or etoposide (topoisomerase II inhibitor). Similarly, E2F1 and E2F7-deficient KCs retained apoptotic responses to etoposide and cisplatin. In contrast, E2F7-deficient murine KCs were selectively sensitised to the cytotoxic actions of doxorubicin whilst E2F1-deficiency protected against doxorubicin. We showed that the sensitivity to doxorubicin was E2F7 specific since the reintroduction of E2F7 in E2F7 deficient KCs had a clear protective effect against doxorubicin. These results indicate that E2F7 regulate cytotoxic responses of SCC to doxorubicin, and doxorubicin toxicity is mediated, in part, by an E2F1-dependent mechanism. We also looked at the possible mechanism to drive E2F7-mediated doxorubicin induced cell death. Our results showed activation of caspase-3 in E2F7 deficient KCs mediates the doxorubicin-induced reduction in cell viability. Since E2F7 modulates sensitivity to doxorubicin, we looked at different SCC cells in their response to cytotoxic action of doxorubicin. The results suggested that SCC cells vary in their sensitivity to doxorubicin and this correlates with E2F7/E2F1 protein expression ratio in these SCC cells. The most sensitive SCC cell lines, KJD, had a very low E2F7/E2F1 ratio whilst the most insensitive SCC cell line, SCC25, had a very high E2F7/E2F1 ratio. These data showed that E2F7/E2F1 ratio is disrupted in SCC and this contributes to the insensitivity to cytotoxic actions of doxorubicin. Combined, these data indicate the existence of a novel, non-redundant and isoform-specific activities of E2F7 in KCs that have implications for our understanding of chemotherapeutic sensitivity in SCC. Our findings would suggest that if we sensitise SCC to the effects of anthracyclines, this would have a significant clinical value and add to therapeutic options available to treat SCC patients where there is a critical need. Citation Format: Mehlika Hazar Rethinam, Nicholas Saunders, Fiona Simpson. E2F7 regulates sensitivity to the cytotoxic actions of anthracyclins in squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4389. doi:10.1158/1538-7445.AM2013-4389

Abstract 3041: A tumor-suppressive role of the IL-21R/Jak/STAT3 pathway in the germinal center B cell subtype of diffuse large B cell lymphomas.

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common type of B cell malignancy in the adult population. Based on their patterns of gene expression, DLBCLs can be divided into two major subgroups: the germinal center (GC) B cell like (GCB-DLBCL) and the activated B cell like (ABC-DLBCL) cases. Current studies suggest that the cell-of-origin for both subgroups is a normal GC B cell. The difference lies in the degree of maturation. While the GCB variant corresponds to the GC centroblasts that abundantly express BCL6 with no NF-kappaB activation, the ABC variant phenotypically resembles activated GC centrocytes and plasma cell precursors that have received activating signals (e.g. IL-21 and CD40L) from follicular T helper (Tfh) cells and thus contain activated STAT3 and NF-kappaB due to mutations in a high percentage of cases. Although it is commonly accepted that pathogenesis of B cell lymphomas such as DLBCL is intimately linked to impaired B cell differentiation, a direct examination of this concept has not been made for GCB-DLBCL. In a previous study, we showed that the IL-21R/Jak/Stat3 signaling pathway plays a critical role in post-GC plasma cell differentiation, while concurrent signals transduced through the CD40/NF-kappaB axis potentiate the activity of Jak/Stat3 through two distinct mechanisms. In the current study, we report that IL-21-triggered Stat3 activation is often defective in GCB-DLBCL cell lines. Impaired STAT3 activation results in failure to commit to the plasma cell fate as evidenced by lack of Blimp-1 induction. The IL-21 insensitive cell lines either have very low levels of IL-21R or lack the Jak3 protein. Aberrant expression of the components of the IL-21R/Jak/STAT3 signaling pathway can also be detected in subsets of primary GCB-DLBCL samples. Lentivirus mediated reconstitution of IL-21R and Jak3 in the defective cell lines restored IL-21 responsiveness and induced a delayed cell death phenotype, indicating that low level expression of a single signaling molecule is responsible for the IL-21R signaling defect in a given cell line. We have sequenced the coding regions of the IL21R, Jak3 and STAT3 genes in 3 cell lines, but did not find any mutations. Subsequent analyses showed that the IL-21R abnormality is a transcriptional defect associated with Sp1 expression changes, while silencing of the Jak3 gene is caused by promoter methylation. Collectively, our results suggest that in GCB-DLBCL, the IL-21R/Jak/STAT3 pathway plays a novel, tumor suppressive role, and its epigenetic inactivation may promote lymphoma development by blocking terminal B cell differentiation. Citation Format: B. Hilda Ye, Enguang Bi, J. Jessica Yu, Yun Mai, B. Belinda Ding, Yiting Yu, Amit Verma, Rita Shaknovich, Wing C. Chan. A tumor-suppressive role of the IL-21R/Jak/STAT3 pathway in the germinal center B cell subtype of diffuse large B cell lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3041. doi:10.1158/1538-7445.AM2013-3041

Abstract 4655: In vitro tyrosine kinase activity profiling to identify molecular targets and predictive biomarkers in gastric cancer cell lines.

Abstract Background: Gastric cancer is among the leading causes of cancer-related deaths worldwide, notably in Asia. Though targeted therapies have been under clinical evaluation, the biological complexity of this cancer type may confer resistance to single-targeted therapies. Especially, scirrhous phenotype is known to be affected by multiple growth factors. To overcome this biological complexity, multi-targeted kinase inhibitors (MTKIs) have been clinically tested and were reported to be effective in a subset of patients. We wanted to elucidate the kinase activity signature of gastric cancer and to explore predictive biomarkers of MTKIs using gastric cancer cell lines by tyrosine kinase activity profiling. Material and methods: The growth-inhibitory effect of the MTKIs sunitinib, sorafenib, pazopanib and the Akt inhibitor MK-2206 was evaluated by the MTT assay. 11 scirrhous and 14 non-scirrhous gastric cancer cell lines were grown until semi-confluent, lysed, aliquoted and stored at -80°C until use. The basal tyrosine kinase activity of the lysates was measured on PamChip® peptide micro-arrays, containing 144 peptides derived from known human phosphorylation sites. Quantification of peptide phosphorylation and data analysis were performed using BioNavigator and SIMCA P+ software. Results: HSC-39, HSC-40A, KATO-III and HSC-43 scirrhous cells and SNU-16 non-scirrhous cells were sensitive to all three MTKIs with IC50 values < 1 μmol/L. Orthogonal partial least square-discriminant analysis (OPLS-DA) of basal tyrosine kinase activity profiles showed a clear segregation between sensitive cell lines and insensitive cell lines. Levels of putative target proteins such as PDGFR, VEGFR, Raf and Kit did not correlate with the sensitivity. All sensitive cell lines turned out to harbor an FGFR2 amplification suggesting that FGFR2 amplification may be a predictive biomarker of response to MTKI treatment. Kinases such as EGFR, EPHA, FGFR, IGF1R, INSR and Src contributed to this segregation suggesting that these are relevant to the sensitivity to MTKIs. MET overexpressing cell lines were resistant to treatment with the four inhibitors. Scirrhous cells were segregated from non-scirrhous cells by OPLS-DA among cell lines with signet-ring cell carcinoma cytology with R2, 0.994; Q2, 0.615. EGFR, ERBB2, 3, 4, EPHAs, FAK, IGF1R, INSR, MAP2Ks, RET and SRC seemed to be responsible for the segregation suggesting that they play an important role in scirrhous gastric cancer biology. Conclusions: Kinase activity profiles reflect the biological complexity of gastric cancer. FGFR2 status correlates with sensitivity to sunitinib, pazopanib and sorafenib, whereas MET status does not. These data imply that tyrosine kinase activity profiling with PamChip® peptide arrays can be utilized to identify molecular targets and predictive biomarkers in gastric cancer and this should be further explored. Citation Format: Yasuhiro Koh, Masakuni Serizawa, Rik de Wijn, Riet Hilhorst, Takako Nakajima, Hirofumi Yasui, Kazuyoshi Yanagihara, Martijn Dankers, Rob Ruijtenbeek, Narikazu Boku. In vitro tyrosine kinase activity profiling to identify molecular targets and predictive biomarkers in gastric cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4655. doi:10.1158/1538-7445.AM2013-4655

Photodynamic therapy induced cell death of hormone insensitive prostate cancer PC-3 cells with autophagic characteristics

The introduction of photodynamic therapy (PDT) to the treatment of advanced prostate cancer can accomplish the eradication of local neoplasm and distant metastases with minimized damage to the adjacent structures. The evidence of PDT efficacy for androgen-refractory prostate cancer will be especially meaningful for the patients resistant to hormone therapy. Pheophorbide a (PhA) as a photosensitizer was employed to evaluate the photodynamic efficacy in androgen-insensitive PC-3 prostate cancer cells in culture by cell viability assay, reactive oxygen species (ROS) measurement and cell cycle test. Characteristics of apoptosis and autophagy were investigated via DNA fragmentation electrophoresis and immune-fluorescence staining, acidic vesicle determination and detection of LC3B in puncta form by fluorescence microscopy, Western blotting of autophagy-related (Atg) proteins and detailed phenotype shown by electron microscopy. PhA exerted significant photo-cytotoxicity toward androgen-insensitive prostate cancer PC-3 cells in photosensitizer-dose and light-dose dependent manners. The photoactivation immediately initiated hyperproduction of ROS, the depolarization of mitochondrial membrane potential and the arrest of the cell cycle in the G0/G1 phase. Autophagy was revealed in PhA-PDT treated PC-3 cells by a significant high amount of acidic vesicular organelles with acridine orange staining, recruitment of LC3B on the membrane of autophagosomes by fluorescent microscopy, double membrane-bound vesicles suggesting autophagosomes by electron microscopy, significant increased Atg proteins such as beclin-1, Atg12-Atg5 conjugation, Atg7 and the conversion of LC3B-I to LC3B-II by Western blot analysis. PhA-mediated PDT induced significant autophagy in hormone-refractory prostate cancer PC-3 cells.

Reversing the Warburg Effect as a Treatment for Glioblastoma

Glioblastoma multiforme (GBM), like most cancers, possesses a unique bioenergetic state of aerobic glycolysis known as the Warburg effect. Here, we documented that methylene blue (MB) reverses the Warburg effect evidenced by the increasing of oxygen consumption and reduction of lactate production in GBM cell lines. MB decreases GBM cell proliferation and halts the cell cycle in S phase. Through activation of AMP-activated protein kinase, MB inactivates downstream acetyl-CoA carboxylase and decreases cyclin expression. Structure-activity relationship analysis demonstrated that toluidine blue O, an MB derivative with similar bioenergetic actions, exerts similar action in GBM cell proliferation. In contrast, two other MB derivatives, 2-chlorophenothiazine and promethazine, exert no effect on cellular bioenergetics and do not inhibit GBM cell proliferation. MB inhibits cell proliferation in both temozolomide-sensitive and -insensitive GBM cell lines. In a human GBM xenograft model, a single daily dosage of MB does not activate AMP-activated protein kinase signaling, and no tumor regression was observed. In summary, the current study provides the first in vitro proof of concept that reversal of Warburg effect might be a novel therapy for GBM.

Identification of plant extracts sensitizing breast cancer cells to TRAIL

Triple-negative breast cancer (TNBC) is an aggressive heterogeneous cancer subgroup with a higher rate of distant recurrence and a poorer prognosis compared to other subgroups. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive molecule that induces cell death in various tumor cells without causing cytotoxicity to normal cells; however, primary or acquired resistance to TRAIL often limits its efficacy in cancer patients. To develop combination therapies to improve TRAIL efficacy and/or to overcome the resistant mechanism, we screened 138 medicinal plant extracts against TRAIL-sensitive and -insensitive TNBC cell lines, MDA-MB-231 and MDA-MB-468. Among them, 5 plant extracts, Uvaria dac, Artemisia vulgaris, Cortia depressa, Dichasia bengalensis and Cinnamomum obtusifolium did not cause apparent cytotoxicity (<20%) as a single regimen, but showed significant synergistic effects in combination with TRAIL against both cell lines. Moreover, Uvaria dac, Artemisia vulgaris and Cinnamomum obtusifolium were found to suppress the phosphorylation of p65 that is involved in TRAIL-resistant mechanisms. These observations suggest that the identified plant extracts in combination with TRAIL could lead to potential therapeutic benefits for cancer patients in the clinical setting.

A gene expression profile indicative of early stage HER2 targeted therapy response

BackgroundEfficacious application of HER2-targetting agents requires the identification of novel predictive biomarkers. Lapatinib, afatinib and neratinib are tyrosine kinase inhibitors (TKIs) of HER2 and EGFR growth factor receptors. A panel of breast cancer cell lines was treated with these agents, trastuzumab, gefitinib and cytotoxic therapies and the expression pattern of a specific panel of genes using RT-PCR was investigated as a potential marker of early drug response to HER2-targeting therapies.ResultsTreatment of HER2 TKI-sensitive SKBR3 and BT474 cell lines with lapatinib, afatinib and neratinib induced an increase in the expression of RB1CC1, ERBB3, FOXO3a and NR3C1. The response directly correlated with the degree of sensitivity. This expression pattern switched from up-regulated to down-regulated in the HER2 expressing, HER2-TKI insensitive cell line MDAMB453. Expression of the CCND1 gene demonstrated an inversely proportional response to drug exposure. A similar expression pattern was observed following the treatment with both neratinib and afatinib. These patterns were retained following exposure to traztuzumab and lapatinib plus capecitabine. In contrast, gefitinib, dasatinib and epirubicin treatment resulted in a completely different expression pattern change.ConclusionsIn these HER2-expressing cell line models, lapatinib, neratinib, afatinib and trastuzumab treatment generated a characteristic and specific gene expression response, proportionate to the sensitivity of the cell lines to the HER2 inhibitor.Characterisation of the induced changes in expression levels of these genes may therefore give a valuable, very early predictor of the likely extent and specificity of tumour HER2 inhibitor response in patients, potentially guiding more specific use of these agents.

Identification, characterization and targeting of Docetaxel-resistant prostate cancer cells

Identification, characterization and targeting of Docetaxel-resistant prostate cancer cells

The Direct Anti-Tumor Activity of Enzastaurin (LY317615.HCl) and Its Primary Metabolite (LY326020.HCl) Is Evident in Preclinical DLBCL Models Regardless of Molecular Subtype

AbstractAbstract 1336Enzastaurin (Enza) is an ATP-competitive inhibitor of PKCβ and related AGC family kinases and blocks signaling through the PI3K/AKT/TOR pathway. Accordingly, in preclinical tumor models, clinically achievable Enza doses suppress phosphorylation of AKT, GSK3β, ribosomal protein S6, mTOR, p70S6 Kinase and 4EBP1.Enza has shown significant activity in Diffuse Large B Cell Lymphoma (DLBCL) patients under clinical study. 4/55 DLBCL patients treated with Enza were progression- free after prolonged, continuous oral enza therapy, with 3/4 confirmed as complete responders (Robertson et al., JCO, 2007). Furthermore, preliminary analyses of intermediate or high-risk DLBCL patients treated with R-CHOP vs. Enza + R-CHOP suggest that Enza improves both progression-free survival and complete response rates (Hainsworth et al., JCO, 29: 2011, suppl; abstract 8016). Enza is currently being investigated in a phase 3 registration trial for DLBCL patients at high risk of relapse following R-CHOP therapy.Gene expression profiling studies have revealed molecular alterations involved in DLBCL. For example, overexpression of PKCβ is particularly evident in recurrent DLBCL and has been associated with reduced patient survival. Similarly, BCL-6, a transcriptional repressor likely involved in the corruption of appropriate B Cell differentiation, is overexpressed in ∼ 70% of DLBCL and the target of translocation in nearly 40% of DLBCL. From these studies, distinct molecular subtypes of DLBCL have emerged. The Activated B Cell (ABC) subtype, which carries the poorest prognosis, is defined by constitutive activation of NFκB whereas the Germinal Center (GC) subtype, which carries a better prognosis, is not. 10% of ABC DLBCLs harbor CARD11 mutations leading to constitutive NFκB activation, while another 20% have mutations in CD79A/B, enabling chronic B Cell receptor signaling. In both ABC and GC subtypes, chronic activation of the B Cell receptor complex signals through PKCβ and the PI3K/AKT pathway.Given its activity in DLBCL patients and its molecular mechanism of action, we chose to investigate the molecular basis for the direct anti-cancer activity of Enza, specifically in DLBCL, and to discern whether sensitivity to Enza is associated with distinct DLBCL subtypes. In multiple DLBCL lines, we show that Enza treatment at clinically achievable concentrations significantly reduces BCL-6 expression. We also show that Enza robustly induces apoptosis in both GC and ABC models, blocking signaling throughout the PI3K/AKT pathway. Moreover, we show in the same DLBCL models that the primary metabolite of enza (LY326020.HCl), which comprises ∼50% of circulating drug levels in patients, more potently inhibits these same signaling pathways and more robustly induces apoptosis. Importantly, we have also identified cell lines that are resistant to both enza and its metabolite. Profiling differences between these resistant and sensitive DLBCL lines, we now show that the pro-apoptotic activity of Enza and LY326020 requires control of the eIF4F translation initiation complex- the complex responsible for enabling translation of critical, malignancy-related mRNAs (e.g. VEGF, BCL-2, c-myc). In insensitive DLBCL cells, 4EBP1 expression levels are low to non-existent, even prior to treatment. In sensitive DLBCL cells, Enza and LY326020 reduce 4EBP1ser65 phosphorylation in a dose and time-dependent manner, increasing 4EBP1: eIF4E binding thereby precluding eIF4E:eIF4G binding and preventing eIF4F complex assembly. Furthermore, selection of DLBCL cells for resistance yields cells with reduced 4EBP1 expression, increased eIF4G expression, or both, highlighting the critical importance of this complex for Enza activity. Collectively, these data show that: 1) Enza and LY326020 have direct anti-cancer activity in DLBCL cells regardless of ABC or GC subtype; 2) Enza and LY326020 directly impact numerous molecular alterations driving DLBCL (BCL-6 expression, PKCβ and PI3K-AKT pathway signaling); 3) the ability to control eIF4F complex assembly is critical for enza and LY326020 –induced apoptosis; and 4) LY326020 is more potent than Enza at blocking signaling and inducing apoptosis. Moreover, these data suggest that baseline expression levels of 4EBP1 and/or levels of phosphorylated eIF4E (an indicator of active eIF4F complex) may be particularly informative in delineating patients most sensitive to treatment. Disclosures:Graff:Eli Lilly and Company: Employment. McNulty:Eli Lilly and Company: Employment. Dumstorf:Eli Lilly and Company: Employment. Konicek:Eli Lilly and Company: Employment. Hall:Eli Lilly and Company: Employment. Parsons:Eli Lilly and Company: Employment.

Targeting STAT5 Expression Resulted in Molecular Response Improvement in Patients with Chronic Phase CML Treated with Imatinib

AbstractAbstract 696 Background.We have previously demonstrated that PPAR-γ agonists (such as pioglitazone) negatively regulate the level of Stat5A and Stat5Bgene expression in normal CD34+ bone marrow progenitors (Prost, Le Dantec et al. 2008). Aim.We investigated whether targeting Stat5 expression with pioglitazone may impact clonogenic activity of Chronic Myelogenous Leukemia (CML) cells in vitro and result in molecular response improvement in vivo. Patients and methods.Preliminary in vitro studies tested the ability of pioglitazone to impact viability and clonogenicity of CD34+ primary cells from CML patients (pts). We conducted clonogenic and LTC-IC studies. Cultivated Ph+ -CD34+cells were characterized by facs and analyzed by CSFE assay. BCR-ABL and Stat5 expression were quantified by real time PCR. Control experiments were conducted using lentiviral vectors and siRNA assay for Stat5 and PPAR-g.CML pts were eligible in the ACTIM trial (EudraCT 2009–011675-79) if they were treated by imatinib for at least 2 years with a stable daily dose for at least 3 months and in major molecular response without having achieved CMR4.5 (defined by a BCR-ABL/ABL IS ratio ≤ 0.0032%). After inclusion, pts received imatinib (no dose modification) and started pioglitazone (Actos®) 30 mg/d during 2 months and 45 mg/d thereafter for 12 months. BCR-ABL transcript level was monitored every 3 months during the study period. Primary objective was the proportion of patients achieving a confirmed undetectable level of BCR-ABL transcript. Secondary objectives included cumulative incidence of CMR4.5 and safety. A companion biologic study evaluated imatinib through levels, Stat5 expression in bone marrow at baseline, months 6 and 12 and clonogenic activity of bone marrow mononuclear cells at baseline, months 6 and 12. Results.From the in vitro studies we first demonstrated that pioglitazone at pharmacological doses inhibited cell growth of the Bcr-Abl positive cell line K562 through the activation of the PPAR-g/STAT5 pathway. We next showed that PPAR-g / STAT5 pathway induced a clonogenic defect in CD34+ cells from CML patients. Moreover, the activation of the PPAR-g / STAT5 pathway also induced a clonogenic and a proliferative defect in CML LTC-IC. We then confirmed that imatinib induced a selection of insensitive quiescent CML cells and showed that this effect was abrogated by the activation of the PPAR-g / STAT5 pathway.Twenty seven pts were enrolled in the clinical trial and 24 were evaluable (1 was excluded in CMR4.5, 1 pt was not in MMR and 1 pt had consent withdrawal). Median age was 61.6 years (24.1–79) and median follow-up after inclusion was 13 months (9.8–21). All evaluable pts started pioglitazone as planned. Seven pts (29.2%) discontinued pioglitazone before 12 months, 6 following investigator decision after the warning of the French ministry of health regarding the risk of bladder cancer and 1 after its own decision. No pt discontinued due to adverse events. Discontinuations occurred between month 3 and month 9. Median duration of pioglitazone therapy was 11.2 months (2.6–15.4) median daily dose was 39.9 mg. No interaction was observed between imatinib and pioglitazone in term of through level before (median 850 ng/ml) and after (median 927 ng/ml) pioglitazone initiation (p=ns). Main adverse events were weight gain and worsening fluid retention in 3 pts. Three pts (14%) obtained a confirmed undetectable level of BCR-ABL transcript. The one year cumulative incidence of CMR4.5 was 57%. Stat5 mRNA quantification was significantly diminished in pt samples at M6 and M12 compared to the baseline values and a reduction of the clonogenic potential was also observed in bone marrow cells at M6 and M12. We collected “control pts” with similar characteristic (n=20). None of these pts obtained a confirmed CMR and the cumulative incidence of CMR4.5 in this control group was 27% as compared to 57% in the pioglitazone group (p=0.02). Conclusion.We have extended our in vitro results showing that PPAR-γ agonists resulted in Stat5 down regulation in CML CD34+ cells and preferentially reduced their clonogenic and long term potency in CFCs and LTC-IC assays. We now demonstrated that these effects translate in vivo by the achievement of MMR in more than half of the pts treated with the combination of pioglitazone and imatinib suggesting that it may be possible to target quiescent CML cells in vivo and supporting the concept of stem cell pool erosion. Disclosures:Off Label Use: pioglitazone in CML. Roy:Novartis, BMS: Speakers Bureau. Legros:Novartis, BMS: Research Funding, Speakers Bureau. Coiteux:Novartis, BMS: Speakers Bureau. Mahon:Novartis, BMS: Consultancy, Research Funding.

Epigenetics-related genes in prostate cancer: expression profile in prostate cancer tissues, androgen-sensitive and -insensitive cell lines

Epigenetic changes have been suggested to drive prostate cancer (PCa) development and progression. Therefore, in this study, we aimed to identify novel epigenetics-related genes in PCa tissues, and to examine their expression in metastatic PCa cell lines. We analyzed the expression of epigenetics-related genes via a clustering analysis based on gene function in moderately and poorly differentiated PCa glands compared to normal glands of the peripheral zone (prostate proper) from PCa patients using Whole Human Genome Oligo Microarrays. Our analysis identified 12 epigenetics-related genes with a more than 2-fold increase or decrease in expression and a p-value <0.01. In moderately differentiated tumors compared to normal glands of the peripheral zone, we found the genes, TDRD1, IGF2, DICER1, ADARB1, HILS1, GLMN and TRIM27, to be upregulated, whereas TNRC6A and DGCR8 were found to be downregulated. In poorly differentiated tumors, we found TDRD1, ADARB and RBM3 to be upregulated, whereas DGCR8, PIWIL2 and BC069781 were downregulated. Our analysis of the expression level for each gene in the metastatic androgen-sensitive VCaP and LNCaP, and -insensitive PC3 and DU-145 PCa cell lines revealed differences in expression among the cell lines which may reflect the different biological properties of each cell line, and the potential role of each gene at different metastatic sites. The novel epigenetics-related genes that we identified in primary PCa tissues may provide further insight into the role that epigenetic changes play in PCa. Moreover, some of the genes that we identified may play important roles in primary PCa and metastasis, in primary PCa only, or in metastasis only. Follow-up studies are required to investigate the functional role and the role that the expression of these genes play in the outcome and progression of PCa using tissue microarrays.

Abstract B31: Expression of Wnt3 activates Wnt/β-catenin pathway and promotes EMT-like phenotype in trastuzumab resistant HER2-overexpressing breast cancer cells.

Abstract To understand the mechanisms leading to trastuzumab resistance in HER2-overexpressing breast tumors we created trastuzumab insensitive cell lines (SKBR3/100-8 and BT474/100-2). The cell lines maintain HER2 receptor overexpression, and show increase in EGFR. Upon trastuzumab treatment, SKBR3/100-8 and BT474/100-2 cell lines displayed increased growth rate and invasiveness. The trastuzumab resistance in SKBR3/100-8 and BT474/100-2 was accompanied with activation of the Wnt/β-catenin signaling pathway. Further investigation found that Wnt3 overexpression played a key role toward the development of trastuzumab resistance. The expression of Wnt3 in trastuzumab resistant cells increased nuclear expression of β-catenin and transactivated expression of EGFR. The increased Wnt3 in the trastuzumab resistant cells also promoted a parental EMT-like transition (epithelial to mesenchymal transition), increased N-cadherin, Twist, SLUG and decreased E-cadherin. Knockdown Wnt3 by siRNA restored cytoplasmic expression of β-catenin, and decreased EGFR expression in trastuzumab resistant cells. Furthermore the EMT markers were decreased, E-cadherin was increased and the cell invasiveness was inhibited in response to the Wnt3 down-regulation. Conversely, SKBR3 cells which had been stably transfected with full-length Wnt3 exhibited EMT-like transition. The Wnt3 transfectants, SKBR3/Wnt3-7 and SKBR3/Wnt3-9, showed significant decrease in E-cadherin and increase in N-cadherin, Twist and SLUG. The cells were less sensitive to trastuzumab compared to parental SKBR3 and vector transfected cells. In summary, our data suggests that Wnt3 overexpression activates Wnt/β-catenin signaling pathway that leads to transactivation of EGFR and promotes EMT-like transition. This could be an important mechanism leading to trastuzumab resistance in HER2 overexpressing breast cancer cells. Citation Format: Yanyuan Wu, Charles Ginther, Juri Kim, Nicole Mosher, Seyung Chung, Dennis Slamon, Jaydutt V. Vadgama. Expression of Wnt3 activates Wnt/β-catenin pathway and promotes EMT-like phenotype in trastuzumab resistant HER2-overexpressing breast cancer cells. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B31.