bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2068: Combination erlotinib and cisplatin causes decrease angiogenesis in lung adenocarcinoma.
Authors: Lee Jasmine G. , Wu Reen
Published: August 2014

Introduction: Platinum based agents such as cisplatin have been traditionally used to for the treatment of lung cancer, and tyrosine kinase inhibitors such as erlotinib are being used more readily. Combinations of anti-cancer agents are becoming more popular due to the anticipation that combination treatment is more effective than individual drug treatments. In this study, we evaluate the anti-cancer effects of combination erlotinib and cisplatin in in vitro cell lines and in in vivo mice models. In addition, as studies of cisplatin and erlotinib in other cancer types show potential to inhibit angiogenesis, we examine whether these agents are able to target angiogenesis in lung adenocarcinoma.

Method: Three individual lung adenocarcinoma cell lines (PC9, A549, and H292) were utilized. Each cell line had four treatment groups: control, erlotinib only, cisplatin only, and combination erlotinib and cisplatin. Alamar Blue Assay was utilized to evaluate for in vitro cell death following two day treatments. PC9 cells were injected into the left flank of nude mice for in vivo experiments. Erlotinib and/or cisplatin treatments were injected directly into the tumor and tumor measurements were made every 2 days for a total of 14 days. Western blot analysis of VEGFR, p-VEGFR, and CD31was performed for both in vitro and in vivo samples. Hematoxylin and eosin (H&E) staining and 3,3′-Diaminobenzidine (DAB) staining for CD31 were done on tumor samples.

Results: Combination treatment in EGFR mutated PC9 cells led to significantly higher cell death compared to other treatment groups (P<0.0001); the effect was synergistic. In EGFR wild type A549 and H292 cell lines there were no significant differences. Similar to results from cell cultures, in vivo studies of combination treatment showed significant inhibition of tumor growth compared to other treatment groups (p=0.001). On western blot analysis, there were significantly lower levels of p-VEGFR (p<0.0001) and CD31 (p=0.0291) in the combination group compared to all other groups. H&E staining shows significantly lower density of blood vessel and DAB staining of CD31in the combination group compared to other groups.

Conclusions: Combination low dose erlotinib and cisplatin is able to induce synergistic cell death. This effect, however, may be specific to EGFR mutated lung adenocarcinoma. Combination treatment also demonstrated the ability to inhibit tumor growth in mice. The inhibition of tumor growth may be secondary to inhibition of angiogenesis.

Citation Format: Jasmine G. Lee, Reen Wu. Combination erlotinib and cisplatin causes decrease angiogenesis in lung adenocarcinoma. [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 2068. doi:10.1158/1538-7445.AM2013-2068
Abstract 2073: The parp inhibitor olaparib (AZD2281) greatly enances the effect of temozolomide in soft tissue sarcoma cell lines.
Authors: Musi Elgilda , Ambrosini Grazia , Schwartz Gary K.
Published: August 2014

Sarcomas are heterogeneous mesenchymal neoplasms which have very limited treatment options. Chemotherapies for advanced sarcoma include DNA damaging agents such as doxorubicin, dacarbazine and gemcitabine with very low response rates. We elected to study the effects of olaparib (AZD2281), a novel, potent orally active PARP inhibitor in combination with standard chemotherapy Temozolomide (TMZ) in various sarcoma subtypes. We examined the effects of olaparib and TMZ in a panel of 7 sarcoma cell lines including leiomyosarcoma (SK-LMS, SK-UT1, SK-UT1b), liposarcoma (LS141, DDLS) and malignant peripheral nerve sheath tumors (MPNST, ST8814). In vitro results have shown inhibition of cell viability with combination treatment in all cell lines at concentrations of 300uM TMZ and 1 uM olaparib. After 48 hrs of drug treatment, we also examined biochemical effects showing PAR inhibition, p53 induction and γH2AX increase, indicative of DNA damage. Flow cytometric analysis of each cell line indicated G2/M cell cycle arrest with combination treatment. These observations correlated with enhanced apoptosis as measured by biochemical PARP cleavage and DNA content (sub-G1 population). Taking these observations and results, there is strong evidence to support combining standard chemotherapy TMZ with the PARP inhibitor olaparib in various sarcoma subtypes.

Citation Format: Elgilda Musi, Grazia Ambrosini, Gary K. Schwartz. The parp inhibitor olaparib (AZD2281) greatly enances the effect of temozolomide in soft tissue sarcoma 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 2073. doi:10.1158/1538-7445.AM2013-2073
Abstract 2077: Downregulation of miR-21 induces differentiation of chemo-resistant colon cancer cells and enhances susceptibility to therapeutic regimens.
Authors: Yu Yingjie , Sarkar Fazlul H. , Majumdar Adhip P.N.
Published: August 2014

MicroRNAs (miRNAs) are endogenous posttranscriptional modulators that negatively control the expression of their target genes and play an important role in the development and progression of colorectal carcinoma. In particular, microRNA-21 (miR-21), whose expression is greatly increased in chemotherapy-resistant (CR) colon cancer cells that are enriched in undifferentiated cancer stem/stem-like cells (CSCs/CSLCs). We hypothesize that miR-21 plays a critical role in regulating differentiation of colon cancer (CR cells). Indeed, we observed that downregulation of miR-21 in CR colon cancer HCT-116 cells by anti-sense-miR-21 induced differentiation, as evidenced by marked increases in CK-20 (cytokeratin-20) expression and alkaline phosphatase activity. These changes were accompanied by a significant reduction in the expression of colon CSC/CSLCs marker CD44, colonospheres formation and TCF/LEF activity and increased expression of the pro-apoptotic gene PDCD4. Induction of differentiation greatly increased the sensitivity of CR cells to the growth inhibitory effects of all three regimens tested: 5- FU+ Oxaliplatin (FUOX), difluorinated curcumin (CDF), an analog of curcumin and the combination of CDF and FUOX. However, the magnitude of inhibition of growth by either CDF (75%) alone or CDF + FUOX (80%) was much higher than that observed with FUOX (40%). Growth inhibition by CDF and CDF + FUOX in differentiating CR colon cancer cells was associated with a 98-99% reduction in the expression of CD44 and EGFR. The current observation suggest that CDF as well as CDF + FUOX are highly effective in inhibiting growth and reducing colon CSCs/CSLCs in anti-miR-21-induced differentiating CR colon cancer cells, which supports our contention that differentiating cells become susceptible to conventional and non-conventional therapeutic regimen.

Citation Format: Yingjie Yu, Fazlul H. Sarkar, Adhip P.N. Majumdar. Downregulation of miR-21 induces differentiation of chemo-resistant colon cancer cells and enhances susceptibility to therapeutic regimens. [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 2077. doi:10.1158/1538-7445.AM2013-2077
Abstract 2079: Antitumoral activity of INC280 against adult glioblastoma brain tumors xenografts.
Authors: Keir Stephen T. , Roskoski Martin A. , Wagner Sabrina , Tiedt Ralph , Bigner Darell D. , Friedman Henry S.
Published: August 2014

INTRODUCTION: INC280 is an oral, highly selective c-Met receptor tyrosine kinase inhibitor. The hepatocyte growth factor (HGF)-c-Met pathway is one of the most frequently dysregulated pathways in human cancers. Aberrant HGF-c-Met signaling has been documented in a wide range of human malignancies. The c-Met pathway can be activated by abnormal HGF and c-Met expression levels, c-Met activating mutations and gene amplifications. At this time, INC280 is in development for the treatment of solid tumors with activation of the c-Met pathway. As a part of this study, we looked at the antitumoral activity of INC280 in adult glioblastoma (GBM) brain tumor xenografts with elevated levels of Met or HGF expression.

METHODS: Met expression and copy number was determined by ELISA and qPCR, HGF mRNA by RT-qPCR. Adult GBM tumor xenografts were grown subcutaneously in athymic BALB/c mice. After tumor size reached 200-500 mm3 subcutaneously, groups of 10 mice were randomly treated with either drug vehicle/control or INC280 (10mg/kg BID) PO for 30 days. Tumor responses for subcutaneous xenografts were assessed by tumor growth delay and regression.

RESULTS: Based on activation of the c-Met pathway, the following two xenograft lines were selected for treatment with INC280: D-09-0337 MG and D-09-0477 MG. As a single agent, INC280 demonstrated statistically significant (p<0.001) growth delays of 40.23 and 30.13 days in D-09-0337 MG and D-09-0477 MG, respectively. In addition, when treated with INC280, tumors implanted with D-09-0337 MG (10 out of 10) or D-09-0477 MG (4 out of 10) regressed.

CONCLUSION: Our results demonstrate the therapeutic efficacy of the inhibition of a c-Met receptor tyrosine kinase in adult brain tumor xenografts with activation of the c-Met pathway. These results warrant further exploration of INC280 in clinical trials for the treatment of adult brain tumor patients with dysregulated proto-oncogene c-Met and its ligand HGF.

Citation Format: Stephen T. Keir, Martin A. Roskoski, Sabrina Wagner, Ralph Tiedt, Darell D. Bigner, Henry S. Friedman. Antitumoral activity of INC280 against adult glioblastoma brain tumors xenografts. [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 2079. doi:10.1158/1538-7445.AM2013-2079
Abstract 2096: Gefitinib analogue V1801 induces apoptosis of T790M EGFR-harboring lung cancer cells by up-regulation of the BH-3 only protein Noxa .
Authors: Zhang Bo , Yao Zhujun , Zhou Guangbiao
Published: August 2014

Treatment of non-small cell lung cancer (NSCLC) with drugs targeting the epidermal growth factor receptor (EGFR), e.g., gefitinib and erlotinib, will eventually fail because of the development of secondary mutations such as T790M in EGFR. Strategies to overcome this resistance are therefore an urgent need. In this study, we synthesized a dozen of novel gefitinib analogues and evaluated their effects on L858R/T790M-EGFR harboring NSCLC cells, and reported that one of these gefitinib mimetics, N-(2-romo-5-(trifluoromethyl)phenyl)-6-methoxy-7-(3-(piperidin-1-yl)propoxy)uinazolin-4-amine (hereafter, V1801), triggered apoptosis of the NSCLC cells and overcame gefitinib-resistance in mice inoculated with NCI-H1975 cells. Though V1801 only moderately inhibited EGFR kinase activity, it markedly induced the expression of the BH3-only protein Noxa, and Noxa silencing significantly reduced V1801-induced apoptosis of NCI-H1975 cells. It is showed that V1801 interfered with the expression of the transcription factor c-Myc and the extracellular signal regulated kinase (Erk) pathway. V1801 in combination with proteasome inhibitor bortezomib exerted enhanced cytotoxicity in NCI-H1975 cells possibly due to potentiated induction of Noxa expression. These data indicate that gefinitib analogues with weak EGFR inhibitory activity may overcome drug-resistance via activation of BH-3 only pro-apoptotic proteins, and V1801 may have therapeutic potentials for NSCLC.

Citation Format: Bo Zhang, Zhujun Yao, Guangbiao Zhou. Gefitinib analogue V1801 induces apoptosis of T790M EGFR-harboring lung cancer cells by up-regulation of the BH-3 only protein Noxa . [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 2096. doi:10.1158/1538-7445.AM2013-2096
Abstract 2099: Novel inhibitors of NFkB inducing kinase (NIK) are cytotoxic for myeloma cell lines with NIK-dependent activation of NFkB.
Authors: Demchenko Yulia N. , Kuehl Walter Michael
Published: August 2014

NFkB activity is critical for survival and proliferation of normal lymphoid cells and many kinds of B-cell tumors, including multiple myeloma (MM). NFkB activating mutations, which are apparent progression events, enable MM tumors to become less dependent on bone marrow signals that activate NFkB. Mutations that activate NFkB-inducing kinase (NIK) protein are the most prevalent among the many kinds of NFkB mutations in MM tumors. NIK is the main activating kinase of the alternative NFkB pathway, although over-expression of NIK also can activate the classical pathway. Three Amgen compounds (two NIK inhibitors and an isomeric control) were tested with human myeloma cell lines. These specific NIK inhibitors are selectively cytotoxic for cells with NIK-dependent activation of NFkB. The combination of NIK-inhibitors with IKKβ-inhibitors or dexamethasone show a synergistic effect that might more efficiently target some MM tumors.

Citation Format: Yulia N. Demchenko, Walter Michael Kuehl. Novel inhibitors of NFkB inducing kinase (NIK) are cytotoxic for myeloma cell lines with NIK-dependent activation of NFkB. [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 2099. doi:10.1158/1538-7445.AM2013-2099

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 21: Survival prognostic significance of TNFRII, TGFa, TIMP1 & CRP in high-risk melanoma patients.
Authors: Tarhini Ahmad A. , Lin Yan , LaFramboise William A. , Lee Sandra , Kirkwood John
Published: August 2014

Background:

The E1694 trial tested the GM2-KLH-QS21 (GMK) vaccine versus high dose interferon-alpha2b as adjuvant therapy for patients with operable high-risk stage IIB/III melanoma in relation to relapse-free survival (RFS) and overall survival (OS). Multiplex analysis of candidate serum analytes utilizing banked specimens from patients participating in this trial was evaluated to develop biomarkers of prognostic or therapeutic predictive value.

Methods:

The Aushon Multiplex Platform was used to simultaneously quantitate the serum levels of 115 candidate analytes selected based on previous data. Univariate proportional hazard (PH) model was used to assess the association between each marker and OS or RFS. The Benjamini and Hochberg's method was used to adjust for multiple testing. Baseline serum samples from 40 patients treated with the GMK vaccine were analyzed. The least absolute shrinkage and selection operator proportional hazard regression (LASSOPH) was used to select markers that are most informative for RFS and OS. We then fit the regular PH models using the markers selected by the LASSOPH. The survival ROC analysis was used to evaluate the ability of the models to predict 1 year RFS and 5 year OS. Leave-one-out cross validations (LOOCVs) were used to avoid over fitting.

Results:

A panel of four markers, Tumor Necrosis Factor alpha Receptor II (TNFRII), Transforming Growth Factor alpha (TGFa), Tissue Inhibitor of Metalloproteinases 1 (TIMP1), and C-reactive protein (CRP), at baseline was found to be most informative to OS after GMK vaccine treatment (high serum levels correlate with worse prognosis). The dichotomized risk score based on the four markers can separate the OS curves well (log rank test p-value=0.0005). When using the 4 marker PH model to predict the 5 year OS, we achieved an AUC of 89% (cross validated AUC=72%). High baseline TNFRII was also identified as the most informative marker of worse RFS after GMK vaccine. The RFS of patients with high (above median) baseline TNFRII was significantly lower than that of patients with low (below median) baseline TNFRII (log rank test p-value=0.01).

Conclusions:

Our modeling analysis yielded a panel of four serum biomarkers that is significantly prognostic of survival in patients with high-risk melanoma and warrants further investigation.

Citation Format: Ahmad A. Tarhini, Yan Lin, William A. LaFramboise, Sandra Lee, John Kirkwood. Survival prognostic significance of TNFRII, TGFa, TIMP1 & CRP in high-risk melanoma patients. [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 21. doi:10.1158/1538-7445.AM2013-21
Abstract 210: GPR56 regulates the turnover of tissue transglutaminase and tumor growth in melanoma.
Authors: Yang Liquan , Mohanty Sonali , Corson Nancy , Xu Lei
Published: August 2014

Biochemical and biophysical properties of extracellular matrix (ECM) regulate multiple aspects of cancer cell behavior during tumor progression. The mechanisms of these regulations are not well defined. We previously reported that an atypical G protein-coupled receptor (GPCR), GPR56, inhibited melanoma growth and metastasis and its N-terminus binds to tissue transglutaminase, TG2, a major cross-linking enzyme in ECM. We thus postulated that GPR56 might suppress melanoma progression by influencing ECM properties through its interaction with TG2. To test this, we examined the effects of GPR56 on melanoma growth in the presence or absence of TG2, using the immunodeficient Tg2+/+ or Tg2−/− mice. Our results showed that the crosslinking enzyme, TG2, promoted melanoma growth, but this tumor-promoting effect was antagonized by GPR56. The mechanisms of this antagonism were revealed through a series of immunohistochemical and biochemical analyses. In GPR56-expressing cells, the extracellular TG2 was internalized by GPR56 and subsequently degraded in a lysome-dependent manner, resulting in a down-regulation of TG2 in ECM. This down-regulation of TG2 by GPR56 led to changes in the deposition of TG2 and other matrix proteins in melanomas and a reduction in melanoma growth. Taken together, we present evidence that a surface receptor on melanoma cells, GPR56, regulates the turnover of the crosslinking enzyme, TG2, in the tumor microenvironment, leading to changes in ECM deposition and melanoma progression.

Citation Format: Liquan Yang, Sonali Mohanty, Nancy Corson, Lei Xu. GPR56 regulates the turnover of tissue transglutaminase and tumor growth in melanoma. [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 210. doi:10.1158/1538-7445.AM2013-210

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2100: Preclinical evaluation of PF-04691502 in nasopharyngeal carcinoma.
Authors: Loong Herbert H. , Wong Eric C H , Hui Connie W C , Lau Cecilia P Y , Hui Edwin P. , Ma Brigette B Y , Chan Anthony T C
Published: August 2014

Nasopharyngeal carcinoma (NPC) is common in Southeast Asia. PF-04691502 (PF) is a novel potent dual inhibitor of PI3K/mTOR capable of inhibiting downstream effectors Akt, S6 and 4E-BP1, resulting in suppression of protein synthesis and cell survival. Five NPC cell lines include two well differentiated cell lines CNE-1 and HK1, two poorly differentiated NPC cell lines CNE-2 and HONE-1, and an undifferentiated NPC cell line C666-1 were selected for this preclinical study. All cell lines expressed basal level of Akt and p70S6K in both phosphorylated and total forms. The cytotoxicity of PF on NPC was evaluated by MTT assay. Cells were incubated in PF with increasing concentration (0.1nM, 50nM, 100nM, 200nM, 500nM, 1μM and 10μM) in culture medium for either 48 hours or 72 hours. Maximum growth inhibition was achieved in over 80% in 72 hours of PF incubation. The IC50 of all cell lines were in hundred nanomolar range (CNE-1 = 276nM, CNE-2 = 84nM, HK1 = 121nM, HONE-1 = 157nM and C666-1 = 395nM). Two most sensitive cell lines (CNE-2 and HK1) together with the least sensitive one (HONE-1) were selected to further evaluate the effect of PF on cell cycle, apoptosis and Akt signaling. Cells treated with either 100nM or 500nM PF for 6 hours and 24 hours were evaluated by cell cycle analysis and G0/G1 arrest was observed at 24 hour time point. Apoptotic effect was indicated by increasing cleaved PARP expression in which 500nM PF treatment for 24 hours induced apoptosis in CNE-2 and HK1 but not in HONE-1. At 48 hours, a lower dose of PF at 100nM could induce apoptosis in all NPC cell lines. The expression of pi-mTOR, pi-p70S6K, pi-Akt(S473), pi-Akt(T308), pi-S6 and pi-4EBP1 was inhibited in by PF, suggesting its effectiveness in anti-proliferation and inhibition of protein synthesis. Antitumor activity was observed in NPC xenograft model after 2 weeks of 10mg/kg PF administration to tumor bearing female athymic nude mice. Both tumor volume and tumor weight in the treatment group were significantly smaller and lighter than that in vehicle group (3.17cm3 vs. 5.58cm3 at 18 days, p=0.005 and 146mg vs. 284mg, p=0.001 respectively) while the body weight between groups were comparable, suggesting this working dose was well-tolerated. The beneficial effects of PF-09641502 in both in vitro and in vivo studies for NPC warrant a further investigation.

Acknowledgement: This work is supported by an independent research grant from Pfizer.

*Co-authorship

Citation Format: Herbert H. Loong, Eric C H Wong, Connie W C Hui, Cecilia P Y Lau, Edwin P. Hui, Brigette B Y Ma, Anthony T C Chan. Preclinical evaluation of PF-04691502 in nasopharyngeal 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 2100. doi:10.1158/1538-7445.AM2013-2100
Abstract 2102: Investigational agent 5F 203 modulates apoptotic regulatory gene expression and induces lysosomal membrane permeabilization in breast cancer cells.
Authors: Brantley Eileen J. , Thorne Gabriell , Daly Devin , Zylstra Dain , McLean Lancelot
Published: August 2014

Despite advances in targeted therapeutic agents for women with breast cancer that either express estrogen receptor (ER) alpha or over-expresses human epidermal growth factor receptor 2 (Her2), nearly 40,000 women die each year due to this malignancy. An investigational anticancer agent 2-(4-amino-3-methylphenyl)-5-benzothiazole (5F 203) potently inhibits the growth of breast cancer cells irrespective of either ER or Her2 status. In our current studies we used the Annexin V/PI assay to determine that 5F 203 induced a dose-dependent increase in the percentage of ER/Her2-positive T47D cells and ER/Her2-negative MDA-MB-468 breast cancer cells in early apoptosis. Cell death was not entirely caspase-dependent as pretreatment with pan-caspase inhibitor z-VAD-fmk only partially suppressed apoptosis. A pathway specific PCR-array revealed the ability for 5F 203 to increase the mRNA expression of pro-apoptotic genes bcl-2-antagonist/killer 1 (BAK1) and lymphotoxin alpha (LTA). Using the Acridine Orange assay, we found that 5F 203-mediated cell death was associated with lysosomal membrane permeabilization in both T47D and MDA-MB-468 cells. Additionally, we determined using the Cathepsin B assay that 5F 203 promoted the release of cathepsin B from the lysosomes in both breast cancer cell types. These data indicate 5F 203 upregulates BAK1 and LTA expression and promotes lysosomal cell death triggering the release of cathepsin B in breast cancer cells to confer its anticancer action.

Citation Format: Eileen J. Brantley, Gabriell Thorne, Devin Daly, Dain Zylstra, Lancelot McLean. Investigational agent 5F 203 modulates apoptotic regulatory gene expression and induces lysosomal membrane permeabilization in breast 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 2102. doi:10.1158/1538-7445.AM2013-2102
Abstract 2105: Advanced glycation end products of type I collagen promote cell proliferation and resistance against doxorubicin in HT-1080 cancer cells.
Authors: Saby Charles , EL Btaouri Hassan , Van Gulick Laurence , Guilbert Marie , Jeannesson Pierre , Morjani Hamid
Published: August 2014

Resistance to apoptosis and chemotherapy is a critical factor in cancer recurrence and patient relapse. Extracellular matrix via its receptors, the integrins, has emerged as a major pathway contributing to cancer cell survival and resistance to chemotherapy (Aoudjit and Vuori, Chemother Res Pract. 2012;2012:283181). However, the effects of extracellular matrix advanced glycation end products (AGE) on cell proliferation and sensitivity to chemotherapy is not well established. In this study, we investigated whether type I collagen and particularly its AGE are involved in cell proliferation and resistance to doxorubicin in human fibrosarcoma HT-1080 cancer cells.

Type I collagen was extracted from rats tail tendons (10 days, 8 and 96 weeks-old) and prepared as already described (Millerot-Serrurot et al., 2010, Cancer Cell Int, 13: 10-26). First, we characterized the glycation pattern of the different collagens using SDS-PAGE electrophoresis and fluorescence spectroscopy. A significant increase in AGE level has been observed in collagen extracted from 8 and 96 weeks-old rats. AGE level was significantly higher in collagen extracted from 96 weeks-old rats when compared to 8 weeks-old ones. Only collagen extracted from 96 weeks-old rats was able to induce 3-fold increase in proliferation of HT-1080 cells. Western-blot analysis showed that activation of MAPK/ERK, but not PI3K/AKT, was required for collagen AGE mediated cell proliferation. The MAPK/ERK inhibitor U0126 abolished this effect. Both collagens were able to inhibit doxorubicin-induced apoptosis in HT-1080 cells. Moreover, protection against apoptosis was significantly higher in the presence of collagen extracted from 96 weeks-old rats.

Our data suggest extracellular matrix AGE signalling as an important promoter of cancer cell proliferation and that this pathway may contribute to a new mechanism of drug resistance.

Citation Format: Charles Saby, Hassan EL Btaouri, Laurence Van Gulick, Marie Guilbert, Pierre Jeannesson, Hamid Morjani. Advanced glycation end products of type I collagen promote cell proliferation and resistance against doxorubicin in HT-1080 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 2105. doi:10.1158/1538-7445.AM2013-2105
Abstract 2109: Activation of multiple cellular stresses by the NO generating antineoplastic agent JS-K.
Authors: Kaur Imit , Terrazas Moises , Kosak Ken M. , Shami Paul J.
Published: August 2014

The NO prodrug O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate or JS-K is active against acute myeloid leukemia (AML), multiple myeloma, hepatocellular carcinoma, prostate cancer, glioma and non-small lung cancer. We have developed a nanoscale micelle formulation for JS-K (P123/JS-K) using Pluronic® P123 polymers to stabilize and solubilize the drug. In the present study, the mechanism of action of JS-K was investigated. We speculated that, being a NO-releasing drug, JS-K causes nitrosative/oxidative stress. We therefore hypothesized that JS-K causes multiple stress responses that ultimately trigger apoptosis in the cell. We also hypothesized that JS-K affects the “intrinsic component” (defined as cell cycle, survival and stress pathways in the cell) of leukemic cells.

In this work, using human myeloid leukemia HL-60 cells, we studied the effect of JS-K on two major cellular stresses, namely ER stress and nitrosative/oxidative stress. On treating HL-60 cells with 5μM JS-K formulated in DMSO (JS-K) or P123/JS-K for 4-8 hours, activation of both GRP-78 and CHOP (markers of ER stress) was observed using western blot analysis. ER stress could also be triggered by phosphorylation of JNK protein. Again, using western blot analysis, JNK phosphorylation was observed when HL60 cells were treated with JS-K or P123/JS-K for 4-6 hours. Oxidative stress was studied by quantifying intracellular levels of reactive oxygen/reactive nitrogen species (ROS/RNS) using the ROS/RNS sensitive fluorophore 5-(and-6)-chloromethyl-2’-7’-dichlorodihydrofluorescein diacetate. Treatment of HL-60 cells with 5μM of JS-K or P123/JS-K for 30, 60 or 120 minutes lead to increased levels of ROS/RNS.

These experiments show that JS-K activates the intrinsic component of cell death by producing ER stress and oxidative stress. JS-K is at an advanced stage of pre-clinical development. Its novel and multiple mechanisms of action make it an attractive therapy for the treatment of AML and other malignancies.

Citation Format: Imit Kaur, Moises Terrazas, Ken M. Kosak, Paul J. Shami. Activation of multiple cellular stresses by the NO generating antineoplastic agent JS-K. [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 2109. doi:10.1158/1538-7445.AM2013-2109
Abstract 2117: Lasonolide A-induced premature chromosome condensation is mediated by the proto-oncogene c-Raf (Raf1).
Authors: Zhang Yong-Wei , Josse Rozenn , Ghosh Arun , Luo Ji , Pommier Yves
Published: August 2014

Lasonolide A (LSA) was originally identified in a screen for novel antitumor compounds from marine organisms with high and selective cytotoxicity against mesenchymal cancer cells. We recently demonstrated that LSA induces rapid and reversible premature chromosome condensation (PCC) associated with profound epigenetic alterations in different cell types, independently of cyclin-dependent kinases activation. In order to identify cellular targets involved in LSA sensitivity, we used a pool of kinases and phosphatases shRNAs in the human colon carcinoma HCT-116 cell line. Among the genes whose inactivation promotes the survival of LSA-treated cells, we observed an enrichment of genes involved in mitotic functions including Raf1, a well-known serine-threonine kinase regulating cell proliferation and survival. LSA induces the rapid and reversible phosphorylation of Raf1 on Ser338 in a time- and a dose-dependent manner. To establish a causal relationship between LSA-induced PCC and Raf1, we first used siRNA targeting Raf1. Knocking-down Raf1 decreased LSA-induced PCC. Furthermore, mouse embryonic fibroblasts depleted in Raf1 were found resistant to LSA-induced PCC. Our results suggest a novel role of Raf1 in chromatin condensation. They also demonstrate that the target of LSA affects the c-Raf pathway and a novel mechanism of action of LSA as an effective inducer of PCC and potential novel anticancer agent.

Citation Format: Yong-Wei Zhang, Rozenn Josse, Arun Ghosh, Ji Luo, Yves Pommier. Lasonolide A-induced premature chromosome condensation is mediated by the proto-oncogene c-Raf (Raf1). [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 2117. doi:10.1158/1538-7445.AM2013-2117

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2122: The role of Rho GTPase in cell stiffness and cisplatin resistance in ovarian cancer cells.
Authors: Santiskulvong Chintda , Sharma Shivani , Bentolila Laurent , Rao Jian Y. , Gimzewski James , Dorigo Oliver
Published: August 2014

Introduction: Cell stiffness (Young's E modulus), as measured via Atomic Force Microscopy (AFM) is a newly recognized characteristic of cancer cells. We previously demonstrated that cisplatin-resistant ovarian cancer cells have a stiffer cell membrane as compared to their syngeneic cisplatin-sensitive counterpart, and that cisplatin increases cell stiffness in cisplatin-sensitive cells. Here we examine the role of Rho GTPase in cell stiffness and cisplatin resistance.

Experimental Procedures: Prior to AFM analysis, cells were stained with Annexin V-FLUOS and only non-apoptotic cells were chosen for measurements. Cell stiffness was measured via AFM on live cells in physiological buffer. The actin cytoskeleton was visualized using phalliodin-ATTO 647N. Cells were imaged with a Leica TCS SP5 STED confocal system or a Nikon Eclispe 90i. Cisplatin sensitivity was determined via the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] cell viability assay. A nonlinear regression curve fit (one phase exponential decay) was used to analyze cisplatin dose response experiments and determine the IC50 (GraphPad Prism). Rho inhibition and activation were accomplished using Rho Inhibitor I and Rho Activator II, respectively (Cytoskeleton, Inc.).

Results: The cisplatin IC50 of cisplatin-sensitive (A2780, OVCAR5, SKOV3) and cisplatin-resistant (CP70, OVCAR5-CisR, SKOV3-CisR, RMG-1) cells correlated with Young's E modulus, with increasing IC50 corresponding to a stiffer cell phenotype. Cells that are resistant to cisplatin displayed an actin cytoskeleton that consisted of long stress fibers that spanned the width of the cell. Rho inhibition in CP70 and OVCAR5-CisR cells abolished this network of actin stress fibers, decreased cell stiffness, and increased cisplatin sensitivity. In contrast, Rho activation in A2780 and OVCAR5 cells induced the reorganization of the actin cytoskeleton into long stress fibers, increased cell stiffness, and decreased cisplatin sensitivity. We demonstrated previously that cisplatin increases cell stiffness in cisplatin-sensitive OVCAR5 cells, and here we show that cisplatin induces actin stress fiber formation in these cells.

Conclusions: Rho-mediated actin stress fiber formation increases cell stiffness and mediates cisplatin resistance in ovarian cancer cells.

Citation Format: Chintda Santiskulvong, Shivani Sharma, Laurent Bentolila, Jian Y. Rao, James Gimzewski, Oliver Dorigo. The role of Rho GTPase in cell stiffness and cisplatin resistance in ovarian 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 2122. doi:10.1158/1538-7445.AM2013-2122
Abstract 2130: Small molecule activation of diaphanous-related formins as a novel anti-cancer strategy.
Authors: Lash Leanne L. , Alberts Arthur S.
Published: August 2014

Formin-based cytoskeletal remodeling contributes to structural rearrangements that support an array of cellular demands, including chemotaxis and cell division. The Diaphanous formin family is managed in cells by Rho small GTP-binding proteins. Rho-GTP binding to the formin interferes with the intramolecular interaction between the Dia-inhibitory and -autoregulatory (DID & DAD) domains that flank the critical formin homology-2 (FH2) domain. The FH2 domain can then interact with microtubules to stabilize microtubule dynamics and loosely associate with barbed ends of polymeric actin filaments to add actin monomers. As a consequence of monomer consumption into filaments, formins also activate the MAL/MRTF-SRF transcription factor axis.

DAD-derived peptides can activate formins in cells by interrupting the DID-DAD autoregulatory mechanism. We screened for drug-like small molecules with in vitro, cellular, and in vivo activities that could mimic the impact of DAD peptides. Two chemically similar molecules, called the Intramimics (IMMs) were identified. Both molecules (IMM-01 and -02) blocked DID-DAD binding in vitro, induced actin/microtubule stabilization in cells, activated SRF, and impaired cell-cycle progression. The IMMs each reduced xenograft tumor growth in mice. Due to their novel activity on formins, the IMMs represent an innovative therapeutic strategy for the study and treatment of diseases impacted by defects in cell structure, including autism, neurodegenerative disease, and cancer.

Citation Format: Leanne L. Lash, Arthur S. Alberts. Small molecule activation of diaphanous-related formins as a novel anti-cancer strategy. [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 2130. doi:10.1158/1538-7445.AM2013-2130
Abstract 2147: Generation of primary cell lines from advanced colon cancer: a tool for screening novel anticancer molecules.
Authors: Nair Rohini , Basu Aninda , Dhandapani Muthu , Majumder Biswanath , Ulaganathan Baraneedharan
Published: August 2014

For diversely heterogeneous diseases like cancer, a major challenge in the development of new drugs is using appropriate in vitro testing platform. Although high throughput screening of new compounds requires a large panel of genetically well characterized cell lines, their numbers are far less than requirement of biophrama. On top of this the translational potential of cell line panel is limited because most of the commercially available cell lines have been generated from metastatic site and highly transformed, therefore do not represent the true state of the disease. In this context, we have developed two early passage patient derived primary colorectal cancer (CRC) cell lines. Using this improved in vitro testing system we tested the efficacy of a number of anticancer agents against CRC.

Cell lines were generated by enzymatic disaggregation method. The tissues were obtained after informed consent from patient. The tumor tissues were sectioned into to small pieces (∼1mm) and were digested using 0.5% Collagenase for 2-4 hrs at 37°C. The single cell suspensions were cultured in DMEM supplemented with 10% fetal bovine serum. The epithelial cells were propagated up to passage 2 and were cryopreserved for further applications. Mutational (K-RAS, BRAF, PIK3CA, PTEN) and standard CRC specific marker (such as mucin) analyses were done in order to confirm the authenticity of these lines. The cell viability was evaluated using MTT assay following treatment with Standard of Care (SOC) agents (both single and combinations). Additionally for screening of selected FDA approved NSAIDs like Aspirin, Etoricoxib, Ibuprofen, Indomethacin, Mefenamic acid, Methotrexate and Nimesulide (repurposing) were performed at their respective CMAX concentrations after 48 hrs of treatment.

Our results showed that, the SOCs when tested as single agents did not show any significant cell death. However, when treated in combination with Irinotecan, Fluorouracil and Leucovorin in the presence and absence of Avastin a significant augmentation of cell death was observed in both the cell lines. Further, Mefenamic acid, Methotrexate, Ibuprofen and Aspirin exhibited a significant cytotoxic effect in both cell lines after 48 hrs of treatment. Together, these results demonstrate that COX-2 inhibitors could be developed as a as an effective anticancer therapeutics for colorectal cancer treatment and primary cell line model provides a rapid and more reliable screening platform.

Citation Format: Rohini Nair, Aninda Basu, Muthu Dhandapani, Biswanath Majumder, Baraneedharan Ulaganathan. Generation of primary cell lines from advanced colon cancer: a tool for screening novel anticancer molecules. [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 2147. doi:10.1158/1538-7445.AM2013-2147
Abstract 2152: The influence of didox on the efficacy and toxicity of doxorubicin.
Authors: Al-Abbasi Fahad A. , Assad Gihan F. , Al-Abd Ahmed M. , Abdel-Naim Ashraf B.
Published: August 2014

Didox, possess antioxidant propertieson the top of its DNA synthesis and repair inhibiting ability. Doxorubicin (DOX) is a major chemotherapy for liver cancer; however it evokes severe cardiotoxic effect which is attributed to DOX derived free radicals. Herein, we assessed the effect didox in enhancing DOX efficacy in liver cancer cells while protecting against its cardiotoxic effects. Combination with didox significantly reduced in the IC50 of DOX in Huh7 and HepG2 liver cancer cell lines. The combination index between DOX and didox indicated additive type interaction (CI-value of 0.81-0.9). Both DOX and didox significantly blocked the cell cycle in S-phase and their combination significantly aggravated that block. In addition, didox with DOX showed higher caspase-3 level compared to DOX alone. This enhancement effect might be attributed to didox induced PARP enzyme inhibition. On the other hand, didox (150 mg/kg daily) significantly protected the cardiomyocyte and decreased the intra cardiac reactive oxygen species induced by DOX treatment (15 mg/kg). This effect was coupled with reverting DOX induced cardiomegaly and cardio-pathological features. Didox significantly prolonged the median survival of DOX treated mice and reduced their mortality risk by 3.7 folds. In conclusion, didox significantly improved the cytotoxic effect of DOX in liver cancer cells and protected from its cardiotoxic side effects.

Citation Format: Fahad A. Al-Abbasi, Gihan F. Assad, Ahmed M. Al-Abd, Ashraf B. Abdel-Naim. The influence of didox on the efficacy and toxicity of doxorubicin. [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 2152. doi:10.1158/1538-7445.AM2013-2152
Abstract 2160: Development of selective MELK kinase inhibitors for cancer treatments.
Authors: Kowalczyk Piotr , Wegrzyn Paulina , Zawadzki Przemyslaw , Palacz Edyta , Trebacz Ewa , Wiklik Katarzyna , Milik Mariusz , Zarebski Adrian , Krawczynska Karolina , Brzózka Krzysztof
Published: August 2014

Maternal embryonic leucine zipper kinase (MELK) is an atypical member of the AMPK family of serine-threonine kinases that has been implicated in stem cell renewal, cell cycle progression, cytokinesis, mRNA splicing and apoptosis. MELK activity is correlated with its phosphorylation level, is cell cycle dependent and maximal during mitosis although direct upstream regulators of MELK kinase activity are unknown. This kinase is highly expressed in several types of solid cancers: colon, breast, ovary, lung, and brain and shows relatively low expression levels in normal tissues. MELK overexpression in patient-derived tumors strongly correlates with poor prognosis in glioblastoma and breast cancer. Moreover, siRNA mediated knockdown of MELK kinase significantly inhibits growth of tumor cell lines both in vitro and in vivo. Therefore, MELK kinase is an emerging and interesting target of significant potential for therapeutic intervention in cancer. In this study, we are reporting results for a series of new MELK kinase inhibitors that were developed at Selvita. Newly synthesized derivatives exert good selectivity and potency in MELK inhibitions with the low sub-micromolar range. Anticancer effects of these compounds were investigated in several cancer cell lines of solid tumor origin where the compounds were shown to induce cell death with low micromolar ED50 values. Compounds were also analyzed for their effects on cell death, proliferation, apoptosis, cell cycle parameters and ADME properties. Taken altogether, the presented data supports our rationale of using inhibitors of MELK kinases as a novel approach for the cancer therapy, especially for the treatment of glioblastoma and breast cancer.

Citation Format: Piotr Kowalczyk, Paulina Wegrzyn, Przemyslaw Zawadzki, Edyta Palacz, Ewa Trebacz, Katarzyna Wiklik, Mariusz Milik, Adrian Zarebski, Karolina Krawczynska, Krzysztof Brzózka. Development of selective MELK kinase inhibitors for cancer treatments. [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 2160. doi:10.1158/1538-7445.AM2013-2160
Abstract 218: R-Spondin (RSPO) signaling drives the growth of multiple human tumor types.
Authors: Gurney Austin , Chartier Cecile , Axelrod Fumiko , Cain Jennifer , Yen Wan-Ching , Murriel Christopher , Raval Janak , Fischer Marcus , Shah Jalpa , Ji May , Bond Christopher , Kapoun Ann , Lewicki John , Hoey Timothy
Published: August 2014

The R-spondin-LGR signaling axis is a recently appreciated pathway that promotes beta-catenin signaling in normal stem cell populations. Beta-catenin signaling had long been considered synonymous with Wnt signaling. We conducted a reporter-based screen for secreted beta-catenin signaling activities produced by a panel of human tumors. This exercise and further molecular characterization efforts discovered RSPO activity produced by numerous human tumors of multiple tumor types including ovarian, pancreatic, colon, breast and non-small cell lung cancer. Specific antibody antagonists of RSPO family members were developed. In minimally passaged human tumor xenograft models, anti-RSPO treatment markedly inhibited tumor growth in several tumor types. Moreover, RPSO blockade promoted tumor differentiation and reduced the frequency of tumor initiating cells. These data highlight the potential for therapeutic intervention with this newly appreciated signaling axis.

Citation Format: Austin Gurney, Cecile Chartier, Fumiko Axelrod, Jennifer Cain, Wan-Ching Yen, Christopher Murriel, Janak Raval, Marcus Fischer, Jalpa Shah, May Ji, Christopher Bond, Ann Kapoun, John Lewicki, Timothy Hoey. R-Spondin (RSPO) signaling drives the growth of multiple human tumor types. [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 218. doi:10.1158/1538-7445.AM2013-218