bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 763: Resistance to cabazitaxel is associated with ABCB1/P-glycoprotein activation, alterations in β-tubulin content and dynamics, reduced BRCA1, and a mesenchymal phenotype in MCF-7 human breast cancer variants
Authors: Duran George E. , Wang Yan C. , Francisco E Brian , Martinez Francisco J. , Sikic Branimir I.
Published: May 2015

In order to identify determinants of cellular response to this new taxane, we derived a resistant variant from the MCF-7 human breast cancer cell line by stepwise selection in cabazitaxel. The 33-fold resistance to cabazitaxel in this variant, MCF-7/CTAX, was associated with ABCB1/P-glycoprotein (P-gp) activation, but also 3-fold residual resistance to taxanes after modulation with the P-gp inhibitor PSC-833, 2 μM. Another variant was established by co-selecting in the presence of PSC-833 (MCF-7/CTAX-P). This variant was negative for ABCB1 transcripts and accumulated parental levels of rhodamine-123, BODIPY-labeled paclitaxel and [3H]-docetaxel, indicating that the taxane resistance (9-fold) observed in this cell line is not mediated by transporters. However, we observed a 34% reduction in bound fluorescent-labeled paclitaxel in MCF-7/CTAX-P relative to parental controls by flow cytometry. These cells are hypersensitive to depolymerizing agents (vincas and colchicine) indicating a change in tubulin dynamic instability, and we observed reduced baseline tubulin polymer in untreated MCF-7/CTAX-P cells, and impaired tubulin polymerization in response to taxane exposure (cabazitaxel or docetaxel) relative to MCF-7 parental cells. Quantitative PCR and immunoblotting confirmed elevated levels of the class III (TUBB3) β-tubulin isotype in both MCF-7/CTAX and MCF-7/CTAX-P. Reduced BRCA1 content was observed in both MCF-7 variants, which could affect taxane response via the regulation of the mitotic spindle checkpoint. Gene silencing with specific siRNAs confirmed that reduced TUBB3 sensitizes cells to cabazitaxel, and reduced BRCA1 results in taxane resistance. In addition, altered epithelial-mesenchymal transition markers (elevated Vimentin, reduced E-cadherin) are associated with cabazitaxel resistance in these MCF-7 variants. In summary, cabazitaxel resistance mechanisms include MDR (although at a lower level than paclitaxel and docetaxel), and alterations in microtubule dynamicity, as manifested by higher expression of TUBB3, decreased BRCA1, and by the induction of EMT.

Citation Format: George E. Duran, Yan C. Wang, E Brian Francisco, Francisco J. Martinez, Branimir I. Sikic. Resistance to cabazitaxel is associated with ABCB1/P-glycoprotein activation, alterations in β-tubulin content and dynamics, reduced BRCA1, and a mesenchymal phenotype in MCF-7 human breast cancer variants. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 763. doi:10.1158/1538-7445.AM2014-763
Abstract 765: Short- and long-term TNF-alpha mediated NF-κB signaling exerts diverging effects on ABCB1/PgP expression
Authors: Walther Wolfgang , Bauer Lisa , Aumann Jutta , Kobelt Dennis , Stein Ulrike S.
Published: May 2015

Multidrug resistance (MDR) still represents one major obstacle for cancer chemotherapy. The ABC transporter ABCB1/PgP is an important factor mediating resistance of tumor cells towards cytostatic drug treatment. Among numerous overcoming strategies, persistent treatment of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) leads to down-regulation of MDR-associated genes, such as ABCB1. The transcription factor NF-κB/p65 is a known key regulator of ABCB1 expression. We analyzed short- and long-term effects of TNF-alpha regarding modulation of NF-κB/p65 signaling and ABCB1/PgP expression in colon carcinoma cells. Highly resistant ABCB1 endogenously overexpressing HCT15 human colon carcinoma cells were subjected to short- (30-120 min) or long-term (24-96 h) TNF-alpha treatment. TNF-alpha-mediated modulation of ABCB1/PgP expression was analyzed by real-time RT-PCR and Western-blot. The effect of TNF-receptor triggered NF-κB/p65 signaling on ABCB1/PgP expression was analyzed by Western blot, ABCB1 promoter reporter assays and by EMSA. The effects of TNF-alpha on drug accumulation and cytotoxicity were determined by FACScan analysis and MTT cytotoxicity assays.

The studies revealed that long-term, but not short-term action of TNF-alpha leads to TNF-receptor 1-mediated down-regulation of ABCB1/PgP expression. It furthermore dampens NF-κB/p65 activation as well as IκBα resynthesis. This is associated with reduced nuclear accumulation of NF-κB/p65 and lack of its binding to the NF-κB/p65 consensus sequence in the ABCB1 promoter. In consequence long-term TNF-alpha treatment mediates significant increase in drug accumulation and sensitization of HCT15 cells towards drug treatment. The study reveals the diverging effects of short- or long-term TNF-alpha action and provides novel insights into mechanism of ABCB1 regulation by this pro-inflammatory cytokine.

Citation Format: Wolfgang Walther, Lisa Bauer, Jutta Aumann, Dennis Kobelt, Ulrike S. Stein. Short- and long-term TNF-alpha mediated NF-κB signaling exerts diverging effects on ABCB1/PgP expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 765. doi:10.1158/1538-7445.AM2014-765
Abstract 766: Human ABCB1 and ABCG2 confer acquired resistance to Polo-like kinase 1 inhibitors, BI 2536, volasertib and GSK641364
Authors: Wu Chung-Pu , Sim Hong-May , Ambudkar Suresh V.
Published: May 2015

The overexpression of the serine/threonine specific polo-like kinase 1 (Plk1) has been detected in various types of cancer, and thus has fast become an attractive therapeutic target for cancer therapy. Plk1 inhibitors BI 2536, volasertib and GSK461364, were designed to selectively inhibit cancer cell proliferation by promoting G2/M cell cycle arrest at nanomolar concentrations. Unfortunately, alike most chemotherapeutic agents, the development of acquired resistance to Plk1 inhibitors is prone to present a significant therapeutic challenge. One of the most common mechanisms for acquired resistance in cancer chemotherapy is associated with the overexpression of ATP-binding cassette (ABC) transporters ABCB1, ABCC1 and ABCG2. Here, we discovered that in human cancer cells, the overexpression of ABCB1 and/or ABCG2 can lead to acquired resistance to three selective Plk1 inhibitors, BI 2536, volasertib and GSK461364. Moreover, these Plk1 inhibitors stimulate the ATPase activity of ABCB1 and ABCG2, as well as competitively inhibit the drug substrate transport mediated by ABCB1 and ABCG2. More significantly, the reduced chemosensitivity and Plk1 inhibitors-mediated G2/M cell cycle arrest in cancer cells overexpressing either ABCB1 or ABCG2 can be significantly restored in the presence of selective inhibitor of ABCB1 and ABCG2. Taken together, our findings indicate that in order to circumvent ABCB1 or ABCG2-mediated acquired resistance to Plk1 inhibitors, a combined regimen of Plk1 inhibitors and modulators or clinically active drugs that potently inhibit the function of ABC drug transporters, should be considered as a potential treatment strategy in the clinic.

Citation Format: Chung-Pu Wu, Hong-May Sim, Suresh V. Ambudkar. Human ABCB1 and ABCG2 confer acquired resistance to Polo-like kinase 1 inhibitors, BI 2536, volasertib and GSK641364. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 766. doi:10.1158/1538-7445.AM2014-766
Abstract 772: CCL2 regulates EMT-mediated chemo-resistance in prostate cancer
Authors: Wang Wenchu , Lu Yi , Zhou Xiaolin , Mizokami Atsushi , Keller Evan , Zhang Jian
Published: May 2015

Prostate cancer is one of the most common cancers and the second leading cause of cancer-related death in western countries. In China, the incidence has increased, unexpectedly and sharply, for the past 20 years. Chemo-resistance, in various types of tumor including prostate cancer, is one of the critical problems in cancer therapy. In prostate cancer, for example, the resistance to taxanes (paclitaxel or docetaxel, DTX) accounts for tumor relapse and progression resulting in skeletal metastasis and high mortality. Epithelial-mesenchymal transition (EMT) has been suggested as a key process that dominates chemo-resistance through unknown mechanisms. In the current study, we reported that CCL2 serves as key molecules in EMT-mediated chemo-resistance in prostate cancer cells using paclitaxel-resistant cells, DU145-TxR, PC-3-TxR and C4-2B-TxR. Parental prostate cancer cells were used as controls. We observed that DU145-TxR, PC-3-TxR and C4-2B-TxR cells produced significantly higher amount of CCL2 compared to their parental cells, determined by cytokine array. In addition, DU145-TxR, PC-3-TxR and C4-2B-TxR cells demonstrated EMT phenotypes including hall maker changes in E-cadherin and Vimentin expression and transcriptional factor (Snail, ZEB1, and Slug) alterations, examined by western blot. These changes were reversed partially by adding neutralizing antibodies against CCL2. Finally DU145-TxR, PC-3-TxR, and C4-2B-TxR or their parental cells were implanted into SCID mice and the mice were given DTX (10mg/kg, biweekly) and tumor growth was monitored for 4 weeks. Both DU145-TxR, PC-3-TxR and C4-2B-TxR tumors grew faster than the parental tumors. Tumor tissues were collected and total RNAs were isolated. Levels of CCL2 expression in these tumors were significantly higher in DU145-TxR, PC-3-TxR and C4-2B-TxR tumors. IHC analysis confirmed corresponding staining of EMT markers such as E-cadherin and Vimentin expression. Together we concluded that CCL2 regulates EMT-mediated chemo-resistance in prostate cancer. These results are of high impact of human prostate cancer chemotherapy. Supported by U.S. Department of Defense PC061231; NSF key project 81130046; NSF projects 81171993 and 81272415.

Citation Format: Wenchu Wang, Yi Lu, Xiaolin Zhou, Atsushi Mizokami, Evan Keller, Jian Zhang. CCL2 regulates EMT-mediated chemo-resistance in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 772. doi:10.1158/1538-7445.AM2014-772
Abstract 773: Restoration of LKB1 defect in EKVX cells leads to resistance to microtubule targeting agents
Authors: Mao Kaisheng , Liu Fakeng , Liu Xiuju , He Yulong , Khuri Fadlo , Marcus Adam , Li Mingsong , Zhou Wei
Published: May 2015

LKB1 is frequently inactivated in non-small cell lung cancers, probably because of its role in suppressing tumor metastasis. Here, we identified a novel LKB1 mutation in the EKVX lung adenocarcinoma cell line, where serine216 in the kinase domain was mutated to phenylalanine. Genomic sequencing revealed this change to be homozygous, suggesting that the other allele was probably eliminated through a loss of heterozygosity mechanism. We also demonstrated that EKVX cells are defective in energetic stress-induced AMPK phosphorylation, which can be restored by the re-establishment of wild-type but not mutant LKB1 expression. This is consistent with the role of LKB1 as an upstream kinase for AMPK in energetic stress conditions. Interestingly, expression of wild-type LKB1 in EKVX cells was found to be resistant to microtubule targeting agents, such as paclitaxel or vinblastine by SRB analysis. Cell cycle analysis indicated that while the parental or LKB1-mutant expressing cells enter mitotic arrest after 25nM paclitaxel treatment, EKVX-LKB1 cells failed to enter G2/M arrest. Immunofluorescence analysis also revealed a decrease in microtubule bundling after 50nM paclitaxel treatment in EKVX cells with wild-type LKB1 expression. This phenomenon, however, was not observed in other natural LKB1-null A549 or H460 cells. Therefore, our data suggest that the re-establishment of LKB1 expression might lead to resistance to microtubule targeting agents in a subset of non-small cell lung cancer cells with LKB1 mutation.

Citation Format: Kaisheng Mao, Fakeng Liu, Xiuju Liu, Yulong He, Fadlo Khuri, Adam Marcus, Mingsong Li, Wei Zhou. Restoration of LKB1 defect in EKVX cells leads to resistance to microtubule targeting agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 773. doi:10.1158/1538-7445.AM2014-773
Abstract 774: EGFR-regulated RGS expression contributes to paclitaxel resistance in human lung cancer cells
Authors: Park Shin-Hyung , Sung Myung-A , Lee Ho-Young
Published: May 2015

Lung cancer is one of the main causes of cancer-related death worldwide, and the 5-year survival of lung cancer is less than 20%. Paclitaxel is a widely used chemotherapeutic agent to treat a variety of cancers, but its therapeutic efficacy tends to be limited due to the development of drug resistance, raising the need to understand the molecular mechanisms underlying paclitaxel resistance. In the current study, we investigated the mechanisms of paclitaxel resistance in non-small cell lung cancers (NSCLCs). First, we established paclitaxel-resistant NSCLC cell lines, including H460 TxR and SK-MES TxR, by pulse- or prolonged treatment of paclitaxel. The IC50 value of paclitaxel-resistant cell lines was 50 fold less than that of parental cells. Upon paclitaxel treatment, H460 TxR and SK-MES TxR sublines experienced markedly greater anchorage-dependent and -independent colony forming abilities with reduced apoptosis than did the parental cell lines. Next, we investigated the underlying molecular mechanism of acquired resistance against paclitaxel. Through microarray analysis, we found 19 genes commonly-increased both in H460 TxR and SK-MES TxR. Among them, the expression of regulator of G-protein signaling (RGS) gene increased the most. Knockdown of RGS gene expression by siRNA sensitized the paclitaxel-resistant cells to the drug treatment in vitro. Furthermore, H460 TxR xenograft mice showed a significant decrease in tumor growth after combined treatment with siRGS and paclitaxel, implying the association of RGS with paclitaxel resistance in NSCLC. To identify the mechanisms involved in increase in RGS expression, we assessed the phosphorylation level of several kinases involved in cell proliferation and survival. Among them, epidermal growth factor receptor (EGFR) showed the most significant phosphorylation. EGFR inhibition by erlotinib down-regulated RGS expression in a transcriptional level, leading to the enhanced sensitivity to paclitaxel in H460 TxR. Taken together, our results indicate that RGS overexpression is a mediator of paclitaxel resistance in NSCLCs and represent the first link between RGS and chemoresistance. We suggest that RGS might be a potential target of therapeutic strategies for lung cancer patients with paclitaxel resistance.

Citation Format: Shin-Hyung Park, Myung-A Sung, Ho-Young Lee. EGFR-regulated RGS expression contributes to paclitaxel resistance in human lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 774. doi:10.1158/1538-7445.AM2014-774
Abstract 777: Efficacy of nab-paclitaxel on 5-fluorouracil (5-FU) resistant human gastric cancer cell lines
Authors: Nukatsuka Mamoru , Kobunai Takashi , Matsuoka Kazuaki , Takechi Teiji
Published: May 2015

Purpose: 5-Fluorouracil (5-FU)-based combination chemotherapy is widely used for the treatment of advanced gastric cancer in Japan. Thus, the development of effective chemotherapy for 5-FU-resistant gastric cancer after 5-FU-based chemotherapy is important. Nab-paclitaxel is an albumin-bound, 130-nm particle form of paclitaxel that exhibits a higher activity and lower toxicity than solvent-based paclitaxel. In the present study, the activity of nab-paclitaxel alone or in combination with 5-FU was evaluated in four 5-FU resistant cell lines.

Method: 5-FU-resistant human gastric cancer cell lines were established from parent cells (MKN-45, MKN-74, NCI-N87, and KATO-III) by continuous exposure to escalating concentrations of 5-FU over a one-year period. The sensitivities of these cell lines to chemotherapeutic agents (5-FU and nab-paclitaxel) were evaluated using the WST-8 colorimetric assay. To evaluate the effects of the combination of 5-FU and nab-paclitaxel, the combination index (CI) value was calculated using CalcuSyn 2.0 (Biosoft, Cambridge, UK). The gene expressions of secreted protein acidic and rich in cysteine (SPARC) in the parent and 5-FU-resistant cell lines were evaluated using a Whole Human Genome 44K oligo DNA microarray (Agilent Technologies, Santa Clara, CA).

Results: The IC50 value of 5-FU ranged from 2.35 to 36.7 μM, and its ratio (resistant cell lines/parent cell lines) ranged from 2.6 to 15.6. The IC50 value of nab-paclitaxel ranged from 4.45 to 54.5 nM, and its ratio ranged from 1.0 to 1.2. Therefore, the 5-FU-resistant cell lines used in this study showed no signs of cross-resistance to nab-paclitaxel. The calculated CI value treated with 5-FU and nab-paclitaxel was approximately 1; therefore, nab-paclitaxel additively inhibited the cell growth when administered in combination with 5-FU. Interestingly, the combination of 5-FU and nab-paclitaxel had an additive cytotoxic activity in the 5-FU-resistant human gastric cell lines in vitro. The relative expression of SPARC mRNA in the resistant cell lines to that in the parent ones ranged from 0.86 to 1.47, and SPARC expression might be weakly correlated with the acquisition of 5-FU resistance. Conclusion: Nab-paclitaxel might be a promising agent for the treatment of 5-FU-resistant human gastric cancer when used alone or in combination with 5-FU. These results suggest that nab-paclitaxel might be a promising chemotherapy for gastric cancer patients who relapse after undergoing 5-FU-based chemotherapy.

Citation Format: Mamoru Nukatsuka, Takashi Kobunai, Kazuaki Matsuoka, Teiji Takechi. Efficacy of nab-paclitaxel on 5-fluorouracil (5-FU) resistant human gastric cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 777. doi:10.1158/1538-7445.AM2014-777
Abstract 778: Antioxidants decrease the apoptotic effect of 5-Fu in colon cancer by regulating Src-dependent caspase-7 phosphorylation
Authors: Fu Yang , Yang Ge , Zhu Feng , Peng Cong , Li Wei , Li Haitao , Kim Hong-Gyum , Bode Ann M. , Dong Ziming , Dong Zigang
Published: May 2015

Although the rate of development of drug resistance remains very high, 5-Fu is still the most common chemotherapeutic drug used for the treatment of colon cancer. A better understanding of the mechanism of why cancers develop resistance to 5-Fu could improve its therapeutic effect. Sometimes antioxidants are used simultaneously with 5-Fu treatment. However, a recent clinical trial showed no advantage or even a harmful effect of combining antioxidants with 5-Fu compared to administration of 5-Fu alone. The mechanism explaining this phenomenon is still poorly understood. In this study, we show that 5-Fu can induce reactive oxygen species-dependent Src activation in colon cancer cells. Mouse embryonic fibroblasts that are deficient in Src showed a clear resistance to 5-Fu, and knocking down Src protein expression in colon cancer cells also decreased 5-Fu-induced apoptosis. We found that Src could interact with and phosphorylate caspase-7 at multiple tyrosine sites. Functionally, the tyrosine phosphorylation of caspase-7 increases its activity, thereby enhancing cellular apoptosis. When using 5-Fu and antioxidants together, Src activation was blocked, resulting in decreased 5-Fu-induced apoptosis. Our results provide a novel explanation as to why 5-Fu is not effective in combination with some antioxidants in colon cancer patients, which is important for clinical chemotherapy.

Keywords: antioxidant; 5-Fu; Src; caspase-7; colon cancer; apoptosis

Citation Format: Yang Fu, Ge Yang, Feng Zhu, Cong Peng, Wei Li, Haitao Li, Hong-Gyum Kim, Ann M. Bode, Ziming Dong, Zigang Dong. Antioxidants decrease the apoptotic effect of 5-Fu in colon cancer by regulating Src-dependent caspase-7 phosphorylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 778. doi:10.1158/1538-7445.AM2014-778
Abstract 78: A novel orthotopic mammary epithelial cell (MEC) transplantation model of breast cancer formation
Authors: Szucs Zoltan , Prater Michael D. , Stingl John , Brindle Kevin M.
Published: May 2015

The pathogenesis of breast cancer is polygenic. Exploring the involvement of different onco- and tumor suppressor genes, and combinations of these genes, in early breast cancer formation is important for understanding the natural history of the disease. Preclinical studies of the fundamental biological and metabolic changes that occur during breast oncogenesis in currently available transgenic mouse models are limited by the requirement for tissue-specific promoters and the random nature of tumor formation.

The mouse mammary gland is a unique organ in that most of its development occurs after birth. Moreover, isolated mammary epithelial cells (MECs) have the capacity to regenerate a functional gland upon orthotopic transplantation into the cleared fat pad of a syngeneic recipient. We describe here a new transplantation system using pure MECs derived from genetically engineered mice harboring floxed oncogenes. First we selected in vitro for a stromal and hematopoietic cell-free MEC preparation. In the MEC donor transgenic strains the activation of oncogenes (K-Ras, c-Myc) or the deletion of tumor suppressors (p53) is dependent on Cre recombinase expression (Cre-lox system). Using an adenoviral Cre-recombinase vector we activated these oncogenic changes in isolated MECs. Colony formation assays demonstrated the viability of these mixed transduced/non-transduced MEC populations. Following transplantation into the cleared fat pad of syngeneic mice the transduced MECs preserved their fat pad repopulating and duct forming potential and the induced oncogenic changes resulted in mammary tumor formation. This proposed system offers tissue specific promoter-free and lactation independent spatial control of oncogenic events in the epithelial cell compartment of breast ducts.

Citation Format: Zoltan Szucs, Michael D. Prater, John Stingl, Kevin M. Brindle. A novel orthotopic mammary epithelial cell (MEC) transplantation model of breast cancer formation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 78. doi:10.1158/1538-7445.AM2014-78
Abstract 782: Transmembrane 2-3 loop domain of the proton coupled folate transporter (PCFT) forms a novel “reentrant loop” structure and is functionally important
Authors: Wilson Michael R. , Hou Zhanjun , Matherly Larry H.
Published: May 2015

The proton coupled folate transporter (PCFT) is a proton-folate symporter with a twelve transmembrane domain (TMD) structure. Human PCFT (hPCFT) is highly expressed in many solid tumors and has an acidic pH optimum, suggesting its high activity in the acidic tumor microenvironment. Our recent studies have focused on exploiting PCFT for targeting solid tumors with novel cytotoxic antifolates. Predicted topology models suggest that the loop domain between TMDs 2 and 3 resides in the cytosol. However, mutations in TMD2-3 loop residues Asp109 or Arg113 result in loss of PCFT activity, suggesting an important role in transport activity. Additionally, TMD2 may itself form a part of the substrate binding domain, suggesting functional importance for TMD2 residues. Using site-directed mutagenesis, we replaced each of the seven cysteine (cys) residues of hPCFT with serine, creating functional “cys-less” PCFT for use in substituted cys accessibility methods (SCAM). We then used site-directed mutagenesis to create 34 single cys mutants spanning TMD2 and the TMD 2-3 loop region (positions 85-118) in a cys-less PCFT background. These mutants were transfected into R1-11 HeLa cells, which express no endogenous folate transporters. We measured [3H]methotrexate uptake at pH 5.5 as a readout of PCFT activity and confirmed PCFT protein expression by western blotting. All 34 mutants were detected on westerns and 29 mutants were active, while W107C, D109C, G112C, R113C, and R114C were inactive. Using functionally active mutants, we performed pull-down assays with 2-aminoethyl methanethiosulfonate-biotin (MTSEA-biotin) and steptavidin beads, in order to determine the aqueous accessibility of each mutant. We found that mutants G93C, F94W, G105C, A106C, S110C, and L116C reacted with MTSEA-biotin, suggesting that these residues are aqueous-accessible. For G93C, F94C, and L116C, MTSEA-biotin labeling was inhibited by pemetrexed pretreatment, and MTSEA-biotin treatment inhibited transport activity of G93C and F94C. Our results suggest that the TMD 2-3 loop domain is aqueous accessible and must form a novel functionally important “reentrant loop” structure. Additionally, residues located in TMD2 (Gly93 and Phe94) appear to play a key role in substrate binding to PCFT. Better understanding of functional and structural determinants of PCFT membrane transport will be critical to the development of novel PCFT-selective antifolate drugs for targeting solid tumors.

Citation Format: Michael R. Wilson, Zhanjun Hou, Larry H. Matherly. Transmembrane 2-3 loop domain of the proton coupled folate transporter (PCFT) forms a novel “reentrant loop” structure and is functionally important. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 782. doi:10.1158/1538-7445.AM2014-782
Abstract 784: Significance of osteopontin in the sensitivity of malignant pleural mesothelioma to pemetrexed
Authors: Takeuchi Susumu , Seike Masahiro , Noro Rintaro , Soeno Chie , Sugano Teppei , Zou Fenfei , Matsumoto Masaru , Miyanaga Akihiko , Minegishi Yuji , Kubota Kaoru , Gemma Akihiko
Published: May 2015

Background: Pemetrexed (PEM) is currently recommended as one of the standard anticancer drugs for malignant pleural mesothelioma (MPM). However, the mechanism of the sensitivity of MPM to PEM remains unclear.

Materials and Methods: We analyzed the antitumor effects of PEM in six MPM cell lines by MTS assay. To identify genes associated with drug sensitivity, we conducted gene expression profiling on the same set of cell lines using GeneChips and pathway analysis.

Results: Three cell lines were sensitive to PEM. Eighteen transcripts and fourteen genes identified by Gene-chips were significantly correlated with sensitivity to PEM. Pathway analysis revealed that osteopontin (SPP1/OPN) was an important target in PEM sensitivity. Overexpression of SPP1/OPN was observed in the sensitive cells by quantitative RT-PCR analysis and Western blotting. Introduction of SPP1/OPN by lentiviral vector significantly enhanced the invasion activities of MPM cells. PEM treatment with SPP1/OPN knockdown inhibited the PEM-induced cell growth-inhibitory effect in PEM-sensitive cells. Expression of SPP1/OPN and AKT phosphorylation significantly decreased after PEM treatment of the PEM sensitive cells. High immunohistochemical expression of SPP1/OPN was observed in two of three MPM patients who had a partial response to PEM-based chemotherapy.

Conclusion: PEM has anti-tumor effects in MPM cells dependent on SPP1/OPN overexpression resulting in AKT activation. Our results suggest that SPP1 may be used as a single predictive biomarker of the effectiveness of PEM treatment in MPM.

Citation Format: Susumu Takeuchi, Masahiro Seike, Rintaro Noro, Chie Soeno, Teppei Sugano, Fenfei Zou, Masaru Matsumoto, Akihiko Miyanaga, Yuji Minegishi, Kaoru Kubota, Akihiko Gemma. Significance of osteopontin in the sensitivity of malignant pleural mesothelioma to pemetrexed. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 784. doi:10.1158/1538-7445.AM2014-784
Abstract 788: Lichen-derived metabolites show synergistic effects in combination with lapatinib and doxorubicin against breast cancer cells
Authors: Bessadottir Margret , Skuladottir Edda A. , Baldursson Sindri , Omarsdottir Sesselja , Ögmundsdottir Helga M.
Published: May 2015

Protolichesterinic acid (PA) is the major biologically active second metabolite of the lichen Cetraria islandica. PA has anti-proliferative effects on several types of cancer cells, but no effect on normal skin fibroblasts and is an inhibitor of 5- and 12-lipoxygenase (LOX). Usnic acid (UA) (from Cladonia arbuscula) has a wide range of biological activities, e.g. anti-inflammatory, anti-viral and anti-bacterial activity. UA affects pH gradients and inhibits growth and proliferation of several cancer cells.

Fatty acid synthase (FASN) is up-regulated in a variety of cancers. Overexpression of human epidermal growth factor receptor 2 (HER2) has been linked to increased translation of FASN. The chemical structure of PA is very similar to the FASN inhibitor, C75. FASN inhibition interacts synergistically with HER2 targeted drugs (e.g. lapatinib) in breast cancer cells and can overcome resistance. Doxorubicin stops DNA replication. The accumulation and distribution of doxorubicin in cells is pH-dependent. Synergistic effects of the lichen compounds and lapatinib or doxorubicin were estimated, using the CalcuSyn software, in the breast cancer cell lines, SK-BR-3 (HER2+, high levels of FASN) T-47D (P53-mutated) and MCF7. Significant synergistic effects were obtained in SK-BR-3 cell line after combined treatment with PA/lapatinib, and in T-47D cells but at higher concentrations. No synergism was seen in MCF7 cells. Treatment with UA/doxorubicin showed marked synergism at all tested concentrations in T-47D cells; none was seen in SK-BR-3 cells. Synergism between a lichen compound and a drug can be related to metabolic effects. Moreover the synergistic effects appear to be cell-type-dependent.

Citation Format: Margret Bessadottir, Edda A. Skuladottir, Sindri Baldursson, Sesselja Omarsdottir, Helga M. Ögmundsdottir. Lichen-derived metabolites show synergistic effects in combination with lapatinib and doxorubicin against breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 788. doi:10.1158/1538-7445.AM2014-788
Abstract 789: Parthenolide sensitizes colorectal cancer cells to TRAIL-induced apoptosis by regulating mitochonrial pathway
Authors: Kim Se Lim , Kim Sang-Wook , Lee Soo-Teik , Kim Seong Hun , Kim In Hee , Lee Seung Ok , Kim Dae Ghon
Published: May 2015

Combination therapy utilizing of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in conjunction with other anticancer agents, is a promising strategy to overcome TRAIL resistance in malignant cells. Recently, parthenolide (PT) has proved to be a promising anticancer agent, and several studies have explored its use in combination therapy. Here, we investigated the molecular mechanisms by which PT sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29 cells with an even higher dose of TRAIL. A combination of PT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29 cells. Consistent with cell growth inhibition, apoptotic cell death was significantly increased by a combination of PT with TRAIL in both of HCT116 and HT-29 cells. Results of flow cytometry analysis demonstrated that TRAIL-sensitive HCT116 cells had much higher death receptor (DR) 5 than TRAIL-resistant HT-29 cells. Interestingly, treatment of PT and/or TRAIL did not affect DR4/DR5, these results indicate that the apoptotic effect of combination is death receptor-independent apoptosis. We observed that the synergistic effect was associated with Bcl-2 family members, p53 and cytochrome C. Moreover, activation of caspase -3, -8 and -9 was increased by combination treatment in both of TRAIL-resistant and -sensitive cells. These results suggest that PT sensitizes TRAIL-induced apoptosis via death receptor-independent and mitochondrial-dependent pathway. Combination treatment using PT and TRAIL might offer an effective strategy to overcome TRAIL resistance in certain CRC cells.

Citation Format: Se Lim Kim, Sang-Wook Kim, Soo-Teik Lee, Seong Hun Kim, In Hee Kim, Seung Ok Lee, Dae Ghon Kim. Parthenolide sensitizes colorectal cancer cells to TRAIL-induced apoptosis by regulating mitochonrial pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2014-789
Abstract 79: Deletion of STAT3 in a mouse model for metastatic melanoma
Authors: Swoboda Alexander , Sobieszek Isabel J. , Schlederer Michaela , Zahma Safia , Marjanovic Jelena , Poli Valeria , Kenner Lukas , Hengstschläger Markus , Mikula Mario , Moriggl Richard
Published: May 2015

The incidence of metastatic melanoma, a very aggressive type of human skin cancer, has steadily increased over the last years and is associated with high mortality. Activating mutations in the N-Ras proto-oncogene are characteristic for cutaneous melanoma. The most frequent mutation found in N-Ras is a lysine substitution of glutamine at codon 61 (Q61K) constitutively activating the protein. Together with other mutations, especially inactivation of the CDKN2A locus, it is sufficient for spontaneous melanoma formation and subsequent metastasis formation in mice.

STAT3 proteins are highly phosphorylated in melanoma. We identified strong STAT3 expression and activation in 70% of patients that show melanoma brain metastases. Here, we investigated whether deletion of this transcription factor in a mouse model expressing mutant N-RasQ61K transgene under the control of the tyrosinase promoter in combination with a homozygous CDKN2A deletion, leads to modified melanoma formation. Interestingly, we found decreased survival in male mice that do not express STAT3 in melanoma cells. We analyzed primary tumor development, metastasis, and survival using histo-pathological examination between groups of STAT3 positive and negative mice.

To further assess whether the enhanced tumor progression upon loss of STAT3 is a cell autonomous effect we established melanoma cell lines derived from mouse tumors with or without STAT3. These novel cell lines will be used to analyze growth and survival properties. We will also test the influence of a functional immune system to question immunoediting by xenotransplantation experiments.

Citation Format: Alexander Swoboda, Isabel J. Sobieszek, Michaela Schlederer, Safia Zahma, Jelena Marjanovic, Valeria Poli, Lukas Kenner, Markus Hengstschläger, Mario Mikula, Richard Moriggl. Deletion of STAT3 in a mouse model for metastatic melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 79. doi:10.1158/1538-7445.AM2014-79
Abstract 790: A high-throughput chemical screen identifies synergistic activity between neurotransmitter receptor inhibitors and crizotinib in ALK-mutated neuroblastoma
Authors: Moore Nathan , Pukma Srunphut , Gray Nathanael , George Rani
Published: May 2015

Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent important therapeutic targets in neuroblastoma, a common childhood cancer that accounts for 15% of all pediatric oncology deaths. One of the more common mutations, ALKF1174L, is sensitive to the sole FDA-approved ALK inhibitor, crizotinib, only at high doses and mediates acquired resistance to crizotinib in ALK-rearranged cancers. To identify compounds that would synergize with crizotinib to increase cytotoxicity in neuroblastoma cells, we devised a high-throughput small molecule synergy screen using the ALKF1174L-dependent SH-SY5Y cell line to compare cell viability between screen compound alone and screen compound in combination with crizotinib. Compounds ranged from those with clinical approval and known therapeutic indications to those with only predicted bioactivity. Of ∼10,000 compounds screened, the most recurrent hits included adrenergic and dopaminergic neurotransmitter receptor inhibitors. Validation studies with a candidate dopamine receptor D2 antagonist, chlorpromazine, resulted in increased cytotoxicity when combined with crizotinib over that of either single agent alone. These studies support further pre-clinical efforts to explore the therapeutic potential of combining crizotinib with neurotransmitter receptor inhibitors in ALKF1174L-mutated neuroblastoma.

Citation Format: Nathan Moore, Srunphut Pukma, Nathanael Gray, Rani George. A high-throughput chemical screen identifies synergistic activity between neurotransmitter receptor inhibitors and crizotinib in ALK-mutated neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 790. doi:10.1158/1538-7445.AM2014-790
Abstract 792: Usefulness of alternate-day administration of S-1 and leucovorin in a xenograft mouse model of colorectal cancer: A shorter drug-free interval leads to more efficient antitumor effects
Authors: Komura Toshihiro , Ohnuma Shinobu , Miura Koh , Shirasaka Tetsuhiko , Kajiwara Taiki , Kudoh Katsuyoshi , Haneda Sho , Toshima Masahide , Kohyama Atsushi , Musha Hiroaki , Naitoh Takeshi , Katayose Yu , Unno Michiaki
Published: May 2015

[Background] A clinical trial of S-1 with leucovorin (S-1/LV) in metastatic colorectal cancer (CRC) patients demonstrated promising efficacy; however, the gastrointestinal toxicities were so severe that it has not been applied in the clinical setting. On the other hand, alternate-day administration of S-1 has been proposed to attenuate the adverse events without reducing its anticancer activity. The aim of this study was to confirm the feasibility of alternate-day administration of S-1/LV in in vivo xenograft tumor models.

[Methods] Mice were treated with S-1/LV either in a daily group (two weeks of administration followed by two weeks of withdrawal) or an alternate-day group (administration on alternate days for four weeks), then the mice of both groups were sacrificed and the xenograft tumors were resected. To assess the adverse reactions, the body weight changes, the condition of feces, mucosal injury of small intestine and myelosuppression were compared between both groups. Furthermore, tumor volume, tumor growth inhibition (TGI) and the expression of Ki67, TUNEL, cIAP2 and XIAP were analyzed to evaluate the antitumor activity and tumor apoptosis.

[Results] Severe weight loss, diarrhea, mucosal injury and myelosuppression were observed only in the daily group; however, no adverse reactions were observed in the alternate-day group, except slight myelosuppression. The TGI in the alternate-day group was better than that in the daily group, possibly resulting from apoptosis of tumor cells due to the suppression of cIAP2.

[Conclusion] Our findings suggest that alternate-day administration of S-1/LV for CRC treatment can achieve higher antitumor activity without severe adverse reactions. Therefore, we propose that clinical trials with this regimen should be conducted in CRC patients.

Citation Format: Toshihiro Komura, Shinobu Ohnuma, Koh Miura, Tetsuhiko Shirasaka, Taiki Kajiwara, Katsuyoshi Kudoh, Sho Haneda, Masahide Toshima, Atsushi Kohyama, Hiroaki Musha, Takeshi Naitoh, Yu Katayose, Michiaki Unno. Usefulness of alternate-day administration of S-1 and leucovorin in a xenograft mouse model of colorectal cancer: A shorter drug-free interval leads to more efficient antitumor effects. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 792. doi:10.1158/1538-7445.AM2014-792
Abstract 795: Metronomic chemotherapy with cyclophosphamide and metformin inhibits tumor and metastasis growth M-406 murine mammary adenocarcinoma
Authors: Asad Antonela S. , Basualdo Jesús , Micheletti Lucía , Gardenal María C. Capello , Perroud Herman A. , Rico María J. , Rozados Viviana R. , Scharovsky O. Graciela
Published: May 2015

Metronomic chemotherapy (MCT) refers to the chronic, equally spaced, delivery of low doses of different chemotherapeutic drugs, without extended interruptions. We have already demonstrated that MCT, as an intervention strategy, of metronomic Cy combined with celecoxib or doxorubicin inhibits mammary tumors growth. Our present aim was to analyze the antitumor and antimetastatic effects of MCT of Cy combined with Met. Inbred CBi mice were s.c. or i.v. challenged with M-406 tumor (Day 0), and treated when the tumors reached a volume of 100 a 150 mm3 (s.c.) or from Day 3 (i.v.) on, with: I) no further treatment (controls); II) Cy in the drinking water (20-30 mg/kg body weight/day); III) Met 3 times/week by gavage (100 mg/kg); IV) Treated as II + III. Tumors were measured and animals weighed twice a week and blood samples for glycemia determination were taken. On day 36 tumor volume in group IV (mm3, mean ± SEM: 929.7 ± 213.8) was lower than in groups I (2880.0 ± 1530.0), II (2896.4 ± 976.9) and III (3772.0 ± 68.0). On day 46, with only two groups remaining, tumor volume in group IV was lower than that in group II (p=0.032). All the groups differed in tumor volume doubling time (days, mean ± SEM; I: 3.8 ± 0.3, II: 5.3 ± 0.7, III: 3.6 ± 0.4, IV: 5.4 ± 0.2) (p<0.05). All the groups differed in survival (days, median, I: 30.5; II: 44; III: 29; IV: 50) (p=0.0063), showing group IV the highest one. There were neither weight losses nor changes in the glycemia with respect to basal values both in s.c. and i.v. experiments. In the i.v. experiment, animals were sacrificed on day 13, when the first mouse showed signs of metastatic illness. The number and diameter of lung metastasis were determined and total metastatic volume (TMV) was calculated. The experimental groups differed in TMV (p<0.0001), being those of groups II (mm3, mean ± SEM: 162.1 ± 41.4) and IV (218.6 ± 31.3) significantly lower (p<0.001 and p<0.01, respectively) than those in groups I (491.3 ± 80.6) and III (432.3 ± 54.5). These results clearly show that metronomic chemotherapy with a combination of cyclophosphamide and metformin significantly inhibits tumor growth and increase survival, also decreasing the development of metastasis of a murine mammary adenocarcinoma, while being devoid of toxicity.

Citation Format: Antonela S. Asad, Jesús Basualdo, Lucía Micheletti, María C. Capello Gardenal, Herman A. Perroud, María J. Rico, Viviana R. Rozados, O. Graciela Scharovsky. Metronomic chemotherapy with cyclophosphamide and metformin inhibits tumor and metastasis growth M-406 murine mammary adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 795. doi:10.1158/1538-7445.AM2014-795
Abstract 796: Histone deacetylase inhibitors restore toxic BH3 domain protein expression in anoikis-resistant mammary and brain cancer stem cells, thereby enhancing the response to anti-ERBB1/ERBB2 therapy
Authors: Cruickshanks Nichola A. , Hamed Hossein A. , Booth Larry A. , Tavallai Seyedmehrad , Sajithlal G B. , Grant Steven , Poklepovic Andrew , Dent Paul
Published: May 2015

Detachment of cells from the extracellular matrix (ECM) results in a form of cell death referred to as anoikis. Anoikis resistance (AR), frequently acquired by malignant cells, is required for tumor metastases. The present study focused on identifying the mechanisms by which drug sensitivity is restored in previously resistant AR breast cancer cells. AR breast cancer cells display reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA and PUMA. In contrast, expression of the protective BCL-2 family proteins BCL-XL and MCL-1 was increased. It has previously been noted that AR cells display resistance to several currently available anti-tumor cell therapies, including a combinational approach with Lapatinib (ERBB1/2 inhibitor) and Obatoclax (MCL-1 inhibitor, exhibiting reduced autophagic flux, despite similarly exhibiting increased levels of LC3II processing. Knockdown of MCL-1 and BCL-XL in AR cells resulted in apoptosis to a greater extent than in their wild-type counterparts. Pre-treatment of AR cells with Valproate (histone deacetylase inhibitor (HDACIs)); for 24hrs increased the levels of toxic BH3 domain proteins whilst simultaneously reducing levels of MCL-1 and re-sensitising AR cells to therapy-mediated cell death. In vivo, pre-treatment of AR breast tumors in the brain with valproate restored chemo-sensitivity and prolonged animal survival. These data argue that pre-treatment with HDACIs may provide a mechanism to enhance the anti-tumor effect of chemotherapy in AR breast cancer cells.

Citation Format: Nichola A. Cruickshanks, Hossein A. Hamed, Larry A. Booth, Seyedmehrad Tavallai, G B. Sajithlal, Steven Grant, Andrew Poklepovic, Paul Dent. Histone deacetylase inhibitors restore toxic BH3 domain protein expression in anoikis-resistant mammary and brain cancer stem cells, thereby enhancing the response to anti-ERBB1/ERBB2 therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 796. doi:10.1158/1538-7445.AM2014-796