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PDF - 489K, Table S1. Antibodies for Western Blot Analyses. Table S2. Identification of proteins differentially expressed in drug-sensitive and -resistant cells. Fig. S1. Expression levels of stathmin, Ser16-P-stathmin (A) and galectin-1 (B) in drug-sensitive MDA231 and Taxol-resistant K20T cells. Fig. S2. Expression levels of CLIC1, Ezrin and NM23-H1 in drug-resistant A549 and Hey cells. Fig. S3. Suppression of galectin-1 expression in drug-resistant cells. Fig. S4. Suppression of galectin-1 expression increased drug sensitivity in both drug-sensitive and drug-resistant Hey cells.

Supplementary Data from Insulin-like Growth Factor 2 Expression Modulates Taxol Resistance and Is a Candidate Biomarker for Reduced Disease-Free Survival in Ovarian Cancer

Purpose: To evaluate the drug combination of discodermolide and Taxol in human ovarian cancer cells and in an in vivo model of ovarian carcinoma.Experimental Design: The combination index method was used to evaluate the interaction of Taxol and discodermolide in human ovarian SKOV-3 carcinoma cells. Data were correlated with alterations in cell cycle distribution and caspase activation. In addition, SKOV-3 xenograft-bearing mice were treated with either Taxol, discodermolide, or a combination of both drugs given concurrently to evaluate the antitumor efficacy and toxicity of this combination. The Matrigel plug assay and CD31 immunohistochemistry were done to assess antiangiogenic effects.Results: Taxol and discodermolide interact synergistically over a range of concentrations and molar ratios that cause drug-induced aneuploidy in ovarian carcinoma cells. In SKOV-3 xenograft-bearing mice, the combination is significantly superior to either single agent, and induces tumor regressions without notable toxicities. Immunohistochemical analysis of CD31 and Matrigel plug analysis show decreased vessel formation in mice treated with the combination relative to either drug alone.Conclusions: The synergistic activity of Taxol and discodermolide in cells is most potent at drug concentrations that result in drug-induced aneuploidy rather than mitotic arrest. Moreover, in an animal model of ovarian carcinoma, this is a well-tolerated combination that induces tumor regressions and suppresses angiogenesis. These data confirm the potency of this combination and support the use of concurrent low doses of Taxol and discodermolide for potential use in cancer therapeutics.

Supplementary Figure S1 from Potentiation of Taxol Efficacy by Discodermolide in Ovarian Carcinoma Xenograft-Bearing Mice

Purpose: This study was undertaken to examine the role of the insulin-like growth factor (IGF) signaling pathway in the response of ovarian cancer cells to Taxol and to evaluate the significance of this pathway in human epithelial ovarian tumors.Experimental Design: The effect of Taxol treatment on AKT activation in A2780 ovarian carcinoma cells was evaluated using antibodies specific for phospho-AKT. To study the drug-resistant phenotype, we developed a Taxol-resistant cell line, HEY-T30, derived from HEY ovarian carcinoma cells. IGF2 expression was measured by real-time PCR. A type 1 IGF receptor (IGF1R) inhibitor, NVP-AEW541, and IGF2 small interfering RNA were used to evaluate the effect of IGF pathway inhibition on proliferation and Taxol sensitivity. IGF2 protein expression was evaluated by immunohistochemistry in 115 epithelial ovarian tumors and analyzed in relation to clinical/pathologic factors using the χ2 or Fisher's exact tests. The influence of IGF2 expression on survival was studied with Cox regression.Results: Taxol-induced AKT phosphorylation required IGF1R tyrosine kinase activity and was associated with upregulation of IGF2. Resistant cells had higher IGF2 expression compared with sensitive cells, and IGF pathway inhibition restored sensitivity to Taxol. High IGF2 tumor expression correlated with advanced stage (P < 0.001) and tumor grade (P < 0.01) and reduced disease-free survival (P < 0.05).Conclusions: IGF2 modulates Taxol resistance, and tumor IGF2 expression is a candidate prognostic biomarker in epithelial ovarian tumors. IGF pathway inhibition sensitizes drug-resistant ovarian carcinoma cells to Taxol. Such novel findings suggest that IGF2 represents a therapeutic target in ovarian cancer, particularly in the setting of Taxol resistance. Clin Cancer Res; 16(11); 2999–3010. ©2010 AACR.

Supplementary Figure S2 from Potentiation of Taxol Efficacy by Discodermolide in Ovarian Carcinoma Xenograft-Bearing Mice

Supplementary Figures S1 & S2 from Enhancement of the Therapeutic Efficacy of Taxol by the Mitogen-Activated Protein Kinase Kinase Inhibitor CI-1040 in Nude Mice Bearing Human Heterotransplants

Supplementary Figure 2 from Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression

Supplementary Figure 1 from Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression

Taxol may contribute to intrinsic chemoresistance by activating the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cytoprotective pathway in human cancer cell lines and tumors. We have previously shown additivity between Taxol and the MEK inhibitor, U0126 in human cancer cell lines. Here, the combination of Taxol with an orally bioavailable MEK inhibitor, CI-1040, was evaluated in human lung tumors heterotransplanted into nude mice. Unlike xenograft models that are derived from cells with multiple genetic alterations due to prolonged passage, heterotransplanted tumor models are more clinically relevant. Combined treatment with both drugs resulted in inhibition of tumor growth in all models and tumor regressions in three of four models tested, supporting our previous observation that Taxol's efficacy is potentiated by MEK inhibition. Concurrent administration was superior to intermittent dosing. Pharmacodynamic assessments of tumors indicated that suppression of MEK was associated with induction of S473 phosphorylated Akt and reduced proliferation in the combination groups relative to single agents, in addition to suppression of fibroblast growth factor–mediated angiogenesis and reduced expression of vascular endothelial growth factor. These findings are significant and indicate that this combination may have broad therapeutic applications in a diverse range of lung tumors with different intrinsic chemosensitivities.

Lung cancer is a genetically heterogeneous disease characterized by the acquisition of somatic mutations in numerous protein kinases, including components of the rat sarcoma viral oncogene homolog (RAS) and AKT signaling cascades. These pathways intersect at various points, rendering this network highly redundant and suggesting that combined mitogen-activated protein/extracellular signal-regulated kinase (MEK) and mammalian target of rapamycin (mTOR) inhibition may be a promising drug combination that can overcome its intrinsic plasticity. The MEK inhibitors, CI-1040 or PD0325901, in combination with the mTOR inhibitor, rapamycin, or its analogue AP23573, exhibited dose-dependent synergism in human lung cancer cell lines that was associated with suppression of proliferation rather than enhancement of cell death. Concurrent suppression of MEK and mTOR inhibited ribosomal biogenesis by 40% within 24 h and was associated with a decreased polysome/monosome ratio that is indicative of reduced protein translation efficiency. Furthermore, the combination of PD0325901 and rapamycin was significantly superior to either drug alone or PD0325901 at the maximum tolerated dose in nude mice bearing human lung tumor xenografts or heterotransplants. Except for a PTEN mutant, all tumor models had sustained tumor regressions and minimal toxicity. These data (a) provide evidence that both pathways converge on factors that regulate translation initiation and (b) support therapeutic strategies in lung cancer that simultaneously suppress the RAS and AKT signaling network. [Cancer Res 2007;67(23):11300–8]

Supplementary Figure S3 from Enhancement of the Therapeutic Efficacy of Taxol by the Mitogen-Activated Protein Kinase Kinase Inhibitor CI-1040 in Nude Mice Bearing Human Heterotransplants

Discodermolide is a new microtubule-targeted antimitotic drug in Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts synergistically with paclitaxel to inhibit proliferation of A549 human lung cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978–1987, 2000). To understand their synergy, we examined the effects of the two drugs singly and in combination in A549 cells and found that, surprisingly, their antiproliferative synergy is related to their ability to synergistically inhibit microtubule dynamic instability and mitosis. The combination of discodermolide and paclitaxel at their antiproliferative IC50s (7 nm for discodermolide and 2 nm for paclitaxel) altered all of the parameters of dynamic instability synergistically except the time-based rescue frequency. For example, together the drugs inhibited overall microtubule dynamicity by 71%, but each drug individually inhibited dynamicity by only 24%, giving a combination index (CI) of 0.23. Discodermolide and paclitaxel also synergistically blocked cell cycle progression at G2-M (41, 9.6, and 16% for both drugs together, for discodermolide alone, and for paclitaxel alone, respectively; CI = 0.59), and they synergistically enhanced apoptosis (CI = 0.85). Microtubules are unique receptors for drugs. The results suggest that ligands that bind to large numbers of binding sites on an individual microtubule can interact in a poorly understood manner to synergistically suppress microtubule dynamic instability and inhibit both mitosis and cell proliferation, with important consequences for combination clinical therapy with microtubule-targeted drugs.

Supplementary Figure 3 from Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression

Supplementary Tables 1-2 from Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells

Purpose: To determine if ixabepilone is a substrate for cytochrome P450 3A4 (CYP3A4) and if its metabolism by this cytochrome is clinically important, we did a clinical drug interaction study in humans using ketoconazole as an inhibitor of CYP3A4. Experimental Design: Human microsomes were used to determine the cytochrome P450 enzyme(s) involved in the metabolism of ixabepilone. Computational docking (CYP3A4) studies were done for epothilone B and ixabepilone. A follow-up clinical study was done in patients with cancer to determine if 400 mg/d ketoconazole (inhibitor of CYP3A4) altered the pharmacokinetics, drug-target interactions, and pharmacodynamics of ixabepilone. Results: Molecular modeling and human microsomal studies predicted ixabepilone to be a good substrate for CYP3A4. In patients, ketoconazole coadministration resulted in a maximum ixabepilone dose administration to 25 mg/m2 when compared with single-agent therapy of 40 mg/m2. Coadministration of ketoconazole with ixabepilone resulted in a 79% increase in AUC0-∞. The relationship of microtubule bundle formation in peripheral blood mononuclear cells to plasma ixabepilone concentration was well described by the Hill equation. Microtubule bundle formation in peripheral blood mononuclear cells correlated with neutropenia. Conclusions: Ixabepilone is a good CYP3A4 substrate in vitro; however, in humans, it is likely to be cleared by multiple mechanisms. Furthermore, our results provide evidence that there is a direct relationship between ixabepilone pharmacokinetics, neutrophil counts, and microtubule bundle formation in PBMCs. Strong inhibitors of CYP3A4 should be used cautiously in the context of ixabepilone dosing.

Lung cancer is a genetically heterogeneous disease characterized by the acquisition of somatic mutations in numerous protein kinases, including components of the rat sarcoma viral oncogene homolog (RAS) and AKT signaling cascades. These pathways intersect at various points, rendering this network highly redundant and suggesting that combined mitogen-activated protein/extracellular signal-regulated kinase (MEK) and mammalian target of rapamycin (mTOR) inhibition may be a promising drug combination that can overcome its intrinsic plasticity. The MEK inhibitors, CI-1040 or PD0325901, in combination with the mTOR inhibitor, rapamycin, or its analogue AP23573, exhibited dose-dependent synergism in human lung cancer cell lines that was associated with suppression of proliferation rather than enhancement of cell death. Concurrent suppression of MEK and mTOR inhibited ribosomal biogenesis by 40% within 24 h and was associated with a decreased polysome/monosome ratio that is indicative of reduced protein translation efficiency. Furthermore, the combination of PD0325901 and rapamycin was significantly superior to either drug alone or PD0325901 at the maximum tolerated dose in nude mice bearing human lung tumor xenografts or heterotransplants. Except for a PTEN mutant, all tumor models had sustained tumor regressions and minimal toxicity. These data (a) provide evidence that both pathways converge on factors that regulate translation initiation and (b) support therapeutic strategies in lung cancer that simultaneously suppress the RAS and AKT signaling network. [Cancer Res 2007;67(23):11300–8]

Purpose: To evaluate the drug combination of discodermolide and Taxol in human ovarian cancer cells and in an in vivo model of ovarian carcinoma. Experimental Design: The combination index method was used to evaluate the interaction of Taxol and discodermolide in human ovarian SKOV-3 carcinoma cells. Data were correlated with alterations in cell cycle distribution and caspase activation. In addition, SKOV-3 xenograft-bearing mice were treated with either Taxol, discodermolide, or a combination of both drugs given concurrently to evaluate the antitumor efficacy and toxicity of this combination. The Matrigel plug assay and CD31 immunohistochemistry were done to assess antiangiogenic effects. Results: Taxol and discodermolide interact synergistically over a range of concentrations and molar ratios that cause drug-induced aneuploidy in ovarian carcinoma cells. In SKOV-3 xenograft-bearing mice, the combination is significantly superior to either single agent, and induces tumor regressions without notable toxicities. Immunohistochemical analysis of CD31 and Matrigel plug analysis show decreased vessel formation in mice treated with the combination relative to either drug alone. Conclusions: The synergistic activity of Taxol and discodermolide in cells is most potent at drug concentrations that result in drug-induced aneuploidy rather than mitotic arrest. Moreover, in an animal model of ovarian carcinoma, this is a well-tolerated combination that induces tumor regressions and suppresses angiogenesis. These data confirm the potency of this combination and support the use of concurrent low doses of Taxol and discodermolide for potential use in cancer therapeutics.

Purpose: Because induction of drug efflux transporters is one of the major underlying mechanisms of drug resistance in cancer chemotherapy, and human pregnane X receptor (hPXR) is one of the principal “xenobiotic” receptors whose activation induces transporter and drug-metabolizing enzyme gene transcription, it would be ideal to develop chemotherapy drugs that do not activate hPXR. This report describes studies undertaken to explore the characteristics of hPXR stimulation and mechanisms of drug-receptor interactions in vitro with new anti-tubulin drugs. Experimental Design: In vitro transient transcription, glutathione S-transferase pull-down assays, and mammalian one-hybrid and two-hybrid systems were used to explore drug-receptor interactions. Loss of righting reflex was used to assess effects of drugs on PXR activity in vivo. Results: The current study showed that paclitaxel, discodermolide, and an analogue of epothilone B, BMS-247550, induced CYP3A4 protein expression in HepG2 hepatoma cells. Transient transcription assays of a luciferase reporter in the presence and absence of a GAL4-steroid and xenobiotic receptor (SXR) plasmid in HepG2 cells showed that these drugs activate hPXR. This was not true for the inactive analogue of paclitaxel, baccatin III, or for an analogue of epothilone A, analogue 5, none of which stabilizes microtubules. To determine the mechanisms by which paclitaxel, discodermolide, and BMS-247550 activate hPXR, a mammalian two-hybrid assay was done using VP16SRC-1 (coactivator) and GAL4-SXR. SRC-1 preferentially augmented the effects of these drugs on hPXR. Expression of SMRT (corepressor) but not NCoR suppressed the drug-induced activation of SXR by ∼50%, indicating a selectivity in corepressor interaction with hPXR. These drugs resulted in shortened duration of loss of righting reflex in vivo, indicating drug-induced activation of PXR in mice. Conclusion: These findings suggest that activation of hPXR with selective displacement of corepressors is an important mechanism by which microtubule-stabilizing drugs induce drug-metabolizing enzymes both in vitro and in vivo.

Next-generation taxanes are known to be active against taxane-resistant cell lines that overexpress P-glycoprotein (P-gp) and/or βIII-tubulin, both of which are associated with drug resistance. In this study, we examined the effect of two potent next-generation taxanes, SB-T-1214 and SB-CST-10202 (C-seco-taxane), on growth of the ovarian cancer cell line Hey and its drug-resistant daughter cell lines that overexpress different levels of P-gp and express increased levels of βIII-tubulin. We found that SB-T-1214 was ∼6-9-fold more potent than SB-CST-10202 against these drug resistant cells. Since the taxanes bind to both tubulin and P-gp, the relative binding affinity of the taxanes to these two cellular targets may influence the effectiveness of the taxanes in resistant cells. To determine the effects of these two taxanes on binding of Taxol® to P-gp and tubulin, a tritium-labeled Taxol® analog, 2-(m-azidobenzoyl)taxol (2-m-AzTax), was used. We have previously demonstrated that 2-m-AzTax photolabels a peptide (amino acids 217-231) in β-tubulin. To study the relative binding affinities of SB-T-1214 and SB-CST-10202, tubulins from bovine brain (BBT) and chicken erythrocytes (CET) were specifically photolabeled with [3H]2-m-AzTax, in the presence and absence of either Taxol®, Taxotere®, SB-T-1214 or SB-CST-10202. β-tubulin isotype content from BBT and CET is different, the former contains βI-, βII-, βIII- and βIV-tubulins, but the latter has only βVI. Taxol® had a minimal inhibitory effect (∼3%) on BBT, but a strong inhibitory effect (99%) on CET. Taxotere® appeared to have a stronger inhibitory effect than Taxol® on photolabeling (31% and 99% for BBT and CET, respectively). The inhibitory effects elicited by the two next-generation taxanes on photolabeling were distinct for β-tubulins from these two sources. SB-T-1214 and SB-CST-10202 inhibited BBT photolabeling by 38% and 32%, respectively. Interestingly, these two drugs caused a 41% and only 6% inhibition, respectively, on photolabeling of CET that does not contain βIII-tubulin. We are currently analyzing the effect of SB-T-1214 on binding of [3H]2-m-AzTax to different tubulin isotypes resolved by isoelectrofocusing. To study photolabeling of P-gp, plasma membranes from a multidrug resistant (MDR) cell line SKVLB1 that expresses very high levels of P-gp were prepared. SB-T-1214 and SB-CST-10202 inhibited photolabeling of P-gp markedly (> 80% inhibition), suggesting that these two taxanes are good substrates for P-gp. They also caused an increase in steady state [3H]2-m-AzTax accumulation in the MDR cell line SKVLB1. Since drug binding to tubulin and P-gp is the primary event that occurs in the cell following drug administration, our data on taxane binding affinity to the two targets will help in understanding the effectiveness of the next-generation taxanes in taxane-resistance. Citation Format: Chia-Ping H. Yang, Changwei Wang, Iwao Ojima, Susan Band Horwitz. The next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibit distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1229.

Class V βeta tubulin isotype (βV-tubulin), was recently found to have a tissue-specific expression pattern in epithelial tissues with secretory function, and deregulated expression in certain tumors. The overall aim of this study was to define the localization of βV-tubulin in the fallopian tube and its expression in pre-malignant dysplasia. Fallopian tube epithelium (FTE) was obtained from patients undergoing salpingectomy for various reasons including familial history that comprised BRCA mutant cases. Additionally, βV-tubulin expression was also examined in serous ovarian neoplasms. Immunohistochemistry, using a novel antibody developed in our lab against human βV-tubulin, was used to evaluate expression in paraffin-embedded sections of the distal fallopian tube (n = 55) and tumors (n = 13), from prospectively selected cases, categorized according to reasons for salpingectomy. Staining with standard markers, Pax-8 and acetylated α-tubulin was done to demarcate the secretory and ciliated cells of the FTE, respectively. Pilot data indicate that in the FTE, βV-tubulin is present in secretory cells and essentially never in ciliated cells. Morphologically “normal” FTE had only rare, scattered βV-tubulin positive cells; whereas, percentage positivity was increased in cases with serous ovarian neoplasms, and BRCA-wildtype familial breast cancer. The highest expression was observed in FTE from patients with BRCA-mutant breast cancers. Four distinct types of FTE atypia were observed in patients with known BRCA mutations. βV-tubulin was highly expressed in serous ovarian neoplasms, with the extent and intensity of staining elevated in high-grade serous carcinomas, compared to serous borderline tumors. In summary, βV-tubulin was localized to secretory cells of the distal FTE and its expression varied according to the clinical diagnosis. The frequency of these cells, and thus expression of βV-tubulin was dramatically enriched in tissue obtained from BRCA mutant cases. BRCA mutants also exhibited pronounced histologic atypia indicative of early dysplasia. Furthermore, elevated expression of βV-tubulin correlated with poor differentiation status in serous ovarian neoplasms. Citation Format: Deepti Mathew, Yanhua Wang, Anne Van Arsdale, Susan Band Horwitz, Hayley McDaid. Atypical expression of βV-tubulin in secretory cells of the fallopian tube as a biomarker for early dysplasia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 482.

Background: Activated MAPK and PI3K/AKT/mTOR pathway signaling are associated with poor prognosis in triple negative Breast cancer (TNBC). Therefore, suppression of both arms of the MAPK/PI3K/AKT/mTOR network is a promising strategy for targeting TNBC. Here we explore the anti-tumor effects of combinations of MEK inhibitor with PI3K, AKT, or mTOR inhibitors with a focus on cell fate and biomarker development in two major subtypes of TNBC, basal-like and mesenchymal. Methods: Combinations of a MEK inhibitor with a PI3K inhibitor, AKT inhibitor, dual mTORC 1/2 inhibitor, or rapalog were evaluated in TNBC cell lines and an orthotopically implanted patient-derived xenograft (PDX) model of TNBC. We utilized reverse-phase protein array (RPPA) and quantitative real-time PCR to interrogate the signaling architecture and transcriptional activity of the treated cell lines. Results: All combinations synergistically suppress the growth of basal-like and mesenchymal TNBC models. RPPA confirms that all combinations suppress common network targets including pERK Th202/T204, pPRAS40 T246, pS6rp S235/236, and p4E-BP1 S65 in all cell lines. Notably, however, comparable repression of MAPK/PI3K/AKT/mTOR signaling produces distinct fates in basal-like and mesenchymal subtypes. Basal-like cell lines preferentially undergo delayed cell death with surviving cells displaying profound growth arrest. In contrast, mesenchymal cell lines respond with uniform quiescence exhibiting little or no cell death. Transcriptional analysis corroborates these phenotypic effects demonstrating differential modulation of genes regulating apoptosis and proliferation in basal-like versus mesenchymal cell lines. Drug treated mesenchymal cells exhibit transcriptional ‘reprogramming’ of epithelial-mesenchymal status as evidenced by reduced mesenchymal gene expression inferring potential effects on invasion and metastasis. In a PDX model of basal-like TNBC that is paclitaxel resistant, early and sustained suppression of tumor growth was achieved by treating once daily with the MEK inhibitor, GDC-0973, in combination with either the PI3K inhibitor, GDC-0941, or the rapalog, Temsirolimus. Concurrent dosing on this intense schedule was associated with some toxicity that could likely be diminished with dose modifications. Conclusions: These data highlight the therapeutic potential of combined MEK and PI3K/AKT/mTOR inhibition in chemo-resistant TNBC. Importantly, they demonstrate innate differences in the response of basal-like and mesenchymal subtypes of TNBC to these combinations, supporting the concept that molecular subtype will be an important predictor of response both in this setting and for other targeted therapies. The combination of GDC-0973 and GDC-0941 performed particularly well in a taxane resistant PDX model and deserves further evaluation in clinical trials for the treatment of TNBC. Citation Format: Sarah J. Schweber, Alicia Rodriguez-Gabin, Jinghang Zhang, Valerie Calvert, Huiping Liu, Emanuel M. Petricoin, III, Eleni Andreopoulou, Susan Band Horwitz, Hayley M. McDaid. Proteomic and transcriptional profiling reveal differential responses to combined MEK and PI3K-mTOR network inhibition in basal-like and mesenchymal subtypes of triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A35.

Objectives: Our objective is to determine the effect of PI3K activity in mediating ligand-independent estrogen signaling and endocrine sensitivity in Uterine Papillary Serous Carcinoma (UPSC). Methods: Drug effect on cell proliferation was calculated via Calcusyn in patient-derived UPSC cell lines ARK 1 and 2. PI3K mutation was analyzed after laser capture microdissection of tumor DNA in 7 consecutive patients with UPSC. Protein expression, via Western Blot, was correlated prospectively with clinical parameters. Results: Table 1 shows cells line baseline characteristics and IC50 to drugs. Fulvestrant, an ER antagonist, rendered the cells significantly more resistant to taxol in ARK1 and 2(p=0.035, 0.021 respectively). The concomitant decrease in pERS167 and pAKTS473 with Fulvestrant treatment is independent of baseline ER expression and PI3K mutation. In ARK2, a cell line that is ER-, disrupting AKT signaling via the PI3K inhibitor GDC-0941, or with the lapatinib is synergistic with Fulvestrant with Combination Index (CI)50 of 0.441 and 0.229, respectively. Independent of HER2 amplification or PI3K mutation, lapatinib and GDC-0941 exhibited synergistic cytotoxicity in both UPSC cell lines (CI50 of 0.577, ARK 1 and 0.233, ARK2). Finally, PI3K mutation and pAKTS473 expression was analyzed in 7 tumor samples. While none harbored PI3K mutation, patients who are chemotherapy resistant had significantly lower expression of baseline pAKTS473 in their tumor samples than those who are chemosensitive, similar to our cell line data. Conclusions: PI3K pathway dysregulation, either via upstream erbB amplification, or downstream constitutive activation of AKT, may be important in mediating endocrine and taxol sensitivity in UPSC. pAKTS473 may be an important biomarker of drug sensitivity. The combination of PI3K inhibitor and lapatinib is synergistic and warrant further therapeutic investigation. Baseline characteristics and drug sensitivities in UPSC cell linesARK1ARK2PI3KCA mutationfExon 9NoERα (WB)YesNopERα167 (WB)YesNopHER (WB)NoYesIC50 nM (mean ± STD)GDC-094193.1±5.9271±116.5LapatinibNo effect135.6±29.2FulvestrantNo effectNo effectTaxol4.6±1.97.8±5.6Cisplatin1103±65.72632.6±1797.9 Citation Format: Kelly S. Levano, Alicia Rodriguez-Gabin, Gary L. Goldberg, Susan B. Horwitz, June Y. Hou. The PI3K inhibitor GDC-0941 is synergistic with lapatinib, and mediates endocrine sensitivity in uterine papillary serous carcinoma. [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 4687. doi:10.1158/1538-7445.AM2014-4687

There are seven β-tubulin isotypes present in distinct quantities in cells of different origin. Altered expression of β-tubulin isotypes were found in cells resistant to microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, ixabepilone and discodermolide, all of which bind to β-tubulin. For example, the level of βIII-tubulin was very low in the human ovarian cancer cell line, Hey, but increased 8- to 15-fold in two drug resistant daughter cell lines, EpoB8 and Ixab80, respectively. The βV-tubulin level was increased by 2-fold in EpoB8 cells. In contrast, levels of βI- and βIV-tubulin were unchanged and decreased, respectively, in EpoB8. Therefore, it is important to measure the binding affinity of MSAs to different β-tubulin isotypes and study their influence on the drug resistant phenotype. To study the relative binding affinities of MSAs, tubulins from HeLa cells, bovine brain, porcine brain and chicken erythrocytes were specifically photolabeled with the tritium labeled 2-(m-azidobenzoyl)taxol, in the presence and absence of Taxol, Taxotere, epothilone B, ixabepilone and discodermolide. β-tubulin isotype content from these sources was different, particularly for chicken erythrocytes that contain only one β-tubulin isotype, βVI. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulins from different sources. Taxol had a minimal inhibitory effect on HeLa, bovine and porcine brain β-tubulin, but a strong inhibitory effect on chicken erythrocyte β-tubulin. Discodermolide exhibited opposite effects on photolabeling, compared to those of Taxol. However, Taxotere appeared to have a stronger inhibitory effect than Taxol on photolabeling of β-tubulins from all sources tested. Ixabepilone had the strongest inhibitory effect on HeLa β-tubulin. The inhibition pattern for β-tubulin from porcine brain correlated very well with that from bovine brain. To determine drug binding affinities for the different β-tubulin isotypes, tubulins from different sources were photolabeled and the isotypes resolved by high resolution isoelectrofocusing (IEF). Approximately 20 bands were seen with bovine and porcine brain tubulin, but only 3-5 bands were resolved with chicken erythrocyte tubulin. Many bands represent posttranslationally modified tubulins. IEF patterns for bovine and porcine brain tubulin were very similar. All bands were analyzed by mass spectrometry following CNBr digestion, and the identity and relative quantity of each β-tubulin isotype determined. After measuring the radioactivity associated with each band, it was found that the binding affinity of 2-(m-azidobenzoyl)taxol to βIII-tubulin was the lowest, compared to other β-tubulin isotypes. Citation Format: Chia-Ping H. Yang, Hui Xiao, Susan Band Horwitz. Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol. [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 833. doi:10.1158/1538-7445.AM2014-833

Eribulin, a microtubule-targeting antitumor drug, is FDA-approved for the treatment of certain patients with advanced or metastatic breast cancer. Some patients treated with this drug have evidence of reduced metastatic dissemination. Triple-negative breast cancer (TNBC), which accounts for approximately 15%-20% of all breast cancers, does not respond to hormone therapies so patients are dependent on chemotherapy. A subset of these patients are intrinsically resistant to chemotherapy, or subsequently relapse and develop metastatic disease, eventually leading to mortality. We explored the mechanistic basis for the antitumor efficacy of eribulin in a panel of triple negative breast cancer cell lines that histologically are classified as basal-like or claudin-low. Specifically, we determined the ability of eribulin to induce cell death versus senescence, and secondly to modulate cancer cell invasion and epithelial to mesenchymal transition (EMT) gene expression. Senescence is a clinically relevant cell fate following chemotherapy that can propel tumor progression and EMT. Eribulin was efficacious at inducing cell death in both subtypes of TNBC, and had a remarkably low propensity to induce senescence in TNBC compared to non-small cell lung cancer and colon cancer cells. A 24-hour treatment with eribulin partially prevented tumor cell invasion in the claudin-low subtype, as assayed by Boyden chamber analysis. Drug-tolerant claudin-low cells that survived high-dose eribulin treatment were also found to have a reduction in EMT markers. In summary, in vitro analysis of cell fate and metastasis indicate that eribulin has an ability to modulate invasiveness and EMT characteristics in preclinical models of TNBC. This research was funded by Eisai Inc. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B165. Citation Format: Jing Hu, Susan Band Horwitz, Hayley M. McDaid. The antitumor efficacy of eribulin is mediated via suppression of invasion and profound induction of cell death in triple negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B165.

Distinct α- and β-tubulin isotypes are present in different quantities in diverse eukaryotic cells. The tritium-labeled photoaffinity Taxol analog, 2-(m-azidobenzoyl)Taxol, was used to photolabel tubulins prepared from HeLa cells, bovine brain and chicken erythrocytes. A 5-fold molar excess of unlabeled compound inhibited the photolabeling of tubulin by >90%, demonstrating the specificity of this photolabeling. Tubulin isotype content varies from one source to another, particularly for chicken erythrocytes that contain only one α-tubulin and one β-tubulin (α1 and βVI). 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulin was carried out in the presence of six different microtubule stabilizing agents, including Taxol, epothilone B, discodermolide, ixabepilone, laulimalide and peloruside A. The inhibitory effects elicited by Taxol, epothilone B, discodermolide and ixabepilone, at a 5-fold molar excess, on photolabeling are different for tubulins prepared from different sources. Taxol had a minimal inhibitory effect on HeLa and bovine brain tubulin, but a strong inhibitory effect on chicken erythrocyte tubulin. Discodermolide exhibited opposite effects on photolabeling, compared to those of Taxol. This is most likely due to the presence of distinct tubulin isotypes in different sources. However, laulimalide and peloruside A that are known to bind to a different site than Taxol in β-tubulin, stimulated photolabeling of tubulins from all three sources. Stathmin (S) plays an important role in the regulation of microtubule dynamics through sequestering tubulin (T) into a T2S complex. When stathmin was incubated, in the presence of 1 mM CaCl2, with chicken erythrocyte tubulin at a tubulin:stathmin ratio of 2:1, followed by incubation with tritium-labeled 2-(m-azidobenzoyl)Taxol in a buffer containing EGTA, photolabeling of β-tubulin was markedly reduced, suggesting that stathmin inhibited binding of Taxol to tubulin. Radiolabeled photoaffinity drug analogs are useful tools for determining drug binding affinities for the different tubulin isotypes. We are currently analyzing binding affinities of 2-(m-azidobenzoyl)Taxol to different bovine brain tubulin isotypes resolved by isoelectrofocusing. Citation Format: Chia-Ping H. Yang, Hui Xiao, Susan Band Horwitz. Differential inhibition by microtubule stabilizing agents of 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulins from different eukaryotic sources. [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 4496. doi:10.1158/1538-7445.AM2013-4496

Taxol and discodermolide are both microtubule stabilizing agents that bind to the β-tubulin subunit of microtubules. Based on our results that the hydrophobic binding pocket of β-tubulin is occupied by the Taxol side chain but not by (+)-discodermolide, and that the two drugs act synergistically in cancer cell lines, a small library of (+)-discodermolide-Taxol hybrids was synthesized (J. Med. Chem. 54:6319, 2011). Cytotoxicity assays demonstrated a 2-9-fold increase in antiproliferative activity by the hybrids compared to discodermolide in the human cancer cell lines, A549 and MCF-7. Hybrids containing a tether of 3 carbons that connect discodermolide with the Taxol side chain, exhibited the best activity. Tubulin assembly assays were performed with the two most potent hybrid molecules. Like discodermolide, these two hybrids increased tubulin assembly rapidly without a lag period. Tubulin polymerization studies using purified bovine brain tubulin demonstrated that they also had the greatest effect on the formation of polymerized microtubules. These results also were observed in intact A549 cells and in 100,000 x g supernatants prepared from these cells. We have previously shown that [3H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-231 of β-tubulin. A 5-fold molar excess of unlabeled compound inhibited the photolabeling of purified bovine brain tubulin by 94%, demonstrating the specificity of this photolabeling. Microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, discodermolide and ixabepilone, each at a 5-fold molar excess, inhibited the photolabeling by 24%, 92%, 100% and 41%, respectively, indicating that discodermolide is the most potent inhibitor of the photolabeling. In contrast, two other MSAs, laulimalide and peloruside that are known to bind to a different site in β-tubulin, exhibited stimulatory effects on the photolabeling. Both drugs, at a 5-fold molar excess, increased the labeling by 30-40%. [3H]2-(m-azidobenzoyl)Taxol (0.5 - 20 µM) was used to study the kinetics of the inhibitory effects of the hybrid molecules on photoaffinity labeling of tubulin. Discodermolide-Taxol hybrids inhibited the photolabeling of bovine brain tubulin in a dose dependent manner. The concentrations that inhibited by 50% were lowest for the two most potent hybrid molecules. Therefore, the tubulin polymerization activity and the binding affinity of the hybrids to β-tubulin correlated with their antiproliferative activity. Other biological properties of the discodermolide-Taxol hybrids including senescence and antitumor activity are being evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2789. doi:1538-7445.AM2012-2789

Microtubule stabilizing drugs, such as Taxol that is used in the treatment of numerous malignancies, cause distinct responses in cancer cells, including apoptosis, mitotic catastrophe, necrosis and accelerated senescence. However, drug resistance, a complex and mutlifactorial phenotype, is a major obstacle to therapy and the foremost cause of disease progression and cancer-related mortality. MiRNAs are small non-coding RNAs that regulate protein expression by targeting their 3′UTRs, and are emerging as key regulators of cancer pathogenesis that can influence sensitivity to chemotherapy and radiotherapy. We have identified miR-7 as a regulator of sensitivity to the microtubule-stabilizing agents, discodermolide and Taxol. Over-expression of miR-7 in the lung cancer cell lines A549 and H2122, strongly inhibited cell proliferation and sensitized the cells to drug-induced apoptosis. Over-expression of miR-7 also increased the number of acidic lysosomes, as measured by β-galactosidase activity, which likely reflects lysosomal stress. It also increased basal and drug-induced caspase activation, which most likely mediate the sensitizing effects of discodermolide and Taxol. In addition, we validated that the transcription factor, Kruppel like factor 4 (KLF4) is a target of miR-7, and that its expression also regulates sensitivity to discodermolide and Taxol. KLF4 is suppressed in discodermolide- and Taxol-treated A549 cells that have a strong senescent phenotype, and is over-expressed in A549 cells that are resistant to either drugs. ShRNA-mediated knockdown of KLF4 in A549 and H2122 also sensitized cells to discodermolide and Taxol though to a lesser degree than miR-7 overexpression, and also inhibited cell proliferation and increased lysosomal β-galactosidase activity and caspase activity. Thus, we hypothesize that suppression of KLF4 partially mediates the potent anti-tumor effects of miR-7 in A549 and H2122 lung cancer cells. In summary, we have identified miR-7 and its novel target, KLF4, as important regulators of sensitivity to discodermolide and Taxol, and suggest that miR-7 is a promising candidate for therapeutic development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4224. doi:1538-7445.AM2012-4224

Discodermolide is a microtubule stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence. AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells. Lentiviral-mediated re-expression of wild type 4E-BP1 in AD32 cells increased the proliferation rate and reverted resistance to discodermolide via restoration of discodermolide-induced accelerated senescence. Consistent with this, cell growth and response to discodermolide was confirmed in vivo using tumor xenograft models. Furthermore, re-introduction of a non-phosphorylatable mutant (Thr 37/46 Ala) of 4E-BP1 was able to partially restore sensitivity and enhance proliferation in AD32 cells, suggesting that these effects are independent of phosphorylation by mTORC1. Microarray profiling of AD32 resistant cells versus sensitive A549 cells, and subsequent unbiased gene ontology analysis, identified molecular pathways and functional groupings of differentially expressed mRNAs implicated in overcoming discodermolide-induced senescence. The most-statistically significant classes of differentially expressed genes included p53 signaling, G2/M checkpoint regulation and genes involved in the role of BRCA1 in the DNA damage response. Consistent with this, p53 protein expression was up-regulated and had increased nuclear localization in AD32 cells relative to parental A549 cells. Furthermore, the stability of p53 was enhanced in AD32 cells. Our studies propose a role for 4E-BP1 as a regulator of discodermolide-induced accelerated senescence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1228. doi:10.1158/1538-7445.AM2011-1228

Tubulins are a family of proteins consisting of 7 α- and 6 β-tubulin isotypes. Many studies have demonstrated that tumors overexpressing βIII-tubulin have a poor response to chemotherapy. Suppression of βIII-tubulin sensitizes non-small cell lung cancer (NSCLC) cells to microtubule-interacting agents, such as Taxol. βIII-tubulin has also been shown to mediate the incidence and progression of lung cancer. Tubulin isotype content of βI-, βIII-, βIV- and βV-tubulin in the human lung carcinoma cell line A549 is 50%, 8%, 36.5%, and 5.5%, respectively. In our study, the ability of six microtubule stabilizing agents, Taxol, epothilone B (EpoB), discodermolide (Disco), Ixabepilone, laulimalide and peloruside A, to polymerize tubulin in a 100,000 × g supernatant prepared from A549 cells was examined. The level of different tubulin isotypes in the drug-polymerized tubulin was determined by Western blot analysis with tubulin isotype specific antibodies. In the absence of drug, the level of polymerized βI-, βIII-, βIV- and βV-tubulin was 55%, 9%, 25%, and 48%, respectively. Ten µM Taxol caused a smalll increase ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 664. doi:10.1158/1538-7445.AM2011-664

Microtubules are an important component of the euraryotic cytoskeleton and are essential for normal cell division. They are also a validated target for antitumor drugs, such as taxol, that bind to the – tubulin subunit in the microtubule polymer. Endogenous resistance or the development of drug resistance during treatment with taxol is a serious clinical problem. Understanding the emergence of taxol resistance and being able to predict patient response would enhance the efficacy of the drug. A number of mechanisms, such as overexpression of P-glycoprotein that acts as an ATP-dependent drug-efflux pump, or mutations in the drug-binding site in tubulin, have been described. Of particular interest to our research, is the aberrant expression of β-tubulin isotypes in cancer. Tubulin is composed of α- and β-tubulin subunits that exist as heterodimers. In humans, there are seven β-tubulins and eight β-tubulins, each the product of a distinct gene located on different chromosomes. Each isotype can undergo extensive posttranslational modifications such as acetylation, phosphorylation, polyglutamylation, polyglycylation and reversible tyrosination, all of which add to the complexity of the tubulin/microtubule system. Most of the divergence between the isotypes is found in the last 15-20 amino acids at the carboxyl end of tubulin and in addition, most posttranslational modifications of tubulin occur in this region. For the most part, tubulin isotypes have been detected at the mRNA level or at the protein level with isotype-specific antibodies. Our laboratory has developed proteomic methods, including highresolution isoelectrofocusing, CNBr cleavage and mass spectrometry that allow us to determine the isotype content and posttranslational modifications of tubulin in cells and tissues in a single experiment. We have utilized taxol, which enhances tubulin polymerization, to isolate sufficient tubulin from cells and are developing methods to quantify tubulin isotype expression by mass spectrometry. The overexpression of βIII-tubulin has been associated with resistance to taxanes in a range of tumor types and in taxol-resistant cells generated in our laboratory. βIII-tubulin is normally expressed only in neuronal cells. However, it is aberrantly expressed in numerous malignant tissues and could be a meaningful prognostic factor for treatment outcome. As has been shown by others in different tumors, in our laboratory an immunohistochemical analysis of 47 tumor specimens from head and neck patients determined that overexpression of β-III tubulin was highly prognostic of survival. Citation Information: Clin Cancer Res 2010;16(14 Suppl):IA9-1.

Microtubule-stabilizing agents (MSAs) comprise a class of drugs that bind to polymerized microtubules (MTs), thereby inhibiting their dynamic properties and leading to arrest in mitosis. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling studies led to the identification of the binding site for Taxol in the β-tubulin subunit of the MT polymer, the effect of the drugs on MT conformation was only recently deciphered in our laboratory using hydrogen deuterium exchange (HDX) coupled to high-pressure liquid chromatography and mass spectrometry (HPLC-MS). We have also utilized this technique to determine the binding mode and conformational effects of discodermolide, another MSA with potential to become a useful anti-tumor drug. We found that in tubulin isolated from chicken erythrocytes, discodermolide bound to an overlapping binding site with Taxol, but had a distinct pose within the pocket, such that it oriented itself away from the M-loop and toward the N-terminal H1-S2 loop of β-tubulin. Evidence of conformational effects on the C-terminal H12 helix in the α-tubulin subunit and of complementary MT stabilization between Taxol and discodermolide was found. These results provide the molecular basis for the ability of the two drugs to modulate interactions with endogenous microtubule associated proteins (MAPs) and for the synergy observed between them in vivo. Preliminary analysis of the data with four other MSAs, epothilone B, ixabepilone, peloruside A, and laulimalide, suggests a very similar mode of MT stabilization by all the drugs in this class, with some drug-specific, and more importantly, binding-mode-specific MT-stabilizing effects. Careful examination and qualitative comparison of the conformational effects of all the aforementioned MSAs is currently in progress, and will allow us to determine a molecular signature for drug action that may correlate with the corresponding therapeutic benefits. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2678.

Overexpression of insulin-like growth factor 2 (IGF2) is associated with worse outcome in ovarian cancer patients; previously, our group has shown that IGF2 is upregulated after Taxol treatment of ovarian cancer cells and leads to activation of AKT. Thus, we hypothesized that IGF2 may be involved in drug resistance. The Taxol-resistant cell line HEY-T30 was developed in our laboratory from chemo-naive, sensitive HEY ovarian cancer cells by repeated Taxol treatment in tissue culture. HEY-T30 is 40-fold resistant to Taxol (IC50: 2.6 nM versus 105.9 nM), as determined by sulforhodamine B cytotoxicity assays. In these cells, IGF2 mRNA levels, measured by quantitative PCR, are increased 8.8-fold compared with its expression in the parental cell line. In addition, HEY-T30 have a genomic amplification of ABCB1 (mdr1; encoding the drug efflux pump p-glycoprotein) detected by array CGH, associated with increased ABCB1 mRNA expression. Alterations in IGF2 and ABCB1 were assessed in an additional drug-resistant ovarian cancer cell line, OVCAR8-D30, developed in our laboratory. OVCAR8-D30 is 2-fold-resistant to discodermolide (IC50: 56 nM versus 115 nM), another microtubule-stabilizing drug with a distinct chemical structure. This resistant cell line also showed an increase (4-fold) in IGF2 expression compared to its parental cell line, as well as a 19-fold increase in ABCB1 expression. To determine the effect of IGF pathway inhibition in Taxol-resistant cells, NVP-AEW541 (a small molecule inhibitor of the IGF1R, the major receptor for IGF2) was used. By concurrent treatment with NVP-AEW541, HEY-T30 cells were significantly re-sensitized to Taxol (IC50 from 105.9 nM to 17.0 nM). The effect of combined Taxol and NVP-AEW541 treatment on cell cycle distribution was evaluated by flow cytometry. Eighteen hours after Taxol (75 nM, 125 nM and 250 nM) and NVP-AEW541 (1 μM) treatment alone or in combination, more cells were arrested in G2/M with combination treatment compared to Taxol treatment alone at the higher Taxol concentrations (75 nM: 23.3% to 26.4% with NVP, 125 nM: 23.8% to 44.0% with NVP, and 250 nM: 34.6% to 74.1% with NVP) as determined by flow cytometry. At this time point, caspase activity analysis by flow cytometry detection of FAM-VAD-FMK FLICA reagent, showed no significant differences between the two treatments (75 nM: 6.7% and 9.5% with NVP, 125 nM: 10.0% and 11.5% with NVP, and 250 nM: 12.4% and 14.9% with NVP). These data suggest that treatment with the IGF1R inhibitor NVP-AEW541 can restore Taxol sensitivity in cells with multiple aberrations that confer drug-resistance (IGF2 and ABCB1 over-expression). We conclude that IGF2 has a prominent role in resistance to microtubule-stabilizing drugs in ovarian cancer cell lines and we therefore propose IGF2 to be a valuable target in future molecular therapies for chemo-resistant ovarian cancer. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A33

Our group recently reported increased insulin-like growth factor 2 (IGF2) expression and AKT activation in ovarian cancer cells following treatment with the microtubule-stabilizing agent Taxol. We hypothesized that IGF2 is a modulator of Taxol resistance. Therefore, we developed cell line models of Taxol-resistance in A2780 and HEY, two ovarian cell lines, by repeated treatment with increasing Taxol concentrations, with or without concurrent verapamil, a p-glycoprotein inhibitor. The involvement of IGF2 in drug resistance was evaluated by real time PCR, immunoblotting, siRNA knockdown of IGF2, and small-molecule inhibition of the IGF1 Receptor (IGF1R; which is the major receptor for IGF2). In both of the resistant cell lines (A2780-Tx8Vp, 3-fold Taxol-resistance; and HEY-T30, 40-fold Taxol-resistance), IGF2 mRNA expression, measured by real time PCR, was significantly elevated compared with the parental cell lines, while IGF1R expression, assessed by immunoblotting, was mildly decreased. IGF1R inhibition by the small molecule NVP-AEW541 dramatically re-sensitized both resistant cell lines to Taxol, and cell cycle analysis demonstrated that concurrent NVP-AEW541 and Taxol significantly enhanced G2/M arrest, compared with Taxol alone. While A2780-Tx8Vp cells did not express detectable mdr1/abcb1 (encoding p-glycoprotein), HEY-T30 cells did over-express mdr1 (and were sensitized to Taxol treatment by verapamil). To exclude a possible interaction with p-glycoprotein, we evaluated IGF2 siRNA as an alternate, more specific approach of targeting the IGF pathway in HEY-T30 cells. It was found that IGF2 depletion by siRNA significantly enhanced the antiproliferative effect of Taxol in these cells. In summary, IGF2 modulates Taxol resistance in these ovarian cancer cell lines. IGF pathway inhibition, by IGF2 depletion or by inhibition of its major receptor IGF1R, reverses Taxol resistance in these cells. Such novel findings suggest that IGF2 could be a therapeutic target for ovarian cancer, particularly in the setting of Taxol resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1809.

OBJECTIVES Epothilone B (EpoB), like Taxol, induces tubulin polymerization and microtubule stabilization resulting in an inhibition of microtubule dynamic instability. The drug is presently being evaluated in phase III clinical trials. An EpoB analog Ixabepilone, has been approved by the FDA for the treatment of metastatic breast cancer. Epithelial cell adhesion antigen (EpCAM) expression is significantly higher in epithelial ovarian cancer cells compared to normal cells. METHODS We used biochemical methods, immunofluorescence and flow cytometry to identify EpCAM expression on the surface of the ovarian cancer cell line, Hey, after exposure to EpoB. We also investigated the relationship between EpoB-mediated surface EpCAM expression and EpoB-induced tubulin acetylation in Hey cells. RESULTS We investigated the effect of EpoB and other microtubule-interacting agents on surface EpCAM expression and found that nanomolar concentrations of EpoB, Taxol, discodermolide or vinblastine caused a marked increase in surface EpCAM expression in an ovarian cancer cell line, Hey. Alpha-tubulin acetylation, a surrogate marker for stable microtubules, was increased following treatment with Taxol, EpoB and discodermolide, but not with vinblastine, indicating that drug-enhanced surface EpCAM expression does not correlate with tubulin acetylation or stabilization. Unexpectedly, EpoB did not have a significant effect on EpCAM mRNA expression, nor did it alter the level of total cellular EpCAM in the human ovarian cancer cell line. CONCLUSIONS Our results suggest that disruption of the microtubule cytoskeleton is associated with the redistribution of cell surface antigens in ovarian cancer cells. The increase in cell surface EpCAM antigen density may facilitate the antibody targeting of EpCAM-positive ovarian cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2512.

The regulation and function of the estrogen receptor alpha (ERα) by the ligand estrogen (ES) in ovarian tumorigenesis is not well established. Expression of ERα in ovarian carcinoma, like breast carcinoma, is regulated epigenetically and also via changes in growth-factor receptor signaling and intracellular signaling pathways, such as the Mitogen-activated Protein Kinase (MAPK). Numerous studies have documented an inverse relationship between MAPK activity and ERα expression. Oncogenic MAPK signaling can occur via mutation of K-RAS and B-RAF, which are not prevalent in ovarian carcinomas, and overexpression of receptor tyrosine kinases such as HER2. The aim of these studies were to elucidate (i) the relationship between MAPK signaling and ERα expression, in a panel of ovarian carcinoma cell lines and (ii) the utility of PD0325901, a selective allosteric inhibitor of MEK1/2, in sensitizing ERα expressing ovarian cancer to antiestrogen therapy. We compared ERα expression in a panel of 5 ovarian carcinoma cell lines (A2780, Hey, SKOV3, OVCAR8, IGROV) under physiologic and ES deprived conditions and also after treatment with PD0325901 by immunoblot analysis. The proliferation rate of all cell lines was reduced in E2-free conditions and the PI3K mutant SKOV3 cell line was most sensitive to ES deprivation. A2780 and SKOV3 (PTEN null and PIK3CA mutant, respectively) expressed ERα. The PTEN and BRCA1-null IGROV cells were MEK-dependent and hypersensitive to PD0325901, whereas SKOV3 cells were highly resistant, due to increased phosphorylation and cross-activation of HER2 by PD0325901. Unlike our previous studies in lung cancer, constitutive activation of PIK3CA /AKT did not predict resistance to MEK-inhibitors. Treatment of SKOV3 cells with PD0325901 was associated with increased expression of ERα and altered mobility by immunoblot analysis. These effects were not observed in A2780 cells that express low levels of ERα. Finally, the interaction of the estrogen receptor antagonist fulvestrant and PD0325901 was also evaluated in ERα expressing SKOV3 and A2780 cells. A greater than additive cytotoxic effect was observed in SKOV3, while an antagonistic / minimally additive interaction was noted for A2780 cells that do not show changes in ERα expression after treatment with PD0325901. Our data support the utility of MEK-inhibitors as modulators of ERα expression and function in ovarian cancer cell lines. Ongoing studies will focus on delineating the mechanism of PD0325901-mediated changes in ERα in various histological subtypes of ovarian carcinoma (such as the clear cell carcinoma cell line SKOV3). This may identify subsets of ovarian cancers that may benefit from therapeutic intervention by estrogen antagonists in conjunction with MEK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4152.