Treatment Of Resistant Cells Research Articles

Aba enhances the apoptotic effect of docetaxel in the multidrug-resistant DU145 prostate cancer cell line

This study aimed to induce drug resistance in DU145 prostate cancer cells by exposing them to docetaxel and mitoxantrone, and to examine the effects of combining docetaxel and abscisic acid (ABA). The IC50 values for docetaxel and mitoxantrone in non-resistant cells were 54.57 nM and 6.25 nM, respectively, rising to 808.53 nM and 50.07 nM after resistance had developed. RT-PCR analysis showed that treatment of resistant cells with 50.07 nM docetaxel and 500 ?M ABA (ABA) resulted in the following changes in gene expression: heat shock protein (HSP) 70 (0.63-fold), glucose-regulated protein 94 (GRP94) 0.33-fold, inositol-requiring transmembrane kinase endoribonuclease-1? (IRE1?) 1.62-fold, ER degradation-enhancing alpha-mannosidase-like 1 (EDEM1) 1.77-fold, X-box binding protein 1 (XBP1) 1.53-fold, p21 (2.53-fold), cellular tumor antigen p53 (p53) 2.49-fold, bcl-2-like protein 4 (Bax) 2.7-fold, and tumor necrosis factor (TNF-?) 6.35-fold. Tali? cytometry analysis showed a 47% increase in apoptotic/necrotic cells with the combined treatment of docetaxel and ABA, compared to a 26% increase with docetaxel alone. Fluorescent staining revealed that co-administration of docetaxel and ABA increases apoptosis in resistant DU145 cells compared to treatment with docetaxel alone. This study suggests that combining ABA with docetaxel could be effective in drug-resistant prostate cancer.

The Role of Cyanidin-3-O-glucoside in Modulating Oxaliplatin Resistance by Reversing Mesenchymal Phenotype in Colorectal Cancer.

Epithelial-mesenchymal transition (EMT) plays an important role in the biological and biochemical processes of cells, and it is a critical process in the malignant transformation, and mobility of cancer. Additionally, EMT is one of the main mechanisms contributing to chemoresistance. Resistance to oxaliplatin (OXA) poses a momentous challenge in the chemotherapy of advanced colorectal cancer (CRC) patients, highlighting the need to reverse drug resistance and improve patient survival. In this study, we explored the response of cyanidin-3-O-glucoside (C3G), the most abundant anthocyanin in plants, on the mechanisms of drug resistance in cancer, with the purpose of overcoming acquired OXA resistance in CRC cell lines. We generated an acquired OXA-resistant cell line, named HCT-116-ROx, by gradually exposing parental HCT-116 cells to increasing concentrations of OXA. To characterize the resistance, we performed cytotoxicity assays and shape factor analyses. The apoptotic rate of both resistant and parental cells was determined using Hoechst 33342/Propidium Iodide (PI) fluorescence staining. Migration capacity was evaluated using a wound-healing assay. The mesenchymal phenotype was assessed through qRT-PCR and immunofluorescence staining, employing E-cadherin, N-cadherin, and Vimentin markers. Resistance characterization announced decreased OXA sensitivity in resistant cells compared to parental cells. Moreover, the resistant cells exhibited a spindle cell morphology, indicative of the mesenchymal phenotype. Combined treatment of C3G and OXA resulted in an augmented apoptotic rate in the resistant cells. The migration capacity of resistant cells was higher than parental cells, while treatment with C3G decreased the migration rate of HCT-116-ROx cells. Analysis of EMT markers showed that HCT-116-ROx cells exhibited loss of the epithelial phenotype (E-cadherin) and gain of the mesenchymal phenotype (N-cadherin and Vimentin) compared to HCT-116 cells. However, treatment of resistant cells with C3G reversed the mesenchymal phenotype. The morphological observations of cells acquiring oxaliplatin resistance indicated the loss of the epithelial phenotype and the acquisition of the mesenchymal phenotype. These findings suggest that EMT may contribute to acquired OXA resistance in CRC. Furthermore, C3G decreased the mobility of resistant cells, and reversed the EMT process, indicating its potential to overcome acquired OXA resistance.

Reversal of Multidrug Resistance by Symmetrical Selenoesters in Colon Adenocarcinoma Cells.

Recently, selenium containing derivatives have attracted more attention in medicinal chemistry. In the present work, the anticancer activity of symmetrical selenoesters was investigated by studying the reversal of efflux pump-related and apoptosis resistance in sensitive and resistant human colon adenocarcinoma cells expressing the ABCB1 protein. The combined effect of the compounds with doxorubicin was demonstrated with a checkerboard assay. The ABCB1 inhibitory and the apoptosis-inducing effects of the derivatives were measured with flow cytometry. Whole transcriptome sequencing was carried out on Illumina platform upon the treatment of resistant cells with the most potent derivatives. One ketone and three methyl ester selenoesters showed synergistic or weak synergistic interaction with doxorubicin, respectively. Ketone selenoesters were the most potent ABCB1 inhibitors and apoptosis inducers. Nitrile selenoesters could induce moderate early and late apoptotic processes that could be explained by their ABCB1 modulating properties. The transcriptome analysis revealed that symmetrical selenoesters may influence the redox state of the cells and interfere with metastasis formation. It can be assumed that these symmetrical selenocompounds possess toxic, DNA-damaging effects due to the presence of two selenium atoms in the molecule, which may be augmented by the presence of symmetrical groups.

Phytochemical profiling, molecular docking, and anti-hepatocellular carcinoid bioactivity of extracts

The ATP-binding cassette is the major class of transporters responsible for the efflux of chemotherapeutic agents from cancer cells, resulting in treatment failures of cancer’s patients. Suaeda vermiculata Forssk. ex. J. F. Gmel. is traditionally known for its liver protective activity. The LC-MS based chemical profilings of the sequentially partitioned sub-extracts obtained from the alcoholic extract of S. vermiculata using n -hexane, chloroform, ethyl acetate, and n -butanol as fractionating solvents, identified a total of thirty six compounds. These sub-extracts were evaluated for their anti-hepatocarcinoma activity against the sensitive HepG2 and doxorubicin (DOX)-resistant, HepG-2/ADR cell lines. A mixture of doxorubicin and sub-extracts at 20 μg/ml doses were also tested for their anti-hepatocarcinoma activity. The exhibited IC 50 values for the chloroform, ethyl acetate, n -hexane, and n -butanol sub-extracts, and the doxorubicin against HepG2, and HepG-2/ADR cell lines were found at 64.5, 66.8, 81.25, 125, 1.3 μg/ml, and 110.1, 91.82, 138.2, 265.7, 4.77 μg/ml levels, respectively. However, the treatment of resistant cells with 20 μg/ml of different sub-extracts in combination with the doxorubicin showed significant improvements in the doxorubicin activity against the resistant cells, and the IC 50 values for DOX + chloroform, DOX + ethyl acetate, DOX + n -hexane, and DOX + n -butanol against resistant cells, were at 1.77, 2.05, 2.66, and 2.71 μg/ml levels, respectively. The IC 50 values exhibited 2.69x, 2.33x, 1.79x and 1.76x-folds reversal of the sensitivity in the resistant cancer cell lines. The molecular docking studies of the compounds identified in the LC-MS chemical profilings, against three ATP-binding cassette proteins i.e. , ABCB1, ABCC1, and ABCG2, showed that flavonoids as the major class of compounds responsible for reversal of the resistant cells sensitivities. The predicted binding affinity for the flavonoids against the above mentioned three ATP-binding cassette proteins’ are in the ranges of ∼−8 to −11 kcal/mol. Our results clearly indicate that the presence of flavonoids, as the major class of compounds in the S. vermiculata is responsible for the chemosensitization of the resistant HCC-cell lines. Moreover, the structures, 21 (5‐O‐methyl visamminol), 22 (N- trans -feruloyl tyramine), 27 (atractylenolide-III), and 32 (ginsenoside-Rh2) were also identified among the potential ATP-binding cassette’s modulators during the current study. These observations put the S. vermiculata in perspective with the traditionally claimed liver protective efficacy of the plant.

Blocking autophagy overcomes resistance to dual histone deacetylase and proteasome inhibition in gynecologic cancer

Histone deacetylase (HDAC) inhibitors and proteasome inhibitors have been approved by the FDA for the treatment of multiple myeloma and lymphoma, respectively, but have not achieved similar activity as single agents in solid tumors. Preclinical studies have demonstrated the activity of the combination of an HDAC inhibitor and a proteasome inhibitor in a variety of tumor models. However, the mechanisms underlying sensitivity and resistance to this combination are not well-understood. This study explores the role of autophagy in adaptive resistance to dual HDAC and proteasome inhibition. Studies focus on ovarian and endometrial gynecologic cancers, two diseases with high mortality and a need for novel treatment approaches. We found that nanomolar concentrations of the proteasome inhibitor ixazomib and HDAC inhibitor romidepsin synergistically induce cell death in the majority of gynecologic cancer cells and patient-derived organoid (PDO) models created using endometrial and ovarian patient tumor tissue. However, some models were not sensitive to this combination, and mechanistic studies implicated autophagy as the main mediator of cell survival in the context of dual HDAC and proteasome inhibition. Whereas the combination of ixazomib and romidepsin reduces autophagy in sensitive gynecologic cancer models, autophagy is induced following drug treatment of resistant cells. Pharmacologic or genetic inhibition of autophagy in resistant cells reverses drug resistance as evidenced by an enhanced anti-tumor response both in vitro and in vivo. Taken together, our findings demonstrate a role for autophagic-mediated cell survival in proteasome inhibitor and HDAC inhibitor-resistant gynecologic cancer cells. These data reveal a new approach to overcome drug resistance by inhibiting the autophagy pathway.

Abstract P6-04-16: Glucocorticoid receptor signaling elicits anti-cancer effects in endocrine resistant breast cancer via induction of AZGP1

Abstract Despite the availability of effective drugs that target ERα-positive breast cancer, ERα-positive disease still accounts for more breast cancer related deaths than any other subtype. Relapse in these patients is largely due to the development of resistance to anti-estrogen therapies such as tamoxifen. While tamoxifen and its resistance mechanisms have been extensively studied, relatively little is known about its active metabolite endoxifen. Our group has provided evidence that endoxifen is a potent and clinically relevant metabolite of tamoxifen. Therefore, characterization of endoxifen resistance mechanisms may be crucial to understanding tamoxifen resistance. We have developed novel endoxifen resistant MCF7 and T47D cell lines through chronic exposure to endoxifen over a period of 12-24 months. Using these models and their respective controls, we compared global gene expression profiles of endoxifen resistant cells to tamoxifen resistant cells and found marked differences between the two models. We subjected endoxifen resistant cells to a genome-wide, CRISPR-mediated knockout screen to identify genes involved in mediating endoxifen resistance. Subsequent analyses revealed that disruption of genes regulated by the glucocorticoid receptor (GR) enhanced cells’ ability to survive and proliferate in the presence of endoxifen, suggesting that GR signaling blocks growth of resistant cells. We provide evidence that GR activation via dexamethasone significantly inhibits the proliferation rates of endoxifen resistant cells by 50-60%, with little to no inhibitory effects in endoxifen sensitive models. Follow-up studies suggested that alpha zinc-glycoprotein 1 (AZGP1), a secreted lipolytic enzyme induced by GR, plays a role in mediating this phenotype. AZGP1 RNA and protein are highly expressed in normal breast tissue, but corresponding levels are lower in ERα-positive breast malignancies. In these tumors, low AZGP1 expression correlates with worse prognosis. Additionally, high expression of AZGP1 is associated with improved outcomes in patients with prostate, pancreatic, gastric and breast cancer. AZGP1 is expressed in endocrine sensitive MCF7 and T47D cells, but is silenced in endoxifen resistant models at the mRNA and protein level. Further, treatment of resistant cells with conditioned medium containing AZGP1 resulted in significant inhibition of cell proliferation and migration, and antibody-mediated depletion of AZGP1 from conditioned medium completely reversed these effects. While there are no reports regarding the molecular functions of AZGP1 in cancer we provide evidence that AZGP1 directly inhibits oncogenic signaling in endocrine resistant breast cancer through interaction with, and inhibition of, β6 integrin. Through the analysis of endoxifen resistant breast cancer cells, we have identified novel roles for GR, AZGP1 and β6 integrin in the development and progression of endocrine resistant breast cancer. We demonstrate that activation of GR may elicit therapeutic benefit in endocrine resistant breast cancer, specifically in tumors expressing AZGP1 and β6 integrin. Further, AZGP1 and β6 integrin may represent novel prognostic and/or predictive biomarkers in endocrine resistant disease. Citation Format: Calley J. Jones, Santiago Acero Bedoya, Anna V. Mykytyn, Matthew P. Goetz, John R. Hawse. Glucocorticoid receptor signaling elicits anti-cancer effects in endocrine resistant breast cancer via induction of AZGP1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-16.

Abstract P5-04-05: Glucocorticoid receptor activation inhibits proliferation of endoxifen resistant breast cancer cells and resensitizes cells to hormonal therapy

Abstract Background: Despite the prevalent treatment options for ERα-positive breast cancer patients, and their initial efficacy for many women, ERα-positive disease still accounts for more breast cancer related deaths than any other subtype. Relapse in these patients is largely due to the development of resistance to anti-estrogen therapies such as tamoxifen. While tamoxifen and its resistance mechanisms have been extensively studied from both the bench and the bedside, relatively little is known about its active metabolite endoxifen. Our group has provided evidence that endoxifen is the most potent and clinically relevant metabolite of tamoxifen, suggesting that its characterization may be crucial to understanding tamoxifen resistance. Methods: We have developed novel endoxifen resistant MCF7 and T47D cell lines through chronic exposure to endoxifen during a period of 12-24 months. Using these models and their respective controls, we compared global gene expression profiles of endoxifen resistant cells to tamoxifen resistant cells and found marked differences between the two models. Additionally, we subjected treatment naïve cells to a genome-wide, CRISPR-mediated knockout screen to identify genes, and their associated pathways, that are likely involved in mediating endoxifen resistance. Results: Analysis of CRISPR guide RNAs enriched or depleted in response to chronic endoxifen treatment revealed that disruption of genes regulated by dexamethasone (Dex), a potent glucocorticoid receptor (GR) agonist, enhanced cells' ability to survive and proliferate in the presence of endoxifen. These data suggest that GR activation may inhibit endoxifen resistance, and that treatment of resistant cells with Dex may restore endoxifen efficacy. Indeed, Dex treatment significantly inhibited the proliferation rates of endoxifen resistant cells by 50-60% with little to no inhibitory effects in endoxifen sensitive models. Further, Dex was shown to synergize with endoxifen in resistant cells to further suppress cell proliferation, implying that Dex treatment could be utilized as an effective therapy for endocrine resistant disease. Conditioned media harvested from cells chronically exposed to Dex also resulted in substantial inhibition of endoxifen resistant cell proliferation rates. To explore potential mechanisms of these effects, we performed RNA-seq on both treatment-naïve and endoxifen resistant cells following Dex treatment. Out of 246 genes significantly regulated by Dex in endoxifen resistant cells, we identified 61 genes that were not differentially regulated in treatment naïve cells. These genes may provide insights into the mechanisms of GR activity specific to endoxifen resistant cells. Conclusions: To our knowledge, we have developed the first models of endoxifen resistance and have demonstrated that global transcriptomic changes that occur during this process are substantially different than those observed in tamoxifen resistant models. We have shown that activation of GR signaling elicits significant growth-inhibitory effects specifically in the setting of endoxifen resistance. These data identify the GR pathway as a potential novel therapeutic target for the treatment of endocrine resistant breast cancer. Citation Format: Jones CJ, Goetz MP, Ingle JN, Hawse JR. Glucocorticoid receptor activation inhibits proliferation of endoxifen resistant breast cancer cells and resensitizes cells to hormonal therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-05.

Correction: Reversion of resistance to oxaliplatin by inhibition of p38 MAPK in colorectal cancer cell lines: involvement of the calpain / Nox1 pathway.

[This corrects the article DOI: 10.18632/oncotarget.21780.].

Mutation in the FGFR1 tyrosine kinase domain or inactivation of PTEN is associated with acquired resistance to FGFR inhibitors in FGFR1‐driven leukemia/lymphomas

Stem cell leukemia/lymphoma syndrome (SCLL) is driven by constitutive activation of chimeric FGFR1 kinases generated by chromosome translocations. We have shown that FGFR inhibitors significantly suppress leukemia and lymphoma development in vivo, and cell viability in vitro. Since resistance to targeted therapies is a major reason for relapse, we developed FGFR1-overexpressing mouse and human cell lines that are resistant to the specific FGFR inhibitors AZD4547 and BGJ398, as well as non-specific inhibitors, such as ponatinib, TKI258 and E3810. Two mutually exclusive mechanisms for resistance were demonstrated; an activating V561M mutation in the FGFR1 kinase domain and mutational inactivation of PTEN resulting in increased PI3K/AKT activity. Ectopic expression of PTEN in the PTEN-mutant cells resensitizes them to FGFR inhibitors. Treatment of resistant cells with BGJ398, in combination with the BEZ235 PI3K inhibitor, shows an additive effect on growth in vitro and prolongs survival in xenograft models in vivo. These studies provide the first direct evidence for both the involvement of the FGFR1 V561M mutation and PTEN inactivation in the development of resistance in leukemias overexpressing chimeric FGFR1. These studies also provide a potential strategy to treat leukemias and lymphomas driven by FGFR1 activation that become resistant to FGFR1 inhibitors.

Targeting the ER-Mitochondrial Interface of Cell Death Sensitizes Leukemia Cells Towards Cytostatics

BackgroundCombination therapy has proved to be a successful strategy and generally benefits from the crosstalk of antileukemic agents. However, the mechanisms of amplification remain mostly elusive and the outcome of relapsed or chemoresistant ALL and AML is still dismal. Therefore, drug discovery is not only challenged to address the identification of novel compounds and targets, but also to enhance the understanding of their molecular interaction pathways in order to exploit their potential for the translation into combination therapies.Screening small molecules that sensitize cancer cells towards Etoposide (VP-16) induced apoptosis, we recently introduced PS89 which targets protein disulfide isomerase (PDI) (Eirich et al, Angew Chem, 2014), a crucial enzyme for the maintenance of ER homeostasis and exciting novel target in cancer research (Xu et al, Drug Discov Today, 2014). Its chemosensitizing profile predestined PS89 for combination therapy and moreover called for the elucidation of critical networks integrated in the synergistic pro-apoptotic signaling.MethodsApoptosis of PS89 in combination with standard cytostatics (Daunorubicin/DNR, Etoposide/VP-16, Vincristine/VCR) was analyzed by flow cytometry in different leukemia cell lines and patient-derived xenograft (PDX) ALL and AML cells (Vick et al, PLoS One, 2014; Terziyska et al, PLoS One, 2012) versus lymphocytes of healthy individuals (at least n=3 biological replicates each). Activity-based protein profiling (ABPP) was applied in leukemic cells to identify the network of PS89 target proteins. Following interaction studies (STRING v10), biological validation of proposed communication pathways and time-dependent analysis of critical stress triggers was performed.ResultsThe apoptosis rate of cytostatics was synergistically enhanced in several cell line models of acute leukemia, notably applying PS89 at subtoxic concentrations in all assays (Jurkat 0.5 µM VP-16 17.7% vs PS89+VP-16 51.8%; HL-60 10 nM DNR 14.4% vs PS89+DNR 43.3%; CCRF-CEM 1 nM VCR 12.8% vs PS89+VCR 46.6%). This approach was confirmed as a therapeutic advancement in the treatment of resistant cells (VCR-resistant CEM 0.5 µM VCR 3.8% vs PS89+VCR 36.1%) as well as Pre-B-ALL and T-ALL PDX cells (n=2 diagnosis samples, 5 nM VCR 14.2% vs PS89+VCR 43.6%) or AML PDX cells (n=4 relapse samples, 20 nM DNR 38.3% vs PS89+DNR 53.6%). While in vivo studies with PDX cells are ongoing, apoptotic effects of PS89 combinations were significantly lower in isolated lymphocytes of healthy individuals which are in line with the good in vivo tolerability of PS89 (30 mg/kg i. p. daily for 2 weeks).In a proteomics screen by ABPP in Jurkat cells, we identified the B-cell receptor-associated protein 31 (Bap31) as a further target of PS89 transducing apoptotic signals from the ER to mitochondria under ER stress (Namba et al, Cell Rep, 2013) and serving as a platform to activate caspase-8 (CASP8) following apoptotic stimuli (Iwasawa et al, EMBO J, 2011). In this regard, we proposed a distinct mechanistic pathway via the Bap31/CASP8 axis mediating the crosstalk of PDI inhibition and signaling of cytostatics.Indeed, apoptosis induced by PS89/VP-16 combination treatment in Jurkat cells was critically dependent on CASP8 activity (4.4-fold reduction with CASP8 inhibitor Z-IETD-FMK) and co-immunoprecipitation revealed that CASP8 cleavage at the Bap31 protein complex was only detectable in the presence of PS89. In turn, active CASP8 is able to cleave Bap31 (shown in PS89/VP-16 treated cells, Western Blot) leading to ER calcium release. We observed moreover, that loss of mitochondrial membrane integrity mediated concurrently by ER calcium and p53-dependent signaling was accompanied by elevation of reactive oxygen species (ROS) levels which provokes further ER stress and finally closes the feedback loop.ConclusionThe strong antileukemic effects of the PDI inhibitor PS89 in combination with cytostatics are orchestrated by its direct target Bap31 which transduces apoptosis signals at the ER-mitochondrial interface and emphasizes the crucial role of this social network of cell death for synergistic drug combinations. Exploiting to tune these mutual amplification loops makes PS89 uniquely applicable at subtoxic concentrations and therefore a valuable novel option to sensitize acute leukemia cells towards established chemotherapeutics. [Display omitted] DisclosuresBraig:Dr Pfleger Foundation: Research Funding.

Structure-Activity Relationships of Di-2-pyridylketone, 2-Benzoylpyridine, and 2-Acetylpyridine Thiosemicarbazones for Overcoming Pgp-Mediated Drug Resistance.

Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) represents a significant impediment to successful cancer treatment. The compound, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), has been shown to induce greater cytotoxicity against resistant cells than their nonresistant counterparts. Herein, the structure-activity relationships of selected thiosemicarbazones are explored and the novel mechanism underlying their ability to overcome resistance is further elucidated. Only thiosemicarbazones with electron-withdrawing substituents at the imine carbon mediated Pgp-dependent potentiated cytotoxicity, which was reversed by Pgp inhibition. Treatment of resistant cells with these thiosemicarbazones resulted in Pgp-dependent lysosomal membrane permeabilization (LMP) that relied on copper (Cu) chelation, reactive oxygen species generation, and increased relative lipophilicity. Hence, this study is the first to demonstrate the structural requirements of these thiosemicarbazones necessary to overcome MDR. We also demonstrate the mechanism that enables the targeting of resistant tumors, whereby thiosemicarbazones "hijack" lysosomal Pgp and form redox-active Cu complexes that mediate LMP and potentiate cytotoxicity.

Abstract 2155: Acquired tamoxifen resistance sensitises breast cancer cells to bisphosphonates

Abstract Acquisition of resistance to endocrine therapies is a major limiting factor to their clinical effectiveness, resulting in disease relapse and poor prognosis. This may be due in part to the observations that acquired resistance to agents such as tamoxifen is accompanied by a gain in aggressive cellular features likely to promote disease spread and survival at metastatic sites. Bisphosphonates (BPs) are potent antiresorptive drugs used to treat osteoporosis and bone metastasis. Recent evidence also suggests that these agents may possess anti-tumour properties independently of their action on osteoclasts in a range of different tumours including breast cancers resulting in suppression of proliferation and migration and an induction of apoptosis. Here we have explored the potential of the bisphosphonate, zoledronic acid (ZOL) to suppress the aggressive phenotype of acquired endocrine-resistant breast cancer models versus their endocrine-sensitive counterparts. ER+, endocrine-sensitive MCF7 cells and their highly-proliferative, ER+, tamoxifen-resistant (‘TamR’) counterparts were exposed to increasing concentrations of ZOL (1-160μM) for 3 days and changes in growth determined by MTT assay and cell counting. Immunohistochemical analysis of Ki67 confirmed cell growth effects whilst cell cycle analysis was performed using FACS and propidium iodide. Changes in signalling pathways were investigated by Western blotting. The proliferative capacity of TamR cells was greatly suppressed by ZOL in a dose dependent manner in contrast to MCF7, where the effect was minimal (IC50 ± SD: 30 ± 4 μM (TamR) versus 150 ± 15 μM (MCF7); p<0.05). This was confirmed with Ki67 analysis which showed a reduction in Ki67 positivity of 40% (TamR) versus 25% (MCF7); p<0.05. ZOL treatment of TamR cells resulted in a significant loss of cells in G1 phase and an accumulation of the cells in S phase. ZOL treatment of TamR cells also resulted in loss of AKT activity, whilst AKT was unaffected in MCF7 cells. Our data suggests that acquisition of resistance to tamoxifen confers a sensitivity to BPs where treatment of resistant cells with agents such as zoledronic acid results in suppression of proliferation and modulation of AKT signalling. These data suggest that BPs may exert direct anti-tumour effects on breast cancers which have relapsed on endocrine treatment. Citation Format: Dionysia Lymperatou, Christopher Smith, Wen Guo Jiang, Bronwen Evans, Stephen Hiscox. Acquired tamoxifen resistance sensitises breast cancer cells to bisphosphonates. [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 2155.

Sustained Epigenetic Drug Delivery Depletes Cholesterol–Sphingomyelin Rafts from Resistant Breast Cancer Cells, Influencing Biophysical Characteristics of Membrane Lipids

Cell-membrane lipid composition can greatly influence biophysical properties of cell membranes, affecting various cellular functions. We previously showed that lipid synthesis becomes altered in the membranes of resistant breast cancer cells (MCF-7/ADR); they form a more rigid, hydrophobic lipid monolayer than do sensitive cell membranes (MCF-7). These changes in membrane lipids of resistant cells, attributed to epigenetic aberration, significantly affected drug transport and endocytic function, thus impacting the efficacy of anticancer drugs. The present study's objective was to determine the effects of the epigenetic drug, 5-aza-2'-deoxycytidine (DAC), delivered in sustained-release nanogels (DAC-NGs), on the composition and biophysical properties of membrane lipids of resistant cells. Resistant and sensitive cells were treated with DAC in solution (DAC-sol) or DAC-NGs, and cell-membrane lipids were isolated and analyzed for lipid composition and biophysical properties. In resistant cells, we found increased formation of cholesterol-sphingomyelin (CHOL-SM) rafts with culturing time, whereas DAC treatment reduced their formation. In general, the effect of DAC-NGs was greater in changing the lipid composition than with DAC-sol. DAC treatment also caused a rise in levels of certain phospholipids and neutral lipids known to increase membrane fluidity, while reducing the levels of certain lipids known to increase membrane rigidity. Isotherm data showed increased lipid membrane fluidity following DAC treatment, attributed to decrease levels of CHOL-SM rafts (lamellar beta [Lβ] structures or ordered gel) and a corresponding increase in lipids that form lamellar alpha-structures (Lα, liquid crystalline phase). Sensitive cells showed marginal or insignificant changes in lipid profile following DAC-treatment, suggesting that epigenetic changes affecting lipid biosynthesis are more specific to resistant cells. Since membrane fluidity plays a major role in drug transport and endocytic function, treatment of resistant cells with epigenetic drugs with altered lipid profile could facilitate anticancer drug transport to overcome acquired drug resistance in a combination therapy.

The broad-spectrum metalloproteinase inhibitor BB-94 inhibits growth, HER3 and Erk activation in fulvestrant-resistant breast cancer cell lines.

Breast cancer cells can switch from estrogen receptor α (ER)- to human epidermal growth factor receptor (HER)-driven cell growth upon acquiring antiestrogen resistance. HER ligands are cleaved by metalloproteinases leading to release of active HER ligands, activation of HER receptors and consequently increased cell growth. In this study, we investigated the importance of HER receptors, in particular HER3, and HER ligand shedding for growth and signaling in human MCF-7 breast cancer cells and MCF-7-derived sublines resistant to the antiestrogen fulvestrant. The HER3/HER4 ligand heregulin 1β induced phosphorylation of HER3, Akt and Erk, and partly rescued fulvestrant-inhibited growth of MCF-7 cells. HER3 ligands were found to be produced and shed from the fulvestrant-resistant cells as conditioned medium from fulvestrant-resistant MCF-7 cells induced phosphorylation of HER3 and Akt in MCF-7 cells. This was prevented by treatment of resistant cells with the metalloproteinase inhibitor TAPI-2. Only the broad-spectrum metalloproteinase inhibitor BB-94, and not the more selective inhibitors GM6001 or TAPI-2, which inhibited shedding of the HER ligands produced by the fulvestrant-resistant cells, was able to inhibit growth and activation of HER3 and Erk in resistant cells. Compared to MCF-7, fulvestrant-resistant cells have increased HER3 phosphorylation, but knockdown of HER3 had no inhibitory effect on resistant cell growth. The EGFR inhibitor gefitinib exhibited only a minor growth inhibition, whereas the pan-HER inhibitor CI-1033 exerted growth arrest. Thus, neither HER3 nor EGFR alone are the main driver of fulvestrant-resistant cell growth and treatment should target both receptors. Ligand shedding is not a treatment target, as receptor activation occurred, independent of release of ligands. Only the broad-spectrum metalloproteinase inhibitor BB-94 could abrogate HER3 and Erk activation in the resistant cells, which stresses the complexity of the resistance mechanisms and the requirement of targeting signaling from HER receptors by multiple strategies.

Abstract C74: Stabilization of mutant BRCA1 confers PARP inhibitor and platinum resistance.

Abstract The BRCA1 gene is commonly mutated in hereditary breast and ovarian cancers. The BRCA1 protein has multiple domains that mediate protein interactions; BRCA1 gene mutations may produce truncated proteins that lose the ability to interact with associated proteins. Additionally, mutations in the BRCT domain of BRCA1 create protein folding defects that result in protease-mediated degradation. Cells that contain dysfunctional BRCA1 proteins are hypersensitive to DNA damaging agents. In particular, BRCA1-deficient cell lines are exquisitely sensitive to poly(ADP-ribose) polymerase (PARP) inhibitor treatment. Despite initial responses of BRCA1 mutant cancers to PARP inhibitor treatment, acquired resistance develops. To study PARP inhibitor resistance, specifically in the setting of BRCA1 C-terminal truncating mutations, we cultured the triple negative breast cancer cell line MDA-MB-436 (5396+1G>A) in the presence of the PARP inhibitor rucaparib. Drug resistant clones emerged approximately 2-4 months after initial exposure. Clones were highly resistant to rucaparib, and cross-resistant to olaparib, as well as cisplatin. No changes in the mutant BRCA1 DNA or mRNA sequences were detected and the PARP enzyme was efficiently inhibited by rucaparib. The mutant BRCA1 protein was undetectable in MDA-MB-436 parent cells, but was abundant in resistant clones by western blot. HSP90 co-immunoprecipitated with the stabilized mutant BRCA1 protein and treatment of resistant cells with the HSP90 inhibitor 17-DMAG reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. The stabilized mutant BRCA1 protein also interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation and PARP inhibitor resistance. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occurs in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum. These results provide evidence for a novel mechanism by which BRCA1-mutant tumors acquire anti-cancer therapy resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C74. Citation Format: Neil Johnson, Shawn F. Johnson, Yifan Wang, Andrea Bernhardy, Wei Yao, Young-Eun Choi, Marzia Capelletti, Kristopher Sarosiek, Lisa Moreau, Dipanjan Chowdhury, Joyce Liu, Alan D'Andrea, Alexander Miron, Anneka Wickramanayake, Maria Harrell, Elizabeth M. Swisher, Geoffrey I. Shapiro. Stabilization of mutant BRCA1 confers PARP inhibitor and platinum resistance. [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 C74.

Sphingosine Kinase 1 Overexpression Contributes to Cetuximab Resistance in Human Colorectal Cancer Models

Although the anti-EGF receptor (EGFR) monoclonal antibody cetuximab is an effective strategy in colorectal cancer therapy, its clinical use is limited by intrinsic or acquired resistance. Alterations in the "sphingolipid rheostat"-the balance between the proapoptotic molecule ceramide and the mitogenic factor sphingosine-1-phosphate (S1P)-due to sphingosine kinase 1 (SphK1) overactivation have been involved in resistance to anticancer-targeted agents. Moreover, cross-talks between SphK1 and EGFR-dependent signaling pathways have been described. We investigated SphK1 contribution to cetuximab resistance in colorectal cancer, in preclinical in vitro/in vivo models, and in tumor specimens from patients. SphK1 was found overexpressed and overactivated in colorectal cancer cells with intrinsic or acquired resistance to cetuximab. SphK1 contribution to resistance was supported by the demonstration that SphK1 inhibition by N,N-dimethyl-sphingosine or silencing via siRNA in resistant cells restores sensitivity to cetuximab, whereas exogenous SphK1 overexpression in sensitive cells confers resistance to these agents. Moreover, treatment of resistant cells with fingolimod (FTY720), a S1P receptor (S1PR) antagonist, resulted in resensitization to cetuximab both in vitro and in vivo, with inhibition of tumor growth, interference with signal transduction, induction of cancer cells apoptosis, and prolongation of mice survival. Finally, a correlation between SphK1 expression and cetuximab response was found in colorectal cancer patients.

Hedgehog Signaling Is a Novel Therapeutic Target in Tamoxifen-Resistant Breast Cancer Aberrantly Activated by PI3K/AKT Pathway

Endocrine resistance is a major challenge in the management of estrogen receptor (ER)-positive breast cancers. Although multiple mechanisms leading to endocrine resistance have been proposed, the poor outcome of patients developing resistance to endocrine therapy warrants additional studies. Here we show that noncanonical Hedgehog (Hh) signaling is an alternative growth promoting mechanism that is activated in tamoxifen-resistant tumors. Importantly, phosphoinositide 3-kinase inhibitor/protein kinase B (PI3K/AKT) pathway plays a key role in regulating Hh signaling by protecting key components of this pathway from proteasomal degradation. The levels of Hh-signaling molecules SMO and GLI1 and the targets were significantly elevated in tamoxifen-resistant MCF-7 cells and T47D cells. Serial passage of the resistant cells in mice resulted in aggressive tumors that metastasized to distant organs with concurrent increases in Hh marker expression and epithelial mesenchymal transition. RNAi-mediated depletion of SMO or GLI1 in the resistant cells resulted in reduced proliferation, clonogenic survival and delayed G(1)-S transition. Notably, treatment of resistant cells with PI3K inhibitors decreased SMO and GLI1 protein levels and activity that was rescued upon blocking GSK3β and proteasomal degradation. Furthermore, treatment of tamoxifen-resistant xenografts with anti-Hh compound GDC-0449 blocked tumor growth in mice. Importantly, high GLI1 expression correlated inversely with disease-free and overall survival in a cohort of 315 patients with breast cancer. In summary, our results describe a signaling event linking PI3K/AKT pathway with Hh signaling that promotes tamoxifen resistance. Targeting Hh pathway alone or in combination with PI3K/AKT pathway could therefore be a novel therapeutic option in treating endocrine-resistant breast cancer.

1651P - Sphingosine Kinase 1 (SPHK1) Overexpression Contributes to Cetuximab Resistance in Human Colorectal Cancer Models

ABSTRACT Purpose Although the anti-Epidermal Growth Factor (EGFR) monoclonal antibody (mAb) cetuximab is an effective strategy in colorectal cancer therapy, its clinical use is limited by intrinsic or acquired resistance. Alterations in the ‘sphingolipid rheostat’ - the balance between the proapoptotic molecule ceramide and the mitogenic factor sphingosine-1-phosphate (S1P) - due to sphingosine kinase 1 (SphK1) overactivation have been involved in resistance to anticancer targeted agents. Moreover, cross-talks between SphK1 and EGFR-dependent signalling pathways have been described. Experimental design: In this study, we investigated SphK1 contribution to cetuximab resistance in colorectal cancer in preclinical in vitro/in vivo models and in tumor specimens from patients. Results Immunohistochemical analysis on colorectal cancer tissues revealed a correlation between SphK1 expression and K-Ras mutations. Moreover, SphK1 was found overexpressed and overactivated in colorectal cancer cells with intrinsic or acquired resistance to cetuximab. SphK1 contribution to resistance was supported by the demonstration that SphK1 inhibition by N,N-dimethyl-sphingosine (DMS) or silencing via siRNA in resistant cells restores sensitivity to cetuximab, whereas exogenous SphK1 overexpression in sensitive cells confers resistance to these agents. Finally, treatment of resistant cells with fingolimod (FTY720), a S1P receptor (S1PR) inhibitor, resulted in re-sensitization to cetuximab both in vitro and in vivo, with significant inhibition of tumor growth, interference with signal transduction, induction of cancer cells apoptosis and prolongation of mice survival. Conclusion Our data could contribute to clarify SphK1 role in cetuximab resistance and may suggest SphK1 inhibition as a part of novel targeting strategies potentially effective also in resistant colorectal cancer patients. Disclosure All authors have declared no conflicts of interest.

Drug Resistance to the HDM-2 Inhibitors MI-63 and Nutlin Is Mediated by Point Mutations of p53, but Can Be Overcome with the p53 Targeting Agent RITA

Abstract 2726 Background:The ubiquitin-proteasome pathway has been validated as a target for multiple myeloma (MM) and mantle cell lymphoma (MCL) by the demonstration of the effectiveness of the proteasome inhibitor bortezomib (BZB) in these malignancies. E3 ubiquitin ligases are involved in ubiquitination of a small subset of cellular proteins and play an integral part in regulation of turnover of cellular proteins. HDM-2 is the E3 ubiquitin ligase responsible for degradation and regulation of the p53 tumor suppressor, and HDM-2 inhibitors such as nutlin (NUT) and MI-63 stabilize p53 and enhance its pro-apoptotic effects in the absence of DNA damage. Methods:To evaluate the mechanisms through which wild type p53 MCL and MM cells could evade the therapeutic effects of HDM-2 inhibitors, we generated cell lines resistant to MI-63 and Nutlin. Resistant lines were generated by growing cells over a period of 3 months in increasing concentrations of either MI-63 or NUT until the cells were resistant to 10 mM of either agent. Results:H929 MM and Granta-519 MCL cells resistant to MI-63 (MI63R) were cross resistant to NUT, and NUT resistant (NUTR) cells displayed resistance to MI-63. Indeed, treatment of resistant cells with either MI-63 or NUT resulted in enhanced proliferation of these cells. MI-63R and NUTR cells showed enhanced resistance to both BZB and doxorubicin, whilst unexpectedly maintaining sensitivity to RITA (reactivation of p53 and induction of tumor cell apoptosis). Immunoblot analysis indicated increased levels of p53 in resistant cells at baseline and upon treatment with MI-63, NUT, or doxorubicin, but these treatments failed to induce p21 and HDM-2. Sequencing of genomic DNA from these cells for both HDM-2 and p53 genes indicated no alteration in the sequence of HDM-2. However, substitution mutations were found within p53 encompassing exons 4 through 8 for both NUTR and MI63R cells. When RITA was used in the resistant cells, an SG2 cell cycle arrest was seen at 48 hours post treatment. A time course analysis indicated that resistant cells treated with RITA displayed up-regulation of HDM-2, PUMA and NOXA from around 12 hours, with a corresponding induction of PARP cleavage from 48 to 72 hours. These data suggested RITA was able to restore wtp53 function resulting in induction of p53 transcriptional targets and thereby overcoming resistance to NUT and MI-63. The combination of either MI-63 or NUT with RITA demonstrated that simultaneous addition of the agents or pretreatment with RITA resulted in an enhancement of cell death over RITA alone. This result indicates that RITA restored the function of mutp53 within MI-63 and NUT resistant cells and thereby resensitized the cells to the effects of MI-63 or NUT. Conclusions:Chronic exposure of MM and MCL cells to HDM-2 inhibitors resulted in cross resistance to other inhibitors in the same class through mutation of p53 as the primary mechanism of resistance. HDM-2 inhibitor resistant cells did remain sensitive to the effects of RITA through restoration of p53 function, and restoring the sensitivity of such cells to agents that promote p53's pro apoptotic effects may be a rational strategy for translation to the clinic. Disclosures:No relevant conflicts of interest to declare.

Abstract 727: NF-κB Mediates Resistance to Rapamycin in Neuroblastoma

Abstract Neuroblastoma is the most common extracranial pediatric solid tumor and is the most prevalent malignancy of infancy. We were interested in the potential use of rapamycin for the treatment of neuroblastoma. However, 7/8 of neuroblastoma cell lines were found to be rapamycin resistant in vitro, independently of MYCN status. The sensitive cell line (CHLA90) and 2 of the most resistant cell lines (SKNAS [non-MYCN amplified] and NB1691 [MYCN amplified]) were further analyzed to characterize their response to rapamycin. Electromobility shift assay analysis showed that treatment of resistant cells with rapamycin activated NF-κB, while NF-κB activation was reduced in sensitive cells. Immunoblotting with specific NF-κB antibodies showed that rapamycin induced p50 but not p65 nuclear translocation in resistant cells, suggesting that the NF-κB complex activated by rapamycin represents p50 homodimers. Furthermore, immunocytochemical analysis showed nuclear translocation the p50 protein following treatment with rapamycin. To attempt to overcome rapamycin resistance, we tested the combination of two different NF-κB inhibitors (Velcade, a proteasome inhibitor and BMS-345541, a highly selective IκB kinase inhibitor) with rapamycin. Sub-lethal concentrations that resulted in NF-κB inhibition of both agents were identified and these doses were used in combination with rapamycin treatment. A synergy index (SI) was calculated and in both cell lines significant synergy (SI<0 and 95% confidence interval<0) between rapamycin and the two NF-κB inhibitory agents tested, was observed. Orthotopic neuroblastoma mouse models were established in the retroperitoneal space of SCID mice. Tumors were sized matched into cohorts of 5-10 mice each. Treatment groups included vehicle only, single agent (Velcade: 0.5mg/kg, 3x/w; rapamycin: 5mg/kg, 3x/w) and combination groups. Mice were sacrificed after 2 weeks of treatment and final tumor volumes calculated. In SKNAS, while Velcade and rapamycin individually had only modest effects on tumor volume (Control 6.6±3.2cm3; Velcade 4.5±1.8cm3; Rapamycin 2.6±0.95cm3), combination treatment was significantly more efficacious than either monotherapy (0.99±0.24cm3) (p<0.0002). In NB1691, Velcade has no effect on tumor size (Control 2.68±0.53cm3; Velcade 2.67±0.58cm3) while rapamycin had a modest effect on tumor volume (1.28±0.34cm3). The combination treatment was significantly more efficacious than either monotherapy (0.71±0.15cm3) (p<0.008). A synergy index (SI) was calculated and in both models significant synergy (SI<0 and 95% confidence interval<0) between rapamycin and Velcade was observed. In conclusion, the efficacy of rapamycin treatment of neuroblastoma may be limited by cellular resistance to the cytostatic and cytotoxic effects of rapamycin. However resistance can be overcome by NF-κB inhibitors, which results in significantly synergistic inhibition of tumor growth in vivo. 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 727. doi:10.1158/1538-7445.AM2011-727