CisR Cells Research Articles

Soft drug-resistant ovarian cancer cells migrate via two distinct mechanisms utilizing myosin II-based contractility

The failure of chemotherapeutic drugs in treatment of various cancers is attributed to the acquisition of drug resistance. However, the migration mechanisms of drug-resistant cancer cells remain incompletely understood. Here we address this question from a biophysical perspective by mapping the phenotypic alterations in ovarian cancer cells (OCCs) resistant to cisplatin and paclitaxel. We show that cisplatin-resistant (CisR), paclitaxel-resistant (PacR) and dual drug-resistant (i.e., resistant to both drugs) OCCs are more contractile and softer than drug-sensitive cells. Protease inhibition suppresses invasion of CisR cells but not of PacR cells, indicative of a protease-dependent mode of migration in CisR cells and a protease-independent mode of migration in PacR. Despite these differences, actomyosin contractility, mediated by the RhoA-ROCK2-Myosin II signaling pathway, regulates both modes of migration. Confined migration experiments establish the role of myosin IIA and IIB in mediating nuclear translocation and regulation of proteolytic activity. Collectively, our results highlight the importance of myosin II as a potential therapeutic target for treatment of drug-resistant ovarian cancer cells.

Abstract 3184: Autophagy regulation by vacuolar-ATPases: A contributing mechanism of chemo-resistance in ovarian cancer

Abstract In ovarian cancer (OVCA), the main obstacle in the clinical usefulness of platinum based drugs is the eventual emergence of chemo-resistant tumors. Exposure of cancer cells to platinum drugs such as cisplatin elicits a stress response that leads to the induction of coping mechanisms favoring cancer cell survival. Autophagy induction is the primary protective mechanism in cancer cells that also contributes to cisplatin resistance. A tightly regulated pH is imperative for autophagy and the role of major pH regulators is therefore vital to this process. Vacuolar ATPases (V-ATPases) are the multi-subunit proton pumps that regulate intra/extra-cellular pH in cancer. Its `a2` isoform (V-ATPase-a2) is located at the plasma membrane and early/late endosomal compartments. We previously found that this isoform is overexpressed on cisplatin resistant cells (cis-R) and its inhibition sensitizes cis-R cells to platinum drugs. In continuation with our efforts, the present study was performed to understand what alterations in the autophagy process occur upon V-ATPAse-a2 inhibition and how these contribute to cisplatin sensitization. Expression profiling of ovarian cancer cells lines (A2780-cis R and TOV-112D- cis R) revealed that upon a2 inhibition, the basal levels of autophagosome membrane protein, LC3B were enhanced compared to control cis-R cells as determined by flow cytometry and western blot analysis. Upon cisplatin treatment, the LC3B levels were further enhanced in a2 inhibited cis-R cells compared to control cells. Further, we observed a significantly increased expression of the autophagy protein, beclin-1 in these cells as compared to control cells. In addition, the inhibition of V-ATPase-a2v increased the protein levels of SQSTM1/p62, a known substrate for the autophagy process. The enhanced LC3B and P62 levels suggest an accumulation of the autophagosomes due to an altered autophagy flux upon V-ATPase-a2v inhibition. The study provides a rationale for the utility of V-ATPase-a2 inhibitors in combination with standard drugs as a novel strategy to improve the treatment efficacy of the chemoresistant ovarian cancer. Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Safaa A. Ibrahim, Alice Gilman-Sachs, Kenneth D. Beaman. Autophagy regulation by vacuolar-ATPases: A contributing mechanism of chemo-resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3184. doi:10.1158/1538-7445.AM2017-3184

P2.01-031 CCL Chemokines May Play an Important Role in Cisplatin Resistance

In the absence of a targetable mutation, cisplatin based chemotherapy is the backbone of NSCLC treatment. However, a diverse patient population combined with complex tumor heterogeneity is hampering its’ clinical utility. Although intrinsic and acquired resistance to cisplatin is common, the mechanisms have not yet been fully elucidated. However, some studies have suggested that inflammatory pathways may play a key role in chemo-resistance. The aim of this project is to increase our understanding of inflammatory mediated cisplatin resistance in NSCLC. A number of isogenic cell line models of NSCLC (adenocarcinoma, squamous cell carcinoma, large cell carcinoma) cisplatin resistance were utilized to assess the role of inflammation in chemo-resistance. These included a sensitive parental cell line (PT) and a matched resistant subtype (CisR). The cell lines were screened for NFKB and a number of inflammatory mediators including chemokines and TLRs at the mRNA (RT-PCR/qPCR) and protein level (Western Blot/ELISA). A specific NFKB inhibitor, DHMEQ, and recombinant chemokines were employed to further characterize inflammatory pathways in PT and CisR cells in terms of cisplatin sensitivity, proliferation (BrdU ELISA), cellular viability (Cytell Cell Imaging System) and DNA damage response (Comet). An in vivo study was also completed using DHMEQ alone and in combination with cisplatin. A number of NFKB targets and responsive pathways are deregulated in CisR cells compared with their matched sensitive PT cell line. Amongst others, CCL2 and CCL5 were altered across all NSCLC subtypes. Preliminary data suggests that DHMEQ enhances cisplatin sensitivity in both PT and CisR cells, conversely recombinant chemokines elicit a protective effect. Additionally, DHMEQ treatment resulted in opposite effects on CCL2 and CCL5 mRNA levels in the PT and CisR cell lines. This may reflect an alternative pathway hierarchy within the cells. Further characterization is ongoing assessing chemokine specific inhibitors. Although, in vivo data suggests a trend of decreased tumor growth in the DHMEQ cohorts compared with vehicle control, the data was not significant. However, tumor samples appeared more necrotic with DHMEQ and are currently being characterized using IHC for necrosis and proliferation. Targeting chemokines downstream of NFKB may provide a means to overcome inflammatory mediated acquired and intrinsic NSCLC chemo-resistance. Given the increased significance of immuno-oncology agents to harness the body’s own immune system in the fight against cancer, these agents may also prove fruitful in re-sensitizing patients to chemotherapy.

MA02.02 A Novel 5-miR Signature Shows Promise as a Diagnostic Tool and as a Predictor of Cisplatin Response in NSCLC

MicroRNAs are a class of small non-coding RNAs that range in size from 19-25 nucleotides. They have been shown to regulate a number of processes within tumor biology, including metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemoresistance in solid tumors, including lung cancer. MicroRNA expression within an isogenic panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines was profiled using the 7th generation miRCURY LNA arrays (Exiqon). Significantly altered miRNAs within the CisR sublines were manipulated using antagomirs (Exiqon) and Pre-miRs (Ambion) and functional studies were carried out in the presence and absence of cisplatin. To examine the translational relevance of these miRNAs, their expression was examined in a cohort of chemo-naïve patient-matched normal and lung tumor tissue and serum from NSCLC patients of different histologies. To create a xenograft model of cisplatin resistance 1x103 cells H460 PT or CisR cells were injected into 5-7week old NOD/SCID mice. Tumor volume was measured over time and harvested once the tumor mass measured 500mm3 and formalin-fixed and paraffin embedded (FFPE). Expression of the 5-miR signature was analysed within FFPE murine tumors and cisplatin resistance was investigated relative to cisplatin sensitive controls. Profiling and subsequent validation revealed a 5-miR signature associated with our model of cisplatin resistance (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286). Inhibition of the miR-30 family and miR-34a-5p reduced clonogenic survival of CisR cells when treated cisplatin. Expression of the miRNA signature was significantly altered in both adenocarcinoma (AD) and squamous cell carcinoma (SCC) relative to matched normal lung tissue and between SCC and AD tissue. miR-4286 was significantly up-regulated in SCC sera compared to normal control and AD sera. Similarly to the cell line expression of the miRNAs, the miR-30 family members and miR-34a-5p were up-regulated in the CisR xenograft FFPE tissue relative to PT. A novel miRNA signature associated with cisplatin resistance was identified in vitro, genetic manipulation of which altered clonogenic response to cisplatin. The 5-miR signature shows both diagnostic and prognostic biomarker potential across a number of diagnostically relevant biological mediums.

P2.03b-096 Utilization of a Novel 3D Culture Technology for the Assessment of Chemo-Resistance in Non-Small Cell Lung Cancer: Topic: Biomarkers

Elucidation of the key mechanisms underlying resistance to chemotherapy is an on-going and complex process. Owing to its suggested increased biological relevance, many are now transitioning from two-dimensional (2D) to three-dimensional (3D) cellular-based assay systems. These systems permit the formation of 3D multicellular structures (MCS). The internal micro-environment of these structures mimics closely those found in vivo. In addition they are considered to provide a more biologically relevant model of chemo-resistance. This study focuses on the utilization of a novel 3D culture technology to compare chemo-resistant models of non-small cell lung cancer (NSCLC) in 3D culture with those cultured in 2D monolayers. Critically, this will provide a valuable tool to determine the biological discrepancies which exist between the two culture methods with the aim to identify novel mechanisms of chemo-resistance in NSCLC. Isogenic NSCLC models of cisplatin resistance, which encompassed sensitive parent (PT) and matched cisplatin resistant (CisR) cell lines, were used for this study. The 3D MCS were cultured in Happy Cell Advanced Suspension Medium™ and 2D monolayers as standard. Both 2D and 3D cultures were exposed to a range of cisplatin concentrations (0 – 100 μM) for a period of 72 h. Subsequently, cellular viability, hypoxia, proteomic and morphological assays were conducted in order to compare the response of both PT and CisR cells in 2D and 3D. High content imaging has identified a central necrotic core within the 3D MCS, which is a feature of the asymmetric growth patterns observed in vivo; that being a decrease in viable cells as you move inwards from the periphery of the MCS. Preliminary data suggests that at equivalent cisplatin concentrations, H460 3D MCS exhibit increased resistance to cisplatin compared with 2D monolayers in both PT and CisR cell lines. Proteomic analysis has also identified diverse pathway modifications in 3D compared with 2D culture, with a number of proteins expressed exclusively in each. Additional cellular characterization and further bioinformatic analysis is on-going. Chemotherapeutic intervention is most frequently employed in the treatment of NSCLC, however many patients exhibit intrinsic and acquired resistance to common chemotherapy drugs, such as cisplatin and targeted agents. As it has been argued that 3D models and their micro-environment are more reflective of the in vivo situation, 3D culture may provide a more accurate in vitro model to elucidate mechanisms of chemo-resistance and possibly aid in the identification of novel targets to re-sensitize and stratify patients for therapy.

P2.03a-063 Small Molecule Cancer Stemness Inhibitor, BBI608, Restores Cisplatin Sensitivity in Resistant NSCLC: Topic: Clinical Trials

The cancer stem cell (CSC) hypothesis is now a well-established and widely investigated field within oncology. It hypothesizes that there is a robustly resistant stem-like population of cells that survive initial chemotherapeutic treatment. These surviving CSCs contribute to the recapitulation of a heterogeneous tumor via a combination of asymmetric and symmetric cell division, subsequently resulting in relapse and therapeutic resistance. BBI608 is a small molecule inhibitor of cancer stemness; it targets STAT3, leading to the inhibition of critical genes required for the maintenance of cancer stemness. To date, preclinical studies investigating BBI608 in in vitro and in vivo models of pancreatic and prostate cancer have shown promise. Aldefluor (Stemcell Technologies) staining and flow cytometry analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified the ALDH1-positive (ALDH1+ve) subpopulation of cells as a key CSC subset across cisplatin resistant NSCLC cell lines. PT and CisR cell lines were treated with BBI608 (1μM) and stemness factors investigated, the presence of the ALDH1+ve CSC population was reassessed by flow cytometry and expression of stemness factors (Nanog, Oct-4, Sox-2, Klf4 and cMyc) were examined by reverse transcriptase PCR. The functional parameters of proliferation, clonogenic survival and apoptosis were investigated with increasing concentrations of cisplatin (0-100μM) in the presence and absence of 1μM BBI608. The NSCLC CisR sublines showed a significantly greater ALDH1+ve CSC population relative to their PT counterparts. Treatment of the CisR sublines with 1μM BBI608 significantly depleted the ALDH1+ve CSC population and decreased gene expression of stemness markers. BBI608 significantly decreased the proliferative capacity and clonogenic survival of the CisR sublines when in combination with cisplatin relative to cisplatin alone. Cisplatin in combination with BBI608 significantly increased cisplatin-induced apoptosis in the CisR sublines indicating restoration of cisplatin sensitivity. To date, BBI608 has not been investigated in terms of a cisplatin resistant CSC population in lung cancer. BBI608, via the inhibition of STAT3, pharmacologically depleted the CSC subpopulation and stemness expression while simultaneously restoring cisplatin sensitivity. There are currently a number of clinical trials recruiting patients to further investigate BBI608. These data suggest a promising role for BBI608 in the treatment of non-responsive or recurrent NSCLC.

Abstract 3537: Cisplatin refractory lung cancer cells exhibit an increased autophagic flux at baseline and following exposure to cisplatin

Abstract Introduction: Lung cancer is the leading cause of cancer-related death. Platinum-based therapy is a standard treatment for advanced lung cancer. However, most patients develop resistance to treatment within few months. Autophagy is a catabolic degradation process whereby cellular proteins and organelles are engulfed by autophagosomes, degraded in lysosomes, and recycled to sustain cellular metabolism. Activation of autophagy in response to cellular stress enables cancer cell survival, which can lead to tumor growth and therapeutic resistance. Materials and Methods: Cisplatin-resistant (cisR A549) lung cancer cells, obtained by treating parental A549 cells with incremental concentrations of cisplatin, were kindly provided by Dr. Martin Barr. To measure autophagy, cells were stained for CytoID and mounted on the Cellometer vision CBA platform, an automated image-based fluorescence microscope equipped with bio-analysis system. Autophagy was confirmed by immunoblotting for LC3II/I and p62 in cell lysates. Lysosomal membrane permeabilization (LMP) was detected after loading lysosomes with FITC-dextran 40KDa and detecting dextran leakage into the cytosol on confocal microscopy. Cell viability was evaluated using CellTiter blue assay. Results: We observed an increase in baseline autophagic flux in cisR A549 lung cancer cells as compared to parental cells in full media condition. This was demonstrated by an increase in LC3II/I ratio, and reduction in p62 level in untreated cells. Compared with untreated cells, cisplatin induced a time-dependent increase in autophagosome formation in cisR A549 cells as manifested by the enhanced cytoID signal. To examine whether the increase in CytoID signal was due to autophagic flux stimulation or autophagosome accumulation secondary to blockage of the last step of autophagy, cells were treated with cisplatin in combination with chloroquine, a known lysosomal inhibitor. This had led to a significant increase in cytoID signal as compared to each drug alone, suggesting that autophagy was induced following exposure to cisplatin in cisR cells. Interestingly, increased CytoID signal following exposure to cisplatin was less pronounced in cisplatin-sensitive cells, suggesting that autophagy stimulation following exposure to the drug is inherent for resistant cells. Inhibition of autophagy using chloroquine sensitized resistant cells to cisplatin. Chloroquine was found to induce LMP. Indeed, neutralization of leaked lysosomal proteases with E64, a pan-protease inhibitor, attenuated the synergistic effect of chloroquine. Further works evaluating whether specific autophagy inhibition employing siRNA ATG3 or 3-MA can restore susceptibility to cisplatin are ongoing. Conclusion: Our findings suggest that increased autophagy may be involved in acquired resistance to cisplatin in lung cancer. Targeting autophagy is a viable strategy to reverse chemoresistance. Citation Format: Magdalena Circu, James Cardelli, Martin Barr, Hazem E. El-Osta. Cisplatin refractory lung cancer cells exhibit an increased autophagic flux at baseline and following exposure to cisplatin. [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 3537.

80P XRCC6BP1: A key DNA repair gene in platinum-resistant NSCLC

Background In the absence of specific treatable mutations, platinum-based doublet chemotherapy remains the gold standard treatment for NSCLC patients. However, its clinical efficacy is hindered in many patients due to both intrinsic and acquired resistance to these agents, in particular, cisplatin. Alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. Methods DNA Repair Pathway RT2 Profiler Arrays were used to elucidate key DNA repair genes in H460 cisplatin resistant (CisR) and corresponding parental (PT) NSCLC cell lines previously generated in our laboratory. DNA repair genes significantly altered in CisR cells were validated using RT-PCR and western blot analysis, respectively. Loss of function studies were carried out using siRNA technology. The effect of gene knockdown on apoptosis was assessed by Annexin V/propidium iodide (PI) staining using the Cytell® Imaging System. DNA damage and repair in response to cisplatin following gene knockdown was investigated using the γH2AX foci formation assay. The translational relevance of these genes was examined in a cohort of chemo-naive matched normal and tumour lung tissues (n = 20). Results We identified a number of important DNA repair genes differentially regulated between H460 PT and CisR NSCLC cells. These included XRCC6BP1, TOP3A, XPA, PMS1 and hSSB1. XRCC6BP1 mRNA was significantly increased (19.4-fold) in H460 CisR cells relative to their PT counterparts. Gene silencing of XRCC6BP1 re-sensitized CisR lung cancer cells to the pro-apoptotic effects of cisplatin and significantly reduced the DNA repair capacity of these cells. Relative to matched normal lung tissues, XRCC6BP1 mRNA was significantly increased in adenocarcinoma tissues (AD), while hSSB1 mRNA was significantly increased in both AD and squamous cell carcinoma (SCC) tissues. Conclusions We have identified XRCC6BP1, in addition to hSSB1, as key DNA repair genes implicated in cisplatin resistant NSCLC. Our data highlights the potential of targeting XRCC6BP1, at least in part, in re-sensitizing chemoresistant lung cancer cells to the cytotoxic effects of cisplatin chemotherapy.

70P Identification of a novel microRNA signature: Potential diagnostic biomarkers and predictors of cisplatin response?

Background In addition to their involvement in mechanisms underlying cancer initiation and progression, emerging evidence has highlighted a key role for microRNAs in resistance to a number of anti-cancer therapies. Methods MicroRNA profiling and validation was carried out on a panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines using 7th generation miRCURY LNA™ arrays (Exiqon). Transient transfections were used to either inhibit or over-express these microRNAs using antagomirs or pre-miRs, respectively. The effects of modulating these miRNAs on cell proliferation, apoptosis and clonogenic survival were examined in response to cisplatin. The translational relevance of these miRNAs in lung cancer was confirmed in a cohort of chemo-naive matched normal and tumour lung tissues and sera from NSCLC patients. MicroRNAs were also assessed in mouse xenograft FFPE tissues from H460 CisR-derived ALDH1+ and ALDH1– cancer stem cell subpopulations. Results A 5-miRNA signature consisting of members of the miR-30 family, miR-34a and miR-4286 was identified and validated. MicroRNAs were significantly up-regulated in all CisR cell lines relative to their parental counterparts, in contrast to miR-4286 which was significantly down-regulated. Inhibition of the miR-30 family and miR-34a and forced over-expression of miR-4286 significantly reduced the clonogenic survival ability of CisR cells in response to cisplatin. The miR-30 family was significantly decreased in AD and SCC tissues. MiR-34a expression was significantly lower in SCC tissues while miR-4286 levels were significantly increased in AD tissues only. A significant-fold increase in miR-4286 was found in sera from SCC lung cancer patients. Similar to that found at the in vitro level, the miR-30 family and miR-34a were up-regulated in mouse xenograft FFPE tissues derived from CisR and PT cell lines in vivo. Conclusions In this study, a 5-microRNA signature was identified using an in vitro model of cisplatin resistance. Differential expression of these microRNAs was found between matched normal and tumour lung tissues from NSCLC patients. These may offer potential as diagnostic markers in NSCLC and in predicting response to cisplatin chemotherapy.

75P Inhibition and exploitation of aldehyde dehydrogenase 1 (ALDH1) as a cancer stem cell marker in cisplatin resistant NSCLC

Background The root of therapeutic resistance is hypothesized to be due to the presence of a rare CSC population within the tumour population. When challenged with chemotherapy, this tumour cell subset survives the insult and has the potential to recapitulate a heterogenic tumour. Aldehyde dehydrogenase 1 (ALDH1) is involved in the catalytic conversion of vitamin A (retinol) to retinoic acid and has been identified as a CSC marker in a number of solid malignancies. Methods FACS analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines of different histologies was used to examine ALDH1 activity. The H460 CisR subline was FACS-sorted (MoFlo®) into ALDH1-positive and ALDH1-negative fractions and subcutaneously injected into NOD/SCID mice to assess tumour initiation and growth. The effects of inhibiting ALDH1 on cell proliferation (BrdU), apoptosis (Annexin-V/propidium iodide) and colony formation (crystal violet) in PT and CisR cell lines was assessed in vitro using two ALDH1 inhibitors, DEAB (N,N-diethylaminobenzaldehyde) and disulfiram (Antabuse), alone and in combination with cisplatin. Lung cancer cells were treated with retinol and All-trans retinoic acid (ATRA) to exploit the role of ALDH1 in the vitamin A/retinoic acid axis in NSCLC. Results ALDH1 activity was significantly increased in all CisR cell lines relative to their parental counterparts in which little or no ALDH1 expression was detected. Xenograft studies using ALDH1+ and ALDH1– fractions from H460 CisR cells showed no significant difference in tumour growth in mice. ALDH1 inhibition, in combination with cisplatin, significantly decreased cell viability, proliferation and clonogenic survival of CisR sublines only and increased apoptotic cell death compared to cells treated with cisplatin alone. Similarly, treatment of cells with retinol or ATRA, in combination with cisplatin, showed similar re-sensitizing effects. Conclusions In this study, we show that inhibition of the CSC marker, ALDH1, re-sensitizes cisplatin resistant NSCLC cells to the cytotoxic effects of cisplatin. Furthermore, our data suggest a role for ALDH1 as a key enzyme in the vitamin A/retinoic acid axis.

Abstract 353: Vorinostat enhances anti-tumor effects of cisplatin in head and neck cancer cells by targeting cancer stem cells

Abstract Objective: Head and neck squamous cell carcinoma (HNSCC) remains a major health care problem worldwide, comprising almost 50% of all malignancies in some developing nations. Even with extensive surgery and full course chemoradiation treatment the survival rates of HNSCC patients have not significantly improved over the last two decades. Acquisition of chemoresistance, locoregional recurrence and metastatic phenotype are the major causes of treatment failure and mortality in these patients. It is therefore imperative that we gain a better understanding of the molecular mechanisms that contribute to the aggressive tumor phenotype in order to develop novel and effective strategies for the treatment of head and neck cancer patients. Recent studies have highlighted the role of histone deacetylases (HDACs) in regulating a number of genes that are involved in cell cycle control, proliferation, survival, DNA repair and differentiation. In this study, we examined if treatment with a HDAC inhibitor, Vorinostat could reverse chemoresistance in head and neck cancer cells and enhance the therapeutic efficacy of cisplatin treatment. Methodology: HDACs expression in tumor samples from HNSCC patients was examined by real time PCR. Cell growth was examined by MTT assay and colony formation assay. Western blot analysis was used to measure changes in protein levels. Cancer stem cell phenotype was assessed by tumorsphere formation. A SCID mouse xenograft model was used to access the in vivo efficacy of Vorinostat. Results: Our results show that HDAC1, 2, and 6 levels are markedly increased in the head and neck cancer cell lines as compared to normal human oral keratinocytes. We took two cisplatin sensitive cell lines and grew them in increasing concentrations of cisplatin to mimic acquired resistance in patients. Both of these cisplatin resistant cell lines (cisR) showed further increase in HDAC1 and 2 expressions as compared to the cispaltin sensitive parental cells. Depletion of HDAC1 and 2 in these cisR cell lines by siRNA significantly enhanced cisplatin sensitivity. Next, we used an HDAC inhibitor, Vorinostat on these cell lines and observed a dose dependent inhibition of cell proliferation (MTT). In addition, Vorinostat treatment significantly decreased tumorsphere formation and the expression of stem cell marker, Nanog. Vorinostat in combination with cisplatin showed a synergistic effect on the inhibition of cell proliferation and tumorsphere formation. We then assessed the efficacy of Vorinostat treatment as a single agent or in combination with cispaltin in SCID mouse xenograft model. Similar to the in vitro results, combination treatment was most effective in reducing the tumor burden in the mice. Conclusions: Taken together, our results suggest that targeting of HDACs with Vorinostat could be an effective treatment strategy for the treatment of HNSCC patients that do not respond to currently used treatment regimens. Citation Format: Bhavna Kumar, Arti Yadav, Theodoros N. Teknos, Pawan Kumar. Vorinostat enhances anti-tumor effects of cisplatin in head and neck cancer cells by targeting cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 353. doi:10.1158/1538-7445.AM2015-353

Suberoylanilide hydroxamic acid (SAHA) reverses chemoresistance in head and neck cancer cells by targeting cancer stem cells via the downregulation of nanog.

Acquisition of chemoresistance and metastatic phenotype are the major causes of treatment failure and mortality in head and neck squamous cell carcinoma (HNSCC) patients. Histone deacetylases (HDACs) have been shown to be overexpressed in many tumor types and directly linked to poor prognosis. In this study, we demonstrate that HDACs are markedly elevated in HNSCC. HDACs expression was further increase in cisplatin resistant cell lines (CisR). In addition, cisplatin-resistant cells showed enhanced stem cell properties and tumor metastasis. Depletion of HDAC1 and 2 in CisR cell lines significantly reversed cisplatin resistance and tumorsphere formation. Next, we tested the efficacy of Suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, by using both in vitro and in vivo models. SAHA significantly inhibited cell proliferation and synergistically enhanced the anti-proliferative effects of cisplatin. In addition, SAHA significantly decreased tumorsphere formation by markedly reducing nanog expression. In a SCID mouse xenograft model, SAHA significantly enhanced the anti-tumor effects of cisplatin treatment with no added systemic toxicity. Furthermore, SAHA and cisplatin combination treatment significantly decreased tumor metastasis and nanog expression, in vivo. Taken together, our results suggest that targeting HDACs with SAHA could be an effective treatment strategy for the treatment of HNSCC patients.

Upregulated expression of FGF13/FHF2 mediates resistance to platinum drugs in cervical cancer cells

Cancer cells often develop drug resistance. In cisplatin-resistant HeLa cisR cells, fibroblast growth factor 13 (FGF13/FHF2) gene and protein expression was strongly upregulated, and intracellular platinum concentrations were kept low. When the FGF13 expression was suppressed, both the cells' resistance to platinum drugs and their ability to keep intracellular platinum low were abolished. Overexpression of FGF13 in parent cells led to greater resistance to cisplatin and reductions in the intracellular platinum concentration. These cisplatin-resistant cells also showed increased resistance to copper. In preoperative cervical cancer biopsy samples from poor prognoses patients after cisplatin chemoradiotherapy, FGF13-positive cells were detected more abundantly than in the biopsy samples from patients with good prognoses. These results suggest that FGF13 plays a pivotal role in mediating resistance to platinum drugs, possibly via a mechanism shared by platinum and copper. Our results point to FGF13 as a novel target and useful prognostic guide for cancer therapy.

Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

IntroductionInherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.MethodsAn isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed.ResultsCharacterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.ConclusionOur results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.

Micro-RNA-21 regulates the sensitivity to cisplatin in human neuroblastoma cells

Background/purposeDrug resistance often causes treatment failure in neuroblastomas. Increasing evidence has implicated that the micro-RNAs (miRNAs) are involved in the development of drug resistance. In this report, we aimed to investigate the role of miRNA in cisplatin resistance of neuroblastoma cells. Materials and methodsThe cell viability of the neuroblastoma cells after cisplatin treatment was analyzed. The expression of the miRNAs and phosphatase and tensin homolog (PTEN) messenger RNA in the neuroblastoma cells was studied by real-time polymerase chain reaction. Overexpression of miRNA or suppression of miRNA expression by antagomir was used to investigate the effects of miRNA on the cisplatin-induced cell death or proliferation. ResultsThe expression of miR-21 was increased in the cisplatin-resistant (CisR) neuroblastoma cells as compared with the parental cells, and the antagomir against miR-21 converted the resistant cells into sensitive ones. Ectopic expression of pre-miR-21 in parental cells resulted in decreased sensitivity to cisplatin treatment. In addition, overexpression of pre-miR-21 markedly increa sed the proliferation rate of neuroblastoma cells. The level of PTEN messenger RNA and protein in the CisR cells was lower than that in the parental cells. Transfection of pre-miR-21 into the parental cells reduced the PTEN expression, and transfection of anti-miR-21 into the CisR cells increased the PTEN expression. ConclusionMicro-RNA-21 regulated the drug resistance and proliferation in neuroblastoma cells.

149P - Microrna Profiling and CDDP-GPG DNA Adduct Formation Analysis in a Panel of Cisplatin Resistant Non-Small Cell Lung Cancer Cell Lines

ABSTRACT Introduction The outcome of cisplatin therapy in NSCLC has reached a plateau, with the development of resistance being a major obstacle in the use of this drug. Understanding the molecular mechanisms underlying this resistance phenotype may aid in the development of novel agents that enhance the sensitivity of cisplatin. In this study, we examined the microRNA profile and levels of cisplatin-DNA adduct formation in a panel of cisplatin resistant NSCLC cell lines. Methods We have previously generated a panel of cisplatin resistant (CisR) cell lines (MOR, H460, A549, SKMES-1, H1299) from original, age-matched parent (PT) cells and extensively characterised these based on a number of functional assays including proliferation (MTT), apoptosis (FACS), cell cycle arrest (PI staining), survival ability (clonogenic assay), DNA copy number gains and losses (CGH arrays) and cancer stem cell marker expression (CD44, CD133, ALDH1, Nanog, Sox-2 and Oct-4) by FACS and Western blot analysis. MicroRNA profiling was carried out using the nCounter miRNA Expression Assay consisting of 749 target miRNA's. Immunofluorescence staining and measurement of specific DNA platination products (CDDP-GpG DNA adducts) was performed at 0, 4, 12 and 24h using immunofluorescence microscopy and quantitative digital image analysis using the ACAS 6.0 CytometryAnalysis System, respectively. Results In a panel of five NSCLC cell lines exhibiting a cisplatin resistant phenotype, a 3- and 13-miRNA signature was deduced, based on differential expression of 3 miRNA's (5/5 cell lines) and 13 miRNA's (4/5 cell lines) in the CisR cell lines relative to their parent counterparts. Levels of CDDP-GpG adducts were greatly reduced in resistant cells up to 24h in response to cisplatin compared to that observed in corresponding parent cells. Conclusion We have identified a distinct miRNA fingerprint in a clinically relevant, isogenic model of cisplatin resistance that may predict resistance to cisplatin in lung cancer. Furthermore, as cisplatin-DNA adducts correlate with cisplatin resistance in vitro, these may be used as a potential predictive marker in patient response to platinum therapy in NSCLC. Disclosure All authors have declared no conflicts of interest.

Prenylation at the indole ring leads to a significant increase of cytotoxicity of tryptophan-containing cyclic dipeptides

Fourteen tryptophan-containing cyclic dipeptides 1a–14a, including all four stereoisomers of cyclo-Trp-Pro and cyclo-Trp-Ala, were converted to their C2-regularly prenylated derivatives 1b–14b in the presence of dimethylallyl diphosphate by using the purified recombinant FtmPT1 as catalyst. The enzyme products were isolated on HPLC in preparative scales and their structures were elucidated by NMR and MS analyses.The cytotoxic effects of the prenylated products and their substrates were tested with human leukemia K562 and ovarian cancer A2780 sens and A2780 CisR cell lines. Preliminary results have been clearly shown that prenylation at C2 led to a significant increase of the cytotoxicity of the tested cyclic dipeptides in all the 14 cases. The second amino acid and the stereochemistry of tryptophan moiety of the cyclic dipeptides showed less influence on the cytotoxicity of the tested compounds.

Synergism from Combination of Cisplatin and Multicentred Platinums Coded as DH6Cl and TH1 in the Human Ovarian Tumour Models

Development of drug resistance and the presence of dose-limiting side-effects remain the two main problems in cancer chemotherapy. Combination of drugs with different mechanisms of action can offer a distinct advantage over monotherapy in overcoming drug resistance and reducing the side-effects. In this study synergism in activity from the combinations of cisplatin (Cis) with two multicentred platinum complexes coded as TH1 and DH6Cl in the human ovarian A2780, A2780cisR and A27800473R cancer cell lines had been investigated. Although Cis, TH1 and DH6Cl all bind with nucleobases in the DNA, they differ in the nature of adducts formed and the non-covalent interactions they may undergo. Whereas Cis binds with nucleobases in the DNA forming mainly intrastrand bifunctional adducts such 1,2-Pt(GG) and 1,2-Pt(AG), TH1 and DH6Cl are expected to form mainly interstrand bifunctional G-Pt.....Pt-G adducts with the DNA. It was found that Cis in combination with TH1 and DH6Cl produced both sequence- and concentration-dependent synergism. Generally greater synergism was produced when the two compounds were added at the same time than with a 4 h time gap. For the combination of Cis with TH1, significant synergism was produced only in the parent A2780 cell line but not in the resistant A2780(cisR) and A2780(0473R) cell lines, thus indicating that the combinations of Cis with TH1 would not offer any advantage in overcoming the drug resistance. In contrast, 0/0 h and 4/0 h combinations of Cis and DH6Cl in A2780(cisR) cell line were found to be synergistic, thus indicating that combinations of Cis with DH6Cl may offer a therapeutic advantage. Although both TH1 and DH6Cl are expected to form a number of long-range interstrand G-Pt......Pt-G adducts with nucleobases in the DNA, TH1 and DHCl are expected to differ in their non-covalent interactions with the DNA due to the presence of two 3-hydroxypyridine ligands bound to the central metal ion in TH1 but not in DH6Cl which instead contains two ammino ligands bound to the central palladium ion.

New pH-dependent complexes, from mononuclear Pd(II) monomer to heteronuclear [Pd(II),K(I)]Polymer: DNA cleavage and cytotoxicity in vitro

Two novel complexes, namely, [Pd(pdc) 2]·(dmp)·6H 2O (1) and [Pd(pdc) 2K(H 2O) 5·3H 2O]n (2) [H 2pdc = pyridine-2,3-dicarboxylate acid, dmp = 2,9-Dimethyl-1,10-phenanthroline] were synthesized and characterized as pH-dependent products. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the pBR 322 plasmid DNA. Values of IC 50 calculated for complexes 1 and 2 show that the two complexes exhibit good cytotoxic activity against different cell lines tested in general, especially more effective against HL-60 and A2780 cisR cell lines.

The relative activity of cisplatin, oxaliplatin and satraplatin in testicular germ cell tumour sensitive and resistant cell lines

Germ cell tumours (GCT) can become resistant to cisplatin, which is associated with a relatively poor prognosis. Oxaliplatin and satraplatin have been developed to overcome cisplatin resistance in other cancers, but their effect in cisplatin resistant (cisR) GCTs is unclear. In this work we address this issue by comparing their efficacy in three paired sensitive and cisR GCT cell lines. Three established cisplatin sensitive (cisS) and resistant cell line pairs were used (GCT27, GCT27r: SUSA, SUSAr: 833k, 833kr). Viability was assessed using a luciferase based ATP assay and EC(50) and EC(80) concentrations were calculated. Western blot analysis and flow cytometry was used for further assessment. Sensitivity to the three platinum compounds was broadly similar in the three cisS lines GCT cell lines (EC(50) = 0.27-0.51 microM for cisplatin, 0.52-0.79 microM for oxaliplatin, 0.31-1.26 microM for satraplatin). EC(50) values for cisplatin in the three cisR sub lines were 1.8- to 3.8-fold higher than in the sensitive parental lines. Cross resistance to satraplatin and oxaliplatin occurred in all three cisR cell lines (resistance factor 1.9-4.4), with the exception of oxaliplatin in the 833Kr (resistance factor 0.9). Differences in the effect of specific drugs on cell cycle distribution, p53, p21 and MDM2 were observed. These data suggest that satraplatin and oxaliplatin could theoretically be used in chemo-naive GCTs and support the further clinical evaluation of these agents in this setting. The mechanism of cross resistance to these drugs appears multifactorial.