Chemotherapy-resistant Cell Lines Research Articles

Discovery and cellular stress pathway analysis of 1,4-naphthoquinone derivatives with novel, highly potent broad-spectrum anticancer activity

BackgroundChemotherapy and targeted therapies have made important strides in cancer treatment yet they often fail and new therapies are still needed. Here, we employed a phenotypic screen to identify and analyze the mechanism of action of novel small molecules that interfere with critical pathways involved in tumor cell growth, using chemoresistant A375 melanoma cells as a model.MethodsCell culture studies were performed in ATCC-recommended media. Compounds, and compound libraries were obtained from Boston University or purchased commercially. Effects on A375 cell viability, proliferation and morphology were determined by Celigo Image Cytometer and viability staining. Anticancer activity of the lead compound was tested in a xenograft nude mouse model. Signaling and cell death pathways were analyzed by SDS-PAGE and immunoblotting, and/or fluorescence microscopy.ResultsAfter evaluating 4477 compounds, one hit compound CB533 was identified that caused significant reduction of A375 cell growth. CB533 is an unexplored 1,4-naphthoquinone (NQ) derivative which unlike 1,4-NQ, induced rapid cell death without generating reactive oxygen species (ROS). Structure-activity relationship analysis showed that a pyrrolidine in the 1,4-NQ nucleus in lead compound Pyr-1 yielded optimal activity. CB533 and Pyr-1 had growth-suppressing effects on a large variety of chemotherapy-resistant cancer cell lines in the nano to picomolar range. Pyr-1 also significantly reduced growth of MDA-MB-231 breast cancer cells in nude mice. Pyr-1 rapidly induced activation of major stress pathways and autophagy, which was efficiently blocked by ERK, and somewhat by PI3K inhibitors.ConclusionCB533 and lead Pyr-1 represent novel broad-spectrum, anticancer compounds that are up to 1000-fold more potent than plumbagin, a natural 1,4-NQ with known anticancer activity. Since the growth suppression activities of CB533 and Pyr-1 are unaffected by the chemotherapy resistance of cancer cells, these compounds have promising therapeutic potential. The pyrrolidine in the 3 position of the 1,4-NQ nucleus of Pyr-1 is a critical component of the pharmacophore. Pyr-1-induced cellular stress was mediated by an ERK, and to a lesser extent by an AKT-dependent pathway without involving apoptosis. Our data suggest that Pyr-1 derives its greatly enhanced antitumor activity via mimicking ROS-induced stress signaling without generating ROS, and likely committing cells to autophagy.

Long non-coding RNA tumor suppressor candidate 7 advances chemotherapy sensitivity of endometrial carcinoma through targeted silencing of miR-23b.

Endometrial carcinoma is the most common malignant tumor of the female genital tract worldwide. TUSC7 (tumor suppressor candidate 7) is an antisense long non-coding RNA and is downregulated and acts as a potential tumor suppressor in several malignant tumors. In this study, the low expression of TUSC7 was confirmed in endometrial carcinoma tissues and was associated with high pathological stages of endometrial carcinoma, which revealed that TUSC7 might be involved in tumorigenesis and progression of endometrial carcinoma. Moreover, the expression of TUSC7 in endometrial carcinoma tissues and cell lines resistant to CDDP and Taxol was lower than that in sensitive endometrial carcinoma tissues and cell lines, which indicated that the TUSC7 expression level was positively correlated with the response of endometrial carcinoma patients to chemotherapy with CDDP and Taxol. TUSC7 upregulation inhibited proliferation, blocked cells at G1 phase, and advanced apoptosis and chemotherapy sensitivity to CDDP and Taxol in HEC1A/CR cell line. Furthermore, miR-23b was upregulated in endometrial carcinoma and negatively correlated with the expression of TUSC7. RNA pull-down assay indicated that TUSC7 could specifically silence the expression of miR-23b in HEC1A/CR cell line; miR-23b was a target gene of TUSC7. MiR-23b upregulation mostly reversed the TUSC7-induced regulatory effects on HEC1A/CR cell line. In summary, long non-coding RNA TUSC7 was underexpressed in endometrial carcinoma, especially in endometrial carcinoma chemotherapy-resistant tissues and cell lines and acted as a potential tumor suppressor gene to inhibit cell growth as well as advance the chemotherapy sensitivity through targeted silencing of miR-23b, which might provide a new therapeutic target to endometrial carcinoma.

Construction of colonic cancer drug-resistant cell line COLO and its relationship with tumor stem cells

Objective To construct a colon cancer chemotherapy-resistant cell line COLO, and study its characteristics and its relationship with tumor stem cells. Methods We constructed two 5-fluorouraci (5-FU)-resistant colon cancer cell line COLO/5-FU-1 and COLO/5-FU-2, which were resistant to 0.10 μmol/ml and 0.20 μmol/ml 5-FU respectively through gradiently increased drug concentration. The cha-racteristics of 5-FU-resistant cell lines were compared with parental colon cancer cell line COLO related to proli-feration, colony forming ability, migration and invasion, sphere forming ability, expression of stemness genes and cross drug-resistance. Results In the cell viability assay, 4 days after regular training, the absorbancy of colon cancer 5-FU-resistant cell lines COLO/5-FU-2, COLO/5-FU-1 and parental colon cancer cell line COLO were 0.61±0.13, 0.54±0.07 and 0.41±0.09 respectively, with significant difference (F=63.43, P=0.033). With the increased concentration of 5-FU, 5-FU-resistant cell lines presented increasing clonality. The cloning efficiency of COLO/5-FU-2, COLO/5-FU-1 and parental colon cancer cell line COLO were (87.6±12.7)%, (65.3±9.7)% and (38.5±7.6)% respectively, with significant difference (F=33.64, P=0.017). In each high power field of vision, the cell numbers of migration through the basement membrane of COLO/5-FU-2, COLO/5-FU-1 and parental colon cancer cell line COLO were 482±39, 434±45 and 373±38 respectively; and the cell numbers of invasion through the basement membrane were 174±42, 112±31 and 87±29 respectively, with significant differences (F=109.61, P=0.009; F=67.31, P=0.032). Compared with parental colon cancer cell line COLO, 5-FU-resistant cell lines had higher expression of stemness genes (F=47.31, P=0.042). 5-FU-resistant cell lines were cross-resistant to other chemotherapeutic drugs such as mitoxantrone. For example, after incubation for 96 hours, inhibition rate of mitoxantrone to parent colon cancer cell line COLO was higher significantly than COLO/5-FU-1 and COLO/5-FU-2 (0.749±0.042, 0.423±0.024, 0.342±0.018), with significant difference (F=12.61, P=0.028). The microsphere forming rates of COLO/5-FU-2, COLO/5-FU-1 and parental colon cancer cell line COLO were (8.90±0.97)%, (6.20±0.75)% and (3.90±0.32)% respectively, with significant difference (F=164.32, P=0.006). Conclusion Colon cancer drug-resistant cell line COLO possess tumor stem cell-like characteristics, which are enriched in cancer stem cells. Key words: Colonic neoplasms; Drug resistance, neoplasms; Neoplastic stem cells; Drug-resistant cell line

Expression of microRNA-30a-5p in drug-resistant and drug-sensitive ovarian cancer cell lines.

The present study aimed to explore the expression of microRNA (miRNA or miR) in drug-resistant and drug-sensitive ovarian cancer cell lines, and to seek the potential therapeutic target of ovarian cancer drug-resistant mechanism in order to improve drug resistance by altering miRNA levels. The drug-resistant characteristics of SKOV3/DDP, SKOV3, COC1/DDP and COC1 cell lines were studied. The miRNAs that were differentially expressed between cisplatin-resistant cells and its parental cells in ovarian cancer were screened with a miRNA chip. The effect of miRNAs was detected, and their drug-resistant mechanism was investigated by transfection and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide methods. Among the expression screening of miRNAs, 41 mRNAs, including Homo sapiens (hsa)-miR-30a-5p and hsa-miR-34c-5p, were highly expressed in the drug-resistant cells, whereas 44 miRNAs, including hsa-miR-96-5p and hsa-miR-200c-3p, were lowly expressed. The expression levels of hsa-miR-30a-5p in two types of ovarian cancer chemotherapy-resistant cell lines were significantly higher than those in chemotherapy-sensitive cell lines, which was associated with ovarian cancer chemotherapy resistance. In conclusion, high expression of miRNA-30a-5p was able to promote cell growth and colony forming ability, and enhance cell migration and invasion. Thus, miRNA-30a-5p is expected to become a meaningful novel target for ovarian cancer resistant treatment.

PI3K/Akt/mTOR Pathway Inhibition in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Introduction: Burkitt lymphoma (BL) is the most common form of B-cell non-Hodgkin lymphoma (B-NHL) in children. Despite significant improvements in survival with de novo disease, treatment of relapsed or refractory BL remains a significant hurdle with survival in only about 20% of patients. Novel therapeutic approaches are necessary to improve outcomes in this group of childhood B-NHL patients with the worst prognosis. Recent literature has identified a high rate of recurrent mutations that result in activation of the PI3K/Akt pathway in BL and have implicated activation of PI3K/Akt in coordination with Myc in BL lymphomagenesis. Our laboratory has developed rituximab and chemotherapy resistant cell line models and subsequently found that these cell lines exhibit increased activation of Akt. We hypothesized that increased activation of Akt may be contributing to chemoresistance and that targeting the PI3K/Akt/mTOR pathway may increase chemoresponsiveness. To that end, we have investigated the effect of inhibiting the PI3K/Akt/mTOR pathway with either the PI3K-delta inhibitor idelalisib or the pan-PI3K/mTOR inhibitor BEZ-235 in cell line models of BL.Methods: The in vitro effect of idelalisib or BEZ-235 was investigated in BL cell lines including Raji, Raji 2R and Raji 4RH (rituximab-chemotherapy resistant), Raji 7R and Raji 8RH (rituximab resistant), Ramos and Daudi. Cell viability following inhibitor exposure was assessed by Alamar blu and cell-titer glo assays. The effect of inhibitor exposure on cell cycle progression was determined by flow cytometry using propidium iodide staining. Inhibition of Akt activation following inhibitor exposure was determined using phospho-flow cytometry. The activity of cytotoxic chemotherapeutic agents following inhibition by idelalisib or BEZ-235 was assessed using Alamar blu and cell titer glo assays.Results: In vitro exposure of BL cell lines to idelalisib in concentrations from 0.1-100µM for 24, 48 or 72 hours resulted in a dose and time-dependent decrease in viable cells in all cell lines tested with IC50 concentrations of 60-300uM. Pre-treatment with the pan-caspase inhibitor QVD resulted in a small reversal in the decrease in cell viability suggesting only a minimal portion of the activity was caspase dependent. When induction of apoptosis was measured using annexin V-propidium iodide staining, little induction of apoptosis was observed with single agent idelalisib at concentrations up to 100uM. Determination of cell cycle progression following exposure to idelalisib at 1, 10, 50 or 100 uM for 24, 48 or 72 hours indicated a time and dose dependent cell cycle arrest in all cell lines. In chemotherapy-sensitive cell lines the arrest was primarily noted in G1, while the chemotherapy-resistant Raji 2R and Raji 4RH cell lines exhibited arrest primarily in G2/M. A significant reduction in cell viability following chemotherapy exposure for 48 hours was noted in chemotherapy resistant Raji 2R cells following pre-treatment for 48 hours with idelalisib 10uM compared to non-idelalisib exposed cells (doxorubicin 10uM 55% vs 77%, p<0.001; vincristine 0.05uM, 48% vs 61%, P<0.001). At higher idelalisib pre-treatment concentrations (50uM) additional synergistic activity was observed in Raji 2R cells (cisplatin 48% vs 61%, p<0.001; dexamethasone 67% vs 87%, p<0.01). To further assess the effect of dual inhibition of PI3K and mTOR, cell lines were exposed to the dual inhibitor BEZ-235. BEZ-235 exhibited a more potent decrease in cell viability compared to idelalisib with activity at nM concentrations. Unlike idelalisib, exposure to BEZ-235 resulted in significant induction of apoptosis by Annexin V-propidium iodide staining. BEZ-235 also exhibited synergistic activity in combination with chemotherapy in all cell lines. At equivalent dosing, BEZ-235 exposure resulted in a more significant decrease in Akt phosphorylation compared to idelalisib as determined by flow cytometry for p-Akt at Ser and Thr phosphorylation sites.Conclusions: Chemotherapy sensitive and resistant BL cell line models are susceptible to inhibition of the PI3K/Akt/mTOR pathway. Targeted inhibition of this pathway leads to a decrease in AKT activation, decrease in cell viability, cell cycle arrest and an increase in sensitivity to cytotoxic chemotherapeutic agents. Broader inhibition of both PI3K and mTOR is more effective than more targeted inhibition of PI3K-delta alone. DisclosuresNo relevant conflicts of interest to declare.

Targeting executioner procaspase-3 with the procaspase-activating compound B-PAC-1 induces apoptosis in multiple myeloma cells

Multiple myeloma (MM) is a plasma cell neoplasm that has a low apoptotic index. We investigated a new class of small molecules that target the terminal apoptosis pathway, called procaspase activating compounds (PACs), in myeloma cells. PAC agents (PAC-1 and B-PAC-1) convert executioner procaspases (procaspase 3, 6, and 7) to active caspases 3, 6, and 7, which cleave target substrates to induce cellular apoptosis cascade. We hypothesized that targeting this terminal step could overcome survival and drug-resistance signals in myeloma cells and induce programmed cell death. Myeloma cells expressed executioner caspases. Additionally, our studies demonstrated that B-PAC-1 is cytotoxic to chemotherapy-resistant or sensitive myeloma cell lines (n = 7) and primary patient cells (n = 11). Exogenous zinc abrogated B-PAC-1-induced cell demise. Apoptosis induced by B-PAC-1 treatment was similar in the presence or absence of growth-promoting cytokines such as interleukin 6 and hepatocyte growth factor. Presence or absence of antiapoptotic proteins such as BCL-2, BCL-XL, or MCL-1 did not impact B-PAC-1-mediated programmed cell death. Collectively, our datademonstrate the proapoptotic effect of B-PAC-1 in MM and suggest that activating terminal executioner procaspases 3, 6, and 7 bypasses survival and drug-resistance signals in myeloma cells. This novel strategy has the potential to become an effective antimyeloma therapy.

Abstract 3103: Cyclin E amplification predicts sensitivity of primary Uterine Serous Carcinoma (USC) cell lines to the cdk2 inhibitor CYC065

Abstract We evaluated the in vitro effectiveness of the cdk2 inhibitor CYC065 on multiple primary chemotherapy-resistant USC cell lines with or without CCNE1 amplification. CCNE1-amplified primary cell lines were significantly more sensitive than wild type USC cell lines to CYC065 in vitro (i.e., IC50: mean±STDV = 61.75±13.22 nM and 103.16± 21.9 nM for CCNE1-amplified USC-ARK-2 and USC-ARK-7 cell lines, respectively and 539.2±182.1 nM for the wild type USC-ARK-4 cell line, p = 0.0048). Consistently, low concentrations of CYC065 (i.e., 100 - 300 nM) caused a dose dependent arrest in the G1 phase of the cell cycle specifically in CCNE1-amplified primary USC cell lines. Importantly, CCNE1 knockdown in the USC-ARK-2 cell line resulted in a 9.29-fold increase in the CYC065 IC50 when compared to the MOCK-transfected USC-ARK-2 cell line (p = 0.021). Finally, when primary CCNE1-amplified USC cell lines also harboring ERBB2 amplification (50% of CCNE1-amplified USC cell lines) were incubated in vitro with the combination of CYC065 and Herceptin (a monoclonal antibody targeting the product of the ERBB2 gene, HER2/neu), an increased inhibitory effect was reported in the combination treatment when compared to Herceptin or CYC065 used as single agent (i.e.,% viable cells: mean±STDV = 71.4±0.85, 65.4±14.2, 42.2±2.1 for the Herceptin, the CYC065 and the combination treatment on USC-ARK-2, respectively; p = 0.014). Together these findings identify CYC065 as a promising drug to be considered alone or in combination in the treatment of patients harboring CCNE1-amplified USC. Citation Format: Emiliano Cocco, Stefania Bellone, Salvatore Lopez, Elena Bonazzoli, Federica Predolini, Jonathan D. Black, Alessandro D. Santin. Cyclin E amplification predicts sensitivity of primary Uterine Serous Carcinoma (USC) cell lines to the cdk2 inhibitor CYC065. [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 3103. doi:10.1158/1538-7445.AM2015-3103

Vorinostat, a Histone Deacetylase (HDAC) Inhibitor, Is Active in Chemotherapy Resistant B-Cell Non-Hodgkin Lymphoma and Enhances the Anti-Tumor Activity of Chemotherapy Agents

Pre-clinical models of chemotherapy resistance and clinical observations derived from the CORAL study suggest that primary refractory/relapsed B-Cell Non-Hodgkin Lymphoma (NHL) is a more aggressive and resistant lymphoma to current available treatments. Pre-clinically, we demonstrated that chemotherapy resistance is associated with a deregulation on the apoptotic machinery rendering lymphoma cells resistant to apoptotic stimuli. There is a dire need to develop agents capable to execute alternative pathways of cell death in an attempt to overcome chemotherapy resistance. Post-transcriptional histone modification plays an important role in regulating gene transcription, and is altered by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs regulate several key cellular functions, including: cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. Given their influence in multiple regulatory pathways, HDAC inhibition is an attractive strategy to evaluate its anti-tumor activity in cancer cells. To this end we studied the anti-tumor activity and mechanisms of action of suberoylanilide hydroxamic acid (SAHA, Vorinostat) in drug-resistant pre-clinical models.A panel of rituximab and chemotherapy resistant cell lines and primary tumor cells isolated from B-cell NHL patients (N=42: Denovo=27, Relapse/Rafractory=15) were exposed to escalating doses of vorinostat. Changes in cell viability were determined by Cell Titer Glo ® (Promega, Fitchburg, WI) luminescent assays. Changes in cell cycle were determined by flow cytometric analysis. Subsequently, protein lysates were isolated from vorinostat exposed cells and changes in members of apoptosis, cell cycle family proteins and the acetylation status of histone H3 were evaluated by Western blotting. In addition, cell lines were pre-exposed to vorinostat for 48 hrs and subsequently plated in 384 well plates and exposed to several chemotherapy agents (cisplatin, etoposide, or gemcitabine) changes in cell viability were determined by Cell Titer Glo and synergistic activity was evaluated using the Calcusm® software.Vorinostat induced dose-and time- dependent cell death in cell lines and in primary tumor cells. In addition, in vitro exposure of lymphoma cells to vorinostat resulted in an increase in p21, decrease cyclin dependent kinase2 (CDK2) and cyclin E; acetylation of histone H3 and was associated with a G1 cell cycle arrest. As expected, PARP cleavage was not observed in cell lines exposed to vorisnostat at IC50 doses, and Caspase inhibitor experiment further showed that blockage of caspase pathway cannot rescue cells and primary patient tumor samples from vorinostat induced cell death, suggesting that alternative cell death pathways were executed (i.e. irreversible cell cycle arrest). Moreover vorinostat was found to enhances the anti-tumor activity of chemotherapy agents.Our data suggests that vorinostat is active in drug-resistant pre-clinical models and that in cell lines with known defective apoptotic machinery, it can active alternative pathway of cell death. Given the multiple pathways affected by HDAC inhibition, vorisnostat can potentially be used to overcome acquired resistant to chemotherapy in aggressive B-cell lymphoma. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) DisclosuresNo relevant conflicts of interest to declare.

Afatinib demonstrates remarkable activity against HER2-amplified uterine serous endometrial cancer in vitro and in vivo.

Background:Uterine serous carcinomas (USCs) are an aggressive form of uterine cancer that may rely on HER2/neu amplification as a driver of proliferation. The objective of this paper is to assess the sensitivity of USC cell lines with and without HER2/neu gene amplification to afatinib, an irreversible ErbB tyrosine kinase inhibitor, and to test the efficacy of afatinib in the treatment of HER2-amplified USC xenografts.Methods:Eight of fifteen primary USC cell lines (four with HER2 amplification and four without) demonstrating similar in vitro growth rates were treated with scalar concentrations of afatinib. Effects on cell growth, signalling and cell cycle distribution were determined by flow cytometry assays. Mice harbouring xenografts of HER2/neu-amplified USC were treated with afatinib by gavage to determine the effect on tumour growth and overall survival.Results:Primary chemotherapy-resistant USC cell lines harbouring HER2/neu gene amplification were exquisitely sensitive to afatinib exposure (mean±s.e.m. IC50=0.0056±0.0006 μM) and significantly more sensitive than HER2/neu-non-amplified USC cell lines (mean±s.e.m. IC50=0.563±0.092 μM, P<0.0001). Afatinib exposure resulted in abrogation of cell survival, inhibition of HER2/neu autophosphorylation and S6 transcription factor phosphorylation in HER2/neu overexpressing USC and inhibited the growth of HER2-amplified tumour xenografts improving overall survival (P=0.0017).Conclusions:Afatinib may be highly effective against HER2/neu-amplified chemotherapy-resistant USC. The investigation of afatinib in patients harbouring HER2/neu-amplified USC is warranted.

Solitomab, an epithelial cell adhesion molecule/CD3 bispecific antibody (BiTE), is highly active against primary chemotherapy-resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo.

Solitomab is a novel, bispecific, single-chain antibody that targets epithelial cell adhesion molecule (EpCAM) on tumor cells and also contains a cluster of differentiation 3 (CD3) (T-cell coreceptor) binding region. The authors evaluated the in vitro activity of solitomab against primary chemotherapy-resistant epithelial ovarian carcinoma cell lines as well as malignant cells in ascites. EpCAM expression was evaluated by flow cytometry in 5 primary ovarian cancer cell lines and in 42 fresh ovarian tumor cell cultures in ascites from patients with mainly advanced or recurrent, chemotherapy-resistant disease. The potential activity of solitomab against EpCAM-positive tumor cells was evaluated by flow cytometry, proliferation, and 4-hour chromium-release, cell-mediated cytotoxicity assays. EpCAM expression was detected by flow cytometry in approximately 80% of the fresh ovarian tumors and primary ovarian tumor cell lines tested. EpCAM-positive, chemotherapy-resistant cell lines were identified as resistant to natural killer cell-mediated or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean±standard error of the mean, 3.6%±0.7% of cells killed after incubation of EpCAM-positive cell lines with control bispecific antibody). In contrast, after incubation with solitomab, EpCAM-positive, chemotherapy-resistant cells became highly sensitive to T-cell cytotoxicity (mean±standard error of the mean, 28.2%±2.05% of cells killed; P<.0001) after exposure to peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with EpCAM-expressing malignant cells in ascites with solitomab resulted in a significant increase in T-cell activation markers and a reduction in the number of viable ovarian tumor cells in ascites (P<.001). Solitomab may represent a novel, potentially effective agent for the treatment of chemotherapy-resistant ovarian cancers that overexpress EpCAM.

Solitomab, an EpCAM/CD3 bispecific antibody (BiTE®), is highly active against primary chemotherapy-resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo

Objectives: Chemotherapy-resistant ovarian cancer remains an incurable disease. Solitomab is a novel bispecific single-chain antibody that targets the epithelial antigen EpCAM on tumor cells and also contains a CD3-binding region. We evaluated the in vitro activity of solitomab against multiple primary chemotherapy-resistant epithelial ovarian carcinoma cell lines and malignant tumor cells in ascitic fluid.

Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

BackgroundExportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. MethodsWe used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5' regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. ResultsThe first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. [Display omitted] ConclusionsOur data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures:Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, Equity Ownership.

TARGETING THE 19S PROTEASOMAL SUBUNIT, RPT4, IN COLON CANCER CELLS INDUCES CELL DEATH AND REDUCES CELLULAR PROLIFERATION IN VITRO AND IN VIVO

IntroductionColorectal cancer (CRC) is the third leading cause of cancer related deaths in the US with half a million deaths worldwide. In recent years much research has centered on the...

932 Targeting the 19S Proteasomal Subunit, Rpt4, in Colon Cancer Cells Induces Cell Death and Reduces Cellular Proliferation In Vitro and In Vivo

Introduction Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the US with half a million deaths worldwide. In recent years much research has centered on the ubiquitin-proteasome pathway (UPP), responsible for the degradation of the majority of intracellular proteins, as a therapeutic target for cancer treatment, with deregulation of the UPP frequently observed in a number of malignancies. Indeed, malignant cells have higher vulnerability to the cytotoxic effects of proteasomal inhibition through their greater dependence on proliferative and anti-apoptotic pathways. Aims/Background Here we investigated the expression of the 19S proteasome subunit, Rpt4, one of six ATPases of the 19S regulatory subunit involved in the recognition of proteasome substrates and their unfolding and entry into the catalytic core, in tumour samples derived from patients with CRC. Results Western blotting and immunohistochemical staining of tissue microarrays demonstrated increased expression of Rpt4 in tumour tissue compared to patient matched normal mucosa. Inhibition of Rpt4 expression using siRNA in HCT-116 colorectal cancer cells showed that inhibition of Rpt4 expression leads to reduced cellular proliferation, increased apoptosis and reduced clonogenic survivalin a p53 independent manner. CRL 1807 non-transformed colonocytes remained largely unaffected suggesting that tumour cells may be selectively sensitive to Rpt4 inhibition. Moreover, chemotherapy resistant cell lines were demonstrated to be sensitive to the effects of Rpt4 inhibition. In addition Rpt4 inhibition acted in concert with 5-FU based chemotherapeutic agents by enhancing levels of induced cell death. Interestingly, Rpt4 inhibition led to decreased proteolytic activity of the proteasome. Finally, we could demonstrate that decreased Rpt4 expression could inhibit tumour growth in vivo. HCT-116 luc2 ULTRA (Caliper LS) colorectal cancer cells transfected ex vivo with Rpt4 siRNA and subcutaneously implanted in Balb/c nu-nu immunodeficient mice had significantly increased survival compared to controls. In the same tumour model a cell penetrating peptide based microparticle delivery system administering Rpt4 siRNA via intratumoural injection, demonstrated that in vivo gene silencing of Rpt4 using 0.1 mg/kg siRNA led to reduced tumour volume growth and statistically significant improvement (Log-rank Mantel Cox Test) in survival in these mice. Conclusion Taken together our data suggest the specific inhibition of Rpt4 function may represent a novel therapeutic target for the treatment of CRC.

Knock-Down of PRAME Increases Retinoic Acid Signaling and Cytotoxic Drug Sensitivity of Hodgkin Lymphoma Cells

The prognosis for patients with Hodgkin lymphoma (HL) has improved in recent decades. On the other hand, not all patients can be cured with the currently established therapy regimes and this therapy is associated with several adverse late effects. Therefore it is necessary to develop new therapy strategies. After treatment of L-540 HL cells with 5′-azacytidine (5AC), we observed increased expression of the preferentially expressed antigen in melanoma (PRAME). In addition, we detected an increased resistance of 5AC-treated cells against cytotoxic drugs. We analyzed the influence of PRAME on cell survival of HL cells by knocking down PRAME in the chemotherapy resistant cell line L-428, a cell line that express PRAME at a high level. After knock-down of PRAME using vector based RNA interference we observed increased sensitivity for cisplatin, etoposide and retinoic acid. DNA microarray analysis of HL cells after PRAME knock-down indicated regulation of several genes including down-regulation of known anti-apoptotic factors. Increased retinoic acid signaling in these cells was revealed by increased expression of the retinoic acid metabolizing cytochrome P450 (CYP26B1), a transcriptional target of retinoic acid signaling. Our data suggest that PRAME inhibits retinoic acid signaling in HL cells and that the knock-down of PRAME might be an interesting option for the development of new therapy strategies for patients with chemo-resistant HL.

Highlights of This Issue

The protease urokinase-type plasminogen activator (uPA) is an established prognostic marker and a potential therapeutic target in cancer. To circumvent the specificity problems inherent in inhibitors of the catalytic activity of proteases, we have generated a highly specific uPA-binding 35 nucleotides long 2′-fluoro-pyrimidine RNA molecule. This aptamer inhibits the conversion of the inactive zymogen form to active uPA, the binding of uPA to its cellular receptor uPAR, and uPA-dependent tumor cell dissemination in an in vivo model system. Our results are of general interest for the design of new modalities for inhibition of cancer dissemination.Rummel and colleagues address the controversy as to whether the breast microenvironment is genetically stable. Using a panel of microsatellite markers, they evaluated the genetic changes in high-quality, research-grade stromal DNA, compared them to data generated from archival specimens, and found that high-quality specimens harbored significantly fewer alterations than did archived tissues. These data, which demonstrate a lack of genetic changes, support the idea that development and use of therapeutics targeting the breast stroma will provide a new avenue of effective treatment for patients with breast cancer.Glioblastoma multiforme (GBM) is an aggressive primary brain tumor that is highly resistant to chemotherapy. Svilar and colleagues conducted a synthetic lethal siRNA screen in a chemotherapy-resistant GBM cell line with the alkylator temozolomide (TMZ). A comparison with alkylation screens done in Escherichia coli and Saccharomyces cerevisiae suggested that alkylation resistance mechanisms were evolutionarily conserved. Functional analysis showed that high-dose TMZ treatment initiated the formation of reactive oxygen species and revealed an epistatic relationship between UNG and UBE3B in response to TMZ. These studies provide potential new targets to overcome alkylating agent resistance.The RAS-activated ERK–MAP kinase pathway is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDA), but the mechanism(s) by which this pathway contributes to the aberrant pathobiology of PDA cells remains obscure. By expression profiling of a panel of PDA cell lines in the absence or presence of pharmacologic pathway blockade, Gysin and colleagues detail the full complexity of mRNAs regulated by this pathway. As expected, mRNAs with protein products that promote the cell division cycle feature prominently. However, surprisingly, many of the antiproliferative effects of RAF–MEK–ERK pathway inhibition can be ascribed to regulatory effects on the c-MYC oncoprotein, a noted master regulator of general mRNA expression within the cell.

Alkylation Sensitivity Screens Reveal a Conserved Cross-species Functionome

To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma-derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored toward "druggable" targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases uracil-DNA glycosylase (UNG) and A/G-specific adenine DNA glycosylase (MYH) as well as methylpurine-DNA glycosylase (MPG) to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in Escherichia coli and Saccharomyces cerevisiae. The conserved biologic processes across all three species compose an alkylation functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance.

Smoothened Antagonists Reverse Taxane Resistance in Ovarian Cancer

The hedgehog pathway has been implicated in the formation and maintenance of a variety of malignancies, including ovarian cancer; however, it is unknown whether hedgehog signaling is involved in ovarian cancer chemoresistance. The goal of this study was to determine the effects of antagonizing the hedgehog receptor, Smoothened (Smo), on chemotherapy response in ovarian cancer. Expression of hedgehog pathway members was assessed in three pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using quantitative PCR and Western blot analysis. Cell lines were exposed to increasing concentrations of two different Smo antagonists (cyclopamine, LDE225) alone and in combination with carboplatin or paclitaxel. Selective knockdown of Smo, Gli1, or Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay. A2780cp20 and SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel, or combination therapy. Chemoresistant cell lines showed higher expression (>2-fold, P < 0.05) of hedgehog signaling components compared with their respective parental lines. Smo antagonists sensitized chemotherapy-resistant cell lines to paclitaxel, but not to carboplatin. LDE225 treatment also increased sensitivity of ALDH-positive cells to paclitaxel. A2780cp20 and SKOV3TRip2 xenografts treated with combined LDE225 and paclitaxel had significantly less tumor burden than those treated with vehicle or either agent alone. Increased taxane sensitivity seems to be mediated by a decrease in P-glycoprotein (MDR1) expression. Selective knockdown of Smo, Gli1, or Gli2 all increased taxane sensitivity. Smo antagonists reverse taxane resistance in chemoresistant ovarian cancer models, suggesting combined anti-hedgehog and chemotherapies could provide a useful therapeutic strategy for ovarian cancer.

Parallel Evolution under Chemotherapy Pressure in 29 Breast Cancer Cell Lines Results in Dissimilar Mechanisms of Resistance

BackgroundDeveloping chemotherapy resistant cell lines can help to identify markers of resistance. Instead of using a panel of highly heterogeneous cell lines, we assumed that truly robust and convergent pattern of resistance can be identified in multiple parallel engineered derivatives of only a few parental cell lines.MethodsParallel cell populations were initiated for two breast cancer cell lines (MDA-MB-231 and MCF-7) and these were treated independently for 18 months with doxorubicin or paclitaxel. IC50 values against 4 chemotherapy agents were determined to measure cross-resistance. Chromosomal instability and karyotypic changes were determined by cytogenetics. TaqMan RT-PCR measurements were performed for resistance-candidate genes. Pgp activity was measured by FACS.ResultsAll together 16 doxorubicin- and 13 paclitaxel-treated cell lines were developed showing 2–46 fold and 3–28 fold increase in resistance, respectively. The RT-PCR and FACS analyses confirmed changes in tubulin isofom composition, TOP2A and MVP expression and activity of transport pumps (ABCB1, ABCG2). Cytogenetics showed less chromosomes but more structural aberrations in the resistant cells.ConclusionWe surpassed previous studies by parallel developing a massive number of cell lines to investigate chemoresistance. While the heterogeneity caused evolution of multiple resistant clones with different resistance characteristics, the activation of only a few mechanisms were sufficient in one cell line to achieve resistance.

A Systems Biology Approach Identifies SART1 as a Novel Determinant of Both 5-Fluorouracil and SN38 Drug Resistance in Colorectal Cancer

Chemotherapy response rates for advanced colorectal cancer remain disappointingly low, primarily because of drug resistance, so there is an urgent need to improve current treatment strategies. To identify novel determinants of resistance to the clinically relevant drugs 5-fluorouracil (5-FU) and SN38 (the active metabolite of irinotecan), transcriptional profiling experiments were carried out on pretreatment metastatic colorectal cancer biopsies and HCT116 parental and chemotherapy-resistant cell line models using a disease-specific DNA microarray. To enrich for potential chemoresistance-determining genes, an unsupervised bioinformatics approach was used, and 50 genes were selected and then functionally assessed using custom-designed short interfering RNA (siRNA) screens. In the primary siRNA screen, silencing of 21 genes sensitized HCT116 cells to either 5-FU or SN38 treatment. Three genes (RAPGEF2, PTRF, and SART1) were selected for further analysis in a panel of 5 colorectal cancer cell lines. Silencing SART1 sensitized all 5 cell lines to 5-FU treatment and 4/5 cell lines to SN38 treatment. However, silencing of RAPGEF2 or PTRF had no significant effect on 5-FU or SN38 sensitivity in the wider cell line panel. Further functional analysis of SART1 showed that its silencing induced apoptosis that was caspase-8 dependent. Furthermore, silencing of SART1 led to a downregulation of the caspase-8 inhibitor, c-FLIP, which we have previously shown is a key determinant of drug resistance in colorectal cancer. This study shows the power of systems biology approaches for identifying novel genes that regulate drug resistance and identifies SART1 as a previously unidentified regulator of c-FLIP and drug-induced activation of caspase-8.