Inhibition Of Cancer Stem Cells Research Articles

Abstract 2506: Citral reduces breast tumor growth by inhibiting cancer stem cell marker ALDH1A3

Abstract Breast cancer stem cells (CSCs) can be identified by increased Aldefluor activity which is primarily due to aldehyde dehydrogenase 1A3 (ALDH1A3) expression. In addition to being a CSC marker, ALDH1A3 regulates genes expression via inducing retinoic acid (RA) signaling and plays an important role in mediating progression of cancers, including breast, melanoma, lung and glioblastoma. Therefore, ALDH1A3 represents a novel druggable anti-cancer target of interest. There are no currently characterized inhibitors of the ALDH1A3 isoform, so twelve general ALDH inhibitors were tested for their effectiveness in targeting ALDH1A3 activity. Ability to directly inhibit ALDH activity was quantified with the Aldefluor assay on a patient tumor xenograft and on breast cancer cell lines with known ALDH isoform activity. Inhibition of RA signaling was quantified by measuring expression of the RA-inducible genes RARRES1 and RARβ. To investigate the anti-cancer properties of these compounds, apoptosis was quantified with the annexin V assay. Citral significantly reduced ALDH1A3- and ALDH2-associated Aldefluor activity in breast cancer cell lines and also reduced Aldefluor activity of a patient-derived xenograft. Citral reduced expression of RA-inducible genes through reducing ALDH1A3 activity. Citral induced apoptosis in vitro in an ALDH-independent manner in breast cancer cell lines and reduced proliferation in the MDA-MB-231 breast cancer cell line. Nanoparticle (NP)-encapsulated citral was generated for in vivo use and administered intravenously to female NOD/SCID mice harbouring MDA-MB-231 ALDH1A3-overexpression tumors. 10mg/kg NP-citral significantly decreased the growth of ALDH1A3-driven MDA-MB-231 tumors. Nanoparticle-encapsulated citral shows promise as an adjuvant therapy for patients with tumors that have a large population of high-ALDH1A3 CSCs. Citation Format: Margaret L. Thomas, Roberto De Antueno, Krysta Coyle, Brianne Cruickshank, Michael Giacomantonio, Roy Duncan, Carman Giacomantonio, Paola Marcato. Citral reduces breast tumor growth by inhibiting cancer stem cell marker ALDH1A3. [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 2506.

High-throughput screening identifies artesunate as selective inhibitor of cancer stemness: Involvement of mitochondrial metabolism

Cancer stem cells (CSCs) have robust systems to maintain cancer stemness and drug resistance. Thus, targeting such robust systems instead of focusing on individual signaling pathways should be the approach allowing the identification of selective CSC inhibitors. Here, we used the alkaline phosphatase (ALP) assay to identify inhibitors for cancer stemness in induced cancer stem-like (iCSCL) cells. We screened several compounds from natural product chemical library and evaluated hit compounds for their efficacy on cancer stemness in iCSCL tumorspheres. We identified artesunate, an antimalarial drug, as a selective inhibitor of cancer stemness. Artesunate induced mitochondrial dysfunction that selectively inhibited cancer stemness of iCSCL cells, indicating an essential role of mitochondrial metabolism in cancer stemness.

Forkhead Box Q1 Is a Novel Target of Breast Cancer Stem Cell Inhibition by Diallyl Trisulfide

Diallyl trisulfide (DATS), a metabolic byproduct of garlic, is known to inhibit the growth of breast cancer cells in vitro and in vivo This study demonstrates that DATS targets breast cancer stem cells (bCSC). Exposure of MCF-7 and SUM159 human breast cancer cells to pharmacological concentrations of DATS (2.5 and 5 μm) resulted in dose-dependent inhibition of bCSC, as evidenced by a mammosphere assay and flow cytometric analysis of aldehyde dehydrogenase 1 (ALDH1) activity and the CD44(high)/CD24(low)/epithelial specific antigen-positive fraction. DATS-mediated inhibition of bCSC was associated with a decrease in the protein level of FoxQ1. Overexpression of FoxQ1 in MCF-7 and SUM159 cells increased ALDH1 activity and the CD49f(+)/CD24(-) fraction. Inhibition of ALDH1 activity and/or mammosphere formation upon DATS treatment was significantly attenuated by overexpression of FoxQ1. In agreement with these results, stable knockdown of FoxQ1 using small hairpin RNA augmented bCSC inhibition by DATS. Expression profiling for cancer stem cell-related genes suggested that FoxQ1 may negatively regulate the expression of Dachshund homolog 1 (DACH1), whose expression is lost in invasive breast cancer. Chromatin immunoprecipitation confirmed recruitment of FoxQ1 at the DACH1 promoter. Moreover, inducible expression of DACH1 augmented DATS-mediated inhibition of bCSC. Expression of FoxQ1 protein was significantly higher in triple-negative breast cancer cases compared with normal mammary tissues. Moreover, an inverse association was observed between FoxQ1 and DACH1 gene expression in breast cancer cell lines and tumors. DATS administration inhibited ALDH1 activity in vivo in SUM159 xenografts. These results indicate that FoxQ1 is a novel target of bCSC inhibition by DATS.

Designing and Development of Novel Curcumin Analogues/Congeners as Inhibitors of Breast Cancer Stem Cells Growth

Curcumin, a polyphenolic yellow pigment of the spice turmeric is a unique, multi-targeted molecule having potential for cancer therapy. The inhibition of cancer stem cell growth is an essential step for breast cancer treatment. The main objective of the present work is to identify a potential target of curcumin against breast cancer stem cells and also to predict potency of designed curcumin analogues/congeners on predicted target. The high expression level of P-glycoprotein (P-gp) in the population of breast cancer stem cells promotes the cancer stem cells growth. The anticancer activity of curcumin is directly associated with the inhibition of P-gp mediated efflux process. This efflux mechanism is considered as a major reason for the failure of multidrug resistance in the cancer treatment. Using computational tools, the strong potency of curcumin against P-gp for the inhibition of breast cancer stem cells growth has been determined. Few analogues of curcumin were found to be more effective than curcumin towards cancer stem cells inhibition. In this way, the present study provides the pathway to design and improve the efficacy of novel and therapeutically important herbal drugs for the inhibition of breast cancer stem cells via inhibiting the P-gp mediated efflux mechanism.

Vitamin D and androgen receptor-targeted therapy for triple-negative breast cancer.

Anti-estrogen and anti-HER2 treatments have been among the first and most successful examples of targeted therapy for breast cancer (BC). However, the treatment of triple-negative BC (TNBC) that lack estrogen receptor expression or HER2 amplification remains a major challenge. We previously discovered that approximately two-thirds of TNBCs express vitamin D receptor (VDR) and/or androgen receptor (AR) and hypothesized that TNBCs co-expressing AR and VDR (HR2-av TNBC) could be treated by targeting both of these hormone receptors. To evaluate the feasibility of VDR/AR-targeted therapy in TNBC, we characterized 15 different BC lines and identified 2 HR2-av TNBC lines and examined the changes in their phenotype, viability, and proliferation after VDR and AR-targeted treatment. Treatment of BC cell lines with VDR or AR agonists inhibited cell viability in a receptor-dependent manner, and their combination appeared to inhibit cell viability additively. Moreover, cell viability was further decreased when AR/VDR agonist hormones were combined with chemotherapeutic drugs. The mechanisms of inhibition by AR/VDR agonist hormones included cell cycle arrest and apoptosis in TNBC cell lines. In addition, AR/VDR agonist hormones induced differentiation and inhibited cancer stem cells (CSCs) measured by reduction in tumorsphere formation efficiency, high aldehyde dehydrogenase activity, and CSC markers. Surprisingly, we found that AR antagonists inhibited proliferation of most BC cell lines in an AR-independent manner, raising questions regarding their mechanism of action. In summary, AR/VDR-targeted agonist hormone therapy can inhibit HR2-av TNBC through multiple mechanisms in a receptor-dependent manner and can be combined with chemotherapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-016-3807-y) contains supplementary material, which is available to authorized users.

Nanomedicines Eradicating Cancer Stem-like Cells in Vivo by pH-Triggered Intracellular Cooperative Action of Loaded Drugs.

Nanomedicines capable of control over drug functions have potential for developing resilient therapies, even against tumors harboring recalcitrant cancer stem cells (CSCs). By coordinating drug interactions within the confined inner compartment of core-shell nanomedicines, we conceived multicomponent nanomedicines directed to achieve synchronized and synergistic drug cooperation within tumor cells as a strategy for enhancing efficacy, overcoming drug resistance, and eradicating CSCs. The approach was validated by using polymeric micellar nanomedicines co-incorporating the pan-kinase inhibitor staurosporine (STS), which was identified as the most potent CSC inhibitor from a panel of signaling-pathway inhibitors, and the cytotoxic agent epirubicin (Epi), through rationally contriving the affinity between the drugs. The micelles released both drugs simultaneously, triggered by acidic endosomal pH, attaining concurrent intracellular delivery, with STS working as a companion for Epi, down-regulating efflux transporters and resistance mechanisms induced by Epi. These features prompted the nanomedicines to eradicate orthotopic xenografts of Epi-resistant mesothelioma bearing a CSC subpopulation.

CD44 targeted chemotherapy for co-eradication of breast cancer stem cells and cancer cells using polymeric nanoparticles of salinomycin and paclitaxel

This combinational therapy is mainly aimed for complete eradication of tumor by killing both cancer cells and cancer stem cells. Salinomycin (SLM) was targeted towards cancer stem cells whereas paclitaxel (PTX) was used to kill cancer cells. Drug loaded poly (lactic-co-glycolic acid) nanoparticles were prepared by emulsion solvent diffusion method using cationic stabilizer. Size of the nanoparticles (below 150nm) was determined by dynamic light scattering technique and transmission electron microscopy. In vitro release study confirmed the sustained release pattern of SLM and PTX from nanoparticles more than a month. Cytotoxicity studies on MCF-7 cells revealed the toxicity potential of nanoparticles over drug solutions. Hyaluronic acid (HA) was coated onto the surface of SLM nanoparticles for targeting CD44 receptors over expressed on cancer stem cells and they showed the highest cytotoxicity with minimum IC50 on breast cancer cells. Synergistic cytotoxic effect was also observed with combination of nanoparticles. Cell uptake studies were carried out using FITC loaded nanoparticles. These particles showed improved cellular uptake over FITC solution and HA coating further enhanced the effect by 1.5 folds. CD44 binding efficiency of nanoparticles was studied by staining MDA-MB-231 cells with anti CD44 human antibody and CD44+ cells were enumerated using flow cytometry. CD44+ cell count was drastically decreased when treated with HA coated SLM nanoparticles indicating their efficiency towards cancer stem cells. Combination of HA coated SLM nanoparticles and PTX nanoparticles showed the highest cytotoxicity against CD44+ cells. Hence combinational therapy using conventional chemotherapeutic drug and cancer stem cell inhibitor could be a promising approach in overcoming cancer recurrence due to resistant cell population.

An iTEP-salinomycin nanoparticle that specifically and effectively inhibits metastases of 4T1 orthotopic breast tumors

Cancer stem cell (CSC) inhibitors are a new category of investigational drugs to treat metastasis. Salinomycin (Sali) is one of most studied CSC inhibitors and has reached clinical tests. Several drug carriers have been developed to improve efficacy of Sali. However, Sali has not been shown to inhibit metastasis from orthotopic tumors, the gold standard for metastasis. To fill this gap, we developed an immune-tolerant, elastin-like polypeptide (iTEP)-based nanoparticle (iTEP-Sali-ABA NP) that released 4-(aminomethyl)benzaldehyde-modified Sali (Sali-ABA) under acidic conditions. We found that the NP increased the area under the curve (AUC) of Sali-ABA by 30-fold and the tumor accumulation by 3.4-fold. Furthermore, no metastasis was detected in any of the mice given the NP. However, all the mice died of primary tumor burdens. To overcome primary tumor growth and improve the overall survival, we applied a combination therapy consisting of the iTEP-Sali-ABA NP and iTEP NP-delivered paclitaxel. This therapy effectively retarded primary tumor growth, and most importantly, improved the overall survival. In conclusion, delivery of Sali-ABA by the NP, alone or in combination with paclitaxel, was more effective than free Sali-ABA in decreasing metastasis and increasing survival. This iTEP-Sali-ABA NP represents a novel and clinically promising therapy to combat metastasis.

Cancer stem cell drugs target K-ras signaling in a stemness context.

Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC.

Overcoming chemo/radio-resistance of pancreatic cancer by inhibiting STAT3 signaling.

Chemo/radio-therapy resistance to the deadly pancreatic cancer is mainly due to the failure to kill pancreatic cancer stem cells (CSCs). Signal transducer and activator of transcription 3 (STAT3) is activated in pancreatic CSCs and, therefore, may be a valid target for overcoming therapeutic resistance. Here we investigated the potential of STAT3 inhibition in sensitizing pancreatic cancer to chemo/radio-therapy. We found that the levels of nuclear pSTAT3 in pancreatic cancer correlated with advanced tumor grade and poor patient outcome. Liposomal delivery of a STAT3 inhibitor FLLL32 (Lip-FLLL32) inhibited STAT3 phosphorylation and STAT3 target genes in pancreatic cancer cells and tumors. Consequently, Lip-FLLL32 suppressed pancreatic cancer cell growth, and exhibited synergetic effects with gemcitabine and radiation treatment in vitro and in vivo. Furthermore, Lip-FLLL32 reduced ALDH1-positive CSC population and modulated several potential stem cell markers. These results demonstrate that Lip-FLLL32 suppresses pancreatic tumor growth and sensitizes pancreatic cancer cells to radiotherapy through inhibition of CSCs in a STAT3-dependent manner. By targeting pancreatic CSCs, Lip-FLLL32 provides a novel strategy for pancreatic cancer therapy via overcoming radioresistance.

Inhibitory Effect of Zinc Sulfide Nanoparticles Towards Breast Cancer Stem Cell Migration and Invasion

Cancer stem cells are demonstrated to be a highly malignant cancer with an extremely high migratory ability and conventional therapies have little effect on preventing cancer migration and invasion. In this study, we investigated the inhibitory effect of zinc sulfide (ZnS) nanoparticles towards the migration and invasion of breast cancer stem cells MCF-7-SC. The cytotoxicity studies and the Hoechst staining experiments suggested that there is no obvious toxicity of ZnS has been observed upto a concentration of 400 µg/mL. ZnS nanoparticles significantly inhibited the wound healing in the MCF-7-SC cells. The cell invasion assay and western blot analysis results suggested that ZnS nanoparticles inhibited the metastasis of MCF-7-SCs in dose-dependent manner by suppressing epithelial-mesenchymal transition process. Overall, our experimental analysis suggested that nano ZnS has the ability to inhibit cancer stem cell migration and invasion, which can open up new insights in the cancer therapy.

Natural (tryptophan) metabolites—mediated activation of aryl hydrocarbon receptor inhibits cancer stem cell signaling: what does it mean to prevent cancer recurrence?

Environmental pollutes and toxicants have long been believed as the drivers for cancer initiation and progression. The aryl hydrocarbon receptor (AhR) is one of the master regulators of the metabolism of the environmental toxicants in the cells. The AhR ligands which act as agonist have been identified in a broad range such as air pollutes of Dioxin (also called TCDD) in mist, Polycyclic aromatic hydrocarbon found in soil and particulate matter suspended in air, benzoapyrene found in atmospheric wood burning, indigo dye and dietary carotenoids (1). Ligand bound AhR can be released from HSP90-AhR complex and enter into the nucleus. As a transcription factor with basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domains, AhR nuclear translocation can promote transcription of several genes whose encoding proteins catalyze the metabolism and conjugation of xenobiotics or carcinogens such as CYP1A1 in which gene promoter embeds dioxin response elements (DREs) (1).

Cancer Stem Cells and Cancer Stem Cell Inhibitors in Gastrointestinal Cancers

Cancer stem cells (CSCs) are a subpopulation of phenotypically distinct cancer cells that may play an important role in tumor pathogenesis. The gastrointestinal (GI) system provides a good example for investigation of the role of CSCs in tumor proliferation; GI CSCs are suitable for study due to their abundance, proliferative potential, and consistent structural arrangement that is maintained under tightly controlled signaling pathways. GI stem cells have a long lifespan and this, combined with their rapid turnover, may predispose them to forming CSCs. Alternative possible sources of GI CSCs include differentiated intestinal cells, bone marrow, and cancer cells. Therapies that specifically target CSCs present an exciting opportunity to treat patients with cancer. Enhanced understanding of CSC markers, such as CD133, CD44, and epithelial cell adhesion molecule (EpCAM), may facilitate development of therapies that target them. Among the stemness pathways that have been targeted are Wnt/β-catenin, STAT, Notch, and Nanog.

Mifepristone Suppresses Basal Triple-Negative Breast Cancer Stem Cells by Down-regulating KLF5 Expression.

Triple-negative breast cancer (TNBC) is currently the most malignant subtype of breast cancers without effective targeted therapies. Mifepristone (MIF), a drug regularly used for abortion, has been reported to have anti-tumor activity in multiple hormone-dependent cancers, including luminal type breast cancers. In this study, we showed that MIF suppressed tumor growth of the TNBC cell lines and patient-derived xenografts in NOD-SCID mice. Furthermore, MIF reduced the TNBC cancer stem cell (CSC) population through down-regulating KLF5 expression, a stem cell transcription factor over-expressed in basal type TNBC and promoting cell proliferation, survival and stemness. Interestingly, MIF suppresses the expression of KLF5 through inducing the expression of miR-153. Consistently, miR-153 decreases CSC and miR-153 inhibitor rescued MIF-induced down-regulation of the KLF5 protein level and CSC ratio. Taken together, our findings suggest that MIF inhibits basal TNBC via the miR-153/KLF5 axis and MIF may be used for the treatment of TNBC.

Targeting Cancer Stem Cell Survival in Plasma Cell Leukemia with a Pan-BCL2 Inhibitor

INTRODUCTIONRepresenting ten percent of all hematologic malignancies, multiple myeloma (MM) is typified by clonal plasma cell proliferation in the bone marrow (BM) and may progress to a therapy-resistant stage characterized by circulating malignant plasma cells, which is termed plasma cell leukemia, (PCL). Notably, copy number amplification involving the myeloid cell leukemia (MCL)-1 locus, and translocations at the BCL2 locus have been described in high-risk MM. Moreover, BCL2 family members, including both MCL1 and BCL2, are highly expressed in relapsed MM. Finally, downregulation of BCL2 family members was reported to increase sensitivity to lenalidomide, a standard treatment for MM, suggesting that BCL2-targeted treatments may obviate therapeutic resistance.Sabutoclax, a potent small molecule inhibitor of 6 anti-apoptotic BCL2 family proteins, was shown to inhibit cancer stem cell (CSC) survival in chronic myeloid leukemia (CML) (Goff et al Cell Stem Cell 2013). Thus, we investigated BCL2 pro-survival splice isoform expression in therapy-resistant MM and PCL and evaluated whether Sabutoclax reduces malignant plasma cell burden in humanized primagraft assays.METHODS AND RESULTSBCL2 pro survival expression in primary myeloma samplesMononuclear cells from MM or PCL samples were isolated by Ficoll-Paque density gradient separation and collected for RNA extraction or FACS Aria purified into CD138+ or CD34-/CD138- cancer stem cells (CSC) subsets. To evaluate BCL2 pro-survival family member expression, splice isoform-specific quantitative PCR was performed to measure expression of pro-survival long isoforms compared with short splice isoforms, which are pro-apoptotic. Interestingly, MM and PCL patients displayed higher levels of BCL2-L, MCL1-L, BCLX-L and BLF1-L compared to normal controls. Moreover, prolonged lenalidomide exposure increased BCL2-L and MCL1-L expression in the myeloma cell line H929, compared to untreated cells.Sabutoclax Treatment of a Novel Humanized Plasma Cell Leukemia Primagraft ModelMononuclear cells from three primary PCL patient samples were stably transduced with a GFP-luciferase lentiviral vector and transplanted intrahepatically in newborn RAG2γ-/- c-/- mice. Engraftment was monitored by peripheral blood free light chain ELISA assays. Flow cytometric analyses revealed robust engraftment of PCL cells in bone marrow, spleen, liver and peripheral blood. Once transplanted mice displayed significant tumor burden above background free light chain levels, animals were randomized by ELISA values in vehicle versus Sabutoclax groups. Sabutoclax was selected because this pan-BCL2 targeted compound, unlike related BCL2 inhibitors such as ABT-199, also inhibits MCL1. Sabutoclax (10mg/kg) was administered intravenously twice weekly for two to four doses. Sabutoclax treated PCL mice showed reduced human plasma cell burden in bone marrow and spleen tissues compared to vehicle controls.CONCLUSIONExpression of pro-survival BCL2 splice isoforms, including BCL2-L and MCL1-L, portends PCL engraftment in immunocompromised mice. Treatment of human PCL engrafted mice with Sabutoclax reduces malignant plasma cell survival in hematopoietic tissues. Thus, selective targeting of pro-survival isoform expressing CSC with a pan-BCL2 inhibitor may abolish BCL2 and MCL1-dependent therapeutic resistance in MM and PCL. DisclosuresJamieson:GlaxoSmithKline: Research Funding; Johnson & Johnson: Research Funding.

Abstract B41: The gelatinases-stimuli nanoparticles reverse docetaxel resistance and epithelial to mesenchymal transition in lung cancer cell line

Abstract Drug resistance is a main obstacle for the successful cancer therapy. Emerging evidence suggests that miR-200c functions as an effective cancer stem cells (CSCs) inhibitor and restores sensitivity to microtubule-targeting drugs. In the present work, we engineered the intelligent gelatinases-stimuli nanoparticles (NPs) to co-delivery miR-200c and antitumor drug docetaxel (DOC) to verify their synergetic effects on inhibition of CSCs and cancer cells. After tumor cells were treated with miR-200c NPs, miR-200c and its targeted gene TUBB3 expression were evaluated. The effects of (miR-200c+DOC) NPs on docetaxel-resistant lung cancer cells viability as well as the expression of E-cadherin and CD44 were studied. We found that the (miR-200c+DOC) NPs significantly overcome DOC resistance, possibly by elevated miR-200c expression,low TUBB3 level, and reversed the EMT through upregulation E-cadherin and inhibition lung CSCs. The (miR-200c+DOC) NPs may provide a new modality for co-delivery of nucleic acid and drugs to inhibit CSCs and reverse drug resistance. Citation Format: Qin Liu, Rutian Li, Baorui Liu. The gelatinases-stimuli nanoparticles reverse docetaxel resistance and epithelial to mesenchymal transition in lung cancer cell line. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B41.

Abstract C21: Generation of Wnt- and mitogenic receptor binding bispecific antibodies to target cancer stem cells

Abstract Background: In colorectal cancer (CRC) and other solid tumors, cancer stem cells (CSC) contribute to tumor progression and resistance to standard chemotherapies. The continuous regeneration of the colon is dependent on strict control of developmental (e.g. Wnt) and mitogenic (e.g. EGF) pathway signaling; dysregulation results in uncontrolled proliferation forming the basis of aggressive tumors with metastatic potential. Here we describe the generation of novel bispecific antibodies designed to target CSC through Wnt signaling receptors and block growth factor signaling. The Wnt targets LGR5, LGR4, ZNRF3 and RNF43 were selected since their expression is modulated in CSC populations. The GPCR family members LGR4/LGR5 are positive Wnt regulators and the transmembrane E3 ligases ZNRF3/RNF43 are negative Wnt regulators. The growth factor receptor EGFR is frequently (>70%) overexpressed in CRC and its blockade has demonstrated clinical benefit in a subgroup of patients. More recently, HER3 pathway activation has been implicated in resistance to EGFR-targeted therapies. Experimental procedures and results: Two parallel strategies were applied to generate panels of common light chain (cLC) Fab against LGR4, LGR5, ZNRF3 and RNF43. Humanized cLC mice (MeMo®) were immunized with recombinant protein or DNA, and materials harvested from these mice used to generate Fab regions against these antigens. The second approach utilized large and diverse synthetic cLC Fab-phagemid libraries. Combined, these methods resulted in ∼1500 unique antigen-specific Fab from which ∼300 were selected for further testing. Bispecific antibodies were produced in a human cLC IgG1 format using substitutions in the IgG Fc regions for coexpression of two different heavy chains resulting in the generation of large panels of pure and stable bispecific IgG suitable for screening. The Wnt target specific Fab were combined with a Tetanus toxoid-specific control Fab arm allowing for stringent ranking of these Wnt-specific panels in a monovalent format for specificity, affinity, stability, and ligand (R-Spondin3) blocking potency. Based on this characterization the 54 most promising Wnt targeting arms were combined with a panel of previously characterized EGFR and HER-3 specific Fab arms resulting in ∼ 500 different cLC bispecific IgG for functional testing. All bispecific IgG were screened for potency of growth inhibition of CSC using novel 3D high content imaging readouts on patient-derived CRC organoids. The organoids are cultured using growth factors that allow for the maintenance and proliferation of healthy and diseased stem cells and their offspring. Functional analysis revealed several bispecific antibodies that inhibited CRC organoid growth much more potently than comparator drugs such as cetuximab or erlotinib. Conclusion: Bispecific antibodies present a biological modality that result in unexpected functional activities by mechanisms possible unique to the architecture of these molecules. Identifying these unique properties requires the rapid generation and screening of large panels of bispecific IgG directly in the therapeutic format in relevant functional assays. Initial screening results of the bispecific antibodies targeting Wnt and HER family members supports the concept of CSC targeting and several leads are currently undergoing more extensive characterization. Citation Format: Berina Eppink, Rob Roovers, Bram Herpers, Wim de Lau, Carina Clements, Vanessa Zondag van der Zande, Abdul Basmeleh, Willem Bartelink, Marc van de Wetering, Robert Vries, Leo Price, John De Kruif, Mark Throsby. Generation of Wnt- and mitogenic receptor binding bispecific antibodies to target cancer stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C21.

Abstract A2-32: Identification of withaferin A as a novel cancer stem cell inhibitor to overcome drug resistance in non-small cell lung carcinoma

Abstract Cancer stem cell (CSC) theory has drawn much attention, with evidence supporting the contribution of stem cells to tumor initiation, relapse, and therapy resistance. In this study, we focus on screening drugs that target CSCs to improve the current treatment outcome and overcome drug resistance in patients with lung cancer. We used publicly available embryonic stem cell and CSC-associated gene signatures to query the Connectivity Map (CMap) for potential drugs that can, at least in part, reverse the gene expression profile of CSCs. High scores were noted for several phenothiazine-like antipsychotic drugs, including trifluoperazine and perphenazine. The combination of gemcitabine with either trifluoperazine or perphenazine shows synergistic effects in anti-NSCLC cells. By using L1000 and CMap analysis, we identified withaferin A (WA) as a potential drug, which shares similar gene expression patterns to trifluoperazine or perphenazine combine with gemcitabine and to reverse differential gene expression of stem-like cell vs. differential cell. Further experiments demonstrated that WA significantly suppressed cancer stemness associated phenotypes, shown by decreasing side population and tumor spheroid formation. Furthermore, WA was accompanied by down-regulation of JAK2/STAT3 signaling pathway. The combination of WA and cisplatin significantly reduced the number of cancer spheroids and attenuated the proportion of ALDH+ cell population in CL97 lung spheroids, which was resistant to cisplatin treatment alone. In vivo study also demonstrated the anti-tumor effect of WA treatment. In conclusion, WA was identified as a potential anti-cancer stem cell inhibitor, which could also inhibit tumorigenesis and overcome cisplatin resistance in lung cancer. Citation Format: Tai-shan Cheng, Yu-Chen Tsai, Alexander T. H. Wu, Peter M. H. Chang, Wei-Hsiang Hsu, Jin-Mei Lai, Chi-Ying Huang. Identification of withaferin A as a novel cancer stem cell inhibitor to overcome drug resistance in non-small cell lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-32.

Detonation nanodiamond complexes with cancer stem cells inhibitors or paracrine products of mesenchymal stem cells as new potential medications

Combined use of complexes of the most active chemotherapeutic drugs and detonation nanodiamonds (DND) is a new trend in cancer therapy, which is probably related to selective chemotherapeutic drug delivery by DND to the zone of so-called cancer stem cells (CSC). Stable DND complexes of 4–5 nm size with salinomycin—a strong CSC inhibitor—have been obtained (as a suspension). It has been demonstrated that a complex administration considerably increases the drug antitumor effect on the transplantable tumor of LLC mice. A similar effect has been observed in CSC models in vivo, obtained by exposure of stem cells of normal mice tissues to a carcinogen 1,2-dimethylhydrazine. It has also been found out, that administration of DND complexes with the conditioned medium from mesenchymal stem cells (MSC) cultures to mice results in a considerable stimulation of stem cell pools in normal mice tissues, which can be used in regenerative medicine.

Doxycycline and therapeutic targeting of the DNA damage response in cancer cells: old drug, new purpose.

There is a small proportion of cells within a tumour with self-renewing properties, which is resistant to conventional therapy, and is responsible for tumour initiation, maintenance and metastasis. These cells are known as cancer stem cells (CSCs) or tumour-initiating cells (TICs) [1]. Recent publications identify several antibiotics, such as salinomycin or doxycycline, as selective CSCs inhibitors [2-4]. However, the mechanisms of action of these antibiotics on CSCs are not fully understood.