anti-CSC Effect Research Articles

PLGA-Nano-Encapsulated Disulfiram Inhibits Cancer Stem Cells and Targets Non-Small Cell Lung Cancer In Vitro and In Vivo.

Cancer stem cells (CSCs) play a key role in non-small cell lung cancer (NSCLC) chemoresistance and metastasis. In this study, we used two NSCLC cell lines to investigate the regulating effect of hypoxia in the induction and maintenance of CSC traits. Our study demonstrated hypoxia-induced stemness and chemoresistance at levels comparable to those in typical CSC sphere culture. Activation of the NF-κB pathway (by transfection of NF-κB-p65) plays a key role in NSCLC CSCs and chemoresistance. Disulfiram (DS), an anti-alcoholism drug, showed a strong in vitro anti-CSC effect. It blocked cancer cell sphere reformation and clonogenicity, synergistically enhanced the cytotoxicity of four anti-NSCLC drugs (doxorubicin, gemcitabine, oxaliplatin and paclitaxel) and reversed hypoxia-induced resistance. The effect of DS on CSCs is copper-dependent. A very short half-life in the bloodstream is the major limitation for the translation of DS into a cancer treatment. Our team previously developed a poly lactic-co-glycolic acid (PLGA) nanoparticle encapsulated DS (DS-PLGA) with a long half-life in the bloodstream. Intra venous injection of DS-PLGA in combination with the oral application of copper gluconate has strong anticancer efficacy in a metastatic NSCLC mouse model. Further study may be able to translate DS-PLGA into cancer applications.

Nimbolide-based nanomedicine inhibits breast cancer stem-like cells by epigenetic reprogramming of DNMTs-SFRP1-Wnt/β-catenin signaling axis

Triple-negative breast cancer (TNBC) harbors a high percentage of breast cancer stem-like cells (BCSCs) that significantly contribute to poor prognosis, metastasis, and relapse of the disease. Thus, targeting BCSCs could be a promising approach to combat TNBC. In this context, we investigated nimbolide (Nim), a limonoid triterpenoid that has potent anticancer properties, but poor pharmacokinetics and low bioavailability limit its therapeutic application. So, to enhance the therapeutic potential of Nim, Nim-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Nim NPs) were formulated and the anticancer stem cell (CSC) effects evaluated invitro and invivo. Invitro studies suggested that Nim NPs significantly inhibited several inherent characteristics of BCSCs, such as stemness, self-renewability, chemoresistance, epithelial-to-mesenchymal transition (EMT), and migration in comparison to native Nim. Next, the mechanism behind the anti-CSC effect of Nim was explored. Mechanistically, we found that Nim epigenetically restores tumor suppressor gene secreted frizzled-related protein 1 (SFRP1) expression by downregulating DNA methyltransferases (DNMTs), leading to Wnt/β-catenin signaling inhibition. Further, invivo results demonstrated that Nim NPs showed enhanced anti-tumor and anti-metastatic effects compared to native Nim in two preclinical models without any systemic toxicity. Overall, these findings provide proof of concept that Nim-based phytonanomedicine can inhibit BCSCs by epigenetic reprogramming of the DNMTs-SFRP1-Wnt/β-catenin signaling axis.

AMPK's double-faced role in advanced stages of prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer deaths in men. Unfortunately, a very limited number of drugs are available for the relapsed and advanced stages of PCa, adding only a few months to survival; therefore, it is vital to develop new drugs. 5´ AMP-activated protein kinase (AMPK) is a master regulator of cell metabolism. It plays a significant role in the metabolism of PCa; hence, it can serve well as a treatment option for the advanced stages of PCa. However, whether this pathway contributes to cancer cell survival or death remains unknown. The present study reviews the possible pathways by which AMPK plays role in the advanced stages of PCa, drug resistance, and metastasis: (1) AMPK has a contradictory role in promoting glycolysis and the Warburg effect which are correlated with cancer stem cells (CSCs) survival and advanced PCa. It exerts its effect by interacting with hypoxia-induced factor 1 (HIF1) α, pyruvate kinase 2 (PKM2), glucose transporter (GLUT) 1 and pyruvate dehydrogenase complex (PDHC), which are key regulators of glycolysis; however, whether it promotes or discourage glycolysis is not conclusive. It can also exert an anti-CSC effect by negative regulation of NANOG and epithelial-mesenchymal transition (EMT) transcription factors, which are the major drivers of CSC maintenance; (2) the regulatory effect of AMPK on autophagy is also noticeable. Androgen receptors' expression increases AMPK activation through Calcium/calmodulin-dependent protein kinase 2 (CaMKK2) and induces autophagy. In addition, AMPK itself increases autophagy by downregulating the mammalian target of rapamycin complex (mTORC). However, whether increased autophagy inhibits or promotes cell death and drug resistance is contradictory. This study reveals that there are numerous pathways other than cell metabolism by which AMPK exerts its effects in the advanced stages of PCa, making it a priceless treatment target. Finally, we mention some drugs developed to treat the advanced stages of PCa by acting on AMPK.

Disulfiram and copper combination therapy targets NPL4, cancer stem cells and extends survival in a medulloblastoma model.

Medulloblastoma (MB) is the most common brain malignancy in children, and is still responsible for significant mortality and morbidity. The aim of this study was to assess the safety and efficacy of Disulfiram (DSF), an FDA-approved inhibitor of Aldehyde-Dehydrogenase (ALDH), and Copper (Cu++) in human SSH-driven and Group 3 MB. The molecular mechanisms, effect on cancer-stem-cells (CSC) and DNA damage were investigated in xenograft models. The cytotoxic and anti-CSC effects of DSF/Cu++ were evaluated with clonogenic assays, flow-cytometry, immunofluorescence, western-blotting. ONS76, UW228 (SHH-driven with Tp53m), D425med, D283 and D341 (Group 3) cell-lines were used. In vivo survival and nuclear protein localization protein-4 (NPL4), Ki67, Cleaved-Caspase-3, GFAP and NeuN expression were assessed in two Group 3 MB xenografts with immunohistochemistry and western-blotting. Significant in vitro cytotoxicity was demonstrated at nanomolar concentrations. DSF/Cu++ induced cell-death through NPL4 accumulation in cell-nucleus and buildup of poly-ubiquitylated proteins. Flow-cytometry demonstrated a significant decrease in ALDH+, Nestin+ and CD133+ following treatment, anti-CSC effect was confirmed in vitro and in vivo. DSF/Cu++ prolonged survival, and increased nuclear NPL4 expression in vivo. Our data suggest that this combination may serve as a novel treatment, as monotherapy or in combination with existing therapies, for aggressive subtypes of pediatric MB.

High dose acetaminophen inhibits STAT3 and has free radical independent anti-cancer stem cell activity

High-dose acetaminophen (AAP) with delayed rescue using n-acetylcysteine (NAC), the FDA-approved antidote to AAP overdose, has demonstrated promising antitumor efficacy in early phase clinical trials. However, the mechanism of action (MOA) of AAP's anticancer effects remains elusive. Using clinically relevant AAP concentrations, we evaluated cancer stem cell (CSC) phenotype in vitro and in vivo in lung cancer and melanoma cells with diverse driver mutations. Associated mechanisms were also studied. Our results demonstrated that AAP inhibited 3D spheroid formation, self-renewal, and expression of CSC markers when human cancer cells were grown in serum-free CSC media. Similarly, anti-CSC activity was demonstrated in vivo in xenograft models - tumor formation following in vitro treatment and ex-vivo spheroid formation following in vivo treatment. Intriguingly, NAC, used to mitigate AAP's liver toxicity, did not rescue cells from AAP's anti-CSC effects, and AAP failed to reduce glutathione levels in tumor xenograft in contrast to mice liver tissue suggesting nonglutathione-related MOA. In fact, AAP mediates its anti-CSC effect via inhibition of STAT3. AAP directly binds to STAT3 with an affinity in the low micromolar range and a high degree of specificity for STAT3 relative to STAT1. These findings have high immediate translational significance concerning advancing AAP with NAC rescue to selectively rescue hepatotoxicity while inhibiting CSCs. The novel mechanism of selective STAT3 inhibition has implications for developing rational anticancer combinations and better patient selection (predictive biomarkers) for clinical studies and developing novel selective STAT3 inhibitors using AAP's molecular scaffold.

Propolis reduces the stemness of head and neck squamous cell carcinoma

ObjectiveTo evaluate the effect of Brazilian propolis on head and neck cancer stem cells in vitro. MethodsHead and neck squamous cell carcinoma (HNSCC) cell lines (UM-SCC-17B and UM-SCC-74A), human keratinocytes (HK), and primary human dermal microvascular endothelial cells (HDMEC) were treated with 0.5, 5.0, or 50 μg/mL green, brown or red Brazilian propolis or vehicle control for 24, 36, and 72 h. Cell viability was evaluated by Sulforhodamine B assay. Western blots evaluated expression of cancer stem cell (CSC) markers (i.e. ALDH, CD44, Oct-4, Bmi-1) and flow cytometry was performed to determine the impact of propolis in the fraction of CSC, defined as ALDHhighCD44high cells. Resultspropolis significantly reduced cell viability of HNSCC and HDMEC cells, but not HK. Notably, red propolis caused a significant reduction in the percentage of CSC, reduced the number of orospheres, and downregulated the expression of stem cell markers. ConclusionsCollectively, our data demonstrate an anti-CSC effect of propolis, and suggest that propolis (i.e. red propolis) might be beneficial for patients with head and neck cancer.

Leukaemia Inhibitory Factor (LIF) Inhibits Cancer Stem Cells Tumorigenic Properties through Hippo Kinases Activation in Gastric Cancer.

Cancer stem cells (CSCs) present chemo-resistance mechanisms contributing to tumour maintenance and recurrence, making their targeting of utmost importance in gastric cancer (GC) therapy. The Hippo pathway has been implicated in gastric CSC properties and was shown to be regulated by leukaemia inhibitory factor receptor (LIFR) and its ligand LIF in breast cancer. This study aimed to determine LIF’s effect on CSC properties in GC cell lines and patient-derived xenograft (PDX) cells, which remains unexplored. LIF’s treatment effect on CSC markers expression and tumoursphere formation was evaluated. The Hippo kinase inhibitor XMU-MP-1 and/or the JAK1 inhibitor Ruxolitinib were used to determine Hippo and canonical JAK/STAT pathway involvement in gastric CSCs’ response to LIF. Results indicate that LIF decreased tumorigenic and chemo-resistant CSCs, in both GC cell lines and PDX cells. In addition, LIF increased activation of LATS1/2 Hippo kinases, thereby decreasing downstream YAP/TAZ nuclear accumulation and TEAD transcriptional activity. LIF’s anti-CSC effect was reversed by XMU-MP-1 but not by Ruxolitinib treatment, highlighting the opposite effects of these two pathways downstream LIFR. In conclusion, LIF displays anti-CSC properties in GC, through Hippo kinases activation, and could in fine constitute a new CSCs-targeting strategy to help decrease relapse cases and bad prognosis in GC.

Synthesis and Characterization of pH-Responsive PEG-Poly(β-Amino Ester) Block Copolymer Micelles as Drug Carriers to Eliminate Cancer Stem Cells

PEG-poly(β-amino ester) (PEG-PBAE), which is an effective pH-responsive copolymer, was mainly synthesized by Michael step polymerization. Thioridazine (Thz), which was reported to selectively eliminate cancer stem cells (CSCs), was loaded into PEG-PBAE micelles (PPM) prepared by self-assembly at low concentrations. The critical micelle concentrations (CMC) of PPM in water were 2.49 μg/mL. The pH-responsive PBAE segment was soluble due to protonated tertiary amine groups when the pH decreased below pH 6.8, but it was insoluble at pH 7.4. The Thz-loaded PEG-PBAE micelle (Thz/PPM) exhibited a spherical shape, and the drug loading was 15.5%. In vitro release of Thz/PPM showed that this pH-sensitivity triggered the rapid release of encapsulated Thz in a weakly acidic environment. The in vitro cytotoxicity and cellular uptake of various formulations at pH 7.4 and 5.5 were evaluated on the mammospheres (MS), which were sorted by MCF-7 human breast cancer cell lines and identified to be a CD44+/CD24− phenotype. The results of the cytotoxicity assay showed that blank micelles were nontoxic and Thz/PPM exhibited a similar anti-CSC effect on MS compared to Thz solution. Stronger fluorescence signal of Coumarin-6 (C6) was observed in MS treated by C6-loaded PPM (C6/PPM) at pH 5.5. The tumor inhibition rate and tumor weight of the free DOX and Thz/PPM groups were significantly different from those of the other groups, which free DOX and Thz/PPM effectively suppressed breast tumor growth in vivo. The above experimental results showed that Thz/PPM is an ideal and effective pH-responsive drug delivery carrier to a targeted therapy of CSCs.

Gamma-Tocotrienol Induces Apoptosis in Prostate Cancer Cells by Targeting the Ang-1/Tie-2 Signalling Pathway.

Emerging evidence suggests that gamma-tocotrienol (γ-T3), a vitamin E isomer, has potent anti-cancer properties against a wide-range of cancers. γ-T3 not only inhibited the growth and survival of cancer cells in vitro, but also suppressed angiogenesis and tumour metastasis under in vivo conditions. Recently, γ-T3 was found to target cancer stem cells (CSCs), leading to suppression of tumour formation and chemosensitisation. Despite its promising anti-cancer potential, the exact mechanisms responsible for the effects of γ-T3 are still largely unknown. Here, we report the identification of Ang-1 (Angiopoietin-1)/Tie-2 as a novel γ-T3 downstream target. In prostate cancer cells, γ-T3 treatment leads to the suppression of Ang-1 at both the mRNA transcript and protein levels. Supplementing the cells with Ang-1 was found to protect them against the anti-CSC effect of γ-T3. Intriguingly, inactivation of Tie-2, a member receptor that mediates the effect of Ang-1, was found to significantly enhance the cytotoxic effect of γ-T3 through activation of AMP-activated protein kinase (AMPK) and subsequent interruption of autophagy. Our results highlighted the therapeutic potential of using γ-T3 in combination with a Tie-2 inhibitor to treat advanced prostate cancer.

Berberine-targeted miR-21 chemosensitizes oral carcinomas stem cells.

Cancer recurrence and chemoresistance are two major obstacles to the treatment of oral squamous cell carcinomas (OSCC). And cancer stem cells (CSCs) have been found to possess tumor initiating, self-renewal and metastasis abilities, resulting in the relapse and chemoresistance of OSCC. In the present study, we investigated the anti-CSCs effect of berberine, a phenanthrene alkaloid isolated from the Berberis genus. Our results demonstrated that berberine dose dependently downregulated the oncogenicity in vitro, including ALDH1 activity, self-renewal property, and colony formation and invasion abilities as well as potentiated chemosensitivity of OSCC-CSCs. In addition, tumor growth in mice was attenuated after oral gavage treatment of berberine. We showed that the expression of miR-21 was suppressed following administration of berberine in OSCC-CSCs. And inhibition of endogenous miR-21 reduced the characteristics of CSCs, including self-renewal, migration, invasion capabilities and ALDH1 activity. Taken together, we demonstrated the anti-CSC effect of berberine in oral cancer and its potential to serve as adjuvant to traditional chemotherapy to improve treatment effect.

GMI ablates cancer stemness and cisplatin resistance in oral carcinomas stem cells through IL-6/Stat3 signaling inhibition.

Cancer stem cells (CSCs) have been identified to exert tumor-initiating ability, resulting in the recurrence, metastasis and chemoresistance of oral squamous cell carcinomas. In the present study, we showed that GMI, an immunomodulatory protein from Ganoderma microsporum, induc ed a cytotoxic effect in oral carcinomas stem cells (OCSCs). Treatment of GMI dose-dependently inhibited the expression of CSC markers, including ALDH1 activity and CD44 positivity. Moreover, GMI suppressed the self-renewal property, colony formation, migration, and invasion abilities as well as potentiated chemo-sensitivity in OCSCs. Our results suggested that the tumor suppressive effect of GMI was mediated through inhibition of IL-6/Stat3 signaling pathway. Furthermore, tumor growth was reduced in mice bearing xenograft tumors after oral administration of GMI. Taken together, we demonstrated the anti-CSC effect of GMI in oral cancer and GMI may serve as a natural cisplatin adjuvant to prevent cancer recurrence.

Selective Targeting of Cancer Stem Cells by 2-Aminodihydroquinoline Analogs.

Many aminodihydroquinoline compounds have been studied to determine their cytotoxicity to cancer cells. However, anti-cancer stem cells (CSCs) activity of aminodihydroquinoline has not been tested in spite that CSC is believed to do an important roles in chemotherapy resistance and recurrence. The CSC selective targeting activities of 10 recently synthesized 2-aminodihydroquinoline analogs were examined on CSCs and bulk culture of a glioblastoma cell line. A diethylaminopropyl substituted aminodihydroquinoline, 5h, showed a strong anti-CSC effect and general cytotoxicity. However, a benzyl substituted aminodihydroquinoline, 5i, displayed the most effective anti-CSC effect, with no or small significant cytotoxic effect in bulk culture conditions. While 5h temporarily enhanced CSC marker-positive cells and eventually suppressed the CSC population, which is similar to other cytotoxic anticancer reagents reported, 5i selectively eliminated CSC marker-positive cells based on fluorescence activated cell sorter (FACS) analysis. 5h also temporarily activated some genes associated with signaling required for CSC, while 5i selectively suppressed these genes supporting that the differential effects are resulted from different molecular responses. In addition, the selective CSC effect is also found against a colon cancer cell line. Collectively, we suggest that these two novel aminodihydroquinoline compounds possess novel anti-CSC effects in colon and brain tumor derived cell lines probably through independent pathways.

Ginsenoside-Rb1 targets chemotherapy-resistant ovarian cancer stem cells via simultaneous inhibition of Wnt/β-catenin signaling and epithelial-to-mesenchymal transition.

Chemoresistance is a major clinical problem compromising the successful treatment of cancer. One exciting approach is the eradication of cancer stem/tumor-initiating cells (jointly CSCs), which account for tumor initiation, progression, and drug resistance. Here we show for the first time, with mechanism-based evidence, that ginsenoside-Rb1, a natural saponin isolated from the rhizome of Panax quinquefolius and notoginseng, exhibits potent cytotoxicity on CSCs. Rb1 and its metabolite compound K could effectively suppress CSC self-renewal without regrowth. Rb1 and compound K treatment also sensitized the CSCs to clinically relevant doses of cisplatin and paclitaxel. These effects were associated with the Wnt/β-catenin signaling pathway by downregulating β-catenin/T-cell factor-dependent transcription and expression of its target genes ATP-binding cassette G2 and P-glycoprotein. We also identified reversal of epithelial-to-mesenchymal transition as a new player in the Rb1 and compound K-mediated inhibition of CSCs. Rb1 and compound K treatment also inhibited the self-renewal of CSCs derived from ovarian carcinoma patients as well as in xenograft tumor model. Moreover, we did not observe toxicity in response to doses of Rb1 and compound K that produced an anti-CSC effect. Therefore, Rb1 should be explored further as a promising nutraceutical prototype of treating refractory tumors.

Abstract 2497: ONC201 targets cancer stem cells in colorectal, prostate and glioblastoma multiforme tumors via modulation of stem cell-related gene expression

Abstract ONC201 is a first-in-class anti-tumor agent that selectively targets cancer cells without observed toxicity. ONC201 is being tested in phase I/II clinical trials for patients with advanced solid tumors and hematological malignancies. Several tumors contain a rare population of cancer stem cells (CSCs) capable of self-renewal that contribute to tumor maintenance and resistance to therapy. We have previously demonstrated that ONC201 targets self-renewing, chemotherapy-resistant colorectal CSCs (Prabhu et al, Cancer Res, 2015). We hypothesized that anti-CSC effects of ONC201 involve changes in stem-cell related gene expression. A targeted network analysis of gene expression profiles of HCT116 p53-null and RKO human colon cancer cells treated with ONC201 revealed that several stem cell-related pathways and proteins are affected with potential implications for anti-CSC effects in diverse tumor types. Specifically, mRNA levels of ID1 (colon/glioblastoma CSC-regulation, 2.5 fold), ID2 (glioma stem cell regulation, 3.2 fold), ID3 (colon/glioma CSC-regulation, 2.9 fold), ALDH7A1 (prostate CSC marker, 2 fold) were significantly downregulated and KLF9 (glioblastoma stem cell inhibitor, 1.5 fold) was significantly upregulated in HCT116 p53-null cells upon ONC201 treatment. Also, mRNA levels of Wnt pathway related genes such as ligand WNT16 (haematopoietic stem cell/prostate cancer resistance-related, 13.5 fold), receptors FZD2 (2.98 fold), FZD4 (3.9 fold) and transcription factor TCF7L2 (3.55 fold) were significantly downregulated. Validation with qRT-PCR indicated that ID2, WNT16 mRNA levels were significantly downregulated while KLF9 mRNA was significantly upregulated in response to ONC201 treatment in HCT116 p53-null cells. Interestingly, ONC201 downregulated mRNA levels of ID1 (2.1 fold) and FZD4 (1.6 fold) in RKO cells but not in ONC201-resistant RKO cells, indicating that CSC-inhibition could serve as a biomarker of ONC201 response. To functionally validate anti-CSC effects of ONC201 in glioblastoma multiforme (GBM) and prostate cancer we determined effects on self-renewal using tumorsphere formation. ONC201 eradicated CSC-enriched 3-dimensional neurosphere culture models of primary GBM samples, including newly diagnosed and recurrent samples. ONC201 potently inhibited in vitro cell proliferation of all 5 lines, with IC50 values of 433 nM (MGG18), 1.46 μM (MGG4), 1.09 μM (MGG8), 3.97 μM (MGG67R) and 688 nM (MGG152). ONC201 significantly reduced tumorsphere formation of 22Rv1, DU145 and PC3 human prostate cancer cells. Additionally, western blotting revealed that Wnt16 was downregulated in LNCaP and 22Rv1 while CSC marker CD44 was downregulated in 22Rv1 cells. Ongoing studies are further validating these potential anti-CSC molecular targets of ONC201 and determining their contribution to the overall anti-CSC and anti-tumor effect of ONC201. Citation Format: Varun Vijay Prabhu, Joshua E. Allen, Dan Zhao, Amriti R. Lulla, Christina Leah B. Kline, A. Pieter J. van den Heuvel, Avital Lev, Tracy T. Batchelor, David T. Dicker, Andrew S. Chi, Wafik S. El-Deiry. ONC201 targets cancer stem cells in colorectal, prostate and glioblastoma multiforme tumors via modulation of stem cell-related gene expression. [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 2497.

Small-Molecule ONC201/TIC10 Targets Chemotherapy-Resistant Colorectal Cancer Stem-like Cells in an Akt/Foxo3a/TRAIL-Dependent Manner.

Self-renewing colorectal cancer stem/progenitor cells (CSC) contribute to tumor maintenance and resistance to therapy. Therapeutic targeting of CSCs could improve treatment response and prolong patient survival. ONC201/TIC10 is a first-in-class antitumor agent that induces TRAIL pathway-mediated cell death in cancer cells without observed toxicity. We have previously described that ONC201/TIC10 exposure leads to transcriptional induction of the TRAIL gene via transcription factor Foxo3a, which is activated by dual inactivation of Akt and ERK. The Akt and ERK pathways serve as important targets in CSCs. Foxo3a is a key mediator of Akt and ERK-mediated CSC regulation. We hypothesized that the potent antitumor effect of ONC201/TIC10 in colorectal cancer involves targeting CSCs and bulk tumor cells. ONC201/TIC10 depletes CD133(+), CD44(+), and Aldefluor(+) cells in vitro and in vivo. TIC10 significantly inhibits colonosphere formation of unsorted and sorted 5-fluorouracil-resistant CSCs. ONC201/TIC10 significantly reduces CSC-initiated xenograft tumor growth in mice and prevents the passage of these tumors. ONC201/TIC10 treatment also decreased xenograft tumor initiation and was superior to 5-fluorouracil treatment. Thus, ONC201/TIC10 inhibits CSC self-renewal in vitro and in vivo. ONC201/TIC10 inhibits Akt and ERK, consequently activating Foxo3a and significantly induces cell surface TRAIL and DR5 expression in both CSCs and non-CSCs. ONC201/TIC10-mediated anti-CSC effect is significantly blocked by the TRAIL sequestering antibody RIK-2. Overexpression of Akt, DR5 knockdown, and Foxo3a knockdown rescues ONC201/TIC10-mediated depletion of CD44(+) cells and colonosphere inhibition. In conclusion, ONC201/TIC10 is a promising agent for colorectal cancer therapy that targets both non-CSCs and CSCs in an Akt-Foxo3a-TRAIL-dependent manner.

ONC201 Depletes Cancer Stem Cells in Refractory Cancer Patient Samples

ONC201 is a small molecule that is being developed as a novel anticancer drug based on its strong anti-tumor efficacy in preclinical models of advanced cancer (Allen et al, Sci Transl Med, 2013). ONC201 has demonstrated strong antitumor effects in vitro and in vivo in a variety of solid and liquid tumors. Cancer stem cells (CSCs) are known to be responsible for tumor propagation and maintenance as well as driving resistance to many cancer therapies. Based on the potent efficacy of ONC201 in preclinical models, we investigated the therapeutic impact of ONC201 on CSCs to rationalize its strong antitumor effects and realize the extent of its therapeutic potential.In primary refractory AML patient samples, ONC201 induced very high levels of apoptosis in leukemia stem/progenitor cells (CD45 dim+/ CD34+/ CD38-) to a similar extent observed in the bulk tumor population. In addition to this effect in hematological malignancies, ONC201 has also exhibited strong anti-CSC efficacy in solid tumors such as colorectal cancer (CRC) and glioblastoma multiforme (GBM). ONC201 eradicated CSC-enriched 3-dimensional neurosphere culture models of primary GBM samples, including newly diagnosed and recurrent samples. ONC201 potently inhibited in vitro cell proliferation of all 5 neurosphere lines tested, with IC50 values of 433 nM (MGG18), 1.46 µM (MGG4), 1.09 µM (MGG8), 3.97 µM (MGG67R) and 688 nM (MGG152). In CRC, ONC201 significantly depleted CD133, CD44 and Aldefluor positive CSCs in HCT116, DLD1 and SW480 cells. ONC201 significantly inhibited colonosphere formation of unsorted and sorted Aldefluor positive 5-Fluorouracil-resistant DLD1 and SW480 cells. ONC201 significantly reduced tumor growth of CSC-initiated tumors and prevented the passage of these tumors. CD44 and CD133 expression was reduced in ONC201-treated tumors. ONC201-treatment decreased tumor initiation relative to 5-Fluorouracil treatment in a limiting dilution tumor initiation assay. The ONC201-mediated anti-CSC effect was significantly blocked by the TRAIL sequestering antibody RIK-2. ONC201-mediated TRAIL-induction was also observed in CSC-initiated tumors. ONC201 inhibited Akt and ERK leading to Foxo3a activation and surface TRAIL induction in sorted CSCs and non-CSCs. Overexpression of myristoylated Akt significantly reduced the induction of surface TRAIL by ONC201 in CSCs and prevented ONC201-mediated depletion of CSCs and colonosphere inhibition.In conclusion, ONC201 cytotoxicity is extended to CSCs in hematological and solid malignancies as well as the bulk tumor population. The ability of ONC201 to deplete CSCs suggests the clinical potential for ONC201 to reduce the incidence of disease relapse, which is frequently a fatal event in hematological malignancies, in addition to its immediately apparent antitumor effects. DisclosuresAllen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. Andreeff:Oncoceutics: Membership on an entity’s Board of Directors or advisory committees. El-Deiry:Oncoceutics: Equity Ownership, Patents & Royalties.

Combination therapy of prostate cancer with HPMA copolymer conjugates containing PI3K/mTOR inhibitor and docetaxel

Combination therapies have been investigated to address the current challenges of anti-cancer therapeutics. In particular, a novel paradigm of combination therapy targeting both cancer stem/progenitor cells and bulk tumor cells is promising to improve the long-term therapeutic benefit against prostate cancer. Among the therapeutic agents with anti-CSC activities, the PI3K/mTOR inhibitors exhibit preferential inhibitory effect on prostate cancer stem/progenitor cells and potent cytotoxicity against bulk tumor cells. The combination of PI3K/mTOR inhibitor and traditional chemotherapy docetaxel may show superior therapeutic effect over single drug treatment. Aiming to further improve the combinational anti-tumor and anti-CSC effect, we developed the combination therapy containing two HPMA copolymer–drug conjugates, incorporated with PI3K/mTOR inhibitor GDC-0980 (P-(GDC-0980)) and docetaxel (P-DTX), respectively. The anti-tumor and anti-CSC effects of the single and combination therapy were investigated in vitro and on PC-3 prostate cancer xenografts in nude mice. Our evaluations showed that P-(GDC-0980) suppressed CD133+ prostate stem/progenitor cell growth even at the low dose which does not cause significant growth inhibition in bulk tumor cells. The combination therapy exhibited effective anti-CSC effect as well as enhanced anti-bulk tumor effect in vitro. Among all the single and combination dosing regimens of free drugs and conjugates, the macromolecular combination therapy showed significantly prolonged mice survival in vivo.

Abstract 3906: Combined inhibition of PI3K isoforms and mTOR kinase is critical for cancer stem cell inhibition by VS-5584

Abstract Cancer stem cells (CSCs) represent a subpopulation of cancer cells that have tumor-initiating capability, are particularly resistant to chemotherapy, and can mediate tumor recurrence both locally and at metastatic sites. As such, these cells represent a critical challenge for effective treatment of cancer. High-throughput screening for small molecules that preferentially target CSCs identified dual inhibitors of the PI3K/mTOR pathway, suggesting the importance of this signaling pathway for CSC biology. Here we demonstrate that ablation of mTOR or individual PI3K isoforms by isoform-specific siRNA is not sufficient to reduce the proportion of CSCs. In contrast, combined knock down of PI3K isoforms and mTOR effectively reduced the proportion of CSCs in tumor cell lines. VS-5584 is a potent and selective pan-PI3K/TORC1/TORC2 inhibitor with equipotency against all four human Class I PI3K isoforms and the mTOR kinase. We demonstrate that VS-5584 preferentially targets CSCs in multiple orthogonal assays both in vitro and in human tumor xenograft models. Cancer stem cells express high levels of aldehyde dehydrogenase, and an Aldefluor assay that measures activity of this enzyme was used to identify CSCs. VS-5584 decreased the percentage of Aldefluor+ cells across multiple breast cancer cell lines. We demonstrated that VS-5584 preferentially induced apoptosis in Aldefluor+ SUM159 cells relative to Aldefluor- cells as measured by Annexin V and Caspase 3/7 assays. In contrast, paclitaxel induced more apoptosis in Aldefluor- than Aldefluor+ cells and enriched the percentage of CSCs. Another characteristic of CSCs is their enhanced ability to efflux cytotoxic agents. This drug-resistant CSC population, called side population (SP), can be monitored by exclusion of Hoechst dye. VS-5584 effectively eliminated the SP CSCs across multiple cancer types, while cisplatin and etoposide increased this subpopulation. Furthermore, ex vivo treatment of primary breast and ovarian tumor specimens with VS-5584 decreased the proportion of CSCs as measured by the Aldefluor assay and cell surface markers. Significantly, VS-5584 also targets CSCs in vivo in MDA-MB-231 triple negative and MCF7 ER+ breast cancer xenograft models as evidenced by decreases in the percentage of Aldefluor+ cells, tumorsphere-forming efficiency, and tumor-initiating capability in an in vivo limiting dilution re-implantation assay. Consistent with the notion that combined inhibition of PI3K isoforms and mTOR is critical for exerting a strong anti-CSC effect, the mTORC1-selective inhibitor everolimus did not reduce CSCs in the MCF7 xenograft model. The potent anti-CSC activities in primary patient cancer tissue and in xenograft models provide strong rationale for the clinical development of VS-5584 in combination with agents targeting the bulk tumor to potentially achieve durable clinical responses for cancer patients. Citation Format: Vihren N. Kolev, Quentin G. Wright, David T. Weaver, Jennifer E. Ring, Christian M. Vidal, Mahesh V. Pavdal, Jonathan A. Pachter, Qunli Xu. Combined inhibition of PI3K isoforms and mTOR kinase is critical for cancer stem cell inhibition by VS-5584. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3906. doi:10.1158/1538-7445.AM2014-3906

OP046: Targeting cancer stem cells (CSC) in oral cavity head and neck squamous cell carcinoma (HNSCC) with dendritic cell (DC) vaccines

Targeting CSCs with a DC vaccine is an innovative and rationale approach that could specifically address the most replicative and therapy-resistant subpopulation of cancer cells within a solid tumor malignancy. Patients with oral cavity squamous cell carcinoma had tumors harvested and the cells sorted for ALDH high and ALDH low cells. ALDH high cells have been shown to possess the properties of cancer stem cells. DCs were generated from the peripheral blood of patients with HNSCC and pulsed in vitro with CSC lysate. DCs generated from the peripheral blood of patients with HNSCC and pulsed with CSC lysate confer greater specific antitumor efficacy by inducing anti-CSC immunity in vitro than DCs pulsed with whole tumor or non-CSC lysate. There was greater specific lysis of ALDH high target cells (vs. ALDH low targets) by CTL co-stimulated with DCs pulsed with ALDH high tumor cells suggesting a specific anti-CSC effect with this stimulation approach. Targeting CSCs in oral cavity HNSCC tumors with a DC vaccine is feasible and demonstrates an antitumor immune response.

Trifluoperazine, an Antipsychotic Agent, Inhibits Cancer Stem Cell Growth and Overcomes Drug Resistance of Lung Cancer

Cancer stem cell (CSC) theory has drawn much attention, with evidence supporting the contribution of stem cells to tumor initiation, relapse, and therapy resistance. To screen 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 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. We then treated lung CSCs with different EGFR mutation status with trifluoperazine to examine its anti-CSC properties. Lung CSCs resistant to epidermal growth factor receptor-tyrosine kinase inhibitor or cisplatin were treated with trifluoperazine plus gefitinib or trifluoperazine plus cisplatin. Animal models were used for in vivo validation of the anti-CSC effect and synergistic effect of trifluoperazine with gefitinib. We demonstrated that trifluoperazine inhibited CSC tumor spheroid formation and down-regulated the expression of CSC markers (CD44/CD133). Trifluoperazine inhibited Wnt/β-catenin signaling in gefitinib-resistant lung cancer spheroids. The combination of trifluoperazine with either gefitinib or cisplatin overcame drug resistance in lung CSCs. Trifluoperazine inhibited the tumor growth and enhanced the inhibitory activity of gefitinib in lung cancer metastatic and orthotopic CSC animal models. Using in silico drug screening by Connectivity Map followed by empirical validations, we repurposed an existing phenothiazine-like antipsychotic drug, trifluoperazine, as a potential anti-CSC agent that could overcome epidermal growth factor receptor-tyrosine kinase inhibitor and chemotherapy resistance.