CSC Population Research Articles

NF-κB Participates in the Stem Cell Phenotype of Ovarian Cancer Cells

NF-κB is a transcription factor involved in cancer stem cells maintenance of many tumors. Little is known about the specific stem-associated upstream regulators of this pathway in ovarian cancer. The Aim of the study was to analyze the role of the canonical and non-canonical NF-κB pathways in stem cells of ovarian cancer cell lines. Stem cells were isolated using sorting cytometry. Western blot and RT-PCR were used to quantify protein and messenger RNA levels. Loss and gain of function assays were performed using siRNAs and dominant-negative proteins, respectively. NF-κB binding activity was measured with a reporter gene assay. The stem phenotype was estimated with clonogenic assays using soft agar, colony formation, ovospheres formation and invivo tumorigenicity assays. The CD44+subpopulation of SKOV3 ovarian cancer cell line presented higher mRNA levels of key stemness genes, an increased tumorigenic capacity and higher expression of the RelA, RelB and IKKα. When the canonical pathway was inhibited by means of a dominant-negative version of IkBα, the stem cell population was reduced, as shown by a reduced CD44+subpopulation, a decrease in the expression of the stemness genes and a reduction of the stem phenotype. In addition, IKKα, the main upstream non-canonical kinase, was highly expressed in the CSC population. Accordingly, when IKKα was inhibited using shRNAs, the expression of the stemness genes was reduced. This report is the first to show the importance of several elements of both NF-κB pathway in maintaining the ovarian cancer stem cell population.

The heterogeneity of cancer stem-like cells at the invasive front

Cancer stem-like cells exhibit the multi-functional roles to survive and persist for a long period in the minimal residual disease after the conventional anti-cancer treatments. Cancer stem-like cells of solid malignant tumors which highly express CD44v8-10, the variant isoform of CD44 generated by alternative splicing, has a resistance to redox stress by the robust production of glutathione mediated by ESRP1-CD44v-xCT (cystine/glutamate antiporter) axis. It has been reported that CD44v and c-Myc tend to show the inversed expression pattern at the invasive front of the aggressive tumors. Given that the accumulation of reactive oxygen species triggers the activation of Wnt/β-catenin signal pathway, it is hypothesized that CD44v causes the negative feedback machinery in the regulation of c-Myc expression via the attenuated ROS-induced Wnt signal pathway. To address the fundamental question whether and how both proliferative and quiescent cancer stem-like cells heterogeneously exist at the invasive/metastatic edge, researchers need to investigate into the E3-ubiquitin ligase activity essential for c-Myc degradation. CSCs heterogeneity at the invasive/metastatic front is expected to demonstrate the dynamic tumor evolution with the selective pressure of anti-cancer treatments. Furthermore, the novel molecular targeting therapeutic strategies would be established to disrupt the finely-regulated c-Myc expression in the heterogeneous CSC population in combination with the typical drug-repositioning with xCT inhibitor.

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.

P2.03a-064 Inhibition and Exploitation of Aldehyde Dehydrogenase 1 as a Cancer Stem Cell Marker to Overcome Cisplatin Resistant NSCLC: Topic: Clinical Trials

The root of therapeutic resistance is hypothesized to be the presence a rare CSC population within the tumor population which survives chemotherapeutic treatment and has the potential to recapitulate a heterogenic tumor. Aldehyde dehydrogenase 1 (ALDH1) is involved the catalytic conversion of vitamin A (retinol) to retinoic acid and has been identified as a CSC marker in a number of solid malignancies. FACS analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified ALDH1 as a promising CSC-marker associated with cisplatin resistance across NSCLC histologies. The H460 CisR subline was separated by FACS into ALDH1-positive and negative subpopulations and subcutaneously injected into NOD/SCID mice to assess tumor initiation and growth. ALDH1 was inhibited in vitro within the cell lines using two pharmacological ALDH1 inhibitors, DEAB and disulfiram, alone and in combination with cisplatin. Cell lines were treated in vitro with retinol and all-trans retinoic acid (ATRA) to exploit the vitamin A/retinoic acid axis in which ALDH1 is involved. The CisR sublines showed significantly greater ALDH1 activity relative to their PT counterparts. In vivo subcutaneous injection of ALDH1-positive and negative subpopulations revealed no significant difference in tumor initiation or growth rate. ALDH1 inhibition in combination with cisplatin significantly decreased clonogenic and proliferative competencies and increased apoptosis cell death compared to cisplatin alone. Vitamin A supplementation and ATRA treatment in combination with cisplatin showed similar re-sensitizing effects. This pharmacological CSC depletion in conjunction with cisplatin treatment resulted in re-sensitization of cisplatin resistant cells to the cytotoxic effects of cisplatin. These data suggest vitamin A supplementation or the addition of ATRA or an ALDH1 inhibitor to the cisplatin-based chemotherapeutic regimen may be of clinical benefit in overcoming tumor recurrence and cisplatin resistance.

STMC-01. NANOPARTICLE DELIVERY OF miRNAs TO INHIBIT GBM STEM CELLS

Cancer cells arise from multiple complementary genomic and epigenomic abnormalities that deregulate pathways that control cell proliferation and tissue homeostasis. Epigenetic modifications, involving deregulation of non-coding RNAs, are emerging as critical determinants of gene expression and essential drivers of neoplastic phenotypes. Our knowledge of how these complex epigenetic mechanisms operate in the context of cancer cell phenotype regulation remains limited. Non-coding RNAs, in particular miRNAs, are emerging as critical epigenetic regulators of cell fate and oncogenesis. MiRNAs act by selectively inhibiting gene expression primarily by targeting mRNA for degradation usually via complementary 3’-UTR seed sequences. Numerous miRNAs have been found to regulate tumorigenesis and cancer cell stemness by virtue of their capacity to target tumor-suppressing or tumor promoting transcripts. We recently showed that the coordinated actions of Oct4 and Sox2 induce a tumor-propagating stem-like state in GBM cells through a mechanism that involves the induction of DNMTs and down-regulation of a network of miRNAs through promoter DNA methylation. Two of the miRNAs repressed by Oct4/Sox2, miR-148a and miR-296-5p, efficiently inhibit the tumor propagating capacity of GBM stem-like, making them excellent candidates for therapeutic intervention. Recent developments in nanomedicine provide new and exciting opportunities to treat and manage brain tumors. Cationic polymers are a class of biomaterials with great promise for targeted molecular therapeutics. We combined this cutting-edge technology with our newly discovered stem cell inhibiting miRNAs to develop nano/miR conjugates to treat gliomas. We show these nano/miR conjugates distribute throughout an established tumor in vivo, and more importantly, delivering these tumor-suppressing miRNAs using PBAE polymers inhibits the growth of established GBM tumor in mouse models. Our findings demonstrate that identifying and validating stem cell-inhibitory in combination with current advances in nanomedicine will undoubtedly impact the development of novel therapies for targeting the CSC population and treating GBM.

Abstract B21: Exostosin 1 regulates cancer cell stemness in breast cancer cells

Abstract Cancer stem cells (CSCs), a group of cancer cells, are associated with resistance to radiation and chemotherapy and are implicated in recurrent of cancer. Exostosin 1 (EXT1) gene is widely reported as tumor suppressor and its indispensable role in elongation of heparan sulfate (HS) can speculate probable role as tumor promotor. In recent years, a number of tumors are reported to over express EXT1. Here, we investigated the role of EXT1 in the maintenance of cancer cell stemness. MCF7/ADR cells developed by exposing MCF7, breast cancer cells, to doxorubicin for several months in culture, showed high resistance to doxorubicin compared to MCF7, parental cell line. Doxorubicin resistant MCF7/ADR cells have enhanced P-glycoprotein (P-gp) expression. In FACs analysis, MCF7/ADR cells were accounted for high number of CSC population; they possessed large number of ALDH+ and CD44+/CD24- population compared to MCF7. CSC marker, CD44, was found overexpressed in MCF7/ADR compared to MCF7. Besides that, in mammosphere culture, MCF7/ADR cells formed large number of mammospheres in contrast to MCF7. In microarray data analysis, several genes were found upregulated in MCF7/ADR cells and EXT1 was selected as a candidate gene. Overexpressed EXT1 in MCF7/ADR cells was confirmed by real time PCR. The critical role of EXT1 in maintaining cancer cell stemness was confirmed by siRNA mediated knockdown of EXT1. Knockdown of EXT1 repressed CSC markers, reduced population of ALDH+ and CD44+/CD24-, and suppressed expression of CD44. As a molecular mechanism, we detected suppressed expression of heparan sulfate, syndecan1(SDC1) with siEXT1. With these results, we can suggest that EXT1 level might be a determining factor for doxorubicin sensitive breast cancer cells and enhanced expression of EXT1 can take part in development of CSC phenotype on exposure to anthracycline based therapy through upregulation of SDC1. Citation Format: Sarala Manandhar, Chang-gu Kim, Su Young Oh, Sun-Hee Lee, Jiyoon Seok, Yuk-Dong Jung, Hye-Eun Lee, Young-Sun Choi, You Mie Lee*. Exostosin 1 regulates cancer cell stemness in breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B21.

Abstract 4748: Targeting colon cancer stem cells: Novel marmelin analog THB suppresses DCLK1 and Notch Signaling

Abstract Background: Despite therapeutic advances, colon cancer remains the second leading cause of death in the United States. Previously, we have reported that the identification of a novel compound, HDNC or Marmelin from Aegle marmelos with potent anti-colon cancer activity. We have now developed a novel marmelin analogue THB and made it water soluble THB using β-cyclodextrin (THBCD). The current study is designed to determine whether THB affects stem cells and to identify a mechanism. Method: Colon cancer cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we have performed HCT116 cells induced tumor xenografts. Immunohistochemistry was determined for stem cell markers and Notch signaling proteins. Results: THB treatment induced a significant dose-dependent inhibition of proliferation and colony formation of the two colon cancer cell lines, but not that of the normal cells. To demonstrate THB effects on stem cells, we performed colosphere assays. THB treatment significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, colon stem cell marker proteins DCLK1, LGR5, and CD44 were also decreased. Further proof was obtained by flow cytometry analyses, where THB reduced the number of DCLK1+ cells. We next determined whether THB affects the Notch signaling pathway, a pathway that is important in maintaining CSC population. Notch receptor and its ligands are up-regulated in human colon cancer tissues. THB treatment significantly downregulated the expression of Notch1, its ligand Jagged1 and downstream target protein Hes1. Notch activation requires cleavage by the γ-secretase complex. THB treatment inhibits the expression of γ-secretase complex proteins Presenilin1, Nicastrin, APH1 and PEN2. Moreover, ectopic expression of the Notch Intracellular domain (NICD) rescued the cells from THB mediated growth suppression. These data demonstrate that THB mediated effects of colon cancer stem cells is in part through downregulating Notch1 activation. To determine the effect of THB on tumor growth in vivo, mice carrying HCT116 tumor xenografts were administered the compound intraperitoneally (5mg/kg bw) every day for 21 days. THB treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition of CSC marker proteins DCLK1, LGR5 and CD44 and also the Notch signaling proteins in the THB-treated xenograft tissues. Conclusion: Together, these data suggest that THB treatment suppresses colon cancer growth that targets stem cells by inhibiting Notch1 signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Prasad R. Dandawate, Ossama W. Tawfik, Roy A. Jensen, Scott J. Weir, Subhash B. Padhye, Shrikant Anant. Targeting colon cancer stem cells: Novel marmelin analog THB suppresses DCLK1 and Notch Signaling. [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 4748.

Abstract 2504: Assessing the efficacy of the anti metabolic drug, CPI-613, for targeting of ovarian cancer stem cells

Abstract Cancer stem cells (CSC) are believed to contribute to the recurrent and often platinum resistant ovarian cancer (OvCa). Moreover, it has been suggested that some anticancer treatments result in an increased frequency of CSC relative to non-CSC. More recently, we have determined that the PARP inhibitor olaparib increased the frequency of CD133, CD117 and ALDH positive cells in the established serous OvCa cell lines OVCAR4 and CaOV4 and in primary cell lines derived from serous carcinomas obtained with IRB approval at the time of initial surgery. We also showed that olaparib treatment increased sphere forming capacity, expression of stemness genes and CSC proliferation suggesting that olaparib has the unintended effect of stimulating CSC function. Previous work has shown that the metabolic inhibitor CPI-613 preferentially targets ovarian CSCs due to their altered metabolism. Our objective here was to determine whether CPI-613 could negate the effect of olaparib on CSC function. OVCAR4 and CaOV4 cells were treated with 50 μM CPI-613 and the relative frequency of CD133+ and CD177+ cells was assessed at 72 hours post treatment by flow cytometry with cell viability analyzed by MTT. Additionally, the sphere forming capacity of CPI-613 treated cells relative to that of vehicle treated cells was determined in an extreme limiting dilution assay. Finally, the expression of stemness genes in the CPI-613 treated and vehicle treated cells was evaluated by RT-PCR. Treatment of OVCAR4 and CaOV4 cells with CPI-613 decreased the frequency of CD133+ and CD117+ positive cells relative to untreated cells (p <0.001) without affecting cell viability. Additionally, sphere forming capacity was reduced in the CPI-613 treated cells. The decrease in both CD133+ and CD117+ cell frequency and sphere forming capacity suggests that CPI-613 is targeting the CSC population in these cells. Dual treatment with olaparib and CPI-613 blunted the increase in CD133+ and CD117+ cell frequency induced by single agent olaparib. Parallel treatment of CSC-rich spheres resulted in a considerable decrease in cell viability and frequency of CD133+ and CD117+ cells Clinical trials with olaparib in OvCa have resulted in increased progression free survival but no significant difference in overall survival has been observed. This raises the question of whether treatment with olaparib selects for the recurrence of more resistant disease, driven in part by the proliferation of CSCs. Our results suggest that CPI-613-mediated disruption of the mitochondrial respiratory pathway preferentially targets CSC and suppresses olaparib's effects on CSC function. In conclusion, targeting CSCs with non-traditional strategies could augment clinical outcomes and CPI-613 represents a promising candidate for combination with FDA approved olaparib treatment in OvCa. Citation Format: Chiara Bellio, Rosemary Foster, Whitfield B. Growdon, Bo R. Rueda. Assessing the efficacy of the anti metabolic drug, CPI-613, for targeting of ovarian cancer stem cells. [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 2504.

Abstract 2534: Low glucose and hypoxia environment promote the enrichment of liver cancer stem cells

Abstract Cancer stem cells (CSCs), a subpopulation possess tumor initiation and self-renewal capacity, are responsible for recurrence, metastases and drug resistance. Thus, it is important to understand their biology for identification of new therapeutic approaches. Liver surgery and/or transarterial (chemo) embolization (TAE/TACE) therapy usually generate a microenvironment that fosters aggressive tumor recurrence. These areas are characterized by chronic hypoxia and possibly low glucose concentration. Here, we tested the hypothesis that whether hypoxia along with low glucose environment promote the expansion of CSC population in HCC cells. We cultured human liver cancer cell line-Huh7 cells in normoxia or hypoxia condition with high or low glucose medium. The EMT markers (N-cadherin and Vimentin) but not stemness genes (CD44 and CD133) were upregulated in hypoxia condition. However, all genes were significantly induced under low glucose/hypoxia condition. In addition, results from sphere assay showed that low glucose/hypoxia condition dramatically improved the sphere formation ability of Huh7 cells, while low glucose or hypoxia along cannot promote sphere formation. Moreover, Huh7 cells cultured in low glucose/hypoxia condition displayed a lower sensitivity to cisplatin and doxorubicin treatment compared to cells maintained in high glucose/normoxia condition. Therefore, our findings suggested that low glucose concentration and hypoxia environment remarkably promote the enrichment of liver CSCs which could be useful for developing novel therapeutic modalities. Citation Format: Chih Chung Lai, Tsu-Hsiang Shia, Yu-Peng Liu, Chia-Hung Yen. Low glucose and hypoxia environment promote the enrichment of liver cancer stem cells. [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 2534.

NOTCH1 inhibition enhances the efficacy of conventional chemotherapeutic agents by targeting head neck cancer stem cell

Cancer stem cells (CSCs) are considered responsible for tumor initiation and chemoresistance. This study was aimed to investigate the possibility of targeting head neck squamous cell carcinoma (HNSCC) by NOTCH1 pathway inhibition and explore the synergistic effect of combining NOTCH inhibition with conventional chemotherapy. NOTCH1/HES1 elevation was found in human HNSCC, especially in tissue post chemotherapy and lymph node metastasis, which is correlated with CSCs markers. NOTCH1 inhibitor DAPT (GSI-IX) significantly reduces CSCs population and tumor self-renewal ability in vitro and in vivo. Flow cytometry analysis showed that NOTCH1 inhibition reduces CSCs frequency either alone or in combination with chemotherapeutic agents, namely, cisplatin, docetaxel, and 5-fluorouracil. The combined strategy of NOTCH1 blockade and chemotherapy synergistically attenuated chemotherapy-enriched CSC population, promising a potential therapeutic exploitation in future clinical trial.

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

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

Epigallocatechin-3-gallate targets cancer stem-like cells and enhances 5-fluorouracil chemosensitivity in colorectal cancer.

Resistance to cytotoxic chemotherapy is a major cause of mortality in colorectal cancer (CRC) patients. A small subset of cancer cells, termed “cancer stem cells” (CSCs), are believed to be key contributors of chemoresistance and tumor recurrence. Recently, epigallocatechin-3-gallate (EGCG), an active catechin present in green tea, has been shown to suppress CSC growth in various cancers, but whether it can specifically target CSCs and subsequently sensitize chemoresistant CRC cells to standard of care chemotherapeutic treatments remains unknown. Herein, we investigated the chemosensitizing effects of EGCG in 5-fluorouracil (5FU)-resistant (5FUR) CRC cells and spheroid-derived CSCs (SDCSCs), and interrogated the underlying molecular mechanisms responsible for its chemopreventive activity. EGCG enhanced 5FU-induced cytotoxicity and inhibited proliferation in 5FUR cell lines through enhancement of apoptosis and cell cycle arrest. The 5FUR cells showed higher spheroid forming capacity compared to parental cells, indicating higher CSC population. EGCG treatment in these cells resulted in suppression of SDCSC formation and enhanced 5FU sensitivity to SDCSCs. Furthermore, EGCG suppressed Notch1, Bmi1, Suz12, and Ezh2, and upregulated self-renewal suppressive-miRNAs, miR-34a, miR-145, and miR-200c, which are some of the key pathways targeted in 5FUR CRC cells. These findings were validated in vivo, wherein EGCG treatment resulted in inhibited tumor growth in a SDCSC xenograft model. Collectively our data provide novel and previously unrecognized evidence for EGCG-induced sensitization to 5FU through targeting of CSCs in CRC. Our data highlight that in addition to its chemopreventive ability, EGCG may serve as an adjunctive treatment to conventional chemotherapeutic drugs in CRC patients.

Methylisoindigo preferentially kills cancer stem cells by interfering cell metabolism via inhibition of LKB1 and activation of AMPK in PDACs

Pancreatic ductal adenocarcinoma (PDAC) clinically has a very poor prognosis. No small molecule is available to reliably achieve cures. Meisoindigo is chemically related to the natural product indirubin and showed substantial efficiency in clinical chemotherapy for CML in China. However, its effect on PDAC is still unknown. Our results showed strong anti-proliferation effect of meisoindigo on gemcitabine-resistant PDACs. Using a recently established primary PDAC cell line, called Jopaca-1 with a larger CSCs population as model, we observed a reduction of CD133+ and ESA+/CD44+/CD24+ populations upon treatment and concomitantly a decreased expression of CSC-associated genes, and reduced cellular mobility and sphere formation. Investigating basic cellular metabolic responses, we detected lower oxygen consumption and glucose uptake, while intracellular ROS levels increased. This was effectively neutralized by the addition of antioxidants, indicating an essential role of the cellular redox balance. Further analysis on energy metabolism related signaling revealed that meisoindigo inhibited LKB1, but activated AMPK. Both of them were involved in cellular apoptosis. Additional in situ hybridization in tissue sections of PDAC patients reproducibly demonstrated co-expression and -localization of LKB1 and CD133 in malignant areas. Finally, we detected that CD133+/CD44+ were more vulnerable to meisoindigo, which could be mimicked by LKB1 siRNAs. Our results provide the first evidence, to our knowledge, that LKB1 sustains the CSC population in PDACs and demonstrate a clear benefit of meisoindigo in treatment of gemcitabine-resistant cells. This novel mechanism may provide a promising new treatment option for PDAC.

Copper enriches efficacy of Dp44mT in breast cancer cells

Neoplastic cells need essential metals, such as iron and copper, for cellular functions and rapid growth. Metal chelation and changes in their redox cycle in favor of oxidative stress may be critical for making these cells vulnerable to cell death. The HER2-overexpressing breast cancer cell line, MCF7-HER2, and its vehicle control, MCF7-vec cells, were treated with CuCl2, FeSO4, and Dp44mT. Reactive oxygen species (ROS) production in response to these chemicals was detected with flow cytometry, and cell viability was measured by MTT assay in the cells. ROS levels were relatively increased by Dp44mT in the cells, and this was reversed by a combination of iron, while a copper combination further induced ROS. Parallel changes were observed in the inhibition of cell growth by Dp44mT, and NAC partially rescued the inhibition. Additionally, copper decreased the CSC population by half relative to the control in MCF7-HER2 cells. Copper supplement enriches ROS production mediated by Dp44mT in MCF7 cells. However, iron addition recovers Dp44mT-depleted iron levels, yet has no effect on ROS generation. Copper treatment also decreases the proportion of CSCs. In this manner, Dp44mT depletes iron and binds copper to form a redox active complex, which leads to oxidative stress. This dual cytotoxic case is significant for the survival of cancer cells.

Targeting Cancer Stem Cells: Promises and Challenges.

Recent discoveries have provided the compelling evidence that stem cell populations within each individual tumor are key contributors of therapy failure regardless of whether these populations are transient or stable. Thus, it is becoming increasingly clear that efficient tumor treatment requires eradication of the entire CSC population. The potential role of CSCs in tumor initiation and relapse has motivated an investigation of the CSCspecific treatments. However, development of the therapeutic strategies targeting CSCs might be challenged by a high diversity and plasticity of CSC features. Moreover, taking in account that an origin of CSC remains controversial and accumulating experimental evidence suggests a possibility of tumor cell reprogramming, efficient anti-cancer treatment should eradicate both CSCs and tumor bulk as well as prevent tumor dedifferentiation. In this article we discuss new insights into the stem cell concept of tumor development, review the treatment strategies eradicating CSCs that have been evaluated in preclinical and clinical studies and summarize the strategies to identify new CSC-targeted therapy.

Effects of NVP-BKM120 on the triple-negative breast cancer cell

To investigatethe effect of NVP-BKM120 on the triple-negative breast cancer cell lines. Breast cancer cell line MDA-MB-231 and Cal51 were divided into control group and experimental group. The inhibitory effects of BKM120 were evaluated by MTT assays. The drug effects on CSC population and characteristics were investigated through mammosphere formation assay and colony formation assay. Western blot was used to observe the expressionof related protein. The BALB/c mice were injected with stem cells (SCs) and different treatments were administered subsequently. In MDA-MB-231 cell lines, IC50 of BKM120 was (20.01±3.46) µmol/L for SCs and (3.07±0.14) µmol/L for total cells. BKM120 significantly inhibit the cell growth, in vitrocloning and microspheres formed of triple-negative breast cancer cells. BKM120 can inhibit tumor growth in nude mice without significant adverse reactions. BKM120 can significantly inhibit the proliferation of the triple-negative breast cancer cell lines.

Targeting CXCR1 on breast cancer stem cells: signaling pathways and clinical application modelling.

In breast cancer it has been proposed that the presence of cancer stem cells may drive tumor initiation, progression and recurrences. IL-8, up-regulated in breast cancer, and associated with poor prognosis, increases CSC self-renewal in cell line models. It signals via two cell surface receptors, CXCR1 and CXCR2. Recently, the IL-8/CXCR1 axis was proposed as an attractive pathway for the design of specific therapies against breast cancer stem cells. Reparixin, a powerful CXCR1 inhibitor, was effective in reducing in vivo the tumour-initiating population in several NOD/SCID mice breast cancer models, showing that the selective targeting of CXCR1 and the combination of reparixin and docetaxel resulted in a concomitant reduction of the bulk tumour mass and CSC population. The available data indicate that IL-8, expressed by tumour cells and induced by chemotherapeutic treatment, is a key regulator of the survival and self-renewal of the population of CXCR1-expressing CSC. Consequently, this investigation on the mechanism of action of the reparixin/paclitaxel combination, was based on the observation that reparixin treatment contained the formation of metastases in several experimental models. However, specific data on the formation of breast cancer brain metastases, which carry remarkable morbidity and mortality to a substantial proportion of advanced breast cancer patients, have not been generated. The obtained data indicate a beneficial use of the drug combination reparixin and paclitaxel to counteract brain tumour metastasis due to CSC, probably due to the combined effects of the two drugs, the pro-apoptotic action of paclitaxel and the cytostatic and anti-migratory effects of reparixin.

Abstract SY27-03: Breast cancer stem cell state transitions mediate therapeutic resistance

Abstract SY27-03: Breast cancer stem cell state transitions mediate therapeutic resistance

Abstract 5079: Parasympathetic signaling suppresses pancreatic cancer development

Abstract Background/Aims: The parasympathetic nervous system plays an important role in the regulation of epithelial homeostasis and has also been postulated to play a role in tumorigenesis. However, the exact role of the vagus nerve in pancreatic carcinogenesis is not well understood. Here we study the effects of muscarinic signaling on pancreatic tumorigenesis in a genetically engineered mouse model of pancreatic cancer (PDx1-Cre/KRasG12D (KC). Methods: Mice were either vagotomized at 8 weeks or treated with a cholinergic agonist. Pancreatic tissue was collected and analyzed by immunohistochemistry and RT-PCR at 20 weeks of age; cells were isolated and assayed for colony and sphere forming assays. Different human (AsPC-1, BxPC-3, Mia PaCa-2, Panc-1) and murine (K-2548 and K-8282) pancreatic cancer cell lines were subjected to cholinergic and anti-cholinergic drugs and assayed by RT-PCR, Western blot and flow cytometry. Results: In pancreatic organoid cultures derived from pancreata harboring an oncogenic KRas mutation, cholinergic agonists suppressed sphere formation significantly. Furthermore, pharmacological inhibition or genetic knockout of the muscarinic M3 receptor abolished this effect in vitro. In human and murine pancreatic cancer cells, anchorage independent growth and tumor sphere forming capacity were reduced by pretreatment with cholinergic agonists. Further evaluation revealed that parasympathetic agonists decrease the CD44+CD24+EpCAM+ proved CSC's population. Vagotomy, when performed in KC mice at 8 weeks resulted in pancreatic cancer development in 20% of the animals at 20 week of age. Treatment with the direct muscarinic agonist bethanechol caused a significant delay of PanIN progression in KC mice. Conclusions: Taken together, our findings suggest that vagal innervation has a regulatory role in pancreatic tumorigenesis via M3 receptor-mediated suppression of CSCs. Since current surgical approaches to resection of PDAC from the head of the pancreas are necessarily associated with a parasympathetic denervation of the pancreas, and thereby a loss of its suppressive effect, this fact may might partly explain the high local recurrence rate of this dismal disease. Additional treatment with cholinergic agonists should be considered in future regimen. Citation Format: Bernhard W. Renz, Marina Macchini, Yoku Hayakawa, C. Benedikt Westphalen, Michael Churchill, Suchetak Kar, Xiaowei Chen, Karan Nagar, Yagnesh Tailor, Daniel L. Worthley, Alina C. Iuga, Ken P. Olive, Timothy C. Wang. Parasympathetic signaling suppresses pancreatic cancer development. [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 5079. doi:10.1158/1538-7445.AM2015-5079

Abstract 974: FAK inhibitors VS-6063 and VS-4718 target cancer stem cells: Implications for TNBC sequential and combination therapies

Abstract Cancer stem cells (CSCs), a subpopulation of tumor cells characterized by tumor-initiating capability and resistance to chemotherapy, are an underlying cause of tumor progression and metastasis. In breast cancer, CSCs can be identified by aldehyde dehydrogenase 1 (ALDH) or CD44+/CD24- expression separately, although combination of ALDH, CD44+, and CD24- together may better describe the CSC population in breast cancer. Neoadjuvant chemotherapy has been shown to lead to an increase in CSCs in locally advanced breast cancer (Alamgeer et al., 2014, Br. Can. Res. 16:R14) which is associated with a significantly worse prognosis. It is also evident that stem-like features of tumor cells are present in metastatic breast cancer (Yu et al., 2013, Science 339:580) indicating that targeting of CSCs may be valuable in metastatic disease. VS-6063 and VS-4718 are orally bioavailable, potent and selective inhibitors of Focal Adhesion Kinase (FAK). These agents preferentially target CSCs in preclinical models and are currently in clinical development. Here we report that VS-6063 and VS-4718 preferentially target CSCs in multiple breast cancer models. Ex vivo treatment of explants from human breast tumors with either VS-6063 or VS-4718 decreased the proportion of CSCs in contrast to paclitaxel treatment which increased the proportion of CSCs. In an MDA-MB-231 mouse xenograft model, in vivo treatment with VS-6063 decreased CSCs by more than 6-fold as demonstrated by limiting dilution transplantation assay. Consistent with the notion that CSCs are responsible for cancer relapse after chemotherapy, VS-6063 and VS-4718 substantially delayed tumor growth following cessation of paclitaxel or cisplatin treatment in models of TNBC, including a patient-derived xenograft. Furthermore, using an imaging-based 4T1-luciferase TNBC orthotopic model, both VS-6063 and VS-4718 inhibited metastatic outgrowth or induced regression of metastases after primary tumor resection, whereas metastases progressed in all animals in the vehicle control group. A multiplex assay for multiple CSC markers (ALDH1, CD44 and CD24) was developed and validated in biopsies of primary patient tumors and matched lymph node biopsies taken pre- and post-neoadjuvant chemotherapy. CSCs, represented by the ALDH+ and/or CD44+/CD24- populations were present at detectable levels in a high fraction of triple negative breast cancer primary tumors. In summary, CSCs are readily detectable in primary breast cancers at surgery, and VS-6063 and VS-4718 diminish the CSC subpopulation in vitro, ex vivo and in xenograft models. This critical subpopulation of CSCs is detectable in residual tumors after neoadjuvant therapy, and may also be present in metastatic disease. CSC-targeted agents such as VS-6063 or VS-4718 should be clinically tested in these breast cancer settings to potentially delay time to relapse and improve patient outcome. Citation Format: Vihren N. Kolev, Kam Sprott, Qunli Xu, Jonathan A. Pachter, David T. Weaver. FAK inhibitors VS-6063 and VS-4718 target cancer stem cells: Implications for TNBC sequential and combination therapies. [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 974. doi:10.1158/1538-7445.AM2015-974