Cancer Stem Cells Surface Research Articles

Suppression of pancreatic adenocarcinoma upregulated factor (PAUF) increases the sensitivity of pancreatic cancer to gemcitabine and 5FU, and inhibits the formation of pancreatic cancer stem like cells.

Pancreatic cancer stem cells (CSCs) play a crucial role in tumorigenesis and chemoresistance of pancreatic ductal adenocarcinoma. Pancreatic adenocarcinoma up-regulated factor (PAUF), a novel secretory protein, has been shown to contribute to cancer progression and metastasis. Because the clinical relationship between PAUF and pancreatic CSCs is largely unknown, we investigated the associations between the functional role of PAUF and pancreatic CSCs. Pancreatic cancer sphere cultured from the CFPAC-1 cells showed elevated expression of PAUF and pluripotent stemness genes (Oct4, Nanog, Stat3, and Sox2), and the mRNA of PAUF were increased in CD44+CD24+ESA+ pancreatic CSCs. PAUF knockdown (shPAUF) CFPAC-1 diminished the number of spheres and decreased stemness genes and CSC surface markers (CD133, c-MET and ALDH1). In addition, siPAUF CFPAC-1 decreased the mRNA expression of multidrug resistant protein 5 (MRP5) and ribonucleotide reductase M2 (RRM2) and were more vulnerable to gemcitabine and 5-FU than negative control (p<0.05). In conclusion, PAUF was increased in pancreatic CSCs and the suppression of PAUF enhances chemotherapeutic response to gemcitabine and 5FU by decreasing MRP5 and RRM2 in pancreatic cancer cells.

The Implications and Future Perspectives of Nanomedicine for Cancer Stem Cell Targeted Therapies.

Cancer stem cells (CSCs) are believed to exhibit distinctive self-renewal, proliferation, and differentiation capabilities, and thus play a significant role in various aspects of cancer. CSCs have significant impacts on the progression of tumors, drug resistance, recurrence and metastasis in different types of malignancies. Due to their primary role, most researchers have focused on developing anti-CSC therapeutic strategies, and tremendous efforts have been put to explore methods for selective eradication of these therapeutically resistant CSCs. In recent years, many reports have shown the use of CSCs-specific approaches such as ATP-binding cassette (ABC) transporters, blockade of self-renewal and survival of CSCs, CSCs surface markers targeted drugs delivery and eradication of the tumor microenvironment. Also, various therapeutic agents such as small molecule drugs, nucleic acids, and antibodies are said to destroy CSCs selectively. Targeted drug delivery holds the key to the success of most of the anti-CSCs based drugs/therapies. The convention CSCs-specific therapeutic agents, suffer from various problems. For instance, limited water solubility, small circulation time and inconsistent stability of conventional therapeutic agents have significantly limited their efficacy. Recent advancement in the drug delivery technology has demonstrated that specially designed nanocarrier-based drug delivery approaches (nanomedicine) can be useful in delivering sufficient amount of drug molecules even in the most interiors of CSCs niches and thus can overcome the limitations associated with the conventional free drug delivery methods. The nanomedicine has also been promising in designing effective therapeutic regime against pump-mediated drug resistance (ATP-driven) and reduces detrimental effects on normal stem cells. Here we focus on the biological processes regulating CSCs' drug resistance and various strategies developed so far to deal with them. We also review the various nanomedicine approaches developed so far to overcome these CSCs related issues and their future perspectives.

Abstract 2194: Synergistic cytotoxicity of targeted liposomes containing doxorubicin and C6-ceramide against nucleolin-overexpressing ovarian cancer cells is supported by the downregulation of the Akt pathway

Abstract The acknowledgment that cancer stem cells (CSC) may originate from non-stem cancer cells, interconverting through an EMT-mediated process has turned these distinct cell subpopulations into two relevant therapeutic targets. Moreover, the PI3K/AKT/mTOR pathway is essential to CSC proliferation and survival and it is often over activated in ovarian cancer. One of the strategies to target these different tumor cell populations, relies on the combination of conventional chemotherapeutic drugs (as tumor debulking agents, targeting non-stem cancer cells) with sphingolipids targeting CSC (at the level of PI3K/Akt/mTOR). Nucleolin overexpression has been demonstrated on the surface of both breast CSC and non-stem breast cancer cells (Fonseca NA, Biomaterials 2015) and endothelial cells from tumor blood vessels. The pH-sensitive lipid-based nanoparticle, functionalized with the nucleolin-binding F3 peptide, was recently modified to encapsulate a synergistic combination of a sphingolipid (C6-ceramide) and doxorubicin (DXR). Following the promising results in breast cancer, the aim of this work was to assess the therapeutic potential of this strategy against nucleolin-overexpressing ovarian cancer cell lines, as well as the underlying mechanism of action at the molecular level. A 12.9-fold increase of association of fluorescently-labelled F3 peptide-targeted liposomes ([F3]L), relative to the non-targeted counterparts, into (bulk) ovarian cancer cell lines (SKOV-3, OVCAR-3 and TOV-112D) was observed by flow cytometry. Importantly, a similar pattern of association by putative CSC enriched population (CD44high/EpCAMhigh) was also observed. These results corroborated the marked increase of cytotoxicity (assessed by the resazurin reduction viability assay) enabled by the targeted drug combinations, relative to [F3]L containing only DXR. In fact, the targeted drug combination enabled a 90% death of SKOV-3 cancer cells, following an 8 h incubation, a level of cell killing not achieved by the counterpart containing only DXR. Against the OVCAR-3 and TOV-112D cell lines, the IC90 of the targeted drug combination, following 1 h of incubation, was 2-fold lower than the one resulting from the incubation with [F3]L containing only DXR. In addition, the effect of nucleolin-mediated intracellular delivery of C6-ceramide at the p-Akt protein levels was further discriminated by western blot analysis in SKOV-3 cells. The observed 3.2-fold downregulation of p-Akt levels supported the C6-ceramide-mediated improved cytotoxicity of the drug combination. Overall, these results pointed out the enhanced efficacy of targeted synergistic drug combinations against ovarian cancer, due to the capability to target nucleolin-overexpressing cancer cells and further inhibit the Akt pathway. Citation Format: Ana Filipa Cruz, Nuno André Fonseca, Susana F. Sampaio, Vera Moura, Ramiro D. Almeida, Sérgio Simões, João Nuno Moreira. Synergistic cytotoxicity of targeted liposomes containing doxorubicin and C6-ceramide against nucleolin-overexpressing ovarian cancer cells is supported by the downregulation of the Akt pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2194. doi:10.1158/1538-7445.AM2017-2194

Abstract 936: Single cell analysis reveals cancer stem cell heterogeneities in hepatocellular carcinoma

Abstract Tumor heterogeneity represents a major obstacle to effective cancer treatment and personalized medicine. Hepatocellular carcinoma (HCC) is clinically and biologically heterogeneous, which is partly attributed to the presence of hepatic cancer stem cells (CSCs). CSC surface makers can have overlapping expression or exclusive expression of different subpopulations of hepatic CSCs, and these cells may contain different oncogenic changes. The existence of various CSC surface-marked subpopulations might pose a problem for CSC targeted therapeutics. Little is known about whether there are shared or distinct pathways in different subpopulations. Moreover, it remains unknown whether heterogeneity exists within certain subpopulations. Thus, we propose to profile the global transcriptome of surface-marked CSCs (EpCAM+, CD133+ and CD24+) at the single-cell level using index flow cytometric sorting and single-cell RNA sequencing technology. We statistically compared mRNA transcriptomes of individual single cells of triple positive (EpCAM+/ CD133+ /CD24+) CSCs and triple negative (EpCAM-/ CD133- /CD24-) cells. Further, we compared the triple positive and the triple negative cells at the single cell level in terms of their self-renew capability in both normoxic and hypoxic conditions. Our results show that at the single cell level, the transcriptomic profiles show a high-degree of heterogeneity in HCC cell lines, meanwhile, there is a dramatic difference between triple positive and triple negative cells in their transcriptomic profiles. There is a high-degree of intra-tumor and inter-tumor heterogeneity observed in single cell transcriptomes of HCC patients’ samples. Triple positive CSC single cells show continuous rather than discrete stemness-related gene expression patterns. In parallel, the self-renewal capability of triple positive cells also exhibits heterogeneity, while triple negative cells show little self-renewal capability. Thus, our single cell analysis study reveals molecular and biological heterogeneity of cancer stem cells in HCC, which may provide insight into the underlying mechanisms of how CSCs contribute to tumor heterogeneity and underscore key pathways and novel targets for hepatic CSC therapy. Citation Format: Hongping Zheng, Yotsawat Pomyen, Maria Hernandez, Caiyi Li, Ferenc Livak, Tim Greten, Anuradha Budhu, Xin Wang. Single cell analysis reveals cancer stem cell heterogeneities in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 936. doi:10.1158/1538-7445.AM2017-936

Cancer stem cell surface markers on normal stem cells.

The cancer stem cell (CSC) hypothesis has captured the attention of many scientists. It is believed that elimination of CSCs could possibly eradicate the whole cancer. CSC surface markers provide molecular targeted therapies for various cancers, using therapeutic antibodies specific for the CSC surface markers. Various CSC surface markers have been identified and published. Interestingly, most of the markers used to identify CSCs are derived from surface markers present on human embryonic stem cells (hESCs) or adult stem cells. In this review, we classify the currently known 40 CSC surface markers into 3 different categories, in terms of their expression in hESCs, adult stem cells, and normal tissue cells. Approximately 73% of current CSC surface markers appear to be present on embryonic or adult stem cells, and they are rarely expressed on normal tissue cells. The remaining CSC surface markers are considerably expressed even in normal tissue cells, and some of them have been extensively validated as CSC surface markers by various research groups. We discuss the significance of the categorized CSC surface markers, and provide insight into why surface markers on hESCs are an attractive source to find novel surface markers on CSCs.

Ultrasensitive Label‐free Electrochemical Immunosensors for Multiple Cell Surface Biomarkers on Liver Cancer Stem Cells

AbstractLiver cancer is one of the most widely spread cancers in the world. Cancer stem cells (CSCs) are a small subpopulation of liver cancer cells that are thought to be responsible for relapse of cancer and the resistance to chemotherapy. Detection of CSCs is highly demanded for screening patients who are at high risk for developing metastatic cancers. However, the current methodologies for CSCs detection are sophisticated, expensive and not reliable. Here, we report the development of a label‐free impedance immunosensors for liver CSC quantification using four established CSC surface biomarkers (CD44, CD90, CD133/2 and OV‐6). The immunosensors were simply fabricated by the covalent attachment of four biomarkers specific antibodies on gold electrodes using cysteamine/phenylene isothiocyanate linker. Electrochemical impedance spectroscopy was employed to detect the binding of the cells to the immunosensors. The binding of the CSCs to the gold electrode surface retards the access of ferri‐ferrocyanide redox molecules to the electrode leading to enhancement in the charge transfer resistance (Rct) which represents the basis of the detection signal. The developed electrochemical immunosensors showed high sensitivity and selectivity for CSC detection with a wide linear range from 1 × 101 to 1× 104 cells/mL with a limit of detection of 1 cell/ml. This work represents a new, accurate, simple and low cost method for the detection of liver CSC that might help in the early diagnosis of metastatic disease and cancer relapse.

CD133 confers cancer stem-like cell properties by stabilizing EGFR-AKT signaling in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the seventh most common malignant tumor and the third leading cause of cancer-related death in the world. Cancer stem cells (CSCs) are small subpopulation of cells within tumors that drive chemoresistance and tumor recurrence in various cancers. We characterized CSCs in primary HCC and identified CD133 as a CSC surface marker. CD133+ HCC cells displayed more stem cell-like properties, tumor spheroid-forming ability, chemoresistance, migration ability, and tumorigenic capacity than CD133- HCC cells. The biological function and molecular mechanism of CD133 remain unclear. HCC cell lines with a high level of CD133 expression overexpressed EGFR, which is overexpressed in approximately 70% of conventional HCCs. CD133 depletion destabilized EGFR by augmenting EGFR internalization and thus inhibited EGFR-AKT signaling. CD133 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Furthermore, EGFR-deficient CD133+ HCC cells manifested greater sensitivity to anticancer drugs and less spheroid-formation capacity than control CD133+ HCC cells. Our results strongly indicate that CD133 facilitates CSC-like properties by stabilizing EGFR-AKT signaling in HCC. It might therefore be feasible to use CD133 as a novel target to sensitize HCC cells that manifest resistance to EGFR-targeted therapy.

Niclosamide and its analogs are potent inhibitors of Wnt/β-catenin, mTOR and STAT3 signaling in ovarian cancer.

Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer mortality worldwide. Platinum-based therapy is the standard first line treatment and while most patients initially respond, resistance to chemotherapy usually arises. Major signaling pathways frequently upregulated in chemoresistant cells and important in the maintenance of cancer stem cells (CSCs) include Wnt/β-catenin, mTOR, and STAT3. The major objective of our study was to investigate the treatment of ovarian cancer with targeted agents that inhibit these three pathways. Here we demonstrate that niclosamide, a salicylamide derivative, and two synthetically manufactured niclosamide analogs (analog 11 and 32) caused significant inhibition of proliferation of two chemoresistant ovarian cancer cell lines (A2780cp20 and SKOV3Trip2), tumorspheres isolated from the ascites of EOC patients, and cells from a chemoresistant patient-derived xenograft (PDX). This work shows that all three agents significantly decreased the expression of proteins in the Wnt/β-catenin, mTOR and STAT3 pathways and preferentially targeted cells that expressed the ovarian CSC surface protein CD133. It also illustrates the potential of drug repurposing for chemoresistant EOC and can serve as a basis for pathway-oriented in vivo studies.

284 - Activation of NRF2 Signaling in Breast Cancer Stem Cell-Enriched Population

Cancer stem cells (CSCs) are known to express high levels of antioxidant proteins and drug efflux transporters. In this study, we investigated the potential involvement of NF-E2-related factor 2 (NRF2) in CSC survival and stress response by using MCF7 breast cancer cells. First, the levels of NRF2 and its target gene expressions were increased in breast CSC-enriched mammosphere culture system. As an underlying mechanism, we demonstrated that enzymatic function and expression of the proteasome are decreased in mammospheres. Additionally, mammospheres retained a higher level of p62 and the silencing of p62 attenuated NRF2 activation in sphere cultured MCF7. Sphere growth and anticancer drugs resistance in mammospheres were diminished in NRF2-knockdown mammospheres. To further confirm NRF2 activation in breast CSC, we isolated CSC surface marker CD44-positive MCF7 cells by flow cytometry. The CD44-positive cells showed increased NRF2 protein and target genes, which were accompanied by CSC markers expression. Furthermore, we observed NRF2 activation in CD44-enriched doxorubicin-resistant MCF7 cells. Collectively, these results indicated that NRF2 plays a role in breast CSCs survival and anticancer drug resistance.

Abstract 1039: Metabolic hormones FGF19/15 promote hepatocellular carcinoma through activation of CSCs in fatty liver

Abstract Background: Fibroblast growth factor 19 (FGF19), a member of the fibroblast growth factor (FGF) family, is a hormone that regulate glucose, lipid, and energy homeostasis in human. It has been shown that FGF19 and its specific receptor FGFR4 play an important role in non-alcoholic steatohepatitis (NASH) which is closely related hepatocellular carcinoma (HCC). Recently, we found FGF19 overexpressed in the HCC tissue and serum in patients. The high level of FGF19 was positively related to cancer stem cells (CSCs) specific surface markers such as CD133 and EpCAM. The aim of this study is to identify potential mechanism in which FGF15 (the FGF19's orthologous protein in mouse)/FGFR4 associated abnormal lipid metabolism initiates HCC progression in a NASH-HCC mouse Model. Methods: In vivo study, male C57L/J mice were injected intraperitoneally with N-nitrosodiethylamine(DEN) when 2 weeks old. Both the DEN and control mice were divided into two groups based on receiving high fat diet (HFD) or control diet (CD). Tumor nodules in the liver were monitored by ultrasound. Body weight, glucose tolerance test (GTT) and insulin tolerance test (ITT) were recorded during the experimental period. The liver weights, tumor volume, histology, alanine transaminase (ALT), alpha fetoprotein (AFP), and triglyceride (TG) were determined at 8, 20 and 32weeks. In vitro study, human HCC cell lines (Hep3B and Huh7) as well as mouse HCC cell lines (Hepa1-6 and FL83B,) were exposed to a long-chain mixture of free fatty acid(FFA) at different concentrations (UT, 0.5mM and 1mM FFA) for 24 and 48 hours, respectively. EpCAM and CD133 were determined by flow cytometery in the isolated tumor cells from HCC tissues. FGF19/15, FGFR4, CSCs surface markers and Wnt signaling pathway (β-catenin, GSK3β) were analyzed by RT-PCR and Western blot. FGFR4 signal was blocked to elucidate the role of FGF19/15 during the CSCs activation in vitro. In addition, FGF19, FGFR4 and EpCAM were also analyzed in human HCC samples. Results: Metabolic disorders were observed when DEN mice fed with HFD. HCC incidence and tumor volume were significantly increased in DEN+HFD group compared to that in DEN+CD group. DEN mice fed with HFD showed increased levels of FGF15/FGFR4 and β-catenin as well as the CSCs surface makers. Increased expression of CSCs surface makers were also found in the HCC cell lines when exposure to FFA in vitro. Blockage the FGF19 signal can abolish Wnt/β-catenin pathway and cell stemness features in the FFA environment. Increased levels of FGF19, FGFR4 and EpCAM were found in human HCC samples. Conclusion: Up-regulated FGF19/15 and FGFR4 promoted the development of HCC by active CSCs signaling in metabolic disorder microenvironment. FFA enhanced the CSCs activation was partially dependent on Wnt/β-catenin signaling. FGF19/FGFR4 could be a potential therapeutic target in HCC patients. Citation Format: Guozhen Cui, Xingkai Liu, Harshul Pandit, Yingbin Yang, Suping Li, Lu Cai, Robert C Martin, Hengjun Zhao, Wei Li, Yan Li. Metabolic hormones FGF19/15 promote hepatocellular carcinoma through activation of CSCs in fatty liver. [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 1039.

Evolving Strategies for Therapeutically Targeting Cancer Stem Cells.

Cancer is a multifactor and multistep process that is affected intrinsically by the genetic and epigenetic makeup of tumor cells and extrinsically by the host microenvironment and immune system. A key component of cancer involves a unique subpopulation of highly malignant cancerous cells referred to as cancer stem cells (CSCs). CSCs are positioned at the apex of the tumor hierarchy with an ability to both self-renew and also generate non-CSC/differentiated progeny, which contribute to the majority of the tumor mass. CSCs undergo functional changes and show plasticity that is stimulated by specific microenvironmental cues and interactions in the tumor niche, which contribute to the complexity and heterogeneity of the CSC population. The prognostic value of CSCs in the clinic is evident since there are many examples in which CSCs serve as markers for poor patient prognosis. CSCs are innately resistant to many standard therapies and they display anoikis resistance, immune evasion, tumor dormancy, and field cancerization, which may result in metastasis and relapse. Many academic laboratories and biotechnology companies are currently focusing on strategies that target CSCs. Combination therapies, epigenetic modifiers, stemness inhibitors, CSC surface marker-based therapies, and immunotherapy-based CSC-targeting drugs are currently undergoing clinical trials. Potential new targets/strategies in CSC-targeted therapy include MDA-9/Syntenin (SDCBP), Patched (PTCH), epigenetic targets, noncoding RNAs, and differentiation induction. Defining ways of targeting and destroying CSCs holds potential to impact significantly on cancer therapy, including prevention of metastasis and cancer recurrence.

Quercetin Mediates β-Catenin in Pancreatic Cancer Stem-Like Cells.

The aim of this study is to explore how quercetin interacts with pancreatic cancer stem-like cells and the mechanism underlying the effective quercetin-mediated suppression. A model of pancreatic cancer stem-like cells was generated by using a sphere formation culture system. A comparative analysis was performed between the parent cells and pancreatic cancer stem-like cells with a related treatment strategy focusing on cancer stem cell (CSC) properties and drug-resistance-related mechanisms in vitro. Our data show that pancreatic cancer stem-like cells have greater resistance to gemcitabine and stronger CSC properties compared with the parent cells. In contrast to the pancreatic cancer stem-like cells, overexpression of β-catenin was observed in the parent cells. Quercetin suppressed proliferation, invasion and self-renewal capacity, and CSC surface markers expression, with alterations of β-catenin in pancreatic cancer stem-like cells. The combination of quercetin and gemcitabine can reduce tumor growth and decrease drug resistance in pancreatic cancer. β-Catenin plays an important role in maintenance and progression of pancreatic cancer. Targeting β-catenin using quercetin combined with gemcitabine may be a treatment strategy to improve prognosis in patients with pancreatic cancer.

Concise Review: Emerging Role of CD44 in Cancer Stem Cells: A Promising Biomarker and Therapeutic Target.

The reception and integration of the plethora of signals a cell receives from its microenvironment determines the cell's fate. CD44 functions as a receptor for hyaluronan and many other extracellular matrix components, as well as a cofactor for growth factors and cytokines, and thus, CD44 is a signaling platform that integrates cellular microenvironmental cues with growth factor and cytokine signals and transduces signals to membrane-associated cytoskeletal proteins or to the nucleus to regulate a variety of gene expression levels related to cell-matrix adhesion, cell migration, proliferation, differentiation, and survival. Accumulating evidence indicates that CD44, especially CD44v isoforms, are cancer stem cell (CSC) markers and critical players in regulating the properties of CSCs, including self-renewal, tumor initiation, metastasis, and chemoradioresistance. Furthermore, there is ample evidence that CD44, especially CD44v isoforms, are valuable prognostic markers in various types of tumors. Therefore, therapies that target CD44 may destroy the CSC population, and this holds great promise for the cure of life-threatening cancers. However, many challenges remain to determining how best to use CD44 as a biomarker and therapeutic target. Here we summarize the current findings concerning the critical role of CD44/CD44v in the regulation of cancer stemness and the research status of CD44/CD44v as biomarkers and therapeutic targets in cancer. We also discuss the current challenges and future directions that may lead to the best use of CD44/CD44v for clinical applications. Mounting evidence indicates that cancer stem cells (CSCs) are mainly responsible for cancer aggressiveness, drug resistance, and tumor relapse. CD44, especially CD44v isoforms, have been identified as CSC surface markers for isolating and enriching CSCs in different types of cancers. The current findings concerning the critical role of CD44/CD44v in regulation of cancer stemness and the research status of CD44/CD44v as biomarkers and therapeutic targets in cancer are summarized. The current challenges and future directions that may lead to best use of CD44/CD44v for clinical applications are also discussed.

Abstract A24: CD133 initiates tumors, induces epithelial-mesenchymal transition, and increases metastasis in pancreatic cancer

Abstract CD133 has been implicated as a cancer stem cell (CSC) surface marker in several malignancies including pancreatic cancer, however, the functional role in the cancer stem cell remains elusive. In this study, we overexpressed CD133 CSC marker in a cell line with minimal endogenous CD133 in order to determine its functional role within a cancer context. We determined that CD133 expression led to increased tumorigenicity down to 10 cells. Progression of tumors derived from cells overexpressing CD133 increased in volume quicker than controls. Additionally, the number of lesions and sites of metastasis were increased in the orthotopic tumors derived from CD133 overexpressing cells. In vitro, CD133 expression induced epithelial-mesenchymal transition (EMT). Mesenchymal marker genes such as SNAI1, ZEB1, CDH2, and VIM increased upon CD133 overexpression and led to increased in vitro invasion by Boyden chamber invasion assay. Furthermore, our results indicate EMT induction and increased invasiveness were mediated by increased NF-κB activation upon CD133 overexpression, as inhibition of NF-κB mitigated these effects. In conclusion, this study demonstrated a functional role of CD133 in pancreatic cancer: its expression increases tumorigenicity; induces EMT; and increases invasion and metastasis. Citation Format: Alice Nomura, Sulagna Banerjee, Veena Sangwan, Rohit Chugh, Vikas Dudeja, Selwyn M. Vickers, Ashok K. Saluja. CD133 initiates tumors, induces epithelial-mesenchymal transition, and increases metastasis in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A24.

Downregulation of PRRX1 Confers Cancer Stem Cell-Like Properties and Predicts Poor Prognosis in Hepatocellular Carcinoma.

Downregulation of paired related homeobox 1 (PRRX1) is associated with the acquisition of cancer stem cell (CSC)-like properties and poor prognosis in cancers. The purpose of this study is to clarify the role of PRRX1 expression in predicting prognosis and mediating CSC-like properties in hepatocellular carcinoma (HCC). The association between PRRX1 expression and overall survival (OS) of patients with HCC was analyzed in three independent datasets: 62 resected primary cases, 242 cases from GSE14520, and 162 cases from The Cancer Genome Atlas (TCGA). A cell line expressing PRRX1 (HuH7) was established for the functional analyses. The ability to form spheres, the expression levels of the hepatic CSC surface markers (CD13, CD133, and EpCAM), in vitro chemosensitivity to 5-fluorouracil (FU), and radiosensitivity were evaluated. Univariate and multivariate analyses showed that the 5-year OS of the low PRRX1 expression group was significantly poorer than that of the high PRRX1 expression group (P = 0.024 and P = 0.045, respectively). Consistent with this, the low PRRX1 expression group in GSE14520 and TCGA datasets showed significantly shorter OS (P = 0.027 and P = 0.010, respectively). Gene set enrichment analysis on GSE14520 and TCGA datasets indicated that downregulation of PRRX1 was correlated with the stemness signature. The number of spheres and the expression levels of CSC markers were significantly decreased when PRRX1 was expressed. Moreover, PRRX1 impaired resistance to 5-FU and radiation. Downregulation of PRRX1 expression contributes to the poor prognosis of patients with HCC through acquisition of CSC-like properties.

Abstract 3865: ATOH8 depletion can reprogram noncancer stem cells into cancer stem cells

Abstract Introduction: The development of hepatocellular carcinoma (HCC), one of the most lethal of human malignancies, is caused by the accumulation of multiple genetic and epigenetic alterations. Recent advances in cancer stem cell (CSC) study have associated the poor prognosis of cancer with CSC. ATOH8 is a nucleus factor with an obvious structural similarity to the Drosophila proneural gene atonal. The aim of this study is to investigated the role of ATOH8 in CSC and its therapeutic potentials for HCC treatment. Experimental procedures: The clinical significance of ATOH8 in a cohort of 242 HCC cases was assessed by clinical correlation and Kaplan-Meier survival analyses. ATOH8-overexpressing and scliencing cell model was generated by Lentivirus system to detect its functional roles in HCC both in vivo and in vitro. iPS technology is used to analyze effects of ATOH8 on CSCs. Result: Downregulation of ATOH8 was detected in over 40% of 242 pairs primary HCC cases, which was significantly associated with disease free survival (P=0.031), high level of AFP (P=0.03) and diferentiation (P=0.01). Functional studies demonstrate the tumor suppressive role of ATOH8 in HCC, including the inhibition of cell growth, cell proliferation, tumor formation in SCID mice and cell motility when ATOH8 was introduced into HCC cells. Further study finds that ATOH8 depletion can enhance the stemness of HCC including the tumorigenicity and abilities for self-renewal, differentiation and chemo-resistance by repressing the transcription of stemness-related genes such as OCT4, NANOG, SOX2, AFP, and HCC cancer stem cell (CSC) surface markers such as CD24 and CD133. One interesting finding in this study is that knockdown of ATOH8 could increase CD133+ cell population in QSG7701 and BEL7402 cells. CD133+ cells induced by ATOH8 depletion were flow-sorted and their CSC property was characterized. The results found that flow-sorted CD133+ cells induced by ATOH8 depletion possessed most characteristics of CSC including the capacity to self-renew, differentiation, sustained proliferation and chemoresistance. In addition, ATOH8 depletion could enhance the frequency of pluripotent stem cells (iPSC) generation induced by 4 factors (OCT4, Sox2, Klf4 and cMyc). All these data strongly suggest that a non-CSC could be reprogrammed into a CSC under some circumstance such as downregulation of ATOH8. Conclusion: This study demonstrates that ATOH8 plays an important role in HCC development, and provides a potential new HCC therapeutic approach that ATOH8 could increase the chemosensitivity of HCC cells to chemotherapeutics as demonstrated in this study. Citation Format: Yangyang Song, Xin-Yuan Guan. ATOH8 depletion can reprogram noncancer stem cells into cancer stem cells. [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 3865. doi:10.1158/1538-7445.AM2014-3865

Abstract 3051: Genome-wide microarray expression and genomic alteration by array-CGH analysis in neuroblastoma stem-like cells

Abstract Neuroblastoma, the most common extracranial malignant solid tumor in childhood has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to molecular differences in tumor cell subpopulations. The cancer stem cells (CSC) model proposes the existence of a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the stem-like cell population content of two commercial neuroblastoma cell lines (SKNDZ and SIMA) by the use of conditioned cell culture media for neurospheres, and compared genome expression and genomic gains and losses appearing in neurospheres versus the alterations appearing in standard tumor cells, by microarray analysis and array-CGH, respectively. Array-CGH did not show any significant differences between standard and neurosphere-forming cell lines, both in SKNDZ and in SIMA. The microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the stem-cell like phenotype. As expected, some signalling pathways detected were involved in self-renewal of normal tissues (Wnt, Notch, Hedgehog and TGF-β) and seem to contribute to CSC phenotype when deregulated. Among them, we focused on the aberrant activation of Hedgehog and TGF-β signalling pathways. We confirmed the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by quantitative PCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We analyzed two CSC surface markers, and found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk of different pathways in neuroblastoma, and the importance of it in stem-like cells, as confirmed by the overexpression observed in JAG1, one of the targets of TGF-β and also the main activator of Notch signalling pathway. Further analysis based on this work could identify possible new targets for molecular CSC therapies against neuroblastoma, highlighting the importance of redirecting current cancer treatments towards CSC to achieve total elimination of tumor cell population and improve treatment effectiveness. Citation Format: Raquel Ordoñez, Gabriel Gallo, Soledad Martínez, Sheila Legarra, Noémie Pata-Merci, Justine Guegan, Giselle Danglot, Xing Fan, Juan A. Rey, Alain Bernheim, Javier S. Castresana. Genome-wide microarray expression and genomic alteration by array-CGH analysis in neuroblastoma stem-like cells. [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 3051. doi:10.1158/1538-7445.AM2014-3051

Abstract 3882: Downregulation of PRRX1 confers cancer stem cell-like properties and poor prognosis in hepatocellular carcinoma

Abstract Background: Recent studies have been focused on the relationship between cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT). Unlike classical EMT inducers, PRRX1 is involved in the acquisition of CSC-like properties when its down-regulation induces mesenchymal-epithelial transition in breast cancer. Meanwhile, hepatocellular carcinoma (HCC) is one of the most intractable malignancies, and it is required to identify a bona-fide molecular target to regulate tumor progression and the treatment resistance. The purpose of this study is to investigate the role of PRRX1 expression on clinical significance and CSC-like properties in HCC. Methods: (1) PRRX1 expression was analyzed in 62 resected primary HCC cases by quantitative RT-PCR (qRT-PCR). Clinicopathological factors and overall survival (OS) according to PRRX1 expression were analyzed. (2) The gene set enrichment analysis (GSEA) on the published clinical data set of 242 HCC samples (GSE14520) was applied to investigate the association of PRRX1 expression levels and the stem cell-like properties. (3) PRRX1 stably expressing HCC cell lines (HuH7) were established using a lentiviral vector. The expression levels of the hepatic CSC surface markers (CD13, CD133 and EpCAM) were analyzed by flowcytometry and qRT-PCR. Furthermore, the ability of sphere formation and in vitro chemosensitivity to 5-FU were evaluated to assess the CSC-like properties. Results: (1) Univariate analysis showed that 5-year OS of the low PRRX1 expression group was significantly shorter than that of the high PRRX1 expression group (49.8% vs. 80.3%, P=0.014). On multivariate analysis, low PRRX1 expression was an independent prognostic factor for HCC (P=0.026). (2) GSEA analysis on the 247 HCC data set indicated that the down-regulation of PRRX1 was significantly correlated with the stem cell signature (P=0.039). Furthermore, using gene sets of polycomb repressive complex 2 (PCR2) targets and H3 lysine-27 trimethylation mediated by PCR2, HCC with low PRRX1 expression showed parallel enrichment patterns with human embryonic stem cells (P=0.002 and P&amp;lt;0.001, respectively). These results suggested that down-regulation of PRRX1 in HCC induces the acquisition of CSC-like properties through modification of chromatin structure. (3) The level of CSC markers (CD13, CD133 and EpCAM) and the number of spheres were significantly decreased in PRRX1-expressing HuH7 cells compared with the controls. Moreover, PRRX1 impaired resistance to 5-FU in HCC cells. Conclusions: The down-regulation of PRRX1 expression contributes to the poor prognosis through the acquisition of CSC-like properties in HCC patients. We might apply the aberrant expression of PRRX1 as a novel clinical biomarker for prediction of the malignant potential in HCC cases. Citation Format: Hidenari Hirata, Keishi Sugimachi, Ryutaro Uchi, Junji Kurashige, Tae Matsumura, Yuki Takano, Hiroki Ueo, Masami Ueda, Shotaro Sakimura, Yoshiaki Shinden, Hidetoshi Eguchi, Tomoya Sudo, Masakazu Hirakawa, Hiroshi Honda, Koshi Mimori. Downregulation of PRRX1 confers cancer stem cell-like properties and poor prognosis in hepatocellular carcinoma. [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 3882. doi:10.1158/1538-7445.AM2014-3882

Comparison of cancer stem cell antigen expression by tumor cell lines and by tumor biopsies from dogs with melanoma and osteosarcoma

Cancer stem cells (CSCs) represent a small subpopulation of tumor cells that play a critical role in initiating and sustaining tumor growth. However, we currently have an incomplete understanding of the expression patterns of CSC antigens in tumors of dogs, nor do we understand how expression of these antigens vary between tumor cell lines and tumor biopsy specimens. Therefore, we used flow cytometry and commonly reported CSC surface and intracellular markers to evaluate the phenotype and overall frequency of CSC subpopulations in tumor cell lines and primary tumor biopsy samples from dogs with melanoma and osteosarcoma. We found that cells expressing common CSC antigens were rare in tumor cell lines, with the exception of tumor cells expressing CD44 and CD90. In contrast, tumor cells expressing conventional CSC antigens such as CD133, CD34, CD44, CD24 and Oct3/4 were much more common in tumor biopsy samples. Notably, the frequency and types of putative CSC subpopulations were very similar in biopsy samples from dogs with either melanoma or osteosarcoma. Our results suggest that the tumor microenvironment significantly influences CSC subpopulations within tumors and that tumor cell lines may not accurately reflect the actual frequency or types of CSC subpopulations present in tumor tissues in vivo.

Using SV119‐Gold Nanocage Conjugates to Eradicate Cancer Stem Cells Through a Combination of Photothermal and Chemo Therapies

Cancer stem cells (CSCs) are believed to be responsible for the long-term growth of a tumor, as well as its metastasis and recurrence after conventional therapies. Here, it is demonstrated that the sigma-2 receptor is overexpressed on the surface of breast CSCs, and thus could serve as a biomarker for the purpose of targeting. Breast CSCs are targeted with Au nanocages (AuNCs) by functionalizing their surfaces with SV119, a synthetic small molecule capable of binding to the sigma-2 receptor with high specificity. The interiors of the AuNCs could also be loaded with an anticancer drug to be selectively delivered to breast CSCs and released in a controllable fashion. The results demonstrate that the SV119-AuNC conjugate can serve as a new platform to carry out photothermal and chemo therapies simultaneously, eradicating breast CSCs more effectively through a synergetic effect.