Therapeutic Targeting Of Cancer Stem Cells Research Articles

Dinactin: A New Antitumor Antibiotic with Cell Cycle Progression and Cancer Stemness Inhibiting Activities in Lung Cancer.

Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the most complex diseases, despite the existence of effective treatments such as chemotherapy and immunotherapy. Since cancer stem cells (CSCs) are responsible for chemo- and radio-resistance, metastasis, and cancer recurrence, finding new therapeutic targets for CSCs is critical. Dinactin is a natural secondary metabolite produced by microorganisms. Recently, dinactin has been revealed as a promising antitumor antibiotic via various mechanisms. However, the evidence relating to cell cycle progression regulation is constrained, and effects on cancer stemness have not been elucidated. Therefore, the aim of this study is to evaluate the new function of dinactin in anti-NSCLC proliferation, focusing on cell cycle progression and cancer stemness properties in Lu99 and A549 cells. Flow cytometry and immunoblotting analyses revealed that 0.1-1 µM of dinactin suppresses cell growth through induction of the G0/G1 phase associated with down-regulation of cyclins A, B, and D3, and cdk2 protein expression. The tumor-sphere forming capacity was used to assess the effect of dinactin on the cancer stemness potential in NSCLC cells. At a concentration of 1 nM, dinactin reduced both the number and size of the tumor-spheres. The quantitative RT-PCR analyses indicated that dinactin suppressed sphere formation by significantly reducing expression of CSC markers (i.e., ALDH1A1, Nanog, Oct4, and Sox2) in Lu99 cells. Consequently, dinactin could be a promising strategy for NSCLC therapy targeting CSCs.

Enhanced O-GlcNAc modification induced by the RAS/MAPK/CDK1 pathway is required for SOX2 protein expression and generation of cancer stem cells

Cancer stem cells (CSCs) have tumour initiation, self-renewal, and long-term tumour repopulation properties, and it is postulated that differentiated somatic cells can be reprogrammed to CSCs by oncogenic signals. We previously showed that oncogenic HRASV12 conferred tumour initiation capacity in tumour suppressor p53-deficient (p53−/−) primary mouse embryonic fibroblasts (MEFs) through transcription factor NF-κB-mediated enhancement of glucose uptake; however, the underlying mechanisms of RAS oncogene-induced CSC reprogramming have not been elucidated. Here, we found that the expression of the reprogramming factor SOX2 was induced by HRASV12 in p53−/− MEFs. Moreover, gene knockout studies revealed that SOX2 is an essential factor for the generation of CSCs by HRASV12 in mouse and human fibroblasts. We demonstrated that HRASV12-induced cyclin-dependent kinase 1 (CDK1) activity and subsequent enhancement of protein O-GlcNAcylation were required for SOX2 induction and CSC generation in these fibroblasts and cancer cell lines containing RAS mutations. Moreover, the CDK inhibitor dinaciclib and O-GlcNAcylation inhibitor OSMI1 reduced the number of CSCs derived from these cells. Taken together, our results reveal a signalling pathway and mechanism for CSC generation by oncogenic RAS and suggest the possibility that this signalling pathway is a therapeutic target for CSCs.

Abstract PR-004: Inhibition of focal adhesion kinase (FAK) improves pancreatic ductal adenocarcinoma’s response to immunotherapy by targeting cancer stem cells (CSCs)

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with a 5-year survival rate less than 10%. Current therapies consisting of cytotoxic chemotherapeutic agents are not effective in most patients with metastatic PDAC, and the impact of new strategies, including immunotherapies, has not been established. Previous studies have shown that inhibition of focal adhesion kinase (FAK) modulates the PDAC immunosuppressive tumor microenvironment (TME), and sensitizes tumors to immune checkpoint blockade. However, the role of FAK in regulating tumor cell intrinsic resistance to immunotherapy is not well understood. We and others previously demonstrated FAK regulates PDAC cancer stem cells (CSCs) activity including self-renewal, tumor initiation, and drug resistance. These CSCs are responsible for metastatic dissemination and resistance to multiple therapies, including immunotherapy. In this study, we evaluated the sensitivity of PDAC cells, including CSCs to cytotoxic T cells, and we also analyzed the impact of FAK inhibition on the immunogenicity and the response to cytotoxic T cell activity of PDAC CSCs. Methods: To understand how FAK regulates tumor intrinsic resistance to antigen-specific cytotoxic T cells, we utilized OT-I T cells (OT-I cells) that specifically recognize a peptide fragment of ovalbumin (ova) within the context of MHC-I. Mouse KPC pancreatic cancer cell lines expressing ova were generated and co-cultured with cytotoxic CD8+ OT-I cells in vitro. We analyzed the effect of FAK in regulating bulk cells sensitivity to antigen-specific cytotoxic T cells and clonogenic growth of CSCs by flow cytometry, MTT assay and colony formation assay using CRISPR-Cas9 and small molecule FAK inhibitors. We also analyzed the expression of surface proteins that mediate interactions with T cells (class I MHC) in bulk cells and CSCs after FAK inhibition. Results: Loss of FAK activity decreased CSCs activity (self-renewal, colony formation) in PDAC mouse cell lines, which is consistent with our previous data. Using antigen-specific OT-I co-culture model, we found that CSCs are resistant to cytotoxic T cells anti-tumor activity compared to bulk tumor cells. We also found inhibition of FAK activity not only enhances PDAC cells to cytotoxic T cell activity, but also sensitized CSCs to T cell killing. Studies in both human and mouse PDAC cell lines demonstrated decreased MHC-I expression in CSCs compared to the bulk tumor cells which could be reversed by FAK inhibition. Conclusion: We have demonstrated that CSCs are resistant to antigen-specific cytotoxic T cells, and FAK inhibition in CSCs can sensitize these cells to anti-tumor T cell activity that is associated with changes in the expression of factors involved in antigen presentation. Our study provides novel insights into the unique immunogenicity of CSCs and the role of CSCs in regulating tumor intrinsic immunotherapy resistance in PDAC. Our findings also lead to the identification of FAK as a novel therapeutic target for CSCs to improve immunotherapy efficacy in PDAC. Citation Format: Yezi Zhu, Lyndsey Sandow, William Matsui. Inhibition of focal adhesion kinase (FAK) improves pancreatic ductal adenocarcinoma’s response to immunotherapy by targeting cancer stem cells (CSCs) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-004.

Cripto-1 as a Potential Target of Cancer Stem Cells for Immunotherapy.

Simple SummaryCancer immunotherapy is gaining attention as a potential fourth treatment following surgery, chemotherapy, and radiation therapy. Cancer stem cells have recently been recognized and validated as a key target for cancer treatment. Cripto-1, which is a GPI-anchored membrane-bound protein that functions as a co-receptor of Nodal, is a marker of cancer stem cells. Since Nodal is a member of the TGF-β family, which performs an important role in stem cells and cancer stem cells, the inhibition of Cripto-1 could be a strategy by which to block Nodal signaling and thereby suppress cancer stem cells. We propose that Cripto-1 may be a novel target for cancer immunotherapy.The immune system has been found to be suppressed in cancer patients. Cancer cells are extremely resistant to chemotherapeutic drugs, conventional immunotherapy, or cancer antigen vaccine therapy. Cancer immunotherapy, which is mainly based on immune checkpoint inhibitors, such as those for PD-1, PD-L1, and CTLA4, is an effective treatment method. However, no immunotherapeutic target has been found that retains validity in the face of tumor diversity. The transforming growth factor (TGF)-β cytokine family possesses broad biological activity and is involved in the induction and/or transdifferentiation of helper T cells, which are important in immunotherapy. Nodal is a member of the TGF-β family playing important roles in tissue stem cells and cancer stem cells (CSCs), interacting with the co-receptor Cripto-1, as well as with Activin type IB (Alk4) and Activin typeIIreceptors, and maintaining stemness and Notch and Wnt/β-catenin signaling in CSCs. In recent years, it has been reported that Cripto-1 could be a potential therapeutic target in CSCs. Here, we review the accumulated literature on the molecular mechanisms by which Cripto-1 functions in CSCs and discuss the potential of Cripto-1 as an immunotherapeutic target in CSCs.

The Role of Extracellular Vesicles in the Development of a Cancer Stem Cell Microenvironment Niche and Potential Therapeutic Targets: A Systematic Review.

Simple SummaryCancer stem cells (CSCs) are cancer cells that possess traits usually attributed to stem cells. An increase in CSCs can lead to more rapid cancer progression, treatment resistance and the increased likelihood of recurrence. To promote CSC survival and associated cancer progression, cancer cells enter into reciprocal crosstalk with the surrounding tissue environment, as well as with distant metastatic sites. This mechanism of communication relies, in part, on secreted factors, of which extracellular vesicles (EVs) are thought to have a critical role. This systematic review evaluates the current knowledge of cancer communication via EVs to alter the microenvironment to increase the survival and maintenance of CSCs. A total of 16 studies spanning the EV content, pathway alterations and CSC-targeting treatments provide new insights into how EVs mediate CSC traits and identify the gaps in our understanding of how modulation of the microenvironment plays a key role.Cancer stem cells (CSCs) have increasingly been shown to be a crucial element of heterogenous tumors. Although a relatively small component of the population, they increase the resistance to treatment and the likelihood of recurrence. In recent years, it has been shown, across multiple cancer types (e.g., colorectal, breast and prostate), that reciprocal communication between cancer and the microenvironment exists, which is, in part, facilitated by extracellular vesicles (EVs). However, the mechanisms of this method of communication and its influence on CSC populations is less well-understood. Therefore, the aim of this systematic review is to determine the evidence that supports the role of EVs in the manipulation of the tumor microenvironment to promote the survival of CSCs. Embase and PubMed were used to identify all studies on the topic, which were screened using PRISMA guidelines, resulting in the inclusion of 16 studies. These 16 studies reported on the EV content, pathways altered by EVs and therapeutic targeting of CSC through EV-mediated changes to the microenvironment. In conclusion, these studies demonstrated the role of EV-facilitated communication in maintaining CSCs via manipulation of the tumor microenvironment, demonstrating the potential of creating therapeutics to target CSCs. However, further works are needed to fully understand the targetable mechanisms upon which future therapeutics can be based.

Expression of Components of the Renin-Angiotensin System by Cancer Stem Cells in Renal Clear Cell Carcinoma.

This study investigated the expression of components of the renin-angiotensin system (RAS) by cancer stem cells (CSCs) we have recently demonstrated in renal clear cell carcinoma (RCCC). Fifteen RCCC tissue samples underwent immunohistochemical staining for components of the RAS: renin, pro-renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme 2 (ACE2), and angiotensin II receptor 2 (AT2R). Immunofluorescence co-staining or double immunohistochemical staining of these components of the RAS with stemness-associated markers OCT4 or KLF4 was performed on two of the samples. Protein and transcript expression of these components of the RAS in six RCCC tissue samples was investigated using western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. In addition, angiotensin II receptor 1 (AT1R) was investigated using RT-qPCR only. Immunohistochemical staining demonstrated expression of renin, PRR, and ACE2 in 11, 13, and 13 out of 15 RCCC samples, respectively, while AT2R was expressed in all 15 samples. ACE was detected in the endothelium of normal vasculature only. Double immunohistochemical staining demonstrated localization of ACE2, but not renin, to the KLF4+ CSCs. Immunofluorescence staining showed localization of PRR and AT2R to the OCT4+ CSCs. Western blotting confirmed protein expression of all components of the RAS except renin. RT-qPCR demonstrated transcript expression of all components of the RAS including AT1R, but not AT2R, in all six RCCC tissue samples. This study demonstrated expression of PRR, ACE2, and AT2R by the CSCs within RCCC. Further studies may lead to novel therapeutic targeting of CSCs by manipulation of the RAS in the treatment of this aggressive cancer.

CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells

Cancer stem cells (CSCs) or tumor-initiating cells are thought to play critical roles in tumorigenesis, metastasis, drug resistance, and tumor recurrence. For the diagnosis and targeted therapy of CSCs, the molecular identity of biomarkers or therapeutic targets for CSCs needs to be clarified. In this study, we identified CD166 as a novel marker expressed in the sphere-forming CSC population of A2780 epithelial ovarian cancer cells and primary ovarian cancer cells. The CD166+ cells isolated from A2780 cells and primary ovarian cancer cells highly expressed CSC markers, including ALDH1a1, OCT4, and SOX2, and ABC transporters, which are implicated in the drug resistance of CSCs. The CD166+ cells exhibited enhanced CSC-like properties, such as increased sphere-forming ability, cell migration and adhesion abilities, resistance to conventional anti-cancer drugs, and high tumorigenic potential in a xenograft mouse model. Knockdown of CD166 expression in the sphere-forming ovarian CSCs abrogated their CSC-like properties. Moreover, silencing of CD166 expression in the sphere-forming CSCs suppressed the phosphorylation of focal adhesion kinase, paxillin, and SRC. These results suggest that CD166 plays a key role in the regulation of CSC-like properties and focal adhesion kinase signaling in ovarian cancer.

Stem cell programs in cancer initiation, progression, and therapy resistance

Over the past few decades, substantial evidence has convincingly revealed the existence of cancer stem cells (CSCs) as a minor subpopulation in cancers, contributing to an aberrantly high degree of cellular heterogeneity within the tumor. CSCs are functionally defined by their abilities of self-renewal and differentiation, often in response to cues from their microenvironment. Biological phenotypes of CSCs are regulated by the integrated transcriptional, post-transcriptional, metabolic, and epigenetic regulatory networks. CSCs contribute to tumor progression, therapeutic resistance, and disease recurrence through their sustained proliferation, invasion into normal tissue, promotion of angiogenesis, evasion of the immune system, and resistance to conventional anticancer therapies. Therefore, elucidation of the molecular mechanisms that drive cancer stem cell maintenance, plasticity, and therapeutic resistance will enhance our ability to improve the effectiveness of targeted therapies for CSCs. In this review, we highlight the key features and mechanisms that regulate CSC function in tumor initiation, progression, and therapy resistance. We discuss factors for CSC therapeutic resistance, such as quiescence, induction of epithelial-to-mesenchymal transition (EMT), and resistance to DNA damage-induced cell death. We evaluate therapeutic approaches for eliminating therapy-resistant CSC subpopulations, including anticancer drugs that target key CSC signaling pathways and cell surface markers, viral therapies, the awakening of quiescent CSCs, and immunotherapy. We also assess the impact of new technologies, such as single-cell sequencing and CRISPR-Cas9 screening, on the investigation of the biological properties of CSCs. Moreover, challenges remain to be addressed in the coming years, including experimental approaches for investigating CSCs and obstacles in therapeutic targeting of CSCs.

Interleukin 6 signaling maintains the stem-like properties of bladder cancer stem cells

Background: The relapse and metastasis of bladder cancer are due to its strong resistance to chemotherapeutic drugs after surgery as a result of the expansion and self-renewal of cancer stem cells (CSCs). However, the molecular mechanisms underlying the biology of bladder CSCs are unknown. This study aimed to investigate the role of interleukin 6 (IL6)/IL6 receptor (IL6R) in the stem-like characteristics of bladder CSCs. Methods: Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect IL6 expression in the supernatant and cells of bladder CSCs, respectively. Following that, self-renewal, stem cell-associated gene expression, invasion, metastasis, and tumorigenicity were assessed by sphere-forming assay, qRT-PCR, invasion and transwell assays, and tumor-forming experiment in NOD/SCID mice, respectively. Finally, Western blot and qRT-PCR were employed to examine the IL6 signaling pathway in regulating the stem-like properties of bladder CSCs. Results: The spheres, originating from the bladder cancer cell lines RT4 and J82, possessed a higher expression of stem-associated genes. The expression levels of IL6 were elevated in the supernatant and cells of the bladder CSCs. IL6R was also up-regulated in the bladder CSCs. Recombinant IL6 promoted the stem-like properties of the bladder CSCs, including self-renewal, expression of stem-associated genes, invasion, migration, and tumorigenicity. Mechanistically, IL6 exerted its biological effects by binding to IL6R, which enhanced the phosphorylation of STAT3 and triggered its activation. Furthermore, these effects were alleviated by the FDA-approved drug tocilizumab. Conclusions: Our findings demonstrate that IL6/IL6R/STAT3 maintains the stem-like properties of bladder CSCs. Furthermore, IL6R may serve as a potential therapeutic target for CSCs in bladder cancer.

Role of autotaxin in cancer stem cells.

Stem cells are a rare subpopulation defined by the potential to self-renew and differentiate into specific cell types. A population of stem-like cells has been reported to possess the ability of self-renewal, invasion, metastasis, and engraftment of distant tissues. This unique cell subpopulation has been designated as cancer stem cells (CSC). CSC were first identified in leukemia, and the contributions of CSC to cancer progression have been reported in many different types of cancers. The cancer stem cell hypothesis attempts to explain tumor cell heterogeneity based on the existence of stem cell-like cells within solid tumors. The elimination of CSC is challenging for most human cancer types due to their heightened genetic instability and increased drug resistance. To combat these inherent abilities of CSC, multi-pronged strategies aimed at multiple aspects of CSC biology are increasingly being recognized as essential for a cure. One of the most challenging aspects of cancer biology is overcoming the chemotherapeutic resistance in CSC. Here, we provide an overview of autotaxin (ATX), lysophosphatidic acid (LPA), and their signaling pathways in CSC. Increasing evidence supports the role of ATX and LPA in cancer progression, metastasis, and therapeutic resistance. Several studies have demonstrated the ATX-LPA axis signaling in different cancers. This lipid mediator regulatory system is a novel potential therapeutic target in CSC. In this review, we summarize the evidence linking ATX-LPA signaling to CSC and its impact on cancer progression and metastasis. We also provide evidence for the efficacy of cancer therapy involving the pharmacological inhibition of this signaling pathway.

XIAP Limits Autophagic Degradation of Sox2 and Is A Therapeutic Target in Nasopharyngeal Carcinoma Stem Cells.

Rationale: Nasopharyngeal carcinoma (NPC) is the most frequent head and neck tumor in South China. The presence of cancer stem cells (CSCs) in NPC contributes to tumor maintenance and therapeutic resistance, while the ability of CSCs to escape from the apoptosis pathway may render them the resistant property to the therapies. Inhibitor of apoptosis proteins family proteins (IAPs), which are overexpressed in nasopharyngeal carcinoma stem cells, may play an important role in maintaining nasopharyngeal cancer stem cell properties. Here, we develop a novel CSC-targeting strategy to treat NPC through inhibiting IAPs.Methods: Human NPC S-18 and S-26 cell lines were used as the model system in vitro and in vivo. Fluorescence activated cell sorting (FACS) assay was used to detect nasopharyngeal SP cells and CD44+ cells. The characteristics of CSCs were defined by sphere suspension culture, colony formation assay and cell migration. The role of XIAP on the regulation of Sox2 protein stability and ERK1-mediated phosphorylation of Sox2 signaling pathway were analyzed using immunoblotting, immunoprecipitation, immunofluorescence, phosphorylation mass spectrometry, siRNA silencing and plasmid overexpression. The correlation between XIAP and Sox2 in NPC biopsies and their role in prognosis was performed by immunohistochemistry. APG-1387 or chemotherapies-induced cell death and apoptosis in S-18 and S-26 were determined by WST, immunoblotting and flow cytometry assay.Results: IAPs, especially X chromosome-linked IAP (XIAP), were elevated in CSCs of NPC, and these proteins were critically involved in the maintenance of CSCs properties by enhancing the stability of Sox2. Mechanistically, ERK1 kinase promoted autophagic degradation of Sox2 via phosphorylation of Sox2 at Ser251 and further SUMOylation of Sox2 at Lys245 in non-CSCs. However, XIAP blocked autophagic degradation of Sox2 by inhibiting ERK1 activation in CSCs. Additionally, XIAP was positively correlated with Sox2 expression in NPC tissues, which were associated with NPC progression. Finally, we discovered that a novel antagonist of IAPs, APG-1387, exerted antitumor effect on CSCs. Also, the combination of APG-1387 with CDDP /5-FU has a synergistic effect on NPC.Conclusion: Our study highlights the importance of IAPs in the maintenance of CSCs in NPC. Thus, XIAP is a promising therapeutic target in CSCs and suggests that NPC patients may benefit from a combination treatment of APG-1387 with conventional chemotherapy.

Abstract 3345: Targeting the YAP1/COX2/SOX2 signaling axis eliminates cancer stem cells in urothelial carcinoma

Abstract Urothelial cancer stem cells (CSCs) contribute to tumor maintenance and resistance to therapy and accumulated evidence suggest that chronic carcinogen exposure induce “stemness”. Therapeutic targeting of CSCs could improve treatment response and prolong patient survival. Here we used our recently published in vitro chronic arsenic (As) exposed models to characterize the property of urothelial CSCs due to arsenic exposure. We hypothesized that urothelial stem cells have a survival selection advantage during carcinogen exposure such as As, and it facilitates their malignant transformation and in acquiring selective phenotypes similar to CSC. Arsenic-exposed cells displayed more aggressive phenotype than arsenic unexposed cells in a time dependent manner as determined by MTT, invasion, and wound healing assay. In gene set enrichment analysis of expression array of chronic arsenic exposed and unexposed cells; EGFR, COX2 and YAP1 were top-ranked oncogenic signature based on enrichment score in As-exposed cells. Further analysis indicated that several known basal cell markers such as KRT5, KRT6A, and KRT6C etc were overexpressed in As exposed cells in comparison with As unexposed cells. Because the presence of urothelial CSCs in basal-type provides a biological explanation for their aggressive behaviors, we assessed the influence of As on generating CSC phenotypes. As exposure was associated with overabundance of potential CSCs characterized by sphere formation, self-renewal capacity, redifferentiation, and chemotherapy resistance. To explore global association of As exposure and CSC generation, we used the Human Stem Cell RT2 Profiler™ PCR Array and found that SOX2 has been gradually overexpressed in line with acquired spheroid formation and self-renewal capacities. SOX2 is frequently overexpressed in numerous urothelial carcinoma (UC) cell lines as well as in arsenic-exposed cells, especially in the spheroid cells. Stable silencing of SOX2 reduces in vitro CSCs properties with reduce expression of CSCs associated molecules such as NANOG,OCT4 etc and also in vivo tumorigenicity. COX2 and YAP1 are also frequently overexpressed in UC cell lines, and inhibition of COX2 or YAP1 reduced SOX2 expression. Interestingly, the inhibition of COX2 and YAP1 expression inversely induced YAP1 and COX2 expression, respectively. Combination treatment with COX2 and YAP1 inhibitors reduced SOX2 expression and suppressed spheroid formation significantly than either inhibitor alone. In xenograft model, the combination treatment suppressed the tumor growth rate, but not either inhibitor alone. In conclusion, chronic As exposure induces CSCs with SOX2 overexpression, one of the most important CSC factor for UC. COX2 and YAP1 coordinately regulate SOX2 expression, and mutually compensate for the reduction of expression of SOX2 to maintain CSCs. Thus targeting the COX2/YAP/SOX2 signaling axis eliminates urothelial CSCs. Citation Format: Akira Oki, Mohammad Hoque. Targeting the YAP1/COX2/SOX2 signaling axis eliminates cancer stem cells in urothelial carcinoma. [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 3345.

Human colorectal CD24+ cancer stem cells are susceptible to epithelial-mesenchymal transition.

Conventional cancer chemotherapy preferentially destroys non-stem cancer cells within a tumor, and a subpopulation of cancer stem cells (CSCs) is more resistant and survives, leading to relapses and metastasis. Howeve, recent studies suggest that CD24 and susceptibility to epithelial-mesenchymal transition (EMT) can serve as markers of CSCs. We report that CD24(+) cells are susceptible to induction of EMT, a phenotype important for cancer metastasis. We studied the responsiveness of CSC markers to TGF-β , an effective EMT inducer. The data on CD24 demonstrated that CD24(+) cells are susceptible to EMT, a phenotype important for cancer metastasis in two colorectal cancer cell lines, the CaR-1 and CCK81. CD24(+) cells expressed Notch 1 in response to exposure to TGF-β in culture and showed higher tumorigenic activity compared to controls. This evidence shows that CD24(+) cells are susceptible to EMT induction and to cancer progression and is indicative of the candidacy of CD24 as a therapeutic target in CSC.

Abstract LB-193: Ganglioside GD2 identifies cancer stem cells and inhibition of GD2 biosynthesis by targeting GD3 synthase exerts antitumor effects

Abstract Currently, only very few markers either as single or in combination are available to identify cancer stem cells (CSCs). In this report, we identified that the ganglioside GD2, a marker known to express on mesenchymal stromal cells (MSCs) is expressed on a small fraction (5.5% ± 3.4%) of transformed human mammary epithelial cells (HMLER) and breast cancer patient tumors. FACS sorted GD2+ cells appear spindle shaped and proliferate 5 fold slower compared to GD2- cells in-vitro. Analysis of breast cancer cell lines (n=12) indicated that GD2 expression varies and that the basal breast cancer cell lines have a higher percentage of GD2+ cells (median 9%, range 1.2-17%, n=6), as compared to their luminal counterparts (median 0.2%, range 0-3%, n=6, p<0.001). Functional analysis revealed that GD2+ HMLER and MDA-MB-231 cells produced 2-5 fold more mammospheres in-vitro (p<0.003) and tumors in-vivo than GD2- cells (p<0.04). Recent reports suggest that breast tumor cells with CD44highCD24low phenotype exhibit CSCs characteristics. Interestingly, the majority (94% ± 3.5%) of GD2+ cells were characterized by the CD44highCD24low phenotype in HMLER cells. Analysis of primary breast cancer samples (n=10) revealed that GD2 is variably expressed in these samples (median 4.35%, range 0.5%-35.8%) and 95.5% ± 2.7% of GD2+ cells co-segregated with CD44highCD24lowCD45- phenotype. In contrast, only 2.4%± 0.4% of GD2- cells displayed this phenotype. Gene-chip and quantitative RT-PCR analysis revealed that GD3 synthase (GD3S), the enzyme responsible for synthesis of GD3, the precursor of GD2, was expressed 10-fold higher in GD2+ cells compared to GD2- HMLER and MDA-MB-231 cells. Analysis of GD2 expression in HMLER cells induced to undergo EMT revealed that the percentage of GD2+ cells increased 6-7 fold and the expression of GD3S > 10 fold. Moreover, we observed spontaneous generation of GD2+ from GD2- cells and vice versa in both in-vitro and in-vivo, suggesting a role of EMT in this process. Stable knock-down of GD3S in MDA-MB-231 cells using shRNA impaired in-vitro matrigel invasion by more than 10-fold and completely abolished tumor growth in-vivo. Importantly, Triptolide, an anti-inflammatory and anti-cancer drug, which was recently shown to inhibit GD3S expression in melanoma cells, also inhibited GD3S expression in MDA-MB-231 and SUM159 cells by >95% in a dose dependent manner and thereby inhibited growth of GD2+ cells in a time dependent manner. Intra-peritoneal administration of Triptolide (0.15mg/Kg/day) in NOD/SCID mice bearing MDA-MB-231 breast tumors completely eliminated tumors in 50% and reduced the tumor volume 7- to 8-fold in 25% of the mice. In conclusion, we identified GD2 as a new CSC specific cell surface marker and GD3 synthase as a potential therapeutic target for CSCs, with the potential of improving survival and cure rates of patients with breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-193. doi:1538-7445.AM2012-LB-193