Potential Cancer Stem Cells Research Articles

Transforming growth factor β signaling regulates the invasiveness of normal mammary epithelial cells and the metastasis formation of tumor cells.

Abstract Breast cancer patients with disseminated metastatic disease still have a very unfavorable prognosis. Investigations into the molecular mechanisms that underlie metastasis formation have a high priority and can possibly result in improved therapeutic interventions. The process of oncogenic epithelial to mesenchymal transition (EMT) has recently become a focus in cancer research because it encompasses many of the phenotypic traits characteristic of metastatic cells, e.g., increased motility, invasion, anoikis resistance, immunosuppression, and cancer stem cell potential. A number of central cellular signaling pathways and transcription factors have been implied in the control of EMT and metastasis formation, among them signal originating from the activation of the transforming growth factor β (TGFβ), epithelial growth factor, Wnt, Notch, and Hedgehog pathways. We have investigated the contribution of TGFβ signaling to metastasis-related cellular properties. TGFβ signaling can have tumor-suppressive and -promoting effects depending on the tumor type and the stage of tumor progression. TGFβ can inhibit the proliferation of mammary epithelial cells (MECs), but it can also induce EMT, invasion, and metastasis, possibly through Smad-independent signaling events. We investigated the effects of TGFβ pathway inhibition on the proliferation, differentiation, and invasion of both normal and malignant MECs. shRNA-mediated downregulation of the Smad4 protein in non-tumorigenic HC11 and tumorigenic 4T1 cells promotes the invasiveness of both cell lines. Mammary gland reconstitution studies, with primary MECs expressing shSmad4, resulted only in subtle effects on the glandular morphogenesis. Orthotopic transplantation of shSmad4-transduced 4T1 tumor cells caused the accelerated growth of mammary tumors and enhanced colonization and macroscopic lung metastases when compared to control cells. Surprisingly, the expression of Smad4 was restored, and a strong activation of Stat3 was found in the metastatic lesions present in the lungs. These lesions express metastatic factors, such as angiopoietin-like-4 and the inhibitor of DNA binding/differentiation 1. We suggest that the downregulation of Smad4 inhibits the tumor-suppressive effects of TGFβ signaling and enhances tumor growth. The downregulation, however, was only transient, and the reactivation of Smad4 expression caused the reversal of EMT, mesenchymal to epithelial transition, and thereby promoted metastasis formation in the lungs.

P1-04-05: T-DM1 and Pertuzumab as New Tools for HER2 Specific Antibody-Therapy Against Breast Cancer Stem Cells in HER2−Positive Mammary Carcinoma.

Abstract Trastuzumab (Herceptin®) has significantly improved the overall survival of patients with HER2−positive breast cancer. The efficacy of this treatment depends significantly on antibody-dependent cell mediated cytotoxicity (ADCC). However, while trastuzumab can direct NK cells towards tumor cells and thereby promote tumor cell lysis, spheroid-forming tumor-initiating cells are largely spared. Consequently, populations of surviving cells showed greatly enhanced clonogenicity in vitro and tumorigenicity in vivo. Low HER2 levels and reduced expression of activating NK cell ligands provide likely explanations for the escape of these cells from HER2−directed NK cell-dependent therapies. Moreover, recent data show that unproductive interactions between immune cells and cancer cells can induce a process called “epithelial-to-mesenchymal transition” (EMT) which enables previously differentiated cancer cells to acquire stem cell-like properties as evidenced by a CD44highCD24low breast cancer stem cell signature and aldehyde dehydrogenase positivity. We thus investigated whether new HER2−specific antibodies could enhance NK-cell mediated target cell lysis or inhibit the induction of tumor stem cells. The antibody pertuzumab also binds to the HER2−receptor. It inhibits homo- and heterodimerization of the HER2 receptor with other member of the HER family, in particular the dimerization of HER2/HER3 receptors. We were able to show that the combination of trastuzumab and pertuzumab leads to increased antibody-binding on breast cancer cells and breast cancer stem cells, resulting in enhanced ADCC. This correlates with the clinically observed synergy between trastuzumab and pertuzumab as observed for example in the Neosphere trial, a neoadjuvant study for HER2−positive breast cancer. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate which combines trastuzumab with the tubulin inhibitor maytansine. Interestingly, treatment with T-DM1 largely prevented the NK cell-dependent induction of the EMT markers Twist and Snail and the characteristic downregulation of E-cadherin. Thus, T-DM1 apparently interferes with EMT and may hence prevent the re-induction of the stem cell phenotype. In line with the presumed effect against cancer stem cells, T-DM1 significantly attenuated the clonogenic potential of FACS-sorted trastuzumab-unresponsive cancer stem cells. Accordingly, our data show that these new therapeutics may be more effective against cancer stem cells and thereby promise a more sustained treatment of HER2−positive breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-05.

Abstract A62: Monensin-induced oxidative stress reduces prostate cancer cell motility and cancer stem cell markers.

Abstract Therapeutic options for prostate cancer are limited and treatment responses to currently existing therapies are often unsatisfactory. Thus, there is an urgent need for novel agents to target advanced and metastatic prostate cancer cells. We have recently carried out a chemical-biological high-throughput screening of 4,910 known drugs and drug-like molecules in four prostate cancer cell models and two non-tumorigenic prostate epithelial cell lines to identify prostate cancer cell growth selective inhibitors. Only four compounds, antibiotic ionophore monensin, aldehyde dehydrogenase (ALDH) inhibitor disulfiram, histone deacetylase inhibitor trichostatin A and fungicide thiram inhibited selectively cancer cell growth at nanomolar concentrations. The mechanistic studies indicated that monensin inhibited prostate cancer cell growth by inducing oxidative stress and apoptosis. In addition, monensin reduced androgen receptor signaling, showed a synergistic anti-proliferative effect with anti-androgens as well as reduced the levels of MYC and ERG oncogenes and reduced the activity of ALDH in prostate cancer cells. Moreover, antioxidant vitamin C rescued the monensin induced growth inhibition, indicating that oxidative stress plays a key role in the antineoplastic effect of monensin in cultured prostate cancer cells. Our previous Connectivity Map results indicated that monensin has agonistic effects to NF-κB inactivator and oxidative stress inducer niclosamide. Here, we show that monensin indeed reduced the activity of NF-κB pathway. NF-κB maintains cellular antioxidant defence capacity and its inhibition induces oxidative stress as well as reduces tumorigenesis, metastasis and cancer stem cell potential. Cancer stem cells have a controlled redox balance system including high ALDH and CD44 expression which protect cancer stem cells from oxidative stress. Our results confirmed that monensin reduced the cancer stem cell markers in prostate cancer cells. Moreover, monensin induced epithelial cell differentiation shown as well as reduced motility in cultured prostate cancer cells, suggesting that monensin inhibits prostate tumorigenesis by multiple ways. Furthermore, the steroid profiling indicated that monensin increases the levels of oxidative stress inducing steroids and reduces androgen precursors in cultured prostate cancer cells. In conclusion, our results suggest that impairing the redox control, which has a crucial role in cancer cells enabling survival under high intracellular ROS, is a potent way to target prostate cancer cells and potentially also prostate cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A62.

Increased Wild-Type N-Ras Activation by Neurofibromin Down-Regulation Increases Human Neuroblastoma Stem Cell Malignancy

Cellular heterogeneity is a well-known feature of human neuroblastoma tumors and cell lines. Of the 3 phenotypes (N-, I-, and S-type) isolated and characterized, the I-type cancer stem cell of neuroblastoma is the most malignant. Here, we report that, although wild-type N-Ras protein is expressed at the same level in all 3 neuroblastoma cell phenotypes, activated N-Ras-GTP level is significantly higher in I-type cancer stem cells. When activated N-Ras levels were decreased by transfection of a dominant-negative N-Ras construct, the malignant potential of I-type cancer stem cells decreased significantly. Conversely, when weakly malignant N-type cells were transfected with a constitutively active N-Ras construct, activated N-Ras levels, and malignant potential, were significantly increased. Thus, high levels of N-Ras-GTP are required for the increased malignancy of I-type neuroblastoma cancer stem cells. Moreover, increased activation of N-Ras results from significant down-regulation of neurofibromin (NF1), an important RasGAP. This specific down-regulation is mediated by an ubiquitin-proteasome-dependent pathway. Thus, decreased expression of NF1 in I-type neuroblastoma cancer stem cells causes a high level of activated N-Ras that is, at least in part, responsible for their higher tumorigenic potential.

Preparation of Epithelial and Mesenchymal Stem Cells from Murine Mammary Gland

The mammary gland is a complex organ consisting of multiple cell types that undergo extensive remodeling during pregnancy and involution, cyclical changes that suggest the existence of a resident stem cell population that is responsible for remarkable tissue regeneration. The basic functional unit of the mammary gland is the terminal duct lobular unit, which invades the stromal tissue (fat, connective tissue, blood vessels, etc.). Luminal epithelial cells line the ducts while outer myoepithelial cells secrete the basal lamina that separates the mammary gland parenchyma from the mesenchymal cells of the stroma. Within the epithelial cell population of the ducts resides the mammary gland stem cells and it is believed that this population is the origin of the mammary gland cancer stem cells as well. In the mouse, epithelial stem cells can be separated from mesenchymal cells on the basis of CD24, CD44, and CD49f expression. This allows for the determination of both normal and cancer stem cell potential of these two populations and permits investigation into their interaction in tumor development.

H+-myo-Inositol Transporter SLC2A13 as a Potential Marker for Cancer Stem Cells in an Oral Squamous Cell Carcinoma

Cancer Stem Cells (CSCs) from tumors of different phenotypes possess a marked capacity for proliferation, self-renewal, and differentiation. They also play a critical role in cancer recurrence. Although CSC has been regarded as a new target for cancer therapy, the fundamental questions in the CSC study have not been resolved mainly due to the lack of proper CSC markers. To find new CSC markers for oral squamous cell carcinoma (OSCC), we cultured the primary tumor cells from OSCC patients the regular culture condition and the sphere-forming culture condition to enrich primary tumor cells and potential CSCs. We compared gene expression profiles between sphere-forming and non-forming cells, thus identifying that 23 membrane protein-coding genes were over-expressed in the sphere-forming cells. Among them, 8 belonged to the solute carrier (SLC) protein family. H⁺-myo-inositol transporter SLC2A13 and monocarbohydrate transporter SLC16A6 genes that were consistently increased in the sphere-forming cells in the primary cultures of OSCC samples. Confocal microscopy revealed that SLC2A13-expressing cells were embedded in the limited areas of tumor tissue as a cluster, while SLC16A6 was uniformly detected in hyperplastic epithelium. Moreover, SLC2A13 an expression was induced in human breast adenocarcinoma MCF7 cells after serum starvation. Taken together, our results suggest that SLC2A13 can be a potential markers for CSC in various tumors.

Is Irradiation of Potential Cancer Stem Cell Niches in the Subventricular Zones Indicated in GBM?

Is Irradiation of Potential Cancer Stem Cell Niches in the Subventricular Zones Indicated in GBM?

Direct In Vivo Evidence for Tumor Propagation by Glioblastoma Cancer Stem Cells

High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC.

Identification of Endothelin‐1 and NR4A2 as CD133‐regulated genes in colon cancer cells

Several in vitro assays have been proposed to identify cancer stem cells (CSCs), including immunophenotyping, sphere assay and side population (SP) assay. CD133 antigen has been proposed as a CSC marker in colon cancer (CC). However, no functional data are available to date and conflicting results have been reported regarding its role as true CSC marker. Here we set out to identify a molecular signature associated with potential CSC. CD133(+) cells isolated from the CaCo-2 CC cell line were analysed by microarray molecular profiling compared to CD133(-) counterparts. Various differentially expressed genes were identified and the most relevant transcripts found to be over-expressed in CD133(+) cells were evaluated by quantitative RT-PCR in the CD133(+) fractions isolated from several CC cell lines. In the attempt to find a correlation between putative CSCs, isolated by means of CD133 immunophenotyping and the SP approach, we demonstrated a significant enrichment of CD133(+) cells within the SP fraction of CC cells, and comparison of the gene expression profiles revealed that Endothelin-1 (END-1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts are highly expressed in both CD133(+) and SP fractions of CC cells. Moreover, depletion of CD133 by siRNA induced a significant attenuation of END-1 and NR4A2 expression levels in CaCo-2 cells, while expression of all three molecules decreased during sodium butyrate-induced differentiation. In conclusion, we have identified a molecular signature associated with potential CSCs and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression in colon cancer cells.

Liposarcoma cells with aldefluor and CD133 activity have a cancer stem cell potential

Aldehyde dehydrogenase (ALDH) has recently been shown to be a marker of cancer stem-like cells (CSCs) across tumour types. The primary goals of this study were to investigate whether ALDH is expressed in liposarcomas, and whether CSCs can be identified in the ALDHhigh subpopulation. We have demonstrated that ALDH is indeed expressed in 10 out of 10 liposarcoma patient samples. Using a liposarcoma xenograft model, we have identified a small population of cells with an inducible stem cell potential, expressing both ALDH and CD133 following culturing in stem cell medium. This potential CSC population, which makes up for 0, 1-1, 7% of the cells, displayed increased self-renewing abilities and increased tumourigenicity, giving tumours in vivo from as few as 100 injected cells.

Abstract LB-113: Non-small cell lung cancer cells with high ALDH1 activity display cancer stem cell properties and show chemoresistance

Abstract Introduction Cancer stem cells (CSC) are believed to possess tissue stem cell-like properties such as self-renewal and toxicity resistance. These characteristics would facilitate tumor propagation and development of chemoresistance. The biology and regulation of CSC have mostly been studied in acute leukemia and certain solid tumors such as breast cancer. Relatively little data are available regarding CSC in non-small cell lung cancer (NSCLC) due to the lack of a specific CSC marker. This study aims to analyze the applicability of candidate CSC markers by utilizing chemotherapy treatment to enhance CSC proliferation in NSCLC cells. Methodology The expression or activity of 4 putative stem cell markers, CD24, CD44, CD133 and aldehyde dehydrogenase (ALDH1) were measured by flow cytometry in the NSCLC cell lines H1650, HCC827 and H358 before and after chemotherapy treatment for 24 hours. Tumor cells with enhanced marker expression after treatment were regarded as potential CSC, and were selected by fluorescence-activated cell sorting (FACS). Functional analysis was used to test for CSC properties in the marker+ and marker-subpopulations of untreated cells. The expression of genes involved in tissue stem cell functions were also compared by quantitative RT-PCR. Results Flow cytometry analysis showed amongst the 4 markers, only ALDH1+ subpopulation was significantly enhanced by chemotherapeutic treatment, suggesting ALDH1 could be a CSC marker. Untreated ALDH1+ cells formed significantly higher numbers and larger cell spheres in non-adherent culture medium than ALDH1− cells. Likewise, ALDH1+ cells formed significantly more and larger colonies in colony formation assay. Furthermore, MTT assay demonstrated higher resistance to cisplatin and etoposide treatments in ALDH1+ than ALDH1− cells. In addition, ALDH1+ cells showed more prominent tumorigenicity than ALDH1− cells in vivo; as few as 500 ALDH1+ cells formed xenograft tumor in SCID mice which were serially transplantable to 2nd and 3rd recipients, while no tumor was formed from ALDH− cells. Finally, expression analysis of sorted cells revealed higher expression of the pluripotency genes, OCT4, NANOG, BMI1 and SOX9, in ALDH1+ cells. Conclusion NSCLC cells expressing ALDH1 displayed higher capacity for cell renewal, tumorigenecity and drug resistance, as well as showed higher expression of pluripotency genes, suggesting that ALDH1 could be a useful CSC marker in NSCLC. Pathways mediated by the differentially expressed genes studied could be involved in CSC maintenance and/or chemoresistance in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2011-LB-113

Abstract 5194: Notch regulates squamous differentiation, cell plasticity and tumor heterogeneity in esophageal carcinoma

Abstract Introduction: The Notch receptor family regulates cell fates and may act as a tumor suppressor in the squamous epithelia. While squamous differentiation is maintained in esophageal squamous cell carcinoma (ESCC) and its precursor lesions, the roles of Notch in esophageal tumor biology remain elusive. Methods: Transformed human esophageal cells expressing EGFR, p53R175H and cyclin D1 (EPC2-T) and ESCC cell lines were stably transduced with ICN1, an active form of NOTCH1 (N1) in a regulatable manner (Tet-On system). Notch was inhibited by dominant negative mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor or gamma-secretase inhibitors (GSI). 8xCSL-luciferase reporter was transiently transfected to assess Notch activity. Short hairpin RNA was stably transduced by lentivirus to knockdown ZEB1 and ZEB2. Squamous epithelium was reconstituted in organotypic (3D) culture, a form of tissue engineering. In addition, cell growth was assessed in soft agar and immunodeficient mice. Quantitative RT-PCR, Western blotting, immunohistochemistry (IHC) and flow cytometry were done to determine gene expression. Primary ESCC tissues (n=20) were analyzed by IHC. Results: In primary ESCC, N1 was localized to well-differentiated tumor cell nests expressing involucrin (IVL), a squamous differentiation marker, while downregulated in poorly-differentiated cells lacking IVL within a single tumor tissue, implying N1 in tumor heterogeneity. In cell lines, ICN1 activated Notch-dependent transcription and squamous differentiation in the absence of DNMAML1. In 3D culture, DNMAML1 and GSI not only blocked squamous differentiation but induced massive invasion and epithelial-to-mesenchymal transition (EMT) with Wnt activation as suggested by loss of E-cadherin and nuclear localization of β-catenin. In agreement, DNMAML1 upregulated LEF1, Zinc finger E-box binding proteins ZEB1 and ZEB2, transcription factors all essential in EMT and maintenance of cancer stem cells, facilitating transforming growth factor (TGF)-β1-induced EMT in EPC2-T cells. Moreover, DNMAML1 enriched a unique subset of CD44-bright and CD24-dim cells and augmented colony formation, tumorigenicity as well as chemotherapeutic drug resistance against Cisplatin. Importantly, Cre-mediated removal of chromosomally integrated floxed DNMAML1 reactivated Notch to allow terminal differentiation with IVL induction. Moreover, ZEB knockdown greatly impaired TGF-β-mediated EMT while restoring chemotherapeutic drug sensitivity. Conclusions: These data indicate that Notch activation promotes squamous differentiation whereas Notch inhibition enriches poorly differentiated tumor cells with cancer stem cell potential with drug resistance, which involves ZEB and Wnt activation, thus providing a novel mechanistic insight into how Notch signaling may contribute to tumor heterogeneity in ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5194. doi:10.1158/1538-7445.AM2011-5194

Abstract 4382: Matricellular protein Cyr61/CCN1 regulates the stemness of oral cancer stem-like cells

Abstract Background: Cysteine-rich 61 (Cyr61 [CCN1]), a matricellular and plasma membrane protein that has disparate function dependent on cell type-specific manner, is closely related to proliferation, invasion, angiogenesis and epithelial-mesenchymal transition (EMT). Besides, EMT promotes stemness properties in normal and neoplastic cells. Therefore, we want to examine whether CCN1 plays a role in our previous enriched oral cancer stem-like cells (OC-SLCs). Methods: Initially, the expression of CCN1 in OC-SLCs translationally was evaluated. Further, the cell surface CCN1 positive cells from oral cancer cells or OC-SLCs were sorted for further study. We will compare the difference of stemness properties and tumorigenicity between CCN1 positive and negative cells. Further, downregulation of CCN1 in oral cancer cells was achieved to evaluate the function of CCN1 on oral cancer tumorigenesis in vitro and in vivo. The molecular mechanisms mediated by CCN1 on regulating the oral cancer tumorigenicity will also be elucidated Results: We found that CCN1 protein was upregulated in OC-SLCs. Comparing to CCN1 negative cells, CCN1 positive cells exhibited higher sphere formation ability and highly expressed stem cell markers, Nanog and Oct-4. Flow cytometry analyses showed that cytoplasmic membrane CCN1 positive OC-SLCs also co-expression with cancer stem-like cell makers, ABCG2, Grp78 and CD133. In addition, CCN1 knockdown cells decreased sphere formation ability and displayed morphological alternation with loss of EMT. Analysis of CCN1 knockdown cells found that EMT related markers were significantly changed. Overall, we demonstrated that CCN1 is involved in maintenance of stemness and acquisition of epithelial-mesenchymal transition in OC-SLCs. Conclusion: Our data suggest that Cyr61 could be a potential oral cancer stem cell marker, and also the key molecule to regulate the stemness of oral cancer stem-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4382. doi:10.1158/1538-7445.AM2011-4382

Abstract 5178: Expression of CD44/CD24 and aldehyde dehydrogenase 1 in resected breast cancer: CD24-positivity predicts early relapse

Abstract Background: CD44+/CD24- or aldehyde dehydrogenase 1 (ALDH1) has been suggested as potential markers for breast cancer stem cells, although little is known about its clinical implications. We evaluated the expression of CD44/CD24 and ALDH1 in breast cancer patients in both ends of the prognostic spectrum. Methods: Of 819 resected breast cancer cohort with positive estrogen receptor, ‘early relapse group’ within 5 years postsurgery and ‘no relapse group’ longer than 10 years postsurgery were defined. Paraffin-embedded tumor tissues were available in 31 early relapsed patients and 68 patients from no relapse group. CD44/CD24 and ALDH1 expression were evaluated with immunohistochemical staining. Positive criteria were designated as more than 15% tumor cell membranous staining for CD44/CD24 and more than 5% tumor cell cytosolic staining for ALDH1. Results: Early relapse group contained higher N stage and higher histologic grade than no relapse group but there were no differences in age, menopauseal status, progesterone receptor status, HER2 status, and T stage between both groups. Phenotypes of CD44/CD24 were CD44+/CD24- in one patient (1%), CD44+/CD24+ in one (1%), CD44-/CD24+ in 12 (12%), and CD44-/CD24- in 67 (68%). Four patients (4%) showed ALDH1-positivity. Due to rarity of CD44-positivity or ALDH1-positivity, staining results were simply dichotomized into CD24-positive (13%, 13/99 patients) and CD24-negative status (87%, 86/99 patients) and only the status of CD24 was further analyzed. CD24-positivity was higher in early relapse group (32%) than in no relapse group (4%). CD24-positivity was associated with negative progesterone receptor (P=0.026), higher N stage (P=0.029), and higher histologic grade (P=0.034). However, in the multivariate logistic regression adjusted by known prognostic factors, CD24-positivity was a still significant predictive factor for early relapse (hazard ratio=8.51; P=0.006). Conclusions: The incidence of CD44+/CD24- and ALDH1-positivity which are potential breast cancer stem cell markers, were low. However, in resected breast cancer, expression of CD24 was a significant predictive factor for early relapse. CD24 is worth further investigation as a novel biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5178. doi:10.1158/1538-7445.AM2011-5178

Abstract 5199: Primary culture of human midgut carcinoid cell lines and isolation of a putative carcinoid stem cell population

Abstract Background: Research in carcinoid and neuroendocrine tumors (NETs) of midgut origin is limited by a lack of cell lines. The existing foregut NET lines, BON and H727, are phenotypically distinct from midgut NETs highlighting the importance of establishing midgut derived NET cell lines. Therapeutic options are limited for midgut NETs. NET cell lines are difficult to culture for long periods (thus few cell lines). We are developing methods to improve primary and prolonged NET cell culture in an attempt to identify novel therapeutic approaches. Along these lines, we are conducting studies to identify a putative cancer stem cell population in human NET specimens to provide new insights regarding potential targets in NETs. Methods: Sterile human midgut NET surgical specimens were digested to a single cell suspension and fibroblast depleted with magnetic beads. The NET cells were maintained on collagen I coated plates for primary culture. A portion of the primary cell culture was used for FACS by the Aldefluor assay to isolate potential cancer stem cells (CSCs). Sorted cells were grown in serum-free media on low attachment plates to assess sphere formation and preserved for RNA isolation and subsequent gene array. Subcutaneous xenografts in nude and NOD/SCID gamma mice using fresh tumor implants and NET cell suspensions were performed as potential means to further propagate NET cells. Results: The NET markers, somatostatin receptor 2 and chromogranin A, were detectable in cultures from a midgut carcinoid liver metastasis at passage 3 and 5, as well as in one mouse xenograft. While retention of NET markers from xenograft implants is promising, tumor growth remains limited and slow, with tumors in 5/59 mice over 4-10 months. We are now studying tumor xenografts in more immunodeficient NOD/SCID gamma mice with current patient samples to potentially enhance xenograft growth. FACS sorting identified an ALDH+ population of NET cells, which formed spheres (a characteristic of CSCs) more frequently than ALDH(−) negative cells. A FACS sorted midgut sample plated at 10k cells/well in sphere forming media produced spheres as early as 8 days in the ALDH+ population, while the ALDH- cells did not form spheres (sham sorted cells formed rare spheres), which was also observed in other samples where viable cells were obtained. Additionally, the ALDH+ cells from this population were cutlured on plastic and remained viable for 4 weeks, while the ALDH- population was not viable in culture. RNA isolation for gene array is underway for the ALDH+ population to characterize these cells in midgut carcinoids. Conclusions: We have developed a system allowing for NET primary culture for up to 4 weeks. We have also identified a putative CSC population in NETs. These techniques are the first step in obtaining cells for in vitro studies and in identifying novel therapeutic targets for NETs. Supported by the The Raymond and Beverly Sackler Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2011-5199

P53 spreads out further: suppression of EMT and stemness by activating miR-200c expression

p53 spreads out further: suppression of EMT and stemness by activating miR-200c expression

The natural products parthenolide and andrographolide exhibit anti-cancer stem cell activity in multiple myeloma

Multiple myeloma (MM) is an incurable plasma cell malignancy where nearly all patients succumb to a relapse. The current preclinical models of MM target the plasma cells, constituting the bulk of the tumor, leaving the cancer stem cells to trigger a relapse. Utilizing a three-dimensional tissue culture system where cells were grown in extracellular matrix designed to reconstruct human bone marrow, we tested the anti-multiple myeloma cancer stem cell (MM-CSC) potential of two natural product inhibitors of nuclear factor κB (NFκB). Here we show that parthenolide and andrographolide are potent anti-MM-CSC agents. Both natural products demonstrated preferential toxicity toward MM-CSCs over non-tumorigenic MM cells. Addition of the bone marrow stromal compartment abrogated andrographolide activity while having no effect on parthenolide cytoxicity. This is the first report of a natural product with anti-CSC activity in myeloma, suggesting that it has the potential to improve the survival of patients with MM by eliminating the relapse-causing MM-CSCs.

Calcineurin Signaling as a Negative Determinant of Keratinocyte Cancer Stem Cell Potential and Carcinogenesis

Calcineurin is the only known serine-threonine phosphatase under calcium-calmodulin control and key regulator of the immune system. Treatment of patients with calcineurin-inhibitory drugs like cyclosporin A and FK506 to prevent graft rejection dramatically increases the risk of cutaneous squamous cell carcinoma, which is a major cause of death after organ transplants. Recent evidence indicates that suppression of calcineurin signaling, together with its impact on the immune system, exerts direct tumor-promoting effects in keratinocytes, enhancing cancer stem cell potential. The underlying mechanism involves interruption of a double negative regulatory axis, whereby calcineurin and nuclear factors of activated T-cell signaling inhibits expression of ATF3, a negative regulator of p53. The resulting suppression of keratinocyte cancer cell senescence is of likely clinical significance for the many patients under treatment with calcineurin inhibitors and may be of relevance for other cancer types in which altered calcium-calcineurin signaling plays a role.

Alternative Lengthening of Telomeres in Human Glioma Stem Cells

Cancer stem cells are increasingly recognized as major therapeutic targets. We report here the isolation of glioma stem cells (GSCs) maintaining telomere length through a telomerase-independent mechanism known as alternative lengthening of telomeres (ALTs). TG20 cells were isolated from a glioblastoma multiforme, which had the ALT phenotype. They have no detectable telomerase activity and extremely long and heterogeneous telomeres colocalizing with promyelocytic leukemia bodies. The cancer stem cell potential of TG20 cells was confirmed based on their expression of neural stem cell markers, their capacity of in vitro long-term proliferation and to form intracranial tumors in immune-deficient mice. Interestingly, we found that both in vitro and in vivo TG20 cells were significantly more resistant to ionizing radiation than GSCs with telomerase activity. Analysis of DNA damage foci, DNA double-strand breaks repair, and chromosome instability suggest that radiation resistance was related to interference of ALT pathway with DNA damage response. Therefore, our data show for the first time that the ALT pathway can confer to cancer stem cells the capacity to sustain long-term proliferation as telomerase activity and importantly may also affect treatment efficiency. TG20 cells are thus the first cellular model of GSCs displaying ALT and should prove to be useful for the development of specific treatment strategies.

Evidence for Epithelial-Mesenchymal Transition in Cancer Stem Cells of Head and Neck Squamous Cell Carcinoma

Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinomas (HNSCC) have been related to the behavior of cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH1) activity. We quantified and enriched ALDH1+ cells within HNSCC cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). Spheroid culture enriched CSC from five HNSCC cell lines by up to 5-fold. In spheroid-derived cells (SDC) and the parental monolayer-derived cell line ALDH1, CD44, CD24, E-Cadherin, α-SMA, and Vimentin expression was compared by flow-cytometry and immunofluorescence together with proliferation and cell cycle analysis. Invasion activity was evaluated by Matrigel assay and expression of stemness-related transcription factors (TF) Nanog, Oct3/4, Sox2 and EMT-related genes Snail1 and 2, and Twist by real-time PCR. All cell lines formed spheroids that could self-renew and be serially re-passaged. ALDH1 expression was significantly higher in SDC. ALDH1+ cells showed increased colony-formation. The proportion of cells with a putative CSC marker constellation of CD44+/CD24− was highly variable (0.5% to 96%) in monolayer and spheroid cultures and overlapped in 0%–33% with the CD44+/CD24−/ALDH1+ cell subset. SDC had significantly higher invading activity. mRNA of the stemness-related genes Sox2, Nanog, and Oct3/4 was significantly increased in SDC of all cell lines. Twist was significantly increased in two while Snail2 showed a significant increase in one and a significant decrease in SDC of two cell lines. SDC had a higher G0 phase proportion, showed high-level expression of α-SMA and Vimentin, but significantly decreased E-Cadherin expression. HNSCC-lines harbor potential CSC, characterized by ALDH1 and stemness marker TF expression as well as properties like invasiveness, quiescence, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.