Cancer Stem Cells Sphere Research Articles

Abstract POSTER-BIOL-1332: Molecular, cellular and clinical interaction between MDSC and ovarian cancer stemness

Abstract Myeloid derived suppressor cells (MDSCs) and cancer stem cells (CSCs) are important cellular components in the cancer microenvironment, and may affect cancer phenotype and patient outcome. The nature of MDSCs and their interaction with CSCs in ovarian carcinoma are unclear. We examined the interaction between MDSCs and CSCs in patients with ovarian carcinoma. We demonstrate that MDSCs inhibit T cell activation, enhance CSC gene expression, sphere formation and cancer metastasis. MDSCs trigger microRNA101 expression in cancer cells; microRNA101 subsequently represses the co-repressor gene C-terminal binding protein-2 (CtBP2), and CtBP2 directly targets stem cell core genes resulting in increased cancer cell stemness, and increasing metastatic and tumorigenic potential. Increased MDSC density and tumor microRNA101 levels, and decreased tumor CtBP2 expression independently predict poor survival. Collectively, the work identifies a novel immune associated cellular, molecular and clinical network involving MDSCs/microRNA101/CtBP2/stem cell core genes, which extrinsically controls cancer stemness and impacts patient outcome. Citation Format: Ilona Kryczek, Tracy X. Cui, Lili Zhao, Linda Vatan, Wojciech Szeliga, Rebecca Liu, Weiping Zou. Molecular, cellular and clinical interaction between MDSC and ovarian cancer stemness [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1332.

Abstract 3117: Hsa-miR-140-5p inhibits colorectal cancer stem cell invasion and metastasis by suppressing Smad2 and autophagy

Abstract Colorectal cancer is one of the most common cancers and frequently metastasizes to the liver. TGF-β signaling has been shown to regulate cancer metastasis by promoting epithelial to mesenchymal transition and cancer stem cell (CSC) phenotypes. Smad2 is a key element downstream of TGF-β. In this study, we showed that hsa-hsa-miR-140-5p-5p directly targets Smad2 and overexpression of hsa-miR-140-5p in colorectal cancer cell lines decreases Smad2 expression level, which leads to decreased level of cell invasion and proliferation, as well as increasing cell cycle arrest. Ectopic expression of hsa-miR-140-5p in colorectal CSCs inhibited CSC growth and sphere formation in vitro by disrupting autophagy, an essential process for cancer cell survival and chemoresistance. We have systematically identified the targets of hsa-miR-140-5p involved in autophagy. Furthermore, overexpression of hsa-miR-140-5p in CSCs abolished the tumor formation as well as metastasis in vivo by using subcutaneous xenografts and tail vein injection of CSCs in NOD/SCID mice. In addition, there is a progressive loss of hsa-miR-140-5p expression from normal colorectal mucosa to primary tumor tissues, with further reduction in liver metastatic tissues, quantified by qRT-PCR. The functional and clinical significance of hsa-miR-140-5p suggests that it is a key regulator in colorectal cancer progression and metastasis. As a result, it may have potential as a novel therapeutic molecule to treat colorectal cancer. Citation Format: Haiyan Zhai, Andrew Fesler, Jingfang Ju. Hsa-miR-140-5p inhibits colorectal cancer stem cell invasion and metastasis by suppressing Smad2 and autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3117. doi:10.1158/1538-7445.AM2015-3117

Abstract A25: WWOX phosphorylation at Ser14 enhances melanoma docking and growth in the lung and liver in mice

Abstract Understanding of the fundamental cell behavior would help develop strategies for cancer stem cell eradication. Metastatic cancer cells tend to have epigenetically inactivated WWOX gene and are highly aggressive. Time-lapse microscopy revealed that they sense the surrounding WWOX-expressing normal or cancer cells, run away from them, and divide rapidly in order to survive. With time of growth and increased space pressure, these cancer cells grow as spheres with the expression of pluripotent stem cell markers such as Oct4, SSEA-4, TRA-1-60, and others. WWOX is re-expressed in the cancer stem cell spheres with S14 phosphorylation (pS14), suggesting that pS14WWOX is a potential cancer stem cell marker. Notably, organs, expressing pS14WWOX, are susceptible for melanoma cell docking and homing in vivo. Suppression of WWOX phosphorylation at Ser14 by micromolar levels of full-length Zfra1-31 or truncated Zfra4-10 prevents melanoma growth in the lung and liver in mice. Zfra stimulates non-T and non-B memory Hyal-2+ CD3- CD19- lymphocytes in the mouse spleens by binding to membrane hyaluronidase Hyal-2 and probably suppressing the TGF-beta/Hyal-2/WWOX/Smad4 signaling. In vitro education of Hyal-2+ CD3- CD19- cells suppresses melanoma growth in vivo. Citation Format: Wan-Pei Su, Wan-Jen Wang, Nan-Shan Chang. WWOX phosphorylation at Ser14 enhances melanoma docking and growth in the lung and liver in mice. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A25.

Inhibition of colorectal cancer stem cell survival and invasive potential by hsa-miR-140-5p mediated suppression of Smad2 and autophagy.

Colorectal cancer (CRC) is the third highest mortality cancer in the United States and frequently metastasizes to liver and lung. Smad2 is a key element downstream of the TGF-β signaling pathway to regulate cancer metastasis by promoting epithelial to mesenchymal transition and maintaining the cancer stem cell (CSC) phenotype. In this study, we show that hsa-miR-140-5p directly targets Smad2 and overexpression of hsa-miR-140-5p in CRC cell lines decreases Smad2 expression levels, leading decreased cell invasion and proliferation, and increasing cell cycle arrest. Ectopic expression of hsa-miR-140-5p in colorectal CSCs inhibited CSC growth and sphere formation in vitro by disrupting autophagy. We have systematically identified targets of hsa-miR-140-5p involved in autophagy. Furthermore, overexpression of hsa-miR-140-5p in CSCs abolished tumor formation and metastasis in vivo. In addition, there is a progressive loss of hsa-miR-140-5p expression from normal colorectal mucosa to primary tumor tissues, with further reduction in liver metastatic tissues. Higher hsa-miR-140 expression is significantly correlated with better survival in stage III and IV colorectal cancer patients.The functional and clinical significance of hsa-miR-140-5p suggests that it is a key regulator in CRC progression and metastasis, and may have potential as a novel therapeutic molecule to treat CRC.

(-)-Liriopein B Suppresses Breast Cancer Progression via Inhibition of Multiple Kinases.

Numerous breast cancer patients who achieve an initial response to HER-targeted therapy rapidly develop resistance within one year, leading to treatment failure. Observations from clinical samples indicate that such resistance correlates with an increase in Src, EGFR, and PI3K/Akt activities and a decrease in PTEN activity. Furthermore, Akt survival signaling activation is also found in tumors treated by toxic chemotherapeutic agents. Because cotreatment with a PI3K inhibitor is a promising strategy to delay acquired resistance by preventing secondary gene activation, we therefore investigated the effects of a newly identified compound, (-)-Liriopein B (LB), on PI3K/Akt signaling activity in breast cancer cells. Our results showed that nontoxic doses of LB are able to inhibit AKT activation in both luminal-like MCF-7 and basal-like MDA-MB-231 breast cancer cells. Low doses of LB also inhibited cell migration, invasion, and cancer-stem cell sphere formation. Suppression of EGF-induced EGFR and ERK1/2 activation by LB might contribute in part to retardation of cancer progression. Furthermore, LB increases sensitivity of MDA-MB-231 cells to gefitinib in vitro, suggesting that EGFR may not be the only target of LB. Finally, a small scale in vitro kinase assay screen demonstrated that LB has a potent inhibitory effect on multiple kinases, including PI3K, Src, EGFR, Tie2, lck, lyn, RTK5, FGFR1, Abl, and Flt. In conclusion, this study demonstrates for the first time that the compound LB improves tumor therapeutic efficacy and suggests LB as a promising candidate for studying new leads in the development of kinase inhibitors.

WWOX Regulation of Cancer Stem Cell Sphere Formation

Time-lapse microscopy revealed that triple negative and WWOX negative breast cancer MDA-MB-231 cells are highly aggressive. They sense the surrounding WWOX-expressing normal or cancer cells, run away from them, and divide rapidly in order to survive. With time of growth and increased space pressure, breast cancer stem cells grow as spheres with the expression of pluripotent stem cell markers such as Oct4, SSEA-4, TRA-1-60, and others. WWOX is re-expressed in the cancer stem cell spheres with S14 phosphorylation (pS14). However pS14 disappears upon release of cells from the spheres. Notably, organs and organ cells, expressing pS14WWOX, are susceptible for cancer cell docking and homing both in vitro and in vivo. A synthetic peptide WWgre, surrounding the S14 area (but without S14 phosphorylation), effectively abolishes the growth of cancer xenografts in nude mice (100% suppression of breast and other cancer) for their entire lifetime, suggesting WWgre eradicates breast cancer stem cells. Scrambled peptides had no blocking effects against breast cancer growth. Together, WWOX plays a critical in controlling the proliferation and differentiation of cancer stem cells. (Supported in part by DoD, USA and MOST and NHRI, Taiwan)

G protein-coupled receptors in stem cell maintenance and somatic reprogramming to pluripotent or cancer stem cells.

G protein-coupled receptors (GPCRs) are a large class of transmembrane receptors categorized into five distinct families: rhodopsin, secretin, adhesion, glutamate, and frizzled. They bind and regulate 80% of all hormones and account for 20-50% of the pharmaceuticals currently on the market. Hundreds of GPCRs integrate and coordinate the functions of individual cells, mediating signaling between various organs. GPCRs are crucial players in tumor progression, adipogenesis, and inflammation. Several studies have also confirmed their central roles in embryonic development and stem cell maintenance. Recently, GPCRs have emerged as key players in the regulation of cell survival, proliferation, migration, and self-renewal in pluripotent (PSCs) and cancer stem cells (CSCs). Our study and other reports have revealed that the expression of many GPCRs is modulated during the generation of induced PSCs (iPSCs) or CSCs as well as during CSC sphere formation. These GPCRs may have crucial roles in the regulation of selfrenewal and other biological properties of iPSCs and CSCs. This review addresses the current understanding of the role of GPCRs in stem cell maintenance and somatic reprogramming to PSCs or CSCs. [BMB Reports 2015; 48(2): 68-80]

Isolation and identification of cancer stem cells from human LACC cell line

To find ways to isolate and culture lacrimal adenoid cystic carcinoma (LACC) stem cells from human LACC cell line and to identify their biological properties. Experimental research. LACC cells were digested, centrifuged and suspended in specific serum free medium (SFM) to get LACC cancer stem cell spheres. Limiting dilution analysis and monoclonal formation assay were performed to determine the proportion of cancer stem cells in LACC cell line. MTT assay was used to determine the proliferation and chemotherapeutic drug resistance of stem cell spheres. The expressions of cell surface markers CD44⁺/CD24⁻ were detected by flow cytometry. Stem cell spheres differentiation were induced by dropping serum medium into SFM and the morphologic changes were observed. The IC₅₀ and UV optical density of two kinds of cells were tested by Student's t-test. About (0.92 ± 0.02) % cells were clonogenic in LACC cell line. They could survive and proliferate to form free-floating cell spheres in SFM, which could stably passaged. The IC₅₀ values of cancer stem cells and LACC cell line were 22.53 mg/L and 11.06 mg/L (t = 37.94, P < 0.05), which suggested that cancer stem cells were more resistant to cis-platinum than LACC cell line. In flow cytometry assay, 84.25% stem cell spheres were CD44⁺/CD24⁻ cells, comparing with 23.77% in LACC cell line. Stem cell spheres could differentiate into normal adenoid cystic carcinoma cells in medium with serum. LACC stem cell spheres, containing a large number of cancer stem cells, could be obtained by serum free suspension culture. This provide us an ideal model system for cancer stem cell research.

Abstract 3027: Stromal cells-derived paracrine factors promote dedifferentiation of human lung carcinoma cells into cancer stem cells

Abstract Tumors are dynamic entities that act like organs in a perfect trading with the entire body. Through the employment of deceiving cellular and molecular strategies, they use the corporal machinery for the promotion of their survival and dissemination. The extensive crosstalk mediated by cytokines and chemokines overcome the inefficiency of the invasion-metastasis cascade, thus allowing the development of the often fatal metastatic disease. As heterogeneous entities, tumors are comprised of distinct cell populations. Cancer stem cells (CSCs) are a subset of notably chemo- and radiotherapy resistant malignant tumor cells with the capacity to drive tumorigenesis and metastases formation and eventually tumor relapse. Lung cancer is a leading cause of death worldwide and its prevalence is increasing mostly due to sustained smoking habits and the accumulation of atmosphere pollutants. In this work hexavalent chromium [Cr(VI)] was the carcinogenic agent used considering its increasing occupational relevance. The non-malignant human bronchial epithelial airway system 2B (BEAS-2B) was malignantly transformed into the RenG2 cell line using low density culture in the presence of Cr(VI). A parallel control cellular system (Cont1) was produced under the same conditions, though, in the absence of Cr(VI). Two additional derivative cell lines, DRenG2 and DDRenG2, were attained following serial rounds of injections in immunocompromised mice. A panoply of techniques was used to characterize the attained cellular systems leading to the hypothesis of CSCs involvement in Cr(VI)-driven BEAS-2B malignant transformation. The sphere-formation assay readily confirmed our hypothesis as CSCs spheres were isolated only from the derivative cell lines (SC-DRenG2 and SC-DDRenG2 cells, respectively). Tumorigenic and RT-qPCR studies further supported the stem potential of the attained systems, suggesting that a dedifferentiation process featured its emergence during RenG2 derivation in nude mice. Using an elegant cell culture model that encompassed the prolonged co-culture of surgically isolated mice lumbar stromal cells with the RenG2 cells, we proved that RenG2 cells’ dedifferentiation was driven by paracrine factors released by the mouse fibroblasts, as they lost the RenG2's molecular signature and acquired a new one more close to that of both DRenG2 and SC-DRenG2. Finally, the key cytokines released by the stromal fibroblasts were identified in the conditioned media of such cultures. CSCs formation boosted the malignant potential of our carcinoma cell population, providing them with increased resistance to therapeutic drugs. Also, the dedifferentiation process revealed to be essentially chemical and specie-unspecific, as human cells were able to dedifferentiate in response to mice cells-released cytokines. Work sponsored by FEDER, POFC-COMPETE and the FCT grants PTDC/BBB-BQB/2450/2012 and SFRH/BD/33884/2009. Citation Format: Carlos F D Rodrigues, Inês P. Rodrigues, Mariana Val, Lina Carvalho, Artur Paiva, Anatoly Zhitkovich, Isabel M. Carreira, Mª Carmen Alpoim. Stromal cells-derived paracrine factors promote dedifferentiation of human lung carcinoma 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 3027. doi:10.1158/1538-7445.AM2014-3027

QKI impairs self-renewal and tumorigenicity of oral cancer cells via repression of SOX2

Cancer stem cells (CSCs) may contribute to tumor initiation, distant metastasis and chemo-resistance. One of RNA-binding proteins, Quaking (QKI), was reported to be a tumor suppressor. Here we showed that reduced QKI levels were observed in many human oral cancer samples. Moreover further reduction of QKI expression in CSCs was detected compared with non-CSCs in oral cancer cell lines. Overexpressing QKI in oral cancer cells significantly reduced CSC sphere formation and stem cell-associated genes. In tumor implanting nude mice model, QKI significantly impeded tumor initiation rates, tumor sizes and lung metastasis rates. As a contrast, knocking down QKI enhanced the above effects. Among the putative CSC target genes, SOX2 expression was negatively affected by QKI, mechanism study revealed that QKI may directly regulate SOX2 expression via specific binding with its 3′UTR in a cis element-dependent way. Loss of SOX2 even completely reversed the sphere forming ability in QKI knockdown cell line. Taken together, these data demonstrated that SOX2 is an important CSC regulator in oral cancer. QKI is a novel CSC inhibitor and impaired multiple oral CSC properties via partial repression of SOX2. Therefore, reduced expression of QKI may provide a novel diagnostic marker for oral cancer.

Hypoxia promotes stem-like properties of laryngeal cancer cell lines by increasing the CD133+ stem cell fraction

Evidence indicates that a hypoxic micro-environment plays an essential role in the regulation of cancer stem cells (CSCs). However, whether hypoxia is able to regulate the stem-like biological properties of laryngeal cancer cells remains unknown. In this study, we investigated the influence of hypoxia on the stemness of two laryngeal cancer cell lines, Hep-2 and AMC-HN-8. We cultured the two cell lines under hypoxia and normoxia and examined the influence of hypoxia on the expression of hypoxia-inducible factors (HIFs) and the cancer stem-like properties of these cells, including cell cycle distribution, expression of stem cell genes (OCT4, SOX2 and NANOG) and laryngeal CSC surface marker (CD133), proliferation, invasion, colony formation and sphere formation capacity. We determined that both of these cell lines, when maintained under hypoxic conditions, showed expanded cells in the G0/G1 phase, exhibited preferential expression of stem cell genes and CD133, and manifested upregulation of HIFs. When treated with hypoxia followed by normoxia exposure, the two cell lines exhibited enhanced capacities for proliferation, invasion, and sphere and colony formation compared with cells maintained consistently under normoxia. Our findings indicate that a hypoxic microenvironment may upgrade the stem-like biological properties of laryngeal cancer cell lines by the expansion of the CD133(+) stem cell fraction.

Global gene expression analysis of canine osteosarcoma stem cells reveals a novel role for COX-2 in tumour initiation.

Osteosarcoma is the most common primary bone tumour of both children and dogs. It is an aggressive tumour in both species with a rapid clinical course leading ultimately to metastasis. In dogs and children distant metastasis occurs in >80% of individuals treated by surgery alone. Both canine and human osteosarcoma has been shown to contain a sub-population of cancer stem cells (CSCs), which may drive tumour growth, recurrence and metastasis, suggesting that naturally occurring canine osteosarcoma could act as a preclinical model for the human disease. Here we report the successful isolation of CSCs from primary canine osteosarcoma, as well as established cell lines. We show that these cells can form tumourspheres, and demonstrate relative resistance to chemotherapy. We demonstrate similar results for the human osteosarcma cell lines, U2OS and SAOS2. Utilizing the Affymetrix canine microarray, we are able to definitively show that there are significant differences in global gene expression profiles of isolated osteosarcoma stem cells and the daughter adherent cells. We identified 13,221 significant differences (p = 0.05), and significantly, COX-2 was expressed 141-fold more in CSC spheres than daughter adherent cells. To study the role of COX-2 expression in CSCs we utilized the COX-2 inhibitors meloxicam and mavacoxib. We found that COX-2 inhibition had no effect on CSC growth, or resistance to chemotherapy. However inhibition of COX-2 in daughter cells prevented sphere formation, indicating a potential significant role for COX-2 in tumour initiation.

Abstract A261: NPV-LDE-225 (Erismodegib) inhibits human prostate cancer stem cell growth in NOD/SCID IL2γnull mice by regulating Bmi-1 and microRNA-128.

Abstract Prostate cancer stem cells (CSCs) are defined by their extensive self-renewal, differentiation, and tumor initiation properties. It is now clear that CSCs are involved in tumor growth and recurrence, and resistance to conventional treatments. Thus, the strategy that suppresses stemness and consequently tumorigenic potential of CSCs could be considered for the management of prostate cancer. The objectives of this study were to examine the molecular mechanisms by which NPV-LDE-225 / Erismodegib (smoothened inhibitor) regulates stem cell characteristics in prostate cancer. Effects of NPV-LDE-225 on CSC's viability, sphere formation, apoptosis, transcriptional activity, and epithelial-mesenchymal transition (EMT) were measured. NPV-LDE-225 inhibited cell viability and spheroid formation, and induced apoptosis by activation of caspase-3 and cleavage of PARP. NPV-LDE-225 induced expression of Bax and Bak, and inhibited the expression of Bcl-2, Bcl-XL, XIAP, cIAP1, cIAP2 and survivin. NPV-LDE-225 inhibited Gli transcriptional activity, Gli-DNA interaction, and the expression of Gli1, Gli2, Patched1 and Patched 2 in prostate CSCs. Interestingly, NPV-LDE-225 induced PDCD4 and apoptosis and inhibited cell viability by suppressing miR-21. Furthermore, NPV-LDE-225 inhibited pluripotency maintaining factors Nanog, Oct4, cMyc and Sox-2. The inhibition of Bmi-1 by NPV-LDE-225 was regulated by up-regulation of miR-128. NPV-LDE-225 suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin, Snail, Slug and Zeb1 through regulating miR-200 family. Finally, NPV-LDE-225 inhibited CSC tumor growth which was associated with the suppression of Gli1, Gli2, Patched-1, Patched-2, Cyclin D1 and PCNA, and cleaved caspase-3 and PARP in tumor tissues derived from NOD/SCID IL2Rϒnull mice. Overall, our findings suggest that inhibition of the Shh signaling pathway in CSCs is a potential therapeutic strategy for prostate cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A261. Citation Format: Rajesh Nanta, Daniel Meeker, Mariana Rodova, Peter J. Van Veldhuizen, Sharmila Shankar, Rakesh K. Srivastava. NPV-LDE-225 (Erismodegib) inhibits human prostate cancer stem cell growth in NOD/SCID IL2γnull mice by regulating Bmi-1 and microRNA-128. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A261.

Myeloid-Derived Suppressor Cells Enhance Stemness of Cancer Cells by Inducing MicroRNA101 and Suppressing the Corepressor CtBP2

Myeloid-derived suppressor cells (MDSCs) and cancer stem cells (CSCs) are important cellular components in the cancer microenvironment and may affect cancer phenotype and patient outcome. The nature of MDSCs and their interaction with CSCs in ovarian carcinoma are unclear. We examined the interaction between MDSCs and CSCs in patients with ovarian carcinoma and showed that MDSCs inhibited Tcell activation and enhanced CSC gene expression, sphere formation, and cancer metastasis. MDSCs triggered miRNA101 expression in cancer cells. miRNA101 subsequently repressesed the corepressor gene C-terminal binding protein-2 (CtBP2), and CtBP2 directly targeted stem cell core genes resulting in increased cancer cell stemness and increasing metastatic and tumorigenic potential. Increased MDSC density and tumor microRNA101 expression predict poor survival, as does decreased tumor CtBP2 expression, independent of each other. Collectively, our work identifies an immune-associated cellular, molecular, and clinical network involving MDSCs-microRNA101-CtBP2-stem cell core genes, which extrinsically controls cancer stemness and impacts patient outcome.

Abstract 3193: NPV-LDE-225 (Erismodegib) inhibits human prostate cancer stem cell growth in NOD/SCID IL2Rγ null mice by regulating Bmi-1 and microRNA-128.

Abstract Prostate cancer stem cells (CSCs) are defined by their extensive self-renewal, differentiation, and tumor initiation properties. It is now clear that CSCs are involved in tumor growth and recurrence, and resistance to conventional treatments. Thus, the strategy that suppresses stemness and consequently tumorigenic potential of CSCs could be considered for the management of prostate cancer. The objectives of this study were to examine the molecular mechanisms by which NPV-LDE-225 / Erismodegib (smoothened inhibitor) regulates stem cell characteristics in prostate cancer. Effects of NPV-LDE-225 on CSC's viability, sphere formation, apoptosis, transcriptional activity, and epithelial-mesenchymal transition (EMT) were measured. NPV-LDE-225 inhibited cell viability and spheroid formation, and induced apoptosis by activation of caspase-3 and cleavage of PARP. NPV-LDE-225 induced expression of Bax and Bak, and inhibited the expression of Bcl-2, Bcl-XL, XIAP, cIAP1, cIAP2 and survivin. NPV-LDE-225 inhibited Gli transcriptional activity, Gli-DNA interaction, and the expression of Gli1, Gli2, Patched1 and Patched 2 in prostate CSCs. Interestingly, NPV-LDE-225 induced PDCD4 and apoptosis and inhibited cell viability by suppressing miR-21. Furthermore, NPV-LDE-225 inhibited pluripotency maintaining factors Nanog, Oct4, cMyc and Sox-2. The inhibition of Bmi-1 by NPV-LDE-225 was regulated by up-regulation of miR-128. NPV-LDE-225 suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin, Snail, Slug and Zeb1 through regulating miR-200 family. Finally, NPV-LDE-225 inhibited CSC tumor growth which was associated with the suppression of Gli1, Gli2, Patched-1, Patched-2, Cyclin D1 and PCNA, and cleaved caspase-3 and PARP in tumor tissues derived from NOD/SCID IL2Rγ null mice. Overall, our findings suggest that inhibition of the Shh signaling pathway in CSCs is a potential therapeutic strategy for prostate cancer. Citation Format: Rajesh Nanta, Sharmila Shankar, Rakesh K. Srivastava. NPV-LDE-225 (Erismodegib) inhibits human prostate cancer stem cell growth in NOD/SCID IL2Rγ null mice by regulating Bmi-1 and microRNA-128. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3193. doi:10.1158/1538-7445.AM2013-3193

Abstract 3738: Knockdown of Annexin A2 by liposomal formulation of Annexin A2 small hairpin RNA induces apoptosis and inhibits angiogenesis in lung cancer stem cells.

Abstract Recent studies support the existence of small populations of pluripotent stem-like cancer cells capable of self-renewal. These cancer stem cells (CSCs) are attributed to the resistance phenotype occurring in many tumors such as invasion and metastasis. Annexin A2 (Anxa2) overexpression is associated with invasion, metastasis and drug resistance in many cancers. We investigated the effect of Anxa2 knockdown by a liposomal (nanosome) formulation of Anxa2 shRNA (shAnxa2) on growth and metastasis in H1650 CSCs. H1650 CSCs were maintained on basement membrane-coated supports in DMEM:F12 media enriched with growth factors. Nanosomes were prepared by solvent evaporation and bath sonication. H1650 CSCs were transfected with lipoplexes of shAnxa2 and varying amounts of nanosome in serum-free media. After 4 hrs of incubation, the medium was replaced with regular media and knockdown of Anxa2 analyzed after 48 hrs by Western blot. Inhibition of H1650 CSC sphere formation over 7 days was investigated in lipidure® plates following treatment with lipoplexes for 4 hrs. CSCs were assessed for induction of apoptosis after treatment with lipoplexes (4 hrs) by acridine orange-ethidium bromide staining. The effect of Anxa2 lipoplexes on proliferation, migration and capillary-like tube formation was investigated in human primary pulmonary artery endothelial cells (HPAEC). The effect of Anxa2 lipoplexes on the expression of Anxa2, EGFR, VEGF, and NFkβ was evaluated by Western blot. Efficient loading of shAnxa2 in nanosomes was achieved with high transfection efficiency compared to lipofectamine. Anxa2 shRNA lipoplex was cytotoxic to H1650 CSC spheroids, inhibiting spheroid growth by 30-60% between day 3 and 7. Early and late apoptosis induction was observed after treatment with shAnxa2 with a 2-fold increase of cells undergoing apoptosis compared to shAnxa2-lipofectamine 2000 complex. Anxa2 knockdown (90%) was observed in H1650 CSCs parallel to decreases in EGFR, VEGF, and NFkβ expression as shown by Western blot. High loading and enhanced transfection of shAnxa2 was achieved with the nanosome formulation. Anxa2 knockdown was achieved in parallel with inhibition of H1650 CSC spheroid growth, angiogenesis and induction of apoptosis. These results altogether support evidence that Anxa2 could be a therapeutic target for treatment of non-small cell lung cancer and that nanosome formulation enhances delivery of shAnxa2 as an anticancer agent. Citation Format: Terrick A. Andey, Srujan Marepally, Pomila Singh, Mandip S. Sachdeva. Knockdown of Annexin A2 by liposomal formulation of Annexin A2 small hairpin RNA induces apoptosis and inhibits angiogenesis in lung cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3738. doi:10.1158/1538-7445.AM2013-3738

NVP-LDE-225 (Erismodegib) inhibits epithelial–mesenchymal transition and human prostate cancer stem cell growth in NOD/SCID IL2Rγ null mice by regulating Bmi-1 and microRNA-128

Prostate cancer stem cells (CSCs) are defined by their extensive self-renewal, differentiation and tumor initiation properties. It is now clear that CSCs are involved in tumor growth and recurrence, and resistance to conventional treatments. The sonic hedgehog (Shh) pathway has a crucial role in stemness and tumorigenesis. Thus, the strategy that suppresses stemness and consequently tumorigenic potential of CSCs could be considered for the management of prostate cancer. The objectives of this study were to examine the molecular mechanisms, by which NVP-LDE-225/Erismodegib (smoothened inhibitor) regulates stem cell characteristics and tumor growth in prostate cancer. The effects of NVP-LDE-225 on CSC's viability, sphere formation, apoptosis, epithelial–mesenchymal transition (EMT) and tumor growth in NOD/SCID IL2Rγ null mice were examined. NVP-LDE-225 inhibited cell viability and spheroid formation, and induced apoptosis by activation of caspase-3 and cleavage of poly-ADP ribose polymerase (PARP). NVP-LDE-225 induced expression of Bax and Bak, and inhibited the expression of Bcl-2, Bcl-XL, XIAP, cIAP1, cIAP2 and survivin. NVP-LDE-225 inhibited Gli transcriptional activity, Gli-DNA interaction and the expression of Gli1, Gli2, Patched1 and Patched-2 in prostate CSCs. Interestingly, NVP-LDE-225 induced PDCD4 and apoptosis and inhibited cell viability by suppressing miR-21. Furthermore, NVP-LDE-225 inhibited pluripotency-maintaining factors Nanog, Oct-4, c-Myc and Sox-2. The inhibition of Bmi-1 by NVP-LDE-225 was regulated by upregulation of miR-128. NVP-LDE-225 suppressed EMT by upregulating E-cadherin and inhibiting N-cadherin, Snail, Slug and Zeb1 by regulating the miR-200 family. Finally, NVP-LDE-225 inhibited CSC tumor growth, which was associated with the suppression of Gli1, Gli2, Patched-1, Patched-2, Cyclin D1, Bmi-1 and PCNA and cleavage of caspase-3 and PARP in tumor tissues derived from NOD/SCID IL2Rγ null mice. Overall, our findings suggest that inhibition of the Shh signaling pathway could therefore be a novel therapeutic option in treating prostate cancer.

Prognostic value of different amounts of cancer stem cells in different molecular subtypes of breast cancer.

To investigate the differences in the amount of cancer stem cells in different breast cancer subtypes. THE SPECIMENS OF BREAST CANCER TISSUES WERE DIVIDED INTO FIVE GROUPS: group A (luminal A type), B (luminal B type), C (HER-2 + type), D (basal-like type) and E (normal-like type) according to their molecular subtypes. The cancer stem cell spheres in each group were measured through colony formation method, and then the relations of the amount of cancer stem cells with the subtypes of breast cancer were analyzed. The numbers of cancer stem cell spheres in group A and B were (1.1±0.2)/1,000 cells and (1.3±0.1)/1,000 cells, respectively, and this had no significant difference (P>0.05). The number of cancer stem cell spheres in group C was (8.6±1.0)/1,000 cells, which was significantly higher than that in group A and B (both P<0.05). The numbers of cancer stem cell spheres in group D and E were (22.4±1.2)/1,000 cells and (17.7±2.0)/1,000 cells, respectively, and this was statistically similar (P>0.05) but either was significantly higher than that in the other three groups (all P<0.05). Breast cancer tissues of different molecular subtypes contain varying amounts of cancer stem cells, and this may have certain significance for predicting the prognosis of breast cancer.

Proteome of human colon cancer stem cells: A comparative analysis

To isolate and identify the biological characteristics of human colon cancer stem cells (SW1116 cells) and further study their proteome. SW1116 cells were isolated and cultured with a serum-free medium (SFM). Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SW1116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction (PCR)-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis (2-DE) and differentially expressed proteins were identified by tandem mass spectrometry (MALDI-TOF/TOF). The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusion-degradation 1-like protein, nuclear chloride channel protein, tubulin β, Raichu404X, stratifin, F-actin capping protein α-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein β polypeptide 2-like 1, respectively. SW1116 cells are biologically characterized by self-renewal, proliferation and differentiation, and the differently expressed proteins in SW1116 cells may be essential for isolating cancer stem cells.

Cytoplasmic accumulation of connexin32 expands cancer stem cell population in human HuH7 hepatoma cells by enhancing its self‐renewal

Although the connexin32 (Cx32)-mediated gap junction is abolished in hepatocellular carcinoma (HCC), the expression of cytoplasmic Cx32 tends to increase in correspondence with the grade of malignancy. Establishing a Tet-off expression system in human nonmetastatic HuH7 HCC cells where cytoplasmic Cx32 was overexpressed by doxycycline (Dox) withdrawal, we previously demonstrated that overexpression of cytoplasmic Cx32 made HuH7 cells metastatic in mice. In our study, hypothesizing that the cytoplasmic Cx32-induced metastasis may involve expansion of the cancer stem cell (CSC) population, we examined whether cytoplasmic Cx32 controlled the size of the side population (SP) in HuH7 Tet-off Cx32 cells. Fluorescence-activated cell sorting revealed that SP was expanded in a Dox-free medium compared with a Dox-supplemented one. Although cytoplasmic Cx32 did not block maturation from SP to non-SP, purified SP reconstituted a larger SP fraction in the Dox-free medium than in the Dox-supplemented one. Furthermore, although SP from HuH7 Tet-off mock cells formed a similar number of CSC spheres of a similar size whether with or without Dox, SP from HuH7 Tet-off Cx32 cells developed a greater number of larger CSC spheres in the Dox-free medium than in the Dox-supplemented one. Taken together, these results suggest that accumulation of cytoplasmic Cx32 should enhance self-renewal of CSC to expand the CSC population in HCC.