Inhibition Of Nanog Research Articles

Limonin inhibits the stemness of cancer stem-like cells derived from colorectal carcinoma cells potentially via blocking STAT3 signaling.

Limonin is one of the most abundant active ingredients of Tetradium ruticarpum. It exerts antitumor effects on several kinds of cancer cells. However, whether limonin exerts antitumor effects on colorectal cancer (CRC) cells and cancer stem-like cells (CSCs), a subpopulation responsible for a poor prognosis, is unclear. To evaluate the effects of limonin on CSCs derived from CRC cells. CSCs were collected by culturing CRC cells in serum-free medium. The cytotoxicity of limonin against CSCs and parental cells (PCs) was determined by cholecystokinin octapeptide-8 assay. The effects of limonin on stemness were detected by measuring stemness hallmarks and sphere formation ability. As expected, limonin exerted inhibitory effects on CRC cell behaviors, including cell proliferation, migration, invasion, colony formation and tumor formation in soft agar. A relatively low concentration of limonin decreased the expression stemness hallmarks, including Nanog and β-catenin, the proportion of aldehyde dehydrogenase 1-positive CSCs, and the sphere formation rate, indicating that limonin inhibits stemness without presenting cytotoxicity. Additionally, limonin treatment inhibited invasion and tumor formation in soft agar and in nude mice. Moreover, limonin treatment significantly inhibited the phosphorylation of STAT3 at Y705 but not S727 and did not affect total STAT3 expression. Inhibition of Nanog and β-catenin expression and sphere formation by limonin was obviously reversed by pretreatment with 2 μmol/L colievlin. Taken together, these results indicate that limonin is a promising compound that targets CSCs and could be used to combat CRC recurrence and metastasis.

Abstract 2616: Stemness factors nanog and oct4 contribute to epithelial-to-mesenchymal transition and are predictive for outcome in esophageal adenocarcinoma

Abstract Background and Aims: The incidence of esophageal adenocarcinoma (EAC) has increased six-fold in Western countries over the last decades, and 5-year survival rates remain low at 5-20%. While multimodality treatment strategies for curative treatment of esophageal cancer, including the CROSS regimen (chemoradiotherapy followed by surgery) have increased median overall survival, the majority of patients develop recurrences after several months. Epithelial to mesenchymal transition (EMT) has been recently shown by our group to be one of the major underlying mechanisms of resistance to therapy. Paradoxically, therapeutic pressure of effective therapies such as the CROSS regimen are found to instruct a mesenchymal, resistant phenotype in models for EAC. In this study, the aim was to delineate the heterogeneity for the propensity to undergo EMT after chemoradiation and which mechanisms underpin this propensity. Methods: A panel of 8 EAC cell lines (5 primary and 3 ATCC cell lines) were treated with chemoradiotherapy and ranked by their propensity to undergo EMT, based on morphology when EMT occurred and protein marker expression. Next, the cell line panel as well as 44 pre-treated esophageal biopsies were RNA-sequenced. Expression data of the cell line panel were linked to their ranked in vitro EMT response. By means of Leave-one-out cross validation with Ridge Regression, EMT score prediction in pre-treated biopsies was validated. Gene expression profiles were related to clinical outcome data to identify markers that associated with propensity for EMT in patients. Results: In the panel of in vitro EAC models, a strong heterogeneity was observed for the propensity to EMT after chemoradiation. For each marker, Ridge regression analysis identified the top 50 highly correlating genes. Combining all positively correlating genes of days to EMT, NCAD and ZEB1, known key transcription factors of pluripotency including NANOG and OCT4 emerged. Expression of NANOG and OCT4 in pre-treatment biopsies was highly predictive for response to neoadjuvant chemoradiation, occurrence of recurrences, and survival in patients. Genetic perturbation by knockout and inhibition of NANOG and OCT4 reduced the onset of EMT and sensitized cells for chemoradiation. Conclusions: In conclusion, we were able to identify patients who are disproportionally prone to develop EMT in response to chemoradiation. Moreover, stemness factors NANOG and OCT4 are crucial regulators in plasticity of EAC and are promising predictive markers in pre-treatment biopsies of patients. By targeting NANOG and OCT4 in vitro, cells were sensitized to chemoradiation, holding promise for stemness inhibition to prevent therapy resistance in EAC. Citation Format: Amber Perenna van der Zalm, Mark P. Dings, Reimer Janssen, Peter Bailey, Jan Koster, Danny Zwijnenburg, Richard Volckmann, Cynthia Waasdorp, Jeroen Blokhuis, César Oyarce, Gerrit Hooijer, Sybren L. Meijer, Jan Paul Medema, Hanneke W. van Laarhoven, Maarten F. Bijlsma. Stemness factors nanog and oct4 contribute to epithelial-to-mesenchymal transition and are predictive for outcome in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2616.

FOXC1 Downregulates Nanog Expression by Recruiting HDAC2 to Its Promoter in F9 Cells Treated by Retinoic Acid.

FOXC1, a transcription factor involved in cell differentiation and embryogenesis, is demonstrated to be a negative regulator of Nanog in this study. FOXC1 is up-regulated in retinoic acid-induced differentiation of F9 Embryonal Carcinoma (EC) cells; furthermore, FOXC1 specifically inhibits the core pluripotency factor Nanog by binding to the proximal promoter. Overexpression of FOXC1 in F9 or knockdown in 3T3 results in the down-regulation or up-regulation of Nanog mRNA and proteins, respectively. In order to explain the mechanism by which FOXC1 inhibits Nanog expression, we identified the co-repressor HDAC2 from the FOXC1 interactome. FOXC1 recruits HDAC2 to Nanog promoter to decrease H3K27ac enrichment, resulting in transcription inhibition of Nanog. To the best of our knowledge, this is the first report that FOXC1 is involved in the epigenetic regulation of gene expression.

Relation between Immune cell response and stemness genes expression in breast cancer; A new approach in NANOG gene and Let7-a expression in breast cancer cell lines

Introduction: Failure and recurrence in breast cancer treatment cause a great obstacle in cancer therapy and identification of cell population named cancer stem cells (CSCs) in the tumor can be led us to define it as target in novel therapeutic strategy. Objectives: The aim of this study is the finding of correlation between stemness and metastatic characteristic, also knowing CSCs as a potential target of therapy because of its developmental behavior and similarities with normal stem cells. Materials and Methods: Here, we focus on the expression of NANOG in breast CSCs, a key molecule in the physiological process of stem cells and the Let-7a that is involved in the differentiation of the cells. Results: In this work, we found that NANOG was highly expressed in SKBR3 and down-regulation of let-7a, as a differentiation miRNA, was found in MDA-MB-468 cells. Conclusion: It will be critical for the developing of effective anti-tumor drugs, utilizing mentioned concepts. Inhibition of NANOG in combination with Let-7a up-regulation can help to decrease the stemness and increase the differentiation of CSCs. The decrease of stemness and increase of differentiation initiate the apoptotic process. So, modification in the mechanism of apoptosis beside anti-cancer drugs provide a good preclinical study goal. However, in order to these drugs become clinical, the problems of their side effects and toxicity must be solved. Differentiation of CSCs provides an optimal condition to activity of immune cells which never let them escape from immune cells by alteration of immunogenicity.

A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG

BackgroundNANOG functions as the gateway for the generation of pluripotent stem cells (PSCs) in mice and humans. NANOG is a transcription factor highly expressed in pig pre-implantation embryos, indicating that it is a conserved pluripotency-associated factor. However, pig NANOG reporter PSCs have yet to be established, and the regulation of pluripotency by NANOG is not fully understood in this animal.MethodsIn this study, pig NANOG tdTomato knock-in reporter positive PC-iPS cells were established using CRISPR/Cas9. The resulting cell line was treated with several cytokines and their corresponding inhibitors to identify pathways that regulate NANOG expression. The pathways examined were LIF (leukemia inhibitory factor)/IL6 (interleukin 6)-STAT3, FGF (fibroblast growth factor)/ERK, IGF1 (insulin-like growth factor 1)/PIP3 (phosphoinositide 3-kinase)-AKT, Activin A/SMAD, and BMP4 (bone morphogenetic proteins)/SMAD.ResultsOur experiments showed that the Activin A/SMAD pathway is directly associated with activation of NANOG expression in the pig, as is also the case in mice and humans. Activin A directly regulates the expression of pig NANOG via SMAD2/3; inhibition of this pathway by SB431542 resulted in inhibition of NANOG expression.ConclusionsOur results show that Activin A plays an important regulatory role in NANOG-mediated pluripotency in pig iPS cells. Activin A treatment may be therefore an effective method for de novo derivation of authentic embryonic stem cells (ESCs) from pig pre-implantation embryos.

Abstract 2335: Targeting the DNA repair as well as survival functions of MCL-1 enhances cancer cell killing

Abstract MCL-1 is a a pro-survival BCL2 protein family member which is over-expressed in drug resistant cancer cells. Our purpose was to assess whether MCL-1 increases genomic instability along with its anti-apoptotic function in cancer cells. Depletion of MCL-1 by treating human small cell lung cancer (SCLC) lines with immunotoxin sensitizes these cells to increased killing by BH3 mimetic - Navitoclax (ABT-263) both in vitro and in mice xenograft models. These SCLC cell lines are resistant to both single agent Immunotoxin or ABT-263. In addition, inhibition of NANOG or NANOGP8 by lentivrus delivered shRNA depletes MCL-1 in human colorectal cancer cells and sensitizes these cells to increased cell killing by venetaclox (ABT-199). This increased killing depended on loss of MCL-1, was caspase-dependent and reversed by re-expression of MCL-1. Depletion of MCL-1 in cancer cells , increases cell sensitivity to ionizing radiation (IR) induced death, which was reversed by expression of MCL-1. In response to IR, MCL-1 depleted cells showed reduced survival even at radiation doses less than 6 Gy, however an increase in caspases 3/7 activity was observed at 6 Gy and above, suggesting that apoptosis is activated only when MCL-1 depleted cells are irradiated with higher IR doses. In order to explain the decreased survival post- irradiation the genomic instability and DNA repair pathways were analyzed in MCL-1 depleted cells. Post-irradiation MCL-1 depleted cells exhibited increased genomic instability as measured by increase in chromosome aberrations at different phases of the cell cycle. The increase in aberrations were significantly higher in G2 and S- phases of the cell cycle suggesting defect in Homologous recombination repair pathway (HR). Moreover, the MCL-1 depleted cells show decreased gamma-H2AX foci at earlier time points (30 and 90 minutes) post-irradiation indicating defect in DNA Damage response (DDR) and higher residual foci at 360 minutes, indicating defective DNA repair. These observations were further confirmed by decreased phosphorylation of ATR suggesting defect in DNA Damage response and higher levels of residual 53BP1 and RIF1 foci in MCL-1 depleted cells , confirming DNA DSB repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1 depleted cells exhibited a reduced frequency of IR-induced MRE11, BRCA1, RPA and Rad51 foci formation, decreased DNA end resection and decreased HR repair in the DR-GFP DSB repair model. Similarly, after Hydoxyurea (HU) induction of stalled replication forks in MCL-1 depleted cells there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR mediated resolution of collapsed forks. In summary, MCL-1 is an important therapeutic target in cancer cells and its depletion increases cell killing by either increase in apoptosis or suppression of HR and increased replication stress. Citation Format: Abid Mattoo, J Milburn Milburn Jessup, Tej K. Pandita. Targeting the DNA repair as well as survival functions of MCL-1 enhances cancer cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2335. doi:10.1158/1538-7445.AM2017-2335

Carboxyamidotriazole Synergizes with Sorafenib to Combat Non-Small Cell Lung Cancer through Inhibition of NANOG and Aggravation of Apoptosis.

Lung cancer is currently the leading cause of cancer-related deaths worldwide. In this study, we investigated the combination of carboxyamidotriazole (CAI) and sorafenib in non-small cell lung cancer (NSCLC) in vitro and in vivo to test whether CAI enhances the antitumor effects of sorafenib and reduces its side effects. The combination index (CI) showed that coadministration of CAI and sorafenib synergistically inhibited the proliferation of NSCLC cells (Lewis lung carcinoma, A549, and NCI-H1975 cells). Cell death as a result of the combination treatment was attributed to apoptosis, which was accompanied by activation of caspase-3 and poly(ADP-ribose) polymerase. In addition, combination therapy induced the accumulation of mitochondrial-associated reactive oxygen species, as well as depolarization of mitochondrial and reduced NANOG (homeobox protein NANOG) mRNA and protein expression. Basic fibroblast growth factor, a stimulator of NANOG, was applied to identify the possible mechanism. The addition of basic fibroblast growth factor followed by combined treatment may stimulate NANOG expression and synchronously rescue the accumulation of reactive oxygen species. C57BL/6J mice bearing Lewis lung carcinoma were randomized to receive vehicle (polyethylene glycol 400), CAI (30 mg/kg), low-dose sorafenib (SFB-L; 10 mg/kg), high-dose sorafenib (SFB-H; 30 mg/kg), or a CAI and SFB-L combination. Tumor growth was significantly suppressed in the combination group, and the efficacy of combination treatment was equivalent to that of the SFB-H monotherapy group. Furthermore, the combination group had reduced side effects compared with the SFB-H group, as indicated by weight preservation in mice. Our study illustrates that CAI enhances the antitumor activity of sorafenib in NSCLC and provides a novel strategy for NSCLC treatment.

Lentivirus-mediated shRNA targeting Nanog inhibits cell proliferation and attenuates cancer stem cell activities in breast cancer

Emerging evidences suggest that cancer stem cells (CSCs) are responsible for tumor growth, metastasis and treatment resistance. Nanog is one of the transcription factors that are essential for stem cellular physiology process. Previous studies reported that Nanog was detected in breast cancer and other solid tumors and indicated that it has oncogenic characteristics. However, expression feature of Nanog in breast cancer stem cells (BCSCs) enriched population and its biological function in BCSCs is poorly understood. In this study, CD44 + CD24− fraction sorting with Fluorescence Activated Cell Sorter and mammosphere culture were used for enriching BCSCs. We report here that Nanog was highly expressed in CSCs-enriched population from the breast cancer cells, as well as stemness-associated genes. In addition, we employed the lentivirus-mediated shRNA targeting Nanog to investigate function of Nanog in BCSCs. We found that targeted inhibition of Nanog could suppress proliferation and colony formation in breast cancer cells. Further studies showed that targeted inhibition of Nanog resulted in a decrease of BCSCs activities, including mammosphere formation, CD44 + CD24− proportion and expressions of stemness-associated genes. These data therefore suggest that Nanog possesses important function in BCSCs and targeted inhibition of Nanog may provide a novel means of targeting and eliminating BCSCs.

Inhibition of NANOG/NANOGP8 downregulates MCL-1 in colorectal cancer cells and enhances the therapeutic efficacy of BH3 mimetics.

High levels of BCL-2 family members in colorectal carcinoma cause resistance to treatment. Inhibition of NANOG or its paralog NANOGP8 reduces the proliferation, stemness, and tumorigenicity of colorectal carcinoma cells. Our hypothesis was that inhibition of NANOG/NANOGP8 enhances the cytotoxic effect of BH3 mimetics targeting BCL-2 family members in colorectal carcinoma cells through reducing expression of MCL-1, a prosurvival BCL-2 protein. Lentiviral vector (LV) shRNA to NANOG (shNG-1) or NANOGP8 (shNp8-1) transduced colorectal carcinoma cells that were also exposed to the BH3 mimetics ABT-737 or ABT-199 in vivo in colorectal carcinoma xenografts and in vitro where proliferation, protein and gene expression, and apoptosis were measured. Clone A and CX-1 were sensitive to ABT-737 and ABT-199 at IC50s of 2 to 9 μmol/L but LS174T was resistant with IC50s of 18 to 30 μmol/L. Resistance was associated with high MCL-1 expression in LS174T. LVshNG-1 or LVshNp8-1 decreased MCL-1 expression, increased apoptosis, and decreased replating efficiency in colorectal carcinoma cells treated with either ABT-737 or ABT-199 compared with the effects of either BH3 mimetic alone. Inhibition or overexpression of MCL-1 alone replicated the effects of LVshNG-1 or LVshNp8-1 in increasing or decreasing the apoptosis caused with the BH3 mimetic. The combination therapy inhibited the growth of LS174T xenografts in vivo compared with untreated controls or treatment with only LV shRNA or ABT-737. Inhibition of NANOGP8 or NANOG enhances the cytotoxicity of BH3 mimetics that target BCL-2 family members. Gene therapy targeting the NANOGs may increase the efficacy of BH3 mimetics in colorectal carcinoma.

Abstract 339: Inhibition of NANOG/NANOGP8 downregulates MCL-1 in colorectal cancer (CRC) cells, enhances the efficacy of BH3 mimetics and inhibits clonogenicity

Abstract Combining lentiviral (LV) delivered shRNA against NANOG (shNG-1) or NANOGP8 (shNp8-1) with BH3 mimetics (ABT-737 and ABT-199) was undertaken to determine whether inhibition of NANOG/NANOGP8 enhances the cytotoxic effect of these BH3 mimetics in Colorectal Cancer (CRC) cells. NANOG is a key transcription factor important for both pluripotency in embryonic stem cells and malignant transformation and progression of colorectal carcinoma (CRC). Inhibition of NANOG or its paralog NANOGP8 reduces the proliferation, stemness and tumorigenicity of CRC cells. The cytotoxic potential of ABT-737 or ABT-199, as single agents, was tested in the CRC cell lines- Clone A, CX-1 and LS174T that have high levels of anti-apoptotic BCL-2 family proteins. Treatment with ABT-737 or ABT-199 showed variable cytotoxicity in the three CRC lines with LS174T being the most resistant. Inhibition of NANOG and NANOGP8 with LVshNG-1 or LVshNp8-1 in these cell lines decreased expression of MCL-1. The combination of LVshNG-1 and/or LVshNp8-1, with ABT-737 produced enhanced killing of the CRC cells by caspase-dependent apoptosis. These results were were confirmed when LV shNG-1 or LV shNp8-1 was used in combination with ABT-199. siRNA targeting MCL-1 reproduced the effects of NANOG/NANOGP8 inhibition. The CRC cells that survived the combination treatment showed lower regrowth potential and reduced clonogenicity when re-plated in fresh media. These results demonstrate that inhibition of NANOGP8 or NANOG enhances the cytotoxicity of BH3 mimetics that target BCL-2 family members through inhibition of MCL-1 expression. Citation Format: Abid R. Mattoo, Jingyu Zhang, Luis A. Espinoza, Snorri S. Thorgeirsson, J. M. Jessup. Inhibition of NANOG/NANOGP8 downregulates MCL-1 in colorectal cancer (CRC) cells, enhances the efficacy of BH3 mimetics and inhibits clonogenicity. [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 339. doi:10.1158/1538-7445.AM2014-339

Abstract 200: EGCG inhibits pancreatic cancer stem cell characteristics in human primary tumors, and carcinogenesis in KrasG12D transgenic mice.

Abstract EGCG, a component of green tea, exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines through diverse mechanisms. However, the mechanisms by which EGCG inhibits cell proliferation and induces apoptosis are poorly understood. The objectives of this study were to examine the molecular mechanisms by which EGCG inhibits stem cell characteristics of pancreatic cancer stem cells (CSCs) derived from human primary tumors and carcinogenesis in KrasG12D transgenic mice. Human pancreatic CSCs (CD133+CD44+CD24+ESA+) are highly tumorigenic and form subcutaneous tumors in NOD/SCID IL2R gamma mice. Human pancreatic CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic cancer cells. Similarly, CSCs from KrasG12D mice express significantly high levels of pluripotency maintaining factors than pancreatic tissues from Pdx-Cre mice. EGCG inhibits the growth (size and weight) and development (PanIN lesions) of pancreatic cancer in KrasG12D mice by suppressing the activation of Kras, ERK, AKT and FoxO. EGCG inhibits the self-renewal capacity of pancreatic CSCs derived from human primary tumors and KrasG12D mice. EGCG inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhances the inhibitory effects of EGCG on self-renewal capacity of CSCs. Finally, EGCG inhibits CSC's migration and invasion and markers of epithelial-mesenchymal transition (Zeb-1, Slug and Snail). These data suggest that EGCG can inhibit pancreatic cancer development in human and KrasG12D transgenic mice by inhibiting stem cell characteristics. In conclusion, EGCG can be used for the management of pancreatic cancer. Citation Format: Luke Marsh, Su-Ni Tang, Dara Nall, Chris Jackman, Sharmila Shankar, Rakesh K. Srivastava. EGCG inhibits pancreatic cancer stem cell characteristics in human primary tumors, and carcinogenesis in KrasG12D transgenic mice. [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 200. doi:10.1158/1538-7445.AM2013-200

Resveratrol promotes proteasome-dependent degradation of Nanog via p53 activation and induces differentiation of glioma stem cells

Glioblastoma is the most common and aggressive primary brain tumor. Glioma stem cells (GSCs) are relatively resistant to chemo-radiotherapy and are responsible for tumor progression and the recurrence of glioblastomas after conventional therapy. Thus, the control of the GSC population is considered key to realizing long-term survival of glioblastoma patients. Here, we identified that resveratrol significantly reduced the self-renewal and tumor-initiating capacity of patient-derived GSCs. Furthermore, resveratrol promoted Nanog suppression via proteasomal degradation, which was inhibited by MG132, a proteasome inhibitor. p53 activation is an important factor in Nanog suppression and treatment with resveratrol was also found to activate the p53/p21 pathway. Importantly, inhibition of Nanog by siRNA provoked inhibitory effects on both the self-renewal and tumor-forming capacity of GSCs. Our findings indicate that Nanog is an essential factor for the retention of stemness and may contribute to the resveratrol-induced differentiation of GSCs. Our results also suggest that targeting GSCs via the p53–Nanog axis, with resveratrol for instance, could be a therapeutic strategy against glioblastoma.

Nanog signaling in cancer promotes stem-like phenotype and immune evasion

Adaptation of tumor cells to the host is a major cause of cancer progression, failure of therapy, and ultimately death. Immune selection drives this adaptation in human cancer by enriching tumor cells with a cancer stem cell-like (CSC-like) phenotype that makes them resistant to CTL-mediated apoptosis; however, the mechanisms that mediate CSC maintenance and proliferation are largely unknown. Here, we report that CTL-mediated immune selection drives the evolution of tumor cells toward a CSC-like phenotype and that the CSC-like phenotype arises through the Akt signaling pathway via transcriptional induction of Tcl1a by Nanog. Furthermore, we found that hyperactivation of the Nanog/Tcl1a/Akt signaling axis was conserved across multiple types of human cancer. Inhibition of Nanog in a murine model of colon cancer rendered tumor cells susceptible to immune-mediated clearance and led to successful, long-term control of the disease. Our findings establish a firm link among immune selection, disease progression, and the development of a stem-like tumor phenotype in human cancer and implicate the Nanog/Tcl1a/Akt pathway as a central molecular target in this process.

Abstract A45: Resveratrol inhibits prostate cancer stem cell characteristics by suppressing Wnt, sonic hedgehog, and Notch pathways

Abstract The existence of cancer stem cells (CSCs) in prostate cancer has profound implications for cancer prevention and/or treatment. The CSC population resides within the prostate tumor mass and they may be responsible for drug resistance, and for the recurrence of prostate cancer. Therefore, development of novel strategies to target CSCs by nontoxic agent resveratrol is an innovative and novel idea for the management of prostate cancer. Activation of Wnt, sonic hedgehog (Shh) and Notch pathways are required for the growth of numerous tissues and organs and recent evidence indicates that these pathways are often recruited to regulate self-renewal and metastasis of CSCs. The objectives of this study were to examine the molecular mechanisms by which resveratrol, an active compound in grapes and red vine inhibits self-renewal capacity of prostate CSCs. Our data demonstrated that resveratrol inhibited the expression of pluripotency maintaining transcription factors (Nanog, c-Myc and Oct-4), and spheroid formation by prostate CSCs. Inhibition of Nanog by shRNA enhanced the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Resveratrol suppressed cell proliferation and induced apoptosis by inhibiting the expression of Bcl-2 and IAPs, and activating caspase-3. Interestingly, resveratrol also inhibited the transcriptional activities TCF/LEF, Gli and RBP-Jk. Furthermore, resveratrol inhibited epithelial-mesenchymal transition (EMT) by suppressing the expression of Snail, Slug and ZEB1 transcription factors, which correlated with significantly reduced CSC's migration and invasion, suggesting the blockade of signaling involved in early metastasis. In conclusion, resveratrol can inhibit prostate cancer CSC characteristics by suppressing various signal transduction pathways, and can be used for the management of prostate cancer. Citation Format: Rakesh K. Srivastava, Mariana Rodova, Junsheng Fu, Dara Nall, Sharmila Shankar. Resveratrol inhibits prostate cancer stem cell characteristics by suppressing Wnt, sonic hedgehog, and Notch pathways [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A45.

Differential expression of nanog1 and nanogp8 in colon cancer cells

Nanog, a homeodomain transcription factor, is an essential regulator for promotion of self-renewal of embryonic stem cells and inhibition of their differentiation. It has been demonstrated that nanog1 as well as nanogp8, a retrogene of nanog1, is preferentially expressed in advanced stages of several types of cancer, suggesting their involvement during cancer progression. Here, we investigated the expression of Nanog in well-characterized colon cancer cell lines. Expression of Nanog was detectable in 5 (HCT116, HT29, RKO, SW48, SW620) out of seven cell lines examined. RNA expression analyses of nanog1 and nanogp8 indicated that, while nanog1 was a major form in SW620 as well as in teratoma cells Tera-2, nanogp8 was preferentially expressed in HT29 and HCT116. In accordance with this, shRNA-mediated knockdown of nanog1 caused the reduction of Nanog in SW620 but not in HT29. Inhibition of Nanog in SW620 cells negatively affected cell proliferation and tumor formation in mouse xenograft. Biochemical subcellular fractionation and immunostaining analyses revealed predominant localization of Nanog in cytoplasm in SW620 and HT29, while it was mainly localized in nucleus in Tera-2. Our data indicate that nanog1 and nanogp8 are differentially expressed in colon cancer cells, and suggest that their expression contributes to proliferation of colon cancer cells.

Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics

Activation of the sonic hedgehog (SHh) pathway is required for the growth of numerous tissues and organs and recent evidence indicates that this pathway is often recruited to stimulate growth of cancer stem cells (CSCs) and to orchestrate the reprogramming of cancer cells via epithelial mesenchymal transition (EMT). The objectives of this study were to examine the molecular mechanisms by which (-)-epigallocatechin-3-gallate (EGCG), an active compound in green tea, inhibits self-renewal capacity of pancreatic CSCs and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Our data demonstrated that EGCG inhibited the expression of pluripotency maintaining transcription factors (Nanog, c-Myc and Oct-4) and self-renewal capacity of pancreatic CSCs. Inhibition of Nanog by shRNA enhanced the inhibitory effects of EGCG on self-renewal capacity of CSCs. EGCG inhibited cell proliferation and induced apoptosis by inhibiting the expression of Bcl-2 and XIAP and activating caspase-3. Interestingly, EGCG also inhibited the components of SHh pathway (smoothened, patched, Gli1 and Gli2) and Gli transcriptional activity. Furthermore, EGCG inhibited EMT by inhibiting the expression of Snail, Slug and ZEB1, and TCF/LEF transcriptional activity, which correlated with significantly reduced CSC's migration and invasion, suggesting the blockade of signaling involved in early metastasis. Furthermore, combination of quercetin with EGCG had synergistic inhibitory effects on self-renewal capacity of CSCs through attenuation of TCF/LEF and Gli activities. Since aberrant SHh signaling occurs in pancreatic tumorigenesis, therapeutics that target SHh pathway may improve the outcomes of patients with pancreatic cancer by targeting CSCs.

Abstract 5206: Nanog family members are essential for spherogenicity and metastasis in colorectal carcinoma

Abstract Liver is the most common site for colorectal cancer (CRC) distant metastasis and hepatic metastasis is one of the main causes of death in CRC patients. Inhibition of liver metastases in patients with CRC may increase the survival of patients. Our postulate is that genes involved in early embryogenesis, Nanog, Oct4 and SOX2, may be reactivated and control the malignant properties of metastasis and resistance to chemo- and radiotherapy. Nanog is a family of 11 pseudogenes with the original parent gene on chromosome 12. In order to identify which gene product is expressed in human CRC, we developed a novel endonuclease restriction enzyme assay, which was used in six human CRC cell lines. Monolayer cultures of CRC cells express Nanog, NanogP8, and NanogP7 but spheroids cultured in serum-free medium only express NanogP8 which was confirmed by direct sequencing. In addition, two potential DNA methylation regions are detected in the promoters of Nanog and NanogP8 by sodium bisulfite sequencing of genomic DNA in hES and CRC CloneA cells. In hES cells, the methylation status of Nanog and NanogP8 was about 30% and 60%, respectively. In contrast, in CloneA cells, it was 65% for Nanog and about 40% for NanogP8 and lower for CX-1 CRC cells. These results suggest that NanogP8 is re-activated in CRC through promoter demethylation in CRC spheroids that are enriched for putative stem cells. Overexpression of NanogP8 in CloneA and CX-1 significantly increased the spherogenicity of single CRC cells. In addition, lentiviral shRNA to Nanog inhibited spherogenicity in vitro and tumorigenicity in vivo whereas shRNA to OCT4 and SOX2 actually increased spherogenicity and tumorigenicity. Lentiviral shRNA inhibition of Nanog prevented liver colonies after intrasplenic injection of CX-1 cells whereas 45% of NOD/SCID mice developed experimental metastases after injection of the untreated CX-1 cells or CX-1 cells treated with negative control shRNA. As a result, targeting Nanog and its family members expressed in CRC may block the progression of CRC. 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 5206. doi:10.1158/1538-7445.AM2011-5206

Resveratrol inhibits pancreatic cancer stem cell characteristics in human and KrasG12D transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition.

BackgroundCancer stem cells (CSCs) can proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which resveratrol inhibits stem cell characteristics of pancreatic CSCs derived from human primary tumors and KrasG12D transgenic mice.Methodology/Principal FindingsHuman pancreatic CSCs (CD133+CD44+CD24+ESA+) are highly tumorigenic and form subcutaneous tumors in NOD/SCID mice. Human pancreatic CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic cancer cells. Similarly, CSCs from KrasG12D mice express significantly higher levels of Nanog and Oct-4 than pancreatic tissues from Pdx-Cre mice. Resveratrol inhibits the growth (size and weight) and development (PanIN lesions) of pancreatic cancer in KrasG12D mice. Resveratrol inhibits the self-renewal capacity of pancreatic CSCs derived from human primary tumors and KrasG12D mice. Resveratrol induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2 and XIAP in human CSCs. Resveratrol inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhances the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Finally, resveratrol inhibits CSC's migration and invasion and markers of epithelial-mesenchymal transition (Zeb-1, Slug and Snail).Conclusions/SignificanceThese data suggest that resveratrol inhibits pancreatic cancer stem cell characteristics in human and KrasG12D transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition. In conclusion, resveratrol can be used for the management of pancreatic cancer.

Sulforaphane synergizes with quercetin to inhibit self-renewal capacity of pancreatic cancer stem cells

According to the cancer stem cell hypothesis, the aggressive growth and early metastasis of cancer may arise through dysregulation of self-renewal of stem cells. The objectives of this study were to examine the molecular mechanisms by which sulforaphane (SFN, an active compound in cruciferous vegetables) inhibits self-renewal capacity of pancreatic cancer stem cells (CSCs), and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Our data demonstrated that SFN inhibited self-renewal capacity of pancreatic CSCs. Inhibition of Nanog by lentiviral-mediated shRNA expression enhanced the inhibitory effects of sulforaphane on self-renewal capacity of CSCs. SFN induced apoptosis by inhibiting the expression of Bcl-2 and XIAP, phosphorylation of FKHR, and activating caspase-3. Moreover, SFN inhibited expression of proteins involved in the epithelial-mesenchymal transition (beta-catenin, vimentin, twist-1, and ZEB1), suggesting the blockade of signaling involved in early metastasis. Furthermore, the combination of quercetin with SFN had synergistic effects on self-renewal capacity of pancreatic CSCs. These data suggest that SFN either alone or in combination with quercetin can eliminate cancer stem cell-characteristics.