Self-renewal Capacity Of Cancer Stem Cells Research Articles

RNA Polymerase II-Associated Factor 1 Regulates Stem Cell Features of Pancreatic Cancer Cells, Independently of the PAF1 Complex, via Interactions With PHF5A and DDX3

It is not clear how pancreatic cancer stem cells (CSCs) are regulated, resulting in ineffective treatments for pancreatic cancer. PAF1, a RNA polymerase II-associated factor 1 complex (PAF1C) component, maintains pluripotency of stem cells, by unclear mechanisms, and is a marker of CSCs. We investigated mechanisms by which PAF1 maintains CSCs and contributes to development of pancreatic tumors. Pancreatic cancer cell lines were engineered to knockdown PAF1 using inducible small hairpin RNAs. These cells were grown as orthotopic tumors in athymic nude mice and PAF1 knockdown was induced by administration of doxycycline in drinking water. Tumor growth and metastasis were monitored via IVIS imaging. CSCs were isolated from pancreatic cancer cell populations using flow cytometry and characterized by tumor sphere formation, tumor formation in nude mice, and expression of CSC markers. Isolated CSCs were depleted of PAF1 using the CRISPR/Cas9 system. PAF1-regulated genes in CSCs were identified via RNA-seq and PCR array analyses of cells with PAF1 knockdown. Proteins that interact with PAF1 in CSCs were identified by immunoprecipitations and mass spectrometry. We performed chromatin immunoprecipitation sequencing of CSCs to confirm the binding of the PAF1 sub-complex to target genes. Pancreatic cancer cells depleted of PAF1 formed smaller and fewer tumor spheres in culture and orthotopic tumors and metastases in mice. Isolated CSCs depleted of PAF1 downregulated markers of self-renewal (NANOG, SOX9, and β-CATENIN), of CSCs (CD44v6, and ALDH1), and the metastasis-associated gene signature, compared to CSCs without knockdown of PAF1. The role of PAF1 in CSC maintenance was independent of its RNA polymerase II-associated factor 1 complex component identity. We identified DDX3 and PHF5A as proteins that interact with PAF1 in CSCs and demonstrated that the PAF1-PHF5A-DDX3 sub-complex bound to the promoter region of Nanog, whose product regulates genes that control stemness. Levels of the PAF1-DDX3 and PAF1-PHF5A were increased and co-localized in human pancreatic tumor specimens, human pancreatic tumor-derived organoids, and organoids derived from tumors of KPC mice, compared with controls. Binding of DDX3 and PAF1 to the Nanog promoter, and the self-renewal capacity of CSCs, were decreased in cells incubated with the DDX3 inhibitor RK-33. CSCs depleted of PAF1 downregulated genes that regulate stem cell features (Flot2, Taz, Epcam, Erbb2, Foxp1, Abcc5, Ddr1, Muc1, Pecam1, Notch3, Aldh1a3, Foxa2, Plat, and Lif). In pancreatic CSCs, PAF1 interacts with DDX3 and PHF5A to regulate expression of NANOG and other genes that regulate stemness. Knockdown of PAF1 reduces the ability of orthotopic pancreatic tumors to develop and progress in mice and their numbers of CSCs. Strategies to target the PAF1-PHF5A-DDX3 complex might be developed to slow or inhibit progression of pancreatic cancer.

Differential mechanisms of autophagy in cancer stem cells: Emphasizing gastrointestinal cancers

Gastrointestinal (GI) cancers are one of the most common forms of malignancies and still are the most important cause of cancer-related mortality worldwide. Autophagy is a conserved catabolic pathway involving lysosomal degradation and recycling of whole cellular components, which is essential for cellular homeostasis. For instance, it acts as a pivotal intracellular quality control and repair mechanism but also implicated in cell reformation during cell differentiation and development. Indeed, GI cancer stem cells (CSCs) are thought to be responsible for tumour initiation, traditional therapies resistance, metastasis and tumour recurrence. Molecular mechanisms of autophagy in normal vs CSCs gain great interest worldwide. Here, we shed light on the role of autophagy in normal stem cells differentiation for embryonic progression and its role in maintaining the activity and self-renewal capacity of CSCs which offer novel viewpoints on promising cancer therapeutic strategies based on the differential roles of autophagy in CSCs.

Abstract PR14: HIF-2alpha regulates self-renewal of MYC dependent cancer stem cells

Abstract Numerous studies report that both hematopoietic and some solid tumors are organized in a hierarchical manner where tumor growth is driven by a small subset of cancer stem cells (CSCs). However, the distinct molecular identity of CSC versus non-CSC is not yet clearly known, which limit our understanding on how CSC self-renewal pathways operate to drive tumorigenicity. Here, we report that MYC and HIF-2alpha co-operate to maintain distinct identity and self-renewal capacity of CSCs. In a transgenic mouse model of conditional MYC driven thymic lymphoma, we identified a Sca-1+ cell population that showed tumor stemness (self-renewal and undifferentiation state) phenotype, including high expression of Nanog, Sox2 and HIF-2alpha. When injected to congenic mice, Sca-1+ cells but not Sca-1- cells exhibited hierarchical organization including self-renewal capacity. ChIP assay revealed direct MYC binding of HIF-2alpha promoter region in Sca-1+ but not in the Sca-1- cells. Inhibition of HIF-2alpha led to increase ROS generation, decrease of GSH synthesis, differentiation, and loss of self-renewal capacity of Sca-1+ cells. In contrast, inhibition of HIF-2alpha had no phenotypic effect on Sca-1- cells. Thus, we have identified a distinct CSC population in MYC dependent cancer cells, where MYC preferentially binds to HIF-2alpha to maintain self-renewal capacity. Citation Format: Bikul Das, Bikul Das, Hong Li, Hong Li, Rashmi Bhuyan, Dean W. Felsher. HIF-2alpha regulates self-renewal of MYC dependent cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR14.

Targeting proapoptotic protein BAD inhibits survival and self-renewal of cancer stem cells.

Emerging evidence suggests that the resistance of cancer stem cells (CSC) to many conventional therapies is one of the major limiting factors of cancer therapy efficacy. Identification of mechanisms responsible for survival and self-renewal of CSC will help design new therapeutic strategies that target and eliminate both differentiated cancer cells and CSC. Here we demonstrated the potential role of proapoptotic protein BAD in the biology of CSC in melanoma, prostate and breast cancers. We enriched CD44(+)/CD24(-) cells (CSC) by tumorosphere formation and purified this population by FACS. Both spheres and CSC exhibited increased potential for proliferation, migration, invasion, sphere formation, anchorage-independent growth, as well as upregulation of several stem cell-associated markers. We showed that the phosphorylation of BAD is essential for the survival of CSC. Conversely, ectopic expression of a phosphorylation-deficient mutant BAD induced apoptosis in CSC. This effect was enhanced by treatment with a BH3-mimetic, ABT-737. Both pharmacological agents that inhibit survival kinases and growth factors that are involved in drug resistance delivered their respective cytotoxic and protective effects by modulating the BAD phosphorylation in CSC. Furthermore, the frequency and self-renewal capacity of CSC was significantly reduced by knocking down the BAD expression. Consistent with our in vitro results, significant phosphorylation of BAD was found in CD44(+) CSC of 83% breast tumor specimens. In addition, we also identified a positive correlation between BAD expression and disease stage in prostate cancer, suggesting a role of BAD in tumor advancement. Our studies unveil the role of BAD in the survival and self-renewal of CSC and propose BAD not only as an attractive target for cancer therapy but also as a marker of tumor progression.

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

Abstract 2593: Procyanidin B2 3,3″-di-O-gallate causes strong efficacy towards prostate cancer stem cells via targeting Notch1 signaling.

Abstract Prostate cancer (PCa) is the most common malignancy in US men; statistical estimates for 2012 indicate 241,740 new PCa cases and 28,170 associated deaths. Epithelial cancers including PCa are now recognized as generated and maintained by a small sub-set of undifferentiated cells known as cancer stem cells (CSC) or tumor initiating cells. Importantly, CSC are inherently resistant to chemo- and radio-therapies, and that has been the major reason for the failure of most of the current therapies. Since an early event in carcinogenesis involves expansion of CSC pool, interventions inducing apoptosis or differentiation with a loss of self-renewal capacity of CSC, represent a rational approach for PCa prevention and treatment. In this regard, the present study was aimed to determine whether grape seed extract (GSE), a natural non-toxic chemopreventive agent and its active constituent Procyanidin B2 3,3”-di-O-gallate (B2G2), have the potential to target CSC in PCa. For this, we determined the effect of these agents on the self-renewal capacity of the CSC population of PCa cell lines. The CSC cell populations were isolated and purified based on CD44+ α2β1highsurface markers in PCa cell lines LNCaP, C4-2B, 22Rv1, PC3, and DU145, and then subjected to sphere cluster formation assays, in the absence or presence of (a single or multi-treatments) GSE (5-125 μg/ml) or B2G2 (25-100μM). The percentage of floating spheroids (prostaspheres) generated in the sphere cluster assays after 1-2 weeks were determined. Results indicated that while a single treatment of GSE is not sufficient to reduce either the number or size of generated spheres in the PCa cell lines, multiple treatments with GSE at low dose was sufficient to cause a significant reduction in the number and size of generated spheres. On the other hand, single treatment of PCa cells with B2G2 significantly reduced the number and size of prostaspheres. As formation of spheroids under specific in vitro conditions is a measure of stemness, these results indicate the potential of GSE and B2G2 to target the self-renewal of CSC in PCa cell lines, though B2G2 was found to be more potent in its efficacy. Subsequent mechanistic studies revealed that both agents strongly decreased the constitutive as well as Jagged1 (Notch1 ligand)-induced protein levels of cleaved Notch and its transcriptional target Hes-1 in PCa cells. The implications of such an effect could be important, since the modulation of Notch signaling among stem cells and progenitor cells in PCa results in the expansion of CSC pool together with an increase in proliferative cell population. Together, our results are significant as they show that both GSE and B2G2 interfere with kinetics of CSC pool expansion via targeting Notch-1 regulatory signals, which eventually could help control PCa growth and progression. Citation Format: Alpna Tyagi, Komal Raina, Sushil Kumar, Rajesh Agarwal, Chapla Agarwal. Procyanidin B2 3,3″-di-O-gallate causes strong efficacy towards prostate cancer stem cells via targeting Notch1 signaling. [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 2593. doi:10.1158/1538-7445.AM2013-2593

Metformin Inhibits Cell Proliferation, Migration and Invasion by Attenuating CSC Function Mediated by Deregulating miRNAs in Pancreatic Cancer Cells

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States, which is, in part, due to intrinsic (de novo) and extrinsic (acquired) resistance to conventional therapeutics, suggesting that innovative treatment strategies are required for overcoming therapeutic resistance to improve overall survival of patients. Oral administration of metformin in patients with diabetes mellitus has been reported to be associated with reduced risk of pancreatic cancer and that metformin has been reported to kill cancer stem cells (CSC); however, the exact molecular mechanism(s) has not been fully elucidated. In the current study, we examined the effect of metformin on cell proliferation, cell migration and invasion, and self-renewal capacity of CSCs and further assessed the expression of CSC marker genes and microRNAs (miRNA) in human pancreatic cancer cells. We found that metformin significantly decreased cell survival, clonogenicity, wound-healing capacity, sphere-forming capacity (pancreatospheres), and increased disintegration of pancreatospheres in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. Metformin also decreased the expression of CSC markers,CD44, EpCAM,EZH2, Notch-1, Nanog and Oct4, and caused reexpression of miRNAs (let-7a,let-7b, miR-26a, miR-101, miR-200b, and miR-200c) that are typically lost in pancreatic cancer and especially in pancreatospheres. We also found that reexpression of miR-26a by transfection led to decreased expression of EZH2 and EpCAM in pancreatic cancer cells. These results clearly suggest that the biologic effects of metformin are mediated through reexpression of miRNAs and decreased expression of CSC-specific genes, suggesting that metformin could be useful for overcoming therapeutic resistance of pancreatic cancer cells.

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.

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.

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.