Embryonic Stem Cell Transcription Factors Research Articles

Hypoxia exposure induces lactylation of Axin1 protein to promote glycolysis of esophageal carcinoma cells

The hypoxic microenvironment in esophageal carcinoma is an important factor promoting the rapid progression of malignant tumor. This study was to investigate the lactylation of Axin1 on glycolysis in esophageal carcinoma cells under hypoxia exposure. Hypoxia treatment increases pan lysine lactylation (pan-kla) levels of both TE1 and EC109 cells. Meanwhile, ECAR, glucose consumption and lactate production were also upregulated in both TE1 and EC109 cells. The expression of embryonic stem cell transcription factors NANOG and SOX2 were enhanced in the hypoxia-treated cells. Axin1 overexpression partly reverses the induction effects of hypoxia treatment in TE1 and EC109 cells. Moreover, lactylation of Axin1 protein at K147 induced by hypoxia treatment promotes ubiquitination modification of Axin1 protein to promote glycolysis and cell stemness of TE1 and EC109 cells. Mutant Axin1 can inhibit ECAR, glucose uptake, lactate secretion, and cell stemness in TE1 and EC109 cells under normal or hypoxia conditions. Meanwhile, mutant Axin1 further enhanced the effects of 2-DG on inhibiting glycolysis and cell stemness. Overexpression of Axin1 also inhibited tumor growth in vivo, and was related to suppressing glycolysis. In conclusion, hypoxia treatment promoted the glycolysis and cell stemness of esophageal carcinoma cells, and increased the lactylation of Axin1 protein. Overexpression of Axin1 functioned as a glycolysis inhibitor, and suppressed the effects of hypoxia exposure in vitro and inhibited tumor growth in vivo. Mechanically, hypoxia induces the lactylation of Axin1 protein and promotes the ubiquitination of Axin1 to degrade the protein, thereby exercising its anti-glycolytic function.

Analysis of regulatory sequences in exosomal DNA of NANOGP8.

Exosomes participate in intercellular communication by transporting functionally active molecules. Such cargo from the original cells comprising proteins, micro-RNA, mRNA, single-stranded (ssDNA) and double-stranded DNA (dsDNA) molecules pleiotropically transforms the target cells. Although cancer cells secrete exosomes carrying a significant level of DNA capable of modulating oncogene expression in a recipient cell, the regulatory mechanism is unknown. We have previously reported that cancer cells produce exosomes containing NANOGP8 DNA. NANOGP8 is an oncogenic paralog of embryonic stem cell transcription factor NANOG and does not express in cells since it is a pseudogene. However, in this study, we evaluated NANOGP8 expression in glioblastoma multiforme (GBM) tissue from a surgically removed brain tumor of a patient. Significantly higher NANOGP8 transcription was observed in GBM cancer stem cells (CSCs) than in GBM cancer cells or neural stem cells (NSCs), despite identical sequences of NANOGP8-upstream genomic region in all the cell lines. This finding suggests that upstream genomic sequences of NANOGP8 may have environment-dependent promoter activity. We also found that the regulatory sequences upstream of exosomal NANOGP8 GBM DNA contain multiple core promoter elements, transcription factor binding sites, and segments of human viruses known for their oncogenic role. The exosomal sequence of NANOGP8-upstream GBM DNA is different from corresponding genomic sequences in CSCs, cancer cells, and NSCs as well as from the sequences reported by NCBI. These sequence dissimilarities suggest that exosomal NANOGP8 GBM DNA may not be a part of the genomic DNA. Exosomes possibly acquire this DNA from other sources where it is synthesized by an unknown mechanism. The significance of exosome-bestowed regulatory elements in the transcription of promoter-less retrogene such as NANOGP8 remains to be determined.

Atlas of regulated target genes of transcription factors (ART-TF) in human ES cells

BackgroundTranscription factors (TFs) play central roles in maintaining “stemness” of embryonic stem (ES) cells and their differentiation into several hundreds of adult cell types. The regulatory competence of TFs is routinely assessed by detecting target genes to which they bind. However, these data do not indicate which target genes are activated, repressed, or not affected by the change of TF abundance. There is a lack of large-scale studies that compare the genome binding of TFs with the expression change of target genes after manipulation of each TF.ResultsIn this paper we associated human TFs with their target genes by two criteria: binding to genes, evaluated from published ChIP-seq data (n = 1868); and change of target gene expression shortly after induction of each TF in human ES cells. Lists of direction- and strength-specific regulated target genes are generated for 311 TFs (out of 351 TFs tested) with expected proportion of false positives less than or equal to 0.30, including 63 new TFs not present in four existing databases of target genes. Our lists of direction-specific targets for 152 TFs (80.0%) are larger that in the TRRUST database. In average, 30.9% of genes that respond greater than or equal to twofold to the induction of TFs are regulated targets. Regulated target genes indicate that the majority of TFs are either strong activators or strong repressors, whereas sets of genes that responded greater than or equal to twofold to the induction of TFs did not show strong asymmetry in the direction of expression change. The majority of human TFs (82.1%) regulated their target genes primarily via binding to enhancers. Repression of target genes is more often mediated by promoter-binding than activation of target genes. Enhancer-promoter loops are more abundant among strong activator and repressor TFs.ConclusionsWe developed an atlas of regulated targets of TFs (ART-TF) in human ES cells by combining data on TF binding with data on gene expression change after manipulation of individual TFs. Sets of regulated gene targets were identified with a controlled rate of false positives. This approach contributes to the understanding of biological functions of TFs and organization of gene regulatory networks. This atlas should be a valuable resource for ES cell-based regenerative medicine studies.

Inhibitors Targeting CDK9 Show High Efficacy against Osimertinib and AMG510 Resistant Lung Adenocarcinoma Cells.

Simple SummaryNon-small cell lung cancer accounts for 80% of all lung cancer cases. While a subset of non-small cell lung cancer patients respond to immunotherapy, those who are treated with chemotherapy or targeted therapy develop resistance to the drugs. Thus, novel therapeutic strategies are needed to combat this disease. Here we show that inhibitors of the cyclin-dependent kinase 9 are highly effective in preventing the growth of a variety of lung cancer cell lines and lung cancer organoids with high potency. These inhibitors suppressed the expression of several genes like Sox2, Sox9, and Mcl1 that promote tumor growth, facilitating growth arrest. Since inhibitors of cyclin-dependent kinase 9 are undergoing clinical trials for hematological malignancies, our studies suggest that these inhibitors would be attractive candidates to combat non-small cell lung cancer.Non-small cell lung cancer has a 5-year survival rate of less than 12–15%, calling for the development of additional therapeutic strategies to combat this disease. Here we tested the efficacy of inhibiting cyclin-dependent kinase 9 (CDK9) on lung cancer cell lines with K-Ras and EGFR mutations and on lung cancer organoids. Three different CDK9 inhibitors reduced the viability and anchorage-independent growth of lung cancer cell lines at very low nanomolar to micromolar concentrations. CDK9 inhibition suppressed the expression of the anti-apoptotic protein, Mcl1, as well as the embryonic stem cell transcription factors, Sox2 and Sox9, which are pro-tumorigenic. In contrast, treatment with CDK9 inhibitors increased the levels of WT p53 and its downstream target p21 in K-Ras mutant cell lines. Furthermore, the CDK9 inhibitors could markedly reduce the viability of Osimertinib-resistant PC9 and AMG510-resistant H23 and H358 cells with comparable efficacy as the parental cells. CDK9 inhibitors could also significantly reduce the growth and viability of lung cancer organoids with high potency. Taken together, the data presented here strongly suggest that CDK9 inhibitors would be efficacious against K-Ras mutant and EGFR mutant NSCLCs, including those that develop resistance to targeted therapies.

Immunocytochemical characterization of primary teeth pulp stem cells from three stages of resorption in serum-free medium.

Dental pulp stem cells from primary teeth cultured in serum-free conditions may have clinical use for the repair and regeneration of teeth as well as other complex tissues and organs. The aim of this study was to test the change in the stem cell markers expression/ stem cell population in human primary pulp cells at the different stages of root resorption. Caries-free human primary canines at defined stages of physiological root resorption were included (n=9). In vitro cultures were established in xeno-free, serum-free Essential 8™ medium with human truncated vitronectin for cell attachment. An embryonic stem cell line (GENEA002) was used as a positive control. The expression of embryonic stem cell markers (Oct4, Nanog and Sox2), neural crest stem cell markers (nestin and Dlx2) and mesenchymal stem cell surface markers (CD90, CD73 and CD105) were investigated by immunocytochemistry. Mesenchymal stem cell markers CD105, CD73 and CD90 and haematopoietic markers: CD45, CD34, CD11b, CD19 and HLA-DR were quantified with flow cytometry. The early neural progenitor markers nestin and Dlx2 were detected in most serum-free cultured dental pulp stem cells, regardless of the tooth resorption stage from which they were harvested. Only isolated cells were found that expressed the embryonic stem cell transcription factors Oct4A, Nanog and Sox2, and in the late stages of resorption, no Oct4A was detected. The majority expressed the mesenchymal stem cell markers CD90, CD73 and CD105. Flow cytometry found positive signals for CD90>97.3%, CD73>99.6% and CD105>82.5%, with no detectable differences between resorption stages. This study identified populations of dental pulp cells in vitro with markers characteristically associated with embryonic stem cells, neural crest-derived cells and mesenchymal stem cells. Flow cytometry found CD105 expressed at lower levels than CD90 and CD73. The consistency of stem cell marker expression in cells cultured from teeth at different resorption stages suggests that pre-exfoliated primary teeth that are free of caries may provide a convenient source of multipotent stem cells for use in regenerative medicine.

Abstract 504: p27 transcriptionally coregulates STAT3 to drive cancer stem cells

Abstract p27 is a cell cycle inhibitor and a tumor suppressor. It can also regulate cellular processes including migration and transcription through mechanisms independent of its CDK-inhibitory role. In cancers, p27 C-terminal phosphorylation by PI3K-activated kinases, AKT, RSK1, and SGK1 at T157 and T198 alters p27 protein-protein interactions and shifts p27 from CDK-inhibitor to an oncogene. Previously, our group showed that CDK-binding defective p27pT157pT198 phosphomimetic (p27CK-DD) upregulates epithelial-mesenchymal transition (EMT) and the metastatic potential of cancer cell lines. In addition to its action to promote EMT, p27 appears to promote CSC expansion and or maintenance. Here we demonstrated that C-terminally phosphorylated p27 increases CSC properties, including tumor sphere formation and CSC markers. p27CK-DD increases the expression of several embryonic stem cell transcription factors (ES-TFs), including SOX2, NANOG and cMYC, which are known to drive embryonic stem cell self-renewal and to promote CSC expansion. A human phospho-kinase array showed Pyk2 is activated by p27CK-DD. We demonstrated that Pyk2 activation and its binding to p27 are dependent on phosphorylation of p27 at T198 and T157. Treatment with a Pyk2 inhibitor, and PYK2-knockdown by siRNA or shRNA revealed that Pyk2 is a key mediator of the increase in tumor spheres, ALDH1 activity and ES-TFs in cancer cells expressing abundant C-terminally phosphorylated p27. Pyk2 enhances STAT3 phosphorylation and activation. We identified that C-terminally phosphorylated p27 recruits STAT3 to form p27-Pyk2-STAT3 complex leading to STAT3 activation. p27/STAT3 complexes co-localize to the nucleus and co-occupy chromatin. We confirmed by ChIP-qPCR that p27/STAT3 bind and activate the cMYC promotor. Altogether, in breast cancer cells with high p27pT157pT198 or expressing p27CK−DD, STAT3 is partly activated by Pyk2 and interacts with p27. p27 is a STAT3 coregulator, whose assembly and chromatin association is governed by p27 phosphorylation. These data reveal a novel mechanism whereby p27-driven Pyk2 activation promotes CSC expansion and tumor progression via transcriptionally activation of the STAT3 and its target genes. Citation Format: Seyedehfatemeh Razavipour, Kibeom Jang, Hyunho Yoon, Minsoon Kim, Miyoung Shin, Dekuang Zhao, Joyce Slingerland. p27 transcriptionally coregulates STAT3 to drive cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 504.

Synergic effects of cancer stem cells markers, CD44 and embryonic stem cell transcription factor Nanog, on bladder cancer prognosis

ABSTRACTBackground: Therapy that targets cancer stem cells has the potential to eradicate cancer and prevent tumour recurrence. Therefore, we hypothesized the combined prognostic significance of stem cell markers CD44 (prevalent in basal layer of urothelial carcinoma) and Nanog (embryonic stem cell transcription factor) in bladder cancer.Material and Methods: CD44 and Nanog expression were determined by immunohistochemistry in 112 bladder cancer cases of which 79 were non-muscle invasive and 33 muscle invasive.Results: A significant correlation was found between CD44 and Nanog expression (r = 0.41, p < 0.001). The bladder cancer patients with high CD44 and Nanog expression had poor recurrence-free survival and poor overall survival (all p < 0.01). Multivariate Cox regression analysis identified lymph node positivity (hazard ratio; HR 3.81, 95% confidence interval; CI 1.66–8.75), CD44 (HR/95%CI 7.03 [3.04–16.22]) and Nanog (HR/95%CI 2.89 [1.23–6.77]) as independent prognostic biomarkers for recurrence-free survival, whilst a combined index of CD44 and Nanog expression (high expression group; HR/95%CI 25.45 [6.71-96.50] for recurrence-free survival) and lymph node positivity (HR/95%CI 3.68 [1.63-8.33] for recurrence-free survival) were independent prognostic biomarkers for recurrence-free survival and overall survival (all p < 0.001).Conclusions: A combined index of CD44 and Nanog expression is a promising prognostic predictor of recurrence-free survival and overall survival in bladder cancer. It may help identification of patients who will benefit from intensive treatment.

Abstract 1398: Super-enhancers: Possible target in pancreatic cancer for therapeutic approaches

Abstract Introduction: Super enhancers (SE) are distinctive areas of the genome that are densely bound by numerous transcription factors and play a pivotal role in the cell identity, tissue specification, and maintenance. Although the exact functions of super-enhancers are not yet well understood, these regions are known for driving high-level transcription. Studies have established most of the oncogenes playing important role in cancer are driven by SEs. It is likely that the peculiar pancreatic-specific tumor phenotype is a consequence of oncogenes hacking the resident tissue regenerative program, thus interfering with super enhancer-driven repair networks might exert a disproportionately disruptive effect on tumor versus normal pancreatic tissue. The hypothesis of this study is that the tumor cells in PDAC acquire super-enhancers at key oncogenes during tumor development, which drive higher levels of transcription of these genes than in healthy cells. Acquired super-enhancers may thus be biomarkers that could be useful for diagnosis and therapeutic intervention. Thus drugs which could affect super enhancers in cancer may have therapeutic effect. Methods: In this study, we have used ChIP-Seq techniques and bioinformatics analysis following qPCR to identify SE in PDAC cell lines and in pancreatic cancer tissues. Immunohistochemistry, proliferation assays, western blot, RNAseq and CETSA (cellular thermal shift assay) have been performed for understanding the role of enhancers and SE in PDAC. Results: H3K27ac marks were identified at enhancer region of numerous genes that act as SE in PDAC. The most prominent super-enhancers identified, based on a high level of H3K27ac marks were associated with c-MYC, MED1, OCT-4, NANOG and SOX2 genes. Differential association of SE in non-cancerous pancreatic cells, cancerous and metastatic cell lines of PDAC have been implicated in comparison normal and PDAC cells. In this study, we found that GZ17-6.02, combinations of natural compounds, affects acetylation of some of the major SE related genes and at a higher dose, a complete reduction in acetylation marks was seen in embryonic stem cell transcription factors. The mRNA sequencing data after GZ17-6.02 treatment also shows a reduction in transcription of, major transcription factors, SHH pathway genes, and stem cell markers both in vitro and in vivo pancreatic cancer orthotopic models. Cancer cells are more sensitive to lower doses of GZ17-6.02 than normal cell; hence normal cells are expected to have less side effects. Conclusions: There is no systematic study showing an association of SE with pancreatic cancer so far. Hence, in the present study, we have established that several super-enhancer marks can be targeted by combination of natural compounds, GZ17-6.02, in PDAC. This study concludes that super-enhancers can be an important therapeutic target for PDAC. Citation Format: Chandrayee Ghosh, Sumedha Gunewardena, Prasad Dandawate, Santanu Paul, Ossama Tawfik, Cameron West, Shrikant Anant, Animesh Dhar. Super-enhancers: Possible target in pancreatic cancer for therapeutic approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1398.

Pluripotency factors functionally premark cell-type-restricted enhancers in ES cells.

While enhancers for embryonic stem cell (ESC)-expressed genes and lineage-determining factors are characterized by conventional marks of enhancer activation in ESCs1,2,3, it remains unclear whether enhancers destined to regulate cell-type-restricted transcription units might also have some currently overlooked, distinct signatures in ESCs. Here, we report that cell-type-restricted enhancers, are unexpectedly premarked and activated as transcription units by the binding of a single, or two, ESC transcription factors, although not exhibiting traditional enhancer epigenetic marks in ESCs, thus uncovering the initial temporal origins of cell-type-restricted enhancers. This premarking is required for future cell-type-restricted enhancer activity in the differentiated cells, with the strength of the ESCs signature being functionally important for subsequent robustness of cell-type-restricted enhancer activation. This model has been experimentally validated in macrophage-restricted enhancers and neural precursor cells (NPCs)-restricted enhancers using ESCs-derived macrophages or NPCs, edited to contain specific ESC transcription factor motif deletions. The ESC transcription factor-determined DNA hydroxyl-methylation of the enhancers in ESCs may serve as a potential molecular memory for subsequent enhancer activation in the mature macrophage. These findings suggest that the massive repertoire of cell-type-restricted enhancers are essentially hierarchically and obligatorily “premarked” by binding of a defining ESC transcription factor in ESCs, dictating robustness of enhancer activation in mature cells.

Abstract LB-A26: Regulation of super-enhancers by GZ17-6.02 has therapeutic effect in pancreatic cancer (PDAC)

Abstract Super enhancers (SE) are defined as unique areas of the genome that are densely bound by numerous transcription factors and play a pivotal role in the cell, including, tissue specification, identity and maintenance. Super enhancers are known to regulate the expression of associated genes and often drive high-level transcription. Studies have established many genes that play an important role in cancer biology as SE-driven oncogenes. It is likely that the peculiar pancreatic-specific tumor phenotype is a consequence of oncogenes hacking the resident tissue regenerative program, thus interfering with super enhancer-driven repair networks exerting a disproportionately disruptive effect on tumor versus normal pancreatic tissue. In this study, we have used ChIP-seq, RNA-seq and bioinformatics analysis to identify super-enhancers in the human metastatic pancreatic cancer cell line S2-007. H3K27ac marks were identified at enhancer region of numerous genes that act as SE in PDAC. The most prominent super-enhancers identified, based on a high level of H3K27ac marks were associated with c-MYC, MED1, OCT-4, NANOG and SOX2 genes. Previously we showed that “isovanillin”, one of the major compound of A. palaestinum, in combination with other anticancer agents termed as ‘GZ17-6.02’ inhibited tumor progression and metastasis in PDAC by suppressing cancer stem cells (CSCs) and sonic hedgehog (SHH) pathway in both in vitro and in vivo systems. In this study, we found that GZ17-6.02 affects acetylation of histones at some of the major SE related genes and at a high dose, a complete reduction in acetylation marks was seen in embryonic stem cell transcription factors. The mRNA sequencing data after GZ17-6.02 treatment also shows a reduction in transcription of, major transcription factors, SHH pathway genes, and stem cell markers. Cancer cells are more sensitive to lower doses of GZ17-6.02 than normal cells; hence normal cells are expected to have fewer side effects. There is no systematic study showing an association of super-enhancers with pancreatic cancer so far. Hence, in the present study, we have established that several super-enhancer targets can be targeted by GZ17-6.02 in PDAC. This study concludes that super-enhancers can be an important therapeutic target for PDAC. Citation Format: Chandrayee Ghosh, Sumedha Gunewardena, Santanu Paul, Cameron West, Animesh Dhar. Regulation of super-enhancers by GZ17-6.02 has therapeutic effect in pancreatic cancer (PDAC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A26.

Abstract B19: The role of ABCB1 genotypes and targeting cancer stem cells in pancreatic cancer: Effects of metformin and dietary agents

Abstract Pancreatic cancer carries a poor prognosis and survival rate. Developing effective agents or repurposing agents with low toxicity, such as metformin, an insulin-lowering drug, has recently been investigated for pancreatic cancer. Metformin has long been associated with decreased cancer risk, particularly in diabetic patients. Cancer stem cells have been identified in pancreatic tumors and have been shown to contribute to its progression and resistance to standard treatments. Pancreatic cancer stem cells are subject to regulation by key embryonic stem cell transcription factors that are known to be aberrantly expressed in pancreatic cancer, such as SOX2, OCT4 and NANOG. Overexpression of OCT4, SOX2 and NANOG together or separately leads to tumor transformation and tumor metastasis. These stem cell factors are known to promote self-renewal by interacting with other transcription factors. Using a human embryonic stem cell RT2 Profiler gene array, numerous stem cell genes were expressed in pancreatic cancer cell lines (MIAPaca2, Panc1). Both cell lines expressed high levels of SOX2, NANOG, CD44, GATA2, POU5F1 (OCT4) and other genes. Panc1 expressed high levels of SOX 17, while MIAPaCa2 expressed high levels of SOX 3, 15 and 17. Treatment of cells with the dietary agent, indole-3-carbinol alone at 100 or 200 µM inhibited or decreased the expression of SOX2, NANOG, CD44, SOX15 and STAT3. However, treatment with metformin alone increased expression of SOX15 and STAT3 in Panc1 cells but not in MIAPaCa2. Combination of metformin and indole-3-carbinol inhibited the expression of SOX2, NANOG, STAT3, and CD44. Expression levels of drug transporters in pancreatic tissue can also affect potential treatment of pancreatic cancer. High level of expression of ABCB1 causes multidrug-resistance. Metformin modulated the ATP-binding cassette gene, ABCB1, expression in pancreatic cancer cell lines with different ABCB1 genotypes. Metformin (100 µM or 200 µM) alone (p=0.0212 or p=0.0161) or in combination with gemcitabine (15nM) (p=0.0248 or p=0.0174) significantly decreased ABCB1 expression in MIAPaCa2 pancreatic cells after 24 hr or 72 hr treatments. This correlated to increased cell death. Metformin in combination with indole-3-carbinol also significantly (p=0.0317) decreased ABCB1 expression. However, a sex or genotype difference was noted when two female pancreatic cell lines, SU86.86 and AsPC1, were used. Metformin did not down-regulate ABCB1 expression and was chemoresistance in these two cell lines. AsPC1 and SU86.86 both carry the 2677GG-3435CC ABCB1 haplotype compared to MiaPaca-2, which carries the 2677TT-3134TT ABCB1 haplotype. The 2677TT and 3435TT genotypes/haplotypes are known to be associated with lower risk of developing pancreatic cancer. These data demonstrated that these genotypes are also more sensitive to metformin treatment compared to the 2677GG or 3435CC genotypes. Further research is needed to ascertain whether sex or genotype is contributing to different efficacy of metformin in pancreatic cancer cells with different genotypes. This study have shown that the dietary agent, indole-3-carbinol alone and in combination with metformin down-regulate critical stem cell genes involved in maintenance of pluripotency and self-renewal of cancer stem cells. Furthermore, downregulation of ABCB1 expression varied with treatment with specificity to certain genotypes. Citation Format: Beverly D. Lyn-Cook, Beverly Word, George Hammons. The role of ABCB1 genotypes and targeting cancer stem cells in pancreatic cancer: Effects of metformin and dietary agents [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B19.

Abstract 1733: Inhibition of embryonic stem cells gene expression in pancreatic cancer: Effects of metformin and indole-3-carbinol

Abstract Pancreatic cancer is among the top five deadliest cancers in developing countries and is rapidly increasing in minority populations, particularly African Americans. Understanding molecular mechanisms involved in the etiology and progression of this cancer can greatly improve patient prognosis. Cancer stem cells have been identified in pancreatic tumors and have been shown to contribute to its progression and resistance to standard treatments. Pancreatic cancer stem cells are subject to regulation by key embryonic stem cell transcription factors that are known to be aberrantly expressed in pancreatic cancer, such as SOX2, OCT4 and NANOG. Overexpression of OCT4, SOX2 and NANOG together or separately led to tumor transformation and tumor metastasis. These stem cell factors are known to promote self-renewal by interacting with other transcription factors. Using a human embryonic stem cell RT2 Profiler gene array, numerous stem cell genes were expressed in pancreatic cancer cell lines (MIAPaca2, Panc1). Both cell lines expressed high levels of SOX2, NANOG, CD44, GATA2, POU5F1 (OCT4) and other genes. Panc1 expressed high levels of SOX 17, while MIAPaCa2 expressed high levels of SOX 3,15 and 17. Treatment of cells with indole-3-carbinol alone at 100 and 200μM inhibited or decreased the expression of SOX2, NANOG, CD44, SOX15 and STAT3. However, treatment with metformin alone increased expression of SOX 15 and STAT3 in Panc1 cells but not in MIAPaCa2. Combination of metformin and indole-3-carbinol inhibited the expression of SOX2, NANOG, STAT3, and CD44. SOX2, POU5F1(OCT4) and NANOG are thought to be important players in various human cancers. OCT4 has been found to be expressed in 69% of PDAC and expression was shown too correlated to status and clinical state. High levels of OCT4 and NANOG has been found to correlate to worse prognosis and furthermore, contribute to multidrug-resistance and metastasis. This study have shown that the dietary agent, indole-3-carbinol alone and in combination with metformin down-regulate critical stem cell genes involved in maintenance of pluripotency and self-renewal of cancer stem cells. Citation Format: Beverly D. Lyn-Cook, Stancy Joseph, Beverly Word, George Hammons. Inhibition of embryonic stem cells gene expression in pancreatic cancer: Effects of metformin and indole-3-carbinol. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1733.

Abstract AS04: Epigenomic characterization of gene regulatory networks in human ovarian cancer stem cells

Abstract Ovarian cancer is the fifth leading cause of cancer mortality among women in the United States. Despite aggressive surgery and chemotherapy, most patients relapse and develop drug-resistant tumors, leading to the &amp;lt;40% chance of 5 year survival in ovarian cancer patients. Evidence suggests that a small subpopulation of cells in the tumor, ovarian cancer stem cells (OvCSCs), are responsible for tumor relapse and drug resistance. OvCSCs have enhanced tumorigenicity, inherent chemoresistance, and stem-like properties such as the ability to self-renew or divide asymmetrically. These latter characteristics give CSCs an analogous role in tumors that healthy stem cells have in developing organs (adult stem cells) or entire organisms (embryonic stem cells).The goal of this study is to determine how the cancer stem cell state is regulated by annotating the gene regulatory network of OvCSCs. In doing so, we will uncover commonalities that exist between OvCSCs and embryonic or progenitor stem cells. The epigenome provides the structural framework for cell-type specific gene regulation. Chromatin modifications that uniquely mark gene regulatory elements, like promoters and enhancers, are easily identified throughout the genome using ChIP-seq. ChIP-seq performed in pluripotent stem cells has revealed many epigenetic features that distinguish them from differentiated cells. In an effort to better understand CSC gene regulatory networks, we are generating comprehensive transcriptomic maps and globally identifying enhancers marked by histone 3, lysine 4 monomethylation (via RNAseq and H3K4me1 ChIP-seq respectively) in OvCSCs and their daughter tumor cells. We are also comparing OvCSCs to previously generated data in human embryonic stem cells (hESCs) to identify shared stemness traits. OvCSCs were acquired from patient biopsies and detailed characterization identified a CD133+ CSC population within the tumor. We are using a mouse xenograft model to expand tumors in vivo. Harvested tumors are depleted of blood lineage cells and CD133+ cells (CSCs) are isolated from the remaining cells in the tumor. Our analysis of RNA-seq and enhancer maps have revealed OvCSC signatures that are absent in tumor cells but present in embryonic stem cells. Many important embryonic stem cell transcription factors are expressed in OvCSCs and have almost identical chromatin enhancer profiles. We identified over 7000 H1 hESC enhancers that have at least 95% overlap with OvCSC enhancers implying a shared regulatory network in these cell types. Motif analysis of nucleosome-free regions within enhancers reveals enrichment for many ESC transcription factors that are also expressed in OvCSCs. Our integrative analysis indicates that OvCSCs share common features of the ESC regulatory network and is beginning to provide novel insight on why CSCs have stem cell properties. We are continuing to build maps for additional histone modifications and genome-wide DNA methylation in OvCSCs to gain a comprehensive view of how the epigenome regulates the cancer stem cell state. Citation Format: Stephanie L. Battle, Antti Larjo, Joling Liao, Harri Lähdesmäki, Andre Lieber, R. David Hawkins. Epigenomic characterization of gene regulatory networks in human ovarian cancer stem cells [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 AS04.

Abstract 1516: YAP1 regulates Oct4 activity and Sox2 expression, facilitating self-renewal and vascular mimicry of stem-like cells

Abstract Non-small cell lung cancer (NSCLC) is highly correlated with smoking and has very low survival rates. Multiple studies have shown that cancer stem-like cells (CSCs) contribute to the genesis and progression of NSCLC. These cells have been shown to form differentiated tumor cells upon receiving appropriate microenvironmental cues and can give rise to multiple components of the tumor, including the vasculature. Earlier work from our laboratory had shown that Hoechst 33342 dye excluding side-population (SP) cells are enriched in cells having stem-like properties. SP cells displayed high self-renewal capacity, were highly drug resistant, and could form metastatic tumors in immunocompromised mice. The present study shows that the transcriptional co-activator YAP1, which is the oncogenic component of the Hippo signaling pathway, is elevated in the stem-like cells from NSCLC and contributes to their self-renewal and ability to form angiogenic tubules in matrigel. Inhibition of YAP1 by a small molecule inhibitor Visudyne or depletion of YAP1 by siRNAs suppressed self-renewal and vascular mimicry of stem-like cells. These effects of YAP1 were mediated through the embryonic stem cell transcription factor Sox2. YAP1 could associate with the Sox2 promoter and could transcriptionally induce Sox2 as seen by qRT-PCRs, ChIP assays and promoter luciferase assays; this induction was through a physical interaction of YAP1 with Oct4 protein. The binding of Oct4 to YAP1 could be detected in cell lines as well as NSCLC tumor tissues as seen by immunofluorescence, proximity ligation assays (PLA), immunoprecipitation-western blot experiments and GST binding assays. Interestingly, the interaction was elevated in NSCLC samples compared to normal tissue as seen by PLA. A peptide that could disrupt the interaction between YAP1 and Oct4 abrogated Sox2 expression, self-renewal and vascular mimicry. Depleting YAP1 reduced the expression of EMT genes and prevented the growth and metastasis of tumor xenografts in mice, while overexpression of Sox2 in YAP1 depleted cells could rescue self-renewal of stem-like cells. In conclusion, our results demonstrate that YAP1 can modulate the stem-like functions of cancer stem cells by regulating Sox2, and this regulatory pathway can potentially be targeted for combating NSCLC. Citation Format: Namrata Bora Singhal, Jonathan Nguyen, Courtney Schaal, Deepak Perumal, Sandeep Singh, Domenico Coppola, Srikumar Chellappan. YAP1 regulates Oct4 activity and Sox2 expression, facilitating self-renewal and vascular mimicry of stem-like cells. [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 1516. doi:10.1158/1538-7445.AM2015-1516

Gli1-Mediated Regulation of Sox2 Facilitates Self-Renewal of Stem-Like Cells and Confers Resistance to EGFR Inhibitors in Non–Small Cell Lung Cancer

Non–small cell lung cancer (NSCLC) patients have very low survival rates because the current therapeutic strategies are not fully effective. Although EGFR tyrosine kinase inhibitors are effective for NSCLC patients harboring EGFR mutations, patients invariably develop resistance to these agents. Alterations in multiple signaling cascades have been associated with the development of resistance to EGFR inhibitors. Sonic Hedgehog and associated Gli transcription factors play a major role in embryonic development and have recently been found to be reactivated in NSCLC, and elevated Gli1 levels correlate with poor prognosis. The Hedgehog pathway has been implicated in the functions of cancer stem cells, although the underlying molecular mechanisms are not clear. In this context, we demonstrate that Gli1 is a strong regulator of embryonic stem cell transcription factor Sox2. Depletion of Gli1 or inhibition of the Hedgehog signaling significantly abrogated the self-renewal of stem-like side-population cells from NSCLCs as well as vascular mimicry of such cells. Gli1 was found to transcriptionally regulate Sox2 through its promoter region, and Gli1 could be detected on the Sox2 promoter. Inhibition of Hedgehog signaling appeared to work cooperatively with EGFR inhibitors in markedly reducing the viability of NSCLC cells as well as the self-renewal of stem-like cells. Thus, our study demonstrates a cooperative functioning of the EGFR signaling and Hedgehog pathways in governing the stem-like functions of NSCLC cancer stem cells and presents a novel therapeutic strategy to combat NSCLC harboring EGFR mutations.

YAP1 Regulates OCT4 Activity and SOX2 Expression to Facilitate Self-Renewal and Vascular Mimicry of Stem-Like Cells.

Non-small cell lung cancer (NSCLC) is highly correlated with smoking and has very low survival rates. Multiple studies have shown that stem-like cells contribute to the genesis and progression of NSCLC. Our results show that the transcriptional coactivator yes-associated protein 1 (YAP1), which is the oncogenic component of the Hippo signaling pathway, is elevated in the stem-like cells from NSCLC and contributes to their self-renewal and ability to form angiogenic tubules. Inhibition of YAP1 by a small molecule or depletion of YAP1 by siRNAs suppressed self-renewal and vascular mimicry of stem-like cells. These effects of YAP1 were mediated through the embryonic stem cell transcription factor, Sox2. YAP1 could transcriptionally induce Sox2 through a physical interaction with Oct4; Sox2 induction occurred independent of TEAD2 transcription factor, which is the predominant mediator of YAP1 functions. The binding of Oct4 to YAP1 could be detected in cell lines as well as tumor tissues; the interaction was elevated in NSCLC samples compared to normal tissue as seen by proximity ligation assays. YAP1 bound to Oct4 through the WW domain, and a peptide corresponding to this region could disrupt the interaction. Delivery of the WW domain peptide to stem-like cells disrupted the interaction and abrogated Sox2 expression, self-renewal, and vascular mimicry. Depleting YAP1 reduced the expression of multiple epithelial-mesenchymal transition genes and prevented the growth and metastasis of tumor xenografts in mice; overexpression of Sox2 in YAP1 null cells rescued these functions. These results demonstrate a novel regulation of stem-like functions by YAP1, through the modulation of Sox2 expression.

Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer.

Advanced cancers display cellular heterogeneity driven by self-renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying and isolating cell populations that possess differences in self-renewal and tumor initiation. To overcome these barriers in triple-negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well-established pluripotency gene NANOG. NANOG-GFP+ cells gave rise to both GFP+ and GFP(-) cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self-renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well-established CSC markers CD24(-) /CD44(+) , CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG-GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule-A (JAM-A). JAM-A was highly expressed in GFP+ cells and patient-derived xenograft ALDH+ CSCs compared with the GFP(-) and ALDH(-) cells, respectively. Depletion of JAM-A compromised self-renewal, whereas JAM-A overexpression induced self-renewal in GFP(-) cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non-CSCs in TNBC that can be used to identify CSC-specific targets for the development of future therapeutic strategies.

Abstract P2-06-04: Proteosome inhibitor bortezomib inhibits NFκB and effectively overcomes cancer stem cell escape triggered by Wnt inhibitor therapy in FOXC1+ basal-Like/claudin-low breast cancer

Abstract Cancer stem cells (CSCs) are considered to be an important contributing factor towards treatment failure, cancer recurrence, and mortality. CSCs are known to be more enriched in the basal-like and claudin-low subtypes of breast cancer. The Wnt/β catenin signaling pathway is well known as a regulator of embryonic development and stem cell biology, and is prominently active in basal-like and claudin-low breast cancers. We have previously reported that transcription factor FOXC1 plays a critical role in mediating aggressive cell traits in basal-like/claudin-low breast cancer. In this study, we sought to investigate the link between Wnt signaling and FOXC1 and its potential in regulating CSC biology in basal-Like/claudin-low breast cancer. We observed that exposure of the MDA-MB-231 basal-like/claudin-low cell line (low constitutive FOXC1 expressor) to Wnt3a (a canonical Wnt signaling ligand), resulted in increased expression of FOXC1. Reciprocally, overexpression of FOXC1 in MCF10A human mammary epithelial cells led to a pronounced increase in Wnt signaling activity, strongly suggestive of a direct or indirect positive feedback loop between Wnt signaling and FOXC1. More importantly, BT549 and HS578t basal-like/claudin-low cells (high constitutive FOXC1 expressors) proved to be more sensitive to treatment with the Wnt inhibitor ICRT3 as evidenced by decreased cell viability when compared to MCF7 (luminal) or SKBR3 (HER2) breast cancer cell lines. Furthermore the decrease in cell viability appeared to be proportionate to the level of FOXC1 expression. Upon pharmacological inhibition with ICRT3 and biological inhibition with siRNA knockdown of LRP6, (a canonical Wnt signaling cell surface receptor) a decrease in FOXC1 expression level was observed in a dose and time dependent manner. This effect was particularly pronounced in mammosphere cultures enriched for BT549 cancer stem-like cells. Inhibition of Wnt signaling reduced mammosphere formation efficiency of BT549 cells, suggesting that Wnt inhibition targets cancer stem cells (CSCs) in the basal-like/claudin-low breast cancer subtype. More importantly, however, after an initial 4 day incubation period, some cells are observed to persist and later display renewed enhancement of mammosphere formation ability. Profiling of such cells interestingly revealed depletion of FOXC1+ve cells but persistence of cells displaying pronounced up regulation of stereotypical embryonic stem cell Transcription Factors OCT4, SOX2 and NANOG, strongly suggestive of a potential primitive stem cell/quiescent cell state escape mechanism. qRT-PCR based pathway activation analysis revealed marked activation of NFκB signaling in the residual cells that withstood Wnt inhibition. Simultaneous pharmacologic inhibition with Wnt inhibitor ICRT3 and the proteasome inhibitor Bortezomib (known to inhibit NFκB signaling) effectively targeted BT549 cancer stem cells in mammosphere culture and prevented the persistence/emergence of any residual cells. Taken together, our findings suggest that combination therapy approaches are likely required to effectively target breast cancer stem cells. Citation Format: Partha S Ray, Connie Y Tsai, Tania Ray, Bomy Kim, Tor W Jensen. Proteosome inhibitor bortezomib inhibits NFκB and effectively overcomes cancer stem cell escape triggered by Wnt inhibitor therapy in FOXC1+ basal-Like/claudin-low breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-04.

Abstract P2-06-01: Development of a fluorescent reporter system to delineate self-renewing cells in triple negative breast cancer

Abstract Background : Advanced cancers including triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, contain a self-renewing, tumorigenic cancer stem cell (CSC) population. CSCs contribute to tumor progression and therapeutic resistance. Despite an effective early response to chemotherapy, TNBC relapses with a highly heterogeneous and refractory metastatic disease enriched in CSCs. Apart from being chemo- and radio-resistant, CSCs display a high degree of multipotency, heterogeneity and plasticity. Due to the complex nature of CSCs, elucidating the characteristics of CSCs in TNBC progression continues to be a challenge. Thus, developing anti-CSC therapies to be integrated into clinical paradigms represents an immediate priority. Rationale : A major obstacle to the identification of CSC regulatory mechanisms is the lack of experimental systems that enable the reliable enrichment of CSCs from non-CSCs for comparative analysis. While CSCs have been isolated from TNBC using CD44+/CD24- and/or ALDH+ phenotype, this enrichment paradigm requires refinement as it is not universally applicable and lacks the ability to study CSCs in real time. The limitations of these systems have excluded their application in studying the molecular heterogeneity among breast cancer tumors. CSCs are molecularly characterized based on the expression of cell surface receptors and the embryonic stem cell transcription factors NANOG, OCT4 and SOX2. NANOG, the master regulator of stem cell self-renewal, has emerged as a pro-carcinogenic factor in cancer cell lines with CSC behavior. Our previous studies have shown that silencing NANOG in cancer cells leads to reduced proliferation and self-renewal based on in vitro and in vivo experiments. Hypothesis : We hypothesized that a Nanog promoter could be used to effectively enrich for TNBC CSCs. Results : We generated two TNBC cell lines (MDA-MB-231 and HCC70) harboring NanogGFP reporter gene by lentiviral transductions. Increased NANOG mRNA and protein expression was observed in flow cytometry-sorted GFP+ cells compared with GFP- cells. GFP+ cells were enriched for the CSC markers CD49f and CD44+/CD24-. GFP+ cells also demonstrated an increased protein expression of the stem cell transcription factors NANOG, SOX2 and OCT4. Limiting dilution analyses revealed increased self-renewal and significantly higher stem cell frequencies in GFP+ cells. GFP+ cells demonstrated a mesenchymal phenotype with increased invasive capacity. Subcutaneous injections of GFP+ cells showed significantly higher in vivo tumor initiation and progression than GFP- cells. Flow cytometry-sorted GFP+ and GFP- cells enriched for CSCs and non-CSCs, respectively. Using this system, we performed a high-throughput flow cytometry screen and identified an additional novel CSC marker for TNBC. Conclusion : We have developed a novel TNBC CSC reporter system using a GFP reporter driven by the Nanog promoter and identified a novel CSC marker. We have defined and validated this robust system wherein we have characterized and monitored the role of CSCs and non-CSCs in TNBC tumor initiation and progression. Using this approach, identifying CSC targets in TNBC could unravel the potential for development of innovative therapeutic strategies. Citation Format: Praveena S Thiagarajan, Masahiro Hitomi, James S Hale, Alvaro G Alvarado, Baltin Otvos, Kevin Stoltz, Maksim Sinyuk, Awad Jarrar, Qiao Zheng, Dustin Thomas, Thomas Egelhoff, Jeremy N Rich, Justin D Lathia, Ofer Reizes. Development of a fluorescent reporter system to delineate self-renewing cells in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-01.

Retroviral transcriptional regulation and embryonic stem cells: war and peace.

Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps "noisy" control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks.