Quiescent Cancer Stem Cells Research Articles

Quiescent Human Glioblastoma Stem Cells Mediate Tumor Initiation, Expansion, and Recurrence Following Chemotherapy

We test the hypothesis that human glioblastoma harbors quiescent cancer stem cells that evade anti-proliferative therapies. Functional characterization of spontaneous glioblastomas from genetically engineered mice reveals essential quiescent stem-like cells that can be directly isolated from tumors. A derived novel quiescent cancer stem cell specific gene expression signature is enriched in pre-formed patient xenograft single cell clusters that lack proliferative gene expression. A refined human 118-gene signature is preserved in quiescent single cell populations from primary and recurrent human glioblastomas. The signature conserved F3 cell surface receptor identifies quiescent tumor cells by antibody immunohistochemistry. F3 antibody sorted quiescent glioblastoma cells exhibit stem cell gene expression, enhanced self-renewal in culture, mediate tumor initiation, serial transplantation, and reconstitute tumor heterogeneity. Upon chemotherapy, the spared cancer stem cell pool becomes activated and accelerates transition to proliferation. These results help explain conventional treatment failure and lay a conceptual framework for alternative therapies.

Markers and Reporters to Reveal the Hierarchy in Heterogeneous Cancer Stem Cells.

A subpopulation within cancer, known as cancer stem cells (CSCs), regulates tumor initiation, chemoresistance, and metastasis. At a closer look, CSCs show functional heterogeneity and hierarchical organization. The present review is an attempt to assign marker profiles to define the functional heterogeneity and hierarchical organization of CSCs, based on a series of single-cell analyses. The evidences show that analogous to stem cell hierarchy, self-renewing Quiescent CSCs give rise to the Progenitor CSCs with limited proliferative capacity, and later to a Progenitor-like CSCs, which differentiates to Proliferating non-CSCs. Functionally, the CSCs can be tumor-initiating cells (TICs), drug-resistant CSCs, or metastasis initiating cells (MICs). Although there are certain marker profiles used to identify CSCs of different cancers, molecules like CD44, CD133, ALDH1A1, ABCG2, and pluripotency markers [Octamer binding transcriptional factor 4 (OCT4), SOX2, and NANOG] are used to mark CSCs of a wide range of cancers, ranging from hematological malignancies to solid tumors. Our analysis of the recent reports showed that a combination of these markers can demarcate the heterogeneous CSCs in solid tumors. Reporter constructs are widely used for easy identification and quantification of marker molecules. In this review, we discuss the suitability of reporters for the widely used CSC markers that can define the heterogeneous CSCs. Since the CSC-specific functions of CD44 and CD133 are regulated at the post-translational level, we do not recommend the reporters for these molecules for the detection of CSCs. A promoter-based reporter for ABCG2 may also be not relevant in CSCs, as the expression of the molecule in cancer is mainly regulated by promoter demethylation. In this context, a dual reporter consisting of one of the pluripotency markers and ALDH1A1 will be useful in marking the heterogeneous CSCs. This system can be easily adapted to high-throughput platforms to screen drugs for eliminating CSCs.

RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence

SummaryRecent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to damage-induced quiescent revival stem cells of the regenerating intestine. In addition, we identify negative regulators of cell cycle, downstream of p53, that we show are indicators of poor prognosis and may be targeted for qCSC abolition in both p53 wild-type and mutant tumors. These data support the temporal inhibition of downstream targets of p53 signaling, in combination with standard-of-care treatments, for the elimination of qCSCs and prevention of relapse in colon cancer.

Reporters of Cancer Stem Cells as a Tool for Drug Discovery.

In view of the importance of cancer stem cells (CSCs) in chemoresistance, metastasis and recurrence, the biology of CSCs were explored in detail. Based on that, several modalities were proposed to target them. In spite of the several clinical trials, a successful CSC-targeting drug is yet to be identified. The number of molecules screened and entered for clinical trial for CSC-targeting is comparatively low, compared to other drugs. The bottle neck is the lack of a high-throughput adaptable screening strategy for CSCs. This review is aimed to identify suitable reporters for CSCs that can be used to identify the heterogeneous CSC populations, including quiescent CSCs, proliferative CSCs, drug resistant CSCs and metastatic CSCs. Analysis of the tumor microenvironment regulating CSCs revealed that the factors in CSC-niche activates effector molecules that function as CSC markers, including pluripotency markers, CD133, ABCG2 and ALDH1A1. Among these factors OCT4, SOX2, NANOG, ABCG2 and ALDH1A1 are ideal for making reporters for CSCs. The pluripotency molecules, like OCT4, SOX2 and NANOG, regulate self-renewal, chemoresistance and metastasis. ABCG2 is a known regulator of drug resistance while ALDH1A1 modulates self-renewal, chemoresistance and metastasis. Considering the heterogeneity of CSCs, including a quiescent population and a proliferative population with metastatic ability, we propose the use of a combination of reporters. A dual reporter consisting of a pluripotency marker and a marker like ALDH1A1 will be useful in screening drugs that target CSCs.

Stem cell quiescence and its clinical relevance.

Quiescent state has been observed in stem cells (SCs), including in adult SCs and in cancer SCs (CSCs). Quiescent status of SCs contributes to SC self-renewal and conduces to averting SC death from harsh external stimuli. In this review, we provide an overview of intrinsic mechanisms and extrinsic factors that regulate adult SC quiescence. The intrinsic mechanisms discussed here include the cell cycle, mitogenic signaling, Notch signaling, epigenetic modification, and metabolism and transcriptional regulation, while the extrinsic factors summarized here include microenvironment cells, extracellular factors, and immune response and inflammation in microenvironment. Quiescent state of CSCs has been known to contribute immensely to therapeutic resistance in multiple cancers. The characteristics and the regulation mechanisms of quiescent CSCs are discussed in detail. Importantly, we also outline the recent advances and controversies in therapeutic strategies targeting CSC quiescence.

Regulatory Role of Quiescence in the Biological Function of Cancer Stem Cells.

Quiescence in cancer cells is considered a therapeutic challenge as it confers dormancy in tumour, hence circumventing inherent anti-neoplastic surveillance system and standard-of-care cancer therapeutics including chemotherapy and radiotherapy. Since majority of the therapeutics target actively proliferating cancer cells, cancer cells eventually develop quiescent nature as mechanism of survival and cancer progression under both niche and therapeutic pressures. Quiescence state in cancer cells, eventually, confers resistant and aggressive nature to conventional cancer therapies, resulting in disease progression and relapse. Therefore, targeting quiescent cancer cells or cancer stem cells is a promising therapeutic approach, however an extensive review of the relevant information is needed in order to device an effective therapy. While the evidence of quiescence regulation in CSCs is rather a complex molecular and cellular network, herein, we aim to provide a comprehensive understanding of both intrinsic and extrinsic regulation in association with the function of CSCs. Findings on induction of quiescent state in CSCs population, its regulation at both cellular and molecular level, key molecular regulators, cellular events and processes including potential targets to develop therapeutics are extensively reviewed. This review also highlights the impact of CSC plasticity on quiescence which capturing the key challenge of targeting the cells in this state. Beyond understanding the mechanisms underlying quiescence nature of cancer cells, this review provides insightful perspective and future direction on insight in targeting these populations, hence collapse the tumour dormancy programme in order to eradicate tumour mass as a whole. Capability of CSCs to establish quiescent state as a mechanism of survival during unfavorable conditions, as well as its impact in cancer progression and subsequent relapse, including the potential therapeutic strategy to eradicate this CSCs sub-population in the tumor mass as an effective cancer therapy.

1942P IL6 pre-treatment promotes chemosensitivity by eliminating quiescent cancer stem cells

The blockade of entering the cell cycle represents a hallmark of chemoresistant cancer cells, especially in chemoresistant cancer stem cells. Cell cycle blockade in quiescent cancer (stem) cells is largely attributed to the natural dormant behaviour of stem cells, and in a natural state the quiescent stem cells respond to stimuli rapidly for activation. Thus, eliminating quiescent chemoresistant cancer (stem) cells by extrinsic stimuli that promote entry into cell cycle provides a potential strategy for overcoming chemoresistance. Lung cancer cells were treated with or without IL6 before cDDP chemotherapy and a lung cancer patient derived single cell ex-vivo system and mice xenograft in-vivo model were developed. Here we report that in lung adenocarcinoma the interleukin 6 (IL6) treatment resulted in the expansion of cancer stem cells (EpCAM+/CD133+/CD44+/CD24-) and reduction of quiescent cancer (stem) cells (Hoechst 33342 and Pyronin γ double negative population, G0 cells) in several primary lung adenocarcinoma patient-derived primary lung cancer cells. IL6 pre-treatment increased cDDP chemosensitivity in lung adenocarcinoma by promoting quiescent lung cancer (stem) cells to enter the cell cycle. In parallel, we showed a mechanism for regulating the sensitivity of the IL6-STAT3 pathway to IL6 treatment, by ChIP-PCR and duo-luciferase reporter assay we identified an AP2 transcription factor TFAP2A activates the expression of IL6 receptor transcriptionally in lung cancer cell lines. Moreover, using a mice tail vein xenograft in vivo chemotherapy model with lung cancer cell lines, we report that IL6 pre-treatment significantly induced more cell death in platinum-based chemotherapy. Our data indicate that IL6 contributes to quiescent lung cancer (stem) cells entering the cell cycle, which significantly undermines chemoresistance and introduces a considerable clinical approach for platinum-based chemotherapy in lung adenocarcinoma.

Aggressiveness Potential of Spontaneous Canine Mucosal Melanoma Can Dictate Distinct Cancer Stem Cell Compartment Behaviors in Regard to Their Initial Size and Expansion Abilities.

Mucosal melanoma represents one of the most highly metastatic and aggressive subtypes of melanoma. The biology of mucosal melanoma is poorly documented, and the lack of experimental models makes it difficult to design and test new therapies. Dogs are frequently affected by melanomas of the oral cavity, making spontaneous canine melanoma a potentially predictable model for their human counterpart. We recently established and characterized two new canine mucosal melanoma cell lines named OCR_OCMM1 and OCR_OCMM2. Here, we identified quiescent cancer stem cell (CSC) subpopulations in both canine cell lines that displayed similarities to human quiescent CSCs: canine melanoma CSCs had the ability to self-renew, produced nonstem cell (SC) progeny, and formed melanospheres that recapitulated the phenotypic profile of the parental tumor. These CSCs also formed melanoma in immunodeficient mice, and the inhibition of PI3K/AKT signaling expanded the CSC pool. A subset of non-CSCs transitioned to become CSCs. OCR_OCMM1 and OCR_OCMM2 displayed different CSC compartment behaviors in regard to their initial size and expansion abilities. Collectively, this study showed that the OCR_OCMM1 and OCR_OCMM2 canine melanoma cell lines are powerful cellular tools to study melanoma SCs, not only for mucosal but also for the more common human cutaneous melanoma.

Abstract A81: Targeting EZH2/DAB2IP/Wnt axis in ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies. Recent data have pointed to the persistence of quiescent ovarian cancer stem cells (OCSCs) not eliminated by chemotherapy and able to regenerate tumors as the main contributor to tumor relapse and metastasis. Downregulation of tumor suppressor DAB2IP significantly correlated with poor patient survival in OC. In the current study, we tested the hypothesis that DAB2IP is downregulated by EZH2 methylation in OC and targeting DAB2IP inhibits OCSCs, which would prevent disease recurrence. Subpopulations of CSC and non-CSC were isolated from OC cell lines by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity, a consistent CSC marker. Expression of DAB2IP in ALDH(+) cells was lower (P<0.05) compared to non-CSC ALDH(-) cells, which can be restored by EZH2 inhibition. Chromatin immunoprecipitation (ChIP) analysis demonstrated enrichment (P<0.05) of H3K27me3 at DAB2IP promoter loci in CSC compared to non-CSC. Furthermore, inhibiting EZH2 decreased H3K27me3 and increased (P<0.05) DAB2IP expression in OC cells, demonstrating that DAB2IP downregulation in CSC was due to EZH2. Knocking out DAB2IP using CRISPR/Cas9 system in OC cell lines upregulated (P<0.05) expression of stemness-related genes and increased (P<0.05) the percentage of ALDH(+) cells. Enforced overexpression of DAB2IP decreased (P<0.05) the number of ALDH(+) cells and inhibited (P<0.05) both spheroid and colony formation. Furthermore, elevated DAB2IP expression decreased (P<0.05) cisplatin IC50 of both OCSCs and OC cells and inhibited (P<0.05) cell migration capacity, suggesting DAB2IP plays a role in regulating OCSC function. OVCAR3 cells and DAB2IP-overexpressing OVCAR3 cells were further analyzed by RNA-sequencing and bioinformatics. Transcriptome analysis revealed that DAB2IP overexpression significantly (FDR < 0.05, fold change > 2) altered expression of 449 genes, including downregulation of ALDH1A1, LGR5, PROM1, and TWIST1, markers strongly associated with CSC phenotypes. Ingenuity Pathway Analysis for upstream regulators of differentially expressed genes revealed Wnt-signaling as a dominant pathway mediating the anti-OCSC effects of DAB2IP. Based on RNA-seq analysis, WNT5B expression decreased (P<0.05) by 3.83 fold, indicating that DAB2IP may negatively regulate Wnt signaling pathway by repressing WNT5B. Furthermore, WNT5B recombinant protein significantly increased (P<0.05) the OCSC population, and reverse phase protein array analysis demonstrated activation of JNK/c-Jun as a possible downstream target of WNT5B. Collectively, our data reveal that DAB2IP, via noncanonical Wnt-mediated signaling pathway, plays a critical role in modulating OCSC properties. Based on these novel findings, we are testing novel combination treatment strategies targeting OCSCs and with the goal of inhibiting tumor relapse and overcoming chemoresistance. Citation Format: Xingyue Zong, Ali Ozes, Weini Wang, Kenneth P. Nephew. Targeting EZH2/DAB2IP/Wnt axis in ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A81.

Abstract B56: Tissue transglutaminase/Frizzled receptor clusters regulate WNT transcriptional activity in ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the most lethal gynecologic cancer, characterized by chemoresistance and fatal tumor recurrence after primary treatment. The metastatic progression and post-chemotherapy recurrence of OC is linked to a small population of cancer stem cells (CSCs) detectable as ALDH+/CD133+ phenotype. In OC, the tumor microenvironment (TME) provides a favorable milieu that protects quiescent CSCs during chemotherapy and supports their tumorigenic functions. The multifunctional protein tissue transglutaminase (TG2), with enzymatic and scaffold functions, is an important molecule secreted in the TME where it modulates oncogenic signaling by interacting with extracellular matrix (ECM) components, such as fibronectin (FN) and integrins. Its aberrant expression is correlated with OC progression and with the tumorigenic OCSC phenotype. Here, we hypothesized that the formation of the TG2/FN/integrins ternary complex at the cell membrane promotes survival of CSCs and supports OC spheroid formation by regulating the Wnt/beta-catenin pathway. Our data demonstrate that TG2, FN, and integrin β1 mRNA expression is markedly increased in OCSCs (ALDH+/CD133+) compared with non-CSC (ALDH-/CD133-) and highly enriched in OC cells grown as spheroids compared with monolayers (p<0.01). Immunofluorescence (IF) staining showed abundant complexes of TG2 with integrin β1 and FN in OC cells grown as spheroids, and the protein-protein interactions were detected in human tumors by proximity ligation assay. TG2 expression was strongly correlated with ITGB1 (R=0.23, P<0.0001) and with FN1 (R=0.39, P<0.0001) in the TCGA ovarian cancer database. Use of function-inhibiting antibody against the FN-binding domain of TG2 (4G3) dramatically suppressed spheroid proliferation, TG2/FN/integrin complex formation, and tumor growth in a xenograft model. Disruption of TG2/FN complex altered key oncogenic signaling pathways, and in particular blocked the canonical Wnt/β-catenin signaling pathway essential to sustaining cancer cell stemness. The Wnt receptor Frizzled genes (Fzd1, Fzd7 and Fzd8) showed the greatest difference in gene expression, indicating a direct correlation between TG2/FN complex and Wnt pathway activation. Co-immunoprecipitation and IF staining demonstrated that TG2 co-localizes and directly binds to Fzd7. Furthermore, protein docking and peptide inhibition demonstrated that the interaction between TG2 and Fzd7 overlapped with the FN-binding domain of TG2. Finally, blockade of TG2/FN complex and shRNA-mediated Fzd7 knockdown attenuated spheroids proliferation and targeted gene expression induced by Wnt3a/7a. This study provides strong evidence supporting a key function of TG2/FN complexes in OCSCs. By demonstrating that TG2 directly binds Fzd7, we identified a novel function of TG2 and a new mechanism promoting CSCs’ proliferation and tumorigenicity. These results point to TG2/FN clusters or the newly discovered TG2/Fzd7 complex as potential new therapeutic CSC targets. Citation Format: Salvatore Condello, Livia Sima, Cristina Ivan, Horacio Cardenas, Gary Schiltz, Rama Mishra, Daniela Matei. Tissue transglutaminase/Frizzled receptor clusters regulate WNT transcriptional activity in ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B56.

Mitochondrial Stress-Mediated Targeting of Quiescent Cancer Stem Cells in Oral Squamous Cell Carcinoma.

IntroductionDespite improved therapeutics in oral squamous cell carcinoma (OSCC), tumor cells that are either quiescent and/or endowed with stem cell–like attributes usually survive treatment and recreate tumor load at relapse. Through this study, we aimed strategically to eliminate these stem cell–like cancer cells using a combination drug approach.MethodsPrimary cultures from 15 well–moderately differentiated OSCC were established, and the existence of cancer cells with stem cell–like characteristics using five cancer stem cell (CSC) specific markers — CD44, CD133, CD147, C166, SOX2 and spheroid assay was ascertained. Next, we assessed quiescence in CSCs under normal and growth factor–deprived conditions using Ki67. Among several gene signatures regulating quiescent cellular state, we evaluated the effect of inhibiting Dyrk1b in combination with topoisomerase II and histone deacetylase inhibitors in targeting quiescent CSCs. Multiple drug-effect analysis was carried out with CompuSyn software to determine combination-index values.ResultsWe observed that CD44+CD133+ showed the highest level of SOX2 expression. CSCs showed varying degrees of quiescence, and inhibition of Dyrk1b decreased quiescence and sensitized CSCs to apoptosis. In the drug-combination study, Dyrk1b inhibitor was combined with topoisomerase II and histone deacetylase inhibitors to target quiescent CSCs. In combination, a synergistic effect was seen even at a 16-fold lower dose than IC50. Furthermore, combined treatment decreased glutathione levels and increased ROS and mitochondrial stress, leading to increased DNA damage and cytochrome c in CSCs.ConclusionWe report marker-based identification of CSC subpopulations and synergy of Dyrk1b inhibitor with topoisomerase II and HDAC inhibitors in primary OSCC. The results provide a new therapeutic strategy to minimize quiescence and target oral CSCs simultaneously.

Chemosensitization of prostate cancer stem cells in mice by angiogenin and plexin-B2 inhibitors

Cancer stem cells (CSCs) are an obstacle in cancer therapy and are a major cause of drug resistance, cancer recurrence, and metastasis. Available treatments, targeting proliferating cancer cells, are not effective in eliminating quiescent CSCs. Identification of CSC regulators will help design therapeutic strategies to sensitize drug-resistant CSCs for chemo-eradication. Here, we show that angiogenin and plexin-B2 regulate the stemness of prostate CSCs, and that inhibitors of angiogenin/plexin-B2 sensitize prostate CSCs to chemotherapy. Prostate CSCs capable of self-renewal, differentiation, and tumor initiation with a single cell inoculation were identified and shown to be regulated by angiogenin/plexin-B2 that promotes quiescence and self-renewal through 5S ribosomal RNA processing and generation of the bioactive 3′-end fragments of 5S ribosomal RNA, which suppress protein translation and restrict cell cycling. Monoclonal antibodies of angiogenin and plexin-B2 decrease the stemness of prostate CSCs and sensitize them to chemotherapeutic agents in vitro and in vivo.

A pre-existing population of ZEB2+ quiescent cells with stemness and mesenchymal features dictate chemoresistance in colorectal cancer

BackgroundQuiescent/slow cycling cells have been identified in several tumors and correlated with therapy resistance. However, the features of chemoresistant populations and the molecular factors linking quiescence to chemoresistance are largely unknown.MethodsA population of chemoresistant quiescent/slow cycling cells was isolated through PKH26 staining (which allows to separate cells on the basis of their proliferation rate) from colorectal cancer (CRC) xenografts and subjected to global gene expression and pathway activation analyses. Factors expressed by the quiescent/slow cycling population were analyzed through lentiviral overexpression approaches for their ability to induce a dormant chemoresistant state both in vitro and in mouse xenografts. The correlation between quiescence-associated factors, CRC consensus molecular subtype and cancer prognosis was analyzed in large patient datasets.ResultsUntreated colorectal tumors contain a population of quiescent/slow cycling cells with stem cell features (quiescent cancer stem cells, QCSCs) characterized by a predetermined mesenchymal-like chemoresistant phenotype. QCSCs expressed increased levels of ZEB2, a transcription factor involved in stem cell plasticity and epithelial-mesenchymal transition (EMT), and of antiapototic factors pCRAF and pASK1. ZEB2 overexpression upregulated pCRAF/pASK1 levels resulting in increased chemoresistance, enrichment of cells with stemness/EMT traits and proliferative slowdown of tumor xenografts. In parallel, chemotherapy treatment of tumor xenografts induced the prevalence of QCSCs with a stemness/EMT phenotype and activation of the ZEB2/pCRAF/pASK1 axis, resulting in a chemotherapy-unresponsive state. In CRC patients, increased ZEB2 levels correlated with worse relapse-free survival and were strongly associated to the consensus molecular subtype 4 (CMS4) characterized by dismal prognosis, decreased proliferative rates and upregulation of EMT genes.ConclusionsThese results show that chemotherapy-naive tumors contain a cell population characterized by a coordinated program of chemoresistance, quiescence, stemness and EMT. Such population becomes prevalent upon drug treatment and is responsible for chemotherapy resistance, thus representing a key target for more effective therapeutic approaches.

Building the case for the calcitonin receptor as a viable target for the treatment of glioblastoma.

Researchers are actively seeking novel targeted therapies for the brain tumour glioblastoma (GBM) as the mean survival is less than 15 months. Here we discuss the proposal that the calcitonin receptor (CT Receptor), expressed in 76–86% of patient biopsies, is expressed by both malignant glioma cells and putative glioma stem cells (GSCs), and therefore represents a potential therapeutic target. Forty-two per cent (42%) of high-grade glioma (HGG; representative of GSCs) cell lines express CT Receptor protein. CT Receptors are widely expressed throughout the life cycle of organisms and in some instances promote apoptosis. Which of the common isoforms of the CT Receptor are predominantly expressed is currently unknown, but a functional response to cell stress of the insert-positive isoform is hypothesised. A model for resistant malignancies is one in which chemotherapy plays a direct role in activating quiescent stem cells for replacement of the tumour tissue hierarchy. The putative role that the CT Receptor plays in maintenance of quiescent cancer stem cells is discussed in view of the activation of the Notch–CT Receptor–collagen V axis in quiescent muscle (satellite) stem cells. The pharmacological CT response profiles of four of the HGG cell lines were reported. Both CT responders and non-responders were sensitive to an immunotoxin based on an anti-CT Receptor antibody. The CALCR mRNA exhibits alternative splicing commonly associated with cancer cells, which could result in the atypical pharmacology exhibited by CT non-responders and an explanation of tumour suppression. Due to the inherent instability of CALCR mRNA, analysis of CT Receptor protein in patient samples will lead to improved data for the expression of CT Receptor in GBM and other cancers, and an understanding of the role and activity of the splice variants. This knowledge will aid the effective targeting of this receptor for treatment of GBM.

Stem cell programs in cancer initiation, progression, and therapy resistance

Over the past few decades, substantial evidence has convincingly revealed the existence of cancer stem cells (CSCs) as a minor subpopulation in cancers, contributing to an aberrantly high degree of cellular heterogeneity within the tumor. CSCs are functionally defined by their abilities of self-renewal and differentiation, often in response to cues from their microenvironment. Biological phenotypes of CSCs are regulated by the integrated transcriptional, post-transcriptional, metabolic, and epigenetic regulatory networks. CSCs contribute to tumor progression, therapeutic resistance, and disease recurrence through their sustained proliferation, invasion into normal tissue, promotion of angiogenesis, evasion of the immune system, and resistance to conventional anticancer therapies. Therefore, elucidation of the molecular mechanisms that drive cancer stem cell maintenance, plasticity, and therapeutic resistance will enhance our ability to improve the effectiveness of targeted therapies for CSCs. In this review, we highlight the key features and mechanisms that regulate CSC function in tumor initiation, progression, and therapy resistance. We discuss factors for CSC therapeutic resistance, such as quiescence, induction of epithelial-to-mesenchymal transition (EMT), and resistance to DNA damage-induced cell death. We evaluate therapeutic approaches for eliminating therapy-resistant CSC subpopulations, including anticancer drugs that target key CSC signaling pathways and cell surface markers, viral therapies, the awakening of quiescent CSCs, and immunotherapy. We also assess the impact of new technologies, such as single-cell sequencing and CRISPR-Cas9 screening, on the investigation of the biological properties of CSCs. Moreover, challenges remain to be addressed in the coming years, including experimental approaches for investigating CSCs and obstacles in therapeutic targeting of CSCs.

MiR-4666-3p and miR-329 Synergistically Suppress the Stemness of Colorectal Cancer Cells via Targeting TGF-β/Smad Pathway.

Quiescent caner stem cells are identified as a subpopulation of colon cancer cells in dormant state and possess strong stem-cell like characteristics. Previously, we have identified this subpopulation in colorectal cancer (quiescent colon cancer stem cells, QCCSCs), and find QCCSCs are sensitive to the apoptotic effect of IFN-γ, which is attributed to their high IFN-γR expression levels. Microarray and bioinformatic analysis indicate miR-4666-3p is low expressed in QCCSCs and target IFN-γR1/2, which is proved by luciferase assay and western-blot. Furthermore, we find miR-4666-3p could also target TGF-βR1 to block the activation of TGF-β1/Smad pathway, therefore function as a tumor suppressor gene to inhibit the stemness of colon cancer cells. Besides, compared with QCCSCs, we find the TGF-β1 expression also decreased with the weakening of stemness properties. In terms of mechanism, our result reveal TGF-β1 is the target gene of miR-329, which is also high expressed in non-QCCSCs. Thereafter, we perform gain- and loss- function experiments to confirm the synergistic effect between miR-4666-3p and miR-329 in blocking the activation of TGF-β/Smad pathway. Finally, we evaluate the expression of both miR-4666-3p and miR-329 in 73 tumor specimens and paired normal tissue, and find both two miRNAs are related to unfavorable prognosis and advanced tumor stage in colorectal cancer. Our study revealed a novel epigenetic regulation mechanism in colon cancer stem cells, which could be exploited as a novel therapeutic strategy for cancer treatment.

Therapeutic Targeting of Cancer Stem Cells in Human Glioblastoma by Manipulating the Renin-Angiotensin System.

Patients with glioblastoma (GB), a highly aggressive brain tumor, have a median survival of 14.6 months following neurosurgical resection and adjuvant chemoradiotherapy. Quiescent GB cancer stem cells (CSCs) invariably cause local recurrence. These GB CSCs can be identified by embryonic stem cell markers, express components of the renin-angiotensin system (RAS) and are associated with circulating CSCs. Despite the presence of circulating CSCs, GB patients rarely develop distant metastasis outside the central nervous system. This paper reviews the current literature on GB growth inhibition in relation to CSCs, circulating CSCs, the RAS and the novel therapeutic approach by repurposing drugs that target the RAS to improve overall symptom-free survival and maintain quality of life.

BMP signaling mediates glioma stem cell quiescence and confers treatment resistance in glioblastoma

Despite advances in therapy, glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells, GSCs) as mediators of therapeutic resistance and tumor progression. In this study, we investigated the role of the transforming growth factor-β (TGF-β) superfamily, which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems, as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-β signaling define divergent molecular and functional identities in glioblastoma, and mark relatively quiescent and proliferative GSCs, respectively. Treatment of GSCs with BMP inhibits cell proliferation, but does not abrogate their stem-ness, as measured by self-renewal and tumorigencity. Further, BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy.

SET Domain-Containing Protein 4 Epigenetically Controls Breast Cancer Stem Cell Quiescence.

Quiescent cancer stem cells (CSC) play important roles in tumorigenesis, relapse, and resistance to chemoradiotherapy. However, the determinants of CSC quiescence and how they sustain themselves to generate tumors and relapse beyond resistance to chemoradiotherapy remains unclear. Here, we found that SET domain-containing protein 4 (SETD4) epigenetically controls breast CSC (BCSC) quiescence by facilitating heterochromatin formation via H4K20me3 catalysis. H4K20me3 localized to the promoter regions and regulated the expression of a set of genes in quiescent BCSCs (qBCSC). SETD4-defined qBCSCs were resistant to chemoradiotherapy and promoted tumor relapse in a mouse model. Upon activation, a SETD4-defined qBCSC sustained itself in a quiescent state by asymmetric division and concurrently produced an active daughter cell that proliferated to produce a cancer cell population. Single-cell sequence analysis indicated that SETD4+ qBCSCs clustered together as a distinct cell type within the heterogeneous BCSC population. SETD4-defined quiescent CSCs were present in multiple cancer types including gastric, cervical, ovarian, liver, and lung cancers and were resistant to chemotherapy. SETD4-defined qBCSCs had a high tumorigenesis potential and correlated with malignancy and chemotherapy resistance in clinical breast cancer patients. Taken together, the results from our previous study and current study on six cancer types reveal an evolutionarily conserved mechanism of cellular quiescence epigenetically controlled by SETD4. Our findings provide insights into the mechanism of tumorigenesis and relapse promoted by SETD4-defined quiescent CSCs and have broad implications for clinical therapies. SIGNIFICANCE: These findings advance our knowledge on the epigenetic determinants of quiescence in cancer stem cell populations and pave the way for future pharmacologic developments aimed at targeting drug-resistant quiescent stem cells.

Abstract 4398: KDM3A and DCLK1 interactions promote stemness and tumorigenesis in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the major leading cause of cancer related human death in USA. There is no possible treatment or target available in PDAC. It was proposed that the cancer stem cells (CSCs) can regulates malignancy in PDAC. DCLK1 is one of the quiescent cancer stem cell marker in PDAC that can regulate tumor progression. Identification of the key factors that influence stemness will help to target PDAC. Histone lysine demethylase KDM3A is an enzyme/protein which can regulate stem cell renewability and tumor progression, thereby KDM3A can interact with DCLK1 for tumor progression. KDM3A influences tumor growth and regulates stemness. Therefore, our goal is to find out the the role of KDM3A in tumor progression in PDAC through interactions with DCLK1. We observed expression of KDM3A in both PDAC patients’ samples and cells. Knockdown and overexpression of KDM3A were executed by using lentiviral vector. Tumor progression were also observed in orthotopic mice model. ChIP and RNA seq were performed to validate the data. KDM3A was overexpressed in human PDAC patient tissues and human pancreatic cancer cells with concomitant increase of CSC marker, DCLK1. Moreover, DCLK1 and KDM3A was found to be co-localized in patient tissue samples and identified binding sites of KDM3A with DCLK1 using ChIP-seq. Knockdown of KDM3A abrogates oncogenic potential whereas, overexpressed KDM3A in transformed HPNE cells showed malignant properties with enhanced invasive property, pancosphere formation, foci formation and tumor formation in mouse. Moreover, ChIP-seq and RNA seq suggested that KDM3A regulated DCLK1 expression in tumor progression. In conclusion, co-expression of DCLK1 with KDM3A influenced stemness and enhance tumor progression in PDAC. Citation Format: Chandrayee Ghosh, Kankaraj Palaniyandi, Santanu Paul, Prasad Dandawate, Sonia Rawal, Dharmalingam Subramaniam, Subhash Padhye, Sumedha Gunewardena, Sufi Thomas, Maura O'Neil, Roy Jensen, Danny Welch, Sally Milisky, Scott Weir, Tomoo Iwakuma, Shrikant Anant, Animesh Dhar. KDM3A and DCLK1 interactions promote stemness and tumorigenesis in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4398.