Maintenance Of Tumor-initiating Cells Research Articles

Abstract 3967: A novel translation program regulates tumor-initiating potential and chemoresistance in multiple myeloma

Abstract Background: Despite the introduction of novel therapies, the majority of the multiple myeloma (MM) patients eventually relapse and develop chemoresistance, and the factors responsible for disease progression are not fully understood. Relapse suggest that MM cells that are resistant to chemotherapy and have the clonogenic growth potential to produce tumor cells exist, and we have found that tumor cells lacking the plasma cell antigen CD138 are enriched in these properties. Understanding the mechanisms regulating these MM cancer stem cells (CSCs) may lead to novel targeting strategies. Aberrant translation is a widespread characteristic of tumor cells and plays a major role in drug resistance. Thus, therapeutic agents that target translation hold promise as novel anti-cancer drugs. MM cells are characterized by heightened protein production in the form of immunoglobulin (Ig) and are highly dependent on proteolytic pathways such as the proteasome to maintain protein homeostasis. As a consequence, proteasome inhibitors (PIs) such as bortezomib (BTZ) have been established as a frontline therapy for the disease. Immature plasma cells with diminished Ig production are resistant to BTZ, and patients with low Ig producing tumors are more likely to relapse. Therefore, low protein synthesis may confer growth advantages to cancer cells such as chemoresistance and enhanced tumor regrowth potential. Methods & Results: To examine alterations in the translational landscape in therapy resistant MM, we treated MM cells with BTZ, and found that eukaryotic translation initiation factor-5 (eIF5) was highly upregulated. Importantly, over-expression of eIF5 led to increased chemotherapy resistance and enhanced clonogenicity. O-propargyl-puromycin incorporation showed a global translational reduction in cells over-expressing eIF5. Matched transcriptional RNA sequencing (RNA-seq) and ribosome profiling revealed that a subset of genes, including MSI2 and EZH1, escaped translation suppression by maintaining efficient translation in eIF5 over-expressing cells. In addition, clonogenic (CD138neg) MM cells exhibited increased eIF5 expression and decreased protein synthesis. Discussion: Understanding the mechanisms of chemoresistance in the cell populations responsible for residual disease and re-growth may lead to novel targeting strategies. Recent studies have indicated that stem cell behavior is coupled to protein synthesis regulation. However, the role of translational control in the maintenance of tumor-initiating cells has been explored only recently. Studies have implicated deregulation of initiation factors in various types of cancer. However, the role of eIF5 in cancer remains elusive. Our data reveals, for the first time, a novel eIF5-directed translational program that enhances chemoresistance and tumor-initiating potential in MM cells, making it a promising therapeutic target. Citation Format: Kiera Rycaj, Eun-Hee Park, Tori Tonn, Qiuju Wang, Can Cenik, Christian Gocke, William Matsui. A novel translation program regulates tumor-initiating potential and chemoresistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3967.

OS06.3A LSD1-directed therapy curtails glioblastoma tumorigenicity by limiting the adaptation of tumor initiating cells to stressful environments

Abstract BACKGROUND Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, therapy resistance and poor blood-brain-barrier penetration hinder the amelioration of the standard-of-care. Included in the GBM mass are the tumor initiating cells (TICs), representing the driver of GBM growth and relapse in virtue of their stem-like traits and therapy-resistance. Being constantly exposed to environmental stress, including nutrients deficiency, hypoxia and therapeutic insults, all GBM cells -and TICs in particular- have to be highly adaptive in order to survive. Thus, their ability to cope with stress could be targeted to curtail TICs maintenance and the whole GBM aggressiveness. The key of TICs adaptation relies, among the others, on their epigenetic plasticity, hence encouraging epigenetic drugs testing. MATERIAL AND METHODS By exploiting patient-derived GBM TICs and orthotopic xenograft models, we tested the antitumorigenic features of a novel, selective, orally bioavailable and brain-penetrant Lysine-specific histone demethylase 1 inhibitor (LSD1i). We confirmed the specificity of its effects by LSD1 genetic targeting. A combination of RNA-seq, Chromatin Immunoprecipitation(ChIP)-seq, Mass Spectrometry and reverse genetic experiments unraveled LSD1 molecular players in GBM TICs. RESULTS We identified LSD1 as a druggable target in human GBM: LSD1i treatment, mirrored by LSD1 genetic targeting, impairs growth, viability, stem-like traits and in vivo tumorigenicity of GBM TICs. Mechanistically, LSD1 is crucial for the expression of the activating transcription factor 4 (ATF4), which coordinates the integrated stress response (ISR) to manage stressful stimuli as nutrient deprivation and endoplasmic reticulum stress. By mimicking these stress cues in vitro, we found that LSD1i triggers a delayed but unabated ATF4 translation which provokes an over-lasting ISR, eventually culminating in GBM TICs apoptosis. Lastly, LSD1 demethylase activity is dispensable for ATF4 induction. Rather, LSD1i exerts its anti-tumorigenic potential by interfering with LSD1 scaffolding function in GBM TICs. CONCLUSION LSD1-directed therapy is likely a promising strategy to hinder GBM. By sensitizing GBM TICs to stress, LSD1i endangers the GBM TICs pool. The effectiveness of LSD1i administration in different patient-derived GBM TICs and xenografts, regardless of their molecular profile, places a strong rationale toward the clinical translation of this approach for GBM management. FUNDING Italian association for Cancer Research (AIRC) and Italian Ministry of Health

CBP-mediated Wnt3a/β-catenin signaling promotes cervical oncogenesis initiated by Piwil2

Our previous work demonstrated that Piwil2 reactivated by the human papillomavirus oncoproteins E6 and E7 may reprogram somatic cells into tumor-initiating cells (TICs), which contribute to cervical neoplasia lesions. Maintaining the stemness of TICs is critical for the progression of cervical lesions. Here, we determined that canonical Wnt signaling was aberrantly activated in HaCaT cells transfected with lentivirus expressing Piwil2 and in cervical lesion specimens of low-grade squamous intraepithelial lesion, high-grade squamous intraepithelial lesion, and invasive carcinoma. Blocking the β-catenin and CREB binding protein interaction with ICG-001 significantly downregulated the reprogramming factors c-Myc, Nanog, Oct4, Sox2, and Klf4, thus leading to cell differentiation and preventing tumorigenicity in Piwil2-overexpressing HaCaT cells. Similarly, Piwil2 also critically regulated the canonical Wnt signaling pathway in cervical cancer. We further demonstrated that ICG-001 increased cisplatin sensitivity and significantly suppressed tumor growth of cervical cancer alone or in combination with cisplatin both in vitro and in vivo. The β-catenin/ CREB binding protein-mediated transcription activated by Piwil2 is essential for the maintenance of TICs, therefore contributing to the progression of cervical oncogenesis.

Abstract 1014: Pharmacological modulation of PDAC stroma to enhance checkpoint blockade immunotherapy

Abstract Sonic hedgehog signaling pathway is an important regulator during the initiation and progression of pancreatic cancer, and sustained activation of this pathway contributes to excessive deposition of tumor stroma and maintenance of tumor-initiating cells. Cyclopamine (CPA) is a potent inhibitor of the SHH pathway. In this work, we encapsulated both paclitaxel (PTX) and CPA in a long-circulating polymeric micelles to obtain M-CPA/PTX. In a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) (KPC model), systemic administration of M-CPA/PTX significantly prolonged the median survival of mice with well-established tumors from 23 days with nab-paclitaxel or gemcitabine to 58 days post-enrollment (p<0.0001). Further studies indicated that M-CPA/PTX modulated PDAC stroma by softening extracellular matrix, increasing blood perfusion, and alleviating hypoxia without depletion of α-smooth muscle actin-positive tumor-associated fibroblasts. Significantly, combination of M-CPA/PTX and anti-PD-1 immunotherapy significantly prolonged the survival of PDAC-bearing KPC mice compare with monotherapies (p<0.01). In an immune-competent, orthotopic PDAC model bearing established murine PDAC, treatments with combined M-CPA/PTX and anti-PD-1 enhanced tumor infiltration of CD8+ T cells and IFNγ expression. Neutralization of either CD8 T cells or INFγ nullified the therapeutic gain achieved with combination therapy. Thus, M-CPA/PTX enhanced checkpoint blockade immunotherapy through modulation of PDAC stroma and enhanced tumor infiltration of effector immune cells. Citation Format: Jun Zhao, Zhilan Xiao, Willem Overwijk, Chun Li. Pharmacological modulation of PDAC stroma to enhance checkpoint blockade immunotherapy [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 1014.

Reactive species balance via GTP cyclohydrolase I regulates glioblastoma growth and tumor initiating cell maintenance.

Depending on the level, differentiation state, and tumor stage, reactive nitrogen and oxygen species inhibit or increase cancer growth and tumor initiating cell maintenance. The rate-limiting enzyme in a pathway that can regulate reactive species production but has not been thoroughly investigated in glioblastoma (GBM; grade IV astrocytoma) is guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1). We sought to define the role of GCH1 in the regulation of GBM growth and brain tumor initiating cell (BTIC) maintenance. We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples, and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production, and survival in orthotopic patient-derived xenograft models were determined. GCH1 was expressed in GBMs with elevated but not exclusive RNA and protein levels in BTICs in comparison to non-BTICs. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In converse experiments, GCH1 knockdown with short hairpin RNA led to GBM cell growth inhibition and reduced self-renewal in association with decreased CD44 expression. GCH1 was critical for controlling reactive species balance, including suppressing reactive oxygen species production, which mediated GCH1 cell growth effects. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence, and worse survival. GCH1 expression in established GBMs is pro-tumorigenic, causing increased growth due, in part, to promotion of BTIC maintenance and suppression of reactive oxygen species.

144 - GTP Cyclohydrolase I in Tumor Initiating Cell Maintenance and Glioblastoma Growth: Functions and Mechanisms

Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree of intratumoral heterogeneity that contributes to treatment failures. One cellular subset enriched for the capacity to propagate tumor growth and promote therapeutic resistance is brain tumor initiating cells (BTICs). The aim of the study was to define the roles of GTP cyclohydrolase 1 (GCH1) in regulation of BTIC phenotypes via reactive species. We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production and in vitro animal survival were determined. We found that GCH1 RNA and protein expression were increased in BTICs in comparison to non-BTICs, but that GCH1 was not exclusive to the BTIC fraction. Indeed, GCH1 was elevated in GBM in comparison to normal brain. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 knockdown with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal models. Mechanistically, GCH1 increased CD44 expression and was critical for controlling reactive species balance, including suppressing reactive oxygen species production. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in BTICs promotes tumor maintenance and is a key regulator of reactive oxygen species in GBM.

Abstract 1925: Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance

Abstract Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy because they are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using a human-derived biomatrix HuBiogelTM and maintained with physiologically relevant microenvironments. Percentages of BITCs were determined based on cell surface marker expression (CD133), label retention (carboxyfluorescein succinimidyl ester; CFSE), and tumorsphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient restricted microtumors significantly increased the percentage of both CD133+ and CFSE+ cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintain neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting. Citation Format: Ashley Gilbert, Kiera Walker, Anh Tran, Yancey Gillespie, Raj Singh, Anita B. Hjelmeland. Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1925. doi:10.1158/1538-7445.AM2017-1925

Glycolytic reprogramming through PCK2 regulates tumor initiation of prostate cancer cells.

Tumor-initiating cells (TICs) play important roles in tumor progression and metastasis. Identifying the factors regulating TICs may open new avenues in cancer therapy. Here, we show that TIC-enriched prostate cancer cell clones use more glucose and secrete more lactate than TIC-low clones. We determined that elevated levels of phosphoenolpyruvate carboxykinase isoform 2 (PCK2) are critical for the metabolic switch and the maintenance of TICs in prostate cancer. Information from prostate cancer patient databases revealed that higher PCK2 levels correlated with more aggressive tumors and lower survival rates. PCK2 knockdown resulted in low TIC numbers, increased cytosolic acetyl-CoA and cellular protein acetylation. Our data suggest PCK2 promotes tumor initiation by lowering acetyl-CoA level through reducing the mitochondrial tricarboxylic acid (TCA) cycle. Thus, PCK2 is a potential therapeutic target for aggressive prostate tumors.

Abstract 1318: Pancreatic cancer therapy using polyion micelles co-loaded with cyclopamine and paclitaxel

Abstract Sonic hedgehog (SHH) signaling pathway is an important regulator during the initiation and progression of pancreatic cancer, and sustained activation of this pathway contributes to excessive deposition of tumor stroma and maintenance of tumor-initiating cells. Cyclopamine, a potent inhibitor of the SHH pathway, has shown promising antitumor efficacy. However, cyclopamine is insoluble in water and is highly toxic, and cyclopamine monotherapy did not effectively inhibit tumor growth in a range of preclinical pancreatic cancer models. We hypothesized that polyion complex polymeric micelles that simultaneously deliver cyclopamine and paclitaxel (M-CPA/PTX) would display greater efficacy against pancreatic tumor than micelles loaded with cyclopamine alone (M-CPA) or paclitaxel alone (M-PTX). We tested this hypothesis in cell cultures and in an orthotopic human pancreatic cancer Miapaca-2 xenograft model and a genetically engineered mouse model of pancreatic ductal carcinoma (PDAC), the KPC model. Our results showed that M-CPA interrupted the SHH pathway by antagonizing the smoothened receptor, reduced expression of Gli-1 transcription factor, and inhibited proliferation of Miapaca-2 cells and stroma-producing human pancreatic stellate cells. In Miapaca-2 cells, M-CPA decreased the fraction of CD133+ cells (cancer stem cells) and decreased tumorsphere formation, whereas M-TXL increased the fraction of CD133+ cells and tumorsphere formation. M-CPA negated the stimulation of human pancreatic stellate cells by exogenous SHH ligands. In vivo, M-CPA/PTX reduced tumor cell proliferation, depleted tumor stroma, and increased tumor vasculature density, leading to effective inhibition of tumor growth in Miapaca-2 xenografts. In the KPC model, M-CPA/PTX significantly prolonged media survival of mice with PDAC compared to non-treatment control mice. In conclusion, M-CPA/PTX was an effective strategy to treat pancreatic cancer. (supported in part by the Viragh Family Foundation and the John S. Dunn Foundation) Citation Format: Jun Zhao, Chunhui Wu, Xiaoxia Wen, Junjie Li, Qian Huang, Ying Ma, Stephen Ullrich, Huamin Wang, Jason Fleming, Chun Li. Pancreatic cancer therapy using polyion micelles co-loaded with cyclopamine and paclitaxel. [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 1318.

Abstract 3352: Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) regulates the tumor initiating cell phenotype through SOX2 expression and modulates treatment response to anti-cancer drugs in lung adenocarcinoma

Abstract Lung cancer is the most lethal of all cancers world-wide and the majority of patients require chemotherapy or targeted therapy at presentation. Survival is compromised by drug resistance through multiple mechanisms such as by-pass mutations, micro-environment activation of survival programs and induction of tumor initiating cells (TIC). Induction of TIC phenotypes could be accompanied by upregulation of embryonic or developmental pathways but molecular mechanisms are incompletely understood. Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) is a multifunctional serine/threonine kinase involved in growth and stress signals integration. A tumor-supportive role has been reported in leukaemia and thyroid cancer but reports on its involvement in TIC regulation are limited. We investigated the role and molecular mechanism of CaMK2A in lung cancer TIC regulation using in vitro and in vivo models. The results showed CaMK2A was overexpressed in lung cancer cell lines and human lung adenocarcinomas compared to normal lung. In cancer cells with stable CaMK2A-knockdown, in vitro reduction in tumor spheres formation, anchorage independent growth and increased reactive oxygen species, as well as in vivo reduction of xenograft tumorigenicity were observed. The opposite effects were observed in cancer cells with stable CaMK2A over-expression. Further, specific pharmacological inhibition of CaMK2A by KN93 led to significantly reduced IC50 for gefitinib in lung cancer cells harboring activating EGFR mutation (HCC827) and in HCC827 exogenously induced for gefitinib resistance. In contrast, IC50 of cisplatin treatment was increased in cancer cells with genetically-induced CaMK2A over-expression. Moreover, CaMK2A knockdown led to reduced mRNA expressions of pluripotency genes (SOX2, NANOG, OCT4) and TIC markers (ALDH, CD166). These results suggested CaMK2A is involved in lung tumorigenicity through TIC regulation. To further investigate the molecular mechanism, we showed tumorigenicity was restored in xenografts with CaMK2A knockdown rescued with SOX2 over-expression. In CaMK2A knockdown cells, reduced SOX2 expression was associated with increased H3K27me3 repressive histone mark, which has not been reported in the literature. Taken together, we have shown CaMK2A plays a role in lung adenocarcinoma and TIC maintenance through histone modification and regulation of SOX2 expression. Targeting TIC through CaMK2A modulation might be useful for overcoming drug resistance and improving long term lung cancer survival. Citation Format: Maria P. Wong, Jing Liu, Zhi-Jie Xiao, Si-Qi Wang, Vicky Pc Tin. Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) regulates the tumor initiating cell phenotype through SOX2 expression and modulates treatment response to anti-cancer drugs in lung adenocarcinoma. [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 3352.

The Wnt signaling pathway: prospects for pharmacological regulation

Wnt cascade is one of the most important signal pathways in the cell, which is required for normal embryonic development, differentiation, maintenance of stem cell phenotype and migration. Mutations in this pathway are associated with tumor growth (especially with colon cancers, hepatocarcinomas and leukemias), where they participate in maintenance of tumor-initiating cells and metastasing. Because of that there is a considerable interest to develop inhibitors of Wnt pathway as anti-tumor agent. But this inhibitors are still only in early stages of clinical investigations. In this article we review the main targets for this anti-cancer agents and their preclinical and clinical status.

Covering the Cover

Covering the Cover

CDC20 maintains tumor initiating cells.

Glioblastoma is the most prevalent and lethal primary intrinsic brain tumor. Glioblastoma displays hierarchical arrangement with a population of self-renewing and tumorigenic glioma tumor initiating cells (TICs), or cancer stem cells. While non-neoplastic neural stem cells are generally quiescent, glioblastoma TICs are often proliferative with mitotic control offering a potential point of fragility. Here, we interrogate the role of cell-division cycle protein 20 (CDC20), an essential activator of anaphase-promoting complex (APC) E3 ubiquitination ligase, in the maintenance of TICs. By chromatin analysis and immunoblotting, CDC20 was preferentially expressed in TICs relative to matched non-TICs. Targeting CDC20 expression by RNA interference attenuated TIC proliferation, self-renewal and in vivo tumor growth. CDC20 disruption mediated its effects through induction of apoptosis and inhibition of cell cycle progression. CDC20 maintains TICs through degradation of p21CIP1/WAF1, a critical negative regulator of TICs. Inhibiting CDC20 stabilized p21CIP1/WAF1, resulting in repression of several genes critical to tumor growth and survival, including CDC25C, c-Myc and Survivin. Transcriptional control of CDC20 is mediated by FOXM1, a central transcription factor in TICs. These results suggest CDC20 is a critical regulator of TIC proliferation and survival, linking two key TIC nodes-FOXM1 and p21CIP1/WAF1-elucidating a potential point for therapeutic intervention.

Autophagy Supports Breast Cancer Stem Cell Maintenance by Regulating IL6 Secretion.

Autophagy is a mechanism by which cells degrade cellular material to provide nutrients and energy for survival during stress. The autophagy is thought to be a critical process for cancer stem cell (CSC) or tumor-initiating cell maintenance but the mechanisms by which autophagy supports survival of CSCs remain poorly understood. In this study, inhibition of autophagy by knockdown of ATG7 or BECN1 modified the CD44(+)/CD24(low/-) population in breast cancer cells by regulating CD24 and IL6 secretion. In a breast cancer cell line that is independent of autophagy for survival, autophagy inhibition increased IL6 secretion to the media. On the other hand, in an autophagy-dependent cell line, autophagy inhibition decreased IL6 secretion, cell survival, and mammosphere formation. In these cells, IL6 treatment or conditioned media from autophagy-competent cells rescued the deficiency in mammosphere formation induced by autophagy inhibition. These results reveal that autophagy regulates breast CSC maintenance in autophagy-dependent breast cancer cells by modulating IL6 secretion implicating autophagy as a potential therapeutic target in breast cancer. Modulation of autophagy in breast cancer has different and even opposite effects, indicating the need for a selection strategy when trying to manipulate autophagy in the context of cancer therapy.

PKCδ maintains phenotypes of tumor initiating cells through cytokine-mediated autocrine loop with positive feedback.

The existence of tumor initiating cells (TICs) has been emerged as a good therapeutic target for treatment of glioblastoma that is the most aggressive brain tumor with poor prognosis. However, the molecular mechanisms that regulate the phenotypes of TICs still remain obscure. In this study, we found that PKCδ, among PKC isoforms, is preferentially activated in TICs and acts as a critical regulator for the maintenance of TICs in glioblastoma. By modulating the expression levels or activity of PKCδ, we demonstrated that PKCδ promotes self-renewal and tumorigenic potentials of TICs. Importantly, we found that the activation of PKCδ persists in TICs through an autocrine loop with positive feedback that was driven by PKCδ/STAT3/IL-23/JAK signaling axis. Moreover, for phenotypes of TICs, we showed that PKCδ activates AKT signaling component by phosphorylation specifically on Ser473. Taken together, we proposed that TICs regulate their own population in glioblastoma through an autocrine loop with positive feedback that is driven by PKCδ-dependent secretion of cytokines.

Abstract 1939: Calcineurin/NFAT signaling regulates tumor initiating cell properties in non-small cell lung cancer

Abstract Background: Targeting specific pathways responsible for Tumor Initiating Cells (TIC) maintenance is postulated to be an important therapeutic strategy for more effective control of tumors. Nuclear Factors of Activated T-cells (NFATs), consisting of 4 homologues (NFATc1-c4), are calcium-dependent transcription factors that are activated upon dephosphorylation by the protein phosphatase calcineurin. NFAT is known to be involved in T cell activation and differentiation. Recently, its roles in regulating embryonic stem cell proliferation and cancer survival have been reported. However, the involvement of Calcineurin/NFAT signaling in TIC maintenance remains unknown. Objective: To study the role of Calcineurin/NFAT in regulating TIC properties in non-small cell lung cancer (NSCLC). Methods and results: Analysis of NFAT mRNA expression in a panel of established and early passage patient derived NSCLC cell lines showed NFATc2 and NFATc4 were up-regulated in 92.6% (25 of 27) and 72% (20 of 27) of cancer cells compared to normal bronchial cell line (BEAS-2B), respectively. To study its role in TIC, tumor spheres or flow cytometry-isolated ALDHhigh/CD44high cells were obtained and NFAT expressions and activities were compared between TIC and non-TIC. The expression of NFAT and downstream targets were significantly higher in tumor spheres and ALDHhigh/CD44high cells compared to the corresponding monolayers and ALDHlow/CD44low cells. Correspondingly, luciferase reporter assay also showed higher NFAT activities in tumor spheres compared to monolayers. To study its functional importance, calcineurin and NFAT were inhibited and effects on TIC properties were analyzed. Inhibition of calcineurin by pharmacological inhibitors cyclosporin A (CSA) and FK506 resulted in reduced sphere formation ability, down-regulation of pluripotent genes (SOX2, OCT4, NANOG) and TIC markers (ALDH1A1, CD133, CD166) expression, as well as decreased TIC proportions (ALDHhigh/CD44high and CD133+) by flow cytometry. These effects were verified using siRNA knockdown of PPP3R1 which encodes one of the calcineurin regulatory subunits. Apart from expression changes, stable knockdown of NFATc2 using lentiviral shRNA inhibited in vitro and in vivo tumorigenecity as shown in soft agar colony formation and mice xenografts compared to shRNA control. To explore the clinical relevance of NFAT, NFAT expression levels were analyzed in human resected lung cancers by quantitative PCR and immunohistochemsitry. Results showed that NFATs were significantly up-regulated in clinical NSCLC compared to corresponding normal lung, and higher NFAT expression predicted shorter recurrence free survival. Conclusions: Calcineurin/NFAT signaling is active in both NSCLC and lung TIC. It plays an important role in TIC maintenance. Inhibition of this pathway is a promising approach for long term lung cancer control. Citation Format: Zhi-Jie Xiao, Jing Liu, Vicky Pui-Chi Tin, Maria Pik Wong. Calcineurin/NFAT signaling regulates tumor initiating cell properties in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1939. doi:10.1158/1538-7445.AM2014-1939

Abstract 1248: The Gemini vitamin D analogue BXL0124 inhibits Notch signaling via HES1, resulting in the reduction of CD44+/CD24-/low subpopulation and proliferation of MCF10DCIS cells

Abstract Activation of Notch signaling is correlated with poor prognosis and decreased survival of breast cancer patients. Recent studies reported that Notch signaling plays an important role for the maintenance of tumor-initiating cells in breast cancer. Using MCF10DCIS.com human breast cancer cells (MCF10DCIS), which contain tumor-initiating subpopulation (CD44+/CD24-/low), we demonstrated that the Gemini vitamin D analog, BXL0124, reduced the tumor-initiating subpopulation in vitro and repressed the growth of MCF10DCIS xenograft tumors in vivo. In the present study, we investigated Notch signaling in CD44+/CD24-/low and CD44+/CD24+ subpopulations of MCF10DCIS cells and determined whether BXL0124 represses tumor-initiating subpopulation of breast cancer by targeting Notch signaling. CD44+/CD24-/low subpopulation showed higher Notch1 activation and cell proliferation than CD44+/CD24+ subpopulation in MCF10DCIS cells. To investigate the effects of BXL0124 on Notch signaling, the protein levels of Notch receptors and their ligands were determined. BXL0124 decreased activated Notch1 (c-Notch1) as well as its ligands, Jagged1, Jagged2 and DLL1. However, total Notch1 was not affected by BXL0124, suggesting that BXL0124 inhibits Notch signaling by decreasing Notch ligands. A downstream target of Notch signaling, cMyc, and cell proliferation were reduced by BXL0124. The inhibition of Notch1 activation by BXL0124 was blocked with knock-down of the vitamin D receptor (VDR), indicating that it is a VDR-dependent event. In a time course study, the effects of BXL0124 on the mRNA expression of Notch1, Notch ligands and downstream targets were investigated. BXL0124 decreased the mRNA levels of Jagged1, Jagged2 and DLL1 starting at 8 h which followed by the repression of cMyc mRNA at 16 h. The mRNA level of a transcriptional repressor HES1, which is a key downstream target of Notch signaling, was rapidly induced by BXL0124 as early as 30 min. To determine the involvement of HES1 in Notch signaling, both knock-down and overexpression of HES1 were performed by using HES1 siRNA and HES1-expression vector, respectively. The inhibition of Notch signaling by BXL0124 was reversed by the knock-down of HES1, while the overexpression of HES1 inhibited Notch activation, reduced CD44+/CD24-/low population and decreased cell proliferation of MCF10DCIS cells. These results suggest that HES1 functions as a repressor of Notch signaling, and BXL0124 inhibits Notch signaling in a HES1-dependent manner. The present study demonstrates that BXL0124 inhibits Notch signaling by rapid induction of HES1 which results in the reduction of CD44+/CD24-/low subpopulation and proliferation of MCF10DCIS cells. Our study suggests the Gemini vitamin D analog, BXL0124, as a potential agent to repress the tumor-initiating subpopulation of breast cancer by targeting Notch signaling. Citation Format: Jae Young So, David M. Salerno, Hubert Maehr, Milan Uskokovic, Nanjoo Suh. The Gemini vitamin D analogue BXL0124 inhibits Notch signaling via HES1, resulting in the reduction of CD44+/CD24-/low subpopulation and proliferation of MCF10DCIS cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1248. doi:10.1158/1538-7445.AM2014-1248

Telomerase inhibition abolishes the tumorigenicity of pediatric ependymoma tumor-initiating cells.

Pediatric ependymomas are highly recurrent tumors resistant to conventional chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless replication, has been found to be critically important for the maintenance of tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the tumor from which they are identified, and are drivers of recurrence in numerous cancers. In this study, telomerase enzymatic activity was directly measured and inhibited to assess the therapeutic potential of targeting telomerase. Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas to determine the prevalence and prognostic potential of telomerase activity or alternative lengthening of telomeres (ALT) as telomere maintenance mechanisms, respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate the effect of telomerase inhibition on proliferation and tumorigenicity in established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT. Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity. Imetelstat inhibited proliferation and self-renewal by shortening telomeres and inducing senescence in vitro. In vivo, Imetelstat significantly reduced subcutaneous xenograft growth by 40 % (p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma TICs in an orthotopic xenograft model. Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1327-6) contains supplementary material, which is available to authorized users.

RNA binding protein RBM14 promotes radio-resistance in glioblastoma by regulating DNA repair and cell differentiation.

Glioblastoma multiforme (GBM) is the most aggressive and lethal type of brain tumor. Standard treatment for GBM patients is surgery followed by radiotherapy and/or chemotherapy, but tumors always recur. Traditional therapies seem to fail because they eliminate only the bulk of the tumors and spare a population of stem-like cells termed tumor-initiating cells. The stem-like state and preferential activation of DNA damage response in the GBM tumor-initiating cells contribute to their radio-resistance and recurrence. The molecular mechanisms underlying this efficient activation of damage response and maintenance of stem-like state remain elusive. Here we show that RBM14 controls DNA repair pathways and also prevents cell differentiation in GBM spheres, causing radio-resistance. Knockdown of RBM14 affects GBM sphere maintenance and sensitizes radio-resistant GBM cells at the cellular level. We demonstrate that RBM14 knockdown blocks GBM regrowth after irradiation in vivo. In addition, RBM14 stimulates DNA repair by controlling the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway. These results reveal unexpected functions of the RNA-binding protein RBM14 in control of DNA repair and maintenance of tumor-initiating cells. Targeting the RBM14-dependent pathway may prevent recurrence of tumors and eradicate the deadly disease completely.

Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells.

Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH(hi)CD44(hi) subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH(hi)CD44(lo), ALDH(lo)CD44(hi), ALDH(lo)CD44(lo) cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH(hi)CD44(hi)xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH+CD44+ compartment. Chemotherapy and targeted therapy resulted in higher AALDH(hi)CD44(hi) subset viability and ALDH(lo)CD44(lo) subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested theALDH(hi)CD44(hi)compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH(hi)CD44(hi)TIC maintenance would be beneficial for the development of long term lung cancer control.