Cancer-initiating Cell Population Research Articles

Abstract 2444: RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion

Abstract Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for ~85% of all lung cancer diagnoses. A sub-population of cells within human NSCLC tumors called cancer stem cells (CSCs) have been found to exhibit distinct properties that contribute to factors associated with poor outcomes in NSCLC patients including metastasis, relapse, and therapeutic resistance. We previously performed RNAseq analysis on bulk cancer cells (BCCs) and CSCs from a panel of primary NSCLC tumors and cell lines to identify signaling mechanisms involved in the tumorigenic behavior of NSCLC CSCs. Our analysis revealed that expression of RAB27B, a small GTPase that plays a critical role in extracellular vesicle (EV) formation and secretion, was significantly upregulated in CSCs when compared to BCCs. Interestingly, RAB27B is overexpressed and predicts patient survival in NSCLC. The goal of this study was to assess the role of RAB27B in the tumorigenic behavior of NSCLC CSCs and determine the mechanism by which RAB27B promotes an aggressive stem-cell like phenotype in NSCLC cells an aspect of RAB27B function that has not been investigated in NSCLC. QPCR and immunoblot analysis was used to compare RAB27B mRNA and protein expression in a panel of NSCLC BCCs and CSCs. Two independent shRNA constructs were used to knockdown (KD) RAB27B in NSCLC CSCs. The effects of RAB27B KD on CSC expansion, transformed growth and invasion were analyzed by clonal expansion, soft agar colony formation and Boyden chamber assays, respectively. Xenograft and tail vein mouse models were used to assess the effects of RAB27B KD on CSC tumor growth and lung colonization. EVs were isolated from the conditioned media of BCCs, CSCs and RAB27B KD CSCs using differential ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblotting according to MISEV guidelines. Our analysis revealed that RAB27B is significantly upregulated in NSCLC CSCs compared to BCCs. RAB27B KD cells exhibited a significant decrease in clonal expansion, transformed growth and invasion when compared to non-target shRNA control CSCs. In vivo studies demonstrated that RAB27B is important for the tumorigenicity of CSCs and promotes tumor cell proliferation, and angiogenesis. EVs were positive for EV-specific markers and negative for an intracellular vesicle marker. CSCs secrete significantly more EVs when compared to BCCs and KD of RAB27B in CSCs led to a decrease in the levels of EVs secreted. Furthermore, delivery of CSC-derived, but not BCC-derived EVs to BCCs induced spheroid growth, clonal expansion, and invasion in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating and invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Citation Format: Kayleah M. Meneses, Jennifer A. Lindemann, Prita Pandya, Dania S. Al-Qasrawi, Ryan Argo, Celeste Weems, Danielle J. Beetler, Irene K. Yan, Joy Wolfram, Tushar C. Patel, Verline Justilien. RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2444.

Abstract 397: The role of heparan sulfate in enhancing the chemotherapeutic response in triple-negative breast cancer

Abstract Among women worldwide, breast cancer has the highest incidence and is the leading cause of cancer-related death. Patients with the triple-negative breast cancer (TNBC) subtype have an inferior prognosis in comparison to other breast cancers because the current therapies do not facilitate long-lasting responses. Thus, there is a critical need for novel therapeutic strategies that induce stronger responses. In our previous research, we discovered that augmenting the concentration of extracellular ATP (eATP) greatly enhances the chemotherapeutic response of TNBC cell lines by activating purinergic receptors (P2RXs), leading to cell death through the induction of non-selective membrane permeability. However, eATP levels are limited by several classes of extracellular ATPases. One endogenous molecule of interest that can inhibit multiple classes of extracellular ATPases is heparan sulfate. Heparan sulfate itself is degraded by heparanase, an enzyme that is known to be highly expressed in various cancers, including breast cancer. Heparan sulfate has previously been shown to regulate several cancer-related processes such as fibroblast growth factor signaling, neoangiogenesis by sequestering vascular endothelial growth factors in the extracellular matrix, hedgehog signaling and cell adhesion. In this project, we identified an additional mechanism for a tumor suppressor role of heparan sulfate: inhibition of extracellular ATPases, leading to augmented levels of eATP. Several heparanase inhibitors have been previously identified, including OGT 2115, suramin, PI-88, and PG 545. We hypothesized that heparanase inhibitors would augment eATP concentrations in TNBC by increasing heparan sulfate in the tumor microenvironment, resulting in increased cell death in response to chemotherapy. We treated TNBC cell lines MDA-MB 231, Hs 578t, and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells with increasing concentrations of the chemotherapeutic agent paclitaxel in the presence of heparan sulfate and/or OGT 2115 with eATP content and cell viability being assessed. Moreover, to confirm that the effects of OGT 2115 are mediated through eATP, we utilized specific antagonists to the purinergic receptors P2RX4 and P2RX7. In addition, the protein expression of heparanase was compared in all the examined cell lines via Western blot analysis. We also evaluated the effects of this combination on the breast cancer-initiating cell population in the treated cells using flow cytometry and tumorsphere formation efficiency assays. These results demonstrate that inhibiting the degradation of heparan sulfate in the tumor microenvironment enhances the susceptibility of TNBC cell lines to chemotherapy by augmenting extracellular ATP concentrations. This strategy could potentially be used to induce deeper and more durable responses in triple-negative breast cancer patients. Citation Format: Jasmine M. Manouchehri, Jharna Datta, Mathew A. Cherian. The role of heparan sulfate in enhancing the chemotherapeutic response in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 397.

Identification of Malignant Cell Populations Associated with Poor Prognosis in High-Grade Serous Ovarian Cancer Using Single-Cell RNA Sequencing.

Simple SummaryOvarian cancer has a high recurrence rate (~75%), and tumor heterogeneity is associated with such tumor recurrence. However, it is still poorly understood in ovarian cancer. To reveal tumor heterogeneity, we performed single-cell RNA sequencing (RNA-seq) of serous ovarian cancer cells from four different patients: two with primary carcinoma, one with recurrent carcinoma, and one with carcinoma treated with interval debulking surgery. As a result, we found two malignant tumor cell subtypes associated with poor prognosis. One malignant population included the earliest cancer cells and cancer stem-like cells. SLC3A1 and PEG10 were identified as the marker genes of cancer-initiating cells. The other malignant population expressing CA125 (MUC16), the most common biomarker for ovarian cancer, is associated with a decrease in the number of tumor-infiltrating cytotoxic T lymphocytes (CTLs). Our findings will offer new markers for diagnosis and choosing treatments targeting the malignant populations in ovarian cancer.To reveal tumor heterogeneity in ovarian cancer, we performed single-cell RNA sequencing (RNA-seq). We obtained The Cancer Genome Atlas (TCGA) survival data and TCGA gene expression data for a Kaplan–Meier plot showing the association of each tumor population with poor prognosis. As a result, we found two malignant tumor cell subtypes associated with poor prognosis. Next, we performed trajectory analysis using scVelo and Monocle3 and cell–cell interaction analysis using CellphoneDB. We found that one malignant population included the earliest cancer cells and cancer stem-like cells. Furthermore, we identified SLC3A1 and PEG10 as the marker genes of cancer-initiating cells. The other malignant population expressing CA125 (MUC16) is associated with a decrease in the number of tumor-infiltrating cytotoxic T lymphocytes (CTLs). Moreover, cell–cell interaction analysis implied that interactions mediated by LGALS9 and GAS6, expressed by this malignant population, caused the CTL suppression. The results of this study suggest that two tumor cell populations, including a cancer-initiating cell population and a population expressing CA125, survive the initial treatment and suppress antitumor immunity, respectively, and are associated with poor prognosis. Our findings offer a new understanding of ovarian cancer heterogeneity and will aid in the development of diagnostic tools and treatments.

Synergism of Proneurogenic miRNAs Provides a More Effective Strategy to Target Glioma Stem Cells.

Simple SummarymiRNAs function as critical regulators of gene expression and have been defined as contributors of cancer phenotypes by acting as oncogenes or tumor suppressors. Based on these findings, miRNA-based therapies have been explored in the treatment of many different malignancies. The use of single miRNAs has faced some challenges and showed limited success. miRNAs cooperate to regulate distinct biological processes and pathways and, therefore, combination of related miRNAs could amplify the repression of oncogenic factors and the effect on cancer relevant pathways. We established that the combination of tumor suppressor miRNAs miR-124, miR-128, and miR-137 is much more effective than single miRNAs in disrupting proliferation and survival of glioma stem cells and neuroblastoma lines and promoting differentiation and response to radiation. Subsequent genomic analyses showed that other combinations of tumor suppressor miRNAs could be equally effective, and its use could provide new routes to target in special cancer-initiating cell populations.Tumor suppressor microRNAs (miRNAs) have been explored as agents to target cancer stem cells. Most strategies use a single miRNA mimic and present many disadvantages, such as the amount of reagent required and the diluted effect on target genes. miRNAs work in a cooperative fashion to regulate distinct biological processes and pathways. Therefore, we propose that miRNA combinations could provide more efficient ways to target cancer stem cells. We have previously shown that miR-124, miR-128, and miR-137 function synergistically to regulate neurogenesis. We used a combination of these three miRNAs to treat glioma stem cells and showed that this treatment was much more effective than single miRNAs in disrupting cell proliferation and survival and promoting differentiation and response to radiation. Transcriptomic analyses indicated that transcription regulation, angiogenesis, metabolism, and neuronal differentiation are among the main biological processes affected by transfection of this miRNA combination. In conclusion, we demonstrated the value of using combinations of neurogenic miRNAs to disrupt cancer phenotypes and glioma stem cell growth. The synergistic effect of these three miRNA amplified the repression of oncogenic factors and the effect on cancer relevant pathways. Future therapeutic approaches would benefit from utilizing miRNA combinations, especially when targeting cancer-initiating cell populations.

Abstract IA30: Using single-cell, high-dimensional approaches to unravel tumor heterogeneity in pediatric cancer

Abstract Introduction: Despite improving responses to up-front therapies for children with cancer, relapse remains a significant barrier to long-term remissions and cure. Curing children of cancer requires effectively targeting all cancer-initiating cell populations. Understanding what cellular populations have initiating capacity and lead to poor clinical outcomes requires examining tumors at the single-cell level. Here we discuss such an approach to predict relapse in B-cell precursor acute lymphoblastic leukemia and response to common ALL therapies including glucocorticoids and chimeric antigen receptor T cells. Methods: Primary diagnostic or relapse bone marrow or peripheral blood samples or patient-derived xenografts were obtained under informed consent and IRB approval. Samples were clinically annotated for relevant prognostic features and clinical outcomes. Cryopreserved samples and healthy control bone marrows were treated in vitro with short-term stimulations to reveal signaling states. Cells were stained with a 40-antibody panel including relevant B-cell developmental proteins and intracellular signaling proteins. Samples were analyzed by mass cytometry (CyTOF). Patient samples underwent developmental classification in which each leukemia cell was assigned its most similar healthy counterpart. Further analysis was performed using various analysis methods, including machine learning modeling of relapse outcomes. Results: Studying over eighty primary patient diagnostic samples, we first organized the heterogeneous single-cell data classifying leukemic cells to 12 different subpopulations of B lymphopoiesis. We identified the transitional populations between early pro-B cells and late pre-B cells are expanded across almost all patients with BCP ALL. Extracting all measured features in these populations, we applied an elastic net model to determine cell populations associated with future relapse. This resulted in identification of early pre-B cells characterized by basally activated pCREB, pS6, pSYK, and p4EBP1 as predictive of future relapse. Further, these cells are apparent also at the time of relapse. Taking a similar approach, we also examined how these cell populations respond to treatment with glucocorticoids, a keystone of BCP ALL therapy. Similarly, we identify the same network activation in glucocorticoid-resistant patients associated also with a differentiation process. Interestingly, we can identify similar resistant phenotype in primary cells from patient treated in vivo with glucocorticoids. Finally, using these tools and methods, we describe CD19neg cells identified prior to treatment with CD19-targeted CAR T cells in patients who go on to suffer CD19neg relapse. Conclusions: Together, we present an approach to organizing single-cell tumor heterogeneity that reveals relapse-associated phenotypes. This highlights the translational potential of such an approach that could be applied to other single-cell studies in diverse tumor types. Citation Format: Kara L. Davis. Using single-cell, high-dimensional approaches to unravel tumor heterogeneity in pediatric cancer [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA30.

P53 functional states are associated with distinct aldehyde dehydrogenase transcriptomic signatures

p53 and aldehyde dehydrogenase (ALDH) have been implicated in key tumorigenesis processes including cancer initiating cell (CIC) maintenance; however, the relationship between these two mediators remains poorly defined. In this study, ALDH isoform expression diversity was revealed in CICs with disparate p53 functional states: gain of function, high risk p53 mutation (p53HRmut) and wildtype p53 (p53WT) inactivated by the human papillomavirus 16 (HPV16) E6 oncogene. Interrogation of head and neck squamous cell carcinoma (HNSCC) cell lines and patient tumors showed that HPV16+/p53WT cases have higher ALDH variance score (AVS), a measure of tumor ALDH isoform expression diversity, compared to HPV−/p53HRmut cases (p = 0.03). AVS and several individual ALDH isoforms were associated with prognosis in HPV16+/p53WT HNSCC but not in HPV−/p53HRmut HNSCC. Knockdown of the dominant ALDH isoform in high AVS HNSCC depleted the CIC pool in vitro and in vivo. Our results demonstrate that p53 functional states are associated with distinct ALDH isoform transcriptomic signatures. Moreover, tumor ALDH profiling may provide insight on which ALDH isoform to target in high AVS HNSCC tumors to deplete the CIC population.

Typhonium flagelliforme extract induce apoptosis in breast cancer stem cells by suppressing survivin.

Breast cancer stem cells (bCSCs) are a small population of cancer-initiating cells within breast cancer, characterized as CD44+ CD24-/low. bCSCs develop apoptosis resistance by expressing survivin and suppressing caspase-9 and caspase-3 expression. Typhonium flagelliforme tuber extract (TFTe) can induce apoptosis in several types of cancer cells; however, the effects of TFTe to induce the bCSCs remain unclear. This study aimed to investigate the effects of TFTe on apoptosis induction in bCSCs through the suppression of survivin and the exhibition of caspase-9 and caspase-3. This study employed a posttest only, control group design. To analyze the apoptotic index, TFTe, at concentrations of 25 (Tf1d), 50.89 (Tf2d), and 100 μg/mL (Tf3d) were used to treat bCSCs for 24 h, in a humidified incubator containing 5% CO2, at 37°C. The control group was exposed to dimethyl sulfoxide. Apoptosis was measured by propidium iodide and acridine orange double-staining, and the expression levels of survivin, caspase-9, and caspase-3 were assessed by immunocytochemistry. Differences were analyzed by the independent Student's t-test, to compare two groups, and the Kruskal-Wallis test, to compare more than two groups. P < 0.05 was considered statistically significant. TFTe inhibited bCSC proliferation, with an IC50 value of 50.89 μg/mL, and significantly induced apoptosis in bCSCs (P < 0.001). TFTe also significantly decreased the expression levels of survivin in bCSCs (P < 0.001) and increased the expression levels of caspase-9 and caspase-3 (P < 0.001). TFTe can induce apoptosis in bCSCs by decreasing survivin expression levels and increasing the levels of caspase-9 and caspase-3.

Dual-targeting nanomicelles with CD133 and CD44 aptamers for enhanced delivery of gefitinib to two populations of lung cancer-initiating cells.

Lung cancer is an aggressive type of cancer that is associated with a high mortality rate. Lung cancer-initiating cells are populations of self-renewing cancer cells with pluripotent differentiation ability. Cancers typically originate from multiple phenotypically distinct cancer-initiating cells. CD133 and CD44 are specific markers that maybe used to distinguish lung cancer-initiating cells. The ability to target a variety of subsets of cancer-initiating cells instead of targeting only one population of cancer initiating-cells has the potential to increase the cancer therapeutic efficacy. In the present study, CD133 and CD44 aptamer-conjugated nanomicelles loaded with gefitinib (CD133/CD44-NM-Gef) were developed to target CD133+ and CD44+ lung cancer-initiating cells. The therapeutic efficacy of CD133/CD44-NM-Gef against lung cancer-initiating cells was assessed by evaluating cell proliferation, tumorsphere formation and detection of CD44+ and CD133+ cells using flow cytometry. The results indicated that CD133/CD44-NM-Gef targeted CD133+ and CD44+ lung cancer-initiating cells and exhibited greater therapeutic efficacy against lung cancer-initiating cells than single-target and non-targeted nanomicelles, suggesting that CD133/CD44-NM-Gef represents a promising treatment for lung cancer by specifically targeting lung cancer-initiating cells. To the best of our knowledge, the present study was the first to report on drug delivery via nanomedicines targeted to multiple populations of cancer-initiating cells using aptamers. As cancer is typically derived from phenotypically distinct cancer-initiating cells, the nanomicelle-based multiple targeting strategy provided is promising for targeting multiple subsets of cancer-initiating cell within a tumor.

Abstract A080: Caveolin-1 detected in tumor-derived exosomes induces cancer stem cell phenotypes in castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is by far the most common male malignancy and the third leading cause of cancer-related death in the US. Regardless of a great response of hormonal therapy for metastatic PCa patients, eventually PCa relapses to an end-stage castration-resistant prostate cancer (CRPC) leading to mortality. Although introducing newer androgen receptor (AR) pathway inhibitors is encouraging, treatment resistance in CRPC continues to remain a clinical problem. Clinical evidence of treatment resistant CRPC indicates carcinoma cell undergoing transdifferentiation. However, the mechanism as well as cell lineage are not fully characterized, which highlights an unmet clinical need to uncover potential therapeutic targets for targeted therapy to improve the overall survival of CRPC patients. Exosome is characterized as nano-grade small extracellular vesicle derived from the endosomal system from many kinds of cells. Therefore, exosome can carry many kinds of message molecules as a delivery vehicle in a paracrine or endocrine manner. Overall, tumor-derived exosomes (TDE) has been shown to play a critical role in promoting cancer progression or drug resistance. Our recent data have shown that TDE from PCa cells is able to induce neuroendocrine differentiation of CRPC, which leads us to further elucidate the role of PCa-derived exosome. Previously by using xenograft model, we have reported that elevated caveolin-1 (Cav-1) in LNCaP cell can accelerate its tumor growth but also increase the tumor incidence of low tumorigenic cells in a paracrine manner, implying that Cav-1 can be secreted extracellualrly. However, the functional role of secreted Cav-1 in PCa is still being debated. The expression of Cav-1 was considered to function as a suppressor of cell transformation and as preventing the development and metastases of mammary tumor; on the other hand, Cav-1 served as an oncogene that correlates with PCa progression. Furthermore, recent studies have shown that Cav-1 expression is associated with chemoresistance in multiple cancers as well as cancer stem cell (CSC) property in lung cancer. CSC theory has raised significant attention in recent years because of its self-renewal and quiescent nature, which underlies its role as a cancer-initiating cell population responsible for recurrent tumor with treatment resistance. In this study, we purified TDE from various PCa cell lines including either Cav-1 positive (Cav-1High) or negative (Cav-1low) cells using membrane-based affinity chromatography, and further characterized its size and number using Zetaview (Particle Matrix, Mebane, NC) and Exocet quantitation kit (SBI, Palo Alto, CA) separately. We found that TDE derived from metastatic PCa cells without AR expression is capable of inducing CSC phenotypes in AR-expressing recipient cells. Further analyses demonstrated that the high accumulation of Cav-1 in TDE appears to be a potential candidate. Indeed, Cav-1High cells or exogenous Cav-1-treated PCa cell lines can increase CSC phenotypes in vitro and in vivo. Mechanistically, the presence of Cav-1 is able to increase stem cell transcription Yamanaka factors, including KLF4, SOX2, OCT4, and Nanog. Furthermore, from microarray screening, we found RPC32 as another potential Cav-1 downstream effector, which is characterized as a subunit of RNA polymerase III (Pol III). RPC32 has two different isoforms (α and β), and only RPC32α is known to be highly regulated and expressed in stem cell and transformed cells. Therefore, we hypothesize that RPC32α is responsible for the elevated Yamanaka factors protein expression leading to CSC development. Nevertheless, we conclude that the presence of Cav-1 in PCa TDE plays an inductive role in the appearance of treatment-resistant CRPC in a paracrine manner, which provides a potential prognostic marker and therapeutic target. Citation Format: Chun-Jung Lin, Andrew Dang, Elizabeth Hernandez, Jer-Tsong Hsieh. Caveolin-1 detected in tumor-derived exosomes induces cancer stem cell phenotypes in castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A080.

Abstract 2008: Stem cell-associated proteins P63 and Bmi-1 may cooperate in maintaining cancer-initiating cell populations in squamous cell carcinomas

Abstract Overexpression of p53-related protein p63, particularly ΔNp63α, is seen in squamous cell carcinomas of the head and neck, lung, cervix and skin. We use primary murine epidermal keratinocytes as a model of human squamous epithelial cells and mimic these elevated levels using adeno- and lentiviral vectors. In previous studies, we established that ΔNp63α inhibits induction of p16ink4a and p19arf, blocks oncogene-induced senescence, and cooperates with activated v-rasHa to enhance conversion of papilloma to carcinoma in a nude mouse grafting model. We evaluated the relationship between ΔNp63α and Bmi-1, a member of the polycomb complex known to repress the INK4a/ARF gene locus, and modulated Bmi-1 expression in the presence or absence of elevated ΔNp63α to assess its impact on p16ink4a and p19arf expression. Bmi-1 protein levels correlate with those of ΔNp63α; however, higher levels of Bmi-1 block p16ink4a but not p19arf. Consistent with this, Bmi-1 is required for the binding of ΔNp63α to the p16ink4a but not the p19arf promoter. These findings may have further important implications in HNSCC as other previously published models have demonstrated that Bmi-1 is important for prevention of senescence in stem cells. Using FACS analysis and cell sorting, we are defining cell populations of keratinocytes with respect to the expression of ΔNp63α, Bmi-1, and other markers of stem cells and senescence and extending these approaches from murine to human HNSCC models with the aim of correlating these distinct patterns with cancer-initiating potential and tumor phenotype. Citation Format: Linan Ha, Devin Reilly, Roshini Ponnamperuma, Andrea George, Wendy C. Weinberg. Stem cell-associated proteins P63 and Bmi-1 may cooperate in maintaining cancer-initiating cell populations in squamous cell carcinomas [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 2008.

NAMPT Is a Potent Oncogene in Colon Cancer Progression that Modulates Cancer Stem Cell Properties and Resistance to Therapy through Sirt1 and PARP.

Purpose: Colorectal cancer is the second most common cancer in women and the third most common in men worldwide. However, despite current progress, many patients with advanced and metastatic tumors still die from the malignancy. Refractory disease often relies on nicotinamide adenine dinucleotide (NAD)-dependent mechanisms. NAD metabolism and a stable NAD regeneration circuit are required to maintain tissue homeostasis and metabolism. However, high levels of NAD confer therapy resistance to tumors.Experimental Design: Ectopic overexpression of nicotinamide phosphoribosil transferase (NAMPT) and shRNAs in colorectal cancer cell lines, tumorigenic and stemness properties and transcription measurement in culture and in vivo Transcriptional analysis in public databases. Therapeutic approaches.Results: NAMPT, the rate-limiting enzyme responsible for the highest source of physiologic NAD biosynthesis, increases tumorigenic properties and induces cancer stem cell-like properties through pathways that control stem cell signaling, thus enriching the cancer-initiating cell (CIC) population. Furthermore, NAMPT expression correlated with high levels of CIC-like cells in colon tumors directly extracted from patients, and transcription meta-analysis revealed that NAMPT is also a key factor that induces cancer stem pathways in colorectal cancer tumors. This effect is mediated by PARP and SIRT1. In addition, we report a novel NAMPT-driven signature that stratifies prognosis from high to low expression groups. The NAMPT signature contained SIRT1 and PARP1 levels as well as other cancer stem cell-related genes. Finally, NAMPT inhibition increased the sensitivity to apoptosis in both NAMPT-expressing cells and tumorspheres.Conclusions: NAMPT represents a novel therapeutic target in colon cancer progression and relapse, particularly the CIC subset of human colon cancers. Clin Cancer Res; 24(5); 1202-15. ©2017 AACR.

NAMPT overexpression induces cancer stemness and defines a novel tumor signature for glioma prognosis.

Gliomas are the most prevalent primary malignant brain tumors associated with poor prognosis. NAMPT, a rate-limiting enzyme that boosts the nicotinamide adenine dinucleotide (NAD) regeneration in the salvage pathway, is commonly expressed in these tumors. NAD metabolism is required to maintain tissue homeostasis. To maintain metabolism, cancer cells require a stable NAD regeneration circuit. However, high levels of NAD confer resistance to therapy to these tumors, usually treated with Temozolomide (TMZ). We report that NAMPT overexpression in glioma cell lines increases tumorigenic properties controlling stem cell pathways and enriching the cancer-initiating cell (CIC) population. Furthermore, NAMPT expression correlated with high levels of Nanog, CD133 and CIC-like cells in glioblastoma directly extracted from patients. Meta-analysis reveals that NAMPT is also a key factor inducing cancer stem pathways in glioma cells. Furthermore, we report a novel NAMPT-driven signature which stratify prognosis within tumor staging. NAMPT signature also correlates directly with EGFR positive and IDH negative tumors. Finally, NAMPT inhibition increases sensitivity to apoptosis in both NAMPT-expressing cells and tumorspheres. Therefore, NAMPT represents a novel therapeutic target in Glioma progression and relapse.

336 - Molecular mechanisms involved in the immunomodulation induced by LIF in cancer

LIF is a pleiotropic cytokine that can act as an immunomodulatory factor in different biological processes, such as embryo implantation, organ transplantation tolerance and multiple sclerosis. Here, we have observed that in tumors expressing LIF, its blockade inhibits tumor growth, and that the anti-tumor effect of LIF neutralization is mediated by the polarization of tumor-associated myeloid cells (TAMCs). LIF sustains the expression of CCL22, MRC1 and CD163 in human healthy monocytes and in TAMCs from mouse syngeneic (non-small cell lung cancer, ovarian cancer and colorectal cancer) models, patient-derived xenograft models and organotypic cultures from glioblastoma. We show that LIF blockade down-regulates CCL22 secretion by TAMCs preventing regulatory T cell and inducing effector T and NK cell tumor infiltration, leading to an increase in tumor cell apoptosis and an anti-tumor response. A positive correlation between M2-like markers and LIF expression has been observed in glioblastoma patients, and high levels of these factors confer poor prognosis. Moreover, we have studied one of the multiple mechanisms through which LIF could modulate the inhibition of effector T and NK cells: the regulation of PD-1/PDL1 axis. We have seen that after anti-LIF treatment of tumor-bearing mice, the expression of PDL1 decreases in tumor cells and in TAMCs. Furthermore, a positive correlation between PDL1 and LIF expression has been observed in glioblastoma patients, and both proteins also correlate with CD44 expression, a marker for cancer-initiating cell (CIC) population. High levels of these three factors confer poor prognosis. Thus, we have found that LIF acts as an immune-suppressor in cancer recapitulating its normal function in other biological processes. LIF is a bridge between the innate and adaptive immune responses through the induction of CCL22 in TAMCs and subsequent regulation of regulatory and effector T and NK cells. Our results identify LIF as a promising immuno-oncology therapeutic target and establish the translational potential of anti-LIF agents.

Sexual dimorphism in cancer.

The incidence of many types of cancer arising in organs with non-reproductive functions is significantly higher in male populations than in female populations, with associated differences in survival. Occupational and/or behavioural factors are well-known underlying determinants. However, cellular and molecular differences between the two sexes are also likely to be important. In this Opinion article, we focus on the complex interplay that sex hormones and sex chromosomes can have in intrinsic control of cancer-initiating cell populations, the tumour microenvironment and systemic determinants of cancer development, such as the immune system and metabolism. A better appreciation of these differences between the two sexes could be of substantial value for cancer prevention as well as treatment.

Abstract 2331: CD271 defines a cancer-initiating cell population in hypopharyngeal cancer

Abstract Cancer stem cells (CSCs) or Cancer initiating cells (CICs) contribute to the malignant phenotypes of a variety of cancers, but markers to identify human hypopharyngeal cancer (HPC) stem cells remain poorly understood. We found that the CD271+ but not CD271- cell population sorted from xenotransplanted HPCs possesses tumor-initiating capability in immunodeficient mice. Tumors generated from the CD271+ cells contained both CD271+ and CD271- cells, indicating that the population could undergo differentiation. Immunohistological analyses of the tumors revealed that the CD271+ cells localized to a perivascular niche near CD34+ vasculature, to invasive fronts, and to the basal layer. In accordance with these characteristics, a stemness marker, Nanog, and matrix metaloproteinases (MMPs), which are implicated in cancer invasion, were significantly up-regulated in the CD271+ compared to the CD271- cell population. Furthermore, using primary HPC specimens, we demonstrated that high CD271 expression was correlated with a poor prognosis for patients. Furthermore, RNA interference of CD271 in CD271+ cells decreased efficiency of tumor initiation and migration phenotypes, suggesting that CD271 is indeed functional. Taken together, our findings indicate that CD271 is a novel marker for cancer initiating cells and for HPC prognosis. Citation Format: Nobuyuki Tanaka, Takayuki Imai, Mai Mochizuki, Keiichi Tamai, Kazunori Yamaguchi, Kennichi Satoh, Kazuto Matsuura, Ikuro Sato, Shigeru Saijo, Kazuo Sugamura. CD271 defines a cancer-initiating cell population in hypopharyngeal cancer. [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 2331. doi:10.1158/1538-7445.AM2015-2331

Role of flavonoids in future anticancer therapy by eliminating the cancer stem cells.

Despite the numerous recent advances made in conventional anticancer therapies, metastasis and recurrence still remain the major problems in cancer management. The current treatment modalities kill the bulk of the tumor, leaving cancer stem cells behind and therefore, the agents specifically targeting this cancer initiating cell population may have important clinical implications. In this review article, the data about the inhibitory action of flavonoids, both natural as well as their synthetic derivatives, on the self-renewal capacity and survival of cancer stem cells of different origins are compiled and analyzed. These data indicate that several plant secondary metabolites, including soy isoflavone genistein, green tea catechins and a widely distributed flavonol quercetin, have the potential to suppress the stemness markers and properties, traits of the epithelial-to-mesenchymal transition and migratory characteristics, being also able to sensitize these cells to the standard chemotherapeutic drugs. These polyphenolic compounds act through multiple signal transduction pathways, providing thus the maximal therapeutic response and offering some promise to be included in the future cancer treatment schemes in combination with the conventional therapies. Such approach may give an important contribution to the shift of cancer management from palliative to curative mode, likely leading to the disease-free survival. Thus, flavonoids can serve as attractive candidates for novel anticancer agents by eliminating the roots of cancer.

Smad4 Decreases the Population of Pancreatic Cancer–Initiating Cells through Transcriptional Repression of ALDH1A1

Cancer progression involves a rare population of undifferentiated cancer-initiating cells that have stem cell-like properties for self-renewal capacity and high tumorigenicity. We investigated how maintenance of pancreatic cancer-initiating cells is influenced by Smad4, which is frequently deleted or mutated in pancreatic cancers cells. Smad4 silencing up-regulated the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA, whereas forced expression of Smad4 in pancreatic cancer cells down-regulated it. Smad4 and ALDH1 expression inversely correlated in some human clinical pancreatic adenocarcinoma tissues, suggesting that ALDH1 in pancreatic cancer cells was associated with decreased Smad4 expression. We then examined whether ALDH1 served as a marker of pancreatic cancer-initiating cells. Pancreatic cancer cells contained ALDH1(hi) cells in 3% to 10% of total cells, with high tumorigenic potential. Because Smad4 is a major mediator of transforming growth factor (TGF)-β family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-β and bone morphogenetic proteins. Treatment with TGF-β attenuated ALDH1(hi) cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-β regulated ALDH1A1 mRNA transcription through binding ofSmad4 to its regulatory sequence. It appears that TGF-β negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer-initiating cells.

Abstract LB-152: Macrophage control of cancer cell stemness and tumor metastasis

Abstract A characteristic feature of cancer is the presence of an abundant inflammatory infiltrate, which includes tumor-associated macrophages. In response to diverse tumor-derived chemoattractants, macrophages invade the tumor microenvironment, promoting angiogenesis, growth, immunosuppression and metastasis. Because they stimulate tumor progression and metastasis, it is possible that tumor-associated macrophages enhance cancer initiating cell populations leading to increased tumorigenicity and metastasis. In the tumor microenvironment, macrophages are characterized into having two phenotypes, classical (M1) or alternative (M2). M2 macrophages suppress T-cell activation and promote angiogenesis and tumor progression, while M1 macrophages produce immunostimulatory cytokines and stimulate T cell mediated anti-tumor immunity. Whether and how macrophages affect cancer cell stemness and metastasis is not well understood. Recently our lab showed that suppression of PI3Kinase-gamma signaling in myeloid cells leads to a reduction in tumor growth, angiogenesis, and metastasis. Genetic or pharmaceutical blockade of PI3Kinase-gamma or its target alpha4beta1 integrin inhibits the trafficking of monocytes to tumors and blocks expression of M2 macrophage factors, such as TGF-beta, Arginase and IL-6. To discover how PI3Kinase-gamma regulates the macrophage phenotype we performed RNA sequencing (RNA-seq) of classical and alternative polarization conditions in PI3Kinase-gamma-deficient and WT macrophages. Using this approach, we identified gene clusters associated with tumor immunity and metastasis that are regulated by PI3Kinase-gamma. Furthermore, we observed that macrophages polarized to the M2 phenotype enhanced the colony formation of breast and lung cancer cell lines in an anchorage independent tumor spheroid outgrowth assay whereas M1 polarization inhibited colony formation. Taken together, our data support the hypotheses that PI3Kinase-gamma expressing M2 macrophages promote cancer cell stemness and tumor metastasis. These studies could lead to new approaches for cancer therapy by encouraging the use of PI3Kinase-gamma antagonists as adjuvants for immunotherapy of human cancer. Citation Format: Sara Gorjestani. Macrophage control of cancer cell stemness and tumor metastasis. [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 LB-152. doi:10.1158/1538-7445.AM2014-LB-152

DCLK1 Marks a Morphologically Distinct Subpopulation of Cells With Stem Cell Properties in Preinvasive Pancreatic Cancer

As in other tumor types, progression of pancreatic cancer may require a functionally unique population of cancer stem cells. Although such cells have been identified in many invasive cancers, it is not clear whether they emerge during early or late stages of tumorigenesis. Using mouse models and human pancreatic cancer cell lines, we investigated whether preinvasive pancreatic neoplasia contains a subpopulation of cells with distinct morphologies and cancer stem cell-like properties. Pancreatic tissue samples were collected from the KC(Pdx1), KPC(Pdx1), and KC(iMist1) mouse models of pancreatic intraepithelial neoplasia (PanIN) and analyzed by confocal and electron microscopy, lineage tracing, and fluorescence-activated cell sorting. Subpopulations of human pancreatic ductal adenocarcinoma (PDAC) cells were similarly analyzed and also used in complementary DNA microarray analyses. The microtubule regulator DCLK1 marked a morphologically distinct and functionally unique population of pancreatic cancer-initiating cells. These cells displayed morphological and molecular features of gastrointestinal tuft cells. Cells that expressed DCLK1 also expressed high levels of ATAT1, HES1, HEY1, IGF1R, and ABL1, and manipulation of these pathways in PDAC cell lines inhibited their clonogenic potential. Pharmacological inhibition of γ-secretase activity reduced the abundance of these cells in murine PanIN in a manner that correlated with inhibition of PanIN progression. Human PDAC cells and pancreatic neoplasms in mice contain morphologically and functionally distinct subpopulations that have cancer stem cell-like properties. These populations can be identified at the earliest stages of pancreatic tumorigenesis and provide new cellular and molecular targets for pancreatic cancer treatment and/or chemoprevention.

The nuclear coactivator amplified in breast cancer 1 maintains tumor-initiating cells during development of ductal carcinoma in situ

The key molecular events required for the formation of Ductal Carcinoma in Situ (DCIS) and its progression to invasive breast carcinoma have not been defined. Here we show that the nuclear receptor coactivator Amplified In Breast cancer 1 (AIB1) is expressed at low levels in normal breast but is highly expressed in DCIS lesions. This is of significance since reduction of AIB1 in human MCFDCIS cells restored a more normal 3D mammary acinar structure. Reduction of AIB1 in MCFDCIS cells, both prior to DCIS development or in existing MCFDCIS lesions in vivo, inhibited tumor growth and led to smaller, necrotic lesions. AIB1 reduction in MCFDCIS cells was correlated with significant reduction in the CD24−/CD44+ Breast Cancer Initiating Cells (BCIC) population, and a decrease in myoepithelial progenitor cells in the DCIS lesions in vitro and in vivo. Loss of AIB1 in MCFDCIS cells was also accompanied by a loss of expression of NOTCH 2, 3 and 4, JAG2, HES1, GATA3, HER2 and HER3 in vivo. These signaling molecules have been associated with differentiation of breast epithelial progenitor cells. These data indicate that AIB1 plays a central role in the initiation and maintenance of DCIS and that reduction of AIB1 causes loss of BCIC, loss of components of the NOTCH, HER2 and HER3 signaling pathways and fewer DCIS myoepithelial progenitor cells in vivo. We propose that increased expression of AIB1, through maintenance of BCIC, facilitates formation of DCIS, a necessary step prior to development of invasive disease.