Putative Tumor-initiating Cells Research Articles

Annexin A1 Is Required for Efficient Tumor Initiation and Cancer Stem Cell Maintenance in a Model of Human Breast Cancer.

Simple SummaryTriple-negative breast cancer (TNBC) has a poor outcome compared to the other major breast cancer subtypes and new therapies are needed. We sought to clarify the functions of a ubiquitous protein, Annexin A1, in the development and progression of TNBC. We found that Annexin A1 expression correlated with poor patient prognosis in basal-like breast tumors and also in the basal like-2 subset of TNBCs. Stable knockdown of Annexin A1 attenuated the growth of SUM149 xenografts, which model basal-like 2 tumors. In a polyoma middle T antigen-driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation, induced epithelial to mesenchymal transition and upregulated basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24+/Sca1− population containing putative tumor-initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in certain TNBC tumors.Triple-negative breast cancer (TNBC) has a poor outcome compared to other breast cancer subtypes, and new therapies that target the molecular alterations driving tumor progression are needed. Annexin A1 is an abundant multi-functional Ca2+ binding and membrane-associated protein. Reported roles of Annexin A1 in breast cancer progression and metastasis are contradictory. Here, we sought to clarify the functions of Annexin A1 in the development and progression of TNBC. The association of Annexin A1 expression with patient prognosis in subtypes of TNBC was examined. Annexin A1 was stably knocked down in a panel of human and murine TNBC cell lines with high endogenous Annexin A1 expression that were then evaluated for orthotopic growth and spontaneous metastasis in vivo and for alterations in cell morphology in vitro. The impact of Annexin A1 knockdown on the expression of genes involved in mammary epithelial cell differentia tion and epithelial to mesenchymal transition was also determined. Annexin A1 mRNA levels correlated with poor patient prognosis in basal-like breast tumors and also in the basal-like 2 subset of TNBCs. Unexpectedly, loss of Annexin A1 expression had no effect on either primary tumor growth or spontaneous metastasis of MDA-MB-231_HM xenografts, but abrogated the growth rate of SUM149 orthotopic tumors. In an MMTV-PyMT driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation in both immuno-competent and immuno-deficient mice and induced epithelial to mesenchymal transition and upregulation of basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24+/Sca1− population containing putative tumor initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in a model of human breast cancer and suggest that some basal-like TNBCs may require high endogenous tumor cell Annexin A1 expression for continued growth.

Abstract 3107: Modeling DICER1 syndrome in cells

Abstract DICER1 is an endoribonuclease central to generating microRNAs (miRNAs), small RNA molecules that downregulate the expression of approximately 30% of protein-coding genes. Germ-line mutations in DICER1 have been identified in patients afflicted with a pleiotropic tumor predisposition syndrome, usually referred to as DICER1 syndrome (OMIM 606241). DICER1 syndrome is thought to originate from a mesenchymal cell bearing a frameshift or nonsense mutation on one allele and a missense mutation affecting the RNase IIIb domain on the other allele. While interest in the role of DICER1 in normal development and pathogenesis has been explored in both the knockout and overexpression context, neither situation is relevant for understanding DICER1 syndrome. We hypothesize that defective DICER1 protein production in a mesenchymal stem cell at a critical time in development leads to altered miRNA populations that, in turn, initiate or prime cells for tumorigenesis. The goal of this study was to develop and characterize a model of the putative tumor-initiating cell namely a mesenchymal stromal cell (MSC) bearing a DICER1 RNase IIIb mutation. SV-40 immortalized mouse mesenchymal stromal cells (MSCs) containing a homozygous floxed exon for DICER1 were ex vivo cre inactivated to produce Dicer-/- MSCs. Into these Dicer-/- MSCs, a FLAG-tagged human DICER1, a FLAG-tagged RNase IIIa mutant DICER1, and a FLAG-tagged RNase IIIb mutant DICER1 were stably introduced. Cells were characterized by Western blot in response to serum starvation and stimulation. Their ability to form anchorage-independent colonies was assessed by soft agar assay pre- and post-oncogene introduction. Transcriptomes were interrogated by gene expression microarray. Results: Alterations in protein expression were observed in cells in response to starvation and stimulation. Certain cell lines were able to produce anchorage-independent colonies. Conclusions: We have created MSCs bearing RNase IIIb-mutated DICER1 to model DICER1 syndrome. We suggest that the ability for an MSC to produce only 3p miRNAs in the presence of an oncogene can allow for anchorage-independent growth. Citation Format: Mona K. Wu. Modeling DICER1 syndrome in cells [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 3107.

Abstract 112: Identifying drugs that target ovarian cancer tumor initiating cells

Abstract Ovarian cancer is the most lethal gynecological cancer, with overall five-year survival for women with advanced disease being at only 25%. The current standard of care is treatment with a platinum-based chemotherapy drug. Unfortunately, a subpopulation of tumor cells often persists, contributing to platinum-based chemotherapy resistance and disease recurrence. In this study we investigated drugs to target these putative tumor-initiating cells (TICs), and explored the pathways of these drugs in both adherent proliferative cell culture conditions and non-adherent TIC-enriched cultures. We conducted a drug screen under both growth conditions, in order to identify compounds that are able to inhibit growth of TICs. Of the drugs identified, we focused further efforts on those involved with inhibiting the NF-kappaB pathway because prior studies have linked NF-kappaB activity with drug resistance and poor survival. Cell viability assays done with the four drugs, bardoxolone methyl, salinomycin, disulfiram, and elesclomol, show these drugs inhibit the growth of ovarian cancer cell lines both as TICs and in corresponding adherent cultures. Disulfiram showed preferential killing of TICs in some of the cell lines tested. All drugs showed some evidence for inhibiting NF-kappaB on Western blot, and ability to decrease CD133/ALDH double-positive TICs on flow cytometry. Carboplatin is known to kill proliferative ovarian cancer cells, and increase the relative percentage of CD133/ALDH positive TICs. Our ongoing studies will address combination of each drug with carboplatin, with the hypothesis that these drugs will increase in vivo efficacy by suppressing TICs, and thereby decrease recurrence of platinum-resistant ovarian cancer. Citation Format: Michelle K. Ozaki, Carrie D. House, Christina M. Annunziata. Identifying drugs that target ovarian cancer tumor initiating cells [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 112. doi:10.1158/1538-7445.AM2017-112

Gain-of-function p53 activates multiple signaling pathways to induce oncogenicity in lung cancer cells

Gain‐of‐function (GOF) mutants of p53 upregulate genes implicated in cell proliferation and oncogenesis. Here, we report that GOF p53 induces tumorigenicity through simultaneous activation of key oncogenic pathways including those controlling putative tumor‐initiating cell functions. We determined that in cells expressing p53‐R273H, GOF p53 simultaneously upregulates genes from multiple signaling pathways by recognizing promoters containing distinct transcription factor (TF) binding sites. Our analytical data support a model in which GOF p53 complexes with two TFs on the promoter—a mediator protein, Med17, and a histone acetyl transferase, activating histone acetylation—and enhances gene expression to signal cell proliferation and oncogenesis. Thus, therapeutic inhibition of one GOF p53‐induced pathway would be insufficient to prevent tumor growth as the oncoprotein activates a multitude of parallel pathways. This discovery suggests enormous selection advantage for cancer cells with GOF p53 to induce oncogenic growth, highlighting the problems of cancer therapy.

KDM4C, a H3K9me3 Histone Demethylase, is Involved in the Maintenance of Human ESCC-Initiating Cells by Epigenetically Enhancing SOX2 Expression

Our studies investigating the existence of tumor-initiating cell (TIC) populations in human esophageal squamous cell carcinoma (ESCC) had identified a subpopulation of cells isolated from ESCC patient-derived tumor specimens marked by an ALDHbri+ phenotype bear stem cell-like features. Importantly, KDM4C, a histone demethylase was enhanced in ALDHbri+ subpopulation, suggesting that strategies interfering with KDM4C may be able to target these putative TICs. In the present study, by genetic and chemical means, we demonstrated that, KDM4C blockade selectively decreased the ESCC ALDHbri+ TICs population in vitro and specifically targeted the TICs in ALDHbri+-derived xenograft, retarding engraftment. Subsequent studies of the KDM4C functional network identified a subset of pluripotency-associated genes (PAGs) and aldehyde dehydrogenase family members to be preferentially down-regulated in KDM4C inhibited ALDHbri+ TICs. We further supported a model in which KDM4C maintains permissive histone modifications with a low level of H3K9 methylation at the promoters of several PAGs. Moreover, ectopic expression of SOX2 restored KDM4C inhibition-dependent ALDHbri+ TIC properties. We further confirmed these findings by showing that the cytoplasmic and nuclear KDM4C staining increased with adverse pathologic phenotypes and poor patient survival. Such staining pattern of intracellular KDM4C appeared to overlap with the expression of SOX2 and ALDH1. Collectively, our findings provided the insights into the development of novel therapeutic strategies based on the inhibition of KDM4C pathway for the eliminating of ESCC TIC compartment.

High expression of TROP2 characterizes different cell subpopulations in androgen-sensitive and androgen-independent prostate cancer cells.

Progression of castration-resistant tumors is frequent in prostate cancer. Current systemic treatments for castration-resistant prostate cancer only produce modest increases in survival time and self-renewing Tumor-Initiating Cells (TICs) are suspected to play an important role in resistance to these treatments. However it remains unclear whether the same TICs display both chemo-resistance and self-renewing abilities throughout progression from early stage lesions to late, castration resistant tumors. Here, we found that treatment of mice bearing LNCaP-derived xenograft tumors with cytotoxic (docetaxel) and anti-androgen (flutamide) compounds enriched for cells that express TROP2, a putative TIC marker. Consistent with a tumor-initiating role, TROP2high cells from androgen-sensitive prostate cancer cell lines displayed an enhanced ability to re-grow in culture following treatment with taxane-based chemotherapy with or without androgen blockade. TROP2 down-regulation in these cells reduced their ability to recur after treatment with docetaxel, in the presence or absence of flutamide. Accordingly, in silico analysis of published clinical data revealed that prostate cancer patients with poor prognosis exhibit significantly elevated TROP2 expression level compared to low-risk patients, particularly in the case of patients diagnosed with early stage tumors. In contrast, in androgen-independent prostate cancer cell lines, TROP2high cells did not exhibit a differential treatment response but were characterized by their high self-renewal ability. Based on these findings we propose that high TROP2 expression identifies distinct cell sub-populations in androgen-sensitive and androgen-independent prostate tumors and that it may be a predictive biomarker for prostate cancer treatment response in androgen-sensitive tumors.

MB-23 * CO-INHIBITION OF THE SONIC HEDGEHOG AND CXCR4 PATHWAYS UNIQUELY BLOCKS TUMOR INITIATING CELL FUNCTION IN MEDULLOBLASTOMA

Medulloblastoma is the most common malignant brain tumor of childhood. Even with current intensive treatments there is significant morbidity and mortality associated with this diagnosis. Recent hope for improved outcome derives from the stratification of medulloblastoma into four molecular subtypes and the promise that subtype-specific targeted therapy will be more efficacious and less toxic. However, even within specific subtypes there can be variable outcomes, suggesting that further subclasification will be required. We previously showed that CXCR4, a G-protein coupled receptor, is differentially expressed in the Sonic hedgehog (Shh) subtype of medulloblastoma and its activation is required for maximal medulloblastoma growth in vitro and in vivo. We investigated the utility of combining inhibition of both the Shh and CXCR4 pathways in a preclinical model of Shh-medulloblastoma. We measured anti-tumor activity and effects on tumor subtransplantation efficiency of single agents and combined treatment with the Shh antagonist GDC-0449 (vismodegib) and the CXCR4 antagonist AMD3100. We found that combined therapy had the greatest anti-tumor effect and that only inhibition of both pathways abrogated tumorigenicity upon subtransplantation. These results indicated that dual-inhibition was affecting the tumor initiating cell (TIC) population. We used flow cytometry to further investigate this possibility and found that dual inhibition resulted in the loss of a subpopulation of cells with high CXCR4 and CD15 surface expression. Concordant with this diminution in the putative TIC population we found decreased expression of the stem cell markers Sox2, Nanog and Oct4 in doubly treated tumors. Decreased expression of these stem cell markers was associated with increased presence of the repressive histone mark H3K27me3 at their promoters. Together these data indicate that the Shh and CXCR4 signaling pathways in medulloblastoma converge in the subpopulation of TICs to regulate stem cell activity. Dual inhibition may therefore represent a powerful new approach to medulloblastoma cure.

Transcriptome analysis of basal and luminal tumor-initiating cells in ErbB2-driven breast cancer.

Breast cancer is the leading cause of cancer-related mortality for females worldwide [1]. Improving early screening strategies and understanding the events that lead to tumor initiation have led to demonstrable improvements in clinical outcome. Our previous work revealed a variance in the tumorigenic capacity between different mammary epithelial cell populations in an MMTV-ErbB2 mouse model. In order to greater understand how different mammary epithelial cells influence the tumorigenic capacity in ErbB2-induced breast cancer, we transplanted different cell populations from pre-neoplastic MMTV-ErbB2 female mice into recipient mice for tumorigenic study. We found that different mammary epithelial cells bear different tumorigenic potentials even when induced by the same ErbB2 proto-oncogene. To understand the difference in tumors formed from different epithelial cells, we performed gene expression profiling using these tumors (GSE64487). Several genes were further validated using real-time reverse transcription polymerase chain reaction (RT-PCR). Here we provide further details on the experimental methods and microarray analysis. This data provides a resource to further understanding how different mammary cell populations can initiate ErbB2-driven tumors and the role of these cell populations as putative tumor-initiating cells (TICs).

Initiate Tumors with Single Cell Spheres Formed in Serum-Containing Medium.

Background: It is difficult to prospectively identify and maintain putative tumor-initiating cells (TICs). Spheres that formed in serum-free media contained more TICs while spheres formed in serum-containing media were not used in tumor-initiating.Methods: Soft-agar was used to isolate colonies. A continuous, static suspension culture using serum-containing media was modified from liquid overlay technique and tumor cell spheres could be maintained by this method for >90 days. Tumor-initiating capacity of these spheres was tested in nude mice. The nuclear staining of OCT4 in sphere cells and the expression profile of hepatic cell lineage related genes were examined.Results: Soft-agar derived HepG2 colonies indicated different properties from their parental cells. The suspension cells of A549 and MCF7 could initiate tumors at 104 cells level. The growth proportions of individual A549, MCF7 and HepG2 spheres with diameter of 101-150 µm were significantly higher than smaller spheres. After suspension culture for 15-27 days, HepG2 and MCF7 spheres could initiate tumors with diameter up to 200 µm; the estimated TIC frequency was 1/1500-1/400. The HepG2 and MCF7 spheres retain tumor-initiating potential for at least 52 days.Conclusion: After 15 days' serum-containing suspension culture small HepG2 and MCF7 cell spheres (diameter ~200 µm) could initiate tumors in nude mice.

Abstract A70: Chemotherapy-resistant subpopulations in a tumor-initiating cell model of human osteosarcoma

Abstract Introduction: Tumor-initiating cells (TICs) are a subpopulation of therapy resistant cells in osteosarcoma. To date, TICs have been experimentally defined based on the chosen isolation technique. Common to all isolation methods, TICs demonstrate a high degree of the multi-drug resistance phenotype, are more invasive, metastasize readily, and have increased capacity for tumorigenesis. Given this phenotype TICs represent a viable therapeutic target. Cultivating cells in low-adherence, serum-deprived conditions results in spherical colony formation. Certain growth factors present in serum drive epithelial-to-mesenchymal transition, and typically confound TIC models of carcinoma. The effect of serum on mesenchymal-based sarcoma TIC models is less clear. Recent literature suggests that merely the presence of low-adherence conditions can enrich in TICs. We have shown that osteosarcoma cells cultured in low-adherence conditions in the presence of 10% fetal bovine serum (FBS) form spheres, exhibit an increased expression of genes implicated in developmental programs and stem cells, are more resistant to chemotherapy, and more readily initiate tumors than adherent cells when injected into mice. Methods: The osteosarcoma cell lines 143B, mHOS, and MG-63 were cultured in low-adherence plates using DMEM or MEM media supplemented with 10% FBS. Spheres were routinely grown for 4-6 days between passages and dissociated using 0.05% trypsin. Relative mRNA expression levels of Nanog, Sox2, Oct4 and Axin2 was assessed using quantitative-PCR (SYBR Green). Resistance to chemotherapy (doxorubicin and cisplatin) was determined by comparing both adherent cells and spheres (in low adherence 96-well plates) grown to at least the second passage. After 72 hours IC50 values were calculated using the CellTitre Glo luminescence assay. To assess in vivo tumorgenicity, adherent and sphere cells were dissociated and injected into the flanks of nude mice at a density of 10,000 cells. Upon tumor formation the mice were euthanized and the tumors re-implanted into a second set of mice to test their ability to serially transplant. Results: Sarcospheres can be successfully and reproducibly grown and passaged in low-adherent culture conditions in media supplemented with 10% FBS. Expression profiling demonstrates increased expression of Sox2, Oct4, Nanog, and Axin2, all genes that have been identified to be associated with developmental programs and stem cells. Sarcospheres also displayed increased chemoresistance compared to adherent cultures to both cisplatin (IC50 change of 1.9, 6.9, and 6.2-fold in 143B, mHOS and MG-63, respectively) and more so to doxorubicin (IC50 change of 12.7, 35 and greater than 74-fold in 143B, mHOS, and MG-63, respectively). Additionally, in a pilot subcutaneous xenograft study, using sphere and adherent cells, differential growth was observed within the TIC-enriched populations demonstrating (1) earlier tumor initiation and (2) a lower overall proliferation rate among the primary and serially transplanted tumors confirming a persistence of the TIC phenotype through in vivo passaging. Conclusions: Osteosarcoma cells cultured in low-adherence conditions in the presence of serum display many of the characteristics of putative tumor-initiating cells including robust sphere formation, upregulation of key stemness mediators, enhanced chemoresistance, and in vivo tumor initiation and serial transplantability. Using this system, we aim to further evaluate these stem-like populations in order to elucidate potential therapies targeting this specialized, chemoresistant niche. Citation Format: Jessica M. Foley, Noel R. Monks, Donald J. Scholten, II, David J. Monsma, Dawna Dylewski, Paula J. Davidson, Matthew R. Steensma. Chemotherapy-resistant subpopulations in a tumor-initiating cell model of human osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A70.

The differentiation of pancreatic tumor-initiating cells by vitronectin can be blocked by cilengitide.

Pancreatic cancer is a leading cancer type and its molecular pathology is poorly understood. The only potentially curative therapeutic option available is complete surgical resection; however, this is inadequate as most of the patients are diagnosed at an advanced or metastatic stage. Tumor-initiating cells (TICs) constitute a subpopulation of cells within a solid tumor that sustain tumor growth, metastasis, and chemo/radioresistance. Within pancreatic cancer, TICs have been identified based on the expression of specific cell surface markers. We use a sphere formation assay to enrich putative TICs and use human serum as a driver of differentiation. We demonstrate by using specific blocking reagents that we can inhibit the differentiation process and maintain TIC-associated markers and genes. We can induce differentiation of pancreatospheres with the addition of human serum, and we identified vitronectin as an inducer of differentiation. We inhibit differentiation by human serum using an arginine-glycine-aspartate-specific peptide, which is Cilengitide; hence, demonstrating this differentiation is mediated via specific integrin receptors. Overall, our studies further the definition of pancreatic TICs and provide further insight into both the maintenance and differentiation of this lethal population.

MiR145 targets the SOX9/ADAM17 axis to inhibit tumor-initiating cells and IL-6-mediated paracrine effects in head and neck cancer.

ALDH1(+)CD44(+) cells are putative tumor-initiating cells (TIC) in head and neck squamous cell carcinomas (HNC). miR-145 regulates tumorigenicity in various cancers but the breadth of its mechanistic contributions and potential therapeutic applications are not completely known. Here, we report that ALDH1(+)CD44(+)-HNC cells express reduced levels of miR145. SPONGE-mediated inhibition of miR-145 (Spg-miR145) was sufficient to drive tumor-initiating characteristics in non-TICs/ALDH1(-)CD44-negative HNC cells. Mechanistic analyses identified SOX9 and ADAM17 as two novel miR145 targets relevant to this process. miR-145 expression repressed TICs in HNC in a manner associated with SOX9 interaction with the ADAM17 promoter, thereby activating ADAM17 expression. Notably, the SOX9/ADAM17 axis dominated the TIC-inducing activity of miR-145. Either miR-145 suppression or ADAM17 overexpression in non-TICs/ALDH1(-)CD44(-)-HNC cells increased expression and secretion of interleukin (IL)-6 and soluble-IL-6 receptor (sIL-6R). Conversely, conditioned medium from Spg-miR145-transfected non-TICs/ALDH1(-)CD44(-)-HNC cells was sufficient to confer tumor-initiating properties in non-TICs/ALDH1(-)CD44(-)-HNC and this effect could be abrogated by an IL-6-neutralizing antibody. We found that curcumin administration increased miR-145 promoter activity, thereby decreasing SOX9/ADAM17 expression and eliminating TICs in HNC cell populations. Delivery of lentivral-miR145 or orally administered curcumin blocked tumor progression in HNC-TICs in murine xenotransplant assays. Finally, immunohistochemical analyses of patient specimens confirmed that an miR-145(low)/SOX9(high)/ADAM17(high) phenotype correlated with poor survival. Collectively, our results show how miR-145 targets the SOX9/ADAM17 axis to regulate TIC properties in HNC, and how altering this pathway may partly explain the anticancer effects of curcumin. By inhibiting IL-6 and sIL-6R as downstream effector cytokines in this pathway, miR-145 seems to suppress a paracrine signaling pathway in the tumor microenvironment that is vital to maintain TICs in HNC.

Protein kinase Cα suppresses Kras-mediated lung tumor formation through activation of a p38 MAPK-TGFβ signaling axis

Protein kinase C alpha (PKCα) can activate both pro- and anti-tumorigenic signaling depending upon cellular context. Here, we investigated the role of PKCα in lung tumorigenesis in vivo. Gene expression data sets revealed that primary human non-small lung cancers (NSCLC) express significantly decreased PKCα levels, indicating that loss of PKCα expression is a recurrent event in NSCLC. We evaluated the functional relevance of PKCα loss during lung tumorigenesis in three murine lung adenocarcinoma models (LSL-Kras, LA2-Kras and urethane exposure). Genetic deletion of PKCα resulted in a significant increase in lung tumor number, size, burden and grade, bypass of oncogene-induced senescence, progression from adenoma to carcinoma and a significant decrease in survival in vivo. The tumor promoting effect of PKCα loss was reflected in enhanced Kras-mediated expansion of bronchio-alveolar stem cells (BASCs), putative tumor-initiating cells, both in vitro and in vivo. LSL-Kras/Prkca(-/-) mice exhibited a decrease in phospho-p38 MAPK in BASCs in vitro and in tumors in vivo, and treatment of LSL-Kras BASCs with a p38 inhibitor resulted in increased colony size indistinguishable from that observed in LSL-Kras/Prkca(-/-) BASCs. In addition, LSL-Kras/Prkca(-/-) BASCs exhibited a modest but reproducible increase in TGFβ1 mRNA, and addition of exogenous TGFβ1 to LSL-Kras BASCs results in enhanced growth similar to untreated BASCs from LSL-Kras/Prkca(-/-) mice. Conversely, a TGFβR1 inhibitor reversed the effects of PKCα loss in LSL-Kras/Prkca(-/-) BASCs. Finally, we identified the inhibitors of DNA binding (Id) Id1-3 and the Wilm's Tumor 1 as potential downstream targets of PKCα-dependent tumor suppressor activity in vitro and in vivo. We conclude that PKCα suppresses tumor initiation and progression, at least in part, through a PKCα-p38MAPK-TGFβ signaling axis that regulates tumor cell proliferation and Kras-induced senescence. Our results provide the first direct evidence that PKCα exhibits tumor suppressor activity in the lung in vivo.

CD8+ T-cell Clones Specific for the 5T4 Antigen Target Renal Cell Carcinoma Tumor-initiating Cells in a Murine Xenograft Model

The tumor antigen 5T4 is frequently expressed at high levels on renal cell carcinoma (RCC) and other epithelial carcinomas. Surveys of normal tissues demonstrate abundant 5T4 expression on placental trophoblast cells with limited expression elsewhere. 5T4 is the target for a therapeutic cancer vaccine (MVA-5T4) that elicits 5T4-specific serological, proliferative, and cytotoxic T lymphocyte (CTL) responses. However, the antitumor activity of 5T4-specific CTL has not been extensively characterized. CD8 T cells from HLA-A2 healthy donors (n=4) or RCC patients (n=2) were stimulated in vitro with the HLA-A2-binding nonamer peptides 5T417-25 or 5T497-105 and screened by flow cytometry with specific tetramers (TET). CD8/TET T-cell clones specific for 5T417-25 or 5T497-105 peptide were isolated from 4/6 and 1/4 donors, respectively. A subset of clones specific for 5T417-25 was cytolytic for MVA-5T4-infected HLA-A2 EBV-transformed lymphoblastoid cell line target cells and for constitutively HLA-A2-expressing and 5T4-expressing RCC tumor cell lines (including A498 RCC). In a xenoengraftment assay, the coinoculation of a representative 5T417-25-specific CTL clone with A498 RCC tumors cells into immune-deficient mice completely prevented growth of A498 tumors. Taken together, these data demonstrate high-avidity CD8 CTL able to recognize the naturally processed 5T417-25 epitope on RCC tumor cells including putative tumor-initiating cells are present in peripheral blood of both healthy donors and RCC patients. CD8T-cell immunity targeting 5T417-25 is therefore of substantial interest both as a potential target for further development of vaccination or adoptive cellular immunotherapy and for immune monitoring studies in association with nonspecific immunotherapies.

Expression of Six1 in luminal breast cancers predicts poor prognosis and promotes increases in tumor initiating cells by activation of extracellular signal-regulated kinase and transforming growth factor-beta signaling pathways

IntroductionMammary-specific overexpression of Six1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. Six1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that Six1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs.MethodsWe examined gene expression datasets to determine the breast cancer subtypes with Six1 overexpression, and then examined its expression in the CD24low/CD44+ putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. Six1 overexpression, or knockdown, was performed in different systems to examine how Six1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which Six1 influences TICs using genetic/inhibitor approaches in vitro and in vivo. Finally, we examined the expression of Six1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers.ResultsHigh levels of Six1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal B subtype. Six1 levels are enriched in the CD24low/CD44+ TIC population in human luminal breast cancers xenografted through mice, and in tumorsphere cultures in MCF7 and T47D luminal breast cancer cells. When overexpressed in MCF7 cells, Six1expands the TIC population through activation of transforming growth factor-beta (TGF-β) and mitogen activated protein kinase (MEK)/ERK signaling. Inhibition of ERK signaling in MCF7-Six1 cells with MEK1/2 inhibitors, U0126 and AZD6244, restores the TIC population of luminal breast cancer cells back to that observed in control cells. Administration of AZD6244 dramatically inhibits tumor formation efficiency and metastasis in cells that express high levels of Six1 ectopically or endogenously. Finally, we demonstrate that Six1 significantly correlates with phosphorylated ERK in human breast cancers.ConclusionsSix1 plays an important role in the TIC population in luminal breast cancers and induces a TIC phenotype by enhancing both TGF-β and ERK signaling. MEK1/2 kinase inhibitors are potential candidates for targeting TICs in breast tumors.

Identification and characterization of the human leiomyoma side population as putative tumor-initiating cells

To isolate and characterize human leiomyoma stem cells by the side population (SP) method. Prospective experimental human and animal study. University research laboratory-affiliated infertility clinic. Women undergoing laparoscopic myomectomy. Female non-obese diabetic severe combined immune deficiency (NOD-SCID) mutation mice. Obtainment of human leiomyoma SP cells as candidate tumor-initiating cells and establishment of two leiomyoma SP lines. Flow cytometry, semiquantitative polymerase chain reaction, clonogenicity assays, cDNA microarrays hybridization, cell culture, karyotype, molecular analysis, immunocytochemistry, invitro differentiation, xenotransplantation assays, immunohistochemistry. SP cells from human leiomyomas were isolated, identified, and characterized. Two leiomyoma's SP cell lines with a normal karyotype were thus established. Undifferentiated status was confirmed by the expression of OCT-4, NANOG, DNMT3B, GDF3. Presence of typical mesenchymal markers (CD90, CD105, CD73) and absence of hematopoietic stem cell markers (CD34, CD45) supported their mesodermal origin. Mesenchymal lineage commitment was also demonstrated by their ability to differentiate invitro into adipogenic and osteogenic lineages. Finally, their functional capability was established in an animal model by leiomyoma tissue reconstruction. SP cells from human leiomyoma exhibit key features of tumor-initiating cells, opening up new possibilities of understanding the origin and developing new nonsurgical approaches for leiomyomas.

Use of an optimized primary ovarian cancer xenograft model to mimic patient tumor biology and heterogeneity.

5036 Background: Current xenograft and transgenic models of ovarian cancer are mainly homogeneous and poorly predict response to therapy. Use of patient tumors may represent a better model for tumor biology and offer potential to test personalized medicine approaches, but poor take rates and questions of recapitulation of patient tumors have limited this approach. We have developed a protocol for improved feasibility of such a model and examined its similarity to the patient tumor. Methods: Under IRB and IACUC approval, 23 metastatic ovarian cancer samples were collected at the time of tumor reductive surgery. Samples were implanted either subcutaneously (SQ), intraperitoneally (IP), in the mammary fat pad (MFP), or in the subrenal capsule (SRC) and monitored for tumor growth. Cohorts from 8 xenolines were treated with combined carboplatin and paclitaxel or vehicle, and response to therapy compared between xenografts and patients. Expression of tumor-initiating cell (TIC) markers ALDH1, CD133, and CD44 was assessed by immunohistochemistry in tumors from patients and treated and untreated xenografts. Results: At least one SQ implanted tumor developed in 91.3% of xenografts, significantly higher than in the MFP (63.6%), IP (23.5%), or SRC (8%). Xenografts were similar in expression of putative TIC’s compared to patient tumors. The patients and the xenografts also have similar responses to chemotherapy in that xenografts from patients with a partial response responded more slowly than those from patients achieving a complete response (45 vs 21 days, p=.004). Treated xenografts were more densely composed of TICs. ALDH1 increased to 36.1% from 16.2% (p=0.002) and CD133 increased to 33.8% from 16.2% (p=0.026). Conclusions: Xenoline development can be achieved at a high rate when tumors collected from metastatic sites are implanted SQ. These xenografts are similar to patient tumors with regard to chemotherapy response and TIC expression.. This model may be a more accurate model for in vivo pre-clinical studies as compared to current models. Also, as treated xenografts become chemoresistant, this model is well positioned to evaluate targeted therapies aimed at the most aggressive populations in a heterogeneous tumor.

S6-5: Obesity Drives Epithelial-to-Mesenchymal Transition and Tumor Progression in a Novel Claudin-Low Mammary Cancer Model.

Abstract Background: Epidemiological evidence suggests a potential role of obesity in regulating clinical subtype, differentiation status, and prognosis of breast cancer. Specifically, an elevated waist-hip ratio is associated with increased risk and progression of basal-like breast cancer, an aggressive form characterized by heterogeneous tumors typically enriched in a putative tumor initiating cell (TIC) population. However, the exact mechanism of obesity-driven tumor progression remains unknown. Therefore, we hypothesized that obesity regulates a plastic population of multipotent malignant cells. Materials, Methods, and Results: To test this hypothesis, we generated and characterized two distinct murine mammary tumor cell lines derived from MMTV-Wnt-1 transgenic mice, designated M-Wnt and E-Wnt. M-Wnt cells displayed a mesenchymal morphology while E-Wnt cells had an epithelial morphology. M-Wnt cells harbored a large CD44+/CD24- putative TIC population (62% +/−7.8), had significant mammosphere forming capacity (>30% of cells form mammospheres, p<0.0001), and increased ALDH activity (7% are ALDH+, p=0.0004). M-Wnt cells have increased migration (scratch assay) and invasion in vitro (226-fold higher after 30h, p<0.0001). As few as 50 unsorted M-Wnt cells, injected into C57BL/6 mice were capable of forming a tumor. Microarray analysis revealed that M-Wnt cells display gene expression profiles virtually identical to human Claudin-low breast tumors, while E-Wnt cells clustered with basal-like tumors. EMT and stem cell gene expression patterns of M-Wnt cells were maintained in vivo, including decreased E-cadherin and increased N-cadherin, fibronectin, vimentin, SNAIL, TWIST, SLUG, FOXC2, and TGF-β (p<0.05 for all). Using these cell lines in vivo, we tested the hypothesis that energy balance modulation, through diet-induced obesity (DIO) and calorie restriction (CR), regulates the TIC population. We found that M-Wnt tumors, transplanted into ovariecomized syngeneic female C57BL/6 mice, grew at significantly different growth rates depending on the diet treatment (DIO > Control, p=0.011; CR < Control, p=0.012), while E-Wnt tumors were only affected by CR (CR < Control p=0.001; no difference between DIO and Control tumors). DIO enhanced M-Wnt tumor progression, adipocyte infiltration, and central necrosis and drove EMT (decreased E-Cadherin and increased fibronectin and N-Cadherin expression) through modulation of key TIC associated genes, including increased TGF-β, SNAIL, FOXC2, and Oct4 (p<0.05 for all). Discussion: In conclusion, we found that the mesenchymal M-Wnt cell line is highly responsive to changes in dietary energy balance status relative to the E-Wnt differentiated epithelial cell line, which demonstrates that clinical subtypes of breast cancer are differentially regulated by energy balance modulation. Additionally, our data demonstrates, for the first time, that energy balance modulation (ie, CR and obesity) directly regulates EMT, in response to local upregulation of TGF-β signaling. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S6-5.

Abstract A215: Antitumor activity of an antibody-drug conjugate targeting 5T4, a putative tumor-initiating cell antigen.

Abstract 5T4 (also known as TPBG) is a transmembrane, tumor-associated protein that rapidly internalizes. In preclinical models of NSCLC, 5T4 was identified as a marker of undifferentiated tumorigenic cells that express properties of tumor-initiating cells (TICs) and was associated with a highly proliferating (Ki67 positive) cell phenotype. In addition, 5T4 has been associated with a worse clinical outcome in NSCLC (Damelin et al, Cancer Res, 2011). We constructed an ADC with a humanized anti-5T4 Ab (A1) linked to the tubulin inhibitor monomethylauristatin F through the noncleavable maleimidocaproyl (mc) linker attached to cysteine residues (A1mcMMAF) with a DAR of approximately 4 in order to target 5T4 expressing tumor cells. Anti-5T4 A1 Ab has a Kd of 0.9 nM against the 5T4 antigen, binds to a broad range of 5T4 expressing cell lines, and rapidly internalizes (66% internalized within 4h). As an ADC, A1mcMMAF retains similar properties to the unconjugated Ab. In vitro, A1mcMMAF exhibited receptor-density dependent cytotoxic activity against a panel of 5T4 + cell lines with an IC50 of 5 ng Ab/ml against high 5T4 expressing cells. A1mcMMAF exerts no inhibitory effect on 5T4 negative cells demonstrating it does not exert off-target activity in vitro. In vivo, A1mcMMAF causes long-term (100 days) tumor regressions at 10 mg Ab/kg in MDAMB435/5T4 tumor xenografts (Q4d×2, high 5T4 expression by IHC) and in the NSCLC patient-derived xenograft 37622A model (Q4d×4, high 5T4 expression by IHC though only a portion of the cells express 5T4). Significant anti-tumor activity was demonstrated at 1 mg/kg. Against the low 5T4 expressing H1975 tumor xenograft, A1mcMMAF causes potent tumor regression in spite of its lack of in vitro inhibitory activity against this cell line. Bioimaging of the H1975 tumor demonstrated good penetration of Alexa 750 labeled A1 Ab into the xenograft. A1mcMMAF exhibited no overt toxicities when administered at 10 mg Ab/kg/cycle × 2 to cynomolgus macques and possessed a half-life of 4 to 7 days. The observation that A1mcMMAF regresses H1975 tumor xenografts while demonstrating poor activity against H1975 in vitro demonstrates the attractiveness of targeting 5T4 in the tumor environment and may be explained by 5T4's principal association with proliferating cells in the tumor. It is also interesting that A1mcMMAF was able to completely regress 37622A xenografts since this patient-derived xenograft presents heterogeneous 5T4 expression, i.e. not all cells demonstrate detectable levels of 5T4. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A215.

Increased expression of DNA repair genes in invasive human pancreatic cancer cells.

Pancreatic cancer was the fourth leading cause of cancer death in the United States in 2010. Recurrence of disease after resection occurs because of neoplastic cell survival. To better understand these highly aggressive cells, gene expression microarrays were performed. Using the established lines HPAC and PANC1 and a Matrigel assay, genome expression arrays were performed to analyze patterns between invasive and total cells. Significant increases in the expression of genes related to DNA repair were observed. A number of the same genes also demonstrated an increase in expression when comparing bulk cells to a putative tumor-initiating cell (TIC) population. The TIC population was isolated using the spheroid technique, and compared with bulk cells, spheroid cells functionally repair breaks in DNA faster after challenge with the drug gemcitabine. Finally, using Oncomine, we observed a significant increase in DNA copy number of BRCA1 and RAD51 in tissue isolated from metastatic pancreatic cancer compared with tissue isolated from the primary site. From these data, we conclude that the most invasive cells within a pancreatic tumor are able to thrive because of their increased genomic stability. These cells have also been linked to the TIC population in a tumor.