Bisrat G. Debeb

ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1

Epithelial–mesenchymal transition (EMT) is associated with characteristics of breast cancer stem cells, including chemoresistance and radioresistance. However, it is unclear whether EMT itself or specific EMT regulators play causal roles in these properties. Here we identify an EMT-inducing transcription factor, zinc finger E-box binding homeobox 1 (ZEB1), as a regulator of radiosensitivity and DNA damage response. Radioresistant subpopulations of breast cancer cells derived from ionizing radiation exhibit hyperactivation of the kinase ATM and upregulation of ZEB1, and the latter promotes tumour cell radioresistance in vitro and in vivo. Mechanistically, ATM phosphorylates and stabilizes ZEB1 in response to DNA damage, ZEB1 in turn directly interacts with USP7 and enhances its ability to deubiquitylate and stabilize CHK1, thereby promoting homologous recombination-dependent DNA repair and resistance to radiation. These findings identify ZEB1 as an ATM substrate linking ATM to CHK1 and the mechanism underlying the association between EMT and radioresistance.

Mesenchymal Stem Cells Promote Mammosphere Formation and Decrease E-Cadherin in Normal and Malignant Breast Cells

Introduction Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as “mammospheres” in three-dimensional cultures. Objective We tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation. Results We found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC. Conclusions MSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy.

Omental adipose tissue-derived stromal cells promote vascularization and growth of endometrial tumors.

Purpose: Adipose tissue contains a population of tumor-tropic mesenchymal progenitors, termed adipose stromal cells (ASC), which engraft in neighboring tumors to form supportive tumor stroma. We hypothesized that intra-abdominal visceral adipose tissue may contain a uniquely tumor-promoting population of ASC to account for the relationship between excess visceral adipose tissue and mortality of intra-abdominal cancers. Experimental Design: To investigate this, we isolated and characterized ASC from intra-abdominal omental adipose tissue (O-ASC) and characterized their effects on endometrial cancer progression as compared with subcutaneous adipose-derived mesenchymal stromal cells (SC-ASC), bone marrow–derived mesenchymal stromal cells (BM-MSC), and lung fibroblasts. To model chronic recruitment of ASC by tumors, cells were injected metronomically into mice bearing Hec1a xenografts. Results: O-ASC expressed cell surface markers characteristic of BM-MSC and differentiated into mesenchymal lineages. Coculture with O-ASC increased endometrial cancer cell proliferation in vitro. Tumor tropism of O-ASC and SC-ASC for human Hec1a endometrial tumor xenografts was comparable, but O-ASC more potently promoted tumor growth. Compared with tumors in SC-ASC–injected mice, tumors in O-ASC–injected mice contained higher numbers of large tortuous desmin-positive blood vessels, which correlated with decreased central tumor necrosis and increased tumor cell proliferation. O-ASC exhibited enhanced motility as compared with SC-ASC in response to Hec1a-secreted factors. Conclusions: Visceral adipose tissue contains a population of multipotent MSCs that promote endometrial tumor growth more potently than MSCs from subcutaneous adipose tissue. We propose that O-ASCs recruited to tumors express specific factors that enhance tumor vascularization, promoting survival and proliferation of tumor cells. Clin Cancer Res; 18(3); 771–82. ©2011 AACR.

miR-205 acts as a tumour radiosensitizer by targeting ZEB1 and Ubc13

Tumor cells associated with therapy resistance (radioresistance and drug resistance) are likely to give rise to local recurrence and distant metastatic relapse. Recent studies revealed microRNA (miRNA)-mediated regulation of metastasis and epithelial-mesenchymal transition; however, whether specific miRNAs regulate tumor radioresistance and can be exploited as radiosensitizing agents remains unclear. Here we find that miR-205 promotes radiosensitivity and is downregulated in radioresistant subpopulations of breast cancer cells, and that loss of miR-205 is highly associated with poor distant relapse-free survival in breast cancer patients. Notably, therapeutic delivery of miR-205 mimics via nanoliposomes can sensitize the tumor to radiation in a xenograft model. Mechanistically, radiation suppresses miR-205 expression through ataxia telangiectasia mutated (ATM) and zinc finger E-box binding homeobox 1 (ZEB1). Moreover, miR-205 inhibits DNA damage repair by targeting ZEB1 and the ubiquitin-conjugating enzyme Ubc13. These findings identify miR-205 as a radiosensitizing miRNA and reveal a new therapeutic strategy for radioresistant tumors.

Histone deacetylase inhibitors stimulate dedifferentiation of human breast cancer cells through WNT/β-catenin signaling

Recent studies have shown that differentiated cancer cells can dedifferentiate into cancer stem cells (CSCs) although to date no studies have reported whether this transition is influenced by systemic anti‐cancer agents. Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor that promotes self‐renewal and expansion of hematopoietic stem cells and facilitates the generation of induced pluripotent stem cells from somatic cells and is currently being investigated in breast cancer clinical trials. We hypothesized that HDAC inhibitors reprogram differentiated cancer cells toward the more resistant stem cell‐like state. Two highly aggressive breast cancer cell lines, SUM159 and MDA‐231, were sorted based on aldehyde dehydrogenase (ALDH) activity and subsequently ALDH‐negative and ALDH‐positive cells were treated with one of two known HDAC inhibitors, VA or suberoylanilide hydroxamic acid. In addition, primary tumor cells from patients with metastatic breast cancer were evaluated for ALDH activity following treatment with HDAC inhibitors. We demonstrate that single‐cell‐sorted ALDH‐negative cells spontaneously generated ALDH‐positive cells in vitro. Treatment of ALDH‐negative cells with HDAC inhibitors promoted the expansion of ALDH‐positive cells and increased mammosphere‐forming efficiency. Most importantly, it significantly increased the tumor‐initiating capacity of ALDH‐negative cells in limiting dilution outgrowth assays. Moreover, while HDAC inhibitors upregulated β‐catenin expression and significantly increased WNT reporter activity, a TCF4 dominant negative construct abolished HDAC‐inhibitor‐induced expansion of CSCs. These results demonstrate that HDAC inhibitors promote the expansion of breast CSCs through dedifferentiation and have important clinical implications for the use of HDAC inhibitors in the treatment of cancer. STEM CELLS2012;30:2366–2377

Isolation of Oct4-expressing extraembryonic endoderm precursor cell lines

Background The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. Methodology/Principal Findings Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines (“XEN-P cell lines”), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. Conclusions/Significance Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation.

Radiation Resistance of Breast Cancer Stem Cells : Understanding the Clinical Framework

Meta-analyses of tens of thousands of women treated with radiation as a component of their breast cancer treatment have shown that radiation improves overall survival from breast cancer in women with early stage and advanced disease. However, data suggest that breast cancer stem/progenitor cells can be enriched after radiation and that breast cancer stem/progenitor clonogens are particularly resistant to radiation. Potentially resistant breast cancer stem/progenitor populations appear to be over-represented in estrogen receptor negative breast cancer and indeed, clinically these cancers are more resistant to radiation than estrogen receptor positive breast cancers. Emerging pre-clinical data suggest that targeting cancer stem/progenitor survival pathways may lead to effective radiosensitization in subgroups of patients with resistant disease. Herein, preclinical studies are reviewed in the context of the clinical framework.

Imaging and Analysis of 3D Tumor Spheroids Enriched for a Cancer Stem Cell Phenotype

Tumors that display a highly metastatic phenotype contain subpopulations of cells that display characteristics similar to embryonic stem cells. These cells exhibit the ability to undergo self-renewal; slowly replicate to retain a nucleoside analog label, leading to their definition as “label-retaining cells”; express specific surface markers such as CD44+/CD24–/low and CD133; and can give rise to cells of different lineages (i.e., they exhibit multipotency). Based on these characteristics, as well as their demonstrated ability to give rise to tumors in vivo, these cells have been defined as tumor-initiating cells (TICs), tumor-propagating cells, or cancer stem cells (CSCs). These cells are highly resistant to chemotherapeutic agents and radiation and are believed to be responsible for the development of both primary tumors and metastatic lesions at sites distant from the primary tumor. Established cancer cell lines contain CSCs, which can be propagated in vitro using defined conditions, to form 3D tumor spheroids. Because the vast majority of studies to identify cancer-associated genes and therapeutic targets use adherent cells grown in 2 dimensions on a plastic substrate, the multicellular composition of these 3D tumor spheroids presents both challenges and opportunities for their imaging and characterization. The authors describe approaches to image and analyze the properties of CSCs within 3D tumor spheroids, which can serve as the basis for defining the gene and protein signatures of CSCs and to develop therapeutic strategies that will effectively target this critically important population of cells that may be responsible for tumor progression.

DIFFERENTIAL RADIOSENSITIZING EFFECT OF VALPROIC ACID IN DIFFERENTIATION VERSUS SELF-RENEWAL PROMOTING CULTURE CONDITIONS

PURPOSE It has been shown that valproic acid (VA) enhances the proliferation and self-renewal of normal hematopoietic stem cells and that breast cancer stem/progenitor cells can be resistant to radiation. From these data, we hypothesized that VA would fail to radiosensitize breast cancer stem/progenitor cells grown to three-dimensional (3D) mammospheres. METHODS AND MATERIALS We used the MCF7 breast cancer cell line grown under stem cell-promoting culture conditions (3D mammosphere) and standard nonstem cell monolayer culture conditions (two-dimensional) to examine the effect of pretreatment with VA on radiation sensitivity in clonogenic survival assays and on the expression of embryonic stem cell transcription factors. RESULTS 3D-cultured MCF-7 cells expressed higher levels of Oct4, Nanog, and Sox2. The 3D passage enriched self-renewal and increased radioresistance in the 3D mammosphere formation assays. VA radiosensitized adherent cells but radioprotected 3D cells in single-fraction clonogenic assays. Moreover, fractionated radiation sensitized VA-treated adherent MCF7 cells but did not have a significant effect on VA-treated single cells grown to mammospheres. CONCLUSION We have concluded that VA might preferentially radiosensitize differentiated cells compared with those expressing stem cell surrogates and that stem cell-promoting culture is a useful tool for in vitro evaluation of novel cancer therapeutic agents and radiosensitizers.

Characterizing cancer cells with cancer stem cell-like features in 293T human embryonic kidney cells

BackgroundSince the first suggestion of prospectively identifiable cancer stem cells in solid tumors, efforts have been made to characterize reported cancer stem cell surrogates in existing cancer cell lines, and cell lines rich with these surrogates have been used to screen for cancer stem cell targeted agents. Although 293T cells were derived from human embryonic kidney, transplantation of these cells into the mammary fat pad yields aggressive tumors that self-renew as evidenced by serial xenograft passages through transplantation. Herein we fully characterize cancer stem cell-like features in 293T human embryonic kidney cells.Results293T cells can be readily cultured and passaged as spheres in serum-free stem cell promoting culture conditions. Cells cultured in vitro as three-dimensional spheres (3D) were shown to contain higher ALDH1 and CD44+/CD24- population compared to monolayer cells. These cells were also resistant to radiation and upregulate stem cell survival signaling including β-catenin, Notch1 and Survivin in response to radiation. Moreover, 3D spheres generated from the 293T cells have increased expression of mesenchymal genes including vimentin, n-cadherin, zeb1, snail and slug as well as pro-metastatic genes RhoC, Tenascin C and MTA1. In addition, microRNAs implicated in self-renewal and metastases were markedly reduced in 3D spheres.Conclusions293T cells exhibit a cancer stem cell-like phenotype when cultured as 3D spheres and represent an important research tool for studying the molecular and biological mechanisms of cancer stem cells and for testing and developing novel targets for cancer therapy.