A mammosphere formation RNAi screen reveals that ATG4A promotes a breast cancer stem-like phenotype
© Wolf et al.; licensee BioMed Central Ltd. 2013
Received: 4 March 2013
Accepted: 31 October 2013
Published: 14 November 2013
Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells in-vitro, they are typically enriched via mammosphere culture. Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties.
We describe a sub-population expressing the stem-like marker CD44+/CD24-/low in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties, we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4A was validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of ATG4A expression on the sub-population expressing the stem-like marker CD44+/CD24low. Next, the tumorigenic potential of SUM149 after up- or down-regulation of ATG4A was examined by xenograft experiments.
Using this method, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity in vivo.
In summary, we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4A.
Breast tumours, like many other solid tumours, consist of highly heterogeneous cell populations with varying phenotypic and functional properties . Similar to the normal mammary gland, these populations include cells with luminal-epithelial, basal and stem-cell-like features . Based on gene expression profiles, basal-like breast cancers have been associated with the surface marker expression CD44+/CD24-/low while luminal epithelial cells have been associated with CD24+/CD44- expression [4, 5]. Stem-like cells with tumour initiating capabilities have been found to be enriched in the CD44+/CD24-/low sub-population of basal breast carcinoma cells . These stem-like cells or cancer stem cells (CSCs) are held responsible for metastasis formation [7, 8] and chemoresistance . Further, it was found that CD44+/CD24-/low breast cancer cells exhibit epithelial-to-mesenchymal transition (EMT) features that might be responsible for their aggressive clinical behaviour . EMT has long been recognised as an important programme for embryonic development  and has more recently been associated with breast CSC regulation [12, 13]. It is hypothesised that differentiated cancer cells can become CSCs as a consequence of EMT, allowing them to migrate, metastasize and survive chemotherapy [7, 14]. In line with these findings, CSCs have been connected to a mesenchymal phenotype , and it was shown that chemoresistant cells display not only CSC but also mesenchymal features . Due to the aggressive nature of CSCs, the identification of genes and pathways that they depend on is an active area of research, fuelled by the promise that combination of conventional chemotherapy with specific CSC inhibitors will increase therapeutic success rates .
A major restraint when studying stem cells as well as CSC is their rareness. One approach to enrich breast stem cells, which has become particularly popular over the past ten years, is culturing cells as mammospheres . It was shown that rare, single-founder stem cells can form multi-cellular sphere structures under serum-free suspension conditions that are enriched for stem and early progenitor cells . Later, it was also found that rare cancer cells possess the ability to form mammospheres enriched for highly tumourigenic CSCs of the CD44+/CD24-/low phenotype [5, 18]. Moreover it was shown that cells enriched in mammospheres had passed through EMT and were chemoresistant  which are two properties typically attributed to CSCs . Here, we utilised the enrichment of CSCs in mammospheres and developed a high-throughput a short hairpin RNA interference (shRNAi) screening system to assay the involvement of over 5,000 genes in the maintenance of a population of cells with CSC properties. The results give an insight into molecular mechanisms underlying CSC maintenance in mammospheres and attribute a previously unrecognised role in this process to the autophagy regulator ATG4A.
Materials and methods
Adherent and mammosphere cell culture
SUM149 cells were obtained from the Kuperwasser Laboratory (Tufts University, Boston) and are commercially available (Asterand, Royston, UK). Cells were cultured in Ham’s F12 (Life Technologies, Darmstadt, Germany) with 5% calf serum, 5 μg/ml bovine insulin (Sigma-Aldrich, Taufkirchen, Germany), and 1 μg/ml hydrocortisone (Sigma-Aldrich). MDA-MB-231 and MCF-7 were cultured in DMEM (Life Technologies) with 10% calf serum. For mammosphere formation, 104 cells/cm2 cells were plated in an ultra-low attachment cell culture flask (Corning, Kaiserslautern, Germany) and cultured in MammoCult medium (StemCell Technologies, Grenoble, France). After 14 days, the mammospheres were counted and pictures were taken. For flow cytometric analysis, spheres were filtered through a 40-μm cell strainer (Becton Dickinson, Heidelberg, Germany) and treated with Accutase (Sigma-Aldrich) to obtain a single cell suspension.
Mammosphere formation RNAi screen
The DECIPHER RNAi library Module 1 (Cellecta, Mountain View, USA)  was used at low multiplicity of infection (MOI = 0.3) to transduce SUM-149 cells, followed by 48 h of selection with 2.5 μg/ml puromycin. Following 48 h recovery in antibiotic-free medium, 1.4 × 108 stably transduced cells were seeded into 180 × 75 cm2 ultra-low attachment cell culture flask (Corning) and cultured in MammoCult (StemCell Technologies). After 14 days, mammospheres larger than 40 μm were extracted by five individual rounds of filtration through 40-μm mesh size cell strainers (Becton Dickinson). About 7 × 106 transduced cells were cultured adherently for the same period of time in Ham’s F12 as a reference. Cells were passaged after reaching 80% confluence. Genomic DNA from adherently cultured cells at the beginning (baseline) and the end of the screen (tadherent) as well as from pooled mammosphere samples (tsphere) was isolated using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Construct-specific barcode sequences were amplified under PCR conditions provided by the manufacturer of the DECIPHER library (Cellecta). Barcode sequences are 18 nucleotide-long DNA sequences that are unique for each of the 27,500 shRNA expression constructs in the DECIPHER library pool. Hence, they can be used as surrogate markers to quantify the number of cells expressing a certain shRNA in a pool of cells. PCR amplification of barcode sequences resulted in ready-to-load sequencing libraries including adaptor sequences for Illumina GA and HiSeq platforms. The barcodes were amplified and sequenced in duplicate on Illumina GAIIx machines and quantified using Barcode Deconvoluter software (Cellecta).
Two separate barcode read-count ratios were calculated. In order to identify shRNAs, which are toxic to adherent cells or mammospheres, the ratios [tadherent/baseline] or [tsphere/baseline] were calculated, respectively. Results are shown in Additional file 1. Ratios from each set of shRNAs (5 to 6) targeting a particular gene were compared to ratios from a set of 21 negative control shRNAs targeting the gene Luciferase via unpaired, two-sided, unequal variance t-test statistics. Calculated mean fold changes from each set of shRNA expression constructs and corresponding P-values for every gene present in the library are shown in Additional file 2.
For target validation, shRNA template sequences identified in the screen were synthesized individually (Sigma-Aldrich) and cloned into the pRSI9 vector backbone. Cloning and virus production were performed following the protocol provided by the manufacturer (Cellecta). Sequences were as follows: shATG4A-1 5′-ACCGGCAGATACAGATGAGTTGGTATGTTAATATTCATAGCATACCAGCTCATCTGTATCTGTTTT-3′; shATG4A-2 5′-ACCGGCCCGGAAAGAAATAGAATAATGTTAATATTCATAGCATTGTTCTATTTCTTTCCGGGTTTT-3′; shATG4A-3 5′- ACCGGGCTGTTGTAATGAGGAAATGGGTTAATATTCATAGCCCATTTCCTCATTGCAGCAGCTTTT-3′ and shCTRL 5′-ACCGGATCACAGAATCGTCGTATGTAGTTAATATTCATAGCTGCATACGACGATTCTGTGATTTTT-3′. For virus production, cloned shRNA plasmids were co-transfected with the packaging plasmids psPAX2 and pMD2.G into HEK293T cells. Viral supernatant was harvested 48 h post transfection and cleared from debris via centrifugation. Cells were transduced with lentivirus for 24 h in cell culture medium containing 8 μg/ml polybrene (Sigma-Aldrich) and selected with 2.5 μg/ml puromycin (Sigma-Aldrich) for 48 h. Following selection cells were recovered for 48 h in antibiotic-free culture medium.
Total RNA was isolated from cells or mammospheres using RNeasy Mini Kit (Qiagen). Reverse transcription and PCR were performed using the One Step Quantifast SYBR Green RT-PCR Kit (Qiagen) with a LightCycler 480 system (Roche, Mannheim, Germany). For target gene amplification, the QuantiTect Primer Assay (Qiagen) was used. Target gene expression was normalised to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
To determine the protein concentration, cells were lysed in TBS containing 1% Triton X-100, 10 mM Na3VO4, 1 mM NaF, 4 mM ethylenediaminetetraacetic acid (EDTA), protease inhibitor mixture (Roche). the protein concentration was measured with the bicinchoninic acid (BCA) Protein Assay (Thermo Fisher Scientific, Bonn, Germany). Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (GE Healthcare, Freiburg, Germany). The membrane was probed with antibodies; peroxidase-conjugated secondary antibodies detected the bands by ECL Plus (ThermoFisher Scientific). Antibodies were: anti-ATG4A (GeneTex, Irvine, USA) and anti-Tubulin (Abcam, Cambridge, UK).
vSingle cell suspension of adherent cells or spheres was stained with CD24-FITC, CD44-PE/Cy7 and EpCAM-APC (Becton Dickinson), E-cadherin-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) and Vimentin-Alex488 (Cell Signaling, Danvers, USA). The cells were measured using a FACS-Canto-II (Becton Dickinson) and data were analysed using FlowJo software (Tree Star, Ashland, USA).
Colony formation assay
We suspended 2,500 cells/cm2 in 0.3% agarose with MammoCult medium (StemCell Technologies) on a 0.8% agar base layer. The culture was covered with 0.5 ml MammoCult medium and cultured for 14 days. For quantification, the wells were imaged using a microscope, and the colonies were analysed using ImageJ software.
Microarray and gene expression analysis
SUM149 cells were cultured adherently and under mammosphere formation conditions in biological triplicates for two weeks. Spheres were filtered using a 40-μm cell strainer (Becton Dickinson) and RNA was isolated from spheres and adherent cells using RNeasy Mini Kit (Qiagen). RNA was analysed on HumanHT-12 v4 Expression BeadChip (Illumina, San Diego, USA) according to manufacturer instructions. Raw data were normalised and grouped using Chipster. Genes with significant gene expression changes (P <10-10) were used for pathway enrichment analysis using DAVID Functional Annotation Tool . Data were uploaded to ArrayExpress  under the accession number E-MTAB-1553.
MACS cell enrichment of sub-population
The described sub-population of SUM149 cell was enriched by depletion of EpCAM-expressing cells using EpCAM MicroBead Kit (Miltenyi Biotec). The depletion was performed according to the manufacturer’s protocol. Enrichment of CD44+/CD24low/EpCAM-/low cells was confirmed via fluorescent-activated cell sorting (FACS).
Cells were transduced with plasmids expressing shATG4A-1 and -2 (shATG4A), the ATG4A open reading frame (ATG4A-OE), or a non-silencing control (shCTRL). This was followed by a selection of transduced cells with puromycin. For each injection, 4 × 104 cells in 15 μl PBS were mixed 1:1 (v/v) with Matrigel (BD Biosciences, Heidelberg, Germany) prior to injection into the second left thoracic mammary fat pad of 8- to 9-week-old NOD SCID gamma (NSG) female mice. Tumour growth was monitored over a period of 15 weeks and tumour size was determined twice a week using a caliper. Significance values from Kaplan-Meier plots were calculated using the Wilcoxon test and GraphPad Prism software. For tissue staining, tumours were collected and embedded into paraffin according to routine procedures. H&E staining was done on 5-μm paraffin sections. Studies were approved by the local ethics committee at Regierungspräsidium Karlsruhe (G74/11, G244/11).
SUM-149 cell line contains a sub-population of cells with cancer stem-cell properties
Cells with cancer stem-cell properties accumulate in mammospheres
It was previously shown that cancer cells with stem-like characteristics become strongly enriched in mammospheres . This enrichment is a result of their ability to grow independently of anchorage, a condition under which most cancer cells undergo anoikis . The resistance to anoikis is commonly attributed to cells that have undergone EMT [13, 26]. As shown in Figure 1E, flow cytometry analysis of mammosphere-derived SUM-149 cells revealed an enrichment of the CD44+/CD24low/EpCAM-/low population compared to adherent cultured cells. In accord with the enrichment of this sub-population in mammospheres, it was found that spheres express lower levels of E-cadherin and higher levels of vimentin when compared to adherent cells (Figure 1F). These data clearly confirmed that a sub-population of cells with CSC properties became enriched during mammosphere formation. Therefore, targeting the survival of these cells should lead to impaired sphere formation. Based on this hypothesis, we established a screening system for the identification of genes that are specifically involved in mammosphere formation.
Negative selection shRNAi screen for specific regulators of mammosphere formation
Pathway enrichment analysis of candidate genes
hsa04914:Progesterone-mediated oocyte maturation
hsa05110:Vibrio cholerae infection
hsa04260:Cardiac muscle contraction
hsa00020:Citrate cycle (TCA cycle)
hsa04630:Jak-STAT signaling pathway
hsa04060:Cytokine-cytokine receptor interaction
hsa04150:mTOR signaling pathway
hsa05223:Non-small cell lung cancer
hsa05200:Pathways in cancer
hsa04062:Chemokine signaling pathway
hsa04640:Hematopoietic cell lineage
Mammospheres express high levels of lysosomal and oxidative phosphorylation genes
In order to further investigate molecular differences between mammospheres and adherently cultured cells, gene expression profiles were compared; the results of a pathway enrichment analysis are summarized in Additional file 4. Genes involved in cell cycle regulation (P = 2 × 10-20) as well as DNA replication (P = 2 × 10-14) were found to be down-regulated in mammospheres, which is in accordance with the reduced growth rate that cells exhibit under serum-free suspension conditions. Interestingly, strongest enrichment of up-regulated genes was seen for lysosome related genes (P = 2 × 10-18) and genes involved in oxidative phosphorylation (P = 3 × 10-14) indicating a requirement of lysosomal activity and energy generation under sphere forming conditions.
ATG4A is upregulated in mammospheres
Modulation of ATG4Aspecifically regulates mammosphere formation
To investigate whether regulation of ATG4A specifically regulates mammosphere formation, the impact of ATG4A modulation on the adherent proliferation, sphere formation and sphere diameter of SUM-149 cells was determined (Figure 3A-C). It was found that inhibition of ATG4A had no impact on cell viability under adherent culture conditions illustrating that ATG4A is not an essential gene for the bulk of SUM-149 cells (Figure 3A). Yet the inhibition of ATG4A led to a decrease in sphere number and size. On average, 33 mammospheres formed from 2,500 cells (1.3%) seeded under serum-free suspension conditions. Inhibition of ATG4A reduced this figure to 18 and 15 spheres, respectively, and overexpression increased the number of spheres formed to 40 (Figure 3B). Mammospheres had an average diameter of 120 μm at fourteen days post seeding of control cells. Inhibition of ATG4A reduced sphere size to 73 μm or 88 μm, respectively, and ATG4A overexpression resulted in significantly larger spheres of 168 μm (Figure 3C). Representative images of mammospheres following ATG4A knockdown or overexpression for fourteen days are shown in Figure 3C. In addition to sphere formation, the colony formation capacity of SUM-149 cells seeded in soft agar was determined after up- or down-regulation of ATG4A. As shown in Figure 3D, inhibition of ATG4A reduced the number of colonies formed, and overexpression slightly increased it. Further, the impact of ATG4A expression on sphere formation of breast cancer cell lines from different sub-types, namely basal MDA-MB-231 (CD44+/CD24-) and luminal MCF-7 cells (CD44-/CD24+)  was analysed. MDA-MB-231 is a highly metastatic cell line with a high tumourigenicity compared to the non-invasive MCF-7 . It was found that ATG4A inhibition reduced sphere formation in MDA-MB-231 cells, whereas its overexpression led to a dramatic increase (Figure 3E). Decreased sphere formation, although to a lesser extent, was also detected in luminal MCF-7 cells following ATG4A inhibition.
ATG4Aexpression maintains sub-population of cells with cancer stem-cell phenotype
Modulation of ATG4A regulates tumourigenic potential of SUM-149 cells in vivo
CSCs are rare cells that are suspected to be responsible for tumour recurrence, formation of metastases and chemoresistance [7–9]. The rareness of these cells makes it particularly hard for researchers to study their function. To date, the only functional possibility to enrich breast CSCs with tumour-initiating properties in vitro is to culture them as mammospheres [17, 18, 31]. We found that SUM-149 mammospheres were enriched for cells that expressed a surface marker signature typical for stem-like breast cancer cells (Figure 1E), passed through EMT (Figure 1B and 1F), were chemoresistant (Figure 1C) and more tumourigenic in NSG mice (Figure 1D). These are properties typically attributed to breast CSCs . Although under adherent conditions this sub-population accounted for approximately 6% of the total population (Figure 5A), it became enriched 5-fold in mammospheres (Figure 5B).
Here, we exploited this enrichment to establish a high-throughput pooled RNAi screening system suitable to identify genes that are specifically involved in the maintenance of those rare cells with CSC properties. The system is based on the comparison of two separate RNAi screens performed under (i) adherent and (ii) mammosphere culture conditions (Figure 2A). With the first screen, genes essential for the survival of the total cell population were identified. The second screen identified genes important for sphere formation and hence, served as a surrogate screen to identify genes that are important for the maintenance of cells with CSC properties. Subtractive analysis finally revealed genes that are primarily important for the survival of cells with CSC properties. The identified genes were used for a pathway enrichment analysis, which returned a number of cancer-related pathways and regulatory processes (Table 1B). In total 22 candidate genes were found to be associated with the Jak-STAT signalling pathway (KEGG), making it the most significant pathway identified (P = 0.00176). A schematic presentation of some of the identified candidate genes acting in the Jak-STAT pathway is shown in Figure 2C. It is known that cytokine signalling via the IL6 receptor, GP130, JAK3, STAT1 and STAT3, as identified in our screen, is a regulator of breast CSC self renewal and differentiation [32, 33]. Further, activated Jak-STAT signalling is essential for the survival of CD44+/CD24-/low stem-like breast cancer cells  and was shown to play an important role during mammosphere formation . Last, STAT3 was identified by another RNAi screen to be a critical player in mammosphere formation and self-renewal of breast CSCs . Taken together, these findings confirm the utility of the presented screening system to identify processes with specific relevance to the maintenance and expansion of CSCs.
Despite the advantages of a functional enrichment, culturing of cells as mammospheres also has some drawbacks when performing a high-throughput screen. For the analysis of the screen, we pooled all spheres bigger than 40 μm. Therefore, we could not distinguish between sphere size and number of spheres. Large spheres are believed to consist of more differentiated cells or early progenitors than smaller spheres. Another concern might be the formation of spheres because of cell aggregation instead of clonal growth. To overcome this hurdle, we chose an appropriate cell density to avoid any sphere fusion. Moreover, we validated our candidates in semi-solid soft agar colony formation assays that guarantee clonal sphere growth.
Besides Jak-STAT signalling, a number of previously uncharacterized candidate genes were identified in this screen, one of which was the positive regulator of autophagy, ATG4A. Autophagy is a lysosome-dependant degradation pathway allowing cells to remove macromolecules and damaged organelles in order to survive stress conditions [37, 38]. Interestingly, it was recently published that autophagy promotes the undifferentiated stem-like CD44+/CD24-/low phenotype in breast cancer cells  and further evidence for the involvement of autophagy in cancer stem-like cell maintenance as well as their differentiation is accumulating rapidly [40–43]. ATG4A is a redox-regulated cysteine protease . ATG4A can cleave ATG8 near its C-terminus allowing the conjugation of ATG8 to phosphatidylethanolamine and subsequently to the membrane of the autophagosome . In a second reaction, ATG4 can delipidate ATG8, releasing it from the autophagosomal membrane [44, 45]. This cleavage marks a final step in autophagy and allows the fusion of autophagosome and lysosome [46, 47]. The emerging role of autophagy in cancer stem cell maintenance together with the activation of lysosomal gene expression (Additional file 4) and upregulation of ATG4A (Figure 4A) in mammospheres strongly suggest an important role for autophagy, or more precisely, ATG4A in breast CSC maintenance. As it was demonstrated here, inhibition of ATG4A led to reduction of a sub-population with CSC properties (Figure 5A). Moreover, the enrichment of this sub-population during sphere formation was almost completely prevented by ATG4A inhibition (Figure 5B). Furthermore, ATG4A levels influenced size (Figure 3C) and numbers of mammospheres formed from breast cancer cell lines of the luminal and, even stronger, the basal type (Figure 3B and 3E). Last, modulation of ATG4A expression affected the tumourigenicity of SUM-149 cells under physiological conditions in the mammary fat pad of NSG mice (Figure 6A) as well as the composition of resulting tumours (Figure 6B). These results clearly demonstrate that ATG4A is involved in carcinogenesis and the maintenance of cells with a CSC phenotype.
In order to develop targeted CSC therapies, it is essential to understand the underlying molecular mechanisms of CSC maintenance. To study those mechanisms, we developed a high-throughput negative selection RNAi screening system and provide evidence that it is suitable to identify genes which, like ATG4A, are involved in the maintenance of cells with CSC properties. Analysis of additional cell lines using the described approach should greatly accelerate the search for novel molecular targets that could be used to tackle the cancer stem cell.
Cancer stem cell
Dulbecco’s modified eagle’s medium
Fluorescence activated cell sorting
Hematoxylin and eosin
Kyoto encyclopedia of genes and genomes
Multiplicity of infection
NOD SCID gamma
Reverse transcription polymerase chain reaction
Short hairpin RNA interference
Signal transducers and activators of transcription.
This work was supported by a grant of the DKFZ intramural funding programme awarded to MB and a DKFZ PhD scholarship awarded to JW. Lentiviral shRNA libraries were kindly provided and developed by Cellecta based on NIH-funded research grant support 44RR024095, 44HG003355. Financial support by the German Federal Ministry of Education and Research (BMBF) as part of the PaCaNet consortium is gratefully acknowledged.
- Marusyk A, Polyak K: Tumor heterogeneity: causes and consequences. Biochim Biophys Acta. 2010, 1805: 105-117.PubMedPubMed CentralGoogle Scholar
- Sarrio D, Franklin CK, Mackay A, Reis-Filho JS, Isacke CM: Epithelial and mesenchymal subpopulations within normal basal breast cell lines exhibit distinct stem cell/progenitor properties. Stem Cells. 2012, 30: 292-303. 10.1002/stem.791.PubMedView ArticleGoogle Scholar
- Honeth G, Bendahl PO, Ringner M, Saal LH, Gruvberger-Saal SK, Lovgren K, Grabau D, Ferno M, Borg A, Hegardt C: The CD44+/CD24- phenotype is enriched in basal-like breast tumors. Breast Cancer Res. 2008, 10: R53-10.1186/bcr2108.PubMedPubMed CentralView ArticleGoogle Scholar
- Shipitsin M, Campbell LL, Argani P, Weremowicz S, Bloushtain-Qimron N, Yao J, Nikolskaya T, Serebryiskaya T, Beroukhim R, Hu M, Halushka MK, Sukumar S, Parker LM, Anderson KS, Harris LN, Garber JE, Richardson AL, Schnitt SJ, Nikolsky Y, Gelman RS, Polyak K: Molecular definition of breast tumor heterogeneity. Cancer Cell. 2007, 11: 259-273. 10.1016/j.ccr.2007.01.013.PubMedView ArticleGoogle Scholar
- Fillmore CM, Kuperwasser C: Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res. 2008, 10: R25-10.1186/bcr1982.PubMedPubMed CentralView ArticleGoogle Scholar
- Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 2003, 100: 3983-3988. 10.1073/pnas.0530291100.PubMedPubMed CentralView ArticleGoogle Scholar
- Chaffer CL, Weinberg RA: A perspective on cancer cell metastasis. Science. 2011, 331: 1559-1564. 10.1126/science.1203543.PubMedView ArticleGoogle Scholar
- Sheridan C, Kishimoto H, Fuchs RK, Mehrotra S, Bhat-Nakshatri P, Turner CH, Goulet R, Badve S, Nakshatri H: CD44+/CD24- breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res. 2006, 8: R59-10.1186/bcr1610.PubMedPubMed CentralView ArticleGoogle Scholar
- Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, Hilsenbeck SG, Pavlick A, Zhang X, Chamness GC, Wong H, Rosen J, Chang JC: Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst. 2008, 100: 672-679. 10.1093/jnci/djn123.PubMedView ArticleGoogle Scholar
- Sarrio D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, Palacios J: Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. Cancer Res. 2008, 68: 989-997. 10.1158/0008-5472.CAN-07-2017.PubMedView ArticleGoogle Scholar
- Thiery JP, Acloque H, Huang RY, Nieto MA: Epithelial-mesenchymal transitions in development and disease. Cell. 2009, 139: 871-890. 10.1016/j.cell.2009.11.007.PubMedView ArticleGoogle Scholar
- May CD, Sphyris N, Evans KW, Werden SJ, Guo W, Mani SA: Epithelial-mesenchymal transition and cancer stem cells: a dangerously dynamic duo in breast cancer progression. Breast Cancer Res. 2011, 13: 202-10.1186/bcr2789.PubMedPubMed CentralView ArticleGoogle Scholar
- Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008, 133: 704-715. 10.1016/j.cell.2008.03.027.PubMedPubMed CentralView ArticleGoogle Scholar
- Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A, Rimm DL, Wong H, Rodriguez A, Herschkowitz JI, Fand C, Zhanga X, Hec X, Pavlicka A, Gutierreza MC, Renshawb L, Larionovb AA, Faratianb D, Hilsenbecka SG, Peroud CM, Lewisa MT, Rosena JM, Chang JC: Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci USA. 2009, 106: 13820-13825. 10.1073/pnas.0905718106.PubMedPubMed CentralView ArticleGoogle Scholar
- Micalizzi DS, Farabaugh SM, Ford HL: Epithelial-mesenchymal transition in cancer: parallels between normal development and tumor progression. J Mammary Gland Biol Neoplasia. 2010, 15: 117-134. 10.1007/s10911-010-9178-9.PubMedPubMed CentralView ArticleGoogle Scholar
- Cardiff RD, Couto S, Bolon B: Three interrelated themes in current breast cancer research: gene addiction, phenotypic plasticity, and cancer stem cells. Breast Cancer Res. 2011, 13: 216-10.1186/bcr2887.PubMedPubMed CentralView ArticleGoogle Scholar
- Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS: In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003, 17: 1253-1270. 10.1101/gad.1061803.PubMedPubMed CentralView ArticleGoogle Scholar
- Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA, Daidone MG: Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res. 2005, 65: 5506-5511. 10.1158/0008-5472.CAN-05-0626.PubMedView ArticleGoogle Scholar
- Guttilla IK, Phoenix KN, Hong X, Tirnauer JS, Claffey KP, White BA: Prolonged mammosphere culture of MCF-7 cells induces an EMT and repression of the estrogen receptor by microRNAs. Breast Cancer Res Treat. 2012, 132: 75-85. 10.1007/s10549-011-1534-y.PubMedView ArticleGoogle Scholar
- The DECIPHER Open Source RNAi Screening Project: [http://www.decipherproject.net/]
- da Huang W, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009, 4: 44-57.PubMedView ArticleGoogle Scholar
- ArrayExpress: http://www.ebi.ac.uk/arrayexpress/,
- Gupta PB, Fillmore CM, Jiang G, Shapira SD, Tao K, Kuperwasser C, Lander ES: Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells. Cell. 2011, 146: 633-644. 10.1016/j.cell.2011.07.026.PubMedView ArticleGoogle Scholar
- Wells A, Yates C, Shepard CR: E-cadherin as an indicator of mesenchymal to epithelial reverting transitions during the metastatic seeding of disseminated carcinomas. Clin Exp Metastasis. 2008, 25: 621-628. 10.1007/s10585-008-9167-1.PubMedPubMed CentralView ArticleGoogle Scholar
- Mendez MG, Kojima S, Goldman RD: Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition. FASEB J. 2010, 24: 1838-1851. 10.1096/fj.09-151639.PubMedPubMed CentralView ArticleGoogle Scholar
- Drasin DJ, Robin TP, Ford HL: Breast cancer epithelial-to-mesenchymal transition: examining the functional consequences of plasticity. Breast Cancer Res. 2011, 13: 226-10.1186/bcr3037.PubMedPubMed CentralView ArticleGoogle Scholar
- Lacroix M, Leclercq G: Relevance of breast cancer cell lines as models for breast tumours: an update. Breast Cancer Res Treat. 2004, 83: 249-289. 10.1023/B:BREA.0000014042.54925.cc.PubMedView ArticleGoogle Scholar
- Leek RD, Landers RJ, Harris AL, Lewis CE: Necrosis correlates with high vascular density and focal macrophage infiltration in invasive carcinoma of the breast. Br J Cancer. 1999, 79: 991-995. 10.1038/sj.bjc.6690158.PubMedPubMed CentralView ArticleGoogle Scholar
- Balkwill F, Charles KA, Mantovani A: Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell. 2005, 7: 211-217. 10.1016/j.ccr.2005.02.013.PubMedView ArticleGoogle Scholar
- Mantovani A, Allavena P, Sica A, Balkwill F: Cancer-related inflammation. Nature. 2008, 454: 436-444. 10.1038/nature07205.PubMedView ArticleGoogle Scholar
- Bruna A, Greenwood W, Le Quesne J, Teschendorff A, Miranda-Saavedra D, Rueda OM, Sandoval JL, Vidakovic AT, Saadi A, Pharoah P, Stingl J, Caldas C: TGFbeta induces the formation of tumour-initiating cells in claudinlow breast cancer. Nat Commun. 2012, 3: 1055-PubMedView ArticleGoogle Scholar
- Liu S, Wicha MS: Targeting breast cancer stem cells. J Clin Oncol. 2010, 28: 4006-4012. 10.1200/JCO.2009.27.5388.PubMedView ArticleGoogle Scholar
- Korkaya H, Liu S, Wicha MS: Breast cancer stem cells, cytokine networks, and the tumor microenvironment. J Clin Invest. 2011, 121: 3804-3809. 10.1172/JCI57099.PubMedPubMed CentralView ArticleGoogle Scholar
- Marotta LL, Almendro V, Marusyk A, Shipitsin M, Schemme J, Walker SR, Bloushtain-Qimron N, Kim JJ, Choudhury SA, Maruyama R, Wu Z, Gönen M, Mulvey LA, Bessarabova MO, Huh SJ, Silver SJ, Kim SY, Park SY, Lee HE, Anderson KS, Richardson AL, Nikolskaya T, Nikolsky Y, X. Liu S, Root DE, Hahn WC, Frank DA, Polyak K: The JAK2/STAT3 signaling pathway is required for growth of CD44(+)CD24(−) stem cell-like breast cancer cells in human tumors. J Clin Invest. 2011, 121: 2723-2735. 10.1172/JCI44745.PubMedPubMed CentralView ArticleGoogle Scholar
- Hernandez-Vargas H, Ouzounova M, Le Calvez-Kelm F, Lambert MP, McKay-Chopin S, Tavtigian SV, Puisieux A, Matar C, Herceg Z: Methylome analysis reveals Jak-STAT pathway deregulation in putative breast cancer stem cells. Epigenetics. 2011, 6: 428-439. 10.4161/epi.6.4.14515.PubMedView ArticleGoogle Scholar
- Dave B, Landis MD, Tweardy DJ, Chang JC, Dobrolecki LE, Wu MF, Zhang X, Westbrook TF, Hilsenbeck SG, Liu D, Lewis MT, Tweardy DJ, Chang JC: Selective small molecule Stat3 inhibitor reduces breast cancer tumor-initiating cells and improves recurrence free survival in a human-xenograft model. Plos One. 2012, 7: e30207-10.1371/journal.pone.0030207.PubMedPubMed CentralView ArticleGoogle Scholar
- Gurusamy N, Das DK: Autophagy, redox signaling, and ventricular remodeling. Antioxid Redox Signal. 2009, 11: 1975-1988. 10.1089/ars.2009.2524.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen N, Karantza-Wadsworth V: Role and regulation of autophagy in cancer. Biochim Biophys Acta. 2009, 1793: 1516-1523. 10.1016/j.bbamcr.2008.12.013.PubMedPubMed CentralView ArticleGoogle Scholar
- Cufi S, Vazquez-Martin A, Oliveras-Ferraros C, Martin-Castillo B, Vellon L, Menendez JA: Autophagy positively regulates the CD44(+) CD24(−/low) breast cancer stem-like phenotype. Cell Cycle. 2011, 10: 3871-3885. 10.4161/cc.10.22.17976.PubMedView ArticleGoogle Scholar
- Gong C, Bauvy C, Tonelli G, Yue W, Delomenie C, Nicolas V, Zhu Y, Domergue V, Marin-Esteban V, Tharinger H, Delbos L, Gary-Gouy H, Morel A-P, Ghavami S, Song E, Codogno P, Mehrpour M: Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like/progenitor cells. Oncogene. 2013, 32: 2261-2272. 10.1038/onc.2012.252.PubMedPubMed CentralView ArticleGoogle Scholar
- Berardi DE, Campodonico PB, Diaz Bessone MI, Urtreger AJ, Todaro LB: Autophagy: friend or foe in breast cancer development, progression, and treatment. Int J Breast Cancer. 2011, 2011: 595092-PubMedPubMed CentralView ArticleGoogle Scholar
- Vessoni AT, Muotri AR, Okamoto OK: Autophagy in stem cell maintenance and differentiation. Stem Cells Dev. 2012, 21: 513-520. 10.1089/scd.2011.0526.PubMedView ArticleGoogle Scholar
- Gong C, Song E, Codogno P, Mehrpour M: The roles of BECN1 and autophagy in cancer are context dependent. Autophagy. 2012, 8: 12-10.4161/auto.21691.View ArticleGoogle Scholar
- Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z: Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J. 2007, 26: 1749-1760. 10.1038/sj.emboj.7601623.PubMedPubMed CentralView ArticleGoogle Scholar
- Marino G, Lopez-Otin C: Autophagy: molecular mechanisms, physiological functions and relevance in human pathology. Cell Mol Life Sci. 2004, 61: 1439-1454.PubMedView ArticleGoogle Scholar
- Yu ZQ, Ni T, Hong B, Wang HY, Jiang FJ, Zou S, Chen Y, Zheng XL, Klionsky DJ, Liang Y, Xie Z: Dual roles of Atg8-PE deconjugation by Atg4 in autophagy. Autophagy. 2012, 8: 883-892. 10.4161/auto.19652.PubMedPubMed CentralView ArticleGoogle Scholar
- Nair U, Yen WL, Mari M, Cao Y, Xie Z, Baba M, Reggiori F, Klionsky DJ: A role for Atg8-PE deconjugation in autophagosome biogenesis. Autophagy. 2012, 8: 780-793. 10.4161/auto.19385.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.