Aggressiveness of non-EMT breast cancer cells relies on FBXO11 activity
Molecular Cancer volume 17, Article number: 171 (2018)
Tumorigenesis is increasingly considered to rely on subclones of cells poised to undergo an epithelial to mesenchymal transition (EMT) program. We and others have provided evidence, however, that the tumorigenesis of human breast cancer is not always restricted to typical EMT cells but is also somewhat paradoxically conveyed by subclones of apparently differentiated, non-EMT cells. Here we characterize such non-EMT-like and EMT-like subclones. Through a loss-of-function screen we found that a member of the E3 ubiquitin ligase complexes, FBXO11, specifically fuels tumor formation of a non-EMT-like clone by restraining the p53/p21 pathway. Interestingly, in the related EMT-like clone, FBXO11 operates through the BCL2 pathway with little or no impact on tumorigenesis. These data command caution in attempts to assess tumorigenesis prospectively based on EMT profiling, and they emphasize the importance of next generation subtyping of tumors, that is at the level of clonal composition.
In recent years, due to its aggressive behavior, much attention has been directed towards the basal-like subtype of breast cancer believed to be caused by cancer stem cell activity often considered synonymous to the concept of EMT. Accordingly, as a consequence of its relatively differentiated, non-EMT-like appearance, the most frequent subtype of breast cancer, the luminal, has been somewhat understudied in this regard. Indeed, the very impact of EMT on breast cancer has been questioned altogether [1, 2]. With the purpose of examining cancer stem cell activity among typical epithelial cells in breast cancer we have previously focused on comparisons between clonally related non-EMT-like and EMT-like cells . To our surprise we found that independently of subtype the non-EMT-like cells are more tumorigenic and tumor-initiating than the EMT-like cells [3, 4]. When we cloned cells with a stem-like profile, we found that they readily generated luminal-like progeny to indicate the existence of a hierarchy which could sustain heterogeneity as previously described for human breast cancer . We also cloned differentiated variants without a typical stem-cell profile, but with a luminal profile which resembles the majority of grade I luminal breast cancers, i.e. polarized luminal-like cells without signs of basal traits. While the latter clones turned out to be both highly tumor-initiating and invasive , the question remained open as to how aggressiveness is maintained in such clones if not by hijacked stem-cell or established EMT-related pathways.
Large-scale loss-of-function screens have been successfully applied to identify tumorigenic mechanisms and in turn have led to discovery of novel targets for drug intervention . Here, we performed an shRNA screen in non-EMT-like and EMT-like clones for identifying differentially depleted shRNAs and found significantly depleted shRNAs in a clone-dependent manner. Based on this, we propose to reappraise pathways of tumorigenesis in non-EMT breast cancer subclones and to emphasize clonal heterogeneity as a supplement to breast cancer subtyping.
Characterization of MCF7 breast cancer cell clones as a model of tumor aggressiveness and FBXO11 as a functional readout
In order to investigate mechanisms alternative to EMT, which facilitate tumor progression, we used an established set of non-EMT-like and EMT-like clones of MCF7 cells known to differentially express CDH1 and TWIST, SNAI2, FN and VIM, respectively . Here, using an RT-qPCR approach different from the one used previously, the EMT properties of these cells are further substantiated (Fig. 1a). Also, we confirm the epithelial properties of the non-EMT-like clone by positive staining for ZO-1, Occludin and E-cadherin, whereas the EMT-like clone expresses little of these markers (Fig. 1b). Notably, transplantation to NOG mice for in vivo imaging resulted in a significant increase in tumor size of the non-EMT-like clone compared to the EMT-like (Fig. 1c and d). Immunofluorescence imaging with keratin 19 further revealed that only non-EMT-like cells formed metastatic colonies in the lungs (Fig. 1e). Since MCF7 breast cancer cells are considered quite stationary in monolayer culture , we did not pursue migration at the single-cell level as a readout of EMT. These findings nevertheless underscore that cancer-cell aggressiveness is not restricted to cells with a typical EMT-like profile, emphasizing the need for alternative explanations to this behavior.
Accordingly, we applied a loss-of-function screen with a lentiviral library of 2365 pLKO shRNA constructs (Fig. 1f and Additional files 1 and 2). While the screen identified several potentially interesting genes significantly depleted in both clones, here we focused on differently depleted shRNAs. Among these were FBXO11, NAT8L and TOX4 in the non-EMT-like and NAP1L4, TCF20 and ID3 in the EMT-like clones. Since the non-EMT-like clones are more tumorigenic and metastatic, we selected FBXO11 for further scrutiny (Fig. 1f).
FBXO11 has been reported to have different cellular roles. On one hand, it has been shown to keep EMT in check by facilitating degradation of SNAI1 and SNAI2 (reviewed in ). On the other hand, FBXO11 has also been reported to neddylate p53 protein, leading to inhibition of its activity . Since p53 is a bona fide tumor suppressor, a consequence of FBXO11 expression in this context would be to promote tumor growth. Accordingly, we show that based on multiple cohorts , FBXO11 is a robust predictor of a poor clinical outcome in breast cancer (Fig. 1g). To validate the relevance of FBXO11 specifically in luminal breast cancer which is the topic of the present study, we examined 30 ER-positive breast cancer biopsies for FBXO11 expression. We found nuclear staining with FBXO11 in the majority of the cancer cells in 18 biopsies and six were highly differentiated, polarized, tubular breast carcinomas (Fig. 1h), providing proof of principle that tumors with FBXO11 expression are not necessarily linked to EMT. These data were supported by multivariate analysis of FBXO11 expression and impact on survival, specifically within the subtype of luminal breast cancer (Additional file 3: Table S1). Based on these findings, FBXO11 signaling was further investigated with respect to tumorigenicity in non-EMT breast cancer cells.
shFBXO11 preferentially attenuates tumor initiation of non-EMT breast cancer cells
We first tested the knockdown efficiency of shRNAs targeting FBXO11 (shFBXO11s). As expected, in both clones FBXO11 was reduced by shFBXO11 (Additional file 3: Figure S1A from which selected lanes were included in Fig. 2a, and S1B). Next, we tested the consequences of silencing FBXO11 in terms of tumorigenicity in vivo. While tumor formation by the non-EMT-like cells was significantly inhibited by shFBXO11, no effect was recorded by the EMT-like cells (Fig. 2a and b). Similar data were obtained with a pGIPZ shFBXO11 (data not shown). To corroborate the effects of shFBXO11 in the non-EMT-like cells, we generated FBXO11 indel clones, using a CRISPR-Cas9 system (Fig. 2c and d), and further validated that FBXO11 is necessary for tumorigenesis (Fig. 2e). In summary, FBXO11 impacts primarily on tumorigenicity of non-EMT cells.
Given the known effect of FBXO11 on p53 and the role of a p53/p21/BCL2 pathway on invasion , we next investigated whether the FBXO11-dependent inhibition of non-EMT-like cells could be explained within such context. While the clinical datasets were too small to resolve a potential impact of FBXO11 on e.g. p53 (Additional file 3: Table S1), in culture, shFBXO11 led to a strong increase in p21 expression in the non-EMT-like cells but not in the EMT-like cells (Fig. 2f upper, and Additional file 3: Figure S1C and D). Similar data of p21 induction were obtained with the non-EMT-like cells deleted for FBXO11 (Fig. 2g). Importantly, the shFBXO11-induced p21 expression was abolished by co-transduction with shp53 (Fig. 2f lower). Also functionally, shFBXO11 significantly reduced the cell number to 37%, which was rescued in part, up to 71%, by concurrent silencing of p53 (Fig. 2h). These data were substantiated with two different FBXO11 indel clones (Fig. 2i). In contrast to the non-EMT cells, the p53/p21 pathway was not appreciably affected by FBXO11 in the EMT cells. Rather in these cells, FBXO11 led to an increase in BCL2, which was not pursued further in the present study (Additional file 3: Figure S1C and D). In summary, FBXO11 specifically fuels tumor formation in non-EMT-like cells by restraining the p53/p21 pathway.
In absence of an obvious EMT-based explanation to tumor aggressiveness, we sought for another mechanism. The most straightforward candidate process for EMT-independent invasion and metastasis is that by collective invasion . Here, we decided to gauge for collective cancer-cell invasion in a basement membrane chamber assay. In an initial experiment we unequivocally demonstrate that deletion of FBXO11 leads to near absence of invasion (Fig. 2j). While this was expected, exactly how the non-EMT-like cells invaded was further investigated by mixing GFP-labeled and non-labeled cells in suspension (Fig. 2k). When the clusters were plated in the invasion assay, cells emerged from single pores as mixed duplets (Fig. 2k), suggesting that indeed non-EMT breast cancer cells spread in a collective manner.
Based on these observations, it is concluded that FBXO11 is a candidate molecular alternative to the canonical EMT-dependent aggressiveness in highly differentiated luminal tumors. In addition, our results suggest a more prominent role for FBXO11 in subclone-behavior than hitherto anticipated. This emphasizes the relevance of considering clonal heterogeneity in future therapeutic strategies in breast cancer.
Clustered regularly interspaced short palindromic repeats-CRISPR associated system 9
Epithelial to mesenchymal transition
F box protein 11
Reverse transcription-quantitative polymerase chain reaction
Scrambled shRNA control
Standard error of the mean
Short hairpin RNA
Ye X, Brabletz T, Kang Y, Longmore GD, Nieto MA, Stanger BZ, et al. Upholding a role for EMT in breast cancer metastasis. Nature. 2017;547(7661):E1–3.
Fischer KR, Durrans A, Lee S, Sheng J, Li F, Wong ST, et al. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature. 2015;527(7579):472–6.
Kim J, Villadsen R, Sørlie T, Fogh L, Grønlund SZ, Fridriksdottir AJ, et al. Tumor initiating but differentiated luminal-like breast cancer cells are highly invasive in the absence of basal-like activity. Proc Natl Acad Sci U S A. 2012;109(16):6124–9.
Leth-Larsen R, Terp MG, Christensen AG, Elias D, Kuhlwein T, Jensen ON, et al. Functional heterogeneity within the CD44 high human breast cancer stem cell-like compartment reveals a gene signature predictive of distant metastasis. Mol Med. 2012;18:1109–21.
Zuber J, Shi J, Wang E, Rappaport AR, Herrmann H, Sison EA, et al. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature. 2011;478(7370):524–8.
Comsa S, Cimpean AM, Raica M. The story of MCF-7 breast Cancer cell line: 40 years of experience in research. Anticancer Res. 2015;35(6):3147–54.
Diaz VM, de Herreros AG. F-box proteins: keeping the epithelial-to-mesenchymal transition (EMT) in check. Semin Cancer Biol. 2016;36:71–9.
Abida WM, Nikolaev A, Zhao W, Zhang W, Gu W. FBXO11 promotes the Neddylation of p53 and inhibits its transcriptional activity. J Biol Chem. 2007;282(3):1797–804.
Gyorffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q, et al. An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat. 2010;123(3):725–31.
Friedl P, Locker J, Sahai E, Segall JE. Classifying collective cancer cell invasion. Nat Cell Biol. 2012;14(8):777–83.
The authors thank Tove Marianne Lund, Lena Kristensen, Anita S. Friismose, and Christa A. Andersen for expert technical assistance; Dr. Vera Timmermans Wielenga (Pathology Department, Rigshospitalet) for providing tumor samples. The authors also thank to Dr. Sanjiv. Sam Gambhir in Stanford University for providing pFU-L2G bioluminescence reporter and Dr. Balazs Gyorffy for a personal communication regarding the usage of the online clinical database.
This work was supported in part by the Novo Nordisk Fonden and Danish Research Council Grant 10–092798 (to DanStem), the Kirsten and Freddy Johansens Fond (to OWP), the Familien Erichsens Mindefond and Vera og Carl Johan Michaelsens Legat (to JK), Læge Sophus Carl Emil Friis og hustru Olga Friis’ Legat, Oda og Hans Svenningsens Fond and Thora og Viggo Groves Mindelegat (to RV) and the Harboefonden, Else og Mogens Wedell-Wedellsborgs Fond and Danish Cancer Society Grant R146-A9257 (to LRJ).
Availability of data and materials
The data supporting the conclusion of this study are included within this manuscript and the Additional files. Please see Additional file 4 for a detailed explanation of all experimental procedures and materials.
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Animal experiments were conducted according to procedures approved by the Animal Experiments Inspectorate, and the use of human material has been reviewed by the Regional Scientific Ethical Committees and approved with reference to H-2-2010-051 and H-3-2010-095.
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pLKO human shRNA library and its validation. (XLS 1013 kb)
Screening summary of normalized reads of shRNAs. (XLSX 379 kb)
Table S1. Clinical outcome as a function of FBXO11 expression in breast cancer subtypes and Figure S1. FBXO11 facilitates protein degradation in a subclone-dependent manner. (PDF 315 kb)
Materials and Methods. (DOCX 33 kb)
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Bagger, S.O., Hopkinson, B.M., Pandey, D.P. et al. Aggressiveness of non-EMT breast cancer cells relies on FBXO11 activity. Mol Cancer 17, 171 (2018). https://doi.org/10.1186/s12943-018-0918-6