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Abstract

Cancer is generally conceived as a dedifferentiation process in which quiescent post-mitotic differentiated cells acquire stem-like properties and the capacity to proliferate. This view holds for the initial stages of carcinogenesis but is more questionable for advanced stages when the cells can transdifferentiate into the contractile phenotype associated to migration and metastasis. Singularly from this perspective, the hallmark of the most aggressive cancers would correspond to a genuine differentiation status, even if it is different from the original one. This seeming paradox could help reconciling discrepancies in the literature about the pro- or anti-tumoral functions of candidate molecules involved in cancer and whose actual effects depend on the tumoral grade. These ambiguities which are likely to concern a myriad of molecules and pathways, are illustrated here with the selected examples of chromatin epigenetics and myocardin-related transcription factors, using the human MCF10A and MCF7 breast cancer cells. Self-renewing stem like cells are characterized by a loose chromatin with low levels of the H3K9 trimetylation, but high levels of this mark can also appear in cancer cells acquiring a contractile-type differentiation state associated to metastasis. Similarly, the myocardin-related transcription factor MRTF-A is involved in metastasis and epithelial–mesenchymal transition, whereas this factor is naturally enriched in the quiescent cells which are precisely the most resistant to cancer: cardiomyocytes. These seeming paradoxes reflect the bistable epigenetic landscape of cancer in which dedifferentiated self-renewing and differentiated migrating states are incompatible at the single cell level, though coexisting at the population level.

Keywords

Breast cancer metastasis transdifferentiation epithelial–mesenchymal transition epigenetic landscape

Introduction

The multifaceted characteristics of cancer have historically been classified into two main classes of activities: immortalization and transformation [12], which can be induced by the same or separate cooperating oncogenes [51]. Immortalization is defined by the capacity of cells to divide in a deregulated manner, sustained by self-mitogenic stimulation. By itself, immortalization alone is rarely fatal since it leads to verrucas or at worst to well delimited primary tumors whose surgical treatment is relatively simple. By contrast, transformation is associated to aggressive metastatic tumors with poor prognosis and covers a wide range of cellular activities such as migration, disruption of the extracellular matrix and chemotaxis. The cellular programs of division and migration are different and incompatible in single cells [5], which implies that they are logically associated with different epigenetic marks and different sets of genetic circuits in which given gene products can play either positive or negative roles in cancer.

Transformation relies on normal cellular processes

In post-mitotic tissues, cellular immortalization generally results from cellular disorders, often resulting from mutations. By contrast, metastasis borrows non-altered cellular programs. Very interestingly, despite the dramatic importance of metastasis in cancer malignancy, metastasis has not been causally associated yet to any genetic alteration [67] and could rely on non-genetic dynamic epigenetics [21]. All the identified cancer-driver mutations, not merely age-dependent and passenger only [64], belong to pathways involved in cellular immortalization, but not in metastasis [67]. The interpretation of these authors is that metastasis could not result from genetic defects, but from normal programs highjacked by cancer cells. Accordingly, all the facets of metastasis can be carried out by completely normal cells without need for oncogenes. As a matter of fact, the experimental displacement of normal cells in ectopic locations is sufficient to induce similar changes [53]. Moreover, the migratory phenotype of aggressive metastatic cells evokes a particular type of differentiation, closely related to that of contractile cells. This view is strikingly supported by the involvement in cancer of factors identified in normal muscle differentiation, exemplified by myocardin-related factors. These factors are also at work in the complex phenomenon of epithelial–mesenchymal transition (EMT, where the T is sometimes used for Transdifferenciation, in agreement with the present proposal) [36,56]. EMT recapitulates metastatic processes [24] including migration, cellular individualization and selfish behaviours, with loss of the initial differentiation markers and a metabolic switch to predominant glycolysis, similar to that of pluripotent stem cells [65]. Although EMT is often presented in the literature as a primary target for fighting cancer, it is also a normal cellular activity during development and healing [15,54]. It is by essence reversible in these situations and also during cancer evolution. Once they reached metastasis-housing tissues, invading cells can initiate the reverse process of mesenchymal-epithelial transition (MET) and recover an epithelial-like state [24], so that fighting specifically EMT could have unwanted consequences and no effect on implanted secondary tumors [46]. Different categories of EMT have been defined, either normal, as in development and wound healing, or pathological in cancer [24,78], but they are in fact likely to share identical genetic programs [78] among which it is illusory to find cancer-specific defects. The propensity of the scientific community to separate and classify cellular phenomena can occult the generality of certain circuits. For example the different terms EMT, fibroblasts to myofibroblasts conversion, myoepithelial cell differentiation, endothelial to mesenchymal transition and metastasis etc. are processes involving common aspects [78], even if they are variable in their details [75]. The conception of metastasis as the result of normal genetic programs leads to counterintuitive conclusions: (i) The most deleterious aspect of cancer progression, corresponds paradoxically to a differentiation process. (ii) Metastatic phenotypes involve genes otherwise associated to normal differentiation, classically assumed to prevent cancer. This situation could be responsible for misunderstandings about the pro- or anti-cancer role of many genes, because the same cellular sub-programs can have opposite issues depending on the context and the balance between proliferation and migration indexes of the tumor. The example of such ambiguous pathway selected here is that of myocardin-related transcription factors MRTFs, which has been associated with both metastasis in cancer and differentiation in muscular tissues which are, ironically, the most refractory to cancer. Ambiguous results have also been reported in the field of chromatin epigenetics.

Contradictory reports on cancer epigenetics

Conflicting proposals coexist in the literature about cancer chromatin epigenetics. Cancer cells generally lose differentiation marks of their original tissue and acquire stem-like features including proliferation and open chromatin. But at the opposite, repressive chromatin characteristic of differentiated cells, has also been considered as a hallmark of cancer [62]. These controversies mirror those existing about the impact on cancer of the enzyme responsible for the formation of the H3K9me3 histone mark: SUV39H1. SUV39H1 is sometimes considered as a therapeutic target [4,76], and inversely as a cancer-preventing molecule whose downregulation has a strong tumorigenic potential [26]. These discrepancies can be clarified by considering the ambiguous nature of cancer. Normal cell differentiation is associated with a profound reorganization of chromatin, including an increase of repressive marks (H3K9me3 and H3K37me3) [13]. By contrast, diffuse facultative heterochomatin and genome-wide gene expression characterize stem cells [7,29,37]. This property has long been exploited as a technical opportunity in mouse genome engineering, for allowing the selection of embryonic stem (ES) cells expressing antibiotic resistance cassettes inserted in virtually any gene. The background of generalized gene expression in stem cells [7] explains the necessity to demetylate H3K9me3 to maintain the self-renewal of embryonic stem cells [31]. Similar observations have been reported for cancer cells which lose de-differentiation markers and acquire globally open chromatin, with prominent histone acetylation and lower H3K9me3 trimethylation [37]. Inversely, this chromatin mark has been shown responsible for the difficulty to reprogram differentiated nuclei in nuclear transfer experiments [1,33]. The massive loss of heterochromatin in cancer is also reflected by the decondensation of Barr bodies (X inactivated) observed in many breast and ovarian cancers [50].

In the globally loose chromatin of undifferentiated or de-differentiated states, beside constitutive heterochromatin, H3K9me3 could be confined to few genetic loci, corresponding notably to tumour-suppressor genes. But in migratory cells, this mark could be more abundant and reflect the innermost nature of contractility as part of a differentiation program. This difference could explain the conflicting conclusions found about the role of SUV39H1 in cancer depending on the tumours considered, either positive [26] or negative [76]. The same reasoning applies to many genetic pathways, including that of myocardin-related transcription factors.

The Yin and Yang of myocardin-related transcription

Among the lot of molecules already incriminated in EMT and tumor transformation, attention will be focused here on myocardin-related transcription factors (MRTFs), because they are representative of genes with bivalent roles in cancer. The switch of tumoral cells from proliferative to migratory behaviours involves MRTFs, linking the cytoskeleton to gene expression. But somewhat paradoxically, MRTFs are also the markers of a particular differentiation state, precisely the most refractory to cancer, cardiomyocytes.

Cardiac departments are missing in cancer institutes

The Holy Grail of the anti-cancer recipe has been sought by researchers in exotic organisms resistant to cancer; but it is in fact not necessary to look as far, as the anti-cancer secret seems to be detained by one of our own organs: the heart. Heart is resistant to cancer despite its relatively high number of cells (300–400 g), compared for example to pancreas (70–100 g), that is highly subject to cancer. The rare tumours found in the heart are either of exogenous origin or derive from the connective tissue (myxomas), but never from cardiomyocytes. To understand this strange cellular specificity, one is tempted to look for genes specifically expressed in these cells and preventing their tumorigenesis. This inquiry readily leads to transcription factors specialised in the synthesis of contractile machineries, including myocardin, specific of cardiomyocytes and smooth muscle cells (SMC) [19,32,35,68,70] and myocardin-related factors, constitutively nuclear and strongly enriched in these cells, but sometimes expressed at lower levels in other cell types where they are subject to nucleocytoplasmic shuttling. The experimental disruption of myocardin leads to the de-differentiation and malignant transformation of smooth muscular cells [38] and conversely, myocardin is an ingredient of a cocktail of factors capable of reprogramming cells towards a cardiac fate [43]. These observations are in radical contrast with the activities of MRTFs reported in cancer.

Common modes of action but opposite effects of myocardin-related transcription factors in cancer and myocytes

Cardiomyocytes and SMCs overexpress actin isoforms and actin-related genes which are governed by serum-responsive elements (SRE), originally identified in the promoter of the immediate early gene c-fos and then extended to muscle-specific elements (named CArG). The selection of the subsets of SRE-dependent genes related to either division or motility, is partly dictated by the competing association with a common surface of SRF [77], with different coactivators [59], namely TCF for division and MRTFs for cytoskeletal dynamics [8,71]. The result of this competition depends on the mode of cellular adhesion [72]. In contractile cells, the poorly efficient SRF transcription factor is strengthened by fixation of MRTFs which work as coactivators because they are endowed with potent transcription activation domains while devoid of an own DNA-binding domain. Some members of the partially redundant MRTF family are specific of cardiomyocytes, such as its founder member Myocardin [68]. Other ones such as MRTF-A/MKL1 are abundant in muscle cells [58,69], but are also found at lower levels in other cell types. MRTFs are constitutively nuclear and active in muscular cells, whereas their nuclear translocation and activity are controlled by stimuli in other cell types [49]. Heart-specific myocardin is not regulatable by actin-mediated nuclear shuttling in spite of the presence of RPEL motifs in its amino-acid sequence [11]. At first glance, the roles of MRTFs appear strikingly different in the different cell types. In epithelial cells, MRTF-A seems to be involved in EMT and cancer progression [30,56] and consistently a factor inhibiting the transcriptional activity of MRTF-A, SCAI, is a suppressor of cancer [2], whereas in SMCs, MRTFs are clearly involved in differentiation [32]. The main interpretation suggested here to reconcile these opposite effects is that these factors are basically involved in the synthesis of actin-related contractile machineries (actin isoforms, myosin light chains, gelsolin, FHL2, etc.). These machineries participate to some extent to the mechanical phases of mitosis, but they are necessary in much larger amounts for the contractile processes of migration and thereby metastasis. Conversely, in the context of differentiated SMC and cardiomyocytes, these molecules participate to a normal differentiation program reinforcing a differentiated state.

Supporting observations with breast cancer cells

The theoretical considerations proposed above can be illustrated with original observations obtained with the widely used human Michigan Cancer Foundation (MCF) breast cancer cell lines, which are poorly aggressive and fail to give tumors in nude mice. Consistent results are obtained using estrogen receptor-negative MCF10A cells and estrogen receptor-positive MCF7 cells. These two cell lines are immortalized but retain certain epithelial markers. This situation facilitates in practice the visualization of a transdifferentiation into a contractile phenotype.

MCF10A cells expressing or not the transforming oncogene src

The MCF10A-Er-Src cell line has been engineered from the parental MCF10A cell line to contain an inducible viral oncogene v-src oncogene [23]. This potent oncogene found in the Rous-sarcoma virus has the well-established capacity to transform the cell through deregulated phosphorylation of many target proteins from the cytoskeleton such as vinculin [16]. Upon addition of the inducer of the ER-src construct (the ER ligand 4OHT), certain cells switch from an epithelial-like to individualized and stellate morphology. This transition is accompanied by series of changes. As shown in Fig. 1, the epithelioid shapes are disrupted and the cells appear more individualized, with a disorganization and a dramatic drop of the expression of E-cadherin and an apparent shrinking of nuclei (top of panel A). This transformation is associated with a profound reorganization of actin with an increase of filamentous actin (Fig. 1A bottom), and a significant increase of the mark H3K9me3 (Fig. 1C). The transcriptional coactivator MRTF-A involved in the expression of actin and contractile machineries also increased globally, with a tendency of nuclear enrichment in certain dish areas where the cells present the most altered morphology (Panel D). Interestingly, these effects are to some extent recovered without induction of ER-src, by merely adjusting the density culture (panel E), consistent with the established influence of cell–cell contacts on SRF/MRTF signalling [3]. This effect of density, in conjunction with serum, has been examined in another widely used cellular tool for studying breast cancer, MCF7 cells.

MCF7 cells

When cultured at confluence, MCF7 mammary cells retain a relatively differentiated epithelial phenotype, which can be disrupted at low density. In our experimental conditions with epithelial cells, the proportion of proliferating and migrating cells can be adjusted experimentally in non-confluent cell cultures (Fig. 2A). Serum is a crude mixture containing some nutrients and, in variable amounts depending on the batches, factors capable of inducing proliferation (such as EGF) and cytoskeletal dynamics (such as TGFβ ). As a consequence, its global effect is complex and should be redefined for every cell type. The drastic effect of serum addition on nuclear translocation is well established in fibroblastic cells [39], but less spectacular in epithelial cells. We first examined the behaviour of MCF7 cells with low or high serum in cell spreading assays after release form confluence (Ibidi^® chambers). The cells initially densely packed are suddenly allowed to evade a dense area and different markers are examined in the spreading area (Fig. 2B). Low serum promotes cell spreading and migration, with a marked decrease of the fraction of cells with a strong nuclear staining of PCNA. In contrast, in presence of high serum, the cells actively proliferate and colonize free areas by increase of cell number instead of net moving. Interestingly, nuclear ERα staining decreased in all conditions in peripheral cells, proliferating as well as migrating (not shown), which indicates that in both cases, they lost their original epithelial differentiation state. Strikingly, the nuclear enrichment of MRTF-A was observed only in migrating cells whereas it remains predominantly cytoplasmic in proliferating cells. This result agrees with the admitted mode of nuclear translocation of MRTF-A, anchored in the cytoplasm by globular non-polymerized actin and released in the nucleus following actin polymerization [41]. In parallel, dramatic changes are observed at the chromatin level. Migrating cells with nuclear MRTF-A were strongly enriched in trimethylated H3K9 whereas acetylated H3K9 strongly increased in proliferative cells. Though simple, this experiment is illuminating with respect to the dual behaviour of cancer cells. H3K9 trimethylation only reflects the differentiation-like nature of migration but is not a primary marker of cancer since it is not increased in proliferative cells. Conversely, H3K9 hyperacetylation and PCNA accumulation are observed only in proliferating cells in which MRTF-A is less enriched in the nucleus. The nuclear enrichment of MRTF-A in presence of low serum is confirmed in homogeneous culture conditions at low density (Fig. 3A). This result seems discordant with the initial reports on nuclear translocation of MRTF-A [39]. We verified that the propensity of MRTF-A to translocate in the nucleus is much higher in highly aggressive cells in which MRTF-A is predominantly nuclear, such as the human breast cancer cells MDA, and the epithelioid Hela cells (data not shown). Hence, using the moderately transformed MCF7 cells, the combination of density and serum allows to separate proliferation and migration. Serum has been shown to induce both programs in NIH 3T3 cells fibroblastic [8], but it would be interesting to precise if they are indeed activated in the same individual cells. Our experiments suggest that these two programs coexist in the same population but are mutually exclusive at the level of individual cells. In our conditions, serum addition stimulates proliferation without triggering massive nuclear accumulation of MRTF-A in MCF7 cells and conversely, low serum favours the nuclear accumulation of MRTF-A.

The selective involvement of MRTF-A in the migratory phenotype can be further visualized by the co-detection of markers at the single cell level. The nuclear fraction of MRTF-A differs from one cell to another within the same cell culture, but the stronger enrichment of nuclear MRTF-A is systematically associated to the exclusion of traditional proliferation markers (Fig. 3B) such as cyclin D1 (cells in G1 phase), proliferating cell nuclear antigen PCNA (cells in S phase) and, at the level of chromatin, acetylated lysine 9 and phosphorylated serine 10 (cells in M phase) of histone H3. Conversely the cytostatic cyclin dependent kinase inhibitor P27 (cells in G0 phase) is increased in MRTF-A positive cells. The transfected cells in which MRTF-A is strongly nuclear correspond precisely to those containing the higher total cellular content of this protein, suggesting the existence of a simultaneous control of MRTF-A translocation and stabilization. As shown in Fig. 3C and quantified in Fig. 3D, the loss of the proliferation marker PCNA is predominant in the cells which acquire the higher contractile phenotype depicted by the peripheral accumulation and organization of the focal adhesion protein FHL2.

To further assess the mutual exclusion between MRTF-A activity and proliferation, we took advantage of a MCF7 cell line described in [10]. This cell line contains a transgene encoding a mutant version of MRTF-A lacking its N-terminal domain called RPEL and responsible for the cytoplasmic anchoring of MRTF-A with globular actin. This mutant version is constitutively active (CA) and nuclear, capable of binding to SRF and activating the expression of SRF-MRTF-A-activated genes. As shown in Fig. 3, the cell cycle is clearly downregulated in these cells as revealed by the traditional proliferation markers Ki-67 at the protein as well as mRNA level. Conversely the mRNA of the cell cycle arrest marker P21 was upregulated (not shown). We conclude that the proliferation of MCF7 cells is stimulated by mitogenic factors of serum and conversely migration is favoured in presence of low serum. The cell cycle is inhibited in the cells where MRTF-A/SRF is most strongly activated (Fig. 3B), consistent with the cytostatic effect of MRTF-A reported in certain cells [74].

Revision of the cancer epigenetic landscape

The transdifferentiation hypothesis described here is expected to correspond to two attractors in a dynamic epigenetic landscape, popularized as the Waddington landscape [22,45,61]. A simplified such landscape is proposed in Fig. 4 to illustrate the situation. In this scheme, two juxtaposed attractors correspond to proliferative and myocytic cells. Compared to the initial differentiated state, cancer cells have the higher potential in this landscape, contrary to that proposed in [62] in which the cells with high H3K9me3 have the less determined epigenetic status. On reflection, it seems logical that self-renewing cells with a less condensed chromatin, are the most pluripotent. In self-renewing pluripotent cells, the selective closure of incompatible genetic circuits did not yet occur, so that these cells retain many possible differentiation fates [44]. Globally more accessible chromatin is also consistent with the intense gene expression necessary for synthesizing the components of new cells.

Attractor switching

Some cells can occasionally leave this intensely biosynthetic stem-like state and commit to differentiate in various cell types which can then be selected by environmental conditions. In particular, a myocytic-like phenotype could underlie metastasis, providing selectable advantages to the cancer cell population. Although migration and proliferation are mutually incompatible at a given moment for a given cell, they coexist in different cells cultured in the same conditions and also in endogenous tumors. They can also coexit in the same cell, but at different times, because migration alone is likely to be not sustainable for individual cells if not periodically interrupted by phases of cellular division. These phases may alternate with a reorganization of chromatin, consistent with the plasticity and reversibility of H3K9me3 heterochromatin [14]. In this picture, chromatin changes are expected to mainly rely on dynamic protein modifications but to be not further locked by DNA methylation, in agreement with the absence of DNA methylation measured during EMT [34], thereby facilitating bidirectional switches between the two cancer attractors drawn in Fig. 3. By nature, Waddington epigenetics is reversible, so that individual cells in the tumoral population can occasionally switch from an attractor to another one, which could contribute to the phenotypic heterogeneity observed in tumors and in cell cultures. The conversions between these attractors could be stochastic or deterministic. Deterministic parameters cover a wide variety of chemical and physical microenvironmental conditions such as the number of neighbouring cells, concentration gradients of extracellular ligands or serum composition and concentration. The transitions can also result from negative feedback-driven oscillations or simply be stochastic, that is to say apparently random and underlain by hidden microscopic causes. The fundamental reversibility of Waddington epigenetics suggests that in theory, the stem-like cells of Fig. 4 could re-differentiate into epithelial cells or virtually any other cell type. This possibility seems to be not forbidden, as suggested by certain experiments of reprogramming of nuclei from maligant cells [18], but it is however likely to be prevented in most cases by no-return mutations with a ratchet effect, or mutations distorting the epigenetic landscapes [20].

The proliferative and migrating states are mutually exclusive but are expected to be close together [5]. The proximity of proliferation and migration is suggested by the dual involvement of SRF in the transcription of mitotic and contractile gene repertoires [8] and to some extent, by the involvement of MRTF-A in proliferation. At the cell population level, the src oncogene as well as serum can increase both the mitotic index and actin cytoskeletal structures, suggesting that they can induce the two facets of cancer (Fig. 4), which generally coexist in the cell population, in variable extents depending on the cancer grade. These two aspects are also gathered together in EMT, showing that EMT corresponds more to a dual cellular competence than to an integral cellular activity strictly speaking. In a single cell, only one of these programs can be activated at a time, as suggested but the exclusion of proliferative markers in the cells in which MRTF-A the most strongly activated (Fig. 3B).

Attractor disjoining and stability

The plasticity of EMT does not forbid the clear separation of the attractors which are individualized by specific self-stabilizing biochemical circuits [44]. The proliferative state is relatively stable as it corresponds to an archaic attractor reminiscent of unicellular organisms and maintained in stem cells present in most tissues [21,25]. The contractile phenotype is also strengthened by many stabilizing positive feedbacks. On the one hand, MRTF-A activities are controlled by actin dynamics and on the other hand, actin genes are key transcriptional targets of MRTF-A, in a typical feedback [55] that can enforce the migratory phenotype in cancer cells. Another example of self-sustained loop stabilizing the migratory phenotype involves the adaptor protein FHL2, closely associated to metastasis [66]. FHL2 is strongly upregulated at the transcriptional level by MRTF-A ([57] and Fig. 3), and in turn, MRTF-A is strongly stabilized by FHL2 [17].

Conclusion

There are many paradoxes in the current literature on cancer, particularly regarding (i) its epigenetic marks, or (ii) the impact of many genes on cancer evolution. Several molecules are perceived as either favouring or preventing cancer depending on the laboratories. A celebrated example is the TGFβ pathway considered as a safeguard signal for quiescent differentiated cells through its anti-mitotic action, but which becomes a deleterious signal aggravating the metastatic phenotype [63]. The same duality is recovered for intracellular mediators of TGFβ signalling such as Smad3, capable of either preventing [28] or aggravating tumoral evolution [73]. Similarly, how to explain that myocardin-related factors are associated to the differentiation of cancer-resistant cardiomyocytes and SMCs, or cell cycle arrest during the differentiation of podocytes [74], whereas they are involved in EMT and tumor transformation [56]. In the same manner, why SUV39H1 is considered depending on the authors as either a pro-tumoral [4] or anti-tumoral [6,26] enzyme. The view of cancer as a trans-differentiation phenomenon can shed light on this virtually endless list of discrepancies. Their commonplace justification in the literature is that they could result from unknown cellular contexts and the use of different cell lines. Indeed, a major lesson of dynamic epigenetics symbolized by Waddington landscapes, is that the precise function of every gene is specified by the collective adjustment of all gene products in gene regulatory circuits. A stationary state in which all these levels of expression are mutually compatible is called an attractor and in theory, the precise function of every gene should be redefined in every attractor, as illustrated by the differential impact of TGFβ on the intact versus injured regions of the epithelium [60]. The harmful activity of TGFβ could selectively target the myocytic-like attractor, in a MRTF-A dependent manner [40,47,48]. As a matter of fact, Smad3 and MRTF collaborate to induce the repressor of the epithelial differentiation state Snail2 [40]. The role of TGFβ on the change of cellular adhesion and shape associated to EMT involves MRTF-A signaling [47] and conversely, the response to TGFβ depends on cellular density [42].

There is a generalized connection, more or less distant, between all the genetic circuits in a global Waddington epigenetic landscape. In particular, the two facets selected here, chromatin epigenetics and MRTF-A signalling, are related because MRTF-A can profoundly alter the degree of chromatin compaction and modifications [10] and it has recently been shown to mediate the formation of permissive chromatin with trimethylated H3K4 at the levels of TGFβ -responsive genes [9]. In the mixed view of cancer presented here, the cellular population distributes over two attractors which are both close together and distinct: an undifferentiated one characterized by low H3K9me3 chromatin and a more differentiated one enriched in H3K9me3 marks, in which contractile functions are activated. In fact, metastatic cells undergoing migration are quiescent, in line with the cytostatic effect of MRTF-A [27] and the low level of PCNA measured in migratory cells (Figs 2A and 3C). But in cancer cells, contrary to cardiomyocytes, this state is not a primary differentiation process but is necessarily subsequent to the previous dedifferentiation of the original differentiation state, epithelial in the example described here. This double jump is typically a transdifferentiation phenomenon. The present hypothesis is aimed at solving inconsistencies in the literature on cancer, but unfortunately does not really simplify the therapeutic approaches. For example, fighting specifically proliferation through depriving cancer cells from nutrients could have deleterious long term effects by selecting the opportunistic nomadic consumption strategy of migratory cells.

Materials and methods

Cell lines and treatments

MCF7 were routinely cultured in DMEM (Dulbecco’ Modified Eagle’ Medium, Invitrogen) supplemented with 10% FBS (biowest) and antibiotics (Invitrogen) at 37 ^∘ C in 5% CO2. MCF7 cells stably expressing MKL1 or MKL1-ΔN200 constructs were established using the T-Rex subclones system [10]. Expression of the proteins of interest was induced by a 48 hours treatment of the MCF7 subclones with tetracyclin, in DMEM supplemented with 2% charcoal-stripped of fetal calf serum (FCS). Migration assay were carried out in IBIDI chambers (Ramcon). MCF7 cells were seeded at confluence in DMEM supplemented with 2% or 10% of FCS into chambers fixed with collagenase to the lamella. After 48 hours, cells were released by gently removing the chambers for 15 hours. MCF10A stably expressing the ER-Src construct were kindly provided by Dr Kevin Struhl from Harvard Medical School, and were maintained in DMEM supplemented 5% of charcoal stripped horse serum (Invitrogen), with 20 ng/ml of EGF, 0.5 μ g/ml of hydrocortisone (Sigma-Aldrich), 100 ng/ml of cholera toxin (Sigma-Aldrich) and 10 μ g/ml of insulin (Sigma-Aldrich). When indicated, MCF10A ER-Src cells were treated with 1 μ M of 4-Hydroxytamoxifen (Sigma-Aldrich).

RNA extraction and qPCR

Total RNA extractions were performed using RNeasy kit (Qiagen) according to the manufacturer’s instructions. Retrotranscription was performed on 1 μ g of RNA with random primers using MMLV reverse transcriptase (Sigma). Quantitative RT-PCRs were performed using the iQ^™ SYBR Green supermix from BioRad (Bio-Rad, Hercules, CA, USA). The primer sequences used for qPCR are ki67 forward 5′ -ATAAACACCCCAACACACACAA-3′ and reverse 5′ -GCCACTTCTTCATCCAGTTAC- 3′ ; fhl2 forward 5′ -GGCAAGAAGTACATCCTGCG-3′ and reverse 5′ -CCACCAGTGAGTTTCTGCAC-3′ .

Immunofluorescence and antibodies

Cells were grown on 10-mm-diameter coverslips in 24-well plates in the presence of DMEM containing 2% charcoal-stripped FCS. Cells were fixed with PBS/4% PFA (paraformaldehyde) for 10 minutes and then permeabilized in PBS/0.3% Triton X-100 for 10 minutes. The antibodies used for immufluorescence analyses where used at 1∕1000 dilution and are indicated as follows: E-cadherin (ab15148; Abcam), MKL1 (ab113264; Abcam), H3K9ac (histone H3 acetylated at Lys 9; ab10812; Abcam), H3K9me3 (histone H3 trimethylated at Lys 9; ab8898, Abcam), H3 (ab1791, Abcam), PCNA (Dako, M0879), FHL2 (PA5-28643, Thermo Scientific), P27 (sc528, Santa Cruz), Cyclin D1 (Santa Cruz), H3pSer10 (Serine 10 Phosphorylated Histone H3, sc8656-K Santa Cruz) and FLAG (M2, Sigma-Aldrich). Images were quantified using ImageJ software.

Quantification and statistical analysis

Immunostaining signals were quantified from fluorescence microscopy images by averaging the intensity of the same area of 50 arbitrarily chosen cells or nuclei obtained from three independent experiments. Stars on histograms correspond to Mann–Whitney U tests. RT-PCR values are expressed as the mean of fold induction compared to controls of four independent experiments; ^∗ P < 0.05 with the Mann–Whitney U test.

Footnotes

Acknowledgements

This work is supported by the Ligue inter-régionale Grand Ouest de lutte contre le cancer.

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Envisioning metastasis as a transdifferentiation phenomenon clarifies discordant results on cancer

Abstract

Keywords

Introduction

Transformation relies on normal cellular processes

Contradictory reports on cancer epigenetics

The Yin and Yang of myocardin-related transcription

Cardiac departments are missing in cancer institutes

Common modes of action but opposite effects of myocardin-related transcription factors in cancer and myocytes

Supporting observations with breast cancer cells

MCF10A cells expressing or not the transforming oncogene src

MCF7 cells

Revision of the cancer epigenetic landscape

Attractor switching

Attractor disjoining and stability

Conclusion

Materials and methods

Cell lines and treatments

RNA extraction and qPCR

Immunofluorescence and antibodies

Quantification and statistical analysis

Footnotes

Acknowledgements

References