1 – The translocation of CD24 from cytosol to membrane is an early event in breast tumor cells under drug stress
Phenotype switching, also commonly referred to as cell plasticity, is an important process observed during treatment of cancer which was repeatedly associated with stemness. Here, using breast cancer cell line, we explored the dynamics of the CSC marker CD24 after doxorubicin treatment. At first, we sought to define the localization of CD24 in MDA-MB-231 cells by extra and intracellular staining. As shown by our results obtained by flow cytometry (Fig 1a,b) only about 5% of cells expressed CD24 in cell membrane corroborating with other studies [23] which explains why MDA-MB-231 is considered as CD24low/-. By contrast, a significant intracellular pool of CD24 was encountered in all cells. Fluorescence microscopy confirmed the presence of both extracellular (yellow arrows) and intracellular CD24 (white arrows) (Fig 1c). After the treatment with doxorubicin at 0,6mM – concentration representing the EC50 after 24h treatment calculated in MDA-MB-231 cell line – a cell phenotype switching occurred which corresponded to an enrichment of the CD24+ subpopulation (Fig 1d). Notably, MFI analysis showed an increase of surface CD24 density during drug treatment (Fig 1e). This phenomenon occurred rapidly since ~42% of cells converted into CD24+ after 2h to finally reach ~96% after 48h of treatment, as visualized by flow cytometry (Fig 1d). Importantly, the majority of cells remained positive even after a pause in the treatment (incubation in drug-free medium for 48h after treatment) as visualized by the last pseudocolor plot in the figure 1d. The fact that this event was detected in the first hours of treatment excludes the possibility of a Darwinian selection of CD24+ cells. These results led us to hypothesize that the intracellular pool of CD24 immediately available might play a role in this process by translocating from the cytosol toward cell surface under doxorubicin treatment. To support these data, MDA-MB-231 cells were sorted into CD24+ and CD24- subpopulations (Fig 1h) by using magnetic beads and the CD24 localization was evaluated in CD24- cells after doxorubicin treatment. As shown in the figure 1i, the translocation of CD24 occurred even in CD24- population obtained after cell sorting since CD24- cells were able to rapidly convert in CD24+ cells. Such data reinforce the idea that CD24+ cells enrichment during drug treatment does not correspond to a pre-selection of clones but to a drug-induced phenotype switching. In order to confirm this theory, we took the opportunity of using brefeldin A, an inhibitor of protein transport from endoplasmic reticulum to Golgi apparatus, to disturb the CD24 traffic after drug treatment. After flow cytometry analysis, we observed that when MDA-MB-231 cells were treated with brefeldin A before doxorubicin, the translocation of CD24 was reduced (Fig 1f). These results were consistent with the fluorescence microscopy images obtained from doxorubicin-treated cells and stained with anti-CD24 without permeabilization to solely detect surface CD24 (Fig 1g). To discard the hypothesis that the increased membrane CD24 expression could be partially due to an increased protein synthesis, we used actinomycin D, a DNA-transcription inhibitor. The incapability of actinomycin D to reduce the CD24+ phenotype enrichment in presence of doxorubicin indicated that the intracellular pool of CD24 was the main source of CD24 traffic in the presence of doxorubicin (data not shown).
Concerning MACL-1 and MGSO-3 breast cell lines, obtained from Brazilian patients, the percentage of CD24+ cells was about 5% and 46% respectively, corroborating with their classification in other study [24]. The presence of intracellular CD24 was also detected in the whole population of both cell lines, like observed in MDA-MB-231 population (Fig 2a,b). As observed in MDA-MB-231 cells under treatment, phenotype switching occurred in both cell lines which corresponded to an enrichment of the CD24+ subpopulation (Fig 2c,d) since ~27% and ~45% of MACL-1 and MGSO3 cells, respectively, converted into CD24+ after 4h of treatment with doxorubicin. After 24h of treatment, the conversion rate in CD24+ cells reached ~90% for MDA-MB-231 and MACL-1 and ~70% for MGSO-3 cells.
Therefore, we propose a new dynamic model of cell transition phenotype under drug stress that could allow each cell of breast cancer to convert into CD24+ cells which involves the translocation of intracellular CD24 accompanied with its increased expression at cell surface.
2 – Translocation of CD24 correlates with the upregulation of Bcl-2 expression leading to chemoresistance in MDA-MB-231 cells treated with doxorubicin
Here, we investigated the molecular identity of the survival cells after 48h of doxorubicin treatment named CD24+/DxR cells. The figure 3a represents the methodology used to obtain CD24+/DxR cells and schematize the steps to perform the following MTT and western blot assays. At first, we tested the sensibility of the CD24+/DxR cells to respond to a second doxorubicin treatment using MTT assay. As shown in the figure 3b, only a slight reduction of CD24+/DxR cell number was noted after a second treatment with doxorubicin while the viability of naïve cells, that received the treatment for the first time, declined below 40%. These data indicated that CD24+/DxR cells became tolerant. So, we sought to identify whether this phenotype change corresponded to a putative cell reprogramming in response to drug stress. By using western blot assay, we compared the protein profile of naïve and CD24+/DxR cells. When we focused on protein expression involved in cell proliferation or death, it was detected a significant increase of Bcl-2 expression which was inversely correlated with Bax expression in CD24+/DxR (Fig 3c). In addition, a remarkable decrease of cyclin D1, a regulator of cell cycle progression, was noted, suggesting that acquisition of drug tolerance controlled by Bcl-2 expression may also require cells to exit the cell cycle (Fig 3c). In previous studies, we have focused on the role of p38 MAPK and ERK1/2 in the proliferation of MDA-MB-231 cells [25,26], so we analyzed their activation profile in the resistant CD24+/DxR cell. Interestingly, this phenotype switching was accompanied by a strong and continuous activation of p38 MAPK at the detriment of another MAPK, ERK1/2 (Fig 3d). Then, we verified the relationship between CD24 and Bcl-2 by silencing CD24 using interference-RNA. In CD24-silenced cells (SiCD24), a decreased Bcl-2 and p38 expression was observed (Fig 3f). This may explain the reduced capacity of SiCD24 cells to resist to drug treatment in all the doxorubicin concentrations tested when compared with control-silenced (SiC) and parental MDA-MB-231 cells (Fig 3e). By contrast, CD24- subpopulation, obtained from magnetic sorting, presented a similar drug sensibility when compared with CD24+ subpopulation and parental MDA-MB-231 cells, probably due to the phenotype switching after drug treatment thanks to CD24 translocation as shown in the figure 1j (Fig 3e).
Next, we sought to treat MDA-MB-231 cells with a drug capable to reduce cell proliferation without inducing immediate death, like doxorubicin does. We tested the efficacy of the TLR7 agonist Imiquimod, previously used in skin cancer treatment and in the treatment of cutaneous metastatic breast cancer [27–29], on MDA-MB-231 cell proliferation. At the concentration of 1mM, a significant decreased in cell proliferation was observed while a total blocking of cell replication was noted at 10mM during the period of experiment (Fig 3g). No significant cell death was observed in the first 48 hours in the presence of both concentrations of Imiquimod. In such context, we tested the capacity of cells to respond to a second treatment 96h after the first dose of Imiquimod. As shown in the figure 3h, where the results are expressed in % of cell viability, we observed a similar pattern when an unique dose or two subsequent treatments with the TLR7 agonist were used indicating that no phenotype change occurred after the first treatment and consequently avoid chemoresistance. When we used CD24 as a phenotypic marker, we confirmed that no switching phenotype of MDA-MB-231 population leading to an enrichment of CD24+ cells occurred (Fig 3i). In accord with this, no upregulation of Bcl-2 expression was detected in cells treated with Imiquimod (Fig 3j).
Taken together these data indicated that translocation of CD24 is a triggering event leading to phenotype change and upregulation of Bcl-2 expression.
3 – CD24 and p38 work in tandem in the chemoresistance acquisition phenotype in breast cancer cells.
According to the figure 3d, p38 phosphorylation was stronger, constitutive and independent on serum in CD24+/DxR cells contrasting with the serum-dependent activation of p38 in MDA-MB-231 cells under normal culture conditions. This suggests that p38 activation in different configurations can cause different outputs and may participate in the phenotype switching.
The next question was whether there was a privileged relationship between CD24 and p38. At first, we explored this point in MDA-MB-231 cells under growing culture conditions. After magnetic sorting, we evaluated by western blot the status of MAPK activation after cell stimulation with serum according to the kinetic presented in the figure 4a. As clearly shown, CD24+ cells phosphorylated p38 in a more pronounced way than CD24- subpopulation. In contrast, a higher phosphorylation of ERK1/2 was observed in the CD24- and parental MDA-MB-231 cells. The results obtained by flow cytometry confirmed the correlation between surface CD24 expression and preferential p38 phosphorylation, as observed by the fact that ~70% of the CD24+ cells phosphorylated p38, while in only ~15% of the CD24- cells the activation of this MAPK was observed (Fig 4b).
Another evidence demonstrating that CD24 and p38 work together is presenting in the figure 4c. According to the western blotting, siRNA-mediated knockdown of CD24 decreased the phosphorylation of p38 when cells were submitted to doxorubicin treatment indicating that the absence of CD24 jeopardized the cell capacity to induce activation of p38 MAPK.
The sustained p38 activation in CD24+/DxR cells makes it a prime target. In this context, we used SB203580, a p38 activity inhibitor [30] to evaluate its impact on drug resistance. According to the results obtained by cell counting, the combination of SB203580 and doxorubicin was more efficient in reducing cell number than doxorubicin alone (Fig 4d). Concerning the results observed by MTT assay, the inhibition of p38 was benefit from two aspects: firstly, SB203580 sensitized MDA-MB-231 cells to the therefore doxorubicin treatment (blue line vs black line) and at second, SB203580 disrupted the resistant-phenotype acquired by the cells that received two consecutives doxorubicin treatments (green line vs red line). The effect of the drug association may be considered as synergistic since the total effect of the combination of SB203580 and doxorubicin was greater than the sum of the individual effects of each drug. Importantly, SB203580 alone was unable to impact on cell viability in the same experimental conditions (Fig 4e).
To visualize these results, we performed the capture of light microscopy images of cells treated with drug pair. These experiments were performed under sub-confluence or confluence conditions to exclude the influence of fluctuating environment. A direct impact of doxorubicin on MDA-MB-231 cells was observed after 24h treatment marked by a decreased cell number and changes in morphology. The association of SB203580 and doxorubicin exacerbated the cell phenotype changes under confluent and sub-confluent conditions. The results confirmed that the efficiency of the drug pair constituted by doxorubicin and SB203580 was superior in killing cells in both plating conditions (Fig 4g).
Consistent with the above results, western blots showed that SB203580 prevented the augment of Bcl-2 expression induced by doxorubicin (Fig 4f). Further, in MDA-MB-231, which has high levels of a mutant p53, it has been described that p53 mutants can contribute to the suppression of apoptosis [31]. In line with this, SB203580 was also able to reduce the expression of p53 in doxorubicin treated cells (Fig 4f).
Epigenetic events drive cell reprogramming and tumor cell plasticity [14,15]. In this context, we have, evaluated whether the state of histones could be altered in CD24+/DxR cells. We focused on the lysine 9 at the histone H3 (H3K9), which can turn genes on or silence them by getting acetylated or methylated [32]. Our western blot analyzes have shown that doxorubicin treatment increased the tri-methylation of H3K9 (H3K9me3) which combined with its deacetylation. Importantly, SB203580 was able to prevent the increase of H3K9me3 induced by doxorubicin treatment indicating that p38 can regulate the methylation state of H3K9 observed under drug pressure (Fig 4h).
Taken together, these results suggest that targeting p38 during the chemotherapy-induced phenotypic cell state transition can overcome adaptive resistance to doxorubicin treatment.
4 – CD24/DxR cells become proliferative after a long-lasting period in dormancy.
The capacity of slow cycling cells to reentry into cell cycle has been a topical debate for quite a time. As reported above, CD24+/DxR cells have adopted a slow-down cell cycle after doxorubicin treatment evidenced by a reduction of cyclin D1 (Fig 3c) and were characterized by an enlarged morphology that are hallmarks of dormant cells (Fig 5b).
So, we sought to monitor CD24+/DxR cells to evaluate the reversibility of their dormant state according to the scenario presented in the figure 5a. CD24+/DxR cells were cultured in drug-free medium for a long period and then were submitted to a serum deprivation for two days followed by the culture in medium contained 10% of serum. In such conditions, we observed the emergence of revertant cells (named DxR/30) which reacquired the ability to proliferate as confirmed by their capacity to incorporate BrdU (Fig 5c).
The percentage of CD24+ cells in the revertant-population recovered to levels observed in naïve MBA-MB-231 cell population (Fig 5d). Importantly, even in the absence of drug, the phosphorylation of p38 in DxR/30 remained strong and constitutive indicating that DxR/30 cells have conserved some features of their precedent states while having eliminated others (Fig 5e).
When we evaluated the drug resistance of DxR/30 cells, more than one month after the first treatment the cells remained tolerant to doxorubicin. Indeed, as shown in the light microscopy images, the morphology of these cells appeared little affected after treatment when compared to naïve MDA-MB-231 treated cells. More surprisingly, DxR/30 cells retained their capacity to proliferate even in the presence of drug (5 days) without entering in slow cycling stage like CD24+/DxR, confirming that DxR/30 cells have acquired a new identity. After 12 days of treatment, they have re-colonized the plastic dishes (Fig 5f). One of the hypothesis is that DxR/30 cells might have a competitive advantage over naïve cells under drug stress due to their constitutive phosphorylation of p38. Finally, we evaluated the migratory capacity of these cells by using the in vitro wound healing assay based on the creation of an artificial gap, so called "scratch", on a confluent cell monolayer. Images were captured every 24 hours during cell migration, the scratch was measured and a comparison of time required to close the scratch between naïve cells and DxR/30 was performed. The incubation time was determined at 48h when the faster moving cells DxR/30 were just about to close the scratch. The confirmation of the migratory capacity of both cells was made by using the software Image J (Fig 5g).
According to our data, slow-cycling cells under stress may reentry into cell cycle leading to cells that possess new properties, including higher drug resistance and higher migratory capacity, reaffirming that they have acquired a new identity.