BCSCs require RAC signaling for their self-renewal maintenance
We have previously shown that Rac1 is required for MaSC self-renewal . In order to elucidate whether RAC signaling is also required for BCSC activity, we used two specific RAC-inhibitors with different modes of action in the mammosphere culture of human breast cancer cell lines that are known to generate proliferation-driven mammospheres . These lines represent different breast cancer subtypes: Luminal-A (MCF7 and T47D), Luminal-B (BT474), and HER2+ (JIMT-1). The inhibitors render RAC proteins in a nucleotide-free inactive state (EHT-1864) or prevent the RAC activation by GEFs (EHop-016) [29, 30]. Interestingly, the mammosphere-forming ability of these cell lines was completely abrogated in the presence of either EHT-1864 or EHop-016 (Figure 1A,B).
Mammosphere formation requires an initial self-renewing division of the stem cell, followed by consecutive rounds of proliferation of their non-stem cell progeny generating all other cells within the mammosphere . Therefore, we asked whether RAC inhibition results in depletion of BCSCs or inhibition of cell proliferation, both of which could prevent mammosphere formation. To address this question, we treated MCF7 cells with RAC inhibitors in the primary mammosphere culture for 5 days and then performed a secondary mammosphere formation assay in the absence of inhibitors. If the effect of RAC inhibition on primary mammosphere formation is due to inhibition of cell proliferation, BCSCs would be able to initiate the secondary mammosphere formation when RAC inhibitors are removed. Our results demonstrated that RAC-inhibited MCF7 cells did not form any secondary mammospheres despite the absence of RAC inhibitors (Figure 1C). This suggests that RAC inhibition results in BCSC depletion.
We also tested whether RAC inhibition affects the proliferation of the non-stem cell progeny of BCSCs. To this end, we added RAC inhibitors to the mammosphere culture of MCF7 cells at the time of plating (0 hours) or 24 hours after plating. Our results showed that the effect of RAC inhibition is restricted to the initial cell divisions of BCSCs that take place within the first 24 hours of culture, whereas the proliferation of non-stem cell progeny of BCSCs does not rely on RAC signalling (Figure 1D). At higher concentrations of RAC inhibitors, there was a cell-shedding phenotype rather than a slow growth of mammospheres. A similar cell-shedding phenotype also occurs when fully formed mammospheres at Day 5 of culture are treated with high concentrations of these inhibitors (data not shown), suggesting a potential inhibition of cell-cell adhesion in the presence of higher concentrations of inhibitors.
Since BCSCs play essential roles in breast tumorigenesis, we examined whether Rac1 is required for breast tumorigenesis in vivo. We generated a double transgenic mouse line bearing floxed-Rac1 allele  and MMTV-Neu-IRES-Cre (NIC) transgene , which allows genetic deletion of Rac1 in the same cells that overexpress Neu oncogene. Tumor latency analysis reveals that heterozygous deletion of Rac1 in Rac1flox/+;MMTV-NIC mice significantly delays the palpable tumor formation compared with Rac1+/+;MMTV-NIC mice (Figure 1E). Although we were able to obtain only two female Rac1flox/flox;MMTV-NIC mice, only one of them has developed palpable tumors during its first year of age (Figure 1E). Since Rac1 is indispensable for early-stage embryogenesis , it is likely that leaky expression from the MMTV promoter during early embryogenesis led to Rac1 deletion and thus embryonic lethality in most of the Rac1flox/flox;MMTV-NIC embryos.
These results reveal that RAC signalling is required for the self-renewal maintenance of BCSCs in vitro, and that loss-of Rac1 function delays or supresses breast tumorigenesis in a dose-dependent manner in vivo.
RAC1B influences BCSC plasticity
Since RAC inhibitors and Cre-mediated genomic deletion of Rac1 result in the loss of both RAC1 and RAC1B functions [35, 36], we decided to investigate to which extent the observed phenotypes would be recapitulated by targeting only RAC1B. Initially, we analysed whether RAC1B is expressed in human breast cancer cell lines, murine mammary gland, and breast tumors of MMTV-NIC mice (Supplementary Figure 1). RAC1B mRNA and protein are present in ER+ cell lines, and low levels of RAC1B protein was also observed in the HER2+ cell line JIMT1. In contrast, RAC1 mRNA and protein are present in all 5 cell lines tested, though RAC1 protein levels are higher in HER2-overexpressing cell lines BT474 and JIMT1. In mice, Rac1b mRNA was detected predominantly in the basal mammary epithelial cells at adult nulliparous and early-pregnancy stages, and in the MMTV-NIC tumor cells (Supplementary Figure 1).
To determine whether variant-specific loss of RAC1b affects BCSCs, we employed CRISPR/Double-nickase method to target the exon3b-encoding genomic sequence in MCF7 cells (Figure 2A). This was followed by single-cell cloning to ensure the genomic homogeneity of clones for further phenotypic analyses. We obtained several single-cell clones that specifically lacked RAC1b mRNA (Figure 2B) and protein (Figure 2C). Sequencing of the targetedgenomic locus in these clones revealed distinct insertion/deletion (indel) mutations in each allele of each clone (Figure 2A). Interestingly, even small deletions within the exon3b-coding sequence resulted in the loss of RAC1B mRNA, suggesting that those deletions may have disrupted the splicing-regulatory sequences required for RAC1B splicing.
We found that the loss of RAC1B function in these MCF7 clones did not alter their mammosphere-forming capacity (Figure 2D), although it caused a significant increase in the frequency of their Aldefluorbright BCSC population as determined by flow cytometry (Figure 2E). To address whether gain of RAC1B function leads to an inverse phenotype, we generated stable transgenic MCF7 clones with doxycycline-inducible expression of RFP-RAC1B fusion protein (Figure 2F). Similar to the RAC1B-null MCF7 clones, the RAC1B overexpression did not alter MCF7 mammosphere-forming capacity (Figure 2G). However, it resulted in a significant increase in the CD44+;CD24- BCSC population (Figure 2H).
Earlier studies have described Aldefluorbright and CD44+;CD24- populations in MCF7 cells as the proliferative epithelial-like and quiescent mesenchymal-like states of BCSCs, respectively, and suggested that the ability to reversibly transit between these states underlies the plasticity within the BCSC pool [37, 38]. Our results therefore suggest that RAC1B regulates the reversible switching between the proliferative versus quiescent states of BCSCs without altering the total BCSC numbers and it is likely to be required for the quiescent BCSC state.
RAC1B function is essential for chemoresistance and in vivo tumor initiating ability of MCF7 cells
Resistance to chemotherapy is a feature often attributed to CSCs. As cytoablative treatments specifically target proliferating cells, quiescent CD44+;CD24- BCSCs are likely to constitute the chemoresistant population of tumor cells. Given that RAC1B function might be required for this particular BCSC subpopulation, we hypothesised that RAC1B may have a crucial role in chemoresistance. We therefore determined the effect of doxorubicin on RAC1B-null and RAC1B-overexpressing MCF7 cells. We treated these cells with 2.5 uM doxorubicin for 24 hours, which led to more than 90% of cell loss, and then measured the recovery as cell growth in the absence of doxorubicin. Parental MCF7 and RAC1B-proficient MCF7 clone (Clone-22) showed a slow but steady recovery during the five-day period after doxorubicin removal (Figure 3A). In contrast, RAC1B-null MCF7 clones did not recover during the same period (Figure 3A) nor up to 3 weeks post-treatment (data not shown). Conversely, the RAC1B-overexpressing cells showed a robust recovery upon doxorubicin withdrawal (Figure 3B) compared with the same cells not treated with doxycycline to induce RAC1B overexpression. These results indicate that RAC1B function plays a crucial role in the chemoresistance of MCF7 cells in vitro, possibly through its effect on BCSC plasticity.
Since impaired BCSC plasticity may alter tumorigenesis, we investigated whether RAC1B is required for tumor-initiating ability of BCSCs in vivo. Xenograft transplantation of parental MCF7 cells resulted in visible tumor formation within 6–7 weeks (Figure 3C). In contrast, RAC1B-null MCF7 clones formed no visible tumors, even up to 100 days post-transplantation. At the experimental endpoint (either maximum tumor burden of 1.25 cm3 or 100 days post-transplantation), tumors/tissues at the site of transplantation were dissected and analysed for the human-origin cells using human-specific antigen CD298 expression by flow cytometry (Figure 3D). Surprisingly, explants obtained from mice transplanted with RAC1B-null MCF7 clones still contained some CD298+ cells, despite the absence of tumor growth. However, unlike parental MCF7 cells recovered from xenograft tumors, RAC1B-null MCF7 cells sorted as CD298+ population from the explants formed neither mammospheres nor monolayer colonies (Figure 3E,F). These results indicate that RAC1B is indispensable for BCSC self-renewal and tumor growth in vivo.
Taken together, our results revealed that RAC1B is essential for BCSC plasticity and chemoresistance of MCF7 cells in vitro, and for BCSC maintenance and tumor-initiating ability in vivo.
Loss of Rac1b function does not alter mammary gland development
Rac1 is indispensable for mammary gland development and function, particularly in MaSCs in nulliparous animals, lobuloalveolar development during pregnancy and tissue remodelling during involution [18, 39-42]. Since the Rac1flox/flox mouse line used in these studies resulted in the loss of both Rac1 and Rac1b, we generated a Rac1b-/- mouse line to identify Rac1b-specific loss-of function phenotypes. In both C57BL/6 and FVB backgrounds, Rac1b-/- mice were born with expected Mendelian ratios and had a normal life span with no apparent health problems.
To determine whether the loss of Rac1b function hampers mammary gland development, we performed whole-mount staining on No4 inguinal mammary glands of both Rac1b-/- mice and their wild-type littermates at different postnatal developmental stages. During pubertal stages, there were no macroscopically obvious differences in ductal outgrowth or ductal branching in 4-, 6-, 8-, and 10-week-old Rac1b-/- mice (Figure 4A,B). Similarly, Rac1b-/- mammary glands were indistinguishable from Rac1b+/+ glands in early and late pregnancy, lactation, and involution stages (Figure 4C,D).
Next, we evaluated whether the loss of Rac1b function affects mammary epithelial lineage diversification and/or MaSC activities. Basal (CD49fhigh;CD24low) and luminal (CD49flow;CD24high) epithelial cell populations showed a similar distribution within the glands of 8-week-old nulliparous Rac1b-/-, Rac1b+/- and Rac1b+/+ mice as determined by flow cytometry (Supplementary Figure 2A-C). When sorted and plated in mammosphere culture, Rac1b-/- luminal and basal epithelial populations were indistinguishable from their Rac1b+/+ counterparts in terms of luminal progenitor-driven acini and MaSC-driven mammosphere formation, respectively (Supplementary Figure 2D,E). These results indicate that Rac1b function is dispensable for luminal progenitor and MaSC activities.
Together, our data demonstrate that mammary gland phenotypes of Rac1-null mice [18, 39, 40] are due to the loss-of-function of Rac1, but not Rac1b. Importantly, Rac1b deficiency does not lead to any obvious alterations in MaSCs or defects in normal mammary gland development.
Rac1b expression marks a substantial subset of BCSCs and is required for BCSC maintenance
Dual loss of Rac1 and Rac1b functions in MMTV-NIC mouse model delays tumor latency in a dose-dependent manner (Figure 1E). To determine whether loss of Rac1b is responsible for the observed tumor latency phenotype, we analysed the impact of Rac1b deficiency on palpable tumor formation using MMTV-NIC mouse model. Our results revealed that the tumor latency in Rac1b-/-;MMTV-NIC or Rac1b+/-;MMTV-NIC mice is similar to Rac1b+/+;MMTV-NIC mice (Figure 5A). This indicates that the delayed tumor latency phenotype observed in Rac1flox/flox;MMTV-NIC and Rac1flox/+;MMTV-NIC mice is due to the loss of Rac1, not Rac1b.
Since the loss of RAC1B in MCF7 cells results in BCSC depletion in vivo (Figure 3F), we examined whether Rac1b is also required for BCSCs in the MMTV-NIC mouse model. We performed mammosphere assay using CD49f+CD24+ tumor cells isolated from Rac1b+/+;MMTV-NIC (or Rac1b+/-;MMTV-NIC) and Rac1b-/-;MMTV-NIC tumors (Figure 5B,C). Our results revealed a 65% decrease in overall mammosphere-forming BCSC frequency in Rac1b-null tumors compared with Rac1b-proficient tumors.
To identify whether BCSCs express Rac1b in the MMTV-NIC tumors, we aimed to generate a transgenic mouse line serving as a surrogate reporter for Rac1b splicing. To this end, we utilised CRISPR-targeting approach, coupled with homology-directed repair (HDR) template, to knock-in a T2A-mRFP cassette in-frame within the exon3b of Rac1 gene. First, we used the murine mammary epithelial cell line, EPH4, to optimise the HDR template design for achieving a successful knock-in without disrupting proper splicing of the transgenic mRNA. Single-cell EPH4 clones, generated after HDR template-coupled CRISPR targeting using three different HDR templates, were genotyped (Supplementary Figure 3A,B) and subsequently verified by sequencing. Flow cytometry analysis of these clones revealed that the insertion of the T2A-mRFP cassette in the middle or at the 5’- end, but not at the 3’-end, of exon3b results in a population of 5–6% of cells expressing mRFP (Supplementary Figure 3C). Importantly, Rac1 expression in these clones was not altered (Supplementary Figure 3D). We then used the HDR-template option-B (knocking-in the T2A-mRFP cassette into the middle of exon3b) for HDR-coupled CRISPR targeting of fertilised mouse embryos. This provided us a new transgenic mouse line, Rac1bRFP/+. RT-PCR analysis of RFP+ and RFP- cells sorted from mammary glands of nulliparous Rac1bRFP/+ mice confirmed that mRFP expression in this mouse line is a valid surrogate reporter for Rac1b splicing (Supplementary Figure 3E,F).
To determine whether Rac1b is expressed by BCSCs, we crossed Rac1bRFP/+ and MMTV-NIC mouse lines and analysed the RFP+ cells from the mammary tumors of double transgenic animals by flow cytometry and mammosphere assay. The RFP+ (i.e. Rac1b-expressing) cells from Rac1bRFP/+;MMTV-NIC tumors constituted a small population of lineage (CD31, CD45, TER119)-negative cells (Figure 6A), which displayed a 4-fold enriched frequency of mammosphere-forming cells compared with the Lin-RFP- population (Figure 6B and Supplementary Figure 4A). Immunostaining of primary mammospheres formed by the Lin-RFP+ cells isolated from Rac1bRFP/+;MMTV-NIC tumors revealed that most of these mammospheres (~90%) were composed of cells expressing CK18 luminal and/or CK14 basal epithelial lineage markers (Figure 6C). These results demonstrate that a substantial subset of BCSCs in MMTV-NIC tumors express Rac1b.
To further define the composition of RFP+ cell populations in Rac1bRFP/+;MMTV-NIC tumors, we immunostained the sorted Lin-RFP+ cells for CK18 and CK14 (Supplementary Figure 4B). Our results revealed that an average of 79.3% of Lin-RFP+ cells were expressing CK18, whereas 2.7% were positive for both CK14 and CK18 (Figure 6D). Furthermore, the flow cytometry analysis showed that an average of 84% of the Lin-RFP+ cells from Rac1bRFP/+;MMTV-NIC tumors were also CD24+ (Figure 6E). These results indicate that in MMTV-NIC tumors Rac1b is expressed in a small population of tumor epithelia that also contains a substantial subset of BCSCs.
Next, we analysed Rac1b-proficient (Rac1bRFP/+;MMTV-NIC) and Rac1b-null (Rac1bRFP/-;MMTV-NIC) tumors to determine whether the observed decrease in overall BCSC pool in Rac1b-/-;MMTV-NIC tumors is due to a change in RFP+ BCSCs. In both genotypes, Lin-RFP+ cells were detected with similar frequency (Figure 6F). However, there were approximately 42% fewer mammosphere-forming BCSCs in the Lin-RFP+ population of Rac1b-null tumors compared with the same population in Rac1b-proficient tumors (Figure 6G). In contrast, mammosphere-forming efficiency of Lin-RFP- cells did not show a significant difference between genotypes.
Collectively, our results demonstrate that Rac1b is expressed in a substantial subset of BCSCs, which requires Rac1b function for their maintenance in vivo.
Loss of Rac1b increases the chemosensitivity of primary breast tumor cells.
Rac1b function is required for the chemoresistance of MCF7 cells (Figure 3A). To investigate whether Rac1b also affects chemoresistance of Neu-driven tumors, we generated primary cell lines from Rac1b+/+;MMTV-NIC and Rac1b-/-;MMTV-NIC tumors and treated them with either 1 uM or 2.5 uM doxorubicin for 24 hours. At both concentrations of doxorubicin, the relative cell loss was significantly higher in Rac1b-null lines compared with Rac1b-proficient lines after 24-hour treatment (Figure 7; Day 0 samples), demonstrating an increased cytotoxic response of Rac1b-null tumor cells to doxorubicin.
The sustained cytotoxic effect of doxorubicin during the first 4 days of recovery after the removal of chemotherapeutic agent was observed in both genotypes but showed a significantly higher cell loss in Rac1b-null lines for the 1uM doxorubicin-treated groups (Figure 7; Day 4 samples). Starting from Day 8 of the recovery period, most samples showed an increase in cell growth. However, the mean values for Rac1b-null lines was consistently lower than the Rac1b-proficient lines during the whole period of recovery, despite not displaying a statistical significance between genotypes due to the heterogeneity of recovery-response by individual cell lines of same genotypes (Supplementary Figure 5). Interestingly, 3 out of 4 Rac1b-null lines treated with 2.5 uM doxorubicin did not show an increase in cell numbers between Day 8 and Day 28 of recovery, whereas only 1 out of 4 Rac1b-proficient lines showed a lack of recovery during the same period (Figure 7C). These results imply that, even at lower concentrations, doxorubicin treatment achieves a higher level of cytotoxic effect in Rac1b-null tumor cells, whereas at higher doxorubicin concentrations the recovery from the chemotherapy treatment is less likely in Rac1b-null tumor lines compared with Rac1b-proficient tumor lines.
These data demonstrate that Rac1b function is required for chemoresistance of primary breast tumor cells similar as observed in MCF7 cells. This reveals that a future strategy for treating breast cancers can involve the combined use of chemotherapeutic agents together with Rac1b-selective inhibitors.