3.1 Decitabine enhances the TNBC response to docetaxel.
The anticancer effect of DAC was shown in several studies, most of which used a high dose . As DAC at high dose rapidly induced DNA damage and cell-apoptosis, it inevitably caused substantial toxicity. To identify a suitable dose of DAC for our study, TNBC cells were treated with various concentrations of DAC. DAC treatment did not significantly decrease cell viability until the concentration exceeded 100 nM (Fig. S1A). Thus, a low dose of DAC (100 nM) was used to further study.
TNBC cells were pretreated with DAC or vehicle (veh) before DOC treatment to investigate whether DAC improves the DOC response of TNBC. Pretreatment of DAC improved the response to DOC in TNBC cells (Fig. 1A). Using flow cytometry, we observed a higher proportion of cell apoptosis in the DAC-pretreatment groups than in the control groups (Fig. 1B). Further, xenograft tumor model was used to evaluate the anticancer effect of DAC in vivo (Fig. 1C). The volume of tumors pretreated with DAC before DOC treatment barely increased during chemotherapeutic treatment, and these tumors grew much slower and smaller than the DOC group (Fig. 1D-F). These data suggested that DAC enhanced the TNBC response to DOC. Although DAC treatment moderately suppressed tumor growth (Fig. 1D), we did not observe a significantly increased proportion of cell apoptosis compared to that in the veh group (Fig. 1G), indicating that the anticancer effect of DAC treatment is independent of cell apoptosis.
3.2 Decitabine inhibits the CSC enrichment induced by DAB2IP hyper-methylation.
Several studies revealed that cells surviving after chemotherapy exhibited high CSC capacities (self-renewal ability and CD44 expression) [9, 15]. To analyze the changes of CSC population under DOC or DAC+DOC treatment, we labeled CSCs and dead cells with anti-CD44 antibodies and PI, respectively. Cells treated with DOC exhibited a higher surface expression of CD44 than the veh group, while DAC-pretreatment decreased the CD44 expression in the DOC-treated cells (Fig. 2A and Fig. S2A). Consistently, the mRNA expression of CD44 was higher in DOC-treated cells than in vehicle-treated cells, and pretreatment with DAC decreased the expression of CD44 (Fig. 2B). Although we did not observe significant changes in the expression of CD24 (Fig. S2A), another CSC-specific marker, previous studies have proven that CD44 expression is adequate for CSC population selection . These data indicated that DOC treatment induced the enrichment of CD44hi population which could be significantly inhibited by DAC treatment. Further, we established DOC-resistant (DOC-R) cell lines (Fig. S2B-C). Compared with DOC-naive (DOC-N) cells, DOC-R cells exhibited higher proportion of CD44hi cells and increased CD44 expression (Fig. 2C-D and Fig. S2D). The DAC treatment attenuated the CD44hi population enrichment and the elevated CD44 expression in DOC-R cells (Fig. 2C-D and Fig. S2D). Using tumor-sphere assay, we observed a higher self-renewal potential in DOC-R cells than in DOC-N cells (Fig. 2E). Importantly, low-dose DAC treatment significantly suppressed self-renewal in DOC-R cells (Fig. 2E).
We further investigated what drives CSC enrichment and how DAC inhibits CSC enrichment. Previous studies suggested that Ras, a driver gene for human cancers, plays a causal role in CSC enrichment, and RAS GAPs are negative regulators for Ras activity [9, 16, 17]. Thus, we examined whether loss of RAS GAPs caused CSC enrichment. We compared the mRNA expression of 13 RAS GAP genes in suspension-cultured TNBC cells with that in attachment-cultured cells. IQGAP2, IQGAP3 and DAB2IP were consistently silenced in suspension-cultured cells (Fig. 2F). DAB2IP was aberrantly silenced in several cancers which caused activation of Ras, and IQGAP 2/3, although do not exhibit RAS GAP activity, were shown to affect the Ras signaling through direct association with Rac1. Further, we collected 10 paired normal mammary and breast cancer tissues and observed that the mRNA level of DAB2IP was significantly decreased in 9/10 tumor tissues Compared to the respective expression levels in normal tissues (Fig. S2E-G). The above findings were also supported by TCGA BRCA dataset (Fig. S2H). Furthermore, we observed a higher DAB2IP protein expression in attachment-cultured cells than in suspension-cultured spheroids (Fig. 2G-H). The expression of DAB2IP negatively correlated with the size of spheroids indicating that the expression of DAB2IP might be negatively correlated with self-renewal capacity in TNBC (Fig. 2H). Further, GSEA showed that DAB2IP expression was positively correlated with a methylated gene signature (Fig. S2I). Using the CCLE database, we observed that the DAB2IP-expression negatively correlated with the DNA-methylation level in breast cancer cell lines (Fig. S2J).
DAB2IP is encoded by 4 most commonly seen transcripts (Supplementary table 3). Although transcription of the four transcripts is regulated by different promoters, 4 functional domains (including C2, RAS GAP, Pleckstrin homology (PH) and DUF3498 Domain) are similarly contained in 4 protein isoforms indicating that all these protein isoforms have the potential to regulate the activity of RAS signaling. Primers were designed to detect methylation status according to the distribution of CpG islands in the promoter region of 4 transcripts (Supplemental table 3). Comparing to attachment-cultured cells, we observed a significantly increased methylation level at the promoter region of the ENST00000259371.6 transcript in suspension-cultured cells (Fig. 2I and Fig. S2K). In TNBC cells, DNA methylation level of DAB2IP (ENST00000259371.6) negatively correlated with mRNA expression that MDA-MB-231 exhibited the lowest level of DNA methylation and highest mRNA level of DAB2IP while HCC1806 exhibited the highest level of DNA methylation and lowest mRNA level of DAB2IP (Fig. S2L). By inhibition of DNA methylation, DAC increased the expression of DAB2IP in TNBC cells (Fig. S2M). Thus, the transcript ENST00000259371.6 is selected to further study. Further, DAC treatment significantly inhibited DNA methylation at DAB2IP (ENST00000259371.6) promoter and increased the expression of DAB2IP in suspension-cultured cells (Fig. 2J-K).
3.3 Loss of DAB2IP enhances cancer stem cell capacity through activation of Erk/β-catenin signaling in TNBC.
DAB2IP is a tumor suppressor in several cancers (breast, liver, prostate and lung cancers) and has been implicated in the regulation of tumor growth, cancer metastasis and drug resistance [11, 19]. GSEA showed that DAB2IP expression was negatively correlated with CSC-related gene signatures (Fig. S3A). To investigate the function of DAB2IP, we established stable cell lines overexpressing/silencing DAB2IP (Fig. S3B). Tumor-sphere culture showed that self-renewal potential was enhanced by DAB2IP-inhibition and inhibited by DAB2IP-overexpression in TNBC cells (Fig. 3A and Fig. S3C). Enrichment of CD44hi population was enhanced in DAB2IP-inhibition cells and attenuated in DAB2IP-overexpression cells (Fig. 3B and Fig. S3D). As key regulators of self-renewal in CSCs, the mRNA levels of Nanog and Sox2 were significantly increased in DAB2IP-silenced cells but decreased in DAB2IP-overexpressing cells (Fig. 3C and Fig. S3E).
To understand the mechanism underlying inhibition of CSC enrichment by DAB2IP, we performed GSEA and observed that DAB2IP expression was associated with repression of the Wnt-target gene signature (Fig. S3A right panel). β-Catenin, the downstream effector of canonical Wnt signaling, is necessary for CSC capacities in BC. Previous study has revealed that phosphorylation of Erk1/2, the main downstream effector of Ras, maintains the expression of β-catenin and subsequently facilitates the nuclear translocation of β-catenin . In TNBC spheroids, cells with DAB2IP-inhibition increased the expression of p-Erk1/2 and activated β-catenin (Fig. 3D). To examine whether DAB2IP-inhibition promoted the nuclear translocation of β-catenin, we stimulated TNBC spheroids with Wnt3a (250 ng/ml), which is a stimulator of β-catenin signaling. At the early phase of Wnt3a treatment (15 min), active β-catenin staining was readily detected in the cytoplasm and nucleus of cells silencing DAB2IP. In control cells, active β-catenin staining was mostly detected at the cell membrane and hardly detected in the cytoplasm and nucleus (Fig. 3D). As we prolonged the interval of Wnt3a treatment to 30 min, confocal analysis showed that active β-catenin staining could only be detected in the nucleus in cells with silenced DAB2IP, indicating the nuclear translocation of β-catenin. In contrast, active β-catenin staining was mostly detected at the cell membrane rather than in the cytoplasm and nucleus in control cells (Fig. 3D). Conversely, DAB2IP overexpression significantly weakened the staining of p-Erk1/2 and active β-catenin and further inhibited the nuclear accumulation of β-catenin in TNBC spheroids (Fig. S3F).
Next, we performed a limiting dilution assay to explore the effect of DAB2IP on the tumor-initiation capacity of TNBC cells. A higher frequency of tumor initiation was observed in cells with DAB2IP-inhibition than in the control cells (Fig. 3E). On the contrary, we observed a decreased incidence of tumor initiation in cells overexpressing DAB2IP compared to that in the control group (Fig. S3G). DAB2IP significantly suppressed the tumor initiation capacity in TNBC (Table 1).
3.4 Loss of DAB2IP induces docetaxel resistance in TNBC.
As loss of DAB2IP induced CSC enrichment, we further investigated whether loss of DAB2IP induces DOC resistance in TNBC. Compared to control cells, DAB2IP-silenced cells exhibited a higher viability accompanied by a lower proportion of cell apoptosis when treated with DOC (Fig. 4A-B). When treated with DOC, DAB2IP-silenced cells exhibited a decreased proportion of cells at the peak of the G2/M phase (Fig. 4C). In contrast, DAB2IP-overexpression significantly improved the cytotoxic effect of DOC in TNBC cells (Fig. S4A-C). Using Western blotting, we observed that DAB2IP expression was negatively correlated with the level of p-Erk1/2, while there were no significant changes in Erk1/2 expression in DAB2IP-silenced or DAB2IP-overexpressing cells (Fig. 4D and Fig. S4D). As downstream targets of Erk1/2, Akt, CCNB1 and CDK1 are key regulators of cell apoptosis and cell cycle (G2/M) transitions. In cells with DAB2IP-inhibition, we detected an increased phosphorylation level of Akt and CDK1 with no significant changes in the protein level (Fig. 4D). In addition, the protein level of CCNB1 was increased in DAB2IP-inhibition cells (Fig. 4D). In contrast, the levels of phosphorylated-Akt, phosphorylated-CDK1 and CCNB1 were decreased in DAB2IP-overexpressing cells (Fig. S4D).
In animal models, tumors with DAB2IP-inhibition grew much faster and larger than those in controls under DOC treatment, while the growth of tumors overexpressing DAB2IP was significantly suppressed by DOC treatment. (Fig. 4E-F and Fig. S4E-G). We also observed that the proportion of cell apoptosis was decreased in tumors with DAB2IP-inhibition, while an increased proportion of cell apoptosis was shown in DAB2IP-overexpression tumors (Fig. 4G and Fig. S4H).
3.5 Low dose of decitabine reverses docetaxel resistance by restoring DAB2IP expression.
We further examined whether DAC overcomes DOC resistance through re-expression of DAB2IP. In Doc-R cells, we observed that the DNA-methylation level of DAB2IP was higher and the expression of DAB2IP was lower than those in DOC-N cells (Fig. 5A and Fig. S5A). When treated with DAC, the DNA-methylation level was decreased in Doc-R cells, and DAB2IP expression was increased (Fig. 5B and Fig. S5B). In Doc-R cells, DAC+DOC treatment induced a higher proportion of cell apoptosis than DOC treatment alone (Fig. 5C). We also observed that DAC treatment could not restore the DAB2IP expression in DAB2IP-silenced cells and DAC pretreatment could not improve the response to DOC in DAB2IP-silenced cells (Fig. S5C-D). These data indicated that DAB2IP is essential for DAC to improve the DOC response in TNBC cells.
In vivo, tumors in the DAC+DOC group grew slower and smaller than the tumors in the DOC group (Fig. 5D and Fig. S5E). The weights of tumors sequentially treated with DAC and DOC were less than those of tumors treated with DOC alone (Fig. 5E). Moreover, we observed stronger DAB2IP staining in tumors treated with DAC than that in tumors without DAC treatment (Fig. 5F). Combination of DAC and DOC increased the proportion of apoptotic cells in DOC-R tumors (Fig. 5G).
3.6 Epigenetic silencing of DAB2IP predicts poor outcomes in TNBC.
Further, we investigated whether the expression or methylation status of DAB2IP acts as a prognostic marker for TNBC. We collected 304 TNBC patient tissues and detected the expression and methylation level of DAB2IP (Fig. 6A-B). Clinical and pathological features of the TNBC patients are listed in Table S1. Low expression of DAB2IP was significantly correlated with a hyper-methylation status in TNBC (Fig. 6B-C). Survival analysis showed that low expression and hypermethylation of DAB2IP correlated with poor outcome in TNBC patients (Fig. 6D). Among patients treated with DOC-based chemotherapy, both low DAB2IP expression and hypermethylation correlated with worse outcomes (Fig. 6E).