FABP7 and ESR1 are differentially expressed in breast cancer cases received NAC
We selected three GEO datasets (GSE21997, GSE32646 and GSE25055), which are genomic expression profiles for breast cancer subjects treated with neoadjuvant anthracycline and taxanes combination. Next, we compared gene expression profiles acquired by microarray analysis from subjects with or without residual tumor after neoadjuvant chemotherapy. A number of genes were identified as the potential predictors at the threshold of fold change≥2 and p-value < 0.05. As shown in Figure 1A-C, a total of 94 genes (61 up-regulated and 33 down-regulated genes) in GSE25055, 66 genes (19 up-regulated and 47 down-regulated genes) in GSE21997, and 30 genes (19 up-regulated and 11 down-regulated genes) in GSE32646 were filtered as differentially expressed Genes. The intersection identified a total of 2 differentially expressed genes, including FABP7 and ESR1, might be essential indicators of chemotherapeutic efficacy in breast cancer (Fig. 1D; Table 3).
Higher level FABP7 is correlated to better chemotherapeutic response, while higher level ESR1 is associated with poorer chemotherapeutic sensitivity
Next, we analyzed the association between either FABP7 or ESR1 mRNA level and chemotherapeutic response. It was found that mRNA level of FABP7 was considerably lower in residual tumor after NAC (GSE21997: p=0.0264; GSE32646: p=0.0075; GSE25055: p=0.0004) (Fig. S2A, S2C and S2E). On the contrary, the mRNA level of ESR1 was much higher in residual tumor after NAC (GSE21997: p=0.0166; GSE32646: p<0.0001; GSE25055: p<0.0001) (Fig. S2B, S2D and S2F). These results suggest that FABP7 might be associated with better chemotherapeutic response, while ESR1 might be related to poorer chemotherapeutic sensitivity.
The expression of FABP7 is significantly lower in breast cancer than normal tissue samples
The analysis in ONCOMINE database demonstrated that the mRNA level of FABP7 was significantly lower in breast cancer than normal-tissue samples across a series of datasets in multiple cancer types (Fig. S3A). The FABP7 mRNA expression in breast cancer samples was lower than that in normal tissues (Fold changes were –21.383, p=2.66E-6 or –8.265, p=3.37E-9) (Fig S3B-C). On the contrary, ESR1 mRNA level was 4.032-fold (p=3.37E-9) increased in breast cancer samples compared with normal tissue samples in Curtis breast statistics (Fig. S3D-S3E). Similar trend (Fold changes were 4.931, p=7.58E-5) was found in The Cancer Genome Atlas (TCGA) breast statistics.
FABP7 is particularly high expressed in ER-negative breast tumor and negatively associated with ESR1, GATA3 and FOXA1
In bc-GenExMiner v4.0, the mRNA level of FABP7 in basal-like subtype tumors was significantly higher than non-basal-like subtype counterparts (Fig. S4A). Similarly, the mRNA level of FABP7 was found significantly higher in triple-negative breast cancer (TNBC) than non-TNBC (Fig. 2A). Moreover, the highest FABP7 expression was observed in basal-like subtypes of breast cancer, while the lowest expression of FABP7 was found in the luminal subtypes (Fig. S4B). The between-group comparisons were shown in Supplementary Table 4. Higher FABP7 mRNA levels were found in patients with ER-negative than ER-positive tumors (Fig. 2B). Gene correlation targeted analysis indicated that higher expression of FABP7 in mRNA level was correlated to lower mRNA level of ESR1 (r=−0.42, p<0.001) (Fig. 2C), GATA3 (r=−0.46, p<0.001) (Fig. 2D) and FOXA1 (r=−0.41, p<0.001), which were typical epithelial biomarkers (Fig. 2E). Correlation maps for all patients among FABP7, ESR1, GATA3 and FOXA1 were showed (Fig. 2F). These results suggested that the mRNA level of FABP7 is particular higher in ER-negative breast tumor than other subtypes and was negatively associated with the mRNA levels of ESR1, GATA3 and FOXA1.
DEGs were mainly involved in cell cycle and drug response
The cBioPortal for Cancer Genomics database (TCGA, provisional) was used to analyze the DEGs (|log Ratio|≥1 and P-value < 0.05) in breast cancer patients with or without FABP7 alterations, which was drawn with online tool (https://paolo.shinyapps.io/ShinyVolcanoPlot/) (Fig. 3A). The gene ontology (GO) enrichment analysis was conducted to identify the functional differences of DEGs and they were classed into three functional groups, including Molecular function (MF), Cellular Component (CC), and Biological Process (BP). The genes in the MF group were primarily enriched in heparin binding and calcium ion binding (Fig. 3B); the genes in the CC group were considerably enriched in cell body fiber, cell body fiber and extracellular exosome (Fig. 3C). The genes in the BP group were predominantly enriched in cell cycle regulation, cellular response to estradiol stimulus, as well as response to drug and cell proliferation (Fig. 3D). These results indicated that these DEGs might mainly be involved in cell cycle and drug response.
Doxorubicin-resistant MDA-MB-231 cells lowly express FABP7 and tend to arrest cell cycle at G0/G1 phrase compared with parental MDA-MB-231 cells
To verify the bioinformatic analysis result, we performed western-blot assay and RT-PCR experiments. The results showed that FABP7 expressed significantly higher in ER-negative subgroups than ER-positive counterparts (Fig. 4A and Fig. 4B), which were consistent with our primary hypothesis. To examine whether the acquisition of doxorubicin resistance is accompanied by morphological changes, we observe the differences in cell morphology. As shown in Fig.4C, MDA-MB-231-ADR cells exhibited rounded morphology and was more likely to cluster, compared to MDA-MB-231 cells, they were elongated spindle. We speculated that MDA-MB-231-ADR cells exhibit decreased mesenchymal phenotype but rather, the Epithelial phenotype.
To verify doxorubicin resistance in the MDA-MB-231-ADR cells, we treated both parental and MDA-MB-231-ADR cells with concentration gradient of doxorubicin and then determined the IC50 value with CCK8 assay (Fig. 4D). According to our data, the sensitivity to doxorubicin of MDA-MB-231-ADR cells was markedly lower as compared to the parental cells. Next, we performed RT-PCR and western blotting assays (Fig. 4E and 4F) to determine the expression of FABP7 and ESR1 in both mRNA and protein levels. We found that the expression in both protein and mRNA levels of FABP7 was significantly lower, which was negatively correlated with ESR1 in the MDA-MB-231-ADR cells than in the parental cells. Moreover, the FABP7 expression was also lower in MDA-MB-231 breast cancer cells treated with doxorubicin than in the control group (Fig. S5).
Moreover, we examined whether the two cell lines (MDA-MB-231 and MDA-MB-231-ADR) influenced cell cycle. Therefore, we performed western blotting and flow cytometer assays to validate the expression level of relevant protein and DNA. As our dates shown, MDA-MB-231-ADR cells compared to parental cells, were more likely to arrest the cell cycle at G0/G1 phase (Fig. 4G). Next, we detected several vital proteins related to the G0/G1 phase, such as Cdh1, Skp2 and p27kip1. The Western blotting results showed that the expression of Cdh1 and p27kip1 were up-regulated while that of Skp2 was down-regulated in MDA-MB-231-ADR cells (Fig. 4F). These data suggest that FABP7 was negatively relative to ESR1 in doxorubicin resistance breast cancer cells. Furthermore, the sensitivity of cells to chemotherapy drugs is related to the cell cycle regulation. Therefore, we hypothesized that FABP7 could predict the drug sensitivity in breast cancer cells through regulating the cell cycle.
FABP7 enhances drug sensitivity by promoting G1/S transition in cell cycle.
We have found that FABP7 was negatively relative to ESR1 expression in MDA-MB-231-ADR cells. To further study the regulating relationship between FABP7 and ESR, we over-expressed FABP7 in MDA-MB-231-ADR cells by transient transfection of PCMV-FABP7 and we transfected small interfering RNA FABP7(siFABP7) or siNC in parental MDA-MB-231 cells. As shown in Supplement Figure 6 and Figure 5A, overexpression of FABP7 could reduce the expression of ESR1 in MDA-MB-231-ADR in both mRNA and protein level. While silencing FABP7 in parental ER negative breast cancer cells, ESR1 expression increased (Fig. 5D).
Furthermore, to investigate whether the over-expression of FABP7 could make a difference in regulating cell cycle, we compared the PCMV-FABP7 and PCMV in MDA-MB-231-ADR cells with western blotting and flow cytometry assays. Our data showed that, compared with control MDA-MB-231-ADR, the high expression of FABP7 decreased the percentage of cells at G0/G1 phrase and increased the percentage of cells at S and G2 phrase (Fig. 5B). Considering the high expression of FABP7 might promote the transition of G1 to S phrase, we examined several related proteins associated with cell cycle with western blotting assays. Our results showed that Cdh1, p27kip1 were decreased while the expression of Skp2 was up-regulated (Fig. 5A). When we silenced the expression of FABP7 in ER negative breast cancer MDA-MB-231 cells, we found exactly the opposite results (Figure 5D), Cdh1 and p27kip1 increased expression, while the Skp2 was up-regulated.
Next, we determined whether the expression level of FABP7 correlated with doxorubicin resistance. We transfected PCMV-FABP7 into MDA-MB-231-ADR cells and siFABP7 into MDA-MB-231 cells. We subsequently measured IC50 value with CCK8 assay to determine their sensitivity to doxorubicin. Our data showed that over-expression of FABP7 could increase the sensitivity of MDA-MB-231-ADR cells to doxorubicin, while silencing FABP7 would decrease the sensitivity of MDA-MB-231 cells to doxorubicin (Fig. 5C and Fig. 5F). Taken together, these data suggest that overexpression of FABP7 increases doxorubicin sensitivity in MDA-MB-231-ADR cells. On the other hand, inhibition of endogenous FABP7 in MDA-MB-231 cells induces doxorubicin resistance. Besides, the expression level of FABP7 could affect cell cycle progress. Thus, FABP7 might enhance drug sensitivity by regulating the cell cycle process.
Increased FABP7 was linked to longer recurrence-free survival (RFS) in BC subjects treated with adjuvant chemotherapy, particularly in those with ER-negative subtype of BC
Survival analysis demonstrated that higher mRNA level of FABP7 was closely linked to longer RFS in all BC subjects (HR=0.64, p=1.1E-14) (Fig. 6A). Subgroup analysis suggested that higher FABP7 mRNA level was significantly related to better RFS in subjects with ER-positive (HR=0.8, p=0.023) (Fig. 6B), ER-negative (HR=0.63, p=0.00017) (Fig. 6C), basal-like (HR=0.48, p=9e-09) (Fig. 6D), Luminal-A (HR=0.63, p=1.6e-07) (Fig. 6E), Luminal-B (HR=0.53, p=8.5e-07) (Fig. 6F) and Her-2 positive tumors (HR=0.62, p=0.029) (Fig. 6G). These results suggest that FABP7 is a strong predictor of favorable prognosis in patients with ER-negative breast cancer.
Moreover, higher expression of FABP7 in mRNA level was significantly correlated to better RFS in subjects who have received treatments including either chemotherapy (HR=0.71, p=0.015) (Fig S7A) or neoadjuvant chemotherapy (HR=0.5, p=0.022) (Fig. S7D). Of noteworthy, higher mRNA level of FABP7 is linked to better RFS only in patients with ER-negative tumor treated with chemotherapy (Fig. S7C) and adjuvant chemotherapy (Fig. S7F), but not in ER-positive breast cancer patients (Fig. S7B and S7E). These results indicate that elevated mRNA level of FABP7 predicts longer RFS in patients with ER-negative subtype of BC treated with chemotherapy or adjuvant chemotherapy.