C/EBPβ regulates the JAK/STAT signaling pathway in triple‐negative breast cancer

C/EBPβ is a member of the CCAAT/enhancer‐binding protein (C/EBP) family, which consists of a number of b‐ZIP transcription factors. Although C/EBPβ has been implicated in the development of certain cancers, including breast cancer, it remains unknown whether dysregulation of C/EBPβ in breast cancer is subtype‐specific. Moreover, the underlying mechanisms by which C/EBPβ regulates breast cancer carcinogenesis are not fully understood. Here, we present evidence that C/EBPβ is specifically overexpressed in human TNBC samples, but not in non‐TNBC samples. C/EBPβ depletion dramatically suppressed TNBC cell growth, migration, invasion, and colony formation ability. A subsequent mechanistic study revealed that the JAK/STAT signaling pathway was upregulated in C/EBPβ_high TNBC samples compared with C/EBPβ_low TNBC samples. C/EBPβ ChIP‐seq and qPCR were performed to demonstrate that C/EBPβ directly binds to and regulates JAK/STAT signaling pathway genes in TNBC. Taken together, our data indicate the oncogenic role of C/EBPβ in human TNBC and reveal a novel mechanism by which C/EBPβ promotes TNBC carcinogenesis.

and thoroughly studied in triple-negative breast cancer (TNBC), and the underlying mechanisms remain largely unknown.
In the present study, we attempted to dissect the potential role of C/EBPb specifically in TNBC and to understand the underlying mechanisms.

Materials and methods
Data source mRNA expression data and clinical phenotype data of breast cancer from The Cancer Genome Atlas (TCGA) were downloaded from the UCSC Xena TCGA Hub. This included 1097 primary breast cancer samples and 113 normal breast tissue samples. According to the clinical phenotypes, 122 cases were TNBC and 617 cases were non-TNBC, whereas the remaining cases had at least one of the progesterone receptor (PR) status, ER status, and HER-2 status missing.

Cell culture and RNAi
The TNBC cell line BT549 was originally purchased from ATCC. BT549 cells were cultured in RPMI 1640 medium (Invitrogen, Invitrogen, CA, USA) supplemented with 10% FBS. The shRNAs targeting C/EBPb and the control scrambled sequence were as follows: C/EBPb-sh1, GCCGC CGCCTGCCTTTAAATC; C/EBPb-sh2, GCCCTGAGTA ATCGCTTAAAG; shControl, TTCTCCGAACGTGTCA CGT. Lentivirus preparation was performed as previously described [20]. The lentivirus was then used to infect BT549 cells to knock down C/EBPb expression followed by selection using puromycin.

Quantitative real-time PCR
RNA extraction, reverse transcription, and quantitative real-time PCR were carried out as previously described [20].
EdU incorporation assay and clonogenicity formation assay BT549 cells were seeded at a density of 3 9 10 3 cells per well in 96-well plates and were incubated overnight. The media was prepared using EdU (5-ethynyl-2'-deoxyuridine) for a final concentration of 50 lM, and the plates were incubated in a cell culture incubator for 2 h. The EdU incorporation assay was then performed using an EdU In vitro Imaging Kit (Ribo Bio, Guangdong, China) following the manufacturer's protocol. The tumor cell colonyforming ability was evaluated as previously described [20].

Wound-healing assay and Transwell cell invasion assay
Wound-healing and Transwell cell invasion assays were performed as previously prescribed [20].

Gene Set Enrichment Analysis
C/EBPb expression in TNBC was divided into C/EBPb_high and C/EBPb_low expression groups using the median value. For the Gene Set Enrichment Analysis (GSEA), upregulated genes in the C/EBPb_high group were used with the clusterProfiler R package.

C/EBPb expression was dysregulated in human triple-negative breast cancer
The study was designed to investigate the potential regulatory role of C/EBPb in TNBC. To this end, we first compared C/EBPb expression levels in TNBC samples (n = 122) versus non-TNBC samples (n = 617) using data obtained from the UCSC Xena data hub. We found that the C/EBPb was aberrantly overexpressed (more than twofold) in TNBC compared with non-TNBC samples (Fig. 1A). Moreover, the C/EBPb mRNA level in TNBC was also significantly higher than that in normal breast tissue samples (Fig. 1B). We also assessed the C/EBPb expression in TNBC using another dataset (GSE58135) and observed a similar overexpression pattern in TNBC samples compared with the noncancerous adjacent samples (Fig. 1C). More importantly, we also observed a significant association between C/EBPb expression and poor survival probability among TNBC patients, indicating the promising prognostic value of C/EBPb in TNBC (Fig. 1D). Taken together, these results indicate that C/ EBPb may play a promoting role specifically in TNBC.

C/EBPb regulates TNBC cell proliferation, colony formation, migration, and invasion
To directly assess the regulatory role of C/EBPb in TNBC, we knocked down C/EBPb expression in BT549 cells. Knockdown efficiency was evaluated by real-time quantitative PCR and western blotting ( Fig. 2A). On the other hand, the EdU proliferation assay was used to assess the regulatory role of C/EBPb in TNBC cell proliferation. As shown in Fig. 2B,C, C/ EBPb knockdown significantly inhibited TNBC cell proliferation. Next, we assessed the effect of C/EBPb knockdown on TNBC cell colony formation using a clonogenic assay. The results showed that C/EBPb knockdown dramatically impaired the colony formation ability of TNBC cells (Fig. 2D,E). To further elucidate the role of C/EBPb in TNBC, we also investigated the regulatory role of C/EBPb in TNBC cell migration and invasion, which are both important processes in breast cancer metastasis. To this end, wound-healing and Transwell cell invasion assays were performed. As shown in Fig. 3A,B, we observed slower migration of TNBC cells upon C/ EBPb knockdown. Furthermore, the Transwell cell invasion assay results demonstrated that C/EBPb knockdown dramatically impaired the invasiveness of TNBC cells (Fig. 3C,D). These results together support the conclusion that C/EBPb plays a role in promoting TNBC.

C/EBPb regulates JAK/STAT signaling pathway genes in TNBC
We next explored the underlying mechanisms by which C/EBPb promotes TNBC carcinogenesis. To this end, we divided the TNBC cases into two groups, the C/ EBPb_high group and the C/EBPb_low group, based on the median C/EBPb expression level in TNBC.
Then, we compared the two groups and identified the differentially expressed genes (DEGs) using the DEse-q2 R package. The gene expression patterns between the C/EBPb_high group and the C/EBPb_low group were evidently distinct (Fig. 4A,B). The upregulated genes in the C/EBPb_high group were then used in the GSEA analysis, and the results indicate that the JAK/ STAT signaling pathway genes were enriched and upregulated in the C/EBPb_high group compared with the C/EBPb_low group (Fig. 4C).
As a transcriptional factor, C/EBPb directly binds to DNA in a sequence-specific manner to regulate the expression of target genes. We were interested in determining whether these JAK/STAT signaling pathway genes were directly bound by C/EBPb at the chromatin level. Since C/EBPb recognizes and binds to specific DNA sequences, we first tried to identify potential C/EBPb-binding sites at the JAK/STAT signaling pathway genes. We used the STAT5A gene as an example in this study. We first obtained a C/EBPbbinding DNA sequence motif from the JASPAR database (Fig. 4D)   software to identify C/EBPb-binding motif sites at the STAT5A gene locus. As seen in Fig. 4E, we identified five potential C/EBPb-binding sites throughout the STAT5A gene body region. However, the existence of a C/EBPb-binding motif sequence does not necessarily mean that C/EBPb is actually bound. The binding of C/EBPb to the motif sequence is regulated by many other factors, and its genome-binding profile varies from cell to cell. To verify whether these sites are actually bound by C/EBPb in TNBC, we analyzed the ChIP-seq data for C/EBPb binding in SUM159PT cells, a TNBC cell line. The results showed that there was a strong C/EBPb-binding signal at the 3 0 end of the STAT5A gene body region, corresponding to the 5 th C/EBPb-binding motif locus identified above (Fig. 4F). Weak binding of C/ EBPb was also observed in the middle region, corresponding to the 2nd C/EBPb-binding motif locus. To further demonstrate the regulatory role of C/EBPb in TNBC cells, we assessed the effect of C/EBPb depletion on the expression of genes involved in the JAK/ STAT pathway in TNBC cells. The results demonstrated that the majority of JAK/STAT signaling pathway genes examined here were downregulated upon C/ EBPb knockdown, including STAT5A (Fig. 4G,H), suggesting a positive regulatory role of C/EBPb in the expression of these genes. Taken together, our results support that C/EBPb directly binds to JAK/STAT signaling pathway genes and regulates their expression in TNBC.

Discussion
TNBC is an aggressive subtype of breast cancer in which the expression of estrogen receptor (ER), PR, and human epidermal growth factor receptor-2 is absent [21,22]. The survival time of TNBC patients is shorter, and the mortality rate is higher compared with that of non-TNBC patients. Moreover, TNBC is highly invasive, and nearly half of TNBC patients will have distant metastasis. Here, we examined the regulatory role of C/EBPb, specifically in TNBC. Our data showed that C/EBPb is significantly upregulated in TNBC samples compared with non-TNBC and normal breast tissue samples. C/EBPb depletion in TNBC cells inhibited their proliferation rate and colony formation ability. Since TNBC is a highly invasive breast cancer subtype, we also investigated the role of C/EBPb in TNBC cell migration and invasion, which are both critical processes in cancer metastasis. Our data demonstrated that C/EBPb knockdown dramatically inhibited TNBC cell migration and invasion. A subsequent mechanistic study showed that C/EBPb binds to JAK/ STAT signaling pathway genes and regulates their expression. Given that the dysregulation of the JAK/ STAT signaling pathway plays an instrumental role in breast cancer development and metastasis as well as in other types of cancer [23][24][25][26][27][28][29], we concluded that C/ EBPb promotes TNBC carcinogenesis probably via the regulation of the JAK/STAT signaling pathway. C/EBPb is an intronless gene, and due to multiple translation start sites within the mRNA, three distinct isoforms of C/EBPb proteins (LAP*, LAP, and LIP) are expressed. The C/EBPb isoform LAP has been previously reported to have an impact on the tumor microenvironment in TNBC [30]. The involvement of LAP in tumor immunity indicates that C/EBPb plays a much broader role in TNBC than we previously thought. Although we observed C/EBPb overexpression in TNBC samples, we do not know which specific isoform is eventually overexpressed at the protein level. Previous studies showed that LAP* was the only C/EBPb protein expressed in normal mammary tissue and was absent from breast cancer cells by contrast. Instead, the isoform LAP was reportedly upregulated in primary breast tumors [17]. Therefore, we hypothesize that LAP is the isoform that is overexpressed in TNBC as a result of upregulated C/EBPb mRNA levels. However, further experimental data are needed to confirm this hypothesis.

Conclusion
In the present study, we observed elevated C/EBPb expression specifically in TNBC samples compared with non-TNBC and normal breast tissue samples. Further studies showed that C/EBPb knockdown inhibited TNBC carcinogenesis. Moreover, a mechanistic study revealed that C/EBPb binds to and regulates JAK/STAT signaling pathway genes in TNBC cells, indicating that C/EBPb may promote TNBC carcinogenesis by regulating the JAK/STAT signaling pathway. In conclusion, our results demonstrate a promoting role of C/EBPb in human TNBC and reveal a novel underlying mechanism.