Decreased NFIL3 mRNA indicates elevated NFIL3 protein in TNBC by screening the aberrant expression of rhythm genes in breast cancer
We first investigated the aberrant expression of 32 rhythm genes in breast cancer based on the TCGA dataset and GTEx dataset by searching the GEPIA database. The expression of six rhythm genes, CRY2, NFIL3, PER1, EGR3, NRID1 and TIMELESS, was significantly changed in breast cancer compared with normal breast tissues (Fig. 1A & Supplementary Fig. 1A). We further compared the mRNA levels of these six rhythm genes between 20 normal breast tissues and 20 breast cancer tissues by using qPCR and confirmed that the aberrant expression of NFIL3 and EGR3 in breast cancer is consistent with the results from the GEPIA database (Fig. 1B). When considering the expression patterns of NFIL3 and EGR3 in different subtypes of breast cancer, we found that EGR3 mRNA was significantly downregulated in the HER2-positive subtype of breast cancer, whereas NFIL3 mRNA was specifically lower in the TNBC subtype than in the other subtypes of breast cancer (Fig. 1C). We next focused on NFIL3 and evaluated the expression of NFIL3 protein between TNBC and other subtypes of breast cancer by using IHC. Surprisingly, we found that the expression of NFIL3 protein was significantly higher in TNBC than in other subtypes of breast cancer (Fig. 1D), which was contrary to the mRNA levels of the NFIL3 expression pattern in TNBC (Fig. 1C). Moreover, we demonstrated that high expression of NFIL3 protein is associated with poor prognosis of TNBC patients (Fig. 1E).
By investigating the expression change of NFIL3 mRNA across cancers, we found that NFIL3 was downregulated in most (13 out of 15) cancer types with significant NFIL3 alterations (Supplementary Fig. 1B). However, it is frequently reported that the expression of NFIL3 is elevated in various cancer types[17, 18]. To confirm the contrary results regarding the aberrant expression of NFIL3 at the mRNA and protein levels in cancer, we detected the expression of NFIL3 at the protein level in four additional cancer types with NFIL3 mRNA downregulation, including LUSC, LUAD, CESC and OV, by performing IHC. Our results showed that NFIL3 protein was significantly overexpressed in these detected cancer types compared with their normal tissues (Supplementary Fig. 1C), suggesting an opposite result regarding the expression of NFIL3 at the mRNA and protein levels in pancancer.
To determine the opposite results regarding NFIL3 expression at both the mRNA and protein levels, we analyzed the transcription factors that potentially bind the promoter region of the NFIL3 gene by searching the Cistrome database. Interestingly, we found that NFIL3 potentially regulates its own transcription (Supplementary Fig. 1D), and several NFIL3 binding peaks within the promoter region of the NFIL3 gene were identified (Supplementary Fig. 1E). Furthermore, we employed ChIP assays (Fig. 1F) and dual-luciferase reporter assays (Fig. 1G) to demonstrate that NFIL3 could bind the promoter region of the NFIL3 gene and suppress its transcription. These results indicate that NFIL3 is a repressor of the transcription of the NFIL3 gene, which may explain the opposite change in NFIL3 at the mRNA and protein levels. To test this hypothesis, we exogenously overexpressed NFIL3 driven by CMV enhancer in two TNBC cell lines (Fig. 1H) and an additional seven cancer cell lines representing six cancer types (Supplementary Fig. 1F-G) and detected the impact of elevated NFIL3 protein on the transcription of the endogenous NFIL3 gene. We showed that pre-mRNA of NFIL3 was significantly downregulated upon increase of exogenous NFIL3 protein in all detected cancer cell lines (Fig. 1I & Supplementary Fig. 1H). These results suggest that NFIL3 is a transcriptional repressor of NFIL3 transcription, and decreased NFIL3 mRNA indicates elevated NFIL3 protein levels across cancers.
NFIL3 protein promotes the proliferation and metastasis of TNBC cells
To detect the potential roles of NFIL3 protein in TNBC progression, we investigated the effects of NFIL3 protein on the proliferation and metastasis of two TNBC cell lines, BT549 and Hs578T, by gain-of-function and loss-of-function strategies, respectively. The stable overexpression of exogenous NFIL3 in two TNBC cell lines at both the mRNA and protein levels was confirmed by qRT–PCR and western blot assays (Supplementary Fig. 2A, B). Cell counting assays and clone formation assays revealed that increasing NFIL3 protein promoted the proliferation of Hs578T cells (Fig. 2A-B) and BT549 cells (Supplementary Fig. 2C, D), whereas knockdown of endogenous NFIL3 (Supplementary Fig. 2E, F) inhibited the proliferation of Hs578T cells (Fig. 2C, D) and BT549 cells (Supplementary Fig. 2G, H) in vitro. Consistently, the subcutaneous tumor formation assay also demonstrated that elevating NFIL3 protein promoted the growth of Hs578T cells (Fig. 2E), whereas decreasing NFIL3 protein inhibited the growth of Hs578T cells in vivo (Fig. 2F). Using wound healing assays and Transwell assays, we demonstrated that overexpression of NFIL3 protein promoted the migration and invasion of Hs578T cells (Fig. 2G-I) and BT549 cells (Supplementary Fig. 2I-K), whereas decrease of NFIL3 protein inhibited the migration and invasion of both cell lines (Fig. 2J-L and Supplementary Fig. 2L-N). Consistently, the mouse tail vein injection model also revealed that elevated NFIL3 protein promoted the lung metastasis of Hs578T cells (Fig. 2M), whereas decrease of NFIL3 protein inhibited the lung metastasis of Hs578T cells (Fig. 2N). These results suggest that NFIL3 protein promotes the progression of TNBC cells in vitro and in vivo.
NFIL3 regulates tumor associated inflammation in breast cancer
To investigate the molecular mechanism by which NFIL3 protein promotes the progression of TNBC cells, we detected global gene expression changes upon NFIL3 knockdown using a mRNA array when NFIL3 was considered a transcription factor. We identified 1990 genes that were significantly changed upon NFIL3 knockdown in Hs578T cells, including 891 upregulated genes and 1099 downregulated genes (Supplementary Fig. 3A, B & Supplementary Table 2). The analysis of functional enrichment in ten hallmarks of cancer showed that both downregulated and upregulated genes were significantly enriched in inflammation (Fig. 3A, B). Then, we analyzed the major inflammatory pathways affected by NFIL3 knockdown and showed that both innate immune signaling pathways (such as Toll-like receptor signaling, RIG-I-like receptor signaling and NOD-like receptor signaling) and adaptive immune signaling pathways (such as T cell receptor signaling and B cell receptor signaling) were significantly enriched (Fig. 3C). By analyzing the correlation between NFIL3 mRNA levels and the infiltration of 22 inflammatory cells in breast cancer tissues based on the TCGA dataset (Fig. 3D-E & Supplementary Fig. 3C, D), we showed that NFIL3 mRNA levels were positively correlated with the infiltration levels of type I macrophages (M1), CD4+ memory T cells, CD8+ T cells, dendritic cells (DCs), follicular T helper cells, M0 cells and neutrophils (Fig. 3D & Supplementary Fig. 3C), while negatively correlated with the levels of Treg cells, type II macrophages (M2), monocytes, resting mast cells, naïve CD4+ T cells, γδ T cells and eosinophils (Fig. 3E & Supplementary Fig. 3D), respectively. Furthermore, we detected the correlation between NFIL3 protein levels and the amount of infiltrated M1, M2 and CD8+ T cells in 60 TNBC tissues and confirmed that NFIL3 protein levels were positively correlated with infiltrated M2 cells whereas negatively correlated with infiltrated M1 cells and CD8 + T cells, respectively (Figure 3F-G). These results suggest that NFIL3 regulates tumor associated inflammation in breast cancer.
NFIL3 enhances the activity of NF-κB signaling by inhibiting the expression of NFKBIA in TNBC cells
The NF-κB signaling pathway, a well-known inflammatory pathway associated with cancer progression, was enriched in the top ten inflammatory signaling pathways regulated by NFIL3 (Fig. 3C). Next, we evaluated the roles of NFIL3 protein in regulating the activity of NF-κB signaling. Translocation of NF-κB from the cytoplasm into the nucleus is required and indicates the activation of NF-κB signaling. By performing an immunofluorescence assay, we detected the distribution changes of NF-κB in Hs578T cells stimulated with TNF-α upon altering the expression of NFIL3. After stimulation with TNF-α, NF-κB is quickly translocated from the cytoplasm to the nucleus, and the overexpression of NFIL3 protein significantly enhances the translocation of NF-κB into the nucleus at different time points, whereas decrease of NFIL3 protein blocks the nuclear translocation of NF-κB (Fig. 4A). Subsequently, we confirmed that overexpression of NFIL3 protein significantly enhances NF-κB activity, whereas knockdown of NFIL3 significantly inhibits NF-κB activity using a dual-luciferase reporter assay (Fig. 4B). These results demonstrated that NFIL3 effectively promotes NF-κB activation in TNBC cells.
In total, six NF-κB signaling pathway-related genes were significantly changed upon NFIL3 knockdown, three of which were downregulated, while three were upregulated (Supplementary Figure 4A), and the expression change of these six genes upon NFIL3 knockdown was confirmed by qPCR (Supplementary Figure 4B). Among the six NFIL3-regulated genes in the NF-κB signaling pathway, four (TRIM25, BCL10, RIPK1 and NFKBIA) are regulators of NF-κB activity, whereas the others (GADD45B and CXCL2) are downstream target genes of NF-κB (Supplementary Figure 4A). The downregulation of BCL10 and RIPK1 and upregulation of NFKBIA may all contribute to suppressing the activity of NF-κB upon NFIL3 knockdown. Considering that NFIL3 is a transcriptional suppressor and NFKBIA, a canonical suppressor of NF-κB activation, is significantly upregulated in NFIL3 knockdown cells, we focused on NFKBIA for further validation as the direct target of NFIL3.
ChIP-sequencing data showed that there are several NFIL3 binding sites in the promoter region of the NFKBIA gene (Supplementary Fig. 4C). We employed ChIP assays and dual-luciferase reporter assays to demonstrate that NFIL3 binds the NFKBIA gene (Fig. 4C) and inhibits its transcription (Fig. 4D) in Hs578T cells. We further showed that NFKBIA was decreased in NFIL3-overexpressing cells but was increased in NFIL3 knockdown cells at both the mRNA and protein levels in both Hs578T cells (Fig. 4E-F) and BT549 cells (Supplementary Fig. 4D-E). Moreover, we detected the protein levels of NFIL3 and NFKBIA in breast cancer tissues using IHC and revealed that the NFIL3 protein level was negatively correlated with the NFKBIA protein level in breast cancer tissues (Fig. 4G). A similar result was obtained in normal breast tissues (Supplementary Fig. 4F). Consistently, we analyzed the correlation between NFIL3 and NFKBIA in the TCGA dataset and found that NFIL3 mRNA was positively correlated with NFKBIA mRNA in both breast cancer (Fig. 4H) and pancancer (Supplementary Fig. 4G). These results show that NFKBIA is a direct target of NFIL3 protein and is transcriptionally repressed by NFIL3 in TNBC cells.
NFKBIA suppresses the activity of NF-κB signaling and blocks the proliferation and metastasis of TNBC cells in vitro and in vivo
We examined the role of NFKBIA in the activity of NF-κB signaling and tumor associated inflammation in breast cancer. First, the stable overexpression (Supplementary Fig. 5A, B) and knockdown (Supplementary Fig. 5C-D) of NFKBIA at both the mRNA and protein levels in BT549 and Hs578T cells were confirmed by qRT–PCR and western blot assays, respectively. Then, we detected the effect of NFKBIA on NF-κB activity. As expected, NFKBIA knockdown enhanced the translocation of NF-κB from the cytoplasm into the nucleus, whereas overexpression of NFKBIA blocked the nuclear translocation of NF-κB in Hs578T cells (Fig. 5A). Consistently, we showed that knockdown of NFKBIA significantly enhances NF-κB activity, whereas overexpression of NFKBIA significantly inhibits NF-κB activity using a dual-luciferase reporter assay (Fig. 5B). Additionally, by analyzing the correlation between NFKBIA mRNA levels and the infiltration of 22 inflammatory cells in breast cancer tissues based on the TCGA dataset (Fig. 5C, D & Supplementary Fig. 5E-F), we showed that NFKBIA mRNA levels were positively correlated with infiltration levels of M1, CD4+ T cells, CD8+ T cells and naïve B cells (Fig. 5C & Supplementary Fig. 5E), whereas negatively correlated with infiltration levels of M2 cells, resting mast cells and eosinphils in breast cancer tissues based on the TCGA dataset (Fig. 5D). The correlation between NFKBIA protein levels and the amount of infiltrated M1, M2 and CD8+ T cells was also confirmed in breast cancer tissues using IHC assays (Fig. 5E, F). These results suggest that NFKBIA is a suppressor of NF-κB signaling and it regulates tumor associated inflammation in TNBC different from NFIL3.
Next, using cell counting and clone formation assays, we found that overexpression of NFKBIA inhibited proliferation, whereas knockdown of NFKBIA promoted the proliferation of Hs578T cells (Fig. 6A-D) and BT549 cells (Supplementary Fig. 6A-D). Consistently, overexpression of NFKBIA inhibited the growth of subcutaneous Hs578T tumors (Fig. 6E), whereas knockdown of NFKBIA promoted the growth of subcutaneous Hs578T tumors in a nude mouse model (Fig. 6F). Furthermore, overexpression of NFKBIA not only inhibited the migration and invasion of Hs578T cells (Fig. 6G-I) and BT549 cells (Supplementary Fig. 6E, F) in vitro but also decreased the lung metastasis of Hs578T cells in a mouse tail vein injection model (Fig. 6J). Knockdown of NFKBIA promoted the migration and invasion of Hs578T cells (Fig. 6K-M) and BT549 cells (Supplementary Fig. 6G, H) in vitro and enhanced the metastasis of Hs578T cells in vivo (Fig. 6N). These results suggest that NFKBIA, unlike oncogenic NFIL3, is a tumor suppressor that inhibits the progression of breast cancer cells.
NFIL3 promotes the activation of NF-κB and the progression of breast cancer by inhibiting NFKBIA
To further evaluate the critical roles of NFKBIA in mediating the effects of NFIL3 on the activation of NF-κB and the progression of TNBC, we simultaneously overexpressed both NFKBIA and NFIL3 in Hs578T (Fig. 7A) and BT549 cells (Supplementary Fig. 7A) and detected the impact of both NFKBIA and NFIL3 overexpression on the activity of NF-κB and the proliferation, migration and invasion of both breast cancer cell lines. We found that rescuing the expression of NFKBIA in breast cancer cells with NFIL3 overexpression blocked the rapid translocation of NF-κB from the cytoplasm into the nucleus triggered by NFIL3 overexpression in Hs578T cells (Fig. 7B). Consistently, we confirmed that rescuing the expression of NFKBIA inhibited the promotional effect of NFIL3 on NF-κB activity using a dual-luciferase reporter assay (Fig. 7C). We further demonstrated that rescuing the expression of NFKBIA partially reversed the promotional effect of NFIL3 on the proliferation of Hs578T cells (Fig. 7D) and BT549 cells (Supplementary Fig. 7B) and on the migration and invasion of Hs578T (Fig. 7E, F) and BT549 cells (Supplementary Fig. 7C, D). Taken together, we showed that NFIL3 promotes the progression of TNBC by suppressing NFKBIA and then enhancing the activity of the NF-κB signaling pathway.
Anti-inflammatory strategies targeting NF-κB activity block the oncogenic role of NFIL3 protein in TNBC
Because activating NF-κB activity by inhibiting the expression of NFKBIA mimics the roles of NFIL3 in the progression of TNBC, we proposed that anti-inflammatory strategies targeting NF-κB activity may block the oncogenic role of NFIL3 protein in TNBC. To test this hypothesis, we evaluated the effects of aspirin and Bay11–7082, two inhibitors of NF-κB activity that inhibit the phosphorylation of IKKB and IKKA, respectively[19, 20], on the roles of NFIL3 protein in stimulating NF-κB activity and promoting the progression of TNBC. As expected, both aspirin and Bay11–7082 significantly blocked the activation of NF-κB signaling mediated by NFIL3 protein, as detected by dual-luciferase reporter assay (Fig. 7G). Moreover, both aspirin and Bay11–7082 also significantly inhibited the promotional effect of NFIL3 on the proliferation of Hs578T cells (Fig. 7H) and BT549 cells (Supplementary Fig. 7E) and on the migration and invasion of Hs578T (Fig. 7I, J) and BT549 cells (Supplementary Fig. 7F, G). Taken together, we showed that strategies targeting NF-κB activity block the oncogenic roles of NFIL3 protein in TNBC.