LINC00173 silence and estrone supply suppress ER+ breast cancer by estrogen receptor a degradation and LITAF activation

Abstract

Persistent activation of estrogen receptor alpha (ERa)-mediated estrogen signaling plays a pivotal role in driving the progression of estrogen receptor positive (ER⁺) breast cancer (BC). In the current study, LINC00173, a long non-coding RNA, was found to bind both ERa and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFa) factor (LITAF), then cooperatively to inhibit ERa protein degradation by impeding the nuclear export of ERa. Concurrently, LITAF was found to attenuate TNFa transcription after binding to LINC00173, and this attenuating transcriptional effect was quite significant under lipopolysaccharide stimulation. Distinct functional disparities between estrogen subtypes emerge, with estradiol synergistically promoting ER⁺ BC cell growth with LINC00173, while estrone (E1) facilitated LITAF-transcriptional activation. In terms of therapeutic significance, silencing LINC00173 alongside moderate addition of E1 heightened TNFa and induced apoptosis, effectively inhibiting ER⁺ BC progression.

Abbreviations
• ASO
• antisense oligonucleotide
• BC
• breast cancer
• CHX
• cycloheximide
• Co-IP
• co-immunoprecipitation
• E1
• estrone
• E2
• estradiol
• ER
• estrogen receptor
• ER⁺
• estrogen receptor positive
• ERa
• estrogen receptor alpha
• ERE
• estrogen response element
• ET
• endocrine therapy
• FISH
• fluorescence in situ hybridization
• IF
• immunofluorescence
• IgG
• immunoglobulin G
• LITAF
• lipopolysaccharide-induced tumor necrosis factor-alpha factor
• LMB
• leptomycin B
• lncRNA
• long noncoding RNA
• LPS
• lipopolysaccharide
• mRNA
• messenger RNA
• miRNA
• microRNA
• NF-?B
• nuclear factor kappa-B
• NGS
• next-generation sequencing
• PARP
• poly ADP-ribose polymerase
• qRT-PCR
• quantitative real-time polymerase chain reaction
• RNA-IP
• RNA-immunoprecipitation
• TLR
• toll-like receptor
• TNFa
• tumor necrosis factor alpha
• WB
• western blot

INTRODUCTION

Breast cancer ranks among the most prevalent malignant cancers, accounting for the second-highest cancer-related mortality in females.¹ Estrogen receptor positive BC represents approximately 80% of all cases.² ERa, a nuclear receptor, on activation, translocates to the nucleus and plays a pivotal role in the genomic signaling pathway, thereby promoting the expression of genes associated with proliferation and survival. Consequently, approximately 95% of ERa protein is localized within the nucleus.³ In the terms of transcriptional regulation, ERa directly binds to the estrogen response element (ERE) in the promoter regions of hormonally regulated genes, initiating the transcriptional activation of target genes involved in cell proliferation and survival,⁴ therefore ERa represents a common target for treatment through endocrine therapies (ETs).⁵

In female patients with diverse physiological or age characteristics, alterations in the estrogen synthesis pathway result in variations in the use of ETs targeting the ERa signaling pathway.^6,7 Additionally, the key differences in the endocrine systems of these patients include changes in the primary estrogen subtypes.⁸ Recent research has uncovered variations in the physiological effects among distinct estrogen subtypes, particularly in the promotion of inflammation.^9,10 These factors highlight potential factors that can be used to develop strategies for ETs, including the synergistic interactions between different estrogen subtypes and medications.

Lipopolysaccharide-induced tumor necrosis factor-alpha factor, also known as PIG7 or SIMPLE, has emerged as a crucial player in the regulation cytokines involved in the response to LPS, such as tumor necrosis factor alpha (TNFa).^11,12 While prior research has predominantly focused on LITAF's role in promoting inflammation in macrophages¹³ and inhibiting progression in specific cancer,¹⁴ its involvement in ER⁺ BC remains underexplored. TNFa can be produced by various cellular sources, including adipose cells, tumor-associated macrophages, and cancer cells themselves.¹⁵ Notably, the expression of TNFa receptors on BC cells suggests that these cells can both produce and respond to the TNFa signaling pathway independently of the immune microenvironment.¹⁶ Previous studies have reported that TNFa induces apoptosis in BC cells through nuclear factor kappa-B (NF-?B), caspase and the poly ADP-ribose polymerase (PARP)-dependent signaling pathway.^15,17 Recent research has also underscored the interplay between ERa and TNFa, potentially contributing to ER⁺ BC progression.⁹ Consequently, gaining insight into the role of TNFa regulation in ER⁺ BC may pave the way for its use as a therapeutic target.

Long noncoding RNAs (lncRNAs), characterized by transcripts exceeding 200 nucleotides that do not encode proteins, have been implicated as pivotal contributors to cancer progression. LINC00173 has emerged as a promoter and biomarker in the progression and chemoresistance of cancer, thereby offering significant potential as a therapeutic target.¹⁸ Most of the reported studies describe the function of LINC00173 as a microRNA (miRNA) sponge, sequestering miRNA such as miR-218, miR-511–5p, and miR-490-3p in lung cancer and triple-negative BC.^19–21 While the oncogenic role of LINC00173 has been preliminarily demonstrated, its expression and function in ER⁺ BC as well as its therapeutic potential remain unexplored in existing literature.

In this study, we identified the collaborative impact of LINC00173 and LITAF in ER⁺ BC, elucidating their roles in activating the ERa signaling pathway and promoting cancer progression, and found the influence of different estrogen subtypes varied considerably.

MATERIALS AND METHODS

Gene expression across distinct cell lines was screened and validated by next-generation sequencing (NGS), quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB). Dual-luciferase assays were utilized to evaluate transcriptional activity. Subcellular localization of molecular was examined using fluorescence in situ hybridization (FISH), immunofluorescence (IF), and nuclear-cytoplasmic fractionation. Binding interactions were confirmed through RNA-immunoprecipitation (RNA-IP) and co-immunoprecipitation (Co-IP). Therapeutic effects on cell line-derived xenograft models were verified through estrone (E1) pellet implantation and intratumoral antisense oligonucleotide injection.

Details of the materials and methods are provided in Appendix S1, and the corresponding primer and probe sequences are given in Tables S1–S5.

RESULTS High LINC00173 levels in ER⁺ BC cells positively correlate with progression, partly because LINC00173 promotes ERa activity

To determine the lncRNA factors driving ER⁺ BC progression, we screened the expression of several lncRNAs across various human-derived breast cell lines and revealed high LINC00173 levels in ER⁺ BC cells comparing to ER-negative breast nontumoral or BC cells (Figure 1A). Using public databases, we also found high LINC00173 levels exhibited in both ER⁺ BC patients and ER⁺ cell lines (Figure 1B). Based on this evidence, we further established cell models in which antisense oligonucleotide-induced LINC00173 knockdown and LINC00173 overexpression, respectively (Figure 1C).

FIGURE 1. Functional characterization of LINC00173 in estrogen receptor positive (ER+) breast cancer (BC) progression and the estrogen receptor alpha (ERa) signaling pathway. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) reveals elevated LINC00173 in ER+ BC cell lines compared to nontumoral and estrogen receptor (ER)-negative counterparts. (B) The public database confirms increased LINC00173 in ER+ BC patients and cell lines relative to ERa-negative cases. (C) qRT-PCR demonstrates the efficiency of LINC00173 knockdown and overexpression. (D) Schematic representation of ERa transcriptional activity on the estrogen response element (ERE) in the target gene. The dual-luciferase reporter assay shows that LINC00173 knockdown impairs ERE transcriptional activity in ER+ BC cells. (E) qRT-PCR demonstrates the correlation between LINC00173 expression and messenger RNA (mRNA) levels of GREB1 and TFF1 in ER+ BC cells. (F) qRT-PCR based on RNA-immunoprecipitation (RNA-IP) reveals the binding of LINC00173 with ERa in ER+ BC cells. (G) The cell viability and colony formation assay show that LINC00173 correlates with the progression of ER+ BC cells. ASO, antisense oligonucleotide. Data are presented as means ± standard deviation. Statistical analysis was performed using two-sided, unpaired t-tests and one-way analysis of variance (ANOVA) as appropriate. ns, not significant; *P [less than] 0.05, **P [less than] 0.01, ***P [less than] 0.001.

Since ERa binds to specific DNA sequences (GGTCAnnnTGACC) called EREs and transactivates gene expression in response to estrogen,⁴ the effect of LINC00173 on ERa transcription activity in ER⁺ BC was investigated. By dual-luciferase assay, EREs were inserted into reporter plasmids and then transfected into MCF7 and T47D cells. Following LINC00173 knockdown, a significant decrease in luciferase activity was observed (Figure 1D). Simultaneously, we evaluated the expression of canonical ERa transcription factor-regulated target genes, GREB1 and TFF1, with or without LINC00173, and found that LINC00173 promoted the expression of these two (Figure 1E).

Subsequently, we discovered binding between ERa and LINC00173 in ER⁺ BC cells by RNA-IP analysis. Cells lysis was immunoprecipitated with ERa specific antibody or immunoglobulin G (IgG) as a control, receptively, and then the messenger RNA (mRNA) was extracted and detected by qRT-PCR. The results showed that enrichment of LINC00173 binding with ERa, confirming the binding between ERa and LINC00173 (Figure 1F). Functional experiments illustrated that the growth and clonogenicity were sustained by LINC00173 (Figure 1G).

LINC00173 decelerates ERa protein degradation and collaborates with E2 to promote ER⁺ BC cell growth

Given the observed effect of LINC00173 on ERa activity and ER⁺ BC cell progression, we directed attention towards examining the effect of LINC00173 on ERa expression. Interesting results indicated that the expression of ERa mRNA remained unaffected following LINC00173 knockdown, whereas a reduction in ERa protein levels was observed (Figure 2A,B). Investigation into LINC00173's post-transcriptional regulation of ERa focused on its influence on ERa protein degradation. After choosing the protein synthesis inhibitor cycloheximide (CHX), WB analysis revealed an acceleration of ERa protein degradation on LINC00173 knockdown in both MCF7 and T47D cells (Figure 2C,D). These results suggest a role for LINC00173 in stabilizing ERa protein.

FIGURE 2. LINC00173 modulates estrogen receptor alpha (ERa) protein stability and collaborates with estradiol (E2) in estrogen receptor positive (ER+) breast cancer (BC) cell growth. (A) Western blot (WB) reveals that LINC00173 knockdown decreases ERa protein levels in ER+ BC cells. (B) Quantitative real-time polymerase chain reaction (qRT-PCR) indicates that LINC00173 knockdown does not influence ERa messenger RNA (mRNA) levels in ER+ BC cells. (C, D) WB with 50 µg/mL cycloheximide (CHX) treatment demonstrates that LINC00173 knockdown accelerates ERa protein degradation in ER+ BC cells. (E) Cell viability shows that LINC00173 exerts a stronger growth-promoting effect with E2 in promoting ER+ BC cell growth. (F) qRT-PCR indicates that LINC00173 exerts a significant synergistic impact with E2 in increasing the mRNA levels of GREB1 and TFF1 in ER+ BC cells. ASO, antisense oligonucleotide; E1, estrone. Data are presented as means ± standard deviation. Statistical analysis was performed using two-sided, unpaired t-tests and one-way analysis of variance (ANOVA) as appropriate. ns, not significant; *P [less than] 0.05, **P [less than] 0.01, ***P [less than] 0.001.

The ERa is a ligand-activated enhancer protein, so the influence of estrogen was also examined. It was found that E2 effectively promoted the growth of MCF7 and T47D cells compared to E1. Further overexpression of LINC00173 in addition to E2 greatly enhanced cell growth, while E1 supplementation moderately slowed this growth (Figure 2E). Notably, a synergistic action between E2 and LINC00173 overexpression in promoting cells growth was evident. Additionally, the transcription of ERa-regulated target genes, GREB1 and TFF1, was promoted by the synergistic action (Figure 2F). The synergistic promotion of E2 with LINC00173 has really caused us concern.

LINC00173 binds another transcription factor LITAF, which decelerates ERa protein degradation to promote ERa activity in ER⁺ BC cells

This study also sought to determine whether LINC00173 binds to other transcription factors that contribute to the regulation of ERa. NGS was used to compare the transcriptomes of nontumoral breast epithelial MCF10A cells and MCF7 cells. Elevated LITAF in MCF7 cells was observed and RNA-protein binding sites on LINC00173 for both ERa and LITAF were individually predicted. RNA-IP was performed on MCF7 and T47D cells using LITAF-specific antibody or IgG as a control, respectively. The enrichment of LINC00173 was detected through qRT-PCR. As predicted, LITAF exhibited a higher level of enrichment in binding with LINC00173 compared to IgG. This indicated the existence of another binding between LINC00173 and LITAF (Figure 3A). The elevated LITAF levels in ER⁺ BC cells relative to MCF10A and ER-negative BC cells were subsequently validated (Figures 3B and S1).

FIGURE 3. Role of lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) in the estrogen receptor alpha (ERa) signaling pathway and estrogen receptor positive (ER+) breast cancer (BC) progression. (A) Prediction from the Vienna RNA website and catRAPID omics indicates the binding sites for ERa and LITAF, respectively, on LINC00173. next-generation sequencing-based transcription factor analysis reveals upregulated LITAF levels in MCF7 compared to MCF10A cells. Quantitative real-time polymerase chain reaction (qRT-PCR) based on RNA-immunoprecipitation (RNA-IP) reveals the binding of LINC00173 with LITAF in ER+ BC cells. (B) qRT-PCR and western blot (WB) demonstrate elevated LITAF levels in ER+ BC compared to MCF10A cells. (C) WB demonstrates the efficiency of LITAF knockdown and overexpression. (D) WB shows ERa overexpression efficiency and dual-luciferase reporter assay demonstrates that elevated LITAF expression enhances the transcriptional activity of estrogen response element in ER+ BC cells, but not in LINC00173-negative MCF10A cells. (E) qRT-PCR shows that elevated LITAF expression enhances messenger RNA (mRNA) levels of GREB1 and TFF1 in ER+ BC cells, but not in LINC00173-negative MCF10A cells. (F) Cell viability and colony formation assay show that LITAF is related to progression of ER+ BC cells. Data are presented as means ±standard deviation. Statistical analysis was performed using two-sided, unpaired t-tests and one-way analysis of variance (ANOVA) as appropriate. ns, not significant; *P [less than] 0.05, **P [less than] 0.01, ***P [less than] 0.001.

A manipulation model for LITAF expression was established and its efficiency was validated (Figure 3C). LINC00173 and ERa expression were lacking in MCF10A cells, while LITAF levels were low. Consequently, ERa was artificially overexpressed in both MCF10A and MCF7 cells. This allowed examination of the impact of LITAF on ERa transcriptional promotion activity in ER⁺ BC, using the ERE reporter plasmids system mentioned above. We observed that LITAF could also increase the activity of ERa as long as ERa was present in the cell and was not overexpressed; once ERa was overexpressed, LITAF could increase the activity of ERa in MCF7 cells. However, this result was not the case in LINC00173-negative MCF10A cells (Figure 3D). Likewise, concerning ERa transcriptional functionality, LITAF increased the transcriptional level of target genes in MCF7, but not MCF10A cells (Figure 3E). Functional experiments illustrated that the growth and clonogenicity are sustained by high LITAF levels in ER⁺ BC cells (Figure 3F).

Again, an interesting result indicated that the expression of ERa mRNA remained unaffected following LITAF knockdown, whereas a reduction in ERa protein levels was observed (Figure 4A,B). Investigation into LITAF's post-transcriptional regulation of ERa focused on ERa protein degradation. After choosing the CHX, WB analysis revealed an acceleration of ERa protein degradation on LITAF knockdown in both MCF7 and T47D cells (Figure 4C,D). These results suggest a role for LITAF in stabilizing ERa protein, as our previous studies had found.

FIGURE 4. Lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) modulates estrogen receptor alpha (ERa) protein stability in estrogen receptor positive (ER+) breast cancer (BC) cells. (A) Western blot (WB) reveals that LITAF knockdown decreases ERa protein levels in ER+ BC cells. (B) Quantitative real-time polymerase chain reaction analysis indicates that LITAF knockdown does not influence ERa messenger RNA (mRNA) levels in ER+ BC cells. (C, D) WB with 50 µg/mL cycloheximide (CHX) treatment demonstrates that LITAF knockdown accelerates ERa protein degradation in ER+ BC cells. Data are presented as means ± standard deviation. Statistical analysis was performed using two-sided, unpaired t-tests and one-way analysis of variance (ANOVA) as appropriate. ns, not significant; *P [less than] 0.05, **P [less than] 0.01.

LINC00173 mediates LITAF and ERa binding, and decelerates ERa protein degradation by inhibiting nuclear export

We have identified and confirmed the binding of LINC00173 to LITAF and ERa, respectively, and the deceleration of ERa protein degradation by LINC00173 and LITAF. To pursue the underlying mechanism, MCF7 cells with high LINC00173 levels and those with LINC00173 knockdown were subjected to SweAMI-FISH and IF staining. Fluorescence demonstrated that LINC00173 (green), along with LITAF (pink) and ERa (red) were located in the nucleus. Correspondingly, partial export of LITAF and complete export of ERa from the nucleus was observed with LINC00173 knockdown (Figure 5A), and this was further verified and confirmed by qRT-PCR and WB on the basis of cytoplasmic isolation in extended cell lines (Figure 5B,C). Moreover, the binding between LITAF and ERa was achieved through WB analysis based on Co-IP with anti-ERa antibody (Figure 5D). Confirmatory Co-IP, conducted with an anti-LITAF antibody based on nucleocytoplasmic isolation, verified that the binding between LITAF and ERa primarily occurs in the nucleus (Figure 5E). Correspondingly, Co-IP experiments using an anti-ERa antibody to immunoprecipitate LITAF showed that LINC00173 knockdown led to a reduction in the binding between ERa and LITAF (Figure 5F). These findings indicate a crucial role for LINC00173 in facilitating the interaction between ERa and LITAF, as well as promoting the enrichment of ERa in the nucleus.

FIGURE 5. LINC00173-mediated regulation of lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) binding to estrogen receptor alpha (ERa) and inhibition of ERa protein degradation through nuclear export inhibition. (A) Representative SweAMI-fluorescence in situ hybridization and immunofluorescence images based on MCF7 (left) and its LINC00173-knockdown (right) cells reveal nuclear enrichment of ERa, and LINC00173 and LINC00173 knockdown relocates ERa outside the nucleus in MCF7 cells. (B) Quantitative real-time polymerase chain reaction based on subcellular isolation demonstrates predominant nuclear localization of LINC00173 in estrogen receptor positive (ER+) breast cancer (BC) cells. (C) Western blot (WB) based on subcellular isolation shows nuclear localization of ERa in ER+ BC cells. (D) WB based on co-immunoprecipitation (Co-IP) demonstrates the binding of ERa with LITAF in ER+ BC cells. (E) WB based on subcellular isolation and Co-IP indicates that LITAF primarily binds with ERa in the nucleus of MCF7 cells. (F) WB based on Co-IP indicates that LINC00173 knockdown reduces the binding of ERa with LITAF in ER+ BC cells. (G, H) WB with 48 h of 50 µg/mL cycloheximide (CHX) treatment demonstrates that knockdown of LINC00173 (G) and LITAF (H) promotes ERa degradation, which is inhibited by additional 2.5 ng/mL leptomycin B (LMB). ASO, antisense oligonucleotide. Data are presented as means ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA) as appropriate. ns, not significant; *P [less than] 0.05, **P [less than] 0.01, ***P [less than] 0.001.

Considering that proteins are typically degraded in proteasomes or lysosomes, the degradation of nucleoproteins should occur after exporting from the nucleus. This was further verified by the treatment of nuclear export inhibitor leptomycin B (LMB) under CHX treatment to assess ERa protein degradation. The results suggest that the decline of ERa protein was due to the release of the inhibition signal of nuclear export after LINC00173 or LITAF was depleted separately or simultaneously (Figure 5G,H). These results suggest that sustained high ERa levels in the nucleus are stabilized by LINC00173 and LITAF through the inhibition of ERa nuclear export, preventing it from being degraded, for example in a proteasome-dependent manner, in ER⁺ BC cells.

The binding of LITAF by LINC00173 decreases LITAF transcription of TNFa, and E1 probably increases LITAF transcription of TNFa

LITAF is responsive to inflammatory stimuli. As a transcription factor of TNFa, LITAF induced by LPS can promote the expression of TNFa and thus necrosis of tumor cells. We have also carried out relevant research on this aspect of LITAF function.^11,13 Since LINC00173 binds LITAF, it is reasonable to assume that LITAF will weaken the TNFa transcription function, leading to the reduction of cell necrosis. To confirm this, we re-analyzed the NGS data and found a reduction in the TNFa in MCF7 cells (Figures 6A,B and S2). Validation through qRT-PCR in MCF10A and ER⁺ BC cell lines confirmed low expression of TNFa mRNA in ER⁺ BC (Figure 6A).

FIGURE 6. LINC00173 decreased the transcriptional regulation of tumor necrosis factor-alpha (TNFa) by lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF), and estrone (E1) enhanced the transcriptional regulation of TNFa by LITAF. (A) Next-generation sequencing-based analysis demonstrates downregulation of TNFa signaling pathway-related genes in MCF7 compared to MCF10A cells, and quantitative real-time polymerase chain reaction (qRT-PCR) shows decreased TNFa messenger RNA (mRNA) levels in estrogen receptor positive (ER+) breast cancer (BC) compared to MCF10A cells. (B) LITAF's transcriptional function on TNFa in response to inflammatory stimulation. (C) qRT-PCR shows that 6 h of lipopolysaccharide (LPS) induces a smaller increase in TNFa mRNA levels in ER+ BC cells compared to MCF10A cells. (D) qRT-PCR indicates that overexpression of LINC00173 inhibits the increase in TNFa mRNA levels under 6 h of LPS stimulation in MCF10A cells, and knockdown of LINC00173 elevates it in ER+ BC cells. (E) Dual-luciferase reporter assay demonstrates that overexpression of LINC00173 impairs the transcriptional activity on the TNFa promoter increased by 6 h of LPS stimulation in both MCF10A and MCF7 cells. Knockdown of LINC00173 enhances the transcriptional activity on the TNFa promoter increased by LPS stimulation, and this effect is counteracted by LITAF knockdown in ER+ BC cells. (F) Western blot (WB) shows that LITAF expression is elevated by 24 h of treatment of E1 but not estradiol (E2) in MCF7 cells. (G) qRT-PCR indicates that 6 h of treatment of E1, but not E2, cooperates with LPS in elevating TNFa mRNA levels in MCF7 cells. (H) Dual-luciferase reporter assay demonstrates that 6 h of treatment of E1 increases LPS-induced TNFa transcription partially by promoting the transcriptional function of LITAF in MCF7 cells. ASO, antisense oligonucleotide. Data are presented as means ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA) as appropriate. ns, not significant; *P [less than] 0.05, **P [less than] 0.01, ***P [less than] 0.001.

High LINC00173 levels in ER⁺ BC suggest that the LITAF was likely involved in binding interactions with LINC00173. It was further hypothesized that the diminished expression of TNFa mRNA resulted from the binding. To verify this, TNFa expression was induced by LPS, and the TNFa transcriptional level was assessed under LINC00173 expression manipulation. The results indicate that TNFa mRNA was suppressed by LINC00173 overexpression (Figure 6C,D). Furthermore, the impact of LINC00173 on LITAF's transcriptional activity for TNFa was examined. In the dual-luciferase assay, promoter sequences of TNFa were inserted into reporter plasmids. The results demonstrate that following LINC00173 overexpression, the detected luciferase activity was reduced in both MCF10A and MCF7 cells (Figure 6E). Conversely, LINC00173 knockdown led to an increase in luciferase activity and simultaneously, the activity was decreased with further LITAF knockdown (Figure 6E).

E1 was found to have a pro-inflammatory function compared to E2.^9,10 Considering the differences between E1 and E2 in promoting cell growth (Figure 2E), the effects of E1 and E2 on LITAF-mediated TNFa transcription were explored. It was found that E1 increased LITAF protein compared to E2 (Figure 6F). Additionally, in terms of TNFa transcription, while E1 alone did not increase TNFa transcription, E1 supplementation had a synergistic effect on increasing TNFa transcription under LPS stimulation (Figure 6G). Furthermore, with LITAF transcriptional activity assay as detailed above, extra E1 led to an increase in LPS-induced LITAF transcriptional activity of TNFa and simultaneously this decreased with additional LITAF knockdown in MCF7 cells (Figure 6H). These results not only reveal the mechanism of the differential effects of E1 and E2 on inflammatory signals but also suggest that E1 may specifically induce TNFa anticancer compared to E2.

Silencing LINC00173, along with E1, promotes apoptosis and inhibits ER⁺ BC progression via TNFa

Finally, we tested the therapeutic potential in ER⁺ BC. We applied flow cytometry to detect the effects of LINC00173 knockdown and E1 on cells, and found that silencing LINC00173 alone or adding E1 alone under LPS could induce apoptosis in vitro. Combined intervention resulted in the most effective cell apoptosis (Figure 7A). Later, a mouse xenograft model was used to test novel therapeutics. Tumor growth was expressively restrained by the combined intervention of LINC00173 knockdown and E1. Furthermore, injecting infliximab, a blocking antibody against TNFa, could partially offset the tumor inhibitory effect after LINC00173 knockdown combined with E1 (Figures 7B and S3). These findings highlight that LINC00173 stabilizes ERa and reduces TNFa in ER⁺ BC, suggesting its potential as a multitargeted therapeutic option (Figure S4). The appropriate combination of silencing LINC00173 and E1 demonstrates effective inhibition of ER⁺ BC.²²

FIGURE 7. Silencing LINC00173, combining with estrone (E1), induces apoptosis and suppresses estrogen receptor positive (ER+) breast cancer (BC) progression via tumor necrosis factor-alpha (TNFa). (A) Flow cytometry assay reveals that both LINC00173 knockdown and the addition of 24 h treatment of E1 increase lipopolysaccharide (LPS)-induced cell apoptosis. The combination achieves a more effective apoptotic effect in ER+ BC cells. Cells were treated with 1 µg/mL LPS for 48 h. (B) cell line-derived xenograft experiment design and outcomes showing that the combination of LINC00173 knockdown and E1 inhibits the progression of ER+ BC. The effect is partially compromised by infliximab (anti-TNFa) treatment. The representative images of mice in each group are shown. ASO, antisense oligonucleotide. Data are presented as means ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA) as appropriate. *P [less than] 0.05, **P [less than] 0.01, ***P [less than] 0.001.

This study demonstrated that high LINC00173 levels and their combination with LITAF promoted the accumulation of ERa and the progression of ER⁺ BC, during which E2 played a synergistic role. At the same time, high LINC00173 levels coupled with binding to LITAF led to reduced TNFa transcription, thereby promoting ER⁺ BC, and E1 plays an antagonistic role in this process (Figure 8).

FIGURE 8. LINC00173 functions as a mediator, facilitating the binding of lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) to estrogen receptor alpha (ERa), ultimately promoting the survival and progression of estrogen receptor positive (ER+) breast cancer (BC) cells (left). Conversely, when LINC00173 is silenced using antisense oligonucleotide, LITAF is liberated from this complex. Consequently, LITAF can resume its role with estrone in promoting lipopolysaccharide-induced tumor necrosis factor-alpha transcription. Concurrently, ERa protein undergoes nuclear export, leading to accelerated degradation and the suppression of its transcriptional activity. This concerted action inhibits progression by inducing apoptosis in ER+ BC cells (right). ASO, antisense oligonucleotide; E2, estradiol; E1, estrone; ERE, estrogen response element.

DISCUSSION

In the context of BC, ERa level regulation primarily hangs on alterations in degradation kinetics.²³ The heightened activity of ERa results from an extended stability attributed to a decelerated degradation rate, a pivotal factor in its functionality. Our investigation specifically reveals that, in ER⁺ BC cells, the collaborative action of LINC00173 with LITAF impedes ERa degradation, thereby amplifying its activity. This interaction mechanism involves a collaborative effort to attenuate ERa degradation and concurrently suppress its nuclear export, similar to the function of TEAD in advancing ER⁺ BC.²⁴ Numerous studies have concentrated on the process of ERa degradation in the cytoplasm following nuclear export, notably through the ubiquitin-proteasome system and lysosomal degradation.^23,25 Referring to ERa's short half-life, this alteration in ERa dynamics corresponds with increased ERa activity, such as that associated with serine-118 phosphorylation.²⁶ Fundamentally, our findings demonstrate that LINC00173, in partnership with LITAF, importantly decelerates the degradation of ERa, thereby crucially augmenting its relative stability and sustaining elevated activity in ER⁺ BC.

In our study, additional E2 promotes growth in BC cells, consistent with literature findings.^23,26 In contrast, E1 exhibits weaker growth-promoting effects on BC cells, suggesting potential variations in estrogen subtypes despite both being ERa ligands. Notably, compared with E2 or estriol, E1 demonstrated weaker affinity for the receptor ERa.²⁷ Further experiments revealed the therapeutic potential of E1 in BC. Previous literature indicates that E1 can activate NF-?B signaling associated with inflammation.²⁸ Our study demonstrated that E1 can assist LITAF in the transcription of TNFa, thereby inhibiting the growth of cells. This observation underscores the potential therapeutic significance of E1 treatment. Notably, this finding aligns with recent reports indicating that the E1 analogues 16AABE and 16BABE can inhibit the proliferation and metastasis of BC cells.²² Overall, our findings highlight the different effects of estrogen types on the development and potential treatment of ER⁺ BC. These results provide crucial preclinical research data for precision drug selection among relevant populations.

Recent studies have elucidated the potential of LPS in sensitizing BC cells to apoptosis-inducing therapies through TLR- and MyD88-mediated mechanisms producing TNFa.²⁹ The presence of TLR in breast cells, particularly when stimulated by LPS, enhances TNFa secretion.³⁰ Moreover, studies have shown that TNFa can reduce the transcriptional activity of ERE.³¹ This aligns with our discovery that overexpression of LITAF also slightly diminishes the transcriptional activity of ERE in MCF10A cells lacking LINC00173. Building on this, our investigation demonstrated LINC00173's pivotal role in further attenuating the production of TNFa induced by LPS. LINC00173 achieves this by impeding the transcriptional function of LITAF. Moreover, our findings align with the NGS data and support the downregulation of TNFa in BC cells, which is supported by the literature.^15,31 This dual line of evidence strengthens the proposition that LINC00173, by modulating TNFa production and associated pathways, emerges as a key regulatory element, influencing its responsiveness to apoptosis-inducing therapies. Regarding triple-negative BC, our observation of reduced expression levels of LINC00173 and LITAF relative to ER⁺ BC cells raises the possibility of a distinct role for LITAF, such as its reported tumor suppressor function.³² These findings highlight the specificity of our study for ER⁺ BC.

Targeting LINC00173 in a therapeutic strategy exhibits a dual anticancer effect, manifested through the simultaneous downregulation of ERa activity and upregulation of LITAF, leading to increased transcription of TNFa. The incorporation of E1 further enhances the overall architecture of the anti-BC process, rendering it more comprehensive. Notably, immune checkpoint blockades stand as the established therapeutic paradigm for tumors, with inflammatory cytokines, including TNFa, emerging as contributors that may amplify the efficacy of this treatment modality.³³ Consequently, our findings represent a contribution to the understanding of how manipulating the LINC00173 axis can optimize the therapeutic outcomes of immune checkpoint blockades, providing a valuable insight into a potentially curative treatment for this disease.

AUTHOR CONTRIBUTIONS

Yu Xie: Conceptualization; data curation; formal analysis; investigation; software; validation; writing – original draft. Meihua Shan: Methodology. Jing Yu: Formal analysis. Yongjun Du: Validation. Chengkun Wu: Writing – review and editing. Shujing Liu: Writing – review and editing. Jiayin Li: Writing – review and editing. Yupeng Xiao: Writing – review and editing. Yan Yan: Funding acquisition. Ning Li: Writing – review and editing. Junfang Qin: Writing – review and editing. Lan Lan: Funding acquisition. Yue Wang: Funding acquisition; project administration; supervision; writing – review and editing.

ACKNOWLEDGMENTS

The authors thank to Natural Science Foundation of China (Nos. 31770968 and 31800661) for the funding support.

FUNDING INFORMATION

This research was supported by the Natural Science Foundation of China (Nos. 31770968 and 31800661).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

Approval of the research protocol by an institutional reviewer board: N/A.

Informed Consent: N/A.

Registry and the Registration No. of the study/trial: N/A.

Animal Studies: All experiments were performed under the approval of the Ethics Committee at the Nankai University.