HLF regulates ferroptosis, development and chemoresistance of triple-negative breast cancer by activating tumor cell-macrophage crosstalk

1To the editor,

2TAMs are major components of the tumor microenvironment that directly affect tumor cell growth, neoangiogenesis and immunosuppression. The crosstalk between tumor cells and TAMs has been studied in mammary tumors; however, the mechanisms underlying the activation of TAMs by tumor cells remain unclear in TNBC.

3Here, we found that HLF, which has been reported as an important regulator in liver fibrosis and hepatocellular carcinoma, was not only increased in TNBC samples but also associated with poor outcome in TNBC patient cohort (Fig. 1a, b, Additional file 1: Fig. S1, Additional file 3: Table S1). Bioinformatics analysis of the HLF promoter revealed a set of potential binding sites for different transcription factors including SMAD3. HLF expression in TNBC cells was decreased by interference with SMAD3 (Fig. 1c, d, Additional file 1: Fig. S2a–e). Moreover, we found that TGF-β1 secreted by TAMs induced HLF expression in TNBC (Fig. 1e, Additional file 1: Fig. S2f–j). ChIP analysis revealed that SMAD3 binds to the HLF promoter (Fig. 1f). Nevertheless, the HLF-Mut luciferase reporter could not be activated by SMAD3 (Additional file 1: Fig. S2k).

4Mechanistically, GGT1 transcription was downregulated in HLF-knockdown cells while increased in HLF overexpression cells, respectively (Fig. 1g–i, Additional file 1: Fig. S3a–e). There was a close correlation between HLF levels and GGT1 expression in TNBC tumor specimens (Additional file 1: Fig. S3f). Significant enrichment of HLF in the promoter region of GGT1 was detected by ChIP assay (Fig. 1j). Nevertheless, the GGT1-Mut luciferase reporter could not be suppressed by HLF interference (Additional file 1: Fig. S3g).

5GGT1 catalyzes the cleavage of extracellular GSH into its components to provide cysteine for the production of intracellular GSH. As expected, the GSH/GSSG ratio was decreased in HLF-knockdown cells (Additional file 1: Fig. S4a). A reduction in the GSH/GSSG ratio causes deactivation of ferroptosis regulator GPX4. Accordingly, the GPX4 activity was downregulated in HLF-knockdown cells (Additional file 1: Fig. S4b). Meanwhile, the iron, MDA and lipid ROS levels were increased in HLF-knockdown cells (Additional file 1: Fig. S4c–e). Notably, the GSH/GSSG ratio and deactivation of GPX4 in HLF-knockdown TNBC cells could be restored through introduction of GGT1 (Fig. 1k, l). Moreover, the HLF depletion-enhanced ferroptosis was reduced by GGT1 overexpression in TNBC cells (Fig. 1m, n, Additional file 1: Fig. S4f). Taken together, these data indicate that HLF inhibits ferroptosis by regulating the GGT1/GSH/GPX4 axis in TNBC cells.

6GGT1, an enzyme localized and bound to the plasma membrane, has been reported to be involved in the regulation of tumorigenesis. Functionally, the cellular proliferation and metastasis were significantly decreased in HLF-knockdown cells in vitro and in vivo (Fig. 1o, p, Additional file 1: Fig. S5a–g). Also, the sensitivity of TNBC cells to cisplatin was increased on stable knockdown of HLF (Fig. 1q, Additional file 1: Fig. S5h–j). Importantly, our data showed that the interference of GGT1 blocked the cellular proliferation, metastasis and cisplatin resistance induced by HLF (Fig. 1r, s, Additional file 1: Fig. S6a–e). Furthermore, HLF depletion-mediated proliferation ability, invasive capacity and cisplatin sensitivity could be recovered by treatment with the ferroptosis inhibitor liproxstatin-1 (Additional file 1: Fig. S6f–j). Clinical investigation revealed that TNBC patients with both elevated HLF and GGT1 predict a worse prognosis (Fig. 1t).

7Human Cytokine Antibody Array (Additional file 2) showed that six cytokines were significantly increased in the conditioned medium (CM) of MB-231 HLF cells (fold-change > 2) (Additional file 1: Fig. S7a). Gene expression analysis further showed that of IL-6 was the most significantly altered (Fig. 2a, b, Additional file 1: Fig. S7b–e). The activation of IL-6 promoter can be attenuated through HLF knockdown, or enhanced via HLF overexpression, respectively (Additional file 1: Fig. S7f, g). Furthermore, a putative homologous HLF binding site within the human IL-6 promoter was detected by ChIP assays (Fig. 2c). As expected, THP-1 cells treated with IL-6 or CM from TNBC control cells became stretched and elongated (Fig. 2d).

8Mechanistically, we found that stimulation of THP-1 cells with IL-6 or CM from TNBC cells resulted in increased expression of p-JAK2 and p-STAT3, whereas treatment with IL-6 neutralizing antibodies or S3I-201 inhibited this pathway (Fig. 2e–g, Additional file 1: Fig. S8a–d). Moreover, the expression of TGF-β1 was significantly increased in THP-1 cells cocultured with IL-6 or CM from TNBC cells, whereas treatment with IL-6 neutralizing antibodies or S3I-201 inhibited it (Fig. 2h, Additional file 1: Fig. S8e–g). A putative homologous STAT3(S727) but not STAT3(Y705) binding site within the human TGF-β1 promoter was identified by ChIP assays (Fig. 2i). Nevertheless, the TGF-β1-Mut luciferase reporter could not be suppressed by STAT3 interference (Additional file 1: Fig. S8h).

9The supernatants from TAMs pre-treated with IL-6, which contained multiple pro-inflammatory cytokines and chemokines as previous study, enhanced the HLF-GGT1 axis, increased the GSH/GSSG ratio and amplified GPX4 activity of TNBC cells, which associated with the suppression of ferroptosis (Additional file 1: Fig. S9a–g). In addition, CM of TAMs pre-treated with IL-6 not only promoted proliferation and metastasis of TNBC cells but also reduced their sensitivity to cisplatin (Fig. 2j, Additional file 1: Fig. S9h–l), and these phenomena were partially reversed by TGF-β receptor inhibitor LY2109761. These results indicate that TAMs educated by IL-6 can promote the ferroptosis resistance, progression and chemoresistance of TNBC cells.

10In summary, we identified that TAM-derived TGF-β1 induced ferroptosis resistance in TNBC cells and hence enhanced progression and chemoresistance by regulating the SMAD3/HLF/GGT1/GPX4 pathway. Reciprocally, elevated IL-6 expression by TAM-educated TNBC cells significantly promoted the recruitment of macrophages via JAK2/STAT3 axis in a feedback way (Fig. 2k).