TLR4 ligand/H₂O₂ enhances TGF-β1 signaling to induce metastatic potential of non-invasive breast cancer cells by activating non-Smad pathways

Abstract

TGF-β1 has the potential to activate multiple signaling pathways required for inducing metastatic potential of tumor cells. However, TGF-β1 was inefficient in inducing metastatic potential of many non-invasive human tumor cells. Here we report that the enhancement of TGF-β1 signaling is required for inducing metastatic potential of non-invasive breast cancer cells. TGF-β1 alone could not efficiently induce the sustained activation of Smad and non-Smad pathways in non-invasive breast cancer cells. TLR4 ligand (LPS) and H₂O₂ cooperated with TGF-β1 to enhance the sustained activation of non-Smad pathways, including p38MAPK, ERK, JNK, PI3K, and NF-κB. The activation of MAPK and PI3K pathways resulted in a positive feed-back effect on TGF-β1 signaling by down-regulating Nm23-H1 expression and up-regulating the expression of TβRI and TβRII, favoring further activation of multiple signaling pathways. Moreover, the enhanced TGF-β1 signaling induced higher expression of SNAI2, which also promoted TβRII expression. Therefore, the sustained activation levels of both Smad and non-Smad pathways were gradually increased after prolonged stimulation with TGF-β1/H₂O₂/LPS. Consistent with the activation pattern of signaling pathways, the invasive capacity and anoikis-resistance of non-invasive breast cancer cells were gradually increased after prolonged stimulation with TGF-β1/H₂O₂/LPS. The metastatic potential induced by TGF-β1/H₂O₂/LPS was sufficient for tumor cells to extravasate and form metastatic foci in an experimental metastasis model in nude mice. The findings in this study suggested that the enhanced signaling is required for inducing higher metastatic capacity of tumor cells, and that targeting one of stimuli or signaling pathways might be potential approach in comprehensive strategy for tumor therapy.

Figure 1. TGF-β1/H₂O₂/LPS facilitates invasive capability of non-invasive breast cancer cells.

(A) MCF-7 and T-47D cells were cultured in absence or presence of TGF-β1 (5 ng/ml)/H₂O₂ (50 µM)/LPS (100 ng/ml) for the indicated time, and then used for Matrigel invasion assay. (B–D) Tumor cells were cultured in absence or presence of TGF-β1, H₂O₂ and LPS for 8 days. The cells were then used for following experiments. (B) The cells were used for Matrigel invasion assay. (C) The expression of αvβ3 was analyzed by real-time RT-PCR and flow cytometry. αvβ3 expression index was calculated as described in Materials and Methods. (D) The cells were then cultured in presence of matrigel for 48 h. The mRNA level of MMP9 was detected by real-time RT-PCR. The MMP-9 in supernatants was detected by zymography assay, and the fold difference of active MMP-9 was calculated after densitometric analysis of the gel. P values, *P<0.05, **P<0.01.

Figure 2. Co-stimulation with TGF-β1/H₂O₂/LPS enhances the sustained activation of non-Smad pathways.

(A) MCF-7 cells were cultured for 7 days in absence or presence of TGF-β1 (5 ng/ml), H₂O₂ (50 µM), and LPS (100 ng/ml). The phosphorylated and un-phosphorylated p38MAPK, ERK, JNK, and Akt were detected by Western blot. The activity of NF-κB was assayed as described in Methods. (B) MCF-7 cells were cultured for the indicated time in absence or presence of TGF-β1 and/or H₂O₂/LPS. The ratio of phosphorylated and un-phosphorylated p38MAPK, ERK, JNK, and Akt was calculated after densitometric analysis of Western blots. The activity of NF-κB was assayed as described in Methods. P values, *P<0.05, **P<0.01.

Figure 3. H₂O₂ and LPS enhance the sustained activation of Smad pathway.

(A) MCF-7 cells were stimulated with TGF-β1 (5 ng/ml). Phospho-Smad2, Smad2, phospho-Smad3 and Smad3 were detected by Western blot at the indicated time points. (B) MCF-7 cells were cultured in absence or presence of TGF-β1, H₂O₂ (50 µM), and LPS (100 ng/ml) for 7 days. Phospho-Smad2, Smad2, phospho-Smad3 and Smad3 were detected by Western blot. (C) MCF-7 cells were cultured in presence of TGF-β1/H₂O₂/LPS. Phospho-Smad2, Smad2, phospho-Smad3, Smad3, Smad7, and β-actin were detected by Western blot at the indicated time points. Smad4 in nuclear extract was detected by Western blot. Lamin B1, a nuclear protein, in nuclear extract was used as control. (D) MCF-7 cells were cultured in absence or presence of TGF-β1/H₂O₂/LPS (left) for the indicated time. Or the cells were cultured for 6 days in absence or presence of TGF-β1, H₂O₂, and LPS (right). The expression of SMAD7 (D) and SNAI2 (E) were detected by real-time RT-PCR. P values, *P<0.05, **P<0.01.

Figure 4. TGF-β1/H₂O₂/LPS down-regulates Nm23-H1 expression and up-regulates the expression of TGF-β receptors.

(A–C) MCF-7 cells were cultured in absence or presence of TGF-β1/H₂O₂/LPS (left) for the indicated time. Or the cells were cultured for 6 days in absence or presence of TGF-β1, H₂O₂, and LPS (right). The expression of TGFBR1 (A), and TGFBR2 (B) was detected by real-time RT-PCR. The expression of TβRI and TβRII was detected by Western blot after 6-d culture (C). (D) MCF-7 cells were cultured for 6 days in absence or presence of TGF-β1, H₂O₂, and LPS. The expression of Nm23-H1 and EDG2 was detected by Western blot. (E) MCF-7 cells were un-stimulated or stimulated for 6 days with TGF-β1/H₂O₂/LPS in absence or presence of wortmannin (WT, 40 nM), SB203580 (SB, 10 µM), PD98059 (PD, 10 µM), SP600125 (SP, 10 µM) or QNZ (40 nM). The expression of TGFBR1, TGFBR2 and NM23-H1 was detected by real-time RT-PCR. (F and G) Control MCF-7 cells and the MCF-7 cells expressing control shRNA or SNAI2 shRNA were untreated or treated with TGF-β1/H₂O₂/LPS for 6 days. SNAI2 expression was detected by Western blot (F). The expression of TGFBR2 was detected by real-time RT-PCR (G). P values, *P<0.05, **P<0.01.

Figure 5. TGF-β1/H₂O₂/LPS promotes anoikis-resistance of non-invasive breast cancer cells.

MCF-7 and T-47D cells were cultured for 8 days in absence or presence of TGF-β1 (5 ng/ml), H₂O₂ (50 µM), and LPS (100 ng/ml). The cells were then used in following experiments. (A) The cells were transferred to poly-HEMA-coated plate and cultured for 24 h. The apoptosis of the cells was analyzed by flow cytometry. (B) The cells were then cultured in soft agar for 3 weeks. The colonies were counted. (C) MCF-7 cells were used for the assay of the expression of Bax, Bim, Bid, Bcl-2, Mcl-1 and c-FLIP. The expression of these genes was detected by real-time RT-PCR and Western blot. P values, *P<0.05, **P<0.01.

Figure 6. TGF-β1/H₂O₂/LPS promotes the capacity of tumor cells to extravasate and form metastatic foci.

(A) MCF-7 and T-47D cells were cultured for 8 days in absence or presence of TGF-β1 (5 ng/ml) and/or H₂O₂ (50 µM)/LPS (100 ng/ml). The cells were then labeled with CFSE and injected to mice via tail vein. Mice were sacrificed 5 h and 24 h after injection. The CFSE-labeled tumor cells in frozen sections were visualized by fluorescence microscopy (left). Fluorescent spots in the frozen sections of lung tissues were counted (right). (B–D) MCF-7 and T-47D cells were untreated or pre-treated for 8 days with TGF-β1/H₂O₂/LPS. The cells were then injected to mice via tail vein. The mice without inoculation with tumor cells were used as control. Mice (n = 6 per group) were sacrificed on d21 after inoculation (B) or at the indicated time after inoculation (C). The sections of lung tissues were prepared and subjected to immunohistochemical analysis. The tissue sections were stained with anti-human MGB1 antibody (B) or anti-human Ki-67 antibody (C). In each photograph in C, 2 representative sites (indicated by arrows) were further amplified to show the expression of Ki-67 in tumor cells (indicated by arrows). The immunohistochemical staining intensity of Ki-67 was measured using the HSCORE scoring system (right) as described in Methods. (D) The mice (n = 6 per group) were sacrificed 8 weeks after inoculation. The sections of lung tissues were prepared and subjected to H&E staining. Insets are the high-power view of metastatic foci in corresponding picture indicated by arrows. Representative photographs are shown (B–D). (E) MCF-7 and T-47D cells were untreated (un) or pre-treated for 8 days with TGF-β1/H₂O₂/LPS. The cells were then cultured in absence of stimuli for indicated time. The expression of Nm23-H1 and EDG2 was detected by Western blot. P values, **P<0.01.