Non-canonical Fzd7 signaling contributes to breast cancer mesenchymal-like stemness involving Col6a1

Mesenchymal-like stemness is characterized by epithelial-mesenchymal transition (EMT). Breast cancer (BC) cell mesenchymal-like stemness is responsible for distal lung metastasis. Interrogation of databases showed that Fzd7 was closely associated with a panel of mesenchymal-related genes and a panel of stemness-related genes. Fzd7 knockdown in mesenchymal-like MDA-MB-231 and Hs578T cells reduced expression of Vimentin, Slug and Zeb1, induced an epithelial-like morphology, inhibited cell motility, impaired mammosphere formation and decreased Lgr5+ subpopulation. In contrast, Fzd7 overexpression in MCF7 cells resulted in opposite changes. Fzd7 knockdown delayed xenograft tumor formation, suppressed tumor growth, and impaired lung metastasis. Mechanistically, Fzd7 combined with Wnt5a/b and modulated expression of phosphorylated Stat3 (p-STAT3), Smad3 and Yes-associated protein 1 (Yap1). Moreover, Fzd7-Wnt5b modulated expression of collagen, type VI, alpha 1 (Col6a1). Both Wnt5b knockdown and Col6a1 knockdown disrupted BC cell mesenchymal phenotype and stemness. Taken together, Fzd7 contributes to BC cell EMT and stemness, inducing tumorigenesis and metastasis, mainly through a non-canonical Wnt5b pathway. Col6a1 is implicated in Fzd7-Wnt5b signaling, and mediates Fzd7-Wnt5b -induced mesenchymal-like stemness. Video Abstract


In silico analysis
Cancer Cell Line Encyclopedia (CCLE) database and GSE12777 database were interrogated for gene expression in human BC cell lines. GSE2603 database was interrogated for gene expression in human BC tissues. Correlation between two genes was analyzed by Pearson statistics. Heat maps were generated by GraphPad Prism 7.0.

Cell transfection
BC cells were transfected with shRNA lentiviruses, and selected with 2 μg/ml puromycin (Invitrogen) 48 h posttransfection. Cells with gene stable knockdown were maintained in DMEM supplemented with 10% FBS and 2 μg/ml puromycin. MCF7 cells were transfected with FZD7 overexpression lentiviruses and selected with 1 μg/ ml puromycin. Cells with FZD7 stable overexpression were cultured in DMEM contained with 10% FBS and 1 μg/ml puromycin.

Immunofluorescence assay
Cells were grown on cover slips for 24 h, then washed twice with PBS and fixed by 4% paraformaldehyde solution. Cytomembranes were penetrated with 1% Triton-100 solution. Primary antibodies (Cell Signaling Technology, E-cadherin, #3195; Vimentin, #5741) were added at 4°C overnight. Cells were then incubated with Alexa fluor 488donkey anti-rabbit IgG(H + L) (Invitrogen, A21206) for 2 h, and with DAPI staining solution (Beyotime) for 5 min at room temperature in the dark. The fluorescence was visualized by a confocal microscope.

Wound healing assay
Cells were cultured in six-well plate. A pipette tip was used to scratch confluent monolayers. Cell debris was washed away with warm PBS. Then cells were cultured for 24 h in serum-free medium. An inverted microscope was used to capture the images.

Cell invasion assay
Cells were resuspended with serum-free medium and made into 1 × 10 5 /ml. 0.2 ml of the cell suspensions (2 × 10 4 cells) were added to the upper chamber of transwell plates (Corning Costar). In the lower chamber, 0.5 ml medium with 10% FBS was added to promote cell movement through the pores of the membrane. After 24 h, the cotton swab was used to clean the inside of the chamber. Migrated cells were fixed with a paraformaldehyde solution for 15 min and stained with the Crystal Violet. Images were captured using an inverted microscope.
Mammosphere assay 1 × 10 4 cells were seeded in 6-well Ultra-Low Attachment Surface Polystyrene culture plates (Corning Costar) added with complete MammoCult™ Human Medium (STEMCE LL Technologies). Cells were cultured at 37°C and 5% CO 2 for 8 days. Analysis was performed by counting the number of mammospheres in 5 randomly selected fields under an inverted microscope.

Flow cytometry
The proportion of Lgr5 + stem cells was determined by flowcytometry. Pre-cooled stain buffer (Invitrogen) was used to wash and resuspend cells. 1 × 10 6 cells were stained with 5 μl PE-conjugated anti-hLgr5 (Invitrogen) antibody on ice for 30 min. Stained populations were sorted and analyzed on BD Accuri C6 Plus (BD Biosciences).

In vivo study
All animal experiments were performed using 8-weekold NOD SCID mice. Mice were maintained in laminar flow rooms with constant temperature and humidity. 5 × 10 5 MDA-MB-231 cells transfected with FZD7 lentiviruses or control lentiviruses were injected into the fat pad of mice. Tumor growth was followed every 7 days by tumor diameter measurements using vernier calipers. Tumor volumes (V) were calculated using 1/2 × length×width 2 . Ten weeks after inoculation, tumors and lungs were obtained, and fixed in 4% paraformaldehyde solution. Lung samples were stained with HE and images were captured by using a positive microscope. All animal experiments were approved by the ethics committee of the China Medical University.

Immunohistochemistry
Immunohistochemistry (IHC) analysis was performed on 4 μm paraffin sections of human BC tissues and mouse xenograft tumors. Xylene and gradient alcohols were performed to deparaffinize and hydrate, respectively. 3% H 2 O 2 was added to the sections to remove endogenous peroxidase. Sections were incubated with citrate buffer to repair antigen, and blocked by BSA. Primary antibodies were used as follows: Fzd7 (Santa Cruz Biotechnology, sc-293,261), Col6a1 (Santa Cruz Biotechnology, sc-377,143), Ki67 (Invitrogen, 14,569,982), Cleaved caspase-3 (Cell Signaling Technology, #9661). After incubated with primary antibodies overnight at 4°C in a wet box, biotinylated secondary antibodies were added. Diaminobenzidine (BOS-TER) was used to stain the sections dissolved in Tris-HCl and H 2 O 2 . Then sections were re-stained in hematoxylin, dehydrated with gradient alcohol and xylene and sealed with cover slides.

Human specimens
Paraffin-embedded BC tissues including 10 invasive ductal BC (IDC), 5 invasive lobular BC (ILC) and 3 ductal BC in situ (DCIS) were obtained from Liaoning Province Tumor Hospital with the informed consent of the patients. The use of these specimens for research purposes was approved by Institutional Research Ethics Committee of China Medical University.

Co-immunoprecipitation
Cell lysates of MDA-MB-231 and Hs578T were centrifuged at 12000 rpm at 4°C. Fifty microliter supernatant was taken as normal sample and 20 μl protein A/G agarose beads (Santa Cruz Biotechnology, sc-2003) were added to the remaining supernatant and incubated for 30 min. After incubating and centrifuging, samples were divided into two tubes. Two microgram Fzd7 (R&D systems) and 2 μg mouse IgG (Santa Cruz Biotechnology) were added, respectively. Each tube sample was combined with 20 μl protein A/G agarose beads overnight. The non-specifically bound proteins were removed by washing the agarose beads. Following procedures were same as Western blot.

Statistical analysis
GraphPad Prism 7.0 was used to analyze the data, and all data were presented as the mean ± s.e.m. Comparison of means within two groups was analyzed using twotailed unpaired Student's t test. P < 0.05 was considered as significant.

Fzd7 is associated with mesenchymal phenotype
To investigate the association of Fzd7 with BC cell mesenchymal phenotype, we first interrogated Cancer Cell Line Encyclopedia (CCLE) database and GSE12777 database. It was shown that expression of FZD7 was positively correlated with that of mesenchymal-related genes, whereas negatively correlated with that of epithelial-related genes, in human BC cell lines ( Fig. 1a-b, Supplementary file 1: Fig. S1A-B). Expression of Fzd7 in some representative cell lines was subsequently determined. As shown by Western blot detection, Fzd7 was highly expressed in several mesenchymal-like cell lines such as MDA-MB-231, Hs578T and BT-549 (Fig. 1c).
Mammosphere formation test was then used to evaluate the role of Fzd7 in BC cell stemness. Fzd7 knockdown impaired mammosphere formation capacity of MDA-MB-231 cells and Hs578T cells, while Fzd7 overexpression promoted mammosphere formation in MCF7 cells (Fig. 2d-e, Supplementary file 1: Fig. S5A). Furthermore, Fzd7 knockdown reduced the fraction of Lgr5 + subpopulation in MDA-MB-231 cells and Hs578T cells, whereas Fzd7 overexpression increased the fraction of Lgr5 + subpopulation in MCF7 cells ( Fig. 2f-g, Supplementary file 1: Fig. S5B). Loss and gain of Fzd7 also affected CD44 expression in BC cells (Supplementary file 1: Fig. S5C-D). Moreover, Fzd7 knockdown had no effect on the subpopulation of aldehyde dehydrogenase 1 (ALDH1) which was recognized as a marker of epithelial-like stemness [11] (data not shown).

Fzd7 knockdown suppresses BC tumorigenesis and metastasis
To evaluate the effect of Fzd7 knockdown on BC tumorigenesis and metastasis in vivo, NOD SCID mice were inoculated with MDA-MB-231 cells transfected with FZD7 shRNA lentiviruses or control shRNA lentiviruses. Xenograft tumors were harvested and lung metastasis was analyzed 10 weeks after inoculation. Tumors derived from FZD7 shRNA-transfected cells grew much slower than those from control shRNA-transfected cells, indicating that Fzd7 knockdown inhibited tumor growth ( Fig. 3a-b). Moreover, Fzd7 knockdown delayed the appearance of xenograft tumors (Fig. 3c). Tumors derived from FZD7 shRNA-transfected cells exhibited less Ki67-positive cells but more Cleaved caspase-3-positive cells (Fig. 3d). Macro-metastasis on lung surface was found in 6 of 8 control mice, and in 3 of 8 mice inoculated with FZD7 shRNA-transfected cells (Fig. 3e). In mice without macro-metastasis, micro-metastasis existed in 2 control mice and 1 mouse inoculated with FZD7 shRNAtransfected cells (Fig. 3f).

Wnt5b is involved in BC cell mesenchymal phenotype and stemness
Since Wnt5b was a potential ligand for Fzd7, we subsequently determined the role of Wnt5b in BC cell mesenchymal phenotype and stemness. Interrogation of databases showed that WNT5B, similar to FZD7, was positively associated with mesenchymal-related genes, whereas negatively associated with epithelial-related genes (Supplementary file 1: Fig. S6A-B). Wnt5b knockdown reduced expression of Vimentin, Slug and Zeb1 in BC cells ( Fig. 5a-b, Supplementary file 1: Fig. S6C-D). Moreover, Wnt5b knockdown suppressed BC cell migration and invasion (Fig. 5c-d). Interrogation of databases also showed that WNT5B was associated with the panel of stemness-related genes (Fig. 5e-f). Consistently, Wnt5b knockdown impaired mammosphere formation capacity, reduced the fraction of Lgr5 + subpopulation and suppressed expression of CD44 in BC cells (Fig. 5g-h, Supplementary file 1: Fig. S7A-C).

Col6a1 is implicated in Fzd7 signaling
It was demonstrated that collagen signaling contributed to BC stemness and metastasis [19][20][21]. By interrogating databases, we identified the implication of Col6a1 in Fzd7 signaling. It was revealed that COL6A1 was associated with FZD7 and WNT5B in both BC cell lines and BC tissues (Fig. 6a-c). Immunohistochemistry detection revealed that Fzd7 and Col6a1 were expressed in a similar pattern in BC tissues (Fig. 6d). Expression of Col6a1 was downregulated in MDA-MB-231 cells with either Fzd7 knockdown or Wnt5b knockdown (Fig. 6e-f). Intriguingly, Col6a1 knockdown inhibited expression of Wnt5a/b in BC cells (Fig. 6g-h). Col6a1 knockdown also reduced expression of p-Stat3, Smad3 and Yap1 (Fig. 6g-h).

Col6a1 maintains BC cell mesenchymal phenotype and stemness
We finally assessed the role of Col6a1 in BC cell mesenchymal phenotype and stemness. Interrogation of databases showed that COL6A1 was positively associated with mesenchymal-related genes, whereas negatively associated with epithelial-related genes (Supplementary file 1: Fig. S8A-B). Col6a1 knockdown reduced expression of Vimentin, Slug and Zeb1 in BC cells (Fig. 7a-b, Supplementary file 1: Fig. S8C-D). Col6a1 knockdown inhibited BC cell migration and invasion (Fig. 7c-d). Interrogation of databases further showed that COL6A1 was also associated with the panel of stemness-related genes (Supplementary file 1: Fig. S8E-F). As expected, Col6a1 knockdown impaired mammosphere formation  (Fig. 7e-f). Moreover, Col6a1 knockdown reduced the fraction of Lgr5 + subpopulation (Fig. 7g-h).
Lgr5 is both a marker of adult stem cells and a modulator of their activity [34]. Lgr5 + mammary epithelial cells were shown sufficient and necessary for postnatal mammary organogenesis [35]. Single-cell analysis demonstrated that early metastatic BC cells highly expressed EMT and stemness -associated genes including LGR5  [36]. BC cell-derived Tenascin C (TNC) promoted Lgr5 expression, stemness and pulmonary metastasis [18]. Interrogation of CCLE and GSE2603 showed that FZD7 was associated with TNC and LGR5 in both BC cell lines and BC tissues. Furthermore, our study indicated that Fzd7 expression modulated Lgr5 + subpopulation. In consistent with our finding, Fzd7 was revealed enriched in Lgr5 + intestinal stem cells. Deletion or blocking of Fzd7 reduced the number of stem cells or impaired the function of stem cells [37,38]. Moreover, Wnt5a was shown to stimulate Lgr5 expression in osteoblasts [39].
Increasing evidence has indicated that collagen signaling promotes BC stemness and metastasis. Discoidin Domain Receptor 1 (DDR1), when exposed to its ligand Collagen I, induced BC cell stemness and lung metastasis in a Stat3-dependent manner [21]. Interrogation of databases showed that FZD7 was closely correlated with COL1A1 (data not shown). Activation of ANTXR1 by its ligand C5a, a fragment of Col6a3, also increased BC cell stemness and lung metastasis [19]. Interrogation of databases showed that FZD7 was associated with ANTXR1. These observations, in combine with our finding that Fzd7-Wnt5b modulated expression of Col6a1, indicate that non-canonical Fzd7 signaling cross-talked with Collagen signaling.

Conclusions
Our study identified that Fzd7 contributed to BC cell EMT and stemness, inducing tumorigenesis and metastasis, via a non-canonical Wnt5b pathway. We also discovered that Col6a1 was implicated in Fzd7-Wnt5b signaling, and mediated Fzd7-Wnt5b -induced mesenchymal-like stemness. These findings linked non-canonical Wnt pathway to Collagen signaling in BC progression. Mechanistically, we proposed an oncogenic network consisting of non-canonical Wnt, Collagen, IL-6/Stat3, TGF-β1/Smad3, EGFR and NOTCH2.