Targeted inhibition of Wnt signaling with a bacterial toxin fragment suppresses breast cancer tumor-initiating/chemo-resistant cells

doi:10.21203/rs.3.rs-58989/v1

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Targeted inhibition of Wnt signaling with a bacterial toxin fragment suppresses breast cancer tumor-initiating/chemo-resistant cells

https://doi.org/10.21203/rs.3.rs-58989/v1

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BRCA1 germ-line mutations are a major cause of hereditary breast cancer and BRCA1-deficient breast cancer shares many characteristics as sporadic basal-like breast cancer (BLBC). Effective therapeutic targets for BRCA1-deficient BLBC remain lacking. By utilizing a BRCA1-deficient BLBC mouse model based on intraductal injection of Krt8-Cre adenovirus to inactivate Brca1 and Trp53 in luminal mammary epithelial cells, here we report that the Wnt receptor Frizzled 7 (FZD7) serves as a biomarker and therapeutic target in the resulting mammary tumor cells and is particularly enriched in cancer stem cells / tumor-initiating cells (CSCs/TICs). Inhibiting FZD7-mediated Wnt signaling using a nontoxic FZD-binding fragment of C. difficile toxin B (TcdBFBD) attenuates growth of BRCA1-deficient tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Finally, FZD1/2/7-positive cells are enriched in chemotherapy-resistant cells in both BLBC and luminal breast tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These findings demonstrate the therapeutic value for targeting FZD1/2/7 in treating breast cancers and establish TcdBFBD as a potential therapeutic agent targeting TICs and chemotherapy-resistant cancer cells.

Cancer Biology

Stem Cell & Developmental Cell Biology

breast cancer

BRCA1

Wnt signaling

mammary tumor cells

Yes there is potential Competing Interest. A patent application on utilizing TcdB fragments for inhibiting Wnt signaling has been filed by Boston Children’s Hospital, with M.D., R.J., L.T., A.H., and P.C. as inventors.

FigS1final.jpg
Expression levels of FZDs in human breast cancers. Expression levels of FZDs in different subtypes of human breast cancer based on bc-GenExMiner online tool.
FigS2.png
Expression levels of FZDs in murine breast cancer models. High expression levels of Fzd7 and Fzd6 in mouse models for human BLBC/TNBC; data was based on GEO accession # GSE25488 (in heatmap, red to blue represents highest to lowest expression levels). The first five from the left side (marked as mammary tumor tissue rep1-5) represent the C3(1)-Tag model.
FigS3.png
The percentage of FZD7+ cells are assessed in three breast cancer models. (A) Schematic diagram showing the generation of a p53/BRAC1-deficient breast cancer model by intraductal injection of Ad-K8-Cre into Trp53L/L;Brca1L/L;R26Y female mice. (B) Kaplan-Meier tumor-free results showing that Trp53L/L;Brca1L/L;R26Y female mice (n=17) injected with Ad-K8-Cre developed malignant mammary tumors over time after Ad-K8-Cre injection. (C) Representative FACS plots of FZD7+ cells from normal mammary glands, p53/BRCA1-deficient, MMTV-PyMT, and C3(1)-Tag tumors.
FigS4.png
TcdBFBD inhibits Wnt signaling in human breast cancer cell lines. (A) Left panel: Phylogenetic analysis of human FZD proteins (15); Right panel: qRT–PCR analysis of Fzds in MDA-MB-231 cells. (B) Cell viability measured by the MTT assay showed that TcdBFBD did not exhibit cytotoxicity to the indicated human cell lines (error bars indicate mean ± SEM, three independent experiments). (C) Representative images of clonogenic growth of PaTu8988s and HPAF-II cells cultured in the presence of TcdBFBD (150 nM) or LGK974 (100 nM). (D) Representative images of sphere formation assay in PaTu8988s and HPAF-II cells cultured in the presence of TcdBFBD (150 nM) or LGK974 (100 nM). (E) Quantitation of colony numbers and sphere formation from (C-D), error bars indicate mean ± SEM, n= 4.
FigS5.png
TcdBFBD inhibits Wnt 956 signaling in p53/BRCA1-deficient mammary tumor cells. (A) Experiments were carried out as described in Fig. 3A. qRT-PCR analysis showing downregulation of Wnt signaling-related genes (Axin2 and Rnf43) and EMT associated genes (Vim and Zeb1) in tumor organoids upon TcdBFBD treatment. (B) Experiments were carried out as described in Fig. 3D. qRT-PCR analysis showing downregulation of Wnt associated genes (Axin2 and Rnf43) and EMT-associated genes (Vim and Zeb1) in TcdBFBD treated xenograft tumors. (C) Experiments were carried out as described in Fig. 3D. Representative immunostaining images of LEF1 expression (green) in different treatment groups are shown (Control, TcdBFBD or TcdBmu). Scale bar = 50 μm.
FigS6.png
TcdBFBD inhibits growth of human TNBC organoids. The viability of two lines of human breast cancer organoids, a luminal tumor line 74T (panel A) and a BL tumor line 86T (panel B), were exposed to the indicated concentrations of TcdBFBD or TcdBmu. Cell viability was assessed using the CellTiter-Glo luminescent assay.
FigS7.png
TICs in p53/BRCA1-deficient mouse mammary tumors are largely FZD7+. (A) FACS analysis using FITC-TcdBFBD and anti-FZD7 antibody showed that primary p53/BRCA1-deficient tumors cells targeted by TcdBFBD are largely FZD7+. (B) Representative images showing the tumorspheres formed from p53/BRCA1-deficient tumor cells. (C) Tumorsphere cells cultured from p53/BRCA1-deficient tumors were examined by FACS analysis using an anti-FZD7 antibody. (D) Representative images showing the tumor organoid formed from p53/BRCA1-deficient tumor cells. (E) FACS analysis of FZD7+ cells in the tumor organoids described in (D) showing that most organoid cells express FZD7.
FigS8.png
TcdBFBD and cisplatin showed synergistic effect in inhibiting BL mammary tumor organoids. (A) Representative images of p53/BRCA1-deficient mouse mammary tumor organoids treated with TcdBFBD alone (150 nM), cisplatin alone (0.2 μM), or a combination of both. (B) Quantitation of organoids sizes for the indicated treatment groups in (A). (C) Quantitation of organoid numbers for the indicated groups in (A). (D) Representative pictures of C3(1)-Tag mouse mammary tumor organoids treated with TcdBFBD alone, cisplatin alone, or a combination of both. (E) Quantitation of organoids sizes for the indicated treatment groups in (D). (F) Quantitation of organoid numbers for the indicated groups in (D). Scale bar = 100 μm.

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Targeted inhibition of Wnt signaling with a bacterial toxin fragment suppresses breast cancer tumor-initiating/chemo-resistant cells

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