Targeted inhibition of Wnt signaling with a Clostridioides difficile toxin B fragment suppresses breast cancer tumor growth

Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of C. difficile toxin B (TcdBFBD), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdBFBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7–positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.

[1]  Jingze Liu,et al.  Low-density lipoprotein receptor-related protein 1 is a CROPs-associated receptor for Clostridioides infection toxin B , 2021, Science China Life Sciences.

[2]  Lan Huang,et al.  Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection , 2021, Nature Communications.

[3]  W. Muller,et al.  Insights from transgenic mouse models of PyMT-induced breast cancer: recapitulating human breast cancer progression in vivo , 2020, Oncogene.

[4]  Myan Do,et al.  Selective activation of FZD7 promotes mesendodermal differentiation of human pluripotent stem cells , 2020, bioRxiv.

[5]  Gang Huang,et al.  A novel humanized Frizzled-7-targeting antibody enhances antitumor effects of Bevacizumab against triple-negative breast cancer via blocking Wnt/β-catenin signaling pathway , 2020, Journal of experimental & clinical cancer research : CR.

[6]  W. Yeh,et al.  Development of Potent, Selective Surrogate WNT Molecules and Their Application in Defining Frizzled Requirements. , 2020, Cell chemical biology.

[7]  D. Virshup,et al.  Wnt Signaling and Drug Resistance in Cancer , 2019, Molecular Pharmacology.

[8]  Y. Shaked.  The pro-tumorigenic host response to cancer therapies , 2019, Nature Reviews Cancer.

[9]  J. Moffat,et al.  Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice , 2019, eLife.

[10]  L. Wessels,et al.  Loss of p53 triggers Wnt-dependent systemic inflammation to drive breast cancer metastasis , 2019, Nature.

[11]  Benjamin G. Bitler,et al.  Activation of Wnt signaling promotes olaparib resistant ovarian cancer , 2019, Molecular carcinogenesis.

[12]  H. Clevers,et al.  Inadequate DNA Damage Repair Promotes Mammary Transdifferentiation, Leading to BRCA1 Breast Cancer , 2019, Cell.

[13]  S. Whelan,et al.  Sulfated glycosaminoglycans and low-density lipoprotein receptor contribute to Clostridium difficile toxin A entry into cells , 2019, Nature Microbiology.

[14]  J. Moffat,et al.  Structure-guided design fine-tunes pharmacokinetics, tolerability, and antitumor profile of multispecific frizzled antibodies , 2019, Proceedings of the National Academy of Sciences.

[15]  M. Iorio,et al.  WNT signaling modulates PD-L1 expression in the stem cell compartment of triple-negative breast cancer , 2019, Oncogene.

[16]  R. Jin,et al.  Structural insight into Wnt signaling inhibition by Clostridium difficile toxin B , 2018, The FEBS journal.

[17]  Gang Huang,et al.  A novel recombinant human Frizzled-7 protein exhibits anti-tumor activity against triple negative breast cancer via abating Wnt/β-catenin pathway. , 2018, The international journal of biochemistry & cell biology.

[18]  T. Reya,et al.  Stem cell fate in cancer growth, progression and therapy resistance , 2018, Nature Reviews Cancer.

[19]  K. Kalland,et al.  Targeting Wnt/β-Catenin Signaling for Cancer Immunotherapy. , 2018, Trends in pharmacological sciences.

[20]  G. Loots,et al.  Sclerostin neutralization unleashes the osteoanabolic effects of Dkk1 inhibition. , 2018, JCI insight.

[21]  Cassandra R Diegel,et al.  Bone loss from Wnt inhibition mitigated by concurrent alendronate therapy , 2018, Bone Research.

[22]  K. Lam,et al.  Structural basis for recognition of frizzled proteins by Clostridium difficile toxin B , 2018, Science.

[23]  Joshua M. Stuart,et al.  Cell-of-Origin Patterns Dominate the Molecular Classification of 10,000 Tumors from 33 Types of Cancer , 2018, Cell.

[24]  Hans Clevers,et al.  A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity , 2018, Cell.

[25]  R. Nusse,et al.  Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities , 2017, Cell.

[26]  Karen Blyth,et al.  In vivo models in breast cancer research: progress, challenges and future directions , 2017, Disease Models & Mechanisms.

[27]  Hans Clevers,et al.  Genome-wide CRISPR screens reveal a Wnt–FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors , 2016, Nature Medicine.

[28]  A. Brass,et al.  Frizzled are colonic epithelial receptors for Clostridium difficile toxin B , 2016, Nature.

[29]  M. Boutros,et al.  Wnt signaling in cancer , 2016, Oncogene.

[30]  N. Rosenfeld,et al.  The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes , 2016, Nature Communications.

[31]  Jinsong Sheng,et al.  Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity , 2015, Proceedings of the National Academy of Sciences.

[32]  T. Gajewski,et al.  Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity , 2015, Nature.

[33]  Annelie Abrahamsson,et al.  CCL2 and CCL5 Are Novel Therapeutic Targets for Estrogen-Dependent Breast Cancer , 2015, Clinical Cancer Research.

[34]  H. Clevers,et al.  Frizzled7 Functions as a Wnt Receptor in Intestinal Epithelial Lgr5+ Stem Cells , 2015, Stem cell reports.

[35]  L. Castellano,et al.  Mammosphere formation assay from human breast cancer tissues and cell lines. , 2015, Journal of visualized experiments : JoVE.

[36]  A. Jimeno,et al.  Targeting the Wnt pathway in human cancers: therapeutic targeting with a focus on OMP-54F28. , 2015, Pharmacology & therapeutics.

[37]  H. Feng,et al.  Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B , 2014, Cell Research.

[38]  Dino Amadori,et al.  ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling , 2014, Nature Cell Biology.

[39]  Michael Kahn,et al.  Can we safely target the WNT pathway? , 2014, Nature Reviews Drug Discovery.

[40]  Zhe Li,et al.  Lineage Tracing of Mammary Epithelial Cells Using Cell-Type-Specific Cre-Expressing Adenoviruses , 2014, Stem cell reports.

[41]  W. Sellers,et al.  Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974 , 2013, Proceedings of the National Academy of Sciences.

[42]  Jason I. Herschkowitz,et al.  Transcriptomic classification of genetically engineered mouse models of breast cancer identifies human subtype counterparts , 2013, Genome Biology.

[43]  J. Seidman,et al.  An RNA‐seq protocol to identify mRNA expression changes in mouse diaphyseal bone: Applications in mice with bone property altering Lrp5 mutations , 2013, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[44]  Xi He,et al.  Frizzled and LRP5/6 receptors for Wnt/β-catenin signaling. , 2012, Cold Spring Harbor perspectives in biology.

[45]  N. Caplen,et al.  Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer , 2012, Breast Cancer Research.

[46]  Steven J. M. Jones,et al.  Comprehensive molecular portraits of human breast tumors , 2012, Nature.

[47]  M. Fischer,et al.  Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors , 2012, Proceedings of the National Academy of Sciences.

[48]  R. Clarke,et al.  A Detailed Mammosphere Assay Protocol for the Quantification of Breast Stem Cell Activity , 2012, Journal of Mammary Gland Biology and Neoplasia.

[49]  F. Markowetz,et al.  The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups , 2012, Nature.

[50]  Ben Tran,et al.  Luminal-B breast cancer and novel therapeutic targets , 2011, Breast Cancer Research.

[51]  K. Zhang,et al.  FZD7 has a critical role in cell proliferation in triple negative breast cancer , 2011, Oncogene.

[52]  E. Lander,et al.  Genetic predisposition directs breast cancer phenotype by dictating progenitor cell fate. , 2011, Cell stem cell.

[53]  M. Pegram,et al.  Triple negative breast cancer: unmet medical needs , 2011, Breast Cancer Research and Treatment.

[54]  A. Ashworth,et al.  BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells. , 2010, Cell stem cell.

[55]  S. Fox,et al.  Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers , 2009, Nature Medicine.

[56]  H. Clevers,et al.  Wnt signaling in the intestinal epithelium: from endoderm to cancer. , 2005, Genes & development.

[57]  R. Tibshirani,et al.  Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[58]  Christian A. Rees,et al.  Molecular portraits of human breast tumours , 2000, Nature.

[59]  Cheryl Jorcyk,et al.  The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma , 2000, Oncogene.

[60]  R. Kurzrock,et al.  Targeting the Wnt/beta-catenin pathway in cancer: Update on effectors and inhibitors. , 2018, Cancer treatment reviews.

[61]  C. Alexander.  The Wnt Signaling Landscape of Mammary Stem Cells and Breast Tumors. , 2018, Progress in molecular biology and translational science.