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Targeted inhibition of Wnt signaling with a Clostridioides difficile toxin B fragment suppresses breast cancer tumor growth [1]

['Aina He', 'Department Of Oncology', 'Shanghai Jiaotong University Affiliated Sixth People S Hospital', 'Shanghai', 'People S Republic Of China', 'Department Of Urology', 'Boston Children S Hospital', 'Harvard Medical School', 'Boston', 'Massachusetts']

Date: 2023-11

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 (TcdB FBD ), 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 TcdB FBD 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 TcdB FBD 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.

Funding: This study was partially supported by St. Baldrick’s Foundation (585350 to A.H.), Burroughs Wellcome Fund (to M.D.), Natural Science Foundation of Shanghai, China (16ZR1425900 to A.H.), National Natural Science Foundation of China (82173358 to A.H.), and US Department of Defense Breast Cancer Research Program Breakthrough Awards (W81XWH-15-1-0100 and W81XWH-18-1-0037 to Z.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All data is available in the main text or supplementary materials. All materials created in this study are available with material transfer agreements approved by Boston Children’s Hospital to any researcher for purposes of reproducing or extending the analysis. Original FCS files are publicly available: http://flowrepository.org/id/FR-FCM-Z6Q4 .

Taking advantage of this specific inhibitor and utilizing several breast cancer mouse models including a novel one that we recently established, we demonstrate that TcdB FBD suppresses tumor growth and cancer stem cell activities and synergizes with chemotherapy agent cisplatin in vivo, without side effects on bone density. These findings demonstrate both the feasibility and therapeutic value for narrow-spectrum Wnt signaling inhibitors for treating a broad range of breast cancers.

TcdB is a large protein (approximately 270 kDa) and recognizes multiple receptors via different domains. Previous studies have demonstrated that TcdB FBD region does not recognize any other potential receptors for TcdB including chondroitin sulfate proteoglycan 4 (CSPG4) [ 26 , 29 , 30 ], poliovirus receptor-related 3 (PVRL3) [ 31 ], and LDL receptor–related protein 1 (LRP1) [ 32 ]. Furthermore, it has been shown binding of TcdB FBD to FZD1/2/7 inhibits Wnt signaling mediated by these FZDs [ 26 ], demonstrating that TcdB FBD can serve as a highly specific narrow-spectrum Wnt signaling inhibitor against FZD1/2/7.

FZD1/2/7 form a subgroup with nearly identical CRDs within the FZD family and FZD7 has been previously shown to be associated with triple-negative breast cancer and targeting FZD7 may reduce growth of breast cancer cells [ 22 – 25 ]. Here, we have developed and evaluated a specific inhibitor targeting Wnt-FZD1/2/7 signaling utilizing a fragment of C. difficile toxin B (TcdB) that binds to FZD1/2/7 [ 26 , 27 ]. Our previous work established that TcdB recognizes FZD1/2/7 subgroup as its major receptors. This recognition is highly specific as only expression of FZD1/2/7 in cells mediated binding of TcdB, but not any other FZD members [ 26 ]. We have also solved the structure of a TcdB fragment containing FZD-binding domain (amino acid residues 1285–1804, designated TcdB FBD ) bound to FZD2-CRD, revealing that TcdB FBD effectively blocks Wnt signaling by targeting a region in CRD that is critical for docking of the palmitate in Wnt [ 27 ]. All Wnts are modified by lipidation through the addition of a palmitoleic acid (PAM) to a conserved serine, which is essential for their secretion and binding to FZDs. Binding of TcdB FBD prevents docking of the Wnt PAM into a hydrophobic groove in CRDs. Key residues for TcdB FBD interactions are conserved in CRD1, 2, and 7, but varies in other FZD members, which are the reasons for the selective high-affinity binding of TcdB FBD to CRD1/2/7 [ 27 , 28 ].

There are 10 FZD members in humans, divided into 4 subgroups (FZD1/2/7, 5/8, 3/6, 4/9/10) [ 15 ]. They contain only 1 relatively small extracellular domain on their N-termini, designated cysteine-rich domain (CRD, approximately 120 to 150 residues), which serves as the binding site for Wnt [ 15 ]. CRDs are highly conserved across all mammals, and therefore, it has been difficult to generate specific antibodies against CRDs by immunization in animals. Instead, antibodies targeting specific CRDs including FZD5-CRD and FZD7-CRD have been previously generated and validated by screening various antibody libraries in vitro [ 16 – 19 ]. The most advanced Wnt-signaling inhibition antibody, OMP-18R5 (developed by OncoMed), which was identified through in vitro phage-display approach and bind 5 FZDs across 2 subgroups (FZD1/2/7 and FZD5/8), went through Phase II clinical trial, but its development has been suspended due to side effects including loss of bone density in patients [ 9 , 10 , 20 ]. Bone density loss is also a primary side effect associated with an Fc fusion protein containing FZD8-CRD [ 13 , 21 ].

Wnt and its receptors, the 7-pass transmembrane protein Frizzled (FZDs), are a large family [ 8 , 15 ]. One way to minimize side effects is to target the specific Wnt-FZD pair(s) enriched in cancer cells. However, such narrow-spectrum inhibitors are difficult to develop due to high degrees of homology among Wnt-FZD members. There are also doubts on whether inhibiting a subgroup of Wnt-FZD pairs is sufficient to achieve any therapeutic effects in vivo.

Wnt/β-catenin signaling plays key roles in stem cell self-renewal and injury repair [ 8 ]. Mutations in components of the Wnt pathways are well-established as a dominant causal factor in colorectal cancer and many other solid tumors [ 8 , 9 ]. The therapeutic value of targeting Wnt signaling in these tumors are well-established using numerous animal models and by a number of broad-spectrum pan-Wnt signaling inhibitors [ 8 – 10 ]. Up-regulation of Wnt signaling without any mutations in the pathways has also been broadly implicated in cancer development, epithelial–mesenchymal transition (EMT), metastasis, chemotherapy-resistance, and immune escape of a broad range of human cancers [ 9 , 11 ], possibly reflecting a universal requirement of Wnt signaling in maintaining the stemness of cancer cells. For example, Wnt pathway activation has been previously reported to be enriched in basal-like breast cancer and predicts poor outcome [ 12 ], and activation of Wnt signaling (without mutations in Wnt pathways) is observed in >50% of human breast cancer cases and is linked to reduced overall survival [ 9 ]. This broad range of tumors potentially can benefit from Wnt signaling inhibition, but the side effect associated with pan-Wnt inhibition often diminishes the therapeutic value [ 13 , 14 ].

Breast cancers are heterogeneous and different subtypes require distinct treatments [ 1 , 2 ]. Targeted therapy in breast cancer is most successful when subtype-specific key pathways that drive cancer cell growth are defined and serve as therapeutic targets. Among breast cancer subtypes, estrogen receptor (ER) + luminal breast cancers are treated by endocrine therapy (e.g., aromatase inhibitor, tamoxifen) that targets the ER signaling pathway, whereas breast cancers with HER2 overexpression can be targeted by Trastuzumab (Herceptin), a monoclonal antibody that blocks HER2 signaling. Basal-like breast cancer largely overlaps with triple-negative breast cancer, which lacks ER and progesterone receptor (PR) expression and HER2 overexpression [ 3 ]. Basal-like/triple-negative breast cancers lack clear driver mutations, as evident from recent sequencing studies [ 4 ]. Thus, treatment of these breast cancers relies on standard chemotherapy, with the worst prognosis among all breast cancer subtypes [ 2 ]. In addition, even among luminal breast cancers, those belonging to the luminal B subtype have high proliferation index and often do not respond to endocrine therapy well; thus, they are treated by chemotherapy as well [ 5 ]. Chemotherapy can eliminate the bulk of cancer cells, but inevitably therapy-resistant cancer cells emerge, which typically possess stem cell-like properties [ 6 , 7 ]. In order to eliminate these cells, it is important to define key programs that sustain their stemness so that therapeutic approaches can be designed to target them.

Results

TcdBFBD inhibits growth of FZD7+ mammary tumors To test whether TcdBFBD could affect mammary tumors with FZD7 expression, we first treated tumor organoids derived from the above-described murine models with TcdBFBD or TcdBmu. Treatment with TcdBFBD, but not TcdBmu, reduced the viability of organoids formed from the p53/BRCA1-deficient or C3(1)-Tag tumor cells; in contrast, TcdBFBD treatment did not affect organoids formed from MMTV-PyMT luminal tumor organoids (Fig 3A). To further determine the specificity of TcdBFBD in inhibiting Wnt signaling, we used CHIR99021, a small-molecule inhibitor of glycogen synthase kinase-3 (GSK3), which activates Wnt/β-catenin signaling downstream of FZDs. We found that the growth inhibition of p53/BRCA1-deficient tumor organoids by TcdBFBD was rescued by CHIR99021 (S10A and S10B Fig), suggesting that TcdBFBD acts on tumor organoids by suppressing Wnt signaling. PPT PowerPoint slide

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TIFF original image Download: Fig 3. TcdBFBD inhibits growth of basal-like mammary tumors. (A) Organoids were seeded in a 48-well plate. TcdBFBD (150 nM) or TcdBmu (150 nM) were added to the culture medium once per day. PBS was used as a vehicle control. After 5 days treatment, the viability of organoids was assessed using the ATP release assay. Error bars indicate mean ± SD; N = 5 for p53/BRCA1-deficient organoid; N = 6 for C3(1)-Tag and MMTV-PyMT organoid; data were considered significant when p-value < 0.01 (Student’s t test). (B) Mice were subcutaneously transplanted with p53/BRCA1-deficient mammary tumor organoid cells (10,000 cells per mice) and treated for the indicated period when tumor volumes reached 50 mm3 (left panel). The averaged tumor volumes over time were measured and plotted (middle panel). Tumor tissues were harvested on day 24 and weighted (right panel). Error bars represent SEM of 8–10 independently injected mice. Numerical values are in S1 Data. https://doi.org/10.1371/journal.pbio.3002353.g003 Our previous study showed that the mutated mammary epithelial cells underwent luminal to basal/mesenchymal cell fate change in p53/BRCA1-deficient breast cancer mouse model [38]. We thus examined expression levels of several EMT-related and Wnt signaling-related genes upon TcdBFBD treatment. We found TcdBFBD-treated p53/BRCA1-deficient tumor organoids exhibited reduced expression of Wnt signaling-related genes (e.g., Axin2, Rnf43) and EMT-related genes (e.g., Vim, Zeb1) compared with the control and TcdBmu-treated organoids (S11A Fig). Next, we utilized TcdBFBD to evaluate whether inhibiting FZD7-mediated Wnt signaling may offer any therapeutic benefits in vivo. Subcutaneous injection of p53/BRCA1-deficient tumor organoid cells (1 × 104 cells) into nude mice resulted in robust tumor growth. When tumors reached approximately 50 mm3, TcdBFBD was injected i.p. into the recipient mice. TcdBFBD administration at a dose of 20 or 50 mg/kg with the intervals indicated in Fig 3B, but not that of TcdBmu, attenuated tumor growth, although the effect was modest (Fig 3B). At molecular levels, genes related to Wnt signaling and EMT were down-regulated in tumors from TcdBFBD-treated mice, compared with those in vehicle or TcdBmu-treated groups (S11B Fig). Consistently, expression of lymphocyte enhancer-binding factor 1 (LEF1), a representative Wnt signaling effector, was reduced at the protein level (S11C Fig). To further evaluate whether TcdBFBD could attenuate growth of human breast cancer cells, we took advantage of a recently established biobank of over 100 primary and metastatic human breast cancer organoid lines [50]. We chose 2 organoid lines from this biobank, 74T and 86T, which represent a luminal and a basal-like breast cancer line with low and high level of a BRCA1-deficiency signature (i.e., signature 3), respectively [50]. The luminal organoid line 74T was insensitive to TcdBFBD treatment (S12A Fig). In contrast, growth of the basal-like line 86T organoids was attenuated by TcdBFBD (S12B Fig). Similar to the p53/BRCA1-deficient xenograft model, the inhibitory effect is rather modest and lacks a dose-dependency for unknown reasons. Nevertheless, these findings suggest that selective inhibition of FZD7-mediated signaling is sufficient to exhibit an inhibitory effect on growth of tumor cells, although Wnt signaling is not likely a driving force but rather one of the contributing factors in tumorigenesis of human breast cancers.

TcdBFBD treatment does not affect the intestine and bones We next analyzed whether inhibiting FZD7-mediated signaling by TcdBFBD at therapeutically effective doses is tolerated in Wnt sensitive tissues such as the intestine and bones. TcdBFBD was injected into mice at 20, 50, or 100 mg/kg twice a week for 5 weeks. These mice showed similar weight gains comparable with the control mice (Fig 4A). To examine the potential impact on the intestine, we injected (i.p.) EdU, which incorporates into replicating DNAs and marks proliferating cells, at the end of the fifth week. The intestine epithelium constantly turns over and newly generated cells are produced from stem cells located the bottom of the crypt region. Wnt signaling is a key pathway regulating intestinal stem cells and a reduction in EdU incorporation would reflect an inhibition on stem cell activity [51,52]. The intestinal tissues were dissected out and EdU incorporation was measured. As shown in Fig 4B, TcdBFBD treatment at 20 and 50 mg/kg levels did not reduce EdU levels in the intestinal tissues. There appears to be a slight reduction at 100 mg/kg, but it did not reach statistical significance. PPT PowerPoint slide

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TIFF original image Download: Fig 4. TcdBFBD treatment in vivo does not affect the intestine and bones. (A) Six weeks old female nude mice (Hsd: Athymic Nude-Foxn1nu) were injected (i.p.) with the TcdBFBD (20, 50, or 100 mg/kg) or TcdBmu (20 mg/kg) twice a week for 5 weeks. Their body weight gains were monitored and plotted. Error bars indicate mean ± SEM, n = 8–10 mice. (B) Mice were treated with TcdBFBD as described in panel A. EdU was injected (i.p., 100 mg/kg) 12 h before euthanization. Intestine tissues were harvested, fixed, and analyzed. The representative images were shown in the left panel and quantification of the percentage of EdU-positive cells per crypt was plotted in the right panel. Scale bar = 200 μm. P = 0.28. (C) Mice were treated with TcdBFBD as described in panel A and their right femur bones were extracted, fixed, and subjected to micro-computed tomography (μCT) analysis. Representative μCT reconstructions of the midshaft femur cortical bone (upper row), distal femur metaphyseal bone (middle row), and entire distal femur with the ventral half of the femur digitally removed to reveal the cancellous compartment (lower row) are shown. N = 6 /dose group. (D) Quantification of the μCT analysis described in panel C for trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and cortical area (Ct.Ar), P = 0.67, 0.63. 0.68, 0.67, respectively. Numerical values are in S1 Data. https://doi.org/10.1371/journal.pbio.3002353.g004 Clinical trials with OMP-18R5, which blocks FZD1/2/7 and 5/8, revealed that bone density loss is the single most critical side effect in humans [13]. Consistently, mice treated with pan-Wnt signaling inhibitors (Porcupine inhibitors LGK974 and ETC-1922159) exhibited loss of bone volume and density even at doses <10 mg/kg [53]. We thus focused our analysis on bone mass and architecture utilizing micro-computed tomography (μCT) technology in mice treated with TcdBFBD at 20, 50, and 100 mg/kg doses for 5 weeks. No differences were observed in cancellous or cortical microstructure in the femur (Fig 4C). Bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) in the distal femur metaphysis were not affected by TcdBFBD, neither were cortical bone parameters, e.g., cortical thickness (Ct.Th) (Fig 4D). These data suggest that TcdBFBD may have a higher therapeutic safety window compared with LGK974, although additional pharmacokinetic studies would be required to fully examine the effective and safety doses of TcdBFBD.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002353

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