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Genetic factors and estrogen deficiency contribute to the development of osteoporosis. The single-nucleotide polymorphism (SNP) rs2887571 is predicted from genome-wide association studies (GWASs) to associate with osteoporosis but has had an unknown mechanism. Analysis of osteoblasts from 110 different individuals who underwent joint replacement revealed that the genotype of rs2887571 correlates with WNT5B expression. Analysis of our ChIP-sequencing data revealed that SNP rs2887571 overlaps with an estrogen receptor alpha (ERα) binding site. Here we show that 17β-estradiol (E2) suppresses WNT5B expression and further demonstrate the mechanism of ERα binding at the enhancer containing rs2887571 to suppress WNT5B expression differentially in each genotype. ERα interacts with NFATc1, which is predicted to bind directly at rs2887571. CRISPR-Cas9 and ChIP-qPCR experiments confirm differential regulation of WNT5B between each allele. Homozygous GG has a higher binding affinity for ERα than homozygous AA and results in greater suppression of WNT5B expression. Functionally, WNT5B represses alkaline phosphatase expression and activity, decreasing osteoblast differentiation and mineralization. Furthermore, WNT5B increases interleukin-6 expression and suppresses E2-induced expression of alkaline phosphatase during osteoblast differentiation. We show that WNT5B suppresses the differentiation of osteoblasts via receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2), which activates DVL2/3/RAC1/CDC42/JNK/SIN3A signaling and inhibits β-catenin activity. Together, our data provide mechanistic insight into how ERα and NFATc1 regulate the non-coding SNP rs2887571, as well as the function of WNT5B on osteoblasts, which could provide alternative therapeutic targets for osteoporosis.

Osteosarcoma is a pediatric bone cancer that has no targeted therapy and has had no treatment advances for the last three decades. Osteosarcoma frequently metastasizes to the lungs, reducing patient survival with metastatic disease to 20% after 5 years. The goal of this study is to describe the function of WNT5B and its signaling pathway in osteosarcoma stem cells, metastasis and chemoresistance and its potential as a therapeutic target. Using RNA sequencing from publicly available datasets and immunohistochemistry on tumor microarrays, we reveal that WNT5B is the most expressed WNT in osteosarcoma patients and correlates with both metastasis and survival. Osteosarcoma is thought to result from a block in differentiation, and based on WNT5B’s role in inhibiting osteoblast differentiation of mesenchymal stem cells from normal bone, we hypothesized a role for WNT5B in osteosarcoma stem cells. In spheroids, we show that both protein and mRNA levels of WNT5B are enhanced in the stem cell population compared to adherent cells. We found that WNT5B upregulates the expression of the stemness gene SOX2 and directs stemness phenotypes, such as sphere forming efficiency, proliferation and migration. We show a reduction in sphere forming efficiency with 143B-WNT5B-knockdown cells compared to parental control cells which can be rescued with re-introduction of WNT5B. Additionally, we show that WNT5B drives proliferation and migration of osteosarcoma stem cells through increasing sphere size and capability to migrate on collagen matrix. Further, WNT5B enhances osteosarcoma chemoresistance to methotrexate. Through revealing a novel role for WNT5B in osteosarcoma cancer stem cells and therapy resistance, we present the WNT5B pathway as a candidate for therapeutically targeting osteosarcoma stem cells in patients. Citation Format: Rachel S. Perkins, Sarocha Suthon, Gustavo A. Miranda-Carboni, Susan A. Krum. WNT5B drives osteosarcoma stemness, metastasis and chemoresistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3527.

Triple-negative breast cancer (TNBC) has the worst survival outcome and the greatest incidence of metastasis among breast cancer subtypes. We have shown that the WNT10B network, composed of β-CATENIN, HMGA2, and EZH2, is predictive of higher rates of TNBC metastasis and poorer overall survival in TNBC. The stroma-tumor microenvironment (TME) interactions in metastatic breast cancer remain poorly defined, and prior to our work, the role of WNT10B signaling in the TME was unknown. Global gene expression profiling of the Wnt10bKO total mammary glands demonstrated a gene signature consistent with altered metabolism and markers promoting adipocyte differentiation. Wnt10b ablation in the mammary gland increases adipogenic differentiation and decreases the number of mammary gland terminal-end buds, and epithelia to stroma/fibroblast cell ratios. Therefore, we questioned the role of WNT10B in stromal cancer-associated fibroblast (CAFs) and cancer-associated adipocytes (CAAs) in the TME of transformed mammary glands. By backcrossing our Wnt10bKO mice to the highly metastatic MMTV-PyMT (PyMT) transgenic mammary tumor model, we demonstrated alteration in the age of tumor onset, changes in the incidence of transformation of thoracic and abdominal-inguinal tumors, and impacts on survival rates. Moreover, adipogenic markers are higher and persist through transformation in PyMTWnt10bKO mice compared to PyMT mice. Proliferation, migration, and invasion are also impacted in the PyMTWnt10bKO CAFs when co-cultured with PyMT tumor cells. The ability of PyMTWnt10bKO CAFs to alter cellular plasticity and generate CAAs is higher than PyMT CAFs. Additionally, PyMTWnt10bKO CAFs lose expression of canonical Wnt markers DVL and LRP and gain non-canonical Wnt signaling via WNT5B/ROR pathways that persist in the KO CAAs. We also observed alterations in chemoresistance in KO tumor epithelium. The resistance to paclitaxel from enriched PyMT epithelia from thoracic tumors was higher than epithelia enriched from the abdominal-inguinal tumors. Interestingly, the Wnt10bKO TME inverted the response to paclitaxel and the abdominal-inguinal epithelial tumor cells were more resistant than those from the thoracic epithelium. Furthermore, resistance to paclitaxel was associated with higher responses to PRI-724, a CBP-β-CATENIN inhibitor. Neither WT nor KO epithelial subtypes were resistant to olaparib exposure. Therefore, we conclude that Wnt10b is essential to educate highly metastatic breast cancer stromal cells to promote growth, metastatic colonization, and resistance to paclitaxel. Citation Format: Hannah R. Kelso, Rachel S. Perkins, Ikbale El Ayachi, Jackelyn A. Alva-Ornelas, Tiffany N. Seagroves, Victoria L. Seewaldt, Susan A. Krum, Gustavo Adolfo Miranda-Carboni. Ablation of WNT10B alters the tumor microenvironment in highly metastatic breast cancer, altering paclitaxel response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 72.

The Wnt signaling ligand WNT5B is implicated in various developmental pathways, both in normal and pathological physiology. Most of the research on WNT5B has been associated with expression analysis and disease states, leaving the signaling pathways underexplored. Here, we review the current understandings of WNT5B's regulation of signal transduction, from receptors to downstream mediators and transcription factors. We also describe its roles in β-catenin-dependent and β-catenin-independent (Planar Cell Polarity and Wnt/Ca2+) Wnt signaling.

Osteosarcoma is a rare but deadly pediatric bone cancer. As it stands, there is no targeted therapy available for osteosarcoma. Therefore, patients are left with chemotherapy and surgical resection as their only treatment options. Further, osteosarcoma frequently metastasizes to the lungs and patients with metastatic disease have a dismal overall survival rate of 20% due to the lack of targeted therapy. Therefore, the goal of this study is to understand the role of the WNT5B signaling pathway in osteosarcoma, as it could be a potential therapeutic target. Using RNA sequencing from publicly available data sets and immunohistochemistry on tumor microarrays, we show that WNT5B is overexpressed in a subset of osteosarcoma patients. In these high expressing patients, WNT5B’s overexpression correlates to metastasis and worse overall survival. Due to the significant increase in WNT5B expression between primary tumors and metastasis, we began looking at the stem cell population of osteosarcoma cells. By selecting for the osteosarcoma stem cells using spheroid assays, we show that both protein and mRNA levels of WNT5B are enhanced in the stem cell population compared to the standard adherent cell population. Then, we looked at stemness markers and we show that WNT5B regulates the stemness gene SOX2. We then performed limiting dilution sphere assays to assess the role of WNT5B in sphere forming. We reveal that WNT5B is directly responsible for the sphere forming efficiency, as evidenced by a 50% reduction in spheres formed between 143B control and 143B WNT5B knockdown cells. Additionally, WNT5B drives proliferation and migration of osteosarcoma cancer stem cells as there is significantly decreased sphere size and migratory distance in WNT5B knockdown cells compared to control cells, and there is significantly increased sphere size and migration with the addition of recombinant WNT5B. Through revealing a novel role for WNT5B in osteosarcoma cancer stem cells, we present the WNT5B pathway as a candidate for therapeutically targeting the clinical implications of cancer stem cells, that is, the development of chemoresistance, metastasis and relapse. Citation Format: Rachel S. Perkins, Sarocha Suthon, Gustavo A. Miranda-Carboni, Susan A. Krum. WNT5B in osteosarcoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3894.