Abstract Background: Enhancer of Zeste Homolog 2 (EZH2) is a histone methyltransferase and catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), responsible for trimethylating histone H3 at lysine 27 (H3K27me3). In human breast cancer, EZH2 overexpression is an independent prognostic marker and is significantly associated with negative ER and PR expression. However, the timing and functional significance of EZH2 overexpression in breast cancer development is still unclear. Towards this, we generated a conditional model of EZH2 overexpression to the mammary gland in FVB mice. Methods: We generated a doxycycline-inducible transgenic mouse model MMTV-rtTA;TetO-EZH2 and appropriate controls in an FVB background to enable targeted overexpression of EZH2 within mammary epithelial cells. We administered doxycycline (2 mg/mL) in the drinking water to 10-week-old female mice for 96 hours and in chow for 6 months, to induce EZH2 overexpression. At this age, the mammary glands are mature. At study endpoints, mammary glands were resected and analyzed using whole-mount carmine alum staining to assess ductal architecture. Mammary glands were embedded in paraffin and studied by histopathology and immunostaining using anti-EZH2 antibodies. Results: Induction of EZH2 expression in adult mice for 96 hours led to increased numbers of ductal branches compared to induced and uninduced controls. Long-term EZH2 induction also resulted in a hyperbranching phenotype and development of mammary epithelial nodules observed in carmine alum stains of whole glands, which were not present in the controls. Additional studies, including histopathological evaluation, immunostaining, and spatial analyses are underway to further characterize the biological and molecular consequences of EZH2 induction. Conclusions: We present the development and initial characterization of a novel inducible EZH2 overexpression model in the mammary glands of adult mice. Preliminary studies show that EZH2 overexpression results in intraductal epithelial hyperplasia and ductal hyperbranching recapitulating human preneoplastic lesions. Future studies will combine this novel model with other breast cancer mouse models to explore the consequences of EZH2 overexpression on tumor initiation and progression. Citation Format: Ahmad Eido, Maria E. Gonzalez, LiJyun Syu, Andrzej A. Dlugosz, Celina G. Kleer. Characterization of a novel doxycycline inducible model of EZH2 overexpression in mammary glands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6096.

Abstract Background: Triple negative breast cancers (TNBC) have proven resistant to conventional and immune therapies with 5-year survival rates of 50%. Among the most aggressive of these is metaplastic breast carcinoma (mBrCA), a unique histopathology with spindle or sarcomatoid (chondroid/osseous) components and low immune cell infiltration with poor responses to immunotherapy. We have reported that mammary epithelial cell-specific Ccn6 knockout mice (MMTV-Cre;Ccn6fl/fl) develop mBrCAs like human spindle mBrCAs, defining CCN6 as a tumor suppressor for spindle mBrCA. Lately we showed CCN6 antagonizes the pro-invasive effects of Wnt/ β-catenin/EZH2 signaling. Here, we tested the hypothesis that CCN6 genetically interacts with EZH2 to induce a phenotypic switch from spindle to sarcomatoid cancer cell phenotype and limits CD8+T cell infiltration. Methods: MDA-MB-231 (TNBC) and Ccn6-KO cells derived from MMTV-Cre;Ccn6fl/fl (Ccn6-KO) TNBC mouse model were transduced with vector or EZH2 shRNA knockdown (KD). Ccn6-KO EZH2 KD cells were rescued with lentivirus containing EZH2-wild-type (WT). To overexpress EZH2 we infected cancer cells with adenovirus with vector or EZH2-MYC construct. We investigated how Ccn6 loss and EZH2 affect mBrCA sarcomatoid by differentiation assays of the breast cancer cells in chondrogenic and osteogenic specialized media compared to controls. In vivo, to recapitulate the bone microenvironment, we injected Ccn6-KO cells into the tibia of FVB mice and treat them with the EZH2 inhibitor EPZ-6438. Flow cytometry analyses of tumors derived from Ccn6-KOshC, shEZH2 and EZH2-wild type rescue cells and IHC of Ccn6-KO tumors treated with EZH2i were used to measure the percentage of CD8+gzmb+IFNg+ T cells. ChIP-sequencing studies in dominant negative (dn)TCF4 mutant vs. controls were used to study regulation of bivalent genes in Ccn6-KO cells. Results: EZH2 overexpression in Ccn6-KO spindle mBrCA cells induces chondrogenic and osteogenic differentiation in cells grown in specialized media and EZH2 inhibition rescues the sarcomatoid phenotype in vivo. We found that EZH2 shRNA KD or pharmacological inhibition increases CD8+T cell infiltration in Ccn6-KO tumors, which is rescued by EZH2 overexpression. WNT/β-catenin signaling blockade in Ccn6-KO cells using dnTCF4 downregulates EZH2 and H3K27me3 mark at the promoter regions of 19 bivalent gene targets with roles in development. Conclusion: These data provide a clear link between CCN6 defects and EZH2 overexpression as inducers of sarcomatoid immune cell-low mBrCAs. We demonstrate the effectiveness of pharmacological inhibition of EZH2 methyltransferase activity in reversing these aggressive phenotypes. These data reveal a novel oncogenic cooperation and provide evidence to support the potential therapeutic value of CCN6 restoration, Wnt pathway and EZH2 inhibition as promising strategies for mBrCA patients Citation Format: Maria E. Gonzalez, Ahmad Eido, Kendall Miller, Celina G. Kleer. Ccn6 loss cooperates with EZH2 overexpression to induce sarcomatoid differentiation in metaplastic breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 608.

Elevated extracellular potassium (K+) in the tumor microenvironment (TME) of breast and other cancers is increasingly recognized as a critical factor influencing tumor progression and immune suppression. Current methods for noninvasive mapping of the potassium distribution in tumors are limited. Here, we employed photoacoustic chemical imaging (PACI) with a solvatochromic dye-based, potassium-sensitive nanoprobe (SDKNP) to quantitatively visualize extracellular potassium levels in an orthotopic metaplastic breast cancer mouse model, Ccn6-KO. Tumors of three distinct sizes (5 mm, 10 mm, and 20 mm) were imaged using multi-wavelength photoacoustic imaging at five laser wavelengths (560, 576, 584, 605, and 625 nm). Potassium concentration maps derived from spectral unmixing of the photoacoustic images at the five laser wavelengths revealed significantly increased potassium levels in larger tumors, confirmed independently by inductively coupled plasma mass spectrometry (ICP-MS). The PACI results matched ICP-MS measurements, validating PACI as a robust, noninvasive imaging modality for potassium mapping in tumors in vivo. This work establishes PACI as a promising tool for studying the chemical properties of the TME and provides a foundation for future studies evaluating the immunotherapy response through ionic biomarker imaging.

An acidic microenvironment is a hallmark of solid tumors, which occurs due to cancer cells' preferential utilization of aerobic glycolysis. Both in vitro and in vivo studies provide evidence that clinicians can exploit this acidity by selecting drugs whose efficacy is particularly affected by the tumor microenvironment (TME). However, noninvasive measurement of tumor pH remains challenging. Photoacoustic chemical imaging enhanced by pH-sensitive nanoparticles as contrast agents is a promising technique to address this need. This imaging technique was employed to quantitatively monitor the TME pH in mice bearing patient-derived xenografts undergoing simultaneous alkalization treatment and chemotherapy. Compared to controls, in alkalinized tumors, paclitaxel chemotherapy significantly reduced tumor size, increased tumor necrosis, and reduced cancer cell proliferation as validated by histopathology and immunohistochemistry. Thus, photoacoustic chemical imaging of TME pH may provide significant predictive value and represents a promising noninvasive, reliable technology for realizing personalized cancer chemotherapy.

PurposeEarly detection and diagnosis of cancer is critical for achieving positive therapeutic outcomes. Biomarkers that can provide clinicians with clues to the outcome of a given therapeutic course are highly desired. Oxygen is a small molecule that is nearly universally present in biological tissues and plays a critical role in the effectiveness of radiotherapies by reacting with DNA radicals and subsequently impairing cellular repair of double strand breaks.Techniques for measuring oxygen in biological tissues often use blood oxygen saturation to approximate the oxygen partial pressure in surrounding tissues despite the complex, nonlinear, and dynamic relationship between these two separate oxygen populations.Methods and MaterialsWe combined a directly oxygen-sensitive, tumor-targeted, chemical contrast nanoelement with the photoacoustic lifetime-based (PALT) oxygen imaging technique to obtain image maps of oxygen in breast cancer tumors in vivo. The oxygen levels of patient-derived xenografts in a mouse model were characterized before and after a course of radiotherapy.ResultsWe show that, independent of tumor size, radiotherapy induced an increase in the overall oxygenation levels of the tumor. Further, this increase in the oxygenation of the tumor significantly correlated with a positive response to radiotherapy, as demonstrated by a reduction in tumor volume over the twenty-day monitoring period following therapy and histological staining.ConclusionOur PALT imaging presented here is simple, fast, and non-invasive. Facilized by the PALT approach, imaging of tumor reoxygenation may be utilized as a simple, early indicator for evaluating cancer response to radiotherapy. Further characterization of the reoxygenation degree, temporal onset, and possible theragnostic implications are warranted.

Abstract Background: Metaplastic breast carcinomas (mBrCAs) are a highly aggressive subtype of triple negative breast cancer with histological evidence of deregulated differentiation towards non-glandular components. Previous studies have demonstrated that human mBrCA often exhibit activation of differentiation pathways, including canonical Wnt/b-catenin and EZH2-mediated transcriptional repression. Our lab has identified CCN6 as a tumor suppressor in mBrCA. MMTV-Cre;Ccn6fl/fl (CCN6KO) mice develop spindle mBrCAs and CCN6 is reduced/lost in 68% of human mBrCAs. Recently, we have demonstrated that the tumor suppressor function of CCN6 in spindle mBrCA requires activation of Wnt/b-catenin signaling pathway. Here, we tested the hypothesis that CCN6 KO leads to the upregulation of EZH2 histone methyltransferase promoting mBrCA. Furthermore, we investigated the requirement for the activation of the Wnt/b-catenin pathway in this mechanism. Methods: To test the effect of CCN6/b-catenin on EZH2 gene and protein expression we performed IHC, IF, qRT-PCR, ChIP-Seq, RNA-seq, invasion and adhesion assays, EZH2 reporter assay and immunoblots in mBrCA cell lines and MMTV-Cre;Ccn6fl/fl tumors. To investigate the role of CCN6KO-induced b-catenin activation on EZH2 activity and neoplastic functions we employed three independent approaches: i) Expression of a dominant-negative Tcf4 (dnTcf4) rescued with EZH2-WT, dSET and dNLS domain mutants versus vector in MMTV-Cre;Ccn6fl/fl tumor-derived cells; ii) Expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus control; iii) Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used for in vivo rescue experiments with rhCCN6 or BSA. To assess the therapeutic benefit of inhibiting EZH2 methyltransferase activity, MMTV-Cre;Ccn6fl/fl tumor cells (CCN6KO cells) were implanted orthotopically or intracardially in FVB mice, followed by treatment with EPZ-6438 (a selective EZH2 methyltransferase inhibitor) or vehicle. We tested CCN6, b-catenin, and EZH2 expression by IHC in a cohort of 27 human mBrCA tumor samples. Results: CCN6KO-induced b-catenin/TCF activation mediates EZH2 transcriptional upregulation and the deposition of repressive H3K27me3 in spindle mBrCAs. We found that the invasion program triggered by CCN6KO-induced Wnt/b-catenin requires EZH2 catalytic activity as WT-EZH2 (but not dSET-EZH2) rescued the reduced invasion of CCN6KO-dnTcf4 cells. RNA-seq and 3H3K27 ChIP-seq of CCN6KO-dnTcf4 cells transduced with EZH2-WT or dSET-EZH2 identify specific CCN6KO-b-catenin/Tcf targets that require EZH2 transcriptional repressor function. In vivo, administration of CCN6 protein to MMTV-Cre;Ccn6fl/fl tumor transplants reduces tumor growth and nuclear b-catenin, EZH2 and 3H3K27 in the tumors. Pharmacologic inhibition of EZH2 reduces the growth and metastasis of CCN6KO mBrCA tumors and improves survival. We identify a subset of human spindle mBrCA (54%) that display a CCN6Low/nuclear b-cat/EZH2High phenotype. Conclusion: We found a critical role for EZH2 activation in CCN6-deficient mBrCA tumor phenotypes via b-catenin/TCF Wnt canonical signaling. We demonstrate the effectiveness of pharmacological inhibition of EZH2 methyltransferase activity in reducing primary tumor growth and distant metastasis in mouse models of spindle mBrCA. In clinical samples, low CCN6 is significantly associated with activated b-catenin and high EZH2 in spindle mBrCAs compared to other subtypes. These data reveal a novel tumor suppressor mechanism of CCN6 and provide compelling evidence supporting the potential therapeutic value of CCN6 restoration, b-catenin or EZH2 inhibition as promising approaches for the treatment of spindle mBrCAs. Citation Format: Maria Gonzalez, Ahmad Eido, GIUSEPPINA AUGIMERI, Celina Kleer. EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-05.

Abstract Metaplastic breast carcinomas (mBrCA) are a highly aggressive subtype of triple-negative breast cancer with histologic evidence of epithelial-to-mesenchymal transition and aberrant differentiation. Inactivation of the tumor suppressor gene cellular communication network factor 6 (CCN6; also known as Wnt1-induced secreted protein 3) is a feature of mBrCAs, and mice with conditional inactivation of Ccn6 in mammary epithelium (Ccn6-KO) develop spindle mBrCAs with epithelial-to-mesenchymal transition. Elucidation of the precise mechanistic details of how CCN6 acts as a tumor suppressor in mBrCA could help identify improved treatment strategies. In this study, we showed that CCN6 interacts with the Wnt receptor FZD8 and coreceptor LRP6 on mBrCA cells to antagonize Wnt-induced activation of β-catenin/TCF-mediated transcription. The histone methyltransferase EZH2 was identified as a β-catenin/TCF transcriptional target in Ccn6-KO mBrCA cells. Inhibiting Wnt/β-catenin/TCF signaling in Ccn6-KO mBrCA cells led to reduced EZH2 expression, decreased histone H3 lysine 27 trimethylation, and deregulation of specific target genes. Pharmacologic inhibition of EZH2 reduced growth and metastasis of Ccn6-KO mBrCA mammary tumors in vivo. Low CCN6 is significantly associated with activated β-catenin and high EZH2 in human spindle mBrCAs compared with other subtypes. Collectively, these findings establish CCN6 as a key negative regulator of a β-catenin/TCF/EZH2 axis and highlight the inhibition of β-catenin or EZH2 as a potential therapeutic approach for patients with spindle mBrCAs. Significance: CCN6 deficiency drives metaplastic breast carcinoma growth and metastasis by increasing Wnt/β-catenin activation to upregulate EZH2, identifying EZH2 inhibition as a mechanistically guided treatment strategy for this deadly form of breast cancer.

Patients with triple-negative breast cancer remain at risk for metastatic disease despite treatment. The acquisition of chemoresistance is a major cause of tumor relapse and death, but the mechanisms are far from understood. We have demonstrated that breast cancer cells (BCCs) can engulf mesenchymal stem/stromal cells (MSCs), leading to enhanced dissemination. Here, we show that clinical samples of primary invasive carcinoma and chemoresistant breast cancer metastasis contain a unique hybrid cancer cell population coexpressing pancytokeratin and the MSC marker fibroblast activation protein-α. We show that hybrid cells form in primary tumors and that they promote breast cancer metastasis and chemoresistance. Using single-cell microfluidics and in vivo models, we found that there are polyploid senescent cells within the hybrid cell population that contribute to metastatic dissemination. Our data reveal that Wnt Family Member 5A (WNT5A) plays a crucial role in supporting the chemoresistance properties of hybrid cells. Furthermore, we identified that WNT5A mediates hybrid cell formation through a phagocytosis-like mechanism that requires BCC-derived IL-6 and MSC-derived C-C Motif Chemokine Ligand 2. These findings reveal hybrid cell formation as a mechanism of chemoresistance and suggest that interrupting this mechanism may be a strategy in overcoming breast cancer drug resistance.

The importance of EZH2 as a key methyltransferase has been well documented theoretically. Practically, the first EZH2 inhibitor Tazemetostat (EPZ6438), was approved by FDA in 2020 and is used in clinic. However, for most solid tumors it is not as effective as desired and the scope of clinical indications is limited, suggesting that targeting its enzymatic activity may not be sufficient. Recent technologies focusing on the degradation of EZH2 protein have drawn attention due to their potential robust effects. This review focuses on the molecular mechanisms that regulate EZH2 protein stability via post-translational modifications (PTMs), mainly including ubiquitination, phosphorylation, and acetylation. In addition, we discuss recent advancements of multiple proteolysis targeting chimeras (PROTACs) strategies and the latest degraders that can downregulate EZH2 protein. We aim to highlight future directions to expand the application of novel EZH2 inhibitors by targeting both EZH2 enzymatic activity and protein stability.

Abstract Background: Triple negative breast carcinomas (TNBC) are highly aggressive tumors with limited effective treatments. Clinical studies showed that targeting low HER2 levels in TNBC with anti-HER2 agents has survival benefit. Enhancer of Zeste Homolog 2 (EZH2) is a lysine methyltransferase component of the Polycomb Repressive Complex2 that regulates differentiation genes in normal and neoplastic cells. EZH2 phosphorylation at T416 was shown to maintain the TNBC phenotype. We have reported that p38-induced EZH2-T367 phosphorylation enhances metastasis and that high pEZH2-T367 expression is significantly associated with human TNBC tumors compared to other breast cancer subtypes. In this study we tested the hypothesis that pEZH2-T367 may regulate the HER2 pathway in TNBC and that inhibition of pEZH2-T367 may upregulate HER2 signaling, with clinical implications. Methods: MDA-MB-231 (mesenchymal-like TNBC) and CCN6KO cells derived from MMTV-Cre;Ccn6fl/fl (CCN6KO) TNBC mouse model generated in our lab were transduced with vector or EZH2 shRNA knockdown (KD). MDA-MB-231 EZH2 KD cells were rescued with lentivirus containing EZH2-wild-type (WT) or T367A phosphorylation-deficient mutant. Gene and protein expression was analyzed by RNA sequencing, q-RT-PCR, immunoblot and immunohistochemistry. Vector and EZH2-KD rescue MDA-MB-231 cells were treated with anti-HER2 (trastuzumab or pertuzumab 100 ug/ml) followed by Hoestch proliferation assays. TNBC cells were treated with vehicle, EZH2 inhibitor (EPZ-6438, 20uM), p38 inhibitor (p38i, SB202190, 20uM), EPZ+p38i, anti-HER2 (100 ug/ml), and EPZ+p38i+anti-HER2, followed by Hoestch cell proliferation assays. Results: EZH2-T367A deregulates the expression of a 94-gene network of HER2 pathway genes including EREG, SERPINB5, MERTK, IL6R, SOCS3, and FASCN1 compared to EZH2-WT. EZH2-T367A increased protein levels of HER2, EREG, and PP2AB, a major HER2 regulator and was sufficient to sensitize TNBC cells to the anti-proliferative effects of anti-HER2. Supporting the role of pEZH2-T367, we found that the combination of p38i and EPZ significantly sensitizes TNBC cells to anti-HER2 therapies. Conclusions: We demonstrate that T367 phosphorylation mediates the effect of EZH2 on HER2 signaling in TNBC. Our data support a role for pEZH2-T367 in maintaining the HER2-negative phenotype in and suggest that blocking this phosphorylation event may render TNBC tumors amenable to anti-HER2 treatment strategies. Citation Format: Maria E. Gonzalez, Ahmad Eido, Celina G. Kleer. p38-mediated EZH2 phosphorylation at T367 regulates HER2 pathway in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6981.

We hereby apply the approach of photoacoustic chemical imaging, performing an in vivo chemical analysis that is spatially resolved (200 μm) and in real time, to predict a given tumor's response to therapy. Using triple negative breast cancer as a model, we took photoacoustic images of tumors' oxygen distributions in patient-derived xenografts (PDXs) in mice using biocompatible, oxygen-sensitive tumor-targeted chemical contrast nanoelements (nanosonophores), which function as contrast agents for photoacoustic imaging. Following radiation therapy, we established a quantitatively significant correlation between the spatial distribution of the initial oxygen levels in the tumor and its spatial distribution of the therapy's efficacy: the lower the local oxygen, the lower the local radiation therapy efficacy. We thus provide a simple, noninvasive, and inexpensive method to both predict the efficacy of radiation therapy for a given tumor and identify treatment-resistant regions within the tumor's microenvironment.

Supplementary Methods from Histone Methyltransferase EZH2 Induces Akt-Dependent Genomic Instability and BRCA1 Inhibition in Breast Cancer

Supplementary Data Legends from Altered Expression of the Early Mitotic Checkpoint Protein, CHFR, in Breast Cancers: Implications for Tumor Suppression

Supplementary Tables 1-2 from Histone Methyltransferase EZH2 Induces Akt-Dependent Genomic Instability and BRCA1 Inhibition in Breast Cancer

Supplementary Figures 1-4 from Blockade of CCN6 (WISP3) Activates Growth Factor–Independent Survival and Resistance to Anoikis in Human Mammary Epithelial Cells

Background: Metaplastic breast carcinomas (mBrCAs) are a rare and highly aggressive subtype of triple negative breast cancer, with histological evidence of non-glandular differentiation and frequent activation of the canonical (-catenin-dependent) Wnt pathway. Our laboratory has reported that CCN6 is expressed in normal mammary epithelium, but CCN6 expression is lost in 68% of spindle mBrCAs. We found mice with mammary epithelial cell-specific conditional deletion of Ccn6 (MMTV-Cre; Ccn6fl/fl mice) develop mammary tumors that recapitulate human spindle mBrCAs, including upregulation of Wnt pathway genes. We investigated if and how secreted CCN6 protein functions in tumor suppression in spindle mBrCA via effects on the canonical Wnt pathway. Methods: To investigate CCN6 binding to the Wnt co-receptors LRP6 and FZD8 proteins, we performed Flag-IPs on MDA-MB231 mesenchymal-like breast cancer cells expressing Flag-CCN6 or vector. Effects of CCN6 on b-catenin subcellular localization and gene and protein expression were studied by IHC, IF, qRT-PCR and immunoblot in human cell lines and MMTV-Cre;Ccn6fl/fl tumors. To test effects of recombinant CCN6 on canonical Wnt signaling, we used the Leading-Light Wnt Reporter Assay and also tested CCN6 effects in WNT3A- and WNT10B-mediated Wnt signaling activation and on MDA-MB231 cell invasion. To study b-catenin/TCF function in invasive growth of CCN6-deficient cancer cells, we employed two independent approaches: i) expression of a dominant-negative Tcf4 (dnTcf4) versus control vector in MMTV-Cre; Ccn6fl/fl tumor-derived cells; and ii) expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus BSA control. Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used in vivo for rescue experiments with i.p. injections of rhCCN6 or BSA. We monitored tumor growth and morphology, and performed IHC to determine b-catenin localization and expression. Results: We found in co-IPs that CCN6 interacts with LRP6 and FZD8 to form a complex that antagonizes canonical Wnt signaling. CCN6 ectopic expression in MDA-MB231 cells led to reduced nuclear and increased membrane localization of b-catenin and decreased invasive growth in vitro. In vivo, CCN6 protein administration to MMTV-Cre; Ccn6fl/fl mice reduced tumor growth and was linked to decreased nuclear b-catenin in the tumors. Conclusion: CCN6 antagonizes canonical Wnt/b-catenin in part by binding Wnt ligands, leading to reduced active b-catenin in the cytoplasm and nucleus. Our data indicate a critical role for b-catenin activation for CCN6-deficient mBrCA tumor phenotypes. In vivo, rhCCN6 protein reduces tumorigenesis in MMTV-Cre; Ccn6fl/fl mBrCA tumors, highlighting how CCN6 restoration or b-catenin inhibition could be new therapeutic approaches for mBrCAs. Citation Format: Maria E. Gonzalez, Eric R. Fearon, Celina Kleer. PD5-06 CCN6 suppresses spindle metaplastic breast carcinoma in part via antagonizing Wnt/β-catenin signaling [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-06.

Supplementary Table 1, Figures 1-5 from High SEPT9_v1 Expression in Human Breast Cancer Cells Is Associated with Oncogenic Phenotypes