Wnt Signaling In Breast Cancer Research Articles

Transmembrane protein 97 exhibits oncogenic properties via enhancing LRP6-mediated Wnt signaling in breast cancer

Upregulation of transmembrane protein 97 (TMEM97) has been associated with progression and poor outcome in multiple human cancers, including breast cancer. Recent studies suggest that TMEM97 may be involved in the activation of the Wnt/β-catenin pathway. However, the molecular mechanism of TMEM97 action on Wnt/β-catenin signaling is completely unclear. In the current study, TMEM97 was identified as an LRP6-interacting protein. TMEM97 could interact with LRP6 intracellular domain and enhance LRP6-mediated Wnt signaling in a CK1δ/ε-dependent manner. The binding of TMEM97 to LRP6 facilitated the recruitment of CK1δ/ε to LRP6 complex, resulting in LRP6 phosphorylation at Ser 1490 and the stabilization of β-catenin. In breast cancer cells, knockout of TMEM97 attenuated the Wnt/β-catenin signaling cascade via regulating LRP6 phosphorylation, leading to a decrease in the expression of Wnt target genes AXIN2, LEF1, and survivin. TMEM97 deficiency also suppressed cell viability, proliferation, colony formation, migration, invasion, and stemness properties in breast cancer cells. Importantly, TMEM97 knockout suppressed tumor growth through downregulating the Wnt/β-catenin signaling pathway in a breast cancer xenograft model. Taken together, our results revealed that TMEM97 is a positive modulator of canonical Wnt signaling. TMEM97-mediated Wnt signaling is implicated in the tumorigenesis of breast cancer, and its targeted inhibition may be a promising therapeutic strategy for breast cancer.

Key Epigenetic Events Involved in the Maintenance of Breast Cancer Stem Cells.

Cancer stem cells (CSCs) are a small subpopulation of cells within tumors and play significant roles in tumorigenesis, metastasis, resistance to treatment, and relapse. They are defined by self-renewal and multi-lineage differentiation, and aggressiveness. Epigenetic modifications, including DNA methylation and acetylation, histone modifications, and non-coding. RNAs (ncRNAs), are partly responsible for CSC potentials and are involved in the modification of key components of crucial pathways such as Notch and Wnt signaling in breast cancer. In this review, we present an overview of the pathways and epigenetic events that lead to the transformation of mammary gland stem cells to breast CSCs (BCSCs). Based on the data presented here, important pathways such as TGF-β/SMAD2 and Wnt/β-catenin and epigenetic modifications, including histone modifications, DNA methylations, and microRNAs, play important roles in BCSC formation and maintenance. Epigenetic events can alter the expression of genes and functional RNAs, resulting in tumor initiation and progression. Thus, a better understanding of epigenetic modifications involved in BCSC maintenance signaling pathways may help to eliminate or suppress BCSCs and overcome cancer by generating more effective and efficient therapeutic agents.

Abstract 2890: MicroRNA-mediated upregulation of the WNT signaling activities in human breast cancer stem cells

Abstract The canonical WNT signaling plays a critical role in many adult stem cells, including those of the breast and intestine. The fact that the canonical WNT signaling is implicated in both stem cell self-renewal and cancer suggests that normal physiological regulator of stem cell functions might be “hijacked” in cancer. Adenomatous polyposis coli (APC) is a component of the destruction complex that destabilizes β-catenin and suppresses the activity of the canonical WNT signaling. MicroRNAs (miRNAs) are important regulators of stem cell functions. We have previously reported a set of 37 miRNAs that are upregulated or downregulated in human breast cancer stem cells (BCSCs, a CD44+CD24-/lowlineage- population of human breast cancer cells) as compared to non-tumorigenic breast cancer cells (NTCs). Among them, miR-200c targets BMI1 that is a critical regulator of the stem cell maintenance, and strongly impairs the functions of human BCSCs in vivo. In this study, we compared the expression profiles of miRNAs, mRNAs and proteins between BCSCs and NTCs isolated from the patient specimens of human breast cancers and patient-derived tumor xenografts (PDXs) established by their transplantation. Luciferase assays were performed using the plasmid in which the 3’UTR region of candidate mRNA was cloned downstream of a luciferase minigene. The effect of miRNAs on the activity of WNT signaling was evaluated using a TCF reporter plasmid. Finally, the abilities to form organoids and to form tumors in immunodeficient mice were evaluated using the human BCSCs infected with the miRNA inhibitor expressing lentivirus. We found that miR-142 was highly upregulated in BCSCs, but was hardly expressed in NTCs in the patient breast cancer specimens. We confirmed that miR-142 targeted the sequence within the 3’UTR of APC mRNA and suppressed APC protein expression. Accordingly, miR-142 activated the canonical WNT signaling pathway in an APC-suppression dependent manner. The results of mRNA and protein expression profiling of the BCSCs isolated from human breast cancer PDXs suggested that the canonical WNT signaling was activated in BCSCs. Finally, inhibition of miR-142 in the BCSCs suppressed the tumor growth in vivo. These results suggest that the miR-142, a miRNA frequently upregulated in human BCSCs, could provide at least a part of the molecular mechanism for aberrant activation of the canonical WNT signaling in breast cancer in which APC mutations are much less frequent than colon cancer. Citation Format: Yohei Shimono, Taichi Isobe, Andrei Turtoi, Junko Mukohyama, Toru Mukohara, Akira Suzuki, Vincent Castronovo, Hironobu Minami. MicroRNA-mediated upregulation of the WNT signaling activities in human breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2017-2890

Epigenetic Regulation of WNT and Hedgehog Oncogenic Signaling in Breast Cancer Cells in Response to Dietary Polyphenols

The Wnt and Hedgehog signaling pathways play roles in embryonic and stem cell developmental processes but can become overactive in human cancers, contributing to cancer cell invasion and metastasis. Crosstalk occurs between these two pathways in cancer due to the ability of Hedgehog signaling proteins to regulate activity of the Wnt pathway, and vice versa. This interdependent regulation allows for cancer therapies to mutually target the activity of both pathways. Interestingly, certain dietary polyphenolic compounds suppress the Hedgehog and Wnt signals in cancer. In our study, polyphenols were shown to modify epigenetic marks in genes positively regulating these pathways including SHH, a Hedgehog signaling protein, GLI2, a transcriptional activator of Hedgehog signaling, and four Wnt signaling ligands including WNT4, WNT6, WNT9A, and WNT10B.Using the polyphenol resveratrol from grapes, we examine the role of DNA methylation in regulation of Hedgehog and Wnt signaling in breast cancer. Non‐invasive MCF‐7 and invasive MCF10CA1a human breast cancer cell lines were used as an experimental model. Following genome‐wide DNA methylation analysis with Illumina 450K array, pyrosequencing and QPCR were performed to assess methylation and expression of the selected positive regulators and downstream targets of Hedgehog and Wnt signaling.We found 6,347 CpG sites differentially methylated upon 9‐day treatment of MCF10CA1a breast cancer cells with 15μM resveratrol compared to untreated cells. A majority of those CpG sites were hypermethylated and located within genes functionally associated with oncogenic signaling pathways, including Hedgehog and Wnt. Resveratrol led to increased methylation of enhancer regions of SHH, GLI2, and WNT4, and CpG island regions of WNT6, WNT9A, and WNT10B. Increase in methylation of these genes was confirmed by pyrosequencing in breast cancer cells with high and low invasive potential. Along with increased methylation of these regions, the expression of these genes decreased following resveratrol treatment. This was associated with downregulation of Hedgehog target gene Bcl‐2 and Wnt target gene EpCam, as well as a mutual target FOXM1, in invasive MCF10CA1a breast cancer cells.Our results provide insight into regulation of oncogenic signals in cancer and support for epigenetic‐targeting strategies as an effective anti‐cancer approach.Support or Funding InformationThis study was supported by the Purdue University Center for Cancer Research, Indiana CTSI (UL1TR001108), Women's Global Health Institute, and USDA National Institute of Food and Agriculture (Hatch project 1005656) granted to BS.

Abstract 2460: APC regulates COX-2 independent of Wnt signaling in breast cancer

Abstract Breast cancer is the leading cause of cancer-related death in non-smoking women, and the most commonly diagnosed cancer in women. The Adenomatous Polyposis Coli (APC) tumor suppressor gene is mutated or silenced by hypermethylation in 18-70% of sporadic breast cancers depending on subtype; however, the molecular mechanisms downstream of APC loss in the breast remain largely unexplored. While inactivation of APC most commonly leads to increased signaling through the Wnt/β-catenin pathway, there are multiple, less-investigated functions of APC, including regulation of GSK3β-mediated signaling, proliferation, epithelial polarity, cytoskeletal organization, and DNA repair. Given that restoration of tumor suppressors is a hurdle in treating tumors arising from loss of gene function, it is critical to investigate and understand the downstream signaling pathways involved in tumorigenesis. Our laboratory is particularly interested in investigating the effect of APC loss or mutation in breast cancer, and the complex Wnt-independent signaling downstream of APC loss in the breast. We previously demonstrated that Apc mutation accelerates the mouse mammary tumor virus-Polyoma middle T antigen (MMTV-PyMT) transgenic model of breast tumorigenesis independently of Wnt/β-catenin signaling. Interestingly, focal adhesion kinase (FAK)/Src/JNK signaling was up-regulated and required for the enhanced proliferation and squamous phenotype mediated by Apc mutation. Despite the lack of Wnt/β-catenin pathway activation, Apc-mutant mammary tumor cell lines demonstrated enhanced expression of Cyclooxygenase-2 (COX-2) and production of prostaglandin E2 (PGE2), known to be important in triple-negative and inflammatory breast cancers, two aggressive breast cancer subtypes. While treatment with the Src inhibitor (PP2) decreases COX-2 protein expression, there is no effect on the level of phosphorylated JNK, indicating a non-linear signaling pathway. Consistent with this, treatment with an inhibitor of JNK (SP600125) has no effect on COX-2 expression. These data suggest that while Src activation downstream of Apc mutation enhances COX-2 in a Wnt-independent manner, the pathway does not travel through activation of JNK. Therefore, the mechanisms by which Apc mutation initiates this stream of events to enhance mammary tumorigenesis, and the contribution of COX-2 to the phenotypes acquired in Apc-mutant breast cancer remain unclear. Citation Format: Monica K. VanKlompenberg, Claire Bedalov, Jenifer R. Prosperi. APC regulates COX-2 independent of Wnt signaling in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2460. doi:10.1158/1538-7445.AM2014-2460

Abstract 2382: SOX17 promoter methylation in CTC and cell free DNA isolated from plasma of breast cancer patients

Abstract Introduction: Molecular characterization of Circulating Tumor Cells (CTC) is a hot topic nowadays. SOX17 plays a very critical role in the regulation of development and stem/precursor cell function, at least partly through repression of Wnt pathway activity. Silencing of SOX17 due to its promoter methylation is a frequent event and may contribute to aberrant activation of Wnt signaling in breast cancer. We have recently shown that the promoter of this gene is methylated in CTC (Chimonidou et al, Clin Chem 2011). The aim of the present study was to evaluate whether SOX17 promoter methylation in CTC, is associated with the methylation pattern of this gene in matched cell free DNA isolated from plasma of breast cancer patients. Patients and methods: We firstly developed a highly sensitive and specific Methylation Specific Real-time PCR method for SOX17. The methylation status of SOX17 was examined in paired specimens of DNA isolated from CTC and cell free DNA (cfDNA) circulating in plasma of the same individuals. 58 patients with operable breast cancer, 21 patients with verified metastasis, and 12 healthy individuals used as controls were enrolled in our study. For CTC analysis, DNA was extracted from EpCAM positive immunomagnetically isolated CTC fraction, while cfDNA was isolated from 200μL of plasma from the same individuals. DNA extracted from isolated CTC and cfDNA was modified by a Sodium Bisulfite (SB) conversion reaction and subjected to methylation specific real time PCR. Results: In CTC, promoter methylation of SOX17 was observed in 22/58 (37.9%) of patients with operable breast cancer, 12/21 (57.1%) of patients with verified metastasis and 1/12 (8.3%) of healthy individuals. In the matched plasma samples, promoter methylation of SOX17 was observed in 23/58 (39.6%) of patients with operable breast cancer, 11/21 (52.4%) of patients with verified metastasis and 1/12 (8.3%) in healthy individuals. By comparing these results we found that promoter methylation of SOX17 in CTC was significantly correlated with promoter methylation of SOX17 in cell free DNA isolated from plasma of the same patients (P<0.005). Conclusion: We show for the first time that promoter methylation of SOX17 in CTC, is associated with the methylation pattern of this gene in matched cell free DNA isolated from plasma of breast cancer patients. Our findings suggest a new dimension to the origin of cell free DNA and indicate a correlation between cell free DNA and CTC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2382. doi:1538-7445.AM2012-2382

Is there more to Wnt signalling in breast cancer than stabilisation of β-catenin?

Increased Wnt signalling has been implicated in the aetiology of many different human cancers, including breast cancers. In most cases, Wnt signalling is thought to drive tumourigenesis through the stabilisation of cytosolic β-catenin and the subsequent changes in the expression of T-cell factor (TCF)-dependent genes. However, this is not necessarily the only mechanism, as Wnt proteins can signal through a number of different intracellular signalling pathways. The ongoing work from Nancy Hynes' laboratory continues to highlight this latter possibility.

Correction: Article on Wnt Signaling in Breast Cancer

Correction: Article on Wnt Signaling in Breast Cancer

Methylation‐associated silencing of SFRP1 with an 8p11‐12 amplification inhibits canonical and non‐canonical WNT pathways in breast cancers

Recently, we analysed the 8p11-12 genomic region for copy number and gene expression changes in a panel of human breast cancer cell lines and primary specimens. We found that SFRP1 (Secreted frizzled related protein 1) is frequently under expressed even in breast tumours with copy number increases in this genomic region. SFRP1 encodes a WNT signalling antagonist, and plays a role in the development of multiple solid tumour types. In this study, we analysed methylation-associated silencing of the SFRP1 gene in breast cancer cells with the 8p11-12 amplicon, and investigated the tumour suppressor properties of SFRP1 in breast cancer cells. SFRP1 expression was markedly reduced in both the breast cancer cell lines and primary tumour specimens relative to normal primary human mammary epithelial cells even when SFRP1 is amplified. Suppression of SFRP1 expression in breast cancer cells with an SFRP1 gene amplification is associated with SFRP1 promoter methylation. Furthermore, restoration of SFRP1 expression suppressed the growth of breast cancer cells in monolayer, and inhibited anchorage independent growth. We also examined the relationship between the silencing of SFRP1 gene and WNT signalling in breast cancer. Ectopic SFRP1 expression in breast cancer cells suppressed both canonical and non-canonical WNT signalling pathways, and SFRP1 expression was negatively associated with the expression of a subset of WNT responsive genes including RET and MSX2. Thus, down-regulation of SFRP1 can be triggered by epigenetic and/or genetic events and may contribute to the tumourigenesis of human breast cancer through both canonical and non-canonical WNT signalling pathways.

Sox17, the canonical Wnt antagonist, is epigenetically inactivated by promoter methylation in human breast cancer

SRY-box 17 (Sox17) is a transcription factor which involved in a variety of developmental processes and can act as an antagonist of canonical Wnt/beta-catenin signaling pathway. However, the relationship between Sox17 gene expression, methylation status, and beta-catenin in breast cancer has not been established. Here we report that the expression level of Sox17 mRNA was dramatically decreased in five different breast cancer cell lines and 23 of 31 primary breast tumor samples, which significantly correlated with its methylation status. After treated with 5-aza-2'-deoxycytidine (5-aza-dC, a demethylation agent), the expression levels of Sox17 mRNA and protein were obviously increased. Restored expression of Sox17 by 5-aza-dC treatment decreased the expression level of beta-catenin in breast cancer cell lines. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Sox17 in SKBR-3 and Bacp-37 cells enhanced beta-catenin expression. In 31 paired tissue samples, a significant difference between the expression level of Sox17 and beta-catenin was also observed (P < 0.001). Clinically, Sox17 methylation was detected in 74.3% breast tumors (84/113) and 31.9% (36/113) paired normal tissues, respectively (P < 0.0001). Sox17 methylation was also associated with tumor stage (P = 0.028) and lymph node metastasis (P = 0.013). These findings indicate that silencing of Sox17 due to promoter hypermethylation is a frequent event and may contribute to aberrant activation of Wnt signaling in breast cancer. Sox17 may be a valuable biomarker for the study of breast cancer carcinogenesis and progression.

Investigating h-Prune activation of Wnt signalling in breast cancer

We have been investigating a novel link between two independent processes linked to breast cancer: Wnt signalling and h-Prune overexpression. The canonical Wnt signalling pathway was activated in 40% to 60% of human breast cancers through mechanisms that are not understood. Similarly, the phosphodiesterase h-Prune was overexpressed or amplified in 54% of breast cancers and was linked to breast tumour progression through unknown mechanisms. We have shown that overexpression of xenopus Prune induced formation of a secondary axis in a standard assay to identify activators of the Wnt signalling pathway. In HEK293 cells, xenopus Prune overexpression induced a 300-fold increase in Wnt/TCF-dependent transcription. Whilst human prune does not appear to be able to activate Wnt signalling as potently as its xenopus homologue, it does synergise with other activators of the pathway to increase TCF-dependent transcription. Here we show whether there is a correlation between overexpression of h-Prune and active Wnt signalling in breast cancer, and whether the synergistic responses described are mediated through the enzymatic activity of prune, or through binding to GSK-3.

Repressor roles for TCF-4 and Sfrp1 in Wnt signaling in breast cancer

Mutations in Wnt pathway genes are rare in human breast cancer, yet activation of the pathway is evident from the misolocalization of beta-catenin. We searched for relationships in the expression of Wnt pathway genes and found that both secreted frizzled related protein 1 (Sfrp1) and TCF-4 transcripts were all highly downregulated in a common subset of breast cancers relative to normal breast tissue. Sfrp1 has been previously characterized as a Wnt inhibitor, and we found that interfering with its expression in the human mammary epithelial cell line MCF10A activated Wnt signaling. Reduction of TCF-4 levels in breast cancer was surprising as it is a transcription factor that is responsive to Wnt signaling. Therefore, we investigated a possible inhibitory role for TCF-4 in human breast cells as well as further characterizing Sfrp1. We identified CD24 as a Wnt target in MCF10A cells and used its expression a marker of Wnt signaling. Interfering with either Sfrp1 or TCF-4 in this cell line enhanced CD24 expression. Furthermore, removal of TCF/LEF binding sites in a CD24-luciferase reporter resulted in elevated reporter gene expression. Our results indicate that both Sfrp1 and TCF-4 repress Wnt signaling in breast tissue and their downregulation contributes to the activation of Wnt signaling.

Wnt signaling in breast cancer: have we come full circle?

Since the original identification of Wnt1 as a mammary oncogene in mouse mammary tumor virus infected mice, questions have been asked about its relevance to human breast cancer. Wnt1 is now known to be one of a large family of Wnt genes encoding structurally similar secreted signaling proteins, several of which are functionally redundant. The principal intracellular signaling pathway activated by these proteins has been elucidated in recent years. Components of this pathway include proto-oncogene products, such as β-catenin, and tumor suppressor proteins such as APC. Although WNT1 itself has not been implicated in human breast neoplasms, it has been reported that other WNT genes are sometimes overexpressed in human breast cancer and there is growing evidence that downstream components of the Wnt signaling pathway are activated in a significant proportion of breast tumors.