CCN6 regulates IGF2BP2 and HMGA2 signaling in metaplastic carcinomas of the breast.

PURPOSE: Metaplastic breast carcinomas are an aggressive subtype of triple-negative breast cancer (TNBC) in which part or all of the adenocarcinoma transforms into a non-glandular component (e.g., spindled, squamous, or heterologous). We discovered that mammary-specific Ccn6/Wisp3 knockout mice develop mammary carcinomas with spindle and squamous differentiation that share upregulation of the oncofetal proteins IGF2BP2 (IMP2) and HMGA2 with human metaplastic carcinomas. Here, we investigated the functional relationship between CCN6, IGF2BP2, and HMGA2 proteins in vitro and in vivo, and their expression in human tissue samples. METHODS: MMTV-cre;Ccn6fl/fl tumors and spindle TNBC cell lines were treated with recombinant CCN6 protein or vehicle. IGF2BP2 was downregulated using shRNAs in HME cells with stable CCN6 shRNA knockdown, and subjected to invasion and adhesion assays. Thirty-one human metaplastic carcinomas were arrayed in a tissue microarray (TMA) and immunostained for CCN6, IGF2BP2, and HMGA2. RESULTS: CCN6 regulates IGF2BP2 and HMGA2 protein expression in MMTV-cre;Ccn6fl/fl tumors, in MDA-MB-231 and - 468, and in HME cells. CCN6 recombinant protein reduced IGF2BP2 and HMGA2 protein expression, and decreased growth of MMTV-cre;Ccn6fl/fl tumors in vivo. IGF2BP2 shRNA knockdown was sufficient to reverse the invasive abilities conferred by CCN6 knockdown in HME cells. Analyses of the TCGA Breast Cancer Cohort (n = 1238) showed that IGF2BP2 and HMGA2 are significantly upregulated in metaplastic carcinoma compared to other breast cancer subtypes. In clinical samples, low CCN6 is frequent in tumors with high IGF2BP2/HMGA2 with spindle and squamous differentiation. CONCLUSIONS: These data shed light into the pathogenesis of metaplastic carcinoma and demonstrate a novel CCN6/IGF2BP2/HMGA2 oncogenic pathway with biomarker and therapeutic implications.

A hybrid breast cancer/mesenchymal stem cell population enhances chemoresistance and metastasis.

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.

Quantitative proteomic landscape of metaplastic breast carcinoma pathological subtypes and their relationship to triple-negative tumors.

Metaplastic breast carcinoma (MBC) is a highly aggressive form of triple-negative cancer (TNBC), defined by the presence of metaplastic components of spindle, squamous, or sarcomatoid histology. The protein profiles underpinning the pathological subtypes and metastatic behavior of MBC are unknown. Using multiplex quantitative tandem mass tag-based proteomics we quantify 5798 proteins in MBC, TNBC, and normal breast from 27 patients. Comparing MBC and TNBC protein profiles we show MBC-specific increases related to epithelial-to-mesenchymal transition and extracellular matrix, and reduced metabolic pathways. MBC subtypes exhibit distinct upregulated profiles, including translation and ribosomal events in spindle, inflammation- and apical junction-related proteins in squamous, and extracellular matrix proteins in sarcomatoid subtypes. Comparison of the proteomes of human spindle MBC with mouse spindle (CCN6 knockout) MBC tumors reveals a shared spindle-specific signature of 17 upregulated proteins involved in translation and 19 downregulated proteins with roles in cell metabolism. These data identify potential subtype specific MBC biomarkers and therapeutic targets.

A reproducible scaffold-free 3D organoid model to study neoplastic progression in breast cancer.

While 3D cellular models are useful to study biological processes, gel-embedded organoids have large variability. This paper describes high-yield production of large (~1 mm diameter), scaffold-free, highly-spherical organoids in a one drop-one organoid format using MCF10A cells, a non-tumorigenic breast cell line. These organoids display a hollow lumen and secondary acini, and express mammary gland-specific and progenitor markers, resembling normal human breast acini. When subjected to treatment with TGF-ß, the hypoxia-mimetic reagent CoCl2, or co-culture with mesenchymal stem/stromal cells (MSC), the organoids increase collagen I production and undergo large phenotypic and morphological changes of neoplastic progression, which were reproducible and quantifiable. Advantages of this scaffold-free, 3D breast organoid model include high consistency and reproducibility, ability to measure cellular collagen I production without noise from exogenous collagen, and capacity to subject the organoid to various stimuli from the microenvironment and exogenous treatments with precise timing without concern of matrix binding. Using this system, we generated organoids from primary metaplastic mammary carcinomas of MMTV-Cre;Ccn6fl/fl mice, which retained the high grade spindle cell morphology of the primary tumors. The platform is envisioned to be useful as a standardized 3D cellular model to study how microenvironmental factors influence breast tumorigenesis, and to potential therapeutics.

Mesenchymal Stem/Stromal Cell Engulfment Reveals Metastatic Advantage in Breast Cancer.

Twenty percent of breast cancer (BC) patients develop distant metastasis for which there is no cure. Mesenchymal stem/stromal cells (MSCs) in the tumor microenvironment were shown to stimulate metastasis, but the mechanisms are unclear. Here, we identified and quantified cancer cells engulfing stromal cells in clinical samples of BC metastasis by dual immunostaining for EZH2 and ALDH1 expression. Using flow cytometry and a microfluidic single-cell paring and retrieval platform, we show that MSC engulfment capacity is associated with BC cell metastatic potential and generates cells with mesenchymal-like, invasion, and stem cell traits. Whole-transcriptome analyses of selectively retrieved engulfing BC cells identify a gene signature of MSC engulfment consisting of WNT5A, MSR1, ELMO1, IL1RL2, ZPLD1, and SIRPB1. These results delineate a mechanism by which MSCs in the tumor microenvironment promote metastasis and provide a microfluidic platform with the potential to predict BC metastasis in clinical samples.

The matricellular protein CCN6 (WISP3) decreases Notch1 and suppresses breast cancer initiating cells.

Increasing evidence supports that the epithelial to mesenchymal transition (EMT) in breast cancer cells generates tumor initiating cells (TICs) but the contribution of the tumor microenvironment to these programs needs further elucidation. CCN6 (WISP3) is a secreted matrix-associated protein (36.9 kDa) of the CCN family (named after CTGF, Cyr61 and Nov) that is reduced or lost in invasive carcinomas of the breast with lymph node metastasis and in inflammatory breast cancer. CCN6 exerts breast cancer growth and invasion inhibitory functions, but the mechanisms remain to be defined. In the present study we discovered that ectopic CCN6 overexpression in triple negative (TN) breast cancer cells and in cells derived from patients is sufficient to induce a mesenchymal to epithelial transition (MET) and to reduce TICs. In vivo, CCN6 overexpression in the TIC population of MDA-MB-231 cells delayed tumor initiation, reduced tumor volume, and inhibited the development of metastasis. Our studies reveal a novel CCN6/Slug signaling axis that regulates Notch1 signaling activation, epithelial cell phenotype and breast TICs, which requires the conserved thrombospondin type 1 (TSP1) motif of CCN6. The relevance of these data to human breast cancer is highlighted by the finding that CCN6 protein levels are inversely correlated with Notch1 intracellular activated form (NICD1) in 69.5% of invasive breast carcinomas. These results demonstrate that CCN6 regulates epithelial and mesenchymal states transition and TIC programs, and pinpoint one responsible mechanism.