Proteolysis-a characteristic of tumor-initiating cells in murine metastatic breast cancer

Abstract

Tumor initiating cells (TICs) have been identified and functionally characterized in hematological malignancies as well as in solid tumors such as breast cancer. In addition to their high tumor-initiating potential, TICs are founder cells for metastasis formation and are involved in chemotherapy resistance. In this study we explored molecular pathways which enable this tumor initiating potential for a cancer cell subset of the transgenic MMTV-PyMT mouse model for metastasizing breast cancer. The cell population, characterized by the marker profile CD24+CD90+CD45−, showed a high tumorigenicity compared to non-CD24+CD90+CD45− cancer cells in colony formation assays, as well as upon orthotopic transplantation into the mammary fat pad of mice. In addition, these orthotopically grown CD24+CD90+CD45− TICs metastasized to the lungs. The transcriptome of TICs freshly isolated from primary tumors by cell sorting was compared with that of sorted non-CD24+CD90+CD45− cancer cells by RNA-seq. In addition to more established TIC signatures, such as epithelial-to-mesenchymal transition or mitogen signaling, an upregulated gene set comprising several classes of proteolytic enzymes was uncovered in the TICs. Accordingly, TICs showed high intra- and extracellular proteolytic activity. Application of a broad range of protease inhibitors to TICs in a colony formation assay reduced anchorage independent growth and had an impact on colony morphology in 3D cell culture assays. We conclude that CD24+CD90+CD45− cells of the MMTV- PyMT mouse model possess an upregulated proteolytic signature which could very well represent a functional hallmark of metastatic TICs from mammary carcinomas.

MMP14 empowers tumor‐initiating breast cancer cells under hypoxic nutrient‐depleted conditions

“…Animal model iTICs, as well as primary whole tumor cells, were generated from primary tumors of FVB/N-TgN(MMTV-PyVT)634Mul/J mice (7) that were used as a model for invasive metastasizing breast cancer, as previously described (10). Female Rag2 2/2 gc 2/2 lymphocyte-deficient mice (25,26) were used for OT and lung colonization assays.…”

“…Isolated tumors and lungs were paraffin embedded, processed, stained with hematoxylin/eosin (both MilliporeSigma, Burlington, MA, USA), as well as Ki67 (Santa Cruz Biotechnology, Heidelberg, Germany), E-cadherin (Cdh1; 610182; BD Biosciences, San Jose, CA, USA), keratin 8 (ab59400; Abcam, Cambridge, United Kingdom), or vimentin (Vim; 550513; BD Biosciences), and analyzed as previously described (10).…”

“…First identified in hematologic malignancies, TICs also reside within solid tumors, specifically human breast cancers (4)(5)(6). In the transgenic mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) mouse model for metastasizing breast cancer (7)(8)(9), we have recently documented the existence of a TIC subpopulation (10). This population was identified by the cell surface marker profile CD24 + CD90 + CD45 2 , which also identifies TICs of the MMTV-Wnt1 mouse model and metastasis-initiating ABBREVIATIONS: 2D, 2-dimensional; 3D, 3-dimensional; Akt, PKB; Cdh1, E-cadherin; Cdh2, N-cadherin; Ctrl, control; ECM, extracellular matrix; EMT, epithelial-to-mesenchymal transition; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; Hif, hypoxia-inducible factor; HSCC, hypoxic stem cell condition; iTIC, immortalized tumor-initiating cell; MIC, metastasisinitiating cell; MMP, matrix metalloproteinase; MMTV-PyMT, mouse mammary tumor virus-polyoma middle T; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; NSC, normoxic standard condition; OT, orthotopic transplantation; PC, principal component; PCA, principal component analysis; PE, phycoerythrin; pPyMT, primary polyoma middle T; pTIC, primary tumor-initiating cell; qRT-PCR, quantitative RT-PCR; sh, short hairpin; shCtrl, short hairpin control; Snai1, Snail homolog 1; TAPI-0, TNF-a protease inhibitor 0; TIC, tumor-initiating cell; Twist1, Twist-related protein 1; Vim, vimentin; Zeb1, zinc finger E-box-binding homeobox 1 cells (MICs) of the MMTV-PyMT mouse model (11,12).…”

“…Furthermore, a significantly up-regulated proteolytic signature was identified in these cells compared with non-CD24 + CD90 + CD45 2 breast cancer cells from the MMTV-PyMT model. The up-regulated proteases included mainly matrix metalloproteinases (MMPs), specifically Mmp2, Mmp13, and Mmp14 (10). Metalloproteinases are crucial players in tumorigenesis, determining the availability of growth and angiogenetic factors, regulating the activity of chemokines and cytokines by post-translational modifications, and altering cell-cell contacts and the extracellular matrix (ECM) composition (13)(14)(15)(16).…”

“…The detailed functional characterization of TICs remains challenging as a result of their small cell population size in primary MMTV-PyMT cancers (10). In addition, commercially available breast cancer cell lines possess only limited stem cell-like characteristics, with serial passaging associated with differentiation of the cells.…”