CD24⁺ ovary cancer cells exhibit an invasive mesenchymal phenotype.

We recently reported that the subset of CD24(+) cells in ovarian cancer possesses various cancer stem cell properties. In this study, we further show that this subpopulation of ovarian cancer cells exhibits an epithelial-mesenchymal transition (EMT) phenotype, high invasive capacity, and CXCR4/SDF-1-mediated chemotactic migration. We evaluated CD24 expression in various ovarian cancer cell lines by flow cytometric analysis. CAOV3 and a primary ovarian cancer cell line Clone 4 were sorted into CD24(+) and CD24(-) subpopulations by FACS and Western blot, cell invasion, adhesion, and in vitro chemotaxis assays were performed with these two subpopulations. We also assessed the effects of shRNA depletion of CD24 in CAOV3 and Clone 4 cells by Western blot and cell invasion assays. CD24 expression in ovarian cancer cell lines correlated with aggressive histologic subtypes of epithelial ovarian cancer. The CD24(+) subpopulation was also more invasive than the CD24(-) subpopulation and showed higher CXCR4/SDF-1-mediated chemotactic migration. CD24(+) cells exhibited an EMT phenotype as characterized by loss of E-cadherin expression and gain of vimentin, Twist, and Snail1 expression. In addition, CD24(+) cells stimulated cell attachment to fibronectin through the activation of ß1 integrin. Depletion of CD24 expression by CD24 shRNA efficiently suppressed cell invasion and induced downregulation of CXCR4 as well as loss of the EMT phenotype. In conclusion, CD24 expression in ovarian cancer may be related to tumor aggressiveness, in particular cell invasion and chemotactic migration. Therefore, CD24 may be a good candidate for a therapeutic target for ovarian cancer.

Invasive breast cancer induces laminin-332 upregulation and integrin ß4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype during tissue remodeling.

INTRODUCTION: Although development of anoikis-resistant myofibroblasts during tissue remodeling is known to be associated with tumor invasion, the mechanism by which myofibroblasts become resistant to anoikis is unknown. We previously demonstrated laminin-332 upregulation in the fibrosis around invasive ductal carcinoma (IDC). Because laminin-332 promotes cell survival through binding to integrins, we hypothesized that invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts by upregulating laminin-332 expression during tissue remodeling. Here, we demonstrate that invasive breast cancer cells induce laminin-332 upregulation and integrin ß4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype. METHODS: Three types of fibroblasts were isolated from the tumor burden, the fibrosis, and normal tissue of patients with early stage IDC (less than 10 mm diameter), designated cancer-associated fibroblasts (CAFs), interface fibroblasts (InFs), and normal breast fibroblasts (NBFs), respectively. To investigate direct and indirect crosstalk with tumor cells, fibroblasts were co-cultured with invasive MDA-MB-231 or noninvasive MCF7 cells or in conditioned medium. Anoikis resistance of fibroblasts was measured by cell viability and caspase-3 activity after incubation on poly-HEMA coated plates for 72 hours. Involvement of laminin-332/integrin α3ß1 or α6ß4 signaling in anoikis resistance was confirmed by treatment with purified laminin-332 or blocking antibodies against laminin-332, integrin ß1, or integrin ß4. RESULTS: MDA-MB-231 cells induced laminin-332 upregulation and integrin ß4 neoexpression in fibroblasts, leading to anoikis resistance. InFs showed a higher endogenous level of laminin-332 than did CAFs and NBFs. After stimulation with MDA-MB-231-conditioned medium, laminin-332 expression of InFs was dramatically increased and maintained under anoikis conditions. Laminin-332 upregulation was also observed in CAFs and NBFs, but at a lower level than in InFs. Laminin-332 induced Akt (Ser473) phosphorylation by binding to integrin α3ß1. Integrin ß4 neoexpression induced laminin-332-independent Rac1 activation and promoted anoikis resistance in fibroblasts approximately twofold more effectively than did laminin-332, regardless of the type of fibroblast. In addition, integrin ß4 expression suppressed fibroblast aggregation in conditions of anoikis. CONCLUSION: Invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts during tissue remodeling by inducing laminin-332 upregulation and integrin ß4 neoexpression. Interface fibroblasts appear to be the primary myofibroblasts that interact with invasive tumor cells during tissue remodeling.

Stromal fibroblasts from the interface zone of human breast carcinomas induce an epithelial-mesenchymal transition-like state in breast cancer cells in vitro.

Fibroblasts were extracted from tissue in tumor burden zones, distal normal zones and interface zones between tumor and normal tissue of human breast carcinomas, and the corresponding fibroblasts were designated as cancer-associated fibroblasts (CAFs), normal zone fibroblasts (NFs) and interface zone fibroblasts (INFs). The crosstalk between three types of fibroblasts and breast cancer cells was evaluated using an in vitro direct co-culture model. We found that INFs grew faster and expressed higher levels of fibroblast activation protein than did NFs and CAFs. Compared with CAFs and NFs, INFs grown with breast cancer cells were significantly more effective in inducing an epithelial-mesenchymal transition (EMT) in cancer cells, as indicated by induction of vimentin and N-cadherin and downregulation of E-cadherin. This EMT process was also accompanied by activation of extracellular signal-regulated kinase (ERK) and modulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Additionally, INFs promoted breast cell migration to a larger extent compared with NFs and CAFs. Taken together, these findings indicate that INFs isolated from the tumor interface zone exhibited more robust biological modulatory activity than did NFs and CAFs isolated from normal and tumor zones of the same tumor tissue, suggesting that the interface zone of the tumor represents a dynamic region vital to tumor progression.

Proteomic molecular portrait of interface zone in breast cancer.

Surgical tumor margins are intended to encompass residual tumor cells but may not always accurately delineate the boundary between tumor and normal tissue. Efforts to define tumor margins based on molecular analysis have achieved limited success. Furthermore, no clinical trials have addressed the scope of the tumor microenvironment. Here, we considered the tumor cell population and surrounding microenvironment in delineating tumor margins, classifying breast cancer into tumor and normal zones, and introducing the concept of an interface zone, the region between the invading tumor front and normal tissue, which develops during tumor invasion and metastasis through remodeling of the tumor microenvironment. Pathological signatures of invasion markers in tumor tissues are most dynamic within the invading tumor front. We compared protein profiles of tumor, normal, and interface zones using MALDI-MS. Proteins upregulated in the interface zone were identified by peptide mass fingerprinting and confirmed by database searching with chemically assisted MALDI-PSD spectra. Upregulation was confirmed for RhoGDIα, CAPG, WDR1, and CK8 by Western and immunohistochemical analyses. Our results demonstrate that the molecular profile of the interface zone is unique and suggest that upregulation of proteins here may be related to progression and metastasis of breast carcinomas.