Krüppel-like factors in cancer progression: three fingers on the steering wheel

Kruppel-like factors (KLFs) comprise a highly conserved family of zinc finger transcription factors, that are involved in a plethora of cellular processes, ranging from proliferation and apoptosis to differentiation, migration and pluripotency. During the last few years, evidence on their role and deregulation in different human cancers has been emerging. This review will discuss current knowledge on Kruppel-like transcription in the epithelial-mesenchymal transition (EMT), invasion and metastasis, with a focus on epithelial cancer biology and the extensive interface with pluripotency. Furthermore, as KLFs are able to mediate different outcomes, important influences of the cellular and microenvironmental context will be highlighted. Finally, we attempt to integrate diverse findings on KLF functions in EMT and stem cell biology to fit in the current model of cellular plasticity as a tool for successful metastatic dissemination

Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays

Background: Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. Methodology/Principal Findings: Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0-100 nM) correlated well with SRB (Rho>0.95) with similar IC50 values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho. 0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. Conclusions/Significance: The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased sensitivity however implies standardized experimental conditions to minimize technical-induced variance

Expression profiling of migrated and invaded breast cancer cells predicts early metastatic relapse and reveals Krüppel-like factor 9 as a potential suppressor of invasive growth in breast cancer

Cell motility and invasion initiate metastasis. However, only a subpopulation of cancer cells within a tumor will ultimately become invasive. Due to this stochastic and transient nature, in an experimental setting, migrating and invading cells need to be isolated from the general population in order to study the gene expression profiles linked to these processes. This report describes microarray analysis on RNA derived from migrated or invaded subpopulations of triple negative breast cancer cells in a Transwell set-up, at two different time points during motility and invasion, pre-determined as “early” and “late” in real-time kinetic assessments. Invasion- and migration-related gene expression signatures were generated through comparison with non-invasive cells, remaining at the upper side of the Transwell membranes. Late-phase signatures of both invasion and migration indicated poor prognosis in a series of breast cancer data sets. Furthermore, evaluation of the genes constituting the prognostic invasion-related gene signature revealed Krüppel-like factor 9 (KLF9) as a putative suppressor of invasive growth in breast cancer. Next to loss in invasive vs non-invasive cell lines, KLF9 also showed significantly lower expression levels in the “early” invasive cell population, in several public expression data sets and in clinical breast cancer samples when compared to normal tissue. Overexpression of EGFP-KLF9 fusion protein significantly altered morphology and blocked invasion and growth of MDA-MB-231 cells in vitro. In addition, KLF9 expression correlated inversely with mitotic activity in clinical samples, indicating anti-proliferative effects

Differential regulation of extracellular matrix protein expression in carcinoma-associated fibroblasts by TGF-β1 regulates cancer cell spreading but not adhesion

Cancer progression is characterized by a complex reciprocity between neoplastic epithelium and adjacent stromal cells. In ductal carcinoma in situ (DCIS) of the breast, both reduced stromal decorin expression and myxoid stroma are correlated with increased recurrence risk. In this study, we aimed to investigate paracrine regulation of expression of decorin and related extracellular matrix (ECM) proteins in cancerassociated fibroblasts (CAFs). Transforming growth factor-β1 (TGF-β1) was identified as a competent ECM modulator, as it reduced decorin and strongly enhanced versican, biglycan and type I collagen expression. Similar but less pronounced effects were observed when fibroblasts were treated with basic fibroblast growth factor (bFGF). Despite this concerted ECM modulation, TGF-β1 and bFGF differentially regulated alpha-smooth muscle actin (α-SMA) expression, which is often proposed as a CAFmarker. Cancer cell-derived secretomes induced versican and biglycan expression in fibroblasts. Immunohistochemistry on twenty DCIS specimens showed a trend toward periductal versican overexpression in DCIS with myxoid stroma. Cancer cell adhesion was inhibited by decorin, but not by CAF-derived matrices. Cancer cells presented significantly enhanced spreading when seeded on matrices derived from TGF-β1-treated CAF. Altogether these data indicate that preinvasive cancerous lesions might modulate the composition of surrounding stroma through TGF-β1 release to obtain an invasion-permissive microenvironment