Marc Bracke

Stromal myofibroblasts are drivers of invasive cancer growth

Tissue integrity is maintained by the stroma in physiology. In cancer, however, tissue invasion is driven by the stroma. Myofibroblasts and cancer‐associated fibroblasts are important components of the tumor stroma. The origin of myofibroblasts remains controversial, although fibroblasts and bone marrow‐derived precursors are considered to be the main progenitor cells. Myofibroblast reactions also occur in fibrosis. Therefore, we wonder whether nontumorous myofibroblasts have different characteristics and different origins as compared to tumor‐associated myofibroblasts. The mutual interaction between cancer cells and myofibroblasts is dependent on multiple invasive growth‐promoting factors, through direct cell–cell contacts and paracrine signals. Since fibrosis is a major side effect of radiotherapy, we address the question how the main methods of cancer management, including chemotherapy, hormonotherapy and surgery affect myofibroblasts and by inference the surrogate endpoints invasion and metastasis.

Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac

Myofibroblasts are present at the invasion front in colon cancer. In an attempt to understand their putative proinvasive activity, we have developed an in vitro model. Myofibroblasts isolated from colon cancer tissue or obtained through transdifferentiation of colon fibroblasts by transforming growth factor (TGF)‐β stimulate invasion of colon cancer cells into collagen type I and Matrigel. We identified two convergent proinvasive agents secreted by myofibroblasts: namely scatter factor/hepatocyte growth factor (SF/HGF) and the TGF‐β‐upregulated extracellular matrix glycoprotein tenascin‐C (TNC), each of which is necessary though not sufficient for invasion. Myofibroblast‐stimulated invasion into collagen type I is characterized by a change from a round, nonmigratory morphotype with high RhoA and low Rac activity to an elongated, migratory morphotype with low RhoA and high Rac activity. RhoA inactivation is determined by the epidermal growth factor (EGF)‐like repeats of TNC through EGF‐receptor signaling that confers a permissive and priming signal for the proinvasive activity of SF/HGF that activates Rac via c‐Met. We confirmed the validity of this mechanism by using pharmacological modulators and dominant negative or constitutive active mutants that interfere with RhoA‐Rho kinase and Rac signaling. Our in vitro results point to a new putative proinvasive signal for colon cancer cells provided by myofibroblasts in the tumor stroma.

The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling

Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.

Molecular and pathological signatures of epithelial–mesenchymal transitions at the cancer invasion front

Reduction of epithelial cell–cell adhesion via the transcriptional repression of cadherins in combination with the acquisition of mesenchymal properties are key determinants of epithelial–mesenchymal transition (EMT). EMT is associated with early stages of carcinogenesis, cancer invasion and recurrence. Furthermore, the tumor stroma dictates EMT through intensive bidirectional communication. The pathological analysis of EMT signatures is critically, especially to determine the presence of cancer cells at the resection margins of a tumor. When diffusion barriers disappear, EMT markers may be detected in sera from cancer patients. The detection of EMT signatures is not only important for diagnosis but can also be exploited to enhance classical chemotherapy treatments. In conclusion, further detailed understanding of the contextual cues and molecular mediators that control EMT will be required in order to develop diagnostic tools and small molecule inhibitors with potential clinical implications.

Dual activities of galectin-3 in human prostate cancer: tumor suppression of nuclear galectin-3 vs tumor promotion of cytoplasmic galectin-3.

Galectin-3, a multifunctional lectin, is involved during cancer progression. Previous observations showed that both cytosolic expression and nuclear exclusion of galectin-3 in human prostate cancer cells were associated to progression of the disease. In this study, we examined the biological roles of galectin-3 when expressed either in the nucleus or in the cytosol. LNCaP, a galectin-3-negative human prostate cancer cell line, was used to generate transfectants expressing galectin-3 either in the nucleus or in the cytosol. No changes in cell morphology, proliferation, attachment to laminin-1 or androgen dependency were observed. Cytoplasmic galectin-3 induced significantly increased Matrigel invasion, anchorage-independent growth and in vivo tumor growth and angiogenesis, and decreased inducible apoptosis. Surprisingly, nuclear galectin-3 affected these parameters in an opposite fashion with an overall antitumoral activity. Thus, our study demonstrates that galectin-3 exerts opposite biological activities according to its cellular localization: nuclear galectin-3 plays antitumor functions and cytoplasmic galectin-3 promotes tumor progression.

Critical role of N-cadherin in myofibroblast invasion and migration in vitro stimulated by colon-cancer-cell-derived TGF-β or wounding

Invasion of stromal host cells, such as myofibroblasts, into the epithelial cancer compartment may precede epithelial cancer invasion into the stroma. We investigated how colon cancer-derived myofibroblasts invade extracellular matrices in vitro in the presence of colon cancer cells. Myofibroblast spheroids invade collagen type I in a stellate pattern to form a dendritic network of extensions upon co-culture with HCT-8/E11 colon cancer cells. Single myofibroblasts also invade Matrigel™ when stimulated by HCT-8/E11 colon cancer cells. The confrontation of cancer cells with extracellular matrices and myofibroblasts, showed that cancer-cell-derived transforming growth factor-β (TGF-β) is required and sufficient for invasion of myofibroblasts. In myofibroblasts, N-cadherin expressed at the tips of filopodia is upregulated by TGF-β. Functional N-cadherin activity is implicated in TGF-β stimulated invasion as evidenced by the neutralizing anti-N-cadherin monoclonal antibody (GC-4 mAb), and specific N-cadherin knock-down by short interference RNA (siRNA). TGF-β1 stimulates Jun N-terminal kinase (also known as stress-activated protein kinase) (JNK) activity in myofibroblasts. Pharmacological inhibition of JNK alleviates TGF-β stimulated invasion, N-cadherin expression and wound healing migration. Neutralization of N-cadherin activity by the GC-4 or by a 10-mer N-cadherin peptide or by siRNA reduces directional migration, filopodia formation, polarization and Golgi-complex reorientation during wound healing. Taken together, our study identifies a new mechanism in which cancer cells contribute to the coordination of invasion of stromal myofibroblasts.

Synthetic and biological activity evaluation studies on novel 1,3-diarylpropenones.

Fourteen novel C-prenylated and O-allylated 1,3-diarylpropenones (chalcones) were synthesized by Claisen-Schmidt condensation reaction of C-prenylated/O-allylated acetophenones with appropriate aldehydes; twelve of these model chalcones were screened in an assay based on the confrontation of invasive human MCF-7/6 mammary carcinoma cells with fragments of normal embryonic chick heart in vitro. Out of the twelve chalcones tested, three were found to exhibit potent anti-invasive activity. Some of these chalcones and their precursor acetophenones were also tested for inhibition of initiation of lipid peroxidation in rat liver microsomes; a prenylated acetophenone carrying two methoxy groups and two free phenolic hydroxy functions was found to be a potential antioxidant.

Effect of the Secretory Small GTPase Rab27B on Breast Cancer Growth, Invasion, and Metastasis

BACKGROUND Secretory GTPases like Rab27B control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment. The expression and role of Rab27B in breast cancer were unknown. METHODS Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided. RESULTS Increased expression of Rab27B promoted G(1) to S phase cell cycle transition, proliferation and invasiveness of cells in culture, and invasive tumor growth and hemorrhagic ascites production in a xenograft mouse model (n = 10; at 10 weeks, survival of MCF-7 GFP- vs GFP-Rab27B-injected mice was 100% vs 62.5%, hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.88, P = .03). Mass spectrometric analysis of purified Rab27B-secretory vesicles identified heat-shock protein 90alpha as key proinvasive growth regulator. Heat-shock protein 90alpha secretion was Rab27B-dependent and was required for matrix metalloproteinase-2 activation. All Rab27B-mediated functional responses were GTP- and geranylgeranyl-dependent. Presence of endogenous Rab27B mRNA and protein, but not of Rab3D or Rab27A mRNA, was associated with lymph node metastasis (P < .001) and differentiation grade (P = .001) in ER-positive human breast tumors. CONCLUSIONS Rab27B regulates invasive growth and metastasis in ER-positive breast cancer cell lines, and increased expression is associated with poor prognosis in humans.

Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography.

A new and reliable HPLC method for the quantitation of daidzein, equol, and genistein in human breast tissue has been developed. The method was applied to biopsies from women undergoing breast reductions, who, prior to surgery, had ingested either a soy isoflavone preparation or a placebo tablet. The results were compared with data collected for urine and serum of the same subjects using standard methods. The limits of detection in the breast tissue homogenate were 24.7 nmol/l for daidzein, 148.0 nmol/l for equol, and 28.4 nmol/l for genistein (S/N of 3). The chromatographic limits of quantitation were 62.5 nmol/l for daidzein and genistein, and 125.0 nmol/l for equol, for which the accuracies were 86.0%, 83.6%, and 81.8%, respectively. The coefficients of variation of these measurements were all below 20% (11.1% for daidzein, 16.4% for genistein, and 13.2% for equol). The sample preparation comprised a concentration step and the absolute limits of quantitation were, therefore, 4.7 nmol/l, 18.8 nmol/l, and 0.94 nmol/l for daidzein and genistein, and 9.4 nmol/l, 37.5 nmol/l, and 1.9 nmol/l for equol in urine, serum, and breast tissue homogenate, respectively. Recoveries were between 70% (+/-5.6%) in breast tissue homogenate and 100% (+/-14.1%) in urine and serum for all three compounds. Equol (less than 1 micromol/l homogenate) was found to be the predominant phytoestrogen in breast tissue and its concentrations exceeded those in serum. The concentrations of phytoestrogens were at least 100-fold higher in urine than in serum and breast tissue.

Antitumor Activity of the Selective MDM2 Antagonist Nutlin-3 Against Chemoresistant Neuroblastoma With Wild-Type p53

BACKGROUND Restoring p53 function by antagonizing its interaction with the negative regulator MDM2 is an appealing nongenotoxic approach to treating tumors with wild-type p53. Mutational inactivation of p53 is rare in neuroblastoma tumors at diagnosis and occurs in only a subset of multidrug-resistant neuroblastomas. METHODS The antiproliferative and cytotoxic effect of nutlin-3, a small-molecule MDM2 antagonist, was examined in chemosensitive (UKF-NB-3) and matched chemoresistant neuroblastoma cells with wild-type p53 (UKF-NB-3(r)DOX20) or with mutant p53 (UKF-NB-3(r)VCR10). Activation of the p53 pathway was assessed by expression analysis of p53 target genes, flow cytometric cell cycle analysis, and apoptosis assays. Mice with established chemoresistant tumor xenografts were treated orally with nutlin-3 or vehicle control (n = 5-10 mice per group) and were used to evaluate effects on tumor growth, p53 pathway activity, and metastatic tumor burden. All statistical tests were two-sided. RESULTS Nutlin-3 induced a similar activation of the p53 pathway in UKF-NB-3 and UKF-NB-3(r)DOX20 cells, as evidenced by increased expression of p53 target genes, G1 cell cycle arrest, and induction of apoptosis. No such response was observed in UKF-NB-3(r)VCR10 cells with mutant p53. Oral administration of nutlin-3 to UKF-NB-3(r)DOX20 xenograft-bearing mice led to inhibition of primary tumor growth (mean tumor volume after 3 weeks of treatment, nutlin-3- vs vehicle-treated mice: 772 vs 1661 mm3, difference = 890 mm3, 95% confidence interval = 469 to 1311 mm3, P < .001), p53 pathway activation, and reduction in the extent of metastatic disease. The growth of UKF-NB-3(r)VCR10 xenografts was unaffected by nutlin-3. CONCLUSIONS Nutlin-3 activates the p53 pathway and suppresses tumor growth in this model system of chemoresistant neuroblastoma, provided that wild-type p53 is present.