Vincent Castronovo

Histone deacetylases inhibitors as anti-angiogenic agents altering vascular endothelial growth factor signaling

Angiogenesis is a complex biological process involving the coordinated modulation of many genes. Histone deacetylases (HDAC) are a growing family of enzymes that mediate the availability of chromatin to the transcriptional machinery. Trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA), two HDAC inhibitors known to relieve gene silencing, were evaluated as potential antiangiogenic agents. TSA and SAHA were shown to prevent vascular endothelial growth factor (VEGF)-stimulated human umbilical cord endothelial cells (HUVEC) from invading a type I collagen gel and forming capillary-like structures. SAHA and TSA inhibited the VEGF-induced formation of a CD31-positive capillary-like network in embryoid bodies and inhibited the VEGF-induced angiogenesis in the CAM assay. TSA also prevented, in a dose-response relationship, the sprouting of capillaries from rat aortic rings. TSA inhibited in a dose-dependent and reversible fashion the VEGF-induced expression of VEGF receptors, VEGFR1, VEGFR2, and neuropilin-1. TSA and SAHA upregulated the expression by HUVEC of semaphorin III, a recently described VEGF competitor, at both mRNA and protein levels. This effect was specific to endothelial cells and was not observed in human fibroblasts neither in vascular smooth muscle cells. These observations provide a conspicuous demonstration that HDAC inhibitors are potent anti-angiogenic factors altering VEGF signaling.

Small Integrin-Binding LIgand N-linked Glycoproteins (SIBLINGs): Multifunctional proteins in cancer

Numerous components and pathways are involved in the complex interplay between cancer cells and their environment. The family of glycophosphoproteins comprising osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein and matrix extracellular phosphoglycoprotein — small integrin-binding ligand N-linked glycoproteins (SIBLINGs) — are emerging as important players in many stages of cancer progression. From their detection in various human cancers to the demonstration of their key functional roles during malignant transformation, invasion and metastasis, the SIBLINGs are proteins with potential as diagnostic and prognostic tools, as well as new therapeutic targets.

DECREASED EXPRESSION OF GALECTIN-3 IS ASSOCIATED WITH PROGRESSION OF HUMAN BREAST CANCER

Galectin‐3, a member of the β‐galactoside‐binding lectin family, is involved in several biological events including binding to the basement membrane glycoprotein laminin. Although the exact role of galectin‐3 during the interactions between cells and laminin is not yet known, it has recently been observed that its expression is down‐regulated at both the protein and the mRNA level in colon cancer tissues in correlation with progression of the disease. This study investigated the possibility that breast cancer cells might also exhibit decreased galectin‐3 expression in association with their aggressiveness. The expression of galectin‐3 was examined by immunoperoxidase staining, using a polyclonal antibody raised against recombinant galectin‐3, in a collection of 98 human breast lesions including 12 fibroadenomas, 15 fibrocystic disease lesions, 22 in situ carcinomas, and 49 infiltrating ductal carcinomas, 19 of which had positive axillary lymph nodes. Normal breast tissue adjacent to the lesions was present in 59 biopsies. Normal breast tissue expressed high levels (3+) of galectin‐3. High expression (2+ to 3+) was also found in most benign lesions examined. The expression of galectin‐3 was significantly decreased in in situ carcinoma and this down‐regulation was more pronounced in invasive ductal carcinoma, particularly when associated with infiltration of axillary lymph nodes. These data constitute the first observation that galectin‐3 is down‐regulated in breast cancer and suggest the decreased expression of this galactoside‐binding lectin is associated with the acquisition of the invasive and metastatic phenotype.

The NF-kappa B transcription factor and cancer: high expression of NF-kappa B- and I kappa B-related proteins in tumor cell lines.

NF-kappa B is a pleiotropic transcription factor which controls the expression of many genes and viruses. To date, there is good evidence, but no definitive proof, for its role in tumor formation and development of metastasis. To investigate the possibility that members of the NF-kappa B family could participate in the molecular control of the transformed and invasive phenotype, we examined the expression of these proteins in a variety of human tumor cell lines. The expression of p50, p65, p52 and I kappa B was quantified at the protein level using western immunoblot and mobility shift assay and at the RNA level by northern blot. We observed high expression of the NF-kappa B inhibitor I kappa B in the ovarian carcinoma cell line OVCAR-3 together with constitutive nuclear NF-kappa B activity. We also studied the colon carcinoma cell line HT-29 and its metastatic counterpart HTM-29 and we observed specific expression of the p52 NF-kappa B-related protein in the metastatic cells. Our data confirm that NF-kappa B could be involved in the genesis of a variety of cancers including solid tumors and provide us with interesting models to explore the exact role of these transcription factors in cancer.

Histone deacetylase HDAC8 associates with smooth muscle alpha-actin and is essential for smooth muscle cell contractility

Although originally characterized as nuclear enzymes controlling the stability of nucleosomes, histone deacetylases (HDACs) may also exert their activity within the cytosol. Recently, we have demonstrated that HDAC8, a class I HDAC, is a novel, prominently cytosolic marker of smooth muscle differentiation. As HDAC8 displays a striking stress fiber‐like pattern of distribution and is coexpressed in vivo with smooth muscle α‐actin (α‐SMA) and smooth muscle myosin heavy chain, we have explored the possible participation of this HDAC in smooth muscle cytoskeleton regulation. Cell fractionation assays performed with primary human smooth muscle cells (HSMCs) showed that HDAC8, in contrast to HDAC1 and HDAC3, was enriched in cytoskeleton‐bound protein fractions and insoluble cell pellets, suggesting an association of HDAC8 with the cystoskeleton. Coimmunoprecipitation experiments using HSMCs, NIH‐3T3 cells, and human prostate tissue lysates further demonstrated that HDAC8 associates with α‐SMA but not with β‐actin. HDAC8 silencing through RNA interference strongly reduced the capacity of HSMCs to contract collagen lattices. Mock transfections had no effect on HSMC contractily, and transfections with small interfering RNAs (siRNAs) specific for HDAC6, a cytosolic HDAC that functions as an α‐tubulin deacetylase, resulted in a weak contraction inhibition. Although mock‐ and HDAC6 siRNA‐transfected HSMCs showed no noticeable morphological changes, HDAC8 siRNA‐transfected HSMCs displayed a size reduction with diminished cell spreading after replating. Altogether, our findings indicate that HDAC8 associates with the smooth muscle actin cytoskeleton and may regulate the contractile capacity of smooth muscle cells.

GALECTIN‐3 AND LAMININ EXPRESSION IN NEOPLASTIC AND NON‐NEOPLASTIC THYROID TISSUE

Galectin‐3 is a 31 kD β‐galactoside‐binding lectin which is expressed by several types of non‐neoplastic and neoplastic cells and which may be involved in cell–extracellular matrix interactions. An immunohistochemical study has been made of the expression of galectin‐3, as well as its ligand, laminin, in a spectrum of benign and malignant thyroid neoplasms and in some non‐neoplastic conditions. Immunohistochemistry with anti‐human recombinant galectin‐3 antibody showed consistent, intense positivity in the neoplastic cells of 18 cases of papillary carcinoma and less intense staining in the five anaplastic carcinomas studied. In addition, two out of three poorly differentiated carcinomas, three out of six medullary carcinomas, and four out of eight follicular carcinomas had less intense or focal positivity. One case of Hürthle cell carcinoma showed scattered strongly positive cells. Eight follicular adenomas, three hyperplastic nodules, five nodular goitres, and normal thyroid tissue were negative. Galectin‐3 mRNA expression was also evaluated in three of the papillary carcinomas, two follicular adenomas, and one hyperplastic nodule with matched normal tissue. Northern blot analysis demonstrated mRNA overexpression in the three cases of papillary carcinomas, whereas normal and benign tissues were negative. Laminin distribution in neoplastic and non‐neoplastic tissue varied with architectural patterns but did not correlate with galectin‐3 immunohistochemical expression. We conclude that expression of galectin‐3 is limited to inflammatory foci in normal and benign thyroid tissue and is a phenotypic feature of malignant thyroid neoplasms, especially papillary carcinomas.

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.

New insights into the metastasis‐associated 67 kD laminin receptor

The interactions between tumor cells and laminin or other components of the extracellular matrix have been shown to play an important role in tumor invasion and metastasis. These interactions are mediated by different cell surface molecules, including the monomeric 67 kD laminin receptor. This molecule appears to be very peculiar since so far only a full‐length gene encoding a 37 kD precursor protein has been isolated and the mechanism by which the precursor reaches the mature form is not understood. Based on clinical data, which clearly demonstrate the importance of the receptor in tumor progression, studies were conducted to define the structure, expression, and function of this laminin receptor as a step toward developing therapeutic strategies that target this molecule. The data suggest that acylation of the precursor is the key mechanism in maturation of the 67 kD form. The function of the membrane receptor is to stabilize the binding of laminin to cell surface integrins, acting as an integrin‐accessory molecule, although homology of the gene encoding the receptor precursor with other genes suggests additional functions. Downregulation of the receptor expression on tumor cells might open new therapeutic approaches to decrease tumor aggressiveness. J. Cell. Biochem. 67:155–165, 1997.

Expression of the 67-kD laminin receptor, galectin-1, and galectin-3 in advanced human uterine adenocarcinoma

Alterations of tumor cell interactions with laminin, a basement membrane glycoprotein, are consistent features of the invasive and metastatic phenotype. Qualitative and quantitative changes in the expression of cell surface laminin-binding proteins have been correlated with the ability of cancer cells to cross basement membranes during the metastatic cascade. Such phenotypic modifications are usually associated with poor prognosis. In this study, the authors examined the possibility that expression of three laminin-binding proteins, the 67-kD laminin receptor (67LR), galectin-1, and galectin-3, is altered in human endometrial cancer in a fashion similar to that reported in other carcinomas, such as breast, colon, and ovarian cancer. Twenty advanced uterine adenocarcinomas were analyzed for expression of these three molecules using immunoperoxidase staining and specific antibodies. The authors found a significant increase in the expression of the 67LR and galectin-1 in cancer cells compared with normal adjacent endometrium (P = .0004 and .0022, respectively). As observed in other carcinomas, a significant down-regulation of galectin-3 expression was found in endometrial cancer cells compared with normal mucosa (P = .02). In the galectin-3 positive tumors, galectin-3 was detected in the cytoplasm and/or nucleus of cancer cells. Interestingly, tumors in which galectin-3 was detected only in the cytoplasm were characterized by deeper invasion of the myometrium than lesions where galectin-3 was found both in nucleus and cytoplasm (P = .02). This study shows an alteration of nonintegrin laminin-binding protein expression in advanced human endometrial cancer. Further studies on larger populations should determine the prognostic value of the detection of these laminin-binding proteins in endometrial carcinoma. Inverse modulation of the 67LR and galectin-3 appears to be a phenotypical feature of invasive carcinoma.

Formation of the 67-kDa laminin receptor by acylation of the precursor

Even though the involvement of the 67‐kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full‐length gene encoding a 37‐kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67‐kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120‐kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP‐40‐lysis buffer whereas the 37LRP is NP‐40‐insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP‐40‐soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67‐ and the 120‐kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross‐reacting molecule. J. Cell. Biochem. 69:244–251, 1998.