Avraham Raz

Galectin-3 in angiogenesis and metastasis

Galectin-3 is a member of the family of β-galactoside-binding lectins characterized by evolutionarily conserved sequences defined by structural similarities in their carbohydrate-recognition domains. Galectin-3 is a unique, chimeric protein consisting of three distinct structural motifs: (i) a short NH2 terminal domain containing a serine phosphorylation site; (ii) a repetitive proline-rich collagen-α-like sequence cleavable by matrix metalloproteases; and (iii) a globular COOH-terminal domain containing a carbohydrate-binding motif and an NWGR anti-death motif. It is ubiquitously expressed and has diverse biological functions depending on its subcellular localization. Galectin-3 is mainly found in the cytoplasm, also seen in the nucleus and can be secreted by non-classical, secretory pathways. In general, secreted galectin-3 mediates cell migration, cell adhesion and cell-cell interactions through the binding with high affinity to galactose-containing glycoproteins on the cell surface. Cytoplasmic galectin-3 exhibits anti-apoptotic activity and regulates several signal transduction pathways, whereas nuclear galectin-3 has been associated with pre-mRNA splicing and gene expression. Its unique chimeric structure enables it to interact with a plethora of ligands and modulate diverse functions such as cell growth, adhesion, migration, invasion, angiogenesis, immune function, apoptosis and endocytosis emphasizing its significance in the process of tumor progression. In this review, we have focused on the role of galectin-3 in tumor metastasis with special emphasis on angiogenesis.

3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

Herceptin failure is a major clinical problem in breast cancer. A subset of breast cancer patients with high HER-2/neu levels eventually experience metastatic disease progression when treated with Herceptin as a single agent. Mechanistic details of development of this aggressive disease are not clear. Therefore, there is a dire need to better understand the mechanisms by which drug resistance develops and to design new combined treatments that benefit patients with aggressive breast cancer and have minimal toxicity. We hypothesized that 3, 3′-diindolylmethane (DIM), a non-toxic agent can be combined with Herceptin to treat breast cancers with high levels of HER-2/neu. Here, we evaluated the effects of Herceptin alone and in combination with DIM on cell viability, apoptosis and clonogenic assays in SKBR3 (HER-2/neu-expressing) and MDA-MB-468 (HER-2/neu negative) breast cancer cells. We found that DIM could enhance the effectiveness of Herceptin by significantly reducing cell viability, which was associated with apoptosis-induction and significant inhibition of colony formation, compared with single agent treatment. These results were consistent with the down-regulation of Akt and NF-kB p65. Mechanistic investigations revealed a significant upregulation of miR-200 and reduction of FoxM1 expression in DIM and Herceptin-treated breast cancer cells. We, therefore, transfected cells with pre-miR-200 or silenced FoxM1 in these cells for understanding the molecular mechanism involved. These results provide experimental evidence, for the first time, that DIM plus Herceptin therapy could be translated to the clinic as a therapeutic modality to improve treatment outcome of patients with breast cancer, particularly for the patients whose tumors express high levels of HER-2/neu.

Nuclear transport of galectin-3 and its therapeutic implications.

Galectin-3, a member of β-galactoside-binding gene family is a multi-functional protein, which regulates pleiotropic biological functions such as cell growth, cell adhesion, cell-cell interactions, apoptosis, angiogenesis and mRNA processing. Its unique structure enables it to interact with a plethora of ligands in a carbohydrate dependent or independent manner. Galectin-3 is mainly a cytosolic protein, but can easily traverse the intracellular and plasma membranes to translocate into the nucleus, mitochondria or get externalized. Depending on the cell type, specific experimental conditions in vitro, cancer type and stage, galectin-3 has been reported to be exclusively cytoplasmic, predominantly nuclear or distributed between the two compartments. In this review we have summarized the dynamics of galectin-3 shuttling between the nucleus and the cytoplasm, the nuclear transport mechanisms of galectin-3, how its specific interactions with the members of β-catenin signaling pathways affect tumor progression, and its implications as a therapeutic target.

Autocrine motility factor promotes HER2 cleavage and signaling in breast cancer cells.

Trastuzumab (Herceptin) is an effective targeted therapy in HER2-overexpressing human breast carcinoma. However, many HER2-positive patients initially or eventually become resistant to this treatment, so elucidating mechanisms of trastuzumab resistance that emerge in breast carcinoma cells is clinically important. Here, we show that autocrine motility factor (AMF) binds to HER2 and induces cleavage to the ectodomain-deleted and constitutively active form p95HER2. Mechanistic investigations indicated that interaction of AMF with HER2 triggers HER2 phosphorylation and metalloprotease-mediated ectodomain shedding, activating phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase signaling and ablating the ability of trastuzumab to inhibit breast carcinoma cell growth. Furthermore, we found that HER2 expression and AMF secretion were inversely related in breast carcinoma cells. On the basis of this evidence that AMF may contribute to HER2-mediated breast cancer progression, our findings suggest that AMF-HER2 interaction might be a novel target for therapeutic management of patients with breast cancer, whose disease is resistant to trastuzumab.

Nucleoporin Nup98 mediates galectin-3 nuclear-cytoplasmic trafficking

Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus-cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3-nucleus translocation rate. Overall, the results show Nup98s involvement in nuclear-cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.

Gp78, an E3 ubiquitin ligase acts as a gatekeeper suppressing nonalcoholic steatohepatitis (NASH) and liver cancer.

Nonalcoholic steatohepatitis (NASH) is related to metabolic dysregulation and the perturbation of endoplasmic reticulum (ER) homeostasis that frequently develops into hepatocellular carcinoma (HCC). Gp78 is E3 ligase, which regulates endoplasmic reticulum-associated degradation (ERAD) by ubiquitinylation of misfolded ER proteins. Here, we report that upon ageing (12 months), gp78-/- mice developed obesity, recapitulating age-related human NASH. Liver histology of gp78-/- mice revealed typical steatosis, hepatic inflammation and fibrosis, followed by progression to hepatocellular tumors. Acute ER stress revealed that loss of gp78 results in up regulation of unfolded protein response (UPR) pathways and SREBP-1 regulating de novo lipogenesis, responsible for fatty liver. Tissue array of human hepatocellular carcinoma (HCC) demonstrated that the expression of gp78 was inversely correlated with clinical grades of cancer. Here, we have described the generation of the first preclinical experimental model system which spontaneously develops age-related NASH and HCC, linking ERAD to hepatosteatosis, cirrhosis, and cancer. It suggests that gp78 is a regulator of normal liver homeostasis and a tumor suppressor in human liver.

Multivalent scaffolds induce galectin-3 aggregation into nanoparticles

Summary Galectin-3 meditates cell surface glycoprotein clustering, cross linking, and lattice formation. In cancer biology, galectin-3 has been reported to play a role in aggregation processes that lead to tumor embolization and survival. Here, we show that lactose-functionalized dendrimers interact with galectin-3 in a multivalent fashion to form aggregates. The glycodendrimer–galectin aggregates were characterized by dynamic light scattering and fluorescence microscopy methodologies and were found to be discrete particles that increased in size as the dendrimer generation was increased. These results show that nucleated aggregation of galectin-3 can be regulated by the nucleating polymer and provide insights that improve the general understanding of the binding and function of sugar-binding proteins.

Autocrine Motility Factor Modulates EGF-Mediated Invasion Signaling

Autocrine motility factor (AMF) enhances invasion by breast cancer cells, but how its secretion and effector signaling are controlled in the tumor microenvironment is not fully understood. In this study, we investigated these issues with a chimeric AMF that is secreted at high levels through a canonical endoplasmic reticulum (ER)/Golgi pathway. Using this tool, we found that AMF enhances tumor cell motility by activating AKT/ERK, altering actin organization, and stimulating β-catenin/TCF and activating protein 1 transcription. EGF enhanced secretion of AMF through its casein kinase II-mediated phosphorylation. RNA interference-mediated attenuation of AMF expression inhibited EGF-induced invasion by suppressing extracellular signal-regulated kinase signaling. Conversely, exogenous AMF overcame the inhibitory effect of EGF receptor inhibitor gefitinib on invasive motility by activating HER2 signaling. Taken together, our findings show how AMF modulates EGF-induced invasion while affecting acquired resistance to cytotoxic drugs in the tumor microenvironment.

Ocimum gratissimum retards breast cancer growth and progression and is a natural inhibitor of matrix metalloproteases

Ocimum genus (a.k.a holy basil or tulsi) is a dietary herb used for its multiple beneficial pharmacologic properties including anti-cancer activity. Here we show that crude extract of Ocimum gratissimum (OG) and its hydrophobic and hydrophilic fractions (HB and HL) differentially inhibit breast cancer cell chemotaxis and chemoinvasion in vitro and retard tumor growth and temporal progression of MCF10ADCIS.com xenografts, a model of human breast comedo-ductal carcinoma in situ (comedo-DCIS). OG-induced inhibition of tumor growth was associated with decreases in basement membrane disintegration, angiogenesis and MMP-2 and MMP-9 activities as confirmed by in situ gelatin zymography and cleavage of galectin-3. There was also decrease in MMP-2 and MMP-9 activities in the conditioned media of OG-treated MCF10AT1 and MCF10AT1-EIII8 premalignant human breast cancer cells as compared with control. The MMP-2 and MMP-9 inhibitory activities of OG were verified in vitro using gelatin, a synthetic fluorogenic peptide and recombinant galectin-3 as MMP substrates. Mice fed on OG-supplemented drinking water showed no adverse effects compared with control. These data suggest that OG is non-toxic and that the anti-cancer therapeutic activity of OG may in part be contributed by its MMP inhibitory activity.

The role of cullin3-mediated ubiquitination of the catalytic subunit of PP2A in TRAIL signaling

Protein phosphatase 2A (PP2A) is the major serine-threonine phosphatase that regulates a number of cell signaling pathways. PP2A activity is controlled partially through protein degradation; however, the underlying mechanism is not fully understood. Here we show that PP2A/C, a catalytic subunit of PP2A, is degraded by the Cullin3 (Cul3) ligase-mediated ubiquitin-proteasome pathway. In response to death receptor signaling by tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL), PP2A/C, caspase-8 and Cul3, a subunit of the cullin family of E3 ligases, are recruited into the death-inducing signaling complex (DISC) where the Cul3 ligase targets PP2A/C for ubiquitination and subsequent degradation. Functionally, knockdown of PP2A/C expression by siRNA or pharmacological inhibition of PP2A activity increases TRAIL-induced apoptosis. In cancer cells that have developed acquired TRAIL resistance, PP2A phosphatase activity is increased, and PP2A/C protein is resistant to TRAIL-induced degradation. Thus, this work identifies a new mechanism by which PP2A/C is regulated by Cul3 ligase-mediated degradation in response to death receptor signaling and suggests that inhibition of PP2A/C degradation may contribute to resistance of cancer cells to death receptor-induced apoptosis.