Tirza Raz

Cleavage of galectin-3 by matrix metalloproteases induces angiogenesis in breast cancer

Galectin‐3 cleavage is related to progression of human breast and prostate cancer and is partly responsible for tumor growth, angiogenesis and apoptosis resistance in mouse models. A functional polymorphism in galectin‐3 gene, determining its susceptibility to cleavage by matrix metalloproteinases (MMPs)‐2/‐9 is related to racial disparity in breast cancer incidence in Asian and Caucasian women. The purpose of our study is to evaluate (i) if cleavage of galectin‐3 could be related to angiogenesis during the progression of human breast cancer, (ii) the role of cleaved galectin‐3 in induction of angiogenesis and (iii) determination of the galectin‐3 domain responsible for induction of angiogenic response. Galectin‐3 null breast cancer cells BT‐459 were transfected with either cleavable full‐length galectin‐3 or its fragmented peptides. Chemotaxis, chemoinvasion, heterotypic aggregation, epithelial‐endothelial cell interactions and angiogenesis were compared to noncleavable galectin‐3. BT‐549‐H64 cells harboring cleavable galectin‐3 exhibited increased chemotaxis, invasion and interactions with endothelial cells resulting in angiogenesis and 3D morphogenesis compared to BT‐549‐P64 cells harboring noncleavable galectin‐3. BT‐549‐H64 cells induced increased migration and phosphorylation of focal adhesion kinase in migrating endothelial cells. Endothelial cells cocultured with BT‐549 cells transfected with galectin‐3 peptides indicate that amino acids 1–62 and 33–250 stimulate migration and morphogenesis of endothelial cells. Immunohistochemical analysis of blood vessel density and galectin‐3 cleavage in a breast cancer progression tissue array support the in vitro findings. We conclude that the cleavage of the N terminus of galectin‐3 followed by its release in the tumor microenvironment in part leads to breast cancer angiogenesis and progression.

Galectin-3 Cleavage: A Novel Surrogate Marker for Matrix Metalloproteinase Activity in Growing Breast Cancers

Failed therapies directed against matrix metalloproteinases (MMP) in cancer patients may be attributed, in part, to lack of diagnostic tools to differentiate between pro-MMPs and active MMPs, which indicate whether a treatment is efficacious or not. Because galectin-3 is cleavable in vitro by MMPs, we have developed differential antibodies recognizing its cleaved and noncleaved forms and tested their clinical utilization as a surrogate diagnostic marker for the presence of active MMPs in growing breast cancers. Wild-type and cleavage-resistant galectin-3 were constructed and expressed in galectin-3-null human breast carcinoma cells (BT-549). Tumorigenic and angiogenic potential of the clones was studied by injections into nude mice. MMP-2, MMP-9, full-length, and cleaved galectin-3 were localized in the xenografts by immunohistochemical analysis of paraffin-embedded sections using specific antibodies. Activities of MMP-2/9 were corroborated by in situ zymography on frozen tissue sections. Galectin-3 cleavage was shown in vivo by differential antibody staining and colocalized with predicted active MMPs both in mouse xenografts and human breast cancer specimens. In situ zymography validated these results. In addition, BT-549 cells harboring noncleavable galectin-3 showed reduced tumor growth and angiogenesis compared with the wild-type. We conclude that galectin-3 cleavage is an active process during tumor progression and could be used as a simple, rapid, and reliable surrogate marker for the activities of MMPs in growing breast cancers.

Racial Disparity in Breast Cancer and Functional Germ Line Mutation in Galectin-3 (rs4644): A Pilot Study

For reasons largely unknown, Caucasian women are at a significantly higher risk of developing breast cancer than Asian women. Over a decade ago, mutations in BRCA1/2 were identified as genetic risk factors; however, the discovery of additional breast cancer genes and genes contributing to racial disparities are lacking. We report a functional germline mutation (polymorphism) in the galectin-3 gene at position 191 (rs4644) substituting proline with histidine (P64H), which results in susceptibility to matrix metalloproteinase cleavage and acquisition of resistance to drug-induced apoptosis. This substitution correlates with incidence of breast cancer and racial disparity. Genotype analysis of 338 Caucasian (194 disease free and 144 breast cancer patients) and 140 Asian (79 disease free and 61 breast cancer patients) women showed that the allele homozygous for H64 exists in disease free Caucasian and Asian women at a frequency of 12% and 5%, respectively, versus 37% and 82% in breast cancer patients. The data indicate that H/H allele is associated with increased breast cancer risk in both races. The data implicate galectin-3 H(64) in breast cancer and explain, in part, the noted racial disparity, thus providing a novel target for diagnosis and treatment.

Differential regulation of phosphoglucose isomerase/autocrine motility factor activities by protein kinase CK2 phosphorylation

Phosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic housekeeping enzyme of the sugar metabolism pathways that plays a key role in both glycolysis and gluconeogenesis. PGI is a multifunctional dimeric protein that extracellularly acts as a cytokine with properties that include autocrine motility factor (AMF)-eliciting mitogenic, motogenic, and differentiation functions, and PGI has been implicated in tumor progression and metastasis. Little is known of the biochemical regulation of PGI/AMF activities, although it is known that human PGI/AMF is phosphorylated at Ser185 by protein kinase CK2 (CK2); however, the physiological significance of this phosphorylation is unknown. Thus, by site-directed mutagenesis, we substituted Ser185 with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation. A Ser-to-Ala mutant protein (S185A) was generated to abolish phosphorylation. Biochemical analyses revealed that the phosphorylation mutant proteins of PGI exhibited decreased enzymatic activity, whereas the S185A mutant PGI protein retained full enzymatic activity. PGI phosphorylation by CK2 also led to down-regulation of enzymatic activity. Furthermore, CK2 knockdown by RNA interference was associated with up-regulation of cellular PGI enzymatic activity. The three recombinant mutant proteins exhibited indistinguishable cytokine activity and receptor-binding affinities compared with the wild-type protein. In both in vitro and in vivo assays, the wild-type and S185A mutant proteins underwent active species dimerization, whereas both the S185D and S185E mutant proteins also formed tetramers. These results demonstrate that phosphorylation affects the allosteric kinetic properties of the enzyme, resulting in a less active form of PGI, whereas non-phosphorylated protein species retain cytokine activity. The process by which phosphorylation modulates the enzymatic activity of PGI thus has an important implication for the understanding of the biological regulation of this key glucose metabolism-regulating enzyme.

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.

Abstract 2017: Galectin-3 SNP rs10148371 in the anti-death motif NWGR is related to the colon cancer incidence in African Americans

Survival after cancer diagnosis is poorer for African Americans than for Caucasians. While some of this survival difference is driven by differences in the stage at diagnosis, even within the stage this trend is evident. For example, 5-year relative survival for colorectal cancer diagnosed as regional stage is 68.5% for Caucasians and only 60.6% for African Americans. Little is known about racial differences in response to chemotherapy and the subsequent impact on survival. A number of recent reports have focused on single nucleotide polymorphisms (SNPs) with the notion that such genetic event(s) can/may contribute to cancer development or progression. We have identified a SNP, rs10148371, which results in the substitution of R183K in the NWGR anti-death motif of the galectin-3 gene. Galectin-3, belonging to the family of carbohydrate-binding galectins is a multi-function tumor associated protein, which regulates cell growth, cell proliferation, cell adhesion, angiogenesis and apoptosis. It contains the NWGR motif characteristic of BH1 domain of Bcl-2 family responsible for protecting cells from apoptosis. To understand the significance of this SNP, we generated point mutation G548 to A in galectin-3 cDNA leading to R183K substitution. The resulting cDNA was stably transfected into low galectin-3 expressing colon carcinoma cells SW480 and RKO. Selected clones were analyzed for cell survival and apoptotic potential against cisplatin, 5 fluoro-uracil and oxaliplatin. The clones containing mutated galectin-3 showed a higher susceptibility to apoptosis compared to the wild type galectin-3. The WT clones showed an up-regulation of β-catenin and its downstream target proteins, which was not seen in the mutant clones. Ability/inability of mutated galectin-3 in the nuclear translocation of β-catenin is being studied. The racial distribution of the SNP rs10148371 in the normal population of the African Americans is 22%, while in Caucasians and Asians disease free population it can be found in less than 1%. To analyze the significance of this SNP in relation to breast and colorectal cancer, we performed genotype analysis with breast cancer (N=124) or colorectal cancer (N=129) patients. Stratified analyses revealed that the Lys variant was marginally significantly inversely associated with advanced tumor stage (p=0.08) and younger ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2017. doi:1538-7445.AM2012-2017