Galectin-3 and cancer stemness

Galectin-3 and cancer stemness Abstract Over the last few decades galectin-3, a carbohydrate binding protein, with affinity for N-acetyllactosamine residues, has been unique due to the regulatory roles it performs in processes associated with tumor progression and metastasis such as cell proliferation, homotypic/heterotypic aggregation, dynamic cellular transformation, migration and invasion, survival and apoptosis. Structure–function association of galectin-3 reveals that it consists of a short amino terminal motif, which regulates its nuclear-cytoplasmic shuttling; a collagen α-like domain, susceptible to cleavage by matrix metalloproteases and prostate specific antigen; accountable for its oligomerization and lattice formation, and a carbohydrate-recognition/binding domain containing the anti-death motif of the Bcl2 protein family. This structural complexity permits galectin-3 to associate with numerous molecules utilizing protein–protein and/or protein–carbohydrate interactions in the extra-cellular as well as intracellular milieu and regulate diverse signaling pathways, a number of which appear directed towards epithelial–mesenchymal transition and cancer stemness. Self-renewal, differentiation, long-term culturing and drug-resistance potential characterize cancer stem cells (CSCs), a small cell subpopulation within the tumor that is thought to be accountable for heterogeneity, recurrence and metastasis of tumors. Despite the fact that association of galectin-3 to the tumor stemness phenomenon is still in its infancy, there is sufficient direct evidence of its regulatory roles in CSC-associated phenotypes and signaling pathways. In this review, we have highlighted the available data on galectin-3 regulated functions pertinent to cancer stemness and explored the opportunities of its exploitation as a CSC marker and a therapeutic target. cancer stemness, EMT, galectin-3, invasion, migration Introduction Galectin-3 belongs to a specialized family of mammalian sugar-binding proteins (galectins) harboring a highly conserved sequence recognizing and binding cellular carbohydrate moieties of glyco-conjugates as well as those that are part of the extra-cellular matrix. To date, the 15 reported members of this family are classified into three groups (i) prototype, containing a single carbohydrate-recognition domain (CRD); (ii) tandem repeats that harbor two CRDs and (iii) chimera. Galectin-3 is the single chimeric protein, comprised of three structurally distinct domains: (i) a short amino terminal consisting of 12 amino acids that contains a serine phosphorylation site responsible for its translocation (Gong et al. 1999); (ii) a ~110 amino acid long collagen-alpha like stretch rich in proline, alanine, glycine; and (iii) the ~140 amino acid C-terminal domain encompassing the CRD. Of note, the collagen-like domain of galectin 3 can be cleaved by matrix metalloproteases (Ochieng et al. 1994; Nangia-Makker et al. 2010) and prostate specific antigen (PSA) (Balan et al. 2012), while the CRD contains the characteristic NWGR anti-death motif of the bcl-2 family (Akahani et al. 1997). Galectin-3 is located within the cytoplasm and the nucleus but can be transported onto the cell surface, extra-cellular space, and the circulation via nonclassical secretory pathways as it is devoid of the secretory signal sequence (Menon and Hughes 1999). Galectin-3 may be secreted via either vesicular release, the exosome-mediated pathway or it may traverse the lipid bilayer (Hughes 1999; Baptiste et al. 2007; Jones et al. 2010; Nakajima, Kho, Yanagawa, Harazono, et al. 2016). Galectin-3 is recycled by the cells and plays a crucial role in apical protein trafficking. After being secreted, it re-enters the cells by nonclathrin mediated endocytosis and acts as a raft for cargo proteins (Honig et al. 2015). In each cellular compartment, galectin-3 interacts with specific binding-partners and is involved with several cellular activities, which include apoptosis, cell migration, proliferation, angiogenesis, etc. These functions of galectin-3 have been the subject of reviews by several authors (Dumic et al. 2006; Nangia-Makker et al. 2008; Haudek et al. 2010; Funasaka et al. 2014a, 2014b; Nakajima, Kho, Yanagawa, Zimel et al. 2016). Here, we have focused on the functional properties of galectin-3 relating to epithelial–mesenchymal transition (EMT) with probable reference to cancer stemness. Cancer stem cells Tumor heterogeneity is defined by three models. According to the stochastic model, the heterogeneity in an initially homogeneous tumor arises due to random genetic events, resulting in a genetic drift and acquisition of dominant genetic changes in various permutations. Keeping with this model, cancer progression is nonlinear and a heterogeneous tumor is established from the cell clones branching out to produce diverse subpopulations within the tumor (Nowell 1976; Wolman and Heppner 1992; Marusyk and Polyak 2010; Marusyk et al. 2012). The cancer stem cell (CSC) model proposes that stem cell-like precursors with self-renewal and pluripotency capabilities, which reside within tissues, acquire mutations and develop into unique populations of cells that differentiate and acquire diverse sets of biological and phenotypical characteristics (Reya et al. 2001; Bapat 2007; Marusyk et al. 2012; Kreso and Dick 2014). The third model suggests inclusion of both these models and posits that a small subpopulation of CSC resides within the primary tumor. These CSC subgroups undergo diverse mutations resulting in intratumoral heterogeneity (Marusyk et al. 2012; Elshamy and Duhe 2013; Marjanovic et al. 2013). Over the last decade, the acceptance of this model has been increasing as the general theory of cancer progression, recurrence and most probably therapeutic resistance. Characteristics of CSCs CSCs are thought to originate from normal stem, progenitor or differentiated cells by acquiring mutations. Like normal stem cells, they acquire essential properties that include (i) self-renewing ability, (ii) pluripotency, (iii) high tumor initiating ability from a few cells and (iv) chemo-resistance. Due to these characteristics, CSCs play a significant role in tumor initiation, progression, invasion, metastasis, drug-resistance and recurrence. Cancer progression culminating in metastasis is an intricate multistep process including migration and invasion of tumor cells through surrounding stroma, intravasation and survival in the blood circulation under anchorage independent settings, extravasation, adherence and proliferation at the secondary site. Only a few cells that have transitioned through the EMT phase would complete these steps successfully (Thiery 2002; Yang and Weinberg 2008). It should be noted, that increasing records support the contention that only cells that have undergone EMT may display CSC-like properties (Mani et al. 2008; Jordan et al. 2011; Krantz et al. 2012; Wu 2011), and EMT can provoke the reversion of tumor cells to CSC-like phenotype (Mani et al. 2008; Polyak and Weinberg 2009). For example, pathways including Wnt, Notch and Hedgehog, which are controlled during normal development, are deregulated in both EMT and CSCs (Huber et al. 2005; Malanchi et al. 2008; Peacock and Watkins 2008; Singh and Settleman 2010). A dynamic plasticity interplay between epithelial and mesenchymal phenotype has also been suggested in the process of metastasis (Liao and Yang 2017). Table I summarizes the signaling pathways that have been associated with embryonic development, and EMT/CSCs. Table I. Signaling pathways in cancer stem cells and role of galectin-3 Pathway  Main players  Role of galectin-3  Wnt  Wnt Signaling pathway involving GSK-3β, β-catenin and TCF/LEF is dysregulated in various cancers. The main player in this pathway is β-catenin, which activates the transcription of certain genes upon translocation to nucleus.  Activates GSK-3β, binds to β-catenin and modulates its nuclear export and transcriptional activity (Shi et al. 2007; Shimura et al. 2004, 2005; Song et al. 2009)  Notch  The Notch pathway involving Snail, Slug and E-cadherin has been implicated in cancer initiation and development, as well as early stages of cancer progression by regulating conserved cellular programs such as EMT. A role for Notch signaling is also reported in osteolytic bone metastasis.  Activates/cleaves Notch transcription of its target proteins (Fermino et al. 2016; Kang et al. 2016; Nakajima et al. 2014) and regulates E-cadherin (Cao et al. 2017)  Hedgehog  Hedgehog (Hh) signaling involving FAK/Akt and targeting MMPs is an evolutionarily conserved pathway essential for self-renewal and cell fate determination. Aberrant Hh signaling is associated with the development and progression of various cancers and is implicated in multiple aspects of tumorigenesis, including the maintenance of CSCs.  Increases Akt activity (Gao et al. 2016; Kobayashi et al. 2011; Nangia-Makker et al. 2010; Zhang et al. 2013)  Transforming growth factor-β  TGF-β signaling pathway is involved in growth, differentiation, apoptosis, cellular homeostasis and other cellular functions in both the adult organism and the developing embryo including EMT. The key players SMADs act as transcription factors and regulate target gene expression. The non-Smad pathways include various branches of MAP kinase pathways, Rho-like GTPase signaling pathways and phosphatidylinositol-3-kinase/Akt pathways.  Stabilizes TGF-βR and plays an important role in FAK, PI3K/Akt pathways (Park et al. 2015)  EGFR/FGFR  Receptor tyrosine kinase binding to its ligand activates PI3K/Akt and MAPK pathways, which play an important role in the regulation of cell growth, proliferation and differentiation. It also induces transcription of EMT markers ZEB2, Snail, FOXC2, etc.  Stabilizes EGFR, and activates EGFR induced activation of K-Ras, PI3K and Akt, which in turn regulate β-catenin transcriptional activity (Elad-Sfadia et al. 2004; Shalom-Feuerstein et al. 2005)  Pathway  Main players  Role of galectin-3  Wnt  Wnt Signaling pathway involving GSK-3β, β-catenin and TCF/LEF is dysregulated in various cancers. The main player in this pathway is β-catenin, which activates the transcription of certain genes upon translocation to nucleus.  Activates GSK-3β, binds to β-catenin and modulates its nuclear export and transcriptional activity (Shi et al. 2007; Shimura et al. 2004, 2005; Song et al. 2009)  Notch  The Notch pathway involving Snail, Slug and E-cadherin has been implicated in cancer initiation and development, as well as early stages of cancer progression by regulating conserved cellular programs such as EMT. A role for Notch signaling is also reported in osteolytic bone metastasis.  Activates/cleaves Notch transcription of its target proteins (Fermino et al. 2016; Kang et al. 2016; Nakajima et al. 2014) and regulates E-cadherin (Cao et al. 2017)  Hedgehog  Hedgehog (Hh) signaling involving FAK/Akt and targeting MMPs is an evolutionarily conserved pathway essential for self-renewal and cell fate determination. Aberrant Hh signaling is associated with the development and progression of various cancers and is implicated in multiple aspects of tumorigenesis, including the maintenance of CSCs.  Increases Akt activity (Gao et al. 2016; Kobayashi et al. 2011; Nangia-Makker et al. 2010; Zhang et al. 2013)  Transforming growth factor-β  TGF-β signaling pathway is involved in growth, differentiation, apoptosis, cellular homeostasis and other cellular functions in both the adult organism and the developing embryo including EMT. The key players SMADs act as transcription factors and regulate target gene expression. The non-Smad pathways include various branches of MAP kinase pathways, Rho-like GTPase signaling pathways and phosphatidylinositol-3-kinase/Akt pathways.  Stabilizes TGF-βR and plays an important role in FAK, PI3K/Akt pathways (Park et al. 2015)  EGFR/FGFR  Receptor tyrosine kinase binding to its ligand activates PI3K/Akt and MAPK pathways, which play an important role in the regulation of cell growth, proliferation and differentiation. It also induces transcription of EMT markers ZEB2, Snail, FOXC2, etc.  Stabilizes EGFR, and activates EGFR induced activation of K-Ras, PI3K and Akt, which in turn regulate β-catenin transcriptional activity (Elad-Sfadia et al. 2004; Shalom-Feuerstein et al. 2005)  Identification and isolation of CSCs accountable for tumor initiation and propagation is a large undertaking due to the complexity in their biology, their heterogeneity that originates from genotypic and phenotypic plasticity, their scarcity in cancers, and diversity of cell surface markers, which differ between tissue types. Commonly identified cell surface markers are: CD44+CD24−/lowALDH1 activity in breast cancer, ALDH+CD44+CD166+EpCAM+ in colorectal cancer, ABCB5+ CD271 in melanoma, CD133+ in brain cancer, CD34+CD38− in acute myeloid lymphoma, CD44+ CD117+ in ovarian cancer, CD44+CD24+ESA+CD133+CD47 in pancreatic cancer, CD44+CD24+α2β1 high in prostate cancer and ABCG2 in retinoblastoma (for further information, see Pakravan et al. 2015). Galectin-3 in EMT, migration and invasion EMT is a biological manifestation whereby epithelial cells transition into mesenchymal cells both during normal development and tumor progression (Thiery 2003). The changes in cellular traits during EMT are decreased expression of epithelial markers such as E-cadherin and upregulation of mesenchymal markers α-smooth muscle actin and vimentin, loss of cell–cell junctions and apicobasolateral polarity resulting in a spindle shaped more migratory and invasive phenotype. Galectin-3 interacts with and mediates the expression of many proteins, which contribute to EMT during tumor cell migration and invasion. Galectin-3 over-expressing cells show an increased migration towards Matrigel (Nangia-Makker et al. 1995, 2000), whereas down-regulation of galectin-3 expression in MDA-MB-435 human breast carcinoma cells results in decreased migratory and invasive characteristics in vitro associated with reduced tumor growth in vivo (Honjo et al. 2001). Extra-cellular galectin-3, secreted by breast cancer cells, was involved with migration of endothelial cells in a dose-dependent fashion (Nangia-Makker et al. 2000) and phosphorylated focal adhesion kinase (pFAK) upregulated at the migratory edge (Nangia-Makker et al. 2010). Galectin-3 promotes focal adhesions turnover by stabilizing FAK because of transmembrane crosstalk between the galectin lattice and tyrosine phosphorylated caveolin (Goetz et al. 2008). Immuno-histochemical analysis of patient breast tumor specimens and xenografts revealed that galectin-3 expression shifted from luminal to peripheral cells in tandem with the progression to comedo-DCIS or invasive carcinomas. It was suggested that this transitional shift in the expression of galectin-3 increases the invasive potential of tumor cells through altered interactions with the stromal counterparts (Shekhar et al. 2004). An increase in E-cadherin expression accompanied with a reduced vascular mimicry in HepG2-Runx2 hepatocellular carcinoma cells was associated with galectin-3 down-regulation (Cao et al. 2017). Galectin-3 induced Hela cell migration via MAPK/ERK-1/2 pathway was sensitive to calcium (Gao et al. 2014), while increased cell migration by galectin-3 in mesenchymal stem cells was due to inhibition of RhoA GTPase activity and increased pERK1/2 and pAkt (Gao et al. 2016). Similarly, galectin-3 mediated tumor cell invasion in pancreatic and tongue cancer cells (Zhang et al. 2013) through the initiation of Akt phosphorylation and increased β-catenin transcriptional activity resulting in increased MMP-2 and MMP-9 activities. Galectin-3 increased the in vitro migration of BV2 microglia associated with integrin-linked-kinase signaling (Wesley et al. 2013) and induced EMT like morphology in a murine epithelial cell line GE11 via β-1 integrin that triggers galectin-3 expression through demethylation of its promoter. Galectin-3, in turn stimulated cell migration and adhesion mediated by β-1 integrin, thus forming a functional feed-back-loop (Margadant et al. 2012). Galectin-3 inhibition induced activation of β-catenin and attenuated lung, hepatic and renal fibrosis, making it a proposed marker for active fibrosis (Mackinnon et al. 2012). Galectin-3 is instrumental in cyclin D1 and c-myc over-expression affected by β-catenin since the NH2 terminus of β-catenin binds to the COOH terminus of galectin-3 forming a nuclear complex prompting the transcriptional activity of Tcf-4, independent of mutations in either APC or β-catenin (Shimura et al. 2004, 2005; Henderson et al. 2008). Moreover, Wnt-2- and galectin-3 synergistically induced apoptosis in colorectal cancer cells (Shi et al. 2007). Reproduction of galectin-3 modulation of β-catenin expression and Tcf-4 activity was also reported in colon cancer cells. In the absence of galectin-3, Akt and GSK-3β were dephosphorylated and increased GSK-3β activity lead to increased phosphorylation of β-catenin and its degradation (Song et al. 2009). Wang et al. (2013) reported an acceleratory effect of galectin-3 on proliferation, migration and invasion of oral tongue squamous cell carcinoma via Wnt/β-catenin dependent pathway and EMT. Galectin-3 correspondingly contributes to the tumor cell’s ability to invade and degrade basement membrane by increasing the activity of MMPs. Silencing galectin-3 reduced secretion of MMP-2 and invasive ability of C8161-c9 melanoma cells (Mourad-Zeidan et al. 2008). Galectin-3 was also reported to bind directly to transcription factor activated AP-1 for MMP-1 (Wang et al. 2012). Galectin-3 oligomerization increased the affinity of elastin to αvβ3 integrin, which in turn regulates melanoma cell invasion (Pocza et al. 2008). Further, binding between integrin and galectin-3 enhanced cell migration (Saravanan et al. 2009) by reducing α2β1 integrin–collagen binding in the extra-cellular matrix (Friedrichs et al. 2008). Due to the galectin-3N-terminal oligomerization property, galectin-3 can cooperate with cell surface glyco-conjugates (glycoproteins and glycolipids). Its binding affinity depends on the number of glycosylation sites and glycosyl transferase activity (Demetriou et al. 2001). The galectin oligomers form bridges between glycoproteins creating a lattice, which increases the interactions of multiple proteins as well as stabilizing them by preventing their endocytosis (for review, see, Nabi et al. 2015). For example, the interactions between galectin-3 and N-glycans on the extra-cellular domains of αvβ3 integrin resulting in its cluster formation modulate KRAS clustering and resistance to EGFR inhibitors (Seguin et al. 2014). Thus, the conclusion was that galectin-3 might regulate receptor tyrosine kinase signaling, cell-motility and formation of fibronectin fibrils under pathological conditions (Akahani et al. 1997; Partridge et al. 2004; Dennis et al. 2009). Galectin-3 and chemo-resistance CSCs employ multiple mechanisms for chemo-resistance such as enhanced drug efflux, aldehyde mediated detoxification, enhanced DNA repair mechanism, quiescence, apoptosis inactivation and activated signaling pathways (Wnt, Hedgehog and Notch) leading to multidrug resistance (MDR) (Thomas et al. 2015). Endogenous galectin-3 inhibits apoptosis in epithelial cell induced by staurosporine and chemotherapeutic agents such as cisplatin, genistein, tumor necrosis factor and nitric oxide (Akahani et al. 1997; Kim et al. 1999; Lin et al. 2000; Yoshii et al. 2002). Anti-apoptotic activity of galectin-3 is dependent on its phosphorylation and nuclear export (Takenaka et al. 2004) and Ser6 is essential for this phosphorylation. Using Ser6Ala or Ser6Glu mutation, Takenaka et al. (2004) demonstrated that mutant galectin-3 was neither phosphorylated nor exported from the nucleus, while the wild-type galectin-3 was phosphorylated and exported from nucleus to the cytoplasm, imparting drug resistance to cancer cells. Additionally, peritoneal macrophages isolated from galectin-3 deficient mice were more sensitive to apoptosis compared with from normal control mice (Sano et al. 2003) and galectin-3 directly inhibited anti-Fas antibody induced T-cell apoptosis (Yang et al. 1996). In metastatic colon cancer cells, cell surface galectin-3 was responsible for resistance to Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL) by blocking the trafficking of death receptors prior to apoptosis (Mazurek et al. 2012). Galectin-3 knockdown stimulated TRAIL-induced apoptosis in human bladder carcinoma cancer cells by down-regulating Akt/PI3K pathway and gemcitabine induced apoptosis in human pancreatic carcinoma cells (Kobayashi et al. 2011). In a recent study, the biliary epithelial cells (BECs) of galectin-3 knockout mice showed increased sensitivity to apoptotic insults and the presence of proinflammatory lymphocytes and dendritic cells (DCs) was more pronounced in the livers of galectin-3 knockout mice compared to the normal mice. This suggests a novel function for galectin-3 as a safeguard of BECs from pathways leading to inflammation (Arsenijevic et al. 2016). Similarly, galectin-3 induces chemo-resistance by modulating signaling pathways activated in CSCs. In Wnt signaling pathway, it regulated GSK-3β activity and the accumulation of β-catenin in the nucleus (Shimura et al. 2004, 2005). Human bone marrow-derived mesenchymal stromal cells hBM-MSCs exhibited cytotoxic drug resistance in acute leukemia cells (ALCs) by galectin-3, which in turn stimulated β-catenin transcriptional activity. Down-regulation of galectin-3 by short hairpin RNA (shRNA) reversed this effect (Hu et al. 2015). Galectin-3 silencing in cultured human osteosarcoma cells reduced expression and activation of invasion mediating proteins FAK, Src, Lyn, PI3K/Akt, ERK1/2 and β-catenin, and potentiated sensitivity to cisplatin (Park et al. 2015). Galectin-3 is also an essential component of the cluster formed between αvβ3 integrin/KRAS at the plasma membrane, facilitating recruitment and hyper-activation of RalB, which in turn drives NFkB via TBK1. This clustering is responsible for lung and pancreatic tumor stemness and resistance to tyrosine kinase 1 inhibitors (Seguin et al. 2014). Galectin-3 also regulates Notch signaling. It binds to Notch1, accelerates its cleavage and regulates bone-remodeling (Nakajima et al. 2014). A crosstalk between galectin-3 and the Jagged/Notch affects the T helper polarization programs (Fermino et al. 2016). In a different tumor system, i.e. ovarian cancer, upregulation of galectin-3 expression was associated with the nuclear translocation and increased NICD1 (Notch 1 intracellular domain) cleavage concomitant with the upregulation of Notch target genes Hes1 and Hey1 (Kang et al. 2016). Further, interaction between galectin-3, Hedgehog and histone deacetylase protein was implicated in liver fibrosis (de Oliveira et al. 2017) and inhibition of TGFβ signaling in TβRII deficient mice, resulted in a reduced galectin-3 expression and associated Akt phosphorylation (Gong et al. 2012). Galectin-3 in differentiation and regeneration Cellular and circulating galectin-3 were found to be significant markers for multipotent progenitor cells of the bone marrow, mesenchymal stem/stromal cells (MSCs), which can differentiate into osteoblasts, chondrocytes, myocytes and adipocytes, i.e. the cells of mesenchymal origin. MSCs play an essential role in T-cell mediated immunological response as well as in wound healing, regeneration in response to ischemia and/or injury (reviewed in Rahimzadeh et al. 2016). Galectin-3 is responsible for the ability of MSCs to suppress cardiac inflammation and fibrosis in hearts with chronic Chagas cardiomyopathy symptoms (Souza et al. 2017). Galectin-3 induction in ALCs by bone marrow-derived MSCs is a prerequisite for drug resistance (Hu et al. 2015). Galectin-3 upregulation and secretion by senescent adipose derived MSCs is critical for their growth stimulatory effect on colorectal cancer LoVo cells by activating the MAPK ERK1/2 pathway (Li et al. 2015). Similarly, galectin-3 is essential for the proliferation of hepatic progenitor cells in response to liver injury (Hsieh et al. 2015), inflammation promotion in hepatitis (Volarevic et al. 2015) and multiple sclerosis (Chen et al. 2014). It should be noted that in galectin-3 knockout mice, the ability of myeloid progenitor cells to differentiate into mature myeloid cells is abrogated, consequently, modifying the bone marrow compartment (Brand et al. 2011). Galectin-3 as a cancer biomarker and therapeutic target There are numerous publications on the altered cellular/extra-cellular distribution and expression of galectin-3 in various cancers using human and xenograft tumor tissues (reviewed by Balan et al. 2010; Dong et al. 2017; Thijssen et al. 2015). In breast cancer, galectin-3 expression was redistributed from luminal to peripheral epithelial cells in the more invasive ducts (Shekhar et al. 2004). An increase in MMP-2 cleaved galectin-3 protein correlated with angiogenesis in progressive stages of breast and prostate cancer (Nangia-Makker et al. 2007, 2010; Wang et al. 2009). While galectin-3 is a diagnostic and/or prognostic marker for thyroid (Liu et al. 2008; Rydlova et al. 2008; Than et al. 2008), gastric (Baldus et al. 2000; Miyazaki et al. 2002), colorectal cancer (Lu et al. 2017) and to some extent pancreatic cancer (Shimamura et al. 2002), melanocytes accumulate galectin-3 in the nucleus as the melanoma progresses (Prieto et al. 2006). Nuclear galectin-3 expression in association with thyroid transcription factor 1 is an independent marker of worst prognosis for lung cancer patients (Puglisi et al. 2004), whereas in colon cancer enhanced cytoplasmic levels of galectin-3 were reported (Lotz et al. 1993; Sanjuan et al. 1997). In brain tumors, galectin-3 is differentially expressed in various tumors (Park et al. 2008) and can be used as a tool to track their origin (Neder et al. 2004; Park et al. 2008). Due to its involvement in diverse biological functions, many attempts have been made to use galectin-3 as a therapeutic target. Modified citrus pectin (MCP) was the first reported natural inhibitor of galectin-3 functions in mouse models of prostate, colon and breast cancer (Platt and Raz 1992; Pienta et al. 1995; Nangia-Makker et al. 2002). Galectin-3 suppresses apoptosis and increases resistance of tumor cells to chemotherapeutic drugs, it has been used as a target to resensitize cancer cells to chemotherapeutic drugs (Glinsky and Raz 2009). Targeting galectin-3 via MCP or lactosyl-l-leucine (LL) sensitized malignant cells to doxorubicin (Johnson et al. 2007), taxol (Glinsky and Raz 2009), cisplatin (Wang et al. 2010), bortezomide and dexamethasone (Chauhan et al. 2005). In a recent study, another polysaccharide inhibitor of galectin-3 RN1 is reported to suppress growth of pancreatic ductal carcinoma in mouse xenografts and patient derived xenografts (Zhang et al. 2017). A truncated, dominant negative form of galectin-3, galectin-3C, is another promising new compound for effective adjuvant therapies in advanced, refractory multiple myeloma and ovarian cancer (John et al. 2003) (Mirandola, Nguyen, et al. 2014; Mirandola, Yu et al. 2014). In addition, galectin-3 inhibitors including peptide antagonists, lactulose amines (Glinskii et al. 2012) and synthetic glycol-conjugates (reviewed in Campo et al. 2016) have also been the focus of several studies. Galectin-3 and cancer stemness Based on the above it is tempting to draw a link between cancer stemness and galectin-3. A proteomic comparative analysis between the increasing culture passages of tumor spheres and monolayer cultures of MCF-7 breast cancer cells revealed an amplification of stem like cell markers associated with functional identifiers such as self-renewal and symmetric cell division in the tumor spheres (Deleyrolle et al. 2011). The spheres as well as subpopulations within the spheres were more tumorigenic than the parental cells implying the emergence and enrichment of CSCs. There was also an increased expression of galectin-3 together with carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-5 and -6, MUC-1, -5 and -6. When the spheres were reseeded in the presence of galectin-3 inhibitor N-acetyllactosamine, a significant reduction in sphere formation was noted (Morrison et al. 2012). In MCF-7 cells, an increased apoptosis-resistance and side-population with CSC properties were attenuated by down-regulating galectin-3 (Guha et al. 2014). Similarly, there was increased expression of galectin-3, -4, -7 and -9 in spheres formed by the human nonsmall cell lung cancer cell line H1299. However, galectin-3 expression was significantly higher than other galectins in the tumor spheres during each successive passage (92). Those spheres also showed increased levels of stem cell markers Oct4, Sox2, Nanog, CXCR4, β-catenin and CD133. A different experimental approach of down-regulating galectin 3 expression by lentivirus-mediated shGal-3 inhibited self-renewal, chemo-resistance and malignancy of the spheres. In sharp contrast, the addition of recombinant-galectin-3 to the culture medium stimulated sphere formation. The authors hypothesized that the extra-cellular galectin-3 may interact with EGF and bFGF present in the stem cell culture medium thus contributing to the generation of spheres, whereby intracellular galectin-3 acts as a cofactor. It may also interact with β-catenin and augment its transcriptional activities resulting in the expression of stemness-related genes and upregulation of ABC transporter genes (Chung et al. 2015). Another experimental approach examining the regulation of lung CSCs by galectin 3 has demonstrated that galectin-3 enhanced lung cancer stemness through the EGFR/c-myc/Sox2 axis via carbohydrate-recognition activities and that Oct 4 promoted galectin-3 expression forming a positive regulatory loop (Kuo et al. 2016). Moreover, amplified expression of galectin-3 protein was reported in glioblastoma stem cells (Ma et al. 2014), whereby the tumor suppressor miR-152 was found to regulate galectin-3 expression in CSC. Over-expression of miRNA-152 reduced the level of galectin-3 by down-regulating KLF4, which binds and activates galectin-3 promoter while inhibiting MEK1/2 and PI3K signaling (Ma et al. 2014). Recently, the ability of galectin-3 to characterize CSC phenotype of human colorectal and pancreatic cancer cell lines was explored. CD44+, CD24+, EpCAM+ and CD166+ cells from colorectal and pancreatic cells were sorted for galectin-3 positive or negative cells and analyzed for their stemness properties. In this study, the level of cell surface expression of galectin-3 correlated with enhanced CSC phenotypes, like sphere forming, tumorigenicity and drug resistance. Down-regulation of galectin-3 expression by shRNA abrogated the levels of stem cell markers and sphere forming ability of CSCs (Ilmer et al. 2016). In lung and pancreatic tumors, presence of galectin-3 was essential for cluster formation with αvβ3 integrin and KRAS, which activated the NFkB pathway and stemness (Seguin et al. 2014). Increased levels of galectin-3, together with MMP-2, alpha actinin-4 and MARCKS in the secretome of pancreatic CSCs compared to parental cell line Panc1 was also noted (Brandi et al. 2016). Similarly, in ovarian CSCs, galetin-3 supports stemness by activation of Notch1 intracellular domain (Kang et al. 2016). Conclusions and future directions Emergence of drug resistant cells is the leading cause of tumor recurrence and metastasis. The cells capable of successfully metastasizing to distant sites are endowed with invasive and migratory properties, which are the essential traits of both EMT and CSC. Galectin-3 expression is necessary for the preservation of the transformed and drug-resistant phenotypes of cancers. Interactions of galectin-3 with diverse signaling pathways associated with EMT and CSC are depicted in Fig. 1. In the tumor microenvironment the galectin-3 molecules that are secreted by macrophages and tumor cells, interact with extra-cellular matrix components and may be cleaved by MMPs affecting homotypic and heterotypic cell recognition and adhesion. The lattice formation by the extra-cellular galectin-3 mediated oligomerization by binding to N-acetyllactosamine residues of cell surface glyco-ligands (EGFR, TGF-βR, integrins, etc.) stabilizes them and prevents their endocytosis and degradation, thus facilitating their downstream events[for review see (Nabi et al. 2015)]. In addition, it activates Notch 1 and enables its cleavage (Kang et al. 2016). Therefore, one must conclude that galectin-3 plays an important role in activating signaling pathways that are crucial for EMT and continuation of stemness. Stabilization of β-catenin by galectin-3 regulates transcription of its target genes which include stem cell markers like c-myc, and CD44 among others (Funasaka et al. 2014b). While galectin-3 is instrumental in activation of MDR gene responsible for drug efflux and resistance to apoptosis (Harazono et al. 2015), it also establishes a positive regulatory loop with the pluripotency genes Klf4 and Oct4 (Ma et al. 2014; Kuo et al. 2016). Based on the above, a few recent studies have tested the presence/over-expression of galectin 3 in CSCs and reported its upregulation in CSCs while its inhibition reduced the stem cell characteristics (Morrison et al. 2012; Chung et al. 2015; Ilmer et al. 2016; Kang et al. 2016). In gastrointestinal cancer, cell surface galectin-3 was identified in a subset of CSCs with heightened stem cell characteristics (Ilmer et al. 2016). Figure 1. View largeDownload slide A schematic representation of the interactions and regulatory role of galectin-3 in stemness-related pathways. Figure 1. View largeDownload slide A schematic representation of the interactions and regulatory role of galectin-3 in stemness-related pathways. Although an elementary understanding of the direct relationship of galectin-3 to cancer stemness is beginning to emerge a more detailed and comprehensive insight will be required to understand its role and possible use as a target to inhibit CSC function. Further, the relationship among members of the galectin family to each other and their alternate compensatory mechanisms should be taken into account. The presence of a SNP (rs4644) in the galectin-3 gene, a substitution of Proline64 with Histidine, results in cells with increased chemotaxis, chemo-invasion, angiogenesis, apoptosis resistance in vitro, tumor formation in vivo and an increased susceptibility to breast cancer (Nangia-Makker et al. 2007; Balan et al. 2008). Subsequent studies reported an association of this SNP to prostate cancer and cervical cancer susceptibility (Meyer, 2013, Fang, 2017), rheumatoid arthritis (Hu, 2011) and drug resistance (Mazurek, 2011) via β-catenin pathway. It would be interesting to establish whether galectin-3 germline mutation is associated with cancer stemness. Funding This work was supported by the Paul Zuckerman Endowment (to A.R.) and by the NCI/NIH, Cancer Center Support Grant (CA-22453). Conflict of interest statement None declared. Abbreviations ALCs, acute leukemia cells; BECs, biliary epithelial cells; CEACAM, carcinoembryonic antigen-related cell adhesion molecule; CRD, carbohydrate-recognition domain; CSCs, cancer stem cells; EMT, epithelial–mesenchymal transition; LL, lactosyl-l-leucine; MCP, modified citrus pectin; MSCs, mesenchymal stem/stromal cells; pFAK, phosphorylated focal adhesion kinase; PSA, prostate specific antigen; TRAIL, tumor necrosis factor Related Apoptosis Inducing Ligand. References Akahani S, Nangia-Makker P, Inohara H, Kim HR, Raz A. 1997. Galectin-3: A novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family. Cancer Res . 57: 5272– 5276. 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Galectin-3 and cancer stemness

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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0959-6658
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10.1093/glycob/cwy001
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Abstract

Abstract Over the last few decades galectin-3, a carbohydrate binding protein, with affinity for N-acetyllactosamine residues, has been unique due to the regulatory roles it performs in processes associated with tumor progression and metastasis such as cell proliferation, homotypic/heterotypic aggregation, dynamic cellular transformation, migration and invasion, survival and apoptosis. Structure–function association of galectin-3 reveals that it consists of a short amino terminal motif, which regulates its nuclear-cytoplasmic shuttling; a collagen α-like domain, susceptible to cleavage by matrix metalloproteases and prostate specific antigen; accountable for its oligomerization and lattice formation, and a carbohydrate-recognition/binding domain containing the anti-death motif of the Bcl2 protein family. This structural complexity permits galectin-3 to associate with numerous molecules utilizing protein–protein and/or protein–carbohydrate interactions in the extra-cellular as well as intracellular milieu and regulate diverse signaling pathways, a number of which appear directed towards epithelial–mesenchymal transition and cancer stemness. Self-renewal, differentiation, long-term culturing and drug-resistance potential characterize cancer stem cells (CSCs), a small cell subpopulation within the tumor that is thought to be accountable for heterogeneity, recurrence and metastasis of tumors. Despite the fact that association of galectin-3 to the tumor stemness phenomenon is still in its infancy, there is sufficient direct evidence of its regulatory roles in CSC-associated phenotypes and signaling pathways. In this review, we have highlighted the available data on galectin-3 regulated functions pertinent to cancer stemness and explored the opportunities of its exploitation as a CSC marker and a therapeutic target. cancer stemness, EMT, galectin-3, invasion, migration Introduction Galectin-3 belongs to a specialized family of mammalian sugar-binding proteins (galectins) harboring a highly conserved sequence recognizing and binding cellular carbohydrate moieties of glyco-conjugates as well as those that are part of the extra-cellular matrix. To date, the 15 reported members of this family are classified into three groups (i) prototype, containing a single carbohydrate-recognition domain (CRD); (ii) tandem repeats that harbor two CRDs and (iii) chimera. Galectin-3 is the single chimeric protein, comprised of three structurally distinct domains: (i) a short amino terminal consisting of 12 amino acids that contains a serine phosphorylation site responsible for its translocation (Gong et al. 1999); (ii) a ~110 amino acid long collagen-alpha like stretch rich in proline, alanine, glycine; and (iii) the ~140 amino acid C-terminal domain encompassing the CRD. Of note, the collagen-like domain of galectin 3 can be cleaved by matrix metalloproteases (Ochieng et al. 1994; Nangia-Makker et al. 2010) and prostate specific antigen (PSA) (Balan et al. 2012), while the CRD contains the characteristic NWGR anti-death motif of the bcl-2 family (Akahani et al. 1997). Galectin-3 is located within the cytoplasm and the nucleus but can be transported onto the cell surface, extra-cellular space, and the circulation via nonclassical secretory pathways as it is devoid of the secretory signal sequence (Menon and Hughes 1999). Galectin-3 may be secreted via either vesicular release, the exosome-mediated pathway or it may traverse the lipid bilayer (Hughes 1999; Baptiste et al. 2007; Jones et al. 2010; Nakajima, Kho, Yanagawa, Harazono, et al. 2016). Galectin-3 is recycled by the cells and plays a crucial role in apical protein trafficking. After being secreted, it re-enters the cells by nonclathrin mediated endocytosis and acts as a raft for cargo proteins (Honig et al. 2015). In each cellular compartment, galectin-3 interacts with specific binding-partners and is involved with several cellular activities, which include apoptosis, cell migration, proliferation, angiogenesis, etc. These functions of galectin-3 have been the subject of reviews by several authors (Dumic et al. 2006; Nangia-Makker et al. 2008; Haudek et al. 2010; Funasaka et al. 2014a, 2014b; Nakajima, Kho, Yanagawa, Zimel et al. 2016). Here, we have focused on the functional properties of galectin-3 relating to epithelial–mesenchymal transition (EMT) with probable reference to cancer stemness. Cancer stem cells Tumor heterogeneity is defined by three models. According to the stochastic model, the heterogeneity in an initially homogeneous tumor arises due to random genetic events, resulting in a genetic drift and acquisition of dominant genetic changes in various permutations. Keeping with this model, cancer progression is nonlinear and a heterogeneous tumor is established from the cell clones branching out to produce diverse subpopulations within the tumor (Nowell 1976; Wolman and Heppner 1992; Marusyk and Polyak 2010; Marusyk et al. 2012). The cancer stem cell (CSC) model proposes that stem cell-like precursors with self-renewal and pluripotency capabilities, which reside within tissues, acquire mutations and develop into unique populations of cells that differentiate and acquire diverse sets of biological and phenotypical characteristics (Reya et al. 2001; Bapat 2007; Marusyk et al. 2012; Kreso and Dick 2014). The third model suggests inclusion of both these models and posits that a small subpopulation of CSC resides within the primary tumor. These CSC subgroups undergo diverse mutations resulting in intratumoral heterogeneity (Marusyk et al. 2012; Elshamy and Duhe 2013; Marjanovic et al. 2013). Over the last decade, the acceptance of this model has been increasing as the general theory of cancer progression, recurrence and most probably therapeutic resistance. Characteristics of CSCs CSCs are thought to originate from normal stem, progenitor or differentiated cells by acquiring mutations. Like normal stem cells, they acquire essential properties that include (i) self-renewing ability, (ii) pluripotency, (iii) high tumor initiating ability from a few cells and (iv) chemo-resistance. Due to these characteristics, CSCs play a significant role in tumor initiation, progression, invasion, metastasis, drug-resistance and recurrence. Cancer progression culminating in metastasis is an intricate multistep process including migration and invasion of tumor cells through surrounding stroma, intravasation and survival in the blood circulation under anchorage independent settings, extravasation, adherence and proliferation at the secondary site. Only a few cells that have transitioned through the EMT phase would complete these steps successfully (Thiery 2002; Yang and Weinberg 2008). It should be noted, that increasing records support the contention that only cells that have undergone EMT may display CSC-like properties (Mani et al. 2008; Jordan et al. 2011; Krantz et al. 2012; Wu 2011), and EMT can provoke the reversion of tumor cells to CSC-like phenotype (Mani et al. 2008; Polyak and Weinberg 2009). For example, pathways including Wnt, Notch and Hedgehog, which are controlled during normal development, are deregulated in both EMT and CSCs (Huber et al. 2005; Malanchi et al. 2008; Peacock and Watkins 2008; Singh and Settleman 2010). A dynamic plasticity interplay between epithelial and mesenchymal phenotype has also been suggested in the process of metastasis (Liao and Yang 2017). Table I summarizes the signaling pathways that have been associated with embryonic development, and EMT/CSCs. Table I. Signaling pathways in cancer stem cells and role of galectin-3 Pathway  Main players  Role of galectin-3  Wnt  Wnt Signaling pathway involving GSK-3β, β-catenin and TCF/LEF is dysregulated in various cancers. The main player in this pathway is β-catenin, which activates the transcription of certain genes upon translocation to nucleus.  Activates GSK-3β, binds to β-catenin and modulates its nuclear export and transcriptional activity (Shi et al. 2007; Shimura et al. 2004, 2005; Song et al. 2009)  Notch  The Notch pathway involving Snail, Slug and E-cadherin has been implicated in cancer initiation and development, as well as early stages of cancer progression by regulating conserved cellular programs such as EMT. A role for Notch signaling is also reported in osteolytic bone metastasis.  Activates/cleaves Notch transcription of its target proteins (Fermino et al. 2016; Kang et al. 2016; Nakajima et al. 2014) and regulates E-cadherin (Cao et al. 2017)  Hedgehog  Hedgehog (Hh) signaling involving FAK/Akt and targeting MMPs is an evolutionarily conserved pathway essential for self-renewal and cell fate determination. Aberrant Hh signaling is associated with the development and progression of various cancers and is implicated in multiple aspects of tumorigenesis, including the maintenance of CSCs.  Increases Akt activity (Gao et al. 2016; Kobayashi et al. 2011; Nangia-Makker et al. 2010; Zhang et al. 2013)  Transforming growth factor-β  TGF-β signaling pathway is involved in growth, differentiation, apoptosis, cellular homeostasis and other cellular functions in both the adult organism and the developing embryo including EMT. The key players SMADs act as transcription factors and regulate target gene expression. The non-Smad pathways include various branches of MAP kinase pathways, Rho-like GTPase signaling pathways and phosphatidylinositol-3-kinase/Akt pathways.  Stabilizes TGF-βR and plays an important role in FAK, PI3K/Akt pathways (Park et al. 2015)  EGFR/FGFR  Receptor tyrosine kinase binding to its ligand activates PI3K/Akt and MAPK pathways, which play an important role in the regulation of cell growth, proliferation and differentiation. It also induces transcription of EMT markers ZEB2, Snail, FOXC2, etc.  Stabilizes EGFR, and activates EGFR induced activation of K-Ras, PI3K and Akt, which in turn regulate β-catenin transcriptional activity (Elad-Sfadia et al. 2004; Shalom-Feuerstein et al. 2005)  Pathway  Main players  Role of galectin-3  Wnt  Wnt Signaling pathway involving GSK-3β, β-catenin and TCF/LEF is dysregulated in various cancers. The main player in this pathway is β-catenin, which activates the transcription of certain genes upon translocation to nucleus.  Activates GSK-3β, binds to β-catenin and modulates its nuclear export and transcriptional activity (Shi et al. 2007; Shimura et al. 2004, 2005; Song et al. 2009)  Notch  The Notch pathway involving Snail, Slug and E-cadherin has been implicated in cancer initiation and development, as well as early stages of cancer progression by regulating conserved cellular programs such as EMT. A role for Notch signaling is also reported in osteolytic bone metastasis.  Activates/cleaves Notch transcription of its target proteins (Fermino et al. 2016; Kang et al. 2016; Nakajima et al. 2014) and regulates E-cadherin (Cao et al. 2017)  Hedgehog  Hedgehog (Hh) signaling involving FAK/Akt and targeting MMPs is an evolutionarily conserved pathway essential for self-renewal and cell fate determination. Aberrant Hh signaling is associated with the development and progression of various cancers and is implicated in multiple aspects of tumorigenesis, including the maintenance of CSCs.  Increases Akt activity (Gao et al. 2016; Kobayashi et al. 2011; Nangia-Makker et al. 2010; Zhang et al. 2013)  Transforming growth factor-β  TGF-β signaling pathway is involved in growth, differentiation, apoptosis, cellular homeostasis and other cellular functions in both the adult organism and the developing embryo including EMT. The key players SMADs act as transcription factors and regulate target gene expression. The non-Smad pathways include various branches of MAP kinase pathways, Rho-like GTPase signaling pathways and phosphatidylinositol-3-kinase/Akt pathways.  Stabilizes TGF-βR and plays an important role in FAK, PI3K/Akt pathways (Park et al. 2015)  EGFR/FGFR  Receptor tyrosine kinase binding to its ligand activates PI3K/Akt and MAPK pathways, which play an important role in the regulation of cell growth, proliferation and differentiation. It also induces transcription of EMT markers ZEB2, Snail, FOXC2, etc.  Stabilizes EGFR, and activates EGFR induced activation of K-Ras, PI3K and Akt, which in turn regulate β-catenin transcriptional activity (Elad-Sfadia et al. 2004; Shalom-Feuerstein et al. 2005)  Identification and isolation of CSCs accountable for tumor initiation and propagation is a large undertaking due to the complexity in their biology, their heterogeneity that originates from genotypic and phenotypic plasticity, their scarcity in cancers, and diversity of cell surface markers, which differ between tissue types. Commonly identified cell surface markers are: CD44+CD24−/lowALDH1 activity in breast cancer, ALDH+CD44+CD166+EpCAM+ in colorectal cancer, ABCB5+ CD271 in melanoma, CD133+ in brain cancer, CD34+CD38− in acute myeloid lymphoma, CD44+ CD117+ in ovarian cancer, CD44+CD24+ESA+CD133+CD47 in pancreatic cancer, CD44+CD24+α2β1 high in prostate cancer and ABCG2 in retinoblastoma (for further information, see Pakravan et al. 2015). Galectin-3 in EMT, migration and invasion EMT is a biological manifestation whereby epithelial cells transition into mesenchymal cells both during normal development and tumor progression (Thiery 2003). The changes in cellular traits during EMT are decreased expression of epithelial markers such as E-cadherin and upregulation of mesenchymal markers α-smooth muscle actin and vimentin, loss of cell–cell junctions and apicobasolateral polarity resulting in a spindle shaped more migratory and invasive phenotype. Galectin-3 interacts with and mediates the expression of many proteins, which contribute to EMT during tumor cell migration and invasion. Galectin-3 over-expressing cells show an increased migration towards Matrigel (Nangia-Makker et al. 1995, 2000), whereas down-regulation of galectin-3 expression in MDA-MB-435 human breast carcinoma cells results in decreased migratory and invasive characteristics in vitro associated with reduced tumor growth in vivo (Honjo et al. 2001). Extra-cellular galectin-3, secreted by breast cancer cells, was involved with migration of endothelial cells in a dose-dependent fashion (Nangia-Makker et al. 2000) and phosphorylated focal adhesion kinase (pFAK) upregulated at the migratory edge (Nangia-Makker et al. 2010). Galectin-3 promotes focal adhesions turnover by stabilizing FAK because of transmembrane crosstalk between the galectin lattice and tyrosine phosphorylated caveolin (Goetz et al. 2008). Immuno-histochemical analysis of patient breast tumor specimens and xenografts revealed that galectin-3 expression shifted from luminal to peripheral cells in tandem with the progression to comedo-DCIS or invasive carcinomas. It was suggested that this transitional shift in the expression of galectin-3 increases the invasive potential of tumor cells through altered interactions with the stromal counterparts (Shekhar et al. 2004). An increase in E-cadherin expression accompanied with a reduced vascular mimicry in HepG2-Runx2 hepatocellular carcinoma cells was associated with galectin-3 down-regulation (Cao et al. 2017). Galectin-3 induced Hela cell migration via MAPK/ERK-1/2 pathway was sensitive to calcium (Gao et al. 2014), while increased cell migration by galectin-3 in mesenchymal stem cells was due to inhibition of RhoA GTPase activity and increased pERK1/2 and pAkt (Gao et al. 2016). Similarly, galectin-3 mediated tumor cell invasion in pancreatic and tongue cancer cells (Zhang et al. 2013) through the initiation of Akt phosphorylation and increased β-catenin transcriptional activity resulting in increased MMP-2 and MMP-9 activities. Galectin-3 increased the in vitro migration of BV2 microglia associated with integrin-linked-kinase signaling (Wesley et al. 2013) and induced EMT like morphology in a murine epithelial cell line GE11 via β-1 integrin that triggers galectin-3 expression through demethylation of its promoter. Galectin-3, in turn stimulated cell migration and adhesion mediated by β-1 integrin, thus forming a functional feed-back-loop (Margadant et al. 2012). Galectin-3 inhibition induced activation of β-catenin and attenuated lung, hepatic and renal fibrosis, making it a proposed marker for active fibrosis (Mackinnon et al. 2012). Galectin-3 is instrumental in cyclin D1 and c-myc over-expression affected by β-catenin since the NH2 terminus of β-catenin binds to the COOH terminus of galectin-3 forming a nuclear complex prompting the transcriptional activity of Tcf-4, independent of mutations in either APC or β-catenin (Shimura et al. 2004, 2005; Henderson et al. 2008). Moreover, Wnt-2- and galectin-3 synergistically induced apoptosis in colorectal cancer cells (Shi et al. 2007). Reproduction of galectin-3 modulation of β-catenin expression and Tcf-4 activity was also reported in colon cancer cells. In the absence of galectin-3, Akt and GSK-3β were dephosphorylated and increased GSK-3β activity lead to increased phosphorylation of β-catenin and its degradation (Song et al. 2009). Wang et al. (2013) reported an acceleratory effect of galectin-3 on proliferation, migration and invasion of oral tongue squamous cell carcinoma via Wnt/β-catenin dependent pathway and EMT. Galectin-3 correspondingly contributes to the tumor cell’s ability to invade and degrade basement membrane by increasing the activity of MMPs. Silencing galectin-3 reduced secretion of MMP-2 and invasive ability of C8161-c9 melanoma cells (Mourad-Zeidan et al. 2008). Galectin-3 was also reported to bind directly to transcription factor activated AP-1 for MMP-1 (Wang et al. 2012). Galectin-3 oligomerization increased the affinity of elastin to αvβ3 integrin, which in turn regulates melanoma cell invasion (Pocza et al. 2008). Further, binding between integrin and galectin-3 enhanced cell migration (Saravanan et al. 2009) by reducing α2β1 integrin–collagen binding in the extra-cellular matrix (Friedrichs et al. 2008). Due to the galectin-3N-terminal oligomerization property, galectin-3 can cooperate with cell surface glyco-conjugates (glycoproteins and glycolipids). Its binding affinity depends on the number of glycosylation sites and glycosyl transferase activity (Demetriou et al. 2001). The galectin oligomers form bridges between glycoproteins creating a lattice, which increases the interactions of multiple proteins as well as stabilizing them by preventing their endocytosis (for review, see, Nabi et al. 2015). For example, the interactions between galectin-3 and N-glycans on the extra-cellular domains of αvβ3 integrin resulting in its cluster formation modulate KRAS clustering and resistance to EGFR inhibitors (Seguin et al. 2014). Thus, the conclusion was that galectin-3 might regulate receptor tyrosine kinase signaling, cell-motility and formation of fibronectin fibrils under pathological conditions (Akahani et al. 1997; Partridge et al. 2004; Dennis et al. 2009). Galectin-3 and chemo-resistance CSCs employ multiple mechanisms for chemo-resistance such as enhanced drug efflux, aldehyde mediated detoxification, enhanced DNA repair mechanism, quiescence, apoptosis inactivation and activated signaling pathways (Wnt, Hedgehog and Notch) leading to multidrug resistance (MDR) (Thomas et al. 2015). Endogenous galectin-3 inhibits apoptosis in epithelial cell induced by staurosporine and chemotherapeutic agents such as cisplatin, genistein, tumor necrosis factor and nitric oxide (Akahani et al. 1997; Kim et al. 1999; Lin et al. 2000; Yoshii et al. 2002). Anti-apoptotic activity of galectin-3 is dependent on its phosphorylation and nuclear export (Takenaka et al. 2004) and Ser6 is essential for this phosphorylation. Using Ser6Ala or Ser6Glu mutation, Takenaka et al. (2004) demonstrated that mutant galectin-3 was neither phosphorylated nor exported from the nucleus, while the wild-type galectin-3 was phosphorylated and exported from nucleus to the cytoplasm, imparting drug resistance to cancer cells. Additionally, peritoneal macrophages isolated from galectin-3 deficient mice were more sensitive to apoptosis compared with from normal control mice (Sano et al. 2003) and galectin-3 directly inhibited anti-Fas antibody induced T-cell apoptosis (Yang et al. 1996). In metastatic colon cancer cells, cell surface galectin-3 was responsible for resistance to Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL) by blocking the trafficking of death receptors prior to apoptosis (Mazurek et al. 2012). Galectin-3 knockdown stimulated TRAIL-induced apoptosis in human bladder carcinoma cancer cells by down-regulating Akt/PI3K pathway and gemcitabine induced apoptosis in human pancreatic carcinoma cells (Kobayashi et al. 2011). In a recent study, the biliary epithelial cells (BECs) of galectin-3 knockout mice showed increased sensitivity to apoptotic insults and the presence of proinflammatory lymphocytes and dendritic cells (DCs) was more pronounced in the livers of galectin-3 knockout mice compared to the normal mice. This suggests a novel function for galectin-3 as a safeguard of BECs from pathways leading to inflammation (Arsenijevic et al. 2016). Similarly, galectin-3 induces chemo-resistance by modulating signaling pathways activated in CSCs. In Wnt signaling pathway, it regulated GSK-3β activity and the accumulation of β-catenin in the nucleus (Shimura et al. 2004, 2005). Human bone marrow-derived mesenchymal stromal cells hBM-MSCs exhibited cytotoxic drug resistance in acute leukemia cells (ALCs) by galectin-3, which in turn stimulated β-catenin transcriptional activity. Down-regulation of galectin-3 by short hairpin RNA (shRNA) reversed this effect (Hu et al. 2015). Galectin-3 silencing in cultured human osteosarcoma cells reduced expression and activation of invasion mediating proteins FAK, Src, Lyn, PI3K/Akt, ERK1/2 and β-catenin, and potentiated sensitivity to cisplatin (Park et al. 2015). Galectin-3 is also an essential component of the cluster formed between αvβ3 integrin/KRAS at the plasma membrane, facilitating recruitment and hyper-activation of RalB, which in turn drives NFkB via TBK1. This clustering is responsible for lung and pancreatic tumor stemness and resistance to tyrosine kinase 1 inhibitors (Seguin et al. 2014). Galectin-3 also regulates Notch signaling. It binds to Notch1, accelerates its cleavage and regulates bone-remodeling (Nakajima et al. 2014). A crosstalk between galectin-3 and the Jagged/Notch affects the T helper polarization programs (Fermino et al. 2016). In a different tumor system, i.e. ovarian cancer, upregulation of galectin-3 expression was associated with the nuclear translocation and increased NICD1 (Notch 1 intracellular domain) cleavage concomitant with the upregulation of Notch target genes Hes1 and Hey1 (Kang et al. 2016). Further, interaction between galectin-3, Hedgehog and histone deacetylase protein was implicated in liver fibrosis (de Oliveira et al. 2017) and inhibition of TGFβ signaling in TβRII deficient mice, resulted in a reduced galectin-3 expression and associated Akt phosphorylation (Gong et al. 2012). Galectin-3 in differentiation and regeneration Cellular and circulating galectin-3 were found to be significant markers for multipotent progenitor cells of the bone marrow, mesenchymal stem/stromal cells (MSCs), which can differentiate into osteoblasts, chondrocytes, myocytes and adipocytes, i.e. the cells of mesenchymal origin. MSCs play an essential role in T-cell mediated immunological response as well as in wound healing, regeneration in response to ischemia and/or injury (reviewed in Rahimzadeh et al. 2016). Galectin-3 is responsible for the ability of MSCs to suppress cardiac inflammation and fibrosis in hearts with chronic Chagas cardiomyopathy symptoms (Souza et al. 2017). Galectin-3 induction in ALCs by bone marrow-derived MSCs is a prerequisite for drug resistance (Hu et al. 2015). Galectin-3 upregulation and secretion by senescent adipose derived MSCs is critical for their growth stimulatory effect on colorectal cancer LoVo cells by activating the MAPK ERK1/2 pathway (Li et al. 2015). Similarly, galectin-3 is essential for the proliferation of hepatic progenitor cells in response to liver injury (Hsieh et al. 2015), inflammation promotion in hepatitis (Volarevic et al. 2015) and multiple sclerosis (Chen et al. 2014). It should be noted that in galectin-3 knockout mice, the ability of myeloid progenitor cells to differentiate into mature myeloid cells is abrogated, consequently, modifying the bone marrow compartment (Brand et al. 2011). Galectin-3 as a cancer biomarker and therapeutic target There are numerous publications on the altered cellular/extra-cellular distribution and expression of galectin-3 in various cancers using human and xenograft tumor tissues (reviewed by Balan et al. 2010; Dong et al. 2017; Thijssen et al. 2015). In breast cancer, galectin-3 expression was redistributed from luminal to peripheral epithelial cells in the more invasive ducts (Shekhar et al. 2004). An increase in MMP-2 cleaved galectin-3 protein correlated with angiogenesis in progressive stages of breast and prostate cancer (Nangia-Makker et al. 2007, 2010; Wang et al. 2009). While galectin-3 is a diagnostic and/or prognostic marker for thyroid (Liu et al. 2008; Rydlova et al. 2008; Than et al. 2008), gastric (Baldus et al. 2000; Miyazaki et al. 2002), colorectal cancer (Lu et al. 2017) and to some extent pancreatic cancer (Shimamura et al. 2002), melanocytes accumulate galectin-3 in the nucleus as the melanoma progresses (Prieto et al. 2006). Nuclear galectin-3 expression in association with thyroid transcription factor 1 is an independent marker of worst prognosis for lung cancer patients (Puglisi et al. 2004), whereas in colon cancer enhanced cytoplasmic levels of galectin-3 were reported (Lotz et al. 1993; Sanjuan et al. 1997). In brain tumors, galectin-3 is differentially expressed in various tumors (Park et al. 2008) and can be used as a tool to track their origin (Neder et al. 2004; Park et al. 2008). Due to its involvement in diverse biological functions, many attempts have been made to use galectin-3 as a therapeutic target. Modified citrus pectin (MCP) was the first reported natural inhibitor of galectin-3 functions in mouse models of prostate, colon and breast cancer (Platt and Raz 1992; Pienta et al. 1995; Nangia-Makker et al. 2002). Galectin-3 suppresses apoptosis and increases resistance of tumor cells to chemotherapeutic drugs, it has been used as a target to resensitize cancer cells to chemotherapeutic drugs (Glinsky and Raz 2009). Targeting galectin-3 via MCP or lactosyl-l-leucine (LL) sensitized malignant cells to doxorubicin (Johnson et al. 2007), taxol (Glinsky and Raz 2009), cisplatin (Wang et al. 2010), bortezomide and dexamethasone (Chauhan et al. 2005). In a recent study, another polysaccharide inhibitor of galectin-3 RN1 is reported to suppress growth of pancreatic ductal carcinoma in mouse xenografts and patient derived xenografts (Zhang et al. 2017). A truncated, dominant negative form of galectin-3, galectin-3C, is another promising new compound for effective adjuvant therapies in advanced, refractory multiple myeloma and ovarian cancer (John et al. 2003) (Mirandola, Nguyen, et al. 2014; Mirandola, Yu et al. 2014). In addition, galectin-3 inhibitors including peptide antagonists, lactulose amines (Glinskii et al. 2012) and synthetic glycol-conjugates (reviewed in Campo et al. 2016) have also been the focus of several studies. Galectin-3 and cancer stemness Based on the above it is tempting to draw a link between cancer stemness and galectin-3. A proteomic comparative analysis between the increasing culture passages of tumor spheres and monolayer cultures of MCF-7 breast cancer cells revealed an amplification of stem like cell markers associated with functional identifiers such as self-renewal and symmetric cell division in the tumor spheres (Deleyrolle et al. 2011). The spheres as well as subpopulations within the spheres were more tumorigenic than the parental cells implying the emergence and enrichment of CSCs. There was also an increased expression of galectin-3 together with carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-5 and -6, MUC-1, -5 and -6. When the spheres were reseeded in the presence of galectin-3 inhibitor N-acetyllactosamine, a significant reduction in sphere formation was noted (Morrison et al. 2012). In MCF-7 cells, an increased apoptosis-resistance and side-population with CSC properties were attenuated by down-regulating galectin-3 (Guha et al. 2014). Similarly, there was increased expression of galectin-3, -4, -7 and -9 in spheres formed by the human nonsmall cell lung cancer cell line H1299. However, galectin-3 expression was significantly higher than other galectins in the tumor spheres during each successive passage (92). Those spheres also showed increased levels of stem cell markers Oct4, Sox2, Nanog, CXCR4, β-catenin and CD133. A different experimental approach of down-regulating galectin 3 expression by lentivirus-mediated shGal-3 inhibited self-renewal, chemo-resistance and malignancy of the spheres. In sharp contrast, the addition of recombinant-galectin-3 to the culture medium stimulated sphere formation. The authors hypothesized that the extra-cellular galectin-3 may interact with EGF and bFGF present in the stem cell culture medium thus contributing to the generation of spheres, whereby intracellular galectin-3 acts as a cofactor. It may also interact with β-catenin and augment its transcriptional activities resulting in the expression of stemness-related genes and upregulation of ABC transporter genes (Chung et al. 2015). Another experimental approach examining the regulation of lung CSCs by galectin 3 has demonstrated that galectin-3 enhanced lung cancer stemness through the EGFR/c-myc/Sox2 axis via carbohydrate-recognition activities and that Oct 4 promoted galectin-3 expression forming a positive regulatory loop (Kuo et al. 2016). Moreover, amplified expression of galectin-3 protein was reported in glioblastoma stem cells (Ma et al. 2014), whereby the tumor suppressor miR-152 was found to regulate galectin-3 expression in CSC. Over-expression of miRNA-152 reduced the level of galectin-3 by down-regulating KLF4, which binds and activates galectin-3 promoter while inhibiting MEK1/2 and PI3K signaling (Ma et al. 2014). Recently, the ability of galectin-3 to characterize CSC phenotype of human colorectal and pancreatic cancer cell lines was explored. CD44+, CD24+, EpCAM+ and CD166+ cells from colorectal and pancreatic cells were sorted for galectin-3 positive or negative cells and analyzed for their stemness properties. In this study, the level of cell surface expression of galectin-3 correlated with enhanced CSC phenotypes, like sphere forming, tumorigenicity and drug resistance. Down-regulation of galectin-3 expression by shRNA abrogated the levels of stem cell markers and sphere forming ability of CSCs (Ilmer et al. 2016). In lung and pancreatic tumors, presence of galectin-3 was essential for cluster formation with αvβ3 integrin and KRAS, which activated the NFkB pathway and stemness (Seguin et al. 2014). Increased levels of galectin-3, together with MMP-2, alpha actinin-4 and MARCKS in the secretome of pancreatic CSCs compared to parental cell line Panc1 was also noted (Brandi et al. 2016). Similarly, in ovarian CSCs, galetin-3 supports stemness by activation of Notch1 intracellular domain (Kang et al. 2016). Conclusions and future directions Emergence of drug resistant cells is the leading cause of tumor recurrence and metastasis. The cells capable of successfully metastasizing to distant sites are endowed with invasive and migratory properties, which are the essential traits of both EMT and CSC. Galectin-3 expression is necessary for the preservation of the transformed and drug-resistant phenotypes of cancers. Interactions of galectin-3 with diverse signaling pathways associated with EMT and CSC are depicted in Fig. 1. In the tumor microenvironment the galectin-3 molecules that are secreted by macrophages and tumor cells, interact with extra-cellular matrix components and may be cleaved by MMPs affecting homotypic and heterotypic cell recognition and adhesion. The lattice formation by the extra-cellular galectin-3 mediated oligomerization by binding to N-acetyllactosamine residues of cell surface glyco-ligands (EGFR, TGF-βR, integrins, etc.) stabilizes them and prevents their endocytosis and degradation, thus facilitating their downstream events[for review see (Nabi et al. 2015)]. In addition, it activates Notch 1 and enables its cleavage (Kang et al. 2016). Therefore, one must conclude that galectin-3 plays an important role in activating signaling pathways that are crucial for EMT and continuation of stemness. Stabilization of β-catenin by galectin-3 regulates transcription of its target genes which include stem cell markers like c-myc, and CD44 among others (Funasaka et al. 2014b). While galectin-3 is instrumental in activation of MDR gene responsible for drug efflux and resistance to apoptosis (Harazono et al. 2015), it also establishes a positive regulatory loop with the pluripotency genes Klf4 and Oct4 (Ma et al. 2014; Kuo et al. 2016). Based on the above, a few recent studies have tested the presence/over-expression of galectin 3 in CSCs and reported its upregulation in CSCs while its inhibition reduced the stem cell characteristics (Morrison et al. 2012; Chung et al. 2015; Ilmer et al. 2016; Kang et al. 2016). In gastrointestinal cancer, cell surface galectin-3 was identified in a subset of CSCs with heightened stem cell characteristics (Ilmer et al. 2016). Figure 1. View largeDownload slide A schematic representation of the interactions and regulatory role of galectin-3 in stemness-related pathways. Figure 1. View largeDownload slide A schematic representation of the interactions and regulatory role of galectin-3 in stemness-related pathways. Although an elementary understanding of the direct relationship of galectin-3 to cancer stemness is beginning to emerge a more detailed and comprehensive insight will be required to understand its role and possible use as a target to inhibit CSC function. Further, the relationship among members of the galectin family to each other and their alternate compensatory mechanisms should be taken into account. The presence of a SNP (rs4644) in the galectin-3 gene, a substitution of Proline64 with Histidine, results in cells with increased chemotaxis, chemo-invasion, angiogenesis, apoptosis resistance in vitro, tumor formation in vivo and an increased susceptibility to breast cancer (Nangia-Makker et al. 2007; Balan et al. 2008). Subsequent studies reported an association of this SNP to prostate cancer and cervical cancer susceptibility (Meyer, 2013, Fang, 2017), rheumatoid arthritis (Hu, 2011) and drug resistance (Mazurek, 2011) via β-catenin pathway. It would be interesting to establish whether galectin-3 germline mutation is associated with cancer stemness. Funding This work was supported by the Paul Zuckerman Endowment (to A.R.) and by the NCI/NIH, Cancer Center Support Grant (CA-22453). Conflict of interest statement None declared. Abbreviations ALCs, acute leukemia cells; BECs, biliary epithelial cells; CEACAM, carcinoembryonic antigen-related cell adhesion molecule; CRD, carbohydrate-recognition domain; CSCs, cancer stem cells; EMT, epithelial–mesenchymal transition; LL, lactosyl-l-leucine; MCP, modified citrus pectin; MSCs, mesenchymal stem/stromal cells; pFAK, phosphorylated focal adhesion kinase; PSA, prostate specific antigen; TRAIL, tumor necrosis factor Related Apoptosis Inducing Ligand. References Akahani S, Nangia-Makker P, Inohara H, Kim HR, Raz A. 1997. Galectin-3: A novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family. Cancer Res . 57: 5272– 5276. 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