Impact of Galectins in Resistance to Anticancer Therapies

Cancer is a multistep process in which cellular reprogramming promotes the major hallmarks associated with malignant transformation, including uncontrolled cell proliferation, metastatic capacity, induction of angiogenesis, and escape from immune surveillance, among others (1). Acquisition of this tumor phenotype takes place via a complex process involving transcriptional control, posttranscriptional regulation, and posttranslational modifications. Although one cannot deny the impact of DNA and RNA dysregulation with respect to processes associated with cancer onset and progression, the contributions associated with posttranslational modification are also crucial, as these mechanisms may serve to control protein stability, activity, subcellular localization, and interactions with other molecules. In particular, changes in protein glycosylation are among the most frequent posttranslational modifications reported in association with neoplastic transformation, and have a strong impact on most aspects of cancer biology (2–4). Mounting research has pointed to alterations in tumor-specific glycosylation and the manner in which these patterns provide cancer cells with advantages, including the ability to metastasize and/or to evade immune surveillance. These findings provide us with novel clinical opportunities for identification of biomarkers and cell-directed therapies (5). The cancer glycome, which is the entire repertoire of glycans found in tumor cells and in their microenvironments, includes mainly modifications associated with N- and O-linked carbohydrate branching, O-glycan truncation, sialylation, and fucosylation (2, 6). Acquisition of these glycosylation patterns by malignant cells or by their associated stroma results in a “cancer glyco-code,” which can be efficiently deciphered by glycan-binding receptors (i.e., lectins), mainly calcium-dependent lectins (C-type), sialic acid-binding Ig-like lectins (siglecs), and galectins (6).

Galectins are a family of 15 (11 in humans) evolutionarily conserved lectins that share a high degree of structural homology in their carbohydrate-recognition domain (CRD) sequence motifs and have affinity for N-acetyllactosamine–containing glycoconjugates (7). They are classified into three subfamilies, according to their structure (Fig. 1); these include “prototype” galectins (Gal1, Gal2, Gal5, Gal7, Gal10, Gal11, Gal13, Gal14, and Gal15) that contain one CRD and form noncovalent homodimers, “chimera-type” galectins (Gal3) that contain a single CRD and an N-terminal sequence involved in protein oligomerization, and “tandem-repeat” galectins (Gal4, Gal6, Gal8, Gal9, and Gal12) that have two CRDs connected in tandem by a linker peptide (Fig. 1). Galectins are widely expressed in a variety of different cell types; they are synthesized in the cytosol and can be translocated to the nucleus or secreted to the extracellular compartment via mechanisms that remain poorly understood, as they lack the typical signal peptide required for externalization (8). Galectin binding to cognate ligands, either intracellularly or extracellularly, promotes a plethora of biological functions, including promotion of cell–cell and cell–extracellular matrix interactions, regulation of cell death and proliferation, and intracellular signal transduction. Hence, is not surprising that cancer cells may coopt galectin expression to increase invasion and metastasis, to promote angiogenesis, and to evade antitumor immunity (9–11). In fact, most tumors exhibit aberrant expression of galectins compared with normal tissue (12, 13). Of particular note, while most earlier studies targeted functions and responses associated with Gal1 and Gal3, other galectins have been the focus of more recent attention (10, 14). Notably, except for Gal1, which is normally overexpressed in cancer and is clearly a marker of poor prognosis, other galectins serve in either pro- or antitumor roles depending on the tumor type (9, 14); this observation highlights the need for a more comprehensive picture of the “galectin signature” together with additional insights into the specific roles played by each galectin in a given tumor microenvironment (TME). Moreover, it is critical to appreciate the fact that different galectins can bind to the same ligands and can display redundant functions; this point needs to be taken into consideration in the design of anticancer inhibitory strategies.

Resistance to therapy is one of the major obstacles hampering the development of cures for patients with cancer; this remains an important challenge in oncology research (15). Recently, emerging evidence has suggested a role for galectins in the development of resistance to distinct anticancer therapies; this effect is related to their capacity to influence one or more of the distinct hallmarks of tumor progression (10, 11). Galectins may have a profound impact on critical factors involved in developing drug resistance, including tumor growth, the nature of the TME (including roles of immune cells, endothelial cells (EC), and cancer-associated fibroblasts), and therapeutic pressure, among others (15). Importantly, despite considerable progress that has been made toward understanding galectin functions with respect to cancer, to the best of our knowledge, there are still no review articles that have successfully integrated our current understanding of galectin functions with their roles in promoting resistance to anticancer therapeutic strategies. To fill this gap, in the next sections, we will discuss the role of individual members of the galectin family with respect to resistance or sensitivity to current major anticancer treatments including chemotherapy, radiotherapy, targeted therapy, antiangiogenic therapy, and immunotherapy (Figs. 2 and 3).

The first use of chemotherapy for cancer treatment dates back to the 1940s, when a patient with advanced non-Hodgkin lymphoma was systemically (and successfully) treated with nitrogen mustard, a cytotoxic DNA alkylating drug. Since that time, the number of drugs approved by the FDA for cancer chemotherapy increased until 1980s, when the focus in molecular oncology changed toward target-driven therapies (16). Notwithstanding, more than 70 years after its first discovery, single or combined chemotherapy remains the first-line treatment for a large number of neoplastic diseases. These drugs as a group are cytotoxic agents that interfere with DNA synthesis in rapidly dividing cells; they include antimetabolites, alkylating agents, anthracyclines, and topoisomerase inhibitors. Unfortunately, chemotherapy has critical limitations, not only because of dosage-limiting toxicity, but even more importantly, due to the fact that tumors frequently are or become resistant to single agents. Supporting the notion that the presence of drug-resistant cancer cells is associated with mutational rates linked to tumor size and growth (17), current chemotherapeutic strategies involve combinations of cytotoxic agents that are administered simultaneously, sequentially, or in alternative sequences (18). Resistance to chemotherapy is triggered by different mechanisms, including increased drug metabolism, interference with drug uptake, overexpression of transporters that favor drug export, inactivation of cell death programs, and activation of prosurvival pathways, among others (18–20).

Mounting evidence has revealed that galectin expression in tumor tissue undergoes modulation in response to chemotherapy (21–54); this observation suggests a role for these proteins in resistance to anticancer therapies (Table 1; Fig. 2). Some reports describe the association between elevated galectin levels and drug resistance, while other studies demonstrate a direct link between galectin expression and the response to chemotherapy. For example, temozolomide, the standard treatment for glioblastoma multiforme (GBM), induces Gal1 overexpression in glioma cells both in vitro and in vivo, a response that confers drug resistance; in contrast, downregulation of Gal1 overcomes this effect (21, 22, 33, 44, 49). Similarly, knockdown of Gal1 in lung cancer cells increases their sensitivity to cisplatin (52, 53), while Gal1 overexpression in hepatocellular carcinoma (HCC) induces sorafenib resistance (26, 27). In the same line, many studies have reported that Gal3 expression promotes resistance to chemotherapy (30–32, 34–43, 45–48). This is illustrated by overexpression of Gal3 in human prostate cancer cells, which inhibits cisplatin- or etoposide-induced apoptosis (30); likewise, Gal3 expression by leukemia cells confers resistance to vincristine (43). While these studies consistently demonstrated positive correlations between Gal1 or Gal3 expression and tumor resistance to chemotherapy; involvement of other galectin family members has been poorly explored and their role in drug response is unclear. For example, Gal4 may function in opposition to the actions of Gal1 and Gal3, as high levels of this protein correlate with higher sensitivity to 5-fluorouracil (5-FU) and oxaliplatin in specific molecular subtypes of gastric cancer (55). Likewise, Gal7 expression has been associated with lower resistance to paclitaxel (56) or to concurrent chemoradiotherapy with cisplatin in cervical carcinoma (57) and urothelial cancer (58). In addition, Gal7 sensitizes oral squamous cell carcinoma (OSCC) cells to chemotherapy (carboplatin, cisplatin, and 5-FU) and/or radiotherapy (59), which correlates with its reported functions as a proapoptotic and tumor suppressor gene (60–63). In contrast, in breast cancer, Gal7 expression confers resistance to doxorubicin (64), suggesting a tissue-specific role for this protein in promoting chemoresistance. Finally, a recent study featuring whole-exome and RNA-sequencing of peritoneal metastatic tumor samples from patients with gastric cancer identified an aggressive mesenchymal-like tumor type that typically responds poorly to chemotherapy and expresses high levels of Gal9; these findings suggest that Gal9 may also play a role in conferring resistance to anticancer drugs (65).

The molecular mechanisms through which galectins confer resistance to chemotherapy have been studied thoroughly, most notably those underlying the actions of Gal1 and Gal3 (Fig. 2). In particular, one key mechanism involved in Gal1-mediated chemoresistance is the overexpression of P-glycoprotein (encoded by the multidrug resistance gene 1, MDR1), which is one of the main ATP-binding cassette transporters involved in expelling cytotoxic drugs from the intracellular space (66). In breast cancer cells, downregulation of Gal1 results in reduced levels of P-glycoprotein via its actions on the Raf-1/AP-1 pathway; this leads to increased sensitivity to paclitaxel or doxorubicin (25). This mechanism has also been reported for Gal3-mediated resistance to doxorubicin in thyroid cancer (34). Moreover, Gal1 and Gal3 modulate survival pathways, which is a common mechanism developed by cancer cells to avoid drug-induced toxicity. To illustrate this concept, Gal1 mediates cisplatin resistance in HCCs by inducing autophagy; this in turn triggers mitophagy and decreases mitochondrial potential loss and apoptosis (54). Similarly, Gal1 knockdown in neuroblastoma cells increases cisplatin sensitivity through autophagy inhibition (23). In contrast, Gal1-induced resistance to the proautophagic drug, temozolomide, is autophagy independent, as this function is mediated by modulation of the unfolded protein response associated with endoplasmic reticulum stress and p53-mediated transcriptional activity in glioma cells (21), or by lysosomal membrane permeabilization and release of cathepsin B into the cytosolic compartment of melanoma cells (50). Moreover, a recent study revealed that Gal1 mediates doxorubicin resistance in breast cancer through binding to β₁ integrin, activation of ERK1/2 and STAT3, and upregulation of survivin (24). A large number of studies have pointed toward an antiapoptotic role for Gal3, which confers chemoresistance to several tumors (67). In particular, cytoplasmic Gal3 protects against mitochondrial membrane disruption and prevents release of cytochrome c; this inhibits activation of caspase 3 and apoptosis in response to cisplatin treatment in both prostate and breast cancer cells (30, 68). Interestingly, Gal3 is the only galectin that contains an NWGR motif, which is highly conserved among members of the BCL-2 gene family and is required for its antiapoptotic function (46, 69). Moreover, several studies have reported that Gal3 phosphorylation, followed by nuclear–cytosol translocation and activation of ERK1/2 and AKT/PI3K signaling pathways, is crucial to prevent apoptosis in response to drug treatment in breast and bladder cancer (47, 70, 71).

In contrast to Gal1 and Gal3, the molecular mechanisms underlying chemoresistance associated with other galectins have not been studied in such detail. In particular, Gal7-mediated resistance to doxorubicin in breast cancer has been attributed to its cytosolic location; this results in impaired nuclear translocation of p53 and decreased p21 expression (64). Conversely, Gal7 sensitizes bladder cancer cells to cisplatin therapy by upregulating reactive oxygen species, promoting JNK signaling, and inducing apoptosis (58). In OSCC, the positive correlation between Gal7 expression and sensitivity to chemoradiotherapy may be related to its cytosolic compartmentalization, which serves to impair cell proliferation (59). Furthermore, Gal7 knockdown in cervical cancer cells revealed a role for this lectin in sensitizing these cells to paclitaxel treatment by targeting the PI3K/AKT pathway (Fig. 2; ref. 56).

Thus, although many studies have elucidated key roles for Gal1 and Gal3 in promoting resistance to various chemotherapeutic modalities via modulation of drug export or interactions with cell death/survival pathways, additional studies are required to generate a more complete understanding of the mechanisms underlying this effect. We also need to have a better appreciation of the contributions of other galectin family members to chemotherapy responses.

The roots of clinical radiotherapy date back to 1896, when Émil Grubbé used X-rays to treat an advanced breast tumor (72); radiotherapy remains central in our approach to cancer treatment. In fact, radiotherapy, alone or in combination with chemotherapy, is among the most effective of the cytotoxic treatments and is frequently used as curative therapy for localized solid tumors. The power of ionizing radiation comes from its capacity to induce extensive DNA damage; this ultimately leads to tumor cell death by apoptosis, necrosis, autophagy, or mitotic catastrophe. Radiotherapy can also modify the TME, boost the immune system, and/or increase drug delivery, which are all the factors that support its use as combined anticancer therapy. However, despite considerable advances that have focused on ways to increase the efficiency and accuracy of this approach, optimization of the balance between effective versus toxic radiation doses with the goal of avoiding harmful side effects in normal tissues remains a major challenge. Moreover, mechanisms of resistance to radiotherapy are mainly associated with elevated levels of DNA repair and survival pathways, induction of hypoxia, and activation of cancer stem cells (73, 74).

Interestingly, there is increasing evidence that suggests that galectins play a significant role in the development of resistance to radiotherapy (Fig. 2). Tumors that are highly resistant to radiation, including glioma, prostate cancer, and melanoma, typically express high levels of Gal1 (75–77). Remarkably, Gal1 expression can be induced under conditions associated with tumor hypoxia (78–81), which is known to be a major cause of radiotherapy failure (82). The contributions of Gal1 to radiotherapy resistance under hypoxic conditions are mediated by a positive feedback loop involving the induction of hypoxia-inducible factor-1α (HIF1α) via Gal1-mediated activation of H-Ras pathway (83). Moreover, several groups have shown that Gal1 is overexpressed in response to ionizing radiation and contributes to induction of radiotherapy resistance in glioma and neuroblastoma, as well as in lung, cervical, and breast cancer (83–88). Interestingly, induction of Gal1 by radiation is not restricted to cancer cells, but also has an impact on the TME. In particular, Gal1 synthesized by ECs after radiation treatment can also confer resistance to radiotherapy (89). In classical Hodgkin lymphoma, multivariate analysis has revealed that high levels of Gal1 detected in the TME correlate with poor prognosis (29). Mechanistically, Gal1-driven resistance to radiotherapy shares some (but not all) features in common with resistance to chemotherapy; while both treatments trigger DNA damage, radiotherapy-specific–resistant mechanisms include immune evasion and stimulation of angiogenesis (83). For example, in cervical cancer cells, radiation-induced Gal1 activates H-Ras, thereby promoting downstream activation of the Raf-1/ERK pathway, which facilitates repair of DNA damage; this mechanism limits the effectiveness of radiation-induced cell death (85). Similarly, preclinical studies focused on lung cancer revealed that Gal1 secreted by tumor cells in response to radiotherapy results in systemic lymphopenia; these events lead to immune evasion and a diminished response to radiotherapy (88). Interestingly, lymphopenia is associated with the poorest prognosis in patients with lung cancer who are undergoing treatment with radiotherapy (90). However, not only Gal1 but also Gal3 levels were found to increase in response to combined treatment with temozolomide and radiotherapy in glioma cells (91); these results suggested that both Gal1 and Gal3 contribute to radio-resistance mechanisms. In fact, the Gal3-specific inhibitor, Td131_1, enhances apoptosis of papillary thyroid cancer cells via inactivation of AKT, ERK1/2, and Bad pathways, thereby improving their sensitivity to radiotherapy (31).

Clinical studies that feature different subtypes of gastric cancer included reports of increased levels of Gal4 in patients who responded to adjuvant 5-FU–based chemoradiation; these findings suggested a role for Gal4 in promoting sensitivity to chemoradiotherapy (55). In the same line of thinking, the extent of Gal7 expression correlated directly with favorable responses to radiotherapy among those diagnosed with OSCC (59) or cervical carcinoma (57); the molecular mechanisms underlying these observations have not yet been elucidated.

In summary, expression of galectins in tumor cells and/or in the TME can modulate response to radiotherapy via distinct, albeit partially overlapping mechanisms (Fig. 2). While emerging data suggest that expression of Gal4 and Gal7 promotes an overall increase in sensitivity to radiotherapy, there is also convincing evidence demonstrating that elevated levels of Gal1 and Gal3, detected specifically in response to treatment, result in impaired clinical efficacy. As such, therapies targeting Gal1 and/or Gal3 have emerged as novel strategies that may help to overcome radiotherapy resistance.

Efforts made since the mid-20th century to understand the molecular mechanisms underlying malignant transformation (1, 92) have led to a new era in cancer treatment with the revolution of target-driven therapies. These therapies represent a significant breakthrough in clinical oncology (15, 16). Compared with chemo- and radiotherapy, targeted therapies are based on drugs with selective effects against tumor cells that have little to at most moderate impact on normal cells and tissues. A seminal work that established the basis for targeted therapies was published by Druker and colleagues (93); these authors demonstrated that 90% of patients achieved complete remission of chronic myeloid leukemia (CML) in response to treatment with imatinib (Glivec). Imatinib is an inhibitor of the tyrosine kinase BCR–ABL, which is a fusion protein detected nearly universally in this type of cancer (93). These studies led to the FDA approval of Glivec (2001), a milestone in target-driven therapy. Since then, many other cancer-specific molecules have been identified; among those introduced into the arsenal of drugs used against tumors, there are now characterized inhibitors of other tyrosine kinases, as well as antagonists of membrane and nuclear receptors, growth factors, oncogenes, signaling, and cell-cycle proteins, as well as apoptosis-related molecules. The success of target-driven therapies is illustrated by their introduction into routine clinical care protocols used to design cancer treatment; among these modalities are antagonists of androgen or estrogen receptors for prostate or breast cancer, respectively, as well as inhibitors of members of the EGFR family for different solid tumors. Importantly, the concept of target-driven therapy has been extended to the TME, including the development and FDA approval of angiogenesis inhibitors, as well as more complex immunotherapeutic modalities. However, considering the substantial relevance and tremendous clinical interest with respect to these two therapeutic approaches, we will focus on them separately in the next sections; here, the role of galectins in targeted therapies directed against cancer cells is highlighted.

Whereas targeted strategies are typically initially successful in promoting disease control, a significant number of patients with cancer develop resistance and ultimately undergo relapse. The molecular mechanisms underlying development of resistance to target-driven therapies can include mutations or amplifications of the target protein that result in drug resistance. Interestingly, these mechanisms are similar to those described for conventional chemotherapy and radiotherapy, and include alterations in the MDR transporters and/or cell death pathways (94). Several galectin family members have been identified as mediators of resistance to targeted anticancer therapies (Table 2; Fig. 2). Illustrating this concept, one recent report revealed that Gal1 confers imatinib resistance in CML by modulating MDR1 expression via activation of p38 MAPK and NF-κB translocation (95). A proteomic analysis using HCC cell lines, identified Gal1 as a key mediator that conferred resistance to the tyrosine kinase inhibitor, sorafenib, which targets VEGFR, platelet-derived growth factor receptor (PDGFR), and the RAF kinases c-Raf and B-Raf (26). Importantly, these data were validated in patients diagnosed with HCC. In these patients, high serum levels of Gal1 correlated with poor responses to sorafenib; these findings highlight the potential value of this protein for stratifying patients for personalized therapeutic approaches (26). Similarly, recent studies revealed that vemurafenib-mediated inhibition of BRAF led to an increase in Gal1 expression in human melanoma cells (96).

A number of studies have served to elucidate the molecular mechanisms underlying Gal3-driven resistance with respect to targeted therapies (Fig. 2). Using in vitro approaches and in vivo xenografts, it was noted that Gal3 promotes PSA transcriptional activity, thereby increasing expression of target genes associated with the androgen receptor (AR); these include transmembrane protease serine 2 (TMPRSS2) and kallikrein-related peptidase 3 (KLK3), which serve to block the effects of anti-AR drugs, MDV3100 and bicalutamide, at their targets (97). Interestingly, increased Gal1 expression has been identified in AR antagonist–resistant prostate cancer (98), although the functional relevance of this finding remains uncertain. Similar to Gal1, Gal3 has also been linked to resistance to tyrosine kinase inhibitors. In fact, high levels of Gal3 serve to activate AKT and ERK1/2 in CML cell lines; this is associated with accumulation of Mcl-1 and drug resistance because of impaired apoptosis (43). In addition, diminished levels of Gal3 identified in esophageal squamous cell carcinoma cells, both in vitro and in vivo, impair EGFR-mediated endocytosis and as such function synergistically with anti-EGFR therapy (gefitinib; ref. 99). However, it is critical to recognize that these observations were context dependent; the opposite effect was observed in colorectal cancer cell lines, as Gal3 was overexpressed in gefitinib-responsive as opposed to -resistant cell lines (100). Likewise, Gal3 blocked the autophagy process in melanoma cells, which sensitized them to anti-B-Raf inhibition with vemurafenib. Cells with low levels of Gal3 expression were resistant to this treatment and responded with increased levels of superoxide production and induction of the endoplasmic reticulum stress response (101). Furthermore, given the importance of Gal3 as an antiapoptotic protein, it has been proposed to play a key role in undermining anti-Bcl-2 therapy in clinical trials, thereby suggesting the need for new combination regimens that target both Gal3 and Bcl-2 (102). In cases of multiple myeloma and lymphoma, administration of the Gal3 inhibitor, GCS-100, induced apoptosis in cells that were resistant to chemotherapy, as well as to the proteasome inhibitor, bortezomib, via activation of caspase-3, caspase-8, and PARP cleavage. Indeed, combination of GCS-100 and PK-11195, the latter is a molecule that targets the peripheral benzodiazepine receptor in the mitochondria and as such, the apoptosis intrinsic pathway, results in synergistic activity against multiple myeloma (103). In the same line, Gal3 may mediate resistance to apoptosis induced by TRAIL, in studies that included breast (104), bladder (71), and colon cancer (105). The molecular mechanisms underlying resistance have not been fully elucidated, although in bladder cancer, Gal3 may induce resistance to TRAIL-induced apoptosis via the activation of AKT (71). Interestingly, Gal3 anchors death receptors in glycan nanoclusters in metastatic colon cancer cells, thereby inhibiting trafficking, and thus apoptosis (105). Moreover, in breast cancer, the His64/Pro64 polymorphism associated with Gal3 serves to regulate sensitivity to TRAIL; this finding underscores the potential of this lectin with respect to stratifying patients for personalized treatment (104).

However, similar to chemotherapy and radiotherapy, we have only limited information on the role of other galectins and their capacity to promote resistance to targeted therapies (Fig. 2). For example, in prostate cancer, c-Myc upregulates the expression of several glycosyltransferases, thus priming for Gal4 binding and inducing HER2 tyrosine kinase activation and cell stemness via modulation of Sox9; this effect leads to castration resistance (106). Moreover, target-driven therapy with the Ras inhibitor, salirasib, in malignant peripheral nerve sheath tumors, induced a shift in galectin expression; Gal7 expression was upregulated and Gal1 was downregulated, thereby counteracting Ras activation and sensitizing cells to apoptosis (61). Finally, expression of Gal9 resulted in increased sensitivity to imatinib in cases of CML; Gal9 was capable of inducing apoptosis via activation of transcription factor 3 (ATF3) and upregulation of the proapoptotic effector Noxa (107).

Taken together, we conclude that galectins play relevant roles in modulating the responses to target-driven therapies for cancer. These findings highlight the importance of delineating the galectin signature associated with each distinct tumor type to facilitate successful design of personalized targeted anticancer therapies.

Antiangiogenic therapies are designed to target abnormal tumor vascularization (108). However, secondary beneficial effects of antiangiogenic therapies include sensitization to other anticancer modalities including radiotherapy, chemotherapy, and immunotherapy. Thus, mechanisms of resistance to antiangiogenic therapies might also compromise other modalities of anticancer treatment (109). Antiangiogenesis was originally conceived as monotherapy to promote nutrient and oxygen starvation; however, the clinical benefits of this strategy have largely appeared to involve combination with other therapeutic modalities (110). Mechanisms of resistance to antiangiogenic therapies include expression of alternative angiogenic factors in the TME, including FGF-2, placental growth factor, platelet-derived growth factor (PDGF), angiopoietin-2, and hepatocyte growth factor (111, 112).

Galectins, particularly Gal1, Gal3, Gal8, and Gal9, are all powerful stimulators of angiogenesis via their association with different EC surface receptors and their capacity to activate distinct signaling pathways and to regulate distinct events associated with the angiogenic cascade (Fig. 3; ref. 113). Gal1 binds to VEGFR2 and neuropilin-1 (NRP-1) via glycosylation-dependent mechanisms; it also modulates receptor segregation and endocytosis, leading to phosphorylation and signaling via the Raf/ERK1/2 and AKT pathways (81, 114–118). Consistent with these findings, targeting Gal1 expression resulted in suppression of aberrant angiogenesis and tumor growth in vivo in experiments featuring numerous tumor types, including melanoma (77, 115, 116), Kaposi sarcoma (81), prostate adenocarcinoma (119), lung adenocarcinoma (116), T-cell lymphoma (116), pancreatic adenocarcinoma (120), glioblastoma (121), and multiple myeloma (122). In contrast, Gal3 promotes vascularization via binding to α_vβ₃ integrin, modulation of VEGFR2 endocytosis, and activation of the Jagged-1–Notch axis (123–126); the latter mechanism is one of the molecular pathways that may promote resistance to anti-VEGF therapies. Moreover, Gal8 triggers EC signaling via its capacity to bind to activated leukocyte cell adhesion molecule (CD166; ref. 127), whereas different Gal9 isoforms play divergent roles, and can either stimulate or impair angiogenesis (128). Remarkably, Gal9 has been described as a biomarker correlating with positive outcomes in a number of human cancers; these findings highlight important therapeutic implications (10). Furthermore, Gal2 and Gal4 induce secretion of EC-derived cytokines and chemokines (G-CSF, IL6, MCP-1, and GROα), which in turn triggers additional EC signaling (129). Moreover, adipose tissue–derived Gal12 has demonstrated proangiogenic activity via its recognition of 3′-fucosylated ligands on ECs (130); these results suggest a role for this lectin in vascular–adipose tissue cross-talk. Whether adipose tissue–derived Gal12 influences tumor angiogenesis and progression remains to be elucidated. Interestingly, galectins also contribute to platelet-derived angiogenesis by inducing release of proangiogenic mediators including VEGF-A and endostatin (131). Thus, galectin–glycan interactions may function to fine-tune EC biology via their capacity to bind to a select repertoire of glycosylated receptors and by activating distinct signaling pathways (Fig. 3).

Mounting evidence indicates that specific interactions between Gal1 and complex N-glycans couple tumor hypoxia to vessel formation (81, 116). Hypoxia induced expression of Gal1 in different tumor types via HIF1α- (78) or NF-κB- (81) dependent mechanisms. Moreover, hypoxia favored a Gal1-permissive glycophenotype in ECs that was characterized by higher β1-6GlcNAc–branched N-glycans and poly-LacNAc structures and lower levels of α2,6-linked sialic acid compared with ECs exposed to normoxic conditions (116). Interestingly, Gal1-induced angiogenesis was found to be independent of canonical proangiogenic factors including VEGF, FGF2, oncostatin M, angiopoietin-like 4 (ANGPTL-4), and PDGF (76, 81, 116), whereas the Gal3 proangiogenic pathway was dependent on VEGF (Fig. 3; ref. 124). Remarkably, galectins can also regulate lymphangiogenesis, which is a critical process in tumor progression. In this regard, Gal8 has been identified as a key mediator linking the actions of podoplanin and VEGFR3 during pathologic lymphangiogenesis (132). Moreover, podoplanin-expressing macrophages promote lymphangiogenesis in breast cancer via interaction with Gal8 expressed on lymphatic ECs (LEC; ref. 133). Furthermore, genome-wide functional analysis identified Gal1 as a central regulator of LEC remodeling (134). Thus, galectins control both ECs and LECs programs; these findings highlight an additional obstacle that functions to limit the efficacy of anticancer therapy.

Interestingly, a glycosylation-based program mediated by Gal1 has been proposed as a mechanism underlying resistance associated with VEGF blockade (116). Gal1 fosters angiogenesis by promoting receptor segregation and retention on the surface of ECs via binding to nonsialylated N-glycans on VEGFR2. This glycosylation-based mechanism leads to phosphorylation and activation of VEGFR2, ERK1/2, and AKT, thereby mimicking the impact of VEGF-like signaling (116). Remarkably, anti-VEGF refractory tumors produced large amounts of Gal1 in response to hypoxia or VEGF blockade and their associated vasculature displayed glycosylation patterns that facilitated Gal1 binding, namely higher β1,6GlcNAc branching, lower α2,6-linked sialic acid, and high levels of poly-LacNAc–extended glycans. In contrast, vessels associated with anti-VEGF–sensitive tumors exhibited higher levels of α2,6 sialylation in response to VEGF blockade, which prevented Gal1 binding and angiogenesis. Accordingly, loss of α2,6-sialylation in tumor-associated vessels reduced their sensitivity to anti-VEGF treatment and favored angiogenesis mediated by Gal1–receptor interactions. In contrast, the absence of β1-6 GlcNAc–branched N-glycans in ECs or silencing of tumor-derived Gal1 converted anti-VEGF–resistant into anti-VEGF–sensitive tumors (116). These findings emphasize the importance of glycosylation-dependent Gal1-driven programs as therapeutic targets that might be exploited to overcome anti-VEGF resistance. Supporting these findings, treatment of tumors with both bevacizumab and the antiangiogenic peptide, anginex, which is known to bind to Gal1, normalized tumor vessels, increased oxygenation, and improved therapeutic responses (135). Moreover, administration of OTX008, a synthetic compound that targets Gal1, potentiated the activity of the tyrosine kinase inhibitor, sunitinib, in a nude mouse tumor xenograft model (136). Interestingly, aflibercept, a chimeric VEGFR1/VEGFR2-based decoy receptor fused to the Fc fragment of IgG1, was capable of neutralizing Gal1 activity independently of VEGF-A (137). Taken together, these results support the use of Gal1-blocking agents to overcome resistance and to maximize the efficacy of conventional antiangiogenic therapies.

mAb-based therapies targeting the immune checkpoint molecules such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and/or programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) have yielded significant clinical benefits, including durable cancer regression and increased overall survival in patients with distinct malignancies; as a group these agents function by unleashing antitumor immunity (138). Whereas significant clinical responses have been observed in some patients, others maintain only short-term benefit as their tumors acquire adaptive resistance by engaging additional immunosuppressive pathways (138).

Galectins can reprogram innate and adaptive immune responses via glycan-dependent or -independent mechanisms (139, 140). There is compelling evidence supporting the concept that Gal1 is capable of thwarting immune responses in a variety of tumors including melanoma (141, 142), lung adenocarcinoma (116, 143, 144), neuroblastoma (145, 146), head and neck squamous carcinoma (HNSC; ref. 147), glioblastoma (148), Hodgkin lymphoma (149), breast adenocarcinomas (150), pancreatic adenocarcinoma (120, 151), ovary carcinoma (152), and neuroendocrine tumors (153). Likewise, Gal3 limits antitumor responses in models of pancreatic adenocarcinoma (154) and melanoma (155), whereas Gal9 is associated with tumor immunosuppression in acute myeloid leukemia (156), pancreatic adenocarcinoma (157), and melanoma (158). Mechanistically, these galectins promote tumor immunosuppression by engaging coinhibitory receptors, disrupting costimulatory pathways, and/or controlling activation, differentiation, and/or survival of immune cells (ref. 140; Fig. 3). Regarding T-cell activation, Gal1 acts by antagonizing T-cell receptor (TCR) signaling (159, 160). Likewise, Gal3 prevents clustering of TCR, CD4, and Lck into GM1-enriched membrane microdomains during T-cell activation (161). Interestingly, Gal3–N-glycan lattices can also promote T-cell anergy of human tumor-infiltrating lymphocytes (TIL) by interrupting the interactions between TCR and CD8 molecules (155). A deeper mechanistic analysis revealed that Gal3 limited the formation of a functional secretory synapse in CD8 TILs, thus preventing optimal lymphocyte-function-associated antigen-1 (LFA-1) triggering (162). Interestingly, treatment with galactomannan GM-CT-01 (Davanat) corrected T-cell dysfunction by interrupting Gal3 and Gal1 functions, whereas administration of 2-deoxy-glucose inhibited Gal3 binding to T cells (163, 164). Moreover, Gal3 counteracts formation of the immunologycal synapse by promoting TCR downmodulation through intracellular mechanisms involving interactions with alix, which is an adaptor endocytic protein (165); these results suggest that different immune inhibitory circuits triggered by these lectins may serve to counteract antitumor responses.

Interestingly, galectins also contribute to cancer-driven immunosuppression via direct engagement of immune checkpoint receptors, transmission of inhibitory signals, and prevention of receptor endocytosis via glycosylation-dependent mechanisms (Fig. 3). In this regard, Gal3 promotes N-glycan–dependent retention of CTLA-4 at the surface of T cells, thus amplifying inhibitory signals transmitted by this immune checkpoint receptor (166). Moreover, Gal3 also impairs antitumor immunity by engaging LAG-3 on the surface of CD8 T cells, whereas Gal9 binds to TIM-3 leading to T-cell exhaustion (154, 167). The later inhibitory activity is counteracted by HLA-B–associated transcript 3 (Bat3; ref. 167). Thus, association of galectins with other immune checkpoint pathways (140, 168) reveal novel therapeutic targets that could be blocked using specific mAbs or glycan-related inhibitors.

Galectin–glycan interactions may also control T-cell viability. Gal1 has been proposed to trigger T-cell apoptosis by engaging a selected repertoire of glycosylated receptors including CD45 and CD43 (169). Interestingly, Gal1 preferentially eliminates terminally differentiated Th1 and Th17 cells that exhibit a repertoire of glycans required for Gal1 binding; in contrast, Th2 cells are protected from Gal1-induced death via α2,6 sialylation of surface glycoproteins (170). Likewise, Gal9 deletes Th1, Th17, and CD8 T cells via binding to TIM-3 (171, 172). Thus, galectins reprogram antitumor T cells through modulation of their activation, signaling, and survival.

Galectins may also control the fate of Foxp3⁺ and Foxp3⁻ T regulatory cells (Treg) within the TME. In fact, Gal1 contributes to differentiation and immunosuppressive activity of Tregs in a breast cancer model (150). Moreover, Gal1 also licenses T cells with a Foxp3⁻ regulatory signature characterized by high levels of IL10 and IL21 expression and modulation of the c-Maf/Aryl hydrocarbon receptor pathway (142). Furthermore, Gal1 licenses γδ T cells with immunosuppressive activity within the ovary TME, which impairs antitumor immunity and accelerates malignant progression (152). Moreover, Gal9 acts synergistically with TGFβ₁ to promote differentiation of inducible Tregs via interaction with CD44 and TGFβRI (173). Likewise, Gal8 promotes Treg differentiation by modulating TGFβ₁ and IL2 signaling (174).

Galectins can also reprogram the myeloid cell compartment within the TME. For example, Gal1 can induce differentiation of IL27⁺ IL10⁺ pSTAT3⁺ tolerogenic dendritic cells (DC; ref. 175). Interestingly, this lectin can also inhibit transendothelial migration of inflammatory, but not tolerogenic, DCs through mechanisms involving differential O-glycosylation of CD43 (176). Notably, tumor-driven expression of the transcription factor, AT-rich sequence-binding protein 1 (Satb1), provides inflammatory DCs with immunosuppressive potential via Gal1-mediated mechanisms (177). In addition, Gal1 can also polarize macrophages toward an anti-inflammatory M2 phenotype (44, 178). Gal3–N-glycan lattices can also enhance the immunosuppressive activity of macrophages by augmenting surface residency of TGFβR and prolonging delivery of TGFβ-dependent inhibitory signals (179). Finally, Gal9 favors expansion of CD11b⁺Ly-6G⁺ myeloid-derived suppressor cells (MDSC) and inhibits antitumor responses through TIM-3–dependent mechanisms (180). Thus, galectins may shape the myeloid cell compartment by inducing tolerogenic DCs, polarizing macrophages toward an M2 phenotype, and promoting expansion of MDSCs (Fig. 3).

On the basis of their immunosuppressive activities within the TME, galectins have shown the capacity to modulate responses to immunotherapy. Gal1 siRNA–loaded chitosan nanoparticles can knockdown expression of Gal1; this manipulation sensitized glioblastoma tumors to PD-1 blockade (44). Moreover, in HNSC models, Gal1 conferred resistance to anti-PD-1 treatment by promoting an immunosuppressive tumor endothelium, which favored T-cell exclusion by expressing high levels of PD-L1 and Gal9. Targeted disruption of Gal1 enhanced T-cell infiltration into tumor tissue and augmented responses to anti-PD-1 therapy either in the absence or presence of radiotherapy (147). In-line with this evidence, Gal1 blockade enhanced T-cell influx induced by anti-VEGF mAb in models of melanoma and lung adenocarcinoma (116). Moreover, in non-Hodgkin lymphoma models, Gal1 expression conferred intrinsic resistance to anti-CD20 therapy by modulating antibody-mediated phagocytosis (181). Furthermore, Gal1 expression levels were associated with resistance to immunotherapy via its capacity to impair the efficacy of a bispecific mAb targeting epithelial cellular adhesion molecule and CD3 in an HCC tumor model (182). Furthermore, in a cohort of patients with non-small cell lung cancer, lymphoid cells expressing TIM-3 and monocytic MDSCs expressing Gal9 have been implicated in resistance to PD-1 blockade (183). Interestingly, Gal9 has also been shown to serve as a ligand for Dectin-1, a C-type lectin receptor in macrophages, favoring their reprogramming in pancreatic ductal adenocarcinoma. Targeting Gal9 or Dectin-1 synergized with PD-1 blockade to increase T-cell activation and revert T-cell exhaustion programs (157). Moreover, Gal9 was upregulated in pancreatic tumor cells resistant to tMUC1-specific chimeric antigen receptor T-cell treatment (184). Finally, higher levels of circulating anti-Gal3 antibodies have been found in patients with melanoma treated with a combination of anti-CTLA-4 (ipilimumab) and anti-VEGF (bevacizumab) mAbs (185), suggesting a role for this lectin in cross-resistance mechanisms. Furthermore, high levels of Gal3 in sera of pretreated patients correlated with poor response to anti-PD-1 therapy (185). Thus, targeting galectins may enhance the overall therapeutic efficacy of different immunostimulatory modalities.

Galectins are multifunctional mediators of tumor progression that are capable of influencing all hallmarks of cancer via both glycan-dependent and -independent mechanisms (11). These proteins may trigger regulatory circuits that modulate sensitivity to different anticancer modalities, including chemotherapy, radiotherapy, targeted therapy, antiangiogenic therapy, and immunotherapy. In this review, we integrate our current understanding of the distinct functions of galectins during tumor progression and discuss their roles in modulating sensitivity to anticancer treatment. Although we discussed the functions of each of these proteins separately with respect to each specific treatment, the mechanisms may converge when two or more therapeutic approaches are combined. For example, induction of Gal1 after radiotherapy may promote tumor immune evasion (83); this observation might help to explain the failure of combined immunotherapy and radiotherapy that has been reported for several tumors. Moreover, overexpression of Gal1 in response to hypoxia may play a role in promoting resistance to radiotherapy and may also mediate cross-resistance observed in response to combined antiangiogenic and immunotherapeutic approaches (186, 187). In the same line, the recently identified role of galectins, particularly Gal1 and Gal3, in supporting cancer stemness in lung, ovary, colorectal, and breast cancer (48, 188–191) may serve as a common mechanism of resistance shared by different cancer therapies, as cancer stem cells exhibit an overall low sensitivity to antitumor drugs, thus limiting the efficacy anticancer therapies and promoting disease recurrence (192).

However, before galectin blockade can be embraced as a rational strategy and direction to circumvent resistance to anticancer therapies, some obstacles need to be overcome. First, most mechanistic studies have been performed in vitro using human cell lines or animal models; there is only limited information available from studies performed in human patients with cancer (25, 31, 37, 52, 106). In this regard, several reports have revealed associations between Gal1, Gal3, Gal7, and Gal9 and responses to therapy in patients with different types of cancer (26, 27, 29, 42, 48, 57–59, 65, 88, 96, 183, 185); these results underscore several novel strategies that might be undertaken with respect to elucidating a role for galectins in promoting sensitivity to treatment and likewise, the use galectin inhibitors in combinatorial modalities. Second, a critical aspect that deserves further attention is the analysis of intrinsic versus acquired resistance mechanisms operating in response to galectin blockade. When viewed simplistically, resistance to cancer therapy can be divided into intrinsic resistance (preexisting in the tumor before treatment) and acquired resistance (induced by drug treatment); however, the true scenario in the case of galectins is much more complex, as both types of resistance can evolve together during tumor development and progression. Illustrating this concept, Gal1 and Gal3 are frequently overexpressed in tumors even before treatment (10, 147, 181, 182), suggesting that they participate in intrinsic resistance; however, both galectins can be upregulated in response to treatment, including chemotherapy (21, 41, 42), radiotherapy (86–89, 91), targeted therapy (96), and antiangiogenic therapy (121, 126). Likewise, there are reports that suggest that Gal9 can induce both intrinsic and adaptive resistance to immunotherapy in both lung and pancreatic cancer (183, 184). Another fundamental question with respect to galectin-driven resistance to cancer therapy relies on the role of glycosylation and its impact on galectin-mediated ligand recognition. Evidence for altered glycosylation and galectin-mediated resistance to target-driven therapies is illustrated by Gal4 induction associated with castration resistance in prostate cancer. In fact, O-glycosylation regulated by the oncoprotein, c-Myc, primed prostate cancer cells to Gal4 binding, which promoted castration resistance and metastasis (106). Changes in glycosylation associated with malignant transformation have also been linked to apoptosis and chemotherapy resistance via Gal1- or Gal3-mediated mechanisms (37, 193, 194); these findings emphasize the associations between aberrant glycosylation, regulation of galectin binding, and resistance to anticancer therapies. Thus, targeting galectins using specific mAbs (116, 157), glycan inhibitors (155, 195), allosteric peptide antagonists such as anginex, 6DBF7, and OTX008 (196–198), or DNA aptamers (144), represents not only a possible direct therapeutic strategy (6), but it may also help overcome resistance to different anticancer therapies, thereby increasing the number of patients who will benefit from the entire range of treatments.

Similar to what has been observed for other immunotherapeutic strategies, adverse effects might arise in response to galectin blockade, including the development of autoimmune manifestations and chronic inflammatory conditions. As one example of this concept, we recently observed age-dependent development of spontaneous autoimmunity in Gal1-deficient (Lgals1^−/−) mice, which presented a Sjögren-like inflammatory disease (160). Notably, this phenotype is similar to aging associated manifestations in mice lacking PD-L1/PD-1 (199). In this sense, immune checkpoint blockade therapies have led the way with respect to the study and ongoing follow-up of immune adverse events (200). Last but not least, intracellular versus extracellular functions of galectins is another important aspect to keep in mind to understand the role of these proteins in anticancer therapy resistance, as well as in the design of galectin-based cancer treatments. Future studies might be aimed at dissecting the precise mechanisms underlying galectin-driven resistance pathways, analyzing the potential role of these lectins with respect to the generation of cross-resistance, and examining their predictive and prognostic values as potential biomarkers of treatment responses.

G.A. Rabinovich has a patent issued for Methods for Modulating Angiogenesis of Cancers Refractory to Anti-VEGF Treatment (publication number: 20200016266). No potential conflicts of interest were disclosed by the other authors.

The work in Navarro's laboratory was supported by grants from the Spanish Ministry of Economy and Competitiveness/ISCIII-FEDER (PI17/00199) and the Generalitat de Catalunya (2017-SGR-225). The work in Rabinovich's laboratory was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT 2017-0494), Fundación Sales, Fundación Bunge & Born, and Richard Lounsbery Foundation. We thank Helene F. Rosenberg for critical reading of the article and Albert Flotats for support with graphic design. We apologize to authors whose articles could not be cited because of space limitations.