Prognostic and diagnostic significance of galectins in pancreatic cancer: a systematic review and meta-analysis

Pancreatic cancer is among the most aggressive malignancies due to limited early diagnosis and therapeutic strategies, ranking as the fourth leading cause of cancer-related deaths [1, 2]. Despite enormous advances in diagnostic and therapeutic strategies made in many other tumours over the past decades, the prognosis of pancreatic cancer remains unsatisfactory, with a 5-year survival rate of approximately 7% [3, 4]. Most patients with pancreatic cancer are diagnosed at advanced stages, and a lack of effective biomarkers for identifying patients with a high risk for recurrence contributes to poor prognosis.

Galectins constitute a family of multifunctional proteins that share similar β-galactoside-binding affinity and carbohydrate-recognition domains (CRDs) [5]. Galectins are divided into three subtypes (prototype, chimeric and tandem-repeat) based on their structural differences [6]. Prototype galectins (galectin-1, 2, 5, 7, 10, 11, 13, 14 and 15) contain one CRD that can homodimerize. Tandem repeat galectins (galectin-4, 6, 8, 9 and 12) consist of two CRDs that are connected by a linker up to 70 amino acids in length. Chimeric galectin (galectin-3) consists of a single CRD fused to unusual tandem repeats of proline- and glycine-rich short stretches (a total of approximately 120 amino acids) [7]. Eleven galectins have been identified in humans, among which, galectin-1, 3 and 9 have been the most widely studied across different fields [8]. Galectins can be localized both inside and outside of cells. Secreted galectins can crosslink with cell-surface glycoconjugates covered with galactose-containing oligosaccharides to induce intracellular signaling, including mitosis, apoptosis and cell-cycle progression. Intracellular galectins shuttle between the cytoplasm and nucleus to participate in processes such as pre-mRNA splicing [7]. In this review, we will discuss four different galectins. Galectin-1 (LGALS1, 22q13.1) belongs to the prototype galectins, and is usually released from stromal cells and endothelial cells [9]. Galectin-3 (LGALS3, 14q22.3) is the only galectin that belongs to the chimeric galectin, and was first described to be localized to the outer membrane of macrophages (Mac-2 antigen) [10]. It is also found in endothelial cells, immune cells and fibroblasts, and can be transported from the nucleus to the cytoplasm to interact with mitochondria and regulate apoptosis [11, 12]. Galectin-4 (LGALS4, 19q13.2) and galectin-9 (LGALS9, 17q11.2) belong to the tandem repeat type galectins and are involved in inflammatory and immune processes [13, 14]. Recent studies have elucidated the biological functions of galectins in tumors, including in the regulation of oncogenesis, cancer cell growth, apoptosis, cell adhesion, migration and immune escape [8].

Evidence has suggested that altered expression of galectins in pancreatic cancer tissues may have a role in tumour carcinogenesis, proliferation, progression, angiogenesis, metastasis and immune response [15‐18]. Recent studies have also raised the possibility of using galectins in diagnostic, prognostic and other clinical characteristics, such as tumour node metastasis (TNM) stage pathological grade [17, 19‐28]. Although many studies have concentrated on the correlation between galectins and pancreatic cancer, conclusions remain controversial. Therefore, we performed a comprehensive meta-analysis to clarify the diagnostic and prognostic role of galectins in pancreatic cancer.

Due to the lack of early diagnosis and the poor survival rate after surgery, efforts have been made to identify novel diagnostic and prognostic biomarkers in patients with pancreatic cancer. Carbohydrate antigen 19-9 (CA19-9) is the most widely used serum biomarker for detecting pancreatic cancer. The correlation between CA19-9 and surgical outcomes has been described in many studies and indicates that CA19-9 could assist in identifying resectable or nonresectable pancreatic cancer. However, the thresholds identified in each study were varied [39]. Our team previously conducted a series of relevant studies in this field [40‐42]. We found that the serum signature of CEA⁺/CA125⁺/CA19-9 > 1000 U/ml was a preoperative indicator for worse surgical outcome in pancreatic cancer, even though the R0-resection was successfully conducted. However, no consensus has been reached on how CA19-9 serum levels change and their predictive value in managing pancreatic cancer patients. A pooled analysis of 6 prospective trials indicated that baseline CA19-9 is prognostic in patients with advanced pancreatic cancer who underwent treatment with gemcitabine-containing regimens. However, reduced CA19-9 after the second cycle of chemotherapy is no longer predictive [43]. Therefore, multiple studies have been conducted to search for novel biomarkers that provide earlier or more accurate prediction for pancreatic cancer.

As part of the lectin superfamily, galectins are soluble proteins widely expressed in a variety of cells, exerting their primary biological functions both intracellularly and extracellularly [44]. In general, galectins are involved in diverse biological process, including regulation of cell signalling, progression of the cell cycle, apoptosis, pre-mRNA splicing and cell motility and adhesion [45, 46]. Consistent with these various biological functions, altered expression levels or dysfunction of galectins have been correlated with the development of diseases, such as cancer. Evidence has suggested the influence of galectins on different hallmarks of pancreatic cancer, including cell proliferation, invasion and migration, immune escape and angiogenesis [17, 47‐49]. As a result, therapeutic strategies have been developed that target galectins in pancreatic cancer. Yao et al. suggested that HH1-1, a novel galectin-3 inhibitor, inhibited the progression of pancreatic cancer both in vitro and in vivo [49]. Shih et al. found that combination treatment of paclitaxel and LLS2, a novel galectin-1 inhibitor, enhanced toxicity in human pancreatic cancer cell lines [50]. However, for clinical applications, most clinical trials on galectin inhibitors combined with chemotherapy for the treatment of different tumors were neither withdrawn nor terminated, and only one trial has completed although no results are currently available (NCT00054977). Thus, the use of galectin inhibitors for the treatment of cancer has received renewed interest. Recently, a novel biomarker on the galectin-9 binding partner, T cell immunoglobulin mucin-3 (TIM-3), was found to be upregulated in response to anti-PD-1 therapy and has been targeted as a novel immune checkpoint in tumor immunotherapy [51]. Although these studies are still in their early stages, anti-TIM-3 agents (e.g., TSR-022, LY3321367) combining anti-PD-1/PD-L1 have been applied in several ongoing clinical trials (NCT02817633; NCT03099109), highlighting their promising role for the treatment of advanced solid tumors.

Many studies have also explored the prognostic role of galectins in cancer. Galectins are thought to be associated with patient outcome. Increasing evidence has suggested that galectin-1 is elevated in cancer tissues and a high expression level of galectin-1 is associated with poor OS and DFS in different cancer types, particularly in digestive cancers [19, 52]. Galectin-1 has also been proved to play oncogenic role by some researches in pancreatic cancer. Galectin-1 could regulate acinar-to-ductal metaplasia by promoting Hedgehog pathway signaling in PDAC cells and tumor-stroma crosstalk [16]. Galectin-1 also reportedly enhances the production of stromal cell-derived factor-1via NF-κB signalling, resulting in increased metastasis in pancreatic cancer both in vitro and in vivo [53]. In addition, a novel galectin-1 inhibitor LLS2 was found to potentiate the antitumor effects of paclitaxel in several human cancer cell lines including pancreatic cancer cells in vitro [50]. Consistent with the oncogenic role in pancreatic carcinogenesis, our results indicated that in patients with pancreatic cancer, high levels of galectin-1 were significantly correlated with poor OS, as complements to some previous meta-analysis in solid tumors, demonstrating that higher expression of galectin-1 was associated with worse prognosis in cancers [52], though more excellently-designed large-sized prospective researches are needed in the future.

However, another subtype of galectins, tandem-repeat galectins, seemed to exhibit the opposite picture in prognostic value. Although only limited tumor types were evaluated, higher galectin-9 expression was reported in a meta-analysis to be related to better prognosis in solid tumors especially in digestive cancers [54]. Due to the limited number of studies about galectin-4 and galectin-9, their biological behaviours and underlying mechanisms in malignancies still remain controversy. Galectin-9 was found to suppress the proliferation of pancreatic cancer cell lines, and metastatic liver cancer cell lines [55, 56]. Conversely, higher serum galectin-9 was observed in PDAC patients [57]. Inhibition of galectin-9 leads to significant tumour regression in a mouse model. Mechanistically, dectin-1 binds to galectin-9, resulting in immunogenic or tolerogenic phenotypes of CD4 + and CD8 + T cells that promote tumour progression in pancreatic cancer [58]. Though only a small number of studies have been performed, low expression of galactin-4 has been described to be significantly correlated with early recurrence and poor survival of pancreatic cancer [27]. Reduced expression of galectin-4 was also described in colorectal cancer, skin cancer and prostate cancer [59‐62]. Our meta-analysis supported the various function of different galectin subtypes in cancer prognosis, that converse to galectin-1, high levels of galectin-4 or galectin-9 predicted better OS and DFS in pancreatic cancer.

The prognostic role of galectin-3 has been widely studied, but appears to be unclear and disparate between different cancer types. For example, although galectin-3 has been proven to be related to poor survival and play an oncogenic role in many types of cancer, such as ovarian, colorectal, and non-small cell lung cancer [63], high expression of galectin-3 appears to better predict survival in patients with gastric cancer [64]. Alterations in galectin-3 expression have also been reported in previous studies wherein it is implicated in cell proliferation, apoptosis, adhesion, and angiogenesis [65]. Therefore, substantial studies have explored its role in pancreatic cancer, and most studies proved evidence to support its oncogenic role. Overexpressed galectin-3 in pancreatic cancer cells induced cell proliferation and invasion by activating Ras signaling [15]. Silencing of galectin-3 decreased pancreatic cancer cell proliferation and cyclin-D1 levels [66]. Zhao et al. found that inhibition of galectin-3 resulted in smaller tumour size and fewer metastases in a co-implanted murine model of pancreatic cancer cells and pancreatic stellate cells (PSCs). Galectin-3 activates the integrin subunit beta 1 on PSCs, resulting in activated NF-κB through integrin-linked kinase, which influences the transcription of interleukin-8 [67]. The controversial role of galectin-3 in prognosis has been addressed in numerous studies, our present study indicated that galectin-3 showed limited prognostic value, with no direct correlation to OS and clinical characteristics in pancreatic cancer.

One reason for the conflict between the oncogenic mechanism and clinical features may be due to the function of galectins likely being dynamic during tumour progression, which is one part of the balance in the tumour microenvironment. Another reason could be that galectins might only play an important role in a certain group of patients, while a large population covering the small group could potentially lead to negative results. Differences in methodologies among these studies may have caused these controversial results; thus, standardization of the evaluation methods used for galectin expression and proper cut-off points are urgently needed. A consensus needs to be reached on research design and analysis of results in future studies, especially for other types of galectins that are less well studied. These findings provide hints for reconsidering the efficacy of galectin-targeting strategies, and identification of a specific population sensitive to galectins should be performed.

Although novel markers are being evaluated for more accurate prediction, a systematic review on serum tumor markers for the detection of recurrent pancreatic cancer reported that although the biomarker CA 19-9 has certain limitations, it remains the most widely used serum biomarker for postoperative surveillance of pancreatic cancer with a sensitivity and specificity 0.73 and 0.83, respectively [68]. Though galectins alone may not be an effective independent prognostic biomarker for pancreatic cancer compared to the performance of traditional clinical biomarkers such as CA19-9 and carcinoembryonic antigen (CEA) [69], the strategy to combine galectins with other biomarkers is worth consideration. A recent study has shown that the overexpression of galectin-3 and ezrin had stronger predictive value than either alone in cervical cancer [70]. In addition, another study in non-small cell lung cancer observed higher expression of cyclin D1 in galectin-3 free tumor tissues [71]. Similarly, to avoid the limitations of a single predictor, galectin-9 was included in a compelling immune biomarker panel to predict cancer-specific survival in pancreatic cancer, which might also benefit future prospective immunotherapy trials [28]. These results suggest that the potential role of galectins in predicting survival outcomes in cancer patients should not be underestimated and that the combination of biomarkers might be a more powerful prognostic tool.

The search for effective diagnostic serum markers of pancreatic cancer remains intense due to its relatively simple and noninvasive features. Various serum markers with potential diagnostic value have been widely investigated, particularly CA19-9, CA125 and CEA [41, 72]. CA 19-9 is still the most widely used diagnostic marker for pancreatic cancer due to its relatively high diagnostic accuracy, with an AUC reaching nearly 87% [73‐75]. However, CA 19-9 is also elevated in some other conditions including other types of cancers as well as nonmalignant pathologies such as pancreatitis and cirrhosis. This has limited the sensitivity and specificity of CA19-9 for early detection. Furthermore, approximately 5–10% patients do not express CA19-9 [39]. Therefore, many studies have focused on the development of novel diagnostic panels to improve diagnostic accuracy based on this marker. Increased levels of circulating galectins have been reported in pancreatic cancer and some other cancer types, which has generated interest in galectins as potential diagnostic markers [76]. Galectin-1 and galectin-3 are intriguing markers for oral squamous cell carcinoma for the screening of higher risk populations [77]. The serum level of galectin-3 could assist as a diagnostic marker in bladder cancer patients [78]. With respect to diagnostic value in pancreatic cancer, our meta-analysis suggested that the pooled DOR of galectin-3 was 5.93, but the 95% CI was 0.96–36.72, indicating an unsatisfactory diagnostic accuracy and substantial heterogeneity. The most probable reason for the heterogeneous diagnostic performance of galectin-3 may be inconsistency in the control groups (healthy volunteer or pancreatitis patients). Some studies have proposed elevated circulating expression of galectin-3 as a potential biomarker for pancreatic cancer, and combined determination of galectin-3, CA19-9, and CA125 provided complementary diagnostic value for pancreatic cancer with a diagnostic sensitivity of 97.5% [22, 79]. However, those studies included a control group of healthy people and ignored patients with pancreatic or liver fibrosis despite the role of galectins in inflammation and collagen production [65]. Another study focused on the diagnostic value of galectin-3 in inflammatory pancreatic disease and indicated that galectin-3 is not an interesting biomarker for the detection of pancreatic adenocarcinoma [23]. Similarly, no significant difference in galectin-3 was observed between cirrhotic and hepatocellular carcinoma patients [80]. Therefore, although elevated galectin-3 has been observed in pancreatic cancer patients, galectin-3 alone might not be a viable diagnostic marker of pancreatic cancer due to its role in inflammatory diseases. Notably recent studies have suggested that the ligands of galectin-3 demonstrated relatively good performance for the diagnosis of cancer [81, 82]. Thus, adding galectin-binding glycoproteins in a galectin-based diagnostic panel might provide a strategy to improve the diagnostic performance of galectins. Hence, these results indicate the potential clinical diagnostic value of galectin-3, although more well-designed studies are needed to reach a definitive conclusion. Combination strategies are worthy of further exploration to improve the diagnostic capability of galectin-3.

Several limitations should be addressed for this meta-analysis. First, this meta-analysis includes a relatively small amount of studies with limited patients, which may have led to insufficient statistical power for analysing the diagnostic and prognostic role of galectins in pancreatic cancer. Second, given the lack of a standard cut-off value for galectins, different cut-off points were applied in the different included studies. Third, some of the HRs with 95% CIs were estimated by data extraction from the survival curves, which might convey certain statistical deviations. Fourth, we found that the different galectins, sample sizes, patient characteristics and cut-off values of the included studies might be potential sources of heterogeneity through subgroup analysis. Fifth, a potential publication bias and flawed methodologic design exists in the smaller studies included in the prognostic analysis. Finally, considering the limitations of the present study, additional well-designed studies with larger sample sizes need to be conducted.

Our current research describes the first meta-analysis to comprehensively and systematically address the prognostic and diagnostic role of galectins in patients with pancreatic cancer. Our meta-analysis found that high expression galectin-1 and a low level of galectin-4 or galectin-9 were associated with worse prognosis, while galectin-3 expression did not show a correlation with prognosis and other clinical characteristics in pancreatic cancer patients. Although galectin-3 exhibited some diagnostic value in patients with pancreatic cancer in this meta-analysis, clinical application prospects remain to be validated.