Nuclear Galectin-1 promotes KRAS-dependent activation of pancreatic cancer stellate cells

Significance

Our findings provide insights into the biology of pancreatic stellate cell (PSC) activation in pancreatic ductal adenocarcinoma (PDAC), a leading cause of cancer mortality. By identifying a nuclear role for Galectin-1 (Gal1) in regulating the transcription of oncogenic genes such as KRAS, we uncover a critical mechanism driving PSC activation and the secretion of protumorigenic factors. Given the central role of tumor-stroma crosstalk in PDAC progression and its contribution to therapy resistance, these results offer a unique perspective on the functional dynamics of the tumor microenvironment. Targeting Gal1 presents a promising therapeutic strategy to reprogram tumor-associated fibroblasts into a more quiescent phenotype, potentially mitigating their protumorigenic effects and improving patients’ outcomes.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, primarily due to its complex tumor microenvironment (TME), which drives both disease progression and therapy resistance. Understanding the molecular mechanisms governing TME dynamics is essential for developing new treatment strategies for this devastating disease. In this study, we uncover an oncogenic role for Galectin-1 (Gal1), a glycan-binding protein abundantly expressed by activated pancreatic stellate cells (PSCs), a key component of the PDAC TME that orchestrates tumor progression. Our findings reveal that Gal1 expression is elevated in the nucleus of human PSCs in both tissue samples and cultured cell lines. Using chromatin immunoprecipitation followed by sequencing analysis (ChIP-seq), we identify Gal1 occupancy at the promoters of several cancer-associated genes, including KRAS, a pivotal oncogene involved in PDAC pathogenesis. We demonstrate that Gal1 binds to the KRAS promoter, sustaining KRAS expression in PSCs, which, in turn, maintains PSC activation and promotes the secretion of protumorigenic cytokines. Mechanistically, Gal1 is required to preserve histone H3 lysine 4 monomethylation levels and to recruit the histone methyltransferase MLL1 to target promoters. Collectively, our findings define a nuclear function of Gal1 in modulating the transcriptional landscape of cancer-associated genes in PSCs within the PDAC TME, mediated through an epigenetic mechanism. These insights enhance our understanding of PDAC pathology and open potential avenues for therapeutic interventions targeting intracellular Gal1.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer, accounting for over 90% of cases. With a five-year relative survival rate of 13% (1), PDAC ranks as the third leading cause of cancer-related deaths and is projected to become the second by 2040 (2). One of the major hallmarks of PDAC is the presence of a dense fibrotic tumor microenvironment (TME), which comprises more than 80% of the tumor mass (3). This aggressive TME is, in part, driven by pancreatic stellate cells (PSCs), the primary cancer-associated fibroblasts in PDAC (4, 5). Activation of PSCs is known to promote PDAC progression, tumor aggressiveness, and resistance to therapies (6). While these biological roles are well documented, the mechanisms underlying PSC activation and their contribution to the aggressive nature of PDAC’s TME remain only partially understood.

In this study, we uncover insights into the role of intracellular Galectin-1 (Gal1) in shaping the biology of PSCs within PDAC. Gal1 belongs to a class of lectins defined by the presence of a highly conserved carbohydrate recognition domain (7) responsible for their affinity for β-galactosides (8). Extracellularly, Gal1 typically functions as a homodimer, enabling the formation of cell surface lattices (9), that mediate homo- and heterotypic cell interactions, cell–extracellular matrix (ECM) binding, as well as interactions between ECM components (10–12), therefore modifying cell adhesion and motility, as well as ECM assembly and remodeling (13, 14). In addition, Gal1 recognition of specific cell-surface proteins can trigger intracellular signaling pathways regulating cell proliferation (15) and differentiation (16). Intracellularly, Gal1 has been detected in both the cytoplasm and nucleus, shuttling between these two compartments (10, 17). In the cytoplasm, Gal1 interacts with HRAS to promote malignant transformation (18), and with protocadherin-24 to retain β-catenin at the plasma membrane, thereby inhibiting cancer cell proliferation (19). While the extracellular and cytoplasmic functions of Gal1 have been well characterized (10, 20), its nuclear role remains largely unexplored, with its only known nuclear function being as a pre-mRNA splicing factor (21). Interestingly, Gal1 is highly expressed in PSCs within PDAC TME, where it plays a pivotal role in tumor-stroma crosstalk by promoting stroma activation, tumor angiogenesis, and immune evasion (22–25). In this study, we show that Gal1 is highly expressed in the nucleus of PSCs, where it regulates gene expression and controls the oncogenic functions of these cells. Together, our findings provide mechanistic insights into the regulation of the TME, laying the groundwork for potential therapeutic strategies targeting PDAC.

Results

Gal1 Is Highly Expressed in PSC Nuclei.

We previously characterized Gal1 expression in pancreatic healthy tissue, pancreatic precursor lesions, and PDAC and found that it was highly expressed in the stromal compartment (26). Using immunohistochemistry on human PDAC tissue samples, we here confirmed that Gal1 is abundantly expressed particularly in tumor-associated PSCs, as identified by α-smooth muscle actin (α-SMA) staining on serial sections (Fig. 1A). Remarkably, Gal1 exhibited strong staining in the nucleus of PSCs (Fig. 1B, arrowheads), although a diffuse cytosolic pattern was also observed. The nuclear location of Gal1 was further confirmed in vitro using immunofluorescence staining of immortalized human PSCs (HPSC) (27), as well as nonimmortalized primary human and mouse PSCs (28) (Fig. 1C and SI Appendix, Fig. S1A). In addition, Western blot analysis revealed high levels of Gal1 in the chromatin-bound fraction of PSC extracts (Fig. 1D). These data demonstrate that Gal1 expression levels are elevated in the nucleus of human PSCs.

Fig. 1.

Galectin-1 is highly expressed in PSC nuclei. (A) Immunohistochemistry of Gal1 (a and b) and α-SMA (c and d) in human PDAC tissue samples. Gal1 was highly detected in tumor stroma. [Scale bars, 80 µm (a and c), and 40 µm (b and d).] (B) Immunohistochemistry of Gal1 in PDAC tissue samples, showing strong signal in PSCs nuclei (arrowheads). [Scale bars, 100 µm (a and b), and 25 µm (b and d).] (C) Immunofluorescence of Gal1 in human PSCs (HPSC) and tumor-derived primary cultured PSCs from human and mouse origin (hFib and mFib, respectively). Nuclei were stained with DAPI. Strong nuclear staining of Gal1 was found in the nucleus of HPSC (arrows). [Scale bars correspond to 50 μm (a–c, e–g, and i–k) and 20 μm (d, h, and l).] (D) Western blot showing the detection of Gal1 in different subcellular compartments in HPSC (cytoplasm, nucleoplasm, and chromatin-bound fractions). Tubulin and Histone H3 were used as controls of proper subcellular fractionation.

Gal1 Binds to Cancer-Related Genes’ Promoter Sequences.

Given Gal1’s nuclear presence, we speculated that it may interact with regulatory elements to modulate PSC gene expression. To test this hypothesis, we performed a chromatin immunoprecipitation followed by sequencing (ChIP-seq) in HPSC to map the genomic localization of Gal1. A total of 7,844 peaks were identified across the PSC genome in samples immunoprecipitated with anti-Gal1 antibody (Fig. 2A). Close to 16% of these peaks were located within promoter sequences, defined as regions between 3,000 base pairs (bp) upstream and 1,000 bp downstream of the transcription start site. These candidate Gal1-target promoter sequences correspond to a set of 1,230 genes (Dataset S1). Functional annotation analysis via DAVID (database for annotation, visualization and integrated discovery), identified several cancer-related pathways, including Rap1 signaling pathway, which governs cell–cell and cell–ECM interactions (29, 30); the Hippo signaling pathway, involved in cell growth and proliferation (31); and the WNT signaling pathway, which plays a major role in carcinogenesis, cell fate determination (32), proliferation and migration (Fig. 2B). Further analysis using the integrated genome browser (IGB) identified KRAS as a strong candidate target of Gal1 (Fig. 2C). We validated the binding of Gal1 to the KRAS promoter by ChIP-qPCR using two independent Gal1 antibodies (Fig. 2D). We observed more than two-fold enrichment of KRAS in α-Gal1 ChIP samples compared to control (IgG), confirming the binding of Gal1 to the KRAS promoter region.

Fig. 2.

Galectin-1 binds to the KRAS promoter and activates its expression in HPSC. (A) Distribution of detected Model-based analysis of ChIP-Seq (MACS) peaks along the human genome [Homo sapiens genome assembly GRCh38 (hg38) from Genome Reference Consortium]. (B) Analysis of the most representative pathways using the DAVID functional annotation tool. Only MACS peaks mapped in promoter regions were included in this analysis. Statistical significance was evaluated by the modified Fisher Exact, P-value < 0.05 (EASE score). (C) IGB MACS Gal1 peak mapped on the KRAS promoter region. (D) ChIP-qPCR validation for Gal1 occupancy at the KRAS promoter using two different anti-Gal1 antibodies (Left, Abcam #138513; Right, Santa Cruz #166618). Represented data were normalized relative to control (IgG Ctl). (E) Gal1 protein expression in HPSC after Gal1 downregulation using two siRNA-specific sequences (siGal1#1 and siGal1#2) or a nontargeting control (siCtl). Tubulin is shown as the normalization control. (F) Gal1 mRNA expression after downregulation using two different Gal1 siRNAs. (G) KRAS mRNA expression levels upon Gal1 downregulation in HPSC using two different Gal1 siRNA. Represented data were normalized relative to the nontarget control (siCtl) (F and G). (H) KRAS mRNA expression levels after transfection of HPSC with expression vector pCMV-Gal1 wild type (Gal1), mutant Gal1 N46D (Gal1-N46D), or empty pCMV vector (Ctl). Deviation is given as SEM of at least three independent experiments. ***P < 0.001; *P < 0.05.

To investigate the functional significance of Gal1 binding to the KRAS promoter, we knocked down Gal1 in HPSCs using siRNA strategies (SI Appendix, Fig. S1B) and assessed the effects on KRAS expression. Gal1 knockdown efficiency was confirmed at both the protein and RNA levels by WB and qPCR, respectively (Fig. 2 E and F). Notably, KRAS mRNA levels were significantly reduced in HPSCs following Gal1 knockdown compared to control cells (siCtl) (Fig. 2G). Finally, to demonstrate that these effects were mediated by nuclear Gal1, we transfected HPSCs with a mutant Gal1 N46D, which enhances the nuclear localization of Gal1 by altering its carbohydrate-binding properties (33). Interestingly, HPSCs overexpressing the mutant Gal1 showed increased KRAS levels compared to cells transfected with wild-type Gal1 or the empty vector (Ctl) (Fig. 2H). Altogether, these results indicate that KRAS is a direct target of nuclear Gal1 and that Gal1 controls its expression through the regulation of the KRAS promoter.

KRAS Downregulation Impairs PSC Proliferation and Activation.

Previous studies have reported the involvement of Gal1 in the activation of PSCs (22–25). To investigate the biological significance of KRAS expression in PSCs and its potential role in Gal1-mediated functions, we used a shRNA strategy to achieve stable knockdown of KRAS and performed functional assays (Fig. 3A). KRAS downregulation significantly reduced α-SMA expression at both the mRNA (Fig. 3B) and protein levels (Fig. 3C), indicating that KRAS plays a role in maintaining the activated status of PSCs. These findings align with our previous results following Gal1 knockdown in HPSC (24), further supporting the idea that KRAS may play an important role in Gal1-driven PSC activation. Next, we evaluated the impact of KRAS depletion on cell morphology and proliferation, major features of PSC activation (34, 35). KRAS knockdown in HPSC led to a shift in cell morphology from a spindle-like shape in control cells to a more epithelioid phenotype, as determined by a significant increase in circularity in KRAS knockdown cells (Fig. 3D). This morphology change mirrors the phenotype observed following Gal1 downregulation (Fig. 3D). Additionally, KRAS downregulation impaired cell proliferation (Fig. 3E). Taken together, these results suggest that KRAS is critical for maintaining the classic markers of PSC activation and may partially mediate Gal1 effects on PSCs within the PDAC stromal compartment.

Fig. 3.

KRAS downregulation impairs PSC activation and proliferation. (A) RT-qPCR KRAS mRNA expression in HPSC control cells (Ctl), cells transfected with two independent shRNAs for KRAS downregulation (shKRAS#1 and shKRAS#2) or with a nontarget control (shCtl). (B) RT-qPCR comparing mRNA levels of the fibroblast activation marker α-SMA in HPSC untreated cells (Ctl) transfected with a scramble shRNA (shCtl) or two different sequences (shKRAS#1 and shKRAS#2). (C) Western blot for α-SMA following KRAS shRNA (shKRAS#1 and shKRAS#2). Vinculin is used for protein normalization. (D) Immunofluorescence of phalloidin (staining actin fibers) (red) in HPSC Ctl or transfected with shCtl, shKRAS#1, shKRAS#2, shGal1#1, or shGal1#2. (Scale bars, 100 μm). Right panel, circularity quantification. (E) MTT assays showing HPSC proliferation of Ctl, shCtl, and shRNA against KRAS. (F) Heatmap showing relative protein expression (Z-score) in HPSC cells transfected with nontarget control (siCtl), or cells transfected with siRNA against Gal1 (siGal1) or KRAS (siKRAS), detected by Olink targeted Immuno-Oncology proteomics panel. Results are shown as mean ± SEM with values normalized to shCtl sample (A and C). *P < 0.05; **P < 0.01; ***P < 0.001

Given the relevance of PSC-secreted cytokines in the crosstalk of tumor cells with other components of the TME (6), we also explored the impact of KRAS knockdown, along with Gal1 knockdown, on the cytokine secretion profile of PSCs. To this end, using an Olink Proximity Extension Assay targeting an immune-oncology panel (92 proteins) (SI Appendix, Table S1), we analyzed the cell culture supernatants from control HPSCs and cells with Gal1 or KRAS downregulated levels. Interestingly, both Gal1 and KRAS downregulation resulted in reduced secretion of protumorigenic, anti-inflammatory cytokines IL-1α, CD244, CRTAM, IL-5, LAG3, IL-13, and TNF. Simultaneously, there was increased secretion of proteins with proinflammatory activity, including MCP-2, IL-12, CXCL11, and IL-2 (Fig. 3F), suggesting a shift towards a less immunosuppressive microenvironment.

Overall, our findings highlight the pivotal role of KRAS not only for maintaining traditional markers of PSC activation but also in regulating the secretion of protumorigenic cytokines. These results further reinforce KRAS’s role in the PSC activation state and its influence on the TME, particularly in promoting immune evasion.

Gal1 Regulates H3K4 Methylation and MLL1 Binding to the KRAS Promoter.

Among the different posttranslational modifications, histone methylation and acetylation are well-known key factors modulating gene expression (36, 37). In particular, the methylation of histone H3 at lysine 4 (H3K4me), lysine K9 (H3K9me), and lysine 27 (H3K27me), as well as the acetylation of H3K27 (H3K27Ac) (38, 39), play significant roles in chromatin regulation. Interestingly, in silico analysis identified the presence of high levels of H3K4me3 and H3K27Ac at the KRAS promoter (Fig. 4A). To investigate whether Gal1-dependent KRAS expression is influenced by these histone modifications, we knocked down Gal1 in PSCs using siRNA and determined the levels of enrichment of H3K4me1/3, H3K9me3, H3K27me3, and H3K27Ac in the KRAS promoter by ChIP-qPCR analysis (Fig. 4B). Gal1 knockdown diminished H3K4me1 levels, without significantly affecting the other chromatin marks at the KRAS promoter, indicating that Gal1 expression is necessary for maintaining an active state of the KRAS promoter, specifically by modulating the H3K4 methylation pattern. Further analysis using ChIP–PCR revealed that MLL1, a histone methyltransferase responsible for placing H3K4me1 (40, 41), was bound to the KRAS promoter. Interestingly, data showed MLL1 binding to the KRAS promoter was dependent on Gal1, as its presence was significantly reduced following Gal1 knockdown (Fig. 4C). Finally, we demonstrated that similar to Gal1, MLL1 levels were elevated in human PSCs derived from PDAC cases (Fig. 4D). These findings suggest that Gal1 expression is required for MLL1 binding to KRAS promoter, favoring H3K4me1 status that contributes to the activation of the KRAS promoter and subsequent gene expression in PSCs.

Fig. 4.

Gal1 is required to maintain lysine 4 monomethylation and MLL1 binding at the KRAS promoter. (A) IGB MACS peak for H3K4me3 (blue) and H3K27Ac (turquoise) mapped on the KRAS promoter region. (B) ChIP-qPCR on the KRAS promoter for histone marks (H3K4me1, H3K4me3, H3K9me3, H3K27Ac, and H3K27me3) in HPSC transfected with siRNA against Gal1 (siGal1) or nontarget control (siCtl). (C) ChIP-qPCR for MLL1 on the KRAS promoter performed in HPSC transfected with siGal1 or nontarget control (siCtl). Data were normalized relative to siRNA control (siCtl) (B and C). (D) qPCR analysis of MLL1 in human healthy and PDAC tissue. Results are represented as mean± SEM of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.0001.

Discussion

Activation of PSCs has been shown to play a key role in PDAC onset and progression (27, 42–45) as well as in resistance to therapies (46–48). Consequently, activated PSCs have become a major focus for the development of novel therapeutic strategies for PDAC. Interestingly, depletion of these cells results in more aggressive tumors (49, 50), while reprogramming PSCs from an activated to a quiescent phenotype has emerged as a promising approach to hinder PDAC progression (51–54). Several candidates have been proposed to “switch off” PSCs in pancreatic cancer, including vitamin D (55), HGF (56), TGF-β (57), or Saa3 (58). Our group, along with others has previously shown that Gal1, expressed by PSCs, plays a crucial role in regulating their activation state (22–25). However, the molecular mechanisms underlying these effects have been poorly characterized.

Our study sheds light on the nuclear functions of Gal1, revealing a role in the regulation of gene expression in PSCs. Gal1-mediated oncogenic functions, such as angiogenesis, cell invasion, or immune evasion, have been attributed to extracellular Gal1 (20) or intracellular cytosolic Gal1, through its interaction with HRAS and activation of RAS-MEK-ERK pathway (59). However, the role of nuclear galectins remains poorly understood. Gal3 has been implicated in the splicing and transport of mRNA by binding to Gemin4 (60, 61) as well as in transcriptional regulation of cancer-related gene expression (62–64). For nuclear Gal1, its role in regulating RNA splicing by interacting with Gemin4 in HeLa cells was the only known function until now (21, 65). Our study provides evidence of a role for Gal1 in PSC nuclei, where it promotes the transcription of target genes such as Hippo, Rap1, and WNT, regulating cell growth, cell adhesion, cell fate, and cell migration, ultimately leading to PSC reprogramming and activation. Interestingly, while we and others have previously shown that Gal1 drive PSC activation in PDAC (22–25), the data presented here provide a mechanistic insight by highlighting its nuclear function in this process.

Interestingly, the KRAS promoter was identified as a Gal1 target by ChIP analysis and luciferase reporter activity using HPSC Gal1 knockdown. KRAS is considered a driver oncogene in PDAC, with mutations in this gene found in more than 95% of tumors. Several studies have shown that activation of the KRAS pathway in PDAC can also be achieved through overexpression of the wild-type allele via EGFR signaling (66–68). In fibroblasts, a comparative study between mutated KRAS versus wild-type KRAS gene overexpression revealed common altered gene expression patterns, indicating that increasing levels of wild-type KRAS gene can also induce tumorigenesis (69). Remarkably, our results strengthen the crucial role of KRAS in PDAC, not only inducing epithelial cancer cell transformation by single-point mutations but also by contributing to the activation of the KRAS signaling pathway in PSC through Gal1-driven KRAS overexpression. While the importance of KRAS in cancer cells for PDAC progression is well established (70), its role in fibroblasts remains poorly understood (69, 71, 72). In this study, using KRAS knockdown in PSCs, we observed a shift toward a less-spindle morphology, a reduction of α-SMA expression, and decreased proliferation, indicating that KRAS may be involved in PSC activation. Indeed, many of these processes mirrored the effects observed following Gal1 downregulation in HPSCs (24), suggesting that KRAS may, at least in part, mediate some of the Gal1-driven effects on HPSC activation. However, the reduction in KRAS levels after Gal1 downregulation was less pronounced compared to direct KRAS knockdown, which may explain why KRAS knockdown leads to decreased proliferation, whereas Gal1 knockdown does not. Furthermore, our results demonstrate that both Gal1 and KRAS downregulation in PSCs led to significant changes in the cytokine secretion profile, further supporting their role in modulating the protumorigenic microenvironment. Our data confirm previous reports linking Gal1 to several anti-inflammatory cytokines, such as IL-5 (73, 74), IL-12 (75), and IL-2 (76–78), and suggest that KRAS might be a key mediator of these effects. This implies that KRAS not only maintains the activated PSC phenotype but also regulates cytokines that influence tumor-stroma crosstalk and immune modulation within the PDAC TME.

Mechanistically, we found that PSC-reprogramming via nuclear Gal1 is achieved through epigenetic control of gene expression, particularly by regulating histone methylation. Posttranslational modifications of histones are well-known mechanisms that can alter chromatin structure (79). Specifically, distinct histone methylation patterns are associated with activated versus repressed genes (36, 37). In this study, we demonstrate that Gal1 expression is necessary to maintain an active pattern of histone methylation on the KRAS promoter. Gal1 downregulation leads to a reduction in H3K4me1, a transcriptional activation mark (38), and an increase in H3K9me3, a well-known repressive histone mark (39). Additionally, the levels of the histone methyltransferase MLL1 at the KRAS promoter were reduced upon Gal1 downregulation by RNAi, strengthening the role of nuclear Gal1 in epigenetic reprogramming. Future studies will help to elucidate whether epigenetic regulation is a common mechanism through which Gal1 regulates transcription of other genes beyond KRAS.

In conclusion, our study identifies a function for nuclear Gal1 in PSCs, revealing its ability to bind different DNA promoter regions and, in the case of KRAS, transcriptionally activate its expression through epigenetic regulation. Our findings not only underscore the pivotal role of KRAS in maintaining the activated state of PSCs but also uncover a nuclear function of Gal1 in modulating the transcriptional landscape of cancer-associated genes in PSCs within the PDAC TME. Importantly, this work suggests therapeutic opportunities for reprogramming the TME by targeting nuclear Gal1 to shift PSCs from an activated to a quiescent state. Current Gal1-targeted therapies primarily focus on extracellular functions by using glycan inhibitors, natural polysaccharides, allosteric peptide antagonists, and monoclonal antibodies (80). However, these approaches mainly target the extracellular roles of galectins in the TME (80, 81), without addressing Gal1’s nuclear activity. Targeting nuclear Gal1 could provide a unique strategy to alter PSC gene expression, thereby reducing the protumorigenic effects of an activated stroma in PDAC. Future research should investigate the mechanisms of Gal1 nuclear transport, and the protein complexes involved in its gene regulatory functions, facilitating the development of pharmacological inhibitors that specifically block Gal1’s nuclear localization and to understand whether similar functions may happen in tumor epithelial cells expressing nuclear Gal1. This approach represents a promising Gal1-based strategy for PDAC treatment, focused on reprogramming the TME to inhibit tumor progression.

Materials and Methods

Cell Lines and Culture Conditions.

HPSCs were kindly provided by Dr. Hwang (The University of Texas, MD Anderson Cancer Center, Houston, TX) (27). The HEK-293 T/17 cell line (82) was obtained from Cancer Cell Line Repository (Hospital del Mar Research Institute, Barcelona, Spain). Both cell lines were cultured in DMEM (Gibco) medium supplemented with 10% FBS, 2 mM L-Glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, at 37 °C and 5% CO₂. Lentivirus production and cell infections were conducted in BioSafety Level 2 facility (BSL-2). The nonimmortalized primary cultured PSCs from human or mouse origin [PDAC075 T-hFib (#075T-hFib) and PDAC018 T-mFib (#018T-mFib), respectively] were generated by Dr. Iovanna [INSERM U1068, Centre de Recherche en Cancérologie de Marseille (CRCM), Marseille, France] and cultured as previously described (28).

MTT Assay.

Cell viability was assessed using the MTT assay as previously described (83). Briefly, cells were incubated with MTT reagent, and the resulting formazan product was measured spectrophotometrically at 570 nm.

Human Samples and Immunohistochemistry.

Tumor and healthy pancreatic human tissue were obtained from Parc de Salut MAR Biobanc (Barcelona). The project was approved by the Ethic Committee of Parc de Salut Mar (CEIm-Parc de Salut Mar, 2020/9067/I) in accordance with the Helsinki Declaration of Principles and all participants read and signed the informed consent. Formalin-fixed pancreatic tissues were embedded in paraffin and microsectioned in 5 μm slides, which were deparaffined and stained with primary antibodies: Gal1 (Abcam Cat# ab138513, RRID:AB_2894851) and α-SMA (Sigma-Aldrich Cat# A2547, RRID:AB_476701) as previously detailed (84). Images were taken using an Olympus BX16 microscope.

Immunofluorescence.

PSCs were plated over sterile coverslips, fixed with 4% paraformaldehyde, and quenched with 0.1 M NH₄Cl for 15 min at RT. Cell permeabilization and blocking were performed simultaneously by incubating with 0.3% Triton® X-100 and 1% BSA for 1 h at RT. Primary antibody incubation with Gal1 1:1,000 (Abcam Cat# ab138513, RRID:AB_2894851) was followed by secondary anti-rabbit Alexa® 488 (Thermo Fisher Scientific Cat# A-11008, RRID:AB_143165). For phalloidin detection, Alexa Fluor^TM 568 phalloidin 1:200 was used (Thermo Fisher Scientific Cat#AA12380). For nuclei detection, cells were incubated with 0.25 pg/mL DAPI (Roche). Slides were mounted using Fluoromount-G® (Southern Biotech) and fluorescent pictures were taken under proper emission filters using a NIKON Eclipse Ni microscope. Cell circularity (4π(area/perimeter²)) was analyzed using ImageJ software analysis from images stained with phalloidin.

Lentivirus Generation and HPSC Infection with Short Hairpin RNA (shRNA).

HEK293T/17 cells were transfected with packaging vectors for lentivirus generation together with pLKO.1-puro vector carrying the shRNA of Gal1 or KRAS (MISSION® RNAi, Merck) or the nontargeting scramble shRNA (Merck, SHC002), as previously detailed (84). Infected HPSCs were selected and expanded in the presence of 0.75 μg/mL puromycin.

siRNA Transfections.

For siRNA studies, HPSC were transfected with two different sequences of siRNA targeting human Gal1 [GeneGlobeID: SI03085453M (siGal1#1); SI02628269 (siGal1#2), Qiagen] using RNAiMax (Thermo Fisher Scientific) following the manufacturer’s protocol (Fig. 2 E–G) or with SMARTpool ON-TARGETplus siRNA (Dharmacon) against Gal1 or KRAS, which combine four specific sequences (Fig. 3F). Nontarget control (AllStars Negative Control, GeneGlobeID: SI03650318) and ON-TARGETplus Nontargeting Pool were used as the negative controls (siCtl), respectively (SI Appendix, Fig. S1B). Cells were harvested 48 h after transfection and knockdown efficiency was confirmed by Western blotting and qPCR as described.

Cell Lysis and Western Blot (WB).

Cells were lysed using RIPA Buffer supplemented with complete^TM EDTA-free protease inhibitor cocktail (Sigma), 5 mM NaF, and 1 mM sodium orthovanadate. Supernatants were collected, quantified with DC^TM Protein Assay Kit (BioRad), and then Laemmli Buffer 5X (250 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 5% 2-mercaptoethanol, 0.5% bromophenol blue) was added to samples at 1X final concentration. After boiling at 98 °C for 10 min, 20 μg of each protein sample were loaded in 6 to 15% polyacrylamide gels and transferred to nitrocellulose membranes (Amershan Protran 0.45 μm pore size, GE Healthcare Life Sciences) which were incubated overnight with primary antibodies: Gal1 (Abcam Cat# ab138513, RRID:AB_2894851), α-Tubulin (Sigma-Aldrich Cat# T9026, RRID:AB_477593), Vinculin (Santa Cruz Biotechnology Cat# sc-73614, RRID:AB_1131294), α-SMA (Sigma-Aldrich Cat# A2547, RRID:AB_476701), Histone-H3 (Abcam Cat# ab1791, RRID:AB_302613), followed by secondary antibodies (anti-rabbit (Agilent Cat# P0448, RRID:AB_2617138), anti-mouse (Agilent Cat# P0260, RRID:AB_2636929) HRP-conjugated immunoglobulin G. Chemiluminescence signal was detected with Pierce^TM ECL Western Blotting Substrate (Thermo Fisher Scientific).

qRT-PCR.

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) following the manufacturer’s protocol and RNA was reverse-transcribed using a high-capacity cDNA synthesis kit (Applied Biosystems) and amplified by qRT-PCR using SYBR® Select Master Mix (Life Technologies). The following sense and antisense primer sets were used (SI Appendix, Table S2). Primers were used at 0.25 mM final concentration. In both cases, 2% DMSO was added to reaction mix for proper analysis using LightCycler® 480 II (Roche). mRNA levels were normalized by using an equally weighted combined housekeeping value calculated from HPRT and TBP mRNA levels in the same sample. The results were calculated following the ΔΔCT method. Quantitative PCR of the ChIP products and genomic input DNA was performed using a set of primers encompassing an area containing Gal1 consensus binding site in the KRAS promoter (SI Appendix, Table S2).

Olink Targeted Proteomics.

The Olink Immuno-oncology panel (SI Appendix, Table S1) was run on cell culture supernatants from HPSC control cells or cells transfected with siRNA against Gal1 or KRAS (72 h posttransfection) on Cobiomics facility [Biomedical Institute of Cordoba (IMIBIC) and University of Cordoba (EBT-UCO)].

Chromatin Immunoprecipitation Assay (ChIP).

HPSC were fixed with 1% formaldehyde solution (Merck), and ChIP was conducted as described previously (85). Briefly, cells were collected in lysis buffer (5 mM PIPES, 85 mM KCl, 0.5% NP-40, 1 mM Tris-HCl pH 8.1; supplemented with protease inhibitors) and vortexed. Nuclei were collected and lysed in 50 mM Tris-HCl pH 8.1, 10 mM EDTA pH 8, 1% SDS. DNA was sonicated obtaining 200 to 500 bp fragments, and samples were 10 times diluted with 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, 1.2 mM EDTA pH 8, 1% Triton X-100, 0.01% SDS. Preclearing was performed with anti-rabbit irrelevant IgGs (Sigma-Aldrich Cat# I5006, RRID:AB_1163659) agarose Protein-A Beads (Sigma). Antibody incubation was performed overnight at 4 °C with antibodies against Gal1 (Santa Cruz Biotechnology Cat# sc-166618, RRID:AB_2281310 or Abcam Cat# ab138513, RRID:AB_2894851), MLL1 (Active Motif Cat# 61295, RRID:AB_2793585), H3K4me1 (Active Motif Cat# 61633, RRID:AB_2793712), H3K4me3 (Millipore Cat# 04-745, RRID:AB_1163444), H3K9me3 (Abcam Cat# ab8898, RRID:AB_306848), H3K27Ac (Abcam Cat# ab4729, RRID:AB_2118291), and H3K27me3 (Abcam Cat# ab192985, RRID:AB_2650559) or IgG from rabbit serum (Sigma-Aldrich Cat# I5006, RRID:AB_1163659). Blocked beads were added to each immunoprecipitation and incubated for 4 h. Unbound fraction was collected after centrifugation. Pelleted beads were washed, and immunoprecipitated DNA and protein complexes were eluted by adding 0.1 M Na₂CO₃ and 1% SDS. Supernatants were collected, and both input and IPs were decrosslinked by adding NaCl 200 mM and incubating at 65 °C. DNA was purified using a MiniElute® PCR Purification kit (Qiagen).

Sequencing Analysis.

Qubit, to ensure high sensitivity quality control assessments, DNA library preparation (Bioanalyzer, Agilent Technologies), and Sanger sequencing were performed by the Genomics Unit at the Centre for Genomic Regulation (CRG), PRBB, Barcelona. ChIP-seq data processing and analysis were conducted using The Galaxy Project platform (https://usegalaxy.org). The quality of immunoprecipitated DNA reads was assessed with the FastQC tool, considering only reads with a quality score above 30 for downstream analysis. Selected reads were aligned to the human reference genome (Homo sapiens b38, hg38) using Bowtie2. Aligned reads from the Gal1 IP sample were treated as the ChIP-seq Tag File, applying a P-value cutoff of 10⁻⁵ for peak detection. The MACS peak calling model was generated with an upper MFOLD range of 32 to identify high-confidence enrichment regions in comparison to background signal (ChIP-seq IgG IP control file). A total of 7,844 peaks were detected and mapped to the reference human genome (hg38, Dec 2013). To determine peak distribution across the PSC genome, mapped regions were classified based on annotations from the UCSC Genome Browser, including 3′UTR, 5′UTR, exonic, and intronic sequences. Promoter regions were defined as spanning from 3,000 bp upstream to 1,000 bp downstream of the transcription start site. To identify key DNA motifs and their associated transcription factors, sequences corresponding to MACS peaks within promoter regions were analyzed using The MEME Suite tool (86).

Statistical Analysis.

Values are expressed as mean ± SEM of at least three independent experiments and normalized relative to corresponding controls. Statistical analysis was performed using GraphPad Prism 5, and P-values were determined by the Student t test. For functional and gene ontology annotation, using DAVID, statistical significance was considered when the modified Fisher Exact (EASE score) P-value was lower than 0.05.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Data, Materials, and Software Availability

ChIP sequencing data are available at Gene Expression Omnibus (GEO Accession No. GSE282020) (87). All study data are included in the article and/or supporting information.