Abstract
Background
Pancreatic adenocarcinoma (PAAD) is one of the most leading causes of cancer-related death across the world with the limited efficiency and response rate of immunotherapy. Protein S-palmitoylation, a powerful post-translational lipid modification, is well-known to regulate the stability and cellular distribution of cancer-related proteins, which is mediated by a family of 23 palmitoyl transferases, namely zinc finger Asp-His-His-Cys-type (ZDHHC). However, whether palmitoyl transferases can determine tumor progression and the efficacy of immunotherapy in PAAD remains unknown.
Methods
Bioinformatics methods were used to identify differential ZDHHCs expression in PAAD. A systematic pan-cancer analysis was conducted to assess the immunological role of ZDHHC3 using RNA sequencing data from The Cancer Genome Atlas database. In vivo Panc 02 subcutaneous tumor model validated the anti-tumor effect of knockdown of ZDHHC3 or intraperitoneal injection of 2-bromopalmitate (2-BP), a typical broad-spectrum palmitoyl transferases inhibitor. Furthermore, we explored therapeutic strategies with combinations of 2-BP with PD-1/PD-L1-targeted immunotherapy in C57BL/6 mice bearing syngeneic Panc 02 pancreatic tumors.
Results
ZDHHC enzymes were associated with distinct prognostic values of pancreatic cancer. We identified that ZDHHC3 expression promotes an immunosuppressive tumor microenvironment in PAAD. 2-BP suppressed pancreatic-tumor cell viability and tumor sphere-forming activities, as well as increased cell apoptosis in vitro, without affecting normal human pancreatic ductal epithelial cells. Furthermore, genetic inactivation of ZDHHC3 or intraperitoneal injection of 2-BP impeded tumor progression in Panc 02 pancreatic tumors with enhanced anti-tumor immunity. 2-BP treatment significantly enhanced the therapeutic efficacy of PD-1/PD-L1 inhibitors in Panc 02 pancreatic tumors.
Conclusion
This study revealed some ZDHHC enzyme genes for predicting the prognosis of pancreatic cancer, and demonstrated that ZDHHC3 plays a critical oncogenic role in pancreatic cancer progression, highlighting its potential as an immunotherapeutic target of pancreatic cancer. In addition, combination therapy of 2-BP and PD-1/PD-L1 achieved synergic therapy effects in a mouse model of pancreatic cancer.
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Introduction
Pancreatic adenocarcinoma (PAAD) is among the most aggressive and lethal human malignancies that occur with poor prognosis, which causes over 331,000 cancer-related death per year worldwide [1]. Long-term survival in resectable disease will be improved by surgical resection and adjuvant chemotherapy, but the majority of PAAD patients are diagnosed at an advanced and fatal stage with consequent loss of therapeutic opportunity [2]. Advanced PAAD is non-operable and notorious for its resistance to chemoradiotherapy, reaching a 5-year overall survival (OS) rate below 10% in the USA [1, 3]. Thus, there remains an urgent requirement to discover novel prognostic biomarkers and develop effective therapeutic strategies for PAAD.
Emerging evidence has indicated that the tumor immune microenvironment differentially participates in the pathogenesis of PAAD and influences the clinical outcomes of patients [4]. PAAD patients are predominantly represented by a “cold” tumor microenvironment (TME) with limited infiltrating immune cells, which is characterized by hypoxia and extensive stromal and matrix deposition (desmoplasia) [4, 5]. Increased percentage of immunosuppressive cells and lack of infiltration of natural killer (NK) cells and cytotoxic T lymphocytes contribute to the highly immunosuppressive TME [6, 7]. Currently, immunotherapeutic strategies (e.g., immune checkpoint blockade therapy, combinatorial approach with other immunotherapeutic agents or targeted molecular agents, etc.) have been applied in many solid tumor types due to its excellent efficacy and clinical safety [8]. Especially immune checkpoint inhibitors targeting programmed cell death-1 (PD-1) and its ligand-1 (PD-L1) have dramatically revolutionized the cancer treatment landscape and represented the cornerstone of immunotherapy [9, 10]. However, PD-1/PD-L1 blockade has so far demonstrated a modestly therapeutic effect in PAAD patients [11]. Thus, there is an urgent unmet clinical need to further explore potential molecular targets for pancreatic cancer initiation and progression and to identify effective therapeutic strategies to enhance PAAD patients’ responsiveness to immunotherapy.
Protein lipidation events are a prototypical form of post-translational modification, in which S-palmitoylation exerts multiple effects to dynamically orchestrate the target protein interaction. It involves the reversible attachment of C16 palmitic acid to cysteine residues through thioester bonds [12]. About 10% of the proteome is prone to S-palmitoylation through palmitoylate screens and prediction [13]. Dynamic S-palmitoylation events provide a vital mechanism for modulating protein stability, localization, conformation, intracellular trafficking, and function by altering membrane affinity [14,15,16]. Of note, a large number of cancer-related mammalian proteins with S-palmitoylated internal cysteine residues have been identified [17]. The majority of S-palmitoylated proteins are catalyzed by a group of zinc finger Asp-His-His-Cys-type (ZDHHC) enzymes [18]. Thus, the key to identifying palmitoyl transferase’s role in pancreatic cancer is the systemic evaluation of ZDHHC family members. Several ZDHHC enzymes are highly expressed in the brain, thus, S-palmitoylation has been widely reported in neuronal systems. More recent works focus on the role of S-palmitoylation in the immune pathways and cancer [12, 19]. Hence, targeting ZDHHC enzymes may provide a novel insight into the underlying molecular mechanisms of immunotherapy resistance and offer strategies for pancreatic cancer immunotherapy.
There is an established link between S-palmitoylation and cancer [20,21,22]. Recently, the application of targeting S-palmitoylation in cancer therapy has been widely studied, for instance, genetic inhibition of ZDHHC family members (e.g., ZDHHC3, ZDHHC5, ZDHHC17, ZDHHC9, and ZDHHC12) have shown promising and enhanced anticancer effects in colon cancer, breast cancer, non-small cell lung cancer, glioma, leukemia, and ovarian cancer [23,24,25,26,27,28,29,38]. Mark Ayers and colleagues established an 18-gene expression signature to assess pre-existing adaptive T cell immune responses [39]. As shown in Fig. 3A, B, ZDHHC3 expression was significantly negatively correlated with the pan-cancer T cell infiltration score (R = − 0.2, P = 0.01), indicating that ZDHHC3 is a potent immunosuppressive molecule. Next, we further delineated the potential immunomodulatory roles of ZDHHC3 in different tumor types to identify candidate types that may benefit from ZDHHC3-targeted therapy. Our findings showed that ZDHHC3 was found to be negatively correlated with a majority of immunomodulators in PAAD (Fig. 3C), the established gene signature of the pro-inflammatory TME [40]. We also investigated the infiltration levels of TIICs using the TISIDB database. Furthermore, ZDHHC3 expression was mutually exclusive of four immune checkpoints, including PD-1, cytotoxic T lymphocyte antigen 4 (CTLA-4), and lymphocyte activation gene 3 (LAG-3) expression in some cancer types, including PAAD (Fig. 3D–G). We further noted that ZDHHC3 was negatively associated with TMB (Tumor mutational burden) and MSI (Microsatellite instability) in several cancer types (Additional file 1: Fig. S7). Notably, ZDHHC3 expression was negatively correlated with a majority of immune cells in some cancer types, including PAAD (Fig. 3H). Together, these results indicate that ZDHHC3 may be used as a potential indicator of tumor immunogenicity in some human cancers.
Correlation among ZDHHC3 gene expression and immunological status in pan-cancer analysis. A, B Pan-cancer analysis of correlations between ZDHHC3 gene expression and T cell inflamed score (A) and the 18 individual component genes from the established T cell inflamed signature (B). The T cell inflamed score is significantly positively correlated with immunotherapy response in cancer. C Heat map images show the correlation between ZDHHC3 gene expression and 124 immunomodulators such as MHC, immunostimulator, chemokine, and chemokine receptors using the TISIDB database. D–G Correlation between ZDHHC3 gene expression and four immune checkpoints, PD-L1 (D), CTLA-4 (E), PD-1 (F), and LAG-3 (G). The different color dots represent different cancer types. The Y-axis indicates the Pearson correlation coefficient and the X-axis indicates -log10P values. H Heat map images show the correlation between ZDHHC3 gene expression and 28 tumor-infiltrating immune cells in the TME. The heat map color indicates the correlation coefficient. Red color, positive correlations; blue color, negative correlations. The horizontal axis indicates the infiltration levels of immune cells, and the vertical axis indicates the different cancer types
We next focused on the immunomodulation of ZDHHC3 in pancreatic cancer. Of these, a majority of MHC protein complex related genes were downregulated in the high-ZDHHC3 group, suggesting a possible impairment of the antigen uptake, processing, and presentation compared to low-ZDHHC3 group. Notably, chemokines including (CCL4, CCL5, and CXCL9), identified to be critical for recruitment of activated CD8+ T cells into the TME, were also downregulated in the high-ZDHHC3 group. Similarly, other chemokines, including CCL2, CCL3, CCL8, CCL11, CCL12, CCL14, CCL16, CCL19, CCL21, and CCL23, and paired receptors including CCR1, CCR2, CCR4, CCR5, CCR6, CCR7, CXCR1, and CXCR6, were found to be negatively correlated with ZDHHC3 expression in PAAD (Fig. 4A; Additional file 1: Fig. S8). These chemokines and receptors are known to orchestrate the traffic of effector TIICs such as NK cells, CD8+ T cells, and antigen-presenting cells to the tumor site, can in turn initiate anti-tumor immunity. A series of stepwise anti-tumor immune response events has been previously established to dissect the complex cancer-immune system interactions [41]. The activities of the majority of the steps in the cancer-immunity cycle were suppressed in the high-ZDHHC3 group, including the release of cancer antigens presentation (Step 2), priming and activation (Step 3), and trafficking of immune cells to tumors (Step 4) (CD4+ T cell recruiting, CD8+ T cell recruiting, dendritic cells (DC) recruiting, macrophage recruiting, monocyte recruiting, NK cell recruiting, TH17 recruiting, and B cell recruiting,) (Fig. 4B). Thus, the decreased activities of these key steps may impede the infiltration of effector immune cells into the tumor bed. We next examined the infiltration level of TIICs using six different independent algorithms [42]. Consistent with the above analysis, expression level of ZDHHC3 was negatively associated with the infiltration of CD8+ T cells, NK cells, macrophages, and DCs in different independent algorithms among PAAD samples (Fig. 4C). Similarly, the majority of these TIICs related effector genes were negatively correlated with ZDHHC3 gene expression (Fig. 4D). Low immune checkpoint inhibitors such as PD-L1/PD-1 expression was one of the most notable features in non-inflamed TME of pancreatic cancer [43]. Of note, ZDHHC3 expression was negatively correlated with a majority of immune checkpoints including PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, CD200, and ADORA2A (Fig. 4E). In summary, ZDHHC3 overexpression may exert an immunosuppressed effect in pancreatic cancer.
ZDHHC3 expression promotes an immunosuppressive TME in PAAD. A Heat map showing different expression levels of published immunomodulators signatures (MHC, immunostimulator, chemokine, and chemokine receptors) between high- and low-ZDHHC3 groups in PAAD patients. B The expression patterns of markers of anti-cancer immunity across a 7-step cancer-immunity cycle between high- and low-ZDHHC3 groups. C The association between ZDHHC3 gene and the infiltration degrees of five TIICs (CD8+ T cells, NK cells, macrophages, Th1 cells, and DC), based on different algorithms. D A differential heat map showing the comparison of the published effector gene profiles of indicated immune cells between high and low ZDHHC3 groups. The heat map color indicates the correlation coefficient. Yellow color, positive correlations; blue color, negative correlations. E Correlation between ZDHHC3 gene expression and 20 inhibitory immune checkpoints in PAAD samples. Color scales visualizes Spearman correlation coefficient values. Color codes in the heat map represent relative expression levels as z-scores. P-values were calculated using Mann–Whitney U test with asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p<0.0001)
Mutation landscape between ZDHHC3-high and ZDHHC3-low pancreatic tumors
PAAD patients with KRAS mutation, TP53 mutation, and CDKN2A deletion were more likely to have faster disease progression and worse clinical outcome [44]. We further evaluated the mutation status among the ZDHHC3-high and ZDHHC3-low groups of TCGA-PAAD cohort. Among these altered genes, TP53, KRAS, and CDKN2A were the most significantly differentially mutant genes (Fig. 5A, B). ZDHHC3-high tumors were found to harbor a higher prevalence of TP53 and KRAS mutation rate, indicating the enhanced tumorigenic potential (Fig. 5A, B). Likewise, as ZDHHC3 expression increased, the frequency of KRAS and TP53 gene mutations increased (Additional file 1: Fig. S9). Meanwhile, when compared to wild type KRAS PAAD samples (27%, 17/63), tumors with high KRAS mutation rate samples were more likely to be ZDHHC3-high (70%, 56/80). Meanwhile, when compared to wild type TP53 PAAD samples (32%, 26/80), tumors with TP53 mutation were more likely to express higher ZDHHC3 level (75%, 47/63) (Fig. 5C, D). Moreover, the GSEA results and KEGG analysis indicated that ZDHHC3 is significantly positively associated with the mutant TP53-upregulated genes, while negatively correlated with the mutant TP53-downregulated genes (Fig. 5E, F; Additional file 1: Fig. S10). Furthermore, we found that PAAD patients with p53 induced genes had lower ZDHHC3 expression, while the PAAD patients with p53 suppressed genes had higher ZDHHC3 expression (Fig. 5G). Taken together, these results demonstrate that ZDHHC3-high tumors harbor a higher TP53 and KRAS mutation rate, which may cooperatively contribute to disease progression and prognosis status in pancreatic cancer.
Comparison of mutation landscape between PAAD patients with high and low ZDHHC3 expression. A Forest plot showing the most significant mutant genes in ZDHHC3-high and ZDHHC3-low PAAD tumors. B Circular plot showing the mutation status of ZDHHC3-high and ZDHHC3-low PAAD tumors. The genomic density of mutation genes in ZDHHC3-high tumors, in ZDHHC3-low tumors. C, D Proportion of PAAD patients harboring wildtype or mutant KRAS (C) or TP53 (D). E, F GSEA of genes up- (E) or downregulated (F) in association with mutated KRAS, based on ZDHHC3 expression. G Heat map showing the correlation of ZDHHC3 gene expression and the expression of p53-induced genes or p53-suppressed genes in PAAD tumors. The heat map color indicates the correlation coefficient. Red color, positive correlations; green color, negative correlations (ns, not significant, *p < 0.05, and ***p < 0.001)
ZDHHC3 knockdown slows Panc 02 tumor growth with enhanced anti-tumor immunity
Based on the above results, we hypothesized that ZDHHC3 may promote the development and progression of pancreatic cancer. We next tested the role of ZDHHC3 on oncogenic phenotypes using the Panc 02 syngeneic pancreatic cancer model. Stable knockdown of ZDHHC3 in Panc 02 cells was achieved using the shRNA approach (shZDHHC3). Knockdown efficiency of endogenous ZDHHC3 was assessed by western blotting analysis (Fig. 6A). We transplanted control (shControl) or ZDHHC3 knockdown (shZDHHC3) Panc 02 cells subcutaneously into the right flank of immunocompetent mice and the local volume changes in tumor growth were monitored every 3 days. We observed that genetic inactivation of ZDHHC3 slowed tumor growth and decreased the final tumor burden when compared to shControl Panc 02 tumors, thus prolonging the survival time of Panc 02 tumor bearing mice (Fig. 6B–D). Data from human pancreatic cancer suggest a potential function for ZDHHC3 in sha** the non-inflamed pancreatic cancer tumor microenvironment. We next assessed whether the ZDHHC3-dependent antitumor activity was associated with the potentiation of an adaptive antitumor immune response, similar to the above results that ZDHHC3 expression promotes an immunosuppressive TME in human pancreatic cancer. Panc 02 tumor samples were analyzed by multicolor flow cytometric analysis. A significant increase in the infiltration levels of anticancer immune cells, including NK cells, CD4+ and CD8+ T cells, DCs, and M1 macrophages in ZDHHC3-knockdown Panc 02 tumors as compared to shControl Panc 02 tumors. No significant difference in the infiltration of M2 macrophages was observed in shZDHHC3 Panc 02 tumors compared to shControl Panc 02 tumors (Fig. 6E–H). Since ZDHHC3 knockdown (KD) induced a characteristic immune profile in Panc 02 tumors, we hypothesized that ZDHHC3 knockdown would lead to a change in the cytokine/chemokine profile of the tumor microenvironment. To test this hypothesis, we used ELISA approach to detect the expression amounts of CCL4, CCL5, CXCL9, and CXCL10 in ZDHHC3-knockdown Panc 02 tumor tissues. CXCL9 protein showed the greatest increase in expression in response to ZDHHC3-KD (3.7-fold versus shControl). Meanwhile, CCL5 and CXCL10 protein increased by 1.5 times in response to ZDHHC3-KD, respectively and we did not detect significant changes in CCL4 expression (Additional file 1: Fig. S11). CXCL9 has been identified as the critical chemokine which recruits immune cells that express its cognate receptor, CXC-chemokine receptor 3 (CXCR3), including activated T cells and NK cells [45]. Meanwhile, the dendritic cells (DC) score was also highly correlated with CXCL9 mRNA levels and previous studies have reported that DCs secrete CXCL9 and thereby contribute to the recruitment of CD8+ T cells and NK cells [45]. Based on our results that a significant increase in the infiltration levels of anticancer immune cells, including NK cells, CD8+ T cells, and DCs in ZDHHC3-knockdown Panc 02 tumors as compared to shControl Panc 02 tumors, it would be very interesting to identify whether the functional role of ZDHCH3 in regulating anti-tumor immunity was CXCL9 mediated in future study.
Knockdown of ZDHHC3 expression suppresses tumor growth and induces immune effector cell infiltration in Panc 02 tumors. A Western blot assay detecting the protein level of ZDHHC3 in Panc 02 cells that were transfected with the control-shRNA or ZDHHC3-shRNA; the stable clones were used in subsequent experiments. B Subcutaneous tumor growth curves: 5 × 106 stably-expressing shZDHHC3 Panc 02 cells or shControl Panc 02 cells were injected in the right flank of C57BL6/J mice (n = 8 mice per group). C, D The tumor weight (C) and survival rate (D) of shControl and shZDHHC3 Panc 02 tumors were recorded at the end of the experiment. E–H The tumor tissues at the end point were digested into single-cell suspension and analyzed by flow cytometry. Then, cells were gated on single cells and selected on Live/Dead dye staining. Only live cells were included in the further analysis. Flow cytometry analysis of the number of NK cells (CD45+ CD3−NK1.1+) (E), CD8+ T cells (CD45+ CD8+), CD4+ T cells (CD45+ CD4+) (F), DCs (CD45+ CD11b+CD11c+) (G), type 1 macrophages (M1) (CD45+ CD11b+ F4/80+ CD206−), and type 2 macrophages (M2) (CD45+ CD11b+ F4/80+ CD206+) (H) infiltrating subcutaneous tumors, namely shControl and shZDHHC3 Panc 02 tumors at day 23. Percentages indicate different immune cell populations amongst live CD45+ immune cells. I Western blot assay detecting the protein level of PD-L1 in control, ZDHHC3-knockdown, and ZDHHC3-knockdown with PD-L1 overexpressed Panc 02 cells; the stable clones were used in subsequent experiments. J Subcutaneous tumor growth curves: 5 × 106 stably-expressing (Control, shZDHHC3, and shZDHHC3 + PD-L1) Panc 02 cells were injected in the right flank of C57BL6/J mice (n = 6 mice per group). K, L The tumor weight (K) and survival rate (L) of (Control, shZDHHC3, and shZDHHC3 + PD-L1) Panc 02 tumors were recorded at the end of the experiment. Survival curves from the subcutaneous Panc 02 tumor model were developed until the animal’s death or the tumor volume reached > 1000 mm3. Mice were euthanized when the calculated tumor size reached ~ 1000 mm3 or when tumors became ulcerated. Survival curves were analyzed using the log-rank (Mantel-Cox) test. Results shown are presented as mean ± SEM. (ns, not significant, *p < 0.05, and **p < 0.01)
We next explored potential key molecular targets for the inhibition of pancreatic cancer growth after knockdown of ZDHHC3. Previous studies have shown that ZDHHC3 palmitoylates PD-L1 to maintain its protein stability in colon cancer and gliomas [23, 46]. We next sought to determine whether ZDHHC3 influences cancer immunity by regulating PD-L1 expression in pancreatic cancer cells. Accordingly, we knockdown endogenous ZDHHC3 using two independent siRNAs in PANC-1 cells and detected PD-L1 expression. As shown in (Additional file 1: Fig. S12A, B), ZDHHC3 knockdown decreased PD-L1 protein expression, while not affecting its mRNA expression. These results were consistent with decreased surface expression of PD-L1 on ZDHHC3 knockdown PANC-1 cells (Additional file 1: Fig. S12C, D). Since palmitoyl transferases are known to interact with their substrates, we next investigated whether ZDHHC3 binds to PD-L1 in PANC-1 cells. Double immunofluorescent staining demonstrated that endogenous ZDHHC3 colocalized with PD-L1 in cultured PANC-1 cells (Additional file 1: Fig. S12E, F). Meanwhile, co-immunoprecipitation indicated physical interactions between PD-L1 and ZDHHC3 that were expressed endogenously in PANC-1 cells (Additional file 1: Fig. S12G). Thus, our findings indicated that ZDHHC3 is a key palmitoyl transferase to stabilize PD-L1 protein expression in pancreatic cancer, suggesting that the ZDHHC3-high tumors may have T cell exhaustion which contribute to immune evasion. Next, we performed animal assays to further investigate the functioning axis between ZDHHC3 and PD-L1. We introduced ZDHHC3-knockdown with PD-L1 overexpression clones (shZDHHC3 + PD-L1) by lentivirus expression system. Western blotting results revealed that PD-L1 expression is reduced in shZDHHC3 Panc 02 cells and successfully overexpression of PD-L1 expression in (shZDHHC3 + PD-L1) Panc 02 cells (Fig. 6I). We next subcutaneously transplanted control, ZDHHC3-knockdown, and ZDHHC3-knockdown with PD-L1 overexpressed Panc 02 cells into the right flank of immunocompetent mice and observed tumor growth. Overexpression of PD-L1 can almost completely reverse ZDHHC3 knockdown-induced inhibition of Panc 02 tumor growth and prolongation of mice survival time (Fig. 6J–L), suggesting that ZDHHC3 promotes pancreatic cancer progression probably via upregulation of PD-L1 expression by palmitoylation. Taken together, combining comprehensive bioinformatics and preclinical animal model results, we show that ZDHHC3 deficiency can inhibit pancreatic cancer progression by enhancing anti-tumor immunity.
2-BP impairs cell viability and sphere-forming activities, as well as increases apoptosis of pancreatic cancer cells
Next, we wanted to determine whether ZDHHCs can be targeted as a therapeutic tool for pancreatic cancer, Panc 02 and PANC-1 pancreatic cancer cell lines were treated with 2-bromopalmitate (2-BP), a ZDHHC-specific inhibitor. We found that 2-BP treatment significantly induced apoptosis of Panc 02 cells in a dose-dependent manner using the Annexin V-FITC/PI staining assay, and significantly early apoptosis of PANC-1 cells could also be observed with the 2-BP treatment at 100 µM concentrations (Fig. 7A, B). Moreover, 2-BP treatment suppressed cell viability of Panc 02 and PANC-1 pancreatic cancer cells in a time-dependent manner (Fig. 7C, D). At the same time, 2-BP can also inhibit the migration of Panc 02 and PANC-1 cells (Fig. 7E, F). Pancreatic cancer cells vary in their tumorigenic properties with the presence of a subset pancreatic cancer stem cells (CSCs), which are more resistant to currently used therapeutic agents [47]. We further examined whether 2-BP could inhibit pancreatic CSCs using the single-cell tumor sphere formation assay. Under free-floating tumor sphere culture conditions, 2-BP significantly reduced the sphere formation capacity of pancreatic CSCs in a dose-dependent manner (Fig. 7G, H). The expression level of stemness markers, ALDH1, Nanog, OCT4, and SOX2 was also downregulated by 100 µM 2-BP treatment in two pancreatic CSCs (Fig. 7I, J). Previous studies have determined that mTOR signaling plays a key role in maintaining stemness of pancreatic cancer stem cells and helps cancer cells to survive [48]. As shown in Fig. 7I, J, 2-BP treatment inhibited the self-renewal ability of pancreatic cancer stem cells by inhibiting phosphorylation of mTOR. To assess whether 2-BP treatment has any effect on normal pancreatic ductal epithelial cell. A human pancreatic ductal epithelial cell line (HPDE6-C7) was assayed for cell viability after exposure to 100 µM 2-BP. 2-BP treatment did not elicit significant apoptosis on HPDE6-C7 cells (Fig. 7K). Meanwhile, no significant effect of 2-BP treatment on HPDE6-C7 cell migration and proliferation, or cell cycle was observed (Fig. 7L–N). Taken together, these results suggest that 2-BP selectively exhibit high toxicity against pancreatic cancer cells but not against normal pancreatic ductal epithelial cell.
2-BP suppresses pancreatic-tumor cell viability and tumor sphere-forming activities, as well as increases apoptosis in vitro. A, B The effect of 2-BP on the apoptosis of Panc 02 (A) and PANC-1 (B) cells were detected by flow cytometry. C, D The Panc 02 (C) and PANC-1 (D) cells were pre-treated with 2-BP for 24 h, then the cell viability was analyzed using the CCK8 cell proliferation assay kit. E, F The effect of 2-BP treatment on the migration of Panc 02 (E) and PANC-1 (F) cells were examined by scratch test. G, H Representative images showing Panc 02 (G) and PANC-1 (H) 3D tumor culture model maintained in tumorsphere conditions for 7 days with indicated 2-BP treatment. I, J Differences in the expression of stem cell markers (ALDH1, Nanog, OCT4, SOX2) and mTOR signaling in Panc 02 (I) and PANC-1 (J) tumor spheres treated with 2-BP was analyzed by Western blotting assay. K The effect of 2-BP on the apoptosis of HPDE6-C7 cells was detected by flow cytometry. L The effect of 2-BP treatment on the migration of HPDE6-C7 cells was examined by scratch test. M The effect of 2-BP treatment on the HPDE6-C7 cell proliferation was examined by CCK8 cell proliferation assay kit. N Cell cycle of HPDE6-C7 cells with or without 2-BP treatment was analyzed by flow cytometry. The percent of G0/G1 phase, S phase, and G2/M phase were determined. Results shown are presented as mean ± SD. (ns, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001)
2-BP shows potent therapeutic effects in Panc 02 tumor model
To get potentially useful information for the development of pharmacological approaches against pancreatic tumor, we assessed the effects of the specific S-palmitoylation inhibitor, 2-BP, on Panc 02 syngeneic pancreatic tumor growth. The treatment was started when the tumor volume reached 100 mm3, and 8 mice were sorted into each group. 2-BP (50 mg kg−1; one injection per day) or vehicle was given intraperitoneally continuously for 12 days and tumor volume was monitored every 3 days (Fig. 8A). Notably, as shown in Fig. 8B–D, 2-BP treatment significantly impeded tumor progression and reduced tumor burden in Panc 02-bearing mice. We observed that the effect of 2-BP injection decreased PD-L1 expression and increased the granzyme B (GZMB) expression, indicating enhanced anti-tumor immunity (Fig. 8E–G). Notably, in comparison to the control group, 2-BP treatment significantly augmented an infiltration of CD8+T cells into Panc 02 tumors (Fig. 8F–I) as well as increased the expression of CCL5, CXCL9, and CXCL10, which are three important inflammatory cytokines accounting for the infiltration of CD8+T cells and resha** the inflammatory TME in pancreatic cancer (Fig. 8J) [49]. We also observed elevated secretion of CCL5 and CXCL9, but not CCL4 and CXCL10 in 2-BP treated Panc 02 cells in vitro (Additional file 1: Fig. S13). Together, our results suggest the use of a specific ZDHHCs inhibitor, 2-BP as a potential pharmacological tool for pancreatic cancer treatment.
2-BP delays tumor growth in Panc 02 tumor model. A–D Individual C57BL/6 mice were subcutaneously injected with 5 × 106 Panc 02 cells and received 2-BP treatment or Vehicle (CTRL). Schematic representation of treatment plan (A). Representative images of the tumors taken 28 days after inoculation, individuals’ representative tumor of each group (B), tumor weight (C), and tumor growth curves (D) of Vehicle (CTRL) or 2-BP treated Panc 02 tumors (n = 8 mice per group). E Western blotting analysis showing the decreased PD-L1 protein expression in Panc 02 tumor tissues of mice treated with 2-BP (50 mg/kg) at the indicated time. F, G H&E and IHC staining (F) of PD-L1, CD8α, and GZMB in Panc 02 tumor tissues of mice treated by 2-BP (50 mg/kg) and quantification (G). H, I Flow cytometry analysis of the number of tumor-infiltrating CD8+ T cells (CD45+ CD8+) (H) in CTRL and 2-BP treated Panc 02 tumors and quantification (I). J Concentrations of CCL4, CCL5, CXCL9, and CXCL10 in CTRL and 2-BP treated Panc 02 tumor tissues were measured by ELISA. Results shown are presented as mean ± SD. (ns, not significant, **p < 0.01, and ***p < 0.001)
2-BP enhances the therapeutic responsiveness of anti–PD-L1/PD-1 therapy in Panc 02 tumor model
We next investigated whether 2-BP could enhance the therapeutic efficacy of PD-1/PD-L1 checkpoint antibody blockade in pancreatic cancer. Therefore, we sought to determine the potential synergistic therapeutic effect of 2-BP and PD-1/L1 inhibitor in poorly immunogenic Panc 02 tumor model, characterized by a low proportion of tumor infiltrating lymphocytes with resistance to anti-PD-1/PD-L1 therapy [50]. An anti-PD-1/L1 antibody or isotype control (IgG2a) was injected (200 µg, intraperitoneally) twice a week after the subcutaneous 5 × 106 Panc 02 cells injection, with 2-BP or vehicle intraperitoneally at a dose of 50 mg/kg daily for 12 consecutive days. Survival time was defined as the day of death or an estimated tumor size > 1000 mm3. Strikingly, while the use of anti-PD-1 or anti-PD-L1 alone had almost no effect on Panc 02 tumor growth and mice survival time, the combination treatment of 2-BP and anti-PD-1 or anti-PD-L1 antibody markedly slowed tumor growth and prolonged the mice survival time (Fig. 9A–F). Importantly, the median survival of untreated Panc 02 mice was approximately 27 days, whereas the median survival of mice in the 2-BP plus anti-PD-1 and 2-BP plus anti-PD-L1 co-treatment groups was 78 and 49 days, respectively (Fig. 9C, F). The combination therapy was well-tolerated and no significant weight loss was observed during the treatment period. Taken together, our results indicated that inhibition of palmitoyl transferases by 2-BP can render nonresponsive Panc 02 tumor-bearing mice responsive to anti-PD-1/PD-L1 therapy.
Improved in vivo anti-tumor effect of 2-BP and PD-1/ PD-L1 blockade cotreatment. A–C Male C57BL/6 mice were injected subcutaneously with 5 × 106 Panc 02 cells, then, the tumor bearing mice were co-treated with 2-BP and anti-PD-1 antibody or IgG isotype control. Schematic representation of experimental plan (A). Tumor volume growth curves (B) and survival curves (C) of the mice bearing Panc 02 tumors with the indicated treatment (n = 6 mice per group). D–F Male C57BL/6 mice were subcutaneously injected with 5 × 106 Panc 02 cells, then, the tumor bearing mice were co-treated with 2-BP and anti-PD-L1 antibody or isotype control IgG. Schematic representation of experimental plan (D). Tumor volume growth curves (E) and survival curves (F) of the mice bearing Panc 02 tumors with the indicated treatment (n = 6 mice per group). Survival curves with time were developed until the animal’s death or the tumor volume reached > 1000 mm3. Differences between survival curves were analyzed using the log-rank (Mantel-Cox) test. Results shown are presented as mean ± SD. (*p < 0.05 and **p < 0.01)
Discussion
The palmitoylation status of specific tumor-related proteins can be regulated by selectively manipulating ZDHHCs to finely promote tumorigenesis or tumor suppression. Therefore, ZDHHCs-mediated palmitoylation that is frequently altered in the tumor microenvironment represents promising targets for cancer therapy. Importantly, genetic inhibition of ZDHHCs (ZDHHC3, 5, 17, 9, 12) has revealed significant therapeutic efficacy in some animal tumor models, including colon cancer, glioblastoma, leukemia, and ovarian cancer. However, the expression profile and function of these individual family members in pancreatic cancer remain undefined. In this work, we first screened the mRNA levels of ZDHHCs according to the TCGA-PAAD dataset. We found that six (ZDHHC3, ZDHHC4, ZDHHC5, ZDHHC7, ZDHHC9, and ZDHHC20) out of 23 ZDHHC family members were highly elevated at both transcriptional and protein levels in pancreatic cancer. Additionally, among these differently expressed ZDHHCs, ZDHHC3/4/7/9/20 expression was closely associated with pancreatic cancer patients’ individual stages. Of note, High expression of ZDHHC3/5/9/18/20 and ZDHHC3/4/5/7/9/20 were associated with poor prognosis in overall survival and relapse-free survival, respectively. Taken together, we identified ZDHHC3/9 as two members of 23 ZDHHCs that were differentially expressed in pancreatic carcinoma and were closely associated with pathological stage and prognostic value. RNA sequencing data from 33 pan-cancerous tissues in the GEPIA database showed that the transcriptional level of ZDHHC3 was selectively elevated in pancreatic cancer, so we further analyzed and validated the potential function of ZDHHC3 in PAAD. The CPTAC dataset and HPA database also validated the significant increase in ZDHHC3 protein levels in human pancreatic cancer tissues. Previous studies have identified ZDHHC3 as a therapeutic target for colon cancer and breast cancer [23, 24]. Mechanistically, ZDHHC3 could enhance tumor progression by increasing the level of palmitoylated modifications of PD-L1 in MC38 tumor models and support breast tumor progression by regulating cellular oxidative stress and senescence. These results and our findings reveal that ZDHHC3 might act as a prognostic biomarker and exert a tumor-promoting property in pancreatic cancer.
After exploring the expression patterns and prognostic values of ZDHHC3, we systematically analyzed its potential roles in the tumor immune microenvironment. In the TCGA-PAAD cohort, analysis of the immunological status revealed that ZDHHC3 expression levels were negatively correlated with the majority of immunostimulatory factors, MHC molecules, chemokines, and their ligands, indicating that ZDHHC3 may play a role in the highly immunosuppressive TME of pancreatic cancer. Cancer-immunity cycle can systematically analyze the multistep processes of the anti-tumor immune response and help to find potential molecular targets to enhance cancer immunotherapy [51]. Here, we noted that the ZDHHC3 gene expression is negatively correlated with several key processes of the tumor immune cycle and some immune cells infiltration including DC cells, NK cells, CD4+ T cells, CD8+ T cells, and macrophages. Meanwhile, ZDHHC3 expression was incompatible with PD-1, CTLA4, and LAG3 expression levels in some tumors, including pancreatic cancer, and suggested that the immunomodulatory effects of ZDHHC3 have the potential to be used in combination with these immune checkpoint blockade. Most patients with pancreatic cancer carry mutations in KRAS and TP53, and the production of these mutations creates a highly immunosuppressive tumor microenvironment specific to pancreatic cancer, including a large infiltration of the regulatory T cells (Treg) and myeloid derived suppressor cells (MDSCs), so current immune checkpoint blockade therapies have very limited efficacy [52, 53]. Our analysis of the mutational status of pancreatic cancer and ZDHHC3 expression revealed that pancreatic cancer patients with high expression of ZDHHC3 had a higher proportion of KRAS and TP53 mutations, and pancreatic cancer patients carrying KRAS and TP53 mutations also had significantly higher levels of ZDHHC3 expression. TP53 mutations in PAAD are associated with increased amplification of oncogenes, enhanced chromosomal instability, and a poor prognosis [54]. Given that ZDHHC3 potentially positively regulates the activities of mutant TP53, thus inhibiting ZDHHC3 may sensitize pancreatic tumors to anti-tumor treatments, including immunotherapy. The association and underlying molecular mechanism between TP53 mutations and high ZDHHC3 activity in pancreatic cancer need to be further investigated.
Further, we validated in a Panc 02 pancreatic cancer model that the knockdown of ZDHHC3 in tumor cells could delay tumor growth, which was associated with enhanced anti-tumor immune response. In line with the previous observation that the knockdown of ZDHHC3 enhanced CD8+ T cell infiltration in MC38 tumors [23], we also observed a similar phenomenon in the Panc 02 tumor model. In addition, we observed significantly increased levels of infiltration of DC cells, NK cells, CD4+ T cells, and M1 macrophages in ZDHHC3-knockdown tumors by flow cytometry analysis, further suggesting the activation of systemic anti-tumor immunity. These immune profiles in Panc 02 tumors are also consistent with our bioinformatics results of human PAAD samples based on ZDHHC3 expression. Interestingly, PD-L1 overexpression almost completely abolished the anti-tumor effects of ZDHHC3-knockdown on Panc 02 tumors, evidenced by increased tumor weight and decreased mice survival time. Our results also indicated that ZDHHC3 directly regulates PD-L1 expression at the protein level in pancreatic cancer cells. More importantly, overexpression of PD-L1 can almost abrogate the anti-cancer effect of ZDHHC3 knockdown on Panc 02 pancreatic tumor model, thus targeting ZDHHC3 may be a potential therapeutic approach to enhance anti-tumor immunotherapy. Future studies should focus on ZDHHC3 knockdown in combination with PD-1 or PD-L1 inhibitors in pancreatic cancer treatment strategies. Meanwhile, the major immune cell types involved in ZDHHC3-mediated tumor control need to be further identified.
Finally, we explored whether a pharmacological approach targeting palmitoyl transferases could be a potential therapeutic option for pancreatic cancer. We found that the palmitoyl transferase inhibitor, 2-BP, suppressed Panc 02/PANC-1 cell viability and tumor sphere-forming activities, as well as increased apoptosis in vitro with minimal effect on human pancreatic ductal epithelial cell line (HPDE6-C7). Meanwhile, intraperitoneal injection of 2-BP significantly delayed the growth of Panc 02 tumors and increased the anti-tumor immune response. More importantly, 2-BP can significantly improve the immune checkpoint blockade efficacy of anti-PD-1 or anti-PD-L1. In the absence of observed tumor suppression by PD-1 or anti-PD-L1 against pancreatic cancer in Panc 02 tumor model, synergistic treatment with 2-BP rendered tumors significantly sensitive to immune checkpoint blockade (ICB). Our results demonstrated that the palmitoyl transferase inhibitor, 2-BP may be a novel and effective pharmacological tool against pancreatic cancer.
However, there are still some limitations in our present study. First, our analysis was based primarily on the transcriptional data of ZDHHCs, the prognostic value of these ZDHHC enzymes and its potential role in TME needs to be further determined in large human pancreatic cancer samples. Second, the Panc 02 subcutaneously transplanted tumor model does not fully mimic changes in tumor progression in human pancreatic cancer, and we plan to further validate the therapeutic value of targeting ZDHHC3 as well as 2-BP treatment in a spontaneous pancreatic cancer tumor model in the future study. Despite these limitations, our results provide insight into the potential function of ZDHHC family members in pancreatic carcinogenesis and progression and pharmacologically validated whether targeting ZDHHCs would significantly increase the efficacy of ICB in pancreatic cancer.
Conclusion
Overall, our results indicated that some of ZDHHC enzyme genes are related to the prognosis of pancreatic cancer, and demonstrated that ZDHHC3 plays a critical oncogenic role in pancreatic cancer progression. Moreover, combined 2-BP and PD-1/PD-L1 inhibition overcomed intrinsic resistance to immune checkpoint blockade in a mouse model of pancreatic cancer.
Availability of data and materials
The datasets generated during and/or analyzed during the current study are available on reasonable request.
Change history
24 April 2024
This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s12967-024-05202-x
Abbreviations
- PAAD:
-
Pancreatic adenocarcinoma
- TCGA:
-
The Cancer Genome Atlas
- TPM:
-
Transcript per million
- HPA:
-
Human Protein Atlas
- OS:
-
Overall survival
- RFS:
-
Relapse-free survival
- GEO:
-
Gene Expression Omnibus
- IHC:
-
Immunohistochemical
- TIICs:
-
Tumor-infiltrating immune cells
- ICB:
-
Immune checkpoint blockade
- 2-BP:
-
2-bromopalmitate
- PD-1:
-
Programmed cell death 1
- PD-L1:
-
Programmed cell death ligand 1
- ssGSEA:
-
Single sample Gene Set Enrichment Analysis
- CPTAC:
-
Clinical Proteomic Tumor Analysis Consortium
- CTLA-4:
-
Cytotoxic T lymphocyte antigen 4
- LAG-3:
-
Lymphocyte activation gene 3
- GSVA:
-
Gene Set Variation Analysis
- NK:
-
Natural killer
- ELISA:
-
Enzyme-linked immunosorbent assay
- co-IP:
-
Co-immunoprecipitation
- RT-qRCR:
-
Real-time quantitative PCR
- IF:
-
Immunofluorescence
- TMB:
-
Tumor mutational burden
- MSI:
-
Microsatellite instability
References
Schizas D, Charalampakis N, Kole C, Economopoulou P, Koustas E, Gkotsis E, Ziogas D, Psyrri A, Karamouzis MV. Immunotherapy for pancreatic cancer: a 2020 update. Cancer Treat Rev. 2020;86: 102016.
Bliss LA, Witkowski ER, Yang CJ, Tseng JF. Outcomes in operative management of pancreatic cancer. J Surg Oncol. 2014;110:592–8.
Ilic M, Ilic I. Epidemiology of pancreatic cancer. World J Gastroenterol. 2016;22:9694–705.
Sahin IH, Askan G, Hu ZI, O’Reilly EM. Immunotherapy in pancreatic ductal adenocarcinoma: an emerging entity? Ann Oncol. 2017;28:2950–61.
Pietrobon V, Marincola FM. Hypoxia and the phenomenon of immune exclusion. J Transl Med. 2021;19:9.
Neesse A, Bauer CA, Öhlund D, Lauth M, Buchholz M, Michl P, Tuveson DA, Gress TM. Stromal biology and therapy in pancreatic cancer: ready for clinical translation? Gut. 2019;68:159–71.
Ullman NA, Burchard PR, Dunne RF, Linehan DC. Immunologic strategies in pancreatic cancer: making cold tumors hot. J Clin Oncol. 2022;40:2789–805.
Maggs L, Ferrone S. Improving the clinical significance of preclinical immunotherapy studies through incorporating tumor microenvironment-like conditions. Clin Cancer Res. 2020;26:4448–53.
Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, Roche PC, Lu J, Zhu G, Tamada K, et al. Tumor-associated B7–H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8:793–800.
Chen L, Han X. Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future. J Clin Invest. 2015;125:3384–91.
O’Reilly EM, Oh DY, Dhani N, Renouf DJ, Lee MA, Sun W, Fisher G, Hezel A, Chang SC, Vlahovic G, et al. Durvalumab with or without tremelimumab for patients with metastatic pancreatic ductal adenocarcinoma: a phase 2 randomized clinical trial. JAMA Oncol. 2019;5:1431–8.
Zhang Y, Qin Z, Sun W, Chu F, Zhou F. Function of protein S-palmitoylation in immunity and immune-related diseases. Front Immunol. 2021;12: 661202.
Blanc M, David F, Abrami L, Migliozzi D, Armand F, Burgi J, van der Goot FG. SwissPalm: protein palmitoylation database. F10000 Res. 2015;4:261.
Fukata Y, Murakami T, Yokoi N, Fukata M. Local palmitoylation cycles and specialized membrane domain organization. Curr Top Membr. 2016;77:97–141.
Greaves J, Chamberlain LH. DHHC palmitoyl transferases: substrate interactions and (patho)physiology. Trends Biochem Sci. 2011;36:245–53.
Lanyon-Hogg T, Faronato M, Serwa RA, Tate EW. Dynamic protein acylation: new substrates, mechanisms, and drug targets. Trends Biochem Sci. 2017;42:566–81.
Sanders SS, Martin DD, Butland SL, Lavallee-Adam M, Calzolari D, Kay C, Yates JR 3rd, Hayden MR. Curation of the mammalian palmitoylome indicates a pivotal role for palmitoylation in diseases and disorders of the nervous system and cancers. PLoS Comput Biol. 2015;11: e1004405.
** J, Zhi X, Wang X, Meng D. Protein palmitoylation and its pathophysiological relevance. J Cell Physiol. 2021;236:3220–33.
Lin H. Protein cysteine palmitoylation in immunity and inflammation. FEBS J. 2021;288:7043–59.
Greaves J, Chamberlain LH. New links between S-acylation and cancer. J Pathol. 2014;233:4–6.
Ko PJ, Dixon SJ: Protein palmitoylation and cancer. EMBO Rep 2018, 19.
Yeste-Velasco M, Linder ME, Lu YJ. Protein S-palmitoylation and cancer. Biochim Biophys Acta. 2015;1856:107–20.
Yao H, Lan J, Li C, Shi H, Brosseau JP, Wang H, Lu H, Fang C, Zhang Y, Liang L, et al. Inhibiting PD-L1 palmitoylation enhances T-cell immune responses against tumours. Nat Biomed Eng. 2019;3:306–17.
Sharma C, Wang HX, Li Q, Knoblich K, Reisenbichler ES, Richardson AL, Hemler ME. Protein acyltransferase DHHC3 regulates breast tumor growth, oxidative stress, and senescence. Cancer Res. 2017;77:6880–90.
Tian H, Lu JY, Shao C, Huffman KE, Carstens RM, Larsen JE, Girard L, Liu H, Rodriguez-Canales J, Frenkel EP, et al. Systematic siRNA screen unmasks NSCLC growth dependence by palmitoyltransferase DHHC5. Mol Cancer Res. 2015;13:784–94.
Chen X, Ma H, Wang Z, Zhang S, Yang H, Fang Z. EZH2 palmitoylation mediated by ZDHHC5 in p53-mutant glioma drives malignant development and progression. Cancer Res. 2017;77:4998–5010.
Chen X, Hao A, Li X, Ye K, Zhao C, Yang H, Ma H, Hu L, Zhao Z, Hu L, et al. Activation of JNK and p38 MAPK mediated by ZDHHC17 drives glioblastoma multiforme development and malignant progression. Theranostics. 2020;10:998–1015.
Liu P, Jiao B, Zhang R, Zhao H, Zhang C, Wu M, Li D, Zhao X, Qiu Q, Li J, Ren R. Palmitoylacyltransferase Zdhhc9 inactivation mitigates leukemogenic potential of oncogenic Nras. Leukemia. 2016;30:1225–8.
Zhang Z, Li X, Yang F, Chen C, Liu P, Ren Y, Sun P, Wang Z, You Y, Zeng YX, Li X. DHHC9-mediated GLUT1 S-palmitoylation promotes glioblastoma glycolysis and tumorigenesis. Nat Commun. 2021;12:5872.
Yuan M, Chen X, Sun Y, Jiang L, **a Z, Ye K, Jiang H, Yang B, Ying M, Cao J, He Q. ZDHHC12-mediated claudin-3 S-palmitoylation determines ovarian cancer progression. Acta Pharm Sin B. 2020;10:1426–39.
Chen S, Zhu B, Yin C, Liu W, Han C, Chen B, Liu T, Li X, Chen X, Li C, et al. Palmitoylation-dependent activation of MC1R prevents melanomagenesis. Nature. 2017;549:399–403.
Tang Z, Li C, Kang B, Gao G, Li C, Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017;45:W98–102.
Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM, Holko M, et al. NCBI GEO: archive for functional genomics data sets–update. Nucleic Acids Res. 2013;41:D991-995.
Chandrashekar DS, Bashel B, Balasubramanya SAH, Creighton CJ, Ponce-Rodriguez I, Chakravarthi B, Varambally S. UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses. Neoplasia. 2017;19:649–58.
Nagy A, Munkacsy G, Gyorffy B. Pancancer survival analysis of cancer hallmark genes. Sci Rep. 2021;11:6047.
Huang K, Lin Z, Ge Y, Chen X, Pan Y, Lv Z, Sun X, Yu H, Chen J, Yao Q. Immunomodulation of MiRNA-223-based nanoplatform for targeted therapy in retinopathy of prematurity. J Control Release. 2022;350:789–802.
Huang K, Liu X, Lv Z, Zhang D, Zhou Y, Lin Z, Guo J. MMP9-responsive graphene oxide quantum dot-based nano-in-micro drug delivery system for combinatorial therapy of choroidal neovascularization. Small. 2023;2023:e2207335.
Wellenstein MD, de Visser KE. Cancer-cell-intrinsic mechanisms sha** the tumor immune landscape. Immunity. 2018;48:399–416.
Ayers M, Lunceford J, Nebozhyn M, Murphy E, Loboda A, Kaufman DR, Albright A, Cheng JD, Kang SP, Shankaran V, et al. IFN-gamma-related mRNA profile predicts clinical response to PD-1 blockade. J Clin Invest. 2017;127:2930–40.
Charoentong P, Finotello F, Angelova M, Mayer C, Efremova M, Rieder D, Hackl H, Trajanoski Z. Pan-cancer immunogenomic analyses reveal genotype-immunophenotype relationships and predictors of response to checkpoint blockade. Cell Rep. 2017;18:248–62.
Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39:1–10.
Hu J, Yu A, Othmane B, Qiu D, Li H, Li C, Liu P, Ren W, Chen M, Gong G, et al. Siglec15 shapes a non-inflamed tumor microenvironment and predicts the molecular subtype in bladder cancer. Theranostics. 2021;11:3089–108.
Gupta R, Amanam I, Chung V. Current and future therapies for advanced pancreatic cancer. J Surg Oncol. 2017;116:25–34.
Lenkiewicz E, Malasi S, Hogenson TL, Flores LF, Barham W, Phillips WJ, Roesler AS, Chambers KR, Rajbhandari N, Hayashi A, et al. Genomic and epigenomic landsca** defines new therapeutic targets for adenosquamous carcinoma of the pancreas. Cancer Res. 2020;80:4324–34.
Spranger S, Dai D, Horton B, Gajewski TF. Tumor-residing Batf3 dendritic cells are required for effector T Cell trafficking and adoptive T cell therapy. Cancer Cell. 2017;31:711-723.e714.
Tang W, Xu N, Zhou J, He Z, Lenahan C, Wang C, Ji H, Liu B, Zou Y, Zeng H, Guo H. ALKBH5 promotes PD-L1-mediated immune escape through m6A modification of ZDHHC3 in glioma. Cell Death Discov. 2022;8:497.
Valle S, Alcala S, Martin-Hijano L, Cabezas-Sainz P, Navarro D, Munoz ER, Yuste L, Tiwary K, Walter K, Ruiz-Canas L, et al. Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells. Nat Commun. 2020;11:5265.
Miyazaki Y, Matsubara S, Ding Q, Tsukasa K, Yoshimitsu M, Kosai K, Takao S. Efficient elimination of pancreatic cancer stem cells by hedgehog/GLI inhibitor GANT61 in combination with mTOR inhibition. Mol Cancer. 2016;15:49.
Romero JM, Grunwald B, Jang GH, Bavi PP, Jhaveri A, Masoomian M, Fischer SE, Zhang A, Denroche RE, Lungu IM, et al. A four-chemokine signature is associated with a T-cell-inflamed phenotype in primary and metastatic pancreatic cancer. Clin Cancer Res. 2020;26:1997–2010.
Lv H, Lv G, Chen C, Zong Q, Jiang G, Ye D, Cui X, He Y, **ang W, Han Q, et al. NAD(+) metabolism maintains inducible PD-L1 expression to drive tumor immune evasion. Cell Metab. 2021;33(110–127): e115.
Chen DS, Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature. 2017;541:321–30.
Hashimoto S, Furukawa S, Hashimoto A, Tsutaho A, Fukao A, Sakamura Y, Parajuli G, Onodera Y, Otsuka Y, Handa H, et al. ARF6 and AMAP1 are major targets of KRAS and TP53 mutations to promote invasion, PD-L1 dynamics, and immune evasion of pancreatic cancer. Proc Natl Acad Sci USA. 2019;116:17450–9.
Zhang X, Mao T, Zhang B, Xu H, Cui J, Jiao F, Chen D, Wang Y, Hu J, **a Q, et al. Characterization of the genomic landscape in large-scale Chinese patients with pancreatic cancer. EBioMedicine. 2022;77: 103897.
Donehower LA, Soussi T, Korkut A, Liu Y, Schultz A, Cardenas M, Li X, Babur O, Hsu TK, Lichtarge O, et al. Integrated analysis of TP53 gene and pathway alterations in the cancer genome atlas. Cell Rep. 2019;28(1370–1384): e1375.
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Funding
This work was supported by the Natural Science Foundation of Sichuan Province (Grant number. 2022NSFSC1400) and Science and Technology Project of Chengdu (Grant number. 2022519).
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KH and ZL designed the project. KH, ZL, ZL, XL performed and analyzed all experiments. KH and ZL conceived the manuscript. All authors read and approved the final manuscript.
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This study was reviewed and approved by the school of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University Ethics Committee. All animal experimental procedures were approved by Laboratory Animal Ethics Committee of Wenzhou Medical University.
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Supplementary Information
Additional file 1: Fig. S1.
Representative immunohistochemical images of ZDHHC family members in human pancreatic cancer tissue from the HPA database. Fig. S2. Relationship between expression levels of ZDHHC3/4/7/9/15/17/20/22 and pathological stages of PAAD patients. A-H The association between mRNA expression of ZDHHCs and patients’ individual cancer stages of PAAD from the GEPIA database; p < 0.05 was considered statistically significant. Fig. S3. Relationship between ZDHHCs gene expression and pathological stages of PAAD patients. A-O The association between mRNA expression of ZDHHCs and patients’ individual cancer stages of PAAD from the GEPIA database. Fig. S4. Univariate and multivariate cox regression were used to test prognostic significance of ZDHHCs in TCGA-PAAD datasets. A-B The effect of ZDHHCs expression on overall survival (OS) (A) and relapse-free survival (RFS) (B) from the K-M plotter database. Fig. S5. Associations between ZDHHC3 expression and overall survival time in pan-cancers based on TCGA database. Fig. S6. Associations between ZDHHC3 expression and disease-specific survival in pan-cancers based on TCGA database. Fig. S7. Correlation between TMB or MSI and ZDHHC3 expression in pan-cancers. A A stick chart showing the correlation between the ZDHHC3 and TMB in different tumor types. The red curve represents the Spearman correlation coefficient, and the black value represents the range. B A stick chart showing the correlation between the ZDHHC3 and MSI in different tumor types. Fig. S8. Differentially expressed chemokines and paired receptors between high- and low-ZDHHC3 PAAD patients. A Boxplot showing the chemokines and paired receptors with significant expression differences between high- and low-ZDHHC3 groups in TCGA-PAAD cohort. B Boxplot showing the chemokines and paired receptors with no significant expression differences between high- and low-ZDHHC3 groups in TCGA-PAAD cohort. The R software ggpubr package was used to draw boxplots and perform Student’s t-test statistical analyses. Results shown are presented as mean ± SD. (ns, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001). Fig. S9. Mutation landscape between high- and low-ZDHHC3 in PAAD. A mutation landscape shows the differences in gene mutation frequencies in each set of PAAD samples using chi-square tests to classify the top 15 genes with the highest mutation frequencies according to the expression of ZDHHC3. Fig. S10. KEGG enrichment and pathway correlation analysis for ZDHHC3 in PAAD. A Top 20 KEGG enrichment pathways in PAAD. B The abscissa represents the distribution of the ZDHHC3 expression, and the ordinate represents the distribution of the pathway score. The density curve on the right represents the trend in distribution of pathway immune score, the upper density curve represents the trend in distribution of the ZDHHC3 expression. The value on the top represents the correlation p value, correlation coefficient and correlation calculation method. Fig. S11. Concentrations of CCL4, CCL5, CXCL9, and CXCL10 in shControl and shZDHHC3 Panc 02 tumor tissues on day 23 after tumor inoculation measured by ELISA. Results shown are presented as mean ± SD. (ns, not significant, *p < 0.05, and **p < 0.01). Fig. S12. ZDHHC3 knockdown inhibits PD-L1 expression at protein level in pancreatic cancer cells. (A-D) PANC-1 cells were transfected with siRNA. After 72 h, cells were harvested for (A) western blotting analysis, (B) RT-qPCR analysis, (C) flow cytometry assay and (D) corresponding quantification. (E) Representative immunofluorescence images of ZDHHC3 (green) and PD-L1 (red)staining of PANC-1 cells (blue, DAPI-labelled cell nuclei) and (F) corresponding quantification (Scale bars, 20 µm). (G) Reciprocal co-IP assay was performed to examine the interaction between endogenously expressed ZDHHC3 and PD-L1 in PANC-1 cells. Results shown are presented as mean ± SD. (ns, not significant and ***p < 0.001). Fig. S13. Relative expression of CCL4, CCL5, CXCL9, and CXCL10 in CTRL and 2-BP treated Panc 02 cells in vitro. Concentrations of CCL4, CCL5, CXCL9, and CXCL10 in CTRL and 2-BP treated Panc 02 cells for 24 h were measured by ELISA. Results shown are presented as mean ± SD. (ns, not significant and ***p < 0.001).
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Lin, Z., Lv, Z., Liu, X. et al. RETRACTED ARTICLE: Palmitoyl transferases act as novel drug targets for pancreatic cancer. J Transl Med 21, 249 (2023). https://doi.org/10.1186/s12967-023-04098-3
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DOI: https://doi.org/10.1186/s12967-023-04098-3