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World Journal of Oncology

Original Article

Volume 15, Number 2, April 2024, pages 169-180

Sphingosine-1-Phosphate Inhibition Increases Endoplasmic Reticulum Stress to Enhance Oxaliplatin Sensitivity in Pancreatic Cancer

The prognosis for pancreatic ductal adenocarcinoma (PDAC) patients remains poor with 5-year overall survival of less than 10% [1]. Unfortunately, standard conventional chemotherapy regimens including FOLFIRINOX and gemcitabine/nab-paclitaxel provide marginal survival benefit of only months as resistance commonly develops [2, 3]. Similarly, PDAC is resistant to immunotherapeutic strategies either as monotherapy [4] or in combination with chemotherapy [5, 6]. Understanding the underlying mechanisms of chemotherapy resistance is essential to develop strategies to improve outcomes for PDAC patients.

Growing evidence suggests that cancers harness metabolic pathways to promote survival [7-10]. Sphingolipids are bioactive, structural components of the cell membrane involved in cell growth, cellular proliferation, and programmed cell death [11]. A concept of a “rheostat” between pro-apoptotic ceramides and pro-proliferative sphingosine-1-phosphate (S1P) [12, 13] has been well described [14], which may drive a shift between a pro-apoptotic or pro-survival phenotype in cancer cells depending on the balance of these two sphingolipid species [15-17]. Studies support the role of S1P in promoting PDAC cell survival and proliferation [18-22]. Of particular clinical relevance is inhibition of the sphingosine kinase-2 (SPHK2)/S1P axis [23] via the small molecule inhibitor ABC294640 (ABC). ABC is the only drug targeting S1P that has been used in an oncology clinical phase I trial, where it induced a partial treatment response as monotherapy in cholangiocarcinoma and maintained stable disease in other advanced solid tumors [24].

Multiple antineoplastic agents such as cisplatin [25], oxaliplatin (Oxa) [26], and 5-fluorouracil (5-FU) [27] can induce cellular stresses leading to endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) [28]. Continued activation of UPR pathways results in apoptosis [29-32] and immunogenic cell death (ICD) [33, 34]. Interestingly, ER stress and the ceramide/S1P axis intersect at multiple crucial junctions. ER stress and protein kinase R-like endoplasmic reticulum kinase (PERK) activation can induce ceramide generation [35]. Conversely, ER stress can induce S1P synthesis through upregulation of SPHK2 [36] and can utilize S1P as part of downstream signaling mechanisms [37]. Evidence from preclinical cancer models demonstrates that cancer cells can upregulate SPHK2 as a survival mechanism in response to ER stress [38], and increased intracellular S1P can promote cell survival in a PERK-dependent fashion [39].

Together, these data support the rationale for a comprehensive understanding of the role of sphingolipid metabolism in PDAC, as well as investigating the role of SPHK2/S1P as a potential therapy target for PDAC. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress and ICD to improve therapeutic response to Oxa in PDAC. Thus, the objectives of this study were to first characterize sphingolipid dysfunction via an in silico approach across a large, combined RNA-seq dataset of normal pancreas and PDAC samples. Considering the relationship between S1P and ER stress, we also explored the impact of in vitro antitumoral effect of S1P inhibition via ER stress on ICD induction, both alone and in combination with Oxa. Finally, we investigated the therapeutic effectiveness of S1P inhibition combined with Oxa using an in vivo orthotopic murine syngeneic PDAC model to understand the impact on anti-tumoral immunity.

Publicly available RNA sequencing data were analyzed using normal pancreatic samples from the GTEx [40] and pancreatic tumor samples from TCGA [41]. The expected RSEM count data from the combined GTEx/TCGA dataset were downloaded from the UCSC Xena platform [42] using UCSCXenaTools v1.4.8 [43]. The RNA sequencing data were back transformed into raw counts and filtered to remove lowly expressed genes and to include only expression data from protein coding genes [44]. An annotation of genes in the sphingolipid metabolic process as well as mediators of the S1P biosynthetic process was curated from Gene Ontology (GO: 0006665) [45] and KEGG (map00600) [46]. The count data for this annotation were converted to log2-counts-per-million values, and unsupervised clustering of these genes was performed in R v4.3.0 using Pearson correlation. Differential gene expression was performed using edgeR v3.42.4 [47] using the quasi-likelihood pipeline with false discovery rate correction at a nominal value of 0.05. The log2 fold change cutoff was set at a nominal value of 0.5.

To understand the clinical association between dysregulated sphingolipid metabolism and clinical outcomes of PDAC, clinical data for PDAC samples from TCGA [41] were analyzed using GEPIA2 [48] for associations between sphingolipid mediator genes and overall survival using Kaplan-Meier estimates.

Institutional Review Board approval is not applicable to this study. This study was conducted in compliance with all the applicable institutional ethical guidelines for the care, welfare, and use of animals (IACUC Protocol #AN-8645).

Human cell lines were obtained directly from the American Type Culture Collection (ATCC). Murine Panc02 cells were obtained from the NCI DCTD Tumor Repository (NCI, Frederick MD) and KPC cells were graciously provided by Dr. Guttridge (Medical University of South Carolina, Charleston, SC). MIAPaCa-2, BxPC-3, HPAC, Capan-2, Panc-1, and AsPC-1 cells were obtained from the ATCC. L3.6pl cells were graciously provided by L. Ellis (MD Anderson, Houston TX). MIAPaCa-2, HPAC, Panc-1, Panc02 and KPC cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (50 IU/mL), streptomycin (50 µg/mL) in a humidified incubator with 5% CO₂ at 37 °C. BxPC-3 and AsPC-1 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% FBS, penicillin (50 IU/mL), streptomycin (50 µg/mL) in a humidified incubator with 5% CO₂ at 37 °C. Capan-2 cells were grown in McCoys A medium supplemented with 10% FBS, penicillin (50 IU/mL), streptomycin (50 µg/mL) in a humidified incubator with 5% CO₂ at 37 °C. All cell lines used in this study were tested for mycoplasma contamination at regular intervals using a PCR-based detection method and submitted for STR authentication at regular intervals.

Oxa was purchased from SelleckChem and dissolved in distilled water. ABC was provided by Apogee Biotechnology Corporation (Hummelstown, PA). ABC was dissolved in DMSO for in vitro treatment and in a formula of 46.6% saline, 46.6% PEG 400 and 6.6% Tween-80 for in vivo animal treatment.

SPHK2 knockdown in human PDAC cells was performed by transfection of small interfering RNA (siRNA) which target human SPHK2 gene (targeted sequence: #1, CCCUGAAACUAAACAAGCUUGGUAC and #2, CGUGCUUCCCAUGAUCUCUGAAGCT, 5′ to 3′) with Lipofectamine^® 3000 DNA Transfection Reagent (Invitrogen) according to the manufacturer’s protocol. A non-target scrambled siRNA (scrambled sequence: UACAGUUUAUUGAUAUUCAAUAAAG, 5′ to 3′) was used as a negative control of siSPHK2. Forty-eight hours after transfection, cells were added with the indicated treatment in a six-well plate. SPHK2 protein level was determined by immunoblotting with the specific antibody.

MTT assay was used to determine cell viability in vitro. Cells were seeded in 96-well plates at 1,000 - 3,000 cells per well for 24 h, followed by indicated treatment for 24 to 72 h. Cells were then incubated with MTT solution (thiazolyl blue tetrazolium bromide, 5 mg/mL) (Tocris Bioscience) at different endpoints at 37 °C for 2 h. After the removal of medium and MTT solution, 100 µL DMSO was added to each well. The absorbance was read at 570/630 nm on a 96-well CLARIOstar (BMG Labtech) plate reader. Each experiment was performed at least in triplicates.

Cells were harvested and lysed in TNN buffer (50 mM Tris, 0.25 M NaCl, 5 mM EDTA, and 0.5% Nonidet P-40), and total proteins of tumor samples were extracted in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS) with protease inhibitor cocktails. After sonication, the protein concentration was measured by BCA assay (Invitrogen). The lysates were boiled in SDS loading buffer, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred to the polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and immunoblotted with described antibodies. After imaging of the immunoblots, optical density (OD) was calculated for the detected protein bands using ImageJ by normalizing the target band against the loading control, and then comparing protein bands of interest against a control protein band.

The following antibodies were used for immunoblotting: anti-SPHK2 antibody (#17096-1-AP, Proteintech), anti-phospho-eIF2α antibody (#3597, Cell Signaling), anti-phospho-PERK antibody (#3179, Cell Signaling), anti-PERK antibody (#sc-377400, Santa Cruz), anti-eIF2α antibody (#sc-133132, Santa Cruz), anti-GAPDH antibody (#sc-25778, Santa Cruz), anti-β-actin antibody (#sc-47778, Santa Cruz), ER stress/UPR antibody pack (#NBP2-52746, Novus), anti-rabbit IgG-HRP (#7074, Cell Signaling) and anti-mouse IgG-HRP (#7076, Cell Signaling).

The Lumit™ HMGB1 (human) immunoassay kit (Promega) was used to detect HMGB1 released from cultured cell environment undergoing immunogenic cell death. Generally, 10,000 - 15,000 cells were seeded in 96-well plate and followed by indicated treatment next day. The bioluminescence signal was measured on a 96-well CLARIOstar (BMG Labtech) plate reader within 45 min after the addition of Lumit™ substrate. Each experiment was performed at least in triplicates.

This study was conducted in compliance with all the applicable institutional ethical guidelines for the care, welfare, and use of animals. Male C57B1/6 mice of 7 to 8 weeks old were purchased from Jackson Laboratory and housed in a pathogen-free, biohazard barrier facility according to protocols approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine. During the surgery, mice were anesthetized, and the left flank was shaved and sterilized with Betadine. A 1-cm subcostal incision was made through the skin, subcutaneous tissues, and peritoneum. The pancreas was exteriorized, and 0.15 × 10⁶ KPC cells in 0.03 mL PBS (Hyclone) were injected into the pancreatic tail using a sterile 0.5 mL insulin syringe with a 28-gauge needle. Mice were administered pain medication for 3 days post-operatively and sacrificed if evidence of pain or suffering was present.

After orthotopic injection of KPC cells was performed, 7 to 10 healthy appearing mice were randomized to four treatment groups. Treatment was started 7 days after tumor implantation. Oxa at 3 mg/kg was intraperitoneally injected twice weekly and 50 mg/kg of ABC dissolved in the formula of 46.6% saline, 46.6% PEG 400 and 6.6% Tween-80 was administrated by oral gavage three times per week. The same amount of formula was given as vehicle control of gavage feeding. Mice were monitored for health during the treatment and euthanized 3 weeks after the treatment to measure tumor burden.

Following sacrifice, tumors were resected, fixed overnight in 10% buffered formalin, set in paraffin blocks, and cut into 5 µm thick sections. Sections were stained with standard hematoxylin and eosin staining. Sections were also stained with Ki67 antibody (1:400 dilution, cat#12202, Cell Signaling), cleaved caspase-3 (CC3) antibody (1:400 dilution, cat#9661, Cell Signaling), HMGB1 antibody (1:1,000 dilution, cat#6893, Cell Signaling), CD3 antibody (1:400 dilution, cat# NB600-1441SS, Novus Biologicals), and CD8 antibody (1:400 dilution, cat#98941s, Cell Signaling). Stained slides were scanned using the EVOS M5000 Imaging System at × 40 magnification after white balance calibration. From each tumor sample, five randomly selected regions chosen by a lab technician blinded to treatment groups were quantified for positively stained nuclei per high power field using QuPath. The intensity of stained nuclei was calculated by thresholding the stained nuclear OD into low, medium, and high intensity staining and converted into an H-score.

Statistical analysis was performed using GraphPad PRISM v9.5.0. The unpaired two-tailed t-test or two-way analysis of variance (ANOVA) were used to compare the differences between experimental groups, where appropriate. The false discovery rate was controlled using the two-stage step-up method of Benjamini, Krieger and Yekutieli [49], with a q-value < 0.05 considered statistically significant. Similarly, for multiple comparisons, Tukey’s honest significant difference (HSD) test was used for multiple comparisons in two-way ANOVA, with an adjusted P-value < 0.05 considered statistically significant.

To evaluate the role of sphingolipid metabolic dysregulation in PDAC, we initially analyzed publicly available RNA sequencing data of normal and pancreatic tumor samples. In total, 167 normal pancreatic tissue samples from GTEx and 183 pancreatic tumor samples from TCGA were included in this analysis. Unsupervised clustering of annotated sphingolipid metabolic genes discriminated between normal pancreatic tissue and PDAC (Fig. 1a). On comparison of PDAC and normal tissue, 100 of the 140 sphingolipid metabolic genes were significantly differentially expressed with a log2 fold change greater or less than 0.5 (Fig. 1b). For example, serine palmitoyltransferase is the initial and also rate-limiting enzyme in global sphingolipid biosynthesis to form 3-ketosphinganine [50]. Two of its isoforms, SPTLC1 and SPTLC2, are significantly upregulated in PDAC. Other S1P biosynthesis pathway genes including ACER1, ACER3, and SPHK1 were significantly upregulated compared to normal pancreatic tissue (Fig. 1b). The immediate downstream receptors of S1P are five G-protein-coupled receptors known as sphingosine-1-phosphate receptors (S1PR1-5) [51]. Four of the five S1P receptors (S1PR1-4) are also significantly upregulated in PDAC compared to normal pancreas. Similarly, a panel of pancreatic cancer cell lines demonstrated strong expression of upstream and downstream S1P mediators (Fig. 2a). Based on the TCGA PDAC patient cohort, multiple significantly overexpressed sphingolipid genes involved in S1P biosynthesis such as KDSR, SPHK1, and SPTLC2 are significantly associated with worsened survival (P < 0.05) (Supplementary Material 1, www.wjon.org). Taken together, these data suggest that S1P biosynthetic pathways (Fig. 2b) are highly dysregulated in PDAC and likely contribute to aggressive biology observed in patients with PDAC.

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Figure 1. In silico sphingolipid profiling in PDAC. (a) Unsupervised clustering of genes included in the sphingolipid metabolic annotation (GO: 0006665) demonstrates accurate discrimination of normal pancreas tissue from pancreatic tumor tissue using samples from GTEx and TCGA. (b) Volcano plot of differentially expressed genes in the sphingolipid metabolic annotation, with S1P biosynthetic pathway members identified. PDAC: pancreatic ductal adenocarcinoma; S1P: sphingosine-1-phosphate.

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Figure 2. Sphingolipid mediator profiling across human and murine PDAC cell lines. (a) Western blot profiling of upstream and downstream mediators of S1P signaling. (b) Diagram of the biosynthetic pathway of S1P synthesis, with significantly differentially expressed members highlighted. PDAC: pancreatic ductal adenocarcinoma; S1P: sphingosine-1-phosphate.

Next, we investigated the antitumoral effects of S1P signaling inhibition in PDAC cell lines. Based on cell viability assay, 48-h knockdown of SPHK2 (siSPHK2) demonstrated no significant change in cell viability compared to controls across multiple PDAC cell lines. However, siSPHK2 significantly enhanced Oxa cytotoxicity in multiple cell lines (Fig. 3a). For example, in the MiaPACA-2 cells, the addition of siSPHK2 to Oxa decreased cell viability by 40% compared to cells treated with control scramble siRNA and Oxa (P-value < 0.001). SPHK2 inhibition successfully sensitized PDAC cells to Oxa treatment at both high and low Oxa doses. The effect of SPHK2 inhibition was confirmed using an additional siRNA clone.

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Figure 3. SPHK2 inhibition enhances cytotoxicity of Oxa in PDAC cell lines. (a) After transfection with either siSPHK2 (black bars) or scramble control (gray bars), MiaPaCa-2 and Panc-1 cells were treated with Oxa for 48 h and tested for cell viability based on MTT assay. Western blots confirming effective knockdown of SPHK2 using siRNA clones are displayed below. (b) PDAC cell lines were treated with combination of 20 µM ABC and Oxa for 48 h. Cell viability was assessed by MTT assay. For cell viability assay, data shown represent at least three biological replicates (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Oxa: oxaliplatin; PDAC: pancreatic ductal adenocarcinoma; S1P: sphingosine-1-phosphate; SPHK2: sphingosine kinase 2.

To further explore the ability of SPHK2-S1P inhibition to sensitize PDAC cells to Oxa, we tested the combination of ABC, a small molecular inhibitor of SPHK2-S1P signaling, and Oxa. While treatment with ABC alone inhibited cell viability, we observed that the combination of ABC and Oxa increased cancer cell cytotoxicity compared to Oxa alone across multiple PDAC cell lines. In Panc-1 and KPC PDAC cell lines, treatment with ABC decreased cell viability by an additional 20% compared to treatment with 20 µM of Oxa alone (P-value < 0.001, Fig. 3b). This effect was apparent at multiple doses of Oxa, similar to the combination of siSPHK2 with Oxa. Taken together, we demonstrate that therapeutic inhibition of SPHK2-S1P signaling can enhance the antitumoral effects of Oxa.

Oxa is a well-described agent that drives ER stress [26] and UPR through phosphorylation [52] of the PERK/eukaryotic initiation factor 2α (eIF2α) axis [26], as well as immune activation through ICD [33, 34]. Considering the enhanced cytotoxicity of combination Oxa and S1P inhibition in PDAC, we explored the role of S1P in mediating response to Oxa and its immunogenic effects. In both human and murine PDAC cell lines, Oxa administration induced ER stress and phosphorylation/activation of the PERK/eIF2α pathway (Fig. 4a). siSPHK2 alone mildly activated the ER stress pathway through PERK/eIF2α axis, with a twofold increase in phosphorylated eIF2α expression and phosphorylated PERK expression across both cell lines based on densitometry analysis (Fig. 4a, b). Further, the combination of Oxa and SPHK2 knockdown induced a greater degree of ER stress and UPR activation compared to either treatment alone. For example, in the KPC cell line, combination treatment produced a 1.3-fold increase in phosphorylated PERK expression and a twofold increase in phosphorylated eIF2α expression compared to the cells treated with control scramble siRNA and Oxa. Similar to the results with siSPHK2, ABC (Fig. 4b) increased expression of ER stress mediators and increased the response to Oxa. While either Oxa or ABC treatment alone of KPC cells both increased protein expression of the PERK/eIF2α pathway, combination therapy leads to a 2.6-fold increase in phosphorylated PERK expression and a fivefold increase in phosphorylated eIF2α expression compared to controls. This, along with our previous data demonstrating the combined antitumoral efficacy of S1P signaling inhibition and Oxa, further supports the concept that inhibition of S1P signaling can increase Oxa-induced ER stress in PDAC.

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Figure 4. SPHK2 inhibition enhances oxaliplatin-induced ER stress in PDAC cell lines. (a) Loss of SPHK2 increases ER stress through eIF2α and PERK phosphorylation in human and murine cell lines. OD values for p-PERK and p-eIF2α are displayed below the relevant bands. (b) The S1P inhibitor ABC increases ER stress through eIF2α and PERK phosphorylation in human and murine cell lines. OD values for p-PERK and p-eIF2α are displayed below the relevant bands. (c) Loss of SPHK2 and (d) ABC administration increase extracellular HMGB1 release compared to control or Oxa, suggestive of increased ICD induction (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). ABC: ABC294640; ER: endoplasmic reticulum; OD: optical density; Oxa: oxaliplatin; PDAC: pancreatic ductal adenocarcinoma; S1P: sphingosine-1-phosphate; SPHK2: sphingosine kinase 2.

Considering the close relationship between ER stress and ICD [33, 34, 53, 54], as well as the strong influence of S1P signaling on ER stress, we investigated the ability of S1P to augment ICD. HMGB1 is a key mediator of ICD [55, 56]. Compared with control scramble siRNA, siSPHK2 resulted in a twofold increase in extracellular HMGB1 after 72 h (Fig. 4b, P-value < 0.0001). Experimental results with ABC inhibiting the SPHK2-S1P pathway produce similar results (Fig. 4c). Compared to DMSO control, treatment with ABC produced an almost threefold increase in HMGB1 release in MIAPaCa-2 cells (P-value < 0.0001) and a twofold increase in KPC cells (P-value < 0.0001). ABC administration also led to significant increases in HMGB1 expression compared to Oxa treatment alone in both MIAPaCa-2 and KPC cell lines (P-values < 0.0001). This effect was sustained with combination treatment, as treatment with ABC with Oxa maintained a twofold increase in HMGB1 release compared to Oxa alone across both cell lines (P-values < 0.0001). The increase in extracellular HMGB1 release is consistent across different doses of Oxa treatment, suggesting that S1P inhibition can potentiate the post-apoptotic release of HMGB1 associated with Oxa treatment [57] to stimulate a more robust ICD response in PDAC. Taken together, our evidence supports the concept that S1P signaling inhibition can potentiate ICD induction from conventional chemotherapy.

Next, we investigated the in vivo antitumor effect of the combination of S1P inhibition inhibitor and Oxa using an orthotopic KPC model (Fig. 5a). Mice with KPC tumors received treatment for 21 days at which time tumors were harvested and analyzed. As expected, Oxa reduced tumor weight compared with vehicle control by 51% (Fig. 5b, P-value = 0.03). The combination of ABC and Oxa reduced mean tumor weight by 17% compared to Oxa, although this was not statistically different (P-value = 0.72). Considering the robust ER stress and ICD signaling in response to combination S1P and Oxa, we further evaluated the ability of ABC to potentiate Oxa effects on proliferation and apoptosis (Fig. 5c, d). Based on immunohistochemical staining, Oxa significantly reduced Ki67 expression compared to the control group (P-value < 0.0001), consistent with the known anti-proliferative effects of Oxa. However, we did not observe significant differences in Ki67 with ABC treatment either as monotherapy compared to control, or with combination treatment compared to Oxa alone (P-values = 0.13 and 0.64, respectively), suggesting that ABC did not exert an in vivo antiproliferative effect. In contrast, the combination of Oxa and ABC significantly increased apoptotic CC3 staining more than twofold compared to control (P-value = 0.025). This effect was not observed with either ABC or Oxa treatment alone, suggesting that ABC can potentiate the therapeutic benefit of Oxa in PDAC.

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Figure 5. SPHK2-S1P inhibition enhances antitumor activity of Oxa in an orthotopic KPC model. Following treatment for 21 days, orthotopic tumors were harvested and analyzed. (a) In vivo experimental schema. After orthotopic injection of KPC cells, mice were randomized to four treatment groups of 7 - 10 mice each, starting 7 days post-operatively. Oxa at 3 mg/kg (red arrows) was intraperitoneally injected twice weekly and 50 mg/kg of ABC (green arrows) was administrated by oral gavage three times per week. Tumors were harvested 3 weeks after tumor implantation. (b) Analysis of harvested tumor weights by treatment group (*P < 0.05, **P < 0.01). (c) Analysis of IHC stained tumor samples of positively stained nuclei/HPF for Ki67, CC3, CD3, and CD8 between treatment groups. Intensity of HMGB1 staining was quantified by calculating an H-score (*P < 0.05, **P < 0.01). (d) Representative images of IHC stained images of tumors in different treatment groups. ABC: ABC294640; HPF: high power field; IHC: immunohistochemical; Oxa: oxaliplatin; S1P: sphingosine-1-phosphate; SPHK2: sphingosine kinase 2.

Considering the findings that SphK2 inhibition enhances cellular ER stress and ICD signaling, we explored whether ABC could enhance Oxa-mediated ICD and immune activation in the orthotopic PDAC model (Fig. 5c). Treatment with Oxa, ABC, or combined Oxa and ABC all lead to significantly increased intensity of HMGB1 staining compared to vehicle control (P-values < 0.001, < 0.03, and < 0.01, respectively), although there was no significant difference within these treatment groups. We also noted a trend toward decreased CD3 staining (P-value = 0.13) as well as a significant 33% decrease in CD8 staining (P-value = 0.028) with Oxa monotherapy treatment compared to vehicle control. However, the combined administration of ABC and Oxa led to a robust increase in CD3 (P-value = 0.006) and CD8 (P-value = 0.04) compared to Oxa alone.

Understanding chemoresistance mechanisms in PDAC is crucial to improving therapeutic strategies to impact patient outcomes [2, 3]. Cancers, including PDAC, effectively harness metabolic mediators such as sphingolipids to promote survival, metastasis, and therapy resistance [8, 14, 21, 22]. Considering the close relationship between SPHK2-S1P signaling and ER stress, we hypothesized that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress and ICD to improve therapeutic and immunologic response to Oxa in PDAC.

In the present study, we demonstrated sphingolipid metabolism is highly dysregulated in PDAC compared to normal pancreatic tissue. The majority of genes in the S1P biosynthetic pathways and immediate downstream S1P receptors are significantly upregulated in PDAC, while key members such as KDSR, SPHK1, and SPTLC2 are associated with worsened survival. Combined with previous studies that observed higher levels of S1P in tumor [18] and its associations with PDAC tumor growth [19] and carcinomatosis [20], our evidence suggests that the ceramide-S1P rheostat [14] is shifted to a pro-survival phenotype, and strongly implicates S1P biosynthetic and signaling pathways as mediators of the aggressive tumor biology of PDAC.

In regard to the relationship between ER stress and SPHK2, we further explored one aspect of sphingolipid and S1P signaling in PDAC by determining its function in mediating chemoresistance. Our experimental data demonstrate that targeting the SPHK2-S1P axis through either siRNA knockdown or administration of the small-molecule inhibitor ABC results in increased chemosensitivity to Oxa at multiple doses and across multiple human and murine PDAC cell lines. We also demonstrate that while both Oxa and S1P inhibition increase ER stress and subsequent phosphorylation of the PERK/eIF2α pathway, the combination of the two therapies induces a greater degree of phosphorylation compared to either treatment alone. Taken together, our data suggest that physiologically, SPHK2-S1P signaling serves to blunt Oxa-induced ER stress and UPR activation. PDAC possesses a hypoxic, nutrient-deprived microenvironment [58] and exhibits a basal amount of ER stress [59] in response to its disadvantageous microenvironmental milieu. However, continual accumulation of misfolded proteins and persistent ER stress without resolution leads to apoptotic cell death [29-32]. Inhibition of the SPHK2-S1P axis may further increase basal and Oxa-induced ER stress, leading to an enhanced pro-apoptotic state as observed in the in vivo experiment. These findings are consistent with similar cancer investigations that have studied the potentiation of Oxa by inducing greater ER stress [60, 61]. The increase in in vivo apoptotic markers in our PDAC tumors is also encouraging and suggests a chemosensitizing role for SPHK2/S1P inhibition.

In addition to chemoresistance, PDAC is plagued by resistance to immunotherapy [4-6, 58]. Induction of ICD has been explored as a potential adjunctive therapy strategy [62-64], and multiple drugs have been investigated but not yet clinically utilized for their ability to induce ICD [65], including Oxa [66]. The potential for S1P inhibition as an immune adjunct is attractive, as the successes seen with immune remodeling from combination in other malignancies such as lung and breast cancer [67, 68] have not been reproduced in PDAC clinical trials [5, 6]. Compared to Oxa alone, in vitro SPHK2 knockdown and ABC administration result in a higher degree of phosphorylation of eIF2α and induce significantly higher levels of extracellular HMGB1 release, both hallmarks of ICD [34, 65]. Our in vivo results show a significant increase in the intensity of HMGB1 staining along with increased CD3 and CD8 marker expression in our tumors treated with ABC and Oxa. We also noted a significant decrease in CD8 staining with Oxa monotherapy compared to vehicle control, which was ameliorated with combination treatment with Oxa and ABC. Oxa administration has been reported to increase the proportion of CD8 T cells in tumors [69-71]. However, in vivo Oxa models have also reported hematological toxicity and suppression of CD8 T cell populations at doses similar to those used in our experiment [72], suggesting that dose and timing optimization is important in designing future chemoimmunotherapy strategies. It is interesting to note that our combination therapy group with Oxa and ABC did not experience this reduction in CD8 staining. These data suggest that ABC may have a protective effect on CD8 tumor immune infiltrates, although the mechanism behind this is not yet elucidated and requires further experiments. Taken together with the increase in apoptotic marker expression seen in the tumors treated with combination therapy, our data argue for a role in SPHK2-S1P signaling inhibition in increasing Oxa-induced ICD and immune activation in PDAC tumors. ICD inducers such as Oxa have also demonstrated the ability to increase PD-1 and PD-L1 expression [66] as well as improved outcomes in combination with immunotherapy in solid tumors [73, 74]. S1P inhibition represents an appealing strategy to potentiate Oxa-induced ICD and may also serve as an immune adjunct to support future chemoimmunotherapy combinations in PDAC.

Our work highlights the significant dysregulation of sphingolipids in PDAC. Furthermore, our evidence implicates sphingolipid metabolism and S1P signaling in mediating chemoresistance to Oxa, as well as highlighting a potential novel therapeutic target for chemosensitizing PDAC. These findings also strongly support future efforts to explore the downstream immune modulatory effects of ER stress and ICD induction following therapeutic targeting of S1P. Finally, the immune activating properties of targeting S1P alone or with chemotherapy point to an exciting strategy to boost chemoimmunotherapeutic strategies for PDAC.

Suppl 1. Differential expression of selected individual sphingolipid mediators with KRAS mutation and survival.

Acknowledgments

This work was supported in part by Merit Review Award #I01 CX001880-01A1 from the United States (US) Department of Veterans Affairs Biomedical Laboratory Research and Development Program (ERC), and by an award from the National Pancreatic Cancer Foundation. This was also supported by the Dan L Duncan Comprehensive Cancer Center Bench to Bedside Research pilot grant by (Camp & Lee: P30 CA125123).

Financial Disclosure

This work was supported in part by Merit Review Award #I01 CX001880-01A1 from the United States (US) Department of Veterans Affairs Biomedical Laboratory Research and Development Program (ERC), and by an award from the National Pancreatic Cancer Foundation.

Conflict of Interest

The authors declare no potential conflict of interest.

Informed Consent

Not applicable.

Author Contributions

ZG: conceptualization, methodology, formal analysis, investigation, writing - original draft preparation, review, and editing, visualization; HJ: methodology, investigation, writing - review and editing; YX: methodology, investigation, writing - review and editing; SWK: investigation, writing - review and editing; JD: methodology, investigation, writing - review and editing; JC: methodology, investigation, writing - review and editing; BO: writing - review and editing; HSL: writing - review and editing, ERC: conceptualization, resources, supervision, project administration, writing - review and editing.

Data Availability

The data supporting the findings of this study are in whole or part based upon data generated by the TCGA Research Network and can be accessed at https://www.cancer.gov/tcga.

Abbreviations

ABC: ABC294640; ICD: immunogenic cell death; OD: optical density; Oxa: oxaliplatin; PDAC: pancreatic ductal adenocarcinoma; S1P: sphingosine-1-phosphate; SPHK2: sphingosine kinase 2

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