Positive feedback in Cav-1-ROS signalling in PSCs mediates metabolic coupling between PSCs and tumour cells.

Caveolin-1 (Cav-1) is the principal structural component of caveolae, and its dysregulation occurs in cancer. However, the role of Cav-1 in pancreatic cancer (PDAC) tumorigenesis and metabolism is largely unknown. In this study, we aimed to investigate the effect of pancreatic stellate cell (PSC) Cav-1 on PDAC metabolism and aggression. We found that Cav-1 is expressed at low levels in PDAC stroma and that the loss of stromal Cav-1 is associated with poor survival. In PSCs, knockdown of Cav-1 promoted the production of reactive oxygen species (ROS), while ROS production further reduced the expression of Cav-1. Positive feedback occurs in Cav-1-ROS signalling in PSCs, which promotes PDAC growth and induces stroma-tumour metabolic coupling in PDAC. In PSCs, positive feedback in Cav-1-ROS signalling induced a shift in energy metabolism to glycolysis, with up-regulated expression of glycolytic enzymes (hexokinase 2 (HK-2), 6-phosphofructokinase (PFKP) and pyruvate kinase isozyme type M2 (PKM2)) and transporter (Glut1) expression and down-regulated expression of oxidative phosphorylation (OXPHOS) enzymes (translocase of outer mitochondrial membrane 20 (TOMM20) and NAD(P)H dehydrogenase [quinone] 1 (NQO1)). These events resulted in high levels of glycolysis products such as lactate, which was secreted by up-regulated monocarboxylate transporter 4 (MCT4) in PSCs. Simultaneously, PDAC cells took up these glycolysis products (lactate) through up-regulated MCT1 to undergo OXPHOS, with down-regulated expression of glycolytic enzymes (HK-2, PFKP and PKM2) and up-regulated expression of OXPHOS enzymes (TOMM20 and NQO1). Interrupting the metabolic coupling between the stroma and tumour cells may be an effective method for tumour therapy.

BACKGROUND

Pancreatic cancer (PDAC) is one of the most malignant tumours, with a five‐year survival rate of <9% due to early metastasis and recurrence, and PDAC is the fourth leading cause of cancer‐associated death in America. Due to a lack of early symptoms, most PDAC cases have progressed to late‐state disease and migrated to distant organs by the time of diagnosis, thus precluding the opportunity to operate. Recently, many studies have indicated that malignant cancer progression is closely associated with the tumour microenvironment, including stromal cells and metabolic changes. , The crosstalk between stromal cells and tumour cells has been found to contribute to cancer progression.

The most abundant stromal cells in the PDAC microenvironment are cancer‐associated fibroblasts, which are derived from quiescent pancreatic stellate cells (Q‐PSCs). Q‐PSCs are activated during PDAC tumorigenesis and become cancer‐associated fibroblasts (activated PSCs, A‐PSCs), thereby promoting the formation of a tumour‐associated microenvironment to facilitate PDAC progression. , , PSCs not only participate in secreting many factors to increase invasion but also regulate metabolism during PDAC development. ,

Caveolin‐1 (Cav‐1) is the principal component of caveolae and acts as a scaffold protein to regulate a variety of physiological processes, such as vesicle trafficking, signal transduction and cholesterol homoeostasis. , , Cav‐1 is highly expressed in terminally differentiated mesenchymal cells, such as fibroblasts, adipocytes and endothelial cells. However, it is down‐regulated in transformed fibroblasts in response to numerous oncogenic stimuli, such as reactive oxygen species (ROS). Increasing evidence indicates that intracellular oxidative stress is inextricably linked to the loss of Cav‐1 expression. In PDAC, knockdown of Cav‐1 in fibroblasts leads to enhanced tumour growth and chemoresistance. Loss of stromal Cav‐1 expression predicts poor clinical outcomes for PDAC, melanoma, colorectal cancer and breast cancer. , , , However, the mechanisms by which Cav‐1 regulates oxidative stress to affect tumorigenesis and metabolism are not clear.

In skeletal muscle, fast‐twitch fibres are glycolytic and export lactate, which is then used as an energy source by slow‐twitch fibres. Similarly, in the brain, astrocytes are glycolytic and secrete lactate, which is then taken up by adjacent neurons. In the brain, this process is known as neuron‐glia metabolic coupling. The vectorial transport of lactate from glycolytic cells (fast‐twitch fibres and astrocytes) to oxidative cells (slow‐twitch fibres and neurons) is accomplished partly through cell‐type‐specific expression of monocarboxylate transporter (MCT) molecules. For example, MCT4 (which exports lactate) is expressed by glycolytic cells. The expression of MCT4, a known hypoxia‐inducible factor‐1α (HIF‐1α) target gene, is induced by hypoxia. In contrast, MCT1/2 transporters promote the uptake of lactate based on their expression in slow‐twitch muscle fibres (MCT1) and neurons (MCT2). Previous studies have shown metabolic coupling between tumour cells and adjacent cancer‐associated fibroblasts in breast cancer. , In the present study, we aimed to determine whether similar metabolic coupling occurs in a subset of human PDACs in which PSCs undergo glycolysis and tumour cells undergo oxidative phosphorylation (OXPHOS).

In this study, we focused on exploring the role of Cav‐1 in PSCs and examining the effects of Cav‐1 on oxidative stress regulation to facilitate PDAC progression. We observed positive feedback in Cav‐1‐ROS signalling in PSCs, which facilitated PSC activation and thus promoted metabolic coupling between tumour cells and PSCs, with PSCs tending to undergo glycolysis, resulting in high levels of glycolysis products such as lactate, which are secreted into the intercellular space and absorbed by adjacent tumour cells, which tend to undergo OXPHOS.

MATERIAL AND METHODS

Patients and specimens

Experimental tissue specimens were obtained from the Department of Hepatobiliary Surgery of the First Affiliated Hospital of Xi'an Jiaotong University. All patients signed a consent form for the use of their tissue specimens, and the Ethical Committee of the First Affiliated Hospital of Xi'an Jiaotong University approved the experimental procedure. All pathological specimens were identified by two independent senior pathologists.

Cell lines and culture

Human PDAC cell lines (BxPC‐3 and Panc‐1) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in proper cell culture medium containing 10% FBS plus 100 µg/mL ampicillin and 100 µg/mL streptomycin. Primary PSCs were isolated from human pancreatic tissue from patients undergoing liver transplantation. All cells were cultured in a 37°C humidified environment with 5% CO2. Written consent from each patient's family was obtained, and the study protocol and consent forms were approved by the relevant ethical committee of the First Affiliated Hospital of Xi'an Jiaotong University in China.

Western blot analysis

Prior to protein extraction, cells were washed three times with pre‐cooled TBS, RIPA was added to isolate the total protein, and the concentration was determined using a BCA protein assay kit (Pierce). The proteins were then subjected to SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After transfer, the PVDF membranes were blocked for 2 hours in 10% fat‐free milk in Tris‐buffered saline‐Tween (TBST). Primary antibodies were incubated with the membranes overnight at 4°C. Secondary horseradish peroxidase (HRP)‐conjugated antibodies were incubated with the membranes for 2 hours at room temperature, and then, TBST was used to wash the membranes three times. The immunocomplexes were detected using an enhanced chemiluminescence kit and a Molecular Imager ChemiDoc XRS System (Bio‐Rad).

Oil red O staining

After coculturing PSCs with PDAC cells for 48 hours, PSCs were washed three times with cold PBS, and then, the cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature. After washing three times again with PBS, the cells were stained using filtered Oil red O solution and incubated for 30 minutes in a 60°C water bath. Subsequently, the cell nuclei were stained with haematoxylin. A light microscope (Nikon Eclipse Ti‐S) at 400× magnification was used to record intracellular lipid accumulation.

Real‐time PCR (qRT‐PCR) analysis

The cells were washed three times with pre‐cooled PBS, and total RNA was extracted using a Fastgen1000 RNA isolation system (Fastgen). Then, a Prime Script RT reagent kit (TaKaRa) was used to synthesize complementary DNA (cDNA) from RNA. Quantitative real‐time PCR was utilized to examine the mRNA expression of target genes. β‐Actin was used as the normalization control to determine the expression of each target gene. The relative gene expression was calculated using the 2−∆∆Ct method.

Tumorigenesis in nude mice

Pancreatic cancer cells (BxPC‐3 or Panc‐1) alone or with PSCs were injected subcutaneously into the flanks of BALB/c nude mice or orthotopically implanted into the pancreas of nude mice. The mice were sacrificed after 6 weeks, and the tumours were collected and analysed. All experimental protocols were approved by the Ethical Committee of the First Affiliated Hospital of Xi'an Jiaotong University.

Immunohistochemistry (IHC)

Tumour samples were removed after the mice were sacrificed and fixed with 4% paraformaldehyde. PDAC tissue samples were used for immunohistochemical tests. Briefly, the tissue sections were incubated with primary antibodies overnight at 4°C and then incubated with the appropriate biotinylated secondary antibodies. Two independent pathologists who were blinded to the clinicopathological data evaluated and scored all slides and reached a consensus. The percentages of positive tumour or stromal cells were categorized as follows: 0 represents less than 10%, 1 represents 10%‐25%, two represents 25%‐50%, three represents 50%‐75%, and four represents >75%. The staining intensity was scored as follows: 0 indicates no staining, 1 indicates light brown, 2 indicates brown, and 3 indicates dark brown. The IHC scores for the percentage of positive tumour or stromal cells and staining intensity were multiplied to achieve a weighted score for each case, and cases with scores ≥3 were defined as positive.

Detection of intracellular ROS

An ROS assay kit was used to measure intracellular ROS. The cells were washed with PBS, pre‐treated with SFN and incubated with 10 μmol/L DCF‐DA in serum‐free DMEM for approximately 30 minutes. The cells were then washed with PBS 3 times and lysed with 1 mL of RIPA buffer. ROS production was then analysed by fluorometric analysis at 510 nm. The final results were normalized to the total protein content.

Lactate production and ATP generation assays

To detect lactate production, after the designated treatments, cells (40 000 cells/well) were seeded into 96‐well plates and cultured with 200 μL medium. After 12 hours of culture, 100 μL of the supernatant from each group was collected and centrifuged at 200 × g for 5 minutes to remove existing cells. Then, an EnzyChrom L‐lactate assay kit (ECLC‐100, BioAssay Systems) or ATP colorimetric assay kit (#K345, BioVision) was utilized to measure lactate production or ATP generation, respectively, in the cell‐free supernatant according to the manufacturer's instructions. Furthermore, the total numbers of cells in each well were calculated and used to normalize lactate production measurements.

Statistical analysis

The results in this study are presented as the means ± standard deviation (SD). Student's t test was used to compare two groups. The difference among more than two groups was analysed by a Kruskal‐Wallis one‐way ANOVA followed by Dunn's multiple comparison tests. All statistical analyses were performed using SPSS 20.0 (SPSS Inc). P < .05 was considered significant. Each experiment was performed at least three times.

RESULTS

Cav‐1 is expressed at low levels in PDAC stroma and is associated with a poor prognosis and stroma‐tumour cell metabolism shift

We quantified Cav‐1 expression in samples using immunohistochemical analysis. As shown in Figure 1A, Cav‐1 is highly expressed in normal pancreatic stroma but has low expression in PDAC stroma. Kaplan‐Meier analysis of PDAC patients showed that patients with negative stromal Cav‐1 had a poorer prognosis than patients with positive stromal Cav‐1 (Figure 1B). In addition, we analysed the relationship between stromal Cav‐1 expression and metabolism and found that stromal pyruvate kinase isozyme type M2 (PKM2) and MCT4, which are proteins involved in glycolysis, were more highly expressed in Cav‐1–negative stromal tissue than in Cav‐1–positive stromal tissue. However, stromal translocase of outer mitochondrial membrane 20 (TOMM20), a protein involved in OXPHOS, was weakly expressed in Cav‐1–negative stromal tissue compared to Cav‐1–positive stromal tissue (Figure 1C). Intriguingly, tumour cells exhibited the opposite results for PKM2, MCT4 and TOMM20 expression. Tumour cell PKM2 and MCT4 expression was lower in Cav‐1–negative stromal tissue than in Cav‐1–positive stromal tissue. However, tumour cells exhibited higher TOMM20 expression in Cav‐1–negative stromal tissue than in Cav‐1–positive stromal tissue (Figure 1C). The results showed that reduced expression of stromal Cav‐1 was associated with a poor PDAC prognosis and correlated with a PDAC stroma‐tumour cell metabolism shift, with stroma cells tending to undergo glycolysis and tumour cells tending to undergo OXPHOS.

FIGURE 1

Loss of Cav‐1 in PDAC stroma is associated with a poor prognosis and stroma‐tumour cell metabolism shift. A, Immunohistochemistry (IHC) analysis of Cav‐1 expression in PDAC stroma. B, Kaplan‐Meier survival curves for patients with positive or negative stromal Cav‐1 expression. C, IHC detection of the expression of metabolic markers (PKM2, MCT4 and TOMM20) in Cav‐1–negative or Cav‐1–positive PDAC tissues

Coculturing PSCs with tumour cells may induce stroma‐tumour cell metabolic coupling

Primary PSCs were isolated from pancreatic tissues of patients undergoing liver transplantation. These Q‐PSCs are a type of fat‐storing cell with abundant vitamin A‐containing intracellular lipids. Once activated by stimuli such as PDAC cells, PSCs lose cytoplasmic lipid droplets. Oil red O staining showed that Q‐PSCs accumulated lipid droplets (Figure 2Ba). Then, Q‐PSCs were cocultured with BxPC‐3 PDAC cells, as shown in Figure 2A, and lipid droplets decreased in A‐PSCs (Figure 2Bb). The expression of α‐smooth muscle actin (SMA) in PSCs was substantially higher under coculture conditions than under single‐culture conditions, indicating that PSCs were activated by coculture with PDAC cells (Figure 2C). However, no significant difference in the level of ATP was found between Q‐PSCs and A‐PSCs (Figure 2D), but the amount of lactate production was obviously increased in A‐PSCs (Figure 2E). After coculture with BxPC‐3 cells, hexokinase 2 (HK‐2) and 6‐phosphofructokinase (PFKP), which are associated with glycolysis, were up‐regulated, and NAD(P)H dehydrogenase [quinone] 1 (NQO1) and TOMM20, which are associated with oxidative phosphorylation (OXPHOS), were down‐regulated in PSCs (Figure 2F,G). Moreover, glucose transporter (Glut1), which is related to glucose uptake, was up‐regulated in PSCs after coculture with cancer cells. MCT4, which is related to the ability of cells to secrete lactate, was up‐regulated, and MCT1, which reflects the ability of cells to take up lactate, was down‐regulated in PSCs. In contrast, the opposite results were found for BxPC‐3 cells under coculture conditions (Figure 2F,G), with down‐regulated expression of glycolytic enzymes (HK‐2 and PFKP) and the transporter enzyme MCT4 and up‐regulated expression of OXPHOS enzymes (TOMM20 and NQO1) and the transporter enzyme MCT1. These results indicate that the metabolism of PSCs and tumour cells changed under coculture conditions. PSCs tended to undergo glycolysis by taking up glucose and secreting lactate, whereas tumour cells tended to undergo OXPHOS by taking up lactate, decreasing glucose intake and increasing OXPHOS‐related enzymes, suggesting that metabolic coupling may occur between PSCs and tumour cells. In addition, Cav‐1 expression in PSCs decreased significantly under coculture conditions compared with single‐culture conditions (Figure 2H). These data indicate Cav‐1 loss during PSC activation.

FIGURE 2

Stroma‐tumour cell metabolic coupling occurs under PSC and PDAC coculture conditions. A, Experimental design of the PSC and PDAC coculture system. Oil red O staining of lipid droplets in quiescent PSCs (Q‐PSCs) (Ba) and activated PSCs (A‐PSCs) (Bb) cocultured with BxPC‐3 PDAC cells. C, Immunofluorescence detection of α‐SMA in PSCs cultured alone (Q‐PSCs) or cocultured with BxPC‐3 cells (A‐PSCs). D, ATP generation in PSCs cultured alone or cocultured with BxPC‐3 cells. E, Lactate production in PSCs and BxPC‐3 cells under different culture conditions. *P < .05. RT‐PCR (F) and Western blot (G) analyses of the expression of glycolytic (HK‐2 and PFKP), OXPHOS (TOMM20, NQO1) and transporter (Glut1, MCT1 and MCT4) enzymes in PSCs cultured alone or cocultured with BxPC‐3 cells. H, Western blot and RT‐PCR analyses of Cav‐1 expression in PSCs cultured alone or cocultured with BxPC‐3 cells

Positive feedback in Cav‐1‐ROS signalling in PSCs promotes PSC activation

To elucidate whether oxidative stress is involved in the loss of Cav‐1 during PSC activation, the reactive oxygen scavenger N‐acetylcysteine (NAC) was added to the coculture system. Cav‐1 protein and mRNA levels were obviously increased in PSCs in the presence of NAC (Figure 3A,B). To further investigate the interaction between Cav‐1 and oxidative stress, Cav‐1 was knocked down by shRNA (Figure 3C,D), and ROS production was measured by DCF‐DA in PSCs. We found that ROS production significantly increased after Cav‐1 knockdown (Figure 3E) and that HIF‐1α mRNA and protein expression obviously increased in PSCs (Figure 3C,D). Moreover, buthionine sulfoximine (BSO), an oxidation inducer, obviously elevated ROS production in PSCs compared with that in the normal group (Figure 3F). In addition, BSO treatment reduced Cav‐1 expression in PSCs, while the expression level of α‐SMA was up‐regulated (Figure 3G). These data show that knockdown of Cav‐1 promotes the production of ROS, while ROS production further reduces the expression of Cav‐1. Thus, positive feedback was identified in Cav‐1‐ROS signalling in PSCs, which may promote PSC activation.

FIGURE 3

Positive feedback in Cav‐1‐ROS signalling in PSCs facilitates PSC activation. Western blot (A) and real‐time PCR (B) analyses of Cav‐1 protein and mRNA expression in PSCs cocultured with BxPC‐3 cells with or without NAC. *P < .05. Western blot (C) and real‐time PCR (D) analyses of Cav‐1 and HIF‐1α protein and mRNA expression in PSCs after Cav‐1 knockdown. E, ROS production in PSCs after Cav‐1 knockdown was evaluated with DCF‐DA and normalized to the total protein content. *P < .05. F, DCF‐DA intensity detection of ROS production in PSCs treated with or without BSO. G, Western blot analysis of the protein expression of Cav‐1 and α‐SMA in PSCs treated with or without BSO. *P < .05

Positive feedback in Cav‐1‐ROS signalling in PSCs regulates stroma‐tumour cell metabolic coupling

To evaluate the role of Cav‐1‐ROS signalling in PDAC metabolism, we detected glycolytic, transporter and OXPHOS enzymes in both PSCs and BxPC‐3 cancer cells. Under coculture conditions, Cav‐1 interference in PSCs significantly increased the expression of glycolytic enzymes (PFKP, HK‐2 and PKM2) and transporter enzymes (MCT4 and Glut1) and decreased the expression of transporter enzyme (MCT1) and OXPHOS enzymes (TOMM20 and NQO1) in the PSCs (Figure 4A‐D). In contrast, the opposite effect was observed in BxPC‐3 cells by coculture with Cav‐1‐knockdown PSCs (Figure 4A‐D), with decreased expression of glycolytic enzymes (PFKP, HK‐2 and PKM2) and transporter enzymes (MCT4 and Glut1) and up‐regulated expression of OXPHOS enzymes (TOMM20 and NQO1) and the transporter enzyme MCT1.

FIGURE 4

Positive feedback in Cav‐1‐ROS signalling in PSCs mediates stroma‐tumour metabolic coupling in vitro. Real‐time PCR detection of the mRNA expression of glycolytic genes (PFKP, HK‐2 and PKM2) (A), transporter genes (MCT4, MCT1 and Glut1) (B) and OXPHOS genes (TOMM20 and NQO1) (C) in Cav‐1‐knockdown PSCs and BxPC‐3 cells under coculture conditions. *P < .05. D, Western blot detection of the protein expression of metabolism‐associated genes (MCT4, MCT1, PKM2 and TOMM20) in Cav‐1‐knockdown PSCs and BxPC‐3 cells under coculture conditions. Real‐time PCR detection of the mRNA expression of glycolytic genes (PFKP, HK‐2 and PKM2) (E), transporter genes (MCT4, MCT1 and Glut1) (F) and OXPHOS genes (TOMM20 and NQO1) (G) in PSCs and BxPC‐3 cells with or without NAC under coculture conditions. *P < .05. H, Western blot detection of the protein expression of metabolism‐associated genes (MCT4, MCT1, PKM2 and TOMM20) in PSCs and BxPC‐3 cells with or without NAC under coculture conditions

Then, we explored the role of the reactive oxygen scavenger NAC on PDAC metabolism. We found that NAC predominantly reduced glycolytic enzyme (PFKP, HK‐2 and PKM2) and transporter enzyme (MCT4 and Glut1) expression and up‐regulated transporter enzyme (MCT1) and OXPHOS enzyme (TOMM20 and NQO1) expression in PSCs in the coculture system (Figure 4E‐H). In contrast, the opposite results were found for BxPC‐3 cells in the coculture system under NAC exposure (Figure 4E‐H), with increased expression of glycolytic enzymes (PFKP, HK‐2 and PKM2) and transporter enzymes (MCT4 and Glut1) and decreased expression of OXPHOS enzymes (TOMM20 and NQO1) and the transporter enzyme MCT1. All these data indicate that positive feedback in Cav‐1‐ROS signalling in PSCs mediates PDAC stroma‐tumour cell metabolic coupling.

Positive feedback in Cav‐1‐ROS signalling in PSCs regulates the formation of the stroma‐tumour metabolic community in vivo

We further evaluated the role of Cav‐1‐ROS signalling in PDAC metabolism in vivo. After Cav‐1 was knocked down in PSCs by shRNA (Figure 5A,B), BxPC‐3 cells were injected into the subcutaneous tissues of nude mice with or without PSCs. The results showed that the tumour volume and growth rate were significantly higher when tumour cells were injected with PSCs or with sh‐Cav‐1‐knockdown PSCs than when tumour cells were injected alone. BxPC‐3 cells injected with sh‐Cav‐1‐knockdown PSCs exhibited maximal tumour volumes and growth rates. However, when PDAC cells were injected with NAC‐pre‐treated PSCs, the tumour volume and growth rate were significantly reduced (Figure 5C,D). In addition, similar results were obtained in orthotopically implanted tumours injected with PSCs and Panc‐1 PDAC cells (Figure 5E,F). Moreover, very little stroma was observed in the BxPC‐3‐ or Panc‐1‐only injection group. However, considerably more stroma surrounding the substantial tumour nests of tumour cells that were injected with PSCs was noted. MCT4 was expressed in both the tumour parenchyma and the stroma in the BxPC‐3‐ or Panc‐1‐only injection group and in the group coinjected with normal PSCs. However, MCT4 was most highly expressed in the stoma in the group that received coinjection with Cav‐1‐knockdown PSCs. In the group coinjected with NAC‐pre‐treated PSCs, MCT4 was most highly expressed in the tumour parenchyma (Figure 5G). Western blot analysis showed that the expression of matrix metalloprotease 2 (MMP2), MMP9 and C‐X‐C chemokine receptor type 4 (CXCR4) (which are correlated with tumour progression) was increased in tumour tissues coinjected with normal PSCs. When Cav‐1 was knocked out in PSCs, MMP2, MMP9 and CXCR4 were further increased. When NAC was used to pre‐treat PSCs, the expression levels of MMP2, MMP9 and CXCR4 obviously decreased (Figure 5H). These data indicate that positive feedback in Cav‐1‐ROS signalling in PSCs mediates stroma‐tumour metabolic coupling formation in vivo and thus promotes PDAC progression.

FIGURE 5

Positive feedback in Cav‐1‐ROS signalling in PSCs mediates stroma‐tumour metabolic coupling in vivo. Western blot (A) and real‐time PCR (B) analyses of Cav‐1 protein and mRNA expression in PSCs after Cav‐1 knockdown by shRNA. *P < .05. C and D, Matrigel mixtures containing 5.5 × 106 BxPC‐3 cells or a mixture of 5 × 106 BxPC‐3 cells and 0.5 × 106 PSCs (knockdown with Cav‐1 shRNA or pre‐treated with NAC) were implanted subcutaneously in the flanks of BALB/c nude mice. Tumour volumes were determined by measuring the width and length of the tumours every 4 d. The mean (n = 6); bars, SD; *P < .05. E and F, Matrigel mixtures containing 5.5 × 106 Panc‐1 cells or a mixture of 5 × 106 Panc‐1 cells and 0.5 × 106 PSCs (knockdown with Cav‐1 shRNA or pre‐treated with NAC) were orthotopically implanted into the pancreas of nude mice. The mean (n = 6); bars, SD; *P < .05. G, IHC detection of MCT4 expression in subcutaneous or orthotopic xenografts. H, Western blot detection of MMP2, MMP9 and CXCR4 protein expression in subcutaneous or orthotopic xenografts

DISCUSSION

Fibroblasts associated with cancer, such as PSCs, usually undergo oxidative stress and autophagy and exhibit glycolytic phenotypes after stimulation by tumour cells. , ROS‐induced oxidative stress transforms the metabolism of fibroblasts, creating a nutrient‐rich microenvironment for tumour cells and further promoting their growth. , , Metabolic changes in PDAC cells play an important role in the progression of PDAC. Here, we showed a novel mechanism by which PSCs enhance PDAC malignant progression that depends on stroma‐tumour metabolic coupling (Figure 6). Moreover, we demonstrated that metabolic coupling is regulated by positive feedback in Cav‐1‐ROS signalling in PSCs. In metabolic coupling, PSCs tend to undergo glycolysis by taking up glucose and secreting lactate, with up‐regulated expression of glycolytic enzymes (HK‐2, PFKP and PKM2) and the transporter enzyme MCT4, and down‐regulated expression of OXPHOS enzymes (TOMM20, NQO1) and the transporter enzyme MCT1; cancer cells tend to undergo OXPHOS by taking up lactate, with down‐regulated expression of glycolytic enzymes (HK‐2, PFKP and PKM2) and the transporter enzyme MCT4 and up‐regulated expression of OXPHOS enzymes (TOMM20 and NQO1) and the transporter enzyme MCT1 (Figure 6).

FIGURE 6

Schematic diagram of stroma‐tumour metabolic coupling. In PSCs, positive feedback in Cav‐1‐ROS signalling induces an energy metabolism shift to glycolysis. Up‐regulated Glut1 expression accelerates the uptake of glucose. Simultaneously, the expression of key glycolysis enzyme (HK‐2, PFKP and PKM2) increases, and the expression of OXPHOS enzyme (TOMM20 and NQO1) decreases. These events induce PSCs to perform glycolysis, resulting in high levels of glycolysis products such as lactate, which are secreted by the up‐regulated transporter MCT4 in PSCs. Then, PDAC cells take up these glycolysis products (lactate) through the up‐regulated transporter MCT1 to perform OXPHOS with down‐regulation of glycolytic enzyme (HK‐2, PFKP and PKM2) expression and up‐regulation of OXPHOS enzyme (TOMM20 and NQO1) expression. This stroma‐tumour metabolic coupling promotes PDAC malignant progression

Abnormal oxidative metabolism in cells is one of the hallmarks of tumorigenesis. Excessive active substances produced during oxidative metabolism promote cell tumorigenesis by inducing gene mutations and activating oncogenic pathways. Cav‐1 mediates the development and progression of a variety of tumours by modulating oxidative stress. Targeted activation of Cav‐1 also removes active substances produced during oxidative stress. The active substances produced by cellular oxidative stress can in turn regulate the expression, degradation, post‐translational modification and mediated membrane transport of Cav‐1. Multiple antioxidants regulate the Cav‐1–mediated signalling pathway in tumour cells to exert their antitumour effects. Our results showed that decreased expression of Cav‐1 promoted the production of ROS, while ROS production further reduced the expression of Cav‐1 in PSCs. Thus, positive feedback occurs in Cav‐1‐ROS signalling in PSCs, which promotes PSC activation and mediates stroma‐tumour cell metabolic coupling.

Cancer cells rapidly proliferate and metastasize, consuming a large amount of energy and nutrients. Loss of Cav‐1 mediates the energy metabolism transformation of PSCs towards glycolysis, resulting in high levels of glycolysis products such as lactate in PSCs. These metabolites are secreted into the intercellular space and absorbed by adjacent tumour cells. The mitochondria of tumour cells directly use these metabolites to accelerate mitochondrial respiration and promote rapid tumour growth. In our study, we showed that positive feedback in Cav‐1‐ROS signalling in PSCs promotes PSC activation and PDAC growth, with up‐regulated MMP2, MMP9 and CXCR4 expression. Moreover, loss of stromal Cav‐1 was associated with a poor PDAC prognosis.

Therefore, the development of treatments targeting the unique energy metabolism of tumour cells and PSCs and disrupting the metabolic coupling between the stroma and tumour cells is expected to yield effective cures for tumours.

CONCLUSION

In summary, this study shows that positive feedback in Cav‐1‐ROS signalling in PSCs promotes PDAC growth and induces stoma‐tumour cell metabolic coupling in PDAC, with PSCs tending to undergo glycolysis, resulting in high levels of glycolysis products such as lactate, which are secreted into the intercellular space and absorbed by adjacent tumour cells, which tend to undergo OXPHOS. Interrupting the metabolic coupling between the stroma and tumour cells may be an effective method for tumour therapy.

CONFLICT OF INTEREST

The authors confirm that no conflicts of interest exist.

AUTHOR CONTRIBUTION

Shan Shao: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Software (equal); Validation (equal); Visualization (equal); Writing‐original draft (equal); Writing‐review & editing (equal). tao qin: Data curation (equal); Formal analysis (equal); Investigation (equal); Software (equal); Supervision (equal); Writing‐original draft (equal). weikun qian: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Software (equal); Validation (equal). yangyang yue: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal). ying xiao: Conceptualization (equal); Data curation (equal); Investigation (equal); Methodology (equal); Software (equal); Validation (equal). xuqi li: Conceptualization (equal); Investigation (equal); Project administration (equal); Resources (equal); Supervision (equal); Validation (equal). dong zhang: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Supervision (equal). zheng wang: Conceptualization (equal); Investigation (equal); Project administration (equal); Resources (equal); Supervision (equal); Validation (equal); Visualization (equal). Qingyong Ma: Conceptualization (equal); Data curation (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Supervision (equal); Validation (equal); Visualization (equal). jianjun lei: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing‐original draft (equal); Writing‐review & editing (equal).

ETHICAL APPROVAL

All patients provided informed consent, and this study was conducted with the approval of Institutional Ethical Review Board of First Affiliated Hospital of Xi'an Jiaotong University. All animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee at First Affiliated Hospital of Xi'an Jiaotong University, and the protocols for animal care and use adhere to national regulations of the administration of laboratory animals (project license number: 2015‐99).

CONSENT FOR PUBLICATION

All contributing authors agree to the publication of this article.