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. Author manuscript; available in PMC: 2026 Jan 24.
Published in final edited form as: Am J Physiol Gastrointest Liver Physiol. 2025 Oct 1;329(4):G546–G556. doi: 10.1152/ajpgi.00025.2025

Misfolding CPA1 mutation accelerates precancerous pancreas lesions in KC mice

Alexandra Demcsák 1, Miklós Sahin-Tóth 1,*
PMCID: PMC12829554  NIHMSID: NIHMS2133372  PMID: 40982355

Abstract

Germline mutations in the CPA1 gene encoding carboxypeptidase A1 were found in association with chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC). The mutations increase pancreatic disease risk, presumably, by causing proenzyme misfolding and endoplasmic reticulum stress. Previously, we showed that CPA1 N256K mice that carry the p.N256K misfolding human CPA1 mutation in the mouse Cpa1 gene develop spontaneous chronic pancreatitis. Here, our aim was to investigate whether CPA1 N256K mice have increased susceptibility to PDAC induced by a Kras mutation. We generated KrasLSL-G12D × p48-Cre (KC) and KrasLSL-G12D × p48-Cre × CPA1 N256K (KC-CPA1) mice and compared the development of pancreas pathology in the two strains at 1, 3, 6, and 12 months of age. We observed progressive parenchymal remodeling in both strains, with more rapid changes in KC-CPA1 mice. Thus, histological analysis revealed loss of normal pancreas parenchyma, extensive fibrosis, and aberrant ductal structures such as acinar-to-ductal metaplasia and precancerous pancreatic intraepithelial neoplasia. At 3 months, these microscopic changes were significantly more abundant in KC-CPA1 versus KC mice. Owing to the massive fibrosis, the pancreas weight of KC and KC-CPA1 mice was significantly increased relative to C57BL/6N and CPA1 N256K controls, with the largest increase observed in 3-month-old KC-CPA1 animals. The observations indicate that a misfolding Cpa1 mutation accelerated the development of precancerous lesions and fibro-inflammatory remodeling in the pancreas of KC mice, providing support for the notion that CPA1 mutations might be risk factors for human PDAC.

Keywords: pancreatic ductal adenocarcinoma, misfolding, endoplasmic reticulum stress, chronic pancreatitis, digestive enzyme

Graphical Abstract

graphic file with name nihms-2133372-f0008.jpg

NEW & NOTEWORTHY

Inborn mutations in the CPA1 gene encoding carboxypeptidase A1 have been proposed to increase the risk of pancreatic ductal adenocarcinoma (PDAC) by causing enzyme misfolding and endoplasmic reticulum stress in the pancreas. Here, we demonstrated in a novel mouse model that a misfolding Cpa1 mutation accelerated the development of precancerous lesions driven by mutant Kras in the pancreas. The observations offer experimental support for the notion that CPA1 mutations are risk factors for human PDAC.

INTRODUCTION

Pancreatic ductal adenocarcinoma (PDAC) is an invasive epithelial neoplasm with ductal differentiation, which is the third (women) and fourth (men) most common cancer-related cause of death in the United States [1]. Somatic KRAS mutations, such as p.G12D, are present in about 90-95% of cases and play a fundamental role in PDAC initiation and maintenance [2, 3]. Mouse strains expressing mutant Kras in the pancreas develop widespread precancerous lesions including low-grade and high-grade pancreatic intraepithelial neoplasia (PanIN) but progression to PDAC is slow, indicating that KRAS mutations are necessary but not sufficient for the development of invasive cancer [4, 5]. Somatic mutations in other genes such as CDKN2A, TP53, and SMAD4 are required to accelerate PDAC [2, 5–7].

Inborn mutations in numerous genes have been found to predispose to PDAC and some of these susceptibility genes code for pancreatic digestive enzymes [8]. Thus, germline mutations in the PRSS1 gene encoding cationic trypsinogen cause hereditary pancreatitis, which is associated with markedly increased risk for PDAC, presumably due to chronic pancreatic inflammation [9–12]. Furthermore, a common loss-of-function deletion variant in the CTRB2 gene encoding chymotrypsinogen B2 was shown to increase PDAC risk [13, 14]. The variant causes misfolding, abolishes CTRB2 secretion and enzyme activity, and induces endoplasmic reticulum (ER) stress in cell culture. A recent mouse model carrying the deletion in the native mouse Ctrb1 gene replicated these biochemical features [15]. Finally, rare, loss-of-function germline mutations in the CPA1 and CPB1 genes encoding carboxypeptidases A1 and B1 were shown to increase PDAC risk on average by 3.65-fold and 9.51-fold, respectively [16, 17]. The variants caused loss of proenzyme secretion and ER stress in cell culture, suggesting that ER stress due to digestive enzyme misfolding may be a common pathway for PDAC risk associated with variants in multiple risk genes.

Previously, we generated a mouse model that carried the p.N256K misfolding human CPA1 mutation in the mouse Cpa1 gene [18]. The p.N256K mutation was identified in pediatric patients with chronic pancreatitis and its propensity to misfold and induce ER stress was demonstrated in cell culture experiments [19, 20]. We reported that CPA1 N256K mice developed progressive chronic pancreatitis with acinar atrophy, fibrosis, macrophage infiltration, acinar-to-ductal metaplasia, plasma amylase elevation, and pancreatic ER stress [18]. No PDAC or precancerous lesions were observed up to 12 months of age. In the present study, we hypothesized that misfolding CPA1 mutations increase PDAC risk by accelerating the KRAS-driven development of PanINs. To test this notion, we crossed the established KC mouse model expressing the p.G12D Kras mutant in the pancreas with the CPA1 N256K strain and investigated whether the KC-CPA1 mice were more prone to develop precancerous pancreatic changes.

MATERIALS & METHODS

Study approval.

Animal experiments were performed at the University of California, Los Angeles (UCLA) with the approval and oversight of the Animal Research Committee, including protocol review and post-approval monitoring. The animal care program is managed in full compliance with the US Animal Welfare Act, the United States Department of Agriculture Animal Welfare Regulations, the US Public Health Service Policy on Humane Care and Use of Laboratory Animals and the National Research Council’s Guide for the Care and Use of Laboratory Animals. The University of California, Los Angeles has approved Animal Welfare Assurance statement (A3196-01) on file with the US Public Health Service, National Institutes of Health, Office of Laboratory Animal Welfare. The institution is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Experiments were performed in accordance with the ARRIVE 2.0 guidelines [21].

Animals.

Experiments were performed on mice with mixed sexes. Wild-type C57BL/6N mice were obtained from Charles River Laboratories (Wilmington, MA) or produced in our breeding facility from the same stock. The generation and phenotypic features of homozygous CPA1 N256K mice on the C57BL/6N genetic background were previously described [18, 22, 23]. This strain carries the human pancreatitis-associated p.N256K CPA1 mutation in the mouse Cpa1 gene. The conditional KrasLSL-G12D [24] and p48-Cre [25] mice were kindly provided by the laboratory of Dr. Guido Eibl at the University of California, Los Angeles (UCLA). The presumed C57BL/6 genetic background of these mice has not been verified experimentally. The KC mice were generated by crossing the KrasLSL-G12D and p48-Cre mice to achieve Cre-mediated pancreas-specific excision of the LoxP-Stop-LoxP (LSL) cassette and expression of the oncogenic KrasG12D allele. To generate KC-CPA1 mice, first we created the KrasLSL-G12D × CPA1 N256K and p48-Cre × CPA1 N256K crosses, in which the CPA1 N256K allele was homozygous. Crossing the KrasLSL-G12D × CPA1 N256K and p48-Cre × CPA1 N256K strains resulted in the desired KrasLSL-G12D × p48-Cre × CPA1 N256K (KC-CPA1) genotype. Thus, KC and KC-CPA1 mice were not littermates but originated from separate breeding.

Genotyping.

Mouse genomic DNA was isolated from tail samples with the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA). The CPA1 N256K allele was genotyped as described previously [18]. The KrasLSL-G12D allele was genotyped with the following primers. Y116-common: 5’- TCC GAA TTC AGT GAC TAC AGA TG -3’, Y117-LSL: 5’- CTA GCC ACC ATG GCT TGA GT -3’, Y118-WT: 5’- ATG TCT TTC CCC AGC ACA GT -3’ [26]. The product sizes for the wild-type and mutant alleles were 450 bp and 327 bp, respectively. The p48-Cre allele was genotyped with the following primers. Cre forward: 5’- TGC TGT TTC ACT GGT TAT GCG G -3’, Cre reverse: 5’- TTG CCC CTG TTT CAC TAT CCA G -3’, which yielded a 671 bp amplicon [26].

Plasma amylase assay.

Blood was collected through cardiac puncture and plasma was isolated by centrifugation at 2,000 g, 4°C for 15 min. Enzyme activity of amylase in blood plasma (1 μL assayed) was then determined with the 2-chloro-p-nitrophenyl-α-D-maltotrioside substrate, as described previously [28]. The rate of substrate cleavage was expressed in mOD/min units.

Histology.

Pancreas tissue was fixed and kept in 10% neutral buffered formalin, paraffin-embedded, sectioned, and stained with hematoxylin-eosin (Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA) or Masson’s trichrome staining (Translational Pathology Core Laboratory, UCLA), as indicated. Immunohistochemistry staining (UCLA) was performed for the ductal marker SRY-box transcription factor 9 (SOX9) with a rabbit monoclonal antibody (catalog number ab185230, Abcam, Cambridge, MA), and the stellate cell marker α-smooth muscle actin (α-SMA) with a rabbit polyclonal antibody (catalog number ab5694, Abcam). Mucin production was visualized by Alcian blue staining (Division of Research Services, UCLA Pathology & Laboratory Medicine). Histology slides were scanned using a Pannoramic Desk scanner (3DHISTECH version 2.1) and viewed with the SlideViewer software (3DHISTECH version 2.6). Histological scoring was performed for acinar cell loss and aberrant duct-like structures. Intact acinar cells were quantitated as percentage of the total tissue area examined. The distribution of duct-like structures was evaluated by assigning each structure to one of the following groups: reactive ducts, acinar-to-ductal metaplasia (ADM), low-grade PanIN (previously PanIN 1a, 1b, and 2) and high-grade PanIN (previously PanIN 3 or carcinoma in situ), following previously published classification systems [4, 29–31] simplified according to the recommendations of Basturk et al. (2015) [32].

Hydroxyproline assay.

To quantitate fibrosis, the hydroxyproline content of pancreas homogenates from 3-month-old mice was determined by measuring the reaction of oxidized hydroxyproline with 4-(dimethylamino)benzaldehyde using a commercial kit (catalog number MAK008, Millipore Sigma), as reported previously [18]. Values were normalized to the total protein concentration and expressed in units of ng hydroxyproline per μg protein.

RNA isolation and reverse transcription PCR.

Expression levels of Hspa5 (BiP) and Ddit3 (CHOP) mRNA were determined by reverse-transcription quantitative PCR. Total RNA was isolated from the pancreas of 1-month-old mice. Freshly excised pancreas tissue (~20 mg) was kept in 500 μL RNAlater solution (catalog number AM7020, Thermo Fisher Scientific) overnight at 4°C and RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Valencia CA). Two μg of RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (catalog number 4368814, Thermo Fisher Scientific). Real-time quantitative PCR was performed with the Taqman Universal PCR Master Mix (catalog number: 4304437, Thermo Fisher Scientific). The following TaqMan assays (Thermo Fisher Scientific) were used: Hspa5-FAM (Mm00517691_m1), Ddit3-FAM (Mm01135937_g1), and Rpl13a-VIC (Mm01612987_g1). Relative expression levels were estimated with the comparative cycle threshold method (ΔΔCT). CT values for Hspa5 or Ddit3 were first normalized to those of the Rpl13a RNA reference gene (ΔCT) for each sample, then to the mean ΔCT value of the C57BL/6N control samples (ΔΔCT). The results were expressed as fold-change calculated with the 2−ΔΔCT formula.

Statistics.

Experimental results were graphed as individual data points, and the mean and standard deviation were indicated. The difference of means between the groups was analyzed by one-way ANOVA followed by Tukey’s post-hoc test or unpaired t-test, as indicated, using Prism 7 software (GraphPad Software, Boston, MA). P<0.05 was considered statistically significant.

RESULTS

Generation of KC-CPA1 mice.

KC mice carrying the homozygous CPA1 N256K allele were generated by crossing the KrasLSL-G12D, p48-Cre, and CPA1 N256K parent strains, as described under Methods. The resulting KC-CPA1 mice were studied in comparison to KC mice and the CPA1 N256K and C57BL/6N control strains. Mice were analyzed at 1, 3, 6, and 12 months of age. In our hands, nearly all KC and KC-CPA1 mice developed pre-cancerous lesions in their pancreas without overt tumors or liver metastases. There was no disease-related mortality. We also collected data on the KrasLSL-G12D and p48-Cre parent strains, and the KrasLSL-G12D × CPA1 N256K cross. These mice showed no signs of pancreatic disease, and their pancreatic phenotype was indistinguishable from that of C57BL/6N mice. For brevity and clarity, therefore, these data are not shown.

Body weight of KC-CPA1 mice.

Body weight can serve as an indirect indicator of pancreas health. Therefore, first we compared the body weight of KC and KC-CPA1 mice to those of C57BL/6N and CPA1 N256K controls (Figure 1A). Over the 1-year time course studied, C57BL/6N mice gained weight steadily. Similarly, CPA1 N256K mice showed continual weight gain albeit the increase was smaller at 6 and 12 months of age, relative to the C57BL/6N animals. In contrast, the weight of KC mice plateaued at 6 months, while that of the KC-CPA1 animals leveled off even earlier, at 3 months of age. Consequently, at 6 and 12 months of age, both KC strains showed signs of cachexia with significantly decreased body weight compared to the control strains.

Figure 1.

Figure 1.

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Body weight and plasma amylase activity of KC-CPA1, KC, CPA1 N256K, and C57BL/6N mice at 1, 3, 6, and 12 months of age. A, Body weight. B, Plasma amylase activity. The number of mice assessed at the 4 time points were as follows: KC-CPA1, n=11, 10, 9, 12; KC, n=10, 9, 10, 8; CPA1 N256K, n=6, 8, 6, 8; C57BL/6N, n=8, 8, 8, 8. Individual values with mean ± SD are shown. The difference of means was analyzed by one-way ANOVA followed by Tukey’s post-hoc test.

Plasma amylase in KC-CPA1 mice.

As a marker of pancreatic disease, we measured amylase activity from blood plasma (Figure 1B). At 1 month of age, CPA1 N256K mice had significantly elevated plasma amylase levels relative to the other three strains. The high plasma amylase activity in this strain is caused by the incipient chronic pancreatitis and has been reported previously [18]. The low plasma amylase levels in the KC-CPA1 mice were surprising and suggested a direct effect of KRAS activity on amylase release or acinar cell damage. The plasma amylase activity in CPA1 N256K mice decreased over time, presumably due to acinar cell atrophy. Despite this decrease, amylase levels in CPA1 N256K mice remained significantly higher at 3 and 6 months of age relative to those of C57BL/6N and KC-CPA1 mice. Interestingly, KC mice but not KC-CPA1 mice showed a small but significant increase in plasma amylase activity at 3 months and 6 months of age, relative to C57BL/6N mice. No significant difference was found among the four strains at 12 months.

Macroscopic changes of the pancreas in KC-CPA1 mice.

Macroscopically, the pancreas of KC and KC-CPA1 mice were enlarged, with firm consistency, and irregular, nodular surface. At 3 months of age, these changes were restricted to the head of the pancreas in KC mice, whilst the entire pancreas was involved in KC-CPA1 animals (Figure 2A). At 6 months of age, the pancreas of both strains showed similar morphology (not shown). By 1 year, KC and KC-CPA1 mice still showed firm tissue consistency throughout the organ, however, the pancreas lost its nodular appearance. Moreover, in some cases, the pancreas of KC-CPA1 mice exhibited softer consistency around the lienal area (not shown).

Figure 2.

Figure 2.

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Macroscopic pancreas morphology and pancreas weight of KC-CPA1, KC, CPA1 N256K, and C57BL/6N mice. A, Representative pancreas images at 3 months of age. For orientation, the spleen was also included. The pancreas weight, exclusive of the spleen, is indicated. B, Pancreas weight at 1, 3, 6, and 12 months of age. The number of mice assessed at the 4 time points were as follows: KC-CPA1, n=11, 10, 9, 12; KC, n=10, 9, 10, 8; CPA1 N256K, n=6, 8, 6, 8; C57BL/6N, n=8, 8, 8, 8. Individual values with mean ± SD are shown. The difference of means was analyzed by one-way ANOVA followed by Tukey’s post-hoc test.

Pancreas hypertrophy and atrophy in KC-CPA1 mice.

The pancreas weight of KC and KC-CPA1 mice were significantly increased compared to the C57BL/6N and CPA1 N256K control strains at 3, 6, and 12 months of age (Figure 2B). Remarkably, the pancreas of 3-month-old KC-CPA1 mice was significantly more enlarged when compared with the KC parent strain of the same age. At 6 and 12 months of age, the pancreas weight of KC-CPA1 mice was lower than that of the KC animals; the difference was statistically significant at 12 months. Interestingly, the pancreas weight of KC-CPA1 mice decreased after the 3-month peak, whereas that of the KC mice increased further and plateaued at 6 months of age. As described previously, the pancreas weight of CPA1 N256K mice did not increase after 3 months of age due to the atrophy associated with their chronic pancreatitis [18].

Pancreas histopathology in KC-CPA1 mice.

To evaluate the histological progression of disease in KC and KC-CPA1 mice, pancreas sections were stained with hematoxylin-eosin at 1, 3, 6, and 12 months of age, and compared to similarly treated pancreas sections from C57BL/6N and CPA1 N256K controls (Figure 3). C57BL/6N mice maintained normal pancreas histology over the time course studied with the characteristic tightly packed acinar tissue and strong eosinophilic staining. As reported previously, CPA1 N256K mice developed slowly progressing, mild chronic pancreatitis with acinar cell dropout, acinar-to-ductal metaplasia, inflammatory cells, and adipose cell infiltration [18, 22, 23]. In contrast, KC and KC-CPA1 mice showed significant remodeling of acinar tissue which was prominent at 3 months of age (Figure 4A). The tissue remodeling resulted in the loss of normal acini, strong fibrosis, dilated ducts, and the appearance of numerous PanINs and other aberrant duct-like structures. In agreement with the higher pancreas weight observed at 3 months, KC-CPA1 mice exhibited more pronounced changes at this age, with 15% intact acini left versus 60% remaining acini in KC mice (Figure 4B). By 6 and 12 months of age, when the pancreas weight of the two strains was comparable, the KC and KC-CPA1 animals showed a similar histological picture, but pathological changes were still more noticeable in the pancreas from KC-CPA1 mice.

Figure 3.

Figure 3.

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Pancreas histology in KC-CPA1, KC, CPA1 N256K, and C57BL/6N mice at 1, 3, 6, and 12 months of age. Representative hematoxylin-eosin-stained pancreas sections are shown. The scale bar corresponds to 100 μm.

Figure 4.

Figure 4.

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Acinar cell loss and aberrant ducts in KC-CPA1, KC, CPA1 N256K, and C57BL/6N mice. A, Representative hematoxylin-eosin-stained pancreas sections from 3-month-old mice. The scale bar corresponds to 200 μm. B, Quantitation of acinar cell loss by histological scoring of pancreas sections. The number of sections analyzed were as follows: KC-CPA1, n=11, 10, 8, 12; KC, n=10, 9, 9, 8; CPA1 N256K, n=5, 8, 6, 8; C57BL/6N, n=8, 8, 8, 8. Individual values with mean ± SD are shown. The difference of means was analyzed by one-way ANOVA followed by Tukey’s post-hoc test. C, Distribution of aberrant duct-like structures in KC-CPA1 and KC mice. ADM, acinar-to-ductal metaplasia, HG, high-grade, LG, low-grade, PanIN, pancreatic intraepithelial neoplasia. Mean values with SD are shown. The difference of means between two groups was analyzed by unpaired t-test.

Since the largest difference in pancreas weight and histological remodeling in the KC and KC-CPA1 mice was seen at 3 months of age, we chose this time point to quantify the various aberrant duct-like structures such as reactive ducts, acinar-to-ductal metaplasia (ADM), and PanINs (Figure 4C). Based on the simplified recommendations of Basturk et al. (2015), PanINs were graded either as low-grade or high-grade [32]. The majority of duct-like histological structures corresponded to low-grade PanINs, followed by ADMs, reactive ducts, and high-grade PanINs. At 3 months, KC-CPA1 mice contained significantly more low-grade (90% versus 60%) and high-grade (2% versus 0.2%) PanINs than KC mice. In contrast, the pancreas of KC animals had more reactive ducts (10% versus 0.03%) and ADMs (25% versus 8%) than KC-CPA1 mice.

The duct-like pancreatic structures were also visualized by immunohistochemical staining for the ductal marker SOX9 (Figure 5A) which confirmed the results of the hematoxylin-eosin staining and showed strong positivity in KC and KC-CPA1 mice, with more signal visible in the latter. As reported previously, the ADM structures in the pancreas of the CPA1 N256K mice were also highlighted by SOX9 staining, whereas the C57BL/6N pancreas sections showed background signal only [18, 23]. To evaluate the mucin-producing capability of the duct-like structures, pancreas sections were stained with Alcian blue (Figure 5B), which resulted in intense staining in KC and KC-CPA1 mice, with stronger positivity in KC-CPA1 animals. No mucin staining was detected in the pancreas sections from C57BL/6N and CPA1 N256K mice.

Figure 5.

Figure 5.

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Visualization of duct-like structures and mucin-producing cells in the pancreas from 3-month-old KC-CPA1, KC, CPA1 N256K, and C57BL/6N mice. A, Immunohistochemistry staining of pancreas sections for SOX9. B, Alcian blue staining of pancreas sections for mucin (blue). The scale bar corresponds to 100 μm.

Pancreatic fibrosis and stellate cell activation in KC-CPA1 mice.

Pancreatic collagen content was first assessed using Masson’s trichrome staining of pancreas sections from 3-month-old animals (Figure 6A). Mild, diffuse fibrosis was present in the pancreas from CPA1 N256K mice while C57BL/6N mice showed minimal staining, as reported previously [18, 23]. In contrast, extensive fibrosis was present in KC and KC-CPA1 mice, with stronger staining in KC-CPA1 animals. To quantitate the fibrosis, we measured pancreatic hydroxyproline content (Figure 6B). The results demonstrated that the highest collagen levels were in the pancreas of KC-CPA1 mice, followed by KC mice, CPA1 N256K and C57BL/6N controls. The pancreas from 3-month-old KC-CPA1 mice contained 17-times, 5.7-times, and 2.8-times more collagen than those from C57BL/6N, CPA1 N256K, and KC animals, respectively. The observations indicate that the leading cause of pancreas weight increase in KC and KC-CPA1 mice was massive fibrosis. We performed immunohistochemistry for α-SMA which is a marker of stellate cell activation (Figure 6C). Only weak and scattered positivity was observed in the pancreas of CPA1 N256K mice while C57BL/6N mice showed almost no pancreatic staining, as reported previously [18]. However, a significant number of α-SMA positive cells appeared in scattered clusters in KC and KC-CPA1 animals, mostly around ADMs and PanINs. In agreement with the stronger fibrosis, stellate cell activation in the pancreas of KC-CPA1 mice was more prominent relative to KC mice.

Figure 6.

Figure 6.

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Fibrosis and stellate cell activation in the pancreas of 3-month-old KC-CPA1, KC, CPA1 N256K, and C57BL/6N mice. A, Masson’s trichrome staining of representative pancreas sections. Fibrosis is highlighted in blue. B, Hydroxyproline content of the pancreas. The number of pancreata analyzed were as follows: KC-CPA1, n=10; KC, n=8; CPA1 N256K, n=4; C57BL/6N, n=4. Individual values with mean ± SD are shown. The difference of means was analyzed by one-way ANOVA followed by Tukey’s post-hoc test. C, Immunohistochemistry staining of pancreas sections for α-SMA as a marker of stellate cell activation. The scale bar corresponds to 100 μm.

Endoplasmic reticulum stress in KC-CPA1 mice.

To determine whether the stronger pathological changes found in KC-CPA1 versus KC mice were associated with higher ER stress, we measured pancreatic mRNA expression for ER stress markers Hspa5 (BiP) and Ddit3 (CHOP) at 1 month of age (Figure 7). Note that at this age the pancreas was still relatively intact in all animals based on the hematoxylin-eosin-stained histological sections. No significant difference was found in pancreatic Hspa5 levels among the four strains (Figure 7A). In contrast, Ddit3 levels were significantly elevated in CPA1 N256K mice relative to C57BL/6N controls or KC mice, and a similar trend was apparent in KC-CPA1 mice (Figure 7B). No difference was found between KC and C57BL/6N mice with respect to pancreatic Ddit3 expression.

Figure 7.

Figure 7.

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Endoplasmic reticulum stress in the pancreas of 1-month-old KC-CPA1, KC, CPA1 N256K, and C57BL/6N mice. Reverse-transcription quantitative PCR was performed to measure mRNA expression, and the results were expressed as fold change relative to the average value of the C57BL/6N data. A, Hspa5 (BiP) mRNA levels. B, Ddit3 (CHOP) mRNA levels. Individual values with the mean ± SD are shown. The difference of means was analyzed by one-way ANOVA followed by Tukey’s post-hoc test.

DISCUSSION

In this study, we investigated whether a misfolding CPA1 mutation would accelerate PDAC development induced by mutant KRAS. Previously, mutations in CPA1 were shown to increase PDAC risk by 3.65-fold [16, 17]. Functional evaluation of the PDAC-associated CPA1 mutations in cell culture experiments demonstrated loss of proenzyme secretion, diminished enzyme activity, and ER stress. Similar findings were reported for CPB1 variants, too [16, 17]. The authors proposed that chronic ER stress associated with misfolding digestive enzymes could increase PDAC risk. Here, we put this notion to the test by measuring the development of precancerous lesions in the pancreas of KC mice with or without a misfolding Cpa1 variant. We used our previously generated and characterized CPA1 N256K mice, which carry the p.N256K mutation originally identified in early-onset cases of chronic pancreatitis [18]. Although this mutation has not been reported in association with PDAC yet, its functional properties with respect to misfolding and ER stress are highly similar to those found in PDAC. The CPA1 N256K mice were reported to develop spontaneous, progressive chronic pancreatitis with relatively mild pathological features but no PDAC or precancerous lesions. We crossed KC mice with CPA1 N256K animals to generate the novel KC-CPA1 strain.

We found that KC-CPA1 mice exhibited similar pancreas pathology as previously reported in the KC animals, characterized by extensive parenchymal remodeling with acinar atrophy, massive fibrosis, and abundant precancerous ductal lesions, as described by previous studies [4, 33, 34]. When pancreas weight, pancreas histology (loss of acini, abnormal duct-like-structures, SOX9 staining, Alcian blue staining), and fibrosis (trichrome staining, α-SMA staining, hydroxyproline content) were compared, KC-CPA1 mice showed more rapid and more severe changes, which was best appreciated at 3 months of age. The ER stress marker Ddit3, which encodes the pro-apoptotic transcription factor CHOP, was elevated in KC-CPA1 mice relative to KC animals at an early age. Interestingly, no significant changes were seen in Hspa5 mRNA levels, which code for the ER master chaperone BiP. Previous studies with CPA1 N256K also described small changes in Hspa5 mRNA but more significant elevations in Ddit3 mRNA [18]. Although Ddit3 does not appear to play a direct role in chronic pancreatitis in CPA1 N256K mice [23], it might contribute to the development of precancerous lesions in KC-CPA1 animals. Ddit3 has been shown to regulate various cellular processes including differentiation and proliferation, however, evidence for its oncogenic potential is limited [35].

Previously, human genetic studies found that rare CPA1 and CPB1 variants that induced ER stress in cell culture were enriched in PDAC cases [16, 17]. Similarly, a common deletion variant of human CTRB2 increased PDAC risk and elicited ER stress in transfected cells [13, 14] and in the pancreas of a mouse model carrying the deletion variant in the mouse Ctrb1 gene [15]. While ER stress may indeed be the pathogenic culprit, there are at least two other potential explanations for the accelerated development of precancerous lesions in the KC-CPA1 mice. Pancreatic inflammation (i.e. acute or chronic pancreatitis) has been reported to facilitate PDAC development both in human studies and in mouse experiments. In humans, chronic pancreatitis, and PRSS1-related hereditary pancreatitis in particular, markedly increase PDAC risk [9–12]. Also, the incidence of PDAC is significantly elevated after an acute attack of pancreatitis [36]. In mice, cerulein-induced pancreatitis is routinely used to facilitate cancer development in genetic models of PDAC [34]. CPA1 N256K mice develop relatively mild but progressive spontaneous chronic pancreatitis. It is possible, even likely, that pre-existing pancreatic inflammation, possibly through ADM formation [37], might drive the more rapid acinar tissue remodeling and appearance of PanINs in KC-CPA1 mice. Another potential reason for the accelerated precancerous pathology in KC-CPA1 mice might be the loss of carboxypeptidase A1 activity in the digestive system. Because the p.N256K mutation completely blocks proenzyme secretion, the homozygous CPA1 N256K mice are expected to become deficient in carboxypeptidase A1 activity in their digestive tract. Whether or not this can indirectly affect KRAS-induced pancreas pathology is unclear but conceivable. Testing the effect of genetic deletion of Cpa1 on PanIN development in KC mice can answer this question.

Loss-of-function variants in CPA1 were originally described in early-onset cases of chronic pancreatitis [19]. Based on cell culture experiments and the characterization of the CPA1 N256K mouse model, mutation-induced proenzyme misfolding and consequent ER stress emerged as the accepted mechanism by which CPA1 variants elicit chronic pancreatitis [18–20, 38]. Other mouse models expressing misfolding lipase variants, such as the p.T221M PNLIP mutant or the hybrid allele of the carboxyl ester lipase (CEL-HYB1), exhibited phenotypes that were strikingly similar to that of CPA1 N256K mice [39–41]. Curiously, most CPA1 variants considered pathogenic in PDAC have not been found in chronic pancreatitis patients. Similarly, variants strongly associated with chronic pancreatitis were typically not detected in PDAC cases. Furthermore, CPB1 variants were not enriched in chronic pancreatitis cases, indicating that CPB1 is not a risk gene for this condition [42]. In contrast, ER-stress causing CPB1 variants were found to increase PDAC risk by 9.51-fold [16, 17]. Recent studies characterizing 63 CPA1 variants in cell culture experiments demonstrated that many variants caused some level of ER stress but only a subset was demonstrably associated with sporadic and hereditary chronic pancreatitis [43, 44]. The pathogenic variants exhibited nearly complete loss of secretion and induced strong ER stress. Based on these observations, it is plausible that the level of ER stress determines the pathological outcome; CPA1 variants with strong ER stress tend to cause chronic pancreatitis whereas CPA1 and CPB1 variants with moderate ER stress increase PDAC risk. It is also conceivable that CPA1 and CPB1 variants which elicit lower levels of ER stress, may cause subclinical chronic pancreatitis that would drive increased susceptibility to PDAC.

In summary, we demonstrated that a misfolding Cpa1 mutant accelerated the development of precancerous lesions in the pancreas of KC mice, providing support for the notion that gene variants that give rise to misfolding digestive enzymes are risk factors for human PDAC.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. Zsanett Jancsó for help with the initiation of the project and colony management, to Dr. Guido Eibl for providing KrasLSL-G12D and p48-Cre mice and for helpful discussions, and to Dr. Máté Sándor for assistance with the revisions.

GRANTS

This work was supported by the Hirshberg Foundation for Pancreatic Cancer Research Seed Grant Awards 20230501 to AD and 20210733 to MST, and by the National Institutes of Health (NIH) grants R01 DK082412 and DK117809 to MST.

Footnotes

DISCLOSURES

The authors declare that they have no competing interests, financial or non-financial.

DATA AVAILABILITY

Materials, experimental data and protocols associated with the current study are available from the corresponding author upon request.

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Associated Data

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Data Availability Statement

Materials, experimental data and protocols associated with the current study are available from the corresponding author upon request.

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