Protease‐activated receptor 1 drives and maintains ductal cell fates in the premalignant pancreas and ductal adenocarcinoma

Pancreatic acinar cells have high plasticity and can transdifferentiate into ductal‐like cells. This acinar‐to‐ductal metaplasia (ADM) contributes to tissue maintenance but may also contribute to the premalignant transformation that can eventually progress to pancreatic ductal adenocarcinoma (PDAC). Macrophages are key players in ADM, and macrophage‐secreted matrix metalloproteinase (MMP)‐9 induces ADM through yet unknown mechanisms. As we previously identified MMP9 as a novel agonist of protease‐activated receptor 1 (PAR1), a receptor that is known to orchestrate the cross‐talk between macrophages and tumor cells in PDAC, we here assessed the contribution of PAR1 to pancreatic cell fates. We found that genetic deficiency for PAR1 increases acinar gene expression programs in the healthy pancreas and that PAR1 deficiency limits ductal transdifferentiation in experimental systems for ADM. Moreover, PAR1 silencing in PDAC cells increases acinar marker expression. Changes in PDAC cell lines were associated with a downregulation of known Myc‐target genes, and Myc inhibition mimics PAR1 deficiency in enhancing acinar programs in healthy organoids and PDAC cells. Overall, we identify the PAR1‐Myc axis as a driver of ductal cell fates in premalignant pancreas and PDAC. Moreover, we show that cellular plasticity is not unique to acinar cells and that ductal regeneration into acinar‐like cells is possible even in the context of oncogenic KRAS activation.

Pancreatic acinar cells have high plasticity and can transdifferentiate into ductal-like cells. This acinar-to-ductal metaplasia (ADM) contributes to tissue maintenance but may also contribute to the premalignant transformation that can eventually progress to pancreatic ductal adenocarcinoma (PDAC). Macrophages are key players in ADM, and macrophage-secreted matrix metalloproteinase (MMP)-9 induces ADM through yet unknown mechanisms. As we previously identified MMP9 as a novel agonist of protease-activated receptor 1 (PAR1), a receptor that is known to orchestrate the cross-talk between macrophages and tumor cells in PDAC, we here assessed the contribution of PAR1 to pancreatic cell fates. We found that genetic deficiency for PAR1 increases acinar gene expression programs in the healthy pancreas and that PAR1 deficiency limits ductal transdifferentiation in experimental systems for ADM. Moreover, PAR1 silencing in PDAC cells increases acinar marker expression. Changes in PDAC cell lines were associated with a downregulation of known Myc-target genes, and Myc inhibition mimics PAR1 deficiency in enhancing acinar programs in healthy organoids and PDAC cells. Overall, we identify the PAR1-Myc axis as a driver of ductal cell fates in premalignant pancreas and PDAC. Moreover, we show that cellular plasticity is not unique to acinar cells and that ductal regeneration into acinar-like cells is possible even in the context of oncogenic KRAS activation.

Introduction
Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer [1]. Fiveyear survival rates of PDAC are around 9%, and despite intense research efforts to improve the poor outcome, there has been little improvement in the last decades [1,2]. Several features of PDAC contribute to the dismal prognosis, including the late stage at which the disease is typically diagnosed [3] and high intrinsic resistance against chemotherapeutics.
In the exocrine pancreas, which facilitates food digestion, two main cell types exist; acinar cells that produce digestive enzymes and epithelial cells that line the ducts through which these enzymes are transported to the duodenum. Despite the strong ductal characteristics and nomenclature of PDAC, the cell of origin of PDAC development remains the subject of debate. In fact, both acinar and ductal cells may give rise to neoplasia leading up to cancerous lesions; nevertheless, acinar cells are reported to be more susceptible to rapid transdifferentiation and neoplasm formation, especially during inflammation (i.e., pancreatitis) [4][5][6][7]. During inflammatory circumstances, the acinar cell state is plastic, and mature acinar cells can de-differentiate into progenitor cells and subsequently differentiate into duct-like cells [8]. This acinar-to-ductal metaplasia (ADM) contributes to the homeostasis of the pancreas. However, oncogenic activations in acinar cells (typically activating mutations in KRAS) and inflammation of the pancreas (pancreatitis) can trigger ADM and give rise to pancreatic intraepithelial neoplasias (PanIN). Additional oncogenic mutations can accumulate in PanIN lesions, which may then progress to PDAC [9]. Studies on genetically modified mouse models have shown TGFa expression and KRAS mutations as drivers of ADM and PanIN formation [10]. Additionally, mouse models of cerulein-induced pancreatitis have shown that inflammation contributes to PDAC development [7]. Furthermore, KRAS mutations can cooperate with the inflammatory cascade to accelerate the formation of malignant lesions [11]. Given the importance of maintaining the appropriate cell fates and lineages for homeostasis and preventing premalignant transformation, acinar differentiation processes are subject to strict regulation. This has typically been attributed to transcription factors with activity in distinct cell populations and (developmental) compartments, but the role of G protein-coupled receptor (GPCR) signaling known to be important in embryonic development remains underexplored.
Protease-activated receptor 1 (PAR1) is a GPCR family member that is widely expressed in various tissues in both healthy and disease states. It is activated by proteolytic cleavage of the N-terminal arm, which liberates a tethered agonist that binds to its activation domain, thereby initiating intracellular signaling. PAR1 was initially described to be activated by the coagulation factor thrombin, but other agonists like MMP1, MMP13, activated protein C, and kallikreins have now been characterized [12]. Increased PAR1 expression is associated with tumor progression and poor prognosis in, among others, breast, lung, and pancreatic cancer [13][14][15][16]. In PDAC, an orthotopic xenograft study revealed that tumor growth is stalled in PAR1-deficient animals [16]. Interestingly, this reduced tumor growth was accompanied by a significant reduction of macrophage influx into the tumor microenvironment, suggesting a functional link between PAR1, macrophages, and PDAC growth [16].
Macrophages that infiltrate the pancreas are critical players in ADM, and macrophage depletion limits the formation of cerulein-induced PanIN lesions [17]. Infiltrated macrophages secrete matrix metalloproteinase 9 (MMP9) that targets acinar cells leading to transdifferentiation into ductal cells. This is particularly interesting as we recently identified macrophage-secreted MMP9 as a novel PAR1 agonist in the setting of PDAC [18]. In the current manuscript, we address the hypothesis that PAR1 plays a more central role in cellular differentiation and that its activation balances acinar and ductal cell fates in the healthy and premalignant pancreas and PDAC.

Animal studies and ethics approval
C57BL/6 mice (Charles River Laboratories) were housed at the animal facility of the Academic Medical Center of Amsterdam. All mice had access to food and water ad libitum. Institutional Animal Care and Use Committee of Academic Medical Center approved all animal experiments according to protocol number DIX107AA.

Isolation of mouse pancreatic acinar cells
Pancreata from wild-type and PAR1-deficient C57BL/ 6 mice were dissected and washed twice with Hank's buffered saline solution (HBSS; Gibco, Thermo Fisher Scientific). Pancreata were cut with a scalpel to 3-to 5-mm pieces, after which acinar cells were isolated, mostly as described before [19]. In detail, the pancreas pieces were centrifuged at 500 g for 2 min to remove debris and supernatant. For dissociation of cells, pancreas pieces were next treated with collagenase IA solution (200 UÁmL À1 collagenase IA, 10 mM HEPES, and 0.25 mgÁmL À1 trypsin inhibitor in HBSS) for 30 min. Dissociated cells were centrifuged at 500 g for 2 min and washed twice with wash solution (HBSS supplemented with 5% FBS and 10 mM HEPES) to stop the collagenase reaction. The cell pellet was resuspended in 1 mL seeding medium (IMDM containing 1% FBS, 0.1 mgÁmL À1 trypsin inhibitor, and 1 µgÁmL À1 dexamethasone), passed through a 100-µm cell strainer, collected in 5% FCS-IMDM medium, seeded in 6-well plates and incubated for 24 h to separate non-adherent acinar cells from adherent epithelial and other contaminant cells. The following day, cells in suspension were centrifuged at 500 g for 2 min and embedded in a collagen mixture (1 mgÁmL À1 collagen, 19 PBS, 0.1 M HEPES, 0.75% sodium bicarbonate, 0.1 M NaOH) in 6-well plates with a double 3D layer of 1 mgÁmL À1 collagen (bottom layer without cells, set prior to seeding).

Viability assessment of acinar spheroids
To assess the viability of collagen-embedded acini, two different methods were used. First, cells were stained with propidium iodide (PI) at 20 µgÁmL À1 (final concentration). After 20 min of incubation, phase-contrast images depicting the explanted acinar cells and Tx-Red channel images identifying PI-positive dead cells were taken. As positive control for cell death, 0.5 mM NaN 3 (sodium azide)-treated spheroids were used. In our second approach, we performed a resazurin to resofurin reduction (CellTiter Blue) experiment to determine the cells' metabolic activity. In viable cells, resazurin (blue) is converted to resofurin (pink) through reductase activity in the mitochondria. The collagen in which the cells are embedded precluded efficient fluorescence read-out, and we have used imaging instead to reveal color conversion in the viable cells. In this case, 0.5 mM NaN 3 (sodium azide)-treated spheroids were used as a negative control. Images were taken in the color mode of phase-contrast imaging. In both methods, pictures were taken with the EVOS FL cell imaging system (Thermo Fisher Scientific) at 49, 109, and 209 magnification.

Induction of ADM in acinar spheroids
Collagen-embedded acinar cells were cultured in IMDM containing 1% FBS, 0.1 mgÁmL À1 trypsin inhibitor, and 1 µgÁmL À1 dexamethasone. According to a previously described protocol for ADM [17], acinar cells were treated with 50 ngÁmL À1 murine EGF (PeproTech) or 1 ngÁmL À1 recombinant MMP9 for 5 days. At the end of the experiment, one batch of cells was visualized with the phase-contrast channel of the EVOS FL cell imaging system (Thermo Fisher Scientific) at 209 magnification, after which cells were used to isolate RNA. The other batch of cells was fixed with 4% formalin and permeabilized with 0.2% Triton-X (Sigma). For fluorescence imaging, F-actin was stained with Alexa488-conjugated phalloidin (ActinGreen 488 ReadyProbes Reagent; Thermo Fischer Scientific), and ductal structures were stained using rhodamine linked DBA (Dolichos Biflorus Agglutinin; Vector Laboratories). After staining, cells were washed with PBS and imaged with the Yellow Fluorescence (YFP) and Texas-Red channels of the EVOS FL cell imaging system (Thermo Fisher Scientific) at 209 magnification.

Immunohistochemistry on paraffinembedded material
Orthotopic KP tumors, isolated murine healthy pancreata, and murine organoids were fixed in formalin, embedded in paraffin, and 4-lm-thick slides were subsequently deparaffinized, rehydrated, and washed in deionized water. Slides were stained with hematoxylin and eosin (H&E) according to routine procedures. For immunohistochemistry (IHC), endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide for 15 min at room temperature, with antigen retrieval for 10 min at 100°C in 10 mM sodium citrate buffer, pH 7.4. Slides were blocked for 10 min with Ultra V block (Thermo Fisher Scientific). Primary antibodies against cytokeratin 19 (SAB4501670; Sigma) and amylase (A8273; Sigma) were added (1 : 1600 dilution) for overnight incubation at 4°C. Slides were subsequently incubated with appropriate HRP-conjugated goat antirabbit IgG (Brightvision, Immunologic, VWR, Radnor, PA, USA), after which DAB (Brightvision, Immunologic, VWR) staining was used to visualize peroxidase activity. Slides were photographed with a microscope equipped with a digital camera (Leica CTR500; Leica Microsystems, Wetzlar, Germany).

RNA-Seq
Total RNA was isolated from shCtrl PANC-1 and shPAR1 PANC-1 cells using the NucleoSpin RNA miniprep kit (Macherey Nagel, D€ uren, Germany). Yield and purity (260 nm : 280 nm) were determined by a Nanodrop ND-1000 (Thermo Fisher Scientific). The integrity [RNA integrity number (RIN) > 9.0] of the resuspended total RNA was determined by using the RNA Nano Chip Kit on the Bioanalyzer 2100 and the 2100 EXPERT software (Agilent, Amstelveen, the Netherlands). RNA-sequencing libraries were prepared from 250 ng total RNA using KAPA RNA HyperPrep with RiboErase (HMR) kits (Roche, Basel, Switzerland). Libraries were sequenced using the Illumina HiSeq4000 instrument (Illumina, San Diego, CA, USA) to generate single reads (50 bp). The sequencing depth was approximately 40 million reads per sample. Read quality was assessed by means of the FASTQC method (v0.11.5; http://www.bioinformatics.babraha m.ac.uk/projects/fastqc/). TRIMMOMATIC version 0.36 [21] was used to trim Illumina adapters and poorquality bases (trimmomatic parameters: leading = 3, trailing = 3, sliding window = 4 : 15, minimum length = 40). The remaining high-quality reads were used to align against the Genome Reference Consortium human genome build 38 (GRCh38) [22]. Mapping was performed by HISAT2 version 2.1.0 [23] with parameters as default. Count data were generated by means of the HTSeq method [24] and analyzed using the DESeq2 method [25] in the R statistical computing environment (R Core Team [26]). Statistically significant differences were defined by Benjamini and Hochberg-adjusted probabilities < 0.05. Gene expression extraction was performed via the R2 microarray analysis and visualization platform (http:// r2.amc.nl). Sequence libraries are publicly available through the National Center for Biotechnology Information (NCBI) gene expression omnibus (GEO) under the following accession numbers: GSE155010.

Quantitative real-time PCR
Total RNA was isolated with the NucleoSpin RNA miniprep kit (Macherey Nagel). cDNA was synthesized from DNase-treated total RNA with M-MLV-RT (Promega) and random hexamers (Qiagen, Hilden, Germany). Real-time quantitative RT-PCR was performed using the Sensifast SYBR No-Rox Kit (Bioline, London, UK) on a LightCycler 480 II (Roche). Relative expression levels were calculated using the comparative threshold cycle (dCt) method and normalized for expression of the reference gene TBP. Primer sequences of the analyzed genes are shown in Table S2.

Statistical analysis
Data were presented as mean AE SEM. Statistical analyses were performed using GRAPHPAD PRISM 8.0 (Graph-Pad Software Inc., La Jolla, CA, USA). Statistically significant differences were considered with a P-value of < 0.05. For further details of the statistical analysis, see figure legends. P-values are indicated by asterisks with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

PAR1 deficiency promotes acinar gene expression programs in the pancreas
In light of the recent identification of macrophagesecreted MMP9 as a driver of ADM [17] and our data pointing to MMP9 as a novel PAR1 agonist [18], we hypothesized that PAR1 is the receptor that mediates macrophage-induced ADM. To test this, we first ascertained whether PAR1 deficiency results in altered cell fates in the pancreas. Expression levels of known acinar and ductal markers were measured in pancreata of wild-type and F2R/PAR1-deficient (PAR1-KO) mice.
Expression levels of the acinar cell marker carboxypeptidase A1 (Cpa1) and acinar cell-fate transcription factors Ptf1a, Nr5a2, and Mist1 were strongly increased in PAR1-deficient pancreata compared with wild-type controls (Fig. 1A). The expression of ductal and centroacinar markers was not different (Fig. 1B). To corroborate the findings in a human setting, we next assessed the association of PAR1 with acinar and ductal gene sets in human (nontumor) pancreas gene expression datasets. Samples were dichotomized for median PAR1 expression (i.e., PAR1-high versus PAR1-low), and gene set enrichment analysis (GSEA) revealed that PAR1 expression negatively correlates with acinar signatures and positively correlates with ductal signatures (Fig. 1C).

ADM requires PAR1 activity in vitro
To evaluate the contribution of PAR1 to exocrine pancreatic cell fate, we isolated acinar cells from wild-type and PAR1-deficient mice and determined expression levels of acinar ( Fig. 2A) and ductal (Fig. 2B) markers. Among all markers tested, only Cpa1 expression was significantly increased in PAR1-deficient acinar cells compared with wild-type cells. This suggests that at steady-state and in isolated cell populations, PAR1 deficiency does not result in an overt phenotype and indicates that additional interactions (as present in vivo such as in Fig. 1C) or cues are required to reveal the contribution of PAR1 to cell fates.
To assess whether PAR1 impacts on ADM, we utilized the same acinar cell isolates using recombinant MMP9 or EGF as positive controls for ADM induction [17]. After 5 days of culturing wild-type acinar spheroids (viability tested, shown in Fig. S1) in collagen-embedded cultures, clusters of acinar cells gave rise to duct-like budding structures (Fig. 2C). The occurrence of these structures markedly increased following the exposure to MMP9 and EGF. To ascertain the ductal identity of these structures, cultures were stained with DBA [40], and additionally, F-actin was stained to reveal cell mass. Indeed, DBA staining is markedly enhanced in MMP9 and EGF-treated cultures. Importantly, in Par1-KO and in vorapaxartreated wild-type spheroids, there was no visible ductal structure formation (also in the presence of MMP9 or EGF; Fig. S2A), and these acinar spheroids were negative for DBA staining (Fig. 2D and Fig. S2B). Subsequent transcript analysis revealed that, after 5 days of ADM induction, expression levels of acinar-specific transcription factors Ptf1a, Nr5a2, and Mist1 and of the acinar marker Cpa1 were decreased in wild-type acinar cells, whereas their levels remained unchanged in PAR1-deficient acinar cells (Fig. 2E). In line with changes in acinar-related genes, MMP9 and EGF treatment of wild-type acinar cells led to increased expression levels of the ductal markers Krt19, Muc1, and Sox9 and the centroacinar marker Stmn1 (Fig. 2F). Interestingly, under these culture conditions, the expression of the acinarspecific markers Nr5a2 and Cpa1 and the ductalspecific transcription factor Sox9 significantly differed between the two genotypes in the absence of additional ADM cues (Fig. 2E,F    ductal gene signatures. GSEA analysis showed increased expression of acinar signatures in most PAR1-low groups, and all sets showed enhanced ductal signatures in the PAR1-high group (Fig. 3A). To substantiate these findings, we performed RNA-Seq and GSEA of previously established shRNA-mediated PAR1-deficient (shPAR1) PANC-1 cells (knockdown efficiency 70%, see Ref. [28]   significantly enhanced in shPAR1 PANC-1 cells, and a trend toward a reduced ductal signature was observed in the PAR1-deficient cells (Fig. 3B,C). Next, we compared expression levels of key acinar-and ductalrelated transcription factors and markers (Fig. 3D,E). Expression levels of acinar cell-fate driving transcription factors NR5A2, MIST1, and GATA4 were significantly increased, whereas the ductal transcription factor SOX9 was markedly decreased in shPAR1 PANC-1 cells.
This was confirmed by shRNA silencing of PAR1 in two additional routinely used PDAC cell lines (MIA PaCa-2 and Capan-2) and one patient-derived primary line (AMC-PDAC-096; 096 in short; knockdown efficiency of PAR1 in the 096 shPAR1 cell line shown in Fig. S3). Transcript analysis of acinar-specific transcription factors MIST1 and PTF1A showed a significant increase in all PAR1-deficient PDAC cell lines (Fig. 4A,B). Moreover, expression of the ductal marker KRT19 and of the transcription factor SOX9 was  significantly decreased in all shPAR1 cell lines (Fig. 4C,D), with the exception of KRT19 in the 096 line. These data not only confirm that PAR1 is instrumental in the maintenance of ductal cell fates in cancer but also suggest that loss of its activity can re-establish acinar cell fates in this context. We next evaluated KRT19 and amylase expression by IHC in orthotopic tumor cell grafts grown from control or shPAR1-transduced mouse KRAS G12D / TRP53 flox/flox (KP) tumor cells [28] (Fig. 5). In shPAR1 tumors, KRT19 expression was decreased compared to control silenced tumors (shCtrl). Interestingly, we observed numerous amylase-positive cell clusters in shPAR1 tumors, which were (largely) absent in PAR1-proficient (shCtrl) tumors (Fig. 5 with quantification shown in Fig. S4). This confirms that PAR1 not only contributes to ductal cell fates but that its absence partly re-establishes acinar cell identities.

PAR1 drives ductal cell fates through Myc activity
To elucidate the pathways downstream of PAR1 that govern cell fates, we performed GSEA analysis with the Hallmark Gene Sets [41] on RNA-Seq data from shPAR1 and shCtrl PANC-1 cells (Fig. 6A). This analysis revealed a substantial decrease in Myc-target genes in shPAR1 cells. This is particularly interesting, given that Myc overexpression in acinar cells has been shown to drive ADM [42]. Consequently, we next assessed whether inhibition of Myc activity impacted on acinar and ductal cell fates. PTF1A, MIST1,   RNA was collected 48 h after treatment. Shown is the mean AE SEM (n = 4); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, Student's t-test. ( Fig. 6B,C). In line with these changes, Myc inhibition led to a decrease in ductal marker expression (Fig. 6D,E).
To establish whether Myc functions downstream of PAR1 in the maintenance of ductal cell fates, we tested Myc inhibition on control and shPAR1 PANC-1 and MIA PaCa-2 cell lines. In control silenced cell lines, Myc inhibition resulted in decreased ductal and increased acinar marker expression (dark gray bars in Fig. 6F-I). However, Myc inhibition did not affect the expression levels of these markers in shPAR1 cell lines (compare purple bars to pink bars). Collectively, these findings suggest that PAR1 activity drives ADM and maintains a ductal phenotype through the activity of Myc.

Inhibition of PAR1 and Myc enhances acinar cell fates in murine ductal organoids
In tumor contexts, loss or inhibition of PAR1 was able to partially reverse the ductal phenotype of PDAC cells. To further determine whether PAR1 deficiency has a similar impact on ductal maintenance or acinar reprogramming in healthy (nontumor) ductal cells, we utilized murine ductal organoids. Ductal organoids were cultured in the presence of the PAR1 inhibitors vorapaxar or P1-pal12 and the Myc inhibitor 10058-F4. The expression of acinar markers Ptf1a, Nr5a2, Mist1, and Cpa1, and ductal-specific markers Krt19, Muc1, and Sox9, and centroacinar marker Stmn1 were measured. Both PAR1 and c-Myc inhibition led to an increase in acinar markers during the course of several weeks of treatment (Fig. 7A). Correspondingly, expression levels of ductal markers gradually decreased over time (Fig. 7B). Overall, these results show that PAR1 activation drives ductal differentiation in acinar cells (Fig. 7C) and, more importantly, PAR1 inactivation or Myc inhibition can re-establish acinar cell identities in ductal cells and shows that such plasticity is not unique to acinar cells.

Discussion
Transdifferentiation of acinar cells to ductal cells is thought to contribute to tissue regeneration after injury in the pancreas [17,44]. However, aberrant transdifferentiation of acinar cells into ductal cells during ADM is considered a prerequisite for malignant transformation [45]. ADM critically depends on macrophage-secreted MMP9 and subsequent NF-jB activation in acinar cells [17]. However, the cellular receptor on acinar cells through which MMP9 activates the NF-jB pathway and subsequent ADM has not yet been identified. In the current manuscript, we addressed the hypothesis that PAR1 acts as a key cellular receptor driving cell fate programs in the exocrine pancreas. This hypothesis is based on previous work that highlights PAR1 as the orchestrating receptor of macrophage-tumor cell interactions in PDAC and on the fact that MMP9 has recently been identified as a novel PAR1 agonist [18]. In line with former findings and with our current hypothesis, we show that genetic ablation of PAR1 increases acinar-related gene expression in the healthy pancreas and that PAR1 deficiency limits ductal transdifferentiation in experimental models for ADM. Moreover, silencing PAR1 in PDAC cells re-establishes acinar cell identities in these ductal cells. Overall, here we identify PAR1 as a novel cellular receptor to orchestrate ADM, and we show that PAR1 governs cellular identity in the (pre)malignant transformed pancreas.
Acinar cells are thought to have the highest plasticity of all pancreatic cell types and, indeed, rather easily de-differentiate into a progenitor phenotype, after which they further differentiate into duct-like cells. Acinar (de)differentiation under benign conditions is reversible, but oncogenic KRAS activation renders this mechanism irreversible and thereby drives ductal metaplasia [8,46]. KRAS silences the acinar transcription factors MIST1 and PTF1A while it concurrently induces expression of the ductal transcription factor SOX9 [17,47]. Silencing the acinar transcription factors predisposes acinar cells to ductal differentiation [48] and accelerates ADM [49]. In the current manuscript, we identify PAR1 as a novel player in acinar (de)differentiation in health and disease. We show that PAR1 inactivation leads to increased expression levels of PTF1A and MIST1, whereas expression levels of SOX9 and KRT19 decrease. Importantly, this PAR1dependent shift toward an acinar phenotype is not only evident in healthy acinar cells, as PAR1 silencing also increases acinar gene expression in cells from established PDAC. This suggests that also ductal cells are plastic and able to regenerate into acinar-like cells, even in the presence of oncogenic KRAS activation. Based on these findings, we suggest that PAR1 is critical in ADM and that PAR1 inhibition may prevent malignant progression.
An intriguing finding in our manuscript is the identification of Myc as a downstream factor of the PAR1driven cellular differentiation program. Myc inhibition in PDAC cells mimics PAR1 inhibition in re-activating the expression of acinar transcription factors (PTF1A, MIST1, and NR5A2). Interestingly, Myc activation is known to drive acinar transdifferentiation. Indeed, acinar-specific Myc overexpression has been described  to enhance ductal metaplasia and induce the formation of neoplasms that display both acinar-like neoplastic cells and duct-like neoplastic cells [42]. In addition to affecting acinar-ductal tissue homeostasis, our data show that Myc inhibition leads to the re-expression of acinar-specific genes in both PDAC and healthy ductal cells. Surprisingly, acinar cell transdifferentiation has not been reported in the range of ductal plasticity in the pancreas [46]. Our findings here suggest that the PAR1-Myc axis may be a key driver that prevents transdifferentiation of ductal cells back into acinar-like cells.
Another interesting, and at first glance, surprising finding of our study is that PAR1 deficiency (either by genetic ablation or by pharmacological inhibition) also limits ADM formation by EGF. However, PAR1 is known to transactivate the epidermal growth factor receptor (EGFR) in various experimental contexts. For instance, PAR1 transactivates EGFR in invasive breast carcinoma, thereby promoting cellular invasion [50]. PAR1-dependent intracellular phosphorylation of EGFR also induces proliferation of vascular smooth muscle cells [51], whereas PAR1 also promotes human colon cancer proliferation through EGFR transactivation [52]. Our data underscore the importance of EGFR-PAR1 cross-talk in cell fate decisions.
PAR1 expression levels are known to correlate with cancer progression and overall survival in different tumor types of epithelial origin [53,54]. Consistent with such clinical data suggesting a tumor-promoting role of PAR1, experimental studies provide solid evidence for PAR1 as a driver of cancer progression. PAR1 induces proliferation, migration, and invasion of cancer cells in in vitro experiments [54,55], whereas tumor cell-specific PAR1 overexpression drives tumor growth in preclinical animal models of breast and prostate cancer [56]. Inline, inhibition of tumor cell PAR1 [57,58], stromal PAR1 depletion [16,59], or pharmacological PAR1 inhibition [14] consistently suppresses tumor growth in experimental animal models. In the current manuscript, we extend this notion by showing that PAR1 not only drives tumor growth but also contributes to the premalignant stages of PDAC. Of note, this effect of PAR1 may be context-dependent and could be specific for PDAC. Indeed, an elegant prior study showed that PAR1 deficiency in Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice results in diminished apoptosis in transformed epithelia with subsequent larger and more aggressive prostate tumors [60]. Similarly, PAR1 deficiency on the adenomatous polyposis coli mutant (APC Min) background resulted in more and larger adenomas suggesting that PAR1 limits premalignant transformation in prostate and intestinal tumors. In breast cancer, PAR1 deficiency does not seem to modify spontaneous tumor formation [61], further strengthening the notion that PAR1 plays a context-dependent role in premalignant transformation.

Conclusion
A picture emerges in which PAR1 is a cellular receptor that governs ductal cell identity. Its perturbation reveals remarkable cellular plasticity in ductal cells. Furthermore, PAR1 represses acinar transcriptional programs to allow regeneration after injury. PAR1 activates the Myc pathway that enhances ADM and locks differentiated acinar cells in a ductal cell state. Downregulating the PAR1-Myc axis, in part, destabilizes the ductal phenotype leading to the transdifferentiation of PDAC cells. Overall, we identify the PAR1-Myc axis as a driver of ductal cell fates in healthy, premalignant pancreas and PDAC. channels. YFP and Tx-Red channels were merged on ImageJ. Scale bars indicate 200 µm. Fig. S3. shRNA knockdown confirmation of the primary 096 PDAC cell line. Shown mRNA expression for PAR1 (F2R) is relative to TBP. Shown is the mean AESEM (n=4); * p<0.05, Student's t-test. Fig. S4. Amylase expression increases in shPar1 tumors in the murine orthotopic KP model. Amylasepositive nuclei were counted on 4X images of 2 independent tumors in both the shCtrl and shPar1 group with the Image J IHC toolbox for DAB staining and nuclei detection (https://imagej.nih.gov/ij/plugins/ihctoolbox/index.html). Shown is the meanAESEM (n=2); ** p<0.01, Student's t-test. Table S1. Culturing media composition and final concentration chart for murine healthy ductal organoids. Table S2. Primer sequences for qPCR analysis of mRNA expression. Appendix S1. Acinar Signature. Acinar cell-fate related genes used for gene expression analysis. Appendix S2. Ductal Signature. Ductal cell-fate related genes used for gene expression analysis.