Oncogene-Induced Senescence Limits the Progression of Pancreatic Neoplasia

Author Manuscript Published OnlineFirst on June 17, 2020; DOI: 10.1158/0008-5472.CAN-19-3763 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Oncogene-induced senescence limits the progression of pancreatic neoplasia

through production of Activin A

Yajie Zhao,1,7 Zhichong Wu,1 Marie Chanal,1 Fabienne Guillaumond,2,3,4,5 Delphine Goehrig,1

Sophie Bachy,1 Moitza Principe,1 Audrey Ziverec,1 Jean-Michel Flaman,1 Guillaume Collin,1

Richard Tomasini,2,3,4,5 Arja Pasternack,6 Olli Ritvos,6 Sophie Vasseur,2,3,4,5 David Bernard,1

Ana Hennino,1 and Philippe Bertolino1

1 Cancer Research Centre of Lyon (CRCL), INSERM U1052, CNRS UMR5286, Claude Bernard

University, Lyon, France. 2 Centre de Recherche en Cancérologie de Marseille (CRCM), Unité

1068, Institut National de la Santé et de la Recherche Médicale, Marseille, France. 3 Institut

Paoli-Calmettes (IPC), Marseille, France. 4 Unité Mixte de Recherche (UMR 7258), Centre national de la Recherche Scientifique (CNRS), Marseille, France. 5 Université Aix-Marseille,

Marseille, France. 6 Department of Physiology, Faculty of Medicine, University of Helsinki,

Helsinki, Finland. 7 Department of Geriatrics, Ruijin Hospital, School of Medecine, Shanghai

Jia Tong University, Shanghai, China.

Running title: Activin A limits pancreatic neoplastic lesions

Keywords: Pancreatic Neoplasia, Senescence, Activin A, SASP, Acinar-to-Ductal-Metaplasia

Conflict of Interest: The authors disclose no conflicts of interest

Correspondence: Philippe Bertolino, Cancer Research Center-Lyon (CRCL), Inserm U1052,

CNRS UMR5286, Univ. Claude Bernard LyonI. Centre Leon Berard, Bat. Cheney D, 28 Rue

Laennec, 69008 Lyon, France. Phone: +33-4-7878-5160, email: [email protected]

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 17, 2020; DOI: 10.1158/0008-5472.CAN-19-3763 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT

Pancreatic ductal adenocarcinoma (PDA) is a deadly and aggressive cancer. Understanding mechanisms that drive pre-neoplastic pancreatic lesions is necessary to improve early- diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase

(ALK4) have been demonstrated to favor PDA onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data-mining and histological analyses performed on human and mouse tumor tissues reveal that Activin A is the major ALK4-ligand that drives PDA initiation. Activin A, which is absent in normal acinar-cells, was strongly induced during acinar-to-duct metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that is induced in precursor lesions. Blocking Activin A-signaling, through the use of a soluble form of Activin receptor IIB (sActRIIB-Fc) and ALK4-knockout

(KO) in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that Activin A acts as a protective senescence-associated secretory phenotype (SASP) factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions.

SIGNIFICANCE

This study identifies Activin A to be a beneficial, senescence-secreted factor induced in pancreatic pre-neoplastic lesions, which limits their proliferation and ultimately slows progression into pancreatic cancers.

INTRODUCTION

Pancreatic ductal adenocarcinomas (PDAs) are associated with some of the worst survival outcomes among all cancers, with a median survival rate of approximately 6 months and a 5- year survival rate < 8% (1). Although several therapeutic options for PDA have emerged over the last decade, their efficiencies remain relatively limited, and approaches designed to circumvent the commonly late diagnoses of those tumors remain lacking (2). Studies exploring the mechanisms and mutations associated with PDA onset have confirmed the major role played by Kras-activating mutations during the formation of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN), which precede PDA development (3,4). Although the progression of these lesions to PDA requires mutations in additional factors, such as the cyclin-dependent kinase inhibitors p16 and p53, the latency prior to the evolution of PanIN in aggressive and invasive tumors supports the existence of some mechanism that limits the transformation of neoplastic cells. Interestingly, although

Kras activating mutations are known to drive PDA onset (5), Kras can also promote oncogenic-induced senescence (OIS), a safeguard program that limits cancer proliferation and initiation (6,7). The regulating role played by OIS during pancreatic tumorigenesis has previously been demonstrated (8-12), and animal models expressing the gain-of-function

KrasG12D mutation support the existence of a senescent tumor-suppressive program that limits the expansion of Kras-mutant cells (13,14). In addition to the OIS-induced arrest of growth in cancer-initiated cells, senescent cells are capable of producing and secreting a complex series of cytokines and molecules that are associated with the senescence- associated secretory phenotype (SASP) (15). A large number of SASP-associated proteins have been identified, among which several are involved in pro-inflammatory responses (16).

Although the exact roles of SASP-associated cytokines and pro-inflammatory components

during PDA initiation remain unclear (17,18), recent evidence supports the importance of

SASP-associated molecules, such as C-X-C motif chemokine ligand 1 (CXCL1) during pancreatic tumorigenesis (18).

Among the SASP candidates that are potentially involved in paracrine senescence, transforming growth factor  (TGF) superfamily ligands, including TGF1, inhibin subunit beta A (INHBA), bone morphogenetic protein 2 (BMP2), and growth differentiation factor 15

(GDF15), have been identified through secretome analyses of cells undergoing OIS (19).

Interestingly INHBA, which encodes Activin A, is targeted by Kras during oncogenic activation in pancreatic duct cells (20), suggesting that Activin A may be a candidate SASP-associated factor that is induced by Kras-OIS in pancreatic neoplastic lesions. Interestingly, although the ablation of the Activin cognate receptor Activin-like kinase 4 (ALK4) favors the development of intraductal papillary mucinous neoplasms (IPMNs) and accelerates the formation of PDA in mice harboring the KrasG12D mutation (21,22), little is known regarding the contributions of ALK4-mediated Activin signaling and its downstream signaling factors, P-Smad2 and P-

Smad3, to the earliest stages of Kras-driven pancreatic cell transformations or the involvement of this pathway to Kras-OIS in low-grade pancreatic neoplastic lesions. Here, we aimed to understand the mechanisms associated with ALK4-mediated signaling during PDA initiation. We report the identification of Activin A as the major ALK4-ligand expressed in pancreatic ductal neoplastic lesions. Our work demonstrates that Activin A, which is absent in normal acinar pancreatic cells, is strongly induced in ADM-lesions, promoted by KrasG12D and pancreatitis, in the mouse and human pancreas. More importantly, we report that

Activin A-induced expression during ADM contributes to the senescence program induced in precursor lesions. Blocking Activin A signaling, through the use of a soluble form of Activin receptor IIB (sActRIIB-Fc) molecules or the genetic targeting of ALK4, we further

demonstrate that Activin A acts as a beneficial SASP factor that limits the proliferation and expansion of pancreatic neoplastic lesions.

METHODS

Mouse Models and Experimental Procedures

All animal maintenance and experiments were performed in accordance with Animal

Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and French laws and were approved by the local Animal Ethics Evaluation Committee. The generation of Acvr1b mutant mice has been previously described (23). Acvr1bflox/flox;LSL-KrasG12D/+;Ptf1a-Cre (termed 4KC) mice were created by breeding Acvr1bflox/flox mice with the previously established LSL-

KrasG12D/+;Ptf1a-Cre (termed KC) model (24). LSL-KrasG12D/+;Ink4a/Arfflox/flox;Pdx1-Cre mice

(termed KIC) have been previously described (25). Acute pancreatitis experiments were performed using a standardized procedure (26). Briefly, pancreatitis was induced in 1.5- month-old wild-type (WT) C57BL/6 mice, using a regimen of 7 hourly intraperitoneal (i.p.) injections of caerulein (50 g/kg body weight per injection; Sigma-Aldrich) dissolved in phosphate-buffered saline (PBS) for 2 consecutive days, followed by pancreas collection 48 hours later. Control littermates received injections of PBS only.

Cell lines

Panc-1 (CRL-1469), Mia Paca-2 (CRL-1420) and Capan-1 (HTB-79) PDA cell lines were purchased from ATCC and grown in the recommended culture media. All cell lines were mycoplasma tested and experiments performed between passages 12-15 (Panc-1), 6-9

(Capan-1) and 8-12 (Mia Paca-2) following their purchase. Activin A (25 g/mL, Peprotech),

TGF (5 g/mL, Peprotech), SB431542 (10 M Sigma-Aldrich, 616464) and sActRIIB-Fc

(0.5g/mL) were added to cells for 24 h, in 2% FCS culture media.

Immunohistological Staining and Analysis

Mouse pancreases were harvested and fixed overnight, in 4% buffered formalin, prior to paraffin embedding and 3-m sectioning. All immunohistological staining procedures, unless otherwise mentioned, were performed following heat-induced epitope retrieval

(antigen-unmasking solution, Vector Laboratories, UK), and primary antibodies were incubated overnight at 4°C. Immunohistochemical (IHC) stains were revealed using 3,3’- diaminobenzidine (DAB kit; Vector Laboratories, UK) and sections were counterstained with hematoxylin. Immunofluorescence (IF) staining was performed using a standard protocol, and sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Vector

Laboratories, UK). All images were acquired on an Eclipse-NiE NIKON microscope and analyzed using NIS-Elements Software. A complete list of the primary antibodies used is provided in Supplementary Table 1. BIC14011a and PA485 human tissue microarrays (TMAs) were obtained from a commercial source (US Biomax, Inc.). BIC14011a is a pancreas array that contains 22 pancreatitis cases, 18 pancreatic intraepithelial neoplasia cases, and 8 pancreatic adenocarcinoma cases. PA485 is a pancreatitis and matching pancreatic adenocarcinoma array, containing 43 cases of pancreatitis and 5 matched pancreatic adenocarcinomas. Senescence-associated beta-galactosidase (SA--gal) whole-mount staining was performed overnight, according to the procedures described in the Senescence

Cells Histochemical Staining Kit (Sigma-Aldrich, #CS0030). All incubations and washes were performed using fresh and filtered solutions. Stained tissues were subsequently washed 3 times in PBS and post-fixed with 4%-paraformaldehyde, prior to paraffin-embedding for

sectioning. Combined SA--gal/IF and SA--gal/IHC staining were performed on 7-M sections, obtained from whole-mount Sa--Gal stained tissues. IF and bright-field images were acquired on a Zeiss Axioimager microscope and merged using ImageJ. Activin A positivity in pancreatitis and PanIN lesions was visually quantified by the identification of at least 1 positive cell per lesion, within each individual TMA spot. The quantification of

ADM/PanIN areas was performed on hematoxylin and eosin (H&E)-stained pancreatic scanned sections (Panoramic Scan 3D HISTECH Ltd., Budapest, Hungary). The surface areas of individual regions containing ADM/PanIN lesions were determined using ImageJ and normalized against the surface areas occupied by normal pancreatic tissues.

Inhibition of Activin A signaling using soluble ActRIIB-Fc

Activin A signaling was inhibited through the use of recombinant sActRIIB-Fc protein, which was produced and used as previously reported (27,28). For in vivo treatments, randomized 1.5-month-old KC mice were injected i.p. with either 5 mg/kg body weight sActRIIB-Fc suspended in PBS or PBS alone, twice a week. Short-term (ST) and long-term (LT) treatments consisted of 5 and 12 injections, respectively, performed over 3 and 6 consecutive weeks, respectively. All pancreases were collected 24 h following the last injection. For in vitro treatments, sActRIIB-Fc was used at a final concentration of 0.5 g/mL.

Acinar 3D Cell Culture

Mouse acinar cells were isolated, as previously reported (29). Resected pancreases were transferred to ice-cold Hank’s balanced salt solution (HBSS 1, Life-Technologies) and subsequently minced/dissociated with 1 mg/mL Collagenase P (Sigma-Aldrich

#11213865001) in 1 HBSS, for 30 minutes at 37°C. Dissociated cells were washed with 1

HBSS, containing 10% fetal bovine serum (FBS) and 10 mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), prior to filtration through a 100-m sterile nylon mesh (VWR, France). Following centrifugation, cells were suspended in 3D-culture medium

(RPMI1 640 medium, Gibco, #72400-021), supplemented with 25mM HEPES, 1% FBS, 1% penicillin/streptomycin (P/S), 1 g/mL dexamethasone, and 0.1 mg/mL soybean trypsin inhibitor (Sigma-Aldrich, #T6522) (29). Three-dimensional (3D) cultures were performed in

24-well tissue culture plates, coated with 3 mg/mL type I collagen from rat tail (Life

Technologies, #A1048301). Acinar-isolated cells were embedded in a 1:1 mix of rat-tail collagen and 3D-culture medium, prior to plating on the prepared collagen layers. The cell- collagen mixture was allowed to solidify for 1 h at 37°C, before covering the collagen discs with 1 mL 3D-culture media. Media was changed on days 1 and 3, and the discs and cells grown on collagen were harvested on day 6, for gene expression analysis, SA--gal whole- mount staining, or paraffin embedding, prior to immunohistological staining.

RNA expression analysis

RNA was extracted using the RNeasy-Kit, according to the manufacturer’s instruction

(Qiagen, Valencia, USA). Real-time polymerase chain reaction (PCR) analyses were performed on a Step-One RT-System (Applied-Biosystem, France). The primer sequences used are detailed in Supplementary Table 2.

Gene expression analysis

Gene expression analysis was performed using the Affymetrix gene expression array dataset, deposited in the Gene Expression Omnibus repository (accession no. GSE61412) (30). Briefly, expression data from 9-week-old KIC (n = 3) or control [LSL-KrasG12D/+;Ink4a/Arfflox/flox

(termed KI, n = 3] mice were normalized using the robust multichip average (RMA) method in Bioconductor package (R 3.4.4 software) (30,31). Heatmap illustrations showing the expression of genes coding ligands that trigger the ALK4 function were generated with

Morpheus software (https://software.broadinstitute.org/morpheus). Each column represents the RMA data for a given gene, obtained from 3 independent control (C9) or PDA

(T9) pancreas samples, derived from 9-week-old KI and KIC mice, respectively. The significant differential expression of genes between KIC and control KI pancreases was determined from normalized RMA dataset, using a Student’s t-test analysis. Genes with a p-value < 0.05 were the sole candidates considered as being significantly dysregulated in KIC-samples and are represented with fold-changes and p-values transformed into log base 2 and –log base

10 scales, respectively.

Statistical Analysis

All analyses were performed using Prism 8 software (GraphPad, USA). Statistical analyses were performed as described in the figure legends. Unpaired Student’s t-tests were used for pairwise comparisons. A one-way analysis of variance (ANOVA), with Tukey’s post hoc test, was used for multiple comparisons. *P < 0.05; **P < 0.01 and ***P < 0.001. The significance of Activin A expression in pancreatitis and PanIN lesions, assessed using TMA analysis, was determined using Fisher’s exact test.

RESULTS

Activin A expression is ectopically induced in acinar cells during ADM

To understand the mechanisms associated with the loss of ALK4-signaling during PDA initiation, we first attempted to identify potential candidate ligands that trigger ALK4

activation and the downstream phosphorylation of its effectors, Smad2 and Smad3, in pancreatic lesions mediated by a KrasG12D mutation. Taking advantage of a previous transcriptomic analysis, performed on the LSL-KrasG12D/+;Ink4a/Arfflox/flox;Pdx1-Cre (termed

KIC) PDA-mouse model (25,30), we used data mining to screen the expression levels of 8 putative ALK4 ligands. Ligand expression levels were analyzed in 9-week-old KIC mice, after the development of PanIN and pancreatic adenocarcinomas (25). Among the tested genes

(Fig. 1A), 6 were found to be either significantly up- (InhbA, InhbB, and Gdf11) or downregulated (Nodal, Gdf1, and Mstn) in KIC mouse pancreases (p < 0.05) compared with age-matched KI control pancreases (Fig. 1B). More interestingly, a fold-change analysis indicated that InhbA and InhbB were the only genes that displayed a greater than 2-fold- change, InhbA, which encodes Activin A, showing the most significant change (Fig. 1B).

These results suggested that Activin A may represent the major ALK4-signaling ligand during pancreatic tumorigenesis. We next used an immunohistological approach to detect Activin A expression within different grades of pancreatic lesions. Although Activin A was not expressed in acinar-, islet- and Ck19-positive duct-cells in the normal pancreas, Activin A expression was significantly increased in all KIC-analyzed lesions, ranging from ADMs to adenocarcinomas (Fig. 1C). A reduced number of Activin A-expressing cells were detected in the PanIN1/2 lining, and the diffuse expression of Activin A in was detected in PanIN3 and adenocarcinoma lesions (Fig. 1D), which led us to speculate that the ectopic induction of

Activin A expression in ADM may represent an acute response to an oncogenic insult, driven by the combined ablation of the Ink4a/Arf allele and the activation of Kras.

We next questioned whether Activin A-induced expression occurred when ADM was induced by other pathological contexts, such as the sole activation of KrasG12D in acinar cells or the inflammatory cues that drive pancreatitis. Using LSL-KrasG12D/+;Ptf1a-Cre (termed KC) and

caerulein injections in WT animals as respective models of low-grade neoplastic lesions (32) and pancreatitis (33), we found that Activin A was strongly induced in acinar-to-duct converting cells, independent of the ADM-triggering signal (Fig. 2A and 2B). Interestingly, as demonstrated in KIC pancreases, we confirmed that following the induction of Activin A expression in ADM, Activin A was only detected in a subset of Ck19-negative cells lining the

PanIN lesions in KC mice (see arrow in Fig. 2A). Finally, using human pancreatic TMA analysis, we further validated the lack of Activin A expression in normal acinar cells and the existence of robust Activin A expression in 60% of ADM-lesions observed in patients (n = 64), whereas Activin A immunoreactivity was reduced and limited to a subset of duct-cells in low- grade PanIN (Fig. 2C).

Targeting Activin A in KC mice accelerates the formation of ADM/PanIN lesions

Because Activin A appears to be ectopically expressed in acinar cells during ADM, we next addressed the consequences of blocking Activin A signaling. We first used sActRIIB‐Fc, a soluble Activin receptor that was previously demonstrated to bind Activin A with high affinity (KD of 35.7 pM) and to inhibit Activin A signaling through ALK4 (27,34). Short-term

(ST) treatments, involving 5 i.p. sActRIIB‐Fc injections, performed over 3 weeks, significantly increased the weights of 1.5-month-old, sActRIIIB-FC-treated KC mouse pancreases compared with those from age-matched WT and non-treated KC controls (Fig. 3A and 3B).

Histological analysis of KC-treated animals confirmed the inhibition of Smad2- phosphorylation (Fig. 3C) and revealed that sActRIIB-Fc administration accelerated the development of ADM/PanIN lesions, as assessed by surface area measurements (Fig. 3D). In contrast, no alterations were observed in the pancreases of WT sActRIIIB-FC-treated mice

(Fig. S1A-C). These observations were subsequently confirmed through the use of an ALK4

conditional KO mouse model, which was previously reported (23), to generate

Acvr1bflox/flox;LSL-KrasG12D/+;Ptf1a-Cre compound-mutant mice (termed 4KC). Although the analysis of 3-week-old pancreases from KC and 4KC animals did not reveal obvious ADM or

PanIN lesions, we found that the pancreases from 1.5-month-old 4KC mice were enlarged, presenting an increased number of ADM lesions and reduced P-Smad2 expression (Fig. 3E,

Fig. S2A-B, and Fig. S3). The accelerated ADM formation observed in 4KC pancreases, compared with KC pancreases, was further confirmed by the identification of an enlarged area of Ck19 immunoreactivity, associated with more pronounced collagen deposition and the increased recruitment of Desmin+ pancreatic-stellate cells (Fig. S2C).

Activin A expression is induced in senescent cells found in ADM lesions

Gene-expression screening performed on cells undergoing OIS has indicated that InhbA may represent a SASP factor candidate (19). Because InhbA has been reported to be a target of

Kras oncogenic activation in pancreatic duct cells (20), we hypothesize that the induced- expression of Activin A during ADM may underline a Kras-OIS process occurring in those lesions. When driven by a KrasG12D mutation or TGF stimulation, primary cultures of pancreatic acinar-cells grown in a 3D-collagen matrix form cystic-duct structures that resemble and recapitulate ADM (29). Using such a model, we found that InhbA expression was induced during the formation of duct-structures generated from cultured KC-acinar cells

(Fig. 4A). Although the loss of acinar markers and increased duct-marker expression confirmed the formation of duct-like structures (Fig. S4A-C), the use of a SA--Gal assay indicated the existence of senescent cells lining the formed structures (Fig. 4B). Real-time

PCR analysis further confirmed the induced expression of the putative senescence- associated cyclin-dependent kinase inhibitors Cdkn1a (p21) and Cdkn2a (p16) and several

SASP-associated genes, such as Il6, Il1a, Ccl20, and Vegfc, in day-6 duct-like structures compared with day-1 acinar cells (Fig. 4C). Having demonstrated that acinar-derived duct- like structures were subjected to senescence and expressed InhbA, we next addressed whether Activin A was produced by senescent cells in ADM lesions. Using pancreases from

KC mice, we first confirmed the induction of a senescence-phenotype in ADM and PanIN lesions (Fig. 4D), as previously described (12). Interestingly, we noted that the patterns of

SA--Gal+ cells detected in both ADM and PanIN-lesions were reminiscent of the pattern of

Activin A+ cells observed in KIC, KC, and caerulein-induced lesions (Fig. 4D). The colocalization of Activin A expression in SA--Gal+ cells and the expression of the senescence markers p21 and -H2A histone family member X (H2AX) was confirmed in ADM lesions (Fig.

4E and F). The colocalization of ALK4 and Activin A in KC lesions further suggests that these mechanisms are cell-autonomous (Fig. S5). Taken together, these results support a role for

Activin A as a SASP component in ADM lesions.

Inhibition of Activin A-signaling reduces OIS in ADM

After demonstrating the Activin A signaling increased ADM/PanIN formation, we next questioned whether this finding reflects an escape to the senescence program, promoted by

KrasG12D activation. The treatment of KC-derived acinar-cells with sActRIIB-Fc confirmed that duct-structures obtained from treated cells were less prone to senescence, demonstrated by reduced SA--Gal staining and the decreased expression levels of the senescence-associated cyclin-dependent kinase inhibitors Cdkn1a and Cdkn2a (Fig. 5A-B and Fig. S6A). Although the expression levels of Ccl20 and Vegfc were reduced following sActRIIB-Fc treatment, the expression levels of Il6, Il1a, Il1b, and Ccl2 were not significantly impacted (Fig. 5B), suggesting that the inhibition of Activin A signaling has moderate impacts on SASP

regulation. Histological analysis confirmed that senescence was reduced in the formed cysts during the differentiation of ADM structures, yet the expression levels of the duct-markers

Ck19 and Sox19 were not impacted following the inhibition of Activin A signaling (Fig. 5C and

Fig. S6B). Finally, we found that the decreased senescence observed in SA--Gal assays was further supported by significant reductions in p21 and H2AX expression levels in Ck19+ cells within the duct-structures formed from KC-sActRIIB-Fc treated and 4KC acinar cells (Fig. 5D-E and Fig. S6C). We confirmed a direct role for Activin A on Cdkn1a and p21 expression, mediated by P-Smad2 in the Panc-1 adenocarcinoma cell line, which was responsive to

Activin A stimulation, unlike in Capan-2 cells that are not responsive to Activin A stimulation

(Fig. S7A-C). Mia-Paca-2 cells were responsive to Activin A stimulation but did not show p21 upregulation. Based on these observations, we speculated that blocking Activin A signaling may not alter the formation of ADM but, instead, impact senescence mechanisms among pre-neoplastic ADM cells. We explored this hypothesis in 4KC and KC animals exposed to ST sActRIIB-Fc treatments. As shown in Figure 5F and figure S8A, we found that blocking Activin

A signaling resulted in reduced ADM and PanIN SA--Gal activity, for both models. Reduced senescence was further associated with significantly decreased expression levels of p21 and

H2AX (Fig. 5G-H and Fig. S8B-C), indicating that in vivo Activin A signaling likely contributes to the senescence cues observed in Kras-mediated ADM and PanIN.

Senescence escape, mediated by Activin A signaling inhibition, results in the formation of proliferative lesions

Senescence bypass is a key feature of tumor progression, and OIS results in the transcriptional repression of proliferation, through the activation of the p53-p21 and/or p16-

Rb suppressor pathways (7). Because blocking Activin A signaling resulted in reduced

senescence and the decreased expression of the cell cycle inhibitor p21, we next determined whether these observations are associated with the promotion of proliferation. The quantification of paraffin-embedded structures confirmed that the chemical and genetic inhibition of Activin A signaling resulted in the formation of large-diameter cysts (Fig. 6A and

Fig. S9A). Using Ki67 as a proliferative marker, large cysts were observed with a larger percentage of Ki67-positive cells per duct following Activin A inhibition compared with controls (Fig. 6B and Fig. S9B). Consistent with these observations, the analysis of 4KC and sActRIIB-Fc treated KC pancreases further confirmed that ADM lesions demonstrated significantly increased proliferation compared with age-matched controls (Fig. 6C and Fig.

S9C). Interestingly, we found that proliferation was sustained in PanIN lesions and in Ck19+ cells expressing ALK4, following sActRIIB-Fc treatment (Fig. 6C and Fig. S10A-D and S11).

These results indicated that the inhibition of Activin A signaling may favor the formation of lesions with increased proliferative potential, in a cell-autonomous manner. The administration of sActRIIB-Fc to 4KC animals did not impact the pancreas volume, Activin A expression levels, or the numbers of Ck19+ pancreatic cells or Ki67+ proliferative cells in ADM and PanIN lesions (Fig. S10).

Senescence escape, mediated by the inhibition of Activin A signaling, results in the accelerated progression of pancreatic tumors and the formation of cystic lesions, in vivo

We next examined the consequences of sustained Activin A inhibition on KrasG12D-expressing acinar cell transformation. The analysis of 14-week- and 30-week-old 4KC mice confirmed the accelerated progression of ADM to PanIN-graded lesions within the entire pancreas (Fig.

7A). 4KC mutant animals developed Ck19+ cystic lesions lacking ALK4, which were reminiscent of the IPMNs reported in the Acvr1bflox/flox;LSL-KrasG12D/+;Pdx1-Cre model

developed by Qiu et al. (22) (Fig. 7A-B and Fig. S12A-C). Subsequent analysis revealed that

4KC pancreases presented significantly increased numbers of proliferating Ki67+/Ck19+ adjacent cells, located within the lining of the cystic lesions (Fig. 7C). To further validate these observations, we explored the consequences of long-term (LT) sActRIIB-Fc treatment in KC mice, by administering i.p. sActRIIB-Fc injections to 1.5-month-old KC mice twice a week for 6 weeks, prior to the histological analysis of pancreatic tissues. Consistent with our finding in aged 4KC mice, the analysis of LT sActRIIB-Fc treatment in KC mice confirmed that the inhibition of Activin A signaling and the subsequent senescence escape of ADM cells resulted in the accelerated formation of both PanIN and Ck19+ cystic lesions in the pancreases of LT sActRIIB-Fc treated mice (Fig. 7D-E). As observed within the 4KC cystic lesions, a significant increase in the number of proliferating Ck19+ cells was observed, which were frequently adjacent to each other and located in the lining of the cystic lesions (Fig.

7F).

DISCUSSION

The contribution of ALK4-mediated signaling to the onset of pancreatic cancer has been demonstrated by the identification of ACVR1B homozygous mutations associated with PDA and the use of ALK4-KO mouse models (22,35,36). Although the role played by ALK4- mediated signaling has previously been demonstrated in the promotion of aggressive pancreas cancers and the formation of IPMNs (22,35,36), little is known regarding the ligands that trigger ALK4 activation during pancreatic tumor initiation. Here, we report that

Activin A was identified as the most prominent ALK4 ligand expressed during the onset of mouse pancreatic tumors. We showed that Activin A expression was strongly induced in

ADM lesions and was subsequently limited to a subset of cells in lesions that progress to

PanIN grades, in both mouse and human tissues. Our observations indicate that Activin A may represent a major SASP factor in the OIS promoted by Kras within ADM. These results further suggest that Activin A production in neoplastic, senescent cells inhibits the proliferation of ADM, in a cell-autonomous manner, limiting progression to PanIN and more advanced lesions, through the modulation of p16 or p21 expression. In addition, our study indicates the importance of Kras-OIS for the limitation of ADM progression to more advanced lesions and further demonstrates that SASP factors could exert beneficial, anti- tumoral effects. These results emphasize that blocking Activin A-signaling through ALK4 reduced senescence, both in vitro and in vivo, leading to the increased proliferation of ADM and PanIN lesions, which is consistent with the reduced survival observed for patients carrying ALK4 mutations (36).

The involvement of Activin A in PDA is supported by clinical studies reporting the expression of Activin A in a subset of human pancreatic cancers and the identification of elevated serum

Activin A levels in PDA patients (37,38). Activin A has been reported to drive the self-renewal of pancreatic cancer stem cells (39,40) and to promote pancreatic cancer-associated cachexia (38,41). Our results indicated that in addition to previously identified pro-tumoral functions in advanced lesions, Activin A signaling plays a suppressive role during the early stages of pancreatic tumor initiation, through SASP-associated functions. This observation supports the existence of anti-tumoral SASP factors, such as Activin A, that exert beneficial in vivo effects during OIS.

Although members of the TGF signaling family have been associated with a pro-fibrotic senescence phenotype (42), in contrast with the pro-inflammatory SASP mediated by nuclear factor (NF)-B, other ligands within the TGF-superfamily have been proposed to be

important actors in the paracrine senescence mediated by Kras (19). The blockade of TGFβ signaling has shown to enhance oncogenic Ras-induced tumorigenesis and metastasis, in mammary epithelial cells, and to reduce senescence, in pancreatic cancer models (19,43).

Proteomic analysis has revealed a dynamic pattern of Ras-related SASPs, indicating a role for

TGF signaling during the early stages of the senescence process (42) and identifying INHBA,

GDF15, BMP2, and BMP6 as major actors during paracrine SASP, whereas TGF1 was only moderately induced compared to the above-mentioned ligands (19). These observations indicate that Activin A may represent a key element in TGF-signaling-mediated paracrine senescence. This hypothesis is supported by the proposed use of Activin A as a potential blood biomarker for senescent cell burden, in vivo (44), and is consistent with the recently published role for Activin A as a prominent autocrine regulator of hepatocyte growth arrest and cellular senescence, through CDKN2B/p15ink4a (45).

Although Activin A downstream signaling, mediated by ALK4 and P-Smad2, appears to limit

ADM/PanIN progression, cell- and non-cell-autonomous molecular targets further downstream of this signaling pathway remain to be further defined. Previous studies have indicated that Activin A induces p21 expression, through ALK4 and Smad-mediated signaling pathways, in various cells, including pancreatic tumor cell lines (36,46-48). Interestingly, p21 has been reported to limit senescence and ADM formation during pancreatitis (49).

Similarly, the suppression of p21 accelerates the progression of Kras-induced pancreatic lesions, through increased proliferation (50). Taken together, these observations indicate that p21 may represent an Activin A target, downstream of ALK4/P-Smad2 activation, which is supported by the combined observations of reduced p21 expression and increased proliferation in ADM/PanIN lesions and the direct regulation of p21 transcript and protein levels observed in Panc-1 cells, following Activin A inhibition.

Our results may have clinical relevance, as targeting Activin A, through the use of sActRIIB-Fc has been proposed to be an effective therapeutic intervention against muscle atrophic conditions, cancer cachexia, and, more recently, senescence-associated aging conditions

(51). Promising therapeutic benefits have been shown for the reversion of cancer cachexia, promoted by Activin A and Myostatin production in ovarian tumor and xenografted lung and colon cancer cell lines (52-54). Our results indicate that the use of such therapy, which may be efficient for many pathological conditions, should be considered with extreme caution for the treatment of pancreatic cancer. Indeed, given that low-grade pancreatic lesions are frequently found in the peri-tumoral regions surrounding PDA, whether blocking Activin A- induced cachexia using sActRIIB-Fc may favor the progression of neoplastic lesions into more advanced grades should be more thoroughly investigated.

In conclusion, our study provides the first demonstration that Activin A signaling through

ALK4 represents one of the first defense mechanisms induced by Kras-OIS to limit the development and progression of PanIN. Although limited to the sole function of Activin A as an anti-tumoral cell-autonomous SASP-factor, our study further emphasizes the necessity of better understanding the complex paracrine functions associated with Activin A and the activities of SASP molecules produced by ADM lesions, particularly how they mediate anti- or pro-tumorigenic effects in both pre-neoplastic cells and their cellular environments.

Understanding the impacts of senescent cells and SASP factors within low-grade pancreatic neoplasm is likely to represent a key step toward developing innovative therapeutic strategies for pancreatic cancer.

ACKNOWLEDGMENTS

The authors thank the animal care staff at ALECS and ANICAN for the maintenance of the transgenic mouse strains. We are grateful to Nicolas Gadot (Research Pathology Platform,

CRCL), Christophe Vanbelle, and Christelle Carreira (IARC), for assistance with histological staining and image acquisition. We thank Ivan Mikaelian, Ulrich Valcourt, and David Vincent for valuable scientific discussions. This study was supported by La Region-Rhone Alpes-

Auvergne, La Ligue de la Loire et du Rhône et la Fondation ARC pour la Recherche sur le

Cancer. YZ and ZW are supported by a Chinese Scholarship Council (CSC) Fellowship.

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FIGURE LEGENDS

Figure 1: Expression of Activin A is induced in pancreatic tumors developed by KIC mice. (A)

Heatmap, illustrating the relative expression levels of ALK4 candidate ligands in 3 independent 9-week-old KIC (T9 rows) and KI (C9 rows) mice. Normalized RMA values for each gene were mapped to colors, using the minimum (blue) and maximum (red) values for each column, independently. (B) Fold-change (FC) of significantly up- or downregulated candidate genes in pancreases from KIC mice compared with those in KI mice, with p < 0.05

(-log10 value of 1.3). Dashed lines delineate the 2-FC threshold (log2 value of 1 or -1). FCs are shown as the mean  SD, as log2 values (n = 3 mice per genotype). The p-values for each gene expression FC (square-markers/gray curve) are represented as -log10 values. (C)

Immunohistochemical staining against Activin A, in WT and KIC formalin-fixed paraffin- embedded (FFPE)-pancreas sections. The right column shows enlarged views of the dashed areas. ADM, PanIN, and adenocarcinoma lesions were stained in pancreas sections, obtained from 10 independent KIC mice. The quantification of the percentage of Activin A-positive cells found in normal pancreata and each lesion is shown as a graph (Normal, n=5; ADM, n=10; PanIN, n=9; PDA, n=4). (D) Representative pictures of Ck19 and Activin A double IF staining, performed in WT and KIC FFPE-pancreas sections. Right columns are enlarged views of dashed areas. Grades and scale bars are indicated. The reproducibility of the staining was validated in 6 independent mice for each genotype.

Figure 2: Activin A is expressed in ADM-lesions induced during pancreatitis and by KrasG12D oncogenic transformation. (A, B) Representative pictures of Ck19 and Activin A double IF staining performed in FFPE-pancreas sections, obtained from age-matched WT (n = 6), LSL-

KrasG12D/+;Ptf1a-Cre (KC) (n = 6), and Caerulein-treated WT mice (n = 3). Right panels show

enlarged views of the dashed areas. Grades and scale bars are indicated. (A) WT and KC mice were analyzed at 1.5 months of age. (B) IF staining was performed on pancreases collected

48 h after the administration of 7 hourly Caerulein or PBS i.p. injections over 2 days. (C)

Immunohistochemical analysis of Activin A expression using human pancreatic tissue microarrays. 2 independent patients shown for each grade. Insets show enlarged views of the dashed areas. The quantification of Activin A immunoreactivity is shown for the

Pancreatitis (n = 46) and PanIN1/2 (n = 18) samples, from 2 independent TMAs (BIC14011a and PA485, US Biomax, Inc.). The significance of Activin A expression in Pancreatitis and

PanIN lesions was analyzed using Fisher’s exact test, and the p-value is indicated.

Figure 3: In vivo inhibition of Activin A signaling favors ADM formation. (A) Schematic image depicting the experimental design for short-term (ST) sActRIIB-Fc treatments. Either 5 mg/kg sActRIIB-Fc or an equal volume of PBS (vehicle) were i.p. injected in 1.5-month-old KC animals, twice a week (n = 5 animals/group). (B) Representative pictures of collected pancreases following ST treatment. The weights of sActRIIB-Fc- and vehicle-treated pancreases are indicated. A representative WT pancreas is shown as a reference. The right panel shows the quantification of pancreas weights (n = 5 per group). (C) Representative

Ck19/P-Smad2 IF staining, demonstrating the efficacy of sActRIIB-Fc-treatments for blocking downstream ALK4 signaling, mediated through Smad2-phosphorylation. (D) Representative

Hematoxylin & Eosin (H&E) staining of KC pancreases, collected from sActRIIB-Fc- or vehicle- treated mice. ADM/PanIN lesions are circled by dashed-lines, and the quantification of each individual are shown in the histogram. (D) Representative H&E staining of age-matched LSL-

KrasG12D/+;Ptf1a-Cre (KC) and Acvr1bflox/flox;LSL-KrasG12D/+;Ptf1a-Cre (4KC) mice. Pictures of 3- week-old and 1.5-month-old pancreases are shown. The surface area quantifications of

ADM/PanIN lesions (dashed lines show representative regions included in the quantification), performed on 1.5-month-old KC (n = 6) and 4KC (n = 9) mice, are shown as the mean  SEM. ∗P < 0.05; ∗∗∗P < 0.005; Student’s t-test.

Figure 4: Activin A is expressed in ADM cells undergoing senescence. (A) Day-1 and day-6 bright-field images of KC isolated acinar cells, grown in 3D-rat collagen medium. Note the duct-like structures that are formed on day 6. The quantification of InhbA gene expression, by real-time quantitative PCR, was analyzed on days 1 and 6 (4 independent experiments were performed). (B) Representative FFPE-sections of whole-mount SA--Gal staining, performed on acinar-cell-derived duct-structures from 1.5-month-old KC pancreases, cultured in 3D-collagen for 6 days. Arrowheads indicate senescent cells positive for SA--Gal.

Senescence- (Cdkn1a and Cdkn2a) and SASP-associated (Il6, Il1a, Il1b, Ccl2, Ccl20, and Vegfc) genes were normalized against Hprt expression. Acinar cell cultures from 1.5-month-old KC mice were analyzed at the indicated time points. Data are presented as the mean 

SEM. ∗P < 0.05; ∗∗∗P < 0.005; ns: non-significant, Student’s t-test. (D) FFPE-sections from whole-mount SA--Gal staining, performed on 1.5-month-old WT and KC pancreases. ADM and PanIN lesions are indicated by arrows or delineated by dashed lines (left panel).

Representative SA--Gal staining, combined with Ck19 IHC staining, is shown in the right panel, to highlight the preponderant senescence associated with ADM and the weak expression of Ck19 in senescent cells in PanIN. The lower row represents magnified views of the dashed-squares. 1.5-month-old KC mice were analyzed. (E) The colocalization of SA--

Gal reactive areas with Activin A expression, in ADM sections from 1.5-month-old KC mice.

Merged Activin A-IF and SA--Gal bright-field images, taken from the same stained section,

are magnified on the right. (F) IHC staining against Activin A and indicated acinar (Amylase),

ADM/Duct (Sox9), and senescence (H2AX and p21) markers are shown in ADM pancreatic sections from KC and WT mice. Scale bars are indicated.

Figure 5: Inhibition of Activin A signaling, mediated by sActRIIB-Fc, reduces Kras-OIS in

ADM. (A) Representative FFPE-sections from whole-mount SA--Gal staining, performed on

KC acinar-cells cultured in 3D-collagen for 6 days, with sActRIIB-Fc (KC + sActRIIB-Fc) or vehicle (KC). All experiments were performed in KC cells, isolated from 1.5-month-old animals, and sActRIIB-Fc-treated and non-treated cells were isolated from the same animal in each experiment (n = 4). The quantification of percent SA--Gal-positive cells in each field is shown. (B) Quantitative RT-PCR of the indicated senescence- and SASP-associated genes.

Acinar cell cultures from 1.5-month-old KC mice, grown in differentiation media supplemented with sActRIIB-Fc or PBS, were analyzed after 6 days of culture. (C-E)

Representative double IF staining for Ck19/Sox9 (C), Ck19/p21 (D), and Ck19/H2AX (E), performed on FFPE-embedded duct-structures, obtained from KC acinar cells cultured in 3D- collagen for 6 days with either sActRIIB-Fc or vehicle. Insets show magnified views of stained-cells within the formed duct-structures. The quantification of relative Sox9 expression (C) and the percentage of p21- and H2AH-positive cell (D and E) are shown for 5 independent experiments. (F) FFPE sections from whole-mount SA--Gal staining, performed on the pancreases of 1.5-month-old KC mice exposed to ST sActRIIB-Fc or vehicle treatment

(ST). Representative pictures of SA--Gal reactivity in PanIN and ADM lesions and the quantification of ADM/PanIN surfaces that are positive for SA--gal are shown (experiments were performed on groups of 5 mice). (G, H) Pictures of p21 and H2AX IHC staining performed on ADM sections obtained from KC mice, following ST treatments with sActRIIB-

Fc or vehicle. Quantifications are shown in right panels (n = 5 animals/group) Scale bars are indicated. Data are presented as the mean  SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.005; ns: non- significant, Student’s t-test.

Figure 6: Inhibition of Activin A signaling promotes cell proliferation in ADM. (A, B) IHC and

IF staining, performed on 3-M sections of FFPE-embedded duct-structures, obtained from

KC acinar cells cultured in 3D-collagen for 6 days, with either sActRIIB-Fc or vehicle. The right column shows magnified views of dashed squares. (A) Representative Ck19 IHC staining, showing the enlarged diameters of duct structures formed from sActRIIB-Fc-treated KC acinar cells. Quantifications are shown. (B) Representative Ck19/Ki67 IF staining, showing the increased proliferation of sActRIIB-Fc-treated cells. Insets show magnified views of stained cells. The quantification of Ki67-positive cells in each individual duct-structure is shown (n = 21-27 structures, from 3 independent experiments). (C) Representative pictures of Ck19/Ki67 double IF staining, performed on KC pancreas sections, showing ADM/PanIN.

Sections were obtained from mice subjected to ST sActRIIB-Fc or vehicle treatments.

Quantifications are shown in the right panels (n = 5 animals/group). Scale bars are indicated.

Data are presented as the mean  SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.005; ns: non-significant,

Student’s t-test.

Figure 7: Targeting Activin A signaling accelerates the progression of ADM into proliferative Ck19+ cystic lesions. (A) Representative H&E staining of pancreas sections, collected from 14- and 30-week-old KC and 4KC mice. (B) Macroscopic picture of a 30-week- old 4KC pancreas, with cystic lesions (black arrowheads). A magnified view of the cystic lesion, indicated by the white arrowhead, is shown. Ck19 IHC staining of the pancreas

indicates the cystic nature of the formed Ck19 ductal lesions. (C) Representative Ck19/Ki67 double IF staining of cystic lesions detected in the pancreases of 3 independent, 30-week- old, 4KC mice. Lower panels are magnified views of the dashed rectangles, showing Ki67- proliferative cells striping the wall of the pancreatic cystic lesions. Quantification of Ki67- proliferative Ck19+ cells is shown (n = 3 mice analyzed/group; number of scored lesions: KC, n = 13; 4KC, n = 14). (D) Experimental design of the long-term (LT) sActRIIB-Fc treatments and H&E staining of the representative cystic lesions that develop in KC sActRIIB-Fc LT- treated mice, compared with vehicle-treated mice. Quantifications of ADM/PanIN and cystic lesions are shown (n = 3 mice were analyzed for each condition) (E) Weights of LT-treated pancreases are indicated for sActRIIB-Fc- (n = 3) or vehicle-treated groups (n = 3).

Representative pancreases for each group are shown. (F) Representative Ck19/Ki67 double

IF staining of the lesions found in the pancreases of LT-treated KC mice. Lower panels show magnified views of dashed squares. Quantifications of Ki67-proliferative Ck19+ cells are shown (n = 3 mice analyzed/group). Both cystic and non-cystic lesions demonstrate increased proliferative capacities. Scale bars are indicated. Data are presented as the mean 

SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.005; ns: non-significant, Student’s t-test.

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Oncogene-induced senescence limits the progression of pancreatic neoplasia through production of Activin A

Yajie Zhao, Zhichong Wu, Marie Chanal, et al.

Cancer Res Published OnlineFirst June 17, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-3763

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/06/17/0008-5472.CAN-19-3763.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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