PACE4 Undergoes an Oncogenic Alternative Splicing Switch in Cancer

Author Manuscript Published OnlineFirst on October 9, 2017; DOI: 10.1158/0008-5472.CAN-17-1397 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Title: PACE4 Undergoes an Oncogenic Alternative Splicing Switch in Cancer

Author list: Frédéric Couture1,2,4, Robert Sabbagh2,4, Anna Kwiatkowska1,2,4, Roxane Desjardins1,2,4, Simon-Pierre Guay3,4,5, Luigi Bouchard3,4,5, Robert Day1, 2,4,6.

Author Affiliations: 1Institut de Pharmacologie de Sherbrooke, 2Department of Surgery, Division of Urology, 3Department of Biochemistry, 4Faculté de médecine et des sciences de la Santé, Université de Sherbrooke, Québec, J1H 5N4, Canada. 5ECOGENE-21 Biocluster, CIUSSS du Saguenay–Lac-St-Jean, Saguenay, Québec, G7H 7K9, Canada. 6Corresponding author.

Running Title: PACE4 splicing in cancer

Keywords: proprotein convertase, alternative splicing, PACE4 isoform, GDF-15, prostate cancer

Contact information: [email protected] Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, 3001, 12e Ave. Nord, Sherbrooke, Québec (J1H 5N4), Canada. Phone: (819) 821-8000 ext. 75428; email: [email protected]

Financial support: To R. Day and R. Sabbagh: Prostate Cancer Canada (grant #2012-951), Movember Foundation (grant #D2013-8), Canadian Cancer Society (grant #701590). To R. Day: Fondation Mon Étoile support. To F. Couture : Prostate Cancer Canada (grant #GS2014-02) and Canadian Institutes of Health Research (CIHR) Graduate Studentship award from and Banting and Charles Best Canada Graduate Scholarships (grant #315690).

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 9, 2017; DOI: 10.1158/0008-5472.CAN-17-1397 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Inhibition of PACE4, a proprotein convertase that is overexpressed in prostate cancer, has been shown to block cancer progression in an androgen-independent manner. However, the basis for its overexpression and how its growth inhibitory effects are mitigated and uncertain. Here we report that PACE4 pre-mRNA undergoes DNA methylation-sensitive alternative splicing of its terminal exon 3' untranslated region, generating an oncogenic, C- terminally modified isoform (PACE4-altCT). We found this isoform to be strongly expressed in prostate cancer cells, where it displayed an enhanced auto-activating process and a distinct intracellular routing that prevented its extracellular secretion. Together these events led to a dramatic increase in processing of the pro-growth differentiation factor pro-GDF15 as the first PACE4 substrate to be identified in prostate cancer. We detected robust expression of PACE4-altCT in other cancer types, suggesting that an oncogenic switch for this pro-enzyme may offer a therapeutic target not only in advanced prostate cancer but perhaps also more broadly in human cancer.

Introduction: Among malignancies, prostate cancer (PCa) remains the most common type of cancer in men, with 233,000 new cases each year in the USA, representing 27% of all new cancer cases, as well as the second most common cause of cancer-related mortality (1). When diagnosed in its early progression stages, clinical interventions are able to circumvent disease progression and yield high survival rates over 5-15 years. However, if tumor initiated metastatic dissemination as already occured at the time of diagnosis or following recurrence, survival rates drop considerably, leading to patient death within 5 years in approximately 75% of cases. The treatment for metastatic PCa involves androgen suppression therapy but remains palliative. Once resistance to castration occurs, the only option remaining is chemotherapy. Novel targeted therapeutic avenues arising from yet unexplored biological pathways may provide a solution, either alone or as co-targets.

Among potential targets that have yet to be fully defined are the proprotein convertases (PCs), which are responsible for the posttranslational processing of pro-protein substrates within the secretory pathway. The PCs are composed of nine members, namely, PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1 and PCSK9. The first seven are calcium-dependent serine proteases that cleave at paired basic residues at the consensus cleavage site R-X-(K/R)-R (2). The PCs have been loosely associated with malignancies because of their ability to enhance the activity of cancer-associated protein substrates, which are overexpressed by tumor cells, e.g. members of the ADAM family of proteases, transforming growth factor-β, MMPs and insulin-like growth factor-1 receptor (IGF1R) family members (3). As PCs display increased expression in tumor cells and are required for enhanced processing to sustain tumorigenesis, they have been proposed as attractive antineoplastic targets (4). At this time the evidence remains indirect as to how oncogenic control by the PCs is executed.

Among the PCs, recent data has made the strongest case yet for PACE4 and its specific association with malignant transformation (5). In PCa, among other PCs, PACE4 is specifically overexpressed and carries non-redundant growth-sustaining functions for cancer cells (6,7). PACE4 inhibition using either silencing tools or the high affinity PACE4 inhibitor: [dL]LLLRVK-amidinobenzylamide (Amba) herein called C23, both prevented PCa tumor progression (8). Despite these important advances, neither the downstream substrates nor the mechanisms associated with sustained PACE4 overexpression in PCa cells have been elucidated. Thus, important questions remain regarding how PACE4 levels can be envisioned as a viable target or have a direct relationship with PCa disease outcomes.

In this present study, we uncovered important post-transcriptional changes that have profound effects on PACE4 mRNA and protein as well as its cell trafficking and substrate processing. Our analysis reveals a yet unreported PACE4 splice variant that results in the expression of a C-terminally modified protein

isoform (PACE4-altCT), increased auto-activation and retention within cells. Compared with its parent isoform, PACE4-altCT strongly regulates sustained cell proliferation, further defining this isoform as the molecular target of the PACE4 inhibitor-mediated response of cancer cells and conciliating the thus far unexplainable requirement of PACE4 inhibitor to reach intracellular compartments. This new isoform is strongly up-regulated at both the mRNA and protein levels in PCa tissues, which is not the case of any other known PACE4 isoforms. A scan in various malignancy types reveals that PACE4-altCT is expressed in other cancer types, notably in pancreatic, lung and thyroid cancers. In PCa cells, this splicing activity is regulated through a distinctive intra-exonic DNA methylation pattern modulating the binding CCCTC- binding factor CTCF and the inclusion of a novel distal terminal exon. A SILAC proteomic approach permitted the identification of pro-growth differentiation factor-15 (pro-GDF-15) as a PACE4-specific substrate in PCa. Using primary tissues and xenografted mice treated with the C23 PACE4-inhibitor, blocking the processing of this growth factor suggested that pro-GDF-15 could serve as a target engagement biomarker. The results of this work define the mechanism behind our assertion that PACE4-altCT is a pharmacologically targetable oncogenic driver of PCa-sustained cell growth.

Material and Methods

Ethics and study approval Patients agreed to participate and freely signed a consent form, and the research protocol was approved by the Institutional Review Committee for the Use of Human Resected Material at the Centre Hospitalier Universitaire de Sherbrooke (approval #10-017). The experimental protocols were approved by the Université de Sherbrooke Ethics Committee for Animal Care and Use in accordance with guidelines established by the Canadian Council on Animal Care.

Cell culture Cell lines (DU145, LNCaP and HT1080) were obtained (American Type Culture Collection; ATCC, Mannasas, USA) and HEK293-FT (Life Technologies Inc.) were cultured only at low passages (<25) from the cryovials generated directly from the original tube cultures under their recommended culture conditions (see Supplementary Material). No re-authentication tests were performed. Mycoplasma testing was done routinely (after every new cryo-tube thawing). Stable knockdown cell lines were the same as reported by Couture et al. 2012 (7). Stably expressing cell lines were never passaged more than 15 times.

DNA constructs, plasmids, transfections and splicing mapping Detailed western blot, PCR, reverse-transcription and qPCR conditions, vector information and procedures for reporter assays can be found in the Supplemental material. Primers sequence and product lengths are found in Supplementary Table S1 and S2.

Proliferation assays For the proliferation assays, the cells were plated in 96-well plates, and the metabolic activity was measured after 72 h as described in (9). For the colony formation assay, the cells were plated at low densities (50 cells for HT1080, 100-200 cells for DU145 and 500-1000 cells for LNCaP) and allowed to form colonies for 10-14 days in complete medium before being stained with crystal violet counted after with a visible/infrared scanner (Odyssey Imager, LI-COR Biosciences). See Supplementary material for detailed procedure.

Activity assays For activity assays, medium collected from stable S2 cell cultures expressing the PACE4 isoforms was collected, concentrated to similar enzyme content (monitored by Western blots) using Amicon-Ultra 30K centrifugal units (Millipore) with PACE4 activity buffer (bis-Tris 20 mM pH 6.5, 1 mM CaCl2). Activity

assays were performed as described in (10). Identical preparations from wild type S2 cells were used as blanks.

Statistics Statistical analyses were performed with GraphPad Prism 7.0 (GraphPad Software), and all data are represented as the mean ± SEM. The one-tailed student’s t test was used for the data analysis, and when paired samples were compared, a matched-pairs t test was used. For correlation analysis, the Spearman correlation test was used. P-values less than 0.05 were considered statistically significant.

Fresh tissue dissection and RNA isolation Prostate tissues used for RNA extraction were freshly (typically within 30 min) dissected from prostate specimens obtained from radical prostatectomies performed at the Centre Hospitalier Universitaire de Sherbrooke. The tissues were frozen at -20˚C with OCT compound (Tissue-Tek; Miles Scientific), and 5- µm slices were cut and immediately fixed in formalin to perform hematoxylin-eosin staining for pathological examination. Tumor zones were delimitated together with the adjacent non-cancerous tissues by a clinical pathologist, and dissection was performed accordingly. Dissected tissues were washed with nano-pure RNase-free water (Wisent) to remove all apparent traces of OCT compound. The tissues were then finely ground in liquid nitrogen, and RNA extraction was performed using QIAgen RNeasy spin columns (QIAgen, Valentia, CA, USA) following the manufacturer’s instructions. RNA integrity was assessed by analysis using an Agilent Bioanalyser with RNA Nano Chips (Agilent Technologies, Palo Alto, CA, USA). Normal RNA standardized preparations were obtained from Clontech Laboratories (Total RNA Master Panel II; Mountain View, CA). Normal and tumoral cDNA samples were obtained from the Origene (Rockville, MD) Cancer Survey cDNA Array covering different cancers across identical qPCR plates. The samples' content can be found at the following URL: http://www.origene.com/assets/documents/TissueScan/CSRT103.xls.

DNA methylation analysis DNA was purified from prostate specimens or from cell pellets using the DNeasy Blood & Tissue Kit (Qiagen, #69504). Pyrosequencing assays were performed as previously described (11). The PCR and sequencing primers shown in Supplementary Table S3 were designed using PyroMark Assay Design software v.2.0.1.15. Overall, nine potentially methylated cytosines (in the context of CpG dinucleotides) were analyzed at the PCSK6 gene locus. The detailed procedure is provided in the Supplemental material.

Immunohistochemistry

Tissues sections (4 µm tick) were stained using Peroxidase Detection Kit (Pierce) counterstained with Harris hematoxylin (Sigma-Aldrich) and scanned with a Nanozoomer (Hamamatsu, Hertfordshire, UK) using Nanozoomer Digital Pathology software. Antibodies details can be found in Supplementary Table S4. For detailed procedure, see Supplementary material. For custom rabbit polyclonal antibodies, antibodies were raised and purified from serum on a peptide-coated chromatographic column (Pacific Immunology, Ramona, CA).

Chromatin immunoprecipitation 350 µg of DNA from sonicated and microccocal nuclease digested nuclei isolated from formaldehyde- crosslinked cells used each IP using Protein A MagneResyn bead incubations. ChIP-isolated DNA was further used for qPCR analyses using input as a standard. See supplementary material for detailed procedure.

Confocal microscopy 48 h after transfection, cells were fixed, permeabilized and stained with the primary antibodies (see Supplementary Table S4) at 4˚C. Fluorescent secondary antibodies (Alexa Fluor 488 and 594 antibodies, Thermo Fisher) were further applied for 1 h incubation at room temperature, followed by DAPI staining and final mounting with SlowFade (Invitrogen). The cells were examined with a Plan Apo 60x oil immersion objective NA 1.42 on an FV1000 inverted spectral scanning confocal microscope (Olympus, Tokyo, Japan). See supplementary material for detailed procedure.

LC-MS/MS analysis Acquisition was performed with a Sciex TripleTOF 5600 (Sciex, Foster City, CA, USA) equipped with an electrospray interface with a 25-μm iD capillary and coupled to an Eksigent μUHPLC (Eksigent, Redwood City, CA, USA). Analyst TF 1.6 software was used to control the instrument and for data processing and acquisition. See Supplementary material for detailed procedure and Supplementary Table S6 and S7 for the data from the IP-MS and the SILAC analysis in secretome respectively..

Peptide synthesis and cleavage analysis ML peptide and its derivatives (Peg8-ML and C23) were synthesized as previously described (12). Synthesis and cleavage analysis of the GDF-15 spanning peptide are described in the Supplementary material.

Xenograft assay Trypsin-harvested LNCaP cells were mixed with ice-cold Matrigel (BD Biosciences, Bedford, MA) and subcutaneously injected into the shoulders of Nu/Nu male mice (Charles River Laboratories, LaSalle,

Canada) as described in (8). PSA and GDF-15 levels were determined using Quantikine ELISA kit (R&D, Minneapolis, USA). Tumor lysates were obtained as described in (7).

Results: PACE4 mRNA levels correlate with tumor aggressiveness in PCa tissue specimens The overexpression of PACE4 in PCa has been documented, however, no correlations with clinical parameters, such as Gleason grading, has been reported (6,13,14). New molecular markers with relevance to PCa progression should show a positive correlation with the established histological prognostic indicator, i.e. Gleason grading, to be considered of prognostic usefulness. Using matched cancerous and adjacent non-cancerous tissues (ANCT), PACE4 mRNA levels were analyzed by real-time quantitative PCR (RT-qPCR). In this cohort (Gleason scores 6 to 9), 34/38 samples (>89%) showed significantly increased PACE4 mRNA levels (Figure 1A). This overexpression pattern was tightly correlated with the tumor Gleason scores (Spearman r: 0.46, P-value: 0.004; Figure 1B). Our results are supported by similar analyses that were performed using the data from two distinct datasets in the cBioPortal for the Cancer Genomics database (Supplementary Figure S1A-B). Immunohistochemistry (IHC) analyses of specimens from different tumor grades with an antibody targeting the catalytic domain of PACE4 (i.e. detecting all PACE4 isoforms) corroborated our results as once again overexpression was visible with increasing levels in higher grade foci (Figure 1C).

PCa exploits a PACE4 mRNA terminal exon splicing event that is specific to tumor cells. Human PACE4 mRNA is encoded by the 186-kbs PCSK6 gene located at the 15q26.3 locus, which is not a locus that has been reported to be susceptible to frequent changes in copy number in PCa specimens (OncomineTM databases). While PACE4 overexpression could be due to increased transcription, another mechanism that has gained enormous importance in cancer biology is alternative splicing as a means to promote the expression of genes that sustain proliferation (15). The link between alternative splicing and proliferation is often observed through a shortening of 3’UTR regions of oncogenes and proto-oncogenes, further allowing the upregulation of gene expression through evasion of post-translational regulatory mechanisms such as repression by miRNAs (16). Using 3’ rapid amplification of cDNA ends (3’ RACE) in LNCaP cell cDNA, the PACE4 mRNA 3’ end revealed the presence of the consensual 1335-bp-long 3’UTR as well as two shorter 3’UTRs (164 and 118 bps; Figure 1D) generated by alternative splicing. This splicing event, confirmed by 3’RACE product sequencing and three-primer PCR (TP-PCR; Supplementary Figure S1C), incorporates a distal terminal exon located 6.3 kbs downstream of the 25th exon consensually used (Figure 1E), substituting the 25th exon with this alternative exon (25-alt) and altering the coding region of the C-terminal end of the protein. Thus, two distinct PACE4 proteins can be generated from this splicing event, namely a PACE4 full-length (PACE4-FL) isoform and a PACE4 isoform with an alternative C-terminal end (PACE4-altCT) that has a novel 30 amino acid sequence with 2 Cys replacing the 32-amino acid sequence that has 4 Cys residues. To our knowledge, this alternative splicing event has not been documented among the reported PACE4 splice variants in the literature (17), but could be retrieved in some expressed sequence tag (EST) databases.

Upon PACE4-knockdown in cell lines (LNCaP and DU145) using a shRNA targeting the 5’ region (exon #2) of PACE4 mRNA, both splice variants were down-regulated (Supplementary Figure S1D). When assessed in paired prostate ANCT and tumor tissues, PACE4-altCT mRNA was primarily detectable in the tumor specimens with very low or undetectable levels in the ANCT matched specimens (Figure 1F). RT-qPCR analyzes showed that PACE4-altCT mRNA displayed 8-fold greater amounts between tumor and normal zones compared to PACE4-FL (Figure 1G), with >95% patients showing increased PACE4- altCT mRNA levels. When stratified by their tumor Gleason scores (Figure 1H), PACE4-altCT mRNA levels displayed a highly reminiscent pattern to the initial observations (Figure 1A). Once translated, PACE4-altCT and PACE4-FL have distinct C-termini (Figure 1E), which were exploited to generate isoform-specific antibodies to validate these observations. IHC on PCa specimens using both antibodies (Figure 1I) showed that, as observed for the mRNA, both isoforms were readily overexpressed in tumor cells, and those levels increased along with the grading of the tumor foci. The cellular distribution pattern for PACE4-altCT appeared predominantly vesicular, which was not the case for PACE4-FL, which particularly accumulated in the peritumoral stroma. However, staining for PACE4-altCT showed a strong positive signal within the tumor epithelium and was absent from the normal epithelium. The difference in PACE4-altCT staining intensities between normal prostate glands and tumor cells was much higher than that observed for PACE4-FL (Figure 1I; Supplementary Figure S2A-B). Using a set of prostate cancer specimens, IHC staining for PACE4 (catalytic domain antibody), PACE4-FL and PACE4-altCT were quantified in normal and cancerous foci using a semi-quantitative scaling (Figure 1J). Normal epithelium was positive in most cases (12/12 and 11/12 for PACE4 and PACE4-FL respectively) whereas it was completely negative for PACE4-altCT (11/12). In cancer foci, whereas PACE4-FL remained constant when comparing with normal epithelium, PACE4-altCT staining was greatly increased, which is consistent with the performed mRNA analysis (Figure 1H).

As PACE4 has been reported to have other splice variants (17), ANCT and PCa tissue pairs were subjected to comparative profiling of PACE4 splicing events by end-point RT-PCR to map all exon-exon junctions (Supplementary Figure S3A-B). Among the reactions, only three potential splice sites were depicted as active (Supplementary Figure S3C-G) namely: (i) exon 4 skipping; (Supplementary Figure S3D) and (ii) exon 18 skipping, known as PACE4-I and PACE4-II; (Supplementary Figure S3E), having or not the 18th exon respectively, and (iii) exon 25 substitution, as observed in Figure 3F. Upon validation by RT-PCR, exon 4 skipping could barely be detected (Supplementary Figure S3G), and skipping of exon 18, which encodes a short protein segment of approximately 1.5 kDa between the RGD motif and the Cys-rich domain of PACE4, was active but with similar levels between ANCT and tumor specimens (Supplementary Figure S3H-I). Only the substitution of exon 25 was confirmed as a splicing event specific to PCa upon comparison to normal tissues (Supplementary Figure S3G).

PACE4-altCT is generated by a stabilized mRNA variant that leads to intracellular retention, enhanced stability and increased auto-activation Consistent with the nature of the 3’UTRs and their roles in regulating mRNA stability, the consensus (PACE4-FL) and alternative (PACE4-altCT) 3’UTR sequences were analyzed for miRNA site prediction using RegRNA and miRDB (18) (see Supplementary Table S5). In the 3’UTR accompanying the alternative terminal exon, 90% less miRNA sites were aligned compared with the consensus 3’UTR (Figure 2A). The PACE4-targetting miRNA regulatory sites predicted by TargetScan for miR-21, which is overexpressed in PCa (19), and miR-124, a tumor-suppressor that is downregulated in PCa (20), were all absent from the transcript after 3’UTR replacement (14) (Figure 2A). To verify that the 3’UTR substitution could increase mRNA stability by escaping miRNA-dependent degradation, actinomycin-D chase followed by RT-qPCR showed that transcripts with the alternative 3’UTR were more stable than those carrying the consensus 3’UTR. Luciferase reporter assays using luciferase constructs harboring the shorter and consensus 3’UTRs showed a 2-fold amplification of protein production when the short 3’UTRs were compared to the consensus form (Figure 2B).

Upon expression as constructs in cell lines, the first major distinction observed between the two isoforms was the lack of secretion of PACE4-altCT accompanied by intracellular retention in comparison to PACE4-FL (Figure 2C and Supplementary Figure S4A-B). This observation is consistent with the distinct vesicular cell distribution observed for each isoform in IHC in PCa tissues (Figure 1I and Supplementary Figure S2A-B). PACE4 has been previously reported to be secreted in the medium, although it could be retained in the extracellular matrix through binding to heparan sulfate proteoglycans by its Cys-rich domain while also being displaceable with heparin (21). Upon addition of heparin to the medium of transfected cells, PACE4-FL, but not PACE4-altCT, was efficiently displaced from the extracellular matrix to the medium (Supplementary Figure S4C-E), further confirming the inability of the intracellular isoform to exit the cells.

It has been previously reported that the C-terminal end of PACE4 encoded by the 25th exon of PACE4-FL negatively regulates its autocatalytic activation and secretion (22). In this context, we tested the auto- activation rates by addition of cycloheximide (CHX) to determine the rate of prodomain removal for both isoforms. Time-course experiments showed that PACE4-altCT had a higher rate of auto-activation than PACE4-FL (Figure 2D and Supplementary Figure S4F-H). Moreover, in all tested cell lines, PACE4-altCT stability was considerably higher (more than two-fold) than that of PACE4-FL (Figure 2D-E and Supplementary Figure S4F-H). Among previously reported PACE4 isoforms, only isoform A (PACE4-FL) is known to be active or processed from pro-PACE4 to PACE4 (23). Enzymatic activity was thus tested using recombinant (r) PACE4-FL and rPACE4-altCT (S2; Supplementary Figure S4I), showed that both

isoforms had similar activity against a fluorogenic PC substrate (pyroERTKR-amido-methylcoumarin) and were equally inhibited by a PACE4 specific inhibitor, i.e., the multi-leucine (ML) peptide (Figure 2F). Taken together, these data demonstrate that the intracellularly retained PACE4-altCT is generated by an mRNA splice variant that is less susceptible to degradation and that the resulting protein isoform is more rapidly activated and is more stable than its parent isoform (PACE4-FL), while maintaining equal activity levels to the parent isoform. For cancer cells, this may result in higher tumor growth.

Since the two PACE4 isoforms are differentially localized, it follows that their intracellular trafficking patterns will be divergent, and it is highly likely that interacting proteins are responsible for this re-routing. To test this hypothesis, we carried out co-immunoprecipitation (co-IP) studies using transiently transfected HEK293-FT cell lysates that were subjected to Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS)-based analysis to investigate differences in intracellular interacting proteins of both isoforms (Figure 2G-H). The proteins identified in each pull-down were compared for their IP enrichment between the two isoforms. We focused on proteins that are typically associated with cell compartments (Figure 2I). Both isoforms pulled-down endoplasmic reticulum (ER) proteins with similar enrichment. The two isoforms clearly had different patterns in terms of endosomal compartment-associated proteins, such as Arf6, Rab13 and Vps16, with the PACE4-altCT pull-down displaying higher enrichment levels (Figure 2I-J). However, the PACE4-FL pull-downs demonstrated a stronger association with exocyst complex-associated proteins, such as Exoc2 and Vps13. Co-IPs performed in transfected LNCaP cells analyzed by Western blot confirmed differences between the isoforms (Supplementary Figure S4J).

Consequentially, we performed confocal microscopy using known markers of cellular trafficking in colocalization studies with either PACE4-FL or PACE4-altCT. Using RCAS1 as a Golgi marker (24), PACE4-FL was clearly better co-localized within the Golgi than did PACE4-altCT (Figure 2K). Using Rab GTPase markers, co-localization analysis showed that compared with PACE4-FL, PACE4-altCT accumulated in Rab5-positive endosomal compartments and in Rab9-associated compartments (Figure 2L-N), suggesting differential routing through an endosomal pathway. The enrichment of secretory pathway-associated proteins from IP-MS studies has been summarized on a cell secretory pathway map together with the immunofluorescence results (Figure 2J). These data support the notion that PACE4-FL and PACE4-altCT use differential trafficking pathways, accounting for their secretion or retention status, respectively. It may be possible that the removal of Cys residues in the protein C-terminus (Figure 1E) leads to complex reorganization of the Cys-rich domain in PACE4-altCT resulting in the striking differences observed.

PACE4 splicing is regulated by intra-exonic DNA methylation

We then focused our attention on the mechanisms regulating PACE4 terminal exon 25 substitution. According to chromatin immunoprecipitation sequencing (ChIP-Seq) data in the UCSC genome browser (Figure 3A), CCCTC-binding factor (CTCF) is found to have three reported binding sites in the vicinity of the segment encoding the 25th alternative exon. CTCF has recently been reported to regulate upstream exon inclusion through the binding of non-methylated CpG dinucleotides in intra-exonic regions (25). The alternative 25th exon genomic segment, which includes the two CTCF binding sites, is located within a DNaseI hyper-sensitivity cluster and includes 7 CpG dinucleotides, which suggested a similar regulatory mechanism. RNA-seq data assessing the transcription levels in cell lines showed that transcription was sustained from the consensus 25th exon to the alternative 25th exon (Figure 3A). It is important to note that the alternative 25th exon is only conserved in primates and is completely absent in the other available tested vertebrates, as it is the case for most splice variants (26).

Using bisulfite-pyrosequencing of DNA from both ANCT and cancerous specimens of PCa, the methylation status of the different CpG dinucleotides found either within the CTCF binding sites in the alternative terminal exon or in the site located in the 24th intron was characterized (Figure 3B). Significant tumor-specific CpG hypomethylation was observed in both intra-exonic and upstream CTCF binding sites (Figure 3C) but not in other surrounding CpGs. This local hypomethylation suggests a tight regulatory mechanism that is most likely mediated by locus-specific recruitment of DNA modifying or binding factors that protect DNA from methylation by DNA methyltransferases. Such tumor-specific regulation of CTCF binding sites by methylation are known (27). It has even been shown that CTCF itself can regulate DNA methylation patterns and that cancerous or immortalized cells, when compared to normal cells, have a distinct CTCF binding landscape in the genome (28), highlighting the complexity of methylation-related regulation, especially when addressed in a locus-specific manner.

Upon CTCF downregulation in cells, the splicing index (i.e., ratio of PACE4-altCT/PACE4-FL mRNA) was reduced by 70% (Figure 3D-F), supporting the regulation of exon 25 substitution by CTCF. When these cells were treated with 5-aza-2’-deoxycytidine (5-aza-dC) to force genome hypomethylation (Supplementary Figure S5A-C), splicing increased in a dose-dependent manner, further reinforcing the relationship between the observed alternative splicing and DNA methylation (Figure 3G-H). After either treating the cells with 5-aza-dC treatment or following CTCF overexpression (Figure 3I-J), PACE4 terminal exon splicing increased significantly (Figure 3K). Combining both 5-aza-dC and CTCF expression resulted in even higher splicing levels. ChIP of endogenous CTCF confirmed the binding to DNA within the intronic regions in the 5’ as well as in the 3’ part of the alternative terminal exon and within the alternative exon itself (Figure 3L), with a clear enrichment in the intra-exonic and the upstream region upon 5-aza-dC treatment (Figure 3L, Supplementary Figure S5D). These findings show the DNA methylation-dependent CTCF binding and its relationship with exon-inclusion.

The expression of PACE4-altCT in normal tissues and various cancer types suggests a common tumor mechanism The question arises as to whether PACE4 alternative splicing is only found in PCa cells, or could also be found in other tissues or cancer types. Thus, we used RNA from normal human tissues and other cancer types to map both PACE4 isoforms. In normal tissues, PACE4-altCT mRNA was only strongly detected in the liver, the testis and the brain/spinal cord, (Figure 4A, Supplementary Figure S6A-F). Interestingly, the liver, testis and brain are known for their higher rates of alternative splicing compared with other human tissues. Moreover, very high splicing indices were observed in fetal tissues, e.g., liver and brain. Splicing indices were determined on an array of cDNA preparations from various tumor types and normal tissues (Figure 4B). Some cancer types other than PCa also displayed enhanced splicing activity in the tumor specimens, notably in lung, thyroid, adrenal and pancreatic cancers. Other PCs were also evaluated in these normal (Supplementary Figure S6H-L) and tumor specimen cDNAs (Figure 4C). Among all PCs, PACE4, furin and PC1/3 were the only ones that displayed frequent overexpression levels across numerous types of cancer (Figure 4C and Supplementary Figure S6H-L).

IHC analyses using normal and tumoral specimens originating from different organs supported the results of the mRNA mapping in normal tissues, with again high levels of PACE4-altCT in colon and testis and the lowest levels observed in lungs, adrenal glands and ovary (Figure 4D and Supplementary Figure S6M). Consistent with the observation at the mRNA level (Figure 4B), the tested placenta as well as lymphoid tissues (thymus, lymph node, lymphomas and tonsil) where all negative for PACE4-altCT by IHC, whereas organs such as the stomach, pancreas, liver and kidney were positive to some extent (Supplementary Figure S6N). Matched tumor specimens also corroborated the expression shift observed at mRNA levels in numerous cancer types, including, among others, lung, esophagus, testicular and thyroid cancers, thus supporting the concept that PACE4 undergoes an oncogenic alternative splicing event in PCa as well as in some other cancer types where it may act as an oncogenic driver.

PACE4-altCT is the main isoform responsible for PCa cell-sustained growth capabilities From a functional point of view, we then asked the question as to the phenotype acquired with each isoform. Thus, isoform-specific features were explored by both overexpression and gene silencing studies in cell-based assays. First, PACE4-FL and PACE4-altCT were stably expressed as untagged proteins in cell lines by stable lentiviral transduction. Despite similar mRNA expression levels, PACE4-altCT protein levels were much higher than PACE4-FL protein levels in whole cell lysates (Figure 5A-B and Supplementary Figure S7A). Moreover, very little increase in secreted PACE4 was detected in the PACE4-altCT-overexpressing cells. When tested in cell proliferation assays, PACE4-FL and PACE4- altCT-overexpressing cells displayed enhanced growth and clonogenic capabilities, as depicted by

proliferation (Supplementary Figure S7B-C) and colony formation assays (Figure 5C). However, PACE4- altCT overexpression yielded stronger effects, especially in LNCaP and HT1080 cells (Figure 5C & Supplementary Figure S7B-C). Levels of cognate PC mRNAs in these stable cell lines were evaluated, as well as varying patterns of PC7 and furin expression, suggesting a cross-talk between the pathways regulating these PCs (Supplementary Figure S7D-F). These findings indicate that the overexpression strategy has some limitations.

Therefore, siRNAs specifically targeting each splice variant were designed to assess the importance of endogenous PACE4-altCT compared with its parent isoform PACE4-FL. Following transfection, each siRNA efficiently silenced its splice variant (i.e., 70-95% knockdown without affecting the other co- expressed PCs; Figure 5D and Supplementary Figure S7G). Despite the lack of western-blot-compatible antibodies to specifically detect PACE4-FL separately from PACE4-altCT, the use of an anti-PACE4 antibody that recognizes the catalytic domain (and thus both isoforms) revealed a pattern that was coherent with observations for the tagged protein (Figure 2C). The siRNA targeting PACE4-altCT resulted in a stronger reduction in intracellular levels of endogenous PACE4 than the siRNA targeting PACE4-FL (i.e., 45% vs 13% reduction, respectively; Figure 5E and Supplementary Figure S7H). Conversely, in the conditioned medium, siRNA targeting PACE4-altCT had minimal effects on secreted PACE4, whereas siRNA targeting PACE4-FL resulted in a very large decrease (i.e., >80%). These data obtained with siRNAs and endogenous PACE4 correlate well with our previous observations concerning the differential secretion of the two isoforms, validating this approach to test the biological significance of PACE4-FL vs PACE4-altCT. Silencing of PACE4-altCT yielded a much stronger reduction in terms of growth and clonogenic capabilities than PACE4-FL silencing, which barely affected these parameters in either LNCaP or DU145 cells (Figure 5F-G and Supplementary Figure S7I-J). These results demonstrate the distinct functions of the two isoforms in sustaining cancer cell growth, in which PACE4-altCT plays a much more important role, allowing the conclusion that (i) the PACE4 knockdown-associated phenotype in PCa cells (7) results from the downregulation of PACE4-altCT (Supplementary Figures S7B) and (ii) efficacy of PACE4 inhibitors and cell permeability requirements are in fact due to PACE4-altCT-specific functions.

Identification of GDF-15 as a specific PACE4 substrate and pharmacological target engagement marker While our work points to the importance of sustained PACE4 activity to maintain PCa cell growth and proliferation (7,8), the substrate(s) of PACE4 that is responsible for these actions remains to be determined. Secreted factors have previously been suggested to be the main effectors of the PACE4- related cancer cell growth phenotype (7). Therefore, we turned to a SILAC-based proteomic approach to analyze the secretome content in both DU145 and LNCaP PCa cells. The secretomes were pooled 1:1 (shNon-Target:shPACE4) and fractionated, and then each fraction was analyzed by tandem LC-MS/MS.

From the protein identified, secreted proteins were retrieved using ProteINSIDE and used to draw a heat map based on light/heavy (L/H; shPACE4/Non-Target) ratio proportions for each cell line (Supplementary Figure S8A). Proteins having PC-based or PC-like processing events, determined by both Uniprot PTM/Processing data or using the ProP 1.0 server (29), were highlighted as potential substrates.

For validation, we chose to perform western blotting arrays of (i) cell lines silenced with shPACE4, shfurin and shPC7 (7), (ii) cell lines stably overexpressing PACE4-FL and PACE4-altCT and (iii) cell lines treated with either the non-selective and irreversible PC inhibitor decanoyl-RVKR-chloromethylketone (CMK) or the PACE4 high-affinity C23 inhibitor. To test our western blot arrays, we selected known PC substrates, namely, IGF1R and integrin alpha-6 (ITGA6), two well-accepted furin substrates (30) (Figure 6A and Supplementary Figure S8B) and E-cadherin, which has been shown to be a furin and PC7 substrate (31). For both IGF1R and ITGA6, only furin knockdown and CMK treatments prevented the processing of their pro-forms, whereas PACE4 knockdown and the C23 PACE4 inhibitor had no effect, further supporting its selectivity toward PACE4 even in cells with concentrations above the selectivity range (Ki: low nanomolar). In contrast, overexpression of PACE4-FL and PACE4-altCT increased the processing of IGF1R and ITGA6 pro-forms, highlighting the cautionary interpretation that is needed in overexpression studies. Regarding E-cadherin, PC7 knockdown showed the best results in blocking the processing of its pro-form (Figure 6A and Supplementary Figure S8B).

Among the candidate proteins with an L/H ratio <1 in either DU145 or LNCaP, which displayed a PC-like cleavage site(s) (highlighted in bold in Supplementary Figure S8A), many were tested by western blotting using antibodies allowing the discrimination of human pro- and mature protein forms, when available. These included low-density lipoprotein receptor-related protein 1 (LRP1), hepatocyte growth factor receptor (HGFR, also known as Met), clusterin (CLU), desmoglein-2 (DSG2), ADAM10, ADAM17 and GDF-15 (Supplementary Figure S8B-E). Some other tested substrates could not be detected adequately by western blotting. Among these potential PACE4 substrates, only GDF-15 (which is only expressed in the LNCaP line) appeared to be a unique PACE4 substrate (Figure 6B), whereas the other substrates (LRP1, HGFR, DSG2 and CLU; Supplementary Figure S8B-E) were by far mostly processed by furin and, to a much lesser extent, by PC7.

GDF-15 (also known as prostate differentiation factor; PDF, or macrophage inhibitory cytokine 1; MIC-1) is synthesized as a 35-kDa proprotein that requires PC-based cleavage at the RRAR196 site to generate a 17-kDa C-terminal mature form that associates as a disulfide-linked dimer, which is further secreted into the medium (32). Virtually no mature GDF-15 was observed in the medium of shPACE4 knockdown or CMK- or C23-treated cells, whereas processed GDF-15 levels were still evident after furin and PC7 knockdown as assessed by both western blotting and ELISA (Figure 6B). In both PACE4-FL and PACE4-

altCT-overexpressing cell lines, the amount of secreted GDF-15 was approximately 5 times higher (see ELISA, Figure 6C) than in the pLenti6 control cells. Under all conditions, the secreted GDF-15 levels inversely correlated with the accumulation of pro-GDF-15 in the cell lysates, confirming that cleavage is a prerequisite for its secretion. These results demonstrate that GDF-15 is cleaved by PACE4 with very limited redundancy with the other endogenously co-expressed PCs, and are in line with a previous report showing that GDF-15 remained uncleaved in PACE4-deficient PC3 cells (10,33). Nevertheless, we also tested the in vitro cleavage of a GDF-15-spanning peptide incorporating the PC site (QAARGRRRARARNG) with recombinant furin and PACE4 enzyme preparations (10). The GDF-15 peptide was cleaved correctly by both PCs (Figure 6D), further demonstrating the importance of the cellular context and/or full length substrates to full comprehend PC functions.

A marked difference in terms of GDF-15 modulation was observed upon PACE4-FL or PACE4-altCT silencing. Following PACE4-FL knockdown, GDF-15 intracellular pro-GDF-15 levels increased by 2.9-fold. However, upon PACE4-altCT depletion, a robust 10.5-fold increase in intracellular pro-GDF-15 levels was observed (Figure 6E), which was also accompanied by a strong increase in GDF-15 mRNA levels that was not visible under the siPACE4-FL condition (Figure 6F) but occurred in the shPACE4 line (in which both splice variants were knocked down, see Supplementary Figure S1D). When treated with the cell- permeable PACE4 peptide inhibitor (ML) and its PEGylated cell-impermeable version (PEG8-ML) (10,34), cleavage of GDF-15 was more susceptible to the ML-peptide, indicating that an important proportion of cleavage was performed inside the cells (Figure 6G). Altogether, these data suggest that pro-GDF-15 mostly requires intracellular PACE4 cleavage (predominantly by PACE4-altCT; Figure 6E), although we cannot exclude the possibility that it can also be retained at the cell surface (35) for potential cleavage by surface PACE4-FL. This phenomenon would be consistent with the data showing that overexpression of PACE4-altCT (mostly retained intracellularly) and PACE4-FL (both secreted and intracellular) resulted in similar increases in pro-GDF-15 processing (Figure 6A-B). GDF-15 is an important growth factor for prostate tumor progression and resistance to cytotoxic agents and radiotherapy in PCa and other cancers (36-41). Upon GDF-15 silencing (Figure 6H), both LNCaP growth and colony formation capabilities were reduced to similar extent to those obtained upon PACE4-altCT silencing (Figure 6I-J), further encompassing the importance of GDF-15 processing/secretion for its activity.

The knowledge that PACE4-altCT expression is strongly elevated in PCa, as well as that it functions as the prominent PC for the processing of pro-GDF-15, provides us with a complete picture from epigenetic and alternative splicing events to substrate activity and biological impact. The highest tissue expression of GDF-15 occurs in the adult prostate gland, and GDF-15 is known to be overexpressed in PCa (42), according to the Human Protein Atlas. However, there is an inverse correlation between pro-GDF-15 deposition in PCa tumors and disease relapse, suggesting that the processing of pro-GDF-15 is an

important factor in patient outcomes (35). In pairs of non-cancerous and tumoral prostate tissues, the processing of pro-GDF-15 increased along with the tumor grading (Figure 7A). It has also been reported that serum GDF-15 levels are elevated in patients with PCa (43), which may serve as a companion biomarker with the prostatic specific antigen (PSA) (44). With the knowledge of GDF-15 as a PACE4- specific substrate, we sought to investigate if it may serve a as target engagement biomarker in vivo to validate PACE4 inhibition in preclinical models. LNCaP-xenografted mice were intraperitoneally treated with 2 or 4 mg/kg of C23 every 24 h. Both doses strongly inhibited tumor progression (Figure 7B), which could also be observed based on plasma PSA levels in the mice (Figure 7C). In the plasma collected at the end of the 28 treatment days, the levels of GDF-15 measured by ELISA displayed a dose-dependent reduction but also correlated with the end-point of tumor size (Figure 7D-E). IHC analyses further confirmed the increases in terms of intracellular GDF-15 levels, which appeared as intracellular puncta that were predominantly visible in the xenograft obtained from treated compared with control animals (Figure 7F). Excised xenografts were either lysed and subjected to western blot analysis or paraffin- embedded for IHC. In tumor lysates, GDF-15 processing displayed a significant reduction along with the PACE4 inhibitor doses (Figure 7G). Moreover, proliferation, cell quiescence and apoptosis markers (Ki67, p27 and cleaved PARPAsp214) showed a dose-response pattern (Figure 7H-J). Through the impact of C23 on GDF-15, these data validate for the first time that C23 pharmacodynamics is mediated by PACE4 inhibition, more precisely the PACE4-altCT isoform.

Discussion: The PCs are regarded as promising targets for the development of cancer therapeutics because of their position upstream of numerous oncogenic pathways (3,45). Through the endoproteolytic processing of proproteins, including mediators involved in most of the key hallmarks of cancer, activation by PCs becomes a rate-limiting step following the overexpression of protein pathway components. Thus, when PC substrates are overexpressed by cancer cells, concomitant increases in terms of PC activity may occur to achieve a maximal biological outcome. PC overexpression has been documented in a number of cancer types (3), but the observed variations are not consistent across all cancer types (Figure 4C and Supplementary Figure S6G), which may be due to differences in tumor types or, alternatively, to insufficient data because many studies commonly assume PC activity to be assigned to a single member, namely furin. With such diverse and sometimes fragmentary information, it remains difficult to ascertain the best PCs to target in cancers or cancer subtypes. Based on our previous work, we identified PACE4 as critical in PCa, providing strong evidence of its overexpression as an indispensable component for sustained tumor growth. Despite this strong proof-of-concept, the molecular mechanisms were not well defined until now.

Discovery of the novel PACE4-altCT isoform, along with its strong expression in PCa specimens, sheds light on an important mechanism of sustained proliferation that exploits PACE4 activity to promote tumor growth. An alternative splicing epigenetic switch generates the PACE4-altCT isoform (Figure 3), which has drastically different characteristics in terms of cellular trafficking, autocatalytic activation rates and stability compared with the PACE4-FL isoform (Figure 2). These processes result in a sophisticated molecular control mechanism that sustains global PACE4 activity through the evasion of several mRNA regulatory elements (Figure 8).

It should be noted that a previous study dating back to the late 90’s reported presumed splice variants (46,47). Many of these encoding truncated isoforms included artefactual/frameshift errors (48,49). Moreover, these splice variants were isolated using cDNA libraries (17). It is unclear if these isoforms are produced in tissues or in disease states, however another study showed that all of these isoforms were tested inactive in vitro because of important domain truncations leading to inactive zymogens retained in the ER (23). Our tumor-oriented splicing analysis did not detect any of these variants nor could we validate their tumor-specific expression (Supplementary Figure S1). The only splice variant that could be validated was the previously not reported PACE4-altCT isoform. This new isoform is readily and actively converted to its active form despite being non-secreted which is a completely novel feature. We believe that PACE4-altCT may be a key element in cancer cell proliferation, since our observations were not limited to PCa cells and tissues. Indeed other cancer types also displayed increased PACE4-

altCT/PACE4-FL mRNA expression ratios and positive PACE4-altCT expression by IHC (Figure 4B-D), suggesting an important, although not ubiquitous, mechanism of action.

The discovery of PACE4-altCT permits us to refine our working model using pharmacological PACE4 inhibitors. In previous studies, we showed that when our ML-peptide inhibitor was modified by the addition of an N-terminal polyethylene glycol moiety, it lost its anti-proliferative activity toward PCa cells, which was correlated with a strong reduction of cell penetration properties (10). However, no apparent biological explanation could conciliate this phenomenon. Our present study shows that the PACE4-altCT isoform is retained inside the cell, while PACE4-FL is secreted or at the cell surface (Figure 2C and Supplementary Figure S4C-E). Since PACE4-altCT exhibits most of the cancer cell-associated growth stimulation capabilities (Figure 5), it is now clear that the anti-proliferative effects of the ML-peptide are in fact due to inhibition of the PACE4-altCT isoform, even if the ML-peptide has equal inhibitory activity toward both isoforms (Figure 2F and Figure 6G).

Unraveling pro-GDF-15 as a PACE4 substrate defines PACE4 activity in the PCa landscape, illustrating how it may sustain tumor progression. As a secreted factor, inhibition of its processing and further secretion likely restrict its action over its surface receptor (presumably TGF- receptor II; (50)), it is though not surprising that PACE4-altCT silencing resulted in similar phenotype as GDF-15 silencing (Figure 6I-J). GDF-15 is an important factor with immunosuppressive characteristics that supports PCa cell growth (41,51), but it is also known to protect cancer cells against radiation-induced cell death (36), to mediate tumor-mediated anorexia and weight loss (50) as well as to sustain tumor neovascularization (37). These observations however do not exclude that PACE4 probably have other substrates contributing to the observed effects. Serum GDF-15, which represents the mature form of this protein, has already been suggested as a potential PCa biomarker, highlighting the relevance of the mechanism leading to its cleavage-dependent secretion (43,44). Our data now extend to the conclusion that measuring pro-GDF-15 processing can serve as a means to monitor PACE4 activity in vivo, where target engagement would be evaluated in pharmacological interventions using PACE4 inhibitors in cancer therapeutic strategies (Figure 7). Having such markers is of great importance regarding the preclinical and clinical evaluation and readout of novel therapeutics (52). In our model, GDF-15 correlates directly with the tumor response, implying that its measurement is a direct reflection of PACE4 inhibition (Figure 7F-G), which is not the case for the other markers of cell cycle/apoptosis (Figure 7H-J) or for the tumor volume monitoring; the latter was not even able to show the dose-dependent activity of C23 (Figure 7B).

The study of PC cell biology has often utilized transient or stable overexpression systems (i.e., of PCs and/or their substrates) due to the lack of selective inhibitors. Our study used both approaches and revealed that cautionary interpretations of the data must take into account misbalanced stoichiometries,

altered levels of other endogenous PCs (Supplementary Figure S7D-F), and altered cellular trafficking. As an example, we clearly observed the cleavage of ITGA6 by overexpressing PACE4 (both FL and altCT), while PACE4 silencing had no effect (Figure 6A). In this same vein, a comparison of in vitro vs in cellulo analysis of substrate cleavage patterns also revealed potential misinterpretations of PC functions due to the lack of appropriate substrate-enzyme cellular compartmentalization (Figure 6D). Previous studies have also made substantial efforts to define PC motifs to distinguish between unique and redundant PC substrates. However, these studies relied solely on in vitro analysis of small peptide cleavages (53), potentially explaining the conclusions of a lack of specific PC functions. As a final point, changes observed with overexpression studies, while useful, do not always reveal underlying mechanisms, as observed in the study by Mahloogi et al., which showed malignant transformation of cells following transfection with PACE4 (PACE4-FL) (5). While we do not dispute this result, we are now convinced that it is the result of an artificial experimental design that would not occur in cancer cells, with the underlying mechanism of malignancy being the genesis of the PACE4-altCT isoform.

While our focus was on the expression of PACE4-altCT in cancer cells and tissues, we also examined the expression of this splice variant in normal tissues. We observed that PACE4-altCT was expressed at levels comparable to those in tumor cells in normal testis, liver and fetal tissues (Figure 4). This finding suggests that the epigenetic PCSK6 splicing switch that yields either PACE4-FL or PACE4-altCT regulates underlying physiological functions that are hijacked by cancer cells to sustain their growth by acting as a proto-oncogene. This hypothesis is also supported by the genomic environment of the 25th alternative exon, which is highly accessible because it is located in the DNase I sensitivity cluster (Figure 3A) and bound by DNA-binding elements such as CTCF according to the DNA methylation status. The finding that these epigenetic modifications are distinct between PCa and closed benign zones of the tested patients illustrates the complexity of the genetic regulation, as well as the cancer-associated switching that occurs to solicit PACE4 alternative splicing (Figure 8). It is noteworthy that PACE4-altCT is not conserved in non-primate species, in which a similar genomic environment was lacking, thus not allowing terminal exon substitution. As this significant element of human PACE4 biology is absent in mice, murine models of PCa, as well as other cancers or potentially other PACE4-related diseases, will require careful interpretation.

Cancer genomic studies using high-throughput approaches have generated ever-growing databases recording complex somatic genomic alterations. In most cases, it is rare to find common acquired mutations that are widespread across all patients with one type of cancer (54), or to find acquired mutations that are widespread across different types of cancer (55). While the PACE4-altCT isoform is not an acquired mutation, it is a significant change that promotes cancer cell proliferation and was detectable (at least at the mRNA level) in most patient samples that were available to us (>95%).

Regarding potential applications, PACE4-altCT (mRNA or protein) could be used as a biomarker to identify PACE4-dependent cancers, which appears to be a non-negligible constituent of many different cancer types (Figure 4B). Coupled with a specific pharmacological inhibitor of PACE4, as we have previously described (8), it may be possible to envisage a potent therapeutic strategy for PACE4-altCT dependent cancers. Despite the observation that this initial discovery oriented us toward its use as a tissue biomarker (IHC analysis), many useful serum biomarker proteins are not secreted but are still detectable in the circulation (e.g., HSP70 (56), S100B (57), CA125 (58)). These proteins are actually secreted by tumor cells upon either tissue structure disorganization, tumor cell necrosis or even apoptosis, which often occurs as hypoxic conditions arise in solid tumors (59). Thus, we believe that both total PACE4 and, most importantly, PACE4-altCT in patient serum could serve as biomarkers for PCa, especially considering the tumor specificity of the alternative isoform and the correlation between its expression and tumor Gleason score (Figure 1A-B&H).

Our growing understanding of the mechanisms that manage the switch from paracrine to autocrine mediation of sustained growth signaling in late androgen-independent stages may provide strategies to circumvent androgen-resistance and/or serve as biomarkers. PACE4 pharmacological targeting could provide an alternative to presently used androgen therapies that eventually fail due to resistance mechanisms, after which the only remaining option is chemotherapy. Alternatively, targeting PACE4 could also be considered as an adjuvant to chemotherapy. Our new understanding of the mechanisms involved in PACE4-associated functions in cancer cells provides strong support for these notions.

Acknowledgments: LB is a junior research scholar from the Fonds de la recherche du Québec en santé (FRQS), and RDay and LB are members of the FRQS-funded Centre de recherche du Centre Hospitalier Universitaire de Sherbrooke.

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Abbreviations: 3’UTR, 3’ untranslated region; 5-aza-dC , 5-aza-deoxy-cytidine; ADAM, A disintegrin and metalloproteinase; Amba, 4’amidinobenzylamide; ANCT, adjacent non-cancerous tissues; ChIP, chromatin immunoprecipitation; CHX, cycloheximide; CLU, clusterin; CTCF, CCCTC-binding factor; CMK, decanoyl-RVKR-chloromethylketone; EST, expressed sequence tag; GDF-15, growth differentiation factor 15; HGFR, hepatocyte growth factor receptor; IGF1R, insulin-like growth factor 1; IHC, immunohistochemistry; IP, immunoprecipitation; ITGA6, integrin alpha-6; LRP1, low-density lipoprotein receptor-related protein 1; MMP, matrix metalloproteinases; ML, multi-Leu peptide; PACE4-altCT, PACE4-alternative C-Terminal; PACE4-FL, PACE4-full length; PC, proprotein convertase; PCa, prostate cancer; PSA, prostate specific antigen r, recombinant; RT-qPCR, real time quantitative PCR; S2, Schneider 2; SILAC, stable isotope labeling by amino acids in cell culture; SWATH-MS, sequential window acquisition of all theoretical mass spectra; TP-PCR, three primers PCR.

Figure legends:

Figure 1 - Alternative splicing of PACE4 terminal exon is strongly enhanced in prostate cancer specimens and correlates with tumor aggressiveness. A – qPCR analyses of PACE4 expression levels in fresh prostate tissues specimens showing a significant correlation between levels and tumor Gleason scores (n= 7, 14, 11 and 6 for Gleason scores 6, 7, 8 and 9 respectively). Data are means  SEM. B- Similar analyses with data retrieved using c-BioPortal for Cancer Genomics using the Broad/Cornel Nature Genetics 2012 and MSKCC Cancer Cell 2010 datasets, (see data in Supplementary Figure S1A- B). r values are Spearman’s correlation coefficients. C – H&E and PACE4 IHC (using catalytic domain targeting antibody) in PCa representative specimens showing the concomitant increase of PACE4 expression at the protein level. Scale bars represent 200µm. D –3’RACE PCR showing 3’UTR lengths (expected: 1564 bps; 3’UTR: 1335 bps) and the two alternative 3’UTR lengths (341 bps; 3’UTR: 164bps, and 397 bps; 3’UTR: 118 bps) in LNCaP cDNA. E – Representation of the 3’ end of PCSK6 on the genome and on the mRNA transcript encompassing the exons, splicing motifs, 3’UTRs positions and lengths, primer design used for the 3’RACE and TP-PCR and the distinctive protein C-termini generated (PACE4-FL in blue and PACE4-altCT in red; see Supplementary Figure S1C for TP-PCR design and validation). F– TP-PCR performed on 10 paired ANCT and PCa tissues. G – RT-qPCR on paired tissues. Data are means  SEM, n= 6, 15, 9, 9 and 6 for Gleason 6, 7, 8 and 9 respectively. (H) Sub-analysis according to tumor Gleason scores. I – IHC analysis of consecutive slides from FFPE prostate specimens using anti-PACE4 antibodies targeting the consensual and alternative C-termini (see Supplementary Figure S2A-B for antibody validation). Scale bars represent 100 µm. Panel J provides a semi-quantitative analysis of epithelial foci (low grade: Gleason 3, high grade: Gleason 4-5) from a set of prostate cancer specimens stained with the catalytic domain antibody (used in panel 1C).

Figure 2 – PACE4 alternative splicing results in 3’UTR shortening and in the generation of an isoform differentially retained by cells with enhanced stability and increased auto-activation A – Actinomycin-D chase performed on LNCaP cells. Data are means  SEM (n=3). The table below the graph indicates the number of miRNA putative sites retrieved in both 3’UTR using miRDB (18) and RegRNA2.0 (http://regrna2.mbc.nctu.edu.tw/) tools (see list in Supplementary Table S5). B – Luciferase reporter assay with the different PACE4 3’UTRs, the length refers to the stop codon used, see Figure 1E (n=2 at least in each conditions performed in duplicate). C – V5-tagged PACE4-FL or PACE4-altCT secretion kinetic in LNCaP (“C” and “M” indicates Cell lysate and Medium respectively). See Supplementary Figure S4A-B for other cell lines and quantifications.

D – Representative blot of a CHX-chase in V5-tagged PACE4-FL or PACE4-altCT transfected LNCaP cell lysates. (E) Quantitative analysis of protein content in lysates during the CHX-chase in HEK293, DU145 and LNCaP (see Supplementary Figure S4F; n=3 at least, one being shown in panel D). F - Enzymatic activity (cleavage kinetic of Pyr-Arg-Thr-Lys-Arg-methylcoumaryl-7-amide) and inhibitory profiles by the ML-peptide inhibitor of both rPACE4 isoforms preparations (Supplementary Figure S4I), identical preparations from non-transfected S2 cells served as a blank. G - V5-IPs performed on the lysates used for IP-MS (n=6). H - PACE4- tryptic peptides integrated area under the curve in the V5-IPs (n=6). I – Fold enrichments of cell compartment-associated proteins from V5-IPs performed in non-denaturating lysates of transiently expressing HEK293-FT cells (n=6). J – Secretory pathway map showing the localization of IF-tested proteins and IP-MS identified proteins with color codes referring to PACE4-FL (red) or PACE4-altCT (green). K – IF confocal images and quantitative co-localization analyzes with V5 and RCAS1, Rab5 (L), Rab7 (M) and Rab9 (N). Data are means  SEM of at least 7 individual cells analyzed.

Figure 3 – PACE4 alternative splicing and polyadenylation is dependent on CTCF-mediated exon inclusion and regulated by intra-exonic DNA methylation. A – UCSC genome browser (human genome GRCh37/hg19 assembly) view of PACE4 terminal exons. B – Diagram encompassing the CpG dinucleotides (in red) within the 3 CTCF enriched regions (represented as red ovals). C – CpG dinucleotides methylation shown in B analyzed in paired tissues. Data are shown in pairs linked by a line (n=13 pairs). Graphic titles refers to panel B. D – CTCF expression by RT-qPCR (n=3 at least). E – CTCF protein levels in siCTCF-transfected cells (n=2 loaded side by side). F – PACE4 exon 25th splicing indices in siRNA transfected cells (n=4 at least). G – Splicing indices determined by RT-qPCR and TP-PCR (H) in cells treated with 5-aza-dC for 72h (n=4 at least). Hypomethylation efficacy was determined (see Supplementary Figure S5A-C) I – CTCF protein levels in LNCaP transfected with myc-CTCF vector treated or not with 5-aza-dC 10 µM for 72h. (n=2 loaded side by side). J – CTCF mRNA levels following overexpression (n=3 at least). Data are presented as means  SEM. K – Splicing indices determined by RT-qPCR on the LNCaP overexpressing CTCF with or without 5-aza- dC treatment (n=3 at least). Data are presented as means  SEM. L – CTCF ChIP in DU145 (and LNCaP; see Supplementary Figure S5D) cells treated or not with 5-aza- dC for 72h (n=3 at least). Data are presented as means  SEM.

Figure 4– Mapping of PACE4-altCT across human tissues and different cancer types reveals a common tumor molecular switch mechanism. A – PACE4 25th exon mRNA splicing analysis in standard RNA preparation from pooled human organs (see material and methods) either by RT-qPCR (splicing index) or by TP-PCR. See other PCs in Supplementary Figure S6A-F. B – Splicing indices measured across cancerous and non-cancerous tissues cDNA (see Supplemental Experimental Procedures; data are means  SEM). Other PCs are shown on Supplementary Figure S6G. C – Quantitation of all tested PCs across the cancer types available reported as mean fold changes between normal and cancerous tissues (see Supplementary Figure S6H-L). D – IHC of PACE4-FL and PACE4-altCT performed on matched normal and cancerous tissues (see Supplementary Experimental Procedures). Selected representative fields shown are aligned for both antibodies tested. Additional tissue pairs can be found in Supplementary Figure S6M. Scale bars represent 200 µm.

Figure 5 – PACE4-altCT is responsible of PACE4-associated sustained growth capabilities in prostate cancer cells. A – PACE4 protein levels (without tags; using a catalytic domain targeting antibody) in stably overexpressing LNCaP cells. See Supplementary Figure S7A for DU145 cells data. B – PACE4 mRNA expression levels in RT-qPCR analyses in stable pLenti6 transfected LNCaP cell lines (Means  SEM, n=3). C- Colony formation assays on stably expressing LNCaP and HT1080 cell lines (see Supplementary Figure S7B for colony size analysis). The image shows representative stained LNCaP wells (n=4 at least). D– PACE4 isoforms and cognate PCs expression levels in siRNA transfected LNCaP cells by RT-qPCR (n=3). See Supplementary Figure S7G for knockdown in DU145. E – PACE4 protein levels (endogenous levels; using a catalytic domain targeting antibody) in siRNA transfected LNCaP cell lysates and their respective serum-free conditioned media. See Supplementary Figure S7H for knockdown in DU145. F – Colony formation assays on transfected LNCaP at a density of 200 cells/well for 12 days (n=2 in duplicate). Representative fields are shown above the relative quantitation relative to siNon-Target transfected cells. See Supplementary Figure S7I for DU145 results. G – XTT proliferation assays performed on LNCaP transfected cells 72h after transfection (n=2 in triplicate). See Supplementary Figure S7J for DU145 results. All data are means  SEM.

Figure 6– Identification and validation of GDF-15 as a PACE4-specific substrate in prostate cancer.

A – Western blots of IGF1R, ITGA6, E-cadherin and GDF-15 processing in DU145 knockdown for each endogenously expressed PC, PACE4-overexpressing and inhibitor treated cells, a representative blot is shown, experiments were carried out twice at least (n=2). B – GDF-15 western blot in LNCaP cell lysates and conditioned medium. Below are indicated processing index (mature/mature+pro) compared to the respective controls. C – GDF-15 concentrations in DU145 and LNCaP conditioned medium by ELISA. D – GDF-15 spanning peptide cleavage by rPACE4 and rfurin monitored by HPLC. Cleavage site is underlined in the spanning peptide sequence. Peptide identity after cleavage were confirmed by mass using MALDI-MS analysis of collected HPLC fractions. A representative experiment is shown from the two performed E – GDF-15 western blots in LNCaP cells transfected with PACE4-isoforms targeting siRNAs. Experiment performed in n=2 (loaded side by side). F – GDF-15 mRNA levels measured by RT-qPCR in the LNCaP cells transfected with PACE4-isoforms targeting siRNAs and in the shPACE4-knockdown cells (both isoforms, see Supplementary Figure S1D). G – GDF-15 cleavage analysis by western blot in LNCaP cells treated with ML-peptide or the cell impermeable version (PEG8-ML) to discriminate intracellular PACE4 contribution to GDF-15 processing. Indicated values are the ratio between mature/pro. H – GDF-15 western blot of siGDF-15 transfected LNCaP cells. I – Colony formation and (J) XTT proliferation assay of siGDF-15 transfected LNCaP cells Data are means  SEM (n=3).

Figure 7– GDF-15 as a PACE4-activity marker in vivo. A – GDF-15 western blot analysis of paired tissues. A blot showing various cancer grades is shown together with the densitometry analysis shown in panel of 19 tissues pairs (n= 6 (3+3), 7 (3+4), 6 (4+3). Data are means  SEM. B – Volume of LNCaP subcutaneous xenograft during treatment with C23 (IP, 0, 2 or 4 mg/kg in saline every 24h). Volumes are normalized in percentage based on their volume at the initiation of treatments. Data are means  SEM, (n=8 ,7 and 10 mice for the 0, 2 and 4 mg/kg groups respectively). C – Plasma PSA levels measured by ELISA from saphena vein bleeds during the treatment phase of the experiment. Data are means  SEM. D – Plasma GDF-15 levels measured in the final bleeding of the mice at the end of the experiment. Data are means  SEM. E – Correlation between final tumor size and plasma GDF-15 levels. F – Representative IHC images from FFPE xenografts sections. The scales represents 200 µm. G – Densitometry analysis of GDF-15 western blot in xenograft tissues lysates. Data are means  SEM. (n= 5, 5 and 4 for the 0, 2 and 4 mg/kg groups respectively loaded side by side).

H – IHC quantitative image analysis for Ki67 proliferation indexes, (I) p27 positivity indexes and (J) cleaved PARPAsp214 apoptosis indexes (n= 7, 6 and 6 for the 0, 2 and 4 mg/kg groups respectively). Data are means  SEM. * indicates P<0.05, ** indicates P<0.01 and *** indicates P<0.001 using student-t-test.

Figure 8 – Summary and working model. In cancer cells, CpG methylation levels in the intra-exonic region of the alternative terminal exon is reduced which favors the binding of the DNA-binding factor CTCF and promote the inclusion of this terminal exon instead of the consensual one. The resulting mRNA then exhibits a distinct and shorter 3’UTR region lacking several of the miRNA sites present on the consensual transcripts. This increases mRNA stability and yields increased protein translation of the resultant isoform PACE4-altCT, which has a higher autocatalytic cleavage rate compared to PACE4-FL and is rerouted in the secretory pathway in a way that its secretion is prevented. The combination of these biochemical differences result in increased cleavage of the PACE4 substrate; pro-GDF-15, and sustain cancer cell growth capabilities.

1 10-2 6 10-3 Author Manuscript Published OnlineFirst on October 9, 2017; DOI: 10.1158/0008-5472.CAN-17-1397 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

4 10-3 Normal

2 10-3 PIN PACE4 mRNA levels relative to actin to relative levels mRNA PACE4

ANCT Gleason 6 Gleason 7 Gleason 8 Gleason 9 B

n r P-value PCaGrade Low This study 38 (pairs) 0.4601 0.0037

BroadCornell, Nature Genetics 20 0.5355 0.0150 2012

MSKCC Cancer Cell 2010 137 (pairs) 0.1799 0.0354 High Grade PCaGrade High D E Genomic DNA 23 24 25 25-alt NEST on F-23+3’RACEcDNA equivalent 4813 bp 6050 bp 6343 bp F-23+3’RACESpanning + Spanning3’RACE + 3’RACE 104 bp 130 bp 113 bp 256 bp

-2036 ACG GTGAGCAG TGACTTTTGTCTTCCAG GGGA -1636 1350- 3’UTR 3’UTR-alt -1018 mRNA (with primer design) 916- UAA AAUAAA UAA AAGAAA Span-25 AAUAAA 500- 23 24 25 25alt 350-

200- F-23 R-25 R-25alt 23 RACE Protein PACE4-FL ADETFCEMVKSNRLCERKLFIQFCCRTCLLAG […] NHTCSN... GDIWQRLETFWVVTTGRMYSHPVGGGQECC F PACE4-altCT

1350- 25 23 24 25 alt I PCa PCa PCa PCa PCa 500- ANCT ANCT ANCT ANCT ANCT Exon 25 alt PACE4-altCT IHC PACE4-FL IHC 200- Exon 25 cons

G H 7.95 fold change 10000 2000 PACE4-FL

1000 PACE4-altCT

1500 Normal

100

1000

10 Tumor/ANCT expression ratio expression Tumor/ANCT 500 1 Tumor/ANCT mRNA expression ratio expression mRNA Tumor/ANCT J

0.1 0 Low gradeLow PCa

ACE4 ACE4 P

Gleason 6 Gleason 7 Gleason 8 Gleason 9 Alternative P Consensual

J PACE4 PACE4-FL PACE4-altCT IHC grading n - lowhigh - lowhigh - lowhigh normal 12 0120 1110111 0 Highgrade PCa PIN 7 0 6 1 1 6 0 4 3 0 Low grade foci 6 0 2 4 0 5 1 0 5 1 High grade foci 1308 5111148 1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. FIGURE 1 Author Manuscript Published OnlineFirst on October 9, 2017; DOI: 10.1158/0008-5472.CAN-17-1397 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A B 3 Long 3'UTR (1335bps) C Short 3'UTR AAUAAA (164bps) PACE4-FL-V5 PACE4-altCT-V5 Short AAGAAA (118bps) Time post- 100 transfection mock 8h 24h 30h 8h 24h 30h

2 C M C M C M C M C M C M C M -PACE4 100- (V5) mRNA half-life 50- (linear fits) 1 -Actin consensual 3'UTR: 7.1h 37- mRNA levels relative to 0h to relative levels mRNA alternative 3'UTR: 24.9h 10 0 2 4 6 8 activity luciferase Normalized 0 Time (h) P 3'UTR (bps) miRDB RegRNA 2.0 PACE4-altCT PACE4-FL consensus 1335 20 108 LNCa DU145 I Thioesterase superfamily member 6 (THEM6) alternative 164 2 7 HEK293FT

ecreted Tight Junction Protein 1 ( ZO-1) s Membrane/ PACE4-FL-V5 PACE4-altCT-V5 F 1500 PACE4-FL D PACE4-altCT Time ADP-Ribosylation Factor-Like 8A (ARL8A) PACE4-FL + 50 M ML post-CHX (h) 0 1 2 4 6 0 1 2 4 6 Vacuolar Protein Sorting 16 Homolog (VPS16) PACE4-altCT + 50 M ML Vacuolar Protein Sorting 4 Homolog A (VPS4A) 1000 100- -PACE4 Ras-related protein Rab-5C (RAB5C) (V5) EH-Domain Containing 4 (EHD4) Endosomal Endosomal

50- compartments Vac14 Homolog (VAC14)

-Actin 500 ADP-Ribosylation Factor 6 (ARF6) 37- Ras-related protein Rab-13 (RAB13) Relative Fluoresence Units (RFU) Units Fluoresence Relative Vacuolar protein sorting-associated protein 13C (VPS13C) E 150 0 Exocyst Complex Component 7 (EXOC7) 0 20 40 60 Time (min) Exocyst Complex Component 2 (EXOC2) Synaptotagmin-Like 4 (SYTL4) 100 Ras-related protein Rab-10 (RAB10) GH exocyst & TGN Input IP 4 107 ADP-Ribosylation Factor 4 (ARF4) (%) ADP-Ribosylation Factor 3 (ARF3) 50 3 107

PACE4-altCT PACE4-FL PACE4-altCT PACE4-FL Coatomer Protein Complex, Subunit Gamma 2 (COP 2) 150- 2 107 Coatomer Protein Complex, Subunit Beta 2 (COP 2)

Percentage of remaining protein signal protein remaining of Percentage -PACE4 0 100- 0 1 2 4 6 Coatomer Protein Complex, Subunit Beta (COP ) Time (h) 7 75- 1 10 Coatomer Protein Complex, Subunit Epsilon (COP )

PACE4-FL PACE4-FL PACE4-FL Binding immunoglobulin protein (BiP) HEK293 DU145 LNCaP intensity integrated peptides PACE4 PACE4-altCT PACE4-altCT PACE4-altCT 0 Sec1 Family Domain Containing 1 (SCFD1) 50- 5 5 Surfeit 4 (SURF4)

Empty Vector

PACE4-FL-V transport ER-Golgi PACE4-altCT-V WAS Protein Family Homolog 4 (WASH4P) Secretion Associated, Ras Related GTPase 1A (SAR1A)

Vesicle-trafficking protein SEC22b (SEC22B) J Green: enriched in PACE4-altCT IP Higher colocalization with PACE4-altCT Yip1 Domain Family, Member 5 (YIPF5) Red: enriched in PACE4-FL IP Higher colocalization with PACE4-FL 1 0 6 1 0.1 100 1000 10 10000 Exocytosis Endocytosis 100000 1 Them6 Ratio over control IP Rab10 Zo-1 Arf6 Clathrin K M Arf6 Sytl4 dependent 80 Caveolin 50 Exoc2 ** V5 (PACE4-FL) V5 (PACE4-altCT) dependent V5 (PACE4-FL) V5 (PACE4-altCT) Rab7 Rab7 RCAS1 RCAS1 Exoc7 Ehd4 Edh4 40 60

Rab13 30 Early endosomes 40 (%)

Recycling (%) Vac14 Endosome 20

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10 colocalizingpixel V5+ V5+ pixel colocalizingpixel V5+ Rab10 Rab13 Trans-Golgi Arf3 Rab9 0 0 Network L T Rab13 Rab9 ACE4-FL PACE4-F P PACE4-altC RCAS1 PACE4-altCT Golgi Arf4 Late Rab7 L N Endosome 30 V5 (PACE4-FL) V5 (PACE4-altCT) COPII * Rab9 Sec22 β-COP 50 Rab9 Yipf5 COPI Vps4 * V5 (PACE4-FL) V5 (PACE4-altCT) Wash4 ε-COP Rab5 Rab5 Scfd1 40 Surfeit4 Scfd1 Rab7 20 BiP 30 (%)

Sar1a (%) Endoplasmic Lysosome 20 10 Reticulum with RAB9+ pixel RAB9+ with Vps4 pixel RAB5 with

10 colocalizingpixel V5+ V5+ pixel colocalizingpixel V5+

Nucleus Arl8 0 0 T

PACE4-FL PACE4-altCT PACE4-FL PACE4-altC Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. FIGURE 2 A B 25th alt UPSTREAM 25th alt BOUNDARY 25th alt DOWNSTREAM 25th cons DOWNSTREAM chr15 (q26.3) p13 15p12 15p11.2 11.1 q11.2 12 15q14 q21.121.215q21.3q22.2 15q23 25.125.2 25.3 q26.1 q26.2q26.3 5’ AACGACATTCTCCGGC[...]ATCGGG ACCGGGG[...]GGCCGAG CYGAGGTTGAGAAAYTCCG CACGGCGGC 3’

Scale 2 kb hg19 chr15: 101,840,500 101,841,000 101,841,500 101,842,000 101,842,500 101,843,000 101,843,500 101,844,000 101,844,500 101,845,000 UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics) AK130759 PCSK6 PCSK6 3’ 5’ PCSK6 25 25 24 ALT Human mRNAs from GenBank Human mRNAs Human ESTs That Have Been Spliced Spliced ESTs UniGene Alignments 6,3 kb intron 6 kb intron UniGene AceView Gene Models With Alt-Splicing AceView Genes DNaseI Hypersensitivity Clusters in 125 cell types from ENCODE (V3) C 25th alt UPSTREAM 25th alt BOUNDARY DNase Clusters * Transcription Factor ChIP-seq (161 factors) from ENCODE with Factorbook Motifs 100 100 CTCF dG1HLhhhUKnnnnoAAe1LKStGIKAgGggggHLUKMMMMMnpAaaaaaabbcgggggggGhhhhhhhhhHLhhhhhhhhUhKMnnnnrsSw RFX5 H RUNX3 G ** 80 90 ZNF143 GK SMC3 GHLK RAD21 G1LKSAG1HLIK 60 PRDM1 H 80 ** CTCF g 40 % CpG methylation CpG % Transcription Levels Assayed by RNA-seq on 9 Cell Lines from ENCODE methylation CpG % Transcription 70 20 1 _ 100 vertebrates conservation by PhastCons 100 Vert. Cons T a T a T a C Ca P PCa P PC PC 0 _ ANC ANC ANCT ANCT ANC Multiz Alignments of 46 Vertebrates 25th alt DOWNSTREAM 25th cons DOWNSTREAM Chimp 100 Orangutan 100 Rhesus Mouse Rat 80 Rabbit 80 Dog Chicken Zebrafish Author Manuscript Published OnlineFirst on October 9, 2017; DOI: 10.1158/0008-5472.CAN-17-1397 60 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 60 % CpG methylation CpG % %CpG methylation %CpG th Alternative 25 exon 40 terminal exon 40 T a a Ca C C PCa P P P ANC ANCT ANCT ANCT

D E F G 0.15 DU145 0.003 DU145 0.15 DU145 LNCaP LNCaP LNCaP DU145 lysate siCTLR siCTCF 0.10

150- 0.002 -CTCF 0.10 0.05 50- index Splicing -actin 37- Splicing index index Splicing (arbitrary units) (arbitrary 0.00 expression levels expression 0.001 LNCaP lysate 0.05 0 5 5 0 . 1 Relative CTCF mRNA mRNA CTCF Relative 2 H 0.25 150- -CTCF 5-aza-dC ( M) [5-aza-dC] 50- 0 0.25 2.5 5 10 -actin (µM) 0.000 37- 0.00 500- 25 23 24 25 alt siCTRL siCTCF siCTRL siCTCF siCTRL siCTCF siCTRL siCTCF Exon 25 alt

200- Exon 25 cons

LNCaP lysates Empty vector I myc-CTCF K L 10 ChIP CTCF (DU145 untreated) 5-aza-dC - + - + ChIP normal IgG (DU145 untreated) 0.08 ChIP CTCF (10 M 5aza-dC 72h) ** ChIP normal IgG (10 M 5aza-dC 72h) ** 150- -CTCF 8 * * 50- 0.06 -actin * 37- 6 0.04 J 1.0 Splicing index Splicing 0.5 0.02 4

0.05 0.00

expression levels expression 2 Relative CTCF mRNA mRNA CTCF Relative Fold enrichment (over normal IgG) normal (over enrichment Fold 0.00 ector + DMSO V ector + 5-aza-dC hCTCF + DMSO V hCTCF + 5-aza-dC 0 n n n xo Vector + DMSO e exo ector + 5-aza-dC hCTCF + DMSO h h V hCTCF + 5-aza-dC t t 25 f t-25 ' o al alt-25th exo 3' of alt-25th 5 f o 5'

Kcnq5 (positive control)

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. FIGURE 3 Author Manuscript Published OnlineFirst on October 9, 2017; DOI: 10.1158/0008-5472.CAN-17-1397 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

D PACE4-altCT PACE4-FL A 0.25

0.20

Lung Lung

0.15

0.10 Thyroid

Splicing index Splicing Thyroid

0.05

Adrenal Gland 0.00 Adrenal Gland -alt -cons

T Y R S S

LIVE HEAR LUNG TESTIS LNCaP KIDNE SPLEEN THYMUSUTERUCOLON (WHOLE) Testis Testis PLACENTAPROSTATE INTESTINE STOMACH FETAL FETALBRAIN LIVER SPINAL CORD BONE MARROWBRAIN SKELETAL MUSCLE BRAIN (CEREBELLUM)

B C Adenosquamous Carcinoma Adenosquamous Carcinoma 3 Adjacent Non-Cancerous Tissue Mean Fold Change TUMOR/NORMAL Tumoral Tissue PACE4 2 consensus alternative splicing PACE4 PACE4 Tumor Site index 1.0 Lungs >100 2,6 >100 Thyroid 15,2 0,3 1,6 Adrenals 14,5 0,5 1,9 Papillary Adenoarcinoma Papillary Adenoarcinoma 0.8 Testis 10,4 0,1 0,5 Endometrium 4,0 1,2 11,3 Pancreas 3,3 1,1 6,5 Splicing index Splicing 0.5 Oesophagus 3,1 1,0 3,0 Prostate 3,0 6,2 2,0 Ovary 2,0 0,7 1,0 0.3 Liver 1,9 0,6 0,8 Breast 1,6 0,7 0,7 Pheochromocytoma Pheochromocytoma Colon 1,0 1,0 1,0 0.0 Stomach 1,0 3,9 2,6 Y S E D Kidney 0,6 1,4 1,4 M US E G EAS AT EAST RIU LIVER UNGS T Bladder 0,4 6,2 9,5 R L ISSU OVARY TESTIS B CERVIXCOLON KIDNE NCR OS OMACH T THYROIBLADDER Cervix 0,3 3,0 0,8 ADRENALS PA PR S ESOPHA OID T NDOMETO H Lymphoid Tissues N/A 3,0 N/A E YMP L Seminoma Seminoma Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. FIGURE 4 Author Manuscript Published OnlineFirst on October 9, 2017; DOI: 10.1158/0008-5472.CAN-17-1397 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

PACE4 PACE4 A “empty” C pLenti6- FL altCT Cell lysate 100- -PACE4

75- pLenti6-empty pLenti6-PACE4-FL pLenti6-PACE4-altCT 50- -Actin 800 pLenti6-empty 37- pLenti6-hPACE4-FL pLenti6-hPACE4-altCT Medium 100- -PACE4 600

75- NS

400

B 0.08 (% of control) of (% 0.06 Number of colonies colonies of Number 200 0.04

0.02

0.00 levels relative to actin to relative levels 0 PACE4 expression mRNA expression PACE4

ACE4-FL

pLenti6-empty HT1080 LNCaP pLenti6-hP pLenti6-hPACE4-ALT D E 2.0 PACE4-FL Furin PACE4-altCT PC7 Cell lysate siCTLR siPACE4-FL siPACE4-altCT 1.5 150- -PACE4 100- 50- 1.0 -Actin 37- relative to actin actin to relative 0.5 Cond. Medium 1.0 0.87 0.55 150- * 100-

Normalized expression level level expression Normalized -PACE4 *** 0.0 75- NS 1.0 0.17 0.72

siNon-Target siPACE4-FL ACE4-altCT siP

F G

1.5 )

3 650nm 1.0 * 2 , Ref; DO Ref; , 0.5

1 550nm (DO Number of colonies of Number (relative to siNon-Target) to (relative

0 siNon-Target to relative signal XTT 0.0

arget T mock

siNon-Target siPACE4-FL ACE4-altCT PACE4-FL siP siNon- si PACE4-altCT si

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research.FIGURE 5 Author Manuscript Published OnlineFirst on October 9, 2017; DOI: 10.1158/0008-5472.CAN-17-1397 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A B

shNon-Targetshfurin shPACE4 shPC7 pLenti6 pLenti6-PACE4-FLpLenti6-PACE4-altCTCMK C23 shNon-Targetshfurin shPACE4 shPC7 pLenti6 pLenti6-PACE4-FLpLenti6-PACE4-altCTCMK C23 DU145 LNCaP 50- Lysate Lysate 250- 37- -proGDF-15 -proIGF1R 150- 25- Medium 37- -proGDF-15 100- 25- -IGF1R 20- 75- 150- -proITGA6 -GDF-15 -ITGA6 100- 1.0 0.55 0.05 0.81 1.0 1.15 1.16 0 0.01 150- -proE-cadherin -E-cadherin C 1000 100- 750 50- 500 -actin 300 200

37- (pg/mL) 100

GDF-15 concentration GDF-15 0 4 E -FL CMK C23 shfurin shPC7 pLenti6 shPAC Non-Target i6-PACE4 D NH t H 2N-QAARGRRRARARNG- 2 800 pLen pLenti6-PACE4-alt

Buﬀer 600 H 2N-QAARGRRRAR-COOH GDF-15 Peptide E F 4 Cell lysate siCTLR siPACE4-FL siPACE4-altCT GDF-15 Peptide + rPACE4 (6h) 37- 3 400 -pro-GDF-15 ) GDF-15 Peptide + rPACE4 (2h) 25- 50- 2 214nm 200 -Actin GDF-15 Peptide + rfurin (6h) 37- 1 Cond. Medium

GDF-15 Peptide + rfurin (2h) mRNA GDF-15 Normalized Signal(OD 0 expression levels relative to actin actin to relative levels expression 0 18 20 22 24 26 28 30 32 -GDF-15 arget arget PACE4 Retention time (min) sh siNon-T siPACE4-FL shNon-T siPACE4-altCT

G LNCaP H IJ ) VHL ML PEG8-ML #1 #2 Lysate 37- 1.5 LNCaP 2.0 650nm -proGDF-15 siGDF-15 siGDF-15 25- Lysate siNon-Target 1.5 1.0 50- 37- -pro-GDF-15 1.0 DO Ref; , -actin 0.5 25- 0.5 50- 550nm 37- siNon-Target to 0.0 Medium -Actin 0.0 (DO 37- relative number Colony arget 20- Cond. Med. 1.0 0.32 0.52 siNon-Target to relative signal XTT PACE4-Fl ACE4-FL si 20- P siNon-Target siGDF-15 #1siGDF-15 #2 -GDF-15 -GDF-15 siNon-T si ACE4-altCTsiGDF-15 #1siGDF-15 #2 siPACE4-altCT siP

1.0 0.62 0.98 1.0 0.12 0.09

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A B Tissue lysates 500 Control (saline) C23 (2mg/kg•24h) IP ANCT PCa ANCT PCa ANCT PCa ANCT PCa ANCT PCa ANCT PCa ANCT PCa 3+3 3+3 4+3 3+4 3+4 4+3 5+3 C23 (4mg/kg•24h) IP 37- 400 -proGDF-15 30

300

20 20- -GDF-15 (%) 200

37- animal volume/ 10

-proGDF-15 tumor normalized Mean ** ** ** Higher exposure 100 ** ** ** *** ** *** *** 25- ** 0 ** * 3+3 3+4 4+3 37- *** GDF-15 processing (mature/proprotein) processing GDF-15 Fold change between tumor and ANCT and tumor between change Fold -actin Tumor Gleason Score 0 0 10 20 30 Time post-treatment initiation (days) C D 1 106 E 100 Control (saline) 10000 VHL (saline) C23 (2mg/kg•24h) IP C23 (4mg/kg•24h) IP 2mg/kg IP 24h

) 4mg/Kg IP 24h 75 3 1 105 1000

50 (pg/mL)

1 104 (mm size Tumor 100 GDF-15 concentration GDF-15

PSA serum levels (ng/mL) serumlevels PSA 25 Pearson r: 0.8429 P-value: <0.0001 10 1 103 100 1000 10000 100000 1 106 0 10 20 30 plasma GDF-15 (pg/mL) Time post-treatment initiation (days)

Control (saline) C23 2mg/kg C23 4mg/kg F G Control (saline) 1.0 *** *** Xenograft Saline treated C23 2mg/kg C23 4mg/kg 0.8 lysates 37- 0.6 -proGDF-15

25- 0.4

(mature/pro-form) -GDF-15 20- GDF-15 processing index index processing GDF-15 0.2 20- C23 2mg/kg -GDF-15 0.0 Higher exposure 37- 24h -actin

Control (saline) 2mg/Kg IP 4mg/Kg IP 24h H I J *** *** 20 *** *** ** 10 70 *** 2)

15 8 C23 4mg/kg 60

cells /tumor mm /tumor cells 6 ** 10 Asp214

(% cells/field) (% 4

50 index Apoptosis p27 positive cell index cell positive p27

(% of positive cells) positive of (% 5

Ki67 proliferation index index proliferation Ki67 2

40 0 PARP cleaved ( 0

C23 2mgkg C23 4mgkg C23 2mgkg C23 4mgkg C23 2mgkg C23 4mgkg Control (saline) Control (saline) Control (saline) Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. FIGURE 7 Author Manuscript Published OnlineFirst on October 9, 2017; DOI: 10.1158/0008-5472.CAN-17-1397 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Normal cells Prostate cancer cells

Local intra-exonic hypomethylation CTCF binding

PACE4 Alternative splicing pre-mRNA Alternative terminal exon inclusion

mRNA stability protein translation auto-activation rate intracellular rerouting pro-GDF-15 processing sustained cell growth

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PACE4 Undergoes an Oncogenic Alternative Splicing Switch in Cancer

Frédéric Couture, Robert Sabbagh, Anna Kwiatkowska, et al.

Cancer Res Published OnlineFirst October 9, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-1397

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2017/10/07/0008-5472.CAN-17-1397.DC1

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

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