Kras/ADAM17-Dependent Jag1-ICD Reverse Signaling Sustains

Published OnlineFirst September 10, 2019; DOI: 10.1158/0008-5472.CAN-19-0145

Cancer Tumor Biology and Immunology Research

Kras/ADAM17-Dependent Jag1-ICD Reverse Signaling Sustains Colorectal Cancer Progression and Chemoresistance Maria Pelullo1, Francesca Nardozza2, Sabrina Zema2, Roberta Quaranta2, Carmine Nicoletti3, Zein Mersini Besharat4, Maria Pia Felli4, Bruna Cerbelli5,Giulia d'Amati5, Rocco Palermo2, Carlo Capalbo2, Claudio Talora2, Lucia Di Marcotullio6, Giuseppe Giannini6, Saula Checquolo7, Isabella Screpanti2, and Diana Bellavia2

Abstract

Colorectal cancer is characterized by well-known genetic tumor growth and epithelial–mesenchymal transition, defects and approximately 50% of cases harbor oncogenic Ras enhancing colorectal cancer progression and chemoresistance mutations. Increased expression of Notch ligand Jagged1 both in vitro and in vivo. These data highlight a novel role for occurs in several human malignancies, including colorectal Jagged1 in colorectal cancer tumor biology that may go cancer, and correlates with cancer progression, poor prognosis, beyond its effect on canonical Notch activation and suggest and recurrence. Herein, we demonstrated that Jagged1 was that Jag1-ICD may behave as an oncogenic driver that is able constitutively processed in colorectal cancer tumors with to sustain tumor pathogenesis and to confer chemoresistance mutant Kras, which ultimately triggered intrinsic reverse sig- through a noncanonical mechanism. naling via its nuclear-targeted intracellular domain Jag1-ICD. This process occurred when Kras/Erk/ADAM17 signaling was Signiﬁcance: These ﬁndings present a novel role of the switched on, demonstrating that Jagged1 is a novel target of transcriptionally active Jag1-ICD fragment to confer and medi- the Kras signaling pathway. Notably, Jag1-ICD promoted ate some of the activity of oncogenic KRAS.

Introduction b-catenin–dependent gene expression (1). In intestinal epithelial cells, constitutive activation of b-catenin/TCF leads to adenoma- Sporadic colorectal cancer development is characterized by tous polyp formation, a first step toward colorectal cancer devel- well-known histopathological changes, resulting from specific opment. In addition, Ras driver mutations, found in about 50% of genetic defects in selected oncogenes and tumor-suppressor all colorectal cancers and in advanced adenomas (2, 3), strongly genes. The most of sporadic colorectal cancers and hereditary sustain the pathogenesis of colorectal cancer, regulating tumor colorectal tumors show loss of APC function, the negative regu- cell proliferation, survival, invasion, metastasis formation, and lator of Wnt signaling, ultimately leading to abnormal drug resistance (2, 4). Constitutive activation of Kras is one of the best-characterized events in colorectal cancer development, able to trigger multiple downstream pathways, including the RAF/ 1Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, MEK/Erk MAPK and the PI3K–AKT effector pathways (5). Several Italy. 2Department of Molecular Medicine, Sapienza University, Rome, Italy. observations suggest an involvement of MEK/Erk signaling in 3 Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Unit intestinal tumorigenesis (6), but the exact molecular mechanisms of Histology and Medical Embryology, Sapienza University, Rome, Italy. 4Depart- remain unclear. Of note, a growing body of evidence shows that ment of Experimental Medicine, Sapienza University, Rome, Italy. 5Department of Radiological, Oncological and Pathological Sciences, Sapienza University, the oncogenic Kras regulates ADAM17 activity and the shedding Rome, Italy. 6Department of Molecular Medicine, Sapienza University, Labora- of several growth factors in a MEK/Erk-dependent manner (4, 7). tory affiliated to Istituto Pasteur Italia, Italy. 7Department of Medico-Surgical Kras mutations confer colorectal cancer resistance to anti-EGFR Sciences and Biotechnology, Sapienza University, Latina, Italy. therapy and are associated with a worse prognosis (8). Current Note: Supplementary data for this article are available at Cancer Research therapeutic options for advanced colorectal cancer have not Online (http://cancerres.aacrjournals.org/). dramatically improved clinical outcomes of patients with meta- M. Pelullo, F. Nardozza, and S. Zema contributed equally to this article. static colorectal cancer. Therefore, a better understanding of molecular mechanisms involved in colorectal cancer develop- Current address for R. Quaranta: Novo Nordisk s.p.a., Clinical Operations, Rome, Italy. ment and progression is imperative for the improvement of therapeutic approaches. Corresponding Authors: Diana Bellavia, Sapienza University, Viale Regina Elena Interestingly, recent studies have revealed that a sustained 291, 00161 Rome, Italy. Phone: 396-4470-0816; Fax: 396-4925-5671; E-mail: [email protected]; and Saula Checquolo, [email protected] activation of b-catenin/TCF is responsible for transcriptional activation of Notch-ligand Jagged1, resulting in an upregulation Cancer Res 2019;79:5575–86 of Jagged1 that is required for tumorigenesis in the intestine (9). doi: 10.1158/0008-5472.CAN-19-0145 High-expression levels of Jagged1 are associated with increased 2019 American Association for Cancer Research. progression, metastatic potential, recurrence, and poor prognosis

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in several human malignancies, as prostate, renal, head and neck nologies). The media were renewal 2 to 3 times per week. Cells cancer and colorectal cancer (10–13). The commonly accepted recovered from frozen aliquots were allowed one passage to reach scenario is based on the idea that Jagged1 ligand is able to exponential growth phase following recovery before being used. contribute to tumorigenesis by activating canonical Notch Cells at passages greater than 10 were not used. signaling (14). An opportune amount of cells was treated with different Jagged1 belongs to the Delta, Serrate, Lag-2 (DSL) family of compounds: 50 mmol/L TAPI-2 (# 55123-66-5; Peptides Interna- single-pass transmembrane ligands, including Delta-like (DLL1, 3 tional Inc.), 200 ng/mL of Phorbol 12-myristate 13-acetate and 4) and Jagged (Jagged 1 and 2) that transactivate the Notch (#P8139, Sigma-Aldrich), 30 mmol/L di U0126 (#662005, Cal- receptors (Notch1–4) in signal-receiving cell (14), through a biochem), with 5-fluorouracil (5FU; #F6627, Sigma-Aldrich) or direct contact. Receptor/ligand interaction renders Notch suscep- irinotecan (#134760, Sigma-Aldrich). tible to proteolytic processes mediated by A-Disintegrin Metallo- protease ADAM-10 and PS/g-secretase protein complex, which Cell-cycle cytofluorimetric analysis ends in the release of its intracellular domain (Notch-ICD). A total of 1 106 HCT15 cells, treated with TAPI-2 compound Notch-ICD moves into the nucleus where it binds to RBP-Jk or vehicle alone, were fixed for 300 in EtOH 70%, washed in PBS, transcription factor and recruits coactivators to form a transcrip- treated with 100 mg/mL RNase A (cat. #R6513, Sigma-Aldrich) for tion-activating complex to activate several downstream effectors, 150 and then incubated with 10 mg/mL propidium iodide (cat.# such as hairy and enhancer of split (Hes). Aberrant activation of P4170) for 300. The stained cells were analyzed on a FACS-Calibur Notch signaling is frequently observed in many human with CellQuest software (BD Biosciences; ref. 24). cancers (15–17), including colorectal cancer (18). Emerging evidences indicate that Jagged1 is processed in a Plasmid construct and generation of stable cell lines fashion similar to Notch by sequential proteolytic cleavages that For generating cell lines stably overexpressing Jag1-ICD, involve two distinct enzymes: ADAM-17/TACE and PS/g-secretase murine Jag1-ICD cDNA was amplified by RT-PCR (Supplemen- complex, ultimately resulting in the release of a nuclear-targeted tary Table S1) and cloned into pcDNA 3.1/V5-His TOPO TA intracellular domain (Jag1-ICD), that may play an important role Expression Kit (#KJ48001-01, Invitrogen by Life Technologies) in tumor development and carcinogenesis (19–21), possibly by following the manufacturer's instructions. V5-Jag1-ICD plas- interacting and/or empowering the activation of other deregu- mid or pcDNA3-Neo was used to transfect HCT15 cell line using lated signaling pathways (22, 23). Lipofectamine 2000 (Life Technologies), according to the man- In this article, we demonstrate that the function of Jagged1 may ufacturer's instructions. Forty-eight hours post-transfection, the go beyond its effect on canonical Notch activation in colon cells were cultured in selection medium containing 800 ng/mL malignancies. Indeed, we observed that in colorectal cancer cells neomycin (#A1720, Sigma-Aldrich) for 4 weeks. pBABE-PURO with Kras activation, the Jagged1 ligand is not only abundantly (#1764) and pBABE K-RAS 12V (#12544) retroviral constructs expressed, but it undergoes a constitutive processing that ends in were purchase from AddGene. Phoenix packaging cells were the aberrant generation of an intracellular fragment (Jag1-ICD), transfected with retroviral vectors by Lipofectamine 2000. After capable to move into the nucleus and to induce intrinsic reverse 48 hours of incubation at 32C, the supernatants containing viral signaling, exerting regulatory effects on colorectal cancer particles were collected and infection of CCD18-Co cells was tumor biology. A Kras/Erk/ADAM17 axis constitutively triggers performed, by using a 2 mgr/mL of Polybrene. Stable clones were Jag1-ICD nuclear accumulation, which favors tumor develop- obtained by using 1.5 mgr/mL for puromycin for one week. ment, progression, and chemoresistance through a noncanonical mechanism. RT-PCR/qRT-PCR Total RNA extraction and reverse transcription-PCR (RT-PCR) were previously described (25, 26). One mg of RNA was processed Materials and Methods for RT-PCR using SensiFAST cDNA Synthesis Kit (Bioline). Anal- Animals ysis of gene expression was realized by qPCR using Taq-Man The 6-week-old female CD1 nude mice were purchased from designed assays (Supplementary Table S1; Dharmacon Inc.) on Charles River Laboratories Italia s.r.l. and were housed in the the StepOnePlus Real-Time PCR System (Applied Biosystems, Life Institute's Animal Care Facilities. Technologies), following the manufacturer's protocol for the All animal experiments were approved by local ethic author- comparative Ct method. Data were analyzed by the DDCt method ities and conducted in accordance with Italian Governing Law and GAPDH was used for normalization (27). (D.Lgs. n.26/2014/Protocol Number: C1368.4) and European Directive 2010/63/UE RNA interference analysis RNA silencing was performed using 100 nmol/L of Jagged1 Cell lines and treatments (cat. #L-011060-00-0005) or Kras (cat. #L-005069-00-0005) The following human colon cell lines CCD18-Co (CRL-1459), ON-TARGET plus SMART pool small interference RNA (siRNA) HT29, HCT15, DLD1, HCT116, LS174T, LoVo, RKO, SW1116, or scrambled (cat. #D-001810-10-20, Dharmacon Inc.), using and SW948 were purchased from the ATCC. Cell lines were Lipofectamine RNAiMAX (Life Technologies), according to the subjected to routine cell line quality controls (e.g., morphology, manufacturer's instructions. Mycoplasma #G238, Abm Inc.) and authenticated by DNA pro- filing (short tandem repeat, STR) by the cell bank prior to Protein extracts, subcellular fractioning, immunoprecipitation, shipping. The culture media were supplemented with 1% Gluta- and immunoblotting mine (ECB3000D, Euroclone), 1% Antibiotics (ECB3001D, Whole-cell extract (WCE; ref. 28), extracellular shed protein Euroclone), and 10% regular FBS (Heat-Inactivated; Life Tech- preparations (29), subcellular fractioning (30), and immunoblot

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assay with the described antibodies (Supplementary Table S2; pre-coated with BD Matrigel matrix (BD Biosciences; ref. 37). The ref. 31) were performed as described elsewhere. Bound antibodies invading cells were fixed with PFA 4%, rinsed with PBS, permea- were detected with enhanced chemiluminescence (ECL kit, bilized with EtOH 100%, stained with 1% Crystal Violet and Amersham, GE Healthcare). To perform immunoprecipitation photographed. Cells were quantified as the average number of assay (32), an equal amount of WCE derived from HCT15 or cells found in five random microscopic fields in three indepen- DLD1 cell lines, treated with the opportune dose of phorbol dent inserts. 12-myristate 13-acetate (PMA) or vehicle, were precleared with Protein A-Agarose (cat. #sc-2001; Santa Cruz Biotechnology); Animal studies immunoprecipitation assay was realized with ADAM17 anti- To establish xenograft tumors, 1 107 HCT15 cells, stably body (Supplementary Table S2) or normal IgG (cat. #sc-2027; transfected with V5-Jag1-ICD expressing vector or negative con- Santa Cruz Biotechnology) overnight at 4C. The complexes were trol, were, respectively, injected subcutaneously into right and left precipitated with Protein A–Agarose, and the post-transductional dorsal flank of CD1 nude mice (n ¼ 6). Conversely, 2 106 DLD1 modifications were evaluated by using anti–phospho-serine cells were injected subcutaneously into the hind leg of 6-week-old antibody (Supplementary Table S2; ref. 33). CD1 nude female (n ¼ 6). When tumor reached a mean volume of 150 mm3, the animals were randomly separated into different Immunohistochemistry groups and treated, respectively, with 5FU at 40–50 mg/kg/ Tissues were fixed in 4% formalin and paraffin embedded. 2–3 days intraperitoneally (n ¼ 4), U0126 25 mmol/kg/ Consecutive sections (2-mm-thick) were stained with hematoxy- 2 days intraperitoneally (n ¼ 4) and TAPI-2 at 2 mg/Kg/ lin and eosin. Immunocytochemical assay was performed using 2 days oral gavage (n ¼ 6), dissolved in 0.2 mL of saline solution. an anti-Jagged1 antibody (Abcam; Supplementary Table S2). The control group received injection/oral gavage of vehicle Detection was carried out with Mouse-to-Mouse HRP (DAB) alone. After 27 days, mice were killed and tumors were excised. staining system (ScyTek Laboratories), according to the manu- Tumor size was measured every 3/4 days with a caliper and facturer's instructions. Images were acquired with a Leica volume was calculated according to the formula: length DM1000 microscope equipped with a ProgRes Speed XTcore 3 width 0.5 (lengthþwidth; ref. 38). Harvested tumor tissues CCD camera and collected using ProgRes CapturePro 2.8 software were subjected to RNA and WCE extraction as described. (Jenoptik Optical Systems GmbH; ref. 34). In silico analysis of colorectal cancer patients' deposited data Chromatin immunoprecipitation Samples from the following cohorts: 545 patients with colo- Chromatin immunoprecipitation (ChIP) was performed as rectal cancer (GEO ID: gse3958283; ref. 39) and 83 patients with described earlier (35). One mg of specific antibodies (Supplemen- colorectal cancer (GEO ID: gse28702; ref. 40) were selected and tary Table S2), or normal IgG (cat. #sc-2027, from Santa Cruz analyzed for the Jagged1 gene expression levels. The expression Biotechnology) was used for immunoprecipitation. In silico anal- values of Jagged1 were filtered in each analysis using the expres- ysis using MatInspector (Genomatix Software GmbH, Munich, sion probe set 209099_x_at. The expression value of Jagged1 is Germany) allowed us to identify predicted binding sites for RBP- given in log2 scale after normalizing data with rma and mas5.0 Jk on human snail1 and snail2 promoters, racing from 1,860 to normalization. GraphPad Prism 6 was used for statistical analysis 1,847 for snail1 and from 5,091 to 5,087 for snail2 (Sup- and P values were calculated using Student t test and one-way plementary Table S1). ANOVA, where appropriate.

Cell growth and soft agar assays Statistical analysis HCT15 cells stably transfected with V5-Jag1-ICD–expressing All results were confirmed in at least three independent vector or pCDNA3-Neo control were plated in 96-well plate experiments and all quantitative data were reported as the (5,000 cells/well) and the MTT solution (Sigma-Aldrich) was mean SD. Student t or ANOVA tests for unpaired samples were used as described elsewhere (36). Spectrophotometric absor- used to assess differences among groups. A P value of <0.05 was bance at 570 nm wavelength was determined by GloMax-Multi considered statistically significant (n.s., nonsignificant, P > 0.05; Detection System (Promega). Colony formation assay was per- , P < 0.05; , P < 0.01; , P < 0.001, and , P < 0.0001). formed by using a 6-well plate pre-coated with 1% of soft agar SeaKEM LE Agarose (LONZA) dissolved in medium, supplemented by 1X glutamine, 1X antibiotics, 20% FBS and 800 ng/mL of Results neomycin. The 3000 cells/mL were plated on the upper layer Jag1-ICD is expressed and localized into the nucleus of (0.7% agarose dissolved in medium plus 1X glutamine, 1X colorectal cancer cell lines antibiotic, 20% of FBS and 800 ng/mL of neomycin). This top On the basis of the observation that Jagged1 transcripts are layer was covered by 1 mL of complete medium. The cell colonies overexpressed in a large number of human colorectal cancers, were fixed with 10% Methanol/10% Acetic Acid for 100 and then while they are undetectable in the adjacent normal tissue (41, 42), stained with a 0.005% Crystal Violet (Sigma-Aldrich). we monitored the expression of Jagged1 transcripts in several human colorectal cancer cell lines by qRT-PCR assays. Accordingly, Wound-healing and invasion assays we found a significant upregulation of Jagged1 mRNA in most Cell migration was analyzed by wound-healing assay. Briefly, colorectal cancer cell lines, compared with the normal colon cell an opportune amount of cells were grown in 6-well plates. Wound line CCD-18Co (Fig. 1A), being HT29 and RKO cells the only injury was made with the tip of a sterile micropipette and cells exceptions. It is well demonstrated that the transmembrane Jag1-FL were allowed to migrate for up to 48 hours. In vitro invasion assay undergoes ADAM17-mediated ectodomain processing, resulting was performed using a 24-well Transwell insert (8-mm-pore size) in the Jag1-ECD shedding, followed by PS/g-secretase–dependent

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Figure 1. Jagged1 expression and constitutive processing in colorectal cancer cell lines. A, qRT-PCR analysis of jagged1 gene expression in normal colon (CCD-18Co) and several colorectal cancer cell lines. Gene expression normalized relative to human GAPDH and is depicted as fold change to CCD-18Co. Data are presented as mean SD. , P < 0.05; , P < 0.01 (Student t test). B, Representative immunoblots of Jag1-FL and Jag1-ICD in WCE of colorectal cancer cell lines. Protein levels normalized relative to b-actin. C, Representative immunoblots of Jag1-ICD protein translocation to the nucleus. Protein levels normalized relative to Lamin B in the nuclear fraction and a-tubulin in the cytoplasmatic fraction. All data are representative of at least three independent experiments, each in triplicate.

intramembrane proteolysis that releases an intracellular fragment shows a strong positivity for Jagged1 immunostaining in (Jag1-ICD; refs. 19–21, 23). Intriguingly, here we provide the first human primary colon cancer specimens and this is consistent evidence of a Jag1-FL aberrant processing in colorectal cancer cell with our preclinical data. lines, which ultimately results in the release of a remarkable Overall, these data indicate that the constitutive expression amount of Jag1-ICD (Fig. 1B), able to translocate into the nucleus, of Jag1-ICD enhances the tumorigenic behavior of colorectal as revealed by subcellular protein fractionation (Fig. 1C). Notably, cancer cells, suggesting that Jag1-ICD possesses an intrinsic onco- as suggested by Supplementary Fig. S1, Jagged1 is strongly genic activity. expressed/processed only in colorectal cancer cell lines presenting simultaneously APC-b-catenin/Kras. Jag1-ICD affects EMT directly controlling the expression of Snail1 and Snail2 Jag1-ICD enhances colorectal cancer cells tumorigenicity via an So far, our results support an intrinsic oncogenic activity of intrinsic oncogenic activity Jag1-ICD, possibly impinging on an invasion/migration pheno- Because Jag1-ICD might have a role in tumor development and type, which is typically associated with epithelial–mesenchymal carcinogenesis (20, 23), we explored whether its overexpression transition (EMT). This is consistent with in silico analysis of a might affect oncogenic properties of colorectal cancer cells. We public dataset (40), which reveals increased Jagged1 expression in found that Jag1-ICD ectopic expression in HCT15 cells (Supple- patients with metastatic colorectal cancer compared with primary mentary Fig. S2A), which express low levels of endogenous tumors (Fig. 3A). Because PMA is known to support EMT with Jagged1 (HCT15-V5Jag1-ICD), determined a significant increase effects on cell migration and tumor formation in colorectal cancer in cellular proliferation, as revealed by the MTT assay (Fig. 2A), cells (43), we assessed the potential role of Jag1-ICD in this induced an increased clonogenic capacity in soft agar colony context. Noteworthy, PMA-treated colorectal cancer cell lines formation assays (Fig. 2B) and sustained cell invasion activity readily acquired a spindle-shaped morphology consistent with in vitro, using Transwell inserts (Fig. 2C). Interestingly, Jag1-ICD mesenchymal transition (Fig. 3B), associated with a strong upre- overexpression was also able to sustain colorectal cancer cells gulation of snail1 and snail2, vimentin and N-cadherin and a invasion/migration ability, as demonstrated by wound-healing downmodulation of E-cadherin observed at the mRNA and/or assays (Fig. 2D). This was associated with an increased expression protein levels (Fig. 3C and D). Interestingly, immunoblotting of invasion-related snail and mmp9 genes, as revealed by qRT-PCR also revealed a time-dependent increase of cleaved Jag1-ICD in (Fig. 2E). PMA-treated HCT15, SW948 and DLD1 cells (Fig. 3D). Altogether To further validate these in vitro results, we xenografted HCT15- these observations support a correlation between Jag1-ICD V5Jag1-ICD- or pcDNA3-Neo empty vector-transfected HCT15 accumulation and PMA-induced EMT in colorectal cancer cell cells, into nude mice. Twenty-seven days after injection, we lines. Indeed, siRNA-mediated Jag1 depletion (Supplementary found that Jag1-ICD expressing clones generated larger tumors Fig. S2B) significantly compromised the migratory activity of when compared with control cells (Fig.2FandG).Importantly, Jag1-silenced HCT15 cells both under basal (DMSO; decreased this was associated to an increased expression of mmp9, snail1, by 40%) or PMA-induced conditions (decreased by 30%; snail2, cyclin D2, and PCNA transcripts in Jag1-ICD tumors, Fig. 3E) and significantly impaired snail mRNA expression when compared with controls (Fig. 2H). In addition, Fig. 2I (Supplementary Fig. S2C). We previously demonstrated that

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Figure 2. Jag1-ICD is required to strengthen tumorigenic behavior of colorectal cancer. HCT15 cells stably expressing the intracellular domain of Jag1 (V5-Jag1-ICD) or control vector (pcDNA3-Neo) were used in vitro and in vivo experiments. A, Cell viability of HCT15 V5-Jag1-ICD and HCT15 pcDNA3-Neo analyzed by MTT assay and graphed as fold changes SD versus control. B, Left, representative image of HCT15-V5-Jag1-ICD and control after performing soft agar assay and subsequently Crystal Violet staining. Right, the number of colonies is graphed as fold of changes SD versus control. C, Left, Matrigel assay for HCT15-V5-Jag1- ICD and control. Right, the amount of invading cells is graphed as percentage of total cells. Scale bar, 50 mm. D, Representative area for wound-healing assay of HCT15-V5-Jag1-ICD cells in respect to the negative control shown after 24 and 48 hours of scratch. Scale bar, 200 mm. E, qRT-PCR analysis of mmp9 and snail1 mRNA in HCT15-V5-Jag1-ICD cells compared with control. Data are reported as fold changes SD after intrasample normalization to the level of GAPDH. F, Representative group of CD1/nude mice used for xenograft tumor formation deriving from subcutaneous ﬂank injection of 1 107 stably transfected HCT15 V5- Jag1-ICD or control cells, at the end point of the experiment. G, Top, the volume measure of xenografted tumors derived from F is graphed. Bottom, representative tumor masses derived from F. H, RNA extracted from snap-frozen xenografts from G and analyzed by qRT-PCR for the expression of cell proliferation markers (PCNA, cyclin D2) and metastatic markers (mmp9, snail1, and snail2). I, Representative histologic pictures of human colonic cancers showing strong positivity of immunohistochemistry for Jagged1. Data are reported as fold changes SD after intrasample normalization to the level of GAPDH. All data are representative of at least three independent experiments, each in triplicate. , P < 0.05; , P < 0.01; , P < 0.001. ns, nonsigniﬁcant.

Jag1-ICD directly interacts with CSL/RBP-Jk transcription factor, around these sites showed a significant recruitment of CSL/RBP-Jk sustaining its transcriptional activation (23). Sequence analysis and Jag1-ICD in PMA-treated HCT15 cells (Fig. 3F). of the human snail1 and snail2 promoters identified consensus Overall, these findings demonstrate that nuclear accumulated CSL/RBP-Jk–binding sites (Supplementary Fig. S2D). ChIP assays Jag1-ICD directly controls the expression of EMT-related genes

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Figure 3. PMA-dependent Jagged1 activation induces EMT. A, jagged1 gene expression levels in primary and patients with metastatic colorectal cancer by an in silico analysis using the probe set 209099_x_at, in a cohort of 83 patients with colorectal cancer (metastasis, n ¼ 27; primary, n ¼ 56; GEO ID: gse28702). Data are presented as log2 scale. Each dot represents a patient. , P < 0.01 (Student t test, one-way ANOVA). B, HCT15, SW948, and DLD1 cells are treated with PMA or DMSO for 4 hours. Representative picture of plate area shows the tapered shape in PMA-treated cells in respect to control. Scale bar, 20 mm. C, qRT-PCR analysis of snail1, snail2,andE-cadherin mRNAs expression in PMA-treated cells. Data are reported as fold changes SD versus DMSO control and normalized against the level of GAPDH. , P < 0.05; , P < 0.01 (Student t test). D, Representative Western blots of Jag1-ICD, Snail, vimentin, and N-cadherin in PMA-treated cells along a time course. Protein levels normalized relative to a-tubulin. E, Left, representative picture of plate area for wound-healing assay shown after 24 hours of scratch in HCT15 cells silenced for Jagged1 or scramble control, upon PMA treatment. The dash lines show the front. Scale bar, 200 mm. Right, the percentage of covered scratched area was graphed as mean SD for each group of treatment. , P < 0.001 (Student t test, one-way ANOVA). F, ChIP of endogenous Jag1-ICD and RBP-Jk from HCT15 cells treated or not with PMA for 4 hours, followed by PCR analysis for snail1 promoter (pSnail1) and snail2 promoter (pSnail2). All data are representative of at least three independent experiments, each in triplicate.

and the migratory activity of colorectal cancer cells, unveiling a before the cleavage of Jag1-ICD by the PS/g–secretase com- tight link between aberrant Jagged1 processing and colorectal plex (19). It is known that PMA enhances ADAM17 sheddase cancer aggressiveness. activity, by directly inducing Erk kinase phosphorylation and activation (44, 45), which is an important prerequisite for Kras/Erk/ADAM17 signaling axis induces the constitutive ADAM17 triggering (44, 46). activation of Jag1-ICD in colorectal cancer tumors First, to assess the phosphorylation status of ADAM17 upon Jagged1 is a substrate of the catalytic activity of ADAM17 that PMA treatment in colorectal cancer cells, we carried out immu- allows the shedding of Jag1-ECD ectodomain, an obligatory step noprecipitation assays of endogenous proteins from DLD1 cell

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Figure 4. Kras-mediated ADAM17 activity triggers a constitutive Jagged1 processing. A, Representative immunoblots of pan-phospho-serine for ADAM17- immunoprecipitated in DLD1 cells, treated with PMA or control, for 150. Protein levels normalized to total ADAM17. B and C, Representative Western blots of Jag1- ECD, Jag1-ICD, pErk, and total Erk in HCT15, LoVo, SW948, and DLD1 cells treated with PMA (B), U0126 (C), or control for 4 hours. Protein levels normalized relative to a-tubulin. D, jag1 gene expression levels obtained by an in silico analysis, using the probe set 209099_x_at, in a cohort of 545 patients with colorectal mut wt cancer (Kras , n ¼ 217; Kras , n ¼ 328; GEO ID: gse39582). Data are presented as log2 scale. Each dot represents a patient. , P < 0.01 (Student t test, one-way ANOVA). E, Representative immunoblots of Kras, Jag1-ECD, and Jag1-ICD in HCT15, SW948, and DLD1 cell lines transiently transfected with Kras siRNA or scramble control for 48 hours. Protein levels normalized relative to a-tubulin. All data are representative of at least three independent experiments, each in triplicate. F, Representative picture of plate area shows the tapered shape in CCD18-Co infected with pBABE-KRAS 12V, then negative control (pBABE). Scale bar, 10 mm. G, Representative immunoblots of Jag1-ICD and pERK in CCD18-Co infected with pBABE-KRAS 12V or negative control (pBABE; left) after 48 hours of TAPI-2 treatment (right). Protein levels normalized relative to a-tubulin.

line. As shown in Fig. 4A, we revealed a rapid induction of Ser- processing, revealed by a significant decrease in Jag1-ECD shed- phosphorylation on ADAM17 within 15 minutes of stimulation. ding and Jag1-ICD release, strongly suggesting a direct link Then, we investigated the effects of PMA on ADAM17 sheddase between Kras activity and Jagged1 processing (Fig. 4E). Consis- activity, by monitoring Jagged1 cleavage in colorectal cancer cell tently, the overexpression of mutant Kras (pBabe Kras 12V), by lines. Interestingly, PMA treatment induced extensive Jagged1 retroviral infection of CCD18-Co cells (CCD18-Co-Kras cell line), processing, revealed by a significant increase of soluble Jag1-ECD causes a drastic change in cell morphology with the appearance of and Jag1-ICD fragments in HCT15, LoVo, SW948 and DLD1 spindle-shaped cells, compared with empty backbone infected colorectal cancer cell lines with different expression levels of cells (pBabe-Puro; Fig. 4F). Notably, pERK was strongly induced Jagged1, associated to an important Erk activation (Fig. 4B). by Kras in CCD18-Co-Kras cell line, which triggers Jag1-ICD Consistently, Erk inhibition via the U0126 antagonist strongly release, compared with CCD18-Co-Puro cells (Fig. 4G, left). Of impaired Jagged1 processing, indicating that Jag1-ICD accumu- note, the Kras-induced Jag1-ICD processing was inhibited by lation is Erk-dependent (Fig. 4C). Notably, ectopic Jag1-ICD, TAPI2 compound (Fig. 4G, right). stably transfected in HCT15-V5Jag1-ICD cells, is sufficient to Altogether these results highlight a Kras/Erk/ADAM17/Jagged1 revert the effect of U0126, as revealed by the sustained activation signaling axis in colorectal cancer cells, whereby Kras activation of EMT-linked target genes, as snail and E-cadherin (Supplemen- leads to Erk-ADAM17-dependent Jagged1 cleavage, resulting in tary Fig. S2E and S2F). In silico analysis of a public dataset (39), the nuclear accumulation of Jag1-ICD. considering a large cohort of patients with colorectal cancer, showed that increased expression of Jagged1 transcripts is signif- Pharmacological inhibition of Jag1-ICD activation impairs icantly associated to Kras mutation-bearing samples compared proliferation and invasiveness of Krasmut colorectal cancer cells with Kras wt tumors (Fig. 4D). Moreover, it is reported that To explore the role of Jag1-ICD in sustaining the tumorigenic oncogenic Kras is able to regulate ADAM17 activity in a MEK/ potential of colorectal cancer cells, we abrogated constitutive ERK-dependent manner (7). Interestingly, Jagged1 is strongly Jagged1 cleavage in HCT15 cells by using the TAPI-2 compound, processed only in colorectal cancer cell lines bearing Kras muta- which is able to inhibit ADAM17 activity (Fig. 5A, left). TAPI-2 tions (Fig. 1B; Supplementary Fig. S1). These observations sup- treatment impaired HCT15 cell growth by 40%, as determined by port the existence of a direct correlation between the aberrant trypan blue cell counting (Fig. 5A, right), associated to a G0–G1 activation of the Kras/Erk pathway and the Jagged1 processing in cell-cycle arrest (Fig. 5B). This was associated to the decrease of the colorectal cancer cells. To clarify this correlation, we investigated endogenous cyclin D2 and PCNA transcripts, as revealed by qRT- the status of Jagged1 protein in response to siRNA-mediated Kras PCR, in TAPI-2–treated when compared with control cells depletion in HCT15, SW948 and DLD1 colorectal cancer cell (Fig. 5C). Consistent with previous results, the inhibition of lines. Kras silencing resulted in a marked impairment of Jagged1 Jag1-ICD release by TAPI-2 significantly decreased HCT15

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Figure 5. Jag1-ICD sustains colorectal cancer proliferation and invasion. The HCT15 cell line was treated with 50 mmol/L of TAPI-2 or vehicle for 48 hours. A, Left, representative immunoblots of Jag1-FL and Jag1-ICD used as control for TAPI-2 treatment. Protein levels normalized relative to a-tubulin. Right, cell growth of HCT15 treated or not with TAPI-2 graphed after Trypan blue staining. Quantiﬁcation depicted as percentage of total cell population SD (error bars) of three independent experiments performed in triplicate. B, Histogram shows the percentage of HCT15 cells treated

with TAPI-2 or EtOH in G0–G1-S-G2–M cell-cycle phases. C, qRT-PCR analysis of PCNA and cyclin D2 mRNA in HCT15 cells treated with TAPI-2 compound compared with control. Gene expression depicted as fold change to vehicle alone after intrasample normalization to the level of GAPDH. D, Left, Matrigel assay for HCT15 treated with TAPI-2 or CTR. Scale bar, 50 mm. Right, the amount of invading cells is graphed as the percentage of total cells. E, qRT-PCRanalysisofmmp9, snail1, and snail2 mRNA showing their reduction in HCT15 cells treated with TAPI-2. Gene expression depicted as fold change to vehicle alone after intrasample normalization to the level of GAPDH. F, Left, representative picture of plate area for wound-healing assay shown after 24 and 48 hours of scratch in HCT15 cells treated with TAPI-2 compound, PMA, or combination. The dash lines show the front. Scale bar, 200 mm. Right, the percentage of covered scratched area after 48 hours was graphed as mean SD for each group of treatment. G, The volume measure of xenografted tumors derived from 2 106 DLD1 injected in the posterior ﬂank of CD1/nude mice treated with vehicle control or TAPI-2 is graphed. H, Representative tumor masses derived from G. I, WCE derived from H were immunoblotted for Jag1-ICD. The amount of total extracts normalized in respect to the a-tubulin. All data are representative of at least three independent experiments, each in triplicate. , P < 0.05; , P < 0.01; , P < 0.001. ns, nonsigniﬁcant.

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invasiveness through the Matrigel (56%; Fig. 5D) and reduced the expression levels of endogenous Jag1-ICD. To confirm such expression of invasion-related transcripts such as mmp9, in vitro results, we xenografted DLD-1 cells into nude mice, treated snail1, and snail2 (Fig. 5E). In addition, we carried out wound- with 5FU or U0126 alone or in combination (5FU/U0126) and healing assays to determine the biological effect of Jag1-ICD on the tumor growth was measured along the time (Fig. 6G). A the migration capability of HCT15 cells, treated with PMA alone significant reduction of Jag1-ICD levels was observed in tumors or co-treated with TAPI-2 compound (Fig. 5F). Notably, treatment treated with U0126 alone, associated to a drastic reduction of with PMA alone strongly determined a time-related increased tumor growth (Fig. 6H and I), with respect to vehicle-treated mice. motility (increased by 40%), when compared with control. As expected, no significant difference was found in the tumor size Intriguingly, PMA effect is delayed in presence of TAPI-2 com- from mice treated with 5FU alone, which further increases the pound (decreased by 30%), which counteracts the PMA-induced release of endogenous Jag1-ICD, when compared with control Jag1-ICD shedding, indicating that Jag1-ICD release is required mice (Fig. 6G–I), sustaining the idea that 5FU is able to induce for colorectal cancer cell migration. Of note, TAPI-2 treatment colorectal cancer resistance by inducing Jag1-ICD shedding. On does not have any impact on HCT15-V5Jag1-ICD cells, expressing the basis of the compelling in vitro evidences, U0126 is not able to Jag1-ICD constitutively, revealed by sustained expression levels of completely counteract the 5FU-dependent Jag1-ICD increase in EMT-target genes (Supplementary Fig. S2G and S2H). To inves- tumors from mice with a combined treatment, 5FU/U0126 tigate the effects of Jag1-ICD on colon cancer in vivo, DLD1 cells (Fig. 6G–I). were injected subcutaneously into the flanks of nude mice, which Overall these data demonstrate that 5FU and irinotecan are were treated with TAPI-2 or control vehicle. The results in Fig. 5G able to strongly sustain the Jagged1 processing, by triggering the and H show that tumor volume was clearly decreased in TAPI-2 Erk/ADAM17 axis, which results in the release of the Jag1-ICD treated with respect to control mice. Western blot results from oncogenic fragment, able to confer chemoresistance to colorectal tumor xenografted samples showed that the expression levels of cancer, both in vitro and in vivo. Jag1-ICD were markedly decreased in samples obtained from mice treated with TAPI-2 (Fig. 5I). Altogether these results indicate that Jag1-ICD plays a role in Discussion regulating malignant features, such as proliferation and invasion/ The Notch ligand Jagged1 is upregulated in a large number of migration ability in colorectal cancer cell lines both in vitro and cancers, where it plays a key role in cell growth, EMT and in vivo. metastatic process (10). An increased expression of Jagged1 has been identified in about 50% of human colorectal cancer (41) Jag1-ICD activation confers chemoresistance in Krasmut where it has been correlated with poor prognosis and recur- colorectal cancer cells rence (13). To date, the most widely accepted scenario suggest Kras mutation is an important predictor of drug resistance in that the increased expression of Jagged1 ligand identified in several cancers and is associated with a worse prognosis (8). colorectal cancer triggers an overactivation of Notch signal- Notably, it is reported that chemotherapy results in a significant ing (42, 49). However, Jagged1 may be processed in a fashion increase of ADAM17 activity and growth factors shedding, which similar to Notch receptor, ultimately resulting in the release of the determine drug resistance in Krasmut colorectal cancer tumors nuclear-targeted intracellular domain Jag1-ICD, thus triggering a (4, 47). Chemoresistance is often associated to acquisition of reverse signaling (19, 23). Herein, we demonstrate that Jag1-ICD EMT (48), the phenotype induced in colorectal cancer cells by the is able to empower the Kras-mediated oncogenic signaling, by Kras/Erk/ADAM17/Jag1-ICD axis that we described above. Inter- sustaining features of malignancies, tumor-cell invasion, migra- estingly, it is reported that high Jagged1 expression levels, com- tion, and resistance to chemotherapy. bined with low E-cadherin expression, in cancer cells of patients Previous data revealed that more than one oncogenic "driver" with colorectal cancer are correlated with poor prognosis, poorer is deregulated in colorectal cancer tumors (1). Mutations in the survival rate and increased risk of recurrence (13). Overall these Wnt pathway cause colon cancer through constitutive activation observations allowed us to speculate about a direct link between of the b-catenin/TCF transcription complex (50). Recent reports an enforced Jag1-ICD shedding and the acquisition of resistance. have shown that b-catenin/TCF is responsible of a direct regu- In keeping with this hypothesis, stable Jag1-ICD overexpression lation of Jagged1 expression, which is required for tumorigenesis in HCT15 cells was sufficient to confer resistance to 5FU and in the intestine (9). In addition, gain-of-function mutations in irinotecan agents, as revealed by a sustained survival rate in colo- RAS gene are present in approximately 50% of colon can- rectal cancer cells, with respect to untransfected cells (Fig. 6A). cers (1, 5). Notably, oncogenic Kras signaling increases the To explore the possibility that the resistance to 5FU and/or b-catenin stability, by modulating its phosphorylation at serine irinotecan may depend on Jag1-ICD, we tested the impact of 51) 552 ). Interestingly, increasing evidence suggests that the both chemotherapic agents on Jagged1 processing. Surprisingly, oncogenic Kras mutations control ADAM17 activity and growth treatment of HCT15 cells with 5FU (Fig. 6B) or irinotecan factor shedding, via regulation of MEK/Erk/Adam17 signaling (Fig. 6C) for 24 hours increased the release of Jag1-ICD in a axis (4). These results are supported by the observation that Erk dose-dependent manner, associated to an increased phosphory- activation phosphorylates and associates with ADAM17 (7, 46). lation status of Erk and ADAM17 (Fig. 6B and C) and correlated In agreement with these data, we provide the first evidence that with the modulation of the EMT-specific markers mmp9, snail1, Krasmut colorectal cancer cells specifically show increased expres- snail2, and E-cadherin (Fig. 6D). Notably, 5FU- or irinotecan- sionof Jagged1,whichisconstitutivelyprocessedbyADAM17, in induced Jag1-ICD processing was significantly decreased by a Kras-dependent manner. Of note, we show on one side that TAPI-2 (Fig. 6E) or by U0126 (Fig. 6F) compounds. Interestingly, Kras-silencing attenuates significantly the Jag1-ICD release and Supplementary Fig. S3A–S3E, shows that the effects above on the other side that Kras ectopic expression directly empowers described are also observed in DLD1, a cell line with high the Jagged1cleavage,supportingthe ideathatJagged1processing

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Figure 6. Jag1-ICD confers 5FU/irinotecan resistances in colorectal cancer. A, Proliferation rate of HCT15 cells stably expressing the intracellular domain of Jag1 (V5-Jag1- ICD) or control vector (pcDNA3-Neo) treated with an increasing amount of 5FU or irinotecan. B and C, Representative immunoblot of Jag1-ICD, pERK, total ERK, and pADAM17 in WCE derived from HCT15 cells treated or not with an increasing amount of 5FU (B) or irinotecan (C) for 24 hours. The protein levels normalized respective to a-tubulin. D, qRT-PCR of HCT15 cell line derived from B and C shows the modulation of mmp9, snail1, snail2,andE-cadherin genes. Data are reported as fold changes SD after intrasample normalization to the level of GAPDH. E and F, Representative Western blot of Jag1-ICD, pERK, and total ERK in WCE derived from HCT15 cells treated with 5FU or irinotecan alone or in combination with TAPI-2 (E) or U0126 (F). G, The volume measure of xenografted tumors derived from 2 106 DLD1 injected in the posterior ﬂank of CD1/nude mice treated with vehicle control, 5FU, U0126, or combination. H, Representative tumor masses derived from G. I, WCE derived from H were immunoblotted for Jag1. The amount of total extracts normalized in respect to the a-tubulin. All data are representative of at least three independent experiments, each in triplicate. , P < 0.05; , P < 0.01; , P < 0.001. ns, nonsigniﬁcant.

is a novel substrate of Kras signaling in colorectal cancer cells. ing signiﬁcant decrease of cell growth and reduction of migration Here, we demonstrate that the constitutive Jagged1 cleavage and invasion phenomena, both in vitro and ex vivo,intumor observed in colorectal cancer cells is dependent upon Erk acti- xenografts experiments. Previous observations suggest that vation, able to phosphorylate ADAM17, as revealed by PMA Jag1-ICD may directly interact with RBP-Jk transcription factor stimulation or on inhibition of Erk activity with U0126 com- (23). For the ﬁrst time, we demonstrate that Jag1-ICD is able pound, both in vitro and in vivo experiments. Noteworthy, the to trigger an intrinsic reverse signaling by regulating snail1 and aberrant PMA-induced Jag1-ICD release is associated to a snail2 promoter activity, via CSL/RBP-Jk. Moreover, pre-clinical marked increase of EMT markers Snail, vimentin, N-cadherin, studies, performed by using HCT15-V5Jag1-ICD xenografts and E-cadherin. On the other side, TAPI-2–mediated ADAM17 experiments, sustain the idea that the persistent expression of inhibition correlates with different biological outcomes, includ- Jag1-ICD plays an oncogenic function also in in vivo models.

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Interestingly, Kras mutations are often associated with a colo- finally improve overall tumor control and reduce tumor rectal cancer worse prognosis (4, 7, 8). Of note, Kras status has recurrence. been correlated with Jagged1 expression in patients with colorectal cancer and associated with a poorer survival rate and Disclosure of Potential Conflicts of Interest increased risk of recurrence, characterized by low cadherin No potential conflicts of interest were disclosed. expression and the induction of EMT, but the molecular mechanism is unknown (13). In addition, it has been reported that Authors' Contributions current chemotherapy acutely activates ADAM17 that plays an Conception and design: M. Pelullo, D. Bellavia important role in drug resistance in colorectal cancer Development of methodology: M. Pelullo, F. Nardozza, S. Zema, R. Quaranta, tumors (7, 47). Emerging evidence associates chemoresistance R. Palermo, C. Capalbo with the development of an EMT-like phenotype in cancer Acquisition of data (provided animals, acquired and managed patients, cells (52), suggesting that EMT, metastasis, and chemoresistance provided facilities, etc.): C. Nicoletti, M.P. Felli, C. Capalbo Analysis and interpretation of data (e.g., statistical analysis, biostatistics, are closely related to each other in tumor progression (48). In computational analysis): M. Pelullo, Z.M. Besharat, B. Cerbelli, G. d'Amati, accordance with these observations, we show that the 5FU or C. Capalbo, C. Talora, L. Di Marcotullio, S. Checquolo, I. Screpanti, D. Bellavia irinotecan treatments increase the endogenous Jag1-ICD Writing, review, and/or revision of the manuscript: Z.M. Besharat, B. Cerbelli, release, via Erk phosphorylation, in vitro or in xenografts experi- G. d'Amati, C. Capalbo, G. Giannini, S. Checquolo, I. Screpanti, D. Bellavia ments and are able to induce EMT, as revealed by modulation Study supervision: I. Screpanti, D. Bellavia of endogenous specific markers. Therefore, our data indicate that the constitutive processing of Jagged1, induced by 5FU or Acknowledgments This article is dedicated to the memory of Prof. Alberto Gulino. This work has by irinotecan, could be a crucial event correlated with increased been supported by Italian Ministry of Education, University and Research— risk of recurrence, poor outcome and resistance to chemotherapy Dipartimenti di Eccellenza—L. 232/2016, Associazione Italiana Ricerca Cancro mut of Kras colorectal cancer. (AIRC) Grants # IG20801 (to L. Di Marcotullio), #IG17734 (to G. Giannini); by In conclusion, we provide evidence that Jagged1 is not only Sapienza University Project Num: RP1181643121DD86 (to D. Bellavia); abundantly expressed but is also constitutively processed in RG116154E2C7A6FB and MIUR PNR 2015-2020 ARS01_00432 (to I. colorectal cancer Kras molecular subtype tumors, via a Kras/ Screpanti). Erk/ADAM17 pathway. The release of Jag1-ICD, in turn is able to empower the oncogenic Kras signaling pathway, via a novel The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in mechanism, which sustains invasion and contributes to chemore- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sistance. Therapies targeted at this definite pathway may provide a novel method to sensitize and/or to disrupt the resistance mech- Received January 11, 2019; revised May 17, 2019; accepted September 6, anism of Kras-mutated colorectal cancer to chemotherapy, to 2019; published first September 10, 2019.

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5586 Cancer Res; 79(21) November 1, 2019 Cancer Research

Kras/ADAM17-Dependent Jag1-ICD Reverse Signaling Sustains Colorectal Cancer Progression and Chemoresistance

Maria Pelullo, Francesca Nardozza, Sabrina Zema, et al.

Cancer Res 2019;79:5575-5586. Published OnlineFirst September 10, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-0145

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