ERK1/2 Signaling Induces Upregulation of ANGPT2 and CXCR4 to Mediate Liver Metastasis in Colon Cancer

Published OnlineFirst August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-4028

CANCER RESEARCH | MOLECULAR CELL BIOLOGY

ERK1/2 Signaling Induces Upregulation of ANGPT2 and CXCR4 to Mediate Liver Metastasis in Colon Cancer A C Jelena Urosevic1,2, María Teresa Blasco1,2, Alicia Llorente1, Anna Bellmunt1, Antoni Berenguer-Llergo3, Marc Guiu1, AdriaCa nellas~ 1,2, Esther Fernandez1, Ivan Burkov1, Maria Clapes 1, Mireia Cartana1, Cristina Figueras-Puig1, Eduard Batlle1,2,4, Angel R. Nebreda1,4, and Roger R. Gomis1,2,4,5

ABSTRACT ◥ Carcinoma development in colorectal cancer is driven by genetic cell lines and then tested in clinical samples. The RAS–ERK1/2 axis alterations in numerous signaling pathways. Alterations in the RAS- controlled expression of the cytokine ANGPT2 and the cytokine ERK1/2 pathway are associated with the shortest overall survival for receptor CXCR4 in colorectal cancer cells, which facilitated devel- patients after diagnosis of colorectal cancer metastatic disease, yet opment of liver but not lung metastases, suggesting that ANGPT2 how RAS–ERK signaling regulates colorectal cancer metastasis and CXCR4 are important for metastatic outgrowth in the liver. remains unknown. In this study, we used an unbiased screening CXCR4 controlled the expression of cytokines IL10 and CXCL1, approach based on selection of highly liver metastatic colorectal providing evidence for a causal role of IL10 in supporting liver cancer cells in vivo to determine genes associated with metastasis. colonization. In summary, these studies demonstrate that amplifi- From this, an ERK1/2-controlled metastatic gene set (EMGS) was cation of ERK1/2 signaling in KRAS-mutated colorectal cancer cells defined. EMGS was associated with increased recurrence and affects the cytokine milieu of the tumors, possibly affecting tumor– reduced survival in patients with colorectal cancer tumors. Higher stroma interactions and favoring liver metastasis formation. levels of EMGS expression were detected in the colorectal cancer subsets consensus molecular subtype (CMS)1 and CMS4. ANGPT2 Significance: These findings identify amplified ERK1/2 signaling and CXCR4, two genes within the EMGS, were subjected to gain-of- in KRAS-mutated colorectal cancer cells as a driver of tumor– function and loss-of-function studies in several colorectal cancer stroma interactions that favor formation of metastases in the liver.

Introduction However, recent clinical data emphasize the importance of MAPK signaling not only in primary colorectal cancer development Progression from normal mucosa to carcinoma in colorectal but also in distant tissue colonization (6). OS after diagnosis of cancer is driven by a sequential order of well-defined genetic metastatic disease is shortest for patients with tumors that present alterations that affect the Wnt, MAPK, PI3K, and TGFb signaling alterations in the RAS pathway (7). In addition, having mutations in pathways (1). Alterations in MAPK signaling occur early during KRAS is associated with a higher risk of recurrence in patients after primary colorectal cancer development (1). Activating mutations in surgical resection of liver metastases from colorectal cancer (8–10). KRAS, NRAS,andBRAF, which are part of the RAS-ERK1/2 MAPK The presence of mutations in both KRAS and BRAF genes can signaling cascade, are detected in nearly 50% of colorectal cancer influence the metastatic pattern of colorectal cancer, as patients cases (2–4). In addition, genetic variations in several members of with colorectal cancer who have a KRAS-mutant tumors have as MAPK signaling pathways are associated with the risk of developing well an increased risk of lung recurrence after primary tumor colorectal cancer, as well as with overall survival (OS) after diag- resection (11), whereas those with a BRAF mutations tend to nosis with colorectal cancer (5). develop metastasis to peritoneum and distant lymph nodes (12). Extensive genomic profiling has detected a high level of concor- dance in mutational status between colorectal cancer primary 1Cancer Science Program, Institute for Research in Biomedicine (IRB Barcelona), tumors and matched metastases (7, 13, 14), thus suggesting that The Barcelona Institute of Science and Technology, Barcelona, Spain. 2CIBER- metastatic development is not driven by the acquisition of addi- ONC, Spain. 3Biostatistics and Bioinformatics Unit, Institute for Research in tional mutational events. How RAS–ERK signaling regulates colo- Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technol- rectal cancer metastasis, and how it determines metastatic patterns 4 ogy, Barcelona, Spain. ICREA, Institucio Catalana de Recerca i Estudis Avancats,¸ is still unknown. Barcelona, Spain. 5School of Medicine, Universitat de Barcelona, Barcelona, Spain. Note: Supplementary data for this article are available at Cancer Research Materials and Methods Online (http://cancerres.aacrjournals.org/). Cell culture J. Urosevic, M.T, Blasco, A. Llorente, and A. Bellmunt contributed equally to the article. The colorectal cancer cell lines were maintained in 5% CO2 at 37 C in DMEM (Gibco) supplemented with glutamine (0.29 mg/mL), Corresponding Author: Roger R. Gomis, Institute for Research in Biomedicine, penicillin (100 U/mL), streptomycin (0.1 mg/mL), and either 5% FBS Baldiri i Reixac 10, Barcelona 08028, Spain. Phone: 349-3403-9959, Fax: 349- 3403-9960; E-mail: [email protected] (for the cell lines SW620-P and SW620-LiM2 derivatives from SW620, SW480, and Colo26) or 10% FBS (for the HCT116 cell line); all Cancer Res 2020;80:4668–80 supplements were purchased from Biological Industries. All cell lines doi: 10.1158/0008-5472.CAN-19-4028 were purchased from the ATCC but Colo26 was gift from the Batlle lab. 2020 American Association for Cancer Research. All cell lines were authenticated for KRAS/BRAF mutations and tested

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routinely (biweekly) for Mycoplasma by PCR. Cell lines were not Microarray processing of data from cell lines passaged more than 50 times. Microarray samples from SW620 cell lines were processed using packages oligo from R and Bioconductor. Raw cell files were normal- Inhibitor treatment ized using RMA background correction and summarization at the core Cells (2.5 106) were seeded and treated for 24 hours with the transcript level. Chip probesets were annotated using the information MEK1/2 inhibitors U0126 (10 mmol/L, Cell Signaling Technology) or provided by Affymetrix (details and references in the Supplementary PD0325901 (100 nmol/L, Tocris) in DMEM supplemented with 0.1% Materials and Methods). of FBS. Enrichment analysis of cell line data Lentiviral production ERK1/2-controlled metastatic gene set (EMGS) genes were evalu- 293T cells were used for lentiviral production. Lentiviral vectors ated for pathway enrichment using a hypergeometric test. Gene sets expressing short hairpin (shRNA) against human CXCR4, derived from the Kyoto Encyclopedia of Genes and Genomes (KEGG) ANGPT2, ETV4, or ETV5 from Mission shRNA Library were pathway database as collected in R packages KEGG.db and org.Hs. purchased from Sigma-Aldrich (see sequences in Supplementary eg.db. were used for these analyses. P values obtained from the Material and Methods). hypergeometric test were corrected by multiple comparisons using the Benjamini–Hochberg FDR method (details and references in the Retroviral production Supplementary Materials and Methods). Retroviruses were produced using 293T cells as described previously (15). Transcriptome datasets of whole tumor samples Transcriptome analyses in human colorectal cancer tumors were Animal studies carried out on 1,485 samples that were available in two public The Ethical Committee of Animal Experimentation of the Gov- repositories listed in the Supplementary Materials. Microarray samples ernment of Catalonia approved all animal work (protocol number were processed separately for each dataset using packages affy and 9317). Intrasplenic injections were done as previously reported, and affyPLM from Bioconductor. Raw cel files were normalized using liver metastasis development was followed twice a week by biolumi- RMA background correction and summarization. Standard quality nescence imaging using the IVIS-200 imaging system from Xenogen controls were performed in order to identify abnormal samples. (Living Image 2.60.1 software; ref. 15). Treatment with IL10 antibody Microarray intensities were corrected separately by metrics PM.IQR, or IgG was initiated 7 days postimplantation of the cells and mice were RMA.IQR, and RNA.DEG as described previously. TCGA RNA-seq treated three times per week with 1.5 mg of antibody. For the exper- expression data were downloaded and processed as detailed in the iment using mouse colorectal cancer organoids, treatment with IL10 Supplementary Material (details and references in the Supplementary antibody or IgG was initiated 3 days after implantation of the cells and Materials and Methods). mice were treated three times per week with 5 mg of antibody. Molecular annotation of tumor samples Western blot analysis When not available in the clinical info, microsatellite instability Protein extracts obtained from whole cell lysates (40 mg) were (MSI) status was imputed in each dataset separately based on the fractionated in SDS-PAGE gels, transferred onto Immobilion-P expression of genes included in a published transcriptomic signature. (Millipore) membranes, and subjected to immunoblot analysis Assignation to MSI or microsatellite stable (MSS) was performed (antibody list in Supplementary Data; ref. 15). according to results of a cluster analysis based on nonparametric density estimation on these correlation coefficients. Human colorectal qRT-PCR analysis cancer samples were annotated according to the consensus molecular Real-time qPCR was performed using TaqMan Gene Expression classification, which was publicly available in the Synapse repository Assay (list in Supplementary Data; ref. 15). for most samples (n ¼ 1,300). KRAS and BRAF mutations were annotated using data provided by the PanCanAtlas project (details Histopathology and IHC and references in the Supplementary Materials and Methods). Tissues were dissected, fixed in 10% buffered formalin (Sigma) and embedded in paraffin. Sections (2–3 mm thick) were stained with Association of gene expression with molecular features in tumor hematoxylin and eosin. Antibodies listed in Supplementary Materials. samples Association with relapse was evaluated using a frailty Cox propor- ELISA assays tional hazards model. For association between gene expression levels Five million cells were seeded in 6-cm diameter plates. The medium and consensus molecular subtypes (CMS) or KRAS/BRAF mutation was changed after 24 hours, and plates were incubated with 3 mL of status, a mixed-effects linear model was used using R packages lme4 media for a further 24 hours. Subsequently, supernatants were col- and lmerTest. Statistical significance was assessed by means of a log- lected and analyzed by ELISA to detect IL10 or CXCL1 following likelihood ratio test, while Wald tests were used for pairwise compar- manufacturer's instructions. isons. Sample groups of low, medium, and high expression levels were defined using the tertiles of the corresponding intensity distribution. Datasets used Accordingly, HRs adjusted group means and their corresponding 95% Four publicly-available Affymetrix microarray datasets where confidence intervals were computed as measures of association. Only downloaded from the NCBI Gene Expression Omnibus repository or samples from patients diagnosed to be at stage I, II, or III were The Cancer Genome Atlas (TCGA) repository: GSE33113, GSE14333, considered for analyses of time to relapse, for a total of 955 samples. GSE39582, and the TCGA colon datasets. The SW620 cell populations’ All analyses were carried out using R (details and references in the dataset can be found at GSE142219. Supplementary Materials and Methods).

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2 activity, and whose expression was upregulated in SW620_LiM2 Results cells compared with SW620_P cells (Fig. 1A). This comparison Genes regulated by RAS–ERK1/2 signaling mediate recurrence provided us with a list of 15 genes (1.75 fold-change and 5% FDR), in colorectal cancer which we named the EMGS(Supplementary Table S1). Next, we We have recently shown that KRAS-mutated colorectal cancer interrogated whether the EMGS was associated with disease progres- cells with increased liver metastatic potential (SW620_LiM2) have sion in colorectal cancer. To this end, we used a pooled cohort of 955 increased ERK1/2 activity as compared with the poorly metastatic stage I–III primary colorectal cancer tumors (GSE14333, GSE33113, parental cells (SW620_P) or with lung potential (SW620_Lu) with the GSE39582, TCGA-COAD) and found that high expression of genes same mutational burden (15). To investigate the role of ERK1/2 in within the EMGS was associated with an increased risk of recurrence metastatic colorectal cancer cells, we first treated SW620_LiM2 cells (Fig. 1B) and mainly occurred at stage II of tumor progression with the MEK1/2 inhibitor U0126 and analyzed the resulting tran- (Supplementary Fig. S1). This association was independent of tumor scriptome for genes whose expression was reduced. These genes were MSI (Fig. 1C). Notably, tumors with annotated mutations in the KRAS then compared with those that were upregulated in metastatic and BRAF genes also had higher expression levels of the EMGS SW620_LiM2 cells as compared with parental (SW620_P) cells (15). (Fig. 1D), thus confirming the association of the EMGS with RAS– Using this approach, we identified genes that were controlled by ERK1/ ERK1/2 signaling.

Figure 1. A B CRC primary tumors stages I,II,III EMGS is associated with recurrence (GSE33113+GSE14333+ GSE39582+TCGA-COAD) in colorectal cancer. A, Schematic SW620 Cells depicting the analyses performed. 1.0 Metacohort (n = 955) B and C, Kaplan–Meier curves representing the proportion of recurrence- 0.8 free patients with colorectal cancer LiM2 LiM2 stratified on the basis of mRNA levels DMSO 0.6 of EMGS. D, EMGS expression in patients stratified on the basis of the mutational status of the KRAS and EMGS 0.4 BRAF genes. E, EMGS expression in P = 0.001 patients stratified on the basis of the 0.2 Parental LiM2 EMGS LOW (n = 323) CMS. , P < 0.001. A Wald test was U0126 EMGS MED (n = 310) Proportion recurrence-free

Gene expression levels – EMGS HIGH (n = 322) used in B D comparing high versus low 0.0 groups. Pairwise test (adjusted) in D. 0 5 10 15 C Time to recurrence (years) CRC primary tumors stages I,II,III (GSE33113+GSE14333+ GSE39582+TCGA-COAD)

1.0 MSI Tumors (n = 186) 1.0 MSS Tumors (n = 768)

0.8 0.8

0.6 0.6

0.4 0.4 P = 0.02 P = 0.009 0.2 EMGS LOW (n = 49) 0.2 EMGS LOW (n = 273) EMGS MED (n = 61) EMGS MED (n = 249) Proportion recurrence-free Proportion recurrence-free EMGS HIGH (n = 76) EMGS HIGH (n = 246) 0.0 0.0 024 681012 0 5 10 15 Time to recurrence (years) Time to recurrence (years)

D *** E *** *** *** 6 *** 6 *** *** 4 4

2 2 EMGS Z-score EMGS Z-score 0 0

−2 −2

KRAS WT KRAS WT KRAS MUT CMS1 CMS2 CMS3 CMS4 BRAF WT BRAF MUT BRAF WT

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Recent large-scale gene expression analyses of six independent ANGPT2 and CXCR4 mediate liver metastasis from colorectal classification systems of colorectal cancer tumors resulted in four cancer CMS, whereby each subtype is characterized by specific biological To functionally validate the role(s) of ANGPT2 and CXCR4 in phenotypes and different clinical and prognostic associations (16). We colorectal cancer metastasis, we performed in vivo assays using found increased expression levels of genes in the EMGS in the CMS1 xenograft mouse models. First, we used shRNAs to downregulate and CMS4 subtypes as compared with the CMS2 and CMS3 subtypes ANGPT2 and CXCR4 expression in SW620_LiM2 cells (Supplemen- (Fig. 1E). CMS1(MSI/immune) patients do not differ significantly in tary Fig. S3A), and then injected the cells intrasplenically into immu- either OS or relapse-free survival (RFS) compared with those with nodeficient BALB/c nude mice to follow the kinetics of liver metastasis CMS2 (epithelial/canonical) or CMS3 (metabolic) tumors, but they do by quantitative bioluminescence imaging. Notably, independent or have a significantly shorter survival after relapse than patients with combined downregulation of ANGPT2 and CXCR4 delayed the onset other CMS subtypes (16). However, of all the subtypes, patients with of liver metastasis (Fig. 3A–C; Supplementary Fig. S3C and S3D). In CMS4 (mesenchymal/stroma) tumors have the worst OS and RFS (16). addition, fewer mice injected with ANGPT2/CXCR4-downregulated Notably, the highest EMGS expression levels were detected in CMS4 SW620_LiM2 cells formed liver metastatic lesions (4/10) than in the tumors, which are further characterized by the upregulation of genes control group (9/10; Fig. 3C), and the lesions that formed were involved in the epithelial-to-mesenchymal transition, as well as in significantly smaller (Fig. 3D). We then used retroviruses to over- tumor–stroma crosstalk. Therefore, the gene set regulated by RAS– express both ANGPT2 and CXCR4 (or the empty vector, as a control) ERK1/2 signaling appears to be associated with both recurrence and in the poorly metastatic SW620_P cell line (Supplementary Fig. S3B) poorer prognosis in colorectal cancer. Of note, the same results were and injected these cells intrasplenically into immunodeficient mice. consistently observed when different fold-change thresholds were used Metastasis formation was followed by in vivo imaging and confirmed for selected genes in the EMGS. Next, we studied whether changes in after the mice were sacrificed. Increased expression of ANGPT2 the expression of the EMGS were associated with specific biological and CXCR4 conferred SW620_P cells with an increased ability to functions. Using a hypergeometric test, we searched the KEGG form liver lesions (observed in 8/10 mice) as compared with the control database for pathways significantly enriched in the EMGS. Our results group (1/9; Fig. 3E and F). However, decreased levels of ANGPT2 and show the highest enrichment with the cytokine–cytokine receptor CXCR4 had no significant impact on the lung metastatic potential of interaction pathway (Supplementary Table S2). The same results were SW620_LiM2 cells (Fig. 3G), in agreement with previous reports obtained for various fold-change thresholds in the differential expres- describing a role for p38 MAPK driving lung metastasis in this sion analysis. Focusing on this gene group, we identified five genes model (15). Similarly, lung metastatic potential from the liver of (AREG, CXCR4, KITLG, NGFR, and ANGPT2) that code for cytokines SW620_P cells was not increased upon expression of either CXCR4 or cytokine receptors in the EMGS. Next, we asked whether the or ANGPT2 (Fig. 3G). Finally, we confirmed that increased expression expression of any of these genes was associated with recurrence in of ANGPT2 and CXCR4 did not confer SW620_P cells with the the pooled cohort of 955 stage I–III primary colorectal cancer tumors. capacity to colonize the lungs when injected via tail vein (Fig. 3H). We found that only increased expression of ANGPT2 (angiopoietin-2) Altogether, these data indicate that ANGPT2 and CXCR4 regulate the and CXCR4 (CXC motif chemokine receptor 4) associated with an ability of colorectal cancer cells to colonize the liver. increased risk of recurrence (Fig. 2A). ANGPT2 is a cytokine crucial for blood vessel remodeling and maturation (17), while CXCR4 is a G RAS–ERK1/2 signaling impinges on the cytokine milieu and protein–coupled receptors, which is known to function as coreceptor cross-talk with the stroma for HIV entry (18, 19). These two proteins are upregulated in numer- We next interrogated the molecular mechanisms that underlie ous human tumors (20–28). liver metastasis formation driven by CXCR4 and ANGPT2. These The highest levels of ANGPT2 and CXCR4 expression were found in experiments were performed in nude mice that lack T cells but retain CMS1 and CMS4 colorectal cancer tumors (Fig. 2B). Subsequently, we the innate immune system. As both ANGPT2 and CXCR4 may validated that the expression levels of both ANGPT2 and CXCR4 contribute to tumor angiogenesis (29–32), we first scored the number þ increased in metastatic SW620_LiM2 cells as compared with the of CD31 endothelial cells in liver lesions developed by SW620_P or SW620_P cells (Fig. 2C; Supplementary Fig. S2A and S2B). SW620_LiM2 cells. A comparison of size-matched lesions revealed an þ increased number of CD31 cells in liver metastasis formed by Expression of ANGPT2 and CXCR4 is regulated by RAS–ERK1/2 SW620_LiM2 cells (Fig. 4A). Downregulation of CXCR4 alone in þ signaling in colorectal cancer cells SW620_LiM2 cells did not affect the number of CD31 cells in liver As both ANGPT2 and CXCR4 mediate extracellular signaling, we metastatic lesions (Fig. 4B). However, ANGPT2 depletion alone or in þ asked whether other genes involved in autocrine signaling were also combination with CXCR4 significantly reduced the number of CD31 differentially expressed in metastatic colorectal cancer cells. However, cells in liver metastasis formed by SW620_LiM2 cells (Fig. 4C and D), the CXCR4 agonist CXCL12 (CXCL12), the ANGPT2 receptor suggesting that ANGPT2 plays a major role in the angiogenesis of liver TIE2 gene (TEK), and the VEGFA were expressed at similar levels in metastasis formed by colorectal cancer cells. between SW620_LiM2 and SW620_P cells (Supplementary Fig. S2C– Colon cancer metastasis follows a two-step hierarchical process to S2E). This finding implies that autocrine signaling is not generally the liver and lung, although EMGS and the combine actions of increased in the highly metastatic cells compared with the poorly ANGPT2 and CXCR4 seem to promote growth in the liver but not metastatic cells. in the lung. The function of ANGPT2 and vascularization in general Finally, treatment of SW620_LiM2 cells with the MEK inhibitors has a broad impact on metastasis. We therefore asked whether CXCR4 PD0325901 or U0126, or treatment of a panel of colorectal cancer cell drives liver metastasis specifically. Indeed, SW620_LiM2 cells in which lines (SW620_P, SW480, HCT116, and Colo26) with U0126, revealed only CXCR4 had been depleted (with two independent shRNAs) that ANGPT2 and CXCR4 expression was dependent on RAS–ERK either progressed slowly to colonize the livers or fail to generate signaling (Fig. 2D–F). Hence, we conclude that expression of ANGPT2 metastasis in mice (Fig. 5A); in contrast, CXCR4 depletion did not and CXCR4 in colorectal cancer cells is regulated by ERK1/2 activity. significantly affect lung colonization (Fig. 5B). As CXCR4 knockdown

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A CRC Primary tumors B C Stages I,II,III Metacohort n = 955 SW620_P 14 1.0 P = n.s. SW620_LiM2 25 0.8 12 *** n = 5 *** 10 20 0.6 ** 8 15 0.4 P = 0.0007 6 10

0.2 ANGPT2 LOW (n = 319) expression ANGPT2 MED (n = 327) ANGPT2 Z-Score 4 5 ANGPT2 HIGH (n = 309) Proportion recurrence-free 0.0 2 0 0 5 10 15 CMS1 CMS2 CMS3 CMS4 Relative mRNA ANGPT2 1.0 P = n.s. 14 *** 0.8 *** 25 12 n = 5 ** 0.6 10 20 CXCR4 8 15 0.4 P = 0.02 6 10 0.2 CXCR4 LOW (n = 305) expression CXCR4 Z-Score CXCR4 MED (n = 312) 4 5 CXCR4 HIGH (n = 338) Proportion recurrence-free 0.0 2

Relative mRNA 0 0 5 10 15 CMS1 CMS2 CMS3 CMS4 Time to recurrence (years)

D SW620_LiM2 ANGPT2 E SW620_LiM2 1.2 1.2 ANGPT2 CXCR4 CXCR4 n = 3 0.8 0.8 n = 3 * ** 0.4 0.4 ** expression expression *** ** *** Relative mRNA 0.0 Relative mRNA 0.0 − −− DMSO: + DMSO: + PD0325901 100 nmol/L: − + U0126 (µmol/L): 01020

P-ERK1 P-ERK1 P-ERK2 P-ERK2

ERK1 ERK1 ERK2 ERK2

Tubulin Tubulin

F SW620_P SW480 HCT116 Colo26 1.2 n = 5 n = 5 n = 5 n = 5 ANGPT2 CXCR4 0.8 ** 0.4 *** *** expression *** *** ** *** Relative mRNA 0.0 *** DMSO: + − + − + − + − U0126 10 µmol/L: − + − + − + − + P-ERK1 P-ERK2 ERK1 ERK2

Tubulin

Figure 2. The expression of ANGPT2 and CXCR4 in colorectal cancer cells is regulated by RAS–ERK1/2 signaling. A, Kaplan–Meier curves representing the proportion of recurrence-free patients with colorectal cancer stratiﬁed upon mRNA levels of expression of ANGPT2 (top) or CXCR4 (bottom). B, Levels of ANGPT2 (top) and CXCR4 (bottom) expression in patients stratiﬁed on the basis of CMS. C, Relative mRNA expression levels of ANGPT2 (top) and CXCR4 (bottom) in SW620 parental and LiM2

cells, n ¼ 5. SW620_ Parentals are for CXCR4 mRNA, Ct ¼ 30.5 and for ANGPT2 mRNA, Ct ¼ 29.8, whereas in SW620_LiM2 are for CXCR4 mRNA, Ct 26.5 and for ANGPT2 mRNA, Ct ¼ 24.6. D, Relative mRNA expression levels of ANGPT2 and CXCR4 (top) and levels of phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 proteins (bottom) after treatment of SW620_LiM2 cells with 100 nmol/L PD0325901. Tubulin was used as a loading control, n ¼ 3. E, Relative mRNA expression levels of ANGPT2 and CXCR4 (top) and levels of P-ERK1/2 and total ERK1/2 proteins (bottom) after treatment of SW620_LiM2 cells with 10 mmol/L or 20 mmol/L U0126. Tubulin was used as a loading control. n ¼ 3. F, Relative mRNA expression levels of ANGPT2 and CXCR4 (top) and levels of P-ERK1/2 and total ERK1/2 proteins (bottom) after treatment of SW620_P, SW480, HCT116, or Colo26 cells with 10 mmol/L U0126. Tubulin was used as a loading control. n ¼ 5 (biological replicates). , P < 0.05; , P < 0.01; , P < 0.001; n.s., nonsigniﬁcant. Statistical signiﬁcance was calculated using two-tailed t test in C–F. Data in D–F plotted as average SD. A Wald-test was used in A comparing high versus low groups and pairwise test (adjusted) in B.

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A SW620_LiM2 B SW620_LiM2

100 100 free P = 0.02 P = 0.017 astasis 50 met 50

f sh Control n = 10 o sh ANGPT2 = 10 y

sh Control = 18 t i sh CXCR4 = 10 l

0 robabi 0 P Probability of metastasis free 0 10 20 30 40 010203040 Days Days

C SW620_LiM2 D P = 0.001 Day 43 n = 10 n = 10 103 (i)

102 100 ) (i) 10 101 sh Control sh Control 0 10 (ii)

P = 0.02 (ii) 50 10−1

sh Control n = 10 photon flux (log −2 sh CXCR4 Normalized ex vivo liver 10 shCXCR4 +shANGPT2

+ sh ANGPT2 n = 10 shCXCR4 +shANGPT2 −3 0 10 Min 5.07e6 Probability of metastasis free 0 2010 30 40 50 Min 1.60e5 Max 5.06e8 Days Max 2.70e8

SW620_P E F P = 0.0006 Day 42 10 3 n = 9 n = 10 (i)

10 2

100 ) 10 P = 0.002 10 1 (ii) MOCK MOCK 10 0 (ii) 50 10−1 − 10 2 (i) photon flux (log MOCK; n = 9 −

Normalized ex vivo liver 3 ANGPT2 + CXCR4 10 ANGPT2 ANGPT2 + CXCR4; n = 10 + CXCR4 0 Min 1.77e5 −4 Probability of metastasis free 0204060 10 Min 4.10e4 Days Max 5.94e7 Max 4.02e8 G H SW620_LiM2 SW620_P SW620_P P = n.s. P = n.s. P = n.s. No mets Lung mets 10 No mets 10 Tail vein injection 10 Lung mets

5 5 5 at day 56 at day 56 Number of mice Number of mice

0 0 0 sh Control shCXCR4 MOCK ANGPT2 MOCK ANGPT2 +shANGPT2 CXCR4 CXCR4

Figure 3. CXCR4 and ANGPT2 mediate liver metastasis formation in colorectal cancer. A–C, Probability of metastasis-free curve and representative images of mice injected intrasplenically with control or ANGPT2- and CXCR4-depleted SW620_LiM2 cells. D, Normalized ex vivo liver photon flux and representative images of livers in C. E, Probability of liver metastasis-free curve and representative images of mice injected intrasplenically with MOCK or ANGPT2- and CXCR4- expressing SW620_ P cells (n ¼ 9, 10). F, Normalized ex vivo liver photon flux and representative images of livers from mice described in F. G, Lung metastasis quantification in C at day 46 and E at day 56. H, Lung metastasis quantification in mice after tail vein injection with MOCK or ANGPT2- and CXCR4-expressing SW620_P cells (n ¼ 10, 10). Data in D and G are represented as whisker plots: midline, median; box, 25th–75th percentile; whisker, minimum to maximum. Statistical significance in A–C and E was calculated using log-rank test while in D, F, G,andH Fisher test was used. n.s., nonsignificant.

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Figure 4. A SW620_Parental CD31 ANGPT2 but not CXCR4 controls angio- SW620_P genesis in colorectal cancer liver metastasis. A, CD31 quantiﬁcation of size-matched P = n.s P = 0.046 liver lesions from mice injected intrasple- n ¼ n = 10 n = 8 nically with SW620_P ( 10 lesions) or 20 150 n = 10 n = 8 n ¼

2 SW620_LiM2 cells ( 8lesions).B, CD31 2 SW620_P SW620_LiM2 quantification of size-matched liver lesions 15 from control or CXCR4-depleted SW620_ 100 SW620_LiM2 LiM2 cells (n ¼ 7 or 8 lesions). C, CD31 fi 10 quanti cation of size-matched liver lesions from control or ANGPT2-depleted SW620_ 50 LiM2 cells (n ¼ 9 or 8 lesions). D, CD31 5 quantification of size-matched liver lesions from mice injected intrasplenically with Total tumor area mm 0 CD31 positive per mm 0 control or ANGPT-2 and CXCR4-depleted CD31 SW620_LiM2 cells (n ¼ 8or11lesions). B SW620_LiM2 sh Control Scale bar, 100 mmol/L. Data in A–C and P = n.s P = n.s D are represented as whisker plots: midline, median; box, 25th–75th percentile; 5 n = 7 n = 8 300 n = 7 n = 8 sh Control whisker, minimum to maximum. Statistical

2 ﬁ 2 sh CXCR4 signi cance was calculated using two- 4 tailed t test. n.s., nonsigniﬁcant. 200 sh CXCR4 3

2 100 1 Total tumor area mm

0 CD31 positive per mm 0 CD31 C SW620_LiM2 sh Control P = n.s P = 0.01

10 n = 9 n = 8 250 n = 9 n = 8 2 2 8 200 sh Control sh ANGPT2 6 150 sh ANGPT2 4 100

2 50 Total tumor area mm 0 CD31 positive per mm 0 D CD31 SW620_LiM2 sh Control P = n.s P = 0.049

5 n = 8 n = 11 250 n = 8 n = 11

2 sh Control 2 sh CXCR4 4 200 + sh ANGPT2 3 150 sh CXCR4 + sh ANGPT2 2 100

1 50 Total tumor area mm 0 CD31 positive per mm 0

in SW620_LiM2 cells did not affect the angiogenesis of liver metastases F4/80 is a well-established marker of murine macrophage populations, (Fig. 4B), we next focused on the role of CXCR4 in liver metastasis including Kupffer cells in the liver (33). Collectively, these results formation. We tested differences in various stromal populations and suggest that CXCR4 and ANGPT2 are downstream effectors of RAS– þ found a signiﬁcant reduction in the inﬁltrating F4/80 cells in liver ERK1/2 signaling in colorectal cancer cells, which support liver metastatic lesions formed by CXCR4-depleted SW620_LiM2 cells as colonization by triggering a supportive stromal response, involving compared with those formed by control SW620_LiM2 cells (Fig. 5C). at least vasculogenesis and macrophage recruitment.

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A P = 0.002 C 25 P = 0.018 ld)

(i) fie 10 3 n = 27 n = 10 n = 17 20

2 sper

) 10 15 sh Control cell 10 1 (i) 10 (ii) 10 0 (iii) 10 F4/80 counts 5 − 10 1 sh CXCR4#1

(% of positive 0 −2 (iii) 10 sh Control sh CXCR4#2 − photon flux (log 10 3 (ii) Normalized ex vivo liver

− sh CXCR4#2 10 4 Min 4.85e5 Max 8.46e8

B P = n.s. sh Control 30

No mets 20 Lung mets

10 CXCR4#2 sh

Number of mice at day 56 0 sh sh sh Control CXCR4#1 CXCR4#2

Figure 5. CXCR4 controls the development of liver but not lung metastasis. A, Normalized ex vivo liver photon flux of mice injected intrasplenically with control or CXCR4- depleted SW620_LiM2 cells. B, Lung ex vivo photon flux of mice described in A. C, F4/80 quantification of liver lesions from mice injected intrasplenically with control or CXCR4-depleted SW620_LiM2 cells (n ¼ 38 fields or n ¼ 56 fields, respectively). Representative images are included. Scale bar, 100 mmol/L. Data in A and B are represented as whisker plots: midline, median; box, 25th–75th percentile; whisker, minimum to maximum. Data in C are represented as scatter dot plot (mean SD). Statistical significance in A–C was calculated using two-tailed Mann–Whitney test. n.s., nonsignificant.

The ETS transcription factor family mediates CXCR4 expression expression of both ETV4 and ETV5, confirming their regulation by downstream of RAS–ERK1/2 signaling RAS–ERK1/2 signaling (Fig. 6C). We also observed that downregula- To address the mechanism by which RAS–ERK1/2 signaling con- tion of either ETV4 or ETV5 in SW620_LiM2 cells decreased CXCR4 trols liver metastasis, we focused on CXCR4, as ANGPT2 has a well- expression (Fig. 6D), whereas ETV4 overexpression increased CXCR4 known role in angiogenesis regulation. We first identified putative expression (Fig. 6E). Collectively, these results support that ETV4 and transcriptional factors whose expression levels correlated with that of ETV5 regulate CXCR4 expression downstream of RAS–ERK1/2 CXCR4 in the pooled cohort of 955 stage I–III primary colorectal signaling. cancer tumors (34, 35). We found that high expression of CXCR4 correlated positively with the expression of the ETV5 transcription CXCR4 promotes liver metastasis through IL10 factor, with a partial correlation coefficient of 0.353 (P <2.22e- Previous data reported that CXCR4 expression is modulated 16; Fig. 6A). Indeed, high expression of ETV5 significantly associated by various cytokines, including TGFb, IL10, IL4, TNFa, and IFNg – with a higher risk of recurrence in patients (P ¼ 2.5 10 5; Fig. 6B). (36–39). As cytokines facilitate tumor cell–microenvironment cross- ETV5 belongs to the ETS transcription factor family, which also talk, which can strengthen the metastatic behavior of tumor cells, we includes ERG, ETV1, and ETV4. ETS transcription factors promote used a cytokine array to study differences in cytokine production by cell proliferation and are involved in critical physiologic processes, SW620_P and SW620_LiM2 cells. We found that IL10 and CXCL1 including early development, organogenesis, and morphogenesis. The were consistently expressed at higher levels in SW620_LiM2 than in Etv4 and Etv5 double knockout mice do not develop kidneys, but this SW620_P cells, at both mRNA and protein levels (Fig. 7A and B), phenotype is not observed in the single knockout mice, suggesting that although they were not included in the EMGS due to the use of a high ETV4 and ETV5 may be functionally redundant in normal physiology. stringent cut-off. Expression of IL10 and CXCL1 was also regulated by We found a small increase in ETV5 expression in SW620_LiM2 cells ERK1/2 signaling, as cells treated with either of two different MEK compared with parental cells (Supplementary Fig. S4A), and treating inhibitors showed reduced mRNA and protein levels of both cytokines SW620_LiM2 cells with MEK inhibitors strongly downregulated the (Fig. 7C and D). Moreover, MEK inhibition reduced the levels of

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A Metacohort (n = 955) B Metacohort (n = 955) 1.0

0.8

0.6

ETV5 P = 5.4e–06 0.4 ETV5 LOW (n = 320) 0.2 ETV5 MEDIUM (n = 311) P = 2.2e−16 Proportion recurrence-free 0.0 ETV5 HIGH (n = 324) 456789 6 8 10 12 0 5 10 15 CXCR4 Time to recurrence (years)

C SW620_LiM2 1.2 1.2 ETV4 ETV4 1.0 ETV5 1.0 ETV5 ETV5 0.8 n = 4 ETV5 0.8 n = 3 pERK1/2 0.6 pERK1/2 0.6 0.4 0.4 ERK1/2 ERK1/2 0.2 0.2 GAPDH Relative mRNA expression

Tubulin Relative mRNA expression *** 0 *** 0 − + − DMSO: + − + − DMSO: + − − + U0126 − + − + PD0325901 + 10 µmol/L: 100 nm:

D SW620_LiM2 E SW620_LiM2 1.2 3 ETV4 1.2 ETV5 n = 3 MOCK 1.0 CXCR4 CXCR4 ETV4-OE 1.0 P = n.s. n = 3 n = 3 *** 0.8 ** 0.8 2 *** P = ns ** ** 0.6 0.6 *** 0.4 *** *** 0.4 1 0.2 0.2 Relative mRNA expression Relative mRNA expression 0 Relative mRNA expression 0 0 sh Control sh ETV4 sh ETV4 sh Control shETV5 shETV5 CXCR4 ANGPT 2 #1 #2 #1 #2

Figure 6. ETV4 and ETV5 transcription factors mediate CXCR4 expression downstream of RAS–ERK1/2 signaling. A, Regression coefficient between ETV5 and CXCR4 expression using a metacohort of 1,485 colorectal cancer primary tumors. B, Kaplan–Meier curve representing the proportion of recurrence-free patients with colorectal cancer stratified upon mRNA levels of expression of ETV5 using metacohort of 955 stage I–III colorectal cancer primary tumors. C, Relative mRNA of ETV4 and ETV5, and protein expression of ETV5, from SW620_LiM2 cells treated with DMSO (as a control; n ¼ 4 biological replicates) or a MEK inhibitor of either 10 mmol/L U0126 (n ¼ 4 biological replicates) or 100 nmol/L PD0325901 (n ¼ 3 biological replicates). D, Relative mRNA expression of ETV4, ETV5, and CXCR4 in SW620_LiM2 cells transduced with control lentiviral particles or with lentiviral particles expressing shRNA against ETV4 (left) or ETV5 (right). Data generated from three biological replicates. E, Relative mRNA expression of ETV4 and CXCR4 in SW620_LiM2 control cells or upon ectopic expression of ETV4 (n ¼ 4 biological replicates). Statistical significance in C–E was calculated using two-tailed t test. Data in C and D plotted as average SD. Wald test was used in B.

CXCL1 and IL10 in the colorectal cancer cell lines SW480, HCT116, aimed to confirm CXCR4 and IL10 cancer epithelial cell dependency in and Colo26 (Supplementary Fig S4B). Furthermore, shRNA-mediated clinical samples. To this end, we used the BRAF mutant–like signa- CXCR4 downregulation led to decreased mRNA and protein expres- ture (40) and the colorectal cancer intrinsic subtypes (CRIS) sub- sion of both CXCL1 and IL10 in SW620_LiM2 cells (Fig. 7D and E)as types (41) to draw cancer cell–dependent associations instead of the well as in SW620_P, SW480, and Colo26 cells (Supplementary CMS subtypes that reflect stromal population infiltration. Overall, we Fig. S4C). Consistently, IL10 expression in stage II primary tumors observed a significant correlation between the ANGPT2, CXCR4, associated with risk of recurrence in patients with colorectal cancer as ETV5, and IL10 genes and the BRAF mutant–like cancer cell signature previously observed for CXCR4 (Supplementary Fig. S4D). Next, we (Supplementary Fig. S5A–S5D). Similarly, CXCR4 expression was

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A SW620 B SW620_Parental C SW620_P SW620_LiM2 SW620_LiM2 CXCL1 IL10 1.2 ** ** 16 n = 5 6 n = 6 1.0 n = 5 n = 5 n = 3 12 0.8 4 8 0.6 CXCL1 2 0.4 IL10

expression 4 SW620_P

expression 0.2 ***

Relative mRNA Relative mRNA *** Relative mRNA Relative mRNA 0 0 0 *** *** 1,500 1.2 n = 3 * n = 3 * 200 1.0 n = 3 n = 3 n = 3 1,000 0.8 0.6

500 100 Relative 0.4 SW620_LiM2

protein levels 0.2 *

Protein (pg/mL) *** ** ****** 0 0 0 DMSO U0126 DMSO U0126 DMSO PD0325901 10 µmol/L 10 µmol/L 100 nmol/L D F SW620_LiM2

1.2 CXCR4 1.2 Ex Vivo CXCL1 TTO 1.0 1.0 5 n = 3 n = 3 10 IgG 0.8 0.8 ** ** 0.6 0.6 LiM2 IgG; n = 7 0.4 0.4 LiM2 IL10 nAb; n = 7

expression *** *** 4 0.2 0.2 10 CXCL1 Relative Relative mRNA Relative mRNA *** *** 0 protein expression 0 sh sh#1 sh#2 sh sh#1 sh#2 P = 0.026 E Control CXCR4 Control CXCR4 103 IL10 nab 1.2 CXCR4 1.2 IL10 1.0 1.0 2 n = 3 n = 3 10 0.8 0.8 0.6 *** 0.6 0.4 ****** *** 0.4 *** *** Normalized ventral photon flux 1 expression 10 0.2 IL10 Relative 0.2 Max = 4.7e08 Relative mRNA

0 protein expression 0 0 510152025 Min = 7.5e05 sh sh#1 sh#2 sh sh#1 sh#2 Days Control CXCR4 Control CXCR4

G H

TTO Ex Vivo 10 6 mIgG CD31

CXCL1 10 5 IL10

P = 0.002 10 4 IL-10nmAb 10 3 CXCR4 10 2 MTO138 mIgG; n = 8 Normalized ventral photon/flux ANGPT2 Max 2.62e8 1 MTO138 IL10 nmAb; n = 8 10 Min 6.39e5 0 5 10152025 EMGS

Days CRC Primary tumor Liver met positive

Figure 7. The expression of IL10 and CXCL1 is controlled by RAS–ERK signaling via CXCR4 in colorectal cancer cells. A, Representative image of cytokine array of supernatants from SW620_P and SW620_LiM2 cells. B, Relative mRNA expression (top) and protein expression (bottom) of CXCL1 (left) and IL10 (right) in SW620_P or SW620_LiM2 cells (n, number of biological replicates). C, Relative mRNA (top) and protein expression (bottom) of CXCL1 and IL10 in SW620_P (left) or SW620_LiM2 cells (right) that had been treated with DMSO (control), the MEK inhibitor U0126 (10 mmol/L) or the MEK inhibitor PD0325901 (100 nmol/L; n, number of biological replicates). D, Relative mRNA (left) and protein (right) expression of CXCL1 in SW620_LiM2 cells transduced with control shRNA lentiviral particles or with those expressing shRNA against CXCR4 (n ¼ 3 biological replicates). E, Relative mRNA and protein expression of IL10 in SW620_LiM2 cells transduced with control lentiviral particles or with those expressing shRNA against CXCR4 (n ¼ 3 biological replicates). F, In vivo photon flux quantification of liver metastasis lesions of mice injected intrasplenically with SW620_LiM2 cells treated with antibody against IL10 or IgG (indicated with arrows; n, number of mice used in each arm of study). G, In vivo photon flux quantification of liver metastasis lesions of mice injected intrasplenically with the colorectal cancer organoid 138 treated with an antibody against mouse IL10 or mouse IgG (indicated with arrows; n, number of mice used in each arm of study). H, Schematic model. Statistical significance in B–E was calculated using two-tailed t test. Statistical significance in F–G was calculated using two-tailed Mann–Whitney test.

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associated with CRIS-A, -B, and -D subtypes (Supplementary liver metastases (Fig. 7H). ETV4 and ETV5 play a role in this Fig. S5E–S5G). Consistently, the CRIS-A subtype captures KRAS metastasis process by controlling CXCR4 expression. Colorectal cancer mutations; the CRIS-B subtype, the TGFb pathway activity, the cells with high levels of CXCR4 tend to migrate toward organs that epithelial–mesenchymal transition, and poor prognosis; and the have high levels of CXCL12, such as the liver or lung. However, our CRIS-D subtype, the WNT pathway activation. Collectively, these results show that an increased ANGPT2 and CXCR4 expression observations suggest that CXCR4 mediates the induction of IL10 and facilitates metastasis development in the liver but not in the lung. In CXCL1 expression by ERK1/2 signaling. addition, downregulation of ANGPT2 and CXCR4 also reduces the We next tested whether blocking IL10 would prevent liver coloni- formation of liver metastases without affecting lung metastases. These zation by the highly metastatic SW620_LiM2 cell line after intrasplenic differences in tissue-specific colonization could be attributed to the injection into nude mice. One week after injection of the cells, mice differences in the structure of liver and lung blood vessels. In this were treated with an antibody against IL10 or control IgGs, and cells regard, liver sinusoidal blood vessels are formed by a discontinuous homing to the liver was followed (Fig. 7F). Notably, we detected a endothelium, which is more permissive for tumor cell extravasation reduction in liver colonization after IL10 antibody treatment as than the tight junctions between the endothelial cells of lung capillar- compared with the IgG-treated control group (Fig. 7F). This was ies. Therefore, ANGPT2 and CXCR4 might not be essential for tumor confirmed by a reduction in metastatic lesions observed ex vivo in livers cell extravasation but control metastatic outgrowth. from mice injected with IL10 antibody (Fig. 7F). To extend this Both ANGPT2 and CXCR4 can control tumor angiogenesis finding, we tested the effect of IL10 inhibition in a colorectal cancer (29–32). However, in our mouse model, the number of endothelial immunocompetent mouse model. We used mouse KRASmut colorectal cells in liver metastatic lesions was dependent exclusively on the cancer organoids derived from Apcfl/fl, KrasLSL-G12D, Tgfbr2fl/fl, expression levels of ANGPT2 but not CXCR4. Of note, the expression Trp53fl/fl, and Lgr5eGFP-creERT2 mice (34) and a neutralizing anti- of VEGF, which has been reported to cooperate with ANGPT2 to body against mouse IL10. As observed using nude mice and human promote tumor growth (47) and to regulate the expression of colorectal cancer cells, we confirmed that anti-IL10 injection impaired CXCR4 (48), was not significantly different between the poorly and liver colonization by colorectal cancer cells in immunocompetent mice highly metastatic cell lines SW620_P and SW620_LiM2, respectively. (Fig. 7G). These results suggest that liver colonization by colorectal The cytokines ANGPT2 and ANGPT1 bind to the TIE2 receptor with cancer cells relies on IL10 production, which is controlled by RAS– similar affinity, but their binding elicits distinct responses in endo- ERK1/2 signaling via CXCR4. thelial cells (49). Namely, ANGPT1 maintains endothelial cells in quiescence, while ANGPT2 promotes vascular remodeling and angiogenesis (49). We detected expression of ANGPT1 neither in SW620_P Discussion nor in SW620_LiM2 cells, which is in line with reports that ANGPT2 Accumulating evidence shows that the activity of signaling path- but not ANGPT1 can be highly expressed in human tumors, and that ways present in the primary tumor cells is modified in metastatic cells. an imbalance in the ANGPT2/ANGPT1 ratio can promote tumor Consequently, this endows metastatic cells with an increased ability to growth (50–54). colonize organs (35). Here we provide evidence that an increased RAS– Increased levels of CXCR4 expression are found in more than ERK signaling in a mutant KRAS background could be a driver of liver 20 human tumor types, including ovarian, prostate, melanoma, neu- metastasis formation in colorectal cancer. We identified a set of genes roblastoma, and colorectal cancer (25–27, 55–58). In addition, controlled by this signaling pathway that is associated with increased CXCR4 has been connected with metastatic spread in breast, recurrence in patients with colorectal cancer (termed EMGS, for prostate, hepatocellular, and colorectal cancers, mostly by regulat- ERK1/2-controlled metastatic gene set). High levels of expression of ing cell migration toward surrounding tissues or toward CXCL12- the EMGS is associated with reduced survival for patients with both enriched organs (26, 57, 59–61). Notably, CXCR4 downregulation þ MSS and microsatellite-instable colorectal cancer tumors, thus imply- in our model was associated with a reduced recruitment of F4/80 ing that therapeutic targeting of ERK1/2 signaling pathway may cells to the metastatic lesions. Our data demonstrate, for the first benefit both groups of patients. time, that CXCR4 controls the expression of the cytokines IL10 and Interestingly, higher levels of EMGS expression were detected in CXCL1, and we also provide evidence for a causal role of IL10 in the colorectal cancer subsets CMS1 and CMS4, which are character- supporting liver colonization. In patients with colorectal cancer, ized by an enrichment in genes that modulate the tumor microenvi- IL10 levels increase as the disease progresses, and high serum levels ronment (16). More precisely, CMS1 tumors have high expression of IL10 correlate with poor survival of these patients (62, 63). In levels of genes associated with immune infiltration, whereas CMS4 breast cancer, CXCL1 promotes lung metastasis; in contrast, in tumors have high expression levels of genes associated with the colon cancer, it may influence the formation of the liver premeta- epithelial-to-mesenchymal transition, the TGFb signaling pathway, static niche (64, 65). The mechanisms through which IL10 and matrix remodeling, and angiogenesis (16). These findings are in CXCL1 promote liver metastasis from colorectal cancer tumors will line with the evidence supporting an effect of oncogenic KRAS be the subject of further investigation.Insummary,wedemonstrate signaling beyond cancer cells and affecting the tumor microenviron- that amplification of ERK1/2 signaling in KRAS-mutated colorectal ment (42). In particular, this pathway may affect the immune cell cancer cells affects the cytokine milieu of these tumors, thus landscape in various kinds of tumor, thereby affecting their growth and probably affecting tumor–stroma interactions and favoring liver progression (43–45). Furthermore, oncogenic KRAS may modulate metastasis formation. tumor-associated angiogenesis by controlling the expression of VEGF or of cytokines such as IL8, CXCL1, and CXCL5 (46). Disclosure of Potential Conflicts of Interest We now provide the evidence that the RAS–ERK1/2 axis controls R.R. Gomis reports other from Inbiomotion SL (member of the board of directors) the expression of the cytokine ANGPT2 and the cytokine receptor outside the submitted work. No potential conflicts of interest were disclosed by the CXCR4 in colorectal cancer cells, which facilitates the development of other authors.

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Authors’ Contributions assistance. J. Urosevic was an AECC (Asociacion Espanola~ Contra el Cancer) Fellow. J. Urosevic: Conceptualization, resources, data curation, formal analysis, A. Llorente, A. Bellmunt., and C. Figueras-Puig were funded by the Spanish investigation, writing-original draft, writing-review and editing. M.T. Blasco: Government (MINECO-Formacion de personal Investigador). I. Burkov was Conceptualization, data curation, formal analysis, investigation, methodology, cofunded by FP7 Marie Curie Actions (COFUND program; grant agreement no. writing-review and editing. A. Llorente: Resources, validation, investigation, IRBPostPro2.0 600404). M.T. Blasco was funded by ISCIII/FEDER-CIBERONC and methodology, writing-original draft. A. Bellmunt: Resources, data curation, by the Spanish Government (Juan de la Cierva Formacion-Postdoctoral Fellowship). formal analysis, investigation, writing-original draft. A. Berenguer-Llergo: R.R. Gomis, E. Batlle., and A.R. Nebreda are supported by the Institucio Catalana de ¸ Data curation, formal analysis, validation, methodology, writing-review and Recerca i Estudis Avancats. Support and structural funds were provided by the editing. M. Guiu: Investigation, methodology, writing-original draft. A. Canellas:~ Generalitat de Catalunya (2014 SGR 535) to R.R. Gomis and A.R. Nebreda, and by the “ ” Investigation. E. Fernandez: Investigation. I. Burkov: Formal analysis, investigation. BBVA Foundation, the ISCIII/FEDER-CIBERONC, the la Caixa Foundation (ID – M. Clapes: Validation, investigation. M. Cartana: Investigation. C. Figueras-Puig: 100010434), under the agreement , the Spanish Ministerio de Econ- – Investigation. E. Batlle: Resources. A.R. Nebreda: Resources, writing-original draft, omia y Competitividad (MINECO) and FEDER funds (CIBEREONC and PID2019 writing-review and editing. R.R. Gomis: Conceptualization, formal analysis, 104948RB-I00) to R.R. Gomis. supervision, funding acquisition, methodology, writing-original draft, writing- review and editing. 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 accordance Acknowledgments with 18 U.S.C. Section 1734 solely to indicate this fact. We thank V. Raker for manuscript editing and IRB Barcelona Functional Genomics (J.I. Pons and D. Fernandez), Histopathology (N. Prats), Advanced Digital Received December 27, 2019; revised June 23, 2020; accepted August 12, 2020; Microscopy (J. Colombelli), and Flow Cytometry (J. Comas) Core Facilities for published ﬁrst August 19, 2020.

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4680 Cancer Res; 80(21) November 1, 2020 CANCER RESEARCH

ERK1/2 Signaling Induces Upregulation of ANGPT2 and CXCR4 to Mediate Liver Metastasis in Colon Cancer

Jelena Urosevic, María Teresa Blasco, Alicia Llorente, et al.

Cancer Res 2020;80:4668-4680. Published OnlineFirst August 19, 2020.

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