Immune-Dependent and Independent Antitumor Activity of GM-CSF Aberrantly Expressed by Mouse and Human Colorectal Tumors

Published OnlineFirst October 29, 2012; DOI: 10.1158/0008-5472.CAN-12-0806

Cancer Tumor and Stem Cell Biology Research

Rocio G. Urdinguio1, Agustin F. Fernandez1, Angela Moncada-Pazos2, Covadonga Huidobro1, Ramon M. Rodriguez1, Cecilia Ferrero1, Pablo Martinez-Camblor4, Alvaro J. Obaya3, Teresa Bernal5, Adolfo Parra-Blanco6, Luis Rodrigo6, Maria Santacana7, Xavier Matias-Guiu7, Beatriz Soldevilla8, Gemma Dominguez8, Felix Bonilla8, Santiago Cal2, Carlos Lopez-Otin2, and Mario F. Fraga1,9

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF2) is a cytokine produced in the hematologic compartment that may enhance antitumor immune responses, mainly by activation of dendritic cells. Here, we show that more than one-third of human colorectal tumors exhibit aberrant DNA demethylation of the GM-CSF promoter and overexpress the cytokine. Mouse engraftment experiments with autologous and homologous colon tumors engineered to repress the ectopic secretion of GM-CSF revealed the tumor-secreted GM-CSF to have an immune-associated antitumor effect. Unexpectedly, an immune-independent antitumor effect was observed that depended on the ectopic expression of GM-CSF receptor subunits by tumors. Cancer cells expressing GM-CSF and its receptor did not develop into tumors when autografted into immunocompetent mice. Similarly, 100% of the patients with human colon tumors that overexpressed GM-CSF and its receptor subunits survived at least 5 years after diagnosis. These data suggest that expression of GM-CSF and its receptor subunits by colon tumors may be a useful marker for prognosis as well as for patient stratiﬁcation in cancer immunotherapy. Cancer Res; 73(1); 395–405. 2012 AACR.

Introduction hematologic cell growth factor, stimulating blood stem cells The immune system can identify and destroy nascent to produce granulocytes and monocytes (4, 5). In addition, tumor cells, in a process termed cancer immunosurveillance, GM-CSF leads to protective immunity, mainly by stimulating which plays an important role in the defense against cancer the recruitment, maturation, and function of dendritic – (1, 2). The ability of the immune system to fight tumor cells cells (6 8), the most potent antigen-presenting cell (9). This has been used in the development of cancer immunotherapy effect of the cytokine on dendritic cells leads to the activa- fi treatments based on enhancing host antitumor responses tion of the immune system against speci cantigens(10). (3). The cytokine granulocyte-macrophage colony stimulat- Therefore, one approach to cancer immunotherapy is vac- ingfactor(GM-CSF,alsoknownasCSF2)functionsasa cination with tumor-irradiated cells engineered to secrete GM-CSF (6, 7, 11–13). In this context, the action of GM-CSF on dendritic cells allows the immune system to be activated fi Authors' Affiliations: 1Cancer Epigenetics Laboratory, HUCA, 2Departa- against the speci c antigens directly provided by the tumor mento de Bioquímica y Biología Molecular, and 4Department of Functional cells (10). This strategy has, for instance, been used in Biology, Institute of Oncology of Asturias (IUOPA), Universidad de Oviedo, vaccination with irradiated GM-CSF–secreting melanoma Oviedo, Spain; 3Unidad de Apoyo a la Investigacion CAIBER, OIB, Oviedo, Spain; 5Servicio de Hematología and 6Department of Gastroenterology, cells resulting in enhanced host responses through improved Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain; 7Depart- tumor antigen presentation by recruited dendritic cells and ment of Pathology and Molecular Genetics and Research Laboratory, Hospital Universitari Arnau de Vilanova, University of Lleida, IRBLLEIDA, macrophages (6). Lleida, Spain; 8Servicio de Oncología Medica, Hospital Universitario Puerta GM-CSF has been shown to be ectopically secreted by cell de Hierro Majadahonda, Facultad de Medicina, Universidad Autonoma de lines derived from a variety of solid tumors (14, 15) but the Madrid, Madrid, Spain; 9Department of Immunology and Oncology, Nation- al Center for Biotechnology, CNB-CSIC, Cantoblanco, Madrid, Spain immune-independent effect of tumor-secreted GM-CSF is still poorly understood. Contradictory results from different Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). groups have shown that GM-CSF could either exert an antiproliferative effect (16, 17) or promote tumor growth Corresponding Author: Mario F. Fraga, Cancer Epigenetics Laboratory – fl (IUOPA), HUCA, C/Dr. Emilio Rodriguez Vigil s/n, Bloque Polivalente A, (18 20). Furthermore, no reproducible in uence on tumor 4 planta, 33006 Oviedo, Spain. Phone: 34-985-109-475; Fax: 34-985-109- growth rate has been reported (21, 22). GM-CSF has also 475; E-mail: [email protected] been reported to be secreted by some primary tumors doi: 10.1158/0008-5472.CAN-12-0806 (23–27) but, to the best of our knowledge, there are no 2012 American Association for Cancer Research. reports to date showing the expression of GM-CSF by

www.aacrjournals.org 395

Urdinguio et al.

primary colon tumor tissues nor its possible effects on the (CIMA), Navarra, Spain] and cultured at 37 Cin5%CO2 in proliferation of this tumor type. Furthermore, the molecular RPMI supplemented with 10% FBS and 50 mmol/L b-mercap- mechanisms involved in the ectopic expression of GM-CSF by toethanol. MC38 and CT26 colon cancer cell were stably tumor cells are still largely unknown. Here, we provide evidence transfected with a HuSH 29-mer shRNA construct against Mus for the possible molecular mechanism involved and describe musculus Csf2 in pGFP-V-RS vector or a HuSH 29-mer Non- the antitumor effect of the process in colorectal cancer. Effective Scrambled pGFP-V-RS vector (OriGene) following the manufacturer's instructions. We selected different constructs Materials and Methods of the 4 provided by the supplier to generate 2 independent qRT-PCR clones from each cell line with Gm-CSf interference as well as a Total RNA was extracted from mouse colon cell lines, scramble. human colon cancer cell lines, and human samples using TRIzol Reagent (Invitrogen) following the manufacturer's In vitro growth experiments procedure. DNA was removed with DNase I treatment with Cell viability was determined by MTT assay on cells stably DNA-free kit (Ambion/Applied Biosystems) and cDNA was transfected with scramble and shGm-CSf vectors as described prepared using SuperScript II Reverse Transcriptase kit by Mosmann and colleagues (28). Ten replicates per condition (Invitrogen) following the manufacturer's recommenda- and time point were assessed. Absorbance at 595 nm was tions. We ran PCR reactions in triplicate using Sybr Green measured with an automated microtitre plate reader Power Master Mix (Applied Biosystems) in a 7900HT Real-Time Wave WS (BioTek). Cell proliferation rate was established by PCR System (Applied Biosystems). We conducted relative cell counting on consecutive days in scramble and shGm-CSf quantification of gene expression based on standard curve cells. Cells were seeded in triplicates in 24-well plates at a transformations of Ct values. Results from each target gene concentration of 1 103 per well. Cells were collected daily for were normalized against their respective ubiquitous house- 5 days and viable cells, as assessed by trypan blue staining, were keeping gene. Primer sequences are listed in Supplementary counted under a microscope in a hemocytometer. Colony Table S2. formation assay was conducted as described in Franken and colleagues (29). Cells were then seeded at different densities Bisulfite pyrosequencing (50, 100, and 200 cells) and after 2 weeks, stable colonies were Genomic DNA isolation was conducted according to a fixed and stained with a mixture of 6.0% Glutaraldehyde and standard phenol-chloroform extraction protocol. Bisulfite 0.5% Crystal Violet in water. After washing and being left to air modification of DNA was conducted with the EZ DNA Meth- dry, colonies were counted. The Plating Efficiency was calcu- ylation-Gold kit (Zymo Research) following the manufacturer's lated as a mean of the Plating Efficiency of the different cell instructions. After PCR amplification of the region of interest dilutions. with specific primers, pyrosequencing was conducted using PyroMark Q24 reagents, vacuum prep workstation, equipment, Tumor transplantation and in vivo treatment studies and software (Qiagen). Primer sequences for murine Gm-CSf For tumor grafting, we subcutaneously (s.c.) injected 1 to 4 and human GM-CSF are shown in Supplementary Table S2. 105 tumor cells into each flank of recipient mice (Nude or NU/ NU, C57BL/6 and BALB/c females, 6 to 8 week old, Charles Human cell lines and human samples River). Anti-PD1 was purchased from BioXCell and adminis- Human colon cancer cell lines were cultured according to tered by intraperitoneal injection of 250 mg. Tumor volumes American Type Culture Collection recommendations. Cell were measured twice or 3 times a week with calipers and lines were authenticated using short tandem repeat profiling calculated using the following formula: tumor volume ¼ 0.4 of an extracted DNA sample using AmpF‘STR Identifiler for (length width2). After sacrifice, tumors were excised and high resolution screening and intraspecies cross-contami- weighed. nation detection by Bio-Synthesis, Inc. Briefly, cell lines were fi grown at 37 Cin5%CO2 in Dulbecco's Modi ed Eagle's Immunohistochemistry Media supplemented with 10% FBS. Human cells were Tissue microarray (TMA) blocks were sectioned at a thick- treated with the demethylating agent 5-aza-20-deoxycytidine ness of 3 mm, dried for 1 hour at 65 before the pretreatment (AdC; Sigma) at various concentrations (2.5 and 5 mmol/L) procedure: deparaffinization, rehydration, and epitope retriev- for 72 hours. Healthy human- and primary tumor-colon al in the Pre-Treatment Module, PT-LINK (DAKO) at 95C for samples were obtained from the Hospital Universitario 20 minutes in 50 Tris/EDTA buffer, pH 9. The antibody Puerta de Hierro (Madrid, Spain) and the Asturias Tumor against CD3 (clone M-20, sc-1127, Santa Cruz Biotechnology) Bank of the Institute of Oncology (Asturias, Spain). Tissue was used to stain the sections, after blocking endogenous collection and analyses were approved by the appropriate peroxidase. Following incubation, the reaction was visualized institutional review boards in accordance with national and with the EnVision Detection Kit (DAKO) using diaminobenzi- EU guidelines. dine chromogen as a substrate. Sections were counterstained with hematoxylin. Appropriate negative controls were also Murine cell lines and shGm-CSf stable transfection tested. For immunofluorescence, TMA sections were pre- Mouse colon cancer cell lines were kindly provided by Dr. treated with sodium-citrate buffer pH 6 (5 minutes, 100C). Ignacio Melero [Centro de Investigacion Medica Aplicada Slides were blocked with 10% donkey serum (2 hours) and

396 Cancer Res; 73(1) January 1, 2013 Cancer Research

Antitumor Activity of GM-CSF in Colorectal Tumors

incubated with the antibody against CSF2RB (LS-B6993, Life- Statistical analyses span Biosciences) and active-beta-catenin (05-665, Millipore) For statistical comparisons we used Student's t test (paired overnight, 4C. After rinsing with Dulbecco's phosphate-buff- and unpaired). We analyzed colon tumor-free survival using ered saline, slides were incubated with Alexa 594 (A11072, Life Kaplan–Meier log-rank test. A P value of less than 0.05 was Technologies) or Alexa 488-conjugated secondary antibody considered statistically significant. (A11017, Life Technologies). DNA was stained with 40,6-dia- midino-2-phenylidole and tissue sections imaged using a dig- Results ital camera connected to a Leica microscope DMRXA. Frequent aberrant promoter demethylation and overexpression of GM-CSF in human colorectal cancer RNA in situ hybridization To characterize GM-CSF expression in colon cancer, we The experiments of RNA in situ hybridization were con- analyzed GM-CSF mRNA levels in 124 paired samples of ducted with the QuantiGene ViewRNA ISH Tissue Assay Kit primary colon tumors and their healthy counterparts, and (Affymetrix) and the QuantiGene ViewRNA TYPE1 # Probe Set included peripheral blood mononuclear cells (PBMC) as a for HUMAN-CSF2-colony stimulating factor 2 (granulocyte- positive control. We found that samples from healthy colon macrophage; Affymetrix) following the manufacturer's instruc- epithelium showed very low, or absent, GM-CSF expression, tions. Images were obtained using a confocal microscope Leica whereasmanytumorsexpressedGM-CSFabovePBMC TCS-SP2-AOBS. levels (Fig. 1A). GM-CSF overexpression by tumors (considered as more than 2-fold increase over the levels from their Cytotoxicity assays corresponding healthy counterparts) was observed in 37.9% After mouse grafting, peripheral lymphocytes were isolated (47 of 124) of patients. We also studied 10 colon cancer cell from blood using Lympholyte-M (Tebu-bio). The protocol for lines (CCL) and found that 6 of them (Caco2, HCT15, the isolation of tumor-infiltrating lymphocytes was adapted HCT116, HT29, RKO, and SW48) did not express GM-CSF, from Radoja and colleagues (30). Cytotoxicity assays were whereas 4 (SW480, DLD1, COLO205, and Co115) showed conducted to determine cell-mediated cytotoxicity against cell expression values similar to, or above, PBMC levels (Fig. 1A lines injected in the mice flanks with CytoTox 96 Non-Radio- and Supplementary Fig. S1A). Our results show that GM-CSF active Cytotoxicity Assay (Promega) following the manufac- is frequently overexpressed in primary colon tumors as well turer's instructions. as in colon cancer cell lines. A large number of molecular alterations in cancer occur at ELISA the epigenetic level (31). Cancer cells show aberrant gain or Culture medium aliquots were frozen in liquid nitrogen loss of DNA methylation at the promoter region of many immediately after extraction and kept at 80C until analysis. genes (32); alterations that are directly involved in tumor- ELISA was conducted with LEGEND MAX Mouse GM-CSF igenesis as they can induce either repression of tumor ELISA kit (BioLegend) for mouse Gm-CSf detection and suppressor genes or activation of oncogenes (32, 33). To Human GM-CSF ELISAPRO kit (Mabtech) for human GM-CSF studythepossibleaberrantepigeneticregulationofGM-CSF detection, following the instructions of the manufacturers. in colon cancer, we determined the methylation status of

Figure 1. GM-CSF overexpression and DNA methylation levels at the GM-CSF promoter in human primary colon cancer. A, GM-CSF mRNA expression levels were determined by quantitative real-time PCR in 124 paired samples of primary colon tumors (Tumor) and their healthy counterparts (HC, healthy colon). Results from PBMCs were included as a positive control of GM-CSF expression. Human GM-CSF mRNA levels are expressed as a ratio in relation to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. B, bisulﬁte pyrosequencing results from 74 colon primary tumors (Tumor) and their 74 paired HC tissues. A schematic representation of the studied region and the methylation values from 2 representative coupled samples are shown on the left. Black depicts methylated CpG and white indicates unmethylated CpG. On the right, a boxplot shows the methylation results of the 74 paired human samples. A signiﬁcant difference in methylation values of tumor samples compared with their healthy counterparts was found (P < 0.001, paired t test).

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 397

Urdinguio et al.

GM-CSF promoter in 10 CCLs. This analysis showed that GM- GM-CSF overexpression leads to secretion of the cytokine by CSF promoter was densely methylated in the 6 CCLs (Caco2, colon cancer cells. HCT15, HCT116, HT29, RKO, and SW48) that had very low levels of GM-CSF expression, whilst it was almost unmethy- Immune-dependent antitumor effect of GM-CSF lated in the 4 CCLs (SW480, DLD1, COLO205, and Co115) ectopically secreted by colon tumors that presented high levels of GM-CSF expression (Supple- Given the antitumor effect of GM-CSF when administered as mentary Fig. S1A and S1B). With the aim of clarifying a vaccine in an immunotherapy context (6, 11), we tested whether GM-CSF promoter demethylation is also a frequent whether the overexpression of this cytokine by colon cancer in vivo event, we analyzed 74 primary colon adenocarcino- cells could mimic this antitumor action. To evaluate a possible mas and their 74 paired healthy colon tissues. Healthy immune-dependent effect of GM-CSF in colon cancer, we tissues presented an average methylation level of 83.5%, knocked down Gm-CSf expression by stable RNA interference whereas the mean methylation level of the tumors was in the murine Gm-CSf–expressing colon cancer cell lines CT26 61.5% (P < 0.001; Fig. 1B). We detected hypomethylation (at and MC38, which are poorly methylated at the Gm-CSf pro- least a 20% change) at the GM-CSF promoter in 38 of the 74 moter (Supplementary Fig. S3A, S3B, S3C, and S4A). To discard tumors (51.4%), thereby confirmingthisasacommonevent a possible immune-independent effect of Gm-CSf, we analyzed in vivo (Fig. 1B). To evaluate whether demethylation of the the expression of the Gm-CSf plasmatic membrane receptor GM-CSF promoter is related to a genome-wide demethyla- subunits Csf2ra and Csf2rb in these same murine colon cancer tion process, something frequently observed in tumor cells cell lines. MC38-derived clones expressed Csf2ra, whereas (34), we analyzed the methylation levels of the LINE-1 CT26-derived clones did not. All clones expressed similar low sequence by bisulfite pyrosequencing (35). A significant levels of Csf2rb (Supplementary Fig. S3D and S4B). Then, we s.c. association (P < 0.001) between LINE-1 and GM-CSF hypo- injected CT26 cells with knocked-down Gm-CSf expression methylation was found (Supplementary Fig. S2), thereby (shGm-CSf) and an isogenic scramble-transfected clone consolidating the notion that DNA hypomethylation plays (scramble) that overexpressed Gm-CSf into a cohort of autol- an important role in the aberrant regulation of GM-CSF in ogous immune-competent BALB/c mice. As CT26 clones do colon cancer. not express Csf2ra (Supplementary Fig. S3D and S4B), this Concurrent GM-CSF overexpression (Fig. 1A) and GM-CSF model allows the comparison of 2 colon cell lines, differing in promoter DNA demethylation (Fig. 1B, right) suggested that their Gm-CSf expression, when they encounter an immune- DNA methylation plays an important role in the regulation competent in vivo environment, excluding immune-indepen- of GM-CSF expression. To get a deeper insight into this dent effects. Abolishing Gm-CSf expression in CT26 cells issue, we analyzed the consequences of in vitro demethy- resulted in a marked increase in tumor formation in vivo (Fig. lation. Firstly, we measured GM-CSF promoter methylation 2A and Supplementary Fig. S4C). At the time the mice were and GM-CSF expression levels in the HCT116 cell line sacrificed, the Gm-CSf expressing CT26 tumors had, on aver- compared with a double DNMT1 and DNMT3B knock-out age, half the volume and weight of their non-Gm-CSf –expres- cell line (DKO) derived from HCT116. We observed genet- sing counterparts (Fig. 2A and Supplementary Fig. S4C). This ically induced GM-CSF promoter hypomethylation and led us to investigate the possible role of the immune system consistent GM-CSF overexpression in the DKO cell line reasoning that it could be responsible for the observed anti- (Supplementary Fig. S1C), supporting the existence of a tumor effect of Gm-CSf (7, 36, 37). We studied T lymphocyte relationship between GM-CSF promoter methylation and its tumor infiltration conducting immunohistochemistry of expression. Furthermore, we analyzed GM-CSF mRNA levels tumors resected from BALB/c mice. On average, we found in 4 colon cancer cell lines incubated with 2 different more T infiltration in Gm-CSf expressing tumors than in their concentrations of the demethylating drug AdC. Treatment silenced counterparts (Fig. 2B). In addition, we measured the with this drug resulted in marked reactivation of GM-CSF specific lysis conducted by lymphocytes isolated from BALB/c expression in a dose-dependent manner in cell lines dis- mice blood and found that lymphocytes from mice carrying playing high levels of GM-CSF promoter methylation (HCT15 Gm-CSf expressing tumors provoked a higher percentage of and RKO), but not in those with low levels of methylation cell lysis than those from mice with nonexpressing tumors (Fig. (Colo205 and Co115; Supplementary Fig. S1D). These data 2C). We further addressed this issue using tumor-infiltrating support the notion that aberrant GM-CSF promoter deme- lymphocytes extracted from tumors generated by scramble or thylation plays an important role in GM-CSF overexpression an additional shGm-CSf clone of CT26 cell line in BALB/c mice. in colon cancer. As shown in Supplementary Fig. S4D, we obtained the minimal To verify that GM-CSF promoter methylation and mRNA required amount of lymphocytes for cytotoxicity assays in only expression levels are related to protein levels and to determine 4 cases. While 4 cases are not sufficient for statistical analysis, whether ectopically overexpressed GM-CSF was secreted by we observed a tendency toward higher cytotoxicity specific cells, we measured GM-CSF protein levels in the culture media lysis from lymphocytes extracted from Gm-CSf–expressing of 2 colon cancer cell lines (HCT116 and DLD1) by ELISA. We tumors (Supplementary Fig. S4E): results that we consider to confirmed that GM-CSF protein levels are directly associated provide evidence for the role of Gm-CSf in activating the with mRNA levels and, inversely related to GM-CSF promoter immune system to limit tumor growth. Thus, ectopic expres- methylation in both colon cancer cell lines (Supplementary sion of this cytokine by tumors might have an antitumor effect Fig. S1E). This suggests that promoter demethylation-related mediated by the immune system.

398 Cancer Res; 73(1) January 1, 2013 Cancer Research

Antitumor Activity of GM-CSF in Colorectal Tumors

Figure 2. Immune-dependent antitumor effects of Gm-CSf on tumor growth in vivo. A, diagram of the in vivo model studied for the analysis of the immune- dependent effects of Gm-CSf showing that BALB/c mice (n ¼ 12) were s.c. injected with scramble or shGm-CSf CT26 cells in both flanks. Macroscopic tumors were observed and regularly measured for 24 days after injection. Evolution of tumor growth measured as tumor volume and a graph with endpoint tumor weights are shown. At the time the mice were sacrificed, scramble CT26 tumors (blue line and blue circles) had significantly higher volume and weight than CT26 tumors lacking Gm-CSf expression (red line and red triangles). Photographs of 1 representative mouse from each analyzed group are þ included. B, IHC of tumors from immune-dependent experiment with BALB/c mice. Two representative images obtained from IHC are shown (anti-CD3 staining appears in brown). The percentage of CD3þ staining was higher in tumors originated from Gm-CSf expressing scramble CT26 cells as shown in the graph on the right. C, percentage of specific lysis carried out by lymphocyte isolated from blood from immune-dependent experiment BALB/c mice (n ¼ 7) when confronting the respective CT26 clones they had been primed for. Lymphocytes extracted from mice with scramble CT26 tumors caused higher specific lysis that reached significance at lower effector:target ratios. Results are expressed as the mean SEM. , P < 0.05; , P < 0.01; , P < 0.001 (Student's t test).

Immune-independent antitumor effect of GM-CSF MC38) could we detect a decrease in cell growth rate ectopically secreted by colon tumors attributable to Gm-CSf (Fig. 3A and Supplementary Fig. We observed in vitro that scramble and shGm-CSf clones S5). This immune-independent effect was confirmed in vivo from CT26 and MC38 cell lines showed variable growth by the s.c. injection of a scramble-transfected Gm-CSf– patterns (Fig. 3A and Supplementary Fig. S5). Reduced expressing MC38 clone and a Gm-CSF–silenced MC38 clone Gm-CSf expression was associated with increased cell via- into immunodeficient nude mice, thereby allowing us to bility, cell growth, and colony formation ability in MC38 cells avoid interference of Gm-CSf effects on the immune system. but its effects were not significant in CT26 cells (Fig. 3A and Gm-CSf expression in scramble MC38 cells resulted in a Supplementary Fig. S5). This observation led us to consider significant reduction in tumor formation in vivo;atthetime that, in addition to the immune-dependent effects described of sacrifice, Gm-CSf expressing MC38 tumors had, on aver- above, Gm-CSf expression may exert an immune-indepen- age, 20% the volume and 20% the weight of those lacking dent effect on tumor growth (16–20). This possibility was Gm-CSf expression (Fig. 3B). Thus, our data suggest that the supported by the direct relationship between growth effects immune-independent antitumor action of tumoral Gm-CSf and the expression of Gm-CSf receptor subunits (38): only depends on the ectopic expressionofGm-CSfreceptorsby when both Gm-CSf receptor subunits were present (in the tumor.

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 399

Urdinguio et al.

Figure 3. Immune-independent antitumor effects of Gm-CSf on tumor growth in vitro and in vivo. A, characterization of in vitro growth of MC38 and CT26 transfected cells. Cell viability was determined by MTT assay on scramble- and shGm-CSf–stably transfected cells. Ten replicates were assessed per time point. Cell proliferation rate was established by taking daily cell counts of scramble and shGm-CSf cells. Three independent replicates were conducted for each time point. Colony formation assay was conducted seeding cells at different densities (50, 100, and 200 cells). Results are represented in the bar diagram as the mean colony-formation units (CFU) SEM of 3 independent replicates for each density. Scramble-transfected cell lines are shown in blue and shGm- CSf–transfected cell lines in red. MC38 derived clones are represented by continuous lines and CT26 derived clones by discontinuous lines. Gm-CSf expression only caused significant differences in cell proliferation and CFU in MC38 derived cells. B, diagram of the in vivo model studied for the analysis of immune-independent effects showing that nude mice (n ¼ 12) were s.c. injected with scramble (on left flank, represented in blue) and shGm-CSf (on right flank, represented in red) MC38 cells. Evolution of tumor growth measured as tumor volume and a graph depicting endpoint tumor weights are also shown. The MC38 scramble cell line produced significantly lower tumor volume and weight in nude mice. A photograph of 1 representative mouse from this experiment is included. Results are expressed as the mean SEM. , P < 0.05; , P < 0.01; , P < 0.001 (Student's t test).

Synergic immune-dependent and immune-independent MC38 clone (which does not express Csf2ra; Supplementary antitumor effect of tumoral GM-CSF Fig. S7), which supports the notion that the immune-indepen- We studied the combinatory effect of Gm-CSf and its dent effect is crucial for the complete suppression of tumor receptor subunits' expression using Gm-CSf–expressing and growth in our mouse model, where the combination of silenced MC38 clones injected into their autologous immune- immune-dependent and immune-independent effects of competent C57BL/6 mice. In this model, where both immune- Gm-CSf leads to the complete loss of tumor formation dependent and immune-independent effects coexist, we found capability. that the MC38 scramble clone was detectable only for the ﬁrst To determine whether our ﬁndings in mice would also hold 10 days before disappearing completely (Fig. 4A and Supple- for human clinical samples, we analyzed GM-CSF, CSF2RA, and mentary Fig. S6). This effect did not occur on the wild-type CSF2RB expression in a set of 124 colorectal cancer patients on

400 Cancer Res; 73(1) January 1, 2013 Cancer Research

Antitumor Activity of GM-CSF in Colorectal Tumors

Figure 4. Combination of immune- dependent and immune- independent antitumor effects in vivo. A, diagram of the in vivo model studied for the analysis of the combination of immune-dependent and immune-independent effects showing that C57BL/6 mice (n ¼ 8) were s.c. injected with scramble (blue line) or shGm-CSf (red line) MC38 cells in both ﬂanks. Evolution of tumor growth measured as tumor volume is shown. The MC38 scramble clone was only detectable for the ﬁrst 10 days, disappearing completely after that. Photographs of 1 representative mouse from each analyzed group are included. Results are expressed as the mean SEM. , P < 0.001 (Student's t test). B, Kaplan–Meier study of overall survival from a cohort of 124 patients in which GM-CSF, CSF2RA, and CSF2RB expression levels were analyzed in both healthy colon tissues and colon cancer samples. Expression of the 3 genes was independent of patient sex (P ¼ 0.828, Mann–Whitney U test), and the age of onset (P ¼ 0.957, Mann– Whitney U test). The group of patients with overexpression of the 3 genes in the tumor sample (green line) shows a 100% overall survival at 5-year follow up (P ¼ 0.021, Kaplan– Meier log-rank test).

whom a long clinical follow-up was available. GM-CSF over- not observed when comparing patients that overexpressed expression (more than 2-fold) was observed in 37.9% (47 of 124) GM-CSF without concurrent changes in receptor subunit of patients. CSF2RA and CSF2RB were overexpressed (more levels, or in patients that overexpressed both receptor subunits than 2-fold) in 22.6% (28 of 124) and 27.2% (34 of 124) of without changes in GM-CSF expression (Supplementary Fig. patients, respectively. Demographic and clinical characteris- S9A and S9B), which sustains the notion that ectopic expres- tics of the patients included in our study are shown in sion of GM-CSF and its receptor subunits by human colorectal Supplementary Table S1. tumors has a strong antitumor effect. Our data show that in To test whether GM-CSF and its receptor were expressed by 100% of cases where the 3 genes were overexpressed overall primary colon cancer cells, we conducted RNA in situ hybrid- survival was dramatically increased. We would thus propose ization of GM-CSF as well as the immunofluorescence detec- that GM-CSF in combination with its receptor subunits should tion of its receptor. As shown in Supplementary Fig. S8, we be considered a valuable marker for diagnosis and prognosis in confirmed that cells from tumor colon epithelia aberrantly clinical studies that may prove helpful in the stratification of express these molecules. As expected, RNA in situ hybridization patients. Furthermore, demethylation-associated GM-CSF of GM-CSF was positively detected in tonsil and undetectable overexpression accompanied by CSF2RA and CSF2RB over- in muscle tissue (Supplementary Fig. S8A). GM-CSF receptor expression is a possible indicator of outcome in colorectal was located in the membrane of colon tumor epithelial cells, cancer patients. and colocalized with beta-catenin staining (which specifically marked epithelial cells, but not stroma cells or infiltrated Anti-PD-1 adjuvant therapy improves the antitumor lymphocytes; Supplementary Fig. S8B). effects of tumoral GM-CSF Concurrent overexpression of GM-CSF, CSF2RA, and Anti-PD-1 is an antibody raised against Programmed death 1 CSF2RB, which was observed in 6.5% (8 of 124) of patients, (PD-1; ref. 39), that enhances T cell activity in chronic pathol- was strongly associated with an increase in overall survival ogies such as cancer (7, 40, 41). The combination of PD-1 rates (Fig. 4B). All the patients with tumors overexpressing, the blockade with GM-CSF–secreting tumor cell immunotherapy 3 genes survived at least 5 years from diagnosis. This effect was has been recently shown to significantly improve antitumor

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 401

Urdinguio et al.

Figure 5. Anti-PD-1 treatment of engrafted tumors in vivo. Diagram of the in vivo model studied for the analysis of anti-PD-1 effect on tumors that differed in their Gm-CSf expression showing that BALB/c mice (n ¼ 16) were s.c. injected with scramble or shGm-CSf CT26 cells in both ﬂanks and treated on days 3, 6, 9, 12, and 24 with intraperitoneal injection of PBS or anti-PD-1. At the time of sacriﬁce, we found a higher percentage of reduction of tumor growth in mice that had Gm-CSf–expressing tumors and were treated with anti-PD-1. Furthermore, there was a 25% remission of tumors in mice treated with anti-PD-1 when the tumor did not express Gm-CSf (red bars), whereas there was a 50% remission, or growth arrest, in mice treated with anti-PD-1 that carried Gm-CSf– expressing scramble cells (blue bars). Photographs of 1 representative mouse from each analyzed group are also included.

responses (41). To determine whether anti-PD-1 also potenti- vaccine is being studied in a phase III trial for patients with ates the antitumor effect of Gm-CSf ectopically secreted by advanced melanoma because of the promising results from tumor cells, we injected Gm-CSf–expressing scramble and previous trials (43). In addition, the GVAX (Cell Genesys) shGm-CSf CT26 cells into their autologous BALB/c mice and approach in immunotherapy involves the administration of administrated PBS or anti-PD-1 treatment. At the moment of irradiated tumor cells that have been engineered to secrete sacrifice, we found a higher percentage of reduced tumor GM-CSF and, in this manner, activate dendritic cells and the growth in mice with Gm-CSf–expressing tumors when they immune system with the antigens provided by the whole tumor were treated with anti-PD-1 (Fig. 5). Furthermore, we found a cells. A significant body of preclinical data supported its 25% remission of tumors in mice developing Gm-CSF–silenced antitumor efficacy, especially in combination with agents such CT26 tumors when they were treated with anti-PD-1, whereas a as anti-CTLA-4, and anti-PD-1 (41, 44). However, phase III 50% remission or reduction of tumor growth progression was clinical trials of GVAX in patients with prostate cancer did not observed when the anti-PD-1 treatment was administered to produce significant results (12). GM-CSF is a valuable tool in mice developing Gm-CSf–secreting CT26 tumors (Fig. 5). cancer immunotherapy but there are still several unanswered questions that need to be addressed before the complexity of Discussion the results from clinical trials can be explained. The cytokine GM-CSF was described because of its ability as Herein, we show that aberrant DNA demethylation of GM- a colony-stimulating factor to promote the formation of gran- CSF can lead to its ectopic secretion by colorectal tumors. ulocytes and monocytes (4). However, GM-CSF also influences However, the natural role of DNA methylation at the GM-CSF dendritic cells and this property is currently being used in promoter is still largely unknown. According to previous DNA various immunotherapy strategies to stimulate the antitumor methylation data from Human Infinium Methylation Array 27k action of the immune system (12). Importantly, the first US (Illumina; ref. 45), GM-CSF promoter is methylated in human Food and Drug Administration approval in history for a stem cells and human healthy primary tissues but not in T þ þ therapeutic cancer vaccine was granted to Sipuleucel-T (Pro- CD4 and T CD8 lymphocytes, that is, it follows a tissue- venge; Dendreon, Inc.) for castration-resistant prostate cancer. specific pattern. Furthermore, comparing this methylation Sipuleucel-T is an autologous dendritic cell-based vaccine in data with available gene expression data from Amazonia which cells are loaded with a fusion protein including GM-CSF. "Human body index" series (46), which comprises human Reinfusion of such activated cells into the patient showed a transcriptome list annotations, we see that only the cells with þ þ 4-month benefit relative to the control group in randomized GM-CSF promoter DNA demethylation (T CD4 and CD8 phase III clinical trials (12, 42). Another strategy, called lymphocytes) are able to express this cytokine after activation OncoVEXGM-CSF (BioVex), involves the administration of (Supplementary Fig. S10). Therefore, GM-CSF expression could GM-CSF through the second generation of oncolitic Herpes be under a double regulatory mechanism with a first level of simplex type 1 virus encoding human GM-CSF. The virus is regulation in which loss of DNA methylation would allow genetically reprogrammed to attack cancerous cells. This further gene expression after cell activation. Thus, GM-CSF

402 Cancer Res; 73(1) January 1, 2013 Cancer Research

Antitumor Activity of GM-CSF in Colorectal Tumors

promoter demethylation might be important in priming this acknowledged to also include epigenetic alterations) in the specific hematopoietic gene for activation/expression in genome of cancer cells during the tumorogenic process (50, response to external stimuli (45). This natural mechanism of 51). Even though driver modifications are considered causa- regulation could suffer aberrant changes in colorectal cancers tive in the development of a tumor, other changes that could leading to its ectopic secretion by the tumor cells. Further be considered passenger modifications, that is, those not studies are necessary to decipher the role of DNA methylation directly related with the tumor development, may become in the natural regulation of GM-CSF and the molecular crucial for tumor progression and be considered driver mechanisms that lead to its loss in colon cancer cells. changes as long as they give tumor advantage against antitu- Our data indicate that tumor-secreted GM-CSF can induce mor therapies. immune-dependent antitumor responses but that it also has a Our results on human primary tumors may have clinical strong direct antitumor action when the tumors express GM- implications, as they suggest that expression of GM-CSF and its CSF receptors. Although the immune-dependent antitumor receptor subunits are indicators of good prognosis in colorec- effect of GM-CSF has been known for some time, hence its use tal cancer. In addition, tumoral expression of the GM-CSF in immunotherapy (12, 47), this is the first time that GM-CSF receptor could also be useful for patient stratification in cancer ectopically secreted by tumor cells is shown to have immune- immunotherapy (i.e., tumors expressing GM-CSF receptor may dependent antitumor effects. This observation might be be better candidates to receive GM-CSF vaccination). The important for the clinical use of GM-CSF vaccines, and further strong immune-independent antitumor activity of GM-CSF studies should determine whether the effects of these vaccines reported in this study suggests that future clinical trials with are affected by tumor-secreted GM-CSF. On the other hand, the GM-CSF–secreting vaccines should also take into account the immune-independent effect of tumor-secreted GM-CSF is still expression of the GM-CSF receptor by tumors. Moreover, poorly understood. Some studies have found it to exert an the results obtained in previous Phase III clinical trials with antiproliferative effect (16, 17), whereas others have shown it to these vaccines (37, 52) should be reevaluated taking into promote growth (18–20) and yet others found it to have no account the tumor expression of GM-CSF receptor subunits. reproducible influence on growth rate (21, 22). Two main In addition, our results indicate that anti-PD-1 treatment may points may explain, to a certain extent, these contradictory enhance the antitumor effect of Gm-CSf ectopically secreted by results: firstly, the dose of GM-CSF used may well be relevant to tumors. Further studies are necessary to determine whether the evaluation of its effects at a physiologically relevant level patients with tumors expressing GM-CSF are better candidates and, secondly, the presence of GM-CSF receptor should also be to receive anti-PD-1 or other enhancers of the clinical activity ascertained to ensure that the studied cells do in fact have the of GM-CSF (53). means to signal GM-CSF presence. Our study goes further in the evaluation of immune-independent effects of GM-CSF as Disclosure of Potential Conflicts of Interest we investigate the effects in vivo and find that tumors with No potential conflicts of interest were disclosed. concurrent expression of Gm-CSf and its receptor do not grow when injected in their autologous immunocompetent mice. Authors' Contributions Conception and design: R.G. Urdinguio, A.F. Fernandez, A.J. Obaya, L. Rodrigo, Furthermore, human colorectal tumors with concurrent over- C. Lopez-Otin, M.F. Fraga expression lead to a very good prognosis and a 100% 5-year Development of methodology: R.G. Urdinguio, A.F. Fernandez, A. Moncada- fi Pazos, C. Huidobro, R.M. Rodriguez, P. Martinez-Camblor, M. Santacana, X. overall survival rate. These ndings support the notion of the Matias-Guiu, M.F. Fraga strong role of the GM-CSF receptor in the immune-indepen- Acquisition of data (provided animals, acquired and managed patients, dent action of GM-CSF on tumor growth and indicate the provided facilities, etc.): R.G. Urdinguio, A.F. Fernandez, C. Huidobro, T. Bernal, A. Parra-Blanco, L. Rodrigo, B. Soldevilla, G. Dominguez, S. Cal, M.F. Fraga importance of verifying the expression of this receptor by Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tumor cells when selecting a strategy for the treatment of computational analysis): R.G. Urdinguio, A.F. Fernandez, A. Moncada-Pazos, C. Ferrero, P. Martinez-Camblor, M. Santacana, X. Matias-Guiu, B. Soldevilla, G. colorectal cancer. On the other hand, a previous report on skin Dominguez, M.F. Fraga cancer (48) showed the tumorigenic role of GM-CSF when this Writing, review, and/or revision of the manuscript: R.G. Urdinguio, A.F. cytokine is combined with G-CSF. Together, these results Fernandez, A. Moncada-Pazos, L. Rodrigo, F. Bonilla, S. Cal, C. Lopez-Otin, M.F. Fraga indicate that the role of GM-CSF in cancer is complex and its Administrative, technical, or material support (i.e., reporting or orga- tumorigenic effect might depend on the tumor context. nizing data, constructing databases): R.G. Urdinguio, A.F. Fernandez, M. Unexpectedly, our data show that promoter demethylation- Santacana, X. Matias-Guiu, M.F. Fraga Study supervision: R.G. Urdinguio, A.F. Fernandez, A.J. Obaya, L. Rodrigo, C. associated expression of GM-CSF in colon cancer cells impairs Lopez-Otin, M.F. Fraga tumorigenesis suggesting that not all the epigenetic alterations occurring in a tumor necessarily favor tumorigenesis. This Acknowledgments possibility is in line with reports showing that, in many gene The authors thank Dr. Ignacio Melero, Juan Dubrot, and Asis Palazon for their de novo advice with mouse cell lines culture and the staff of the Animal Facilities at the promoters, aberrant hypermethylation is not respon- University of Oviedo for technical and animal care assistance. sible for gene repression as these genes are already repressed in healthy tissues (49). Collectively, these data imply that a Grant Support number of DNA methylation alterations in cancer are not This work has been financially supported by IUOPA (R.G. Urdinguio, A.F. subject to growth selection (49), which is in consonance with Fernandez, and R.M. Rodriguez); Fundacion Cientifica de la aecc (R.G. Urdin- fi guio); FIS (FI07/00380 to C. Huidobro); the Spanish Ministry of Health (PI061267; the differentiation between "driver" and "passenger" modi ca- PS09/024549 to M.F. F Fraga); the FIS/FEDER (PI11/01728 to A.F. Fernandez); tions (previously referred to as mutations, but nowadays the ISCIII (CP11/00131 to A.F. Fernandez); the Spanish National Research

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 403

Urdinguio et al.

Council (CSIC; 200820I172 to M.F. Fraga); and the Community of Asturias advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this (FYCIT IB09-106 to M.F. Fraga). The IUOPA is supported by the Obra Social fact. Cajastur, Spain. The costs of publication of this article were defrayed in part by the Received March 2, 2012; revised September 28, 2012; accepted September 28, payment of page charges. This article must therefore be hereby marked 2012; published OnlineFirst October 29, 2012.

References 1. Vesely MD, Kershaw MH, Schreiber RD, Smyth MJ. Natural innate and 21. Berdel WE, Zafferani M, Senekowitsch R, Kreuser ED, Thiel E. Effect of adaptive immunity to cancer. Annu Rev Immunol 2011;29:235–71. interleukin-3 and granulocyte-macrophage colony-stimulating factor 2. Zitvogel L, Tesniere A, Kroemer G. Cancer despite immunosurveil- on growth of xenotransplanted human tumour cell lines in nude mice. lance: immunoselection and immunosubversion. Nat Rev Immunol Eur J Cancer 1992;28:377–80. 2006;6:715–27. 22. Foulke RS, Marshall MH, Trotta PP, Von Hoff DD. In vitro assess- 3. Dougan M, Dranoff G. Immune therapy for cancer. Annu Rev Immunol ment of the effects of granulocyte-macrophage colony-stimulating 2009;27:83–117. factor on primary human tumors and derived lines. Cancer Res 4. Metcalf D, Begley CG, Johnson GR, Nicola NA, Vadas MA, Lopez AF, 1990;50:6264–7. et al. Biologic properties in vitro of a recombinant human granulocyte- 23. Chen Z, Malhotra PS, Thomas GR, Ondrey FG, Duffey DC, Smith macrophage colony-stimulating factor. Blood 1986;67:37–45. CW, et al. Expression of proinflammatory and proangiogenic cyto- 5. Smith BR. Regulation of hematopoiesis. Yale J Biol Med 1990;63: kines in patients with head and neck cancer. Clin Cancer Res 1999; 371–80. 5:1369–79. 6. Dranoff G. GM-CSF-secreting melanoma vaccines. Oncogene 2003; 24. Braun B, Lange M, Oeckler R, Mueller MM. Expression of G-CSF and 22:3188–92. GM-CSF in human meningiomas correlates with increased tumor 7. Li B, Simmons A, Du T, Lin C, Moskalenko M, Gonzalez-Edick M, et al. proliferation and vascularization. J Neurooncol 2004;68:131–40. Allogeneic GM-CSF-secreting tumor cell immunotherapies generate 25. Mueller MM, Herold-Mende CC, Riede D, Lange M, Steiner HH, potent anti-tumor responses comparable to autologous tumor cell Fusenig NE. Autocrine growth regulation by granulocyte colony-stim- immunotherapies. Clin Immunol 2009;133:184–97. ulating factor and granulocyte macrophage colony-stimulating factor 8. Mach N, Gillessen S, Wilson SB, Sheehan C, Mihm M, Dranoff G. in human gliomas with tumor progression. Am J Pathol 1999; Differences in dendritic cells stimulated in vivo by tumors engineered to 155:1557–67. secrete granulocyte-macrophage colony-stimulating factor or Flt3- 26. Mueller MM, Fusenig NE. Constitutive expression of G-CSF and GM- ligand. Cancer Res 2000;60:3239–46. CSF in human skin carcinoma cells with functional consequence for 9. Huang AY, Golumbek P, Ahmadzadeh M, Jaffee E, Pardoll D, Levitsky tumor progression. Int J Cancer 1999;83:780–9. H. Role of bone marrow-derived cells in presenting MHC class I- 27. Revoltella RP, Menicagli M, Campani D. Granulocyte-macrophage restricted tumor antigens. Science 1994;264:961–5. colony-stimulating factor as an autocrine survival-growth factor in 10. Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nat human gliomas. Cytokine 2012;57:347–59. Rev Cancer 2004;4:11–22. 28. Mosmann T. Rapid colorimetric assay for cellular growth and survival: 11. Nagai E, Ogawa T, Kielian T, Ikubo A, Suzuki T. Irradiated tumor cells application to proliferation and cytotoxicity assays. J Immunol Meth- adenovirally engineered to secrete granulocyte/macrophage-colony- ods 1983;65:55–63. stimulating factor establish antitumor immunity and eliminate pre- 29. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. existing tumors in syngeneic mice. Cancer Immunol Immunother Clonogenic assay of cells in vitro. Nat Protoc 2006;1:2315–9. 1998;47:72–80. 30. Radoja S, Saio M, Frey AB. CD8þ tumor-infiltrating lymphocytes are 12. Le DT, Pardoll DM, Jaffee EM. Cellular vaccine approaches. Cancer primed for Fas-mediated activation-induced cell death but are not J 2010;16:304–10. apoptotic in situ. J Immunol 2001;166:6074–83. 13. Nemunaitis J, Sterman D, Jablons D, Smith JW II, Fox B, Maples P, 31. Fraga MF, Esteller M. Epigenetics and aging: the targets and the et al. Granulocyte-macrophage colony-stimulating factor gene-mod- marks. Trends Genet 2007;23:413–8. ified autologous tumor vaccines in non-small-cell lung cancer. J Natl 32. Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer Inst 2004;96:326–31. Cancer 2004;4:143–53. 14. Lahm H, Wyniger J, Hertig S, Yilmaz A, Fischer JR, Givel JC, et al. 33. Caffarelli E, Filetici P. Epigenetic regulation in cancer development. Secretion of bioactive granulocyte-macrophage colony-stimulating Front Biosci 2011;17:2682–94. factor by human colorectal carcinoma cells. Cancer Res 1994;54: 34. Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of 3700–2. some human cancers from their normal counterparts. Nature 1983; 15. Steube KG, Meyer C, Drexler HG. Secretion of functional hematopoi- 301:89–92. etic growth factors by human carcinoma cell lines. Int J Cancer 1998; 35. Estecio MR, Gharibyan V, Shen L, Ibrahim AE, Doshi K, He R, et al. 78:120–4. LINE-1 hypomethylation in cancer is highly variable and inversely 16. Ruff MR, Farrar WL, Pert CB. Interferon gamma and granulocyte/ correlated with microsatellite instability. PLoS One 2007;2:e399. macrophage colony-stimulating factor inhibit growth and induce anti- 36. Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, et al. gens characteristic of myeloid differentiation in small-cell lung cancer Vaccination with irradiated tumor cells engineered to secrete murine cell lines. Proc Natl Acad Sci U S A 1986;83:6613–7. granulocyte-macrophage colony-stimulating factor stimulates potent, 17. Yamashita Y, Nara N, Aoki N. Antiproliferative and differentiative effect specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S of granulocyte-macrophage colony-stimulating factor on a variant A 1993;90:3539–43. human small cell lung cancer cell line. Cancer Res 1989;49:5334–8. 37. Simmons AD, Li B, Gonzalez-Edick M, Lin C, Moskalenko M, Du T, et al. 18. Berdel WE, Danhauser-Riedl S, Steinhauser G, Winton EF. Various GM-CSF-secreting cancer immunotherapies: preclinical analysis of human hematopoietic growth factors (interleukin-3, GM-CSF, G-CSF) the mechanism of action. Cancer Immunol Immunother 2007;56: stimulate clonal growth of nonhematopoietic tumor cells. Blood 1653–65. 1989;73:80–3. 38. Matsuguchi T, Zhao Y, Lilly MB, Kraft AS. The cytoplasmic domain of 19. Dedhar S, Gaboury L, Galloway P, Eaves C. Human granulocyte- granulocyte-macrophage colony-stimulating factor (GM-CSF) recep- macrophage colony-stimulating factor is a growth factor active on a tor alpha subunit is essential for both GM-CSF-mediated growth and variety of cell types of nonhemopoietic origin. Proc Natl Acad Sci U S A differentiation. J Biol Chem 1997;272:17450–9. 1988;85:9253–7. 39. Yamazaki T, Akiba H, Iwai H, Matsuda H, Aoki M, Tanno Y, et al. 20. Onetto N. Extra hematopoietic effect of colony-stimulating factors. Expression of programmed death 1 ligands by murine T cells and APC. Blood 1989;74:1446–7. J Immunol 2002;169:5538–45.

404 Cancer Res; 73(1) January 1, 2013 Cancer Research

Antitumor Activity of GM-CSF in Colorectal Tumors

40. Curran MA, Allison JP. Tumor vaccines expressing flt3 ligand syner- 46. Le Carrour T, Assou S, Tondeur S, Lhermitte L, Lamb N, Reme T, gize with ctla-4 blockade to reject preimplanted tumors. Cancer Res et al. Amazonia!: an online resource to google and visualize public 2009;69:7747–55. humanwholegenomeexpression data. Open Bioinformatics 41. Li B, VanRoey M, Wang C, Chen TH, Korman A, Jooss K. Anti- J 2010;4:5–10. programmed death-1 synergizes with granulocyte macrophage colo- 47. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of ny-stimulating factor–secreting tumor cell immunotherapy providing age. Nature 2011;480:480–9. therapeutic benefit to mice with established tumors. Clin Cancer Res 48. Obermueller E, Vosseler S, Fusenig NE, Mueller MM. Cooperative 2009;15:1623–34. autocrine and paracrine functions of granulocyte colony-stimulating 42. Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, factor and granulocyte-macrophage colony-stimulating factor in et al. Sipuleucel-T immunotherapy for castration-resistant prostate the progression of skin carcinoma cells. Cancer Res 2004;64: cancer. N Engl J Med 2010;363:411–22. 7801–12. 43. Kaufman HL, Bines SD. OPTIM trial: a Phase III trial of an oncolytic 49. Keshet I, Schlesinger Y, Farkash S, Rand E, Hecht M, Segal E, et al. herpes virus encoding GM-CSF for unresectable stage III or IV mel- Evidence for an instructive mechanism of de novo methylation in anoma. Future Oncol 2010;6:941–9. cancer cells. Nat Genet 2006;38:149–53. 44. van Elsas A, Hurwitz AA, Allison JP. Combination immunotherapy of 50. Kalari S, Pfeifer GP. Identification of driver and passenger DNA meth- B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen ylation in cancer by epigenomic analysis. Adv Genet 2010;70:277–308. 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor 51. Torkamani A, Verkhivker G, Schork NJ. Cancer driver mutations in (GM-CSF)-producing vaccines induces rejection of subcutaneous and protein kinase genes. Cancer Lett 2009;281:117–27. metastatic tumors accompanied by autoimmune depigmentation. 52. Gupta R, Emens LA. GM-CSF-secreting vaccines for solid tumors: J Exp Med 1999;190:355–66. moving forward. Discov Med 2010;10:52–60. 45. Calvanese V, Fernandez AF, Urdinguio RG, Suarez-Alvarez B, Mangas 53. Jinushi M, Hodi FS, Dranoff G. Enhancing the clinical activity of C, Perez-Garcia V, et al. A promoter DNA demethylation landscape of granulocyte-macrophage colony-stimulating factor-secreting tumor human hematopoietic differentiation. Nucleic Acids Res 2011. cell vaccines. Immunol Rev 2008;222:287–98.

www.aacrjournals.org Cancer Res; 73(1) January 1, 2013 405

Immune-Dependent and Independent Antitumor Activity of GM-CSF Aberrantly Expressed by Mouse and Human Colorectal Tumors

Rocio G. Urdinguio, Agustin F. Fernandez, Angela Moncada-Pazos, et al.

Cancer Res 2013;73:395-405. Published OnlineFirst October 29, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-0806

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2012/11/01/0008-5472.CAN-12-0806.DC1

Cited articles This article cites 52 articles, 19 of which you can access for free at: http://cancerres.aacrjournals.org/content/73/1/395.full#ref-list-1

Citing articles This article has been cited by 8 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/73/1/395.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/73/1/395. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.