micrornas reprogram normal fibroblasts into cancer … · micrornas (mirna) are small noncoding rna...

1100 | CANCER DISCOVERY�DECEMBER 2012 www.aacrjournals.org

RESEARCH BRIEF

ABSTRACT Cancer-associated fi broblasts (CAF) are a major constituent of the tumor stroma, but little is known about how cancer cells transform normal fi broblasts into CAFs.

microRNAs (miRNA) are small noncoding RNA molecules that negatively regulate gene expression at a posttranscriptional level. Although it is clearly established that miRNAs are deregulated in human can-cers, it is not known whether miRNA expression in resident fi broblasts is affected by their interaction with cancer cells. We found that in ovarian CAFs, miR-31 and miR-214 were downregulated, whereas miR-155 was upregulated when compared with normal or tumor-adjacent fi broblasts. Mimicking this deregulation by transfecting miRNAs and miRNA inhibitors induced a functional conversion of normal fi broblasts into CAFs, and the reverse experiment resulted in the reversion of CAFs into normal fi brob-lasts. The miRNA-reprogrammed normal fi broblasts and patient-derived CAFs shared a large number of upregulated genes highly enriched in chemokines, which are known to be important for CAF function. The most highly upregulated chemokine, CCL5, (C-C motif ligand 5) was found to be a direct target of miR-214. These results indicate that ovarian cancer cells reprogram fi broblasts to become CAFs through the action of miRNAs. Targeting these miRNAs in stromal cells could have therapeutic benefi t.

SIGNIFICANCE: The mechanism by which quiescent fi broblasts are converted into CAFs is unclear. The present study identifi es a set of 3 miRNAs that reprogram normal fi broblasts to CAFs. These miRNAs may represent novel therapeutic targets in the tumor microenvironment. Cancer Discov; 2(12); 1100–8.©2012 AACR.

Authors’ Affi liations: 1 Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, The University of Chicago; and 2 Division of Hematology/Oncology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois

Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

M.E. Peter and E. Lengyel share last authorship.

Corresponding Authors: Ernst Lengyel, University of Chicago, 5841 South Maryland Avenue, MC 2050, Chicago, IL 60637. Phone: 773-702-6722; Fax: 1-773-702-5411; E-mail: [email protected] ; and Marcus Peter, Northwestern University Feinberg School of Medicine, 303 E Superior Street, Lurie 6-123, Chicago, IL 60611. Phone: 312-503-1291; Fax: 1-312-503-0189; E-mail: [email protected] doi: 10.1158/2159-8290.CD-12-0206 ©2012 American Association for Cancer Research.

MicroRNAs Reprogram Normal Fibroblasts into Cancer-Associated Fibroblasts in Ovarian Cancer Anirban K. Mitra 1 , Marion Zillhardt 1 , Youjia Hua 2 , Payal Tiwari 1 , Andrea E. Murmann 2 , Marcus E. Peter 2 , and Ernst Lengyel 1

on March 13, 2021. © 2012 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst November 21, 2012; DOI: 10.1158/2159-8290.CD-12-0206

http://cancerdiscovery.aacrjournals.org/

DECEMBER 2012�CANCER DISCOVERY | 1101

miRNAs Regulate CAFs RESEARCH BRIEF

INTRODUCTION

During invasion and metastasis, cancer cells change the normal stroma into a “reactive” environment, which promotes the growth and viability of tumor cells ( 1 ). Upon interaction with cancer cells, quiescent resident fi broblasts, which are the predominant cell type in normal stroma, are transformed into cancer-associated fi broblasts (CAF), which become a major component of the tumor stroma. CAFs promote cancer cell invasion, proliferation, and metastasis by secreting cytokines and chemokines, which stimulate receptor tyrosine kinase signaling and epithelial–mesenchymal transition (EMT) pro-grams ( 1 ). Moreover, CAFs secrete a distinctive extracellular matrix that promotes the attachment and invasion of tumor cells ( 2 ). Several steps of the bidirectional signaling between cancer cells and fi broblasts have been elucidated. Neoplastic cells secrete cytokines such as interleukin (IL)-6, IL-8, and IL-1β to activate fi broblasts and stimulate their proliferation ( 3 ). The CAFs, in return, secrete cancer-activating chemokines such as SDF-1α, thereby stabilizing and promoting tumori-genesis ( 4 ). However, it is currently not clear how cancer cells reprogram quiescent fi broblasts to become CAFs.

MicroRNAs (miRNA) are small noncoding RNA mol-ecules that negatively regulate gene expression at a post-transcriptional level ( 5 ). miRNAs are powerful regulators of cellular differentiation, as they affect the expression of many genes and are deregulated in cancer cells ( 6 ). It is not clear at present whether endogenous miRNAs are involved in the conversion of resident fi broblasts to CAFs. Several reports have shown that miRNAs can reprogram various somatic cells to become pluripotent stem cells ( 7 ). A combi-nation of miR-124 and the transcription factors MYT1L and BRN2 reprograms primary human dermal fi broblasts into functional neurons that exhibit typical neural morphology, fi re action potentials, and produce functional synapses ( 8 ). miR-15 and mir-16, are downregulated in fi broblasts sur-rounding prostate tumors ( 9 ), but their functional role in promoting tumor growth is unclear. Recently, miR-511-3p has been reported to prevent the tumor-promoting activity of tumor-associated macrophages ( 10 ).

Epithelial serous ovarian carcinoma has a unique pat-tern of metastasis that remains largely restricted to the abdominal cavity, in which the omentum, a large fat pad in front of the bowel, is a major site of colonization ( 11 ). Ovarian tumors and metastases have a substantial stromal component of which CAFs are an important constituent ( 3 ). Although the role of CAFs in ovarian cancer progression is well established, the mechanism of CAF formation remains unclear ( 12 ).

Most miRNA studies have focused on the tumor cell, but little is known about miRNA expression in the tumor microenvironment. Given the broad regulatory role of miRNAs, we have now investigated whether miRNAs are involved in the reprogramming of normal fibroblasts to CAFs, thereby promoting tumorigenesis. We report here that the combination of 3 miRNAs induces normal human omental fibroblasts to become CAFs, leading to the upregulation of chemokines important in invasion and metastasis.

RESULTS

Identifi cation of miRNAs Deregulated in Cancer-Associated Fibroblasts

The miRNA expression in CAFs was compared with that in primary human normal omental fi broblasts (NOF) using 2 independent, unbiased approaches. In one approach, pri-mary human CAFs were isolated from the omental metas-tases of patients with metastatic serous ovarian cancer and compared with tumor-adjacent NOFs extracted from a nor-mal area of the omentum, at least 1 inch from the tumor, from the same patients. In the other approach, NOFs from the omentum of cancer-free patients operated on for benign gynecologic disease (e.g., fi broids) were compared with NOFs cocultured with HeyA8 ovarian cancer cells ( Fig. 1A ). While the former approach identifi es the miRNA changes that occur during tumor progression, the latter detects the early changes induced exclusively by cancer cells. Confocal time-lapse micro-scopy revealed that CAFs were more migratory (Supplemen-tary Fig. S1A and S1B) and induced the invasion of different ovarian cancer cell lines more effi ciently than either NOFs or tumor-adjacent NOFs (Supplementary Fig. S2A). The induced CAFs, generated by a 7-day coculture of NOFs with ovarian cancer cells, had an enhanced ability to promote tumor cell invasiveness (Supplementary Fig. S2B), suggesting that cancer cells impart CAF-like properties to these cells.

Therefore, the miRNA expression of CAFs was compared with that of adjacent NOFs and the miRNA expression of NOFs was compared with that of induced CAFs ( Fig. 1A ). A miRNA array analysis of RNA isolated from these cells iden-tifi ed 19 signifi cantly expressed miRNAs that were upregu-lated ( P < 0.05) and 15 miRNAs that were downregulated in the cocultured, induced CAFs (Supplementary Table S1). Of these miRNAs, 1 was found to be upregulated and 4 down-regulated in CAFs compared with matching adjacent NOFs from 6 patients with postmenopausal, advanced serous ovar-ian carcinoma ( Fig. 1B and Supplementary Table S1). In both induced CAFs and primary patient-derived CAFs, the miRNAs that were most signifi cantly up- and downregulated were miR-155 and miR-214 , respectively, and therefore these were selected for further testing. miR-31 was included because it was the second most signifi cantly downregulated miRNA in induced CAFs and because it was reported as downregulated in fi broblast cell lines derived from patients with endometrial cancer ( 13 ). Using quantitative real-time PCR (qRT-PCR), downregulation of miR-31 and miR-214 and upregulation of miR-155 was confi rmed in all CAF and induced CAF samples (Supplementary Fig. S3A). In situ hybridization of an omental metastasis from a patient with ovarian cancer confi rmed that the expression of miR-214 was lost in CAFs and high in adjacent NOFs; an opposite expres-sion pattern was observed for miR-155 (Supplementary Fig. S3B).

Reversible Conversion of Normal Fibroblasts into CAFs

Triple transfection of NOFs with anti- miR-31 , anti- miR-214 , and pre- miR-155 (to generate miR-CAFs) enhanced fi broblast migration as well as the invasion and colony formation of cocultured HeyA8 and SKOV3ip1 cells ( Fig. 1C–E ), suggesting





Mitra et al.RESEARCH BRIEF

Figure 1. Identifi cation of miRNAs that are both markers and regulators of CAFs. A, left, isolation of CAFs and tumor-adjacent NOFs from partially tumor-transformed omentum of patients with serous ovarian cancer. Right, generation of induced CAFs by coculturing fl uorescently labeled NOFs from the omentum of noncancer patients with ovarian cancer cell lines. B, list of upregulated or downregulated miRNAs. miRNAs were sorted according to the P value of correlation. The most upregulated miRNA, miR-155 (green), and 2 most downregulated miRNAs, miR-214 and miR-31 (red), are highlighted. C, migration of NOFs triple transfected with anti- miR-31 , anti- miR-214 , and pre- miR-155 (to generate miR-CAFs) and CAFs triple transfected with pre- miR-214 , pre- miR-31 , and anti- miR-155 (to generate NOFs) compared with matching scrambled controls. D and E, tumor cell invasion and colony formation of HeyA8 (D) and SKOV3ip1 (E) cells in the presence of triple-transfected NOFs/CAFs. Data are shown as fold change normalized to NOFs transfected with matching scrambled controls. Error bars represent SD of 3 independent experiments. One-tailed t tests were conducted. F, subcutane-ous tumor growth in mice (10 mice/group) injected with either HeyA8 cells expressing luciferase alone, or HeyA8 cells coinjected with immortalized NOFs transiently expressing either scrambled control (scr) or anti- miR-31 , anti- miR-214 , and pre- miR-155 , or with immortalized CAFs. Luciferase activity was quantifi ed after 14 days and normalized to HeyA8 cells alone and the fold change in radiance plotted. One-way ANOVA was conducted comparing all groups to HeyA8 + scrambled control NOFs (scr). *, P < 0.05. G, immunohistochemistry (Ki-67 staining for proliferation, F4/80 staining for macrophages, H&E) of tumors isolated from mice injected with either HeyA8 cells + NOFs transiently expressing scrambled control (scr) or miR-31 , miR-214 , and anti- miR-155 to induce CAFs (miR-CAFs). Ki-67 staining was quantifi ed. Inset, mouse spleen as positive control for F4/80 staining. Scale bar, 50 μm.

Ex vivoA

C

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hsa-miR-155hsa-miR-214hsa-miR-31hsa-miR-199a-5phsa-let-7b

1.47

−1.22−1.22−1.28−1.26

1.88

−2.22−1.79−1.86−1.43

0.004

0.0260.0430.0310.013

0.002

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that deregulating the expression of the 3 miRNAs could convert NOFs to CAFs. The reverse was also true, as transfection of CAFs with pre-miR-31, pre-miR-214, and anti-miR-155 reduced their migration and also reduced the invasion and colony formation of cocultured ovarian cancer cells to levels similar to those of ovarian cancer cells cocultured with NOFs ( Fig. 1C–E ). miRNA

transfection was validated by qRT-PCR (Supplementary Fig. S4A, B). To assess the contribution of each of the miRNAs to the functional effects of miR-CAFs on cancer cells (migra-tion, invasion, and colony formation), NOFs or CAFs were transfected with individual miRNAs or their inhibitors. miR-214 was active in regulating fi broblast migration and cancer






cell invasiveness, miR-155 predominantly affected cancer cell invasiveness, and miR-31 affected colony formation of the can-cer cells (Supplementary Fig. S5A–S5F).

To test the in vivo activity of the identifi ed miRNAs, miR-CAFs or NOFs transfected with scrambled RNA were coin-jected with luciferase-expressing HeyA8 cells subcutaneously into mice. Coinjected CAFs were used as a positive control. Both miR-CAFs and CAFs signifi cantly enhanced the growth of tumor cells when compared with NOFs ( Fig. 1F ), which was also refl ected in increased expression of the proliferation marker Ki-67 ( Fig. 1G ). This growth enhancement was a direct effect of fi broblasts on tumor cells because the number of infi l-trating macrophages or neutrophilic granulocytes was similar in all experimental groups ( Fig. 1G and data not shown).

Chemokines Are the Most Highly Deregulated Genes in CAFs, Induced CAFs, and miR-CAFs

To determine whether altering the expression of the 3 miRNAs in NOFs to generate mir-CAFs mimicked the dif-

ferentiation of NOFs into CAFs in patients, tumor-adjacent NOFs and CAFs were subjected to gene array analysis. These data were compared with the gene expression profi le obtained from miR-CAFs, which were reprogrammed NOFs derived from patients without cancer through transfection with anti- miR-214/31 and pre- miR-155 . The successful transfection of the miRNAs/miRNA inhibitors and their functional effects on fi broblasts was confi rmed using RT-PCR and invasion assays (data not shown). Supplementary Table S2 lists the genes that were upregulated more than 1.5-fold in CAFs (when compared with adjacent NOFs from the same patients) and in miR-CAFs (when compared with matching NOFs). Surprisingly, 7 of the 10 most highly upregulated genes were chemokines. Most of these chemokines were also found to be upregulated when CAFs were compared with NOFs and miR-CAFs to adjacent NOFs (Supplementary Table S3 and data not shown).

To determine the extent of the reprogramming by miRNAs, we compared gene expression changes between patient-derived CAFs, induced CAFs, and miR-CAFs and their respective

Figure 2. Chemokines are the most highly upregulated genes in CAFs, induced CAFs (iCAF), and miR-CAFs. A, comparison of genes upregulated (green) and downregulated (red) in patient-derived CAFs [compared with matched adjacent NOFs (aNOF)], induced CAFs derived from a 7-day coculture of NOFs with HeyA8 cells (compared with matched NOFs) and miR-CAFs 2 days after triple transfection (compared with matched NOFs). B, ranked list of genes found to be up- (green) and downregulated (red) in CAFs, induced CAFs, and miR-CAFs. C, qRT-PCR to assess upregulation of the 3 highest upregulated chemokines for each array analysis. Error bars represent SD of a triplicate experiment.

1 4.66 CCL202 4.29 CCL53 4.82 IL8/CXCL84 3.95 BCL2A15 3.81 LOC4010976 3.78 CXCR47 3.73 CA28 3.29 TGFA9 3.28 IL1B

10 2.89 NFATC211 2.60 TRIM1412 2.53 CTSS13 2.22 PMAIP1

Rank Fold change Gene

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7.2%17

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584

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1234

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A






normal fi broblasts ( Fig. 2A ). The overlap in induced gene changes between miR-CAFs and CAFs was 15.3% compared with 23.2% between induced CAFs and CAFs. Interestingly, only 13 genes were upregulated and 4 genes downregulated in the CAFs, induced CAFs, and miR-CAFs from all patients, and again, the top 3 upregulated genes were chemokines ( Fig. 2B ). The upregulation of these chemokines on the mRNA level was confi rmed by qRT-PCR for each array analysis ( Fig. 2C ).

Identifi cation of CCL5 as a Target of miR-214 To determine whether miR-CAFs, generated by alter-

ing miRNA expression, secreted chemokines, a protein array was constructed for 10 of the most highly upregu-lated cytokines found on the gene array. In this assay, the most highly induced chemokine on the protein level was CCL5/RANTES, followed by CXCL10 (C-X-C motif chem-okine 10), CCL7, and CCL8 ( Fig. 3A ). To understand how miRNAs regulate expression of chemokines in CAFs, we analyzed the 3′-UTR of all 9 chemokines upregulated in

miR-CAFs and CAFs for possible miRNA seed matches using miRanda, TargetScan, and PicTar software. Only 1 chemo-kine was predicted to contain seed matches for any of the 3 miRNAs. Overlapping seed matches for miR-214 and miR-31 were found in the CCL5 3′-UTR ( Fig. 3B ).

Although triple-transfected NOFs secreted most CCL5, anti-miR-214 was effi cient in inducing expression of CCL5 when transfected alone ( Fig. 3C ), suggesting that CCL5 is a direct target of miR-214 . Luciferase constructs were used with the CCL5 wild-type 3′-UTR and 2 different CCL5 3′-UTR mutants with substitutions in the miR-214 or the miR-31 seed matches ( Fig. 3B ). Cotransfection of miR-214 or miR-31 into 293T cells, together with the luciferase constructs, showed that miR-214 was more potent in targeting the CCL5 3′-UTR than miR-31 ( Fig. 3D ), and this activity was greatly diminished when the miR-214 seed match was mutated. This was confi rmed for endog-enous miRNAs by transfecting the reporter constructs into either primary CAFs or NOFs. Luciferase activity was signifi -cantly reduced in miR-214/miR-31 high-expressing NOFs when

Figure 3. Identifi cation of CCL5 as a miR-214 target secreted by CAFs. A, the culture medium of NOFs triple transfected with anti- miR-214 , anti- miR-3 1, and pre- miR-155 (to generate miR-CAFs) was analyzed 3 days after transfection using a custom array designed to detect 10 cytokines. Shown is one of 2 independent analyses, which gave similar results. B, schematic of the miR-214 and miR-31 seed matches in the human CCL5 3′-UTR and mutated binding sites introduced into luciferase 3′-UTR constructs. The miRNA seed sequences are indicated in teal blue and the mutated bases in the CCL5 3’UTR miRNA seed matches are in red. C, detection of the chemokine CCL5 by ELISA in culture supernatant of NOFs transfected with the indicated anti-miRs or pre-miRs, thereby creating miR-CAFs. D, 293T cells were cotransfected with the CCL5 3′-UTR [wild-type (wt), miR-214 mutant, miR-31 mutant] and either pre- miR-31 or pre- miR-214 . Changes in repression of luciferase activity are shown. E, changes in luciferase activity in NOFs or CAFs transfected with the different CCL5 3′-UTR constructs. Error bars represent SD of triplicate experiments. One-tailed t tests were conducted.

3′ ACGGACAGACACGGACG-A 5′

3′ GAUACGGUCGUAGAACGG 5′

825 bp miR-214

B

miR-31

| | :miR-214

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CCL5 (wt)

5′ uuugccugUUUCUGCUUGC-UCCUUUAcu 3′

5′ uuUGCCUGUUUCUGAUCGA-Ucuuguccu 3′

5′ uuUGCCUGUUUCUGCUUGC-UCUUGUCcu 3′

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compared with CAFs, with the miR-214 site mutant show-ing the greatest loss of repression ( Fig. 3E ). Consistent with miR-214 targeting CCL5 , an alignment of the 3′-UTRs of human, mouse, and rat CCL5 revealed that the region of highest identity contains the seed match for miR-214 , whereas the sequence for miR-31 is less well conserved (Supplementary Fig. S6).

CCL5 Is Critical for the Activity of Both CAFs and miRNA Reprogrammed CAFs

To determine the in vivo relevance of CCL5 in the activ-ity of miR-CAFs, miR-CAFs were generated by transiently transfecting NOFs with anti- miR-31 , anti- miR-214 , and pre- miR-155 . Using an orthotopic ovarian cancer mouse model, luciferase-expressing HeyA8 cells were coinjected with the miR-CAFs into the ovaries of nude mice and tumor growth was monitored by bioluminescence ( Fig. 4A and Supplemen-tary Fig. S7A). Reprogrammed fi broblasts clearly increased

the growth of coinjected HeyA8 cells, an effect that could be blocked by injections with a neutralizing anti-CCL5 anti-body. This indicates that the CCL5 secreted by miR-CAFs is a key tumor-promoting factor. To determine the in vivo signifi cance of CCL5 in the activity of patient isolated CAFs, luciferase-expressing HeyA8 cells were coinjected orthotopi-cally with either NOFs or CAFs ( Fig. 4B and Supplemen-tary Fig. S7B). The neutralizing anti-CCL5 antibody again inhibited the augmented growth of HeyA8 promoted by coinjected CAFs. The histology of the ovaries from mice coinjected with either NOFs, miR-CAFs, or CAFs ( Fig. 4C ) showed that all tumors were invasive, high-grade, and were primarily composed of cancer cells. Tumor cells coinjected with either miR-CAFs or CAFs showed increased invasive growth replacing normal ovarian structures such as follicles and fallopian tube more effi ciently as compared with coin-jected NOFs. The cancer cells in both the miR-CAF IgG and

Figure 4. CCL5 is critical for the activity of CAFs and miR-CAFs. A, HeyA8 cells expressing luciferase were coinjected orthotopically into the mouse ovary with either NOFs triple transfected with anti- miR-31 , anti- miR-214 , and pre- miR-155 (miR-CAF) or with equivalent scrambled controls (NOF). Mice were injected with either control immunoglobulin (IgG) or a neutralizing anti-CCL5 antibody (α-CCL5). Tumor growth was monitored by bioluminescence using Xeno-gen IVIS Spectrum and the fold change in radiance plotted. One-way ANOVA was conducted comparing all groups with HeyA8+miR-CAFs treated with IgG for the same time point (*, P < 0.05). Number of mice per group is indicated at the base of the columns. B, HeyA8 cells expressing luciferase were coinjected ortho-topically with either NOFs or CAFs. Mice were injected twice with either control IgG or a neutralizing anti-CCL5 antibody (α-CCL5). Tumor growth was monitored by bioluminescence and the fold change in radiance plotted. Number of mice per group is indicated at the base of the columns. One-way ANOVA was conducted comparing all groups with HeyA8 + CAFs treated with IgG for the same time point (*, P < 0.05). C, H&E staining of ovaries from IgG-injected mice shown in A and (B). Scale bar, 1,500 μm. Insets show close-up of tumor tissues (labeled by box), scale bar in insets, 200 μm. D, plug-homing assay. Matrigel plugs were embedded with NOFs transfected with anti- miR-31 and anti- miR-214 , scrambled control (scr) or no cells (/) and placed equidistant in the same culture dish and overlaid with GFP-expressing HeyA8 cells. Half of the wells were treated with an IgG control antibody and half were treated with a neutralizing anti-CCL5 antibody (α-CCL5). Homing of ovarian cancer cells to the Matrigel plugs was imaged (left) and quantifi ed (right). D–H, all data are normalized to the corresponding control Matrigel plugs with no cells. E, quantifi cation of the homing assay shown in D conducted with GFP-expressing SKOV3ip1 cells. (continued on following page)

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CAF IgG group had more Ki-67 staining (Supplementary Fig. S7C and S7D) indicating increased proliferation that could be inhibited by the anti-CCL5 antibody. Consistent with the histologic analysis of the subcutaneously injected tumors ( Fig. 1G ), there was no difference in infi ltrating immune cells (data not shown).

Our data suggest that CCL5 is an important factor in the provision of a tumor-promoting environment for ovarian can-cer cells. However, the in vivo experiments did not exclude the possibility that CCL5 acted by enhancing the conversion of NOFs to CAFs rather than by affecting the cancer cells. To test this possibility, a novel homing assay was developed in which Matrigel plugs containing the different fi broblasts were placed equidistantly in the same culture dish and overlaid with media containing fl uorescently labeled ovarian cancer cells. Two ovar-ian cancer cell lines were attracted by NOFs cotransfected with

both anti- miR-31 and anti- miR-214 ( Fig. 4D and E ) as well as to induced CAFs ( Fig. 4F ), an effect that was inhibited by neutral-izing CCL5 antibody. These data suggested that CCL5 acts by binding to one of its cognate receptors (CCR1, 3, and 5) on the surface of the cancer cells. Analysis of a data set comparing ovarian cancer samples and normal ovarian surface epithelial cells ( 14 ) revealed that CCR1 is upregulated in primary ovar-ian cancer (data not shown). Consistently, we found CCR1 to be expressed in both HeyA8 and SKOV3ip1 cells (Supple-mentary Fig. S8A), and treatment of the ovarian cancer cells with the CCR1/3 inhibitor J113863 blocked the homing of either HeyA8 or SKOV3ip1 cells to NOFs transfected with anti- miR-214 and anti- miR-31 ( Fig. 4G and H ). Finally, ectopic expression of CCL5 in NOFs (Supplementary Fig. S8B) signifi -cantly increased their ability to promote HeyA8 cell coinvasion, an effect blocked by a CCL5-neutralizing antibody ( Fig. 4I ).

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P = 0.02

Ctr CCR1/3 Inh.

Ctr CCR1/3 Inh.

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miR-155

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Normal fibroblasts CAFs OvCas

Figure 4. (Continued) F, Matrigel plugs were embedded with induced CAFs [iCAF (NOFs cocultured with HeyA8)], corresponding parental NOFs [control (Ctr)], or no cells (/) and placed equidistant in the same culture dish and overlaid with GFP-expressing HeyA8 cells. Half of the wells were treated with an IgG control antibody and half were treated with a neutralizing anti-CCL5 antibody (α-CCL5). Homing of ovarian cancer cells to the Matrigel plugs was imaged and quantifi ed. G, Matrigel plugs were embedded with NOFs transfected with anti- miR-31 and anti- miR-214 , scrambled control (scr) or no cells (/), and placed equidistant in the same culture dish and overlaid with GFP-expressing HeyA8 cells. Wells were treated with the CCR1/3 inhibitor J113863 (CCR1/3 Inh) or dimethyl sulfoxide (DMSO) control (Ctr). Homing of cancer cells to the Matrigel plugs was imaged and quantifi ed. E–G, SEs are shown for 3 independent experiments using fi broblasts from 3 to 4 different patients. One-tailed t tests were conducted. H, homing assay shown in G conducted with GFP-expressing SKOV3ip1 cells. SEs are shown for 3 independent experiments. One-tailed t tests were conducted. I, Coinvasion of HeyA8 with NOFs transfected with human CCL5 and treated with a CCL5-blocking antibody (NOF-CCL5+αCCL5) or IgG control (NOF-CCL5) were compared with coinvasion of HeyA8 with NOFs transfected with vector control (NOF-vect). Coinvasion of HeyA8 with CAFs treated with a CCL5-blocking antibody (CAF + αCCL5) or IgG control (CAF). All SEs represent at least 3 independent experiments. One-tailed t tests were conducted. J, schematic of miRNA induced reprogramming of NOFs to CAFs in ovarian cancer. Cancer cells induce a change in expression of miR-214 , miR-31 , and miR-155 in NOFs resulting in reprogramming into CAFs, which then promote tumor growth and invasion through increased secretion of cytokines (e.g., CCL5). Ovcas, ovarian cancers.






DISCUSSION

Although the cancer-promoting role of CAFs is unambigu-ously established, it is less clear how quiescent fi broblasts are transformed into CAFs ( 1 ). We tackled this question with 2 complementary approaches: comparing primary human fi broblasts from patients with and without ovarian cancer and a 3-dimensional organotypic coculture model. The results indicate that the downregulation of miR-214 and miR-31 and the upregulation of miR-155 can rapidly reprogram normal fi broblasts into CAFs. These 3 miRNAs combined are able to activate tumor-promoting functions, including migration, invasion, and colony formation, in normal fi broblasts. To the best of our knowledge, this is the fi rst report that shows that 3 miRNAs cooperate in reprogramming quiescent fi broblasts into cancer growth–promoting active fi broblasts, suggesting that resting fi broblasts possess the plasticity to become CAFs.

Our data suggest that CCL5 is a miRNA-regulated candidate effector molecule in CAFs, contributing to tumor cell recruit-ment and growth. CCL5 is abundant in the serum of ovarian cancer patients with advanced, metastatic disease ( 15 ). It is also secreted by mesenchymal stem cells, which have similar func-tional properties to fi broblasts ( 16 ). Of note, the overexpression of CCL5 in fi broblasts was suffi cient to promote the metasta-sis of admixed breast cancer cells ( 17 ) in a manner similar to our results, which showed that contact with CCL5-transfected NOFs increased the invasion of ovarian cancer cells.

CAFs at least partially maintain their characteristics when cultured in the absence of malignant cells, suggesting that genetic or epigenetic alterations have occurred. Although genetic alterations have been reported in CAFs from breast and ovarian cancer ( 18, 19 ), careful analysis showed that such genetic alterations are extremely rare ( 20 ). It is more likely that the stable phenotype of CAFs is regulated by epigenetic changes, as suggested by a genome-wide analysis of breast cancer stroma ( 21 ). Our results also lend support to a model in which tumor cells directly reprogram normal residential fi broblasts to become CAFs in the absence of permanently acquired mutations by changing miRNA expression in the fi broblasts ( Fig. 4J ). The tumor-promoting role of CAFs, together with their genetic stability, makes them an attractive therapeutic target in treatments aimed at removing tumor-supporting factors from the microenvironment. The model for miRNA-induced NOF → CAF transformation presented here opens the possibility of a treatment approach targeting the tumor stroma with miRNA and miRNA inhibitors.

METHODS Additional methods are included in the Supplementary Data.

Fibroblast Isolation and Characterization CAFs were isolated from the tumor-transformed omentum and

adjacent NOFs from the normal part of the omentum. All patients had newly diagnosed advanced, metastatic high-grade serous ovar-ian carcinoma and were undergoing primary debulking surgery by a gynecologic oncologist at the University of Chicago (Chicago, IL). NOFs were from omentum of female patients undergoing surgery for benign reasons. CAFs, adjacent NOFs, and NOFs were isolated as described ( 22, 23 ). CAFs were characterized by the expression of α-smooth muscle actin (α-SMA) , which was not expressed by the

adjacent NOFs or NOFs (Supplementary Fig. S9A–C). The miR-CAFs did not express α-SMA, which was expected, as it is not a target of the 3 miRNAs used to reprogram NOFs. Of note, α-SMA is not expressed in all CAFs even though these CAFs are function-ally active ( 24 ). CAF activity was validated through functional experiments (increased invasion, migration, colony formation, and tumor growth when coinjected with ovarian cancer cells in vivo ). Induced CAFs were generated by coculture of NOFs with ovarian cancer cells, and miR-CAFs were generated by transfection with locked nucleic acid (LNA) anti- miR-214 , anti- miR-31 (Exiqon), and pre- miR-155 (Ambion).

Cell Lines Human ovarian cancer cell lines SKOV3ip1, OVCAR5, and HeyA8

were validated by short tandem repeat (STR) DNA fi ngerprint-ing using the AmpFℓSTR Identifi er kit (Applied Biosystems) and compared with known American Type Culture Collection fi nger-prints, the Cell Line Integrated Molecular Authentication database (CLIMA), and the University of Texas MD Anderson Cancer Center fi ngerprint database.

miRNA and Gene Array Analysis Total RNA was isolated and subjected to both miRNA array

(miRCURY LNA array, v. 10.0, Exiqon) and gene array analyses using GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). All gene array data are available through Gene Expression Omnibus (GEO) accession number GSE35364.

Invasion and Migration Fibroblasts labeled with cell tracker red (CMTPX, Invitrogen) and

GFP-expressing tumor cells were mixed and seeded in 96-well glass-bottom plates coated with growth factor–reduced Matrigel. Thereaf-ter, upward invasion of cells through Matrigel towards Dulbecco’s modifi ed Eagle’s medium with 10% FBS was followed with time-lapse confocal microscopy (Supplementary Fig. S2). The speed of the invading cancer cells was analyzed with Imaris Software (Bitplane Inc.). Migration was quantifi ed using Transwell assays ( 23 ).

Plug-Homing Assay NOFs transfected with anti- miR-214 and miR-31 or with equimolar

scrambled controls were mixed with 20 μL of growth factor–reduced Matrigel. Plugs of miR-214 + miR-31 or control-transfected NOFs or Matrigel alone were placed equidistant in the same culture dish. Alternatively, plugs of induced CAFs or NOFs or Matrigel alone were used. Homing of GFP-labeled cancer cells towards the plugs was monitored using an Axio-observer A1 fl uorescent microscope (Carl Zeiss) and quantifi ed using Image J software (NIH).

Xenograft Experiments NOFs (1 × 10 5 cells) transiently transfected with anti- miR-31 ,

anti- miR-214 , and pre- miR-155 , or with the scrambled controls for each, were mixed with 0.5 × 10 5 HeyA8-Luc cGFP cells and then injected subcutaneously into the fl anks of female athymic nude mice. Alternatively, 50,000 CAFs, NOFs, anti- miR-214 , anti- miR-31 - and pre- miR-155 –transfected NOFs, or scrambled control–transfected NOFs were coinjected with 25,000 HeyA8-Luc cGFP cells into the right ovary of nude mice with 2 μg/mL of CCL5-blocking antibody or nonspecifi c mouse IgG. Mice were subsequently injected with 1 mg/kg CCL5 antibody or IgG control intraperitoneally 3 and 6 days thereafter. Tumor growth was quantifi ed using the Xenogen IVIS 200 Imaging System. Radiance was measured for tumors on each fl ank for the subcutaneous model. Total radiance from the tumors in each mouse was measured for the orthotopic, intra-abdominal model. Sig-nifi cance was calculated comparing all groups using 1-way ANOVA.






Disclosure of Potential Confl icts of Interest No potential confl icts of interest were disclosed by the authors.

Authors’ Contributions Conception and design: A.K. Mitra, M.E. Peter, E. Lengyel Development of methodology: A.K. Mitra Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.K. Mitra, M. Zillhardt, P. Tiwari, A.E. Murmann, E. Lengyel Analysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): A.K. Mitra, Y.-J. Hua, E. Lengyel Writing, review, and/or revision of the manuscript: A.K. Mitra, M.E. Peter, E. Lengyel Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P. Tiwari, A.E. Murmann Study supervision: A.K. Mitra, M.E. Peter, E. Lengyel

Acknowledgments The authors thank Dr. Cap (Arthur) Haney (University of Chicago,

Department of Obstetrics & Gynecology) for collecting normal omen-tal biopsies and Dr. Anthony Montag (University of Chicago, Depart-ment of Pathology) for help with the histologic analyses. The authors are grateful to Gail Isenberg for editing the manuscript.

Grant Support This work was supported by the Burroughs Wellcome Fund (to

E. Lengyel), and the Ovarian Cancer Research Fund to A.K. Mitra, E. Lengyel and M.E. Peter.

Received May 8, 2012; revised August 8, 2012; accepted September 7, 2012; published OnlineFirst November 21, 2012.

REFERENCES 1. Hanahan D , Coussens LM . Accessories to the crime: Functions of cell

recruited to the tumor microenvironment . Cancer Cell 2012 ; 21 : 309 – 22 . 2. Barkan D , Green JE , Chambers AF . Extracellular matrix: A gatekeeper

in the transition from dormancy to metastatic growth . Eur J Cancer 2010 ; 46 : 1181 – 8 .

3. Schauer IG , Sood AK , Mok S , Liu J . Cancer-associated fi broblasts and their putative role in potentiating the initiation and development of epithelial ovarian cancer . Neoplasia 2011 ; 13 : 393 – 405 .

4. Orimo A , Gupta P , Sgroi D , Arenzana-Seisdedos F , Delaunay T , Naeem R , et al. Stromal fi broblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis throught ele-vated SDF-1/CXCL 12 secretion . Cell 2005 ; 121 : 335 – 48 .

5. Ambros V . The functions of animal microRNAs . Nature 2004 ; 431 : 350 – 5 . 6. Schickel R , Boyerinas B , Park SM , Peter ME . MicroRNAs: Key players

in the immune system, differentiation, tumorigenesis and cell death . Oncogene 2008 ; 27 : 5959 – 74 .

7. Ambasudhan R , Talantova M , Coleman R , Yuam X , Zhu S , Lipton SA , et al. Direct reprogramming of adult human fi broblasts to functional neurons under defi ned conditions . Cell Stem Cell 2011 ; 9 : 113 – 8 .

8. Kim KS . Converting human skin cells to neurons: A new tool to study and treat brain disorders? Cell Stem Cell 2011 ; 9 : 179 – 81 .

9. Musumeci M , Coppola V , Addario A , Patrizii M , Maugeri-Sacca M , Memeo L , et al. Control of tumor and microenvironment cross-talk by miR-15a and miR-16 in prostate cancer . Oncogene 2011 ; 30 : 4231 – 42 .

10. Squadrito ML , Pucci F , Magri L , Moi D , Gilfi llan GD , Ranghetti A , et al. mir-511-3p modulates genetic programs of tumor-associated macrophages . Cell Rep 2012 ; 1 : 141 – 54 .

11. Lengyel E . Ovarian cancer development and metastasis . Am J Pathol 2010 ; 177 : 1053 – 64 .

12. Martinez-Outschoorn UE , Whitaker-Menezes D , Pavlides S , Chiava-rina B , Bonuccelli G , Trimmer C , et al. The autophagic tumor stroma model of cancer or “battery-operated tumor growth” a simple solu-tion to the autophagy paradox . Cell Cycle 2010 ; 9 : 4297 – 306 .

13. Aprelikova O , Yu X , Palla J , Wei BR , John S , Yi M , et al. The role of miR-31 and its target gene SATB2 in cancer-associated fi broblasts . Cell Cycle 2010 ; 9 : 4387 – 610 .

14. Bowen NJ , Walker LD , Matyunina LV , Logani S , Totten KA , Benigno BB , et al. Gene expression profi ling supports the hypothesis that human ovarian surface epithelia are multipotent and capable of serving as ovarian cancer initiating cells . BMC Med Genomics 2009 ; 2 : 71 .

15. Tsukishiro S , Suzumori NNH , Aarakawa A , Suzumori K . Elevated serum RANTES levels in patients with ovarian cancer correlate with the extent of the disorder . Gynecol Oncol 2006 ; 102 : 542 – 5 .

16. Haniffa MA , Wang XN , Holtick U , Rae M , Isaacs JD , Dickinson AM , et al. Adult human fi broblasts are potent immunoregulatory cells and functionally equivalent to mesenchymal stem cells . J Immunol 2007 ; 179 : 1595 – 604 .

17. Karnoub A , Dash AB , Vo AP , Sullivan A , Brooks MW , Bell GW , et al. Mesenchymal stem cells within tumour stroma promote breast can-cer metastasis . Nature 2007 ; 449 : 557 – 63 .

18. Patocs A , Zhang L , Xu Y , Weber F , Caldes T , Mutter GL , et al. Breast-cancer stromal cells with TP53 mutations and nodal metastases. N Engl J Med 2007 ; 357 : 2543 – 51 .

19. Tuhkanen H , Anttila M , Kosma VM , Heinonen S , Juhola M , Helisalmi S , et al. Frequent gene dosage alterations in stromal cells of epithelial ovarian carcinomas . Int J Cancer 2006 ; 119 : 1345 – 53 .

20. Qiu W , Hu M , Sridhar A , Opeskin K , Fox S , Shipitsin M , et al. No evidence of clonal somatic genetic alterations in cancer-associated fi broblasts from human breast and ovarian carcinomas . Nat Genet 2008 ; 40 : 650 – 5 .

21. Hu M , Yao J , Cai L , Bachman KE , van den Brule F , Velculescu V , et al. Distinct epigenetic changes in the stromal cells of breast cancers . Nat Genet 2005 ; 37 : 899 – 905 .

22. Proia DA , Kuperwasser C . Reconstruction of human mammary tis-sues in a mouse model . Nat Protoc 2006 ; 1 : 206 – 14 .

23. Kenny HA , Krausz T , Yamada SD , Lengyel E . Use of a novel 3D cul-ture model to elucidate the role of mesothelial cells, fi broblasts and extra-cellular matrices on adhesion and invasion of ovarian cancer cells . Int J Cancer 2007 ; 121 : 1463 – 72 .

24. Erez N , Truitt M , Olson P , Arron ST , Hanahan D . Cancer-associated fi broblasts are activated in incipient neoplasia to orchestrate tumor-promoting infl ammation in an NF-kappaB-dependent manner . Cancer Cell 2010 ; 17 : 135 – 47 .




2012;2:1100-1108. Published OnlineFirst November 21, 2012.Cancer Discovery Anirban K. Mitra, Marion Zillhardt, Youjia Hua, et al. Fibroblasts in Ovarian CancerMicroRNAs Reprogram Normal Fibroblasts into Cancer-Associated

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