Transcriptional Activity of ATF3 in the Stromal Compartment of Tumors Promotes Cancer Progression

Carcinogenesis vol.32 no.12 pp.1749–1757, 2011 doi:10.1093/carcin/bgr203 Advance Access publication September 7, 2011 Transcriptional activity of ATF3 in the stromal compartment of tumors promotes cancer progression

Yosef Buganimy, Shalom Madary, Yoach Rais, targets executes cell fate decisions such as cell cycle arrest or Leslie Pomeraniec, Einav Harel, Hilla Solomon, Eyal Kalo, apoptosis (9–14). Ido Goldstein, Ran Brosh, Ora Haimov, Camila Avivi1, During tumorigenesis, ATF3 might promote cancer or hinder its Sylvie Polak-Charcon1, Naomi Goldﬁnger, Iris Barshack1 progression depending on the tumor type and its genetic makeup (15). and Varda Rotter However, a possible role for ATF3 in the tumor microenvironment has not been proposed, although several studies have shown that ATF3 Department of Molecular Cell Biology, Weizmann Institute of Science, might affect potential members of the stromal milieu such as endo- Rehovot 76100, Israel and 1Department of Pathology, The Chaim Sheba thelial cells, smooth muscle cells and macrophages. For example, Medical Center, Tel-Hashomer, Ramat Gan 52662, Israel

ATF3 was shown to protect endothelial cells from tumor necrosis Downloaded from https://academic.oup.com/carcin/article/32/12/1749/2463391 by guest on 28 September 2021 To whom correspondence should be addressed. Tel: þ972 8 9344070; factor a-induced cell death (16). Furthermore, ATF3 induction in Fax: þ972 8 9465265; endothelial cells endowed them with the ability to form tubule struc- Email: [email protected] tures (17). A more recent study revealed that ATF3 increases the survival and enhances the migration of vascular smooth muscle cells Compelling evidences have rendered the tumor microenviron- (18). These findings imply that ATF3 might be involved in cancer ment a crucial determinant in cancer outcome. Activating tran- progression through the ability to promote the survival and formation scription factor 3 (ATF3), a stress response transcription factor, is of blood vessels in the vicinity of the tumor. ATF3 was also exten- known to have a dichotomous role in tumor cells, acting either as sively studied in the context of the innate immune response regulation a tumor suppressor or an oncogene in a context-dependent man- (15). Accordingly, it was shown to be a transcriptional regulator of ner. However, its expression and possible role in the tumor micro- Toll Like Receptors signaling in macrophages (19,20). The fact that environment are hitherto unknown. Here we show that ATF3 is ATF3 restrains the immune response might be utilized by the adjacent upregulated in the stromal compartment of several types of can- tumor cells to evade immunosurveillance. cer. Accordingly, Cancer-associated fibroblasts (CAFs) ectopi- In light of these findings, the primary motivation of this work was cally expressing ATF3 proliferated faster as indicated by to investigate the possible role of ATF3 in the stromal compartment of increased colony-forming capacity and promoted the growth of tumors. We found that ATF3, which is kept at low levels in normal adjacent tumor cells when co-injected into nude mice. Utilizing cells, is highly expressed in the stromal compartment of various can- a genome-wide profiling approach, we unraveled a robust gene cers. Our findings suggest that stromal ATF3 assumes an active role in expression program induced by ATF3 in CAFs. Focusing on a spe- cancer progression by (i) granting stromal cells with a growth advan- cific subset of genes, we found that the ability of stromal ATF3 to tage via transcriptional regulation of LIF, CLDN1, SERPINE2, promote cancer progression is mediated by transcriptional re- HSD17B2, ITGA7 and PODXL and by (ii) enhancing tumor cell pression of CLDN1 and induction of CXCL12 and RGS4. In addi- growth through transcriptional repression of CLDN1 and activation tion, regulation of LIF, CLDN1, SERPINE2, HSD17B2, ITGA7 of CXCL12 and RGS4. and PODXL by ATF3 mediated the increased proliferation capacity of CAFs. In sum, our findings implicate ATF3 as a novel stro- Materials and methods mal tumor promoter and suggest that targeting ATF3 pathway might be beneficial for anticancer therapy. Cell lines Cancer-associated fibroblasts (CAFs) from prostate, designated PF-179, and breast and lung CAFs designated ZB-06 and HK-3, respectively (a kind gift from Dr Jair Bar) were isolated from the stromal compartments of tumor Introduction specimens and grown in culture. The letter ‘T’ is used to denote that the designated cell line was immortalized with the human telomerase catalytic In recent years, a growing body of evidence has shaped the notion that subunit (hTERT). CAFs and WI-38 human embryonic lung fibroblasts were tumorigenesis is governed by a complex network of tumor cells, grown in minimum essential medium supplemented with 10% fetal bovine stromal residents such as fibroblasts, endothelial cell and infiltrating serum, 2 mM L-glutamine, 1 mM sodium pyruvate and antibiotics. Cancer cell immune cells and the extracellular matrix (1–3). The cancer-promot- lines (MDA-231, MDA-468, MCF-7, SKBR-3, SW-480, HT-29, HCT-116, ing communication between tumor cells and their surrounding stroma HepG-2, PC-3, LNCaP, DU-145, H-460, H-1299, NHBET and HT-1080) were escalates throughout the transformation process and has been termed grown according to ATCC protocols. Cells were maintained in a humidified the ‘vicious cycle’ (4). Anticancer drugs, specifically designed to incubator at 37°C and 5% CO2. target this interaction, are being developed, and an increasing number Tissue arrays of such drugs is currently under clinical trials (5–7). It is therefore Two commercial cancer tissue arrays (MC2081 and MC803, US-Biomax, crucial to identify key players that operate in the stroma and to Rockville, MD) consisting of biopsies from several types of cancer (colon, decipher the mechanism by which they exert their tumorigenic effect. rectum, breast, prostate, lung, retinoblastoma, nephroblastoma and neuroblas- The activating transcription factor 3 (ATF3) gene encodes a member toma), as well as a set of biopsies from 107 lung cancers (Sheba Medical of the ATF/cyclic adenosine monophosphate response element- Center), were stained with an anti-ATF3 antibody (HPA001562, Sigma, binding family of transcription factors, which share the basic Rehovot, Israel) as described below. MC2081 was also stained with an anti- region/leucine zipper DNA-binding motif and bind to the CREB/ CXCL12 antibody (MAB350, R&D Systems, Minneapolis, MN) ATF and AP-1 consensus sequences (8). ATF3 is a stress response Sarcomas samples protein, which in normal cells responds to a plethora of insults such as RNA samples were isolated from various human sarcoma specimens. Lipo- Endoplasmic Reticulum stress, DNA damage, tissue injury and met- sarcomas (5) and various sarcomas as follows: fibrosarcomas (3), leiomyosar- abolic stress and either through induction or repression of numerous coma (5), neurofibrosarcoma, dermatofibrosarcoma and rhabdomyosarcoma. Samples were kindly provided by Dr Paul Meltzer (National Institutes of Health, Bethesda, MD). Abbreviations: ATF3, activating transcription factor 3; CAF, cancer-associated fibroblast; mRNA, messenger RNA; NAF, normal-associated fibroblast; PCR, Subcutaneous injections of tumor cells polymerase chain reaction. All in vivo experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Weizmann institute (Permit numbers: yThese authors contributed equally to this work 80109-2 and 310110-1). Ten immunodeficient mice (CD-1-Hfh11nu Harlan

Laboratories, Rehovot, Israel) were anesthetized with a mixture of 100 mg/kg prepared with passive lysis buffer (Promega) and luciferase and Renilla activities ketamine and 10 mg/kg xylazin (Ketaset). Cells (2 106/50 ll per injection, in were determined using a Victor luminometer (Wallac, Waltham, MA). phosphate-buffered saline) were then subcutaneously injected into each hind limb using a 30 gauge needle and a 0.1 ml syringe. Mice were sacrificed when Statistical analysis tumor volume reached 1 cm3. Tumors were then removed, photographed and Unless stated otherwise, an unpaired one-tailed student’s t-test was performed; weighed. For co-injection experiments, the same setup was used with a mixture the resulted P-value is shown. All quantitative real-time PCR experiments were of 2 106 fibroblasts and 1 106 tumor cells/50 ll phosphate-buffered saline. repeated at least three times, and the results were normalized to the expression of GAPDH and presented as a mean ± standard deviation of two duplicate runs Western blot analysis from a typical experiment. Total cell extracts were fractionated by gel electrophoresis; proteins were transferred to nitrocellulose membranes and immunoblotted with Rabbit Polyclonal Chromatin immunoprecipitation Anti-ATF3 (Sc-188, Santa Cruz Biotechnology, Santa Cruz, California) and anti- Chromatin immunoprecipitation was conducted as described (23). GAPDH (MAB374; Chemicon, Billerica, MA). The protein–antibody complexes were detected by horseradish peroxidase-conjugated secondary antibodies Primers list followed by the enhanced SuperSignal west pico chemiluminescent substrate Specific primers for quantitative real-time PCR, Chromatin immunoprecipita- (Thermo Scientific, Huntsville, IL). tion and overexpression are listed in the Supplementary Materials and methods (available at Carcinogenesis Online). Downloaded from https://academic.oup.com/carcin/article/32/12/1749/2463391 by guest on 28 September 2021 Genes overexpression Overexpression plasmids were constructed by cloning the desired open reading frames using reverse transcription–polymerase chain reaction (PCR) with Results specific primers (see Supplementary Materials and methods, available at Carcinogenesis Online). Products were transferred into the pGEMT-easy ATF3 is upregulated in the stromal compartment of human tumors vector (Promega, Madison, WI) and then restricted with EcoRI and inserted ATF3 expression was reported previously to be elevated in tumors into either pBABE-Neo or PLXSN-Neo-expressing vectors. Cells were then (24,25). Accordingly, we found ATF3 to be upregulated both at the retrovirally infected as described elsewhere (21). messenger RNA (mRNA) and the protein levels in diverse types of Gene knockdown tumors (Supplementary Figure S1A–D is available at Carcinogenesis Expression of the specified genes was knocked down using Human pLKO.1 Online), whereas its levels in normal tissues were low. Also in agree- lentiviral short hairpin RNA target gene sets (Open Biosystems, Thermo Fisher ment with previous studies (25–31), modulation of ATF3 in various Scientific, Huntsville, AL) according to the manufacturer protocol and was cancer cell lines had cell-specific effects, ranging from suppression to compared with an empty vector. promotion of tumor growth in vivo (Supplementary Table S1 and Figure Isolation of total RNA and quantitative real-time PCR S1E and F are available at Carcinogenesis Online). Interestingly, in Total RNA was isolated using the RNAeasy kit (Qiagen) according to the man- addition to the frequent overexpression of ATF3 in primary tumors, ufacturer’s protocol. A 2 lg aliquot was reverse transcribed using MMLV-RT elevation of ATF3 protein levels was also evident in the tumor micro- (Bio-RT) and random hexamer primers. Quantitative real-time PCR was environment of the colon, lung and breast cancer samples (Figure 1A, performed on an ABI 7300 instrument (Applied Biosystems, Foster City, CA) right panel), with a frequency of 46, 25 and 29% positive biopsies, using Platinum SYBR Green and qPCR SuperMix (Invitrogen, Carlsbad, CA). respectively. The colon and lung samples exhibited both nuclear and Microarray cytoplasmatic expression of stromal ATF3, whereas the breast samples showed a cytoplasmatic and a more diffusible expression pattern. In Total RNA was extracted using Tri-Reagent (MRC, London, England) according to manufacturer’s protocol and sent to the MicroArray unit (Weizmann Institute contrast, only a small portion of the rectum and prostate specimens had of Science, Rehovot, Israel). The procedure was performed using Affymetrix HU- positive staining for stromal ATF3. As a control, a corresponding non- GENE1.0st oligonucleotide arrays with technical duplicates. Raw analyses were malignant tissue located 1.5 cm from the tumor was stained and re- performed using BRB-ArrayTools developed by Dr Richard Simon and BRB- vealed no traces of ATF3 in the stromal compartment (Figure 1A, ArrayTools development team (22). For ATF3 differential expression, a class left panel). Moreover, as depicted in Figure 1B and C, ATF3 was comparison analysis was performed, comparing ATF3-expressing cells to empty upregulated in CAFs dissected from a prostatic tumor margins com- control and ATF3DZip, with a threshold of P , 0.05 and log 1.2-fold change. pared with distal fibroblasts (normal-associated fibroblasts, NAFs). Fur- Colony-forming efficiency assay thermore, ATF3 levels were significantly elevated in cancer-associated stroma removed from breast cancer patients compared with normal Cells were seeded in triplicates at a cell density of 20 cells per square centi- meter. Medium was replaced every 4 days. Two weeks after plating, plates stroma, in a data set retrieved from the ‘Ocnomine’ database (32,33) were stained with crystal violet and scanned and colonies were counted. (Figure 1D). Since ATF3 is upregulated in the vicinity of tumors, we reasoned Immunostaining that cancer cells might benefit from the expression of ATF3 in the Formalin-fixed and paraffin-embedded sections of lung tissue microarray blocks, stroma and thus induce its expression in the stromal compartment. 4 lm thick, were dewaxed in xylene and rehydrated. Antigen retrieval was To address this question, we cloned ATF3 promoter into a retroviral performed using a pressure cooker (Milestone, Microwave Laboratory Systems, luciferase reporter plasmid and inserted it into immortalized Peine, Germany) at 120°C for 5 min in citrate buffer, pH 6, and slides were human lung CAFs (HK-3T). Subsequently, the fibroblasts were rinsed with Tris-buffered saline buffer. Subsequently, an endogenous peroxidase cocultivated with an increasing number of human lung cancer cell block was performed for 10 min in 3% H2O2/phosphate-buffered saline. After Tris-buffered saline rinses, sections were blocked with 10% goat serum for lines, either H-1299 or H-460, or with normal lung bronchial cells 30 min at room temperature and subsequently incubated with the relevant and non-infected fibroblasts as a control. ATF3 promoter activity was primary antibody (ATF3, CXCL12 described above or Ki-67—MU297-UC, elevated in a cell number-dependent manner in the fibroblasts when Biogenex, San Ramon, CA) overnight at 4°C. Detection was performed with cocultivated with both lung cancer cell lines, but not with the the Histostain SP Broad Spectrum kit (Zymed Laboratories, San Francisco, CA, controls—fibroblasts or normal lung cells (Figure 1E). Combined, Invitrogen, Carlsbad, CA). Briefly, sections were incubated with a biotinylated these results demonstrate that signals communicated from the cancer secondary antibody and subsequently, after Tris-buffered saline rinse, with cells to their stroma can induce stromal ATF3 transcription. horseradish peroxidase–streptavidin for 30 min at room temperature. Visualiza- tion was performed with the substrate–chromogen AEC, counterstained with hematoxylin and coverslipped with an aqueous mounting fluid (glycergel). Ectopic expression of ATF3 in CAFs promotes their proliferation and induces the growth of adjacent tumor cells Transfections and reporter assays To evaluate the impact of stromal ATF3 expression, we ectopically HT-1080 cells were plated at a density of 2 104 cells per well in a six well plate for 24 h prior to transfection. Cells were then transfected using FuGene HD expressed its coding sequence in three different types of fibroblasts, (Roche, Mannheim, Germany) with 1 microgram per well of CXCL12 pro- originating from prostate, breast and lung tumors, as well as in the moter–luciferase reporter construct and 400 nanograms per well pLXSN-Renilla WI-38 lung immortalized fibroblasts, which often serve as a tool to expression vector for normalization of transfection efficiency. Cell extracts were study the behavior of fibroblasts in cancer (34). The cells were 1750 Stromal ATF3 promotes cancer progression Downloaded from https://academic.oup.com/carcin/article/32/12/1749/2463391 by guest on 28 September 2021

Fig. 1. ATF3 expression in the tumor microenvironment. (A) (Right panel) Cancer samples from colon, lung and breast cancer patients were stained with an anti- ATF3 antibody and photographed. Arrows point at stromal cells with high ATF3 staining. (Left panel) Non-malignant tissue located 1.5 cm away from the tumor served as a control for the corresponding tumor type. (B) NAFs and CAFs originating from prostate cancer (38) were analyzed for ATF3 expression by (B) western blot and (C) quantitative real-time PCR, P 5 0.001. (D) The Oncomine database was queried for studies in which ATF3 expression significantly differs between NAFs and breast CAFs (32,33). The P-value designates the probability that the observed difference in ATF3 expression between normal and cancer samples is due to random chance. The bars represent 25–75% interval of normalized ATF3 expression levels in each group. The deviation lines represent 10–90% percentiles, and dots represent samples with minimum and maximum expression level in a given group. (E) Lung fibroblasts (HK-3T) expressing a luciferase gene downstream of ATF3 promoter were cocultured with growing amounts of fibroblasts, normal lung bronchial cells (NHBET) or the lung cancer cell lines H-460 and H-1299. ATF3 promoter activity is indicated by normalized luciferase luminescence intensity. P , 0.05. introduced with an empty vector, ATF3 or ATF3 alternative isoform proliferating cells as can be inferred from the percentage of Ki67- (ATF3DZip)—which lacks the leucine zipper DNA-binding domain positive cells. This result may account for their increased tumor size. (35) and therefore serves as a negative control for ATF3 transcrip- Our results indicate that overexpression of ATF3 in CAFs confers tional activity (Figure 2A and Supplementary Figure S2 is available at them with a survival advantage and promotes the growth of adjacent Carcinogenesis Online). First, in a colony formation assay, all four tumor cells in a non-cell autonomous manner. fibroblastic cell lines overexpressing ATF3 displayed an increased capacity to form colonies when seeded at low cell density, which indicates that these cells gained a growth advantage under unfavor- ATF3 facilitates a cancer-associated transcriptional program in able conditions (Figure 2B). As mentioned above, ATF3 was elevated fibroblasts in the stromal compartment of clinical tumor samples (Figure 1A), In an attempt to understand the molecular mechanism underlying ATF3 which suggests that stromal ATF3 might promote tumor growth. To activity in the tumor–stroma interaction, we utilized microarrays to test this hypothesis, we injected lung fibroblasts overexpressing ATF3 globally analyze mRNA expression of the two sets of the aforemen- (HK-3TATF3) together with H-1299 human lung carcinoma cells into tioned lung stromal fibroblasts, namely HK-3T and WI-38T, expressing the hind limb of 10 nude mice. As a control, the parallel hind limb of ATF3, ATF3DZip or an empty vector. As illustrated in Figure 3A, ATF3 each mouse was injected with HK-3T-expressing empty vector and expression in HK-3T lung fibroblasts resulted in upregulation of 80 H-1299 cells, generating a comparative setup, in which the capability genes and downregulation of 269 genes compared with the empty vector of the fibroblasts to promote tumor growth can be measured. As or ATF3DZip cells. In the WI-38T lung fibroblasts, ATF3 expression depicted in Figure 2C, HK-3TATF3 promoted the growth of the co- resulted in upregulation of 147 genes and downregulation of 172 genes. injected carcinoma cells, giving rise to larger tumors. Since H-1299 Notably, a large number of genes that were influenced by ATF3 expres- cells are highly tumorigenic and have an unstable genomic makeup sion (36 in the upregulated gene cluster and 131 in the downregulated (36), we wished to test whether stromal ATF3 can promote the growth gene cluster) overlapped between the two lung fibroblasts, as depicted in of less aggressive tumor cells. To this end, we co-injected WI-38T a Venn diagram (Figure 3A, right panels), implying that the transcrip- cells expressing ATF3 (WI-38TATF3) or an empty vector, with a lung tional regulation of these genes by ATF3 is not cell line specific and fibrosarcoma-forming cell line (WI-38TTransformed) that was generated might represent a general transcription program facilitated by ATF3 in previously in our laboratory (31). In agreement with the results ob- fibroblasts. A gene ontology clustering program [GeneDecks (37)] re- tained with HK-3TATF3 cells, WI-38TATF3 cells promoted the growth vealed that these subsets of genes were significantly enriched with of the adjacent WI-38TTransformed cells (Figure 2C). Examination of cancer-related clusters such as ’tumors’, ’metastasis’ and ’positive the resulting tumor sections revealed malignant and rapidly growing regulation of cellular proliferation’, as well as with extracellular local- tumors with a growth pattern that is cohesive and expansile (Figure ization (Supplementary Figure S3A is available at Carcinogenesis 2D). Furthermore, as shown in Figure 2D and E, tumors that were co- Online). Thus, ATF3 facilitates a general, cancer-related gene expres- injected with ATF3-expressing stroma had a significant enrichment in sion program in fibroblasts. 1751 Y.Buganim et al. Downloaded from https://academic.oup.com/carcin/article/32/12/1749/2463391 by guest on 28 September 2021

Fig. 2. ATF3 promotes fibroblast colony-forming ability and tumor growth. (A) The designated fibroblast cultures were introduced with a retroviral empty vector or retroviruses encoding ATF3 or ATF3DZip. Western blot analysis was conducted for ATF3 and GAPDH as loading control. (B) Colony-forming efficiency assay. The designated cell cultures were seeded at a low cell density, and following 14 days, plates were stained with crystal violet. ‘’Denotes a significant increase in colony formation in the ATF3 sample versus the empty control vector, P , 0.05. (C) (Left panel) HK-3TATF3 or an empty vector were co-injected with H-1299 cells into 10 Nude mice. The bars represent 25–75% interval of tumor weight in milligrams, and the contrasted bars denote the median. Dots represent minimum and maximum weight in a given group. The same experiment was performed using WI-38TATF3 and transformed WI-38TTransformed (right panel). (D) H-1299/HK-3TATF3-generated tumors were fixed, sectioned and stained with either hematoxylin and eosin or anti-Ki67. (E) Several 70 70 lm fields of view were scanned and the percentage of Ki67-positive cells were calculated, P 5 0.008. For further information, see also Supplementary Figure S2 (available at Carcinogenesis Online).

1752 Stromal ATF3 promotes cancer progression Downloaded from https://academic.oup.com/carcin/article/32/12/1749/2463391 by guest on 28 September 2021

Fig. 3. ATF3 facilitates a cancer-related transcriptional program in fibroblasts. (A) WI-38T and HK-3T were introduced with an empty vector or vectors encoding ATF3 or ATF3DZip and subjected to a complementary DNA microarray analysis. ATF3-induced/repressed gene clusters are shown. The numbers of the differentially expressed genes and the overlap between the two cell lines are shown in a Venn diagram (lower panels). Hypergeometric probabilities for the overlap between the gene lists are presented above the diagrams. (B) The expression level of 12 genes (six upregulated and six downregulated) was measured by quantitative real-time PCR and compared between WI-38T and HK-3T expressing ATF3, ATF3DZip or empty vector (control), P , 0.01. (C) A complementary DNA microarray analysis conducted by Finak et al. (32) comparing NAFs and CAFs was queried using the ‘Oncomine’ website (32,33) for the expression of the indicated genes. P-value for differential expression between NAFs and CAFs for all genes is smaller than 4 103.(D) A chromatin immunoprecipitation was done using an antibody against ATF3 or a control IgG antibody in HK-3TATF3 cells. Quantitative real-time PCR was performed for precipitated DNA with primers that amplify regions surrounding the predicted ATF3/CREB-binding sites in the designated loci. Significant promoter enrichment was measured by two-tailed paired t-test. ‘’Denotes P-value ,0.02. (E) Avector encoding luciferase under the control of CXCL12 promoter and the indicated amounts of an expression vector encoding ATF3 were transfected into HT-1080 cells. The chart depicts normalized CXCL12 promoter activity calculated by dividing the luciferase activity in each well by the Renilla activity of co-transfected plasmid. (F) Tumors (WI-38Transformed co-injected with WI-38TATF3) were removed, embedded in paraffin and sectioned. Sections were stained with antibodies against CXCL12 or ATF3. Nuclei were visualized with 4#,6-diamidino-2-phenylindole (DAPI). (G) Breast cancer slides consisting of 47 samples were stained for ATF3 and CXCL12. (upper panel) AVenn diagram depicting the calculated overlap between ATF3 and CXCL12 stromal-positive samples. (lower panel) Representative images taken from matching samples stained for ATF3 and CXCL12, either negative (left hand) or positive (right hand). Supportive evidence is shown in Supplementary Figure S3 (available at Carcinogenesis Online).

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To identify specific targets that might mediate the cancer-promot- antibodies against ATF3 and CXCL12. Strikingly, CXCL12 staining ing activity of ATF3, we chose to focus on a group of 12 genes (six was evident exclusively in the Extra Cellular Matrix surrounding the upregulated and six downregulated) that demonstrated a robust tran- ATF3-expressing cells (Figure 3F). As CXCL12 was reported pre- scriptional response to the modulation of ATF3 in lung fibroblasts viously to be elevated in breast cancer (41), we stained two consec- (Figure 3B), as well as in the two other fibroblastic systems, namely utive slides consisting of 48 breast cancer specimens with a-ATF3 and breast cancer-derived and prostate cancer-derived fibroblasts (supple- a-CXCL12 antibodies. We found stromal expression of CXCL12 in mentary Figure S3B is available at Carcinogenesis Online). Impor- 21 of the samples and 14 ATF3-positive stromal samples. More im- tantly, in accordance with our in vitro analysis, five of the 12 genes portantly, as depicted in Figure 3G, an overlap between the positive were coregulated with ATF3 in a clinical study presented in the ‘Oc- samples was evident in 13 cases (hypergeometric function, P 5 1.5 nomine’ database, comparing NAFs and breast CAFs (32,33) (Figure 105), further substantiating the correlation between ATF3 and 3C). Furthermore, revisiting our previously published study compar- CXCL12 expression. ing NAFs and CAFs, we found that ATF3, CLDN1, HSD17B2 and CXCL12 exhibited the same expression pattern in prostate CAFs (38) (Supplementary Figure S3C is available at Carcinogenesis Online). Stromal ATF3 promotes cancer progression through the regulation of A summary of the function of these genes and the number of their CXCL12, RGS4 and CLDN1 Downloaded from https://academic.oup.com/carcin/article/32/12/1749/2463391 by guest on 28 September 2021 cancer-related publications is described in Table 1. Interestingly, most To determine the identity of the most prominent mediators of ATF3 of the genes (10 out of 12) were already investigated in the context of tumor-promoting activity, we reversed the expression of ATF3-regu- cancer, whereas the involvement of two genes, FIBIN and CNIH3,in lated genes by either overexpression or short hairpin-mediated knock- tumorigenesis is first proposed herein. In addition, this set of genes down in HK-3TATF3. These manipulations were performed on the consists of secreted proteins (CXCL12, LIF, FIBIN and SERPINE2) background of high ATF3 levels, in an attempt to reverse ATF3 tu- and cell surface proteins (RGS4 and CLDN1) that can alter the tumor mor-promoting capacity. Figure 4A illustrates the mRNA levels of the microenvironment scenery and may account for ATF3 non-cell auton- genes, following stable infections. Due to the fact that we could not omous mediated outcome. To investigate whether ATF3 effect on the observe any reduction in CNIH3 mRNA following infection of four expression of the 12 selected genes is mediated through DNA binding, different short hairpin RNA constructs, we decided to exclude it from we performed a Chromatin immunoprecipitation assay using HK- the experiment. First, we tested whether reversing the transcriptional 3TATF3. For the majority of the genes (10 out of 12), we detected levels of ATF3 target genes will affect the ability of the stromal cells binding of ATF3 to a predicted ATF3/CREB-binding sites (described to form colony in unfavorable condition (as shown in Figure 2B). in Supplementary Figure S3D and E is available at Carcinogenesis Indeed, reversing the transcriptional levels of the ATF3 targets LIF, Online) in their promoters (Figure 3D). As CXCL12 (SDF-1a) is one CLDN1, SERPINE2, HSD17B2 PODXL and ITGA7 attenuated the of the key factors in the tumor–stroma interaction (40), we decided to colony-forming capacity of HK-3TATF3 cells (Figure 4B), suggesting perform an in-depth validation as for its regulation by ATF3, as a proof that these genes mediate the growth-facilitating effect of ATF3 in the of concept. To this end, we cloned the promoter of CXCL12 into stroma. More importantly, utilizing the aforementioned co-injection a luciferase reporter vector and co-transfected it into HT-1080 cells experimental setup (Figure 2C), we found that CAFs overexpressing with an ATF3-expressing vector or an empty vector. Indeed, CXCL12 CLDN1 and CAFs with a knocked down expression of RGS4 and promoter was activated by ATF3 in a dose-dependent manner (Figure CXCL12 profoundly reduced the adjacent tumor size (Figure 4C). 3E). In addition, the aforementioned tumors, which were generated by Reversal of the remaining genes did not have a significant effect on co-injecting tumor and stromal cells, were sectioned and stained with tumor size (data not shown). These results indicate that by repressing

Table 1. ATF3 target genes summary

Gene symbol Description/functiona Cancer-related references

ATF3-upregulated genes CXCL12 A stromal cell-derived alpha chemokine, member of .30 the intercrine family. PODXL Binds in a membrane protein complex with Naþ/Hþ 4 exchanger, expressed in vascular endothelium cells. RGS4 GTPase-activating proteins for G alpha subunits of 5 heterotrimeric G proteins. ITGA7 Belongs to the integrin alpha chain family receptor 5 for the basement membrane protein laminin-1. CNIH3 Selective transport and maturation of TGFa family 0 proteins. Upregulated in certain human mesenchy- mal stem cells. SLITRK1 Integral membrane proteins that are characterized by 2 two N-terminal leucine-rich repeat domains. ATF3-downregulated genes LIF A pleiotropic cytokine involved in the induction of .10 hematopoietic and neuronal differentiation. FIBIN A secreted lateral mesoderm signal protein essential 0 for ﬁn initiation in zebra ﬁsh. TSPAN12 A cell surface protein, belongs to the transmembrane 1 4 superfamily SERPINE2 A secreted Serpin peptidase inhibitor. 7 HSD17B2 Hydroxysteroid (17-beta) dehydrogenase 2, respon- 4 sible for the inactivation of estradiol. CLDN1 An integral membrane protein and a component of 16 tight junction complexes aGene descriptions/functions were taken from National Center for Biotechnology Information ‘Gene’ tool (39). 1754 Stromal ATF3 promotes cancer progression Downloaded from https://academic.oup.com/carcin/article/32/12/1749/2463391 by guest on 28 September 2021

Fig. 4. ATF3 effect on tumor growth is mediated via a subset of genes. HK-3TATF3 was introduced either with a vector encoding an ATF3-downregulated target or with a vector expressing a short hairpin against an ATF3-upregulated target. Corresponding empty vectors were used as controls for each of the cases. (A) Quantitative real- time PCR analysis was performed to measure the designated genes’ expression, P , 0.05 (B) Colony-forming efﬁciency assay. The designated cell cultures were seeded at a low cell density and following 14 days plates were stained with crystal violet. (C) HK-3TATF3 cells were co-injected with H-1299 cells into 10 Nude mice. Bars represent 25–75% interval of tumor weight in milligrams, and the contrasted bars denote the median. Dots represent minimum and maximum weight in a given group.

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CLDN1 expression and by transactivating CXCL12 and RGS4, stromal ATF3 exerts a tumor-promoting activity.

Discussion Although ATF3 has been thoroughly investigated in tumor cells, up to date, the possible role of ATF3 in the tumor stroma has not been reported. In our current work, we chose to pursue the possible in- fluence of ATF3 in the stromal microenvironment. We demonstrate here for the first time that ATF3 is frequently expressed in the stroma of several cancers, which extends the spectrum of ATF3 activity to the vicinity of the tumor and raises new possibilities as for its involvement in the process of tumorigenesis. Our findings invoke several intriguing questions that are addressed in this discussion; among them are ATF3 deregulation in cancer, the identity and consistency of its targets and Downloaded from https://academic.oup.com/carcin/article/32/12/1749/2463391 by guest on 28 September 2021 the alluring potential of targeting stromal ATF3, as a mean to control cancer progression.

ATF3 deregulation in cancer The ample data regarding ATF3 portray it as a dichotomous protein, which may act as a tumor suppressor, protecting normal and cancer cells from further transformation, but can also facilitate tumor progression (15,25). Several mechanisms may account for ATF3 dichotomy; among them are its different heterodimerization partners, which may lead to the activation or repression of different target Fig. 5. A schematic model summarizing stromal ATF3 activity in cancer. genes. In addition, as a transcription factor, it might be greatly af- fected by a given chromatin availability state, which can be tumor stage or type dependent. As a stress response protein, all normal reversing their expression had a noticeable impact on both the cells benefit from ATF3 upregulation in response to various insults. colony-forming capacity and tumor-promoting phenotype. However, its constitutive expression in a large portion of cancers suggests that in the long run, ATF3 may exert oncogenic properties, ATF3 as a potential target for anticancer therapy which are positively selected by the tumor cells, and according to In contrast to a wide range of transcriptionally activated and repressed our present findings, in their surrounding stroma, as well. Indeed, targets of ATF3 in different tumors (15), which renders ATF3 tran- ATF3 was reported to be overexpressed in cancer due to increased scriptional activity as ‘cell-type specific’, the identity of ATF3 targets genomic copy number (24,25). The transition between the tumor- in various stromal cells seems to be consistent. This incongruity might suppressive and oncogenic state could be attributed to posttranslational stem from the frequent genomic instability observed in cancer com- modifications. Notably, ATF3 amino acid sequence is enriched with pared with the relatively stable genomes of stromal cells (7,44). Since potential phosphorylation, acetylation, methylation, ubiquitination and stromal ATF3 overexpression occurs in a relatively stable genomic sumoylation sites, spanning 20% of the protein (42). Future experimen- background and since it executes a general transcriptional program in tal studies should be aimed at understanding whether ATF3 activity fibroblasts, which is not dependent on the specific tumor, it might be is mediated via posttranslational modifications—variations which a suitable candidate for stromal-targeted therapy in multiple cancers. may account for its dichotomy in cancer both in the tumor and its Moreover, CXCL12, which was reported previously to be expressed surrounding cells. in many cancer-related stromal cells (32,38), is described here as a novel target of ATF3. CXCL12 is a cancer-related chemokine that The identity of ATF3#s targets binds CXCR4 and CXCR7 receptors (45–48) and regulates cell sur- Two principal selective engines shape the behavior and gene expression vival, proliferation and chemotaxis (49). More importantly, Orimo of tumor-associated stromal cells. The first is increased survival and et al. (40), reported that CAFs promoted tumor growth through secre- enhanced growth rate often exhibited by CAFs (38,43), which grant the tion of CXCL12, which on the one hand induce angiogenesis and on cells with a growth advantage over other inhabitants of the tumor stroma the other hand directly enhance tumor growth by binding and activat- milieu. A second mechanism is the selective force imposed by the de- ing CXCR4 receptor on the surface of tumor cells. Accordingly, veloping tumor, which favors only the stromal cells that can supply its CXCL12 and its interaction with downstream components are cur- growing demands. For example, in a seminal work by Hill et al. (43), rently being targeted in stromal-related cancer therapy, and two such tumor cells were shown to actively select for highly prolific fibroblasts, drugs (AMD3100 and CTCE-9908) had been approved for clinical which harbor p53 mutations. As depicted in Figure 5, our findings use (47,50). As ATF3 was shown to promote endothelial survival and suggest that the identity of ATF3 targets in the stromal cells is governed sprouting (17) and as it is a regulator of CXCL12, it is tantalizing to by these two selective forces. On the one hand, LIF, CLDN1, speculate that ATF3 inhibition will result in attenuation of tumor SERPINE2, HSD17B2, ITGA7 and PODXL mediate the increased growth as a result of insufficient angiogenesis. colony-forming ability of the fibroblasts following ATF3 expression. In contrast, despite not affecting the immediate growth of stromal cells, Supplementary material CXCL12 and RGS4 induction by ATF3 enhanced tumor growth in a non-cell autonomous manner. Moreover, these genes were also found Supplementary Table S1 and Figures S1–S5 can be found at http:// to be co-expressed with ATF3 in cancer-associated stromal cells that carcin.oxfordjournals.org/. were isolated from clinical samples. Thus, tumor selection may account for their ATF3-mediated upregulation. ATF3 seems to activate a pleth- Funding ora of genes and pathways all funneled into the regulation of cancer progression. This could stem from the fact that most of these genes (10 Center of Excellence grant from Flight Attendant Medical Research out of 12) were shown to exert a substantial effect (either suppression Institute, Yad Abraham Center for Cancer Diagnosis and Therapy and or promotion) on tumor progression independently of ATF3. Thus, by European Community FP7-INFLACARE number-223151. 1756 Stromal ATF3 promotes cancer progression

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