HOXB7 Promotes Malignant Progression by Activating the Tgfb

Published OnlineFirst December 26, 2014; DOI: 10.1158/0008-5472.CAN-14-3100

Cancer Molecular and Cellular Pathobiology Research

HOXB7 Promotes Malignant Progression by Activating the TGFb Signaling Pathway Shou Liu1,2, Kideok Jin3,Yvonne Hui4, Jie Fu1,2, Chunfa Jie5, Sheng Feng6, David Reisman1,2, Qian Wang6, Daping Fan4, Saraswati Sukumar3, and Hexin Chen1,2

Abstract

Overexpression of HOXB7 in breast cancer cells induces an expression in breast cancer cells were reversed by knockdown of epithelial–mesenchymal transition and promotes tumor pro- TGFb2 or pharmacologic inhibition of TGFb signaling. Further- gression and lung metastasis. However, the underlying more, knockdown of TGFb2 in HOXB7-overexpressing MDA- mechanisms for HOXB7-induced aggressive phenotypes in MB-231 breast cancer cells dramatically inhibited metastasis to breast cancer remain largely unknown. Here, we report that the lung. Interestingly, HOXB7 overexpression also induced phosphorylation of SMAD3 was detected in a higher percentage tumor-associated macrophage (TAM) recruitment and acquisi- in primary mammary tumor tissues from double-transgenic tion of an M2 tumor-promoting phenotype. TGFb2mediated MMTV-Hoxb7/Her2 mice than tumors from single-transgenic HOXB7-induced activation of macrophages, suggesting that Her2/neu mice, suggesting activation of TGFb/SMAD3 signal- TAMs may contribute to HOXB7-promoted tumor metastasis. ing by HOXB7 in breast tumor tissues. As predicted, TGFb2 Providing clinical relevance to these ﬁndings, by real-time washighinfourMMTV-Hoxb7/Her2 transgenic mouse tumor PCR analysis, there was a strong correlation between HOXB7 cell lines and two breast cancer cell lines transfected with and TGFb2 expression in primary breast carcinomas. Taken HOXB7, whereas TGFb2 was low in HOXB7-depleted cells. together, our results suggest that HOXB7 promotes tumor HOXB7 directly bound to and activated the TGFb2 promoter progression in a cell-autonomous and non–cell-autonomous in luciferase and chromatin immunoprecipitation assays. manner through activation of the TGFb signaling pathway. Increased migration and invasion as a result of HOXB7 over- Cancer Res; 75(4); 709–19. 2014 AACR.

Introduction binding sequence-specific DNA motifs (ATTA/TAAT; refs. 5, 6). HOX genes have been identified as master transcriptional The HOX family of homeobox-containing genes encodes regulators controlling the coordinated expression of genes transcription factors that are highly conserved from Drosophila involved in development and differentiation (7). Recently, a to Homo sapiens (1–3). The homeobox, a characteristic feature growing body of literature has emerged on the involvement of of this family of genes, is an 180-bp DNA sequence encoding HOX genes in the pathogenesis of cancers (8). Recently, a few a trihelical 60 amino acid homeodomain (3, 4). It is usually linesofevidencewerepresented to suggest that HOXB7 also located at a terminal or subterminal position of the correspond- plays a role in tumorigenesis. First, HOXB7 was found to be ing homeoprotein and is responsible for recognizing and frequently overexpressed in melanoma, ovarian, and breast cancer cell lines as well as primary tumor cells (9–11). Second, overexpression of HOXB7 in the breast cancer cell line SKBR3 1Department of Biological Science, University of South Carolina, increased proliferation and angiogenesis by upregulating basic 2 Columbia, South Carolina. Center for Colon Cancer Research, Uni- fibroblast growth factor (bFGF; refs. 9, 12, 13). In addition, versity of South Carolina, Columbia, South Carolina. 3Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland. overexpression of HOXB7 in breast cancer cells induced epithe- 4Department of Cell Biology and Anatomy, University of South Car- lial–mesenchymal transition (EMT) and rendered breast cancer olina School of Medicine, Columbia, South Carolina. 5Department of cells resistant to tamoxifen treatment through activation of the Surgery, Transplant Surgery Division, Feinberg School of Medicine, Northwestern University, Chicago, Illinois. 6Department of Chemistry EGFR pathway (14, 15). and Biochemistry, University of South Carolina, Columbia, South To study the role of Hoxb7 in breast tumorigenesis, our lab Carolina. generated an MMTV-Hoxb7 FVB/N transgenic mouse model Note: Supplementary data for this article are available at Cancer Research where expression of HOXB7 is regulated by the mouse mammary Online (http://cancerres.aacrjournals.org/). tumor virus (MMTV) promoter (16). Although overexpression of fi Corresponding Authors: Hexin Chen, Department of Biological Sciences, Uni- HOXB7 alone was not suf cient to cause tumor formation, in versity of South Carolina, 715 Sumter Street, PSC621, Columbia, SC 29205. crosses of MMTV-Hoxb7 mice with MMTV-Her2/neu transgenic Phone: 803-777-2928; Fax: 803-777-4002; E-mail: [email protected]; and mice, it dramatically impacted oncogene Her2/neu-induced Saraswati Sukumar, The Johns Hopkins School of Medicine, 1650 Orleans Street, tumorigenesis. In double-transgenic mice, overexpression of CRB 1, Room 143, Baltimore, MD 21231-1000. Phone: 410-614-2479; Fax: 410-614- HOXB7 delayed tumor onset and lowered tumor multiplicity 4073; E-mail: [email protected] (16), but promoted tumor progression and metastasis. This con- doi: 10.1158/0008-5472.CAN-14-3100 trasting phenotype was intriguing and reminiscent of the dual role 2014 American Association for Cancer Research. of TGFb in breast cancer. Siegel and colleagues used transgenic

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mouse models to demonstrate that TGFb signaling suppressed HOXB7-overexpressing MDA-MB-231 cells were further trans- Her2/neu-induced mammary tumor growth while promoting fected with pSIH1-TGFb2 to generate the TGFb2knockdown subsequent lung metastasis (17). This led us to hypothesize that cell line (MDA-B7-b2KD). HOXB7-overexpressing H605 cell HOXB7 may directly or indirectly regulate TGFb signaling. line was established by transfection of Her2 transgenic mouse In line with this hypothesis, we have now demonstrated that mammary tumor cells, H605 with pcDNA3-HOXB7 or empty overexpression of HOXB7 induces the expression of TGFb2in vector plasmids followed by G418 selection for establishing both mouse and human breast cancer cell lines, leading to stable clones. increased cell motility and invasiveness, and recruitment and b activation of macrophages. Expression of HOXB7 and TGF 2is Animal experiments strongly correlated in primary breast cancer tissues and is asso- All procedures were conducted in accordance with NIH regula- ciated with advanced stages of tumor progression. Overall, our tions, and approved by the Institutional Animal Care and Use results suggest that HOXB7 may be a potential therapeutic target Committee of the University of South Carolina. In the tail-vein in invasive and metastatic breast cancer. injection experiments, a total of 1 106 MDA-MB-231 cells (MDA-con), MDA-MB-231/HOXB7 (MDA-B7) per animal were Materials and Methods suspended in sterile PBS and injected into the tail vein of six NOD-SCID mice, 6 to 8 weeks of age. At 60 days after injection, Primary tissue samples and cell culture the mice were killed and the lungs were photographed under Human breast cancer tissue samples were obtained through fluorescent microscope, and then fixed in 10% formalin. Hema- the South Carolina Tissue Bank with approval from the Insti- toxylin and eosin (H&E) staining of the entire paraffin-embedded tutional Review Board at the University of South Carolina lung tissue was performed. The metastatic foci were counted (Columbia, SC). Tissue samples were randomly collected from under 200 magnification (ten randomly selected high-power patients who were diagnosed with invasive breast ductal car- fields). In the orthotopic transplantation experiments, 1 105 cinoma between 2003 and 2007. Their clinicopathologic char- H605 cells (mammary tumor cell line from MMTV-Her2 trans- acteristics are summarized in Supplementary Table S1. Adjacent genic mice) per site were injected into the mammary fat pad of 6- normal tissues that were at least 2 mm away from the tumor to 8-week-old female FVB/N mice. At 30 days after injection, the fi margins and con rmed to be free of tumor deposits were used tumors were collected and processed for FACS assay and immu- as normal control in this study. The isolation of carcinoma cells nofluorescence staining analysis. from tumors developing in MMTV-Her2/neu transgenic mice and establishment of the primary HER2 tumor cell line, H605, fl were described previously (18). All human breast cancer cell Immunohistochemistry and immuno uorescent assay lines were obtained from ATCC, and with the exception of Immunohistochemical (IHC) staining of formaldehyde- fi fi MCF10A, were maintained in DMEM with 10% FBS. MCF10A xed, paraf n-embedded primary or xenograft tumor tissues was maintained in DMEM/F12 containing 5% horse serum, was carried out as previously reported (14), using the human 10 mg/mL human insulin, 0.5 mg/mL hydrocortisone, 100 ng/mL p-SMAD3 antibody at 1:100 dilution. Stained slices were cholera toxin, and 20 ng/ml EGF. scored according to intensity of staining and percentage of tumor cells staining positive for p-SMAD3 [0%, IHC level 0; – þ – þ > Plasmids and stable cell lines 1% 30%, IHC level 1 ;31% 70%, IHC level 2 ;and 70%, þ To generate luciferase reporter plasmids containing the TGFb2 IHC level 3 ]. fl promoter sequences, DNA fragments containing different lengths For immuno uorescence staining of macrophages, fresh tumor of the TGFb2 promoter sequence (nucleotide from 1015 to tissues were embedded in OCT gel and kept at 80 C. Frozen m fi þ68) were amplified using genomic DNA. The PCR products were tissue sections (10- m slices) were xed in 4% paraformaldehyde. cloned into pGL3-promoter vector. The plasmids for TGFb2-183 Immunostaining was performed using FITC-conjugated anti- and TGFb2-25 were generated by cutting plasmid TGFb2-1083 F4/80 (dilution, 1:200) and PE-conjugated anti-CD206 (dilution, with Kpn I and Sac I, respectively. To construct HOXB7-over- 1:200). The sections were then counterstained with DAPI. expressing plasmids, mouse HOXB7 cDNA was inserted into pcDNA3, and human HOXB7 cDNA was inserted into pMSCV- Treatment of macrophages with tumor cells conditioned IRES-GFP. To generate HOXB7 knockdown and TGFb2 knock- media down plasmids, shRNA for human HOXB7 and TGFb2 were To obtain conditioned media, stable cell lines MDA-B7, inserted into pSIH1-puro. The sequences for HOXB7 knockdown MDA-B7-b2KD, and the control MDA-con were seeded in a and TGFb2 knockdown are ATCTTGATCTGTCTTTCCG and 10-cm dish at 5 106 cells per dish and cultured till 90% GAACTAGAAGCAAGATTTG, respectively. confluence. The media was then replaced with serum-free To generate HOXB7-overexpressing or knockdown stable cell DMEM. After 24 hours, the supernatants were collected and lines, 293T cells were cotransfected with retroviral constructs of filtered through a 0.22-mm filter. To obtain mouse macrophages, pMSCV-HOXB7 or pSIH1-HOXB7 along with the respective mice were injected intraperitoneally with 3 mL of 3% thiogly- package system plasmids for 2 days. The supernatant was collateinsterilePBS.Threedayslater,micewereeuthanizedand collected to infect different human breast cancer cell lines for the peritoneal macrophages were harvested by lavaging the 2 days, and then cells were collected for RT-PCR and Western peritoneal cavity with 2 10 mL of PBS. Cells were suspended blot analysis. To generate HOXB7-overexpressing MDA-MB- with DMEM media containing 10% FBS, and plated in 6-well 231 (MDA-B7), plasmid pBI-HOXB7-eGFP and the empty plates. After 1 hour, the nonadherent cells were removed by PBS, vector were transfected into MDA-MB-231 cells. The GFP-pos- and the adhered macrophages were further cultured in serum- itive cells were flow sorted and used in the experiments. The free DMEM for 24 hours, followed by treatment with control

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HOXB7 Regulates the TGFb Signaling Pathway

DMEM or indicated tumor cell conditioned media for another gested that HOXB7 may promote tumor progression and 24 hours. metastasis via enhancement of the TGFb–SMAD3 pathway.

Other methods HOXB7 upregulates TGFb2 expression in mammary tumors Quantitative real-time RT-PCR analysis, chromatin immuno- and breast cancer cells precipitation (ChIP) assay, Western blot analysis, wound-healing Given that HOXB7 is a classical transcription factor, we per- assay, Matrigel invasion assay, and FACS assay were performed formed RT-PCR analysis to screen the TGFb signaling pathway using standard protocol (14, 16, 19). See Supplementary Methods genes in primary tumor cells with, and without HOXB7 over- for more details. expression. Semiquantitative RT-PCR analysis demonstrated an increase in TGFb2 mRNA in all of the HOXB7-overexpressing primary mouse tumors compared with Her2/neu tumors. In Statistical analyses contrast, no significant changes in the expression of TGFb1 and The statistical analyses were conducted with R and GraphPad TGFb3 were observed (Fig. 1C). We next determined whether software packages (GraphPad). The association between the increased TGFb2 mRNA in HOXB7-overexpressing primary level of p-SMAD3 and overexpression of HOXB7 in mammary tumors cells was reflected in increased TGFb2 protein. TGFb2 tumors was assessed with the Fisher exact test using R software. mRNA encodes a precursor protein of approximately 40 kDa, AStudentt test or ANOVA test was used for comparison of which is cleaved to an approximately 12 kDa active secreted quantitative data. The linear correlations between HOXB7 and protein. We found increased production of pro-TGFb2in TGFb ligand expression in primary breast cancer tissues were HOXB7-overexpressing tumor cells in comparison with control evaluated with Pearson correlation coefficient analysis. Values cells (Fig. 1D). TGFb signaling activation is associated with the of P < 0.05 were considered significant. activation (via phosphorylation) of SMAD3; indeed, we found increased phosphorylation of SMAD3 in HOXB7-overexpressing Results Her2 tumor cells compared with Her2 tumor cells (Fig. 1D). To further determine whether HOXB7 can activate TGFb2 Activation of TGFb signaling in HOXB7-overexpressing expression in human breast cancer cells, human HOXB7-expres- mammary tumors sing plasmids were transiently transduced into two breast cancer The dual role of HOXB7 in Her2/neu-induced tumorigen- cell lines (MDA-MB-231 and SKBR3) with low levels of HOXB7 esis led us to hypothesize that HOXB7 may activate TGFb expression (14). Semiquantitative RT-PCR and Western blot signaling, which is known to inhibit tumor onset and promote analysis were performed to examine the expression of HOXB7 tumor progression in several types of cancers (16, 17). TGFb and TGFb2. Consistent with the results obtained in mouse tumor has been shown to signal most commonly through the SMAD cells, overexpression of HOXB7 in two human breast cancer cell family signaling pathway (20). The activation of the trans- lines, MDA-MB-231 and SKBR3, increased TGFb2 expression at membrane TGFb receptor leads to the phosphorylation and both mRNA and protein levels (Fig. 1E and 1F), and induced an activation of SMAD3, which then forms a complex with autocrine TGFb signaling as evidenced by increased phosphory- SMAD4 to regulate the transcription of target genes by binding lation of SMAD3 in HOXB7-overexpressing cells compared with to DNA at specific SMAD-binding elements(20).Toassessthe control cells (Fig. 1E). Conversely, HOXB7 knockdown in two involvement of the TGFb signaling in both mammary tumor HOXB7-expressing breast cancer cell lines (MDA-MB-468 and formation and subsequent metastasis, we examined the phos- T47D) suppressed TGFb2 expression at both mRNA and protein phorylation status of SMAD3 in primary tumors arising in levels (Fig. 1G and H), with concomitant decrease of phosphor- MMTV-Her2/neu and MMTV-Hoxb7/neu transgenic mice. IHC ylation of SMAD3 (Fig. 1H). These data indicated that HOXB7 can staining analysis of SMAD3 phosphorylation revealed a strik- upregulate TGFb2 expression in breast cancer cells. ing difference between these two groups of tumors (Fig. 1A). The majority of primary mammary tumors from MMTV-Her2/ neu mice showed undetectable (level 0) or weakly detectable HOXB7 directly binds to the TGFb2 promoter and upregulates (level 1þ) staining signal and about 15% of tumors display its transcription activity moderate (level 2þ)orstrong(level3þ)p-SMAD3staining.In All of the HOX proteins bind to the consensus binding site contrast, all of the tumors from MMTV-Hoxb7/neu double- containing the ATTA/TAAT motif in in vitro assays (8, 21). The transgenic mice were positive for p-SMAD3 staining. MMTV- in vivo binding affinity and specificity is determined by the Hoxb7/neu mammary tumors (54.6%) showed strong staining adjacent sequences and/or binding cofactors (21). Searching (level 3þ). Overall, overexpression of HOXB7 in mammary through the upstream sequence of TGFb2promoterallowedus tumors was strongly associated with increased phosphoryla- to identify 10 potential HOXB7-binding sites within the prox- tion of SMAD3 (P ¼ 0.048). imal 1.5 Kb of TGFb2 50 flanking region (relative to the Our previous studies have shown that overexpression of transcription start site). To investigate whether HOXB7 binds HOXB7 promotes lung metastasis (16). We therefore exam- to the TGFb2 promoter directly (by interacting with the cis ined whether there is a correlation between the phosphoryla- elements) or indirectly (by influencing another protein that tion level of SMAD3 and the number of metastatic foci in the interacts with the cis elements) in vivo,wefirst performed the lung. As shown in Fig. 1B, tumors with higher phosphorylation ChIP assay. Cell lysates were sonicated to generate approxi- level of SMAD3 were more likely to metastasize to lung. The mately 200-bp chromatin fragments before immunoprecipita- number of metastasis foci was significantly higher in tumors tion. Chromatin that coimmunoprecipitated with anti-HOXB7 with p-SMAD3 staining at the IHC level 3þ than in p-SMAD3– (or control IgG) antibody was amplified by real-time PCR negative tumors (P ¼ 0.036). Together, these findings sug- using primers flanking the proximal (126 to 354 bp) or

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A Mouse mammary tumor B 30 phospho- neu neu/Hoxb7 P a P = 0.036 IHC 0 IHC 1 SMAD3 (n = 13) (n = 11)

8 0 n ) IHC level 0 (61.5%) (0%) 20 3 3 IHC level 1 (23.1%) (27.3%) 1 2 0.048 10

IHC 2 IHC 3 IHC level 2 (7.7%) (19.2%) foci Metastatic

1 6 ( lung per animal IHC level 3 (7.7%) (54.6%) 0 5/13 11/11 0 1 2 3 (38.5%) (100%) Total p-SMAD3 IHC level

C Neu tumor Neu/Hoxb7 tumor D Neu tumor Neu/Hoxb7 tumor 317 318 321 326 236 301 302 305 317 318 321 326 236 301 302 305 HOXB7 HOXB7 TGFβ1 TGFβ2 TGFβ2 p-SMAD3 TGFβ3 SMAD3 β-Actin GAPDH

EFMDA-MB-231 SKBR3 MDA-MB-231 SKBR3 G MDA-MB-468 T47D H MDA-MB-468 T47D V B7 V B7 V B7 V B7 Scr. shB7 Scr. shB7 Scr. shB7 Scr. shB7 HOXB7 . . HOXB7 . HOXB7 HOXB7 TGFβ1 TGFβ2 TGFβ1 TGFβ2 TGFβ2 p-SMAD3 TGFβ2 p-SMAD3 TGFβ3 SMAD3 TGFβ3 SMAD3 β-Actin GAPDH β-Actin GAPDH

Figure 1. Overexpression of HOXB7 increases TGFb2 expression and phosphorylation level of SMAD3 in mouse primary MMTV-Her2/HoxB7 bitransgenic mammary tumors and human breast cancer cells. A, overexpression of HOXB7 is associated with increased phosphorylation level of SMAD3 in the mouse mammary tumors. Representative IHC images of primary mouse mammary tumors stained with antibody against p-SMAD3 are presented. IHC 0, negative; IHC 1, 1%–30% weak positive; IHC 2, 31%–70% positive; IHC 3, >70% strong positive. aFisher exact test with R software. B, correlation between p-SMAD3 level in primary tumors and the number of metastatic foci in lung. C, RT-PCR analysis of TGFb1, TGFb2, TGFb3,andHOXB7 mRNA expression in primary MMTV- neu and MMTV-Hoxb7/neu transgenic mouse mammary tumor tissues. D, Western blot analysis of HOXB7, TGFb2, SMAD3, and p-SMAD3 levels in primary mammary tumors described above. E and G, RT-PCR analysis of TGFb1, TGFb2, TGFb3,andHOXB7 mRNA expressions in HOXB7-overexpressing (E) and HOXB7 knockdown (G) human breast cancer cells. Different breast cancer cells were transduced with HOXB7-overexpressing retroviruses or HOXB7 knockdown lentiviruses for 48 hours before harvesting for RT-PCR analysis of mRNA expression. F and H, Western blot analysis of HOXB7, TGFb2, SMAD3, and p-SMAD3 levels in transiently HOXB7-overexpressing (F) and HOXB7 knockdown (H) human breast cancer cells described above. V, vector; B7, HOXB7; Scr., Scrambled shRNA; shB7, HOXB7 shRNA.

two distal (628 to 848 bp and 1210 to 1392 bp) putative We then investigated the effect of HOXB7 overexpression on HOXB7 binding site–containing regions (Fig. 2A, top). We activity of the TGFb2 promoter. For these studies, a series of found that only the proximal binding site–containing region reporter gene constructs based on the potential binding sites were (site 3) specifically coprecipitated with HOXB7 from HOXB7- generated, representing truncations of the TGFb2 50 flanking overexpressing cells. In contrast, the upstream binding sites region (Fig. 2D, left). These reporter constructs (or empty reporter (sites 1 and 2) did not show significant HOXB7-binding capa- vector) were cotransfected into MCF7 cells with a plasmid over- bilities (Fig. 2A). Interaction of HOXB7 with this proximal expressing HOXB7 (or empty vector). HOXB7 overexpression region is about 15-fold greater in HOXB7-overexpressing cells significantly increased reporter gene expression from all, except than in control cells (Fig. 2B). Because HOX proteins frequently the T-25 construct (Fig. 2D, right). The significant increase of the bind to DNA as a heterodimer with Pbx or Meis proteins (21), reporter activity from the T-183 demonstrates that the other nine we investigated whether these cofactors can be recruited to the upstream binding sites may only contribute minimally to the TGFb2 promoter as well. Real-time ChIP results showed that induction of TGFb2 by HOXB7 (Fig. 2D, right), which is consis- HOXB7 may form complexes with either PBX2 or MEIS-1, but tent with the results of ChIP assays. Overall, these results sug- notPBX1.NcoRwasnotrecruitedtothesamesiteandservedas gested that HOXB7 directly binds to, and activates the TGFb2 a negative control in this experiment (Fig. 2C). promoter in breast cancer cells.

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HOXB7 Regulates the TGFb Signaling Pathway

A B 20 Figure 2. Vector ** HOXB7 directly binds to TGFb2 15 HOXB7 –1400 –1200 –1000 –800 –600 –400 –200 promoter and upregulates its transcriptional activity. A, ChIP assay +1 10 for the TGFb2 promoter. Diagram –1392 to –1210 –848 to –628 –354 to –126 5 representing the position of primers site1 site2 site3 TGFb2

used for PCR in the promoter Fold enrichment region (top). MCF-7 cells were 0 transduced with HOXB7- Site 1 Site 2 Site 3 Site 1 Site 2 Site 3 overexpressing retroviruses or V B7 V B7 V B7 C control, and ChIP was performed by 6 Vector ** immunoprecipitation with either anti- Input HOXB7 HOXB7 antibody or control IgG 4 ** (lower). V, vector; B7, HOXB7. B, real- HOXB7 time PCR analysis of relative binding 2 afﬁnity of HOXB7 to the three IgG binding sites in TGFb2 promoter P < 0 region. , 0.01. C, real-time PCR Fold enrichment analysis of recruitment of HOXB7 PBX-1 PBX-2 MEIS-1 NCOR cofactors to the site 3 of TGFb2 D promoter. , P < 0.01. D, luciferase HOXB7 0 mg activity of truncated constructs of –1400 –1200 –1000 –800 –600 –400 –200 8 HOXB7 0.2 mg TGFb2 promoter. Schematic diagram +1 +68 m presenting the potential recognition HOXB7 0.4 g site (ATTA or TAAT) of HOXB7 in T-1083 luciferase 6 HOXB7 0.8 mg TGFb2 promoter and the truncated constructs of TGFb2 promoter (left). T-493 luciferase 4 The luciferase activity of four T-183 luciferase truncated constructs was measured 2 when cotransfected with increasing Potential recognition site T-25 luciferase amounts of HOXB7-overexpressing (ATTA or TAAT) plasmid (right). 0 T-1083 T-493 T-183 T-25 Relative luciferase activities

TGFb2 induction is critical for HOXB7-mediated cell migration SB431542. Western blots of cell lysates were probed with and invasion antibodies to total and p-SMAD3 to confirm the blockage of Our previous study had shown that overexpression of the TGFb signaling pathway. As expected, the phosphorylation HOXB7 promotes cell migration and invasion (14). We next level of SMAD3 was decreased by SB431542 treatment of determined whether TGFb2 mediates the effect of HOXB7 on HOXB7-overexpressing MDA-MB-231 cells (Supplementary cell migration and invasion in breast cancer cells. We first Fig. S1A). More importantly, the inhibition of TGFb signaling utilized the TGFb2-specific shRNA to knock down TGFb2 significantly attenuated cell migration and invasion abilities in expression in HOXB7-overexpressing (MDA-B7) and control HOXB7-overexpressing cells, but only slightly reduced cell (MDA-con) MDA-MB-231 cells. Western blot analysis showed migration and invasion abilities in vector cells (Supplementary that TGFb2 shRNAs achieves a knockdown of TGFb2 expression Fig. S1B–S1D). This is consistent with our hypothesis that in HOXB7-overexpressing MDA-MB-231 cells by 50% to 60% autocrine TGFb2 production induced by HOXB7 in breast (Fig. 3A). Using these TGFb2 knockdown cells, we performed cancer cells stimulates cell migration and invasion through cell invasion assay and found that cell invasion in HOXB7- activation of the classical TGFb2–SMAD3 signaling pathway. overexpressing MDA-MB-231 cells was significantly greater than in vector cells (Fig. 3B and C). The invasive response was decreased significantly in HOXB7-overexpressing cells after Knockdown of TGFb2 expression impairs HOXB7-induced transfection with TGFb2 shRNA (Fig. 3C). Although transfec- tumor metastasis in vivo tion of TGFb2 shRNA also decreased invasion of control cells, To further investigate whether upregulation of TGFb2 is critical the percentage of decrease was significantly smaller, compared for HOXB7-induced metastasis in vivo, we utilized shRNA to stably with HOXB7-overexpressing cells (45.5 11.1% for control knockdown TGFb2 expression in HOXB7-overexpressing MDA- cells vs. 80.1 6.2% for HOXB7-overexpressing cells; Fig. 3C). MB-231 (MDA-B7-b2KD) cells (Fig. 4A). HOXB7 overexpression We also performed wound-healing assays under the same had no effect on cellular morphology and growth rate of these conditions. The transfection of TGFb2 shRNA into vector cells MDA-MB-231 cells (Supplementary Fig. S2B). To determine lung slightly slowed down migration, whereas a knockdown of colonization ability of these cells, the stable cell lines (MDA-B7 TGFb2 expression in HOXB7-overexpressing cells dramatically and MDA-B7-b2KD) and control MDA-MB-231(MDA-con) cells inhibited cell migration (Fig. 3D). To further confirm that the were injected through the tail vein into NOD-SCID mice. As TGFb2–SMAD3 signaling pathway is critical for HOXB7-medi- showed in (Fig. 4B), MDA-B7 cells formed more lung metastases ated cell migration and invasion, we treated HOXB7-overex- than MDA-B7-b2 and MDA-con cells after 2 months. The number pressing or control cells with TGFb receptor I inhibitor of metastatic foci in the lungs of mice injected with MDA-B7 cells

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24 h A MDA-con MDA-B7 D 0 h 6 h 12 h shRNA Scr. b2 Scr. b2 HOXB7 MDA-con + b TGF 2 Scramble p-SMAD3 SMAD3 b-Actin

MDA-con + B MDA-con MDA-B7 TGFb2 shRNA Scramble

MDA-B7 + TGFb2 Scramble shRNA

C MDA-con MDA-B7 + 80 ** ** MDA-B7 TGFb2 shRNA 60

40 20

0 Scramble TGFb2 shRNA Number of cells per field

Figure 3. HOXB7 promotes breast cancer cell migration and invasion via upregulation of TGFb2 expression. A, Western blot analysis of the efficiency of TGFb2 knockdown in HOXB7-overexpressing MDA-MB-231 cells. HOXB7-overexpressing (MDA-B7) and control MDA-MB-231 (MDA-con) cells were transduced with scrambled shRNA or TGFb2–specific shRNA-expressing retroviruses for 48 hours, then lysed for Western blot analysis. Scr., scrambled shRNA; b2, TGFb2 shRNA. B, TGFb2 knockdown impaired HOXB7-induced cell invasion. Cell invasion was examined using Matrigel invasion chamber assay. The images were taken under 200 magnification. C, the quantitative representation of cell invasion described in Fig. 4B. The invaded cells were counted under 200 magnification (ten randomly selected fields). D, the effect of TGFb2 knockdown on HOXB7-induced cell migration. Cell migration was monitored at the indicated time points.

was almost 4-fold higher than in mice injected with MDA-con overexpressed HOXB7 for transplantation into immunocom- cells (11.7 2.2 vs. 3.0 0.6; P ¼ 0.0037). More importantly, petent syngeneic mice (Supplementary Fig. S3A). HOXB7 over- knocking down TGFb2 dramatically reduced the number of expression marginally increased the proliferation rate of H605 metastatic foci in the lung (11.7 2.2 vs. 2.5 0.7; P ¼ cells (Supplementary Figs. S3B and S3C; P ¼ 0.058). HOXB7- 0.0028; Fig. 4C). Therefore, TGFb2 plays a pivotal role in overexpressing and control H605 cells were injected into mam- HOXB7-promoted metastasis of MDA-MB-231 cells to the lung. mary fat pad of syngeneic FVB/N mice. The size of tumors derived from HOXB7-overexpressing H605 cells was moder- TGFb2 cytokine secreted by HOXB7-overexpressing breast ately larger than that in control H605 cells injected mice, but cancer cells directly stimulates peritoneal macrophages to thedifferencewasnotstatisticallysignificant (Supplementary acquire M2 features Fig. S3D; P ¼ 0.059). The recruitment of macrophage pheno- It is well documented that tumor-associated macrophages, type was examined by immunofluorescence staining (Fig. 5A) especially M2 phenotype, promote tumor growth and metas- and FACS analysis (Fig. 5B). Significantly more F4/80 (marker tasis by secreting many cytokines, chemokines, and proteases for mouse macrophage) and CD206 (specificmarkerformouse (22–27). It is reported that TGFb2 can induce macrophages to M2 phenotype) double-staining positive cells were detected in acquire a M2 phenotype (28). Besides inducing cell migration tumors derived from overexpressing HOXB7 cells than control and invasion via tumor cell–autonomous manner, we wanted cells (Fig. 5A). As showed in Fig. 5B and C (top), the average þ to test whether HOXB7 can also promote tumor progression percentage of infiltrating macrophages (F4/80 )inHOXB7- through regulation of tumor microenvironment such as recruit- overexpressing tumors was more than 2-fold higher than ment and activation of macrophages. To examine the effect of H605 control tumors (13.1 1.9 vs. 4.8 0.5; P ¼ þ þ HOXB7 on recruitment of macrophages in vivo, we generated a 0.0031). In addition, the M2 population (F4/80 /CD206 ) MMTV-Her2 mouse mammary tumor cell line H605 that stably was also increased by nearly 3-fold (9.8 1.8 vs. 2.8 0.4;

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HOXB7 Regulates the TGFb Signaling Pathway

A B

MDA-con MDA-B7 MDA-B7-b2KD HOXB7

TGFb2

p-SMAD3

SMAD3

b-Actin C 15 * *

Number of metastatic foci MDA-con MDA-B7 MDA-B7-b2KD

Figure 4. TGFb2 is required for HOXB7-induced tumor metastasis in vivo. A, establishment of a stable TGFb2 knockdown cell line. Western blot analysis was performed to validate the knockdown efficiency. MDA-con, control MDA-MB-231cells, MDA-B7, HOXB7-overexpressing cells; MDA-B7-b2KD, HOXB7-overexpressing, and TGFb2 knocked down cells. B, examination of lung metastasis in tail-vein injected animals. MDA-con, MDA-B7, and MDA-B7-b2KD cells were injected via tail vein into NOD/SCID mice (n ¼ 6/group). The mice were sacrificed 2 months later, and the lungs were photographed under fluorescence microscope and then processed for H&E staining. Representative photos are showed. Arrows, metastatic foci. C, quantification of the metastatic foci in lungs described in B. The metastatic foci were counted under 200 magnification (ten randomly selected high-power fields for each sample; n ¼ 6). , P < 0.05.

P ¼ 0.0045; Fig. 5B and C, right). These data strongly suggest expression levels of HOXB7 and the TGFb ligands, we found a that HOXB7 promotes recruitment and induction of macro- significant linear correlation between HOXB7 and TGFb2 (R ¼ phages into the M2 phenotype. 0.267, P ¼ 0.0002) in primary breast cancer tissues (Fig. 6C). We determine whether TGFb2 is involved in HOXB7-mediated However, we did not observe such an association between the induction of M2 phenotype by using an additional model system. expression of HOXB7 and TGFb1 (Fig.6B).Theseresultssup- We treated na€ve mouse peritoneal macrophages with growth port our finding that HOXB7 and TGFb2 are coexpressed in medium conditioned by MDA-B7, MDA-B7-b2, and MDA-con primary breast cancers and our findings in model systems find a cells for 48 hours. Because the proliferation rates of MDA-B7, close parallel in clinical samples. MDA-B7-b2, and MDA-con cells are almost identical in tissue A large number of reports have shown that TGFb can pro- culture, this approach enabled us to compare the effects of tumor mote tumor progression at later stages of breast cancer (29, 30). cell–secreted factors on macrophages. As shown by Western blot To investigate whether the activation of TGFb2 expression by analysis in Fig. 5D, expression of typical M2 markers, Arg1 and HOXB7 contributes to tumor progression, we calculated the YM1,was strongly induced in mouse peritoneal macrophages after correlation coefficient between the expression of HOXB7 and stimulation with conditioned medium by MDA-B7 cells than with TGFb2 in subgroups stratified by tumor grade and stage MDA-con cells, whereas knockdown TGFb2 expression dimin- (Table 1). Interestingly, the linear correlation between HOXB7 ished the upregulation of Ym1 and Arg1 (Fig. 5D). These findings and TGFb2 expression is especially pronounced in higher suggest that HOXB7-overexpressig cancer cells stimulate na€ve grade tumors, for example, grade 2 tumors (R ¼ 0.485; P ¼ mouse peritoneal macrophages to acquire features of M2 macro- 0.001) and in stage III/IV (R ¼ 0.769; P ¼ 0.01). Furthermore, a phages by secreting cytokines, among which an important con- significant correlation is only observed in lymph node–positive stituent is TGFb2. tumors (R ¼ 0.297; P ¼ 0.007), but not in the lymph node– negative tumors (R ¼ 0.168; P ¼ 0.092). In line with our HOXB7 and TGFb2 expression in primary breast cancer is previous observation that HOXB7 is a prognostic marker in associated with aggressive phenotypes HER2-positive tumors (16), the positive correlation between To explore the clinical relevance of HOXB7 regulation of HOXB7 and TGFb2 is significant in HER2-positive tumors as TGFb2 expression in breast cancer, we performed quantitative well (R ¼ 0.453; P ¼ 0.016). Together, our results support a RT-PCR analysis of cDNAs generated from 46 clinically and relationship between HOXB7 and TGFb2 with effects on breast pathologically annotated cases of breast cancer. Compared with cancer progression and metastasis. the adjacent normal tissues, primary breast cancers expressed higher levels of HOXB7 (P ¼ 0.03) and TGFb2 (P ¼ 0.015). TGFb1 mRNA levels, on the other hand, were comparable Discussion between breast carcinomas and adjacent normal tissues (Fig. This study contributes to our understanding of the molecular 6A). After calculating the correlation coefficients between the mechanism by which overexpression of HOXB7 in breast cancers

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A B Control HOXB7 0.7% 3.0% 5.4% 8.4% H605-control H605-HOXB7

DAPI F4/80

CD206

C 20 F4/80 * 15 * 15

cells (%) cells 10 +

cells (%) 10 + 5

5 /CD206 + F4/80 CD206 0 0 Control HOXB7 F4/80 Control HOXB7 D Conditioned media

Merged YM1

Arg1

Actin

Figure 5. Overexpression of HOXB7 enhances recruitment and activation of macrophages to acquire M2 phenotypes. A, overexpression of HOXB7 enhances recruitment and activation of macrophages in orthotropic transplantation models of breast cancer. HOXB7-overexpressing and control mouse mammary tumor cell lines were injected into the mammary fat pad of syngeneic FVB/N mice (n ¼ 6/group). One month later, the tumors were harvested for IHC staining using FITC-conjugated anti-F4/80 and PE-conjugated anti-CD206 antibodies. The slices were counterstained with DAPI and photographed. B, the representative pictures of FACS analysis of the inﬁltrated macrophages in the transplanted tumors. The tumor tissues described in A were digested into single cells and subject to FACS analysis with the FITC-conjugated anti-F4/80 and PE-conjugated anti-CD206 antibodies. C, quantitative analysis of percentage of total and M2 macrophages in the transplanted tumors (n ¼ 5); , P < 0.05. D, HOXB7 induces peritoneal macrophages to acquire M2 phenotypes. Conditioned media from MDA-con, MDA-B7, and MDA-B7-b2KD and fresh DMEM media were used to treat primary mouse peritoneal macrophages for 24 hours. Expression levels of Arg1 and YM1 were examined by Western blot analysis. Arg1, Arginase, type I.

promotes tumor progression. Our data suggested that upregula- same target region. Promoter deletion analysis indicated that tion of TGFb2 by HOXB7 is responsible for both tumor cell– overexpression of HOXB7 alone was sufficient to activate pro- autonomous and tumor–stromal interaction mechanisms, lead- moter activity. It is also noteworthy that two other HOX proteins, ing to an aggressive phenotype and tumor progression to metas- HOXA10 and HOXB9, can activate TGFb2 expression as well tasis. As a classical transcriptional factor, HOXB7 may directly or (31, 32). Although HOXB9-binding sites in TGFb2 promoter indirectly regulate the transcription of target genes. Among many have not been identified (32), HOXA10 binds to two cis elements candidate genes we screened, TGFb2 expression was selectively located at the proximal (410 to 385) and distal (1506 to upregulated by HOXB7 in both human breast cancer cell lines and 1478) regions in the TGFb2 promoter (31), which are different mouse primary mammary tumors. Further statistical analysis from the main HOXB7-binding site (75 to 78). Meanwhile, indicated that there was a positive correlation between expression the ATTA/TAAT motif is also present in TGFb1 promoter region; of HOXB7 and TGFb2 in the mouse mammary tumors. Although however, overexpression or knockdown of HOXB7 in breast many genes have been shown to be induced or activated as a result cancer cells seems to have no effect on its expression and there of HOXB7 overexpression in cancer cells, only a few genes have is no correlation between the expression of HOXB7 and TGFb1 in been identified as direct targets (directly binding of HOXB7 the clinical samples either. All of these lines of evidence suggested protein to the promoter of target genes; refs. 9, 12, 13, 21). This that HOXB7 can specifically regulate its target gene expression in a is largely due to the ambiguous short binding motif containing context-dependent manner. It remains unknown whether the ATTA or TAAT sequence, which is present in almost any gene binding specificity is determined by the adjacent sequences or promoter and can be shared by 38 other HOX proteins (21). To unknown cofactors. demonstrate that TGFb2 is one of the novel direct targets of Another question is whether TGFb2 is important for the func- HOXB7, we performed quantitative ChIP assay and found that tions of HOXB7 in cancer progression. HOXB7 has been shown to HOXB7 protein directly binds to the two proximal potential promote cell migration and invasion, and induce EMT and binding sites in the TGFb2 promoter. The direct regulation of angiogenesis (9, 12–14). A large number of reports indicate that TGFb2 transcription by HOXB7 was corroborated by the obser- TGFb can play very similar roles (33, 34). For example, TGFb vation that HOX cofactor Pbx2 and Meis were recruited to the expression is higher in invasive breast carcinoma than in ductal

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A HOXB7 TGFb1 TGFb2 40 150 P = 0.03 15 P = 0.015 P = 0.15 30 10 100 20 Figure 6. The coordinate expression of HOXB7 and 4 5 50 TGFb2 in primary human breast cancers. A, real-time RT-PCR analysis of HOXB7, 2 TGFb1, and TGFb2 in primary human breast cancer tissues and normal adjacent tissues. Relative mRNA expression 0 Relative mRNA expression 0 Relative mRNA expression 0 B, correlation analysis of HOXB7 and TGFb1 Normal Tumor Normal Tumor Normal Tumor expression in primary invasive breast cancers (n ¼ 46). Pearson correlation BCR = 0.015 coefﬁcient was calculated on the basis of 4 10 R = 0.267 P = 0.42 the relative mRNA expression levels of P = 0.0002 HOXB7 and TGFb1. C, correlation analysis of 2 5 HOXB7 and TGFb2 expression in primary ) ) n ¼ P < 0 2 breast cancer ( 46). Values of 0.05 2 0 were considered signiﬁcant. –2 (log (log –5 –4 Expression of TGF b 2

Expression of TGF b 1 –6 –10 –4 –2 0 2 4 6 –4 –2 0 2 4 6

Expression of HOXB7 (log2) Expression of HOXB7 (log2)

carcinoma in situ (35, 36). Although most studies analyzed TGFb1 cating that upregulation of TGFb2 expression at least partially expression, TGFb2 may also favor invasiveness of breast cancer accounts for the function of HOXB7 in promotion of tumor because both isoforms share similar biologic activities (29, 30). progression in vivo. In line with this finding, the linear correlation This hypothesis is supported by data that TGFb2 was identified as between HOXB7 and TGFb2 expression tends to be more pro- one of the genes differentially expressed in highly metastatic nounced in later stage and more aggressive breast tumors. tumors in a mouse model of spontaneous breast cancer metastasis It is well known that macrophages play very important roles in to bone (37). We therefore proposed that HOXB7 promotes progression of tumors. Tumor cells recruit macrophages via tumor progression through upregulation of TGFb2 gene in breast chemokines and in turn activate them to acquire M2 phenotype carcinomas. Indeed, our data confirmed that chemical inhibition by secreting cytokines (22, 25, 38–40). M2 macrophages are of TGFb signaling or knockdown of TGFb2 expression can dra- strongly associated with poor outcomes in patients with a variety matically decrease HOXB7-mediated migration and invasion of cancers (40). M2 macrophages are believed to promote tumor abilities. Further in vivo assay demonstrated that HOXB7-promot- cell migration, invasion, and intravasation by releasing growth ed metastasis to lung was blocked by TGFb2 knockdown, indi- factors such as VEGF and bFGF to induce angiogenesis and by producing proteases such as MMP9 to degrade extracellular matrix (41, 42). Recently, it was reported that TGFb2 can induce macro- Table 1 Correlation of HOXB7 and TGFb2 in primary breast tumors phages into M2 phenotype (28), which led us to explore the effect Group N (%) R2 Pa of HOXB7 on activation of macrophages. We found that macro- fi All 46 (100%) 0.267 0.0002 phages were attracted more ef ciently into HOXB7-overexpres- Tumor grade sing tumors than the control tumors, and more importantly, 1 4 (8.6%) 0.023 0.848 acquired M2 macrophage phenotypes in HOXB7-overexpressing 2 19 (41.3%) 0.485 0.001 tumors. Further in vitro analysis showed that TGFb2 secreted by 3 23 (50.0%) 0.218 0.025 HOXB7-overexpressing breast cancer cells was directly responsi- Clinical stage ble the induction of M2 macrophage phenotypes. Recruitment I 9 (19.6%) 0.442 0.051 II 27 (58.6%) 0.165 0.040 and activation of macrophages adds another new mechanism to III and IV 7 (15.2%) 0.769 0.010 HOXB7-mediated promotion of tumor progression. Missing 3 (6.5%) 0.939 0.159 Lymph node status Disclosure of Potential Conflicts of Interest Negative 18 (39.1%) 0.168 0.092 No potential conflicts of interest were disclosed. Positive 24 (52.2%) 0.291 0.007 Missing 4 (8.6%) 0.970 0.015 HER2 Status Authors' Contributions Negative 22 (47.8%) 0.098 0.157 Conception and design: S. Liu, K. Jin, Q. Wang, H. Chen Positive 12 (26.1%) 0.453 0.016 Development of methodology: K. Jin, J. Fu, D. Fan, H. Chen Missing 12 (26.1%) 0.381 0.032 Acquisition of data (provided animals, acquired and managed patients, aValues in bold are statistically significant (P < 0.05). provided facilities, etc.): S. Liu, J. Fu, S. Feng, D. Reisman, D. Fan, S. Sukumar

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Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Grant Support computational analysis): S. Liu, C. Jie, D. Fan, S. Sukumar This work was supported by the American Cancer Society Research Award Writing, review, and/or revision of the manuscript: S. Liu, S. Feng, D. Reisman, (RSG-10-067-01-TBE; H. Chen), Susan G. Komen Postdoctoral Fellow- D. Fan, S. Sukumar, H. Chen ship (KG101506; K. Jin), and Susan G. Komen Leadership Grants Grant Administrative, technical, or material support (i.e., reporting or organizing (# SAC110050; S. Sukumar). data, constructing databases): Y. Hui, Q. Wang, The costs of publication of this article were defrayed in part by the pay- Study supervision: Q. Wang, H. Chen ment of page charges. This article must therefore be hereby marked advertise- ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments The authors thank Dr. Meredith Ray for providing assistance on statistical Received October 20, 2014; revised December 16, 2014; accepted December analysis and Ella S. Weinkle for help on processing breast cancer tissues. 17, 2014; published OnlineFirst December 26, 2014.

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HOXB7 Promotes Malignant Progression by Activating the TGFβ Signaling Pathway

Shou Liu, Kideok Jin, Yvonne Hui, et al.

Cancer Res 2015;75:709-719. Published OnlineFirst December 26, 2014.

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