HIF-1 regulates CD47 expression in breast cancer cells PNAS PLUS to promote evasion of and maintenance of cancer stem cells

Huimin Zhanga,b,c, Haiquan Lub,c, Lisha Xiangb,c, John W. Bullenb,c, Chuanzhao Zhangb,c, Debangshu Samantab,c, Daniele M. Gilkesd, Jianjun Hea, and Gregg L. Semenzab,c,d,e,f,g,h,1

aDepartment of Breast Surgery, The First Affiliated Hospital of Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061, China; bInstitute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; cMcKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; dDepartment of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; eDepartment of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; fDepartment of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; gDepartment of Radiation Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and hDepartment of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205

Contributed by Gregg L. Semenza, October 9, 2015 (sent for review September 18, 2015; reviewed by Shinae Kizaka-Kondoh and Michail V. Sitkovsky) Increased expression of CD47 has been reported to enable cancer phenotype (19, 20) through functional and physical interactions cells to evade phagocytosis by and to promote the of HIF-1 with the coactivator TAZ (20, 21) and by HIF-dependent cancer stem cell phenotype, but the molecular mechanisms regulat- expression of pluripotency factors (22). Hypoxia also induces im- ing CD47 expression have not been determined. Here we report mune evasion by several HIF-dependent mechanisms (23, 24). that hypoxia-inducible factor 1 (HIF-1) directly activates transcription A major mechanism by which cancer cells evade the innate im- CD47 of the in hypoxic breast cancer cells. Knockdown of HIF mune system is by expression of CD47, which is a cell-surface activity or CD47 expression increased the phagocytosis of breast that interacts with signal regulatory protein α (SIRPα)on cancer cells by bone marrow-derived macrophages. CD47 expression was increased in mammosphere cultures, which are enriched for the surface of macrophages to block phagocytosis (25, 26). Ex- cancer stem cells, and CD47 deficiency led to cancer stem cell de- pression of (CRT) on the surface of cancer cells is pletion. Analysis of datasets derived from thousands of patients the primary trigger for phagocytosis by binding to low-density with breast cancer revealed that CD47 expression was correlated lipoprotein-related protein (LRP) on the surface of macro- with HIF target and with patient mortality. Thus, phages (27). The prophagocytic signal triggered by CRT–LRP CD47 expression contributes to the lethal breast cancer phenotype ligation is counterbalanced by the antiphagocytic signal trig- that is mediated by HIF-1. gered by CD47–SIRPα ligation (28). Analysis of circulating tumor cells isolated from the blood of patients with breast antitumor immunity | immune evasion | tumor-initiating cells | cancer revealed that CD47 expression identified a subpopula- “ ’ ” | don t eat me signal tion of cells with the capability to generate tumor xenografts in mice (29). Here, we report that CD47 expression is induced in a he pathogenesis of breast cancer reflects not only the con- HIF-dependent manner when human breast cancer cells are Tsequence of somatic mutations that dysregulate oncogenes and tumor suppressor , but also the effect of the changing exposed to hypoxia. Modest inhibition of CD47 expression was tumor microenvironment, particularly the development of intra- sufficient to increase the phagocytosis of breast cancer cells and decrease the number of breast CSCs. Human breast cancer tumoral hypoxia. The median pO2 within primary breast cancers is 10 mmHg (1.4% O2) compared with 65 mmHg (9.3% O2)in database analysis revealed that high CD47 expression is corre- normal breast tissue (1). Exposure of breast cancer cells to re- lated with increased HIF target gene expression and decreased duced O2 availability induces the activity of hypoxia-inducible patient survival. factors (HIFs), which are heterodimeric transcriptional activators, α α α consisting of an O2-regulated HIF-1 ,HIF-2 , or HIF-3 subunit Significance and a constitutively expressed HIF-1β subunit, that control the expression of hundreds of target genes (2). Increased expression of α Uncontrolled cell proliferation and abnormal blood vessel for- the HIF-1 subunit, detected by immunohistochemistry in primary mation result in regions of breast cancers that are hypoxic breast cancer biopsies, is associated with a significantly increased (deprived of oxygen). Hypoxia-inducible factors (HIFs) stimu- risk of and mortality (2). late the expression of genes that enable cancer cells to invade MEDICAL SCIENCES Preclinical studies in mouse models have demonstrated that α α and metastasize, leading to patient mortality. In this paper, we loss of HIF-1 or HIF-2 expression impairs the metastasis of report that HIFs stimulate the production of CD47, a protein on breast cancer cells to axillary lymph nodes (3), lungs (4, 5), and the cell surface that enables cancer cells to avoid destruction by bone (6, 7). Specific HIF target genes have been identified that macrophages. CD47 is also important for maintaining cancer are induced by hypoxia in breast cancer cells and promote stem cells, which are a small population of cells that are re- critical steps in the metastatic process, including stromal cell quired for the formation of primary tumors and metastases. recruitment (8, 9), cancer (10), invasion and Reduction of HIF activity or CD47 levels in breast cancer cells – intravasation (11 15), margination and extravasation (5), and led to increased killing by macrophages and depletion of can- premetastatic niche formation (12, 16). Increased expression of cer stem cells. HIF target genes in primary breast cancers is associated with increased patient mortality (17, 18). Author contributions: H.Z. and G.L.S. designed research; H.Z., H.L., L.X., J.W.B., C.Z., D.S., To give rise to a primary tumor, a tumor relapse, or a meta- and D.M.G. performed research; J.H. contributed new reagents/analytic tools; H.Z. and static tumor, a breast cancer cell must possess two important G.L.S. analyzed data; and H.Z. and G.L.S. wrote the paper. characteristics: first, the cell must avoid destruction by the im- Reviewers: S.K.-K., Tokyo Institute of Technology; and M.V.S., Northeastern University. mune system and, second, the cell must possess stem-cell–like The authors declare no conflict of interest. properties. Hypoxia induces the breast cancer stem cell (CSC) 1To whom correspondence should be addressed. Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1520032112 PNAS | Published online October 28, 2015 | E6215–E6223 Downloaded by guest on September 26, 2021 Results is an immortalized but nontumorigenic mammary epithelial line; + + MCF7 is ER PR and tumorigenic but nonmetastatic; HCC1954 is Hypoxia Induces Increased CD47 Expression in a HIF-Dependent Manner. + HER2 and tumorigenic but nonmetastatic; and MDA-MB-231, Breast cancers are classified based on their expression of the estrogen − − − receptor (ER), progesterone receptor (PR), and human epidermal MDA-MB-435, and SUM159 are ER PR HER2 (i.e., triple growth factor receptor 2 (HER2); they are also classified according to negative), tumorigenic and metastatic. Reverse transcription (RT) expression of a 50-mRNA signature (PAM50) into luminal A, lu- and quantitative real-time PCR (qPCR) assays revealed that minal B, HER2-enriched, basal-like, and normal-like subgroups (30). expression of CD47 mRNA was significantly induced by We analyzed the effect of hypoxia (1% O2 for 24 h) on CD47 hypoxia in MCF7, HCC1954, MDA-MB-435, and SUM159 mRNA levels in six different human breast cell lines: MCF10A cells but not in MCF10A or MDA-MB-231 cells (Fig. 1A).

ABCMCF7 HCC1954

MCF7 HCC1954 SUM159 D SUM159 E

FGHMCF7 HCC1954 SUM159

20% O2 1% O2 20% O2 1% O2

NTC NTC NTC NTC 20% O2 DKD DKD DKD DKD 1% O2 ISO ISO ISO ISO ISO % of Max

Fig. 1. Hypoxia induces CD47 expression in a HIF-dependent manner. (A) Reverse transcription (RT) and quantitative real-time PCR (qPCR) were performed to

determine CD47 mRNA levels in human breast cell lines following exposure to 20% or 1% O2 for 24 h. For each sample, the expression of CD47 mRNA was quantified relative to 18S rRNA and then normalized to the result obtained from MCF-10A cells at 20% O2 (mean ± SEM; n = 3). Significant increase at 1% O2 compared with 20% O2:*P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test). (B and C) CD47 mRNA expression was analyzed by RT-qPCR in MCF7 (B) and HCC1954 (C) subclones expressing shRNA targeting HIF-1α (sh1α), HIF-2α (sh2α), or HIF-1α and HIF-2α (DKD), or expressing a nontargeting control shRNA (NTC), which were ## ### exposed to 20% or 1% O2 for 24 h. Data were normalized to NTC at 20% O2 (mean ± SEM; n = 3). **P < 0.01, ***P < 0.001 vs. NTC at 20% O2; P < 0.01, P < 0.001 vs. NTC at 1% O2 (two-way ANOVA with Bonferroni posttest). (D) SUM159 cells were treated with vehicle (Con) or 2.5 μM acriflavine (ACF), exposed to 20% or ## 1% O2 for 48 h, and expression of CD47 mRNA was assayed by RT-qPCR. ***P < 0.001 vs. Con at 20% O2; P < 0.01 vs. Con at 1% O2 (two-way ANOVA with Bonferroni posttest). (E) Immunoblot assays were performed to analyze HIF-2α,HIF-1α, CD47, and Actin protein expression in whole cell lysates prepared from MCF7

(Left) and HCC1954 (Middle) subclones exposed to 20% O2 (N) or 1% O2 (H) for 24 h, and lysates from SUM159 cells (Right), which were treated with vehicle (Con) or 2.5 μM acriflavine (ACF) and exposed to 20% or 1% O2 for 48 h. (F–H)MCF7(F), HCC1954 (G), and SUM159 (H) cells were exposed to 20% or 1% O2 for 24 h and CD47 expression on the cell surface was determined by flow cytometry using anti-CD47 antibody or isotype control antibody (ISO) (Upper). The anti-CD47 median fluorescence intensity (MFI) was determined and normalized to lane 1 in each bar graph (Lower; mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. NTC at ## ### 20% O2; P < 0.01, P < 0.001 vs. NTC at 1% O2 (two-way ANOVA with Bonferroni posttest for MCF7 and HCC1954 subclones; Student’s t test for SUM159 cells).

E6216 | www.pnas.org/cgi/doi/10.1073/pnas.1520032112 Zhang et al. Downloaded by guest on September 26, 2021 To determine whether HIF-1α or HIF-2α was required for CD47 CD47 Is a Direct HIF-1 Target Gene. To determine whether HIF-1 PNAS PLUS expression under hypoxic conditions, we analyzed MCF7 and directly regulates CD47 gene transcription, the HCC1954 subclones, which were stably transfected with an expression sequence was searched for matches to the consensus HIF bind- vector encoding short hairpin RNA (shRNA) targeting HIF-1α ing site 5′-(A/G)CGTG-3′ (32) that were located within DNase (sh1α)orHIF-2α (sh2α)orbothHIF-1α and HIF-2α (double I-hypersensitive domains at the CD47 . Chromatin immu- knockdown, DKD) or a nontargeting control shRNA (NTC). noprecipitation (ChIP) assays revealed that a DNA sequence, Hypoxia-induced CD47 mRNA expression was lost in MCF7 sh1α which encompassed 5′-GCGTG-3′ at −239 bp (site 1) and 5′- and DKD subclones (Fig. 1B) and in HCC1954 sh1α,sh2α,and CACGC-3′ (5′-GCGTG-3′ on the antisense strand) at −200 bp DKD subclones (Fig. 1C). Acriflavine inhibits the dimerization of (site 2), relative to the CD47 transcription start site (Fig. 2A, α α β HIF-1 or HIF-2 with HIF-1 (31), leading to the degradation of Top), was enriched by immunoprecipitation of chromatin from α α > undimerized HIF-1 or HIF-2 subunits during prolonged ( 24 h) hypoxic MCF7, HCC1954, and SUM159 cells with HIF-1α or incubation (18). Treatment of SUM159 cells with acriflavine HIF-1β antibodies (Fig. 2B), indicating that hypoxia induces di- blocked hypoxia-induced CD47 mRNA expression (Fig. 1D). rect binding of HIF-1 to the CD47 promoter. Hypoxic induction of CD47 protein expression in whole cell To determine whether DNA sequences encompassing either lysates was also abrogated in MCF7-DKD and HCC1954-DKD site alone functioned as a hypoxia response element (HRE), a subclones and in acriflavine-treated SUM159 cells (Fig. 1E). 55-bp oligonucleotide spanning HIF binding site 1 or site 2 (Fig. Because cell-surface expression of CD47 is required for its 2A, Bottom) was inserted into pGL2-promoter, which contains a antiphagocytic capacity, flow cytometry was performed. MCF7- DKD and HCC1954-DKD subclones showed decreased CD47 basal SV40 promoter driving expression of firefly luciferase, to cell-surface expression compared with the respective NTC sub- generate reporters pGL2-HRE1 and pGL2-HRE2, respectively. As a negative control, we used pSV-Renilla, which encodes clones under both hypoxic and nonhypoxic (20% O2) conditions (Fig. 1 F and G). In HCC1954-NTC and SUM159 cells, hypoxia Renilla luciferase driven by the SV40 promoter alone. Hypoxia increased CD47 cell-surface expression (Fig. 1H). It was not significantly increased the ratio of firefly:Renilla luciferase in possible to perform flow cytometry on acriflavine-treated cells HCC1954 and SUM159 cells that were cotransfected with either due to the inherent fluorescence of the drug. Taken together, the pGL2-HRE1 or pGL2-HRE2 and pSV-Renilla (Fig. 2C), dem- data in Fig. 1 demonstrate that CD47 mRNA and protein ex- onstrating that each of the 55-bp oligonucleotides functions as pression are induced by hypoxia in three breast cancer cell lines an HRE. Taken together, the data presented in Fig. 2 indicate in a HIF-dependent manner, leading to increased cell-surface that HIF-1 binds directly to the CD47 promoter to activate gene expression of CD47 in HCC1954 and SUM159 cells. transcription.

A

B MCF7 HCC1954 SUM159

C HCC1954 SUM159 HCC1954 SUM159 MEDICAL SCIENCES

pGL2-HRE1 pGL2-HRE2

Fig. 2. CD47 is a direct HIF-1 target gene. (A) Two candidate HIF-1 binding sites in the 5′-flanking region of the human CD47 gene were identified: 5′-GCGTG-3′ (site 1, located 239 bp 5′ to the transcription start site) and 5′-CACGC-3′ (site2,located200bp5′ to the transcription start site), which is the complementary sequence of 5′-

GCGTG-3′ on the antisense strand, are shown in red. (B)MCF-7(Left), HCC1954 (Middle), and SUM159 (Right) cells were exposed to 20% or 1% O2 for 16 h and chromatin immunoprecipitation (ChIP) assays were performed using IgG or antibodies against HIF-1α or HIF-1β. Primers flanking the entire sequence shown in A were used for qPCR

and results were normalized to IgG at 20% O2 (mean ± SEM; n = 3). *P < 0.05, **P < 0.01 vs. 20% O2 (Student’s t test). (C) Cells were cotransfected with pSV-Renilla and firefly luciferase reporter pGL2-HRE1 or pGL2-HRE2, containing an oligonucleotide encompassing HIF binding site 1 or site 2, respectively, and exposed to 20% or 1% O2 for 24 h. Luciferase activity (firely:Renilla luciferase ratio) was determined and normalized to 20% O2 (mean ± SEM; n = 3). **P < 0.01 vs. 20% O2 (Student’s t test).

Zhang et al. PNAS | Published online October 28, 2015 | E6217 Downloaded by guest on September 26, 2021 A CD47 Deficiency Increases the Phagocytosis of Breast Cancer Cells. To determine whether CD47 mediates protection of breast cancer cells against phagocytosis, we transfected SUM159 cells with an expression vector encoding one of five different shRNAs tar- geting CD47. Remarkably, we were not able to establish stable subclones that expressed three of the shRNAs and expression of the other two shRNAs (shCD47-2 and shCD47-4) caused rela- tively modest (50–75%) inhibition of CD47 mRNA (Fig. 4A)and cell-surface protein (Fig. 4B) expression. However, phagocytosis was significantly increased in the CD47-knockdown subclones (Fig. 4C) and CD47 levels were inversely correlated with the ex- tent of phagocytosis. Taken together, the data presented in Fig. 4 demonstrate that CD47 expression protects breast cancer cells

F4/80-APC against phagocytosis by bone marrow-derived macrophages.

CD47 Promotes the Breast CSC Phenotype. CD47 is preferentially expressed on bladder, liver, and pancreatic CSCs compared with the bulk cancer cells (non-CSCs) (33–36). To investigate whether CD47 plays a role in breast CSCs, we first analyzed CD47 mRNA levels in SUM159 cells, which were cultured as standard adherent monolayers or as nonadherent spheroids (mammo- spheres), which are highly enriched for CSCs (37). CD47 mRNA levels were twofold higher in mammosphere cultures (Fig. 5A). Exposure of breast cancer cells to hypoxia for 3 d induces in- CFSE creased mammosphere formation in a HIF-1α–dependent man- ner (20). Knockdown of CD47 expression significantly decreased mammosphere formation, with the greatest reduction observed BCHCC1954 MCF7 in the shCD47-4 subclone (Fig. 5B), which had the greatest in- hibition of CD47 expression (Fig. 4 A and B). Analysis of alde- hyde dehydrogenase (ALDH) activity in breast cancer cells also identifies a subpopulation that is enriched for CSCs (38) and the + percentage of ALDH cells increases in response to hypoxia (19, + 20). Hypoxia markedly increased the percentage of ALDH cells in the NTC subclone, whereas knockdown of CD47 expression + significantly decreased the percentage of ALDH CSCs at both 20% and 1% O2 (Fig. 5C). Taken together, the data presented in Fig. 5 demonstrate that CD47 expression plays an important role in promoting the breast CSC phenotype. We and others have α Fig. 3. Increased phagocytosis of HIF-deficient breast cancer cells. (A) HCC1954 previously reported that knockdown of HIF-1 blocks hypoxia-

NTC and DKD subclones were exposed to 20% or 1% O2 for 24 h, stained with induced enrichment of breast CSCs, as determined by ALDH CFSE, incubated with mouse bone marrow-derived macrophages for 2 h, expression or mammosphere formation assay (19, 20). Thus, stained with F4/80-APC antibody, and analyzed by flow cytometry. (B and C) CD47 loss-of-function phenocopies HIF-1α loss of function with Phagocytosis assays were performed with HCC1954 (B)andMCF7(C) subclones + + respect to CSC maintenance. as described above and the percentage of CFSE APC phagocytosed cancer cells ± = < < was normalized to NTC at 20% O2 (mean SEM; n 3). **P 0.01, ***P CD47 Expression Is Associated with HIF Target Gene Expression and ## < ### < 0.001 vs. NTC at 20% O2; P 0.01, P 0.001 vs. NTC at 1% O2 (two-way Patient Mortality. To test whether the results obtained from the ANOVA with Bonferroni posttest). analysis of breast cancer cell lines are relevant to patients with breast cancer, we analyzed gene expression data from 1,040 primary human HIF Deficiency Increases the Phagocytosis of Breast Cancer Cells. We breast cancer samples that are publicly available in The Cancer observed decreased expression of CD47 on the surface of HIF- Genome Atlas (TCGA) database (39). To determine whether HIFs deficient human breast cancer cells (Fig. 1 F and G), leading us regulate CD47 gene expression in human breast cancers, we com- to hypothesize that HIF inhibition may promote phagocytosis of pared CD47 mRNA levels with the levels of P4HA1, P4HA2, breast cancer cells by macrophages. To test this hypothesis, we ANGPTL4, SLC2A1, CXCR3, VEGFA, PLOD1, PLOD2, L1CAM, and MET mRNA, which are all products of genes that are HIF performed in vitro phagocytosis assays on HCC1954 and MCF7 regulated in breast cancer cells (18, 20). CD47 mRNA levels, like subclones. The breast cancer cells were labeled with the fluo- those of other HIF target genes, were most highly expressed in breast rescent dye CFSE, exposed to 20% or 1% O for 24 h, cocultured 2 cancers of the basal-like molecular subtype (Fig. 6A). Statistical with bone marrow-derived macrophages for 2 h, incubated with analysis revealed that CD47 mRNA levels were significantly corre- allophycocyanin (APC)-labeled F4/80 antibody, and analyzed by + + lated with the levels of 8 of 10 mRNAs encoded by HIF target genes flow cytometry to detect APC CFSE cells, which represent (Fig. 6B), which is comparable to correlations between members of macrophages that have phagocytosed breast cancer cells (Upper this group (10, 15, 20). Expression of CD44, which is a marker of Right quadrant [Q2] of scatter plots in Fig. 3A). Phagocytosis was breast CSCs (40) and a HIF target gene (41), was also significantly significantly increased in MCF7-DKD and HCC1954-DKD cells correlated with CD47 expression in the 1,040 breast cancers (Fig. 6B). compared with the respective NTC subclone, under both hypoxic HIF target gene expression in human breast cancers is sig- and nonhypoxic conditions (Fig. 3 B and C). Hypoxia modestly nificantly associated with patient mortality (18). To determine if increased phagocytosis of both NTC and DKD subclones, which CD47 mRNA expression was also a prognostic factor in human suggests the existence of a prophagocytic signal that is induced by breast cancer, we used two published datasets that contain both hypoxia in a HIF-independent manner. gene expression and patient survival data (42, 43). Patients were

E6218 | www.pnas.org/cgi/doi/10.1073/pnas.1520032112 Zhang et al. Downloaded by guest on September 26, 2021 A stratified into two groups based on whether CD47 mRNA levels PNAS PLUS in the primary tumor were greater or less than the median value. Increased CD47 mRNA expression levels were associated with a significantly decreased probability of overall survival in the two independent datasets, which together comprised 1,954 patients with breast cancer (Fig. 6C). Discussion Growing evidence indicates that in various human cancers, CD47 expression is required to avoid innate immune surveillance and elimination by phagocytosis. Blocking the interaction of CD47 with its receptor, SIRPα, on macrophages enables phagocytosis and inhibits tumor growth (35, 36, 44–46). However, the mo- lecular mechanisms regulating the expression of CD47 by cancer B cells have not been delineated. In this study, we demonstrate that 20% O2 1% O2 hypoxia, which is a critical microenvironmental stimulus in ad- vanced breast cancers, induced HIF-dependent expression of NTC NTC CD47, leading to decreased phagocytosis of cancer cells by sh-2 sh-2 macrophages and induction of the breast CSC phenotype, which sh-4 sh-4 promote cancer progression and patient mortality (Fig. 6D). ISO ISO Despite the recent interest in targeting CD47 for cancer therapy (46), remarkably little is known about the transcriptional regulation

% of Max of the CD47 gene. The results presented here represent to our knowledge the first identification of a transcriptional regulator of CD47 expression in breast cancer cells and further studies are re- quired to determine whether HIF-1 cooperates with other tran- Anti-CD47-PE scription factors that are induced by the tumor microenvironment, such as CREB, NF-κB, SMAD2, or STAT3. Increased NF-κB activity was observed in hepatocellular carcinoma cells that de- veloped sorafenib resistance (36), which is of interest because the antiangiogenic effects of sorafenib induce intratumoral hypoxia that causes HIF-1–dependent induction of breast CSCs (19). Im- munosuppressive functional interactions of HIF-1 and CREB have been proposed in T cells (47), but may also occur in cancer cells. Intratumoral hypoxia is a common finding in breast cancer and HIFs activate the transcription of a large battery of genes encoding that promote multiple steps in tumor progression, including tumor growth and vascularization, stromal cell recruitment, extracellular matrix remodeling, premetastatic niche formation, cell motility, margination and extravasation of circulating tumor cells, and CSC specification/maintenance (2). Here, we find that HIFs also play an important role in immune evasion by activating the expression of CD47, an antiphagocytic signal. In three different breast cancer cell + lines, which represent luminal/ER (MCF7), HER2-enriched (HCC1954), and basal-like/triple-negative (SUM159) subtypes of C breast cancer, the expression of CD47 mRNA and protein was in- duced by hypoxia in a HIF-dependent manner. A recent study reported that CD47 protein was detected in 5% of hormone re- − ceptor positive, HER2 breast cancers (48). Our bioinformatic analysis suggests that CD47 expression is likely to be higher among

triple-negative breast cancers, most of which fall into the basal-like MEDICAL SCIENCES molecular subtype that is characterized by increased expression of the HIF transcriptome (18, 39). Hypoxia did not induce increased CD47 expression in MCF10A or MDA-MB-231 cells. This heterogeneous transcriptional re- sponse of breast cell lines to hypoxia is commonly observed. For example, analysis of mRNAs encoding lysyl oxidase (LOX) and LOX-like proteins (LOXL1–4) revealed that hypoxia induced

Fig. 4. CD47 deficiency increases the phagocytosis of breast cancer cells. (A)CD47 mRNA levels were analyzed by RT-qPCR in SUM159 subclones expressing either of two different shRNAs targeting CD47 (shCD47) or a nontargeting control shRNA ANOVA with Bonferroni posttest). (C) SUM159 subclones were exposed to

(NTC). Results were normalized to NTC (mean ± SEM; n = 3). ***P < 0.001 vs. NTC 20% or 1% O2 for 24 h, stained with CFSE, incubated with bone marrow- (one-way ANOVA with Bonferroni posttest). (B) CD47 protein expression on the cell derived macrophages for 2 h, stained with F4/80-APC antibody, subjected to + + surface was determined by flow cytometry of SUM159 subclones exposed to 20% or flow cytometry, and the percentage of CFSE APC phagocytosed cancer cells

1% O2 for 24 h (Upper). The anti-CD47 median fluorescence intensity (MFI) was was determined and normalized to the NTC subclone at 20% O2 (mean ± # ## determined and normalized to NTC at 20% O2 (Lower; mean ± SEM; n = 3). **P < SEM; n = 3). **P < 0.01 vs. NTC at 20% O2; P < 0.05, P < 0.01 vs. NTC at 1% ### 0.01, ***P < 0.001 vs. NTC at 20% O2; P < 0.001 vs. NTC at 1% O2 (two-way O2 (two-way ANOVA with Bonferroni posttest).

Zhang et al. PNAS | Published online October 28, 2015 | E6219 Downloaded by guest on September 26, 2021 A B

Monolayer Mammosphere

C

Fig. 5. CD47 expression promotes the breast cancer stem cell phenotype. (A) CD47 mRNA expression relative to 18S rRNA was analyzed by RT-qPCR using total RNA isolated from SUM159 cells that were grown in conventional adherent monolayer culture or as nonadherent mammospheres (described below).

The results were normalized to monolayer cells (mean ± SEM; n = 3). ***P < 0.001 (Student’s t test). (B) SUM159 subclones were exposed to 20% or 1% O2 for 72 h in monolayer culture, harvested, seeded at a density of 5,000 cells per well on six-well ultra-low attachment culture plates, and cultured for 5din

the presence of 20% O2 (Upper). Mammospheres that were greater than 50 μm in diameter were counted by automated image analysis (Lower; mean ± SEM; ### n = 3). **P < 0.01 vs. NTC at 20% O2; P < 0.001 vs. NTC at 1% O2 (two-way ANOVA with Bonferroni posttest). (C) SUM159 subclones were exposed to 20% or + 1% O2 for 72 h in monolayer culture and the percentage of cells expressing aldehyde dehydrogenase (ALDH ) was determined by flow cytometry (mean ± ## ### SEM; n = 3). *P < 0.05, **P < 0.01 vs. NTC at 20% O2; P < 0.01, P < 0.001 vs. NTC at 1% O2 (two-way ANOVA with Bonferroni posttest).

increased expression of LOX and LOXL4 in MDA-MB-231, suggesting that the increased phagocytosis of NTC and DKD whereas LOXL2 expression was induced in MDA-MB-435 subclones after hypoxic exposure may be due to HIF-inde- cells (12). We also observed heterogeneity with respect to re- pendent CRT expression, but further studies are required to quirements for HIF-α subunits, as CD47 induction required only test this hypothesis. A recent report demonstrated that CD47 HIF-1α in MCF7 cells, but both HIF-1α and HIF-2α in HCC1954 blockade also promotes T-cell–mediated elimination of im- cells. Similarly, we previously reported that hypoxia-induced munogenic tumors (52) and HIF-1 is known to inhibit effector LOXL2 expression required only HIF-1α in MDA-MB-435 cells, T cells through the production of adenosine (23, 47), sug- whereas expression of LOX and LOXL4 in MDA-MB-231 cells re- gesting that HIF inhibitors may improve the response to CD47 quired HIF-1α and HIF-2α (12). Heterogeneity at the level of gene blockade both by inhibiting CD47 expression and by dis- expression is recognized as a major obstacle to successful cancer inhibiting T-cell–mediated antitumor immunity. therapy. However, in the cases described above, treatment with a HIF Two findings in our study suggested that hypoxia-induced ex- inhibitor, such as acriflavine, successfully blocked all hypoxia-induced pression of CD47 plays an important role that is independent of gene expression (Fig. 1D). its antiphagocytic function. First, although CD47 mRNA and Cells express varying levels of prophagocytic and antiphagocytic protein expression were induced by hypoxia in all three breast signaling proteins, and it is the integration of both signals that de- cancer cell lines analyzed, increased CD47 cell-surface expres- termines whether the target cell will be phagocytosed. The relatively sion and decreased phagocytosis were only induced by hypoxia in modest changes in CD47 cell surface levels that resulted from ex- SUM159 cells. These data suggest that hypoxia-induced CD47 pression of shRNA targeting HIF-1α and HIF-2α or CD47 led to may fulfill another function that does not require cell-surface significant changes in phagocytosis, indicating a fine balance between expression of the protein. Second, attempts at generating CD47- pro- and antiphagocytic signaling, such that a hypoxic tumor micro- deficient cells were only successful in establishing subclones with environment may significantly tip the balance toward immune eva- modest knockdown of CD47, suggesting that expression of the sion. Several prophagocytic signals have been identified, including protein was required for clonal expansion. Using two established cell-surface expression of phosphatidylserine and CRT (49, 50). CRT assays for breast CSCs, we demonstrated that CD47 expression is required for the phagocytosis of cancer cells when CD47–SIRPα was increased in breast CSCs (relative to bulk cancer cells) and interaction is blocked (28). CRT expression was induced by ex- was required for breast CSC maintenance, as decreased CD47 posure of cardiomyocytes to hypoxia and reoxygenation (51), expression led to significantly reduced numbers of CSCs in a

E6220 | www.pnas.org/cgi/doi/10.1073/pnas.1520032112 Zhang et al. Downloaded by guest on September 26, 2021 PNAS PLUS ABGENE CD47 P4HA1 * P4HA2 n.s. ANGPTL4 n.s. SLC2A1 *** CXCR3 *** VEGFA * PLOD1 *** PLOD2 *** L1CAM *** MET *** CD44 ***

C D Intratumoral Hypoxia

HIF-1

CD47

Evasion of Specification and/or phagocytosis maintenance of CSCs

Cancer progression

Patient mortality

Fig. 6. CD47 expression in primary human breast cancers is associated with HIF target gene expression and decreased patient survival. (A) The relative expression levels of 10 mRNAs encoded by HIF target genes, as well as CD44 and CD47 mRNAs, are shown for 1,040 primary breast cancer tissue samples from The Cancer Genome Atlas (TCGA), which were grouped according to the expression levels of 50 mRNAs (PAM50) that define the luminal A, luminal B, HER2+, basal-like, and normal-like breast cancer molecular subtypes (57). Color code for mRNA expression levels are as follows: red, greater than median; green, less than median. (B) CD47 mRNA expression in each primary breast cancer specimen was compared with the expression of CD44 mRNA and HIF-regulated mRNAs using Pearson’s correlation test. *P < 0.05, ***P < 0.001; n.s., not significant. (C)Kaplan–Meier curves were constructed to analyze the association of CD47 mRNA expression in the primary tumor with overall survival (OS) of patients with breast cancer using two independent datasets (42, 43). Statistical analysis was performed using log- tests. (D) Intratumoral hypoxia induces HIF-1–dependent ex- pression of CD47 in breast cancer cells, leading to decreased phagocytosis of cancer cells by macrophages and induction of the cancer stem cell (CSC) phenotype, which promote cancer progression and patient mortality. MEDICAL SCIENCES dose-dependent manner. Further studies are required to de- also found that CD47 mRNA expression in primary human breast termine whether CD47 must be expressed at the cell surface to cancers was significantly associated with patient mortality in two contribute to the maintenance of breast CSCs. large and independent datasets (Fig. 6C). Similar results were To generate a primary, recurrent, or metastatic tumor, a breast recently reported regarding the immunohistochemical detection of cancer cell must avoid immune destruction and give rise to both CD47 protein expression in breast cancer biopsies (48). CSCs and differentiated breast cancer cells. A recent study found We have recently demonstrated that exposure of breast cancer that among circulating tumor cells in the blood of women with cells to hypoxia or chemotherapy induces the breast CSC phe- metastatic breast cancer, the cells that were capable of initiating notype through multiple HIF-dependent molecular pathways tumors when injected into mice expressed the cell surface proteins (18, 20, 54). Induction of CD47 expression provides another CD47, CD44, and MET (29). Remarkably, as in the case of CD47, mechanism by which hypoxia induces the CSC phenotype. In the expression of CD44 and MET is induced by hypoxia in a HIF- addition to interacting with SIRPα, CD47 engages in several dependent manner (41, 53). Furthermore, our analysis of gene other functional interactions (55) and further studies are re- expression data from over 1,000 human breast cancers revealed quired to determine the molecular mechanism by which the ex- that CD47 expression was significantly correlated with the ex- pression of CD47, localized to the cell surface or perhaps an pression of CD44, MET, and other HIF target genes (Fig. 6B). We intracellular compartment, promotes the specification and/or

Zhang et al. PNAS | Published online October 28, 2015 | E6221 Downloaded by guest on September 26, 2021 maintenance of CSCs. Genetic or pharmacologic inhibition of TGA GGT CCC CGG CGA GCG TGG GAA CAC AGG GTT CAG CCT CCT GC-3′ HIF activity reduced the percentage of CSCs (18–20, 56), which and 5′-TCG AGC AGG AGG CTG AAC CCT GTG TTC CCA CGC TCG CCG GGG may be due in part to the inhibition of CD47 expression. In- ACC TCA TTG CAC CCG TT-3′;HRE2,5′-GAT CGG TTC AGC CTC CTG CGG ′ ′ creased expression of HIF-1α mRNA or protein, or HIF target CGG GCG AGC ACG CGG ACC CCA GGG GCG GGC GGG TGC GA-3 and 5 - TCG ATC GCA CCC GCC CGC CCC TGG GGT CCG CGT GCT CGC CCG CCG gene mRNAs, in primary breast cancer biopsies is associated CAG GAG GCT GAA CC-3′. At 24 h after transfection, cells were exposed to with increased patient mortality (2, 18). Our data suggest that 20% or 1% O2 for 24 h and lysed. Luciferase activities were determined with a addition of HIF inhibitors to current treatment regimens may multiwell luminescence reader (PerkinElmer) using the Dual-Luciferase Re- improve outcome in such high-risk patients in part by attacking porter Assay System (Promega). CSCs and disinhibiting innate immunity. Cell Surface Expression of CD47. Cells were harvested after 24-h exposure to

Materials and Methods 20% or 1% O2 and a single cell suspension was prepared. Cells were in- Cell Lines and Culture. Human breast cell lines MCF10A, MCF7, HCC1954, cubated with Fc Block (BD Pharmingen) for 10 min, then stained with phy- SUM159, MDA-MB-231, and MDA-MB-435 were cultured as previously de- coerythrin (PE)-conjugated CD47 antibody or isotype control (R&D Systems),

scribed (18, 20, 21, 54). All cells were maintained at 37 °C in a 5% CO2 and and subjected to flow cytometry (FACScalibur, BD Biosciences). Dead cells 95% air (20% O2) incubator. For hypoxia exposure, cells were placed in a were gated out by side-scatter and forward-scatter analysis. modular incubator chamber (Billups–Rothenberg) that was flushed with a

gas mixture containing 1% O2,5%CO2, and 94% N2. Isolation of Bone Marrow-Derived Macrophages. Bone marrow cells were isolated from mouse long bones and cultured for 7 d in RPMI-1640 supple- shRNA, Lentiviruses, and Transduction. All lentiviral vectors contained a puro- mented with 10% (vol/vol) FBS, 1% penicillin/streptomycin, and 30 ng/mL mycin resistance gene. Vectors encoding shRNA targeting HIF-1α and HIF-2α CSF1 (R&D Systems) as previously described (9). were described previously (5). Vectors encoding CD47 shRNAs were purchased from Sigma-Aldrich; the clone ID and nucleotide sequences coding for shRNA Phagocytosis Assay. Macrophages were plated (5 × 104 per well) in a 24-well are as follows: shCD47-2: NM_001777.2–1024s1c1; 5′-CCG GGC TTC CAA TCA tissue-culture plate in complete RPMI-1640 medium, which was supple- GAA GAC TAT ACT CGA GTA TAG TCT TCT GAT TGG AAG CTT TTT-3′; shCD47-4: mented with CSF1, 24 h before the experiment. Breast cancer cells were NM_001777.2–988s1c1; 5′-CCG GGC ACA ATT ACT TGG ACT AGT TCT CGA stained with 2.5-μM carboxyfluorescein succinimidyl ester (CFSE) according GAA CTA GTC CAA GTA ATT GTG CTT TTT-3′. Lentiviruses were packaged in to the manufacturer’s protocol (Invitrogen). Macrophages were incubated in 293T cells by cotransfection with plasmid pCMV-dR8.91 and plasmid encoding serum-free medium for 2 h before adding 2 × 105 CFSE-labeled breast cancer vesicular stomatitis virus G protein using PolyJet (SignaGen). Supernatant cells. After coculture for 2 h at 37 °C, cells were harvested, macrophages containing viral particles was collected 48 h posttransfection, filtered (0.45-μm were stained with APC-conjugated F4/80 (Novus Biologicals), and flow pore size), and transduced into MCF7, HCC1954, or SUM159 cells in the pres- cytometry (FACScalibur, BD Biosciences) was performed. A total of 10,000 μ ence of 8 g/mL of Polybrene (Sigma-Aldrich). After 24 h, cells were main- cells in each sample were analyzed. Unstained control and single stained μ tained in medium containing 0.5 g/mL puromycin (Sigma-Aldrich). cells were prepared for gating. Phagocytosis was calculated as the per- + + + centage of F4/80 CFSE cells (Q2) among CSFE cells (Q2 + Q3): phagocytosis RT-qPCR. Total RNA was extracted, cDNA was synthesized by RT, and qPCR (%) = [Q2/(Q2 + Q3)] × 100%. analysis was performed as described (18), using PCR primers with the fol- ′ ′ lowing sequences: CD47, 5 -AGA AGG TGA AAC GAT CAT CGA GC-3 and Mammosphere Assay. Cells were exposed to 20% or 1% O in monolayer ′ ′ ′ 2 5 -CTC ATC CAT ACC ACC GGA TCT-3 ; and 18S rRNA, 5 -CGG CGA CGA CCC adherent culture for 72 h. Single cells were plated in six-well ultra-low at- ′ ′ ′ ATT CGA AC-3 and 5 -GAA TCG AAC CCT GAT TCC CCG TC-3 . tachment culture plates (Corning) at a density of 5,000 cells per well in complete mammocult medium (Stem Cell Technologies). Mammospheres Immunoblot Assay. Whole-cell lysates were prepared in modified RIPA buffer; (diameter ≥50 μm) were counted after 5 d. Mammosphere cultures were im- proteins were separated by SDS/PAGE, blotted onto nitrocellulose membranes, aged using an Olympus phase-contrast microscope and mammosphere diam- and probed with primary antibodies against HIF-1α,HIF-2α, and CD47 (Novus eters were determined using ImageJ software (National Institutes of Health). Biologicals). HRP-conjugated secondary antibodies (GE Healthcare) were used. Chemiluminescent signals were detected using ECL Plus (GE Healthcare). Blots ALDH Assay. Cells were exposed to 20% or 1% O2 for 72 h and harvested for were stripped and reprobed with antiactin antibody (Santa Cruz). Aldefluor assay (Stem Cell Technologies). Cells were suspended in assay buffer containing 1 μmol/L BODIPY-aminoacetaldehyde and incubated for ChIP Assay. Cells were exposed to 20% or 1% O2 for 16 h, then cross-linked in 45 min at 37 °C. As a negative control, for each sample an aliquot of cells was the presence of 3.7% (vol/vol) formaldehyde for 10 min, quenched in treated with the ALDH inhibitor diethylaminobenzaldehyde (50 mM). Sam- 0.125 M glycine for 5 min, and lysed with SDS lysis buffer. Chromatin was ples were subjected to flow cytometry analysis (FACScalibur, BD Biosciences). sheared by sonication, and lysates were precleared with salmon sperm DNA/ protein A-agarose slurry (Millipore) and incubated with IgG or antibodies Statistical Analysis. Data are expressed as mean ± SEM and were analyzed against HIF-1α (Santa Cruz) or HIF-1β (Novus Biologicals) in the presence of using Student’s t test for two groups or ANOVA with Bonferroni posttest for protein A-conjugated agarose beads overnight. After sequential washes of multiple groups. Kaplan–Meier curves were generated using two indepen- the beads, DNA was eluted in 1% SDS with 0.1 M NaHCO , and cross-links 3 dent datasets containing gene expression and overall survival data on pa- were reversed by addition of 0.2 M NaCl. DNA was purified by phenol- tients with breast cancer (42, 43). The log rank test was performed to chloroform extraction and ethanol precipitation. Candidate binding sites determine whether observed differences between groups were statistically were analyzed by qPCR using flanking primers with the following sequences: significant. P values <0.05 were considered significant. 5′-AGG AAC GGG TGC AAT GAG-3′ and 5-CTT CCA GGT CAC GTC CTG TC-3′. ACKNOWLEDGMENTS. We thank Karen Padgett of Novus Biologicals for Luciferase Reporter Assays. A total of 10,000 cells plated onto 48-well plates providing APC-F4/80, CD47, and HIF-1β antibodies. This work was supported were cotransfected with pGL2-HRE1 or pGL2-HRE2 and pSV-Renilla. The in part by American Cancer Society Grant 122437-RP-12-090-01-COUN. G.L.S. sequences of the double-strand oligonucleotides used for pGL2-HRE is an American Cancer Society Research Professor and the C. Michael Armstrong plasmid construction are as follows: HRE1, 5′-GAT CAA CGG GTG CAA Professor at The Johns Hopkins University School of Medicine.

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