The S100A7-C-Jun Activation Domain Binding Protein 1 Pathway Enhances Prosurvival Pathways in Breast Cancer

Research Article

The S100A7-c-Jun Activation Domain Binding Protein 1 Pathway Enhances Prosurvival Pathways in Breast Cancer

Ethan D. Emberley,1,3 Yulian Niu,2,3 Linda Curtis,2,3 Sandra Troup,2,3 Sanat K. Mandal,4 Jeffery N. Myers,5 Spencer B. Gibson,1,3 Leigh C. Murphy,1,3 and Peter H. Watson2,3

Departments of 1Biochemistry and Medical Genetics and 2Pathology, University of Manitoba; 3Manitoba Institute of Cell Biology, Winnipeg, Manitoba, Canada; 4Basic Medical Sciences, Memorial University of Newfoundland and College of the North Atlantic, Clarenville Campus, Clarenville, Newfoundland, Canada; and 5Department of Head and Neck Surgery, University of Texas M.D. Anderson Cancer Center, Houston, Texas

Abstract expression occurs in parallel with early stages of tumor progression, S100A7 is among the most highly expressed genes in and S100A7 can be among the most highly expressed proteins in preinvasive breast cancer, is a marker of poor survival when preinvasive carcinoma in situ in both skin (3, 7) and breast (8, 9). expressed in invasive disease, and promotes breast tumor With progression to invasive carcinoma, expression is often progression in experimental models. To explore the mecha- diminished, but persistent expression can occur in subsets of nism of action, we examined the role of S100A7 in cell survival invasive breast carcinomas where S100A7 is associated with a and found that overexpression of S100A7 in MDA-MB-231 cell worse clinical outcome (10). The action of S100A7 in breast cells may be exerted through an lines promotes survival under conditions of anchorage- independent growth. This effect is paralleled by increased interaction with the multifunctional signaling molecule c-Jun activity of nuclear factor-KB (3-fold) and phospho-Akt (4- activation domain-binding protein 1 (Jab1). S100A7 expression in fold), which are known to mediate prosurvival pathways. the MDA-MB-231 cell model is associated with altered activity of S100A7 and phospho-Akt are also correlated in breast tumors activator protein-1 (AP-1) and hypoxia-inducible factor-1 (HIF-1) examined by immunohistochemistry (n = 142; P < 0.0001; transcription factors and their downstream genes, consistent with r = 0.34). To explore the underlying mechanism, we examined the downstream pathways believed to be regulated by Jab1 (11). the role of a putative c-Jun activation domain-binding protein 1 These molecular changes associated with expression of S100A7 are (Jab1)–binding domain within S100A7 using a panel of MDA- correlated with changes in several biological features in vitro, MB-231 breast cell lines stably transfected with either S100A7 including decreased adhesion, increased growth, and increased invasiveness. However, the magnitude of these in vitro effects is or S100A7 mutated at the Jab1 domain. Structural analysis by three-dimensional protein modeling, immunoprecipitation, relatively modest in comparison with a significant overall effect of and yeast two-hybrid assay and functional analysis using S100A7 on tumorigenesis in an in vivo xenograft model (11). This transfected reporter gene and Western blot assays revealed suggests that S100A7 may exert other effects that are more that the in vitro effects of S100A7 on phospho-Akt and the important and primarily manifested in the context of the more nuclear factor-KB pathway are dependent on the Jab1-binding complex and three-dimensional in vivo environment. site and the interaction with Jab1. Enhanced epidermal While considering the function of S100A7, we have observed that growth factor receptor signaling was also found to correlate S100A7 expression can be prominent within areas of tumors that with the increased phospho-Akt. Furthermore, the Jab1- show loss of adhesion and increased apoptosis and necrosis. These binding domain is also necessary for the enhanced tumorige- are particularly prominent features within high-grade ductal nicity conferred by S100A7 expression in murine xenograft carcinoma in situ (DCIS), but dyskeratosis and loss of cell adhesion tumors in vivo. We conclude that the S100A7-Jab1 pathway are also common features within preinvasive epidermal squamous acts to enhance survival under conditions of cellular stress, carcinoma and inflammatory skin lesions, including psoriasis and other idiopathic dermatoses, where S100A7 is highly expressed (12). such as anoikis, which may promote progression of breast cancer. (Cancer Res 2005; 65(13): 5696-702) In breast cells, overexpression is associated with diminished adhesion (11) and S100A7 is induced under conditions of cellular stress, including loss of adhesion (8). In this study, we have examined Introduction the relationship between S100A7 and cellular response to stress S100A7 is relatively unique among the S100 gene family in terms related to loss of adhesion and the possible role of the S100A7-Jab1 of structure and its prominent association with early stages of pathway in mediating a prosurvival effect in breast cancer. carcinoma (1). Originally associated with abnormally differentiating keratinocytes in psoriasis, ‘‘psoriasin’’ (S100A7) has since been found to be expressed in association with neoplasia in several Materials and Methods tissues, including squamous carcinomas in skin (2, 3) and bladder Mutation of the c-Jun activation domain-binding protein 1–binding (4), as well as adenocarcinomas of the breast (5, 6). Although the site in S100A7 and molecular modeling. The complete open reading protein is not expressed in normal luminal epithelium, increased frame sequence for S100A7 was cloned previously into the pcDNA3.1 Zeo (Invitrogen, Canada) expression vector (11). Site-directed mutagenesis was done using the Transformer Site-Directed Mutagenesis kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions. The Requests for reprints: Peter H. Watson, Department of Pathology, University of three amino acids (Asp58, Leu80, and Gln90) comprising the putative Jab1- Manitoba, D212-770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0W3. 58 80 90 Phone: 204-789-3435; Fax: 204-789-3931; E-mail: [email protected]. binding domain (13) were altered to encode Gly ,Met, and Lys , I2005 American Association for Cancer Research. respectively, using mutagenic oligonucleotides to produce the plasmid

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. S100A7 Stimulates Survival Pathways in Breast Cancer pcDNA3.1mut-psor. All mutations were confirmed by DNA sequencing. Beverly, MA); PTEN (gift from C. Eng, Ohio State University); and h-actin Three-dimensional models of S100A7 and mutant S100A7 (S100A7mut) were (Sigma). The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 built based on the X-ray structures (Protein Data Bank codes 3PSR and (Calbiochem, 15 Amol/L for 24 hours) or the EGFR inhibitor BPIQ (Alexis, 2PSR) and compared with the experimental findings (14). CAChe Work- Switzerland, 0.29 nmol/L for 24 hours) was added to the cell culture in some System Pro 6.1.1 (Fujitsu, Beaverton, OR) software was used to substitute experiments. the residues and to build the three-dimensional models. The energy of the Reporter gene assays. MDA-MB-231 wild-type cells and clones express- resulting molecules was optimized using molecular mechanics (MM2) and ing S100A7 or S100A7mut were transfected with a nuclear factor-nB (NF-nB)– optimization was continued until the energy change was <0.001 kcal/mol. driven luciferase reporter plasmid together with a constitutively expressing Cell culture and stable transfection. Human breast carcinoma cell Renilla luciferase expression vector (Promega, Canada) in a 10:1 ratio in lines MDA-MB-231 and MDA-MB-468 were cultured as described previously triplicate experiments using Effectene. Dual luciferase signals were measured (9). The former cell line is negative for S100A7, whereas the latter expresses from each cell lysate using the Stop and Glow kit (Promega) 18 hours after S100A7 mRNA and protein [confirmed by reverse transcription-PCR (RT- transfection. Firefly luciferase activity for each sample was standardized to PCR) and Western blot; data not shown]. MDA-MB-231 clones stably the corresponding Renilla luciferase activity for that sample. expressing S100A7 were described previously (11). MDA-MB-231 clones In vivo mouse tumorigenic studies. The tumorigenic capability of stably expressing mutated S100A7 (S100A7mut) were generated by trans- MDA-MB-231 cells expressing S100A7mut was tested by establishing fecting the plasmid pcDNA3.1mut-psor using Effectene transfection reagent xenografts in nude mice as described previously (11). Briefly, breast cancer (Qiagen, Canada) followed by zeocin selection (Invitrogen). Two MDA-MB- cells (five experimental groups comprising MDA-MB-231 parental cells, 231 clones were isolated that express S100A7mut (designated as 231-PTM.2 231-HP1, 231-HP2, 231-PTM.2, and 231-PTM.14) were grown in culture; and 231-PTM.14; PTM = psoriasin triple mutant). This was determined by then, 5 Â 105 cells were suspended in 0.2 mL PBS before injection into Western blot using a specific polyclonal anti-S100A7 antibody (10, 11) mammary fat pads of female nude mice according to a protocol approved directed to the COOH-terminal region of S100A7 that detects both wild-type by the University of Manitoba Animal Care Committee. Each experimental and mutant proteins. group included five animals, each animal with bilateral s.c. injections into Anchorage-independent growth viability assay. Cells were grown to mammary fad pads. Mice were monitored for occurrence of tumors and 40% confluence in 100 mm tissue culture dishes, trypsinized, and individual mice were euthanized at 8 weeks or when tumor diameter resuspended in complete medium. Suspended cells were continuously reached 1.5 cm as required by the protocol. rocked back and forth in 100 mm sterile bacterial dishes at 40 rpm in a Tissue microarray and immunohistochemistry. A tissue microarray standard tissue culture incubator for 72 hours at 37jC. Nonviable cells were was constructed based on a cohort of 142 estrogen receptor (ER)–negative defined according to morphologic assessment of chromatin condensation invasive carcinomas with ductal or lobular histology selected from the using fluorescent dyes that bind DNA (15). Briefly, cells are stained for Manitoba Breast Tumor Bank. Duplicate 0.6 mm tissue cores were removed 10 minutes in 5 AL PBS with 5 Ag/mL acridine orange (Calbiochem, San from the central portion of a representative paraffin block from each tumor Diego, CA) and 5 Ag/mL ethidium bromide (Sigma, Canada). A 10 AL using a Beecher Instruments (Sun Prairie, WI) tissue arrayer. Immunohis- aliquot of cells was placed on a microscope slide and viewed with a tochemical staining for S100A7 and phospho-Akt was done using an fluorescent microscope using a wide-band fluorescein filter. Nonviable cells automated tissue immunostainer (Ventana Medical Systems, Tucson, AZ) were defined as having condensed chromatin, which lines the nuclear and bulk reagents supplied by manufacturer (10). Primary S100A7 antibody membrane in crescent shapes. The relative viability for each line was (1:3,000) or phospho-Akt (Ser473) antibody (1:150) was incubated for 32 calculated and compared with the MDA-MB-231 parental line. The minutes. Tumor cell staining was scored as described (10) to generate a calculated relative viability reflects the proportion of viable cells for 300 semiquantitative immunostaining score (immunohistochemistry score = % cells counted per assay. Triplicate assays were done. positive neoplastic epithelial cells Â intensity) that ranged from 0 to 300. Yeast two-hybrid system. Studies were conducted essentially as For categorical statistical analysis, S100A7 immunohistochemistry score of described previously (11). The coding region of full-length human S100A7 >0 was regarded as positive for S100A7 (10, 16). For phospho-Akt, and the COOH-terminal half (encoding amino acids 43-101) were fused in- immunohistochemistry scores of >0, z20 (median score), and z60 (upper frame with the GAL4 DNA-binding domain of the pGBT9 vector (Clontech) quartile score) were used to define positive status in separate analyses to generate pGBT9-psor and pGBT9-Cterm-psor, respectively (both because a wide range of cut points have been used in the literature (17, 18). tryptophan selection). Additional versions of each plasmid were also Statistical analysis was done with Prism GraphPad software and using generated with three mutations within the potential Jab1-binding domain Spearman correlation and Fisher’s exact tests as appropriate. as described above. The resulting bait plasmids were found to produce Reverse transcription-PCR. Total RNA was isolated using TRIzol fusion proteins and tested for the ability to interact with the Jab1 fusion (Sigma) from each clone. RT-PCR reactions were done as described protein produced from the prey plasmid pACT2-Jab1 (leucine selection). previously (11). Briefly, total RNA (1 Ag) was reverse transcribed in duplicate Control medium was deficient in tryptophan and leucine only. Different from triplicate samples. PCR was done using specific primers for EGF: EGF- combinations of prey and bait plasmids were tested for their ability to upper (sense) 5V-CACAACCCAAGGCAGAAGAT and EGF-lower (antisense) activate the histidine reporter gene (assayed by growth) in Saccharomyces 5V-ACATTGCGTGGACAGGAA or glyceraldehyde-3-phosphate dehydroge- cerevisiae strain KGY37 (gift from R.D. Gietz, University of Manitoba, nase (GAPDH): GAPDH-upper (sense) 5V-ACCCACTCCTCCACCTTTG and Winnipeg, Manitoba, Canada) when grown on selective medium that was GAPDH-lower (antisense) 5V-CTCTTGTGCTCTTGCTTGTTGG. Reactions Trp-Leu-His- and containing 5 mmol/L 3-amino-1,2,4-triazole (Sigma). were stopped during the log-linear stage of PCR amplification and samples Immunoprecipitation and Western blot. Human breast cancer cell lines were electrophoresed through a 1% agarose gel that was poststained with expressing mutant and unmodified S100A7 were lysed and protein ethidium bromide for DNA visualization. Images were captured using a LCD complexes were immunoprecipitated with Jab1 antibody as described camera and MCID software (Imaging Research, Canada). previously (11). Coimmunoprecipitated S100A7 proteins were detected by immunoblotting using an S100A7-specific antibody. Culture cells were lysed and protein was harvested after being allowed to grow for 48 hours in a Results 60 mm culture dish and cell density was between 70% and 80%. Protein S100A7 expression is associated with increased survival concentration of samples was determined using the Micro BCA Protein Assay under anchorage-independent conditions. We observed that Kit (Pierce, Rockford, IL). Antibodies used for Western blot were Jab1 and epidermal growth factor (EGF) receptor (EGFR; Santa Cruz Biotechnology, S100A7 expression in some lung and breast tumors can be pro- Inc., Santa Cruz, CA); p27Kip1 (clone 57, Becton Dickinson, Canada); minent in apparently detached but morphologically viable tumor phospho-c-Jun (Ser63), total c-Jun, phospho-Akt (Ser473), total Akt, InBa, cells adjacent to and within regions of necrosis and/or increased 183 185 and phospho-c-Jun NH2-terminal kinase (JNK; Thr /Tyr ; Cell Signaling, apoptosis (Fig. 1A). To determine if S100A7 enhanced viability under www.aacrjournals.org 5697 Cancer Res 2005; 65: (13). July 1, 2005

Figure 1. S100A7 expression is found in regions of detached cells in vivo and confers increased survival under anchorage-independent conditions in vitro. A, S100A7 expression detected by immunohistochemistry is prominent in regions of tumors where there is cell detachment and apoptosis (asterisk) in tumors from breast (left) and lung (middle). Right, H&E-stained serial section of the lung (middle) for reference. B, representative images showing nuclear architecture of parental MDA-MB-231 cells and 231-HP2 cells expressing S100A7 after being subjected to 72 hours of anchorage-independent growth (detached) and the same cells before treatment (attached). C, relative viability after treatment for MDA-MB-231 cells expressing S100A7 (231-HP1 and 231-HP2) and mutant S100A7 (231-PTM.2 and 231-PTM.14) compared with parental (231) and control transfected (231-neo) cells. Columns, mean of triplicate independent experiments; bars, SD. Ps were determined by t test.

conditions of decreased cellular adhesion, MDA-MB-231 cells square is calculated when the three residues Asp58/Gly58, Leu80/ expressing S100A7 were assessed after 72 hours of growth under Met80, and Gln90/Lys90 are superimposed on each other. Similarly, anchorage-independent conditions (Fig. 1B). Two independent cell changes occur around the metal sites for Ca2+ and Zn2+ and clones (231-PTM.2 and 231-PTM.14) were characterized and shown structural error of 0.3981 is predicted on metal sites when the three to express comparable levels of S100A7mut to the level of S100A7 in metal cations are superimposed (data not shown). These three- the previously established MDA-MB-231 clone 231-HP2 (Fig. 2A), dimensional modeling data predict that mutation of the three key but both S100A7mut clones showed no difference in growth rate amino acids within the Jab1-binding domain would not substan- when compared with parental cells (data not shown). The two tially alter overall protein structure but would result in significant independent clones constitutively expressing S100A7 (231-HP1 regional changes in conformation. and 231-HP2) had a similar 1.32 F 0.053–fold increase (mean F To next assess the effect of mutation of this domain on the Jab1 SD, n = 3) and 1.34 F 0.036–fold increase, respectively, in viability interaction, we mutated the putative Jab1-binding domain in both compared with nonexpressing control (231-neo) and MDA-MB-231 the full-length S100A7 cDNA and a cDNA representing the COOH- parental cells (Fig. 1C). Two clones expressing S100A7mut were also terminal portion alone and coexpressed these proteins separately assessed under the same conditions. They exhibited a partial with Jab1 in the yeast two-hybrid assay. We found that in both increase in their relative viability, with rates that were less than contexts these mutations abolished the interaction between those shown by S100A7-expressing lines (Fig. 1C). The mean F SD S100A7 and Jab1 (Fig. 3B). To further test the role of this same (n = 3) relative loss of viability for 231-PTM.2 and 231-PTM.14 were domain within human breast cells and to enable subsequent 1.174 F 0.034 and 1.193 F 0.022, respectively. These data suggest functional analysis, we then developed MDA-MB-231 cells with that S100A7 confers a survival advantage in cells subjected to stable expression of the full-length mutant S100A7 cDNA anchorage-independent conditions. (S100A7mut). The clones 231-PTM.2 and 231-PTM.14 were then Mutation of the c-Jun activation domain-binding protein assayed for an interaction between S100A7mut protein and Jab1 by 1–binding domain of S100A7 abrogates the interaction with coimmunoprecipitation (Fig. 3C). Under the same conditions c-Jun activation domain-binding protein 1. We have identified whereby endogenous and transgene-derived S100A7 protein could previously an interaction between S100A7 and Jab1 and an be coimmunoprecipitated with Jab1 from MDA-MB-468 and 231- association between S100A7 expression and alteration of AP-1 HP2 cell clones, respectively, mutation of the Jab1-binding domain and HIF-1 activities. We therefore postulated that the S100A7-Jab1 within S100A7 significantly reduced the association between interaction might be necessary for the effect on survival pathways. S100A7mut and Jab1 in the 231-PTM.2 and 231-PTM.14 clones. Sequence analysis of S100A7 reveals a domain within the COOH- However, higher levels of expression seen in 231-PTM.14 cells terminal region that is present in other proteins that physically (Fig. 3A) revealed a limited residual interaction (Fig. 3C). interact with Jab1 (11, 13). To test the role of this putative Jab1- The S100A7-c-Jun activation domain-binding protein 1 binding domain in mediating the interaction with S100A7, we first interaction can stimulate activation of Akt and nuclear did three-dimensional protein modeling of the potential effect of factor-KB activity. The activation of the Akt kinase is thought mutation of the three key amino acids within this domain (13) to be a major factor in promoting survival in epithelial cells under using the known resolved structure for the S100A7 protein (14). anchorage-independent conditions (19). We therefore assessed Akt Comparison of the three-dimensional models for S100A7 and expression and phosphorylation status in MDA-MB-231 cell clones S100A7mut proteins confirmed that mutation within this domain expressing S100A7 and S100A7mut proteins by Western blot. Total does not significantly alter the overall protein structure. However, Akt levels were similar in all cell clones; however, a 4.3 F 0.4–fold regional changes in conformation are predicted to occur (Fig. 3A). increase (mean F SD, n = 3) in the relative amount of phospho-Akt A minimal structural error of 0.1767 as determined by root mean (Ser473) was observed in both clones expressing S100A7 when

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Figure 2. S100A7 expression stimulates Akt and NF-nB pathways. A, relative expression of S100A7 or S100A7mut proteins in a panel of MDA-MB-231 cell lines as indicated by the key. S100A7 expression is associated with increased amounts of phospho-Akt (without any change in total Akt or PTEN levels), a reduction in InBa, and a reduction in p27. S100A7-expressing cells were also found to have higher levels of total and phospho-c-Jun without any change in phospho-JNK levels. In comparison, S100A7mut expression confers no similar changes in the above protein levels. B, relative NF-nB activity in the same panel of MDA-MB-231 cell lines as determined by transient reporter gene transfection assay. Columns, mean of triplicate independent experiments; bars, SD. Ps were determined by t test.

compared with parental and 231-neo control cells, but no increase An intact c-Jun activation domain-binding protein 1– in phospho-Akt was present in either clone expressing S100A7mut binding domain in S100A7 is required for enhanced tumor- (Fig. 2A). igenesis in vivo. To determine if abrogation of the S100A7-Jab1 NF-nB is also a major factor in promoting cell survival (20) and interaction affects promotion of tumorigenesis in vivo,the Akt can influence NF-nB via phosphorylation of the NF-nB repressor S100A7mut-expressing MDA-MB-231 cell lines were injected into InBa (21). Western blot analysis of MDA-MB-231 cell clones shows the mammary fat pad of nude mice and the generation of tumors that there is a reduction in InBa in the presence of S100A7 but not was assessed in comparison with S100A7-expressing lines and S100A7mut protein expression (Fig. 2A). We then assessed NF-nB control parental cells (Fig. 5). All cell lines generated tumors that activity by transient transfection of a NF-nB reporter plasmid. Cells were detectable in most animals by 3 weeks. However, S100A7 cells expressing S100A7 protein show a 3.4 F 0.7–fold induction (mean F developed larger tumors significantly more rapidly than the SD, n = 4) of NF-nB activity (Fig. 2B). By comparison, no such S100A7mut cells, which in turn developed tumors similarly to and increase is observed in cells expressing S100A7mut. showed no significant difference from parental controls (Fig. 5). At We have reported previously that S100A7 expression correlates 6 weeks postinjection, tumors >1.5 cm had developed in 5 of 5 and with induction of c-Jun-related AP-1 activity and down-regulation 3 of 5 231-HP1 and 231-HP2 cell-injected mice, respectively, but in 0 of p27Kip1 (11). We observed similar changes here with reduction in of 5, 1 of 5, and 0 of 5 for 231-PTM.2, 231-PTM.14, and parental p27Kip1 and induction of phospho-c-Jun associated with S100A7 but control MDA-MB-231 cell-injected mice. This same difference was not S100A7mut protein expression (Fig. 2A). The increase in reflected in a significantly (P = 0.0039) worse overall survival time phospho-c-Jun in S100A7 clones is paralleled by a similar increase for animals injected with S100A7 cells compared with parental in total c-Jun levels. This is not attributable to an increase in the control cells as shown previously (11). However, tumors derived phosphorylation status of JNK, as JNK is not altered by S100A7 or from both S100A7mut cells showed no significant difference from S100A7mut expression (Fig. 2A), but JNK phosphorylation can be the parental control (Fig. 5). increased in these same cells by transfection of mitogen-activated S100A7 induction of phospho-Akt is dependent on phos- protein kinase kinase kinase 1 (data not shown). This is compatible phatidylinositol 3-kinase activity. Akt activation is typically the with Jab1 stabilization of c-Jun and through direct phosphorylation consequence of extracellular signals that act through a range of cell by a JNK-independent mechanism (22). surface receptors to stimulate PI3K, the upstream positive These data suggest that S100A7 expression is associated with regulator of Akt. Alternative mechanisms also exist, including induction of both phospho-Akt and NF-nB activity and that the reduced activity of PTEN, a negative regulator of Akt. We observed induction of these prosurvival pathways as well as AP-1 are that PTEN levels were unaffected by the expression of S100A7 or dependent on an intact Jab1-binding domain within S100A7. S100A7mut (Fig. 2A), but inhibition of PI3K by LY294002 S100A7 is associated with an increase in phospho-Akt in significantly reduced or abolished the expression of phospho-Akt breast tumors. To examine if a similar relationship between associated with S100A7 (Fig. 6A). We next considered the possible S100A7 and phospho-Akt could be detected in vivo within human role of the EGFR signaling pathway that is one of many pathways breast tumors, we examined their expression within a tumor tissue that might act to stimulate PI3K, because AP-1 is essential for the array. S100A7 expression was seen in 78 of 142 invasive carcinomas transcriptional regulation of the EGFR ligand, EGF (23). We and phospho-Akt was expressed in 108 of 142 tumors, comparable observed an increase in EGF mRNA levels associated with S100A7 with the frequencies observed previously within breast tumors by but not with S100A7mut (Fig. 6B) and this correlated with induction ourselves (10) and others (17) for these proteins. Overall, there was of p21 protein, a downstream marker of EGFR signaling in the a significant correlation between levels of expression for S100A7 same cells. Additional experiments confirmed that all cell clones and phospho-Akt within this cohort (P < 0.0001; r = 0.34; Spearman express EGFR at equivalent levels (Fig. 6C). Treatment of the correlation). The association between S100A7 and phospho-Akt different cell clones with a specific EGFR inhibitor (BPIQ) revealed status was also evident in categorical analysis and remained that both baseline and induced phospho-Akt amounts in all cell significant (P < 0.0001, Fisher’s exact test) for all of the three cut clones could be significantly reduced regardless of whether S100A7 points used to distinguish positive from negative phospho-Akt was present or was mutated (Fig. 6D). These data suggest that in status (Fig. 4). the MDA-MB-231 cell line model S100A7 induces phospho-Akt www.aacrjournals.org 5699 Cancer Res 2005; 65: (13). July 1, 2005

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Cancer Research through a classic PI3K-dependent pathway. This may in part be attributable to the autocrine effect of AP-1 induction of EGF leading to an increase in the stimulation of EGFR. When EGFR activity is elevated, it promotes the stimulation of PI3K through phosphorylation events, which in turn result in an enhancement in the phosphorylation status of Akt.

Discussion In neoplasia, S100A7 is expressed in several tumor types. In addition, S100A7 is induced and sometimes highly expressed with abnormal differentiation in inflammatory dermatoses (12) and in response to diverse stress stimuli, including loss of substrate attachment (8) and UV-B radiation (24) in either breast or skin Figure 4. S100A7 expression is correlated with increased phospho-Akt in human breast tumors. A, histogram representation of the relative levels of phospho-Akt expression (p-Akt immunohistochemistry score) among S100A7-negative (n = 64) and S100A7-positive (n = 78) tumor subgroups as determined by immunohistochemistry in a tumor tissue microarray. B, contingency table analysis of S100A7 and phospho-Akt status. S100A7 and phospho-Akt immunohistochemistry scores of >0 were used to define a tumor as positive and statistical significance was tested by Fisher’s exact test.

systems. Stress is also a prominent factor within the neoplastic epithelium and particularly in preinvasive lesions where there is cellular crowding, loss of attachment, and necrosis in a restricted but expanding cell population, disconnected from the nutrient and mechanical support of the stroma by an intact basement membrane. We and others (8) have speculated that up-regulation of S100A7 in preinvasive carcinoma is indicative of this stress. S100A7 may play a role as a secreted chemotactic factor (25), yet more recent data also point to an intracellular action. S100A7 has been associated with several proteins in vitro (11, 26–29). However, only the S100A7-Jab1 interaction has yet been associated with any functional effect (11). Jab1 was originally identified as a factor influencing c-Jun stimulated transcription of AP-1-regulated genes (30). It is now known that Jab1 is a component of a multimeric protein complex (the COP9 signalosome) and that Jab1 interacts with many components of cell signaling pathways (31). This may be in the context of either phosphorylation or proteasomal activities, as Jab1 is also the only known deneddylating protein active in control of the SCF-cullin ubiquitin ligases (32). We have now shown that S100A7 can promote survival under anchorage-independent conditions in breast cancer cells (33). This survival function, like growth and invasion, is coupled to up- regulation of Jab1 downstream pathways, including AP-1 [as described previously (16)] and NF-nB, and induction of phospho- Akt via a PI3K-dependent mechanism. We have also now established that the above effects are largely dependent on an intact Jab1-binding site within the S100A7 protein. This binding site was originally identified through site-directed mutagenesis as present in both p27 and c-Jun and also necessary for their interaction with Jab1 (13). In p27, triple mutations at the key amino acids within this site (Asp108, Leu130, and Asn140) were shown not to Figure 3. The putative Jab1-binding domain in S100A7 is required for the substantially alter the gross protein conformation but reduced the interaction with Jab1. A, three-dimensional ribbon model to illustrate the overall similarity and regional differences between the predicted molecular structures of binding ability with Jab1 to 15% of the wild-type (13). Our data here S100A7mut compared with S100A7 protein. Structures are aligned in the same also show that mutation of the three key amino acids that define orientation. B, yeast two-hybrid control plate (left) shows that the presence of this same motif in S100A7 leaves the overall predicted three- both a bait and a prey plasmid are required for growth in the absence of tryptophan and leucine. However, both the full-length and the COOH-terminal dimensional protein structure unchanged but causes subtle half of S100A7 can interact with Jab1 (middle), but mutation of the Jab1-binding regional changes in particular around the metal-binding sites. domain prevents an association between these proteins as determined by inactivation of the histidine reporter gene resulting in the absence of growth Nevertheless, this is sufficient to significantly reduce the in vitro (middle). Right, plate sector key diagram. C, coimmunoprecipitation reveals that and in vivo interaction with Jab1 and significantly diminishes although S100A7 and Jab1 interact in human breast cell lines that express signaling (e.g., induction of phospho-c-Jun, Akt, and NF-nB activity) endogenous (MDA-MB-468) or transgene-derived S100A7 (231-HP1 and 231- HP2) no significant interaction is seen in cells expressing S100A7 with mutation and biological (e.g., tumorigenesis in mice) effects of S100A7. The of the Jab1-binding domain (231-PTM.2 and 231-PTM.14, respectively). difference in tumorigenic capacity observed in vivo between

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under anoikis conditions (38, 39); however, phosphorylation of Akt may not necessarily be critical for enhanced survival (40). A link between Jab1 and Akt has not been identified previously. We can speculate that in breast cells S100A7-mediated induction of phospho-Akt is attributable to increased production of AP-1 and possibly also NF-nB-regulated growth factors and cytokines that can act in an autocrine fashion to stimulate PI3K through their cognate receptors. One such pathway, based on our current data, may be the EGF-EGFR-PI3K signaling pathway. A similar autocrine effect has recently been recognized in relation to overexpression of another EGFR ligand in breast cells (41). We have shown here that the in vitro relation between S100A7 and increased phospho-Akt can also be identified in vivo in ERa-negative tumors. Akt activation is known to promote localization of p27 to the cytoplasm (42) and Figure 5. S100A7 but not S100A7mut promotes cell growth and enhanced reduced p27 has been observed in ERa-negative tumors (43). We survival in vivo. Survival plot showing that cells expressing S100A7mut show have also shown previously that S100A7 is associated with reduced no significant difference from parental control cells (MDA-MB-231) with p27 in DCIS and invasive breast cancer (16). respect to tumor growth and survival compared with cells expressing S100A7. MDA-MB-231 cell clones were injected into the mammary fat pads of nude mice. Jab1 might also influence NF-nB activity directly by binding to Three animal groups represent mice injected with MDA-MB-231 parental cells the Bcl-3 protein, which enhances p50-Bcl-3-DNA complex (n = 5), S100A7mut cells (n = 10), and S100A7 cells (n = 10). Statistical n significance was tested by log-rank test. formation, thereby potentially influencing NF- B activity (44). The effect of S100A7 on NF-nB is consistent with the known role of S100A7- and S100A7mut-expressing cells is attributable to the intact NF-nB in promoting cell survival in breast cancer (45). S100A7 Jab1-binding domain in the former cells. Whereas S100A7- expression is strongly correlated with the ERa-negative phenotype expressing cells were found previously to have a modest 1.3-fold in both DCIS and invasive disease (9). In ERa-negative breast cell increase in their growth rate in vitro (11), S100A7mut-expressing lines, a critical role for EGFR-NF-nB signaling has been defined in cells showed no difference in growth rate compared with MDA- mediating cell cycle progression (46) and resistance to chemother- MB-231 parental cells (data not shown). It is therefore likely that apeutic agent–induced apoptosis (47). ErbB-2 signaling can also the significantly enhanced tumorigenicity related to S100A7 alter NF-nB activity, which correlates with reduced apoptosis. More expression that is seen in the in vivo setting is related to other recently, it has also been shown that an ERa-negative subset of properties than only the increased growth rate. The S100A7-Jab1 invasive tumors shows increased activation of NF-nB. In this small interaction results in changes in several signaling pathways and the series (45), 56% of ER-negative invasive tumors manifested higher activation of prosurvival pathways related to Akt and NF-nB may levels of NF-nB activation, which is similar to the frequency of play an important additional role in promoting tumorigenesis S100A7 expression in the same tumor phenotype. under in vivo conditions. Promoting cell survival is a role previously suspected for several To understand the possible connections between S100A7 other S100 family proteins and S100A7 is one of many similar expression and survival in breast cancer cells, it may be relevant factors that may influence the balance between survival and to consider similar pathways in keratinocytes in the skin under apoptosis in breast cancer. However, S100A7 is important because, circumstances where S100A7 is also highly expressed. As already as several groups have confirmed using distinct technical noted, these include inflammatory dermatoses (34) and the approaches, S100A7 is among the most highly expressed mRNAs response to UV-B-related stress (24), resistance to anoikis, and and proteins in DCIS and is frequently differentially expressed response to wound healing, where altered signaling pathways have during the transition to invasive carcinoma in skin and breast (6–8, been relatively well defined (23, 35–37). In these circumstances, 48, 49). The S100A7-Jab1 prosurvival pathway therefore has the induction of Akt, NF-nB, and AP-1 are often critical components. In potential to have a significant impact in promoting breast cancer at breast cells, Akt and NF-nB have also been implicated in cell survival a critical stage in tumor progression.

Figure 6. S100A7 activation of Akt is dependent on PI3K and correlates with increased EGFR signaling as indicated by increased EGF and p21. A, Western blot analysis shows that both enhanced expression of phospho-Akt in cells expressing S100A7 and baseline phospho-Akt expression in parental cells and cells expressing S100A7mut can be significantly reduced or eliminated by treatment with a specific PI3K inhibitor, LY294002. B, RT-PCR analysis shows increased levels of EGF mRNA in clones expressing S100A7 but not S100A7mut. C, Western blot analysis shows increased levels of p21 in clones expressing S100A7 but not S100A7mut. D, Western blot showing phospho-Akt levels can be equalized and decreased in all clones regardless of S100A7 expression by pretreatment with the EGFR inhibitor BPIQ. Controls used are GAPDH mRNA and h-actin protein. See Fig. 2 for key to cell lines.

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Acknowledgments Research Studentship (E.D. Emberley). The Manitoba Breast Tumor Bank is also supported by the CIHR and CancerCare Manitoba. Received 11/2/2004; revised 3/21/2005; accepted 4/13/2005. The costs of publication of this article were defrayed in part by the payment of page Grant support: Canadian Institutes for Health Research (CIHR), CIHR New charges. This article must therefore be hereby marked advertisement in accordance Investigator Award (S.B. Gibson), and National Cancer Institute of Canada Terry Fox with 18 U.S.C. Section 1734 solely to indicate this fact.

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Ethan D. Emberley, Yulian Niu, Linda Curtis, et al.

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