Oncogene (2008) 27, 3527–3538 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Reciprocal negative regulation between S100A7/psoriasin and b-catenin signaling plays an important role in tumor progression of squamous cell carcinoma of oral cavity

G Zhou1, T-X Xie1, M Zhao1, SA Jasser1, MN Younes1, D Sano1, J Lin1, ME Kupferman1, AA Santillan1, VPatel 2, JS Gutkind2, AK EI-Naggar3, ED Emberley4, PH Watson4, S-I Matsuzawa5, JC Reed5 and JN Myers1,6

1Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA; 3Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 4Deeley Research Center, BC Cancer Agency, Victoria, British Columbia, Canada; 5Burnham Institute for Medical Research, La Jolla, CA, USA and 6Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Overexpression of S100A7 (psoriasin), a small calcium- Introduction binding , has been associated with the development of andcarcinomas in different types of epithelia, S100A7 (psoriasin), a low-molecular weight (11.4 kDa) but its precise functions are still unknown. Using human Ca2 þ -binding protein, is a member of the S100 family of tissue specimens, culturedcell lines, anda mouse model, and was originally identified through its brisk we foundthat S100A7 is highly expressedin preinvasive, overexpression in the skin of patients suffering from well-differentiated and early staged human squamous cell psoriasis, a hyperproliferative skin disease characterized carcinoma of the oral cavity (SCCOC), but little or no by abnormal differentiation. Like many other members expression was found in poorly differentiated, later-staged of the S100 family, S100A7 is located within the invasive tumors. Interestingly, our results showedthat ‘epidermal differentiation complex’ on human chromo- S100A7 inhibits both SCCOC cell proliferation in vitro some 1q21 and its protein is believed to be implicated in andtumor growth/invasion in vivo. Furthermore, we Ca2 þ -dependent and -independent regulation of a demonstratedthat S100A7 is associatedwith the variety of intracellular and extracellular activities b-catenin complex, andinhibits b-catenin signaling (Donato, 2001, 2003; Heizmann et al., 2002). It has by targeting b-catenin degradation via a noncanonical also been found that S100A7 is highly overexpressed in mechanism that is independent of GSK3b-mediated wound healing, and many inflammatory and hyperpro- phosphorylation. More importantly, our results also liferative epidermal diseases, including psoriasis, atopic indicated that b-catenin signaling negatively regulates dermatitis, mycosis fungoides, Darier’s disease and S100A7 expression. Thus, this reciprocal negative regula- lichen sclerosus et atrophicus as well as skin cancers tion between S100A7 and b-catenin signaling implies their and breast cancers. However, the exact role of S100A7 important roles in tumor development and progression. in these diseases is still unclear. Despite its high levels of expression in early stage S100A7 overexpression during skin and breast tumor- SCCOC tumorigenesis, S100A7 actually inhibits SCCOC igenesis suggests that S100A7 might have an ‘oncogenic’ tumor growth/invasion as well as tumor progression. role in tumor development. However, a close examination Downregulation of S100A7 in later stages of tumorigen- of the expression profile of S100A7 during tumor esis increases b-catenin signaling, leading to promotion of progression suggests a more complex picture of functional tumor growth andtumor progression. roles for S100A7 in tumor development. Though its Oncogene (2008) 27, 3527–3538; doi:10.1038/sj.onc.1211015; expression in skin is highly induced in keratinocytes under published online 28 January 2008 specific pathological circumstances, including hyperplasia associated with inflammatory skin lesions and dysplasia Keywords: S100A7; psoriasin; b-catenin; oral squamous associated with neoplastic progression (Alowami et al., cell carcinoma; head and neck squamous cell carcinoma 2003; Emberley et al., 2003a), S100A7 actually first emerged in breast cancer when identified as a cDNA downregulated in a nodal metastasis relative to a primary invasive breast tumor (Moog-Lutz et al., 1995). Further- Correspondence: Professor JN Myers, Department of Head and Neck more, analysis in breast cancers showed that S100A7 Surgery, University of Texas MD Anderson Cancer Center, 1515 expression is relatively low or undetectable in normal, Holcombe Boulevard, Unit 441, Houston, TX 77030-4009, USA. benign and atypical hyperplastic proliferative ductal E-mail: [email protected] Received 17 July 2007; revised 12 November 2007; accepted 3 December lesions, but is often highly induced in the ductal epithelial 2007; published online 28 January 2008 cells of premalignant, preinvasive ductal carcinoma in situ S100A7 inhibits SCCOC progression G Zhou et al 3528 (DCIS) (Leygue et al., 1996; Enerback et al., 2002) and patterns of S100A7 in 27 archival human tongue tissue then its expression is diminished in invasive carcinomas specimens, including those of histologically normal and (Leygue et al., 1996; Emberley et al., 2003b). Such dysplastic squamous mucosa and invasive SCCOC. In ‘biphasic expression’ suggests that its overexpression is normal oral epithelia, no expression of S100A7 was associated with altered differentiation in glandular epithe- observed in dividing basal layer keratinocytes, whereas lium of DCIS, that S100A7 may only have a role in early some low-level, scattered expression of S100A7 was seen breast tumor progression (Watson et al., 1998), and that in the differentiated suprabasal layers (Figure 1a). the concomitant loss of S100A7 expression in the later S100A7 expression was increased in dysplastic lesions stage of invasive carcinomas may be required for tumor and in well-differentiated SCCOC, but in the later stages progression. In other words, its overexpression may of poorly differentiated invasive SCCOC, expression actually ‘suppress’ the tumor progression to later stages was greatly reduced or absent (Figure 1a). Of the 27 of invasive carcinomas. Consistent with the putative SCCOC specimens we evaluated, 74% (20/27) were well ‘tumor suppressive’ role are the observations that the differentiated or moderately differentiated carcinomas expression of S100A7 is relatively low or undetectable in and they were all positive for S100A7 expression, most highly proliferating keratinocytes and tumor cells whereas none of the seven poorly differentiated invasive in vitro, but it can be induced by agents that stimulate cell carcinomas expressed S100A7 (Figure 1b). In addition, differentiation such as retinoic acid (Tavakkol et al., 1994), S100A7 expression level was inversely correlated with calcium (Hoffmann et al., 1994), UVlight (Di Nuzzo et al., histological grade, with higher expression seen in the 2000), DNA damaging agents (Kennedy et al., 2005), loss well-differentiated and preinvasive carcinomas, less in of cell attachment to extracellular matrix, growth factor the moderately differentiated carcinomas, and none in starvation and cell confluent culture conditions (Enerback the poorly differentiated invasive carcinomas (Figures et al., 2002; Martinsson et al., 2005). In addition, in 1a and b). We also examined the expression of S100A7 normal epithelium, S100A7 has been shown to be in orthotopic SCCOC tumor models in nude mice expressedinthesuperficial,differentiatedregionofthe generated from three isogenic human SCCOC cell lines epithelium rather than in the highly proliferative basal (Tu167, JMAR and DM14) with varying tumor growth region, and its expression correlates with the degree of and metastatic potentials (Supplementary Materials). As keratinocyte differentiation (Broome et al., 2003). In skin shown in Figure 1c, the Tu167-derived tongue tumors tumors, it is absent in undifferentiated basalioma and (less malignant) exhibited a more differentiated mor- strongly expressed in carcinoma in situ,aswellasin phology and a higher level of S100A7 expression, keratoacanthoma and differentiated squamous cell carci- whereas JMAR-derived tongue tumors (moderately noma. These findings support the hypothesis that S100A7 malignant) demonstrated reduced S100A7 expression is involved in epithelial differentiation (Martinsson et al., with only scattered staining detected. In the most 2005) rather than in cell proliferation. malignant DM14 tumors, however, S100A7 expression So far very little is known about molecular mechanisms was almost entirely lost. Together, these results demon- of S100A7’s function. To study the potential biological strate an inverse relationship between levels of S100A7 role of S100A7 in oral squamous epithelial tumor expression and tumor progression in both human progression, we first evaluated S100A7 expression in both SCCOC and orthotopic tumors in nude mice. human and mouse specimens of human squamous cell carcinoma of the oral cavity (SCCOC) at different stages Expression of S100A7 inhibits SCCOC cell proliferation of tumor development. We then used both adenoviral- in vitro and tumor growthand tumor progression in vivo and lentiviral-mediated gene overexpression and under- While in vivo expression of S100A7 is very high in expression strategies in human SCCOC cell lines, which early staged, well-differentiated SCCOC, its expression identified a critical role of S100A7 in inhibiting tumor in vitro SCCOC cells (for example, Tu167, JMAR and growth and progression of SCCOC. We further estab- DM14) is very low or undetectable (Supplementary lished that S100A7 is an important negative modulator of Figure S1B), but can be induced by cell confluence or b-catenin signaling through targeting b-catenin for cell detachment (Figure 6a) as previously reported in degradation, and that b-catenin in turn can inhibit the other cell lines (Enerback et al., 2002). To elucidate the expression of S100A7. Our results suggest that although role of S100A7 in cell growth and tumorigenesis of S100A7 is overexpressed in early stages of tumorigenesis, SCCOC, we generated an adenoviral mediated expres- this protein actually plays an inhibitory role in tumor sion system to overexpress S100A7 (Figure 2a). As progression of SCCOC, and loss of S100A7 expression is shown in Figure 2b and Supplementary Figure S1A, associated with more advanced disease. when S100A7 and control green fluorescent protein (GFP) virus were used to infect Tu167 and JMAR cells, cell proliferation was inhibited by S100A7 overexpres- Results sion in a dose-dependent manner, Similarly, when Tu167 or immortalized human keratinocytes HOK16B S100A7 is overexpressed in early stages of preinvasive, were infected with the virus, then subjected to a colony well-differentiated SCCOC but is lost in later stages of formation and cell migration assays, the number of tumor development colonies and cell migration were greatly suppressed by To investigate the role of S100A7 in the development of S100A7 overexpression (Figures 2c–e). To further human SCCOC, we first evaluated the expression extrapolate these findings in vivo, we used an orthotopic

Oncogene S100A7 inhibits SCCOC progression G Zhou et al 3529

Cornified layer Suprabasal layer Basal layer

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Tu167 Tumor JMAR Tumor DM14 Tumor

Tumor malignancy Figure 1 S100A7 is overexpressed in the early stages of well-differentiated, preinvasive squamous cell carcinoma of the oral cavity (SCCOC). (a) Representative results from immunohistochemical (IHC) staining of human SCCOC specimens with S100A7 antibody. (b) Summary of S100A7 IHC staining from 27 of human SCCOC specimens. *Po0.05, Wilcoxon rank-sum test. (c) Representative results from IHC staining with S100A7 antibody of orthotopic tongue SCCOC tumor specimens in nude mice derived from three human SCCOC cell lines. tongue tumor model, in which JMAR cells were infected in JMAR-S100A7-Ad tumors, PCNA staining was with the adenovirus and then orthotopically inoculated restricted to cells at the periphery of the tumor into tongues of nude mice. Consistent with our islands that did not express S100A7, while tumor cells observation in vitro, JMAR tumor growth was sup- expressing S100A7 exhibited no PCNA staining as pressed by S100A7 overexpression by B5-fold as shown in consecutive sections (Figure 2g). This inverse measured by tumor volume (Figure 2f). correlation in vivo between S100A7 and PCNA staining In addition, histological analysis revealed that tumors further highlights that overexpression of S100A7 in- derived from control cells and S100A7 cells had different hibits tumor cell proliferation, tumor cell dissemination tumor morphologies (Figure 2g). In the control group and invasion. (JMAR-GFP-Ad), tumors consisted of sheets of poorly differentiated and highly infiltrative cells. In contrast, Overexpression of S100A7 inhibits b-catenin signaling S100A7-infected JMAR tumor cells (JMAR-S100A7- To study the possible mechanisms involved in S100A7- Ad) grew as well-defined, organized islands that were mediated tumor growth inhibition, we examined the well demarcated from surrounding mesenchymal tissue effects of overexpression of S100A7 on multiple signal- and well differentiated, as shown by the presence of ing pathways including b-catenin signaling. We first keratin pearls. An immunofluorescent assay using Flag noticed that the tyrosine phosphorylation of b-catenin tag antibody further demonstrated that exogenous induced by epidermal growth factor (EGF) stimulation S100A7 was indeed expressed in JMAR-S100A7-Ad was concomitantly inhibited by adenovirus-mediated tumors (Figure 2h). In addition, in control tumors, expression of S100A7 in JMAR cells (Figure 3a). immunohistochemical (IHC) staining demonstrated a Further analysis indicated that the reduced levels of high cellular proliferative index, as determined by phosphorylated b-catenin could be attributed to an extensive proliferating cell nuclear antigen (PCNA) overall decrease in the level of b-catenin protein staining throughout the tumor (Figure 2g). However, secondary to S100A7 overexpression (Figures 3b and c).

Oncogene S100A7 inhibits SCCOC progression G Zhou et al 3530

Figure 2 Overexpression of S100A7 inhibits cell proliferation in vitro and tumor growth in vivo.(a) Generation of recombinant adenovirus expressing Flag-tagged S100A7. (top) Schematic of adenoviral construct encoding S100A7. (bottom left) S100A7 adenovirus-infected JMAR cells (10 multiplicity of infection) marked by green fluorescent protein (GFP) fluorescence. (bottom right) Western blotting of JMAR cells infected with S100A7 and control GFP adenoviruses. (b) MTT assay for cell proliferation of Tu167 cells infected with S100A7 or GFP adenovirus. (c and d) Colony formation assay of Tu167 and HOK16B cells infected with different doses of S100A7 or GFP adenovirus. (e) Cell migration assay of Tu167 cells infected with the virus. (f) Orthotopic tongue tumor volumes derived from JMAR cells infected with or without virus. In all the above-mentioned cases, error bars represent standard deviation. *Po0.05. (g) Consecutive tumor sections of immunohistochemical (IHC) staining of tumors derived from control GFP (JMAR-GFP-Ad) and S100A7 virus-infected (JMAR-S100A7-Ad) tumors using antibodies as indicated (insets  200). (h) Immunofluorescent staining of frozen sections of control and JMAR-S100A7-Ad tumors using Flag antibody as indicated. In each group,  200 images on right hand side magnify box areas of the  50 images on left-hand side. Hematoxylin and eosin (H&E) section is the consecutive slide of fluorescent image on the top.

The finding that S100A7 decreases b-catenin expression 1997) were co-transfected into 293T, Tu167 and JMAR led us to hypothesize that S100A7 expression could cells together with b-catenin and S100A7 expression negatively regulate b-catenin signaling through nuclear vectors. As shown in Figure 3d, the b-catenin-activated translocation and activation of the T-cell factor (TCF)/ luciferase activity was inhibited in a dose-dependent b-catenin transcription complex and subsequent activation manner by S100A7 overexpression in all three cell lines. of TCF-regulated including c-Myc (He et al., 1998). Moreover, both basal and EGF-induced expressions of To test this hypothesis, a b-catenin/TCF-dependent c-Myc were suppressed by S100A7 overexpression in JMAR luciferase reporter TopFlash and its b-catenin/TCF- cells (Figure 3e). Together, these findings support a role for binding site mutant control FopFlash (Korinek et al., S100A7 expression in inhibition of b-catenin signaling.

Oncogene S100A7 inhibits SCCOC progression G Zhou et al 3531

GFP-Ad S100A7-Ad EGF - + - + IB: P-Tyr IgG GFP-Ad S100A7-Ad IP: β-Catenin β-Catenin p63 S100A7 S100A7 β-Catenin Merge Actin Actin Lysate

5 293T 3 Tu167 JMAR 3 4 GFP-Ad S100A7-Ad 2 0 0.5 1 2 0 0.5 1 2 EGF (h) 3 2 c-Myc 2 S100A7 1 1 1 Actin

Relative Luc Activity JMAR 18234567 9111210 13 14 15 16 17 18 FopFlash +++----- + ---- + ---- TopFlash ---+++++ - ++++ - + +++ β-Catenin -++-++++ ++++- + - +++ S100A7 --+------Figure 3 Overexpression of S100A7 inhibits b-catenin signaling. (a) Immunoprecipitation (IP) with b-catenin antibody followed by immunoblotting (IB) with phosphotyrosine (P-Tyr) in the absence and presence of epidermal growth factor (EGF; 50 ng mlÀ1) stimulation (30 min) in serum-starved JMAR cells infected with control green fluorescent protein (GFP) or S100A7 adenovirus. (b) Western blotting of b-catenin protein in JMAR cells after S100A7 and control GFP virus infection. (c) Representative confocal microscopic images of immunofluorescent staining of JMAR cells infected with S100A7 adenovirus. (d) Luciferase assay of b-catenin- dependent reporter TopFlash in 293T, Tu167 and JMAR cells. (e) Western blotting of c-Myc protein in serum-starved JMAR cells infected with virus after EGF (50 ng mlÀ1) stimulation for different time (h).

S100A7 targets b-catenin degradation via a noncanonical inhibited by proteasome inhibitor MG132 (compare mechanism that is independent of GSK3b-mediated lane 4 with 3, and lane 7 with 6). Taken together, all phosphorylation these results demonstrated the specificity of S100A7- To further investigate the mechanism involved in the mediated b-catenin inhibition and strongly suggested inhibition of b-catenin signaling by S100A7, we that it might be due to the post-transcriptional regula- examined the subcellular localization of S100A7. To tion, which is dependent on proteasome-mediated visualize endogenous S100A7, we used breast cancer protein degradation. MDA-MB-468 cells because this cell line has high levels Because the degradation of b-catenin is controlled by of S100A7 expression (Supplementary Figure S1B). As a GSK3b-mediated phosphorylation-dependent (Hart shown in Figure 4a, most of the S100A7 was localized in et al., 1999; Kitagawa et al., 1999) (Moon et al., 2004; the cytoplasm as well as the cell cortex region. Willert and Jones, 2006) as well as Siah-1/Sip-mediated Reciprocal immunoprecipitation (IP) assays showed GSK3b phosphorylation-independent pathways (Liu that S100A7 and b-catenin were co-precipitated from et al., 2001; Matsuzawa and Reed, 2001; Fukushima MDA-MB-468 and confluent JMAR cell lysates et al., 2006), we used both luciferase assay and western (Figure 4b), indicating that S100A7 interacts with the blotting to test the possible mechanisms involved. As b-catenin complex in these cells. shown in Figure 4d, S100A7 did not appear to influence To understand whether the downregulation of b- the activity of GSK3b, which regulates b-catenin levels catenin is due to transcriptional or post-transcriptional by targeting it for degradation. However, when S100A7 regulation, CMVpromoter-driven Akt or b-catenin was transfected together with Siah-1/Sip into 293T constructs were transfected with S100A7 into 293T cells. cells, it further decreased b-catenin protein levels and As shown in Figure 4c, the expression of both b-catenin-dependent luciferase activity (Figure 4d). endogenous and transfected b-catenin was suppressed Furthermore, when a constitutively active mutant by S100A7 expression (compare lane 3 with 1, 2 and b-catenin that is resistant to GSK3b-mediated phos- lane 6 with 5), whereas transfected Akt expression was phorylation and degradation (Bienz and Clevers, 2000; not affected (compare lanes 3, 4 with 2). Moreover, Matsuzawa and Reed, 2001) was transfected together the S100A7-mediated suppression of b-catenin was with S100A7 into cells, it was also inhibited by S100A7

Oncogene S100A7 inhibits SCCOC progression G Zhou et al 3532

S100A7 E-Cadherin Merge A7

-CateninIB:IP: IgG IB: IP: IgG  S100 S100A7 -Catenin IgG IgG JMAR MDA-MB-468

MG132 ---+ --+ 5 S100A7 - --+ +++ 4 HA-Akt - + ++--- -Catenin - ---+ ++ 3 -Catenin 2 HA (Akt) 1 S100A7 Relative Luc Activity

Actin Top Flash + + + + + + + + +  1 234567 -Catenin - ++++++++ S100A7 - - + - +++- -  - - - ++ - - - - S100A7 GSK3 Siah-1 - - - - - ++ ++ MT--Catenin Sip ------++ WT--Catenin Flag--Catenin Flag--Catenin S100A7 Actin Myc-GSK3

Flag-Siah-1

Myc-Sip

Actin

Figure 4 S100A7 targets b-catenin degradation. (a) Representative confocal microscopic images of fluorescent immunostaining with S100A7 (green) and E-cadherin (red) antibodies in MDA-MB-468 cells. (b) Immunoprecipitation (IP) assays using S100A7 or b-catenin antibodies as indicated. Cell lysates were from serum-starved (12 h) confluent JMAR or MDA-MB-468 cells as indicated. IB, immunoblotting. (c) Western blotting of 293T cells 48 h after transfection with indicated DNA. In lanes 4 and 7, transfected cells were treated with 10 mM MG132 for 3 h before harvest. (d) (top) Luciferase assay and (bottom) western blotting of 293T cells after transfection with indicated DNA in 12-well culture plate. (e) Western blotting of 293T cell transfected with wild-type (WT) or constitutive active mutant (MT) b-catenin in the presence or absence of increasing amounts of S100A7.

overexpression in a dose-dependent manner even though 1999; Moon et al., 2004; Willert and Jones, 2006). As this mutant form of b-catenin is more stable than wild- shown in Figure 5a, upon treatment with LiCl, the levels type b-catenin (Figure 4e). Together, these results of S100A7 protein were significantly reduced in both cell strongly indicate that S100A7 targets b-catenin for lines, and this inhibition appeared to be independent of degradation via a noncanonical mechanism that is proteasome-mediated protein degradation since protea- independent of GSK3b-mediated phosphorylation. some inhibitor MG132 did not affect the downregula- tion of S100A7 by LiCl. Consistent with this, real-time b-Catenin negatively regulates S100A7 expression RT-PCR analyses demonstrated that mRNA levels of Since our data indicate that S100A7 inhibits b-catenin S100A7 were inhibited by LiCl (Figure 5b). Since the signaling, and Wnt/b-catenin signaling functions as a inhibition of GSK3b by LiCl releases the suppression of ‘master switch’ for proliferation versus differentiation b-catenin signaling, we hypothesized that b-catenin (van de Wetering et al., 2002), we set out to determine signaling may be directly implicated in the regulation whether b-catenin signaling could in turn regulate of S100A7 expression. To test this, we overexpressed a S100A7 expression in SCCOC cells. To assess this, we constitutively active mutant of b-catenin in MDA-MB- first treated both JMAR and MDA-MB-468 cells with 468 and JMAR cells, As shown in Figure 5c, retrovirus- LiCl, an inhibitor of GSK3b that negatively regulates mediated overexpression of b-catenin in stable clones b-catenin signaling (Hart et al., 1999; Kitagawa et al., reduced the S100A7 expression in these cells, suggesting

Oncogene S100A7 inhibits SCCOC progression G Zhou et al 3533 - LiCl 1.2 + LiCl 1

MDA-MB-468 JMAR 0.8

- - + + --++ vels of S100A7 LiCl 0.6 - + - +-++ - MG132

S100A7 RNA le 0.4

Actin 0.2

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Cat -Cat -  

Ctr shRNA shRNA-B shRNA-A pBabe pBabe- pBabe pBabe- -Catenin -Catenin -Catenin

S100A7 S100A7 S100A7

Actin Actin Actin JMAR MDA 468 JMAR Figure 5 b-Catenin signaling inhibits S100A7 expression. (a) Western blotting of MDA-MB-468, and confluent JMAR cells in the absence (À) or presence ( þ ) of LiCl (40 mM) and/or MG132 (10 mM) for 6 h. (b) Relative levels of S100A7 mRNA measured by real- time RT-PCR. (c) Western blotting of stable MDA-MB-468, and confluent JMAR cells generated from control pBabe retrovirus and pBabe-b-cat retrovirus expressing a constitutive active mutant b-catenin. Stable cells are polyclonal pools established from selection with puromycin (2 mgmlÀ1) after retroviral infection. (d) Western blotting of stable JMAR cells infected with control (Ctr) and b-catenin (A and B) shRNA-expressing lentivirus (see ‘Materials and methods’). Stable cells are pools of green fluorescent protein (GFP)-positive cells sorted by fluorescence-activated cell sorting (FACS). that activation of b-catenin signaling is sufficient to inhibit was found to be markedly diminished and localized to S100A7 expression in these cell lines. Conversely, when two the cell membrane when compared with the adjacent independent lentivirus-mediated b-catenin shRNAs (that is, S100A7-negative areas that exhibited higher levels of no. A and B) were introduced into JMAR cells to PCNA and b-catenin staining both in cytoplasm and downregulate b-catenin, S100A7 expression was induced nuclei (Figure 6b). The inverse correlation between under confluent culture condition (Figure 5d). Moreover, S100A7 and b-catenin staining in tissue specimens lends levels of S100A7 expression were inversely correlated with further support to the hypothesis that these proteins are levels of b-catenin expression (Figure 5d). Taken together, reciprocally regulated. our results have demonstrated that S100A7 inhibits b-catenin levels, and that b-catenin signaling in turn, inhibits the expression of S100A7 in a negative feedback loop. Decreased expression of S100A7 enhances b-catenin signaling and promotes tumor growthin an orthotopic SCCOC xenograft mouse model Expression of S100A7 and b-catenin is inversely To further study the physiological role of S100A7, we correlated witheachotherin both in vitro and in vivo generated recombinant lentivirus expressing three in- To further evaluate the reciprocal regulation of S100A7 dependent shRNAs targeting different regions of and b-catenin, we examined the expression pattern of S100A7 gene (Supplementary Materials and methods). S100A7 and b-catenin both in vitro and in vivo. Since the MDA-MB-468 cell line has a high level of Although Tu167, JMAR and DM14 cells exhibit S100A7 expression (Supplementary Figure S1B), we first different levels of b-catenin expression in vitro (Supple- used it to establish stable polyclonal cell lines to test mentary Figure S1B), b-catenin expression was found to whether these shRNAs can suppress S100A7 expression. be downregulated by detachment from the extracellular As shown in Figure 7a, in stable polyclonal cell matrix, a condition which leads to elevated levels of populations generated from shRNAs nos. 2 and 3, S100A7 (Figure 6a). Similarly, in both naturally S100A7 expression was inhibited by more than 80% occurring human SCCOC and orthotopic tongue tumor when compared to that in cells transfected with samples, b-catenin staining in S100A7-expressing areas control shRNAs. More importantly, siRNA-mediated

Oncogene S100A7 inhibits SCCOC progression G Zhou et al 3534 Tu167 JMAR DM14 Cell Detachment - + - + - + S100A7

β-Catenin

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S100A7β-Catenin S100A7 β-Catenin

X 50

X 200 Human SCCOC

Case 1 Case 2

H&E S100A7β-Catenin PCNA

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Tu167 Tumor Tu167 X 200

Orthotopic SCCOC X 50 JMAR Tumor X 200

Figure 6 Expression of S100A7 and b-catenin is inversely correlated in both in vitro and in vivo. (a) Western blotting of Tu167, JMAR and DM14 cells treated with cell detachment. Confluent cells were treated with ( þ ) or without (À) cell detachment for 24 h. (b) Representative immunohistochemical (IHC) images of consecutive tumor sections of human squamous cell carcinoma of the oral cavity (SCCOC) and orthotopic Tu167, JMAR tongue tumors. In each group, consecutive tumor sections stained with different antibodies are shown as indicated, in which  200 images at the bottom magnify box areas of the  50 images on the top. Note that S100A7-expressing area has a reduced staining of b-catenin (mainly on the cell membranes) and proliferating cell nuclear antigen (PCNA), when compared with adjacent S100A7-negative areas that has a higher level of both b-catenin (in both cytoplasm and nuclei) and PCNA staining.

suppression of S100A7 in shRNA polyclonal cells (nos. MDA-MB468 cells, these cells had decreased levels of 2 and 3) resulted in increased b-catenin as well as c-Myc S100A7, but elevated levels of b-catenin expression expression when compared with the control cells (no. 5) relative to control stable cells when examined under (Figure 7b). In addition, our results indicate that confluent culture conditions (Figure 7c). After ortho- decreased expression of S100A7 also induces snail topic injection of these cells into the tongues of nude expression and inhibits E-cadherin-mediated cell–cell mice, JMAR cells with S100A7 shRNA (nos. 2 and 3) adhesion (Supplementary Figure S2). These data are grew faster than control cells as measured by the tumor consistent with the notion that S100A7 negatively volume (Figure 7d), despite that no obvious morpholo- regulates levels of b-catenin as well as its downstream gical differences between two groups were observed target genes in this cell line. (Supplementary Figure S3). However, since Tu167 As shown in Figure 1c, Tu167 and JMAR tumors tumor exhibits a more differentiated phenotype and a exhibit different levels of differentiation, malignancy higher level of S100A7 expression than JMAR tumor and S100A7 expression in vivo. However the level of in vivo (Figures 1c and 6b), our result demonstrated S100A7 expression in these two cell lines was virtually that downregulated S100A7 by S100A7 shRNA had undetectable in vitro under subconfluent culture condi- a more impact on Tu167 tumor morphology, and that tion (Supplementary Figure S1B) unless induced by cell S100A7 shRNA Tu167 tumors exhibit a less differen- confluence or cell detachment (Figure 6a). In spite of tiated phenotype when compared with the control this, we infected these cells with S100A7 shRNA (Figure 7e). In addition, an inverse correlation between lentivirus and established stable shRNA clones of both S100A7 and b-catenin, PCNA was also found in both JMAR and Tu167 cells. Consistent with results in control and S100A7 shRNA tumors (Figure 7e), further

Oncogene S100A7 inhibits SCCOC progression G Zhou et al 3535 JMAR 90 shRNA #3 S100A7

Ctr ) shRNA #2 shRNA shRNA Control S100A7 Control S100A7 3 80 ctrl shRNA #4 S100A7 Cont r o l #5 #3 #2 shRNA shRNA shRNA shRNA 70 shRNA shRNA * #4 #2 #3 #5 #2 60 S100A7 50 * #1 #2 #3 #4 #5 S100A7 40 S100A7 β-Catenin (mm olum β 30 c-Myc -Catenin Actin 20 10

Actin V Tumor MDA-MB468 Actin 0 MDA-MB468 0 7 9 12 14 JMAR Tu167 Day Post Cell Inoculation

Tu167 control shRNA #5 Tu167 S100A7 shRNA #2 H&E

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β

PCNA

X50 X200 X50 X200 Figure 7 Decreased expression of S100A7 leads to increased levels of b-catenin, c-Myc and promotes tumor growth in an orthotopic squamous cell carcinoma of the oral cavity (SCCOC) xenograft mouse model. (a) Western blotting of lentiviral shRNA stably transfected MDA-MB468 cell lines. Each stable cell line was generated from pools of green fluorescent protein (GFP)-positive cells from different S100A7 shRNAs (nos. 1, 2 and 3) and control shRNA (nos. 4 and 5) lentivirus. (b) Western blotting of lentiviral shRNA transfected MDA-MB468 cells with different antibodies as indicated. (c) Western blotting of confluent Tu167 and JMAR shRNA stable polyclones by indicated antibodies. (d) Tumor volumes derived from JMAR shRNA stable polyclones. *Po0.05. (e) Representative immunohistochemical (IHC) images of consecutive sections of Tu167 tumors derived from S100A7 shRNA (no. 2) and control (no. 5) stable polyclones. In each panel, consecutive sections stained with different antibodies are shown as indicated, in which  200 images on right hand side magnify box areas of the  50 images on left hand side. Note that S100A7-expressing area has a reduced staining of b-catenin (mainly on the cell membranes) and proliferating cell nuclear antigen (PCNA), when compared with adjacent S100A7-negative areas that has higher levels of both b-catenin (in both cytoplasm and nuclei) and PCNA staining. supporting that S100A7 expression inhibits b-catenin basis of our results, we have proposed a working model signaling and tumor cell proliferation in vivo. Therefore, for S100A7 function, which is summarized in Figure 8. from our vitro and in vivo analyses of overexpression In this model, S100A7 functions as a putative and downregulation of S100A7, we conclude that ‘tumor suppressor’ inhibiting tumor growth and tumor S100A7 negatively regulates tumor growth and progres- progression, despite its overexpression in the early sion by targeting b-catenin signaling. stage of tumor development. In support of this model are our results showing that S100A7 inhibits SCCOC cell proliferation in vitro, promotes tumor differentia- Discussion tion and suppresses tumor growth, tumor cell invasion in vivo in an orthotopic xenograft SCCOC In this study, we have shown that the reciprocal negative tumor model. Moreover, we have demonstrated that regulation of S100A7 and b-catenin signaling plays an S100A7 is associated with the b-catenin complex, and it important role in tumor progression of SCCOC. On the negatively regulates b-catenin signaling by targeting its

Oncogene S100A7 inhibits SCCOC progression G Zhou et al 3536 Pre-malignancy Malignancy constitutively expresses extremely high levels of S100A7 carcinoma in situ Invasive carcinoma protein (Supplementary Figure S1B) (Enerback et al., 2002; Emberley et al., 2003a), and exhibits a poorly tumorigenic phenotype in subcutaneous mouse model (Krop et al., 2005) when compared with our orthotopic S100A7 S100A7 SCCOC model (10 versus 2 weeks). Although our results indicate that decreased expression of S100A7 in MDA- β-Catenin MB-468 cells increased b-catenin signaling in vitro β-Catenin (Figures 7a and b), how this will impact tumor growth Tumor Progression in vivo in this particular cell line needs to be further EMT investigated. However, given its unusually high levels of Degradation S100A7 expression when compared with most in vitro tumor cell lines, it is quite possible that MDA-MB-468 X β-Catenin cells have developed certain genetic or epigenetic TCF TCF c-myc modifications that render them resistant to growth cyclin D1 inhibition by S100A7 overexpression. etc. It has been shown that activation of Wnt/b-catenin Differentiation Proliferation signaling functions as a master switch for proliferation Invasion versus differentiation (van de Wetering et al., 2002), and Metastasis this plays a pivotal role in the initiation and progression of Figure 8 Working model of S100A7 in oral cancer tumor cancers (Morin and Weeraratna, 2003; Taketo, 2004). In progression. Despite its overexpression in premalignant tumors, S100A7 actually suppresses tumor progression by targeting b- normal epithelium, Wnt/b-catenin signaling pathways catenin signaling. Conversely, its downregulation by b-catenin have been shown to control the specification, mainte- signaling and/or other unknown mechanisms promotes tumor nance, and activation of stem cells, and dysregulation of progression. the pathway often results in the development of familial and/or sporadic epithelial cancers (Blanpain et al., 2007). Our finding that S100A7 negatively regulates b-catenin- degradation via a noncanonical mechanism that is signaling pathways suggests that S100A7 functions independent of GSK3b-mediated phosphorylation. through these pathways to inhibit cell proliferation and Conversely, downregulation of S100A7 expression by promote cell differentiation. In further support of this siRNA was shown to enhance b-catenin signaling, hypothesis, we have shown that S100A7 overexpression including increased b-catenin and c-Myc, promoting inhibited expression of the b-catenin target gene, c-Myc tumor growth and tumor progression in vivo. Finally, (Figure 3e). c-Myc, an oncogenic protein, has the ability we demonstrated that not only does S100A7 inhibit to drive unrestricted cell proliferation, inhibit cell b-catenin, but b-catenin signaling can negatively regulate differentiation, stimulate cell growth and vasculogenesis, S100A7 expression to form a negative feedback loop. reduce cell adhesion and promote metastasis and genomic Therefore, the reciprocal negative regulation between instability (Patel et al., 2004; Adhikary and Eilers, 2005). S100A7 and b-catenin signaling highlights their impor- Conversely, the loss of Myc proteins not only inhibits cell tant roles in tumor development and tumor progression. proliferation and growth but also accelerates differentia- In contrast to our findings are previous studies of tion and increases cell adhesion (Patel et al., 2004; breast cancer MDA-MB-231 and MDA-MB-468 cells Adhikary and Eilers, 2005). Therefore, the tumor that suggested that S100A7 expression promotes tumor suppressive phenotype of S100A7 overexpression shown growth in nude mice (Emberley et al., 2003a, 2005; Krop in our study is consistent with the phenotype derived from et al., 2005). This discrepancy may be attributed to the the loss of Myc function, supporting the involvement of biological differences between squamous and secretory S100A7 in the negative regulation of the Wnt/b-catenin/ epithelial cells, which are derived from distinct stem cell Myc pathways. It should be noted that despite S100A7 lineages. It should be noted that in agreement with our overexpression in skin cells of patients suffering from current results, a prior study of MDA-MB-468 cells psoriasis, a hyperproliferative skin disease characterized indicated that S100A7 expression inhibited cell ancho- by abnormal differentiation, its role in psoriasis is largely -independent growth, motility and invasion in unknown. On the basis of our observation, however, we in vitro assays (Krop et al., 2005). However, in the same believed that it plays a role in ‘abnormal differentiation’ study, downregulation of S100A7 expression by rather than ‘proliferation’ in this disease. Consistent with shRNAs did not increase MDA-MB-468 cell tumor- this, both decreased b-catenin and lack of activation of igenicity in nude mice. The different in vitro and in vivo Wnt/b-catenin signaling were observed in psoriatic skin phenotypic behaviors of MDA-MB-468 cells remain lesions (Reischl et al., 2007). unexplained. Unlike most cultured tumor cell lines that While our preliminary results indicate that S100A7 do not normally demonstrate constitutive S100A7 might interact with the b-catenin complex and target b- expression unless induced by differentiation stimuli catenin degradation in a phosphorylation-independent (Hoffmann et al., 1994; Tavakkol et al., 1994; Di Nuzzo manner (Figure 4), the detailed mechanisms involved are et al., 2000; Enerback et al., 2002; Kennedy et al., 2005), still largely unknown. Previously, other members of MDA-MB-468 is a relatively differentiated cell line that S100A7 proteins such as S100A1, S100A6, S100A12,

Oncogene S100A7 inhibits SCCOC progression G Zhou et al 3537 S100B and S100P, were shown to interact with SIP in a Cell culture, transient DNA and siRNA transfections calcium-dependent manner (Filipek et al., 2002). It Human SCCOC cell lines Tu167 and JMAR were cultured in remains to be determined whether S100A7 can also Dulbecco’s modified Eagle’s medium, and human mammary interact with SIP and participate in Siah-SIP-mediated adenocarcinoma cell line MDA-MB468 was cultured in RPMI b-catenin degradation as previously reported (Liu et al., 1640 medium. The spontaneously immortalized human oral keratinocyte cell line HOK16B was maintained in keratinocyte 2001; Matsuzawa and Reed, 2001; Fukushima et al., serum-free medium (Life Technologies, Bethesda, MD, USA) 2006). Alternatively, S100A7 may target additional supplemented with EGF and bovine pituitary extract. The components of b-catenin-signaling or other important background information of these cells and transfection cell signaling pathways, which remain to be identified. methods are described in Supplementary Materials. Furthermore, although our results unambiguously demonstrate that b-catenin signaling provides negative feedback to inhibit S100A7 expression (Figure 5), the Generation of recombinant adenovirus, retrovirus and lentivirus The detail for generation of adenovirus, retrovirus and mechanisms involved are still unknown. Interestingly, lentivirus for overexpression or suppression of S100A7 and c-Myc, one of downstream targets of b-catenin signal- b-catenin is described in Supplementary Materials. ing, was recently shown to directly inhibit S100A7 expression (Kennedy et al., 2005). However, whether c-Myc or/and other downstream targets are also Cell proliferation, cell migration and colony formation assays implicated in the inhibition of S100A7 expression by Cell proliferation was determined using the tetrazolium-based b-catenin signaling still remain to be further investigated. (that is, MTT) assay as described in Yigitbasi et al. (2004) or The exact mechanisms by which the biphasic expres- cell counting. Cell migration and colony formation assays are described in Supplementary Materials sion (up- and downregulation) of S100A7 during the SCCOC tumorigenesis is achieved and their biological significance also remain to be determined. However, Luciferase reporter assay through this study, we have identified a critical role of Luciferase reporter assay is described in Supplementary S100A7 in a negative regulation of b-catenin signaling, Materials. and vice versa, in head and neck cancer cells. On the basis of our results, we believe that the initial increase in Western blotting, IP and quantitative real-time RT-PCR S100A7 expression in premalignant/highly differentiated The details of western blotting, IP and quantitative real-time tumors may represent a natural defense mechanism by RT-PCR are described in Supplementary Materials. which the cells may attempt to promote differentiation and restrict cell migration. When increased levels of S100A7 are lost by activated b-catenin signaling, the Orthotopic injections of oral tumors cells are then relieved from S100A7’s pro-differentiat- The procedures for cell injection, tumor measurements and subsequent analyses were conducted as described in Yigitbasi ing/growth inhibitory effect, thereby unleashing their et al. (2004) and Supplementary Materials. growth and invasive capacity. Therefore, despite its high levels of expression in early stage SCCOC tumorigen- esis, S100A7 appears to function as a tumor suppressor Tumor specimens, IHC and immunofluorescence microscopy by inhibiting tumor growth and tumor progression. The detail of tumor specimens, IHC and immunofluorescence Together, our findings contribute to elucidating the microscopy is described in Supplementary Materials. mechanistic roles of S100A7 and b-catenin signaling during tumor development and progression of SCCOC, Acknowledgements and may help us to eventually develop diagnostic and therapeutic strategies for SCCOC. This work was supported by the National Institute of Dental and Craniofacial Research—NIH grant RO1DE01461301 Materials andmethods and the NIH Cancer Center support grant CA16672 and CA69381. We thank Mrs Carol Johnston for the excellent Materials and Plasmids technical assistance. We thank Dr PD McCrea, Dr M-C Hung, Materials and plasmids are described in Supplementary Dr X-W Wu, Dr E Fearon and Dr GP Nolan for providing Materials. plasmids.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene