Tetraspanin CD151 Plays a Key Role in Skin Squamous Cell Carcinoma

ORIGINAL ARTICLE Tetraspanin CD151 plays a key role in skin squamous cell carcinoma

QLi1, XH Yang2,FXu1, C Sharma1, H-X Wang1, K Knoblich1, I Rabinovitz3, SR Granter4 and ME Hemler1

Here we provide the first evidence that tetraspanin CD151 can support de novo carcinogenesis. During two-stage mouse skin chemical carcinogenesis, CD151 reduces tumor lag time and increases incidence, multiplicity, size and progression to malignant squamous cell carcinoma (SCC), while supporting both cell survival during tumor initiation and cell proliferation during the promotion phase. In human skin SCC, CD151 expression is selectively elevated compared with other skin cancer types. CD151 support of keratinocyte survival and proliferation may depend on activation of transcription factor STAT3 (signal transducers and activators of transcription), a regulator of cell proliferation and apoptosis. CD151 also supports protein kinase C (PKC)a–a6b4 integrin association and PKC-dependent b4 S1424 phosphorylation, while regulating a6b4 distribution. CD151–PKCa effects on integrin b4 phosphorylation and subcellular localization are consistent with epithelial disruption to a less polarized, more invasive state. CD151 ablation, while minimally affecting normal cell and normal mouse functions, markedly sensitized mouse skin and epidermoid cells to chemicals/drugs including 7,12-dimethylbenz[a]anthracene (mutagen) and camptothecin (topoisomerase inhibitor), as well as to agents targeting epidermal growth factor receptor, PKC, Jak2/Tyk2 and STAT3. Hence, CD151 ‘co-targeting’ may be therapeutically beneficial. These findings not only support CD151 as a potential tumor target, but also should apply to other cancers utilizing CD151/laminin-binding integrin complexes.

Oncogene (2013) 32, 1772–1783; doi:10.1038/onc.2012.205; published online 23 July 2012 Keywords: tetraspanin CD151; skin squamous cell carcinoma; chemical carcinogenesis; integrin a6b4; STAT3; PKCa

INTRODUCTION and breast tumor xenograft growth in vivo.32,33 Also, CD151 in 34 Cell surface protein CD151, a tetraspanin protein family member, is host tissues facilitates tumor angiogenesis, and tumor cell expressed in epithelial, endothelial, Schwann and dendritic cells, and CD151 supports ErbB2 drug resistance in cells plated on specific 35 in skeletal, smooth and cardiac muscle.1 CD151 associates closely laminin isoforms. CD151 has not previously been reported to with laminin-binding integrins (that is, a3b1, a6b1, a6b4anda7b1) affect de novo carcinogenesis or specific stages of carcinogenesis and modulates their functions.2–12 CD151-null mice are mostly (that is, initiation, promotion, progression). Furthermore, there is viable, healthy and fertile.13 However, humans and mice (in little precedent from studies of related molecules (that is, other particular strains, and under specific conditions) lacking CD151 tetraspanin proteins) for regulation of early carcinogenesis stages. have kidney and skin deficiencies.14–17 Absence of laminin-binding To evaluate CD151’s role during de novo carcinogenesis, we 36 integrins also causes kidney and skin pathology,18 consistent with used two-stage skin chemical carcinogenesis. During skin tumor CD151 being functionally linked to laminin-binding integrins. CD151 initiation, carcinogen DMBA (7,12-dimethylbenz[a]anthracene) is may affect functions of laminin-binding integrins by regulating metabolized to reactive diol-epoxides that bind to DNA and integrin diffusion,19 glycosylation20 and/or internalization.21 Also, mutate target keratinocytes. Initiated cells escaping DNA repair CD151 can recruit proteins such as phosphoinositide 4-kinase, and apoptosis may clonally expand during TPA (12-O- protein kinase C (PKC) and other tetraspanins (for example, CD9, tetradecanoyl phorbol 13-acetate)-induced tumor promotion. CD81, CD82, CD63) into complexes with laminin-binding This model is ideally suited for investigating CD151 functions in integrins,11,22,23 which potentially affects integrin functions.24 de novo carcinogenesis at specific carcinogenesis stages. Because laminin and laminin-binding integrins contribute Furthermore, mouse skin chemical carcinogenesis results are both negatively and positively to carcinogenesis,25–28 CD151 relevant to human skin SCC.36,37 Among epidermoid carcinomas, conceivably could also play negative and/or positive roles. For squamous cell carcinoma (SCC) is one of the most common forms example, integrin a6b4 suppresses tumor-initiating mouse skin of cancer in the United States. There are B250,000 new cases/ cell growth29 and induces apoptosis in carcinoma cells expressing year,38 with 2–9.9% metastasis incidence, which leads to poor p53.30 Hence, while supporting a6b4, CD151 might inhibit early long-term prognosis.39 carcinogenesis stages. Alternatively, studies with cancer cell lines We show significant and selective CD151 upregulation on show CD151 contributing to epidermoid carcinoma cell migration human skin SCC samples. Also CD151 knockout mice, subjected to and metastasis,31 breast cancer cell invasion and migration in vitro, skin chemical carcinogenesis, showed extended tumor latency

1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA; 2Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, KY, USA; 3Department of Pathology, Division of Cancer Biology and Angiogenesis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA and 4Department of Pathology, Harvard Medical School, Brigham and Women’s Hospital, Boston, MA, USA. Correspondence: Dr ME Hemler, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Harvard Medical School, Room D1430, 44 Binney Street, Boston, MA 02115, USA. E-mail: [email protected] Received 6 November 2011; revised 30 March 2012; accepted 23 April 2012; published online 23 July 2012 CD151 in human and mouse skin cancer QLiet al 1773 and decreased tumor incidence, multiplicity and size. Further- keratinocytes also showed increased DMBA-induced apoptosis more, CD151 contributed during the skin tumor initiation, (Figure 3C). Another type of DNA-damaging agent, camptothecin, promotion and progression stages, and supported chemical/drug also increased apoptosis in CD151-null keratinocytes (Figure 3C). resistance. Also we provide mechanistic insights involving CD151 Thus, CD151–/– keratinocytes appear sensitized to chemical stress. effects on STAT3, PKCa and integrin a6b4. These results are To study tumor promotion, mice were treated four times with relevant not only toward understanding human skin SCC, but also TPA, over 2 weeks, without prior DMBA treatment. Epidermal for other epithelial cancers similarly using CD151–integrin thickness increased to a lesser extent in CD151–/– mice (Figures complexes. 4a and b). Likewise, CD151–/– mice incorporated 45% less bromodeoxyuridine (BrdU; Figures 4c and d). Thus, reduced tumor development in CD151-null skin may arise from changes in both RESULTS DMBA-induced apoptosis and TPA-induced proliferation. CD151 elevation in human skin SCC To assess CD151 protein expression in human skin tumors, we examined 83 skin cancer microarray samples. Representative CD151 affects STAT3 activation images (Figures 1a–c) show abundant CD151 staining of human STAT3, a transcription factor constitutively active in many human skin SCC samples, especially at cell membranes, with also some malignancies,41 is essential for epidermal survival and proliferation intracellular staining (Figure 1b). In normal human skin, CD151 is during skin carcinogenesis leading to SCC.42,43 Mouse skin cell abundant only in basal and parabasal cell layers (Figure 1c, left lines derived from CD151–/– papillomas showed 40–60% less panel). Most human skin SCC samples showed elevated CD151 activated STAT3, while total STAT3 was unchanged (Figure 5a). staining (67% with score of 41; Figure 1d). Samples from grade II After 1-h TPA stimulation, À / À skin cell lines also showed SCC trended toward more elevated CD151 staining (score ¼ 3, diminished increase in STAT3 activation (B1.5-fold, compared n ¼ 14), compared with grade I SCC (score ¼ 2.35, n ¼ 36, P ¼ 0.1). with 3-fold for þ / þ cells; Supplementary Figure S1A). Contrasting with skin SCC, other skin cancer types showed weak In unstimulated human A431 epidermoid carcinoma cells, staining. Staining scores were 1 (Figure 1d) for most basal cell CD151 knockdown minimally affected STAT3 activation carcinomas (92%), metastatic melanoma (91%) and dermatofi- (Figure 5b, Supplementary Figure S1B). However, CD151-ablated brosarcoma protuberans (100%) samples. Overall, CD151 is cells showed markedly less STAT3 activation after TPA 5–30 min selectively and significantly elevated in human skin SCC compared stimulation, and showed almost complete STAT3 inactivation after with other skin tumors (Figure 1d; mean scores in right panel), and 30–60 min. Total STAT3 levels were unchanged (Figure 5b, thus is well positioned for significant functional contributions. Supplementary Figure S1B). These results indicate a possible combination of diminished kinase and enhanced tyrosine Response of CD151-null mice to two-stage carcinogenesis phosphatase activities affecting STAT3 in CD151 À / À cells. Following treatment with DMBA alone (as in Figure 3A), mouse To address CD151 functions, we used a two-stage mouse chemical skin samples were lysed and analyzed for STAT3 activation. Again, carcinogenesis protocol, which models human skin SCC initiation, 36 STAT3 was less activated in samples from CD151–/– mice promotion and progression. CD151 wild-type, heterozygous and (Figure 5c), suggesting that loss of STAT3 activation contributes null mice (129/Sv strain) were backcrossed four generations into to increased apoptosis (Figures 3A and B). After mouse skin FVB/N background, which is substantially more sensitive to skin treatment with TPA alone (as in Figure 4), STAT3 activation tumor formation.40 Mice were treated once with initiating agent substantially increased in CD151 þ / þ , but not CD151–/– mouse DMBA (100 nmol), and then weekly with 8 nmol TPA. Figure 2a samples (Figure 5d). Thus, failure to increase STAT3 activation may shows markedly reduced tumor development in CD151-null mice, contribute to diminished epidermal proliferation in CD151–/– skin. compared with þ / þ and þ / À mice. Tumors appeared in most The presence or absence of CD151 minimally affected total STAT3 þ / þ mice by 7 weeks, in significantly greater numbers (up to 14 levels (Figures 5c and d). per mouse). By contrast, –/– mouse tumors appeared later (after 9 CD151-null cultured primary keratinocytes showed diminished weeks) and less frequently (2–4 per mouse; Figures 2a–c), and proliferation (measured two different ways; Figure 6a), likely due were smaller (Figure 2d). Tumor appearance, multiplicity and size to diminished response to EGF in keratinocyte growth media. This were only partly impaired in CD151 þ /– compared with CD151 difference disappeared upon cell treatment (for 2–3 days) with þ / þ mice (Figures 2a–d). After 20 weeks, 6.4% of þ / þ mouse nifuroxazide, which inhibits Jak2 and Tyk2 kinases immediately tumors showed conversion to malignant SCC. By contrast, upstream of STAT3,45 consistent with CD151 functions depending malignant conversion was significantly reduced for þ / À (2.3%) on STAT3. Results in Figure 6b confirm that nifuroxazide indeed and À / À (0%) mouse tumors, as judged by histological analysis inhibits STAT3 activation in both CD151-null and control cells. (Table 1). In summary, CD151 contributes to mouse skin tumor Next we found that CD151 affects the entire epidermal growth appearance, multiplicity, size and malignant conversion. factor receptor (EGFR)–Jak2/Tyk2–STAT3 signaling pathway, as evidenced by enhanced drug sensitivity in CD151-ablated cells, CD151 contributions during tumor initiation and promotion stages using a STAT3 activation readout. Indeed, in papilloma-derived Before treatment, þ / þ and –/– mouse skin differed minimally mouse skin cell lines lacking CD151, potency of nifuroxazide in apoptosis (seen by caspase staining of epidermis; Figures 3A inhibition of STAT3 activation was enhanced (Supplementary and B), epidermal proliferation (Figures 4c and d) and epidermal Figure S1A). To explore this further, we analyzed STAT3 activation STAT3 activation (Figures 5c and d). Also, morphology and in A431 cells. In untreated cells, CD151 ablation minimally affected epidermal thickness was essentially identical, as seen in hematox- STAT3 activation. However, CD151 absence markedly enhanced ylin and eosin-stained cross-sections of dermis and epidermis sensitivity to nifuroxazide. STAT3 activation in control A431 cells (Figure 4a). In addition, untreated þ / þ and –/– primary was relatively unaffected by 2–5 mM nifuroxazide (Figure 6c, keratinocytes showed essentially identical background apoptosis Supplementary Figure S2A). By contrast, CD151-ablated cells were (Figure 3C). sensitized, showing markedly diminished STAT3 activation, To determine CD151 effects on keratinocytes during tumor especially 5 h after treatment at the higher nifuroxazide dose. initiation, we treated mouse skin with DMBA alone, and then Using EGFR inhibitor lapatinib,46 we again saw CD151 ablation intact skin sections were assessed for apoptosis by caspase-3 increasing drug sensitivity, resulting in greater loss of constitutive staining. DMBA treatment stimulated more keratinocyte apoptosis STAT3 activation (Figure 6d, top panel, lanes 4 and 6). Total STAT3 in CD151-null mice (Figures 3A and B). Cultured CD151-null levels were unaffected (second panel). Although EGFR activation

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Figure 1. CD151 is elevated in human skin tissues. (a) Staining of human skin SCC samples (five left columns) and normal skin (right column). (b) Enlarged CD151 staining of a human SCC sample. Red arrows indicate representative intracellular and peripheral CD151 staining. (c) Immunohistochemical images show CD151 in normal skin (left panel) and skin SCC tumors with representative scoring 1–4 (see Materials and methods). (d), Quantitation of results from a panel of different human skin cancer samples, such as shown in a and b. The right panel shows mean scores for 51 skin SCC and 32 combined basal cell carcinomas, metastatic melanoma (MM), and dermatofibrosarcoma protuberans (DFSP) samples±95% confidence interval. *Po0.0001; Mann–Whitney U-test.

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Figure 2. CD151 contribution to mouse skin tumor formation. (a) Skin tumors in CD151 þ / þ , þ /– and –/– mice 20 weeks after DMBA initiation. (b) Delayed tumor appearance in CD151–/– mice. *Po0.0001 (log-rank test). (c) Average tumor numbers per mouse are indicated (±95% conﬁdence interval). For data obtained after 20 weeks: **Po0.0001; *P ¼ 0.002 (Mann–Whitney U-test). (d) For each mouse group, tumor sizes are separated into three categories. Statistical analyses are based on two categories (large; medium þ small). For wild type (WT) vs null, P ¼ 0.001; For heterozygous (Het) vs null, P ¼ 0.009 (Fisher’s exact test).

Table 1. Histological analysis of skin tumors at 20 weeks CD151–/– cells were less well spread, and less tightly clustered (Figure 7a). Similarly, cultured primary CD151–/– keratinocyte b4was CD151 genotypes þ / þþ/ ÀÀ/ À more distributed to the cell periphery (Figure 7b). During keratinocyte carcinogenesis, EGF/TPA stimulates b4serine Number of mice 19 32 20 phosphorylation at key sites, including S142452 and S1356.50,53 Total tumors 235 306 66 Benign tumors 220 299 66 This enables b4 dissociation from the intermediate filament cytoskeleton, as hemidesmosomes are disrupted and epithelial SCC tumors 15 7 0 50,52,53 SCC tumors/total tumors 6.4% 2.3% 0% cells lose polarity while becoming more invasive. Reagents aP ( þ / þ vs þ / À , À / À ) — 0.026 0.048 were unavailable for analyzing mouse serine site-specific b4 aP ( þ / À vs À / À ) — — 0.61 phosphorylation. However in CD151-ablated human A431 cells, S1424 phosphorylation was notably impaired in response to TPA Abbreviation: SCC, squamous cell carcinoma. aFisher’s exact test. (38% of wild type) or EGF (51% of wild type; Figure 7c). By contrast, b4 S1356 phosphorylation was similarly stimulated by TPA and EGF, decreased with higher lapatinib doses, this was unaffected by regardless of CD151 presence (Figure 7c, second row). CD151 (Figure 6d, third panel). A direct inhibitor of STAT3, ST3–01,44 again preferentially inhibited STAT3 activation in A431 CD151 affects conventional PKC location and function cells lacking CD151 (Figure 6e, Supplementary Figure S2B). Phosphorylation of b4 at S1424,52 but not S1356,54 requires CD151 has been suggested to affect c-MET signaling, b4 conventional PKC (cPKC) isoforms. Integrin b4 in A431 cells integrin collaboration and support of tumor xenograft growth,47 coimmunoprecipitated with a cPKC isoform (PKCa) after cell and c-MET may signal through STAT3.48 However, using c-MET stimulation with phorbol ester or EGF for 5–60 min. However, inhibitor su11274,49 we did not see altered STAT3 activation PKCa–a6b4 association was markedly diminished (especially 5 min ±CD151 ablation (Supplementary Figure S3A). A consistent after TPA or EGF stimulation) if CD151 was ablated, even though 25–30% decrease in c-MET activation affirms that su11274 is equal amounts of b4 and PKCa were retained (Figure 8a). These functional (Supplementary Figure S3A, third panel). Furthermore, results are consistent with CD151 linking PKCa to a6b4. We also diminished stimulation of STAT3 activation in CD151-ablated cells addressed cPKC effects on STAT3 activation. As indicated (Supplementary Figures S3B and S3C, top panels) was not (Figure 8b), cPKC inhibitor Go6976 markedly inhibited STAT3 accompanied by altered c-MET activation (Supplementary activation in CD151-ablated A431 cells, but not in control cells. The Figures S3B and S3C, third panel). PKC inhibitor did not affect total levels of STAT3 and PKCa.

CD151 affects b4 integrin phosphorylation Possible indirect effects of CD151 on skin tumor formation Among laminin-binding integrins, a6b4 makes critical contributions As CD151 can support tumor angiogenesis,34 we considered that during skin cancer progression to SCC.29,50,51 CD151 affects CD151 ablation may diminish chemical carcinogenesis due to subcellular distribution of tumor cell a6b4.33 Indeed, within decreased angiogenesis. However, mouse skin tumor microarray CD151–/– skin tumor cells, b4 integrin was considerably more analyses revealed no decrease, in CD151-null tumors, in VEGF or punctate and/or distributed toward the cell periphery. By contrast, CD31 expression (Supplementary Table 1). Furthermore, wild-type CD151 þ / þ cell b4 was distributed more diffusely (Figure 7a). Also, and null tumor vessel densities did not differ.

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Figure 3. DMBA-induced apoptosis in CD151 deﬁcient mice. (A) Representative active caspase-3 staining of epidermis from (a) untreated þ / þ , (b) þ / þ mice with DMBA, (c) untreated –/– and (d) –/– mice with DMBA. Caspase-3-positive cells appear in both hair follicle bulge regions (arrows) and interfollicular epidermis (arrowhead); bar ¼ 50 mm. (B) Quantitation of caspase-3-positive cells per unit area from data as in A. Shown are means±s.e.m., n ¼ 5; *Po0.001. (C) Quantitation of keratinocyte apoptosis, after 4 h of chemical treatment, based on accumulation of cytoplasmic histone-associated DNA fragments (see ‘Materials and methods’). Shown are means±s.e.m., n ¼ 5; *Po0.005; **Po0.006 (Student’s t-test). KO, knockout; WT, wild type.

Cytokines (for example, interleukin (IL)-6, IL-12, IL-17, IL-23) play CD151 and skin SCC 55–59 key roles in skin disease, SCC and/or during STAT3 activation. Although many epithelial tumor types upregulate CD151,32,33,61–63 However, levels of cytokines (IL-6, IL-12, IL-17, IL-23) in skin did not appearance on human skin SCC had not been reported. Tissue change upon CD151 ablation (Supplementary Table 2). Further- microarray data now show that CD151 selectively upregulated in more, CD151 ablation caused no decrease in IL-6-stimulated human skin SCC, but not in other human skin tumors (basal activation of TYK2 (Supplementary Figure 1c), a major cytokine- cell carcinoma, metastatic melanoma, dermatoﬁbrosarcoma 60 responsive STAT3-activating kinase. Likewise, IL-6-induced protuberans). Laminin-binding integrins are not known to play STAT3 activation was undiminished in mouse keratinocytes major roles in those other skin cancers, which may explain lack of lacking CD151 (Supplementary Figure 1c). Hence, CD151 removal CD151 upregulation. Although CD151 trended toward elevated does not perturb major cytokine networks important for regulat- expression in higher grade skin SCC, patient prognosis data were ing STAT3 and skin carcinogenesis. unavailable. However, CD151 associates with poor patient prognosis in breast cancer and other cancers,32,61–63 and correlates with metastasis in esophageal SCC.47 CD151 is not DISCUSSION only present, but also is a key contributor in the genesis of skin SCC, as seen using a mouse model that has previously yielded On the basis of the prior studies (see Introduction), it was unclear many results relevant to multistage genesis of human epithelial whether CD151 would positively or negatively affect de novo cancers, including skin SCC.36,37 tumor formation. Here we show that during mouse chemical carcinogenesis CD151: reduced tumor lag time; promoted tumor incidence, multiplicity, size and progression to SCC; contributed to CD151 effects during the tumor initiation stage both tumor initiation and promotion stages, and markedly altered The absence of CD151 markedly enhanced apoptosis induced by signaling through b4 integrin, EGFR, PKCa and STAT3. DMBA (during carcinogenesis initiation) and by camptothecin

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Figure 4. TPA-induced epidermal proliferation. (a) Mice (n ¼ 5 per group) were treated with TPA and killed 24 h after the last treatment. BrdU was injected 2 h prior to killing. Hematoxylin and eosin staining of epidermis from (a) untreated þ / þ ,(b) þ / þ treated with TPA, (c) untreated –/– and (d) –/– mice treated with TPA. Bar ¼ 50 mm. (b) Quantitation of epidermal thickness from wild-type (black bar) and CD151-null mice (white bar). n ¼ 5; *Po0.02. (c) Immunohistochemical analysis of proliferation marker BrdU in dorsal skin after TPA treatment. (d) Quantitation of BrdU-positive cells in the epidermis of wild-type (black bar) and CD151-null mice (white bar) after treatment with TPA. n ¼ 5; *Po0.001. KO, knockout; WT, wild type.

(a chemotherapeutic agent). Thus, CD151 absence has a general during two-stage carcinogenesis. Mouse skin TPA responses sensitizing effect to different types of chemical agents. CD151 acts require a functioning EGFR.69 Integrin a6b4 may associate by modulating functions of laminin-binding integrins, including physically and/or functionally with the EGFR,70 with CD151 also a6b4, which plays a particularly important role during skin SCC supporting EGFR functions.33 In this regard, CD151 ablation pathogenesis.29,51 CD151 can affect a6 distribution,33 adhesion diminished b4 phosphorylation responses to both TPA and EGF strengthening,64 diffusion mode65 and sometimes cell adhesion.21 (Figure 7c). Hence EGFR disruption, due to CD151 removal, may On CD151-null cultured mouse keratinocytes and tumor-derived explain diminished TPA responses. cells, a6b4 became less diffusely distributed, while showing more SCC tumor promotion during chemical carcinogenesis requires punctate and/or peripheral staining. Changes in molecular STAT3 activation.43 Thus, impaired TPA-induced STAT3 activation (and/ organization of a6b4, possibly coupled with altered laminin or enhanced TPA-induced STAT3 inactivation) may be central to adhesion, likely contribute to chemical sensitization. In this regard, diminished tumor promotion and skin tumor formation in CD151-null integrins, including laminin-binding integrins,35,66 support cell mice. Because the EGFR makes major contributions to STAT3 activation resistance to a variety of drugs/chemicals. during tumor promotion,69 loss of CD151–integrin regulation of the Increased DMBA-induced apoptosis in CD151-null keratinocytes EGFR should contribute to diminished STAT3 activation. Disrupted was accompanied by decreased STAT3 activation. This result helps recruitment of PKCa also likely contributes to diminished TPA induction explain reduced CD151-null skin tumor formation, because STAT3 of STAT3 activation in CD151-ablated cells (next section). activation plays a major role during chemical carcinogenesis 43 initiation. Decreased STAT3 activation accompanies decreased CD151 and tumor progression 67 resistance to multiple types of cellular stresses. In this regard, CD151 CD151-null mouse tumors did not progress to SCC. As activated removal enhanced sensitivity to DMBA (a mutagen), camptothecin STAT3 promotes malignant progression during chemical carcino- (an inducer of DNA fragmentation) and to STAT3 pathway inhibitors, genesis,42 reduced STAT3 activation likely underlies absence of thus indicating general chemical stress sensitization. Decreased CD151-null tumor progression. SCC may arise from keratinocyte STAT3 activity in keratinocytes might arise not only from decreased stem cells,71 implying that stem cell oncogenesis could be activation, but also from enhanced deactivation, due to 68 deﬁcient in CD151-null mice. As a6 integrins serve as phosphatases, which would be consistent with our results. keratinocyte stem cell markers,72 CD151 is well positioned to possibly contribute to stem cell oncogenesis. CD151 and tumor promotion Mice (and cells) lacking CD151 showed major decreases in TPA- CD151 effects on PKCa induced epidermal hyperplasia, epidermal cell proliferation and Besides disrupting a6b4–EGFR collaborations, CD151 ablation also STAT3 activation, thus further explaining diminished tumor yield impairs a6b4–PKC connections. TPA stimulation activates cPKC

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Figure 5. CD151 inﬂuences STAT3 activation. (a) Mouse-derived skin cell lines were lysed in 1% Triton X-100, and blotted for pSTAT3 (Tyr705, mAb from Cell Signaling), total STAT3 (rabbit polyclonal Ab, Santa Cruz) and b-actin. (b) CD151 was stably ablated in human A431 cells. Knockdown (KD) and control cells were treated with TPA (50 ng/ml) for indicated times, lysed, and then blotted as in a. CD151 was detected using mAb 1 A5. CD151 knockdown was 490% (bottom panel). Bar graphs, based on densitometry scans, show relative STAT3 activation. (c) Epidermis from þ / þ and –/– mice was isolated 24 h following DMBA treatment, and lysate proteins were blotted for activated STAT3, total STAT3 and b-actin. Bar graphs show relative STAT3 activation; n ¼ 5, *Po0.01. (d) Epidermis from þ / þ and –/– mice was isolated 24 h following the last TPA treatment, and lysate proteins were blotted for activated STAT3, total STAT3 and b-actin. Bar graphs show relative STAT3 activation, n ¼ 5, *Po0.02. WT, wild type.

isoforms, such as PKCa in skin.73 Hence, PKCa dysregulation could functions (that is, epidermal proliferation and STAT3 activation— underlie deficient TPA stimulation in the absence of CD151. TPA see Figure 9). (and EGF) stimulate PKCa-dependent phosphorylation of b4at 52 S1424. Phosphorylation of b4 S1424 (and three other serines) CD151 and STAT3 contributes to b4 dissociation from intermediate filament CD151 had not previously been linked to STAT3 signaling. Integrin hemidesmosomes, while switching to an actin-connected a6b4 may signal through STAT3 in ErBB2-driven breast cancer invasive state.50,53,74 Hence, diminished S1424 phosphorylation, cells, but this did not involve proliferation or apoptosis.26 Hence, due to CD151 absence, is consistent with the maintenance of our results uncover a new link between CD151–a6b4 complexes a6b4 in a more intermediate filament-anchored state, less and STAT3 signaling during the tumor initiation and promotion susceptible to epithelial disruption and less likely to increase stages. invasion. Shifting of b4 in keratinocytes and mouse tumor lines Several findings point to CD151 acting through STAT3 during toward a peripheral/punctate staining pattern appears consistent skin tumor formation. First, activated STAT3 is essential for tumor with diminished hemidesmosomes association. Unfortunately, progression in the DMBA/TPA model.42 Hence, decreased STAT3 antibody specific for mouse b4 S1424 was not available to activation, due to CD151 removal, must affect tumor progression. confirm expected loss of S1424 phosphorylation in mouse cells. Second, inhibition of STAT3 eliminated CD151 effects on Phosphorylation of b4 at S1356, which is independent of cPKC proliferation (see Figure 6a), thus linking CD151 functions to isoforms,54 was unaffected by CD151 absence, thus emphasizing STAT3. Third, STAT3 is required for skin tumor initiation and tumor selective CD151 support for PKCa function. Only when CD151 was promotion.43 Therefore, diminished STAT3 activation caused by present did we observe TPA stimulation of PKCa association with CD151 absence can explain contributions of CD151 during tumor a6b4, thus supporting a role for CD151 in recruiting PKCa into initiation and tumor promotion stages. proximity with a6b4. Previously CD151 was shown to recruit, upon TPA stimulation, cPKC isoform PKCbII into proximity with laminin-binding integrins.23 Selective loss of PKCa recruitment Are CD151 effects ‘keratinocyte-specific’? to a6 integrins could explain not only diminished b4 S1424 CD151 effects on skin tumor formation appear to be largely phosphorylation, but also other impaired TPA-stimulated ‘keratinocyte-specific’. Decades of research show that skin

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Figure 6. CD151 and STAT3 inhibition effects. (a) Equal numbers of freshly isolated primary mouse keratinocytes were cultured±5 mM nifuroxazide. After 2 days, cell proliferation was assessed using the MTT (3-(4,5]dimethylthiazol-2,5-diphenyltetrazolium bromide) assay, which measures metabolic energy (left panel) and after 3 days total cell numbers were counted (right panel). Bars represent mean±s.d. for three independent experiments. *Po0.05; **Po0.001. (b) STAT3 activation (pSTAT3-Y705) is shown for primary mouse keratinocytes treated for 2 days with 5 mM nifuroxazide. (c) CD151 knockdown (KO) and control A431 cells were treated with nifuroxazide (2 or 5 mM) for indicated times, lysed, and then pSTAT3 (Y705) and total STAT3 were blotted, and ratios were determined from densitometric quantitation of blot intensities. See blots in Supplementary Figure S2A. (d) A431 cells±CD151 were treated with lapatinib, and then blotted for pSTAT3, total STAT3, pEGFR and CD151 as indicated. Densitometric quantitation showed CD151 ablation causing pSTAT3/STAT3 ratios to decrease by 0, 64 and 84% (with 44 0, 1, 5 mM lapatinib, respectively). (e) A341 cells±CD151 were treated with STAT3 inhibitor ST3–01, and then STAT3 activity was determined as indicated in Supplementary Figure S2B. WT, wild type. chemical carcinogenesis initiation and promotion occur entirely in C57Bl6 mice,13,34 but plays a critical role during pathological through keratinocyte mutation and selection.36,37 Furthermore, events such as tumor angiogenesis,34 skin wound healing15 and CD151 ablation from purified epidermoid cell populations skin tumor formation (shown here). Because CD151 effects are definitively alters cell survival, proliferation, STAT3 activation, more obvious in pathological compared to normal situations, STAT3 drug sensitivity, integrin b4 distribution and phos- CD151 prospects as a tumor target are enhanced. phorylation, and b4–PKC association (consistent with altered TPA Neoplastic transformation in many cell types is supported by response). activated STAT341 and by a6 integrins (for example, Guo et al.26), We considered that CD151 on other cell types might affect skin which invariably associate with CD151. Hence, CD151 should tumor formation by altering angiogenesis,34 immune cell contribute to de novo tumor formation, progression and STAT3 functions75 or cytokines that activate STAT3.55–59 However, key activation in many other epithelial cancers. CD151 contributions to cytokine levels in skin were unchanged, and evidence obtained (a) cell survival during tumor initiation, (b) cell proliferation during thus far does not support altered angiogenesis. In this regard, tumor promotion and (c) tumor progression, represent major implanted melanoma cells did not show altered angiogenesis additions to the growing list of CD151 cancer roles, during when CD151 was absent from the host.34,65 Furthermore, immune angiogenesis,34 tumor metastasis,31 tumor xenograft growth32,33 cells in CD151-ablated tumor-bearing mice were unaltered with and on stem-like prostate tumor-initiating cells.76 Consequently, respect to tumor infiltration, subset sizes, activation or targeting CD151 should have multi-pronged anticancer effects. trafficking.65 In conclusion, available data point to keratinocyte Also, because CD151 removal sensitizes tumor cells to agents CD151 as being largely responsible for supporting chemical targeting EGFR/ErbB2, STAT3 and PKCa, co-targeting of CD151 carcinogenesis. may enhance effectiveness of other agents directed at a6 integrins and associated signaling pathways. Summary and broader implications CD151 absence had no detectable effect on normal mouse skin 13,17 morphology or histology, as seen previously and confirmed MATERIALS AND METHODS here. However, CD151 absence markedly diminished cell survival Human tissue arrays during tumor initiation, proliferation during the promotion phase Paraffin sections containing 106 human skin tissue samples (SK483 and and progression to SCC. A pattern has emerged in which CD151 BC21011, US Biomax, Rockville, MD, USA) were stained with anti-CD151 may be dispensable for normal physiological processes, while (clone RLM30) by Brigham and Women’s Hospital Histopathology Core. contributing to pathological events. For example, CD151 is CD151 expression score (0–4) combines %CD151-positive tumor area and unnecessary for normal development of blood vessels and skin graded positive area intensity, each determined by visual inspection.

& 2013 Macmillan Publishers Limited Oncogene (2013) 1772 – 1783 CD151 in human and mouse skin cancer QLiet al 1780

Figure 7. CD151 affects b4 distribution and phosphorylation. (a) Mouse tumor-derived cell lines were permeabilized and stained for integrin b4. (b) Keratinocytes were isolated from wild-type and null mice and then cultured for a few weeks before integrin b4 subunit staining, bar ¼ 5 mm. (a and b) Linear staining intensity, across the diameter of representative cells, was measured using the Image J program (from NIH). (c) Human A431 cells, stably ablated for CD151, were treated with TPA (50 ng/ml) or EGF (100 ng/ml) for 5 min and 1 h, and then blotted for the indicated proteins, using antibodies for b4-S1424, b4-S1356 and total b4. KD, knockdown; WT, wild type.

Cells, antibodies, chemicals and short hairpin RNA pathology complications.14 All mice used were viable, fertile, and lacked A431 cells were maintained in Dulbecco’s modified Eagle’s medium, 10% obvious defects in kidneys or elsewhere. Supplementary Figure S4 shows representative diagnostic PCR image and primer sequences used for fetal calf serum, at 37 1C in humidified 5% CO2. Mouse tumor-derived cell lines were cultured in Dulbecco’s modified Eagle’s medium/F12, containing CD151 genotyping. For two-stage carcinogenesis, 7-week-old female 5% fetal calf serum, 5 mg/ml insulin, 100 ng/ml Cholera toxin, 10 mM HEPES, mouse dorsal skin was shaved, and after 1 week treated once with DMBA 1 mg/ml hydrocortisone and 10 ng/ml EGF. Anti-b-actin was from Sigma- (100 nmol in 0.2 ml acetone). After another week, mice were treated with Aldrich (St Louis, MO, USA), anti-STAT3 and anti-PKCa were from Santa Cruz TPA (8 nmol in 0.2 ml acetone) twice weekly for 19 more weeks. Control Biotechnology (Santa Cruz, CA, USA), and anti-pSTAT3(Tyr705)wasfromCell mice were treated with 0.2 ml acetone for 20 weeks. Mice were examined Signaling Technologies (Beverly, MA, USA). Monoclonal antibodies to CD151 weekly. Growths 41 mm diameter, persisting for X2 weeks, were were 5C11,11 1A5 (gift from A Zijlstra) and RLM30 (Leica Microsystems, recorded as tumors. Buffalo Grove, IL, USA). Anti-phospho-EGFR (to activated EGFR) was from BD For ‘initiation only’ experiments, mice were treated on dorsal skin once Biosciences (San Jose, CA, USA), anti-c-MET was from Santa Cruz, and anti- (DMBA, 100 nmol) or with acetone alone and killed after 24 h. To assess pTyr antibody was from EMD Millipore (Billerica, MA, USA). Monoclonal and apoptosis, skin sections were stained with antiactive caspase-3 (R&D polyclonal anti-integrin b4 were from EMD Millipore, and polyclonal anti-b4 Systems, Minneapolis, MN, USA) followed by biotinylated anti-rabbit S1356 and b4 S1424 were described.50,52 DMBA, BrdU, camptothecin and immunoglobin-G and horseradish peroxidase-conjugated ABC reagent TPA were from Sigma-Aldrich. Nifuroxazide45 and ST3–01 (also called (Vector Laboratories, Burlingame, CA, USA). For ‘promotion only’ experi- ML116)44 were from Dr DA Frank, Dana-Farber Institute. Lapatinib was ments, 8-week-old female mice were treated twice weekly for 2 weeks with from LC Laboratories (Woburn, MA, USA), and SU11274 was from TPA (8 nmol) or acetone vehicle and killed after another 24 h. Some mice Sigma-Aldrich. CD151 was stably ablated from A431 cells as described.21 were injected intraperitoneally with BrdU (2 mg per mouse) in phosphate- CD151-null cells were negatively selected by flow cytometry (using mAb buffered saline 2 h prior to killing. Dorsal skin was formalin-fixed, paraffin- 5C11). Knockdown efficiency was evaluated by flow cytometry and embedded, sectioned at 4 mm, stained with hematoxylin and eosin and immunoblotting using mAb 1A5. anti-BrdU antibody (BD Biosciences-Pharmingen, San Jose, CA, USA), and then with biotinylated anti-mouse immunoglobin-G and horseradish peroxidase-conjugated ABC reagent (Vector Laboratories). Hematoxylin Skin carcinogenesis experiments and histological examinations and eosin-stained skin thickness was measured by micrometer. To CD151-null mice in Sv129 background34 were crossed four generations determine epidermal cell proliferation, BrdU-positive cells were counted, onto FVB/N background, to increase chemical carcinogenesis sensitivity.40 per unit area. Paraffin sectioning and hematoxylin and eosin and CD151–/– mice were not further backcrossed into FVB/N to avoid kidney immunohistochemical staining were performed by Harvard Medical School

Oncogene (2013) 1772 – 1783 & 2013 Macmillan Publishers Limited CD151 in human and mouse skin cancer QLiet al 1781 Histology Core. All treatments were approved by Dana-Farber Animal Care Committee and followed NIH guidelines.

In vitro tumor and keratinocyte cultures To establish mouse tumor lines, specimens were collected (after 20 weeks DMBA/TPA treatment), minced and resuspended in collagenase (Worthing- ton Biochemical, Lakewood, NJ, USA, CLS3 type; 200 U/ml) in Medium-199 plus gentamycin (5 mg/ml). After 4 h at 37 1C, suspended cells were cultured in Dulbecco’s modified Eagle’s medium/F12 medium. Mouse primary keratinocytes were isolated and cultured as described.77 Briefly, newborn mouse skin specimens were trypsinized (0.25% trypsin, Invitrogen, Eugene, OR, USA) for 8–10 h at 4 1C, and epidermis was separated from the dermis. Isolated keratinocytes were plated (60 cm dishes), precoated with collagen type I (BD Biosciences), and containing keratinocyte medium (EMEM; Fisher Scientific, Cambridge, MA, USA) supplemented with 0.1 mM monoethanolamine, 0.1 mM phosphoryl ethanolamine, 0.5 mM hydrocortisone at 37 1Cin5%CO2. After 5 h, unattached cells were removed by phosphate-buffered saline washing, and attached cells were further cultured in media minus growth factors. Twenty-four hours later, DMBA (30 nM) was added. Apoptosis was determined using Cell Death Detection ELISA kit (Roche Molecular Biochemicals, Mannheim, Germany) and proliferation was determined using MTT (3-(4,5]dimethylthiazol-2,5-diphenyltetrazolium bromide) assay (Roche Diagnostics, Indianapolis, IN, USA), and by cell counting (Countless automated counter, Invitrogen).

Figure 8. CD151 affects cPKC localization and function. (a) A431 cells Protein lysate preparation were stimulated with TPA (50 ng/ml) or EGF (5 min and 1 h) and then Cell lines were lysed in 50 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1% Triton lysed in 1% Brij 56 detergent. PKCa and integrin b4 were X-100 and protease inhibitor cocktail. Excised mouse dorsal skin (2 Â 2 immunoprecipitated (IP), followed by immunoblotting (IB) with cm2) was placed on ice-cold glass, epidermis was removed with a blade, antibodies to b4 and PKCa as indicated. WCL, whole cell lysates. and then tissue was lysed in buffer containing 50 mM Tris–HCl (pH 8.6), 1% *Numbers below the right panel represent mean density values for NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM pheny- PKCa (associated with b4) from three independent experiments, methylsulfonyl ﬂuoride and protease inhibitor cocktail (Roche Diagnostics, including the one shown in the top right panel. (b) A431 cells were Indianapolis, IN). After 10 min at 4 1C, lysates were snap-frozen in liquid stimulated with cPKC inhibitor Go 6976 for 2 h, and then cells were nitrogen, thawed and centrifuged (14,000g, 15 min, 4 1C). Supernatant was lysed and blotted for pSTAT3, total STAT3, total PKC and CD151. KD, resolved on 8–12% SDS/polyacrylamide gels, prior to immunoblotting. knockdown; WT, wild type. Immunoﬂuorescence microscopy For confocal analyses, cells on coverslips were stained with primary, then secondary antibody (Alexa 488 or Alexa 594–conjugated goat anti-mouse or anti-rat) alone or combined (Invitrogen). Cells were visualized using Leica SP5X laser-scanning confocal microscope (Leica Microsystems, Chicago, IL, USA).

CONFLICT OF INTEREST The authors declare no conﬂict of interest.

ACKNOWLEDGEMENTS We thank Dr R Bronson for assistance with mouse histopathology, Dr Julie Aldridge for assistance with statistical analyses, and Drs David Frank, Sarah Walker and coworkers for nifuroxazide, ST3–01, assistance with STAT3 functional assay and helpful discussions. This work was supported by NIH grant CA42368 (to MEH), and a SG Komen Career Catalyst Award (to XHY).

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