Loss of integrin α3 prevents skin tumor formation bypromoting epidermal turnover and depletion ofslow-cycling cellsNorman Sachsa,1,2, Pablo Secadesa,1, Laura van Hulsta, Maaike Krefta, Ji-Ying Songb, and Arnoud Sonnenberga,3

aDivision of Cell Biology and bDepartment of Experimental Animal Pathology, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands

Edited* by Richard O. Hynes, Massachusetts Institute of Technology, Cambridge, MA, and approved November 15, 2012 (received for review March 19, 2012)

Progression through the various stages of skin tumorigenesis iscorrelated with an altered expression of the integrin α3β1, sug-gesting that it plays an important role in the tumorigenic process.Using epidermis-specific Itga3 KO mice subjected to the 7,12-dime-thylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-ace-tate two-stage skin carcinogenesis protocol, we demonstrate thatefficient tumor development is critically dependent on the pres-ence of α3β1. In the absence of α3β1, tumor initiation is dramati-cally decreased because of increased epidermal turnover, leading toa loss of DMBA-initiated label-retaining keratinocytes. Lineage trac-ing revealed emigration of α3-deficient keratinocytes residing inthe bulge of the hair follicle toward the interfollicular epidermis.Furthermore, tumor growth and cell proliferation were stronglyreduced in mice with an epidermis-specific deletion of Itga3. How-ever, the rate of progression of α3β1-null squamous cell carcinomasto undifferentiated, invasive carcinomas was increased. Therefore,α3β1 critically affects skin carcinogenesis with opposing effectsearly and late in tumorigenesis.

skin cancer | cell adhesion | cell migration | laminin receptor | hair cycling

Skin cancer is the most common form of cancer among whitepopulations, with basal cell carcinomas and squamous cell

carcinomas (SSCs) being the most common subtypes. Althoughearly detection and surgical resection can prevent most complica-tions associated with this disease, SCCs frequently metastasize andthen cannot be effectively treated. Understanding the molecularbasis of skin tumorigenesis is a prerequisite for future preventionand therapy. The well-characterized 7,12-dimethylbenzanthracene(DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) protocolmodels the multistep nature of human skin carcinogenesis in mice.Oncogenic mutations (e.g., Hras), induced by a single treatmentwith the carcinogen DMBA confer growth advantage to the ini-tiated cells, which form benign papillomas under repetitive tumor-promoting treatments with the phorbol ester TPA. Subsequentprogression to SCCs involves mutation of Trp53 and trisomizationof chromosomes 6 and 7 (1–5).Integrins are αβ heterodimeric adhesion receptors that play an

important role in maintaining epithelial integrity. In the skin, themajor integrins α2β1, α3β1, and α6β4 connect the cytoskeleton ofbasal keratinocytes to the underlying basement membrane (6).Besides their key function in skin physiology, these integrins alsohave been implicated in the development and progression ofSCCs (7). Mouse models in which different integrins are eitheroverexpressed in the suprabasal epidermis or mutated in thewhole animal showed altered susceptibilities to chemically in-duced skin tumorigenesis (8–10). Increased expression of α2β1,α3β1, and α6β4 has been observed in hyperproliferating humancancers of the head and neck (11). Integrins thus seem to playa role in initiation and promotion of tumors. Surprisingly, therole of α3β1 in basal keratinocytes in skin tumorigenesis has notbeen investigated experimentally. To study its contribution toinitiation, growth, and malignant progression of skin tumors,we subjected epidermis-specific Itga3 KO mice (Itga3 eKO) tochemically induced skin carcinogenesis.

ResultsReduced Two-Stage Skin Carcinogenesis in the Absence of Itga3. Skincarcinogenesis of epidermis-specific Itga3 KO mice (Itga3fl/fl;K14-Cre+, referred to as Itga3 eKO) and Cre-negative litter-mates (Itga3fl/fl; K14-Cre−, referred to as WT) was induced oncewith DMBA and promoted twice weekly with TPA (1) (Fig. 1A).Whereas WT mice readily developed tumors, Itga3 eKO miceshowed a significant reduction in tumor number and volume(Fig. 1B; Fig. S1). The percentage of mice bearing tumors witha diameter of more than 1 mm was the same in the two groups,whereas tumors larger than 3 mm in diameter occurred almostexclusively in WT mice (Fig. 2A). Ki67 labeling showed reducedtumor cell proliferation in the DMBA/TPA-treated Itga3 eKOmice compared with that in WT littermates (Fig. 2B). Histo-logical analysis revealed that most of the tumors formed in WTand Itga3 eKO mice were benign papillomas (>99%) and kera-toacanthomas (Fig. 2C). As expected, the majority of papillomastested carried the activating c.182A > T mutation in codon 61of the Hras1 proto-oncogene (Fig. S2) (3). Malignant tumorssuch as SCCs or spindle cell carcinomas occurred at a frequencyof <1% and were restricted to WT mice (Fig. S3). Malignantconversion in the Itga3 eKO group may have remained undetectedbecause of the low overall total tumor number (<100). Togetherthese data show that Itga3 eKO mice rarely develop tumors inresponse to DMBA/TPA, and if formed, proliferation is decreased.

Exit of Label-Retaining Cells from Their Niches and IncreasedEpidermal Turnover in Itga3 eKO Mice. The efficacy of chemicallyinduced skin carcinogenesis is critically dependent on the haircycle phase in which the mice are treated with DMBA (12). Wefound no difference in the duration of the first two synchronizedhair cycles between WT and Itga3 eKO mice. (Fig. S4 A and B).A second hypothesis that could explain the difference in tumordevelopment concerns the fate of slow-cycling, label-retainingcells (LRCs) of hair follicles (HFs) and interfollicular epidermis(IFE). These cells are thought to be the primary source ofchemically induced skin tumors, as DMBA-initiated cells persistand can efficiently advance to tumors even after extended peri-ods of time (13, 14). LRCs have indeed been shown to retainmutagenic DMBA-DNA adducts (15) and proliferate on TPAtreatment (16). Using a BrdU label retention assay, we found thenumber of BrdU-positive LRCs to be strongly reduced in theHFs of Itga3 eKOmice compared with that in WT mice (Fig. 3A).

Author contributions: N.S., P.S., and A.S. designed research; N.S., P.S., L.v.H., and M.K.performed research; N.S., P.S., J.Y.S., and A.S. analyzed data; and N.S. and A.S. wrotethe paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1N.S. and P.S. contributed equally to this work.2Present address: Hubrecht Institute for Developmental Biology and Stem Cell Research(KNAW), 3584 CT Utrecht, The Netherlands.

3To whom correspondence should be addressed. E-mail: a.sonnenberg@nki.nl.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1204614110/-/DCSupplemental.

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No difference was observed immediately after BrdU injection(Fig. S5A). Although the number of LRCs in the IFE did notdiffer significantly between the two groups, LRCs were regularlyfound suprabasally in Itga3 eKO mice en route to terminal dif-ferentiation (Fig. 3A; Fig. S5A). Using double-label pulse-chase(5-ethynyl-2-deoxyuridine (EdU), 4 wk; BrdU, 2 h) experiments,we also showed that several LRCs in the IFE were mitoticallyactive (Fig. 3B). Virtually no LRCs were found in whole-mountedHFs of Itga3 eKO mouse tails after a single dose of TPA (Fig.3C). Based on these results, we hypothesized that epidermalturnover may be increased in the absence of Itga3 and foundthis indeed to be the case for the basal proliferative layer (14-d BrdU pulse-chase; Fig. 3D; Fig. S5B), as well as the upper-most cornified layer (dansyl chloride painting; Fig. 3E; Fig. S5C).Interfollicular epidermal width and proliferation were equal inItga3 eKO and WT mice regardless of whether short-termDMBA/TPA treatments were applied (Fig. 3F). The number of

apoptotic cells 24 h after DMBA was negligible and did notdiffer significantly between the two groups (Fig. 3F). In contrast,follicular proliferation was increased in the absence of α3 (Fig.3G). To determine whether loss of LRCs has functional con-sequences on HF homeostasis, we treated the back skin of Itga3eKO and WT mice on a C57BL/6 background semiweekly withTPA for 5 mo. Hair growth and HF density were diminished inItga3 eKO mice (Fig. S4C).

Lineage Tracing of Keratin 15–Positive Cells. Immunofluorescentanalysis of Itga3 eKO skin revealed the presence of a substantialnumber of keratin 15–positive (Krt15+) keratinocytes in the in-fundibulum and IFE, whereas other HF markers were normallyexpressed (Fig. 4A; Fig. S6A). TPA treatment increased thisnumber and caused a temporary efflux of Krt15+ keratinocytesfrom the HFs of WT littermates (Fig. 4B). In line with ourprevious data, we also found LRCs and transit amplifying cells

Fig. 1. Impaired skin carcinogenesis of Itga3 eKO mice. (A)From left to right: Western blot showing the absence of α3in the epidermis of Itga3 eKO mice, timeline of the appliedtwo-stage skin carcinogenesis protocol, and macroscopic im-age of three littermates at the end of the experiment. (B)Tumor number and volume are significantly diminished inItga3 eKO mice compared with those in WT littermates afterDMBA/TPA-induced skin carcinogenesis (*P < 0.05, **P < 0.01,***P < 0.001).

Fig. 2. Incidence and histology of benign tumors. (A) WTand Itga3 eKO mice develop at least one tumor with a di-ameter larger than 1 mm. In contrast, tumors with a diameterlarger than 3 mm occur in virtually all WT but only in 25% ofItga3 eKO mice. (B) Proliferation is significantly diminished inpapillomas of DMBA/TPA-treated Itga3 eKO mice comparedwith those of WT littermates. (Scale bars, 100 μm.) (Insetmagnification, 5×; *P < 0.05.) (C) Histology of representativepapillomas and keratoacanthomas. Tumors are histologicallysimilar but are larger in WT mice than in Itga3 eKO mice.(Scale bars, 500 μm.)

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among the Krt15+ IFE cell population (Fig. 4C). Microarrayanalysis showed identical Krt15 mRNA levels in the IFE of WTand Itga3 eKO mice (Fig. S7), making it unlikely that IFE ker-atinocytes expressed Krt15 de novo. The presence of Krt15+

keratinocytes in the adult IFE has thus far only been observedduring wound healing (Fig. S8) (17, 18). Because adolescentItga3 eKO mice occasionally display microblisters in the IFE, theloss of LRCs might be the result of a wound healing response(19, 20). To test this, we specifically deleted Itga3 in Krt15+

keratinocytes of telogen HFs and traced their progeny using afluorescent Cre-based reporter system (21, 22). As shown in Fig.4D, 10 d after Cre expression was induced in Krt15+ keratino-cytes with the progesterone analog RU486, GFP+ cells werefound exclusively in HFs of Itga3+/+; mTmG+/−; Krt1-15-CrePR1+mice. In contrast, in similarly treated Itga3fl/fl; mTmG+/−; Krt1-15-CrePR1+ littermates, GFP+ cells were not only present in HFsbut also in the IFE. Keratinocytes in the bulge of HFs lackingα3β1 can thus exit their niche and contribute to epidermal dif-ferentiation. Even though these cells remain positive for keratin15 in the IFE, de novo expression of IFE-keratins 1, 5, and 10and loss of HF-keratin 6 suggest that normal differentiation in-deed occurs (Fig. S6B). In the context of skin carcinogenesis, itseems thus plausible that DMBA-initiated cells lacking α3β1 exittheir compartment and terminally differentiate before they canacquire additional mutations that would lead to the onset of cancer.

α3β1 Affects Migration, Adhesion, and Proliferation of (Transformed)Keratinocytes. To directly assess the role of α3β1 in keratinocytemigration, we seeded normal mouse keratinocytes (MKs) ± Itga3sparsely on laminin-332 and observed that the Itga3-null kerati-nocytes migrated faster throughout the experiment, with a tem-porary increase in the migration speed in response to TPA (Fig.5A). As previously suggested (20), we noted a decrease in adhe-sion strength of keratinocytes lacking α3 (Fig. 5A). We confirmed

the negative effect of α3β1 on migration in the precancerouspapilloma cell line P1. On stable knockdown of Itga3 (Fig. S9),P1 cells migrated more efficiently through matrigel-coated filters(Fig. 5B). In addition, P1 cells lacking α3 formed smaller sphe-roids when grown in 3D matrigel (Fig. 5C). Similarly, mousesquamous carcinoma cells (MSCCs) lacking Itga3 generated 40%smaller tumors upon s.c. injection in mice than WT parental cells(Fig. 5D; Fig. S10F). As shown earlier, proliferation of un-transformed keratinocytes is not affected by the absence of α3(20) (Fig. 3F), indicating that only transformed keratinocytesrequire the presence of α3β1 for efficient proliferation.

Loss of α3 Potentiates Tumor Progression. Because the incidenceof tumor progression to SCCs after DMBA/TPA treatmentwas too low to deduce a function of α3β1, we subjected WT andItga3 eKO mice to the complete carcinogenesis protocol of weeklyDMBA applications. As in the DMBA/TPA model, we observedsignificantly fewer tumors in Itga3 eKO mice than in WT litter-mates (Fig. 6A), with the great majority of tumors being histolog-ically classified as SCCs in both groups. However, the malignancygrade of tumors arising in Itga3 eKO mice was significantlyhigher (Fig. 6 B and C; Fig. S11). In cell lines representative ofseveral stages of skin carcinogenesis (23, 24), we found α3 pro-tein levels to be inversely correlated with the progression stage(Fig. 6D). Of note, in these cell lines, α3 was a more preciseindicator of the malignancy grade than E-cadherin (Fig. 6D).SCCs of WT mice remained well differentiated until they reacheda diameter of 5.0 ± 0.78 mm, whereas well-differentiated SCCsof Itga3 eKO mice only reached 2.1 ± 0.05 mm (P < 0.003,Student t test) before progressing. Dedifferentiation in the ab-sence of α3 thus occurs fairly early during tumorigenesis. Ourfindings indicate a requirement for α3β1 in tumor initiation andearly growth, whereas loss of α3β1 enhances progression of SCCsat later stages.

Fig. 3. Loss of label-retaining cells and increased epider-mal turnover in Itga3 eKO mice. (A) The number of BrdU-LRCs is significantly reduced in back skin HFs of 8- and 14-wk-old Itga3 eKO mice compared with that in HFs of WTmice. LRCs occur almost twice less frequently in the IFE ofItga3 eKO mice (*P < 0.05). (B) LRCs are present in the IFE ofItga3 eKO mice and can proliferate (arrowheads) [non-specific staining in the sebaceous glands (SGs)]. (Scale bar,50 μm.) (C) Whole mounts of tail epidermis 24 h after asingle application of TPA. Whereas a large number of LRCsare still present in the HF bulge of a WT mouse, very fewLRCs remain in the HF of an Itga3 eKO littermate (non-specific staining in SG). (Scale bars, 50 μm.) (D and E) In-creased epidermal turnover and desquamation in Itga3eKO mice is shown by fewer BrdU+ cells in the Itga3-null IFE14 d after pulse and the accelerated loss of dansyl chloridefrom the epidermis of Itga3 eKO mice, respectively (*P <0.05, **P < 0.01, ***P < 0.001). (Scale bar, 20 μm.) (F) Short-term treatment of mouse back skin with TPA induceshyperproliferation, which does not significantly differ be-tween Itga3 eKO mice and WT littermates. DMBA treat-ment causes apoptosis in very few cells of the IFE of WT andItga3 eKO mice. Treatments were a single dose of TPA,effect analyzed 24 h later; a single dose of DMBA followedby four doses of TPA spread evenly over 14 d, effect ana-lyzed 24 h later; the respective vehicle controls (acetone);treated and analyzed in parallel; a single dose of DMBA,number of apoptotic cells (active caspase-3 IHC) analyzed24 h later. (G) Increased proliferation in HFs of Itga3 eKOcompared with that in HFs of WT mice (*P < 0.05). (Scalebar, 50 μm.)

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DiscussionIn this study, we investigated the role of α3β1 in a two-stage skincarcinogenesis model and show that mice lacking the epidermalintegrin α3β1 are considerably less susceptible to skin carcinogenesisthan WT mice. Whereas all mice developed benign tumors (papil-lomas and keratoacanthomas), the total number of tumors formedin the Itga3 eKOmice was significantly decreased. Furthermore, thefew tumors formed in Itga3 eKOmice weremuch smaller than thoseformed in WT mice. These results suggest a role of α3β1 in bothtumor initiation and proliferation of transformed cells.

As an underlying cause for the effect of α3β1 on tumor initi-ation, we excluded altered hair cycling in Itga3 eKO mice andsubsequently focused on the fate of slow-cycling LRCs as theproposed target of DMBA and on the cell of origin of thetumors. In the absence of epidermal α3β1, the number of LRCswas significantly reduced, and LRCs were regularly foundsuprabasally. After short-term treatment (48 h) with TPA, LRCswere virtually absent in Itga3 eKO mice. We therefore suggestthat the DMBA-initiated cells lacking α3β1 leave their com-partment and terminally differentiate, reducing the probability toaccumulate the necessary mutations required for tumor onset.Indeed, we found many Krt15+ keratinocytes outside their nor-mal niche (the HF bulge) in Itga3 eKO mice, as shown by lineagetracing experiments of α3-deficient Krt15+ HF keratinocytes.The increased mobility and subsequent loss of Krt15+ label-retaining HF cells lacking α3 led to a decrease in HF density withage, which was aggravated after long-term TPA treatment. No-tably, TPA was able to induce Krt15+ cell efflux from HFs in WTmice, possibly because of hemidesmosome instability followingphosphorylation of the β4 subunit of integrin α6β4 leading todecreased adhesion strength and increased migration (25, 26).TPA indeed temporarily increased the velocity of WT kerati-nocytes in vitro. In the absence of α3β1, both the efflux of Krt15+

cells in vivo and their migration speed in vitro were increasedunder all conditions. In line with previously published data, weprovide evidence that the increased migration speed of Itga3 KOkeratinocytes is caused by decreased adhesion strength of thesecells to the underlying matrix (20). Together these findings em-phasize the importance of α3β1 in epidermal turnover and the long-term maintenance of Krt15+ LRCs keratinocytes in the HF bulge.The fact that efficient formation of large tumors depends on

α3β1 indicates that the integrin confers a proliferative growthadvantage to transformed keratinocytes. Indeed, Itga3 KO pap-illomas contained significantly fewer proliferating Ki67+ kerati-nocytes than WT papillomas. Furthermore, we found that SCCslacking α3β1 give rise to significantly smaller tumors when injec-ted s.c. into nude mice. The positive influence of α3β1 on tumorgrowth is most likely direct, because efficient spheroid formationof pure papilloma cell populations in vitro critically depends onthe presence of α3β1. A similar effect on proliferation of skintumors has been observed in β1 mutant mice (8). A role of α3β1in supporting cell proliferation has already been suggested inprevious reports. For example, it was shown that the expressionof α3β1 in suprabasal keratinocytes increases during hyper-proliferative pathological conditions (6, 11). Furthermore, engi-neered suprabasal expression of α3β1 in mice increased BrdUincorporation on TPA treatment (9), whereas deletion of α3β1from transformed keratinocytes caused reduced tumor growth(27). Although the exact mechanism responsible for the contri-bution of α3β1 to tumor cell proliferation remains to be deter-mined, the importance of integrins and their associated signalingpathways in skin carcinogenesis is further demonstrated by thefact that FAK and Src, two kinases that are activated downstreamof integrin engagement, critically contribute to experimentalmouseskin carcinogenesis (8, 28). Another example of transformedkeratinocytes being critically dependent on integrin signaling isthat the laminin-binding integrin α6β4 confers proliferative ad-vantage to Ras-transformed keratinocytes (29), whereas the lackof β4 renders Ras-IκBα–transformed keratinocytes resistant totumor formation (30).Unfortunately we could not deduce a function of α3β1 during

tumor progression from the results of the initial DMBA/TPAexperiment because the rate of progression to malignant SCCswas too low. We were, however, able to investigate the role ofα3β1 in the development and progression of SCCs using the fullcarcinogenesis protocol of DMBA-only treatments. Tumor numberwas again significantly reduced in Itga3 eKO mice, which we attri-bute to a loss ofDMBA-initiated LRCs. The fact that the differencein the number of tumors is less pronounced than after DMBA/TPAtreatments is consistent with the finding that the efflux of Krt15+

Fig. 4. Keratin 15–positive cells from the HF bulge exit their niche in theabsence of Itga3. (A) HF and IFE of whole-mounted Itga3-null tails are Krt15+,whereas this cell population is confined to the HF bulge in WT littermates(Ist., isthmus). (Scale bar, 100 μm.) (B) Keratin 15 stainings of whole-mountedtail HFs at indicated time points after single TPA doses. Twenty-four hoursafter TPA, Krt15+ cells have migrated out of the bulge into the isthmus ofWT HFs. In Itga3 eKO mice, basal efflux of Krt15+ cells increases further onTPA stimulation. (Scale bar, 50 μm.) (C) (Left) EdU+ LRC in IFE is Krt15+.(Center and Right) Forty-eight hours after pulse, EdU+ Krt15+ cells arepresent in the isthmus and IFE. (Scale bar, 50 μm.) (D) HF bulge cells from theback skin of Itga3fl/fl; mTmG+/−; K1-15-CrePR1+ mice leave their compart-ment (asterisk) and migrate into the IFE (arrowheads). They remain in thebulge region (asterisk) in WT littermates. (Scale bar, 100 μm.)

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cells lacking α3β1 following TPA treatment is increased. We foundthat the absence of α3β1 strongly promotes the malignant pro-gression of SCCs. The few well-differentiated Itga3-null SCCswere much smaller than those in WT mice, indicating that de-differentiation occurs at an earlier time point during tumorigenesis.

In cell lines representative of several stages of skin carcinogenesis(23, 24), α3 protein levels are inversely correlated with progressionstage. We postulate that α3β1, as a constitutively expressed basalkeratinocyte integrin, allows transformed keratinocytes in SCCsof WT mice to maintain their relative integrity and permit the

Fig. 5. Absence of α3 affects migration, adhesion, and pro-liferation in vitro. (A) Single cell migration (Left) and adhesionassay (Right) of untransformed MKs on laminin-332. MKslacking α3 migrate faster than their parental cells, whereasboth lines transiently increase their migration speed on TPAtreatment. Adhesion to laminin-332 after 24 h is significantlydiminished in the absence of α3 (Τ50 refers to the force atwhich 50% of cells detached, ***P < 0.001). (B and C) Stableknockdown of Itga3 increased migration of the papilloma cellline P1 through a matrigel-coated Boyden chamber and de-creased spheroid formation in matrigel (cell lines character-ized in Fig. S9; *P < 0.05, ***P < 0.001). (Scale bar, 50 μm.) (D)MSCCs lacking Itga3 (characterized in Fig. S10) develop smallertumors than the parental Itga3fl/fl MSCC line when injecteds.c. into nude mice.

Fig. 6. Loss of Itga3 results in fewer, but less differenti-ated, carcinomas after DMBA tumorigenesis. (A) Fewertumors are formed in Itga3 eKO mice during a 25-wk reg-imen of DMBA-only carcinogenesis compared with WTlittermates (*P < 0.05). (B) Pie chart of the tumor differ-entiation showing a high percentage of poorly differenti-ated Itga3-null carcinomas. The difference in malignancygrades of tumors in WT and Itga3 eKO mice is statisticallysignificant (P < 0.001; χ2 test; see Fig. S11 for details). (C)(Upper) Representative histological examples of well,moderately, and poorly differentiated SCCs found in WTand Itga3 eKO mice after complete carcinogenesis. Withthe exception of larger well-differentiated tumors in WTmice, no differences were observed. (Lower) Histology ofspindle cell and anaplastic carcinomas found in Itga3 eKOmice after a complete carcinogenesis regimen. (Scale bar,100 μm.) (D) Western blot analysis showing high α3 proteinlevels in the papilloma cell line P1, moderate levels in thesquamous carcinoma cell line B9, and virtually no α3 in thespindle carcinoma cell line A5. E-cadherin and β-tubulin areshown as controls.

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formation of larger clusters of more differentiated cells comparedwith SCC keratinocytes of Itga3 eKOmice, whose adhesion to thebasement membrane is destabilized. Altogether, our studiesidentify α3β1 as a critical factor for the initiation and progressionof chemically induced skin tumors.

Materials and MethodsAnimal Experiments. According to Mouse Genome Informatics (JacksonLaboratory), the names of epidermal Itga3 KO mice are Itga3tm1Son/tm1Son;Krt14tm1(cre)Wbm on FVB(N6) or C57BL/6(N10) (31, 32). Krt15+ cell-specificItga3 KO mice carrying the mTmG reporter were Itga3tm1Son/tm1Son; mTmG+/−;Tg(Krt1-15-cre/PGR)22Cot on FVB(N3) (21, 22). For DMBA/TPA-induced carci-nogenesis, the backs of 7-wk-old mice were shaved and treated with a singledose of DMBA (30 μg in 200 μL acetone; Sigma) followed by semiweeklyapplications of TPA (12.34 μg in 200 μL acetone; Sigma) for 20 wk. For DMBA-only carcinogenesis, the backs of 7-wk-old mice were shaved and treated withweekly doses of DMBA (30 μg in 200 μL acetone) for up to 25 wk. For LRCtracing, mice were injected i.p. with 6 × 50 μg BrdU or EdU every 12 h frompostnatal day 3 and chased for up to 14 wk (33). Detailed descriptions of allother animal experiments can be found in SI Materials and Methods.

Cell Lines.All cell lines were grown at 37 °C in a humidified atmosphere of 5%CO2 in air. Migration and adhesion assays of MK Itga3fl/fl and MK Itga3−/− (6)on a laminin-332 rich matrix deposited by Rac11-P cells were performed aspreviously described (20, 34–36). Murine skin cancer cell lines P1, B9, and A5were cultured as described (23, 24) (SI Materials and Methods). Stable Itga3knockdown P1 cells were generated by lentiviral transduction of short RNAhairpins cloned into pLKO.1 vectors (clone 1: TRCN0000065998; clone 2:TRCN0000066002; Thermo Scientific Dharmacon RNAi Technologies) andsorted three times for lack of cell surface α3. Standard matrigel (BD)spheroid formation and Boyden chamber assays were performed as de-scribed in SI Materials and Methods. MSCC Itga3fl/fl were isolated from the

sentinel lymph node of a SCC-bearing Itga3fl/fl mouse, treated with DMBA/TPA by mechanical disruption followed by collagenase digestion, and cul-tured in DMEM containing 10% FCS (Gibco). α3 was deleted using Adeno-Cre (37) to generate MSCC Itga3−/−.

Immunohistochemistry, Immunofluorescence, Immunoblotting, and FACS. Weapplied a standard methodology that can be found in SI Materials andMethods and Table S1.

Supporting Information. SI Materials and Methods includes detailed materialsand methods. Fig. S1 shows most DMBA/TPA-treated WT and Itga3 eKO miceat the end of the carcinogenic protocol. Fig. S2 shows Hras1 status of pap-illomas. Fig. S3 depicts histological representations of progressed tumorsfollowing DMBA/TPA treatment of WT mice. Fig. S4 illustrates the synchro-nized hair cycle of WT and Itga3 eKO mice and the decrease of HF density inthe absence of Itga3 after long-term TPA treatment. Fig. S5 shows histo-logical examples of BrdU pulse-chase and dansyl chloride desquamationexperiments. Fig. S6 shows immunofluorescent analysis of HF markers in WTand Itga3 eKO mice, as well as differentiation markers in GFP+ cells fromItga3fl/fl; mTmG+/−; Krt1-15-CrePR1+ mice. Fig. S7 shows the MA plot ofa microarray comparing the IFE of WT and Itga3 eKO mice. Fig. S8 depictsthe contribution of Krt15+ HF bulge cells to reepithelialization afterwounding. Figs. S9 and S10 show characterization of P1 cells and MSCCs ±Itga3. Fig. S11 lists the detailed number of malignancies after the completecarcinogenesis protocol.

ACKNOWLEDGMENTS. We thank Drs. W. Birchmeier, G. Cotsarelis, and L. Luofor providing mouse strains. We also thank all staff members of the facilitiesfor animal maintenance, histology, digital microscopy, and flow cytometry atThe Netherlands Cancer Institute for excellent technical assistance; and Drs. A.Berns and M. Ports for critically reading the manuscript. This work wassupported by a grant from the Dutch Cancer Society.

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