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  • Loss of integrin α3 prevents skin tumor formation by promoting epidermal turnover and depletion of slow-cycling cells Norman 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 is correlated 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 that efficient 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 to a loss of DMBA-initiated label-retaining keratinocytes. Lineage trac- ing revealed emigration of α3-deficient keratinocytes residing in the bulge of the hair follicle toward the interfollicular epidermis. Furthermore, tumor growth and cell proliferation were strongly reduced in mice with an epidermis-specific deletion of Itga3. How- ever, the rate of progression of α3β1-null squamous cell carcinomas to undifferentiated, invasive carcinomas was increased. Therefore, α3β1 critically affects skin carcinogenesis with opposing effects early 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. Although early detection and surgical resection can prevent most complica- tions associated with this disease, SCCs frequently metastasize and then cannot be effectively treated. Understanding the molecular basis of skin tumorigenesis is a prerequisite for future prevention and therapy. The well-characterized 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) protocol models the multistep nature of human skin carcinogenesis in mice. Oncogenic mutations (e.g., Hras), induced by a single treatment with 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. Subsequent progression to SCCs involves mutation of Trp53 and trisomization of chromosomes 6 and 7 (1–5). Integrins are αβ heterodimeric adhesion receptors that play an

    important role in maintaining epithelial integrity. In the skin, the major integrins α2β1, α3β1, and α6β4 connect the cytoskeleton of basal keratinocytes to the underlying basement membrane (6). Besides their key function in skin physiology, these integrins also have been implicated in the development and progression of SCCs (7). Mouse models in which different integrins are either overexpressed in the suprabasal epidermis or mutated in the whole animal showed altered susceptibilities to chemically in- duced skin tumorigenesis (8–10). Increased expression of α2β1, α3β1, and α6β4 has been observed in hyperproliferating human cancers of the head and neck (11). Integrins thus seem to play a role in initiation and promotion of tumors. Surprisingly, the role of α3β1 in basal keratinocytes in skin tumorigenesis has not been investigated experimentally. To study its contribution to initiation, growth, and malignant progression of skin tumors, we subjected epidermis-specific Itga3 KO mice (Itga3 eKO) to chemically induced skin carcinogenesis.

    Results Reduced Two-Stage Skin Carcinogenesis in the Absence of Itga3. Skin carcinogenesis 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 once with DMBA and promoted twice weekly with TPA (1) (Fig. 1A). Whereas WT mice readily developed tumors, Itga3 eKO mice showed a significant reduction in tumor number and volume (Fig. 1B; Fig. S1). The percentage of mice bearing tumors with a diameter of more than 1 mm was the same in the two groups, whereas tumors larger than 3 mm in diameter occurred almost exclusively in WT mice (Fig. 2A). Ki67 labeling showed reduced tumor cell proliferation in the DMBA/TPA-treated Itga3 eKO mice compared with that in WT littermates (Fig. 2B). Histo- logical analysis revealed that most of the tumors formed in WT and Itga3 eKO mice were benign papillomas (>99%) and kera- toacanthomas (Fig. 2C). As expected, the majority of papillomas tested carried the activating c.182A > T mutation in codon 61 of the Hras1 proto-oncogene (Fig. S2) (3). Malignant tumors such as SCCs or spindle cell carcinomas occurred at a frequency of

  • No difference was observed immediately after BrdU injection (Fig. S5A). Although the number of LRCs in the IFE did not differ significantly between the two groups, LRCs were regularly found 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 mitotically active (Fig. 3B). Virtually no LRCs were found in whole-mounted HFs of Itga3 eKO mouse tails after a single dose of TPA (Fig. 3C). Based on these results, we hypothesized that epidermal turnover may be increased in the absence of Itga3 and found this 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 in Itga3 eKO and WT mice regardless of whether short-term DMBA/TPA treatments were applied (Fig. 3F). The number of

    apoptotic cells 24 h after DMBA was negligible and did not differ 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 Itga3 eKO and WT mice on a C57BL/6 background semiweekly with TPA for 5 mo. Hair growth and HF density were diminished in Itga3 eKO mice (Fig. S4C).

    Lineage Tracing of Keratin 15–Positive Cells. Immunofluorescent analysis of Itga3 eKO skin revealed the presence of a substantial number of keratin 15–positive (Krt15+) keratinocytes in the in- fundibulum and IFE, whereas other HF markers were normally expressed (Fig. 4A; Fig. S6A). TPA treatment increased this number and caused a temporary efflux of Krt15+ keratinocytes from the HFs of WT littermates (Fig. 4B). In line with our previous 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 α3 in the epidermis of Itga3 eKO mice, timeline of the applied two-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 in Itga3 eKO mice compared with those in WT littermates after DMBA/TPA-induced skin carcinogenesis (*P < 0.05, **P < 0.01, ***P < 0.001).

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

    Sachs et al. PNAS | December 26, 2012 | vol. 109 | no. 52 | 21469

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  • among the Krt15+ IFE cell population (Fig. 4C). Microarray analysis showed identical Krt15 mRNA levels in the IFE of WT and 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 observed during wound healing (Fig. S8) (17, 18). Because adolescent Itga3 eKO mice occasionally display microblisters in the IFE, the loss 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 a flu