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Tumor-Initiating Stem Cells of Squamous Cell Carcinomas and Their Control by TGF-Β and Integrin/Focal Adhesion Kinase (FAK) Signaling

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Tumor-Initiating Stem Cells of Squamous Cell Carcinomas and Their Control by TGF-Β and Integrin/Focal Adhesion Kinase (FAK) Signaling Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-β and integrin/focal adhesion kinase (FAK) signaling Markus Schobera and Elaine Fuchsa,b,1 aLaboratory of Mammalian Cell Biology and Development and bThe Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065 Contributed by Elaine Fuchs, May 17, 2011 (sent for review May 2, 2011) Cancer stem cells (CSCs) sustain tumor growth through their ability of 7,12-dimethylbenz[α]anthracene (DMBA) and 12-o-tetrade- to self-renew and to generate differentiated progeny. These func- canoylphorbol-13 acetate (TPA) is a well-established chemical tions endow CSCs with the potential to initiate secondary tumors carcinogen treatment that leads primarily to papillomas in the bearing characteristics similar to those of the parent. Recently the skin. Recently, it was shown that, like hair follicles (HFs), tumors hair follicle stem cell marker CD34 was used to purify a CSC-like cell formed by this chemical regimen contain a small population of population from early skin tumors arising from treatment with 7,12- cells that express the cell-surface glycoprotein CD34, a marker – dimethylbenz[α]anthracene/12-o-tetradecanoylphorbol-13-acetate, expressed by a variety of adult SCs. In a 500 50,000 cell serial + fi which typically generates benign papillomas that occasionally prog- transplant assay, CD34 cells puri ed from these tumors were ress to squamous cell carcinomas (SCCs). In the present study, we shown to possess increased tumor-initiating ability compared with identify and characterize CSCs purified from malignant SCCs. We unfractionated tumor cells (7). The extent to which CD34 defines CSCs is currently unknown. Also poorly understood are show that SCCs contain two highly tumorigenic CSC populations that – differ in CD34 levels but are enriched for integrins and coexist at how CSCs self-renew, how they differentiate into non tumor- initiating progeny in cutaneous SCC, and how they compare with the SCC–stroma interface. Intriguingly, whether CD34lo or CD34hi, stem cells and progenitor cells in normal tissue. These questions α6hiβ1hi populations can initiate secondary tumors by serial limit- α loβ lo are pivotal to address for the development of therapies. dilution transplantation assays, but 6 1 populations cannot. The TGF-β pathway is commonly deregulated in human can- Moreover, secondary tumors generated from a single CSC of either cers, including SCCs, where TGF-β functions initially as a tumor lo hi α hiβ hi subtype contain both CD34 and CD34 6 1 CSCs, indicating suppressor but promotes metastasis in late-stage carcinogenesis fi their nonhierarchical organization. Genomic pro ling and hierarchi- (8, 9). TGF-β receptor II (TβRII) is an essential component of cal cluster analysis show that these two CSC subtypes share a molec- β − the TGF- pathway, and its conditional ablation in skin epithe- ular signature distinct from either the CD34 epidermal or the lium (TβRIIKO) accelerates the development of aggressive SCCs hi CD34 hair follicle stem cell signature. Although closely related, upon exposure to the chemical carcinogen DMBA (9). Con- hi hi lo hi hi hi α6 β1 CD34 and α6 β1 CD34 CSCs differ in cell-cycle gene ex- comitant with TβRII loss in keratinocytes is the hyperactivation pression and proliferation characteristics. Indeed, proliferation and of integrins and the integrin signal transducer focal adhesion expansion of α6hiβ1hiCD34hi CSCs is sensitive to whether they can kinase (FAK) (9), features that promote cell proliferation, cell initiate a TGF-β receptor II–mediated response to counterbalance survival, and carcinogenesis (10–13). Indeed, integrins and elevated focal adhesion kinase-mediated integrin signaling within FAK are commonly up-regulated and are critical for the de- the tumor. Overall, the coexistence and interconvertibility of CSCs velopment of mouse and human solid tumors, including SCCs with differing sensitivities to their microenvironment pose chal- (10–15). The potent effects of TGF-β/TβRII and integrin/FAK lenges and opportunities for SCC cancer therapies. signaling on SCC formation are particularly intriguing, given that normal stem cells (SCs) of epidermis and HFs are responsive to cancer stem cell signature | epithelial–mesenchymal interactions | TGF-β signaling and display elevated integrin levels relative to skin cancer their committed progeny (16–19). These features provide an ideal platform for exploring the consequences of perturbing these pathways on the characteristics of SCC tumors and their ancers develop when cells acquire mutations in tumor- associated CSCs. Csuppressor genes and proto-oncogenes that favor growth- promoting over growth-restricting processes, thereby unbalanc- Results ing tissue homeostasis (1, 2). Indeed, cancer cells are generally β fi proliferative, refractory to apoptotic cell death, and deficient in FAK Function Is Critical for SCC Tumor Susceptibility in T RII-De cient normal cellular differentiation. However, solid tumors such as Mice. Mice lacking FAK are more refractory to SCC formation, β cutaneous squamous cell carcinomas (SCCs) are not simply whereas those lacking T RII show enhanced tumor susceptibility. cancer cell clones but rather are complex structures composed of To investigate whether FAK/integrin signaling is critical for the β KO multiple cell types in unique microenvironments (3). How tumor development of T RII SCCs, we generated mice whose skin ep- β architecture develops and how it is maintained over time is still ithelium was conditionally null for both T RII and FAK (dKO). As β KO KO poorly understood for most cancers. Integral to these issues is were T RII (9) and FAK (20), dKO skins were asymptomatic. whether deregulated proto-oncogenes and tumor-suppressor genes affect all cancer cells equally or perform specific functions within distinct cellular compartments of the tumor. Of particular Author contributions: M.S. and E.F. designed research; M.S. performed research; M.S. importance is how these mutations affect those cancer cells that analyzed data; and M.S. and E.F. wrote the paper. ensure long-term growth and survival of the tumor. The authors declare no conflict of interest. Cancer stem cells (CSCs) sustain tumor growth through their Freely available online through the PNAS open access option. ability to self-renew and differentiate into hierarchically orga- Data deposition: The data discussed in this article have been deposited in the Gene nized cancerous tissue (4, 5). These functions endow CSCs with Expression Omnibus (GEO) database (accession no. GSE29328)(http://www.ncbi.nlm.nih. the potential to initiate secondary tumors bearing characteristics gov/geo/query/acc.cgi?acc=GSE29328). similar to those of the parent. In SCCs, actively proliferating 1To whom correspondence should be addressed. E-mail: [email protected]. cancer cells reside at the tumor–stroma interface and differen- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tiate into nontumorigenic pearls in the tumor center (6). The use 1073/pnas.1107807108/-/DCSupplemental. 10544–10549 | PNAS | June 28, 2011 | vol. 108 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1107807108 Downloaded by guest on September 27, 2021 However, complete carcinogenesis by topical DMBA treatments occasional newly developing tumors regressed in both control and (two times per week) induced cutaneous SCCs in all genotypes, FAKKO mice, suggesting either that they failed to generate CSCs with some SCCs well contained but invasive and others less differ- for sustained long-term growth or that CSC self-renewal had been entiated and very invasive. SCC initiation appeared sooner in restricted by elevated suppressive activities within benign tumors TβRIIKO mice and later in FAKKO mice than in their wild-type lit- (Fig. 1C) (2). By contrast, tumor regression was not observed in termates. Interestingly, the accelerated tumor initiation in TβRIIKO TβRIIKO mice and was rare in dKO animals. Together, these data β β mice was not seen in dKO mice, which developed SCCs at rates suggest that T RII/TGF- and integrin/FAK signaling interact in indistinguishable from those of wild-type littermates (Fig. 1A). controlling not only tumor initiation and growth but also the fre- Once initiated, TβRIIKO SCCs grew faster than control SCCs quency with which benign tumors regress, persist, or progress to (Fig. 1B). Additionally, although all SCCs executed a program malignant SCCs. resembling disorganized epidermal wound repair, TβRIIKO SCCs were the most poorly differentiated (Fig. S1). Such signs are Fractionating SCC Populations by Their Surface CD34 and Integrins typical of highly aggressive SCCs (11, 14). and Functionally Testing Them for Self-Renewing Capacity in Vitro. FAK function appeared to be critical for the accelerated growth For the present study, we focused on tumors that progressed to of TβRIIKO SCCs, because growth rates in dKO SCCs were com- SCCs in each of the four genetic backgrounds. Based on the no- parable to those of FAKKO and control SCCs (Fig. 1B). Moreover, tion that CSCs should reside within the relatively undifferentiated keratin 5(K5)/ keratin 14+ proliferative cells at the tumor–stroma interface (6), we posited that CSCs of SCCs should display abundant integrins. Indeed, all SCC cells located at the tumor– A B C stroma interface expressed high levels of the hemidesmosomal α6 and β4 integrins and the focal adhesion marker β1 integrin, but 100 WT 15 40 only a fraction of these were CD34+ (Fig. 1D and Fig. S2). When 80 TβRII dKO 30 coupled with the genotype-specific differences in SCC charac- 60 FAK 10 teristics, these spatial differences in the intensity of CD34 and 20 – 40 integrin staining at the tumor stroma interface were suggestive of 5 fl β (N=28) a heterogeneity that might be in uenced by T RII and/or FAK- (N=87) (N=92) 10 (N=109) 20 functions. 0 0 0 Tumor regression (%) To place this heterogeneity in the context of proliferative po- Tumor free mice % 0 10 20 30 Tumor diameter (mm) 1234 56 WT tential, we fractionated these cancer cells from genotypically weeks after DMBA weeks dKOFAK CELL BIOLOGY TβRII distinct primary SCCs by FACS.
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