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Tumor suppressor identity can contribute to heterogeneity of phenotype in hair follicle stem cell‐induced squamous cell carcinoma

Experimental Dermatology2016Vol. 25(9), pp. 733–735

Citations Over TimeTop 13% of 2016 papers

Aimee Flores, William D. Grant, Andrew C. White, Phillip Scumpia, Rie Takahashi, William E. Lowry

Abstract

Pathological examination of cutaneous squamous cell carcinoma (cSCC) can identify up to 12 distinct features of this disease 1. An outstanding question in the field is how so many different flavours of cSCC can arise. Direct sequencing of bulk tumors has uncovered generally 10 consistent gain or loss of function mutations common to SCC, with Ras and p53 mutations overrepresented 2. Years of effort have implicated Hras and Kras mutations in the aetiology of SCC 3-5, and murine models of SCC strongly suggest that Ras activation is critical to SCC formation 5. Work from our laboratory demonstrated that hair follicle stem cells (HFSCs) can serve as cells of origin for SCC and that gain of KrasG12D mutation and loss of p53 are sufficient to generate high grade SCC typical of spindle cell carcinoma 6. These results suggest that SCC can be generated by mutations in Ras and p53 in HFSCs, but this particular model does not yield the full spectrum of phenotypes known to be present in human SCC. We sought to identify sources of tumor heterogeneity by fixing both the cell of origin and the initial oncogenic driver, while altering the identity of the second genomic hit, the tumor suppressor. In doing so, we could identify whether a specific population of cells within the epidermis (HFSCs) has the ability to generate different types of tumors, while also demonstrating the role tumor suppressors play in tumor heterogeneity. We used mice transgenic for lox-stop-lox-KrasG12D to enable Cre recombinase-mediated induction of constitutively active Ras expressed from its endogenous locus. These mice were crossed to mice expressing inducible Cre (Cre-PR) under control of a promoter active specifically in HFSCs of the skin (Keratin 15). Double transgenic mice were then crossed to mice with floxed alleles of either p53, Rb or PTEN to produce mice that would induce expression of constitutively active Kras coupled with loss of a single tumor suppressor only upon treatment with a progesterone antagonist (mifepristone). These experiments have potential clinical relevance as mutations in KRAS, TP53, RB1 and PTEN have all been shown to occur in human cutaneous SCC at varying rates (Fig. 1a) 3, 7. Mice were allowed to reach adulthood, and Cre-mediated recombination was induced just prior to the second adult hair cycle which is synchronized at postnatal day 50. Between 8 and 10 weeks following delivery of genetic hits and activation of the hair cycle, tumors became apparent macroscopically. To demonstrate the efficiency of this transgenic system to delete various tumor suppressors, we immunostained each of the targeted tumor suppressors on skin from animals of all genotypes (Fig. 1b). In addition, there were not large differences in the rate of tumor initiation from either the time transgene expression was induced or from the beginning of the next hair cycle (Fig. 1c). This is important, as we previously showed that initiation of phenotype by induction of KrasG12D depends on the onset of the telogen-to-anagen transition 8. Strikingly, we observed a profound difference in the type of squamous tumors formed between the animals with different deletions of tumor suppressors (Figs 1d and S1). While we ideally would look to human SCC samples for a similar correlation, mutations in some of the genes examined here are rare in human SCC (Fig. 1a), and human SCC is thought to derive from the interfollicular epidermis, not the hair, precluding simple identification of a similar correlation in human. As described previously 8, K15CrePR;KrasG12D;p53fl/fl mice developed significant phenotypes and eventually progressed to high grade SCC (n > 15) (Fig. 1d). In mice where PTEN was deleted instead of p53 (K15CrePR;KrasG12D;Ptenfl/fl), hyperplasia developed over a similar time course but did not progress to the point of SCC within 15 weeks after induction (the time by which all animals had to be sacrificed due to tumor/phenotype burden) (n = 6) 8. Deletion of PTEN along with induction of oncogenic Kras led to a different type of squamous hyperplasia (Figure S1). While we previously characterized phenotypes of K15CrePR;KrasG12D;p53fl/fl mice 6, 8, we provide a more complete description of phenotypes in K15CrePR;KrasG12D;Ptenfl/fl and K15CrePR;KrasG12D;Rbfl/fl mice in Figure S2. In K15CrePR;KrasG12D;Rbfl/fl mice, the hyperplasia generated appeared to move upwards towards the infundibulum and the interfollicular epidermis (n = 4). This hyperplasia also did not progress to the point of bona fide SCC, but instead produced benign papilloma (Figs 1d and S3). In summary, when controlling for the cell of origin and the initial oncogenic stimulus, the identity of the tumor suppressor appeared to be significantly related to the type of squamous phenotype that developed. To characterize the phenotypic disparity between the genotypes tested, we assessed proliferation, growth control, MAP kinase signalling, dedifferentiation and immune response in each genotype. Staining for Ki67 showed that tumors without p53 or PTEN had extensive proliferation, whereas those made without Rb had significantly less proliferation once the tumors were formed. Coincident with this, staining for p16, a protein known to be induced where growth control is needed, was induced in tumors without p53 and PTEN, but less so in tumors lacking Rb. In addition, staining for phospho-Erk, a marker of activated MAP kinase signalling, showed significant activity only in tumors lacking p53. Keratin 8 is only expressed in postnatal keratinocytes upon dedifferentiation to SCC. Staining for Keratin 8 across the various genotypes presented demonstrated that only mice with deletion of p53 showed significant dedifferentiation (Fig. 2). The skin is highly sensitive to inflammation, and numerous inflammatory cells migrate to the skin in cases of wounding, infection or tumorigenesis. Myeloid-derived suppressor cells (MDSCs) are thought to suppress proliferation and contain tumor growth 9. MDSCs are defined by immunostaining with antibodies for Gr1 and Cd11b. Attempts to identify MDSCs with these markers only reliably uncovered them in tumors formed from K15CrePR;KrasG12D;p53fl/fl mice (Fig. 2). To further characterize the immune response in tumors generated by loss of various tumor suppressors, we performed a complete pathological examination of immune cells present in the tumors characterized in Figure S1 (Figure S4). While some driver mutations have been identified consistently across SCCs, it is unclear whether the heterogeneity of phenotypes is due to variability across mutations, cell of origin, immune response or variable genomic instability. Our data show that HFSCs can drive a variety of SCC phenotypes that correlate strictly with the type of tumor suppressor deleted. This work was supported by NIH (5R01AR057409-05 and TCBT32CA09056), a CIRM Training Grant (TG2-01169), a fellowship from the Broad Center for Regenerative Medicine (UCLA, AF), MARC program (UCLA, WG). AF, WG, ACW, PS and RT performed the research and analysed the data. WL wrote the manuscript. The authors have declared no conflicting interests. Figure S1. Blinded pathological analysis of tumor phenotype across different genotypes. Figure S2. Phenotypic characteristics of KrasG12D/Pten mutant backskin. Figure S3. Phenotypic characteristics of KrasG12D/Rb mutant backskin. Figure S4. Pathological examination of immune cells in tumor models. Appendix S1. Materials and Methods. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Citations Over TimeTop 13% of 2016 papers

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