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Exploiting Mouse Models to Study Ras-Induced Cutaneous Squamous Cell Carcinoma

  • William E. Lowry
    Correspondence
    Correspondence: William E. Lowry, University of California Los Angeles, 621 Charles Young Drive South, Los Angeles, California 90095, USA.
    Affiliations
    Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, USA

    Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California, USA

    Eli and Edythe Broad Center for Regenerative Medicine, University of California Los Angeles, Los Angeles, California, USA

    Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, USA
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  • Aimee Flores
    Affiliations
    Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, USA

    Eli and Edythe Broad Center for Regenerative Medicine, University of California Los Angeles, Los Angeles, California, USA

    Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, USA
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  • Andrew C. White
    Affiliations
    Cornell University Stem Cell Program, Cornell University, Ithaca, New York, USA
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Open ArchivePublished:May 07, 2016DOI:https://doi.org/10.1016/j.jid.2016.03.017
      Recently developed methods have allowed for the delivery of cancer-causing genetic mutations to particular cell types in the epidermis in an inducible fashion. These methods have allowed for sophisticated explorations on the cellular and molecular origins of squamous cell carcinoma due to oncogenic mutations in Ras. These experiments have provided insights into whether cancer is initiated by stem or more specified cells under various conditions, and have highlighted the ability of particular genetic hits to serve as tumor initiators or promoters. Here we provide a summary of data from our lab and others that demonstrate the ability of hair follicle stem cells to serve as cancer cells of origin, and the ability of various molecular players to drive heterogeneity of tumor cell types. A synthesis of these studies potentially could provide unique insights into the process by which Ras can initiate squamous cell carcinoma in human patients and could eventually inform treatment strategies.

      Abbreviations:

      K15 (keratin 15), SCC (squamous cell carcinoma)

      Introduction

      Enormous progress has been made in the last 20 years to understand the cellular and molecular basis of squamous cell carcinoma (SCC) using genetically engineered mouse models (
      • Bailleul B.
      • Surani M.A.
      • White S.
      • Barton S.C.
      • Brown K.
      • Blessing M.
      • et al.
      Skin hyperkeratosis and papilloma formation in transgenic mice expressing a ras oncogene from a suprabasal keratin promoter.
      ,
      • Brown K.
      • Strathdee D.
      • Bryson S.
      • Lambie W.
      • Balmain A.
      The malignant capacity of skin tumours induced by expression of a mutant H-ras transgene depends on the cell type targeted.
      ,
      • French J.E.
      • Libbus B.L.
      • Hansen L.
      • Spalding J.
      • Tice R.R.
      • Mahler J.
      • et al.
      Cytogenetic analysis of malignant skin tumors induced in chemically treated TG-AC transgenic mice.
      ,
      • Greenhalgh D.A.
      • Rothnagel J.A.
      • Quintanilla M.I.
      • Orengo C.C.
      • Gagne T.A.
      • Bundman D.S.
      • et al.
      Induction of epidermal hyperplasia, hyperkeratosis, and papillomas in transgenic mice by a targeted v-Ha-ras oncogene.
      ,
      • Guo L.
      • Yu Q.C.
      • Fuchs E.
      Targeting expression of keratinocyte growth factor to keratinocytes elicits striking changes in epithelial differentiation in transgenic mice.
      ,
      • Owens D.M.
      • Spalding J.W.
      • Tennant R.W.
      • Smart R.C.
      Genetic alterations cooperate with v-Ha-ras to accelerate multistage carcinogenesis in TG.AC transgenic mouse skin.
      ,
      • Vitale-Cross L.
      • Amornphimoltham P.
      • Fisher G.
      • Molinolo A.A.
      • Gutkind J.S.
      Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis.
      ). A similar effort has been made to define the etiology of another nonmelanoma skin cancer, namely basal cell carcinoma, which is described elsewhere (
      • Donovan J.
      Review of the hair follicle origin hypothesis for basal cell carcinoma.
      ). Pathological studies of SCC typically implicate squamous cells of the interfollicular epidermis as cells of origin based on the localization of lesions and histological staining patterns. Although there is no evidence to directly dispute this interpretation, experimental evidence from murine models of SCC suggests, at a minimum, that cells from the hair follicle can serve as cells of origin (
      • da Silva-Diz V.
      • Sole-Sanchez S.
      • Valdes-Gutierrez A.
      • Urpi M.
      • Riba-Artes D.
      • Penin R.M.
      • et al.
      Progeny of Lgr5-expressing hair follicle stem cell contributes to papillomavirus-induced tumor development in epidermis.
      ,
      • Lapouge G.
      • Youssef K.K.
      • Vokaer B.
      • Achouri Y.
      • Michaux C.
      • Sotiropoulou P.A.
      • et al.
      Identifying the cellular origin of squamous skin tumors.
      ,
      • Li S.
      • Park H.
      • Trempus C.S.
      • Gordon D.
      • Liu Y.
      • Cotsarelis G.
      • et al.
      A keratin 15 containing stem cell population from the hair follicle contributes to squamous papilloma development in the mouse.
      ,
      • White A.C.
      • Tran K.
      • Khuu J.
      • Dang C.
      • Cui Y.
      • Binder S.W.
      • et al.
      Defining the origins of Ras/p53-mediated squamous cell carcinoma.
      ). Indeed, inducible cell type-specific delivery of oncogenic stimuli indicates that hair follicle stem cells in particular are better able to serve as cancer cells of origin, at least under noninflammatory conditions (
      • Lapouge G.
      • Youssef K.K.
      • Vokaer B.
      • Achouri Y.
      • Michaux C.
      • Sotiropoulou P.A.
      • et al.
      Identifying the cellular origin of squamous skin tumors.
      ,
      • White A.C.
      • Tran K.
      • Khuu J.
      • Dang C.
      • Cui Y.
      • Binder S.W.
      • et al.
      Defining the origins of Ras/p53-mediated squamous cell carcinoma.
      ). The role of inflammation on the identity of cancer cells of origin has also been described elsewhere (
      • White A.C.
      • Lowry W.E.
      Refining the role for adult stem cells as cancer cells of origin.
      ), so this review will instead focus on models of SCC initiation under homeostatic conditions. In fact, whether the study of such models under homeostatic conditions is actually relevant for human SCC is a matter of some debate, as has been discussed (
      • Schwarz M.
      • Munzel P.A.
      • Braeuning A.
      Non-melanoma skin cancer in mouse and man.
      ). In spite of that, the identification of cancer cells of origin has been the topic of intense investigation recently with the advent of cell type-specific molecular genetics.
      Recent genomic sequencing approaches have identified numerous mutations that consistently appear in human cutaneous SCC (
      • Durinck S.
      • Ho C.
      • Wang N.J.
      • Liao W.
      • Jakkula L.R.
      • Collisson E.A.
      • et al.
      Temporal dissection of tumorigenesis in primary cancers.
      ,
      • Pickering C.R.
      • Zhou J.H.
      • Lee J.J.
      • Drummond J.A.
      • Peng S.A.
      • Saade R.E.
      • et al.
      Mutational landscape of aggressive cutaneous squamous cell carcinoma.
      ,
      • South A.P.
      • Purdie K.J.
      • Watt S.A.
      • Haldenby S.
      • den Breems N.Y.
      • Dimon M.
      • et al.
      NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis.
      ). These studies have identified mutations in the Notch pathway as the most highly prevalent (>50%), whereas mutations in Hras and Kras mutations were found less often (
      • Pickering C.R.
      • Zhou J.H.
      • Lee J.J.
      • Drummond J.A.
      • Peng S.A.
      • Saade R.E.
      • et al.
      Mutational landscape of aggressive cutaneous squamous cell carcinoma.
      ,
      • South A.P.
      • Purdie K.J.
      • Watt S.A.
      • Haldenby S.
      • den Breems N.Y.
      • Dimon M.
      • et al.
      NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis.
      ). In one study of UV-induced cutaneous SCC, mutations in Hras were found in 20% of samples, 75% of which were activating mutations (for Kras, mutations were found in 10% of samples, half of which were activating) (
      • South A.P.
      • Purdie K.J.
      • Watt S.A.
      • Haldenby S.
      • den Breems N.Y.
      • Dimon M.
      • et al.
      NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis.
      ). In a larger study of 91 cSCCs, Hras was found to be mutated in 16% of samples, 30% of which were activating (
      • Pickering C.R.
      • Zhou J.H.
      • Lee J.J.
      • Drummond J.A.
      • Peng S.A.
      • Saade R.E.
      • et al.
      Mutational landscape of aggressive cutaneous squamous cell carcinoma.
      ,
      • South A.P.
      • Purdie K.J.
      • Watt S.A.
      • Haldenby S.
      • den Breems N.Y.
      • Dimon M.
      • et al.
      NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis.
      ). Experimentally, decades of work in murine models identified mutations in Ras as key to SCC initiation and progression. Experimental gain and loss of function for Notch in the epidermis argues for a role as a tumor suppressor in cSCC (
      • Demehri S.
      • Liu Z.
      • Lee J.
      • Lin M.H.
      • Crosby S.D.
      • Roberts C.J.
      • et al.
      Notch-deficient skin induces a lethal systemic B-lymphoproliferative disorder by secreting TSLP, a sentinel for epidermal integrity.
      ,
      • Demehri S.
      • Turkoz A.
      • Kopan R.
      Epidermal Notch1 loss promotes skin tumorigenesis by impacting the stromal microenvironment.
      ,
      • Lee J.
      • Basak J.M.
      • Demehri S.
      • Kopan R.
      Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes.
      ,
      • Nicolas M.
      • Wolfer A.
      • Raj K.
      • Kummer J.A.
      • Mill P.
      • van Noort M.
      • et al.
      Notch1 functions as a tumor suppressor in mouse skin.
      ), albeit through non-cell autonomous mechanisms such as skin barrier disruption, despite the fact that Notch mutations have been shown to be oncogenic in most other tissues. In this review, we will focus on experimental data gathered from murine models of Ras-initiated SCC where the consequence is known to be activation of this proto-oncogene.

      Identification of Multiple Progenitor Pools Within the Follicle With Tumorigenic Potential

      Various pathological and molecular analyses have uncovered a wealth of cellular diversity within the hair follicle. For instance, recent data have demonstrated that instead of a single homogenous pool of stem cells localized to the bulge region, there are in fact at least four distinct pools of cells within various portions of the follicle with proposed stem cell activity. These cells have been identified by molecular lineage tracing experiments with ever more specific promoters driving reporter genes such as green fluorescent protein. Within the follicle, promoters for keratin 15 (K15), Lgr5, Lrig, Gli1, and Lgr6 have been suggested to label either all (K15) or just the top (Gli1, Lgr6, and Lrig) or bottom (Lgr5) of this stem cell niche (
      • Brownell I.
      • Guevara E.
      • Bai C.B.
      • Loomis C.A.
      • Joyner A.L.
      Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells.
      ,
      • Grachtchouk M.
      • Pero J.
      • Yang S.H.
      • Ermilov A.N.
      • Michael L.E.
      • Wang A.
      • et al.
      Basal cell carcinomas in mice arise from hair follicle stem cells and multiple epithelial progenitor populations.
      ,
      • Jaks V.
      • Barker N.
      • Kasper M.
      • van Es J.H.
      • Snippert H.J.
      • Clevers H.
      • et al.
      Lgr5 marks cycling, yet long-lived, hair follicle stem cells.
      ,
      • Jensen K.B.
      • Collins C.A.
      • Nascimento E.
      • Tan D.W.
      • Frye M.
      • Itami S.
      • et al.
      Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis.
      ,
      • Kasper M.
      • Jaks V.
      • Are A.
      • Bergstrom A.
      • Schwager A.
      • Barker N.
      • et al.
      Wounding enhances epidermal tumorigenesis by recruiting hair follicle keratinocytes.
      ,
      • Morris R.J.
      • Liu Y.
      • Marles L.
      • Yang Z.
      • Trempus C.
      • Li S.
      • et al.
      Capturing and profiling adult hair follicle stem cells.
      ,
      • Snippert H.J.
      • Haegebarth A.
      • Kasper M.
      • Jaks V.
      • van Es J.H.
      • Barker N.
      • et al.
      Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin.
      ) (Figure 1). Both lineage tracing and immunostaining have shown that Lgr6, Lrig, Blimp1, and Mts24 in fact mark populations above the bulge, in the isthmus portion of the follicle. Despite their location outside the bulge, these populations clearly show progenitor potential as implicated by lineage tracing (
      • Fullgrabe A.
      • Joost S.
      • Are A.
      • Jacob T.
      • Sivan U.
      • Haegebarth A.
      • et al.
      Dynamics of Lgr6 progenitor cells in the hair follicle, sebaceous gland, and interfollicular epidermis.
      ,
      • Horsley V.
      • O'Carroll D.
      • Tooze R.
      • Ohinata Y.
      • Saitou M.
      • Obukhanych T.
      • et al.
      Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland.
      ,
      • Jensen K.B.
      • Collins C.A.
      • Nascimento E.
      • Tan D.W.
      • Frye M.
      • Itami S.
      • et al.
      Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis.
      ,
      • Nijhof J.G.
      • Braun K.M.
      • Giangreco A.
      • van Pelt C.
      • Kawamoto H.
      • Boyd R.L.
      • et al.
      The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells.
      ). Lrig, Lgr6, and Blimp1 lineage tracing mark sebaceous gland progenitors, a target of adenoma formation that can proceed to SCC and is clearly derived from hair follicles. The fact that multiple cells within the follicle show progenitor capability is consistent with the idea that there could be multiple cells of origin for SCC within the follicle.
      Figure 1
      Figure 1This image depicts several scenarios for modeling tumor initiation by various transgenic means. (1) Constitutive Kras is induced and p53 is deleted in Lgr5+ cells of the bulge, but there is no effect in the short term, even after a telogen to anagen transition. (2) Induction of Hras and deletion of p53 in Krt15+ cells leads to only minor follicular hyperplasia. (3) Induction of constitutive Hras and deletion of p53 in Lgr6+ cells drives infundibular hyperplasia. (4) Induction of constitutive Kras and deletion of p53 in Lgr6+ cells generates infundibular hyperplasia and papilloma. (5) Induction of constitutive Kras and deletion of p53 in telogen has no effect until the next hair cycle. (6) Induction of Kras and deletion of p53 before or during a telogen-to-anagen transition drives tumor initiation, and eventually leads to (7) frank carcinoma. Note that only cell types with defined Cre lineage drivers are depicted in the image, and that the image does not fully depict all the types of phenotype that arise from the indicated genotype.

      K15 Versus Lgr5 Versus Lgr6 Cells as Cell of Origin for SCC

      In an effort to define the cell of origin for SCC amongst hair follicle cells, we compared delivery of KrasG12D and loss of p53 specifically to cells active for K15CrePR, Lgr5CreER, and Lgr6CreER. Activation of the K15CrePR allele with mifepristone just before the start of a new hair cycle in these mice led to profound induction of hyperplasia, epithelial to mesenchymal transition, cysts, and eventually high-grade SCC routinely within 5–10 weeks (
      • White A.C.
      • Tran K.
      • Khuu J.
      • Dang C.
      • Cui Y.
      • Binder S.W.
      • et al.
      Defining the origins of Ras/p53-mediated squamous cell carcinoma.
      ,
      • White A.C.
      • Khuu J.K.
      • Dang C.Y.
      • Hu J.
      • Tran K.V.
      • Liu A.
      • et al.
      Stem cell quiescence acts as a tumour suppressor in squamous tumours.
      ). Surprisingly, initiation from the Lgr5CreER-positive cells did not generate phenotypes until subsequent hair cycles, where eventually SCC did develop (
      • White A.C.
      • Khuu J.K.
      • Dang C.Y.
      • Hu J.
      • Tran K.V.
      • Liu A.
      • et al.
      Stem cell quiescence acts as a tumour suppressor in squamous tumours.
      and unpublished data). These contrasting results were consistent with the idea that the follicular cell of origin for SCC resides within the K15+/Lgr5− population of cells in the upper portion of the bulge (
      • White A.C.
      • Khuu J.K.
      • Dang C.Y.
      • Hu J.
      • Tran K.V.
      • Liu A.
      • et al.
      Stem cell quiescence acts as a tumour suppressor in squamous tumours.
      ) (Figure 1). We presume that the reason for the long latency for Lgr5CreER-induced tumors is the fact that Lgr5+ cells of the lower bulge can be relocated to upper bulge regions after anagen cycles (
      • Jaks V.
      • Barker N.
      • Kasper M.
      • van Es J.H.
      • Snippert H.J.
      • Clevers H.
      • et al.
      Lgr5 marks cycling, yet long-lived, hair follicle stem cells.
      ). In addition, the nature of the experimental design is essentially a lineage trace where KrasG12D is induced in the Lgr5+ cells and all their progeny. As a result, when those bulge cells go on to make a new hair follicle during anagen, nearly all of the cells of the new follicle will express KrasG12D. At that point, any of the cells of any portion of the bulge (or elsewhere in the follicle for that matter) have the potential to induce tumorigenesis. Therefore, the true follicular cells of origin for SCC probably reside in the upper portion of the bulge, which interestingly is more likely to harbor label-retaining cells as a result of quiescence (
      • Jaks V.
      • Barker N.
      • Kasper M.
      • van Es J.H.
      • Snippert H.J.
      • Clevers H.
      • et al.
      Lgr5 marks cycling, yet long-lived, hair follicle stem cells.
      ,
      • Tumbar T.
      • Guasch G.
      • Greco V.
      • Blanpain C.
      • Lowry W.E.
      • Rendl M.
      • et al.
      Defining the epithelial stem cell niche in skin.
      ).
      When appropriate genetic mutations were delivered to Lgr6+ cells, the phenotypes were typically upper follicle hyperplasia, sebaceous hyperplasia, and sometimes benign papillomas (
      • White A.C.
      • Khuu J.K.
      • Dang C.Y.
      • Hu J.
      • Tran K.V.
      • Liu A.
      • et al.
      Stem cell quiescence acts as a tumour suppressor in squamous tumours.
      and unpublished data). Because Lgr6 is expressed in a portion of interfollicular epidermis-, it is possible that papillomas can arise from this site.
      We also found that although K15+ cells can serve as cells of origin, they will only do so when in a permissive, activated state. For instance, if combinations of genetic hits are delivered to hair follicle stem cells during telogen, the follicles do not respond until the initiation of a new hair cycle. In fact, weeks can go by with oncogenic Ras expression in HFSCs, but no phenotype develops until a telogen-anagen transition begins (
      • White A.C.
      • Khuu J.K.
      • Dang C.Y.
      • Hu J.
      • Tran K.V.
      • Liu A.
      • et al.
      Stem cell quiescence acts as a tumour suppressor in squamous tumours.
      ). This observation was consistent with previous efforts showing a dependence on the hair cycle for 7,12-dimethylbenz[a]anthracene-12-O-tetradecanoylphorbol-13-acetate-induced SCC (
      • Miller S.J.
      • Wei Z.G.
      • Wilson C.
      • Dzubow L.
      • Sun T.T.
      • Lavker R.M.
      Mouse skin is particularly susceptible to tumor initiation during early anagen of the hair cycle: possible involvement of hair follicle stem cells.
      ). We showed that quiescent HFSCs use the phosphatase Pten to blunt the response to activated Ras, as deletion of this tumor suppressor allowed for Ras-induced tumor formation at any point in the hair cycle.

      Diversity of Genetic Hits

      Some of the first transgenic animals created employed expression of constitutively active Hras to generate hyperplasia and tumors from various promoters such as K1 (spinous layers), K5, and K14 (both strong in basal epidermis, weaker in differentiated cells) (
      • Bailleul B.
      • Surani M.A.
      • White S.
      • Barton S.C.
      • Brown K.
      • Blessing M.
      • et al.
      Skin hyperkeratosis and papilloma formation in transgenic mice expressing a ras oncogene from a suprabasal keratin promoter.
      ,
      • Greenhalgh D.A.
      • Rothnagel J.A.
      • Quintanilla M.I.
      • Orengo C.C.
      • Gagne T.A.
      • Bundman D.S.
      • et al.
      Induction of epidermal hyperplasia, hyperkeratosis, and papillomas in transgenic mice by a targeted v-Ha-ras oncogene.
      ,
      • Hansen L.A.
      • Tennant R.W.
      Follicular origin of epidermal papillomas in v-Ha-ras transgenic TG.AC mouse skin.
      ,
      • Owens D.M.
      • Spalding J.W.
      • Tennant R.W.
      • Smart R.C.
      Genetic alterations cooperate with v-Ha-ras to accelerate multistage carcinogenesis in TG.AC transgenic mouse skin.
      ). The published work suggested that overexpression of oncogenic Hras was capable of generating SCC in all these models suggesting that a variety of cell types could potentially serve as cancer cells of origin. However, interpretation of these results is complicated by the fact that the transgenic approach used (typically direct transgene injection into blastocysts) potentially led to supraphysiological doses of the oncogenic Hras due to multiple integration sites into the genome.
      Subsequently, new genetic knockin animals were generated that harbored constitutively active alleles of Hras and Kras within their native loci by homologous recombination (
      • Jackson E.L.
      • Willis N.
      • Mercer K.
      • Bronson R.T.
      • Crowley D.
      • Montoya R.
      • et al.
      Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.
      ,
      • Tuveson D.A.
      • Shaw A.T.
      • Willis N.A.
      • Silver D.P.
      • Jackson E.L.
      • Chang S.
      • et al.
      Endogenous oncogenic K-ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects.
      ). These alleles were preceded by a floxed stop signal rendering the allele silent until Cre recombinase was delivered to the cell. This approach had the advantage that oncogenic Kras or Hras could be expressed from their native loci, as would typically happen in a patient with SCC, where the oncogenic mutation is typically thought to be due to constitutive activation of the GTPase function of the protein and not its overexpression. These transgenic alleles, termed LSL-KrasG12D and LSL-HrasG12V, are then expressed in cells with active Cre recombinase. The Cre recombinase is typically expressed under the control of a cell type-specific promoter, and made to be inducible by the addition of the ligand binding domain of the progesterone or estrogen receptor (
      • Metzger D.
      • Clifford J.
      • Chiba H.
      • Chambon P.
      Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase.
      ).
      We and others have coupled the LSL-KrasG12D allele with various inducible Cre transgenic lines (K14-CreER, K15Cre-PR, Lgr5-CreER, Lgr6-CreER, and K5-CreER) to determine which hair follicle lineages are able to give rise to tumors or bona fide cancer (
      • Caulin C.
      • Nguyen T.
      • Longley M.A.
      • Zhou Z.
      • Wang X.J.
      • Roop D.R.
      Inducible activation of oncogenic K-ras results in tumor formation in the oral cavity.
      ,
      • da Silva-Diz V.
      • Sole-Sanchez S.
      • Valdes-Gutierrez A.
      • Urpi M.
      • Riba-Artes D.
      • Penin R.M.
      • et al.
      Progeny of Lgr5-expressing hair follicle stem cell contributes to papillomavirus-induced tumor development in epidermis.
      ,
      • Faurschou A.
      • Haedersdal M.
      • Poulsen T.
      • Wulf H.C.
      Squamous cell carcinoma induced by ultraviolet radiation originates from cells of the hair follicle in mice.
      ,
      • Vitale-Cross L.
      • Amornphimoltham P.
      • Fisher G.
      • Molinolo A.A.
      • Gutkind J.S.
      Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis.
      ,
      • White A.C.
      • Tran K.
      • Khuu J.
      • Dang C.
      • Cui Y.
      • Binder S.W.
      • et al.
      Defining the origins of Ras/p53-mediated squamous cell carcinoma.
      ,
      • White A.C.
      • Khuu J.K.
      • Dang C.Y.
      • Hu J.
      • Tran K.V.
      • Liu A.
      • et al.
      Stem cell quiescence acts as a tumour suppressor in squamous tumours.
      ). This approach originally met with some resistance from those who argue that HrasG12V is the clinically relevant mutation to study, and not KrasG12D. Despite this criticism, numerous studies showed that delivery of the KrasG12D to hair follicle cells could drive tumor formation, and coupled with inducible loss of a tumor suppressor (floxed), bona fide cancer (
      • Lapouge G.
      • Youssef K.K.
      • Vokaer B.
      • Achouri Y.
      • Michaux C.
      • Sotiropoulou P.A.
      • et al.
      Identifying the cellular origin of squamous skin tumors.
      ,
      • Mukhopadhyay A.
      • Krishnaswami S.R.
      • Yu B.D.
      Activated Kras alters epidermal homeostasis of mouse skin, resulting in redundant skin and defective hair cycling.
      ,
      • Vitale-Cross L.
      • Amornphimoltham P.
      • Fisher G.
      • Molinolo A.A.
      • Gutkind J.S.
      Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis.
      ,
      • White A.C.
      • Tran K.
      • Khuu J.
      • Dang C.
      • Cui Y.
      • Binder S.W.
      • et al.
      Defining the origins of Ras/p53-mediated squamous cell carcinoma.
      ). In fact, recent studies of the mutational landscape of 7,12-dimethylbenz[a]anthracene-TPA tumors (
      • Nassar D.
      • Latil M.
      • Boeckx B.
      • Lambrechts D.
      • Blanpain C.
      Genomic landscape of carcinogen-induced and genetically induced mouse skin squamous cell carcinoma.
      ) and patient tumor sequencing (COSMIC, TCGA) have in fact clearly demonstrated that activating mutations in Kras are present in a significant proportion of SCCs (
      • Flores A.
      • Grant W.
      • White A.C.
      • Scumpia P.
      • Takahashi R.
      • Lowry W.E.
      Tumor suppressor identity can contribute to heterogeneity of phenotype in hair follicle stem cell induced squamous carcinoma.
      ). Furthermore, in the mouse model, there are studies suggesting that mutations in Kras can complement and drive tumors on experimental deletion of Hras and 7,12-dimethylbenz[a]anthracene/TPA treatment (
      • McCreery M.Q.
      • Halliwill K.D.
      • Chin D.
      • Delrosario R.
      • Hirst G.
      • Vuong P.
      • et al.
      Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers.
      ,
      • Wong C.E.
      • Yu J.S.
      • Quigley D.A.
      • To M.D.
      • Jen K.Y.
      • Huang P.Y.
      • et al.
      Inflammation and Hras signaling control epithelial-mesenchymal transition during skin tumor progression.
      ).
      On the other hand, Hras is still mutated in a larger proportion of human SCC (and in 7,12-dimethylbenz[a]anthracene-TPA mice) (
      • Lieu F.M.
      • Yamanishi K.
      • Konishi K.
      • Kishimoto S.
      • Yasuno H.
      Low incidence of Ha-ras oncogene mutations in human epidermal tumors.
      ,
      • Nassar D.
      • Latil M.
      • Boeckx B.
      • Lambrechts D.
      • Blanpain C.
      Genomic landscape of carcinogen-induced and genetically induced mouse skin squamous cell carcinoma.
      ,
      • Pierceall W.E.
      • Goldberg L.H.
      • Tainsky M.A.
      • Mukhopadhyay T.
      • Ananthaswamy H.N.
      Ras gene mutation and amplification in human nonmelanoma skin cancers.
      ,
      • South A.P.
      • Purdie K.J.
      • Watt S.A.
      • Haldenby S.
      • den Breems N.Y.
      • Dimon M.
      • et al.
      NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis.
      ,
      • van der Schroeff J.G.
      • Evers L.M.
      • Boot A.J.
      • Bos J.L.
      Ras oncogene mutations in basal cell carcinomas and squamous cell carcinomas of human skin.
      ), and therefore it would be expected that the LSL-HrasG12V should promote tumorigenesis when expressed in the epidermis. However, despite its availability for a number of years, relatively few papers have taken advantage of this allele for murine tumorigenesis. Those that have shown LSL-HrasG12V to promote tumorigenesis in the epidermis have coupled this genetic hit with others, as expression of one copy of this allele appears to just promote modest proliferation and does not generate even benign tumors as one copy of LSL-KrasG12D has been shown to do extensively (
      • Beronja S.
      • Janki P.
      • Heller E.
      • Lien W.H.
      • Keyes B.E.
      • Oshimori N.
      • et al.
      RNAi screens in mice identify physiological regulators of oncogenic growth.
      ).
      We have performed similar experiments in our lab recently with a variety of inducible Cre lineages to determine whether HFSCs respond differently to KrasG12D versus HrasG12V. As shown in Figure 1, delivery of HrasG12V generates barely detectable phenotypes in the hair follicle, even after extended periods of time. Data from transgenic mice are shown in Supplementary Figure S1 online. This was the case whether K15CrePR or Lgr5CreER was used, suggesting that, in murine models, HFSCs are not particularly sensitive to mutations of Hras, as opposed to Kras (
      • Lapouge G.
      • Youssef K.K.
      • Vokaer B.
      • Achouri Y.
      • Michaux C.
      • Sotiropoulou P.A.
      • et al.
      Identifying the cellular origin of squamous skin tumors.
      ,
      • White A.C.
      • Tran K.
      • Khuu J.
      • Dang C.
      • Cui Y.
      • Binder S.W.
      • et al.
      Defining the origins of Ras/p53-mediated squamous cell carcinoma.
      ). Expression of HrasG12V in LGR6+ cells of the isthmus, sebaceous gland, and interfollicular epidermis also showed only minor hyperplasia of upper portions of the follicle and the IFE (Figure 1 and Supplementary Figure S1) as opposed to expression of KrasG12D in the same population that presented with prominent upper follicle hyperplasia and occasional papilloma (
      • White A.C.
      • Khuu J.K.
      • Dang C.Y.
      • Hu J.
      • Tran K.V.
      • Liu A.
      • et al.
      Stem cell quiescence acts as a tumour suppressor in squamous tumours.
      ). This could either be due to a differential response to these two oncogenes, or potentially different levels of expression of the oncogenes driven by their endogenous promoters.

      Sources of Tumor Diversity

      Within SCC, there are at least 12 different diagnoses that are routinely made by pathology and histological examination. What is the source of this diversity? Various hypotheses have been made from the cell of origin, to the identity of the genetic hits, contribution of an immune response, vascularization, hypoxia, etc., but little experimental evidence exists to provide concrete answers. Pathological studies have argued for a consistent cell of origin coupled with a variety of tumor initiating genetic hits. However, most consider the IFE to be the prime source for cells of origin, and Hras mutations to be the key oncogenic driver for SCC, so why do so many different flavors of SCC still arise?
      We sought to test the possibility that tumor suppressors could play a role in creating tumor diversity using the inducible mouse model to drive tumorigenesis in a setting whereby both the cell of origin and the first genetic hit were fixed (K15CrePR; LSLKrasG12D). We then varied the identity of the tumor suppressor coupled to oncogene activation. We and others have shown that KrasG12D coupled with deletion of p53 in HFSCs leads to a high-grade SCC, with spindle cell phenotypes (
      • Lapouge G.
      • Youssef K.K.
      • Vokaer B.
      • Achouri Y.
      • Michaux C.
      • Sotiropoulou P.A.
      • et al.
      Identifying the cellular origin of squamous skin tumors.
      ,
      • White A.C.
      • Tran K.
      • Khuu J.
      • Dang C.
      • Cui Y.
      • Binder S.W.
      • et al.
      Defining the origins of Ras/p53-mediated squamous cell carcinoma.
      ,
      • White A.C.
      • Khuu J.K.
      • Dang C.Y.
      • Hu J.
      • Tran K.V.
      • Liu A.
      • et al.
      Stem cell quiescence acts as a tumour suppressor in squamous tumours.
      ,
      • White A.C.
      • Lowry W.E.
      Exploiting the origins of Ras mediated squamous cell carcinoma to develop novel therapeutic interventions.
      ). Both Kras and p53 are mutated in human SCCs (
      • Anderson J.A.
      • Irish J.C.
      • Ngan B.Y.
      Prevalence of RAS oncogene mutation in head and neck carcinomas.
      ,
      • Nassar D.
      • Latil M.
      • Boeckx B.
      • Lambrechts D.
      • Blanpain C.
      Genomic landscape of carcinogen-induced and genetically induced mouse skin squamous cell carcinoma.
      ,
      • Nichols A.C.
      • Yoo J.
      • Palma D.A.
      • Fung K.
      • Franklin J.H.
      • Koropatnick J.
      • et al.
      Frequent mutations in TP53 and CDKN2A found by next-generation sequencing of head and neck cancer cell lines.
      ,
      • Pierceall W.E.
      • Mukhopadhyay T.
      • Goldberg L.H.
      • Ananthaswamy H.N.
      Mutations in the p53 tumor suppressor gene in human cutaneous squamous cell carcinomas.
      ,
      • South A.P.
      • Purdie K.J.
      • Watt S.A.
      • Haldenby S.
      • den Breems N.Y.
      • Dimon M.
      • et al.
      NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis.
      ,
      • Stransky N.
      • Egloff A.M.
      • Tward A.D.
      • Kostic A.D.
      • Cibulskis K.
      • Sivachenko A.
      • et al.
      The mutational landscape of head and neck squamous cell carcinoma.
      ,
      • Su F.
      • Viros A.
      • Milagre C.
      • Trunzer K.
      • Bollag G.
      • Spleiss O.
      • et al.
      RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.
      ,
      • van der Schroeff J.G.
      • Evers L.M.
      • Boot A.J.
      • Bos J.L.
      Ras oncogene mutations in basal cell carcinomas and squamous cell carcinomas of human skin.
      ,
      • Yoneda K.
      • Yokoyama T.
      • Yamamoto T.
      • Hatabe T.
      • Osaki T.
      p53 gene mutations and p21 protein expression induced independently of p53, by TGF-beta and gamma-rays in squamous cell carcinoma cells.
      ,
      • Ziegler A.
      • Jonason A.S.
      • Leffell D.J.
      • Simon J.A.
      • Sharma H.W.
      • Kimmelman J.
      • et al.
      Sunburn and p53 in the onset of skin cancer.
      ), so it is surprising that in the mouse model delivery of these two hits led to a variety of SCCs rarely found in humans. Of course, we cannot rule out the possibility that the spindle cell phenotype arose in mice because the cancer was initiated from HFSCs as opposed to IFE cells, as is thought to happen in human cSCC.
      More recently, we found that the same oncogene expressed in the same cell of origin leads to a distinct SCC phenotype if instead coupled to loss of Pten or Rb. In
      • Flores A.
      • Grant W.
      • White A.C.
      • Scumpia P.
      • Takahashi R.
      • Lowry W.E.
      Tumor suppressor identity can contribute to heterogeneity of phenotype in hair follicle stem cell induced squamous carcinoma.
      , we have found that coupling KrasG12D with loss of Pten leads to crateriform proliferation of hyalinized keratinocytes and keratoacanthoma. Furthermore, coupling KrasG12D with loss of Rb led to follicular nevi, infundibular and sebaceous hypertrophy, and papillomas. In the case of a Kras/Rb model, it was interesting to observe that the phenotypes appeared to move upward toward the IFE as opposed to the Kras coupled to either p53 or Pten. Coupled with the pathological differences between genotypes, we also observed distinct patterns of proliferation, EMT, and immune response (
      • Flores A.
      • Grant W.
      • White A.C.
      • Scumpia P.
      • Takahashi R.
      • Lowry W.E.
      Tumor suppressor identity can contribute to heterogeneity of phenotype in hair follicle stem cell induced squamous carcinoma.
      ). Our data clearly showed that the combination of KrasG12D with loss of p53 led to the highest grade tumors, which presumably explains the pronounced difference in proliferation observed across tumors from different genotypes. However, the difference in immune response and general cellular diversity across genotypes is not easily explained by the aggressiveness of the tumors generated.
      Together, these data suggest that the identity of the tumor suppressor can affect the diversity of cellular phenotypes that arise in the tumor. This previously unappreciated source of tumor diversity could be simply due to the identity of genetic hits, unrelated to the cell of origin or interactions with the tumor-stroma microenvironment. It is also not clear whether these data point toward a novel source of diversity in human cutaneous SCC, except that these same genetic hits are found in human SCC with high variability. Going forward, it will be necessary to genotype human SCC samples and couple this analysis with phenotyping to see if genotype can at least correlate with phenotype when it comes to the identity of tumor suppressors lost in human cancers. So far this type of deep sequencing of tumor tissues coupled with detailed pathological examination has not been described in the literature as it has been for other types of cancer.

      Summary

      Taking all the data from murine transgenic models of SCC together, one can posit a model by which SCC develops in human skin (Figure 1). In the murine model, it seems clear that HFSCs are cells of origin for SCC, and that KrasG12D can be a key oncogene in this setting. We also have evidence to suggest that the diversity of tumor phenotypes that can be observed in this model is affected by the identity of the genetic hits delivered to HFSCs, particularly the secondary loss of tumor suppressors. Translating this model to the process in which human cutaneous SCC arises remains a challenge. However, cutaneous SCC is one of the most common forms of cancer, and because of the available tools, it is safe to say that cutaneous SCC is one of the best-studied models for cancer available. Also, many of the phenotypes and secondary processes that appear at a tumor origin in these models appear to be highly similar to other epithelial cancers both in mouse and human. Therefore, we would argue that murine transgenic models of SCC are very useful to facilitate the development of novel treatment strategies going forward.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We would like to thank all the Lowry lab members who over the years helped carry out much of the work described in this review, including Joan Khuu, Mallory Neebe, Christine Deng, Kathy Tran, Sarah Gomez, Yongyan Cui, and Jeanny Hu. We apologize to those whose work we did not cite due to space constraints. This effort was supported by NIH (NIAMS 5R01AR057409), the Jonsson Comprehensive Cancer Center, and training grants to ACW and AF (CIRM, NIH-Tumor Cell Biology, Broad Center for Regenerative Medicine at UCLA).

      Supplementary Material

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