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Cancer Stem Cells in Squamous Cell Carcinoma

  • Zhe Jian
    Affiliations
    Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA

    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China
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  • Alexander Strait
    Affiliations
    Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA
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  • Antonio Jimeno
    Affiliations
    Department of Medicine, Division of Medical Oncology, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA
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  • Xiao-Jing Wang
    Correspondence
    Correspondence: Xiao-Jing Wang, Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Building RC1-N, RoomP18-5128, Mail Stop 8104, Aurora, Colorado 80045-0508, USA.
    Affiliations
    Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA
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Open ArchivePublished:September 16, 2016DOI:https://doi.org/10.1016/j.jid.2016.07.033
      Cancer stem cells (CSCs) are found in many cancer types, including squamous cell carcinoma (SCC). CSCs initiate cancer formation and are linked to metastasis and resistance to therapies. Studies have revealed that several distinct CSC populations coexist in SCC and that tumor initiation and metastatic potential of these populations can be uncoupled. Therefore, it is critical to understand CSC biology to develop novel CSC-targeted therapies for patients with SCC with poor prognoses. This review compares the properties of CSCs in SCC with normal stem cells in the skin, summarizes current advances and characteristics of CSCs, and considers the challenges for CSC-targeted treatment of SCC.

      Abbreviations:

      ABC (ATP binding cassette), ALDH (aldehyde dehydrogenase), CSC (cancer stem cell), K15 (keratin 15), SC (stem cell), SCC (squamous cell carcinoma), SP (side population)

      Introduction

      Nonmelanoma skin cancers, including basal cell carcinoma and squamous cell carcinoma (SCC), are the most common skin cancer types and have increased dramatically worldwide in recent years (
      • Moore S.P.
      • Antoni S.
      • Colquhoun A.
      • Healy B.
      • Ellison-Loschmann L.
      • Potter J.D.
      • et al.
      Cancer incidence in indigenous people in Australia, New Zealand, Canada, and the USA: a comparative population-based study.
      ,
      • Narayanan D.L.
      • Saladi R.N.
      • Fox J.L.
      Ultraviolet radiation and skin cancer.
      ). SCC can metastasize to ectopic sites (
      • Klein C.A.
      Selection and adaptation during metastatic cancer progression.
      ), and advanced SCCs have high mortality rates and are often refractory to conventional therapy (
      • Geissler E.K.
      Skin cancer in solid organ transplant recipients: are mTOR inhibitors a game changer?.
      ). SCCs contain subpopulations of cells with cancer stem cell (CSC) properties that are linked to SCC initiation, metastasis, and resistance to chemo- and radiotherapy (
      • Biddle A.
      • Liang X.
      • Gammon L.
      • Fazil B.
      • Harper L.J.
      • Emich H.
      • et al.
      Cancer stem cells in squamous cell carcinoma switch between two distinct phenotypes that are preferentially migratory or proliferative.
      ,
      • da Silva-Diz V.
      • Simon-Extremera P.
      • Bernat-Peguera A.
      • de Sostoa J.
      • Urpi M.
      • Penin R.M.
      • et al.
      Cancer stem-like cells act via distinct signaling pathways in promoting late stages of malignant progression.
      ,
      • Oshimori N.
      • Oristian D.
      • Fuchs E.
      TGF-beta promotes heterogeneity and drug resistance in squamous cell carcinoma.
      ,
      • Schober M.
      • Fuchs E.
      Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-beta and integrin/focal adhesion kinase (FAK) signaling.
      ,
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ,
      • Zhang Q.
      • Shi S.
      • Yen Y.
      • Brown J.
      • Ta J.Q.
      • Le A.D.
      A subpopulation of CD133(+) cancer stem-like cells characterized in human oral squamous cell carcinoma confer resistance to chemotherapy.
      ). Therefore, characterizing SCC CSCs will provide new insights into SCC treatment. This review covers similarities and differences between SCC CSCs and normal stem cells (SCs) in the skin and discusses therapeutic strategies to target CSCs.

      SCs Versus SCC CSCs in the Skin

      SCs are responsible for regenerating and maintaining tissues and have unique defining characteristics (Figure 1). First, normal SCs are capable of self-renewal. Each SC typically undergoes asymmetrical cell division to produce two daughter cells: one SC and one differentiating cell. Second, normal SCs are usually slow cycling with low proliferation rates, retaining tritium thymidine or BrdU labeling for long periods of time (also known as label retaining cells), yet maintain the capacity for clonogenic growth (
      • Bickenbach J.R.
      Identification and behavior of label-retaining cells in oral mucosa and skin.
      ,
      • Morris R.J.
      • Potten C.S.
      Slowly cycling (label-retaining) epidermal cells behave like clonogenic stem cells in vitro.
      ). Third, they are rare in most tissues. Fourth, they are undifferentiated but can give rise to one or more cell lineages (multipotency or pluripotency). Fifth, normal SCs have a much longer lifespan than their progeny. Finally, normal SCs often have specific locations determined by their microenvironment (niche).
      Figure 1
      Figure 1Venn diagram showing stem cell and cancer stem cell characteristics. ABC, ATP binding cassette.
      Epidermal SCs are located in the bulge of hair follicles, the basal layer of the interfollicular epidermis, and the base of the sebaceous gland (
      • Levy V.
      • Lindon C.
      • Harfe B.D.
      • Morgan B.A.
      Distinct stem cell populations regenerate the follicle and interfollicular epidermis.
      ). Hair germ cells, thought to arise from bulge cells, also contain BrdU label retaining cells (
      • Ito M.
      • Kizawa K.
      • Hamada K.
      • Cotsarelis G.
      Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen.
      ). Although distinctive, the pattern of gene expression in hair germ cells is more similar to bulge cells than to transiently amplifying follicular matrix cells (
      • Greco V.
      • Chen T.
      • Rendl M.
      • Schober M.
      • Pasolli H.A.
      • Stokes N.
      • et al.
      A two-step mechanism for stem cell activation during hair regeneration.
      ). Studies suggest that bulge cells and possibly hair germ cells contain multipotent follicular SCs that normally generate hair follicles, but can also regenerate the epidermis and sebaceous glands in response to skin injury (
      • Ito M.
      • Liu Y.
      • Yang Z.
      • Nguyen J.
      • Liang F.
      • Morris R.J.
      • et al.
      Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis.
      ,
      • 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.
      ,
      • Levy V.
      • Lindon C.
      • Harfe B.D.
      • Morgan B.A.
      Distinct stem cell populations regenerate the follicle and interfollicular epidermis.
      ,
      • Levy V.
      • Lindon C.
      • Zheng Y.
      • Harfe B.D.
      • Morgan B.A.
      Epidermal stem cells arise from the hair follicle after wounding.
      ,
      • Morris R.J.
      • Liu Y.
      • Marles L.
      • Yang Z.
      • Trempus C.
      • Li S.
      • et al.
      Capturing and profiling adult hair follicle stem cells.
      ). Under normal conditions, SCs in the interfollicular epidermis and sebaceous glands are lineage specific, and generate their respective tissues without recruitment of SCs from the bulge (
      • Claudinot S.
      • Nicolas M.
      • Oshima H.
      • Rochat A.
      • Barrandon Y.
      Long-term renewal of hair follicles from clonogenic multipotent stem cells.
      ,
      • Clayton E.
      • Doupe D.P.
      • Klein A.M.
      • Winton D.J.
      • Simons B.D.
      • Jones P.H.
      A single type of progenitor cell maintains normal 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.
      ,
      • Ito M.
      • Liu Y.
      • Yang Z.
      • Nguyen J.
      • Liang F.
      • Morris R.J.
      • et al.
      Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis.
      ,
      • Levy V.
      • Lindon C.
      • Harfe B.D.
      • Morgan B.A.
      Distinct stem cell populations regenerate the follicle and interfollicular epidermis.
      ,
      • Morris R.J.
      • Liu Y.
      • Marles L.
      • Yang Z.
      • Trempus C.
      • Li S.
      • et al.
      Capturing and profiling adult hair follicle stem cells.
      ).
      CSCs are certain tumor cells exhibiting stem cell-like properties. Whereas normal SCs have several distinct characteristics as described above, CSCs are primarily defined by one criterion: the ability to initiate tumors, and the term CSC is often used interchangeably with “tumor-initiating cell.” CSCs can be derived from SCs (
      • Morris R.J.
      • Fischer S.M.
      • Slaga T.J.
      Evidence that a slowly cycling subpopulation of adult murine epidermal cells retains carcinogen.
      ) or from nonstem cells that acquire the capacity to self-renew (
      • Jamieson C.H.
      • Ailles L.E.
      • Dylla S.J.
      • Muijtjens M.
      • Jones C.
      • Zehnder J.L.
      • et al.
      Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML.
      ). Unlike normal SCs, CSCs may not be multipotent, leading to single lineage tumors, such as SCC (epidermal lineage), various follicular tumor types (hair follicle lineage), or sebaceous gland tumors (sebaceous lineage). Furthermore, CSCs may not be quiescent. For example, normal slow cycling bulge SCs can acquire genetic mutations, such as Kras mutations or Smad4 deletions, that drive them into hyperproliferation (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ). Finally, the number of CSCs varies widely, ranging from ≤1% to approximately 20% in SCCs, depending on tumor types and experimental models used to assess tumor initiation, such as the severity of immune suppression of recipient mice in xenografts (
      • Quintana E.
      • Shackleton M.
      • Sabel M.S.
      • Fullen D.R.
      • Johnson T.M.
      • Morrison S.J.
      Efficient tumour formation by single human melanoma cells.
      ,
      • Song J.
      • Chang I.
      • Chen Z.
      • Kang M.
      • Wang C.Y.
      Characterization of side populations in HNSCC: highly invasive, chemoresistant and abnormal Wnt signaling.
      ,
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ). For example, in our SCC mouse model, CSCs were rare in primary SCCs, but their numbers dramatically increased in metastatic SCCs and SCCs with epithelial to mesenchymal transition (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ).
      SCs and CSCs also share characteristics, such as the capacity for self-renewal, high levels of ATP binding cassette (ABC) transporters, certain cell surface markers, and being influenced by their niche. Also, CSCs share certain regulators with normal SCs. For example, SCC CSCs express factors that regulate self-renewal in embryonic SCs, such as SOX2, MYC, and OCT-4 (
      • Bose B.
      • Shenoy S.P.
      Stem cell versus cancer and cancer stem cell: intricate balance decides their respective usefulness or harmfulness in the biological system.
      ,
      • Boumahdi S.
      • Driessens G.
      • Lapouge G.
      • Rorive S.
      • Nassar D.
      • Le Mercier M.
      • et al.
      SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma.
      ,
      • Lim W.
      • Choi H.
      • Kim J.
      • Kim S.
      • Jeon S.
      • Ni K.
      • et al.
      Expression of cancer stem cell marker during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.
      ). Similarly, some common “stemness” pathways are activated in follicular SCs and CSCs, such as Wnt signaling (
      • Malanchi I.
      • Peinado H.
      • Kassen D.
      • Hussenet T.
      • Metzger D.
      • Chambon P.
      • et al.
      Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling.
      ,
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ).

      Sorting CSCs

      CSCs can be sorted by putative cell surface markers that can be either shared with or distinct from normal SCs depending on tumor type. CD34, a cell surface marker for mouse bulge SCs (
      • Trempus C.S.
      • Morris R.J.
      • Bortner C.D.
      • Cotsarelis G.
      • Faircloth R.S.
      • Reece J.M.
      • et al.
      Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34.
      ), also serves as a CSC marker in mouse SCCs (
      • Trempus C.S.
      • Morris R.J.
      • Ehinger M.
      • Elmore A.
      • Bortner C.D.
      • Ito M.
      • et al.
      CD34 expression by hair follicle stem cells is required for skin tumor development in mice.
      ) but is not expressed in human bulge SCs (
      • Ohyama M.
      • Terunuma A.
      • Tock C.L.
      • Radonovich M.F.
      • Pise-Masison C.A.
      • Hopping S.B.
      • et al.
      Characterization and isolation of stem cell-enriched human hair follicle bulge cells.
      ). CD200, a cell surface marker expressed in both mouse and human bulge SCs (
      • Ohyama M.
      • Terunuma A.
      • Tock C.L.
      • Radonovich M.F.
      • Pise-Masison C.A.
      • Hopping S.B.
      • et al.
      Characterization and isolation of stem cell-enriched human hair follicle bulge cells.
      ), is also enriched in metastatic SCC (
      • Stumpfova M.
      • Ratner D.
      • Desciak E.B.
      • Eliezri Y.D.
      • Owens D.M.
      The immunosuppressive surface ligand CD200 augments the metastatic capacity of squamous cell carcinoma.
      ). CD49f is a surface marker of quiescent label retaining cells, including bulge and interfollicular SCs (
      • Blanpain C.
      • Lowry W.E.
      • Geoghegan A.
      • Polak L.
      • Fuchs E.
      Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche.
      ,
      • Jiang S.
      • Zhao L.
      • Purandare B.
      • Hantash B.M.
      Differential expression of stem cell markers in human follicular bulge and interfollicular epidermal compartments.
      ,
      • Terunuma A.
      • Jackson K.L.
      • Kapoor V.
      • Telford W.G.
      • Vogel J.C.
      Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells.
      ), and also serves as an SCC CSC marker (
      • Schober M.
      • Fuchs E.
      Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-beta and integrin/focal adhesion kinase (FAK) signaling.
      ,
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ). CD44 is high in SCC CSCs (
      • Lapouge G.
      • Beck B.
      • Nassar D.
      • Dubois C.
      • Dekoninck S.
      • Blanpain C.
      Skin squamous cell carcinoma propagating cells increase with tumour progression and invasiveness.
      ,
      • Malanchi I.
      • Peinado H.
      • Kassen D.
      • Hussenet T.
      • Metzger D.
      • Chambon P.
      • et al.
      Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling.
      ,
      • Prince M.E.
      • Sivanandan R.
      • Kaczorowski A.
      • Wolf G.T.
      • Kaplan M.J.
      • Dalerba P.
      • et al.
      Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma.
      ). CD133, a cell surface marker specific for hematopoietic SCs (
      • Yin A.H.
      • Miraglia S.
      • Zanjani E.D.
      • Almeida-Porada G.
      • Ogawa M.
      • Leary A.G.
      • et al.
      AC133, a novel marker for human hematopoietic stem and progenitor cells.
      ), was the first cell surface marker used to define tumor-initiating cells in human cutaneous SCC (
      • Patel G.K.
      • Yee C.L.
      • Terunuma A.
      • Telford W.G.
      • Voong N.
      • Yuspa S.H.
      • et al.
      Identification and characterization of tumor-initiating cells in human primary cutaneous squamous cell carcinoma.
      ). In addition to cell surface markers, CSCs in SCCs can be sorted based on their aldehyde dehydrogenase (ALDH) and ABC transporter activity (
      • Clay M.R.
      • Tabor M.
      • Owen J.H.
      • Carey T.E.
      • Bradford C.R.
      • Wolf G.T.
      • et al.
      Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehydrogenase.
      ,
      • Yang L.
      • Ren Y.
      • Yu X.
      • Qian F.
      • Bian B.S.
      • Xiao H.L.
      • et al.
      ALDH1A1 defines invasive cancer stem-like cells and predicts poor prognosis in patients with esophageal squamous cell carcinoma.
      ). The side population (SP) assay identifies stem-like cells based on their ability to pump out Hoechst dye and chemotherapeutic drugs via ABC transporters (
      • Goodell M.A.
      • Brose K.
      • Paradis G.
      • Conner A.S.
      • Mulligan R.C.
      Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.
      ,
      • Zhang P.
      • Zhang Y.
      • Mao L.
      • Zhang Z.
      • Chen W.
      Side population in oral squamous cell carcinoma possesses tumor stem cell phenotypes.
      ), and has been used as a marker for both normal skin SCs and SCC CSCs (
      • Larderet G.
      • Fortunel N.O.
      • Vaigot P.
      • Cegalerba M.
      • Maltere P.
      • Zobiri O.
      • et al.
      Human side population keratinocytes exhibit long-term proliferative potential and a specific gene expression profile and can form a pluristratified epidermis.
      ,
      • Song J.
      • Chang I.
      • Chen Z.
      • Kang M.
      • Wang C.Y.
      Characterization of side populations in HNSCC: highly invasive, chemoresistant and abnormal Wnt signaling.
      ,
      • Tabor M.H.
      • Clay M.R.
      • Owen J.H.
      • Bradford C.R.
      • Carey T.E.
      • Wolf G.T.
      • et al.
      Head and neck cancer stem cells: the side population.
      ,
      • Wan G.
      • Zhou L.
      • Xie M.
      • Chen H.
      • Tian J.
      Characterization of side population cells from laryngeal cancer cell lines.
      ,
      • Zhang P.
      • Zhang Y.
      • Mao L.
      • Zhang Z.
      • Chen W.
      Side population in oral squamous cell carcinoma possesses tumor stem cell phenotypes.
      ). SP cells are distinct from CD4f+ keratinocyte stem cells (
      • Terunuma A.
      • Jackson K.L.
      • Kapoor V.
      • Telford W.G.
      • Vogel J.C.
      Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells.
      ) and SCC CSCs (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ). As discussed below, CSC markers can be altered by CSC plasticity and interactions with their niche. For instance, CD44+ SCC CSCs express other CSC markers with a high degree of variability (
      • Krishnamurthy S.
      • Dong Z.
      • Vodopyanov D.
      • Imai A.
      • Helman J.I.
      • Prince M.E.
      • et al.
      Endothelial cell-initiated signaling promotes the survival and self-renewal of cancer stem cells.
      ).

      Determinants of Skin SC and CSC Behavior

      Genetic and epigenetic modification

      Within the same SC compartment, different genetic mutations have distinct effects on CSC behavior. For example, a KrasG12D mutation in keratin 15 (K15)+ bulge SCs initiates benign papillomas in genetically engineered mouse models, but requires the loss of an additional tumor suppressor to induce SCC (
      • Lapouge G.
      • Youssef K.K.
      • Vokaer B.
      • Achouri Y.
      • Michaux C.
      • Sotiropoulou P.A.
      • et al.
      Identifying the cellular origin of squamous skin tumors.
      ,
      • Nassar D.
      • Latil M.
      • Boeckx B.
      • Lambrechts D.
      • Blanpain C.
      Genomic landscape of carcinogen-induced and genetically induced mouse skin squamous cell carcinoma.
      ,
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ). When combined with radiation, a heterozygous Ptch deletion is sufficient to induce basal cell carcinoma in K15+ cells, and is exacerbated by the additional loss of p53 (
      • Wang G.Y.
      • Wang J.
      • Mancianti M.L.
      • Epstein Jr., E.H.
      Basal cell carcinomas arise from hair follicle stem cells in Ptch1(+/−) mice.
      ). We found that a KrasG12D mutation in combination with Smad4 deletion not only caused metastatic SCCs from K15+ cells, but also produced tumors of other lineages, such as basal cell carcinomas, trichoepitheliomas, and sebaceous adenomas (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ). However, because the K15 promoter could create leaky Cre expression, targeted mutations may not be limited to bulge stem cells. Nevertheless, not all genetic mutations caused multilineage tumor types despite being driven by the same K15 promoter, suggesting that specific stem cell mutations play an important role in determining tumor lineages. To validate if bulge stem cells are the source of tumor-initiating cells, additional bulge stem cell markers described in the Sorting CSCs section are used in studies summarized in Table 1.
      Table 1Different tumor types develop in mouse models with genetic alteration in K15+ cells
      Genetic alteration in K15+ cellsTumor type
      KrasG12DPapilloma (
      • Lapouge G.
      • Youssef K.K.
      • Vokaer B.
      • Achouri Y.
      • Michaux C.
      • Sotiropoulou P.A.
      • et al.
      Identifying the cellular origin of squamous skin tumors.
      )
      KrasG12D/p53−/−SCC (
      • 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.
      )
      KrasG12D/p53−/−/Pten−/−SCC (
      • Nassar D.
      • Latil M.
      • Boeckx B.
      • Lambrechts D.
      • Blanpain C.
      Genomic landscape of carcinogen-induced and genetically induced mouse skin squamous cell carcinoma.
      )
      Ptch+/− + irradiationBCC (
      • Wang G.Y.
      • Wang J.
      • Mancianti M.L.
      • Epstein Jr., E.H.
      Basal cell carcinomas arise from hair follicle stem cells in Ptch1(+/−) mice.
      )
      KrasG12D/Smad4−/−SCC and metastases, BCC, sebaceous adenoma (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      )
      Abbreviations: BCC, basal cell carcinoma; K15, keratin 15; SCC, squamous cell carcinoma.
      Epigenetic regulation, including DNA methylation, histone acetylation, and miRNA expression, also plays an important role in skin SC and CSC behaviors. For example, enhancer of zeste homolog 2 is a major epigenetic component of polycomb repressive complex 2 and is required for epidermal CSC survival, migration, invasion, and tumor formation (
      • Adhikary G.
      • Grun D.
      • Balasubramanian S.
      • Kerr C.
      • Huang J.M.
      • Eckert R.L.
      Survival of skin cancer stem cells requires the Ezh2 polycomb group protein.
      ,
      • Banerjee R.
      • Mani R.S.
      • Russo N.
      • Scanlon C.S.
      • Tsodikov A.
      • Jing X.
      • et al.
      The tumor suppressor gene rap1GAP is silenced by miR-101-mediated EZH2 overexpression in invasive squamous cell carcinoma.
      ). miRNAs can also maintain SC populations. For example, miR-205 enhances phosphoinositide 3-kinase (PI3K) signaling and is required for the expansion of neonatal skin SCs (
      • Wang D.
      • Zhang Z.
      • O'Loughlin E.
      • Wang L.
      • Fan X.
      • Lai E.C.
      • et al.
      MicroRNA-205 controls neonatal expansion of skin stem cells by modulating the PI(3)K pathway.
      ). miR-203, the most abundant miRNA in normal skin, is downregulated by the Hras oncogene, and silencing of miR-203 is an early event in mouse and human SCC (
      • Riemondy K.
      • Wang X.J.
      • Torchia E.C.
      • Roop D.R.
      • Yi R.
      MicroRNA-203 represses selection and expansion of oncogenic Hras transformed tumor initiating cells.
      ). miR-203 limits cell division in both early embryonic skin development and SCC CSCs, and its loss caused an expansion of CSCs, resulting in increased tumorigenesis in an experimental skin carcinogenesis model (
      • Riemondy K.
      • Wang X.J.
      • Torchia E.C.
      • Roop D.R.
      • Yi R.
      MicroRNA-203 represses selection and expansion of oncogenic Hras transformed tumor initiating cells.
      ). Furthermore, we found that miR-9 overexpression contributes to the expansion of metastasis-associated CSCs by inhibiting α-catenin and subsequent Wnt activation (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ).

      Location and microenvironment

      As discussed above, mutations in hair follicle bulge SCs potentially cause tumor formation representing lineages of the epidermis, hair follicles, and sebaceous glands, whereas lineage-committed mutant SCs only generate tumor types from that lineage. For instance, mutant interfollicular SCs typically generate SCCs, mutant sebaceous gland SCs cause sebaceous tumors, and mutant transit-amplifying cells of the hair follicles cause hair follicle tumors (
      • Owens D.M.
      • Watt F.M.
      Contribution of stem cells and differentiated cells to epidermal tumours.
      ). The microenvironment controls CSC fate via cell-cell interactions between CSCs, tumor cells, and the neighboring stroma, including immune cells, cancer-associated fibroblasts, and endothelial cells. A mouse model of SCC showed that CSCs exist in a vascular niche, and vascular endothelial growth factor secreted by endothelial cells is associated with their expansion (
      • Beck B.
      • Driessens G.
      • Goossens S.
      • Youssef K.K.
      • Kuchnio A.
      • Caauwe A.
      • et al.
      A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours.
      ). SCC CSCs in close proximity to endothelial tumor cells often express high levels of SOX2, which promotes the expansion of SCC CSCs along the tumor-stroma interface (
      • Siegle J.M.
      • Basin A.
      • Sastre-Perona A.
      • Yonekubo Y.
      • Brown J.
      • Sennett R.
      • et al.
      SOX2 is a cancer-specific regulator of tumour initiating potential in cutaneous squamous cell carcinoma.
      ). Neighboring stromal cells provide cues to regulate the cell cycle of SCC CSCs (
      • Schober M.
      • Fuchs E.
      Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-beta and integrin/focal adhesion kinase (FAK) signaling.
      ), demonstrated by a study showing that transforming growth factor-β secreted by neighboring endothelial and stromal cells bestowed slower cycling properties to SCC CSCs in mice (
      • Oshimori N.
      • Oristian D.
      • Fuchs E.
      TGF-beta promotes heterogeneity and drug resistance in squamous cell carcinoma.
      ).

      Quiescence paradox

      Adult SCs and certain CSC populations are typically quiescent (
      • Fuchs E.
      The tortoise and the hair: slow-cycling cells in the stem cell race.
      ). Quiescence in normal SCs limits proliferation and protects genomic integrity (
      • Coller H.A.
      • Sang L.
      • Roberts J.M.
      A new description of cellular quiescence.
      ,
      • Sang L.
      • Coller H.A.
      • Roberts J.M.
      Control of the reversibility of cellular quiescence by the transcriptional repressor HES1.
      ,
      • Viatour P.
      • Somervaille T.C.
      • Venkatasubrahmanyam S.
      • Kogan S.
      • McLaughlin M.E.
      • Weissman I.L.
      • et al.
      Hematopoietic stem cell quiescence is maintained by compound contributions of the retinoblastoma gene family.
      ,
      • 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.
      ). The tumor suppressor phosphatase and tensin homolog (PTEN) plays a role in maintaining quiescence in hair follicle SCs, even in the presence of tumorigenic stimuli (
      • 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.
      ). However, quiescent CSCs may contribute to cancer progression by increasing epithelial to mesenchymal transition, enhancing colony formation, invasion, and tumor initiation (
      • Moore N.
      • Lyle S.
      Quiescent, slow-cycling stem cell populations in cancer: a review of the evidence and discussion of significance.
      ). In a chemical carcinogenesis SCC model, tumors grow from the skin after rapidly proliferating epidermal cells were killed by the chemotherapeutic agent 5-fluorouracil, suggesting that tumors rise from quiescent CSCs (
      • Morris R.J.
      • Coulter K.
      • Tryson K.
      • Steinberg S.R.
      Evidence that cutaneous carcinogen-initiated epithelial cells from mice are quiescent rather than actively cycling.
      ). Quiescent CSCs are delayed in entering late S phase and have high DNA repair activity, making them more resistant to therapeutics that inhibit cell cycle progression or promote DNA damage-induced cell death, encompassing the mechanisms of many chemotherapeutic drugs and radiation therapy (
      • Ahsan A.
      • Hiniker S.M.
      • Davis M.A.
      • Lawrence T.S.
      • Nyati M.K.
      Role of cell cycle in epidermal growth factor receptor inhibitor-mediated radiosensitization.
      ,
      • Masunaga S.
      • Ono K.
      • Abe M.
      A method for the selective measurement of the radiosensitivity of quiescent cells in solid tumors—combination of immunofluorescence staining to BrdU and micronucleus assay.
      ,
      • Oshimori N.
      • Oristian D.
      • Fuchs E.
      TGF-beta promotes heterogeneity and drug resistance in squamous cell carcinoma.
      ). Indeed, rescuing hematopoietic SCs from a quiescent state increased their sensitivity to 5-fluorouracil (
      • Essers M.A.
      • Offner S.
      • Blanco-Bose W.E.
      • Waibler Z.
      • Kalinke U.
      • Duchosal M.A.
      • et al.
      IFNalpha activates dormant haematopoietic stem cells in vivo.
      ).

      CSC Plasticity and Heterogeneity

      CSCs display functional heterogeneity that is less location dependent than normal SCs. In the dynamic CSC model, CSCs and non-CSCs can interconvert in response to environmental cues, such as secreted factors within the niche that activate downstream signaling. For instance, hepatocyte growth factor secreted from myofibroblasts activates Wnt signaling that converted non-CSCs to CSCs in a human colon cancer transplant model (
      • Vermeulen L.
      • De Sousa E.M.F.
      • van der Heijden M.
      • Cameron K.
      • de Jong J.H.
      • Borovski T.
      • et al.
      Wnt activity defines colon cancer stem cells and is regulated by the microenvironment.
      ). In human breast cancer CSC transplant models, IL-6 secretion converts non-CSCs to CSCs (
      • Iliopoulos D.
      • Hirsch H.A.
      • Wang G.
      • Struhl K.
      Inducible formation of breast cancer stem cells and their dynamic equilibrium with non-stem cancer cells via IL6 secretion.
      ), and transforming growth factor-β activates Zeb1 transcription, causing conversion of CD44low cells to CD44high CSCs (
      • Chaffer C.L.
      • Marjanovic N.D.
      • Lee T.
      • Bell G.
      • Kleer C.G.
      • Reinhardt F.
      • et al.
      Poised chromatin at the ZEB1 promoter enables breast cancer cell plasticity and enhances tumorigenicity.
      ). Because of this “dynamic stemness,” SCC tumors likely contain multiple CSC populations with distinct characteristics. One study found that CSCs from human SCCs fall into two phenotypes: one was similar to normal epithelial SCs and associated with growth and proliferation, whereas the other became migratory (
      • Biddle A.
      • Liang X.
      • Gammon L.
      • Fazil B.
      • Harper L.J.
      • Emich H.
      • et al.
      Cancer stem cells in squamous cell carcinoma switch between two distinct phenotypes that are preferentially migratory or proliferative.
      ). Subpopulations of metastatic CSCs have also been identified, suggesting that CSCs might be the “lethal seeds” responsible for metastasis (
      • Hermann P.C.
      • Huber S.L.
      • Herrler T.
      • Aicher A.
      • Ellwart J.W.
      • Guba M.
      • et al.
      Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.
      ,
      • Pang R.
      • Law W.L.
      • Chu A.C.
      • Poon J.T.
      • Lam C.S.
      • Chow A.K.
      • et al.
      A subpopulation of CD26+ cancer stem cells with metastatic capacity in human colorectal cancer.
      ,
      • Patel S.A.
      • Dave M.A.
      • Murthy R.G.
      • Helmy K.Y.
      • Rameshwar P.
      Metastatic breast cancer cells in the bone marrow microenvironment: novel insights into oncoprotection.
      ,
      • Sun S.
      • Wang Z.
      ALDH high adenoid cystic carcinoma cells display cancer stem cell properties and are responsible for mediating metastasis.
      ). We found two distinct CSC populations (SP and CD34+CD49f+) in a mouse SCC model developed by Smad4 deletion and KrasG12D activation in K15+ SCs (K15.KrasG12D.Smad4−/−). Although both SP and CD34+/CD49f+ CSC populations were tumorigenic, only tumors initiated by SP cells underwent epithelial to mesenchymal transition and metastasized to the lung (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ).

      Modeling Human SCC in Mice

      The most widely used SCC models are genetically engineered mouse models and human tumor xenograft models. Genetically engineered mouse models (examples in Table 1) phenotypically and histologically mimic human SCC and are powerful tools for dissecting driver mutations that contribute to tumorigenesis and metastasis. Some driver mutations found in human SCCs are required to initiate SCCs in mouse models (Table 1), but some oncogenic mutations, such as p53, are insufficient to cause SCC. This is also observed in human skin, which normally harbors many oncogenic driver mutations (
      • Martincorena I.
      • Roshan A.
      • Gerstung M.
      • Ellis P.
      • Van Loo P.
      • McLaren S.
      • et al.
      Tumor evolution. High burden and pervasive positive selection of somatic mutations in normal human skin.
      ). Therefore, mouse models provide a valuable tool to identify which combinations of driver mutations initiate SCC (examples in Table 1). Compared with the mutation burden of 2–6 mutations/Mb/cell in chronically UV damaged and aged skin (
      • Martincorena I.
      • Roshan A.
      • Gerstung M.
      • Ellis P.
      • Van Loo P.
      • McLaren S.
      • et al.
      Tumor evolution. High burden and pervasive positive selection of somatic mutations in normal human skin.
      ), the mutation burden in cutaneous SCC ranges from 1 to 380 mutations/Mb or averaged 61.2 mutations/Mb depending on sample sources (
      • Martincorena I.
      • Roshan A.
      • Gerstung M.
      • Ellis P.
      • Van Loo P.
      • McLaren S.
      • et al.
      Tumor evolution. High burden and pervasive positive selection of somatic mutations in normal human skin.
      ,
      • 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.
      ). Similarly, mouse SCCs initiated by few oncogenic driver mutations typically harbor numerous subsequent genetic alterations (
      • Bornstein S.
      • White R.
      • Malkoski S.
      • Oka M.
      • Han G.
      • Cleaver T.
      • et al.
      Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation.
      ,
      • Torchia E.C.
      • Caulin C.
      • Acin S.
      • Terzian T.
      • Kubick B.J.
      • Box N.F.
      • et al.
      Myc, Aurora Kinase A, and mutant p53(R172H) co-operate in a mouse model of metastatic skin carcinoma.
      ). UVB signature p53 mutations were found in approximately 58% of cutaneous SCCs (
      • Brash D.E.
      • Rudolph J.A.
      • Simon J.A.
      • Lin A.
      • McKenna G.J.
      • Baden H.P.
      • et al.
      A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.
      ), and Ras mutations (9% Hras, 7% Nras, and 5% Kras) were detected in 21% of cutaneous SCCs (
      • Bamford S.
      • Dawson E.
      • Forbes S.
      • Clements J.
      • Pettett R.
      • Dogan A.
      • et al.
      The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website.
      ). In mice, the combination of KrasG12D and p53 deletion in bulge SCs or their hair follicle progeny is required to induce skin SCC (
      • Lapouge G.
      • Youssef K.K.
      • Vokaer B.
      • Achouri Y.
      • Michaux C.
      • Sotiropoulou P.A.
      • et al.
      Identifying the cellular origin of squamous skin tumors.
      ). Smad4 expression is lost in 70% of patients with SCC (
      • Hoot K.E.
      • Lighthall J.
      • Han G.
      • Lu S.L.
      • Li A.
      • Ju W.
      • et al.
      Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression.
      ), and K15.KrasG12D.Smad4−/− mice developed metastatic SCCs with stage-specific histotypes including epithelial hyperplasia, dysplasia, and primary and metastatic SCC, which are comparable to human lesions and tumor progression (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ).
      Although it is commonly believed that cutaneous SCC arises from the interfollecular epidermis, follicular SCC, which is derived from a pre-existing hair follicle structure (e.g., from the scalp), may arise from bulge SCs (
      • Shendrik I.
      • Crowson A.N.
      • Magro C.M.
      Follicular cutaneous squamous cell carcinoma: an under-recognized neoplasm arising from hair appendage structures.
      ). Because it is difficult to confirm the origin of these tumors in patients, mouse models of SCC arising from mutations in bulge SCs provide unique tools for lineage tracing of these mutant bulge cells. Among them, K15.KrasG12D.Smad4−/− mice bearing SCCs developed lung metastases (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ). However, when Smad4 deletions were targeted to the epidermis by promoters not restricted to bulge SCs, such as K5, K14, and mouse mammary tumor virus (MMTV), the resulting SCCs failed to metastasize to the lung, even in the presence of spontaneous Ras activating mutations (
      • Bornstein S.
      • White R.
      • Malkoski S.
      • Oka M.
      • Han G.
      • Cleaver T.
      • et al.
      Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation.
      ,
      • Owens P.
      • Engelking E.
      • Han G.
      • Haeger S.M.
      • Wang X.J.
      Epidermal Smad4 deletion results in aberrant wound healing.
      ,
      • Qiao W.
      • Li A.G.
      • Owens P.
      • Xu X.
      • Wang X.J.
      • Deng C.X.
      Hair follicle defects and squamous cell carcinoma formation in Smad4 conditional knockout mouse skin.
      ,
      • Yang L.
      • Li W.
      • Wang S.
      • Wang L.
      • Li Y.
      • Yang X.
      • et al.
      Smad4 disruption accelerates keratinocyte reepithelialization in murine cutaneous wound repair.
      ). In those models, SCCs mimic interfollicular SCCs or oral SCCs in human patients, which can arise from many different progenitor cell locations. Taken together, genetic alterations in bulge SCs appear to result in more aggressive SCCs than the same genetic alterations in broader keratinocyte populations.
      Human SCC xenografts are commonly used to characterize CSCs. However, these models are limited by a lack of native tumor stroma and interactions with an intact immune system. To overcome these constraints, humanized xenograft models are being developed. One example is XactMice, in which the bone marrow of nonobese diabetic/severe combined immunodeficiency (NOD/SCID)/IL2rg−/− mice is replaced with human hematopoietic stem and progenitor cells (
      • Morton J.J.
      • Bird G.
      • Keysar S.B.
      • Astling D.P.
      • Lyons T.R.
      • Anderson R.T.
      • et al.
      XactMice: humanizing mouse bone marrow enables microenvironment reconstitution in a patient-derived xenograft model of head and neck cancer.
      ). Tumors grown in XactMice contained infiltrated human CD45+ hematopoietic cells, including CD3+ T cells, CD4+ T-helper cells, CD19+ B cells, and α-smooth muscle actin-positive cells (activated fibroblasts) (
      • Morton J.J.
      • Bird G.
      • Keysar S.B.
      • Astling D.P.
      • Lyons T.R.
      • Anderson R.T.
      • et al.
      XactMice: humanizing mouse bone marrow enables microenvironment reconstitution in a patient-derived xenograft model of head and neck cancer.
      ). Moreover, tumors can at least partially reverse the genetic drift observed in classical patient-derived xenograft models (
      • Tentler J.J.
      • Tan A.C.
      • Weekes C.D.
      • Jimeno A.
      • Leong S.
      • Pitts T.M.
      • et al.
      Patient-derived tumour xenografts as models for oncology drug development.
      ), and RNA sequencing data suggested that stromal signatures in XactMice SCCs revert back to signatures similar to primary SCCs (
      • Morton J.J.
      • Bird G.
      • Keysar S.B.
      • Astling D.P.
      • Lyons T.R.
      • Anderson R.T.
      • et al.
      XactMice: humanizing mouse bone marrow enables microenvironment reconstitution in a patient-derived xenograft model of head and neck cancer.
      ). Thus, XactMice provide an advanced model to study human SCC CSCs and experimental therapeutics within a native stromal environment.

      Therapeutically Targeting CSCs

      Strategies to target CSCs are being explored using experimental therapeutics and clinical trials in the following areas.

      Targeting ABC transporter proteins

      ABC transporter overexpression leads to higher drug efflux and therapeutic resistance in SCC. SCC SP cells display increased ABCG2 expression and activity, which may mediate resistance to diverse cancer drugs including platinum compounds, bortezomib, and 5-fluorouracil (
      • Sun G.
      • Fujii M.
      • Sonoda A.
      • Tokumaru Y.
      • Matsunaga T.
      • Habu N.
      Identification of stem-like cells in head and neck cancer cell lines.
      ,
      • Tabor M.H.
      • Clay M.R.
      • Owen J.H.
      • Bradford C.R.
      • Carey T.E.
      • Wolf G.T.
      • et al.
      Head and neck cancer stem cells: the side population.
      ,
      • Yajima T.
      • Ochiai H.
      • Uchiyama T.
      • Takano N.
      • Shibahara T.
      • Azuma T.
      Resistance to cytotoxic chemotherapy-induced apoptosis in side population cells of human oral squamous cell carcinoma cell line Ho-1-N-1.
      ,
      • Yanamoto S.
      • Kawasaki G.
      • Yamada S.
      • Yoshitomi I.
      • Kawano T.
      • Yonezawa H.
      • et al.
      Isolation and characterization of cancer stem-like side population cells in human oral cancer cells.
      ). Indeed, SCC cells selected for high cisplatin resistance show enhanced ABCG2 expression and a CSC-like phenotype (
      • Tsai L.L.
      • Yu C.C.
      • Chang Y.C.
      • Yu C.H.
      • Chou M.Y.
      Markedly increased Oct4 and Nanog expression correlates with cisplatin resistance in oral squamous cell carcinoma.
      ), and SCC SP cells become sensitized to chemotherapy on general inhibition of ABC transporters by the calcium channel blocker verapamil (
      • Loebinger M.R.
      • Giangreco A.
      • Groot K.R.
      • Prichard L.
      • Allen K.
      • Simpson C.
      • et al.
      Squamous cell cancers contain a side population of stem-like cells that are made chemosensitive by ABC transporter blockade.
      ). Our observation that SP cells were associated with SCC metastasis prompted us to investigate how to therapeutically target them. In our study, K15.KrasG12D.Smad4−/− SCCs were treated with docetaxel, a first-line chemotherapeutic for SCC, alone or in combination with verapamil. Although docetaxel alone had no effect, the combination with verapamil significantly reduced lung metastasis (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ). Although a number of other inhibitors specific for ABC transporters have been identified, including MS-209, PSC833, VX710, and the third-generation inhibitor tariquidar (
      • Dean M.
      • Fojo T.
      • Bates S.
      Tumour stem cells and drug resistance.
      ,
      • Modok S.
      • Mellor H.R.
      • Callaghan R.
      Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer.
      ), their use in clinical trials has yielded largely negative results or been terminated early because of an increased incidence of adverse effects (
      • Pusztai L.
      • Wagner P.
      • Ibrahim N.
      • Rivera E.
      • Theriault R.
      • Booser D.
      • et al.
      Phase II study of tariquidar, a selective P-glycoprotein inhibitor, in patients with chemotherapy-resistant, advanced breast carcinoma.
      ). Because CSCs rely on several mechanisms to escape drug sensitivity, ABC transporter inhibitors will need to be combined with other strategies to efficiently eliminate CSCs in vivo, and safer ABC transporter inhibitors need to be developed.

      Inhibiting Wnt signaling

      Wnt activation plays an important role in SCC CSC maintenance as depleting β-catenin, a key component of Wnt signaling that is activated in human SCCs, in mouse SCCs initiated by CD34+ CSCs resulted in tumor regression (
      • Malanchi I.
      • Peinado H.
      • Kassen D.
      • Hussenet T.
      • Metzger D.
      • Chambon P.
      • et al.
      Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling.
      ,
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ). Further, we have found that Wnt signaling is linked to metastasis-associated CSCs because knocking down miR-9, a miRNA responsible for activating Wnt signaling and expanding SP CSCs, reduced SCC lung metastases in mice (
      • White R.A.
      • Neiman J.M.
      • Reddi A.
      • Han G.
      • Birlea S.
      • Mitra D.
      • et al.
      Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.
      ). Currently, although no Wnt antagonists are in clinical trials for treating SCC, the Wnt inhibitor LGK974 is undergoing a phase I trial to treat a variety of other cancers, such as melanoma, breast cancer, and pancreatic adenocarcinoma (http://clinicaltrials.gov/show/NCT01351103), and could provide insight for future SCC treatment strategies.

      Enhancing antitumor immunity

      Immune escape is defined by the inability of the immune system to recognize and eliminate transformed cells during disease progression. Immune escape is essential for SCC development, and organ transplant recipients with immune suppression demonstrated a 50- to 100-fold increased risk of SCC (
      • Harwood C.A.
      • McGregor J.M.
      • Swale V.J.
      • Proby C.M.
      • Leigh I.M.
      • Newton R.
      • et al.
      High frequency and diversity of cutaneous appendageal tumors in organ transplant recipients.
      ). Additionally, high expression of ABC transporters, such as ABCB5, have immune suppressive effects in addition to their role in chemoresistance as mentioned above (
      • Schatton T.
      • Yang J.
      • Kleffel S.
      • Uehara M.
      • Barthel S.R.
      • Schlapbach C.
      • et al.
      ABCB5 identifies immunoregulatory dermal cells.
      ). Thus, targeting ABCB5 may reduce both chemoresistance and immune evasion. ALDH is a potential CD8+ T-cell antigen in SCC (
      • Visus C.
      • Ito D.
      • Amoscato A.
      • Maciejewska-Franczak M.
      • Abdelsalem A.
      • Dhir R.
      • et al.
      Identification of human aldehyde dehydrogenase 1 family member A1 as a novel CD8+ T-cell-defined tumor antigen in squamous cell carcinoma of the head and neck.
      ), and ALDHhigh CD8+ T cells were effective against models of nonsmall cell lung cancer (
      • Luo H.
      • Zeng C.
      • Fang C.
      • Seeruttun S.R.
      • Lv L.
      • Wang W.
      A new strategy using ALDHhigh-CD8+T cells to inhibit tumorigenesis.
      ). Because ALDH is also a SCC CSC biomarker (
      • Adhikary G.
      • Grun D.
      • Kerr C.
      • Balasubramanian S.
      • Rorke E.A.
      • Vemuri M.
      • et al.
      Identification of a population of epidermal squamous cell carcinoma cells with enhanced potential for tumor formation.
      ), stimulating a CD8+ T-cell response against ALDH antigens may preferentially eliminate CSCs. The development of cancer vaccines that target CSCs through dendritic and other antigen-presenting cells is also promising (
      • Li Q.
      • Prince M.E.
      • Moyer J.S.
      Immunotherapy for head and neck squamous cell carcinoma.
      ,
      • Ning N.
      • Pan Q.
      • Zheng F.
      • Teitz-Tennenbaum S.
      • Egenti M.
      • Yet J.
      • et al.
      Cancer stem cell vaccination confers significant antitumor immunity.
      ,
      • Xu Q.
      • Liu G.
      • Yuan X.
      • Xu M.
      • Wang H.
      • Ji J.
      • et al.
      Antigen-specific T-cell response from dendritic cell vaccination using cancer stem-like cell-associated antigens.
      ), but it remains to be determined if immunotherapy will be effective at eradicating CSCs in SCCs.

      Conclusions

      Our understanding of CSCs in cutaneous SCC can be summarized as follows: first, although SCs and CSCs share some features, CSC heterogeneity is less location dependent when compared with normal SCs. Second, genetic alterations in bulge SCs may cause more aggressive SCCs than the same genetic alterations in broader keratinocyte populations. Third, the capacity for tumor initiation by CSCs may not always be linked to their metastatic potential. Finally, although CSCs may be difficult to eradicate, therapeutic interventions can be designed to target specific functions of CSC populations responsible for metastasis. We foresee that these research discoveries will translate into CSC-targeted therapeutic interventions in the near future.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by NIH grants DE015953, CA87849, and DE024371. ZJ is a visiting scholar supported by the National Natural Science Foundation of China (No. 81402599).

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