Epithelial stem cell mutations that promote squamous cell carcinoma metastasis
Ruth A. White, … , Dennis R. Roop, Xiao-Jing Wang
Ruth A. White, … , Dennis R. Roop, Xiao-Jing Wang
Published September 3, 2013
Citation Information: J Clin Invest. 2013;123(10):4390-4404. https://doi.org/10.1172/JCI65856.
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Research Article Oncology

Epithelial stem cell mutations that promote squamous cell carcinoma metastasis

Abstract

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene KrasG12D activation and Smad4 deletion, to mouse keratin 15–expressing (K15+) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell–enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9–transduced human SCC cells exhibited increased invasion. We identified α-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with α-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with KrasG12D activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs.

Authors

Ruth A. White, Jill M. Neiman, Anand Reddi, Gangwen Han, Stanca Birlea, Doyel Mitra, Laikuan Dionne, Pam Fernandez, Kazutoshi Murao, Li Bian, Stephen B. Keysar, Nathaniel B. Goldstein, Ningjing Song, Sophia Bornstein, Zheyi Han, Xian Lu, Joshua Wisell, Fulun Li, John Song, Shi-Long Lu, Antonio Jimeno, Dennis R. Roop, Xiao-Jing Wang

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Figure 2

K15.KrasG12D.Smad4–/– SCCs contained CSC-rich populations.

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K15.KrasG12D.Smad4–/– SCCs contained CSC-rich populations. (A) Immuno...
(A) Immunofluorescence staining of K15.KrasG12D.Smad4–/– tumors shows nests of K15+ cells within K14-expressing tumor cells. Middle panel: K15+ (green) cells are largely overlapping with proliferating PCNA+ cells (red). Bottom panel: K15+ (green) cells are distinct from differentiated K1-expressing keratinocytes (red). Scale bar: 50 μm. (B) Flow cytometry of the Hoechst dye–effluxing SP. SP cells are gated as those cells that are negative for Hoechst dye (left panel). Verapamil blocks the ABC transporter, eradicating the SP+ cells (right panel). (C) The non-SP cells were further sorted for the CD34+/CD49f+ population and a population negative for CSC cells (non-SP/CD34–/CD49f–). (D) Grafting of 10,000 SP+ cells formed tumors 3 weeks later, while 10,000 non-SP/CD34–/CD49f– cells did not form tumors. (E) Grafting of 10,000 CD34+/CD49f+ cells also formed tumors 3 weeks later. (F) A tumor derived from sorted SP cells (B and D) gave rise to both SP+ cells and CD34+/CD49+ populations. (G) A tumor derived from sorted CD34+/CD49+ cells (C and E) gave rise to both SP+ cells and CD34+/CD49+ populations.