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 3

Increased SP cells, not CD34+/CD49f+ cells, in K15.KrasG12D.Smad4–/– SCCs correlate with metastasis, and SP cells in passaged K15.KrasG12D.Smad4–/– SCCs exhibit increased expression levels of miR-9 and Abcb1a.

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Increased SP cells, not CD34+/CD49f+ cells, in K15.KrasG12D.Smad4–/– SCC...
(A) Analysis of the SP size (percentage of viable cells sorted) in metastatic (n = 15) and nonmetastatic (n = 9) tumors. Diamonds indicate SP sizes of individual tumors and bars represent averages. *P = 0.009 compared with SP sizes in nonmetastatic SCCs. (B) Analysis of the CD34+/CD49f+ population size (percentage of viable cells sorted) in metastatic (n = 15) and nonmetastatic (n = 9) tumors. No significant difference was seen. (C) Increased miR-9 expression correlates with an increase in SP+ cells in passaged tumors (assayed by qRT-PCR). Expression values are displayed as normalized miR-9 cycle threshold values raised to the –1 power (nCt–1). Black diamonds represent miR-9 expression in individual tumor SP+ cells, and gray bars represent the average miR-9 expression within each group. *P = 0.002 compared with the primary SP. (D) miR-9 expression levels correlate with the SP sizes of tumors. Significance was determined by calculating the correlation coefficient (R2 = 0.87). (E) Increased Abcb1a expression in SP+ cells of passaged tumors (n = 8). *P = 0.03 compared with the primary SP. (F) IHC showing an increase in clustered MDR1+ cells in a passaged SCC compared with sporadic MDR1+ cells in a primary SCC. Scale bars: 50 μm (upper panels), 16 μm (lower panels).