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 4

miR-9 promotes invasion and metastasis in mouse and human SCC cells.

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miR-9 promotes invasion and metastasis in mouse and human SCC cells. (A)...
(A) Increased invasion in FaDu-miR-9 cells compared with FaDu-GFP cells (*P = 0.005). (B) Reduced invasion in B931-Zip-9 cells compared with B931-GFP control cells (*P = 0.005). All invasion assays were performed in triplicate. (C) Immunofluorescence of membrane-associated E-cadherin and α-catenin in tumors derived from FaDu cells transduced with GFP or miR-9. K14 was used to counterstain. Scale bar: 30 μm. (D) H&E staining showing a representative metastasis derived from FaDu-GFP cells exhibiting clustered epithelial cell morphology, whereas a metastasis from FaDu-miR-9 cells displayed poorly differentiated and EMT morphology (more obvious in the magnified panels). Scale bars: 50 μm (upper panels), 8 μm (lower panels). (E) Quantification of metastases showed reduced numbers of metastases in B931-Zip-9 mice (n = 5) compared with B931-GFP control mice (n = 6). *P = 0.04 for both chest wall and lung metastases. Metastases were identified grossly and confirmed by H&E staining. (F) Examples of multiple metastases in the chest wall and lung developed from a B931-GFP mouse compared with a single chest wall metastasis in a B931-Zip-9 mouse. Arrowheads point to metastases in the thoracic cavity.