Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation
Sophia Bornstein, Ruth White, Stephen Malkoski, Masako Oka, Gangwen Han, Timothy Cleaver, Douglas Reh, Peter Andersen, Neil Gross, Susan Olson, Chuxia Deng, Shi-Long Lu, Xiao-Jing Wang
Sophia Bornstein, Ruth White, Stephen Malkoski, Masako Oka, Gangwen Han, Timothy Cleaver, Douglas Reh, Peter Andersen, Neil Gross, Susan Olson, Chuxia Deng, Shi-Long Lu, Xiao-Jing Wang
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Research Article

Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation

Abstract

Smad4 is a central mediator of TGF-β signaling, and its expression is downregulated or lost at the malignant stage in several cancer types. In this study, we found that Smad4 was frequently downregulated not only in human head and neck squamous cell carcinoma (HNSCC) malignant lesions, but also in grossly normal adjacent buccal mucosa. To gain insight into the importance of this observation, we generated mice in which Smad4 was deleted in head and neck epithelia (referred to herein as HN-Smad4–/– mice) and found that they developed spontaneous HNSCC. Interestingly, both normal head and neck tissue and HNSCC from HN-Smad4–/– mice exhibited increased genomic instability, which correlated with downregulated expression and function of genes encoding proteins in the Fanconi anemia/Brca (Fanc/Brca) DNA repair pathway linked to HNSCC susceptibility in humans. Consistent with this, further analysis revealed a correlation between downregulation of Smad4 protein and downregulation of the Brca1 and Rad51 proteins in human HNSCC. In addition to the above changes in tumor epithelia, both normal head and neck tissue and HNSCC from HN-Smad4–/– mice exhibited severe inflammation, which was associated with increased expression of TGF-β1 and activated Smad3. We present what we believe to be the first single gene–knockout model for HNSCC, in which both HNSCC formation and invasion occurred as a result of Smad4 deletion. Our results reveal an intriguing connection between Smad4 and the Fanc/Brca pathway and highlight the impact of epithelial Smad4 loss on inflammation.

Authors

Sophia Bornstein, Ruth White, Stephen Malkoski, Masako Oka, Gangwen Han, Timothy Cleaver, Douglas Reh, Peter Andersen, Neil Gross, Susan Olson, Chuxia Deng, Shi-Long Lu, Xiao-Jing Wang

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Figure 4

Abnormal centrosomes, increased genomic aberrations, and increased MMC sensitivity in HN-Smad4–/– mucosa and HNSCC.

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Abnormal centrosomes, increased genomic aberrations, and increased MMC s...
(A) Immunofluorescence for pericentrin (green or yellow). HN-Smad4–/– mucosa and HNSCCs have increased abnormal centrosome numbers compared with HN-Smad4+/+ mucosa and control HN-Smad4+/+ tumors (HN-K-rasG12D papillomas), respectively. K14 (red) was used to counterstain epithelial cells. 3–5 samples per group were analyzed, and a representative image is presented. Arrows highlight cells with at least 3 centrosomes. The histogram summarizes quantification of centrosome numbers. 100–200 cells per group were analyzed. Error bars indicate SEM. †P < 0.05 versus HN-Smad4+/+ tumors; *P < 0.05 versus HN-Smad4+/+ mucosa. Scale bar: 10 μm (all panels). (B) Chromosome 4 aCGH of 3 HN-Smad4–/– HNSCCs indicates that HN-Smad4–/– HNSCCs have several consistent genomic aberrations. The boxed region represents 2 copies of loss at chromosome 4qA5. (C) MMC sensitivity assay. Percent cell viability at increasing MMC concentrations indicates that Smad4–/– cells were significantly more sensitive to MMC than Smad4+/+ cells. The experiment was run in triplicate, and error bars indicate SEM. P < 0.05 for all data points, other than 0 ng/ml, versus Smad4+/+ cells. (D) Chromosome breakage assay. Plot of average chromosome breaks per cell for Smad4+/+ and Smad4–/– cells at increasing MMC concentrations indicates that Smad4–/– cells have increased chromosome breaks compared with Smad4+/+ cells. The experiments were run in triplicate, and error bars indicate SEM. P < 0.001 for all data points, other than 0 ng/ml, versus Smad4+/+ cells.