Pilot Award (2004 Ballantyne Award) – Interim Report
- Role of Smad4 in HNSCC Formation and Progression
- Principal Investigator: Douglas D. Reh, MD
- Institution: Oregon Health & Science University
Loss of expression of Smad4, one of the major intracellular mediators for signal transduction of the transforming growth factor b (TGFb), frequently occurs in human HNSCCs. However, the exact role of this molecular alteration in HNSCC carcinogenesis remains unknown. We observed that loss of expression of Smad4 occurring at pre-transcriptional level is common with as high as 63% (22/35) in human HNSCC samples as examined by quantitative PCR. We then set up a K5CrePR1/Smad4 knockout system in which Smad4 can be inducibly deleted in head and neck epithelia. A total of 25 Smad4 homozygous (K5CreSmad4f/f), 16 heterozygous (K5CreSmad4f/w) and 18 monogenic (either K5CrePR1, Smad4f/f, Smad4f/w) were included in our study. We observed spontaneous epithelial proliferation in mouse head and neck epithelia with homozygous Smad4 deletion as early as 4 weeks after RU486 treatment. Deletion-specific PCR using DNA extracted from the tongue, buccal mucosa, and esophagus confirmed Smad4 deletion in all these tissues of K5.CrePR1/Smad4-f/f. Smad4 deletion in the K5CreSmad4f/f mice blocked TGFb-mediated growth inhibition resulting in epithelia hyperplasia confirmed on histologic analysis and by increased BrdU labeling index. However Smad4 homozygous deletion had no effect on apoptosis (as revealed by Tunel staining).
The Smad4 homozygous mice spontaneously developed HNSCC at 6 months. This spontaneous HSNCC formation occurred without treating the mice with an initiator such as DMBA, an environmental carcinogen that we used previously to induce HNSCC formation in the homozygous mice. In total about 70% of the Smad4 homozygous mice developed HNSCC, while none of the Smad4 heterozygous mice or monogenic mice developed HNSCC after 16 months of observation. About 26% of the affected mice developed metastatic HNSCC to jugular lymph nodes. Tumors arising from Smad4 knockout mice exhibited characteristics that recapitulate those of HNSCCs in humans. These characteristics include aberrant differentiation (loss of Keratin 13 and K1, and increased K18 expression), enlarged nuclei and massive mitosis in tumor cells, inflammation (as revealed by leukocytes marker staining), angiogenesis (as revealed by CD31 staining) and perineural invasion. Further characterization of the Smad4 knockout tumors showed an increased genetic instability, and activation of Stat3. The detailed molecular mechanisms are currently under investigation. Our result suggests a tumor suppressor role of Smad4 in HNSCC carcinogenesis in vivo. The result also shows the feasibility of developing metastatic HNSCC models using our inducible knockout system. We hope that these models can be used to test therapeutic approaches for HNSCCs in the future.
Expenditures for the First Year
Animal expense: 3000$
Taqman probes: 2000$
PCR/ enzymes/primers: 2000$