Effects of BMP-2 and -7 on Oral Squamous Carcinoma Cells 1 University of Michigan School of Dentistry, Ann Arbor, Michigan, USA; 2 Washington University School of Medicine, St. Louis, Missouri, USA 1 Steven Z. Edlund, DDS 2 Brian Nussenbaum, MD 1 Wilbur Tong, MS 1 Paul H. Krebsbach, DDS, PhD

Background Bone morphogenetic proteins (BMPs) are molecules in the TGF-β superfamily that were discovered based on their osteoinductive activity by Marshall Urist in 1965 rhBMP-2 and -7 are FDA-approved for limited orthopedic indications (in absence of malignancy) based on phase III multicenter randomized clinical trials Surgical ablation of oral squamous cell carcinoma (SCCA) often results in a segmental bone defect requiring reconstruction. Current methods of bone reconstruction are centered around free tissue transfer, but limitations still exist Tissue engineering approaches using BMP protein or gene therapy in the head and neck region would be quite appealing, but the effects of BMPs on SCCA are unknown.

Statement of Purpose To determine the effects of BMP-2 and -7 on oral squamous carcinoma cells

Experimental Design In Vitro –Determine baseline expression profile of BMP-2, BMP-7, and BMP receptors –Measure the effects of BMP protein or gene therapy on proliferation of human oral SCCA –Determine the effects of BMP protein or gene therapy on the expression of pro-angiogenic factors by oral SCCA

Experimental Design In Vivo –Implantation into SCID mice of oral squamous carcinoma cells genetically modified to express BMP-7 –Harvest of implanted sites and histopathologic evaluation

Experimental Design Cell lines studied: –UMSCC 14A: poorly differentiated T1N0M0 recurrent floor of mouth lesion –UM-SCC 74A: poorly differentiated T3N0M0 recurrent lesion of the tongue Gene therapy vectors used: –Replication-defective adenovirus with the cDNA for BMP-2 or BMP-7.

Expression profile for cell lines M 74A 14A Mg63 SaOS BMP-2 BMP-7 BMPR-IA BMPR-IB BMPR-II ActR-I ActR-IIA ActR-IIB  -actin 500 bp > > > > > > > > > PCR data:

74A treated with Ad BMP-7 I didn’t include a slide here because, while the data shows what we want, it is scant and requires a bit of explaining with regards to why there is baseline expression of BMP 7 in untreated cells that don’t express BMP on PCR. I also have evidence (also low numbers) uptace of lacZ in 74A and no uptake od lacZ in 14A. My plan was to mention briefly that we have shown uptake and expression in 74A and we have shown no uptake and even cell death with exposure to the adenovirus.

74A treated with rhBMP-7 or AdBMP-7 MTT assay: rhBMP-7 AdBMP-7

14A treated with rhBMP-7 or AdBMP-7 MTT assay: rhBMP-7 AdBMP-7

IL-8 and VEGF production in response to rhBMP-7 or AdBMP7 IL-8 IL-8VEGF 14A BMP-7 No difference (ND) ND 14A AdBMP-7 NDND 14A ADMT NDND 74A BMP-7 NDND 74A AdBMP-7 NDND 74A ADMT NDND IL-8 and VEGF measured by ELISA in supernatant from treated cells

In vivo study Oral SCCA cell line 74A treated with AdBMP-7 Fibroblasts treated with AdBMP-7 1 x 10 6 cells suspended in a collagen matrix subcutaneously injected into the flank of a SCID mouse Mice sacrificed at 3 weeks

74A treated with Ad BMP-7 Bone - BMP - SCCA interactions Bone/tumor coexist Marrow replaced by SCCA Increased vascular response, increased osteoclasts

m b AdBMP-7 treated fibroblasts Cell express BMP-7 and Induce bone formation

Conclusions The oral carcinoma cells tested did not have baseline expression of BMP-2 or -7 but expressed BMP- receptors. Protein or gene therapy using BMP-2 or -7 did not affect in vitro cellular proliferation or stimulate secretion of pro- angiogenic molecules. Genetically modified oral SCCA can secrete biologically active BMPs. The in vivo effects on proliferation are still being studied. These are significant findings in the context of using BMP therapies (recombinant protein or gene therapy) for bone tissue engineering in oral cancer defects that might harbor microscopic residual disease