Effects of BMP-2 and -7 on Oral Effects of BMP-2 and -7 on Oral Squamous Carcinoma CellsSquamous Carcinoma Cells

1University of Michigan School of Dentistry, Ann Arbor, Michigan, USA; 2Washington University School of Medicine, St. Louis,

Missouri, USA

1Steven Z. Edlund, DDS

2Brian Nussenbaum, MD

1Wilbur Tong, MS

1Paul H. Krebsbach, DDS, PhD

BackgroundBackgroundBone morphogenetic proteins (BMPs) are molecules in the Bone morphogenetic proteins (BMPs) are molecules in the TGF-TGF-ββ superfamily that were discovered based on their superfamily that were discovered based on their osteoinductive activity by Marshall Urist in 1965osteoinductive activity by Marshall Urist in 1965

rhBMP-2 and -7 are FDA-approved for limited orthopedic rhBMP-2 and -7 are FDA-approved for limited orthopedic indications (in absence of malignancy) based on phase III indications (in absence of malignancy) based on phase III multicenter randomized clinical trials multicenter randomized clinical trials

Surgical ablation of oral squamous cell carcinoma (SCCA) Surgical ablation of oral squamous cell carcinoma (SCCA) often results in a segmental bone defect requiring often results in a segmental bone defect requiring reconstruction. Current methods of bone reconstruction are reconstruction. Current methods of bone reconstruction are centered around free tissue transfer, but limitations still existcentered around free tissue transfer, but limitations still exist

Tissue engineering approaches using BMP protein or gene Tissue engineering approaches using BMP protein or gene therapy in the head and neck region would be quite therapy in the head and neck region would be quite appealing, but the effects of BMPs on SCCA are unknown.appealing, but the effects of BMPs on SCCA are unknown.

Statement of PurposeStatement of Purpose

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

Experimental DesignExperimental Design

In VitroIn Vitro

– Determine baseline expression profile of Determine baseline expression profile of BMP-2, BMP-7, and BMP receptorsBMP-2, BMP-7, and BMP receptors

– Measure the effects of BMP protein or gene Measure the effects of BMP protein or gene therapy on proliferation of human oral SCCAtherapy on proliferation of human oral SCCA

– Determine the effects of BMP protein or gene Determine the effects of BMP protein or gene therapy on the expression of pro-angiogenic therapy on the expression of pro-angiogenic factors by oral SCCAfactors by oral SCCA

Experimental DesignExperimental Design

In VivoIn Vivo

– Implantation into SCID mice of oral Implantation into SCID mice of oral squamous carcinoma cells genetically squamous carcinoma cells genetically modified to express BMP-7 modified to express BMP-7

– Harvest of implanted sites and Harvest of implanted sites and histopathologic evaluationhistopathologic evaluation

Experimental DesignExperimental Design

Cell lines studied:Cell lines studied:– UMSCC 14A: poorly differentiated T1N0M0 UMSCC 14A: poorly differentiated T1N0M0

recurrent floor of mouth lesionrecurrent floor of mouth lesion– UM-SCC 74A: poorly differentiated T3N0M0 UM-SCC 74A: poorly differentiated T3N0M0

recurrent lesion of the tongue recurrent lesion of the tongue

Gene therapy vectors used:Gene therapy vectors used:

– Replication-defective adenovirus with the Replication-defective adenovirus with the cDNA for BMP-2 or BMP-7. cDNA for BMP-2 or BMP-7.

Expression profile for cell linesExpression profile for cell lines

M 74A

14A

Mg6

3Sa

OS

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-774A treated with Ad BMP-7

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

74A treated with rhBMP-7 or AdBMP-774A treated with rhBMP-7 or AdBMP-7

MTT assay: rhBMP-7

AdBMP-7

1 2 3 40

1

2

30 moi

10 moi

100 moi

500 moi

1000 moi

time (days)

1 2 3 40

1

2

30 ng/m l

10 ng/ml

50 ng/ml

100 ng/ml

time (days)

14A treated with rhBMP-7 or AdBMP-714A treated with rhBMP-7 or AdBMP-7

MTT assay:

rhBMP-7

AdBMP-7

1 2 3 40

1

20 moi

10 moi

100 moi

500 moi

1000 moi

time (days)

1.0 2.0 3.0 4.00.0

0.5

1.0

1.50 ng/ml

10 ng/ml

50 ng/ml

100 ng/ml

time (days)

IL-8 and VEGF production IL-8 and VEGF production in response to rhBMP-7 or AdBMP7in response to rhBMP-7 or AdBMP7

IL-8IL-8 VEGFVEGF

14A BMP-714A BMP-7 No difference No difference (ND)(ND) NDND

14A AdBMP-714A AdBMP-7 NDND NDND

14A ADMT14A ADMT NDND NDND

74A BMP-774A BMP-7 NDND NDND

74A AdBMP-774A AdBMP-7 NDND NDND

74A ADMT74A ADMT NDND NDND

IL-8 and VEGF measured by ELISA in supernatant from treated

cells

In vivoIn vivo study study

Oral SCCA cell line 74A treated with AdBMP-7Oral SCCA cell line 74A treated with AdBMP-7

Fibroblasts treated with AdBMP-7Fibroblasts treated with AdBMP-7

1 x 101 x 1066 cells suspended in a collagen matrix cells suspended in a collagen matrix subcutaneously injected into the flank of a SCID subcutaneously injected into the flank of a SCID mousemouse

Mice sacrificed at 3 weeksMice sacrificed at 3 weeks

74A treated with Ad BMP-7 74A treated with Ad BMP-7 Bone - BMP - SCCA interactionsBone - BMP - SCCA interactions

Bone/tumor coexistBone/tumor coexistMarrow replaced by SCCAMarrow replaced by SCCAIncreased vascular response, increased osteoclastsIncreased vascular response, increased osteoclasts

m

b

AdBMP-7 treated fibroblastsAdBMP-7 treated fibroblasts

Cell express BMP-7 andInduce bone formation

ConclusionsConclusions

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