Dr. Francis Y. Lee joined the Orthopaedic Surgery department in 1999 after completing a Musculoskeletal Tumor Fellowship at the Massachusetts General Hospital and Boston Children's Hospital, and a Pediatric Orthopedic Surgery Fellowship at the Hospital for Sick Children in Toronto. He is currently an Assistant Professor of Orthopedic Surgery at Columbia University, specializing in pediatric orthopedic surgery including spine deformity and complex developmental orthopedic disorders, musculoskeletal tumors and bone disease. He serves as the Chief of the Tumor & Bone Disease service and Director of Center for Orthopedic Research. He is conducting both basic and clinical studies on musculoskeletal disorders. He is a recipient of American Academy of Orthopaedic Surgeons / Orthopaedic Research Education and Foundation Clinician Scientist Traveling Fellowship Award (2004-2005).
Dr. Lee was recently appointed Vice Chairman of Research in the Department of Orthpedic Surgery, and is one of a handful of orthopaedic surgeons with an NIH-R01 research grant.
CURRENT RESEARCH PROGRAMS
Dr. Lee, as an orthopaedic surgeon who has been involved in the care of patients with bone and soft tissue sarcomas, naturally thinks about a new paradigm for more efficacious treatments. There are two fundamental issues in the musculoskeletal oncology practice. The first issue is reconstruction of massive skeletal or muscular defect after complete surgical removal of tumor. The second issue is life saving with respect to tumor recurrence, and spread of tumor to lungs and death. These two multidisciplinary research programs are innovative, human disease-oriented translational research projects which meet the goals of the new NIH Roadmap.
Basic Research
Enhancement of Prosthesis Longevity: A new perspective
Most commonly, orthopaedic oncologists use tumor prostheses in order to save the limb and provide function for daily life. However, the prostheses do not last long due to inadequate bone support and implant loosening. In order to challenge this implant loosening problem, Dr. Lee is going to explore a new paradigm of implant wear particle-induced inflammation at a molecular level. The study is designed based on compelling preliminary data. This research program is supported by the Orthopaedic Research and Education Foundation Career Development Award (2006 – 2009) and NIH R01 grant (2007 – 2011). Two central hypotheses are:
Molecular targeting of inflammatory pathways can prevent inflammatory bone loss.
Specific transcriptional pathways regulate physical load-induced cytokine production. Answers to these two fundamental questions will provide a novel insight into a new way of preventing inflammatory bone loss and enhancing bone quality by optimal physical loading. This research program is conceptually innovative in that Dr. Lee is going to dissect the molecular mechanism of implant wear particle-induced inflammation under physiologic and superphysiologic mechanical perturbations. The study is technically innovative in that he is going to undertake mechanistic studies of gain-of-function and loss-of-function using genetically altered mice, pharmacologic inhibition, gene overexpression, gene silencing with small interfering RNA (siRNA) and custom-made mechanical loading device. Successful completion of this study will open two new fields of molecular mechanobiology (study on the effect of physical force on cells) and molecular tribology (study on wear particles). Dr. Lee will prepare competitive renewal NIH R01 grants in 2010-2011. Any novel findings from this research will be subject to patent application by Columbia University.
Summary of Current Molecular Tribology / Mechanobiology Program (Excerpt from NIH R01EB006834-01A1: Priority score 137; Percentile score: 1.7%):
Our long-range goal is to elucidate the mechanobiological mechanism responsible for hip prosthesis loosening. A hip arthroplasty using biomaterials is a widely accepted treatment for advanced osteoarthritis, rheumatoid arthritis, avascular necrosis and hip fractures. The long-term clinical success of hip arthoplasties is limited by prosthesis loosening which is associated with ultrahigh molecular weight polyethylene (UHMWPE) wear particle-induced inflammatory bone loss and loss of host bone-prosthesis integration. While polyethylene wear particles are known to stimulate macrophages and osteoblasts to produce tumor necrosis factor alpha (TNF-α) and other pro-osteoclastogenic cytokines, clinical observations have implicated mechanical perturbations such as increased deformational strains and pressures in the periprosthetic space as possible causes for the implant loosening. There is a knowledge gap in the mechanobiological mechanism by which mechanical instability at the host bone-prosthesis interface amplifies UHMWPE wear particle-induced inflammatory signaling at a molecular level.
The objective is to delineate the role of the calcineurin/nuclear factor of activated T cells (NFAT) axis as a mechanobiological mediator in periprosthetic inflammatory bone loss. Our preliminary data indicate that clinically relevant UHMWPE wear particles, deformational strains and pressures activate NFAT and induce the TNF-α, MSCF and COX-2 gene in bone marrow-derived human mesenchymal cells (hMSC) and macrophages. Our central hypothesis is that converging signals from UHMWPE wear particles and mechanical perturbation amplify inflammatory cytokine gene expression, activate macrophages, augment osteoclastogenesis, and promote the loss of osteoblastic phenotypes by co-activating the calcineurin/NFAT axis. We will use the clinically relevant UHMWPE wear particles which were generated from a hip joint simulator. Clinical periprosthetic mechanical perturbations will be simulated by applying deformational strains which were measured from human femora with hip prosthesises, clinically relevant fluctuating fluid pressures which were measured in periprosthetic space in human patients, and contact pressures which were measured in implanted hip prosthesis in humans. We propose the following 3 Specific Aims:
Specific Aim 1: to verify that UHMWPE wear particles and mechanical perturbation amplify TNF-α production by co-activating the calcineurin/NFATc1 axis in human and mouse osteoblasts. We hypothesize that UHMWPE wear particles and mechanical perturbation activate calcineurin, NFATc and TNF-α gene induction in hMSCs and mouse osteoblasts. We will apply clinically relevant deformational strains (Aim 1A) and pressures which are derived from periprosthetic space and implanted femoral prosthesis (Aim 1B). We will measure calcineurin/NFATc activity, TNF-α gene promoter activity, NFAT binding with the TNF-α gene by chromatin immunoprecipitation, TNF-α mRNA expression, and TNF-α secretion. We will conduct a series of loss-of-function studies using pharmacologic inhibitors, hMSC, calcineurin Aβ knockout mice and NFATc1 -/- mouse embryonal fibroblasts. We will also conduct gain-of-function experiments by overexpressing NFATc1 cDNA.
Specific Aim 2: to verify that UHMWPE wear particles and mechanical perturbation activate macrophgages and enhance RANKL-supported osteoclastogenesis by co-activating the calcineurin/NFATc1 axis. We hypothesize that activation of the calcineurin/NFATc1 axis by UHMWPE wear particles and mechanical perturbation activate macrophages and augment RANKL-supported osteoclastogenesis. We will measure tartrate resistant acid phosphatase (TRAP) activity, cytokine gene expression, NFATc1 binding with TNF-α gene promoter and osteoclast numbers in the presence of UHMWPE wear particles and clinically relevant mechanical perturbations.
Specific Aim 3: to determine the combinatorial effect of UHMWPE wear particles and mechanical perturbations on loss of osteoblastic phenotypes. One of the critical findings in pathologic specimens is formation of fibrous tissue instead of bone around the prosthesis. We hypothesize that UHMWPE wear particles and mechanical perturbations cooperatively inhibit osteoblastic activity and promote the loss of osteoblastic phenotypes by activating a TNF-α signaling pathway. We will quantify mineral nodules and alkaline phosphatase activity in osteoblasts under the influence of UHMWPE wear particles and clinical mechanical perturbations.
The rationale is that once we delineate the mechanobiological interaction between biomaterials, micromechanical environments, and the host immune system, we can prevent or treat the biomaterial-induced inflammatory bone loss by targeting pro-inflammatory pathways.
Sarcoma Research Program
A Novel Treatment for Sarcomas using small interfering RNA and mesenchymal stem cells
Sarcomas are cancers in connective tissues such as bone, muscle and soft tissues. Patients with sarcomas die from metastasis of sarcoma cells. Sarcomas in general do not respond to chemotherapy or radiation. Ewing sarcomas and osteosarcomas respond better than other types of sarcomas but many patients suffer from recurrence, amputation, lung metastasis and death even after chemotherapy and radiation. Therefore, much is needed to improve the care for patients with sarcomas. How can this be accomplished? One such way is to utilize recent advances in molecular biology, stem cell research and conventional treatment. A very simple concept is to prolong the life of patients with sarcomas by enhancing the efficacy of current treatments, reducing recurrence, preventing metastasis and avoiding amputation. This ideal scenario seems to be very possible in the 21st century.
Cell death and survival are tightly regulated by specific genes such as cell death promoting genes (pro-apoptotic genes) and cell survival genes (anti-apoptotic genes). For example, we may enhance cancer cell death by increasing the activity of death-promoting genes. However, the increased activity of cell death-promoting genes is not sufficient to control sarcomas. Cell survival genes are often activated in sarcomas after radiation or chemotherapy. In 2006, the Nobel Prize in Medicine was awarded to two scientists who discovered RNA interference (RNAi) or gene knockdown. This revolutionary technique knocks down target genes by delivering sequence-specific RNA. RNA is an intermediate molecule which deciphers DNAs and makes proteins. Stem cells can be utilized as a carrier of target-specific RNAi. One particular property of stem cells is homing to the site of sarcomas. For example, if stem cells are injected in mice bearing a tumor, stem cells travel and then gather together around the tumor. By combining techniques using stem cells and RNAi knocking down cell survival genes, we may improve the efficacy of chemotherapy and radiation in the treatment of sarcomas which are refractory to chemo- and radiotherapy.
This new research program is an extension of Dr. Lee’s previous projects and publications. Through the support of the Aircast Foundation ($100,000, 2004-2006, Principal Investigator: Francis Y. Lee, M.D.), Dr. Lee proved the therapeutic concept of siRNA as a radio- and chemosensitizer using human sarcoma cells. These compelling results were published in the Journal of Orthopaedic Research in March 7th, 2007. Briefly, human chondrosarcoma cells make more cell survival genes when sarcoma cells are exposed to radiation. This finding indicates that chondrosarcoma cells escape cell death by activating the cell survival pathway. This is a very plausible mechanism of radioresistance of sarcomas. Then, silencing cell survival genes such as Bcl-2, Bcl-xL and XIAP increased radiosensitivity and chemosensitivity of sarcomas. In addition, a therapeutic effect is more pronounced by targeting two cell survival genes. This discovery was filed for patent application by the Columbia University in 2007. Dr. Lee is now in the process of confirming the delivery of small interfering RNA using stem cells and developing an animal model bearing human osteosarcoma and Ewing sarcoma cells through the support of Columbia University Venture Fund. He will collaborate with Dr. Peter Brian and Dr. Ira Cohen, State University of New York at Stony Brook, N.Y., who have expertise in stem cell-based delivery of siRNA. Over the next two years, Dr. Lee is planning to prepare a NIH R01 grant which focuses on therapeutic application of stem cells and siRNA for the treatment of sarcomas. The central hypothesis is that mesenchymal stem cell-based delivery of shRNA will enhance radiosensitivity and chemosensitivity in sarcomas.
Specific Aim 1: to determine the delivery of shRNA from mesenchymal stem cells to sarcoma cells in vitro. We hypothesize that mesenchymal stem cells overexpressing shRNA targeting cell survival genes can delivery shRNA to sarcoma cells through gap junctions. We will co-culture human mesenchymal stem cells with a fluorescence-tagged shRNA and sarcoma cells. We will verify the presence of fluorescence in sarcoma cells in vitro.
Experiments are being conducted to determine whether siRNA can be delivered from mesenchymal stem cells to sarcoma cells through gap junctions in vitro.
Specific Aim 2: to determine the delivery of shRNA from mesenchymal stem cells to sarcoma cells in vivo. We hypothesize that mesenchymal stem cells are homed to the site of sarcoma. In order to determine the migration of stem cells toward sarcoma cells, we will conduct cell migration in vitro and will verify homing of stem cells to the site of sarcoma in a mouse sarcoma model. We will then verify Bcl-2, Bcl-xL and XIAP gene silencing effect in sarcoma cells.
Specific Aim 3: to determine the effect of stem cell-based delivery shRNA on the radiosensititivty and chemotherapy of sarcomas in vivo. We hypothesize that stem cell-based knockdown of cell survival genes will increase radiosensitivity and chemosensitivity. We will knockdown cell survival genes in sarcomas and we will treat sarcomas with radiation and doxorubicin. We will measure tumor viability, cell death and tumor growth 2, 4, 6 and 8 weeks after gene silencing and radiation/chemotherapy.
Radiation will be applied to control tumors (control; stem cells without siRNA; mesenchymal stem cells with nonsilencing siRNA) and experimental tumors (stem cell-based delivery of siRNA targeting anti-apoptotic genes). Sarcoma cell death in the tibia and lung metastasis will be assessed by in vivo imaging and histopathology.
Chemotherapy (conventional agent such as doxorubicin or methotrexate; new experimental agents) will be applied to aforementioned groups and outcome analysis will be done as described.
It is envisioned that patients with sarcomas will benefit from improved treatment methods utilizing stem cells and gene silencing.
Discovery of Oncogenes Responsible for Sarcomas
The goal of this research is to identify critical genes which are either activated or inhibited during the development of sarcomas using a cDNA microarray analysis technique. Differential gene expression profiling was completed using normal epiphyseal chondrocytes and human chondrosarcomas. A similar series of experiments will be conducted using osteosarcomas and pleomorphic sarcomas. It is expected that a candidate pool of genes will be identified. Functionality of target genes will be verified using loss-of-function (siRNA) and gain-of-function (overexpression) experiments in vitro and in vivo.
Dr. Francis Y. Lee joined the Orthopaedic Surgery department in 1999 after completing a Musculoskeletal Tumor Fellowship at the Massachusetts General Hospital and Boston Children's Hospital, and a Pediatric Orthopedic Surgery Fellowship at the Hospital for Sick Children in Toronto. He is currently an Assistant Professor of Orthopedic Surgery at Columbia University, specializing in pediatric orthopedic surgery including spine deformity and complex developmental orthopedic disorders, musculoskeletal tumors and bone disease. He serves as the Chief of the Tumor & Bone Disease service and Director of Center for Orthopedic Research. He is conducting both basic and clinical studies on musculoskeletal disorders. He is a recipient of American Academy of Orthopaedic Surgeons / Orthopaedic Research Education and Foundation Clinician Scientist Traveling Fellowship Award (2004-2005).
