Novel Cytosine Deaminase Fusion Gene Enhances the Effect of Radiation on Breast Cancer in Bone by Reducing Tumor Burden, Osteolysis, and Skeletal Fracture

Abstract

Background: Painful breast carcinoma metastases in bone are a common manifestation of malignant disease. Eradication of these tumors can be evasive, and as a result, skeletal morbidity increases with disease progression. Experimental Design: The treatment potential of cytosine deaminase (CD) gene therapy combined with radiation treatment was evaluated in vitro and in vivo using a 4T1 murine breast carcinoma model. 4T1 carcinoma cells were transduced with a fusion gene encoding the extracellular and transmembrane domains of the human nerve growth factor receptor and the cytoplasmic portion of the yeast CD gene (NGFR-CD y ). Results and Conclusions: CD-expressing tumor cells (4TCD y ) were highly sensitive to treatment by 5-fluorocytosine prodrug (P < 0.0001). 5-Fluorocytosine treatment of 4TCD y , but not 4T1 cells, enhanced the effects of radiation in vitro (P < 0.0001). 5-Fluorocytosine prodrug treatment also increased the therapeutic potential of radiation in vivo. Mice with 4TCD y intrafemoral tumors showed increased effectiveness of radiation based on improved reductions in tumor size, reductions in tumorigenic osteolysis, and a decrease in skeletal fractures (P < 0.01).Enzyme/prodrug gene therapy involves expression of a nonmammalian enzyme that converts a nontoxic prodrug to a cytotoxic agent at sites of tumor. The cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug system converts 5-FC to the highly cytotoxic compound 5-fluorouracil (5-FU) and is a promising therapeutic approach for the treatment of tumors because of its potential for sensitizing tumor cells to radiation treatment. Deamination of 5-FC by CD-expressing tumor cells produces 5-FU. Intracellular 5-FU is then converted to the active metabolites 5-fluoro-2-dUMP (FdUMP) or 5-fluoro-2-dUTP. 5-FU can passively diffuse across the cell membrane and enter adjacent cells where intracellular conversion of 5-FU to FdUMP is done by thymidine kinase. FdUMP inhibits cellular DNA synthesis by binding to and inhibiting thymidylate synthase (1, 2). Although 5-FU has both DNA-directed and RNA-directed effects, its radiationenhancing effects result from DNA-directed mechanisms (3). These include lethal influences on radioresistant S-phase cells and reduction in DNA repair after radiation-induced DNA injury (1).Because 5-FU has been shown to enhance the effect of radiation on tumor cells, the CD/5-FC enzyme/prodrug system is attractive as an adjuvant for treating tumors that will receive radiation treatment (4). The CD/5-FC enzyme/prodrug system has been used in conjunction with radiation treatment in experimental animal models and clinical trials for treating softtissue cancers. Soft-tissue cancers studied with this concomitant therapy include colon, nasopharyngeal, prostate, esophageal, and breast cancers, malignant gliomas and sarcomas, and epidermoid carcinoma (4 -10). Treatment of soft-tissue cancers in experimental models has involved delivery of the CD gene via tumor cell transduction or delivery of adenovirus containing the CD gene fo...

Evaluating distant metastases in breast cancer: from biology to outcomes

“…Trastuzumab, a monoclonal antibody directed against HER2/neu oncogene, has been shown to inhibit skeletal metastases in breast cancer [120], but trastuzumab has also been associated with cardiac dysfunction, congestive heart failure, and pulmonary toxicity [121]. New agents are being developed to treat skeletal metastases (Table 2) [122][123][124][125][126][127][128][129][130][131], but they also have some safety and specificity drawbacks. Receptor activator of nuclear factor-κB ligand (RANKL) mediates osteoclast differentiation and is found to be elevated in breast cancer cells causing excessive bone resorption.…”

Osteoclasts Direct Bystander Killing of Bone Cancer

“…5-FC treatment of Tg/NCD mice caused a profound reduction in the capacity of bone marrow cells collected 4 and 7 days after 5-FC treatment to form osteoclasts in vitro, but did not have a significant effect on osteoclast number. This apparent paradox of effects on osteoclast precursors but not on mature osteoclasts most likely reflects the fact that osteoclast precursor cells are engaged in cell division and thus more sensitive to 5-FU, whereas the mature osteoclasts are quiescent and less sensitive to 5-FU (24).…”

“…Radiation-activated promoters could also be considered as bone cancer is commonly treated with radiation, and 5-FU can function as a radiation enhancer (34)(35)(36). In fact, recent work describes the CD/5-FC enzyme/prodrug system as capable of enhancing the killing effect of radiation on breast cancer tumors in bone (24,36).…”

Animal Models of Cancer Pain

“…176 When sarcoma cells (10 5 in 20 μl) were implanted into mouse femurs to examine the effects of 20-Gy and 30-Gy radiation as a means to control cancer pain, these radiation therapies effectively decreased cancer-induced osteolysis, reduced tumor size by 75%, and decreased bone cancerrelated pain. [57][58][59] Radiation therapy also successfully controlled pain and skeletal fractures associated with femoral implantation of 4T1 breast carcinoma cells into mice. 59 These studies demonstrated that radiation therapy effectively decreased cancer-induced pain by direct effects on tumor cells.…”

“…[57][58][59] Radiation therapy also successfully controlled pain and skeletal fractures associated with femoral implantation of 4T1 breast carcinoma cells into mice. 59 These studies demonstrated that radiation therapy effectively decreased cancer-induced pain by direct effects on tumor cells. 57,58 Support for the effects of radiation therapy also comes from studies in which transplantable hepatocellular carcinoma cells, HCa-1, were injected into the periosteal membrane of the foot dorsum in C3H/ HeJ mice.…”