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Arming a replicating adenovirus with osteoprotegerin reduces the tumor burden in a murine model of osteolytic bone metastases of breast cancer.

Cody JJ, Rivera AA, Lyons GR, Yang SW, Wang M, Sarver DB, Wang D, Selander KS, Kuo HC, Meleth S, Feng X, Siegal GP, Siegall GP, Douglas JT - Cancer Gene Ther. (2010)

Bottom Line: Enhanced infection of cells expressing low levels of the primary Ad5 receptor was conferred by incorporating an arginine-glycine-aspartic acid (RGD) peptide sequence into the fiber knob to mediate binding to α(v) integrins.After characterization of the armed CRAd, we demonstrated that infection of breast cancer cells by Ad5-Δ24-sOPG-Fc-RGD both killed the infected cells by oncolysis and inhibited the formation of osteoclasts in an in vitro co-culture model.In a murine model of osteolytic bone metastases of breast cancer, the CRAd armed with shortened OPG (sOPG)-Fc reduced tumor burden in the bone and inhibited osteoclast formation more effectively than an unarmed CRAd.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Most patients with advanced breast cancer develop osteolytic bone metastases, which have numerous complications. Because current therapies are not curative, new treatments are needed. Conditionally replicating adenoviruses (CRAds) are anticancer agents designed to infect and lyse tumor cells. However, in spite of their promise as selective cancer therapeutics, replicating adenoviruses have shown limited efficacy in the clinical setting. We hypothesized that a CRAd armed with osteoprotegerin (OPG) would eradicate bone metastases of breast cancer both directly, by oncolysis, and indirectly, by inhibiting osteoclastic bone resorption, and thus reducing the tumor burden. We constructed an armed CRAd (Ad5-Δ24-sOPG-Fc-RGD) by replacing viral E3B genes with a fusion of the ligand-binding domains of OPG and the Fc portion of human IgG1. Conditional replication was conferred by a 24-base pair deletion within E1A (Δ24), which prevents the binding of E1A to the retinoblastoma tumor suppressor/cell cycle regulator protein and limits replication in normal cells. Enhanced infection of cells expressing low levels of the primary Ad5 receptor was conferred by incorporating an arginine-glycine-aspartic acid (RGD) peptide sequence into the fiber knob to mediate binding to α(v) integrins. After characterization of the armed CRAd, we demonstrated that infection of breast cancer cells by Ad5-Δ24-sOPG-Fc-RGD both killed the infected cells by oncolysis and inhibited the formation of osteoclasts in an in vitro co-culture model. In a murine model of osteolytic bone metastases of breast cancer, the CRAd armed with shortened OPG (sOPG)-Fc reduced tumor burden in the bone and inhibited osteoclast formation more effectively than an unarmed CRAd.

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CRAd armed with sOPG-Fc lyses cancer cells and inhibits osteoclast formation in vitro. MDA-MB-435 cells were infected with Ad5-Δ24-sOPG-Fc-RGD, Ad5-Δ24RGD or replication-defective vectors and grown for 9 days on inserts overlaying cocultures of murine osteoclast precursors and ST2 bone marrow stromal cells or human osteoclast precursors and RANKL. A, Cancer cells on the inserts were fixed and stained with crystal violet. The osteoclast marker protein TRAP5b was assayed by ELISA in the murine (B) or human (C) systems. Results are means ± SD of duplicate determinations. Representative results of 2 separate experiments are shown. Significant differences for sOPG-Fc-armed CRAd vs. uninfected (*) and unarmed CRAd (†) are indicated; P ≤ 0.05, determined by Student’s-Fisher t test. D, expression of sOPG-Fc in the medium of the murine system was quantified in an ELISA.
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Figure 5: CRAd armed with sOPG-Fc lyses cancer cells and inhibits osteoclast formation in vitro. MDA-MB-435 cells were infected with Ad5-Δ24-sOPG-Fc-RGD, Ad5-Δ24RGD or replication-defective vectors and grown for 9 days on inserts overlaying cocultures of murine osteoclast precursors and ST2 bone marrow stromal cells or human osteoclast precursors and RANKL. A, Cancer cells on the inserts were fixed and stained with crystal violet. The osteoclast marker protein TRAP5b was assayed by ELISA in the murine (B) or human (C) systems. Results are means ± SD of duplicate determinations. Representative results of 2 separate experiments are shown. Significant differences for sOPG-Fc-armed CRAd vs. uninfected (*) and unarmed CRAd (†) are indicated; P ≤ 0.05, determined by Student’s-Fisher t test. D, expression of sOPG-Fc in the medium of the murine system was quantified in an ELISA.

Mentions: In the model systems, the breast cancer cells were lysed by the unarmed and sOPG-armed CRAds, whereas the replication-defective vectors expressing OPG failed to lyse the breast cancer cells (MDA-MB-435 cells shown in Fig 5A). The level of TRAP5b in the medium was significantly reduced in both the murine (Fig. 5B) and human (Fig. 5C) coculture systems overlaid with MDA-MB-435 breast cancer cells infected by the armed CRAds compared with the unarmed CRAd platform, indicating a reduction in the number of osteoclasts (P < 0.05 for all pair-wise comparisons within an experiment, by Student’s-Fisher t test). This reduction in osteoclast formation correlated with the expression of sOPG-Fc by the armed CRAds (shown for murine system in Fig. 5D). Similar results were obtained when MDA-MB-231 cells were infected. Hence, the sOPG-Fc-armed CRAds could inhibit osteoclast formation while simultaneously lysing breast cancer cells.


Arming a replicating adenovirus with osteoprotegerin reduces the tumor burden in a murine model of osteolytic bone metastases of breast cancer.

Cody JJ, Rivera AA, Lyons GR, Yang SW, Wang M, Sarver DB, Wang D, Selander KS, Kuo HC, Meleth S, Feng X, Siegal GP, Siegall GP, Douglas JT - Cancer Gene Ther. (2010)

CRAd armed with sOPG-Fc lyses cancer cells and inhibits osteoclast formation in vitro. MDA-MB-435 cells were infected with Ad5-Δ24-sOPG-Fc-RGD, Ad5-Δ24RGD or replication-defective vectors and grown for 9 days on inserts overlaying cocultures of murine osteoclast precursors and ST2 bone marrow stromal cells or human osteoclast precursors and RANKL. A, Cancer cells on the inserts were fixed and stained with crystal violet. The osteoclast marker protein TRAP5b was assayed by ELISA in the murine (B) or human (C) systems. Results are means ± SD of duplicate determinations. Representative results of 2 separate experiments are shown. Significant differences for sOPG-Fc-armed CRAd vs. uninfected (*) and unarmed CRAd (†) are indicated; P ≤ 0.05, determined by Student’s-Fisher t test. D, expression of sOPG-Fc in the medium of the murine system was quantified in an ELISA.
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Related In: Results  -  Collection

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Figure 5: CRAd armed with sOPG-Fc lyses cancer cells and inhibits osteoclast formation in vitro. MDA-MB-435 cells were infected with Ad5-Δ24-sOPG-Fc-RGD, Ad5-Δ24RGD or replication-defective vectors and grown for 9 days on inserts overlaying cocultures of murine osteoclast precursors and ST2 bone marrow stromal cells or human osteoclast precursors and RANKL. A, Cancer cells on the inserts were fixed and stained with crystal violet. The osteoclast marker protein TRAP5b was assayed by ELISA in the murine (B) or human (C) systems. Results are means ± SD of duplicate determinations. Representative results of 2 separate experiments are shown. Significant differences for sOPG-Fc-armed CRAd vs. uninfected (*) and unarmed CRAd (†) are indicated; P ≤ 0.05, determined by Student’s-Fisher t test. D, expression of sOPG-Fc in the medium of the murine system was quantified in an ELISA.
Mentions: In the model systems, the breast cancer cells were lysed by the unarmed and sOPG-armed CRAds, whereas the replication-defective vectors expressing OPG failed to lyse the breast cancer cells (MDA-MB-435 cells shown in Fig 5A). The level of TRAP5b in the medium was significantly reduced in both the murine (Fig. 5B) and human (Fig. 5C) coculture systems overlaid with MDA-MB-435 breast cancer cells infected by the armed CRAds compared with the unarmed CRAd platform, indicating a reduction in the number of osteoclasts (P < 0.05 for all pair-wise comparisons within an experiment, by Student’s-Fisher t test). This reduction in osteoclast formation correlated with the expression of sOPG-Fc by the armed CRAds (shown for murine system in Fig. 5D). Similar results were obtained when MDA-MB-231 cells were infected. Hence, the sOPG-Fc-armed CRAds could inhibit osteoclast formation while simultaneously lysing breast cancer cells.

Bottom Line: Enhanced infection of cells expressing low levels of the primary Ad5 receptor was conferred by incorporating an arginine-glycine-aspartic acid (RGD) peptide sequence into the fiber knob to mediate binding to α(v) integrins.After characterization of the armed CRAd, we demonstrated that infection of breast cancer cells by Ad5-Δ24-sOPG-Fc-RGD both killed the infected cells by oncolysis and inhibited the formation of osteoclasts in an in vitro co-culture model.In a murine model of osteolytic bone metastases of breast cancer, the CRAd armed with shortened OPG (sOPG)-Fc reduced tumor burden in the bone and inhibited osteoclast formation more effectively than an unarmed CRAd.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Most patients with advanced breast cancer develop osteolytic bone metastases, which have numerous complications. Because current therapies are not curative, new treatments are needed. Conditionally replicating adenoviruses (CRAds) are anticancer agents designed to infect and lyse tumor cells. However, in spite of their promise as selective cancer therapeutics, replicating adenoviruses have shown limited efficacy in the clinical setting. We hypothesized that a CRAd armed with osteoprotegerin (OPG) would eradicate bone metastases of breast cancer both directly, by oncolysis, and indirectly, by inhibiting osteoclastic bone resorption, and thus reducing the tumor burden. We constructed an armed CRAd (Ad5-Δ24-sOPG-Fc-RGD) by replacing viral E3B genes with a fusion of the ligand-binding domains of OPG and the Fc portion of human IgG1. Conditional replication was conferred by a 24-base pair deletion within E1A (Δ24), which prevents the binding of E1A to the retinoblastoma tumor suppressor/cell cycle regulator protein and limits replication in normal cells. Enhanced infection of cells expressing low levels of the primary Ad5 receptor was conferred by incorporating an arginine-glycine-aspartic acid (RGD) peptide sequence into the fiber knob to mediate binding to α(v) integrins. After characterization of the armed CRAd, we demonstrated that infection of breast cancer cells by Ad5-Δ24-sOPG-Fc-RGD both killed the infected cells by oncolysis and inhibited the formation of osteoclasts in an in vitro co-culture model. In a murine model of osteolytic bone metastases of breast cancer, the CRAd armed with shortened OPG (sOPG)-Fc reduced tumor burden in the bone and inhibited osteoclast formation more effectively than an unarmed CRAd.

Show MeSH
Related in: MedlinePlus