Interaction of Metastatic Breast Cancer Cells with Osteoblasts

Open Access
Author:
Shuman, Laurie Ann
Graduate Program:
Immunology and Infectious Diseases
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
April 16, 2009
Committee Members:
  • Andrea Marie Mastro, Dissertation Advisor
  • Andrea Marie Mastro, Committee Chair
  • Adam Bleier Glick, Committee Member
  • Rosalyn Bryson Irby, Committee Member
  • Robert Paulson, Committee Member
  • Danny Welch, Committee Member
Keywords:
  • transcription factor
  • cytokine
  • metastasis
  • osteoblast
  • Breast cancer
Abstract:
A majority of cancer deaths (90%) are a result of the dissemination of tumor cells from the primary site to a secondary site, not due to the primary tumor itself. Therefore, it is necessary to investigate mechanisms utilized by tumor cells to promote growth in and destruction of the secondary sites. Advances in microarray technology allow for rapid screening and identification of potential genetic targets involved in host response to cancers, however there is a need for high-throughput screening of the identified targets to determine their efficacy. In vitro systems that more accurately represent in vivo microenvironments would be a useful tool to screen potential genetic targets as treatment of diseases. Here we examined the interaction of breast cancer cells with osteoblasts in a specialized culture device (bioreactor). Breast cancer preferentially metastasizes to the bone resulting in the formation of osteolytic lesions. While administration of osteoclast-inhibiting drugs, such as bisphosphonates, slow further lesion formation, existing lesions do not heal. Therefore, osteoblasts appear functionally disabled in the presence of metastatic breast cancer cells. Previous studies in this laboratory have shown that breast cancer cells alter osteoblast adhesion and morphology, increase osteoblast apoptosis, and decrease the expression of osteoblast differentiation genes when exposed to metastatic breast cancer cell conditioned medium for extended periods (5 – 35 days). In order to examine the interaction of metastatic breast cancer cells with osteoblasts apart from osteoclasts, a specialized bioreactor culture system was utilized. In this culture device, osteoblasts grew and differentiated into a multiple-cell-layer, three-dimensional mineralizing tissue. Co-culture of metastatic breast cancer cells with osteoblasts in a bioreactor were compared to co-culture in conventional cell culture. The breast cancer cells not only attached and grew on the osteoblast tissue in the bioreactor culture system, but also formed distinct colonies that aligned in the same axis as the osteoblasts, similar to “Indian filing” seen in authentic pathological tissue. Moreover, in this culture system, the breast cancer cells penetrated the osteoblast tissue, a phenomenon not apparent with conventional cell culture methods. Metastatic breast cancer cells also interfered with the differentiation of osteoblasts as evidenced by a decrease in production of proteins, such as osteocalcin and collagen. In addition, co-culture of the metastatic breast cancer cells with osteoblasts resulted in increased production of the inflammatory cytokine, IL-6, a known activator of osteoclasts. The bioreactor culture system is advantageous over conventional cell culture systems in emulating the bone microenvironment and for studying the interaction of metastatic breast cancer cells with osteoblasts. Additionally, in this report, transcription factor targets within MC3T3-E1 cells treated with MDA-MB-231 human metastatic breast cancer cell conditioned medium were identified. Expression of a particular transcription factor, Early Growth Response – 1 (EGR-1), was silenced in MC3T3-E1 cells using shRNA targeting murine EGR-1. Osteoblast differentiation and cytokine production was measured in the presence or absence of MDA-MB-231 metastatic breast cancer cell conditioned medium. Osteoblasts having decreased expression of EGR-1 differentiated in culture and when exposed to metastatic breast cancer cell conditioned medium showed reduced cytokine production. However, the reduction in osteoblast-derived cytokines did not interfere with the ability of the breast cancer cells to attach or colonize the osteoblast layer. When osteoblasts having decreased EGR-1 expression were cultured in metastatic breast cancer cell conditioned medium for extended periods (19 – 37 days), differentiation was still inhibited as indicated by decreased alkaline phosphatase staining and RNA expression of type I collagen and osteocalcin. Therefore, these data indicate that decreased EGR-1 expression in MC3T3-E1 cells leads to decreased inflammatory cytokine production by these cells when treated with metastatic breast cancer cell conditioned medium. However, decreased EGR-1 expression in osteoblasts does not reverse the block in osteoblast differentiation caused by the metastatic breast cancer cell conditioned medium. Therefore, these data imply that EGR-1 expression in osteoblasts is directly related to inflammatory cytokine production by, and not differentiation of, osteoblasts stimulated with metastatic breast cancer cell conditioned medium. Combined together, these studies provide useful information for future studies focusing on identification and screening of potential therapeutic targets to treat breast cancer cell metastasis to the bone. The first study validates the use of a bioreactor culture system as an in vitro culture device to better examine the interaction of breast cancer cells with osteoblasts, while the second study identifies a potential therapeutic target in osteoblasts for inflammatory cytokine production induced by breast cancer metastasis.