Tennessee Valley Healthcare System VISN

Tennessee, United States

Tennessee Valley Healthcare System VISN

Tennessee, United States
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Li X.,Vanderbilt Ingram Cancer Center | Sterling J.A.,Vanderbilt Ingram Cancer Center | Sterling J.A.,Center for Bone Biology | Sterling J.A.,Tennessee Valley Healthcare System VISN | And 8 more authors.
Molecular Cancer Research | Year: 2012

Loss of TGF-β type II receptor (TβRII, encoded by Tgfbr2) expression in the prostate stroma contributes to prostate cancer initiation, progression, and invasion. We evaluated whether TβRII loss also affected prostate cancer bone metastatic growth. Immunohistologic analysis revealed that TβRII expression was lost in cancer-associated fibroblasts in human prostate cancer bone metastatic tissues. We recapitulated the human situation with a conditional stromal Tgfbr2 knockout (Tgfbr2-KO) mouse model. Conditioned media from primary cultured Tgfbr2-KO or control Tgfbr2-flox prostatic fibroblasts (koPFCM or wtPFCM, respectively) were applied to C4-2B prostate cancer cells before grafting the cells tibially. We found that koPFCM promoted prostate cancer cell growth in the bone and development of early mixed osteoblastic/osteolytic bone lesions. Furthermore, the koPFCM promoted greater C4-2B adhesion to type-I collagen, the major component of bone matrix, compared to wtPFCM-treated C4-2B. Cytokine antibody array analysis revealed that koPFCM had more than two-fold elevation in granulocyte colony-stimulating factor and CXCL1, CXCL16, and CXCL5 expression relative to wtPFCM. Interestingly, neutralizing antibodies of CXCL16 or CXCL1 were able to reduce koPFCM-associated C4-2B type-I collagen adhesion to that comparable with wtPFCM-mediated adhesion. Collectively, our data indicate that loss of TGF-β responsiveness in prostatic fibroblasts results in upregulation of CXCL16 and CXCL1 and that these paracrine signals increase prostate cancer cell adhesion in the bone matrix. These microenvironment changes at the primary tumor site can mediate early establishment of prostate cancer cells in the bone and support subsequent tumor development at the metastatic site. ©2012 AACR.


Preston Campbell J.,Vanderbilt University | Merkel A.R.,Vanderbilt University | Merkel A.R.,Tennessee Valley Healthcare System VISN | Kathryn Masood-Campbell S.,Vanderbilt University | And 3 more authors.
Journal of Visualized Experiments | Year: 2012

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality 1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio 2, that has since been transfected for GFP expression and re-cloned by our group 3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis 2, in certain instances intratibial 4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6. This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses. © JoVE 2006-2012. All Rights Reserved.


Guelcher S.A.,Vanderbilt University | Sterling J.A.,Vanderbilt University | Sterling J.A.,Tennessee Valley Healthcare System VISN
Cancer Microenvironment | Year: 2011

Certain tumors, such as breast, frequently metastasize to bone where they can induce bone destruction. Currently, it is well-accepted that the tumor cells are influenced by other cells and growth factors present in the bone microenvironment that lead to tumor-induced bone disease. Over the past 20 years, many groups have studied this process and determined the major contributing factors; however, these results do not fully explain the changes in gene expression and cell behavior that occur when tumor cells metastasize to bone. More recently, groups studying metastasis from soft tissue sites have determined that the rigidity of the microenvironment, which increases during tumor progression in soft tissue, can regulate tumor cell behavior and gene expression. Therefore, we began to investigate the role of the rigid bone extracellular matrix in the regulation of genes that stimulate tumor-induced bone disease. We found that the rigidity of bone specifically regulates parathyroid hormone-related protein (PTHrP) and Gli2 expression in a transforming growth factor β (TGF-β) and mechanotransduction-dependent mechanism In this review, we summarize the mechanotransduction signaling pathway and how this influences TGF-β signaling and osteolytic gene expression. © 2011 Springer Science+Business Media B.V.


Sterling J.A.,Tennessee Valley Healthcare System VISN | Sterling J.A.,Vanderbilt University | Johnson R.W.,Tennessee Valley Healthcare System VISN | Johnson R.W.,Vanderbilt University
International Journal of Cancer Research and Prevention | Year: 2012

Breast cancer frequently metastasizes to distant organs such as lung and bone where tumors cause changes in the local micro-environment. These alterations often lead to bone destruction or bone formation and can cause pain, hypercalcemia, and increased fracture rates in patients. Several small animal models have been developed that are capable of producing pathogenesis strikingly similar to the clinical condition in both tumor burden and bone disease. In order to fully investigate these animal models, many imaging techniques have been applied with most focusing on either imaging the tumor or changes in bone. Radiography using Faxitron analysis has been the central tool for researchers for 20 years, but other techniques have developed that allow for more comprehensive analysis. Over the past 10 years, imaging tumors in bone has primarily been performed using tumor cells over-expressing fluorescent proteins or luciferase. Both approaches have been very successful due to rapid data output, no radiation, and cost-effectiveness, but are both limited in resolution and in the ability to detect small tumors that are deep in the bone. Therefore, new technologies are emerging that utilize the near-infrared (NIR) spectra, which has a longer wavelength and allows for imaging tumors deeper within the body and has lower background than fluorescent imaging. Not only can NIR proteins be transfected like fluorescent proteins, but antibodies can also be conjugated with NIR to detect the expression of specific proteins in vivo. Other emerging techniques for imaging tumors in bone are positron emission tomography (PET) imaging and magnetic resonance imaging (MRI), both of which are commonly used clinically for diagnosing tumors in bone, but are not applied frequently for animal studies due to high cost and limited throughput. However, both approaches can give valuable information in animal studies. For imaging tumor-induced effects on bone, Faxitron analysis still remains the primary method of analysis, but in recent years micro-Computed Tomography (μCT) analyses have gained popularity due to the ability to obtain high resolution 3-dimensional images. While the highest resolution images can be obtained by ex vivo scanners, such as the Scanco μCT, the utility of these scanners is obviously limited to endpoint analyses. This limitation has recently stimulated the advancement of in vivo μCT analyses in breast cancer-induced osteolysis which can be performed longitudinally without affecting tumor cell growth due to high-dose radiation. Live animal μCT can also be combined with other imaging techniques such as MRI, μPET, and μSPECT (single photon emission computed tomography). Additionally, both μSPECT and μPET technologies have been applied to bone to image bone turnover and allow investigators to analyze changes in bone that have been caused by the presence of tumor. Finally, investigators are currently developing technologies to fuse multiple imaging modalities together to give a comprehensive understanding of tumor-induced bone disease. While there remains significant room for improvement, current imaging modalities can, in combination, provide an accurate and dynamic reflection of breast tumor burden and bone destruction in small animal models and have helped propagate significant advances in our understanding of tumor metastasis to bone. © Nova Science Publishers, Inc.


Johnson R.W.,Tennessee Valley Healthcare System VISN | Johnson R.W.,Vanderbilt University | Merkel A.R.,Tennessee Valley Healthcare System VISN | Merkel A.R.,Vanderbilt University | And 6 more authors.
Anticancer Research | Year: 2011

Background: Breast cancer cells frequently metastasize to bone, where they up-regulate their expression of the transcription factor GLI2 and the downstream osteolytic factor parathyroid hormone-related protein (PTHrP). The guanosine nucleotide 6-thioguanine (6-TG) inhibits PTHrP expression and blocks osteolytic bone destruction in mice inoculated with bone metastatic cells; however, the mechanism by which 6-TG inhibits PTHrP remains unclear. We hypothesized that 6-TG inhibition of PTHrP is mediated through GL12 signaling. Materials and Methods: Human MDA-MB-231 breast cancer cells and RWGT2 squamous-cell lung carcinoma cells were treated with 100 μM 6-TG and examined for GL12 mRNA expression and stability by Q-PCR, promoter activity by luciferase assay, and protein expression by Western blot. Results: 6-TG significantly blocked GLI2 mRNA and protein expression, but did not affect stability. Additionally, 6-TG directly inhibited GLI2 promoter activity, and when cells were transfected with constitutively expressed GL12, the inhibitory effect of 6-TG on PTHrP expression was abolished. Conclusion: Taken together, these data indicate that 6-TG regulates PTHrP in part through GLI2 transcription, and therefore the clinical use of 6-TG or other guanosine nucleotides may be a viable therapeutic option in tumor types expressing elevated levels of GLI proteins.


Johnson R.W.,Tennessee Valley Healthcare System VISN | Johnson R.W.,Vanderbilt University | Merkel A.R.,Tennessee Valley Healthcare System VISN | Merkel A.R.,Vanderbilt University | And 5 more authors.
Clinical and Experimental Metastasis | Year: 2014

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-β) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF-β in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by β-catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF-β and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched β-catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a β-catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF-β signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF-β signaling. © 2014, US Government.


Sterling J.A.,Tennessee Valley Healthcare System VISN | Sterling J.A.,Vanderbilt University | Guelcher S.A.,Vanderbilt University
Current Osteoporosis Reports | Year: 2014

Healing fractures resulting from osteoporosis or cancer remains a significant clinical challenge. In these populations, healing is often impaired not only due to age and disease, but also by other therapeutic interventions such as radiation, steroids, and chemotherapy. Despite substantial improvements in the treatment of osteoporosis over the last few decades, osteoporotic fractures are still a major clinical challenge in the elderly population due to impaired healing. Similar fractures with impaired healing are also prevalent in cancer patients, especially those with tumor growing in bone. Treatment options for cancer patients are further complicated by the fact that bone anabolic therapies are contraindicated in patients with tumors. Therefore, many patients undergo surgery to repair the fracture, and bone grafts are often used to stabilize orthopedic implants and provide a scaffold for ingrowth of new bone. Both synthetic and naturally occurring biomaterials have been investigated as bone grafts for repair of osteoporotic fractures, including calcium phosphate bone cements, resorbable polymers, and allograft or autograft bone. In order to re-establish normal bone repair, bone grafts have been augmented with anabolic agents, such as mesenchymal stem cells or recombinant human bone morphogenetic protein-2. These developing approaches to bone grafting are anticipated to improve the clinical management of osteoporotic and cancer-induced fractures. © 2014 Springer Science+Business Media.


PubMed | Tennessee Valley Healthcare System VISN
Type: Journal Article | Journal: Anticancer research | Year: 2011

Breast cancer cells frequently metastasize to bone, where they up-regulate their expression of the transcription factor GLI2 and the downstream osteolytic factor parathyroid hormone-related protein (PTHrP). The guanosine nucleotide 6-thioguanine (6-TG) inhibits PTHrP expression and blocks osteolytic bone destruction in mice inoculated with bone metastatic cells; however, the mechanism by which 6-TG inhibits PTHrP remains unclear. We hypothesized that 6-TG inhibition of PTHrP is mediated through GLI2 signaling.Human MDA-MB-231 breast cancer cells and RWGT2 squamous-cell lung carcinoma cells were treated with 100 M 6-TG and examined for GLI2 mRNA expression and stability by Q-PCR, promoter activity by luciferase assay, and protein expression by Western blot.6-TG significantly blocked GLI2 mRNA and protein expression, but did not affect stability. Additionally, 6-TG directly inhibited GLI2 promoter activity, and when cells were transfected with constitutively expressed GLI2, the inhibitory effect of 6-TG on PTHrP expression was abolished.Taken together, these data indicate that 6-TG regulates PTHrP in part through GLI2 transcription, and therefore the clinical use of 6-TG or other guanosine nucleotides may be a viable therapeutic option in tumor types expressing elevated levels of GLI proteins.


PubMed | Tennessee Valley Healthcare System VISN
Type: Journal Article | Journal: Clinical & experimental metastasis | Year: 2014

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone destruction in bone metastatic tumors. Transforming growth factor-beta (TGF-) stimulates PTHrP production in part through the transcription factor Gli2, which is regulated independent of the Hedgehog signaling pathway in osteolytic cancer cells. However, inhibition of TGF- in vivo does not fully inhibit tumor growth in bone or tumor-induced bone destruction, suggesting other pathways are involved. While Wnt signaling regulates Gli2 in development, the role of Wnt signaling in bone metastasis is unknown. Therefore, we investigated whether Wnt signaling regulates Gli2 expression in tumor cells that induce bone destruction. We report here that Wnt activation by -catenin/T cell factor 4 (TCF4) over-expression or lithium chloride (LiCl) treatment increased Gli2 and PTHrP expression in osteolytic cancer cells. This was mediated through the TCF and Smad binding sites within the Gli2 promoter as determined by promoter mutation studies, suggesting cross-talk between TGF- and Wnt signaling. Culture of tumor cells on substrates with bone-like rigidity increased Gli2 and PTHrP production, enhanced autocrine Wnt activity and led to an increase in the TCF/Wnt signaling reporter (TOPFlash), enriched -catenin nuclear accumulation, and elevated Wnt-related genes by PCR-array. Stromal cells serve as an additional paracrine source of Wnt ligands and enhanced Gli2 and PTHrP mRNA levels in MDA-MB-231 and RWGT2 cells in vitro and promoted tumor-induced bone destruction in vivo in a -catenin/Wnt3a-dependent mechanism. These data indicate that a combination of matrix rigidity and stromal-secreted factors stimulate Gli2 and PTHrP through Wnt signaling in osteolytic breast cancer cells, and there is significant cross-talk between the Wnt and TGF- signaling pathways. This suggests that the Wnt signaling pathway may be a potential therapeutic target for inhibiting tumor cell response to the bone microenvironment and at the very least should be considered in clinical regimens targeting TGF- signaling.

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