A Histomorphometric Analysis of Radiation Damage in an Isogenic Murine Model of Distraction Osteogenesis

Authors

Alexander R. Zheutlin, BS, Sagar S. Deshpande, BS, Noah S. Nelson, BS, Yekaterina Polyatskaya, MD, Jose J. Rodriguez, MD, Alexis Donneys, MD, MS, Steven R. Buchman, MD

Abstract

Purpose The devastation radiation therapy (XRT) causes to endogenous tissue in head and neck cancer (HNC) patients can be a prohibitive obstacle in reconstruction of the mandible, demanding a better understanding of XRT-induced damage and options for reconstruction. Our study investigates the cellular damage caused by radiation in an isogenic murine model of mandibular distraction osteogenesis (DO). We posit that radiation will result in reduced osteocytes, with elevated empty lacunae and immature osteoid.

Methods Twenty Lewis rats were randomly assigned to two groups: DO (n=10) and XRT/DO (n=10). Both groups underwent an osteotomy and mandibular DO across a 5.1 mm gap. XRT was administered to the XRT/DO group at a fractionated, human equivalent dose of 35 Gy prior to surgery. Animals were sacrificed on postoperative day 40 and mandibles were harvested and sectioned for histological analysis.

Results Bone that underwent radiation revealed a significantly decreased osteocyte count and complementary increase in empty lacunae when compared to non-XRT bone (p=0.019, p=0.000). Additionally, XRT bone demonstrated increased immature osteoid and decreased mature woven bone when compared to non-radiated bone (p=0.001 and p=0.003, respectively). Furthermore, analysis of the ratio of immature osteoid to woven bone volume exhibited a significant increase in the XRT bone, further revealing the devastating damage brought by XRT (p=0.001).

Conclusion These results clearly demonstrate the cellular diminution that occurs as a result of radiation. This foundational study provides the groundwork upon which to investigate cellular therapies in an immunoprivileged model of mandibular DO.

Link To Article

http://dx.doi.org/10.1016/j.joms.2015.08.002

The TGF-β Signaling Regulator PMEPA1 Suppresses Prostate Cancer Metastases to Bone

Authors

Pierrick G.J. Fournier, Patricia Juárez, Guanglong Jiang, Gregory A. Clines, Maria Niewolna, Hun Soo Kim, Holly W. Walton, Xiang Hong Peng, Yunlong Liu, Khalid S. Mohammad, Clark D. Wells, John M. Chirgwin, Theresa A. Guise

Abstract

Transforming growth factor-β (TGF-β) regulates the expression of genes supporting breast cancer cells in bone, but little is known about prostate cancer bone metastases and TGF-β. Our study reveals that the TGFBR1 inhibitor SD208 effectively reduces prostate cancer bone metastases. TGF-β upregulates in prostate cancer cells a set of genes associated with cancer aggressiveness and bone metastases, and the most upregulated gene was PMEPA1. In patients, PMEPA1 expression decreased in metastatic prostate cancer and low Pmepa1 correlated with decreased metastasis-free survival. Only membrane-anchored isoforms of PMEPA1 interacted with R-SMADs and ubiquitin ligases, blocking TGF-β signaling independently of the proteasome. Interrupting this negative feedback loop by PMEPA1 knockdown increased prometastatic gene expression and bone metastases in a mouse prostate cancer model.

Link To Article

http://dx.doi.org/10.1016/j.ccell.2015.04.009

Single-Limb Irradiation Induces Local and Systemic Bone Loss in a Murine Model

Authors

Laura E. Wright Ph.D., Jeroen T. Buijs Ph.D., Hun-Soo Kim M.D., Laura E. Coats M.D., Anne M. Scheidler M.D., Sutha K. John M.S., Yun She B.A., Sreemala Murthy M.S., Ning Ma M.D., Helen J. Chin- Sinex B.S., Teresita M. Bellido Ph.D., Ted A. Bateman Ph.D., Marc S. Mendonca Ph.D., Khalid S. Mohammad M.D., Ph.D. and Theresa A. Guise M.D.

Abstract

Increased fracture risk is commonly reported in cancer patients receiving radiotherapy, particularly at sites within the field of treatment. The direct and systemic effects of ionizing radiation on bone at a therapeutic dose are not well characterized in clinically relevant animal models. Using twenty-week male C57Bl/6 mice, effects of irradiation (right hindlimb; 2 Gy) on bone volume and microarchitecture were evaluated prospectively by microcomputed tomography and histomorphometry and compared to contralateral-shielded bone (left hindlimb) and non-irradiated control bone. One-week post-irradiation, trabecular bone volume declined in irradiated tibiae (-22%; p < 0.0001) and femora (-14%; p = 0.0586) and microarchitectural parameters were compromised. Trabecular bone volume declined in contralateral tibiae (-17%; p = 0.003), and no loss was detected at the femur. Osteoclast number, apoptotic osteocyte number and marrow adiposity were increased in irradiated bone relative to contralateral and non-irradiated bone, while osteoblast number was unchanged. Despite no change in osteoblast number one-week post-irradiation, dynamic bone formation indices revealed a reduction in mineralized bone surface and a concomitant increase in unmineralized osteoid surface area in irradiated bone relative to contralateral and non-irradiated control bone. Further, dose- and time-dependent calvarial culture and in vitro assays confirmed that calvarial osteoblasts and osteoblast-like MC3T3 cells were relatively radioresistant, while calvarial osteocyte and osteocyte-like MLO-Y4 cell apoptosis was induced as early as 48h post-irradiation (4 Gy). In osteoclastogenesis assays, radiation exposure (8 Gy) stimulated murine macrophage RAW264.7 cell differentiation and co-culture of irradiated RAW264.7 cells with MLO-Y4 or murine bone marrow cells enhanced this effect. These studies highlight the multi-faceted nature of radiation-induced bone loss by demonstrating direct and systemic effects on bone and its many cell types using clinically relevant doses and have important implications for bone health in patients treated with radiation therapy.

Link To Article

http://dx.doi.org/10.1002/jbmr.2458

Prostate Cancer Cells Preferentially Home To Osteoblast-Rich Areas In The Early Stages Of Bone Metastasis – Evidence From In Vivo Models

Authors

Ning Wang Ph.D, Freyja E Docherty PhD, Hannah K Brown PhD, Kimberley J Reeves PhD, Anne CM Fowles MSc, Penelope D Ottewell PhD, T. Neil Dear PhD, Ingunn Holen PhD, Peter I Croucher PhD andColby L Eaton PhD

Abstract

It has been suggested that metastasis-initiating cells gain a foothold in bone by homing to a metastastatic microenvironment (or ‘niche’). Whereas the precise nature of this niche remains to be established, it is likely to contain bone cell populations including osteoblasts and osteoclasts. In the mouse tibia, the distribution of osteoblasts on endocortical bone surfaces is non-uniform and we hypothesize that studying co-localization of individual tumour cells with resident cell populations will reveal the identity of critical cellular components of the niche. In this study, we have mapped the distribution of three human prostate cancer cell lines (PC3-NW1, LN-CaP, and C4 2B4) colonising the tibias of athymic mice following intracardiac injection and evaluated their interaction with potential metastatic niches. Prostate cancer cells labelled with the fluorescent cell membrane dye (Vybrant DiD) were found by two-photon microscopy to be engrafted in the tibiae in close proximity (∼40 µm) to bone surfaces and 70% more cancer cells were detected in the lateral compared to the medial endocortical bone regions. This was associated with a 5-fold higher number of osteoblasts and 7-fold higher bone formation rate on the lateral endocortical bone surface compared to the medial side. By disrupting cellular interactions mediated by the chemokine (C-X-C motif) receptor 4 (CXCR4)/chemokine ligand 12 (CXCL12) axis with the CXCR4 inhibitor AMD3100, the preferential homing pattern of prostate cancer cells to osteoblast-rich bone surfaces was disrupted. In this study, we map the location of prostate cancer cells that home to endocortical regions in bone and our data demonstrate that homing of prostate cancer cells is associated with the presence and activity of osteoblast lineage cells, and suggest that therapies targeting osteoblast niches should be considered to prevent development of incurable prostate cancer bone metastases.

Link To Article

http://dx.doi.org/10.1002/jbmr.2300

RUNX3 Facilitates Growth of Ewing Sarcoma Cells

Authors

Krista L. Bledsoe, Meghan E. McGee-Lawrence, Emily T. Camilleri, Xiaoke Wang, Andre J. van Wijnen, Andre M. Oliveira and Jennifer J. Westendorf

Abstract

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt-domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal-derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI-responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma.

Link To Article

http://dx.doi.org/10.1002/jcp.24663

Curcumin Inhibits Prostate Cancer Bone Metastasis by Up-Regulating Bone Morphogenic Protein-7 in Vivo

Authors

Thambi Dorai, Janane Diouri, Orla O’Shea, Stephen B. Doty

Abstract

A number of studies have focused on the beneficial properties of Curcumin (diferuloyl methane, used in South Asian cuisine and traditional medicine) such as the chemoprevention of cancer. Recent studies have also indicated that this material has significant benefits for the treatment of cancer and is currently undergoing several clinical trials. We have been interested in the application of this compound as a therapeutic agent for advanced prostate cancer, particularly the skeletal complications in this malignancy. Our earlier work indicated that this compound could inhibit the osteomimetic properties which occur in castration resistant prostate cancer cells, by interfering with the common denominators between these cancer cells and the bone cells in the metastatic tumor microenvironment, namely the osteoblasts and the osteoclast. We predicted that curcumin could break the vicious cycle of reciprocal stimulation that results in uncontrolled osteolysis in the bony matrix. In this work, we have evaluated the potential of this compound in inhibiting the bone metastasis of hormone refractory prostate cancer cells in an established animal model. Our results strongly suggest that curcumin modulates the TGF-β signaling that occurs due to bone matrix degradation by up-regulating the metastasis inhibitory bone morphogenic protein-7 (BMP-7). This enhancement of BMP-7 in the context of TGF-β in the tumor microenvironment is shown to enhance the mesenchymal-to-epithelial transition. Most importantly, we show that as a result of BMP-7 up-regulation, a novel brown/beige adipogenic differentiation program is also up-regulated which plays a role in the inhibition of bone metastasis. Our results suggest that curcumin may subvert the TGF-β signaling to an alternative adipogenic differentiation program in addition to the previously established interference with the osteomimetic properties, thus inhibiting the bone metastatic processes in a chemopreventive as well as therapeutic setting.

Link To Article

http://dx.doi.org/10.4236/jct.2014.54044