Hedgehog Signaling Regulates PTHrP Expression in Osteolytic Breast Tumor Metastases:

Mouse Bearing MDA 231 Breast Tumor Cells. These tumors frequently metasticize to the hind limbs. Left: Flourescent image of the hind limb showing the tumor cells in bone. Right: X-Ray showing osteolysis. Note the tumor and osteolysis overlap.

The skeleton is the most common site of metastasis in patients with advanced breast cancer.   Once established in the skeleton, breast tumors have catastrophic effects on the skeleton inducing a vicious cycle in which tumor produced factors stimulate bone resorption resulting in the release of growth factors from the bone which further stimulate tumor growth and bone destruction.  One such factor is parathyroid hormone-related peptide (PTHrP) secreted by breast tumors that have metastasized to bone leads to tumor induced osteolysis and bone destruction.  It has been demonstrated previously that inhibition of PTHrP transcription by small molecules reduces bone lesions and tumor burden. It is therefore clearly important to understand the molecular mechanisms responsible for PTHrP expression by breast cancer cells.  However, the mechanisms responsible for increased PTHrP expression by breast cancer cells remains largely unknown. We have found that the Gli family of transcriptional regulators are responsible for regulating PTHrP transcription in breast cancer.  Specifically we have found that Gli2 mRNA expression in various different cancer cell lines.  We found that Gli2 was expressed in osteolytic cell lines that express PTHrP (MDA-MB-231, RWGT2, and PC-3), but not in non-osteolytic cell lines which do not secrete PTHrP (MCF-7, ZR-75, and T47D).   When breast cancer cell lines were transiently transfected with a PTHrP promoter luciferase constructs and expression vectors for the Gli family, we found that Gli2 specifically increased PTHrP promoter activity, while Gli3 and Gli1 did not significantly affect basal promoter activity.  Using a proteasomally processed, repressor form of Gli3 or a dominant negative Gli2 construct (Gli2-EnR) suppressed the Gli2 stimulated-PTHrP transcription in human MDA-MB-231 breast cancer cells. Furthermore, when MDA-MB-231 human breast cancer cells stably overexpressing Gli2 were inoculated into the left cardiac ventricle of athymic nude mice, enhanced osteolysis as determined by lesion area and trabecular bone volume was observed when compared with mice injected with the empty vector control.  Taken together, these data indicate that the Gli2 regulates PTHrP expression and subsequent tumor-induced osteolysis.   

Small Animal Imaging For Monitoring Osteolytic Bone Metastases:

Our laboratory utilizes a well established mouse model of breast cancer metastasis to bone in order to identify molecular targets for the development of better therapeutics. Standard practice has been to monitor bone metastases using standard radiography and bone histomorphometry performed at the end of the experiments.  Osteolytic lesions are visible by radiography approximately 14-21 days after tumor cell inoculation via the left cardiac ventricle.  However, at this time point, tumors are well established, and there are regions of the skeleton which we are not able to visualize lesions by faxitron analysis.  Furthermore, it is not possible to monitor tumor burden in bone in living animals throughout the course of the experiment with current techniques.  In order to better understand the relationship between tumor burden and osteolysis, other more sensitive techniques are clearly needed and were utilized in this study.  In breast cancer patients, 18[F]-FDG PET scans and 99mTc-MDP–SPECT have been used to track tumor growth, bone metastases and response to therapy. 

We hypothesized that 18[F]-FDG MicroPET scans and 99mTc-MDP–MicroSPECT with MicroCT anatomical co-registration would allow us to detect tumors at earlier time points and to detect smaller bone metastases than possible with radiography alone.  Here, we present the first use of MicroPET, MicroSPECT, and MicroCT to track tumor burden and bone metastases in a mouse model of breast cancer bone metastases.  

Using this model we were able to detect tumor in bone as early as 7 days post tumor cell inoculation using 18[F]-FDG MicroPET, which is much earlier than by radiography.  In addition, we are able to detect decreased 99mTC-MDP uptake using MicroSPECT/CT in mice with more severe osteolytic lesions. Furthermore, we were able to detect osteolytic lesions by MicroCT that would otherwise have been undetectable by radiography because of their size and by histological methods because of their site.  These techniques will allow us to follow tumor burden and osteolysis through out the course of metastases in the same animals at multiple time points.  The ability to do this will greatly enhance our ability to monitor the effect of drug treatments and other interventions on bone metastases.

Hedgehog Signaling Regulates Growth Plate Chondrocytes:

Parathyroid hormone related peptide (PTHrP) plays an important physiological role in the regulation of chondrocyte proliferation in the growth plate.  Its expression is controlled by Indian hedgehog (Ihh), and both Ihh and PTHrP null mutant mice have severe and overlapping growth plate abnormalities, with premature differentiation and decreased proliferation of growth plate chondrocytes. Although these data suggest that Ihh controls PTHrP expression in cartilage cells, the mediators responsible are unknown. Hedgehog signaling is mediated by the Gli family of transcriptional regulators and Gli2 null mice exhibit increased hypertrophic chondrocytes and decreased PTHrP expression (Miao et al., 2004), indicating that the Gli family has an important and non-redundant role in chondrocyte regulation and regulation of PTHrP.  We therefore reasoned that Gli2 may therefore regulate PTHrP expression in chondrocyte cell lines, and to examine this we performed transfections of the growth plate chondrocyte cell line TMC-23 with a 1.1kb PTHrP promoter construct containing the P2 and P3 promoters (Cataisson et al., 2004) and Gli expression constructs. Using a PTHrP promoter luciferase construct we demonstrated that Gli2 specifically increased PTHrP promoter activity by approximately 4 fold, while other Gli family members had no effect on PTHrP promoter activity. A Gli2 construct, which acts as a dominant negative by blocking Gli2 binding to DNA, decreased PTHrP promoter activity.  Furthermore, Gli2 siRNA duplexes that specifically decrease Gli2 expression by >90% dramatically blocked the Gli2-mediated PTHrP promoter activity, suggesting that Gli2 is a physiologic regulator of PTHrP expression in cartilage cells.  We next analyzed the PTHrP promoters for consensus Gli binding sites. The site with the closest homology was a 6/9 match, which by EMSA did not bind Gli2, indicating that the effects of Gli2 on the PTHrP promoter are likely indirect.  Furthermore, we used a full length (4kb) PTHrP promoter luciferase construct that contains the P1 promoter region in addition to the P2 and P3 promoter.  There was no additional effect of Gli2 on the 4kb promoter over the response observed with the 1.1kb promoter.  The effect of Gli2 on the PTHrP promoter was abolisheshed when constructs containing only the P2 or P3 region of the promoter were utilized in transfections.  However, the effect of Gli2 was restored when a construct containing the 3’ region of P2 and the 5’region of the P3 promoter (-822 to -465).  Taken together, our data suggest that Gli2 is an important regulator of PTHrP transcription in growth plate chondrocyte and that Gli2 mediates the effects of Hh proteins on PTHrP expression and subsequent endochondral bone formation.

Research Techniques:

Promoter Studies (Luciferase Assays, transfections)
Animal Models of Bone Metastasis (intracardiac inoculation)
Small animal imaging (GFP, micro-SPECT, micro-CT, micro-PET, X-ray)
Effect of gene alterations on tumor cell metastasis (stable transfections, SiRNA) 

Research Support:

National Institutes of Health
September 1, 2004 - September 1, 2007
Kirschstein-NRSA Individual Fellowship (F32 AR51639-01)
Regulation of PTHrP expression in chondrocytes by Gli2

The purpose of this fellowship is to determine the molecular mechanisms by which Gli2 stimulates the PTHrP promoter and mediates Hh signaling in chondrocytes and to elucidate the role of the E3 ligase β-TrCP in the regulation of PTHrP expression and growth plate development in vivo.

San Antonio Area Foundation Grant
June 1, 2005 - December 1, 2006
Inhibition of Osteolytic Bone Metastases by Blocking Hedgehog Signaling

Manuscripts:

J. A. Sterling, B. O. Oyajobi , B. Grubbs, S. S. Padalecki, A. Gupta, B.  Story, S. Munoz, Ming Zhao, and Gregory R. Mundy.  The Hedgehog Signaling Molecule Gli2 Induces PTHrP Expression and Osteolysis In Metastatic Human Breast Cancer Cells.  Cancer Research 66 (2006).

J. A. Sterling, L. Wu, and S. S. Banerji. Transcriptional Regulation of TGF-β Receptor Type II by a Novel Inverted CCAAT Box Binding Protein in the ER Positive Breast Cancer Cell Line, MCF-7. Anticancer Research 26 (2006). 1893-1901.

Abstracts and Meetings:

J. A. Sterling, L. Z. Sun, B. Grubbs, M. Zhao, B. O. Oyajobi, S. S. Padalecki, G. R. Mundy.  TGF-β Stimulation of PTHrP Expression in Breast Cancer Cells Is Regulated by Gli2.  American Society for Bone and Mineral Research 27th Annual Meeting.  Nashville, Tennessee. 2005.  Plenary Poster. 

J. A. Sterling, B. Goins, B. Grubbs, M. Amurao, C. Zavaleta, A. Soundararajan, Z. Wang, W. Phillips, B. Oyajobi, G. Mundy, S. S. Padalecki.  Functional and Anatomical Imaging Allows for More Sensitive Monitoring of Tumor Burden and Osteolysis in a Mouse Model of Breast Cancer Metastasis to Bone.  American Society for Bone and Mineral Research 27th Annual Meeting.  Nashville, Tennessee. 2005.

J. A. Sterling, M. Zhao, B. G. Grubbs, B. O. Oyajobi, and G. R. Mundy.  PTHrP Expression in Cartilage Cells is Regulated by Gli2.  2nd Joint Meeting of the International Bone and Mineral Society (IBMS) and the European Society for Calcified Tissue (ECTS).  Geneva, Switzerland.  2005.  Short Oral Presentation.

Vth International Conference on Cancer-Induced Bone Diseases.  Davos, Switzerland.  2005.  J. A. Sterling, S. S. Padalecki, B. G. Grubbs, M. Zhao, B. O. Oyajobi, B. Goins, G. R. Mundy.  Gli2 Regulates PTHrP Expression and Tumor Induced Osteolysis by Metastatic Breast Cancer Cells.  Oral Presentation.

13th Annual San Antonio Cancer Institute Research Symposium.  San Antonio, Texas. 2004.  J. A. Sterling, S. S. Padalecki, B. Goins, B Grubbs, C Zavaleta, A. Soundararajan, W. Phillips, B. O. Oyajobi, G. R. Mundy.  Use of MicroPET and MicroSPECT to Monitor Bone Metastases in a Mouse Model of Breast Cancer Metastases to Bone. 

13th Annual San Antonio Cancer Institute Research Symposium.  San Antonio, Texas.  2004.  J. A. Sterling, M. Zhao, B., B. O. Oyajobi, B. Grubbs, S. S. Padalecki, A. Gupta, G. R. Mundy.  Regulation of PTHrP Expression and Osteolysis by Human Breast Cancer Cells by Gli2.   

American Society for Bone and Mineral Research 26th Annual Meeting.  Seattle, Washington. 2004.  J. A. Sterling, B. O. Oyajobi, M. Zhao, A. Gupta, M. Banerjee*, B. Grubbs, G. R. Mundy Regulation of PTH-rP Expression and Osteolysis by Human Breast Cancer Cells by Gli2 and Gli3.  Poster Session III.

American Society for Bone and Mineral Research 26th Annual Meeting.  Seattle, Washington. 2004. J. A. Sterling, B. O. Oyajobi, M. Zhao, A. Gupta, M. Banerjee, G. R. Mundy.PTH-rP Expression in Cartilage Cells is Regulated by the Gli Family of Transcription Factors.  Plenary Poster.