Deletion of the gene encoding calcitonin and calcitonin gene-related peptide α does not affect the outcome of severe infection in mice.

Procalcitonin (PCT) is expressed in nonthryoidal tissues of humans during severe infections. Serum PCT levels are measured to diagnose and guide therapy, and there is some evidence that PCT may also contribute to the pathogenesis of sepsis. We tested whether disruption of the gene encoding PCT in mice affected the course of sepsis. Mice with exons 2-5 of the gene encoding calcitonin/calcitonin gene-related polypeptide α (Calca) knocked out and congenic C57BL/6J control mice were challenged with aerosolized Streptococcus pneumoniae or Pseudomonas aeruginosa, or injected intraperitoneally with S. pneumoniae. There were no significant differences in the survival of knockout and control mice in the two pneumonia models, and no significant differences in weight loss, splenic bacterial counts, or blood leukocyte levels in the peritoneal sepsis model. To verify disruption of the Calca gene in knockout mice, the absence of calcitonin in the serum of knockout mice and its presence and inducibility in control mice were confirmed. To evaluate PCT expression in nonthyroidal tissues of control mice, transcripts were measured in multiple organs. PCT transcripts were not significantly expressed in liver or spleen of control mice challenged with aerosolized P. aeruginosa or intraperitoneal endotoxin, and were expressed in lung only at low levels, even though serum IL-6 rose 3,548-fold. We conclude that mice are not an ideal loss-of-function model to test the role of PCT in the pathogenesis of sepsis because of low nonendocrine PCT expression during infection and inflammation. Nonetheless, our studies demonstrate that nonendocrine PCT expression is not necessary for adverse outcomes from sepsis.

Hypercalcemia in disseminated coccidioidomycosis: expression of parathyroid hormone-related peptide is characteristic of granulomatous inflammation.

BACKGROUND: Hypercalcemia is an uncommon complication of disseminated granulomatous infections. The pathogenesis of hypercalcemia associated with infection is not clear. METHODS: We investigated a case of disseminated coccidioidomycosis with hypercalcemia. We used a sensitive radioimmunoassay to measure serum parathyroid hormone-related peptide (PTHrP) and a mouse monoclonal antibody to PTHrP to immunostain biopsies. RESULTS: We found elevated serum levels of PTHrP while the patient was hypercalcemic that became undetectable when serum calcium normalized. We also found that the inflammatory cells and some surrounding tissues in skin biopsies expressed PTHrP. PTHrP was expressed by all biopsied lesions of patients with coccidioidomycosis that we examined, whether localized to the lung or disseminated, but no other cases were hypercalcemic. PTHrP was also expressed by the 3 mycobacterial granulomas we examined, and in a lymph node from a patient with sarcoidosis. CONCLUSIONS: The expression of PTHrP is a property of infectious granulomas regardless of etiology or the tissue involved, suggesting that PTHrP expression is part of the normal granulomatous immune response. Hypercalcemia may result if there is disseminated infection and multiple granulomas. We propose that excess production of PTHrP is the cause of hypercalcemia in granulomatous infections.

Knockdown of the ß(1) integrin subunit reduces primary tumor growth and inhibits pancreatic cancer metastasis.

To address the role of ß(1) integrins in pancreatic cancer progression, we stably knocked down ß(1) integrin subunit expression in human FG-RFP pancreatic cancer cells using lentiviral-based RNA interference. We then examined the effects of ß(1) integrin subunit knockdown on pancreatic cancer cell adhesion, migration and proliferation on tumor microenvironment-specific extracellular matrix proteins in vitro and on tumor progression in vivo using a clinically relevant fluorescent orthotopic mouse model of pancreatic cancer. Knockdown of the ß(1) integrin subunit inhibited cell adhesion, migration and proliferation on types I and IV collagen, fibronectin and laminin in vitro. In vivo, knockdown of the ß(1) integrin subunit reduced primary tumor growth by 50% and completely inhibited spontaneously occurring metastasis. These observations indicate a critical role for the ß(1) integrin subunit in pancreatic cancer progression and metastasis in particular. Our results suggest the ß(1) integrin subunit as a therapeutic target for the treatment of pancreatic cancer, especially in the adjuvant setting to prevent metastasis of this highly aggressive cancer.

Parathyroid hormone-related peptide is a naturally occurring, protein kinase A-dependent angiogenesis inhibitor.

Angiogenesis is a highly regulated process that results from the sequential actions of naturally occurring stimulators and inhibitors. Here, we show that parathyroid hormone-related peptide, a peptide hormone derived from normal and tumor cells that regulates bone metabolism and vascular tone, is a naturally occurring angiogenesis inhibitor. Parathyroid hormone-related peptide or a ten-amino-acid peptide from its N terminus inhibits endothelial cell migration in vitro and angiogenesis in vivo by activating endothelial cell protein kinase A. Activation of protein kinase A inhibits cell migration and angiogenesis by inhibiting the small GTPase Rac. In contrast, inhibition of protein kinase A reverses the anti-migratory and anti-angiogenic properties of parathyroid hormone-related peptide. These studies show that parathyroid hormone-related peptide is a naturally occurring angiogenesis inhibitor that functions by activation of protein kinase A.

Cell cycle actions of parathyroid hormone-related protein in non-small cell lung carcinoma.

Parathyroid hormone-related protein (PTHrP), a paraneoplastic protein expressed by two-thirds of human non-small cell lung cancers, has been reported to slow progression of lung carcinomas in mouse models and to lengthen survival of patients with lung cancer. This study investigated the effects of ectopic expression of PTHrP on proliferation and cell cycle progression of two human lung adenocarcinoma cell lines that are normally PTHrP negative. Stable transfection with PTHrP decreased H1944 cell DNA synthesis, measured by thymidine incorporation, bromodeoxyuridine uptake, and MTT proliferation assay. A substantial fraction of PTHrP-positive cells was arrested in or slowly progressing through G1. Cyclin D2 and cyclin A2 protein levels were 60-70% lower in PTHrP-expressing cells compared with control cells (P < 0.05, N = 3 independent clones per group), while expression of p27(Kip1), a cyclin-dependent kinase inhibitor, was increased by 35 +/- 9% (mean +/- SE, P < 0.05) in the presence of PTHrP. Expression of other cyclins, including cyclins D1 and D3, and cyclin-dependent kinases was unaffected by PTHrP. PTHrP did not alter the phosphorylation state of Rb, but decreased cyclin-dependent kinase (CDK) 2-cyclin A2 complex formation. Ectopic expression of PTHrP stimulated ERK phosphorylation. In MV522 cells, PTHrP had similar effects on DNA synthesis, cyclin A2 expression, pRb levels, CDK2-cyclin A2 association, and ERK activation. In summary, PTHrP appears to slow progression of lung cancer cells into S phase, possibly by decreasing activation of CDK2. Slower cancer cell proliferation could contribute to slower tumor progression and increased survival of patients with PTHrP-positive lung cancer.

Integrin-mediated laminin-1 adhesion upregulates CXCR4 and IL-8 expression in pancreatic cancer cells.

BACKGROUND: We have shown recently that alpha(2)beta(1) integrin-mediated type I collagen adhesion promotes a more malignant phenotype in pancreatic cancer cell lines than other extracellular matrix (ECM) proteins. MiaPaCa-2 cells, by contrast, do not express collagen-binding integrins, but are metastatic in our orthotopic mouse model and migrate maximally on laminin-1 (Ln-1). It has also been shown that CXCR4 and IL-8 expression correlates directly with metastasis in pancreatic cancer in vivo. We therefore examined the potential of the ECM to regulate CXCR4 and IL-8 expression in pancreatic cancer cells. METHODS: We cultured 8 pancreatic cancer cell lines on fibronectin (Fn), types I and IV collagen, Ln-1 and vitronectin (Vn), and examined cell lysates for CXCR4 by immunoblotting and media for IL-8 by ELISA. We also conducted cell migration assays with stromal-derived factor-1 (SDF-1) as the chemoattractant to examine integrin-binding specificity and CXCR4 function. RESULTS: All cell lines expressed CXCR4 protein. MiaPaCa-2 cell growth on Ln-1 increased significantly CXCR4 and IL-8 expression relative to other ECM proteins. Migration inhibition studies showed that both the alpha(6)beta(1) and alpha(3)beta(1) integrins mediate MiaPaCa-2 migration on Ln-1. Growth studies showed further that CXCR4 expression on Ln-1 was mediated by the alpha(6)beta(1) integrin whereas IL-8 expression was mediated by both the alpha(6)beta(1) and alpha(3)beta(1) integrins. The expression of functional CXCR4 was also shown in migration assays, where SDF-1 significantly increased pancreatic cancer cell chemotaxis on Ln-1. CONCLUSIONS: These data indicate that integrin-mediated Ln-1 adhesion upregulates CXCR4 and IL-8 expression and may play a mechanistic role in pancreatic cancer metastases.

Sex-specific survival advantage with parathyroid hormone-related protein in non-small cell lung carcinoma patients.

PURPOSE: Parathyroid hormone-related protein (PTHrP) is commonly expressed in non-small cell lung carcinomas (NSCLC). Expression of the protein could have implications for progression of the disease because it regulates cancer cell growth, apoptosis, and angiogenesis. However, its relationship with survival has not been evaluated in a large-scale investigation. EXPERIMENTAL DESIGN: PTHrP expression was assessed in paraffin-embedded tumor samples from 407 patients with NSCLC by immunohistochemistry. A pathologist unaware of the clinical history classified specimens as PTHrP positive or PTHrP negative. The log-rank test was used to compare survivals of PTHrP-positive and PTHrP-negative groups, and Cox regression was used to adjust for additional covariates. RESULTS: Median survival was 55 versus 22 months (P < 0.001) in female patients with and without tumor PTHrP, respectively. Male survival was 38 months independent of PTHrP status. Stage, histology, age, and smoking history were also associated with increased longevity. PTHrP remained a significant predictor of survival for female patients after controlling for stage, histology, and age. CONCLUSIONS: In this study, PTHrP expression was associated with a survival advantage in female patients. Additional investigations must be done to ascertain whether the result is reproducible and independent of potential confounding covariates. Sex-dependent effects of PTHrP in lung cancer would open new avenues of research into the role of sex in cancer progression.

The bisphosphonate olpadronate inhibits skeletal prostate cancer progression in a green fluorescent protein nude mouse model.

PURPOSE: Metastatic bone disease is one of the major causes of morbidity and mortality in prostate cancer patients. Bisphosphonates are currently used to inhibit bone resorption and reduce tumor-induced skeletal complications. More effective bisphosphonates would enhance their clinical value. EXPERIMENTAL DESIGN: We tested several bisphosphonates in a green fluorescent protein (GFP)-expressing human prostate cancer nude mouse model. The in vivo effects of four bisphosphonates, including pamidronate, etidronic acid, and olpadronate, on bone tumor burden in mice intratibially inoculated with PC-3-GFP human prostate cancer cells were visualized by whole-body fluorescence imaging and X-ray. RESULTS: The PC-3-GFP cells produced extensive bone lesions when injected into the tibia of immunocompromised mice. The skeletal progression of the PC-3-GFP cell growth was monitored by GFP fluorescence and the bone destruction was evaluated by X-ray. We showed that 3,3-dimethylaminopropane-1-hydroxy-1,1-diphosphonic acid (olpadronate) was the most effective bisphosphonate treatment in reducing tumor burden as assessed by GFP imaging and radiography. The GFP tumor area and X-ray score significantly correlated. Reduced tumor growth in the bone was accompanied by reduced serum calcium, parathyroid hormone-related protein, and osteoprotegerin. CONCLUSIONS: The serum calcium, parathyroid hormone-related protein, and osteoprotegerin levels were significantly correlated with GFP area and X-ray scores. Treatment with olpadronate reduced tumor growth in the bone measured by GFP and X-ray imaging procedures. Imaging of GFP expression enables monitoring of tumor growth in the bone and the GFP results complement the X-ray assessment of bone disease. The data in this report suggest that olpadronate has potential as an effective inhibitor of the skeletal progression of clinical prostate cancer.

A carboxyl leucine-rich region of parathyroid hormone-related protein is critical for nuclear export.

PTHrP is an oncofetal protein with distinct proliferative and antiapoptotic roles that are affected by nucleocytoplasmic shuttling. The protein's nuclear export is sensitive to leptomycin B, consistent with a chromosome region maintenance protein 1-dependent pathway. We determined that the 109-139 region of PTHrP was involved in its nuclear export by demonstrating that a C-terminal truncation mutant, residues 1-108, exports at a reduced rate, compared with the wild-type 139 amino acid isoform. We searched for potential nuclear export sequences within the 109-139 region, which is leucine rich. Comparisons with established nuclear export sequences identified a putative consensus signal at residues 126-136. Deletion of this region resulted in nuclear export characteristics that closely matched those of the C-terminal truncation mutant. Confocal microscopic analyses of transfected 293, COS-1, and HeLa cells showed that steady-state nuclear levels of the truncated and deletion mutants were significantly greater than levels of wild-type PTHrP and were unaffected by leptomycin B, unlike the wild-type protein. In addition, both mutants demonstrated greatly reduced nuclear export with assays using nuclear preparations and intact cells. Based on these results, we conclude that the 126-136 amino acid sequence closely approximates the structure of a chromosome region maintenance protein 1-dependent leucine-rich nuclear export signal and is critical for nuclear export of PTHrP.

Identification of DU 145 prostate cancer cell proteins that bind to the carboxy-terminal peptide of human PTHrP in vitro.

Peptides spanning the range of human parathyroid hormone-related protein (PTHrP) have been shown to bind heat shock protein-70 expressed on the surface of cancer cells with cytoprotective consequences in vitro. The present study focused on identification of intracellular proteins that interact with the carboxy-terminal peptide of human PTHrP. Using affinity chromatography, we applied extracts of DU 145 prostate cancer cells over PTHrP (140-173)-Sepharose and eluted with 8 M urea. After concentration and electrophoresis, protein bands were excised and subjected to mass spectroscopy analyses. Proteins identified included those associated with protection from oxidative stress, DNA repair, protection from apoptosis, and proteins involved in membrane trafficking and cytoskeletal rearrangement. These novel protein-protein interactions further support the hypothesis that the carboxy-terminus of PTHrP plays a role in cell survival.

Heat shock protein-70 expressed on the surface of cancer cells binds parathyroid hormone-related protein in vitro.

Recent studies have shown that the functions of PTH-related protein (PTHrP) and its derived peptides cannot be attributed solely to PTH/PTHrP receptor binding. The present study focused on the identification of other proteins that might bind PTHrP at the cell surface. Using affinity chromatography, we applied extracts of cell-surface biotinylated proteins from cancer and normal cell lines over Sepharose beads coupled with different PTHrP-derived peptides. Elution with the corresponding free peptide revealed a major protein of about 70 kDa that was present in all of the PTHrP peptide eluates from cancer cell extracts but not from normal breast cell extracts. Mass spectroscopy analysis and immunoblotting identified this PTHrP-binding protein as heat shock protein-70 (HSP70). Using a recently published algorithm that predicts HSP70 binding sites within proteins, we found that all four PTHrP peptides used in these studies contain amino acid motifs with high probabilities for HSP70 binding in vivo. Cell culture studies in the presence of a polyclonal anti-HSP70 antibody demonstrated increased PTHrP secretion, decreased total cellular protein, and differentially regulated proliferation. Taken together, these studies demonstrate a novel and biologically relevant interaction between cell surface-expressed HSP70 and PTHrP in cancer.

Hypercalcemia and local production of parathyroid hormone-related protein by a perisellar rhabdomyosarcoma after remote pituitary irradiation.

OBJECTIVE: To describe a case of metastatic rhabdomyosarcoma originating from the sphenoid sinus in a patient previously treated with conventional irradiation for a prolactinoma, presenting as hypercalcemia in the setting of a normal level of serum parathyroid hormone-related protein (PTHrP). METHODS: We report the case of a patient who underwent remote pituitary irradiation for a prolactinoma and then presented decades later with hypercalcemia of unknown cause. His clinical course, the initial biochemical and radiologic investigations, and the results of examination of pathology specimens are reviewed. RESULTS: The patient was found to have a mass in the sphenoid sinus. The pathologic features were consistent with alveolar rhabdomyosarcoma. Although he had a normal serum PTHrP level, staining of his tumor with an antibody against PTHrP revealed local production of PTHrP at the tumor margins. His bone marrow biopsy specimen showed 100% involvement with rhabdomyosarcoma. CONCLUSION: PTHrP staining of pathology specimens might explain hypercalcemia of undetermined cause in patients with a known malignant lesion, in whom elevated serum PTHrP levels cannot be demonstrated.

Type I collagen and divalent cation shifts disrupt cell-cell adhesion, increase migration, and decrease PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells.

BACKGROUND: We have shown in FG pancreatic cancer cells that alpha2beta1 integrin-mediated type I collagen adhesion decreases parathyroid hormone-related protein (PTHrP), interleukin-6 (IL-6), and interleukin-8 (IL-8) expression, decreases the localization of E-cadherin and beta-catenin in cell-cell contacts, increases cell migration, and increases glycogen synthase kinase 3 (GSK3) and protein kinase B (PKB/Akt) phosphorylation states relative to alpha5beta1 integrin-mediated fibronectin (Fn) adhesion. AIM OF THE STUDY: To extend our observations in FG cells to other pancreatic cancer cell lines, and to determine whether E-cadherin-mediated cell-cell adhesion and its downstream effectors were functionally involved in the ECM-mediated regulation of PTHrP, IL-6, and IL-8. METHODS: We used standard biochemical techniques to determine ECM-specific differences in E-cadherin and beta-catenin localization, GSK3 and PKB/Akt phosphorylation, haptokinetic cell migration, and cytokine expression in pancreatic cancer cells. We also conducted functional studies using pharmacological inhibitors for GSK3 and PKB/Akt, as well as elevated Mg2+/Ca2+ ratios similar to pancreatic juice, and examined their effects on cytokine expression. RESULTS: Differences in E-cadherin and beta-catenin localization along with GSK3 and PKB/Akt phosphorylation occur in multiple pancreatic cancer cell lines, resulting in differences in ECM-mediated haptokinesis and cytokine expression that are generally consistent with previous observations in FG cells. Our functional studies also suggest that E-cadherin-mediated cell-cell adhesion and downstream effectors are involved in PTHrP, IL-6, and IL-8 expression. CONCLUSIONS: These data indicate that alpha2beta1 integrin-mediated type I collagen adhesion disrupts cell-cell adhesion architecture, resulting in increased migration and decreased PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells.

Hypercalcemia in malignant and inflammatory diseases.

Hypercalcemia is one of the most common metabolic abnormalities in human disease. Although there are many causes, most cases are due to neoplasia. Understanding the pathophysiology can lead to correct diagnosis and effective therapy for most patients.

Parathyroid hormone-related protein as a novel tumor marker in pancreatic adenocarcinoma.

INTRODUCTION: Parathyroid hormone-related protein (PTHrP) can act as an oncoprotein to regulate the growth and proliferation of many common malignancies, including pancreatic cancer. Previous studies have shown that PTHrP is produced by human pancreatic cancer cell lines, can be shown in the cytoplasm and nucleus of paraffin-embedded pancreatic adenocarcinoma tumor specimens, and is secreted into the media of cultured pancreatic adenocarcinoma cells. We hypothesized that PTHrP could serve as a tumor-marker for growth of pancreatic cancer in vivo. AIM AND METHODOLOGY: To test this hypothesis, we used an orthotopic model developed in our laboratory of the PTHrP-producing human pancreatic cancer line, BxPC-3. This tumor was stably transduced with green fluorescence protein (GFP) to facilitate visualization of tumor growth and metastases. At early (5 weeks) and late (13 weeks) time points after surgical orthotopic implantation, serum PTHrP was measured and primary and metastatic tumor burden was determined for each mouse by assessing GFP expression. RESULTS: By 5 weeks after surgical orthotopic implantation (early group), the mean serum PTHrP level was 33.3 pg/mL. In contrast, by 13 weeks after surgical orthotopic implantation (late group), the mean serum PTHrP level increased to 158.5 pg/mL. These differences were highly significant (p < 0.001, Student t test). Numerous metastatic lesions were readily visualized by GFP in the late group. Serum PTHrP levels measured by immunoassay correlated with primary pancreatic tumor weights and serum calcium levels (p <0.01). PTHrP levels were not detectable (<21 pg/mL) in any of the 10 control mice with no tumor. Western blotting of BxPC-3-GFP tumor lysates confirmed the presence of PTHrP. BxPC-3-GFP tumor tissue stained with antibody to PTHrP. CONCLUSION: These results indicate that PTHrP can serve as a tumor marker in animal models of pancreatic cancer and may be a useful tumor marker for clinical pancreatic adenocarcinoma.

Thymic parathyroid carcinoma and postoperative hungry bone syndrome.

OBJECTIVE: To describe a case of parathyroid carcinoma localized to ectopic parathyroid tissue within the thymus and the development of hungry bone syndrome postoperatively. METHODS: We present pertinent clinical, radiologic, pathologic, and laboratory details of the study patient and discuss the relevant literature. RESULTS: A 33-year-old man who presented with only symptoms of fatigue and depression was found to have high serum calcium, parathyroid hormone, and alkaline phosphatase levels. A sestamibi scan revealed evidence of activity adjacent to the sternum. He underwent neck exploration after selective staining of the parathyroid glands with methylene blue dye. A mass was found in the thymic bed, and the pathologic features were consistent with parathyroid carcinoma. Postoperatively, symptoms and signs of hypocalcemia developed despite normal serum calcium levels. CONCLUSION: Physicians should be aware that (1) parathyroid carcinoma may originate wherever ectopic parathyroid tissue is found and patients may have only nonspecific symptoms, (2) wide local excision is imperative if carcinoma is suspected, and (3) symptoms and signs of hypocalcemia may be evident even when the serum calcium concentration is normal, if it has rapidly declined from a high level.

Metachronous double parathyroid adenomas involving two different cell types: chief cell and oxyphil cell.

OBJECTIVE: To describe a patient with the rare occurrence of metachronous double parathyroid adenomas involving two different cell types. METHODS: We present a case report and a summary of the related literature. RESULTS: Double parathyroid adenomas are rare, occurring in 1.7 to 9% of patients with primary hyperparathyroidism. Most double parathyroid adenomas are synchronous lesions; few metachronous double parathyroid adenomas have previously been reported. Most parathyroid adenomas are of the chief cell variety, with oxyphil cell adenomas occurring in less than 1% of cases. In a 73-year-old man with no prior history or known family history of endocrine disease, primary hyperparathyroidism developed, and cervical ultrasonography demonstrated a mass in the right side of the neck. Subsequent parathyroidectomy revealed a right superior chief cell adenoma. Postoperatively, the patient's parathyroid hormone and serum calcium levels returned to normal and remained so for at least 9 years. Twelve years after the first operation, recurrent hyperparathyroidism prompted repeated surgical exploration of the neck, which disclosed a large left superior parathyroid mass. Surgical excision and histologic examination revealed the lesion to be an oxyphil cell adenoma. CONCLUSION: To our knowledge, this is the first reported case of metachronous double parathyroid adenomas involving two different cell types: chief cell and oxyphil cell.

Stability of a transdermal salmon calcitonin formulation.

This study was designed to monitor the stability of salmon calcitonin during storage conditions, under the electric fields generated during iontophoresis and electroporation, in contact with transdermal glass diffusion cells, and during transport through skin. The formulation in a citrate buffer (pH 4.0) was stable in storage for short-term studies but degraded significantly on extended storage. Albumin was able to minimize adsorption in contact with glass surfaces, and aprotinin was able to minimize proteolytic degradation in contact with skin. The formulation was stable under electric field, but there was a loss due to adsorption if salt bridges were used.

Parathyroid hormone related-protein promotes epithelial-to-mesenchymal transition in prostate cancer.

Parathyroid hormone-related protein (PTHrP) possesses a variety of physiological and developmental functions and is also known to facilitate the progression of many common cancers, notably their skeletal invasion, primarily by increasing bone resorption. The purpose of this study was to determine whether PTHrP could promote epithelial-to-mesenchymal transition (EMT), a process implicated in cancer stem cells that is critically involved in cancer invasion and metastasis. EMT was observed in DU 145 prostate cancer cells stably overexpressing either the 1-141 or 1-173 isoform of PTHrP, where there was upregulation of Snail and vimentin and downregulation of E-cadherin relative to parental DU 145. By contrast, the opposite effect was observed in PC-3 prostate cancer cells where high levels of PTHrP were knocked-down via lentiviral siRNA transduction. Increased tumor progression was observed in PTHrP-overexpressing DU 145 cells while decreased progression was observed in PTHrP-knockdown PC-3 cells. PTHrP-overexpressing DU 145 formed larger tumors when implanted orthoptopically into nude mice and in one case resulted in spinal metastasis, an effect not observed among mice injected with parental DU 145 cells. PTHrP-overexpressing DU 145 cells also caused significant bone destruction when injected into the tibiae of nude mice, while parental DU 145 cells caused little to no destruction of bone. Together, these results suggest that PTHrP may work through EMT to promote an aggressive and metastatic phenotype in prostate cancer, a pathway of importance in cancer stem cells. Thus, continued efforts to elucidate the pathways involved in PTHrP-induced EMT as well as to develop ways to specifically target PTHrP signaling may lead to more effective therapies for prostate cancer.