Notch Signaling and ERK Activation Are Important for the Osteomimetic Properties of Prostate Cancer Bone Metastatic Cell Lines

Abstract

Prostate cancer bone metastases are characterized by their ability to induce osteoblastic lesions and local bone formation. It has been suggested that bone metastatic prostate cancer cells are osteomimetic and capable of expressing genes and proteins typically expressed by osteoblasts. The ability of preosteoblasts to differentiate and express osteoblastic genes depends on several pathways, including Notch and MAPK. Here we show that notch1 expression is increased 4 -5 times in C4-2B and MDA PCa 2b cells (osteoblastic skeletal prostate metastatic cancer cell lines) when compared with nonskeletal metastatic cell lines (LNCaP and DU145). Notch1 ligand, dll1, is expressed only in C4-2B cells. Immunohistochemical studies demonstrate that Notch1 is present in both human clinical samples from prostate cancer bone metastases and the C4-2B cell line. To determine whether prostate cancer bone metastases respond to osteogenic induction similar to osteoblasts, C4-2B cells were cultured in osteogenic medium that promotes mineralization. C4-2B cells mineralize and express HES-1 (a downstream target of Notch), an effect that is completely inhibited by L-685,458, a Notch activity inhibitor. Furthermore, osteogenic induction increases ERK activation, runx2 expression, and nuclear localization, independent of Notch signaling. Finally, we show that Notch and ERK activation are essential for Runx2 DNA binding activity and osteocalcin gene expression in C4-2B cells in response to osteogenic induction. These studies demonstrate that prostate cancer bone metastatic cell lines acquire osteoblastic properties through independent activation of ERK and Notch signaling; presumably, both pathways are activated in the bone microenvironment.Prostate carcinoma most commonly metastasizes to the lymph nodes and to bone with a 70 -80% frequency as determined by autopsy studies. A unique characteristic of skeletal metastases of human prostate cancer is their ability to induce osteoblastic lesions, characterized by new woven bone formation and variable degrees of osteoclastic bone resorption (1). It has been proposed that factors produced by prostate cells, both normal and neoplastic, have the potential to stimulate new bone formation by effects on both osteoblasts and osteoclasts. Included in this group are parathyroid hormone-related peptide (2), transforming growth factor-␤ (3), urokinase-type plasminogen activator (4), bone morphogenetic proteins (5, 6), and endothelin-1 (7). The production of these factors by prostate cancer cells, accompanied by the release of growth factors during osteolysis, is proposed to result in stimulating new woven bone formation by increasing the differentiation of osteoblasts in the bone. Interestingly, there is increasing evidence that prostate cancer bone metastases express genes and proteins typically associated with osteoblasts, including RANK ligand, osteoprotegerin (8), sialoproteins, osteopontin (9), Runx2 (Runt-related transcription factor 2) (10

Conditional Disruption of Calcineurin B1 in Osteoblasts Increases Bone Formation and Reduces Bone Resorption

“…At least 10 randomly selected microscopic fields were examined using 10ϫ and 40ϫ objectives. Photos were taken using a Nikon digital camera (23).…”

“…Whole Cell Protein Extraction and Western Blot AnalysisCells were lysed in 0.5% Nonidet P-40 lysis buffer supplemented with protease and phosphatase inhibitors (23). Samples were then centrifuged at 14,000 ϫ g for 30 min at 4°C, and the supernatant protein concentration was measured by the Bio-Rad DC protein assay (23).…”

Hes1: a key role in stemness, metastasis and multidrug resistance

“…[34][35][36][37] Hes1 loss of function via a dominant-negative mastermind (dnMAM) in an osteosarcoma xenograft model, which then results in a significant reduction of lung metastasis neoplasm, while dnMAM has no effect on tumor latency or tumor growth rates. 38 Evidence from 56 samples obtained from patients who did well with osteosarcoma and died from metastasis reveals that Hes1 is inversely correlated with survival.…”

“…42,43 When examined in prostate tumor bone metastasis, Runx2 DNA-binding activity is enhanced in response to osteogenic induction and is inhibited by Notch-Hes1 inhibition, verifying that Hes1-Runx2 signaling functions critically in tumor bone metastasis. 44 Furthermore, EMT process allows the loss of junction with adjacent cells, the polarization of epithelial cells and subsequent acquisition of mesenchymal features. During EMT, cells undergo multiple biologic changes including enhanced motility capacity, invasiveness and resistance to apoptosis has been well established to have an intimate connection with tumor metastasis and also shows its deep relationship to cancer stem cells.…”

The pathological significance of Notch1 in oral squamous cell carcinoma

“…10 The dysregulated expression of Notch receptors, ligands and targets is also observed in solid tumors, including lung, pancreatic, cervical and prostate carcinomas. [11][12][13][14] High-level expressions of Notch1 and JAG1 are associated with poor prognoses in patients with breast cancer or metastasis of prostate cancer. 15,16 On the other hand, there is growing evidence that Notch signaling activation may have growthsuppressive functions in other hematopoietic cells, hepatocytes, the pancreatic epithelium and skin.…”