Calcitonin increases invasiveness of prostate cancer cells: Role for cyclic AMP-dependent protein kinase A in calcitonin action

Abstract

Calcitonin (CT) is synthesized and secreted in prostate epithelium, and its secretion from malignant prostates is several-fold higher than from benign prostates. CT receptor (CTR) is expressed in malignant prostate epithelium, and its activation stimulates growth of prostate cancer (PC) cells via activation of adenylyl cyclase and calcium/phospholipid pathways. To identify the role of ''CT System'' in prostate cancer, we tested the expression of CT and CTR mRNAs in invading tumor cells of prostate cancer specimens. The effect of CT on in vitro invasion of PC cell lines and on activation of gelatinases was also examined. The cells of primary tumors and those invading stroma co-expressed CT/CTR mRNAs. Exogenously added CT increased in vitro invasion of PC cell lines and caused a rapid, several-fold but transient increase in protein kinase A activity. In contrast, anti-CT serum caused a dose-dependent inhibition of in vitro invasion of PC-3M cells. CT also increased the concentration and activities of MMP-2 and MMP-9. Rp.cAMP, a competitive inhibitor of cAMP-dependent protein kinase A, myristoylated protein kinase A inhibitory peptide (PKI) as well as the expression of dominant negative form of PKA all attenuated basal in vitro invasion of PC-3M cells, and CT could not increase in vitro invasiveness in their presence. These results suggest that overexpression of ''CT System'' in invasive PC tumors significantly contributes to increased invasiveness of prostate cancer cells. The action of CT may be mediated by protein kinase A signaling, which subsequently leads to increased cell invasion and secretion of gelatinases. ' 2005 Wiley-Liss, Inc.Key words: prostate cancer; invasion; calcitonin; PKA; metastasis Calcitonin (CT), a 32 amino acid peptide produced by C cells of mammalian thyroid, has also been detected in human brain, the pituitary gland, lung carcinoma as well as the conditioned media from the cultures of primary prostate epithelial cells.1-6 Although most well-known functions of CT include inhibition of bone resorption, renal regulation of sodium, phosphate, calcium and neuromodulation, 6 CT has also been shown to regulate secretory and proliferative functions of several organs. [7][8][9] Previous studies from this laboratory have shown that immunoreactive CT (iCT) is secreted by primary prostate epithelial cells, and its secretion from cancer-derived primary prostate cells is several-fold higher than that from the cells of benign prostatic hyperplasia origin. 1 We have reported that mRNAs for CT and its receptor (CTR) are selectively localized to the basal epithelium of benign prostates but are distributed throughout the epithelium of malignant prostates. High-affinity CT receptors have been identified in membrane preparations of primary tumors as well as androgen-responsive LNCaP and highly metastatic but androgen refractory PC-3M prostate cancer cells.10 Exogenously added CT stimulates the proliferation of LNCaP cells by stimulating cAMP accumulation and increasing cytoplasmic Ca 21 transients, and CT al...

Calcitonin stimulates the secretion of urokinase‐type plasminogen activator from prostate cancer cells: Its possible implications on tumor cell invasion

“…The profile of these results is almost identical to that of CT-induced increase in invasion, as described previously. 23 A second major high-molecular-weight band (140 kDa) may correspond to PAI-1-uPA-uPA receptor complex as suggested by a recent report. 40 …”

“…The increase was at least 3-fold in response to 10 nM CT (the lowest tested concentration), and increased to 7-fold when treated with 1 lM CT (the highest tested concentration). Since our previous studies have shown that CT increases invasiveness of various PC cell lines through the activation of protein kinase A, 37 we tested a possibility that CT may stimulate uPA secretion by the same mechanism. We examined the effect of Rp.cAMP (100 lM), a membrane-permeable competitive inhibitor of cyclic AMPdependent protein kinase A, on CT-induced uPA secretion by PC-3M cells.…”

“…16 In general, tumor cells of several human malignancies express detectable cell surface-associated uPA and plasmin activity, and this positively correlates with their invasiveness. 17,[44][45][46][47][48][49] Since CT stimulates the invasion of several PC cell lines, 23 we examined the actions of CT on the secretion of uPA by immunoassay and fibrin zymography. Present results have shown that CT markedly increased the secretion of uPA in the conditioned media.…”

“…23 Since CT also stimulates the secretion of active uPA in PC-3M cells, we tested whether proinvasive actions of CT are mediated by uPA. We tested this by examining the effect of CT (50 nM, maximal effective dose 23 ) on the invasion of PC-3M cells in Matrigel in the presence or absence of uPA neutralizing antibodies (50 ng/ml). It can be seen from Figure 4a that the tested dose of uPA antibody did not affect the invasiveness of untreated PC-3M cells, but abolished CT-induced increase.…”

“…[19][20][21][22] Exogenously added CT stimulates the proliferation of LNCaP cells by stimulating cAMP accumulation and increasing cytoplasmic Ca 21 transients, and stimulates invasion by activating protein kinase A-mediated mechanisms. 19,23 Urokinase-type plasminogen activator (uPA), a member of the serine protease family, is strongly implicated as a promoter of tumor progression in various human malignancies, including breast cancer and PC. 24 The ability of uPA to break down various components of the extracellular matrix, including laminin, fibronectin and collagen, through the activation of plasminogen to plasmin may facilitate the invasion of tumor cells.…”