Predicting the Diagnosis of Prostate Cancer with a Novel Blood-Based Biomarker: Comparison of Its Performance with Prostate-Specific Antigen.

The purpose of this study was to assess the efficacy, specificity, and predictive value of a newly discovered biomarker, Zinc finger-like1 protein (referred to as neuroendocrine marker, NEM) for the detection of prostate cancer (PCa). We retrospectively analyzed banked plasma samples from 508 men, with a median age of 67 years (range 48-97), to compare the performance of NEM and PSA in predicting subsequent histologic PCa. The cohort consisted of four groups of patients visiting a urology clinic: (1) patients not diagnosed with either benign prostatic disease or prostate cancer (PCa) were defined as normal; (2) patients diagnosed with benign hyperplasia (BPH) but not PCa; (3) patients with confirmed PCa; and (4) patients with cancer other than PCa. The normal men displayed a mean NEM plasma level of 0.948 ± 0.051 ng/mL, which increased to 1.813 ± 0.315 ng/mL in men with BPH, 86.49 ± 15.51 ng/mL in men with PCa, and 10.47 ± 1.029 ng/mL in men with other Ca. The corresponding concentrations of prostate-specific antigen (PSA) in these subjects were 1.787 ± 0.135, 5.405 ± 0.699, 35.77 ± 11.48 ng/mL, and 8.036 ± 0.518, respectively. Receiver operating characteristic (ROC) curve analysis was performed to compare NEM and PSA performance, and the Jouden Index for each biomarker was calculated to determine cut-off points for each biomarker. The area under the ROC curve to predict PCa was 0.99 for NEM and 0.81 for PSA (p < 0.0001). The cut-off for NEM was at 1.9 ng/mL, with sensitivity of 98% and specificity of 97%. The corresponding PSA values were 4.4 ng/mL, with sensitivity of 76% and specificity of 95%. The predictive value of each biomarker in a patient was matched with his pathologic data to determine the accuracy of each biomarker. NEM was more accurate than PSA in differentiating cancer from benign conditions, such as BPH or prostatitis. In conclusion, NEM was a better predictor of PCa than PSA in patients visiting urology clinics. NEM tests, either alone or in conjunction with other biomarkers, provide a reliable, non-invasive, and inexpensive test to remarkably reduce false positives and thereby reduce the number of diagnostic biopsies and associated painful procedures and the loss of quality of life.

Zinc finger protein­like 1 is a novel neuroendocrine biomarker for prostate cancer.

Prostate­derived calcitonin (CT) and its receptor induce tumorigenicity and increase metastatic potential of prostate cancer (PC). CT­inducible genes in human prostate were identified by subtraction hybridization. Among these genes, zinc finger protein like 1 (ZFPL1) protein was interesting since it was abundantly expressed in malignant prostates but was almost absent in benign prostates. ZFPL1 expression was upregulated by CT and androgens, and ZFPL1 protein was secreted by prostate tumor cells through exosomal secretion. Serum levels of ZFPL1 in cancer patients were at least 4­fold higher than those in the sera of cancer­free individuals. Cell biology of ZFPL1 suggests its localization in Golgi bodies and exosomes, and its colocalization with chromogranin A and CD44. These results suggested that ZFPL1 is secreted by tumor cells of neuroendocrine (NE)/stem cell phenotype. The knockdown of endogenous ZFPL1 in (PC) cells led to a remarkable decrease in cell proliferation, and invasion while increasing their apoptosis. As expected, the overexpression of ZFPL1 in prostate cells had an opposite effect on these functions. The knockdown of ZFPL1 in PC cells also decreased Akt phosphorylation, suggesting the actions of ZFPL1 may be mediated through the PI3K­Akt pathway. Moreover, the present results revealed that ZFPL1 is released by tumors cells of NE or androgen­independent phenotype and its serum levels are significantly higher in cancer patients, suggesting that it may serve as a blood­based non­invasive biomarker of aggressive PC.

Calcitonin induces stem cell-like phenotype in prostate cancer cells.

Stem cell-like-cancer cells are key drivers of tumor growth, metastasis, and relapse of cancer following remission. Prostate stem cell-like cancer cells isolated from human prostate cancer (PC) biopsies express CD44+/α2ß1 hi/CD133+ cell surface markers and can self-renew in vitro. Expression of calcitonin (CT) and its receptor (CTR) is frequently elevated in PCs and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. We investigated whether CT-CTR autocrine axis induces stem cell-like phenotype in two PC cell lines. CT-CTR axis in these cell lines was activated by enforced expression of CTR. The cells were then examined for the changes in the expression of CD44 and CD133, collagen adherence, tumorigenic, metastatic and repopulating characteristics. The activation of CT-CTR axis led to a large increase in adherence to collagen and a remarkable increase of CD44 and CD133 in PC-3 and LNCaP cells. This was accompanied by a strong increase in tumorigenic, metastatic and repopulation properties of PC cells. However, the mutation of CTR-C PDZ-binding site in CTR almost abolished CTR-mediated increases in stem cell-like characteristics of PC cells. These results support an important role for CT-CTR axis in the progression of PC from localized cancer to an aggressive form, and a majority of proinvasive CTR actions may be mediated through its interaction with its partner protein at the PDZ-binding site. These results suggest that CT/CTR can serve as a valuable target to prevent the generation of stem-like PC cells.

Calcitonin Receptor-Zonula Occludens-1 Interaction Is Critical for Calcitonin-Stimulated Prostate Cancer Metastasis.

The role of neuroendocrine peptide calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. Here we show by yeast-two hybrid screens that CTR associates with the tight junction protein Zonula Occludens-1 (ZO-1) via the interaction between the type 1 PDZ motif at the carboxy-terminus of CTR and the PDZ3 domain of ZO-1. Mutation of either the CTR C-PDZ-binding motif or the ZO-1-PDZ3 domain did not affect binding of CTR with its ligand or G-protein-mediated signaling but abrogated destabilizing actions of CT on tight junctions and formation of distant metastases by orthotopically implanted PC cells in nude mice, indicating that these PDZ domain interactions were pathologically relevant. Further, we observed CTR-ZO-1 interactions in PC specimens by proximity ligation immunohistochemistry, and identified that the number of interactions in metastatic PC specimens was several-fold larger than in non-metastatic PC. Our results for the first time demonstrate a mechanism by which PDZ-mediated interaction between CTR and ZO1 is required for CT-stimulated metastasis of prostate cancer. Since many receptors contain PDZ-binding motifs, this would suggest that PDZ-binding motif-adaptor protein interactions constitute a common mechanism for cancer metastasis.

A-kinase anchoring protein 2 is required for calcitonin-mediated invasion of cancer cells.

Expression of neuropeptide calcitonin (CT) and its receptor (CTR) is frequently elevated in prostate cancers (PCs) and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. Specific, cell-permeable inhibitors of protein kinase A abolish CTR-stimulated invasion of PC cells. Since PKA is ubiquitously distributed in cells, the present study examined the mechanism(s) by which CTR-stimulated PKA activity is regulated in time and space. CT reduced cell adhesion but increased invasion of PC cells. Both these actions were abolished by st-Ht31 inhibitory peptide suggesting the involvement of an A-kinase anchoring protein (AKAP) in CT action. Next, we identified the AKAP associated with CT action by the subtraction of potential AKAP candidates using siRNAs. Knock-down of membrane-associated AKAP2, but not other AKAPs, abolished CT-stimulated invasion. Stable knock-down of AKAP2 in PC3-CTR cells remarkably decreased their cell proliferation, invasion, clonogenicity and ability to form orthotopic tumors and distant metastases in nude mice. Re-expression of AKAP2-wt restored these characteristics. Primary PC specimens displayed remarkable upregulation of CTR/AKAP2 expression as compared to benign prostates. Metastatic cancers displayed significantly higher CTR/AKAP2 expression than localized cancers. These results for the first time demonstrate that AKAP2 is expressed in human prostates, its expression is elevated in metastatic prostate cancer, and the knock-down of its expression remarkably decreased tumorigenicity and metastatic ability of prostate cancer cells. AKAP2 may serve as a critical component of CTR-mediated oncogenic actions.

Measurement of serum prostate cancer markers using a nanopore thin film based optofluidic chip.

Currently used cancer marker for prostate adenocarcinoma (PC), serum prostate-specific antigen (PSA), greatly overestimates PC population. Patients with high PSA levels have to undergo unnecessary but physically painful and expensive procedure such as prostate biopsies repeatedly. The reliability of PC test can be greatly increased by finding a protein that is secreted selectively by malignant, but not normal, prostate cells. A recently discovered novel protein, referred as neuroendocrine marker (NEM), is secreted only by malignant prostate cells and released in blood circulation. Although NEM seems to be significantly more reliable based on the data obtained from a limited cohort, currently available NEM ELISA is not suitable for undertaking a large study. Therefore, the goal of the present study was to develop an alternative, label-free assay system that can reliably measure NEM and PSA in patient samples. Herein an optofluidic chip that can reliably detect PSA as well as NEM in patient samples has been developed. The optofluidic chip, which consists of arrayed nanopore-based sensors fabricated from anodic aluminum oxide (AAO) thin film, offers improved sensitivity upon the optimization of the concentration of the detector antibodies immobilized on the sensor surface. The results demonstrate that the chip is reliable, extremely sensitive and requires just 1 µl of patient serum (or even less) to measure PSA and NEM even in a non-cancer individual. Compared with the traditional ELISA for PSA, the nanopore-based sensor assay is 50-100 fold more sensitive, and offers many advantages such as elimination of labeled antigen, need for sophisticated equipment and highly trained individuals. These advantages, along with the low cost, should make the technology suitable for point-of-care application to screen elderly male populations for PC and to monitor the progress of patients undergoing PC treatment.

Profiling of the calcitonin-calcitonin receptor axis in primary prostate cancer: clinical implications and molecular correlates.

Expression of the neuroendocrine peptide calcitonin (CT) and its receptor (CTR) is frequently elevated in prostate cancers (PCs), and activation of the CT-CTR axis in non-invasive PC cells induces an invasive phenotype. We aimed to link CT/CTR expression in prostate specimens to clinicopathological parameters of PC. We analyzed CT and CTR expression in cohorts of benign prostates and primary PCs with/without metastatic disease by immunohistochemistry. Furthermore, we correlated CT/CTR expression with several clinicopathological parameters. CT/CTR immunostaining in benign prostate acini was predominantly localized to basal epithelium. However, this spatial specificity was lost in malignant prostates. PC sections displayed a remarkable increase in cell populations expressing CT/CTR and their staining intensity. Tumors with higher CT/CTR expression consistently displayed metastatic disease and poor clinical outcome. High CT/CTR expression in primary prostate tumors may serve as a prognostic indicator of disease aggressiveness and poor clinical outcome.

Methods for evaluation of structural and biological properties of antiinvasive natural products.

Prostate cancer is considered the most common cancer form among males in Western countries. Very limited options are available for the treatment of advanced metastatic prostate cancer. More than 50% of today's anticancer drugs are natural products or derived from a natural origin. To discover new entities with potential to treat prostate cancer at androgen-refractory stages, 36 structurally diverse natural products were screened using functional-based assays. The tested compounds were selected broadly from major secondary metabolites of plants, marine invertebrates, and fungi. These diverse entities were prescreened for their antiinvasive ability against prostate cancer cells, PC-3M, using spheroid disaggregation assay. Active representatives including three selected structural classes, a macrolide, a ß-carboline alkaloid, and a phenylmethylene hydantoin (PMH), were then tested for their ability to stabilize junctional complexes and enhance cell-cell adhesion of androgen independent prostate cancer cells. Transepithelial resistance (TER) and paracellular permeability assays were used to elicit the aforementioned properties. These studies led to the emergence of PMHs as a small molecule class from the marine sponge Hemimycale arabica with a unique potential to attenuate CT-stimulated prostate cancer growth, metastasis, paracellular permeability, and enhance TER and cell-cell adhesion of prostate cancer cells. The unique activities of PMHs were validated using several in vitro assays followed by in vivo testing in two mice models. A 3D QSAR was established using SYBYL 8.1-Comparative Molecular Field Analysis (CoMFA) model. This chapter includes the methodology for evaluation of structural and biological properties of new antiinvasive molecules with an exceptional potential to stabilize junctional complexes from diverse natural product sources.

A new mixed-backbone oligonucleotide against glucosylceramide synthase sensitizes multidrug-resistant tumors to apoptosis.

Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS) with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C(18)-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent.

Identification of a small molecule class to enhance cell-cell adhesion and attenuate prostate tumor growth and metastasis.

Expression of calcitonin (CT) and its receptor (CTR) is elevated in advanced prostate cancer, and activated CT-CTR autocrine axis plays a pivotal role in tumorigenicity and metastatic potential of multiple prostate cancer cell lines. Recent studies suggest that CT promotes prostate cancer metastasis by reducing cell-cell adhesion through the disassembly of tight and adherens junctions and activation of beta-catenin signaling. We attempted to identify a class of molecules that enhances cell-cell adhesion of prostate cells and reverses the disruptive actions of CT on tight and adherens junctions. Screening several compounds led to the emergence of phenyl-methylene hydantoin (PMH) as a lead candidate that can augment cell-cell adhesion and abolish disruptive actions of CT on junctional complexes. PMH reduced invasiveness of PC-3M cells and abolished proinvasive actions of CT. Importantly, PMH did not display significant cytotoxicity on PC-3M cells at the tested doses. I.p. administered PMH and its S-ethyl derivative remarkably decreased orthotopic tumor growth and inhibited the formation of tumor micrometastases in distant organs of nude mice. PMH treatment also reduced the growth of spontaneous tumors in LPB-Tag mice to a significant extent without any obvious cytotoxic effects. By virtue of its ability to stabilize cell junctions, PMH could reverse the effect of CT on junctional disruption and metastasis, which strengthens the possibility of using PMH as a potential drug candidate for CT-positive androgen-independent prostate cancers.

Discovery, design, and synthesis of anti-metastatic lead phenylmethylene hydantoins inspired by marine natural products.

The Red Sea sponge Hemimycale arabica afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin (1), (R)-5-(4-hydroxybenzyl)hydantoin (2), and (Z)-5-((6-bromo-1H-indol-3-yl)methylene)-hydantoin (3). The natural phenylmethylene hydantoin (PMH) 1 and the synthetic (Z)-5-(4-(ethylthio)benzylidene)-hydantoin (4) showed potent in vitro anti-growth and anti-invasive properties against PC-3M prostate cancer cells in MTT and spheroid disaggregation assays. PMHs 1 and 4 also showed significant anti-invasive activities in orthotopic xenograft and transgenic mice models. To study the effect of electronic and lipophilic parameters on the activity, a wide array of several substituted aldehydes possessing electron-withdrawing (+sigma), lipophilic (+pi), electron-donating (-sigma), and less lipophilic substituents (-pi) were used to synthesize several PMHs. Few des-phenylmethylenehydantoins and 2-thiohydanoins were also synthesized and the anti-invasive activities of all compounds were evaluated. Comparative molecular field analysis (CoMFA) was then used to study the 3D QSAR. Predictive 3D QSAR model with conventional r(2) and cross validated coefficient (q(2)) values up to 0.910 and 0.651 were established. In conclusion, PMH is a novel antimetastatic lead class with potential to control metastatic prostate cancer.

Cadherin switching and activation of beta-catenin signaling underlie proinvasive actions of calcitonin-calcitonin receptor axis in prostate cancer.

Calcitonin, a neuroendocrine peptide, and its receptor are localized in the basal epithelium of benign prostate but in the secretory epithelium of malignant prostates. The abundance of calcitonin and calcitonin receptor mRNA displays positive correlation with the Gleason grade of primary prostate cancers. Moreover, calcitonin increases tumorigenicity and invasiveness of multiple prostate cancer cell lines by cyclic AMP-dependent protein kinase-mediated actions. These actions include increased secretion of matrix metalloproteinases and urokinase-type plasminogen activator and an increase in prostate cancer cell invasion. Activation of calcitonin-calcitonin receptor autocrine loop in prostate cancer cell lines led to the loss of cell-cell adhesion, destabilization of tight and adherens junctions, and internalization of key integral membrane proteins. In addition, the activation of calcitonin-calcitonin receptor axis induced epithelial-mesenchymal transition of prostate cancer cells as characterized by cadherin switch and the expression of the mesenchymal marker, vimentin. The activated calcitonin receptor phosphorylated glycogen synthase kinase-3, a key regulator of cytosolic beta-catenin degradation within the WNT signaling pathway. This resulted in the accumulation of intracellular beta-catenin, its translocation in the nucleus, and transactivation of beta-catenin-responsive genes. These results for the first time identify actions of calcitonin-calcitonin receptor axis on prostate cancer cells that lead to the destabilization of cell-cell junctions, epithelial-to-mesenchymal transition, and activation of WNT/beta-catenin signaling. The results also suggest that cyclic AMP-dependent protein kinase plays a key role in calcitonin receptor-induced destabilization of cell-cell junctions and activation of WNT-beta-catenin signaling.

Calcitonin promotes in vivo metastasis of prostate cancer cells by altering cell signaling, adhesion, and inflammatory pathways.

Expression of calcitonin (CT) and its receptor (CTR) is elevated in advanced prostate cancer (PC). Although the significance of CT-CTR axis in PC cell growth, invasion, and epithelial to mesenchymal transition has been established, its role in tumor metastasis has not been examined. To examine the role of CT-CTR axis in tumor metastasis, we employed stable CT-CTR activated and silenced system of three PC cell lines, LNCaP cells that lack endogenous CT, PC-3 cells that lack endogenous CTR, and PC-3M cells that co-express CT and CTR. Enforced expression of CT in LNCaP cells and CTR in PC-3 cells increased their ability to form orthotopic tumors and distant metastases in multiple organs. By contrast, silencing of CT expression in PC-3M cells not only reduced their tumorigenicity, but also completely abrogated their metastatic potential. To investigate the effect of in vivo silencing of CT expression on tumor growth, we employed recombinant adeno-associated virus (rAAV) to deliver anti-CT ribozymes in preexisting tumors of nude mice and large probasin promoter (LPB)-Tag transgenic mice. rAAV-CT(-) treatment not only abrogated the growth of pre-implanted tumors in nude mice, but also significantly reduced the growth of spontaneous tumors in LPB-Tag mice. Analysis of CT upregulated and silenced PC-3M transcriptomes revealed 105 genes affected by the modulation of CT expression. These CT signature genes generated survival, adhesion, pro-inflammatory, and pro-metastatic pathways. Added together, these data indicate a pivotal role for CT-CTR axis in PC metastasis and may serve as a potential therapeutic target for advanced PC.

Biocatalytic and semisynthetic optimization of the anti-invasive tobacco (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol.

Tobacco cembranoids were reported to inhibit tumorigenesis. Biocatalysis of (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (1) using the symbiotic Bacillus sp. NC5, Bacillus sp. NK8, and Bacillus sp. NK7, isolated from the Red Sea sponge Negombata magnifica, afforded two new and four known hydroxylated metabolites 3-8. The use of symbiotic marine bacteria as biocatalysts for bioactive natural product scaffolds is very rare. Cembranoid 1 carbamate analogs 9-11 were prepared by its reaction with corresponding isocyanates. Cembranoid 1 and its bioconversion and carabamate products show anti-invasive activity against the human highly metastatic prostate PC-3M cancer cell line at 10-50 nM doses in Matrigel assay.

Knock-down of calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells.

Calcitonin (CT) and its receptor (CTR) are expressed only in basal epithelium of benign prostate and in whole epithelium of malignant prostates. Also, CT and CTR mRNA levels in prostate cancers increase with an increase in tumor grade. We tested the role of the CT/CTR autocrine axis on the tumorigenicity of prostate cancer cells. We enforced the expression of CTR in CT-positive/CTR-deficient PC-3 cells. In contrast, we knocked down CTR expression in CT/CTR-positive PC-3M cells. The effect of CTR modulation on the oncogenicity was evaluated by the rate of cell proliferation, invasion, colony formation and in vivo growth in nude mice. Up-regulation of CTR in PC-3 cells and its down-regulation in PC-3M cells significantly altered their tumorigenicity. Intratumorally administered CTR RNAi in preexisting PC-3M xenografts markedly attenuated their further growth. This treatment also led to a remarkable decrease in endothelial cell populations in the tumors and increase in apoptotic, PCNA-negative cell populations. Tumors receiving CTR RNAi treatment displayed markedly lower levels of urokinase-type plasminogen activator, phospho-Akt and survivin, suggesting CTR activates uPA-uPAR axis and PI-3-kinase-Akt-survivin pathway. These results suggest an important role for CT-CTR autocrine axis in the progression of localized prostate tumor to a metastatic phenotype, and offer a potential therapeutic option for invasive cancers.

A synthetic peptide derived from mouse pituitary calcitonin cDNA sequence exhibits potent inhibition of prolactin secretion and prolactin mRNA abundance in primary mouse pituitary cells.

We have shown that gonadotrophs synthesize and secrete immunoreactive calcitonin (CT)-like peptide, and CT is a potent inhibitor of prolactin (PRL) secretion and gene transcription. CT cDNA cloned from LssT2 cells (pit-CT cDNA) exhibits 99% homology with mouse CT cDNA sequence, but exhibits four mismatches in the coding region of CT peptide (347-485 bp) with consequent changes in the amino acids at positions 5 and 17 of mouse CT. We have synthesized a putative 23 amino acid pit-CT peptide based on pit-CT cDNA sequence, and tested its effect on PRL secretion and mRNA abundance in primary mouse pituitary cells. The results suggest that synthetic pit-CT attenuates PRL mRNA abundance and inhibits PRL release from mouse anterior pituitary cells. Moreover, pit-CT is remarkably more potent than salmon (S)CT in attenuating PRL mRNA abundance. These results raise a possibility that this endogenous pituitary peptide may potentially serve as a therapeutic molecule for the treatment of prolactinomas.

Calcitonin receptor-stimulated migration of prostate cancer cells is mediated by urokinase receptor-integrin signaling.

Abundance of calcitonin (CT) and calcitonin receptor (CTR) mRNA in primary prostate tumors positively correlates with tumor grade, and exogenously added CT increases the invasion of prostate cancer cell lines. We examined acute and chronic actions of CT on migration of highly metastatic PC-3M cells and poorly invasive LNCaP cells on several extracellular matrices in a spheroid disaggregation/migration assay. While PC-3M spheroids displayed maximum disaggregation/migration on vitronectin (VN), LNCaP spheroids preferred collagen but also migrated significantly on VN. Up-regulation of CT significantly enhanced disaggregation/migration of PC-3M spheroids on VN, but not on fibronectin. In contrast, down-regulation of CT, CTR, protein kinase A or urokinase-type plasminogen activator receptor (uPAR) led to amelioration of PC-3M spheroid disaggregation/migration. CT selectively increased surface activity of alpha v beta 3 or alpha 6 beta 5 integrins in PC-3M and LNCaP cell lines, respectively, and uPAR-integrin association. Finally, either CT or urokinase could completely restore migration of CT-knock-down PC-3M spheroids. But, only forced expression of urokinase receptor coupled with exogenous addition of urokinase restored migration of CTR-knock-down spheroids. These results support our hypothesis that up-regulation of CT biosynthesis and activation of CT-CTR axis in primary prostate tumors may have direct relevance in their progression to the metastatic phenotype.

The predominant CD44 splice variant in prostate cancer binds fibronectin, and calcitonin stimulates its expression.

BACKGROUND: Prostate cancer (PC) consistently overexpresses variant the (v) isoform of the cell adhesion protein CD44, and loses expression of the standard (s) isoform. MATERIALS AND METHODS: We re-expressed CD44 full-length (exons 1-20) or standard (exons 1-5 + 16-20) or enforced stable RNAi against CD44v, and the examined functional effects on PC. The effect of stable knockout of calcitonin, a paracrine factor, or its receptor, on CD44 was assessed. RESULTS: Re-expression of full-length CD44 or CD44s increased the total CD44 mRNA and CD44s protein while suppressing CD44v. These approaches, and RNAi to CD44v, decreased invasion. In adhesion assays, benign prostate cells bound mainly to hyaluronan, whereas PC lost affinity for hyaluronan but bound more strongly to fibronectin. Re-expressing CD44s restored predominant hyaluronan binding. Knockout of the calcitonin receptor in PC-3 derived cells caused marked loss of CD44v expression and reversion to CD44s expression. CONCLUSION: Calcitonin influenced PC's balance between CD44s and CD44v. CD44v controlled invasiveness, altered ligand binding, and provides a target for therapeutic intervention.

Paracrine calcitonin in prostate cancer is linked to CD44 variant expression and invasion.

BACKGROUND: Calcitonin (CT) exerts an autocrine/paracrine influence on prostatic tumor invasion through coupling to transduction protein Gsalpha. Cell adhesion glycoprotein CD44 variant v7-v10 also faciliates invasion, but its modulation by the CT-Gsalpha system was unexplored. MATERIALS AND METHODS: LnCaP, PC-3 and metastasis-derived PC-3M cell lines were studied, including cells modified therefrom: Gsalpha-QL, expressing mutant constitutively active Gsalpha protein, and CT+, overexpressing CT. CD44 variant expression was evaluated in vivo after orthotopic implantion into nude mice, and in vitro by real-time RT-PCR and Western blotting. RESULTS: Both mRNA and protein levels of the CD44 variant were minimal in PC-3M tumor implants, but elevated in Gsalpha-QL. Exogenous CT stimulated invasion into Matrigel strongly in LnCaP and CT+, and less in PC-3 and Gsalpha-QL. By Western blot analysis, untreated Gsalpha-QL and CT+ cells overexpressed CD44 variant compared with LnCaP or PC-3. By quantitative RT-PCR, exogenous CT dose-dependently increased CD44 variant mRNA to seven-fold. Pharmacologic agents that stimulated or inhibited Gsalpha activity or stimulated adenylyl cyclase produced proportionate dose-dependent effects on both CD44 variant expression and Matrigel invasion. CONCLUSION: This paracrine factor, acting though cyclic AMP, regulates the expression of CD44v7-10, which modulates the tumor phenotype.

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

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.