Neuropeptides Induce Mr 92,000 Type IV Collagenase (Matrix Metalloprotease-9) Activity in Human Prostate Cancer Cell Lines1

(CANCER RESEARCH 58, 4288-4291. October I. 1998] Advances in Brief

Inder Sehgal and Timothy C. Thompson2

Sam Department of Urology //. 5.. T. C. TI. Department of Cell Biologv, and Department of Radtotherapv Â¡T.C TI, Ba\lor College of Medicine, Houston, Texas 77030

Abstract sion, as well as insight into the clinical significance of neuropeptides in prostate cancer progression. The type IV collagenases matrix metalloprotease (MMI'l-2 and MMP-9 are linked with a wide array of biological activities, including tumor Materials and Methods invasion, metastasis, and angiogenesis. Here, we report that neuropeptide hormones, which are present in prostatic adenocarcinomas, can stimulate Cell Culture secreted activity of MMP-9 in human prostate cancer cell lines. Northern The human prostate cancer cell lines DU 145 (acquired from ATCC). blotting analyses demonstrated that neuropeptide stimulation lead to Tsu-Prl (from Dr. Marco Marcelli. Baylor College of Medicine), and LNCaP elevated mRNA levels of MMP-9 but not MMP-2. Further assays of (from ATCC) were subcultured in MEM, DMEM, and RPMI 1640 (Life MMP-9 promoter activation and a nuclear run-off indicated that neu Technologies, Inc.. Gaithersburg, MD). respectively, with 10% PCS, 100 ropeptide induction of MMP-9 expression occurs at the level of transcrip units/ml penicillin. 100 jug/ml streptomycin, and 1.0 mM L-glutamine. Cell tion. These data indicate that neuropeptides can regulate MMP activity, cultures to be treated with neuropeptides for Northern blotting. CAT assay, or which, in turn, could facilitate prostate cancer progression. nuclear run-off were grown to 50% confluency and then incubated without Introduction serum overnight, followed by the addition of neuropeptides (10 jujn) for 24 h in DMEM-0.1% BSA. For zymography, cells were cultured with neuropep The type IV collagenase/gelatinases MMP-23 and MMP-9 have tides or in DMEM alone for 48 h. All neuropeptides were purchased from been widely investigated as mediators of tumor metastasis, and the either Bachern Peptides or Peninsula Laboratories. activities of these type IV collagenases are tightly controlled during synthesis and following secretion (1, 2). Although MMP-2 is regu Zymography lated by relatively few polypeptide factors (3, 4), MMP-9 has been Culture medium, conditioned for 48 h in the presence or absence of reported to be induced by several growth factors, cytokines, and neuropeptides, was collected, concentrated, and assayed for type IV gelatinase/ hormones associated with human cancers (3, 5, 6). Neuropeptides. collagenase activity through acrylamide gel zymography as described (6). including calcitonin, serotonin, somatostatin, bombesin/gastrin-releasing peptide, thyroid-stimulating hormone, and neurotensin (7, 8), Northern Blotting are often expressed in prostate cancers (reviewed in Ref. 7) and are MMP-9. Total RNA was extracted from all cell cultures using Ultraspec secreted by foci of prostate cells with neuroendocrine differentiation. RNA reagent (Biotex Laboratories Inc., Houston. TX), then purified to Clusters of such neuroendocrine cells are present in most if not all poly(A)+ RNA (Oligotex mRNA Midi Kit; Qiagen). Samples of poly(A)+ prostatic adenocarcinomas, with incidences ranging up to 100%; in RNA (2 Â¿ig)were denatured and electrophoresed through formaldehyde-1.0% addition, the prevalence of neuroendocrine cells is correlated with agarose gels (SeaKem; FMC Bioproducts) and then transferred onto Zeta poor prognosis and high-grade disease (7). Although it is likely that Probe membranes (Bio-Rad) as described previously (13). Membranes were prehybridized. hybridized, and washed as according to the manufacturer's these small peptides play multiple roles in prostate cancer, most studies have focused primarily on growth or signal transduction instructions. Hybridizations were performed using a riboprobe (Boehringer mechanisms as end points of neuropeptide stimulation (9-11). How Mannheim In Vitro Transcription Kit) generated from a pBluescript KS + vector containing a linearized 2440-bp fragment of the M, 92,000 collagenase ever, Hoosein et al. (12) demonstrated that bombesin or vasoactive (MMP-9) cDNA (a gift from Dr. Barry Marnier. Washington University. St. intestinal peptide increased in vitro invasion through a reconstituted Louis, MO). Blots were either stripped and reprobed or cohybridized with a basement membrane in either PC-3 and LNCaP human prostate can random primed probe for GAPDH. MMP-9 mRNA and nuclear runoff tran cer lines, thus associating neuropeptide stimulus in at least some script levels were quantitated using a Bio-Rad model 620 Video Densitometer prostate lines, with a phenotypic characteristic more directly related to and 1-D Analyst Macintosh data analysis software. invasion or metastasis than growth. Here, we report that neuropeptides MMP-2. Total RNA was extracted as above and directly electrophoresed can stimulate expression and activity of the type IV collagenase through formaldehyde-1.0% agarose gels. Random primed probes were gen MMP-9 in human prostate cancer cells. We further demonstrate that erated using a 3-kb fragment of M, 72,000 collagenase (MMP-2) cDNA (a gift this regulation occurs in part through transcriptional activation. This from Dr. Barry Marmer). study provides a mechanistic link between neuropeptides and inva- Nuclear Runoff

Received 5/26/98; accepted 8/13/98. Adherent cells were washed twice with cold PBS. scrape-collected into The costs of publication of this article were defrayed in part by the payment of page 15-ml conical tubes in PBS, then pelleted, lysed, and stored as described (13). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Nuclear runoff transcriptions were carried out as described in detail (13). after 1This study was supported by NIH Grant CA50588 (lo T. C. T.) and N1H Postdoctoral which samples were extracted, treated with DNase and proteinase K, and Fellowship F32 CA66323 (to I. S.). hybridized for 3 days. 2 To whom requests for reprints should be addressed, at Scott Department of Urology. 6560 Fannin. Suite 1040, Houston. TX 77030. Phone: (713)799-8718; Fax: (713)799- 8712; E-mail: [email protected]. CAT Assays 3 The abbreviations used are: MMP. matrix metalloproteinase; ATCC, American Type Culture Collection; CAT. chloramphenicol acelyltransferase; GAPDH. glyceraldehyde-3- Prostate cancer cell lines, grown to 50% confluency, were subcultured phosphate dehydrogenase. overnight in standard medium without serum and then transfected (Lipo- 4288

feet AMINE; Life Technologies. Inc.) for an 8-h period with 6 p.Â°of a CsCl, purified plasmid containing the full-length human MMP-9 promoter fused to a DU 145 Tsu-Prl CAT reporter gene (3, 14). Transfected cultures were treated without or with rX Â£> ti L neuropeptides for 24 h and then lysed by repeated free/.e/thawing. CAT assays Â£-ii s H g o tL, were performed as described (14. 15) and normalized according to protein o 15 o H 2 o o -a p concentration and transfection efficiency based upon expression of a ÃŸ-galac- Z U PQ Z Z Z ffl U H tosidase vector. Results MMP-9â€” Type IV Collagenase MMP-9 Is Induced in Human Prostate Cancer Cell Lines following Stimulation with Neuropeptides. Se lected neuropeptides were tested for their capacity to modulate levels of secreted type IV collagenases. Of these small peptides. bombesin. calcitonin, neurotensin, and neuromedin N were found to up-regulate secreted MMP-9 levels in the DU 145 cell line (Fig. IA), and two, GAPDHâ€” bombesin and calcitonin (but not neurotensin and neuromedin N; data not shown), induced secreted MMP-9 levels in the Tsu-Prl line (Fig. IÃŸ)during a 48-h period of stimulation. Other neuropeptides tested in Relative Intensity 1.0 6.0 8.3 3.3 6.6 1.0 3.8 31.5 4.1 these assays that did not appear to stimulate MMP-9 in these cell lines included vasoactive intestinal peptide, substance P, and somatostatin B (data not shown). DU 145 Cells Several oncogenes and tumor necrosis factor a have been shown to up-regulate levels of MMP-9, at least in part, through transcriptional _ K iâ€”Ho activation (3, 14); therefore, we began to test potential mechanisms of o o

72kDa- Discussion

Fig. 1. Neuropeptide Stimulation of human prostate cancer cells induces MMP-9 Neuropeptide family members have been extensively studied as activity. Gelatin zymography was performed on media conditioned for 2 days from growth regulators in multiple malignancies, including prostate can DU 145 (A) and Tsu-Prl (ÃŸ)prostate cancer cells. Lane No Tx., untreated controls; Lane Bomb., bombesin-lreated cultures; Lane Caldi., calcitonin-trealed cultures; Lane NT, cers, but have not been previously associated with regulation of neurotensin-treated cultures; Lane NMN, neuromedin N-lreated cultures. extracellular matrix collagenases. Here, we document that specific 4289

Four neuropeptides induced MMP-9 in the DU 145 cell line, and two of these also induced similar activity in the Tsu-Prl line. The DU 145 Tsu-Prl cells did not respond to either neurotensin or neuromedin N, Fold Induction of CPMs vs. control and therefore, it is likely that responses to specific neuropeptides will vary with the cell line and could relate to neuropeptide receptor expression, endopeptidase proteolytic activities, or neuropeptide sig nal transduction pathways. It is interesting to note that the DU 145 cell line is derived from a brain metastasis (ATCC catalogue) and the Tsu-Prl cell is derived from a neuroendocrine tumor (17). Of the four neuropeptides tested, bombesin, the amphibian homo logue of gastrin-releasing peptide has perhaps been the most widely studied neuropeptide in prostate cancer cells. This peptide is reported to regulate Ca2+ mobili/.ation. growth, and invasion in various pros tate cancer cell lines (9. 10, 12). In addition, clinically elevated urine bombesin-likc immunorcactivity has been reported to identify sub 0.0 1.0 2.0 3.0 4.0 S.O 6.0 groups of patients with advanced disease (androgen-independent dis ease and bone metastasis) and to predict shorter survival (18). Neu rotensin has also been associated with prostate cancer as a growth B factor (11, 19), as well as with small cell lung, non-small cell lung, TsuPrl pancreatic, and colon cancers (20, 21, 22). Neuromedin N. a peptide I "Id Induction of CPMs vs. control that is structurally related to neurotensin, is derived from the neuro tensin precursor (23). Calcitonin, commonly associated with medul t'ontrol- lary thyroid tumors (24), has also been found in clinical prostate tumor tissues (7). These studies have addressed the role of neuropeptides in mediating MMP-9 levels in human prostate cancer cells; however, neuropeptide responses are associated with several other malignancies, including astrocytomas, colon cancer, breast cancer, and small cell lung cancer (20, 22, 25, 26). It is. therefore, possible that neuropeptides also regulate MMP-9 or perhaps other MMPs in other tumor types. We Culcitonin have used a breast cancer cell line expressing somatostatin receptors (25), the T47D line and have found that, in this cell line, somatostatin stimulation induces MMP-9 promoter activity â€”¿3-fold(data not

Fig. 3. Analysis for MMP-9 promoter-induced CAT activity by neuropeplides. DU 145 shown). There was, however, no effect of somatostatin on MMP-9 I/I) and Tsu-Prl (B) cell cultures were transiently transfected with an MMP-9 wild-type levels in the prostate cancer lines. (670 hpi human promoter CAT construct and then treated without (D) or with (â€¢)the We have previously introduced the hypothesis that androgen dep indicated neuropeptides. Triplicate measurements of CAT activity obtained for each sample are expressed as a fold increase over the untreated samples (equal to 1.0); bars, rivation could increase the bioavailability of neuropeptides and po means Â±SE. tentially result in the generation of alternative growth-stimulatory pathways involving these neuropeptides. This hypothesis was based neuropeptides associated with prostate cancer can induce MMP-9 type on the observation that androgen-deprivation of the hormone-sensitive IV collagenase levels and activities through transcriptional regulation. human prostate cancer line LNCaP, leads to down-regulation of a Because MMP-9 is associated with tumor invasion, progression, me neuropeptide-degrading proteinase facilitating the accumulation of tastasis, and angiogenesis (1.6) and is required for hematogenous neurotensin and that this neuropeptide stimulated growth in cultures metastasis in an experimental mouse prostate cancer model (16), our of hormone-deprived but not hormone-stimulated cells (11). These results establish a novel potential biological pathway in prostate results now further suggest hormone deprivation and the subsequent cancer progression. availability of neuropeptides could, in addition, facilitate the invasion Although there are multiple levels at which MMP-9 expression may and/or metastasis of responsive populations of prostate tumor cells. be modulated, several oncogenes and at least one polypeptide. tumor Interestingly, a recent study has likely identified the neuropeptide necrosis factor a, regulate this collagenase transcriptionally (3, 14). This study now demonstrates that neuropeptides can also induce MMP-9 in human prostate cancer cells, at least in part, through o transcriptional activation. This conclusions are based upon CAT as Z 24 Hours +Bombesin says with the wild-type 670-bp MMP-9 promoter and by runoff assays GAPDH â€”¿ for de novo RNA synthesis. These transcriptional studies suggest that there may be specific neuropeptide responsive elements within the MMP-9â€” â€”¿ MMP-9 promoter and that activation by bombesin and perhaps other pBS â€”¿ neuropeptides occurs within 1 h but is not a sustained response. The RelativeIntensity 1.0 2.1 0.8 1.0 0.9 level of direct transcriptional activity observed at l h was quantita tively less than that observed with bombesin stimulation of the pro- Fig. 4. Nuclear runoff assay of transcription in isolated nuclei from control or bombesin-lreated DU 145 cultures. Lineari/ed pBluescripl plasmids containing cDNAs for moter-CAT construct; however, reporter assays reflect indirect enzy GAPDH. MMP-9. or pBluescripl alone (5 jig) were hybridized with |':P|UTP-labeled matic activity (CAT) that has accumulated over a 24-h period in RNA transcripts from control or bombesin-treated DU 145 nuclei. Separate controls were viable cells rather than transcriptional activity over a 30-min period in used for the I- and S-h lime points versus the 24-h lime points to correct for potential variations in transcription as a function of culture time. Ntimhrrs. relative intensities of the vitro. MMP-9 transcripts (in reference to GAPDH). 4290

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1998 American Association for Cancer Research. Neuropeptides Induce Mr 92,000 Type IV Collagenase (Matrix Metalloprotease-9) Activity in Human Prostate Cancer Cell Lines

Inder Sehgal and Timothy C. Thompson

Cancer Res 1998;58:4288-4291.

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