c-MET expression in carcinoma of the prostate. In this investigation, we assessed the expression of. HGF and c-MET in prostatic tissues and cell lines and.
Amenican Journal of Pathblogy, Vol. 147 No. 2, Auigust 1995 Copyight © American Society for Investigative Pathology
Hepatocyte Growth Factor and Its Receptor (c-MET) in Prostatic Carcinoma
Peter A. Humphrey,* Xiaopei Zhu,* Reza Zarnegar,t Paul E. Swanson,* Timothy L. Ratliff,*t Robin T. Vollmer,§ and Mark L. Dayt From the Department of Pathology * and Division of Urology, Department of Surgery,* Washington University Medical Center, St. Louis, Missouri; the Department of Pathology,t University of Pittsburgh School ofMedicine, Pittsburgh, Pennsylvania; and the Department of Pathology,5 Duke University Medical Center, Durham, North
Carolina
Hepatocyte growthfactor (scatterfactor) and its receptor, the c-met proto-oncogene product (cMET), have been implicated in embryogenesis, tissue reorganization, and tumorprogression. Little is known, however, of the expression andfunctional significance ofthese molecules in prostatic ceUs and tissue. In this investigation, we assessed the expression of hepatocyte growth factor (HGF) and c-MET in prostatic tissues and cell lines and also determined the effect of purified recombinant HGF on ceU proliferation and scattering ofprostatic carcinoma ceU lines. HGF was expressed by human prostatic stromal myofibroblasts in primary culture but not by three human prostatic carcinoma ceU lines (LNCaP, DU 145, and PC-3) as assessed by Northern blot analysis. HGF was also detected by reverse transcriptasepolymerase chain reaction in both benign and malignant tissuesfrom radicalprostatectomy specimens. c-MET transcripts were identified by Northern blot in two androgen-insensitive human prostatic carcinoma ceU lines (DU 145 and PC-3) but not the androgen-sensitive LNCaP ceU line. Additional evidence of linkage of androgen responsiveness and c-METwas provided by experiments in which androgen deprivation of normal rat prostates via castration produced a marked upregulation of c-MET expression as determined by Northern blot and immunohistochemistry. c-MET protein was detected by immunohistochemical analysis in a substantialpercentage (58 of 128 or 45%) of prostatic carcinomas and was found
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more often in metastatic growths of human prostatic carcinoma (15 of 20 patients) compared with primary tumors (43 of 108 patients; P < 0. 005). Moreover, in Dunning R-332 7 ratprostatic carcinoma cell lines, c-MET expression was highest in the androgen-insensitive subline with the highest metastatic capacity. Purified recombinant human HGF induced dose-dependent celularproliferation and scattering in the DU145 carcinoma cell line. These data indicate thatHGFmay function in the prostate gland as a paracrine growthfactor, with synthesis by stromal cels and with biological target ceUs being the epithelial cells. Expression of the HGF receptor, c-MET, is up-regulated by androgen deprivation and c-MET appears to be preferentialy expressed on androgen-insensitive, metastatic cells, suggesting
apossible linkage ofc-METexpression with pros(Am J Pathol 1995, 147:386-396)
tatic carcinoma progression.
Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a pleiotropic polypeptide growth factor with a number of biological activities, including cell
scattering, stimulation of cell motility, mitogenesis, morphogenesis, angiogenesis, and cellular invasiveness (for reviews see Refs. 1-8). In vivo, HGF and its receptor, the c-met proto-oncogene product,2'3'8-12 are thought to be involved in embryogenesis, tissue reorganization, and tumor progression. HGF and c-MET cDNAs have exhibited transforming activity; HGF is capable of transforming immortalized mouse liver epithelial cells13 and the mouse met protooncogene under SV40 promoter control transforms NIH3T3 cells.14 Additionally, co-transfection of Supported by National Cancer Institute Grant P20CA58193, National Institutes of Health Grant NS29955 (PAH), American Cancer Society Grant CN 55 (RZ), and US Public Health Service Grant RO1 ES-06109 (RZ). Accepted for publication April 10, 1995. Address reprint requests to Dr. Peter A. Humphrey, Department of Pathology, Washington University Medical Center, Box 8118, St. Louis, MO 63110.
HGF and c-MET in Prostate Cancer 387 AJP Auigust 1995, Vol. 147, No. 2
NIH3T3 cells with methu and HGF/SFhu resulted in efficient tumorigenesis.15 HGF and c-MET have been suggested as a model for a paracrine signaling system in the context of mesenchymal cell-epithelial cell interactions.3 In this model, HGF is synthesized by mesenchymal cells, with the target cells being nearby epithelial cells expressing c-MET. The prostate gland would appear to be an organ well suited for such a mechanism of growth as prominent epithelial and stromal components are present. In particular, stromal-epithelial interaction has been hypothesized to be important for prostatic carcinoma growth and progression.16 Little is known, however, of HGF and c-MET expression in carcinoma of the prostate. In this investigation, we assessed the expression of HGF and c-MET in prostatic tissues and cell lines and also examined the effect of purified recombinant HGF on the cell proliferation and scattering of prostate carcinoma cell lines. The data show that HGF may influence prostatic carcinoma cell growth and scattering via a paracrine mode of action, with HGF synthesis by stromal cells and with c-MET-expressing carcinoma cells being the target cells. Additionally, the linkage of c-MET expression in prostatic carcinoma cells with androgen insensitivity and metastasis suggests that c-MET expression may be related to prostatic carcinoma progression.
Materials and Methods Cell Lines and Tissues The three human prostatic carcinoma cell lines LNCaP,17 DU 145,18 and PC-3,19 were maintained in Dr. Ratliff's laboratory. Three sublines of Dunning R-3327 rat prostatic carcinoma cells20 (Dunning G, HI-F, and MAT-LyLu) were from Dr. W.D.W. Heston, Memorial-Sloan Kettering Cancer Center, New York, NY. The human prostatic stromal myofibroblastic cell line was from a patient with benign prostatic hypertrophy and was provided by Dr. Chung Lee, Northwestern University, Chicago, IL. The myofibroblastic nature of this cell line was documented by immunophenotypic analysis and electron microscopy. Briefly, with the immunohistochemical techniques described below, these cells exhibited immunoreactivity for vimentin and muscle-specific actin but not for the epithelial and prostatic epithelial markers cytokeratin and prostate-specific antigen, respectively. Electron microscopy, performed on cultured cells harvested in 3% buffered glutaraldehyde and processed in a methacrylate resin, confirmed these immunophenotypic data; elongate mesenchymal cells with abundant rough endoplasmic reticulum and mitochondria
contained scattered skeins of microfilaments punctuated by dense bodies. Pinocytotic vesicles were evident as were sporadic plasmalemmal plaques. Fresh human prostatic tissues were from radical prostatectomies performed at Washington University Medical Center and were snap-frozen in liquid nitrogen. Benign and carcinomatous tissues were selected by examining frozen sections stained with hematoxylin and eosin. The carcinomatous tissue samples had greater than 50% involvement by tumor cells. Human prostatic tissues utilized for immunohistochemical analysis were archival, formalin-fixed, and paraffin-embedded cases from the Division of Surgical Pathology, Department of Pathology, Washington University Medical Center. A total of 108 cases were of primary prostatic carcinomas in radical prostatectomy tissues, and 20 cases of metastatic disease were studied. The primary tumors and metastatic carcinomas were not matched (that is, were not from the same patient). The metastases were present in pelvic lymph nodes (17 cases) and bone marrow (3 cases).
Northern Analysis Cell lines were grown to 70% confluence and total cellular RNA was extracted as described.21 Total RNA was prepared from snap-frozen human and rat prostatic tissue and human placenta as described.22 Total RNA was quantitated by spectrophotometry at 260 nm. For Northern blot analysis, 15 pg of total RNA were denatured in sample buffer (20 mmol/L MOPS buffer, pH 7.0, 5 mmol/L sodium acetate, 1 mmol/L EDTA, 50% deionized formamide, and 6.5% formaldehyde). Samples were resolved on denaturing 1.2% agarose gels containing 20 mmol/L MOPS, pH 7.0, 5 mmol/L sodium acetate, and 1 mmol/L EDTA, 6.5% formaldehyde at 100 V for 4 hours. Equivalence of RNA load was assessed by both ethidium bromide staining and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) hybridization, with total concordance observed for these two measures of RNA load. RNA was transferred by capillary blotting to nylon membranes (ONCOR, Gaithersburg, MD) in 1 X SSC (0.15 mol/L sodium citrate and 1.5 mol/L NaCI, pH 7.0) overnight and was cross-linked to the membranes by UV irradiation. The utilized cDNA probes were as follows. An EcoR fragment of human HGF cDNA (nucleotides 122-969) corresponding to the kringles 1 and 2 of the a-chain of HGF was generated as described23 and used as a human HGF probe. An 837-bp rat c-MET cDNA probe was prepared by reverse transcriptase-polymerase chain reaction (RT-
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PCR) as described24 and cloned into the pCR 11 plasmid (Invitrogen San Diego, CA); this cDNA encompasses nucleotides 142-979, corresponding to the 5' coding region of the human and mouse c-MET cDNA. Human c-MET cDNA probes were combined 32P-labeled met-D and met-H, from ONCOR. GAPDH cDNA probe was a 144-bp insert in PSK (kindly provided by Dr. Lisa Olson, Washington University Medical Center). Radiolabeled cDNA probes were prepared by the random primer method with [32P]-dCTP (Amersham, Arlington Heights, IL) and the random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). Labeled probes were purified by Sephadex G-50 chromatography. Only probes with specific activities of 1 x 109 cpm/pg or greater were used for hybridizations. Membranes were prehybridized in hybridization buffer (45% formamide 5X SSC 0.075 mol/L sodium citrate, 0.75 mol/L NaCI, pH 7, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), and 100 pg/ml calf thymus DNA) at 45°C (c-MET) or 480C (HGF) for 1 hour. Probes were denatured by boiling in hybridization buffer for 10 minutes, and 1 x 106 cpm/ml of probe was added to the membranes. Hybridization was for 16 hours at 42°C (c-MET) or 480C (HGF). Filters were washed in successive solutions of 0.1% SDS with 2X to 0.1X SSC at 520C (c-MET) and 650C (HGF) and exposed to Hyperfilm-MP (Amersham) with intensifying screens at -700C.
RT-PCR RT-PCR for HGF expression in human prostatic tissue was carried out with total RNA isolated as described above. Reverse transcriptase was primed on 1 pg of total RNA with random hexamers (Perkin-Elmer Cetus, Foster City, CA) and 20 U of reverse transcriptase (Perkin-Elmer Cetus) in 20 pl total volume. The PCR amplification reaction mixture was composed of 20 pl of the first strand cDNA reaction mixture and 80 pl of 2 mmol/L MgCI2, 1 X PCR buffer (Perkin-Elmer Cetus), 0.2 pmol/L HGF primers (forward: 5' ATC AGA CAC CAC ACC GGC ACA AAT 3'; reverse: 5' GAA ATA GGG CAA TAA TCC CAA GGA A 3'), and 2.5 U of Amplitaq DNA polymerase (Perkin-Elmer Cetus). These primers have previously been shown to specifically amplify a 691 -bp product of HGF sequences, as assessed by direct sequencing performed by Dr. Zarnegar's laboratory. The reaction mixture was overlaid with 100 pl of mineral oil. After an initial denaturation step at 950C for 2 minutes, 35 cycles of denaturation at 950C for 1 minute, annealing at 55°C for 1 minute, and extension at 720C for 3 minutes were run
on a Perkin-Elmer Cetus 480 DNA thermal cycler. PCR samples (10 pl each) were analyzed by electrophoresis on 2% agarose gels and visualized by ethidium
bromide (Sigma Chemical Co., St. Louis, MO)
staining.
Immunohistochemical Analysis for c-MET Immunohistochemical studies were performed on
5-pm sections that had been cut from paraffin blocks, mounted on acid-cleaned glass slides, and heated for 1 hour at 550C. Slides were dewaxed and dehydrated and then immersed for 30 minutes in absolute methanol containing 0.6% (v/v) hydrogen peroxide to quench endogenous peroxidase activity. After rehydration through graded ethanols and distilled water, sections were immersed in 10 mmol/L citrate buffer (10 mmol/L citrate monohydrate in distilled water, pH 6.0) in plastic Coplin jars and subjected to microwave irradiation (600 W, 2450 MHz) for 4 minutes. After cooling to room temperature, slides were removed to phosphate-buffered saline (pH 7.4) for 10 minutes. Sections were then overlaid with rabbit affinitypurified polyclonal antibody against either human c-MET (catalog No. SC-161; 1.25 pg/ml) or mouse c-MET, which is cross-reactive with rat c-MET (catalog No. SC-162; 0.1 pg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) and incubated for 18 hours in moisture chambers at 4°C. The avidin-biotin-peroxidase complex method was performed in all cases (VectaStain ABC elite mouse universal kit; Vector Laboratories, Burlingame, CA).25 Chromogenic development resulted from immersion of slides in 3,3'diaminobenzidine tetrahydrochloride (Sigma Chemical Co.; 0.5 mg/ml, containing 0.003% (v/v) hydrogen peroxide) for
