Peptidylglycine alpha-amidating monooxygenase- and proadrenomedullin-derived peptide-associated neuroendocrine differentiation are induced by androgen deprivation in the neoplastic prostate.

Most PCs show NE differentiation. Several studies have tried to correlate NE expression with disease status, but the reported findings have been contradictory. Prostatic NE cells synthesize peptides with a wide spectrum of potential functions. Some of these active peptides, such as PAMP, are amidated. PAM is the only carboxy-terminal peptide-amidating enzyme identified. We studied expression of PAMP and PAM in normal prostate and prostatic tumors (clinical specimens and human xenograft models) with or without prior androgen-deprivation therapy and found a wide distribution of both molecules in NE subpopulations of all kinds. Although the correlation of either marker to tumor grade, clinical progression or disease prognosis did not reach statistical significance, PAMP- or PAM-immunoreactive cells were induced after androgen-blockade therapy. In the PC-310 and PC-295 androgen-dependent models, PAMP or PAM NE differentiation was induced after castration in different ways, being higher in PC-310, which might explain its long-term survival after androgen deprivation. We show induction of expression of 2 new NE markers in clinical specimens and xenografted PC after endocrine therapy.

Differential expression of the early lung cancer detection marker, heterogeneous nuclear ribonucleoprotein-A2/B1 (hnRNP-A2/B1) in normal breast and neoplastic breast cancer.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP-A2/B1) is highly expressed during critical stages of lung development and carcinogenesis. To determine if the expression of hnRNP-A2/B1 is an informative biomarker in breast carcinogenesis, we analyzed hnRNP-A2/B1 overexpression by immunohistochemistry in archived specimens. Expression was detected in 48/85 (56.5%) primary invasive breast cancers and 7/72 (9.7%) specimens of normal breast tissue. Northern analysis of breast cancer cells also demonstrated higher level of hnRNP-A2/B1 expression compared to normal or transformed breast cells. Expression of hnRNP-A2/B1 in breast cancer cells was decreased by exposure to retinoids coordinately with decreased cell growth. These results warrant further evaluation of hnRNP-A2/B1 as a marker of breast carcinogenesis.

Autocrine growth loops dependent on peptidyl alpha-amidating enzyme as targets for novel tumor cell growth inhibitors.

Many small cell lung tumors are dependent in vitro and in vivo on autocrine growth loops. The prototypical small cell lung cancer autocrine growth factor, gastrin-releasing peptide (GRP), is one of many peptide hormones which require post-translational carboxy-terminal alpha-amidation for bioactivity. We have reported that neuroendocrine human lung tumor cell lines express the bifunctional enzyme PAM which catalyzes the biosynthesis of alpha-amidated peptides in a two-step process, and have recently shown that non-small cell lung cancer cell lines and tumors, generally considered to be non-endocrine in nature, also express PAM. We have also shown that two chemical classes of PAM inhibitors, substrate analogues and specific copper chelators, inhibit amidating enzyme activity in cell-free extracts. Here we demonstrate in vitro growth inhibition of lung cancer tumor cell lines by both these classes of PAM inhibitors using the MTT assay and the clonogenic assay. Growth inhibition in a small cell lung cancer cell line can be overcome by exogenous addition of synthetic alpha-amidated GRP. Similar growth-suppressive effects are seen in cell lines stably transfected with a vector expressing antisense PAM RNA. These data support the mechanism of inhibition for a new type of chemotherapeutic/intervention agent, directed at synthesis and activation of peptide growth factors, and support our postulate that alpha-amidated peptide hormones are a common component in lung tumor autocrine growth biology which can be inhibited by targeting the biochemical mechanisms necessary for growth factor synthesis.

Relationship of arachidonic acid metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective biochemical inhibitors.

Arachidonic acid (AA) metabolizing enzymes are emerging as significant mediators of growth stimulation for epithelial cells. The relative contribution of the various family members of AA metabolizing enzymes to epithelial cancer cell growth is not known. To study this question, we first analyzed a series of epithelial cancer cells to establish the relative frequency of expression for the various enzymes. We analyzed the expression of five AA metabolizing enzymes as well as 5-lipoxygenase activating protein (FLAP) in a panel of human epithelial cancer cell lines (n = 20) using reverse transcription-PCR. From this analysis, we found that cyclooxygenase-1 (COX-1), 5-lipoxygenase (5-LOX), and FLAP were universally expressed in all cancer cell lines tested. For the remaining enzymes, the expression of COX-2, 12-LOX, and 15-LOX varied among cell lines, 60, 35, and 90%, respectively. Although the pattern of expression varied among the different cell types, all of the enzymes were expressed in all major cancer histologies. Using a panel of selective biochemical AA metabolizing enzyme inhibitors, we then evaluated the effect of these agents on cell lines with known expression status for the AA metabolizing enzymes. For the enzymes that were not universally expressed, growth inhibition by selective biochemical inhibitors did not closely correlate with the expression status of specific enzymes (P > 0.05). For the universally expressed enzymes, the LOX inhibitors were more potent growth inhibitors than the COX inhibitors. The frequent expression of the AA metabolizing enzymes suggests that AA metabolism pathway may be modulated in response to xenobiotic exposure during carcinogenesis. Although establishing a priori AA metabolizing enzyme status was not consistently informative about what AA metabolizing enzyme inhibition would be most growth inhibitory, the frequent inhibition of many epithelial cancers by these biochemical inhibitors opens a new avenue for cancer therapy and intervention in carcinogenesis.

Immunocytochemical mapping of the amidating enzyme PAM in the developing and adult mouse lung.

The enzyme PAM is required for activation of many peptide hormones. In adult mouse lung, immunostaining for PAM was located in Clara cells, which constitute most of the epithelial cells of the mouse bronchial/bronchiolar tree. Immunoreactivity appeared for the first time in the epithelium on gestational Day 16, being slight and mostly restricted to the apical cytoplasm. As the lung developed, the labeling became gradually stronger and extended throughout the cell. Smooth muscle of airways and blood vessels, and some parenchymal cells, probably macrophages, also showed PAM immunoreactivity. Of the two enzymatically active domains of PAM, only PHM and not PAL immunoreactivity was found at all stages studied. The early appearance of PAM in developing mouse lung, as well as its presence in a variety of tissues, probably indicates a complex role of this enzyme in pulmonary development and function.

Expression of heterogeneous nuclear ribonucleoprotein A2/B1 changes with critical stages of mammalian lung development.

Recent reports have demostrated a link between expression of members of the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and cancer. Overexpression of hnRNP A2/B1 correlated with the eventual development of lung cancer in three different clinical cohorts. We have studied the expression of hnRNP A2/B1 messenger RNA (mRNA) and protein during mammalian development. The expression of hnRNP A2/B1 mRNA and protein are parallel but change dynamically during critical periods in mouse pulmonary development. hnRNP A2/B1 is first detected in the lung in the early pseudoglandular period, peaks at the beginning of the canalicular period, and remains high during the saccular (alveolar) period. In mouse and rat, hnRNP A2/B1 expression is first evident in the earliest lung buds. As lung development progresses, the cuboidal epithelial cells of the distal primitive alveoli show high levels of the ribonucleoprotein, which is almost undetectable in the proximal conducting airways. The expression of hnRNP A2/ B1 is restricted in mature lung. Similar dynamic pattern of expression through lung development was also found in rat and human lung. Upregulated expression of hnRNP A2/B1 at critical periods of lung development was comparable to the level of expression found in lung cancers and preneoplastic lesions and is consistent with hnRNP A2/B1 overexpression playing an oncodevelopmental role.

Peptide-amidating enzymes are expressed in the stellate epithelial cells of the thymic medulla.

C-terminal amidation is a post-translational processing step necessary to convey biological activity to a large number of regulatory peptides. In this study we have demonstrated that the peptidyl-glycine alpha-amidating monooxygenase enzyme complex (PAM) responsible for this activity is located in the medullary stellate epithelial cells of the thymus and in cultured epithelial cells bearing a medullary phenotype, using Northern blot, immunocytochemistry, in situ hybridization, and enzyme assays. Immunocytochemical localization revealed a granular pattern in the cytoplasm of the stellate cells, which were also positive for cytokeratins and a B-lymphocyte-associated antigen. The presence of PAM activity in medium conditioned by thymic epithelial cell lines suggests that PAM is a secreted product of these cells. Among the four epithelial cell lines examined, there was a direct correlation between PAM activity and content of oxytocin, an amidated peptide. Taken together, these data provide convincing evidence that thymic epithelial cells have the capacity to generate amidated peptides that may influence T-cell differentiation and suggest that the amidating enzymes could play an important role in the regulation of thymic physiology.

Recent molecular advances in the approach to early lung cancer detection and intervention.

Lung cancer is a major contributor to overall cancer mortality. Detecting lung cancer while it is still a localized process is a long-cherished goal for improving the outcome of this disease. Recent developments suggest that we are approaching this capability. We next have to think about how to implement a change in our approach to lung cancer management to derive the benefit of better detection capability. This is an area in which our growing understanding of lung cancer biology is providing clues on improving the inhibition of cancer progression.

New approaches to the integrated management of early lung cancer.

We have briefly surveyed some the developments in the field of molecular diagnostics that provide a basis for cautious optimism about progress in population-based early lung cancer screening. The sound lung cancer management strategies that were formulated several decades ago failed in clinical trials because the necessary tools to implement the strategies were not yet available. Technology is beginning to emerge that makes population-based screening achievable. This same technology may be used to define a comprehensive marker panel including the most informative markers from the long list of candidate markers. Validation studies will define more clearly the strengths and limitations of new molecular diagnostics and provide leads for further research attention. The clinical community can expedite this process if these validation efforts are aggressively pursued. Parallel developments are clearly needed in refining the range of therapeutic intervention for early cancer management. The success of both diagnostic and intervention tools is interwoven in the ultimate goal of reducing lung cancer mortality. This article is an invitation to think expansively about new approaches to cancer care that integrate the fruits of our hard-learned lessons.

Expression of peptidyl-glycine alpha-amidating mono-oxygenase (PAM) enzymes in morphological abnormalities adjacent to pulmonary tumors.

Carboxyl-terminal amidated peptide hormones are known to be autocrine growth factors for lung tumors and tumor cell lines. Expression of the enzymes necessary for the biosynthesis of active amidated peptide hormones is therefore necessary for autocrine growth stimulation in lung tumors and possibly in the early proliferative stages of lung carcinogenesis. The peptidyl amidating enzymes have previously been identified in cell lines of all histological types of lung cancer and in lung tumors by immunohistochemistry and in situ hybridization. In this study we analyzed the expression of the peptidyl amidating enzymes in histological abnormalities found in the proximity of pulmonary tumors from a series of 59 patients. Most of the lesions in both the proximal airways (basal cell hyperplasia, carcinoma in situ, and some squamous metaplasia) and the alveoli (type II cell hyperplasia, bronchiolization of the alveoli, atypical alveolar hyperplasia, and isolated atypias) had a high proportion of cells strongly positive for the peptidyl amidating enzymes. The intense expression of peptidyl amidating enzymes in type II cell hyperplasia and atypical alveolar cells, together with the high frequency of these abnormalities in the alveoli, which is an area that does not express these enzymes in normal lung, points to the involvement of peptide hormones in the growth biology of pulmonary tumors. These findings suggest that peptide hormone stimulation of mitogenesis is an early event in tumor progression and merits additional investigation as a target for early detection and chemo-intervention of lung carcinogenesis.

Peptide amidating activity in human bronchoalveolar lavage fluid.

Monitoring respiratory epithelial biology may reveal individuals with incipient lung cancer. The expression of neuroendocrine (NE) markers in pulmonary epithelium is thought to be central to lung development, repair of injury and may contribute to carcinogenesis. In this study, we evaluate several candidate NE markers to determine the feasibility of prospective analysis of clinical specimens. The potential NE markers include the enzyme L-DOPA decarboxylase (DDC), the neuropeptide gastrin releasing peptide (GRP), and peptidyl-glycine alpha-amidating monooxygenase (PAM), the bifunctional enzyme responsible for the final bioactivation step of many neuropeptides. A comparison of PAM activity and DDC levels in 30 lung cancer cell lines indicated that peptide amidating activity may be an indicator of NE status. Bronchoalveolar lavage (BAL) fluid from subjects at risk of developing second primary lung cancer and from volunteers was obtained. The activity of the first PAM enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), ranged from not detectable to 507 pmol/h/mg protein in 57 specimens. The second PAM enzyme, peptidylamidoglycolate lyase (PAL), ranged from not detectable to 414 pmol/h/mg protein in 56 specimens. Using cluster analysis by the average linkage method, a group of enzyme values with PHM greater than 230 pmol/h/mg protein was determined. Long-term follow-up of these patients for new second primary lung cancers may help to determine the potential predictive value of PAM detected in the BAL fluid.

Purification and characterization of a protein that permits early detection of lung cancer. Identification of heterogeneous nuclear ribonucleoprotein-A2/B1 as the antigen for monoclonal antibody 703D4.

We have reported that a mouse monoclonal antibody 703D4, detects lung cancer 2 years earlier than routine chest x-ray or cytomorphology. We purified the 703D4 antigen to elucidate its role in early lung cancer biology, using Western blot detection after SDS-polyacrylamide gel electrophoresis. Purification steps included anion exchange chromatography, preparative isoelectric focusing, polymer-based C18-like, and analytical C4 reverse phase high performance liquid chromatography. After 25-50,000-fold purification, the principal immunostaining protein was > 95% pure by Coomassie staining. The NH2 terminus was blocked, so CNBr digestion was used to generate internal peptides. Three sequences, including one across a site of alternate exon splicing, all identified a single protein, heterogeneous nuclear ribonucleoprotein-A2 (hnRNP-A2). A minor co-purifying immunoreactive protein resolved at the final C4 high performance liquid chromatography step is the splice variant hnRNP-B1. Northern analysis of RNA from primary normal bronchial epithelial cells demonstrated a low level of hnRNP-A2/B1 expression, consistent with immunohistochemical staining of clinical samples, and increased hnRNP-A2/B1 expression was found in lung cancer cells. hnRNP-A2/B1 expression is under proliferation-dependent control in normal bronchial epithelial cell primary cultures, but not in SV40-transformed bronchial epithelial cells or tumor cell lines. With our clinical data, this information suggests that hnRNP-A2/B1 is an early marker of lung epithelial transformation and carcinogenesis.

Insulin-like growth factor expression in human cancer cell lines.

The insulin-like growth factors (IGFs), IGF-I and IGF-II, are potent mitogens for human lung and other epithelial cancer cell lines. Previous studies in defined medium lacking added IGF or insulin suggest that an IGF-related ligand can act as an autocrine growth factor for many cancer cell lines through action via the type I IGF receptor (IGF-R). Analysis of RNA isolated from human lung and breast cancer cell lines by reverse transcription of mRNA and polymerase chain reaction reveal that IGF-I and IGF-II mRNAs were co-expressed with IGF-R in the majority of cell lines. IGF-I mRNA was detected in 11/12 small cell lung cancer cell lines (SCLC), 13/14 nonsmall cell lung cancer (NSCLC) cell lines, and 1/2 breast cancer cell lines. IGF-II mRNA was detected in 8/10 SCLC, 11/12 NSCLC cell lines, and 2/2 breast lines. All cell lines expressed IGF-R. For analysis of IGF peptide secretion, cell lines were adapted to growth in serum/hormone-free culture medium (R0), and to avoid interference by IGF-binding proteins, secreted IGF peptides were isolated under acidic conditions and analyzed by Western blotting. Based upon measurement of the sensitivity of the anti-IGF antibodies for detection of recombinant human IGFs, IGF peptides accumulated in conditioned medium at greater than picomolar concentrations should have been readily detected. In three cell lines (two lung and one breast) secreted IGF immunoreactivity was detected as three molecular mass species of 23, 14, and 6 kDa. Isolation and NH2-terminal sequencing of each of these species definitively identified them as differentially processed forms of the IGF-II prohormone. Despite the high frequency of IGF-I gene expression detected by reverse transcription-polymerase chain reaction analysis, only one lung cancer cell line, NCI-N417d, was found that unequivocally secreted IGF-I peptide. This direct sequence determination unambiguously identifies IGF-II as the predominant IGF involved in the autocrine growth stimulation of human lung and breast epithelial tumor cell lines and supports a growing body of literature that implicates IGF-II/IGF-R autocrine loops as a common growth mechanism in epithelial carcinogenesis.

Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling.

Signal transduction pathways shared by different autocrine growth factors may provide an efficient approach to accomplish clinically significant control of lung cancer growth. In this study, we demonstrate that two autocrine growth factors activate 5-lipoxygenase action of the arachidonic acid metabolic pathway in lung cancer cell lines. Both growth factors increased the production of 5(S)-hydrooxyeicosa-6E,8Z,11Z,14Z-tetraeno ic acid (5-HETE), a major early 5-lipoxygenase metabolic product. Exogenously added 5-HETE stimulated lung cancer cell growth in vitro. Inhibition of 5-lipoxygenase metabolism by selective antagonists resulted in significant growth reduction for a number of lung cancer cell lines. Primary clinical specimens and lung cancer cell lines express the message for the 5-lipoxygenase enzymes responsible for the generation of active metabolites. In vivo evaluation demonstrated that interruption of 5-lipoxygenase signaling resulted in enhanced levels of programmed cell death. These findings demonstrate that 5-lipoxygenase activation is involved with growth factor-mediated growth stimulation for lung cancer cell lines. Pharmacological intervention with lipoxygenase inhibitors may be an important new clinical strategy to regulate growth factor-dependent stages of lung carcinogenesis.

Expression in human lung cancer cell lines of genes of prohormone processing and the neuroendocrine phenotype.

Lung tumor cells and cell lines, principally the histologically classified small cell lung cancer, are characterized by the expression of neuroendocrine (NE) features including AADC (aromatic amino acid decarboxylase, previously called DOPA decarboxylase) and the production of many peptide hormones. The general mechanisms by which most aspects of the NE phenotype affect the clinical behavior of lung tumor cells are unknown, but it is well recognized that peptide hormones can have systemic effects (paraneoplastic syndromes) and several have been shown to be autocrine growth factors for cancer cells. In order to determine the relationship between expression of different aspects of the NE phenotype in lung cancer cell lines, we have compared expression of a gene required for biosynthesis of some active peptide hormones (PAM, peptidylglycine alpha-amidating monooxygenase) to the gene for AADC in 32 lung cancer cell lines. Expression of these genes was quantified by both steady state Northern blot analysis and radiochemical enzymatic activity measurements. To ensure a range of expression of NE markers, non-small cell lung cancer (NSCLC) cell lines were chosen to include several which had previously been shown to express NE markers, and several small cell lung cancer (SCLC) cell lines with previous low levels of AADC were included. PAM enzyme activity and Northern blot analysis showed a two to three log variation in levels of expression in both the small cell and non-small cell lines. A smaller range was found for AADC expression. Using the highly sensitive PAM enzyme assays, all cell lines were found to express detectable PAM. PAM activities were secreted into the growth medium of all cell lines. There was no simple correlation apparent between AADC and PAM gene expression in the lung cancer cell lines. However, classic small cell lines demonstrated high levels of expression of both PAM and AADC genes, as did the carcinoid subset of the NSCLC lines. NSCLC lines expressed levels of PAM mRNA and enzyme activities equivalent to those of SCLC but had infrequent expression of AADC (principally only carcinoid NSCLC expressed AADC). These data demonstrate that separate aspects of the NE phenotype can be differentially expressed in lung cancer histological sub-types. Expression of PAM enzymes in all sub-types of lung cancer suggests that peptide prohormone activation may be a common mechanism for autocrine growth stimulation even in non-Ne NSCLC cell lines, or may reflect maintenance in cell lines of a common pathway of lung tumor promotion.

Human peptidylglycine alpha-amidating monooxygenase transcripts derived by alternative mRNA splicing of an unreported exon.

We are characterizing the alternatively spliced human peptidylglycine alpha-amidating monooxygenase (hPAM)-encoding mRNA transcripts expressed by human cells. Reverse transcription coupled to the polymerase chain reaction (RT-PCR) has been used to identify four alternatively spliced variants that differ in the region joining the two catalytic domains. Two of the transcripts represent previously reported splice variants differentiated by the presence (hPAM-A) or absence (hPAM-B) of a 321-nucleotide (nt) linker (optional exon A) which in the rat produce functionally distinct enzymes. Different mRNAs represent two splice variants, hPAM-C and hPAM-D, that show the presence of an exon unreported for PAM in any other species. This new exon, designated exon C, is 54 nt in length, encodes an 18-amino-acid (aa) peptide containing a conserved dibasic aa endoproteolytic processing motif, and is located 3' of exon A in human genomic DNA. We propose that cell-specific regulation of mRNA splicing would provide a mechanism for control of prohormone activation by these variants of the PAM enzyme.

Isolation and functional expression of human pancreatic peptidylglycine alpha-amidating monooxygenase.

Pancreastatin (PST) is processed from chromogranin A and the C-terminal amide of the peptide is an absolute requirement for biological activities. Human pancreatic carcinoma cells QGP-1 which produce both chromogranin A and PST were used to isolate cDNAs encoding two forms of peptidylglycine alpha-amidating monooxygenase (PAM). The two forms are a full length bifunctional enzyme and a variant lacking the transmembrane domain-coding region. When the cDNAs of these two forms were expressed in COS-7 cells, cells transfected with the predicted soluble form released into the culture medium a very much higher amidating activity which converts human chromogranin A-(273-302) to PST-29. The optimal pH for amidating activity was 5.4 and Cu2+, ascorbate and catalase were required as cofactors for the both forms of PAM. Km values for the membrane-bound and the soluble forms of PAM were 15.7 +/- 3.1 microM and 12.4 +/- 1.6 microM, respectively. These results demonstrate that both forms of PAM can function in the posttranslational processing of chromogranin A to PST in the environment of a secretory vesicle.

Peptidylglycine alpha-amidating monooxygenase (PAM) immunoreactivity and messenger RNA in human pituitary and increased expression in pituitary tumours.

Bioactivity of many peptides depends upon post-translational alpha-amidation of inactive precursors by two enzyme activities known collectively as peptidylglycine alpha-amidating monooxygenase (PAM). PAM enzymes are particularly abundant in the pituitary. The distribution of PAM immunoreactivity and messenger ribonucleic acid (mRNA) in the adult human pituitary and in pituitary tumours was investigated by use of immunocytochemistry and in situ hybridisation. Immunoreactivity was present in numerous cells of the anterior lobe: staining was intense in a proportion of gonadotrophs and folliculo-stellate cells, but weaker in the majority of somatotrophs and lactotrophs, a few corticotrophs and occasional thyrotrophs. PAM staining was also present in nerves, pituicytes and some endocrine cells within the posterior lobe (the human intermediate zone). Forty pituitary tumours of various types were immunoreactive for PAM; more intensely and uniformly stained than normal anterior lobe. In situ hybridisation with digoxigenin-labelled probes demonstrated intense labelling for PAM mRNA in numerous cells in normal anterior pituitary and in tumours. Many regulatory peptides that require amidation for activity, potential targets for PAM, are present in the pituitary. Many tumour growth factors also require amidation and PAM may regulate these mitogenic peptides in tumours.