Cancer and the Vasopressin Gene: Radioimmunoassay Values and Commentary on Copeptin as a Plasma Marker.

Background: Vasopressin gene expression has been demonstrated to be a common feature of all small-cell lung cancer (SCLC) and breast cancer. Provasopressin (ProVP) is a component of the cancer cell membrane and a likely target for treatment. However, a measurable fraction of this cancer provasopressin is also normally processed and products are released into the circulation. Vasopressin (VP) and vasopressin-associated human neurophysin (VP-HNP), two of three products of processing, were earlier shown to be reliable plasma markers for determining the presence of SCLC and monitoring response to treatment. Material and Methods: In this study, copeptin, the third product of provasopressin processing, was preliminarily evaluated as a plasma marker for SCLC or breast cancer using radioimmunoassay (RIA). Antibodies directed against the 18 residue C-terminal peptide fragment of copeptin were used to avoid interference from the large-carbohydrate component of this endogenous glycopeptide. Results: The levels of copeptin in 8 male and 6 female patients with SCLC before treatment ranged from 16 to 319 pmol/L, and these levels were elevated (>2.5 times) in 10 of 14 cases (70%) when compared with healthy volunteers (normal mean, 18 ± 6 pmol/L). Volunteer values for males were smaller than for females (15± 4 pmol/L and 20± 9 pmol/L), but numbers were small. Patients with breast cancer had plasma levels ranging from 12 to 68 pmol/L, with only three of the six elevated. Conclusion: While cancer patients displayed a wide range of plasma copeptin levels over 70% with SCLC and 50% with breast cancer had clearly elevated levels. This finding indicates that for such patients, plasma copeptin, like plasma VP and VP-HNP, could be used to detect disease. The control values found for healthy volunteers using our RIA were in a range predictable from established normal plasma levels of both VP and VP-HNP.

Small-cell lung cancer growth inhibition: synergism between NMDA receptor blockade and chemotherapy.

BACKGROUND: Small-cell lung cancer (SCLC) has a poor prognosis since there is currently no effective therapy for commonly recurring disease. In our previous study, both primary and recurrent human tumors have been shown to express functional N-methyl-D-aspartate (NMDA) receptors, and blockade of these receptors with GluN1 and GluN2B antagonists decreased tumor cell viability in vitro, and growth of tumor xenografts in nu/nu mice. MATERIALS AND METHODS: In this study, we examine the influence of the GluN2B antagonist ifenprodil and the channel-blocker antagonist memantine, on cell viability and growth of tumor xenografts of recurrent SCLC (rSCLC) in mice. RESULTS: Both antagonists significantly reduced cell viability and levels of components of the ERK1/2 pathway, increased apoptosis, and at very safe levels significantly reduced the growth of tumors in mice. Each antagonist and topotecan had additive effects to reduce cell viability with significant synergy demonstrated for the case of memantine. More significantly, combination treatments of xenografts in mice with ifenprodil and the chemotherapeutic agent topotecan produced clear additive effects that completely stopped tumor growth. Moreover, the ifenprodil and topotecan combination showed excellent supra-addition or synergy of inhibition for tumors ≤300 mm in size (P=4.7E-4). Combination treatment of memantine with topotecan also showed clear addition but, unlike ifenprodil, no synergy for the doses chosen. CONCLUSION: Since topotecan is a drug of choice for treatment of rSCLC, our findings suggest that combining this agent with NMDA receptor blockade using the GluN2B antagonist, ifenprodil, will significantly improve patient outcomes.

NMDA receptors are important regulators of pancreatic cancer and are potential targets for treatment.

Pancreatic cancer, particularly adenocarcinoma of the pancreas, is a common disease with a poor prognosis. In this study, the importance of N-methyl-D-aspartate (NMDA) receptors for the growth and survival of pancreatic cancer was investigated. Immunohistochemistry performed with antibodies against GluN1 and GluN2B revealed that all invasive adenocarcinoma and neuroendocrine pancreatic tumors likely express these two NMDA receptor proteins. These proteins were found to be membrane components of pancreatic cancer cell lines, and both channel-blocker antagonist and GluN2B antagonist significantly reduced cell viability in vitro. Both types of antagonists caused an internalization of the receptors. Dizocilpine maleate (MK-801) and ifenprodil hemitartrate both significantly inhibited the growth of pancreatic tumor xenografts in nu/nu mice. These findings predict that, as for other solid tumors investigated by us, pancreatic cancer could be successfully treated, alone or in combination, with NMDA receptor antagonists or other receptor-inhibiting blocking agents.

NMDA receptors are expressed in human ovarian cancer tissues and human ovarian cancer cell lines.

We have earlier demonstrated that breast cancer and small-cell lung cancer express functional NMDA receptors that can be targeted to promote cancer cell death. Human ovarian cancer tissues and human ovarian cancer cell lines (SKOV3, A2008, and A2780) have now been shown to also express NMDA-receptor subunit 1 (GluN1) and subunit 2B (GluN2B). Seventeen ovarian cancers in two arrays were screened by immunohistochemistry using polyclonal antibodies that recognize an extracellular moiety on GluN1 and on GluN2B. These specimens comprised malignant tissue with pathology diagnoses of serous papillary cystadenocarcinoma, endometrioid adenocarcinoma, and clear-cell carcinoma. Additionally, archival tissues defined as ovarian adenocarcinoma from ten patients treated at this institute were also evaluated. All of the cancerous tissues demonstrated positive staining patterns with the NMDA-receptor antibodies, while no staining was found for tumor-adjacent normal tissues or sections of normal ovarian tissue. Human ovarian adenocarcinoma cell lines (A2008, A2780, SKOV3) were demonstrated to express GluN1 by Western blotting, but displayed different levels of expression. Through immunocytochemistry utilizing GluN1 antibodies and imaging using a confocal microscope, we were able to demonstrate that GluN1 protein is expressed on the surface of these cells. In addition to these findings, GluN2B protein was demonstrated to be expressed using polyclonal antibodies against this protein. Treatment of all ovarian cell lines with antibodies against GluN1 was found to result in decreased cell viability (P<0.001), with decreases to 10%-25% that of untreated cells. Treatment of control HEK293 cells with various dilutions of GluN1 antibodies had no effect on cell viability. The GluN1 antagonist MK-801 (dizocilpine maleate) and the GluN2B antagonist ifenprodil, like antibodies, dramatically decreased the viability of A2780 ovarian tumor cells (P<0.01). Treatment of A2780 tumor xenografts with ifenprodil (2.5 mg/kg body weight/day) significantly reduced tumor growth in nu/nu mice. Our findings suggest that both GluN1 and GluN2B proteins as membrane components could be readily available targets for the treatment of most ovarian cancers.

Growth Impairment of Small-Cell Cancer by Targeting Pro-Vasopressin with MAG-1 Antibody.

Previously we demonstrated that human small-cell lung cancer (SCLC) seems to universally express the vasopressin gene, and this leads to the presence of a cell surface marker representing the entire pro-hormone precursor. In this study, we show this marker can be targeted with MAG-1, a mouse monoclonal antibody against a C-terminal moiety on pro-vasopressin. In vitro targeting of cell lines derived from primary and recurrent disease demonstrates attachment of antibody to the cell surface followed by internalization. In vivo targeting with (99)Tc-labeled Fab fragments of MAG-1 shows selective attachment to xenografts. In vivo treatment of tumors from classical cell line, NCI H345, with either ~1.65 mCi (~1.65 mg)/kg body weight (BW) of (90)Yttrium-labeled MAG-1, or ~1.65 mg/kg BW native MAG-1, delivered every second day for 6 days produced similar reductions in the growth rate to ~50% (p < 0.03). When dosing with native MAG-1 was escalated to daily amounts of ~3.3 mg/kg BW over 16 days, tumor growth rates fell to ~33% of saline controls (p < 0.005). Examination of tumors treated with this higher dosing demonstrated the presence in several of extensive apoptosis. Normal tissues seemed to be unaffected. A larger dosage of MAG-1 (~6.6 mg/kg BW) given daily for 14 days was used to treat xenografts of the variant cell line NCI H82 representing recurrent disease. This treatment decreased the rate of increase in tumor size by half, and doubling time ~3-fold. Increases in cleaved PARP supported increased apoptosis with antibody treatment. We believe these data provide evidence that the growth rate of SCLC tumors can be extensively reduced by treatment with MAG-1 antibody, and that a humanized form of this antibody could, in future, be potentially used for targeting therapy onto recurrent SCLC in patients.

Native MAG-1 antibody almost destroys human breast cancer xenografts.

A native form of mouse monoclonal IgG1 antibody called MAG-1, which recognizes an epitope on provasopressin, has been found to shrink and produce extensive necrosis of human breast tumor xenografts in nu/nu mice. We examined the ability of (90)Yttrium-labeled and native MAG-1 to affect the growth in nu/nu mice of cancer xenografts that were estrogen-responsive (from MCF-7 cells) and triple-negative (from MDA-MB231 cells). The growth rates of treated cells were compared to those receiving saline vehicle and those receiving (90)Yttrium-labeled and native forms of the ubiquitous antibody, MOPC21. Short-term treatments (4 doses over 6 days) not only with (90)Yttrium-MAG-1 but also native MAG-1 produced large reductions in size of rapidly growing tumors of both types, while both (90)Yttrium- MOPC21 and native MOPC21 had no effect. Native and (90)Yttrium-MAG-1 effects were similar, and arrested tumors recommenced growing soon after treatments stopped. Increasing native MAG-1 treatment to single dosing for 16 consecutive days shrank tumors of both types with no regrowth apparent over a 20-day post-treatment period of observation. Pathological examination of such tumors revealed they had undergone very extensive (>66%) necrosis.

Detection of Provasopressin in Invasive and Non-invasive (DCIS) Human Breast Cancer Using a Monoclonal Antibody Directed Against the C-terminus (MAG1).

The provasopressin protein (proAVP) is expressed by invasive breast cancer and non-invasive breast cancer, or ductal carcinoma in situ (DCIS). Here we demonstrate the ability of the monoclonal antibody MAG1 directed against the C-terminal end of proAVP to identify proAVP in all cases examined of human invasive cancer and DCIS (35 and 26, respectively). Tissues were chosen to represent a relevant variation in tumor type, grade, patient age, and menopausal status. By comparison, there was 65% positive staining for estrogen receptor, 61% for progesterone receptor, 67% for nuclear p53, and 39% for c-Erb-B2 with the invasive breast cancer sections. Reaction with the normal tissue types examined (67) was restricted to the vasopressinergic magnocellular neurons of the hypothalamus, where provasopressin is normally produced, and the posterior pituitary, where these neurons terminate. The breast epithelial tissue sections on the tissue microarray did not react with MAG1. Previously, we demonstrated that polyclonal antibodies to proAVP detected that protein in all breast cancer samples examined, but there was no reaction with breast tissue containing fibrocystic disease. The results presented here not only expand upon those earlier results, but they also demonstrate the specificity and effectiveness of what may be considered a more clinically-relevant agent. Thus, proAVP appears to be an attractive target for the detection of invasive breast cancer and DCIS, and these results suggest that MAG1 may be a beneficial tool for use in the development of such strategies.

NMDA receptors are expressed by small-cell lung cancer and are potential targets for effective treatment.

We previously showed that functional N-methyl-D-aspartate (NMDA) receptors are expressed by human neuroblastoma cells. In this study we demonstrate functional NMDAR1 and NMDAR2 receptors are expressed by small-cell lung cancer (SCLC) classical cell lines NCI H146, NCI H345, and DMS 53, by variant cell line NCI H82, and by most SCLC tumors, and that these receptors are important for the growth of human SCLC tumor xenografts in mice. Reverse transcription-polymerase chain reaction demonstrated mRNA for both receptors, with sequences identical to those for human mRNAs, are expressed in all four cell lines, and these generated proteins of the expected sizes 120 and 170 kDa. Cell viability tests showed cell growth was significantly (P < 0.0001) impaired by NMDAR1 antagonists MK-801 and memantine. Ifenprodil and Ro25-6981, NMDAR2B antagonists at the polyamine site, also significantly (P < 0.001) inhibited the growth/survival of these cells. Alternatively, the glycine-binding antagonist, L701, 324, increased viability to 140% and 120% in NCI H345 and NCI H82 cells after 48 hours of incubation. Immunohistochemistry of SCLC tumors with our polyclonal antibodies gave specific positive staining for the NMDAR1 receptor in 8 of 10 tissues examined. Small amounts of these same antibodies significantly reduced the growth of NCI-H345 cells up to 25% (P < 0.001). When NCI H345 cells were grown as tumor xenografts in mice, the growth of these tumors was reduced by 60% (P < 0.001) by treatments with MK-801 over five days. All of these data point to active NMDAR receptors possibly having an important influence on SCLC growth and survival.

Breast cancer expresses functional NMDA receptors.

We demonstrate here that functional NMDAR1 and NMDAR2 receptors are expressed by Mcf-7 and SKBR3 breast cancer cell lines, and possibly by most or all high-grade breast tumors, and that these receptors are important for the growth of human breast cancer xenografts in mice. RT-PCR demonstrated mRNA for both NMDAR1 and NMDAR2 receptors are expressed in both Mcf-7 and SKBR3 cell lines, and these messages likely have sequences identical to those reported for human mRNAs. Proteins of the expected respective sizes 120 and 170 kD are generated from these mRNAs by the tumor cells. Cell growth was found to be significantly (P < 0.0001) impaired down to 10% of normal growth by the irreversible NMDAR1 antagonists MK-801 and memantine with IC 50s ranging from 600 to >800 microM and from 200 to 300 microM for the two lines. Paradoxically, memantine with a lower binding affinity had the greater influence of the two inhibitors on cell viability. Immunohistochemical examination of high-grade invasive ductal and lobular breast cancer with our polyclonal antibodies against a peptide (-Met-Ser-Ile-Tyr-Ser-Asp-Lys-Ser-Ile-His-) in the extracellular domain of the NMDAR1 receptor gave specific positive staining for the receptor in all 10 cases examined. Positive staining was chiefly concentrated at the membranes of these tumor tissues. No staining with these antibodies was found for normal breast and kidney tissues. When Mcf-7 cells were grown as tumor xenografts in nu/nu mice, the growth of these tumors was completely arrested by daily treatments with MK-801 over 5 days. All of these data point to active NMDAR receptors being expressed by most breast cancers, and having an important influence on their survival.

Provasopressin expression by breast cancer cells: implications for growth and novel treatment strategies.

The arginine vasopressin (AVP) gene is expressed in certain cancers such as breast cancer, where it is believed to act as an autocrine growth factor. However, little is known about the regulation of the AVP protein precursor (proAVP) or AVP-mediated signaling in breast cancer and this study was undertaken to address some of the basic issues. The cultured cell lines examined (Mcf7, Skbr3, BT474, ZR75, Mcf10a) and human breast cancer tissue extract were found to express proAVP mRNA. Western analysis revealed multiple forms of proAVP protein were present in cell lysates, corresponding to those detected in human hypothalamus extracts. Monoclonal antibodies directed against different regions of proAVP bound to intact live Mcf7 and Skbr3 cells. Dexamethasone increased the amount of proAVP-associated glycopeptide (VAG) secreted by Skbr3 cells and a combination of dexamethasone, IBMX and 8br-cAMP increased cellular levels of VAG. Exogenous AVP (1, 10, and 100 nM) elevated phospho-ERK1/2 levels, and increased cell proliferation was observed in the presence of 10 nM AVP. Concurrent treatment with the V1a receptor antagonist SR49059 reduced the effects of AVP on proliferation in Mcf7 cells, and abolished it in Skbr3 cells. Results here show that proAVP components are found at the surface of Skbr3 and Mcf7 cells and are also secreted from these cells. In addition, they show that AVP promotes cancer cell growth, apparently through a V1-type receptor-mediated pathway and subsequent ERK1/2 activation. Thus, strategies for targeting proAVP should be examined for their effectiveness in diagnosing and treating breast cancer.

Immunohistochemical detection of NRSA on small cell lung cancer with a monoclonal antibody (MAG-1) that recognizes the carboxyl terminus of provasopressin.

A single monoclonal antibody (MAG-1) directed against the C-terminal 18-amino acid region (VAGc18) of provasopressin was examined as an agent for recognizing the tumor-specific NRSA marker common to small cell lung cancer (SCLC) in formalin-fixed tissues with ABC immunohistochemistry. SCLC tumors were obtained from several tissue locations and included primary, metastatic, and recurrent disease. Positive staining was found in 91% of cases (53/58). All five of the unreactive tumors were of the lungs or chest wall, and there did not appear to be an association of this negativity with disease stage, age, or sex. Alternatively, almost all primary lesions, almost all metastatic lesions, and all recurrent lesions examined gave a positive reaction with MAG-1. For this study, vasopressin-producing cells of the human anterior hypothalamus served as a positive control, while negative controls comprised normal lung tissue, tumor that received MAG-1 in the presence of an excess of antigen (VAGc18 peptide), or tumor reacted with a commercial IgG1 isotype as primary antibody. All of the results indicate that MAG-1 can be effectively used to selectively identify the NRSA marker on almost all SCLC tumors, at all disease stages, and at all locations. Since all four tumors tested showing no reactivity with MAG-1 gave a positive reaction for synaptophysin, it is proposed that a combined use of MAG-1 with synaptophysin antibodies could allow all SCLC tumors to be detected by ABC immunohistochemistry.

Immunohistochemical evaluation of vasopressin expression in breast fibrocystic disease and ductal carcinoma in situ (DCIS).

We previously found that expression of the vasopressin gene is a common feature of human breast cancer. In the present study we first examined 21 different cases of benign fibrocystic breast disease for vasopressin expression using immunohistochemistry and antibodies directed against vasopressin (anti-VP) and against vasopressin-associated glycopeptide (anti-VAG). All cases examined were negative for vasopressin gene expression using these antibodies. Alternatively, we examined 16 cases of breast ductal carcinoma in situ (DCIS) using the second of these antibodies (anti-VAG), and all of these cases were positive for vasopressin gene expression. Our results suggest that products of vasopressin gene expression are not markers of cellular proliferation in the breast, and might rather represent an early part of the carcinogenic process in this tissue.

Targeting the neurophysin-related cell surface antigen on small cell lung cancer cells using a monoclonal antibody against the glycopeptide region (MAG-1) of provasopressin.

The vasopressin (VP) gene is largely expressed in hypothalamic neurons, where the resultant pro-VP protein is enzymatically cleaved into its peptide hormone components, which include the neuropeptide VP, VP-associated neurophysin, and VP-associated glycopeptide (VAG). Small cell lung cancer (SCLC) tumors also express the VP gene, but the tumor pro-VP protein can remain intact and localize to the cell surface membrane. Previous studies have shown that polyclonal antibodies directed against different regions of the pro-VP protein bind specifically to the surface of cultured SCLC cells and recognize proteins of approximately 20 and approximately 40 kDa in cultured SCLC whole-cell lysate. Thus, these proteins have been designated neurophysin-related cell surface antigen (NRSA). A monoclonal antibody (mAb) designated MAG-1 was raised in this laboratory using a synthetic peptide representing the COOH-terminal sequence of VAG. The MAG-1 mAb recognizes NRSA in SCLC cell and tissue lysates by Western analysis, whereas immunofluorescent cytometric and microscopic analyses indicate that MAG-1 reacts specifically with NRSA on the surface of viable SCLC cells of both the classical and the variant subtype. Immunohistochemical analysis demonstrates that MAG-1 reacts with human SCLC tumor, but not with normal pulmonary epithelial cells in lung tissue. Additionally, a MAG-1 Fab fragment was generated that was also able to recognize NRSA. This is the first study to demonstrate that a mAb directed to the VAG region of the pro-VP protein has the potential for development into an in vivo diagnostic and therapeutic tool that targets plasma membrane-incorporated NRSA.