Jon R. Wiener

Lysophospholipid Growth Factors in the Initiation, Progression, Metastases, and Management of Ovarian Cancer

Abstract: Levels of lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are elevated in the plasma and ascites of ovarian cancer patients, but not in most other tumor types. LPA increases cell proliferation, cell survival, resistance to cisplatin, cell shrinkage, and production of vascular endothelial growth factor, urokinase plasminogen activator, and LPA itself in ovarian cancer cells, but not in normal ovarian surface epithelial cells. PSP24 and members of the endothelial differentiation gene (EDG) family (EDG1, EDG2, EDG4, and EDG7) of G protein‐coupled receptors mediate LPA signaling. Ovarian cancer cell lines do not express EDG1 mRNA, have variable EDG2 mRNA and protein levels, and frequently exhibit elevated levels of EDG4 mRNA and protein, suggesting that EDG4 may contribute to the deleterious effects of LPA in ovarian cancer. In contrast, activation of the EDG2 LPA receptor on ovarian cancer cells may lead to apoptosis and counter the effects of other LPA receptors. Thus, the development of agonists and antagonists for the appropriate spectrum of LPA receptors may alter proliferation, apoptosis, or response to therapy of ovarian cancer cells. Indeed, over 60% of all current drugs target the G protein‐coupled family of receptors, making the LPA receptor family a “drugable” target. LPC, although not as thoroughly studied, increases cellular proliferation and mediates multiple other functions through unique signaling pathways.

Critical Role of Lysophospholipids in the Pathophysiology, Diagnosis, and Management of Ovarian Cancer

Lysophosphatidic acid (LPA), the simplest of all phospholipids, exhibits pleiomorphic functions in multiple cell lineages. The effects of LPA appear to be mediated by binding of LPA to specific members of the endothelial differentiation gene (Edg) family of G protein-coupled receptors (GPCR). Edg 2, Edg4, and Edg7 are high affinity receptors for LPA, and Edg1 may be a low affinity receptor for LPA. PSP24 has been shown to be responsive to LPA in Xenopus oocytes, however, its role in mammalian cells is unclear. The specific biochemical events initiated by the different Edg receptors, as well as the biological outcomes of activation of the individual receptors, are only beginning to be determined. LPA levels are consistently elevated in the plasma and ascites of ovarian cancer patients, but not in most other epithelial tumors, with the exception of cervix and endometrium, suggesting that LPA may be of particular importance in the pathophysiology of ovarian cancer. In support of this concept, ovarian cancer cells constitutively and inducibly produce high levels of LPA and demonstrate markedly different responses to LPA than normal ovarian surface epithelium. Edg4 and Edg7 levels are consistently increased in malignant ovarian epithelial cells contributing to the aberrant response of ovarian cancer cells to LPA. Edg2 may represent a negative regulatory LPA receptor inducing apoptosis in ovarian cancer cells. Thus, increased levels of LPA, altered receptor expression and altered responses to LPA may contribute to the initiation, progression or outcome of ovarian cancer. Over 40% of known drugs target GPCR, making LPA receptors attractive targets for molecular therapeutics. Indeed, using the structure-function relationship of LPA in model systems, we have identified selective Edg2 anatgonists, as well as Edg4 and Edg7 agonists. These lead compounds are being assessed in preclinical model systems. Understanding the mechanisms regulating LPA production, metabolism and function could lead to improved methods for early detection and to new targets for therapy in ovarian cancer.

The sequences of reovirus serotype 3 genome segments M1 and M3 encoding the minor protein μ2 and the major nonstructural protein μNS, respectively

The sequences of the M1 and M3 genome segments of reovirus serotype 3 strain Dearing, which encode protein mu 2, a minor capsid, component, and protein mu NS, one of the two nonstructural proteins, are reported. They are 2304 and 2235 base pairs long, respectively, and proteins mu 2 and mu NS comprise 736 and 719 amino acids, respectively. This completes the sequencing of the reovirus serotype 3 genome: it comprises 23,549 basepairs. Neither protein mu 2 nor protein mu NS possesses any sequence similarity to any protein sequence in gene banks, nor any of the commonly recognized motifs indicative of specialized function. Protein mu 2 has a higher alpha-helix content (36%) than other capsid proteins; for it, the ratio of amino acids in alpha-helix/beta-sheet configuration is 1.2, whereas that of typical reovirus capsid proteins ranges from 0.5 to 0.9. Thus it is not a typical capsid protein. Protein mu NS has a very high alpha-helix content (about 50%; alpha-helix/beta-sheet ratio 2.5), which is very similar to that of the other nonstructural reovirus protein, protein sigma NS. The C-terminal regions of mu NS and various myosins exhibit periodic sequence similarity elements indicative of helical structure. Protein mu NS exists in two forms in infected cells: protein mu NS and a protein, mu NSC, which lacks a region of about 5 kDa at its N-terminus. Pulse-chase analysis in vivo suggests that protein mu NSC is not a cleavage product of protein mu NS; further, protein mu NSC is formed along with protein mu NS in in vitro protein synthesizing systems, whereas protein mu 1C, the cleavage product of protein mu 1, is not. It is likely, therefore, that protein mu NSC is a primary translation product, formed either by ribosomes reading through the first initiation codon of m1 messenger RNA at position 14 and initiating at codon 42, or by de novo internal initiation at this codon.

Overexpression of the tyrosine phosphatase PTP1B is associated with human ovarian carcinomas

OBJECTIVE Our purpose was to determine whether protein tyrosine phosphatase 1B is overexpressed in ovarian cancers, possibly altering the balance of intracellular tyrosine phosphorylation. STUDY DESIGN The expression of protein tyrosine phosphatase 1B was assayed in frozen sections from 54 human ovarian carcinomas and seven normal ovaries by immunochemical staining with monoclonal antibody AE4-2J, which is specific for protein tyrosine phosphatase 1B. The expression of protein tyrosine phosphatase 1B-specific messenger ribonucleic acid in tumors was determined by Northern analysis. The results were analyzed statistically by means of Fishers exact test. RESULTS Minimal staining was observed in normal ovarian epithelium. In contrast, 43 of 54 (79.6%) tumors displayed increased protein tyrosine phosphatase 1B expression, which is statistically associated with malignancy. Overexpression was associated with the expression of the p185c-erbB-2, p170EGFR, and p165mCSFR growth factor receptor protein tyrosine kinases. Protein tyrosine phosphatase 1B messenger ribonucleic acid expression was inconsistently increased in tumor cells. CONCLUSION Increased expression of protein tyrosine phosphatase 1B in ovarian cancers that also express protein tyrosine kinases suggests that protein tyrosine phosphatase 1B may play a role in the growth regulation of ovarian cancers.

The sequences of the reovirus serotype 1, 2, and 3 L1 genome segments and analysis of the mode of divergence of the reovirus serotypes

We report the sequence of the L1 genome segment of reovirus serotype 3 strain Dearing, which encodes the minor core component protein lambda 3. It is 3854 bp long, with a long open reading frame starting at position 19 that is 1267 codons long. Protein lambda 3 is not detectably related to any other protein, nor does it appear to possess motifs indicative of recognized specialized functions. We have also sequenced the L1 genome segments of reovirus serotypes 1 and 2. The serotype 1 and 3 L1 genome segments are extremely closely related; there are only 154 mismatches (4.1%), 80% of which are in third base codon positions, so that these two lambda 3 proteins are 98.3% related (only 22 mismatches out of 1267). The serotype 2 L1 genome segment is only 75% related to the serotype 1 and 3 genome segments, and the serotype 2 lambda 3 protein is 92% related to the serotype 1 and 3 lambda 3 proteins. We have also analyzed the divergence patterns by which the various reovirus genome segments evolved into the three serotype forms. It appears that serotype 2 separated from the serotype 1/3 precursor long before serotypes 1 and 3 themselves diverged. In all cases the third base codon positions in the various genome segments have diverged about 80% toward randomness. The first and second base codon positions have diverged much less and to varying degree, depending, presumably, on each proteins ability to accept changes without significant loss of function. For the separation into the serotype 1 and 3 forms, the extent of divergence of the various genome varies over a very wide range. The S1 genome segments have again diverged most extensively, the extent of divergence in the first, second, and third base codon positions being about 50, 35 and 75%, respectively. For seven other genome segments that we examined the extent of third base codon position divergence is 56, 53, 48, 29, 22, 13, and 6%, whereas first and second base codon position divergence ranges from no more than 6 to 2 and 3 to less than 1%, respectively. The most likely explanation of these patterns is that the separation of the various genome segments into the present-day serotype 1 and 3 associated forms occurred at different times during evolution, from progenitors that were genome segment reassortants with survival rates as high as or higher than those of homologous genome segment sets.

Relative cytotoxic activity of immunotoxins reactive with different epitopes on the extracellular domain of the c-erbB-2 (HER-2/neu) gene product p185

Different epitopes on the extracellular domain of the HER‐2 receptor can serve as distinct targets for immunotoxins. To determine the optimal epitope target for immunotoxin therapy, 7 anti‐HER‐2 ricin A chain murine monoclonal immunotoxins, each reactive with different epitopes of HER‐2 receptor, were tested for cytotoxic activity. The immunotoxins produced 1.2–4.6 logs of cytotoxicity in limiting dilution clonogenic assays with 2 breast cancer cell lines that overexpressed HER‐2. Cytotoxicity did not correlate with immunoglobulin isotype, binding affinity, relative position of epitopes or internalization of the anti‐HER‐2 immunotoxins. Interestingly, the most and least effective immunotoxins bound to epitopes in very close proximity. Competitive binding assays with unconjugated antibodies have previously indicated that our antibodies recognized epitopes that are arranged in a linear array. To orient this relative epitope map, deletions were prepared in the HER‐2/neu gene and these mutant constructs were expressed in NIH3T3 cells. Epitope expression was determined by antibody binding and radioimmunoassay. Epitopes targeted by the PB3, 454C11 and NB3 antibodies are localized N‐terminal to the epitopes recognized by ID5, BD5, 741F8 and 520C9 antibodies. The 2 non‐conformational epitopes PB3 and NB3 were localized to regions corresponding to amino acides 78–242 of the p185HER‐2 protein. Int. J. Cancer 82:525–531, 1999.

Evolution of reovirus genes: A comparison of serotype 1, 2, and 3 m2 genome segments, which encode the major structural capsid protein μ1c

The sequences of the M2 genome segments of reovirus serotypes 1 and 2 are presented and compared with that of the cognate genome segment of reovirus serotype 3. Genome segment M2 encodes protein mu 1, a cleavage product of which, mu 1C, is the major constituent of reovirus particles. The three M2 genome segments exhibit a serotype 1:3 relatedness pattern: the serotype 1 and 3, 1 and 2, and 2 and 3 genome segment pairs exhibit 15, 23, and 23% nucleotide mismatches, respectively. The vast majority of these mismatches (about 87%) occur in third base codon positions that do not cause amino acid changes; as a result the three mu 1 proteins are very highly related (about 97%). The mu 1 proteins are acidic proteins, low in cysteine, histidine, and methionine, and rich in proline; and they possess a rather low predicted alpha-helix content of 27%. The site where protein mu 1 is cleaved to mu 1C is predicted to be between residues 42 and 43 in a highly conserved portion of the molecule. The three M2 genome segments are related significantly more closely than the three S3 genome segments, and much more closely than the three S1 genome segments. We have analyzed the evolutionary divergence patterns of these three genome segments. The rate of mismatch accumulation in third base codon positions is roughly the same for all three genome segments, but the rates of mismatch accumulations in first, and particularly in second, base codon positions are quite different. For the S1 genome segments there is little difference between the rates of mismatch accumulations in all three codon positions, which indicates that retention of function is compatible with very extensive structural flexibility. By contrast, the rates of mismatch accumulations in first and second base codon positions are far less for the S3 genome segments, which encode the nonstructural protein sigma NS, and much lower still for the M2 genome segments, which suggests the existence of very stringent structural constraints for protein mu 1C, which might be expected of a protein that is a capsomer component.

Comparison of the reovirus serotype 1, 2, and 3 S3 genome segments encoding the nonstructural protein σNS

The sequences of the S3 genome segments of reovirus serotype 1 and 2 prototype strains are presented and these sequences are compared with the sequence of the serotype 3 S3 genome segment. The S3 genome segment encodes the nonstructural protein sigma NS which possesses affinity for ssRNA and appears to function in reovirus morphogenesis. The three S3 genome segments are closely related: all are 1198 nucleotides long and possess a single long open reading frame 366 codons long. They exhibit a serotype 1:3 relatedness pattern: there are only 13% mismatches between the S3 genome segments of serotypes 1 and 3, but 27 and 26% mismatches, respectively, between those of serotype 1 and 2 and serotype 3 and 2. The amino acid mismatches for the three sigma NS proteins are much lower (2.7, 13.9, and 13.7%, respectively), because the majority of nucleotide mismatches are in third base codon positions. The three sigma NS proteins possess a conserved secondary structure that is rich in alpha-helix content; in fact, the predicted alpha-helix content of these nonstructural proteins (about 50%) is much higher than that of the three other sigma size class proteins (about 20%), all of which are structural proteins. We also sequenced the S3 genome segment of a ts mutant of serotype 3 generated by treatment with nitrous acid and found a single nucleotide change that specifies an amino acid change that introduces a five-residue-long beta-sheet prone configuration into a long (80 amino acids) highly conserved alpha-helix in the C-terminal half of sigma NS. This change could account for this mutants ts character. Finally, the three sigma NS proteins have diverged in only about 10% of positions, whereas the three sigma 1 proteins have diverged in about 70%. The rapid evolutionary divergence of the latter may be a result of several factors, including (i) the fact that sigma 1, but not sigma NS, is under immunologic selective pressure; (ii) the fact that the functions of sigma 1 (antigenicity and cell attachment) probably reside in two rather small domains that are not restricted spatially with respect to each other; and (iii) the fact that the functions of sigma NS, namely RNA binding and protein binding (during morphogenesis), require a highly specific overall protein configuration that may permit little variation.

Expression of cell regulatory proteins in ovarian borderline tumors

Tumors of borderline malignancy are still a controversial subgroup of ovarian neoplasms. The expression of several cell regulatory proteins was studied to characterize the molecular phenotype of these tumors, and to compare them with their benign and malignant counterparts.

A LINEAR REGION OF A MONOCLONAL ANTIBODY CONFORMATIONAL EPITOPE MAPPED ON P185HER2 ONCOPROTEIN

Analysis of epitopes recognized by therapeutic monoclonal antibodies (mAb) is critical in clinical applications and in structure/function studies of target antigen. mAb MGr6 recognizes the extracellular domain of the p185HER2 oncoprotein and is a promising candidate for cancer immunodiagnosis and immunotherapy. Thus, epitope location and structure on p185HER2 need to be investigated. The use of MGr6-selected phage-displayed peptides for epitope analysis served to dissect the MGr6 epitope into at least two subregions, mimicked by CHSDC- and (L)P-(L)K(L) phage displayed peptides, respectively. Comparison of amino acid sequences of CHSDC peptides with the p185HER2 protein sequence and analysis of MGr6 reactivity with p185HER2 deletion mutants identified the linear subregion CCHEQCAAG of the MGr6 epitope, corresponding to amino acids 235-243 of the p185HER2 protein. This continuous subregion is part of a larger conformational epitope, and other amino acids, including a proline, a lysine and leucine residues contained in (L)P-(L)K(L) phage-displayed peptides appear to contribute to the formation of the MGr6 epitope surface.