Henry C. Krutzsch

Proteomic analysis and identification of new biomarkers and therapeutic targets for invasive ovarian cancer

Epithelial ovarian cancer kills almost 16 000 women each year in part due to late stage of presentation and lack of reliable biomarkers for disease detection. CA‐125, the currently accepted serum marker, alone lacks the sensitivity for early stage diagnosis, as only 50% of early stage cases are detected with this marker. Although more early stage cases may be detected by lysophosphatidic acid, this marker is also elevated in other cancers. One major objective of the NCI‐FDA Tissue Proteomics Initiative has been to combine the technique of laser capture microdissection (LCM) of epithelial tumor cells in human tissue specimens with two‐dimensional gel electrophoresis (2‐D PAGE) to identify proteins that may serve as invasive ovarian cancer‐specific biomarkers for early detection and/or new therapeutic targets. We performed 2‐D PAGE on lysates from five microdissected ovarian tumors (three invasive ovarian cancers and two noninvasive, low malignant potential (LMP) ovarian tumors). We then compared silver stained 2‐D gels created from microdissected lysates with SYPRO‐Ruby stained 2‐D PAGE profiles of the patient‐matched undissected bulk tumor lysates from all five patients. Twenty‐three proteins were consistently differentially expressed between both the LMP and three invasive ovarian tumors in the limited study set. Thirteen were uniquely present in all three of the invasive ovarian cancer cases and absent or underexpressed in the two LMP cases. Ten were uniquely present in the LMP cases but absent or underexpressed in all invasive ovarian cancer cases. Credentialing and preliminary target validation of the mass spectrometry identified proteins cut from the Ruby‐red stained gels was performed by LCM coupled Western blot and reverse‐phase array technology in a study set of six cases (the aforementioned five cases used in the 2‐D PAGE profiling component of the study plus one additional LMP case). The analysis revealed that the 52 kDa FK506 binding protein, Rho G‐protein dissociation inhibitor (RhoGDI), and glyoxalase I are found to be uniquely overexpressed in invasive human ovarian cancer when compared to the LMP form of this cancer. The direct comparison of LCM generated proteomic profiles of invasive vs. LMP ovarian cancer may more directly generate important markers for early detection and/or therapeutic targets unique to the invasive phenotype.

Inhibition of Angiogenesis by Thrombospondin-1 Is Mediated by 2 Independent Regions Within the Type 1 Repeats

BACKGROUND Suppression of tumor growth by thrombospondin-1 (TSP-1) has been associated with its ability to inhibit neovascularization. The antiangiogenic activity of TSP-1, as defined by cornea pocket assays, was previously mapped to the amino-terminal portion of the protein within the procollagen region and the type 1 repeats. METHODS AND RESULTS We evaluated the specificity and efficacy of different regions of TSP-1 using recombinant fragments of the protein on chorioallantoic membrane (CAM) angiogenesis and endothelial cell proliferation assays. In both assays, fragments containing the second and third type 1 repeats but not the procollagen region inhibited angiogenesis and endothelial cell proliferation. To further define the sequences responsible for the angiostatic effect of TSP-1, we used synthetic peptides. The CAM assay defined 2 sequences that independently suppressed angiogenesis. The amino-terminal end of the type 1 repeats showed higher potency for inhibiting angiogenesis driven by basic fibroblast growth factor (FGF-2), whereas the second region equally blocked angiogenesis driven by either FGF-2 or vascular endothelial growth factor (VEGF). Modifications of the active peptides revealed the specific amino acids required for the inhibitory response. One sequence included the conserved tryptophan residues in the amino-terminal end of the second and third type 1 repeats, and the other involved the amino acids that follow the CSVTCG sequence in the carboxy-terminus of these repeats. Both inhibition in the CAM assay and inhibition of breast tumor xenograft growth in nude mice were independent of the TGF-beta-activating sequence located in the second type 1 repeat. CONCLUSIONS These results indicate that the type 1 repeats of TSP-1 contain 2 subdomains that may independently inhibit neovascularization. They also identify 2 independent pathways by which TSP-1 can block FGF-2 and VEGF angiogenic signals on endothelial cells.

Interactions of thrombospondins with α4β1 integrin and CD47 differentially modulate T cell behavior

Thrombospondin (TSP)-1 has been reported to modulate T cell behavior both positively and negatively. We found that these opposing responses arise from interactions of TSP1 with two different T cell receptors. The integrin α4β1 recognizes an LDVP sequence in the NH2-terminal domain of TSP1 and was required for stimulation of T cell adhesion, chemotaxis, and matrix metalloproteinase gene expression by TSP1. Recognition of TSP1 by T cells depended on the activation state of α4β1 integrin, and TSP1 inhibited interaction of activated α4β1 integrin on T cells with its counter receptor vascular cell adhesion molecule-1. The α4β1 integrin recognition site is conserved in TSP2. A recombinant piece of TSP2 containing this sequence replicated the α4β1 integrin–dependent activities of TSP1. The β1 integrin recognition sites in TSP1, however, were neither necessary nor sufficient for inhibition of T cell proliferation and T cell antigen receptor signaling by TSP1. A second TSP1 receptor, CD47, was not required for some stimulatory responses to TSP1 but played a significant role in its T cell antigen receptor antagonist and antiproliferative activities. Modulating the relative expression or function of these two TSP receptors could therefore alter the direction or magnitude of T cell responses to TSPs.

Cell surface binding of TIMP-2 and pro-MMP-2/TIMP-2 complex

Tissue inhibitor of metalloproteinases (TIMP‐2) is a low molecular weight proteinase inhibitor capable of inhibiting activated matrix metalloproteinases (MMPs). TIMP‐2 is found both free and in a 1:1 stoichiometric complex with the pro‐enzyme form of MMP‐2 (pro‐MMP‐2/TIMP‐2 complex). We have measured the binding of recombinant TIMP‐2 to intact HT‐1080 and MCF‐7 cells. HT‐1080 cells in suspension bound 125I‐labeled rTIMP‐2 with a K d of 2.5 nM and 30,000 sites/cell. Monolayers of MCF‐7 cells were similarly found to bind [125I]rTIMP‐2 with a K d of 1.6 nM and 25,000 sites/cell. Specific binding of MMP‐2 alone to HT‐1080 cells was not observed; however, pro‐MMP‐2/TIMP‐2 complex was capable of binding to the surface of HT‐1080 cells in a TIMP‐2‐dependent manner. Binding of rTIMP‐2 was not competed by the presence of TIMP‐1. These results suggest that rTIMP‐2 alone binds directly to the cell surface of HT‐1080 and MCF‐7 cell lines, and TIMP‐2 is capable of localizing MMP‐2 to the surface of HT‐1080 cells via interaction with a specific binding site.

Insulin-like growth factors stimulate chemotaxis in human melanoma cells

Insulin and insulin-like growth factors stimulate motility in the highly metastatic human melanoma cell line, A2058. Insulin-like growth factor-I (IGF-I) is the most potent with a maximal response at a concentration of 10 nM compared to the activities of insulin and insulin-like growth factor-II (IGF-II) which peak at 300-400 nM. Using checkerboard analysis, the responses to IGF-I and insulin are predominantly chemotactic, although insulin had a significant chemokinetic component. Pertussis toxin does not inhibit the response to any of these polypeptides. However, in previous studies, it was shown that the motile response to autocrine motility factor from these same A2058 cells was markedly inhibited by pertussis toxin. 125I-labelled IGF-I binds saturably and specifically to the A2058 cells. Scatchard analysis indicates a high binding affinity (Kd approximately 3 x 10(-10) M) and an estimated 5000 receptors/cell. These studies indicate that in addition to their mitogenic properties, certain growth factors may profoundly enhance metastasis of tumor cells by their ability to induce motility.

α4β1 Integrin Mediates Selective Endothelial Cell Responses to Thrombospondins 1 and 2 In Vitro and Modulates Angiogenesis In Vivo

Abstract— We examined the function of &agr;4&bgr;1 integrin in angiogenesis and in mediating endothelial cell responses to the angiogenesis modulators, thrombospondin-1 and thrombospondin-2. &agr;4&bgr;1 supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1, vascular cell adhesion molecule-1, or recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by &agr;4&bgr;1, whereas antagonism of fibroblast growth factor-2–stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an &agr;4&bgr;1-dependent manner. Soluble &agr;4&bgr;1 antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1–dependent and not observed in explants from thrombospondin-1−/− mice. Antagonizing &agr;4&bgr;1 may in part block proangiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the &agr;4&bgr;1 binding sequence stimulate angiogenesis in vivo. Therefore, &agr;4&bgr;1 is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis.

Biosynthesis of the 67 kDa high affinity laminin receptor

High affinity interactions between cells and laminin are mediated, at least in part, by the 67 kDa laminin receptor (67 LR). A 37 kDa nascent polypeptide (37 LRP), predicted by a full length cDNA clone and obtained by in vitro translation of hybrid-selected laminin receptor mRNA, has been immunologically identified in cancer cell extracts as the putative precursor of the 67 LR. In this study, we used affinity purified antibodies developed against cDNA-deduced 37 LRP synthetic peptides in pulse chase experiments and demonstrated a precursor-product relationship between the 37 LRP and the 67 LR. Immunoblot, pulse chase and immunofluorescence experiments showed that transient transfection of the full length 37 LRP cDNA clone induced a dramatic increase in the synthesis of the 37 LRP but not of the mature 67 LR. We propose that the 67 LR results from the association of two gene products: the 37 LRP and a polypeptide yet to be identified.

Recognition of the N-terminal Modules of Thrombospondin-1 and Thrombospondin-2 by α6β1 Integrin

In addition to its recognition by α3β1 and α4β1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for α6β1 integrin. α6β1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. α6β1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an α6β1-binding sequence in thrombospondin-1. This peptide inhibited α6β1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by α6β1, but not by α4β1. α6β1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of α6β1 integrin in microvascular endothelial cells by the anti-β1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of α4β1 and α6β1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.

Measurement of cell proliferation in microculture using Hoechst 33342 for the rapid semiautomated microfluorimetric determination of chromatin DNA.

We report the development and characterization of a semiautomated method for measurement of cell proliferation in microculture using Hoechst 33342, a non-toxic specific vital stain for DNA. In this assay, fluorescence resulting from interaction of cell chromatin DNA with Hoechst 33342 dye was measured by an instrument that automatically reads the fluorescence of each well of a 96-well microtiter plate within 1 min. Each cell line examined was shown to require different Hoechst 33342 concentrations and time of incubation with the dye to attain optimum fluorescence in the assay. In all cell lines, cell chromatin-enhanced Hoechst 33342 fluorescence was shown to be a linear function of the number of cells or cell nuclei per well when optimum assay conditions were employed. Because of this linear relation, equivalent cell doubling times were calculated from growth curves based on changes in cell counts or changes in Hoechst/DNA fluorescence and the fluorimetric assay was shown to be useful for the direct assay of the influence of growth factors on cell proliferation. The fluorimetric assay also provided a means for normalizing the incorporation of tritiated thymidine ( [3H] TdR) into DNA; normalized values of DPM per fluorescence unit closely paralleled values of percent 3H-labelled nuclei when DNA synthesis was studied as a function of the concentration of rat serum in the medium. In summary, the chromatin-enhanced Hoechst 33342 fluorimetric assay provides a rapid, simple, and reproducible means for estimating cell proliferation by direct measurement of changes in cell fluorescence or by measurement of changes in the normalized incorporation of thymidine into DNA.

Identification of an α3β1 Integrin Recognition Sequence in Thrombospondin-1

A synthetic peptide containing amino acid residues 190–201 of thrombospondin-1 (TSP1) promoted adhesion of MDA-MB-435 breast carcinoma cells when immobilized and inhibited adhesion of the same cells to TSP1 when added in solution. Adhesion to this peptide was enhanced by a β1 integrin-activating antibody, Mn2+, and insulin-like growth factor I and was inhibited by an α3β1 integrin function-blocking antibody. The soluble peptide inhibited adhesion of cells to the immobilized TSP1 peptide or spreading on intact TSP1 but at the same concentrations did not inhibit attachment or spreading on type IV collagen or fibronectin. Substitution of several residues in the TSP1 peptide with Ala residues abolished or diminished the inhibitory activity of the peptide in solution, but only substitution of Arg-198 completely inactivated the adhesive activity of the immobilized peptide. The essential residues for activity of the peptide as a soluble inhibitor are Asn-196, Val-197, and Arg-198, but flanking residues enhance the inhibitory activity of this core sequence, either by altering the conformation of the active sequence or by interacting with the integrin. This functional sequence is conserved in all known mammalian TSP1 sequences and in TSP1 from Xenopus laevis. The TSP1 peptide also inhibited adhesion of MDA-MB-435 cells to the laminin-1 peptide GD6, which contains a potential integrin-recognition sequence Asn-Leu-Arg and is derived from a similar position in a pentraxin module. Adhesion studies using recombinant TSP1 fragments also localized β1 integrin-dependent adhesion to residues 175–242 of this region, which contain the active sequence.