Ligand-directed surface profiling of human cancer cells with combinatorial peptide libraries

A collection of 60 cell lines derived from human tumors (NCI-60) has been widely explored as a tool for anticancer drug discovery. Here, we profiled the cell surface of the NCI-60 by high-throughput screening of a phage-displayed random peptide library and classified the cell lines according to the binding selectivity of 26,031 recovered tripeptide motifs. By analyzing selected cell-homing peptide motifs and their NCI-60 recognition patterns, we established that some of these motifs (a) are similar to domains of human proteins known as ligands for tumor cell receptors and (b) segregate among the NCI-60 in a pattern correlating with expression profiles of the corresponding receptors. We biochemically validated some of the motifs as mimic peptides of native ligands for the epidermal growth factor receptor. Our results indicate that ligand-directed profiling of tumor cell lines can select functional peptides from combinatorial libraries based on the expression of tumor cell surface molecules, which in turn could be exploited as "druggable" receptors in specific types of cancer.     

A novel DNA methyltransferase I-derived peptide eluted from soluble HLA-A*0201 induces peptide-specific, tumor-directed cytotoxic T cells

MHC peptides derived from tumor-associated antigens (TAAs) can serve as the basis for the development of immunotherapeutics to treat human malignancies. Previously, we identified novel HLA-A*0201 (HLA-A2)-restricted peptides recovered from soluble HLA molecules secreted by human tumor cell lines, transfected with truncated genes of HLA-A2 and HLA-B7. Here, 4 candidate peptides eluted from soluble HLA-A2 were selected on the basis of their precursor proteins being TAAs. Peptide p1028 (GLIEKNIEL), derived from DNA methyltransferase I (DNMT-1), which is overexpressed in various human tumors, showed the highest affinity to HLA-A2 and was relatively abundant in the sMHC/peptide complexes of all transfected breast, ovarian and prostate cancer cell lines. Peptide p1028-specific CTLs were generated in vitro and shown to efficiently lyse not only target cells pulsed with the peptide but also HLA-A2-positive breast cancer cell lines MDA-231 and MCF-7. The peptide induced IFN-gamma production in CTLs, which were selectively stained by a p1028 tetramer. Since DNMT-1 is a widely expressed tumor-associated enzyme, the novel DNMT-1-derived, HLA-A2-restricted peptide GLIEKNIEL identified here may provide a suitable candidate for a therapeutic cancer vaccine.     

Breast cancer growth inhibition by delivery of the MDGI-derived peptide P108

Mammary derived growth inhibitor (MDGI) is a member of the family of cytoplasmic fatty acid binding proteins (FABPs), which bind hydrophobic ligands such as fatty acids, retinoids, eicosanoids and prostaglandines. MDGI and an 11 amino acid MDGI-derived conserved C-terminal peptide (P108) inhibits growth of normal mammary epithelial cells in tissue and organ culture, but fails to inhibit proliferation of many breast cancer cell lines in vitro. Here, the effects of peptide P108 on tumor growth of MCF-7, MDA-MB468 and MDA-MB231 human breast cancer cell lines in nude mice were tested. To deliver P108 into tumors, a novel peptide production system was applied for expression and secretion of small bioactive peptides in mammalian cells. Functional differentiation was observed in MCF-7 and MDA-MB468 cells upon P108 expression. In addition, EGF-dependent colony formation in soft agar by MDA-MB468 cells was inhibited by secreted P108. Tumor growth in athymic nude mice was suppressed in all three cell lines tested. Furthermore, P108 expressed by MCF-7/P108 cells caused paracrine tumor growth inhibition of MDA-MB231 cells. These results indicate that breast cancer inhibition by P108 is independent of binding to hydrophobic ligands and is perhaps mediated by interference with EGF-dependent signaling pathways.     

Analysis of <Emphasis Type="Italic">CUL-5</Emphasis> expression in breast epithelial cells,breast cancer cell lines,normal tissues and tumor tissues

Background  

The chromosomal location of CUL-5 (11q 22-23) is associated with LOH in breast cancer, suggesting that CUL-5 may be a tumor suppressor. The purpose of this research was to determine if there is differential expression of CUL-5 in breast epithelial cells versus breast cancer cell lines, and normal human tissues versus human tumors. The expression of CUL-5 in breast epithelial cells (HMEC, MCF-10A), and breast cancer cells (MCF-7, MDA-MB-231) was examined using RT-PCR, Northern blot analysis, and Western blot analysis. The expression of mRNA for other CUL family members (CUL-1, -2, -3, -4A, and -4B) in these cells was evaluated by RT-PCR. A normal human tissue expression array and a cancer profiling array were used to examine CUL-5 expression in normal human tissues and matched normal tissues versus tumor tissues, respectively.     

Inhibition of proliferative and invasive capacities of breast cancer cells by arginine-glycine-aspartic acid peptide in vitro

The Arg-Gly-Asp (RGD) sequence was selected by using phage-display peptides to target tumors, focusing on targeting alpha(v) integrins in tumor blood vessels. Recent studies suggest that peptides containing the RGD sequence can bind to tumor cells, as well as tumor endothelial cells. To investigate whether the RGD peptide has other effects on tumor cells expressing alpha(v) integrins, besides its tumor targeting capability, we designed and synthesized a 10-amino peptide that contained the RGD sequence in a cyclic conformation with a disulfide bond, which specifically bound to breast cancer cell lines MDA-MB-231 and MCF-7. We found that this RGD peptide, GCGGRGDGGC, inhibited tumor cell proliferation in a dose-dependent manner, and also induced apoptosis and G1-phase cell cycle arrest in both of the cell lines that bound and internalized the peptide. Normal ovarian epithelial cells, which did not bind the RGD peptide, were unaffected. RGD peptide treatment also reduced cell invasiveness in both cell lines in vitro. This study suggests that the RGD peptide not only possesses tumor targeting capacity, but also has direct tumor cytotoxic and invasiveness inhibition effects dependent on the blockage of alpha(v) integrin activity, which would make it more efficient in tumor targeting therapy.     

The Molecular Heterogeneity of Nonspecific Cross-reacting Antigen Synthesized by Tumor Cells and Granulocytes

The molecular heterogeneity of nonspecific cross-reacting antigen (NCA) was examined. Metabolically-labeled glycoproteins were precipitated from cell lysates of human tumor cell lines and of normal peripheral granulocytes with antibodies specific for NCA, and analyzed by SDS-PAGE. NCA components synthesized by three tumor cell lines, QGP-1 (pancreas), HLC-1 (lung) and CAOV-2 (ovary) showed slightly different migration patterns on SDS-PAGE, but the molecular weights of their unglycosylated peptides synthesized in the presence of tunicamycin were all found to be 35K. On the other hand, two molecular species of NCA were identified in normal granulocytes: an 80K mature form derived from a 69K precursor peptide and a 58K mature form from a 41K precursor peptide. Upon SDS-PAGE, the migration pattern of the unglycosylated NCA peptides from tumor cells was affected by the presence of 2-mercaptoethanol, while that of the peptides of granulocytes was not. All the NCAs identified in this study possessed antigenic determinants common to carcinoembryonic antigen as well as those unique to NCA. These results suggest that the molecular heterogeneity of NCA observed thus far resulted from diverse glycosylation of the three fundamental molecular forms of unglycosylated peptides: one with a molecular weight of 35K produced by tumor cells and two with molecular weights of 69K and 41K produced by granulocytes.     

The molecular heterogeneity of nonspecific cross-reacting antigen synthesized by tumor cells and granulocytes

The molecular heterogeneity of nonspecific cross-reacting antigen (NCA) was examined. Metabolically-labeled glycoproteins were precipitated from cell lysates of human tumor cell lines and of normal peripheral granulocytes with antibodies specific for NCA, and analyzed by SDS-PAGE. NCA components synthesized by three tumor cell lines, QGP-1 (pancreas), HLC-1 (lung) and CAOV-2 (ovary) showed slightly different migration patterns on SDS-PAGE, but the molecular weights of their unglycosylated peptides synthesized in the presence of tunicamycin were all found to be 35K. On the other hand, two molecular species of NCA were identified in normal granulocytes: an 80K mature form derived from a 69K precursor peptide and a 58K mature form from a 41K precursor peptide. Upon SDS-PAGE, the migration pattern of the unglycosylated NCA peptides from tumor cells was affected by the presence of 2-mercaptoethanol, while that of the peptides of granulocytes was not. All the NCAs identified in this study possessed antigenic determinants common to carcinoembryonic antigen as well as those unique to NCA. These results suggest that the molecular heterogeneity of NCA observed thus far resulted from diverse glycosylation of the three fundamental molecular forms of unglycosylated peptides: one with a molecular weight of 35K produced by tumor cells and two with molecular weights of 69K and 41K produced by granulocytes.     

Peptides Derived from Type IV Collagen,CXC Chemokines,and Thrombospondin-1 Domain-Containing Proteins Inhibit Neovascularization and Suppress Tumor Growth in MDA-MB-231 Breast Cancer Xenografts

Angiogenesis or neovascularization, the process of new blood vessel formation from preexisting microvasculature, involves interactions among several cell types including parenchymal, endothelial cells, and immune cells. The formation of new vessels is tightly regulated by a balance between endogenous proangiogenic and antiangiogenic factors to maintain homeostasis in tissue; tumor progression and metastasis in breast cancer have been shown to be angiogenesis-dependent. We previously introduced a systematic methodology to identify putative endogenous antiangiogenic peptides and validated these predictions in vitro in human umbilical vein endothelial cell proliferation and migration assays. These peptides are derived from several protein families including type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins. On the basis of the results from the in vitro screening, we have evaluated the ability of one peptide selected from each family named pentastatin-1, chemokinostatin-1, and properdistatin, respectively, to suppress angiogenesis in an MDA-MB-231 human breast cancer orthotopic xenograft model in severe combined immunodeficient mice. Peptides were administered intraperitoneally once per day. We have demonstrated significant suppression of tumor growth in vivo and subsequent reductions in microvascular density, indicating the potential of these peptides as therapeutic agents for breast cancer.     

Expression of WNT14 and WNT14B mRNAs in human cancer, up-regulation of WNT14 by IFNgamma and up-regulation of WNT14B by beta-estradiol.

WNT proteins play key roles in carcinogenesis. We have previously cloned and characterized WNT14 and WNT14B/WNT15. WNT14 and WNT3A genes are clustered on human chromosome 1q42, while WNT14B and WNT3 genes are clustered on human chromosome 17q21. Here, we investigated expression of WNT14 and WNT14B mRNAs in human cancer. WNT14 was significantly up-regulated in 1 out of 9 cases of primary breast cancer. WNT14B was not expressed in primary breast, gastric and colorectal cancers. Among 3 human breast cancer cell lines, WNT14 mRNA was expressed in T-47D cells, and weakly expressed in MCF-7 cells. WNT14 mRNA was also detected in 7 out of 7 pancreatic cancer cell lines, 12 out of 12 esophageal cancer cell lines, 4 out of 4 cervical cancer cell lines, and 5 out of 7 brain tumor cell lines by using cDNA-PCR. These results indicate that WNT14 rather than WNT14B is preferentially expressed in various types of human cancer, such as breast cancer, gastric cancer, and pancreatic cancer. WNT14 mRNA was up-regulated by interferon gamma (IFNgamma), but not by tumor necrosis factor alpha (TNFalpha), in MKN45 cells derived from gastric cancer, while expression of WNT14B mRNA was not affected by IFNgamma and TNFalpha in MKN45 cells. Although expression of WNT14 mRNA was not affected by beta-estradiol in MCF-7 cells, WNT14B mRNA was transiently up-regulated by beta-estradiol in MCF-7 cells. These results indicate that WNT14 is a target gene of IFNgamma in MKN45 cells, and that WNT14B is a target gene of estrogen in MCF-7 cells.     

Generation of cytotoxic T lymphocytes against native and altered peptides of human leukocyte antigen-A*0201 restricted epitopes from the human epithelial cell adhesion molecule.

A growing number of human tumor antigens have been described that can be recognized by CTLs in a MHC class I restricted fashion. The epithelial cell adhesion molecule (Ep-CAM) is expressed in a variety of human tumors and has attracted attention as a therapeutic target for monoclonal antibody serotherapy. We have identified immunogenic peptides derived from Ep-CAM, that bind to human leukocyte antigen-A*0201 and elicit strong peptide-specific human CTL responses, demonstrating that there is an effective T-cell repertoire against these Ep-CAM-derived peptides that can be recruited. Alterations to these peptides were made to increase their binding affinity to MHC class I molecules. The use of such "heteroclitic" peptides allowed generation of cytotoxic T cells that demonstrated increased killing of target cells pulsed not only with the heteroclitic but also with the native peptide. Most important, CTL cell lines that are generated against these peptides specifically lyse epithelial tumor cells expressing Ep-CAM but not normal hematopoietic or bronchial epithelial cells.     

Demonstration of adiponectin receptors 1 and 2 mRNA expression in human breast cancer cells

Recently, we have shown that low adiponectin levels are significantly associated with an increased breast cancer risk. It seems to be very important to study the expression of adiponectin receptor 1 (AdipoR1) and receptor 2 (AdipoR2) in the human breast epithelial cells and breast cancer cells in order to clarify whether or not adiponectin exerts its effects directly on these cells. Expression of adiponectin, AdipoR1, and AdipoR2 mRNA was determined by RT-PCR assay using the RNA samples obtained from human breast cancer cell lines (MCF-7, T47D, SKBR3, and MDA-MB231), HMEC (primary culture of normal human mammary epithelial cells), adipose tissues (axilla) as well as breast cancer cells and normal breast epithelial cells selectively collected from breast cancer tissues by laser microdissection (LMD). Adiponectin mRNA expression was observed only in the adipose tissues. On the other hand, AdipoR1 and AdipoR2 mRNA expression was observed in all four breast cancer cell lines, HMEC, adipose tissues as well as breast cancer cells and normal breast epithelial cells selectively collected by LMD. In addition, AdipoR1 and AdipoR2 expression in both normal breast epithelial cells and breast cancer cells was confirmed by immunohistochemistry. These results suggest a possibility that adiponectin might modulate the growth of normal breast epithelial cells and breast cancer cells directly through AdipoR1 and AdipoR2 receptors, and that the association of low serum adiponectin levels with a high breast cancer risk might be explained, at least in part, by the direct effect of adiponectin on the breast epithelial cells.     

Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells

A gradual loss of functional gap junction between tumor cells has been reported with colorectal cancer (CRC) progression. Here, we explored if colon cancer cells could also affect gap junctions in blood capillary cells. Human microvascular endothelial cells (HMEC) were cultured with two CRC cell lines established from a unique patient. SW480 cells, derived from the primary tumor, migrate much faster across HMEC monolayer than SW620 cells derived from a metastatic site. The motile SW480 cells highly express and release HSP27 that increases gap junction formation with HMEC. Soluble HSP27 phosphorylates the connexin Cx43 on serine residues and induces its interaction with the oncoprotein 14-3-3, which promotes Cx43 delivery at the plasma membrane. The factors secreted by less motile SW620 cells do not affect Cx43 expression but up-regulate the expression of the connexin Cx32 through an activation of the chemokine receptor CXCR2. In turn, SW620 secreted factors induce tubulogenesis and ATP release. Altogether, cell lines derived from CRC primary tumor and metastasis differentially adapt endothelial cell functions by modulating connexin expression through released mediators.     

Preferential induction of necrosis in human breast cancer cells by a p53 peptide derived from the MDM2 binding site

p53 is the most frequently altered gene in human cancer and therefore represents an ideal target for cancer therapy. Several amino terminal p53-derived synthetic peptides were tested for their antiproliferative effects on breast cancer cell lines MDA-MB-468 (mutant p53), MCF-7 (overexpressed wild-type p53), and MDA-MB-157 (null p53). p53(15)Ant peptide representing the majority of the mouse double minute clone 2 binding site on p53 (amino acids 12-26) fused to the Drosophila carrier protein Antennapedia was the most effective. p53(15)Ant peptide induced rapid, nonapoptotic cell death resembling necrosis in all breast cancer cells; however, minimal cytotoxicity was observed in the nonmalignant breast epithelial cells MCF-10-2A and MCF-10F. Bioinformatic/biophysical analysis utilizing hydrophobic moment and secondary structure predictions as well as circular dichroism spectroscopy revealed an alpha-helical hydrophobic peptide structure with membrane disruptive potential. Based on these findings, p53(15)Ant peptide may be a novel peptide cancer therapeutic because it induces necrotic cell death and not apoptosis, which is uncommon in traditional cancer therapy.     

CD47 agonist peptide PKHB1 induces immunogenic cell death in T‐cell acute lymphoblastic leukemia cells

T‐cell acute lymphoblastic leukemia (T‐ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin‐1‐derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first‐described serum‐stable CD47‐agonist peptide) on CEM and MOLT‐4 human cell lines (T‐ALL) and on one T‐murine tumor lymphoblast cell‐line (L5178Y‐R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non‐tumor cells after CD47 activation. In vivo, we determined that PKHB1‐treatment in mice bearing the L5178Y‐R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage‐associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high‐mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase‐independent and calcium‐dependent cell death in leukemic cells while sparing non‐tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.     

Two peptides derived from ras-p21 induce either phenotypic reversion or tumor cell necrosis of ras-transformed human cancer cells

PURPOSE: We investigated the effects of two peptides from the ras-p21 protein, corresponding to residues 35-47 (PNC-7) and 96-110 (PNC-2), on two ras-transformed human cancer cell lines, HT1080 fibrosarcoma and MIAPaCa-2 pancreatic cancer cell lines. In prior studies, we found that both peptides block oncogenic, but not insulin-activated wild-type, ras-p21-induced oocyte maturation. When linked to a transporter penetratin peptide, these peptides induce reversion of ras-transformed rat pancreatic cancer cells (TUC-3) to the untransformed phenotype. METHODS: These peptides and a control peptide, linked to a penetratin peptide, were incubated with each cell lines. Cell counts were obtained over several weeks. The cause of cell death was determined by measuring caspase as an indicator of apoptosis and lactate dehydrogenase (LDH) as marker of necrosis. Since both peptides block the phosphorylation of jun-N-terminal kinase (JNK) in oocytes, we blotted cell lysates of the two cancer cell lines for the levels of phosphorylated JNK to determine if the peptides reduced these levels. RESULTS: We find that both peptides, but not control peptides linked to the penetratin sequence, induce phenotypic reversion of the HT-1080 cell line but cause tumor cell necrosis of the MIA-PaCa-2 cell line. On the other hand, neither peptide has any effect on the viability of an untransformed pancreatic acinar cell line, BMRPA1. We find that, while total JNK levels remain constant during peptide treatment, phosphorylated JNK levels decrease dramatically, consistent with the mechanisms of action of these peptides. CONCLUSION: We conclude that these peptides block tumor but not normal cell growth likely by blocking oncogenic ras-p21-induced phosphorylation of JNK, an essential step on the oncogenic ras-p21-protein pathway. These peptides are therefore promising as possible anti-tumor agents.     

Tropomyosins of human mammary epithelial cells: consistent defects of expression in mammary carcinoma cell lines

Suppression of synthesis of specific tropomyosin (TM) isoforms occurs commonly in human, murine, and avian fibroblasts transformed by retroviral oncogenes or other modalities. The resulting deficiency or altered distribution of TMs may predispose the cells to microfilament instability and contribute to expression of the transformed phenotype. In this study we have asked whether defects in TM expression had relevance to human neoplasia, which arises most often in cells of the epithelial lineage rather than in fibroblasts and often is unrelated to demonstrable expression of oncogenes. TMs were characterized in normal primary human mammary epithelial cells (HMEC) and in an immortalized nontumorigenic cell line derived from them. Seven TM isoforms were identified in primary HMEC, two of which may be unique to epithelial cells. Immortalized nontumorigenic HMEC expressed the same array of isoforms. Of six established human breast carcinoma cell lines studied, all failed to express the Mr 39,000 TM isoform and five of six also lacked expression of either the Mr 38,000 or 35,000 isoform. Northern blot analysis with probes specific for the 1.1-kilobase mRNA of fibroblast TM1 detected a mRNA of this size in normal HMEC. This mRNA, which probably encodes the Mr 39,000 TM missing from all the carcinoma lines, was absent from five of the six breast cancer cell lines. These results indicate that abnormalities in TM expression in neoplastic cells are not limited to fibroblasts. The high frequency and consistent nature of such abnormalities among cell lines derived from human breast cancer raises the possibility that such abnormalities in expression of a major cytoskeletal protein may play a role in human neoplasia.     

Selective Cytotoxicity of a Red Grape Wine Flavonoid Fraction Against MCF-7 Cells

Red wine is a rich source of polyphenolic components such as anthocyanins and flavonoids. The inhibitory effects of red wine polyphenolics on human breast cancer cells have been demonstrated earlier, but their effects on normal cells have not been fully established. Red wine (Merlot) was fractionated by hydrophobic interaction chromatography and different flavonoid fractions with increasing hydrophobicity were obtained. These fractions were tested for their inhibitory effect on human breast cancer cells (MCF-7), normal human mammary epithelial cells (HMEC), and a non-tumorigenic MCF-10A cell line. By contrast to the authentic flavonoids such as quercetin, naringenin and catechin which inhibited the growth of HMEC much more than that of MCF-7 cancer cells, a red wine fraction, that was comprised mainly of the flavonoid aglycones, showed maximal inhibition of the growth of breast cancer cells, with relatively low cytotoxicity towards HMEC and MCF-10A cells. In the presence of this flavonoid fraction, the normal cells grew normally, whereas the breast cancer cells underwent a change in morphology into spherical forms. Cytotoxicity analyses suggested that these cells had become apoptotic. The efficiency of inhibition of cell proliferation by various flavonoid fractions appeared to be related to their inhibition of calcium and calmodulin-promoted phosphodiesterase activity, suggesting that flavonoids may interfere with calcium second messenger function. The results suggest that certain grape wine ingredients have anticancer properties and these ingredients may be helpful for developing designer functional foods with cancer-preventive properties.