Macrophage inflammatory factors promote epithelial-mesenchymal transition in breast cancer.

The majority of breast cancers (90-95%) arise due to mediators distinct from inherited genetic mutations. One major mediator of breast cancer involves chronic inflammation. M1 macrophages are an integral component of chronic inflammation and the breast cancer tumor microenvironment (TME). Previous studies have demonstrated that up to 50% of the breast tumor comprise of tumor-associated macrophages (TAMs) and increased TAM infiltration has been associated with poor patient prognosis. Furthermore, breast cancer associated deaths are predominantly attributed to invasive cancers and metastasis with epithelial-mesenchymal transition (EMT) being implicated. In this study, we investigated the effects of cellular crosstalk between TAMs and breast cancer using an in vitro model system. M1 polarized THP-1 macrophage conditioned media (CM) was generated and used to evaluate cellular and functional changes of breast cancer lines T47D and MCF-7. We observed that T47D and MCF-7 exhibited a partial EMT phenotype in the presence of activated THP-1 CM. Additionally, MCF-7 displayed a significant increase in migratory and invasive properties. We conclude that M1 secretory factors can promote a partial EMT of epithelial-like breast cancer cells. The targeting of M1 macrophages or their secretory components may inhibit EMT and limit the invasive potential of breast cancer.

Synthetic Toll like receptor-4 (TLR-4) agonist peptides as a novel class of adjuvants.

BACKGROUND: Adjuvants serve as catalysts of the innate immune response by initiating a localized site of inflammation that is mitigated by the interactions between antigens and toll like receptor (TLR) proteins. Currently, the majority of vaccines are formulated with aluminum based adjuvants, which are associated with various side effects. In an effort to develop a new class of adjuvants, agonists of TLR proteins, such as bacterial products, would be natural candidates. Lipopolysaccharide (LPS), a major structural component of gram negative bacteria cell walls, induces the systemic inflammation observed in septic shock by interacting with TLR-4. The use of synthetic peptides of LPS or TLR-4 agonists, which mimic the interaction between TLR-4 and LPS, can potentially regulate cellular signal transduction pathways such that a localized inflammatory response is achieved similar to that generated by adjuvants. METHODOLOGY/PRINCIPAL FINDINGS: We report the identification and activity of several peptides isolated using phage display combinatorial peptide technology, which functionally mimicked LPS. The activity of the LPS-TLR-4 interaction was assessed by NF-κB nuclear translocation analyses in HEK-BLUE™-4 cells, a cell culture model that expresses only TLR-4, and the murine macrophage cell line, RAW264.7. Furthermore, the LPS peptide mimics were capable of inducing inflammatory cytokine secretion from RAW264.7 cells. Lastly, ELISA analysis of serum from vaccinated BALB/c mice revealed that the LPS peptide mimics act as a functional adjuvant. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate the identification of synthetic peptides that mimic LPS by interacting with TLR-4. This LPS mimotope-TLR-4 interaction will allow for the development and use of these peptides as a new class of adjuvants, namely TLR-4 agonists.

Endothelial progenitor cell biology in disease and tissue regeneration.

Endothelial progenitor cells are increasingly being studied in various diseases ranging from ischemia, diabetic retinopathy, and in cancer. The discovery that these cells can be mobilized from their bone marrow niche to sites of inflammation and tumor to induce neovasculogenesis has afforded a novel opportunity to understand the tissue microenvironment and specific cell-cell interactive pathways. This review provides a comprehensive up-to-date understanding of the physiological function and therapeutic utility of these cells. The emphasis is on the systemic factors that modulate their differentiation/mobilization and survival and presents the challenges of its potential therapeutic clinical utility as a diagnostic and prognostic reagent.

Identification of PSA peptide mimotopes using phage display peptide library.

Prostate cancer (PCa) is one of the most common types of cancer in men in the United States and is the second leading cause of cancer related death in men. Clinically, secreted prostate specific antigen (PSA) has gained recognition because of its proteolytic activity being directly linked to PCa cell proliferation leading to disease initiation and progression. Using phage display technology, we identified four distinct cyclical peptides. These peptides apart from differences in their amino acid sequence, elicited minimal cross reactive antibody responses against each other. One of the four peptides analyzed produced an antibody response that recognizes the PSA protein. We demonstrate that the synthetic PSA peptide mimics identified in our study are immunologically active and produce neutralizing activity and this has relevance and utility for prostate cancer disease progression.

Identification of peptide mimotopes of gp96 using single-chain antibody library.

Heat shock proteins such as gp96 are immunogenic and are widely used as vaccines in immunotherapy of cancers. The present study focuses on the use of peptide mimotopes as immunotherapeutic vaccines for prostate cancer. To this end, we developed a 15-mer gp96 peptide mimotope specifically reactive to MAT-LyLu gp96-peptide complex using combinatorial single-chain antibody and peptide phage display library. The immunogenicity of the synthesized gp96 mimotope was analyzed initially in normal BALB/c mice in combination with various adjuvants such as complete Freund's adjuvant (CFA), aluminum salts (ALUM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and liposome, of which CFA served as a positive control. The antibody response was determined and found that the gp96 mimotope with ALUM showed a significant increase in antibody titer, followed by GM-CSF and liposomes. Further, the T cell (CD4(+) and CD8(+)) populations from splenocytes, as well as IgG isotypes, interleukin-4, and interleukin-5 of gp96 mimotope with ALUM-immunized animals, were analyzed. The results suggest that the gp96 mimotope may elicit a potent and effective antitumor antibody response. Further, the study identifies ALUM and GM-CSF as adjuvant options to drive an appropriate protective immune response as these adjuvants have prior use in humans.

Sialic acid content of tissue-specific gp96 and its potential role in modulating gp96-macrophage interactions.

Cancer-derived heat shock protein gp96 induces a tumor-specific protective immune response primarily mediated by cytotoxic T lymphocytes (CTL) directed toward cancer-associated peptides associated with gp96. Both innate and adaptive immune responses have been demonstrated using a cell culture-based signaling mechanism. When used as an extraneous vaccine, one critical interaction which must occur for an immune response to be generated is the interaction between gp96 and the antigen presenting cell (APC) surface receptors (CD91, SR-A, TLR-2, and TLR-4). Our previous study concluded that gp96 purified from various rat and human prostate cancers is differentially glycosylated based on the amino and neutral monosaccharide content, and it was postulated that the monosaccharides may play a role in its biological activity. In this report, we report differences in the cancer-specific sialic acid content of gp96 purified from normal rat prostate compared to two rat prostate cancers, MAT-LyLu and Dunning G, as well as between two human prostate cancer cells, LnCaP and DU145. We also examined the modulatory effect of sialic acid residues on the binding of gp96 to APCs and its subsequent activation. Our results supported the contention that significant differences in the sialic acid content exist between Dunning G, MAT-LyLu, and normal rat prostate gp96, which affected its binding and biochemical activity to APCs. We therefore postulate that varied glycans of HPS96, a hitherto neglected structural component, may play a pivotal role in its anticancer activity. We suggest that construction of the glycan tree is a key to identification of the necessary and sufficient elements in the structure-function activity of HSP96.

Targeting the immune system in cancer.

The concept of cancer immunotherapy provides a fresh perspective as it is not associated with many of the drawbacks of conventional therapies such as chemotherapy, radiotherapy and surgery. When fully activated the immune system has immense potential as is evident from mis-matched transplanted organs undergoing rapid immunological attack and rejection. However, the development of immune strategies for cancer therapy has been associated with challenges of their own. Early attempts at cancer vaccination were carried out in an empirical manner that did not always lead to reproducibility. This led to a search of tumor associated antigens with the belief that specific targeting of these antigens would lead to successful tumor elimination. Active vaccination with TAA peptides or passive vaccination with specific lymphocytes against these TAAs did not however demonstrate encouraging results in clinical trials. This was mainly because of the lack of an activating immune response which is required for continuous stimulation of lymphocytes and also because of the selection of tumor escape variants that did not express the particular TAA. On the positive side, attempts at characterizing TAAs illuminated the molecular changes that attribute a malignant phenotype to cancer cells. Attempts at cytokine therapy were also met with challenges of high systemic toxicity and a lack of specific lymphocyte activation. It was therefore realized that an ideal vaccinating agent should be able to combine the effects of both these therapeutic strategies, i.e., it should be able to induce an innate immune response which can be tailored to a tumor specific adaptive immune response. By this, the immunosuppressive tumor environment can be altered to become immune activating, thus facilitating the infiltration of myeloid and lymphoid cells that can act in concert leading to tumor regression. In this regard, immunotherapeutic approaches such as DNA vaccines, dendritic cell based vaccines, HSP based vaccines and gene transfer technology, are being developed and further refined to overcome their inherent limitations. Animal experiments with these therapeutic modalities have demonstrated exciting results, although their evaluation in clinical trials has not indicated exceptional tumor protection in a large percentage of the patients. These observations only further underscore the multivariate and dynamic nature of the immune system and the many ways in which tumor cells modulate themselves and their surroundings to escape immune surveillance. Assessment of successful therapeutic intervention will require periodic evaluations of the suppressive nature of the tumor microenvironment accompanied by qualitative and quantitative measurements of lymphocyte responses in patients. With the development of advanced genetic technologies and continuous identification of tumor antigens, the field of cancer immunotherapy is progressing at an exciting pace giving us hope for the advent of effective treatment modalities that will prolong tumor free survival and enhance the quality of life in patients with malignant disease.

Degenerate binding of tyrosinase peptides to MHC II Ad/Ed molecules.

Recently we defined the tyrosinase233-247IPYWDWRDAEKCDIC peptide as the pentadecamer sequence hosting the linear determinant of the anti-tyrosinase MAb T311. In order to understand the molecular features that render epitopic the amino acid tyrosinase233-247 sequence, we 1) analyzed the MHC II affinity of the tyrosinase233-247IPYWDWRDAEKCDIC peptide using RankPep as a binding prediction program, and 2) experimentally verified the actual peptide-MHC II interaction by carrying out peptide electrophoretic mobility shift assays. Nine random tyrosinase peptides were used as controls. Under the conditions used in this study, it was found that all of the ten tyrosinase peptides were capable of binding to Ad/Ed molecules in the gel-shift binding assay. Moreover, there was no correlation between the theoretical predicted Ad/Ed affinity and the experimental actual peptide binding. We conclude that Ad/Ed binding potential appears influential in the defining of tyrosinase233-247IPYWDWRDAEKCDIC peptide epitopicity.

Non-redundant peptidomes from DAPs: towards "the vaccine"?

Experimental analyses and literature survey reveal low-redundancy to the host proteins as a common denominator of immunogenic sequences mapped along tumor-, autoimmune-, and infectious disease-associated-proteins. The hypothesis that immunogenicity of peptide sequences is linked to proteomic redundancy is discussed.

Preliminary data on Pemphigus vulgaris treatment by a proteomics-defined peptide: a case report.

BACKGROUND: Although described by Hippocrates in 400 B.C., pemphigus disease still needs a safe therapeutical approach, given that the currently used therapies (i.e. corticosteroids and immunosuppressive drugs) often provoke collateral effects. Here we present preliminary data on the possible use of a proteomics derived desmoglein peptide which appears promising in halting disease progression without adverse effects. METHODS: The low-similarity Dsg349-60REWVKFAKPCRE peptide was topically applied for 1 wk onto a lesion in a patient with a late-stage Pemphigus vulgaris (PV) complicated by diabetes and cataract disease. The peptide was applied as an adjuvant in combination with the standard corticosteroid-based immunosuppressive treatment. RESULTS: After 1 wk, the treated PV eroded lesion appeared dimensionally reduced and with an increased rate of re-epithelization when compared to adjacent non-treated lesions. Short-term benefits were: decrease of anti-Dsg antibody titer and reduction of the corticosteroid dosage. Long-term benefits: after two years following the unique 1-wk topical treatment, the decrease of anti-Dsg antibody titer persists. The patient is still at the low cortisone dosage. Adverse effects: no adverse effect could be monitored. CONCLUSION: With the limits inherent to any preliminary study, this case report indicates that topical treatment with Dsg349-60REWVKFAKPCRE peptide may represent a feasible first step in the search for a simple, effective and safe treatment of PV.

Proteomic definition of a desmoglein linear determinant common to Pemphigus vulgaris and Pemphigus foliaceous.

BACKGROUND: A number of autoimmune diseases have been clinically and pathologically characterized. In contrast, target antigens have been identified only in a few cases and, in these few cases, the knowledge of the exact epitopic antigenic sequence is still lacking. Thus the major objective of current work in the autoimmunity field is the identification of the epitopic sequences that are related to autoimmune reactions. Our labs propose that autoantigen peptide epitopes able to evoke humoral (auto)immune response are defined by the sequence similarity to the host proteome. The underlying scientific rationale is that antigen peptides acquire immunoreactivity in the context of their proteomic similarity level. Sequences uniquely owned by a protein will have high potential to evoke an immune reaction, whereas motifs with high proteomic redundancy should be immunogenically silenced by the tolerance phenomenon. The relationship between sequence redundancy and peptide immunoreactivity has been successfully validated in a number of experimental models. Here the hypothesis has been applied to pemphigus diseases and the corresponding desmoglein autoantigens. METHODS: Desmoglein 3 sequence similarity analysis to the human proteome followed by dot-blot/NMR immunoassays were carried out to identify and validate possible epitopic sequences. RESULTS: Computational analysis led to identifying a linear immunodominant desmoglein-3 epitope highly reactive with the sera from Pemphigus vulgaris as well as Pemphigus foliaceous. The epitopic peptide corresponded to the amino acid REWVKFAKPCRE sequence, was located in the extreme N-terminal region (residues 49 to 60), and had low redundancy to the human proteome. Sequence alignment showed that human desmoglein 1 and 3 share the REW-KFAK-RE sequence as a common motif with 75% residue identity. CONCLUSION: This study 1) validates sequence redundancy to autoproteome as a main factor in shaping desmoglein peptide immunogenicity; 2) offers a molecular mechanicistic basis in analyzing the commonality of autoimmune responses exhibited by the two forms of pemphigus; 3) indicates possible peptide-immunotherapeutical approaches for pemphigus diseases.

Synthetic dimer of indole-3-carbinol: second generation diet derived anti-cancer agent in hormone sensitive prostate cancer.

BACKGROUND: Cruciferous vegetables have been found to have anti-prostate cancer effects. The active compounds mediating these effects include indoles such as indole-3-carbinol (I3C) and isothiocyanates. I3C is unstable having tissue tropic effects and clinical utility has been partly addressed by the synthesis of a more stable dimer diindolylmethane (DIM). METHODS: Anti-proliferative activity was measured by XTT assay and cytosolic proteins quantitated by Western blot analysis. RESULTS: DIM (IC(50) 50 microM) is a better anti-proliferative agent than I3C (IC(50) 150 microM) in androgen dependent LNCaP cells, inhibits DNA synthesis, and growth of R1881 stimulated LNCaP cells. Androgen receptor (AR), cyclin D1, and cdk4, induced by R1881, are downregulated by DIM. DIM downregulates phosphorylated Akt and phosphatidyl inositol 3-kinase and downstream inhibition of cyclin D1 and cdk4. CONCLUSION: These studies provide evidence that DIM is a second-generation chemopreventive agent with a viable cellular target and has clinical potential as an anti-prostate cancer chemopreventive.

3,3'-Diindolylmethane downregulates pro-survival pathway in hormone independent prostate cancer.

Epidemiological evidences suggest that the progression and promotion of prostate cancer (CaP) can be modulated by diet. Since all men die with prostate cancer rather than of the disease, it is of particular interest to prevent or delay the progression of the disease by chemopreventive strategies. We have been studying the anticancer properties of compounds present in cruciferous vegetables such as indole-3-carbinol (I3C). Diindolylmethane (DIM) is a dimer of I3C that is formed under acidic conditions and unlike I3C is more stable with higher anti-cancer effects. In the present report, we demonstrate that DIM is a potent anti-proliferative agent compared to I3C in the hormone independent DU 145 CaP cells. The anti-prostate cancer effect is mediated by the inhibition of the Akt signal transduction pathway as DIM, in sharp contrast to I3C, induces the downregulation of Akt, p-Akt, and PI3 kinase. DIM also induced a G1 arrest in DU 145 cells by flow cytometry and downstream concurrent inhibition of cell cycle parameters such as cyclin D1, cdk4, and cdk6. Our data suggest a need for further development of DIM, as a chemopreventive agent for CaP, which justifies epidemiological evidences and molecular targets that are determinants for CaP dissemination/progression. The ingestion of DIM may benefit CaP patients and reduce disease recurrence by eliminating micro-metastases that may be present in patients who undergo radical prostatectomy.

Proteomic scan for tyrosinase peptide antigenic pattern in vitiligo and melanoma: role of sequence similarity and HLA-DR1 affinity.

Immune responses contribute to the pathogenesis of vitiligo and target melanoma sometimes associated with vitiligo-like depigmentation in some melanoma patients. We analyzed the sera from patients with vitiligo and cutaneous melanoma for reactivity toward tyrosinase peptide sequences 1) endowed with low level of similarity to human proteome, and 2) potentially able to bind HLA-DR1 Ags. We report that the tyrosinase autoantigen was immunorecognized with the same molecular pattern by sera from vitiligo and melanoma patients. Five autoantigen peptides composed the immunodominant anti-tyrosinase response: aa95-104FMGFNCGNCK; aa175-182 LFVWMHYY; aa176-190FVWMHYYVSMDALLG; aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC. All of the five antigenic peptides were characterized by being (or containing) a sequence with low similarity level to the self proteome. Sera from healthy subjects were responsive to aa95-104FMGFNCGNCK, aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC, but did not react with the aa175-182LFVWMHYY and aa176-190FVWMHYYVSMDALLG peptide sequences containing the copper-binding His180 and the oculocutaneous albinism I-A variant position F176. Our results indicate a clear-cut link between peptide immunogenicity and low similarity level of the corresponding amino acid sequence, and are an example of a comparative analysis that might allow to comprehensively distinguish the epitopic peptide sequences within a disease from those associated to natural autoantibodies. In particular, these data, for the first time, delineate the linear B epitope pattern on tyrosinase autoantigen and provide definitive evidence of humoral immune responses against tyrosinase.

Differences in glycosylation patterns of heat shock protein, gp96: implications for prostate cancer prevention.

Heat shock protein gp96 induces a tumor-specific protective immunity in a variety of experimental tumor models. Because the primary sequences of the glycoprotein, gp96 are identical between tumor and normal tissues, the peptides associated with gp96 and/or the posttranslational modifications of gp96, determine its immunogenicity. Gp96-associated peptides constitute the antigenic repertoire of the source tissue; thus, purified gp96-peptide complexes have clinical significance as autologous cancer vaccines. However, the role of altered glycosylation and its contribution in the biological as well as immunologic activity of gp96 still remains uncharacterized. We examined the cancer-specific glycosylation patterns of gp96. To this end, monosaccharide compositions of gp96 were compared between normal rat prostate and two cancerous rat prostate tissues, nonmetastatic/androgen-dependent Dunning G and metastatic/androgen-independent MAT-LyLu, as well as two human nonmetastatic prostate cancer cell lines, androgen-dependent LnCaP and androgen-independent DU145. Marked differences were observed between the gp96 monosaccharide compositions of the normal and cancerous tissues. Furthermore, gp96 molecules from more aggressive cellular transformations were found to carry decreasing quantities of several monosaccharides as well as sum total content of neutral and amino sugars. We believe that the unique glycosylation patterns contribute to cellular phenotype and that the posttranslational modifications of gp96 may affect its functional attributes.

Definition of anti-tyrosinase MAb T311 linear determinant by proteome-based similarity analysis.

Using non-self discrimination as a driving force in generating peptide immunogenicity, we have developed a computer-assisted proteomic analysis in order to identify the protein antigenic regions that have evoked humoral response. The purpose of this study was to further validate the computational analysis for melanoma-associated antigens and, at the same time, to assess the efficacy of the methodology in defining antigenic regions of autoantigens associated to autoimmune diseases. To achieve this two-fold objective, we have examined the enzyme tyrosinase, a protein that represents an important autoantigen in patients with vitiligo or melanoma. Here, we report that the antigenic linear determinant of the monoclonal antibody (Mab) T311 raised against the melanoma/vitiligo tyrosinase autoantigen is located in the low similarity 15-mer amino acid sequence tyrosinase 233-247 IPYWDWRDAEKCDIC, within the fragment 237-247. These data confirm non-similarity to the host proteome as a factor that participates in shaping peptide immune reactivity and may be a first step towards designing tyrosinase antigenic peptides to be used for (i) direct neutralization of harmful melanocytes-attacking autoantibodies in vitiligo, or (ii) production of antibodies against tyrosinase-positive melanomas. Moreover tyrosinase peptide antigens might be used as key tools in studying the boundaries between self-tolerance and autoimmunity phenomena.

Gene therapy in cancer: the missing point.

Over the last century cancer research has produced data leading to a composite picture where gene mutations and epigenetic phenomena strictly relate and overlap. This complexity has repercussions on the anti-cancer therapeutical strategies. The therapeutic pathway paved by the kochian one-cause/one disease principle fails in front of a multigenic multiphenomena disease like cancer. We still do not know what target(s) to hit/modify in order to prevent/stop the carcinogenic progression. On the light of cancer statistics 2005, we discuss the need of exactly defining the cancer targets in order to exploit the high potential of gene therapy.

Characterization of polyclonal antibodies raised against a linear peptide determinant of desmoglein-3.

Our labs are pursueing the goal of exactly defining the qualities of peptide antigenicity, immunogenicity and pathogenicity in order to develop safe specific immunotherapies by utilizing specific portions of disease-associated-proteins (DAPs). Using as a model the Pemphigus vulgaris antigen (PVA) desmoglein 3 (Dsg3), we have studied the murine humoral response against the Dsg3 amino acid 49-60 peptide sequence, previously characterized as sequence having low similarity to the mouse proteome. The results show that the low-similarity Dsg3(49-60)REWVKFAKPCRE peptide does not elicit pathogenic antibodies.

Aging-associated changes in gene expression in the ACI rat prostate: Implications for carcinogenesis.

BACKGROUND: Prostate cancer is the most frequently diagnosed neoplasm and the second leading cause of cancer-related mortality in men. Although the incidence of prostate cancer increases with age, the link between aging and prostate cancer is poorly understood. METHODS: Affymetrix oligonucleotide microarrays were used to analyze the mRNA expression levels in the dorsolateral prostates from 6- and 18-month-old ACI rats. Real-time RT-PCR and immunohistochemistry was performed to validate microarray data in a select set of genes. RESULTS: Microarray analysis revealed changes in gene expression associated with inflammation, oxidative stress, tissue remodeling, and energy metabolism. Most of these changes have been related to increased proliferative status of the prostate, anti-apoptosis, activated stroma, and alteration of the energy metabolism. CONCLUSIONS: Age-associated alterations in the gene expression profile may put the aging prostate in risk for the initiation, promotion, and progression of neoplastic transformation in both our animal model and humans.