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PDF file, 2456K, Proliferation curves of HUVEC as generated by xCELLigence RTCA seeding 2x103 cells/well during a period of 72 hours, in the presence of 8% FBS, or 40ng/ml VEGF plus or minus 10 muM ERFR, 10nM RERF or 10 muM RERF. Data are the mean+SD of one experiment, performed in quadruplicate.

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer–mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy. Mol Cancer Ther; 13(3); 752–62. ©2013 AACR.

PDF file - 470K, Figure S1. UN1 mAb did not affect the proliferation rate, cell cycle, viability and apoptosis of HPB-ALL cells. Figure S2. The 2/165 phage clone and synthetic peptides do not inhibit the binding of mAb W6/32 to HPB-ALL cells. Supplementary Figure S3. Competition of the UN1 mAb-binding to UN1-positive breast and gastric cancer tissues by the 2/165 phage and G23 peptide. Figure S4. Characterization of affinity-purified antibodies from sera of 2/165 phagotopeimmunized mice.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration, and uPAR88–92 is the minimal sequence required to induce cell motility. We previously showed that soluble forms of uPAR elicit angiogenic responses through their uPAR88–92 chemotactic sequence and that the synthetic peptide SRSRY exerts similar effects. By a drug design approach, based on the conformational analysis of the uPAR88–92 sequence, we developed peptides (pERERY, RERY, and RERF) that potently inhibit signaling triggered by uPAR88–92. In this study, we present evidence that these peptides are endowed also with a clear-cut antiangiogenic activity, although to different extents. The most active, RERF, prevents tube formation by human endothelial cells exposed to SRSRY. RERF also inhibits VEGF-triggered endothelial cell migration and cord-like formation in a dose-dependent manner, starting in the femtomolar range. RERF prevents F-actin polymerization, recruitment of αvβ3 integrin at focal adhesions, and αvβ3/VEGFR2 complex formation in endothelial cells exposed to VEGF. At molecular level, the inhibitory effect of RERF on VEGF signaling is shown by the decreased amount of phospho-FAK and phospho-Akt in VEGF-treated cells. In vivo, RERF prevents VEGF-dependent capillary sprouts originating from the host vessels that invaded angioreactors implanted in mice and neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Consistently, RERF reduced the growth and vascularization rate of tumors formed by HT1080 cells injected subcutaneously in the flanks of nude mice, indicating that RERF is a promising therapeutic agent for the control of diseases fuelled by excessive angiogenesis such as cancer. Mol Cancer Ther; 12(10); 1981–93. ©2013 AACR.

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Video Description from Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

Supplementary Data to Figure 3 from Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

RERF Conformational Preferenc from Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

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Dattabase Information from Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

Supplementary Figure S2 from Haploinsufficiency of the Hmga1 Gene Causes Cardiac Hypertrophy and Myelo-Lymphoproliferative Disorders in Mice

Supplementary Figure 1 from Deletion of Yin Yang 1 Protein in Osteosarcoma Cells on Cell Invasion and CXCR4/Angiogenesis and Metastasis

The HMGA1 protein is a major factor in chromatin architecture and gene control. It plays a critical role in neoplastic transformation. In fact, blockage of HMGA1 synthesis prevents rat thyroid cell transformation by murine transforming retroviruses, and an adenovirus carrying the HMGA1 gene in the antisense orientation induces apoptotic cell death in anaplastic human thyroid carcinoma cell lines, but not in normal thyroid cells. Moreover, both in vitro and in vivo studies have established the oncogenic role of the HMGA1 gene. In this study, to define HMGA1 function in vivo, we examined the consequences of disrupting the Hmga1 gene in mice. Both heterozygous and homozygous mice for the Hmga1-null allele show cardiac hypertrophy due to the direct role of HMGA1 on cardiomyocytic cell growth regulation. These mice also developed hematologic malignancies, including B cell lymphoma and myeloid granuloerythroblastic leukemia. The B cell expansion and the increased expression of the RAG1/2 endonuclease, observed in HMGA1-knockout spleen tissues, might be responsible for the high rate of abnormal IgH rearrangements observed in these neoplasias. Therefore, the data reported here indicate the critical role of HMGA1 in heart development and growth, and reveal an unsuspected antioncogenic potential for this gene in hematologic malignancies. (Cancer Res 2006; 66(5): 2536-43)

We know that the Yin Yang 1 protein (YY1) overexpression is positively and strongly correlated with the degree of malignancy of bone tumors. Therefore, we questioned whether we could influence cell invasiveness by deleting YY1 in human osteosarcoma cells (SaOs-2), as tested in Matrigel-coated filters and metastasis implantation of such osteosarcoma cells in vivo, by serial analysis with nuclear magnetic resonance. Moreover, we focused our work on the chemokine receptor CXCR4 and its inhibition by T22 antibody, as well as on systemic (direct in vivo assay) and computer-assisted imaging of angiogenesis-related metastasis. Results showed that cell invasiveness and metastasis implantation by wild-type SaOs-2 cells, as evaluated by histology and immunohistochemistry, are associated with up-regulation of CXCR4 expression, which in turn was significantly reduced by T22. In addition, deletion of YY1 (siRNAYY1-SaOs-2) induced a significant decrease of cell invasion and metastasis growth. This phenomenon was associated with decreased vascular endothelial growth factor (VEGF)/angiogenesis and a complex rearrangement of the gene expression profile as evaluated by microarray analysis. In conclusion, YY1 and VEGF/CXCR4 seem to intervene in the pathogenesis of the malignant phenotype of osteosarcoma by acting on cell invasiveness and metastasis growth. [Cancer Res 2008;68(6):1797–808]

Supplementary Table from Identification of the Genes Up- and Down-Regulated by the High Mobility Group A1 (HMGA1) Proteins

Supplementary Tables 1-2 from Deletion of Yin Yang 1 Protein in Osteosarcoma Cells on Cell Invasion and CXCR4/Angiogenesis and Metastasis

Materials and Methods, Suppl. References, Table S1, and Legends to Figures S1 & S2.

High mobility group A (HMGA) proteins are chromatinic proteins that do not have transcriptional activity per se, however, by interacting with the transcription machinery, they regulate, negatively or positively, the expression of several genes. We searched for genes regulated by HMGA1 proteins using microarray analysis in embryonic stem (ES) cells bearing one or two disrupted hmga1 alleles. We identified 87 transcripts increased and 163 transcripts decreased of at least 4-fold in hmga1−/− ES cells. For some of them, a HMGA1-dose dependency was observed, because an intermediate level was observed in the heterozygous ES cells. When the expression analysis of these genes was extended to embryonic fibroblasts and adult tissues such as heart, spleen, and liver from hmga1-knockout mice, contrasting results were obtained. In fact, aside some genes showing the same HMGA1 regulation observed in ES cells, there were some genes that did not modify their expression, and others showing a HMGA1-mediated regulation but in an opposite direction. These results clearly indicate that HMGA1-mediated gene regulation depends on the cellular context. Finally for a couple of analyzed HMGA1-regulated genes, electrophoretic mobility shift assay and chromatin immunoprecipitation revealed a direct binding of HMGA1 proteins to their promoters, suggesting a HMGA1-direct regulation of their expression.

Supplementary Figure S1 from Haploinsufficiency of the Hmga1 Gene Causes Cardiac Hypertrophy and Myelo-Lymphoproliferative Disorders in Mice

Non-small cell lung cancer (NSCLC), the leading cause of cancer death worldwide, is still an unmet medical problem due to the lack of both effective therapies against advanced stages and markers to allow a diagnosis of the disease at early stages before its progression. Immunotherapy targeting the PD-1/PD-L1 checkpoint is promising for many cancers, including NSCLC, but its success depends on the tumor expression of PD-L1. PATZ1 is an emerging cancer-related transcriptional regulator and diagnostic/prognostic biomarker in different malignant tumors, but its role in lung cancer is still obscure. Here we investigated expression and role of PATZ1 in NSCLC, in correlation with NSCLC subtypes and PD-L1 expression. A cohort of 104 NSCLCs, including lung squamous cell carcinomas (LUSCs) and adenocarcinomas (LUADs), was retrospectively analyzed by immunohistochemistry for the expression of PATZ1 and PD-L1. The results were correlated with each other and with the clinical characteristics, showing on the one hand a positive correlation between the high expression of PATZ1 and the LUSC subtype and, on the other hand, a negative correlation between PATZ1 and PD-L1, validated at the mRNA level in independent NSCLC datasets. Consistently, two NSCLC cell lines transfected with a PATZ1-overexpressing plasmid showed PD-L1 downregulation, suggesting a role for PATZ1 in the negative regulation of PD-L1. We also showed that PATZ1 overexpression inhibits NSCLC cell proliferation, migration, and invasion, and that Patz1-knockout mice develop LUAD. Overall, this suggests that PATZ1 may act as a tumor suppressor in NSCLC.

Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we discovered that APAF-1 is negatively regulated by MITF in resistant tumors. This study identifies the MITF/APAF-1 axis as a molecular driver of MAPK inhibitor resistance. A drug-repositioning screen identified quinacrine and methylbenzethonium as potent activators of apoptosis in a context that mimics drug resistance mediated by APAF-1 inactivation. The compounds showed anti-tumor activity in invitro and invivo models, linked to suppression of MITF function. Both drugs profoundly sensitize melanoma cells to MAPK inhibitors, regulating key signaling networks in melanoma, including the MITF/APAF-1 axis. Significant activity of the two compounds in inhibiting specific epigenetic modulators of MITF/APAF-1 expression, such as histone deacetylases, was observed. In summary, we demonstrate that targeting the MITF/APAF-1 axis may overcome resistance and could be exploited as a potential therapeutic approach to treat resistant melanoma.