Francesc Mitjans

In vivo therapy of malignant melanoma by means of antagonists of αv integrins

Integrin αvβ3 (vitronectin receptor) has been implicated in human malignant melanoma progression and angiogenesis as a receptor that provides survival signals. However, little is known about the therapeutic potential of antagonists of αvβ3. In this report, we characterize the activities of 2 antagonists of αvβ3 integrins: a human specific monoclonal antibody (MAb), 17E6, and a cyclic RGD peptide that blocked cell adhesion and induced detachment of previously substrate‐attached cells in vitro. In vivo, αvβ3 antagonists behaved as anti‐tumor drugs in a dose‐ and time‐dependent manner. Moreover, different therapeutic treatments proved to be effective even in the therapy of established macroscopic tumor masses, thus supporting the use of these antagonists in clinical therapy. Using a panel of 6 human melanomas and 5 carcinomas, MAb 17E6 efficiently blocked the in vivo tumor growth of melanomas expressing αvβ3 as xenografts but did not affect the αvβ3‐negative (although αv integrin‐positive) tumors. This demonstrated that αvβ3 is a pivotal integrin for the growth of human melanomas. Furthermore, since MAb 17E6 does not recognize murine αvβ3, the effect is due only to the direct anti‐tumor activity and not to the well‐known anti‐angiogenic activity of αv‐integrin antagonists. Taken together, our results confirm the essential role of αvβ3 integrin in the growth of human malignant melanoma in vivo and provide strong evidence of the therapeutic potential of αv‐integrin antagonists for the treatment of such tumors. Int. J. Cancer 87:716–723, 2000.

RGD Peptides and monoclonal antibodies, antagonists of αv-Integrin, enter the cells by independent endocytic pathways

Cyclic synthetic peptides containing the arginine-glycine-aspartate motif (cRGD) and monoclonal antibodies (mAbs) targeted for individual integrins have been developed as potential therapeutic drugs for the treatment of several diseases. We showed that a cRGD peptide targeted for αvβ3 was internalized in αv-integrin expressing and nonexpressing melanoma cells by an integrin independent fluid-phase endocytosis pathway that does not alter the number of functional integrin receptors at the cell surface. In contrast, a blocking mAb directed to αv was internalized by an integrin-dependent endocytosis pathway that reduced the number of functional integrin receptors at the cell surface. We prove that melanoma cells pretreated with the mAb do not readhere to the substrate, whereas cells pretreated with cRGD peptide retain their readhesion capacity. Given the growing importance of RGD peptides, knowledge of these cellular mechanisms is required to improve the development of antiangiogenic and anti-inflammatory drugs.

Use of siRNAs and Antisense Oligonucleotides Against Survivin RNA to Inhibit Steps Leading to Tumor Angiogenesis

The antiapoptotic protein survivin is an attractive target in cancer therapy because it is expressed differently in tumors and normal tissues and it is potentially required for cancer cells to remain viable. Given that survivin is also overexpressed in endothelial cells (ECs) of newly formed blood vessels found in tumors, its RNA targeting might compromise EC viability and interfere with tumor angiogenesis. We used two antisense strategies against survivin expression, antisense oligonucleotides (aODN) and small interfering RNA (siRNA), to study in ECs the contribution of survivin in various steps leading to tumor angiogenesis. A 21-mer phosphorothioate aODN and two siRNA oligonucleotides against survivin mRNA were designed to downregulate survivin expression. Survivin targeting caused (1) a strong growth-inhibitory effect, (2) a 4-fold increase in apoptosis, (3) an accumulation of cells in the S phase and a decrease in G2/M phase, (4) a dose-dependent inhibition of EC migration on Vitronectin, and (5) a decrease in capillary formation. Control oligonucleotides, an unrelated oligonucleotide, and one with four mismatches, had no significant effect. All these results show that survivin is a suitable target in cancer therapy because its inhibition in EC causes both a proapoptotic effect and an interruption of tumor angiogenesis. The two strategies used, classic aODN and siRNA technology, were very effective. Moreover, the latter can be used in the low nanomolar range, thus increasing the sensitivity of the treatment.

Therapeutic Targeting of Tumor Growth and Angiogenesis with a Novel Anti-S100A4 Monoclonal Antibody

S100A4, a member of the S100 calcium-binding protein family secreted by tumor and stromal cells, supports tumorigenesis by stimulating angiogenesis. We demonstrated that S100A4 synergizes with vascular endothelial growth factor (VEGF), via the RAGE receptor, in promoting endothelial cell migration by increasing KDR expression and MMP-9 activity. In vivo overexpression of S100A4 led to a significant increase in tumor growth and vascularization in a human melanoma xenograft M21 model. Conversely, when silencing S100A4 by shRNA technology, a dramatic decrease in tumor development of the pancreatic MiaPACA-2 cell line was observed. Based on these results we developed 5C3, a neutralizing monoclonal antibody against S100A4. This antibody abolished endothelial cell migration, tumor growth and angiogenesis in immunodeficient mouse xenograft models of MiaPACA-2 and M21-S100A4 cells. It is concluded that extracellular S100A4 inhibition is an attractive approach for the treatment of human cancer.

Alpha v integrin antagonists induce the disassembly of focal contacts in melanoma cells

In recent years, several antagonists of alpha(v)beta3 have been used to develop therapeutic approaches to the treatment of melanoma neoplasia. We studied the effects of anti-alpha(v)-integrin-blocking antibodies on attached M21 melanoma cells, the cellular distribution of alpha(v)-integrin and the molecular organization of focal structures. Anti-alpha(v)-integrin-blocking antibodies 17E6 and LM609, and an anti-alpha(v)beta3-integrin antagonist peptide cRGD 85189 induced detachment of M21 melanoma cells cultured for 24 hours on various substrates. cRGD was the most effective antagonist, reducing the number of adherent cells by 80%, while 17E6 reduced adhesion by only 30%. Light- and electron microscopy revealed attached cells with a flat shape and well-formed actin cytoskeleton. After treatment, cells became rounded and detached from the culture dish. alpha(v)-Integrins and focal-contact proteins were observed at adhesion sites in focal structures by immunocytochemistry. After treatment, however, cell rounding was accompanied by disorganization of the actin filaments and redistribution of alpha(v)-integrins and most of the focal proteins studied, except vinculin and tensin. Our results indicate that treatment of M21 melanoma cells with a(v)-integrin antagonists disrupts the actin cytoskeleton, redistributes a(v)-integrin and induces molecular disassembly of focal contacts.

Anti-migratory and anti-angiogenic effect of p16: A novel localization at membrane ruffles and lamellipodia in endothelial cells

Recent evidence has established different functions for the tumor suppressor protein, p16INK4A aside from controlling the cell cycle. Here we report that cdk4/6 inhibition blocked both human umbilical vein endothelial cells (HUVEC) spreading on a vitronectin matrix and HUVEC migration on vitronectin. p16 can also act as an anti-angiogenic molecule in vitro since HUVEC and HMEC cells transfected with Ad-p16 or treated with Antennapedia p16 peptides are unable to differentiate on a Matrigel matrix. Both, p16, cyclin D1, cdk4 and cdk6 were immuno-colocalized with Ezrin, Rac, Vinculin, αv-integrin, and FAK proteins in the ruffles and lamellipodia of migratory cells. Our results indicate that p16 is a key component of a new cytoplasmic pathway controlling angiogenesis of endothelial cells via the αvβ3-integrin-mediated migration.

S100P antibody-mediated therapy as a new promising strategy for the treatment of pancreatic cancer

Despite progresses in diagnosis and treatment, pancreatic cancer continues to have the worst prognosis of all solid malignant tumors. Recent evidences suggest that the metastasis-promoting protein S100P stimulates pancreatic tumor proliferation, survival, invasion and metastasis progression through extracellular functions. Moreover, its expression is strongly correlated with poor prognosis in patients with several types of cancer although the entire molecular mechanism responsible for the diverse biological functions is not fully understood. We showed that extracellular S100P stimulates pancreatic carcinoma BxPC3 cell line by promoting cell proliferation. We also demonstrated that S100P induces, in this cell line, the phosphorylation of IκBα and the secretion of matrix metalloproteinase 9 (MMP-9). In addition, treatment with S100P protected cells from injuries induced by the cytotoxic agent Gemcitabine. On the basis of these results, we developed function-blocking anti-S100P monoclonal antibodies (mAbs) that abolished all of its in vitro activities. Furthermore, in vivo treatment with the candidate 2H8 antibody decreased tumor growth and liver metastasis formation in a subcutaneous and orthotopic BxPC3 tumor model. We conclude here that a therapeutic strategy blocking the extracellular activity of S100P by means of specific mAbs could be an attractive therapeutic approach as a single agent or in combination with target-directed or chemotherapeutic drugs to treat pancreatic cancer.

Synthesis and biological evaluation of novel bisindolylmaleimides that inhibit vascular endothelial cell proliferation.

A novel class of bisindolylmaleimides were synthesized and antiproliferative activities (HUVECs and three tumor cell lines) of these compounds were investigated. Two water-soluble derivatives, 10 and 12, possessing a dimethylaminoalkoxy side chain in their structure, showed interesting activity and selectivity on HUVECs proliferation.

Tackling lipophilicity of peptide drugs: replacement of the backbone N-methyl group of cilengitide by N-oligoethylene glycol (N-OEG) chains.

Cilengitide is an RGD-peptide of sequence cyclo[RGDfNMeV] that was was developed as a highly active and selective ligand for the αvβ3 and αvβ5 integrin receptors. We describe the synthesis of three analogues of this peptide in which the N-Me group has been replaced by N-oligoethylene glycol (N-OEG) chains of increasing size: namely N-OEG2, N-OEG11, and N-OEG23, which are respectively composed of 2, 11, and 23 ethylene oxide monomer units. The different N-OEG cyclopeptides and the original peptide were compared with respect to lipophilicity and biological activity. The N-OEG2 analogue was straightforward to synthesize in solid phase using an Fmoc-N-OEG2 building block. The syntheses of the N-OEG11 and N-OEG23 cyclopeptides are hampered by the increased steric hindrance of the N-substituent, and could only be achieved by segment coupling, which takes place with epimerization and thus requires extensive product purification. All the N-OEG analogues were found to be more hydrophobic than the parent peptide, and their hydrophobicity was systematically enhanced upon increasing the length of the OEG chain. The N-OEG2 cyclopeptide displayed the same capacity as Cilengitide to inhibit the integrin-mediated adhesion of HUVEC endothelial, DAOY gliobastoma, and HT-29 colon cancer cells to their ligands vitronectin and fibrinogen. The N-OEG11 and N-OEG23 analogues also inhibited cell adhesion to these immobilized ligands, but their IC50 values dropped by 1 order of magnitude with respect to the parent peptide. These results indicate that replacement of the backbone N-Me group of Cilengitide by a short N-OEG chain provides a more lipophilic analogue with a similar biological activity. Upon increasing the size of the N-OEG chain, liophilicity is enhanced, but synthetic yields drop and the longer polymer chains may impede targeted binding.

Amide-to-ester substitution allows fine-tuning of the cyclopeptide conformational ensemble.

Natural or designed peptide ligands rarely bind to cognate receptors in their most stable conformation when in solution. The receptor, through reciprocal induced fitting, applies pressure on the conformational ensemble of the peptide to select its complementary conformation. Given the flexible nature of the peptides’ modular architecture, a bioactive sequence must often be primed for recognition of its target to achieve high binding affinity or specificity. For rational peptide design, cyclization or structure-inducing residues have been successfully used to accomplish ligand preorganization. However, deconvolution of the averaged NMR spectra of strained peptides or other macrocycles had shown that the bound-state conformations are poorly populated in the free uncomplexed state. In small cyclic peptides, the stereochemistry of the backbone, rather than interactions with or among side chains, determines the conformational ensemble by establishing a defined pattern of local torsional preferences. Intramolecular H bonds act on the equilibrium distribution of the conformational ensemble and favor specific conformations. Thus, we envisioned amide-to-ester substitution or “Ester Scan” as an interesting modification for the peptide backbone, which influences the conformational ensemble as well as its equilibrium distribution; this influence is achieved through modulation of the backbone torsional preferences and H-bonding pattern, respectively. As a model we choose cilengitide (CIL), which is an ArgGly-Asp (RGD) peptide of sequence cyclo[RGDfNMeV] (see Scheme 1), with well-characterized biological and conformational properties. CIL displays nanomolar inhibition of vitronectin binding to the isolated avb3 and avb5 integrin receptors, and it blocks integrin-dependent adhesion of tumor and endothelial cells to immobilized extracellular matrix (ECM) proteins and reduces angiogenesis and tumor growth in vivo. Modulation of the internal H-bond pattern of nonmethylated CIL precursors—achieved by changing the flexibility or the chirality of the backbone—was found to influence the antagonist activity on the vitronectin (VN) and fibrinogen (FB) receptors, and has been proposed to control laminin P1 vs. vitronectin receptor specificity. We synthesized all five depsi-analogues of the depsipeptide CIL (D1–D5) by stepwise assembling of the linear precursors on 2-CTC resin (Fmoc/tBu strategy) and cyclization in solution. For the introduction of the a-hydroxy acid residues onto the growing peptide chain, their HFA-activated/protected derivatives were used (Scheme 1). For the acylation of the free hydroxy group, DIPDCI/DMAP-activation was used. For conventional peptide cyclization, the optimal site for macrocycle formation is between the Gly (acting as the C terminus) and Asp (acting as the N terminus) residues because Gly cannot epimerize. This strategy was used for the synthesis of parent CIL, D1, and D2 depsipeptides with PyBOP/HOAt. The macrolactonization required for the preparation of D3 was successful with MSNT activation and NMI as the base. For the depsipeptides D4 and D5, certain particularities of the ester bond had to be taken into account. Our attempts to synthesize the linear precursor of D4 (OGly analogue) starting from OGly as the C terminus, resulted in low yields, and was likely because of cleavage of the ester bond that was mediated by base during repeated treatment with piperidine. Therefore, NMeVal was chosen as the C terminus. For the preparation of D5, the cyclization was performed at the (apparently) less attractive position between (d)Phe and NMeVal. Macrolactamization at less hindered sites was impeded by intramolecular nucleophilic attack on the ester bond, which occurred during peptide chain elongation, and eliminated the (D)Phe–NMeVal couple as dioxopiperazine. Given the increased steric demand of the N-terminal NMeVal, we chose the more reactive PyAOP as the coupling reagent and HOAt as the additive. After cleavage from the solid support, all linear peptide and depsipeptide precursors were obtained in over 85% yield. Head-to-tail cyclization was performed in solution and gave good yields in all cases and, finally, the protecting groups on the side chains were removed using [Pd(PPh3)4]/phenylsilane [*] T. Cupido, Dr. J. Spengler, Dr. J. Ruiz-Rodriguez, Prof. F. Albericio Institute for Research in Biomedicine (IRB), Barcelona Science Park (PCB) and CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine PCB Baldiri Reixac 10, 08028 Barcelona (Spain) Fax: (+34)93-403-7126 E-mail: tommaso.cupido@irbbarcelona.org albericio@irbbarcelona.org