Sequence-specific peptide aptamers,interacting with the intracellular domain of the epidermal growth factor receptor,interfere with Stat3 activation and inhibit the growth of tumor cells

Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1.EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.     

In vitro selection of specific RNA aptamers for the NFAT DNA binding domain

Nuclear factor of activated T cells (NFAT) plays a central role in the immune response, and the immuno-suppressive drugs, cyclosporin A and FK-506, have been developed to inhibit it. However, due to the toxic effects of these drugs, which derive from their ability to inhibit calcineurin in non-immune tissues, the identification of small compounds that target NFAT directly could be an approach to developing less toxic immunosuppressive therapy. Using an in vitro selection technology termed SELEX on a combinatorial RNA library with 40 nucleotide-long random sequences, we have isolated two RNA aptamers to the NFAT DNA binding domain (DBD). Gel retardation assays and surface plasmon resonance measurements showed that the aptamers have a specific and high affinity (apparent KD~10 to 100 nM) for the NFAT DBD. Enzymatic probing analysis showed that the two RNA aptamers have similar structures and share a sequence that forms an apical loop. Moreover, RNase footprinting analysis showed that the shared sequence (GATATGAAGGA/ TGTG/AGAGAG) is critical for binding to both NFATp DBD and NFATc DBD. These results suggest that short RNAs identified in this study is a specific aptamer to NFAT DBD, and hence could be applied not only for the delineation of NFAT functions but for the development of potent immune modulating lead compounds.     

Characterization of the dimerization domain in the FNR transcription factor

The global anaerobic regulator FNR from Escherichia coli is a dimeric Fe-S protein that is inactivated by O(2) through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activity in vivo. Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization. Because hydrophobic side chains are known to stabilize subunit-subunit interactions between alpha-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an alpha-helix that constitutes a dimerization interface. This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face. In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face. These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O(2) availability.     

A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.     

Alphavbeta3 integrin blocking inhibits apoptosis and induces autophagy in murine breast tumor cells

Integrins are cell receptors that mediate adhesion to the extracellular matrix (ECM) and regulate cell migration, a crucial process in tumor invasion. The α_vβ₃ integrin recognizes the arginine-glycine-aspartic acid (RGD) motif in ECM proteins and it can be antagonized by RGD-peptides, resulting in decreased cell migration and invasion. RGD-based drugs have shown disappointing results in clinical trials; however, the reasons for their lack of activity are still obscure. Aiming to contribute to a better understanding of the molecular consequences of integrin inhibition, we tested a recombinant RGD-disintegrin (DisBa-01) in two types of murine cell lines, breast tumor 4T1BM2 cells and L929 fibroblasts. Only tumor cells showed decreased motility and adhesion, as well as morphologic alterations upon DisBa-01 treatment (100 and 1000 nM). This result was attributed to the higher levels of α_vβ₃ integrin in 4T1BM2 cells compared to L929 fibroblasts making the former more sensitive to DisBa-01 blocking. DisBa-01 induced cell cycle arrest at the S phase in 4T1BM2 cells, but it did not induce apoptosis, which was consistent with the decrease in caspase-3, 8 and 9 expression at mRNA and protein levels. DisBa-01 increases PI3K, Beclin-1 and LC3B expression in tumor cells, indicators of autophagic induction. In conclusion, α_vβ₃ integrin blocking by DisBa-01 results in inhibition of adhesion and migration and in the activation of an autophagy program, allowing prolonged survival and avoiding immediate apoptotic death. These observations suggest new insights into the effects of RGD-based inhibitors considering their importance in drug development for human health.     

A specific cell-penetrating peptide induces apoptosis in SKOV3 cells by down-regulation of Bcl-2

Peptides are emerging as pharmaceutical agents in cancer therapy. The peptide, TLSGAFELSRDK (TLS) is a targeting ligand that can specifically triggers cellular uptake by binding to SKOV3 cells. Cell surface proteins and the C-terminal basic residues of the TLS are required for effective cell penetration, and the uptake process is energy-dependent. It inhibited the proliferation of SKOV3 cells and induced early-stage apoptosis by down-regulation of Bcl-2 expression mediated through a caspase-dependent pathway. The synergistic anti-proliferative effects of the peptide TLS and doxorubicin on SKOV3 cells were further investigated. Taken together, TLS, acting as a combination of a targeted ligand and a therapeutic agent, was a promising candidate for the development of peptide-based therapies in ovarian cancer.     

A novel platinum compound inhibits constitutive Stat3 signaling and induces cell cycle arrest and apoptosis of malignant cells

Previous studies have established constitutive activation of Stat3 protein as one of the molecular changes required for tumorigenesis. To develop novel therapeutics for tumors harboring constitutively active Stat3, compounds from the NCI 2000 diversity set were evaluated for inhibition of Stat3 DNA-binding activity in vitro. Of these, a novel platinum (IV) compound, IS3 295, interacted with Stat3 and inhibited its binding to specific DNA-response elements. Further analysis suggested noncompetitive-type kinetics for the inhibition of Stat3 binding to DNA. In human and mouse tumor cell lines with constitutively active Stat3, IS3 295 selectively attenuated Stat3 signaling, thereby inducing cell growth arrest at G0/G1 phase and apoptosis. Moreover, in transformed cells, IS3 295 repressed expression of cyclin D1 and bcl-xL, two of the known Stat3-regulated genes that are overexpressed in malignant cells, suggesting that IS3 295 mediates anti-tumor cell activity in part by blocking Stat3-mediated sub-version of cell growth and apoptotic signals. Together, our findings provide evidence for the inhibition of Stat3 activity and biological functions by IS3 295 through interaction with Stat3 protein. This study represents a significant advance in small molecule-based approaches to target Stat3 and suggests potential new applications for platinum (IV) complexes as modulators of the Stat3 pathway for cancer therapy.