REPORT
CIGB-552: A new penetrating peptide with antitumor action mediated by the increased levels of the COMMD1 protein in cancer cell lines
CIGB-552: nuevo péptido penetrador con acción antitumoral mediada por el aumento de los niveles de la proteína COMMD1 en líneas celulares de cáncer
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Guerra-Vallespi M1, Fernández-Massó JR1, Oliva-Argüelles B1, Reyes-Acosta O1, Garay-Pérez HE1, Delgado-Roche L2, Cabrales-Rico A1, Pimentel G3, Garza J3, Basaco T3, Sánchez I3, Calderón C3, Rodríguez JC3, Tejeda-Gómez Y1, Mendoza-Fuentes O1, Soria Y1, Guillen-Pérez I1, Palenzuela-Gardon D1, Vázquez-Blomquist D1, Musacchio-Lasa A1, Astrada S4, Bollati-Fogolín M4, Novoa-Perez LI1, Gómez-Rodríguez Y1, Rivera-Markelova M5, Fichtner I5
1 Dirección de Investigaciones Biomédicas, Centro de Ingeniería Genética y Biotecnología, CIGB. Ave. 31 e/ 158 y 190, Cubanacán, Playa, CP 11600, La Habana, Cuba.
2 Center of Studies for Research and Biological Evaluations, Pharmacy and Food Sciences College, University of Havana, Havana, Cuba.
3 Department of Nuclear Medicine, Institute of Oncology and Radiobiology. 29 and F Street, Vedado, Havana, Cuba.
4 Cell Biology Unit, Institut Pasteur of Montevideo. Mataojo 2020, Montevideo, 11400, Uruguay.
5 Experimental Pharmacology and Oncology, Berlin-Buch GmbH. Robert-Rössle-Str.10, D-13122, Berlin, Germany.
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ABSTRACT
A second-generation peptide CIGB-552, with cell-penetrating capacity, was developed by the modification of the primary structure of the L-2 peptide. The molecular mechanism underlying its cytotoxic activity remains partially unknown. In this study, it was shown that CIGB-552 binds and increases the levels of COMMD1, a protein involved in copper homeostasis, sodium transport, and the NF-kB signaling pathway. We found that CIGB-552 induces ubiquitination of RelA and inhibits the antiapoptotic activity regulated by NF-κβ, whereas the knockdown of COMMD1 blocks this effect. We also found that CIGB-552 increases the levels of reactive oxygen species (ROS), decreases the cellular antioxidant capacity and induces the peroxidation of proteins and lipids in tumor cells. Altogether, our results bring new insights into the mechanism of action of CIGB-552. Moreover, its anti-tumoral effect was explored by subcutaneous administration in a therapeutic schedule in syngeneic murine tumors and patient-derived xenograft models. Outstandingly, a significant delay of tumor growth was observed after the administration of CIGB-552 in these experimental settings. Our data reinforce the perspectives of CIGB-552 for targeted therapy against cancer. This research granted the 2014 Award of the Cuban National Academy of Sciences.
Keywords: CIGB-552, COMMD1, cytotoxic peptide, oxidative stress, NF-κ&beta, cancer animal models.
Se desarrolló un péptido con capacidad penetradora en las células, denominado CIGB-552, mediante la modificación de la estructura primaria del péptido L-2. Los mecanismos moleculares de su actividad citotóxica han sido caracterizados parcialmente. En este estudio se demostró que el CIGB-552 incrementa los niveles de la proteína COMMD1, involucrada en la homeostasis celular del cobre, el transporte de sodio y en la ruta de señalización de NF-κ&beta. Se halló que el CIGB-552 induce la ubiquitinación de RelA e inhibe la actividad antiapoptótica regulada por NF-κ&beta, mientras que la eliminación de COMMD1 bloquea dicho efecto. También se encontró que CIGB-552 incrementa los niveles de especies reactivas de oxígeno (ERO), disminuye la capacidad antioxidante celular e induce peroxidación de proteínas y lípidos en las células tumorales. De conjunto, nuestros resultados permitieron profundizar en el mecanismo de acción del CIGB-552. Además, se exploró su efecto antitumoral, mediante la administración subcutánea en un esquema terapéutico en el modelo de tumores singénicos murinos y en el de xenotrasplante derivado de pacientes. De forma muy relevante, se observó una demora significativa en el crecimiento tumoral tras la administración del CIGB-552 en condiciones experimentales. Estos resultados refuerzan las perspectivas del uso del péptido CIGB-552 en la terapia dirigida contra el cáncer. Esta investigación mereció el Premio de la Academia de Ciencias de Cuba en 2014.
Palabras clave: CIGB-552, COMMD1, péptido citotóxico, estrés oxidativo, NF-κ&beta, modelos animales de cáncer.
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INTRODUCTION
The development of new, increasingly-selective and effective drugs against cancer, that minimize toxicity and able to be used in combination with standard therapy is a challenge today. Therapies directed against molecular targets important in survival, proliferation and spread of tumor cells are revolutionizing the paradigm of cancer treatment and will probably be used in most patients in the next ten years [1].
The Antimicrobial Peptides (AMP), derived from plants and animals, are one of such potential source of new antitumor agents. The cyclic peptide LALF31-52, derived from horseshoe crab (Limulus polyphemus) LALF protein, was initially identified by its ability to bind LPS and the inhibition of the coagulation cascade mediated by the activation of endotoxins [2]. Studies published by Vallespi et al. identified that the linear variant, the LALF32-51 peptide, exhibited antiviral activity mediated by the induction of α and γ interferons. Furthermore, the effectiveness of the peptide was demonstrated in murine models of bacteria-mediated sepsis [3, 4].
Based on the relationship between the primary structure and the biological function of LALF32-51, a chemical library of peptides was studied by alanine-scanning. The essential amino acids conferring LPS-binding capacity in LALF31-52 and determining or influencing its cytotoxic activity were identified, leading to the design of the L-2 peptide, devoid of LPS-binding activity and showing a stronger cytotoxic effect than the original AMP LALF32-51 peptide. Then, a second generation peptide, named CIGB-552, was generated by replacing amino acids at positions 6 and 12 for their respective D-amino acids in L-2, and blocking its N-terminus by acylation, making it resistant to proteolytic degradation [5, 6].
Subsequently, the molecular basis of the CIGB- 552 peptide antitumor effect was established, the COMMD1 protein being identified as a new molecular target in cancer therapy. These results support the capacity of CIGB-552 to act against cancer cells through apoptosis-mediated mechanisms and the inhibition of tumor angiogenesis. From a practical point of view, the results of pharmacological and toxicological studies provide evidences on the safety of CIGB-552 for its use in humans. This research granted the 2014 Award of the Cuban National Academy of Sciences.
RESULTS AND DISCUSSION
Design of a family of second-generation peptides and evaluation of its cytotoxic activity
The proteolytic stability of natural peptides is a major limitation for its use as drug candidates. Therefore, amino acids substitutions were performed introducing D-amino acids (D-aa.) in specific positions in the original sequence of the L2 peptide, as shown in Table 1. In one case, the N-terminal end was further blocking by acetylation.
]]> Subsequently, the proliferative capacity of tumor cells from different origins was challenged with different amount of the peptides. Cells were seeded into 96-well plates and incubated with increasing amounts of the peptides.Cytotoxicity was estimated with Sulforhodamine B and IC50 values were calculated using the Calcusyn software (Biosoft®, United Kingdom). As shown in figure 1, the CIGB-552 peptide displayed the best cytotoxic effect on tumor cell lines as evidenced by the IC50 values. This finding suggests that the incorporation of unnatural amino acids in the sequence may have improved the metabolic stability of the peptide.
Identification of proteins interacting with the CIGB-552 peptide
To identify proteins that interact with CIGB-552 peptide, a yeast-two-hybrid assay was performed. For that purpose, 38 positive clones were identified and sequenced. Similarity analysis of the sequences of the clones evidenced an expected value with an exponential lower than -25 (Table 2).
Among the proteins identified, alpha-2-HS-glycoprotein (AHGS) is produced abundantly in the liver and is present in serum and in the extracellular matrix. High levels of expression in liver explain the identification of three independent clones in the two hybrid assay, since the cDNA library used was derived from a normal human liver tissue (Clontech, USA).
The AHSG protein is capable of binding to cytokines of the Transforming Growth Factor (TGF) family, through a domain which has homology to the extracellular TGF-receptor (TGFR) domain. In fact, it has been shown that high levels of expression of AHSG contribute to the inhibition of tumor progression in different models [7], this protein mainly located in the extracellular space. The need for internalization of the CIGB-552 family of peptides to exert its effect makes it unlikely that the antitumor mechanism could be mediated by the AHSG-TGFR interaction [6]. The implications of that specific interaction should be properly addressed in further experimental settings.
The second identified interaction corresponded to the COMMD1 protein. COMMD1 is the prototype protein of the COMMD protein family. The ten members of that family are highly conserved and ubiquitously expressed in multicellular organisms [8], their biological functions mostly unknown. COMMD1 function has been linked to copper homeostasis, sodium transport, the signaling mediated by transcription factor NF-κ&beta and the hypoxia-induced factor 1 (HIF-1), among others processes. In the case of the COMMD1 protein, it inhibits the transcription factor NF-κ&beta, an important element activating genes involved in resistance to apoptosis in the cancer cells, stimulates oncogene activation, angiogenesis and cell proliferation. COMMD1 also regulates HIF-1, this protein playing a key role in cell survival in hypoxic tissue, a common feature of tumor [9]. In line with our results, it was recently discovered that COMMD1 is expressed but at very low levels in cancer cells, this also associated with more invasive tumor types [10].
Identification of pathways associated with the cytotoxic effect
Analysis of data obtained from a microarray study, by comparing treated vs. untreated NCI-H460 cells at 0, 2, 4 and 8 h at the CIGB-552 IC50 (25 µM), identified up to 349 differentially-expressed genes using the relative expression criteria of the modular value of fold change (|FC| ≥ 1.5 and adjusted p values by the Benjamini and Hochberg method [11] lower than 0.05. Bioinformatics analysis of the experimental data revealed pathways modulated by CIGB-552 treatment (Figure 2).
The pathway enrichment in differentially-expressed genes in the cell line NCI-H460 treated with the CIGB-552 peptide, identified pathways related with the immune system, which comprise genes associated with the inflammatory response. These pathways correspond to the previously described immunomodulatory action of the LALF32-51 peptide and are common to all AMPs [2, 3]. Surprisingly, the oxidative stress response was found associated with the mechanism of cytotoxicity of the CIGB-552 peptide. This was unprecedented, in spite of the intense debate on the relationship between cancer and oxidative stress [12]. The cellular oxidative damage has been associated with carcinogenesis and apoptosis. Additionally, increased levels of oxidative stress have been considered a successful strategy to induce apoptosis in malignant cells [13, 14].
]]> Moreover, other identified pathways as those triggered by MAPK kinase and NF-κ&beta signaling are also modulated by the oxidative stress. Overall, the results allowed us to identify oxidative stress as a key mechanism mediating the activation of multiple signaling pathways (MAPK, TP53, NF-κ&beta), that explain the cytotoxicity of the CIGB-552 peptide.CIGB-552 accumulates COMMD1 in human cancer cells and promotes ubiquitination and degradation of the RelA subunit of NF-κ&beta
Since COMMD1 was identified as a protein that is associated with antitumor peptides, the role of COMMD1 was studied in the mechanism of action of CIGB-552. First, COMMD1 expression in lysates of human cancer cells of different histological origin was determined using Western blot analysis. These experiments revealed an increase in COMMD1 levels after 5 h of treatment with the peptide. COMMD1 cellular accumulation was also identified by immunofluorescence in human cancer cells [4]. Both cell lines tested, MCF7 and HT29, showed accumulation of COMMD1 after 5 h of treatment with the CIGB-552.
COMMD1 overexpression accelerated the ubiquitination and degradation of RelA subunit of NF-κ&beta. Given the above data, the effect of CIGB-552 levels of ubiquitinated RelA was addressed. Immunoprecipitation of endogenous RelA was performed using mouse anti-RelA, followed by Western Blot with an anti-ubiquitin antibody. Increased amounts of ubiquitinated RelA in response to the peptide was found as early as 2 h after treatment. Then, the possible induction of ubiquitination by CIGB-552 and the subsequent degradation of RelA through a proteasome-dependent process were evaluated through the effect of MG132 and CIGB-552 in the basal levels of RelA. The treatment with CIGB-552 decreases the basal levels of RelA. This result suggests that CIGB- 552 induces ubiquitination of RelA and promotes its proteasomal degradation [4].
The functional role of COMMD1 accumulation in the antitumor activity of CIGB-552 was confirmed by transduction of H460 cells with a retrovirus expressing a shRNA targeting COMMD1. In this cell line, the cytotoxic activity of CIGB-552 decreased compared to control cells, possibly with decreased ubiquitination and downstream degradation of RelA [4].
The CIGB-552 alters the redox state in H-460 cells
Since microarray studies indicated that the CIGB-552 modulates the expression of oxidative stress-related genes, the relevance of COMMD1 accumulation on the oxidative stress balance was assessed by measuring the redox balance in NCI-H460 cells.
We observed that treatment with CIGB-552 caused an increase in the levels of reactive oxygen species (ROS) O2•− and •ON, which explains the activation of genes involved in the process of oxidative stress response observed in the experiments of differential gene expression. This is in agreement with previous findings which describe COMMD1 as an interaction partner of superoxide dismutase 1 (SOD1) [15]. Maturation and activation of SOD1 are highly re-gulated processes requiring several posttranslational modifications, with COMMD1 deteriorating the SOD1 activity by reducing the expression levels of enzymatically-active homodimers in the process of SOD1 posttranslational maturation. A relevant finding was the decrease in the total antioxidant capacity by treating cells with CIGB-552. The increased ROS concentrations and the overall decrease of total antioxidant capacity in cells after the treatment at the IC50 of CIGB-552 are shown in Figure 3. In summary, these results could explain the observed increase in the oxidation of lipids and proteins and the reduction of the mitochondrial membrane potential inducing the apoptosis as part of the cytotoxic mechanism.
Synergistic effect of the combination of CIGB-552 and standard chemotherapy
For this assay, tumor cells lines HT-29 and H-125 were added with culture medium containing the CIGB-552 peptide in a dose range from 9 to 300 µM. The cytostatic 5-FU and cisplatin were added to cell cultures at ten-fold and 1:10 dilutions of their respective IC50 as reported for each cell line. The effect of concomitant treatment-cytostatic peptide was analyzed by the CalcuSyn computer program for the study of drug combinations.
]]> As shown in table 3, the peptide-cytostatic combinations reduced the amount of cytostatic required, given by the values shown in the Reduction Index (RI). These results indicate that the peptide can be administered together with standard chemotherapy to provide an effective treatment (the fraction of affected cells: 89-94 %) with minor amounts of each standard drug. Combining 5-FU with CIGB-552 allows a 20-fold reduction (RI) in the amount of 5-FU in the cell line HT-29 required. For cisplatin, its combination with CIGB-552 allowed a 5-fold reduction in the cytostatic concentration required to attain the effect in H-125 cells. These results indicate that CIGB-552 can be ad-ministered in combination with standard treatment for human colorectal and lung cancer (HT-29 and H-125 cell lines, respectively), facilitating a reduction in the dose of cytostatic. This may reduce the adverse effects associated with chemotherapy.Efficacy of CIGB-552 in the therapy of solid tumors in mice
To evaluate the in vivo antitumor activity of CIGB- 552, it was administered subcutaneously in the murine model of tumor cells CT-26 in BALB/c mice. Both the tumor accumulation of the 131I-labeled CIGB- 552 and the inhibition of tumor growth after treatment with CIGB-552 were evaluated. A human colon tumor xenograft model in nude mice was also used to further evaluate the anticancer effect of CIGB-552.
The results demonstrated that two injections a week of CIGB-552 for 2 weeks elicited an antitumor activity mediated by the inhibition of tumor growth (50 % inhibition). In addition, the peptide reduces the density of blood vessels in the tumors [16]. Together, these data prove that the second-generation peptide CIGB-552 is able to induce a significant antitumor response after systemic delivery.
SCIENTIFIC RELEVANCE OF THE STUDY
The study of the differential gene expression identified the oxidative stress response among the routes and biological processes being modulated by the CIGB-552 in the lung cancer cell line NCI-H460. It was shown that the CIGB-552 causes the increase of ROS, causing damage to mitochondria and increasing the oxidative damage of lipids and proteins. Moreover, proteomic studies revealed that the CIGB-552 interacts with COMMD1, the treatment with CIGB- 552 increasing the expression of COMMD1 in cell lines which leads to apoptosis-mediated cell death. Altogether, these findings provide the molecular basis to fully unravel the cytotoxic mechanism of CIGB- 552 in cancer cells.
From a practical point of view, the results of the pharmacological and toxicological studies support the safety of CIGB-552 for use in humans. The preclinical studies have demonstrated: 1) the antitumor effect of CIGB-552 in animal models of lung and colon cancer; 2) the antiangiogenic activity; 3) the treatment tolerability through the monitoring of body weight in treated mice; and 4) the accumulation of the peptide in tumors, supporting the potential safety based on the bioavailability of the CIGB-552 peptide in Phase I clinical trials in patients with solid tumors. These results support the therapeutic possibilities of this new therapeutic candidate for the treatment of cancer. Significantly, metabolic pathways activated by CIGB-552 peptide makes it unique, constituting the first synthetic peptide aimed at stabilizing COMMD1 as a way to downregulate NF-κ&beta and ultimately lead to cancer cell cytotoxicity.
ACKNOWLEDGEMENTS
]]> The authors thank to Gerardo Guillén Nieto, Raimundo Ubieta Gomez, Amanda Colarte Gonzalez, Tamara Díaz Argudín, Héctor Santana Milian, H. Zarate, Arielis Rodríguez Ulloa, Leovaldo Alvarez, Ariel Sánchez Puente, Yassel Ramos Gomez, Dayana García Lines, Luis Javier González, Vladimir Besada Perez, T. Núñez, Isabel Apezteguía Rodríguez, Ever Pérez Hernández, Reinier Acosta Martín, Antonio Antequera Guevara, Juan Hernández Pinero, Mercedes Arencibia Leyva, Yamilet Pérez Chabas, Mariela Vazquez Castillo, Yamilka Rodriguez Barrera, Yeny Cascaret Nápoles, Regla Estrada Vazquez and José A. Silva Guirado, from the CIGB for their collaboration.
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Received in August, 2015.
Accepted in September, 2015.
Maribel Guerra-Vallespi. Dirección de Investigaciones Biomédicas, Centro de Ingeniería Genética y Biotecnología, CIGB. Ave. 31 e/ 158 y 190, Cubanacán, Playa, CP 11600, La Habana, Cuba. E-mail: maribel.guerra@cigb.edu.cu.
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