Jesus Manuel Perez

The Use of Therapeutic Peptides to Target and to Kill Cancer Cells

Peptide therapeutics is a promising field for emerging anti-cancer agents. Benefits include the ease and rapid synthesis of peptides and capacity for modifications. An existing and vast knowledge base of protein structure and function can be exploited for novel peptide design. Current research focuses on developing peptides that can (1) serve as tumor targeting moieties and (2) permeabilize membranes with cytotoxic consequences. A survey of recent findings reveals significant trends. Amphiphilic peptides with clusters of hydrophobic and cationic residues are features of anti-microbial peptides that confer the ability to eradicate microbes and show considerable anti-cancer toxicity. Peptides that assemble and form pores can disrupt cell or organelle membranes and cause apoptotic or necrotic death. Cell permeable and tumor-homing peptides can carry biologically active cargo to tumors or tumor vasculature. The challenge lies in developing the clinical application of therapeutic peptides. Improving delivery to tumors, minimizing non-specific toxic effects and discerning pharmacokinetic properties are high among the needs to produce a powerful therapeutic peptide for cancer treatment.

The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption

Metastasis accounts for most deaths from breast cancer, driving the need for new therapeutics that can impede disease progression. Rationally designed peptides that take advantage of cancer-specific differences in cellular physiology are an emerging technology that offer promise as a treatment for metastatic breast cancer. We developed CT20p, a hydrophobic peptide based on the C terminus of Bax that exhibits similarities with antimicrobial peptides, and previously reported that CT20p has unique cytotoxic actions independent of full-length Bax. In this study, we identified the intracellular actions of CT20p which precede cancer cell-specific detachment and death. Previously, we found that CT20p migrated in the heavy membrane fractions of cancer cell lysates. Here, using MDA-MB-231 breast cancer cells, we demonstrated that CT20p localizes to the mitochondria, leading to fusion-like aggregation and mitochondrial membrane hyperpolarization. As a result, the distribution and movement of mitochondria in CT20p-treated MDA-MB-231 cells was markedly impaired, particularly in cell protrusions. In contrast, CT20p did not associate with the mitochondria of normal breast epithelial MCF-10A cells, causing little change in the mitochondrial membrane potential, morphology or localization. In MDA-MB-231 cells, CT20p triggered cell detachment that was preceded by decreased levels of α5β1 integrins and reduced F-actin polymerization. Using folate-targeted nanoparticles to encapsulate and deliver CT20p to murine tumors, we achieved significant tumor regression within days of peptide treatment. These results suggest that CT20p has application in the treatment of metastatic disease as a cancer-specific therapeutic peptide that perturbs mitochondrial morphology and movement ultimately culminating in disruption of the actin cytoskeleton, cell detachment, and loss of cell viability.

Chaperonin Containing-TCP-1 Protein Level in Breast Cancer Cells Predicts Therapeutic Application of a Cytotoxic Peptide

Purpose: Metastatic disease is a leading cause of death for patients with breast cancer, driving the need for new therapies. CT20p is a peptide previously discovered by our group that displays cancer-specific cytotoxicity. To design the optimal therapeutic use of the peptide, we identified the intracellular target of CT20p in breast cancer cells, correlating expression patterns of the target with susceptibility to CT20p. Experimental Design: Using polymeric nanoparticles to deliver CT20p, we assessed cytoskeletal changes, cell migration, adhesion, and viability in cells treated with the peptide. Protein pull-down experiments, coupled to mass spectrometry, enabled identification of the peptides intracellular target. Biochemical and histologic techniques validated target identity in human cell lines and breast cancer tissue microarrays and revealed susceptibility patterns to CT20p. Results: Chaperonin containing TCP-1 (CCT) was identified as the intracellular target of CT20p. Cancer cells susceptible to CT20p had increased CCT, and overexpression of CCTβ, a subunit of the CCT complex, enhanced susceptibility to CT20p. Susceptible cells displayed reduced tubulin, a substrate of CCT, and inhibition of migration upon CT20p treatment. CCTβ levels were higher in invasive ductal carcinomas than in cancer adjacent tissues and increased with breast cancer stage. Decreased breast cancer patient survival correlated with genomic alternations in CCTβ and higher levels of the chaperone. Conclusions: Increased CCT protein in breast cancer cells underlies the cytotoxicity of CT20p. CCT is thus a potential target for therapeutic intervention and serves as a companion diagnostic to personalize the therapeutic use of CT20p for breast cancer treatment. Clin Cancer Res; 22(17); 4366–79. ©2016 AACR.

Abstract P5-03-02: The dynamic duo: A breast cancer-targeting nanoparticle loaded with a cytotoxic peptide as a treatment for metastatic disease

Metastatic breast cancer is a uniformly fatal disease with a 5-year survival rate of 15 percent. To date there are no effective approaches for targeted therapy. To develop a treatment for metastatic cancer, our group discovered a novel cytotoxic peptide, CT20p, and developed a nanotechnology-based platform to deliver and concentrate CT20p in breast tumors. CT20p was derived from Bax, a member of the Bcl-2 family. Unlike the parent protein, CT20p does not cause apoptosis and its cytotoxicity is independent of caspases and Bcl-2 overexpression. Rather, the intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT), which is required for the folding of actin and tubulin into their native forms. Inhibition of CCT activity by CT20p, indicated by decreased F-actin and tubulin, impaired the polymerization of microfilaments and microtubules, causing loss of cell migration and adhesion that promoted breast cancer cell death. In contrast, normal, non-transformed cells were resistant to the cytotoxicity of CT20p. On its own, CT20p is not membrane-permeable. To deliver the peptide to cells, we used nanoparticles formed with a novel aliphatic hyperbranched polyester polymer (HBPE-NPs). The surface of HBPE-NPs retains carboxylic acid groups for labeling of targeting ligands to enable accumulation in tumors. To concentrate on breast cancer, we functionalized the HBPE-NPs with either folate (FOL) or glutamate (GLU), which target the folate receptor (FR) or the metabotropic glutamate receptor (GRM-1) respectively. FR and GRM-2 are essential metabolic components that are highly expressed in solid tumors like breast cancer. In vitro targeting studies using triple negative breast cancer cell (TNBC) lines established that folate FOL or GLU-HBPE-NPs loaded with fluorescent dyes were readily up taken at high efficiency by TNBC cells. HBPE-NPs also contain unique hydrophobic cavities especially suited for encapsulating CT20p. We found that once the CT20p-HBPE-NPs were taken up by cancer cells, the peptide was released inside cells under acidic conditions (e.g. endosomes) and directly interacted with its intracellular target, CCT. Studies using primary cells derived from human breast tumors confirmed the targeted uptake of HBPE-NPs as well as demonstrated the cancer-specific cytotoxicity of CT20p. We treated a murine TNBC xenograft model with nanomolar amounts of FOL-CT20p-HBPE-NPs and achieved 100% regression of established tumors as well as prevented tumor growth. These studies indicated that CT20p is a potent and specific anti-cancer agent due to its inhibition of CCT, an essential molecular complex highly expressed in cancer cells, and that the peptide can be efficiently delivered to tumor sites using HBPE-NPs decorated with ligands to receptors, such FR or GRM-1, found on tumor cells. Citation Format: Vishnubhotla P, Khaled AR, Khaled AS, Perez JM, Bassiouni R, Flores O, Nierenberg D. The dynamic duo: A breast cancer-targeting nanoparticle loaded with a cytotoxic peptide as a treatment for metastatic disease. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-02.