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 peptide's 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.

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.

<p>Supplemental figure with genomic data and results from tissue microarrays</p>

<p>Supplemental figure showing IHC scoring scheme</p>

<div>Abstract<p><b>Purpose:</b> 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.</p><p><b>Experimental Design:</b> 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 peptide's intracellular target. Biochemical and histologic techniques validated target identity in human cell lines and breast cancer tissue microarrays and revealed susceptibility patterns to CT20p.</p><p><b>Results:</b> 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.</p><p><b>Conclusions:</b> 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. <i>Clin Cancer Res; 22(17); 4366–79. ©2016 AACR</i>.</p></div>