Abstract A47: Use of a cytotoxic peptide that induces immunogenic cell death to engage innate immunity in the treatment of metastatic breast cancer
Authors: Limaye Arati , Bassiouni Rania , Oyer Jeremiah , Igarashi Robert W. , Flores Orielyz , Perez J. Manuel , Copik Alijca , Khaled Annette R. 
Published: October 2015

Abstract
The median survival rate for women with metastatic breast cancer is 1-2 years, and the 5-year survival rate is only 15 percent. Early detection of metastatic disease is often ineffective, since once the cancer recurs at a distant site, it may be refractory to conventional treatments. A promising strategy for targeting metastatic breast cancer is engaging the immune system to destroy disseminated breast cancer cells. Immunotherapeutic approaches are effective treatments for many types of cancer, such as melanoma. However, breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. Moreover, immunotherapy can cause adverse side effects such as colitis. There is an unmet need to develop new, targeted therapies for breast cancer that stimulate the immune system to fight metastatic disease. One potential approach is to generate dying breast cancer cells that operate like a vaccine to induce a tumor-specific immune response, which can eradicate residual cancer cells. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes like dendritic cells (DCs) that in turn activate tumor-specific cytotoxic T cells. While not well-understood, ICD may also sensitize tumor cells to killing by natural killer (NK) cells. With the discovery of new ICD-inducing agents, interest in this approach is increasing. However, the current doses of drugs, such as anthracyclines, used to induce ICD may be too high to translate into clinically relevant regimens. To address these problems, our group developed a novel cytotoxic peptide, CT20p, and a nanotechnology-based platform to deliver and concentrate CT20p in breast tumors. We found that treatment of mice with nanomolar amounts of CT20p, encapsulated in nanoparticles formed with a novel hyperbranched polyester polymer (HBPE-NPs), resulted in regression of breast cancer tumors, and that dying breast cancer cells expressed markers characteristic of ICD, such as the pre-mortem exposure of calreticulin (Crt). The intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT) which is essential for the folding of actin and tubulin and other critical proteins into their native forms. We found CCT to be overexpressed in breast cancer cells susceptible to the peptide. Inhibition of CCT activity by CT20p, indicated by decreased F-actin, disrupted the cytoskeleton, causing loss of cell motility and adhesion that led to breast cancer cell death. Disruption of CCT could cause endoplasmic reticulum (ER) stress through the accumulation of misfolded proteins. ER stress in turn initiates intracellular pathways that can generate the danger signals associate with ICD. To this end, we observed that breast cancer cells treated with CT20p displayed alterations in PERK (protein kinase RNA-like ER kinase), one of the key mediators of the unfolded protein response (UPR). As a result, CT20p-treated breast cancer cells were more readily phagocytosed. This also suggests that peptide treatment could potentially have an impact on NK cell cytotoxicity by inducing the expression of activating ligands on cancer cells. In our experiments normal breast epithelial cells, macrophages or NK cells were unaffected by CT20p treatment due reduced levels and activity of CCT. These studies indicate that CT20p, by targeting CCT, can be used to induce ICD and stimulate essential innate immune functions like NK cell cytotoxic activity and leukocyte phagocytosis that are necessary for promoting a robust anti-cancer adaptive immune response in order to mitigate the development of lethal metastatic disease.
Citation Format: Arati Limaye, Rania Bassiouni, Jeremiah Oyer, Robert W. Igarashi, Orielyz Flores, J. Manuel Perez, Alijca Copik, Annette R. Khaled. Use of a cytotoxic peptide that induces immunogenic cell death to engage innate immunity in the treatment of metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A47.