The CT20 peptide as an agent for cancer treatment

Due to cancer recurrence and the development of drug resistance, metastatic breast cancer is a leading cause of death in women. In the search for a new therapeutic to treat metastatic disease, we discovered CT20p, an amphipathic peptide based on the C-terminus of Bax. Due to inherent properties of its sequence and similarity to antimicrobial peptides, CT20p is a promising cytotoxic agent whose activity is distinct from the parent protein (e.g. does not cause apoptosis). CT20p is not membrane permeable but can be introduced to cells using polymeric nanoparticles, a method that promotes efficient delivery of the peptide into the intracellular environment. We demonstrated that CT20p was cytotoxic using triple negative breast cancer (TNBC) cell lines, primary breast tumor tissue, and breast tumor murine xenografts. Importantly, normal breast epithelial cells and normal primary breast cells were resistant to the lethal effects of the peptide. Examination of multiple cellular processes showed that CT20p causes cell death by promoting cytoskeletal disruption, cell detachment, and loss of substrate-mediated survival signals. In order to identify the intracellular target of CT20p, we performed pull-down experiments using a biotinylated peptide and found that CT20p binds directly to a type II chaperonin called chaperonin containing T-complex (CCT), which is essential for the folding of actin and tubulin into their native forms. The resulting effect of CT20p upon the cytoskeleton of cancer cells is disruption of vital cellular processes such as migration and adhesion. CCT gene expression and protein levels were examined across several breast cancer cell lines, and we found that susceptibility to CT20p correlated with higher CCT levels. Using human cancer tissue microarrays, we determined that CCT was present in significantly higher amounts in tumor tissues compared to normal tissues and that expression often increased with advanced cancer stage. These results indicate that CCT is a promising therapeutic target for the treatment of metastatic breast cancer and suggest that the use of cancer-targeted nanoparticles loaded with CT20p is a novel and effective therapeutic strategy for cancers, such as TNBC, that recur and are refractory to current treatments

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 peptide\u27s 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 CCTb, 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. CCTb 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 CCTb 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

Psma-Targeted Theranostic Nanocarrier For Prostate Cancer

Herein, we report the use of a theranostic nanocarrier (Folate-HBPE(CT20p)) to deliver a therapeutic peptide to prostate cancer tumors that express PSMA (folate hydrolase 1). The therapeutic peptide (CT20p) targets and inhibits the chaperonin-containing TCP-1 (CCT) protein-folding complex, is selectively cytotoxic to cancer cells, and is non-toxic to normal tissue. With the delivery of CT20p to prostate cancer cells via PSMA, a dual level of cancer specificity is achieved: (1) selective targeting to PSMA-expressing prostate tumors, and (2) specific cytotoxicity to cancer cells with minimal toxicity to normal cells. The PSMA-targeting theranostic nanocarrier can image PSMA-expressing cells and tumors when a near infrared dye is used as cargo. Meanwhile, it can be used to treat PSMA-expressing tumors when a therapeutic, such as the CT20p peptide, is encapsulated within the nanocarrier. Even when these PSMA-targeting nanocarriers are taken up by macrophages, minimal cell death is observed in these cells, in contrast with doxorubicin-based therapeutics that result in significant macrophage death. Incubation of PSMA-expressing prostate cancer cells with the Folate-HBPE(CT20p) nanocarriers induces considerable changes in cell morphology, reduction in the levels of integrin ß1, and lower cell adhesion, eventually resulting in cell death. These results are relevant as integrin ß1 plays a key role in prostate cancer invasion and metastatic potential. In addition, the use of the developed PSMA-targeting nanocarrier facilitates the selective in vivo delivery of CT20p to PSMA-positive tumor, inducing significant reduction in tumor size

Targeting Chaperonin Containing Tcp1 (Cct) As A Molecular Therapeutic For Small Cell Lung Cancer

Identifying new druggable targets is desired to meet the needs for effectivecancer treatments. To this end, we previously reported the efficacy of a therapeuticpeptide called CT20p that displays selective cytotoxicity through inhibition of a multisubunit,protein-folding complex called Chaperonin-Containing TCP-1 (CCT). Toinvestigate the role of CCT in cancer progression, we examined protein levels of CCTsubunits in liver, prostate, and lung cancer using human tissue microarrays. We foundthat these cancers expressed higher levels of CCT2 as compared to normal tissues.Small cell lung cancer (SCLC) stood out as having statistically significant difference inCCT2. Higher levels of CCT2 in tumors from lung cancer patients were also associatedwith decreased survival. Using SCLC cell lines, we observed detectable amounts ofCCT subunits and cells were susceptible to killing by CT20p. Treatment with CT20p,delivered to cells using polymeric nanoparticles, was cytotoxic to all SCLC cell lines,decreasing the levels of CCT client proteins like STAT3. In contrast, treatment witha STAT3 inhibitor was effective in one of the SCLC cell lines. While we found thatCCT levels could vary in cell lines, normal tissues had low levels of CCT and minimaltoxicity to liver or kidney function was observed in mice treated with CT20p. Theseresults indicate that in SCLC, changes in CCT levels could be used as a biomarker fordiagnosis and that targeting CCT for inhibition with CT20p is a promising treatmentApproach for those cancers such as SCLC that currently lack targeted therapeutics

Alveolar type I cells can give rise to KRAS-induced lung adenocarcinoma

Summary: Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer and presents clinically with a high degree of biological heterogeneity and distinct clinical outcomes. The current paradigm of LUAD etiology posits alveolar epithelial type II (AT2) cells as the primary cell of origin, while the role of AT1 cells in LUAD oncogenesis remains unknown. Here, we examine oncogenic transformation in mouse Gram-domain containing 2 (Gramd2)+ AT1 cells via oncogenic KRASG12D. Activation of KRASG12D in AT1 cells induces multifocal LUAD, primarily of papillary histology. Furthermore, KRT8+ intermediate cell states were observed in both AT2- and AT1-derived LUAD, but SCGB3A2+, another intermediate cell marker, was primarily associated with AT1 cells, suggesting different mechanisms of tumor evolution. Collectively, our study reveals that Gramd2+ AT1 cells can serve as a cell of origin for LUAD and suggests that distinct subtypes of LUAD based on cell of origin be considered in the development of therapeutics

Alveolar type I cells can give rise to KRAS-induced lung adenocarcinoma

Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer and presents clinically with a high degree of biological heterogeneity and distinct clinical outcomes. The current paradigm of LUAD etiology posits alveolar epithelial type II (AT2) cells as the primary cell of origin, while the role of AT1 cells in LUAD oncogenesis remains unknown. Here, we examine oncogenic transformation in mouse Gram-domain containing 2 (Gramd2)+ AT1 cells via oncogenic KRASG12D. Activation of KRASG12D in AT1 cells induces multifocal LUAD, primarily of papillary histology. Furthermore, KRT8+ intermediate cell states were observed in both AT2- and AT1-derived LUAD, but SCGB3A2+, another intermediate cell marker, was primarily associated with AT1 cells, suggesting different mechanisms of tumor evolution. Collectively, our study reveals that Gramd2+ AT1 cells can serve as a cell of origin for LUAD and suggests that distinct subtypes of LUAD based on cell of origin be considered in the development of therapeutics