A novel multifunctional radioprotective strategy using P7C3 as a countermeasure against ionizing radiation-induced bone loss.

Radiotherapy is a critical component of cancer care but can cause osteoporosis and pathological insufficiency fractures in surrounding and otherwise healthy bone. Presently, no effective countermeasure exists, and ionizing radiation-induced bone damage continues to be a substantial source of pain and morbidity. The purpose of this study was to investigate a small molecule aminopropyl carbazole named P7C3 as a novel radioprotective strategy. Our studies revealed that P7C3 repressed ionizing radiation (IR)-induced osteoclastic activity, inhibited adipogenesis, and promoted osteoblastogenesis and mineral deposition in vitro. We also demonstrated that rodents exposed to clinically equivalent hypofractionated levels of IR in vivo develop weakened, osteoporotic bone. However, the administration of P7C3 significantly inhibited osteoclastic activity, lipid formation and bone marrow adiposity and mitigated tissue loss such that bone maintained its area, architecture, and mechanical strength. Our findings revealed significant enhancement of cellular macromolecule metabolic processes, myeloid cell differentiation, and the proteins LRP-4, TAGLN, ILK, and Tollip, with downregulation of GDF-3, SH2B1, and CD200. These proteins are key in favoring osteoblast over adipogenic progenitor differentiation, cell matrix interactions, and shape and motility, facilitating inflammatory resolution, and suppressing osteoclastogenesis, potentially via Wnt/ß-catenin signaling. A concern was whether P7C3 afforded similar protection to cancer cells. Preliminarily, and remarkably, at the same protective P7C3 dose, a significant reduction in triple-negative breast cancer and osteosarcoma cell metabolic activity was found in vitro. Together, these results indicate that P7C3 is a previously undiscovered key regulator of adipo-osteogenic progenitor lineage commitment and may serve as a novel multifunctional therapeutic strategy, leaving IR an effective clinical tool while diminishing the risk of adverse post-IR complications. Our data uncover a new approach for the prevention of radiation-induced bone damage, and further work is needed to investigate its ability to selectively drive cancer cell death.

Macromolecules Absorbed from Influenza Infection-Based Sera Modulate the Cellular Uptake of Polymeric Nanoparticles.

Optimizing the biological identity of nanoparticles (NPs) for efficient tumor uptake remains challenging. The controlled formation of a protein corona on NPs through protein absorption from biofluids could favor a biological identity that enables tumor accumulation. To increase the diversity of proteins absorbed by NPs, sera derived from Influenza A virus (IAV)-infected mice were used to pre-coat NPs formed using a hyperbranched polyester polymer (HBPE-NPs). HBPE-NPs, encapsulating a tracking dye or cancer drug, were treated with sera from days 3-6 of IAV infection (VS3-6), and uptake of HBPE-NPs by breast cancer cells was examined. Cancer cells demonstrated better uptake of HBPE-NPs pre-treated with VS3-6 over polyethylene glycol (PEG)-HBPE-NPs, a standard NP surface modification. The uptake of VS5 pre-treated HBPE-NPs by monocytic cells (THP-1) was decreased over PEG-HBPE-NPs. VS5-treated HBPE-NPs delivered a cancer drug more efficiently and displayed better in vivo distribution over controls, remaining stable even after interacting with endothelial cells. Using a proteomics approach, proteins absorbed from sera-treated HBPE-NPs were identified, such as thrombospondin-1 (TSP-1), that could bind multiple cancer cell receptors. Our findings indicate that serum collected during an immune response to infection is a rich source of macromolecules that are absorbed by NPs and modulate their biological identity, achieving rationally designed uptake by targeted cell types.

Chaperonin containing TCP-1 (CCT/TRiC) is a novel therapeutic and diagnostic target for neuroblastoma.

Chaperonin containing TCP1 (CCT/TRiC) is a multi-subunit protein folding complex that enables the cancer phenotype to emerge from the mutational landscape that drives oncogenesis. We and others linked increased expression of CCT subunits to advanced tumor stage and invasiveness that inversely correlates with cancer patient outcomes. In this study, we examined the expression of the second CCT subunit, CCT2, using genomic databases of adult and pediatric tumors and normal tissues, and found that it was highly expressed in pediatric cancers, showing a significant difference compared to normal tissues. Histologic staining confirmed that CCT subunits are highly expressed in tumor tissues, which was exemplified in neuroblastoma. Using two neuroblastoma cells, MYCN-amplified, IMR-32 cells, and non-amplified, SK-N-AS cells, we assessed baseline levels for CCT subunits and found expressions comparable to the highly invasive triple-negative breast cancer (TNBC) cell line, MDA-MB-231. Exogenous expression of CCT2 in both SK-N-AS and IMR-32 cells resulted in morphological changes, such as larger cell size and increased adherence, with significant increases in the CCT substrates, actin, and tubulin, as well as increased migration. Depletion of CCT2 reversed these effects and reduced cell viability. We evaluated CCT as a therapeutic target in IMR-32 cells by testing a novel peptide CCT inhibitor, CT20p. Treatment with CT20p induced cell death in these neuroblastoma cells. The use of CCT2 as a biological indicator for detection of neuroblastoma cells shed in blood was examined by spiking IMR-32 cells into human blood and using an anti-CCT2 antibody for the identification of spiked cancer cells with the CellSearch system. Results showed that using CCT2 for the detection of neuroblastoma cells in blood was more effective than the conventional approach of using epithelial markers like cytokeratins. CCT2 plays an essential role in promoting the invasive capacity of neuroblastoma cells and thus offers the potential to act as a molecular target in the development of novel therapeutics and diagnostics for pediatric cancers.

Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood.

Herein we report the use of Chaperonin-Containing TCP-1 (CCT or TRiC) as a marker to detect circulating tumor cells (CTCs) that are shed from tumors during oncogenesis. Most detection methods used in liquid biopsy approaches for enumeration of CTCs from blood, employ epithelial markers like cytokeratin (CK). However, such markers provide little information on the potential of these shed tumor cells, which are normally short-lived, to seed metastatic sites. To identify a marker that could go beyond enumeration and provide actionable data on CTCs, we evaluated CCT. CCT is a protein-folding complex composed of eight subunits. Previously, we found that expression of the second subunit (CCT2 or CCTß) inversely correlated with cancer patient survival and was essential for tumorigenesis in mice, driving tumor-promoting processes like proliferation and anchorage-independent growth. In this study, we examined CCT2 expression in cancer compared to normal tissues and found statistically significant increases in tumors. Because not all blood samples from cancer patients contain detectable CTCs, we used the approach of spiking a known number of cancer cells into blood from healthy donors to test a liquid biopsy approach using CCT2 to distinguish rare cancer cells from the large number of non-cancer cells in blood. Using a clinically validated method for capturing CTCs, we evaluated detection of intracellular CCT2 staining for visualization of breast cancer and small cell lung (SCLC) cancer cells. We demonstrated that CCT2 staining could be incorporated into a CTC capture and staining protocol, providing biologically relevant information to improve detection of cancer cells shed in blood. These results were confirmed with a pilot study of blood from SCLC patients. Our studies demonstrate that detection of CCT2 could identify rare cancer cells in blood and has application in liquid biopsy approaches to enhance the use of minimally invasive methods for cancer diagnosis.

The TRiCky Business of Protein Folding in Health and Disease.

Maintenance of the cellular proteome or proteostasis is an essential process that when deregulated leads to diseases like neurological disorders and cancer. Central to proteostasis are the molecular chaperones that fold proteins into functional 3-dimensional (3D) shapes and prevent protein aggregation. Chaperonins, a family of chaperones found in all lineages of organisms, are efficient machines that fold proteins within central cavities. The eukaryotic Chaperonin Containing TCP1 (CCT), also known as Tailless complex polypeptide 1 (TCP-1) Ring Complex (TRiC), is a multi-subunit molecular complex that folds the obligate substrates, actin, and tubulin. But more than folding cytoskeletal proteins, CCT differs from most chaperones in its ability to fold proteins larger than its central folding chamber and in a sequential manner that enables it to tackle proteins with complex topologies or very large proteins and complexes. Unique features of CCT include an asymmetry of charges and ATP affinities across the eight subunits that form the hetero-oligomeric complex. Variable substrate binding capacities endow CCT with a plasticity that developed as the chaperonin evolved with eukaryotes and acquired functional capacity in the densely packed intracellular environment. Given the decades of discovery on the structure and function of CCT, much remains unknown such as the scope of its interactome. New findings on the role of CCT in disease, and potential for diagnostic and therapeutic uses, heighten the need to better understand the function of this essential molecular chaperone. Clues as to how CCT causes cancer or neurological disorders lie in the early studies of the chaperonin that form a foundational knowledgebase. In this review, we span the decades of CCT discoveries to provide critical context to the continued research on the diverse capacities in health and disease of this essential protein-folding complex.

Chaperonin-Containing TCP1 Complex (CCT) Promotes Breast Cancer Growth Through Correlations With Key Cell Cycle Regulators.

Uncontrolled proliferation as a result of dysregulated cell cycling is one of the hallmarks of cancer. Therapeutically targeting pathways that control the cell cycle would improve patient outcomes. However, the development of drug resistance and a limited number of inhibitors that target multiple cell cycle modulators are challenges that impede stopping the deregulated growth that leads to malignancy. To advance the discovery of new druggable targets for cell cycle inhibition, we investigated the role of Chaperonin-Containing TCP1 (CCT or TRiC) in breast cancer cells. CCT, a type II chaperonin, is a multi-subunit protein-folding complex that interacts with many oncoproteins and mutant tumor suppressors. CCT subunits are highly expressed in a number of cancers, including breast cancer. We found that expression of one of the CCT subunits, CCT2, inversely correlates with breast cancer patient survival and is subject to copy number alterations through genomic amplification. To investigate a role for CCT2 in the regulation of the cell cycle, we expressed an exogenous CCT2-FLAG construct in T47D and MCF7 luminal A breast cancer cells and examined cell proliferation under conditions of two-dimensional (2D) monolayer and three-dimensional (3D) spheroid cultures. Exogenous CCT2 increased the proliferation of cancer cells, resulting in larger and multiple spheroids as compared to control cells. CCT2-expressing cells were also able to undergo spheroid growth reversal, re-attaching, and resuming growth in 2D cultures. Such cells gained anchorage-independent growth. CCT2 expression in cells correlated with increased expression of MYC, especially in spheroid cultures, and other cell cycle regulators like CCND1 and CDK2, indicative of a novel activity that could contribute to the increase in cell growth. Statistically significant correlations between CCT2, MYC, and CCND1 were shown. Since CCT2 is located on chromosome 12q15, an amplicon frequently found in soft tissue cancers as well as breast cancer, CCT2 may have the basic characteristics of an oncogene. Our findings suggest that CCT2 could be an essential driver of cell division that may be a node through which pathways involving MYC, cyclin D1 and other proliferative factors could converge. Hence the therapeutic inhibition of CCT2 may have the potential to achieve multi-target inhibition, overcoming the limitations associated with single agent inhibitors.

Polymeric Nanoparticles with a Sera-Derived Coating for Efficient Cancer Cell Uptake and Killing.

Nanoparticle-mediated cancer drug delivery remains an inefficient process. The protein corona formed on nanoparticles (NPs) controls their biological identity and, if optimized, could enhance cancer cell uptake. In this study, a hyperbranched polyester polymer (HBPE) was synthesized from diethyl malonate and used to generate NPs that were subsequently coated with normal sera (NS) collected from mice. Cellular uptake of NS-treated HBPE-NPs was compared to PEGylated HBPE-NPs and was assessed using MDA-MB-231 triple-negative breast cancer (TNBC) cells as well as endothelial and monocytic cell lines. NS-treated HBPE-NPs were taken up by TNBC cells more efficiently than PEGylated HBPE-NPs, while evasion of monocyte uptake was comparable. NS coatings facilitated cancer cell uptake of HBPE-NPs, even after prior interaction of the particles with an endothelial layer. NS-treated HBPE-NPs were not inherently toxic, did not induce the migration of endothelial cells that could lead to angiogenesis, and could efficiently deliver cytotoxic doses of paclitaxel (taxol) to TNBC cells. These findings suggest that HBPE-NPs may adsorb select sera proteins that improve uptake by cancer cells, and such NPs could be used to advance the discovery of novel factors that improve the bioavailability and tissue distribution of drug-loaded polymeric NPs.

Investigating Chaperonin-Containing TCP-1 subunit 2 as an essential component of the chaperonin complex for tumorigenesis.

Chaperonin-containing TCP-1 (CCT or TRiC) is a multi-subunit complex that folds many of the proteins essential for cancer development. CCT is expressed in diverse cancers and could be an ideal therapeutic target if not for the fact that the complex is encoded by eight distinct genes, complicating the development of inhibitors. Few definitive studies addressed the role of specific subunits in promoting the chaperonin's function in cancer. To this end, we investigated the activity of CCT2 (CCTß) by overexpressing or depleting the subunit in breast epithelial and breast cancer cells. We found that increasing total CCT2 in cells by 1.3-1.8-fold using a lentiviral system, also caused CCT3, CCT4, and CCT5 levels to increase. Likewise, silencing cct2 gene expression by ~50% caused other CCT subunits to decrease. Cells expressing CCT2 were more invasive and had a higher proliferative index. CCT2 depletion in a syngeneic murine model of triple negative breast cancer (TNBC) prevented tumor growth. These results indicate that the CCT2 subunit is integral to the activity of the chaperonin and is needed for tumorigenesis. Hence CCT2 could be a viable target for therapeutic development in breast and other cancers.

Cypate and Cypate-Glucosamine as Near-Infrared Fluorescent Probes for In Vivo Tumor Imaging.

Near-infrared (NIR) imaging is a promising technique for use as a noninvasive and sensitive diagnostic tool. Although the NIR fluorescently labeled glucose analog glucosamine (cypate-glucosamine) has applications in preclinical imaging, the transport pathways and fate of this probe in tissues remain unaddressed. Here, we have synthesized and characterized cypate and cypate-glucosamine conjugate (cy-2-glu), and investigated the probable transport pathways of these probes in vitro and in vivo. We compared uptake of the probes in the presence and absence of excess d-glucose, "saturated cypate" and palmitic acid in two normal-cancer cell line pairs: lung cancer (A549)-normal (MRC9) and prostate cancer (DU145)-normal (BPH). Breast cancer (MDA-MB-231) and liver cancer (HepG2) cell lines were also examined. Results support use of the glucose transport pathway by cy-2-glu and fatty acid transport pathway by cypate. Mass spectrometry data on the in vitro extracts revealed deamidation of cy-2-glu in prostate and liver cells, suggesting release of glucosamine. In vivo biodistribution studies in mice engrafted with breast tumors showed a distinct accumulation of cy-2-glu in liver and tumors, and to a lesser extent in kidneys and spleen. A negligible accumulation of cypate alone in tumors was observed. Analysis of urine extracts revealed renal excretion of the cy-2-glu probe in the form of free cypate, indicating deamidation of cy-2-glu in tissues. Thus, investigation of the metabolic pathways used by NIR probes such as cy-2-glu advances their use in the detection and monitoring of tumor progression in preclinical animal studies.

Elucidation of Therapeutic Peptide Binding Partners from Isolated Mitochondria.

CT20p is a protein derived from the C-terminus of Bax. It has selective cytotoxicity for cancer cells, such as the sensitive triple-negative MDA-MB-231 breast adenocarcinoma cells, but not normal cells like the resistant MCF-10A epithelial breast cells. To understand the reason for the peptide's selective toxicity, a "pull-down" experiment with biotinylated CT20p (biotin-CT20p) and whole-cell protein lysates from breast cancer and normal cells were performed. These studies revealed that CT20p binds to a cytosolic protein called chaperonin-containing TCP-1 (CCT), a molecular chaperone that folds actin and tubulin. However, this method could not detect possible rare interactions made by CT20p with mitochondrial proteins. To determine whether CT20p is associated with mitochondrial proteins as part of the mechanism by which it induces cell death, mitochondrial protein lysates from MDA-MB-231 and MCF-10A cells were isolated and a streptavidin-agarose pulldown procedure using biotin-CT20p was performed. Protein interactions were visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining. The results of the experimental procedure showed that biotin-CT20p did not "pull down" any observable mitochondrial proteins from the sensitive MDA-MB-231 cells, indicating that the peptide may not interact with mitochondrial proteins in breast cancer cells. Rather, the interactions observed with biotin-CT20p were with mitochondrial proteins derived from resistant MCF-10A cells, indicating that these interactions were not driving the cancer-selective cell death process. The absence of CT20p-associated proteins from the mitochondrial lysates of MDA-MB-231 breast cancer cells supports the hypothesis that CT20p, unlike the parent protein, Bax, exerts its cytotoxic effects via a cytosolic protein.

Formation of a protein corona influences the biological identity of nanomaterials.

The development and testing of nanomaterials is an area of interest due to promising diagnostic and therapeutic applications in the treatment of diseases like cancer or cardiovascular disease. While extensive studies of the physicochemical properties of nanoparticles (NPs) are available, the investigation of the protein corona (PC) that is formed on NPs in biofluids is a relatively new area of research. The fact that few NPs are in clinical use indicates that the biological identity of NPs, which is in large part due to the PC formed in blood or other bodily fluids, may be altered in ways yet to be fully understood. Herein, we review the recent advances in PC research with the intent to highlight the current state of the field. We discuss the dynamic processes that control the formation of the PC on NPs, which involve the transient soft corona and more stable hard corona. Critical factors, like the environment and disease-state that affect the composition and stability of the PC are presented, with the intent of showcasing promising applications for utilizing the PC for disease diagnosis and the identification of disease-related biomarkers. This review summarizes the unique challenges presented by the nanoparticle corona and indicates future directions for investigation.

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.

Cytokines in immunogenic cell death: Applications for cancer immunotherapy.

Despite advances in treatments like chemotherapy and radiotherapy, metastatic cancer remains a leading cause of death for cancer patients. While many chemotherapeutic agents can efficiently eliminate cancer cells, long-term protection against cancer is not achieved and many patients experience cancer recurrence. Mobilizing and stimulating the immune system against tumor cells is one of the most effective ways to protect against cancers that recur and/or metastasize. Activated tumor specific cytotoxic T lymphocytes (CTLs) can seek out and destroy metastatic tumor cells and reduce tumor lesions. Natural Killer (NK) cells are a front-line defense against drug-resistant tumors and can provide tumoricidal activity to enhance tumor immune surveillance. Cytokines like IFN-γ or TNF play a crucial role in creating an immunogenic microenvironment and therefore are key players in the fight against metastatic cancer. To this end, a group of anthracyclines or treatments like photodynamic therapy (PDT) exert their effects on cancer cells in a manner that activates the immune system. This process, known as immunogenic cell death (ICD), is characterized by the release of membrane-bound and soluble factors that boost the function of immune cells. This review will explore different types of ICD inducers, some in clinical trials, to demonstrate that optimizing the cytokine response brought about by treatments with ICD-inducing agents is central to promoting anti-cancer immunity that provides long-lasting protection against disease recurrence and metastasis.

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 effective cancer treatments. To this end, we previously reported the efficacy of a therapeutic peptide called CT20p that displays selective cytotoxicity through inhibition of a multi-subunit, protein-folding complex called Chaperonin-Containing TCP-1 (CCT). To investigate the role of CCT in cancer progression, we examined protein levels of CCT subunits in liver, prostate, and lung cancer using human tissue microarrays. We found that these cancers expressed higher levels of CCT2 as compared to normal tissues. Small cell lung cancer (SCLC) stood out as having statistically significant difference in CCT2. Higher levels of CCT2 in tumors from lung cancer patients were also associated with decreased survival. Using SCLC cell lines, we observed detectable amounts of CCT 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 with a STAT3 inhibitor was effective in one of the SCLC cell lines. While we found that CCT levels could vary in cell lines, normal tissues had low levels of CCT and minimal toxicity to liver or kidney function was observed in mice treated with CT20p. These results indicate that in SCLC, changes in CCT levels could be used as a biomarker for diagnosis and that targeting CCT for inhibition with CT20p is a promising treatment approach for those cancers such as SCLC that currently lack targeted therapeutics.

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'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 CCTß, 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. CCTß 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 CCTß 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.

Transmembrane pore formation by the carboxyl terminus of Bax protein.

Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal "helix 9" of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide-peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents.

Molecular basis for membrane pore formation by Bax protein carboxyl terminus.

Bax protein plays a key role in mitochondrial membrane permeabilization and cytochrome c release upon apoptosis. Our recent data have indicated that the 20-residue C-terminal peptide of Bax (BaxC-KK; VTIFVAGVLTASLTIWKKMG), when expressed intracellularly, translocates to the mitochondria and exerts lethal effect on cancer cells. Moreover, the BaxC-KK peptide, as well as two mutants where the two lysines are replaced with glutamate (BaxC-EE) or leucine (BaxC-LL), have been shown to form relatively large pores in lipid membranes, composed of up to eight peptide molecules per pore. Here the pore structure is analyzed by polarized Fourier transform infrared, circular dichroism, and fluorescence experiments on the peptides reconstituted in phospholipid membranes. The peptides assume an α/ß-type secondary structure within membranes. Both ß-strands and α-helices are significantly (by 30-60 deg) tilted relative to the membrane normal. The tryptophan residue embeds into zwitterionic membranes at 8-9 Å from the membrane center. The membrane anionic charge causes a deeper insertion of tryptophan for BaxC-KK and BaxC-LL but not for BaxC-EE. Combined with the pore stoichiometry determined earlier, these structural constraints allow construction of a model of the pore where eight peptide molecules form an "α/ß-ring" structure within the membrane. These results identify a strong membranotropic activity of Bax C-terminus and propose a new mechanism by which peptides can efficiently perforate cell membranes. Knowledge on the pore forming mechanism of the peptide may facilitate development of peptide-based therapies to kill cancer or other detrimental cells such as bacteria or fungi.

The major isoforms of Bim contribute to distinct biological activities that govern the processes of autophagy and apoptosis in interleukin-7 dependent lymphocytes.

Bim is a BH3-only member of the Bcl-2 family that enables the death of T-cells. Partial rescue of cytokine-deprived T-cells occurs when Bim and the receptor for the T-cell growth factor, interleukin-7, are deleted, implicating Bim as a possible target of interleukin-7-mediated signaling. Alternative splicing yields three major isoforms: BimEL, BimL and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on interleukin-7 for growth, were used. Loss of total Bim in interleukin-7-deprived T-cells resulted in delayed apoptosis. However, loss of Bim also impeded the later degradative phase of autophagy. p62, an autophagy-adaptor protein which is normally degraded, accumulated in Bim deficient cells. To explain this, BimL was found to support acidification of lysosomes that later may associate with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon interleukin-7 withdrawal only in Bim-containing T-cells. intereukin-7 dependent T-cells lacking Bim were less sensitive to inhibition of lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosome-tracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, promoted intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of degradative autolysosomes.

Rational development of a cytotoxic peptide to trigger cell death.

Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify new agents that kill cancer cells by alternative mechanisms. To this end, we examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminal, alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties. Like many antimicrobial peptides, CT20p contains clusters of hydrophobic and cationic residues that could enable the peptide to associate with lipid membranes. CT20p caused the release of calcein from mitochondrial-like lipid vesicles without disrupting vesicle integrity and, when expressed as a fusion protein in cells, localized to mitochondria. The amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs) that have the capacity to harbor targeting molecules, dyes or drugs. The resulting CT20p-NPs proved an effective killer, in vitro, of colon and breast cancer cells, and in vivo, using a murine breast cancer tumor model. By introducing CT20p to Bax deficient cells, we demonstrated that the peptide's lethal activity was independent of endogenous Bax. CT20p also caused an increase in the mitochondrial membrane potential that was followed by plasma membrane rupture and cell death, without the characteristic membrane asymmetry associated with apoptosis. We determined that cell death triggered by the CT20p-NPs was minimally dependent on effector caspases and resistant to Bcl-2 overexpression, suggesting that it acts independently of the intrinsic apoptotic death pathway. Furthermore, use of CT20p with the apoptosis-inducing drug, cisplatin, resulted in additive toxicity. These results reveal the novel features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies to activate multiple death pathways in cancer cells.

JunD/AP-1-mediated gene expression promotes lymphocyte growth dependent on interleukin-7 signal transduction.

Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting immune reconstitution. This feature makes IL-7 an ideal candidate for therapeutic development. As with other cytokines, signaling through the IL-7 receptor induces the JAK/STAT pathway. However, the broad scope of IL-7 regulatory targets likely necessitates the use of other signaling components whose identities remain poorly defined. To this end, we used an IL-7 dependent T-cell line to examine how expression of the glycolytic enzyme, Hexokinase II (HXKII) was regulated by IL-7 in a STAT5-independent manner. Our studies revealed that IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Gel shifts showed that the AP-1 complex induced by IL-7 contained JunD but not c-Fos or c-Jun. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. Because others had shown that JunD was a negative regulator of cell growth, we performed a bioinformatics analysis to uncover possible JunD-regulated gene targets. Our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism. One of these growth promoters was the oncogene, Pim-1. Pim-1 is an IL-7-induced protein that was inhibited when the activities of JNK or JunD were blocked, showing that in IL-7 dependent T-cells JunD can promote positive signals transduced through Pim-1. This was confirmed when the IL-7-induced proliferation of CD8 T-cells was impaired upon JunD inhibition. These results show that engagement of the IL-7 receptor drives a signal that is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth stimulation through the expression of metabolic and signaling factors like HXKII and Pim-1.