Rebecca Boohaker

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.

Apoptosis-induced alkalinization by the Na+/H+ exchanger isoform 1 is mediated through phosphorylation of amino acids Ser726 and Ser729

Apoptosis is a complex process essential for normal tissue development and cellular homeostasis. While biochemical events that occur late in the apoptotic process are better characterized, early physiological changes that initiate the progression of cell death remain poorly understood. Previously, we observed that lymphocytes, undergoing apoptosis in response to growth factor withdrawal, experienced a rapid and transient rise in cytosolic pH. We found that the protein responsible was the pH-regulating, plasma membrane protein Na(+)/H(+) exchanger isoform 1 (NHE1), and that its activity was impeded by inhibition of the stress-activated kinase, p38 MAP kinase. In the current study, we examined how NHE1 is activated during apoptosis. We identified the phosphorylation sites on NHE1 that regulate its alkalinizing activity in response to a cell death stimulus. Performing targeted mutagenesis, we observed that substitution of Ser726 and Ser729 for alanines produced a mutant form of NHE1 that did not alkalinize in response to an apoptotic stimulus, and expression of which protected cells from serum withdrawal- induced death. In contrast, substitution of Ser726 and Ser729 for glutamic acids raised the basal pH and induced susceptibility to death. Analysis of serine phosphorylation showed that phosphorylation of NHE1 during apoptosis decreased upon mutation of Ser726 and Ser729. Our findings thus confirm a necessary function for NHE1 during apoptosis and reveal the critical regulatory sites that when phosphorylated mediate the alkalinizing activity of NHE1 in the early stages of a cell death response.

BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells

The dual functionality of the tumor suppressor BAX is implied by the nonapoptotic functions of other members of the BCL-2 family. To explore this, mitochondrial metabolism was examined in BAX-deficient HCT-116 cells as well as primary hepatocytes from BAX-deficient mice. Although mitochondrial density and mitochondrial DNA content were the same in BAX-containing and BAX-deficient cells, MitoTracker staining patterns differed, suggesting the existence of BAX-dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in BAX-deficient cells, while glycolysis was increased. These results suggested that cells lacking BAX have a deficiency in the ability to generate ATP through cellular respiration. This conclusion was supported by detection of reduced citrate synthase activity in BAX-deficient cells. In nonapoptotic cells, a portion of BAX associated with mitochondria and a sequestered, protease-resistant form was detected. Inhibition of BAX with small interfering RNAs reduced intracellular ATP content in BAX-containing cells. Expression of either full-length or COOH-terminal-truncated BAX in BAX-deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of BAX was not dependent upon its COOH-terminal helix. Expression of BCL-2 in BAX-containing cells resulted in a subsequent loss of ATP measured, implying that, even under nonapoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX is necessary to maintain energy production via aerobic respiration.

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 peptides 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.

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.

Abstract 5138: Structural insights into the dynamic functionality of Bax through molecular dynamics simulations.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The Bcl-2 protein family modulates the balance between cellular life and death. Bax, a pro-apoptotic member of this family, participates in mitochondria-mediated apoptosis but also supports mitochondrial bioenergetics. In previous work, we showed that a peptide derived from the alpha 9-helix of Bax is cytotoxic, specifically when used to treat breast cancer cells. Such structural and biochemical studies are useful for identifying functional domains within Bax that can be developed into therapeutic peptides, but are limiting when ascribing function to domains at a molecular level. Computational modeling and molecular dynamics simulations of Bax provides a high-power analysis of the subtle changes that modulate the regulatory and effector activities of the protein. This study seeks to elucidate the consequence of alterations in micro-domains that confer activity, apoptotic or otherwise, to the protein. Molecular dynamics simulation was used to determine how the trans-membrane region of Bax conferred stability to the protein, identifying regions of high flexibility and differential exposure. Computationally rotating the Ψ angle at Pro168 altered the position of the α9-helix of Bax, mimicking the “active” and “inactive” forms of Bax. We found that “active” Bax, but not “inactive” Bax, presented a more relaxed structure which altered the conformation and exposure of the BH3 domain, potentially enhancing interactions among the family of Bcl-2 proteins. Since dysregulation of Bcl-2 proteins is implicated in a number of cancers, examining the basic structural dynamics at a molecular level yielded information that can aid in the design novel Bcl-2 family-targeting chemotherapeutics. Citation Format: Annette Khaled, Rebecca Boohaker, Craig Finch, Sergio Tafur. Structural insights into the dynamic functionality of Bax through molecular dynamics simulations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5138. doi:10.1158/1538-7445.AM2013-5138

Abstract P6-04-13: Preclinical testing using a novel CT20p peptide-nanoparticle combination in breast cancer

BACKGROUND: The marked difference in metabolism observed between tumor and normal cells could contribute to the development of invasive and metastatic forms of breast cancer. The problem is that while patients diagnosed with invasive forms of breast cancer may be initially responsive to treatment, a significant number develop relapsing and even metastatic disease. There is a critical unmet need to develop new therapeutic approaches for patients diagnosed with invasive forms of breast cancer that are effective given the unique metabolism of tumor cells. METHODS: We examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminus of Bax. For delivery to cells, the amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs). NPs were made using aliphatic hyperbranched polyester (HBPE) that incorporated surface carboxylic groups and interior hydrophobic cavities for encapsulation of CT20p. To examine the cytotoxic potential and targeting capacity of CT20p-HBPE-NPs, we treated MDA-MB-231 breast cancer cells and MCF-10A breast epithelial cells with the peptide-nanoparticle combination and measured changes in mitochondrial function, cell metabolism and induction of apoptotic and non-apoptotic cell death. The ability of CT20p-NP-HBPE to cause tumor regression was examined by subcutaneously implanting MDA-MB-231 cells in nude mice. RESULTS: Initial studies showed that 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. While the peptide alone had little effect upon intact cells, likely not penetrating the plasma membrane, when encapsulated and delivered by nanoparticles, CT20p-HBPE-NPs proved an effective killer of breast cancer cells. CT20p-HBPE-NPs initiated non-apoptotic cell death within 3 hours of treatment by targeting mitochondria and deregulating cellular metabolism. Nanoparticles alone or nanoparticles encapsulating a control peptide had minimal effects. The cytotoxicity of CT20p-HPBE-NPs was most pronounced in breast cancer cells, sparing normal, epithelial cells. In implanted breast tumors, CT20p-HBPE-NPs accumulated in tumors within 24 hours and reduced tumor burden by 50-80%. CONCLUSION: These results reveal the innovative features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies that target the unique metabolism of cancer cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-13.

Abstract 3794: BAX maintains mitochondrial bioenergetics in colon cancer cells

The dual functionality of the tumor suppressor, BAX, is implied by the non-apoptotic functions of other pro-apoptotic BCL-2 family proteins. To explore this, the mitochondrial morphology and ATP producing capability in both BAX-containing and BAX-deficient HCT-116 colon cancer cells were examined. While BAX deficient cells maintain the same mitochondrial mass as their BAX containing counterparts, these mitochondria were visibly smaller, more compact, and unable to maintain an organized network. Oxygen consumption and ATP levels were also reduced in these cells, a phenomenon that was replicated upon treatment of BAX-containing cells with the uncoupling agent, FCCP. These results, coupled with a marked decrease in citrate synthase activity, suggested that cells lacking BAX have a deficiency in the ability to generate ATP through oxidative phosphorylation. Inhibition of endogenous BAX through treatment with small interfering RNAs reinforced the necessity of BAX activity in the maintenance of aerobic respiration and the production of ATP. An examination of the localization of full length and C-terminal truncated BAX in non-apoptotic, BAX deficient cells, indicated that a small fraction of the existing cytosolic BAX is needed at the mitochondrial membrane to maintain ATP production. However, this function is not dependent upon the C-terminal helix. Co-expression of BAX and BCL-2 resulted in a subsequent loss of ATP production, implying that, even under non-apoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX associated with the mitochondria is necessary to maintain mitochondrial bioenergetics. The discovery of this novel, non-apoptotic role for BAX in the maintenance of ATP synthesis and suggests that complete abrogation of BAX would be detrimental to cancer cells dependent on energy production via aerobic respiration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3794. doi:10.1158/1538-7445.AM2011-3794

Biophysical Insights into Bax Oligomerization and Membrane Insertion

The BCL2 family of proteins tightly regulates the delicate balance between life and death. Bax, a proapoptotic member of this family, acts as the penultimate factor in the apoptotic cascade by releasing apoptogenic factors such as Cytochrome C from the mitochondrial lumen. The mechanism of mitochrondrial permeabilization by BAXis not well defined. What is known is that BAX translocates to and aggregates at the outer mitochondrial membrane before cytochrome C is released, implying the insertion of the protein occurs after the aggregation event. In this work, we have evaluated the function of the oligomerization state of BAX on the insertion of the protein into artificial membranes.

Compartmentalization of BCL2 Family Proteins Mediated by Organelle Lipid Membranes

Cancer is defined by a pronounced inhibition of cell death. The BCL2 family of proteins tightly regulates the delicate balance between life and death. One method of regulation is the compartmentalization of antagonistic members. For example, Bax, a pro-apoptotic member of this family, acts as the penultimate factor in the apoptotic cascade by releasing apoptogenic factors such as Cytochrome C from the mitochondrial lumen. The normally cytosolic protein translocates from one internal compartment to another through an elusive mechanism. Individual organelles are defined not only by function (mediated by specific membrane bound proteins), but by the unique composition of their phospholipid membranes. In this work, we have evaluated the contribution of organelle lipids to the localization of of BCL2 proteins.