Daniel J. Toft

Induction of Cancer Cell Death by Self-assembling Nanostructures Incorporating a Cytotoxic Peptide

Nanotechnology offers novel delivery vehicles for cancer therapeutics. Potential advantages of nanoscale platforms include improved pharmacokinetics, encapsulation of cytotoxic agents, enhanced accumulation of therapeutics in the tumor microenvironment, and improved therapeutic structures and bioactivity. Here, we report the design of a novel amphiphilic molecule that self-assembles into nanostructures for intracellular delivery of cytotoxic peptides. Specifically, a cationic alpha-helical (KLAKLAK)(2) peptide that is known to induce cancer cell death by membrane disruption was integrated into a peptide amphiphile (PA) that self-assembles into bioactive, cylindrical nanofibers. PAs are composed of a hydrophobic alkyl tail, a beta-sheet forming peptide, and a bioactive peptide that is displayed on the surface of the nanofiber after self-assembly. PA nanostructures that included (KLAKLAK)(2) were readily internalized by breast cancer cells, in contrast to the (KLAKLAK)(2) peptide that on its own was not cell permeable. (KLAKLAK)(2) nanostructures, but not the peptides alone, also induced breast cancer cell death by caspase-independent and Bax/Bak-independent mechanisms associated with membrane disruption. Significantly, (KLAKLAK)(2) nanostructures induced cell death more robustly in transformed breast epithelial cells than in untransformed cells, suggesting a degree of tumor selectivity. Our results provide proof-of-principle that self-assembling PAs can be rationally designed to generate nanostructures that can efficiently deliver cytotoxic peptides to cancer cells.

Antitumor activity of peptide amphiphile nanofiber-encapsulated camptothecin

Self-assembling peptide amphiphile (PA) nanofibers were used to encapsulate camptothecin (CPT), a naturally occurring hydrophobic chemotherapy agent, using a solvent evaporation technique. Encapsulation by PA nanofibers was found to improve the aqueous solubility of the CPT molecule by more than 50-fold. PAs self-assembled into nanofibers in the presence of CPT as demonstrated by transmission electron microscopy. Small-angle X-ray scattering results suggest a slight increase in diameter of the nanofiber to accommodate the hydrophobic cargo. In vitro studies using human breast cancer cells show an enhancement in antitumor activity of the CPT when encapsulated by the PA nanofibers. In addition, using a mouse orthotopic model of human breast cancer, treatment with PA nanofiber-encapsulated CPT inhibited tumor growth. These results highlight the potential of this model PA system to be adapted for delivery of hydrophobic therapies to treat a variety of diseases including cancer.

Minireview: Basal-like breast cancer: from molecular profiles to targeted therapies.

The classification of breast cancer into molecular subtypes with distinctive gene expression signatures that predict treatment response and prognosis has ushered in a new era of personalized medicine for this remarkably heterogeneous and deadly disease. Basal-like breast cancer (BLBC) is a particularly aggressive molecular subtype defined by a robust cluster of genes expressed by epithelial cells in the basal or outer layer of the adult mammary gland. BLBC is a major clinical challenge because these tumors are prevalent in young woman, often relapsing rapidly. Additionally, most (but not all) basal-like tumors lack expression of steroid hormone receptors (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor 2, limiting targeted therapeutic options for these predominantly triple-negative breast cancers. This minireview will focus on new insights into the molecular etiology of these poor-prognosis tumors that underlie their intrinsic genomic instability, deregulated cell proliferation and apoptosis, and invasive tumor biology. We will also review ongoing efforts to translate these fundamental insights into improved therapies for women with BLBC.

pH and Amphiphilic Structure Direct Supramolecular Behavior in Biofunctional Assemblies

Supramolecular self-assembly offers promising new ways to control nanostructure morphology and respond to external stimuli. A pH-sensitive self-assembled system was developed to both control nanostructure shape and respond to the acidic microenvironment of tumors using self-assembling peptide amphiphiles (PAs). By incorporating an oligo-histidine H6 sequence, we developed two PAs that self-assembled into distinct morphologies on the nanoscale, either as nanofibers or spherical micelles, based on the incorporation of the aliphatic tail on the N-terminus or near the C-terminus, respectively. Both cylinder and sphere-forming PAs demonstrated reversible disassembly between pH 6.0 and 6.5 upon protonation of the histidine residues in acidic solutions. These PAs were then characterized and assessed for their potential to encapsulate hydrophobic chemotherapies. The H6-based nanofiber assemblies encapsulated camptothecin (CPT) with up to 60% efficiency, a 7-fold increase in CPT encapsulation relative to spherical micelles. Additionally, pH-sensitive nanofibers showed improved tumor accumulation over both spherical micelles and nanofibers that did not change morphologies in acidic environments. We have demonstrated that the morphological transitions upon changes in pH of supramolecular nanostructures affect drug encapsulation and tumor accumulation. Our findings also suggest that these supramolecular events can be tuned by molecular design to improve the pharmacologic properties of nanomedicines.

Reactivation of proliferin gene expression is associated with increased angiogenesis in a cell culture model of fibrosarcoma tumor progression

Proliferin (PLF) is an angiogenic placental hormone. We now report that PLF gene expression can also occur in a progressive fibrosarcoma mouse tumor cell model. PLF mRNA and protein are detectable at very low levels in cell lines derived from the mild noninvasive stage of tumor development. Expression is greatly augmented in cell lines from the aggressively invasive stage of development, a stage at which the tumor becomes highly angiogenic, and PLF expression remains high in cell lines from the end stage of fibrosarcoma. Activator protein 1 factors present at high levels in the more invasive stages of the tumor may in part allow for increased PLF expression, as cells from the mild stage in which c-jun and junB are stably expressed secrete levels of PLF comparable to that of the advanced stages. Secreted PLF protein is functionally important in tumor cell angiogenic activity, as demonstrated by the reduction of angiogenic activity in fibrosarcoma cell culture medium by immunodepletion of PLF. These results suggest that an extraembryonic genetic program, which has evolved to support fetal growth, may be reactivated in certain tumors and contribute to tumor growth.

Identification of Three Prolactin-Related Hormones as Markers of Invasive Trophoblasts in the Rat

Abstract An expressed-sequence tag database search has identified three rat cDNA clones in the prolactin/growth hormone family, including a homologue of mouse proliferin-related protein (PRP). The encoded proteins of the two novel clones, designated prolactin-like proteins L (PLP-L) and M (PLP-M), are predicted to be synthesized as precursors of 229 and 227 amino acids, modified by N-linked glycosylation, and secreted as mature glycoproteins of 199 and 200 residues, respectively. Murine homologues to PLP-L and PLP-M were also identified. The open reading frame of rat PRP encodes a precursor protein of 245 amino acids and predicts a secreted 215-amino acid glycoprotein with 81% identity to mouse PRP. All three rat mRNAs are expressed in the placenta, and expression is not detected in other tissues. PLP-L mRNA expression is observed from Days 11–20, with highest levels at Day 13; highest levels of PLP-M are observed from Day 11 until parturition, with peak levels also on Day 13; and highest levels of PRP are also observed from Day 11 until term, with maximal expression on Day 17. All three genes are most highly expressed in invasive trophoblast cells lining the central placental vessel. The identification of molecular markers for endovascular trophoblasts serves to highlight the invasive nature of rodent placentation and may prove useful for future studies of placental function.

Prolactin (PRL)-Like Protein J, a Novel Member of the PRL/Growth Hormone Family, Is Exclusively Expressed in Maternal Decidua

A search of a nonmouse, nonhuman, expressed sequence tag database for messenger RNAs in the PRL/GH family has identified a novel rat complementary DNA clone. The encoded protein, designated PRL-like protein J (PLP-J), is predicted to be synthesized as a precursor of 211 amino acids, modified by N-linked glycosylation, and secreted as a mature glycoprotein of 182 residues. PLP-J messenger RNA synthesis is limited to early pregnancy with abundant expression on day 7, slightly declining expression on day 9, and no detectable expression by day 11. Unlike most other PRL family members, PLP-J does not appear to be synthesized by placental trophoblasts but, rather, by decidual cells surrounding the implantation site. By sequence similarity to rat PLP-J, a murine clone was identified in a mouse expressed sequence tag database. Mouse PLP-J was used to map the gene to a 700-kb region of mouse chromosome 13 that includes other members of the PRL/GH family.

Primary CNS lymphoma with bilateral symmetric hypothalamic lesions presenting with panhypopituitarism and diabetes insipidus

We present an unusual case of primary central nervous system (CNS) lymphoma presenting with bilateral symmetric hypothalamic lesions causing diabetes insipidus and hypopituitarism. A 50-year-old male presented initially with mental status changes, polyuria and polydipsia. The patient was determined to have diabetes insipidus (DI) and significant anterior pituitary deficiencies resulting in symptomatic pleural and pericardial effusions. Brain MRI with contrast demonstrated bilateral enhancement of his hypothalamus extending to the optic tract. The extensive diagnostic workup that ensued on his initial presentation was non-diagnostic as he had no obvious site of involvement that was easily accessible to biopsy. With close follow-up, the patient had rapid radiographic progression of his disease to his cerebral hemispheres. He therefore underwent brain biopsy and was diagnosed with primary CNS large B cell lymphoma. Chemotherapy has resulted in disease remission with resolution of MRI findings, but the patient has not had resolution of the hypopituitarism or DI. This case highlights the unique diagnostic challenge of patients with isolated hypothalamic lesions.

Minireview: Basal-Like Breast Cancer: From Molecular Profiles to Targeted Therapies

The classification of breast cancer into molecular subtypes with distinctive gene expression signatures that predict treatment response and prognosis has ushered in a new era of personalized medicine for this remarkably heterogeneous and deadly disease. Basal-like breast cancer (BLBC) is a particularly aggressive molecular subtype defined by a robust cluster of genes expressed by epithelial cells in the basal or outer layer of the adult mammary gland. BLBC is a major clinical challenge because these tumors are prevalent in young woman, often relapsing rapidly. Additionally, most (but not all) basal-like tumors lack expression of steroid hormone receptors (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor 2, limiting targeted therapeutic options for these predominantly triple-negative breast cancers. This minireview will focus on new insights into the molecular etiology of these poor-prognosis tumors that underlie their intrinsic genomic instability, deregulated cell proliferation and apoptosis, and invasive tumor biology. We will also review ongoing efforts to translate these fundamental insights into improved therapies for women with BLBC. (Molecular Endocrinology 25: 199–211, 2011)

Medical Management of Pheochromocytoma

Patients with catecholamine-secreting pheochromocytomas and paragangliomas require medical treatment to control blood pressure and tachyarrhythmias and to prepare the patient for surgical tumor resection. The mainstay of medical treatment is α-adrenergic receptor blockade, and both nonselective and α1-selective adrenergic receptor blockers can be used. Once blood pressure control is achieved, β-adrenergic receptor blockers can be added to control tachycardia and arrhythmias. When blood pressure cannot be achieved by α-blockade alone, calcium channel blockers and metyrosine can be added. Such treatment, along with fluid and salt replacement, should be carried out for 7–14 days prior to surgery to avoid postoperative hypotension. Uncommonly, patients may present with hypertensive emergencies and require ICU admission with intravenous α-adrenergic receptor blockade with phentolamine and other antihypertensive agents. Another uncommon presentation is acute heart failure which can rapidly progress to cardiogenic shock; such patients also require ICU admission and rapid institution of α- and then β-adrenergic receptor blockade. Patients with metastatic disease may require chronic adrenergic receptor blockade, and new chemotherapeutic agents have varying success. Overall, medical therapy with subsequent surgery of patients with pheochromocytomas is quite successful, and most patients do very well.