Kellye C. Kirkbride

Cortactin: a multifunctional regulator of cellular invasiveness.

Branched actin assembly is critical for a variety of cellular processes that underlie cell motility and invasion, including cellular protrusion formation and membrane trafficking. Activation of branched actin assembly occurs at various subcellular locations via site-specific activation of distinct WASp family proteins and the Arp2/3 complex. A key branched actin regulator that promotes cell motility and links signaling, cytoskeletal and membrane trafficking proteins is the Src kinase substrate and Arp2/3 binding protein cortactin. Due to its frequent overexpression in advanced, invasive cancers and its general role in regulating branched actin assembly at multiple cellular locations, cortactin has been the subject of intense study. Recent studies suggest that cortactin has a complex role in cellular migration and invasion, promoting both on-site actin polymerization and modulation of autocrine secretion. Diverse cellular activities may derive from the interaction of cortactin with site-specific binding partners.

Cortactin promotes exosome secretion by controlling branched actin dynamics.

Sinha et al. show that the cytoskeletal and tumor-overexpressed protein cortactin promotes secretion of exosomes from cancer cells by stabilizing dynamic cortical actin docking sites for multivesicular endosomes, suggesting a potential mechanism by which cortactin may promote tumor aggressiveness.

Tuning PEGylation of mixed micelles to overcome intracellular and systemic siRNA delivery barriers.

A series of endosomolytic mixed micelles was synthesized from two diblock polymers, poly[ethylene glycol-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (PEG-b-pDPB) and poly[dimethylaminoethyl methacrylate-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (pD-b-pDPB), and used to determine the impact of both surface PEG density and PEG molecular weight on overcoming both intracellular and systemic siRNA delivery barriers. As expected, the percent PEG composition and PEG molecular weight in the corona had an inverse relationship with mixed micelle zeta potential and rate of cellular internalization. Although mixed micelles were internalized more slowly, they generally produced similar gene silencing bioactivity (∼ 80% or greater) in MDA-MB-231 breast cancer cells as the micelles containing no PEG (100 D/no PEG). The mechanistic explanation for the potent bioactivity of the promising 50 mol% PEG-b-DPB/50 mol% pD-b-pDPB (50 D) mixed micelle formulation, despite its relatively low rate of cellular internalization, was further investigated as a function of PEG molecular weight (5 k, 10 k, or 20 k PEG). Results indicated that, although larger molecular weight PEG decreased cellular internalization, it improved cytoplasmic bioavailability due to increased intracellular unpackaging (quantitatively measured via FRET) and endosomal release. When delivered intravenously in vivo, 50 D mixed micelles with a larger molecular weight PEG in the corona also demonstrated significantly improved blood circulation half-life (17.8 min for 20 k PEG micelles vs. 4.6 min for 5 kDa PEG micelles) and a 4-fold decrease in lung accumulation. These studies provide new mechanistic insights into the functional effects of mixed micelle-based approaches to nanocarrier surface PEGylation. Furthermore, the ideal mixed micelle formulation identified (50 D/20 k PEG) demonstrated desirable intracellular and systemic pharmacokinetics and thus has strong potential for in vivo therapeutic use.

Dual MMP7-Proximity-Activated and Folate Receptor-Targeted Nanoparticles for siRNA Delivery

A dual-targeted siRNA nanocarrier has been synthesized and validated that is selectively activated in environments where there is colocalization of two breast cancer hallmarks, elevated matrix metalloproteinase (MMP) activity and folate receptor overexpression. This siRNA nanocarrier is self-assembled from two polymers containing the same pH-responsive, endosomolytic core-forming block but varying hydrophilic, corona-forming blocks. The corona block of one polymer consists of a 2 kDa PEG attached to a terminal folic acid (FA); the second polymer contains a larger (Y-shaped, 20 kDa) PEG attached to the core block by a proximity-activated targeting (PAT), MMP7-cleavable peptide. In mixed micelle smart polymer nanoparticles (SPNs) formed from the FA- and PAT-based polymers, the proteolytically removable PEG on the PAT polymers shields nonspecific SPN interactions with cells or proteins. When the PAT element is cleaved within an MMP-rich environment, the PEG shielding is removed, exposing the underlying FA and making it accessible for folate receptor-mediated SPN uptake. Characterization of mixed micelles prepared from these two polymers revealed that uptake and siRNA knockdown bioactivity of a 50% FA/50% PAT formulation was dependent on both proteolytic activation and FA receptor engagement. MMP activation and delivery of this formulation to breast cancer cells expressing the FA receptor achieved greater than 50% protein-level knockdown of a model gene with undetectable cytotoxicity. This modular nanoparticle design represents a new paradigm in cell-selective siRNA delivery and allows for stoichiometric tuning of dual-targeting components to achieve superior targeting specificity.

Regulation of late endosomal/lysosomal maturation and trafficking by cortactin affects Golgi morphology

Cortactin is a branched actin regulator and tumor‐overexpressed protein that promotes vesicular trafficking at a variety of cellular sites, including endosomes and the trans‐Golgi network. To better understand its role in secretory trafficking, we investigated its function in Golgi homeostasis. Here, we report that knockdown (KD) of cortactin leads to a dramatic change in Golgi morphology by light microscopy, dependent on binding the Arp2/3 actin‐nucleating complex. Surprisingly, there was little effect of cortactin‐KD on anterograde trafficking of the constitutive cargo vesicular stomatitis virus glycoprotein (VSVG), Golgi assembly from endoplasmic reticulum membranes upon Brefeldin A washout, or Golgi ultrastructure. Instead, electron microscopy studies revealed that cortactin‐KD cells contained a large number of immature‐appearing late endosomal/lysosomal (LE/Lys) hybrid organelles, similar to those found in lysosomal storage diseases. Consistent with a defect in LE/Lys trafficking, cortactin‐KD cells also exhibited accumulation of free cholesterol and retention of the retrograde Golgi cargo mannose‐6‐phosphate receptor in LE. Inhibition of LE maturation by treatment of control cells with Rab7 siRNA or chloroquine led to a compact Golgi morphology similar to that observed in cortactin‐KD cells. Furthermore, the Golgi morphology defects of cortactin‐KD cells could be rescued by removal of cholesterol‐containing lipids from the media, suggesting that buildup of cholesterol‐rich membranes in immature LE/Lys induced disturbances in retrograde trafficking. Taken together, these data reveal that LE/Lys maturation and trafficking are highly sensitive to cortactin‐regulated branched actin assembly and suggests that cytoskeletal‐induced Golgi morphology changes can be a consequence of altered trafficking at late endosomes.

Combinatorial optimization of PEG architecture and hydrophobic content improves ternary siRNA polyplex stability, pharmacokinetics, and potency in vivo

&NA; A rationally‐designed library of ternary siRNA polyplexes was developed and screened for gene silencing efficacy in vitro and in vivo with the goal of overcoming both cell‐level and systemic delivery barriers. [2‐(dimethylamino)ethyl methacrylate] (DMAEMA) was homopolymerized or copolymerized (50 mol% each) with butyl methacrylate (BMA) from a reversible addition – fragmentation chain transfer (RAFT) chain transfer agent, with and without pre‐conjugation to polyethylene glycol (PEG). Both single block polymers were tested as core‐forming units, and both PEGylated, diblock polymers were screened as corona‐forming units. Ternary siRNA polyplexes were assembled with varied amounts and ratios of core‐forming polymers to PEGylated corona‐forming polymers. The impact of polymer composition/ratio, hydrophobe (BMA) placement, and surface PEGylation density was correlated to important outcomes such as polyplex size, stability, pH‐dependent membrane disruptive activity, biocompatibility, and gene silencing efficiency. The lead formulation, DB4‐PDB12, was optimally PEGylated not only to ensure colloidal stability (no change in size by DLS between 0 and 24 h) and neutral surface charge (0.139 mV) but also to maintain higher cell uptake (> 90% positive cells) than the most densely PEGylated particles. The DB4‐PDB12 polyplexes also incorporated BMA in both the polyplex core‐ and corona‐forming polymers, resulting in robust endosomolysis and in vitro siRNA silencing (˜ 85% protein level knockdown) of the model gene luciferase across multiple cell types. Further, the DB4‐PDB12 polyplexes exhibited greater stability, increased blood circulation time, reduced renal clearance, increased tumor biodistribution, and greater silencing of luciferase compared to our previously‐optimized, binary parent formulation following intravenous (i.v.) delivery. This polyplex library approach enabled concomitant optimization of the composition and ratio of core‐ and corona‐forming polymers (indirectly tuning PEGylation density) and identification of a ternary nanomedicine optimized to overcome important siRNA delivery barriers in vitro and in vivo. Graphical abstract Figure. No caption available.

Human-in-mouse modeling of primary head and neck squamous cell carcinoma.

To develop a reliable modeling system for head and neck squamous cell carcinoma (HNSCC).

Abstract 720: Dual MMP-7-proximity-activated and folate-targeted nanoparticles for siRNA delivery

We have developed a dual, folic acid (FA) and matrix metalloproteinase (MMP), proximity-activated targeting (PAT) smart polymeric nanoparticle (SPN) for tumor-specific siRNA delivery. The nanocarrier was designed to expose concealed FA ligands specifically in regions of elevated MMP activity, resulting in targeted uptake by folate receptor-expressing cells. Site-selective chemistry and reversible addition-fragmentation chain transfer (RAFT) polymerization were used to prepare diblock copolymers {p(DMAEMA)-b-p(DMAEMA-PAA-BMA) (SPN)} terminated with either MMP-7 peptide coupled to a Y-shaped 20 kDa PEG (PAT-SPN) or a 2 kDa linear PEG coupled to FA (FA-SPN). These amphiphilic polymers were co-assembled in aqueous media, forming mixed micelles consisting of 0, 25, 50, 75, 100 mol% FA and presenting zeta potentials of -0.21, -0.20, +1.75, +6.71 and +12.2 mV, respectively. Cleavage by MMP-7 was confirmed by decreased particle diameter (e.g., 25% FA micelle diameter decreased from 54 to 48 nm) and simultaneous increase in zeta potential (-0.20 to +8.11 mV). These data suggest that MMP-7 removal of the PEG corona from the nanocarriers revealed the underlying polycationic charge of the pDMAEMA core. Intracellular uptake of PAT/FA-SPNs was investigated in FA receptor-expressing breast cancer cells by confocal microscopy and flow cytometry. Pre-treatment with exogenous MMP-7 to mimic the proteolytically active microenvironment of human breast cancers, elevated cellular uptake 2.3-fold for single targeting PAT-SPN and 4.6-fold for dual-targeting PAT/FA-SPN compared to intact PAT-SPN. Cell uptake of uncleaved PAT/FA-SPN was statistically equivalent to untreated cells. These data suggest that the 20 kDa Y-shaped PEG effectively shields both the underlying cationic charge and the FA, and also blocks nonspecific cellular interactions of the SPN. 75-100% FA-SPN polymer reduced the dependency of cellular uptake on MMP-7 activity. Excess FA reduced the uptake of FA-SPN-containing micelles, consistent with FA receptor-dependent uptake following MMP activation. Functional protein knockdown delivered by PAT/FA-SPN was validated using luciferase-expressing MDA-MB-231 cells. 50% PAT/FA-SPN, pre-activated with MMP-7and loaded with luciferase siRNA, resulted in significant suppression of luciferase expression relative to controls (no MMP activation or competitive inhibition with free FA or scrambled siRNA), confirming the efficient dual PAT/FA targeting and endosomal escape of this designed nanoparticle. Unlike other similar technologies, this micelle design is optimized for use in vivo. Animal studies confirm an extended blood half-life, appropriate organ distribution, minimal toxicity and significant siRNA activity in mammary tumors. 1. Li, H. et al. Adv. Func. Mater. 2013. 10.1002/adfm.201202215 Support DoD CDMRP (W81XWH-10-1-0445/0446); Vanderbilt Institute of Nanoscale Science & Engineering Citation Format: Todd D. Giorgio, Hongmei Li, Martina Miteva, Kellye C. Kirkbride, Ming Cheng, Chris E. Nelson, Craig L. Duvall. Dual MMP-7-proximity-activated and folate-targeted nanoparticles for siRNA delivery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 720. doi:10.1158/1538-7445.AM2014-720

Abstract 3972: The role of the TGF-β type III receptor in colon carcinogenesis

The loss of the TGF-β type III receptor (TβRIII), a TGF-β superfamily co-receptor, correlates with disease progression in multiple cancer types, suggesting that TβRIII plays a role in regulating tumor progression. TGF-β superfamily signaling is frequently dysregulated in colon cancer but the specific role of TβRIII in colon carcinogenesis has not been examined. In contrast to other cancer types, TβRIII mRNA expression is not altered in colon cancer and there is not a significant change in TβRIII protein expression in tumors compared to normal colon tissue. HT29 colon cancer cells overexpressing TβRIII (HT29-RIII) exhibit an increase in proliferation in response to TGF-β and BMP2 stimulation compared to HT29-Neo cells. This increase in proliferation is mediated through the down-regulation of p21. In a soft agar assay HT29-RIII cells demonstrate increased colony formation in response to ligand treatment. Additionally, HT29-RIII cells are resistant to anoikis and 5FU-induced apoptosis. Colon cancer cells expressing TβRIII also exhibit increased migration potential and this increase in migration can be abrogated by knockdown of TβRIII expression. Furthermore, cells over-expressing TβRIII show alterations in E-cadherin and actin organization. Taken together, these data suggest that TβRIII may have a pro-tumorigenic role in colon cancer tumorigenesis as it mediates ligand stimulated proliferation, resistance to apoptosis and increased migration. Further work is currently underway to more fully elucidate the role of TβRIII in colon cancer progression in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3972.