Devin T. Rosenthal

The Wnt5A/Protein Kinase C Pathway Mediates Motility in Melanoma Cells via the Inhibition of Metastasis Suppressors and Initiation of an Epithelial to Mesenchymal Transition

We have shown that Wnt5A increases the motility of melanoma cells. To explore cellular pathways involving Wnt5A, we compared gain-of-function (WNT5A stable transfectants) versus loss-of-function (siRNA knockdown) of WNT5A by microarray analysis. Increasing WNT5A suppressed the expression of several genes, which were re-expressed after small interference RNA-mediated knockdown of WNT5A. Genes affected by WNT5A include KISS-1, a metastasis suppressor, and CD44, involved in tumor cell homing during metastasis. This could be validated at the protein level using both small interference RNA and recombinant Wnt5A (rWnt5A). Among the genes up-regulated by WNT5A was the gene vimentin, associated with an epithelial to mesenchymal transition (EMT), which involves decreases in E-cadherin, due to up-regulation of the transcriptional repressor, Snail. rWnt5A treatment increases Snail and vimentin expression, and decreases E-cadherin, even in the presence of dominant-negativeTCF4, suggesting that this activation is independent of Wnt/β-catenin signaling. Because Wnt5A can signal via protein kinase C (PKC), the role of PKC in Wnt5A-mediated motility and EMT was also assessed using PKC inhibition and activation studies. Treating cells expressing low levels of Wnt5A with phorbol ester increased Snail expression inhibiting PKC in cells expressing high levels of Wnt5A decreased Snail. Furthermore, inhibition of PKC before Wnt5A treatment blocked Snail expression, implying that Wnt5A can potentiate melanoma metastasis via the induction of EMT in a PKC-dependent manner.

Characterization of the Roles of RHOC and RHOA GTPases in Invasion, Motility, and Matrix Adhesion in Inflammatory and Aggressive Breast Cancers*

The 2 closely related small GTPases, RHOC and RHOA, are involved in mammary gland carcinogenesis; however, their specific roles in determining cancer cell adhesion and invasion have not been elucidated.

Pomegranate Fruit Extract Impairs Invasion and Motility in Human Breast Cancer

Purpose. Pomegranate fruit extracts (PFEs) possess polyphenolic and other compounds with antiproliferative, pro-apoptotic and anti-inflammatory effects in prostate, lung, and other cancers. Because nuclear transcription factor-kB (NF-kB) is known to regulate cell survival, proliferation, tumorigenesis, and inflammation, it was postulated that PFEs may exert anticancer effects at least in part by modulating NF-kB activity. Experimental design. The authors investigated the effect of a novel, defined PFE consisting of both fermented juice and seed oil on the NF-kB pathway, which is constitutively active in aggressive breast cancer cell lines. The effects of the PFE on NF-kB—regulated cellular processes such as cell survival, proliferation, and invasion were also examined. Results. Analytical characterization of the bioactive components of the PFE revealed active constituents, mainly ellagitannins and phenolic acids in the aqueous PFE and conjugated octadecatrienoic acids in the lipid PFE derived from seeds.The aqueous PFE dose-dependently inhibited NF-kB—dependent reporter gene expression associated with proliferation, invasion, and motility in aggressive breast cancer phenotypes while decreasing RhoC and RhoA protein expression. Conclusion. Inhibition of motility and invasion by PFEs, coincident with suppressed RhoC and RhoA protein expression, suggests a role for these defined extracts in lowering the metastatic potential of aggressive breast cancer species.

p38γ Promotes Breast Cancer Cell Motility and Metastasis through Regulation of RhoC GTPase, Cytoskeletal Architecture, and a Novel Leading Edge Behavior

Understanding the molecular alterations that confer cancer cells with motile, metastatic properties is needed to improve patient survival. Here, we report that p38γ motogen-activated protein kinase regulates breast cancer cell motility and metastasis, in part, by controlling expression of the metastasis-associated small GTPase RhoC. This p38γ-RhoC regulatory connection was mediated by a novel mechanism of modulating RhoC ubiquitination. This relationship persisted across multiple cell lines and in clinical breast cancer specimens. Using a computational mechanical model based on the finite element method, we showed that p38γ-mediated cytoskeletal changes are sufficient to control cell motility. This model predicted novel dynamics of leading edge actin protrusions, which were experimentally verified and established to be closely related to cell shape and cytoskeletal morphology. Clinical relevance was supported by evidence that elevated expression of p38γ is associated with lower overall survival of patients with breast cancer. Taken together, our results offer a detailed characterization of how p38γ contributes to breast cancer progression. Herein we present a new mechanics-based analysis of cell motility, and report on the discovery of a leading edge behavior in motile cells to accommodate modified cytoskeletal architecture. In summary, these findings not only identify a novel mechanism for regulating RhoC expression but also advance p38γ as a candidate therapeutic target.

RhoC Impacts the Metastatic Potential and Abundance of Breast Cancer Stem Cells

Cancer stem cells (CSCs) have been shown to promote tumorigenesis of many tumor types, including breast, although their relevance to cancer metastasis remains unclear. While subpopulations of CSCs required for metastasis have been identified, to date there are no known molecular regulators of breast CSC (BCSC) metastasis. Here we identify RhoC GTPase as an important regulator of BCSC metastasis, and present evidence suggesting that RhoC also modulates the frequency of BCSCs within a population. Using an orthotopic xenograft model of spontaneous metastasis we discover that RhoC is both necessary and sufficient to promote SUM149 and MCF-10A BCSC metastasis–often independent from primary tumor formation–and can even induce metastasis of non-BCSCs within these cell lines. The relationship between RhoC and BCSCs persists in breast cancer patients, as expression of RhoC and the BCSC marker ALDH1 are highly correlated in clinical specimens. These results suggest new avenues to combating the deadliest cells driving the most lethal stage of breast cancer progression.

Antiangiogenic Tetrathiomolybdate Protects against Her2/neu-Induced Breast Carcinoma by Hypoplastic Remodeling of the Mammary Gland

Purpose: The objective of the present study was to delineate the efficacy of tetrathiomolybdate (TM), a novel antiangiogenic anticancer agent, as a chemopreventative agent. Experimental Design: Nulliparous Her2/neu transgenic mice were treated with water or TM for 180 days and observed for tumor development during treatment and for 180 days after treatment. Mammary gland composition and architecture were also observed following TM treatment of Her2/neu transgenic and normal FVB mice. Results: At the 1-year follow-up, 86.7% of control and 40% of TM-treated Her2/neu mice had palpable mammary tumors with a median time to tumor development of 234 days (95% confidence interval, 202-279 days) for control and >460 days for TM-treated mice (P < 0.0005, n = 15). The mammary glands from TM-treated Her2/neu and FVB mice showed a blunted epithelial ductal branching system due to a significant decrease in the number of secondary branches and total number of differentiated mammary epithelial cells. Microvessel density in Her2/neu and FVB mammary glands was lowered by 65.6 ± 6.2% and 50.9 ± 4.5% (P < 0.005), respectively, following TM therapy, consistent with the antiangiogenic effect of TM. Lastly, TM treatment resulted in a 2-fold increase in the absolute number of aldehyde dehydrogenase–positive mammary stem cells in Her2/neu and FVB mammary glands. Conclusion: Taken together, these results strongly support that TM is a potent chemopreventative agent as a consequence of hypoplastic remodeling of the mammary gland through modulation of the mammary stem cell compartment. (Clin Cancer Res 2009;15(23):7441–6)

A subset of presympathetic-premotor neurons within the centrally projecting Edinger-Westphal nucleus expresses urocortin-1

Numerous motivated behaviors require simultaneous activation of somatomotor and autonomic functions. We have previously characterized the organization of brain circuits that may mediate this integration. Presympathetic premotor neurons (PSPMNs) that are part of such circuits are distributed across multiple brain regions, which mediate stress-elicited behavioral and physiological responses, including the Edinger-Westphal nucleus (EW). Based on its connectivity and function, EW has recently been re-classified into a preganglionic (EWpg) and a centrally projecting (EWcp) population. Neurons within EWcp are the major source of urocortin 1 (Ucn-1), an analog of the corticotropin-releasing factor that binds the CRFR1 and CRFR2 receptors and has been implicated in mediating homeostatic responses to stress. We hypothesized that a subset of EWcp PSPMNs expresses Ucn-1. Utilizing dual-label immunofluorescence, we initially mapped the distribution of Ucn-1 and cholinergic neurons within EW in colchicine pre-treated rats. Based on this labeling we divided EWcp into three neuroanatomical levels. To examine connections of EWcp neurons to the gastrocnemius muscle and the adrenal gland, we next employed trans-synaptic tract-tracing in a second group of rats, utilizing two pseudorabies virus (PRV) recombinants that express unique reporter proteins. Using multi-label immunofluorescent staining, we identified the presence of Ucn-1-positive PSPMNs, dually labeled with PRV and present throughout the entire extent of EWcp and intermingled with Ucn-1 neurons infected with one or neither of the viral recombinants. Compared to rats pretreated with colchicine, we observed significantly fewer Ucn-1 neurons in animals that received PRV injections. Post hoc analyses revealed significantly fewer Ucn-1 neurons at the rostral level as compared to the caudal and middle levels. These data suggest functional and anatomic heterogeneity within EWcp; this organization may coordinate various aspects of stress-elicited and emotionally salient behaviors.

Rho Proteins in Cancer

From the earliest stages of embryonic development through the metastatic spread of cancer cells, cell motility is a critical feature of life. The Ras homology, or Rho, family of small GTPases plays a broad and essential role in all stages of cell motility. Rac and Cdc42 remodel the actin cytoskeleton at the leading edge of the cell, resulting in membrane ruffles and protrusions, while Rho is largely responsible for orchestrating focal adhesion assembly and generating contractile forces at the rear of the cell, thus permitting cell movement across these adhesive contacts and subsequent detachment by the trailing end of the cell. Activation and inactivation of these small GTPases is modulated by a group of regulatory proteins - guanine nucleotide exchange factors (GEFs) activate, while GTPase activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs) inhibit GTPase activity. Rho family proteins do not typically directly exert their effects on cell motility, but instead operate through a multitude of effector proteins, which each carry out a subset of motility-related functions. Because of their crucial roles in cell motility, Rho family protein expression and activation is altered in almost all types of cancer. As the body of knowledge surrounding Rho proteins, their interactions, and their regulation grows, so do the options for therapeutic intervention at an essential point of metastatic dissemination.

From in vitro to in silico and back again: Using biological and mathematical synergy to decipher breast cancer cell motility

The complexity of biological systems is often prohibitive in testing specific hypotheses from first physical principles. To circumvent these limitations we used biological data to inform a mathematical model of breast cancer cell motility. Using this in silico model we were able to accurately assess the influence of actin cytoskeletal architecture on the motility of a genetically modified breast cancer cell line. Furthermore, using the in silico model revealed a biological phenomenon that has not been previously described in live cell movement. Fusing biology and mathematics as presented here represents a new direction for biomedical research in which advances in each field synergistically drive discoveries in the other.

PKC and Breast Cancer

PKC expression is intimately associated with breast cancer initiation, progression, and therapy responsiveness, and these effects are highly isozyme-specific. PKC isozymes play key roles in proliferation and apoptosis of breast cancer cells and exert important modulatory roles in cell cycle progression. A close relationship exists between specific PKC isozymes and estrogen signaling.