Gino B. Ferraro

Myelin-Associated Inhibitors Regulate Cofilin Phosphorylation and Neuronal Inhibition through LIM Kinase and Slingshot Phosphatase

Myelin-associated inhibitors (MAIs) signal through a tripartate receptor complex on neurons to limit axon regeneration in the CNS. Inhibitory influences ultimately converge on the cytoskeleton to mediate growth cone collapse and neurite outgrowth inhibition. Rho GTPase and its downstream effector Rho kinase are key signaling intermediates in response to MAIs; however, the links between Rho and the actin cytoskeleton have not been fully defined. We found that Nogo-66, a potent inhibitory fragment of Nogo-A, signals through LIM (LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3) kinase and Slingshot (SSH) phosphatase to regulate the phosphorylation profile of the actin depolymerization factor cofilin. Blockade of LIMK1 activation and subsequent cofilin phosphorylation circumvents myelin-dependent inhibition in chick dorsal root ganglion neurons, suggesting that phosphorylation and inactivation of cofilin is critical for neuronal inhibitory responses. Subsequent activation of SSH1 phosphatase mediates cofilin dephosphorylation and reactivation. Overexpression of SSH1 does not mimic the neurite outgrowth inhibitory effects of myelin, suggesting an alternative role in MAI inhibition. We speculate that SSH-mediated persistent cofilin activation may be responsible for maintaining an inhibited neuronal phenotype in response to myelin inhibitors.

LGI1 Is a Nogo Receptor 1 Ligand that Antagonizes Myelin-Based Growth Inhibition

Mutations in leucine-rich glioma inactivated (LGI1) are a genetic cause of autosomal dominant temporal lobe epilepsy with auditory features. LGI1 is a secreted protein that shares homology with members of the SLIT family, ligands that direct axonal repulsion and growth cone collapse, and we therefore considered the possibility that LGI1 may regulate neuronal process extension or growth cone collapse. Here we report that LGI1 does not affect growth directly but instead enhances neuronal growth on myelin-based inhibitory substrates and antagonizes myelin-induced growth cone collapse. We show that LGI1 mediates this effect by functioning as a specific Nogo receptor 1 (NgR1) ligand that antagonizes the action of myelin-based inhibitory cues. Finally, we demonstrate that NgR1 and ADAM22 physically associate to form a receptor complex in which NgR1 facilitates LGI1 binding to ADAM22.

Emerging Strategies for Treating Brain Metastases from Breast Cancer

Brain metastasis is an end stage in breast cancer progression. Traditional treatment options have minimal efficacy, and overall survival is on the order of months. The incidence of brain metastatic disease is increasing with the improved management of systemic disease and prolongation of survival. Unfortunately, the targeted therapies that control systemic disease have diminished efficacy against brain lesions. There are reasons to be optimistic, however, as emerging therapies have shown promise in preclinical and early clinical settings. This review discusses recent advances in breast cancer brain metastasis therapy and potential approaches for successful treatment.

Membrane-type Matrix Metalloproteinase-3 Regulates Neuronal Responsiveness to Myelin through Nogo-66 Receptor 1 Cleavage

Nogo-66 receptor 1 (NgR1) is a glycosylphosphatidylinositol-anchored receptor for myelin-associated inhibitors that restricts plasticity and axonal regrowth in the CNS. NgR1 is cleaved from the cell surface of SH-SY5Y neuroblastoma cells in a metalloproteinase-dependent manner; however, the mechanism and physiological consequence of NgR1 shedding have not been explored. We now demonstrate that NgR1 is shed from multiple populations of primary neurons. Through a loss-of-function approach, we found that membrane-type matrix metalloproteinase-3 (MT3-MMP) regulates endogenous NgR1 shedding in primary neurons. Neuronal knockdown of MT3-MMP resulted in the accumulation of NgR1 at the cell surface and reduced the accumulation of the NgR1 cleavage fragment in medium conditioned by cortical neurons. Recombinant MT1-, MT2-, MT3-, and MT5-MMPs promoted NgR1 shedding from the surface of primary neurons, and this treatment rendered neurons resistant to myelin-associated inhibitors. Introduction of a cleavage-resistant form of NgR1 reconstitutes the neuronal response to these inhibitors, demonstrating that specific metalloproteinases attenuate neuronal responses to myelin in an NgR1-dependent manner.

14-3-3 Proteins Regulate Protein Kinase A Activity to Modulate Growth Cone Turning Responses

Growth cones regulate the speed and direction of neuronal outgrowth during development and regeneration. How the growth cone spatially and temporally regulates signals from guidance cues is poorly understood. Through a proteomic analysis of purified growth cones we identified isoforms of the 14-3-3 family of adaptor proteins as major constituents of the growth cone. Disruption of 14-3-3 via the R18 antagonist or knockdown of individual 14-3-3 isoforms switches nerve growth factor- and myelin-associated glycoprotein-dependent repulsion to attraction in embryonic day 13 chick and postnatal day 5 rat DRG neurons. These effects are reminiscent of switching responses observed in response to elevated cAMP. Intriguingly, R18-dependent switching is blocked by inhibitors of protein kinase A (PKA), suggesting that 14-3-3 proteins regulate PKA. Consistently, 14-3-3 proteins interact with PKA and R18 activates PKA by dissociating its regulatory and catalytic subunits. Thus, 14-3-3 heterodimers regulate the PKA holoenzyme and this activity plays a critical role in modulating neuronal responses to repellent cues.

Preclinical Efficacy of Ado-trastuzumab Emtansine in the Brain Microenvironment

BACKGROUND Central nervous system (CNS) metastases represent a major problem in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer because of the disappointing efficacy of HER2-targeted therapies against brain lesions. The antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. Here, we tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. METHODS We treated female nude mice bearing BT474 or MDA-MB-361 brain metastases (n = 9-11 per group) or cancer cells grown in organotypic brain slice cultures with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, and HER2 signaling. Data were analyzed with one-way analysis of variance (ANOVA), Kaplan-Meier analysis, and Coefficient of Determination. All statistical tests were two-sided. RESULTS T-DM1 delayed the growth of HER2-positive breast cancer brain metastases compared with trastuzumab. These findings were consistent between HER2-driven and PI3K-driven tumors. The activity of T-DM1 resulted in a survival benefit (median survival for BT474 tumors: 28 days for trastuzumab vs 112 days for T-DM1, hazard ratio = 6.2, 95% confidence interval = 6.1 to 85.84, P < .001). No difference in drug distribution or HER2-signaling was revealed between the two groups. However, T-DM1 led to a statistically significant increase in tumor cell apoptosis (one-way ANOVA for ApopTag, P < .001), which was associated with mitotic catastrophe. CONCLUSIONS T-DM1 can overcome resistance to trastuzumab therapy in HER2-driven or PI3K-driven breast cancer brain lesions due to the cytotoxicity of the DM1 component. Clinical investigation of T-DM1 for patients with CNS metastases from HER2-positive breast cancer is warranted.

Molecular targets to promote central nervous system regeneration.

Trauma in the adult mammalian central nervous system (CNS) results in devastating clinical consequences due to the failure of injured axons to spontaneously regenerate. This regenerative failure can be attributed to both a lack of positive cues and to the presence of inhibitory cues that actively prevent regeneration. Substantial progress has been made in elucidating the molecular identity of negative cues present at the CNS injury site following injury. In the past several years, multiple myelin-associated inhibitors including Nogo, Myelin-associated glycoprotein and Oligodendrocyte-myelin glycoprotein have been characterized. Furthermore a neuronal receptor complex and several intracellular substrates leading to outgrowth inhibition have been identified. Rapid progress has also been made in identifying the role of neurotrophins and other positive cues in promoting axonal regrowth. The most recent advances in our understanding of positive stimuli for axon regeneration come from transplantation studies at the CNS lesion site. A number of artificial substrates, tissues, and cells including fetal cells, neural stem cells, Schwann cells and olfactory-ensheathing cells have been tested in animal models of CNS injury. Based on our expanded knowledge of inhibitory influences and on the positive characteristics of various transplants, a number of interventions have been tested to promote recovery in models of CNS trauma. These advances represent the first steps in developing a viable therapy to promote axon regeneration following CNS trauma.

The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation

The brain microenvironment triggers HER3-dependent de novo resistance to therapies targeting PI3K or HER2 in HER2-positive and/or PIK3CA-mutant breast cancer cells. No safe haven for metastases Although targeted therapies for cancer offer great promise, they are often much less effective against brain metastases than against peripheral tumors. This is generally attributed to the drugs’ difficulty in penetrating the blood-brain barrier, but Kodack et al. now demonstrate that this is not the only reason. The authors discovered that, at least in breast cancer, the brain microenvironment itself plays a role in treatment resistance in metastatic tumors. Using mouse models and human cancer samples, the researchers found increased expression of human epidermal growth factor receptor 3 (HER3) in breast cancer–associated brain lesions and showed that it facilitates the tumors’ survival in the presence of targeted treatment and that inhibiting can help overcome resistance to therapy. Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases.

Treatment of Breast Cancer Brain Metastases.

Approximately 10% to 15% of women with metastatic breast cancer will develop brain metastases. Treatment options for these women remain limited, particularly at the time of central nervous system (CNS) relapse following completion of initial CNS-directed therapy. Historically, prior studies have broadly examined systemic treatments for breast cancer brain metastases with mixed, but overall disappointing, results. More recently, studies have increasingly selected patients based on breast cancer subtype and have examined novel, targeted agents that have preclinical suggestion of blood–brain barrier penetration. Correlative science objectives, with both tissue-based and novel imaging endpoints, are more frequently incorporated into trials of this nature, with the goal of enhancing our understanding of possible predictors of response. This review summarizes the current and emerging data on systemic therapy for breast cancer brain metastases and provides a framework for future directions in treating this clinically-challenging entity.

A cerebellar window for intravital imaging of normal and disease states in mice

The cerebellum is a prominent part of the vertebrate hindbrain that is critically involved in the regulation of important body functions such as movement coordination, maintenance of balance and posture, and motor control. Here, we describe a cerebellar window that provides access to the mouse cerebellum for intravital imaging, thereby allowing for a detailed characterization of the dynamic processes in this region of the brain. First, the skull overlying the cerebellum is removed, and then the window is applied to the region of interest. Windows may be exchanged depending on the desired imaging modality. This technique has a variety of applications. In the setting of medulloblastoma, spontaneous or orthotopically implanted lesions can be imaged, and tumor morphology and size can be monitored using ultrasonography. Multiphoton laser-scanning microscopy (MPLSM) or optical-frequency-domain imaging (OFDI) can be applied for in vivo visualization and analysis of cellular and vascular structures in a variety of disease states, including malignancies and ataxia telangiectasia. This protocol describes a novel and rapid method for cerebellar window construction that can be set up in under an hour.