Inna Solomonov

Multilevel regulation of matrix metalloproteinases in tissue homeostasis indicates their molecular specificity in vivo.

The matrix metalloproteinases (MMPs) play a crucial role in irreversible remodeling of the extracellular matrix (ECM) in normal homeostasis and pathological states. Accumulating data from various studies strongly suggest that MMPs are tightly regulated, starting from the level of gene expression all the way to zymogen activation and endogenous inhibition, with each level controlled by multiple factors. Recent in vivo findings indicate that cell-ECM and cell-cell interactions, as well as ECM bio-active products, contribute an additional layer of regulation at all levels, indicating that individual MMP expression and activity in vivo are highly coordinated and tissue specific processes.

Enzymatic turnover of macromolecules generates long-lasting protein–water-coupled motions beyond reaction steady state

Significance The solvent in biological reactions plays an active role in protein function; however, correlating solvation dynamics with specific biological scenarios remains a scientific challenge. Here, we followed time-dependent changes in solvation dynamics using terahertz absorption spectroscopy during proteolysis of collagen substrates by a metalloproteinase. Unexpectedly, we revealed that solvation dynamics do not follow the traditional enzymatic steady-state kinetic theory but generate long-lasting protein–water-coupled motions that last longer than a single catalytic cycle and are substrate-specific. These prolonged solvation dynamics contribute to the net enzyme reactivity impacting substrate binding, positional catalysis, and product release. The main focus of enzymology is on the enzyme rates, substrate structures, and reactivity, whereas the role of solvent dynamics in mediating the biological reaction is often left aside owing to its complex molecular behavior. We used integrated X-ray– and terahertz- based time-resolved spectroscopic tools to study protein–water dynamics during proteolysis of collagen-like substrates by a matrix metalloproteinase. We show equilibration of structural kinetic transitions in the millisecond timescale during degradation of the two model substrates collagen and gelatin, which have different supersecondary structure and flexibility. Unexpectedly, the detected changes in collective enzyme–substrate–water-coupled motions persisted well beyond steady state for both substrates while displaying substrate-specific behaviors. Molecular dynamics simulations further showed that a hydration funnel (i.e., a gradient in retardation of hydrogen bond (HB) dynamics toward the active site) is substrate-dependent, exhibiting a steeper gradient for the more complex enzyme–collagen system. The long-lasting changes in protein–water dynamics reflect a collection of local energetic equilibrium states specifically formed during substrate conversion. Thus, the observed long-lasting water dynamics contribute to the net enzyme reactivity, impacting substrate binding, positional catalysis, and product release.

Modeling invasive breast cancer: growth factors propel progression of HER2-positive premalignant lesions

The HER2/neu oncogene encodes a receptor-like tyrosine kinase whose overexpression in breast cancer predicts poor prognosis and resistance to conventional therapies. However, the mechanisms underlying aggressiveness of HER2 (human epidermal growth factor receptor 2)-overexpressing tumors remain incompletely understood. Because it assists epidermal growth factor (EGF) and neuregulin receptors, we overexpressed HER2 in MCF10A mammary cells and applied growth factors. HER2-overexpressing cells grown in extracellular matrix formed filled spheroids, which protruded outgrowths upon growth factor stimulation. Our transcriptome analyses imply a two-hit model for invasive growth: HER2-induced proliferation and evasion from anoikis generate filled structures, which are morphologically and transcriptionally analogous to preinvasive patients’ lesions. In the second hit, EGF escalates signaling and transcriptional responses leading to invasive growth. Consistent with clinical relevance, a gene expression signature based on the HER2/EGF-activated transcriptional program can predict poorer prognosis of a subgroup of HER2-overexpressing patients. In conclusion, the integration of a three-dimensional cellular model and clinical data attributes progression of HER2-overexpressing lesions to EGF-like growth factors acting in the context of the tumors microenvironment.

Zn2+-Aβ40 Complexes Form Metastable Quasi-spherical Oligomers That Are Cytotoxic to Cultured Hippocampal Neurons

Background: The mechanism by which interaction between Aβ and Zn2+ induces Aβ aggregation and cell toxicity is elusive. Results: Zn2+ and Aβ40 form metastable neurotoxic oligomers. Conclusion: Aβ40 binding to Zn2+ leads to formation of small neurotoxic oligomers that become benign upon further self-assembly. Significance: We provide a structure-function analysis of Zn2+-stabilized Aβ40, a neurotoxic species that may contribute to the pathology in AD. The roles of metal ions in promoting amyloid β-protein (Aβ) oligomerization associated with Alzheimer disease are increasingly recognized. However, the detailed structures dictating toxicity remain elusive for Aβ oligomers stabilized by metal ions. Here, we show that small Zn2+-bound Aβ1–40 (Zn2+-Aβ40) oligomers formed in cell culture medium exhibit quasi-spherical structures similar to native amylospheroids isolated recently from Alzheimer disease patients. These quasi-spherical Zn2+-Aβ40 oligomers irreversibly inhibit spontaneous neuronal activity and cause massive cell death in primary hippocampal neurons. Spectroscopic and x-ray diffraction structural analyses indicate that despite their non-fibrillar morphology, the metastable Zn2+-Aβ40 oligomers are rich in β-sheet and cross-β structures. Thus, Zn2+ promotes Aβ40 neurotoxicity by structural organization mechanisms mediated by coordination chemistry.

Tumor cell invasion can be blocked by modulators of collagen fibril alignment that control assembly of the extracellular matrix

Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR.

Introduction of correlative light and airSEM TM microscopy imaging for tissue research under ambient conditions

A complete fingerprint of a tissue sample requires a detailed description of its cellular and extracellular components while minimizing artifacts. We introduce the application of a novel scanning electron microscope (airSEMTM) in conjunction with light microscopy for functional analysis of tissue preparations at nanometric resolution (<10 nm) and under ambient conditions. Our metal-staining protocols enable easy and detailed visualization of tissues and their extracellular scaffolds. A multimodality imaging setup, featuring airSEMTM and a light microscope on the same platform, provides a convenient and easy-to-use system for obtaining structural and functional correlative data. The airSEMTM imaging station complements other existing imaging solutions and shows great potential for studies of complex biological systems.

Ovarian Dendritic Cells Act as a Double-Edged Pro- Ovulatory and Anti-Inflammatory Sword

Ovulation and inflammation share common attributes, including immune cell invasion into the ovary. The present study aims at deciphering the role of dendritic cells (DCs) in ovulation and corpus luteum formation. Using a CD11c-EYFP transgenic mouse model, ovarian transplantation experiments, and fluorescence-activated cell sorting analyses, we demonstrate that CD11c-positive, F4/80-negative cells, representing DCs, are recruited to the ovary under gonadotropin regulation. By conditional ablation of these cells in CD11c-DTR transgenic mice, we revealed that they are essential for expansion of the cumulus-oocyte complex, release of the ovum from the ovarian follicle, formation of a functional corpus luteum, and enhanced lymphangiogenesis. These experiments were complemented by allogeneic DC transplantation after conditional ablation of CD11c-positive cells that rescued ovulation. The pro-ovulatory effects of these cells were mediated by up-regulation of ovulation-essential genes. Interestingly, we detected a remarkable anti-inflammatory capacity of ovarian DCs, which seemingly serves to restrict the ovulatory-associated inflammation. In addition to discovering the role of DCs in ovulation, this study implies the extended capabilities of these cells, beyond their classic immunologic role, which is relevant also to other biological systems.

Distinct biological events generated by ECM proteolysis by two homologous collagenases

Significance Extracellular matrix (ECM) proteolysis is an abundant biochemical process. Here we describe the multilayered biological complexity generated by structurally homologous collagenases (matrix metalloproteinase-1 and matrix metalloproteinase-13) in collagen-rich, native ECM, that may prove central to tissue homeostasis and pathology. The events induced by these two collagenases generate microenvironments characterized by distinct chemical, biomechanical, and morphological ECM properties that lead to different cellular behaviors. Our findings improve the fundamental understanding of selective ECM degradation by homologous collagenases and its impact on cell behavior. It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM–cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.

Abstract 4724: Targeting matrix metalloproteinases (MMP) for anti-metastatic therapy: Blocking active MMP9 abrogates metastatic niche formation and prevents metastatic seeding in a breast cancer model

Currently there is no cure for a metastatic disease and it is therefore critical to target the early events that foster metastasis. It is now also recognized that a favorable microenvironment in the metastatic site, primed by the tumor, is crucial for metastasis. Our study is geared towards deciphering cellular and molecular mechanisms governing the metastatic niche that may lead to novel targeted anti-metastatic therapeutics. We utilize the multi-stage MMTV-PyMT breast cancer mouse model, which shares significant similarities with human breast cancer. By injecting a reporter metastatic cell line into hyperplasia-bearing mice, we were able to probe the susceptibility of the lung microenvironment to metastatic seeding. We demonstrate that early during mammary tumorigenesis, before metastasis has occurred, a metastatic niche is formed in the lung microenvironment. This niche is initiated in part by tumor-induced systemic pro-inflammatory factors and local extracellular matrix remodelers, as matrix metalloproteinases (MMPs). We show that the metastatic niche is associated with MMP9-expressing CD11b+Gr1+ and other lung stromal cells. MMP9, which is also expressed by tumor cells, appears as a pivotal player in the process and is therefore considered as a desirable therapeutic target. To examine the role of MMP9 activity in lung metastatic colonization, we utilized novel endogenous-like, function-specific antibodies (SDS3) that block the transiently-activated enzyme conformation of MMP9, which presumably contributes to disease progression. The therapeutic potential of SDS3 has been demonstrated in models of inflammatory bowel disease. We show that metastatic seeding within the lung microenvironment can be inhibited by SDS3, not only in experimental metastasis models but also when the lung microenvironment is primed by hyperplastic mammary tumors. Primary tumor burden was not changed with SDS3, suggesting that blocking active MMP9 is effective in preventing early metastasis rather than established tumors. To study the biodistribution and pharmacokinetics of SDS3, we utilized whole-body bioluminescence, intravital and ex-vivo live microscopy as well as flow cytometry. We show that SDS3 is retained in myeloid cells within the microenvironment of mammary tumors and lung metastatic foci. In situ zymography shows high MMP activity in premetastaic MMTV-PyMT lungs, which is reduced after SDS3. SDS3 also inhibits colony formation of cultured metastatic cells. Our results suggest that a metastatic niche is present in the lungs of hyperplastic mammary tumor-bearing mice and that it can be targeted by blocking MMP9 activity. Our study offers new insights into effectively blocking the in vivo activity of dysregulated MMPs as early anti-metastatic therapy of various cancers. Citation Format: Vicki Plaks, Jonathan Chou, Carrie Maynard, Nguyen H. Nguyen, Niwen Kong, Inna Solomonov, Dalit Talmi-Frank, Caroline Bonnans, Irit Sagi, Zena Werb. Targeting matrix metalloproteinases (MMP) for anti-metastatic therapy: Blocking active MMP9 abrogates metastatic niche formation and prevents metastatic seeding in a breast cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4724. doi:10.1158/1538-7445.AM2015-4724