Mihaela Lorger

Capturing Changes in the Brain Microenvironment during Initial Steps of Breast Cancer Brain Metastasis

Brain metastases are difficult to treat and mostly develop late during progressive metastatic disease. Patients at risk would benefit from the development of prevention and improved treatments. This requires knowledge of the initial events that lead to brain metastasis. The present study reveals cellular events during the initiation of brain metastasis by breast cancer cells and documents the earliest host responses to incoming cancer cells after carotid artery injection in immunodeficient and immunocompetent mouse models. Our findings capture and characterize heterogeneous astrocytic and microglial reactions to the arrest and extravasation of cancer cells in the brain, showing immediate and drastic changes in the brain microenvironment on arrival of individual cancer cells. We identified reactive astrocytes as the most active host cell population that immediately localizes to individual invading tumor cells and continuously associates with growing metastatic lesions. Up-regulation of matrix metalloproteinase-9 associated with astrocyte activation in the immediate vicinity of extravasating cancer cells might support their progression. Early involvement of different host cell types indicates environmental clues that might codetermine whether a single cancer cell progresses to macrometastasis or remains dormant. Thus, information on the initial interplay between brain homing tumor cells and reactive host cells may help develop strategies for prevention and treatment of symptomatic breast cancer brain metastases.

Activation of tumor cell integrin αvβ3 controls angiogenesis and metastatic growth in the brain

The incidence of brain metastasis is rising and poses a severe clinical problem, as we lack effective therapies and knowledge of mechanisms that control metastatic growth in the brain. Here we demonstrate a crucial role for high-affinity tumor cell integrin αvβ3 in brain metastatic growth and recruitment of blood vessels. Although αvβ3 is frequently up-regulated in primary brain tumors and metastatic lesions of brain homing cancers, we show that it is the αvβ3 activation state that is critical for brain lesion growth. Activated, but not non-activated, tumor cell αvβ3 supports efficient brain metastatic growth through continuous up-regulation of vascular endothelial growth factor (VEGF) protein under normoxic conditions. In metastatic brain lesions carrying activated αvβ3, VEGF expression is controlled at the post-transcriptional level and involves phosphorylation and inhibition of translational respressor 4E-binding protein (4E-BP1). In contrast, tumor cells with non-activated αvβ3 depend on hypoxia for VEGF induction, resulting in reduced angiogenesis, tumor cell apoptosis, and inefficient intracranial growth. Importantly, the microenvironment critically influences the effects that activated tumor cell αvβ3 exerts on tumor cell growth. Although it strongly promoted intracranial growth, the activation state of the receptor did not influence tumor growth in the mammary fat pad as a primary site. Thus, we identified a mechanism by which metastatic cells thrive in the brain microenvironment and use the high-affinity form of an adhesion receptor to grow and secure host support for proliferation. Targeting this molecular mechanism could prove valuable for the inhibition of brain metastasis.

An RNAi Screen Identifies TRRAP as a Regulator of Brain Tumor-Initiating Cell Differentiation

Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. In particular, knockdown of the adaptor protein TRRAP significantly increased differentiation of cultured BTICs, sensitized the cells to apoptotic stimuli, and negatively affected cell cycle progression. TRRAP knockdown also significantly suppressed tumor formation upon intracranial BTIC implantation into mice. Together, these findings support a critical role for TRRAP in maintaining a tumorigenic, stem cell-like state.

Targeting the variable surface of African trypanosomes with variant surface glycoprotein-specific, serum-stable RNA aptamers.

ABSTRACT African trypanosomes cause sleeping sickness in humans and Nagana in cattle. The parasites multiply in the blood and escape the immune response of the infected host by antigenic variation. Antigenic variation is characterized by a periodic change of the parasite protein surface, which consists of a variant glycoprotein known as variant surface glycoprotein (VSG). Using a SELEX (systematic evolution of ligands by exponential enrichment) approach, we report the selection of small, serum-stable RNAs, so-called aptamers, that bind to VSGs with subnanomolar affinity. The RNAs are able to recognize different VSG variants and bind to the surface of live trypanosomes. Aptamers tethered to an antigenic side group are capable of directing antibodies to the surface of the parasite in vitro. In this manner, the RNAs might provide a new strategy for a therapeutic intervention to fight sleeping sickness.

Tumor Microenvironment in the Brain

In addition to malignant cancer cells, tumors contain a variety of different stromal cells that constitute the tumor microenvironment. Some of these cell types provide crucial support for tumor growth, while others have been suggested to actually inhibit tumor progression. The composition of tumor microenvironment varies depending on the tumor site. The brain in particular consists of numerous specialized cell types such as microglia, astrocytes, and brain endothelial cells. In addition to these brain-resident cells, primary and metastatic brain tumors have also been shown to be infiltrated by different populations of bone marrow-derived cells. The role of different cell types that constitute tumor microenvironment in the progression of brain malignancies is only poorly understood. Tumor microenvironment has been shown to be a promising therapeutic target and diagnostic marker in extracranial malignancies. A better understanding of tumor microenvironment in the brain would therefore be expected to contribute to the development of improved therapies for brain tumors that are urgently required due to a poor availability of treatments for these malignancies. This review summarizes some of the known interactions between brain tumors and different stromal cells, and also discusses potential therapeutic approaches within this context.

Regulation of epithelial wound closure and intercellular adhesion by interaction of AF6 with actin cytoskeleton

AF6 is a human multi-domain protein involved in signaling and organization of cell junctions during embryogenesis. Its homologue in rat is called afadin. Three different AF6 transcripts are known, but only isoform 1 (AF6i1) has been characterized as protein. We focused on the AF6 isoform 3 (AF6i3), which differs from the AF6i1 by an additional C-terminal F-actin-binding site. Knockdown of AF6i3 in epithelial cells, which express only this isoform, resulted in impaired E-cadherin-dependent intercellular adhesion due to concomitantly reduced association of E-cadherin with F-actin and p120-catenin. Impaired intercellular adhesion also accelerated wound closure due to increased directionality of cell migration and delayed de novo formation of cell junctions. In contrast to AF6i3, the AF6i1 displayed a reduced association with the actin cytoskeleton and did not stabilize intercellular adhesion. Therefore, we propose that the AF6i3 protein stabilizes E-cadherin-dependent adhesion during dynamic processes, such as wound closure and formation of cell junctions, by linking the E-cadherin-catenin complex to the actin cytoskeleton via its F-actin-binding site.

A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis

During angiogenesis, Rho-GTPases influence endothelial cell migration and cell–cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell–cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis.

Activated tumor cell integrin αvβ3 cooperates with platelets to promote extravasation and metastasis from the blood stream

Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease.

Comparison of in vitro and in vivo approaches to studying brain colonization by breast cancer cells

Brain metastases occur in 20 to 40% of patients with metastatic breast cancer. The process is complex and depends on successful cancer cell evasion from the primary tumor, distribution and survival within the blood stream and cerebral microvasculature, penetration of the blood brain barrier and proliferation within the brain microenvironment. The initial steps of brain colonization are difficult to study in vivo. Therefore, in vitro assays have been developed to mimic this process. Most commonly, in vitro studies of brain colonization focus on tumor cell adhesion to brain endothelial cells and transendothelial migration. We previously investigated breast cancer brain colonization from the blood stream in vivo and defined the time and process of brain entry for five different cancer cell lines in a mouse model. We now investigated if in vitro approaches can reliably emulate the initial steps that determine successful brain colonization in vivo. To this end, we optimized an in vitro model of the vascular blood brain barrier and compared the brain invasion properties of the in vivo characterized cell models with their ability to interact with and penetrate the blood brain barrier model in vitro. Our results show that the in vitro findings correlate only poorly with the vivo results. The limitations of the in vitro approaches are discussed in light of the in vivo processes. We conclude that investigation of mechanisms supporting the earliest steps of breast cancer brain metastasis from the blood stream will depend on in vivo analyses.

c-Src-Mediated Epithelial Cell Migration and Invasion Regulated by PDZ Binding Site

ABSTRACT c-Src tyrosine kinase controls proliferation, cell adhesion, and cell migration and is highly regulated. A novel regulatory mechanism to control c-Src function that has recently been identified involves the C-terminal amino acid sequence Gly-Glu-Asn-Leu (GENL) of c-Src as ligand for PDZ domains. Herein, we determined the biological relevance of this c-Src regulation in human breast epithelial cells. The intact GENL sequence maintained c-Src in an inactive state in starved cells and restricted c-Src functions that might lead to metastatic transformation under normal growth conditions. c-Src with a C-terminal Leu/Ala mutation in GENL (Src-A) promoted the activation and translocation of cortactin and focal adhesion kinase and increased the motility and persistence of cell migration on the basement membrane. Src-A promoted increased extracellular proteolytic activity, and in acinar cultures, it led to the escape of cells through the basement membrane into the surrounding matrix. We ascribe the regulatory function of C-terminal Leu to the role of GENL in modulating c-Src activity downstream of cell matrix adhesion. We propose that the C terminus of c-Src via its GENL sequence presents a mechanism that restricts c-Src in epithelia and prevents progression toward an invasive phenotype.