Hepatocyte Stress Increases Expression of Yes-Associated Protein and Transcriptional Coactivator With PDZ-Binding Motif in Hepatocytes to Promote Parenchymal Inflammation and Fibrosis.

BACKGROUND AND AIMS: Activated hepatocytes are hypothesized to be a major source of signals that drive cirrhosis, but the biochemical pathways that convert hepatocytes into such a state are unclear. We examined the role of the Hippo pathway transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in hepatocytes to facilitate cell-cell interactions that stimulate liver inflammation and fibrosis. APPROACH AND RESULTS: Using a variety of genetic, metabolic, and liver injury models in mice, we manipulated Hippo signaling in hepatocytes and examined its effects in nonparenchymal cells to promote liver inflammation and fibrosis. YAP-expressing hepatocytes rapidly and potently activate the expression of proteins that promote fibrosis (collagen type I alpha 1 chain, tissue inhibitor of metalloproteinase 1, platelet-derived growth factor c, transforming growth factor ß2) and inflammation (tumor necrosis factor, interleukin 1ß). They stimulate expansion of myofibroblasts and immune cells, followed by aggressive liver fibrosis. In contrast, hepatocyte-specific YAP and YAP/TAZ knockouts exhibit limited myofibroblast expansion, less inflammation, and decreased fibrosis after CCl4 injury despite a similar degree of necrosis as controls. We identified cellular communication network factor 1 (CYR61) as a chemokine that is up-regulated by hepatocytes during liver injury but is expressed at significantly lower levels in mice with hepatocyte-specific deletion of YAP or TAZ. Gain-of-function and loss-of-function experiments with CYR61 in vivo point to it being a key chemokine controlling liver fibrosis and inflammation in the context of YAP/TAZ. There is a direct correlation between levels of YAP/TAZ and CYR61 in liver tissues of patients with high-grade nonalcoholic steatohepatitis. CONCLUSIONS: Liver injury in mice and humans increases levels of YAP/TAZ/CYR61 in hepatocytes, thus attracting macrophages to the liver to promote inflammation and fibrosis.

Pregnancy-associated breast cancers are driven by differences in adipose stromal cells present during lactation.

INTRODUCTION: The prognosis of breast cancer is strongly influenced by the developmental stage of the breast when the tumor is diagnosed. Pregnancy-associated breast cancers (PABCs), cancers diagnosed during pregnancy, lactation, or in the first postpartum year, are typically found at an advanced stage, are more aggressive and have a poorer prognosis. Although the systemic and microenvironmental changes that occur during post-partum involution have been best recognized for their role in the pathogenesis of PABCs, epidemiological data indicate that PABCs diagnosed during lactation have an overall poorer prognosis than those diagnosed during involution. Thus, the physiologic and/or biological events during lactation may have a significant and unrecognized role in the pathobiology of PABCs. METHODS: Syngeneic in vivo mouse models of PABC were used to examine the effects of system and stromal factors during pregnancy, lactation and involution on mammary tumorigenesis. Mammary adipose stromal cell (ASC) populations were isolated from mammary glands and examined by using a combination of in vitro and in vivo functional assays, gene expression analysis, and molecular and cellular assays. Specific findings were further investigated by immunohistochemistry in mammary glands of mice as well as in functional studies using ASCs from lactating mammary glands. Additional findings were further investigated using human clinical samples, human stromal cells and using in vivo xenograft assays. RESULTS: ASCs present during lactation (ASC-Ls), but not during other mammary developmental stages, promote the growth of carcinoma cells and angiogenesis. ASCs-Ls are distinguished by their elevated expression of cellular retinoic acid binding protein-1 (crabp1), which regulates their ability to retain lipid. Human breast carcinoma-associated fibroblasts (CAFs) exhibit traits of ASC-Ls and express crabp1. Inhibition of crabp1in CAFs or in ASC-Ls abolished their tumor-promoting activity and also restored their ability to accumulate lipid. CONCLUSIONS: These findings imply that (1) PABC is a complex disease, which likely has different etiologies when diagnosed during different stages of pregnancy; (2) both systemic and local factors are important for the pathobiology of PABCs; and (3) the stromal changes during lactation play a distinct and important role in the etiology and pathogenesis of PABCs that differ from those during post-lactational involution.

Extracellular Hsp90 Binds to and Aligns Collagen-1 to Enhance Breast Cancer Cell Invasiveness.

Cancer cell-secreted eHsp90 binds and activates proteins in the tumor microenvironment crucial in cancer invasion. Therefore, targeting eHsp90 could inhibit invasion, preventing metastasis-the leading cause of cancer-related mortality. Previous eHsp90 studies have solely focused on its role in cancer invasion through the 2D basement membrane (BM), a form of extracellular matrix (ECM) that lines the epithelial compartment. However, its role in cancer invasion through the 3D Interstitial Matrix (IM), an ECM beyond the BM, remains unexplored. Using a Collagen-1 binding assay and second harmonic generation (SHG) imaging, we demonstrate that eHsp90 directly binds and aligns Collagen-1 fibers, the primary component of IM. Furthermore, we show that eHsp90 enhances Collagen-1 invasion of breast cancer cells in the Transwell assay. Using Hsp90 conformation mutants and inhibitors, we established that the Hsp90 dimer binds to Collagen-1 via its N-domain. We also demonstrated that while Collagen-1 binding and alignment are not influenced by Hsp90's ATPase activity attributed to the N-domain, its open conformation is crucial for increasing Collagen-1 alignment and promoting breast cancer cell invasion. These findings unveil a novel role for eHsp90 in invasion through the IM and offer valuable mechanistic insights into potential therapeutic approaches for inhibiting Hsp90 to suppress invasion and metastasis.

Functional proteomic screens reveal an essential extracellular role for hsp90 alpha in cancer cell invasiveness.

Tumour cell invasiveness is crucial for cancer metastasis and is not yet understood. Here we describe two functional screens for proteins required for the invasion of fibrosarcoma cells that identified the molecular chaperone heat shock protein 90 (hsp90). The hsp90 alpha isoform, but not hsp90 beta, is expressed extracellularly where it interacts with the matrix metalloproteinase 2 (MMP2). Inhibition of extracellular hsp90 alpha decreases both MMP2 activity and invasiveness. This role for extracellular hsp90 alpha in MMP2 activation indicates that cell-impermeant anti-hsp90 drugs might decrease invasiveness without the concerns inherent in inhibiting intracellular hsp90.

Inhibition of Necl-5 (CD155/PVR) reduces glioblastoma dispersal and decreases MMP-2 expression and activity.

Patients afflicted with glioblastoma (GBM) have poor survival due to dispersive invasion throughout the brain. Necl-5, a cell surface receptor for vitronectin, is expressed in GBM but not normal brain. In several GBM cell lines Necl-5 promotes migration and invasion but the mechanism is poorly understood. In this study, we show that knockdown of Necl-5 by RNAi results in markedly decreased invasion of A172 GBM cells in a 3-dimensional matrix. There is a concomitant decrease in the expression and activity of matrix metalloproteinase-2 (MMP-2), a known factor in GBM invasion and disease severity. Knockdown of Necl-5 diminishes basal activation of Akt, an established mediator of MMP-2 expression in gliomas. Knockdown of Necl-5 also limits the maximal Akt activation in response to vitronectin, which requires the activity of Integrin-linked kinase (ILK). During migration, Necl-5, Akt and ILK co-localize at focal contacts at the leading edge of the plasma membrane, suggesting that these molecules may act to integrate Akt signaling at the leading edge to induce MMP-2 expression. By virtue of its restricted expression in GBM and its role in invasion, Necl-5 may be an attractive target for limiting MMP-2 production in glioblastoma, and therefore limiting dispersal.

Secretion of extracellular hsp90alpha via exosomes increases cancer cell motility: a role for plasminogen activation.

BACKGROUND: Metastasis is a multi-step process that is responsible for the majority of deaths in cancer patients. Current treatments are not effective in targeting metastasis. The molecular chaperone hsp90alpha is secreted from invasive cancer cells and activates MMP-2 to enhance invasiveness, required for the first step in metastasis. METHODS: We analyzed the morphology and motility of invasive cancer cells that were treated with exogenous exosomes in the presence or absence of hsp90alpha. We performed mass spectrometry and immunoprecipitation to identify plasminogen as a potential client protein of extracellular hsp90alpha. Plasmin activation assays and migration assays were performed to test if plasminogen is activated by extracellular hsp90alpha and has a role in migration. RESULTS: We found that hsp90alpha is secreted in exosomes in invasive cancer cells and it contributes to their invasive nature. We identified a novel interaction between hsp90alpha and tissue plasminogen activator that together with annexin II, also found in exosomes, activates plasmin. Extracellular hsp90alpha promotes plasmin activation as well as increases plasmin dependent cell motility. CONCLUSIONS: Our data indicate that hsp90alpha is released by invasive cancer cells via exosomes and implicates hsp90alpha in activating plasmin, a second protease that acts in cancer cell invasion.

Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone.

The myosin family of motor proteins is implicated in mediating actin-based growth cone motility, but the roles of many myosins remain unclear. We previously implicated myosin 1c (M1c; formerly myosin I beta) in the retention of lamellipodia (Wang et al., 1996). Here we address the role of myosin II (MII) in chick dorsal root ganglion neuronal growth cone motility and the contribution of M1c and MII to retrograde F-actin flow using chromophore-assisted laser inactivation (CALI). CALI of MII reduced neurite outgrowth and growth cone area by 25%, suggesting a role for MII in lamellipodial expansion. Micro-CALI of MII caused a rapid reduction in local lamellipodial protrusion in growth cones with no effects on filopodial dynamics. This is opposite to micro-CALI of M1c, which caused an increase in lamellipodial protrusion. We used fiduciary beads (Forscher et al., 1992) to observe retrograde F-actin flow during the acute loss of M1c or MII. Micro-CALI of M1c reduced retrograde bead flow by 76%, whereas micro-CALI of MII or the MIIB isoform did not. Thus, M1c and MIIB serve opposite and nonredundant roles in regulating lamellipodial dynamics, and M1c activity is specifically required for retrograde F-actin flow.

Dynactin is essential for growth cone advance.

Dynactin is a multi-subunit complex that serves as a critical cofactor of the microtubule motor cytoplasmic dynein. We previously identified dynactin in the nerve growth cone. However, the function of dynactin in the growth cone is still unclear. Here we show that dynactin in the growth cone is required for constant forward movement of the growth cone. Chromophore-assisted laser inactivation (CALI) of dynamitin, a dynactin subunit, within the growth cone markedly decreases the rate of growth cone advance. CALI of dynamitin in vitro dissociates another dynactin subunit, p150(Glued), from dynamitin. These results indicate that dynactin, especially the interaction between dynamitin and p150(Glued), plays an essential role in growth cone advance.

CD155/PVR enhances glioma cell dispersal by regulating adhesion signaling and focal adhesion dynamics.

We recently identified the immunoglobulin-CAM CD155/PVR (the poliovirus receptor) as a regulator of cancer invasiveness and glioma migration, but the mechanism through which CD155/PVR controls these processes is unknown. Here, we show that expression of CD155/PVR in rat glioma cells that normally lack this protein enhances their dispersal both in vitro and on primary brain tissue. CD155/PVR expression also reduced substrate adhesion, cell spreading, focal adhesion density, and the number of actin stress fibers in a substrate-dependent manner. Furthermore, we found that expression of CD155/PVR increased Src/focal adhesion kinase signaling in a substrate-dependent manner, enhancing the adhesion-induced activation of paxillin and p130Cas in cells adhering to vitronectin. Conversely, depletion of endogenous CD155/PVR from human glioma cells inhibited their migration, increased cell spreading, and down-regulated the same signaling pathway. These findings implicate CD155/PVR as a regulator of adhesion signaling and suggest a pathway through which glioma and other cancer cells may acquire a dispersive phenotype.

Functional proteomic screen identifies a modulating role for CD44 in death receptor-mediated apoptosis.

Apoptotic evasion is a hallmark of cancer and its resistance to chemotherapeutic drugs. Identification of cellular proteins that mediate apoptotic programs is a critical step toward the development of therapeutics aimed at overcoming apoptosis resistance. We developed an innovative high-throughput screen to identify proteins that modulate Fas ligand-mediated apoptosis using fluorophore-assisted light inactivation (HTS-FALIpop). The FALI protein knockdown strategy was coupled to a caspase activity assay with the ability to detect both proapoptotic and antiapoptotic surface molecules expressed by HT-1080 human fibrosarcoma cells. FALI of the Fas receptor (Fas/CD95) using a fluorescein-conjugated anti-Fas antibody abrogated Fas ligand-mediated caspase activation. Ninety-six single-chain variable fragment antibodies (scFv), selected for binding to the surface of HT-1080 cells, were screened by HTS-FALIpop. Three of the scFvs caused decreases in caspase induction after FALI of their protein targets. One of the targets of these positive scFvs was identified as CD44 and was validated by performing FALI using a CD44-specific monoclonal antibody, which resulted in similar protection from Fas apoptosis. CD44-targeted FALI was antiapoptotic in multiple human cancer cell lines, including both Fas signaling type I and II cells, and was also protective against other ligands of the tumor necrosis factor death receptor family. FALI of CD44 inhibited formation and activation of the death-inducing signaling complex, suggesting that CD44 regulates Fas at the cell surface. This mechanism of death receptor regulation represents a novel means of apoptosis modulation that could be exploited by pharmacologic agents.

Emerging Roles of Extracellular Hsp90 in Cancer.

Heat shock protein 90 (Hsp90) is a highly expressed chaperone that modulates the function and stability of hundreds of cellular client proteins. In this capacity, Hsp90 impacts human health in myriad ways and it is accordingly a high-interest molecular target in the oncology setting. This interest has led to a large number of clinical trials to evaluate the potential benefit of Hsp90 inhibitors in cancer treatment and, more recently, in combination with chemotherapeutic agents. Although these studies are still ongoing, some issues have arisen, such as toxicity effects associated with administration of these agents. We and others have identified a novel role for Hsp90 outside of cancer cells. This extracellular Hsp90 (eHsp90) was shown to be critical for the regulation of tumor invasiveness and metastasis, central processes associated with cancer lethality. Since these initial papers, a considerable cohort of studies has expanded upon this role, implicating eHsp90 in the activation of a number of proteins that support tumor cell invasion. As eHsp90 is preferentially detected on the surface of tumor cells, and within their surrounding microenvironment, it is possible that drugs capable of selectively targeting eHsp90 may exploit this differential expression. This selectivity may, in turn, enable treatment regimens with reduced target-related toxicity. This review will focus on our current understanding of eHsp90, particularly in cancer, and we will discuss the relevance of eHsp90 as a biomarker for invasive cancer and its potential as a drug target.

Inactivation of myelin-associated glycoprotein enhances optic nerve regeneration.

CNS regeneration in higher vertebrates is a long sought after goal in neuroscience. The lack of regeneration is attributable in part to inhibitory factors found in myelin (Caroni and Schwab, 1988a). Myelin-associated glycoprotein (MAG) is an abundant myelin protein that inhibits neurite outgrowth in vitro (McKerracher et al., 1994; Mukhopadhyay et al., 1994), but its role in regeneration remains controversial. To address this role, we performed nerve crush on embryonic day 15 chick retina-optic nerve explants and then acutely eliminated MAG function along the nerve using chromophore-assisted laser inactivation (CALI). CALI of MAG permitted significant regrowth of retinal axons past the site of lesion containing CNS myelin in contrast to various control treatments. Electron microscopy of the site of nerve crush shows abundant regenerating axons crossing the gap. When crushed optic nerve was retrogradely labeled at the nerve stump, no labeling of retinal neurons was observed. In contrast, labeling of CALI of MAG-treated crushed optic nerve showed significant retinal labeling (89 +/- 16 cells per square millimeter), a value indistinguishable from that seen with non-crushed nerve (98 +/- 13 cells per square millimeter). These findings implicate MAG as an important component of the myelin-derived inhibition of nerve regeneration. The acute loss of MAG function can promote significant axon growth across a site of CNS nerve damage.

Toward understanding the mechanism of chromophore-assisted laser inactivation--evidence for the primary photochemical steps.

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins of interest to elucidate their biological function. CALI has potential applications to a wide array of biological questions, and its efficiency allows for high-throughput application. A solid understanding of its underlying photochemical mechanism is still missing. In this study, we address the CALI mechanism using a simplified model system consisting of the enzyme beta-galactosidase as target protein and the common dye fluorescein. We demonstrate that protein photoinactivation is independent from dye photobleaching and provide evidence that the first singlet state of the chromophore is the relevant transient state for the initiation of CALI. Furthermore, the inactivation process was shown to be dependent on oxygen and likely to be based on photooxidation of the target protein via singlet oxygen. The simple model system used in this study may be further applied to identify and optimize other CALI chromophores.

Global high-throughput screens for cellular function.

There is a critical need for global methods that allow for high-throughput assessment of cellular function for clinical and basic scientists working in both academia and the pharmaceutical industry. These methods typically couple systematic inactivation strategies with high-throughput cell-based assays that facilitate rapid target validation. We present here a survey of these technologies and their applications. We discuss their promise and limitations in addressing the vast number of candidate molecules of disease relevance that are emerging from genomics and proteomics.

CD155/PVR plays a key role in cell motility during tumor cell invasion and migration.

BACKGROUND: Invasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration. METHODS: We developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion. RESULTS: Using this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and alphav-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate. CONCLUSION: These results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis.

An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein.

Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion.

Extracellular heat shock protein (Hsp)70 and Hsp90α assist in matrix metalloproteinase-2 activation and breast cancer cell migration and invasion.

Breast cancer is second only to lung cancer in cancer-related deaths in women, and the majority of these deaths are caused by metastases. Obtaining a better understanding of migration and invasion, two early steps in metastasis, is critical for the development of treatments that inhibit breast cancer metastasis. In a functional proteomic screen for proteins required for invasion, extracellular heat shock protein 90 alpha (Hsp90α) was identified and shown to activate matrix metalloproteinase 2 (MMP-2). The mechanism of MMP-2 activation by Hsp90α is unknown. Intracellular Hsp90α commonly functions with a complex of co-chaperones, leading to our hypothesis that Hsp90α functions similarly outside of the cell. In this study, we show that a complex of co-chaperones outside of breast cancer cells assists Hsp90α mediated activation of MMP-2. We demonstrate that the co-chaperones Hsp70, Hop, Hsp40, and p23 are present outside of breast cancer cells and co-immunoprecipitate with Hsp90α in vitro and in breast cancer conditioned media. These co-chaperones also increase the association of Hsp90α and MMP-2 in vitro. This co-chaperone complex enhances Hsp90α-mediated activation of MMP-2 in vitro, while inhibition of Hsp70 in conditioned media reduces this activation and decreases cancer cell migration and invasion. Together, these findings support a model in which MMP-2 activation by an extracellular co-chaperone complex mediated by Hsp90α increases breast cancer cell migration and invasion. Our studies provide insight into a novel pathway for MMP-2 activation and suggest Hsp70 as an additional extracellular target for anti-metastatic drug development.

Identification of CD44 as a surface biomarker for drug resistance by surface proteome signature technology.

We developed surface proteome signatures (SPS) for identification of new biomarkers playing a role in cancer drug resistance. SPS compares surface antigen expression of different cell lines by immunocytochemistry of a phage display antibody library directed to surface antigens of HT1080 fibrosarcoma cells. We applied SPS to compare the surface proteomes of two epithelial derived cancer cell lines, MCF7 and NCI/ADR-RES, which is drug resistant because of overexpression of the P-glycoprotein (P-gp) drug efflux pump. Surface proteomic profiling identified CD44 as an additional biomarker that distinguishes between these two cell lines. CD44 immunohistochemistry can distinguish between tumors derived from these lines and predict tumor response to doxorubicin in vivo. We further show that CD44 plays a role in drug resistance, independently of P-gp, in NCI/ADR-RES cells and increases expression of the antiapoptotic protein Bcl-xL. Our findings illustrate the utility of SPS to distinguish between cancer cell lines and their derived tumors and identify novel biomarkers involved in drug resistance.

Chromophore-assisted laser inactivation.

The major challenge of the post-genome world is ascribing in situ function to the myriad of proteins expressed in the proteome. This challenge is met by an arsenal of inactivation strategies that include RNAi and genetic knockout. These are powerful approaches but are indirect with respect to protein function and are subject to time delays before onset and possible genetic compensation. This chapter describes two protein-based inactivation approaches called chromophore-assisted laser inactivation (CALI) and fluorophore-assisted light inactivation (FALI). For CALI and FALI, light inactivation is targeted via photosensitizers that are localized to proteins of interest through antibody binding or expressed domains that are fluorescent or bind fluorescent probes. Inactivation occurs when and where the cells or tissues are irradiated and thus CALI and FALI provide an unprecedented level of spatial and temporal resolution of protein inactivation. Here we provide methods for the labeling of antibodies and setup of light sources and discuss controls, advantages of the technology, and potential pitfalls. We conclude with a discussion on a number of new technologies derived from CALI that combine molecular genetic approaches with light-induced inactivation that provide new tools to address in situ protein function.