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Lipids contribute to the structure, development, and function of healthy brains. Dysregulated lipid metabolism is linked to aging and diseased brains. However, our understanding of lipid metabolism in aging brains remains limited. Here we examined the brain lipidome of mice across their lifespan using untargeted lipidomics. Co-expression network analysis highlighted a progressive decrease in 3-sulfogalactosyl diacylglycerols (SGDGs) and SGDG pathway members, including the potential degradation products lyso-SGDGs. SGDGs show an age-related decline specifically in the central nervous system and are associated with myelination. We also found that an SGDG dramatically suppresses LPS-induced gene expression and release of pro-inflammatory cytokines from macrophages and microglia by acting on the NF-κB pathway. The detection of SGDGs in human and macaque brains establishes their evolutionary conservation. This work enhances interest in SGDGs regarding their roles in aging and inflammatory diseases and highlights the complexity of the brain lipidome and potential biological functions in aging.

Reliable approaches to identify stem cell mechanisms that mediate aggressive cancer could have great therapeutic value, based on the growing evidence of embryonic signatures in metastatic cancers. However, how to best identify and target stem-like mechanisms aberrantly acquired by cancer cells has been challenging. We harnessed the power of reprogramming to examine GRP78, a chaperone protein generally restricted to the endoplasmic reticulum in normal tissues, but which is expressed on the cell surface of human embryonic stem cells and many cancer types. We have discovered that (1) cell surface GRP78 (sGRP78) is expressed on iPSCs and is important in reprogramming, (2) sGRP78 promotes cellular functions in both pluripotent and breast cancer cells (3) overexpression of GRP78 in breast cancer cells leads to an induction of a CD24−/CD44+ tumor initiating cell (TIC) population (4) sGRP78+ breast cancer cells are enriched for stemness genes and appear to be a subset of TICs (5) sGRP78+ breast cancer cells show an enhanced ability to seed metastatic organ sites in vivo. These collective findings show that GRP78 has important functions in regulating both pluripotency and oncogenesis, and suggest that sGRP78 marks a stem-like population in breast cancer cells that has increased metastatic potential in vivo.

There exists a set of factors termed oncofetal proteins that play key roles in ontogeny before they decline or disappear as the organism’s tissues achieve homeostasis, only to then re-emerge in cancer. Although the unique therapeutic potential presented by such factors has been recognized for more than a century, their clinical utility has yet to be fully realized1. This review highlights the small signaling protein CRIPTO encoded by the tumor derived growth factor 1 (TDGF1/Tdgf1) gene, an oft cited oncofetal protein whose presence in the cancer literature as a tumor promoter, diagnostic marker and viable therapeutic target continues to grow. We touch lightly on features well established and well-reviewed since its discovery more than 30 years ago, including CRIPTO’s early developmental roles and modulation of SMAD2/3 activation by a selected set of transforming growth factor β (TGF-β) family ligands. We predominantly focus instead on more recent and less well understood additions to the CRIPTO signaling repertoire, on its potential upstream regulators and on new conceptual ground for understanding its mode of action in the multicellular and often stressful contexts of neoplastic transformation and progression. We ask whence it re-emerges in cancer and where it ‘hides’ between the time of its fetal activity and its oncogenic reemergence. In this regard, we examine CRIPTO’s restriction to rare cells in the adult, its potential for paracrine crosstalk, and its emerging role in inflammation and tissue regeneration—roles it may reprise in tumorigenesis, acting on subsets of tumor cells to foster cancer initiation and progression. We also consider critical gaps in knowledge and resources that stand between the recent, exciting momentum in the CRIPTO field and highly actionable CRIPTO manipulation for cancer therapy and beyond.

Background CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4L75A-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells. Methods We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4L75A-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4L75A-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4L75A-Fc, which represents a candidate therapeutic approach. Results ALK4L75A-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4L75A-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden. Conclusions Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4L75A-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.

Cripto is a small glycosylphosphatidylinisitol (GPI)-anchored and secreted oncofetal protein that plays important roles in regulating normal physiological processes, including stem cell differentiation, embryonal development, and tissue growth and remodeling, as well as pathological processes such as tumor initiation and progression. Cripto functions as a co-receptor for TGF-β ligands such as Nodal, GDF1, and GDF3. Soluble and secreted forms of Cripto also exhibit growth factor-like activity and activate SRC/MAPK/PI3K/AKT pathways. Glucose-Regulated Protein 78 kDa (GRP78) binds Cripto at the cell surface and has been shown to be required for Cripto signaling via both TGF-β and SRC/MAPK/PI3K/AKT pathways. To provide a comprehensive overview of the scientific literature related to Cripto, we performed, for the first time, a bibliometric analysis of the biological roles of Cripto as reported in the scientific literature covering the last 10 years. We present different fields of knowledge in comprehensive areas of research on Cripto, ranging from basic to translational research, using a keyword-driven approach. Our ultimate aim is to aid the scientific community in conducting targeted research by identifying areas where research has been conducted so far and, perhaps more importantly, where critical knowledge is still missing.

Background CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4L75A-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells. Methods We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4L75A-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4L75A-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4L75A-Fc, which represents a candidate therapeutic approach. Results ALK4L75A-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4L75A-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden. Conclusions Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4L75A-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe a novel androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harboursBRCA2 and CHD1somatic mutations, shows anSPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modelledin vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe an androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harbors BRCA2 and CHD1 somatic mutations, shows an SPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modeled in vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds., To date, patients still succumb to cancer, due to tumors not responding to therapy or ultimately acquiring resistance. Here the authors show that by exploiting patient derived organoids and a treatment-naïve patient derived xenograft, patient therapy can be personalized.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre-loxP-controlled lentiviral vectors expressing CRIPTO in cell line-derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft-derived ex vivo tumour slices. CRIPTO-overexpressing patient-derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial-to-mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2-CRIPTO cells formed tumours when injected into immune-compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High-level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO-expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The tumor microenvironment (TME) has been recognized as an integral component of malignancies in breast and prostate tissues, contributing in confounding ways to tumor progression, metastasis, therapy resistance, and disease recurrence. Major components of the TME are immune cells, fibroblasts, pericytes, endothelial cells, mesenchymal stroma/stem cells (MSCs), and extracellular matrix (ECM) components. Herein, we discuss the molecular and cellular heterogeneity within the TME and how this presents unique challenges and opportunities for treating breast and prostate cancers.

Reliable approaches to identify stem cell mechanisms that mediate aggressive cancer could have great therapeutic value, based on the growing evidence of embryonic signatures in metastatic cancers. However, how to best identify and target stem-like mechanisms aberrantly acquired by cancer cells has been challenging. We harnessed the power of reprogramming to examine GRP78, a chaperone protein generally restricted to the endoplasmic reticulum in normal tissues, but which is expressed on the cell surface of human embryonic stem cells and many cancer types. We have discovered that (1) cell surface GRP78 (sGRP78) is expressed on iPSCs and is important in reprogramming, (2) sGRP78 promotes cellular functions in both pluripotent and breast cancer cells (3) overexpression of GRP78 in breast cancer cells leads to an induction of a CD24-/CD44+ tumor initiating cell (TIC) population (4) sGRP78+ breast cancer cells are enriched for stemness genes and appear to be a subset of TICs (5) sGRP78+ breast cancer cells show an enhanced ability to seed metastatic organ sites in vivo. These collective findings show that GRP78 has important functions in regulating both pluripotency and oncogenesis, and suggest that sGRP78 marks a stem-like population in breast cancer cells that has increased metastatic potential in vivo.

To grow and cause disease, intracellular pathogens modulate host cell processes. Identifying these processes as well as the mechanisms used by the pathogens to manipulate them is important for the development of more effective therapeutics. As an example, the intracellular parasite Toxoplasma gondii induces a wide variety of changes to its host cell, including altered membrane trafficking, cytoskeletal reorganization, and differential gene expression. Although several parasite molecules and their host targets have been identified that mediate- these changes, few are known to be required for parasite replication. One exception is the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), which is required for parasite replication in an oxygen-dependent manner. Toxoplasma activates HIF-1 by stabilizing the HIF-1α subunit, and this is dependent on the signaling from the Activin-Like Kinase (ALK) receptor superfamily. Here, we demonstrate that specific overexpression of the ALK family member, ALK4, increased HIF-1 activity in Toxoplasma-infected cells, and this increase required ALK4 kinase activity. Moreover, Toxoplasma stimulated ALK4 to dimerize with its co-receptor, ActRII, and also increased ALK4 kinase activity, thereby demonstrating that Toxoplasma activates the ALK4 receptor. ALK4 activation of HIF-1 was independent of canonical SMAD signaling but rather was dependent on the non-canonical Rho GTPase and JNK MAP kinase signaling pathways. Finally, Toxoplasma increased rates of ALK4 ubiquitination and turnover. These data provide the first evidence indicating that ALK4 signaling is a target for a microbial pathogen to manipulate its host cell.

Background Little is known about the relationship between the metabolite profile of plasma from pre-operative prostate cancer (PCa) patients and the risk of PCa progression. In this study we investigated the association between pre-operative plasma metabolites and risk of biochemical-, local- and metastatic-recurrence, with the aim of improving patient stratification. Methods We conducted a case-control study within a cohort of PCa patients recruited between 1996 and 2015. The age-matched primary cases (n = 33) were stratified in low risk, high risk without progression and high risk with progression as defined by the National Comprehensive Cancer Network. These samples were compared to metastatic (n = 9) and healthy controls (n = 10). The pre-operative plasma from primary cases and the plasma from metastatic patients and controls were assessed with untargeted metabolomics by LC-MS. The association between risk of progression and metabolite abundance was calculated using multivariate Cox proportional-hazard regression and the relationship between metabolites and outcome was calculated using median cut-off normalized values of metabolite abundance by Log-Rank test using the Kaplan Meier method. Results Medium-chain acylcarnitines (C6-C12) were positively associated with the risk of PSA progression (p = 0.036, median cut-off) while long-chain acylcarnitines (C14-C16) were inversely associated with local (p = 0.034) and bone progression (p = 0.0033). In primary cases, medium-chain acylcarnitines were positively associated with suberic acid, which also correlated with the risk of PSA progression (p = 0.032, Log-Rank test). In the metastatic samples, this effect was consistent for hexanoylcarnitine, L.octanoylcarnitine and decanoylcarnitine. Medium-chain acylcarnitines and suberic acid displayed the same inverse association with tryptophan, while indoleacetic acid, a breakdown product of tryptophan metabolism was strongly associated with PSA (p = 0.0081, Log-Rank test) and lymph node progression (p = 0.025, Log-Rank test). These data were consistent with the increased expression of indoleamine 2,3 dioxygenase (IDO1) in metastatic versus primary samples (p = 0.014). Finally, functional experiments revealed a synergistic effect of long chain fatty acids in combination with dihydrotestosterone administration on the transcription of androgen responsive genes. Conclusions This study strengthens the emerging link between fatty acid metabolism and PCa progression and suggests that measuring levels of medium- and long-chain acylcarnitines in pre-operative patient plasma may provide a basis for improving patient stratification.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre-loxP-controlled lentiviral vectors expressing CRIPTO in cell line-derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft-derived ex vivo tumour slices. CRIPTO-overexpressing patient-derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial-to-mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2-CRIPTO cells formed tumours when injected into immune-compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High-level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO-expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

CRIPTO (CR-1, TDGF1) is a cell surface/secreted oncoprotein actively involved in development and cancer. Here, we report that high expression of CRIPTO correlates with poor survival in stratified risk groups of prostate cancer (PCa) patients. CRIPTO and its signaling partner glucose-regulated protein 78 (GRP78) are highly expressed in PCa metastases and display higher levels in the metastatic ALDH(high) sub-population of PC-3M-Pro4Luc2 PCa cells compared with non-metastatic ALDH(low). Coculture of the osteotropic PC-3M-Pro4Luc2 PCa cells with differentiated primary human osteoblasts induced CRIPTO and GRP78 expression in cancer cells and increases the size of the ALDH(high) sub-population. Additionally, CRIPTO or GRP78 knockdown decreases proliferation, migration, clonogenicity and the size of the metastasis-initiating ALDH(high) sub-population. CRIPTO knockdown reduces the invasion of PC-3M-Pro4Luc2 cells in zebrafish and inhibits bone metastasis in a preclinical mouse model. These results highlight a functional role for CRIPTO and GRP78 in PCa metastasis and suggest that targeting CRIPTO/GRP78 signaling may have significant therapeutic potential.

Recombinant bone morphogenetic protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 A that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue, which, when mutated to the corresponding BMP2 residue, resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union bone fractures.

Context: Uterine leiomyomas are highly prevalent benign tumors of premenopausal women and the most common indication for hysterectomy. However, the exact etiology of this tumor is not fully understood. Objective: The objective of the study was to evaluate the role of activin-A and myostatin and their signaling pathways in human myometrial and leiomyoma cells. Design: This was a laboratory study. Setting: Myometrial and leiomyoma cells (primary and cell lines) were cultured in vitro. Patients: The study included premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Interventions: Primary myometrial and leiomyoma cells and/or cell lines were treated with activin-A (4 nM) and myostatin (4 nM) for different days of interval (to measure proliferation rate) or 30 minutes (to measure signaling molecules) or 48 hours to measure proliferating markers, extracellular matrix mRNA, and/or protein expression by real-time PCR, Western blot, and/or immunocytochemistry. Results: We found th...

Summary Little is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell-surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers., Graphical Abstract, Highlights • CRIPTO/GRP78 signaling activates PI3K/AKT in fetal mammary epithelial cells ex vivo • Cell-surface GRP78 marks a CRIPTO-responsive adult mammary stem cell population • An antagonist, ALK4L75A-Fc, blocks soluble CRIPTO growth-factor-like activity • CRIPTO promotes and ALK4L75A-Fc inhibits mammary stem cell maintenance ex vivo, Little is known about the niche factors governing mammary stem cell function and if they are conserved throughout mammary gland development and homeostasis. Here, Gray and colleagues identify CRIPTO as a stem cell maintenance factor in fetal and adult mammary stem/progenitor cells and show that its cell-surface receptor, GRP78, is a mammary stem cell marker and determinant of CRIPTO responsiveness.

Nodal, a member of the TGF-β superfamily, plays an important role in vertebrate and invertebrate early development. The biochemical study of Nodal and its signaling pathway has been a challenge, mainly because of difficulties in producing the protein in sufficient quantities. We have developed a library of stable, chemically refoldable Nodal/BMP2 chimeric ligands (NB2 library). Three chimeras, named NB250, NB260, and NB264, show Nodal-like signaling properties including dependence on the co-receptor Cripto and activation of the Smad2 pathway. NB250, like Nodal, alters heart looping during the establishment of embryonic left-right asymmetry, and both NB250 and NB260, as well as Nodal, induce chondrogenic differentiation of human adipose-derived stem cells. This Nodal-induced differentiation is shown to be more efficient than BPM2-induced differentiation. Interestingly, the crystal structure of NB250 shows a backbone scaffold similar to that of BMP2. Our results show that these chimeric ligands may have therapeutic implications in cartilage injuries.

Cripto is a small, GPI-anchored signaling protein that regulates cellular survival, proliferation, differentiation and migration during normal developmental processes and tumorigenesis. Cripto functions as an obligatory co-receptor for the TGF-β ligands Nodal, GDF1 and GDF3 but attenuates signaling of others such as activin-A, activin-B and TGF-β1. Soluble, secreted forms of Cripto also activate Src, ras/raf/MAPK and PI3K/Akt pathways via a mechanism that remains largely obscure. This review describes the biological roles and signaling mechanisms of Cripto, highlighting our identification of the 78 kDa glucose regulated protein (GRP78) as a cell surface receptor/co-factor required for Cripto signaling via both TGF-β and Src/MAPK/PI3K pathways. We discuss emerging evidence indicating that Cripto/GRP78 signaling regulates normal somatic stem cells and their tumorigenic counterparts.

Raf kinase inhibitor protein (RKIP) regulates growth and differentiation signaling of mitogen-activated protein kinases (MAPK), GRK2 and NF-kappaB pathways each of which regulates cytotrophoblast differentiation and normal placental development. We show here that RKIP is expressed in human normal and preeclampic placentas as detected by immunostaining. RKIP was detected in villous cytotrophoblast in normal placenta and switched to syncytiotrophoblast in pre-eclampsia (PE)-complicated pregnancies. RKIP was also localized in extravillous cytotrophoblast of cell islands and cell columns both in normal and in PE placentas, although staining was less uniform in the latter specimens. In order to test RKIP involvement in cytotrophoblast function, we performed in vitro studies on HTR-8/SVneo cells, a first trimester cytotrophoblast cell line. We show that the RKIP inhibitor locostatin reduces ERK phosphorylation and impairs HTR-8/SV neo cells motility in wound closure experiments. We also document the presence of GRK2 mRNA, the reduction of phosphorylated RKIP expression by locostatin and the induction of PAI mRNA expression in HTR-8/SV neo cells, suggesting the involvement of GRK2 and NF-kappaB pathways in these cells. In conclusion, our work provides evidence that RKIP is a novel factor expressed in cytotrophoblast cells where it likely regulates cell migration.

Background Growth factors are proteins secreted by a number of cell types that are capable of modulating cellular growth, proliferation and cellular differentiation. It is well accepted that uterine cellular events such as proliferation and differentiation are regulated by sex steroids and their actions in target tissues are mediated by local production of growth factors acting through paracrine and/or autocrine mechanisms. Myometrial mass is ultimately modified in pregnancy as well as in tumour conditions such as leiomyoma and leiomyosarcoma. Leiomyomas, also known as fibroids, are benign tumours of the uterus, considered to be one of the most frequent causes of infertility in reproductive years in women. Methods For this review, we searched the database MEDLINE and Google Scholar for articles with content related to growth factors acting on myometrium; the findings are hereby reviewed and discussed. Results Different growth factors such as epidermal growth factor (EGF), transforming growth factor-α (TGF-α), heparin-binding EGF (HB-EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and TGF-β perform actions in myometrium and in leiomyomas. In addition to these growth factors, activin and myostatin have been recently identified in myometrium and leiomyoma. Conclusions Growth factors play an important role in the mechanisms involved in myometrial patho-physiology.

Introduction: Androgen-deprivation therapy is the standard treatment for prostate cancer. Despite the initial response, a fraction of cases manifest progression to castration-resistant prostate cancer. The paradigm of PCa recurrence is the existence of androgen-independent cancer stem cells, which differentiate into androgen-dependent cancer cells that reinitiate tumor growth. The phenotypic properties of cancer stem cells versus highly proliferative progenitor cells/facultative stem cells remain to be further characterized in terms of androgen receptor signaling, quiescence/proliferation, tumorigenicity, and therapy resistance. Methods: To address the molecular basis of CSC switch from androgen-dependency to independency we have employed two patient-derived xenograft models (LAPC-9 and BM-18) that have different androgen sensitivity properties. We have performed microarray and proteomic analysis of BM-18 tumor tissues prior to and following castration as well as androgen replacement. To characterize the cancer stem cells in these models we have isolated different subpopulations based on combination of selected markers and assessed the transcriptomic changes, proteomic profile, and in vivo self-renewal. Results: Castration induces a rapid tumor volume decrease in the BM-18 and a stabilization of tumor burden in the LAPC-9, reflecting different androgen-dependent cell states. Proteomic analysis of bulk BM-18 tumors (intact, 14 days post castration, 50 days post androgen replacement) indicates an enrichment of stem cell markers upon castration; CD44, NKX3.1, and ALDH1 isoforms. Microarray analysis has confirmed upregulation of CD44 and ALDH1A1 at castrated state, and downregulation of expression upon androgen replacement at early time points (24 hours). We have isolated CD44+/-ALDHhigh/low subpopulation by flow cytometry and analyzed their transcriptome and proteomic changes. Castration in the BM-18 model induces an increase in the subpopulations of CD44+/ALDHlow (from 0.44% to 2.3%) and CD44+/ALDHhigh (from 0.06% to 0.38%). However, the same subpopulations are decreased in the LAPC-9 model upon castration, while only the CD44-/ALDHhigh subset is enriched (from 3.5% to 7.1%). Conclusions: Different androgen-independent cancer stem cell subpopulations may be distinguished by the ALDH activity status in combination with CD44. The androgen-independent cells in the BM-18 androgen-dependent model are reflected by enrichment of CD44 expression, as well as a rare double positive population. In the androgen-independent LAPC-9 model, CD44 expression is contrastingly decreased, potentially reflecting androgen-dependent CD44+ cells, and ALDH activity increased in CD44- cells. Ongoing analysis may elucidate the molecular mechanisms controlling the different cancer stem cell fates and their androgen sensitivity. Cancer stem cell potential of the different cell populations will have to be elucidated by transplantation experiments. Citation Format: Sofia Karkampouna, Marta de Menna, Markus Germann, Jöel Grosjean, Peter C. Gray, George N. Thalmann, Marianna Kruithof-de Julio. Molecular characterization of cancer stem cells from patient-derived xenografts of advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B060.

Reliable approaches to identify and target stem-cell mechanisms that mediate aggressive cancer could have great therapeutic value, based on the growing evidence of embryonic signatures in metastatic cancers. However, how to best identify and target stem-like mechanisms aberrantly utilized by cancer cells has been challenging. We harnessed the power of induced pluripotent stem cells (iPSCs) to identify embryonic mechanisms exploited by cancer. A screen comparing the cell surface proteome of iPSCs and breast cancer cells identified GRP78, a heat shock protein that is normally ER-restricted, but has been shown to be aberrantly expressed on the cell surface of several cancers, where it can act as a signaling molecule by poorly understood mechanisms. Although cell surface GRP78 (sGRP78) has emerged as an attractive chemotherapeutic target, understanding how sGRP78 is functioning in cancer has been complicated by the fact that GRP78 can function to regulate a variety of cellular responses, using a diverse array of reported binding partners, which can vary by cell type. Therefore, without insight into the specific GRP78-dependent mechanisms that are responsible for mediating aggressive cancer, it will be difficult to determine how to best target GRP78. We have discovered that (1) sGRP78 is expressed on iPSCs (but not their somatic parental populations) and plays an important role in reprogramming, (2) sGRP78 promotes cellular functions such as proliferation/survival and migration in both stem cells and breast cancer cells (3) overexpression of GRP78 in breast cancer cells leads to an induction of a previously established CD24-/CD44+ 'cancer stem cell' (CSC) population (4) sGRP78+ breast cancer cell populations are enriched for genes involved in stemness and appear to be a subset of previously established CSCs (5) sGRP78+ breast cancer cell populations show a significantly enhanced ability to seed metastatic organ sites in vivo (6) GRP78 interacts with Dermcidin (DCD) at the cell surface of cancer cells and iPSCs, where it is important in regulating stem cell and cancer cell migration and survival/proliferation. These collective findings suggest that sGRP78 marks a stem-like population in breast cancer cells that has increased metastatic potential in vivo, and that sGRP78 and DCD cooperate to regulate key cellular functions important in mediating tumorigenesis. Overall, this work has implications for understanding how cancer cells exploit embryonic-like mechanisms, which could provide novel strategies for chemotherapeutic targeting of aggressive breast cancer cell populations. Citation Format: Tyson W. Lager, Henry C. Conner, Ian H. Guldner, Michael Z. Wu, Yuriko Hishida, Tomoaki Hishida, Sergio Ruiz, Amanda E. Yamasaki, Juan Carlos Izpisua Belmonte, Peter C. Gray, Jonathan A. Kelber, Siyuan Zhang, Athanasia D. Panopoulos. Aberrant cell surface expression of GRP78 in breast cancer cells marks a stem-like population that has increased metastatic potential in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1990.

Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation. Here we have developed a reproducible and efficient chondrogenic protocol to redifferentiate chondrocytes isolated from osteoarthritis (OA) patients. We used morphological, histological and immunological analysis together with a RT-PCR detection of collagen I and collagen II gene expression to show that chondrocytes isolated from articular cartilage biopsies of patients and subjected to long-term culture undergo dedifferentiation and that these cells can be redifferentiated following treatment with the chimeric Activin A/BMP2 ligand AB235. Examination of AB235-treated cell pellets in both in vitro and in vivo experiments revealed that redifferentiated chondrocytes synthesized a cartilage-specific extracellular matrix (ECM), primarily consisting of vertically-orientated collagen fibres and cartilage-specific proteoglycans. AB235-treated cell pellets also integrated into the surrounding subcutaneous tissue following transplantation in mice as demonstrated by their dramatic increase in size while non-treated control pellets disintegrated upon transplantation. Thus, our findings describe an effective protocol for the promotion of redifferentiation of autologous chondrocytes obtained from OA patients and the formation of a cartilage-like ECM that can integrate into the surrounding tissue in vivo.

Cripto-1 (CR-1) is a multifunctional embryonic protein that is re-expressed during inflammation, wound repair, and malignant transformation. CR-1 can function either as a tethered co-receptor or shed as a free ligand underpinning its flexible role in cell physiology. CR-1 has been shown to mediate cell growth, migration, invasion, and induce epithelial to mesenchymal transition (EMT). The main signaling pathways mediating CR-1 effects include Nodal-dependent (Smad2/3) and Nodal-independent (Src/p44/42/Akt) signaling transduction pathways. In addition, there are several naturally occurring binding partner proteins (BPPs) for CR-1 that can either agonize or antagonize its bioactivity. We will review the collective role of CR-1 as an extracellular protein, discuss caveats to consider in developing a quantitation assay, define possible mechanistic avenues applicable for drug discovery, and report on our experimental approaches to overcome these problematic issues.

Betaglycan is a co-receptor that mediates signaling by transforming growth factor beta (TGFbeta) superfamily members, including the distinct and often opposed actions of TGFbetas and inhibins. Loss of betaglycan expression, or abrogation of betaglycan function, is implicated in several human and animal diseases, although both betaglycan actions and the ligands involved in these disease states remain unclear. Here we identify a domain spanning amino acids 591-700 of the betaglycan extracellular domain as the only inhibin-binding region in betaglycan. This binding site is within the betaglycan ZP domain, but inhibin binding is not integral to the ZP motif of other proteins. We show that the inhibin and TGFbeta-binding residues of this domain overlap and identify individual amino acids essential for binding of each ligand. Mutation of Val614 to Tyr abolishes both inhibin and TGFbeta binding to this domain. Full-length betaglycan V614Y, and other mutations, retain TGFbeta binding activity via a distinct site, but are unable to bind inhibin-A. These betaglycan mutants fail to mediate inhibin antagonism of activin signaling but can present TGFbeta to TbetaRII. Separating the co-receptor actions of betaglycan toward inhibin and TGFbeta will allow the clarification of the role of betaglycan in disease states such as renal cell carcinoma and endometrial adenocarcinoma.

Activins are members of the transforming growth factor-beta (TGF-beta) superfamily that control many physiological processes such as cell proliferation and differentiation, immune responses, wound repair and various endocrine activities. Activins elicit these diverse biological responses by signaling via type I and type II receptor serine kinases. Recent studies have revealed details of the roles of inhibin, betaglycan, follistatin and its related protein follistatin-related gene (FLRG), Cripto and BAMBI in antagonizing activin action, and exogenous antagonists against the activin type I (SB-431542 and SB-505124) and type II (activin-M108A) receptors have been developed. Understanding how activin signaling is controlled extracellularly is the first step in providing treatment for wound healing and for disorders such as cachexia and cancer, which result from a deregulated activin pathway.

Activins, like other members of the transforming growth factor-beta (TGF-beta) superfamily, initiate signaling by assembling a complex of two types of transmembrane serine/threonine receptor kinases classified as type II (ActRII or ActRIIB) and type I (ALK4). A kinase-deleted version of ALK4 can form an inactive complex with activin and ActRII/IIB and thereby acts in a dominant negative manner to block activin signaling. Using the complex structure of bone morphogenetic protein-2 bound to its type I receptor (ALK3) as a guide, we introduced extracellular domain mutations in the context of the truncated ALK4 (ALK4-trunc) construct and assessed the ability of the mutants to inhibit activin function. We have identified five hydrophobic amino acid residues on the ALK4 extracellular domain (Leu40, Ile70, Val73, Leu75, and Pro77) that, when mutated to alanine, have substantial effects on ALK4-trunc dominant negative activity. In addition, eleven mutants partially affected activin binding to ALK4. Together, these residues likely constitute the binding surface for activin on ALK4. Cross-linking studies measuring binding of 125I-activin-A to the ALK4-trunc mutants in the presence of ActRII implicated the same residues. Our results indicate that there is only a partial overlap of the binding sites on ALK4 and ALK3 for activin-A and bone morphogenetic protein-2, respectively. In addition three of the residues required for activin binding to ALK4 are conserved on the type I TGF-beta receptor ALK5, suggesting the corresponding region on ALK5 may be important for TGF-beta binding.

Activins and bone morphogenetic proteins (BMPs) elicit diverse biological responses by signaling through two pairs of structurally related type I and type II receptors. Here we report the crystal structure of BMP7 in complex with the extracellular domain (ECD) of the activin type II receptor. Our structure produces a compelling four-receptor model, revealing that the types I and II receptor ECDs make no direct contacts. Nevertheless, we find that truncated receptors lacking their cytoplasmic domain retain the ability to cooperatively assemble in the cell membrane. Also, the affinity of BMP7 for its low-affinity type I receptor ECD increases 5-fold in the presence of its type II receptor ECD. Taken together, our results provide a view of the ligand-mediated cooperative assembly of BMP and activin receptors that does not rely on receptor-receptor contacts.

Activin and inhibin research has provided important insight into reproductive physiology as well as many areas involving regulation of cell growth, differentiation and function. Progress in understanding the roles of these hormones in various cell and tissue types has been complimented by novel discoveries at the molecular level that have shed light on ligand/receptor interactions, signaling mechanisms and regulation. While the receptors and signaling pathway for activin are now well characterized, the molecular basis for inhibin action has remained relatively unclear. Here we summarize recent advances in understanding inhibin’s mode of action focusing on our recent identification of betaglycan as an inhibin co-receptor capable of mediating inhibin action. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

Prostate Cancer (PCa) is the most frequent cancer in males and the second leading cause of death from cancer in men. When PCa progress from androgen-responsive to castration resistance, the formation of incurable metastases, mainly in the bone, is almost inevitable. Therefore, understanding the factors that regulate homing and survival of metastatic cancer cells in the bone is important for the identification of new therapeutic targets. High CD146 expression has been measured in the stroma of lytic and blastic lesions in preclinical models of PCa bone metastasis. The objective of this study is to characterize the role of CD146 in the maintenance of the aggressive and invasive phenotype in human PCa. We used shRNAs to knockdown the expression of CD146 in the lytic PC-3M-Pro4Luc2dTomato and in the blastic C4-2BdTomato PCa cell lines. We validated the knockdown at protein level and tested the effect with functional assays such as migration, proliferation. We used RT-qPCR to test CD146 knockdown on EMT markers. We measured the effect of the knockdown on the maintenance of cancer stem/progenitor-like cells by ALDEFLUOR assay. CD146 knockdown reduced proliferation in PC-3M-Pro4Luc2dTomato PCa cells and resulted in increased E-Cadherin expression. Conversely, no effect on proliferation was measured on C4-2BdTomato cells. It has been described that metastatic human PCa cells target the hematopoietic stem cell (HSC) niche in the bone marrow at the level of an “endosteal/osteoblast” niche and a “vascular/perivascular” niche. We optimized an in vitro model of “osteoblast niche” to study the behavior of prostate cancer cells upon co-culture with osteoblasts and to measure the resulting effects on cancer stem/progenitor-like markers. Our results showed that CD146 is required for the osteoblast-mediated induction of ALDH activity on PCa cells and CD146 knockdown prevented the increase in the size of the ALDHhigh subpopulation in the tumor cells, mediated by human osteoblasts. Additionally, CD146 knockdown in PCa cells co-cultured with osteoblast, reduced the amount of CD146 expressed by osteoblasts compared to non-targeted control. Finally, we showed that CD146 is significantly increased in the highly metastatic ALDHhigh cells and identified a new subset of ALDHhigh / CD146high cells which could be depleted upon CD146 knockdown. In Conclusion, we detected a novel subset of ALDHhigh/CD146high cells and found that CD146 influences the maintenance of an aggressive-mesenchymal phenotype in human PCa. Therefore, CD146 represents a promising molecule to modulate the behavior of aggressive PCa cells. Note: This abstract was not presented at the meeting. Citation Format: Eugenio Zoni, Letizia Astrologo, Janine Melsen, Sofia Karkampouna, Irena Klima, Joël Grosjean, Peter C. Gray, Gabri van der Pluijm, Marco G. Cecchini, Marianna Kruithof-de Julio, George N. Thalmann. Cd146 modulates the malignant phenotype in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4344. doi:10.1158/1538-7445.AM2017-4344

e16576 Background: Prostate cancer is the second most common cancer in men worldwide. Lethality is normally associated with the consequences of metastasis rather than the primary tumor. In particular, bone is the most frequent site of metastasis and once prostate tumor cells are engrafted in the skeleton, curative therapy is no longer possible. Bone morphogenetic proteins (BMPs) play a critical role in bone physiology and pathology. However, little is known about the role of BMP9 and its signaling receptors, ALK1 and ALK2, in prostate cancer and bone metastasis. In this context, we investigate the impact of BMP9 on primary prostate cancer and derived bone metastasis. Methods: The human ALK1 extracellular domain (ECD) binds BMP9 and BMP10 with high affinity. In order to study the effect of BMP9 in vitro and in vivo we use a soluble chimeric protein, consisting of ALK1 ECD fused to human Fc (ALK1Fc), for preventing the activation of endogenous signaling. ALK1Fc sequesters BMP9 and BMP10, preserving the activation of ALK1 through other ligands. Results: We show that ALK1Fc reduces BMP9-mediated signaling and decreases proliferation of highly metastatic and tumor initiating human prostate cancer cells in vitro. In line with these observations, we demonstrate that ALK1Fc reduces tumor growth in vivo in an orthotopic transplantation model. The propensity of the primary prostate cancer to metastasize to the bone is also investigated. In particular, we report how the ALK1Fc influences the prostate cancer cells in vitro and in vivo when these are probed in different bone settings (co-culture with bone cells and intraosseous transplantation in mice). Conclusions: Our study provides the first demonstration that ALK1Fc inhibits prostate cancer cells growth identifying BMP9 as a putative therapeutic target and ALK1Fc as a potential therapy. All together, these findings justify the ongoing clinical development of drugs blocking ALK1 and ALK2 receptor activity.

TGF-β superfamily ligands govern normal tissue development and homeostasis, and their dysfunction is a hallmark of many diseases. These ligands are also well defined both structurally and functionally. This review focuses on TGF-β superfamily ligand engineering for therapeutic purposes, in particular for regenerative medicine and musculoskeletal disorders. We describe the key discovery that structure-guided mutation of receptor-binding epitopes, especially swapping of these epitopes between ligands, results in new ligands with unique functional properties that can be harnessed clinically. Given the promising results with prototypical engineered TGF-β superfamily ligands, and the vast number of such molecules that remain to be produced and tested, this strategy is likely to hold great promise for the development of new biologics.

Activins and bone morphogenetic proteins (BMPs) share activin type 2 signaling receptors but utilize different type 1 receptors and Smads. We designed AB215, a potent BMP2-like Activin A/BMP2 chimera incorporating the high-affinity type 2 receptor-binding epitope of Activin A. In this study, we compare the signaling properties of AB215 and BMP2 in HEK293T cells and gonadotroph LβT2 cells in which Activin A and BMP2 synergistically induce FSHβ. In HEK293T cells, AB215 is more potent than BMP2 and competitively blocks Activin A signaling, while BMP2 has a partial blocking activity. Activin A signaling is insensitive to BMP pathway antagonism in HEK293T cells but is strongly inhibited by constitutively active (CA) BMP type 1 receptors. By contrast, the potencies of AB215 and BMP2 are indistinguishable in LβT2 cells and although AB215 blocks Activin A signaling, BMP2 has no inhibitory effect. Unlike HEK293T, Activin A signaling is strongly inhibited by BMP pathway antagonism in LβT2 cells but is largely unaffected by CA BMP type 1 receptors. BMP2 increases phospho-Smad3 levels in LβT2 cells, in both the absence and the presence of Activin A treatment, and augments Activin A-induced FSHβ. AB215 has the opposite effect and sharply decreases basal phospho-Smad3 levels and blocks Smad2 phosphorylation and FSHβ induction resulting from Activin A treatment. These findings together demonstrate that while AB215 activates the BMP pathway, it has opposing effects to those of BMP2 on FSHβ induction in LβT2 cells apparently due to its ability to block Activin A signaling.

Glucose Regulated Protein (GRP) 94 and GRP78 are critical molecular chaperones and regulators of signaling. Conditional knockout mouse models have revealed tissue specific requirements for GRP94 and GRP78, including selection for allele retention in specific cell types. Here we report the consequences of mammary-targeted knockout of these GRPs. Our studies revealed that MMTV-Cre, Grp94(f/f) mammary glands, despite GRP94 deficiency, exhibited normal proliferation and ductal morphogenesis. Interestingly, MMTV-Cre, Grp78(f/f) mammary glands displayed only slightly reduced GRP78 protein levels, associating with the retention of the non-recombined Grp78 floxed alleles in isolated mammary epithelial cells and displayed phenotypes comparable to wild-type glands. In contrast, transduction of isolated Grp78(f/f) mammary epithelial stem/progenitor cells with adenovirus expressing GFP and Cre-recombinase was successful in GRP78 ablation, and the GFP sorted cells failed to give rise to repopulated mammary glands in de-epithelialized recipient mice. These studies imply GRP78, but not GRP94, is required for mammary gland development.

Backround : Transesophageal echocardiography (TEE) is a useful procedure to evaluate selected stroke patients for cardiac sources of embolism. To date, noninfective valvular vegetations have not been described in large studies using transesophageal echocardiography to detect cardiac sources of embolism. We sought to investigate the frequency of noninfective valvular vegetations in patients with unexplained stroke referred for TEE and to determine the relationship of these vegetations to unrecognized thrombophilic disorders. Methods : We evaluated 641 consecutive patients referred for TEE as a result of unexplained stroke or transient ischemic attack for the presence of valvular vegetations. Of those with vegetations identified, serial blood cultures were obtained to evaluate for an infectious etiology. Patients also had serum testing for thrombophilic disorders and selected patients underwent cerebral angiography. Results : Thirteen patients (2%) who underwent TEE evaluation for unexplained stroke or transient ischemic attack were found to have noninfective valvular vegetations, all involving the mitral valve; none were identified by transthoracic echocardiography. Antiphospholipid antibodies were identified in 8 of these 13 patients (62%) and a protein C deficiency in 1 patient (8%). Conclusions : Noninfective valvular vegetations are a potential cardiac source of embolism in patients with unexplained stroke that can be better identified using transesophageal echocardiography. A large percentage of these individuals have a previously unrecognized thrombophilic disorder.

Subcellular targeting of the cAMP-dependent protein kinase is achieved, in part, through association with A-kinase anchoring proteins (AKAPs). Recent evidence suggests that specific AKAPs direct the kinase to submembrane sites to facilitate phosphorylation and modulation of a variety of ion channels. A new membrane-anchored AKAP targets cAMP-dependent protein kinase to calcium channels and enhances their regulation in multiple cell types.

Rapid, voltage-dependent potentiation of skeletal muscle L-type calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A kinase anchoring protein (AKAP). Here we report the isolation, primary sequence determination, and functional characterization of AKAP15, a lipid-anchored protein of 81 amino acid residues with a single amphipathic helix that binds PKA. AKAP15 colocalizes with L-type calcium channels in transverse tubules and is associated with L-type calcium channels in transfected cells. A peptide fragment of AKAP15 encompassing the RII-binding domain blocks voltage-dependent potentiation. These results indicate that AKAP15 targets PKA to the calcium channel and plays a critical role in voltage-dependent potentiation and regulation of skeletal muscle contraction. The expression of AKAP15 in the brain and heart suggests that it may mediate rapid PKA regulation of L-type calcium channels in neurons and cardiac myocytes.