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Membrane rafts play a crucial role in the regulation of many important biological processes. Our data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft-domain formation and regulation, including in cells with low expression of MPP1. We sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and MS technique. Thereby EFR3A was identified as a candidate protein which interacts with flotillin-2. This was further confirmed via immunoblotting using anti-EFR3A antibody and via co-immunoprecipitation (Co-IP). Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergentresistant membrane (DRM) fraction of HeLa cells, and its presence was sensitive to removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles (GPMVs) derived from KnD cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor (EGFR) and phospholipase C gamma (PLCγ) phosphorylation and affect EGF-dependent cytosolic Ca2+ concentration. Altogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which appears to be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling.

Membrane rafts are heterogeneous and dynamic domains that are characterized by tight packing of lipids. They are enriched in cholesterol, sphingolipids, and certain types of proteins. Among these are various cell signaling proteins, which indicate that rafts play an important role in cell signal transduction pathways, including some involved in cancer development, progression, and invasiveness. Due to their increased cholesterol content, raft domains exhibit lower fluidity than the surrounding membrane. The cell membranes of some solid tumors, such as breast and prostate cancer, contain higher levels of cholesterol, which means larger raft domain can form in those membranes. This may stimulate signaling pathways to promote tumor growth and progression. This review focuses on the known raft-dependent regulatory mechanisms that promote prostate cancer progression.Impact statementProstate cancer remains the most common malignancy and second most frequent cause of cancer-related death in men. Cholesterol levels are usually higher in prostate cancer cells. This affects the cell membrane composition, with cholesterol and sphingolipid-containing raft membrane domains becoming a greater component. In addition to polar lipids, these domains recruit and regulate certain types of protein, including various cell signaling proteins that are critical to cancer cell survival and invasiveness. This suggests that membrane rafts have a regulatory role in tumor progression, making them a potential target in prostate cancer treatment.

Inhibition of the protein neddylation process by the small-molecule inhibitor MLN4924 has been recently indicated as a promising direction for cancer treatment. However, the knowledge of all biological consequences of MLN4924 for cancer cells is still incomplete. Here, we report that MLN4924 inhibits tumor necrosis factor-alpha (TNF-α)-induced matrix metalloproteinase 9 (MMP9)-driven cell migration. Using real-time polymerase chain reaction (PCR) and gelatin zymography, we found that MLN4924 inhibited expression and activity of MMP9 at the messenger RNA (mRNA) and protein levels in both resting cells and cells stimulated with TNF-α, and this inhibition was closely related to impaired cell migration. We also revealed that MLN4924, similar to TNF-α, induced phosphorylation of inhibitor of nuclear factor kappa B-alpha (IκB-α). However, contrary to TNF-α, MLN4924 did not induce IκB-α degradation in treated cells. In coimmunoprecipitation experiments, nuclear IκB-α which formed complexes with nuclear factor kappa B p65 subunit (NFκB/p65) was found to be highly phosphorylated at Ser32 in the cells treated with MLN4924, but not in the cells treated with TNF-α alone. Moreover, in the presence of MLN4924, nuclear NFκB/p65 complexes were found to be enriched in c-Jun and cyclin dependent kinase inhibitor 1 A (CDKN1A/p21) proteins. In these cells, NFκB/p65 was unable to bind to the MMP9 gene promoter, which was confirmed by the chromatin immunoprecipitation (ChIP) assay. Taken together, our findings identified MLN4924 as a suppressor of TNF-α-induced MMP9-driven cell migration in esophageal squamous cell carcinoma (ESCC), likely acting by affecting the nuclear ubiquitin–proteasome system that governs NFκB/p65 complex formation and its DNA binding activity in regard to the MMP9 promoter, suggesting that inhibition of neddylation might be a new therapeutic strategy to prevent invasion/metastasis in ESCC patients.

172 Background: Mechanisms of tumor invasion are not well defined. PTEN, a key tumor suppressor frequently inactivated in epithelial cancers, acts as a central node that controls tumor invasion. Despite PI-3 kinase-phospho-Akt pathway activation resulting in enhanced tumor growth, prostate tumors with PTEN loss undergo p53-mediated senescence that restricts tumor invasion. Methods: ABI1 downregulation is associated with epithelial-mesenchymal transition in highly invasive prostate tumors; these tumors frequently loose PTEN; therefore we set to examine genetic interaction of ABI1 and PTEN using novel mouse model of prostate cancer. We analyzed the correlation of ABI1 and PTEN expression in human PCa tumor tissue. Results: Here, using Abi1/Pten KO mouse model we identified a novel mechanism that guards tumor invasion. In Pten-null tumors upregulation of Abi1 leads to sequestration of activated Src kinase. In the absence of Abi1, this regulation is lost leading to activation of non-canonical WNT-SRC-STAT3 axis and enhanced invasion through activation of MMP2 activity. This molecular mechanism explains progression of tumors with Pten loss from PIN to invasive carcinoma upon concomitant Abi1 inactivation. In human tumors with low Abi1 and Pten are associated with aggressive phenotype, biochemical recurrence and metastasis. Conclusions: ABI1 acts as failsafe mechanism in PTEN null tumors by restricting SRC-mediated tumor invasion. ABI1 might have a predictive value in clinical setting in context of PTEN levels.

We describe a novel method of urethral stricture treatment using liquid buccal mucosal grafts to augment direct vision internal urethrotomy.A rabbit stricture model was used to test this method. In phase 1 the concept of endoscopic liquid buccal mucosal graft implantation was tested by performing direct vision internal urethrotomy in 3 rabbits with immediate intraurethral injection of autologous liquid buccal mucosal grafts suspended in fibrin glue. Animals were sacrificed at 2 to 3 weeks and the urethras were examined for the presence of buccal mucosa engraftment. In phase 2 strictures were induced by electroresection in 9 rabbits divided into 2 groups, including 1) 6 rabbits treated with direct vision internal urethrotomy and liquid buccal mucosal grafts, and 2) 3 controls that underwent direct vision internal urethrotomy and injection of fibrin glue only. Two treated and 1 control animals were sacrificed at 8, 16 and 24 weeks each. Prior to sacrifice the animals underwent retrograde urethrograms and urethroscopy. Histological specimens were examined for the presence of buccal mucosal engraftment.In phase 1, 2 of the 3 rabbits demonstrated engraftment of buccal mucosa in the urethra after injection of liquid buccal mucosal grafts. In phase 2 all 6 treated animals demonstrated engraftment with resolution/improvement of strictures on retrograde urethrograms and urethroscopy. Controls had no buccal engraftment and showed fibrosis and chronic inflammation. One of the 3 controls had persistent stricture on retrograde urethrograms and cystoscopy.This proof of concept study demonstrated the feasibility of using liquid buccal mucosal grafts for endoscopic urethral stricture repair. Such a method may allow for wide application of this novel concept of using liquid buccal mucosal grafts to augment direct vision internal urethrotomy.

Background Prostate cancer development involves various mechanisms, which are poorly understood but pointing to epithelial mesenchymal transition (EMT) as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, acts as tumor suppressor in prostate cancer but the role of ABI1 in EMT is not clear. Methods To investigate the molecular mechanism by which loss of ABI1 contributes to tumor progression, we disrupted the ABI1 gene in the benign prostate epithelial RWPE-1 cell line and determined its phenotype. Levels of ABI1 expression in prostate organoid tumor cell lines was evaluated by Western blotting and RNA sequencing. ABI1 expression and its association with prostate tumor grade was evaluated in a TMA cohort of 505 patients and metastatic cell lines. Results Low ABI1 expression is associated with biochemical recurrence, metastasis and death (p = 0.038). Moreover, ABI1 expression was significantly decreased in Gleason pattern 5 vs. pattern 4 (p = 0.0025) and 3 (p = 0.0012), indicating an association between low ABI1 expression and highly invasive prostate tumors. Disruption of ABI1 gene in RWPE-1 cell line resulted in gain of an invasive phenotype, which was characterized by a loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 spheroids. Through RNA sequencing and protein expression analysis, we discovered that ABI1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of ABI1. Furthermore, an increase in STAT3 phosphorylation upon ABI1 inactivation and the evidence of a high-affinity interaction between the FYN SH2 domain and ABI1 pY421 support a model in which ABI1 acts as a gatekeeper of non-canonical WNT-EMT pathway activation downstream of the FZD2 receptor. Conclusions ABI1 controls prostate tumor progression and epithelial plasticity through regulation of EMT-WNT pathway. Here we discovered that ABI1 inhibits EMT through suppressing FYN-STAT3 activation downstream from non-canonical WNT signaling thus providing a novel mechanism of prostate tumor suppression.

Biological membranes are organized in various microdomains, one of the best known being called membrane rafts. The major function of these is thought to organize signaling partners into functional complexes. An important protein found in membrane raft microdomains of erythroid and other blood cells is MPP1 (membrane palmitoylated protein 1)/p55. MPP1 (p55) belongs to the MAGUK (membrane-associated guanylate kinase homolog) family and it is a major target of palmitoylation in the red blood cells (RBCs) membrane. The well-known function of this protein is to participate in formation of the junctional complex of the erythrocyte mem-brane skeleton. However, its function as a "raft organizer" is not well understood. In this review we focus on recent reports concerning MPP1 participation in membrane rafts organization in erythroid cells, including its role in signal transduction. Currently it is not known whether MPP1 could have a similar role in cell types other than erythroid lineage. We present also preliminary data regarding the expression level of MPP1 gene in several non-erythroid cell lines.

The invasive nature of many cancer cells involves the formation of F-actin-based, lipid-raft-enriched membrane protrusions known as invadopodia or, more broadly, invadosomes. Invadopodia are specialized adhesive structures arising from ventral cell surface within cell-extracellular matrix (ECM) contacts and concentrate high proteolytic activities that allow cells to overcome the dense scaffold of local microenvironment, comprising a natural barrier to cell spreading. This degradative activity distinguishes invadopodia from other adhesive structures like focal adhesions, lamellipodia or filopodia, and is believed to drive cancer progression.

Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of

Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of Abi1 in a novel mouse model leads to development of an MPN-like phenotype resembling human PMF. Abi1 loss resulted in a significant increase in the activity of the Src family kinases (SFKs), STAT3, and NF-κB signaling. We also observed impairment of hematopoietic stem cell self-renewal and fitness, as evidenced in noncompetitive and competitive bone marrow transplant experiments. CD34+ hematopoietic progenitors and granulocytes from patients with PMF showed decreased levels of ABI1 transcript as well as increased activity of SFKs, STAT3, and NF-κB. In aggregate, our data link the loss of Abi-1 function to hyperactive SFKs/STAT3/NF-κB signaling and suggest that this signaling axis may represent a regulatory module involved in the molecular pathophysiology of PMF.

Metastasis is the leading cause of mortality in patients with highly invasive cancers and, as such, is a major problem for medicine. It has been increasingly recognized that cancer-related inflammation plays an important role in promoting invasion and the metastatic process in which cell motility and upregulation of proteolytic enzymes are crucial events. TNFα is a proinflammatory cytokine known to stimulate synthesis of MMP9, a zinc- and calcium-dependent endopeptidase contributing to the regulation of ECM remodeling and cell signaling. However, the precise molecular mechanism of TNFα-induced MMP9 gene expression in cancers is still not fully understood. This study shows that TNFα-induced cell migration and invasion involve ERK1/2-dependent up-regulation of CDKN1A/p21 expression in highly aggressive breast cancer cells and that CDKN1A/p21 plays an important regulatory role in TNFα-induced MMP9 gene expression, indicating an unknown function of CDKN1A/p21 as a regulator of proteolytic activity in cancer cells.

To better understand the role of membrane-associated proteolytic systems in the development of esophageal cancer, we studied the expression of two serine proteases, fibroblast activation protein-α (FAP-α) and dipeptidyl peptidase IV (DPPIV) and three metalloproteinases, matrix metalloproteinase (MMP)-2, MMP-9 and MT1-MMP in 24 primary esophageal squamous cell carcinoma (ESCC) tissues and paired non-cancer tissues. Using reverse-transcription PCR, western blotting and zymography, we showed that both serine proteases and all three metalloproteinases were highly altered in ESCC. A positive correlation between the expression of FAP-α and DPPIV and the activity of both gelatinases was found. This may indicate that these proteolytic systems are tightly linked to each other and collectively are involved in the process of ECM degradation that facilitates cancer cell invasion and metastasis.

The spectrin-based membrane skeleton is crucial for the mechanical stability and resilience of erythrocytes. It mainly contributes to membrane integrity, protein organization and trafficking. Two transmembrane protein macro-complexes that are linked together by spectrin tetramers play a crucial role in attaching the membrane skeleton to the cell membrane, but they are not exclusive. Considerable experimental data have shown that direct interactions between spectrin and membrane lipids are important for cell membrane cohesion. Spectrin is a multidomain, multifunctional protein with several distinctive structural regions, including lipid-binding sites within CH tandem domains, a PH domain, and triple helical segments, which are excellent examples of ligand specificity hidden in a regular repetitive structure, as recently shown for the ankyrin-sensitive lipid-binding domain of beta spectrin. In this review, we summarize the state of knowledge about interactions between spectrin and membrane lipids.

280 Background: Prostate cancer is characterized by heterogeneity of mechanisms which are poorly understood but pointing to epithelial plasticity as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, is proposed to act as tumor suppressor in prostate cancer, but the mechanism of tumor progression due to Abi1 loss is not clear. Methods: To address Abi1’s role in prostate cancer we used CRISPR-based gene editing and retroviral expression to manipulate Abi1 levels in prostate cancer cell lines. Levels of Abi1 expression in prostate organoid tumor cell lines were evaluated by Western blotting and/or RNA sequencing. Association of Abi1 loss with tumor grade was evaluated by immunohistochemistry. Results: Abi1 expression is downregulated in tumor organoid cell lines from metastatic bone and lymph node biopsies. Moreover, low Abi1 expression is associated with high-grade prostate tumors (GG3 or higher, p < 0.001). Disruption of Abi1 gene in a benign prostate epithelial cell line RWPE-1 resulted in a gain of invasive phenotype, which is characterized by loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 Abi1 KO spheroids. Through RNA sequencing and protein expression analysis we discovered that Abi1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of Abi1. Furthermore, increase in STAT3 phosphorylation upon Abi1 inactivation and evidence for high affinity interaction of FYN-SH2 domain with Abi1 pY421 support the model that Abi1 acts as a gatekeeper of the non-canonical WNT-EMT pathway activation downstream from FZD2 receptor. The gene expression profile of Abi1-EMT-WNT pathway overlaps with the reported gene signature of high-risk prostate tumors. Conclusions: Abi1 contributes to prostate cancer progression and epithelial plasticity through regulation of EMT-WNT pathways. Understanding of Abi1’s role may provide more mechanistic understanding of prostate cancer tumor progression.

American Cancer Society predicts over 160,000 new prostate cancer cases in 2017, which amounts to about 20 percent of all cancer diagnoses in men. Despite several treatment options being available, poor prognosis of high-risk and metastasis remains a major concern for prostate cancer (PCa) patients. Therefore, it is important to understand molecular mechanisms of PCa progression to aggressive disease. Our previous research identified Abelson interactor 1 (Abi1), a member of WAVE complex, as a tumor suppressor in human prostate. Not only did we find mutations and deletions in the gene in prostate tumor patient samples, but WAVE complex levels were also found to be inversely correlated to disease recurrence and castration resistance. Moreover, Abi1 is downregulated in majority of prostate cancer cell lines, and patient organoid cultures derived from metastatic bone and lymph node biopsies. These data point to the clinical significance of defining Abi1-dependent tumor suppression mechanisms. To understand these mechanisms we generated CRISPR-mediated Abi1 KO in the RWPE-1 non-tumor human prostate cell line. When plated in 3D cultures in matrigel, we observed striking phenotypic differences in the appearance and behavior of the Abi1 KO spheroids compared to the parental cells. While the parental RWPE-1 grew into spherical organoids with tight cell-cell boundaries, the Abi1 KO cells grew as loose irregular-shaped spheres, with many cells migrating out of the organoids. Hence, the loss of Abi1 in these non-tumor cells lead to gain of an invasive phenotype. Western Blots of Abi1-KO cells demonstrated concurrent disruption of WAVE complex and deregulation of WAVE complex protein levels. Moreover, cell-cell adhesion proteins such as E-cadherin and β-catenin showed modest decrease and/or a substantial loss of membrane localization. Rescue experiments of Abi1 re-expression in the KO cells reversed the phenotype indicating that the observed phenotype is Abi1-dependent. Analyses of RNA sequencing of cells obtained from 3D cultures demonstrated differences in expression signatures characteristic of altered cell-cell and cell-matrix adhesion pathways and indicated upregulation of pathways associated with the invasive phenotype of cancer cells. We propose that Abi1 loss and WAVE complex deregulation represents key mediators of invasive phenotype in PCa. [Supported by NCI R01 CA161018 and NYS Department of Health Prostate Cancer Hypothesis Development RFA #1410200115] Citation Format: Disharee Das, Ming Chen, Claudia Mondragon, Dawn Post, Alexander Nappi, Heidi Hehnly, Anita Hryniewicz-Jankowska, Pier Paolo Pandolfi, Gennady Bratslavsky, Leszek Kotula. Modeling of Abi1 loss using spheroid cultures to investigate mechanisms of prostate tumorigenesis [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 1403.

Using two-dimensional difference gel electrophoresis (2D DIGE) we have analyzed monocytes derived from 10 sickle cell disease patients (5 males and 5 females ages 12–18) to generate hypotheses regarding signature proteins that appear most positively and negatively correlated with vasoocclusive event rate. Signature proteins have been identified by tandem mass spectrometry. Based on the limited number of samples analyzed, the most negatively correlated proteins related to crises rate were transketolase and coronin in the membrane fraction and heat shock 70 kDa protein cognate 4, and adenylate kinase isoenzyme 2, mitochondrial found in the cytosolic fraction. The protein spots that were most positively correlated with crisis rate in the cytoplasmic fraction were far upstream element-binding protein and Alpha actinin 1 or Alpha actinin 4. Utilizing StepSIM analysis, vinculin was able to classify all samples from the combined set and the membrane-only set, and cytosolic leukotriene A-4 hydrolase and phosphoglycerate kinase were also identified as important indicators for differentiating between low and high vasoocclusive event rates.

The object of this paper is to review briefly the studies on the interactions of erythroid and non-erythroid spectrins with lipids in model and natural membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Another important issue is the occurrence of spectrins in membrane rafts, how they are attached to the raft and what is their function in rafts.

Prostate cancer accounts for 20-25% of cancer cases every year and is predicted at over 180,000 new cases in 2016. However, the mechanisms of prostate cancer initiation and castration resistance still remains poorly understood. Our previous research shows mutations and deletions in Abelson interactor 1 (Abi1), an integral part of WAVE complex, in prostate tumor samples. Moreover, WAVE complex levels are inversely correlated to prostate cancer recurrence and castration resistance, outlining their clinical significance. A common phenomenon found early on in most cancers, including prostate cancer, is dysregulated cell-cell adhesion. This points towards a possible tumor suppressor mechanism, since Abi1 and the WAVE complex are in fact the primary regulators of branched actin polymerization via the Arp2/3 complex, and are critical for the formation and maintenance of cell-cell adhesion complexes. Hence, to study Abi1 in prostate cancer, we generated a prostate-specific conditional Abi1 knock-out (KO) mouse. In these mice, we observed evidence of prostatic intraepithelial neoplasia (PIN), a precursor of invasive carcinoma, with changes in proliferation and cell-cell adhesion markers. The goal of this study is to elucidate the mechanism by which the loss of Abi1 leads to the development of PIN in these mice. We are leading this analysis by primary 3D cultures with cells from our Abi1 KO prostates, and 2D cultures with mouse embryonic fibroblasts (MEFs). Loss of Abi1 leads to increase in size of the cultured spheroids, which indicates increased proliferation and dysregulated cell-cell adhesion. Also, adherens junction (AJ) markers E-cadherin and β-catenin levels are lower in western blots and immunostaining, suggesting defects in cell-cell adhesion. In MEFs, we observe loss of membrane localization of AJ proteins in Abi1 KO cells. Loss of Abi1 is often associated with upregulation of Abi2, which may be a contributing factor to the phenotype. To validate our findings in a human system, we have recently developed a CRISPR-mediated KO of Abi1 in the RWPE-1 non-tumor prostate cell line. We will use this KO cell line to determine cell-cell adhesion formation and maintenance in 2D and 3D cultures. In conclusion, loss of Abi1 leads to dysregulation of cell-cell adhesion in prostate epithelial cells, which may be a key mechanism for tumor development and progression in prostate cancer. Supported by NCI R01 CA161018 (LK). Citation Format: Disharee Das, Anita Hryniewicz-Jankowska, Heidi Hehnly, Dawn Post, Gennady Bratslavsky, Leszek Kotula. The key role of Abi1 loss in dysregulating cell-cell adhesion during prostate cancer tumorigenesis [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 4466. doi:10.1158/1538-7445.AM2017-4466

A hallmark of most cancer cells is an altered metabolism involving a shift to aerobic glycolysis with lactate production coupled with a higher uptake of glucose as the main source of energy. Lactate dehydrogenase 5 (LDH-5) catalyzes the reduction of pyruvate by NADH to form lactate, thus determining the availability of NAD(+) to maintain the continuity of glycolysis. It is therefore an important control point in the system of cellular energy release. Its upregulation is common in many malignant tumors. Inhibiting LDH-5 activity has an anti-proliferative effect on cancer cells. It may reverse their resistance to conventional chemo- and radiotherapy. Recent research has renewed interest in LDH-5 as an anticancer drug target. This review summarizes recent studies exploring the role of LDH-5 in cancer growth, its utility as a tumor marker, and developments made in identifying and designing anti-LDH-5 therapeutic agents.

Primary myelofibrosis (PMF) is an age-related myeloproliferative neoplasm (MPN) characterized by progressive bone marrow fibrosis, increasingly ineffective hematopoiesis, vascular and inflammatory complications, and progression to acute myeloid leukemia (AML) in 20% of cases. PMF is the rarest and the most severe of MPNs, with unfavorable prognosis and challenging treatment. Constitutive activation of the JAK-STAT pathway is an essential contributor to the pathogenesis of PMF. Nonetheless, clinically available JAK inhibitors do not show consistent disease-modifying effects at the molecular and malignant stem cell levels, underscoring the need to identify other targetable pathogenic mechanisms in PMF and related MPNs. We recently described a new signaling cascade involved in the maintenance of the malignant phenotype of stem cells in chronic myeloid leukemia that affects their adhesiveness, interaction with the microenvironment and resistance to imatinib (Chorzalska et al., Leukemia 2014). These phenotypic changes were caused by loss of Abelson interactor-1 (Abi-1) which regulates integrin α4 and the Src family kinases (SFKs) signaling pathways. To further delineate how organ-specific absence of Abi-1 affects hematopoiesis and hematopoietic stem cells, we generated a conditional, inducible bone marrow-specific Abi-1 transgenic mouse knockout model. For this purpose, we crossed conditional Abi-1(fl/fl) knockout mice with transgenic Tg(Mx1-cre+/-) mice (B6.Cg-Tg(Mx1-cre+ 1Cgn/J) that express Cre recombinase under the control of the polyinosinic:polycytidylic acid (poly(I:C)) or interferon-inducible Mx1 promoter. Obtained Abi-1(fl/fl);Tg(Mx1- cre(+)) animals were subjected to bone marrow-specific Abi-1 deletion induced by intraperitoneal administration of poly(I:C), resulting in organ-specific Abi-1 knockout (Abi-1 KO). Abi-1(fl/fl);Tg(Mx1- cre(-)) animals subjected to the same poly(I:C) treatment (Abi-1 WT) were used as controls in all experiments (Figure 1A). We confirmed the absence of Abi-1 protein in the Abi-1 KO bone marrow by immunoblotting (Figure 1B). Abi-1 KO mice developed deranged hematopoiesis with progressive anemia, thrombocythemia, megakaryocytosis, and myeloid hyperplasia (Figure 1C). They also developed progressive splenomegaly with 1.4x, 2.4x, and 4.8x enlargement of the spleen at 4, 28, and 50 weeks after recombination, respectively, and progressive femur pallor (Figure 1D) with bone marrow fibrosis confirmed by trichrome and silver staining (Figure 1D). Using fluorescence-activated cell sorting, we determined that Abi-1 KO resulted in more than two-fold expansion of the most primitive long-term hematopoietic stem cells (Lin-CD34-Sca-1+c-Kit+ CD135-) in the bone marrow and 3.75-fold increase of LSK cells (Lin- Sca-1+c-Kit+) in the peripheral blood compared with controls (Figure 1E). Increased activities of the SFKs and NFκB were observed in Abi-1 KO bone marrow, and confirmed in human CD34+ cells isolated from the marrow of patients with PMF or AML (Figure 1F,G). Furthermore, CD34+ hematopoietic progenitors and granulocytes from PMF patients, as well as CD34+ hematopoietic progenitors from AML patients, showed decreased levels of ABI1 transcript (Figure 1H). Taken together, our results indicate that a targeted deactivation of Abi-1 in the bone marrow causes expansion of hematopoietic stem cells, myeloid hyperplasia with thrombocytosis and overproduction of collagen and reticulin fibers. The Abi-1 KO murine phenotype meets the Mouse Models of Human Cancers Consortium criteria for a myeloproliferative neoplasm with features resembling human PMF (Kogan et al., Blood 2002). These data indicate that loss of Abi-1 is sufficient for initiation of bone marrow fibrosis, and that Abi-1-SFKs-NFκB cross-talk may represent a new molecular target for the treatment of PMF. Bone marrow-specific, conditional Abi-1 KO animals represent a new model of PMF offering a unique opportunity to interrogate the molecular details of the Abi-1-driven mechanism that leads to the development of stem cell-derived MPNs, and to elucidate potential novel therapeutic approaches that directly address their pathogenesis. Disclosures Olszewski: Bristol-Myers Squibb: Consultancy; Genentech: Research Funding; TG Therapeutics: Research Funding. Reagan:Alexion: Honoraria; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

Membrane rafts are distinct plasma membrane microdomains that are enriched in sphingolipids and cholesterol. They organize receptors and their downstream molecules and regulate a number of intracellular signaling pathways. This review presents information on the dependence of several growth factor receptor signaling pathways on membrane rafts. It also discusses the involvement of rafts in the regulation of differentiation, apoptosis and cell migration connected with invasiveness and metastasis. Examples of known synthetic and naturally occurring substances that are known to affect lateral membrane organization in tumor cell growth are discussed as potential or actual therapeutics.

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of β-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482–94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na+K+ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the β-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.

The results of many studies indicate that many cytoskeletal proteins interact with lipids, or are regulated by phosphoinositides. Proteins may associate with membranes through specific domains, amphipathic helices and undefined motifs that interact through electrostatic or hydrophobic interactions. The interaction between specific proteins and certain lipids affect stabilization of lipid microdomains, which may provide an anchor for cytoskeletal proteins. In vitro, proteins that sever or depolymerize actin filaments, such as gelsolin, villin, cofilin and profilin, are inactivated by PI(4,5)P2. Conversely, proteins like e.g. vinculin, talin, alpha-actinin, ezrin, N-WASP, WAVE that promote actin polymerization and link actin filaments to each other, are activated by this lipid. The major components of red blood cell membrane skeleton: spectrin and protein 4.1, also their nonerythroid counterparts interact with lipids, and those interactions may be regulated by phosphoinositides.

The object of the chapter is to review the studies on the interactions of erythroid and nonerythroid spectrins and other membrane-skeletal proteins with lipids in model membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Important issue is the mechanism(s) involved in these interactions.

We have determined the variability of the monocyte proteome and identified those proteins that demonstrate the greatest variation in the general control population. Monocytes were isolated from 18 healthy (9 male and 9 female) donors ages 18-50 and with no known genetic or blood disorder. A combination of Ficoll-Paque PLUS density centrifugation of cells found in the buffy coat and positive selection with monoclonal antibodies against CD14, coupled to magnetic beads, led to98% purity of monocytes. A 100,000 g microsomal membrane fraction or 100,000 g supernatant fraction from a control subject was compared to the equivalent fractions from a distinct control subject by two-dimensional differential gel electrophoresis (2D DIGE). Those protein spots that demonstrated Cy3-/Cy5- ratios greater than 2.5-fold in at least one experiment were selected for further statistical analysis. We determined variability for 31 cytosolic and 12 membrane protein spots. Proteins have been identified for 27 of the cytosolic protein spots and 9 of the microsomal membrane protein spots by in-gel digestion with trypsin followed by reverse-phase high-performance liquid chromatography in line with tandem mass spectrometry. We identified 24 distinct monocyte proteins that demonstrated the greatest variability in this general control population. The proteins demonstrating the greatest variance in the cytosolic fraction were enolase-1 and WD (tryptophan-aspartate) repeat-containing protein 1, and in the membrane fraction they were lamin B1 and L-plastin. This study demonstrates the importance of considering variance in the control population when performing future protein profiling comparisons of monocytes derived from disease versus control populations.

It is known that erythroid and non-erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially-expressed recombinant peptides representing betaII(brain)-spectrin's ankyrin-binding domain and its truncated mutants showed lipid-binding activity, although only those containing a full-length amino terminal fragment showed high to moderate affinity towards phospholipid mono- and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on betaI-spectrin's ankyrin-binding domain [Hryniewicz-Jankowska A, et al. Mapping of ankyrin-sensitive, PE/PC mono- and bilayer binding site in erythroid beta-spectrin. Biochem J 2004;382:677-85]. Moreover, we tested also the effect of transient transfection of living cells of several cell-lines with vectors coding for GFP-conjugates including betaII and also betaI full-length ankyrin-binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full-length ankyrin-binding domain of betaII and betaI spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin-binding domain truncated at their N-terminal region. Our results therefore indicate the importance of N-terminal region for lipid-binding activity of the beta-spectrin ankyrin-binding domain and its substantial role in maintaining the spectrin-based skeleton distribution.

The object of this paper is to review briefly the studies on the interactions of erythroid and non-erythroid spectrins with lipids in model and natural membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Another important issue is the occurrence of spectrins in membrane rafts, how they are attached to the raft and what is their function in rafts.

It has been shown previously that binding of vesicles and monolayers containing PE (phosphatidylethanolamine) by either erythroid or non-erythroid spectrin proved sensitive to inhibition by purified erythrocyte ankyrin. We tested the lipid-binding affinities of the purified ankyrin-binding domain of β-spectrin and of its truncated mutants in four ways, by analysing: (1) penetration of ‘loose’ PE/PC (phosphatidylcholine) monolayers; (2) binding to liposomes in suspension; (3) competition with spectrin for liposomes; and (4) binding of a PE/PC monolayer in a surface plasmon resonance system. The results obtained indicated that the full-length ankyrin-binding domain bound PE/PC mono- and bi-layers with moderate affinity, penetrated monolayers and competed with spectrin for liposomes. Moreover, its truncated mutants that retained the N-terminal part, in contrast with those lacking eight or 38 N-terminal residues (which bound lipid mono- and bi-layers with lower affinity), bound PE/PC mono- and bi-layers with an affinity and capacity comparable with those of the full-length ankyrin-binding domain, and this activity was inhibited by purified erythrocyte ankyrin. The full-length domain, in contrast with the mutant lacking 38 N-terminal residues, induced a small increase in the fluidity of PE/PC membranes when probed with 5′-doxyl stearate, similar to the effect of purified spectrin. Therefore we conclude that the binding site for PE-rich lipids, which is sensitive to ankyrin inhibition, is located in a 38-residue N-terminal fragment of the β-spectrin ankyrin-binding domain, and that the first eight residues play a key role in this activity.

We found that a protein of molecular weight close to 65 kDa, present in Escherichia coli cells, reacts with anti-β spectrin antibodies. A method of purification of this protein was designed. The method consists of the following: nonionic detergent extraction, gel filtration chromatography, ion-exchange chromatography using DEAE–Servacell, and two FPLC ion-exchange chromatography runs: the first without urea, the second in its presence. This method allowed us to obtain a highly purified protein. The results of mass spectrometry analysis suggest that the investigated protein is GroEL (Hsp60 Class). Using computer programs, by sequence analysis of both proteins we tried to explain why GroEL isolated from E. coli reacts with anti-β spectrin antibodies. Both proteins may share a single epitope for the antibodies on their surfaces. Additionally, such an assumption is supported by the results of experiments in which antibodies interacting with GroEL were obtained from anti-β spectrin serum and were shown to react with both GroEL and β spectrins.

Ankyrins, originally discovered as components of the erythrocyte membrane appeared to be a family of animal proteins encoded in mammalian cells by three related genes. Developmentally regulated, tissue specific posttranscriptional processing generates a great variety of isoforms which seem to play specific role in various cells and subcellular structures, being involved, for example, in membrane skeleton organisation, ionic transport, maintenance of cell polarity as well as cell-cell adhesion regulation. The interaction between the membrane skeleton and cytoplasmic domains of transmembrane proteins plays a fundamental role in membrane integrity and stability as well as in many cellular processes. Once the cDNA sequence of red blood cell ankyrin was determined it became clear that "ankyrin-repeat" motifs are present in many proteins whose function is rather unrelated to the membrane skeleton, e.g. transcription factors. Ankyrins are a multigene family of intracellular, structural proteins that link several integral membrane proteins and the spectrin-based membrane cytoskeleton. The anion exchanger, Na+-K+ ATPase, a voltage dependent Na+ channel, an Na+/Ca2+-exchanger, and adhesion molecules have been reported to interact with ankyrin in nonerythroid cells. Ankyrin was first found to link integral membrane proteins to the underlying spectrin-actin based membrane skeleton in the human erythrocyte. It was subsequently described in a variety of vertebrate cells and tissues, including brain, epithelia, and skeletal muscle. Variable cellular localisation of these membrane proteins may be possible due to different relative affinities of various isoforms of ankyrin for target proteins.

To invade adjacent tissues, cancer cells form special membrane protrusions with high proteolytic activity, named invadopodia. To better understand the role of invadopodia-associated proteolytic systems in the growth of esophageal cancer, we studied the expression of two serine proteases, FAP-alpha and DPPIV, and three metalloproteinases, MMP-2, MMP-9 and MT1-MMP, in a primary ESCC. The enzyme profile was evaluated in relation to the synthesis of cortactin as an important control factor in invadopodia formation and function. The expression and activity of proteases and cortactin in cancer tissues paired with non-cancer tissues were analyzed by RT-PCR, WB and zymography. Tissue samples were obtained from 24 patients who were undergoing surgical resection for esophageal carcinoma. All the tumors had been classified as ESCC and diagnosed as T3 or T4, N1-N3, and M0-M1 according to the TNM classification. The non-cancer tissue samples were taken from the distal area (5-10 cm from the macroscopic changes) and confirmed in histological examinations to be R0. We found an average 3-fold increase in DPPIV expression and an average five-fold higher level of FAP-alpha expression in ESCC over the control tissues. Up-regulation of DPPIV positively correlates with the FAP-alpha expression ratio. FAP-alpha transcripts and DPPIV mRNAs were observed in all the analyzed cancer and non-cancer tissue samples. We found a much higher level of the active MT1-MMP form in cancers relative to paired non-cancer tissues. The correlation between the level of MT1-MMP active form and MMP-2 activity observed in this study shows a tight connection between the two enzymes. We observed enhanced synthesis and activation of both MMP-2 and MMP-9 in cancer tissues. The cancer to normal ratios of the two active gelatinases correlated positively with the expression levels of both serine proteases. We examined the levels of CTTN in the cancer and non-cancer tissues of esophagus in order to find out if CTTN expression is associated with the expression and/or activity of invadopodia proteases. The increased level of CTTN was identified in 35% of ESCC tissues but the differences between groups were not statistically significant. No significant correlation was found between CTTN expression level and any other factors evaluated in our study. In conclusion, we showed that invadopodia-associated proteases, including FAP-alpha, DPPIV, MT1-MMP, MMP-2 and MMP-9, are significantly up-regulated in ESCC and that it might contribute to a more aggressive esophageal cancer. The presence of FAP-alpha in the cancer-free surgical margin suggests that alterations in the stromal environment may extend much father into surrounding tumor tissues than is usually considered. Citation Format: Katarzyna Augoff, Anita Hryniewicz-Jankowska, Renata Tabola, Leszek Czapla, Piotr Szelachowski, Krzysztof Grabowski, Aleksander F. Sikorski. Up-regulated expression and activation of invadopodia-associated proteases in esophageal squamous cell carcinoma (ESCC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5056. doi:10.1158/1538-7445.AM2013-5056

This review focuses on structure and functions of spectrin as a major component of the membrane skeleton. Recent advances on spectrin function as an interface for signal transduction mediation and a number of data concerning interaction of spectrin with membrane channels, adhesion molecules, receptors and transporters draw a picture of multifaceted protein. Here, we attempted to show the current depiction of multitask role of spectrin in cell physiology. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Purpose Tumor progression is associated with cell migration, invasion and metastasis. These processes are accompanied by the activation of specific proteases that are either linked to cellular membranes or are secreted into extracellular spaces. TNF-α is known to play an important role in various aspects of tumor progression. The aim of this work was to assess the effect of TNF-α on the migration of breast cancer cells and, in addition, to assess its association with the location of membrane-associated proteases in lipid rafts. Methods Wound scratch healing and Transwell migration assays were used to study the effect of TNF-α on the migration of both hormone-dependent and hormone-independent breast cancer-derived cells, i.e., MCF7 and MDA-MB-231, respectively. The expression and secretion of three matrix metalloproteases, MMP9, MMP2 and MT1-MMP, and two dipeptidyl peptidases, CD26 and FAP-α, was investigated using RT-PCR, Western blotting and gelatin zymography. In addition, activation of the MAPK/ERK signaling pathway was investigated by Western blotting. Results We found that a TNF-α-induced enhancement of breast cancer cell migration was accompanied by an increased secretion of MMP9, but not MMP2, into the culture media. We also found that TNF-α upregulated the expression of the dipeptidyl peptidases CD26 and FAP-α in a dose-dependent manner and, in addition, enhanced the concentration of all five proteases in lipid rafts in the breast cancer-derived cells tested, regardless of cell type. Furthermore, we found that TNF-α activated the MAPK/ERK signaling pathway by increasing the ERK1/2 phosphorylation level. Application of the MEK/ERK1/2 inhibitor U-0126 resulted in down-regulation of TNF-α-induced MMP9 secretion and abrogation of the enhanced concentration of proteases in the lipid rafts. Conclusions From our results we conclude that TNF-α-induced activation of the MAPK/ERK signaling pathway may promote breast cancer cell migration via both upregulation of MMP9, CD26 and FAP-α and concentration of these proteases, as also MT1-MMP and MMP2, in the lipid rafts. TNF-α may serve as a potential therapeutic target in breast cancers susceptible to TNF-α stimulation.