Formation Of Focal Adhesion Complexes Research Articles

Abstract 5259: Mechanistic evaluations of the immunological and cell biological effects that distinguish eribulin from other microtubule-targeted chemotherapeutics

Abstract Microtubule-targeted agents (MTAs) are some of the most widely used and effective chemotherapeutics and they continue to find utility as the payloads of antibody-drug conjugates and in combination with targeted agents to improve response, including with immune checkpoint inhibitors. While all MTAs have shared antiproliferative effects, over the last decade it has become increasingly clear that individual drugs of this class have distinct effects on microtubule-dependent signaling and trafficking that are critical for their anticancer efficacy. We and others have demonstrated that eribulin is unique from other MTAs in its ability to promote vascular remodeling, reverse epithelial-to-mesenchymal transition (EMT), and increase immune infiltration into tumors both in animal models and in the clinic. However, the mechanism linking eribulin-mediated microtubule destabilization to these phenotypes, which are associated with improved overall survival of eribulin-treated patients, is unclear. We found that acute eribulin treatment at clinically relevant concentrations promotes the production of antitumor cytokines in both immune and cancer cells and synergizes with direct STING agonists to promote enhanced immune infiltration and improved antitumor efficacy in vivo in both breast and bladder cancer models. This effect was shown to involve activation of natural killer cells and is associated with TBK1-mediated IRF3 phosphorylation, which is regulated downstream of the microtubule associated GTPase GEF-H1. Intriguingly, GEF-H1 release from destabilized microtubules has also been shown to enhance the formation of focal adhesions and promote the differentiation of mesenchymal cells to a more epithelial phenotype. We show that eribulin is indeed sufficient to promote the formation and activation of focal adhesion complexes in mesenchymal triple-negative breast cancer cells, which was not observed when cells were subjected to treatment with microtubule stabilizing chemotherapeutics. Both genetic and pharmacological inhibitors of GEF-H1 were utilized to interrogate the relationship between eribulin-mediated microtubule destabilization and TBK1/IRF3-dependent immunological activation as well as the formation of RhoA-dependent focal adhesions and reversal of EMT-associated phenotypes. Together, our data provide a mechanistic rationale for unique effects of eribulin as compared to other classes of MTAs that could inform on the use of these widely used, but mechanistically underappreciated drugs in a more personalized manner and inform on their rational combination with targeted agents, including immunotherapy. Citation Format: Zaineb Hassouneh, Nancy Wilkinson, Leila Takahashi-Ruiz, Neelam Mukherjee, April L. Risinger. Mechanistic evaluations of the immunological and cell biological effects that distinguish eribulin from other microtubule-targeted chemotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5259.

Abstract P6-14-17: Endomucins and their expression in breast cancer and the cellular and therapeutic impact

Abstract Introduction. Endomucin-1 and endomucin-2 are two proteins structurally related, yet with low homology. The membrane bound sialoglycoproteins appear to play a key role in interfering with the formation of focal adhesion complexes (FAC) and matrix adhesiveness of cells, by mechanisms independent of the MUC1 repeat, which the endomucins do not possess. Endomucins are thought to be expressed at high levels in endothelium and haematopietic cell lineages, although the levels in mammary tissues also seem high. Despite the seeming importance of endomucins in the adhesion and migration of cells, including cancer cells, the clinical value of endomucin in clinical cancer, including breast cancer, is largely unknown. The present study examined the expression profile of endomucins, together with the focal adhesion kinase FAK, in breast cancer and aimed to explore the cellular impact of endomucin on cancer cells. Methods. Human breast cancer cells MCF-7 and MDA MB-231 and a range of other cell types were used. An endomucin overexpression cell model was created and subsequently used to evaluate the function of the cells. The expression profile of the endomucin-1 and endomucin-2 transcripts and FAK, in an existing fresh frozen breast cancer tissue cohort, were quantified. Results. High levels of endomucins, particularly endomucin-1, are good indicators for the overall survival of the patient, p=0.021 for endomucin-1 and p=0.15 for endomucin-2. When expression levels of FAK were integrated into the survival analysis model, patients with high levels of both endomucins and low levels of FAK had the most favourable outcome, compared with those with most unfavourable outcome who had low level of endomucin and high FAK (survival during the follow up period respectively at 100% and 54%, p=0.013, Harzoud Ratio 0.298). Together with the Nottingham Prognostic Index, which independently predicts a poor outcome (p=0.009, HR=7.6), the integrated expression profile of endomucin/FAK represents an independent prognostic indicator for favourable overall survival (p=0.003, HR=0.13), and indeed for a favourable disease free survival (p=0.008, HR=0.17). Mammary tissues, and indeed breast cancer cell lines, expressed high levels of endomucin-2 transcripts and low levels of endomucin-1 transcripts. High levels of endomucin-2 were also seen in fibroblasts and vascular endothelial cells. We created a breast cancer cell submodel with MCF-7, by overexpressing endomucin. It was shown that although endomucin over-expression had some marginal impact on the adhesiveness of breast cancer cells, the over-expression however, had significant impact on cells’ sensitivity to FAK inhibitor, with a markedly reduced adhesiveness to matrix (p< 0.001 control versus endomucin overexpression cells). Discussion. Endomucins have a reduced expression in breast cancers and the reduction, together with low levels of focal adhesion kinase, facilitate a favourable outcome for the patients. Together with the findings of in vitro cell models, it would suggest that the expression profile of endomucins and FAK may be a good indicator, not only for evaluating clinical outcomes, but also for choice of target therapies. Citation Format: Amber Xinyu Li, Tracey A. Martin, Lin Ye, Andrew J. Sanders, Fiona Ruge, Jane Lane, Eleri Davies, Wen G. Jiang. Endomucins and their expression in breast cancer and the cellular and therapeutic impact [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-17.

The interaction of CFLAR with p130Cas promotes cell migration

CASP8 and FADD Like Apoptosis Regulator (CFLAR) is a key anti-apoptotic regulator for resistance to apoptosis mediated by Fas and TRAIL. In addition to its anti-apoptotic function, CFLAR is also an important mediator of tumor growth. High level of CFLAR expression correlates with a more aggressive tumor. However, the mechanism of CFLAR signaling in malignant progression is not clear. Here we report a novel CFLAR-associated protein p130Cas, which is a general regulator of cell growth and cell migration. CFLAR-p130Cas association is mediated by the DED domain of CFLAR and the SD domain of p130Cas. Immunofluorescence observation showed that CFLAR had the colocalization with p130Cas at the focal adhesion of cell membrane. CFLAR overexpression promoted p130Cas phosphorylation and the formation of focal adhesion complex. Moreover, the enhancement of cell migration induced by CFLAR overexpression was obviously inhibited by p130Cas siRNA. In silico analysis on human database suggests high expressions of CFLAR or/and p130Cas are associated with poor prognosis of patients with lung cancer. Together, our results suggest a new mechanism for CFLAR involved in tumor development via association with p130Cas.

Silicon Nanoneedle-Induced Nuclear Deformation: Implications for Human Somatic and Stem Cell Nuclear Mechanics.

Cell nuclear size and shape are strictly regulated, with aberrations often leading to or being indicative of disease. Nuclear mechanics are critically responsible for intracellular responses to extracellular cues, such as the nanotopography of the external environment. Silicon nanoneedle (SiNN) arrays are tunable, engineered cell culture substrates that permit precise, nanoscale modifications to a cell's external environment to probe mechanotransduction and intracellular signaling. We use a library of four different SiNN arrays to investigate the immediate and downstream effects of controlled geometries of nanotopographical cues on the nuclear integrity/dynamics of human immortalized somatic and renewing stem cell types. We quantify the significant, albeit different, nuclear shape changes that both cell types undergo, which suggest that cellular responses to SiNN arrays are more comparable to three-dimensional (3D) environments than traditional flat cultureware. We show that nanotopography-induced effects on nuclear envelope integrity, protein localization, and focal adhesion complex formation are cell-dependent. Migration is shown to be dramatically impeded for human neural progenitor cells (hNPCs) on nanotopographies compared to flat substrates but not for somatic cells. Our results indicate an additional layer of complexity in cellular mechanotransduction, which warrants closer attention in the context of engineered substrates and scaffolds for clinical applications.

Application of Bio-Active Elastin-like Polypeptide on Regulation of Human Mesenchymal Stem Cell Behavior

Regenerative medicine using stem cells offers promising strategies for treating a variety of degenerative diseases. Regulation of stem cell behavior and rejuvenate senescence are required for stem cells to be clinically effective. The extracellular matrix (ECM) components have a significant impact on the stem cell’s function and fate mimicking the local environment to maintain cells or generate a distinct phenotype. Here, human elastin-like polypeptide-based ECM-mimic biopolymer was designed by incorporating various cell-adhesion ligands, such as RGD and YIGSR. The significant effects of bioactive fusion ELPs named R-ELP, Y-ELP, and RY-ELP were analyzed for human bone-marrow-derived stem cell adhesion, proliferation, maintenance of stemness properties, and differentiation. Multivalent presentation of variable cell-adhesive ligands on RY-ELP polymers indeed promote efficient cell attachment and proliferation of human fibroblast cells dose-dependently. Similarly, surface modified with RY-ELP promoted strong mesenchymal stem cell (MSCs) attachment with greater focal adhesion (FA) complex formation at 6 h post-incubation. The rate of cell proliferation, migration, population doubling time, and collagen I deposition were significantly enhanced in the presence of RY-ELP compared with other fusion ELPs. Together, the expression of multipotent markers and differentiation capacity of MSCs remained unaffected, clearly demonstrating that stemness properties of MSCs were well preserved when cultured on a RY-ELP-modified surface. Hence, bioactive RY-ELP offers an anchorage support system and effectively induces stimulatory response to support stem cell proliferation.

CXCL12-driven thymocyte migration is increased by thymic epithelial cells treated with prolactin in vitro

The prolactin hormone (PRL), in addition to its known effects on breast development and lactation, exerts effects on the immune system, including pleiotropic effects on the thymus. The aim of this study was to evaluate the influence of PRL on the epithelial compartment of the thymus. Thymic epithelial cells (TECs) (2BH4 cells) and fresh thymocytes were used. Immunofluorescence assay revealed that PRL treatment (10 ng/mL) increases the deposition of laminin and expression of the chemokine CXCL12 in 2BH4 cells. However, no change was observed in the deposition of fibronectin. Moreover, PRL altered F-actin polymerisation, allowing the formation of focal adhesion complexes in treated cells. When 2BH4 cells were pre-treated with PRL, thymocyte adhesion was not altered. However, in the cell migration assay, pre-treatment with PRL potentiated the chemotactic effect of CXCL12 on the migration of total, double-positive, CD4-positive, and CD8-positive thymocytes. Together, the results of this study demonstrate the effect of PRL on thymic epithelial cells, particularly on CXCL12-driven thymocyte migration, confirming that this hormone is a regulator of thymic physiology.

Mg-Fe layered double hydroxides modified titanium enhanced the adhesion of human gingival fibroblasts through regulation of local pH level

The durability of dental implants is closely related to osseointegration and surrounding soft tissue sealing. Appropriate local pH favors fibroblasts adhesion and contributes to soft tissue sealing. Layered double hydroxides (LDHs) are characterized by adjustable alkalinity, offering a possibility to investigate the influence of pH on cellular behaviors. Herein, we fabricated MgFe LDHs modified titanium. During calcination, the local pH value of LDHs increase, without altering other physics and chemical properties via OH− exchange mechanism. In vitro studies showed that LDHs films calcined at 250 °C for 2 h provide a local pH of 10.17, which promote early adhesion, proliferation, and type I collagen expression of human gingival fibroblasts (hGFs) through the formation of focal adhesion complex and activation of focal adhesion kinase related signaling pathways. In conclusion, endowing the titanium surface with appropriate alkalinity by MgFe LDHs films enhances the adhesion of hGFs, providing a new strategy of designing multifunctional biomaterials for soft tissue sealing around dental implants.

Pitavastatin stimulates retinal angiogenesis via HMG-CoA reductase-independent activation of RhoA-mediated pathways and focal adhesion.

Excessive angiogenesis of the retina is a key component of irreversible causes of blindness in many ocular diseases. Pitavastatin is a cholesterol-lowering drug used to reduce the risk of cardiovascular diseases. Various studies have shown the effects of pitavastatin on angiogenesis but the conclusions are contradictory. The effects of pitavastatin on retinal angiogenesis have not been revealed. This study investigated the effects of pitavastatin at clinically relevant concentrations on retinal angiogenesis and its underlying mechanisms using retinal microvascular endothelial cells (RMECs). The effects of pitavastatin on retinal angiogenesis were determined using in vitro model of retinal angiogenesis, endothelial cell migration, adhesion, proliferation, and apoptosis assays. The mechanism studies were conducted using immunoblotting and stress fiber staining. Pitavastatin stimulated capillary network formation of RMECs in a similar manner as vascular endothelial growth factor (VEGF) and lipopolysaccharide (LPS). Pitavastatin also increased RMEC migration, adhesion to Matrigel, growth, and survival. The combination of pitavastatin with VEGF or LPS was more effective than VEGF or LPS alone in stimulating biological activities of RMECs, suggesting that pitavastatin can enhance the stimulatory effects of VEGF and LPS on retinal angiogenesis. Pitavastatin acted on RMECs in a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-independent manner. In contrast, pitavastatin activated pro-angiogenic microenvironment via promoting the secretion of VEGF and stimulated retinal angiogenesis via multiple mechanisms including activation of RhoA-mediated pathways, induction of focal adhesion complex formation, and activation of ERK pathway. Our work provides a preclinical evidence on the pro-angiogenic effect of pitavastatin in retina via multiple mechanisms that are irrelevant to mevalonate pathway.

C-Jun N-terminal kinase (JNK) pathway activation is essential for dental papilla cells polarization.

During tooth development, dental papilla cells differentiate into odontoblasts with polarized morphology and cell function. Our previous study indicated that the C-Jun N-terminal kinase (JNK) pathway regulates human dental papilla cell adhesion, migration, and formation of focal adhesion complexes. The aim of this study was to further examine the role of the JNK pathway in dental papilla cell polarity formation. Histological staining, qPCR, and Western Blot suggested the activation of JNK signaling in polarized mouse dental papilla tissue. After performing an in vitro tooth germ organ culture and cell culture, we found that JNK inhibitor SP600125 postponed tooth germ development and reduced the polarization, migration and differentiation of mouse dental papilla cells (mDPCs). Next, we screened up-regulated polarity-related genes during dental papilla development and mDPCs or A11 differentiation. We found that Prickle3, Golga2, Golga5, and RhoA were all up-regulated, which is consistent with JNK signaling activation. Further, constitutively active RhoA mutant (RhoA Q63L) partly rescued the inhibition of SP600125 on cell differentiation and polarity formation of mDPCs. To sum up, this study suggests that JNK signaling has a positive role in the formation of dental papilla cell polarization.

Heregulin-induced cell migration is prevented by trastuzumab and trastuzumab-emtansine in HER2+ breast cancer.

Heregulin (HRG) signaling has been implicated in the development of an aggressive phenotype in breast cancer (BC) cells, and HER2 overexpression has been associated with a worse prognosis in BC patients. Nevertheless, the molecular mechanisms through which HRG affects the efficiency of anti-HER2 therapies such as trastuzumab (Tz) and trastuzumab-emtansine (T-DM1) are currently unknown. In the present study, we evaluate the molecular action of HRG toward fundamental scaffold proteins and several kinases in the signal transduction pathways triggered via HER2/HER3, which integrate precise and sequential steps to promote changes in cell morphology to impulse BC cell migration. In addition, we evaluate the effectiveness of Tz and T-DM1 on the control of key proteins involved in BC cell motility, since the acquisition of a migratory phenotype is essential to promote invasion and metastasis. We show that HRG induces actin cytoskeleton reorganization and focal adhesion complex formation, and promotes actin nucleation in BT-474 BC cells. This signaling is triggered by HER2/HER3 to c-Src, FAK and paxillin. When paxillin is phosphorylated, it recruits PAK1, which then phosphorylates cortactin. In parallel, paxillin signals to N-WASP, and both signalings regulate Arp2/3 complex, leading to the local reorganization of actin fibers. Our findings reveal an original mechanism by which HRG increases HER2+ BC cell motility, and show that the latter can be abolished by Tz and T-DM1 treatments. These results provide evidence for the molecular mechanisms involved in cell motility and drug resistance. They will be useful to develop new and more specific therapeutic schemes that interfere with the progression and metastasis of HER2+ BC.

Confirming whether KLHL23 deficiency potentiates migration in urothelial carcinoma.

Epithelial-mesenchymal transition (EMT) is associated with malignant tumors. In a previous study, we found that KLHL23 is a tumor suppressor gene that inhibits EMT and cancer dissemination. However, the correlation between its expression and cancer progression in urothelial carcinoma (UC) remains unknown. This study showed that the deficiency of KLHL23 in the invasive leading cancer cells is important for improving cell migration in UC. Currently, little is known about the underlying mechanisms of KLHL23-mediated cytoskeleton remodeling in the metastatic leading cells of tumors. Our findings showed that silencing of KLHL23 promotes cell migration in UC by regulating the translocation of focal adhesion proteins. Lack of KLHL23 causes abnormal formation of lamellipodia and increases the EMT phenotype and migration. Wound healing assay revealed that KLHL23 potentiates the actin bundles and intracellular focal adhesion protein formation in the invasive leading cells. Knockdown of KLHL23 abolishes the formation of actin stress fibers and translocalizes vinculin to the perimembrane, which enhances the mobility of cancer cells. To elucidate the mechanism, we found that during migration, KLHL23 appears in the leading cells in large numbers and binds to the actin stress fibers. A large amount of vinculin accumulated at both ends of the KLHL23/actin fibers, indicating an increase in cell anchorage. Thus, KLHL23 might play a critical role in enhancing actin fibers and promoting focal adhesion complex formation in the invasive leading cells. Analysis of the overall survival revealed that low KLHL23 is associated with poor survival in patients with bladder UC, indicating its clinical significance. We hypothesize that KLHL23 is involved in the formation of actin stress fibers and focal adhesion complexes in the invasive leading cells and may be associated with EMT progression and prognosis in UC patients.

Mitigative Effect of Sipjeondaebo-tang on RhoA Activation in Cold-Exposed Vascular Cells

Objective To investigate the vasodilatory effect of Sipjeondaebo-tang by inhibiting RhoA activity in vascular cells during cold exposure. Methods Human vascular endothelial cells and pericytes were pretreated with Sipjeondaebo-tang for 30 min, followed by incubation at 37 °C (control) or 25 °C (cold exposure) for 30 min. Activation of endothelin-1-mediated RhoA in pericytes was assessed by pretreating the cells with Sipjeondaebo-tang for 30 min, followed by incubation with endothelin-1 at 37 °C for 30 min. Western blotting was performed to measure the expression of active RhoA. Endothelin-1 and nitric oxide release from endothelial cells was examined with enzyme-linked immunosorbent assay kits. The formation of stress fibers and focal adhesion complexes was analyzed by immunocytochemistry. Results Cold treatment activated RhoA in both pericytes and vascular endothelial cells, whereas Sipjeondaebo-tang treatment inhibited this activation. Sipjeondaebo-tang treatment also reversed the cold-mediated production of endothelin-1 and nitric oxide. Cold exposure promoted the formation of stress fibers and focal adhesion complexes by increasing the expression of phospho-focal adhesion complex kinase, whereas Sipjeondaebo-tang treatment suppressed this response. Conclusions These findings suggested that Sipjeondaebo-tang inhibits cold-induced RhoA activation and its related pathway components, including endothelin-1 and nitric oxide, in vascular cells. Therefore, Sipjeondaebo-tang could be beneficial for the treatment of Raynaud’s phenomenon. Keywords: Raynaud’s phenomenon, cold, rhoA, endothelin-1, nitric oxide, Sipjeondaebo-tang

Inhibition of Pathological Retinal Neovascularization by a Small Peptide Derived from Human Tissue-Type Plasminogen Kringle 2.

Retinal neovascularization is a hallmark pathological process of numerous ocular diseases which comprise the most common causes of blindness and affect millions of people from infants to the elderly. Compared to large proteins, small peptides have advantages for therapeutic application in ocular diseases, especially for retinal diseases. In this study, we investigated a small peptide derived from human tissue-type plasminogen kringle 2 (t-PA kringle 2), named TKII-12, and investigated the effect of TKII-12 on various aspects of vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro and in vivo. Our results showed that TKII-12 effectively inhibited VEGF-induced human retinal microvascular endothelial cell proliferation, migration and tube formation on Matrigel dose-dependently as well as sequence-dependently. TKII-12 inhibited VEGF-induced formation of actin stress fibers and focal adhesions in vascular endothelial cells. Moreover, TKII-12 effectively inhibited retinal neovascularization in a mouse oxygen-induced retinopathy (OIR) model. Our study demonstrated that TKII-12 could effectively inhibit retinal angiogenesis in vitro and in vivo by eliminating the formation of focal adhesion complexes and the organization of actin stress fibers. TKII-12 can serve as a prototype for retinal angiogenesis inhibitory drug development.

Force-Directed "Mechanointeractome" of Talin-Integrin.

Mechanotransduction from the extracellular matrix into the cell is primarily supervised by a transmembrane receptor, integrin, and a cytosolic protein, talin. Integrin binds ligands on the extracellular side, whereas talin couples integrin receptors to the actin cytoskeleton and later acts as a "force buffer". Talin and integrin together form a mechanosensitive signaling hub that regulates crucial cellular processes and pathways, including cell signaling and formation of focal adhesion complexes, which help cells to sense their mechano-environment and transduce the signal accordingly. Although both proteins function in tandem, most literature focuses on them individually. Here, we provide a focused review of the talin-integrin mechano-interactome network in light of its role in the process of mechanotransduction and its connection to diseases. While working under force, these proteins drive numerous biomolecular interactions and form adhesion complexes, which in turn control many physiological processes such as cell migration; thus, they are invariably associated with several diseases from leukocyte adhesion deficiency to cancer. Gaining insights into their role in the occurrence of these pathological disorders might lead us to establish treatment methods and therapeutic techniques.

Niclosamide reverses adipocyte induced epithelial-mesenchymal transition in breast cancer cells via suppression of the interleukin-6/STAT3 signalling axis

The microenvironment of breast cancer comprises predominantly of adipocytes. Adipocytes drive cancer progression through the secretion adipocytokines. Adipocytes induce epithelial mesenchymal transition of breast cancer cells through paracrine IL-6/Stat3 signalling. Treatment approaches that can target adipocytes in the microenvironment and abrogate paracrine signals that drive breast cancer growth and metastasis are urgently needed. Repositioning of old drugs has become an effective approach for discovering new cancer drugs. In this study, niclosamide, an FDA approved anthelminthic drug was evaluated for its anti-breast cancer activity and its ability to inhibit adipocytes induced EMT. Niclosamide potently inhibited proliferation, migration and invasion at low concentration and induced significant apoptosis at high concentrations in human breast cancer cell lines MDA-MB-468 and MCF-7. Additionally, niclosamide reversed adipocyte-induced EMT with a correlated inhibition of IL-6/Stat3 activation and downregulation of EMT-TFs TWIST and SNAIL. Moreover, niclosamide markedly impaired MDA-MB-468 and MCF-7 migration and invasion. We further found that the inhibitory effects of niclosamide on MDA-MB-468 and MCF-7 motility was closely related to destabilization of focal adhesion complex formation. With decreased co-localization of focal adhesion kinase (FAK) and phosphorylated paxillin (pPAX). Collectively, these results demonstrate that niclosamide could be used to inhibit adipocyte-induced breast cancer growth and metastasis.

Cyclin Dependent Kinase 1 (CDK1) Activates Cardiac Fibroblasts via Directly Phosphorylating Paxillin at Ser244.

Atrial fibrillation has caused severe burden for people worldwide. Differentiation of fibroblasts into myofibroblasts, and consequent progress in atrial structural remodeling have been considered the basis for persistent atrial fibrillation, yet little is known about the molecular mechanisms underlying the process. Here, we show that cyclin-dependent kinase 1 (CDK1) is activated in atrial fibroblasts from patients with atrial fibrillation (AFPAF) and in platelet derived growth factor BB (PDGF-BB)-treated atrial fibroblasts from patients with sinus rhythm (AFPSR). We also demonstrate that inhibition of CDK1 suppresses fibroblast differentiation and focal adhesion (FA) complex formation. The FA protein paxillin is phosphorylated directly at Ser244 by CDK1. Importantly, transfection of a paxillin construct harboring a Ser to Ala mutation causes FA complex disassembly and greatly inhibits fibroblast activation. AFPSRs applied with a lentiviral vector carrying the shRNA sequence of paxillin dramatically prevents PDGF-BB induced functional activation. Taken together, all these results suggest that phosphorylation of paxillin at Ser244 by CDK1 is a key mechanism in fibroblast differentiation and could eventually assist atrial fibrosis.

Molecular Actions of Thyroid Hormone on Breast Cancer Cell Migration and Invasion via Cortactin/N-WASP

The thyroid hormone triiodothyronine (T3) plays a fundamental role in growth regulation, differentiation, metabolism and cellular movement. These processes are particularly important considering that deregulation of T3 levels could promote abnormal responsiveness of mammary epithelial cells, which may lead to the development and progression of breast cancer (BC). Once cells migrate and invade different tissues, BC metastasis is the main cause of cancer-related death because it is particularly difficult to revert this multistep process. Cell migration integrates several steps that induce changes in cell structure and morphology to promote BC cell invasion. These sequential steps include actin cytoskeleton remodeling, focal adhesion complex formation and, finally, the turnover of branched actin filament networks. In this article, we demonstrate that T3 has the ability to modify the Epithelial-Mesenchymal Transition process. In addition, we show that T3 induces actin cytoskeleton reorganization, triggers focal adhesion formation and, as a consequence, promotes actin nucleation via non-genomic pathway. These events are specifically modulated by T3 via integrin αvβ3 to FAK/paxillin/cortactin/N-WASP/Arp2/3 complex signaling pathway, increasing cell adhesion, migration and invasion of T-47D BC cells. We suggest that T3 influences the progression of tumor metastasis by controlling signaling pathways that converge in cell motility. This knowledge is crucial for the development of novel therapeutic strategies for BC treatment.

O-GlcNAcylation regulates integrin-mediated cell adhesion and migration via formation of focal adhesion complexes

O-GlcNAcylation is a post-translational modification of a protein serine or threonine residue catalyzed by O-GlcNAc transferase (OGT) in the nucleus and cytoplasm. O-GlcNAcylation plays important roles in the cellular signaling that affect the different biological functions of cells, depending upon cell type. However, whether or not O-GlcNAcylation regulates cell adhesion and migration remains unclear. Here, we used the doxycycline-inducible short hairpin RNA (shRNA) system to establish an OGT knockdown (KD) HeLa cell line and found that O-GlcNAcylation is a key regulator for cell adhesion, migration, and focal adhesion (FA) complex formation. The expression levels of OGT and O-GlcNAcylation were remarkably suppressed 24 h after induction of doxycycline. Knockdown of OGT significantly promoted cell adhesion, but it suppressed the cell migration on fibronectin. The immunostaining with paxillin, a marker for FA plaque, clearly showed that the number of FAs was increased in the KD cells compared with that in the control cells. The O-GlcNAcylation levels of paxillin, talin, and focal adhesion kinase were down-regulated in KD cells. Interestingly, the complex formation between integrin β1, focal adhesion kinase, paxillin, and talin was greatly increased in KD cells. Consistently, levels of active integrin β1 were significantly enhanced in KD cells, whereas they were decreased in cells overexpressing OGT. The data suggest a novel regulatory mechanism for O-GlcNAcylation during FA complex formation, which thereby affects integrin activation and integrin-mediated functions such as cell adhesion and migration.

Multiform TiO2 nano-network enhances biological response to titanium surface for dental implant applications

Our objective in this study was to evaluate biological responses to a unique multiform nano-network of TiO2 on a titanium (Ti) surface for dental implant applications. A novel multiform TiO2 nano-network was formed on the anodized Ti surface using a simple, rapid electrochemical anodization process. The size of the pores in the nano-network ranged from a few nanometers to a few hundred nanometers. Human bone marrow mesenchymal stem cells (hBMSCs) were used as a model in this study. The multiform nanostructure, which is on the scale of various biological species, was shown to enhance protein adsorption, cell motility, cell proliferation, cell differentiation, and surface bioactivity, as well as stimulate outside-in signals via integrins and the formation of focal adhesion complex. It was also shown to promote hBMSCs toward the osteogenic differentiation pathway and convert the Ti surface from bioinert to bioactive. This is a clear indication that the proposed multilayer TiO2 nano-network has excellent biocompatibility and surface bioactivity. The bioactive TiO2 nano-network structure introduced in this study has considerable potential for applications in implant dentistry.

Ulipristal Acetate Interferes With Actin Remodeling Induced by 17β-Estradiol and Progesterone in Human Endometrial Stromal Cells.

Ulipristal acetate (UPA) is a selective progesterone receptor modulator (SPRM) used for emergency contraception and for the medical management of symptomatic uterine fibroids (UF). Treatment with UPA turns in amenorrhea and UF volume reduction. Treatment with UPA is associated with the frequent development of benign, transitory endometrial changes known as SPRM-associated endometrial changes (PAECs). Why PAECs develop and their biological or cellular basis is unknown. Sex steroids, including estrogen and progesterone, are established modulators of the actin cytoskeleton in various cells, including endometrial cells. This explains several morphological and functional changes in endometrial cells. We thus hypothesized that UPA may alter the appearance of the endometrium by interfering with the actions of 17β-estradiol (E2) or progesterone (P4) on actin dynamics. We isolated and cultured human endometrial stromal cells (ESC) from endometrial biopsies from healthy fertile women. Treatment with E2 or P4 stimulated visible actin rearrangements with actin remodeling toward the membrane. Activation through phosphorylation of the actin regulatory proteins, Moesin, and focal adhesion kinase (FAK), hacked actin remodeling induced by E2 and P4. Membrane re-localization of Paxillin and Vinculin were also induced by E2 and P4, showing the formation of focal adhesion complexes. All these E2 and P4 actions were inhibited by co-treatment with UPA, which was otherwise inactive if given alone. The cytoskeletal changes induced by E2 and P4 turned into increased motility of ESC, and UPA again blocked the actions E2 and P4. In conclusion, we find that UPA interferes with the cytoskeletal actions of E2 and P4 in ESC. This finding helps understanding the mode of actions of SPRMs in the endometrium and may be relevant for other potential clinical applications of UPA.