Intracellular Actin Filaments Research Articles

Mechanics of cell-cell junctions

Tissue cells in epithelial or endothelial monolayers are connected through cell-cell junctions, which are stabilized by transmembrane E-cadherin bonds and intracellular actin filaments. These bonds and junctions play a crucial role in maintaining the barrier function of epithelia and endothelia and are believed to transmit forces between cells. Additionally, E-cadherin bonds can impact the shape of cell-cell junctions. In this study, we develop a continuum mechanical model of the cell-cell junction by explicitly incorporating the cell membrane, distributions of E-cadherin bonds, cytoplasmic fluid pressure, and F-actin dynamics. The static force-balanced version of the model is able to analyze the influences of cell cortical tension, actin dynamics, and cytoplasmic pressure on the junction shape and E-cadherin bonds. Furthermore, an extended model that incorporates fluid flow, across the cell boundary as well as around the cell, is also examined. This model can couple cell-shape changes with cell cortical tension and fluid flow, and predicts the additional effect of fluid motion on cell-cell junction mechanics. Taken together, our models serve as an intermediate link between molecular-scale models of cell-junction molecules and cell-scale models of tissue and epithelia.

Cortactin expression in muscle-invasive and nonmuscle-invasive urinary bladder carcinoma in patients live in Sohag City, Egypt

Abstract Background Bladder carcinoma (BC) is the ninth most prevalent cancer worldwide. However, it represents the most frequently occurring cancer in Egypt, where infection by Schistosoma haematobium is an endemic health problem. BCs are clinically subdivided into different stages according to invasion of the muscle layer and distant metastasis. Invasion of the surrounding tissues and metastasis are characteristic issues in malignant tumors. Malignant cells rearrange the intracellular actin filaments in the form of an assembly of branched network in the direction of cell movement. The well-formed actin assembly must be anchored by an adhesive-like protein named cortactin (CTTN). CTTN overexpression has been detected in various human malignant neoplasms. The aim of this study was to evaluate the expression of CTTN in BC and to correlate such expression with different parameters such as depth of invasion, nodal metastasis, and bilharzial infestation. Patients and methods The current study enrolled 66 patients with BCs. Formalin-fixed and paraffin-embedded tissue blocks were prepared from each specimen. Hematoxylin and eosin tissue sections were prepared to confirm the diagnosis of BCs, determine tumor phenotype, assess degree of invasion, and detect any evidence of bilharzial infestation. Corresponding tissue sections were stained by antihuman CTTN antibody using the immunohistochemical approach. Results Overexpression of CTTN was associated with muscle invasion by neoplastic cells (P<0.001), advanced pathological T category (P=0.002), and presence of metastatic deposits in the regional lymph nodes (P<0.001). Conclusion Overexpression of CTTN in BC is associated with increased tumor invasion and nodal metastasis.

A Comprehensive Review of Duchenne Muscular Dystrophy: Genetics, Clinical Presentation, Diagnosis, and Treatment

Duchenne Muscular Dystrophy (DMD) is a genetic disorder involving progressive muscle deterioration leading to loss of mobility, cardiomyopathy, and respiratory complications leading to an early death by the fourth decade of life. Males are affected more often as DMD results from a mutation in the dystrophin gene residing on the X chromosome. The DMD genetic mutation results in a complete functional lack of dystrophin, which culminates as an inadequate connection between the intracellular actin filaments and the extracellular skeleton of muscle. Boys affected by DMD clinically present with muscle weakness before age five, are often wheelchair-bound by age 12, and rarely survive beyond the third decade of life.
 Traditional treatment strategies have focused primarily on quality-of-life improvement and have included the use of glucocorticoids and physical therapy. No cure currently exists, however many novel treatments for DMD are currently being explored. Some of these involve gene therapy, exon skipping, stop codon skipping, CRISPR technology interventions, and the use of a retinal dystrophin isoform. In this comprehensive review, we recapitulate the literature findings to summarize the history, epidemiology, genetics, clinical presentation, diagnosis, and current and future strategies for the treatment of Duchenne Muscular Dystrophy.

7,8-Dihydroxy-3-(4′-hydroxyphenyl)coumarin inhibits invasion and migration of osteosarcoma cells

Advances in pharmacy and medicine have led to the development of many anti-cancer and molecular targeted agents; however, there are few agents capable of suppressing metastasis. To prevent cancer recurrence, it is essential to develop novel agents for inhibiting metastasis. Coumarin-based compounds have multiple pharmacological activities including anti-cancer effects. We screened a compound library constructed at Kyoto Pharmaceutical University and showed that 7,8-dihydroxy-3-(4′-hydroxyphenyl)coumarin (DHC) inhibited invasion and migration of LM8 mouse osteosarcoma cells and 143B human osteosarcoma cells in a concentration-dependent manner. DHC decreased intracellular actin filament formation by downregulating Rho small GTP-binding proteins such as RHOA, RAC1, and CDC42, which regulate actin reorganization. However, DHC did not downregulate the corresponding mRNA transcripts, whereas it downregulated Rho small GTP-binding proteins in the presence of cycloheximide, suggesting that DHC enhances the degradation of these proteins. DHC treatment inhibited metastasis and prolonged overall survival in a spontaneous metastasis mouse model. These results indicate that DHC has the potential to suppress metastasis of osteosarcoma cells by downregulating Rho small GTP-binding proteins.

Modeling Paracrine Noncanonical Wnt Signaling <em>In Vitro</em>

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This process requires complex and coordinated paracrine interactions between signal-sending and signal-receiving cells. Given that these interactions can occur between various types of cells from different lineages, in vivo evaluation of cell-specific defects can be challenging. The present study describes a highly reproducible method to evaluate paracrine noncanonical Wnt signaling in vitro. This protocol was designed with the ability to (1) conduct functional and molecular assessments of noncanonical Wnt signaling between any two cell types of interest; (2) dissect the role of signal-sending versus signal-receiving molecules in the noncanonical Wnt signaling pathway; and (3) perform phenotypic rescue experiments with standard molecular or pharmacologic approaches. This protocol was used to evaluate neural crest cell (NCC)-mediated noncanonical Wnt signaling in myoblasts. The presence of NCCs is associated with an increased number of phalloidin-positive cytoplasmic filopodia and lamellipodia in myoblasts and improved myoblast migration in a wound-healing assay. The Wnt5a-ROR2 axis was identified as a crucial noncanonical Wnt signaling pathway between NCC and second heart field (SHF) cardiomyoblast progenitors. In conclusion, this is a highly tractable protocol to study paracrine noncanonical Wnt signaling mechanisms in vitro.

Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM-based single-cell force spectroscopy

Cadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. In-vitro single molecule force spectroscopy experiments can reveal cadherin–cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin-presenting cell homophilic and heterophilic binding in the proteins’ native conformational and functional states in living cells has rarely been done. Here, we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N- (neural), OB- (osteoblast) and E- (epithelial) cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e., between cells and cadherins of the same and different types. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E-cadherin homophilic binding events are stronger than N/OB heterophilic binding events. Disassembly of intracellular actin filaments affects the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements, but particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins’ homophilic bonds.

Negative Interdomain Coupling of Dystrophin Spectrin Repeats

Spectrin repeat domains are a highly conserved biological motif found in many human structural proteins. Dystrophin contains 24 tandem spectrin repeats which provide structural support through mediating interactions between intracellular actin filaments and the extracellular matrix. However, the molecular mechanism by which dystrophin provides this support is unknown. Understanding this underlying structure/function relationship is important because mutations in dystrophin directly cause muscular dystrophy. Thus far, the following constructs have been expressed in E. coli, purified using chromatography, and thermodynamically characterized: S17, S17-18, S17-19. Parameters were determined through globally fitting thermal denaturation signals from Fluorescence Spectroscopy (FS), Circular Dichroism (CD), and Fluorescence Lifetime Spectroscopy (FLT) to a two-state model of unfolding. Fits were constrained using ΔCp values determined using Differential Scanning Calorimetry (DSC). This parameterization then allowed for determination of the free energy of stability (ΔGunfolding) of each construct. Results indicate that the ΔGunfolding of S17 is nearly double that of S17-19. This pronounced non-additivity indicates that tandem spectrin repeats mutually destabilize each other, termed negative coupling. Additionally, the comparison of Electron Paramagnetic Resonance (EPR) spectra of S17 and S17-19 indicate that the trimer exhibits greater local confirmational flexibility, consistent with decreased stability. To further test this negative coupling hypothesis, we are purifying S19 for thermodynamic characterization. This will allow for the comparison of the sum of S19 and S17-18's ΔGunfolding values with that of the trimer, helping further reveal the energetic and structural basis of dystrophin's mechanism.

The c-terminal region of BLT2 restricts its localization to the lateral membrane in a LIN7C-dependent manner.

Leukotriene B4 receptor type 2 (BLT2) is a G protein-coupled receptor (GPCR) mainly expressed in epithelial cells, where it enhances barrier function. A unique characteristic of BLT2 is its restricted localization to the lateral membrane. However, the molecular mechanism underlying the localization of BLT2 to the lateral membrane and the physiological roles of laterally localized BLT2 are unknown. BLT1 is the most homologous GPCR to BLT2 and localizes to both the apical and lateral membranes. In this study, we generated chimeric receptors of BLT2 and BLT1 as well as deletion mutants of BLT2 to determine the region(s) of BLT2 responsible for its localization. Chimeric receptors containing the C-terminal domain of BLT2 localized only to the lateral membrane, and the C-terminal deletion mutant of BLT2 accumulated at the Golgi apparatus. Furthermore, the middle and C-terminal regions of BLT2 were important for maintaining epithelial barrier function. Proteomics analysis using the chimeric BLT-ascorbate peroxidase 2 biotinylation method showed that some proteins involved in intracellular protein transport, cell-cell junctions, and actin filament binding were located very close to the C-terminal domain of BLT2. Knockdown of lin-7 homolog C (LIN7C), a membrane trafficking protein, led to accumulation of BLT2 in the Golgi apparatus, resulting in diminished epithelial barrier function. These results suggest that the C-terminal region of BLT2 plays an important role in the transport of BLT2 from the Golgi apparatus to the plasma membrane in a LIN7C-dependent manner.

Smoking effect on the ultrastructural properties of cultured lung myofibroblasts

ABSTRACT This study aimed at an ultrastructural characterization of myofibroblasts cultured from different compartments of lung from never-smokers and smokers with or without COPD. In addition, we evaluated the expression of alpha smooth muscle actin (α-SMA), a marker for myofibroblasts, and contractile properties. Stromal cells cultured from central and corresponding peripheral or only from peripheral lung of never-smokers, smokers without COPD and COPD patients were analyzed by transmission electron microscopy (TEM), immunoelectron microscopy (IEM), Western analysis and/or by collagen gel contraction assay. TEM revealed that myofibroblasts cultured from smokers and COPD had less prominent intracellular actin filaments. We also examined fibronexus (FNX), which is a typical ultrastructural feature of myofibroblasts, and observed that patients with COPD more frequently had tandem-like FNX as compared to other samples. Western analysis showed that the samples derived from the central lung of never-smokers expressed higher levels of α-SMA than those of smokers and COPD patients. Cells from central lung were less contractile than those from peripheral lung. We conclude that myofibroblasts have variable ultrastructural and functional properties based on their localization in the lung and, moreover, these properties are affected by both smoking history and COPD.

Convolutional neural network for cell classification using microscope images of intracellular actin networks.

Automated cell classification is an important yet a challenging computer vision task with significant benefits to biomedicine. In recent years, there have been several studies attempted to build an artificial intelligence-based cell classifier using label-free cellular images obtained from an optical microscope. Although these studies showed promising results, such classifiers were not able to reflect the biological diversity of different types of cell. While in terms of malignant cell, it is well-known that intracellular actin filaments are altered substantially. This is thought to be closely related to the abnormal growth features of tumor cells, their ability to invade surrounding tissues and also to metastasize. Therefore, being able to classify different types of cell based on their biological behaviors using automated technique is more advantageous. This article reveals the difference in the actin cytoskeleton structures between breast normal and cancer cells, which may provide new information regarding malignant changes and be used as additional diagnostic marker. Since the features cannot be well detected by human eyes, we proposed the application of convolutional neural network (CNN) in cell classification based on actin-labeled fluorescence microscopy images. The CNN was evaluated on a large number of actin-labeled fluorescence microscopy images of one human normal breast epithelial cell line and two types of human breast cancer cell line with different levels of aggressiveness. The study revealed that the CNN performed better in the cell classification task compared to a human expert.

Synthetic peptide TEKKRRETVEREKE derived from ezrin induces differentiation of NIH/3T3 fibroblasts

Synthetic 14 AA peptide (Gepon) derived from the hinge region of ezrin, a protein that links cell surface molecules to intracellular actin filaments, accelerates and facilitates wound and ulcer healing in clinical applications. However, the molecular mechanisms underlying this phenomenon and involved in enhanced healing of wounds with Gepon are not yet understood. The purpose of current study was to investigate intracellular signaling pathways involved in the effect of this peptide on wild type and genetically modified (CD44 KO) NIH/3T3 embryonic mouse fibroblasts. Gepon treatment of NIH/3T3 cells resulted in morphological and biochemical changes, characteristic of differentiated fibroblasts. While treatment of NIH/3T3 cells with TGF-β1 triggered the activation of both canonical and non-canonical signaling pathways, exposure of fibroblasts to Gepon activated only the ERK1/2 dependent pathway without modulating SMAD dependent signaling pathway. Knocking out hyaluronic acid CD44 receptor did not change Gepon or TGF-β1 dependent activation of intracellular signaling pathways and assembling of α-SMA-positive filaments. Gepon dependent differentiation of NIH/3T3 fibroblasts is based on activation of ERK1/2 kinase, non-canonical intracellular signaling pathway. Our data suggest that the treatment of fibroblasts with Gepon triggers activation of the non-canonical (SMAD independent) intracellular signaling pathway that involves ERK1/2kinase phosphorylation. Activation of the MAPK signaling pathway and the increase in formation of α-SMA containing stress filaments induced by Gepon were independent on presence of CD44 receptor in NIH/3T3 fibroblasts. Thus, our observation designates the significance and sufficiency of MAPK pathway mediated activation of fibroblasts with Gepon for healing of erosion, ulcers and wounds.

Graphene Oxide Nanosheets Retard Cellular Migration via Disruption of Actin Cytoskeleton.

Graphene and graphene-based nanomaterials are broadly used for various biomedical applications due to their unique physiochemical properties. However, how graphene-based nanomaterials interact with biological systems has not been thoroughly studied. This study shows that graphene oxide (GO) nanosheets retard A549 lung carcinoma cell migration through nanosheet-mediated disruption of intracellular actin filaments. After GO nanosheets treatment, A549 cells display slower migration and the structure of the intracellular actin filaments is dramatically changed. It is found that GO nanosheets are capable of absorbing large amount of actin and changing the secondary structures of actin monomers. Large-scale all-atom molecular dynamics simulations further reveal the interactions between GO nanosheets and actin filaments at molecular details. GO nanosheets can insert into the interstrand gap of actin tetramer (helical repeating unit of actin filament) and cause the separation of the tetramer which eventually leads to the disruption of actin filaments. These findings offer a novel mechanism of GO nanosheet induced biophysical responses and provide more insights into their potential for biomedical applications.

Cultured tumor model inspection for anticancer drugs using acoustic impedance microscope

High-frequency ultrasonic microscope is useful to observe intracellular structure of cultured living cells. We have observed the dynamical change of intracellular actin filaments in several cells, and reported the difference of stabilities between cancer cells and ordinary cells. The dynamics of actin filaments are deeply related to cancer cell viability. In this study, we applied this acoustic microscopy to antitumor drug screening. One of the cultured brain cancer models was established with both normal glial cells and C6 glioma cells. Normal glial cells were labeled by endogenous fluorescent protein to be identified. Both cells were co-cultured on a 75 micrometer-thick polystyrene substrate. Acoustic pulse wave with 320 MHz was focused on the interface between the cell and the substrate, and sent through the substrate. Either classical anticancer drug, cytochalasin B, or novel clinical anticancer drug, temozolomide, was applied to the cultured cancer model, and observed their intracellular acoustic cha...

An Unroofing Method to Observe the Cytoskeleton Directly at Molecular Resolution Using Atomic Force Microscopy.

An improved unroofing method enabled the cantilever of an atomic force microscope (AFM) to reach directly into a cell to visualize the intracellular cytoskeletal actin filaments, microtubules, clathrin coats, and caveolae in phosphate-buffered saline (PBS) at a higher resolution than conventional electron microscopy. All of the actin filaments clearly exhibited a short periodicity of approximately 5–6 nm, which was derived from globular actins linked to each other to form filaments, as well as a long helical periodicity. The polarity of the actin filaments appeared to be determined by the shape of the periodic striations. Microtubules were identified based on their thickness. Clathrin coats and caveolae were observed on the cytoplasmic surface of cell membranes. The area containing clathrin molecules and their terminal domains was directly visualized. Characteristic ridge structures located at the surface of the caveolae were observed at high resolution, similar to those observed with electron microscopy (EM). Overall, unroofing allowed intracellular AFM imaging in a liquid environment with a level of quality equivalent or superior to that of EM. Thus, AFMs are anticipated to provide cutting-edge findings in cell biology and histology.

Human recombinant RNASET2: A potential anti-cancer drug.

The roles of cell motility and angiogenetic processes in metastatic spread and tumor aggressiveness are well established and must be simultaneously targeted to maximize antitumor drug potency. This work evaluated the antitumorigenic capacities of human recombinant RNASET2 (hrRNASET2), a homologue of the Aspergillus niger T2RNase ACTIBIND, which has been shown to display both antitumorigenic and antiangiogenic activities. hrRNASET2 disrupted intracellular actin filament and actin-rich extracellular extrusion organization in both CT29 colon cancer and A375SM melanoma cells and induced a significant dose-dependent inhibition of A375SM cell migration. hrRNASET2 also induced full arrest of angiogenin-induced tube formation and brought to a three-fold lower relative HT29 colorectal and A375SM melanoma tumor volume, when compared to Avastin-treated animals. In parallel, mean blood vessel counts were 36.9% lower in hrRNASET2-vs. Avastin-treated mice and survival rates of hrRNASET2-treated mice were 50% at 73 days post-treatment, while the median survival time for untreated animals was 22 days. Moreover, a 60-day hrRNASET2 treatment period reduced mean A375SM lung metastasis foci counts by three-fold when compared to untreated animals. Taken together, the combined antiangiogenic and antitumorigenic capacities of hrRNASET2, seemingly arising from its direct interaction with intercellular and extracellular matrices, render it an attractive anticancer therapy candidate.

Length adaptation of smooth muscle contractile filaments in response to sustained activation

Airway and bladder smooth muscles are known to undergo length adaptation under sustained contraction. This adaptation process entails a remodelling of the intracellular actin and myosin filaments which shifts the peak of the active force–length curve towards the current length. Smooth muscles are therefore able to generate the maximum force over a wide range of lengths. In contrast, length adaptation of vascular smooth muscle has attracted very little attention and only a handful of studies have been reported. Although their results are conflicting on the existence of a length adaptation process in vascular smooth muscle, it seems that, at least, peripheral arteries and arterioles undergo such adaptation. This is of interest since peripheral vessels are responsible for pressure regulation, and a length adaptation will affect the function of the cardiovascular system. It has, e.g., been suggested that the inward remodelling of resistance vessels associated with hypertension disorders may be related to smooth muscle adaptation. In this study we develop a continuum mechanical model for vascular smooth muscle length adaptation by assuming that the muscle cells remodel the actomyosin network such that the peak of the active stress–stretch curve is shifted towards the operating point. The model is specialised to hamster cheek pouch arterioles and the simulated response to stepwise length changes under contraction. The results show that the model is able to recover the salient features of length adaptation reported in the literature.

Amyloid precursor-like protein 2 (APLP2) affects the actin cytoskeleton and increases pancreatic cancer growth and metastasis.

Amyloid precursor-like protein 2 (APLP2) is aberrantly expressed in pancreatic cancer. Here we showed that APLP2 is increased in pancreatic cancer metastases, particularly in metastatic lesions found in the diaphragm and intestine. Examination of matched human primary tumor-liver metastasis pairs showed that 38.1% of the patients had positive APLP2 expression in both the primary tumor and the corresponding liver metastasis. Stable knock-down of APLP2 expression (with inducible shRNA) in pancreatic cancer cells reduced the ability of these cells to migrate and invade. Loss of APLP2 decreased cortical actin and increased intracellular actin filaments in pancreatic cancer cells. Down-regulation of APLP2 decreased the weight and metastasis of orthotopically transplanted pancreatic tumors in nude mice.

Detection of cellular damage by hydrogen peroxide using SV40-T2 cells on shear horizontal surface acoustic wave (SH-SAW) sensor

The rat lung epithelial cell line SV40-T2 was used to develop a cellular biosensing system to assay for environmental toxicants. The novel approach on which this system is based involves direct attachment of cultured rat or human cells onto a cell-adhesive matrix on the device through which shear horizontal surface acoustic waves (SH-SAW) are transmitted using 50MHz SAW resonator. This novel design enables sensitive monitoring of changes of the electrophysical characteristics of cells, such as their conductivity and relative permittivity. A time-dependent change of phase of SAW and change of insertion loss (change of amplitude) were observed when the cells were treated with 0.5 or 1.0mM H2O2. The change of insertion loss was biphasic, with an early phase (1–3h) and a late phase (3–6h). The late phase coincided with the destruction of cell–cell tight junctions detected by measurement of the transepithelial electrical resistance and paracellular permeability; in contrast, the early phase coincided with the destruction of intracellular actin filaments by H2O2. The early-phase effect of H2O2 on phase shift may be attributable to the change of intracellular permittivity by a change of cellular polarity. Immunofluorescence microscopy showed the disappearance of zonula occludens protein 1 from the region of cell–cell contact. These results suggest the correlation between the change of insertion loss as an SAW parameter and the destruction of tight junctions of the cells on the SH-SAW device in the late phase.

Impulses and pressure waves cause excitement and conduction in the nervous system

It is general accepted, that nerval excitement and conduction is caused by voltage changes.However, the influx of fluid into an elastical tube releases impulses or pressure waves. Therefore an influx of ion currents, respectively fluid motions into the elastic neuronal cells and fibres also induce impulses. This motion of charge carriers are measured by voltage devices as oscillations or action potentials, but the voltage changes may be an epiphenomenon of the (mechanical) impulses.Impulse waves can have a high speed. As stiffer or inelastic a tube wall, the greater is the speed of the impulse. Myelin sheaths cause a significant stiffening of the nerve fibre wall and myelinated fibres have a conduction velocity up to 120m/s. The influx of fluid at the nodes of Ranvier intensifies periodically the impulse wave in the nerve fibres.The authors suggest that also the muscle end-plate acts as a conductor of axonal impulses to the inner of the muscle fibres and that the exocytosis of acetylcholine into the synaptic cleft may be an amplifier of the axonal impulse. It is discussed that intracellular actin filaments may also influence motions at the neuronal membrane.Many sensory nerve cells are excited due to exogenous or endogenous mechanical impulses. It may plausible that such impulses are conducted directly to the sensory nerve cell bodies in the dorsal root ganglia without the transformation in electric energy. Excitation conduction happens without noteworthy energy consumption because the flow of ion currents through the membranes takes place equivalent to the concentration gradient.Impulse waves cause short extensions of the lipid membranes of the cell- and fibres walls and therefore they can induce opening and closing of the included ion channels. This mechanism acts to “voltage gated” and “ligand-gated” channels likewise.The concept of neuronal impulses can be helpful to the understanding of other points of neurophysiology or neuronal diseases. This includes e.g., the brain concussion and pathohistological findings in Alzheimer dementia.To verify the concept of (mechanical) impulses in the nervous system it is necessary to carry out biophysical or mechanical investigations in very small dimensions and the authors hope to give for this a sufficient stimulus.

Abstract 400: Coordinated loss of CD9 and IGSF8 expression on the prostate cancer cell surface contributes to metastasis and hormone responsiveness.

Abstract The tetraspanin web is a complex multi-molecular cell structure that tethers cell surface signalling proteins (i.e. integrins) to their intermediate intracellular signalling partners (i.e. small GTPases) and anchors actin filaments. Loss of certain components of the web (CD9, CD82 or CD316) are associated with increased invasive and metastatic behaviors of prostate cancer (PCa) cells and here we demonstrate that two of these tetraspanin web components, CD9 and CD316, also play a role in regulating androgen signaling in PCa cells. Additionally, we utilized human prostate cancer tissue microarrays (TMAs) containing 261 prostate/PCa specimens of differing stages, grades and states of progression (including 60 castration resistant tumors [CRPCs]) to study the relationship between CD9 and CD316 expressions and progression to CRPC. Using androgen-dependent LNCaP cells as a model, we found that androgen deprivation (AD) suppressed expression of CD9 protein and mRNA without affecting CD316 expression. FACS analysis showed that the presence of CD9 on the cell surface was coordinately reduced by AD as was cell surface CD316 which was relocalized to the cytoplasm. This effect was reversed by addition of R1881. Knockdown of CD9 or CD316 in LNCaP cells using siRNA reduced expression of androgen receptor (AR) mRNA but increased expression of PSA, an AR target gene. CD9 or CD316 knockdown also increased LNCaP growth in fetal bovine serum (FBS) containing medium but not in charcoal-stripped FBS (CS-FBS) whereas dual-knockdown further increased growth in FBS as well as in CS-FBS. TMAs immunostained for CD9 or CD316 were assessed for correlation to clinicopathological parameters. For untreated, primary tumors, CD9 and CD316 expression were inversely correlated with Gleason scores (R2=0.9612). CD9 expression was diminished during progression to metastatic (p<0.0038, compared to non-metastatic) and further in CRPC (p<0.040, compared to metastatic non-treated) disease. Conversely, CD316 expression was increased in primary (non-metastatic) as well as in metastatic untreated disease but then reduced (p<0.046) during progression to metastatic CRPC. We propose that androgen deprivation (first line therapy for advanced PCa) may potentially trigger loss of both CD9 and IGSF8 expression on the cell surface that contributes to metastatic behaviors as well as to responsiveness to androgen ablation therapies. This work is supported by the Cancer Research Society (Canada). Citation Format: Na Li, Ladan Fazli, Estelle Li, Ralph Buttyan. Coordinated loss of CD9 and IGSF8 expression on the prostate cancer cell surface contributes to metastasis and hormone responsiveness. [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 400. doi:10.1158/1538-7445.AM2013-400