integrin-FAK Signaling Research Articles

Abstract 5447: Microtubule-associated protein MAP2 promotes drug resistance and cancer stemness in hepatocellular carcinoma through integrin dysregulation

Abstract Integrins are adhesion molecules that mediate mechanical and chemical signal transduction pathways to support cell survival and proliferation. Dysregulated integrin signaling empowers tumor cells to drive oncogenic stemness functions, including tumor initiation, epithelial plasticity, metastatic reactivation, and resistance to therapies. However, the interplay between cancer stemness and integrin signaling in hepatocellular carcinoma (HCC) remains poorly understood. HCC cells marked by CD133 represent an important functional subset of HCC tumors, displaying a dedifferentiated status with stem cell traits. In this study, transcriptome profiling reveals specific downregulation of integrin α family genes and integrin signaling in ‘HCC’ CD133+ cells isolated from NRAS+AKT-driven HCC, but not epithelial-specific ‘normal’ CD133+ cells isolated from regenerating liver. Of note, one of the most differentially upregulated genes identified in CD133+ HCC cell profiling, microtubule-associated protein MAP2, demonstrates the ability to suppress integrin expression. MAP2 overexpression is frequently observed in HCC and correlates with aggressive clinical and stemness features, including survival, tumor stage, and stemness signatures. Epigenetic modifications by H3K27Ac contribute to MAP2 upregulation. Functionally, MAP2 promotes cancer stemness and cell invasion, and confers resistance to the targeted therapy sorafenib. Mechanistically, the inhibition of collagen-binding integrin α subunits by MAP2 subsequently ablates integrin β1-mediated cell adhesion and FAK signaling. Estramustine Phosphate (EMP), previously reported to inhibit the interaction of MAP2 with actin filaments, attains a synergistic effect in suppressing tumor initiation and growth of HCC cell lines, HCC patient-derived organoids and NRAS+AKT HCC mouse model when used in combination with sorafenib. In summary, MAP2 inhibition may represent a potential novel therapy for HCC by targeting its cancer stemness roots and altering integrin signaling. Ongoing work is focused on the study of MAP2 regulation of integrin signaling and cell behaviours in the maintenance of a more stemness state in HCC. Citation Format: Ut Kei Lou, Ka-Hei Lam, Huajian Yu, Jia Jian Loh, Ki-Fong Man, Lei Zhou, Yuan Gao, Tin-Lok Wong, Cheng-Han Yu, Stephanie Ma. Microtubule-associated protein MAP2 promotes drug resistance and cancer stemness in hepatocellular carcinoma through integrin dysregulation [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 5447.

Osteopontin modulates microglial activation states and attenuates inflammatory responses after subarachnoid hemorrhage in rats

AimsOsteopontin (OPN) has demonstrated neuroprotective effects in various stroke models. Its role in neuroinflammation after brain injury remains to be elucidated. This study aims to clarify the effect of OPN on neuroinflammation, particularly on the functional states of microglia after subarachnoid hemorrhage (SAH). Methods77 rats were randomly divided into the following groups: Sham, SAH 24 h, SAH + rOPN, SAH + Vehicle (PBS), SAH + OPN siRNA, and SAH + Scr siRNA, SAH + rOPN+Fib-14 and SAH + rOPN+DMSO. Modified Garcia and beam balance tests were used to evaluate neurobehavioral outcomes. Semi-quantitative immunofluorescence staining was performed to measure expression of myeloperoxidase (MPO) and microglia activation state markers CD16, CD206 after SAH and recombinant OPN treatment. The quantification of microglia activation and functional markers CD16, CD206, TNF-α and IL-10 were further evaluated using Western-blotting. ResultsNasal administration of rOPN improved neurological dysfunction, attenuated neutrophil infiltration, and decreased expression of phenotypic and functional markers of pro-inflammatory microglia CD16 and TNF-α. It also promoted an anti-inflammatory microglial state, as evidenced by increased expression of CD206 and IL-10. Furthermore, after blocking the phosphorylation of FAK signaling, the effects of rOPN on microglial activation states were partially reversed. The downstream pathways of STAT3 and NF-κB also exhibited consistent changes, suggesting the involvement of the STAT3 and NF-κB pathways in OPN's modulation of microglial activation via integrin-FAK signaling. ConclusionOPN attenuates inflammatory responses after SAH by promoting an anti-inflammatory microglial state, potentially mediated through the integrin-FAK-STAT3 and NF-κB signaling pathways.

A Ubiquitin-Dependent Switch on MEF2D Senses Pro-Metastatic Niche Signals to Facilitate Intrahepatic Metastasis of Liver Cancer.

Effective treatment for metastasis, a leading cause of cancer-associated death, is still lacking. To seed on a distal organ, disseminated cancer cells (DCCs) must adapt to the local tissue microenvironment. However, it remains elusive how DCCs respond the pro-metastatic niche signals. Here, systemic motif-enrichment identified myocyte enhancer factor 2D (MEF2D) as a critical sensor of niche signals to regulate DCCs adhesion and colonization, leading to intrahepatic metastasis and recurrence of liver cancer. In this context, MEF2D transactivates Itgb1 (coding β1-integrin) and Itgb4 (coding β4-integrin) to execute temporally unique functions, where ITGB1 recognizes extracellular matrix for early seeding, and ITGB4 acts as a novel sensor of neutrophil extracellular traps-DNA (NETs-DNA) for subsequent chemotaxis and colonization. In turn, an integrin-FAK circuit promotes a phosphorylation-dependent USP14-orchastrated deubiquitination switch to stabilize MEF2D via circumventing degradation by the E3-ubiquitin-ligase MDM2. Clinically, the USP14(pS432)-MEF2D-ITGB1/4 feedback loop is often hyper-active and indicative of inferior outcomes in human malignancies, while its blockade abrogated intrahepatic metastasis of DCCs. Together, DCCs exploit a deubiquitination-dependent switch on MEF2D to integrate niche signals in the liver mesenchyme, thereby amplifying the pro-metastatic integrin-FAK signaling. Disruption of this feedback loop is clinically applicable with fast-track potential to block microenvironmental cues driving metastasis.

Fibronectin fragments generated by pancreatic trypsin act as endogenous inhibitors of pancreatic tumor growth

BackgroundThe pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches.Methods and resultsHere we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5β1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models.ConclusionsThis study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer.

Epigenetic suppression of PGC1α (PPARGC1A) causes collateral sensitivity to HMGCR-inhibitors within BRAF-treatment resistant melanomas

While targeted treatment against BRAF(V600E) improve survival for melanoma patients, many will see their cancer recur. Here we provide data indicating that epigenetic suppression of PGC1α defines an aggressive subset of chronic BRAF-inhibitor treated melanomas. A metabolism-centered pharmacological screen further identifies statins (HMGCR inhibitors) as a collateral vulnerability within PGC1α-suppressed BRAF-inhibitor resistant melanomas. Lower PGC1α levels mechanistically causes reduced RAB6B and RAB27A expression, whereby their combined re-expression reverses statin vulnerability. BRAF-inhibitor resistant cells with reduced PGC1α have increased integrin-FAK signaling and improved extracellular matrix detached survival cues that helps explain their increased metastatic ability. Statin treatment blocks cell growth by lowering RAB6B and RAB27A prenylation that reduces their membrane association and affects integrin localization and downstream signaling required for growth. These results suggest that chronic adaptation to BRAF-targeted treatments drive novel collateral metabolic vulnerabilities, and that HMGCR inhibitors may offer a strategy to treat melanomas recurring with suppressed PGC1α expression.

Down-Regulating Scar Formation by Microneedles Directly via a Mechanical Communication Pathway.

Excessive extracellular matrix deposition drives fibroblasts into a state of high mechanical stress, exacerbating pathological fibrosis and hypertrophic scar formation, leading to tissue dysfunction. This study reports a minimally invasive and convenient approach to obtaining scarless tissue using a silk fibroin microneedle patch (SF MNs). We found that by tuning the MN size and density only, the biocompatible MNs significantly decreased the scar elevation index in the rabbit ear hypertrophic scar model and increased ultimate tensile strength close to regular skin. To advance our understanding of this recent approach, we built a fibroblast-populated collagen lattice system and finite element model to study MN-mediated cellular behavior of fibroblasts. We found that the MNs reduced the fibroblasts generated contraction and mechanical stress, as indicated by decreased expression of the mechanical sensitive gene ANKRD1. Specifically, SF MNs attenuated the integrin-FAK signaling and consequently down-regulated the expression of TGF-β1, α-SMA, collagen I, and fibronectin. It resulted in a low-stress microenvironment that helps to reduce scar formation significantly. Microneedles’ physical intervention via the mechanotherapeutic strategy is promising for scar-free wound healing.

Insulin-like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism.

Simple SummaryInsulin-like growth factor-1 (IGF-1) is a growth hormone and is implicated in prostate cancer progression. Most prostate cancers begin in an androgen-dependent state so that androgen deprivation therapy results in improved clinical outcome. However, some cancerous cells may survive androgen deprivation, growing into therapy-resistant, androgen-independent prostate cancer. The present study investigated the influence of IGF-1 on tumor growth and migration properties using androgen-dependent LNCaP and VCaP and androgen-independent PC3 and DU145 prostate cancer cells. Stimulation with IGF-1 activated growth in all cell lines. There were changes in transmembrane receptors (integrins) that bind cells to each other and changes in focal adhesion kinase that controls cell motility. Intracellular Akt/mTOR signaling, regulating cell division, was also activated. Thus, it seems that prostate cancer progression is controlled by a fine-tuned network between IGF-1-driven integrin-FAK signaling and the Akt-mTOR pathway. Concerted targeting of both pathways may, therefore, help prevent cancer dissemination.Insulin-like growth factor-1 (IGF-1)-related signaling is associated with prostate cancer progression. Links were explored between IGF-1 and expression of integrin adhesion receptors to evaluate relevance for growth and migration. Androgen-resistant PC3 and DU145 and androgen-sensitive LNCaP and VCaP prostate cancer cells were stimulated with IGF-1 and tumor growth (all cell lines), adhesion and chemotaxis (PC3, DU145) were determined. Evaluation of Akt/mTOR-related proteins, focal adhesion kinase (FAK) and integrin α and β subtype expression followed. Akt knock-down was used to investigate its influence on integrin expression, while FAK blockade served to evaluate its influence on mTOR signaling. Integrin knock-down served to investigate its influence on tumor growth and chemotaxis. Stimulation with IGF-1 activated growth in PC3, DU145, and VCaP cells, and altered adhesion and chemotactic properties of DU145 and PC3 cells. This was associated with time-dependent alterations of the integrins α3, α5, αV, and β1, FAK phosphorylation and Akt/mTOR signaling. Integrin blockade or integrin knock-down in DU145 and PC3 cells altered tumor growth, adhesion, and chemotaxis. Akt knock-down (DU145 cells) cancelled the effect of IGF-1 on α3, α5, and αV integrins, whereas FAK blockade cancelled the effect of IGF-1 on mTOR signaling (DU145 cells). Prostate cancer growth and invasion are thus controlled by a fine-tuned network between IGF-1 driven integrin-FAK signaling and the Akt-mTOR pathway. Concerted targeting of integrin subtypes along with Akt-mTOR signaling could, therefore, open options to prevent progressive dissemination of prostate cancer.

Tumor Necrosis Factor-alpha utilizes MAPK/NFκB pathways to induce cholesterol-25 hydroxylase for amplifying pro-inflammatory response via 25-hydroxycholesterol-integrin-FAK pathway.

Exaggerated inflammatory response results in pathogenesis of various inflammatory diseases. Tumor Necrosis Factor-alpha (TNF) is a multi-functional pro-inflammatory cytokine regulating a wide spectrum of physiological, biological, and cellular processes. TNF induces Focal Adhesion Kinase (FAK) for various activities including induction of pro-inflammatory response. The mechanism of FAK activation by TNF is unknown and the involvement of cell surface integrins in modulating TNF response has not been determined. In the current study, we have identified an oxysterol 25-hydroxycholesterol (25HC) as a soluble extracellular lipid amplifying TNF mediated innate immune pro-inflammatory response. Our results demonstrated that 25HC-integrin-FAK pathway amplifies and optimizes TNF-mediated pro-inflammatory response. 25HC generating enzyme cholesterol 25-hydroxylase (C25H) was induced by TNF via NFκB and MAPK pathways. Specifically, chromatin immunoprecipitation assay identified binding of AP-1 (Activator Protein-1) transcription factor ATF2 (Activating Transcription Factor 2) to the C25H promoter following TNF stimulation. Furthermore, loss of C25H, FAK and α5 integrin expression and inhibition of FAK and α5β1 integrin with inhibitor and blocking antibody, respectively, led to diminished TNF-mediated pro-inflammatory response. Thus, our studies show extracellular 25HC linking TNF pathway with integrin-FAK signaling for optimal pro-inflammatory activity and MAPK/NFκB-C25H-25HC-integrin-FAK signaling network playing an essential role to amplify TNF dependent pro-inflammatory response. Thus, we have identified 25HC as the key factor involved in FAK activation during TNF mediated response and further demonstrated a role of cell surface integrins in positively regulating TNF dependent pro-inflammatory response.

Focal Adhesion Kinase Fine Tunes Multifaced Signals toward Breast Cancer Progression.

Simple SummaryBreast cancer is the most common diagnosed malignancy and the main leading cause of tumor-related death among women worldwide. Thus, several studies have been carried out in order to identify valuable molecular biomarkers for the prognosis and prediction of therapeutic responses in breast tumor patients. Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase overexpressed in diverse tumors, including breast cancer. Here, we review previous evidence dealing with the role of FAK in the growth and metastatic features of breast tumors, its action as a driver of cancer stem cell phenotype and function as a mechanotransducer, and FAK activity within the breast tumor microenvironment and critical prognostic value of FAK expression in breast malignancy. In addition, we recapitulated the usefulness of FAK inhibitors in breast cancer treatment.Breast cancer represents the most common diagnosed malignancy and the main leading cause of tumor-related death among women worldwide. Therefore, several efforts have been made in order to identify valuable molecular biomarkers for the prognosis and prediction of therapeutic responses in breast tumor patients. In this context, emerging discoveries have indicated that focal adhesion kinase (FAK), a non-receptor tyrosine kinase, might represent a promising target involved in breast tumorigenesis. Of note, high FAK expression and activity have been tightly correlated with a poor clinical outcome and metastatic features in several tumors, including breast cancer. Recently, a role for the integrin-FAK signaling in mechanotransduction has been suggested and the function of FAK within the breast tumor microenvironment has been ascertained toward tumor angiogenesis and vascular permeability. FAK has been also involved in cancer stem cells (CSCs)-mediated initiation, maintenance and therapeutic responses of breast tumors. In addition, the potential of FAK to elicit breast tumor-promoting effects has been even associated with the capability to modulate immune responses. On the basis of these findings, several agents targeting FAK have been exploited in diverse preclinical tumor models. Here, we recapitulate the multifaceted action exerted by FAK and its prognostic significance in breast cancer. Moreover, we highlight the recent clinical evidence regarding the usefulness of FAK inhibitors in the treatment of breast tumors.

CD81 Controls Beige Fat Progenitor Cell Growth and Energy Balance via FAK Signaling

Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/β1 and αV/β5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.

Fak silencing impairs osteogenic differentiation of bone mesenchymal stem cells induced by uniaxial mechanical stretch

Background/purposeMechanical stretch plays a key role in promoting proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) in distraction osteogenesis (DO). A better understanding of how the extracellular biomechanical stimulation is transferred to intracellular signal expression will benefit DO. Focal adhesion kinase (FAK) is a key factor in integrin signaling pathway. However, little is known about the effect of integrin-FAK signaling during the process of stretch induced osteogenic differentiation of BMSCs. Materials and methodsA specific short hairpin RNAs (shRNAs) lentiviral expression vector was used to silence Fak gene and a well-established in vitro uniaxial dynamic stretching device was applied to stimulate DO. Fak silencing was confirmed by fluorescence microscopy and the detection of Fak mRNA and FAK, p-FAK protein expression. Alkaline phosphatase (ALP) activity, expression of osteogenic differentiation markers - runt-related transcription factor 2 (RUNX2/Runx2) and alkaline phosphatase (Alp) together with integrin upstream signal transduction molecules integrin beta-1 (ITGB1/Itgb1) and downstream signal transduction molecules integrin-linked kinase (ILK) were detected after the stretch. ResultsThe results showed that mechanical stretch in control groups significantly induced the osteogenic differentiation of BMSCs with increased ALP activity, expression of RUNX2/Runx2 and Alp, together with upregulated ITGB1/Itgb1 and ILK, which all vanished in Fak silencing group. ConclusionSilencing of the Fak gene inhibited the osteogenic differentiation of rat BMSCs induced by in vitro mechanical stretch through integrin signaling pathway.

Direct Interactions With Cancer-Associated Fibroblasts Lead to Enhanced Pancreatic Cancer Stem Cell Function.

Cancer-associated fibroblasts (CAFs) play an important role in the progression of pancreatic ductal adenocarcinoma (PDAC) by promoting tumor cell migration and drug resistance. We determined the impact of CAFs on PDAC cancer stem cells (CSCs). Fibroblast cell lines from patients' tumors were cocultured with PDAC cells and examined for clonogenic growth and self-renewal using colony-forming assays and migration in vitro. Changes in the frequency of CSCs was determined by flow cytometry. The effect of integrin-focal adhesion kinase (FAK) signaling on CAF-mediated clonogenic growth was evaluated using short hairpin RNAs against β1 integrin and FAK as well as a small-molecule FAK inhibitor. Cancer-associated fibroblasts enhanced PDAC clonogenic growth, self-renewal, and migration that was associated with an increase in the frequency of CSCs. These fibroblast cells were activated by PDAC cells and increased collagen synthesis resulting in FAK activation in PDAC cells. Knockdown of β1-integrin and FAK or the inhibition of FAK kinase activity in PDAC cells abrogated the impact of CAFs on clonogenic growth. Therefore, CAFs enhance PDAC clonogenic growth, self-renewal, and the frequency of CSCs through type I collagen production that enhances integrin-FAK signaling in PDAC cells.

Regulation of osteoblast functions on titanium surfaces with different micro/nanotopographies and compositions

Surface modification of medical implants is considered as an effective method to improve cellular behaviors and the integration of tissues with materials. Titanium (Ti)-based materials with four different micro/nano-structures and compositions were prepared by acid etching, electrochemical anodization and alkali-heat treatment. The surface morphologies and compositions of the different surface-modified Ti materials were characterized by field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM) and X-ray diffraction (XRD). The effects of the micro/nano structured and compositions of the surfaces on cellular responses were investigated in vitro by observing the morphology, adhesion, proliferation and osteogenic differentiation of osteoblasts. To further investigate the underlying mechanisms, an RT-PCR assay was performed to analyze the expression levels of cell adhesion-related genes. Our results indicated that the nanosized structure and anatase composition could promote the adhesion and proliferation of MC3T3-E1 pre-osteoblast, as well as alkaline phosphatase activity and extracellular matrix mineralization via the integrin-FAK signaling pathway. Taken together, our innovation presented in this work demonstrated that the surface nano-structure design and composition of biomedical implants can be modified of for future orthopaedic applications.

Promotion of tumor progression and cancer stemness by MUC15 in thyroid cancer via the GPCR/ERK and integrin-FAK signaling pathways

Thyroid cancer is the fifth most common cancer diagnosed in women worldwide. Notwithstanding advancements in the prognosis and treatment of thyroid cancer, 10–20% of thyroid cancer patients develops chemotherapeutic resistance and experience relapse. According to previous reports and TCGA database, MUC15 (MUCIN 15) upregulation is highly correlated with thyroid cancer progression. However, the role of MUC15 in tumor progression and metastasis is unclear. This study aimed to investigate factors mediating cancer stemness in thyroid cancer. MUC15 plays an important role in sphere formation, as an evident from the expression of stemness markers including SOX2, KLF4, ALDH1A3, and IL6. Furthermore, ectopic expression of MUC15 activated extracellular signal-regulated kinase (ERK) signaling via G-protein–coupled receptor (GPCR)/cyclic AMP (cAMP) and integrin/focal adhesion kinase pathways. Interestingly, ectopic expression of MUC15 did not affect RAF/mitogen-activated protein kinase kinase (MEK)-mediated ERK activation. The present findings may provide novel insights into the development of diagnostic, prognostic, and therapeutic applications of MUC15 in thyroid cancer.

A Feedforward Mechanism Mediated by Mechanosensitive Ion Channel PIEZO1 and Tissue Mechanics Promotes Glioma Aggression

Alteration of tissue mechanical properties is a physical hallmark of solid tumors including gliomas. How tumor cells sense and regulate tissue mechanics is largely unknown. Here, we show that mechanosensitive ion channel Piezo regulates mitosis and tissue stiffness of Drosophila gliomas, but not non-transformed brains. PIEZO1 is overexpressed in aggressive humangliomas and its expression inversely correlates with patient survival. Deleting PIEZO1 suppresses the growth of glioblastoma stem cells, inhibits tumor development, and prolongs mouse survival. Focal mechanical force activates prominent PIEZO1-dependent currents from glioma cell processes, but not soma. PIEZO1 localizes at focal adhesions to activate integrin-FAK signaling, regulate extracellular matrix, and reinforce tissue stiffening. In turn, a stiffer mechanical microenvironment elevates PIEZO1 expression to promote glioma aggression. Therefore, glioma cells are mechanosensory in a PIEZO1-dependent manner, and targeting PIEZO1 represents a strategy to break the reciprocal, disease-aggravating feedforward circuit between tumor cell mechanotransduction and the aberrant tissue mechanics. VIDEO ABSTRACT.

IL-32\u03b3 attenuates airway fibrosis by modulating the integrin-FAK signaling pathway in fibroblasts

BackgroundFibrosis in severe asthma often leads to irreversible organ dysfunction. However, the mechanism that regulates fibrosis remains poorly understood. Interleukin (IL)-32 plays a role in several chronic inflammatory diseases, including severe asthma. In this study, we investigated whether IL-32 is involved in fibrosis progression in the lungs.MethodsMurine models of chronic airway inflammation induced by ovalbumin and Aspergillus melleus protease and bleomycin-induced pulmonary fibrosis were employed. We evaluated the degree of tissue fibrosis after treatment with recombinant IL-32γ (rIL-32γ). Expression of fibronectin and α-smooth muscle actin (α-SMA) was examined and the transforming growth factor (TGF)-β-related signaling pathways was evaluated in activated human lung fibroblasts (MRC-5 cells) treated with rIL-32γ.ResultsrIL-32γ significantly attenuated collagen deposition and α-SMA production in both mouse models. rIL-32γ inhibited the production of fibronectin and α-SMA in MRC-5 cells stimulated with TGF-β. Additionally, rIL-32γ suppressed activation of the integrin-FAK-paxillin signaling axis but had no effect on the Smad and non-Smad signaling pathways. rIL-32γ localized outside of MRC-5 cells and inhibited the interaction between integrins and the extracellular matrix without directly binding to intracellular FAK and paxillin.ConclusionsThese results demonstrate that IL-32γ has anti-fibrotic effects and is a novel target for preventing fibrosis.

Lung squamous cell carcinoma cells express non-canonically glycosylated IgG that activates integrin-FAK signaling

It is increasingly recognized that many human carcinomas express immunoglobulin (Ig) molecules that are distinct from B-cell-derived Ig and play important roles in cancer initiation, progression, and metastasis. However, the molecular mechanisms underlying the functions of cancer-derived Ig remain elusive. Here, we report that lung squamous cell carcinoma (LSCC) cells frequently express high levels of cancer IgG (CIgG) that is specifically recognized by a monoclonal antibody RP215. RP215 recognizes CIgG via a novel epitope that involves an N-glycan modification at a non-consensus site within the CH1 domain. We demonstrate that RP215 recognized CIgG (RP215-CIgG) promotes survival, migration and in vivo growth of LSCC cells, and these oncogenic activities are strongly inhibited by RP215. Mechanistically, RP215-CIgG executes its oncogenic function through interacting with the integrin α6β4 complex and activating the FAK and Src pathways. Notably, the CIgG-integrin-FAK signaling depends on the N-glycan epitope, which is inhibited by RP215. Together, our studies identified a novel CIgG molecule that activates the oncogenic integrin-FAK signaling in LSCC cells. In addition, the activity of CIgG is inhibited by RP215, providing an attractive target for antibody-based therapy of LSCC.

Blood vitronectin is a major activator of LIF and IL-6 in the brain through integrin-FAK and uPAR signaling.

We defined how blood-derived vitronectin (VTN) rapidly and potently activates leukemia inhibitory factor (LIF) and pro-inflammatory interleukin 6 (IL-6) in vitro and after vascular injury in the brain. Treatment with VTN (but not fibrinogen, fibronectin, laminin-111 or collagen-I) substantially increased LIF and IL-6 within 4 h in C6-astroglioma cells, while VTN-/- mouse plasma was less effective than that from wild-type mice. LIF and IL-6 were induced by intracerebral injection of recombinant human (rh)VTN in mice, but induction seen upon intracerebral hemorrhage was less in VTN-/- mice than in wild-type littermates. In vitro, VTN effects were inhibited by RGD, αvβ3 and αvβ5 integrin-blocking peptides and antibodies. VTN activated focal adhesion kinase (FAK; also known as PTK2), whereas pharmacological- or siRNA-mediated inhibition of FAK, but not PYK2, reduced the expression of LIF and IL-6 in C6 and endothelial cells and after traumatic cell injury. Dominant-negative FAK (Y397F) reduced the amount of injury-induced LIF and IL-6. Pharmacological inhibition or knockdown of uPAR (also known as PLAUR), which binds VTN, also reduced cytokine expression, possibly through a common target of uPAR and integrins. We propose that VTN leakage into tissues promotes inflammation. Integrin-FAK signaling is therefore a novel IL-6 and LIF regulation mechanism relevant to the inflammation and stem cell fields.

Abstract 5877: The functional role(s) of serum amyloid A1 (SAA1) polymorphisms in integrin-mediated cell adhesion in nasopharyngeal carcinoma (NPC) cells

Abstract Serum amyloid A1 (SAA1) was previously identified as a tumor suppressor gene with anti-angiogenic activities in nasopharyngeal carcinoma (NPC). Three SAA1 isoforms (SAA1.1, 1.3, and 1.5) were observed with disproportionate frequencies among the NPC patients and healthy people. SAA1.1 and 1.3 are the functional isoforms to inhibit angiogenesis whereas SAA1.5 was the defective gene. Our immunohistochemical results showed that the loss of SAA1 staining in the metastatic NPC tissues was significantly associated with tumor progression. We aim to investigate the functional roles of the three SAA1 isoforms in NPC metastasis in the present study. We want to investigate whether the functional SAA1.1 and 1.3 isoforms can suppress tumor metastasis by antagonizing the integrin-FAK signaling pathway in the NPC tumor cells. The focal adhesion assay was performed by seeding the tumor cells with or without the integrin alphaVbeta3/beta5 ligand vitronectin. Both the vector-alone control and the SAA1.5-expressing NPC cells began to spread out and adhered to the bottom of the culture dish in the presence of vitronectin from 7 to 9 hours, whereas the SAA1.1 and SAA1.3-cells remained in the round-up morphology with minimal attachment. Interestingly, after 48 hours the SAA1.1 and SAA1.3-expressing cells formed adherents junction among the cells. In order to study the viability of the effects of the three SAA1 isoforms on NPC cells, MTT viability assay was performed. The results showed that the viability of the SAA1.1 and SAA1.3-expressing cells were around 50 % lower than both the vector-alone and the SAA1.5-expressing cells. It is likely that the loss of focal adhesion after seeding of the SAA1.1 and SAA1.3-expressing cells will affect the survival of NPC cells. Furthermore, we found that the presence of the recombinant SAA1.1 and SAA 1.3 proteins could reduce the number of viable NPC cells compared with the solvent control and the SAA1.5 protein. We previously reported that the SAA1 proteins can physically interact with the integrin alphaVbeta3. Taken together, we suggested that the secreted SAA1 proteins from the NPC cells could directly affect the NPC focal adhesion as well as the cell viability by blocking the integrin on the NPC cell surface. We acknowledge the financial support of the General Research Fund (grant number HKBU17115114 to HLL) of the Research Grants Council of the Hong Kong Special Administrative Region. Citation Format: On Ying Man, Hong Lok Lung. The functional role(s) of serum amyloid A1 (SAA1) polymorphisms in integrin-mediated cell adhesion in nasopharyngeal carcinoma (NPC) cells [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 5877. doi:10.1158/1538-7445.AM2017-5877

Metformin inhibits SUV39H1-mediated migration of prostate cancer cells

Prostate cancer (PCa) is a leading cause of cancer-related death among men, largely due to incurable distant metastases. Metformin, the most common used anti-type-2 diabetes medicine, has been linked to reduced cancer risk and better diagnosis. We found that metformin was able to inhibit PCa cell migration, which correlates with tumor metastatic capability. The pathogenesis and progression of tumors are closely related to dysregulated gene expression in tumor cells through epigenetic alterations such as DNA methylation and histone modifications. We found that the level of SUV39H1, a histone methyltransferase of H3 Lys9, was reduced in metformin-treated PCa cells in a time-dependent manner. SUV39H1 overexpression increased PCa migration, whereas SUV39H1 depletion suppressed PCa cell migration. There is a positive correlation between SUV39H1 expression and PCa pathological stages. We further showed that both metformin treatment and SUV39H1 knockout in PCa cells can reduce integrin αV and β1 proteins, as well as their downstream phosphorylated focal adhesion kinase (FAK) levels, which is essential for functional adhesion signaling and tumor cell migration. Taken together, metformin reduced SUV39H1 to inhibit migration of PCa cells via disturbing the integrin-FAK signaling. Our study suggests SUV39H1 as a novel target to inhibit PCa cell migration.