FAK Activation Research Articles

White spot syndrome virus IE1 protein blocks the integrin-FAK signaling to enhance viral infection in shrimp

DNA viruses commonly utilize immediate-early proteins to manipulate cellular signaling pathways in order to facilitate their infection. Our previous research has suggested that IE1, an immediate-early protein encoded by the white spot syndrome virus (WSSV), may modulate the shrimp integrin-FAK signaling pathway. However, the specific molecular mechanism and role of IE1 in regulating this signaling pathway remain unclear. In this study, we demonstrated that IE1 competes for binding to the cytoplasmic tail of Penaeus vannamei integrin-α5, resulting in the inhibition of the integrin-α5-FAK interaction, thereby suppressing FAK activation and cell adhesion. Furthermore, we observed a significant increase in the expression of P. vannamei integrin-α5 and FAK following WSSV infection. Additionally, knockdown of integrin-α5 or FAK through RNA interference has been shown to reduce cell adhesion and enhance WSSV infection. In conclusion, our findings reveal that IE1 disrupts integrin-FAK signaling to inhibit cell adhesion, ultimately promoting WSSV infection in shrimp.

Inducible FAK Deletion but not FAK Inhibition in Endothelial Cells Activates p53 to Suppress Tumor Growth in PYK2-null Mice.

PYK2-null combined with endothelial cell-specific FAK transgenic mouse models show that loss of FAK activity limits tumor spread and that genetic or chemical degradation preventing combined FAK-PYK2 expression may be an approach to induce a p53-associated anti-tumor response.

MiR-126 accelerates renal injury induced by UUO via inhibition PI3K/ IRS-1/ FAK signaling induced M2 polarization and endocytosis in macrophages

To investigate the role and molecular mechanism of miR-126 in unilateral ureteral occlusion (UUO). We used bioinformatics to analyse miRNAs specifically expressed in UUO. The mouse model of UUO was established using RAW264.7 cells cultured in vitro and in vivo. The mice were divided into control group, miR-126-NC (negative control) group and miR-126-KD (knockdown) group. Then the relative expression of miR-126 was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the renal fibrosis was detected by Masson staining, and the protein expression of CD68, collagen I and collagen III in the kidney was detected by immunofluorescence assay. Immunohistochemistry detects α-SMA expression. Moreover, Western blotting was performed to measure the expressions of p-PI3K, CD163, CD206, CD86, iNOS, IL-1β, p-FAK, p-Rac-1, p-IRS-1 and MMP9. The relative fluorescence intensity of F-actin and p-FAK was detected by immunofluorescence assay, and the phagocytosis ability of macrophages was determined by phagocytosis assay with fluorescent microspheres. Bioinformatics analysis reveals miR-126-specific overexpression in UUO. Successful transfection of miR-126-NC and miR-126-KD was confirmed by RT-PCR. The selective reduction of miR-126 was validated by Masson, immunohistochemistry and immunofluorescence staining to decrease the area of UUO-induced renal fibrosis and to lower the expression of CD68, α-SMA, collagen I, and collagen III. The reduction of iNOS expression may also be achieved with selective knockdown of miR-126, as verified by cell tests. enhances the phagocytic ability of macrophages and the expression of p-PI3K, CD206, p-FAK, F-actin, p-Rac-1, p-IRS-1 and MMP9. MiR-126 can inhibit the PI3K signaling pathway, promote M1 macrophage polarization, and suppress the activation of FAK and Rac-1, thus accelerating the progression of UUO.

The expression level of VEGFR2 regulates mechanotransduction, tumor growth and metastasis of high grade serous ovarian cancer cells

Recent data shows that alterations in the expression and/or activation of the vascular endothelial growth factor receptor 2 (VEGFR2) in high grade serous ovarian cancer (HGSOC) modulate tumor progression. However, controversial results have been obtained, showing that in some cases VEGFR2 inhibition can promote tumorigenesis and metastasis. Thus, it is urgent to better define the role of the VEGF/VEGFR2 system to understand/predict the effects of its inhibitors administered as anti-angiogenic in HGSOC. Here, we modulated the expression levels of VEGFR2 and analyzed the effects in two cellular models of HGSOC. VEGFR2 silencing (or its pharmacological inhibition) promote the growth and invasive potential of OVCAR3 cells in vitro and in vivo. Consistent with this, the low levels of VEGFR2 in OV7 cells are associated with more pronounced proliferative and motile phenotypes when compared to OVCAR3 cells, and VEGFR2 overexpression in OV7 cells inhibits cell growth. In vitro data confirmed that VEGFR2 silencing in OVCAR3 cells favors the acquisition of an invasive phenotype by loosening cell-ECM contacts, reducing the size and the signaling of focal adhesion contacts (FAs). This is translated into a reduced FAK activity at FAs, ECM-dependent alterations of mechanical forces through FAs and YAP nuclear translocation. Together, the data show that low expression, silencing or inhibition of VEGFR2 in HGSOC cells alter mechanotransduction and lead to the acquisition of a pro-proliferative/invasive phenotype which explains the need for a more cautious use of anti-VEGFR2 drugs in ovarian cancer.

EP.07F.01 Extracellular Matrix in Early Lung Tumors Drive FAK Activation and Tumor Progression

EP.07F.01 Extracellular Matrix in Early Lung Tumors Drive FAK Activation and Tumor Progression

Chemical Composition, Antioxidant Capacity, and Anticancerous Effects against Human Lung Cancer Cells of a Terpenoid-Rich Fraction of Inula viscosa.

Inula viscosa is a widely used plant in traditional Mediterranean and Middle Eastern medicine for various illnesses. I. viscosa has been shown to have anticancer effects against various cancers, but its effects against lung cancer have been under limited investigation. At the same time, I. viscosa is rich in terpenoids whose anti-lung cancer effects have been poorly investigated. This study aimed to examine the potential anticancer properties of methanolic and aqueous extracts of stems and leaves of I. viscosa and its terpenoid-rich fraction against human lung cancer A549 cells. Results showed that the methanolic extracts of I. viscosa had significantly higher polyphenol and flavonoid content and radical scavenging capacity than the aqueous extracts. In addition, leaves methanolic extracts (IVLM) caused the highest reduction in viability of A549 cells among all the extracts. IVLM also reduced the viability of human ovarian SK-OV-3, breast MCF-7, liver HepG2, and colorectal HCT116 cancer cells. A terpenoid-rich I. viscosa fraction (IVL DCM), prepared by liquid-liquid separation of IVLM in dichloromethane (DCM), displayed a substantial reduction in the viability of A549 cells (IC50 = 27.8 ± 1.5 µg/mL at 48 h) and the panel of tested cancerous cell lines but was not cytotoxic to normal human embryonic fibroblasts (HDFn). The assessment of IVL DCM phytochemical constituents using GC-MS analysis revealed 21 metabolites, highlighting an enrichment in terpenoids, such as lupeol and its derivatives, caryophyllene oxide, betulin, and isopulegol, known to exhibit proapoptotic and antimetastatic functions. IVL DCM also showed robust antioxidant capacity and decent polyphenol and flavonoid contents. Furthermore, Western blotting analysis indicated that IVL DCM reduced proliferation (reduction of proliferation marker Ki67 and induction of proliferation inhibitor proteins P21 and P27), contaminant with P38 MAP kinase activation, and induced the intrinsic apoptotic pathway (P53/BCL2/BAX/Caspase3/PARP) in A549 cells. IVL DCM also reduced the migration of A549 cells, potentially by reducing FAK activation. Future identification of anticancer metabolites of IVL DCM, especially terpenoids, is recommended. These data place I. viscosa as a new resource of herbal anticancer agents.

Integrin-β1 aggravates paraquat-induced pulmonary fibrosis by activation of FAK/ ERK1/2 pathway depending on fibrotic ECM

BackgroundIrreversible pulmonary fibrosis induced by paraquat is the most prevalent cause of death in patients with paraquat poisoning. Pulmonary fibrosis is characterized by abnormal deposition of extracellular matrix (ECM). Currently, the role of fibrotic ECM microenvironment in paraquat-induced pulmonary fibrosis has not been established. MethodsRat pulmonary fibrosis model was induced by paraquat, ATN-161 (an integrin-β1 antagonist) was given to investigate their effect on Rat survival and pulmonary fibrosis. Lungs were decellularized to generate normal and fibrotic acellular ECM scaffolds using Triton and SDS. Fibroblasts were cocultured with ECM scaffolds to established 3D culture systems to investigate the relationship between fibrotic ECM and the differentiation of fibroblasts. Then we explored the effect of fibrotic ECM microenvironment systematically promoting on integrin-β1/FAK/ERK1/2 pathway and established 3D culture systems to investigate the relationship between fibrotic ECM and the differentiation of fibroblasts. ResultsAntagonism of integrin-β1 could alleviate paraquat-induced pulmonary fibrosis and ameliorate survival status of rats. Compared to normal ECM, fibrotic extracellular microenvironment promoted the differentiation of fibroblasts to myofibroblasts. Antagonism of integrin-β1 could also ameliorate the promotion of fibrotic extracellular microenvironment on differentiation of fibroblasts to myofibroblasts. Fibrotic ECM microenvironment promotes fibroblasts transforming into myofibroblasts through integrin-β1/FAK/ERK1/2 signaling pathway. Moreover, this phenomenon holds independent on exogenous integrin-β1. ConclusionsActivation of integrin-β1/FAK/ERK1/2 pathway aggravates paraquat-induced pulmonary fibrosis depend on fibrotic ECM and integrin-β1 may be a prospective therapeutic target for paraquat-induced pulmonary fibrosis in the future.

Focal adhesion kinase signaling - tumor vulnerabilities and clinical opportunities.

Focal adhesion kinase (FAK; encoded by PTK2) was discovered over 30 years ago as a cytoplasmic protein tyrosine kinase that is localized to cell adhesion sites, where it is activated by integrin receptor binding to extracellular matrix proteins. FAK is ubiquitously expressed and functions as a signaling scaffold for a variety of proteins at adhesions and in the cell cytoplasm, and with transcription factors in the nucleus. FAK expression and intrinsic activity are essential for mouse development, with molecular connections to cell motility, cell survival and gene expression. Notably, elevated FAK tyrosine phosphorylation is common in tumors, including pancreatic and ovarian cancers, where it is associated with decreased survival. Small molecule and orally available FAK inhibitors show on-target inhibition in tumor and stromal cells with effects on chemotherapy resistance, stromal fibrosis and tumor microenvironment immune function. Herein, we discuss recent insights regarding mechanisms of FAK activation and signaling, its roles as a cytoplasmic and nuclear scaffold, and the tumor-intrinsic and -extrinsic effects of FAK inhibitors. We also discuss results from ongoing and advanced clinical trials targeting FAK in low- and high-grade serous ovarian cancers, where FAK acts as a master regulator of drug resistance. Although FAK is not known to be mutationally activated, preventing FAK activity has revealed multiple tumor vulnerabilities that support expanding clinical combinatorial targeting possibilities.

Phytoconstituent-derived zingerone nanoparticles disrupt the cell adhesion mechanism and suppress cell motility in melanoma B16F10 cells

Combining phytochemicals and nanotechnology to improve the unfavorable innate properties of phytochemicals and develop them into potent nanomedicines to enhance antitumor efficacy has become a novel strategy for cancer chemoprevention. Melanoma is the most aggressive, metastatic, and deadly disease of the primary cutaneous neoplasms. In this study, we fabricated phytoconstituent-derived zingerone nanoparticles (NPs) and validated their effects on cell adhesion and motility in melanoma B16F10 cells. Our data indicated that zingerone NPs significantly induced cytotoxicity and anti-colony formation and inhibited cell migration and invasion. Moreover, zingerone NPs dramatically interfered with the cytoskeletal reorganization and markedly delayed the period of cell adhesion. Our results also revealed that zingerone NPs-mediated downregulation of MMPs (matrix metalloproteinases) activity is associated with inhibiting cell adhesion and motility. We further evaluated the effects of zingerone NPs on Src/FAK /Paxillin signaling, our data showed that zingerone NPs significantly inhibited the protein activities of Src, FAK, and Paxillin, indicating that they play important roles in zingerone NP-mediated anti-motility and anti-invasion in melanoma cells. Accordingly, the phytoconstituent-zingerone NPs can strengthen the inhibition of tumor growth, invasion, and metastasis in malignant melanoma. Altogether, these multi-pharmacological benefits of zingerone NPs will effectively achieve the purpose of melanoma prevention and invasion inhibition.

Updated study results of novel FAK/ALK/ROS1 inhibitor APG-2449 in patients (pts) with non-small-cell lung cancer (NSCLC) resistant to second-generation ALK inhibitors.

3124 Background: ALK inhibitors increase FAK pathway gene expression in ALK+ NSCLC cell lines, with the highest induced expression in drug-tolerant persister cells. This suggests that FAK pathway activation is involved in ALK inhibitor-induced early adaptive tolerance in ALK+ NSCLC. Investigational APG-2449 is a novel, orally active FAK inhibitor and a third-generation (3G) ALK/ROS1 tyrosine kinase inhibitor (TKI) with potent activity in preclinical models. APG-2449 is well tolerated at the recommended phase 2 dose (RP2D) and showed preliminary efficacy in pts resistant to 2G ALK TKIs. Here, we provide further safety and efficacy results and demonstrate associations between this therapy and FAK signaling pathways. Methods: After the RP2D was determined as 1,200 mg daily (QD), pts with NSCLC were enrolled into 2 dose expansion cohorts. Cohort 1 included pts who were resistant to 2G ALK TKIs. Cohort 2 included those who were ALK or ROS1 TKI naïve. Results: As of December 9, 2023, 144 pts (median [range] age, 53 [21-78] years; 53.5% female) with NSCLC, mesothelioma, or ovarian cancer were treated with APG-2449 at doses of 150 to 1,500 mg. A total of 129 (89.6%) pts had treatment-related adverse events (TRAEs), the most frequent of which were elevated serum creatinine (49.3%), ALT (42.4%), and AST (36.1%); nausea (28.5%); vomiting (23.6%); diarrhea (22.9%); and decreased leukocyte count (22.2%). In all, 20 (13.9%) TRAEs were grade ≥ 3. Of pts with TKI-naïve NSCLC (n = 36) treated at the RP2D, the overall response (ORR) and disease control rates (DCR = CR + PR + SD) were 68.2% (15/22) and 90.1% (20/22) in ROS1 TKI-naïve pts; and 78.6% (11/14) and 100% (14/14) in ALK TKI-naïve pts. Median progression-free survival (mPFS) in ALK TKI-naïve pts was not reached. Of 22 pts with NSCLC resistant to 2G ALK inhibitors and without targetable bypass gene mutations (e.g., KRAS G12C, BRAF V600E), 9 had PRs (9/22; 40.9%) and the mPFS during APG-2449 treatment at RP2D was 11.86 months. Nine of 12 pts in the RP2D group achieved intracranial PR, resulting in an intracranial ORR of 75.0%. Before APG-2449 administration, 17 tumor tissue samples were collected from pts with NSCLC resistant to 2G ALK inhibitors (in the RP2D group), and IHC was used to assess protein levels of phosphorylated FAK (pFAK). The PFS of pts treated with APG-2449 correlated with pFAK levels in baseline tumor tissue, and pts with higher pFAK levels were more likely to benefit from APG-2449 treatment. Conclusions: APG-2449 demonstrated preliminary efficacy in pts with NSCLC whose disease was TKI naïve and resistant to 2G ALK inhibitors, especially in brain metastases. High pFAK expression levels in baseline tumor tissue correlated with improved APG-2449 treatment responses in pts with NSCLC resistant to 2G ALK inhibitors. Internal study identifier: APG2449XC101. Clinical trial information: NCT03917043 .

Platelet Spreading and Clot Retraction are Regulated by two Distinct αIIbβ3 Outside-in Signaling Pathways.

Bi-directional signaling through platelet integrin αIIbβ3 is essential in hemostasis and thrombosis. In quiescent platelets αIIbβ3 is in a low-affinity ligand binding state. However, upon platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the αIIbβ3 induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of PI3K, PLC, PKC, and FAK strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading. Whereas inhibition of Rac1, ROCK, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases (SFKs) are required for platelet spreading and FAK activation but suppresses clot retraction since their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through αIIbβ3 outside-in signaling and should not be used interchangeably as readout for αIIbβ3 outside-in signaling assessment. Significance Statement Current anti-platelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel anti-platelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of αIIbβ3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling is poorly understood. Our work included in this manuscript delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.

Effect of TiO2 Nanotubes on Biological Activity of Osteoblasts and Focal Adhesion Kinase/Osteopontin Level

Osteoblasts are important cells for bone formation and play a major part in bone diseases and bone defects. Clinically, we usually adopt bone implants for related diseases. Also, nanotechnology is important in bones and joints. This study assessed the effects of TiO2 nanotubes of different diameters on osteoblast activity, FAK and OPN levels, aiming to provide an experimental foundation for selection of clinical bone implant materials. The morphology of MG-63 human osteosarcoma cells changed with expansion of TiO2 nanotubes’ diameter. From the biological activity, the cell proliferation and adhesion were enhanced as the diameter of the TiO2 nanotube was increased and its proliferation and adhesion were highest in the 100 nm TiO2 nanotube, which is related to increased ALP activity, FAK and OPN protein and mRNA expression. ELISA detected ALP activity and found that MG-63 cells cultured with 70 nm nanotube had strongest activity. Immune blotting and PCR results showed that, FAK and OPN activities were highest in 70 nm TiO2 nanotube cells. In summary, TiO2 nanotubes increased cell proliferation and adhesion by up-regulating the activities of FAK and OPN in a concentration-dependent relationship.

Abstract 5717: CDK4 inhibition suppresses Rab11 mediated collective cell migration in colon cancer cells

Abstract In tumor progression, cell proliferation and cell adhesion are critical for tumor growth and metastasis. It has been demonstrated cell division which associated with microtubule organization also has impact on tubulin mediated focal adhesion turnover, however, the mechanism is not clear in the cell cohort migration which is accompanied with both cell proliferation and cell migration. This study is to investigate the effect of cell cycle inhibition in the collective cell migration. HT-29 colon cancer cells were treated with CDK4 inhibitor CINK4, paclitaxel and focal adhesion inhibitor PF573228. The cell proliferation rate was determined by MTT assay, cell migration ability was analyzed by wound healing assay. Rab11, E-cadherin and focal adhesion kinase were revealed by immuno-confocal microscopy. When HT-29 cells were treated with lower dose of CDK4 inhibitor CINK4, cell migration was suppressed. Meanwhile, in the result of immunostaining, the recycling endosomal protein Rab11which is involved in E-cadherin turnover in collective cell migration was not distributed in the cell membrane after CINK4 treatment. Furthermore, when cellular FAK activity were inhibited by PF573228 and tubulin disassembly was inhibited by paclitaxel, the cell area was increased with stable attachment, however, the membrane translocation of Rab11 was not affected. This study demonstrated cell cycle inhibition in G1 phase affected Rab11 mediated collective cell migration through tubulin regulated focal adhesion turnover for cell attachment. Keywords: CDK4, cell cycle, focal adhesion kinase, paclitaxel, colon cancer Citation Format: Chun Pu Cheng, Chia-En Ku, Shin-Ru Lee, Yu-Chun Wang, Yu-Chien Ching, Hui-Chun Chen, Wei-Ting Chao. CDK4 inhibition suppresses Rab11 mediated collective cell migration in colon cancer cells [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 5717.

Abstract 4384: Differential epitope tagging of oncogenic GNAQ mutants produces distinct effects on downstream signaling of GNAQ

Abstract GNAQ Q209P and Q209L are common driver mutations in uveal melanoma, leading to constitutive activation of downstream signaling of GNAQ. It is not fully understood whether the two GNAQ mutants have similar effects on downstream signaling pathways. Due to lack of available specific GNAQ antibody, epitope tagging is a well-established tool for protein interaction studies using mass spectrometry. Several studies used an epitope tag inserted into an internal region or the c-terminus of GNAQ to study its function. However, the functional consequences of these modalities on effector engagement are not fully elucidated. Here, we engineered both GNAQ wild-type and GNAQ mutants (Q209L and Q209P) without epitope tag as well as three different commonly used tagging modalities. A 3xflag tag of 22 amino acids was inserted after residue 122 of GNAQ. A MYC/DDK tag consisting of 18 amino acids was incorporated before the stop codon of GNAQ. An internal Glu-Glu epitope tag (EE tag) of 6 amino acids was introduced internally by mutating residues AYLPTQ(171-176) of GNAQ to EYMPTE. To assess the impact of the different modalities on the function of GNAQ mutants, we introduced them into 293FT cells and examined the known oncogenic signaling outputs downstream of GNAQ: MAPK, PKC and FAK signaling. We found that all three tags of both GNAQQ209L and GNAQQ209P activated MAPK signaling at similar levels as the corresponding untagged GNAQ mutants as evidenced by increases of pERK and pp90RSK level compared to GNAQ wild-type controls. By contrast, both GNAQQ209L and GNAQQ209P tagged with MYC/DDK induced lower p-PKD (a readout of PKC) and p-FAK (a readout of FAK activation) compared to EE-tagged or untagged, suggesting that MYC/DDK tag fused to the c-terminus of GNAQ affects specific effectors downstream of GNAQ. Furthermore, GNAQQ209P with the 3xflag tag induced lower levels of p-PKD and p-FAK than 3xflag tagged GNAQQ209L did, indicating that GNAQQ209P may have a different three-dimensional conformation compared to GNAQQ209L with different downstream effectors engagement. Moreover, the GNAQ/11 inhibitor YM-254890 inhibited phosphorylation of ERK, PKD, FAK in a concentration dependent manner in 293FT cells transfected with GNAQ mutants tagged with MYC/DDK but had no effect on MAPK, PKC and FAK signaling in cells expressing EE-tagged GNAQ mutants. These data suggest that EE tag has no effect on the function of GNAQ , but interferes with YM-254890 binding. Our findings highlight unappreciated consequences of epitope-tagging of the oncogene GNAQ that are relevant for studies on the interactome of mutant GNAQ and drug response. Citation Format: Chinchu Jagadan Ushakumari, Aashish Manglik, Boris C Bastian, Xu Chen. Differential epitope tagging of oncogenic GNAQ mutants produces distinct effects on downstream signaling of GNAQ [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 4384.

PCDHA9 as a candidate gene for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. To identify additional genetic factors, we analyzed exome sequences in a large cohort of Chinese ALS patients and found a homozygous variant (p.L700P) in PCDHA9 in three unrelated patients. We generated Pcdhα9 mutant mice harboring either orthologous point mutation or deletion mutation. These mice develop progressive spinal motor loss, muscle atrophy, and structural/functional abnormalities of the neuromuscular junction, leading to paralysis and early lethality. TDP-43 pathology is detected in the spinal motor neurons of aged mutant mice. Mechanistically, we demonstrate that Pcdha9 mutation causes aberrant activation of FAK and PYK2 in aging spinal cord, and dramatically reduced NKA-α1 expression in motor neurons. Our single nucleus multi-omics analysis reveals disturbed signaling involved in cell adhesion, ion transport, synapse organization, and neuronal survival in aged mutant mice. Together, our results present PCDHA9 as a potential ALS gene and provide insights into its pathogenesis.

ZDHHC5-mediated S-palmitoylation of FAK promotes its membrane localization and epithelial-mesenchymal transition in glioma

BackgroundAbnormal activation of FAK is associated with tumor development and metastasis. Through interactions with other intracellular signalling molecules, FAK influences cytoskeletal remodelling, modulation of adhesion signalling, and activation of transcription factors, promoting migration and invasion of tumor cells. However, the exact mechanism that regulates these processes remains unresolved. Herein, our findings indicate that the S-palmitoylation of FAK is crucial for both its membrane localization and activation.MethodsThe palmitoylation of FAK in U251 and T98G cells was assessed by an acyl-PEG exchange (APE) assay and a metabolic incorporation assay. Cellular palmitoylation was inhibited using 2-bromopalmitate, and the palmitoylation status and cellular localization of FAK were determined. A metabolic incorporation assay was used to identify the potential palmitoyl acyltransferase and the palmitoylation site of FAK. Cell Counting Kit-8 (CCK8) assays, colony formation assays, and Transwell assays were conducted to assess the impact of ZDHHC5 in GBM. Additionally, intracranial GBM xenografts were utilized to investigate the effects of genetically silencing ZDHHC5 on tumor growth.ResultsInhibiting FAK palmitoylation leads to its redistribution from the membrane to the cytoplasm and a decrease in its phosphorylation. Moreover, ZDHHC5, a protein-acyl-transferase (PAT), catalyzes this key modification of FAK at C456. Knockdown of ZDHHC5 abrogates the S-palmitoylation and membrane distribution of FAK and impairs cell proliferation, invasion, and epithelial-mesenchymal transition (EMT). Taken together, our research reveals the crucial role of ZDHHC5 as a PAT responsible for FAK S-palmitoylation, membrane localization, and activation.ConclusionsThese results imply that targeting the ZDHHC5/FAK axis has the potential to be a promising strategy for therapeutic interventions for glioblastoma (GBM).DK35umxSfc6RJzPPrNqiMTVideo

In Vitro Evaluation of Wound Healing, Stemness Potentiation, Antioxidant Activity, and Phytochemical Profile of Cucurbita moschata Duchesne Fruit Pulp Ethanolic Extract.

Wound healing comprises an intricate process to repair damaged tissue. Research on plant extracts with properties to expedite wound healing has been of interest, particularly their ability to enhance the stemness of keratinocyte stem cells. Hence, the present study aims to determine the wound healing and stemness potentiation properties of an ethanolic extract derived from Cucurbita moschata fruit pulp (PKE). Human keratinocytes (HaCaT) and primary skin fibroblast cells were used in this study. The migration of the cells was examined by using a scratch wound healing assay, and spheroid behavior was determined by using a spheroid formation assay. The proteins related to migration and stemness were further measured by using Western blotting to explore the mechanism of action of PKE. The methods used to evaluate PKE's antioxidant properties were 2,2-diphenyl-2-picrylhydrazyl (DPPH) scavenging, ABTS radical scavenging activity, and superoxide anion radical scavenging (SOSA) assays. The phytochemistry of the PKE was investigated using phytochemical screening and high-performance liquid chromatography (HPLC) analysis. The results of this study indicate that nontoxic concentrations of PKE increase the rate of migration and spheroid formation. Mechanistically, PKE increased the expression of the migratory-related protein active FAK (phosphorylated FAK), and the subsequence increased the level of p-AKT. The expression of stem cell marker CD133, upstream protein signaling β-catenin, and self-renewal transcription factor Nanog was increased. The PKE also possessed scavenging properties against DPPH, ABTS, and SOSA. The phytochemistry analyses exhibited the presence of alkaloids, glycosides, xanthones, triterpenes, and steroids. Additionally, bioactive compounds such as ɑ-tocopherol, riboflavin, protocatechuic acid, β-carotene, and luteolin were detected. The presence of these chemicals in PKE may contribute to its antioxidant, stem cell potentiation, and wound-healing effects. The findings could be beneficial in the identification of valuable natural resources that possess the capacity to be used in the process of wound healing through the potentiation of stemness via a readily detectable molecular mechanism.

Loss of MTA2-mediated downregulation of PTK7 inhibits hepatocellular carcinoma metastasis progression by modulating the FAK-MMP7 axis.

The expression of metastasis tumor-associated protein 2 (MTA2) and protein tyrosine kinase 7 (PTK7) is associated with hepatocellular carcinoma (HCC) progression. However, the functional effect and mechanism through which MTA2 regulates PTK7-mediated HCC progression remains unclear. Here, we found that MTA2 knockdown significantly down-regulated PTK7 expression in HCC cells (SK-Hep-1 and PLC/PRF/5). Data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases show that the PTK7 expression level was higher in HCC tissues than in normal liver tissues. In HCC patients, the PTK7 expression level clearly correlated with tumor stage and grade, lower overall survival (OS) correlated positively with MTA2 level, and PTK7 expression acted as a downstream factor for MTA2 expression. In addition, matrix metalloproteinase 7 (MMP7) expression was closely regulated by PTK7, and the mRNA and protein expression levels of MTA2 and PTK7 correlated positively with lower OS. MMP7 downregulation by PTK7 knockdown clearly decreased the migration and invasion abilities of HCC cells. In HCC cells, recombinant human MMP7 reversed the PTK7 knockdown-induced suppression of migration and invasion. Furthermore, deactivation of FAK using siFAK or FAK inhibitor (PF-573228, PF) synergistically contributed to PTK7 knockdown-inhibited FAK activity, MMP7 expression, and the migration and invasion abilities of HCC cells. Collectively, our findings show that PTK7 mediates HCC progression by regulating the MTA2-FAK-MMP7 axis and may be a diagnostic value for HCC patients.

Accelerated osteogenesis of bone graft by optimizing the bone microenvironment formed by electrical signals dependent on driving micro vibration stimulation

The strategy of coupling the micro-vibration mechanical field with Ca/P ceramics to optimize the osteogenic microenvironment and enhance the functional activity of the cells can significantly improve the bone regeneration of the graft. However, the regulation mode and mechanism of this coupling strategy are not fully understood at present. This study investigated the influence of different waveforms of the electrical signals driving Microvibration Stimulation (MVS) on this coupling effect. The results showed that there were notable variances in calcium phosphate dissolution and redeposition, protein adsorption, phosphorylation of ERK1/2 and FAK signal pathways and activation of calcium channels such as TRPV1/Piezo1/Piezo2 in osteogenic microenvironment under the coupling action of hydroxyapatite (HA) ceramics and MVS driven by different electrical signal waveforms. Ultimately, these differences affected the osteogenic differentiation process of cells by a way of time-sequential regulation. Square wave-MVS coupled with HA ceramic can significantly delay the high expression time of characteristic genes (such as Runx2, Col-I and OCN) in MC3T3-E1 cells during in vitro the early, middle and late stage of differentiation, while maintain the high proliferative activity of MC3T3-E1 cells. Triangle wave signal-MVS coupled with HA ceramic promoted the osteogenic differentiation of cells in the early and late stages. Sine wave-MVS shows the effect on the process of osteogenic differentiation in the middle stage (such as the up-regulation of ALP synthesis and Col-I gene expression in the early stage of stimulation). In addition, Square wave-MVS showed the best coupling effect. The bone graft constructed under square wave-MVS formed new bone tissue and mature blood vessels only 2 weeks after subcutaneous implantation in nude mice. Our study provides a new non-invasive regulation model for precisely optimizing the osteogenic microenvironment, which can accelerate bone regeneration in bone grafts more safely, accurately and reliably.

Abstract C125: Development of attenuated orf virus as a new generation oncolytic viral vector

Abstract The orf virus (ORFV) possesses remarkable qualities that make it a potential viral vector for cancer therapy. Recently, the cancer-killing properties of ORFV have been demonstrated in a variety of human cancers, including nasopharyngeal carcinoma (NPC). Initially, we found that the ORFV-bearing wild-type genome was successful in eliminating two NPC cell lines derived from patients in Hong Kong and Taiwan, HK-1 and TW02, respectively. To make viral vectors safer for medical use, the oncolytic effects of two live attenuated ORFVs, which were created by removing one of the virulent factors, VEGF or CBP, were further evaluated in these two cell lines. Despite this attenuation, the two recombinant ORFVs were still able to effectively infect and kill NPC cells, particularly HK-1 cells. ORFV infection caused pyroptosis in NPC cells through GSDME cleavage, resulting in significant cell detachment and reduction in FAK and AKT activation during the early stages of infection. The innate immune response is also affected by ORFV infection, as shown by the increased chemotaxis of NK cells and synergistic effect of NK on the cytotoxicity of NPC cells caused by ORFV infection. Infection with both wild-type and CBP-deleted ORFVs significantly reduced HK-1 tumor growth in a xenograft mouse model, suggesting its potential as a therapeutic agent for NPC treatment. To maximize its therapeutic use, it is essential to comprehensively understand the mechanisms that drive the oncolytic activity of ORFV. Citation Format: Yumiko Yamada, Wei-Li Hsu, Yu-Chih Wang, Hao-Ping Liu. Development of attenuated orf virus as a new generation oncolytic viral vector [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C125.