Focal Adhesion Kinase Overexpression Research Articles

Abstract 4569: APG-2449, a novel focal adhesion kinase (FAK) inhibitor, inhibits metastasis and enhances the antitumor efficacy of PEGylated liposome doxorubicin (PLD) in epithelial ovarian cancer (EOC)

Abstract Ovarian cancer is the leading cause of cancer-related death in women and most cases are diagnosed at later stages, with distant metastasis. Standard-of-care therapy is surgery followed by platinum and paclitaxel; however, most patients who initially respond to therapy eventually relapse after developing metastatic disease and chemoresistance. FAK overexpression or activation occurs in a substantial proportion of EOCs and is predictive of poor clinical outcomes. Recent studies have reported that FAK plays an important role in cell migration and chemoresistance, rendering FAK inhibition a promising treatment approach to reduce metastasis of tumor cells and sensitize them to chemotherapy. The aim of this study was to evaluate the antitumor efficacy of investigational APG-2449, a novel FAK inhibitor, combined with PLD, a commonly used chemotherapy, in relapsed or refractory ovarian cancer. Four EOC cell lines (OVCAR-3, SK-OV-3, A2780, and Kuramochi) were used for in vitro experiments. Cell proliferation was measured by CellTiter-Glo assays. Cell migration ability was assessed by wound healing assays. OVCAR-3 xenograft and luciferase-tagged ID8 (ID8-Luc) peritoneal syngeneic models were established to evaluate the synergistic antitumor efficacy of combined APG-2449 and PLD in vivo. APG-2449 combined with doxorubicin showed synergistic antiproliferative effects in both platinum-resistant (OVCAR-3 and SK-OV-3) and platinum-sensitive (A2780 and Kuramochi) cell lines. In wound healing assays, FAK inhibition via APG-2449 alone attenuated migration of ovarian cancer cells (SK-OV-3, OVCAR-3, and ID8-Luc) in a dose-dependent manner. In the OVCAR-3 xenograft model, combined APG-2449 and PLD demonstrated enhanced antitumor effects compared with either APG-2449 or PLD monotherapy (synergistic index, 1.33). In the ID8-Luc peritoneal syngeneic model, the combination regimen outperformed PLD or APG-2449 monotherapy in tumor growth inhibition. This inhibitory effect persisted more than 1 month after stopping combination treatment, whereas the monotherapy groups experienced rapid disease relapse. The combination group also exhibited the longest ascites-free and survival times compared with PLD or APG-2449 monotherapy groups. Overall, our data suggest that inhibition of FAK by APG-2449 enhances the antitumor effect of PLD in EOC cell lines and the OVCAR-3 xenograft mouse model. The combination regimen can also prolong ascites-free and survival times, as shown in the ID8-Luc peritoneal syngeneic model. These promising results support future clinical development of this combination treatment for ovarian cancer. Citation Format: Zhou Yu, Eric Liang, Daojie Liu, Bingxing Wu, Xinyi Yao, Yanqiu Ji, Dajun Yang, Yifan Zhai. APG-2449, a novel focal adhesion kinase (FAK) inhibitor, inhibits metastasis and enhances the antitumor efficacy of PEGylated liposome doxorubicin (PLD) in epithelial ovarian cancer (EOC) [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 4569.

Arctigenin hinders the invasion and metastasis of cervical cancer cells via the FAK/paxillin pathway

ContextCervical cancer is the most common gynecological pernicious tumor with high morbidity and mortality worldwide, especially in developing countries. Arctigenin (ARG), a nature-derived component, has exhibited anti-tumor activity in various tumors. ObjectiveTo explore the effect of ARG on cervical cancer. Materials and methodsThe effect and mechanism of ARG on cervical cancer cells were explored by cell counting kit-8 (CCK-8), flow cytometry, transwell and Western blot assays. Additionally, in vivo experiment was conducted in xenografted mice by immunohistochemistry (IHC), terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) and Western blot assays. ResultsARG treatment induced both concentration-dependent and time-dependent reductions in the cell viability of SiHa and HeLa cells with a IC50 value of 9.34 μM and 14.45 μM, respectively. ARG increased the apoptosis rate and the protein levels of cleaved-caspase 3 and E-cadherin, but decreased the invaded cell numbers and the protein levels of Vimentin and N-cadherin in vitro. Mechanically, ARG inhibited the expression of focal adhesion kinase (FAK)/paxillin pathway, which was confirmed by the overexpression of FAK in SiHa cells. The inhibitory role of overexpression of FAK in proliferation and invasion, as well as its promoted role in apoptosis were reversed with ARG treatment. Meanwhile, ARG suppressed growth and metastasis, and enhanced apoptosis in vivo. Consistently, ARG administration reduced the relative protein level of p-FAK/FAK and p-paxillin/paxillin in tumor tissues of xenografted mice. ConclusionARG inhibited proliferation, invasion and metastasis, but enhanced apoptosis of cervical cancer via the FAK/paxillin axis.

Peptide deregulated in hypertrophic scar-1 alleviates hypertrophic scar fibrosis by targeting focal adhesion kinase and pyruvate kinase M2 and remodeling the metabolic landscape

Hypertrophic scarring is a fibrotic skin disease characterized by excessive deposition of collagens. Emerging evidence has suggested important roles for peptides in fibrosis-related diseases. Here, we demonstrate that a skin-derived endogenous peptide, peptide deregulated in hypertrophic scar-1 (PDHS1), with the sequence IATTTASAATAAAIGATPRAK, inhibits cell proliferation, promotes apoptosis, decreases the proportion of cells in S phase, and decreases collagen synthesis in hypertrophic scar fibroblasts. Additionally, treatment with PDHPS1 alleviates hypertrophic scarring in a rabbit ear model. PDHPS1 was found to bind to focal adhesion kinase (FAK) and to decrease its activity. PDHPS1 was also shown to bind to pyruvate kinase M2 (PKM2) and to decreased its expression. Smad2 phosphorylation is also inhibited by treatment with PDHPS1. Overexpression of FAK rescues the decreased expression of COL3A1 induced by PDHPS1 treatment. Targeted metabolomics revealed that PDHPS1 reprogramed metabolism that related to amino acid synthesis, leading to decreases of the key glycolysis intermediates glucose-6-phosphate and fructose-6-phosphate. These results demonstrated that the endogenous peptide PDHPS1 alleviates hypertrophic scar fibrosis in vitro and in vivo by targeting FAK and PKM2 and remodeling the metabolic landscape. Overall, treatment with PDHPS1 is a potential therapeutic strategy for hypertrophic scarring.

Nintedanib alleviates pulmonary fibrosis in vitro and in vivo by inhibiting the FAK/ERK/S100A4 signalling pathway.

Nintedanib is an effective treatment for pulmonary fibrosis (PF), but the exact mechanism by which this agent works to delay the progression of PF remains unclear. In this study, we explored whether nintedanib alleviates PF at least partially by inhibiting the focal adhesion kinase (FAK)/ERK/S100A4 signalling pathway. Bleomycin (BLM) was used to induce PF in a mouse model, and human fetal lung fibroblast 1 (HFL-1) cells were exposed to transforming growth factor-β 1 (TGF-β1) to create an in vitro model of PF. In both models, nintedanib was administered either alone or in conjunction with a FAK vector. In mouse lung tissues, histopathology, inflammatory factor levels, and collagen content were assessed; in HFL-1 cells, HFL-1 activity was assessed, along with collagen I, collagen III, and α-SMA levels. Both mouse tissue and HFL-1 cells were examined for levels of indices associated with extracellular matrix and the FAK/ERK/S100A4 signalling pathway. In mice exposed to BLM, lung inflammation and extracellular matrix deposition were significantly increased. These factors were alleviated by nintedanib treatment but were aggravated by overexpression of FAK. In HFL-1 cells, nintedanib inhibited HFL-1 activity and collagen I, collagen III, and α-SMA levels, whereas overexpression of FAK produced the opposite effect. In both tissues and cells, the FAK/ERK/S100A4 signalling pathway was activated, but nintedanib was able to suppress this pathway. These results suggest that nintedanib alleviates PF by inhibiting the FAK/ERK/S100A4 signalling pathway both in vivo and in vitro.

FAK-Mediated Signaling Controls Amyloid Beta Overload, Learning and Memory Deficits in a Mouse Model of Alzheimer's Disease.

The non-receptor focal adhesion kinase (FAK) is highly expressed in the central nervous system during development, where it regulates neurite outgrowth and axon guidance, but its role in the adult healthy and diseased brain, specifically in Alzheimer’s disease (AD), is largely unknown. Using the 3xTg-AD mouse model, which carries three mutations associated with familial Alzheimer’s disease (APP KM670/671NL Swedish, PSEN1 M146V, MAPT P301L) and develops age-related progressive neuropathology including amyloid plaques and Tau tangles, we describe here, for the first time, the in vivo role of FAK in AD pathology. Our data demonstrate that while site-specific knockdown in the hippocampi of 3xTg-AD mice has no effect on learning and memory, hippocampal overexpression of the protein leads to a significant decrease in learning and memory capabilities, which is accompanied by a significant increase in amyloid β (Aβ) load. Furthermore, neuronal morphology is altered following hippocampal overexpression of FAK in these mice. High-throughput proteomics analysis of total and phosphorylated proteins in the hippocampi of FAK overexpressing mice indicates that FAK controls AD-like phenotypes by inhibiting cytoskeletal remodeling in neurons which results in morphological changes, by increasing Tau hyperphosphorylation, and by blocking astrocyte differentiation. FAK activates cell cycle re-entry and consequent cell death while downregulating insulin signaling, thereby increasing insulin resistance and leading to oxidative stress. Our data provide an overview of the signaling networks by which FAK regulates AD pathology and identify FAK as a novel therapeutic target for treating AD.

Phosphorylation of Focal Adhesion Kinase at Y925: Role in Glia-Dependent and Independent Migration through Regulating Cofilin and N-Cadherin.

The adult neocortex is a six-layered structure, consisting of nearly continuous layers of neurons that are generated in a temporally strictly coordinated order. During development, cortical neurons originating from the ventricular zone migrate toward the Reelin-containing marginal zone in an inside-out arrangement. Focal adhesion kinase (FAK), one tyrosine kinase localizing to focal adhesions, has been shown to be phosphorylated at tyrosine 925 (Y925) by Src, an important downstream molecule of Reelin signaling. Up to date, the precise molecular mechanisms of FAK and its phosphorylation at Y925 during neuronal migration are still unclear. Combining in utero electroporation with immunohistochemistry and live imaging, we examined the function of FAK in regulating neuronal migration. We show that phosphorylated FAK is colocalized with Reelinpositive Cajal-Retzius cells in the developing neocortex and hippocampus. Phosphorylation of FAK at Y925 is significantly reduced in reeler mice. Overexpression and dephosphorylation of FAK impair locomotion and translocation, resulting in migration inhibition and dislocation of both late-born and early-born neurons. These migration defects are highly correlated to the function of FAK in regulating cofilin phosphorylation and N-Cadherin expression, both are involved in Reelin signaling pathway. Thus, fine-tuned phosphorylation of focal adhesion kinase at Y925 is crucial for both glia-dependent and independent neuronal migration.

The molecular mechanism of baicalein repressing progression of gastric cancer mediating miR-7/FAK/AKT signaling pathway

BackgroundBaicalein (BAI) has a significant anti-cancerous function in the treatment of gastric cancer (GC). Focal adhesion kinase (FAK) is a key regulatory molecule in integrin and growth factor receptor mediated signaling. MicroRNA-7 (miR-7), has been considered as a potential tumor suppressor in a variety of cancers. However, the possible mechanisms by which BAI inhibiting progression of gastric cancer mediating miR-7/FAK/AKT signaling pathway remain unclear. PurposeTo investigate the molecular mechanism and effects of BAI inhibiting progression of gastric cancer mediating miR-7/FAK/AKT signaling pathway. MethodsGastric cancer cell lines with FAK knockdown and overexpression were constructed by lentivirus transfection. After BAI treatment, the effects of FAK protein on proliferation, metastasis and angiogenesis of gastric cancer cells were detected by MTT, EdU, colony formation, wound healing, transwell and Matrigel tube formation assays. In vivo experiment was performed by xenograft model. Immunofluorescence and western blot assay were used to detect the effects of FAK protein on the expression levels of EMT markers and PI3K/AKT signaling pathway related proteins. qRT-PCR and luciferase reporter assay were used to clarify the targeting relationship between miR-7 and FAK. ResultsBAI can regulate FAK to affect proliferation, metastasis and angiogenesis of gastric cancer cells through PI3K/AKT signaling pathway. qRT-PCR showed BAI can upregulated the expression of miR-7 and luciferase reporter assay showed the targeting relationship between miR-7 and FAK. Additionally, miR-7 mediates cell proliferation, metastasis and angiogenesis by directly targeting FAK 3′UTR to inhibit FAK expression. ConclusionBAI repressing progression of gastric cancer mediating miR-7/FAK/AKT signaling pathway.

Focal adhesion kinase splicing and protein activation in papillary thyroid carcinoma progression.

Papillary thyroid carcinoma (PTC), a common endocrine malignancy, presents a challenge from a prognostic standpoint. Molecular alterations underlying PTC progression include deregulation of focal adhesion kinase (FAK) at post-transcriptional and post-translational levels. Searching for candidate markers of PTC progression, we investigated the prognostic significance of FAK alterations on mRNA/protein level. The expression levels and subcellular localisation of auto-phosphorylated FAK (pY397-FAK) were determined by western blot (WB) and immunohistochemistry. The quantity of total FAK mRNA, alternatively spliced FAK-Del26 and FAK-Del33 variants were analysed by RT-qPCR and related to pY397-FAK expression and subcellular distribution. The results were correlated with clinicopathological parameters of the patients. The expression of pY397-FAK was significantly elevated in malignant samples. Active FAK showed predominant cytoplasmic distribution with co-occurrence along the membrane, while nuclear staining was found less frequently. Expression of pY397-FAK in separate cellular compartments correlated with adverse clinicopathological parameters, but the strongest association was found when their mean scores were calculated. Alternatively spliced FAK-Del33 and total FAK transcripts positively correlated to pY397-FAK protein levels as well as to characteristics of PTC advancement. Over-expression of FAK on mRNA (total and Del-33) and activated protein (pY397-FAK) levels is a feature of PTC advanced stages. Of the analysed alterations, the mean pY397-FAK IHC score showed the best predictive performance. Correlation between mRNA FAK-Del33 and pY397-FAK expression implies a regulatory role of alternative splicing in PTC patients.

Mycobacterium tuberculosis Exploits Focal Adhesion Kinase to Induce Necrotic Cell Death and Inhibit Reactive Oxygen Species Production.

Tuberculosis is a deadly, contagious respiratory disease that is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Mtb is adept at manipulating and evading host immunity by hijacking alveolar macrophages, the first line of defense against inhaled pathogens, by regulating the mode and timing of host cell death. It is established that Mtb infection actively blocks apoptosis and instead induces necrotic-like modes of cell death to promote disease progression. This survival strategy shields the bacteria from destruction by the immune system and antibiotics while allowing for the spread of bacteria at opportunistic times. As such, it is critical to understand how Mtb interacts with host macrophages to manipulate the mode of cell death. Herein, we demonstrate that Mtb infection triggers a time-dependent reduction in the expression of focal adhesion kinase (FAK) in human macrophages. Using pharmacological perturbations, we show that inhibition of FAK (FAKi) triggers an increase in a necrotic form of cell death during Mtb infection. In contrast, genetic overexpression of FAK (FAK+) completely blocked macrophage cell death during Mtb infection. Using specific inhibitors of necrotic cell death, we show that FAK-mediated cell death during Mtb infection occurs in a RIPK1-depedent, and to a lesser extent, RIPK3-MLKL-dependent mechanism. Consistent with these findings, FAKi results in uncontrolled replication of Mtb, whereas FAK+ reduces the intracellular survival of Mtb in macrophages. In addition, we demonstrate that enhanced control of intracellular Mtb replication by FAK+ macrophages is a result of increased production of antibacterial reactive oxygen species (ROS) as inhibitors of ROS production restored Mtb burden in FAK+ macrophages to same levels as in wild-type cells. Collectively, our data establishes FAK as an important host protective response during Mtb infection to block necrotic cell death and induce ROS production, which are required to restrict the survival of Mtb.

FAK inhibitor PF-431396 suppresses IgE-mediated mast cell activation and allergic inflammation in mice

Mast cells (MCs) initiate and maintain allergic inflammation. Upon being stimulated with immunoglobulin (Ig)E and antigen (Ag), MCs exhibit FcεRI (high-affinity IgE) receptor-mediated degranulation, cytokine secretion, and increased focal adhesion kinase (FAK) activity. The aims of this study were to examine mechanisms of FAK regulation in IgE-mediated MC activation and the effects of FAK inhibition on MC-mediated allergic responses. FAK activity was manipulated with short hairpin RNA (shRNA) knockdown, FAK overexpression, and the FAK inhibitor PF-431396 (PF). Gene expression and kinase activation were analyzed with quantitative molecular biology assays. PF effects were tested in the passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), and allergic conjunctivitis (AC) mouse models. Our results showed that FAK overexpression increased IgE-mediated degranulation and reduced the dexamethasone inhibitory effect on MCs activation. The FAK inhibitor PF diminished MC release of β-hexosaminidase (β-hex), histamine, and inflammatory cytokines, via a mechanism that involves MAPK and NF-κB signaling pathways. CaMKII was identified as a robust FAK-associating protein. Inhibition of CaMKII activation by KN-93 suppressed FAK activity and its downstream pathway. PF attenuated inflammatory responses in our PCA and ASA models, and relieved signs of allergic disease in AC model mice. In conclusions, MC degranulation and production of inflammatory mediators in allergic disease may be consequent to FcεRI crosslinking inducing CaMKII-mediated activation of FAK activity. FAK inhibition may represent a new MC-suppressing treatment strategy for the treatment of allergic diseases.

Abstract 968: Focal adhesion kinase (FAK) inhibitor APG-2449 sensitizes ovarian tumors to chemotherapy via CD44 downregulation

Abstract Ovarian cancer is one of the deadliest malignancies in women, and up to 70% of patients with epithelial ovarian cancer have FAK overexpression, amplification, or activation. FAK plays an important role in cellular migration, growth factor signaling, cell cycle progression, cellular survival and chemoresistance. This biomarker is also significantly associated with higher tumor stage, metastasis, and shorter overall survival in patients with ovarian cancer. Inhibition of FAK is therefore emerging as a promising treatment target. APG-2449 is a clinical stage FAK/ALK/ROS1 multi-kinase inhibitor. In this study we investigated antitumor activity of APG-2449 combined with standard-of-care chemotherapeutics in ovarian cancer in the preclinical setting. In a mouse xenograft tumor model derived from ovarian cancer cell line OVCAR-3, which was resistant to platinum-based therapies, APG-2449 combined with paclitaxel, and paclitaxel plus carboplatin, synergistically enhanced antitumor activity, whereas the chemotherapeutics showed no activity. Synergistic antitumor activity was also observed in multiple patient-derived xenograft (PDX) models derived from women with chemoinsensitive ovarian cancer, which also frequently expresses high levels of FAK. By comparing gene expression profiles of PDX tumors obtained from responders and nonresponders to the combined therapy, we identified CD44 (a marker for cancer stem cells) as a potentially predictive biomarker. Western blot analysis confirmed higher protein levels of CD44 in pretreated tumors of responders. Interestingly, downregulation of CD44 levels was observed in combination-treated tumors, suggesting that these combinations reduced cancer stem cell populations in ovarian cancer. Accordingly, in ovarian cancer cells exposed to APG-2449 alone or combined with paclitaxel, numbers of cells positive for CD44 or aldehyde dehydrogenase 1 (ALDH1; another marker for cancer stem cells) decreased in a dose-dependent manner. In summary, our data suggest that FAK inhibition by APG-2449 sensitizes ovarian tumors to chemotherapeutics in preclinical tumor models of ovarian cancer. The synergistic antitumor activity was mediated by downregulation of CD44+ or ALDH1+ cancer stem cell populations. These findings encourage clinical development of APG-2449 in combination with chemotherapies for treatment of ovarian cancer. Citation Format: Ran Tao, Douglas D. Fang, Yuanbao Li, Kaixiang Zhang, Chunhua Xu, Xu Fang, Qixin Wang, Dajun Yang, Yifan Zhai. Focal adhesion kinase (FAK) inhibitor APG-2449 sensitizes ovarian tumors to chemotherapy via CD44 downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 968.

Focal adhesion kinase (FAK) promotes cholangiocarcinoma development and progression via YAP activation

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is upregulated in many tumor types and is a promising target for cancer therapy. Herein, we elucidated the functional role of FAK in intrahepatic cholangiocarcinoma (iCCA) development and progression. Expression levels and activation status of FAK were determined in human iCCA samples. The functional contribution of FAK to Akt/YAP murine iCCA initiation and progression was investigated using conditional Fak knockout mice and constitutive Cre or inducible Cre mice, respectively. The oncogenic potential of FAK was further examined via overexpression of FAK in mice. Invitro cell line studies and invivo drug treatment were applied to address the therapeutic potential of targeting FAK for iCCA treatment. FAK was ubiquitously upregulated and activated in iCCA lesions. Ablation of FAK strongly delayed Akt/YAP-driven mouse iCCA initiation. FAK overexpression synergized with activated AKT to promote iCCA development and accelerated Akt/Jag1-driven cholangiocarcinogenesis. Mechanistically, FAK was required for YAP(Y357) phosphorylation, supporting the role of FAK as a central YAP regulator in iCCA. Significantly, ablation of FAK after Akt/YAP-dependent iCCA formation strongly suppressed tumor progression in mice. Furthermore, a remarkable iCCA growth reduction was achieved when a FAK inhibitor and palbociclib, a CDK4/6 inhibitor, were administered simultaneously in human iCCA cell lines and Akt/YAP mice. FAK activation contributes to the initiation and progression of iCCA by inducing the YAP proto-oncogene. Targeting FAK, either alone or in combination with anti-CDK4/6 inhibitors, may be an effective strategy for iCCA treatment. We found that the protein FAK (focal adhesion kinase) is upregulated and activated in human and mouse intrahepatic cholangiocarcinoma samples. FAK promotes intrahepatic cholangiocarcinoma development, whereas deletion of FAK strongly suppresses its initiation and progression. Combined FAK and CDK4/6 inhibitor treatment had a strong anti-cancer effect in invitro and invivo models. This combination therapy might represent a valuable and novel treatment against human intrahepatic cholangiocarcinoma.

Clinical trial in progress: Phase II trial of defactinib (VS-6063) combined with VS-6766 (CH5126766) in patients with metastatic uveal melanoma.

TPS9588 Background: Despite successful treatment of primary uveal melanomas (UM), up to 50% of patients subsequently develop systemic metastasis, with the liver involved in up to 90% of patients. Currently there is no US FDA-approved treatment for metastatic uveal melanoma (MUM). Activating mutations in genes encoding alpha subunits of the heterotrimeric G proteins, GNAQ and GNA11, are found in 80-90% of UM. Recent information suggests that GNAQ/GNA11-oncogenic signaling involves a non-canonical pathway conferring the activation of YAP1, distinct from the activation of PLCβ and PKC-MEK-ERK, which may explain the failure of MEK inhibitors in MUM patients. Focal Adhesion Kinase (FAK) is a tyrosine kinase that provides a direct link between Gαq and tyrosine phosphorylation networks controlling YAP and UM growth. Interestingly, UM represents the human cancer harboring the highest level of FAK overexpression. Recent kinome-wide CRISPR-Cas9 screens revealed that FAK and RAF/MEK co-targeting may provide a new network-based precision therapeutic strategy for MUM treatment. Methods: This is an investigator-initiated, prospective, single arm, single-institution, phase II trial evaluating the combination of a FAK inhibitor (defactinib, VS-6063) with a RAF/MEK inhibitor (VS-6766, CH5126766) for the treatment of patients with metastatic uveal melanoma [NCT04720417]. The primary endpoint of the study is disease control rate (DCR) of 50% including complete response (CR), partial response (PR), and stable disease (SD) as determined by RECIST criteria version 1.1. Secondary endpoints include progression free survival, overall survival, and causality of adverse events. Exploratory endpoints include analysis of the pharmacodynamic profile, mechanism of resistance to the combination, and investigation of circulating free DNA as a biomarker. The efficacy of this combination treatment will be assessed using the Simon’s two stage design. In stage I, a total number of 8 patients are accrued and if there are 2 or fewer overall responses among these 8 patients, further enrollment of patients may be stopped with the conclusion that DCR cannot be 50% or greater. Otherwise, an additional 10 patients will be accrued in stage II, resulting in a total sample size of 18 patients. Patients at 18 years or older with metastases from uveal melanoma will be eligible (any line of therapy). Defactinib (200 mg) will be administered orally twice a day in combination with VS-6766 (3.2 mg) administered orally twice a week for 3 weeks, in 28-day cycles. Dose modification will be considered based on toxicity. Treatment will be continued until maximum clinical benefit is obtained; disease progression or the development of intolerable side effects. Enrollment to stage 1 began in February 2021. Clinical trial information: NCT04720417.

EB1 Restricts Breast Cancer Cell Invadopodia Formation and Matrix Proteolysis via FAK

Regulation of microtubule dynamics by plus-end tracking proteins (+TIPs) plays an essential role in cancer cell migration. However, the role of +TIPs in cancer cell invasion has been poorly addressed. Invadopodia, actin-rich protrusions specialized in extracellular matrix degradation, are essential for cancer cell invasion and metastasis, the leading cause of death in breast cancer. We, therefore, investigated the role of the End Binding protein, EB1, a major hub of the +TIP network, in invadopodia functions. EB1 silencing increased matrix degradation by breast cancer cells. This was recapitulated by depletion of two additional +TIPs and EB1 partners, APC and ACF7, but not by the knockdown of other +TIPs, such as CLASP1/2 or CLIP170. The knockdown of Focal Adhesion Kinase (FAK) was previously proposed to similarly promote invadopodia formation as a consequence of a switch of the Src kinase from focal adhesions to invadopodia. Interestingly, EB1-, APC-, or ACF7-depleted cells had decreased expression/activation of FAK. Remarkably, overexpression of wild type FAK, but not of FAK mutated to prevent Src recruitment, prevented the increased degradative activity induced by EB1 depletion. Overall, we propose that EB1 restricts invadopodia formation through the control of FAK and, consequently, the spatial regulation of Src activity.

Retracted] Epigenetic silencing of miR‑130a ameliorates hemangioma by targeting tissue factor pathway inhibitor2 through FAK/PI3K/Rac1/mdm2 signaling.

Following the publication of this paper, it was drawn to the authors' attention by an interested reader that some tumours featured in Fig.6A of the above paper were strikingly similar to those featured in Fig.8A of an article appearing in the same journal [Fan F‑Y, Deng R, Yi H, Sun H‑P, Zeng Y, HeG‑C and SuY: The inhibitory effect of MEG3/miR‑214/AIFM2 axis on the growth of T‑cell lymphoblastic lymphoma. Int JOncol 51:316‑326, 2017]. Furthermore, flow cytometric images featured in Fig.2G of the above paper were strikingly similar to data featured in the following article [ZhangH‑j, Wei Q‑f, WangS‑j, Zhang H‑j, Zhang X‑y, Geng Q, CuiY‑h and WangX‑h:LncRNA HOTAIR alleviates rheumatoid arthritis by targeting miR‑138 and inactivating NF‑κB pathway. Int Immunopharmacol 50:283‑290, 2017]. The Editor asked the authors for an explanation to account for the appearance of strikingly similar data in their paper independently, and they responded to request that the paper be retracted from International Journal of Oncology. All the authors agreed that the article should be retracted. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in International Journal of Oncology 50: 1821‑1831, 2017; DOI: 10.3892/ijo.2017.3943].

Progress in the Development of Small Molecular Inhibitors of Focal Adhesion Kinase (FAK)

Focal adhesion kinase (FAK) is a nonreceptor intracellular tyrosine kinase that plays an essential role in cancer cell adhesion, survival, proliferation, and migration through both its enzymatic activities and scaffolding functions. Overexpression of FAK has been found in many human cancer cells from different origins, which promotes tumor progression and influences clinical outcomes in different classes of human tumors. Therefore, FAK has been considered as a promising target for small molecule anticancer drug development. Many FAK inhibitors targeting different domains of FAK with various mechanisms of functions have been reported, including kinase domain inhibitors, FERM domain inhibitors, and FAT domain inhibitors. In addition, FAK-targeting PROTACs, which can induce the degradation of FAK, have also been developed. In this Perspective, we summarized the progress in the development of small molecular FAK inhibitors and proposed the perspectives for the future development of agents targeting FAK.

Focal adhesion kinase promotes BMP2-induced osteogenic differentiation of human urinary stem cells via AMPK and Wnt signaling pathways.

Human urine-derived stem cells (hUSCs) serve as favorable candidates for bone transplants due to their efficient proliferative and multipotent differentiation abilities, as well as the capacity to secrete a variety of vasoactive agents to facilitate tissue engineering. The present study aimed to explore the role of focal adhesion kinase (FAK) in bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation of hUSCs and to investigate the underlying mechanism. The degree of osteogenic differentiation and the correlated signals, following BMP2 overexpression and siRNA-mediated silencing of FAK, were determined in vitro. Moreover, hUSCs induced bone formation in a rat model with cranial defects, in vivo. Our findings revealed that alkaline phosphatase production, calcium deposits, osteocalcin and osteopontin expression, and bone formation were upregulated in vitro and in vivo following BMP2-induced osteogenic differentiation, and AMPK and Wnt signaling pathway activation by FAK could effectively regulate BMP2-enhanced osteogenic differentiation of hUSCs. Taken together, these findings indicated that FAK could mediate BMP2-enhanced osteogenic differentiation of hUSCs through activating adenosine 5'-monophosphate-activated protein kinase and Wnt signaling pathways.

Focal Adhesion Kinase (FAK) Overexpression and Phosphorylation in Oral Squamous Cell Carcinoma and their Clinicopathological Significance.

Focal adhesion kinase (FAK) is involved in progression of various cancers, and FAK overexpression has been associated with cancer invasion and metastasis. However, the involvement of FAK expression in the clinicopathological malignancy of oral squamous cell carcinoma (OSCC) remains unknown. In addition, there is no consensus regarding the role of p16 expression in OSCC. In this study, the immunohistochemically measured expression of FAK, phosphorylated FAK (FAKpY397) and p16 expressions and their associations with clinicopathological features and 5-year survival rates were examined in surgical samples from 70 patients with primary OSCC. FAK and FAKpY397 were expressed at high levels in 42 cases (60.0%) and 34 cases (48.6%), respectively, and 9 cases (12.9%) were positive for p16. FAK expression was significantly correlated with local recurrence, subsequent metastasis, and the mode of invasion. FAKpY397 expression was significantly correlated with both N classification and the mode of invasion. p16 expression was significantly correlated with clinical stage only. Patients having high expression of FAK, FAKpY397, or both showed significantly worse prognosis, but p16 expression showed no significant relation to prognosis. The results suggested that overexpression and phosphorylation of FAK in OSCC may affect cancer progression, such as local invasion and lymph node metastasis, and thereby contribute to life prognosis.

Focal Adhesion Kinase and β-Catenin Cooperate to Induce Hepatocellular Carcinoma.

There is an urgent need to understand the molecular signaling pathways that drive or mediate the development of hepatocellular carcinoma (HCC). The focal adhesion kinase (FAK) gene protein tyrosine kinase 2 is amplified in 16.4% of The Cancer Genome Atlas HCC specimens, and its amplification leads to increased FAK mRNA expression. It is not known whether the overexpression of FAK alone is sufficient to induce HCC or whether it must cooperate in some ways with other oncogenes. In this study, we found that 34.8% of human HCC samples with FAK amplification also show β-catenin mutations, suggesting a co-occurrence of FAK overexpression and β-catenin mutations in HCC. We overexpressed FAK alone, constitutively active forms of β-catenin (CAT) alone, or a combination of FAK and CAT in the livers of C57/BL6 mice. We found that overexpression of both FAK and CAT, but neither FAK nor CAT alone, in mouse livers was sufficient to lead to tumorigenesis. We further demonstrated that FAK's kinase activity is required for FAK/CAT-induced tumorigenesis. Furthermore, we performed RNA-sequencing analysis to identify the genes/signaling pathways regulated by FAK, CAT, or FAK/CAT. We found that FAK overexpression dramatically enhances binding of β-catenin to the promoter of androgen receptor (AR), which leads to increased expression of AR in mouse livers. Moreover, ASC-J9, an AR degradation enhancer, suppressed FAK/CAT-induced HCC formation. Conclusion: FAK overexpression and β-catenin mutations often co-occur in human HCC tissues. Co-overexpression of FAK and CAT leads to HCC formation in mice through increased expression of AR; this mouse model may be useful for further studies of the molecular mechanisms in the pathogenesis of HCC and could lead to the identification of therapeutic targets.

Phycocyanin Enhances the Cytotoxic Profile of Cisplatin in Platinum‐Resistant Ovarian Cancer Cells by Targeting Focal Adhesion Kinase

Platinum‐based therapy is one of the major treatment approaches in ovarian cancer. Although greater than 80% of advanced stage ovarian cancer patients initially achieve remission with platinum therapy; the majority of this cohort eventually relapse, and develop drug‐resistant disease. Resistance to chemotherapeutic drugs such as cisplatin has been attributed in part to the overexpression of Focal Adhesion Kinase (FAK), which is known to promote cancer cell survival and metastasis. In recent years, there has been heightened interest in targeting focal adhesion kinase in platinum resistant cancer as a new modality in ovarian cancer treatment. In previous studies, we have reported that Phycocyanin (C‐PC), a major accessory pigment in the Limnothrix sp. 37‐2‐1, demonstrates cytotoxicity towards multiple cancer cell lines. Therefore, in the present study we evaluated the ability of C‐PC to sensitize platinum resistant OVCAR3 cells to cisplatin treatment. We also examined the response of FAK activation by phosphorylation, to treatment with cisplatin and CP‐C alone, and in combination (C‐PC +cisplatin). Cell viability was determined by MTT assay, and DNA fragmentation was used to confirm apoptotic cell death. The expression of phosphorylated FAK (pFAK), total FAK, pAKT, as well as the key apoptotic regulators p21, p53, Bax, and Bcl‐2 in response to the treatments were determined through western blotting. The results from our study confirm that C‐PC significantly enhances the cytotoxic profile of cisplatin in OVCAR‐3 cells through the induction of apoptosis as confirmed by the presence of DNA fragmentation patterns in all three treatment groups. The expression of activated pFAK was also significantly reduced in the CPC and CPC + cisplatin treatment groups and this corresponded with increased p53, p21 and Bax expression, while pAkt and Bcl‐2 expression was downregulated. These results confirm a novel effect of CPC on FAK phosphorylation in platinum resistant ovarian cancer, which can be further explored towards the treatment of advanced stage disease through combination strategies.Support or Funding InformationThis project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, FloridaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.