Tyrphostin A9 attenuates glioblastoma growth by suppressing PYK2/EGFR-ERK signaling pathway.

PURPOSE: Glioblastoma (GBM) is a fatal primary brain tumor with extremely poor clinical outcomes. The anticancer efficiency of tyrosine kinase inhibitors (TKIs) has been shown in GBM and other cancer, with limited therapeutic outcomes. In the current study, we aimed to investigate the clinical impact of active proline-rich tyrosine kinase-2 (PYK2) and epidermal growth factor receptor (EGFR) in GBM and evaluate its druggability by a synthetic TKI-Tyrphostin A9 (TYR A9). METHODS: The expression profile of PYK2 and EGFR in astrocytoma biopsies (n = 48) and GBM cell lines were evaluated through quantitative PCR, western blots, and immunohistochemistry. The clinical association of phospho-PYK2 and EGFR was analyzed with various clinicopathological features and the Kaplan-Meier survival curve. The phospho-PYK2 and EGFR druggability and subsequent anticancer efficacy of TYR A9 was evaluated in GBM cell lines and intracranial C6 glioma model. RESULTS: Our expression data revealed an increased phospho-PYK2, and EGFR expression aggravates astrocytoma malignancy and is associated with patients' poor survival. The mRNA and protein correlation analysis showed a positive association between phospho-PYK2 and EGFR in GBM tissues. The in-vitro studies demonstrated that TYR A9 reduced GBM cell growth, cell migration, and induced apoptosis by attenuating PYK2/EGFR-ERK signaling. The in-vivo data showed TYR A9 treatment dramatically reduced glioma growth with augmented animal survival by repressing PYK2/EGFR-ERK signaling. CONCLUSION: Altogether, this study report that increased phospho-PYK2 and EGFR expression in astrocytoma was associated with poor prognosis. The in-vitro and in-vivo evidence underlined translational implication of TYR A9 by suppressing PYK2/EGFR-ERK modulated signaling pathway. The schematic diagram displayed proof of concept of the current study indicating activated PYK2 either through the Ca2+/Calmodulin-dependent protein kinase II (CAMKII) signaling pathway or autophosphorylation at Tyr402 induces association to the SH2 domain of c-Src that leads to c-Src activation. Activated c-Src in turn activates PYK2 at other tyrosine residues that recruit Grb2/SOS complex and trigger ERK½ activation. Besides, PYK2 interaction with c-Src acts as an upstream of EGFR transactivator that can activate the ERK½ signaling pathway, which induces cell proliferation and cell survival by increasing anti-apoptotic proteins or inhibiting pro-apoptotic proteins. TYR A9 treatment attenuate GBM cell proliferation and migration; and induce GBM cell death by inhibiting PYK2 and EGFR-induced ERK activation.

Novel selective inhibitors of macropinocytosis-dependent growth in pancreatic ductal carcinoma.

Macropinocytosis is a cellular process that enables cells to engulf extracellular material, such as nutrients, growth factors, and even whole cells. It is involved in several physiological functions as well as pathological conditions. In cancer cells, macropinocytosis plays a crucial role in promoting tumor growth and survival under nutrient-limited conditions. In particular KRAS mutations have been identified as main drivers of macropinocytosis in pancreatic, breast, and non-small cell lung cancers. We performed a high-content screening to identify inhibitors of macropinocytosis in pancreatic ductal adenocarcinoma (PDAC)-derived cells, aiming to prevent nutrient scavenging of PDAC tumors. The screening campaign was conducted in a well-known pancreatic KRAS-mutated cell line (MIAPaCa-2) cultured under nutrient deprivation and using FITC-dextran to precisely quantify macropinocytosis. We assembled a collection of 3584 small molecules, including drugs approved by the Food and Drug Administration (FDA), drug-like molecules against molecular targets, kinase-targeted compounds, and molecules designed to hamper protein-protein interactions. We identified 28 molecules that inhibited macropinocytosis, with potency ranging from 0.4 to 29.9 µM (EC50). A few of them interfered with other endocytic pathways, while 11 compounds did not and were therefore considered specific "bona fide" macropinocytosis inhibitors and further characterized. Four compounds (Ivermectin, Tyrphostin A9, LY2090314, and Pyrvinium Pamoate) selectively hampered nutrient scavenging in KRAS-mutated cancer cells. Their ability to impair albumin-dependent proliferation was replicated both in different 2D cell culture systems and 3D organotypic models. These findings provide a new set of compounds specifically targeting macropinocytosis, which could have therapeutic applications in cancer and infectious diseases.

Tyrphostin A9 protects axons in experimental autoimmune encephalomyelitis through activation of ERKs.

AIMS: Small molecule compound tyrphostin A9 (A9), an inhibitor of platelet-derived growth factor (PDGF) receptor, was previously reported by our group to stimulate extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2) in neuronal cells in a PDGF receptor-irrelevant manner. The study aimed to investigate whether A9 could protect axons in experimental autoimmune encephalomyelitis through activation of ERKs. MAIN METHODS: A9 treatment on the protection on neurite outgrowth in SH-SY5Y neuroblastoma cells and primary substantia nigra neuron cultures from the neurotoxin MPP+ were analyzed. Then, clinical symptoms as well as ERK1/2 activation, axonal protection induction, and the abundance increases of the regeneration biomarker GAP-43 in the CNS in the relapsing-remitting experimental autoimmune encephalomyelitis (EAE) model were verified. KEY FINDINGS: A9 treatment could stimulate neurite outgrowth in SH-SY5Y neuroblastoma cells and protect primary substantia nigra neuron cultures from the neurotoxin MPP+. In the relapsing-remitting EAE model, oral administration of A9 successfully ameliorated clinical symptoms, activated ERK1/2, induced axonal protection, and increased the abundance of the regeneration biomarker GAP-43 in the CNS. Interestingly, gene deficiency of ERK1 or ERK2 disrupted the beneficial effects of A9 in MOG-35-55-induced EAE. SIGNIFICANCE: These results demonstrated that small molecule compounds that stimulate persistent ERK activation in vitro and in vivo may be useful in protective or restorative treatment for neurodegenerative diseases.

The involvement of mitochondrial fission in maintenance of the stemness of bone marrow mesenchymal stem cells.

IMPACT STATEMENT: How to maintain the stemness of bone marrow mesenchymal stem cells (BMSCs) in cultures is a long-standing question. The present study found that mitochondrial dynamics affects the stemness of BMSCs in cultures and the retaining of mitochondrial fission enhances the stemness of BMSCs. This work thus provides a novel insight into strategic approaches to maintain the stemness of BMSCs in cultures in relation to the clinical application of bone-marrow stem cells.

Tyrphostin A9 improves blastocyst development in porcine embryos through induction of dynamin-related protein 1-dependent mitochondrial fission.

Mitochondrial dynamics are associated with the development of porcine embryos. However, little is known about the effects of mitochondrial dynamics-related genes (Drp1 and pDrp1-Ser616) on early porcine embryo development. Here, we investigated the effect of Drp1-dependent mitochondrial fission signaling on the development of porcine embryos using the mitochondrial fission inducer, tyrphostin A9 (TA9). We determined that TA9 (1µM) treated embryos were increased the mitochondrial functions, blastocyst development rate and quality, as well as decreased mitochondria-specific superoxide and mitochondrial apoptosis. Thus, TA9-induced appropriate mitochondrial fission improved the developmental competence via maintenance of a balance in mitochondrial dynamics in porcine embryo.