Receptor Tyrosine Kinase Signal Transduction Research Articles

383 (PB371): Characterization of cancer cell lines selected for resistance to growth factor receptor tyrosine kinase inhibitors and signal transduction therapies

383 (PB371): Characterization of cancer cell lines selected for resistance to growth factor receptor tyrosine kinase inhibitors and signal transduction therapies

Effects of SU5416 on angiogenesis and the ERK-VEGF/MMP-9 pathway in rat endometriosis.

SU5416 is a small molecule vascular endothelial growth factor (VEGF) receptor signal transduction inhibitor, which can block the VEGF re-ceptor autophosphorylation and inhibit receptor tyrosine kinase signal trans-duction, thereby reducing VEGF activity. However, there are few reports about the correlation of SU5416 to the occurrence and angiogenesis in endometrio-sis. In this study, we observed the effects of VEGF receptor inhibitor SU5416 on angiogenesis in endometriosis in rats. Thirty femalespecific-pathogen-free Sprague-Dawley rats were randomly divided into sham operation group (SOG), model group (MG), and SU5416 group (n=10 for each group). In the SOG, only the uterus was cut and sutured, and endometriosis models were established in the MG and SU5416 group by autologous transplantation. The SU5416 group was injected with 15 mg/kg SU5416 intraperitoneally, and the SOG and MG were intraperitoneally injected with an equal volume of normal saline for 6 weeks. The volume of ectopic lesions was lower in the SU5416 group at 42 d postoperatively thanin the MG (p<0.05). The proportion of CD31-positive cells in the endometrial tissue of the SU5416 group was lower than that of the MG (p<0.05); angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), laminin-5γ2 (LN-5γ2) and phosphorylation of ERK (P-ERK), VEGF, matrix metalloproteinase (MMP)-2, and MMP-9 protein expressions were lower in the SU5416 groupthan in the MG (p<0.05).VEGFreceptor inhibitor SU5416 can inhibit endometrio-sis angiogenesis and reduce inflammatory response in rats, and its mechanism of action may be related to the down-regulation of the ERK-VEGF/MMP-9 path-way expression.

Network models of protein phosphorylation, acetylation, and ubiquitination connect metabolic and cell signaling pathways in lung cancer

We analyzed large-scale post-translational modification (PTM) data to outline cell signaling pathways affected by tyrosine kinase inhibitors (TKIs) in ten lung cancer cell lines. Tyrosine phosphorylated, lysine ubiquitinated, and lysine acetylated proteins were concomitantly identified using sequential enrichment of post translational modification (SEPTM) proteomics. Machine learning was used to identify PTM clusters that represent functional modules that respond to TKIs. To model lung cancer signaling at the protein level, PTM clusters were used to create a co-cluster correlation network (CCCN) and select protein-protein interactions (PPIs) from a large network of curated PPIs to create a cluster-filtered network (CFN). Next, we constructed a Pathway Crosstalk Network (PCN) by connecting pathways from NCATS BioPlanet whose member proteins have PTMs that co-cluster. Interrogating the CCCN, CFN, and PCN individually and in combination yields insights into the response of lung cancer cells to TKIs. We highlight examples where cell signaling pathways involving EGFR and ALK exhibit crosstalk with BioPlanet pathways: Transmembrane transport of small molecules; and Glycolysis and gluconeogenesis. These data identify known and previously unappreciated connections between receptor tyrosine kinase (RTK) signal transduction and oncogenic metabolic reprogramming in lung cancer. Comparison to a CFN generated from a previous multi-PTM analysis of lung cancer cell lines reveals a common core of PPIs involving heat shock/chaperone proteins, metabolic enzymes, cytoskeletal components, and RNA-binding proteins. Elucidation of points of crosstalk among signaling pathways employing different PTMs reveals new potential drug targets and candidates for synergistic attack through combination drug therapy.

Quantitative fret studies of EphA2-Lyn interactions.

The regulation of the receptor tyrosine kinase (RTK) signal transduction pathways is critical for proper cellular function. RTK activation occurs in response to ligand binding, which induces RTK dimerization or oligomerization to allow cross-phosphorylation of specific tyrosines in the kinase domains of the RTK. Downstream signals are then propagated upon the recruitment of soluble kinases or adaptor proteins to these phoshorylated tyrosines. The EphA2 receptor tyrosine kinase is a member of the largest RTK family and has been reported to initiate downstream signaling by recruiting Lyn, a member of the Src family of non-receptor tyrosine kinases. Contrary to expectations, quantitative FRET experiments demonstrate that ligand-induced EphA2 tyrosine phosphorylation does not lead to Lyn recruitment to EphA2. In control experiments, Lyn is efficiently recruited by a different RTK, EGFR, upon ligand stimulation. The differential interactions of Lyn with EphA2 and EGFR may be a means of signal diversification. Current work is focused on the mechanism behind the unexpected lack of interactions between EphA2 and Lyn upon EphA2 clustering and phosphorylation.

CSIG-17. SCD5 PROTECTS GLIOBLASTOMA STEM CELLS FROM DEATH AND DIFFERENTIATION BY MODULATING INTRACELLULAR LIPID COMPOSITION

Abstract Glioblastoma (GBM) is the most common malignant brain cancer in adults, enriched in a small subpopulation of glioma stem cells (GSC), which can drive tumor recurrence and therapeutic resistance. Considerable evidence suggests that the endogenous levels of unsaturated fatty acids (FA) are crucial regulators of GSCs survival and self-renewal. Stearoyl-CoA desaturase-1 (SCD-1) is the most abundant desaturase in humans. We have previously shown that SCD1 activity is required for GSCs self-renewal and brain tumor initiation. However, SCD1 orthologous isoform, SCD5, has been poorly characterized and its potential role in GBM has not been previously reported. We have observed that SCD5 is highly enriched in GSC both at the mRNA and protein levels. Genetic downregulation of SCD5 in GSCs led to a remarkable decrease in stem cell markers, impaired cell viability and the ability to form neurospheres. Further, the downregulation of SCD5 in GSCs orthotopically implanted in mice resulted in delayed tumor growth and extended overall survival. Shotgun lipidomics in GSCs after either SCD1 or SCD5 knock-down revealed a largely distinctive lipidome profile, highlighting the divergent role of these two isoforms in GBM lipid metabolism. Surprisingly, lipidomics analysis showed that both SCD1 and SCD5 are required to synthesize a variety of lipid species involved in receptor tyrosine kinase (RTKs) and GPCRs signal transduction, directly linking FA synthesis with the oncogenic signaling. We confirmed these results by immunoblot analysis. Using specific tagging and immunofluorescence analysis, we observed that, despite a spatial overlap in SCD1 and SCD5 expression, SCD5 is uniquely present in some subcellular locations. This suggests that different functions of these isoforms could be related to different subcellular localization. Altogether, our results underscore a novel function of SCD isoforms in GSCs metabolism and highlight SCD5 as a potential therapeutic target for GBM.

In unity, there is strength: Phase separation controls receptor tyrosine kinase signal transduction

Lin etal. (2022) discover that FGFR2 undergoes liquid-liquid phase separation with its downstream effectorsSHP2 and PLCγ1, and the formation of phase separated condensates is essential for signaling competency.

Quantifying the strength of heterointeractions among receptor tyrosine kinases from different subfamilies: Implications for cell signaling

Receptor tyrosine kinases (RTKs) are single-pass membrane proteins that control vital cell processes such as cell growth, survival, and differentiation. There is a growing body of evidence that RTKs from different subfamilies can interact and that these diverse interactions can have important biological consequences. However, these heterointeractions are often ignored, and their strengths are unknown. In this work, we studied the heterointeractions of nine RTK pairs, epidermal growth factor receptor (EGFR)-EPH receptor A2 (EPHA2), EGFR-vascular endothelial growth factor receptor 2 (VEGFR2), EPHA2-VEGFR2, EPHA2-fibroblast growth factor receptor 1 (FGFR1), EPHA2-FGFR2, EPHA2-FGFR3, VEGFR2-FGFR1, VEGFR2-FGFR2, and VEGFR2-FGFR3, using a FRET-based method. Surprisingly, we found that RTK heterodimerization and homodimerization strengths can be similar, underscoring the significance of RTK heterointeractions in signaling. We discuss how these heterointeractions can contribute to the complexity of RTK signal transduction, and we highlight the utility of quantitative FRET for probing multiple interactions in the plasma membrane.

Receptor Tyrosine Kinase Ubiquitination and De-Ubiquitination in Signal Transduction and Receptor Trafficking

Receptor tyrosine kinases (RTKs) are membrane-based sensors that enable rapid communication between cells and their environment. Evidence is now emerging that interdependent regulatory mechanisms, such as membrane trafficking, ubiquitination, proteolysis and gene expression, have substantial effects on RTK signal transduction and cellular responses. Different RTKs exhibit both basal and ligand-stimulated ubiquitination, linked to trafficking through different intracellular compartments including the secretory pathway, plasma membrane, endosomes and lysosomes. The ubiquitin ligase superfamily comprising the E1, E2 and E3 enzymes are increasingly implicated in this post-translational modification by adding mono- and polyubiquitin tags to RTKs. Conversely, removal of these ubiquitin tags by proteases called de-ubiquitinases (DUBs) enables RTK recycling for another round of ligand sensing and signal transduction. The endocytosis of basal and activated RTKs from the plasma membrane is closely linked to controlled proteolysis after trafficking and delivery to late endosomes and lysosomes. Proteolytic RTK fragments can also have the capacity to move to compartments such as the nucleus and regulate gene expression. Such mechanistic diversity now provides new opportunities for modulating RTK-regulated cellular responses in health and disease states.

Parallels and Distinctions in FGFR, VEGFR, and EGFR Mechanisms of Transmembrane Signaling.

Receptor tyrosine kinase (RTK) signal transduction is essential in human skeletal, nervous, and vascular development, in homeostasis, and in disease. RTKs are activated by dimerization in the plasma membrane. The mechanisms of receptor dimerization and activation are multifaceted and complex, and unraveling them remains challenging. Most studies of RTKs have been devoted to crystallographic analysis of their isolated extracellular domain and biochemical analysis of the catalytic domain. However, the past few years have seen direct biophysical studies of (intact) RTK dimerization in native membranes lead to significant progress in our fundamental understanding of the mechanisms of their signal transduction across the plasma membrane. This perspective focuses on recent insights into the mechanisms of fibroblast growth factor receptor and vascular endothelial growth factor receptor transmembrane signaling, derived from studies of wild-type and mutant RTKs in a number of environments, including plasma membrane-derived vesicles. These insights reveal distinct steps in and factors of RTK signaling across the plasma membrane that can guide the drug discovery process for RTK targeting therapeutics.

Abstract IA06: Spatial systems biology of cancer

Abstract International efforts have now defined the genomic landscapes of most major human cancer types. The genomic landscape of breast cancer is particularly well described in several recent publications. The high level view of this disease suggests a remarkable level of inter- and intra-tumor heterogeneity, the presence of a few recurrent and many rare genomic aberrations and substantial genomic and epigenomic heterogeneity. The challenge now is to understand how these aberrations and tumor extrinsic influences from the microenvironment collaborate to deregulate aspects of cell differentiation, proliferation, apoptosis, DNA repair, senescence, motility and multiscale architecture that include cancer progression and response to therapy. To this end, the OHSU Center for Spatial Systems Biomedicine has deployed a suite of measurement technologies that enable high throughput assessment of changes in these endpoints to siRNA knockdown of genes found by the TCGA and international genomics efforts to be aberrant and/or involved in regulatory networks that are associated with differentiation subtype and/or differential response to therapy. These studies have revealed both recurrent and rare aberrations that strongly influence these endpoints as well as a range of molecular architectures that influence aspects of receptor tyrosine kinase signal transduction. This presentation will summarize the results of the siRNA knockdown experiments and focus on the concept that analysis of the architectural features influenced by genomic and epigenomic abnormalities will suggest new therapeutic approaches. Illustrative examples of architectural features revealed using advanced imaging approaches that suggest new approaches to the design of RTK targeted therapies include (a) Ras/Raf multimers associated with signal transduction, (b) actin associated microfilaments involved in ERBB2 signal transduction, and (c) intratumor heterogeneity in differentiation status that influences responses to a range of kinase inhibitors. Citation Format: Joe Gray. Spatial systems biology of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA06.

Clinical trials of receptor protein-tyrosine kinase inhabitors in malignant glioma

The occurrence and development of malignant glioma are closely related to abnormal overexpression and activation of receptor tyrosine kinase (RTK) signal transduction pathways.Targeted therapeutic drugs such as RTK inhibitors,RTK downstream signaling pathway inhibitors and multi-target inhibitors can targeting treat malignant glioma at molecular level,some of which have been investigated in clinical trials and achieved good therapeutic effects. Key words: Glioma; Clinical trial; Receptor protein-tyrosine kinase inhabitor

Transdifferentiation of pancreatic acinar cells, and the role of stem cell factor and c-kit receptor tyrosine kinase signal transduction system

Transdifferentiation of pancreatic acinar cells, and the role of stem cell factor and c-kit receptor tyrosine kinase signal transduction system

Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2, but not Akt, in transfected Ba/F3 and UT-7 cells and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal tandem duplications of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation, the downstream Erk signaling pathway, and cell proliferation.

Mediators of receptor tyrosine kinase activation in infantile fibrosarcoma: a Children's Oncology Group study

Infantile fibrosarcoma (IFS; also known as cellular congenital mesoblastic nephroma, CMN, when in the kidney) is a rare, undifferentiated tumour often characterized by the ETV6-NTRK3 fusion transcript. Our goal was to identify downstream pathways, diagnostic markers and potential therapeutic targets for IFS/CMN. Global gene expression, reverse-phase protein array and ETV6-NTRK3 fusion analyses were performed on 14 IFS/CMN and compared with 41 other paediatric renal tumours. These analyses confirm significant receptor tyrosine kinase (RTK) activation, with evidence of PI3-Akt, MAPK and SRC activation. In particular, GAB2 docking protein, STAT5-pTyr-694, STAT3-pSer-729 and YAP-pSer-127 were elevated, and TAZ-pSer-89 was decreased. This provides mRNA and proteomic evidence that GAB2, STAT activation and phosphorylation of the Hippo pathway transcription co-activators YAP and TAZ contribute to the RTK signal transduction in IFS/CMN. All IFS/CMN tumours displayed a distinctive gene expression pattern that may be diagnostically useful. Unexpectedly, abundant ETV6-NTRK3 transcript copies were present in only 7/14 IFS, with very low copy number in 3/14. An additional 4/14 were negative by RT-PCR and absence of ETV6-NTRK3 was confirmed by FISH for both ETV6 and NTRK3. Therefore, molecular mechanisms other than ETV6-NTRK3 fusion are responsible for the development of some IFS/CMNs and the absence of ETV6-NTRK3 fusion products should not exclude IFS/CMN as a diagnosis.

Structural basis for autoinhibition and phosphorylation-dependent activation of c-Cbl

Cbls are RING ubiquitin ligases that attenuate receptor tyrosine kinase (RTK) signal transduction. Cbl ubiquitination activity is stimulated by phosphorylation of a linker helix region (LHR) tyrosine residue. To elucidate the mechanism of activation, we determined the structures of human CBL, a CBL-substrate peptide complex and a phosphorylated-Tyr371-CBL-E2-substrate peptide complex, and we compared them with the known structure of a CBL-E2-substrate peptide complex. Structural and biochemical analyses show that CBL adopts an autoinhibited RING conformation, where the RING's E2-binding surface associates with CBL to reduce E2 affinity. Tyr371 phosphorylation activates CBL by inducing LHR conformational changes that eliminate autoinhibition, flip the RING domain and E2 into proximity of the substrate-binding site and transform the RING domain into an enhanced E2-binding module. This activation is required for RTK ubiquitination. Our results present a mechanism for regulation of c-Cbl's activity by autoinhibition and phosphorylation-induced activation.

Characterization of the Src/Abl Hybrid Kinase SmTK6 of Schistosoma mansoni

Cellular protein-tyrosine kinases play key roles in signal transduction processes in eukaryotes. SmTK4 was the first Syk kinase identified in a parasite and found to be tissue-specifically transcribed in the gonads of adult Schistosoma mansoni. Functional analyses confirmed its role in oogenesis and spermatogenesis. As an SmTK4 upstream binding partner, the cellular protein-tyrosine kinase SmTK6 was isolated from a yeast two-hybrid library. Phylogenetic analyses performed in this study confirmed the first suggestions of a hybrid character of SmTK6. Biochemical studies made in Xenopus oocytes using inhibitors against Src (herbimycin A) and Abl (imatinib) kinases exhibited a biochemical inhibition profile of SmTK6, which was intermediate of Src and Abl kinases. As SmTK6 upstream interaction partners, we identified among others the known Src kinase SmTK3 and the Venus kinase receptor SmVKR1 of S. mansoni by yeast two-hybrid analyses, all of which co-localized in the gonads. Co-immunoprecipitation experiments confirmed interactions between SmTK6 and SmTK3 or SmVKR1. In Xenopus oocytes, it was finally shown that SmVKR1 but also SmTK3 were able to activate SmTK6 enzymatic activity indicating its functions in a receptor tyrosine kinase signal transduction cascade. These results not only demonstrate an intermediate but Src-biased profile of the unusual kinase SmTK6. They also strongly substantiate previous indications for a kinase complex, consisting of a receptor tyrosine kinase, Syk and Src kinases, which has been hypothesized to be involved in proliferation and differentiation processes in the gonads of schistosomes.

Focal adhesion kinase inhibitors are potent anti-angiogenic agents

Focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase and scaffold protein localized to focal adhesions, is uniquely positioned at the convergence point of integrin and receptor tyrosine kinase signal transduction pathways. FAK is overexpressed in many tumor cells, hence various inhibitors targeting its activity have been tested for anti-tumor activity. However, the direct effects of these pharmacologic agents on the endothelial cells of the vasculature have not been examined. Using primary human umbilical vein endothelial cells (HUVEC), we characterized the effects of two FAK inhibitors, PF-573,228 and FAK Inhibitor 14 on essential processes for angiogenesis, such as migration, proliferation, viability and endothelial cell tube formation. We observed that treatment with either FAK Inhibitor 14 or PF-573,228 resulted in reduced HUVEC viability, migration and tube formation in response to vascular endothelial growth factor (VEGF). Furthermore, we found that PF-573,228 had the added ability to induce apoptosis of endothelial cells within 36 h post-drug administration even in the continued presence of VEGF stimulation. FAK inhibitors also resulted in modification of the actin cytoskeleton within HUVEC, with observed increased stress fiber formation in the presence of drug. Given that endothelial cells were sensitive to FAK inhibitors at concentrations well below those reported to inhibit tumor cell migration, we confirmed their ability to inhibit endothelial-derived FAK autophosphorylation and FAK-mediated phosphorylation of recombinant paxillin at these doses. Taken together, our data indicate that small molecule inhibitors of FAK are potent anti-angiogenic agents and suggest their utility in combinatorial therapeutic approaches targeting tumor angiogenesis.

HIV-1 Tat binds to SH3 domains: Cellular and viral outcome of Tat/Grb2 interaction

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.

P66Shc, a multifaceted protein linking Erk signalling, glucose metabolism, and oxidative stress

p66Shc, a 66 kDa proto-oncogene Src collagen homologue (Shc) adaptor protein, is classically known as a signalling protein implicated in receptor tyrosine kinase signal transduction. The p66Shc isoform exerts a physiologically relevant, inhibitory signalling effect on the Erk pathway in skeletal muscle myoblasts, which is necessary for actin cytoskeleton polymerization and normal glucose transport responses. More recently, p66Shc has been also identified as a sensor of oxidative stress-induced apoptosis and as a longevity protein in mammals, actions which require Ser36 phosphorylation of the protein and consequent accumulation of intracellular reactive oxygen species. Oxidative stress plays a key role in dysfunction of several organs and tissues, and this is of interest in metabolic diseases such as type 2 diabetes. Thus changes in p66Shc expression and/or function may play an important role in the pathogenesis of type 2 diabetes and potentially serve as an effective target for its treatment.

Specific inhibition of a pathogenic receptor tyrosine kinase by its transmembrane domain

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) are believed to be important players in RTK signal transduction. However, the degree of specificity and promiscuity of RTK TM domain lateral interactions in mammalian membranes has not been assessed in detail in the literature. A technique to probe the occurrence of interactions between TM domains and their biological significance is to evaluate the propensity for formation of heterodimers of a full-length RTK and its TM domain. Here we examine if the inhibition of two RTK pathogenic mutants, Neu/V664E and FGFR3/A391E, can be achieved by the TM domains of Neu, Neu/V664E, FGFR3 and FGFR3/A391E. We show that the TM domain of Neu/V664E specifically inhibits the phosphorylation of full-length Neu/V664E, while the wild-type Neu TM domain does not. In addition, Neu/V664E TM domain does not affect the phosphorylation levels of full-length FGFR3/A391E. The results suggest that TM domain peptides could be exploited in the future for the development of specific inhibitors of mutant RTKs.