Receptor Tyrosine Kinase Signaling Pathways Research Articles

Raf-mediated cardiac hypertrophy in adultDrosophila

SUMMARYIn response to stress and extracellular signals, the heart undergoes a process called cardiac hypertrophy during which cardiomyocytes increase in size. If untreated, cardiac hypertrophy can progress to overt heart failure that causes significant morbidity and mortality. The identification of molecular signals that cause or modify cardiomyopathies is necessary to understand how the normal heart progresses to cardiac hypertrophy and heart failure. Receptor tyrosine kinase (RTK) signaling is essential for normal human cardiac function, and the inhibition of RTKs can cause dilated cardiomyopathies. However, neither investigations of activated RTK signaling pathways nor the characterization of hypertrophic cardiomyopathy in the adult fly heart has been previously described. Therefore, we developed strategies using Drosophila as a model to circumvent some of the complexities associated with mammalian models of cardiovascular disease. Transgenes encoding activated EGFRA887T, Ras85DV12 and Ras85DV12S35, which preferentially signal to Raf, or constitutively active human or fly Raf caused hypertrophic cardiomyopathy as determined by decreased end diastolic lumen dimensions, abnormal cardiomyocyte fiber morphology and increased heart wall thicknesses. There were no changes in cardiomyocyte cell numbers. Additionally, activated Raf also induced an increase in cardiomyocyte ploidy compared with control hearts. However, preventing increases in cardiomyocyte ploidy using fizzy-related (Fzr) RNAi did not rescue Raf-mediated cardiac hypertrophy, suggesting that Raf-mediated polyploidization is not required for cardiac hypertrophy. Similar to mammals, the cardiac-specific expression of RNAi directed against MEK or ERK rescued Raf-mediated cardiac hypertrophy. However, the cardiac-specific expression of activated ERKD334N, which promotes hyperplasia in non-cardiac tissues, did not cause myocyte hypertrophy. These results suggest that ERK is necessary, but not sufficient, for Raf-mediated cardiac hypertrophy.

Inhibition of platelet-derived growth factor receptor tyrosine kinase and downstream signaling pathways by Compound C

AMP-activated protein kinase (AMPK) has been implicated in anti-proliferative actions in a range of cell systems. Recently, it was observed that Compound C, an inhibitor of AMPK, also reduced the cell viability in human diploid fibroblasts (HDFs). Compound C-induced growth arrest was associated with a decrease in the cell cycle regulatory proteins, such as proliferating cell nuclear antigen, phosphorylated pRB, cyclin-dependent protein kinases (Cdk 2 and 4), cyclins (D and E), and the Cdk inhibitors (p21, p16, and p27). Therefore, the present study examined the molecular mechanism of the antiproliferative effects of Compound C. Although Compound C inhibited serum-induced phosphorylation of Akt and its substrate, glycogen synthase kinase-3β, it did not affect the Akt activity in vitro. Compound C significantly inhibited the receptor tyrosine phosphorylation and the activity of downstream signaling molecules, such as p85 phosphoinositide 3-kinase, phospholipase C-γ1, and extracellular signal-regulated kinase 1/2, induced by platelet-derived growth factor (PDGF) but not by epidermal growth factor- and insulin-like growth factor. In vitro growth factor receptor tyrosine kinase activity profiling revealed the IC50 for PDGF receptor-β (PDGFRβ) to be 5.07μM, whereas the IC50 for the epidermal growth factor receptor and insulin-like growth factor receptor was ≥100μM. The inhibitory effect of Compound C on PDGFRβ and Akt was also observed in AMPKα1/α2-knockout mouse embryonic fibroblasts, indicating that its inhibitory effect is independent of the AMPK activity. The inhibitory effect of Compound C on cell proliferation and PDGFRβ tyrosine phosphorylation was also demonstrated in various PDGFR-expressing cells, including MRC-5, BEAS-2B, rat aortic vascular smooth muscle cells, and A172 glioblastoma cells. These results indicate that Compound C can be used as a potential antiproliferative agent for PDGF- or PDGFR-associated diseases, such as cancer, atherosclerosis, and fibrosis.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Estrogen Regulates MAPK-Related Genes through Genomic and Nongenomic Interactions between IGF-I Receptor Tyrosine Kinase and Estrogen Receptor-Alpha Signaling Pathways in Human Uterine Leiomyoma Cells

Estrogen and growth factors play a major role in uterine leiomyoma (UtLM) growth possibly through interactions of receptor tyrosine kinases (RTKs) and estrogen receptor-alpha (ERα) signaling. We determined the genomic and nongenomic effects of 17β-estradiol (E2) on IGF-IR/MAPKp44/42 signaling and gene expression in human UtLM cells with intact or silenced IGF-IR. Analysis by RT2 Profiler PCR-array showed genes involved in IGF-IR/MAPK signaling were upregulated in UtLM cells by E2 including cyclin D kinases, MAPKs, and MAPK kinases; RTK signaling mediator, GRB2; transcriptional factors ELK1 and E2F1; CCNB2 involved in cell cycle progression, proliferation, and survival; and COL1A1 associated with collagen synthesis. Silencing (si)IGF-IR attenuated the above effects and resulted in upregulation of different genes, such as transcriptional factor ETS2; the tyrosine kinase receptor, EGFR; and DLK1 involved in fibrosis. E2 rapidly activated IGF-IR/MAPKp44/42 signaling nongenomically and induced phosphorylation of ERα at ser118 in cells with a functional IGF-IR versus those without. E2 also upregulated IGF-I gene and protein expression through a prolonged genomic event. These results suggest a pivotal role of IGF-IR and possibly other RTKs in mediating genomic and nongenomic hormone receptor interactions and signaling in fibroids and provide novel genes and targets for future intervention and prevention strategies.

The SAM Domain of Human TEL2 Can Abrogate Transcriptional Output from TEL1 (ETV-6) and ETS1/ETS2

Regulation of gene expression downstream of the Receptor Tyrosine Kinase signaling pathway in Drosophila relies on a transcriptional effector network featuring two conserved Ets family proteins, Yan and Pointed, known as TEL1 (ETV6) and ETS1/ETS2, respectively, in mammals. As in Drosophila, both TEL1 and ETS1/ETS2 operate as Ras pathway transcriptional effectors and misregulated activity of either factor has been implicated in many human leukemias and solid tumors. Providing essential regulation to the Drosophila network, direct interactions with the SAM domain protein Mae attenuate both Yan-mediated repression and PointedP2-mediated transcriptional activation. Given the critical contributions of Mae to the Drosophila circuitry, we investigated whether the human Ets factors TEL1 and ETS1/ETS2 could be subject to analogous regulation. Here we demonstrate that the SAM domain of human TEL2 can inhibit the transcriptional activities of ETS1/2 and TEL1. Drosophila Mae can also attenuate human ETS1/ETS2 function, suggesting there could be cross-species conservation of underlying mechanism. In contrast, Mae is not an effective inhibitor of TEL1, suggesting the mode of TEL2SAM-mediated inhibition of TEL1 may be distinct from how Drosophila Mae antagonizes Yan. Together our results reveal both further similarities and new differences between the mammalian and Drosophila networks and more broadly suggest that SAM domain-mediated interactions could provide an effective mechanism for modulating output from the TEL1 and ETS1/2 oncogenes.

Abstract 2165: Sprouty loss leads to aberrant regulation of receptor tyrosine kinase signaling pathways and accelerates mammary tumorigenesis

Abstract The SPROUTY (SPRY) proteins, key negative regulators of receptor tyrosine kinase (RTK) signaling, control developmental processes such as organ branching and maintain cellular homeostasis. The SPRY proteins inhibit critical signaling pathways that are downstream of RTKs, including the Ras-MAPK, PI3K-AKT, and the PLCα-PKC pathway. These pathways are highly deregulated in various cancers including HER2 overexpressing and PIK3CA mutant breast cancers. Importantly, the SPRY genes are significantly down-regulated in many cancers including 90% of human breast cancer cases, suggesting that they may function as putative tumor suppressors. To understand the role of Spry in normal mammary development, we examined the mammary glands of our Spry1 mutant mice at various developmental stages. In virgin Spry1-/- mice we observed a trend towards more branching of developing breast ducts. Upon pregnancy there was striking overgrowth of the mammary epithelium in Spry1+/− mice (Spry1-/- mice are sterile), suggesting that Spry regulates mammary development. To investigate the role of Spry in breast cancer we explored the consequence of Spry loss in vitro and in vivo. We found that Spry deficient cells possess accelerated cell growth, enhanced cell survival, and increased tumorigenic potential. In addition these cells were hypersensitive to a number of growth factors including EGF, showing enhanced and prolonged activation of the MAPK and PI3K-AKT pathways. To examine the role of Spry loss in promoting mammary tumorigenesis in vivo, we crossed Spry1 mutant mice with mice in which we can induce HER2/neu or PIK3CA expression. We found that Spry1 loss cooperates with oncogenic activation of HER2/neu or PIK3CA to increase cell proliferation and accelerate mammary hyperplasia. Breast tissues from the Spry1-/- mice with oncogenic activation of HER2/neu exhibited a trend towards increased levels of activated Mapk and Akt compared to their Spry1+/+ counterparts. These Spry1-/- breast tissues also accumulated significantly more DNA damage. Finally, microarray gene expression analysis of these tissues identified that genes differentially expressed in Spry1-/- breasts are involved in cell cycle regulation and the DNA damage response. Collectively, this data suggests that Spry functions as a tumor suppressor in breast cancer opposing HER2/neu and PIK3CA by suppressing MAPK and AKT activation to prevent DNA damage. Our work demonstrates that the misregulation of critical signaling pathways, through loss of Spry, leads to increased cell growth, enhanced cell survival, and accelerated tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2165. doi:1538-7445.AM2012-2165

Abstract 1716: FGFR1 gene amplification predicts the antitumor response of FGFR inhibitor LY2874455 in human lung cancer cells

Abstract Lung cancer is the leading cause of cancer-related death for both men and women in the world. FGFR receptor tyrosine kinase signaling pathway has been implicated in tumor cell growth, survival, angiogenesis, and metastasis. Activation of the FGFR pathway via FGFR over-expression, and chromosomal amplification and translocation as well as genetic mutation contributes to the tumor progression and the resistance to anti-EGFR and -VEGF treatment in lung cancer. Targeting FGFR pathway is therefore an attractive therapeutic strategy for lung cancer. We have discovered and developed a novel, ATP competitive and orally active small molecule inhibitor of FGFR, LY2874455, currently in clinic development. LY2874455 is a potent inhibitor against four FGFRs, and demonstrates a broad-spectrum of anti-tumor activity in a number of tumor xenograft models. In the current study, we evaluated the anti-tumor activity of LY2874455 in lung tumor cells with or without FGFR1 gene amplification. FGFR1 is known to be amplified in human lung cancer cells NCI-H1581, NCI-H520 and DMS-114, but not in NCI-H460, A549 and NCI-H1299. The mRNA expression levels of FGFR1 are shown to be higher in the FGFR1 amplified cells than those in the FGFR1 non-amplified cells. Consistent with this, the FGFR1 amplified cells exhibited the much more robust b-FGF-induced phosphorylation of FRS2 and ERK, which are also more sensitive to the inhibition by LY2874455 than that in the FGFR1 non-amplified cells. Furthermore, the FGFR1 amplified cells exhibit the better responses to the growth inhibition and apoptosis induction by LY2874455 than those in the FGFR1 non-amplified cells. Therefore, these findings suggest that FGFR1 plays an important role in lung cancer cell growth and survival, and that LY2874455 may provide a promising treatment option for some lung cancer patients whose tumor harbors FGFR1 gene amplification. The in vivo anti-tumor response of LY2874455 is being further investigated in the tumor xenograft models derived from these FGFR1 amplified or non-amplified human lung cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1716. doi:1538-7445.AM2012-1716

Abstract SY15-01: Cellular context-specific tumor suppression by PTPN11

Cartilage tumors, accounting for 22% of skeletal system tumors, are characterized by the formation of exostoses, enchondroma(s) or both, and cause significant morbidity and mortality. Both benign and malignant cartilaginous tumors can develop at various ages, and can show autosomal dominant inheritance or occur sporadically. The molecular mechanism underlying the development and progression of these cartilaginous lesions remains incompletely understood. Shp2, encoded by the Ptpn11 gene, is one of two Src homology 2 domain-containing protein-tyrosine phosphatases, and is required for most, if not all, receptor tyrosine kinase (RTK), cytokine, and integrin signaling pathways. Global deletion of Shp2 in mice results in early embryonic lethality, whereas postnatal Shp2 deficiency in various tissues/cells has diverse effects on their development and function. Several human malignancies, most notably childhood myeloproliferative disorders, are associated with Ptpn11 gain-of-function (GOF) mutations. Several lines of evidence indicate that Shp2 plays an important role in skeletal development and homoeostasis; however, little is known about its role in vivo. Recently Ptpn11 truncated mutations (presumably protein null) are reported to cause human metachondromatosis, a benign cartilage tumor syndrome with malignant potentials. By taking a tissue-specific gene knockout approach, we report here that Shp2 loss-of-function mutation, in contrast to its GOF mutants in other tissues/cells, causes widespread and severe cartilaginous tumors, strongly suggesting that Shp2 has a tissue specific-tumor suppressor function. These studies also identify the target of cartilage malignancies caused by Shp2 deficiency as a novel cell population with stem/progenitor properties located within the groove of Ranvier. Materials and Methods: Mice carrying Ptpn11 floxed (fl) or cathepsin K-Cre (Ctsk-Cre) were described previously. To generate Ctsk-expressing cell specific Shp2 deficient mice and study the role of Shp2 in these cells to regulate skeletal development and homeostasis, Ptpn11 floxed mice were bred to Ctsk-Cre mice to generate Ptpn11fl/fl;Ctsk-Cre (KO) and Ptpn11fl/+;Ctsk-Cre (Control) animals. To track the fate of Ctsk + cartilaginous cells in vivo in the presence or absence of Shp2, Roza26lacZ (R26lacZ) and Roza26EYFP (R26YFP) reporter alleles were bred to Control and KO mice to generate Control/R26YFP, KO/R26YFP, Control/R26lacZ and KO/R26lacZ compound mice, respectively. YFP-positive cartilaginous cells were isolated by serial enzymatic digestions of epiphyseal cartilage with hyaluronidase/Trypsin/ collagenase D and FACS sorting. Cell surface marker expression was determined by FACS analysis after staining with fluorescence-labeled antibodies. For histological analysis, skeletal tissues were removed from mice after euthanasia and fixed in 4% paraformaldehyde; after decalcification, embedding, and sectioning, they were stained with HELysMCre mcie, revealed that the cartilage phenotype in KO mice was not osteoclast-autonomous. Therefore to identify the cellular origin of the cartilage lesions in KO mice, we conducted a reporter study by using Ctsk-Cre mediated R26lacZ or YFP expression. Surprisingly, we found that the Ctsk promoter is active not only in mature osteoclasts, but also in a subset of cells that live in perichondrial groove of Ranvier. Deficiency of Shp2 in these cells causes unrestrained proliferation, chondrogenic differentiation, and exostoses/enchondroma(s) formation. These cells were found to share several mesenchymal/chondroprogenitor markers, such as CD44, CD90, Stro1 and jagged1. Biochemical analysis shows that Shp2 deficiency impaired Erk activation and caused elevated expression of Indian hedgehog (Ihh) and PTHrP in cartilage lesions. In summary, we specifically ablated Shp2 in Ctsk-expressing cells (primarily in mature osteoclasts and subsets of cartilaginous cells) in mice and found that Shp2, in addition to having a role in osteoclastogenesis in vitro and in vivo, negatively regulates the proliferation and chondrogenic differentiation of a unique population of ctsk+ perichondrial cartilaginous cells. Loss of Shp2 in these cells causes cell proliferation, chondrogenic differentiation, and cartilage tumorigenesis at the metaphyses of tubular bones. This phenotype is strikingly similar to the human disease metachondromatosis. Mechanistically, we believe that this pathogenic process is triggered by ectopic and/or elevated Ihh expression due to impaired Erk activation in Shp2- deficient perichondrial cells. If confirmed, these data identify hedgehog pathway inhibitors as potential therapeutic agents in metachromatosis patients. Our results, in concert with previous studies, show that, depending on the cellular context, Shp2 can act as an oncogene or a tumor suppressor gene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY15-01. doi:1538-7445.AM2012-SY15-01

Semaphorin 3A overcomes cancer hypoxia and metastatic dissemination induced by antiangiogenic treatment in mice

Cancer development, progression, and metastasis are highly dependent on angiogenesis. The use of antiangiogenic drugs has been proposed as a novel strategy to interfere with tumor growth, but cancer cells respond by developing strategies to escape these treatments. In particular, animal models show that antiangiogenic drugs currently used in clinical settings reduce tumor tissue oxygenation and trigger molecular events that foster cancer resistance to therapy. Here, we show that semaphorin 3A (Sema3A) expression overcomes the proinvasive and prometastatic resistance observed upon angiogenesis reduction by the small-molecule tyrosine inhibitor sunitinib in both pancreatic neuroendocrine tumors (PNETs) in RIP-Tag2 mice and cervical carcinomas in HPV16/E2 mice. By improving cancer tissue oxygenation and extending the normalization window, Sema3A counteracted sunitinib-induced activation of HIF-1α, Met tyrosine kinase receptor, epithelial-mesenchymal transition (EMT), and other hypoxia-dependent signaling pathways. Sema3A also reduced tumor hypoxia and halted cancer dissemination induced by DC101, a specific inhibitor of the VEGF pathway. As a result, reexpressing Sema3A in cancer cells converts metastatic PNETs and cervical carcinomas into benign lesions. We therefore suggest that this strategy could be developed to safely harnesses the therapeutic potential of the antiangiogenic treatment.

Novel regulation of PDGFRα activation in Glioblastoma

Glioblastoma (GBM) is characterized by abnormal activation of receptor tyrosine kinase (RTK) signaling pathways. We demonstrate that the extracellular sulfatase, SULF2, an enzyme that regulates multiple heparan sulfate proteoglycan‐dependent RTK signaling pathways, is a novel regulator of RTK‐signaling in GBM. In primary human GBM SULF2 protein is expressed in 29/57 (50%) tumors and using data from The Cancer Genome Atlas SULF2 transcript is increased in 197/424 (46%) tumors (http://cancergenome.nih.gov). To determine the functional role of SULF2 in GBM we knocked down SULF2 in GBM cell lines and generated murine malignant astrocytomas from Sulf2−/− tumorigenic neurospheres. These data demonstrated a striking SULF2‐dependence in activity of PDGFRα, a major signaling pathway in GBM. Ablation of SULF2 resulted in decreased PDGFRα phosphorylation and decreased downstream MAPK signaling activity. In addition, in vivo tumor growth was decreased. Interestingly, the proneural GBM subtype, characterized by aberrations in PDGFRα, demonstrated the strongest SULF2 expression. SULF2 may help identify a subset of GBMs that may be more dependent on exogenous growth factor‐mediated signaling.This work was supported by funds from the National Institutes of Health (K08 NS063456 to J.J.P.) and the Preuss Foundation.

Interference with platelet‐derived growth factor‐induced activation of receptor tyrosine kinase and downstream signaling pathways by compound C

Although AMP‐activated protein kinase (AMPK) activation has been implicated in anti‐proliferative actions in various cell systems, we have observed that compound C, an inhibitor of AMPK, reduced the cell viability of human diploid fibroblasts (HDFs). Flow cytometry revealed blocking of cell cycle progression by compound C. Preincubation of HDFs with compound C abrogated PDGF‐induced Akt phosphorylation. Compound C did not affect Akt activity in vitro. Compound C completely inhibited PDGF‐induced phosphorylation of PDGF receptor‐β, which thereby could block downstream PDGF‐induced PI3K, Akt, and ERK1/2. Compound C did not affect EGF‐ and IGF‐induced receptor tyrosine phosphorylation and downstream signaling. In vitro receptor tyrosine kinase activity profiling revealed that IC50 for PDGFR was 5.07 μM for PDGFRβ, whereas IC50 for EGFR and IGF1R was over 100 μM. The inhibitory effect of compound C on PDGF receptor‐β and Akt phosphorylation was also observed in AMPKα1/α2‐knockout MEF cells, indicating that the inhibitory effect of compound C is independent of regulating the AMPK activity. These results suggest that the inhibitory effect of compound C on the PDGF‐BB‐induced proliferation of HDFs is mediated by blocking tyrosine kinase activity of PDGFRβ and downstream signaling pathways, which is independent of regulating AMPK activity [Research support: KRF # 2010‐000‐ 4564 and # 2011‐000‐5951].

The Capicua repressor – a general sensor of RTK signaling in development and disease

Receptor tyrosine kinase (RTK) signaling pathways control multiple cellular decisions in metazoans, often by regulating the expression of downstream genes. In Drosophila melanogaster and other systems, E-twenty-six (ETS) transcription factors are considered to be the predominant nuclear effectors of RTK pathways. Here, we highlight recent progress in identifying the HMG-box protein Capicua (CIC) as a key sensor of RTK signaling in both Drosophila and mammals. Several studies have shown that CIC functions as a repressor of RTK-responsive genes, keeping them silent in the absence of signaling. Following the activation of RTK signaling, CIC repression is relieved, and this allows the expression of the targeted gene in response to local or ubiquitous activators. This regulatory switch is essential for several RTK responses in Drosophila, from the determination of cell fate to cell proliferation. Furthermore, increasing evidence supports the notion that this mechanism is conserved in mammals, where CIC has been implicated in cancer and neurodegeneration. In addition to summarizing our current knowledge on CIC, we also discuss the implications of these findings for our understanding of RTK signaling specificity in different biological processes.

The role of the small GTPase Rap in Drosophila R7 photoreceptor specification

The Drosophila R7 photoreceptor provides an excellent model system with which to study how cells receive and "decode" signals that specify cell fate. R7 is specified by the combined actions of the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways. These pathways interact in a complex manner that includes antagonistic effects on photoreceptor specification: RTK promotes the photoreceptor fate, whereas N inhibits. Although other photoreceptors are subject to only mild N activation, R7 experiences a high-level N signal. To counter this effect and to ensure that the cell is specified as a photoreceptor, a high RTK signal is transduced in the cell. Thus, there are two levels of RTK transduction in the photoreceptors: in R7 it is high, whereas in others it is low. Here, we address how this high-level RTK signal is transduced in R7 and find that, in addition to Ras, another small GTPase, Rap, is also engaged. Thus, when N activity is high, a robust RTK signal operates that uses both Ras and Rap, but when N activity is low, only a mild RTK signal is transduced and Ras alone suffices for the purpose.

Spry1 and Spry2 Are Essential for Development of the Temporomandibular Joint

The temporomandibular joint (TMJ) is a specialized synovial joint essential for the function of the mammalian jaw. The main components of the TMJ are the mandibular condyle, the glenoid fossa of the temporal bone, and a fibrocartilagenous disc interposed between them. The genetic program for the development of the TMJ remains poorly understood. Here we show the crucial role of sprouty (Spry) genes in TMJ development. Sprouty genes encode intracellular inhibitors of receptor tyrosine kinase (RTK) signaling pathways, including those triggered by fibroblast growth factors (Fgfs). Using in situ hybridization, we show that Spry1 and Spry2 are highly expressed in muscles attached to the TMJ, including the lateral pterygoid and temporalis muscles. The combined inactivation of Spry1 and Spry2 results in overgrowth of these muscles, which disrupts normal development of the glenoid fossa. Remarkably, condyle and disc formation are not affected in these mutants, demonstrating that the glenoid fossa is not required for development of these structures. Our findings demonstrate the importance of regulated RTK signaling during TMJ development and suggest multiple skeletal origins for the fossa. Notably, our work provides the evidence that the TMJ condyle and disc develop independently of the mandibular fossa.

Heparan sulfate sulfatase SULF2 regulates PDGFRα signaling and growth in human and mouse malignant glioma

Glioblastoma (GBM), a uniformly lethal brain cancer, is characterized by diffuse invasion and abnormal activation of multiple receptor tyrosine kinase (RTK) signaling pathways, presenting a major challenge to effective therapy. The activation of many RTK pathways is regulated by extracellular heparan sulfate proteoglycans (HSPG), suggesting these molecules may be effective targets in the tumor microenvironment. In this study, we demonstrated that the extracellular sulfatase, SULF2, an enzyme that regulates multiple HSPG-dependent RTK signaling pathways, was expressed in primary human GBM tumors and cell lines. Knockdown of SULF2 in human GBM cell lines and generation of gliomas from Sulf2(-/-) tumorigenic neurospheres resulted in decreased growth in vivo in mice. We found a striking SULF2 dependence in activity of PDGFRα, a major signaling pathway in GBM. Ablation of SULF2 resulted in decreased PDGFRα phosphorylation and decreased downstream MAPK signaling activity. Interestingly, in a survey of SULF2 levels in different subtypes of GBM, the proneural subtype, characterized by aberrations in PDGFRα, demonstrated the strongest SULF2 expression. Therefore, in addition to its potential as an upstream target for therapy of GBM, SULF2 may help identify a subset of GBMs that are more dependent on exogenous growth factor-mediated signaling. Our results suggest the bioavailability of growth factors from the microenvironment is a significant contributor to tumor growth in a major subset of human GBM.

Initial report on differential expression of sprouty proteins 1 and 2 in human epithelial ovarian cancer cell lines.

Sprouty (Spry) proteins, modulators of receptor tyrosine kinase signaling pathways, have been shown to be deregulated in a variety of pathological conditions including cancer. In the present study we investigated the expression of Spry1 and Spry2 isoforms in a panel of human ovarian cancer cell lines in vitro. Our western blot analysis showed nonuniform patterns of Spry expression in the cancer cells, none of which conformed to the pattern observed in the normal ovarian epithelial cells employed as the control. Among the seven cancer cell lines studied, Spry1 was expressed lower in four cell lines and higher in one as compared with the control. As for Spry2, four cell lines showed lower and two exhibited higher expression. Results from RT-PCR assay raised the possibility that Spry protein levels may not necessarily correspond with its expression at mRNA level. Our immunostaining study revealed that Spry2 was predominantly distributed within the whole cytoplasm in vesicular structures whereas Spry1 was found in both the cytoplasm and nucleus. This might provide clues to further investigation of Spry mode of action and/or function. Collectively, our study unveiled the differential expression of Spry1 and Spry2 proteins in various ovarian cancer cell lines.

Transcriptional regulation of Profilin during wound closure in Drosophila larvae

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. Here, we show that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. We found that JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, we show that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. Our results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized.

The eye of Drosophila as a model system for studying intracellular signaling in ontogenesis and pathogenesis

Many human diseases are caused by malfunction of basic types of cellular activity such as proliferation, differentiation, apoptosis, cell polarization, and migration. In turn, these processes are associated with different routes of intracellular signal transduction. A number of model systems have been designed to study normal and abnormal cellular and molecular processes associated with pathogenesis. The developing eye of the fruit fly Drosophila melanogaster is one of these systems. The sequential development of compound eyes of this insect makes it possible to model human neurodegenerative diseases and mechanisms of carcinogenesis. In this paper we overview the program of the eye development in Drosophila, with emphasis on intracellular signaling pathways that regulate this complex process. We discuss in detail the roles of the Notch, Hedgehog, TGFβ, Wnt, and receptor tyrosine kinase signaling pathways in Drosophila eye development and human pathology. We also briefly describe the modern methods of experimentation with this model organism to analyze the function of human pathogenic proteins.

Primary cilia and coordination of receptor tyrosine kinase (RTK) signalling.

Primary cilia are microtubule-based sensory organelles that coordinate signalling pathways in cell-cycle control, migration, differentiation and other cellular processes critical during development and for tissue homeostasis. Accordingly, defects in assembly or function of primary cilia lead to a plethora of developmental disorders and pathological conditions now known as ciliopathies. In this review, we summarize the current status of the role of primary cilia in coordinating receptor tyrosine kinase (RTK) signalling pathways. Further, we present potential mechanisms of signalling crosstalk and networking in the primary cilium and discuss how defects in ciliary RTK signalling are linked to human diseases and disorders.

Biological mechanisms and clinical implications of endocrine resistance in breast cancer

Endocrine therapy represents the first and most efficacious targeted treatment for women with estrogen receptor-positive (ER+) breast cancer. In the last four decades several hormonal agents have been successfully introduced in clinical practice as both palliative therapy for advanced disease and adjuvant treatment for prevention of tumor relapse. Nevertheless, the intrinsic and acquired resistance occurs in a significant proportion of patients, limiting the efficacy of endocrine treatments. Several molecular mechanisms have been proposed to be responsible for endocrine resistance. Loss of ER expression, altered activity of ER coregulators, deregulation of apoptosis and cell cycle signaling, and hyperactive receptor tyrosine kinase (RTK) and stress/cell kinase pathways can collectively orchestrate the development and sustenance of pharmacologic resistance to endocrine therapy. Preclinical and clinical evidence documents the plasticity in ER expression levels and signaling. As such, ER can either drive gene transcription and tumor progression directly or crosstalk with alternate RTK and cellular kinase signaling pathways, resulting in modulation of its own expression levels and transcriptional program. For this reason a therapeutic approach based on the combination of agents targeting both ER and RTK signaling represents a promising strategy to be tested. Among many RTKs, EGFR, HER2, and PI3K have been found to be viable targets for the combination therapy strategy, at least in the preclinical setting. However, early results from clinical trials testing combination strategies have been discordant, suggesting the need for better approaches to simultaneously inhibit multiple escape pathways and to select the patients who may benefit the most from these strategies.