Role Of Receptor Tyrosine Kinases Research Articles

Ectopic repression of receptor tyrosine kinase-like orphan receptor 2 inhibits malignant transformation of ovarian cancer cells by reversing epithelial-mesenchymal transition.

Receptor tyrosine kinase-like orphan receptor 2 is an enzyme-linked receptor which specifically modulates WNT5A signaling and plays an important role in tumorigenesis, invasion, and metastasis; however, the precise role of receptor tyrosine kinase-like orphan receptor 2 in cancer is controversial. The purpose of this study was to investigate the expression and role of receptor tyrosine kinase-like orphan receptor 2 in ovarian carcinoma and clarify the biological functions and interactions of receptor tyrosine kinase-like orphan receptor 2 with non-canonical Wnt pathways in ovarian cancer. The result of the human ovary tissue microarray revealed that the receptor tyrosine kinase-like orphan receptor 2-positive rate increased in malignant epithelial ovarian cancers and was extremely higher in the metastatic tumor tissues, which was also higher than that in the malignant ovarian tumor tissues. In addition, high expression of receptor tyrosine kinase-like orphan receptor 2 was closely related with ovarian cancer grading. The expression of receptor tyrosine kinase-like orphan receptor 2 protein was higher in SKOV3 and A2780 cells than OVCAR3 and 3AO cells. Knockdown of receptor tyrosine kinase-like orphan receptor 2 inhibited ovarian cancer cell proliferation, migration, invasion, and induced morphologic as well as digestive state alterations in stably transfected SKOV3 cells. Detailed study further revealed that silencing of receptor tyrosine kinase-like orphan receptor 2 reversed the epithelial-mesenchymal transition and inhibited non-canonical Wnt signaling. Our findings suggest that receptor tyrosine kinase-like orphan receptor 2 may be an important regulator of epithelial-mesenchymal transition, primarily regulated the non-canonical Wnt signaling pathway in ovarian cancer cells, and may display a promising therapeutic target for ovarian cancer.

The role of HER2, EGFR, and other receptor tyrosine kinases in breast cancer

Breast cancer affects approximately 1 in 8 women, and it is estimated that over 246,660 women in the USA will be diagnosed with breast cancer in 2016. Breast cancer mortality has decline over the last two decades due to early detection and improved treatment. Over the last few years, there is mounting evidence to demonstrate the prominent role of receptor tyrosine kinases (RTKs) in tumor initiation and progression, and targeted therapies against the RTKs have been developed, evaluated in clinical trials, and approved for many cancer types, including breast cancer. However, not all breast cancers are the same as evidenced by the multiple subtypes of the disease, with some more aggressive than others, showing differential treatment response to different types of drugs. Moreover, in addition to canonical signaling from the cell surface, many RTKs can be trafficked to various subcellular compartments, e.g., the multivesicular body and nucleus, where they carry out critical cellular functions, such as cell proliferation, DNA replication and repair, and therapeutic resistance. In this review, we provide a brief summary on the role of a selected number of RTKs in breast cancer and describe some mechanisms of resistanceto targeted therapies.

4P IGF-IR, but not EGFR, regulates DNA damage response in HeLa cells following irradiation

Legal entity responsible for the study The roles of receptor tyrosine kinases in DNA damage response (DDR) are still largely unknown. In this study, we examined the possible involvement of insulin-like growth factor I receptor (IGF-IR) and epidermal growth factor receptor (EGFR) in the DNA damage response (DDR) following irradiation. Methods HeLa cells expressing the fluorescence ubiquitination-based cell cycle indicator (Fucci) probes (HeLa-Fucci cells) were used in this study. Kinetics of the Fucci fluorescence were detected by FACS and time-lapse imaging. NVP-AEW541, Tyrphostin AG1478, LY294002, PD98059, NU7026 were used as specific inhibitors for IGF-IR, EGFR, PI3-K, MEK, and DNA-PKcs, respectively. Phosphorylation of ERK1/2 and Akt was detected by western blotting. Double strand breaks (DSBs) were detected by immunofluorescence staining for 53BP1. Cells were irradiated using an RX-650 Cabinet X-radiator system (Faxitron). Results To investigate the possible involvement of EGFR and IGF-IR in G2 arrest, FACS analysis and time-lapse imaging of Fucci fluorescence were performed using specific inhibitors. Results showed that inhibition of IGF-IR, but not that of EGFR, prolonged G2 arrest, which was irrespective of cell cycle phases at irradiation, i.e., red (G1 phase) or green (S/G2 phases) phase in the Fucci system. Similarly, only inhibition of IGF-IR decreased the DSB repair activity. Hereafter, further analysis was focused on IGF-IR-associated events. We next attempted to identify the responsible IGF-IR- downstream signaling pathways. Irradiation phosphorylated both Akt and ERK; however, inhibition of IGF-IR abrogated activation of only Akt. Moreover, inhibition of PI3-K/Akt, but not that of MEK/ERK, prolonged G2 arrest, which mimicked that of IGF-IR. Inhibition of DNA-PKcs, a major factor of non-homologous end joining (NHEJ), also prolonged G2 arrest. Conclusions We conclude that irradiation is likely to preferentially activate the IGF- IR/PI3-K/Akt signaling pathway in HeLa-Fucci cells, which may enhance DSB repair, eventually contributing to reduction of G2 arrest in the DDR following irradiation. Legal entity responsible for the study Tokyo Medical and Dental University Funding Tokyo Medical and Dental University, MEXT Japan Disclosure All authors have declared no conflicts of interest.

Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-small cell lung cancer

Aberrant increased expression and activation of receptor tyrosine kinases occur frequently in human carcinomas. Several small molecules targeting receptor tyrosine kinases, which have crucial roles in the growth factor signaling that promote tumor progression in various malignancies, including non-small cell lung cancer (NSCLC), are currently in clinical development. Therapeutic strategies include inhibition of growth factor tyrosine kinase function. Drugs of this type include those that target the epidermal growth factor receptor tyrosine kinase, those that target vascular endothelial growth factor receptors tyrosine kinase and those that target anaplastic lymphoma receptor tyrosine kinase. In this review we first discuss the role of receptor tyrosine kinases in human malignancies, and focus on discussing the potential use of epidermal growth factor receptor tyrosine kinase inhibitors and the vascular endothelial growth factor receptors tyrosine kinase inhibitors in NSCLC. In addition, we discuss the contribution of growth factor receptor tyrosine kinase inhibitors to the clinically observed resistance, and toxicity.

531 KIT Signaling Promotes Tumor Growth in a Subset of Human Colon Cancers

Background & Aims: The role of receptor tyrosine kinases (RTKs) in the regulation of colon cancer growth is unclear even though an RTK inhibitor has been approved for metastatic colon cancer. Previously, we found that the RTK KIT (CD117) marks a subset of crypt base goblet cells that contributes to the stem cell niche in normal murine colon. In human colon, KIT expression has been reported in colon cancer cell lines. We sought to assess the role of KIT in human colon cancers and hypothesized that KIT promotes colon cancer growth. Methods: We performed immunohistochemistry on an array of 316 colon tumor sections to assess the expression of KIT and its ligand, KITLG, in human colon cancers. We generated stable, lentivirally-transduced shRNA knockdowns of KIT and KITLG in colon cancer cell lines and xenografts, and conducted bulk and single-cell gene expression analysis, immunostaining, flow cytometry, and functional assays to assess the roles of KIT in tumor growth in-vitro and in-vivo. We treated KIT+ colon cancers with the KIT inhibitor imatinib and tracked in vivo growth using bioluminescence. We assessed differences in clonogenicity and tumorigenicity using limiting dilution analysis, and queried associated gene expression changes using quantitative RT-PCR. Results: KIT and KITLG are heterogeneously expressed by >50% of our tumor sections. KIT and KITLG immunoreactivity was detected in tumor cells, tumor stroma, or both. Stable lentiviral knockdown of KIT decreased tumor growth in vivo by >60%, increased expression of enterocyte markers (e.g. KRT20, CEACAM1), decreased expression of cycling genes (e.g. MKI67, TOP2A), and unexpectedly increased expression of some stem cell-associated genes (e.g. LGR5, CD44). Interestingly, no activating KIT mutations were detected in the KIT+ cell lines and xenografts, suggesting that KIT signaling occurs through its ligand KITLG. Knockdown of KITLG decreased tumor growth, and single-cell gene expression analysis of tumorigenic (CD44+) cells suggested that KIT may promote growth via KITLG autocrine and/or paracrine signaling. The RTK inhibitor imatinib also inhibited the growth of KIT+ colon cancer xenografts in vitro and in vivo. Finally, KIT-expression enriched for tumorigenic cells. Conclusions: KIT signaling in tumorigenic cells promotes growth of a subset of colon cancers through its ligand KITLG in an autocrine or paracrine manner. KIT+ tumor cells in the tumorigenic CD44+ compartment may be more tumorigenic than KITcells, although both populations can generate tumors. These data suggest that patients with KIT-expressing colon tumors may benefit from KIT inhibition by RTK inhibitors that target KIT.

The UPs and DOWNs of MITF in melanoma resistance.

The identification of mutations in the B-Raf proto-oncogene, serine–threoninekinase (BRAF V600E or V600K) as themost common mutation in melanomahas propelled efforts to develop thera-pies to inhibit BRAF kinase activity.While currently available BRAF inhibi-tors (BRAFi) offer unprecedentedimprovement of patient survival andquality of life, those changes are gener-ally transient due to the high occurrenceof BRAFi-resistant tumorsThe quest to understand mechanismsunderlying BRAFi resistance in mela-noma has prompted a large campaigndriven by both clinical and researchcommunities, which has led to identifi-cation of a common denominator under-lying resistance, namely, reactivation ofmitogen-activated protein kinase(MAPK) signaling by diverse mecha-nisms, including mutations in NRAS orMEK, BRAF splicing anomalies, or over-expression of tyrosine kinase receptors,including PDGFR or EGFR. These find-ings have led to clinical trials employingcombinations of MEK inhibitors (MEKi)and BRAFi; these approaches representan improvement over monotherapy butdo not significantly sustain survival, asresistant tumors eventually arise (Flah-erty et al., 2012; Morris et al., 2013).Thus, identification of additional mecha-nisms underlying resistance to MAPKpathway inhibitors is important toenable creation of more effective thera-pies.A recent paper from Dr. Peeper’sgroup reports a novel approach to iden-tify mechanisms conferring resistanceto MAPK inhibitors. In this study, inser-tional mutagenesis was employed toselect for genes whose elevatedexpression conferred resistance to theERK inhibitor (ERKi; SCH772984).Among key hits identified in the screenwas the master regulator of melaninbiogenesis, microphthalmia-associatedtranscription factor (MITF). This findingis consistent with an earlier report iden-tifying MITF overexpression as a mech-anism underlying resistance to MAPKpathway inhibition (Johannessen et al.,2013).Given the link between MITF overex-pression and MAPK resistance, Peeperand his colleagues next assessed MITFfunction under acquired resistance con-ditions in cultured melanoma cells sub-jected to BRAFi. Surprisingly, greaterthan half of the established BRAFi-resis-tant melanoma cell lines had lost MITFexpression, in contrast to upregulatedMITF expression seen in response toacute treatment with MAPKi seen in theremainder. The seemingly contradictoryfinding that both high and low expressionof MITF (MITF hi or MITF lo, hereafter)are associated with resistance pheno-types was confirmed in a cohort ofBRAFi-resistant tumor samples takenfrom patients, who were evaluated priorto and following treatment. Some sam-ples exhibited increased MITF expres-sion following treatment, while othersshowed MITF decreases. Furthermore,assessment of individual clones derivedfrom one patient revealed that someclones exhibited MITF upregulation,while others showed downregulation(Muller et al., 2014).How can one explain such variation inMITF expression and how could thisimpact on resistance mechanisms?Among plausible answers is phenotypicswitching, a dynamic change in a geneexpression signature previously reportedin melanoma and dependent on MITFexpression. Interestingly, in melanomacells, ‘MITF hi’ is associated with prolif-eration, whereas ‘MITF lo’ cells showincreased invasion and chemoresistance(Hoek and Goding, 2010; Saez-Ayalaet al., 2013). Correspondingly, as shownby Dr. Peeper’s laboratory, acquiredBRAFi-resistant melanoma cells downre-gulating MITF expression exhibitincreased invasiveness with correspond-ing increases in expression of markers ofthe epithelial-to-mesenchymal transition(EMT), in agreement with increased inva-siveness observed in melanoma cellsresistant to MAPK inhibition and withchemoresistance exhibited by stem-likecells (Vultur et al., 2014). Interestingly,MITF lo cells also show resistance to abroader spectrum of MAPK inhibitors orcombinations including those drugs, asreported recently by the Garraway labo-ratory (Konieczkowski, et al., 2014).Both the Pee-per and the Garr-away studiesnote that geneswhose expres-sion increases inMITF lo cells areassociated withcell survival, sug-gesting a mecha-nism underlyingMAPKi resis-tance. Dr. Garr-away’s groupidentified NF-kB as a key player promot-ing resistance in the MITF lo cells.Intriguingly, both studies also highlightthe importance of elevated expressionof receptor tyrosine kinases (RTKs)including AXL, EGFR, and PDGFR inMITF lo, in innate and acquired BRAFi-resistant cells, and both note AXL inparticular as a key RTK that confersresistance. It is noteworthy that AXL,as well as other RTKs, was upregulatedin melanomas that exhibit either intrin-sic or acquired resistance phenotypes,noting their possible negative regulationby MITF. The role of RTKs in resistancephenotypes was confirmed by eradica-tion of cells from intrinsically resistantcultures following treatment with acombination of drugs inhibiting AXL,PDGFR, and EGFR (Muller et al., 2014).Both studies also found that in acquiredresistance, AXL inhibition combinedwith BRAFi treatment more effectivelyimpaired viability of BRAFi-resistant cul-tures than did treatment with BRAFialone. Importantly, an inverse correla-tion between MITF and AXL expressionwas also seen in NRAS mutant melano-mas (Muller et al., 2014). Correspond-ingly, as shown by the Peeperlaboratory, combination treatment withinhibitors of AXL and ERK eliminatedgrowth of intrinsically MAPKi-resistantNRAS mutant melanoma cultures.Coverage on: Muller et al., (2014) LowMITF/AXL ratio predicts early resistanceto multiple targeted drugs in melanoma,Nature Communications 5, 5712, doi:10.1038/ncomms6712.Konieczkowski, et al. (2014). A mela-noma cell state distinction influencessensitivity to MAPK pathway inhibitors.Cancer Discovery 4, 816-27.

Abstract 5355: Twist1 regulates the cell cycle and proliferative capacity of keratinocytes during tumor promotion

Abstract A number of studies have established the role of receptor tyrosine kinases (RTKs) and their downstream signaling targets, including PI3K/Akt/mTOR and Stat3, as important regulatory factors of epidermal proliferative capacity during both chemical (two-stage) and UV skin carcinogenesis. We have previously shown that total protein levels of Twist1 can be regulated by Stat3 in the two-stage skin carcinogenesis model and that nuclear localization of Twist1 was restricted to the proliferative compartment. Twist1, a protein commonly associated with the induction of EMT, has previously been shown to associate with cell cycle effectors, such as p53 and p21, as well as impact cell cycle progression. The cell cycle regulatory functions of Twist1 have been linked to p53 via phosphorylation of Twist1 at Ser42 by Akt. To investigate the role of Twist1 in cellular proliferation, primary keratinocytes were infected with lentiviral particles to create Twist1 knockdown or overexpressing cells, and were used, together with wild-type primary keratinocytes, to analyze the role of Twist1 during cell cycle progression. Western blot analysis of lysates from primary keratinocytes indicated that the expression of cell cycle regulatory proteins Cyclin E1, NF-κB, and c-Myc, coincided with the decrease or increase of Twist1. Protein levels of p21 exhibited an inverse correlation to Twist1 expression. Additionally, ChIP analysis identified Cyclin D1, Cyclin E1, c-Myc, and NF-κB as novel transcriptional targets of Twist1. To further investigate the role of Twist1, we have established transgenic mice with deletion of Twist1 in the basal compartment of the epidermis using BK5.Cre (i.e. BK5.Cre x Twistflox/flox mice). BK5.Cre x Twistflox/flox mice exhibited a significant reduction in epidermal thickness and labeling index (LI) compared to wild-type mice following treatment with TPA. In comparison to wild-type mice, acetone treated Bk5.Cre x Twistflox/flox mice also displayed a significant reduction of epidermal LI. Western blot analysis of epidermal lysates collected from BK5.Cre x Twistflox/flox mice treated with TPA demonstrated a reduction of Cyclin E1 and reduced phosphorylation of Rb at Ser795 compared to wild-type mice, further implicating the regulatory capacity of Twist1 during keratinocyte proliferation. Twist1 Ser42 phosphorylation also correlated with expression of the aforementioned cell cycle proteins. Collectively, these results, combined with other data to be presented, suggest that Twist1 plays a key role in keratinocyte proliferation during skin tumor promotion. Research supported by CA76520 and T32 ES007247. Citation Format: Jaya Srivastava, Everardo Macias, Kaoru Kiguchi, John DiGiovanni. Twist1 regulates the cell cycle and proliferative capacity of keratinocytes during tumor promotion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5355. doi:10.1158/1538-7445.AM2014-5355

Genetic dissection of tie pathway in mouse lymphatic maturation and valve development.

The genetic program underlying lymphatic development is still incompletely understood. This study aims to dissect the role of receptor tyrosine kinase with immunoglobulin-like and EGF (epidermal growth factor)-like domains 1 (Tie1) and Tie2 in lymphatic formation using genetically modified mouse models. We generated conditional knockout mouse models targeting Tie1, Tie2, and angiopoietin-2 in this study. Tie1(ΔICD/ΔICD) mice, with its intracellular domain targeted, appeared normal at E10.5 but displayed subcutaneous edema by E13.5. Lymph sac formation occurred in Tie1(ΔICD/ΔICD) mice, but they had defects with the remodeling of primary lymphatic network to form collecting vessels and valvulogenesis. Consistently, induced deletion of Tie1-ICD postnatally using a ubiquitous Cre deleter led to abnormal lymphangiogenesis and valve formation in Tie1-ICD(iUCKO/-) mice. In comparison with the lymphatic phenotype of Tie1 mutants, we found that the diameter of lymphatic capillaries was significantly less in mice deficient of angiopoietin-2, besides the disruption of collecting lymphatic vessel formation as previously reported. There was also no lymphedema observed in Ang2(-/-) mice during embryonic development, which differs from that of Tie1(ΔICD/ΔICD) mice. We further investigated whether Tie1 exerted its function via Tie2 during lymphatic development. To our surprise, genetic deletion of Tie2 (Tie2(iUCKO/-)) in neonate mice did not affect lymphatic vessel growth and maturation. In contrast to the important role of Tie2 in the regulation of blood vascular development, Tie1 is crucial in the process of lymphatic remodeling and maturation, which is independent of Tie2.

Laminin α1 Regulates Age-Related Mesangial Cell Proliferation and Mesangial Matrix Accumulation through the TGF-β Pathway

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-β1-induced Smad2 phosphorylation, and inhibitors of TGF-β1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-β1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-β/Smad signaling.

Role of Receptor Tyrosine Kinases and Their Ligands in Glioblastoma

Glioblastoma multiforme is the most frequent, aggressive and fatal type of brain tumor. Glioblastomas are characterized by their infiltrating nature, high proliferation rate and resistance to chemotherapy and radiation. Recently, oncologic therapy experienced a rapid evolution towards “targeted therapy,” which is the employment of drugs directed against particular targets that play essential roles in proliferation, survival and invasiveness of cancer cells. A number of molecules involved in signal transduction pathways are used as molecular targets for the treatment of various tumors. In fact, inhibitors of these molecules have already entered the clinic or are undergoing clinical trials. Cellular receptors are clear examples of such targets and in the case of glioblastoma multiforme, some of these receptors and their ligands have become relevant. In this review, the importance of glioblastoma multiforme in signaling pathways initiated by extracellular tyrosine kinase receptors such as EGFR, PDGFR and IGF-1R will be discussed. We will describe their ligands, family members, structure, activation mechanism, downstream molecules, as well as the interaction among these pathways. Lastly, we will provide an up-to-date review of the current targeted therapies in cancer, in particular glioblastoma that employ inhibitors of these pathways and their benefits.

Semaphorin receptors meet receptor tyrosine kinases on the way of tumor progression

Semaphorins are extracellular signals known to guide migrating cells during developmental morphogenesis and in adult tissues. Semaphorin receptors, that is plexins and neuropilins, have been found in association with diverse receptor tyrosine kinases (RTKs), such as Met, ErbB2 and VEGFR2. These receptor complexes are formed in a cell-specific manner and can mediate distinctive signalling cascades, sometimes leading to divergent functional outcomes. This is particularly intriguing in cancer, since the same semaphorin has been found to mediate either tumor-promoting or tumor-suppressing functions, depending on the cancer type and cellular context. We will therefore review the current understanding about the role of RTKs in neuropilin and plexin signalling, putatively accounting for the multifaceted role of semaphorins in cancer.

Tyrosine Kinase Inhibitors in Pulmonary Arterial Hypertension: A Double-Edge Sword?

New treatments for pulmonary arterial hypertension (PAH) are a crucial need. The increased proliferation, migration, and survival of pulmonary vascular cells within the pulmonary artery wall in PAH have allowed successful transposition of pathophysiological elements from oncologic researches. Next steps will require translation of these biological advances in PAH therapeutic arsenal and guidelines. This review synthesizes recent data concerning the role of receptor tyrosine kinases and their inhibitors in PAH, with implications in animal models and humans. Results of clinical trials are now accumulating to establish beneficial role of tyrosine kinase inhibitors (TKIs) in PAH and further findings are expected in the near future. Beside this curative approach, evidences of a possible TKI-induced cardiotoxicity are emerging. These safety issues raise concern about a potential amplified harmful effect in PAH, a pathology characterized by an underlying cardiac dysfunction. In addition, analyses of PAH registries shed light on a selective pulmonary vascular toxicity triggered by TKIs, especially dasatinib. These possible dual effects of the TKIs in PAH need to be taken in account for future pharmacological development of this therapeutic class in PAH.

From an orphan disease to a generalized molecular mechanism: PTPN11 loss-of-function mutations in the pathogenesis of metachondromatosis.

Recently, loss-of-function mutations in PTPN11 were linked to the cartilage tumor syndrome metachondromatosis (MC), a rare inherited disorder featuring osteochondromas, endochondromas and skeletal deformation. However, the underlying molecular and cellular mechanism for MC remained incompletely understood. By studying the role of the Src homology-2 domain-containing protein tyrosine phosphatase Shp2 (encoded by mouse Ptpn11) in cathepsin K-expressing cells, we identified a novel cell population in the perichondrial groove of Ranvier. In the absence of Shp2, these cells exhibit elevated Indian hedgehog (Ihh) signaling, proliferate excessively and cause ectopic cartilage formation and tumors. Our findings establish a critical role for a protein-tyrosine phosphatase (PTP) family member, in addition to the well-known roles of receptor tyrosine kinases (RTKs), in cartilage development and homeostasis. However, whether Shp2 deficiency in other epiphyseal chondroid cells and whether signaling pathways in addition to the IHH/Parathyroid Hormone–related Peptide (PTHrP) axis attribute to the formation of enchondromas and osteochondromas remains elusive. Understanding how chondrogenic events are regulated by SHP2 could aid in the development of novel therapeutic approaches to prevent and treat cartilage diseases, such as MC and osteoarthritis (OA).

Evidence for a cell cycle checkpoint that senses branched actin in the lamellipodium

Recent evidence indicates that branched actin might control cell progression through G1 in addition to lamellipodium protrusion.

Abstract 1861: EPHB4 as a potential therapeutic target for esophageal cancer

Abstract Despite substantial improvements in screening, diagnosis, and treatment of esophageal cancer, the prognosis of this disease remains bleak. Survival at 5 years for all esophageal cancer patients taken together, with amenable treatments and with or without surgery ranges from 5-20% (Cancer Facts and Figures. American Cancer Society 2011). Although a multidisciplinary approach to include surgery, radiation, and chemotherapy, alone or in combination, attempts to improve the survival of this aggressive disease, these statistics underscore the continued need for attention to this disease and for identification of new targets in its treatment. We have been studying the role of receptor tyrosine kinases (RTKs) in cancer, particularly the EphB4 receptor, which has become increasingly associated with the pathobiology of adult cancers over the past several years. It has been shown to be aberrantly expressed and/or to play an oncogenic role in cancers of the breast, bladder, ovary, uterus, colon, head and neck, and prostate through its effects on cellular motility, growth, and migration, as well as on tumors’ ability to induce neoangiogenesis. To date, its role and potential as a target for therapy in different cancers have not been thoroughly investigated and remain poorly understood. Targeted inactivation of EphB4 and its ligand Ephrin-B2 have demonstrated that both are essential for angiogenic remodeling and embryonic survival (Adams and Klein 2000; Kim, Hu et al. 2008). In this report, we determine that EphB4 is overexpressed, has increased gene copy number and is involved in enhanced motility and migration in esophageal cancer. Archival patient samples consisting of 93 squamous cell carcinoma, 100 adenocarcinoma and 25 adjacent normal control samples as well as four adenocarcinoma and nine squamous cell carcinoma cell lines were used for the studies. Extensive mouse modeling of esophageal cancer was also used. We have reported that there is consistently higher expression of EphB4 in both squamous and adenocarcinoma compared to adjacent normal tissue with a statistically significant correlation between EphB4 expression and higher grades of squamous cell carcinoma. In a chemically induced esophageal squamous cell carcinoma model in mice, EphB4 was found overexpressed compared to normal controls. This study identifies EphB4 to be an important pathway in esophageal neoplastic lesions. It would now be useful to bring this to clinical fruition. 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 1861. doi:1538-7445.AM2012-1861

Correction: Molecular Imaging of TGFβ-Induced Smad2/3 Phosphorylation Reveals a Role for Receptor Tyrosine Kinases in Modulating TGFβ Signaling

Correction: Molecular Imaging of TGFβ-Induced Smad2/3 Phosphorylation Reveals a Role for Receptor Tyrosine Kinases in Modulating TGFβ Signaling

Molecular Imaging of TGFβ-Induced Smad2/3 Phosphorylation Reveals a Role for Receptor Tyrosine Kinases in Modulating TGFβ Signaling

The dual modality of TGFβ, both as a potent tumor suppressor and a stimulator of tumor progression, invasion, and metastasis, make it a critical target for therapeutic intervention in human cancers. The ability to carry out real-time, noninvasive imaging of TGFβ-activated Smad signaling in live cells and animal models would significantly improve our understanding of the regulation of this unique signaling cascade. To advance these efforts, we developed a highly sensitive molecular imaging tool that repetitively, noninvasively, and dynamically reports on TGFBR1 kinase activity. The bioluminescent TGFβR1 reporter construct was developed using a split firefly luciferase gene containing a functional sensor of Smad2 phosphorylation, wherein inhibition of TGFβ receptor1 kinase activity leads to an increase in reporter signaling. The reporter was stably transfected into mammalian cells and used to image in vivo and in vitro bioluminescent activity as a surrogate for monitoring TGFBR1 kinase activity. The reporter was successfully used to monitor direct and indirect inhibition of TGFβ-induced Smad2 and SMAD3 phosphorylation in live cells and tumor xenografts and adapted for high-throughput screening, to identify a role for receptor tyrosine kinase inhibitors as modulators of TGFβ signaling. The reporter is a dynamic, noninvasive imaging modality for monitoring TGFβ-induced Smad2 signaling in live cells and tumor xenografts. It has immense potential for identifying novel effectors of R-Smad phosphorylation, for validating drug-target interaction, and for studying TGFβ signaling in different metastasis models.

Adding to the toolbox: Receptor tyrosine kinases as potential targets in the treatment of hepatitis C

Adding to the toolbox: Receptor tyrosine kinases as potential targets in the treatment of hepatitis C

The Kaposi's sarcoma-associated herpesvirus E3 ubiquitin ligase K5 acts as a novel oncogene, altering cellular metabolism and signaling: implications for tumorigenesis

While it is clear that Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8) is the causative agent of a number of malignancies including multicentric Castleman’s disease, primary effusion lymphoma, and Kaposi’s sarcoma (KS), the molecular mechanisms of tumor induction by this virus are still unclear. In part, KS lesion presentation is thought to be driven primarily through paracrine mechanisms and is mainly observed in immunocompromised patients. Monocyte subsets, including dendritic cells and macrophages, are crucial to immune system functionality and are also skewed in KS patients. The goals of this study were to investigate a potential role for the E3 ubiquitin ligase K5 of KSHV, which plays a role in viral immune evasion, in altering monocyte functionality, thereby contributing to KSHV-driven tumorigenesis. A series of wild-type (WT) and mutant K5-expressing stable cell lines were generated in THP-1 monocytic cell line and examined. Surprisingly, these cells demonstrated a serum-dependent increased growth rate and a propensity to acidify the growth medium as compared to vector-THP-1 cells. Biochemical examination indicated that K5 induced aerobic glycolysis and other hallmarks of the “Warburg Effect,” including increased lactate production and glucose uptake. Observed increases in Akt and total cellular tyrosine phosphorylation, combined with the serum-dependence, suggested a role for receptor tyrosine kinases (RTKs). A human-RTK array demonstrated increased activation of the Flt-3, Axl, PDGFR-s, and Flt-4 receptors in K5-expressing versus vector cell lines. Subsequent testing demonstrated increased sensitivity of K5-expressing THP-1 cells to growth arrest and apoptosis by sunitinib and increased ligand-dependent signaling. Dynamin inhibitor studies showed that K5 can target these RTKs from the surface to increase intracellular signaling. Additional molecular details will be presented. Overall, our studies demonstrate that the KSHV K5 protein is acting as a novel oncogene – the first viral protein of its kind – to drive monocyte subset expansion and alter cellular metabolism, contributing to a pro-tumorigenic microenvironment. Intriguingly, the metabolic changes observed are caused by a single KSHV protein and thus serve as a useful model to study different aspects of KS pathology and its overall regulation of cellular metabolism. These studies also provide additional rationale for the currently ongoing clinical trials of sunitinib, Gleevec, and rapamycin for the treatment of KSHV-driven neoplasias. Finally, the ability of this viral E3 ubiquitin ligase to drive metabolic changes provides the tantalizing suggestion that a cellular ligase may be acting in a similar, either physiologic or oncogenic, manner.

Endothelial cells regulate cardiomyocyte development from embryonic stem cells

The molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes are largely unknown. Here, we determined a critical role of receptor tyrosine kinase, EphB4, in regulating cardiomyocyte generation from embryonic stem (ES) cells through endothelial cells. The number of spontaneous contracting cardiomyocytes, and the expression of cardiac-specific genes, including alpha-MHC and MLC-2V, was significantly decreased in EphB4-null ES cells. EphB4 was expressed in endothelial cells underneath contracting cardiomyocytes, but not in cardiomyocytes. Angiogenic inhibitors, including endostatin and angiostatin, inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells. Generation of functional cardiomyocytes and the expression of cardiac-specific genes were significantly enhanced by co-culture of ES cells with human endothelial cells. Furthermore, the defects of cardiomyocyte differentiation in EphB4-deficient ES cells were rescued by human endothelial cells. For the first time, our study demonstrated that endothelial cells play an essential role in facilitating cardiomyocyte differentiation from pluripotent stem cells. EphB4 signaling is a critical component of the endothelial niche to regulate regeneration of cardiomyocytes.