Receptor Tyrosine Kinase Signaling Research Articles

Usp22 Overexpression Leads to Aberrant Signal Transduction of Cancer-Related Pathways but Is Not Sufficient to Drive Tumor Formation in Mice.

Simple SummaryIncreased levels of the Usp22 deubiquitinase have been observed in several types of human cancer, particularly highly aggressive, therapy-resistant tumors. However, the role of Usp22 overexpression in cancer etiology is not known. To address whether Usp22 overexpression is sufficient to induce tumors in vivo, we created a mouse that expresses high levels of Usp22 in all tissues beginning in embryogenesis through to adulthood. Analyses of these mice clearly show that while Usp22 overexpression alters several tumor-related signaling pathways and induces associated morphological changes such as hyperbranching of the mammary gland, Usp22 overexpression alone is not sufficient to induce tumorigenesis. These findings indicate that Usp22 likely enhances tumor formation and progression through cooperation with oncoproteins that exacerbate abnormal signal transduction.Usp22 overexpression is observed in several human cancers and is correlated with poor patient outcomes. The molecular basis underlying this correlation is not clear. Usp22 is the catalytic subunit of the deubiquitylation module in the SAGA histone-modifying complex, which regulates gene transcription. Our previous work demonstrated that the loss of Usp22 in mice leads to decreased expression of several components of receptor tyrosine kinase and TGFβ signaling pathways. To determine whether these pathways are upregulated when Usp22 is overexpressed, we created a mouse model that expresses high levels of Usp22 in all tissues. Phenotypic characterization of these mice revealed over-branching of the mammary glands in females. Transcriptomic analyses indicate the upregulation of key pathways involved in mammary gland branching in mammary epithelial cells derived from the Usp22-overexpressing mice, including estrogen receptor, ERK/MAPK, and TGFβ signaling. However, Usp22 overexpression did not lead to increased tumorigenesis in any tissue. Our findings indicate that elevated levels of Usp22 are not sufficient to induce tumors, but it may enhance signaling abnormalities associated with oncogenesis.

A Comprehensive Comparison of Early-Onset and Average-Onset Colorectal Cancers.

The causative factors for the recent increase in early-onset colorectal cancer (EO-CRC) incidence are unknown. We sought to determine if early-onset disease is clinically or genomically distinct from average-onset colorectal cancer (AO-CRC). Clinical, histopathologic, and genomic characteristics of EO-CRC patients (2014-2019), divided into age 35 years and younger and 36-49 years at diagnosis, were compared with AO-CRC (50 years and older). Patients with mismatch repair deficient tumors, CRC-related hereditary syndromes, and inflammatory bowel disease were excluded from all but the germline analysis. All statistical tests were 2-sided. In total, 759 patients with EO-CRC (35 years, n = 151; 36-49 years, n = 608) and AO-CRC (n = 687) were included. Left-sided tumors (35 years and younger = 80.8%; 36-49 years = 83.7%; AO = 63.9%; P < .001 for both comparisons), rectal bleeding (35 years and younger = 41.1%; 36-49 years = 41.0%; AO = 25.9%; P = .001 and P < .001, respectively), and abdominal pain (35 years and younger = 37.1%; 36-49 years = 34.0%; AO = 26.8%; P = .01 and P = .005, respectively) were more common in EO-CRC. Among microsatellite stable tumors, we found no differences in histopathologic tumor characteristics. Initially, differences in TP53 and Receptor Tyrosine Kinase signaling pathway (RTK-RAS)alterations were noted by age. However, on multivariate analysis including somatic gene analysis and tumor sidedness, no statistically significant differences at the gene or pathway level were demonstrated. Among advanced microsatellite stable CRCs, chemotherapy response and survival were equivalent by age cohorts. Pathogenic germline variants were identified in 23.3% of patients 35 years and younger vs 14.1% of AO-CRC (P = .01). EO-CRCs are more commonly left-sided and present with rectal bleeding and abdominal pain but are otherwise clinically and genomically indistinguishable from AO-CRCs. Aggressive treatment regimens based solely on the age at CRC diagnosis are not warranted.

Lymphatic-specific intracellular modulation of receptor tyrosine kinase signaling improves lymphatic growth and function.

Exogenous administration of lymphangiogenic growth factors is widely used to study changes in lymphatic function in pathophysiology. However, this approach can result in off-target effects, thereby generating conflicting data. To circumvent this issue, we modulated intracellular VEGF-C signaling by conditionally knocking out the lipid phosphatase PTEN using the Vegfr3 promoter to drive the expression of Cre-lox in lymphatic endothelial cells (LECs). PTEN is an intracellular brake that inhibits the downstream effects of the activation of VEGFR3 by VEGF-C. Activation of Cre-lox recombination in adult mice resulted in an expanded functional lymphatic network due to LEC proliferation that was independent of lymphangiogenic growth factor production. Furthermore, compared with lymphangiogenesis induced by VEGF-C injection, LECPTEN animals had mature, nonleaky lymphatics with intact cell-cell junctions and reduced local tissue inflammation. Last, compared with wild-type or VEGF-C-injected mice, LECPTEN animals had an improved capacity to resolve inflammatory responses. Our findings indicate that intracellular modulation of lymphangiogenesis is effective in inducing functional lymphatic networks and has no off-target inflammatory effects.

Glycoprotein non–metastatic melanoma protein B functions with growth factor signaling to induce tumorigenesis through its serine phosphorylation

Breast cancer is the most common cancer among women. Glycoprotein non–metastatic melanoma protein B (GPNMB), a type I transmembrane protein that is highly expressed in many cancers, including breast cancer, has been shown to be a prognostic factor. We previously reported that GPNMB overexpression confers tumorigenic potential, as evidenced by invasive tumor growth in vivo, sphere formation, and cellular migration and invasion to non–tumorigenic mammary epithelial cells. In this study, we focused on the serine (S) residue in the intracellular domain of GPNMB (S530 in human isoform b and S546 in mouse), which is predicted to be a phosphorylation site. To investigate the roles of this serine residue, we made an antibody specific for S530‐phosphorylated human GPNMB and a point mutant in which S530 is replaced by an alanine (A) residue, GPNMB(SA). Established GPNMB(SA) overexpressing cells showed a significant reduction in sphere formation in vitro and tumor growth in vivo as a result of decreased stemness‐related gene expression compared to that in GPNMB(WT)‐expressing cells. In addition, GPNMB(SA) impaired GPNMB‐mediated cellular migration. Furthermore, we found that tyrosine kinase receptor signaling triggered by epidermal growth factor or fibroblast growth factor 2 induces the serine phosphorylation of GPNMB through activation of downstream oncoproteins RAS and RAF.

Coordination among multiple receptor tyrosine kinase signals controls Drosophila developmental timing and body size.

SUMMARYIn holometabolous insects, metamorphic timing and body size are controlled by a neuroendocrine axis composed of the ecdysone-producing prothoracic gland (PG) and its presynaptic neurons (PGNs) producing PTTH. Although PTTH/Torso signaling is considered the primary mediator of metamorphic timing, recent studies indicate that other unidentified PGN-derived factors also affect timing. Here, we demonstrate that the receptor tyrosine kinases anaplastic lymphoma kinase (Alk) and PDGF and VEGF receptor-related (Pvr), function in coordination with PTTH/Torso signaling to regulate pupariation timing and body size. Both Alk and Pvr trigger Ras/Erk signaling in the PG to upregulate expression of ecdysone biosynthetic enzymes, while Alk also suppresses autophagy by activating phosphatidylinositol 3-kinase (PI3K)/Akt. The Alk ligand Jelly belly (Jeb) is produced by the PGNs and serves as a second PGN-derived tropic factor, while Pvr activation mainly relies on autocrine signaling by PG-derived Pvf2 and Pvf3. These findings illustrate that a combination of juxtacrine and autocrine signaling regulates metamorphic timing, the defining event of holometabolous development.

Pancreatic Adenocarcinoma Therapeutics Targeting RTK and TGF Beta Receptor.

Despite the improved overall survival rates in most cancers, pancreatic cancer remains one of the deadliest cancers in this decade. The rigid microenvironment, which majorly comprises cancer-associated fibroblasts (CAFs), plays an important role in the obstruction of pancreatic cancer therapy. To overcome this predicament, the signaling of receptor tyrosine kinases (RTKs) and TGF beta receptor (TGFβR) in both pancreatic cancer cell and supporting CAF should be considered as the therapeutic target. The activation of receptors has been reported to be aberrant to cell cycle regulation, and signal transduction pathways, such as growth-factor induced proliferation, and can also influence the apoptotic sensitivity of tumor cells. In this article, the regulation of RTKs/TGFβR between pancreatic ductal adenocarcinoma (PDAC) and CAFs, as well as the RTKs/TGFβR inhibitor-based clinical trials on pancreatic cancer are reviewed.

Proteomic study on the damage of learning and memory ability of rat offspring caused by chronic stress during pregnancy

Objective: To study the differential protein and signal pathway related to the impairment of learning and memory ability of offspring caused by chronic stress during pregnancy and explore the possible mechanism. Methods: From July to October 2019, sixteen SPF free female SD rats aged 80-90 days, weighing (200±20) g. Twelve SPF grade male SD rats aged 90-100 days, weighing (220±20) g. After a week of adaptive feeding, the female rats were randomly divided into control group and model group (8 rats in each group) , male rats were divided into control mating group (n=8) and model mating group (n=4) . Chronic unpredictable mild stress (CUMS) model was established and stimulated continuously for 21 days. One day before stress, the first, seventh, fourteenth and 21th day after stress, the blood was collected from the inner canthus vein of the female rats, and the content of corticosterone was determined. Morris water maze test was used to detect the spatial learning and memory ability of offspring rats. The morphological changes of hippocampus were observed by HE and Nissl staining. The proteomic correlation analysis of offspring rats' hippocampus was performed by isobaric tags for relative and absolute quantification (iTRAQ) technique. Results: Compared with the control group, the content of plasma corticosterone in the model group was significantly higher (F=7.717, P<0.05) , and the model was successfully established. In Morris water maze test, compared with the control offspring group, the escape latency was longer, the average swimming speed was lower, the number of crossing platform was less, and the target quadrant run was shorter in the model offspring group (P<0.05) . The pathological results showed that the morphology of cells in the hippocampal tissue of the model offspring group was irregular, the number of neurons was small, Nissl body was unevenly distributed, the volume was small and the number was small. Mass spectrometry analysis showed that a total of 5065 proteins were screened out in the two offspring groups, and 26 proteins were differentially expressed (P<0.05) , of which 19 proteins were up-regulated and 7 proteins were down regulated. The differential proteins were mainly involved in 23 biological processes, 14 cellular components and 9 molecular functions. Kyoto Encyclopedia of genes and genomes (KEGG) enrichment analysis showed that 57 pathways were enriched, of which 8 signaling pathways were significantly enriched (P<0.05) . There were 5 signaling pathways that might be involved in the impairment of learning and memory ability of offspring, including neuroactive ligand receptor interaction, cGMP-PKG signaling pathway, adhesion and connection, adhesion and connection FoxO signaling pathway and Notch signaling pathway, mainly including tyrosine protein kinase receptor, tyrosine kinase receptor and Notch signaling pathway, and α2A adrenergic receptor, cGMP dependent protein kinase and other differential proteins may be involved in the injury process. Conclusion: The damage of learning and memory ability of offspring may be caused by chronic stress during pregnancy rats. The enriched signal pathway and key differential proteins of proteomics may play an important role in the process of damage.

New Target for Precision Medicine Treatment of Giant-Cell Tumor of Bone: Sunitinib Is Effective in the Treatment of Neoplastic Stromal Cells with Activated PDGFRβ Signaling.

Simple SummaryThe purpose of this study was to analyze differential cell signaling in response to denosumab treatment to identify and subsequently inhibit molecular targets in the neoplastic stromal cell population, which poses a risk for tumor recurrence. Using phosphoprotein arrays, a distinct signaling profile was detected in GCTB tissues treated with denosumab, a specific RANKL antibody, which coincided with the RTK profile in derived cell lines. PDGFRβ was selected as a promising receptor target, and its inhibition by the small-molecule inhibitor sunitinib resulted in potent inhibition of cell proliferation in vitro. The addition of sunitinib to denosumab resulted in the disappearance of both multinuclear giant cells and neoplastic stromal cells, as reported here. Thus, sunitinib could become an effective addition to denosumab in the treatment of GCTB with activated PDGFRβ.Giant-cell tumor of bone (GCTB) is an intermediate type of primary bone tumor characterized by locally aggressive growth with metastatic potential. The aim of this study was to identify new druggable targets among the cell signaling molecules involved in GCTB tumorigenesis. Profiles of activated signaling proteins in fresh-frozen tumor samples and tumor-derived cell lines were determined using phosphoprotein arrays. Analysis of the obtained data revealed epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor beta (PDGFRβ) as potential targets, but only the PDGFR inhibitor sunitinib caused a considerable decrease in stromal cell viability in vitro. Furthermore, in the case of a 17-year-old patient suffering from GCTB, we showed that the addition of sunitinib to the standard treatment of GCTB with the monoclonal antibody denosumab resulted in the complete depletion of multinucleated giant cells and mononuclear stromal cells in the tumor tissue. To summarize, the obtained data showed that a specific receptor tyrosine kinase (RTK) signaling pattern is activated in GCTB cells and plays an important role in the regulation of cell proliferation. Thus, activated RTKs and their downstream signaling pathways represent useful targets for precision treatment with low-molecular-weight inhibitors or with other types of modern biological therapy.

Abstract 1979: JAK2/STAT3 and TrkA pathways are frequently co-activated in triple-negative and HER2-enriched breast cancers and the co-activation correlates with an increased potential of metastasis

Abstract Breast cancer is the leading cause of cancer-related deaths in American women. Despite the current standard-of-care, which implements tumor resection, radiotherapy, and chemotherapy, triple-negative and HER2-positive breast cancer patients often relapse and present with recurrent disease, for which there is no cure. Thus, there is an urgent need to identify new molecular targets to improve patient response to anti-cancer therapies. The JAK2-STAT3 and TrkA receptor tyrosine kinase signaling pathways have been separately implicated in metastatic breast cancers, but information about their crosstalk remains limited. Activated Janus kinase 2 (JAK2) receptor tyrosine kinase phosphorylates signal transducer and activator of transcription 3 (STAT3) transcription factor, inducing STAT3 nuclear translocation and transcriptional activity. Abnormal activation of STAT3-mediated transcription has been identified as a pro-tumorigenic event in breast cancers and promotes progression through induction of breast cancer stem cells, invasion, and angiogenesis. Tropomyosin receptor kinase A (TrkA) frequently forms oncogenic fusion proteins and its overexpression has been shown to drive malignant transformation of breast cancer cells. JAK2 inhibitors, while approved for treatment of myeloproliferative neoplasms, are currently in clinical trials for triple-negative breast cancers. Currently, there are two FDA-approved TrkA inhibitors for treatment of NTRK1 fusion-positive solid tumors, one of which is considered a tumor-agnostic inhibitor. However, whether these two important pathways are co-activated in breast cancers and whether the co-activation is associated with overall progression of breast cancer have not been investigated. Herein, we report that STAT3 and TrkA are more co-activated in triple-negative and HER2-enriched breast cancers, compared to luminal subtypes, as determined by immunohistochemical staining of 33 invasive breast carcinomas and datamining data of over 1,500 breast cancer patients. We also observed that high co-activation of both JAK2-STAT3 and TrkA pathways significantly shortens overall metastasis-free survival of both triple-negative and HER2-enriched breast cancer patients when compared to patients with low co-activation. Similarly, we also find that JAK2-STAT3 and TrkA co-activation also significantly shortens brain, lung, and bone metastasis-free survival in both of these breast cancer subtypes, suggesting a critical role for JAK2-STAT3 and TrkA in distant metastasis of aggressive breast cancers. Taken together, our findings indicate a novel signaling crosstalk between JAK2-STAT3 and TrkA pathways in triple-negative and HER2-enriched breast cancers, and the use of their co-activation as a prognostic indicator for metastatic breast cancers of both subtypes. Citation Format: Angelina T. Regua, Noah R. Aguayo, Sara Abu Jalboush, Daniel L. Doheny, Sara G. Manore, Dongqin Zhu, Grace L. Wong, Austin Arrigo, Calvin J. Wagner, Yang Yu, Karen Baylon, Alexandra Thomas, Michael D. Chan, Jimmy Ruiz, Guangxu Jin, Roy E. Strowd, Peiqing Sun, Linda J. Metheny-Barlow, Jiayuh Lin, Hui-Wen Lo. JAK2/STAT3 and TrkA pathways are frequently co-activated in triple-negative and HER2-enriched breast cancers and the co-activation correlates with an increased potential of metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1979.

Abstract 2937: Nedd9 controls autophagy to limit non-small cell lung cancer (NSCLC) growth

Abstract The goal of this study is to define the role of NEDD9 in formation of progression of non-small cell lung cancer (NSCLC). NSCLC has a low survival rate, with metastasis contributing to the vast majority of deaths. NEDD9 expression has been shown to be repressed by LKB1, and upregulated in LKB1-mutated NSCLC; in these tumors, elevated NEDD9 promotes metastasis, serving as a scaffolding intermediate in the integrin and receptor tyrosine kinase signaling cascades, promoting pro-invasive signaling. In this study, we for the first time assessed the consequences for cancer formation after introduction of a constitutive null allele of Nedd9 (KPN mice) in the Krastm4Tyj/J/Trp53tm1Brn/J (KP mice) Adeno-cre inducible murine model of NSCLC, in the context of intact Lkb1. Unexpectedly, KPN mice presented with significantly accelerated NSCLC tumorigenesis and enhanced tumor invasion. Orthotopic re-implantation of KP and KPN tumor into syngeneic mice with wt and null Nedd9 genotypes demonstrated that the effect of Nedd9 loss on promoting tumor growth is cell-autonomous. Proteomic analysis of KPN versus KP tumors revealed that more aggressive phenotypes occurred in spite of depressed activity of the pro-proliferative, pro-invasive Nedd9 partner proteins Src and Fak. Treatment of mice with the Src inhibitor dasatinib significantly reduced, but did not eliminate, tumor growth in KP and KPN mice. As an alternative hypothesis, we considered that Ras-dependent NSCLC tumors require active autophagy, a process dependent on activation of the kinases Lkb1 and Ampk; given Lkb1 represses Nedd9, we investigated whether Nedd9 reciprocally regulates Lkb1. We found that KPN tumors specifically and significantly upregulated active Lkb1 based on a mechanism of post-transcriptional activation, causing increased expression of the Lkb1 substrate Ampk and downstream effectors of autophagy, including elevated appearance of LC3-positive autophagosomes in KPN NSCLC tumor tissue. NEDD9 depletion in human NSCLC cell lines similarly elevated LKB1 activation. In vivo treatment of KPN and KP mice with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors, emphasizing the importance of autophagy for the growth differential. In sum, these data for the first time identify and define a previously unknown role for the pro-metastatic protein NEDD9 in control of autophagy, based on a role as a negative regulator of the Lkb1-Ampk signaling axis in NSCLC. Citation Format: Alexander Y. Deneka, Meghan Kopp, Anna S. Nikonova, Anna Gaponova, Anna Kiseleva, Douglas Flieder, Ilya Serebriiskii, Harvey Hensley, Erica Golemis. Nedd9 controls autophagy to limit non-small cell lung cancer (NSCLC) growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2937.

Nationwide Survey of Pediatric Hypodiploid Acute Lymphoblastic Leukemia in Japan

BackgroundPloidy is a highly significant prognostic factor in childhood acute lymphoblastic leukemia (ALL). Children with hypodiploid ALL continue to have inferior outcomes despite current intensive chemotherapy treatment. Hypodiploid ALL is divided into two groups based on genetic alterations. Near-haploid ALL with 24-31 chromosomes harbors alterations targeting receptor tyrosine kinase signaling, RAS signaling, and the lymphoid transcription factor gene IKZF3 and low-hypodiploid ALL with 32-39 chromosomes is characterized by alterations in TP53 that are commonly present in non-tumor cells, IKZF2 and RB1 . There is little information about children with hypodiploid ALL in Asia including Japan, although ethnic differences seem to exist. The aim of this study was to clarify the clinical characteristics and outcome of pediatric hypodiploid ALL in Japanese patients.MethodsPatients with fewer than 46 chromosomes were defined as hypodiploid ALL in this study. All reports of chromosomal analysis were re-reviewed by two independent investigators and 117 cases of hypodiploid ALL were identified out of 4261 patients who were registered in clinical trials between 1997 and 2012. A total of 64 patients were excluded due to the lack of follow-up data (n = 39), only a single cell exhibiting a chromosome number less than 46 in G-banding experiments (n = 18), incorrect diagnosis (n = 4), or congenital chromosomal aberrations such as Down syndrome (n = 1) and Robertson translocations (n = 2). Seventeen patients whose karyotypes at remission were not evaluable were also excluded.ResultsIn total 66 male (56.4%) and 51 female (43.6%) patients' data was evaluated with a median age of 6 years (range 1 to 15 years). There were 101, 8, 2, and 3 patients with chromosome numbers of 45, 44, 43, and 36, respectively. One patient exhibited each with 27, 28, and 29 chromosomes. No patients had a modal chromosome number of 30-35 or 37-42. Among the 117, 110 (94%) had B-precursor ALL and 105 (90%) had CD10 positivity. All T-ALL patients (n = 7) had 45 chromosomes. Out of 117 patients, 22 were TEL-AML1 fusion positive of which 19 had 45 chromosomes, 2 had 44 chromosomes, and 1 had 43 chromosomes. There were no patients with central nervous system (CNS) infiltration at the time of diagnosis. All but one patient who was diagnosed with T-ALL achieved a complete remission (CR) with a CR rate of 98.3%. Overall survival at 5 years varied significantly at 86.0%, 87.5%, and 62.5% in patients with 45, 44, and fewer than 44 chromosomes, respectively (P=0.037). There were no patients with CNS relapse in both the 44 and fewer than 44 chromosomes groups. Five patients out of 8 with fewer than 44 chromosomes had bone marrow relapse, even though 1 patient had undergone hematopoietic stem cell transplantation (HSCT) during his first CR. Four patients underwent HSCT after relapse, but only 1 was cured. In the 44 chromosome group, 2 of 8 patients underwent HSCT during their first CR and did not show relapse. However, one of them developed secondary renal cell carcinoma 10 years after treatment. One patient with 44 chromosomes underwent HSCT after relapse and failed to be salvaged. We also analyzed some variables that influenced event-free and overall survival in the 45 chromosome group: monosomy 7, sex chromosome abnormality, and 9p21.3 deletion. We were not able to identify any significant prognostic factor among patients with 45 chromosomes.ConclusionsAlthough the number of patients was small, patients with 44 chromosomes without HSCT showed better prognosis than those reported previously. However, the outcome of Japanese patients with fewer than 44 chromosomes was as poor as previously reported in other countries. The effect of HSCT in patients with hypodiploid ALL needs to be the subject of future studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization

Overexpression of the receptor tyrosine kinase HER2 plays a critical role in the development of various tumors. Biparatopic designed ankyrin repeat proteins (bipDARPins) potently induce apoptosis in HER2-addicted breast cancer cell lines. Here, we have investigated how the spatiotemporal receptor organization at the cell surface is modulated by these agents and is distinguished from other molecules, which do not elicit apoptosis. Binding of conventional antibodies is accompanied by moderate reduction of receptor mobility, in agreement with HER2 being dimerized by the bivalent IgG. In contrast, the most potent apoptosis-inducing bipDARPins lead to a dramatic arrest of HER2. Dual-color single-molecule tracking revealed that the HER2 “lockdown” by these bipDARPins is caused by the formation of HER2-DARPin oligomer chains, which are trapped in nanoscopic membrane domains. Our findings establish that efficient neutralization of receptor tyrosine kinase signaling can be achieved through intermolecular bipDARPin crosslinking alone, resulting in inactivated, locked-down bipDARPin-HER2 complexes.

Targeted mass-spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP kinase, and AKT signaling

SummaryA primary goal of the US National Cancer Institute's Ras initiative at the Frederick National Laboratory for Cancer Research is to develop methods to quantify RAS signaling to facilitate development of novel cancer therapeutics. We use targeted proteomics technologies to develop a community resource consisting of 256 validated multiple reaction monitoring (MRM)-based, multiplexed assays for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. As proof of concept, we quantify the response of melanoma (A375 and SK-MEL-2) and colorectal cancer (HCT-116 and HT-29) cell lines to BRAF inhibition by PLX4720. These assays replace over 60 western blots with quantitative mass-spectrometry-based assays of high molecular specificity and quantitative precision, showing the value of these methods for pharmacodynamic measurements and mechanism-of-action studies. Methods, fit-for-purpose validation, and results are publicly available as a resource for the community at assays.cancer.gov.

All Good Things Must End: Termination of Receptor Tyrosine Kinase Signal.

Receptor tyrosine kinases (RTKs) are membrane receptors that regulate many fundamental cellular processes. A tight regulation of RTK signaling is fundamental for development and survival, and an altered signaling by RTKs can cause cancer. RTKs are localized at the plasma membrane (PM) and the major regulatory mechanism of signaling of RTKs is their endocytosis and degradation. In fact, RTKs at the cell surface bind ligands with their extracellular domain, become active, and are rapidly internalized where the temporal extent of signaling, attenuation, and downregulation are modulated. However, other mechanisms of signal attenuation and termination are known. Indeed, inhibition of RTKs’ activity may occur through the modulation of the phosphorylation state of RTKs and the interaction with specific proteins, whereas antagonist ligands can inhibit the biological responses mediated by the receptor. Another mechanism concerns the expression of endogenous inactive receptor variants that are deficient in RTK activity and take part to inactive heterodimers or hetero-oligomers. The downregulation of RTK signals is fundamental for several cellular functions and the homeostasis of the cell. Here, we will review the mechanisms of signal attenuation and termination of RTKs, focusing on FGFRs.

Receptor tyrosine kinases and cancer: oncogenic mechanisms and therapeutic approaches.

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease, notably cancer. Since their discovery, several mechanisms of RTK dysregulation have been identified, resulting in multiple cancer types displaying 'oncogenic addiction' to RTKs. As a result, RTKs have represented a major class for targeted therapeutics over the past two decades, with numerous small molecule-based tyrosine kinase inhibitor (TKI) therapeutics having been developed and clinically approved for several cancers. However, many of the current RTK inhibitor treatments eventually result in the rapid development of acquired resistance and subsequent tumor relapse. Recent technological advances and tools are being generated for the identification of novel RTK small molecule therapeutics. These newer technologies will be important for the identification of diverse types of RTK inhibitors, targeting both the receptors themselves as well as key cellular factors that play important roles in the RTK signaling cascade.

Involvement of plasminogen activator inhibitor-1 and its related molecules in atrial fibrosis in patients with atrial fibrillation.

Atrial fibrillation is the most common form of cardiac arrhythmia. Atrial fibrosis is a significant feature of atrial fibrillation though its mechanism is not well understood. We searched the Gene Expression Omnibus database to compare mRNA expression patterns between atrial fibrillation and sinus rhythm samples; one hundred and forty eight differentially expressed genes were identified. Most of these genes were significantly enriched in the extracellular matrix organization process and collagen-activated tyrosine kinase receptor signaling pathway. To screen hub genes involved in atrial fibrosis, we constructed a protein-protein interaction network and found that three hub genes (SERPINE1/plasminogen activator inhibitor-1/PAI-1, TIMP Metallopeptidase Inhibitor 3/TIMP3 and decorin/DCN) play vital roles in atrial fibrosis, especially plasminogen activator inhibitor-1. Elevated plasminogen activator inhibitor-1 expression was positively correlated with the p53 signaling pathway. Plasminogen activator inhibitor-1 and p53 protein expression levels were verified in patients with sinus rhythm and atrial fibrillation by Western blot analysis. Compared with the sinus rhythm controls, p53 and plasminogen activator inhibitor-1 protein expressions were upregulated in the atrial tissues of patients with atrial fibrillation. p53 was also found to regulate plasminogen activator inhibitor-1 based on the results of cellular and molecular experiments. Thus, the p53/plasminogen activator inhibitor-1 signaling axis may participate in the pathophysiological processes of atrial fibrillation, and plasminogen activator inhibitor-1 may serve as a new therapeutic biomarker in atrial fibrillation.

Copper: An Intracellular Achilles' Heel Allowing the Targeting of Epigenetics, Kinase Pathways, and Cell Metabolism in Cancer Therapeutics.

Copper is an essential transition metal frequently increased in cancer known to strongly influence essential cellular processes. Targeted therapy protocols utilizing both novel and repurposed drug agents initially demonstrate strong efficacy, before failing in advanced cancers as drug resistance develops and relapse occurs. Overcoming this limitation involves the development of strategies and protocols aimed at a wider targeting of the underlying molecular changes. Receptor Tyrosine Kinase signaling pathways, epigenetic mechanisms and cell metabolism are among the most common therapeutic targets, with molecular investigations increasingly demonstrating the strong influence each mechanism exerts on the others. Interestingly, all these mechanisms can be influenced by intracellular copper. We propose that copper chelating agents, already in clinical trial for multiple cancers, may simultaneously target these mechanisms across a wide variety of cancers, serving as an excellent candidate for targeted combination therapy. This review summarizes the known links between these mechanisms, copper, and copper chelation therapy.

A transitory signaling center controls timing of primordial germ cell differentiation.

Organogenesis requires exquisite spatiotemporal coordination of cell morphogenesis, migration, proliferation, and differentiation of multiple cell types. For gonads, this involves complex interactions between somatic and germline tissues. During Drosophila ovary morphogenesis, primordial germ cells (PGCs) either are sequestered in stem cell niches and are maintained in an undifferentiated germline stem cell state or transition directly toward differentiation. Here, we identify a mechanism that links hormonal triggers of somatic tissue morphogenesis with PGC differentiation. An early ecdysone pulse initiates somatic swarm cell (SwC) migration, positioning these cells close to PGCs. A second hormone peak activates Torso-like signal in SwCs, which stimulates the Torso receptor tyrosine kinase (RTK) signaling pathway in PGCs promoting their differentiation by de-repression of the differentiation gene, bag of marbles. Thus, systemic temporal cues generate a transitory signaling center that coordinates ovarian morphogenesis with stem cell self-renewal and differentiation programs, highlighting a more general role for such centers in reproductive and developmental biology.

Cell surface integrin α5ß1 clustering negatively regulates receptor tyrosine kinase signaling in colorectal cancer cells via glycogen synthase kinase 3.

As a key process within the tissue microenvironment, integrin signaling can influence cell functional responses to growth factor stimuli. We show here that clustering of integrin α5ß1 at the plasma membrane of colorectal cancer-derived epithelial cells modulates their ability to respond to stimulation by receptor tyrosine kinase (RTK)-activating growth factors EGF, NRG and HGF, through GSK3-mediated suppression of Akt pathway. We observed that integrin α5ß1 is lost from the membrane of poorly organized human colorectal tumors and that treatment with the integrin-clustering antibody P4G11 is sufficient to induce polarity in a mouse tumor xenograft model. While adding RTK growth factors (EGF, NRG and HGF) to polarized colorectal cancer cells induced invasion and loss of monolayer formation in 2D and 3D, this pathological behavior could be blocked by P4G11. Phosphorylation of ErbB family members as well as MET following EGF, NRG and HGF treatment was diminished in cells pretreated with P4G11. Focusing on EGFR, we found that blockade of integrin α5ß1 increased EGFR phosphorylation. Since activity of multiple downstream kinase pathways were altered by these various treatments, we employed computational machine learning techniques to ascertain the most important effects. Partial least-squares discriminant analysis identified GSK3 as a major regulator of EGFR pathway activities influenced by integrin α5ß1. Moreover, we used partial correlation analysis to examine signaling pathway crosstalk downstream of EGF stimulation and found that integrin α5ß1 acts as a negative regulator of the AKT signaling cascade downstream of EGFR, with GSK3 acting as a key mediator. We experimentally validated these computational inferences by confirming that blockade of GSK3 activity is sufficient to induce loss of polarity and increase of oncogenic signaling in the colonic epithelial cells.

Cionin, a vertebrate cholecystokinin/gastrin homolog, induces ovulation in the ascidian Ciona intestinalis type A

Cionin is a homolog of vertebrate cholecystokinin/gastrin that has been identified in the ascidian Ciona intestinalis type A. The phylogenetic position of ascidians as the closest living relatives of vertebrates suggests that cionin can provide clues to the evolution of endocrine/neuroendocrine systems throughout chordates. Here, we show the biological role of cionin in the regulation of ovulation. In situ hybridization demonstrated that the mRNA of the cionin receptor, Cior2, was expressed specifically in the inner follicular cells of pre-ovulatory follicles in the Ciona ovary. Cionin was found to significantly stimulate ovulation after 24-h incubation. Transcriptome and subsequent Real-time PCR analyses confirmed that the expression levels of receptor tyrosine kinase (RTK) signaling genes and a matrix metalloproteinase (MMP) gene were significantly elevated in the cionin-treated follicles. Of particular interest is that an RTK inhibitor and MMP inhibitor markedly suppressed the stimulatory effect of cionin on ovulation. Furthermore, inhibition of RTK signaling reduced the MMP gene expression in the cionin-treated follicles. These results provide evidence that cionin induces ovulation by stimulating MMP gene expression via the RTK signaling pathway. This is the first report on the endogenous roles of cionin and the induction of ovulation by cholecystokinin/gastrin family peptides in an organism.