Phosphorylation Antibody Array Research Articles

Exosomes from the Uterine Cavity Mediate Immune Dysregulation via Inhibiting the JNK Signal Pathway in Endometriosis.

Endometriosis is a chronic inflammatory disease with an uncertain pathogenesis. Peritoneal immune dysregulation plays an important role in the pathogenesis of endometriosis. Exosomes are messengers of intercellular communication. This study mainly investigated the role of exosomes from the uterine cavity in endometriosis. Exosomes of the uterine aspirate fluid were isolated and cocultured with macrophages for 48 h. Flow cytometry was used to detect macrophage polarization. A Human MAPK Phosphorylation Antibody Array and Western blot were used to detect the phosphorylation of the MAPK pathway. A microRNA sequencing analysis was used to detect differentially expressed miRNAs. Our research found that exosomes of the uterine aspirate fluid from endometriosis could reduce the proportion of CD80+ macrophages. Additionally, it could inhibit the expression of P-JNK in macrophages. However, the JNK activator anisomycin could increase the proportion of CD80+ macrophages. In addition, exosomes of the uterine aspirate fluid from endometriosis could promote the migration and invasion of endometrial stromal cells by acting on macrophages. The expression of miR-210-3p was increased in both exosomes and the eutopic endometrium in patients with endometriosis through miRNA sequencing, which could also reduce the proportion of CD80+ macrophages. In summary, we propose that exosomes from the uterine cavity in patients with endometriosis may affect the phenotype of macrophages by inhibiting the JNK signaling pathway, thus mediating the formation of an immunological microenvironment conducive to the development of endometriosis.

PSAT158 Dimeric [Cys25]PTH(1-34) and Monomeric PTH(1-34) Differentially Regulate the Expression of Genes Related to Calcium Homeostasis in Bone and Kidney

Abstract Osteoporosis and its resulting fractures threaten health and life of many patients worldwide; therefore, the development of effective and safe drugs for osteoporosis is an urgent need. In our previous report, dimeric [Cys25]PTH(1-34) peptide with arginine-to-cysteine mutation at position 25 (Dimer) showed a substantial osteo-anabolic effect similar or superior to that of PTH(1-34) in several mouse models. Surprisingly, cAMP was not elevated in serum or urine the Dimer-treated group unlike in the PTH(1-34)-treated group. From this, we hypothesized that the Dimer stimulates anabolic responses through a signaling pathway different from the cAMP-PKA pathway. To test this hypothesis, we assessed responses down-stream of the activated signal cascade from PTHR1 using GPCR Phospho Antibody Array in cells, as well as the expression levels of mRNAs and proteins related to calcium metabolism in bones and kidneys of mice. Cell lysates obtained from GP2.3 cells (treated with PTH(1-34) and Dimer with PBS as a control) were applied to the GPCR-MAPK Pathway Phosphorylation Antibody Array (Full Moon BioSystems, USA), containing 193 antibodies. Each of the antibodies has 6 replicates that are printed on standard-size coated glass microscope slides. The results of the Phospho-antibody array were further confirmed by Western Blot assay. Eighteen CD1 mice of 9 WO were divided into three groups: control, PTH(1-34)-treated, and Dimer-treated groups. 40 µg/kg of PTH(1-34) and 80 µg/kg of the Dimer were subcutaneously injected for each of the experimental groups, respectively. Then, two hours after the injection, the animals were sacrificed, and their kidneys and tibiae were harvested. Total mRNA samples from kidneys and tibiae were extracted using trizol reagent. Protein samples were extracted via protein extraction buffer. We estimated mRNA and protein levels of biomarkers related to calcium regulation via RT-PCR and ELISA with harvested bone and kidney tissues. Several changes of phosphorylation of signaling proteins in GP2.3 cells were detected between Dimer-treated group and PTH(1-34)-treated group. Expression of mRNA and protein levels of RANKL was lower in the Dimer-treated group compared to the PTH(1-34)-treated group. In contrast, expression of mRNA level of OPG in tibia samples was lower in the Dimer-treated group. In kidney, the mRNA and protein levels of 1α(OH)ase in the Dimer-treated group were lower than in the PTH(1-34)-treated group. However, mRNA level of 24(OH)ase was higher in the Dimer-treated group than in the PTH(1-34)-treated group. Other parameters were not significantly different between the Dimer-treated and PTH(1-34)-treated groups. We speculate that the osteoanabolic effect of Dimer uses a signaling pathway different from the cAMP-PKA pathway, regulating different expression parameters related to calcium metabolism in both the bone and kidney. Further studies are required to define exact signaling pathway that Dimer acts on. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Early Development of Cardiac Fibrosis in Young Old-Father Offspring.

Cardiac aging is characterized by progressive fibrosis. Epidemiological studies have found that advanced paternal age is associated with an increased risk of heart failure in the next generation. This study is aimed at evaluating the effect of paternal age, in the young male rat progeny, on cardiac phenotype under circulatory stress conditions. Offspring rats were obtained by mating old males (24 months old) with young females (two months old) and by mating young males (two months old) with the same young females. Hypertension was induced in old father offspring (OFO) rats and young old father (YFO) offspring rats using L-NAME (N(ω)-nitro-L-arginine methyl ester). The OFO L-NAME rats showed a high blood pressure phenotype associated with substantial cardiac hypertrophy and an exacerbation of cardiac fibrosis compared to the YFO L-NAME rats. Histological analysis of heart tissue showed an expansion of the extracellular matrix, with fibroblasts displaying markers of epicardial origin (Tcf21, Tbx18, and Wt1) in the OFO group. Moreover, western blot and protein phosphorylation antibody array identified the TGF-β2 receptor pathway as preferentially activated in aged hearts as well as in OFO cardiac tissue treated with L-NAME. In addition, old father offspring rats (OFO+OFO L-NAME) had increased cardiac DNA methylation. In young hypertensive progeny, advanced paternal age at conception may be a risk factor for early progression towards cardiac fibrosis. An intergenerational transmission may be behind the paternal age-related cardiac remodeling in the young offspring.

Identification of a novel HOOK3-FGFR1 fusion gene involved in activation of the NF-kappaB pathway

BackgroundRearrangements involving the fibroblast growth factor receptor 1 (FGFR1) gene result in 8p11 myeloproliferative syndrome (EMS), which is a rare and aggressive hematological malignancy that is often initially diagnosed as myelodysplastic syndrome (MDS). Clinical outcomes are typically poor due to relative resistance to tyrosine kinase inhibitors (TKIs) and rapid transformation to acute leukemia. Deciphering the transcriptomic signature of FGFR1 fusions may open new treatment strategies for FGFR1 rearrangement patients.MethodsDNA sequencing (DNA-seq) was performed for 20 MDS patients and whole exome sequencing (WES) was performed for one HOOK3-FGFR1 fusion positive patient. RNA sequencing (RNA-seq) was performed for 20 MDS patients and 8 healthy donors. Fusion genes were detected using the STAR-Fusion tool. Fluorescence in situ hybridization (FISH), quantitative real-time PCR (qRT-PCR), and Sanger sequencing were used to confirm the HOOK3-FGFR1 fusion gene. The phosphorylation antibody array was performed to validate the activation of nuclear factor-kappaB (NF-kappaB) signaling.ResultsWe identified frequently recurrent mutations of ASXL1 and U2AF1 in the MDS cohort, which is consistent with previous reports. We also identified a novel in-frame HOOK3-FGFR1 fusion gene in one MDS case with abnormal monoclonal B-cell lymphocytosis and ring chromosome 8. FISH analysis detected the FGFR1 break-apart signal in myeloid blasts only. qRT-PCR and Sanger sequencing confirmed the HOOK3-FGFR1 fusion transcript with breakpoints located at the 11th exon of HOOK3 and 10th exon of FGFR1, and Western blot detected the chimeric HOOK3-FGFR1 fusion protein that is presumed to retain the entire tyrosine kinase domain of FGFR1. The transcriptional feature of HOOK3-FGFR1 fusion was characterized by the significant enrichment of the NF-kappaB pathway by comparing the expression profiling of FGFR1 fusion positive MDS with 8 healthy donors and FGFR1 fusion negative MDS patients. Further validation by phosphorylation antibody array also showed NF-kappaB activation, as evidenced by increased phosphorylation of p65 (Ser 536) and of IKBalpha (Ser 32).ConclusionsThe HOOK3-FGFR1 fusion gene may contribute to the pathogenesis of MDS and activate the NF-kappaB pathway. These findings highlight a potential novel approach for combination therapy for FGFR1 rearrangement patients.

CHMFL-BMX-078, a BMX inhibitor, overcomes the resistance of melanoma to vemurafenib via inhibiting AKT pathway

Our recent study demonstrated eIF3a loss contributes to vemurafenib resistance in melanoma by activating ERK. However, overexpression of eIF3a in the clinic is not feasible to produce vemurafenib re-sensitization, and ERK inhibitors combined with vemurafenib still exhibit limited effectiveness in the treatment of melanoma. Here, using the human receptor tyrosine kinase phosphorylation antibody array, we observed that silencing eIF3a could activate BMX, a tyrosine kinase. The BMX inhibitor CHMFL-BMX-078 could significantly suppress proliferation and induce cell cycle arrest in vemurafenib resistant melanoma cell line A375 (A375R), however, it was hypotoxic in immortal keratinocytes, melanoma cells, and other solid cancer cells such as glioma and breast cancer cells. Furthermore, the combined treatment of CHMFL-BMX-078 and vemurafenib synergistically reduced cell viability and restored the sensitivity of resistant cells to vemurafenib. The reversal of the resistant phenotype by CHMFL-BMX-078 was associated with the AKT signaling pathway, as co-treatment with the AKT activator SC-79 or up-regulation of AKT attenuated the anti-proliferation effect of CHMFL-BMX-078 and vemurafenib. Lastly, we demonstrated that CHMFL-BMX-078 could significantly enhance vemurafenib efficacy in a xenograft model of A375R cells without producing additive toxicity. In conclusion, these findings reveal that the BMX inhibitor CHMFL-BMX-078 may reverse vemurafenib resistance in melanoma by suppressing the AKT signaling pathway, implying that CHMFL-BMX-078 may be a promising compound for overcoming vemurafenib resistance.

Abstract P1-18-22: Unlocking Her2 as a target in invasive lobular cancer and endocrine resistant invasive ductal carcinoma

Abstract Background: Invasive Lobular Carcinoma (ILC) is a molecular subtype of breast cancer that is usually hormone receptor (HR) positive and E-cadherin negative. Ninety percent of ILC carry CDH1 mutations. Although ILC patients have a seemingly favorable outlook at diagnosis, they suffer from late recurrences and hence the long-term outcomes are not as favorable suggesting resistance to conventional therapies. In vitro and in vivo studies have demonstrated cross talk between EGFR/HER2 with HR in inducing endocrine resistance1. Increase in HER2 expression at the time of recurrence has been reported, while HER2 activating mutations occur in approximately 15.4% of ILC’s2. Importantly, HER2 mutations are associated with significantly worse prognosis in patients with CDH1 mutated ILC3. We believe that HER2 is a viable target in ILC, and endocrine resistant IDC when combined with endocrine therapy. HER2 targeted therapy, such as Trastuzumab is not currently used to treat patients with normal HER2 expression as a recent clinical trial NSAPB B-47 found that Trastuzumab has very little effect on these patients. We hypothesize that ONT-380 (Tucatinib), a tyrosine kinase inhibitor (TKI) that is currently in clinical trials for HER2 overexpressing metastatic breast cancer, will be effective in treating normal HER2 expressing ILC and HR positive IDC that are relatively endocrine resistant. Combined treatment with TKI and anti-estrogen could improve responses to endocrine therapy. Methods: We have studied effects of ONT-380 in combination with Tamoxifen on cell proliferation using in vitro cell culture models. We used ERα positive cell lines with varying levels of HER2 expression BT474 (HER2+), and cell lines expressing basal level of HER2, ZR-75-1 (IDC), T47D (IDC), SUM44PE (ILC), and MDA-MB-134-VI (ILC) to understand the effects of simultaneous targeting of HER2 and ERα. We have used MTT assay (Promega) to compare cell proliferation in drug treated and untreated cells. Western blot analysis was used to measure the levels of pHER2Y877, pHER2Y1221/1222,pAKTS473, total HER2 and total AKT. Basal level and drug induced alteration in phosphorylation status of downstream targets of HER2 and ERα signaling will be assessed using Human RTK (Receptor Tyrosine Kinase) phosphorylation antibody array (Abcam). Results: Western blot analysis of whole cell extracts showed that the designated HER2 positive cells (BT474) have higher level of HER2 protein, while the other cells (ZR-75-1, T47D, SUM44PE, MDA-MB-134) have significantly lower level of HER2 protein. HER2 was not detectable in MCF7 cells. Similarly, pAKT, a downstream effector of EGFR/ HER2 signaling was higher in the HER2 positive cell lines. Treatment of ILC cell line MDA-MB-134-VI with ONT-380 (100nM) or Tamoxifen (10 μM) alone or in combination showed additive inhibition of cell proliferation when the two drugs were combined as opposed to single drug treatment. We have observed similar effect of ONT-380 and Tamoxifen on SUM44PE, BT474 and ZR-75-1 cells. Furthermore, treatment of ZR-75-1 cells with ONT-380 and Tamoxifen markedly reduced pAKT level, while the effect of treatment with individual drugs was barely detectable. Similar studies are underway for endocrine resistant ILC and additional IDC cell lines. Conclusion: Targeting HER2 kinase activity in normal HER2 expressing ILC and endocrine resistant IDC with small molecule inhibitors of tyrosine kinase could be a viable option for treating these cancers. Further in vitro and in vivo studies are underway to study the efficacy of this drug combination. 1. Schafer et.al. 2002 (PMID: 12650703) 2. Deniziaut et.al. 2016 (PMID: 27602491) 3. Ping et.al. 2016 (PMID: 27811364) Citation Format: Nikhil Pramod, Sivanesan Dhandayuthapani, Sarmila Majumder, Bhuvaneswari Ramaswamy. Unlocking Her2 as a target in invasive lobular cancer and endocrine resistant invasive ductal carcinoma [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-22.

Protein tyrosine kinase 2: a novel therapeutic target to overcome acquired EGFR-TKI resistance in non-small cell lung cancer

BackgroundProtein tyrosine kinase 2 (PTK2) expression has been reported in various types of human epithelial cancers including lung cancer; however, the role of PTK2 in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) has not been elucidated. We previously reported that pemetrexed-resistant NSCLC cell line PC-9/PEM also acquired EGFR-TKI resistance with constitutive Akt activation, but we could not find a therapeutic target.MethodsCell viability in EGFR-mutant NSCLC cell lines was measured by the WST-8 assay. Phosphorylation antibody array assay for receptor tyrosine kinases was performed in PC-9 and PC-9/PEM cell lines. We evaluated the efficacy of EGFR and PTK2 co-inhibition in EGFR-TKI-resistant NSCLC in vitro. Oral defactinib and osimertinib were administered in mice bearing subcutaneous xenografts to evaluate the efficacy of the treatment combination in vivo. Both the PTK2 phosphorylation and the treatment combination efficacy were evaluated in erlotinib-resistant EGFR-mutant NSCLC cell lines.ResultsPTK2 was hyperphosphorylated in PC-9/PEM. Defactinib (PTK2 inhibitor) and PD173074 (FGFR inhibitor) inhibited PTK2 phosphorylation. Combination of PTK2 inhibitor and EGFR-TKI inhibited Akt and induced apoptosis in PC-9/PEM. The combination treatment showed improved in vivo therapeutic efficacy compared to the single-agent treatments. Furthermore, erlotinib-resistant NSCLC cell lines showed PTK2 hyperphosphorylation. PTK2 inhibition in the PTK2 hyperphosphorylated erlotinib-resistant cell lines also recovered EGFR-TKI sensitivity.ConclusionPTK2 hyperphosphorylation occurs in various EGFR-TKI-resistant NSCLCs. Combination of PTK2 inhibitor and EGFR-TKI (defactinib and osimertinib) recovered EGFR-TKI sensitivity in the EGFR-TKI-resistant NSCLC. Our study result suggests that this combination therapy may be a viable option to overcome EGFR-TKI resistance in NSCLC.

PRSS1 mutation: a possible pathomechanism of pancreatic carcinogenesis and pancreatic cancer

BackgroundPrevious studies revealed somatic mutations of the cationic trypsinogen gene (PRSS1) in patients with chronic pancreatitis and pancreatic cancer. However, whether PRSS1 mutations trigger pancreatic cancer and/or promote malignant proliferation and metastasis in pancreatic cancer remains largely unclear, as well as the potential underlying mechanisms.MethodsIn the present study, whole-exome sequencing was applied for screening, and the R116C mutation was validated by Sanger sequencing. Phosphorylation antibody array, RNA-Seq, and RT-qPCR were adopted to screen and validate that R116C mutation promoted pancreatic cancer progression via the JAK1-STAT5 pathway.ResultsIt showed that migration and invasion were significantly increased in R116C-bearing PANC-1 cells compared with wild type counterparts. In a transgenic mouse model of iZEG-PRSS1_R116C, primary pancreatic intraepithelial neoplasia (PanINs) was observed in the pancreatic duct.ConclusionsThese findings suggested a novel pathway mediating pancreatic cancer development, with PRSS1 mutation and overexpression playing an “inside job” role in pancreatic carcinogenesis and tumor development.

Abstract 1331: Receptor tyrosine kinases are differentially phosphorylated in metastatic castration resistant prostate cancer

Abstract Metastatic castration resistant prostate cancer (mCRPC) is the leading cause of death among prostate cancer (PCa) patients. Patients of African American (AA) origin are particularly disproportionately affected as they are less responsive to current therapies, mainly androgen deprivation therapy (ADT) that target androgen receptor signalling, which is less expressed in AA patients. Receptor tyrosine kinases (RTKs) have been implicated in cancer development and progression and inhibition of their activities has been effective in inhibiting cancer progression in other cancers. However, targeting RTK signalling has not yielded satisfactory results in PCas. The objective of this study was to identify precise RTKs that may be targetable in mCRPCs. An in-vitro model for primary vs mCRPC was developed from AA PCa cell lines: RC77T and RC43T. Briefly, invasive RC77T and RC43T cells were isolated in a cell invasion chamber. The invasive sublines were gradually exposed to increasing concentration (up to 10µM) of MDV3100 over a nine-month period to develop castration resistance. The resulting invasive and castration resistant sublines: RCMI77-CR3 and RCMI43-CR3 were subsequently cultured and maintained in K-SFM containing 10µM of MDV3100. Human Receptor Tyrosine Kinase Phosphorylation Antibody Array and Co-Immunoprecipitation protocols were used to identify differences in RTK activation. TCGA patient data was analysed using R to identify correlation between RTK mRNA expression and PCa progressiveness. MTT assay was used to evaluate effect of RTK inhibitors and MDV3100 treatment on cell survival. The RC77T and RC43T cells were epithelial in appearance, while RCMI77-CR3 and RCMI43-CR3 cells tended to be mesenchymal. The RCMI77-CR3 and RCMI43-CR3 sublines were more resistance to MDV3100 compared to their primary PCa counterparts. Human RTK Array identified four RTKs: KIT, LYN, ITK and RYK to be hyperphosphorylated in RCMI77-CR3 and RCMI43-CR3 relative to RC77T and RC43T, while three RTKs including EphA3, EphB1 and ErbB3 were hypo-phosphorylated. Immunoprecipitation and western blot analysis revealed differential expression of phosphotyrosine kinases between RC77T and RCMI77-CR3 cells. Analysis of TCGA patient data showed, mRNA expression level of only LYN and RYK differed significantly (p<0.05) significantly with disease progression. Analysis of effect of tyrosine kinase inhibitors on cell proliferation showed RCMI77-CR3 to be more responsive to dasatinib and axitinib compared to RC77T. No similar differences in response was observed for the inhibitors; SU6656, IWR-1, ICG-001 and BMS. The current study suggests, activation of specific RTKs are involved survival and drug resistance in mCRPC cells. Inhibiting these specific RTK signalling pathways in segments of patients with specific RTK hyperactivation could significantly improve survival. Citation Format: Raven A. Williams, Joakin Mori, Hui-Xian Lin, Alahni Becks, Clayton Yates, Honghe Wang. Receptor tyrosine kinases are differentially phosphorylated in metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1331.

MicroRNA-143 is Associated With Pathological Complete Response and Regulates Multiple Signaling Proteins in Breast Cancer.

Almost 55% to 80% of patients with breast cancer have an unfavorable pathological complete response to chemotherapy. MicroRNAs are small noncoding RNAs involved in cancer progression; however, their utility as predictors of pathological complete response to neoadjuvant chemotherapy is unclear. Here, we investigated if miR-143 could discriminate between pathological complete response and no-polymerase chain reaction of patients with locally advanced triple negative breast cancer that have received a fluorouracil-cisplatin/paclitaxel-based neoadjuvant treatment. Data showed that miR-143 exhibited a significant low expression (P < .0006) in patients that achieved pathological complete response in comparison to nonresponder group. Receiver operating characteristic curve analysis suggested that miR-143 could be a good predictor of pathological complete response (area under curve = 0.849, P < .0006). Moreover, Kaplan-Meier analysis indicated that before neoadjuvant therapy low levels of miR-143 were associated to increased disease free survival. To gain insights into cellular functions of miR-143, we firstly showed that miR-143 was severely repressed in breast cancer cell lines and tumors in comparison to normal mammary cells and tissues. Ectopic restoration of miR-143 using RNA mimics inhibited both cell proliferation and migration and sensitized breast cancer cells to cisplatin therapy in vitro. To decipher the signaling networks regulated by miR-143, we used a high-throughput enzyme-linked immunosorbent assay-based phosphorylation antibody array. Phospho-proteomic profiling revealed that miR-143 coordinately reduced the protein levels and phosphorylation status of multiple oncoproteins involved in AKT, WNT/β-catenin, SAPK/JNK, FAK, and JAK/STAT signaling pathways. Moreover, low miR-143 and high GSK3-β, RAF1, paxillin, and p21CIP1 expression levels in a large cohort of patients with breast cancer were associated with worst outcome. In summary, miR-143 could be a potential predictor of response to neoadjuvant therapy and it may function as a divergent regulator of diverse signaling networks to suppress cell proliferation and migration in breast cancer.

Cooperative multi-targeting of signaling networks by angiomiR-204 inhibits vasculogenic mimicry in breast cancer cells

RNA-based multi-target therapies focused in the blocking of signaling pathways represent an attractive approach in cancer. Here, we uncovered a miR-204 cooperative targeting of multiple signaling transducers involved in vasculogenic mimicry (VM). Our data showed that invasive triple negative MDA-MB-231 and Hs-578T breast cancer cells, but not poorly invasive MCF-7 cells, efficiently undergoes matrix-associated VM under hypoxia. Ectopic restoration of miR-204 in MDA-MB-231 cells leads to a potent inhibition of VM and reduction of number of branch points and patterned 3D channels. Further analysis of activation state of multiple signaling pathways using Phosphorylation Antibody Arrays revealed that miR-204 reduced the expression and phosphorylation levels of 13 proteins involved in PI3K/AKT, RAF1/MAPK, VEGF, and FAK/SRC signaling. In agreement with phospho-proteomic profiling, VM was impaired following pharmacological administration of PI3K and SRC inhibitors. Mechanistic studies confirmed that miR-204 exerts a negative post-transcriptional regulation of PI3K-α and c-SRC proto-oncogenes. Moreover, overall survival analysis of a large cohort of breast cancer patients indicates that low miR-204 and high FAK/SRC levels were associated with worst outcomes. In conclusion, our study provides novel lines of evidence indicating that miR-204 may exerts a fine-tuning regulation of the synergistic transduction of PI3K/AKT/FAK mediators critical in VM formation.

Expression of the serotonin receptor 2B in uveal melanoma and effects of an antagonist on cell lines.

Uveal melanoma (UM) is the most common primary tumor in the adult, and disseminates to the liver in half of patients. A 15-gene expression profile prognostic assay allows to determine the likelihood of metastasis in patients using their ocular tumor DNA, but a cure still remains to be discovered. The serotonin receptor 2B represents the discriminant gene of this molecular signature with the greatest impact on the prognosis of UM. However, its contribution to the metastatic potential of UM remains unexplored. The purpose of this study was to investigate the effects of a selective serotonin receptor 2B antagonist on cellular and molecular behaviours of UM cells. UM cell lines expressing high level of serotonin receptor 2B proteins were selected by Western blotting. The selective serotonin receptor 2B antagonist PRX-08066 was evaluated for its impact on UM cells using viability assays, phosphorylated histone H3 immunostainings, clonogenic assays, migration assays, invasion assays and membrane-based protein kinase phosphorylation antibody arrays. The pharmacological inhibition of the serotonin receptor 2B reduced the viability of UM cells and the population in mitosis, and impaired their clonogenicity and potential of migration. It also decreased the phosphorylation of kinases from signaling pathways classically activated by the serotonin receptor 2B, as well as kinases β-catenin, Proline-rich tyrosine kinase 2, and Signal transducer and activator of transcription 5. Our findings support a role for the serotonin receptor 2B in the proliferation and migration of UM cells, through activation of many signaling pathways such as WNT, Focal adhesion kinase and Janus kinase/STAT.

The Significance of MMP-1 in EGFR-TKI-Resistant Lung Adenocarcinoma: Potential for Therapeutic Targeting.

Epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI) resistance is one of the most important problems in lung cancer therapy. Lung adenocarcinoma with EGFR-TKI resistance was reported to have higher abilities of invasion and migration than cancers sensitive to EGFR-TKI, but the function of matrix metalloproteinases (MMPs) has not been explored in EGFR-TKI–resistant lung adenocarcinoma. This study aims to clarify the significance of MMP-1 in EGFR-TKI–resistant lung adenocarcinoma. From the results of in vitro studies of migration and invasion assays using EGFR-TKI–sensitive and –resistant cell lines and phosphorylation antibody arrays using EGF and rapamycin, we first demonstrate that overexpression of MMP-1, which might follow activation of a mammalian target of rapamycin (mTOR) pathway, plays an important role in the migration and invasion abilities of EGFR-TKI–resistant lung adenocarcinoma. Additionally, immunohistochemical studies using 89 cases of lung adenocarcinoma demonstrate that high expression of MMP-1 is significantly correlated with poor prognosis and factors such as smoking history and the subtype of invasive mucinous adenocarcinoma. These are consistent with the results of this in vitro study. To conclude, this study provides insights into the development of a possible alternative therapy manipulating MMP-1 and the mTOR signaling pathway in EGFR-TKI–resistant lung adenocarcinoma.

Peiminine serves as an adriamycin chemosensitizer in gastric cancer by modulating the EGFR/FAK pathway.

Gastric cancer (GC) is one of the most common malignancies of the digestive tract. Adriamycin (ADR) has been widely utilized in various chemotherapy regimens for treating GC, yet its long-term application may increase drug resistance resulting in treatment failure. Increasing evidence shows that bioactive natural products can be used as chemotherapeutic sensitizers that can significantly improve chemotherapy sensitivity. Peiminine(PMI) is a biologically active component extracted from Fritillaria walujewii Regel. Thus, in the present study, we aimed to investigate whether peiminine (PMI) alters the chemosensitivity of GC to adriamycin (ADR). GC cells were treated with ADR with or without PMI. MTT assay, flow cytometry and a nude mouse tumor xenograft model of SGC7901 cells were used to evaluate the chemosensitization activity of PMI combined with ADR. Western blotting was used to examine the expression of cyclinD1 and cleaved PARP. The RayBio® Human RTK phosphorylation antibody array kit was used to test the differential protein expression. Compared with the ADR group, PMI combined with ADR significantly suppressed cell proliferation and induced cell apoptosis invitro. The growth curve and tumor weight of the tumor xenografts were significantly decreased in mice treated with the combination of PMI and ADR. However, the organs showed no obvious abnormality after treatment with PMI plus ADR. The expression of cyclinD1 was decreased and the level of cleaved PARP was increased after treatment with PMI and ADR. The expression of p-EGFR and p-FAK was downregulated in cells treated with PMI and ADR, and the validation of p-EGFR and p-FAK was in accordance with the result of the phosphorylation antibody array kit. PMI may serve as a new chemosensitizer by inhibiting the proliferation and inducing the apoptosis to enhance the chemotherapeutic drug sensitivity of ADR in GC.

Non-Genomic Action of Androgens is Mediated by Rapid Phosphorylation and Regulation of Androgen Receptor Trafficking

Background: Testosterone is critical for maintaining spermatogenesis and male fertility. The accomplishment of these processes requires the synergistic actions of the classical and non-classical signaling pathways of androgens. Methods: A murine testicular Sertoli cell line, TM4 cell was used to examine androgen actions in Sertoli cells. Western blot analysis and immunofluorescence assay were employed to study the testosterone-induced Androgen receptor (AR) translocation. Protein phosphorylation antibody array was applied to identify the phosphorylation sites under testosterone treatment, and these findings were verified by Western blot analysis. Results: We found that a physiological dose of testosterone induced fast membrane association of AR. By using a phosphorylation antibody array, several phosphorylation sites, such as MEK1/2 (Ser217/221), Akt (Ser473), and Erk1/2 (Thr202/Tyr204) were rapidly phosphorylated within 5 min of testosterone treatment. Inhibition of the MEK and Akt signaling pathways prevented AR trafficking. Blocking of AR by flutamide eliminated the stimulation effect of testosterone on kinase phosphorylation. Testosterone induced kinase Src phosphorylation, and inhibition of Src restricted AR translocation to the membrane and the nucleus. Conclusion: Findings suggested that the membrane association of AR was mediated by the MEK and Akt phosphorylation signaling pathways, which resulted in Src activation and was initiated by testosterone binding to the membrane-localized AR. This study provides new insights into the testosterone signaling pathway in Sertoli cells, which mediate spermatogenesis. In addition, the study can be used in the diagnosis and treatment of male infertility caused by disorders in spermatogenesis.

The Osmopressor-Induced Angiopoietin-1 Secretion in Plasma and Subsequent Activation of the Tie-2/Akt/eNOS Signaling Pathway in Red Blood Cell.

Water ingestion induces the osmopressor response, which typically presents as increased total peripheral vascular resistance in young healthy subjects. A previous study has suggested that the RBC membrane receptor is involved in osmopressor stress. Recent studies have indicated nitric oxide synthase phosphorylation in RBCs. However, the main process in signaling pathway activation to elicit such a response is unknown. Herein, we hypothesized that hypo-osmotic stress following water ingestion modulates the eNOS/NO pathway, thereby alternating vascular resistance. We included 24 young, healthy subjects. Physiological parameters and blood samples were collected at 5 minutes before and 25 and 50 minutes after 50 ml water, 500 ml water, or 500 ml normal saline ingestion. A human receptor tyrosine kinase (RTK) phosphorylation antibody array was used to simultaneously detect and monitor the biological activation pathways in RBCs. Of the 71 RTKs assayed during the osmopressor response, several RTKs were significantly upregulated, including Tie-2 and Tie-1. Plasma angiopoietin-1 levels significantly increased at 25 minutes after 500 ml water ingestion compared to those at baseline. Simultaneous phosphorylation of Tie-2, Akt, and eNOS in RBCs occurred. RBCs in vitro were stimulated with angiopoietin-1, Tie-2, or 0.8% saline and showed significant increase in Tie-2, Akt, and eNOS phosphorylation upon angiopoietin-1 treatment and enhanced activation upon cotreatment of angiopoietin-1 and 0.8% saline. The hypo-osmotic stimulus of water ingestion increases angiopoietin-1 secretion and subsequently activates the Tie-2/Akt/eNOS signaling pathway in RBCs, thereby revealing a novel biological mechanism simultaneously occurring with the osmopressor response.

Pharmacologic Co-Blockade of IFNγR and IL6R Pathways to Prevent and Treat GvHD

The therapeutic benefits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematologic malignancies are primarily derived from an anti-leukemia effect that is mediated by T cells in donor grafts. Unfortunately, these T cells also mediate graft-versus-host disease (GvHD), the major complication of allo-HSCT.We have previously published that in vivo administration of JAK1/JAK2 inhibitors to murine allo-HSCT recipients of interferon gamma receptor deficient (IFNγR-/-) T cells results in 100% survival in a fully MHC-mismatched B6 to Balb/c allo-HSCT model (Choi et al., 2014, PLoS ONE). Since the infusion of IFNγR-/- T cells alone is associated with only ~70% survival, we hypothesize that JAK1/JAK2 inhibitors have either additional off-target effects or are inhibiting other non-IFNγR signaling pathways which are themselves dependent on JAK1/JAK2. The major other cytokine receptor signaling pathway mediated via both JAK1 and JAK2 is the interleukin 6 receptor (IL6R) signaling pathway. Thus, it is possible that JAK1/JAK2 inhibitors also block signaling through IL6R in addition to IFNγR. In addition, Alam et al. have recently reported that single nucleotide polymorphisms of donor IFNγ and IL6 are closely linked to gastrointestinal GvHD in patients (2015, BMT). Therefore, we examined if blockade of both IFNγR and IL6R signaling results in complete elimination of GvHD after a fully MHC-mismatched allo-HSCT in which B6 (H-2b) T cell-depleted bone marrow cells (5x106) along with B6 pan T cells (5x105) are intravenously injected into lethally irradiated Balb/c mice (H-2d). As shown in Fig. 1, we have found that blocking both IFNγR (IFNγR-/- T cells) and IL6R (α-IL6R Ab) signaling dramatically reduces GvHD and results in >95% survival. In addition, we found that blocking both IFNγR and IL6R signaling significantly increased regulatory T cells (Tregs) in peripheral blood (23.2% Foxp3+ in CD4+ T cells (n=17) vs 2.5% in WT T cell control (n=16) at day 27 after allo-HSCT), suggesting that increase in Tregs might be a potential mechanism underlying the reduced GvHD after dual blockade of IFNγR and IL6R signaling.Baricitinib is a potent and balanced JAK1/JAK2 inhibitor currently being clinically developed by Eli Lilly for the treatment of inflammatory diseases. We hypothesize that baricitinib will optimally block both IFNγR and IL6R signaling pathways and prevent GvHD. We found that that baricitinib is a potent suppressor of GvHD in B6 to Balb/c allo-HSCT models (100% survival), superior to ruxolitinib and similar to blockade of both IFNγR and IL6R signaling (Fig. 2A). Baricitinib increases Tregs in vivo (Fig. 2B) and reduces the ratio of IL5 (Th2 cytokine) to IL2 (cytokine for Treg induction) in plasma (p=0.0046), a potential diagnostic marker for GvHD (Fujii et al., 2006, Int J Mol Med), significantly better than ruxolitinib. Lastly, we found that baricitinib inhibits the expression of T-bet (Fig. 2C), which is the master transcription factor of Th1 cells, that are primary effector T cells in inducing GvHD. These data suggest that the suppression of GvHD by baricitinib results from increased Tregs and decreased Th1 and Th2 cells.We next examined if the prevention of GvHD by baricitinib is dependent on natural regulatory T cells (Tregs) in donor grafts. Tregs were depleted from donor pan T cells before allo-HSCT (B6 to Balb/c). We found that in vivo administration of baricitinib resulted in 70% survival (0% control, p<0.0001; 100% Treg-replete T cells + baricitinib. In addition, based on clinical GvHD score when recipients of Treg-replete T cells were compared with those of Treg-deplete T cells, the beneficial effect of Tregs in the donor grafts for the prevention of GvHD was observed only for the first two weeks after allo-HSCT (p≤0.01).Lastly, we examined whether baricitinib can cure ongoing GvHD by administering baricitinib starting at day 10 after allo-HSCT when GvHD is established (B6 to Balb/c). We found that baricitinib treatment results in a significant reduction of GvHD and 100% survival (10% control, p<0.0001).All of these data suggest that pharmacologic co-blockade of IFNγR and IL6R pathways is a promising therapeutic strategy to prevent and effectively treat established GvHD. The inhibitory effect of baricitinib, ruxolitinib, and blockade of IFNγR and IL6R on JAK-STAT signaling using JAK/STAT phosphorylation antibody arrays is currently being investigated and will be presented. [Display omitted] [Display omitted] DisclosuresDiPersio:Incyte Corporation: Research Funding.

Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells.

Tumor hypoxia is one of the important features of lung adenocarcinoma. Chemokines might mediate the effects caused by tumor hypoxia. As confirmed in tumor tissue and serum of patients, CC chemokine 28 (CCL28) was the only hypoxia induced chemokine in lung adenocarcinoma cells. CCL28 could promote tube formation, migration and proliferation of endothelial cells. In addition, angiogenesis was promoted by CCL28 in the chick chorioallantoic membrane and matrigel implanted in dorsal back of athymic nude mice (CByJ.Cg-Foxn1nu/J). Tumors formed by lung adenocarcinoma cells with high expression of CCL28 grew faster and had a higher vascular density, whereas tumor formation rate of lung adenocarcinoma cells with CCL28 expression knockdown was quite low and had a lower vascular density. CCR3, receptor of CCL28, was highly expressed in vascular endothelial cells in lung adenocarcinoma when examining by immunohistochemistry. Further signaling pathways in endothelial cells, modulated by CCL28, were analyzed by Phosphorylation Antibody Array. CCL28/CCR3 signaling pathway could bypass that of VEGF/VEGFR on the levels of PI3K-Akt, p38 MAPK and PLC gamma. The effects could be neutralized by antibody against CCR3. In conclusion, CCL28, as a chemokine induced by tumor hypoxia, could promote angiogenesis in lung adenocarcinoma through targeting CCR3 on microvascular endothelial cells.

Inhibition of ABCB1 Overcomes Cancer Stem Cell–like Properties and Acquired Resistance to MET Inhibitors in Non–Small Cell Lung Cancer

Patients with non-small cell lung cancer (NSCLC) EGFR mutations have shown a dramatic response to EGFR inhibitors (EGFR-TKI). EGFR T790M mutation and MET amplification have been recognized as major mechanisms of acquired resistance to EGFR-TKI. Therefore, MET inhibitors have recently been used in NSCLC patients in clinical trials. In this study, we tried to identify the mechanism of acquired resistance to MET inhibitors. We analyzed the antitumor effects of two MET inhibitors, PHA-665752 and crizotinib, in 10 NSCLC cell lines. EBC-1 cells with MET amplification were the only cells that were sensitive to both MET inhibitors. We established PHA-665752-resistant EBC-1 cells, namely EBC-1R cells. Activation of KRAS, EGFR, and FGFR2 signaling was observed in EBC-1R cells by FISH and receptor tyrosine kinase phosphorylation antibody arrays. EBC-1R cells also showed overexpression of ATP-binding cassette subfamily B member 1 (ABCB1) as well as phosphorylation of MET. EBC-1R cells grew as cell spheres that exhibited cancer stem cell-like (CSC) properties and epithelial-mesenchymal transition (EMT). The level of miR-138 that targeted ABCB1 was decreased in EBC-1R cells. ABCB1 siRNA and the ABCB1 inhibitor elacridar could reduce sphere numbers and suppress EMT. Elacridar could also reverse resistance to PHA-665752 in EBC-1R cells. Our study demonstrated that ABCB1 overexpression, which was associated with CSC properties and EMT, was involved in the acquired resistance to MET inhibitors. Inhibition of ABCB1 might be a novel therapeutic strategy for NSCLC patients with acquired resistance to MET inhibitors.

PH sensitive nano layered double hydroxides reduce the hematotoxicity and enhance the anticancer efficacy of etoposide on non-small cell lung cancer

Etoposide (VP16), used for the treatment of many carcinomas, can cause leukopenia, thrombocytopenia and hair loss. To overcome the side effects and achieve target therapy, layered double hydroxides (LDHs), a pH sensitive layered double hydroxide nanohybrid, was used here as a nano-carrier. The functions of LDHs-VP16 on non-small cell lung cancer (NSCLC) were firstly explored both in vitro and in vivo. In A549 cell line, LDH-VP16 induced apoptosis 2.3-fold as that of plain VP16 by targeting to mitochondrial, stocking cells in G1 phase. The cellular uptake demonstrated the delivery of LDH for VP16 to pass through the membrane and accumulate in mitochondria. As a carrier, LDH greatly decreased the liver toxicity and hematotoxicity of VP16. The detected liver parameters, including glutamic-oxaloacetic transaminase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), were all turn back to normal range after the delivery of LDH, except ALP. In vivo, LDH-VP16 reduced A549 tumor growth significantly by 60.5%, whereas native VP16 exerted no significant anticancer activity. In LDH-VP16 treated mice, the AUC was increased by 6.26 folds as the native drug, and t1/2 of LDH-VP16 was prolonged from 6.68 to 98.78h. LDH-VP16 showed a targeting effect, which largely increase the concentration in tumor and lung. The phosphorylation antibody array and Western Blot of proteins from xenografts revealed that PI3K–AKT signaling was suppressed in the LDH-VP16 treated tumor, while in VP16 treated mice, ERBB signaling pathway was involved. These results suggested that LDH-VP16 diminishes hematotoxicity, targets NSCLC tumor, performs more effectively than VP16, and different signaling pathway is involved compared to VP16. Statement of significanceThis paper explored that nano-sized layered double hydroxide (LDH) could be used as a pH sensitive delivery system to overcome hematotoxicity and enhance the bioavailability and anticancer efficacy of etoposide (VP16) against non small cell lung cancer, which was not reported before, as the best of our knowledge. We found that the liver and hematotoxicity is nearly recovered after the loading of VP16 in pH sensitive LDH, which prongs the half time from 6.68h to 98h, helps target VP16 to tumor and lung, and protects white blood cells by its pH sensitive and nano-size property. LDH-VP16 achieve markedly performance on non-small cell lung cancer by targeting to mitochondria of A549 cells in vitro and effectively inhibiting the PI3K–AKT signaling pathway in vivo. The inhibition ratio of VP16 on A549 tumor growth is increased from less than 20% (no significance compared to control) to 60.5% after the delivery of LDH. This work provides a novel system for the safe and efficient use of etoposide on non-small cell lung cancer and explores the mechanism of the function of nano carrier in cancer therapy both in vitro and in vivo.