Src Pathways Research Articles

Abstract P3-11-09: ARISTACAT - Aromatase inhibition plus minus saracatinib as advanced breast cancer therapy: A randomised phase II study of aromatase inhibition plus/minus the Src-inhibitor AZD0530 in post-menopausal women with advanced breast cancer

Abstract Background: Src, the first proto-oncogene to be described, is a non-receptor tyrosine kinase interacting with multiple oncogenic pathways, including cell proliferation, survival, angiogenesis and osteoclast activity. Src activation occurs in up to 40% of ER+ breast cancers, is linked with poor prognosis, and pre-clinically has been strongly implicated in endocrine resistance. Saracatinib (AZD0530) is a potent, oral selective Src inhibitor that enhances the anti-proliferative effect of endocrine agents in pre-clinical breast cancer models, preventing the development of endocrine-resistance and restoring endocrine sensitivity. In a murine model of prostate bony metastases saracatinib inhibits osteoclast activity, reducing bone resorption and the development and progression of bone lesions. Clinically, saracatinib is generally well-tolerated with mainly gastro-intestinal adverse events (AEs), clinically meaningful durations of stable disease as monotherapy, and partial responses observed in combination with other therapies. Methods: Multi-centre, placebo-controlled, double-blind, randomised phase II trial. Post-menopausal women with advanced/metastatic breast cancer suitable for 1st or 2nd second line hormonal treatment, were randomised to receive an aromatase inhibitor (AI) plus saracatinib 175mg mg/day or placebo. Patients were stratified as either (i) “AI-sensitive/naïve”, who received anastrazole 1mg daily ± saracatinib, or (ii) “prior-AI”, if they had progressed on a non-steroidal AI but were considered likely to retain some endocrine sensitivity, who received exemestane 25mg daily ± saracatinib. Other stratification factors were bone metastases, bisphosphonate use, performance status, and treatment centre. Treatment was until progression, intolerable toxicity or at the patient’s request. The primary endpoint was PFS; secondary endpoints were toxicity, ORR & OS; exploratory endpoints explored molecular correlates in on-treatment samples (including optional biopsies). Results: 140 patients were enrolled from 22 UK sites between August 2012 and April 2015 of whom 69 were in the “AI-sensitive/naïve” group and 71 in the “prior-AI” group; 134 patients were eligible for efficacy evaluation and 136 for safety. Interim safety data analyses by an IDMC identified no new safety signals. Treatment emergent AEs were generally low grade, the most frequent being fatigue (70%) and nausea (49%); AEs occurring significantly (p<0.05) more often with saracatinib were hypophosphataemia (40% vs 6%), rash (34% vs 12%), anorexia (43% vs 19%), vomiting (33% vs 15%) and diarrhoea (31% vs 16%). More patients receiving saracatinib had a dose reduction or stopped treatment due to toxicity (16% vs 10% and 5% vs 3%, respectively; n.s.). There was no indication of greater efficacy from the addition of saracatinib with respect to PFS (3.7 v 5.6 months; n.s.) or OS (24 vs 23 months; n.s.). ORR appeared lower with saracatinib (11% vs 31%) even when excluding non-evaluable patients. Effects were consistent in the “AI-sensitive/naïve” and “prior-AI” groups. Patients receiving saracatinib were no less likely to progress with bone metastases, while bisphosphonate use was associated with an increased PFS and OS across the trial population. Translational work underway includes assessing Src pathway suppression. Conclusions: Saractinib did not improve outcomes in post-menopausal women with advanced breast cancer treated with exemestane with numerically inferior p response rate and PFS, and no apparent beneficial effect on bone metastases. These data do not support further evaluation of saracatinib in combination with AIs. ARISTACAT - Abstract - San Antonio 2019 Citation Format: David Cameron, Stefan Symeonides, Murray Brunt, Peter Schmid, Simon Waters, Christopher Twelves, Peter Barrett-Lee, John Bartlett, Tammy Piper, Karen McAdam, Eve Macdonald Chisholm, Michelle Welsh, Robert Hill. ARISTACAT - Aromatase inhibition plus minus saracatinib as advanced breast cancer therapy: A randomised phase II study of aromatase inhibition plus/minus the Src-inhibitor AZD0530 in post-menopausal women with advanced breast cancer [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 P3-11-09.

Salvianolic acid A suppresses MMP-2 expression and restrains cancer cell invasion through ERK signaling in human nasopharyngeal carcinoma

Ethnopharmacological relevanceSalvia miltiorrhiza Bunge, as known as Danshen, has used for the prevention and treatment of cardiovascular diseases clinically and anti-cancer activities. Salvianolic acid A (SAA), one of the most abundant ingredients, hydrophilic derivatives of Salvia miltiorrhiza Bunge, exerts a variety of pharmacological actions, such as anti-oxidative, anti-inflammatory and anti-cancer activities. However, the impact of SAA on nasopharyngeal carcinoma (NPC) invasion and metastasis remains unexplored. Aim of the studyTo investigate the potential of SAA to prevent migration and invasion on NPC cell. Materials and methodsMTT assay and Boyden chamber assay were performed to determine cell proliferation, migration and invasion abilities, respectively. The activity and protein expression of matrix metalloproteinase-2 (MMP-2) were determined by gelatin zymography and western blotting. ResultsHere, we showed that SAA considerably suppressed the migrative and invasive activity of human NPC cells but not rendered cytotoxicity. In SAA-treated NPC cells, the activity and expression of matrix metalloproteinase-2 (MMP-2), a key regulator of cancer cell invasion, were reduced. Additionally, the presence of high concentrations of SAA dramatically abolished the activation of focal adhesion kinase (FAK) and moderately inhibited the phosphorylation of Src and ERK in NPC cells. ConclusionsOur results demonstrated that SAA inhibited the migration and invasion of NPC cells, accompanied by downregulation of MMP-2 and inactivation of FAK, Src, and ERK pathways. These findings indicate a usefulness of SAA on restraining NPC invasion and metastasis.

Convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer

BackgroundAberrant activation of STAT3 is frequently encountered and promotes survival, cellular proliferation, migration, invasion and angiogenesis in tumor cell. Convallatoxin, triterpenoid ingredient, exhibits anticancer pharmacological properties. PurposeIn this work, we investigated the anticancer potential of convallatoxin and explored whether convallatoxin mediates its effect through interference with the STAT3 activation in colorectal cancer cells. MethodsIn vitro, the underlying mechanisms of convallatoxin at inhibiting STAT3 activation were investigated by homology modeling and molecular docking, luciferase reporter assay, MTT assay, RT-PCR, Western blotting and immunofluorescence assays. Changes in cellular proliferation, apoptosis, migration, invasion and angiogenesis were analyzed by EdU labeling assay, colony formation assay, flow cytometry assay, wound-healing assay, matrigel transwell invasion assay and tube formation assays. And in vivo, antitumor activity of convallatoxin was assessed in a murine xenograft model of HCT116 cells. ResultsConvallatoxin decreased the viability of colorectal cancer lines. Moreover, convallatoxin reduced the P-STAT3 (T705) via the JAK1, JAK2, and Src pathways and inhibited serine-727 phosphorylation of STAT3 via the PI3K-AKT-mTOR-STAT3 pathways in colorectal cancer cells. Interestingly, we discovered the crosstalk between mTOR and JAK2 in mTOR/STAT3 and JAK/STAT3 pathways, which collaboratively regulated STAT3 activation and convallatoxin play a role in it. Convallatoxin also downregulated the expression of target genes involved cell survival (e.g., Survivin, Bcl-xl, Bcl-2), proliferation (e.g., Cyclin D1), metastasis (e.g., MMP-9), and angiogenesis (e.g., VEGF). Indeed, we found that convallatoxin inhibited tube formation, migration, and invasion of endothelial cells, and inhibited the proliferation. Finally, in vivo observations were confirmed by showing antitumor activity of convallatoxin in a murine xenograft model. ConclusionThe result of the current study show that convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer cells and indicate that convallatoxin could be a valuable candidate for the development of colorectal cancer therapeutic.

Synergistic activation of Src, ERK and STAT pathways in PBMCs for Staphylococcal enterotoxin A induced production of cytokines and chemokines.

Staphylococcal enterotoxin A (SEA) is a well-known superantigen and stimulates human peripheral blood mononuclear cells (PBMCs) involving in the pathogenesis of inflammatory disorders and cancer. To better understand the biological activities of SEA and the possible intracellular mechanisms by which SEA plays its roles in conditions like staphylococcal inflammatory and/or autoimmune disorders and immunotherapy. Recombinant SEA (rSEA) was expressed in a prokaryotic expression system and its effects on the cytokine and chemokine production was examined by Enzyme-linked Immunospot (ELISpot) Assay and ELISA analysis. In vitro experiments showed rSEA could significantly enhance secretion of a broad spectrum of cytokines and chemokines from PBMCs dose-dependently. Increased secretion of cytokines and chemokines from rSEA stimulated PBMCs was barely affected by C-C motif chemokine receptor 2 (CCR2) antagonist INCB3344. However, Src, ERK and STAT pathway inhibitors were able to successfully block the enhanced secretion of most of cytokines and chemokines produced by rSEA stimulated PBMCs. Our work suggested that rSEA serves as a potent stimulant of PBMCs, and induces the release of cytokines and chemokines through Src, ERK and STAT pathways upon a relatively independent network. Our work also strongly supported that Src, ERK and STAT signaling inhibitors could be effective therapeutic agents against diseases like toxic shock syndrome or infection by microbes resistant to antibiotics.

Netrin-1 works with UNC5B to regulate angiogenesis in diabetic kidney disease.

Netrin-1, an axon guidance factor, and its receptor UNC5B play important roles in axonal development and angiogenesis. This study examined netrin-1 and UNC5B expression in kidneys with diabetic kidney disease (DKD) and investigated their roles in angiogenesis. Netrin-1 and UNC5B were upregulated in streptozotocininduced DKD Wistar rats, and their expression was compared with that in healthy controls. However, exogenous netrin-1 in UNC5B-depleted human renal glomerular endothelial cells (HRGECs) inhibited cell migration and tubulogenesis. This effect was likely associated with SRC pathway deactivation. Netrin-1 treatment also eliminated the pro-angiogenic effects of exogenous VEGF-165 on UNC5B-silenced HRGECs. These results indicate that UNC5B antagonizes netrin-1 and that UNC5B upregulation contributes partly to enhancing angiogenesis in DKD. Therefore, introducing exogenous netrin-1 and depleting endogenous UNC5B are potential strategies for reducing the incidence of early angiogenesis and mitigating kidney injury in DKD.

Scutellarin Prevents Angiogenesis in Diabetic Retinopathy by Downregulating VEGF/ERK/FAK/Src Pathway Signaling.

Background Diabetic retinopathy (DR) is a serious microvascular complication of diabetes. This study demonstrates the antiangiogenic effects of scutellarin (SCU) on high glucose- and hypoxia-stimulated human retinal endothelial cells (HRECs) and on a diabetic rat model by oral administration. The antiangiogenic mechanisms of SCU in vitro and in vivo were investigated. Method HRECs were cultured in high glucose- (30 mM D-glucose) and hypoxia (cobalt chloride-treated)-stimulated diabetic condition to evaluate the antiangiogenic effects of SCU by CCK-8 test, cell migration experiment (wound healing and transwell), and tube formation experiment. A streptozotocin-induced type II diabetic rat model was established to measure the effects of oral administration of SCU on protecting retinal microvascular dysfunction by Doppler waveforms and HE staining. We further used western blot, luciferase reporter assay, and immunofluorescence staining to study the antiangiogenic mechanism of SCU. The protein levels of phospho-ERK, phospho-FAK, phospho-Src, VEGF, and PEDF were examined in HRECs and retina of diabetic rats. Result Our results indicated that SCU attenuated diabetes-induced HREC proliferation, migration, and tube formation and decreased neovascularization and resistive index in the retina of diabetic rats by oral administration. SCU suppressed the crosstalk of phospho-ERK, phospho-FAK, phospho-Src, and VEGF in vivo and in vitro. Conclusions These results suggested that SCU can be an oral drug to alleviate microvascular dysfunction of DR and exerts its antiangiogenic effects by inhibiting the expression of the crosstalk of VEGF, p-ERK, p-FAK, and p-Src.

Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer.

Advanced prostate cancer is a very heterogeneous disease reflecting in diverse regulations of oncogenic signaling pathways. Aberrant spatial dynamics of epidermal growth factor receptor (EGFR) promote their dimerization and clustering, leading to constitutive activation in oncogenesis. The EphB2 and Src signaling pathways are associated with the reorganization of the cytoskeleton leading to malignancy, but their roles in regulating EGFR dynamics and activation are scarcely reported. Using single-particle tracking techniques, we found that highly phosphorylated EGFR in the advanced prostate cancer cell line, PC3, was associated with higher EGFR diffusivity, as compared with LNCaP and less aggressive DU145. The increased EGFR activation and biophysical dynamics were consistent with high proliferation, migration, and invasion. After performing single-cell RNA-seq on prostate cancer cell lines and circulating tumor cells from patients, we identified that upregulated gene expression in the EphB2 and Src pathways are associated with advanced malignancy. After dasatinib treatment or siRNA knockdowns of EphB2 or Src, the PC3 cells exhibited significantly lower EGFR dynamics, cell motility, and invasion. Partial inhibitory effects were also found in DU145 cells. The upregulation of parts of the EphB2 and Src pathways also predicts poor prognosis in the prostate cancer patient cohort of The Cancer Genome Atlas. Our results provide evidence that overexpression of the EphB2 and Src signaling pathways regulate EGFR dynamics and cellular aggressiveness in some advanced prostate cancer cells.

RON Receptor Tyrosine Kinase Regulates Epithelial Mesenchymal Transition and the Expression of Pro-Fibrotic Markers via Src/Smad Signaling in HK-2 and NRK49F Cells.

Receptor tyrosine kinases (RTKs) play important roles in the pathogenic processes of kidney fibrosis. However, the pathophysiological roles of recepteur d’origine nantais (RON), one of the receptor tyrosine kinases, have not yet been defined. We investigated whether the activation or sequence-specific small interfering RNA (siRNA) suppression of RON could regulate epithelial mesenchymal transition (EMT) and the expression of pro-fibrotic markers, and its underlying molecular mechanisms. Stable cell lines and transient transfection for RON and the transfected cells of siRNA for RON were developed to investigate the molecular mechanisms in human kidney proximal tubular epithelial (HK-2) and interstitial fibroblasts (NRK49F) cells. RON overexpression induced EMT and increased expression of fibrosis-related proteins such as N-cadherin, vimentin, transforming growth factor-β (TGFβ), αSMA, and fibronectin in HK-2 and NRK49F cells. RON overexpression increased various RTKs and the phosphorylation of Src (Y416) and Smad, while inhibition of RON by siRNA attenuated the expression of EMT- and fibrosis-related proteins and decreased RTKs such as insulin-like growth factor receptor (IGFR), fibroblast growth factor receptor 1 (FGFR1), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR), as well as the phosphorylation of Src and Smad pathways. siRNA silencing of Src also attenuated the expression of IGFR, FGFR1, VEGFR, and PDGFR. Inhibition of RON can exert an anti-fibrotic effect by the inhibition of EMT and other RTKs through control of Src and Smad pathways in HK-2 and NRK49F cells.

I.19 New Insights of EGFR Inhibition: How to Overcome Resistance & New Agents

The main mechanism of resistance to first-generation EGFR TKIs is a single-nucleotide transition mutation in EGFR, cytosine to thymine (C>T) at position 2369 (ACG/ATG) causing threonine to methionine amino acid change at codon 790 (T790M). The T790M mutation occurs in cis with the original driver mutation and leads to steric hindrance and increased binding affinity for ATP. Activation-induced cytosine deaminase (AICDA) is expressed in germinal center B-lymphocytes upon antigen exposure. AICDA translocates to the nucleus, deaminates cytosine in single stranded DNA and converts it to uracil. Gefitinib, afatinib or osimertinib increase AICDA expression. A significant increase in the AICDA expression occurs in the PC9 and other EGFR-mutant cell lines upon treatment with different EGFR TKIs. AICDA is induced through the non-canonical nuclear factor-ĸB (NFĸB) (Figure). NFĸB was noted to be a mechanism of resistance to erlotinib and further studies of the Bivona group have further demonstrated that NFĸB is activated in EGFR-mutant LADCs. AICDA knockdown decreases the frequency of T790M and targeting AICDA activity directly or indirectly with NFĸB inhibitors, as well as DNA damage response inhibitors, could delay or prevent the appearance of T790M. The function of AICDA supports the rationale for an NFĸB-driven generation of T790M. It was demonstrated that, regardless of the use of gefitinib or osimertinib, NFĸB, STAT3 and YAP1 were activated in EGFR-mutant cell lines, including the H1975 (that harbors T790M). The downstream effectors were inhibited when the EGFR TKI was combined with blockers of the STAT3 and Src pathway. Osimertinib showed a PFS of 10.1 months versus 4.4 with platinum-pemetrexed chemotherapy in EGFR-mutant patients with T790M. Screening for EGFR mutations is of great usefulness in customizing therapy in LADCs. However, combinatory therapies with EGFR TKIs is warranted to avoid the mechanisms of drug resistance that occur following single EGFR TKIs. The activation of STAT3 occurred a few hours after treatment with EGFR TKIs. AKT pathway is commonly activated following treatment with first- or third-generation EGFR TKIs (Figure). In addition, co-expression of other receptor tyrosine kinases is commonly present with EGFR mutations. We demonstrated that AXL expression can be driven by the Src- yes-associated protein 1 (YAP1) activation, providing a glimpse for adequate combinatory approaches. Recent articles identify AXL as a mechanism of intrinsic and acquired resistance to osimertinib. YAP1 directly regulates the expression of PD-L1 in EGFR TKI-resistant LADCs. Another downstream central effector is the protein tyrosine phosphatase SHP2 (PTPN11). The urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1) correlates with poor prognosis in EGFR-mutant LADCs (Figure). The suppression of CPS1 potentiates the effects of EGFR inhibition and, therefore, opens an additional angle for optimizing therapy in EGFR-mutant LADCs that could most likely be extrapolated to other, oncogene driven LADCs. Among the transcriptional targets of YAP1 and its effector Forkhead box protein M1 (FOXM1), are the aurora kinases A and B (AURKA and AURKB) (Figure). Both AURKA and AURKB have been associated with resistance to EGFR TKIs. EGFR-mutant LADCs do not respond to immune checkpoint inhibitors. NFĸB, T790M, STAT3, CPS1, LADCs

Mouse models of thyroid cancer: Bridging pathogenesis and novel therapeutics.

Due to a global increase in the incidence of thyroid cancer, numerous novel mouse models were established to reveal thyroid cancer pathogenesis and test promising therapeutic strategies, necessitating a comprehensive review of translational medicine that covers (i) the role of mouse models in the research of thyroid cancer pathogenesis, and (ii) preclinical testing of potential anti-thyroid cancer therapeutics. The present review article aims to: (i) describe the current approaches for mouse modeling of thyroid cancer, (ii) provide insight into the biology and genetics of thyroid cancers, and (iii) offer guidance on the use of mouse models for testing potential therapeutics in preclinical settings. Based on research with mouse models of thyroid cancer pathogenesis involving the RTK, RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, SRC, and JAK-STAT signaling pathways, inhibitors of VEGFR, MEK, mTOR, SRC, and STAT3 have been developed as anti-thyroid cancer drugs for "bench-to-bedside" translation. In the future, mouse models of thyroid cancer will be designed to be ''humanized" and "patient-like," offering opportunities to: (i) investigate the pathogenesis of thyroid cancer through target screening based on the CRISPR/Cas system, (ii) test drugs based on new mouse models, and (iii) explore the underlying mechanisms based on multi-omics.

Leptin Promotes Expression of EMT-Related Transcription Factors and Invasion in a Src and FAK-Dependent Pathway in MCF10A Mammary Epithelial Cells.

Leptin is one of the main adipokines secreted in breast tissue. Leptin promotes epithelial–mesenchymal transition (EMT), cell migration and invasion in epithelial breast cells, leading to tumor progression. Although, the molecular mechanisms that underlie these events are not fully understood, the activation of different signaling pathways appears to be essential. In this sense, the effects of leptin on the activation of kinases like Src and FAK, which regulate signaling pathways that activate the EMT program, are not completely described. Therefore, we investigated the involvement of these kinases using an in vitro model for leptin-induced EMT process in the non-tumorigenic MCF10A cell line. To this end, MCF10A cells were stimulated with leptin, and Src and FAK activation was assessed. Specific events occurring during EMT were also evaluated in the presence or absence of the kinases’ chemical inhibitors PP2 and PF-573228. For instance, we tested the expression and subcellular localization of the EMT-related transcription factors Twist and β-catenin, by western blot and immunofluorescence. We also evaluated the secretion and activation of matrix metalloproteases (MMP-2 and MMP-9) by gelatin zymography. Invasiveness properties of leptin-stimulated cells were determined by invadopodia formation assays, and by the Transwell chamber method. Our results showed that leptin promotes EMT through Src and FAK activation, which leads to the secretion and activation of MMP-2 and MMP-9, invadopodia formation and cell invasion in MCF10A cells. In conclusion, our data suggest that leptin promotes an increase in the expression levels of Twist and β-catenin, the secretion of MMP-2, MMP-9, the invadopodia formation and invasion in MCF10A cells in a Src and FAK-dependent manner.

Effects of Lidocaine and Src Inhibition on Metastasis in a Murine Model of Breast Cancer Surgery.

Breast cancer recurs in 20% of patients following intended curative resection. In vitro data indicates that amide local anaesthetics, including lidocaine, inhibit cancer cell metastasis by inhibiting the tyrosine kinase enzyme Src. In a murine breast cancer surgery model, systemic lidocaine reduces postoperative pulmonary metastases. We investigated whether the additional administration of bosutinib (a known Src inhibitor) influences lidocaine’s observed beneficial effect in this in vivo model. Female BALB/c mice (n = 95) were inoculated with 25,000 4T1 cells into the mammary fad pad and after 7 days the resulting tumours were excised under sevoflurane anaesthesia. Experimental animals were randomized to one of four treatments administered intravenously prior to excision: lidocaine, bosutinib, both lidocaine and bosutinib in combination, or saline. Animals were euthanized 14 days post-surgery and lung and liver metastatic colonies were evaluated. Post-mortem serum was analysed for MMP-2 and MMP-9, pro-metastatic enzymes whose expression is influenced by the Src pathway. Lidocaine reduced lung, but not liver metastatic colonies versus sevoflurane alone (p = 0.041), but bosutinib alone had no metastasis-inhibiting effect. When combined with lidocaine, bosutinib reversed the anti-metastatic effect observed with lidocaine on sevoflurane anaesthesia. Only lidocaine alone reduced MMP-2 versus sevoflurane (p = 0.044). Both bosutinib (p = 0.001) and bosutinib/lidocaine combined (p = 0.001) reduced MMP-9 versus sevoflurane, whereas lidocaine alone did not. In a murine surgical breast cancer model, the anti-metastatic effects of lidocaine under sevoflurane anaesthesia are abolished by the Src inhibitor bosutinib, and lidocaine reduces serum MMP-2. These results suggest that lidocaine may act, at least partly, via an inhibitory effect on MMP-2 expression to reduce pulmonary metastasis, but whether this is due to an effect on Src or via another pathway remains unclear.

SRC and MEK Co-inhibition Synergistically Enhances the Anti-tumor Effect in Both Non-small-cell Lung Cancer (NSCLC) and Erlotinib-Resistant NSCLC

Non-small-cell lung cancer (NSCLC) is the predominant form of lung cancer, and it is regulated by a complex signal transduction network. Single-agent targeted therapy often results in acquired resistance, which leads to treatment failure. In this study, we demonstrated that a combination of the kinase inhibitors trametinib and bosutinib can synergistically suppress the growth of NSCLC by inhibiting both the mitogen-activated protein kinase (MAPK) and proto-oncogene tyrosine-protein kinase (SRC) pathways. The combination was profiled against a panel of 22 NSCLC cell lines, including one erlotinib-resistant cell line, and this combination was found to show synergistic effects against 16 cell lines. NSCLC cell lines (HCC827, HCC827-erlotinib-resistant, and H1650) were treated with trametinib, bosutinib, or a combination of these drugs. The drug combination inhibited colony formation and induced cell apoptosis. A mechanism study showed that the phosphorylation of multiple kinases in the epidermal growth factor receptor (EGFR) signaling pathway in NSCLC was down-regulated. In addition, the combination significantly attenuated tumor growth of HCC827 xenografts with low toxicity. Our findings provide a theoretical basis for further study of the combination of MAPK and SRC pathway inhibitors in NSCLC, especially in the treatment of erlotinib-resistant NSCLC.

Abstract 173: Upregulated EPHB2 and SRC pathways modulate spatial EGFR dynamics and malignant phenotypes and predict poor prognosis in prostate cancer

Abstract Dysregulated dynamics and trafficking of receptor tyrosine kinases (RTK) have been linked to oncogenesis and metastasis. The mechanisms for disfunction of RTK dynamics are emerging and still not clear. Using single-particle tracking (SPT) techniques, we studied the dynamics and trafficking patterns of epidermal growth factor receptor (EGFR) and underlying mechanisms using prostate cancer cells (PCa) (LNCaP, DU145 and PC3) exhibiting variant metastatic capability. Through SPT and super-resolution imaging, it was revealed that cortical actin disorganization modulates increased dynamics of EGFRs, including high EGFR diffusivity, enlarged EGFR confinement size on the plasma membrane and faster EGFR internalization in advanced invasive cells. The elevated EGFR dynamics were associated with advanced aggressive behaviors, and highly upregulated EGFR, EPHB and SRC signaling as identified using single cell RNA-seq. Strong EGFR activation with relatively low EGFR expression in PC3 implicated some other mechanisms, likely biophysical, beyond EGFR quantity. Moreover, the upregulated EPHB and SRC pathways have been shown to regulate actin organization and metastasis and our in silico analysis indicated that genes in the two pathways predict poor prognosis for disease free and survival statuses in The Cancer Genome Atlas prostate cancer patient cohort. To interrogate the roles of EPHB and SRC pathways in actin organization and EGFR dynamics, a series of drug inhibitions and siRNA knockdown were applied to PCa cells followed by mRNA profiling, evaluations of EGFR dynamics, and cell behaviors. Functional knockdown of EPHB2 and SRC led to decreased EGFR dynamics, cell proliferation, migration and invasion. The loss-of-function effects were more profound in advanced invasive PCa. In this study, we discovered the roles of EPHB and SRC pathways in modulating actin organization and EGFR dynamics and leading to aggressive metastatic phenotypes. Additionally, EGFR dynamics were potential biophysical parameters to differentiate the highly-invasive from the non- and less-invasive PCa. Thus, we believe that the SPT-based EGFR dynamics can serve as a new biophysical assay to probe the metastatic malignancy of cancer cells and to monitor their response to anti-cancer drug treatment. Citation Format: Yen-Liang Liu, Aaron M. Horning, Che-Kuang Lin, Brandon Lieberman, Chia-Nung Hung, Chih-Wei Chou, Chiou-Miin Wang, Michael A. Liss, Mirae Kim, Rohan Vasisht, Evan P. Perillo, Katherine Blocher, Hannah Horng, Xi Tian, Chun-Lin Lin, Andrew K. Dunn, Tim H.-M. Huang, Hsin-Chih Yeh, Chun-Liang Chen. Upregulated EPHB2 and SRC pathways modulate spatial EGFR dynamics and malignant phenotypes and predict poor prognosis in 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 173.

Abstract 4683: Targeting GPCR signaling in HER2+ breast cancer suppresses cancer stem cell tumorigenicity and sensitizes HER2-targeted therapies

Abstract G protein coupled receptors (GPCRs) are the largest family of cell surface receptors with over 800 members. Aberrant activation of GPCRs has been implicated in cancer initiation and development. However, many of GPCRs are overexpressed in cancer cells, making it difficult to target individual GPCRs for cancer treatment. In this study, we have tested a new approach of targeting GPCRs in HER2+ breast cancer by blocking their common pathways downstream of G proteins. By mammary gland-specific expression of the pertussis toxin (PTx) catalytic subunit, a selective inhibitor that uncouples a subgroup of GPCRs from activating Gi/o proteins, we demonstrated that blocking GPCR downstream signaling delayed the onset of HER2-driven spontaneous mammary tumor formation and suppressed lung metastasis. Moreover, we showed that aberrant HER2 signaling upregulated Gi/o-GPCR expression in breast cancer cells, which in turn activated the ErbB/HER family of protein-tyrosine kinases via Src and PI3K pathways to maintain cancer stem cell tumorigenicity. Targeting Gi/o-GPCR signaling by PTx, Src or PI3K inhibitors sensitizes HER2+ breast cancer to HER2-targeted therapies. Together, our data demonstrate that targeting GPCR downstream signal pathways may represent a new approach to ablate CSCs to block tumor progression and augment HER2-targeted therapeutics. Citation Format: Wei Wang, Dharmendra Bhargava, Yuan Chao Ye, Songhai Chen. Targeting GPCR signaling in HER2+ breast cancer suppresses cancer stem cell tumorigenicity and sensitizes HER2-targeted therapies [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 4683.

Abstract 3448: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in a preclinical model of TNBC

Abstract Background: The combination of a poly ADP ribose polymerase (PARP) inhibitor with a DNA-damaging agent has shown promise in treating triple-negative breast cancer (TNBC); however, not all patients respond to this combination (Rugo HS et al. The New England Journal of Medicine. 2016). The Src protein kinase modulates multiple cancer cell properties and plays a key role in tumorigenic processes. However, Src inhibitors as single agents have shown limited effects in solid tumors (Finn RS et al. Clinical Cancer Research. 2011). In this study, we examined the antitumor effects of the Src inhibitor dasatinib, the PARP inhibitor veliparib, and the DNA-damaging agent carboplatin in TNBC cells in vitro and tumors in vivo to try to identify the combination with the most clinical potential. Methods: The KRAS/BRAF-mutated TNBC cell line, MDA-MB-231, was used in this study. For in vitro studies, proliferation, cellular apoptosis, and 3D on-top clonogenic growth were measured, tube formation assays and western blot analysis were performed. For in vivo studies, a xenograft model was used to examine treatment responses, and immunohistochemical analysis was performed. Results: Surprisingly, treatment with the combination of veliparib plus carboplatin led to an increase in Src phosphorylation. Importantly, addition of dasatinib attenuated Src overexpression induced by veliparib plus carboplatin and further inhibited the downstream signaling of Src, thereby enhancing the antitumor efficacy of veliparib plus carboplatin. In an MDA-MB-231 xenograft model, the triple combination of dasatinib with veliparib plus carboplatin showed greater tumor growth inhibitory effects compared with single agents or double combinations. No systemic toxicity was observed in mice treated with the triple combination. Conclusions: Here, we provide novel evidence that veliparib combined with carboplatin drives Src activation in the preclinical model tested. Moreover, we provide provocative in vitro and in vivo data highlighting the potential for increasing therapeutic efficacy by combining dasatinib with veliparib and carboplatin in patients with KRAS/BRAF-mutated TNBC. Citation Format: Yuliang Sun, Xiaoqian Lin, Jennifer C. Aske, Casey Williams, Mark Abramovitz, Brian Leyland-Jones. Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in a preclinical model of TNBC [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 3448.

Kisspeptin regulation of human decidual stromal cells motility via FAK-Src intracellular tyrosine kinases.

Can of Clinical Genetics, Maastricht University Medical Centre, Maastricht kisspeptin and its analogues regulate the motility of human decidual stromal cells and what intracellular signaling pathways are involved? Kisspeptin analogue-mediated cell motility in human decidual stromal cells via the focal adhesion kinase (FAK)-steroid receptor coactivator (Src) pathway suggesting that kisspeptin may modulate embryo implantation and decidual programming in human pregnancy. The extravillous trophoblast invades the maternal decidua during embryo implantation and placentation. The motile behavior and invasive potential of decidual stromal cells regulate embryo implantation and programming of human pregnancy. Human decidual stromal cells were isolated from healthy women undergoing elective termination of a normal pregnancy at 6- to 12-week gestation, after informed consent. Kisspeptin analogues were synthetic peptides. Cell motility was estimated by an invasion and migration assay. Immunoblot analysis was performed to investigate the expression of kisspeptin receptor and the effects of kisspeptin analogues on the phosphorylation of FAK and Src. Small interfering RNAs (siRNAs) were used to knock down the expression of kisspeptin receptor, FAK, Src, matrix metallo-proteinases (MMPs) 2 and 9, and extracellular signal-regulated protein kinase (ERK) 1/2. The kisspeptin receptor was expressed in human decidual stromal cells. Kisspeptin agonist decreased, but antagonist increased, cell motility. Kisspeptin agonist decreased the phosphorylation of FAK and Src tyrosine kinases, whereas antagonist increased it. These effects on phosphorylation were abolished by kisspeptin receptor siRNA. The activation of cell motility by kisspeptin analogues was suppressed by siRNA knockdown of endogenous FAK (decreased 66%), Src (decreased 60%), kisspeptin receptor (decreased 26%), MMP-2 (decreased 36%), MMP-9 (decreased 23%), and ERK 1/2 inhibitor (decreased 27%). Human decidual stromal cells were obtained from women having terminations after 6-12weeks of pregnancy and differences in timing could affect their properties. Kisspeptin acting within the endometrium has a potential modulatory role on embryo implantation and decidual programming of human pregnancy. This work was supported by grant NSC-104-2314-B-182A-146-MY2 (to H.-M.W.) from the Ministry of Science and Technology, Taiwan, and grants CMRPG3E0401 and CMRPG3E0402 (to H.-M.W.). This work was also supported by grants from the Canadian Institutes of Health Research to P.C.K.L. P.C.K.L. is the recipient of a Child & Family Research Institute Distinguished Investigator Award. The authors have no conflicts of interest to disclose. N/A.

Attenuation of Macrophage Migration Inhibitory Factor-Stimulated Signaling via S-Nitrosylation.

Nitric oxide (NO) is a key signaling molecule that has various effects via S-nitrosylation, a reversible post-translational modification that affects the enzymatic activity, localization, and metabolism of target proteins. As chronic nitrosative stress correlates with neurodegeneration, the targets have received focused attention. Macrophage migration inhibitory factor (MIF) plays a pivotal role in the induction of gene expression to control inflammatory responses. MIF acts as a ligand for CD74 receptor and activates the Src-p38 mitogen-activated protein kinase (MAPK) cascade. MIF also elevates the expression of brain-derived neurotrophic factor (BDNF), which contributes to the viability of neurons. Here, we show that MIF is S-nitrosylated by a physiological NO donor. Interestingly, the induction of S-nitrosylation resulted in a loss of MIF activity following stimulation of the Src and p38 MAPK signaling pathways and the induction of BDNF expression. Our results shed light on the pathogenic mechanisms of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

Increasing Antitumor Activity of JAK Inhibitor by Simultaneous Blocking Multiple Survival Signaling Pathways in Human Ovarian Cancer

Many signaling pathways, including the JAK/STAT3 pathway, are aberrantly activated and associated with ovarian cancer growth and progression. However, inhibition of STAT3 pathway alone was not sufficient to effectively block human ovarian cancer cell survival in vitro, which could be due to the activation and compensation of multiple survival pathways. In this study, we investigated a strategy that can enhance antitumor activity of JAK/STAT3 inhibitor by combining with inhibitors targeting other growth and survival pathways. We found that the in vitro activity of JAKi was remarkably increased when additional survival pathway was blocked. Blocking SRC pathway with SRC inhibitor (SRCi) increased the efficacy of JAKi more effectively than blocking AKT or MAPK pathway. The increased activity of JAKi in combination with SRCi is synergistic and associated with attenuation of p-STAT3, p-SRC, p-AKT and p-MAPK and increased inhibition of p-AKT. Simultaneous blockade of multiple survival pathways by combining JAKi with both AKT inhibitor (AKTi) and MEK inhibitor (MEKi) also resulted in a synergistic inhibition of cell survival. Furthermore, the combined treatment of JAKi and SRCi led to an increased apoptosis and greater inhibition of tumor growth and ascites formation. Taken together, our results demonstrate that the antitumor efficacy of JAKi is improved most effectively when combined with SRCi, providing a potential combination strategy for the treatment of advanced ovarian cancer.

Identification of biomarkers and their functions in dasatinib-resistant pancreatic cancer using bioinformatics analysis.

Dasatinib is a tyrosine kinase inhibitor, which inhibits tumor proliferation by blocking SRC pathways and is considered as a potential treatment of various epithelial neoplasms, including pancreatic cancer. However, dasatinib efficacy is largely limited due to drug resistance. In the present study, bioinformatics strategies were used to investigate the potential mechanisms of dasatinib-resistance in pancreatic cancer. The gene expression profiles of the Panc0403, Panc0504, Panc1005 (dasatinib-sensitive), SU8686, MiaPaCa2 and Panc1 (acquired dasatinib-resistant) cell lines were obtained from the gene expression omnibus database. The differentially expressed genes (DEGs) were then selected using R software. In addition, gene ontology (GO) and pathway enrichment analysis were performed through the Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction (PPI) network was constructed and analyzed to determine the hub genes using the Search Tool for the Retrieval of Interacting Genes database. A total of 472 DEGs, including vimentin, transmembrane 4 l six family member 18 and S100 calcium binding protein P, were identified. Enrichment analysis by GO function demonstrated that DEGs were associated with extracellular components, signal regulation and binding factors. The analysis of the Kyoto Encyclopedia of Genes and Genomes demonstrated that several adenocarcinoma pathways were enriched, including the phosphoinositide 3-kinases/protein kinase B and mitogen-activated protein kinase signaling pathways. Some hub genes were highlighted following the PPI network construction, including Rac family small GTPase 1, laminin subunit α3, integrin subunit β4, integrin subunit α2, collagen type VI α1 chain, collagen type I α2 chain, arrestin β1 and synaptotagmin 1, which may be associated with pancreatic adenocarcinoma prognosis. A total of five out of eight hub genes were highly associated with the overall survival rate (P<0.05). In conclusion, the present study reported novel insights into the mechanisms of dasatinib resistance. Identification of these hub genes may be considered as potential novel treatment targets for dasatinib-resistance in pancreatic cancer.