Sustained ERK Activation Research Articles

Oncogenic Kras induces spatiotemporally specific tissue deformation through converting pulsatile into sustained ERK activation.

Tissue regeneration and maintenance rely on coordinated stem cell behaviours. This orchestration can be impaired by oncogenic mutations leading to cancer. However, it is largely unclear how oncogenes perturb stem cells' orchestration to disrupt tissue. Here we used intravital imaging to investigate the mechanisms by which oncogenic Kras mutation causes tissue disruption in the hair follicle. Through longitudinally tracking hair follicles in live mice, we found that KrasG12D, a mutation that can lead to squamous cell carcinoma, induces epithelial tissue deformation in a spatiotemporally specific manner, linked with abnormal cell division and migration. Using a reporter mouse capture real-time ERK signal dynamics at the single-cell level, we discovered that KrasG12D, but not a closely related mutation HrasG12V, converts ERK signal in stem cells from pulsatile to sustained. Finally, we demonstrated that interrupting sustained ERK signal reverts KrasG12D-induced tissue deformation through modulating specific features of cell migration and division.

TP53 gain-of-function mutations promote osimertinib resistance via TNF-α–NF-κB signaling in EGFR-mutated lung cancer

EGFR tyrosine kinase inhibitors (TKIs) are effective against EGFR-mutated lung cancer, but tumors eventually develop resistance to these drugs. Although TP53 gain-of-function (GOF) mutations promote carcinogenesis, their effect on EGFR-TKI efficacy has remained unclear. We here established EGFR-mutated lung cancer cell lines that express wild-type (WT) or various mutant p53 proteins with CRISPR-Cas9 technology and found that TP53-GOF mutations promote early development of resistance to the EGFR-TKI osimertinib associated with sustained activation of ERK and expression of c-Myc. Gene expression analysis revealed that osimertinib activates TNF-α–NF-κB signaling specifically in TP53-GOF mutant cells. In such cells, osimertinib promoted interaction of p53 with the NF-κB subunit p65, translocation of the resulting complex to the nucleus and its binding to the TNF promoter, and TNF-α production. Concurrent treatment of TP53-GOF mutant cells with the TNF-α inhibitor infliximab suppressed acquisition of osimertinib resistance as well as restored osimertinib sensitivity in resistant cells in association with attenuation of ERK activation and c-Myc expression. Our findings indicate that induction of TNF-α expression by osimertinib in TP53-GOF mutant cells contributes to the early development of osimertinib resistance, and that TNF-α inhibition may therefore be an effective strategy to overcome such resistance in EGFR-mutant lung cancer with TP53-GOF mutations.

Abstract 3876: Kinetics of RTK activation determine ERK signaling dynamics and resistance to BRAF and MEK inhibitors

Abstract Combination treatment of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) produces remarkable response rates and is expected to prevent ERK activation in BRAF-mutant melanoma. However, sporadic ERK reactivation occurs in melanoma models, and efficacy is severely limited by the emergence of drug resistance. The underlying molecular mechanisms that drive ERK reactivation and adaptation to BRAFi/MEKi remain elusive. Here, using single-cell imaging and optogenetic approaches, we show that the strength and duration of receptor tyrosine kinase (RTK) activation determines ERK reactivation and the development of BRAFi/MEKi-resistant cells. Single-cell data reveal that despite global RTK activation by the same external stimuli, only small subsets of melanoma cells induce effective RTK and ERK activation to develop BRAFi/MEKi resistance. Furthermore, by directly controlling RTK activity, we demonstrate that the kinetics of RTK activation determine ERK signaling dynamics and the percentage of drug-resistant cells. While these drug-resistant cells are rare during early stages of drug treatment, they eventually become the dominant population through sustained RTK-mediated ERK activation that drives cell proliferation. Thus, RTK activation in established drug-resistant cells causes effective ERK activation and cell proliferation. Importantly, limiting RTK signaling activation reverses the BRAFi/MEKi-resistant state. We show that targeting SHP2, a mediator of RTK signaling, effectively suppresses RTK-mediated ERK activation and cell proliferation in BRAFi/MEKi-resistant cells. Our results provide mechanistic insights into the contribution of heterogeneity in RTK activation kinetics to ERK reactivation and BRAFi/MEKi resistance, also highlighting SHP2 activity as a promising target to overcome drug resistance in BRAF-mutant melanoma. Citation Format: Sungsoo Kim, Richard Carvajal, Minah Kim, Hee Won Yang. Kinetics of RTK activation determine ERK signaling dynamics and resistance to BRAF and MEK inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3876.

473 DUSP4-DUSP6 axis contributes to sustained ERK activity and high proliferation of the melanoma cells

A subset of dual-specificity phosphatases (DUSPs) act as major negative regulators of mitogen-activated protein kinases (MAPKs), and their possible involvement in tumorigenesis has been suggested. Among them, DUSP4 preferentially dephosphorylates ERK1/2 and Jnk over p38. In the present study, we aimed to identify the possible role of DUSP4 in melanomagenesis. Examination of publicly available large-scale data on cancer cell-lines revealed a remarkably high DUSP4 expression and dependency of most melanoma cell lines compared with other tumor-derived cell lines. No such high gene dependency specific to melanoma cells was evident for the other 24 DUSPs encoded in the human genome. Using two melanoma cell lines SK-MEL-28 and A375 as representatives, we confirmed that DUSP4 depletion impaired cell growth without notably inducing apoptosis for up to several days. Immunoblotting and kinase translocation reporter assay showed that the depletion simultaneously induced a decrease, not an increase, in ERK1/2 phosphorylation and an increase in DUSP6 protein level and that it barely affected Jnk phosphorylation. This observation indicates that neither ERK nor Jnk was a direct target of DUSP4 in our experimental setting. Quantitation of DUSP6 mRNA suggested that change in DUSP6 protein level mainly occurred through a post-transcriptional process, although it is not clear whether this took place by an increase in translation rate or inhibition of degradation kinetics. CRISPR/CAS9-mediated knock-out of DUSP6 mostly eliminated a decrease in the ERK phosphorylation and growth retardation following DUSP4 depletion suggesting the existence of the DUSP4-DUSP6-ERK serial regulation pathway. Our data support a model that DUSP4 plays a role in maintaining high ERK1/2 activity by negatively regulating DUSP6, thus contributing to the survival and growth of melanoma cells.

Hematopoietic transcription factor GFI1 promotes anchorage independence by sustaining ERK activity in cancer cells.

The switch from anchorage-dependent to anchorage-independent growth is essential for epithelial metastasis. The underlying mechanism, however, is not fully understood. In this study, we identified growth factor independent-1 (GFI1), a transcription factor that drives the transition from adherent endothelial cells to suspended hematopoietic cells during hematopoiesis, as a critical regulator of anchorage independence in lung cancer cells. GFI1 elevated the numbers of circulating and lung-infiltrating tumor cells in xenograft models and predicted poor prognosis of patients with lung cancer. Mechanistically, GFI1 inhibited the expression of multiple adhesion molecules and facilitated substrate detachment. Concomitantly, GFI1 reconfigured the chromatin structure of the RASGRP2 gene and increased its expression, causing Rap1 activation and subsequent sustained ERK activation upon detachment, and this led to ERK signaling dependency in tumor cells. Our studies unveiled a mechanism by which carcinoma cells hijacked a hematopoietic factor to gain anchorage independence and suggested that the intervention of ERK signaling may suppress metastasis and improve the therapeutic outcome of patients with GFI1-positive lung cancer.

LB1011 Oncogenic ras mutation induces spatiotemporally specific tissue deformation through converting fluctuated into sustained ERK activation

Tissue regeneration and maintenance rely on coordinated stem cell behaviors. This orchestration can be impaired by oncogenic mutations leading to tissue architecture disruption and ultimately cancer formation. However, it is still largely unclear how oncogenes perturb stem cells’ functions to break tissue architecture. Here, we utilized intravital imaging and novel signaling reporter to investigate the mechanisms of oncogenic Kras-induced tissue disruption in the hair follicle. Through longitudinally tracking the same hair follicles in live mice, we found KrasG12D, a mutation that can lead to squamous cell carcinoma, induces epithelial tissue deformation in a spatiotemporally specific manner.

IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis.

Interleukin-11 (IL11) is important for fibrosis and inflammation, but its role in the pancreas is unclear. In pancreatitis, fibrosis, inflammation and organ dysfunction are associated with pancreatic stellate cell (PSC)-to-myofibroblast transformation. Here, we show that IL11 stimulation of PSCs, which specifically express IL11RA in the pancreas, results in transient STAT3 phosphorylation, sustained ERK activation and PSC activation. In contrast, IL6 stimulation of PSCs caused sustained STAT3 phosphorylation but did not result in ERK activation or PSC transformation. Pancreatitis factors, including TGFβ, CTGF and PDGF, induced IL11 secretion from PSCs and a neutralising IL11RA antibody prevented PSC activation by these stimuli. This revealed an important ERK-dependent role for autocrine IL11 activity in PSCs. In mice, IL11 was increased in the pancreas after pancreatic duct ligation, and in humans, IL11 and IL11RA levels were elevated in chronic pancreatitis. Following pancreatic duct ligation, administration of anti-IL11RA to mice reduced pathologic (ERK, STAT, NF-κB) signalling, pancreatic atrophy, fibrosis and pro-inflammatory cytokine (TNFα, IL6 and IL1β) levels. This is the first description of IL11-mediated activation of PSCs, and the data suggest IL11 as a stromal therapeutic target in pancreatitis.

CRAF dimerization with ARAF regulates KRAS-driven tumor growth

KRAS, which is mutated in ∼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulatingMAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.

Amphiregulin-EGFR Autocrine Signaling Mediates Neutrophil Elastase-Triggered Prostate Cancer Progression

Abstract Neutrophil elastase (NE) is a serine protease stored in neutrophil azurophilic granules. Growing evidence indicates that NE is intimately involved in the activities of proinflammatory cytokines / chemokines, growth factors, and cell surface receptors. These molecular regulations can modulate innate immune responses as well as directly promote cancer cell outgrowth. To date, however, little is known regarding the molecular mechanisms underlying the stimulatory properties of NE in cancer cells. Here we examine NE effects on prostate cells, demonstrating that NE triggers proliferative signals and cell migration in six prostate cell lines representing the spectrum of prostate cell malignancy, including normal prostatic epithelium, benign prostatic hypertrophy, and metastatic prostate cancer. Using ERK activation as a read-out, we show that NE promotes ERK phosphorylation in a dose dependent manner, and time course study further reveal a sustained ERK activation upon NE treatment. Western blot evaluation demonstrates strong EGFR expression in cell lines derived from normal and benign prostatic gland, and preferential expression in hormone resistant versus hormone responsive cells. In agreement with EGFR-dependent mitogenic signaling, activation of ERK is abrogated by siRNA-mediated EGFR knockdown, as well as by pretreatment of cells with irreversible EGFR inhibitor AG1478. Importantly, NE evokes cancer cell migration at a lower range of NE concentrations relative to nonneoplastic cells. In prostate cells, from a total of seven EGFR ligands, amphiregulin (AREG) is predominantly expressed, and the addition of NE results in the release of AREG. Moreover, AREG gene silencing by siRNA or inhibition of AREG biological activity by neutralizing antibody, prevents NE-induced ERK phosphorylation and cell migration. Together, our study reveals a distinct and essential role of AREG-EGFR signaling axis in NE-triggered prostatic cellular response.

MAPK activity dynamics regulate non-cell autonomous effects of oncogene expression.

A large fraction of human cancers contain genetic alterations within the Mitogen Activated Protein Kinase (MAPK) signaling network that promote unpredictable phenotypes. Previous studies have shown that the temporal patterns of MAPK activity (i.e. signaling dynamics) differentially regulate cell behavior. However, the role of signaling dynamics in mediating the effects of cancer driving mutations has not been systematically explored. Here, we show that oncogene expression leads to either pulsatile or sustained ERK activity that correlate with opposing cellular behaviors (i.e. proliferation vs. cell cycle arrest, respectively). Moreover, sustained-but not pulsatile-ERK activity triggers ERK activity waves in unperturbed neighboring cells that depend on the membrane metalloprotease ADAM17 and EGFR activity. Interestingly, the ADAM17-EGFR signaling axis coordinates neighboring cell migration toward oncogenic cells and is required for oncogenic cell extrusion. Overall, our data suggests that the temporal patterns of MAPK activity differentially regulate cell autonomous and non-cell autonomous effects of oncogene expression.

Reactivation of BMP signaling by suboptimal concentrations of MEK inhibitor and FK506 reduces organ-specific breast cancer metastasis.

TGFβ-SMAD3 signaling is a major driving force for cancer metastasis, while BMP-SMAD1/5 signaling can counteract this response. Analysis of gene expression profiles revealed that an increased TGFβ-SMAD3 and a reduced BMP-SMAD1/5 targeted gene expression signature correlated with shortened distant metastasis free survival and overall survival of patients. At molecular levels, we discovered that TGFβ abolished BMP-induced SMAD1/5 activation in the highly-invasive breast cancer MDA-MB-231cells, but to a less extent in the non-invasive cancer and normal breast cells. This suggests an inverse correlation between BMP signaling and invasiveness of tumor cells and TGFβ signaling acts in a double whammy fashion in driving cancer invasion and metastasis. Sustained ERK activation by TGFβ was specifically observed in MDA-MB-231cells, and MEK inhibitor (MEKi) treatment restored BMP-SMAD1/5 signaling while not affecting SMAD2/3 activation. FK506 potently activated BMP, but not TGFβ signaling in breast cancer cells. MEKi or FK506 alone inhibited MDA-MB-231 extravasation in a zebrafish xenograft cancer model. Importantly, when administrated at suboptimal concentrations MEKi and FK506 strongly synergized in promoting BMP-SMAD1/5 signaling and inhibiting cancer cell extravasation. Furthermore, this combination of suboptimal concentrations treatment in a mouse tumor model resulted in real-time reduction of BMP-SMAD1/5 signaling in live tumors, and consequently potently inhibited tumor self-seeding, liver and bone metastasis, but not lung and brain metastasis. Mechanistically, it is the first time to identify BMP-SMAD1/5 signaling as an underlying molecular driver for organ-specific metastasis. Combining of MEKi and FK506, or their analogues, may be explored for clinical development of breast cancer.

Signaling Diversity Enabled by Rap1 and cAMP/PKA‐Regulated Plasma Membrane ERK with Distinct Temporal Dynamics

A variety of different signals induce specific responses through a common, ERK‐dependent kinase cascade. It has been suggested that signaling specificity can be achieved through precise temporal regulation of ERK activity. Given the wide distrubution of ERK susbtrates across different subcellular compartments, it is important to understand how ERK activity is temporally regulated at specific subcellular locations. To address this question, we expanded the toolbox of FRET‐based ERK biosensors by creating a series of improved biosensors targeted to various subcellular regions via sequence specific motifs to measure spatiotemporal changes in ERK enzymatic activity. Using these sensors, we showed that EGF induces sustained ERK activity near the plasma membrane in sharp contrast to the transient activity observed in the cytopolasm and nucleus. Furthermore, EGF‐induced plasma membrane ERK activity is regulated by Rap1, a noncanonical activator, and the cAMP/PKA signaling axis. We report that plasma membrane ERK controls cell morphology and EGF‐induced membrane protrusion dynamics. Our work strongly supports that spatial and temporal regulation of ERK activity is integrated to control signaling specificity from a single extracellular signal to multiple cellular processes.Support or Funding InformationThis work is supported by NIH R35 CA197622, R01 DK073368 (to J.Z). J.K. and O.M.I are supported by a post‐doctoral fellowship from NIH/NIGMS (K12GM068524). The development of Rap1 sensors at CISMM was supported by 5P41‐EB002025.

Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

BackgroundMedium-chain fatty acids and their 3-hydroxy derivatives are metabolites endogenously produced in humans, food-derived or originating from bacteria. They activate G protein-coupled receptors, including GPR84 and HCA3, which regulate metabolism and immune functions. Although both receptors are coupled to Gi proteins, share at least one agonist and show overlapping tissue expression, GPR84 exerts pro-inflammatory effects whereas HCA3 is involved in anti-inflammatory responses. Here, we analyzed signaling kinetics of both HCA3 and GPR84, to unravel signal transduction components that may explain their physiological differences.MethodsTo study the signaling kinetics and components involved in signal transduction of both receptors we applied the label-free dynamic mass redistribution technology in combination with classical cAMP, ERK signaling and β-arrestin-2 recruitment assays. For phenotypical analyses, we used spheroid cell culture models.ResultsWe present strong evidence for a natural biased signaling of structurally highly similar agonists at HCA3 and GPR84. We show that HCA3 signaling and trafficking depends on dynamin-2 function. Activation of HCA3 by 3-hydroxyoctanoic acid but not 3-hydroxydecanoic acid leads to β-arrestin-2 recruitment, which is relevant for cell-cell adhesion. GPR84 stimulation with 3-hydroxydecanoic acid causes a sustained ERK activation but activation of GPR84 is not followed by β-arrestin-2 recruitment.ConclusionsIn summary, our results highlight that biased agonism is a physiological property of HCA3 and GPR84 with relevance for innate immune functions potentially to differentiate between endogenous, non-pathogenic compounds and compounds originating from e.g. pathogenic bacteria.9ZbwrhpX5Yj3XBGuPG7oW-Video .Graphical abstract

Crosstalk between SHH and FGFR Signaling Pathways Controls Tissue Invasion in Medulloblastoma.

In the Sonic Hedgehog (SHH) subgroup of medulloblastoma (MB), tumor initiation and progression are in part driven by smoothened (SMO) and fibroblast growth factor (FGF)-receptor (FGFR) signaling, respectively. We investigated the impact of the SMO-FGFR crosstalk on tumor growth and invasiveness in MB. We found that FGFR signaling represses GLI1 expression downstream of activated SMO in the SHH MB line DAOY and induces MKI67, HES1, and BMI1 in DAOY and in the group 3 MB line HD-MBO3. FGFR repression of GLI1 does not affect proliferation or viability, whereas inhibition of FGFR is necessary to release SMO-driven invasiveness. Conversely, SMO activation represses FGFR-driven sustained activation of nuclear ERK. Parallel activation of FGFR and SMO in ex vivo tumor cell-cerebellum slice co-cultures reduced invasion of tumor cells without affecting proliferation. In contrast, treatment of the cells with the SMO antagonist Sonidegib (LDE225) blocked invasion and proliferation in cerebellar slices. Thus, sustained, low-level SMO activation is necessary for proliferation and tissue invasion, whereas acute, pronounced activation of SMO can repress FGFR-driven invasiveness. This suggests that the tumor cell response is dependent on the relative local abundance of the two factors and indicates a paradigm of microenvironmental control of invasion in SHH MB through mutual control of SHH and FGFR signaling.

S-allyl cysteine inhibits TNF-α-induced inflammation in HaCaT keratinocytes by inhibition of NF- κB-dependent gene expression via sustained ERK activation.

Tumor necrosis factor-α (TNF-α)-induced keratinocyte inflammation plays a key role in the pathogenesis of multiple inflammatory skin diseases. Here we investigated the anti-inflammatory effect of S-allyl cysteine (SAC) on TNF-α-induced HaCaT keratinocyte cells and the mechanism behind its anti-inflammatory potential. SAC was found to inhibit TNF-α-stimulated cytokine expression. Further, SAC was found to inhibit TNF-α-induced activation of p38, JNK and NF-κB pathways. Interestingly, SAC was found to differentially regulate ERK MAP kinase in cells. TNF-α-induced transient ERK activation and SAC treatment resulted in sustained ERK activation both in the presence and absence of TNF-α. Additionally, SAC failed to inhibit the TNF-α-induced expression of the pro-inflammatory cytokines TNF-α and IL-1β when cells were treated with the MEK inhibitor PD98059, suggesting that the anti-inflammatory effect of SAC is via sustained activation of the ERK pathway. Since ERK activation has been reported to negatively regulate NF-κB-driven gene expression and we find that SAC activates ERK and negatively regulates NF-κB, we investigated whether there existed any crosstalk between the ERK and the NF-κB pathways. NF-κB-dependent reporter assay, visualization of the nuclear translocation of NF-κB-p65 subunit and determination of the cellular levels of I-κB, the inhibitor of NF-κB, revealed that SAC inhibited TNF-α-induced NF-κB activation, and PD98059 treatment reversed this effect. These results collectively suggest that SAC inhibits TNF-α-induced inflammation in HaCaT cells via a combined effect entailing the inhibition of the p38 and the JNK pathways and NF-κB pathway via the sustained activation of ERK.

Entrectinib resistance mechanisms in ROS1-rearranged non-small cell lung cancer

SummaryEntrectinib is a pan-tyrosine-kinase inhibitor that targets oncogenic rearrangements in NTRK, ROS1 and ALK. The combined results of two clinical trials demonstrated the efficacy of entrectinib in ROS1-rearranged NSCLC. Because the development of drug resistance is inevitable, it would be helpful to determine the mechanisms of entrectinib resistance in a ROS1-rearranged tumor model so that future therapeutic strategies can be developed. Here, we characterized the molecular basis of resistance in entrectinib-resistant ROS1-rearranged HCC78 cells (HCC78ER cells). These cells were analyzed by next-generation sequencing and genetic profiling, which revealed the acquisition of KRAS G12C and the amplification of KRAS and FGF3. However, there were no secondary mutations in the ROS1 kinase domain. We also found that sustained ERK activation was involved in entrectinib resistance, and that combined treatment with selumetinib resensitized HCC78ER cells to entrectinib in cell viability and colony formation assays. Our data suggest that activation of the RAS signaling pathway can cause entrectinib resistance in ROS1-rearranged NSCLC, and is unlikely to be overcome by sequential single agent ROS1-targeting strategies against such tumors. Instead, co-targeting ROS1 and MEK may be an effective strategy for overcoming entrectinib resistance in ROS1-rearranged NSCLC.

Modulation of macrophage subtypes by IRF5 determines osteoclastogenic potential.

Bone-resorbing osteoclasts are differentiated from macrophages (MΦ) by M-CSF and RANKL. MΦ can be mainly classified into M1 and M2 MΦ, which are proinflammatory and anti-inflammatory, respectively, but little is known about their osteoclastogenic potential. Here, we investigated the osteoclastogenic potential of MΦ subtypes. When the two MΦ subtypes were differentiated into osteoclasts using M-CSF and RANKL, M2 MΦ more potently differentiated into osteoclasts than M1 MΦ. M2 MΦ generated with IL-4 or IL-10 also showed enhanced osteoclast differentiation compared with M1 MΦ induced by IFN-γ and lipopolysaccharide. In addition, robust bone-resorptive capacity and giant actin rings, which are features of mature osteoclasts, were observed in M2, but not M1 MΦ, under the osteoclast differentiation condition. Osteoclast differentiation was significantly increased in CD206+ M2 MΦ but not in CD86+ M1 MΦ. Compared with M1 MΦ, c-Fms and RANK were highly expressed in M2 MΦ. Enhanced osteoclastogenesis of M2 MΦ was mediated through sustained ERK activation, followed by efficient c-Fos and NFATc1 induction. Notably, the osteoclastogenic potential of M1 MΦ converted into M2 MΦ by exposure to M-CSF was higher than that of M2 MΦ converted into M1 MΦ by exposure to GM-CSF. Silencing IRF5, which is responsible for M1 MΦ polarization, increased osteoclast differentiation by enhancing c-Fms expression and activation of ERK, c-Fos, CREB, and NFATc1, which was inhibited by overexpression of IRF5. Collectively, M2 MΦ are suggested to be more efficient osteoclast precursors than M1 MΦ because of the attenuated expression of IRF5.

SUN-003 Targeting Androgen Receptor-CXCR7-MARK Signaling Axis in CRPC

Prostate cancer (PC) is the most commonly diagnosed cancer among males in the US. The standard of care for patients with metastatic PC is based on androgen-depletion therapy (ADT), including a first-line chemical castration, and a second-line, treatment with high-affinity antagonists for androgen receptor (AR), e.g. enzalutamide (Enz). Unfortunately, most of the patients eventually relapse with castration-resistant metastatic PC (CRPC). Further, use of Enz is thought to cause a growing concern for prevalence of highly aggressive AR-independent CRPC. Therefore, the molecular mechanisms of Enz-resistant (EnzR) CRPC are need to be understood urgently in order to identify novel targeted therapies for deadly CRPC. To approach Enz-resistant CRPC we have created EnzRPC cell lines by passaging cells in the presence of sub-lethal doses of Enz for over 6 months Analyzing the global profiles of expressed genes, we determined chemokine receptor CXCR7 (also known as atypical chemokine receptor ACKR3) as one of the top upregulated genes in EnzRcells. Utilizing molecular techniques we found that AR directly binds to promotor and enhancer regions of CXCR7 gene and inhibits its expression. EnzR PC cells depends on CXCR7-activated signaling in order to survive anti-AR therapy in vitroand in vivowhen injected in immuno-deficient mice. Moreover, we found that patients with metastatic CRPC increase CXCR7 expression comparing to localized PC tumors, and patients who relapse on second-line ADT further increase CXCR7 expression. CXCR7 is a 7-transmembrane (7TM) receptor, which when activated recruits ARRB2, a cytoplasmic scaffold protein. This leads to receptor internalization and activation of MAPK kinases. Normally, CXCR7 is activated by CXCL12 (SDF1a), CXCL11 (ITAC), and MIF. However, we observed that in EnzR PC cells upregulated CXCR7 signaling is constitutive, causing sustained ERK activation. Thus, when we combined MAPKK small molecule inhibitor with enzalutamide we observed superior therapeutic efficacy for CRPC in pre-clinical studies. Besides MAPK, CXCR7 can cross-activate other pro-survival signaling cascades, such as Akt and EGFR. Moreover, GSEA analysis reveals that EnzRPC cells further reduce AR signaling upon CXCR7 knock down suggesting another level of communication between CXCR7 and AR. Currently, CXCR7 lacks known potent antagonists, which would entirely cancel downstream signal activation, thereby, we are searching for novel small molecule CXCR7 inhibitors. Taken together our data suggest that AR and CXCR7 form a multilevel signaling axis, important for EnzR CRPC progression. Currently, we are focusing on further understanding the molecular mechanisms of CXCR7 tumor-promoting activity. In parallel we are developing novel anti-CXCR7 therapies that will be translatable into the clinics as a targeted therapy for patients with deadly CRPC.

Overactive IGF1/Insulin Receptors and NRASQ61R Mutation Drive Mechanisms of Resistance to Pazopanib and Define Rational Combination Strategies to Treat Synovial Sarcoma.

Pazopanib is approved for treatment of advanced soft tissue sarcomas, but primary and secondary drug resistance limits its clinical utility. We investigated the molecular mechanisms mediating pazopanib resistance in human synovial sarcoma (SS) models. We found reduced cell sensitivity to pazopanib associated with inefficient inhibition of the two critical signaling nodes, AKT and ERKs, despite strong inhibition of the main drug target, PDGFRα. In the CME-1 cell line, overactivation of IGF1 and Insulin receptors (IGF1R/InsR) sustained AKT activation and pazopanib resistance, which was overcome by a combination treatment with the double IGF1R/InsR inhibitor BMS754807. In the highly pazopanib resistant MoJo cell line, NRASQ61R mutation sustained constitutive ERK activation. Transfection of the NRAS mutant in the pazopanib sensitive SYO-1 cell line increased the drug IC50. MoJo cells treatment with pazopanib in combination with the MEK inhibitor trametinib restored ERK inhibition, synergistically inhibited cell growth, and induced apoptosis. The combination significantly enhanced the antitumor efficacy against MoJo orthotopic xenograft abrogating growth in 38% of mice. These findings identified two different mechanisms of intrinsic pazopanib resistance in SS cells, supporting molecular/immunohistochemical profiling of tumor specimens as a valuable approach to selecting patients who may benefit from rational drug combinations.

The inositol phosphatase SHIP2 enables sustained ERK activation downstream of FGF receptors by recruiting Src kinases.

Sustained activation of extracellular signal-regulated kinase (ERK) drives pathologies caused by mutations in fibroblast growth factor receptors (FGFRs). We previously identified the inositol phosphatase SHIP2 (also known as INPPL1) as an FGFR-interacting protein and a target of the tyrosine kinase activities of FGFR1, FGFR3, and FGFR4. We report that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued cell phenotypes triggered by pathologic FGFR-ERK signaling. Mutant forms of SHIP2 lacking phosphoinositide phosphatase activity still associated with FGFRs and did not prevent FGF-induced sustained ERK activation, demonstrating that the adaptor rather than the catalytic activity of SHIP2 was required. SHIP2 recruited Src family kinases to the FGFRs, which promoted FGFR-mediated phosphorylation and assembly of protein complexes that relayed signaling to ERK. SHIP2 interacted with FGFRs, was phosphorylated by active FGFRs, and promoted FGFR-ERK signaling at the level of phosphorylation of the adaptor FRS2 and recruitment of the tyrosine phosphatase PTPN11. Thus, SHIP2 is an essential component of canonical FGF-FGFR signal transduction and a potential therapeutic target in FGFR-related disorders.