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Signaling by urokinase-type plasminogen activator receptor (uPAR) can cause epithelial-mesenchymal transition (EMT) in cultured breast cancer cells. In this report, we show that uPAR signaling can also induce cancer stem cell (CSC)–like properties. Ectopic overexpression of uPAR in human MDA-MB-468 breast cancer cells promoted the emergence of a CD24−/CD44+ phenotype, characteristic of CSCs, while increasing the cell surface abundance of integrin subunits β1/CD29 and α6/CD49f that represent putative mammary gland stem cell biomarkers. uPAR overexpression increased mammosphere formation in vitro and tumor formation in an immunocompromized severe combined immunodeficient (SCID) mouse model of orthotopic breast cancer. Hypoxic conditions that are known to induce EMT in MDA-MB-468 cells also increased cell surface β1/CD29, mimicking the effects of uPAR overexpression. Antagonizing uPAR effector signaling pathways reversed the increase in cell surface integrin expression. Whereas uPAR overexpression did not induce EMT in MCF-7 breast cancer cells, CSC-like properties were nevertheless still induced along with an increase in tumor initiation and growth in the orthotopic setting in SCID mice. Notably, in MCF-7 cell mammospheres, which display a well-defined acinus-like structure with polarized expression of E-cadherin and β1-integrin, cell collapse into the central cavity was decreased by uPAR overexpression, suggesting that uPAR signaling may stabilize epithelial morphology. In summary, our findings show that uPAR signaling can induce CSC-like properties in breast cancer cells, either concomitantly with or separately from EMT. Cancer Res; 70(21); 8948–58. ©2010 AACR.

Supplementary Figure 1 from Cell Signaling by Urokinase-type Plasminogen Activator Receptor Induces Stem Cell–like Properties in Breast Cancer Cells

Supplementary Table 2 from Cell Signaling by Urokinase-type Plasminogen Activator Receptor Induces Stem Cell–like Properties in Breast Cancer Cells

Supplementary Figure Legend from Cell Signaling by Urokinase-type Plasminogen Activator Receptor Induces Stem Cell–like Properties in Breast Cancer Cells

Aneuploidy arises from persistent chromosome segregation errors, or chromosomal instability. Although it has long been known as a hallmark of cancer cells, reduced cellular fitness upon induced ploidy alterations hinders the understanding of how aneuploidy relates to cancer development in the body. In this study, we used FISH analysis targeting centromeres to indicate ploidy changes, and quantitatively evaluated the ploidy statuses of gastric tumors derived from a total of 214 patients, ranging from early to advanced disease. We found that cancer cells reveal a marked elevation of aneuploid population, increasingly in cases diagnosed in advanced stages. The expansion of the aneuploid population is well associated with p53 deficiency, consistent with its essential role in genome maintenance. Comparisons among multiple locations within the tumor, or between the primary and metastatic tumors, indicated that cancer cells mostly retain their ploidy alterations throughout primary tumors, but metastatic tumors may consist of cells with either increased or decreased levels of aneuploidy. We also found that a notable proportion of polyploid cells are often already present in chronic gastritis epithelia. These observations underscore that chromosome-level variations are widespread in gastric cancers, shaping their genetic heterogeneity and malignant properties.

본 연구에서는 O3형 층상계산화물의 전구체에 나이오븀 코팅 실시하였고 이 방법이 O3형 층상계산화물의 전기화학적 특성을 크게 향상 시킨다는 것을 확인하였다. 코팅된 층상계산화물은 0.2 C에서 충/방전 100 사이클 이후 83 mAh g-1의 우수한 용량을 보이고 5C의 고율의 충/방전에서는 코팅이 안된 층상계산화물에 비해 약 2.5 배 큰 97.6 mAh g-1의 용량을 보여주었다.

Aneuploidy is a widespread feature of malignant tumors that arises through persistent chromosome mis‐segregation in mitosis associated with a pathological condition called chromosomal instability, or CIN. Since CIN is known to have a causal relationship with poor prognosis accompanying by multi‐drug resistance, tumor relapse, and metastasis, many research groups have endeavored to understand the mechanisms underlying CIN. In this review, we overview possible etiologies of CIN. The key processes to achieve faithful chromosome segregation include the regulation of sister chromatid cohesion, kinetochore‐microtubule attachment, bipolar spindle formation, spindle‐assembly checkpoint, and the activity of separase. Aberrant chromosome structures during DNA replication might also be a potential cause of CIN. Defective regulation in these processes can lead to chromosome mis‐segregation, manifested by lagging chromosomes, and DNA bridges in anaphase, leading to gross chromosome rearrangements. Investigation into the molecular etiologies of CIN should allow us to explore novel strategies to intervene in CIN to control cancers., There are two major pathways in mitosis that lead cells to chromosome mis‐segregation. Defective correction of erroneous microtubule attachments causes lagging chromosomes and defective SAC inactivation and subsequent separase activation causes chromosomal bridges.

Conjugation of antisense oligonucleotide (ASO) with a variety of distinct lipophilic moieties like fatty acids and cholesterol increases ASO accumulation and activity in multiple tissues. While lipid conjugation increases tissue exposure in mice and reduces excretion of ASO in urine, histological review of skeletal and cardiac muscle indicates that the increased tissue accumulation of lipid conjugated ASO is isolated to the interstitium. Administration of palmitic acid-conjugated ASO (Palm-ASO) in mice results in a rapid and substantial accumulation in the interstitium of muscle tissue followed by relatively rapid clearance and only slight increases in intracellular accumulation in myocytes. We propose a model whereby increased affinity for lipid particles, albumin, and other plasma proteins by lipid-conjugation facilitates ASO transport across endothelial barriers into tissue interstitium. However, this increased affinity for lipid particles and plasma proteins also facilitates the transport of ASO from the interstitium to the lymph and back into circulation. The cumulative effect is only a slight (∼2-fold) increase in tissue accumulation and similar increase in ASO activity. To support this proposal, we demonstrate that the activity of lipid conjugated ASO was reduced in two mouse models with defects in endothelial transport of macromolecules: caveolin-1 knockout (Cav1−/−) and FcRn knockout (FcRn−/−).

Antisense oligonucleotides (ASOs) are a novel therapeutic approach to target difficult-to-drug protein classes by targeting their corresponding mRNAs. Significantly enhanced ASO activity has been achieved by the targeted delivery of ASOs to selected tissues. One example is the targeted delivery of ASOs to hepatocytes, achieved with N-acetylgalactosamine (GalNAc) conjugation to ASO, which results in selective uptake by asialoglycoprotein receptor (ASGR). Here we have evaluated the potential of GalNAc-conjugated ASOs as a therapeutic approach to targeting difficult-to-drug pathways in hepatocellular carcinoma (HCC). The activity of GalNAc-conjugated ASOs was superior to that of the unconjugated parental ASO in ASGR (+) human HCC cells in vitro, but not in ASGR (−) cells. Both human- and mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5- to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitrosamine-induced HCC tumor model. Finally, GalNAc conjugation enhanced ASO activity in human circulating tumor cells from HCC patients, demonstrating the potential of this approach in primary human HCC tumor cells. Taken together, these results provide a strong rationale for a potential therapeutic use of GalNAc-conjugated ASOs for the treatment of HCC., Kim et al. report that the activity of antisense oligonucleotides (ASOs) can be significantly enhanced in hepatocellular carcinoma (HCC) through conjugation with N-acetylgalactosamine (GalNAc), a ligand for asialoglycoprotein receptor (ASGR) expressed highly in hepatocytes, demonstrating the potential application of targeted delivery of ASOs to treat HCC.

Red light-sensitized squaraine (SQ) dyes were developed and incorporated into dye-sensitized catalysts (DSCs) with the formula of SQ/TiO2/Cat, and their efficacies were evaluated in terms of performance on either water or carbon dioxide reduction. Pt nanoparticles or fac-[Re(4,4′-bis-(diethoxyphosphorylmethyl)-2,2′-bipyridine)(CO)3Cl] were used as each catalytic center within the DSC frame of SQ/TiO2/Pt (Type I) or SQ/TiO2/Re(I) (Type II). In order to convey the potential utility of SQ in low energy sensitization, the following catalytic reductions were carried out under selective lower energy irradiation (>500 nm). Type I and II showed different catalytic performances, primarily due to the choice of solvent for each catalytic condition: hydrogenation was carried out in H2O, but CO2 reduction in dimethylformamide (DMF), and SQ was more stable in aqueous acid conditions for hydrogen generation than CO2 reduction in DMF. A suspension of Type I in 3 mL water containing 0.1 M ascorbic acid (pH = 2.66) resulted in efficient photocatalytic hydrogen evolution, producing 37 μmol of H2 for 4 h. However, in photocatalysis of Type II (SQ/TiO2/Re(I)) in 3 mL DMF containing 0.1 M 1,3-dimethyl-2-phenyl-1,3-dihydrobenzimidazole, the TiO2-bound SQ dyes were not capable of working as a low energy sensitizer because SQ was susceptible to dye decomposition in nucleophilic DMF conditions, resulting in DSC deactivation for the CO2 reduction. Even with the limitation of solvent, the DSC conditions for the utility of SQ have been established: the anchoring group effect of SQ with either phosphonic acid or carboxylic acid onto the TiO2 surface; energy alignment of SQ with the flat band potentials (Efb) of TiO2 semiconductors and the reduction power of electron donors; and the wavelength range of the light source used, particularly when >500 nm.

The spindle-assembly checkpoint facilitates mitotic fidelity by delaying anaphase onset in response to microtubule vacancy at kinetochores. Following microtubule attachment, kinetochores receive microtubule-derived force, which causes kinetochores to undergo repetitive cycles of deformation; this phenomenon is referred to as kinetochore stretching. The nature of the forces and the relevance relating this deformation are not well understood. Here, we show that kinetochore stretching occurs within a framework of single end-on attached kinetochores, irrespective of microtubule poleward pulling force. An experimental method to conditionally interfere with the stretching allowed us to determine that kinetochore stretching comprises an essential process of checkpoint silencing by promoting PP1 phosphatase recruitment after the establishment of end-on attachments and removal of the majority of checkpoint-activating kinase Mps1 from kinetochores. Remarkably, we found that a lower frequency of kinetochore stretching largely correlates with a prolonged metaphase in cancer cell lines with chromosomal instability. Perturbation of kinetochore stretching and checkpoint silencing in chromosomally stable cells produced anaphase bridges, which can be alleviated by reducing chromosome-loaded cohesin. These observations indicate that kinetochore stretching-mediated checkpoint silencing provides an unanticipated etiology underlying chromosomal instability and underscores the importance of a rapid metaphase-to-anaphase transition in sustaining mitotic fidelity.

The spindle-assembly checkpoint facilitates mitotic fidelity by delaying anaphase onset in response to microtubule vacancy at kinetochores. Following microtubule attachment, kinetochores receive microtubule-derived forces, which causes kinetochores to undergo repetitive cycles of deformations, the motion referred to as kinetochore stretching. The nature of forces and the relevance relating this motion are not well understood. Here we show that kinetochore stretching occurs within a framework of single end-on attached kinetochores, irrespectively of microtubule poleward pulling-force. An experimental setup to conditionally interfere with the stretching allowed us to define that kinetochore stretching comprises an essential process of checkpoint silencing by promoting PP1 phosphatase recruitment, after establishing of end-on attachments and removing the majority of checkpoint-activating kinase Mps1 from kinetochores. Remarkably, we found that the lower frequency of kinetochore stretching correlates with prolonged metaphase and also with the degree of chromosomal instability in cancer cell lines. Perturbation of kinetochore stretching and checkpoint silencing in chromosomally stable cells produced anaphase bridges which can be alleviated by reducing chromosome-loaded cohesin. These observations indicate that kinetochore stretching-mediated checkpoint silencing provides an unanticipated etiology underlying chromosomal instability, and underscore the significance of a rapid metaphase-to-anaphase transition in sustaining mitotic fidelity.

Additional file 1. Previous studies for microbial tyrosine production and nucleotide sequence used in this studies were summarized. In addition, additional results including stoichiometry and intracellular PEP concentration are presented to help understand the our engineering strategy.

Background The Janus kinase (JAK) and signal transduction and activation of transcription (STAT) signaling pathway is an attractive target in multiple cancers. Activation of the JAK-STAT pathway is important in both tumorigenesis and activation of immune responses. In diffuse large B-cell lymphoma (DLBCL), the transcription factor STAT3 has been associated with aggressive disease phenotype and worse overall survival. While multiple therapies inhibit upstream signaling, there has been limited success in selectively targeting STAT3 in patients. Antisense oligonucleotides (ASOs) represent a compelling therapeutic approach to target difficult to drug proteins such as STAT3 through of mRNA targeting. We report the evaluation of a next generation STAT3 ASO (AZD9150) in a non-Hodgkin’s lymphoma population, primarily consisting of patients with DLBCL. Methods Patients with relapsed or treatment refractory lymphoma were enrolled in this expansion cohort. AZD9150 was administered at 2 mg/kg and the 3 mg/kg (MTD determined by escalation cohort) dose levels with initial loading doses in the first week on days 1, 3, and 5 followed by weekly dosing. Patients were eligible to remain on therapy until unacceptable toxicity or progression. Blood was collected pre- and post-treatment for analysis of peripheral immune cells. Results Thirty patients were enrolled, 10 at 2 mg/kg and 20 at 3 mg/kg dose levels. Twenty-seven patients had DLBCL. AZD9150 was safe and well tolerated at both doses. Common drug-related adverse events included transaminitis, fatigue, and thrombocytopenia. The 3 mg/kg dose level is the recommended phase 2 dose. All responses were seen among DLBCL patients, including 2 complete responses with median duration of response 10.7 months and 2 partial responses. Peripheral blood cell analysis of three patients without a clinical response to therapy revealed a relative increase in proportion of macrophages, CD4+, and CD8+ T cells; this trend did not reach statistical significance. Conclusions AZD9150 was well tolerated and demonstrated efficacy in a subset of heavily pretreated patients with DLBCL. Studies in combination with checkpoint immunotherapies are ongoing. Trial registration Registered at ClinicalTrials.gov: NCT01563302. First submitted 2/13/2012. Electronic supplementary material The online version of this article (10.1186/s40425-018-0436-5) contains supplementary material, which is available to authorized users.

Semi-analytical solutions to the optimal control problem for 3-dimensional fuel-efficient planetary landing trajectories with constant exhaust velocity and limited mass-flow rate in a drag-existing central Newtonian field are presented. The first-order optimality conditions reduce the problem to a Hamiltonian canonical system for the design and synthesis of feasible and extremal planetary entry, descent, and landing trajectories. The proposed solutions allow us to describe the state vector and Lagrange multipliers in terms of time and switching function's characteristics to determine the number and sequence of thrust arcs. These solutions can be adjusted for manoeuvres near other celestial bodies for which a central gravitational acceleration can be dominant. The paper describes new extremal trajectories, which are based on the analysis of first- and second-order optimality conditions. The results of this work can be used to support mission design analysis and synthesis of fuel-efficient trajectories.

Chromosomal instability (CIN) is a pathological condition where cells continuously mis-segregate chromosomes, producing aneuploid cells. CIN has been recognized as a hallmark of cancer, and its correlation with biological malignancy has been pointed out. Glioma cell line often reveals karyotype aberrations, and a variety of ploidy in the tumor tissue. However, several studies have indicated that aneuploidy is disadvantageous for proliferation or tumorigenesis, and these paradox prompt us to address the role of aneuploid cells in tumor specimens. Here, we adopted mouse glioma stem cell lineages and found that aneuploid population is increased in glioma stem cells in vitro. We also examined Aurora B at centromeres which is crucial for failsafe chromosome segregation and found its reduced activity in glioma stem cells, suggesting that insufficient Aurora B activity plays a causative role in CIN in glioma stem cells. Next, to investigate the tumorigenicity of aneuploid cells, we sorted the glioma stem cells depending on the karyotype pattern, and allografted into mouse brain. We found that the growth rate of diploid glioma stem cells was higher than others in vitro, and the probability of survival after allogeneic transplant was significantly lower in diploid groups. We will discuss the role of ploidy in glioma cells populations.

Purpose : The purpose of this study was to investigate the effects of flexibility and foot pressure on stretching exercise of hamstring muscle with and without pelvis neutral position. Methods : This study was performed on 30 subjects. Thirty subjects were divided into two group; hamstring passive stretching exercise with pelvis neutral position(n

The Hippo pathway plays important roles in tissue homeostasis. When dysregulated in adult tissues, however, it contributes to the development of a variety of cancers including hepatocellular carcinoma (HCC). YAP1 is the final regulator of the Hippo pathway and is associated with poor prognosis of HCC patients, and more recently has been suggested to be involved in tumor immune evasion. Here, we employed next-generation constrained ethyl (cEt) antisense oligonucleotides (ASOs) to selectively target YAP1 in the genetically engineered mouse (GEM) model of HCC (Salvador KO mice) as well as in a spontaneous carcinogen-induced model of HCC (diethylnitrosamine, DEN-induced HCC). In YAP1-activated Salvador KO mice with established HCC tumors, systemic treatment with YAP1 ASO at 25 mg/kg resulted in a marked reduction in YAP1 protein level in tumor (>90%) and regression of existing tumors, in contrast to the tumors in vehicle or control ASO-treated animals that significantly increased in size. In the DEN-induced HCC model, YAP1 was strongly activated in tumors as demonstrated by nuclear localization of YAP1 protein and the gene signature we previously identified (SOH). YAP1 ASOs produced either complete tumor stasis when DEN tumors were grown in immune-deficient mice, or complete regressions when tumors were grown in immune-competent animals. In accordance with this, YAP1 ASO treatment resulted in significant infiltration of T cells and myeloid lineage cells in the tumors in immune-competent mice. Importantly, when tumor-bearing animals were taken off the treatment after tumor regression was observed with YAP1 ASO, the antitumor response was sustained for more than 1 year, with 4 out of 16 animals remaining tumor-free in YAP1 ASO group. Furthermore, YAP1 ASO in combination with anti-PD1 antibody led to enhanced antitumor activity in the same tumor model. Collectively, these results suggest a potential therapeutic use of YAP1 ASO in the treatment of HCC with YAP1 activation as a single agent or in combination with immuno-oncology drugs. Broad screens have identified the human YAP1 ASO clinical development compound YAP1RX. Citation Format: Youngsoo Kim, Joanna Schmidt, Minji Jo, Jing Yi, Xiaolin Luo, Rachel Fleming, Sun Young Yim, Ju-Seog Lee, Brett P. Mornia, Randy L. Johnson, Gordon Mills, A. Robert MacLeod. Selective depletion of YAP1 with next-generation (constrained ethyl-cEt) antisense oligonucleotides results in tumor regression in mouse models of HCC with YAP1 activation [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B09.

Table S1. Genomic analysis of pre-treatment tumor in complete responder with DLBCL. Table S2. Peripheral blood cell counts of patients reported in Fig. 3 on days of peripheral blood mononuclear cell analysis with fold-change in absolute number. (DOCX 16 kb)

Cancer cells condition macrophages and other inflammatory cells in the tumor microenvironment so that these cells are more permissive for cancer growth and metastasis. Conditioning of inflammatory cells reflects, at least in part, soluble mediators (such as transforming growth factor β and IL-4) that are released by cancer cells and alter the phenotype of cells of the innate immune system. Signaling pathways in cancer cells that potentiate this activity are incompletely understood. The urokinase receptor (uPAR) is a cell-signaling receptor known to promote cancer cell survival, proliferation, metastasis, and cancer stem cell–like properties. The present findings show that uPAR expression in diverse cancer cells, including breast cancer, pancreatic cancer, and glioblastoma cells, promotes the ability of these cells to condition co-cultured bone marrow–derived macrophages so that the macrophages express significantly increased levels of arginase 1, a biomarker of the alternatively activated M2 macrophage phenotype. Expression of transforming growth factor β was substantially increased in uPAR-expressing cancer cells via a mechanism that requires uPA-initiated cell signaling. uPAR also controlled expression of IL-4 in cancer cells via a mechanism that involves activation of ERK1/2. The ability of uPAR to induce expression of factors that condition macrophages in the tumor microenvironment may constitute an important mechanism by which uPAR promotes cancer progression.

Recruitment of monocytes into sites of inflammation is essential in the immune response. In cancer, recruited monocytes promote invasion, metastasis, and possibly angiogenesis. LDL receptor-related protein (LRP1) is an endocytic and cell-signaling receptor that regulates cell migration. In this study, we isografted PanO2 pancreatic carcinoma cells into mice in which LRP1 was deleted in myeloid lineage cells. Recruitment of monocytes into orthotopic and subcutaneous tumors was significantly increased in these mice, compared with control mice. LRP1-deficient bone marrow–derived macrophages (BMDM) expressed higher levels of multiple chemokines, including, most prominently, macrophage inflammatory protein-1α/CCL3, which is known to amplify inflammation. Increased levels of CCL3 were detected in LRP1-deficient tumor-associated macrophages (TAM), isolated from PanO2 tumors, and in RAW 264.7 macrophage-like cells in which LRP1 was silenced. LRP1-deficient BMDMs migrated more rapidly than LRP1-expressing cells in vitro. The difference in migration was reversed by CCL3-neutralizing antibody, by CCR5-neutralizing antibody, and by inhibiting NF-κB with JSH-23. Inhibiting NF-κB reversed the increase in CCL3 expression associated with LRP1 gene silencing in RAW 264.7 cells. Tumors formed in mice with LRP1-deficient myeloid cells showed increased angiogenesis. Although VEGF mRNA expression was not increased in LRP1-deficient TAMs, at the single-cell level, the increase in TAM density in tumors with LRP1-deficient myeloid cells may have allowed these TAMs to contribute an increased amount of VEGF to the tumor microenvironment. Our results show that macrophage density in tumors is correlated with cancer angiogenesis in a novel model system. Myeloid cell LRP1 may be an important regulator of cancer progression. Cancer Res; 73(13); 3902–12. ©2013 AACR.

Pseudopodium-enriched atypical kinase 1 (PEAK1) is a recently described tyrosine kinase that associates with the actin cytoskeleton and focal adhesion (FA) in migrating cells. PEAK1 is known to promote cell migration, but the responsible mechanisms remain unclear. Here, we show that PEAK1 controls FA assembly and disassembly in a dynamic pathway controlled by PEAK1 phosphorylation at Tyr-665. Knockdown of endogenous PEAK1 inhibits random cell migration. In PEAK1-deficient cells, FA lifetimes are decreased, FA assembly times are shortened, and FA disassembly times are extended. Phosphorylation of Tyr-665 in PEAK1 is essential for normal PEAK1 localization and its function in the regulation of FAs; however, constitutive phosphorylation of PEAK1 Tyr-665 is also disruptive of its function, indicating a requirement for precise spatiotemporal regulation of PEAK1. Src family kinases are required for normal PEAK1 localization and function. Finally, we provide evidence that PEAK1 promotes cancer cell invasion through Matrigel by a mechanism that requires dynamic regulation of Tyr-665 phosphorylation.

Binding of urokinase-type plasminogen activator (uPA)1 to its receptor, uPAR, in estrogen receptor-α (ERα) expressing breast cancer cells, transiently activates ERK downstream of FAK, Src family kinases, and H-Ras. Herein, we show that when uPAR is over-expressed, in two separate ERα-positive breast cancer cell lines, ERK activation occurs autonomously of uPA and is sustained. Autonomous ERK activation by uPAR requires H-Ras and Rac1. A mutated form of uPAR, which does not bind vitronectin (uPAR-W32A), failed to induce autonomous ERK activation. Expression of human uPAR or mouse uPAR but not uPARW32A in MCF-7 cells provided a selection advantage when these cells were deprived of estrogen in cell culture for two weeks. Similarly, MCF-7 cells that express mouse uPAR formed xenografts in SCID mice that survived and increased in volume in the absence of estrogen supplementation, probably reflecting the pro-survival activity of phospho-ERK. Autonomous uPAR signaling to ERK was sensitive to the EGFR tyrosine kinase inhibitors, Erlotinib and Gefitinib. The transition in uPAR signaling from uPA-dependent and transient to autonomous and sustained is reminiscent of the transformation in ErbB2/ HER2 signaling observed when this gene is amplified in breast cancer. uPAR over-expression may provide a pathway for escape of breast cancer cells from ERα-targeting therapeutics.

Next-generation sequencing technologies have greatly expanded our understanding of cancer genetics. Antisense technology is an attractive platform with the potential to translate these advances into improved cancer therapeutics, because antisense oligonucleotide (ASO) inhibitors can be designed on the basis of gene sequence information alone. Recent human clinical data have demonstrated the potent activity of systemically administered ASOs targeted to genes expressed in the liver. We describe the preclinical activity and initial clinical evaluation of a class of ASOs containing constrained ethyl modifications for targeting the gene encoding the transcription factor STAT3, a notoriously difficult protein to inhibit therapeutically. Systemic delivery of the unformulated ASO, AZD9150, decreased STAT3 expression in a broad range of preclinical cancer models and showed antitumor activity in lymphoma and lung cancer models. AZD9150 preclinical activity translated into single-agent antitumor activity in patients with highly treatment-refractory lymphoma and non-small cell lung cancer in a phase 1 dose-escalation study.

A truncated and constitutively active form of the EGF receptor, variant III (EGFRvIII), is a major determinant of tumor growth and progression in glioblastoma multiforme (GBM). Extensive bidirectional crosstalk occurs in the cell-signaling pathways downstream of the EGFR and the urokinase-type plasminogen activator receptor (uPAR); however, crosstalk between EGFRvIII and uPAR has not been examined. Here, we show that uPAR does not regulate ERK activation in EGFRvIII-expressing GBM cells; however, in GBM cells isolated from four separate xenografts in which EGFRvIII expression was down-regulated in vivo, uPAR assumed a major role in sustaining ERK activation. Phosphorylation of Tyr-845 in the EGFR, which is mediated by Src family kinases, depended on uPAR in EGFRvIII-expressing GBM cells. Activation of the mitogenic and prosurvival transcription factor, STAT5b, downstream of EGFRvIII, also required uPAR. The EGFR-selective tyrosine kinase inhibitors, erlotinib and gefitinib, blocked not only EGFRvIII signaling to ERK but also uPAR-dependent STAT5b activation. uPAR gene silencing in EGFRvIII-expressing GBM cells and in cells from tumors that escaped dependency on EGFRvIII decreased cell survival and proliferation. Xenografts of EGFRvIII-expressing cancer cell lines and a human GBM, which was propagated as a xenograft, were robustly immunopositive for uPAR and phospho–Tyr-845 by immunohistochemistry. A human GBM in which the EGFR gene was amplified without truncation was immunonegative for both uPAR and phospho–Tyr-845. These studies identify distinct cell-signaling activities for uPAR in GBM cells that express EGFRvIII and in cells released from dormancy when EGFRvIII is neutralized. uPAR and its crosstalk pathways with EGFRvIII emerge as logical targets for therapeutics development in GBM.

Signaling by urokinase-type plasminogen activator receptor (uPAR) can cause epithelial-mesenchymal transition (EMT) in cultured breast cancer cells. In this report, we show that uPAR signaling can also induce cancer stem cell (CSC)–like properties. Ectopic overexpression of uPAR in human MDA-MB-468 breast cancer cells promoted the emergence of a CD24−/CD44+ phenotype, characteristic of CSCs, while increasing the cell surface abundance of integrin subunits β1/CD29 and α6/CD49f that represent putative mammary gland stem cell biomarkers. uPAR overexpression increased mammosphere formation in vitro and tumor formation in an immunocompromized severe combined immunodeficient (SCID) mouse model of orthotopic breast cancer. Hypoxic conditions that are known to induce EMT in MDA-MB-468 cells also increased cell surface β1/CD29, mimicking the effects of uPAR overexpression. Antagonizing uPAR effector signaling pathways reversed the increase in cell surface integrin expression. Whereas uPAR overexpression did not induce EMT in MCF-7 breast cancer cells, CSC-like properties were nevertheless still induced along with an increase in tumor initiation and growth in the orthotopic setting in SCID mice. Notably, in MCF-7 cell mammospheres, which display a well-defined acinus-like structure with polarized expression of E-cadherin and β1-integrin, cell collapse into the central cavity was decreased by uPAR overexpression, suggesting that uPAR signaling may stabilize epithelial morphology. In summary, our findings show that uPAR signaling can induce CSC-like properties in breast cancer cells, either concomitantly with or separately from EMT. Cancer Res; 70(21); 8948–58. ©2010 AACR.

The Hippo pathway plays important roles in maintaining tissue homeostasis. When dysregulated, however, it contributes to the development of a variety of cancers including hepatocellular carcinoma (HCC). YAP1 is the final regulator of the pathway and its causal role in promoting HCC growth has been demonstrated previously in multiple animal models. YAP1 is therefore considered an attractive drug target for HCC but as a transcription coactivator is difficult to inhibit by conventional approaches. We investigated how selective downregulation of YAP1 would affect the growth of established HCC using optimized Gen2.5 antisense oligonucleotides (ASOs) for mouse YAP1. Here, we describe the antitumor activity of YAP1 ASOs in 2 mouse models of HCC, which appears to be mediated by both tumor autonomous and immune modulatory functions of YAP1. In YAP1-activated Salvador KO mice with established HCC, systemic treatment of YAP1 ASO for 2 months at 75 mg/kg/week resulted in a marked reduction (>90%) in tumor YAP1 levels and regression of HCC tumors, which is in contrast to the tumors in the control ASO-treated animals that significantly increased in size. Proliferation of tumor cells was drastically reduced in YAP1 ASO-treated animals as demonstrated by low Ki-67 staining along with a decrease in Cyr61, a downstream YAP1 target. YAP1 ASOs were also tested in the diethylnitrosamine-induced HCC model. Although the model has been widely used in HCC research, lack of reliable biomarkers and high variability in tumor take between animals make it less ideal for an efficacy study. To overcome these limitations, we established a subcutaneous model by in vivo passaging tumors excised from the liver in either nude or immune-competent C57BL/6 mice. Activation of YAP1 in this model was demonstrated by strong nuclear localization of YAP1 protein in tumors. While the effect of YAP1 ASO on tumor was primarily delaying its growth in nude mice, the same YAP1 ASO induced tumor regression when DEN tumors were grown in immune competent mice, suggesting an immune component for activity. Strong infiltration of myeloid lineage cells was observed in tumors of YAP1 ASO-treated animals at the end of the 6 week study, suggesting potential activation of immune cells as a result of YAP1 depletion. Significant infiltration of T cells and myeloid lineage cells into YAP1 ASO-treated tumors was also observed at an early time point as demonstrated by increases in CD3 and F4/80 signals by IHC. Immune cell infiltration into tumors was more pronounced in the combination of ASOs for YAP1 and its paralog mouse TAZ than YAP1 ASO alone; however, the combination group exhibited notable signs of inflammation on the skin and in the liver. Importantly, when tumor-bearing animals were taken off the treatment after tumor regression was observed with YAP1 ASO, the antitumor response was sustained for more than 6 weeks with 2 out of 8 animals remaining tumor free in YAP1 ASO group. Further, safety of ASO-mediated YAP1 inhibition was confirmed in both normal mice with no notable toxicity and tumor-bearing mice with improvement in liver transaminases despite near complete depletion of YAP1 in the liver. Collectively, these results provide evidence for immune activation as a result of YAP1 downregulation and also suggest a potential therapeutic use of YAP1 ASO in the treatment of HCC with YAP1 activation. Citation Format: Youngsoo Kim, Joanna Schmidt, Minji Jo, Jing Yi, Tianyuan Zhou, Xiaolin Luo, Sun Young Yim, Ju-Seog Lee, Randy L. Johnson, Brett P. Monia, Gordon Mills, A. Robert MacLeod. Selective depletion of YAP1 with next-generation antisense oligonucleotides leads to immune cell infiltration and tumor regression in mouse models of HCC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B197.

Hypoxia induces expression of the urokinase receptor (uPAR) and activates uPAR-dependent cell signaling in cancer cells. This process promotes epithelial-mesenchymal transition (EMT). uPAR overexpression in cancer cells also promotes EMT. In this study, we tested whether uPAR may be targeted to reverse cancer cell EMT. When MDA-MB 468 breast cancer cells were cultured in 1% O(2), uPAR expression increased, as anticipated. Cell-cell junctions were disrupted, vimentin expression increased, and E-cadherin was lost from cell surfaces, indicating EMT. Transferring these cells back to 21% O(2) decreased uPAR expression and reversed the signs of EMT. In uPAR-overexpressing MDA-MB 468 cells, EMT was reversed by silencing expression of endogenously produced urokinase-type plasminogen activator (uPA), which is necessary for uPAR-dependent cell signaling, or by targeting uPAR-activated cell signaling factors, including phosphatidylinositol 3-kinase, Src family kinases, and extracellular signal-regulated kinase. MDA-MB 231 breast cancer cells express high levels of uPA and uPAR and demonstrate mesenchymal cell morphology under normoxic culture conditions (21% O(2)). Silencing uPA expression in MDA-MB-231 cells decreased expression of vimentin and Snail, and induced changes in morphology characteristic of epithelial cells. These results demonstrate that uPAR-initiated cell signaling may be targeted to reverse EMT in cancer.

Liver regeneration after partial hepatectomy (PHx) is a complex and well-orchestrated biological process in which synchronized cell proliferation is induced in response to the loss of liver mass. To define long noncoding RNAs (lncRNAs) that participate in the regulation of liver regeneration, we performed microarray analysis and identified more than 400 lncRNAs exhibiting significantly altered expression. Of these, one lncRNA, LncPHx2 (Long noncoding RNA induced by PHx 2), was highly upregulated during liver regeneration. Depletion of LncPHx2 during liver regeneration using antisense oligonucleotides led to a transient increase in hepatocyte proliferation and more rapid liver regeneration. Gene expression analysis showed that LncPHx2 depletion resulted in upregulation of mRNAs encoding proteins known to promote cell proliferation, including MCM components, DNA polymerases, histone proteins, and transcription factors. LncPHx2 interacts with the mRNAs of MCM components, making it a candidate to regulate the expression of MCMs and other genes post-transcriptionally. Collectively, our data demonstrate that LncPHx2 is a key lncRNA that participates in a negative feedback loop modulating hepatocyte proliferation through RNA-RNA interactions.

Epidermal growth factor (EGF) expresses mitogenic activity by a mechanism that requires the EGF receptor (EGFR). We report that murine embryonic fibroblasts (MEFs) proliferate in response to EGF only when these cells express the urokinase receptor (uPAR). EGFR expression was equivalent in uPAR-/- and uPAR+/+ MEFs. In response to EGF, these cells demonstrated equivalent overall EGFR tyrosine phosphorylation and ERK/MAP kinase activation; however, phosphorylation of Tyr-845 in the EGFR, which has been implicated in cell growth, was substantially decreased in uPAR-/- MEFs. STAT5b activation also was decreased. As Tyr-845 is a c-Src target, we overexpressed c-Src in uPAR-/- MEFs and rescued EGF mitogenic activity. Rescue also was achieved by expressing murine but not human uPAR, suggesting a role for autocrine uPAR cell-signaling. In MDA-MB 231 breast cancer cells, EGF mitogenic activity was blocked by uPAR gene silencing, with antibodies that block uPA-binding to uPAR, and with a synthetic peptide that disrupts uPAR-dependent cell signaling. Again, c-Src overexpression rescued the mitogenic activity of EGF. We conclude that uPAR-dependent cell-signaling may prime cells to proliferate in response to EGF by promoting Tyr-845 phosphorylation and STAT5b activation. The importance of this pathway depends on the c-Src level in the cell.

Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression.

Binding of the urokinase-type plasminogen activator (uPA) to its receptor activates diverse cell signaling pathways. How these signals are integrated so that cell physiology is altered remains unclear. In this study, we demonstrated that migration of MCF-7 breast cancer cells and HT-1080 fibrosarcoma cells on serum-coated surfaces is stimulated by agents that activate ERK, including uPA, epidermal growth factor, and constitutively active MEK1. The promigratory activity of these agents was entirely blocked not only by the MEK-specific antagonist PD098059, but also by antagonists of the Rho-Rho kinase pathway, including Y-27632 and dominant-negative RhoA (RhoA-N19). uPA did not significantly increase the level of GTP-bound RhoA, suggesting that the constitutive activity of the Rho-Rho kinase pathway may be sufficient to support ERK-stimulated cell migration. Paradoxically, Y-27632 and RhoA-N19 increased ERK phosphorylation in MCF-7 cells, providing further evidence that ERK activation alone does not promote cell migration when Rho kinase is antagonized. When MCF-7 cell migration was stimulated by ERK-independent processes such as expression of the beta(3) integrin subunit or changing the substratum to type I collagen, Y-27632 and RhoA-N19 failed to inhibit the response. This study supports a model in which the Ras-ERK and Rho-Rho kinase pathways cooperate to promote cell migration. Neutralizing either pathway is sufficient to block the response to agents that stimulate cell migration by activating ERK.

In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.

We have previously shown that rat liver epithelial cells (RLEC) transfected with and constitutively expressing transforming growth factor-α (TGF-α) have an enhanced mitogenic response to hepatocyte growth factor (HGF). In the study reported here, we examined tumor clones derived from the TGF-α transfectants with respect to mitogenic response to HGF. Tumor cell lines that expressed TGF-α responded to HGF with a greater increase in DNA synthesis than did the nontransfected parental RLEC (pRLEC). The tumor clones had also acquired a lower threshold for HGF response, which enabled them to undergo significant DNA synthesis at a low concentration of HGF that did not evoke a response in the pRLEC or TGF-α transfectants. We investigated the mechanisms by which TGF-α expression may influence the HGF/c-met pathway. We showed that most TGF-α transfectants and tumor cells displayed increases in c-met mRNA and protein, indicating that the enhanced HGF response may be due in part to an increase in the amount of receptor present. However, in all transfectants and tumor clones that constitutively expressed TGF-α, c-met was tyrosine phosphorylated in the absence of ligand (HGF) or another exogenous growth factors. These data suggest that induction of c-met mRNA and transactivation of c-met may be a sequela of the constitutive expression of TGF-α and that constitutive activation of the epidermal growth factor receptor pathway leads to phosphorylation and activation of c-met. These studies provide evidence for a novel mechanism of communication between epidermal growth factor receptor and c-met pathways that may partially explain the synergistic effects reported between TGF-α and HGF. Mol. Carcinog. 18:244–255, 1997. © 1997 Wiley-Liss, Inc.

LDL receptor-related protein (LRP1) is an endocytic receptor for multiple ligands, including proteases, growth factors, apolipoproteins, and extracellular matrix proteins. In some cell types, including neurons, neuron-like cells, and Schwann cells, ligand-binding to LRP1 triggers robust cell-signaling. This “direct” pathway by which LRP1 regulates cell-signaling promotes cell survival and cell migration. LRP1 also regulates the composition of the plasma membrane proteome. Although multiple mechanisms are involved, LRP1 and receptors in the same gene family facilitate the endocytosis of other plasma membrane proteins. When LRP1 regulates the abundance or trafficking of another cell-signaling receptor in the plasma membrane, activation of important cell-signaling pathways may be controlled “indirectly” by LRP1. The urokinase receptor (uPAR) was the first cell-signaling receptor identified as a member of the LRP1-regulated plasma membrane proteome. Because LRP1 down-regulates cell-surface uPAR by facilitating its endocytosis, under some conditions, uPAR-initiated cell-signaling may be inhibited by LRP1. However, the relationship between LRP1 and uPAR is complicated because uPAR endocytosis may be necessary for sustained uPAR-initiated cell-signaling. Certain cell-signaling factors, including ERK, phosphatidylinositol 3-kinase, and Rac1 are regulated by LRP1, directly, and indirectly through uPAR. Thus, the predominant effect of LRP1 on cell-signaling, in different cell types, may depend on the abundance of LRP1 and uPAR and on the availability of ligands for LRP1 and uPAR. Opportunities for targeting the uPAR-LRP1 system through drug discovery are discussed.

LDL receptor-related protein (LRP1) is expressed by Schwann cells in vivo mainly after injury to the peripheral nervous system (PNS). Schwann cells in primary culture, which provide a model of Schwann cells in the injured PNS, also express abundant LRP1. Herein, we show that LRP1 gene-silencing or treatment with receptor-associated protein (RAP) promotes Schwann cell adhesion and inhibits cell migration on fibronectin. LRP1 gene-silencing also resulted in the formation of prominent focal adhesions and actin stress fibers. These changes, which were induced by loss of LRP1 expression or activity, were explained mechanistically by an increase in activated RhoA, coupled with a decrease in activated Rac1. Known LRP1 ligands, including matrix metalloprotease-9, tissue-type plasminogen activator, and alpha(2)-macroglobulin activated Rac1 in LRP1-expressing Schwann cells. An inhibitor of Rac1 activation promoted Schwann cell adhesion. Conversely, in cells in which LRP1 was silenced, a Rho kinase inhibitor promoted migration and inhibited adhesion. These results demonstrate that direct binding of ligands to LRP1 controls activation of small Rho family GTPases. The effects of LRP1 gene-silencing and RAP implicate autocrine pathways involving endogenously produced LRP1 ligands. Regulation of Schwann cell migration by LRP1 may be important in PNS injury.

The urokinase receptor (uPAR) promotes metastasis of human malignancies; however, its mechanism of action remains incompletely understood. Established models focus on the ability of uPAR to bind urokinase-type plasminogen activator (uPA) and promote protease activation in the tumor cell microenvironment; however, uPAR also regulates cell signaling and migration by both uPA-dependent and -independent mechanisms in vitro. The significance of uPAR as a cell-signaling receptor in vivo remains unclear. In this study, we expressed either human or mouse uPAR in human embryonic kidney (HEK-293) cells. We selected HEK-293 cells because, unlike most cancer cells, they do not express uPA or uPAR endogenously. Both mouse and human uPAR increased cell adhesion and migration on vitronectin. Rac1 was activated and responsible for the increase in cell migration. HEK-293 cells that did not express uPAR formed palpable tumors in severe combined immunodeficient mice; however, metastases were exceedingly rare. The xenografts contained abundant mouse uPA, produced by infiltrating mouse cells, but no human uPA. Mouse uPA bound only to mouse uPAR and not human uPAR and, thus, could not interact with human uPAR-expressing HEK-293 cells in xenografts. Nevertheless, both mouse and human uPAR significantly increased HEK-293 cell metastasis into the lungs. The activity of human uPAR suggests that uPAR may promote cancer metastasis independent of uPA. Candidate mechanisms include its effects on adhesion, migration, and Rac1 activation.

The worldwide rate of hepatocellular carcinoma (HCC) occurrence is rising rapidly; however, outside of transplantation, few effective treatment options are available. Hepatocellular carcinogenesis is a complex, multistep process involving many genetic alterations, often occurring in targets that are considered “undruggable” by conventional therapeutic modalities. Antisense oligonucleotide (ASO) technology offers the potential to target these undruggable pathways in HCC tumors in vivo and to determine the sensitivity of HCC to the ablation of these pathways. Using either the diethylnitrosamine (DEN) induced mouse HCC model or DEN in combination with the profibrotic agent carbon tetrachloride (CCl4), we first examined gene expression changes in induced HCC tumors. DEN-induced HCC has a very long latency period (∼8 months). We demonstrate that mice injected with DEN and CCl4 developed HCC with a shorter latency and a higher tumor incidence. We also identified several genes of interest that could potentially be HCC drivers and targets for ASO therapy. Conjugation of ASOs with N-acetyl D-galactosamine (GalNAc) has been shown to increase ASO activity >10x in normal liver. Here we demonstrate that GalNAc conjugated ASOs show similar increased potency in HCC tumors and the complete depletion of Myd88 protein levels. Importantly, in addition to the ability to potently inhibit Myd88 levels in tumors, GalNAc-conjugated Myd88 ASOs also resulted in significantly reduced tumor burden. These data demonstrate that GalNAc-conjugated Gen 2.5 ASOs have potent activity in HCC tumors and that Myd88 maybe be an attractive therapeutic target in this disease. Citation Format: Joanna Schmidt, Minji Jo, Tianyuan Zhou, Youngsoo Kim, A. R. MacLeod. Characterization of GalNAc-conjugated generation 2.5 ASOs in DEN and DEN/CCL4-induced HCC tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4359. doi:10.1158/1538-7445.AM2015-4359