Growth Regulatory Proteins Research Articles

Serine 319 phosphorylation is necessary and sufficient to induce a Cx37 conformation that leads to arrested cell cycling.

Connexin 37 (Cx37; protein product of GJA4) expression profoundly suppresses proliferation of rat insulinoma (Rin) cells in a manner dependent on gap junction channel (GJCh) functionality and the presence and phosphorylation status of its C-terminus (CT). In Rin cells, growth is arrested upon induced Cx37 expression and serine 319 (S319) is frequently phosphorylated. Here, we show that preventing phosphorylation at this site (alanine substitution; S319A) relieved Cx37 of its growth-suppressive effect whereas mimicking phosphorylation at this site (aspartate substitution; S319D) enhanced the growth-suppressive properties of Cx37. Like wild-type Cx37 (Cx37-WT), Cx37-S319D GJChs and hemichannels (HChs) preferred the closed state, rarely opening fully, and gated slowly. In contrast, Cx37-S319A channels preferred open states, opened fully and gated rapidly. These data indicate that phosphorylation-dependent conformational differences in Cx37 protein and channel function underlie Cx37-induced growth arrest versus growth-permissive phenotypes. That the closed state of Cx37-WT and Cx37-S319D GJChs and HChs favors growth arrest suggests that rather than specific permeants mediating cell cycle arrest, the closed conformation instead supports interaction of Cx37 with growth regulatory proteins that result in growth arrest.

Tyrosine Threonine Kinase Inhibition Eliminates Lung Cancers by Augmenting Apoptosis and Polyploidy

The spindle assembly checkpoint maintains genomic integrity. A key component is tyrosine threonine kinase (TTK, also known as Mps1). TTK antagonism is hypothesized to cause genomic instability and cell death. Interrogating The Cancer Genome Atlas revealed high TTK expression in lung adenocarcinomas and squamous cell cancers versus the normal lung (P < 0.001). This correlated with an unfavorable prognosis in examined lung adenocarcinoma cases (P = 0.007). TTK expression profiles in lung tumors were independently assessed by RNA in situ hybridization. CFI-402257 is a highly selective TTK inhibitor. Its potent antineoplastic effects are reported here against a panel of well-characterized murine and human lung cancer cell lines. Significant antitumorigenic activity followed independent treatments of athymic mice bearing human lung cancer xenografts (6.5 mg/kg, P < 0.05; 8.5 mg/kg, P < 0.01) and immunocompetent mice with syngeneic lung cancers (P < 0.001). CFI-402257 antineoplastic mechanisms were explored. CFI-402257 triggered aneuploidy and apoptotic death of lung cancer cells without changing centrosome number. Reverse phase protein arrays (RPPA) of vehicle versus CFI-402257-treated lung cancers were examined using more than 300 critical growth-regulatory proteins. RPPA bioinformatic analyses discovered CFI-402257 enhanced MAPK signaling, implicating MAPK antagonism in augmenting TTK inhibitory effects. This was independently confirmed using genetic and pharmacologic repression of MAPK that promoted CFI-402257 anticancer actions. TTK antagonism exerted marked antineoplastic effects against lung cancers and MAPK inhibition cooperated. Future work should determine whether CFI-402257 treatment alone or with a MAPK inhibitor is active in the lung cancer clinic.

Decoding WW domain tandem-mediated target recognitions in tissue growth and cell polarity.

WW domain tandem-containing proteins such as KIBRA, YAP, and MAGI play critical roles in cell growth and polarity via binding to and positioning target proteins in specific subcellular regions. An immense disparity exists between promiscuity of WW domain-mediated target bindings and specific roles of WW domain proteins in cell growth regulation. Here, we discovered that WW domain tandems of KIBRA and MAGI, but not YAP, bind to specific target proteins with extremely high affinity and exquisite sequence specificity. Via systematic structural biology and biochemistry approaches, we decoded the target binding rules of WW domain tandems from cell growth regulatory proteins and uncovered a list of previously unknown WW tandem binding proteins including β-Dystroglycan, JCAD, and PTPN21. The WW tandem-mediated target recognition mechanisms elucidated here can guide functional studies of WW domain proteins in cell growth and polarity as well as in other cellular processes including neuronal synaptic signaling.

Neurospora crassa NADPH Oxidase NOX-1 Is Localized in the Vacuolar System and the Plasma Membrane

The NADPH oxidases (NOX) catalyze the production of superoxide by transferring electrons from NADPH to O2, in a regulated manner. In Neurospora crassa NOX-1 is required for normal growth of hyphae, development of aerial mycelium and asexual spores, and it is essential for sexual differentiation and cell-cell fusion. Determining the subcellular localization of NOX-1 is a critical step in understanding the mechanisms by which this enzyme can regulate all these different processes. Using fully functional versions of NOX-1 tagged with mCherry, we show that in growing hyphae NOX-1 shows only a minor association with the endoplasmic reticulum (ER) markers Ca2+-ATPase NCA-1 and an ER lumen-targeted GFP. Likewise, NOX-1 shows minor co-localization with early endosomes labeled with YPT-52, a GTPase of the Rab5 family. In contrast, NOX-1 shows extensive co-localization with two independent markers of the entire vacuolar system; the vacuolar ATPase subunit VMA-1 and the fluorescent molecule carboxy-DFFDA. In addition, part of NOX-1 was detected at the plasma membrane. The NOX-1 regulatory subunit NOR-1 displays a very different pattern of localization, showing a fine granular distribution along the entire hypha and some accumulation at the hyphal tip. In older hyphal regions, germinating conidia, and conidiophores it forms larger and discrete puncta some of which appear localized at the plasma membrane and septa. Notably, co-localization of NOX-1 and NOR-1 was mainly observed under conidial cell-cell fusion conditions in discrete vesicular structures. NOX functions in fungi have been evaluated mainly in mutants that completely lacked this protein, also eliminating interactions between hyphal growth regulatory proteins NOR-1, the GTPase RAC-1 and the scaffold protein BEM-1. To dissect NOX-1 roles as scaffold and as ROS-producing enzyme, we analyzed the function of NOX-1::mCherry proteins carrying proline 382 by histidine (P382H) or cysteine 524 by arginine (C524R) substitutions, predicted to only affect NADPH-binding. Without notably affecting NOX-1 localization or protein levels, each of these substitutions resulted in lack of function phenotypes, indicating that NOX-1 multiple functions are all dependent on its oxidase activity. Our results open new interpretations to possible NOX functions, as components of the fungal vacuolar system and the plasma membrane, as well as to new vacuolar functions.

Abstract 2072: Selinexor in combination with chemotherapy or idelalisib elicits a synergistic cytotoxic effect in primary CLL cells

Abstract Despite the therapeutic efficacy of new targeted drugs in chronic lymphocytic leukemia (CLL), treatment of high-risk patients remains an unmet clinical need. Tumor suppressor proteins (TSPs) and growth regulatory proteins (e.g. p53, p21, FoxO3a - the effector of Akt signal transduction, and IκB - the endogenous inhibitor of NF-κB) bind the nuclear export protein XPO1 and are carried through the nuclear pore complex into the cell cytoplasm. XPO1 overexpression plays a pathogenic role in CLL by mislocalizing TSPs and other cargoes in the cytoplasm. Selinexor is an oral inhibitor of XPO1, which is active as single agent in different hematologic malignancies. The purpose of this study was to evaluate the in vitro cytotoxic effects of selinexor, used in combination with chemotherapy drugs or with the PI3K inhibitor, idelalisib, against primary CLL cells. Specifically, this study aimed at identifying combination regimens that might overcome single agent resistance. Purified CLL cells from 29 patients were exposed to selinexor (Sel) in combination with fludarabine (F-ara-A), bendamustine (Ben) or idelalisib (Ide). Drug concentrations and exposure times were selected based on data obtained in an initial cohort of 5 patients. In selected experiments, the M2-10B4 murine stromal cell (SC) line was used in co-culture with CLL primary cells. Cell viability was analysed by Annexin-V/propidium Iodide immunostaining and flow cytometry. Combination index (CI) was determined using Calcusyn software. NF-kB and Akt activity was tested through an ELISA assay. After 72-hour culture, a significant decrease in CLL cell viability was observed when samples were treated with drug combinations (i.e. Sel 0.1 µM + F-ara-A 1 µM/F-ara-A 10 µM/Ben 30 µM/Ben 50 µM/Ide 0.1 µM/Ide 1 µM/ Ide 10 µM; Sel 1 µM + F-ara-A 1 µM/Ben 30 µM/Ben 50 µM) compared to the single compounds. The majority of combinations exerted synergistic cytotoxic effects. Interestingly, the addition of selinexor was effective in restoring the fludarabine sensitivity of CLL cells showing in vitro resistance to fludarabine. Sel was also capable to overcome the SC-mediated drug resistance, as shown by the significantly increased and synergistic cytotoxicity exerted by drug combinations, compared to single agents, toward leukemic cells co-cultured with SC. From the molecular standpoint, preliminary data showed that Sel significantly potentiated the inhibitory effect exerted by single-agent idelalisib on NF-kB and Akt activity. Our data demonstrate that the combination of Sel with chemotherapy or idelalisib has synergistic cytotoxic effects, also counteracting intrinsic or SC-mediated drug resistance. In vivo experiments in the Eμ-TCL-1 mouse model are currently ongoing to further corroborate the efficacy of selected drug combinations. Citation Format: Maria Todaro, Valentina Griggio, Chiara Salvetti, Chiara Riganti, Yosef Landesman, Mario Boccadoro, Candida Vitale, Marta Coscia. Selinexor in combination with chemotherapy or idelalisib elicits a synergistic cytotoxic effect in primary CLL cells [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 2072.

Abstract 129: Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-alpha and MMP-9

Abstract Tumor-associated macrophages (TAMs) are major components of tumor microenvironment that promote invasion and metastasis of cancer cells. In this study, we investigated the effect of TAMs on phenotypic conversion of non-neoplastic MCF10A human breast epithelial cells using an indirect co-culture system. Co-culture with TAMs induced epithelial-to-mesenchymal transition, invasive phenotype, and MMP-9 upregulation in MCF10A cells. Comparative proteomic analysis revealed that endoplasmic reticulum oxidoreductase (ERO)1-alpha was increased in MCF10A cells co-cultured with TAMs compared to that in mono-cultured cells. ERO1-alpha was crucial for TAMs-induced invasive phenotype and MMP-9 upregulation involving transcription factors c-fos and c-Jun. Cytokine array analysis showed that levels of interleukin (IL)-6, C-X-C motif ligand (CXCL)1, C-C motif ligand (CCL)2, growth-regulated protein (GRO), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in conditioned media of co-cultured cells. Among these cytokines increased in conditioned media of co-cultured cells, CCL2 was secreted from TAMs, leading to induction of ERO1-alpha, MMP-9 upregulation, and invasiveness in MCF10A cells. Our findings elucidated a molecular mechanism underlying the aggressive phenotypic change of non-neoplastic breast cells by co-culture with TAMs, providing useful information for prevention or treatment of recurrent breast cancer Note: This abstract was not presented at the meeting. Citation Format: Seungeun Lee, Eunhye Lee, EunYi Ko, Mina Ham, Hye Min Lee, Eun-Sook Kim, Minsoo Koh, Hyun Kyung Lim, Joohee Jung, So Yeon Park, Aree Moon. Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-alpha and MMP-9 [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 129.

Abstract 1861: Aviscumine (ME-503) suppresses the growth of melanoma by potentially targeting c-Myc pathway

Abstract Aviscumine, a recombinant mistletoe lectin I, is produced in E. coli and has been evaluated for its antitumor activities in various experimental in-vitro and in-vivo tumor models as well as in clinical trials. A phase II trial demonstrated the safety and efficacy of Aviscumine in pretreated patients with metastatic melanoma (stage IV). However, the mechanism(s) underlying the effect of Aviscumine in melanoma is inconclusive. Here, the antitumor activities and relevant mode of actions of Aviscumine were investigated in human melanoma A375, mouse melanoma Yummer and B16 cells as well as their relevant xenograft animal models. Cells were treated with Aviscumine (0-20 ng/mL) for 48 and 72 hrs, and cell proliferation was measured by MTT assay. Alteration of cell growth regulatory proteins and cell signaling proteins were determined by Reverse Phase Proteomic Array (RPPA) and validated with western blotting. Aviscumine exerted much stronger anti-proliferative activity in both A375 and Yummer cells with IC50 of 0.18 ± 0.03 ng/ml and 0.41 ± 0.06 ng/ml, respectively than that of B16 cells (IC50 15.93 ± 3.48 ng/ml). In A375 cells, Aviscumine significantly increased subG0/G1 population suggesting Aviscumine treatment led to apoptotic and necrotic cell death. Additionally, downregulation of various proteins associated with apoptotic cell death (pCDK1, pRB, MCl-1) was also observed by RPPA. Furthermore, Aviscumine significantly decreased c-Myc protein expression in a concentration-dependent manner measured by both RPPA and western blot. Interestingly, baseline c-Myc protein expression was much higher in the Aviscumine-sensitive A375 and Yummer cells than that in the Aviscumine-resistant B16 cells. Finally, the effects of Aviscumine on tumor growth were tested via subcutaneous injection (30 ng/kg, twice per week for three weeks) in mice bearing A375 or B16 melanoma. At 3 weeks, A375 tumors were markedly smaller in Aviscumune treated mice (383.8 ± 102.2 mg) than in the control-treated group (922.4 ± 296.1 mg). c-Myc protein expression was also significantly reduced (62% vs. control) in Aviscumine treated A375 tumors, as were levels of multiple downstream metabolites (pyruvate, malate, and glutamate) (p&amp;lt; 005). In contrast, similar to the in vitro data, Aviscumine treatment showed no tumor growth inhibitory effects in B16 tumor-bearing mice evidenced by minimum reduction on terminal tumor weight between vehicle control (1455 ± 430 mg) and Aviscumine treated group (1323 ± 270 mg). Together, these preliminary data implicate c-Myc as both a target and predictive biomarker for Aviscumine elicited antitumor activity. Citation Format: Peiying Yang, Tara Conway, Patrea Rhea, Dongmei Chen, Bo Wei, Jibin Ding, Hans Lentzen, Jennifer McQuade. Aviscumine (ME-503) suppresses the growth of melanoma by potentially targeting c-Myc pathway [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 1861.

Abstract 4407: Next generation CDK2/9 inhibitor CYC065 triggers anaphase catastrophe in diverse aneuploid cancers and markedly inhibits growth and metastasis

Abstract Genetically-unstable cancer cells have supernumerary centrosomes and aneuploidy (a hallmark of cancer). CDK2 antagonism inhibits clustering of excessive centrosomes at mitosis leading to multipolar cell division and apoptotic death. This process is called anaphase catastrophe. Anaphase catastrophe affects preferentially aneuploid cancer cells while sparing normal cells with two centrosomes. CYC065 (Cyclacel) is a next-generation CDK2/9 inhibitor that is undergoing clinical trials. Here, we explored CYC065 activity against aneuploid lung and other cancers. CYC065 substantially inhibited growth, triggered apoptosis, and induced anaphase catastrophe in murine (ED1, LKR13, and 393P) and human (Hop62, A549, and H1299) lung cancer cells. In marked contrast, these effects were largely unseen in bipolar immortalized pulmonary epithelial (murine C10 and human BEAS-2B) cells. Notably, anaphase catastrophe was induced by CYC065-treatment even in pancreatic and colon cancers that are often driven by the KRAS oncoprotein. This suggests that engaging anaphase catastrophe is a general antineoplastic mechanism. Comprehensive expression analysis of nearly 300 growth-regulatory proteins was performed after CYC065 treatment in lung cancer cells using Reverse Phase Protein Arrays (RPPAs). In addition to known CDK targets (such as phosphorylated retinoblastoma protein), FAK phosphorylation and SAC phosphorylation that are known to regulate metastasis were unexpectedly down-regulated. Consistent with this finding, in vitro migration and invasion CYC065 treatment assays markedly inhibited migration and invasion of lung cancer cells. CYC065 anti-neoplastic effects were interrogated in mice. CYC065 significantly inhibited tumor growth in several lung cancer syngeneic xenografts and patient-derived xenografts (PDXs) and reduced metastasis of 344SQ lung cancer cells in a syngeneic tail-vein injection model. Immunohistochemistry (IHC) studies of resected PDX tumors revealed downregulation of p-CDK2 (Thr160) and MCL1 expression after CYC065 treatment which is consistent with CDK2/9 antagonism, respectively. In summary, CYC065 engages anaphase catastrophe and elicits marked antineoplastic effects in diverse cancers. These effects are due to mechanisms that confer after CDK2/9 inhibition, anaphase catastrophe, MCL1 down-regulation and suppression of proteins that regulate metastasis. Taken together, findings reveal that anaphase catastrophe is a general antineoplastic mechanism engaged in aneuploid cancers. Intriguingly, this mechanism is activated after CDK2/9 inhibition of lung and other cancers, including those driven by the KRAS oncoprotein. Citation Format: Masanori Kawakami, Lisa Maria Mustachio, Yulong Chen, Zibo Chen, Jason Roszik, Xi Liu, Ethan Dmitrovsky. Next generation CDK2/9 inhibitor CYC065 triggers anaphase catastrophe in diverse aneuploid cancers and markedly inhibits growth and metastasis [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 4407.

Abstract 170: Loss of the ubiquitin-specific peptidase 18 (USP18) reduces lung cancer metastasis

Abstract Metastasis is a major cause of cancer-related mortality. Yet, current antineoplastic strategies do not effectively repress metastasis. Thus, there is a pressing need to explore targets that can reduce metastasis, especially in lung cancer that is the most common cause of cancer mortality. Our prior work reported that expression of the ubiquitin-specific peptidase 18 (USP18) was augmented in diverse cancers (including lung cancers) and USP18 knock-down suppressed in vitro and in vivo growth of murine and human lung cancers. This deubiquitinase stabilizes target proteins by removing interferon-stimulated gene 15 (ISG15) from substrate proteins. We explored here whether engineered loss of USP18 decreased lung cancer metastasis. Stable USP18 knockdown was independently achieved in human (A549 and H1299) and murine (344SQ and KC2) lung cancer cells by use of small hairpin RNAs (shRNAs). Notably, knockdown of USP18 expression reduced lung cancer cellular proliferation, migration and invasion versus control shRNA transfectants (p &amp;lt; 0.001). Lung cancer cells selected for high metastatic potential (344SQ and KC2) were independently injected via tail vein or subcutaneously to establish metastatic mouse models. USP18 knockdown statistically-significantly decreased lung cancer metastases as compared to control shRNA transfectants. To elucidate engaged mechanisms, Reverse Phase Protein Array (RPPA) analysis was performed to interrogate over 300 growth-regulatory proteins in human (A549 and H1299) and murine (344SQ and KC2) lung cancer cells having engineered USP18 knockdown. Results were compared to control shRNA transfectants. Ingenuity pathway analysis highlighted fatty acid synthase (FASN) as a potential USP18 target that regulated lung cancer metastasis. Independent immunoblot analysis confirmed FASN expression was reduced in cancers once USP18 knockdown was achieved. Functional validation of FASN in lung metastasis is underway. These and other findings that will be presented provide a strong rationale for targeting the deubiquitinase USP18 to reduce lung cancer metastasis and combat lung cancers. Citation Format: Zibo Chen, Lin Zheng, Yulong Chen, Masanori Kawakami, Lisa Mustachio, Jason Roszik, Xin Liu, Jonathan Kurie, Xi Liu, Ethan Dmitrovsky. Loss of the ubiquitin-specific peptidase 18 (USP18) reduces lung cancer metastasis [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 170.

Annexin A1-derived peptide Ac2-26 in a pilocarpine-induced status epilepticus model: anti-inflammatory and neuroprotective effects

BackgroundThe inflammatory process has been described as a crucial mechanism in the pathophysiology of temporal lobe epilepsy. The anti-inflammatory protein annexin A1 (ANXA1) represents an interesting target in the regulation of neuroinflammation through the inhibition of leukocyte transmigration and the release of proinflammatory mediators. In this study, the role of the ANXA1-derived peptide Ac2-26 in an experimental model of status epilepticus (SE) was evaluated.MethodsMale Wistar rats were divided into Naive, Sham, SE and SE+Ac2-26 groups, and SE was induced by intrahippocampal injection of pilocarpine. In Sham animals, saline was applied into the hippocampus, and Naive rats were only handled. Three doses of Ac2-26 (1 mg/kg) were administered intraperitoneally (i.p.) after 2, 8 and 14 h of SE induction. Finally, 24 h after the experiment-onset, rats were euthanized for analyses of neuronal lesion and inflammation.ResultsPilocarpine induced generalised SE in all animals, causing neuronal damage, and systemic treatment with Ac2-26 decreased neuronal degeneration and albumin levels in the hippocampus. Also, both SE groups showed an intense influx of microglia, which was corroborated by high levels of ionised calcium binding adaptor molecule 1(Iba-1) and monocyte chemoattractant protein-1 (MCP-1) in the hippocampus. Ac2-26 reduced the astrocyte marker (glial fibrillary acidic protein; GFAP) levels, as well as interleukin-1β (IL-1β), interleukin-6 (IL-6) and growth-regulated alpha protein (GRO/KC). These effects of the peptide were associated with the modulation of the levels of formyl peptide receptor 2, a G-protein-coupled receptor that binds to Ac2-26, and the phosphorylated extracellular signal-regulated kinase (ERK) in the hippocampal neurons.ConclusionsThe data suggest a neuroprotective effect of Ac2-26 in the epileptogenic processes through downregulation of inflammatory mediators and neuronal loss.

Uterine double-conditional inactivation of Smad2 and Smad3 in mice causes endometrial dysregulation, infertility, and uterine cancer

SMAD2 and SMAD3 are downstream proteins in the transforming growth factor-β (TGF β) signaling pathway that translocate signals from the cell membrane to the nucleus, bind DNA, and control the expression of target genes. While SMAD2/3 have important roles in the ovary, we do not fully understand the roles of SMAD2/3 in the uterus and their implications in the reproductive system. To avoid deleterious effects of global deletion, and given previous data showing redundant function of Smad2 and Smad3, a double-conditional knockout was generated using progesterone receptor-cre (Smad2/3 cKO) mice. Smad2/3 cKO mice were infertile due to endometrial hyperproliferation observed as early as 6 weeks of postnatal life. Endometrial hyperplasia worsened with age, and all Smad2/3 cKO mice ultimately developed bulky endometrioid-type uterine cancers with 100% mortality by 8 months of age. The phenotype was hormone-dependent and could be prevented with removal of the ovaries at 6 weeks of age but not at 12 weeks. Uterine tumor epithelium was associated with decreased expression of steroid biosynthesis genes, increased expression of inflammatory response genes, and abnormal expression of cell cycle checkpoint genes. Our results indicate the crucial role of SMAD2/3 in maintaining normal endometrial function and confirm the hormone-dependent nature of SMAD2/3 in the uterus. The hyperproliferation of the endometrium affected both implantation and maintenance of pregnancy. Our findings generate a mouse model to study the roles of SMAD2/3 in the uterus and serve to provide insight into the mechanism by which the endometrium can escape the plethora of growth regulatory proteins.

Novel Biomarkers for the Precisive Diagnosis and Activity Classification of Takayasu Arteritis.

Establishing the diagnosis and determining disease activity of Takayasu arteritis (TA) remains challenging. Novel biomarkers might help to solve this problem. In the screening phase, by using large-scale protein arrays detecting samples from 90 subjects (TA active, 29; TA inactive 31; and controls, 30). In the validation phase, by using enzyme-linked immunosorbent assay (ELISA), potential biomarkers for TA diagnosis, and activity classification were measured in independent cohorts, respectively. In the screening phase, 18 cytokines significantly differentially enriched between TA patients and controls and another 15 cytokines significantly differentially enriched between TA patient in active and inactive status were identified (adjusted P<0.05). In the validation phase, TIMP (tissue inhibitor of metalloproteinases)-1 was identified as a specific biomarker for TA diagnosis that a cutoff value of 221.86 μg/L could provide a specificity of 89.58% and a positive predictive value of 0.92. Meanwhile, we found it unreliable to use a single biomarker for TA activity classification. Considering this, we further built a logistic regression model based on multiple cytokines, including CA (cancer antigen) 125, FLRG (follistatin-related protein), IGFBP (insulin-like growth factor-binding protein)-2, CA15-3, GROa (growth-regulated alpha protein), LYVE (lymphatic vessel endothelial hyaluronic acid receptor)-1, ULBP (UL16-binding protein)-2, and CD (cluster of differentiation) 99, with an area under the curve reaching 0.909 for discriminating TA activity status. This study suggested TIMP-1 as a specific biomarker for TA diagnosis with a cutoff value of 221.86 μg/L. Furthermore, we provided a logistic regression model based on 8 biomarkers for the precisive activity classification of TA with an area under the curve of 0.909.

Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-α and MMP-9

Tumor-associated macrophages (TAMs) are major components of tumor microenvironment that promote invasion and metastasis of cancer cells. In this study, we investigated the effect of TAMs on phenotypic conversion of non-neoplastic MCF10A human breast epithelial cells using an indirect co-culture system. Co-culture with TAMs induced epithelial-to-mesenchymal transition, invasive phenotype, and MMP-9 upregulation in MCF10A cells. Comparative proteomic analysis revealed that endoplasmic reticulum oxidoreductase (ERO)1-α was increased in MCF10A cells co-cultured with TAMs compared to that in mono-cultured cells. ERO1-α was crucial for TAMs-induced invasive phenotype and MMP-9 upregulation involving transcription factors c-fos and c-Jun. Cytokine array analysis showed that levels of interleukin (IL)-6, C-X-C motif ligand (CXCL)1, C-C motif ligand (CCL)2, growth-regulated protein (GRO), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in conditioned media of co-cultured cells. Among these cytokines increased in conditioned media of co-cultured cells, CCL2 was secreted from TAMs, leading to induction of ERO1-α, MMP-9 upregulation, and invasiveness in MCF10A cells. Our findings elucidated a molecular mechanism underlying the aggressive phenotypic change of non-neoplastic breast cells by co-culture with TAMs, providing useful information for prevention or treatment of recurrent breast cancer.

Anti-tumor efficacy of Selinexor (KPT-330) in gastric cancer is dependent on nuclear accumulation of p53 tumor suppressor

Exportin-1 (XPO1) controls the nucleo-cytoplasmic trafficking of several key growth regulatory and tumor suppressor proteins. Nuclear export blockade through XPO1 inhibition is a target for therapeutic inhibition in many cancers. Studies have suggested XPO1 upregulation as an indicator of poor prognosis in gastric cancer. In the current study, we investigated the anti-tumor efficacy of selective inhibitors of nuclear export (SINE) compounds KPT-185, KTP-276 and clinical stage selinexor (KPT-330) in gastric cancer. XPO1 was found to be overexpressed in gastric cancer as compared to adjacent normal tissues and was correlated with poor survival outcomes. Among the 3 SINE compounds, in vitro targeting of XPO1 with selinexor resulted in greatest potency with significant anti-proliferative effects at nano molar concentrations. XPO1 inhibition by selinexor resulted in nuclear accumulation of p53, causing cell cycle arrest and apoptosis. Also, inhibition of XPO1 lead to the cytoplasmic retention of p21 and suppression of survivin. Orally administered selienxor caused significant inhibition of tumor growth in xenograft models of gastric cancer. Furthermore, combination of selinexor with irinotecan exhibited greater anti-tumor effect compared to individual treatment. Taken together, our study underscores the therapeutic utility of XPO1 targeting in gastric cancer and suggests the potential benefits of XPO1 inhibition in-combination with chemotherapy.

Beta2 -adrenoceptor agonist salbutamol increases protein turnover rates and alters signalling in skeletal muscle after resistance exercise in young men.

Animal models have shown that beta2 -adrenoceptor stimulation increases protein synthesis and attenuates breakdown processes in skeletal muscle. Thus, the beta2 -adrenoceptor is a potential target in the treatment of disuse-, disease- and age-related muscle atrophy. In the present study, we show that a few days of oral treatment with the commonly prescribed beta2 -adrenoceptor agonist, salbutamol, increased skeletal muscle protein synthesis and breakdown during the first 5h after resistance exercise in young men. Salbutamol also counteracted a negative net protein balance in skeletal muscle after resistance exercise. Changes in protein turnover rates induced by salbutamol were associated with protein kinase A-signalling, activation of Akt2 and modulation of mRNA levels of growth-regulating proteins in skeletal muscle. These findings indicate that protein turnover rates can be augmented by beta2 -adrenoceptor agonist treatment during recovery from resistance exercise in humans. The effect of beta2 -adrenoceptor stimulation on skeletal muscle protein turnover and intracellular signalling is insufficiently explored in humans, particularly in association with exercise. In a randomized, placebo-controlled, cross-over study investigating 12 trained men, the effects of beta2 -agonist (6×4mg oral salbutamol) on protein turnover rates, intracellular signalling and mRNA response in skeletal muscle were investigated 0.5-5h after quadriceps resistance exercise. Each trial was preceded by a 4-day lead-in treatment period. Leg protein turnover rates were assessed by infusion of [13 C6 ]-phenylalanine and sampling of arterial and venous blood, as well as vastus lateralis muscle biopsies 0.5 and 5h after exercise. Furthermore, myofibrillar fractional synthesis rate, intracellular signalling and mRNA response were measured in muscle biopsies. The mean (95% confidence interval) myofibrillar fractional synthesis rate was higher for salbutamol than placebo [0.079(95% CI, 0.064 to 0.093) vs. 0.066(95% CI, 0.056 to 0.075%)×h-1 ] (P<0.05). Mean net leg phenylalanine balance 0.5-5h after exercise was higher for salbutamol than placebo [3.6(95% CI, 1.0 to 6.2nmol)×min-1 ×100 gLeg Lean Mass-1 ] (P<0.01). Phosphorylation of Akt2, cAMP response element binding protein and PKA substrate 0.5 and 5h after exercise, as well as phosphorylation of eEF2 5h after exercise, was higher (P<0.05) for salbutamol than placebo. Calpain-1, Forkhead box protein O1, myostatin and Smad3 mRNA content was higher (P<0.01) for salbutamol than placebo 0.5h after exercise, as well as Forkhead box protein O1 and myostatin mRNA content 5h after exercise, whereas ActivinRIIB mRNA content was lower (P<0.01) for salbutamol 5h after exercise. These observations suggest that beta2 -agonist increases protein turnover rates in skeletal muscle after resistance exercise in humans, with concomitant cAMP/PKA and Akt2 signalling, as well as modulation of mRNA response of growth-regulating proteins.

Osteogenic Differentiation Modulates the Cytokine, Chemokine, and Growth Factor Profile of ASCs and SHED.

Great efforts have been made to improve bone regeneration techniques owing to a growing variety of sources of stem cells suitable for autologous transplants. Specifically, adipose-derived stem cells (ASCs) and stems cells from human exfoliated deciduous teeth (SHED) hold great potential for bone tissue engineering and cell therapy. After a preliminary characterization of the main biomolecules ASCs and SHED released in their conditioned media, cells were kept both in normal and osteo-inducing conditions. Conventional assays were performed to prove their osteogenic potential such as quantitative real-time polymerase chain reaction (qRT-PCR) (for RUNX-2, collagen type I, osteopontin and osteonectin), alkaline phosphatase activity, osteocalcin production, and von Kossa staining. Conditioned media were tested again after the osteogenic induction and compared to maintaining condition both at base line and after 14 days of culture. The osteogenic condition inhibited the release of all the biomolecules, with the exception, concerning SHED, of growth-regulated alpha protein precursor (GROα), and, to a lesser extent, interleukin (IL)-8. In conclusion, our data support that undifferentiated ASCs and SHED may be preferable to committed ones for general cell therapy approaches, due to their higher paracrine activity. Osteoinduction significantly affects the cytokine, chemokine, and growth factor profile in a differential way, as SHED kept a more pronounced pro-angiogenic signature than ASCs.

Evidence for the ISG15-Specific Deubiquitinase USP18 as an Antineoplastic Target.

Ubiquitination and ubiquitin-like posttranslational modifications (PTM) regulate activity and stability of oncoproteins and tumor suppressors. This implicates PTMs as antineoplastic targets. One way to alter PTMs is to inhibit activity of deubiquitinases (DUB) that remove ubiquitin or ubiquitin-like proteins from substrate proteins. Roles of DUBs in carcinogenesis have been intensively studied, yet few inhibitors exist. Prior work provides a basis for the ubiquitin-specific protease 18 (USP18) as an antineoplastic target. USP18 is the major DUB that removes IFN-stimulated gene 15 (ISG15) from conjugated proteins. Prior work discovered that engineered loss of USP18 increases ISGylation and in contrast to its gain decreases cancer growth by destabilizing growth-regulatory proteins. Loss of USP18 reduced cancer cell growth by triggering apoptosis. Genetic loss of USP18 repressed cancer formation in engineered murine lung cancer models. The translational relevance of USP18 was confirmed by finding its expression was deregulated in malignant versus normal tissues. Notably, the recent elucidation of the USP18 crystal structure offers a framework for developing an inhibitor to this DUB. This review summarizes strong evidence for USP18 as a previously unrecognized pharmacologic target in oncology. Cancer Res; 78(3); 587-92. ©2018 AACR.

Abstract LB-B14: Pediatric Preclinical Testing Consortium evaluation of the second-generation selective inhibitor of nuclear export (SINE) compound KPT-8602

Abstract Introduction: KPT-8602 is a second-generation SINE compound that specifically blocks exportin-1 (XPO1) cargo interactions. XPO1 is a nuclear export receptor, a critical regulator of cell proliferation and survival, that is characterized as the sole transporter of several tumor suppressor and growth regulatory proteins, including p53, p21, p27, BCR-ABL, FOXO and STAT3. Overexpression of XPO1 has been correlated with high-risk disease and poor survival in multiple pediatric cancers, including leukemia and neuroblastoma (NB). Our prior work demonstrated that selinexor, a closely-related SINE compound, induced complete remission (CR) or maintained CR (MCR) in 2 of 8 ALL patient-derived xenografts (PDXs) tested while it did not induce tumor regressions in 6 neuroblastoma models (Attiyeh EF, et al. Pediatr Blood Cancer. 2016;63:276-86). While selinexor has shown activity in a variety of adult malignancies and is being tested in pediatric phase 1 trials, KPT-8602 shows similar pharmacokinetic properties to selinexor and improved tolerability in preclinical models, and thus it was tested against ALL and NB models by the PPTC. Methods: KPT-8602 was tested against 6 NB and 12 ALL pediatric PDX models using 10 and 8 mice/arm, respectively. Based on tolerability, a dose of 12.5 mg/kg was selected and administered once daily via oral gavage 5 days per week for 4 weeks. Events were defined as quadrupling of tumor volume from day 0 (for NB) or as the proportion of human CD45+ cells in the peripheral blood (%huCD45+) ≥25% (for ALL). The Kaplan-Meier method was used to compare time-to-event between treated and control groups. Objective response categories were assigned as progressive disease (PD, which is subdivided into progressive disease without or with growth delay, PD1 or PD2, respectively), stable disease (SD), partial response (PR), CR, and MCR [Houghton PJ, et al. Pediatr Blood Cancer, 2007]. KPT-8602 was developed and provided for testing by Karyopharm Therapeutics, Inc. Results: KPT-8602 was generally well tolerated, but with weight loss during treatment of approximately 15% and 2% for the neuroblastoma and ALL models, respectively. KPT-8602 induced significant differences in event free survival (EFS) distribution compared to control in 16 of the 18 (88.9%) evaluable xenograft lines studied. Four NB and 10 ALL PDX models showed a &amp;gt;2-fold extension in median EFS for treated compared to control animals. For the 6 NB PDX models, 3 had a minimum relative tumor volume (minRTV) of &amp;lt;1, and 2 achieved PR while 1 was PD2. For the ALL panel, 3 achieved a CR and 3 more achieved a MCR. Among the ALL PDXs achieving CR/MCR were 4 B-cell ALL (including 2 Ph-like ALL) and 2 T-ALL. The neuroblastoma PDX showing the most favorable response to selinexor showed a PR to KPT-8602, and the 2 ALL PDXs with CR/MCR responses to selinexor showed MCR responses to KPT-8602. Conclusions: These data indicate that KPT-8602 demonstrates significant anti-cancer efficacy and tolerability in preclinical models of childhood ALL and NB. Our results support further preclinical testing of KPT-8602 in combination with other targeted therapies for ALL and NB, as well as testing against additional pediatric cancer models in the PPTC. (Supported by NCI Grants: CA199222; CA199000; CA199287) Citation Format: Kateryna Krytska, Kathryn Evans, Tara Pritchard, Matthew Tsang, Raymond Yung, Yael P. Mosse, Stephen W. Erickson, Yuelong Guo, Erkan Baloglu, Yosef Landesman, Kyle A. Jensen, William Senapedis, Beverly A. Teicher, Malcolm A. Smith, Richard B. Lock, John M. Maris. Pediatric Preclinical Testing Consortium evaluation of the second-generation selective inhibitor of nuclear export (SINE) compound KPT-8602 [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 LB-B14.

The IL-1β signalling pathway and its role in regulating pro-inflammatory and pro-labour mediators in human primary myometrial cells

Interleukin (IL)-1β plays a central role in the processes of human labour and delivery. The adaptor proteins involved in the IL-1β signalling pathway in human myometrium are not known. This study sought to determine the role of the adaptor proteins myeloid differentiation primary response 88 (MyD88), tumour necrosis factor receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinase 4 (IRAK4) and transforming growth factor beta-activated kinase 1 (TAK1) in IL-1β-induced formation of pro-inflammatory and pro-labour mediators in human myometrium. Human primary myometrial cells were transfected with siRNA against MyD88 (siMYD88), TRAF6 (siTRAF6), IRAK4 (siIRAK4) or TAK1 (siTAK1), treated with IL-1β, and assayed for the mRNA expression and or secretion of pro-inflammatory and pro-labour mediators. Transfection of primary myometrial cells with siMYD88, siTRAF6, siIRAK4 and siTAK1 significantly decreased IL-1β-induced IL-1α, IL-6, growth-regulated alpha protein (GRO-α), IL-8, monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1 and cyclooxygenase (COX)-2 mRNA expression and release of IL-6, GRO-α, IL-8, MCP-1, ICAM-1 and prostaglandin PGF2α. The expression and secretion of the extracellular matrix remodelling enzyme matrix metalloproteinase (MMP)-9 was significantly lower with siMYD88 and siTRAF6. Finally, IL-1β-induced nuclear factor κB (NF-κB) transcriptional activity was significantly attenuated by transfection with siMyD88, siTRAF6 and siIRAK4; there was no effect of siTAK1 transfection on NF-κB transcriptional activity. Collectively, these findings suggest that MyD88, TRAF6, IRAK4 and TAK1 are involved in IL-1β signalling in human myometrium. Further studies are required to determine if inhibition of these proteins can prevent preterm birth.

Proteomic profiling of human HepG2 cells treated with hesperidin using antibody array.

Protein array technology not only identifies a large number of proteins but also determines their expression levels. In the present study, antibody array analysis is used to decipher the proteins involved in hesperidin-induced cell death in HepG2 cells. Altered proteins in hesperidin treated cells were compared with that of untreated control cells by using a RayBio® Label-based (L series) human antibody array kit. The identified proteins were further confirmed using western blot analysis. STRING software based analysis was used to determine the protein-protein interactions. Many proteins related to signal transduction, cellular mechanisms, cell growth and proliferation regulatory proteins were identified. Among the proteins identified Hsp90, Smac/DIABLO, Prdx6 and FRK were significantly reduced in hesperidin treated cells. To the best of the authors' knowledge, the present study is the first to use antibody array for identifying proteins marker in hesperidin-induced cell death in HepG2 cells. The present study provides a novel insight into the anticancer mechanism of hesperidin.