Cell Context-dependent Manner Research Articles

Thiol-ene "Click" Synthesis and Pharmacological Evaluation of C-Glycoside sp2-Iminosugar Glycolipids.

The unique stereoelectronic properties of sp2-iminosugars enable their participation in glycosylation reactions, thereby behaving as true carbohydrate chemical mimics. Among sp2-iminosugar conjugates, the sp2-iminosugar glycolipids (sp2-IGLs) have shown a variety of interesting pharmacological properties ranging from glycosidase inhibition to antiproliferative, antiparasitic, and anti-inflammatory activities. Developing strategies compatible with molecular diversity-oriented strategies for structure–activity relationship studies was therefore highly wanted. Here we show that a reaction sequence consisting in stereoselective C-allylation followed by thiol-ene “click” coupling provides a very convenient access to α-C-glycoside sp2-IGLs. Both the glycone moiety and the aglycone tail can be modified by using sp2-iminosugar precursors with different configurational profiles (d-gluco or d-galacto in this work) and varied thiols, as well as by oxidation of the sulfide adducts (to the corresponding sulfones in this work). A series of derivatives was prepared in this manner and their glycosidase inhibitory, antiproliferative and antileishmanial activities were evaluated in different settings. The results confirm that the inhibition of glycosidases, particularly α-glucosidase, and the antitumor/leishmanicidal activities are unrelated. The data are also consistent with the two later activities arising from the ability of the sp2-IGLs to interfere in the immune system response in a cell line and cell context dependent manner.

Synthesis of polyfluoroalkyl sp2-iminosugar glycolipids and evaluation of their immunomodulatory properties towards anti-tumor, anti-leishmanial and anti-inflammatory therapies

Immunomodulatory glycolipids, among which α-galactosylceramide (KRN7000) is an iconic example, have shown strong therapeutic potential in a variety of conditions ranging from cancer and infection to autoimmune or neurodegenerative diseases. A main difficulty for those channels is that they often provoke a cytokine storm comprising both pro- and anti-inflammatory mediators that antagonize each other and negatively affect the immune response. The synthesis of analogues with narrower cytokine secretion-inducing capabilities is hampered by the intrinsic difficulty at controlling the stereochemical outcome in glycosidation reactions, particularly if targeting the α-anomer, which seriously hampers drug optimization strategies. Here we show that replacing the monosaccharide glycone by a sp2-iminosugar glycomimetic moiety allows accessing N-linked sp2-iminosugar glycolipids (sp2-IGLs) with total α-stereocontrol in a single step with no need of protecting groups or glycosidation promotors. The lipid tail has been then readily tailored by incorporating polyfluoroalkyl segments of varied lengths in view of favouring binding to the lipid binding site of the master p38 mitogen activated protein kinase (p38 MAPK), thereby polarizing the immune response in a cell-context dependent manner. The compounds have been evaluated for their antiproliferative, anti-leishmanial and anti-inflammatory activities in different cell assays. The size of the fluorous segment was found to be critical for the biological activity, probably by regulating the aggregation and membrane-crossing properties, whereas the hydroxylation profile (gluco or galacto-like) was less relevant. Biochemical and computational data further support a mechanism of action implying binding to the allosteric lipid binding site of p38 MAPK and subsequent activation of the noncanonical autophosphorylation route. The ensemble of results provide a proof of concept of the potential of sp2-IGLs as immunoregulators.

SIX4 acts as a master regulator of oncogenes that promotes tumorigenesis in non-small-cell lung cancer cells

A number of homeobox genes are implicated in the malignancy of various cancers. Here, we investigated the role of the homeobox gene SIX4 in non-small-cell lung cancer (NSCLC). The sine oculis homeobox (SIX4) gene was found to be highly expressed at both mRNA and protein levels in NSCLC tumor tissues as compared with matching normal counterparts. In this study, the SIX4 gene of two human NSCLC cell lines (A549 and PC9) was overexpressed or silenced using the lentiviral system. We evaluated the malignancy-associated phenotype of transfected cells, which demonstrated that exogenous expression of the SIX4 gene greatly enhanced the proliferation, migration, and invasion of NSCLC cells. The opposite was true in the SIX4-silenced cells. Transcriptomic profiling analysis revealed that the SIX4 gene modulated the expression of hundreds of downstream target genes in a cell context-dependent manner. Most notably, the SIX4 gene controls the expression of crucial genes with evidently oncogenic function. We conclude that SIX4 plays an oncogenic role and may be potentially utilized as a diagnostic and therapeutic marker for NSCLC.

Targeting B7-H1 (PD-L1) sensitizes cancer cells to chemotherapy

Development of resistance to chemotherapy is a major obstacle in extending the survival of patients with cancer. Although originally defined as an immune checkpoint molecule, B7-H1 (also named as PD-L1 or CD274) was found to play a role in cancer chemoresistance; however, the underlying mechanism of action of B7-H1 in regulation of chemotherapy sensitivity remains unclear in cancer cells. Here we show that development of chemoresistance depends on an increased activation of ERK in cancer cells overexpressing B7-H1. Conversely, B7-H1 knockout (KO) by CRISPR/Cas9 renders human cancer cells susceptible to chemotherapy in a cell-context dependent manner through a reduced activation of p38 MAPK. B7-H1 was found to associate with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and this association promoted or maintained the activation of ERK or p38 MAPK in cancer cells. Importantly, we found that targeting B7-H1 by anti-B7-H1 monoclonal antibody (H1A) increased the sensitivity of human triple negative breast cancer cells to cisplatin therapy in vivo. Our results suggest that targeting B7-H1 by an antibody capable of disrupting B7-H1 signals may be a new approach to sensitize cancer cells to chemotherapy.

Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

AC0010 is a pyrrolopyrimidine-based irreversible inhibitor of epidermal growth factor receptor (EGFR), structurally distinct from previously reported pyrimidine-based irreversible EGFR inhibitors such as osimertinib and rociletinib. AC0010 selectively inhibits EGFR T790M mutation in both preclinical and clinical studies. However, AC0010 treatment eventually triggers drug resistance with unknown mechanism. To this end, we established two H1975 NSCLC-derived lines resistant to AC0010 after a series of drug exposure and selection in either nude-mice xenograft tumor (H1975-P) or cell culture (H1975-AVR) settings. Both lines obtained 100-fold resistance to AC0010 as compared to the parental lines. To elucidate underlying mechanism, we performed unbiased RNAseq-based profiling analysis and found that H1975-P cells had c-MET overexpression, whereas H1975-AVR cells had BCL-2 overexpression. AC0010 resistance was partially abrogated by targeting c-MET or BCL-2 using either pharmacological (small molecule inhibitors) and/or genetic (siRNA-based knockdown) approach, respectively. Our study shows that drug resistance to AC0010 can be developed via the different mechanism in a cell context–dependent manner and provides the proof-of-concept evidence for rational drug combinations to overcome resistance for maximal therapeutic efficacy.

Fractionated radiotherapy might induce epithelial-mesenchymal transition and radioresistance in a cellular context manner.

Despite the fact that radiotherapy is a main therapeutic modality in cancer treatment, recent evidence suggests that fractionated radiotherapy (FR) might confer radioresistance through epithelial-mesenchymal transition (EMT). Nevertheless, the effects of FR on EMT phenotype and the potential link between EMT induction and radioresistance development yet to be clarified. The aim of this study was to assess whether FR could promote EMT, and to elucidate if induction of EMT contributes to the acquisition of radioresistance. To this end, two human cancer cell lines (A549 and HT-29) were irradiated (2 Gy/day) and analyzed using wound healing, transwell migration and invasion assays, real-time polymerase chain reaction (for E-cadherin, N-cadherin, Vimentin, CD44, CD133, Snail, and Twist), clonogenic assay, Annexin V/PI, and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Irradiation of A549 (for 5 or 10 consecutive days) resulted in morphological changes including elongation of cytoplasm and nuclei and pleomorphic nuclei. Also, irradiation-enhanced migratory and invasive potential of A549. These phenotypic changes were in agreement with decreased expression of the epithelial marker (E-cadherin), enhanced expression of mesenchymal markers (N-cadherin, Vimentin, Snail, and Twist) and increased stemness factors (CD44 and CD133). Moreover, induction of EMT phenotype was accompanied with enhanced radioresistance and proliferation of irradiated A549. However, FR (for 5 consecutive days) did not increase HT-29 motility. Furthermore, molecular alterations did not resemble EMT phenotype (downregulation of E-cadherin, Vimentin, ALDH, CD44, CD133, and Snail). Eventually, FR led to enhanced radiosensitivity and decreased proliferation of HT-29. Altogether, our findings suggest that FR might induce EMT and confer radioresistance in a cell context-dependent manner.

Siva-1 emerges as a tissue-specific oncogene beyond its classic role of a proapoptotic gene

Siva-1 is a typical apoptotic protein commonly activated by the p53 tumor suppressor protein and should therefore participate in a barrier against the development of cancer. It has proapoptotic activities in various cell systems. Recent findings suggest that Siva-1 possesses several other apoptosis-independent functions and interacts with many other proteins not directly involved in apoptosis. It harbors the ARF E3 ubiquitin protein ligase activity, a property that is clearly prooncogenic and leads to p53 degradation through the upregulation of the Hdm2 protein level. Surprisingly, recent evidence shows that Siva-1 absence prevents the development of non-small cell lung carcinomas in a mouse model and reveals the oncogenic roles in the same types of human cells, indicating its unique function as an oncogene in the cell context-dependent manner. Herein, we review reported activities of Siva-1 in various experimental settings and comment on its ambiguous function in tumor biology.

3032 - The Cell Polarity and Scaffolding Protein, PAR3, Acts as A Tumour Suppressor in Acute Myeloid Leukemia Through Regulation of the Hippo Pathway

A key feature of epithelial cancers is loss of cell polarity. Polarity in epithelial cells is maintained by three complexes, the Par, Scribble, and Crumbs complexes. PAR3, a key component of the Par polarity complex, acts as a tumour suppressor or promoter in a cell and physiological context dependent manner in epithelial cancers. However, the role of PAR3 in hematopoiesis and hematopoietic malignancies is unknown. We utilised different Par3 conditional knockout mouse models in conjunction with an extensive phenotypic labelling regime and flow cytometry to investigate the role of PAR3 in leukemogenesis. We show loss of PAR3 in experimental mice conferred a strong predisposition to acute myeloid leukemia (AML) as evidenced by a significant increase in Granulocyte/Macrophage progenitors (GMP). In line with these results, interrogation of the TCGA data set revealed AML patients with low levels of PARD3 have a significantly worse prognosis. Reverse Phase Protein Array (RPPA) analyses revealed significant up-regulation of the Hippo downstream transcriptional activator, Yes-associated protein (YAP), in experimental animals compared to controls. Immunohistochemistry confirmed RPPA results and showed YAP was significantly up-regulated in the bone marrow of experimental mice compared to controls and coincided with a significant increase in proliferation as indicated by Ki67 staining. PAR3 and YAP predominantly co-localised in the nucleus further implicating PAR3 as an important component of the Hippo pathway. RNAseq in conjunction with PCR arrays are being conducted to reveal other Hippo family members deregulated in experimental mice compared to controls and we are using 7-color multiplex immunohistochemistry to identify alterations in the spatial distribution and physiology of GMP in the bone marrow upon loss of PAR3. Collectively, our studies will enhance our understanding of how PAR3 suppresses AML and may reveal potential therapeutic targets.

RIP kinase 1-dependent endothelial necroptosis underlies systemic inflammatory response syndrome.

Receptor interacting protein kinase 1 (RIPK1) has important kinase-dependent and kinase-independent scaffolding functions that activate or prevent apoptosis or necroptosis in a cell context-dependent manner. The kinase activity of RIPK1 mediates hypothermia and lethality in a mouse model of TNF-induced shock, reflecting the hyperinflammatory state of systemic inflammatory response syndrome (SIRS), where the proinflammatory "cytokine storm" has long been viewed as detrimental. Here, we demonstrate that cytokine and chemokine levels did not predict survival and, importantly, that kinase-inactive Ripk1D138N/D138N hematopoietic cells afforded little protection from TNF- or TNF/zVAD-induced shock in reconstituted mice. Unexpectedly, RIPK1 kinase-inactive mice transplanted with WT hematopoietic cells remained resistant to TNF-induced shock, revealing that a nonhematopoietic lineage mediated protection. TNF-treated Ripk1D138N/D138N mice exhibited no significant increases in intestinal or vascular permeability, nor did they activate the clotting cascade. We show that TNF administration damaged the liver vascular endothelium and induced phosphorylated mixed lineage kinase domain-like (phospho-MLKL) reactivity in endothelial cells isolated from TNF/zVAD-treated WT, but not Ripk1D138N/D138N, mice. These data reveal that the tissue damage present in this SIRS model is reflected, in part, by breaks in the vasculature due to endothelial cell necroptosis and thereby predict that RIPK1 kinase inhibitors may provide clinical benefit to shock and/or sepsis patients.

The regulatory roles of Notch in osteocyte differentiation via the crosstalk with canonical Wnt pathways during the transition of osteoblasts to osteocytes.

Osteocytes comprise more than 90% of the cells in bone and are differentiated from osteoblasts via an unknown mechanism. Recently, it was shown that Notch signaling plays an important role in osteocyte functions. To gain insights into the mechanisms underlying the functions of Notch in regulating the transition of osteoblasts to osteocytes, we performed a luciferase assay by cloning the proximal E11 and dentin matrix acidic phosphoprotein 1 (DMP1) promotor regions into pGluc-Basic 2 vectors, which were subsequently transfected into the IDG-SW3 (osteocytes), MC3T3 (osteoblasts) and 293T (non-osteoblastic cells) cell lines. Two approaches were used to activate Notch signaling in vitro. One was a Notch1 extracellular antibody-coated cell culture plate, and the other was transfection of a Hairy/Enhancer of Split 1 (Hes1) overexpression vector. The interaction between the Notch and Wnt signaling pathways was probed by assessing the expression of a series of phosphorylated proteins involved in the cascade of both signaling pathways. Our data suggested that Notch signaling regulates E11 expression through Hes1 activity, while Hes1 solely did not initiate the expression of DMP1. The regulatory function of E11 by Hes1 was not observed in the 293T cell line, indicating a cell context-dependent manner of the Notch signaling pathway. Additionally, we found that Notch inhibited Wnt signaling at the late differentiation stage of osteocytes by both directly repressing phosphorylated Akt and preventing the nuclear aggregation of β-catenin. These findings provide profound understandings of Notch's regulatory function in osteocyte differentiation.

Crosstalk between TGF-β signaling and epigenome.

The transforming growth factor beta (TGF-β) family of ligands plays major roles in embryonic development, tissue homeostasis, adult immunity, and wound repair. Dysregulation of TGF-β signaling pathway leads to severe diseases. Its key components have been revealed over the past two decades. This family of cytokines acts by activating receptor activated SMAD (R-SMAD) transcription factors, which in turn modulate the expression of specific sets of target genes. Cells of a multicellular organism have the same genetic information, yet they show structural and functional differences owing to differential expression of their genes. Studies have demonstrated that epigenetic regulation, an integral part of the TGF-β signaling, enables cells to sense and respond to TGF-β signaling in a cell context-dependent manner. R-SMAD, as the central transcription factor of TGF-β signaling, can recruit various epigenetic regulators to shape the transcriptome. In this review, we focus on epigenetic regulatory mechanisms in the TGF-β signaling during mammalian development and diseases and discuss the central role of the interaction between R-SMAD and various epigenetic regulators in this epigenetic regulation. The crosstalk between TGF-β signaling and the epigenome could serve as a versatile fine-tuning mechanism for transcriptional regulation during embryonic development and progression of diseases, particularly cancer.

EphA2 signaling is impacted by carcinoembryonic antigen cell adhesion molecule 1-L expression in colorectal cancer liver metastasis in a cell context-dependent manner.

We have shown that carcinoembryonic antigen cell adhesion molecule 1 long isoform (CEACAM1-L) expression in MC38 metastatic colorectal cancer (CRC) cells results in liver metastasis inhibition via CCL2 and STAT3 signaling. But other molecular mechanisms orchestrating CEACAM1-L-mediated metastasis inhibition remain to be defined. We screened a panel of mouse and human CRC cells and evaluated their metastatic outcome after CEACAM1 overexpression or downregulation. An unbiased transcript profiling and a phospho-receptor tyrosine kinase screen comparing MC38 CEACAM1-L-expressing and non-expressing (CT) CRC cells revealed reduced ephrin type-A receptor 2 (EPHA2) expression and activity. An EPHA2-specific inhibitor reduced EPHA2 downstream signaling in CT cells similar to that in CEACAM1-L cells with decreased proliferation and migration. Human CRC patients exhibiting high CEACAM1 in combination with low EPHA2 expression benefited from longer time to first recurrence/metastasis compared to those with high EPHA2 expression. With the added interaction of CEACAM6, we denoted that CEACAM1 high- and EPHA2 low-expressing patient samples with lower CEACAM6 expression also exhibited a longer time to first recurrence/metastasis. In HT29 human CRC cells, down-regulation of CEACAM1 along with CEA and CEACAM6 up-regulation led to higher metastatic burden. Overall, CEACAM1-L expression in poorly differentiated CRC can inhibit liver metastasis through cell context-dependent EPHA2-mediated signaling. However, CEACAM1’s role should be considered in the presence of other CEACAM family members.

MTOR signaling in immune cells and its implications for cancer immunotherapy

The realization that cellular metabolism dictates immune cell development, differentiation and function affords metabolic intervention as a potential venue for cancer immunotherapy. mTOR signaling, as a metabolic master regulator, controls immune cell biology in a cell type-specific and context dependent manner. Furthermore, mTOR activity needs to be fine-tuned to maintain proper immune function. These properties may be exploited for therapeutic purpose, yet caution should be taken against radical changes of mTOR activity. Here, the intricate and complex mTOR regulation in different immune cells is reviewed, highlighting latest discoveries and opportunities for cancer immunotherapy.

Molecular genetics and targeted therapy of WNT-related human diseases (Review).

Canonical WNT signaling through Frizzled and LRP5/6 receptors is transduced to the WNT/β-catenin and WNT/stabilization of proteins (STOP) signaling cascades to regulate cell fate and proliferation, whereas non-canonical WNT signaling through Frizzled or ROR receptors is transduced to the WNT/planar cell polarity (PCP), WNT/G protein-coupled receptor (GPCR) and WNT/receptor tyrosine kinase (RTK) signaling cascades to regulate cytoskeletal dynamics and directional cell movement. WNT/β-catenin signaling cascade crosstalks with RTK/SRK and GPCR-cAMP-PKA signaling cascades to regulate β-catenin phosphorylation and β-catenin-dependent transcription. Germline mutations in WNT signaling molecules cause hereditary colorectal cancer, bone diseases, exudative vitreoretinopathy, intellectual disability syndrome and PCP-related diseases. APC or CTNNB1 mutations in colorectal, endometrial and prostate cancers activate the WNT/β-catenin signaling cascade. RNF43, ZNRF3, RSPO2 or RSPO3 alterations in breast, colorectal, gastric, pancreatic and other cancers activate the WNT/β-catenin, WNT/STOP and other WNT signaling cascades. ROR1 upregulation in B-cell leukemia and solid tumors and ROR2 upregulation in melanoma induce invasion, metastasis and therapeutic resistance through Rho-ROCK, Rac-JNK, PI3K-AKT and YAP signaling activation. WNT signaling in cancer, stromal and immune cells dynamically orchestrate immune evasion and antitumor immunity in a cell context-dependent manner. Porcupine (PORCN), RSPO3, WNT2B, FZD5, FZD10, ROR1, tankyrase and β-catenin are targets of anti-WNT signaling therapy, and ETC-159, LGK974, OMP-18R5 (vantictumab), OMP-54F28 (ipafricept), OMP-131R10 (rosmantuzumab), PRI-724 and UC-961 (cirmtuzumab) are in clinical trials for cancer patients. Different classes of anti-WNT signaling therapeutics are necessary for the treatment of APC/CTNNB1-, RNF43/ZNRF3/RSPO2/RSPO3- and ROR1-types of human cancers. By contrast, Dickkopf-related protein 1 (DKK1), SOST and glycogen synthase kinase 3β (GSK3β) are targets of pro-WNT signaling therapy, and anti-DKK1 (BHQ880 and DKN-01) and anti-SOST (blosozumab, BPS804 and romosozumab) monoclonal antibodies are being tested in clinical trials for cancer patients and osteoporotic post-menopausal women. WNT-targeting therapeutics have also been applied as reagents for in vitro stem-cell processing in the field of regenerative medicine.

The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/β-catenin pathway by inhibiting the proteasome

UBE3A is a HECT domain E3 ubiquitin ligase whose dysfunction is linked to autism, Angelman syndrome, and cancer. Recently, we characterized a de novo autism-linked UBE3A mutant (UBE3AT485A) that disrupts phosphorylation control of UBE3A activity. Through quantitative proteomics and reporter assays, we found that the UBE3AT485A protein ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear β-catenin, and stimulates canonical Wnt signaling more effectively than wild-type UBE3A. We also found that UBE3AT485A activates Wnt signaling to a greater extent in cells with low levels of ongoing Wnt signaling, suggesting that cells with low basal Wnt activity are particularly vulnerable to UBE3AT485A mutation. Ligase-dead UBE3A did not stimulate Wnt pathway activation. Overexpression of several proteasome subunits reversed the effect of UBE3AT485A on Wnt signaling. We also observed that subunits that interact with UBE3A and affect Wnt signaling are located along one side of the 19S regulatory particle, indicating a previously unrecognized spatial organization to the proteasome. Altogether, our findings indicate that UBE3A regulates Wnt signaling in a cell context-dependent manner and that an autism-linked mutation exacerbates these signaling effects. Our study has broad implications for human disorders associated with UBE3A gain or loss of function and suggests that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.

Notch3 signaling-mediated melanoma–endothelial crosstalk regulates melanoma stem-like cell homeostasis and niche morphogenesis

Melanoma is among the most virulent cancers, owing to its propensity to metastasize and its resistance to current therapies. The treatment failure is largely attributed to tumor heterogeneity, particularly subpopulations possessing stem cell-like properties, ie, melanoma stem-like cells (MSLCs). Evidence indicates that the MSLC phenotype is malleable and may be acquired by non-MSLCs through phenotypic switching upon appropriate stimuli, the so–called ‘dynamic stemness'. Since the phenotypic characteristics and functional integrity of MSLCs depend on their vascular niche, using a two-dimensional (2D) melanoma–endothelium co-culture model, where the MSLC niche is recapitulated in vitro, we identified Notch3 signaling pathway as a micro-environmental cue governing MSLC phenotypic plasticity via pathway-specific gene expression arrays. Accordingly, lentiviral shRNA-mediated Notch3 knockdown (KD) in melanoma cell lines exhibiting high levels of endogenous Notch3 led to retarded/abolished tumorigenicity in vivo through both depleting MSLC fractions, evinced by MSLC marker downregulation (eg, CD133 and CD271); and impeding the MSLC niche, corroborated by the attenuated tumor angiogenesis as well as vasculogenic mimicry. In contrast, Notch3 KD affected neither tumor growth nor MSLC subsets in a melanoma cell line with relatively low endogenous Notch3 expression. Thus, Notch3 signaling may facilitate MSLC plasticity and niche morphogenesis in a cell context-dependent manner. Our findings illustrate Notch3 as a molecular switch driving melanoma heterogeneity, and provide the biological rationale for Notch inhibition as a promising therapeutic option.

CBFβ-SMMHC regulates ribosomal gene transcription and alters ribosome biogenesis.

The core-binding factor (CBF) complex is a heterodimeric transcription factor comprising a CBFβ subunit and a variable DNA-binding RUNX subunit, usually RUNX1 in hematopoietic cells. Aside from its critical hematopoietic functions, CBF regulates the expression of ribosomal protein genes and ribosomal RNA (rRNA) in a cell context-dependent manner.1, 2, 3 Intriguingly, this function may have implications for the pathogenesis of acute myeloid leukemia (AML), as reduced ribosome biogenesis in RUNX1-deficient hematopoietic stem cells has recently been proposed to confer a survival advantage that favors outgrowth of preleukemic RUNX1-deficient clones.3 Furthermore, AML-associated fusion proteins that arise from translocations of CBF subunit genes have been shown to occupy nucleolar organizing regions at mitotic chromosomes,4, 5 suggesting that ribosomal homeostasis might be altered in CBF-AML.

Differential roles of PKC isoforms (PKCs) and Ca2+ in GnRH and phorbol 12-myristate 13-acetate (PMA) stimulation of p38MAPK phosphorylation in immortalized gonadotrope cells

We examined the role of PKCs and Ca2+ in GnRH-stimulated p38MAPK phosphorylation in the gonadotrope derived αT3-1 and LβT2 cell lines. GnRH induced a slow and rapid increase in p38MAPK phosphorylation in αT3-1 and LβT2 cells respectively, while PMA gave a slow response. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs), has revealed differential role for PKCα, PKCβII, PKCδ and PKCε in p38MAPK phosphorylation in a ligand-and cell context-dependent manner. The paradoxical findings that PKCs activated by GnRH and PMA play a differential role in p38MAPK phosphorylation may be explained by differential localization of the PKCs. Basal, GnRH- and PMA- stimulation of p38MAPK phosphorylation in αT3-1 cells is mediated by Ca2+ influx via voltage-gated Ca2+ channels and Ca2+ mobilization, while in the differentiated LβT2 gonadotrope cells it is mediated only by Ca2+ mobilization. p38MAPK resides in the cell membrane and is relocated to the nucleus by GnRH (∼5 min). Thus, we have identified the PKCs and the Ca2+ pools involved in GnRH stimulated p38MAPK phosphorylation.

Abstract B132: Importance and mechanism of poly(A) tail length-mediated translational control in different immune cells

Abstract Translational control is an essential regulatory mechanism in immune cells. Although it has been suggested that poly(A) tail length-mediated translational control plays important roles in regulating select cytokine production in memory T cells and macrophages, whether this is a transcriptome-wide regulatory principle in all immune cells remains unknown. We set out to perform RNA-seq, ribosome-footprint profiling and poly(A) tail length profiling across major types of immune cells to examine the relationship between poly(A) tail length and translational efficiency in these systems. We also plan to delineate the transcriptome-wide landscape of poly(A) tail length before and after immune activation, and investigate if the coupling of poly(A) tail length and translational efficiency plays a regulatory role in this process. Meanwhile, to understand the general mechanistic details of how poly(A) tail length affects translational efficiency in a cell context-dependent manner, we carried out RNA pull-down experiments and isolated proteins from known coupled (Xenopus oocytes) and uncoupled (rabbit reticulocyte lysates) systems. By quantitative mass spectrometry analysis, we identified key factors that are responsible for coupling translational efficiency to poly(A) tail length. Among these candidates, poly(A) binding protein (PABPC1) plays an essential role. Overexpression of PABPC1 in Xenopus oocytes renders the translation of reporter RNAs with either a short poly(A) tail or a long poly(A) tail equally well, turning a coupled system into an uncoupled system. We have also generated an effective degron-induced PABPC1-knockdown cell line and are performing analysis to see if the coupling can be established in a normally uncoupled system. Our results suggest that cells can modify the expression of PABPC1 to help determine if poly(A) tail length regulates translational efficiency. Citation Format: Kehui Xiang, David Bartel. Importance and mechanism of poly(A) tail length-mediated translational control in different immune cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B132.

CAP1 (Cyclase-Associated Protein 1) Exerts Distinct Functions in the Proliferation and Metastatic Potential of Breast Cancer Cells Mediated by ERK.

The actin-regulating protein CAP1 is implicated in the invasiveness of human cancers. However, the exact role remains elusive and controversial given lines of conflicting evidence. Moreover, a potential role in the proliferative transformation has largely been overlooked. Further establishing the role and dissecting underlying mechanisms are imperative before targeting CAP1 can become a possibility for cancer treatment. Here we report our findings that CAP1 exerts cell type-dependent functions in the invasiveness of breast cancer cells. Depletion of CAP1 in the metastatic MDA-MB-231 and BT-549 cancer cells stimulated the metastatic potential while it actually inhibited it in the non-metastatic MCF-7 cancer cells or in normal cells. Moreover, we demonstrate functions for CAP1 in cancer cell proliferation and anchorage-independent growth, again in a cell context-dependent manner. Importantly, we identify pivotal roles for the ERK-centered signaling in mediating both CAP1 functions. Phosphor mutants of CAP1 at the S307/S309 regulatory site had compromised rescue effects for both the invasiveness and proliferation in CAP1-knockdown cells, suggesting that CAP1 likely mediates upstream cell signals to control both functions. These novel mechanistic insights may ultimately open up avenues for strategies targeting CAP1 in the treatment of breast cancer, tailored for specific types of the highly diverse disease.