Regulation Of Cellular Proliferation Research Articles

The BH3 only Bcl-2 family member BNIP3 regulates cellular proliferation.

The BH3-only family member BNIP3 has been described as either promoting cell survival or cell death. This depends upon the level of BNIP3 expression and its cellular localization. Increased BNIP3 expression under hypoxia contributes to cell death through increased mitochondrial dysfunction. Furthermore, mice lacking BNIP3 show inhibition of ischemic cardiomyocyte apoptosis. In contrast, nuclear localization of BNIP3 contributes to blockage of apoptosis in glioma cells through repression of pro-apoptotic genes. We have discovered that mouse embryonic fibroblasts (MEFs) lacking BNIP3 expression show increased proliferation and cell number compared to wild-type cells. Furthermore, the cells lacking BNIP3 showed increased MAPK activation. Increased proliferation was not due to decreased cell death as oxidative stress induced cell death in BNIP3 null MEFs. In addition, we isolated astrocytes from wild-type or embryonic mice lacking expression of BNIP3. There was increased density and cell number in the astrocytes lacking BNIP3 expression. To confirm these results in human cells, we inducibly expressed BNIP3 in human embryonic kidney (HEK293) cells and found that induced BNIP3 reduced cell proliferation and failed to change background cell death levels. Transient over-expression of BNIP3 in the nucleus of HEK293 cells also reduced DNA synthesis. Finally, to determine whether this increased proliferation occurs in mice lacking BNIP3, we isolated brains from wild-type mice or those lacking BNIP3 expression. The mice lacking BNIP3 had increased cellularity in the brain of embryonic and adult mice. Taken together, our study describes a new function for BNIP3 in the regulation of cellular proliferation.

Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins.

Long noncoding RNAs constitute a major fraction of the eukaryotic transcriptome, and together with proteins, they intricately fine-tune various growth regulatory signals to control cellular homeostasis. Here, we describe the functional characterisation of a novel pair of long intergenic noncoding RNAs (lincRNAs) comprised of complementary, fully overlapping sense and antisense transcripts Genomic Instability Inducing RNA (Ginir) and antisense RNA of Ginir (Giniras), respectively, from mouse cells. This transcript pair is expressed in a spatiotemporal manner during embryonic development. The individual levels of the sense and antisense transcripts are finely balanced during embryonic growth and in adult tissues. Functional studies of the individual transcripts performed using overexpression and knock-down strategies in mouse cells has led to the discovery that Ginir RNA is a regulator of cellular proliferation and can act as an oncogene having a preeminent role in malignant transformation. Mechanistically, we demonstrate that the oncogenic function of Ginir is mediated by its interaction with centrosomal protein 112 (Cep112). Additionally, we establish here a specific interaction between Cep112 with breast cancer type 1 susceptibility protein (Brca1), another centrosome-associated protein. Next, we prove that the mutual interaction between Cep112 with Brca1 is significant for mitotic regulation and maintenance of genomic stability. Furthermore, we demonstrate that the Cep112 protein interaction with Brca1 protein is impaired when an elevated level of Ginir RNA is present in the cells, resulting in severe deregulation and abnormality in mitosis, leading to malignant transformation. Inhibiting the Ginir RNA function in transformed cells attenuates transformation and restores genomic stability. Together, these findings unravel, to our knowledge, a hitherto-unknown mechanism of oncogenesis mediated by a long noncoding RNA and establishes a unique role of Cep112–Brca1 interaction being modulated by Ginir RNA in maintaining mitotic fidelity.

Dual-functionality of RASSF1A overexpression in A375 cells is mediated by activation of IL-6/STAT3 regulatory loop.

The RASSF1A, a microtubule associated protein, is a well-known tumor suppressor silenced in various cancer via promoter hypermethylation. RASSF1A is implicated in the regulation of cellular proliferation and apoptosis. However, its role in melanoma A375 cells invasion and metastasis remain unclear. Here, we report an unusual dual function role of ectopic RASSF1A in A375 cells. RASSF1A suppressed A375 cells proliferation but enhanced cells migration, invasiveness and metastatic potential in vivo. We demonstrated RASSF1A simultaneously up-regulated p21 and vimentin expression in A375 cells. Increase of vimentin expression contributes to RASSF1A mediated enhancement of cells mobility and invasion. Transcriptome assay unclosed that RASSF1A promoted IL-6 expression in A375 cells, which in turn activate JAK2/STAT3 signaling. Treatment with recombinant IL-6 enhanced both p21 and vimentin protein level in the empty vector transfected A375 cells to similar level as RASSF1A expressing cells. In contrast, knockdown IL-6 expression by siRNAs decreased p21 and vimentin level in RASSF1A expressing cells. Blockade of JAK2/STAT3 signaling by use of JAK2 inhibitor WP1066 led to decrease of IL-6, p21 and vimentin protein in RASSF1A expressing cells. Our findings unclosed a unusual dual functionality of ectopic RASSF1A overexpression in A375 cells by regulating IL-6/STAT3 regulatory loop, suggesting it should be cautious about the safety of RASSF1A-based gene therapy.

Translocator Protein (TSPO) as a Potential Biomarker in Human Cancers.

TSPO is a receptor involved in the regulation of cellular proliferation, apoptosis and mitochondrial functions. Previous studies showed that the expression of TSPO protein correlated positively with tumour malignancy and negatively with patient survival. The aim of this study was to determine the transcription of Tspo mRNA in various types of normal and cancer tissues. In situ hybridization was performed to localise the Tspo mRNA in various human normal and cancer tissues. The relative level of Tspo mRNA was quantified using fluorescent intensity and visual estimation of colorimetric staining. RT-PCR was used to confirm these mRNA levels in normal lung, lung cancer, liver cancer, and cervical cancer cell lines. There was a significant increase in the level of transcription in liver, prostate, kidney, and brain cancers while a significant decrease was observed in cancers of the colon and lung. Quantitative RT-PCR confirmed that the mRNA levels of Tspo are higher in a normal lung cell line than in a lung cancer cell line. An increase in the expression levels of Tspo mRNA is not necessarily a good diagnostic biomarker in most cancers with changes not being large enough to be significantly different when detected by in situ hybridisation.

PPAR-γ Agonist Alleviates Liver and Spleen Pathology via Inducing Treg Cells during Schistosoma japonicum Infection.

Background Peroxisome proliferator-activated receptor- (PPAR-) γ plays critical roles in human metabolic disorders and has recently been implicated as a regulator of cellular proliferation and inflammatory responses. Regulatory T cells (Tregs), which express high levels of PPAR-γ protein, have the ability to maintain immune tolerance to self-antigens and regulate immune response to Schistosoma infection. However, mechanisms involved in the resolution of these responses are elusive. Methods Liver and spleen tissue samples in Schistosoma japonicum-infected mice after administration of pioglitazone (a PPAR-γ agonist) were collected. The hepatic and splenic pathologies were detected by H&E and Masson staining. The percentages of Th1/2 and Treg cells in the liver and spleen of each mouse were determined using flow cytometry. Levels of gene expression of PPAR-γ and Foxp3 in tissues or cells were determined using real-time PCR (RT-PCR). Macrophages were treated with pioglitazone in vitro or cocultured with normal purified CD4+ T cells for detecting Treg cells by flow cytometry. The interactions of PPAR-γ with Foxp3 in CD4+ T cells were detected by coimmunoprecipitation. Results Administration of pioglitazone resulted in the prevention of the development of hepatic and splenic pathologies. Activation of PPAR-γ by pioglitazone resulted in increased percentages of CD4+CD25+Foxp3+ Treg cells and decreased percentages of CD3+CD4+IFN-γ+ and CD3+CD4+IL-4+ cells in the liver and spleen of Schistosoma japonicum-infected mice. In addition, the PPAR-γ agonist can induce Treg cells in vitro directly or by modulating the macrophage's function indirectly. Furthermore, through interaction with Foxp3 in CD4+ T cells, the PPAR-γ agonist can promote the expression of Foxp3; however, the inhibitor of PPAR-γ weakened the expression of Foxp3 by modifying the coexpression of Foxp3 and PPAR-γ. Conclusions Our study reveals a previously unrecognized role for PPAR-γ/Foxp3 signaling in regulating the immunopathology that occurs during Schistosoma infection through induction of Treg cells.

Abstract 303: Novel role of nuclear BH3-only protein BNIP3 in regulation of cellular proliferation

Abstract The objective of this study is to investigate the role of Bcl-2 nineteen kilodalton interacting protein 3 (BNIP3) in regulating cellular proliferation and identifying the proliferation pathways mediated by BNIP3. BNIP3 is a BH3-only proapoptotic member of Bcl-2 family of cell death regulating proteins. The function of BNIP3 is dependent on its localization and expression levels. Under normal conditions, low levels of BNIP3 are present in the nucleus of cells. Low oxygen or hypoxic conditions lead to increased expression of BNIP3, and high levels of BNIP3 are found in the cytoplasm. Cytoplasmic BNIP3 is associated with caspase-independent apoptotic cell death through mitochondria. If, however, BNIP3 levels are increased in the nucleus, it promotes survival of cells by transcriptionally inhibiting several apoptosis-inducing factors. High levels of BNIP3 in nucleus are seen in majority of glioblastoma tumors, where hypoxic environment of tumor core causes an increase in expression of BNIP3 but high amounts of BNIP3 localize to the nucleus, possibly aiding in the survival of the tumor cells. In this study, we found that BNIP3 knockout mice brains have increased cellularity compared to wild-type brains. We also found that both mouse primary astrocytes and embryonic fibroblasts (MEFs) lacking BNIP3 expression have an increased capacity for proliferation compared to wild-type MEFs. Overexpressing BNIP3 in the nucleus of HEK 293 cells also resulted in reduced proliferation. In contrast, both astrocytes and MEFs lacking BNIP3 failed to show differences in cell death compared to wild-type cells. This reveals a novel function for BNIP3 in regulating cellular proliferation that may lead to new targets for cancer therapy. In future studies, we will identity genes regulated by nuclear BNIP3 that may lead to suppression of cellular proliferation in cancer cells. Citation Format: Amandeep Singh, Meghan Azad, Spencer Gibson. Novel role of nuclear BH3-only protein BNIP3 in regulation of cellular proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 303.

The impact of phosphatases on proliferative and survival signaling in cancer.

The dynamic and stringent coordination of kinase and phosphatase activity controls a myriad of physiologic processes. Aberrations that disrupt the balance of this interplay represent the basis of numerous diseases. For a variety of reasons, early work in this area portrayed kinases as the dominant actors in these signaling events with phosphatases playing a secondary role. In oncology, these efforts led to breakthroughs that have dramatically altered the course of certain diseases and directed vast resources toward the development of additional kinase-targeted therapies. Yet, more recent scientific efforts have demonstrated a prominent and sometimes driving role for phosphatases across numerous malignancies. This maturation of the phosphatase field has brought with it the promise of further therapeutic advances in the field of oncology. In this review, we discuss the role of phosphatases in the regulation of cellular proliferation and survival signaling using the examples of the MAPK and PI3K/AKT pathways, c-Myc and the apoptosis machinery. Emphasis is placed on instances where these signaling networks are perturbed by dysregulation of specific phosphatases to favor growth and persistence of human cancer.

Abstract 614: From Genotype to Phenotype: Molecular and Functional Characterization of the CVD-associated SNP rs11574 in ID3

Atherosclerosis is the primary pathological process of CVD and is strongly controlled by heritable factors; however, the mechanisms and pathways through which many of these genetic components regulate plaque development are poorly understood. Recently, a SNP in the coding region of the gene inhibitor of differentiation 3 ( ID3 ) at rs11574 was identified in multiple studies to be associated with CVD. Mutation of rs11574 from the major allele (G) to the minor allele (A) changes the 105 th amino acid of ID3 from an alanine to a threonine and is associated with an attenuated ability of ID3 to antagonize bHLH transcription factors and to prevent them from binding to DNA and activating transcription. The current study utilized co-immunoprecipitation to demonstrate that the minor allele of rs11574 specifically impairs the ability of ID3 to bind to and sequester E12 and no other member of the bHLH family. ChIP studies further demonstrated that the impaired regulatory ability of the ID3 minor allele variant increases E12 occupancy at promoter regions and enhances transcription of E12 target genes including smooth muscle alpha actin ( Acta2 ) and p21 in murine vascular smooth muscle cells (VSMCs). To study the role of this SNP in human cells in intact chromatin under endogenous regulation, we genome edited human 293T cells using CRISPR/Cas9 to produce the allelic variants of rs11574 in the ID3 gene (GG, AG, and AA). Edited cells showed significant changes in cellular proliferation, gene expression, and promoter occupancy depending upon genotype at rs11574. Cells containing the minor allele of rs11574 displayed significantly increased p21 expression and proliferated more slowly than cells containing the major allele of rs11574; whereas, cells heterozygote at rs11574 displayed an intermediate phenotype. These data implicate rs11574 in the regulation of cellular proliferation and mature VSMC marker expression, key phenotypes regulated in the processes of lesion development and formation.

The changes in the transcriptomic profiling of subcutaneous adipose tissue after bariatric surgery depend on the insulin resistance state

BackgroundThe changes that are produced in the gene expression of subcutaneous adipose tissue after Roux-en-Y gastric bypass are not yet fully known. ObjectiveTo identify the changes in the subcutaneous adipose tissue gene expression of morbidly obese women with low insulin resistance (MO-low-IR) and high insulin resistance (MO-high-IR) to find a relationship with measured obesity-related co-morbidities. SettingA university hospital. MethodsSubcutaneous adipose tissue samples were assessed by microarray analysis before and 2 years after Roux-en-Y gastric bypass in MO-low-IR and MO-high-IR patients. ResultsThere is a group of shared differentially expressed genes (DEG) in both MO-low-IR and MO-high-IR, also there is a group of exclusive DEG in MO-low-IR and another group in MO-high-IR. In MO-high-IR, the downexpressed DEG are related to the regulation of transcription and are involved in the pathways related to cytokine-cytokine receptor interaction, cancer, phosphatidylinositol 3-kinase-protein kinase B signaling, human T-lymphotropic virus I infection, chemokine signaling, and Janus kinase/signal transducers and activators of transcription signaling. In MO-low-IR, the overexpressed DEG are related to carbohydrate metabolic processes, the downexpressed DEG to the glycosaminoglycan metabolic process and regulation of translation, and the pathways are related to phosphatidylinositol 3-kinase-protein kinase B signaling and metabolic pathways. The fold change of DEG mainly correlates with the percentage of change (Δ) of waist, Δhip, Δglucose, and Δtriglycerides. These DEG were mainly related to cancer, inflammation/immune regulation, metabolic pathways, ribonucleic acid/deoxyribonucleic acid regulation, virus infection, and regulation of cellular proliferation. ConclusionsThis study suggests a potential association between high insulin resistance and the expression of genes related to cancer and chronic immune activation/inflammation.

Proline‐Rich Tyrosine Kinase Phosphorylation's Effect On the Na+/H+ Exchanger Isoform 1

The Na+‐H+ exchanger isoform 1 (NHE1) is a 12‐pass transmembrane protein that functions by exchanging one extracellular sodium ion for one intracellular proton. The transmembrane domain of NHE1 is involved in ion exchange, and the cytoplasmic tail is the regulatory domain which includes a series of phosphorylation sites as well as lipid and proteins binding sites that alter transport activity. NHE1 is a central driver of the hallmarks of cancer through its involvement in the regulation of cellular proliferation, migration, and invasion. While the exact number of phosphorylation sites on the cytoplasmic domain of NHE1 is still unclear, many studies have implicated 22 phosphorylation sites and 12 different kinases. One of the kinases believed to directly or indirectly lead to the phosphorylation of NHE1 at S602 and S605 is proline rich tyrosine kinase 2 (PyK2). To evaluate the role of PyK2 in NHE1 regulation we prepared three cell lines derived from Chinese hamster lung fibroblast lacking NHE1 expression (PS120 cells). These cell lines are PSN (PS120 cells expressing human NHE1), PSN S602A/S605A (PSN cells with serine to alanine mutations of the reported PyK2 phosphorylation sites) and PSN S602D/S605D (PSN cells with serine to aspartic acid mutations of the reported PyK2 phosphorylation sites to mimic phosphorylation). Some studies have found that PyK2 works inversely on NHE1 and causes inhibition upon phosphorylation, while other studies have shown PyK2 to enhance NHE1 activity. Here we evaluate the impact of PyK2 inhibition and mutation of the PyK2 phosphorylation sites on proliferation and migration. Western blot analysis is used to evaluate changes in Pyk2 expression and phosphorylation over time under low and normal serum conditions. Cell proliferation and cell migration are both evaluated. Our results suggest that serum stimulation increases Pyk2 phosphorylation and enhances both cell proliferation and cell migration. Inhibition of PyK2 by PF43I396 or the mutation of the Pyk2 phosphorylation sites on NHE1 decreases proliferation and migration rates in serum stimulated cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Regulation of Cellular Proliferation by VACM‐1/CUL5 is Dependent on its Post‐translational Modifications by NEDD8

VACM‐1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase complex formation and subsequent protein degradation by ubiquitin‐dependent pathways. In vivo, VACM‐1/CUL5 is expressed in endothelial cells, and when expressed in vitro it attenuates cellular growth. While the specific mechanism by which VACM‐1/CUL5 suppresses cell proliferation is unknown, its antiproliferative effect is dependent on its post‐translational modification by an ubiquitin‐like protein, NEDD8. To investigate the effect of NEDD8 on the structure and function of VACM‐1/CUL5, we have mutated its cDNA sequence at the putative neddylation sites and expressed the mutated construct in Rat Adrenal Medulla Endothelial Cells (RAMEC) and COS‐1 cells in vitro. Our results demonstrate that the antiproliferative effect of wt VACM/CUL5 was attenuated in cells transfected with the mutated VACM‐1/CUL5 cDNA. Furthermore, the mutated VACM‐1/CUL5 affected the anti‐angiogenic phenotype of wt VACM‐1 when expressed in RAMEC cells and grown on Matrigel® support. Finally, the immunoblotting results show that the mutant is modified at multiple sites by NEDD8 in vitro. These results suggest that antiproliferative effect of VACM‐1/CUL5 is regulated by its neddylation status.Support or Funding InformationThis work is supported by the Paul A. Schaap Fellowship and Hope College Department of Biology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Physiologic and pathophysiologic roles of extra renal CYP27b1: Case report and review

Although the kidney was initially thought to be the sole organ responsible for the production of 1,25(OH)2D via the enzyme CYP27b1, it is now appreciated that the expression of CYP27b1 in tissues other than the kidney is wide spread. However, the kidney is the major source for circulating 1,25(OH)2D. Only in certain granulomatous diseases such as sarcoidosis does the extra renal tissue produce sufficient 1,25(OH)2D to contribute to the circulating levels, generally associated with hypercalcemia, as illustrated by the case report preceding the review. Therefore the expression of CYP27b1 outside the kidney under normal circumstances begs the question why, and in particular whether the extra renal production of 1,25(OH)2D has physiologic importance. In this chapter this question will be discussed. First we discuss the sites for extra renal 1,25(OH)2D production. This is followed by a discussion of the regulation of CYP27b1 expression and activity in extra renal tissues, pointing out that such regulation is tissue specific and different from that of CYP27b1 in the kidney. Finally the physiologic significance of extra renal 1,25(OH)2D3 production is examined, with special focus on the role of CYP27b1 in regulation of cellular proliferation and differentiation, hormone secretion, and immune function. At this point the data do not clearly demonstrate an essential role for CYP27b1 expression in any tissue outside the kidney, but several examples pointing in this direction are provided. With the availability of the mouse enabling tissue specific deletion of CYP27b1, the role of extra renal CYP27b1 expression in normal and pathologic states can now be addressed definitively.

Involvement of Nuclear Receptor REV-ERBβ in Formation of Neurites and Proliferation of Cultured Adult Neural Stem Cells.

Neural stem cells (NSCs) serve as the source of both neurons and support cells, and neurogenesis is reportedly linked to the circadian clock. This study aimed to clarify the functional role of the circadian rhythm-related nuclear receptor, REV-ERBβ, in neurogenesis of NSCs from adult brain. Accordingly, Rev-erbβ expression and the effect of Rev-erbβ gene-specific knockdown on neurogenesis in vitro was examined in adult rodent NSCs. Initial experiments confirmed REV-ERBβ expression in cultured adult NSCs, while subsequent gene expression and gene ontogeny analyses identified functional genes upregulated or downregulated by REV-ERBβ. In particular, expression levels of factors associated with proliferation, stemness, and neural differentiation were affected. Knockdown of Rev-erbβ showed involvement of REV-ERBβ in regulation of cellular proliferation and self-renewal of cultured adult NSCs. Moreover, Rev-erbβ-knockdown cells formed neurons with a slightly shrunken morphology, fewer new primary neurites, and reduced length and branch formation of neurites. Altogether, this suggests that REV-ERBβ is involved in neurite formation during neuronal differentiation of cultured adult NSCs. In summary, REV-ERBβ is a known circadian regulatory protein that appears to be involved in neurogenesis via regulation of networks for cell proliferation and neural differentiation/maturation in adult NSCs.

Overexpression of S-adenosylmethionine decarboxylase impacts polyamine homeostasis during development of Dictyostelium discoideum.

The polyamines putrescine, spermidine and spermine are essential polycations involved in the regulation of cellular proliferation. They exert dynamic effects on nucleic acids and macromolecular synthesis in vitro but their specific functions in vivo are poorly understood. Here, we have modulated the spermidine levels either by overexpressing the S-adenosylmethionine decarboxylase (samdc) gene or treating the cells with methylglyoxal-bis (guanylhydrazone) (MGBG), an inhibitor of SAMDC. In Dictyostelium, overexpression of SAMDC slowed cell proliferation, delayed development and arrested cells in the S-phase of the cell cycle. Treatment with MGBG reduced cell proliferation and stimulated development, but in samdcOE cells, it increased cell proliferation suggesting critical levels of spermidine to be important. In samdcOE cells, spermidine levels remained high throughout development but only small changes in the spermine levels were observed. Initial putrescine levels did increase but reverted to wild-type levels after the mound stage. As tight regulation of polyamine homeostasis is required, we identified genes that could be involved in its maintenance. In conclusion, we characterised samdcOE cells and observed the maintenance of polyamine homeostasis during the development of Dictyostelium cells.

Multi-targeted effects of G4-aptamers and their antiproliferative activity against cancer cells

We selected and investigated nine G-quadruplex (G4)-forming aptamers originally designed against different proteins involved in the regulation of cellular proliferation (STAT3, nucleolin, TOP1, SP1, VEGF, and SHP-2) and considered to be potential anticancer agents. We showed that under physiological conditions all the aptamers form stable G4s of different topology. G4 aptamers designed against STAT3, nucleolin and SP1 inhibit STAT3 transcriptional activity in human breast adenocarcinoma MCF-7 cells, and all the studied aptamers inhibit TOP1-mediated relaxation of supercoiled plasmid DNA. STAT3 inhibition by G4 aptamer designed against SP1 protein provides a new explanation for the SP1 and STAT3 crosstalk described recently. We found some correlation between G4-mediated inhibition of the DNA replication and TOP1 activity. Four G4 aptamers from our dataset that appeared to be the strongest TOP1 inhibitors most efficiently decreased de novo DNA synthesis, by up to 79–87%. Seven G4 aptamers demonstrated significantly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells than on immortalized mammary epithelial MCF-10A cells. Pleiotropic properties of G4 aptamers and their high specificity against cancer cells observed for the majority of the studied G4 aptamers allowed us to present them as promising candidates for multi-targeted cancer therapy.

Ptn functions downstream of C/EBPβ to mediate the effects of cAMP on uterine stromal cell differentiation through targeting Hand2 in response to progesterone.

Ptn is a pleiotropic growth factor involving in the regulation of cellular proliferation and differentiation, but its biological function in uterine decidualization remains unknown. Here, we showed that Ptn was highly expressed in the decidual cells, and could induce the proliferation of uterine stromal cells and expression of Prl8a2 and Prl3c1 which were two well-established differentiation markers for decidualization, suggesting an important role of Ptn in decidualization. In the uterine stromal cells, progesterone stimulated the expression of Ptn accompanied with an accumulation of intracellular cAMP level. Silencing of Ptn impeded the induction of progesterone and cAMP on the differentiation of uterine stromal cells. Administration of PKA inhibitor H89 resulted in a blockage of progesterone on Ptn expression. Further analysis evidenced that regulation of progesterone and cAMP on Ptn was mediated by C/EBPβ. During in vitro decidualization, knockdown of Ptn could weaken the up-regulation of Prl8a2 and Prl3c1 elicited by C/EBPβ overexpression, while constitutive activation of Ptn reversed the repressive effects of C/EBPβ siRNA on the expression of Prl8a2 and Prl3c1. Meanwhile, Ptn might mediate the regulation of C/EBPβ on Hand2 which was a downstream target of Ptn in the differentiation of uterine stromal cells. Attenuation of Ptn or C/EBPβ by specific siRNA blocked the stimulation of Hand2 by progesterone and cAMP. Collectively, Ptn may play a vital role in the progesterone-induced decidualization pathway.

Novel Caffeic Acid Phenethyl Ester (Cape) Analogues as Inducers of Heme Oxygenase-1.

Heme Oxygenase-1 (HO-1) is a metabolic enzyme strongly involved in processes including cytoprotection, modulation of inflammatory response, anti-oxidative functions, regulation of cellular proliferation, angiogenesis, cardiovascular homeostasis, and immuno-modulation. HO-1 induction and/or activation is able to counterbalance, at least in part, oxidative stress and inflammation. For this reason, HO-1 can be regarded as an attractive target to ameliorate different stress-related pathologies. Caffeic acid phenethyl ester, a natural polyphenolic compound, behaves as HO-1 inducer and possesses a plethora of beneficial effects under oxidative stress conditions. A small focused series of caffeic acid phenethyl ester (Cape) analogues was designed and synthesized with the aim of obtaining more potent HO-1 inducers. The capacity of these new compounds to modify the levels of HO-1 was evaluated in human mesenchymal stem cells (hMSCs) derived from bone marrow. Some of tested compounds were found to be good HO-1 inducers and 3-(3,4- dihydroxyphenyl)-(2E)-2-propenoic acid 2-(3,4-dimethoxyphenyl) ethyl ester (VP961) was the most potent. VP961 tested to measure HO-1 protein expression and HO activity in in vitro system resulted more potent than the parent compound Cape both as inducer and as direct activator of the enzyme. VP961, selected as lead compound for further characterization, showed antioxidant properties in a model of H2O2-mediated ROS production and cytoprotective effects in a model of H2O2 cells viability impairment. To the best of our knowledge, VP961 is the first known compound able to activate directly HO-1 enzyme and to induce at the same time its protein expression.

Abstract 4452: Study of the mechanisms of emetine dihydrochloride on inhibiting NSCLC tumor progression and attenuating EGFR signaling pathway

Abstract Epidermal growth factor receptor (EGFR) is a transmembrane receptor protein and can mediate the cellular biological responses including regulation of cellular proliferation, differentiation, migration and survival. More than 60 % of non-small cell Lung cancers (NSCLCs) express relatively high level of EGFR. Therefore, EGFR has become an important therapeutic target for the treatment of NSCLC. However, almost all NSCLC patients ultimately develop drug resistance to targeted therapy agents. The most common secondary mutation is found to harbor EGFR mutation in T790M, which is acquired resistance to EGFR TKIs. In order to overcome the resistance of T790M mutation in lung cancer, we used EGFR pY1068 ELISA as drug-screening platform to screen the candidate compounds with the capacity to suppress EGFR activity from the compound library of more than 300 plant active ingredients. Among the compounds identified, the emetine could most significantly restrain EGFR pY1068 activity and reduce cancer cell viability. Moreover, the Western blotting results showed that emetine treatment can repress EGFR phosphorylation and expression in a dose-dependent manner as well as inhibit its downstream substrates including PI3K, STAT3 and Src in three different Iressa-resistant cell lines. In addition, emetine also could inhibit cell proliferation, migration, invasion, and clonogenicity in vitro and xenograft tumor growth in vivo. Taken together, our data suggest that emetine might be a novel inhibitory modulator of EGFR activity and might be through suppressing the phosphorylation and expression of PI3K, STAT3, and Src to inhibit lung cancer progression. These findings will help us to develop the anti-cancer drugs for lung cancer in the future. Citation Format: Yi-Hua Lai, Wan-Ting Huang, Sheng-Yi Lin, Yao-Ting Huang, Yuh-Ling Chen, Hsuan-Yu Chen, Huei-Wen Chen, Jeremy J.W. Chen. Study of the mechanisms of emetine dihydrochloride on inhibiting NSCLC tumor progression and attenuating EGFR signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4452. doi:10.1158/1538-7445.AM2017-4452

Altered expression of cellular proliferation, apoptosis and the cell cycle-related genes in lung cancer cells with acquired resistance to EGFR tyrosine kinase inhibitors.

Non-small cell lung cancers harboring somatic gain-of-function mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain respond well to treatment with EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib. However, all patients who experience a marked improvement with these drugs eventually develop disease progression due to the acquisition of drug resistance. Approximately half of the cases with acquired resistance to EGFR TKIs can be accounted for by a second-site mutation in exon 20 of the EGFR kinase domain (T790M). However, the changes of gene expression involved in EGFR TKI resistance due to the T790M mutation remain poorly defined. The present study established lung cancer cell lines that were resistant to gefitinib or erlotinib, and these cell lines were verified to contain the EGFR T790M mutation. The differential expression of genes associated with acquired resistance was verified in the present study by mRNA microarray analysis. Among the genes whose expression was significantly altered, genes whose expression was altered in gefitinib- and erlotinib-resistant cells were focused on. Notably, a total of 1,617 genes were identified as being differentially expressed in gefitinib- and erlotinib-resistant cells. Indeed, Gene ontology analysis revealed altered expression of genes involved in the regulation of cellular proliferation, apoptosis, and the cell cycle in EGFR TKI-resistant cells. The present results demonstrate distinctive gene expression patterns of EGFR TKI-resistant lung cancer cells with the EGFR T790M mutation. The present study can provide key insights into gene expression profiles involved in conferring resistance to EGFR TKI therapy in lung cancer cells.

Rapamycin enhances the antiproliferative effect of transforming growth factor-β on MCF-7 human breast cancer cells.

Transforming growth factor-β (TGF-β), a well-known cytokine with pleiotropic biological functions, has an important role in the regulation of cellular proliferation. Rapamycin has specific antagonistic activity on the function of the mammalian target of the rapamycin signaling pathway. The cooperation of TGF-β and rapamycin on the proliferation of Michigan Cancer Foundation (MCF)-7 human breast cancer cells is unclear. The present study demonstrated that TGF-β had a growth-arresting effect on MCF-7 cancer cells. TGF-β stimulation resulted in the upregulation of several cyclin-dependent kinase inhibitors, including p14ARF, p15INK4b, p16INK4a and p21WAF1/CIP1. The present study also demonstrated that rapamycin enhances the antiproliferative effect of TGF-β. The combination of rapamycin and TGF-β induced apoptosis of MCF-7 tumor cells. These findings advance the current understanding of the biological effects of TGF-β and rapamycin.