Growth Suppressor Research Articles

Gas1 up-regulation is inducible and contributes to cell apoptosis in reactive astrocytes in the substantia nigra of LPS and MPTP models.

BackgroundReactive astrogliosis is a remarkable pathogenetic hallmark of the brains of Parkinson’s disease (PD) patients, but its progressive fate and regulation mechanisms are poorly understood. In this study, growth arrest specific 1 (Gas1), a tumor growth suppressor oncogene, was identified as a novel modulator of the cell apoptosis of reactive astrocytes in primary culture and the injured substantia nigra.MethodsAnimal models and cell cultures were utilized in the present study. Lipopolysaccharide (LPS)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated animal models were used to detect Gas1 expression in the brain via immunohistochemistry and western blot. Cell cultures were performed to analyze Gas1 functions in the viability and apoptosis of reactive astrocytes and SH-SY5Y cells by double labeling, CCK-8, LDH, TUNEL, flow cytometry, and siRNA knockdown methods.ResultsGas1 expressions were significantly elevated in the majority of the reactive astrocytes of the brains with LPS or MPTP insults. In the injured substantia nigras, GFAP-positive astrocytes exhibited higher levels of cleaved caspase-3. In cell culture, the up-regulated Gas1 expression induced apoptosis of reactive astrocytes that were insulted by LPS in combination with interferon-γ and tumor necrosis factor-a. This effect was confirmed through siRNA knockdown of Gas1 gene expression. Finally and interestingly, the potential underlying signaling pathways were evidently related to an increase in the Bax/Bcl-2 ratio, the abundant generation of reactive oxygen species and the activation of cleaved caspase-3.ConclusionsThis study demonstrated that the up-regulation of inducible Gas1 contributed to the apoptosis of reactive astrocytes in the injured nigra. Gas1 signaling may function as a novel regulator of astrogliosis and is thus a potential intervention target for inflammatory events in PD conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0643-2) contains supplementary material, which is available to authorized users.

TSC22D2 interacts with PKM2 and inhibits cell growth in colorectal cancer.

We previously identified TSC22D2 (transforming growth factor β-stimulated clone 22 domain family, member2) as a novel cancer-associated gene in a rare multi-cancer family. However, its role in tumor development remains completely unknown. In this study, we found that TSC22D2 was significantly downregulated in colorectal cancer (CRC) and that TSC22D2 overexpression inhibited cell growth. Using a co-immunoprecipitation (co-IP) assay combined with mass spectrometry analysis to identify TSC22D2-interacting proteins, we demonstrated that TSC22D2 interacts with pyruvate kinase isoform M2 (PKM2). These findings were confirmed by the results of immunoprecipitation and immunofluorescence assays. Moreover, overexpression of TSC22D2 reduced the level of nuclear PKM2 and suppressed cyclinD1 expression. Collectively, our study reveals a growth suppressor function of TSC22D2 that is at least partially dependent on the TSC22D2-PKM2-cyclinD1 regulatory axis. In addition, our data provide important clues that might contribute to future studies evaluating the role of TSC22D2.

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk-3).

Reduced expression in immortalized cells (REIC/Dkk-3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk-3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk-3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk-3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk-3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk-3 represents a potential molecular target for the development of therapies against feline mammary cancers.

Targeting leiomyomas with all-trans-retinoic acid at phosphoinositide 3-kinase pathway suppression: Effective roles of β-catenin and of signaling interactions.

Leiomyomas, monoclonal tumors developed by the transformation of myometrium somatic stem cells, are a major health concern that can severely impair quality of life. Pathological alterations of signaling pathways have been recognized as a key feature in a variety of human diseases. Our objective was to analyze treatment with all-trans-retinoic acid (ATRA) by suppression of the phosphoinositide 3-kinase (PI3K) pathway on growth, signaling pattern and interactions among PI3K/B-cell lymphoma 2 (Bcl2)/retinol leiomyoma proteins. Cultures of paired myometrium and leiomyoma cells from premenopausal women undergoing hysterectomy were collected. Western blot and analysis of variance were used for analysis. Significant differences were detected between treatment with ATRA alone or with LY294002 (a PI3K growth suppressor) in response to treatment and among cell samples and cell numbers. Leiomyoma cells were less affected. Immunochemical analysis of signaling patterns demonstrated that treatments affected most of the examined protein levels differently. Significant differences between the cell type responses to treatment in pyruvate phosphate dikinase 1 (pPDK1), Bad and pβ-catenin levels were identified. The pβ-catenin level showed highly significant interaction between response to treatment and cell type. ATRA treatment on PI3K pathway suppression significantly affected growth, signaling pattern and interactions among PI3K/Bcl2/retinol proteins involved in the growth, survival and apoptosis of leiomyomas. Interpretation of our results suggests that increasing knowledge of the role of signaling interplay in the pathogenesis of leiomyomas may present an opportunity to use specific signal transduction inhibitors for treating and preventing this disorder.

Chromatin remodeling geneARID2targets cyclin D1 and cyclin E1 to suppress hepatoma cell progression

Exome and whole-genome sequencing studies have drawn attention to the role of somatic mutations in SWI/SNF chromatin remodeling complexes in the carcinogenesis of hepatocellular carcinoma (HCC). Here, we explored the molecular mechanisms underlying the biological roles of AT-rich interactive domain 2 (ARID2) in the pathogenesis of HCC. We found that ARID2 expression was significantly downregulated in HCC tissues compared with non-tumorous tissues. Restoration of ARID2 expression in hepatoma cells was sufficient to suppress cell proliferation and tumor growth in mice, whereas ARID2 knockdown contributed to the enhancement of cellular proliferation and tumorigenicity. Suppression of ARID2 expression accelerated G1/S transition associated with upregulation of cyclin D1, cyclin E1, CDK4, and phosphorylation of the retinoblastoma protein (Rb). Furthermore, we demonstrated that ARID2 physically interacts with E2F1 and decreases binding of E2F1/RNA Pol II to the promoters of CCND1 and CCNE1. Taken together, these results demonstrate that ARID2 suppresses tumor cell growth through repression of cyclin D1 and cyclin E1 expression, thereby retarding cell cycle progression and cell proliferation in hepatoma cells. These findings highlight the potential role of ARID2 as a tumor growth suppressor in HCC.

A functional microRNA library screen reveals miR-410 as a novel anti-apoptotic regulator of cholangiocarcinoma.

BackgroundCholangiocarcinoma is characterized by late diagnosis and a poor survival rate. MicroRNAs have been involved in the pathogenesis of different cancer types, including cholangiocarcinoma. Our aim was to identify novel microRNAs regulating cholangiocarcinoma cell growth in vitro and in vivo.MethodsA functional microRNA library screen was performed in human cholangiocarcinoma cells to identify microRNAs that regulate cholangiocarcinoma cell growth. Real-time PCR analysis evaluated miR-9 and XIAP mRNA levels in cholangiocarcinoma cells and tumors.ResultsThe screen identified 21 microRNAs that regulated >50 % cholangiocarcinoma cell growth. MiR-410 was identified as the top suppressor of growth, while its overexpression significantly inhibited the invasion and colony formation ability of cholangiocarcinoma cells. Bioinformatics analysis revealed that microRNA-410 exerts its effects through the direct regulation of the X-linked inhibitor of apoptosis protein (XIAP). Furthermore, overexpression of miR-410 significantly reduced cholangiocarcinoma tumor growth in a xenograft mouse model through induction of apoptosis. In addition, we identified an inverse relationship between miR-410 and XIAP mRNA levels in human cholangiocarcinomas.ConclusionsTaken together, our study revealed a novel microRNA signaling pathway involved in cholangiocarcinoma and suggests that manipulation of the miR-410/XIAP pathway could have a therapeutic potential for cholangiocarcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2384-0) contains supplementary material, which is available to authorized users.

Two-Step Forward Genetic Screen in Mice Identifies Ral GTPase-Activating Proteins as Suppressors of Hepatocellular Carcinoma

High-throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinomas (HCCs). However, high intratumor and intertumor heterogeneity, combined with large numbers of passenger mutations, have made it difficult to identify driver mutations that contribute to the development of HCC. We combined transposon mutagenesis with a high-throughput screen of a small-hairpin RNA (shRNA) library to identify genes and pathways that contribute to HCC development. Sleeping beauty transposons were mobilized in livers of transgenic mice predisposed to develop hepatocellular adenoma and HCC owing to expression of the hepatitis B virus surface antigen. This whole-genome mutagenesis technique was used to generate an unbiased catalogue of candidate cancer genes (CCGs). Pooled shRNA libraries targeting 250 selected CCGs then were introduced into immortalized mouse liver cells and the cells were monitored for their tumor-forming ability after injection into nude mice. Transposon-mediated mutagenesis identified 1917 high-confident CCGs and highlighted the importance of Ras signaling in the development of HCC. Subsequent pooled shRNA library screening of 250 selected CCGs validated 27 HCC tumor-suppressor genes. Individual shRNA knockdown of 4 of these genes (Acaa2, Hbs1l, Ralgapa2, and Ubr2) increased the proliferation of multiple human HCC cell lines in culture and accelerated the formation of xenograft tumors in nude mice. The ability of Ralgapa2 to promote HCC cell proliferation and tumor formation required its inhibition of Rala and Ralb. Dual inhibition of Ras signaling via Ral and Raf, using a combination of small-molecule inhibitor RBC8 and sorafenib, reduced the proliferation of HCC cells in culture and completely inhibited their growth as xenograft tumors in nude mice. In a 2-step forward genetic screen in mice, we identified members of the Ral guanosine triphosphatase-activating protein pathway and other proteins as suppressors of HCC cell proliferation and tumor growth. These proteins might serve as therapeutic targets for liver cancer.

Regulatory Differences in Natal Down Development between Altricial Zebra Finch and Precocial Chicken

Birds can be classified into altricial and precocial. The hatchlings of altricial birds are almost naked, whereas those of precocial birds are covered with natal down. This regulatory divergence is thought to reflect environmental adaptation, but the molecular basis of the divergence is unclear. To address this issue, we chose the altricial zebra finch and the precocial chicken as the model animals. We noted that zebra finch hatchlings show natal down growth suppressed anterior dorsal (AD) skin but partially down-covered posterior dorsal (PD) skin. Comparing the transcriptomes of AD and PD skins, we found that the feather growth promoter SHH (sonic hedgehog) was expressed higher in PD skin than in AD skin. Moreover, the data suggested that the FGF (fibroblast growth factor)/Mitogen-activated protein kinase (MAPK) signaling pathway is involved in natal down growth suppression and that FGF16 is a candidate upstream signaling suppressor. Ectopic expression of FGF16 on chicken leg skin showed downregulation of SHH, upregulation of the feather growth suppressor FGF10, and suppression of feather bud elongation, similar to the phenotype found in zebra finch embryonic AD skin. Therefore, we propose that FGF16-related signals suppress natal down elongation and cause the naked AD skin in zebra finch. Our study provides insights into the regulatory divergence in natal down formation between precocial and altricial birds.

Withaferin A inhibits in vivo growth of breast cancer cells accelerated by Notch2 knockdown.

The present study offers novel insights into the molecular circuitry of accelerated in vivo tumor growth by Notch2 knockdown in triple-negative breast cancer (TNBC) cells. Therapeutic vulnerability of Notch2-altered growth to a small molecule (withaferin A, WA) is also demonstrated. MDA-MB-231 and SUM159 cells were used for the xenograft studies. A variety of technologies were deployed to elucidate the mechanisms underlying tumor growth augmentation by Notch2 knockdown and its reversal by WA, including Fluorescence Molecular Tomography for measurement of tumor angiogenesis in live mice, Seahorse Flux analyzer for ex vivo measurement of tumor metabolism, proteomics, and Luminex-based cytokine profiling. Stable knockdown of Notch2 resulted in accelerated in vivo tumor growth in both cells reflected by tumor volume and/or latency. For example, the wet tumor weight from mice bearing Notch2 knockdown MDA-MB-231 cells was about 7.1-fold higher compared with control (P<0.0001). Accelerated tumor growth by Notch2 knockdown was highly sensitive to inhibition by a promising steroidal lactone (WA) derived from a medicinal plant. Molecular underpinnings for tumor growth intensification by Notch2 knockdown included compensatory increase in Notch1 activation, increased cellular proliferation and/or angiogenesis, and increased plasma or tumor levels of growth stimulatory cytokines. WA administration reversed many of these effects providing explanation for its remarkable anti-cancer efficacy. Notch2 functions as a tumor growth suppressor in TNBC and WA offers a novel therapeutic strategy for restoring this function.

Inhibition of PHLPP2/cyclin D1 protein translation contributes to the tumor suppressive effect of NFκB2 (p100).

Although the precursor protein of NFκB2 (p100) is thought to act as a tumor suppressor in mammalian cells, the molecular mechanism of its anti-tumor activity is far from clear. Here, we are, for the first time, to report that p100 protein expression was dramatically decreased in bladder cancers of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice and human patients. Knockdown of p100 in cultured human bladder cancer cells promoted anchorage-independent growth accompanied with elevating abundance of cell-cycle-related proteins and accelerated cell-cycle progression. Above effects could be completely reversed by ectopically expression of p100, but not p52. Mechanistically, p100 inhibited Cyclin D1 protein translation by activating the transcription of LARP7 and its hosted miR-302d, which could directly bind to 3′-UTR of cyclin d1 mRNA and inhibited its protein translation. Furthermore, p100 suppressed the expression of PHLPP2 (PH domain and leucine-rich repeat protein phosphatases 2), thus promoting CREB phosphorylation at Ser133 and subsequently leading to miR-302d transcription. Taken together, our studies not only for the first time establish p100 as a key tumor suppressor of bladder cancer growth, but also identify a novel molecular cascade of PHLPP2/CREB/miR-302d that mediates the tumor suppressive function of p100.

Downregulation of YAP-dependent Nupr1 promotes tumor-repopulating cell growth in soft matrices.

Despite decades of significant progress in understanding the molecular mechanisms of malignant tumorigenic cells, it remains elusive what these tumorigenic cells are and what controls the growth of these malignant cells. Recently, we have mechanically selected and grown highly malignant and tumorigenic tumor-repopulating cells (TRCs), a small sub-population of cancer cells, by culturing single cancer cells in soft fibrin matrices. However, it is unclear what regulates TRC growth besides Sox2. Here we show that nuclear protein 1 (Nupr1), a protein independent of Sox2, is downregulated in TRCs of melanoma, ovarian cancer and breast cancer cultured in soft fibrin matrices. Nupr1 expression depends on nuclear translocation of YAP that is enriched at the Nupr1 promoter sites; YAP is controlled by Cdc42-mediated F-actin and Lats1 interactions. Nupr1 regulates tumor-suppressor p53 and negatively regulates Nestin and Tert that are independent of Sox2 and promote TRC growth. Silencing Nupr1 increases TRC growth and Nupr1 overexpression inhibits TRC growth in culture and in immune-competent mice. Our results suggest that Nupr1 is a suppressor of growth of highly tumorigenic TRCs and may have a critical role in cancer progression.

Magnetic Studies of MgB2Prepared by Internal Magnesium Diffusion With Various Doping

MgB2 wires doped with carbon and SiO2 nanoparticles prepared by the modified internal magnesium diffusion method were studied and compared with an undoped reference sample. It was found that doping of boron powder has a substantial effect on the diffusion reaction and that carbon acts as a MgB2 growth suppressor. The highest critical current density was exhibited by the wire with SMI+C boron powder heat-treated at 650 °C/1 h, with almost 10 000 $\text{A}\cdot\text{cm}^{-2}$ at 10 T and 4.2 K. From the point of view of pinning behavior, this wire showed the lowest pinning energies, indicating that high $J_{c}$ is due to an increased number of pinning sites. The other C-doped wire with the PVZ+C boron powder showed field-direction-dependent pinning energies with higher energies for the parallel direction, in good agreement with the $J_{c}$ results. The SiO2-doped wires exhibited a wide flux jump region and a quickly declining field dependence of $J_{c}$ .

MT1-MMP sheds LYVE-1 on lymphatic endothelial cells and suppresses VEGF-C production to inhibit lymphangiogenesis.

Lymphangiogensis is involved in various pathological conditions, such as arthritis and cancer metastasis. Although many factors have been identified to stimulate lymphatic vessel growth, little is known about lymphangiogenesis inhibitors. Here we report that membrane type 1-matrix metalloproteinase (MT1-MMP) is an endogenous suppressor of lymphatic vessel growth. MT1-MMP-deficient mice exhibit spontaneous corneal lymphangiogenesis without concomitant changes in angiogenesis. Mice lacking MT1-MMP in either lymphatic endothelial cells or macrophages recapitulate corneal lymphangiogenic phenotypes observed in Mmp14−/− mice, suggesting that the spontaneous lymphangiogenesis is both lymphatic endothelial cells autonomous and macrophage associated. Mechanistically, MT1-MMP directly cleaves LYVE-1 on lymphatic endothelial cells to inhibit LYVE-1-mediated lymphangiogenic responses. In addition, MT1-MMP-mediated PI3Kδ signalling restrains the production of VEGF-C from prolymphangiogenic macrophages through repressing the activation of NF-κB signalling. Thus, we identify MT1-MMP as an endogenous inhibitor of physiological lymphangiogenesis.

Abstract B33: BRCA1 RING domain mutations and function-based tools to predict risk for the development of TNBC

Abstract Breast cancer is the second leading cause of cancer-related deaths amongst women with 231,840 new cases projected for 2015. Majority of young African American women with BRCA1 mutations have a so-called Triple negative breast cancer with an aggressive phenotype. Currently there is no targeted therapy for TNBC. BRCA1 encodes a large protein and the two highly conserved regions are located at both ends, an N-terminal RING domain and two BRCT repeats at the C-terminus. The RING domain through interaction with BARD1 contains E3 ubiquitin ligase activity. Various BRCA1 mutations have been found throughout BRCA1 coding region, but the majority of cancer-associated mutations are nonsense or frame shift mutations which lead to chain termination. The C61G mutation has been linked to both breast and ovarian cancer development. In people from Ashkenazi Jewish descent, the most common mutations are 185delAG and 5382 ins C in BRCA1 and is 1:40. Our lab has cloned two naturally occurring splice variants of BRCA1 (BRCA1a and BRCA1b). These isoforms are the most evolutionary conserved of all the splice variants and code for multifunctional proteins. Our group has previously reported that BRCA1 RING domain, unlike K109R and cancer-predisposing mutant C61G BRCA1 proteins interact with the sole SUMO E2-conjugating enzyme Ubc9 and this facilitates both the entry of BRCA1 proteins to the nucleus and mediates ubiquitination of ER-alpha. The disease associated mutants do not bind Ubc9, remain stalled in the cytoplasm and have lost their growth suppressor function. We have identified a new nuclear trafficking pathway and malfunction of this by BRCA1 dysfunction can result in TNBC. The BARD1-dependent -E3 ubiquitin ligase activity of BRCA1 has been predicted to be required for its tumor suppressor function, as certain cancer –associated mutations in BRCA1 RING domain ablate this activity. Several mutations in BRCA1 RING domain have been identified however; their role in TNBC has yet to be elucidated. This work is based on the hypothesis that BRCA1 is a tumor suppressor gene and its RING domain can harbor several mutations some of which can result in loss of BRCA1 function resulting in TNBC and others can be gain of function mutants similar to WT BRCA1. We tested this hypothesis by introducing C61G, K109R and I26A mutations into TNBC cells and studied their growth inhibitory activity using colony suppression assays. Our results demonstrate for the first time that BRCA1 I26A to be a gain of function mutant similar to WT BRCA1. I26A mutant associates with Ubc9, lacks E3 ubiquitin ligase activity and suppresses growth of BRCA1 mutant TNBC and sporadic TNBC cells unlike K109R and C61G mutants. Clinically, the ability to predict which of these mutations can result in TNBC offers unprecedented prospects for early detection and cancer prevention. This is the first study demonstrating the physiological link between Ubc9 binding, loss of BARD1- dependent E3 ubiquitin ligase activity and growth suppression of I26A mutant BRCA1 protein in TNBC cells. BRCA1, by turning off or on Ubc9 binding, regulates growth of TNBC. This study will accelerate precision medicine and reduce cancer health disparities in health outcomes. Work supported in part by Georgia Cancer Coalition Distinguished Cancer Scholar award, NIH-NCRR-RCMI grant G-12-RR003034, U54 RR02613, 5P20RR11104 and NIHMD research endowment grant 2S21MD000101, MSM/TU/UAB CCC Partnership/U54 CA118638 and ING foundation to V.N.R. Patent issued No:8372,580; 2013. Citation Format: Jingyao Xu, Shanekkia Black, Kartik Aysola, Yunlong Qin, Vaishali Reddy, Karan Singh, Joel Okoli, Derrick Beech, Uma Krishnamurthy, Gabriela Oprea, Valerie M. Rice, E Shyam Reddy, Douglas Moellering, Yuchang Fu, Veena N. Rao. BRCA1 RING domain mutations and function-based tools to predict risk for the development of TNBC. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B33.

Loss of Glycogen Debranching Enzyme AGL Drives Bladder Tumor Growth via Induction of Hyaluronic Acid Synthesis.

We demonstrated that amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL) is a tumor growth suppressor and prognostic marker in human bladder cancer. Here we determine how AGL loss enhances tumor growth, hoping to find therapeutically tractable targets/pathways that could be used in patients with low AGL-expressing tumors. We transcriptionally profiled bladder cell lines with different AGL expression. By focusing on transcripts overexpressed as a function of low AGL and associated with adverse clinicopathologic variables in human bladder tumors, we sought to increase the chances of discovering novel therapeutic opportunities. One such transcript was hyaluronic acid synthase 2 (HAS2), an enzyme responsible for hyaluronic acid (HA) synthesis. HAS2 expression was inversely proportional to that of AGL in bladder cancer cells and immortalized and normal urothelium. HAS2-driven HA synthesis was enhanced in bladder cancer cells with low AGL, and this drove anchorage-dependent and independent growth. siRNA-mediated depletion of HAS2 or inhibition of HA synthesis by 4-methylumbelliferone (4MU) abrogated in vitro and xenograft growth of bladder cancer cells with low AGL. AGL and HAS2 mRNA expression in human tumors was inversely correlated in patient datasets. Patients with high HAS2 and low AGL tumor mRNA expression had poor survival, lending clinical support to xenograft findings that HAS2 drives growth of tumors with low AGL. Our study establishes HAS2-mediated HA synthesis as a driver of growth of bladder cancer with low AGL and provides preclinical rationale for personalized targeting of HAS2/HA signaling in patients with low AGL-expressing tumors.

The Plasma microRNA miR-1914* and -1915 Suppresses Chemoresistant in Colorectal Cancer Patients by Down-regulating NFIX.

We investigated mechanisms of colorectal cancer (CRC) chemoresistance to first-line chemotherapy (capecitabine plus oxaliplatin (XELOX)) and identified two putative chemoresistant microRNAs, miR-1914* and -1915, that are downregulated in plasma samples from patients with chemoresistant CRC. A number of plasma samples from CRC patients were analyzed for the levels of miR-1914* and - 1915. Effects of stable and transient expression of 2 microRNAs in human chemoresistant CRC cell lines were analyzed. Tumor formation and chemoresistance in HCT116/5-Fu/OXA that did or did not express 2 microRNAs were analyzed in mice. Nuclear factor I/X (NFIX) was predicted to target the gene of 2 miRNAs and verified in vivo and in vitro. Plasma levels of miR-1914* and -1915 in chemoresistant CRC patients were different than levels in responders, and associated with clinical response. Overexpression of miR-1914* and -1915 in chemoresistant CRC cells reduced resistance to 5-FU and Oxaliplatin in vitro. The microRNAs suppressed chemoresistance in CRC tumors in mice by affecting cell growth, invasion, apoptosis and tumor suppressor function. miR-1914* and -1915 interacted with the 3'-untranslated region of NFIX and reduced NFIX its level in chemoresistant CRC cells. Overexpression of NFIX did not inhibit chemoresistant CRC cell motility and chemoresistant proteins when miR-1914* and -1915 were transfected. Plasma miR-1914* and -1915 interact with NFIX RNA and reduce its level in chemoresistant CRC cells to first-line chemotherapy. Up-regulation of miR-1914* and -1915 decreased the chemoresistance abilities of chemoresistant CRC cells. The plasma miR-1914* and -1915 may play a role in colorectal cancer therapy and diagnosis.

ERβ up-regulation was involved in silibinin-induced growth inhibition of human breast cancer MCF-7 cells

We previously reported that silibinin induced a loss of cell viability in breast cancer (MCF-7) cells by ERα down-regulation. But whether this cytotoxicity depends on another estrogen receptor, ERβ, has yet to be elucidated. Therefore, we sought to explore the effects of ERβ modulation on cell viability by using an ERβ-selective agonist (Diarylprepionitrile, DPN) and an antagonist (PHTPP). Our data demonstrated that ERβ served as a growth suppressor in MCF-7 cells, and the incubation of silibinin, elevated ERβ expression, resulting in the tumor growth inhibition. The cytotoxic effect of silibinin was diminished by PHTPP and enhanced by DPN. Silencing of ERβ by siRNA confirmed these results. Apoptotic cascades, including the sequential activation of caspase-9 and -6, and finally the cleavage of caspase substrates, PARP and ICAD, caused by treatment with silibinin, were all repressed by PHTPP pre-treatment but exacerbated by DPN. Unlike ERα, ERβ did not involve autophagic process in the regulation, since neither autophagic inhibitor (3-MA) nor the inducer (rapamycin) affected the cell survival rates regardless ERβ activity. Taken together, silibinin induced apoptosis through mitochondrial pathway by up-regulating ERβ pathways in MCF-7 cells without the involvement of autophagy.

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways

N-MYC downstream-regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways, including the TGF-β pathway, protein kinase B (AKT)/PI3K pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), was examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2, and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream MAPKK in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2, and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, but was independent of this metastasis suppressor for di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors.

The Mir-193 Family Antagonizes Stem Cell Pathways and Is a Potent Tumor Suppressor in Childhood and Adult Acute Myeloid Leukemia

MicroRNAs (miRNAs) are essential for maintenance and differentiation of normal hematopoietic cells and their dysregulation is strongly implicated in leukemias. In order to identify tumor suppressor miRNAs in the context of hematological malignancies, we performed two complementary miRNA expression screenings in normal hematopoiesis as well as in childhood and adult acute myeloid leukemias (AML). We reasoned that tumor suppressor miRNAs are upregulated in mature myeloid cells, as compared to normal hematopoietic stem and progenitor cells (HSPCs), and downregulated in AML.Based on this screening strategy, we identified the miR-193 family members as potent suppressors of HSPC activity and AML growth. During normal hematopoiesis mmu-miR-193a-3p is exclusively expressed in mature myeloid cells and absent in normal HSPCs. Accordingly, in a cohort of 165 pediatric AML patients hsa-miR-193b-3p was broadly repressed throughout the cytogenetically characterized subgroups. In addition, in a cohort of 43 adult AML patients, its homolog hsa-miR-193a-3p was significantly upregulated in APL cases (p=0.0025, n=7) compared to bone marrow from healthy donors (n=5).To assess the impact of the miR-193 family members on AML maintenance and development, we lentivirally expressed miR-193a/b in the MLL-rearranged cell lines ML2 and THP1, which induced monocytic differentiation in concert with calcitriol treatment, measured by CD11b/CD14 expression (p=0.024). Consistently, enforced miR-193-expression led to a significant growth disadvantage in ML2 and THP1 cells (p=<0.001 and p=0.02, respectively) as well as to reduced colony formation (p=0.008) in methylcellulose-based colony-forming unit (CFU) assays. Noteworthy, these effects were not restricted to MLL-rearranged AML cell lines only, but were also evident in six other AML cell lines representing the most common AML subgroups, such as t(8;21) and t(15;17). Beyond the growth-suppressive and differentiation-inductive effect of miR-193 in human AML cell lines, overexpression of miR-193a caused a significant decrease of proliferation in murine bone marrow cells immortalized in vitro by retroviral expression of Hoxa9 or Hoxa9 and Meis1 (p=0.019 and p=0.008, respectively).Based on these findings in AML, we further investigated the impact of the miR-193 family on normal hematopoiesis. We retrovirally expressed miR-193a in 32D cells treated with granulocyte-colony stimulating factor (G-CSF), which resulted in a strong induction of myeloid differentiation already after day 2 (p=0.006) as assessed by CD11b/Gr-1 surface marker expression. We lentivirally transduced mouse lineage negative (Lin-) HSPCs and transplanted them into irradiated isogenic recipients. Bleedings performed on weeks 4, 8 and 11, as well as the examination of the bone marrow on week 11, showed a severe competitive disadvantage of miR-193-transduced cells (week 11: 2% GFP+ miR-193- vs. 25% GFP+ miR-NSC-transduced cells). These results were further refined using highly purified ESLAM (CD45+ EPCR+ CD48− CD150+) HSCs which failed to reconstitute hematopoiesis when overexpressing miR-193a, indicated by the absence of miR-193a/GFP+ cells at week 8 post transplantation. These observations might be explained by a potent G1 cell cycle arrest in HSPCs when overexpressing miR-193a/b (4-fold decrease in the S phase population) and induction of apoptosis.Our results in normal and malignant hematopoiesis suggested that the miR-193 family acts globally through targeting relevant stem cell pathways. To validate this hypothesis we quantified the knockdown of ten predicted miR-193 target genes. qRT-PCR analysis confirmed the down regulation of KIT, KRAS, SOS2 (key components of the MAPK signaling pathway) and CCND1, a CDK regulator of G1/S phase transition. We propose a dual regulatory platform where firstly, miR-193 targets CCND1 and controls the cell cycle kinetics of stem cells. Secondly, miR-193 interferes with the KIT proto-oncogene and the RAS pathway thereby inhibiting crucial pro-proliferation and anti-apoptotic signaling cascades.Taken together, we identified the miR-193 family as a pan-tumor suppressor in childhood and adult AML. Its anti-leukemic effect is mediated by targeting the stem cell KIT/SOS2/RAS/RAF axis. DisclosuresNo relevant conflicts of interest to declare.

CDKN2 Gene Deletion As Poor Prognosis Predictor Involving in the Progression of Adult B-Lineage Acute Lymphoblastic Leukemia Patients

Background: Genes CDKN2A (MTS1) and CDKN2B (MTS2), which encode for p16INK4a/p14ARF and p15INK4b respectively from the 9p21.3 locus, are thought to be an important growth suppressor gene. INK4a/ARF regulates p53 tumor suppressor function by interacting with MDM2. P15/INK4b works as a CDK inhibitor, which inhibits phosphorylation of Rb by inactivating cyclin/ CDK-4/6 complexes and thereby controls cell-cycle G1 progression. Deletion of CDKN2A/B was well known in many hematologic malignancies, but only few report investigated this deletion effect on clinical prognosis.Methods: This study performed analysis of the CDKN2 deletion in 215 adult B-ALL patients, and relation with cytogenetic prognostic factors (BCR/ABL; E2A/PBXl; TEL/AML1; MLL rearrangement; MYC, IGH translocation).Results: The prevalence of CDKN2 deletions in all study population was 28.4%. There is no difference between patients with CDKN2 deletion and wild-type patients in sex, age, white blood cells(WBC) count, BM blast percentage,extra infiltration and induction complete remission(CR) rate. Analysis at relapse patients revealed increased rate of CDKN2 loss (44.6%) compared with the rate in all patients (28.4%) (p=0.006). Deletions of CDKN2 were significantly associated with poor outcomes including decreased overall survival (OS) (p<0.001), lower disease free-survival (DFS) (p<0.001), and increased cumulative incidence of relapse (p=0.002); Also, CDKN2 deletions were strongly associated with IGH translocation (p=0.021); and had an adverse effect on patients who with BCR-ABL fusion gene or MLL rearranged. Among of patients with the CDKN2 deletions , the subgroup of received Allo-HSCT treatment had a better OS and DFS compared with the subgroup received chemotherapy only (OS: p<0.000; DFS: p<0.000).Conclusion: Patients with CDKN2 gene deletion were benefited from allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Deletion of CDKN2 gene was commonly observed through leukemia progression and was a possible poor prognostic marker in long-term outcomes. DisclosuresNo relevant conflicts of interest to declare.