Growth Repression Research Articles

Mouse 4T1 Breast Cancer Cell-Derived Exosomes Induce Proinflammatory Cytokine Production in Macrophages via miR-183.

Tumor-associated macrophages (TAMs) play a critical role in the tumor inflammatory microenvironment and facilitate tumor growth and metastasis. Most types of tumors aberrantly express microRNAs (miRNAs), which can be transferred between cells by exosomes and can regulate gene expression in recipient cells, but it remains unclear whether tumor-derived miRNAs are transferred by exosomes and regulate the TAM phenotype. We report that mouse 4T1 breast cancer cell-derived exosomes enhanced TAM expression of IL-1β, IL-6, and TNF-α and that inhibition of 4T1-cell exosome secretion through short hairpin RNA-mediated Rab27a/b depletion repressed tumor growth and metastasis and markedly downregulated IL-1β, IL-6, and TNF-α in a 4T1 breast tumor model. Furthermore, miRNA expression profiling revealed that three miRNAs (miR-100-5p, miR-183-5p, and miR-125b-1-3p) were considerably more abundant in 4T1 cell exosomes than in mouse bone marrow-derived macrophages, indicating potential exosome-mediated transfer of the miRNAs, and, notably, miR-183-5p was found to be transferred from 4T1 cells to macrophages through exosomes. Moreover, PPP2CA was verified as an miR-183-5p target gene, and PPP2CA downregulation enhanced NF-κB signaling and promoted macrophage expression of IL-1β, IL-6, and TNF-α. Lastly, when miR-183-5p was downregulated in exosomes through miR-183-5p sponge expression in 4T1 cells, these 4T1-derived exosomes triggered diminished p65 phosphorylation and IL-1β, IL-6, and TNF-α secretion, and the miRNA downregulation also led to repression of tumor growth and metastasis in the 4T1 breast tumor model in vivo. Thus, miR-183-5p expressed in tumor cells was transferred to macrophages by exosomes and promoted the secretion of proinflammatory cytokines by inhibiting PPP2CA expression, which contributed to tumor progression in a breast cancer model.

Two Brassica napus cultivars differ in gene expression, but not in their response to submergence.

Heavy rainfall causes flooding of natural ecosystems as well as farmland, negatively affecting plant performance. While the responses of the wild model organism Arabidopsis thaliana to such stress conditions is well understood, little is known about the responses of its relative, the important oil crop plant Brassica napus. For the first time, we analyzed the molecular response of Brassica napus seedlings to full submergence in a natural light-dark cycle. We used two cultivars in this study, a European hybrid cultivar and an Asian flood-tolerant cultivar. Despite their genomic differences, those genotypes showed no major differences in their responses to submergence. The molecular responses to submergence included the induction of defense- and hormone-related pathways and the repression of biosynthetic processes. Furthermore, RNAseq revealed a strong carbohydrate-starvation response under submergence in daylight, which corresponded with a fast depletion of sugars. Consequently, both B. napus cultivars exhibited a strong growth repression under water, but there was no indication of a low-oxygen response. The ability of the European hybrid cultivar to form a short-lived leaf gas film neither increased underwater net photosynthesis, underwater dark respiration nor growth during submergence. Due to the high sensitivity of both cultivars, the analysis of other cultivars or related species with higher submergence tolerance is required in order to improve flood tolerance of this crop species. One major target could be the improvement of underwater photosynthesis efficiency in order to enhance submergence survival.

Curcumin analogs exhibit anti-cancer activity by selectively targeting G-quadruplex forming c-myc promoter sequence

Curcumin exhibits a broad spectrum of beneficial health properties that include anti-tumor and anti-cancer activities. The down-regulation of c-myc transcription via stabilizing the G-quadruplex structure formed at the promoter region of the human c-myc gene allows the repression in cancer growth. Small molecules can bind and stabilize this structure to provide an exciting and promising strategy for anti-cancer therapeutics. Herein, we investigated the interaction of Curcumin and its synthetic analogs with G-quadruplex DNA formed at the c-myc promoter by using various biophysical and biochemical assays. Further, its cytotoxic effect and mechanistic insights were explored in various cancer cell lines as well as in multicellular tumor spheroid (MCTS) model. The MCTS possesses almost similar microenvironment as avascular tumors, and micro-metastases can be used as a suitable model for the small molecule-based therapeutics development. Our study provides an expanded overview of the anti-cancer effect of a new Curcumin analog via targeting G-quadruplex structures formed at the promoter region of the human c-myc gene.

Chain-shattering Pt(IV)-backboned polymeric nanoplatform for efficient CRISPR/Cas9 gene editing to enhance synergistic cancer therapy

CRISPR/Cas9 system has become a promising gene editing tool for cancer treatment. However, development of a simple and effective nanocarrier to incorporate CRISPR/Cas9 system and chemotherapeutic drugs to concurrently tackle the biological safety and packaging capacity of viral vectors and combine gene editing-chemo for cancer therapy still remains challenges. Herein, a chain-shattering Pt(IV)-backboned polymeric nanoplatform is developed for the delivery of EZH2-targeted CRISPR/Cas9 system (NPCSPt/pEZH2) and synergistic treatment of prostate cancer. The pEZH2/Pt(II) could be effectively triggered to unpack/release from NPCSPt/pEZH2 in a chain-shattering manner in cancer cells. The EZH2 gene disruption efficiency could be achieved up to 32.2% of PC-3 cells in vitro and 21.3% of tumor tissues in vivo, leading to effective suppression of EZH2 protein expression. Moreover, significant H3K27me3 downregulation could occur after EZH2 suppression, resulting in a more permissive chromatin structure that increases the accessibility of released Pt(II) to nuclear DNA for enhanced apoptosis. Taken together, substantial proliferation inhibition of prostate cancer cells and further 85.4% growth repression against subcutaneous xenograft tumor could be achieved. This chain-shattering Pt(IV)-backboned polymeric nanoplatform system not only provides a prospective nanocarrier for CRISPR/Cas9 system delivery, but also broadens the potential of combining gene editing-chemo synergistic cancer therapy.

HbARF2 and HbARF16.3 function as negative regulators for the radial trunk growth of rubber tree

Auxin plays pivotal roles in the regulation of plant growth mainly via the auxin response factors (ARF), pin-formed efflux carriers (PIN) and auxin influx carriers (LAX).To identify potential responses of the expression of these genes to tapping-caused repression of radial growth, the expression of 17 ARF, 4 LAX and 4 PIN genes in tapped and untapped rubber trees (Hevea brasiliensis Muell. Arg.) and among germplasm with differential annual increment of trunk girth from six hybrid combinations was analysed by using quantitative real-time PCR (qPCR). HbARF2 and HbARF16.3 were up-regulated by tapping. The transcript levels were much higher in Reyan8–79 than that in Reken525 whereas radial trunk growth was strongly reduced in Reyan8–79 in comparison with that in Reken525 after tapping. The transcript levels negatively correlated with the annual increment in trunk girth of offsprings. Overexpression of HbARF2 or HbARF16.3 inhibited the growth of transgenic tobacco (Nicotiana tabacum L.) plants. The repressed growth caused by the HbARF2 was involved in the reduced cell sizes of pith, cell size and proliferation of the vascular bundle while the growth inhibition caused by HbARF16.3 attributed to cell proliferation within the pith of transgenic tobacco. The results suggest that HbARF2 and HbARF16.3 function as negative regulators for trunk growth and are related to the tapping-induced inhibition of trunk growth of rubber trees.

SiRNA-mediated lncRNA ANRIL knockdown enhances the sensitivity of gastric cancer cells to doxorubicin

Background: Long non-coding RNAs (lncRNAs) play pivotal roles in carcinogenesis and the development of drug resistance in various malignancies. Objectives: The current study aimed to explore the impact of antisense non-coding RNA in the INK4 locus (ANRIL) silencing on proliferation and the sensitivity of KATO III gastric cells to the doxorubicin as a common chemotherapeutic agent. Methods: The KATO III cells were transfected with ANRIL siRNA (si-ANRIL) using Lipofectamine™2000 reagent. Following that, the relative ANRIL levels were determined by quantitative Real-Time PCR. Trypan blue assay was conducted to observe the tumor cell proliferation following the transfection. Moreover, the MTT assay was performed to identify the cytotoxic effects of doxorubicin and si-ANRIL alone or in combination on KATO III cells. The effects of si-ANRIL/doxorubicin on KATO III cells migration and apoptosis were assessed by wound healing assay and ELISA cell death method, respectively. Results: The results showed that the si-ANRIL significantly diminished ANRIL expression level in a time-dependent manner contributing to the distinct repression of cell growth and enhanced apoptosis. Furthermore, the si-ANRIL synergistically elevated the cytotoxic impacts of doxorubicin. Additionally, the ANRIL down-regulation dramatically promoted its induction of apoptosis. Moreover, KATO III cells transfected with si-ANRIL and exposed with doxorubicin revealed significantly reduced invasion capability and enhanced apoptosis rate. Conclusion: These results demonstrated that the knock-down of lncRNA ANRIL could be a potential therapeutic strategy to trigger apoptosis and circumvent doxorubicin-resistance.

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness.

With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X-press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (qS ) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy-chain single-domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS are used to control productivity and leakiness. In the X-press strain, extracellular SpA and VHH titer reach up to 349 and 19.6mg g-1 , respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.

Antifungal effect of nerol via transcriptome analysis and cell growth repression in sweet potato spoilage fungi Ceratocystis fimbriata

Abstract Ceratocystis fimbriata is the most devastating phytopathogen causing significant losses in post-harvest sweet potato. In this study, monoterpene nerol (NEL), the active compound in neroli essential oil, was found to dose-dependently inhibit the mycelial growth and spore germination of C. fimbriata at a minimum inhibitory concentration (MIC) of 0.25 mL L-1. NEL vapor treatments significantly reduced the incidence and lesion diameter of black rot in sweet potato infected by the fungus and regulated the defense-related enzyme activity of phenylalanine ammonia lyase (PAL). Using RNA sequencing (RNA-seq) and biochemical assays, it was demonstrated that NEL treatment impaired cell membrane integrity via down-regulating the expression of ergosterol synthesis genes and reduced the ergosterol content. Moreover, an analysis of a series of apoptotic events revealed that NEL treatment caused mitochondrial membrane damage by reducing the mitochondrial membrane potential (MMP, Δψm), which led to down-regulation of genes involved in ATP production, then induced accumulation of intracellular reactive oxygen species (ROS) generation. Simultaneously, NEL caused nuclear chromatin condensation and concomitant DNA cleavage, which led to the up-regulation of DNA repair genes expression, and the cell-cycle arrest principally occurred at the G2/M phase in C. fimbriata. Altogether, these findings provide information about the underlying antifungal mechanism of NEL against C. fimbriata and suggest that NEL could be a useful alternative for controlling C. fimbriata in post-harvest spoilage of sweet potato.

561 ENHANCING RADIATION-INDUCED IMMUNOGENICITY IN THE MULTIMODAL TREATMENT OF ESOPHAGEAL CANCER: IS HYPOFRACTIONATION THE ANSWER?

Abstract Esophageal adenocarcinoma (EAC) is increasing exponentially year on year in Western civilisation, linked epidemiologically to GERD and obesity. It is characterized by a highly immunosuppressive tumour microenvironment (TME) and evasion of immune surveillance. A novel treatment option is upregulating immune mediated anti-tumour response via Immune Checkpoint Blockers (ICB). Methods The effects of immune checkpoint blockers were characterised in terms of proliferation, cytolysis and cancer cell viability. The basal expression of immune checkpoints (TIGIT, PD-1, PD-L1, PD-L2) and Damage Associated Molecular Patterns (Calreticulin, HMGB1) in EAC patients was profiled ex vivo using fresh tumor, blood and lymph-node tissue (n = 10) by flow cytometry. In an in-vitro study, T-lymphocytes were isolated and treated with Nivolumab, Pembrolizumab or Atezolizumab, activated and co-cultured for 48 hours with a panel of four esophageal cancer cell lines; OE33P, OE33R, FLO-1, FLO-1LM treated with 1.8Gy and 3.6Gy of radiation (Fig 1). Cytolysis was measured using a CCK8 assay.(n = 6). Results The expression of TIGIT, TIM-3, PD-1 and its ligands (PD-L1, PD-L2) were higher (p < 0.001) in EAC patients compared to age matched healthy controls. Similarly, when mimicking conditions of the TME including nutrient deprivation and hypoxia, this results in a significant (p < 0.001) increase in DAMP and ICB expression on CD3,4 and CD8 T cells in the TME when treated with radiation. T-lymphocytes induced by checkpoint blockers plus ionising radiation directly to the tumor resulted in the best repression of tumour growth with 3.6Gy inducing the highest rate of cytolysis (p < 0.001). ICB and radiation resulted in reduced cancer cell viability and proliferation. Conclusion Fractionated radiation can enhance immunologic function. In combination with ICB, this symbiotic relationship enhances the cytotoxic potential of T lymphocytes with conventional dosing, however, with hypofractionation, this signifies true immunogenicity, and as such this provides a basis for advocating for potential combination strategies with ICB in the multimodal treatment of EAC.

Insights into the role of energy source in hormesis through diauxic growth of bacteria in mixed cultivation systems

Hormesis, a biphasic dose-response relationship characterized by low-dose stimulation and high-dose inhibition, has been reported to be closely related to energy sources in cultivation systems. However, few studies have clarified how the energy source influences hormesis. In this study, based on the typical diauxic patterns of Escherichia coli (E. coli) growth in mixed cultivation media containing 1.0 g L−1 glucose and Luria-Bertani broth, the hormetic response of sulfonamides (SAs) to E. coli growth was investigated under this diauxic growth condition to thoroughly explain the close relationship between hormesis and energy sources in cultivation systems. The results indicated that SAs trigger time-dependent hormetic effects on E. coli growth over the span of 24 h, in which the biphasic dose-response occurs only during the second lag and the earlier stage of the second log phase of diauxic growth. Mechanistic exploration reveals that SAs can bind with adenylate cyclase at a low dose and dihydropteroate synthase at a high dose, respectively, activating the stimulatory and inhibitory signaling pathway to influence carbon catabolite repression in diauxic growth, which can interfere with the metabolism of tryptone and yeast extract to ultimately trigger hormesis. Moreover, the stimulatory and inhibitory effects of SAs are changed by the variations in metabolic status at different growth phases, resulting in time-dependent hormesis. This study proposes an induced mechanistic explanation of hormesis in mixed cultivation media based on the energy source’s metabolism, which may not only reflect the generalizability of hormesis but also further promote its application in production activities.

To grow or not to grow ….

To grow or not to grow ….

Novel goose-origin astrovirus infection in geese: the effect of age at infection

Since 2017, a serious infectious disease characterized by visceral gout has emerged in China's main goose-producing regions. The disease has caused huge economic losses to China's goose industry. In our previous study, we determined that the pathogen causing gout in goslings is a novel goose-origin astrovirus, designated as AStV/SDPY/Goose/1116/17 (AStV-SDPY) strain. To investigate the effect of host age on the outcome of novel goose-origin astrovirus infection, 200 1-day-old healthy goslings were selected to be experimentally infected at 1, 5, 15, 25, and 35 D of age. It was shown in experimental infection that the AStV-SDPY strain was highly pathogenic in goslings aged 1 to 15 D, causing growth repression, severe visceral urate deposition, and even death, whereas goslings infected at 25 and 35 D of age showed mild symptoms. Histopathologic examination indicated that lesions occurred mainly in the kidney and liver of infected goslings, which is correlated to the severity of clinical signs and gross lesions. Viral RNA was detected in all representative tissues, and virus shedding was detected continuously within 15 D after inoculation. Higher viral copy number, especially in vital organs such as the liver and kidney, was developed in the goslings infected at 1 to 15 D of age than older geese. In addition, clinical chemistry and inflammatory cytokines showed that younger geese are more sensitive to AStV infection. Overall, our study demonstrates that the pathogenicity of AStV-SDPY in goslings is partly associated with the age of infection, laying a foundation for further study of the pathogenic mechanism of this virus.

Estrogen Signaling and Its Potential as a Target for Therapy in Ovarian Cancer.

The estrogen receptor (ER) has functionality in selected ovarian cancer subtypes and represents a potential target for therapy. The majority (>80%) of high grade serous, low grade serous and endometrioid carcinomas and many granulosa cell tumors express ER-alpha (ERα), and these tumor types have demonstrated responses to endocrine therapy (tamoxifen and aromatase inhibitors) in multiple clinical studies. Biomarkers of responses to these drugs are actively being sought to help identify responsive cancers. Evidence for both pro-proliferative and pro-migratory roles for ERα has been obtained in model systems. ER-beta (ERβ) is generally considered to have a tumor suppressor role in ovarian cancer cells, being associated with the repression of cell growth and invasion. The differential expression of the specific ERβ isoforms may determine functionality within ovarian cancer cells. The more recently identified G protein-coupled receptor (GPER1; GPR30) has been shown to mediate both tumor-suppressive and tumor-promoting action in ovarian cancer cells, suggesting a more complex role. This review will summarize recent findings in this field.

Leveraging genome-enabled growth models to study shoot growth responses to water deficit in rice.

Elucidating genotype-by-environment interactions and partitioning its contribution to phenotypic variation remains a challenge for plant scientists. We propose a framework that utilizes genome-wide markers to model genotype-specific shoot growth trajectories as a function of time and soil water availability. A rice diversity panel was phenotyped daily for 21 d using an automated, high-throughput image-based, phenotyping platform that enabled estimation of daily shoot biomass and soil water content. Using these data, we modeled shoot growth as a function of time and soil water content, and were able to determine the time point where an inflection in the growth trajectory occurred. We found that larger, more vigorous plants exhibited an earlier repression in growth compared with smaller, slow-growing plants, indicating a trade-off between early vigor and tolerance to prolonged water deficits. Genomic inference for model parameters and time of inflection (TOI) identified several candidate genes. This study is the first to utilize a genome-enabled growth model to study drought responses in rice, and presents a new approach to jointly model dynamic morpho-physiological responses and environmental covariates.

The miR-26b-5p/KPNA2 Axis Is an Important Regulator of Burkitt Lymphoma Cell Growth.

The expression of several microRNAs (miRNAs) is known to be changed in Burkitt lymphoma (BL), compared to its normal counterparts. Although for some miRNAs, a role in BL was demonstrated, for most of them, their function is unclear. In this study, we aimed to identify miRNAs that control BL cell growth. Two BL cell lines were infected with lentiviral pools containing either 58 miRNA inhibitors or 44 miRNA overexpression constructs. Eighteen constructs showed significant changes in abundance over time, indicating that they affected BL growth. The screening results were validated by individual green fluorescent protein (GFP) growth competition assays for fifteen of the eighteen constructs. For functional follow-up studies, we focused on miR-26b-5p, whose overexpression inhibited BL cell growth. Argonaute 2 RNA immunoprecipitation (Ago2-IP) in two BL cell lines revealed 47 potential target genes of miR-26b-5p. Overlapping the list of putative targets with genes showing a growth repression phenotype in a genome-wide CRISPR/Cas9 knockout screen, revealed eight genes. The top-5 candidates included EZH2, COPS2, KPNA2, MRPL15, and NOL12. EZH2 is a known target of miR-26b-5p, with oncogenic properties in BL. The relevance of the latter four targets was confirmed using sgRNAs targeting these genes in individual GFP growth competition assays. Luciferase reporter assay confirmed binding of miR-26b-5p to the predicted target site for KPNA2, but not to the other genes. In summary, we identified 18 miRNAs that affected BL cell growth in a loss- or gain-of-function screening. A tumor suppressor role was confirmed for miR-26b-5p, and this effect could at least in part be attributed to KPNA2, a known regulator of OCT4, c-jun, and MYC.

Glucosinolate Transporter1 involves in salt-induced jasmonate signaling and alleviates the repression of lateral root growth by salt in Arabidopsis

Salt stress has negative impact on plant development and growth. Jasmonic acid (JA), a phytohormone, has been shown to involve in salt-induced inhibition of primary root growth. The Arabidopsis Glucosinolate transporter1 (GTR1/NPF2.10) is characterized as a JA-Ile, a bioactive form of JA, transporter. However, whether GTR1 participates in salt responses is not clear. In this study, we confirmed that GTR1 is induced by both JA and salinity. Salt-induced JA signaling is affected in gtr1 mutant. The JA responsive genes, JAZ1, JAZ5, MYC2, LOX3, are down-regulated in gtr1 mutant. Phenotypic analyses showed that the salinity-induced lateral root growth inhibition is enhanced in gtr1 mutant, suggesting that GTR1 plays a positive role in lateral root development under salt stress. Interestingly, the expression of a Na+ transporter, HKT1, is upregulated in gtr1. Since HKT1 is a negative regulator for lateral root development under salt stress, we proposed that GTR1 alleviates the repression of lateral root development by salt stress by mediating JA signaling and repressing HKT1 expression. This study demonstrates that GTR1 is the molecular link between salt stress, JA signaling, and lateral root development.

SOX9-activated PXN-AS1 promotes the tumorigenesis of glioblastoma by EZH2-mediated methylation of DKK1.

Increasing evidence has validated the essential regulation of long non‐coding RNAs (lncRNAs) in the biological process of tumours. LncRNA PXN‐AS1 has been discovered to be as a tumour suppressor in pancreatic cancer; however, its function and mechanism remain greatly unknown in glioblastoma (GBM). Our present study indicated that PXN‐AS1 was highly expressed in GBM tissues and cells. Besides, the knock‐down of PXN‐AS1 was closely associated with the inhibitory proliferation and inducing apoptosis of GBM cells. PXN‐AS1 inhibition was also found to restrain GBM tumour growth. Importantly, SOX9 functioned as a transcription factor and activated PXN‐AS1 expression, and overexpressed PXN‐AS1 rescued the inhibitory role of down‐regulated SOX9 in GBM cell growth. Subsequently, it was discovered that PXN‐AS1 activated Wnt/β‐catenin pathway. DKK1 was widely known as an inhibitor gene of Wnt/β‐catenin pathway, and its expression was negatively associated with PXN‐AS1 and SOX9. Interestingly, we found that PXN‐AS1 could recruit EZH2 to mediate the H3K27me3 level of DKK1 promoter. Restoration experiments manifested that DKK1 knock‐down counteracted PXN‐AS1 depletion‐mediated repression in GBM cell growth. All facts pointed out that PXN‐AS1 might be of importance in exploring the therapeutic strategies of GBM.

Cyclin C Regulated Transcriptional Response to Stress

When cells are exposed to extracellular stress, there is a coordinated shift in cellular and molecular actions that determine the fate of the cell. An important aspect of this response involves transcriptional reprogramming to either mitigate the effects of the stress or commit to a cell death pathway. This altered transcriptome causes disruption of normal cellular functions, such as growth and proliferation. It also promotes the transcription of genes involved in stress response and cell death. Our previous findings from RNA sequencing (RNA‐seq) studies has shown that the conserved transcription factor, cyclin C, regulates the stress responsive transcriptome in mouse embryonic fibroblast (MEF) cells. Cyclin C is a member of the Cdk8‐kinase module (CKM) which, depending on the locus, can function to either induce or repress transcription. The genes induced by cyclin C prior to stress include those that promote growth and proliferation. In contrast, the genes repressed by cyclin C prior to stress include those that promote autophagy and apoptosis. After exposure to stress, the role of cyclin C is disrupted on each group of genes. This transcriptional reprogramming event results in the repression of the growth and proliferation genes, and induction of the autophagy and apoptosis genes. In the present study, chromatin immunoprecipitation (ChIP) studies show that cyclin C is associated with the promoter region of genes identified through RNA‐seq analysis. ChIP was used to determine promoter occupancy by cyclin C and transcriptional changes occurring from exposure to either oxidative stress or Torin1 to induce apoptosis and autophagy, respectively. Stressed cells show significantly reduced, or complete loss of cyclin C occupancy at specific genes. Cyclin C is also recruited to the promoter of other genes following exposure to stress to induce transcription. The genes identified in this study are involved in the cellular responses upon exposure to stress; specifically, the repression of growth and proliferation and the induction of autophagy and apoptosis. These findings demonstrate a direct role for cyclin C in the transcriptional changes occurring following exposure to stress, and subsequently contribution to cell fate determination.

TIFA‐NF‐κB axis modulates inflammatory microenvironment to offset sorafenib cytotoxicity in hepatocellular carcinoma

Sorafenib is the promising first‐line drug to treat advanced hepatocellular carcinoma (HCC), with the acquired resistance within 6 months in most treated patients. The resistance to sorafenib treatment has been correlated to inflammation or viral hepatitis, but the underlying molecular mechanism remains elusive. We previously reported that the TRAF‐interacting protein TIFA sustains an inflammatory feedback loop of TNFα and NF‐κB to drive a survival pathway. Here we investigated how the inflammatory microenvironment is modulated by TIFA‐NF‐κB axis to confer a sorafenib‐resistant niche for HCC recurrence. The prognostic analysis showed that severe hepatitis was highly associated with poorer clinical responses to sorafenib treatment, suggesting that the sorafenib‐resistant HCC involves chronic inflammation. In support, inflammatory cytokines TNF‐α protected HCC lines form sorafenib cytotoxicity, while inhibition of TIFA‐NF‐κB axis reversed the effect. In addition, we showed that TIFA‐NF‐κB axis offset sorafenib cytotoxicity by supporting BCL‐2‐dependent survival signaling. To further explore the therapeutic relevance, we targeted TIFA‐NF‐κB axis using a liver‐tropic adeno‐associated virus to deliver shRNAs in an orthotopic xenograft HCC model, and result showed significant repression of tumor growth and prolonged animal survival in addition to sorafenib treatment. More intriguingly, such inhibition also abrogated the liver‐infiltration of tumor‐associated macrophages, a mechanistic event proven to be triggered by the TIFA‐NF‐κB‐regulated secretion of damage‐associated molecular pattern molecules S100A8/A9 in response to sorafenib, thereby recruiting precursor monocytes. As consequences of monocyte/macrophage infiltration, the TIFA‐NF‐κB axis in HCC cells compromised sorafenib cytotoxicity in alliance with pro‐tumor M2 macrophages, and harnessed their release of IL‐7 to enhance proliferation, sorafenib resistance, and directional migration of HCC cells. Our results collectively demonstrated that TIFA‐NF‐κB axis supports inflammatory responses to offset sorafenib cytotoxicity, and that its targeting provides therapeutically intervention concomitant with sorafenib treatment in HCC.Support or Funding InformationThis study was supported by Academia Sinica and the Taiwan Protein Project funded by Ministry of Science and Technology (MOST; Grant No. AS‐KPQ‐105‐TPP), and in part by grants MOST 104‐2314‐B‐182A‐060 and Chang Gung Memorial Hospital CMRPG3G0561.

Transcription factor WRKY23 is involved in ammonium-induced repression of Arabidopsis primary root growth under ammonium toxicity

Although WRKY transcription factors (TFs) are known to be involved in the regulation of plant root development, the mechanisms by which these TFs regulate plant tolerance to ammonium (NH4+) toxicity remain unclear. To identify the molecular mechanisms underlying NH4+-induced repression of primary root growth and NH4+ sensitivity in Arabidopsis, wild-type (Col-0) and mutant (wrky23) plants were treated with 10 mM KNO3 (control) or 5 mM (NH4)2SO4 (NH4+ toxicity) for 7 days. Under NH4+ toxicity, the fresh weight of wrky23 mutant was significantly lower than that of Col-0 plants, and the NH4+ concentration in wrky23 roots was significantly higher than that in Col-0 roots. However, we observed no significant differences between the two genotypes under the control treatment. Ammonium transporter AMT1;2 expression was induced in wrky23 roots but not in Col-0 roots. The transcript levels of cytosolic glutamine synthetase-encoding genes and activity of glutamine synthetase did not differ significantly between wrky23 and Col-0. Furthermore, the fluorescence and staining patterns of DR5::GFP and DR5::GUS, respectively, were more pronounced under NH4+ toxicity than under the control treatment. Collectively, our results indicate that AMT1;2 expression was induced in the wrky23 mutant in response to NH4+ toxicity, leading to NH4+ accumulation in the roots and primary root growth repression. Under NH4+ toxicity, both auxin transport and distribution were affected, and auxin accumulation in the root tips inhibited primary root growth in the wrky23 mutant. Our study provides important insights into the molecular mechanisms by which WRKY23 TF regulates plant responses to NH4+ toxicity.