Different emotional states engage distinct descending pathways from the prefrontal cortex.

Emotion regulation, essential for adaptive behavior, depends on the brain's capacity to process a range of emotions. Current research has largely focused on individual emotional circuits without fully exploring how their interaction influences physiological responses or understanding the neural mechanisms that differentiate emotional valence. Using in vivo calcium imaging, electrophysiology, and optogenetics, we examined neural circuit dynamics in the medial prefrontal cortex (mPFC), targeting two key areas: the basal lateral amygdala (BLA) and nucleus accumbens (NAc). Our results demonstrate distinct activation patterns in the mPFC→BLA and mPFC→NAc pathways in response to social stimuli, indicating a mechanism for discriminating emotions: increased mPFC→BLA activity signals anxiety, while heightened mPFC→NAc responses are linked to exploration. Additionally, chronic emotional states amplify activity in these pathways-positivity enhances mPFC→NAc, while negativity boosts mPFC→BLA. This study sheds light on the nuanced neural circuitry involved in emotion regulation, revealing the pivotal roles of mPFC projections in emotional processing. Identifying these specific circuits engaged by varied emotional states advances our understanding of emotional regulation's biological underpinnings and highlights potential targets for addressing emotional dysregulation in psychiatric conditions. Significance statement: While existing circuitry studies have underscored the significance of emotional circuits, the majority of research has concentrated on individual circuits. The assessment of whether and how the balance among multiple circuits influences overall physiological outcomes is often overlooked. This study delves into the neural underpinnings of emotion regulation, focusing on how positive and negative valences are discriminated and managed. By examining the specific pathways from the medial prefrontal cortex (mPFC) to key emotional centers-the basal lateral amygdala (BLA) for negative valence and the nucleus accumbens (NAc) for positive one-we uncovered a novel dual-balanced neural circuit mechanism that enables this essential aspect of human cognition.

CPAP promotes angiogenesis and metastasis by enhancing STAT3 activity.

Centrosomal P4.1-associated protein (CPAP) is overexpressed in hepatocellular carcinoma (HCC) and positively correlated with recurrence and vascular invasion. Here, we found that CPAP plays an important role in HCC malignancies. Functional characterization indicated that CPAP overexpression increases tumor growth, angiogenesis, and metastasis ex vivo and in vivo. In addition, overexpressed CPAP contributes to sorafenib resistance. Mechanical investigation showed that the expression level of CPAP is positively correlated with activated STAT3 in HCC. CPAP acts as a transcriptional coactivator of STAT3 by directly binding with STAT3. Interrupting the interaction between CPAP and STAT3 attenuates STAT3-mediated tumor growth and angiogenesis. Overexpression of CPAP upregulates several STAT3 target genes such as IL-8 and CD44 that are involved in angiogenesis, and CPAP mRNA expression is positively correlated with the levels of both mRNAs in HCC. Knocked-down expression of CPAP impairs IL-6-mediated STAT3 activation, target gene expression, cell migration, and invasion abilities. IL-6/STAT3-mediated angiogenesis is significantly increased by CPAP overexpression and can be blocked by decreased expression of IL-8. Our findings not only shed light on the importance of CPAP in HCC malignancies, but also provide potential therapeutic strategies for inhibiting the angiogenesis pathway and treating metastatic HCC.

A selective Aurora-A 5'-UTR siRNA inhibits tumor growth and metastasis.

Many Aurora-A inhibitors have been developed for cancer therapy; however, the specificity and safety of Aurora-A inhibitors remain uncertain. The Aurora-A mRNA yields nine different 5'-UTR isoforms, which result from mRNA alternative splicing. Interestingly, we found that the exon 2-containing Aurora-A mRNA isoforms are predominantly expressed in cancer cell lines as well as human colorectal cancer tissues, making the Aurora-A mRNA exon 2 a promising treatment target in Aurora-A-overexpressing cancers. In this study, a selective siRNA, siRNA-2, which targets Aurora-A mRNA exon 2, was designed to translationally inhibit the expression of Aurora-A in cancer cells but not normal cells; locked nucleic acid (LNA)-modified siRNA-2 showed improved efficacy in inhibiting Aurora-A mRNA translation and tumor growth. Xenograft animal models combined with noninvasion in vivo imaging system (IVIS) analysis further confirmed the anticancer effect of LNA-siRNA-2 with improved efficiency and safety and reduced side effects. Mice orthotopically injected with colorectal cancer cells, LNA-siRNA-2 treatment not only inhibited the tumor growth but also blocked liver and lung metastasis. The results of our study suggest that LNA-siRNA-2 has the potential to be a novel therapeutic agent for cancer treatment.

The EGF/hnRNP Q1 axis is involved in tumorigenesis via the regulation of cell cycle-related genes.

Heterogeneous nuclear ribonucleoprotein (hnRNP) Q1, an RNA-binding protein, has been implicated in many post-transcriptional processes, including RNA metabolism and mRNA splicing and translation. However, the role of hnRNP Q1 in tumorigenesis remains unclear. We previously performed RNA immunoprecipitation (RIP)-seq analysis to identify hnRNP Q1-interacting mRNAs and found that hnRNP Q1 targets a group of genes that are involved in mitotic regulation, including Aurora-A. Here, we demonstrate that altering the hnRNP Q1 level influences the expression of the Aurora-A protein, but not its mRNA. Stimulation with epidermal growth factor (EGF) enhances both binding between hnRNP Q1 and Aurora-A mRNA as well as the efficacy of the hnRNP Q1-induced translation of Aurora-A mRNA. The EGF/hnRNP Q1-induced translation of Aurora-A mRNA is mediated by the mTOR and ERK pathways. In addition, we show that hnRNP Q1 up-regulates the translation of a group of spindle assembly checkpoint (SAC) genes. hnRNP Q1 overexpression is positively correlated with the levels of Aurora-A and the SAC genes in human colorectal cancer tissues. In summary, our data suggest that hnRNP Q1 plays an important role in regulating the expression of a group of cell cycle-related genes. Therefore, it may contribute to tumorigenesis by up-regulating the translation of these genes in colorectal cancer.

Translational upregulation of Aurora-A by hnRNP Q1 contributes to cell proliferation and tumorigenesis in colorectal cancer.

By using RNA-immunoprecipitation assay following next-generation sequencing, a group of cell cycle-related genes targeted by hnRNP Q1 were identified, including Aurora-A kinase. Overexpressed hnRNP Q1 can upregulate Aurora-A protein, but not alter the mRNA level, through enhancing the translational efficiency of Aurora-A mRNA, either in a cap-dependent or -independent manner, by interacting with the 5'-UTR of Aurora-A mRNA through its RNA-binding domains (RBDs) 2 and 3. By ribosomal profiling assay further confirmed the translational regulation of Aurora-A mRNA by hnRNP Q1. Overexpression of hnRNP Q1 promotes cell proliferation and tumor growth. HnRNP Q1/ΔRBD23-truncated mutant, which loses the binding ability and translational regulation of Aurora-A mRNA, has no effect on promoting tumor growth. The expression level of hnRNP Q1 is positively correlated with Aurora-A in colorectal cancer. Taken together, our data indicate that hnRNP Q1 is a novel trans-acting factor that binds to Aurora-A mRNA 5'-UTRs and regulates its translation, which increases cell proliferation and contributes to tumorigenesis in colorectal cancer.

Epstein-Barr virus latent membrane protein-1 up-regulates cytokines and correlates with older age and poorer prognosis in Hodgkin lymphoma.

AIMS: Previously, we reported an association between Epstein-Barr virus (EBV)-positive Hodgkin lymphoma (HL), older age, and poorer prognosis. The aim of this study was to investigate the mechanisms underlying this association. METHODS AND RESULTS: Transfection of HL cell lines with EBV latent membrane protein-1 (LMP1) resulted in up-regulation of many cytokine genes as assessed by the use of oligonucleotide microarrays. The up-regulation of cytokines was validated by using an inflammatory cytokine protein array: macrophage inflammatory protein (MIP)-1α, MIP-1ß, and interleukin (IL)-13. Immunostaining of HL samples (n = 104) showed that expression of MIP-1α, MIP-1ß and IL-13 correlated with EBV infection and LMP1 expression. Combined expression of these cytokines was more common in patients aged >60 years (P < 0.001), and was associated with a poorer prognosis (P = 0.042). In another cohort, serum levels of MIP-1α, MIP-1ß and IL-13 were increased in HL patients (n = 53) and highest in EBV-positive HL patients as compared with healthy controls (n = 40). Xenograft mice injected with EBV-positive HL cells had higher serum levels of MIP-1α, MIP-1ß and IL-13 than mice injected with EBV-negative HL cells, although there was no difference in growth. CONCLUSIONS: EBV infection appears to promote the release of cytokines in HL patients, and negatively impacts on patient survival. Physiological immunosenescence probably explains the association between EBV infection and older age. Cytokine modulation is a potential therapeutic target for EBV-positive HL patients.

ON 01910.Na inhibits growth of diffuse large B-cell lymphoma by cytoplasmic sequestration of sumoylated C-MYB/TRAF6 complex.

Diffuse large B-cell lymphoma (DLBCL), the most common lymphoma, shows either no response or development of resistance to further treatment in 30% of the patients that warrants the development of novel drugs. We have reported that ON 01910.Na (rigosertib), a multikinase inhibitor, is selectively cytotoxic for DLBCL and induces more hyperphosphorylation and sumoylation of Ran GTPase-activating protein 1 (RanGAP1) in DLBCL cells than in non-neoplastic lymphoblastoid cell line. However, the exact mechanism of rigosertib-induced cell death in DLBCL remains to be clarified. Here, we analyzed the efficacy of rigosertib against DLBCL cells in vitro and in vivo and its molecular effects on tumor biology. We found for the first time that rigosertib attenuated expression of unmodified and sumoylated tumor necrosis factor receptor-associated factor 6 (TRAF6) and c-Myb and inhibited nuclear entry of sumoylated RanGAP1, TRAF6, and c-Myb that was confirmed by immunofluorescence. Moreover, co-immunoprecipitation showed that rigosertib induced sequestration of c-Myb and TRAF6 in the cytoplasm by stimulating their sumoylation through the RanGAP1*SUMO1/Ubc9 pathway. Specific knockdown of c-Myb and TRAF6 induced tumor cell apoptosis and cell cycle arrest at G1 phase. Xenograft mice bearing lymphoma cells also exhibited effective tumor regression on rigosertib treatment along with cytoplasmic expression of c-Myb and TRAF6. Nuclear expression of c-Myb in clinical cases of DLBCL correlated with a poor prognosis. Thus, suppression of c-Myb and TRAF6 activity may have therapeutic implication in DLBCL. These data support the clinical development of rigosertib in DLBCL.

Ran GTPase-activating protein 1 is a therapeutic target in diffuse large B-cell lymphoma.

Lymphoma-specific biomarkers contribute to therapeutic strategies and the study of tumorigenesis. Diffuse large B-cell lymphoma (DLBCL) is the most common type of malignant lymphoma. However, only 50% of patients experience long-term survival after current treatment; therefore, developing novel therapeutic strategies is warranted. Comparative proteomic analysis of two DLBCL lines with a B-lymphoblastoid cell line (LCL) showed differential expression of Ran GTPase-activating protein 1 (RanGAP1) between them, which was confirmed using immunoblotting. Immunostaining showed that the majority of DLBCLs (92%, 46/50) were RanGAP1(+), while reactive lymphoid hyperplasia (n = 12) was RanGAP1(+) predominantly in germinal centers. RanGAP1 was also highly expressed in other B-cell lymphomas (BCL, n = 180) with brisk mitotic activity (B-lymphoblastic lymphoma/leukemia: 93%, and Burkitt lymphoma: 95%) or cell-cycle dysregulation (mantle cell lymphoma: 83%, and Hodgkin's lymphoma 91%). Interestingly, serum RanGAP1 level was higher in patients with high-grade BCL (1.71 ± 2.28 ng/mL, n = 62) than in low-grade BCL (0.75 ± 2.12 ng/mL, n = 52) and healthy controls (0.55 ± 1.58 ng/mL, n = 75) (high-grade BCL vs. low-grade BCL, p = 0.002; high-grade BCL vs. control, p < 0.001, Mann-Whitney U test). In vitro, RNA interference of RanGAP1 showed no effect on LCL but enhanced DLBCL cell death (41% vs. 60%; p = 0.035) and cell-cycle arrest (G0/G1: 39% vs. 49%, G2/M: 19.0% vs. 7.5%; p = 0.030) along with decreased expression of TPX2 and Aurora kinases, the central regulators of mitotic cell division. Furthermore, ON 01910.Na (Estybon), a multikinase inhibitor induced cell death, mitotic cell arrest, and hyperphosphorylation of RanGAP1 in DLBCL cell lines but no effects in normal B and T cells. Therefore, RanGAP1 is a promising marker and therapeutic target for aggressive B-cell lymphoma, especially DLBCL.

SUMOylated CPAP is required for IKK-mediated NF-κB activation and enhances HBx-induced NF-κB signaling in HCC.

BACKGROUND & AIMS: Constitutive activation of NF-κB is an important event involved in chronic inflammation in hepatocellular carcinoma (HCC). CPAP, which plays important roles in centrosomal functions, was previously identified as the transcriptional co-activator of NF-κB. However, the molecular mechanism is unclear. The goal of this study was to investigate the role of CPAP in activating the NF-κB pathway in HCC. METHODS: SK-Hep1, HuH7, HepG2, HepG2X, Hep3B, and Hep3BX cells with CPAP overexpression or CPAP siRNA were used to evaluate activation of NF-κB under TNF-α stimulation by reporter assay, RT-PCR, Q-PCR, and Western blot analysis. In vivo SUMO modification of CPAP was demonstrated by an in situ PLA assay. Human HCC tissues were used to perform Q-PCR, Western blot, and IHC. RESULTS: CPAP siRNA abolished the interaction between IKKß and NF-κB, whereas overexpression of CPAP enhanced this interaction and finally led to augmented NF-κB activation by increasing the phosphorylation of NF-κB. CPAP could enter nuclei by associating with NF-κB. Furthermore, CPAP was SUMO-1 modified upon TNF-α stimulus, and this is essential for its NF-κB co-activator activity. SUMO-1-deficient CPAP mutant lost its NF-κB co-activator activity and failed to enter nuclei. Importantly, SUMOylated CPAP could synergistically increase the HBx-induced NF-κB activity. CONCLUSIONS: CPAP is essential for the recruitment of the IKK complex to inactivated NF-κB upon TNF-α treatment. Expression of CPAP was positively correlated with a poor prognosis in HBV-HCC. CPAP has the potential to serve as a therapeutic target for inflammation and inflammation-related diseases.

Translational up-regulation of Aurora-A in EGFR-overexpressed cancer.

Abnormal expression of Aurora-A and epidermal growth factor receptor (EGFR) is observed in different kinds of cancer and associated with poor prognosis in cancer patients. However, the relationship between Aurora-A and EGFR in tumour development was not clear. In previous reports, we found that EGFR translocates to nucleus to activate Aurora-A expression after EGF treatment in EGFR-overexpressed cells. However, we also observed that not all the EGFR-overexpressed cells have the nuclear EGFR pathway to mediate the Aurora-A expression. In this study, we demonstrated that EGF signalling increased the Aurora-A protein expression in EGFR-overexpressed colorectal cancer cell lines via increasing the translational efficiency. In addition, the overexpression of EGFR was also associated with higher expression of Aurora-A in clinical colorectal samples. Activation of the PI3K/Akt/mTOR and MEK/ERK pathways mediated the effect of EGF-induced translational up-regulation. Besides, only the splicing variants containing exon 2 of Aurora-A mRNA showed increased interaction with the translational complex to synthesize Aurora-A protein under EGF stimulus. Besides, the exon 2 containing splicing variants were the major Aurora-A splicing forms expressed in human colorectal cancers. Taken together, our results propose a novel regulatory mechanism for the abnormal expression of Aurora-A in EGFR-overexpressed cancers, and highlight the importance of alternative 5'-UTR splicing variants in regulating Aurora-A expression. Furthermore, the specific expression of exon 2 containing splicing variants in cancer tissues may serve as a potential target for cancer therapy in the future.