TGF-ß Tumor Suppression through a Lethal EMT.

TGF-ß signaling can be pro-tumorigenic or tumor suppressive. We investigated this duality in pancreatic ductal adenocarcinoma (PDA), which, with other gastrointestinal cancers, exhibits frequent inactivation of the TGF-ß mediator Smad4. We show that TGF-ß induces an epithelial-mesenchymal transition (EMT), generally considered a pro-tumorigenic event. However, in TGF-ß-sensitive PDA cells, EMT becomes lethal by converting TGF-ß-induced Sox4 from an enforcer of tumorigenesis into a promoter of apoptosis. This is the result of an EMT-linked remodeling of the cellular transcription factor landscape, including the repression of the gastrointestinal lineage-master regulator Klf5. Klf5 cooperates with Sox4 in oncogenesis and prevents Sox4-induced apoptosis. Smad4 is required for EMT but dispensable for Sox4 induction by TGF-ß. TGF-ß-induced Sox4 is thus geared to bolster progenitor identity, whereas simultaneous Smad4-dependent EMT strips Sox4 of an essential partner in oncogenesis. Our work demonstrates that TGF-ß tumor suppression functions through an EMT-mediated disruption of a lineage-specific transcriptional network.

SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

ABSTRACT: Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several hematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Coimmunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner, which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

The sterile alpha motif and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several haematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Co-immunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

Age-induced accumulation of methylmalonic acid promotes tumour progression.

The risk of cancer and associated mortality increases substantially in humans from the age of 65 years onwards1-6. Nonetheless, our understanding of the complex relationship between age and cancer is still in its infancy2,3,7,8. For decades, this link has largely been attributed to increased exposure time to mutagens in older individuals. However, this view does not account for the established role of diet, exercise and small molecules that target the pace of metabolic ageing9-12. Here we show that metabolic alterations that occur with age can produce a systemic environment that favours the progression and aggressiveness of tumours. Specifically, we show that methylmalonic acid (MMA), a by-product of propionate metabolism, is upregulated in the serum of older people and functions as a mediator of tumour progression. We traced this to the ability of MMA to induce SOX4 expression and consequently to elicit transcriptional reprogramming that can endow cancer cells with aggressive properties. Thus, the accumulation of MMA represents a link between ageing and cancer progression, suggesting that MMA is a promising therapeutic target for advanced carcinomas.

Nucleosome-bound SOX2 and SOX11 structures elucidate pioneer factor function.

'Pioneer' transcription factors are required for stem-cell pluripotency, cell differentiation and cell reprogramming1,2. Pioneer factors can bind nucleosomal DNA to enable gene expression from regions of the genome with closed chromatin. SOX2 is a prominent pioneer factor that is essential for pluripotency and self-renewal of embryonic stem cells3. Here we report cryo-electron microscopy structures of the DNA-binding domains of SOX2 and its close homologue SOX11 bound to nucleosomes. The structures show that SOX factors can bind and locally distort DNA at superhelical location 2. The factors also facilitate detachment of terminal nucleosomal DNA from the histone octamer, which increases DNA accessibility. SOX-factor binding to the nucleosome can also lead to a repositioning of the N-terminal tail of histone H4 that includes residue lysine 16. We speculate that this repositioning is incompatible with higher-order nucleosome stacking, which involves contacts of the H4 tail with a neighbouring nucleosome. Our results indicate that pioneer transcription factors can use binding energy to initiate chromatin opening, and thereby facilitate nucleosome remodelling and subsequent transcription.

SoxC transcription factors shape the epigenetic landscape to establish competence for sensory differentiation in the mammalian organ of Corti.

The auditory organ of Corti is comprised of only two major cell types-the mechanosensory hair cells and their associated supporting cells-both specified from a single pool of prosensory progenitors in the cochlear duct. Here, we show that competence to respond to Atoh1, a transcriptional master regulator necessary and sufficient for induction of mechanosensory hair cells, is established in the prosensory progenitors between E12.0 and 13.5. The transition to the competent state is rapid and is associated with extensive remodeling of the epigenetic landscape controlled by the SoxC group of transcription factors. Conditional loss of Sox4 and Sox11-the two homologous family members transiently expressed in the inner ear at the time of competence establishment-blocks the ability of prosensory progenitors to differentiate as hair cells. Mechanistically, we show that Sox4 binds to and establishes accessibility of early sensory lineage-specific regulatory elements, including ones associated with Atoh1 and its direct downstream targets. Consistent with these observations, overexpression of Sox4 or Sox11 prior to developmental establishment of competence precociously induces hair cell differentiation in the cochlear progenitors. Further, reintroducing Sox4 or Sox11 expression restores the ability of postnatal supporting cells to differentiate as hair cells in vitro and in vivo. Our findings demonstrate the pivotal role of SoxC family members as agents of epigenetic and transcriptional changes necessary for establishing competence for sensory receptor differentiation in the inner ear.

Mitotic WNT signalling orchestrates neurogenesis in the developing neocortex.

The role of WNT/ß-catenin signalling in mouse neocortex development remains ambiguous. Most studies demonstrate that WNT/ß-catenin regulates progenitor self-renewal but others suggest it can also promote differentiation. Here we explore the role of WNT/STOP signalling, which stabilizes proteins during G2/M by inhibiting glycogen synthase kinase (GSK3)-mediated protein degradation. We show that mice mutant for cyclin Y and cyclin Y-like 1 (Ccny/l1), key regulators of WNT/STOP signalling, display reduced neurogenesis in the developing neocortex. Specifically, basal progenitors, which exhibit delayed cell cycle progression, were drastically decreased. Ccny/l1-deficient apical progenitors show reduced asymmetric division due to an increase in apical-basal astral microtubules. We identify the neurogenic transcription factors Sox4 and Sox11 as direct GSK3 targets that are stabilized by WNT/STOP signalling in basal progenitors during mitosis and that promote neuron generation. Our work reveals that WNT/STOP signalling drives cortical neurogenesis and identifies mitosis as a critical phase for neural progenitor fate.

Developmental programming and lineage branching of early human telencephalon.

The dorsal and ventral human telencephalons contain different neuronal subtypes, including glutamatergic, GABAergic, and cholinergic neurons, and how these neurons are generated during early development is not well understood. Using scRNA-seq and stringent validations, we reveal here a developmental roadmap for human telencephalic neurons. Both dorsal and ventral telencephalic radial glial cells (RGs) differentiate into neurons via dividing intermediate progenitor cells (IPCs_div) and early postmitotic neuroblasts (eNBs). The transcription factor ASCL1 plays a key role in promoting fate transition from RGs to IPCs_div in both regions. RGs from the regionalized neuroectoderm show heterogeneity, with restricted glutamatergic, GABAergic, and cholinergic differentiation potencies. During neurogenesis, IPCs_div gradually exit the cell cycle and branch into sister eNBs to generate distinct neuronal subtypes. Our findings highlight a general RGs-IPCs_div-eNBs developmental scheme for human telencephalic progenitors and support that the major neuronal fates of human telencephalon are predetermined during dorsoventral regionalization with neuronal diversity being further shaped during neurogenesis and neural circuit integration.

SOX4 induces cytoskeleton remodeling and promotes cell motility via N-wasp/ARP2/3 pathway in colorectal cancer cells.

Filopodia are thin, actin-rich projection from the plasma membrane that promote cancer cell invasion and migration. Sex-determining region Y-related high-mobility group-box 4 (SOX4) is a crucial transcription factor that plays a role in the development and metastasis of colorectal cancer (CRC). However, the involvement of SOX4 in cytoskeleton remodeling in CRC remains unknown. For the first time, we demonstrate that SOX4 is a potent regulator of filopodia formation in CRC cells. Overexpression of SOX4 protein enhances both migration and invasion ability of HCT116, and CACO2 cells, which is relevant to the metastasis. Furthermore, through phalloidin staining, cytoskeleton re-assembly was observed in SOX4-modified cell lines. Enhanced expression of SOX4 increased the number and length of filopodia on cell surface. In contrast, silencing SOX4 in SW620 cells with higher endogenous expression of SOX4, impeded the filopodia formation. Moreover, SOX4 was found to be positively regulating the expression of central regulators of actin cytoskeleton - N-Wiskott-Aldrich syndrome protein (N-WASP); WAVE2; Actin related proteins, ARP2 and ARP3. Inhibiting the N-WASP/ARP2/3 pathway diminishes the filopodia formation and the migration of CRC cells. These results indicate the crucial role of SOX4 in the regulation of filopodia formation mediated by N-WASP/ARP2/3 pathway in CRC cells.

Expression of SOX4 Significantly Predicts the Risk of Lymph Node Metastasis for Patients With Early-Stage Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma stands as a notably aggressive malignancy within the digestive system. In cases of early esophageal cancer without lymph node metastasis, endoscopic surgical resection offers a viable alternative, often resulting in improved patient quality of life. However, the paucity of methods to preoperatively ascertain lymph node involvement complicates surgical planning. SOX4 gene was previously found to be highly associated with invasive metastasis in our work through single-cell RNA sequencing on 5 paired tumor/peritumor tissues. This research included the collection of 124 tissue samples from 106 patients (106 tumor and 18 lymph node specimens). Samples were methodically arranged into a tissue microarray and treated with immunohistochemical staining. Statistical analysis was conducted to assess the relationship between them. In the univariate analysis, 3 factors were identified as statistically significant in relation to lymph node metastasis: T category (P = .014), vascular invasion (P < .001), and SOX4 intensity (P = .001). Additionally, when evaluating SOX4 intensity alongside other clinical indicators, SOX4 was shown to independently influence lymph node metastasis. Further, the multivariate analysis revealed that vascular invasion (P < .001) and SOX4 intensity (P = .003) were significantly associated with lymph node metastasis, exhibiting hazard ratios of 10.174 and 7.142, respectively. The results of our study indicate that both SOX4 expression and vascular invasion serve as predictors of lymph node metastasis in patients diagnosed with category T1 esophageal squamous cell carcinoma, underscoring the potential utility of SOX4 in prognostic evaluations.

SOX4 regulates proliferation and apoptosis of human ovarian granulosa-like tumor cell line KGN through the Hippo pathway.

The proliferation and apoptosis of ovarian granulosa cells are important for folliculogenesis. As a transcription factor, SRY-box transcription factor 4 (SOX4) has important roles in regulating cellular proliferation and apoptosis. Nonetheless, the regulatory mechanisms of SOX4 on proliferation and apoptosis of granulosa cells remain elusive. Therefore, a stably overexpressed SOX4 ovarian granulosa cell line KGN was generated by lentivirus encapsulation. We observed that overexpression of SOX4 inhibits apoptosis, promotes proliferation and migration of KGN cells. Comparative analysis of the transcriptome revealed 868 upregulated and 696 downregulated DEGs in LV-SOX4 in comparison with LV-CON KGN cell lines. Afterward, further assessments were performed to explore the possible functions about these DEGs. The data showed their involvement in many biological processes, particularly the Hippo signaling pathway. Moreover, the expression levels of YAP1, WWTR1, WTIP, DLG3, CCN2, and AMOT, which were associated with the Hippo signaling pathway, were further validated by qRT-PCR. In addition, the protein expression levels of YAP1 were markedly elevated, while p-YAP1 were notably reduced after overexpression of SOX4 in KGN cells. Thus, these results suggested that SOX4 regulates apoptosis, proliferation and migration of KGN cells, at least partly, through activation of the Hippo signaling pathway, which might be implicated in mammalian follicle development.

The SOX4/EZH2/SLC7A11 signaling axis mediates ferroptosis in calcium oxalate crystal deposition-induced kidney injury.

Epigenetic regulation is reported to play a significant role in the pathogenesis of various kidney diseases, including renal cell carcinoma, acute kidney injury, renal fibrosis, diabetic nephropathy, and lupus nephritis. However, the role of epigenetic regulation in calcium oxalate (CaOx) crystal deposition-induced kidney injury remains unclear. Our study demonstrated that the upregulation of enhancer of zeste homolog 2 (EZH2)-mediated ferroptosis facilitates CaOx-induced kidney injury. CaOx crystal deposition promoted ferroptosis in vivo and in vitro. Usage of liproxstatin-1 (Lip-1), a ferroptosis inhibitor, mitigated CaOx-induced kidney damage. Single-nucleus RNA-sequencing, RNA-sequencing, immunohistochemical and western blotting analyses revealed that EZH2 was upregulated in kidney stone patients, kidney stone mice, and oxalate-stimulated HK-2 cells. Experiments involving in vivo EZH2 knockout, in vitro EZH2 knockdown, and in vivo GSK-126 (an EZH2 inhibitor) treatment confirmed the protective effects of EZH2 inhibition on kidney injury and ferroptosis. Mechanistically, the results of RNA-sequencing and chromatin immunoprecipitation assays demonstrated that EZH2 regulates ferroptosis by suppressing solute carrier family 7, member 11 (SLC7A11) expression through trimethylation of histone H3 lysine 27 (H3K27me3) modification. Additionally, SOX4 regulated ferroptosis by directly modulating EZH2 expression. Thus, this study demonstrated that SOX4 facilitates ferroptosis in CaOx-induced kidney injury through EZH2/H3K27me3-mediated suppression of SLC7A11.

The transcription factor Sox4 is a downstream target of signaling by the cytokine TGF-ß and suppresses T(H)2 differentiation.

Sox4 is a transcription factor that regulates various developmental processes. Here we show that Sox4 was induced by TGF-ß and negatively regulated the transcription factor GATA-3, the master regulator of function of T helper type 2 (T(H)2) cells, by two distinct mechanisms. First, Sox4 bound directly to GATA-3, preventing its binding to GATA-3 consensus DNA sequences. Second, Sox4 bound to the promoter region of the gene encoding interleukin 5 (IL-5), a T(H)2 cytokine, and prevented binding of GATA-3 to this promoter. T(H)2 cell-driven airway inflammation was modulated by alterations in Sox4 expression. Thus, Sox4 acted as a downstream target of TGF-ß to inhibit GATA-3 function, T(H)2 differentiation and T(H)2 cell-mediated inflammation.

Biological functions of PCAT-1/ SOX4 in cell proliferation and invasion of breast cancer.

Breast cancer (BC) is one of the most common fatal cancers. Recent studies have identified the vital role of long noncoding RNA (lncRNAs) in the development and progression of BC. In this research, lncRNA PCAT-1 was studied to identify how it functioned in the metastasis of BC. PCAT-1 expression of tissues was detected by real-time quantitative polymerase chain reaction (RT-qPCR) in 50 BC patients. Cell proliferation, wound healing assay and transwell assay were used to observe the biological behavior changes of BC cells through knockdown or overexpression of PCAT-1. In addition, RT-qPCR and Western blot assay were performed to discover the potential target protein of PCAT-1 in BC. PCAT-1 expression level in BC samples was higher than that of adjacent ones. Besides, cell proliferation, migrated ability and cell invaded ability of BC cells were inhibited after PCAT-1 was silenced. Cell proliferation, migration and invasion of BC cells were promoted after PCAT-1 was overexpressed. In addition, SOX4 was downregulated after silence of PCAT-1 in BC cells, while SOX4 was upregulated after overexpression of PCAT-1 in BC cells. Furthermore, SOX4 was upregulated in BC tissues and was positively associated with PCAT-1. Our study uncovers a new oncogene in BC and suggests that PCAT-1 could enhance BC cell proliferation, migration and invasion via targeting SOX4, which provided a novel therapeutic target for BC patients.

circ_JMJD1C expedites breast cancer progression by regulating miR-182-5p/JMJD1C/SOX4 axis.

Circular RNAs (circRNAs) are engaged in various types of cancers. This study aimed to investigate the roles of circ_0006743 (circ_JMJD1C) in breast cancer. The downstream of circ_JMJD1C and their interaction network was determined by bioinformatic analyses. Gene expression were analyzed through western blot and qRT-PCR assays. Functional assays were conducted in vitro and in vivo to verify circ_JMJD1C role in BC. FISH and confocal analysis indicated the cellular distribution of circ_JMJD1C. Luciferase reporter, RNA immune-precipitation (RIP) assays, as well as Pearson's correlation analysis, were implemented to test the relation of miR-182-5p, JMJD1C and circ_JMJD1C. Circ_JMJD1C and JMJD1C expression were both elevated, and their expression was positively correlated in BC. Circ_ JMJD1C knockdown hindered BC cell proliferation, invasion, and migration, along with epithelial-mesenchymal transition (EMT) in vitro and in vivo. Circ_JMJD1C facilitated BC progression by the miR-182-5p-JMJD1C axis. Circ_JMJD1C epigenetically upregulated SOX4. Circ_JMJD1C promotes the aggressiveness of BC via regulating miR-182-5p/JMJD1C/SOX4 axis. This may provide a novel and promising therapy targeting BC.

SOX4 silencing alleviates renal injury in rats with acute renal failure by inhibiting the NF-κB signaling pathway and reducing apoptosis and oxidative stress.

Acute renal failure (ARF) is a huge threat to the lives of most patients in intensive care units, and there is currently no satisfactory treatment strategy. SRY-box transcription factor 4 (SOX4) plays a key role in the development of various diseases, but its effect on ARF is unknown. Therefore, this study aimed to explore the relationship between SOX4 and ARF. Blood samples were collected from 20 ARF patients and 20 healthy volunteers. We also established an ARF rat model by excising the right kidney and ligating the left renal artery, and SOX4 knockdown in ARF rats was achieved down by means of lentiviral infection. Subsequently, we used quantitative polymerase chain reaction and western bolt assays to detect the expression levels of SOX4 and nuclear factor-κB (NF-κB) signaling pathway-related proteins in human blood or rat renal tissue and hematoxylin and eosin and terminal deoxynucleotidyl transferase (TdT) 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labeling staining to observe the pathological changes and apoptosis of renal tissue. Enzyme-linked immunosorbent assay and biochemical kits were used to measure the levels of renal function-related indicators (blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin) and inflammatory factors (interleukin [IL]-1ß, IL-6, and tumor necrosis factor-alpha), as well as changes in oxidative stress-related indicators (malondialdehyde [MDA], superoxide dismutase [SOD], and reactive oxygen species [ROS]) in rat serum. SOX4 expression levels in blood samples from ARF patients and renal tissue from ARF rats were significantly higher compared with those in healthy volunteers and control rats, respectively. ARF model rats displayed the typical ARF phenotype, while SOX4 silencing significantly improved pathological injury and apoptosis of renal tissue in ARF rats. Moreover, SOX4 silencing significantly inhibited increased levels of renal function-related indicators and inflammatory factors and reduced the level of excessive oxidative stress (MDA and ROS were upregulated, and SOD was downregulated) in ARF rats. SOX4 also reduced the activity of the NF-κB signaling pathway in ARF samples. Thus, SOX4 knockdown may reduce oxidative stress, the inflammatory response, and apoptosis by reducing the activity of the NF-κB signaling pathway, thereby improving renal injury in ARF rats.

SOX4/HDAC2 Axis Enhances Cell Survivability and Reduces Apoptosis by Activating AKT/MAPK Signaling in Colorectal Cancer.

BACKGROUND: Increased SOX4 (SRY-related HMG-box) activity aids cellular transformation and metastasis. However, its specific functions and downstream targets remain to be completely elusive in colorectal cancer (CRC). AIMS: To investigate the role of SOX4 in CRC progression and the underlying mechanism. METHODS: In the current study, online available datasets of CRC patients were explored to check the expression status of SOX4. To investigate the further functions, SOX4 was overexpressed and knocked down in CRC cells. Colony formation assay, flowcytometry analysis, and MTT assay were used to check for proliferation and apoptosis. Acridine orange staining was done to check the role of SOX4 in autophagy induction. Furthermore, western blot, qRT-PCR, and bioinformatic analysis was done to elucidate the downstream molecular mechanism of SOX4. RESULTS: GEPIA database showed enhanced expression of SOX4 mRNA in CRC tumor, and the human protein atlas (HPA) showed strong staining of SOX4 protein in tumor when compared to the normal tissue. Ectopic expression of SOX4 enhanced colony formation ability as well as rescued cells from apoptosis. SOX4 overexpressed cells showed the formation of acidic vesicular organelles (AVOs) which indicated autophagy. Further results revealed the activation of p-AKT/MAPK molecules upon overexpression of SOX4. SOX4 expression was found to be positively correlated with histone deacetylase 2 (HDAC2). Knockdown of SOX4 or HDAC2 inhibition induced apoptosis, revealed by decrease in BCL2 and increase in BAX expression, and inactivated the p-AKT/MAPK signaling. CONCLUSION: The study uncovers that SOX4/HDAC2 axis improves cell survivability and reduces apoptosis via activation of the p-AKT/MAPK pathway.

The efficient induction of human retinal ganglion-like cells provides a platform for studying optic neuropathies.

Retinal ganglion cells (RGCs) are essential for vision perception. In glaucoma and other optic neuropathies, RGCs and their optic axons undergo degenerative change and cell death; this can result in irreversible vision loss. Here we developed a rapid protocol for directly inducing RGC differentiation from human induced pluripotent stem cells (hiPSCs) by the overexpression of ATOH7, BRN3B, and SOX4. The hiPSC-derived RGC-like cells (iRGCs) show robust expression of various RGC-specific markers by whole transcriptome profiling. A functional assessment was also carried out and this demonstrated that these iRGCs display stimulus-induced neuronal activity, as well as spontaneous neuronal activity. Ethambutol (EMB), an effective first-line anti-tuberculosis agent, is known to cause serious visual impairment and irreversible vision loss due to the RGC degeneration in a significant number of treated patients. Using our iRGCs, EMB was found to induce significant dose-dependent and time-dependent increases in cell death and neurite degeneration. Western blot analysis revealed that the expression levels of p62 and LC3-II were upregulated, and further investigations revealed that EMB caused a blockade of lysosome-autophagosome fusion; this indicates that impairment of autophagic flux is one of the adverse effects of that EMB has on iRGCs. In addition, EMB was found to elevate intracellular reactive oxygen species (ROS) levels increasing apoptotic cell death. This could be partially rescued by the co-treatment with the ROS scavenger NAC. Taken together, our findings suggest that this iRGC model, which achieves both high yield and high purity, is suitable for investigating optic neuropathies, as well as being useful when searching for potential drugs for therapeutic treatment and/or disease prevention.

PLAC8 contributes to the malignant behaviors of cervical cancer cells by activating the SOX4-mediated AKT pathway.

Cervical cancer (CC) is the primary cancer-related cause of morbidity and mortality in women. Previous studies have shown that placenta-specific 8 (PLAC8) has different functions in multiple malignancies. This study aimed to explore the function and regulatory mechanism of PLAC8 in CC. Bioinformatics and immunohistochemical analyses demonstrated that PLAC8 was significantly upregulated in CC tissues compared with normal tissues. Gain/loss-of-function experiments showed that siRNA-mediated knockdown of PLAC8 suppressed cell migration and invasion, while PLAC8 overexpression promoted cell motility. Moreover, PLAC8 was revealed to affect the epithelial-mesenchymal transition (EMT) process by upregulating epithelial (E)-cadherin and decreasing the expression of mesenchymal markers of EMT, including vimentin, zinc finger E-box binding homeobox 1 (ZEB1), neural (N)-cadherin, matrix metalloproteinase-9 (MMP-9), and MMP-2 in PLAC8-silenced cells. PLAC8 activated the AKT pathway, as proven by the downregulation of p-AKTSer473 and p-AKTThr308 expression after PLAC8 knockdown. Furthermore, PLAC8 overexpression upregulated the expression of sex-determining region Y-related high-mobility group box transcription factor 4 (SOX4), which is reported to mediate the activation of the AKT pathway, and SOX4 deficiency reversed the cellular functions caused by PLAC8 overexpression. Overall, the present study indicates that PLAC8 may facilitate CC development by activating the SOX4-mediated AKT pathway, suggesting that PLAC8 may serve as a potential biomarker for CC treatment.

TGF-ß3 in differentiation and function of Tph-like cells and its relevance to disease activity in patients with systemic lupus erythematosus.

OBJECTIVES: T peripheral helper (Tph) cells have major roles in pathological processes in SLE. We sought to clarify the mechanisms of Tph cell differentiation and their relevance to clinical features in patients with SLE. METHOD: Phenotypes and functions of Tph cell-related markers in human CD4+ T cells purified from volunteers or patients were analysed using flow cytometry and quantitative PCR. Renal biopsy specimens from patients with LN were probed by multicolour immunofluorescence staining. RESULTS: Among multiple cytokines, transforming growth factor (TGF)-ß3 characteristically induced programmed cell death protein 1 (PD-1)hi musculoaponeurotic fibrosarcoma (MAF)+, IL-21+IL-10+ Tph-like cells with a marked upregulation of related genes including PDCD-1, MAF, SOX4 and CXCL13. The induction of Tph-like cells by TGF-ß3 was suppressed by the neutralization of TGF-ß type II receptor (TGF-ßR2). TGF-ß3-induced Tph-like cells efficiently promoted the differentiation of class-switch memory B cells into plasmocytes, resulting in enhanced antibody production. The proportion of Tph cells in the peripheral blood was significantly increased in patients with SLE than in healthy volunteers in concordance with disease activity and severity of organ manifestations such as LN. TGF-ß3 was strongly expressed on macrophages, which was associated with the accumulation of CD4+ C-X-C chemokine receptor (CXCR5)-PD-1+ Tph cells, in the renal tissue of patients with active LN. CONCLUSION: The induction of Tph-like cells by TGF-ß3 mainly produced from tissue macrophages plays a pivotal role in the pathological processes of active LN by enhancing B-cell differentiation in patients with SLE.