BACKGROUND: The roles of the transcriptional factor SIX2 have been identified in several tumors. However, its roles in gastric cancer (GC) progression have not yet been revealed. Our objective is to explore the impact and underlying mechanisms of SIX2 on the stemness of GC cells.
METHODS: Lentivirus infection was employed to establish stable expression SIX2 or PFN2 in GC cells. Gain- and loss-of-function experiments were conducted to detect changes of stemness markers, flow cytometry profiles, tumor spheroid formation, and tumor-initiating ability. ChIP, RNA-sequencing, tissue microarray, and bioinformatics analysis were performed to reveal the correlation between SIX2 and PFN2. The mechanisms underlying the SIX2/PFN2 loop-mediated effects were elucidated through tissue microarray analysis, RNA stability assay, IP-MS, Co-Immunoprecipitation, and inhibition of the JNK signaling pathway.
RESULTS: The stemness of GC cells was enhanced by SIX2. Mechanistically, SIX2 directly bound to PFN2\'s promoter and promoted PFN2 activity. PFN2, in turn, promoted the mRNA stability of SIX2 by recruiting RNA binding protein YBX-1, subsequently activating the downstream MAPK/JNK pathway.
CONCLUSIONS: This study unveils the roles of SIX2 in governing GC cell stemness, defining a novel SIX2/PFN2 regulatory loop responsible for this regulation. This suggests the potential of targeting the SIX2/PFN2 loop for GC treatment (Graphical Abstracts).

Parkinson\'s disease (PD) is a severe neurodegenerative disease associated with the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Although its pathogenesis remains unclear, microglia-mediated neuroinflammation significantly contributes to the development of PD. Here we showed that the sine oculis homeobox (SIX) homologue family transcription factors SIX2 exerted significant effects on neuroinflammation. The SIX2 protein, which is silenced during development, was reactivated in lipopolysaccharide (LPS)-treated microglia. The reactivated SIX2 in microglia mitigated the LPS induced inflammatory effects, and then reduced the toxic effect of conditioned media (CM) of microglia on co-cultured MES23.5 DA cells. Using the LPS-stimulated Cx3cr1-CreERT2 mouse model, we also demonstrated that the highly-expressed SIX2 in microglia obviously attenuated neuroinflammation and protected the DA neurons in SN. Further RNA-Seq analysis on the inflammatory activated microglia revealed that the SIX2 exerted these effects via up-regulating the FXYD domain containing ion transport regulator 2 (FXYD2). Taken together, our study demonstrated that SIX2 was an endogenous anti-inflammatory factor in microglia, and it exerted anti-neuroinflammatory effects by regulating the expression of FXYD2, which provides new ideas for anti-neuroinflammation in PD.

The critical roles of NF-κB Inducing Kinase (NIK) in tumor progression have been elucidated in various tumors; however, its effects on hepatocellular carcinoma (HCC) progression are still confusing. Here, we found that NIK level was upregulated in HCC tissues compared to that of normal tissues, and positively correlated with the levels of cancer stem cell (CSC) markers. Then we established HCC cells with NIK-stable knockdown and found that NIK knockdown suppressed the CSC-like traits of HCC cells through in vivo and in vitro experiments. Mechanistically, we revealed that SIX2 protein level, but not its mRNA level, was significantly reduced in HCC cells with NIK knockdown, which was rescued by MG132 treatment. Furthermore, NIK knockdown promoted the ubiquitination level of SIX2 and decreased its protein stability. Moreover, Six2 overexpression partially reversed the inhibition of NIK knockdown on the CSC-like traits of HCC cells. This study identified a novel NIK/SIX2 axis conferring HCC stemness.

Glial cell line-derived neurotrophic factor (GDNF) played critical roles in the survival and repair of dopaminergic (DA) neurons. Transcription factor Six2 could repair injured DA cells by promoting the expression of GDNF, however, the underlying molecular mechanisms remain largely unknown. In this study, we screened forty-three proteins that interacted with Six2 in MES23.5 DA cells treated with 6-OHDA by liquid chromatography - electrospray - ionization tandem mass spectrometry (LC-ESI-ITMS/MS). Among these proteins, Smarcd1 is a member of SWI/SNF chromatin-remodeling complex family. Our results confirmed that Smarcd1 formed a transcription complex with Six2, and Smarcd1 mainly binded to the 2840 bp-2933 bp region of the GDNF promoter. Furthermore, knockdown of Smarcd1 inhibited the effect of Six2 on GDNF expression, and resulted in decreased cell viability and increased the apoptosis of injured DA neurons, and the result of overexpression of Smarcd1 is opposite to knockdown. Taken together, our results indicate that smarcd1 can be recruited to the promoter region of GDNF by transcription factor Six2 to promote the effect of Six2 on GDNF expression and protect injured MES23.5 DA cells, which could be useful in identifying potential drug targets for promoting endogenous GDNF expression.

Breast cancer (BC) is a common cancer in women worldwide. Emerging evidence has indicated that circular RNA hsa-circ_0007255 (circ_0007255) is a prognostic mediator in BC progression. However, the functional role of circ_0007255 needs to be determined.
The expression of circ_0007255, microRNA (miR)-335-5p, and SIX Homeobox 2 (SIX2) was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assay. Actinomycin D and RNase R treatment was performed to analyze the stability of circ_0007255. Additionally, Seahorse extracellular flux, colony formation and transwell analyses were carried out to detect oxygen consumption ratio (OCR), colony formation and cell mobility, respectively. The interaction between miR-335-5p and circ_0007255 or SIX2 was confirmed via dual-luciferase reporter assay. A xenograft tumor model was established to explore the role of circ_0007255 in vivo.
Circ_0007255 and SIX2 were overexpressed, but miR-335-5p was diminished in BC tissues and cells. Circ_0007255 absence inhibited oxygen consumption, colony formation, cell migration and invasion, and these effects were particularly abrogated via miR-335-5p upregulation in BC cells. Moreover, SIX2 deficiency eliminated the promotion effects of miR-335-5p inhibitor on oxygen consumption, colony formation, and cell mobility in BC cells. Importantly, circ_0007255 inhibited tumor growth in vivo. Mechanically, circ_0007255 was a sponge of miR-335-5p to regulate SIX2 expression in BC progression.
Circ_0007255 functioned as a novel oncogene in the progression of BC by regulating miR-335-5p/SIX2 axis, and might be a promising biomarker for BC treatment.
Significant findings of the study: Levels of circ_0007255 and SIX2 were upregulated, but miR-335-5p was diminished in BC tissues and cells. Circ_0007255 was an oncogene in BC development and exerted its function via miR-335-5p/SIX2 axis in BC. Tumor growth was reduced by circ_0007255 absence.
Circ_0007255 functioned as a novel oncogene in the progression of BC by regulating miR-335-5p/SIX2 axis, and might be a promising biomarker for BC treatment.

Injured neurons can initiate their own neurotoxin-induced repair mechanisms by expressing protective genes and activating specific intracellular signal transduction pathways. Although glial cell-derived neurotrophic factor (GDNF) plays a key role in the repair of dopaminergic (DA) neurons, whether there is high expression of GDNF in DA neurons at an early stage of injury has not yet been reported. In this study, neurotoxin-induced GDNF overexpression was detected for the first time in MES23.5 DA immortalized neuroblastoma (MES23.5 DA) cells soon after 6-hydroxydopamine (6-OHDA) treatment. We also observed that the phosphorylation of Akt1, a member of the protein kinase B family, was increased. Further studies showed that activated Akt1 increased the phosphorylation of the protein phosphatase Eya1, which is a member of the eyes absent (Eya) family of transcriptional cofactors. Then, activated Eya1 decreased the phosphorylation of the sine oculis-related homeobox 2 (Six2) transcription factor. In addition, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction (ChIP-qPCR) revealed that Six2 promoted GDNF transcription in MES23.5 DA cells by directly binding to the GDNF promoter. Finally, we showed that inhibiting neurotoxin-induced GDNF overexpression increased MES23.5 DA cell death, while promoting GDNF expression via Six2 overexpression decreased DA neuronal death. These results suggest that MES23.5 DA cells with early 6-OHDA-induced injury can promote the overexpression of GDNF by activating the Akt1/Eya1/Six2 signaling pathway, and this overexpression of GDNF has protective effects on injured MES23.5 DA cells. Hence, this study highlights a new target for drug development for the treatment of Parkinson\'s disease.

BACKGROUND: Increasing evidence indicates that Six2 contributes to tumorigenesis in various tumor including hepatocellular carcinoma (HCC). This study aimed to determine the role of Six2 in HCC and to elucidate the association of Six2 with clinical pathological characteristics.
METHODS: The expressions of Six2 in HCC tumor, para-tumor tissue and portal vein tumor thrombus (PVTT) were detected by tissue microarray technique, immunohistochemistry, real-time RT-PCR and Western blotting. Chi-square and Kaplan-Meier analysis were used to analyze the correlation between Six2 expression and prognosis of HCC patients. Lentivirus mediated Six2 knockdown, spheroid formation assay, proliferation assay and subcutaneous tumor implantation were performed to determine the function of Six2.
RESULTS: In 274 HCC samples, Six2 was strongly expressed. Kaplan-Meier analysis revealed that high expression of Six2 was correlated with a shorter overall survival (OS) and disease-free survival (DFS). Moreover, Six2 expression was associated with sex, alpha-fetoprotein, tumor size and portal vein invasion. Six2 was highly expressed in PVTT. Six2 knockdown inhibited HCC cell lines proliferation, migration, and self-renewal in vitro and in vivo. In addition, low-expression of Six2 weakened TGF-β induced Smad4 activation and epithelial-mesenchymal transition in HCC cell lines.
CONCLUSIONS: Elevated Six2 expression in HCC tumor patients was associated with negative prognosis. Upregulated Six2 promoted tumor growth and facilitated HCC metastasis via TGF-β/Smad signal pathway.

Renal cell carcinoma (RCC) accounts for 2-3% of adult malignant tumors, and the incidence of RCC worldwide has increased by about 2% over the past two decades. The homeobox protein Six2 has been shown to promote the stemness of breast cancer cells and play a role in kidney development, but its involvement in RCC progression has not previously been investigated. Here, we found that six2 expression was significantly increased in RCC tissues and negatively correlated with the overall survival of patients with RCC. In addition, six2 expression exhibited a remarkably higher level relative to that in normal renal cells. Functional experiments showed that six2 knockdown attenuated the stemness of RCC cells, which was evident by decreased spheroid formation ability and stemness marker (sox2 and nanog) expression. Mechanistic studies indicated that Six2 directly bound to the enhancer of sox2, promoting sox2 expression and downstream effector expression of nanog. Furthermore, overexpression of sox2 rescued the inhibitory effects of six2 on the stemness of RCC cells. Notably, six2 expression is positively correlated with sox2 and nanog expression in RCC tissues. Collectively, our results point toward a six2/sox2 axis responsible for RCC cell stemness.

OBJECTIVE: The roles and related mechanisms of six2 in regulating non-small cell lung cancer (NSCLC) cells progression are unclear. This work aimed to explore the roles of six2 in NSCLC cell stemness.
METHODS: Kaplan-Meier plotter analysis was used to examine the correlation between six2 expression and the survival of NSCLC patients. Quantitative reverse transcription PCR and Western blot were performed to detect six2 expression in clinical samples. Moreover, transwell migration, tumour spheroid formation and in vivo tumour formation assays were used to examine the effects of six2 on NSCLC cell progression. Additionally, methylation analysis was carried out to measure E-cadherin methylation level in different cells. Finally, cell viability assay was performed to explore the effects of six2 on chemotherapeutic sensitivity of NSCLC cells.
RESULTS: Lung cancer patients with a higher six2 expression level displayed a shorter overall survival. Six2 expression was higher in lung cancer tissues than in normal adjacent tissues. Additionally, six2 knockdown suppressed NSCLC cell stemness. Mechanistically, six2 overexpression inhibited epithelial marker E-cadherin expression via stimulating its promoter methylation. And E-cadherin knockdown rescued six2 knockdown-induced decrease of NSCLC cancer cell stemness. Notably, six2 knockdown enhanced cisplatin sensitivity in parental NSCLC cells and attenuated cisplatin resistance in cisplatin-resistant NSCLC cells.
CONCLUSIONS: Our results suggest that six2 facilitates NSCLC cell stemness and attenuates chemotherapeutic sensitivity via suppressing E-cadherin expression.

The expression of CYP4Z1 and the pseudogene CYP4Z2P has been shown to be specifically increased in breast cancer by our group and others. Additionally, we previously revealed the roles of the competitive endogenous RNA (ceRNA) network mediated by these genes (ceRNET_CC) in breast cancer angiogenesis, apoptosis, and tamoxifen resistance. However, the roles of ceRNET_CC in regulating the stemness of breast cancer cells and the mechanisms through which ceRNET_CC is regulated remain unclear.
Transcriptional factor six2, CYP4Z1-3\'UTR, and CYP4Z2P-3\'UTR were stably overexpressed or knocked down in breast cancer cells via lentivirus infection. ChIP-sequencing and RNA-sequencing analysis were performed to reveal the mechanism through which ceRNET_CC is regulated and the transcriptome change mediated by ceRNET_CC. Clinical samples were used to validate the correlation between six2 and ceRNET_CC. Finally, the effects of the six2/ceRNET_CC axis on the stemness of breast cancer cells and chemotherapy sensitivity were evaluated by in vitro and in vivo experiments.
We revealed that ceRNET_CC promoted the stemness of breast cancer cells. Mechanistically, six2 activated ceRNET_CC by directly binding to their promoters, thus activating the downstream PI3K/Akt and ERK1/2 pathways. Finally, we demonstrated that the six2/ceRNET_CC axis was involved in chemoresistance.
Our results uncover the mechanism through which ceRNET_CC is regulated, identify novel roles for the six2/ceRNET_CC axis in regulating the stemness of breast cancer cells, and propose the possibility of targeting the six2/ceRNET_CC axis to inhibit breast cancer stem cell (CSC) traits.