RNF6 promotes gastric cancer progression by regulating CCNA1/CREBBP transcription.

Ring finger protein 6 (RNF6) is a member of the E3 ubiquitin ligase family. Previous studies have reported the involvement of RNF6 as a ubiquitin ligase in the progression of gastric cancer (GC). However, this study found that RNF6 has a clear localization in the nucleus of GC, indicating a role other than ubiquitin ligase. Further chromatin immunoprecipitation sequencing (ChIP-seq) analysis revealed that RNF6 has DNA binding and transcriptional regulatory effects and is involved in important pathways such as tumor cell cycle and apoptosis. Cyclin A1 (CCNA1) and CREB binding protein (CREBBP) are downstream targets for RNF6 transcription regulation in GC. RNF6 binds to the promoter region of CCNA1/CREBBP and is actively regulating their expression in GC cells. Silencing CCNA1/CREBBP partially reversed the promoting effect of RNF6 overexpression on the biological function of GC cells. Our study suggests that RNF6 promotes the progression of GC by regulating CCNA1/CREBBP transcription.

RNF6 activates TGF-ß1/c-Myb pathway to promote EMT in esophageal squamous cell carcinoma.

Objective: This study aimed to investigate RING-Finger Protein 6 (RNF6) expression in esophageal squamous cell carcinoma (ESCC) cells and whether it affects cell proliferation, invasion, and migration by regulating the TGF-ß1/c-Myb pathway. Methods: TCGA database was used to analyze RNF6 expression in normal tissues and esophageal cancer tissues. Kaplan-Meier method was used to examine the correlation between RNF6 expression and patient prognosis. SiRNA interference vector and RNF6 overexpression plasmid were constructed, and RNF6 was transfected into Eca-109 and KYSE-150 esophageal cancer cell line. In vitro scratch assay and Transwell assay were conducted to investigate the effects of RNF6 on the migration and invasion of Eca-109 and KYSE-150 cells. RT-PCR detected the expression of Snail, E-cadherin, and N-cadherin, and TUNEL detected the apoptosis of cells. Results: RNF6 up-regulation promoted the progression of esophageal cancer and predicted poor prognosis. RNF6 also enhanced the migration and invasion of ESCC cells in vitro. RNF6 silencing inhibited the migration and invasion of ESCC cells. TGF-ß inhibitors reversed the oncogenic effects of RNF6. RNF6 regulated the migration and invasion of ESCC cells by activating the TGF-ß pathway. RNF6/TGF-ß1 promoted esophageal cancer progression through c-Myb. Conclusion: RNF6 promotes the proliferation, invasion, and migration of ESCC cells possibly by activating the TGF-ß1/c-Myb pathway and affects the progression of ESCC.

Induction of zinc finger protein RNF6 auto-ubiquitination for the treatment of myeloma and chronic myeloid leukemia.

The zinc finger ubiquitin ligase RNF6 has been proposed as a potential therapeutic target in several cancers, but understanding its molecular mechanism of degradation has been elusive. In the present study, we find that RNF6 is degraded via auto-ubiquitination in a manner dependent on its Really Interesting New Gene (RING) domain. We determine that when the RING domain is deleted (ΔRING) or the core cysteine residues in the zinc finger are mutated (C632S/C635S), the WT protein, but not the ΔRING or mutant RNF6 protein, undergoes polyubiquitination. We also identify USP7 as a deubiquitinase of RNF6 by tandem mass spectrometry. We show that USP7 interacts with RNF6 and abolishes its K48-linked polyubiquitination, thereby preventing its degradation. In contrast, we found a USP7-specific inhibitor promotes RNF6 polyubiquitination, degradation, and cell death. Furthermore, we demonstrate the anti-leukemic drug Nilotinib and anti-myeloma drug Panobinostat (LBH589) induce RNF6 K48-linked polyubiquitination and degradation in both multiple myeloma (MM) and leukemia cells. In agreement with our hypothesis on the mode of RNF6 degradation, we show these drugs promote RNF6 auto-ubiquitination in an in vitro ubiquitination system without other E3 ligases. Consistently, reexpression of RNF6 ablates drug-induced MM and leukemia cell apoptosis. Therefore, our results reveal that RNF6 is a RING E3 ligase that undergoes auto-ubiquitination, which could be abolished by USP7 and induced by anti-cancer drugs. We propose that chemical induction of RNF6 auto-ubiquitination and degradation could be a novel strategy for the treatment of hematological malignancies including MM and leukemia.

E3 ubiquitin ligase RNF6 promotes antiviral immune responses through enhancing the expression of interferon stimulated genes in myeloid cells.

Interferon signaling is closely associated with clearance of viral infections as well as the development of systemic lupus erythematosus (SLE). Therefore, from a clinical perspective, it is important to identify the key regulators involved in interferon signaling pathways. In this study, we identified that RNF6, as an interferon inducible E3 ubiquitin ligase, promoted the interferon-dependent antiviral response. Knock-down of RNF6 greatly attenuated expression of ISGs and the transcriptional activity of ISRE. Specifically, increased RNF6 expression in myeloid cells of patients with SLE correlated with high expression of ISGs. Our results uncover RNF6 as a positive mediator in the antiviral immune responses and suggest that RNF6 may contribute to predict interferon signaling in SLE.

Glaucocalyxin A Inhibits the Malignancies of Gastric Cancer Cells by Downregulating MDM2 and RNF6 via MiR-3658 and the SMG1-UPF mRNA Decay Pathway.

Gastric cancer (GC) ranks as the most common gastrointestinal cancer and is among the leading causes of cancer death worldwide. Glaucocalyxin A (GLA), an entkauranoid diterpene isolated from Rab-dosia japonica var., possesses various bioactivities. To date, the data on the effect of GLA on GC are still minimal, and the molecular mechanisms remain largely unknown. Herein, we found that GLA could significantly inhibit the proliferation, cell adhesion, and invasion of HGT-1, SNU-1, SNU-6, and NCI-N87 GC cells in a dose-dependent manner. GLA enhanced the apoptosis of the GC cells as evidenced by the increased caspase-3 activity and the elevated levels of cleaved caspase-3 and cleaved PARP in GC cells in the presence of GLA. We then showed that the downregulation of Murine Double Minute Clone 2 (MDM2) and Ring Finger Protein 6 (RNF6) by GLA was implicated in the GLA-induced inhibition of the GC cells. Furthermore, MDM2 and RNF6 were identified as the targets of miR-3658 that was downregulated in the GC cells and upregulated by GLA. Moreover, it was shown that miR-3658 was hypermethylated in the GC cells, and GLA could rescue the expression of miR-3658 via demethylation by abrogating EZH2-mediated epigenetic silencing. In addition to the miR-3658-MDM2/RNF6 regulatory axis, activation of the SMG1-UPF mRNA decay pathway contributed to the downregulation of MDM2 and RNF6 by GLA in the GC cells. The inhibitory effect of GLA on gastric cancer and the expression of MDM2 and RNF6 was also validated in in vivo study. Our findings suggest that has the therapeutic potential for GC by downregulating oncogenes via posttranscriptional regulation.

Regulation of p27 (Kip1) by Ubiquitin E3 Ligase RNF6.

The cyclin-dependent kinase inhibitor p27 (Kip1) is an important regulator of the G1/S checkpoint. It is degraded by the SCF-SKP2 complex in late G1 thereby allowing cells to progress to the S phase. Here we investigated the role of the E3 ubiquitin ligase RNF6 (Ring Finger Protein 6) in cell cycle progression in prostate cancer cells. Our data demonstrate that RNF6 can promote cell cycle progression by reducing the levels of p27. Knockdown of RNF6 led to an increase in the stability of p27 and to the arrest of cells in the G1 phase. RNF6 interacted with p27 via its KIL domain and this interaction was found to be phosphorylation independent. RNF6 enhanced ubiquitination and subsequent degradation of p27 in the early G0/G1 phase of the cell cycle. Knockdown of RNF6 expression by short hairpin RNA led to inhibition of the CDK2/Cyclin E complex thereby reducing phosphorylation of Retinoblastoma protein (Rb) and to a subsequent decrease in cell cycle progression and proliferation. Our data suggest that RNF6 acts as a negative regulator for p27kip1 leading to its proteasome-dependent degradation in the early G0/G1 phase of the cell cycle.

RNF6 Targeted by miR-26a-5p Protects Pancreatic ß-Cell Function Against Type 2 Diabetes.

BACKGROUND: Type 2 diabetes (T2D) is characterized by progressive ß-cell dysfunction. Regulatory microRNAs (miRNAs) may be associated with this. METHODS: Serum miR-26a-5p and RNF6 levels were detected in T2D patients and healthy volunteers via qRT-PCR. Subsequently, the role of specific dysregulated miR-26a-5p or RNF6 in regulating insulin content, cell proliferation, and apoptosis was studied in INS-1 cells. The targeting correlation between miR-26a-5p and RNF6 was detected using a luciferase assay. RESULTS: RNF6 expression was significantly decreased in T2D individuals and INS-1 cells treated with high glucose, while miR-26a-5p expression was increased. In INS-1 cells, RNF6 overexpression or miR-26a-5p downregulation significantly increased insulin content and secretion, induced proliferation, and inhibited apoptosis. RNF6 has been identified as an miR-26a-5p target, which negatively regulates RNF6 to worsen INS-1 cell function. CONCLUSION: RNF6 promoted insulin secretion and induced cell proliferation in INS-1 cells. This may be related to miR-26a-5p targeting and negatively regulating T2D pathogenesis.

RNF6 promotes colorectal cancer invasion and migration via the Wnt/ß-catenin pathway by inhibiting GSK3ß activity.

BACKGROUND: The purpose of this study was to explore the molecular mechanism underlying the interaction between ring finger protein 6 (RNF6) and glycogen synthase kinase 3ß (GSK3ß) in colorectal cancer (CRC). METHODS: In this study, cell models of overexpressed or silenced RNF6 were established by liposome transfection, and IM-12 was used as the inhibitor of GSK3ß. Real-time quantitative PCR and western blots were used to detect the expression of RNF6, p-GSK3ß, GSK3ß, and ß-catenin, and MTT assays were used to quantify cell proliferation. The tumorigenicity of cells was observed by plate clonal formation assay; the invasiveness of cells was examined in Transwell Boyden chambers, and the migratory capacity of cells was tested by scratch wound assays. The rat CRC model was induced by AOM/DSS, in which we verified activity in the Wnt/ß-catenin pathway by examining GSK3ß phosphorylation. RESULTS: RNF6 was upregulated in CRC samples and cell lines. Silencing or overexpressing RNF6 in colorectal cancer cells inhibited or promoted, respectively, the proliferation, tumorigenicity, invasion and migration of CRC cells, as well as expression of p-GSK3ß, GSK3ß and ß-catenin. IM-12 reversed the Wnt/ß-catenin-activated state change induced by RNF6 silencing and the inhibition of cell proliferation, tumorigenicity, invasion and migration. The same results were observed in vivo in the rat CRC model. CONCLUSIONS: Overexpression of RNF6 in CRC increased the GSK3ß phosphorylation level, which led to activation of the Wnt/ß-catenin pathway and promoted the invasion and migration of CRC cells, suggesting that RNF6 may be a novel target for the treatment of CRC.

RING-finger protein 6 enhances c-Myc-mediated Warburg effect by promoting MAD1 degradation to facilitate pancreatic cancer metastasis.

Aerobic glycolysis (the Warburg effect) promotes tumor metastasis; hence, drugs targeting its regulators are being developed. c-Myc, a critical transcription factor that regulates the Warburg effect, is involved in the tumorigenesis of many cancers, including pancreatic cancer (PC). However, the upstream regulating mechanisms of c-Myc in PC are unclear. Herein, we reported that E3 ubiquitin ligase RING-finger protein 6 (RNF6) was upregulated in PC tissues, and an elevated RNF6 level was closely associated with metastasis and poor prognosis in patients with PC. In functional experiments, RNF6 over-expression accelerated the metastatic ability of PC cells, whereas RNF6 knockdown impaired PC cell motility and invasiveness along with metastasis in an orthotopic mouse model. Furthermore, we found that RNF6 promoted PC cell metastasis by enhancing c-Myc-mediated aerobic glycolysis. Mechanistically, RNF6 increased the expression level of c-Myc by catalyzing the ubiquitination of Max-dimerization protein-1 (MAD1), a cellular antagonist of c-Myc. Lastly, RNF6 promoted the degradation of MAD1 via the ubiquitin-proteasome pathway, and this reduction in the MAD1 levels enabled c-Myc to promote the Warburg effect in PC. Our results demonstrate that RNF6 may be a novel biomarker in PC carcinogenesis, thereby indicating that targeting the RNF6/MAD1/c-Myc axis is a potential strategy for PC therapy.

Knockdown of RNF6 inhibits HeLa cervical cancer cell growth via suppression of MAPK/ERK signaling.

Ring finger protein 6 (RNF6) is implicated in various human malignancies, but its function in cervical cancer (CC) is incompletely understood. Here, we explored the biological significance of RNF6 in HeLa CC cells and the underlying regulatory mechanisms. The expression of RNF6 was observed to be high in both primary tissues and CC cells. RNF6 promoted HeLa CC cell growth. Knockdown of RNF6 in CC cells resulted in suppression of proliferation and promotion of apoptosis. Moreover, elevation of RNF6 had an adverse effect on the prognosis of CC. Subsequent analyses showed that these effects may be mediated via activation of ERK signaling. These findings provide evidence that the knockdown of RNF6 suppresses the MAPK/ERK pathway to regulate the growth of CC cells, which suggests that RNF6 may have potential as a target for diagnosis and treatment for CC.

Gene commander in the trash heap: Transcriptional regulation and ubiquitination modification mediated by RNF6 in carcinogenesis.

RING finger protein 6 (RNF6), a RING finger protein, has been identified as a potential tumor promoter in several cancers. However, the exact mechanism of RNF6 in cancer remains elusive. As in various diseases, RNF6 may be involved in regulating cell growth, cell proliferation, invasion, cell cycle progression, apoptosis and cell adhesion through E3 ligase-mediated ubiquitination. Thus, the research on RNF6 is mainly focused on the ubiquitination of RNF6 in recent years. This article summarizes the role of RNF6 ubiquitination in various physiological and pathological mechanisms, such as Akt/mTOR signaling pathway, Wnt/ß-catenin pathway, RNF6/ERα/Bcl-xL axis, and provides knowledge and understanding for the treatment of diseases.

Ellagic acid induces esophageal squamous cell carcinoma cell apoptosis by modulating SHP-1/STAT3 signaling.

Ellagic acid (EA) has been reported to have antiproliferative and antioxidant properties, but its function in esophageal squamous cell carcinoma (ESCC) has not been investigated yet. In the current study, EA was found have a significant anti-tumor activity in ESCC. In specific, EA inhibited ESCC cell survival in both of a concentration- and time-dependent manner. And our results showed that EA promoted ESCC cell apoptosis, including inducing the cleavages of PARP, and inhibiting the expression of anti-apoptotic proteins. In mechanistic, EA markedly suppressed STAT3-driven luciferase activity, and inhibited both of the endogenous and cytokines-induced STAT3 activation in ESCC cells. Further investigations indicated that EA could significantly upregulate SHP-1 expression, a negative modulator of STAT3 signaling. In contrast, knockdown of SHP-1 could attenuate the effects of EA on inhibiting ESCC cell survival. Moreover, we found that EA could inhibit RNF6 expression, an E3 of SHP-1, and overexpressing RNF6 could also significantly attenuate the effects of EA on inhibiting ESCC cell survival, which further revealed that EA could inhibit STAT3 signaling by modulating RNF6/SHP-1 axis. Our present study indicated that EA could be as a novel STAT3 inhibitor for the treatment of ESCC.

Potential Influences of RNF6 on Prognosis and Metastasis of Colorectal Cancer: A Clinical Analysis.

INTRODUCTION: Ring finger protein 6 (RNF6) locates on the chromatin 13q.12.13, where amplification is frequently occurred in colorectal cancer (CRC). Previous studies have reported the role of RNF6 to accelerate the progression and metastasis of CRC. METHODS: In this paper, we mainly analyzed the potential of RNF6 to predict the prognosis and metastasis of CRC. Based on the cut-off value of RNF6, enrolled CRC patients were assigned into high- and low-level group. Correlation between RNF6 level and survival of CRC patients was assessed. RESULTS: Our findings revealed that RNF6 was upregulated in CRC tissues. IHC staining demonstrated higher positive expression of RNF6 in CRC tissues. Nearly 61.2% CRC patients had a positive expression of RNF6. Moreover, RNF6 was closely linked to lymphovascular invasion (LV) (P=0.006), invasion depth (P=0.001), metastasis (P<0.001) and TNM staging (P<0.001). In CRC tissues, RNF6 level was negatively correlated to that of E-cadherin (r=-0.7093, P<0.0001). OS (overall survival) and RFS (recurrence-free survival) were worse in CRC patients with high-level RNF6, and tumor cell metastasis was believed to be the major reason. CONCLUSION: Therefore, RNF6 was confirmed to be a hallmark predicting the prognosis and metastasis in CRC patients.

RNF6 promotes myeloma cell proliferation and survival by inducing glucocorticoid receptor polyubiquitination.

RNF6, a RING-type ubiquitin ligase, has been identified as an oncogene in various cancers but its role in multiple myeloma (MM) remains elusive. In the present study we first showed that the expression levels of RNF6 in MM were significantly elevated compared with the bone marrow cells of healthy donors. Overexpression of RNF6 in LP1 and PRMI-8266 MM cell lines promoted cell proliferation, whereas knockdown of RNF6 led to apoptosis of MM cells. Furthermore, we revealed that RNF6, as a ubiquitin ligase, interacted with glucocorticoid receptor (GR) and induced its K63-linked polyubiquitination. Different from current knowledge, RNF6 increased GR stability at both endogenous and exogenous contexts. Such an action greatly promoted GR transcriptional activity, which was confirmed by luciferase assays and by the increased expression levels of prosurvival genes including Bcl-xL and Mcl-1, two typical downstream genes of the GR pathway. Consistent with these findings, ectopic expression of RNF6 in MM cells conferred resistance to dexamethasone, a typical anti-myeloma agent. In conclusion, we demonstrate that RNF6 promotes MM cell proliferation and survival by inducing atypical polyubiquitination to GR, and RNF6 could be a promising therapeutic target for the treatment of MM.

RNF6 as an Oncogene and Potential Therapeutic Target-A Review.

The RNF6 gene encodes Ring Finger Protein 6 (RNF6), which functions as a ubiquitin ligase. Its functions are not entirely known, but research shows that it is involved in human cancer development. Initially, this gene was considered to be a tumor suppressor. Numerous statistical analyses on cell lines and animals indicate, however, that RNF6 functions as an oncogene, involved in signaling pathways, including SHP1/STAT3, AKT/mTOR, Wnt/ß-catenin, or ERα/Bcl-xL. Due to this fact, it has become a potential prognostic factor and therapeutic target. Studies in tumor cells and model organisms using inhibitors such as total saponins from Paris forrestii (TSPf), ellagic acid, or microRNA molecules show the effectiveness of inhibiting RNF6, and through it, the pathways of tumor cell proliferation. The results of the currently available studies are promising, but the function of RNF6 is not fully understood. More research is needed to assess the role of RNF6 and to check the safety and efficacy of inhibitors.

MicroRNA-26a-5p inhibits breast cancer cell growth by suppressing RNF6 expression.

MicroRNA-26a-5p (miR-26a-5p) has been reported to be involved in the tumorigenesis of several tumors, but its function in breast cancer is still unknown. In this study, miR-26a-5p was found significantly downregulated in both of the breast cancer tissues and cell lines, and low expression of miR-26a-5p predicted a poor prognosis for breast cancer patients. Overexpression of miR-26a-5p could significantly inhibit breast cancer cell growth. Further studies revealed that overexpression of miR-26a-5p downregulated the protein levels of Cyclin D1, CDK4, and CDK6, but upregulated the expression levels of p21, p27, and p53. In mechanism, miR-26a-5p targeted the 3'UTR of ring finger protein 6 (RNF6) mRNA and inhibited RNF6 expression in breast cancer cells. Moreover, overexpression of miR-26a-5p inhibited RNF6/ERα/Bcl-xL axis in breast cancer cells. In contrast, inhibiting miR-26a-5p upregulated RNF6/ERα/Bcl-xL axis. Further studies indicated that miR-26a-5p mediated RNF6/ERα/Bcl-xL axis through regulating the stability of ERα protein. Collectively, downregulation of miR-26a-5p plays essential roles in breast cancer by mediating RNF6/ERα/Bcl-xL axis, which might provide important implications for the therapeutics of breast cancer.

RNF6 facilitates metastasis and radioresistance in hepatocellular carcinoma through ubiquitination of FoxA1.

Ring finger protein 6 (RNF6), a RING-domain E3 ubiquitin Ligase, functions as a potential tumor promoter in several caners. However, the function and clinical significance of RNF6 in hepatocellular carcinoma (HCC) remains unclear. Here, we found that high RNF6 expression was detected frequently in primary HCC tissues, and was closely associated with malignant phenotype and shorter survival among the HCC patients. Multivariate analyses also revealed that RNF6 overexpression was independent prognostic factors for poor outcome of patients with HCC. Further, RNF6 knockdown notably inhibited the metastasis abilities of HCC in vivo and in vitro. RNF6 silence also suppressed the epithelial-mesenchymal transition (EMT) and radioresistance in HCC. Mechanistically, our results demonstrated that RNF6 directly bound and ubiquitylated Forkhead box protein A1 (FoxA1), an important transcriptional repressor of EMT process. Additionally, our data shown that the oncogenic effect of RNF6 in HCC is partially dependent on FoxA1 degradation. Collectively, our data suggest that RNF6 plays a crucial oncogenic role in HCC tumorigenesis, and we provide a novel evidence that RNF6 may be serve as a prognostic and therapeutic target for HCC progression.

Knockdown of RNF6 inhibits gastric cancer cell growth by suppressing STAT3 signaling.

BACKGROUND AND OBJECTIVE: RNF6, an E3 ligase, has been reported to play an important role in the tumorigenesis in several tissues, but its role in gastric cancer is still unknown. In this study, we aimed to investigate the biological function and molecular mechanisms of RNF6 in gastric cancer. MATERIALS AND METHODS: The expression levels of RNF6 were detected by quantitative real-time PCR (qRT-PCR) and immunoblotting in gastric cancer tissues and cell lines. Cell Counting Kit-8 assay was performed to evaluate cell proliferation. Cell apoptosis was analyzed by flow cytometer and immunoblotting. Luciferase assay, immunoblotting and qRT-PCR were performed to explore the activation of STAT3. Immunoprecipitation was performed to evaluate the ubiquitination of SHP-1. RESULTS: In this study, RNF6 was found to be upregulated in both primary tissues and cell lines of gastric cancer. Knockdown or overexpression of RNF6 inhibited or promoted cell growth of gastric cancer cells. Knockdown of RNF6 also induced the cleavage of PARP and promoted cell apoptosis in gastric cancer cells. In addition, knockdown of RNF6 also increased the cytotoxicity of doxorubicin against gastric cancer. Moreover, knockdown of RNF6 inhibited STAT3-derived luciferase activity and downregulated the phosphorylation of STAT3, but upregulated the protein level of SHP-1. Knockdown of RNF6 downregulated the expression of MCL1 and XIAP, which are target genes of STAT3. Further studies showed that RNF6 regulated the stability of SHP-1 by inducing its polyubiquitination. CONCLUSION: These results demonstrated that RNF6 was highly expressed in gastric cancer and regulated the growth of gastric cancer cells by affecting SHP-1/STAT3 signaling, which suggested that RNF6 could be a novel target for gastric cancer therapy.

Saponins From Paris forrestii (Takht.) H. Li Display Potent Activity Against Acute Myeloid Leukemia by Suppressing the RNF6/AKT/mTOR Signaling Pathway.

Saponins are amphipathic glycosides found in traditional Chinese medicines. In the present study, we isolated a panel of saponins from Paris forrestii (Takht.) H. Li, a unique plant found in Tibet and Yunnan provinces, China. By examining their activities in suppressing acute myeloid leukemia (AML) cell proliferation, total saponins from Paris forrestii (TSPf) displayed more potent activity than individual ones. TSPf induced more than 40% AML cell apoptosis and decreased the viability of all leukemia cell lines. TSPf-induced apoptosis was confirmed by both Annexin V staining and caspase-3 activation. In line with these findings, TSPf downregulated pro-survival proteins Mcl-1, Bcl-xL, and Bcl-2 but upregulated the expression of tumor suppressor proteins p53, p27, Bax, and Beclin 1. The AKT/mTOR signaling pathway is frequently overactivated in various AML cells, and TSPf was found to suppress the activation of both AKT and mTOR, but had no effects on their total protein expression. This was further confirmed by the inactivation of 4EBP-1 and p70S6K, two typical downstream signal molecules in the AKT/mTOR pathway. Moreover, TSPf-inactivated AKT/mTOR signaling was found to be associated with downregulated RNF6, a recently identified oncogene in AML. RNF6 activated AKT/mTOR, and consistently, knockdown of RNF6 led to inactivation of the AKT/mTOR pathway. Furthermore, TSPf suppressed the growth of AML xenografts in nude mice models. Oral administration of TSPf almost fully suppressed tumor growth without gross toxicity. Consistent with the findings in cultured cell lines, TSPf also downregulated RNF6 expression along with inactivated AKT/mTOR signaling in tumor tissues. This study thus demonstrated that TSPf displays potent anti-AML activity by suppressing the RNF6/AKT/mTOR pathway. Given its low toxicity, TSPf could be developed for the treatment of AML.

The Ring Finger Protein RNF6 Induces Leukemia Cell Proliferation as a Direct Target of Pre-B-cell Leukemia Homeobox 1.

RNF6 is a little-studied ring finger protein. In the present study, we found that RNF6 was overexpressed in various leukemia cells and that it accelerated leukemia cell proliferation, whereas knockdown of RNF6 delayed tumor growth in xenografts. To find out the mechanism of RNF6 overexpression in leukemia, we designed a series of truncated constructs of RNF6 regulatory regions in the luciferase reporter system. The results revealed that the region between -144 and -99 upstream of the RNF6 transcription start site was critical and that this region contained a PBX1 recognition element (PRE). PBX1 modulated RNF6 expression by binding to the specific PRE. When PRE was mutated, RNF6 transcription was completely abolished. Further studies showed that PBX1 collaborated with PREP1 but not MEIS1 to modulate RNF6 expression. Moreover, RNF6 expression could be suppressed by doxorubicin, a major anti-leukemia agent, via down-regulating PBX1. This study thus suggests that RNF6 overexpression in leukemia is under the direction of PBX1 and that the PBX1/RNF6 axis can be developed as a novel therapeutic target of leukemia.