RhoGDIß inhibition via miR-200c/AUF1/SOX2/miR-137 axis contributed to lncRNA MEG3 downregulation-mediated malignant transformation of human bronchial epithelial cells.

Nickel pollution is a recognized factor contributing to lung cancer. Understanding the molecular mechanisms of its carcinogenic effects is crucial for lung cancer prevention and treatment. Our previous research identified the downregulation of a long noncoding RNA, maternally expressed gene 3 (MEG3), as a key factor in transforming human bronchial epithelial cells (HBECs) into malignant cells following nickel exposure. In our study, we found that deletion of MEG3 also reduced the expression of RhoGDIß. Notably, artificially increasing RhoGDIß levels counteracted the malignant transformation caused by MEG3 deletion in HBECs. This indicates that the reduction in RhoGDIß contributes to the transformation of HBECs due to MEG3 deletion. Further exploration revealed that MEG3 downregulation led to enhanced c-Jun activity, which in turn promoted miR-200c transcription. High levels of miR-200c subsequently increased the translation of AUF1 protein, stabilizing SOX2 messenger RNA (mRNA). This stabilization affected the regulation of miR-137, SP-1 protein translation, and the suppression of RhoGDIß mRNA transcription and protein expression, leading to cell transformation. Our study underscores the co-regulation of RhoGDIß expression by long noncoding RNA MEG3, multiple microRNAs (miR-200c and miR-137), and RNA-regulated transcription factors (c-Jun, SOX2, and SP1). This intricate network of molecular events sheds light on the nature of lung tumorigenesis. These novel findings pave the way for developing targeted strategies for the prevention and treatment of human lung cancer based on the MEG3/RhoGDIß pathway.

Mitochondria-targeted catalase induced cell malignant transformation by the downregulation of p53 protein stability via USP28/miR-200b/PP2A-Cα axis.

Antioxidants exert a paradoxical influence on cancer prevention. The latest explanation for this paradox is the different target sites of antioxidants. However, it remains unclear how mitochondria-targeted antioxidants trigger specific p53-dependent pathways in malignant transformation models. Our study revealed that overexpression of mitochondria-targeted catalase (mCAT) instigated such malignant transformation via mouse double minute 2 homolog (MDM2) -mediated p53 degradation. In mouse epithelial JB6 Cl41 cells, the stable expression of mCAT resulted in MDM2-mediated p53 degradation, unlike in catalase-overexpressed Cl41 cells. Further, we demonstrated that mCAT overexpression upregulated ubiquitin-specific protease 28 (USP28) expression, which in turn stabilized c-Jun protein levels. This alteration initiated the activation of the miR-200b promoter transcription activity and a subsequent increase in miR-200b expression. Furthermore, elevated miR-200b levels then promoted its binding to the 3'-untranslated region of protein phosphatase 2A catalytic subunit (PP2A-C) α-isoform mRNA, consequently resulting in PP2A-C protein downregulation. This cascade of events ultimately contributed to increased MDM2 phosphorylation and p53 protein degradation. Thus, the mCAT overexpression triggers MDM2/p53-dependent malignant transformation through USP28/miR-200b/PP2A-Cα pathway, which may provide a new information for understanding mitochondria-targeted antioxidants facilitate the progression to the tumorigenic state.

Isorhapontigenin inhibition of basal muscle-invasive bladder cancer attributed to its downregulation of SNHG1 and DNMT3b.

BACKGROUND: Bladder cancer (BC) is among the most prevalent malignant urothelial tumors globally, yet the prognosis for patients with muscle-invasive bladder cancer (MIBC) remains dismal, with a very poor 5-year survival rate. Consequently, identifying more effective and less toxic chemotherapeutic alternatives is critical for enhancing clinical outcomes for BC patients. Isorhapontigenin (ISO), a novel stilbene isolated from a Gnetum found in certain provinces of China, has shown potential as an anticancer agent due to its diverse anticancer activities. Despite its promising profile, the specific anticancer effects of ISO on BC and the underlying mechanisms are still largely unexplored. METHODS: The anchorage-independent growth, migration and invasion of BC cells were assessed by soft agar and transwell invasion assays, respectively. The RNA levels of SOX2, miR-129 and SNHG1 were quantified by qRT-PCR, while the protein expression levels were validated through Western blotting. Furthermore, methylation-specific PCR was employed to assess the methylation status of the miR-129 promoter. Functional assays utilized siRNA knockdown, plasmid-mediated overexpression, and chemical inhibition approaches. RESULTS: Our study demonstrated that ISO treatment significantly reduced SNHG1 expression in a dose- and time-dependent manner in BC cells, leading to the inhibition of anchorage-independent growth and invasion in human basal MIBC cells. This effect was accompanied by the downregulation of MMP-2 and MMP-9 and the upregulation of the tumor suppressor PTEN. Further mechanistic investigations revealed that SOX2, a key upstream regulator of SNHG1, played a crucial role in mediating the ISO-induced transcriptional suppression of SNHG1. Additionally, we found that ISO treatment led to a decrease in DNMT3b protein levels, which in turn mediated the hypomethylation of the miR-129 promoter and the subsequent suppression of SOX2 mRNA 3'-UTR activity, highlighting a novel pathway through which ISO exerts its anticancer effects. CONCLUSIONS: Collectively, our study highlights the critical role of SNHG1 downregulation as well as its upstream DNMT3b/miR-129/SOX2 axis in mediating ISO anticancer activity. These findings not only elucidate the mechanism of action of ISO but also suggest novel targets for BC therapy.

DNMT3a-mediated upregulation of the stress inducible protein sestrin-2 contributes to malignant transformation of human bronchial epithelial cells following nickel exposure.

BACKGROUND: Nickel is a confirmed human lung carcinogen. Nonetheless, the molecular mechanisms driving its carcinogenic impact on lung tissue remain poorly defined. In this study, we assessed SESN2 expression and the signaling pathways responsible for cellular transformation in human bronchial epithelial cells (HBECs) as a result of nickel exposure. METHODS: We employed the Western blotting to determine the induction of SESN2 by nickel. To clarify the signaling pathways leading to cellular transformation following nickel exposure, we applied techniques such as gene knockdown, methylation-specific PCR, and chromatin immunoprecipitation. RESULT: Exposure to nickel results in the upregulation of SESN2 and the initiation of autophagy in human bronchial epithelial cells (HBECs). This leads to degradation of HUR protein and consequently downregulation of USP28 mRNA, PP2AC protein, ß-catenin protein, and diminished VHL transcription, culminating in the accumulation of hypoxia-inducible factor-1α (HIF-1α) and the malignant transformation of these cells. Mechanistic studies revealed that the increased expression of SESN2 is attributed to the demethylation of the SESN2 promoter induced by nickel, a process facilitated by decreased DNA methyl-transferase 3 A (DNMT3a) expression, while The downregulation of VHL transcription is linked to the suppression of the PP2A-C/GSK3ß/ß-Catenin/C-Myc pathway. Additionally, we discovered that SESN2-mediated autophagy triggers the degradation of HUR protein, which subsequently reduces the stability of USP28 mRNA and inhibits the PP2A-C/GSK3ß/ß-Catenin pathway and c-Myc transcription in HBECs post nickel exposure. CONCLUSION: Our results reveal that nickel exposure leads to the downregulation of DNMT3a, resulting in the hypomethylation of the SESN2 promoter and its protein induction. This triggers autophagy-dependent suppression of the HUR/USP28/PP2A/ß-Catenin/c-Myc pathway, subsequently leading to reduced VHL transcription, accumulation of HIF-1α protein, and the malignant transformation of human bronchial epithelial cells (HBECs). Our research offers novel insights into the molecular mechanisms that underlie the lung carcinogenic effects of nickel exposure. Specifically, nickel induces aberrant DNA methylation in the SESN2 promoter region through the decrease of DNMT3a levels, which ultimately leads to HIF-1α protein accumulation and the malignant transformation of HBECs. Specifically, nickel initiates DNA-methylation of the SESN2 promoter region by decreasing DNMT3a, ultimately resulting in HIF-1α protein accumulation and malignant transformation of HBECs. This study highlights DNMT3a as a potential prognostic biomarker or therapeutic target to improve clinical outcomes in lung cancer patients.

Differential functions of RhoGDIß in malignant transformation and progression of urothelial cell following N-butyl-N-(4-hydmoxybutyl) nitrosamine exposure.

BACKGROUND: Functional role of Rho GDP-dissociation inhibitor beta (RhoGDIß) in tumor biology appears to be contradictory across various studies. Thus, the exploration of the molecular mechanisms underlying the differential functions of this protein in urinary bladder carcinogenesis is highly significant in the field. Here, RhoGDIß expression patterns, biological functions, and mechanisms leading to transformation and progression of human urothelial cells (UROtsa cells) were evaluated following varying lengths of exposure to the bladder carcinogen N-butyl-N-(4-hydmoxybutyl) nitrosamine (BBN). RESULTS: It was seen that compared to expression in vehicle-treated control cells, RhoGDIß protein expression was downregulated after 2-month of BBN exposure, but upregulated after 6-month of exposure. Assessments of cell function showed that RhoGDIß inhibited UROtsa cell growth in cells with BBN for 2-month exposure, whereas it promoted the invasion of cells treated with BBN for 6 months. Mechanistic studies revealed that 2-month of BBN exposure markedly attenuated DNMT3a abundance, and this led to reduced miR-219a promoter methylation, increased miR-219a binding to the RhoGDIß mRNA 3'UTR, and reduced RhoGDIß protein translation. While after 6-mo of BBN treatment, the cells showed increased PP2A/JNK/C-Jun axis phosphorylation and this in turn mediated overall RhoGDIß mRNA transcription and protein expression as well as invasion. CONCLUSIONS: These findings indicate that RhoGDIß is likely to inhibit the transformation of human urothelial cells during the early phase of BBN exposure, whereas it promotes invasion of the transformed/progressed urothelial cells in the late stage of BBN exposure. The studies also suggest that RhoGDIß may be a useful biomarker for evaluating the progression of human bladder cancers.

LINC00955 suppresses colorectal cancer growth by acting as a molecular scaffold of TRIM25 and Sp1 to Inhibit DNMT3B-mediated methylation of the PHIP promoter.

BACKGROUND: Long non-coding RNAs play an important role in the development of colorectal cancer (CRC), while many CRC-related lncRNAs have not yet been identified. METHODS: The relationship between the expression of LINC00955 (Long Intergenic Non-protein Coding RNA 955) and the prognosis of colorectal cancer patients was analyzed using the sequencing results of the TCGA database. LINC00955 expression levels were measured using qRT-PCR. The anti-proliferative activity of LINC00955 was evaluated using CRC cell lines in vitro and xenograft models in nude mice in vivo. The interaction of TRIM25-Sp1-DNMT3B-PHIP-CDK2 was analyzed by western blotting, protein degradation experiment, luciferase, RNA-IP, RNA pull-down assays and immunohistochemically analysis. The biological roles of LINC00955, tripartite motif containing 25 (TRIM25), Sp1 transcription factor (Sp1), DNA methyltransferase 3 beta (DNMT3B), pleckstrin homology domain interacting protein (PHIP), cyclin dependent kinase 2 (CDK2) in colorectal cancer cells were analyzed using ATP assays, Soft agar experiments and EdU assays. RESULTS: The present study showed that LINC00955 is downregulated in CRC tissues, and such downregulation is associated with poor prognosis of CRC patients. We found that LINC00955 can inhibit CRC cell growth both in vitro and in vivo. Evaluation of its mechanism of action showed that LINC00955 acts as a scaffold molecule that directly promotes the binding of TRIM25 to Sp1, and promotes ubiquitination and degradation of Sp1, thereby attenuating transcription and expression of DNMT3B. DNMT3B inhibition results in hypomethylation of the PHIP promoter, in turn increasing PHIP transcription and promoting ubiquitination and degradation of CDK2, ultimately leading to G0/G1 growth arrest and inhibition of CRC cell growth. CONCLUSIONS: These findings indicate that downregulation of LINC00955 in CRC cells promotes tumor growth through the TRIM25/Sp1/DNMT3B/PHIP/CDK2 regulatory axis, suggesting that LINC00955 may be a potential target for the therapy of CRC.

ATG7 upregulation contributes to malignant transformation of human bronchial epithelial cells by B[a]PDE via DNMT3B protein degradation and miR-494 promoter methylation.

Lung cancer primarily arises from exposure to various environmental factors, particularly airborne pollutants. Among the various lung carcinogens, benzo(a)pyrene and its metabolite B[a]PDE are the strongest ones that actively contribute to lung cancer development. ATG7 is an E1-like activating enzyme and contributes to activating autophagic responses in mammal cells. However, the potential alterations of ATG7 and its role in B[a]PDE-caused lung carcinogenesis remain unknown. Here, we found that B[a]PDE exposure promoted ATG7 expression in mouse lung tissues, while B[a]PDE exposure resulted in ATG7 induction in human normal bronchial epithelial cells. Our studies also demonstrated a significant correlation between high ATG7 expression levels and poor overall survival in lung cancer patients. ATG7 knockdown significantly repressed Beas-2B cell transformation upon B[a]PDE exposure, and such promotive effect of ATG7 on cell transformation mediated the p27 translation inhibition. Further studies revealed that miR-373 inhibition was required to stabilize ATG7 mRNA, therefore increasing ATG7 expression following B[a]PDE exposure, while ATG7 induction led to the autophagic degradation of the DNA methyltransferase 3 Beta (DNMT3B) protein, in turn promoted miR-494 transcription via its promoter region methylation status suppression. We also found that the miR-494 upregulation inhibited p27 protein translation and promoted bronchial epithelial cell transformation via its directly targeting p27 mRNA 3'-UTR region. Current studies, to the best of our knowledge, are for the first time to identify that ATG7 induction and its mediated autophagy is critical for B[a]PDE-induced transformation of human normal epithelial cells.

SOX2 Promotes Invasion in Human Bladder Cancers through MMP2 Upregulation and FOXO1 Downregulation.

SOX2, a member of the SRY-related HMG-box (SOX) family, is abnormally expressed in many tumors and associated with cancer stem cell-like properties. Previous reports have shown that SOX2 is a biomarker for cancer stem cells in human bladder cancer (BC), and our most recent study has indicated that the inhibition of SOX2 by anticancer compound ChlA-F attenuates human BC cell invasion. We now investigated the mechanisms through which SOX2 promotes the invasive ability of BC cells. Our studies revealed that SOX2 promoted SKP2 transcription and increased SKP2-accelerated Sp1 protein degradation. As Sp1 is a transcriptionally regulated gene, HUR transcription was thereby attenuated, and, in the absence of HUR, FOXO1 mRNA was degraded fast, which promoted BC cell invasion. In addition, SOX2 promoted BC invasion through the upregulation of nucleolin transcription, which resulted in increased MMP2 mRNA stability and expression. Collectively, our findings show that SOX2 promotes BC invasion through both SKP2-Sp1-HUR-FOXO1 and nucleolin-MMP2 dual axes.

Isorhapontigenin (ISO) inhibits stem cell-like properties and invasion of bladder cancer cell by attenuating CD44 expression.

Cancer stem cells (CSC) are highly associated with poor prognosis in cancer patients. Our previous studies report that isorhapontigenin (ISO) down-regulates SOX2-mediated cyclin D1 induction and stem-like cell properties in glioma stem-like cells. The present study revealed that ISO could inhibit stem cell-like phenotypes and invasivity of human bladder cancer (BC) by specific attenuation of expression of CD44 but not SOX-2, at both the protein transcription and degradation levels. On one hand, ISO inhibited cd44 mRNA expression through decreases in Sp1 direct binding to its promoter region-binding site, resulting in attenuation of its transcription. On the other hand, ISO also down-regulated USP28 expression, which in turn reduced CD44 protein stability. Further studies showed that ISO treatment induced miR-4295, which specific bound to 3'-UTR activity of usp28 mRNA and inhibited its translation and expression, while miR-4295 induction was mediated by increased Dicer protein to enhance miR-4295 maturation upon ISO treatment. Our results provide the first evidence that ISO has a profound inhibitory effect on human BC stem cell-like phenotypes and invasivity through the mechanisms distinct from those previously noted in glioma stem-like cells.

The RING domain in the anti-apoptotic protein XIAP stabilizes c-Myc protein and preserves anchorage-independent growth of bladder cancer cells.

X-linked inhibitor of apoptosis protein (XIAP) suppresses apoptosis and plays key roles in the development, growth, migration, and invasion of cancer cells. Therefore, XIAP has recently attracted much attention as a potential antineoplastic therapeutic target, requiring elucidation of the molecular mechanisms underlying its biological activities. Here, using shRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, anchorage-independent growth assay, and invasive assay, we found that XIAP's RING domain, but not its BIR domain, is crucial for XIAP-mediated up-regulation of c-Myc protein expression in human bladder cancer (BC) cells. Mechanistically, we observed that the RING domain stabilizes c-Myc by inhibiting its phosphorylation at Thr-58 and that this inhibition is due to activated ERK1/2-mediated phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser-9. Functional studies further revealed that c-Myc protein promotes anchorage-independent growth and invasion stimulated by the XIAP RING domain in human BC cells. Collectively, the findings in our study uncover that the RING domain of XIAP supports c-Myc protein stability, providing insight into the molecular mechanism and role of c-Myc overexpression in cancer progression. Our observations support the notion of targeting XIAP's RING domain and c-Myc in cancer therapy.

Inhibition of UBE2N-dependent CDK6 protein degradation by miR-934 promotes human bladder cancer cell growth.

Because bladder cancer (BC) is one of the most common malignant cancers of the urinary system, identification of BC cell growth-associated effectors is of great significance. Cyclin-dependent kinase (CDK)6 is a member of the CDK family of cell cycle-related proteins and plays an important role in cancer cell growth. This is borne out by the fact that a CDK6 inhibitor had been approved to treat several types of cancers. Nevertheless, underlying molecular mechanisms concerning how to regulate CDK6 expression in BC remains unclear. In the present study, it was observed that miR-934 was much higher in human BCs and human BC cell lines as well. The results also revealed that miR-934 inhibition dramatically decreased human BC cell monolayer growth in vitro and xenograft tumor growth in vivo; the outcomes were accompanied by CDK6 protein down-regulation and G0-G1 cell cycle arrest. Moreover, overexpression of CDK6 reversed the inhibition of BC cell growth induced by miR-934. Further studies showed that miR-934 binds to a 3'-UTR of ubiquitin-conjugating enzyme 2N (ube2n) mRNA, down-regulated UBE2N protein expression; this, in turn, attenuated CDK6 protein degradation and led to CDK6 protein accumulation as well as the promotion of BC tumor growth. Collectively, this study not only establishes a novel regulatory axis of miR-934/UBE2N of CDK6 but also provides data suggesting that miR-934 and UBE2N may be potentially promising targets for therapeutic strategies against BC.-Yan, H., Ren, S., Lin, Q., Yu, Y., Chen, C., Hua, X., Jin, H., Lu, Y., Zhang, H., Xie, Q., Huang, C., Huang, H. Inhibition of UBE2N-dependent CDK6 protein degradation by miR-934 promotes human bladder cancer cell growth.

miR-3687 Overexpression Promotes Bladder Cancer Cell Growth by Inhibiting the Negative Effect of FOXP1 on Cyclin E2 Transcription.

Cyclin E2, a member of the cyclin family, is a key cell cycle-related protein. This protein plays essential roles in cancer progression, and, as such, an inhibitor of cyclin E2 has been approved to treat several types of cancers. Even so, mechanisms underlying how to regulate cyclin E2 expression in cancer remain largely unknown. In the current study, miR-3687 was upregulated in clinical bladder cancer (BC) tumor tissues, The Cancer Genome Atlas (TCGA) database, and human BC cell lines. Inhibition of miR-3687 expression significantly reduced human BC cell proliferation in vitro and tumor growth in vivo, which coincided with the induction of G0/G1 cell cycle arrest and downregulation of cyclin E2 protein expression. Interestingly, overexpression of cyclin E2 reversed the inhibition of BC proliferation induced by miR-3687. Mechanistic studies suggested that miR-3687 binds to the 3' UTR of foxp1 mRNA, downregulates FOXP1 protein expression, and in turn promotes the transcription of cyclin E2, thereby promoting the growth of BC cells. Collectively, the current study not only establishes a novel regulatory axis of miR-3687/FOXP1 regarding regulation of cyclin E2 expression in BC cells, but also provides strong suggestive evidence that miR-3687 and FOXP1 may be promising targets in therapeutic strategies for human BC.

Genomic and Functional Characterization of the Endophytic Bacillus subtilis 7PJ-16 Strain, a Potential Biocontrol Agent of Mulberry Fruit Sclerotiniose.

Bacillus sp. 7PJ-16, an endophytic bacterium isolated from a healthy mulberry stem and previously identified as Bacillus tequilensis 7PJ-16, exhibits strong antifungal activity and has the capacity to promote plant growth. This strain was studied for its effectiveness as a biocontrol agent to reduce mulberry fruit sclerotiniose in the field and as a growth-promoting agent for mulberry in the greenhouse. In field studies, the cell suspension and supernatant of strain 7PJ-16 exhibited biocontrol efficacy and the lowest disease incidence was reduced down to only 0.80%. In greenhouse experiments, the cell suspension (1.0 × 106 and 1.0 × 105 CFU/mL) and the cell-free supernatant (100-fold and 1000-fold dilution) stimulated mulberry seed germination and promoted mulberry seedling growth. In addition, to accurately identify the 7PJ-16 strain and further explore the mechanisms of its antifungal and growth-promoting properties, the complete genome of this strain was sequenced and annotated. The 7PJ-16 genome is comprised of two circular plasmids and a 4,209,045-bp circular chromosome, containing 4492 protein-coding genes and 116 RNA genes. This strain was ultimately designed as Bacillus subtilis based on core genome sequence analyses using a phylogenomic approach. In this genome, we identified a series of gene clusters that function in the synthesis of non-ribosomal peptides (surfactin, fengycin, bacillibactin, and bacilysin) as well as the ribosome-dependent synthesis of tasA and bacteriocins (subtilin, subtilosin A), which are responsible for the biosynthesis of numerous antimicrobial metabolites. Additionally, several genes with function that promote plant growth, such as indole-3-acetic acid biosynthesis, the production of volatile substances, and siderophores synthesis, were also identified. The information described in this study has established a good foundation for understanding the beneficial interactions between endophytes and host plants, and facilitates the further application of B. subtilis 7PJ-16 as an agricultural biofertilizer and biocontrol agent.

Transcriptional and post-transcriptional upregulation of p27 mediates growth inhibition of isorhapontigenin (ISO) on human bladder cancer cells.

There are few approved drugs available for the treatment of muscle-invasive bladder cancer (MIBC). Recently, we have demonstrated that isorhapontigenin (ISO), a new derivative isolated from the Chinese herb Gnetum cleistostachyum, effectively induces cell-cycle arrest at the G0/G1 phase and inhibits anchorage-independent cell growth through the miR-137/Sp1/cyclin D1 axis in human MIBC cells. Herein, we found that treatment of bladder cancer (BC) cells with ISO resulted in a significant upregulation of p27, which was also observed in ISO-treated mouse BCs that were induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Importantly, knockdown of p27 caused a decline in the ISO-induced G0-G1 growth arrest and reversed ISO suppression of anchorage-independent growth in BC cells. Mechanistic studies revealed that ISO promoted p27 expression at mRNA transcription level through increasing direct binding of forkhead box class O1 (FOXO1) to its promoter, while knockdown of FOXO1 attenuated ISO inhibition of BC cell growth. On the other hand, ISO upregulated the 3'-untranslated region (3'-UTR) activity of p27, which was accompanied by a reduction of miR-182 expression. In line with these observations, ectopic expression of miR-182 significantly blocked p27 3'-UTR activity, whereas mutation of the miR-182-binding site at p27 mRNA 3'-UTR effectively reversed this inhibition. Accordingly, ectopic expression of miR-182 also attenuated ISO upregulation of p27 expression and impaired ISO inhibition of BC cell growth. Our results not only provide novel insight into understanding of the underlying mechanism related to regulation of MIBC cell growth but also identify new roles and mechanisms underlying ISO inhibition of BC cell growth.

p63α protein up-regulates heat shock protein 70 expression via E2F1 transcription factor 1, promoting Wasf3/Wave3/MMP9 signaling and bladder cancer invasion.

Bladder cancer (BC) is the sixth most common cancer in the United States and is the number one cause of death among patients with urinary system malignancies. This makes the identification of invasive regulator(s)/effector(s) as the potential therapeutic targets for managing BC a high priority. p63 is a member of the p53 family of tumor suppressor genes/proteins, plays a role in the differentiation of epithelial tissues, and is believed to function as a tumor suppressor. However, it remains unclear whether and how p63 functions in BC cell invasion after tumorigenesis. Here, we show that p63α protein levels were much higher in mouse high-invasive BC tissues than in normal tissues. Our results also revealed that p63α is crucial for heat shock protein 70 (Hsp70) expression and subsequently increases the ability of BC invasion. Mechanistic experiments demonstrated that p63α can transcriptionally up-regulate Hsp70 expression, thereby promoting BC cell invasion via the Hsp70/Wasf3/Wave3/MMP-9 axis. We further show that E2F transcription factor 1 (E2F1) mediates p63α overexpression-induced Hsp70 transcription. We also found that p63α overexpression activates E2F1 transcription, which appears to be stimulated by p63α together with E2F1. Collectively, our results demonstrate that p63α is a positive regulator of BC cell invasion after tumorigenesis, providing significant insights into the biological function of p63α in BC and supporting the notion that p63α might be a potential target for invasive BC therapy.

XIAP overexpression promotes bladder cancer invasion in vitro and lung metastasis in vivo via enhancing nucleolin-mediated Rho-GDIß mRNA stability.

Our recent studies demonstrate that X-linked inhibitor of apoptosis protein (XIAP) is essential for regulating colorectal cancer invasion. Here, we discovered that RhoGDIß was a key XIAP downstream effector mediating bladder cancer (BC) invasion in vitro and in vivo. We found that both XIAP and RhoGDIß expressions were consistently elevated in BCs of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice in comparison to bladder tissues from vehicle-treated mice and human BCs in comparison to the paired adjacent normal bladder tissues. Knockdown of XIAP attenuated RhoGDIß expression and reduced cancer cell invasion, whereas RhoGDIß expression was attenuated in BBN-treated urothelium of RING-deletion knockin mice. Mechanistically, XIAP stabilized RhoGDIß mRNA by its positively regulating nucleolin mRNA stability via Erks-dependent manner. Moreover, ectopic expression of GFP-RhoGDIß in T24T(shXIAP) cells restored its lung metastasis in nude mice. Our results demonstrate that XIAP-regulated Erks/nucleolin/RhoGDIß axis promoted BC invasion and lung metastasis.

A novel post-translational modification of nucleolin, SUMOylation at Lys-294, mediates arsenite-induced cell death by regulating gadd45α mRNA stability.

Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications, including phosphorylation, methylation, and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of recently verified forms of post-translational modifications and exerts various effects on the target proteins. In the studies reported here, we discovered SUMOylational modification of human nucleolin protein at Lys-294, which facilitated the mRNA binding property of nucleolin by maintaining its nuclear localization. In response to arsenic exposure, nucleolin-SUMO was induced and promoted its binding with gadd45α mRNA, which increased gadd45α mRNA stability and protein expression, subsequently causing GADD45α-mediated cell death. On the other hand, ectopic expression of Mn-SOD attenuated the arsenite-generated superoxide radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced apoptosis by reducing GADD45α expression. Collectively, our results for the first time demonstrate that nucleolin-SUMO at K294R plays a critical role in its nucleus sequestration and gadd45α mRNA binding activity. This novel biological function of nucleolin is distinct from its conventional role as a proto-oncogene. Therefore, our findings here not only reveal a new modification of nucleolin protein and its novel functional paradigm in mRNA metabolism but also expand our understanding of the dichotomous roles of nucleolin in terms of cancer development, which are dependent on multiple intracellular conditions and consequently the appropriate regulations of its modifications, including SUMOylation.

Loss of p27 upregulates MnSOD in a STAT3-dependent manner, disrupts intracellular redox activity and enhances cell migration.

Cell migration is a dynamic process that is central to a variety of physiological functions as well as disease pathogenesis. The modulation of cell migration by p27 (officially known as CDKN1B) has been reported, but the exact mechanism(s) whereby p27 interacts with downstream effectors that control cell migration have not been elucidated. By systematically comparing p27(+/+) mouse embryonic fibroblasts (MEFs) with genetically ablated p27(-/-) MEFs using wound-healing, transwell and time-lapse microscopic analyses, we provide direct evidence that p27 inhibits both directional and random cell migration. Identical results were obtained with normal and cancer epithelial cells using complementary knockdown and overexpression approaches. Additional studies revealed that overexpression of manganese superoxide dismutase (MnSOD, officially known as SOD2) and reduced intracellular oxidation played a key role in increased cell migration in p27-deficient cells. Furthermore, we identified signal transducer and activator of transcription 3 (STAT3) as the transcription factor responsible for p27-regulated MnSOD expression, which was further mediated by ERK- and ATF1-dependent transactivation of the cAMP response element (CRE) within the Stat3 promoter. Collectively, our data strongly indicate that p27 plays a crucial negative role in cell migration by inhibiting MnSOD expression in a STAT3-dependent manner.

The Chinese herb isolate yuanhuacine (YHL-14) induces G2/M arrest in human cancer cells by up-regulating p21 protein expression through an p53 protein-independent cascade.

Yuanhuacine (YHL-14), the major component of daphnane diterpene ester isolated from the flower buds of Daphne genkwa, has been reported to have activity against cell proliferation in various cancer cell lines. Nevertheless, the potential mechanism has not been explored yet. Here we demonstrate that YHL-14 inhibits bladder and colon cancer cell growth through up-regulation of p21 expression in an Sp1-dependent manner. We found that YHL-14 treatment resulted in up-regulation of p21 expression and a significant G2/M phase arrest in T24T and HCT116 cells without affecting p53 protein expression and activation. Further studies indicate that p21 induction by YHL-14 occurs at the transcriptional level via up-regulation of Sp1 protein expression. Moreover, our results show that p38 is essential for YHL-14-mediated Sp1 protein stabilization, G2/M growth arrest induction, and anchorage-independent growth inhibition of cancer cells. Taken together, our studies demonstrate a novel mechanism of YHL-14 against cancer cell growth in bladder and colon cancer cell lines, which provides valuable information for the design and synthesis of other new conformation-constrained derivatives on the basis of the structure of YHL-14 for cancer therapy.