Faculty Bibliography

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-3β (GSK-3β) 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.

Over half a million US residents are suffering with bladder cancer (BC), which costs a total $4 billion in treatment annually. Although recent studies report that autophagy-related gene 7 (ATG7) is overexpressed in BCs, the regulatory effects of ATG7 on cancer stem-like phenotypes and invasion have not been explored yet. Current studies demonstrated that the deficiency of ATG7 by its shRNA dramatically reduced sphere formation and invasion in vitro, as well as lung metastasis in vivo in human invasive BC cells. Further studies indicated that the knockdown of ATG7 attenuated the expression of CD44 standard (CD44s), while ectopic introduction of CD44s, was capable of completely restoring sphere formation, invasion, and lung metastasis in T24T(shATG7) cells. Mechanistic studies revealed that ATG7 overexpression stabilized CD44s proteins accompanied with upregulating USP28 proteins. Upregulated USP28 was able to bind to CD44s and remove the ubiquitin group from CD44s' protein, resulting in the stabilization of CD44s protein. Moreover, ATG7 inhibition stabilized AUF1 protein and thereby reduced tet1 mRNA stability and expression, which was able to demethylate usp28 promoter, reduced USP28 expression, finally promoting CD44s degradation. In addition, CD44s was defined to inhibit degradation of RhoGDIβ, which in turn promotes BC invasion. Our results demonstrate that CD44s is a key ATG7 downstream regulator of the sphere formation, invasion, and lung metastasis of BCs, providing significant insight into understanding the BC invasions, metastasis, and stem-like properties.

Programmed cell death protein 1 (PD-1) and its ligand PD-L1 blockade have been identified to target immune checkpoints to treat human cancers with durable clinical benefit. Several studies reveal that the response to PD-1-PD-L1 blockade might correlate with PD-L1 expression levels in tumor cells. However, the mechanistic pathways that regulate PD-L1 protein expression are not understood. Here, we reported that PD-L1 protein is regulated by ATG7-autophagy with an ATG7-initiated positive feedback loop in bladder cancer (BC). Mechanistic studies revealed that ATG7 overexpression elevates PD-L1 protein level mainly through promoting autophagy-mediated degradation of FOXO3a, thereby inhibiting its initiated miR-145 transcription. The lower expression of miR-145 increases pd-l1 mRNA stability due to the reduction of its direct binding to 3'-UTR of pd-l1 mRNA, in turn leading to increasing in pd-l1 mRNA stability and expression, and finally enhancing stem-like property and invasion of BC cells. Notably, overexpression of PD-L1 in ATG7 knockdown cells can reverse the defect of autophagy activation, FOXO3A degradation, and miR-145 transcription attenuation. Collectively, our results revealed a positive feedback loop to promoting PD-L1 expression in human BC cells. Our study uncovers a novel molecular mechanism for regulating pd-l1 mRNA stability and expression via ATG7/autophagy/FOXO3A/miR-145 axis and reveals the potential for using combination treatment with autophagy inhibitors and PD-1/PD-L1 immune checkpoint blockade to enhance therapeutic efficacy for human BCs.

PHLPP1 (PH domain and leucine rich repeat protein phosphatase 1) is a newly identified family of Ser/Thr phosphatases that catalyzes the dephosphorylation of a conserved regulatory motif of the AGC kinases resulting in a tumor suppressive function, while CDKN1B/p27 also acts as a tumor suppressor by regulating cell cycle, senescence, apoptosis, and cell motility. Our most recent studies reveal that CDKN1B is required for PHLPP1 abundance, which contributes to the inhibition of carcinogenic arsenite-induced cell malignant transformation through inhibition of RPS6-mediated Hif1a translation. However, nothing is known about the mechanisms underlying the crosstalk between these 2 key tumor suppressors in intact cells. Here, for the first time to the best of our knowledge, we show that CDKN1B is able to promote PHLPP1 protein translation by attenuating the abundance of Mir6981, which binds directly to the 5'untranslated region (UTR) of Phlpp1 mRNA. Further studies indicate that the attenuation of Mir6981 expression is due to macroautophagy/autophagy-mediated degradation of Mir6981 in an SQSTM1/p62-dependent fashion. Moreover, we have determined that Sqstm1 is upregulated by CDKN1B at the level of transcription via enhancing SP1 protein stability in an HSP90-depdendent manner. Collectively, our studies prove that: 1) SQSTM1 is a CDKN1B downstream effector responsible for CDKN1B-mediated autophagy; 2) by promoting the autophagy-mediated degradation of Mir6981, CDKN1B exerts a positive regulatory effect on PHLPP1 translation; 3) Mir6981 suppresses PHLPP1 translation by binding directly to its mRNA 5'-UTR, rather than classical binding to the 3'-UTR. These findings provide significant insight into understanding the crosstalk between CDKN1B and PHLPP1.

Muscle-invasive and metastatic bladder cancer have an extremely poor 5-year survival rate of 5%. In comparison, all other bladder cancers (BCs) have a 5-year survival rate of 77%. This striking contrast indicates that one of the therapeutic kernels for bladder cancer is to elucidate the molecular mechanisms underlying its invasiveness and metastasis. In the current study, we demonstrated that maternally expressed gene 3 (MEG3) is significantly downregulated in human invasive bladder cancers in comparison to non-invasive bladder cancers, and that ectopic expression of MEG3 dramatically inhibits the invasiveness of human bladder cancer cells. Consistently, ectopic expression of MEG3 also attenuates metastatic ability of T24T cells, a cell line derived from T24 cells, in the lungs of nude mice. Our mechanistic studies reveal that MEG3, as a ceRNA, inhibits the invasiveness of human bladder cancer cells via negative regulation of c-Myc by competing with PHLPP2 mRNA for miR-27a. These findings not only provide a novel insight into understanding the mechanisms behind the MEG3 inhibition of bladder cancer cell invasion, but also reveal the potential for use of MEG3 as a tool for the prevention and therapy of invasive bladder cancer.

ChlA-F is a novel conformation-derivative of Cheliensisin A, styryl-lactone isolates that show potent anti-tumor potential in vivo and vitro. However, the anti-cancer activity and its potential mechanisms underlying ChlA-F action have never been explored. In the present study, we evaluated the potency of ChlA-F on autophagy-mediated anchorage-independent growth inhibition in human high-grade invasive bladder cancer (BC) cells. We found that ChlA-F treatment significantly inhibited anchorage-independent growth of human BC cells by inducing autophagy in a Sestrin-2 (SESN2)-dependent fashion. Our results revealed that ChlA-F treatment specifically induced SESN2 expression via increasing its transcription and mRNA stability. On one hand, ChlA-F treatment markedly attenuated Dicer protein abundance, in turn abolishing miR-27a maturation and further relieving miR-27a binding directly to SESN2 mRNA 3'UTR, thereby promoting SESN2 mRNA stabilization. On the other hand, ChlA-F treatment promoted Sp1 abundance and consequently mediated SESN2 transcription. These results demonstrate that its activation of the autophagic pathway through specifically promoting SESN2 expression mediates the anti-cancer effect of ChlA-F, which offers insights into the novel anti-cancer effect of ChlA-F on BC, as well as providing therapeutic alternatives against human BC.

Bladder cancer (BC) ranks as the sixth most common cancer in the United States and is the leading cause of death in patients with urinary malignancies. p63 is a member of the p53 family and is believed to function as a tumor suppressor in human BCs. Our most recent studies reveal a previously unknown function of the RING of XIAP in promoting miR-4295 transcription, thereby reducing p63α protein translation and enhancing normal urothelial transformation, whereas p63α upregulates hsp70 transcription, subsequently activating the HSP70/Wasf3/Wave3/MMP-9 axis, and promoting BC cell invasion via initiating the transcription factor E2F1. Here we found that p63α inhibited Cyclin D1 protein expression, subsequently decreasing the ability of BC cell anchorage-independent growth in vitro and tumorigenicity in vivo Mechanistic studies demonstrate that p63α expression is able to down-regulate cyclin d1 transcription through attenuation of c-myc mRNA stability. We further show that the reduction of miR-141-3p expression by p63α directly releases its inhibition of 3'-UTR activity of AU-rich element RNA-binding factor 1 (AUF1) mRNA, thereby increasing AUF1 protein translation and further resulting in degradation of c-myc mRNA which in turn reduces cyclin d1 transcription and BC cell anchorage-independent growth. Collectively, our results demonstrate that p63α is a negative regulator of BC cell tumorigenic growth, a distinctly different function than its promotion of BC invasion; thus providing further new insight into the understanding double faces of p63α in regulation of BC cell tumorigenic growth and progression/invasion.

Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) is a tumor suppressor that catalyzes the de-phosphorylation of the AGC kinases, while p27 acts as a tumor suppressor that regulates cell cycle, apoptosis, and cell motility. Our previous studies have identified that PHLPP2 participates in inhibition of transformation of human bronchial epithelial cells following lung carcinogen B[a]P/B[a]PDE exposure. However, nothing was known about the association of p27 with regulation of PHLPP2 expression and the role of PHLPP2 in bladder cancer (BC) invasion. In our current studies, we demonstrated that PHLPP2 inhibited BC invasion through promoting MMP2 degradation via p62-mediated autophagy; and p27 expression was able to stabilize PHLPP2 protein by inhibiting protein degradation of Hsp90, which could directly bind to PHLPP2 and protect it from degradation. More in-depth studies discovered that stabilization of Hsp90 by p27 was mediated by calpain1 proteolysis system, whereas p27 inhibited calpain1 gene transcription by attenuating Jak1/Stat1 cascade in human invasive BC cells. Collectively, we for the first time revealed PHLPP2 downregulation in BCs and its participating in promotion of BC invasion, as well as novel role of p27 and mechanisms underlying its regulation of PHLPP2 protein degradation through Hsp90-dependent manner. Our findings improve our understanding of p27 and PHLPP2 roles and their crosstalk in regulation of BC invasion, which further contributes to improve the current strategy for invasive bladder cancer therapy.

Our most recent studies demonstrate that miR-137 is downregulated in human bladder cancer (BC) tissues, while treatment of human BC cells with isorhapontigenin (ISO) elevates miR-137 abundance. Since ISO showed a strong inhibition of invasive BC formation in the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced invasive BC mouse model, the elucidation of a potential biological effect of miR-137 on antagonizing BC invasion and molecular mechanisms underlying ISO upregulation of miR-137 are very important. Here we discovered that ectopic expression of miR-137 led to specific inhibition of BC invasion in human high-grade BC T24T and UMUC3 cells, while miR-137 deletion promoted the invasion of both cells, indicating the inhibitory effect of miR-137 on human BC invasion. Mechanistic studies revealed that ISO treatment induced miR-137 transcription by promoting c-Jun phosphorylation and, in turn, abolishing matrix metalloproteinase-2 (MMP-2) abundance and invasion in BC cells. Moreover, miR-137 was able to directly bind to the 3' UTR of Glycogen synthase kinase-3β (GSK3β) mRNA and inhibit GSK3β protein translation, consequently leading to a reduction of heat shock protein-70 (HSP70) translation via targeting the mTOR/S6 axis. Collectively, our studies discover an unknown function of miR-137, directly targeting the 3' UTR of GSK3β mRNA and, thereby, inhibiting GSK3β protein translation, mTOR/S6 activation, and HSP70 protein translation and, consequently, attenuating HSP70-mediated MMP-2 expression and invasion in human BC cells. These novel discoveries provide a deep insight into understanding the biomedical significance of miR-137 downregulation in invasive human BCs and the anti-cancer effect of ISO treatment on mouse invasive BC formation.

Although several previous studies have reported the implication of various microRNAs (miRNAs) in regulation of human bladder cancer (BC) development, alterations and function of many miRNAs in bladder cancer growth are not explored yet at present. Here, we screened 1,900 known miRNAs and first discovered that miR-411 was one of the major miRNAs, which was down-regulated in n-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCs. This miR-411 down-regulation was also observed in human BC tissues and cell lines. The results from evaluating the relationship between miR-411 and patient survival in BC using the TCGA (The Cancer Genome Atlas) database indicated that miR-411 was positively correlated with DFS (disease-free survival). Our studies also showed that miR-411 inhibited tumor growth of human BC cells in a xenograft animal model. Mechanistic studies revealed that overexpression of miR-411 repressed the expression of ALL1-fused gene from the chromosome 1q (AF1q) (MLLT11) by binding to the 3' untranslated region (UTR) of mllt11 mRNA and in turn induced p21 expression and caused cell cycle arrest at the G2/M phase, further inhibiting BC tumor growth. Collectively, our results improve our understanding of the role of miR-411 in BC tumor growth and suggest miR-411 and MLLT11 as potential new targets for the treatment of BC patients.