Master kinase PDK1 in tumorigenesis.

3-phosphoinositide-dependent protein kinase 1 (PDK1) is considered as master kinase regulating AGC kinase family members such as AKT, SGK, PLK, S6K and RSK. Although autophosphorylation regulates PDK1 activity, accumulating evidence suggests that PDK1 is manipulated by many other mechanisms, including S6K-mediated phosphorylation, and the E3 ligase SPOP-mediated ubiquitination and degradation. Dysregulation of these upstream regulators or downstream signals involves in cancer development, as PDK1 regulating cell growth, metastasis, invasion, apoptosis and survival time. Meanwhile, overexpression of PDK1 is also exposed in a plethora of cancers, whereas inhibition of PDK1 reduces cell size and inhibits tumor growth and progression. More importantly, PDK1 also modulates the tumor microenvironments and markedly influences tumor immunotherapies. In summary, we comprehensively summarize the downstream signals, upstream regulators, mouse models, inhibitors, tumor microenvironment and clinical treatments for PDK1, and highlight PDK1 as a potential cancer therapeutic target.

RA-RAR signaling promotes mouse vaginal opening through increasing ß-catenin expression and vaginal epithelial cell apoptosis.

BACKGROUND: Retinoic acid (RA) plays important role in the maintenance and differentiation of the Müllerian ducts during the embryonic stage via RA receptors (RARs). However, the function and mechanism of RA-RAR signaling in the vaginal opening are unknown. METHOD: We used the Rarα knockout mouse model and the wild-type ovariectomized mouse models with subcutaneous injection of RA (2.5 mg/kg) or E2 (0.1 µg/kg) to study the role and mechanism of RA-RAR signaling on the vaginal opening. The effects of Rarα deletion on Ctnnb1 mRNA levels and cell apoptosis in the vaginas were analyzed by real-time PCR and immunofluorescence, respectively. The effects of RA on the expression of ß-catenin and apoptosis in the vaginas were analyzed by real-time PCR and western blotting. The effects of E2 on RA signaling molecules were analyzed by real-time PCR and western blotting. RESULTS: RA signaling molecules were expressed in vaginal epithelial cells, and the mRNA and/or protein levels of RALDH2, RALDH3, RARα and RARγ reached a peak at the time of vaginal opening. The deletion of Rarα resulted in 25.0% of females infertility due to vaginal closure, in which the mRNA (Ctnnb1, Bak and Bax) and protein (Cleaved Caspase-3) levels were significantly decreased, and Bcl2 mRNA levels were significantly increased in the vaginas. The percentage of vaginal epithelium with TUNEL- and Cleaved Caspase-3-positive signals were also significantly decreased in Rarα-/- females with vaginal closure. Furthermore, RA supplementation of ovariectomized wild-type (WT) females significantly increased the expression of ß-catenin, active ß-catenin, BAK and BAX, and significantly decreased BCL2 expression in the vaginas. Thus, the deletion of Rarα prevents vaginal opening by reducing the vaginal ß-catenin expression and epithelial cell apoptosis. The deletion of Rarα also resulted in significant decreases in serum estradiol (E2) and vagina Raldh2/3 mRNA levels. E2 supplementation of ovariectomized WT females significantly increased the expression of RA signaling molecules in the vaginas, suggesting that the up-regulation of RA signaling molecules in the vaginas is dependent on E2 stimulation. CONCLUSION: Taken together, we propose that RA-RAR signaling in the vaginas promotes vaginal opening through increasing ß-catenin expression and vaginal epithelial cell apoptosis.

Associations of timing of physical activity with all-cause and cause-specific mortality in a prospective cohort study.

There is a growing interest in the role of timing of daily behaviors in improving health. However, little is known about the optimal timing of physical activity to maximize health benefits. We perform a cohort study of 92,139 UK Biobank participants with valid accelerometer data and all-cause and cause-specific mortality outcomes, comprising over 7 years of median follow-up (638,825 person-years). Moderate-to-vigorous intensity physical activity (MVPA) at any time of day is associated with lower risks for all-cause, cardiovascular disease, and cancer mortality. In addition, compared with morning group (>50% of daily MVPA during 05:00-11:00), midday-afternoon (11:00-17:00) and mixed MVPA timing groups, but not evening group (17:00-24:00), have lower risks of all-cause and cardiovascular disease mortality. These protective associations are more pronounced among the elderly, males, less physically active participants, or those with preexisting cardiovascular diseases. Here, we show that MVPA timing may have the potential to improve public health.

Lithium treatment promotes the activation of primordial follicles through PI3K/Akt signaling†.

In mammals, dormant primordial follicles represent the ovarian reserve throughout reproductive life. In vitro activation of dormant primordial follicles has been used to treat patients with premature ovarian insufficiency (POI). However, there remains a lack of effective strategies to stimulate follicle activation in vivo. In this study, we used an in vitro ovarian culture system and intraperitoneal injection to study the effect of lithium treatment on primordial follicle activation. Lithium increased the number of growing follicles in cultured mouse ovaries and promoted pre-granulosa cell proliferation. Furthermore, lithium significantly increased the levels of phosphorylated protein kinase B (Akt) and the number of oocytes with forkhead Box O3a (FOXO3a) nuclear export. Inhibition of the phosphatidylinositol 3 kinase (PI3K)/Akt pathway by LY294002 reversed lithium-promoted mouse primordial follicle activation. These results suggest that lithium promotes mouse primordial follicle activation by the PI3K/Akt signaling. Lithium also promoted primordial follicle activation and increased the levels of p-Akt in mouse ovaries in vivo and in human ovarian tissue cultured in vitro. Taken together, lithium promotes primordial follicle activation in mice and humans by the PI3K/Akt signaling. Lithium might be a potential oral drug for treating infertility in POI patients with residual dormant primordial follicles.

Enhanced glycolysis in granulosa cells promotes the activation of primordial follicles through mTOR signaling.

In mammals, nonrenewable primordial follicles are activated in an orderly manner to maintain the longevity of reproductive life. Mammalian target of rapamycin (mTOR)-KIT ligand (KITL) signaling in pre-granulosa cells and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-forkhead Box O3a (FOXO3a) signaling in oocytes are important for primordial follicle activation. The activation process is accompanied by the enhancement of energy metabolism, but the causal relationship is unclear. In the present study, the levels of glycolysis-related proteins GLUT4, HK1, PFKL, and PKM2 were significantly increased in granulosa cells but were decreased in oocytes during the mouse primordial-to-primary follicle transition. Both short-term pyruvate deprivation in vitro and acute fasting in vivo increased the glycolysis-related gene and protein levels, decreased AMPK activity, and increased mTOR activity in mouse ovaries. The downstream pathways Akt and FOXO3a were phosphorylated, resulting in mouse primordial follicle activation. The blockade of glycolysis by 2-deoxyglucose (2-DG), but not the blockade of the communication network between pre-granulosa cells and oocyte by KIT inhibitor ISCK03, decreased short-term pyruvate deprivation-promoted mTOR activity. Glycolysis was also increased in human granulosa cells during the primordial-to-primary follicle transition, and short-term pyruvate deprivation promoted the activation of human primordial follicles by increasing the glycolysis-related protein levels and mTOR activity in ovarian tissues. Taken together, the enhanced glycolysis in granulosa cells promotes the activation of primordial follicles through mTOR signaling. These findings provide new insight into the relationship between glycolytic disorders and POI/PCOS.

SPOP-mediated ubiquitination and degradation of PDK1 suppresses AKT kinase activity and oncogenic functions.

BACKGROUND: 3-phosphoinositide-dependent protein kinase-1 (PDK1) acts as a master kinase of protein kinase A, G, and C family (AGC) kinase to predominantly govern cell survival, proliferation, and metabolic homeostasis. Although the regulations to PDK1 downstream substrates such as protein kinase B (AKT) and ribosomal protein S6 kinase beta (S6K) have been well established, the upstream regulators of PDK1, especially its degrader, has not been defined yet. METHOD: A clustered regularly interspaced short palindromic repeats (CRISPR)-based E3 ligase screening approach was employed to identify the E3 ubiquitin ligase for degrading PDK1. Western blotting, immunoprecipitation assays and immunofluorescence (IF) staining were performed to detect the interaction or location of PDK1 with speckle-type POZ protein (SPOP). Immunohistochemistry (IHC) staining was used to study the expression of PDK1 and SPOP in prostate cancer tissues. In vivo and in vitro ubiquitination assays were performed to measure the ubiquitination conjugation of PDK1 by SPOP. In vitro kinase assays and mass spectrometry approach were carried out to identify casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)-mediated PDK1 phosphorylation. The biological effects of PDK1 mutations and correlation with SPOP mutations were performed with colony formation, soft agar assays and in vivo xenograft mouse models. RESULTS: We identified that PDK1 underwent SPOP-mediated ubiquitination and subsequent proteasome-dependent degradation. Specifically, SPOP directly bound PDK1 by the consensus degron in a CK1/GSK3ß-mediated phosphorylation dependent manner. Pathologically, prostate cancer patients associated mutations of SPOP impaired PDK1 degradation and thus activated the AKT kinase, resulting in tumor malignancies. Meanwhile, mutations that occurred around or within the PDK1 degron, by either blocking SPOP to bind the degron or inhibiting CK1 or GSK3ß-mediated PDK1 phosphorylation, could markedly evade SPOP-mediated PDK1 degradation, and played potently oncogenic roles via activating the AKT kinase. CONCLUSIONS: Our results not only reveal a physiological regulation of PDK1 by E3 ligase SPOP, but also highlight the oncogenic roles of loss-of-function mutations of SPOP or gain-of-function mutations of PDK1 in tumorigenesis through activating the AKT kinase.

Copper Promotes Tumorigenesis by Activating the PDK1-AKT Oncogenic Pathway in a Copper Transporter 1 Dependent Manner.

Copper plays pivotal roles in metabolic homoeostasis, but its potential role in human tumorigenesis is not well defined. Here, it is revealed that copper activates the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB, also termed AKT) oncogenic signaling pathway to facilitate tumorigenesis. Mechanistically, copper binds 3-phosphoinositide dependent protein kinase 1 (PDK1), in turn promotes PDK1 binding and subsequently activates its downstream substrate AKT to facilitate tumorigenesis. Blocking the copper transporter 1 (CTR1)-copper axis by either depleting CTR1 or through the use of copper chelators diminishes the AKT signaling and reduces tumorigenesis. In support of an oncogenic role for CTR1, the authors find that CTR1 is abnormally elevated in breast cancer, and is subjected by NEDD4 like E3 ubiquitin protein ligase (Nedd4l)-mediated negative regulation through ubiquitination and subsequent degradation. Accordingly, Nedd4l displays a tumor suppressive function by suppressing the CTR1-AKT signaling. Thus, the findings identify a novel regulatory crosstalk between the Nedd4l-CTR1-copper axis and the PDK1-AKT oncogenic signaling, and highlight the therapeutic relevance of targeting the CTR1-copper node for the treatment of hyperactive AKT-driven cancers.

USP7 stabilizes EZH2 and enhances cancer malignant progression.

EZH2, a histone methylase, plays a critical role in the tumor progression via regulation of progenitor genes. However, the detailed molecular mechanism of EZH2 in cancer malignant progression remains unknown. Therefore, we aimed to investigate how EZH2 is regulated in human cancer. We used numerous approaches, including Co-immunoprecipitation (Co-IP), Transfection, RT-PCR, Western blotting, Transwell assays, and animal studies, to determine the deubiquitination mechanism of EZH2 in cancer cells. We demonstrated that USP7 regulated EZH2 in human cancer cells and in vivo in mouse models. Overexpression of USP7 promoted the expression of EZH2 protein, but overexpression of a USP7 mutant did not change the EZH2 level. Consistently, knockdown of USP7 resulted in a striking decrease in EZH2 protein levels in human cancer cells. Functionally, USP7 overexpression promoted cell growth and invasion via deubiquitination of EZH2. Consistently, downregulation of USP7 inhibited cell migration and invasion in cancer. More importantly, knockdown of USP7 inhibited tumor growth, while USP7 overexpression exhibited opposed effect in mice. Our results indicate that USP7 regulates EZH2 via its deubiquitination and stabilization. The USP7/EZH2 axis could present a new promising therapeutic target for cancer patients.

AKT methylation by SETDB1 promotes AKT kinase activity and oncogenic functions.

Aberrant activation of AKT disturbs the proliferation, survival and metabolic homeostasis of various human cancers. Thus, it is critical to understand the upstream signalling pathways governing AKT activation. Here, we report that AKT undergoes SETDB1-mediated lysine methylation to promote its activation, which is antagonized by the Jumonji-family demethylase KDM4B. Notably, compared with wild-type mice, mice harbouring non-methylated mutant Akt1 not only exhibited reduced body size but were also less prone to carcinogen-induced skin tumours, in part due to reduced AKT activation. Mechanistically, the interaction of phosphatidylinositol (3,4,5)-trisphosphate with AKT facilitates its interaction with SETDB1 for subsequent AKT methylation, which in turn sustains AKT phosphorylation. Pathologically, genetic alterations, including SETDB1 amplification, aberrantly promote AKT methylation to facilitate its activation and oncogenic functions. Thus, AKT methylation is an important step, synergizing with PI3K signalling to control AKT activation. This suggests that targeting SETDB1 signalling could be a potential therapeutic strategy for combatting hyperactive AKT-driven cancers.

Rottlerin inhibits cell growth and invasion via down-regulation of EZH2 in prostate cancer.

Rottlerin as a natural agent, which is isolated from Mallotus philippinensis, has been identified to play a critical role in tumor inhibition. However, the molecular mechanism of rottlerin-mediated anti-tumor activity is still ambiguous. It has been reported that EZH2 exhibits oncogenic functions in a variety of human cancers. Therefore, inhibition of EZH2 could be a promising strategy for the treatment of human cancers. In this study, we aim to explore whether rottlerin could inhibit tumorigenesis via suppression of EZH2 in prostate cancer cells. Multiple approaches such as FACS, Transwell invasion assay, RT-PCR, Western blotting, and transfection were performed to determine our aim. We found that rottlerin treatment led to inhibition of cell growth, migration and invasion, but induction of apoptosis in prostate cancer cells. Importantly, we defined that rottlerin decreased the expression of EZH2 and H3K27me3 in prostate cancer cells. Moreover, overexpression of EZH2 abrogated the rottlerin-induced inhibition of cell growth, migration, and invasion in prostate cancer cells. Consistently, down-regulation of EZH2 enhanced rottlerin-triggered anti-tumor function. Collectively, our work demonstrated that rottlerin exerted its tumor suppressive function via inhibition of EZH2 expression in prostate cancer cells. Our findings indicated that rottlerin might be a potential therapeutic compound for treating patients with prostate cancer.

MAPK3/1 participates in the activation of primordial follicles through mTORC1-KITL signaling.

The majority of ovarian primordial follicles are preserved in a dormant state to maintain the female reproductive lifespan, and only a few primordial follicles are activated to enter the growing follicle pool in each wave. Recent studies have shown that primordial follicular activation depends on mammalian target of rapamycin complex 1 (mTORC1)-KIT ligand (KITL) signaling in pre-granulosa cells and its receptor (KIT)-phosphoinositol 3 kinase (PI3K) signaling in oocytes. However, the upstream regulator of mTORC1 signaling is unclear. The results of the present study showed that the phosphorylated mitogen-activated protein kinase3/1 (MAPK3/1) protein is expressed in some primordial follicles and all growing follicles. Culture of 3 days post-parturition (dpp) ovaries with the MAPK3/1 signaling inhibitor U0126 significantly reduced the number of activated follicles and was accompanied by dramatically reduced granulosa cell proliferation and increased oocyte apoptosis. Western blot and immunofluorescence analyses showed that U0126 significantly decreased the phosphorylation levels of Tsc2, S6K1, and rpS6 and the expression of KITL, indicating that U0126 inhibits mTORC1-KITL signaling. Furthermore, U0126 decreased the phosphorylation levels of Akt, resulting in a decreased number of oocytes with Foxo3 nuclear export. To further investigate MAPK3/1 signaling in primordial follicle activation, we used phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor bpV(HOpic) to promote primordial follicle activation. In this model, U0126 also inhibited the activation of primordial follicles and mTORC1 signaling. Thus, these results suggest that MAPK3/1 participates in primordial follicle activation through mTORC1-KITL signaling.

Inhibition of Notch-1 pathway is involved in rottlerin-induced tumor suppressive function in nasopharyngeal carcinoma cells.

Recent studies have revealed that rottlerin is a natural chemical drug to exert its anti-cancer activity. However, the molecular mechanisms of rottlerin-induced tumor suppressive function have not been fully elucidated. Notch signaling pathway has been characterized to play a crucial role in tumorigenesis. Therefore, regulation of Notch pathway could be beneficial for the treatment of human cancer. The aims of our current study were to explore whether rottlerin could suppress Notch-1 expression, which leads to inhibition of cell proliferation, migration and invasion in nasopharyngeal carcinoma cells. We performed several approaches, such as CTG, Flow cytometry, scratch healing assay, transwell and Western blotting. Our results showed that rottlerin treatment inhibited cell growth, migration and invasion, and triggered apoptosis, and arrested cell cycle to G1 phase. Moreover, the expression of Notch-1 was obvious decreased in nasopharyngeal carcinoma cells after rottlerin treatment. Importantly, overexpression of Notch-1 promoted cell growth and invasion, whereas down-regulation of Notch-1 inhibited cell growth and invasion in nasopharyngeal carcinoma cells. Notably, we found the over-expression of Notch-1 could abrogate the anti-cancer function induced by rottlerin. Strikingly, our study implied that Notch-1 could be a useful target of rottlerin for the prevention and treatment of human nasopharyngeal carcinoma.

MiR-26b reverses temozolomide resistance via targeting Wee1 in glioma cells.

Emerging evidence has demonstrated that microRNAs (miRNA) play a critical role in chemotherapy-induced epithelial-mesenchymal transition (EMT) in glioma. However, the underlying mechanism of chemotherapy-triggered EMT has not been fully understood. In the current study, we determined the role of miR-26b in regulation of EMT in stable temozolomide (TMZ)-resistant (TR) glioma cells, which have displayed mesenchymal features. Our results illustrated that miR-26b was significantly downregulated in TR cells. Moreover, ectopic expression of miR-26b by its mimics reversed the phenotype of EMT in TR cells. Furthermore, we found that miR-26b governed TR-mediate EMT partly due to governing its target Wee1. Notably, overexpression of miR-26b sensitized TR cells to TMZ. These findings suggest that upregulation of miR-26b or targeting Wee1 could serve as novel approaches to reverse chemotherapy resistance in glioma.

The effects of curcumin on proliferation, apoptosis, invasion, and NEDD4 expression in pancreatic cancer.

Pancreatic cancer (PC) is one of the most fatal cancers worldwide. The incidence and death rates are still increasing for PC. Curcumin is the biologically active diarylheptanoid constituent of the spice turmeric, which exerts its anticancer properties in various human cancers including PC. In particular, accumulating evidence has proved that curcumin targets numerous therapeutically important proteins in cell signaling pathways. The neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) is an E3 HECT ubiquitin ligase and is frequently over-expressed in various cancers. It has reported that NEDD4 might facilitate tumorigenesis via targeting and degradation of multiple tumor suppressor proteins including PTEN. Hence, in the present study we explore whether curcumin inhibits NEDD4, resulting in the suppression of cell growth, migration and invasion in PC cells. We found that curcumin inhibited cell proliferation and triggered apoptosis in PC, which is associated with increased expression of PTEN and p73. These results suggested that inhibition of NEDD4 might be beneficial to the antitumor properties of curcumin on PC treatments.

OncomicroRNAs-Mediated Tumorigenesis: Implication in Cancer Diagnosis and Targeted Therapy.

MicroRNAs (miRNAs) control the expression of approximately 60% of protein-coding genes and regulate cell metabolism, proliferation, differentiation, and apoptosis. Notably, aberrant expression of miRNAs contributes to several diseases including cancer. Accumulating evidence indicates that miRNAs play important roles in EMT, genesis of cancer stem cells, cancer metabolism and carcinogenesis. Aberrant expression of miRNAs triggers tumor initiation, progression and poor prognosis of cancer patients. Accordingly, oncogenic miRNAs have emerged as diagnostic biomarkers and targets for novel anti-cancer drug discovery. However, the mechanisms of miRNAs contriving tumorigenesis are not completely understood. This review aims to clarify the identification of tumorspecific miRNAs, verification of oncogenic miRNA signatures, and dynamic study of oncogenic miRNAs in cancer initiation and development. Despite sound progress in miRNA-mediated anticancer therapy, several barriers like drug stability, immunogenicity, off-target effects and toxicities still remain. We hope our review could stimulate the further study of miRNAs in cancer research field, which may lead to new insights into the mechanisms of carcinogenesis and create new avenues for targeted cancer therapy.

Rottlerin exhibits antitumor activity via down-regulation of TAZ in non-small cell lung cancer.

Rottlerin, a polyphenolic compound derived from Mallotus philipinensis, has been reported to exhibit anti-tumor activities in a variety of human malignancies including NSCLC (non-small cell lung cancer). TAZ (transcriptional co-activator with PDZ-binding motif), one of the key activators in Hippo pathway, has been characterized as an oncoprotein. Therefore, inhibition of TAZ could be useful for the treatment of human cancers. In the current study, we aimed to explore whether rottlerin inhibits the expression of TAZ in NSCLC, leading to its anti-cancer activity. Multiple approaches were applied for determining the mechanism of rottlerin-mediated anti-tumor function, including cell growth assay, Flow cytometry, wound healing assay, invasion assay, Western blotting, and transfection. We found that rottlerin inhibited cell growth, triggered apoptosis, arrested cell cycle, and retarded cell invasion in NSCLC cells. Moreover, our results showed that overexpression of TAZ enhanced cell growth, stimulated apoptosis, and promoted cell migration and invasion. Consistently, inhibition of TAZ exhibited anti-tumor activity in NSCLC cells. Notably, we validated that rottlerin exerted its tumor suppressive function via inactivation of TAZ in NSCLC cells. Taken together, our study indicates that inhibition of TAZ by rottlerin could be a promising strategy for the prevention and therapy of NSCLC.

Targeting of multiple senescence-promoting genes and signaling pathways by triptonide induces complete senescence of acute myeloid leukemia cells.

Leukemia cells aberrantly overexpress senescence-suppression telomerase reverse transcriptase (TERT) and down-regulate key senescence-promoting genes to escape complete senescence, resulting in immortalization and malignant progression. Accordingly, induction of complete senescence is a sensible strategy for anti-leukemia therapy. However, effective senescence-based anti-leukemia drugs with low toxicity are currently lacking. In this study, we found that triptonide (chemical name diterpene triepoxide), a small molecule derived from the herb Tripterygium wilfordii Hook, strongly induced complete senescence in cultured acute myeloid leukemia (AML) cell lines, and potently inhibited growth and colony formation of U937 and HL-60 AML cell line with IC50 values of 7.5 and 12nM, respectively. Strikingly, triptonide (4mg/kg) nearly completely suppressed human leukemia cell tumorigenicity (>99%) without obvious toxicity in a mouse xenograft model. Mechanistic studies showed that triptonide induced senescence followed by apoptosis mainly by suppressing transcription of TERT and oncogenic c-Myc, while concomitantly promoting transcription of senescence-promoting genes p16 and p21 and the pro-apoptotic gene encoding DNA damage-inducible transcript 3. These effects of triptonide are mediated by selective mitogen-activated protein kinase kinase-3/p38 signaling pathway activation. Our study provides a conceptual framework for inducing complete senescence as an effective anti-leukemia therapeutic strategy through a "multiple-hits" model and supports further development of triptonide as an anti-cancer agent.