B7H3 increases ferroptosis resistance by inhibiting cholesterol metabolism in colorectal cancer.

Ferroptosis, a newly discovered form of regulated cell death, has been reported to be associated with multiple cancers, including colorectal cancer (CRC). However, the underlying molecular mechanism is still unclear. In this study, we identified B7H3 as a potential regulator of ferroptosis resistance in CRC. B7H3 knockdown decreased but B7H3 overexpression increased the ferroptosis resistance of CRC cells, as evidenced by the expression of ferroptosis-associated genes (PTGS2, FTL, FTH, and GPX4) and the levels of important indicators of ferroptosis (malondialdehyde, iron load). Moreover, B7H3 promoted ferroptosis resistance by regulating sterol regulatory element binding protein 2 (SREBP2)-mediated cholesterol metabolism. Both exogenous cholesterol supplementation and treatment with the SREBP2 inhibitor betulin reversed the effect of B7H3 on ferroptosis in CRC cells. Furthermore, we verified that B7H3 downregulated SREBP2 expression by activating the AKT pathway. Additionally, multiplex immunohistochemistry was carried out to show the expression of B7H3, prostaglandin-endoperoxide synthase 2, and SREBP2 in CRC tumor tissues, which was associated with the prognosis of patients with CRC. In summary, our findings reveal a role for B7H3 in regulating ferroptosis by controlling cholesterol metabolism in CRC.

B7-H3 confers stemness characteristics to gastric cancer cells by promoting glutathione metabolism through AKT/pAKT/Nrf2 pathway.

BACKGROUND: Cancer stem-like cells (CSCs) are a small subset of cells in tumors that exhibit self-renewal and differentiation properties. CSCs play a vital role in tumor formation, progression, relapse, and therapeutic resistance. B7-H3, an immunoregulatory protein, has many protumor functions. However, little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer (GC) stemness. Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism. METHODS: GC stemness influenced by B7-H3 was detected both in vitro and in vivo . The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction, Western blotting, and flow cytometry. Sphere formation assay was used to detect the sphere-forming ability. The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments. The signaling pathway (Protein kinase B [Akt]/Nuclear factor erythroid 2-related factor 2 [Nrf2] pathway) of B7-H3 on the regulation of glutathione (GSH) metabolism was examined by Western blotting assay. Multi-color immunohistochemistry (mIHC) was used to detect the expression of B7-H3, cluster of differentiation 44 (CD44), and Nrf2 on human GC tissues. Student's t -test was used to compare the difference between two groups. Pearson correlation analysis was used to analyze the relationship between two molecules. The Kaplan-Meier method was used for survival analysis. RESULTS: B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo . Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells, which was further confirmed by the experimental results. Meanwhile, stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism. Furthermore, Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway, and inhibition of AKT signaling pathway could suppress not only GSH metabolism but also GC stemness. mIHC showed that B7-H3 was highly expressed in GC tissues and was positively correlated with the expression of CD44 and Nrf2. Importantly, GC patients with high expression of B7-H3, CD44, and Nrf2 had worse prognosis ( P = 0.02). CONCLUSIONS: B7-H3 has a regulatory effect on GC stemness and the regulatory effect is achieved through the AKT/Nrf2/GSH pathway. Inhibiting B7-H3 expression may be a new therapeutic strategy against GC.

The way of interaction between Vγ9Vδ2 T cells and tumor cells.

Immunotherapy has been a promising, emerging treatment for various cancers. Gamma delta (γδ) T cells own a T cell receptor composed of γ- and δ- chain and act as crucial players in the anti-tumor immune effect. Currently, Vγ9Vδ2 T cells, the predominate γδ T cell subset in human peripheral blood, has been shown to exert multiple biological functions. In addition, a growing body of evidence notes that Vγ9Vδ2 T cells interact with tumor cells in many ways, such as TCR-mediated nonpeptidic-phosphorylated phosphoantigens (pAgs) recognization, NKG2D/NKG2D ligand (NKG2DL) pathway, Fas-FasL axis and antibody-dependent cellular cytotoxicity (ADCC) as well as exosome. More importantly, clinical studies with Vγ9Vδ2 T cells in cancers have propelled several clinical applications to investigate their safety and efficacy. Herein, this review summarized the underlying ways and mechanisms of interplay cancer cells and Vγ9Vδ2 T cells, which may help us to generate new strategies for tumor immunotherapy in the future.

MARCKSL1-2 reverses docetaxel-resistance of lung adenocarcinoma cells by recruiting SUZ12 to suppress HDAC1 and elevate miR-200b.

BACKGROUND: Long non-coding RNAs (lncRNAs) are implicated in the development of multiple cancers. In our previous study, we demonstrated that HDAC1/4-mediated silencing of microRNA-200b (miR-200b) enhances docetaxel (DTX)-resistance of human lung adenocarcinoma (LAD) cells. METHODS AND RESULTS: Herein, we probed the function of LncRNA MARCKSL1-2 (MARCKSL1-transcript variant 2, NR_052852.1) in DTX resistance of LAD cells. It was found that MARCKSL1-2 expression was markedly reduced in DTX-resistant LAD cells. Through gain- or loss- of function assays, colony formation assay, EdU assay, TUNEL assay, and flow cytometry analysis, we found that MARCKSL1-2 suppressed the growth and DTX resistance of both parental and DTX-resistant LAD cells. Moreover, we found that MARCKSL1-2 functioned in LAD through increasing miR-200b expression and repressing HDAC1. Mechanistically, MARCKSL1-2 recruited the suppressor of zeste 12 (SUZ12) to the promoter of histone deacetylase 1 (HDAC1) to strengthen histone H3 lysine 27 trimethylation (H3K27me3) of HDAC1 promoter, thereby reducing HDAC1 expression. MARCKSL1-2 up-regulated miR-200b by blocking the suppressive effect of HDAC1 on the histone acetylation modification at miR-200b promoter. Furthermore, in vivo analysis using mouse xenograft tumor model supported that overexpression of MARCKSL1-2 attenuated the DTX resistance in LAD tumors. CONCLUSIONS: We confirmed that MARCKSL1-2 alleviated DTX resistance in LAD cells by abolishing the inhibitory effect of HDAC1 on miR-200b via the recruitment of SUZ12. MARCKSL1-2 could be a promising target to improve the chemotherapy of LAD.

The potential roles and mechanisms of non-coding RNAs in cancer anoikis resistance.

Increasing evidence indicates that anoikis resistance is a critical process for metastasis of cancer cells, making it the attractive therapeutic target for cancer benefit. Anoikis resistance is widely regulated by various factors, such as signaling pathways, integrins switch, and non-coding RNAs (ncRNAs). ncRNAs composed of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), are frequently dysregulated in a variety of human malignancies and are closely related to anoikis resistance of cancer cells. Based on the available literature, we reviewed the molecular basis underlying ncRNAs modulating cancer cells anoikis resistance, which may contribute to a better understanding of cancer metastasis and provide new beneficial therapeutic strategies against cancer.

Tilianin inhibits the human ovarian cancer (PA-1) cell proliferation via blocking cell cycle, inducing apoptosis and inhibiting JAK2/STAT3 signaling pathway.

Ovarian cancer is one of the deadliest gynecologic malignancies and is the seventh leading cause of mortalities and morbidities globally. Although there are various therapeutic strategies, a major challenge for scientific community is to come up with effective strategy to treat ovarian cancer. Tilianin, a polyphenol flavonoid is well known for its extensive biological actions like cardioprotective, neuroprotective, anti-oxidant, anti-inflammatory, anti-diabetic and anti-tumor properties. The current study is designed to investigate the anti-cancer action of Tilianin in ovarian cancer (PA-1) cells. The findings of this study revealed that Tilianin treatment results in significant and concentration dependent decrease in cell viability. The growth inhibiting action of Tilianin is associated with apoptosis which was confirmed by DAPI and AO/EtBr staining. The Tilianin-triggered apoptosis in PA-1 cells was correlated with elevated generation of ROS, loss of mitochondrial membrane potential, alterations in pro-apoptotic (upregulated mRNA expression of Bax) and anti-apoptotic (downregulated mRNA expression of Bcl2) factors and activation of caspase-8, -9 and -3. Cell cycle analysis revealed that Tilianin treatment prevented G1/S transition through reduced mRNA expression of cyclin D1. Additionally, the findings of this study also showed Tilianin inhibited JAK2/STAT3 signaling (downregulated expression of pJAK2, JAK2, pSTAT3, and STAT3) with no change in mRNA expression level of ERK indicating its non-involvement in the apoptotic and/or growth inhibition of ovarian cancer cells. In conclusion, the findings of this exploration provided clear evidence of anti-cancer effects of Tilianin in PA-1 cells through its anti-proliferative action, ability to induce apoptosis both through extrinsic and intrinsic pathways, cell cycle (G1/S) arrest and JAK2/STAT3 signaling inhibition.

B7-H5 blockade enhances CD8+ T-cell-mediated antitumor immunity in colorectal cancer.

Negative immune checkpoint blockade immunotherapy has shown potential for multiple malignancies including colorectal cancer (CRC). B7-H5, a novel negative immune checkpoint regulator, is highly expressed in tumor tissues and promotes tumor immune escape. However, the clinical significance of B7-H5 expression in CRC and the role of B7-H5 in the tumor microenvironment (TME) has not been fully clarified. In this study, we observed that high B7-H5 expression in CRC tissues was significantly correlated with the lymph node involvement, AJCC stage, and survival of CRC patients. A significant inverse correlation was also observed between B7-H5 expression and CD8+ T-cell infiltration in CRC tissues. Kaplan-Meier analysis showed that patients with high B7-H5 expression and low CD8+ T-cell infiltration had the worst prognosis in our cohort of CRC patients. Remarkably, both high B7-H5 expression and low CD8+ T infiltration were risk factors for overall survival. Additionally, B7-H5 blockade using a B7-H5 monoclonal antibody (B7-H5 mAb) effectively suppressed the growth of MC38 colon cancer tumors by enhancing the infiltration and Granzyme B production of CD8+ T cells. Importantly, the depletion of CD8+ T cells obviously abolished the antitumor effect of B7-H5 blockade in the MC38 tumors. In sum, our findings suggest that B7-H5 may be a valuably prognostic marker for CRC and a potential target for CRC immunotherapy.

B7-H3 suppresses doxorubicin-induced senescence-like growth arrest in colorectal cancer through the AKT/TM4SF1/SIRT1 pathway.

Emerging evidence suggests that cellular senescence induced by chemotherapy has been recognized as a new weapon for cancer therapy. This study aimed to research novel functions of B7-H3 in cellular senescence induced by a low dose of doxorubicin (DOX) in colorectal cancer (CRC). Here, our results demonstrated that B7-H3 knockdown promoted, while B7-H3 overexpression inhibited, DOX-induced cellular senescence. B7-H3 knockdown dramatically enhanced the growth arrest of CRC cells after low-dose DOX treatment, but B7-H3 overexpression had the opposite effect. By RNA-seq analysis and western blot, we showed that B7-H3 prevented cellular senescence and growth arrest through the AKT/TM4SF1/SIRT1 pathway. Blocking the AKT/TM4SF1/SIRT1 pathway dramatically reversed B7-H3-induced resistance to cellular senescence. More importantly, B7-H3 inhibited DOX-induced cellular senescence of CRC cells in vivo. Therefore, targeting B7-H3 or the B7-H3/AKT/TM4SF1/SIRT1 pathway might be a new strategy for promoting cellular senescence-like growth arrest during drug treatment in CRC.

MICA enhances sensitivity to cisplatin in patients with extensive small cell lung cancer via downregulation of ABCG2.

Immunotherapy utilizing natural killer cell-activated receptor natural-killer group-2 member D ligands (NKG2DLs) has had preclinical success in the treatment of small cell lung cancer. The present study aimed to investigate the association between NKG2Ls and chemoresistance. The mRNA expression of six NKG2DLs associated with progression-free survival time (PFS) and first-line chemotherapy were assessed in the present study. Major histocompatibility complex class I polypeptide-related sequence A (MICA)-overexpressing NCI-H446 cell line was constructed, and the mRNA expression levels of 11 genes associated with chemotherapy sensitivity were determined. The results demonstrated that MICA was positively and significantly associated with PFS. Furthermore, MICA expression was 1.6 times higher in patients with prolonged PFS compared with the rapid chemoresistance group. ATP binding cassette subfamily G member 2 (ABCG2) mRNA expression was associated with chemotherapy resistance and significantly downregulated in the cell line overexpressing MICA. Moreover, following addition of nicardipine (an ABCG2 inhibitor), chemotherapeutic sensitivity increased in the MICA-overexpressing cell line. Taken together, the results of the present study suggested that MICA may enhance the chemosensitivity of patients with extensive small cell lung cancer by downregulating ABCG2.

Transforming the spleen into a liver-like organ in vivo.

Regenerating human organs remains an unmet medical challenge. Suitable transplants are scarce, while engineered tissues have a long way to go toward clinical use. Here, we demonstrate a different strategy that successfully transformed an existing, functionally dispensable organ to regenerate another functionally vital one in the body. Specifically, we injected a tumor extract into the mouse spleen to remodel its tissue structure into an immunosuppressive and proregenerative microenvironment. We implanted autologous, allogeneic, or xenogeneic liver cells (either primary or immortalized), which survived and proliferated in the remodeled spleen, without exerting adverse responses. Notably, the allografted primary liver cells exerted typical hepatic functions to rescue the host mice from severe liver damages including 90% hepatectomy. Our approach shows its competence in overcoming the key challenges in tissue regeneration, including insufficient transplants, immune rejection, and poor vascularization. It may be ready for translation into new therapies to regenerate large, complex human tissue/organs.

Prognostic value of the systemic inflammation response index (SIRI) before and after surgery in operable breast cancer patients.

BACKGROUND: The new systemic inflammation response index (SIRI) constructed based on neutrophil, monocyte and lymphocyte counts in peripheral blood is considered to be related to the prognosis of a variety of tumours. OBJECTIVE: To evaluate the prognostic value of the SIRI in operable breast cancer patients and establish a nomogram to predict the survival of breast cancer patients. METHODS: A total of 949 patients with operable breast cancer were enrolled in the present study. RESULTS: The overall survival (OS) of breast cancer patients with SIRI ⩽ 0.65 was significantly higher than that of breast cancer patients with SIRI > 0.65 (P< 0.001). A nomogram generated based on SIRI, grade and TNM stage and SIRI predicted the 5- and 10-year survival rates of breast cancer patients more accurately than TNM stage alone. In addition, the change in SIRI relative to baseline at 4 weeks after surgery was closely related to the survival of breast cancer patients. Compared with those with no SIRI changes (absolute value of variation < 25%), breast cancer patients with an increase in SIRI > 75% or 25-75% had worse OS (P< 0.001). CONCLUSIONS: The SIRI before and after surgery is closely related to the prognosis of breast cancer patients.

Clinical significance of long non-coding RNA ZEB2-AS1 in locally advanced colorectal cancer.

OBJECTIVE: The aim of this study was to investigate the clinical significance of differential expression of long non-coding RNA (lncRNA) ZEB2-AS1 in patients with colorectal cancer (CRC). METHODS: mRNA expression of lncRNA ZEB2-AS1 was evaluated by real-time quantitative PCR on eighty-seven cancerous tissues and adjacent normal mucosal tissues from patients with CRC tissue. Correlation between the lncRNA ZEB2-AS1 expression and clinicopathological characteristics of the colorectal cancer patients was evaluated, and five-year overall survival (OS) was also analyzed according to the lncRNA ZEB2-AS1 expression of the CRC patients. Moreover, Cox Regression Analysis was performed in screening prognosis factors. RESULTS: A significantly upregulated lncRNA ZEB2-AS1 expression, with a fold change of 18.75, was found in CRC tissue compared to the normal tissue. lncRNA ZEB2-AS1 expression in CRC was correlated with death (P<0.001). The five-year OS was 43.2% and 76.7%, respectively, in patients with higher and lower lncRNA ZEB2-AS1 expression. Cox regression analysis showed that location (P=0.020), N1 staging (P=0.021) and lncRNA ZEB2-AS1 lower expression (P<0.001) were independent prognosis factors associated with a better OS. CONCLUSION: Expression of lncRNA ZEB2-AS1 was significantly upregulated in stage III CRC patients and affects the prognosis.

ZNF281 Promotes Growth and Invasion of Pancreatic Cancer Cells by Activating Wnt/ß-Catenin Signaling.

BACKGROUND: Zinc finger protein 281 (ZNF281) has been identified to be involved in embryonic stem cell differentiation and tissue development. Also, ZNF281 was found in various types of cancers. However, its biological functions and clinical significance in pancreatic cancer remain elusive. AIMS: To explore the role of ZNF281 in pancreatic cancer cells proliferation and invasion. METHODS: ZNF281 expression was examined in public database Oncomine and cBioPortal. The correlation between ZNF281 and clinicopathological features was measured, and Kaplan-Meier method was used to measure the overall survival and recurrence-free survival in the TCGA cohort. Ectopic expression and knockdown of ZNF281 were performed to measure the impact on cell proliferation and invasion. Western blot and immunoprecipitation were further used to identify the ZNF281 interacting proteins. Topflash luciferase assay was used to detect the Wnt/ß-catenin signaling activation. RESULTS: ZNF281 was predominantly up-regulated in pancreatic cancer tissues and significantly associated with advanced stage. Meanwhile, the high expression of ZNF281 indicated shorter overall survival and recurrence-free survival and ZNF281 could be an independent prognostic factor of pancreatic cancer. ZNF281 promoted cell proliferation and invasion in vitro. Mechanically, ZNF281 activated Wnt/ß-catenin signaling and induced the downstream gene expression by directly binding with ß-catenin and decreasing the polyubiquitination. CONCLUSIONS: ZNF281 promotes pancreatic cancer cells proliferation and invasion by interacting and up-regulating ß-catenin, highlighting the role of ZNF281 in pancreatic cancer progression.

Sox9 mediated transcriptional activation of FOXK2 is critical for colorectal cancer cells proliferation.

FOXK2, which belongs to the fork head DNA binding protein family, has been shown to play a critical role in tumorigenesis. Here, we detected FOXK2 expression and its clinical significance in colorectal cancer, which has not been fully investigated before. Results from public database and our cohort indicated that FOXK2 was transcriptionally activated in colorectal cancer tissues compared to non-cancer tissues. High expression of FOXK2 was significantly correlated with poor survival. In vitro cell experiments suggested that FOXK2 promoted cell proliferation. Furthermore, we found that oncogene SOX9 was responsible for the up-regulation of FOXK2 by directly binding on its promoter. Depletion of FOXK2 attenuated SOX9 induced cell growth. In addition, we observed that the expression of FOXK2 was significantly associated with the expression of SOX9 both in the public database and our colorectal cancer tissues. The patients with SOX9+FOXK2+ had a poor overall survival (p = 0.0084). In conclusion, our data suggested that SOX9 transcriptionally activated FOXK2 was involved in the pathogenesis of colorectal cancer and might be a novel target for colorectal cancer therapy.

Depression induces poor prognosis associates with the down-regulation brain derived neurotrophic factor of serum in advanced small cell lung cancer.

Patients with lung cancer often experience a state of depression, and these conditions may severely affect their quality of life (QoL) and prescription compliance. The current study was conducted to delineate the complex links between depression and the prognosis of patients with small cell lung cancer (SCLC) and the underlying mechanism was also explored.186 patients who received platinum-based chemotherapy for newly diagnosed stage III or stage IV SCLC were enrolled. The Self-Rating Depression Scale (SDS) questionnaire was completed the day before the start of chemotherapy to assess the depression status of the patients. Patients with stage IV SCLC or lower BMI have higher depression scores. In terms of the adverse effects of chemotherapy, depression severely decreases patient tolerance to chemotherapy and their QoL score (R2 = 0.2385) and is also associated with severe vomiting (P < 0.001), leukopenia (R2 = 0.2332), and prolonged hospital stay (R2 = 0.1961). More importantly, severe depression reduces the PFS (R2 = 0.1943) and OS (P < 0.01) of the patients. We found that patients with severe depression displayed a downregulated level of serum BDNF and that the level of serum BDNF was highly correlated with the OS of the patients (R2 = 0.2292). Using the MTT cell viability assay in vitro, we observed that cotreatment with BDNF clearly enhanced the chemosensitivity of NCI-H69 tumor cells to Cisplatin and induced the downregulation of ABCG2.Based on this evidence, it appears that a relationship does exist between depression and prognosis in SCLC and that the mechanism by which depression affects prognosis is achieved via the downregulation of BDNF expression.

Cholesterol reduces the sensitivity to platinum-based chemotherapy via upregulating ABCG2 in lung adenocarcinoma.

Inoperable lung adenocarcinoma is currently treated with platinum-based chemotherapy. However, the effectiveness of these chemotherapeutic agents is not the same for all patients. Patients either show quick chemoresistance (QCR) or delayed chemoresistance (DCR), which are defined by 87 and 242 days of progression-free survival (PFS) after initial platinum-based treatment, respectively. We found that QCR patients displayed an elevated level of serum cholesterol and that their tumors showed upregulated ABCG2 expression. We propose that chemoresistance may be attributed to cholesterol-induced ABCG2 expression and hypothesize that blocking ABCG2 may increase the efficacy of platinum-based chemotherapeutic agents. Using the MTT cell viability assay, we observed that cotreatment with ABCG2 blocker Nicardipine and platinum-based drugs Cisplatin, Oxaliplatin or Carboplatin significantly decreased cell viability of tumor cells. Importantly, our results also showed that incubating cells with cholesterol prior to chemotherapy treatment or cotreatment increased cell viability of tumor cells relative to the controls.

Clinical implication of Sox9 and activated Akt expression in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most leading causes of cancer-related death. Cancer stem cell is responsible for tumor initiation, metastasis and relapse. Sox9 is a pancreatic stem cell marker. PI3K/PTEN/Akt/mTORC is an important signal for maintaining stem cells. The purpose of this study is to determine the expression pattern of Sox9 and p-Akt in human PDAC and its correlation with prognosis. Immunohistochemical analysis was used to explore the expression of Sox9 and p-Akt in 88 human PDAC patients. The Pearson's test was used to compare the clinicopathological parameters between negative and positive expressors. The Pearson's correlation analysis was used to explore the relationship between Sox9 and p-Akt expression. Kaplan-Meier's method and Cox regression analysis were used to analyze patients' survival. The results showed that Sox9 and p-Akt overactivated in PDAC (p = 0.011, p = 0.008). Sox9-positive expression is significantly associated with distant metastasis (p = 0.046). p-Akt-positive expression is significantly associated with distant metastasis (p = 0.000), TNM stage (0.001) and PCNA expression (p = 0.000). Sox9 expression is positively correlated with p-Akt expression (r = 0.314, p = 0.003). In 54 patients with survival information, both Sox9- and p-Akt-positive expressions are associated with unfavorable prognosis (p = 0.002, p = 0.000). Sox9 and p-Akt double-positive expressor showed much poorer prognosis (p = 0.000). Cox regression analysis showed that Sox9- or p-Akt-positive expression and LN metastasis were independent prognostic factors. This study provides the first evidence that Sox9 and p-Akt are both relevant to distant metastasis and proliferation. Our data suggest the potential of Sox9 and p-Akt as prognostic biomarkers for PDAC.

KLF9, a transcription factor induced in flutamide-caused cell apoptosis, inhibits AKT activation and suppresses tumor growth of prostate cancer cells.

BACKGROUND: Kruppel-like factors (KLFs) are involved in various biological processes; emerging studies have indicated that KLF9 plays a critical role in regulating tumorigenesis. The role of KLF9 in prostate cancer (PCa), however, has not yet been investigated. METHODS: The expression of KLF members, AKT- and apoptosis-related proteins were analyzed by Western blot or qRT-PCR. Tet-On inducible KLF9 expression was established for the evaluation of the effects of KLF9 on cell proliferation, apoptosis, and xenograft tumor growth in nude mice. Cell cycle and apoptosis were determined by flow cytometry. RESULTS: KLF9 was induced in a time-dependent manner in flutamide-caused apoptosis, and knockdown of KLF9 significantly decreased flutamide-induced growth inhibition and apoptosis in LNCaP cells. The levels of KLF9 were relatively lower in PCa cell lines, particularly in androgen-independent cell lines compared with those in nontumorous prostate epithelial cell lines. Overexpression of KLF9 dramatically suppressed cell proliferation and caused cell cycle arrest in the G2/M phase and cell apoptosis in the androgen-independent cell lines, PC3 and DU145. Intriguingly, KLF9 expression severely suppressed the activation of AKT and its downstream targets. AKT reactivation partially rescued the KLF9-mediated inhibitory effects on the proliferation of PCa cells. More importantly, we found that KLF9 overexpression efficiently inhibited the xenograft tumor growth of PCa cells. CONCLUSIONS: These data collectively showing that KLF9 substantially inhibits AKT activation and abrogates tumor growth of PCa cells, suggest the potential of either genetic or pharmacological activation of KLF9 in the therapeutic treatment of castration-resistant PCa.

Growth arrest and DNA damage 45G down-regulation contributes to Janus kinase/signal transducer and activator of transcription 3 activation and cellular senescence evasion in hepatocellular carcinoma.

UNLABELLED: Growth arrest and DNA damage 45G (GADD45G), a stress sensor with multiple implications in various biological processes, is down-regulated in a broad spectrum of cancers. However, little is known about the biological effects of GADD45G on hepatocellular carcinoma (HCC) cells and the related mechanisms. In the present study, we found that GADD45G was commonly down-regulated in oncogene-transformed mouse liver cells and in human and mouse HCC. Ectopic expression of GADD45G robustly elicited senescence in HCC cells and suppressed tumor growth in vivo. Furthermore, GADD45G-induced senescence occurred in HCC cells independently of p53, p16(INK4a) (p16), and retinoblastoma (Rb). Instead, the prompt inhibition of Janus kinase 2 (Jak2), tyrosine kinase 2 (Tyk2), and signal transducer and activator of transcription 3 (Stat3) activation was observed in cells undergoing senescence. Impairment of Jak-Stat3 activation caused by GADD45G expression was associated with activation of SH2 domain-containing protein tyrosine phosphatase-2 (Shp2). Expression of constitutively activated Stat3 or human telomerase reverse transcriptase (hTERT), as well as knockdown of Shp2f, efficiently counteracted GADD45G-induced senescence. More important, in clinical HCC specimens, we found that GADD45G expression was inversely correlated with phosphorylated Stat3 expression in tumor cells and disease progression. CONCLUSION: GADD45G functions as a negative regulator of the Jak-Stat3 pathway and inhibits HCC by inducing cellular senescence. The decrease or absence of GADD45G expression may be a key event for tumor cells or premalignant liver cells to bypass cellular senescence.

AKT hyperactivation confers a Th1 phenotype in thymic Treg cells deficient in TGF-ß receptor II signaling.

The generation of CD4⁺Foxp3⁺ Treg cells in the thymus is crucial for immune homeostasis and self-tolerance. Recent studies have shown Treg-cell plasticity when Th-related transcriptional factors and cytokines are present. However, the mechanisms that maintain the stability of Treg cells are poorly understood. Here, using mice with a T-cell-specific deletion of the transforming growth factor-ß receptor 2 (Tgfbr2⁻/⁻ mice), we identify the restriction of AKT activation as a key event for the control of Treg-cell stability in Th1 inflammation. AKT regulation was evident in thymic CD4⁺Foxp3⁺ Treg cells before they egressed to peripheral tissues. CD4⁺Foxp3⁺ thymocytes from mice with the Tgfbr2 deletion expressed high levels of CXCR3 and T-bet, and produced IFN-γ and TNF-α. Thymic Tgfbr2⁻/⁻ Treg cells also showed an increase in the activation of AKT pathway. Enhanced AKT activity induced the expression of IFN-γ both in natural and inducible Treg cells. Inhibition of AKT activity markedly attenuated the expression of IFN-γ and TNF-α in thymic Tgfbr2⁻/⁻ Treg cells in vivo. In addition, mixed bone marrow transplantation showed that TGF-ß signaling maintained Treg-cell stability in an intrinsic manner. Our results demonstrate that AKT hyperactivation contributes to the conversion of Treg cells to a Th1 phenotype.