Targeting JMJD1C to selectively disrupt tumor Treg cell fitness enhances antitumor immunity.

Regulatory T (Treg) cells are critical for immune tolerance but also form a barrier to antitumor immunity. As therapeutic strategies involving Treg cell depletion are limited by concurrent autoimmune disorders, identification of intratumoral Treg cell-specific regulatory mechanisms is needed for selective targeting. Epigenetic modulators can be targeted with small compounds, but intratumoral Treg cell-specific epigenetic regulators have been unexplored. Here, we show that JMJD1C, a histone demethylase upregulated by cytokines in the tumor microenvironment, is essential for tumor Treg cell fitness but dispensable for systemic immune homeostasis. JMJD1C deletion enhanced AKT signals in a manner dependent on histone H3 lysine 9 dimethylation (H3K9me2) demethylase and STAT3 signals independently of H3K9me2 demethylase, leading to robust interferon-γ production and tumor Treg cell fragility. We have also developed an oral JMJD1C inhibitor that suppresses tumor growth by targeting intratumoral Treg cells. Overall, this study identifies JMJD1C as an epigenetic hub that can integrate signals to establish tumor Treg cell fitness, and we present a specific JMJD1C inhibitor that can target tumor Treg cells without affecting systemic immune homeostasis.

Generation of a Murine Model for c-MYC and BCL2 Co-expression B Cell Lymphomas.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoma in adults, and is characterized as clinically and biologically heterogeneous lymphomas with diverse response to therapy and variation in clinical behavior. It's well-established that c-MYC and BCL2 play important roles in normal B-cell differentiation and tumorigenesis. B cell lymphoma with dual expression of c-MYC and BCL2 (double-expressor lymphoma, DEL) accounts for approximately one-third of DLBCL cases. DEL patients have poor outcomes after chemoimmunotherapy or autologous stem-cell transplantation. Lack of a genetic mouse tool for DEL hinders us from understanding the lymphogenesis mechanism and developing therapeutic strategies. Here, we investigated whether ectopic expression of c-MYC and BCL2 in different stages of B cells could lead to lymphoma and generate a mouse model for DEL. We observed that Co-expression of c-MYC and BCL2 in germinal center (GC) B cells, or pan-B cells could induce B cell lymphomas. The tumor-bearing mice have enlarged lymphoid organs, and B cells massively infiltrate into non-lymphoid organs including lung, liver and kidney. The tumor-bearing mice also manifested significantly shorter lifespan than the controls. In addition, adoptive transfer of Co-expression B cells leads to B cell lymphoma and host mice death. This model will provide us a tool to further explore the pathogenesis and treatment approaches for DEL.

Histone methyltransferase Nsd2 is required for follicular helper T cell differentiation.

Follicular helper T (Tfh) cells provide essential help for humoral immune response. Transcriptional factor Bcl6 is the master regulator for Tfh generation and is induced very early after T cell activation in a CD28-dependent manner, but how CD28 signal promotes Bcl6 early expression remains unknown. Here we found that CD28 signal quickly induces expression of the H3K36me2 methytransferase Nsd2, which is required for Bcl6 expression as early as the first cell division after T cell activation. Nsd2 deficiency in T cells leads to decreased Bcl6 expression, impaired Tfh generation, compromised germinal center response, and delayed virus clearance. Ectopic Bcl6 expression rescues the Tfh defect of Nsd2 KO cells. ICOS signal is dispensable for early Nsd2 induction but required for sustained Nsd2 expression, which is critical for Tfh maintenance. Overexpression of Nsd2 increases Bcl6 expression and enhances Tfh generation; 4-mo-old mice even develop spontaneous Tfh. Overall, our study reveals Nsd2 as a critical epigenetic regulator for Tfh differentiation.

Uhrf1 regulates germinal center B cell expansion and affinity maturation to control viral infection.

The production of high-affinity antibody is essential for pathogen clearance. Antibody affinity is increased through germinal center (GC) affinity maturation, which relies on BCR somatic hypermutation (SHM) followed by antigen-based selection. GC B cell proliferation is essentially involved in these processes; it provides enough templates for SHM and also serves as a critical mechanism of positive selection. In this study, we show that expression of epigenetic regulator ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) was markedly up-regulated by c-Myc-AP4 in GC B cells, and it was required for GC response. Uhrf1 regulates cell proliferation-associated genes including cdkn1a, slfn1, and slfn2 by DNA methylation, and its deficiency inhibited the GC B cell cycle at G1-S phase. Subsequently, GC B cell SHM and affinity maturation were impaired, and Uhrf1 GC B knockout mice were unable to control chronic virus infection. Collectively, our data suggest that Uhrf1 regulates GC B cell proliferation and affinity maturation, and its expression in GC B cells is required for virus clearance.

p70S6K promotes IL-6-induced epithelial-mesenchymal transition and metastasis of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer worldwide and a common cause of cancer-related death, with a 5-year survival rate of less than 60%. IL-6 has been suggested to play an important role in cancer metastasis, but its mechanism in HNSCC has not been fully clarified. p70S6K has been reported to induce epithelial-mesenchymal transition (EMT) of ovarian cancer, but its role in HNSCC remains unknown. In this study, we found that p70S6K and IL-6 were upregulated in high-metastatic HNSCC cell lines that underwent EMT when compared to paired low-metastatic cell lines. Overexpression of p70S6K promoted EMT and migration of HNSCC cells, while downregulation of p70S6K attenuated IL-6-induced EMT and cell migration. Furthermore, IL-6-induced p70S6K activation was attenuated by inhibitors of the PI3K/Akt/mTOR, MAPK/ERK, and JAK/STAT3 signaling pathways, suggesting that it located downstream of these pathways. These findings suggest that p70S6K promotes IL-6-induced EMT and metastasis of HNSCC. Targeting p70S6K for HNSCC therapy may benefit patients through the inhibition of tumor growth, as well as metastasis.

AEG-1/MTDH-activated autophagy enhances human malignant glioma susceptibility to TGF-ß1-triggered epithelial-mesenchymal transition.

Autophagy is a tightly regulated process activated in response to metabolic stress and other microenvironmental changes. Astrocyte elevated gene 1 (AEG-1) reportedly induces protective autophagy. Our results indicate that AEG-1 also enhances the susceptibility of malignant glioma cells to TGF-ß1-triggered epithelial-mesenchymal transition (EMT) through induction of autophagy. TGF-ß1 induced autophagy and activated AEG-1 via Smad2/3 phosphorylation in malignant glioma cells. Also increased was oncogene cyclin D1 and EMT markers, which promoted tumor progression. Inhibition of autophagy using siRNA-BECN1 and siRNA-AEG-1 suppressed EMT. In tumor samples from patients with malignant glioma, immunohistochemical assays showed that expression levels of TGF-ß1, AEG-1, and markers of autophagy and EMT, all gradually increase with glioblastoma progression. In vivo siRNA-AEG-1 administration to rats implanted with C6 glioma cells inhibited tumor growth and increased the incidence of apoptosis among tumor cells. These findings shed light on the mechanisms underlying the invasiveness and progression of malignant gliomas.

Oncogenic miR-9 is a target of erlotinib in NSCLCs.

EGFR-targeted cancer therapy is a breakthrough in non-small cell carcinoma. miRNAs have been proved to play important roles in cancer. Currently, for the role of miRNAs in EGFR-targeted cancer therapy is unclear. In this study, first we found that erlotinib reduced the expression of miR-9. MiR-9 expression was increased in human lung cancer tissues compared with peripheral normal tissues, and miR-9 promoted the growth of NSCLC cells. Overexpression of miR-9 decreased the growth inhibitory effect of erlotinib. Second, miR-9 decreased FoxO1 expression by directly inhibition of its mRNA translation. Adenovirus-mediated overexpression of FoxO1 or siRNA-mediated downregulation of FoxO1 negatively regulated cell growth. And exogenous overexpression FoxO1 reduced the pro-growth effect of miR-9. Finally, we found that erlotinib upregulated FoxO1 protein expression. Moreover, overexpression of miR-9 decreased erlotinib-induced FoxO1 expression, and overexpression of FoxO1 enhanced the growth inhibitory effects of erlotinib. Additionally, we found that erlotinib downregulates miR-9 expression through suppressing the transcrption of miR-9-1 and enhanced DNA methylation maybe involved. These findings suggest that oncogenic miR-9 targeted FoxO1 to promote cell growth, and downregulation of this axis was involved in erlotinib's growth inhibitory effects. Clarifying the regulation of miRNAs by erlotinib may indicate novel strategies for enhancing EGFR-targeted cancer therapy.

AEG-1 is a target of perifosine and is over-expressed in gastric dysplasia and cancers.

BACKGROUND: Perifosine, an alkylphospholipid, is an Akt inhibitor which inhibits the growth of diverse cancer cells. We have reported its inhibitory effects on the growth of gastric cancer cells recently, but its molecular mechanisms are still largely unknown. AIMS: The purpose of this study was to investigate the effect and regulatory mechanism of perifosine in gastric cancer. METHODS: Cell viability was determined by sulforhodamine B assay after transiently transfected with AEG-1 specific siRNAs. qRT-PCR and western blot assay were used to determine the mRNA expression and proteins levels of cell signaling molecules examined. Immunohistochemistry was used to detect the AEG-1 expression in 87 gastric carcinomas, 60 dysplasia, and 47 normal gastric mucosa. RESULTS: Perifosine decreased AEG-1 gene expression along with inhibition of Akt/GSK3ß/C-MYC signaling pathway. Knockdown of AEG-1 using siRNA led to significant down-regulation of cyclin D1 expression at both mRNA level and protein level, and inhibited the growth of gastric cancer cells. AEG-1 expression was elevated in gastric dysplasia and cancer tissues compared to normal gastric mucosa (P < 0.01). AEG-1 over-expression correlated with diffuse type of gastric cancer and advanced tumor stages. CONCLUSIONS: Perifosine inhibits the growth of gastric cancer cells possibly through inhibition of the Akt/GSK3ß/C-MYC signaling pathway-mediated down-regulation of AEG-1 that subsequently down-regulated cyclin D1. AEG-1 may play an important role in the carcinogenesis and progression of gastric cancer and could be a therapeutic target of perifosine.

Oncogenic MicroRNA-27a is a target for genistein in ovarian cancer cells.

MicroRNAs (miRNAs) are emerging as important regulators in various pathobiological processes in cancer. Genistein, as a major isoflavonoid isolated from dietary soybean, possesses a wide variety of biological activities particularly in cancer prevention. However, the molecular mechanisms by which genistein elicits its effects on ovarian cancer cells have not been fully elucidated. In this study, we reported that expression of miR-27a was higher in human ovarian cancer relative to benign ovarian tissues. Meanwhile, transfection of SKOV3 cells with the inhibitor of miR-27a suppressed growth and migration of tumor cells. Our study also found that treatment of ovarian cancer cells with genistein caused an inhibition of ovarian cancer cell growth and migration. Further cellular mechanistic studies revealed that genistein down-regulated miR-27a expression, which was accompanied by significantly increased expression of Sprouty2, a putative miR-27a target gene. Taken together, our findings reveal that oncogenic miR-27a plays an important role in ovarian cancer cell growth and metastasis, and genistein, as nontoxic inactivators of miRNA, can block ovarian cancer cell growth and migration, offering novel insights into the mechanisms of genistein therapeutic actions.

Perifosine enhances mTORC1-targeted cancer therapy by activation of GSK3ß in NSCLC cells.

mTORC1 inhibitors, including rapamycin and its analogs, have been actively studied both pre-clinically and clinically. However, the single treatment of mTORC1 inhibitors has been modest in most cancer types. We have previously demonstrated that the activation of PI3K/Akt and MEK/ERK signaling pathways attenuates the anticancer efficacy of mTORC1 inhibitors. In this study, we report that mTORC1 inhibition also phosphorylates and inactivates GSK3ß, which is a tumor suppressor in lung cancer. Moreover, we show that perifosine, as an Akt inhibitor, decreases rapamycin-induced phosphorylation of GSK3ß and elevated p-GSK3ß levels in rapamycin-resistant cell lines. Combination of perifosine with mTORC1 inhibitors showed enhanced anticancer efficacy both in cell cultures and in a xenograft mouse model. In addition, perifosine inhibits the growth of both rapamycin sensitive and resistant A549 cells. However, inhibition of GSK3ß by a selective inhibitor- LiCl, or downregulation of GSK3ß expression by siRNA, reverses the growth inhibitory effects of perifosine on rapamycin resistant cells, suggesting the important role of GSK3ß activation in enhancing mTORC1 inhibitors efficacy by perifosine. Thus, our results provide a potential therapeutic strategy to enhance mTORC1-targeted cancer therapy by using perifosine or targeting GSK3ß.

MicroRNA-9 suppresses uveal melanoma cell migration and invasion through the NF-κB1 pathway.

The aggressive course of uveal melanoma is believed to reflect its unusually invasive and metastatic nature, which is associated with the nuclear factor kappaB (NF-κB) pathway. MicroRNAs (miRNAs) have been implicated in the regulation of various biological and pathological processes in cancer, however, the special role of miR-9 in uveal melanoma metastasis is largely unknown. In the present study, we showed that miR-9 is significantly reduced in highly invasive uveal melanoma cell lines, and suppressed migration and invasion of highly invasive cells. Furthermore, miR-9 negatively modulated NF-κB1 expression by direct targeting at its 3'-UTRs. Additionally, downstream targets of NF-κB1, such as MMP-2, MMP-9 and VEGFA, were regulated by miR-9 in the same pattern as NF-κB1. Therefore, miR-9 suppresses uveal melanoma cell migration and invasion partly through downregulation of the NF-κB1 signaling pathway.

Downregulation of IRS-1 promotes metastasis of head and neck squamous cell carcinoma.

Lymph node metastasis is responsible for the high morbidity of head and neck squamous cell carcinoma (HNSCC). To date, the role of insulin receptor substrate 1 (IRS-1) in tumorigenesis and metastasis of head and neck cancer has not been elucidated. In this study, we found a negative correlation of IRS-1 expression with tumor metastasis both in human tissue samples and in cell lines. Furthermore, we found that knockdown of IRS-1 expression enhanced cell invasive potency and induced EMT in parallel with upregulation of miR-9 expression. We propose that IRS-1 suppresses metastasis of head and neck cancer possibly through miR-9.

Association between IRS-2 G1057D polymorphism and risk of gastric cancer.

AIM: To investigate the relationship between insulin receptor substrate-2 (IRS-2) G1057D polymorphism and the risk of gastric cancer (GC) in a Chinese population. METHODS: A case-control study with 197 GC patients and 156 age- and sex- matched control subjects was conducted. The genotypes of polymorphism were assessed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The genotype frequencies of IRS-2 G1057D polymorphism in cases were obviously different from those in the control group (P = 0.031). Compared with GG genotype carriers, the risk for GC was significantly higher (adjusted odds ratio = 2.32, 95% CI: 1.03-5.23, P = 0.042) in the individuals with the IRS-2 DD genotype. Furthermore, stratified analysis was performed based on age, sex, smoking status and residence, but no significant difference between the two groups was found. In addition, no significant association between genotypes and clinicopathological features was observed either. CONCLUSION: This study demonstrates that IRS-2 G1057D is involved in susceptibility to GC, although further large-sample studies are still needed.