SUMMARY

Both H4K16 acetylation and H3K4 tri-methylation are required for gene activation. However, it is still largely unclear how these modifications are orchestrated by transcriptional factors. Here we analyzed the mechanism of the transcriptional activation by FOXP3, an X-linked suppressor of autoimmune diseases and cancers. FOXP3 binds near transcriptional start sites of its target genes. By recruiting MOF and displacing histone H3K4 demethylase PLU-1, FOXP3 increases both H4K16 acetylation and H3K4 tri-methylation at the FOXP3-associated chromatins of multiple FOXP3-activated genes. RNAi-mediated silencing of MOF reduced both gene activation and tumor suppression by FOXP3, while both somatic mutations in clinical cancer samples and targeted mutation of FOXP3 in mouse prostate epithelial disrupted nuclear localization of MOF. Our data demonstrate a pull-push model in which a single transcription factor orchestrates two epigenetic alterations necessary for gene activation and provide a mechanism for somatic inactivation of the FOXP3 protein function in cancer cells.

Naïve T cells receive stimulation from the positive selecting ligand in the periphery for their survival. This stimulation does not normally lead to overt activation of T cells, as the T cells remain largely quiescent until they receive either antigenic or lymphopenic stimuli. The underlying mechanism responsible for survival and quiescence of the naïve T cells remain largely unknown. Here we report that T cell-specific deletion of Tsc1, a negative regulator of mTOR, resulted in both spontaneous losses of quiescence and cellularity, especially within the CD8 subset. The Tsc1-deficient T cells have increased cell proliferation and apoptosis. Tsc1 deletion affects the survival and quiescence of T cells in the absence of antigenic stimulation. Loss of quiescence but not cellularity was inhibited by rapamycin. Our data demonstrate that TSC-mTOR maintains quiescence and survival of T cells.

A large number of risk alleles have been identified for multiple sclerosis (MS). However, how genetic variations may affect pathogenesis remains largely unknown for most risk alleles. Through direct sequencing of CD24 promoter region, we identified a cluster of 7 new single nucleotide polymorphisms in the CD24 promoter. A hypermorphic haplotype consisting of 3 SNPs was identified through association studies consisting of 935 control and 764 MS patients (P=0.001, odds ratio 1.3). The variant is also associated with more rapid progression of MS (P=0.016, log rank test). In cells that are heterozygous for the risk allele, chromatin immunoprecipitation revealed that risk allele specifically bind to a transcription factor SP1, which is selectively required for the hypermorphic promoter activity of the variant. In MS patients, the CD24 transcript levels associate with the SP1-binding variant in a dose-dependent manner (P=7x10-4). Our data revealed a potential role for SP1-mediated transcriptional regulation in MS pathogenesis.

The iNKT cells have emerged as an important regulator for immunity to infection, cancer as well as autoimmune diseases. The iNKT cells can be activated by glycolipids binding CD1d. The most effective iNKT ligand reported to date is α-galactosylceramide (α-GalCer) which stimulates iNKT cells to secrete both Th-1 and Th-2 cytokines. Indiscriminative induction of both types of cytokines may limit the therapeutic potential of iNKT ligands, as Th-1 and Th-2 cytokines play different roles under physiological and pathological conditions. Therefore a ligand with a biased cytokine release profile is highly desirable. Here we report the synthesis and biological activity of α-lactosylceramide (α-LacCer). Our data demonstrates that the α-LacCer can stimulate the iNKT cells to proliferate and release cytokines, both in vitro and in vivo. Interestingly, while α-LacCer is approximately 1,000-fold less efficient in inducing Th-1 cytokine, it is as potent as α-GalCer in the induction of Th-2 cytokine. Thus, α-LacCer is a novel compound that induces a biased cytokine release. The processing by β-glycosidase was critical for α-LacCer activity. Moreover, in experimental therapies, α-LacCer is at least as potent as α-GalCer in the treatment of tumors and experimental autoimmune encephalomyelitis.

Molecular targeting of cancer stem cells has therapeutic potential for efficient treatment of cancer although relatively few specific targets have so far been identified. Hypoxia-inducible factor was recently shown to regulate tumorigenic capacity of glioma stem cells under hypoxic condition. Surprisingly, we found that, under normoxia, HIF1α signaling was selectively activated in the stem cells of mouse lymphoma and human acute myeloid leukemia (AML). HIF1a ShRNA and HIF inhibitors abrogated the colony forming unit activity of mouse lymphoma and human AML CSCs. Importantly, the HIF inhibitor echinomycin efficiently eradicated mouse lymphoma and serially transplantable human AML in xenogeneic model by preferential elimination of CSCs. HIF1α maintains mouse lymphoma CSCs by repressing a negative feedback loop in the Notch pathway. Taken together, our results demonstrate an essential function of HIF1α-Notch interaction in maintaining CSCs and provide an effective approach to target CSCs for therapy of hematological malignancies.

Summary

Defective expression of LATS2, a negative regulator of YAP onco-protein, has been reported in cancer of prostate, breast, liver, brain and blood origins. However, no transcriptional regulators for the LATS2 gene have been identified. Defective expression of LATS2, a negative regulator of YAP oncoprotein, has been reported in prostate, breast, liver, brain and blood cancers. However, the basis for LATS2 dysregulation in cancer is undefined. Here we report that spontaneous mutation of the transcription factor FOXP3 reduces expression of the LATS2 gene in mammary epithelial cells. shRNA-mediated silencing of FOXP3 in normal or malignant mammary epithelial cells of mouse and human origin repressed LATS2 expression and increased YAP protein levels. LATS2 induction required binding of FOXP3 to a specific sequence in the LATS2 promoter, and this interaction contributed to FOXP3-mediated growth inhibition of tumor cells. In support of these results, reduced expression and somatic mutations of FOXP3 correlated strongly with defective LATS2 expression in microdissected prostate cancer tissues. Thus, defective expression of LATS2 is attributable to FOXP3 defects and may be a major independent determinant of YAP protein elevation in cancer. Our findings identify a novel mechanism of LATS2 downregulation in cancer and reveal an important tumor suppressor relay between the FOXP3 and HIPPO pathways which are widely implicated in human cancer.

Summary

Recognition of pathogens-associated molecular patterns (PAMPs) by Toll-like receptors (TLR), NOD-like receptors (NLR) and RIG-I-like receptors (RLR) plays a critical role in protecting host against pathogens. In addition, TLR and NLR also recognize danger-associated molecular patterns (DAMPs) to initiate limited innate immune responses. While innate immune response to DAMPs may be important for tissue repairs and wound healing, it is normally well controlled to avoid autoimmune destruction. Recent data support a role for sialoside-based pattern recognition by members of the Siglec family to attenuate innate immunity. In particular, since CD24-Siglec 10/G interaction selectively dampens host response to DAMPs but not PAMPs, this sialoside-based pattern recognition may serve as a foundation to discriminate PAMPs from DAMPs.

The FOXP3 (forkhead box P3) gene is a member of forkhead winged helix family transcription factors and functions as both a transcriptional activator and a repressor. FOXP3 dysfunction is responsible for an X-linked autoimmune syndrome: immune dysregulation, polyendopathy, enterophathy, X-linked syndrome. In addition to its role as an essential transcription factor in regulatory T cells, the FOXP3 gene is an epithelial cell-intrinsic tumor suppressor for breast and prostate cancers. We will focus on the FOXP3 signalling pathway in epithelial cells and discuss how genetic and/or epigenetic inactivation of the FOXP3 contributes to the malignant transformation of cells.

Unlike autosomal genes, the majority of X-linked genes are subject to dosage compensation. As a result, female tissues are comprised of cells exclusively expressing X-linked genes from one or the other parent. The implication of having only one allele of active X-linked genes in cancer pathogenesis, i.e. somatic single-hit inactivation and dominant inheritance has not been explored extensively. Recent studies identified FOXP3 and WTX as two X-linked tumor suppressor genes that are somatically inactivated by single genetic hits. Because the predicted dominant inheritance of cancer risk has not been demonstrated in human, we discuss possible conditions that might prevent such dominant inheritance. We also argue that the existence of a genetically intact allele in cancer cells in women, together with apparent abnormal X-inactivation in cancer cells, might provide an opportunity to selectively reactivate tumor suppressor genes for cancer therapy.

It is now well accepted that the innate immune system recognizes both damage (or danger)- and pathogen-associated molecular patterns (DAMP and PAMP, respectively) through pattern recognition receptors, such as Toll-like receptors (TLR) and/or Nod-like receptors (NLR). Less clear are whether and how the response to PAMP and DAMP are differentially regulated. The answers may reveal whether the primary goal of the immune system is to defend against infections or to alert the host of tissue injuries. We demonstrated recently that the host response to DAMP is controlled by a DAMP-CD24-Siglec axis. Here we propose a key role for the CD24-Siglec pathway in discriminating between DAMPs and PAMPs.

The mammalian target of rapamycin (mTOR) is a signaling molecule that senses environmental cues, such as nutrient status and oxygen supply, to regulate cell growth, proliferation, and other functions. Unchecked, sustained mTOR activity results in defects in HSC function. Inflammatory conditions, such as autoimmune disease, are often associated with defective hematopoiesis. Here, we investigated whether hyperactivation of mTOR in HSCs contributes to hematopoietic defects in autoimmunity and inflammation. We found that in mice deficient in Foxp3 (scurfy mice), a model of autoimmunity, the development of autoimmune disease correlated with progressive bone marrow loss and impaired regenerative capacity of HSCs in competitive bone marrow transplantation. Similarly, LPS-mediated inflammation in C57BL/6 mice led to massive bone marrow cell death and impaired HSC function. Importantly, treatment with rapamycin in both models corrected bone marrow hypocellularity and partially restored hematopoietic activity. In cultured mouse bone marrow cells, treatment with either of the inflammatory cytokines IL-6 or TNF-α was sufficient to activate mTOR, while preventing mTOR activation in vivo required simultaneous inhibition of CCL2, IL-6, and TNF-α. These data strongly suggest that mTOR activation in HSCs by inflammatory cytokines underlies defective hematopoiesis in autoimmune disease and inflammation.

P1A is the first known tumor rejection antigen. It is expressed in embryonic stem cells and multiple tumors but is silent in adult tissues except for the testis and placenta. Therefore, P1A represents a prototype for onco-fetal antigens. To test the potential function of P1A in tumorigenesis, we used a transgenic mouse expressing P1A in lymphoid cells. We observed that immunodeficient host P1A transgenic mice developed thymic tumors after 7 months of age and had shorter survival rates compared to control groups. Most of the 7 examined tumors displayed B cell lineage markers. The P1A transgenic bone marrow cells had higher proliferation ability and more potential progenitors compared to control bone marrow cells. To our knowledge, our data provided the first example that onco-fetal antigen can promote tumorigenesis.

Despite clear epidemiological and genetic evidence for X-linked prostate cancer risk, all prostate cancer genes identified are autosomal. Here we report somatic inactivating mutations and deletion of the X-linked FOXP3 gene residing at Xp11.23 in human prostate cancer. Lineage-specific ablation of FoxP3 in the mouse prostate epithelial cells leads to prostate hyperplasia and prostate intraepithelial neoplasia. In both normal and malignant prostate tissues, FOXP3 is both necessary and sufficient to transcriptionally repress cMYC, the most commonly over-expressed oncogene in prostate cancer as well as among the aggregates of other cancers. FOXP3 is an X-linked prostate tumor suppressor in the male. Since the male has only one X chromosome, our data represents a paradigm of “single-genetic-hit” inactivation-mediated carcinogenesis.

The majority of the Lafora's disease (LD) is caused by defect in the EPM2A gene, including missense and nonsense mutations and deletions. These defects mainly occur in the carbohydrate-binding domain, and how these mutations cause neuronal defects is under active investigation. Here, we report that the mutant proteins encoded by all missense mutations and most deletions tested are unstable, insoluble and ubiquitinated, and are accumulated in aggresome-like structures. The effect of apparent ‘gain-of-function’ mutations can be corrected by co-transfection of wild-type EPM2A cDNA, which is consistent with the recessive nature of these mutations in LD patients. In a neuronal cell line, these mutant aggregates exacerbate endoplasm reticulum (ER) stress and make the cells susceptible to the apoptosis induced by ER stressor, thapsigargin. The chemical chaperon, 4-phenylbutyrate, increased the mutant solubility, reduced the ER stress and dulled the sensitivity of mutant neuronal cells to apoptosis induced by thapsigargin and the mutant laforin proteins. The increased sensitivity to ER stress-induced apoptosis may contribute to LD pathogenesis.

FOXP3 is inactivated in breast cancer cells by a number of mechanisms, including somatic mutations, deletion and epigenetic silencing. Since the mutation and deletion are usually heterozygous in the cancer samples, it is of interest to determine whether the gene can be induced for the purpose of cancer therapy. Here we report that anisomycin, a potent activator of ATF2, and JNK, induces expression of FoxP3 in both normal and malignant mammary epithelial cells. The induction is mediated by ATF2 and c-Jun. Targeted mutation of ATF2 abrogates both constitutive and inducible expression of FoxP3 in normal epithelial cells. Both ATF2 and c-Jun interact with a novel enhancer in the intron 1 of the FoxP3 locus. Moreover, shRNA silencing of ATF2 and FoxP3 reveals an important role of ATF2-FoxP3 pathway in the anisomycin-induced apoptosis of breast cancer cells. A low dose of anisomycin was also remarkably effective in treating established mammary tumor in the mice. Our data demonstrated that FoxP3 can be reactivated for cancer therapy.

p21-loss has been implicated in conferring oncogenic activity to known tumor suppressor gene KLF4 and cancer drug tamoxifen. Regulators of p21 therefore play critical roles in tumorigenesis. Here we report that X-linked tumor suppressor FOXP3 is essential for p21 expression in normal epithelia and that lack of FOXP3 associated with p21 down-regulation in breast cancer samples. A specific FOXP3 binding site in the intron 1 is essential for p21 induction by FOXP3. FOXP3 specifically inhibited binding of histone deacetylase (HDAC) 2 and 4 to the site and increased local histone H3 acetylation. ShRNA silencing of either HDAC2 or HDAC4 is sufficient to induce p21 expression. Our data provides a novel mechanism for transcriptional activation by FOXP3 and a genetic mechanism for lack of p21 in a large proportion of breast cancer.

Purpose

In prostate cancer bearing host, regulatory T cells restrain activity of tumor antigen specific T cells. Since B7:CD28 interactions are needed for both function of CD4+CD25+ Treg cells and the CD8+ effective T cells, targeting this pathway may help to overcome the immunotherapy barriers.

Experimental design

The anti-B7−1/B7−2 mAbs were administrated to a transgenic mouse model of prostate cancer (TRAMP) ectopically expressing SV40 large T antigen (TAg) in different tumor development stages for prevention and therapy of prostate cancer. The treatment was also tested in treating transplanted MC38 colon adenocarcinoma in mice.

Results

Here we showed that short-term administration of anti-B7−1/B7−2 mAbs in TRAMP mice leads to significant inhibited primary tumor growth and the size of metastatic lesions. The treatment is effective to inhibit MC38 colon cancer growth. Correspondingly, this treatment results in a transient reduction of Treg in both thymus and the periphery. In vivo cytotoxicity assay revealed TAg-specific CTL effectors in anti-B7 treated, but not control IgG-treated TRAMP mice.

Conclusions

Transient blockade of B7−1/2 alters the balance between Treg and cancer-reactive T cells to enhance cancer immunotherapy.

Summary

How regulatory T cells (Treg) control autoreactive T cells has not been analyzed in animals with a normal T cell repertoire. Using endogenous viral superantigens (VSAg) as the primary self antigens and mice with the Scurfy mutation of FoxP3, we show here that the Treg defect causes preferential accumulation of autoreactive T cells. Interestingly, in the Scurfy mice, the proliferation of VSAg-reactive T cells was no more vigorous than that of non-VSAg-reactive T cells, which indicated that the preferential accumulation is not due to preferential proliferation. In contrast, VSAg-reactive T cells disappears in WT host despite their preferential proliferation. Importantly, when adoptively transferred into the newborn Scurfy mice, the Treg selectively kill autoreactive T cells without affecting their proliferation. The selective elimination is due to increased susceptibility of autoreactive T cells to Treg-mediated killing.

Patten recognition receptors, which recognize pathogens or components of injured cells (danger), trigger activation of the innate immune system. Whether and how the host distinguishes between danger- versus pathogen-associated molecular patterns remains unresolved. We report that CD24-deficient mice exhibit increased susceptibility to danger- but not pathogen-associated molecular patterns. CD24 associates with high mobility group box 1 (HMGB1), heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90), negatively regulates their stimulatory activity and inhibits nuclear factor-kappa B (NF-κB) activation. This occurs at least in part through CD24 association with Siglec-10 in humans or Siglec-G in mice. Our results reveal that the CD24-Siglec G pathway protects the host against a lethal response to pathological cell death and discriminates danger- versus pathogen-associated molecular patterns.

Glycogen synthase kinase 3β (GSK-3β) represses cell cycle progression by directly phosphorylating cyclin D1 and indirectly regulating cyclin D1 transcription by inhibiting Wnt signaling. Recently, we reported that the Epm2a-encoded laforin is a GSK-3β phosphatase and a tumor suppressor. The cellular mechanism for its tumor suppression remains unknown. Using ex vivo thymocytes and primary embryonic fibroblasts from Epm2a−/− mice, we show here a general function of laforin in the cell cycle regulation and repression of cyclin D1 expression. Moreover, targeted mutation of Epm2a increased the phosphorylation of Ser9 on GSK-3β while having no effect on the phosphorylation of Ser21 on GSK-3α. In the GSK-3β+/+ but not the GSK-3β−/− cells, Epm2a small interfering RNA significantly enhanced cell growth. Consistent with an increased level of cyclin D1, the phosphorylation of retinoblastoma protein (Rb) and the levels of Rb-E2F-regulated genes cyclin A, cyclin E, MCM3, and PCNA are also elevated. Inhibitors of GSK-3β selectively increased the cell growth of Epm2a+/+ but not of Epm2a−/− cells. Taken together, our data demonstrate that laforin is a selective phosphatase for GSK-3β and regulates cell cycle progression by GSK-3β-dependent mechanisms. These data provide a cellular basis for the tumor suppression activity of laforin.

A long-standing but poorly understood observation in experimental cancer therapy is the heterogeneity in cancer susceptibility to energy deprivation. Here we show that the hexose kinase inhibitor, 2-deoxyl glucose (2-dG), preferentially kills cancer cells with defective Laforin expression and significantly increases the survival of mice with aggressive lymphoma due to a genetic defect of the Laforin-encoding Epm2a gene. Normal cells from Epm2a−/− mice also had greatly increased susceptibility to 2-dG. Thus, Laforin is a novel regulator for cellular response to energy-deprivation and its defects in cancer cells may be targeted for cancer therapy.

Dogma that the Treg prevents catastrophic autoimmunity throughout the lifespan relies on the assumption that the FoxP3 locus is transcribed exclusively in Treg. To test the assumption, we used the Rag2−/− and the Rag2−/− mice with the Scurfy mutation (FoxP3sf/y or FoxP3sf/sf) to evaluate FoxP3 expression outside of lymphoid system. Immunohistochemistry and real-time PCR revealed FoxP3 expression in breast epithelial cells, lung respiratory epithelial cells and in prostate secretory epithelial cells, although not in liver, heart and intestine. The specificity of the assays is confirmed as the signals were ablated by the Scurfy mutation of the FoxP3 gene. Using mice with green fluorescence protein (GFP) open-reading frame knocked into the 3′ untranslated region of the FoxP3 locus, we showed that the locus is transcribed broadly in epithelial cells of multiple organs. These results refute an essential underlying assumption of the dogma and question the specificity of FoxP3-based Treg depletion.

The tuberous sclerosis complex (TSC)–mammalian target of rapamycin (mTOR) pathway is a key regulator of cellular metabolism. We used conditional deletion of Tsc1 to address how quiescence is associated with the function of hematopoietic stem cells (HSCs). We demonstrate that Tsc1 deletion in the HSCs drives them from quiescence into rapid cycling, with increased mitochondrial biogenesis and elevated levels of reactive oxygen species (ROS). Importantly, this deletion dramatically reduced both hematopoiesis and self-renewal of HSCs, as revealed by serial and competitive bone marrow transplantation. In vivo treatment with an ROS antagonist restored HSC numbers and functions. These data demonstrated that the TSC–mTOR pathway maintains the quiescence and function of HSCs by repressing ROS production. The detrimental effect of up-regulated ROS in metabolically active HSCs may explain the well-documented association between quiescence and the “stemness” of HSCs.

This review analyzes the current dogma that FoxP3 functions exclusively in the regulatory T cells (Treg) and that FoxP3+ Treg is indispensable for survival of immune competent mice. We outline evidence that FoxP3 is expressed well beyond Treg and that the FoxP3 mutation in thymic stromal cells causes defective thymopoiesis, which in turn leads to increased homeostatic proliferation. We argue that the lethal autoimmune disease in mice with germline mutation of FoxP3 is due to both lack of Treg and enhanced homeostatic proliferation.

Background

It is well established that chronic tumor growth results in functional inactivation of T cells and NK cells. It is less clear, however, whether lymphopoeisis is affected by tumor growth.

Principal Findings

In our efforts of analyzing the impact of tumor growth on NK cell development, we observed a major reduction of NK cell numbers in mice bearing multiple lineages of tumor cells. The decrease in NK cell numbers was not due to increased apoptosis or decreased proliferation in the NK compartment. In addition, transgenic expression of IL-15 also failed to rescue the defective production of NK cells. Our systematic characterization of lymphopoeisis in tumor-bearing mice indicated that the number of the common lymphoid progenitor was significantly reduced in tumor-bearing mice.

The number of B cells also decreased substantially in tumor bearing mice.

Conclusions and Significance

Our data reveal a novel mechanism for tumor evasion of host immunity and suggest a new interpretation for the altered myeloid and lymphoid ratio in tumor bearing hosts.