Lizhong Wang

FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2

S-phase kinase-associated protein 2 (SKP2) is a component of the E3 ubiquitin ligase SKP1-Cul1-Fbox complex. Overexpression of SKP2 results in cell cycle dysregulation and carcinogenesis; however, the genetic lesions that cause this upregulation are poorly understood. We recently demonstrated that forkhead box P3 (FOXP3) is an X-linked breast cancer suppressor and an important repressor of the oncogene ERBB2/HER2. Since FOXP3 suppresses tumor growth regardless of whether the tumors overexpress ERBB2/HER2, additional FOXP3 targets may be involved in its tumor suppressor activity. Here, we show that mammary carcinomas from mice heterozygous for a Foxp3 mutation exhibited increased Skp2 expression. Ectopic expression of FOXP3 in mouse mammary cancer cells repressed SKP2 expression with a corresponding increase in p27 and polyploidy. Conversely, siRNA silencing of the FOXP3 gene in human mammary epithelial cells increased SKP2 expression. We also show that Foxp3 directly interacted with and repressed the Skp2 promoter. Moreover, the analysis of over 200 primary breast cancer samples revealed an inverse correlation between FOXP3 and SKP2 levels. Finally, we demonstrated that downregulation of SKP2 was critical for FOXP3-mediated growth inhibition in breast cancer cells that do not overexpress ERBB2/HER2. Our data provide genetic, biochemical, and functional evidence that FOXP3 is a novel transcriptional repressor for the oncogene SKP2.

Cutting Edge: Broad Expression of the FoxP3 Locus in Epithelial Cells: A Caution against Early Interpretation of Fatal Inflammatory Diseases following In Vivo Depletion of FoxP3-Expressing Cells

Dogma that the regulatory T cell (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 (sf) mutation (FoxP3sf/Y or FoxP3sf/sf) to evaluate FoxP3 expression outside of the lymphoid system. Immunohistochemistry and real-time PCR revealed FoxP3 expression in breast epithelial cells, lung respiratory epithelial cells, and prostate epithelial cells, although not in liver, heart, and intestine. The specificity of the assays was confirmed, as the signals were ablated by the Scurfy mutation of the FoxP3 gene. Using mice with a green fluorescence protein 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.

FOXP3 up-regulates p21 expression by site-specific inhibition of histone deacetylase 2/histone deacetylase 4 association to the locus.

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 is 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 2 (HDAC2) and HDAC4 to the site and increased local histone H3 acetylation. Short hairpin RNA silencing of either HDAC2 or HDAC4 is sufficient to induce p21 expression. Our data provides a novel mechanism for transcription activation by FOXP3 and a genetic mechanism for lack of p21 in a large proportion of breast cancer.

A Dinucleotide Deletion in CD24 Confers Protection against Autoimmune Diseases

It is generally believed that susceptibility to both organ-specific and systemic autoimmune diseases is under polygenic control. Although multiple genes have been implicated in each type of autoimmune disease, few are known to have a significant impact on both. Here, we investigated the significance of polymorphisms in the human gene CD24 and the susceptibility to multiple sclerosis (MS) and systemic lupus erythematosus (SLE). We used cases/control studies to determine the association between CD24 polymorphism and the risk of MS and SLE. In addition, we also considered transmission disequilibrium tests using family data from two cohorts consisting of a total of 150 pedigrees of MS families and 187 pedigrees of SLE families. Our analyses revealed that a dinucleotide deletion at position 1527∼1528 (P1527del) from the CD24 mRNA translation start site is associated with a significantly reduced risk (odds ratio = 0.54 with 95% confidence interval = 0.34–0.82) and delayed progression (p = 0.0188) of MS. Among the SLE cohort, we found a similar reduction of risk with the same polymorphism (odds ratio = 0.38, confidence interval = 0.22–0.62). More importantly, using 150 pedigrees of MS families from two independent cohorts and the TRANSMIT software, we found that the P1527del allele was preferentially transmitted to unaffected individuals (p = 0.002). Likewise, an analysis of 187 SLE families revealed the dinucleotide-deleted allele was preferentially transmitted to unaffected individuals (p = 0.002). The mRNA levels for the dinucleotide-deletion allele were 2.5-fold less than that of the wild-type allele. The dinucleotide deletion significantly reduced the stability of CD24 mRNA. Our results demonstrate that a destabilizing dinucleotide deletion in the 3′ UTR of CD24 mRNA conveys significant protection against both MS and SLE.

Identification of a Tumor Suppressor Relay between the FOXP3 and the Hippo Pathways in Breast and Prostate Cancers

Defective expression of LATS2, a negative regulator of YAP oncoprotein, has been reported in cancer of prostate, breast, liver, brain, and blood origins. However, no transcriptional regulators for the LATS2 gene have been identified. 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.

FOXP3 orchestrates H4K16 acetylation and H3K4 trimethylation for activation of multiple genes by recruiting MOF and causing displacement of PLU-1.

Both H4K16 acetylation and H3K4 trimethylation 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 trimethylation 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 cells 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.

FOXP3 Controls an miR-146/NF-κB Negative Feedback Loop That Inhibits Apoptosis in Breast Cancer Cells

FOXP3 functions not only as the master regulator in regulatory T cells, but also as an X-linked tumor suppressor. The tumor-suppressive activity of FOXP3 has been observed in tumor initiation, but its role during tumor progression remains controversial. Moreover, the mechanism of FOXP3-mediated tumor-suppressive activity remains largely unknown. Using chromatin immunoprecipitation (ChIP) sequencing, we identified a series of potential FOXP3-targeted miRNAs in MCF7 cells. Notably, FOXP3 significantly induced the expression of miR-146a/b. In vitro, FOXP3-induced miR-146a/b prevented tumor cell proliferation and enhanced apoptosis. Functional analyses in vitro and in vivo revealed that FOXP3-induced miR-146a/b negatively regulates NF-κB activation by inhibiting the expression of IRAK1 and TRAF6. In ChIP assays, FOXP3 directly bound the promoter region of miR-146a but not of miR-146b, and FOXP3 interacted directly with NF-κB p65 to regulate an miR-146-NF-κB negative feedback regulation loop in normal breast epithelial and tumor cells, as demonstrated with luciferase reporter assays. Although FOXP3 significantly inhibited breast tumor growth and migration in vitro and metastasis in vivo, FOXP3-induced miR-146a/b contributed only to the inhibition of breast tumor growth. These data suggest that miR-146a/b contributes to FOXP3-mediated tumor suppression during tumor growth by triggering apoptosis. The identification of a FOXP3-miR-146-NF-κB axis provides an underlying mechanism for disruption of miR-146 family member expression and constitutive NF-κB activation in breast cancer cells. Linking the tumor suppressor function of FOXP3 to NF-κB activation reveals a potential therapeutic approach for cancers with FOXP3 defects.

CD24 polymorphisms affect risk and progression of chronic hepatitis B virus infection

T‐cell immunity to hepatitis B virus (HBV) is involved in both viral clearance and the pathogenesis of cirrhosis and hepatocellular carcinoma following chronic HBV infection. It is therefore of great interest to analyze whether genetic polymorphism of genes involved in the immune response may determine the outcomes of chronic HBV infection. Here we report that CD24 polymorphisms affect the risk and progression of chronic HBV infection. Thus the CD24 P170T allele, which is expressed at a higher level, is associated with an increased risk of chronic HBV infection. Among the chronic HBV patients this allele shows recessive association with more rapid progression to liver cirrhosis and hepatocellular carcinoma in comparison to the P170C allele. In contrast, a dinucleotide deletion at position 1527–1528 (P1527del), which reduces CD24 expression, is associated with a significantly reduced risk of chronic HBV infection. To confirm the role for CD24 in liver carcinogenesis, we compared the size of liver tumor developed in CD24−/− and CD24+/− HBV transgenic mice. Our data demonstrate that targeted mutation of CD24 drastically reduced the sizes of spontaneous liver cancer in the HBV transgenic mice. Conclusion: These data demonstrate that genetic variation of CD24 may be an important determinant for the outcome of chronic HBV infection. (HEPATOLOGY 2009.)

Different Lineages of P1A-Expressing Cancer Cells Use Divergent Modes of Immune Evasion for T-Cell Adoptive Therapy

Tumor evasion of T-cell immunity remains a significant obstacle to adoptive T-cell therapy. It is unknown whether the mode of immune evasion is dictated by the cancer cells or by the tumor antigens. Taking advantage of the fact that multiple lineages of tumor cells share the tumor antigen P1A, we adoptively transferred transgenic T cells specific for P1A (P1CTL) into mice with established P1A-expressing tumors, including mastocytoma P815, plasmocytoma J558, and fibrosarcoma Meth A. Although P1CTL conferred partial protection, tumors recurred in almost all mice. Analysis of the status of the tumor antigen revealed that all J558 tumors underwent antigenic drift whereas all P815 tumors experienced antigenic loss. Interestingly, although Meth A cells are capable of both antigenic loss and antigenic drift, the majority of recurrent Meth A tumors retained P1A antigen. The ability of Meth A to induce apoptosis of P1CTL in vivo alleviated the need for antigenic drift and antigenic loss. Our data showed that, in spite of their shared tumor antigen, different lineages of cancer cells use different mechanisms to evade T-cell therapy.

Intracellular CD24 disrupts the ARF–NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation

CD24 is overexpressed in nearly 70% human cancers, whereas TP53 is the most frequently mutated tumour-suppressor gene that functions in a context-dependent manner. Here we show that both targeted mutation and short hairpin RNA (shRNA) silencing of CD24 retard the growth, progression and metastasis of prostate cancer. CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A. CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen. In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels. These data provide a general mechanism for functional inactivation of ARF and reveal an important cellular context for genetic and viral inactivation of TP53.