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Although c-MYC and mTOR are frequently activated proteins in prostate cancer, any interaction between the two is largely untested. Here, we characterize the functional cross-talk between FOXP3–c-MYC and TSC1–mTOR signaling during tumor progression. Deletion of Tsc1 in mouse embryonic fibroblasts (MEF) decreased phosphorylation of c-MYC at threonine 58 (pT58) and increased phosphorylation at serine 62 (pS62), an observation validated in prostate cancer cells. Conversely, inhibition of mTOR increased pT58 but decreased pS62. Loss of both FOXP3 and TSC1 in prostate cancer cells synergistically enhanced c-MYC expression via regulation of c-Myc transcription and protein phosphorylation. This crosstalk between FOXP3 and TSC1 appeared to be mediated by both the mTOR–4EBP1–c-MYC and FOXP3–c-MYC pathways. In mice, Tsc1 and Foxp3 double deletions in the prostate led to prostate carcinomas at an early age; this did not occur in these mice with an added c-Myc deletion. In addition, we observed synergistic antitumor effects of cotreating mice with inhibitors of mTOR and c-MYC in prostate cancer cells and in Foxp3 and Tsc1 double-mutant mice. In human prostate cancer, loss of nuclear FOXP3 is often accompanied by low expression of TSC1. Because loss of FOXP3 transcriptionally induces c-Myc expression and loss of TSC1 activates mTOR signaling, these data suggest cross-talk between FOXP3–c-MYC and TSC1–mTOR signaling that converges on c-MYC to regulate tumor progression. Coadministration of c-MYC and mTOR inhibitors may overcome the resistance to mTOR inhibition commonly observed in prostate cancer cells.Significance:These results establish the principle of a synergistic action of TSC1 and FOXP3 during prostate cancer progression and provide new therapeutic targets for patients who have prostate cancer with two signaling defects.

Supplementary Figure S1: Loss of nuclear FOXP3 accompanying gene defects in human primary prostate cancers; Supplementary Figure S2: Diagram of the floxed Foxp3 and Tsc1 loci and the position of the primers; Supplementary Figure S3: Tumor development and progression in mouse prostate of single, double, and triple mutant mice; Supplementary Figure S4: Targeted deletion of c-Myc in the mouse prostate; Supplementary Figure S5: Relative mRNA levels of the c-Myc gene in micro-dissected prostate epithelial cells; Supplementary Figure S6: Representative H&E staining of anterior lobes of mouse prostate in the mice; Supplementary Figure S7: Public datasets-based analysis of genetic alterations for FOXP3, TSC1, AKT1 and FOXP1 loci in prostate adenocarcinoma; Supplementary Table S1: Specific primary antibodies used in this study; Supplementary Table S2: The clinicopathological characteristics of patients with prostate cancer; Supplementary Table S3: The clinicopathological characteristics of patients with prostate cancer; Supplementary Table S4: Primer and siRNA sequence used in this study; Supplementary Table S5: Characterization of genetic mouse models of prostate cancer; Supplementary Table S6: Association between protein expressions of FOXP3, TSC1, and c-MYC in prostate cancer; Supplementary Table S7: Association between protein expressions of FOXP3, TSC1, and c-MYC in prostate cancer.

Supplementary Data Table S8: Original data for the gene copy differences of candidate genes between nFOXP3+ and FOXP3- human prostate tumors in Supplementary Figure S1B.

Purpose: In prostate cancer cells, there is CD24-dependent inactivation of mutant p53, but the mechanism and its significance remain largely unknown. Here, we validated this observation and explored the therapeutic potential of targeting CD24 in TP53 mutant prostate cancer cells.Experimental Design: Overall, 553 prostate cancers (522 formalin-fixed paraffin-embedded and 31 frozen tissues) were assessed for protein or mRNA expression of CD24 and TP53. The effects of CD24 on p53-dependent transcriptional regulation, cancer cell growth, the cell cycle, apoptosis, and mutant p53 restoration were also determined.Results: As determined with three sample cohorts, CD24 and p53 were not expressed in prostate epithelial cells but in prostate cancer cells in 48% of cases for CD24 and 16% of cases for p53 (mutant form). Expressions of CD24 and mutant p53 were more frequently observed in late-stage and metastatic prostate tumors. Mutant p53 accompanied with CD24 was expressed in most cases (91.6%, 76/83). Silencing of CD24 increased the transcriptional activity of p53 target genes, such as CDKNA1, VDR, and TP53INP1, leading to suppression of p53-dependent cell growth, cell-cycle arrest, and apoptosis in most TP53-mutant prostate cancer cells. Silencing of CD24 enhanced restoration of PRIMA-1–induced mutant p53 in endogenous TP53P223L/V274F DU145 cells and in PC3 cells transfected with TP53R273H.Conclusions: In human prostate cancers, there is CD24-dependent inactivation of mutant p53. The coexpression of CD24 and p53 may help identify aggressive cancers. Targeting CD24 provides a strategy to enhance mutant p53-restoring therapies, especially in patients with TP53R273H prostate cancer. Clin Cancer Res; 22(10); 2545–54. ©2015 AACR.

The tumor-suppressive activity of FOXP3 has been observed in tumor initiation, but the underlying mechanism still remains largely unknown. Here, we identified a FOXP3–microRNA-146 (miR-146)–NF-κB axis in vitro and in vivo in prostate cancer cells. We observed that FOXP3 dramatically induced the expression of miR-146a/b, which contributed to transcriptional inhibition of IRAK1 and TRAF6, in prostate cancer cell lines. Tissue-specific deletion of Foxp3 in mouse prostate caused a significant reduction of miR-146a and upregulation of NF-κB activation. In addition, prostatic intraepithelial neoplasia lesions were observed in miR-146a–mutant mice as well as in Foxp3-mutant mice. Notably, the NF-κB inhibitor bortezomib inhibited cell proliferation and induced apoptosis in prostate epithelial cells, attenuating prostatic intraepithelial neoplasia formation in Foxp3-mutant mice. Our data suggest that the FOXP3–miR-146–NF-κB axis has a functional role during tumor initiation in prostate cancer. Targeting the miR-146–NF-κB axis may provide a new therapeutic approach for prostate cancers with FOXP3 defects. Cancer Res; 75(8); 1714–24. ©2015 AACR.

Supplemental Figures 1-9 and Tables 1 and 2. Supplemental Table 1: Identification of potential FOXP3-targeted microRNAs in breast cancer cells. Supplemental Table 2: Primer sequences used in this study. Supplemental Figure 1: Expression profiles of IRAK1 and TRAF6 in MCF10A cells with miR-146a/b inhibitors. Supplemental Figure 2: Expression of nuclear FOXP3 in FOXP3-Tet-off MCF7 cells. Supplemental Figure 3: The expression profile of NF-κB target genes after FOXP3 induction in FOXP3-Tet-off MCF7 cells. Supplemental Figure 4: Identification of potential FOXP3-binding sites in promoter regions of miR-146a/b in the FOXP3-Tet-off MCF7 cells by whole-genome ChIP-seq. Supplemental Figure 5: Quantitative analysis of FOXP3-binding sites in the potential promoter region of human miR-146b in FOXP3-Tet-off MCF7 cells. Supplemental Figure 6: Specific binding of FOXP3 to the second forkhead-binding motif (GCAAATA) in the miR-146a promoter. Supplemental Figure 7: MiR-146a/b upregulation by FOXP3 transfection in MDA-MB231 cells. Supplemental Figure 8: The mRNA expression levels of IRAK1and TRAF6 in metastatic breast tumor cells after administration of miR-146a/b inhibitors. Supplemental Figure 9: A potential mechanism by which FOXP3 regulates the NF-κB-miR-146a/b feedback loop through IRAK1 and TRAF6 in tumor growth or regulates CXCR4/MMP9 in tumor metastasis.

Supplemental Table 1: Primer sequences used in this study. Supplemental Figure 1: The effect of FOXP3, NF-κB p65-siRNA, and NF-κB inhibitor bortezomib on the growth of DU145 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. Cancer Res; 75(8); 1703–13. ©2015 AACR.

Cancer is one of the most dreaded diseases of the 21st century for humans. Currently, the human cancer mortality rate is approximately 11-25%. However, for some animals, such as elephants, the cancer mortality rate is lower than 5%. p53 is a significant cellular regulator in human cancers, and its important role in response to DNA damage has been highlighted by the discovery of p53 retrogene found in elephants (19 copies) but not humans (0 copies). Therefore, the main question is whether (1) more copies of p53 are better at combating cancer development or (2) is elephant p53 is better than human p53 in p53-mediated function? Herein, we demonstrate for the first time that N-terminal and C-terminal elephant-to-human p53 variants and truncations alter p53 transactivation. First, we created elephant-to-human p53 variants (such as T387R, L383P, LSTEL, etc.) and truncations at the N-terminus and C-terminus of human p53. Secondly, R175H and R273H p53 mutants (conformational and contact mutants, respectively) were used as negative control. Using a p53 (14X) response element LUC and several natural p53 downstream gene promoters, we demonstrated that several N-terminal and C-terminal p53 variants and truncations increases p53 transactivation compared to WT p53. Moreover, we found that deletion of the region between aa374 and aa382 is integral for p53 transactivation. Taken together, our preliminary results demonstrate that several N-terminal and C-terminal elephant-to-human p53 variants enhance p53 transactivation and possible stability. This data is the first step to support the notion that elephant p53 is better than human p53 in p53-mediated function. Citation Format: William H. Yang, Wei-Hsiung Yang. N-terminal and C-terminal elephant-to-human p53 variants and truncations alter p53 transactivation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3735.

JDP2 and ATF3 belong to the family of AP-1 protein. JDP2, a transcription factor, can bind to AP-1 site as well as cAMP responsive element (CRE) site in numerous cis-elements of the target genes. JDP2 has been shown to be involved in cancer development and cell-cycle regulation. Previously, we had shown that JDP2 activates Mc2r transcriptional activity. Since ATF3 and JDP2 share high similarity in C-terminal domains with identities of 65% and ATF3 can bind to p53 and regulate p53 transcriptional activity and stability, we hypothesized that JDP2 may have ability to regulate p53 and MDM2 (the main regulator of p53). Herein, we demonstrate for the first time the relationship between JDP2 and MDM2. First, we found that JDP2 can directly bind to MDM2. Secondly, though ATF3 dose-dependently increases MDM2 level, JDP2 dose-dependently decreases MDM2 level. Moreover, the C-terminus of JDP2 is required for regulation of MDM2 level. Finally, using p53RE-Luc (14X) for p53 transactivation study, while MDM2 decreases p53 transactivation, JDP2 dose-dependently abolishes MDM2-mediated p53 repression. Taken together, our results demonstrate that JDP2 directly binds to MDM2 and reduces MDM2-mediated p53 repression, suggesting that JDP2 is a novel regulator of MDM2. Since p53-MDM2 pathway is an important regulator in endocrinology and reproduction, our finding provides a new layer of regulatory mechanism for fertility, tumor suppression, and longevity. Presentation: No date and time listed

Activating transcription factor 3 (ATF3), a transcription factor and acute stress sensor, is rapidly induced by a variety of pathophysiological signals and is essential in the complex processes in cellular stress response. FOXP3, a well-known breast and prostate tumor suppressor from the X chromosome, is a novel transcriptional repressor for several oncogenes. However, it remains unknown whether ATF3 is the target protein of FOXP3. Herein, we demonstrate that ATF3 expression is regulated by FOXP3. Firstly, we observed that overexpression of FOXP3 reduced ATF3 protein level. Moreover, knockdown FOXP3 by siRNA increased ATF3 expression. Secondly, FOXP3 dose-dependently reduced ATF3 promoter activity in the luciferase reporter assay. Since FOXP3 is regulated by post-translational modifications (PTMs), we next investigated whether PTMs affect FOXP3-mediated ATF3 expression. Interestingly, we observed that phosphorylation mutation on FOXP3 (Y342F) significantly abolished FOXP3-mediated ATF3 expression. However, other PTM mutations on FOXP3, including S418 phosphorylation, K263 acetylation and ubiquitination, and K268 acetylation and ubiquitination, did not alter FOXP3-mediated ATF3 expression. Finally, the FOXP3 binding site was found on ATF3 promoter region by deletion and mutagenesis analysis. Taken together, our results suggest that FOXP3 functions as a novel regulator of ATF3 and that this novel event may be involved in tumor development and progression.

USAAP-1 protein mainly consists of proteins belonging to c-Jun, c-Fos, activating transcription factor (ATF), and Jun dimerization protein (JDP) family. In addition to AP-1 site, JDP2 also binds to cAMP responsive element (CRE) site in numerous cis-elements of the target genes. JDP2 has been shown to be involved in cancer development and cell-cycle regulation, suppression of adipocyte differentiation, and controlling bone homeostasis and antibacterial immunity. ATF3 and JDP2 share high similarity in C-terminal domains with identities of 65%. Previous studies have demonstrated that ATF3 can bind to p53 and regulate p53 transcriptional activity and stability. Though couple of studies have shown that JDP2 inhibits expression of p53 and p53 represses the transcriptional activity of JDP2 promoter; however, whether JDP2 directly interacts and regulates p53 transactivation remains largely unknown. Herein, we demonstrate for the first time that JDP2 can directly interact with p53. First, we found that both ATF3 and JDP2 can bind to p53 and the C-terminus of JDP2 is required for p53 binding. Secondly, in p53-null H1299 cells, JDP2 dose-dependently enhances p53 transactivation using p53RE-Luc (14X). Moreover, we demonstrated that JDP2 dose-dependently decreases p53 protein level in consistence with the previous reports. Finally, JDP2 increases p53 stability in CHX chase experiments. Taken together, our results demonstrate that JDP2 directly binds to p53 and enhances p53 transactivation and stability, suggesting that JDP2 is a novel regulator of p53. Citation Format: Wei-Hsiung Yang, Kasey Price, Leticia Cardoso, William Yang, Chiung-Min Wang, Richard Yang. JDP2 interacts with p53 and enhances p53 transactivation and stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5723.

Steroidogenic Factor 1 (SF-1/NR5A1), an orphan nuclear receptor, is important for sexual differentiation and the development of multiple endocrine organs, as well as cell proliferation in cancer cells. Activating transcription factor 3 (ATF3) is a transcriptional repressor, and its expression is rapidly induced by DNA damage and oncogenic stimuli. Since both NR5A1 and ATF3 can regulate and cooperate with several transcription factors, we hypothesized that NR5A1 may interact with ATF3 and plays a functional role in cancer development. First, we found that NR5A1 physically interacts with ATF3. We further demonstrated that ATF3 expression is up-regulated by NR5A1. Moreover, the promoter activity of the ATF3 is activated by NR5A1 in a dose-dependent manner in several cell lines. By mapping the ATF3 promoter as well as the site-directed mutagenesis analysis, we provide evidence that NR5A1 response elements (&minus, 695 bp and &minus, 665 bp) are required for ATF3 expression by NR5A1. It is well known that the transcriptional activities of NR5A1 are modulated by post-translational modifications, such as small ubiquitin-related modifier (SUMO) modification and phosphorylation. Notably, we found that both SUMOylation and phosphorylation of NR5A1 play roles, at least in part, for NR5A1-mediated ATF3 expression. Overall, our results provide the first evidence of a novel relationship between NR5A1 and ATF3.

Members of the microRNA (miR)-200 family, which are involved in tumor metastasis, have potential as cancer biomarkers, but their regulatory mechanisms remain elusive. We investigated FOXP3-inducible breast cancer cells, Foxp3 heterozygous Scurfy mutant (Foxp3 sf/+ ) female mice, and patients with breast cancer for characterization of the formation and regulation of the miR-200 family in breast cancer cells and circulation. Participants (259), including patients with breast cancer or benign breast tumors, members of breast cancer families, and healthy controls, were assessed for tumor and circulating levels of the miR-200 family. First, we identified a FOXP3-KAT2B-miR-200c/141 axis in breast cancer cells. Second, aging Foxp3 sf/+ female mice developed spontaneous breast cancers and lung metastases. Levels of miR-200c and miR-141 were lower in Foxp3 sf/+ tumor cells than in normal breast epithelial cells, but plasma levels of miR-200c and miR-141 in the Foxp3 sf/+ mice increased during tumor progression and metastasis. Third, in patients with breast cancer, the levels of miR-200c and 141 were lower in FOXP3 low relative to those with FOXP3 high breast cancer cells, especially in late-stage and metastatic cancer cells. The levels of miR-200c and miR-141 were higher in plasma from patients with metastatic breast cancer than in plasma from those with localized breast cancer, with benign breast tumors, with a family history of breast cancer, or from healthy controls. Finally, in Foxp3 sf/+ mice, plasma miR-200c and miR-141 appeared to be released from tumor cells. miR-200c and miR-141 are regulated by a FOXP3-KAT2B axis in breast cancer cells, and circulating levels of miR-200c and miR-141 are potential biomarkers for early detection of breast cancer metastases.

BACKGROUND Using a functional analysis of prostate cancer cells, we found a CD24-dependent inactivation of mutant p53, but the clinical significance of this observation remained uncertain. Here, we validated these results with samples of human prostate cancer and explored the role of a CD24-p53 axis in racial disparities of prostate cancer. METHODS Samples of formalin-fixed, paraffin-embedded prostate cancer from 141 European Americans (EAs) and 147 African Americans (AAs) in two independent sample cohorts were assessed for protein expression of CD24, mutant p53, mouse double minute 2 human homolog (MDM2), and cyclin dependent kinase inhibitor 2A (ARF) using immunohistochemical analyses. All samples were analyzed for TP53R175H and TP53R273H . RESULTS CD24, mutant p53, MDM2, and ARF proteins were expressed in 55%, 24%, 39%, and 68% of prostate cancer samples, respectively. CD24 and mutant p53 were present more frequently in late-stage and metastatic prostate cancer. The presence of CD24 was associated with a greater than fourfold risk of metastasis, which included lymph node and distant metastases. H score analysis showed positive correlations of CD24 expression with mutant p53 (r = .308, P

Fibrinogen is an extracellular matrix protein composed of three polypeptide chains with fibrinogen alpha (FGA), beta (FGB) and gamma (FGG). Although fibrinogen and its related fragments are involved in tumor angiogenesis and metastasis, their functional roles are incompatible. A recent genome-scale screening reveals that loss of FGA affects the acceleration of tumor growth and metastasis of lung cancer, but the mechanism remains elusive. We used CRISPR/Cas9 genome editing to knockout (KO) FGA in human lung adenocarcinoma (LUAD) cell lines A549 and H1299. By colony formation, transwell migration and matrix invasion assays, FGA KO increased cell proliferation, migration, and invasion but decreased the expressions of epithelial–mesenchymal transition marker E-cadherin and cytokeratin 5/8 in A549 and H1299 cells. However, administration of FGA inhibited cell proliferation and migration but induced apoptosis in A549 cells. Of note, FGA KO cells indirectly cocultured by transwells with FGA wild-type cells increased FGA in the culture medium, leading to decreased migration of FGA KO cells. Furthermore, our functional analysis identified a direct interaction of FGA with integrin α5 as well as FGA–integrin signaling that regulated the AKT–mTOR signaling pathway in A549 cells. In addition, we validated that FGA KO increased tumor growth and metastasis through activation of AKT signaling in an A549 xenograft model. Implications: These findings demonstrate that that loss of FGA facilities tumor growth and metastasis through the integrin–AKT signaling pathway in lung cancer.

Programmed Cell Death Protein 4 (PDCD4), a well-known tumor suppressor, acts to inhibit translation initiation and cap-dependent translation by inhibiting the helicase activity of EIF4A. EIF4A tends to target mRNAs with structured 5'-UTR. PDCD4 can also prevent tumorigenesis by inhibiting tumor promoter-induced neoplastic transformation, and studies indicate that PDCD4 may inhibit translation of certain mRNAs via directly binding. A previous study has demonstrated that PDCD4 inhibits translation of p53 mRNA and treatment with DNA-damaging agents down-regulates PDCD4 expression but activates p53 expression. The study further demonstrated that treatment with DNA-damaging agents resulted in down-regulation of PDCD4 expression and an increase in p53 expression, suggesting a potential mechanism in which p53 regulates expression of Pdcd4. However, whether p53 directly regulates PDCD4 remains unknown. Herein, we demonstrate for the first time that p53 regulates PDCD4 expression. First, we found that overexpression of p53 in p53-null cells (H1299 and Saos2 cells) decreased PDCD4 protein level. Secondly, p53 dose-dependently decreased PDCD4 promoter activity in gene reporter assays. Moreover, we demonstrated that mutations on p53 (R273H: contact mutant, hotspot and R175H: conformational mutant, hotspot) abolished p53-mediated PDCD4 repression. Furthermore, using Tet-on expression system, PDCD4 protein level was decreased when WT p53, but not hotspot mutants of p53, was expressed in H1299 cells. Finally, mutations on DNA-binding domain, but not C-terminal regulatory domain, of p53 disrupted p53-mediated PDCD4 repression. Taken together, our results suggest that p53 functions as a novel regulator of PDCD4 and the relationship between of p53 and PDCD4 may be involved in tumor development and progression. Citation Format: Wei-Hsiung Yang, Andrew P. George, William H. Yang, Chiung-Min Wang, Richard H. Yang. Tumor suppressor p53 down-regulates programmed cell death protein 4 (PDCD4) expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2160.

Purpose: In prostate cancer cells, there is CD24-dependent inactivation of mutant p53, but the mechanism and its significance remain largely unknown. Here, we validated this observation and explored the therapeutic potential of targeting CD24 in TP53 mutant prostate cancer cells. Experimental Design: Overall, 553 prostate cancers (522 formalin-fixed paraffin-embedded and 31 frozen tissues) were assessed for protein or mRNA expression of CD24 and TP53. The effects of CD24 on p53-dependent transcriptional regulation, cancer cell growth, the cell cycle, apoptosis, and mutant p53 restoration were also determined. Results: As determined with three sample cohorts, CD24 and p53 were not expressed in prostate epithelial cells but in prostate cancer cells in 48% of cases for CD24 and 16% of cases for p53 (mutant form). Expressions of CD24 and mutant p53 were more frequently observed in late-stage and metastatic prostate tumors. Mutant p53 accompanied with CD24 was expressed in most cases (91.6%, 76/83). Silencing of CD24 increased the transcriptional activity of p53 target genes, such as CDKNA1, VDR, and TP53INP1, leading to suppression of p53-dependent cell growth, cell-cycle arrest, and apoptosis in most TP53-mutant prostate cancer cells. Silencing of CD24 enhanced restoration of PRIMA-1–induced mutant p53 in endogenous TP53P223L/V274F DU145 cells and in PC3 cells transfected with TP53R273H. Conclusions: In human prostate cancers, there is CD24-dependent inactivation of mutant p53. The coexpression of CD24 and p53 may help identify aggressive cancers. Targeting CD24 provides a strategy to enhance mutant p53-restoring therapies, especially in patients with TP53R273H prostate cancer. Clin Cancer Res; 22(10); 2545–54. ©2015 AACR.

Activating transcription factor 3 (ATF3), a stress sensor, is rapidly induced by a variety of physiological and pathological signals, including those from cancer cells, and is essential in the complex processes of stress response. Several lines of evidence have demonstrated that ATF3 is involved in host defense and cancer development. Several studies also provide the relationship between ATF3 and p53 indicating that ATF3 binds to and stabilizes p53 while ATF3 is a negative regulator of TP53 gene expression. FOXP3, a well-known breast and prostate tumor suppressor, is a novel transcriptional repressor for several oncogenes including SKP2. However, whether ATF3 is the target protein of FOXP3 remains unknown. Herein, we demonstrate that ATF3 expression is regulated by FOXP3. First, we observed that overexpression of FOXP3 reduced ATF3 protein level in several cancer cell lines (H1299, HepG2, and Ishikawa cells). Secondly, FOXP3 dose-dependently reduced ATF3 promoter activity in gene reporter assay in several cell lines (MCF7, H1299, MDAMB231, and Saos2 cells). Since FOXP3 is regulated by post-translational modifications (PTMs), we next investigated whether PTMs affect FOXP3-mediated ATF3 expression. Interestingly, we observed that phosphorylation mutation on FOXP3 (Y342F) abolished FOXP3-mediated ATF3 expression. However, other PTM mutations on FOXP3, including S418 phosphorylation, K263 acetylation and ubiquitination, and K268 acetylation and ubiquitination, did not alter FOXP3-mediated ATF3 expression. Finally, FOXP3 binding site was found on ATF3 promoter region, around -870 bp region. Taken together, our results suggest that FOXP3 functions as a novel regulator of ATF3 and this novel event may be involved in tumor development and progression. Citation Format: Wei-Hsiung Yang, Chiung-Min Wang, William Yang. Transcription factors FOXP3 regulate ATF3 expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 310A.

Forkhead Box Protein P3 (FOXP3), a transcription factor of the FOX protein family, is essentially involved in the development of regulatory T (Treg) cells, and functions as a tumor suppressor. Although FOXP3 has been widely studied in immune system and cancer development, its function in the regulation of the UBC9 gene (for the sole E2 enzyme of SUMOylation) is unknown. Herein, we find that the overexpression of FOXP3 in human MCF7 breast cancer cells increases the level of UBC9 mRNA. Moreover, the level of UBC9 protein dose-dependently increases in the FOXP3-Tet-off MCF7 cells. Notably, the promoter activity of the UBC9 is activated by FOXP3 in a dose-dependent manner in both the MCF7 and HEK293 cells. Next, by mapping the UBC9 promoter as well as the site-directed mutagenesis and ChIP analysis, we show that the FOXP3 response element at the &minus, 310 bp region, but not the &minus, 2182 bp region, is mainly required for UBC9 activation by FOXP3. Finally, we demonstrate that the removal of phosphorylation (S418A and Y342F) and the removal of acetylation/ubiquitination (K263R and K263RK268R) of the FOXP3 result in attenuated transcriptional activity of UBC9. Taken together, FOXP3 acts as a novel transcriptional activator of the human UBC9 gene, suggesting that FOXP3 may have physiological functions as a novel player in global SUMOylation, as well as other post-translational modification systems.

Transcription factor forkhead box protein P2 (FOXP2) plays an essential role in the development of language and speech. However, the transcriptional activity of FOXP2 regulated by the post-translational modifications remains unknown. Here, we demonstrated that FOXP2 is clearly defined as a SUMO target protein at the cellular levels as FOXP2 is covalently modified by both SUMO1 and SUMO3. Furthermore, SUMOylation of FOXP2 was significantly decreased by SENP2 (a specific SUMOylation protease). We further showed that FOXP2 is selectively SUMOylated in vivo on a phylogenetically conserved lysine 674 but the SUMOylation does not alter subcellular localization and stability of FOXP2. Interestingly, we observed that human etiological FOXP2 R553H mutation robustly reduces its SUMOylation potential as compared to wild-type FOXP2. In addition, the acidic residues downstream the core SUMO motif on FOXP2 are required for its full SUMOylation capacity. Finally, our functional analysis using reporter gene assays showed that SUMOylation may modulate transcriptional activity of FOXP2 in regulating downstream target genes (DISC1, SRPX2, and MiR200c). Altogether, we provide the first evidence that FOXP2 is a substrate for SUMOylation and SUMOylation of FOXP2 plays a functional role in regulating its transcriptional activity.

The tumor-suppressive activity of FOXP3 has been observed in tumor initiation, but the underlying mechanism still remains largely unknown. Here, we identified a FOXP3–microRNA-146 (miR-146)–NF-κB axis in vitro and in vivo in prostate cancer cells. We observed that FOXP3 dramatically induced the expression of miR-146a/b, which contributed to transcriptional inhibition of IRAK1 and TRAF6, in prostate cancer cell lines. Tissue-specific deletion of Foxp3 in mouse prostate caused a significant reduction of miR-146a and upregulation of NF-κB activation. In addition, prostatic intraepithelial neoplasia lesions were observed in miR-146a–mutant mice as well as in Foxp3-mutant mice. Notably, the NF-κB inhibitor bortezomib inhibited cell proliferation and induced apoptosis in prostate epithelial cells, attenuating prostatic intraepithelial neoplasia formation in Foxp3-mutant mice. Our data suggest that the FOXP3–miR-146–NF-κB axis has a functional role during tumor initiation in prostate cancer. Targeting the miR-146–NF-κB axis may provide a new therapeutic approach for prostate cancers with FOXP3 defects. Cancer Res; 75(8); 1714–24. ©2015 AACR.

Transcription factor Forkhead Box Protein M1 (FOXM1) is a well-known master regulator in controlling cell-cycle pathways essential for DNA replication and mitosis, as well as cell proliferation. Among the three major isoforms of FOXM1, FOXM1B is highly associated with tumor growth and metastasis. The activities of FOXM1B are modulated by post-translational modifications (PTMs), such as phosphorylation, but whether it is modified by small ubiquitin-related modifier (SUMO) remains unknown. The aim of the current study was to determine whether FOXM1B is post-translationally modified by SUMO proteins and also to identify SUMOylation of FOXM1B on its target gene transcription activity. Here we report that FOXM1B is clearly defined as a SUMO target protein at the cellular levels. Moreover, a SUMOylation protease, SENP2, significantly decreased SUMOylation of FOXM1B. Notably, FOXM1B is selectively SUMOylated at lysine residue 463. While SUMOylation of FOXM1B is required for full repression of its target genes MiR-200b/c and p21, SUMOylation of FOXM1B is essential for full activation of JNK1 gene. Overall, we provide evidence that FOXM1B is post-translationally modified by SUMO and SUMOylation of FOXM1B plays a functional role in regulation of its target gene activities.

S-phase kinase-associated protein 2 (SKP2), a component of the E3 ubiquitin ligase SKP1-CUL1-Fbox complex, acts as an oncogene/oncoprotein as evidence suggests that overexpression of SKP2 results in cell cycle dysregulation and tumorigenesis as well as associates with poor prognosis in numerous cancers. The unique peptidyl-propyl isomerase PIN1, which isomerizes the cis-trans peptide bond between pSer/pThr and proline, regulates its target proteins’ cell localization, stability, and function. However, whether SKP2 is the target protein of PIN1 remains unknown. Herein, we demonstrate that SKP2 expression is regulated by PIN1 and FOXP3. First, we observed that knock-down PIN1 using siRNAs reduced SKP2 protein level in several cancer cell lines (MCF7, PC3, DU145, HepG2, and JEG3 cells). Secondly, we found that overexpression of PIN1 increased SKP2 protein level. Furthermore, PIN1 dose-dependently enhanced SKP2 promoter activity in gene reporter assay. Since FOXP3 is a known transcriptional repressor for the oncogene SKP2, we investigated whether PIN1 affects FOXP3-mediated SKP2 expression. Interestingly, we observed that PIN1 dose-dependently counteracted FOXP3-mediated SKP2 promoter activity. Finally, we demonstrated that mutations on PIN1 (W34A) and FOXP3 (Y342F) abolished PIN1-mediated and FOXP3-mediated SKP2 expression, respectively. Taken together, our results suggest that PIN1 functions as a novel regulator of SKP2 and with FOXP3 may be involved in tumor development and progression. Citation Format: Wei-Hsiung Yang, Chiung-Min Wang, William H. Yang. PIN1 and FOXP3 regulate SKP2 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 181A.

Primer and siRNA sequence used in this study. Figure S1. Levels of miR-200c and miR-141 in normal immortalized human epithelial cell line MCF10A. Figure S2. Associations between expression of FOXP3 and miR-200 s in TCGA breast cancer samples. Figure S3. Potential binding signals of FOXP3 in the promoter regions of the miR-200 family and KAT2B gene. Figure S4. The transcriptional activity of miR-200b/a/429 on miR-200 cluster 1 after FOXP3 induction and KAT2B silencing in breast cancer cells. Figure S5. Association of expression levels of miR-200 s with ER/PR/HER2 status in breast cancer cells. Figure S6. Levels of plasma miR-200b, 200a, and 429 in Foxp3sf/+ female mice during tumor progression. Figure S7. Plasma levels of miR-141 and 200c in breast cancer patients with clinical parameters. Figure S8. Levels of miR-200b, 200a, and 429 in FOXP3-Tet-off MCF7 cells, culture medium, and exosomes. Figure S9. Expression of miR-200b, 200a, and 429 in blood cells of Foxp3sf/+ female mice. (DOCX 910 kb)

Jun dimerization protein 2 (JDP2), a basic leucine zipper transcription factor, is involved in numerous biological and cellular processes such as cancer development and regulation, cell-cycle regulation, skeletal muscle and osteoclast differentiation, progesterone receptor signaling, and antibacterial immunity. Though JDP2 is widely expressed in mammalian tissues, its function in gonads and adrenals (such as regulation of steroidogenesis and adrenal development) is largely unknown. Herein, we find that JDP2 mRNA and proteins are expressed in mouse adrenal gland tissues. Moreover, overexpression of JDP2 in Y1 mouse adrenocortical cancer cells increases the level of melanocortin 2 receptor (MC2R) protein. Notably, Mc2r promoter activity is activated by JDP2 in a dose-dependent manner. Next, by mapping the Mc2r promoter, we show that cAMP response elements (between -1320 and -720-bp) are mainly required for Mc2r activation by JDP2 and demonstrate that -830-bp is the major JDP2 binding site by real-time chromatin immunoprecipitation (ChIP) analysis. Mutations of cAMP response elements on Mc2r promoter disrupts JDP2 effect. Furthermore, we demonstrate that removal of phosphorylation of JDP2 results in attenuated transcriptional activity of Mc2r. Finally, we show that JDP2 is a candidate for SUMOylation and SUMOylation affects JDP2-mediated Mc2r transcriptional activity. Taken together, JDP2 acts as a novel transcriptional activator of the mouse Mc2r gene, suggesting that JDP2 may have physiological functions as a novel player in MC2R-mediated steroidogenesis as well as cell signaling in adrenal glands.

Forkhead box protein L2 (FOXL2) is the earliest ovarian marker and plays an important role in the regulation of cholesterol and steroid metabolism, inflammation, apoptosis, and ovarian development and function. Mutations and deficiencies of the human FOXL2 gene have been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome as well as premature ovarian failure. Although Foxl2 interacts with steroidogenic factor 1 (Nr5a1) and up-regulates cyp19a1a gene transcription in fish, FOXL2 represses the transcriptional activity of the gene that codes for steroidogenic acute regulatory protein (Star) in mice. Most of the recent studies have heavily focused on the FOXL2 target genes (Star and Cyp19a1) in the ovaries. Hence, it is of importance to search for other downstream targets of FOXL2 and for the possibility of FOXL2 expression in nonovarian tissues. Herein, we demonstrate that the interplay between FOXL2 and NR5A1 regulates Star and melanocortin 2 receptor (Mc2r) gene expression in mammalian systems. Both FOXL2 and NR5A1 are expressed in ovarian and adrenal gland tissues. As expected, FOXL2 represses and NR5A1 enhances the promoter activity of Star. Notably, the promoter activity of Mc2r is activated by FOXL2 in a dose-dependent manner. Surprisingly, we found that FOXL2 and NR5A1 synergistically up-regulate the transcriptional activity of Mc2r. By mapping the Mc2r promoter, we provide evidence that distal NR5A1 response elements (-1410 and -975) are required for synergistic activation by FOXL2 and NR5A1. These results suggest that the interplay between FOXL2 and NR5A1 on the Mc2r promoter functions as a novel mechanism for regulating MC2R-mediated cell signaling as well as steroidogenesis in adrenal glands.

The nuclear receptor steroidogenic factor 1 (SF-1) is essential for adrenal development and steroidogenesis. The atypical orphan nuclear receptor Dax-1 binds to SF-1 and represses SF-1 target genes. Paradoxically, however, loss-of-function mutations of Dax-1 also cause adrenal hypoplasia, suggesting that Dax-1 may function as an SF-1 coactivator under some circumstances. Indeed, we found that Dax-1 can function as a dosage-dependent SF-1 coactivator. Both SF-1 and Dax-1 bind to steroid receptor RNA activator (SRA), a coactivator that functions as an RNA. The coactivator TIF2 also associates with Dax-1 and synergistically coactivates SF-1 target gene transcription. A naturally occurring Dax-1 mutation inhibits this transactivation, and the mutant Dax-1-TIF2 complex mislocalizes in living cells. Coactivation by Dax-1 is abolished by SRA knockdown. The expression of the steroidogenic gene products steroidogenic acute regulatory protein (StAR) and melanocortin 2 receptor is reduced in adrenal Y1 cells following the knockdown of endogenous SRA. Similarly, the knockdown of endogenous Dax-1 downregulates the expression of the steroidogenic gene products CYP11A1 and StAR in both H295R adrenal and MA-10 Leydig cells. These findings reveal novel functions of SRA and Dax-1 in steroidogenesis and adrenal biology.

Transcription factor forkhead box protein P2 (FOXP2) plays an essential role in the development of language and speech. However, the transcriptional activity of FOXP2 regulated by the post-translational modifications remains unknown. Here, we demonstrated that FOXP2 is clearly defined as a SUMO target protein at the cellular levels as FOXP2 is covalently modified by both SUMO1 and SUMO3. Furthermore, SUMOylation of FOXP2 was significantly decreased by SENP2 (a specific SUMOylation protease). We further showed that FOXP2 is selectively SUMOylated in vivo on a phylogenetically conserved lysine 674 but the SUMOylation does not alter subcellular localization and stability of FOXP2. Interestingly, we observed that human etiological FOXP2 R553H mutation robustly reduces its SUMOylation potential as compared to wild-type FOXP2. In addition, the acidic residues downstream the core SUMO motif on FOXP2 are required for its full SUMOylation capacity. Finally, our functional analysis using reporter gene assays showed that SUMOylation may modulate transcriptional activity of FOXP2 in regulating downstream target genes (DISC1, SRPX2, and MiR200c). Altogether, we provide the first evidence that FOXP2 is a substrate for SUMOylation and SUMOylation of FOXP2 plays a functional role in regulating its transcriptional activity.

In this review, we introduce the IPEX syndrome and its relationship with germline mutations of the FOXP3 gene. We then describe the multiple functional roles of FOXP3 in regulatory T cells and epithelial cells as well as in IPEX syndrome and tumor progression. Potential mechanisms of FOXP3 inactivation and transcriptional regulation are discussed with recent advances. Finally, we point out current issues and a potential FOXP3-mediated therapeutic strategy as well as the reactivation of FOXP3 in patients with IPEX syndrome and cancer.

Problem Biological effectiveness of targeted cytotoxins is dependent on their stability, circulating half-life, receptor binding ability, and cytotoxicity. The objective of this study was to compare stability of gonadotropin-releasing hormone (GnRH)-toxin conjugates made with disulfide linkers to those using a maleimidodibutyryl (mb) linkage. Method of study We developed a sandwich enzyme-linked immunoabsorbent assay recognizing both GnRH analog and cytotoxin to ensure the conjugate measured was intact. Anti-D-Leu6-GnRH was used for capture and anti-pokeweed antiviral protein (anti-PAP) or anti-RNase for quantification. Specificity was verified by lack of reactivity with ovine FSH and LH, PAP, RNase, and D-Lys6-GnRH. Results Conjugates prepared using disulfide linkages were not stable in serum in vitro (half-lives 2 hr. Clearance of mbGnRH-PAP from the circulation of sheep was rapid (t1/2

The enzyme CYP17 primarily regulates androgen production by mediating four reactions: conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively (17alpha-hydroxylase activity), followed by conversion of the 17-hydroxylated steroids to dehydroepiandrosterone and androstenedione, respectively (17,20-lyase activity). Most mammalian CYP17 isoforms have high 17alpha-hydroxylase relative to 17,20-lyase activities and preferentially mediate one of the two 17,20-lyase reactions. In contrast, Xenopus laevis CYP17 potently regulates all four reactions in the frog ovary. CYP17 isoforms generally rely on the cofactor cytochrome b(5) for the 17,20-lyase reaction, suggesting that the high lyase activity of Xenopus CYP17 might be due to a lesser dependence on b(5). The kinetics of Xenopus CYP17 expressed in yeast microsomes were therefore examined in the absence and presence of Xenopus on human b(5). Xenopus CYP17 mediated both 17,20-lyase reactions in the absence of b(5), confirming that the activity did not require b(5). However, both Xenopus and human b(5) slightly enhanced Xenopus CYP17-mediated lyase activity, indicating that the enzyme was still at least partially responsive to b(5). Surprisingly, only the human b(5) cofactor enhanced human CYP17-mediated lyase activity, implying that the human enzyme had more specific cofactor requirements than Xenopus CYP17. Studies using human/Xenopus chimeric b(5) proteins revealed that human b(5) residues 16-41 were important for the specific regulation of the lyase activity of HuCYP17, possibly serving as an interacting domain with the enzyme. CYP17 may therefore have evolved from a general producer of sex steroids in lower vertebrates to a more tightly regulated producer of both sex steroids and glucocorticoids in mammals.

Progesterone has long been considered the primary mediator of Xenopus oocyte maturation. We have recently shown, however, that androgens, which are equal or more potent promoters of maturation and are present at higher levels in ovulating frogs, may also be playing an important physiologic role in mediating maturation. Here, we examined the role of CYP17, a key enzyme mediating sex steroid synthesis, in Xenopus ovarian androgen production. We found that the 17,20-lyase activities ofXenopus CYP17 exceeded the 17α-hydroxylase activities in both the Δ4 and Δ5 pathways; thus, Xenopus CYP17 rapidly converted pregnenolone and progesterone to dehydroepiandrosterone (DHEA) and androstenedione, respectively. This remarkably robust activity exceeds that of CYP17 from most higher vertebrates, and likely explains why virtually no progesterone is detected in ovulating frogs. Additionally, ovarian CYP17 activity was present exclusively in oocytes, although all other enzymes involved in sex steroid production were expressed almost entirely in surrounding follicular cells. This compartmentalization suggests a “two-cell” model whereby Xenopus ovarian androgen production requires both follicular cells and oocytes themselves. The requirement of oocytes for ovarian androgen production further introduces the unusual paradigm whereby germ cells may be responsible for producing important steroids used to mediate their own maturation.

Rates of internalization of the murine GnRH receptor fused via its C-terminus to green fluorescent protein (GnRH-R-GFP) were examined in Chinese hamster ovary cells (CHO cells) and compared to those of native murine GnRH-R in a clonal murine gonadotroph cell line (LbetaT2 cells). The resulting rates of internalization of murine receptors were then compared with those of sheep GnRH-R in ovine gonadotrophs. Cells were incubated with radioiodinated [D-Ala6]GnRH on ice for 4 h to allow binding of the ligand to GnRH-R, then cells were warmed to 37 degrees C to permit internalization. Surface-bound radioligand began to decrease as soon as the cells were warmed and had decreased significantly within 20 min. A steady-state level of surface-bound radioligand was achieved after 60 min in both CHO cells and LbetaT2 cells (38% and 41%, respectively, of initial value; P < 0.05). Internalization of radioligand began immediately after warming the cells to 37 degrees C, and a significant proportion of surface ligand had been internalized by 20 min. A steady-state maximum of internalization was reached after 60 min in both CHO cells and LbetaT2 cells (29% and 28%, respectively, of total cell-associated ligand; P < 0.05). Changes in surface-bound radioligand and internalized radioligand in sheep pituitary cells were similar to those in CHO cells and LbetaT2 cells, but the amount of radioligand internalized after 60 min (40% of total cell-associated ligand) was 1.4 times higher than in CHO cells and LbetaT2 cells (P < 0.05). In a separate experiment, the effect of estradiol on the rate of internalization of GnRH-R in ovine pituitary cells was examined. Although treatment of ovine pituitary cells with estradiol approximately doubled the number of GnRH receptors, it did not alter either the rate or extent of receptor internalization. These results show that rates of internalization of recombinant murine GnRH-R-GFP in CHO cells and native murine and ovine GnRH-R in LbetaT2 cells and in sheep pituitary cells, respectively, are similar, but amounts of ovine GnRH-R internalized are greater than those for murine GnRH-R. Further, the rate of internalization of occupied receptor is similar in gonadotroph and nongonadotroph cells, and the addition of GFP to the C-terminus of the murine GnRH-R does not alter the rate of internalization.

The purpose of this study was to determine concurrent ovarian and endometrial prostaglandin E 2 (PGE 2 ) receptor concentrations throughout the hamster estrous cycle. The effect of progesterone (P 4 ) on PGE 2 receptor in these two tissues was also investigated during in vitro culture. Estrous cycles of mature female hamsters were monitored according to the appearance of the vaginal discharge on cycle day 1 (D1). Ovaries and uteri were removed from cyclic hamsters at 10:00 a.m. on each day of the cycle and at 6:00 p.m. on D4 (proestrus). Ovarian and endometrial cell membranes were collected and assayed for the specific PGE 2 binding by Scatchard plot analysis, using seven different concentrations of 3 H-PGE 2 (0.72–9.1 nM) with or without the presence of unlabelled PGE 2 (9.1 μM). Ovarian and endometrial tissues of the cyclic hamsters were shown to contain a saturable, specific binding site with K d =4.69 ±0.55, and 5.7 ± 0.4 nM for ovary and endometrium, respectively. Relative binding activity of PGE 1 for the PGE 2 binding site was about 28%. PGF 2α and PGA 1 did not compete for the PGE 2 binding site. In ovaries, the PGE 2 receptor levels started to increase sharply in the evening of D4 and reached maximum in the morning of D1. A precipitous drop of PGE 2 receptor was observed on D2 followed by gradual decreases on D3 and D4. The PGE 2 receptor concentrations in endometrium were the lowest in the morning of D4, and increased thereafter until a maximal level was reached on D2. Progesterone (10 nM) augmented PGE 2 receptors in ovarian but not in endometrial tissue during 24-h in vitro incubation.

Steroidogenic factor 1 (NR5A1/SF1) is a well-known master regulator in controlling adrenal and sexual development, as well as regulating numerous genes involved in adrenal and gonadal steroidogenesis. Several studies including ours have demonstrated that NR5A1 can be SUMOylated on lysine 194 (K194, the major site) and lysine 119 (K119, the minor site), and the cycle of SUMOylation regulates NR5A1’s transcriptional activity. An extended consensus negatively charged amino acid-dependent SUMOylation motif (NDSM) enhances the specificity of substrate modification by SUMO has been reported, however, the mechanism of NDSM for NR5A1 remains to be clarified. In this study, we investigated the functional significance of the acidic residue located downstream from the core consensus SUMO site of NR5A1. Here we report that E199A (glutamic acid was replaced with alanine) of NR5A1 reduced, but not completely abolished, its SUMOylation level. We next characterized the functional role of NR5A1 E199A on target gene expression and protein levels. We found that E199A alone, as well as combination with K194R, increased Mc2r and Cyp19a1 reporter activities. Moreover, E199A alone as well as combination with K194R enhanced NR5A1-mediated STAR protein levels in mouse adrenocortical cancer Y1 cells. We also observed that E199A increased interaction of NR5A1 with CDK7 and SRC1. Overall, we provide the evidence that the acidic residue (E199) located downstream from the core consensus SUMO site of NR5A1 is, at least in part, required for SUMOylation of NR5A1 and for its mediated target gene and protein expression.

SUMOylation plays an important role in regulating a wide range of cellular processes. Previously, we showed that ATF3, a stress response mediator, can be SUMOylated and lysine 42 is the major SUMO site. However, the significance of ATF3 SUMOylation in biological processes is still poorly understood. In the present study, we investigated the role of ATF3 SUMOylation on CCND activity and cellular proliferation in human prostate cancer cells. First, we showed that ATF3 can be SUMOylated endogenously in the overexpression system, and lysine 42 is the major SUMO site. Unlike normal prostate tissue and androgen-responsive LNCaP cancer cells, androgen-independent PC3 and DU145 cancer cells did not express ATF3 endogenously. Overexpression of ATF3 increased CCND1/2 expression in PC3 and DU145 cancer cells. Interestingly, we observed that SUMOylation is essential for ATF3-mediated CCND1/2 activation. Finally, we observed that SUMOylation plays a functional role in ATF3-mediated cellular proliferation in PC3 and DU145 cells. Taken together, our results demonstrate that SUMO modification of ATF3 influences CCND1/2 activity and cellular proliferation of prostate cancer PC3 and DU145 cells and explains at least in part how ATF3 functions to regulate cancer development.

The biopotencies of pituitary gonadotropins (GTHs) from various vertebrate classes were examined in an in vitro rat Sertoli cell bioassay which was previously established for mammalian follicle-stimulating hormones (FSHs). Potencies of the gonadotropins were determined by incubation of Sertoli cells obtained from 10-day-old rats, with increasing doses of GTHs, which resulted in dose-related and parallel estradiol-17β formation converted from added 19-hydroxy-androstenedione. In general, mammalian (human and ovine) FSHs were most potent, avian (chicken, turkey, and ostrich) FSHs were intermediate, and reptilian (snapping turtle) and amphibian (bullfrog) FSHs were the least potent. By contrast, mammalian and bullfrog luteinizing hormones (LHs) were inactive or negligibly active in this assay; avian LHs possessed one-fifth to one-half of the potency of the FSH preparations of the same species. The data suggest that the assay is specific for FSHs from mammalian and amphibian species and is relatively specific for FSHs from avian species. Both snapping turtle FSH and LH exhibited low and similar potencies in this bioassay. Black silver carp GTH was also active in this assay, although its potency was much lower. The present study has demonstrated that the immature rat Sertoli cell aromatase assay in vitro is useful for measurement of FSH contents in mammalian species and of FSH activity in diverse nonmammalian species. It also provides an approach for the investigation of structure–function relations of gonadotropin in diverse vertebrate species in relation to phylogenic patterns and specificity of hormone–receptor interaction. The findings from the present study imply that the binding sites of vertebrate FSHs share a certain degree of homology and that the binding sites of FSH receptors on Sertoli cells from immature rat have a relatively low degree of animal class specificity.

Cyclic AMP-dependent transcription factor-3 (ATF3), a stress sensor, plays an essential role in cells to maintain homeostasis and has diverse functions in cellular survival and death signal pathways. ATF3 is a novel regulator of p53 protein stability and function. The activities of ATF3 are modulated by post-translational modifications (PTMs), such as ubiquitination, but whether it is modified by small ubiquitin-related modifier (SUMO) remains unknown. The aim of this study was to investigate whether ATF3 is post-translationally modified by SUMO proteins and also to elucidate SUMOylation of ATF3 on TP53 gene activity. Here we report that ATF3 is clearly defined as a SUMO target protein both in vitro SUMOylation assay using recombinant proteins and at the cellular levels. Furthermore, ATF3 interacted with UBE2I, the only SUMO E2 enzyme found so far. In addition, PIAS3β (a SUMO E3 ligase) enhanced and SENP2 and SENP7 (two SUMOylation proteases) decreased SUMOylation of ATF3, respectively. Finally, we found that ATF3 is selectively SUMOylated at lysine residue 42 but the SUMOylation does not alter subcellular localization of ATF3. We then characterized the functional role of ATF3 SUMOylation on TP53 gene expression. We found that SUMOylation of ATF3 is required for full repression of TP53 gene. Overall, we provide the first evidence that ATF3 is post-translationally modified by SUMO and SUMOylation of ATF3 plays a functional role in regulation of TP53 gene activity.

Adrenocortical carcinomas are rare but present with extremely poor prognosis. One of the approaches to control cancer progression and reduce cancer risk is prevention through diet. Bitter melon is widely consumed as a vegetable and especially as a traditional medicine in many countries. In this study, we have used human and mouse adrenocortical cancer cells as an in vitro model to assess the efficacy of bitter melon extract (BME) as an anticancer agent. The protein concentrations of BME and other extracts were measured before use. First, BME treatment of adrenocortical cancer cells resulted in a significantly dose-dependent decrease in cell proliferation. However, we did not observe an antiproliferative effect in adrenocortical cancer cells treated with extracts from blueberry, zucchini, and acorn squash. Second, apoptosis of adrenocortical cancer cells was accompanied by increased caspase-3 activation and poly(ADP-ribose) polymerase cleavage. BME treatment enhanced cellular tumor antigen p53, cyclin-dependent kinase inhibitor 1A (also called p21), and cyclic AMP-dependent transcription factor-3 levels and inhibited G1/S-specific cyclin D1, D2, and D3, and mitogen-activated protein kinase 8 (also called Janus kinase) expression, suggesting an additional mechanism involving cell cycle regulation and cell survival. Third, BME treatment decreased the key proteins involved in steroidogenesis in adrenocortical cancer cells. BME treatment decreased the level of phosphorylation of cyclin-dependent kinase 7, which is required, at least in part, for steroidogenic factor 1 activation. Finally, we observed that BME treatment significantly reduced the level of insulin-like growth factor 1 receptor and its downstream signaling pathway as evidenced by lower levels of phosphorylated RAC-α serine/threonine-protein kinase. Taken together, these data illustrate the inhibitory effect of bitter melon on cell proliferation of adrenocortical cancer through modulation of diverse mechanisms.

Scientists have long hypothesized the existence of tissue-specific (somatic) stem cells and have searched for their location in different organs. The theory that adrenocortical organ homeostasis is maintained by undifferentiated stem or progenitor cells can be traced back nearly a century. Similar to other organ systems, it is widely believed that these rare cells of the adrenal cortex remain relatively undifferentiated and quiescent until needed to replenish the organ, at which time they undergo proliferation and terminal differentiation. Historical studies examining cell cycle activation by label retention assays and regenerative potential by organ transplantation experiments suggested that the adrenocortical progenitors reside in the outer periphery of the adrenal gland. Over the past decade, the Hammer laboratory, building on this hypothesis and these observations, has endeavored to understand the mechanisms of adrenocortical development and organ maintenance. In this review, we summarize the current knowledge of adrenal organogenesis. We present evidence for the existence and location of adrenocortical stem/progenitor cells and their potential contribution to adrenocortical carcinomas. Data described herein come primarily from studies conducted in the Hammer laboratory with incorporation of important related studies from other investigators. Together, the work provides a framework for the emerging somatic stem cell field as it relates to the adrenal gland.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor selectively expressed in the adrenal cortex and gonads, where it mediates the hormonal stimulation of multiple genes involved in steroid hormone biosynthesis. SF-1 is the target of both phosphorylation and SUMOylation, but how these modifications interact or contribute to SF-1 regulation of endogenous genes remains poorly defined. We found that SF-1 is selectively SUMOylated at K194 in Y1 adrenocarcinoma cells and that although SUMOylation does not alter the subcellular localization of SF-1, the modification inhibits the ability of SF-1 to activate target genes. Notably, whereas SF-1 SUMOylation is independent of S203 phosphorylation and is unaffected by adrenocorticotropin (ACTH) treatment, loss of SUMOylation leads to enhanced SF-1 phosphorylation at serine 203. Furthermore, preventing SF-1 SUMOylation increases the mRNA and protein levels of multiple steroidogenic enzyme genes. Analysis of the StAR promoter indicates that blockade of SF-1 SUMOylation leads to an increase in overall promoter occupancy but does not alter the oscillatory recruitment dynamics in response to ACTH. Notably, we find that CDK7 binds preferentially to the SUMOylation-deficient form of SF-1 and that CDK7 inhibition reduces phosphorylation of SF-1. Based on these observations, we propose a coordinated modification model in which inhibition of SF-1-mediated transcription by SUMOylation in adrenocortical cancer cells is mediated through reduced CDK7-induced phosphorylation of SF-1.

Pokeweed antiviral protein (PAP), a 29-kDa ribosome-inactivating protein isolated from the leaves of Phytolacca americana, has potent cytotoxic activity once it enters the cytoplasm of a cell. It is incapable of entering cells by itself. Therefore, our objective was to determine whether a GnRH analog could be used to deliver PAP specifically to cells expressing GnRH receptors. D-Lys(6)-GnRH-Pro(9)-ethylamide was conjugated to PAP (GnRH-PAP). Chinese hamster ovary cells stably transfected with cDNA for the murine GnRH receptor and a mouse gonadotroph tumor cell line that expresses endogenous GnRH receptors (alphaT3-1 cells) were used to evaluate the cytotoxic effects of GnRH-PAP. We also examined cytotoxicity of GnRH-PAP using human endometrial, breast, and prostate cancer cell lines. Treatment of GnRH receptor-positive cells with GnRH-PAP resulted in dose-dependent cytotoxicity. Cytotoxicity of GnRH-PAP was dependent on number of GnRH receptors (r(2) = 0.871, P < 0.05) and duration of exposure of GnRH-PAP to the cells. In contrast, GnRH-PAP was not cytotoxic to Chinese hamster ovary cells not harboring GnRH receptors. Moreover, the cytotoxic activity of GnRH-PAP could be inhibited by addition of excess GnRH analog. Neither PAP nor GnRH analog alone was cytotoxic. These results suggest that GnRH analogs can be used to specifically deliver toxin molecules to cells that express GnRH receptors. Thus, a new class of biomedicines that act as hormonotoxins against cells expressing GnRH receptors provides a novel approach for inhibiting reproduction and treating cancers that are dependent on reproductive hormones.

Cyclic AMP-dependent transcription factor-3 (ATF3), a stress sensor/mediator, plays an essential role in cells to maintain homeostasis and has diverse functions in cellular death and survival signal pathways. ATF3 binds to the consensus ATF/cAMP response element (CRE) to regulate its downstream target genes, such as CCNE2 (Cyclin E2), CD82 (KAI1), DDIT3 (GADD153), LDLR (LDL receptor), SNAI1, TP53, and TWIST1. A previous report has shown that the activities of ATF3 are modulated by ubiquitination; however, whether it is modified by small ubiquitin-related modifier (SUMO), an important modification in regulating a wide range of cellular processes, remains unknown. The aim of this study was to investigate whether ATF3 is post-translationally modified by SUMO proteins and also to determine the role of SUMOylation of ATF3 on MC2R, NR5A1, STAR, and TP53 gene activities. Here we demonstrate that ATF3 is clearly defined as a SUMO target protein both in vitro SUMOylation assay using recombinant proteins and at the cellular levels. Furthermore, ATF3 interacted with UBE2I (Ubc9), the only SUMO E2 enzyme found so far. In addition, PIAS3β (a SUMO E3 ligase) enhanced and SENP2 and SENP7 (two SUMOylation proteases) decreased SUMOylation of ATF3, respectively. In addition, we found that ATF3 is selectively SUMOylated at lysine residue 42 but the SUMOylation/deSUMOylation does not alter subcellular localization of ATF3. We next characterized the functional role of ATF3 SUMOylation on MC2R, NR5A1, STAR, and TP53 gene expression. We found that SUMOylation of ATF3 is required for full repression of NR5A1 and TP53 genes. While SUMOylation of ATF3 is required for full activation of MC2R gene activity, SUMOylation of ATF3 further enhances repression of STAR gene activity. Overall, we provide the first evidence that ATF3 is post-translationally modified by SUMO to regulate MC2R, NR5A1, STAR, and TP53 gene activities. Citation Format: Chiung-Min Wang, Victoria Brennan, Ninoska Gutierrez, Xirui Wang, Lizhong Wang, Wei-Hsiung Yang. SUMOylation of ATF3 alters MC2R, NR5A1, STAR, and TP53 gene activities. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 763A. doi:10.1158/1538-7445.AM2013-763A

Post-translational modifications to proteins are essential mechanisms for controlling functions of proteins and subsequently for regulating cell fate. SUMO modification (SUMOylation) has emerged as a critical regulatory pathway in cellular function and biological processes. DeSUMOylation (removal of SUMO conjugation) by members of SUMO-specific proteases (SENPs) family makes SUMO modification highly dynamic. In this mini-review, we briefly introduce the current knowledge regarding the regulatory pathway of deSUMOylation and focus on the recent progress of functions of SENPs in biological progresses. [N A J Med Sci. 2012;5(2):71-77.]