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Hepatocellular carcinoma (HCC) is one of the deadliest cancers. The retinoblastoma protein (RB1), a regulator of cell proliferation, is functionally inactivated in HCC by CYCLIN D/E-mediated phosphorylation. However, the mechanism of RB1-inactivation is unclear because only small percentages of HCCs exhibit amplification of CYCLIN D/E or mutations in the CDK-inhibitory genes. We show that FOXM1, which is overexpressed and critical for HCC, plays essential roles in inactivating RB1 and suppressing RB1-induced senescence of the HCC cells. Mechanistically, FOXM1 binds RB1 and DNMT3B to repress the expression of FOXO1, leading to a decrease in the levels of the CDK-inhibitors, creating an environment for phosphorylation and inactivation of RB1. Consistent with that, inhibition of FOXM1 causes increased expression of FOXO1 with consequent activation of RB1, leading to senescence of the HCC cells, in vitro and in vivo. Also, repression-deficient mutants of FOXM1 induce senescence that is blocked by depletion of RB1 or FOXO1. We provide evidence that human HCCs rely upon this FOXM1-FOXO1 axis for phosphorylation and inactivation of RB1. The observations demonstrate the existence of a new autoregulatory loop of RB1-inactivation in HCC involving a FOXM1-FOXO1 axis that is required for phosphorylation of RB1 and for aggressive progression of HCC.

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in breast cancers and correlates with poor prognosis. Mechanistically, FoxM1 associates with CBP to activate transcription and with Rb to repress transcription. Although the activating function of FoxM1 in breast cancer has been well documented, the significance of its repressive activity is poorly understood. Using CRISPR–Cas9 engineering, we generated a mouse model that expresses FoxM1-harboring point mutations that block binding to Rb while retaining its ability to bind CBP. Unlike FoxM1-null mice, mice harboring Rb-binding mutant FoxM1 did not exhibit significant developmental defects. The mutant mouse line developed PyMT-driven mammary tumors that were deficient in lung metastasis, which was tumor cell-intrinsic. Single-cell RNA-seq of the tumors revealed a deficiency in prometastatic tumor cells and an expansion of differentiated alveolar type tumor cells, and further investigation identified that loss of the FoxM1/Rb interaction caused enhancement of the mammary alveolar differentiation program. The FoxM1 mutant tumors also showed increased Pten expression, and FoxM1/Rb was found to activate Akt signaling by repressing Pten. In human breast cancers, expression of FoxM1 negatively correlated with Pten mRNA. Furthermore, the lack of tumor-infiltrating cells in FoxM1 mutant tumors appeared related to decreases in pro-metastatic tumor cells that express factors required for infiltration. These observations demonstrate that the FoxM1/Rb-regulated transcriptome is critical for the plasticity of breast cancer cells that drive metastasis, identifying a prometastatic role of Rb when bound to FoxM1. Significance: This work provides new insights into how the interaction between FoxM1 and Rb facilitates the evolution of metastatic breast cancer cells by altering the transcriptome.

PDF file - 107 KB, A, Representative pictures and quantification of soft agar colonies of CreERT2, Foxm1 fl/fl and p53 -/- sarcoma cells following control, 400nM and 800nM 4OH-tamoxifen treatment. B, Representative pictures and quantification of foci formation assay of CreERT2, Foxm1 fl/fl and p53 -/- sarcoma cells following control, 400nM and 800nM 4OH-tamoxifen treatment.

PDF file - 52 KB, A, Representative picture of tumor mass in kidney. B, Tumor mass of kidneys dissected from PBS, MUT and WT peptide treated SCID mice. Representative two mice from each group are shown.

PDF file - 103 KB, A, CreERT2 Foxm1 fl/fl and p53 -/- thymic lymphoma cell viability at 0h and 24 hours following non, 5�M of ARF 26-44 peptide, 5�M of ARF 37-44 peptide or PBS treatment. B, Foci formation of CreERT2 Foxm1 fl/fl and p53 -/- sarcoma cells following PBS, 25�M of ARF 27-44 peptide, or ARF 36-44 peptide treatment. C, Cleaved-caspase 3 staining of CreERT2 Foxm1 fl/fl and p53 -/- sarcoma cells 24 hours after 25�M of ARF 27-44 peptide treatment.

The forkhead box transcription factor FOXM1 is considered to be a promising target for cancer therapy. However, the significance of FOXM1 in tumors harboring mutation in p53, which is very common, is unclear. In this study, we investigated the efficacy of FoxM1 targeting in spontaneous p53-null tumors using genetic ablation as well as using a peptide inhibitor of FOXM1. We show that conditional deletion of FoxM1 inhibits growth of the p53-null thymic lymphoma and sarcoma cells. In addition, deletion of FoxM1 induces apoptotic cell death of the p53-null tumors, accompanied by reduced expression of the FOXM1 target genes survivin and Bmi1. An ARF-derived peptide that inhibits the activity of FOXM1, by targeting it to the nucleolus, also induces apoptosis in the p53-null sarcoma and lymphoma, leading to a strong inhibition of their metastatic colonization. Together, our observations suggest that FOXM1 is critical for survival and growth of the p53-null lymphoma and sarcoma and provide proof-of-principle that FOXM1 is an effective therapeutic target for sarcoma and lymphoma carrying loss of function mutation in p53. Mol Cancer Ther; 12(5); 759–67. ©2013 AACR.

PDF file - 216 KB, A, FoxM1 IHC staining of 5 micron allografted sarcoma tissue of control group. B, tamoxifen treated group. C, thymic lymphoma control group. D, thymic lymphoma tamoxifen group. E-F and I-J, Representative TUNEL and DAPI staining of tumor sections from the subcutaneously inoculated CreERT2 Foxm1 fl/fl and p53 -/- sarcoma after tamoxifen or control treatment are shown. G-H and K-L, Representative TUNEL and DAPI staining of tumor sections from the subcutaneously inoculated CreERT2 Foxm1 fl/fl and p53 -/- thymic lymphoma after tamoxifen or control treatment are shown. M, quantification of number of positive cleaved-caspase 3 (Asp175) cells per field of thymic lymphoma is shown. N, quantification of number of positive cleaved-PARP (Asp214) cells per field of thymic lymphoma is shown.

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PDF file - 93 KB, Box plot of FoxM1 mRNA expression level in tumors harboring mutations in p53 or having wild type p53. Datasets were extracted from ONCOMINE database. P values were calculated using Student's t test.

Supplementary Data from Transcriptional Repression by FoxM1 Suppresses Tumor Differentiation and Promotes Metastasis of Breast Cancer

Deregulation of the Wnt/β-catenin signaling pathway drives the development of colorectal cancer, but understanding of this pathway remains incomplete. Here, we report that the damage-specific DNA-binding protein DDB2 is critical for β-catenin–mediated activation of RNF43, which restricts Wnt signaling by removing Wnt receptors from the cell surface. Reduced expression of DDB2 and RNF43 was observed in human hyperplastic colonic foci. DDB2 recruited EZH2 and β-catenin at an upstream site in the Rnf43 gene, enabling functional interaction with distant TCF4/β-catenin–binding sites in the intron of Rnf43. This novel activity of DDB2 was required for RNF43 function as a negative feedback regulator of Wnt signaling. Mice genetically deficient in DDB2 exhibited increased susceptibility to colon tumor development in a manner associated with higher abundance of the Wnt receptor–expressing cells and greater activation of the downstream Wnt pathway. Our results identify DDB2 as both a partner and regulator of Wnt signaling, with an important role in suppressing colon cancer development. Cancer Res; 77(23); 6562–75. ©2017 AACR.

Table S1: Sequences of DsiRNA; Table S2: Primers for ChIP assays; Table S3: Primers for gene expression assays; Table S4: Correlation analysis of DDB2 and RNF43 expression in patient colonic samples; Table S5:Primers and probes for 3C assay

Figure S6: Association of DDB2, TCF4 and β-catenin on Rnf43 upstream region and intron regions.Figure S7: EZH2 and PAF are required for Rnf43 expression.Figure S8: DDB2 is required for the RNF43-mediated sequestration of TCF4 to the nuclear membrane.Figure S9: WES showed missense mutation in Ctnnb1 gene.Figure S10: DDB2 regulates RNF43 and Wnt target gene expression in normal colonic tissues.Figure S11: Model of how DDB2 regulates Wnt pathway and colon cancer development through RNF43.

Supplementary methods of Protein Extraction, Immunoblotting and Co-immunoprecipitations, Chromatin Conformation Capture Assay, Immunofluorescent Staining and Whole Exome Sequencing (WES)

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in breast cancers and correlates with poor prognosis. Mechanistically, FoxM1 associates with CBP to activate transcription and with Rb to repress transcription. Although the activating function of FoxM1 in breast cancer has been well documented, the significance of its repressive activity is poorly understood. Using CRISPR–Cas9 engineering, we generated a mouse model that expresses FoxM1-harboring point mutations that block binding to Rb while retaining its ability to bind CBP. Unlike FoxM1-null mice, mice harboring Rb-binding mutant FoxM1 did not exhibit significant developmental defects. The mutant mouse line developed PyMT-driven mammary tumors that were deficient in lung metastasis, which was tumor cell-intrinsic. Single-cell RNA-seq of the tumors revealed a deficiency in prometastatic tumor cells and an expansion of differentiated alveolar type tumor cells, and further investigation identified that loss of the FoxM1/Rb interaction caused enhancement of the mammary alveolar differentiation program. The FoxM1 mutant tumors also showed increased Pten expression, and FoxM1/Rb was found to activate Akt signaling by repressing Pten. In human breast cancers, expression of FoxM1 negatively correlated with Pten mRNA. Furthermore, the lack of tumor-infiltrating cells in FoxM1 mutant tumors appeared related to decreases in pro-metastatic tumor cells that express factors required for infiltration. These observations demonstrate that the FoxM1/Rb-regulated transcriptome is critical for the plasticity of breast cancer cells that drive metastasis, identifying a prometastatic role of Rb when bound to FoxM1.Significance:This work provides new insights into how the interaction between FoxM1 and Rb facilitates the evolution of metastatic breast cancer cells by altering the transcriptome.

Supplementary Figure Legends 1-4 from FoxM1 in Tumorigenicity of the Neuroblastoma Cells and Renewal of the Neural Progenitors

The regulation of androgen receptor (AR) expression in prostate cancer is still poorly understood. The activation of the epidermal growth factor receptor (EGFR) in prostate cancer cells was previously shown to lower AR expression by a rapamycin-sensitive, posttranscriptional mechanism involving the AR mRNA 5′-untranslated region (5′-UTR). In a search for an intermediate within the EGFR/phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway that regulates AR at this site, we identified the nucleic acid–binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNP-K), by mass spectrometric analysis of Akt immune complexes from lipid raft–enriched subcellular fractions. We show here that hnRNP-K is a novel inhibitor of AR mRNA translation that regulates androgen-responsive gene expression and prostate cancer cell proliferation. A functional hnRNP-K binding site involved in down-regulating AR protein levels was identified in the AR mRNA 5′-UTR. Further analysis revealed that hnRNP-K is also able to inhibit AR translation in the absence of the 5′-UTR, consistent with the presence of additional predicted hnRNP-K binding sites within the AR open reading frame and in the 3′-UTR. Immunohistochemical analysis of a human prostate cancer tissue microarray revealed an inverse correlation between hnRNP-K expression and AR protein levels in organ-confined prostate tumors and a substantial decline in cytoplasmic hnRNP-K in metastases, despite an overall increase in hnRNP-K levels in metastatic tumors. These data suggest that translational inhibition of AR by hnRNP-K may occur in organ-confined tumors but possibly at a reduced level in metastases. HnRNP-K is the first protein identified that directly interacts with and regulates the AR translational apparatus. [Cancer Res 2009;69(6):2210–8]

Supplementary Figure 5 from Heterogeneous Nuclear Ribonucleoprotein K Is a Novel Regulator of Androgen Receptor Translation

Supplementary Figure 2 from Heterogeneous Nuclear Ribonucleoprotein K Is a Novel Regulator of Androgen Receptor Translation

Supplementary Figure 3 from Heterogeneous Nuclear Ribonucleoprotein K Is a Novel Regulator of Androgen Receptor Translation

PDF file - 13093K, Supplementary Fig.1: Loss of DDB2 expression in human colon cancer. Supplementary Fig.2: Low magnification IHC data showing reduced expression of DDB2 in high grade colon cancer samples. Supplementary Fig.3: Depletion of DDB2 increases invasivness of colon cancer cells. Supplementary Fig.4: Polyclonal DDB2 knockdown colon cancer cells exhibit increased tumorigenicity Supplementary Fig.5: Polyclonal DDB2 knockdown colon cancer cells exhibit EMT Supplementary Fig.6: DDB2 knockdown colon cancer cells generate aggressive tumors in nude mice Supplementary Fig.7: Low magnification picture showing EMT in xenografted tumors Supplementary Fig.8: DDB2 expression blocks EMT induced by hypoxia and TGF-beta Supplementary Fig.9: Chromatin-IP using T7-antibody demonstrating interaction of T7-DDB2 with the VEGF, Snail and Zeb1 promoters. Supplementary Fig.10: Transcription factor binding sites in the promoter fragments bound by DDB2 Supplementary Fig.11: Western blot analyses for the DDB2-associated proteins and XPC in DDB2-depleted cells. Supplementary Fig.12: Experimental metastasis assay with polyclonal DDB2 knockdown cells. Supplementary Fig.13: Expression of DDB2 does not inhibit proliferation in SW620 cells.

Supplementary Figure 4 from Heterogeneous Nuclear Ribonucleoprotein K Is a Novel Regulator of Androgen Receptor Translation

Supplementary Figure 1 from Heterogeneous Nuclear Ribonucleoprotein K Is a Novel Regulator of Androgen Receptor Translation

Supplementary Figure 1 from FoxM1 in Tumorigenicity of the Neuroblastoma Cells and Renewal of the Neural Progenitors

Supplementary Figure 2 from FoxM1 in Tumorigenicity of the Neuroblastoma Cells and Renewal of the Neural Progenitors

Supplementary Figure 4 from FoxM1 in Tumorigenicity of the Neuroblastoma Cells and Renewal of the Neural Progenitors

Supplementary Legends for Figures 1-5 from Heterogeneous Nuclear Ribonucleoprotein K Is a Novel Regulator of Androgen Receptor Translation

Supplementary Figure 3 from FoxM1 in Tumorigenicity of the Neuroblastoma Cells and Renewal of the Neural Progenitors

Objectives 46, XY difference/disorder of sex development (DSD) is a relatively uncommon group of heterogeneous disorders with varying degree of underandrogenization of male genitalia. Such patients should be approached systematically to reach an aetiological diagnosis. However, we lack, at present, a clinical practice guideline on diagnostic approach in 46, XY DSD from this part of the globe. Moreover, debate persists regarding the timing and cut-offs of different hormonal tests, performed in these cases. The consensus committee consisting of 34 highly experienced endocrinologists with interest and experience in managing DSD discussed and drafted a consensus statement on the diagnostic approach to 46, XY DSD focussing on relevant history, clinical examination, biochemical evaluation, imaging and genetic analysis. Content The consensus was guided by systematic reviews of existing literature followed by discussion. An initial draft was prepared and distributed among the members. The members provided their scientific inputs, and all the relevant suggestions were incorporated. The final draft was approved by the committee members. Summary The diagnostic approach in 46, XY DSD should be multidisciplinary although coordinated by an experienced endocrinologist. We recommend formal Karyotyping, even if Y chromosome material has been detected by other methods. Meticulous history taking and thorough head-to-toe examination should initially be performed with focus on external genitalia, including location of gonads. Decision regarding hormonal and other biochemical investigations should be made according to the age and interpreted according to age-appropriate norms Although LC-MS/MS is the preferred mode of steroid hormone measurements, immunoassays, which are widely available and less expensive, are acceptable alternatives. All patients with 46, XY DSD should undergo abdominopelvic ultrasonography by a trained radiologist. MRI of the abdomen and/or laparoscopy may be used to demonstrate the Mullerian structure and/or to localize the gonads. Genetic studies, which include copy number variation (CNV) or molecular testing of a candidate gene or next generation sequencing then should be ordered in a stepwise manner depending on the clinical, biochemical, hormonal, and radiological findings. Outlook The members of the committee believe that patients with 46, XY DSD need to be approached systematically. The proposed diagnostic algorithm, provided in the consensus statement, is cost effective and when supplemented with appropriate genetic studies, may help to reach an aetiological diagnosis in majority of such cases.

Diabetic kidney disease (DKD) occurs in approximately 20–40% of patients with type 2 diabetes mellitus. Patients with DKD have a higher risk of cardiovascular and all-cause mortality. Angiotensin-converting enzyme inhibitors or angiotensin receptor blockers and antihyperglycemic drugs form the mainstay of DKD management and aim to restrict progression to more severe stages of DKD. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) control hyperglycemia by blocking renal glucose reabsorption in addition to preventing inflammation, thereby improving endothelial function and reducing oxidative stress; consequently, this class of prescription medicines is emerging as an important addition to the therapeutic armamentarium. The EMPA-REG OUTCOME, DECLARE TIMI 58, and CANVAS trials demonstrated the renoprotective effects of SGLT2i, such as restricting decline in glomerular filtration rate, in the progression of albuminuria, and in death due to renal causes. The renoprotection provided by SGLT2i was further confirmed in the CREDENCE study, which showed a 30% reduction in progression of chronic kidney disease, and in the DELIGHT study, which demonstrated a reduction in albuminuria with dapagliflozin compared with placebo (− 21.0%, confidence interval [CI] − 34.1 to − 5.2, p = 0.011). Furthermore, a meta-analysis demonstrated a reduced risk of dialysis, transplantation, or death due to kidney disease (relative risk 0.67; 95% CI 0.52–0.86; p = 0.0019) and a 45% risk reduction in worsening of renal function, end-stage renal disease, or renal death (hazard ratio 0.55, CI 0.48–0.64, p

Cancers in the oral/head & neck region (HNSCC) are aggressive due to high incidence of recurrence and distant metastasis. One prominent feature of aggressive HNSCC is the presence of severely hypoxic regions in tumors and activation of hypoxia-inducible factors (HIFs). In this study, we report that the XPE gene product DDB2 (damaged DNA binding protein 2), a nucleotide excision repair protein, is upregulated by hypoxia. Moreover, DDB2 inhibits HIF1α in HNSCC cells. It inhibits HIF1α in both normoxia and hypoxia by reducing mRNA expression. Knockdown of DDB2 enhances the expression of angiogenic markers and promotes tumor growth in a xenograft model. We show that DDB2 binds to an upstream promoter element in the HIF1Α gene and promotes histone H3K9 trimethylation around the binding site by recruiting Suv39h1. Also, we provide evidence that DDB2 has a significant suppressive effect on expression of the endogenous markers of hypoxia that are also prognostic indicators in HNSCC. Together, these results describe a new mechanism of hypoxia regulation that opposes expression of HIF1Α mRNA and the hypoxia-response genes.

DDB2 is a sensor of DNA damage and it plays an important role in Global Genomic Repair (GG-NER). Our previous studies show that DDB2 is involved in the regulation of metastasis in colon adenocarcinoma. Squamous Cell Carcinomas in the Oral/Head & Neck region (HNSCC) are particularly aggressive due to high incidence of recurrence and distant metastasis. In this study, we show that DDB2 expression is downregulated in advanced HNSCCs and loss of DDB2 expression coincides with reduced survival. Recent meta-analysis of gene expression data characterized the mesenchymal-type (EMT-type) as one most aggressive cancer cluster in HNSCC. Here, we report that DDB2 constitutively represses mRNA expression of the EMT- regulatory transcription factors SNAIL, ZEB1, and angiogenic factor VEGF in HNSCC cells. As a result, re-expression of DDB2 in metastatic cells reversed EMT with transcriptional upregulation of epithelial marker E-cadherin, and downregulation of mesenchymal markers N-cadherin, Vimentin, and Fibronectin. Interestingly, in a reverse assay, depletion of DDB2 in non-metastatic cells induced expression of the same EMT-regulatory transcription factors. TGFβs are major regulators of Snail and Zeb1, and we observed that DDB2 transcriptionally regulates expression of TGFB2 in HNSCC cells. Re-expression of DDB2 in mouse embryonic fibroblasts (MEFs) isolated from Ddb2 (−/−) knockout-mice resulted in repression of EMT-regulatory factors Zeb1, Snail and Tgfb2. Taken together, these results support the active role of DDB2 as a candidate suppressor of the EMT-process in HNSCC. Early detection leads to significantly higher survival in HNSCC and DDB2 expression in tumors can be a predictor of EMT progression.

Arf, a well-established tumor suppressor, is either mutated or downregulated in a wide array of cancers. However, its role in hepatocellular carcinoma (HCC) progression is controversial. Conflicting observations have been published regarding its expression in HCC. In this study, we provide clear genetic evidence demonstrating a protective role of p19Arf in hepatocarcinogenesis. Using Ras-induced mouse model, we show that p19Arf deficiency accelerates progression of aggressive HCC in vivo. To investigate the role of p14ARF in human liver cancers, we analyzed its expression in human HCC using immunohistochemistry (IHC). We observe lack of nucleolar p14ARF in 43.02% of human HCC samples and that low expression of p14ARF strongly correlates with the early onset of HCC. Importantly, cirrhotic livers that did not progress to HCC harbor higher expression of the p14ARF protein in hepatocytes compared with that in cirrhotic livers with HCC. These results are significant because they suggest that nucleolar p14ARF can be used as early prognostic marker in chronic liver disease to reliably identify patients with high risk for developing liver cancer. Currently, there is no effective systemic therapy for advanced liver cancer; hence, more efficient patient screening and early detection of HCC would significantly contribute to the eradication of this devastating disease.

Deregulation of the Wnt/β-catenin signaling pathway drives the development of colorectal cancer, but understanding of this pathway remains incomplete. Here, we report that the damage-specific DNA-binding protein DDB2 is critical for β-catenin–mediated activation of RNF43, which restricts Wnt signaling by removing Wnt receptors from the cell surface. Reduced expression of DDB2 and RNF43 was observed in human hyperplastic colonic foci. DDB2 recruited EZH2 and β-catenin at an upstream site in the Rnf43 gene, enabling functional interaction with distant TCF4/β-catenin–binding sites in the intron of Rnf43. This novel activity of DDB2 was required for RNF43 function as a negative feedback regulator of Wnt signaling. Mice genetically deficient in DDB2 exhibited increased susceptibility to colon tumor development in a manner associated with higher abundance of the Wnt receptor–expressing cells and greater activation of the downstream Wnt pathway. Our results identify DDB2 as both a partner and regulator of Wnt signaling, with an important role in suppressing colon cancer development. Cancer Res; 77(23); 6562–75. ©2017 AACR.

DDB2 is a multifunctional protein that participates in both nucleotide excision repair and regulation of gene transcription. In colon cancer cells, chromatin association of XRCC5/6, in the absence of DNA damage, depends on DDB2, and the DDB2–XRCC5/6 interaction promotes the transcription of the antiangiogenic gene SEMA3A., Damaged DNA-binding protein 2 (DDB2), a nuclear protein, participates in both nucleotide excision repair and mRNA transcription. The transcriptional regulatory function of DDB2 is significant in colon cancer, as it regulates metastasis. To characterize the mechanism by which DDB2 participates in transcription, we investigated the protein partners in colon cancer cells. Here we show that DDB2 abundantly associates with XRCC5/6, not involving CUL4 and DNA-PKcs. A DNA-damaging agent that induces DNA double-stranded breaks (DSBs) does not affect the interaction between DDB2 and XRCC5. In addition, DSB-induced nuclear enrichment or chromatin association of XRCC5 does not involve DDB2, suggesting that the DDB2/XRCC5/6 complex represents a distinct pool of XRCC5/6 that is not directly involved in DNA break repair (NHEJ). In the absence of DNA damage, on the other hand, chromatin association of XRCC5 requires DDB2. We show that DDB2 recruits XRCC5 onto the promoter of SEMA3A, a DDB2-stimulated gene. Moreover, depletion of XRCC5 inhibits SEMA3A expression without affecting expression of VEGFA, a repression target of DDB2. Together our results show that DDB2 is critical for chromatin association of XRCC5/6 in the absence of DNA damage and provide evidence that XRCC5/6 are functional partners of DDB2 in its transcriptional stimulatory activity.

The forkhead box transcription factor FoxM1 is essential for hepatocellular carcinoma (HCC) development, and its overexpression coincides with poor prognosis. Here, we show that the mechanisms by which FoxM1 drives HCC progression involve overcoming the inhibitory effects of the liver differentiation gene FoxA2. First, the expression patterns of FoxM1 and FoxA2 in human HCC are opposite. We show that FoxM1 represses expression of FoxA2 in G1 phase. Repression of FoxA2 in G1 phase is important, as it is capable of inhibiting expression of the pluripotency genes that are expressed mainly in S–G2 phases. Using a transgenic mouse model for oncogenic Ras-driven HCC, we provide genetic evidence for a repression of FoxA2 by FoxM1. Conversely, FoxA2 inhibits expression of FoxM1 and inhibits FoxM1-induced tumorigenicity. Also, FoxA2 inhibits Ras-induced HCC progression that involves FoxM1. Implications: The observations provide strong genetic evidence for an opposing role of FoxM1 and FoxA2 in HCC progression. Moreover, FoxM1 drives high-grade HCC progression partly by inhibiting the hepatocyte differentiation gene FoxA2.

Rationale Forkhead box M1 (FoxM1) is a transcription factor that promotes cell proliferation by regulating a broad spectrum of genes that participate in cell cycle regulation, such as Cyclin B, CDC25B, and Aurora B Kinase. We have shown that hypoxia, a well-known stimulus for pulmonary hypertension (PH), induces FoxM1 in pulmonary artery smooth muscle cells (PASMC) in a HIF-dependent pathway, resulting in PASMC proliferation, while the suppression of FoxM1 prevents hypoxia-induced PASMC proliferation. However, the implications of FoxM1 in the development of PH remain less known. Methods We determined FoxM1 levels in the lung samples of idiopathic PAH (pulmonary arterial hypertension) (IPAH) patients and hypoxia-induced PH mice. We generated constitutive and inducible smooth muscle cell (SMC)-specific FoxM1 knockdown or knockout mice as well as FoxM1 transgenic mice which overexpress FoxM1, and exposed them to hypoxia (10% O2, 90% N2) or normoxia (Room air, 21% oxygen) for four weeks, and measured PH indices. We also isolated mouse PASMC (mPASMC) and mouse embryonic fibroblasts (MEF) from these mice to examine the cell proliferation and expression levels of SMC contractile proteins. Results We showed that in hypertensive human lungs or mouse lungs, FoxM1 levels were elevated. Constitutive knockout of FoxM1 in mouse SMC caused early lethality, whereas constitutive knockdown of FoxM1 in mouse SMC prevented hypoxia-induced PH and PASMC proliferation. Inducible knockout of FoxM1 in SMC reversed hypoxia-induced pulmonary artery wall remodeling in existing PH. Overexpression of FoxM1 enhanced hypoxia-induced pulmonary artery wall remodeling and right ventricular hypertrophy in mice. Alteration of FoxM1 status did not affect hypoxia-induced hypoxia-inducible factor (HIF) activity in mice. Knockout of FoxM1 decreased PASMC proliferation and induced expression of SMC contractile proteins and TGF-β/Smad3 signaling. Conclusions Our studies provide clear evidence that altered FoxM1 expression in PASMC contributes to PH and uncover a correlation between Smad3-dependent signaling in FoxM1-mediated proliferation and de-differentiation of PASMC.

The fork-head box transcription factor FoxMl is essential for hepatocellular carcinoma (HCC) development and its overexpression coincides with poor prognosis. Here, we show that the mechanisms by which FoxM1 drives HCC progression involve overcoming the inhibitory effects of the liver differentiation gene FoxA2. First, the expression patterns of FoxM1 and FoxA2 in human HCC are opposite. We show that FoxM1 represses expression of FoxA2 in G1 phase, a phase in the cell cycle in which cells can undergo differentiation. Repression of FoxA2 in G1 phase is important, as it is capable of inhibiting expression of the pluripotency genes that are expressed mainly in S/G2 phases. Using a transgenic mouse model for oncogenic Ras-driven HCC, we provide genetic evidence for a repression of FoxA2 by FoxM1. Conversely, FoxA2 inhibits expression of FoxM1, and inhibits FoxM1-induced tumorigenicity of HCC cells. Moreover, expression of FoxA2 in mouse liver expressing activated Ras inhibits FoxM1 expression and inhibits HCC progression. The observations provide strong genetic evidence for an opposing role of FoxM1 and FoxA2 in HCC progression.AUTHOR SUMMARYLiver cancer remains untreatable because it is diagnosed at a stage when the cancer is aggressive and resistant to therapeutics. The mechanism that drives aggressive liver cancer is poorly understood. These cancers are made up of poorly differentiated cancer cells. Interestingly, the FoxM1 gene is overexpressed in the aggressive liver cancers. Although FoxM1 is important for expression of the proliferation genes, it does not explain why it is overexpressed mainly in the undifferentiated cancers. The current study addresses this puzzle. Our previous studies demonstrated that FoxM1 increases expression of the pluripotency genes that are expressed mainly in the stem-like cells. In the current manuscript we show that, in addition to activating the pluripotency genes, FoxM1 inhibits expression of the liver differentiation gene FoxA2. Overexpression of FoxM1 is important for this inhibition function, as it involves the retinoblastoma family of proteins, which are often inactivated in cancer cells, and thus, are of low-abundance. Moreover, the inhibition of FoxA2 is significant because FoxA2 could inhibit expression of the pluripotency genes as well as FoxM1. The observations provide new insights into how FoxM1 drives progression of aggressive liver cancer.

Background & Aims Overexpression of FoxM1 correlates with poor prognosis in hepatocellular carcinoma (HCC). Moreover, the Ras-signaling pathway is found to be ubiquitously activated in HCC through epigenetic silencing of the Ras-regulators. We investigated the roles of FoxM1 in Ras-driven HCC, and on HCC cells with stem-like features. Methods We employed a transgenic mouse model that expresses the oncogenic Ras in the liver. That strain was crossed with a strain that harbor floxed alleles of FoxM1 and the MxCre gene that allows conditional deletion of FoxM1 . FoxM1 alleles were deleted after development of HCC, and the effects on the tumors were analyzed. Also, FoxM1 siRNA was used in human HCC cell lines to determine its role in the survival of the HCC cells with stem cell features. Results Ras-driven tumors overexpress FoxM1. Deletion of FoxM1 inhibits HCC progression. There was increased accumulation of reactive oxygen species (ROS) in the FoxM1 deleted HCC cells. Moreover, FoxM1 deletion caused a disproportionate loss of the CD44+ and EpCAM+ HCC cells in the tumors. We show that FoxM1 directly activates expression of CD44 in human HCC cells. Moreover, the human HCC cells with stem cell features are addicted to FoxM1 for ROS-regulation and survival. Conclusion Our results provide genetic evidence for an essential role of FoxM1 in the progression of Ras-driven HCC. In addition, FoxM1 is required for the expression of CD44 in HCC cells. Moreover, FoxM1 plays a critical role in the survival of the HCC cells with stem cell features by regulating ROS.

Reactive oxygen species (ROS) is critical for premature senescence, a process significant in tumor suppression and cancer therapy. Here, we reveal a novel function of the nucleotide excision repair protein DDB2 in the accumulation of ROS in a manner that is essential for premature senescence. DDB2-deficient cells fail to undergo premature senescence induced by culture shock, exogenous oxidative stress, oncogenic stress, or DNA damage. These cells do not accumulate ROS following DNA damage. The lack of ROS accumulation in DDB2 deficiency results from high-level expression of the antioxidant genes in vitro and in vivo. DDB2 represses antioxidant genes by recruiting Cul4A and Suv39h and by increasing histone-H3K9 trimethylation. Moreover, expression of DDB2 also is induced by ROS. Together, our results show that, upon oxidative stress, DDB2 functions in a positive feedback loop by repressing the antioxidant genes to cause persistent accumulation of ROS and induce premature senescence.

FoxM1b is a cell cycle-regulated transcription factor, whose over-expression is a marker for poor outcome in cancers. Its transcriptional activation function requires phosphorylation by Cdk1 or Cdk2 that primes FoxM1b for phosphorylation by Plk1, which triggers association with the co-activator CBP. FoxM1b also possesses transcriptional repression function. It represses the mammary differentiation gene GATA3 involving DNMT3b and Rb. We investigated what determines the two distinct functions of FoxM1b: activation and repression. We show that Rb binds to the C-terminal activation domain of FoxM1b. Analyses with phospho-defective and phospho-mimetic mutants of FoxM1b identified a critical role of the Plk1 phosphorylation sites in regulating the binding of FoxM1b to Rb and DNMT3b. That is opposite of what was seen for the interaction of FoxM1b with CBP. We show that, in addition to GATA3, FoxM1b also represses the mammary luminal differentiation marker FoxA1 by promoter-methylation, and that is regulated by the Plk1 phosphorylation sites in FoxM1b. Our results show that the Plk1 phosphorylation sites in FoxM1b serve as a regulator for its repressor function, and they provide insights into how FoxM1b inhibits differentiation genes and activates proliferation genes during cancer progression.

Background Angiotensinogen (AGT) enzyme comprises a vital module of RAAS system that effectively controls the blood pressure and related cardiovascular functions. Ample association studies have reported the importance of AGT variants in cardiovascular and non-cardiovascular adversities. But lately, owing to the complexity of the many anomalies, the haplotype based examination of genetic variation that facilitates the identification of polymorphic sites which are located in the vicinity of the causative polymorphic site, gets greater appreciation. Methods In the present study, we have done genotype and haplotype analysis of AGT gene in reference to hypertension to confirm the association of the two in an Indian population. To accomplish this, we performed candidate SNPs analysis and construct possible haplotypes across the AGT promoter and gene region in 414 subjects (256 Hypertensive cases and 158 controls). Results We found four SNPs (rs11568020: A-152G and rs5050: A-20C in promoter; rs4762 and rs699 in exon2) and 3 haplotypes (H4, H7 and H8) that showed a stronger positive association with hypertension. The haplotype H2 was showing protective association with hypertension. Conclusion The results of the present study confirmed and reestablished the role of AGT gene variants and their haplotypes in the causation of hypertension in Indian population and showed that haplotypes can provide stronger evidence of association. Electronic supplementary material The online version of this article (doi:10.1186/s40885-017-0069-x) contains supplementary material, which is available to authorized users.