Inactivation of VRK1 sensitizes ovarian cancer to PARP inhibition through regulating DNA-PK stability.

Ovarian cancer is the leading cause of gynecologic cancer death. Among the most innovative anti-cancer approaches, the genetic concept of synthetic lethality is that mutations in multiple genes work synergistically to effect cell death. Previous studies found that although vaccinia-related kinase-1 (VRK1) associates with DNA damage repair proteins, its underlying mechanisms remain unclear. Here, we found high VRK1 expression in ovarian tumors, and that VRK1 depletion can significantly promote apoptosis and cell cycle arrest. The effect of VRK1 knockdown on apoptosis was manifested by increased DNA damage, genomic instability, and apoptosis, and also blocked non-homologous end joining (NHEJ) by destabilizing DNA-PK. Further, we verified that VRK1 depletion enhanced sensitivity to a PARP inhibitor (PARPi), olaparib, promoting apoptosis through DNA damage, especially in ovarian cancer cell lines with high VRK1 expression. Proteins implicated in DNA damage responses are suitable targets for the development of new anti-cancer therapeutic strategies, and their combination could represent an alternative form of synthetic lethality. Therefore, normal protective DNA damage responses are impaired by combining olaparib with elimination of VRK1 and could be used to reduce drug dose and its associated toxicity. In summary, VRK1 represents both a potential biomarker for PARPi sensitivity, and a new DDR-associated therapeutic target, in ovarian cancer.

Degradation of AZGP1 suppresses apoptosis and facilitates cholangiocarcinoma tumorigenesis via TRIM25.

Alpha-2-Glycoprotein 1, Zinc-binding (AZGP1, ZAG) is a secreted protein that is synthesized by adipocytes and epithelial cells; it is downregulated in several malignancies such as breast, prostate, liver and lung cancers. However, its function remains unclear in cholangiocarcinoma (CCA). Here, we evaluated the impact AZGP1 in CCA using Gene Expression Omnibus (GEO) and GEPIA. In addition, we analysed AZGP1 expression using quantitative reverse transcription PCR and western blotting. Expression of AZGP1 was nearly deficient in CCA patients and cell lines and was associated with poor prognosis. AZGP1 overexpression upregulated apoptosis markers. Co-immunoprecipitation experiments showed that AZGP1 interacts with tripartite motif-containing protein 25 (TRIM25), and tissue microarray and bioinformatic analysis showed that AZGP1 is negatively correlated with TRIM25 expression in CCA. Thereafter, TRIM25 knockdown led to AZGP1 upregulation and induced cancer cell apoptosis. TRIM25 targets AZGP1 for degradation by catalysing its ubiquitination. AZGP1 overexpression significantly suppressed tumour growth in a xenograft mouse model. This study findings suggest that AZGP1 is a potential therapeutic target or a diagnostic biomarker for treating patients with CCA.

DGG-300273, a novel WNT/ß-catenin inhibitor, induces apoptotic cell death by activating ROS-BIM signaling in a Wnt-dependent manner in colon cancer cells.

Dysregulated Wnt signaling is associated with malignant oncogenic transformation, especially in colon cancer. Recently, numerous drugs have been developed based on tumorigenesis biomarkers, thus having high potential as drug targets. Likewise, WNT/ß-catenin pathway members are attractive therapeutic targets for colon cancer and are currently in various stages of development. However, although inhibitors of proteins regulating the WNT/ß-catenin signaling pathway have been extensively studied, they have yet to be clinically approved, and the underlying molecular mechanism(s) of their anticancer effects remain poorly understood. Herein, we show that a novel WNT/ß-catenin inhibitor, DGG-300273, inhibits colon cancer cell growth in a Wnt-dependent manner due to upregulation of the BCL2-family protein Bim and caspase-dependent apoptotic cell death. Additionally, DGG-300273-mediated cell death occurs by increased reactive oxygen species (ROS), as shown by abrogation of apoptotic cell death and ROS production following pretreatment with the antioxidant N-acetylcysteine. These results suggest that DGG-300273 represents a promising investigational drug for the treatment of Wnt-associated cancer, thus warranting further characterization and study.

Role of p53 in transcriptional repression of SVCT2.

SVCT2, Sodium-dependent Vitamin C Transporter 2, uniquely transports ascorbic acid (also known as vitamin C and ascorbate) into all types of cells. Vitamin C is an essential nutrient that must be obtained through the diet and plasma levels are tightly regulated by transporter activity. Vitamin C plays an important role in antioxidant defenses and is a cofactor for many enzymes that enable hormone synthesis, oxygen sensing, collagen synthesis and epigenetic pathways. Although SVCT2 has various functions, regulation of its expression/activity remains poorly understood. We found a p53-binding site, within the SVCT2 promoter, using a transcription factor binding-site prediction tool. In this study, we show that p53 can directly repress SVCT2 transcription by binding a proximal- (~-185 to -171 bp) and a distal- (~-1800 to -1787 bp) p53-responsive element (PRE), Chromatin immunoprecipitation assays showed that PRE-bound p53 interacts with the corepressor-histone deacetylase 3 (HDAC3), resulting in deacetylation of histones Ac-H4, at the proximal promoter, resulting in transcriptional silencing of SVCT2. Overall, our data suggests that p53 is a potent transcriptional repressor of SVCT2, a critical transporter of diet-derived ascorbic acid, across the plasma membranes of numerous essential tissue cell types.

Temporal and differential regulation of KAISO-controlled transcription by phosphorylated and acetylated p53 highlights a crucial regulatory role of apoptosis.

Transcriptional regulator KAISO plays a critical role in cell cycle arrest and apoptosis through modulation of p53 acetylation by histone acetyltransferase p300. KAISO potently stimulates apoptosis in cells expressing WT p53, but not in p53-mutant or p53-null cells. Here, we investigated how KAISO transcription is regulated by p53, finding four potential p53-binding sites (p53-responsive DNA elements; p53REs) located in a distal 5'-upstream regulatory element, intron 1, exon 2 coding sequence, and a 3'-UTR region. Transient transcription assays of pG5-p53RE-Luc constructs with various p53REs revealed that p53 activates KAISO (ZBTB33) transcription by acting on p53RE1 (-4326 to -4227) of the 5'-upstream region and on p53RE3 (+2929 to +2959) of the exon 2 coding region during early DNA damage responses (DDRs). ChIP and oligonucleotide pulldown assays further disclosed that p53 binds to the p53RE1 and p53RE3 sites. Moreover, ataxia telangiectasia mutated (ATM) or ATM-Rad3-related (ATR) kinase-mediated p53 phosphorylation at Ser-15 or Ser-37 residues activated KAISO transcription by binding its p53RE1 or p53RE3 sites during early DDR. p53RE1 uniquely contained three p53-binding half-sites, a structural feature important for transcriptional activation by phosphorylated p53 Ser-15·Ser-37. During the later DDR phase, a KAISO-mediated acetylated p53 form (represented by a p53QRQ acetyl-mimic) robustly activated transcription by acting on p53RE1 in which this structural feature is not significant, but it provided sufficient KAISO levels to confer a p53 "apoptotic code." These results suggest that the critical apoptosis regulator KAISO is a p53 target gene that is differently regulated by phosphorylated p53 or acetylated p53, depending on DDR stage.

Reciprocal negative regulation between the tumor suppressor protein p53 and B cell CLL/lymphoma 6 (BCL6) via control of caspase-1 expression.

Even in the face of physiological DNA damage or expression of the tumor suppressor protein p53, B cell CLL/lymphoma 6 (BCL6) increases proliferation and antagonizes apoptotic responses in B cells. BCL6 represses TP53 transcription and also appears to inactivate p53 at the protein level, and additional findings have suggested negative mutual regulation between BCL6 and p53. Here, using Bcl6-/- knockout mice, HEK293A and HCT116 p53-/- cells, and site-directed mutagenesis, we found that BCL6 interacts with p53 and thereby inhibits acetylation of Lys-132 in p53 by E1A-binding protein p300 (p300), a modification that normally occurs upon DNA damage-induced cellular stress and whose abrogation by BCL6 diminished transcriptional activation of p53 target genes, including that encoding caspase-1. Conversely, we also found that BCL6 protein is degraded via p53-induced, caspase-mediated proteolytic cleavage, and the formation of a BCL6-p53-caspase-1 complex. Our results suggest that p53 may block oncogenic transformation by decreasing BCL6 stability via caspase-1 up-regulation, whereas aberrant BCL6 expression inactivates transactivation of p53 target genes, either by inhibiting p53 acetylation by p300 or repressing TP53 gene transcription. These findings have implications for B cell development and lymphomagenesis.

Cell fate decisions by c-Myc depend on ZBTB5 and p53.

The oncoprotein, c-Myc, not only promotes cell proliferation, but can also induce or sensitize cells to apoptosis. However, how c-Myc decides cell fate and which c-Myc downstream target genes are involved remain unknown. Previously, we showed that ZBTB5 (zinc finger and BTB domain-containing 5) is a proto-oncogene that stimulates cell proliferation. ZBTB5 represses p21/CDKN1A by competing with p53 and recruiting corepressor histone deacetylase complexes. Herein, we found that c-Myc directly activates the transcription of ZBTB5. In the absence of p53, ZBTB5 is acetylated at K597 by interacting with p300, and activates transcription of NOXA, which induces apoptosis. In contrast, in the presence of p53, ZBTB5 interacts with p53 and acetylation at ZBTB5 K597 is blocked. ZBTB5 without K597 acetylation interacts with mSin3A/HDAC1 to repress p21/CDKN1A transcription and promote cell proliferation. Cell fate decisions by c-Myc depend on ZBTB5, p53 and p300, and acetylation of ZBTB5 K597.

Inhibition of JAK1/2 can overcome EGFR-TKI resistance in human NSCLC.

Non-small lung cancer (NSCLC) is the most common cancer in the world. The epidermal growth factor receptor (EGFR) gene is mutated in approximately 10% of lung cancer cases in the US and 50% of lung cancer in Asia. The representative target therapeutic agent, erlotinib (EGFR tyrosine kinase inhibitor; EGFR TKI), is effective in inactivating EGFR in lung cancer patients. However, approximately 50-60% of patients are resistant to EGFR TKI. These populations are associated with the EGFR mutation. To overcome resistance to EGFR TKI, we discovered a JAK1 inhibitor, CJ14939. We investigated the efficacy of CJ14939 in human NSCLC cell lines in vitro and in vivo. Our results showed that CJ14939 induced the inhibition of cell growth. Moreover, we demonstrated that combination treatment with erlotinib and CJ14939 induced cell death in vitro and inhibited tumor growth in vivo. In addition, we confirmed the suppression of phosphorylated EGFR, JAK1, and Stat3 expression in erlotinib and CJ14939-treated human NSCLC cell lines. Our results provide evidence that JAK inhibition overcomes resistance to EGFR TKI in human NSCLCs.

Chemosensitivity to HM90822, a novel synthetic IAP antagonist, is determined by p-AKT-inducible XIAP phosphorylation in human pancreatic cancer cells.

Inhibitor of apoptosis proteins (IAPs) are overexpressed in the majority of cancers and prevent apoptosis by inhibiting caspases. IAPs have therefore attracted considerable attention as potential targets for anticancer therapy. Here, we demonstrated that HM90822 (abbreviated HM822; a new synthetic IAP antagonist) induced apoptotic cell death via proteasome-dependent degradation of BIR2/3 domain-containing IAPs in human pancreatic cancer cells. HM822 inhibited the expression of XIAP and cIAP1/2 proteins in Panc-1 and BxPC-3 cells, which are sensitive to HM822. HM822 also induced IAP ubiquitination and promoted proteasome-dependent IAP degradation. However, cells expressing phospho-XIAP (Ser87) and AKT exhibited resistance to HM822. In other words, the overexpression of AKT-CA (constitutive active form for AKT) or AKT-WT induced resistance to HM822. In addition, in Panc-1 xenograft and orthotopic mouse models, we revealed that tumor growth was suppressed by the administration of HM822. Taken together, these results suggest that HM822 induces apoptosis through ubiquitin/proteasome-dependent degradation of BIR3 domain-containing IAPs. These findings suggest that phospho-XIAP and phospho-AKT may be used as biomarkers for predicting the efficacy of HM822 in pancreatic cancer patients.

Zbtb7c is a molecular 'off' and 'on' switch of Mmp gene transcription.

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases that play roles in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. The expression of MMP gene is tightly regulated and shows cell- and tissue-specific expression patterns. Despite their differential expression, MMP genes have AP-1 (activator protein-1) binding elements within their promoters. Interestingly, c-JUN phosphorylation by cytokine signaling decreased its interaction with NCoR, but increased its interaction with p300, resulting in activation of MMP gene transcription. Here, we found that Zbtb7c (Kr-pok) is a critical component of a transcriptional repressor complex containing c-Jun and NCoR. c-Jun, bound at AP-1, interacts with Zbtb7c, which in turn recruits an NCoR/Hdac3 complex to repress several Mmp (-8, -10, -13, and -16) genes. The molecular interaction between c-Jun and Zbtb7c also prevents phosphorylation of c-Jun by p-Jnk, However, Zbtb7c phosphorylation by p-Jnk (induced by TNFα), and its (Zbtb7c) subsequent degradation by the ubiquitin-mediated proteasomal pathway, leads to c-Jun phosphorylation by p-Jnk. Promoter-bound p-c-Jun then recruits the coactivator p300 to upregulate Mmp gene. Overall, these findings show that Zbtb7c is a key molecule that recruits an NCoR/Hdac3 complex to inhibit phosphorylation of c-Jun, and thereby repress Mmp gene expression.

ZBTB2 increases PDK4 expression by transcriptional repression of RelA/p65.

The NF-κB is found in almost all animal cell types and is involved in a myriad of cellular responses. Aberrant expression of NF-κB has been linked to cancer, inflammatory diseases and improper development. Little is known about transcriptional regulation of the NF-κB family member gene RelA/p65. Sp1 plays a key role in the expression of the RelA/p65 gene. ZBTB2 represses transcription of the gene by inhibiting Sp1 binding to a Sp1-binding GC-box in the RelA/p65 proximal promoter (bp, -31 to -21). Moreover, recent studies revealed that RelA/p65 directly binds to the peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α) to decrease transcriptional activation of the PGC1α target gene PDK4, whose gene product inhibits pyruvate dehydrogenase (PDH), a key regulator of TCA cycle flux. Accordingly, we observed that RelA/p65 repression by ZBTB2 indirectly results in increased PDK4 expression, which inhibits PDH. Consequently, in cells with ectopic ZBTB2, the concentrations of pyruvate and lactate were higher than those in normal cells, indicating changes in glucose metabolism flux favoring glycolysis over the TCA cycle. Knockdown of ZBTB2 in mouse xenografts decreased tumor growth. ZBTB2 may increase cell proliferation by reprogramming glucose metabolic pathways to favor glycolysis by upregulating PDK4 expression via repression of RelA/p65 expression.

KAISO, a critical regulator of p53-mediated transcription of CDKN1A and apoptotic genes.

An unresolved issue in genotoxic stress response is identification of induced regulatory proteins and how these activate tumor suppressor p53 to determine appropriate cell responses. Transcription factor KAISO was previously described to repress transcription following binding to methylated DNA. In this study, we show that KAISO is induced by DNA damage in p53-expressing cells and then interacts with the p53-p300 complex to increase acetylation of p53 K320 and K382 residues, although decreasing K381 acetylation. Moreover, the p53 with this particular acetylation pattern shows increased DNA binding and potently induces cell cycle arrest and apoptosis by activating transcription of CDKN1A (cyclin-dependent kinase inhibitor 1) and various apoptotic genes. Analogously, in Kaiso KO mouse embryonic fibroblast cells, p53-to-promoter binding and up-regulation of p21 and apoptosis gene expression is significantly compromised. KAISO may therefore be a critical regulator of p53-mediated cell cycle arrest and apoptosis in response to various genotoxic stresses in mammalian cells.

Transcriptional activation of APAF1 by KAISO (ZBTB33) and p53 is attenuated by RelA/p65.

KAISO, a member of the POK protein family, is induced by DNA-damaging agents to enhance apoptosis in a p53-dependent manner. Previously, we found that p53 interacts with KAISO, and acetylation of p53 lysine residues by p300 is modulated by KAISO. APAF1, the core molecule of the apoptosome, is transcriptionally activated by KAISO only in cells expressing p53, which binds to APAF1 promoter p53-response elements (p53REs). APAF1 transcriptional upregulation is further enhanced by KAISO augmentation of p53 binding to the APAF1 promoter distal p53RE#1 (bp, -765 to -739). Interestingly, a NF-κB response element, located close to the p53RE#1, mediates APAF1 transcriptional repression by affecting interaction between KAISO and p53. Ectopic RelA/p65 expression led to depletion of nuclear KAISO, with KAISO being mainly detected in the cytoplasm. RelA/p65 cytoplasmic sequestration of KAISO prevents its nuclear interaction with p53, decreasing APAF1 transcriptional activation by a p53-KAISO-p300 complex in cells exposed to genotoxic stresses. While KAISO enhances p53-dependent apoptosis by increasing APAF1 gene expression, RelA/p65 decreases apoptosis by blocking interaction between KAISO and p53. These findings have relevance to the phenomenon of cancer cells' diminished apoptotic capacity and the onset of chemotherapy resistance.

Two ZNF509 (ZBTB49) isoforms induce cell-cycle arrest by activating transcription of p21/CDKN1A and RB upon exposure to genotoxic stress.

ZNF509 is unique among POK family proteins in that four isoforms are generated by alternative splicing. Short ZNF509 (ZNF509S1, -S2 and -S3) isoforms contain one or two out of the seven zinc-fingers contained in long ZNF509 (ZNF509L). Here, we investigated the functions of ZNF509 isoforms in response to DNA damage, showing isoforms to be induced by p53. Intriguingly, to inhibit proliferation of HCT116 and HEK293 cells, we found that ZNF509L activates p21/CDKN1A transcription, while ZNF509S1 induces RB. ZNF509L binds to the p21/CDKN1A promoter either alone or by interacting with MIZ-1 to recruit the co-activator p300 to activate p21/CDKN1A transcription. In contrast, ZNF509S1 binds to the distal RB promoter to interact and interfere with the MIZF repressor, resulting in derepression and transcription of RB. Immunohistochemical analysis revealed that ZNF509 is highly expressed in normal epithelial cells, but was completely repressed in tumor tissues of the colon, lung and skin, indicating a possible role as a tumor suppressor.

Promyelocytic leukemia zinc finger-retinoic acid receptor α (PLZF-RARα), an oncogenic transcriptional repressor of cyclin-dependent kinase inhibitor 1A (p21WAF/CDKN1A) and tumor protein p53 (TP53) genes.

Promyelocytic leukemia zinc finger-retinoic acid receptor α (PLZF-RARα) is an oncogene transcriptional repressor that is generated by a chromosomal translocation between the PLZF and RARα genes in acute promyelocytic leukemia (APL-type) patients. The molecular interaction between PLZF-RARα and the histone deacetylase corepressor was proposed to be important in leukemogenesis. We found that PLZF-RARα can repress transcription of the p21WAF/CDKN1A gene, which encodes the negative cell cycle regulator p21 by binding to its proximal promoter Sp1-binding GC-boxes 3, 4, 5/6, a retinoic acid response element (RARE), and distal p53-responsive elements (p53REs). PLZF-RARα also acts as a competitive transcriptional repressor of p53, RARα, and Sp1. PLZF-RARα interacts with co-repressors such as mSin3A, NCoR, and SMRT, thereby deacetylating histones Ac-H3 and Ac-H4 at the CDKN1A promoter. PLZF-RARα also interacts with the MBD3-NuRD complex, leading to epigenetic silencing of CDKN1A through DNA methylation. Furthermore, PLZF-RARα represses TP53 and increases p53 protein degradation by ubiquitination, further repressing p21 expression. Resultantly, PLZF-RARα promotes cell proliferation and significantly increases the number of cells in S-phase.

Human Kruppel-related 3 (HKR3) is a novel transcription activator of alternate reading frame (ARF) gene.

HKR3 (Human Krüppel-related 3) is a novel POK (POZ-domain Krüppel-like zinc-finger) family transcription factor. Recently, some of the POK (POZ-domain Krüppel-like zinc finger) family proteins have been shown to play roles in cell cycle arrest, apoptosis, cell proliferation, and oncogenesis. We investigated whether HKR3, an inhibitor of cell proliferation and an uncharacterized POK family protein, could regulate the cell cycle by controlling expression of genes within the p53 pathway (ARF-MDM2-TP53-p21WAF/CDKN1A). HKR3 potently activated the transcription of the tumor suppressor gene ARF by acting on the proximal promoter region (bp, -149∼+53), which contains Sp1 and FBI-1 binding elements (FREs). HKR3 interacted with the co-activator p300 to activate ARF transcription, which increased the acetylation of histones H3 and H4 within the proximal promoter. Oligonucleotide pull-down assays and ChIP assays revealed that HKR3 interferes with the binding of the proto-oncogenic transcription repressor FBI-1 to proximal FREs, thus derepressing ARF transcription.

Role of promyelocytic leukemia zinc finger (PLZF) in cell proliferation and cyclin-dependent kinase inhibitor 1A (p21WAF/CDKN1A) gene repression.

Promyelocytic leukemia zinc finger (PLZF) is a transcription repressor that was initially isolated as a fusion protein with retinoic acid receptor α. PLZF is aberrantly overexpressed in various human solid tumors, such as clear cell renal carcinoma, glioblastoma, and seminoma. PLZF causes cellular transformation of NIH3T3 cells and increases cell proliferation in several cell types. PLZF also increases tumor growth in the mouse xenograft tumor model. PLZF may stimulate cell proliferation by controlling expression of the genes of the p53 pathway (ARF, TP53, and CDKN1A). We found that PLZF can directly repress transcription of CDKN1A encoding p21, a negative regulator of cell cycle progression. PLZF binds to the proximal Sp1-binding GC-box 5/6 and the distal p53-responsive elements of the CDKN1A promoter to repress transcription. Interestingly, PLZF interacts with Sp1 or p53 and competes with Sp1 or p53. PLZF interacts with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylates Ac-H3 and Ac-H4 histones at the CDKN1A promoter, which indicated the involvement of the corepressor·HDACs complex in transcription repression by PLZF. Also, PLZF represses transcription of TP53 and also decreases p53 protein stability by ubiquitination. PLZF may act as a potential proto-oncoprotein in various cell types.

The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation.

The tumour-suppressor gene CDKN1A (encoding p21Waf/Cip1) is thought to be epigenetically repressed in cancer cells. FBI-1 (ZBTB7A) is a proto-oncogenic transcription factor repressing the alternative reading frame and p21WAF/CDKN1A genes of the p53 pathway. FBI-1 interacts directly with MBD3 (methyl-CpG-binding domain protein 3) in the nucleus. We demonstrated that FBI-1 binds both non-methylated and methylated DNA and that MBD3 is recruited to the CDKN1A promoter through its interaction with FBI-1, where it enhances transcriptional repression by FBI-1. FBI-1 also interacts with the co-repressors nuclear receptor corepressor (NCoR), silencing mediator for retinoid and thyroid receptors (SMRT) and BCL-6 corepressor (BCoR) to repress transcription. MBD3 regulates a molecular interaction between the co-repressor and FBI-1. MBD3 decreases the interaction between FBI-1 and NCoR/SMRT but increases the interaction between FBI-1 and BCoR. Because MBD3 is a subunit of the Mi-2 autoantigen (Mi-2)/nucleosome remodelling and histone deacetylase (NuRD)-HDAC complex, FBI-1 recruits the Mi-2/NuRD-HDAC complex via MBD3. BCoR interacts with the Mi-2/NuRD-HDAC complex, DNMTs and HP1. MBD3 and BCoR play a significant role in the recruitment of the Mi-2/NuRD-HDAC complex- and the NuRD complex-associated proteins, DNMTs and HP. By recruiting DNMTs and HP1, Mi-2/NuRD-HDAC complex appears to play key roles in epigenetic repression of CDKN1A by DNA methylation.

Regulation of the cyclin-dependent kinase inhibitor 1A gene (CDKN1A) by the repressor BOZF1 through inhibition of p53 acetylation and transcription factor Sp1 binding.

The human POZ domain and Krüppel-like zinc finger (POK) family proteins play important roles in the regulation of apoptosis, cell proliferation, differentiation, development, oncogenesis, and tumor suppression. A novel POK family transcription factor, BTB/POZ and zinc finger domains factor on chromosome 1 (BOZF-1; also called ZBTB8A), contains a POZ domain and two C2H2-type Krüppel-like zinc fingers and is localized at nuclear speckles. Compared with paired normal tissues, BOZF1 expression is increased in cancer tissues of the prostate, breast, and cervix. BOZF1 repressed the transcription of p21WAF/CDKN1A by acting on the proximal promoter concentrated with Sp1-binding GC boxes. BOZF1 competed with Sp1 in binding to GC boxes 1-5/6 of the CDKN1A proximal promoter. In addition, BOZF1 interacted with p53 and decreased the acetylation of p53 by p300, which reduced the DNA binding activity of p53 at the far distal p53-binding element. BOZF1 blocked the two major molecular events that are important in both constitutive and inducible transcription activation of CDKN1A. BOZF1 is unique in that it bound to all the proximal GC boxes to repress transcription, and it inhibited p53 acetylation without affecting p53 stability. BOZF1 might be a novel proto-oncoprotein that stimulates cell proliferation.

The Immune Suppressor IGSF1 as a Potential Target for Cancer Immunotherapy.

The development of first-generation immune-checkpoint inhibitors targeting PD-1/PD-L1 and CTLA-4 ushered in a new era in anticancer therapy. Although immune-checkpoint blockade therapies have shown clinical success, a substantial number of patients yet fail to benefit. Many studies are under way to discover next-generation immunotherapeutic targets. Immunoglobulin superfamily member 1 (IGSF1) is a membrane glycoprotein proposed to regulate thyroid function. Despite containing 12 immunoglobin domains, a possible role for IGSF1, in immune response, remains unknown. Here, our studies revealed that IGSF1 is predominantly expressed in tumors but not normal tissues, and increased expression is observed in PD-L1low non-small cell lung cancer (NSCLC) cells as compared with PD-L1high cells. Subsequently, we developed and characterized an IGSF1-specific human monoclonal antibody, WM-A1, that effectively promoted antitumor immunity and overcame the limitations of first-generation immune-checkpoint inhibitors, likely via a distinct mechanism of action. We further demonstrated high WM-A1 efficacy in humanized peripheral blood mononuclear cells (PBMC), and syngeneic mouse models, finding additive efficacy in combination with an anti-PD-1 (a well-characterized checkpoint inhibitor). These findings support IGSF1 as an immune target that might complement existing cancer immunotherapeutics.