NER-factor DDB2 regulates HIF1α and hypoxia-response genes in HNSCC.

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 regulates Epithelial-to-Mesenchymal Transition (EMT) in Oral/Head and Neck Squamous Cell Carcinoma.

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. TGFßs 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.

DDB2 Is a Novel Regulator of Wnt Signaling in Colon Cancer.

Deregulation of the Wnt/ß-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 ß-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 ß-catenin at an upstream site in the Rnf43 gene, enabling functional interaction with distant TCF4/ß-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.

Chromatin association of XRCC5/6 in the absence of DNA damage depends on the XPE gene product DDB2.

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.

DDB2 suppresses epithelial-to-mesenchymal transition in colon cancer.

Colon cancer is one of the deadliest cancers worldwide because of its metastasis to other essential organs. Metastasis of colon cancer involves a complex set of events, including epithelial-to-mesenchymal transition (EMT) that increases invasiveness of the tumor cells. Here, we show that the xeroderma pigmentosum group E (XPE) gene product, damaged DNA-binding protein (DDB)-2, is downregulated in high-grade colon cancers, and it plays a dominant role in the suppression of EMT of the colon cancer cells. Depletion of DDB2 promotes mesenchymal phenotype, whereas expression of DDB2 promotes epithelial phenotype. DDB2 constitutively represses genes that are the key activators of EMT, indicating that DDB2 is a master regulator of EMT of the colon cancer cells. Moreover, we observed evidence that DDB2 functions as a barrier for EMT induced by hypoxia and TGF-ß. Also, we provide evidence that DDB2 inhibits metastasis of colon cancer. The results presented here identify a transcriptional regulatory pathway of DDB2 that is directly linked to the mechanisms that suppress metastasis of colon cancer.

Targeting FoxM1 effectively retards p53-null lymphoma and sarcoma.

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.

Tumor regression by phenethyl isothiocyanate involves DDB2.

Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive agent commonly found in edible cruciferous vegetables. It has been implicated also for therapy, and is in clinical trial for lung cancer. Here, we provide evidence that the tumor suppressive effect of PEITC is related to its ability to induce expression of damaged DNA binding protein 2 (DDB2), a DNA repair protein involved also in apoptosis and premature senescence. DDB2 expression is attenuated in a wide variety of cancers including the aggressive colon cancers. We show that, in colon cancer cells, reactive oxygen species, which are induced by PEITC, augment expression of DDB2 through the p38MAPK/JNK pathway, independently of p53. PEITC-induced expression of DDB2 is critical for inhibition of tumor progression by PEITC. Tumors derived from DDB2-deficient colon cancer cells are refractory to PEITC-treatments, resulting from deficiencies in apoptosis and senescence. The DDB2-proficient tumors, on the other hand, respond effectively to PEITC. The results show that PEITC can be used to induce expression of DDB2, and that expression of DDB2 is critical for effective response of tumors to PEITC.

Damaged DNA binding protein 2 in reactive oxygen species (ROS) regulation and premature senescence.

Premature senescence induced by DNA damage or oncogene is a critical mechanism of tumor suppression. Reactive oxygen species (ROS) have been implicated in the induction of premature senescence response. Several pathological disorders such as cancer, aging and age related neurological abnormalities have been linked to ROS deregulation. Here, we discuss how Damaged DNA binding Protein-2 (DDB2), a nucleotide excision repair protein, plays an important role in ROS regulation by epigenetically repressing the antioxidant genes MnSOD and Catalase. We further revisit a model in which DDB2 plays an instrumental role in DNA damage induced ROS accumulation, ROS induced premature senescence and inhibition of skin tumorigenesis.

p21 cooperates with DDB2 protein in suppression of ultraviolet ray-induced skin malignancies.

Exposure to ultraviolet rays (UV) in sunlight is the main cause of skin cancer. Here, we show that the p53-induced genes DDB2 and p21 are down-regulated in skin cancer, and in the mouse model they functionally cooperate to prevent UV-induced skin cancer. Our previous studies demonstrated an antagonistic role of DDB2 and p21 in nucleotide excision repair and apoptosis. Surprisingly, we find that the loss of p21 restores nucleotide excision repair and apoptosis in Ddb2(-/-) mice, but it does not protect from UV-mediated skin carcinogenesis. In contrast, Ddb2(-/-)p21(-/-) mice are significantly more susceptible to UV-induced skin cancer than the Ddb2(-/-) or the p21(-/-) mice. We provide evidence that p21 deletion in the Ddb2(-/-) background causes a strong increase in cell proliferation. The increased proliferation in the Ddb2(-/-)p21(-/-) background is related to a severe deficiency in UV-induced premature senescence. Also, the oncogenic pro-proliferation transcription factor FOXM1 is overexpressed in the p21(-/-) background. Our results show that the anti-proliferative and the pro-senescence pathways of DDB2 and p21 are critical protection mechanisms against skin malignancies.

FoxM1 in tumorigenicity of the neuroblastoma cells and renewal of the neural progenitors.

Malignant neuroblastomas contain stem-like cells. These tumors also overexpress the Forkhead box transcription factor FoxM1. In this study, we investigated the roles of FoxM1 in the tumorigenicity of neuroblastoma. We showed that depletion of FoxM1 inhibits anchorage-independent growth and tumorigenicity in mouse xenografts. Moreover, knockdown of FoxM1 induces differentiation in neuroblastoma cells, suggesting that FoxM1 plays a role in the maintenance of the undifferentiated progenitor population. We showed that inhibition of FoxM1 in malignant neuroblastoma cells leads to the downregulation of the pluripotency genes sex determining region Y box 2 (Sox2) and Bmi1. We provided evidence that FoxM1 directly activates expression of Sox2 in neuroblastoma cells. By using a conditional deletion system and neurosphere cultures, we showed that FoxM1 is important for expression of Sox2 and Bmi1 in the mouse neural stem/progenitor cells and is critical for its self-renewal. Together, our observations suggested that FoxM1 plays an important role in the tumorigenicity of the aggressive neuroblastoma cells through maintenance of the undifferentiated state.

Cul4A is essential for spermatogenesis and male fertility.

The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A-/- strain lacking exons 4-8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A-/- mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.

DDB2, an essential mediator of premature senescence.

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.

Damaged-DNA Binding Protein-2 Drives Apoptosis Following DNA Damage.

Apoptosis induced by DNA damage is an important mechanism of tumor suppression and it is significant also in cancer chemotherapy. Mammalian cells activate the pathways of p53 to induce apoptosis of cells harboring irreparable DNA damages. While p53 induces expression of various pro-apoptotic genes and directly participates in the disruption of mitochondrial membrane polarization, it also increases expression of the cell cycle inhibitor p21 that is a dominant inhibitor of caspase-activation and apoptosis. Here we discuss how Damaged-DNA Binding Protein-2 (DDB2) subdues the level of p21 in cells harboring irreparable DNA damage to support activation of the caspases. We speculate a model in which DDB2 detects and couples the presence of un-repaired DNA damages to the proteolysis of p21, leading to the induction of apoptosis.

Destabilization of Rb by human papillomavirus E7 is cell cycle dependent: E2-25K is involved in the proteolysis.

The HPV oncoprotein E7 promotes proteasomal degradation of the tumor suppressor protein Rb. In this study, we analyzed the regulation of E7-induced Rb proteolysis in HPV-containing Caski cervical cancer cells. We show that the Rb proteolysis is cell cycle dependent; in S phase Rb is stable while in post-mitotic early G1 phase cells and in differentiated cells, Rb is unstable. Similarly, the in vivo Rb/E7 interaction is not detected in S-phase cells, but is readily detected in differentiating Caski cells. The ubiquitinating enzymes involved in Rb proteolysis have not been identified. We find that the E3 ligase MDM2 is not involved in the Rb proteolysis in Caski cells. An in vivo analysis using multiple catalytic site mutant dominant negative E2 enzymes show that the C92A E2-25K most effectively blocks E7-induced Rb proteolysis. Taken together, these results show that E7 induces Rb proteolysis in growth-arrested cells and E2-25K is involved in the proteolysis.

DDB2 (damaged-DNA binding protein 2) in nucleotide excision repair and DNA damage response.

DDB2 was identified as a protein involved in the Nucleotide Excision Repair (NER), a major DNA repair mechanism that repairs UV damage to prevent accumulation of mutations and tumorigenesis. However, recent studies indicated additional functions of DDB2 in the DNA damage response pathway. Herein, we discuss the proposed mechanisms by which DDB2 activates NER and programmed cell death upon DNA damage through its E3 ligase activity.

DDB2 decides cell fate following DNA damage.

The xeroderma pigmentosum complementation group E (XP-E) gene product damaged-DNA binding protein 2 (DDB2) plays important roles in nucleotide excision repair (NER). Previously, we showed that DDB2 participates in NER by regulating the level of p21(Waf1/Cip1). Here we show that the p21(Waf1/Cip1) -regulatory function of DDB2 plays a central role in defining the response (apoptosis or arrest) to DNA damage. The DDB2-deficient cells are resistant to apoptosis in response to a variety of DNA-damaging agents, despite activation of p53 and the pro-apoptotic genes. Instead, these cells undergo cell cycle arrest. Also, the DDB2-deficient cells are resistant to E2F1-induced apoptosis. The resistance to apoptosis of the DDB2-deficient cells is caused by an increased accumulation of p21(Waf1/Cip1) after DNA damage. We provide evidence that DDB2 targets p21(Waf1/Cip1) for proteolysis. The resistance to apoptosis in DDB2-deficient cells also involves Mdm2 in a manner that is distinct from the p53-regulatory activity of Mdm2. Our results provide evidence for a new regulatory loop involving the NER protein DDB2, Mdm2, and p21(Waf1/Cip1) that is critical in deciding cell fate (apoptosis or arrest) upon DNA damage.

Deregulation of eIF4E: 4E-BP1 in differentiated human papillomavirus-containing cells leads to high levels of expression of the E7 oncoprotein.

Infections with high-risk human papillomaviruses (HPVs) are linked to more than 95% of cervical cancers. HPVs replicate exclusively in differentiated cells and the function of the HPV E7 oncoprotein is essential for viral replication. In this study, we investigated the mechanism that regulates E7 expression in differentiated cells. The level of E7 protein was strongly induced in HPV-containing Caski, HOK-16B, and BaP-T cells during growth in methylcellulose-containing medium, a condition that induces differentiation. Enhanced expression of E7 was observed between 4 and 8 h of culturing in methylcellulose and was maintained for up to 24 h. The increase was not due to altered stability of the E7 protein or an increase in the steady-state level of the E7 mRNA. Instead, the translation of the E7 mRNA was enhanced during differentiation. More than 70 to 80% of the E7 mRNA was found in the polysome fractions in the differentiated cells. Consistent with this observation, higher levels of the phosphorylated translator inhibitor 4E-BP1 were observed in differentiated HPV-containing cells but not in differentiated non-HPV tumor cells or primary keratinocytes. The mTOR kinase inhibitor rapamycin blocked phosphorylation of 4E-BP1 and significantly decreased the level of E7 protein in Caski cells, suggesting that phosphorylation of 4E-BP1 is linked to E7 expression. Prevailing models for the molecular mechanisms underlying E7 expression have focused largely on transcriptional regulation. The results presented in this study demonstrate a significant role of the cellular translation machinery to maintain a high level of E7 protein in differentiated cells.

Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome.

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.

Cul4A physically associates with MDM2 and participates in the proteolysis of p53.

The cullin 4A (Cul4A) gene is amplified and overexpressed in breast and hepatocellular carcinomas. Cul4A functions as an E3 ligase and participates in the proteolysis of several regulatory proteins through the ubiquitin-proteasome pathway. Here, we show that Cul4A associates with MDM2 and p53. Depletion of Cul4A leads to an accumulation of p53. Moreover, expression of Cul4A increases the decay-rate of p53 and delays the accumulation of p53 in response to DNA damage. Cul4A fails to increase the decay of p53 in mouse embryonic fibroblasts lacking MDM2. In addition, the Cul4A-mediated rapid decay of p53 is blocked by p19ARF. The results provide evidence for a role of Cul4A in the MDM2-mediated proteolysis of p53.