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RNF144A is a DNA damage-induced E3 ubiquitin ligase that targets proteins involved in genome instability for degradation, e.g., DNA-PKcs and BMI1. RNF144A is frequently mutated or epigenetically silenced in cancer, providing the rationale to evaluate RNF144A loss of function in tumorigenesis. Here we report that RNF144A-deficient mice are more prone to the development of bladder tumors upon carcinogen exposure. In addition to DNA-PKcs and BMI1, we identify the immune checkpoint protein PD-L1 as a novel degradation target of RNF144A, since these proteins are expressed at higher levels in Rnf144a KO tumors. RNF144A interacts with PD-L1 in the plasma membrane and intracellular vesicles and promotes poly-ubiquitination and degradation of PD-L1. Therefore, Rnf144a KO stabilizes PD-L1 and leads to a reduction of tumor-infiltrating CD8+ T cell populations in the BBN-induced bladder tumors. The bladder tumors developed in WT and Rnf144a KO mice primarily express CK5 and CK14, markers of basal cancer subtype, as expected in BBN-induced bladder tumors. Intriguingly, the Rnf144a KO tumors also express GATA3, a marker for the luminal subtype, suggesting that RNF144A loss of function promotes features of cellular differentiation. Such differentiation features in Rnf144a KO tumors likely result from a decrease of EGFR expression, consistent with the reported role of RNF144A in maintaining EGFR expression. In summary, for the first time our study demonstrates the in vivo tumor suppressor activity of RNF144A upon carcinogenic insult. Loss of RNF144A promotes the expression of DNA-PKcs, BMI1 and PD-L1, likely contributing to the carcinogen-induced bladder tumorigenesis.

About one-fourth of recurrent estrogen receptor–positive (ER+) breast cancers lose ER expression, leading to endocrine therapy failure. However, the mechanisms underlying ER loss remain to be fully explored. We now show that 14-3-3τ, up-regulated in ∼60% of breast cancer, drives the conversion of ER+ to ER– and epithelial-to-mesenchymal transition (EMT). We identify ERα36, an isoform of ERα66, as a downstream effector of 14-3-3τ. Overexpression of 14-3-3τ induces ERα36 in xenografts and tumor spheroids. The regulation is further supported by a positive correlation between ERα36 and 14-3-3τ expression in human breast cancers. ERα36 can antagonize ERα66 and inhibit ERα66 expression. Isoform-specific depletion of ERα36 blocks the ER conversion and EMT induced by 14-3-3τ overexpression in tumor spheroids, thus establishing ERα36 as a key mediator in 14-3-3τ-driven ER loss and EMT. ERα36 promoter is repressed by GATA3, which can be phosphorylated by AKT at consensus binding sites for 14-3-3. Upon AKT activation, 14-3-3τ binds phosphorylated GATA3 and facilitates the degradation of GATA3 causing GATA3 to lose transcriptional control over its target genes ERα66 and ERα36. We also demonstrate a role for the collaboration between 14-3-3τ and AKT in ERα36 induction and endocrine therapy resistance by three-dimensional spheroid and tamoxifen treatment models in MCF7 and T47D ER+ breast cancer cells. Thus, the 14-3-3τ-ERα36 regulation provides a previously unrecognized mechanism for ER loss and endocrine therapy failure.

Cisplatin is a mainstay of systemic therapy for a variety of cancers, such as lung cancer, head and neck cancer, and ovarian cancer. However, resistance to cisplatin represents one of the most significant barriers for patient outcome improvement. Actin-like 6A (ACTL6A) is a component of several chromatin remodeling complexes, including SWI/SNF, NuA4/TIP60 histone acetylase, and INO80. Amplification of ACTL6A gene is often seen in lung squamous cell carcinoma, ovarian cancer, and esophageal cancer, but its significance remains to be fully determined. Here we identify ACTL6A overexpression as a novel cause for platinum resistance. High levels of ACTL6A are associated with chemoresistance in several types of human cancer. We show that overexpression of ACTL6A leads to increased repair of cisplatin-DNA adducts and resistance to cisplatin treatment. In contrast, depletion of ACTL6A inhibits the repair of cisplatin-induced DNA lesions, and increases cisplatin sensitivity in cisplatin-resistant ovarian cancer cells. The regulation of repair by ACTL6A is mediated through the SWI/SNF chromatin remodeling complex. Treatment with a histone deacetylase inhibitor can reverse the effect of ACTL6A overexpression on the repair of cisplatin-induced DNA damage and render cancer cells more sensitive to cisplatin treatment in a xenograft mouse model. Taken together, our study uncovers a novel role for ACTL6A in platinum resistance, and provides evidence supporting the feasibility of using HDAC inhibitors for platinum resistant tumors.

Oxidative stress is a ubiquitous threat to all aerobic organisms and has been implicated in numerous pathological conditions such as cancer. Here we demonstrate a pivotal role for E2F1, a cell cycle regulatory transcription factor, in cell tolerance of oxidative stress. Cells lacking E2F1 are hypersensitive to oxidative stress due to the defects in cell cycle arrest. Oxidative stress inhibits E2F1 transcriptional activity, independent of changes in association with Rb and without decreasing its DNA-binding activity. Upon oxidative insult, SUMO2 is extensively conjugated to E2F1 mainly at lysine 266 residue, which specifically modulates E2F1 transcriptional activity to enhance cell cycle arrest for cell survival. We identify SENP3, a desumoylating enzyme, as an E2F1-interacting partner. Oxidative stress inhibits the interaction between E2F1 and SENP3, which leads to accumulation of sumoylated E2F1. SENP3-deficient cells exhibit hypersumoylation of E2F1 and are resistant to oxidative insult. High levels of SENP3 in breast cancer are associated with elevated levels of E2F targets, high tumor grade, and poor survival. Given the prevalence of elevated levels of SENP3 across numerous cancer types, the SENP3-E2F1 axis may serve as an avenue for therapeutic intervention in cancer.

Cdk2-dependent TopBP1-treslin interaction is critical for DNA replication initiation. However, it remains unclear how this association is terminated after replication initiation is finished. Here, we demonstrate that phosphorylation of TopBP1 by Akt coincides with cyclin A activation during S and G(2) phases and switches the TopBP1-interacting partner from treslin to E2F1, which results in the termination of replication initiation. Premature activation of Akt in G(1) phase causes an early switch and inhibits DNA replication. TopBP1 is often overexpressed in cancer and can bypass control by Cdk2 to interact with treslin, leading to enhanced DNA replication. Consistent with this notion, reducing the levels of TopBP1 in cancer cells restores sensitivity to a Cdk2 inhibitor. Together, our study links Cdk2 and Akt pathways to the control of DNA replication through the regulation of TopBP1-treslin interaction. These data also suggest an important role for TopBP1 in driving abnormal DNA replication in cancer.

Topoisomerase IIβ-binding protein 1 (TopBP1) is involved in cellular replication among other functions and is known to activate ATR/Chk1 during replicative stress. TopBP1 is also expressed at high levels in many cancers. However, the impact of TopBP1 overexpression on ATR/Chk1 activation and cancer development has not been investigated. Here we demonstrate that the degree of ATR/Chk1 activation is regulated by TopBP1 in a biphasic, concentration-dependent manner in a nontransformed MCF10A cell line and several cancer cell lines, including H1299, MDA-MB468, and U2OS. At low levels, TopBP1 activates ATR/Chk1, but once TopBP1 protein accumulates above an optimal level, it paradoxically leads to lower activation of ATR/Chk1. This is due to the perturbation of ATR–TopBP1 interaction and ATR chromatin loading by excessive TopBP1. Overexpression of TopBP1 thus hinders the ATR/Chk1 checkpoint response, leading to the impairment of genome integrity as demonstrated by the cytokinesis-block micronucleus assay. In contrast, moderate depletion of TopBP1 by shRNA in TopBP1-overexpressing cancer cells enhanced ATR/Chk1 activation and S-phase checkpoint response after replicative stress. The clinical significance of these findings is supported by an association between TopBP1 overexpression and genome instability in many types of human cancer. Taken together, our study illustrates an unexpected relationship between the levels of TopBP1 and the final functional outcome and suggests TopBP1 overexpression as a new mechanism directly contributing to genomic instability during tumorigenesis.

Additional file 7: Figure S7. Knockout of CGRRF1 enhances cell proliferation. (A) The growth rate of CGRRF1-knockout MCF7 cell line (sgCGRRF1#2–5) was determined by MTT assay. Error bars represent mean ± SD (n = 6). *p

RNF144A, an E3 ubiquitin ligase for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), can promote DNA damage-induced cell apoptosis. Here we characterize an important regulation of RNF144A through its transmembrane (TM) domain. The TM domain of RNF144A is highly conserved among species. Deletion of the TM domain abolishes its membrane localization and also significantly reduces its ubiquitin ligase activity. Further evidence shows that the TM domain is required for RNF144A self-association and that the self-association may be partially mediated through a classic GXXXG interaction motif. A mutant RNF144A-G252L/G256L (in the G(252)XXXG(256) motif) preserves membrane localization but is defective in self-association and ubiquitin ligase activity. On the other hand, a membrane localization loss mutant of RNF144A still retains self-association and E3 ligase activity, which can be blocked by additional G252L/G256L mutations. Therefore, our data demonstrate that the TM domain of RNF144A has at least two independent roles, membrane localization and E3 ligase activation, to regulate its physiological function. This regulatory mechanism may be applicable to other RBR (RING1-IBR-RING2) E3 ubiquitin ligases because, first, RNF144B also self-associates. Second, all five TM-containing RBR E3 ligases, including RNF144A and RNF144B, RNF19A/Dorfin, RNF19B, and RNF217, have the RBR-TM(GXXXG) superstructure. Mutations of the GXXXG motifs in RNF144A and RNF217 have also be found in human cancers, including a G252D mutation of RNF144A. Interestingly, RNF144A-G252D still preserves self-association and ubiquitin ligase activity but loses membrane localization and is turned over rapidly. In conclusion, both proper membrane localization and self-association are important for RNF144A function.

Accumulating evidence supports the gain-of-function of mutant forms of p53 (mutp53s). However, whether mutp53 directly perturbs the DNA replication checkpoint remains unclear. Previously, we have demonstrated that TopBP1 forms a complex with mutp53s and mediates their gain-of-function through NF-Y and p63/p73. Akt phosphorylates TopBP1 and induces its oligomerization, which inhibits its ATR-activating function. Here we show that various contact and conformational mutp53s bypass Akt to induce TopBP1 oligomerization and attenuate ATR checkpoint response during replication stress. The effect on ATR response caused by mutp53 can be exploited in a synthetic lethality strategy, as depletion of another ATR activator, DNA2, in mutp53-R273H–expressing cancer cells renders cells hypersensitive to cisplatin. Expression of mutp53-R273H also makes cancer cells more sensitive to DNA2 depletion or DNA2 inhibitors. In addition to ATR-activating function during replication stress, TopBP1 interacts with Treslin in a Cdk-dependent manner to initiate DNA replication during normal growth. We find that mutp53 also interferes with TopBP1 replication function. Several contact, but not conformational, mutp53s enhance the interaction between TopBP1 and Treslin and promote DNA replication despite the presence of a Cdk2 inhibitor. Together, these data uncover two distinct mechanisms by which mutp53 enhances DNA replication: ( i ) Both contact and conformational mutp53s can bind TopBP1 and attenuate the checkpoint response to replication stress, and ( ii ) during normal growth, contact (but not conformational) mutp53s can override the Cdk2 requirement to promote replication by facilitating the TopBP1/Treslin interaction.

TRIP6 is an adaptor protein that regulates cell motility and antiapoptotic signaling. Although it has been implicated in tumorigenesis, the underlying mechanism remains largely unknown. Here we provide evidence that TRIP6 promotes tumorigenesis by serving as a bridge to promote the recruitment of p27(KIP1) to AKT in the cytosol. TRIP6 regulates the membrane translocation and activation of AKT and facilitates AKT-mediated recognition and phosphorylation of p27(KIP1) specifically at T157, thereby promoting the cytosolic mislocalization of p27(KIP1). This is required for p27(KIP1) to enhance lysophosphatidic acid (LPA)-induced ovarian cancer cell migration. TRIP6 also promotes serum-induced reduction of nuclear p27(KIP1) expression levels through Skp2-dependent and -independent mechanisms. Consequently, knockdown of TRIP6 in glioblastoma or ovarian cancer xenografts restores nuclear p27(KIP1) expression and impairs tumor proliferation. As TRIP6 is upregulated in gliomas and its levels correlate with poor clinical outcomes in a dose-dependent manner, it may represent a novel prognostic marker and therapeutic target in gliomas.

E2F1 is a eukaryotic transcription factor that is known to regulate various cellular pathways such as cell cycle progression, DNA replication, DNA damage responses and induction of apoptosis. Given its versatile roles, a precise and tight regulation of E2F1 is very critical to maintain genomic stability. E2F1 is regulated both at transcriptional and posttranslational levels during cell cycle and upon DNA damage. After S phase, E2F1 is targeted for degradation and is kept at low levels or in an inactive state until the next G 1/S phase transition. Our studies show that APC/C ubiquitin ligase in conjunction with its co-activator Cdh1 (APC/C (Cdh1) ) can downregulate E2F1. We also identify an APC/C subunit APC5 that binds to E2F1 and is essential for E2F1 ubiquitination. We confirm an interaction between E2F1 and Cdh1 as well as an interaction between E2F1 and APC5 both in vivo and in vitro. In vitro GST pull-down assays have mapped the C-terminal 79 a.a. of E2F1 as Cdh1 interacting residues. Ectopically expressed Cdh1 downregulates the expression of E2F1-4. Our studies have also shown for the first time that E2F1 can be modified by K11-linkage specific ubiquitin chain formation (Ub-K11). The formation of Ub-K11 chains on E2F1 is increased in the presence of Cdh1 and accumulated in the presence of proteasome inhibitor, suggesting that APC/C (Cdh1) targets E2F1 for degradation by forming Ub-K11 chains. We also show that the effect of Cdh1 on E2F1 degradation is blocked upon DNA damage. Interestingly, Ub-K11-linked E2F1 accumulates after treatment of DNA damaging agents. The data suggest that DNA damage signaling processes do not inhibit APC/C (Cdh1) to ubiquitinate E2F1. Instead, they block the proteasomal degradation of Ub-K11-linked E2F1, and therefore lead to its accumulation.

Nearly half of human cancers harbor p53 mutations, which can promote cancerous growth, metastasis, and resistance to therapy. The gain of function of mutant p53 is partly mediated by its ability to form a complex with NF-Y or p63/p73. Here, we demonstrate that TopBP1 mediates these activities in cancer, and we provide both in vitro and in vivo evidence to support its role. We show that TopBP1 interacts with p53 hot spot mutants and NF-YA and promotes mutant p53 and p300 recruitment to NF-Y target gene promoters. TopBP1 also facilitates mutant p53 interaction with and inhibition of the transcriptional activities of p63/p73. Depletion of TopBP1 in mutant p53 cancer cells leads to downregulation of NF-Y target genes cyclin A and Cdk1 and upregulation of p63/p73 target genes such as Bax and Noxa. Mutant p53-mediated resistance to chemotherapeutic agents depends on TopBP1. The growth-promoting activity of mutant p53 in a xenograft model also requires TopBP1. Thus, TopBP1 mediates mutant p53 gain of function in cancer. Since TopBP1 is often overexpressed in cancer cells and is recruited to cooperate with mutant p53 for tumor progression, TopBP1/mutant p53 interaction may be a new therapeutic target in cancer.

The EDD (E3 identified by differential display) gene, first identified as a progestin-induced gene in T-47D breast cancer cells, encodes an E3 ubiquitin ligase with a HECT domain. It was reported that EDD is involved in the G(2)/M progression through ubiquitination of phospho-katanin p60. Previous study has also shown that EDD can act as a transcription cofactor independently of its E3 ligase activity. In this study, we uncover a new role for EDD during cell cycle progression in an E3 ligase-independent manner. We demonstrate that EDD can physically interact with p53 and that this interaction blocks the phosphorylation of p53 by ataxia telangiectasia mutated (ATM). Silencing of EDD induces phosphorylation of p53 at Ser(15) and activates p53 target genes in fibroblasts and some transformed cells without activation of DNA damage response. The G(1)/S arrest induced by EDD depletion depends on p53. On the other hand, overexpression of EDD inhibits p53-Ser(15) phosphorylation and suppresses the induction of p53 target genes during DNA damage, and this effect does not require its E3 ligase activity. Thus, through binding to p53, EDD actively inhibits p53 phosphorylation by ATM and plays a role in ensuring smooth G(1)/S progression.

14-3-3 proteins regulate many cellular functions, including proliferation. However, the detailed mechanisms by which they control the cell cycle remain to be fully elucidated. We report that one of the 14-3-3 isoforms, 14-3-3tau, is required for the G(1)/S transition through its role in ubiquitin-independent proteasomal degradation of p21. 14-3-3tau binds to p21, MDM2, and the C8 subunit of the 20S proteasome in G(1) phase and facilitates proteasomal targeting of p21. This function of 14-3-3tau may be deregulated in cancer. The overexpression of 14-3-3tau is frequently found in primary human breast cancer and correlates with lower levels of p21 and shorter patient survival. Tenascin-C, an extracellular matrix protein involved in tumor initiation and progression and a known 14-3-3tau inducer, decreases p21 and abrogates adriamycin-induced G(1)/S arrest. It has been known that p21 is required for a proper tamoxifen response in breast cancer. We show that the overexpression of 14-3-3tau inhibits tamoxifen-induced p21 induction and growth arrest in MCF7 cells. Together, the findings of our studies strongly suggest a novel oncogenic role of 14-3-3tau by downregulating p21 in breast cancer. Therefore, 14-3-3tau may be a potential therapeutic target in breast cancer.

The elevated lysophosphatidic acid signaling has been causally linked to cancer-associated inflammation and tumorigenesis through upregulation of nuclear factor-κB signaling. However, how this signaling event is regulated has not yet been fully understood. Here we demonstrate that TRIP6, an LPA2 receptor-interacting adaptor protein, functions as a positive regulator of nuclear factor-κB and JNK signaling through direct binding to and activation of the E3 ligase TRAF6. Upon lysophosphatidic acid stimulation, TRIP6 recruits TRAF6 to the LPA2 receptor and promotes lysophosphatidic acid-induced JNK and nuclear factor-κB activation in a TRAF6-dependent manner. TRIP6 antagonizes the recruitment of deubiquitinases A20 and CYLD to TRAF6, thus sustaining the E3 ligase activity of TRAF6 and augmenting lysophosphatidic acid-activated nuclear factor-κB signaling. In contrast, depletion of TRIP6 by TRIP6-specific shRNA or Cas9/sgRNA greatly enhances the association of TRAF6 with A20 and CYLD, and attenuates lysophosphatidic acid-induced muclear factor-κB and JNK/p38 activation in ovarian cancer cells. On the other hand, TRAF6 also regulates TRIP6 by facilitating its binding to nuclear factor-κB p65 and phosphorylation by c-Src. Together, TRIP6 cooperates with TRAF6 to regulate the LPA2 receptor signaling, which may ultimately contribute to chronic inflammation, apoptotic resistance and cell invasion.

Lysophosphatidic acid (LPA) promotes cell survival through the activation of G protein-coupled LPA receptors. However, whether different LPA receptors activate distinct anti-apoptotic signaling pathways is not yet clear. Here we report a novel mechanism by which the LPA(2) receptor targets the proapoptotic Siva-1 protein for LPA-dependent degradation, thereby attenuating Siva-1 function in DNA damage response. The carboxyl-terminal tail of the LPA(2) receptor, but not LPA(1) or LPA(3) receptor, specifically associates with the carboxyl cysteine-rich domain of Siva-1. Prolonged LPA stimulation promotes the association of Siva-1 with the LPA(2) receptor and targets both proteins for ubiquitination and degradation. As a result, adriamycin-induced Siva-1 protein stabilization is attenuated by LPA in an LPA(2)-dependent manner, and the function of Siva-1 in promoting DNA damage-induced apoptosis is inhibited by LPA pretreatment. Consistent with this result, inhibition of the LPA(2) receptor expression increases Siva-1 protein levels and augments adriamycin-induced caspase-3 cleavage and apoptosis. Together, these findings reveal a critical and specific role for the LPA(2) receptor through which LPA directly inactivates a critical component of the death machinery to promote cell survival.

The LIM domain-containing TRIP6 (Thyroid Hormone Receptor-interacting Protein 6) is a focal adhesion molecule known to regulate lysophosphatidic acid (LPA)-induced cell migration through interaction with the LPA2 receptor. LPA stimulation targets TRIP6 to the focal adhesion complexes and promotes c-Src-dependent phosphorylation of TRIP6 at Tyr-55, which creates a docking site for the Crk Src homology 2 domain, thereby promoting LPA-induced morphological changes and cell migration. Here we further demonstrate that a switch from c-Src-mediated phosphorylation to PTPL1/Fas-associated phosphatase-1-dependent dephosphorylation serves as an inhibitory feedback control mechanism of TRIP6 function in LPA-induced cell migration. PTPL1 dephosphorylates phosphotyrosine 55 of TRIP6 in vitro and inhibits LPA-induced tyrosine phosphorylation of TRIP6 in cells. This negative regulation requires a direct protein-protein interaction between these two molecules and the phosphatase activity of PTPL1. In contrast to c-Src, PTPL1 prevents TRIP6 turnover at the sites of adhesions. As a result, LPA-induced association of TRIP6 with Crk and the function of TRIP6 to promote LPA-induced morphological changes and cell migration are inhibited by PTPL1. Together, these results reveal a novel mechanism by which PTPL1 phosphatase plays a counteracting role in regulating TRIP6 function in LPA-induced cell migration.

E2F1 is tightly controlled by multiple mechanisms, but whether ubiquitination regulates its transcriptional activity remains unknown. Here we identify UCH37 as the first, to our knowledge, deubiquitinating enzyme for E2F1. UCH37 does not deubiquitinate UbK48 chains or affect E2F1 protein stability. Instead, UCH37, but not a catalytically dead mutant, decreases the Lys-63-linked ubiquitination of E2F1 and activates its transcriptional activity. UCH37 depletion reduces the gene expression of both proliferative and pro-apoptotic E2F1 target genes. UCH37 depletion also decreases both cell proliferation and apoptosis induction in functional assays. Interestingly, UCH37 expression is induced by E2F1, and its level rises in G1/S transition and S phase, suggesting a positive feedback loop between UCH37 and E2F1. UCH37 protein and mRNA levels are also induced after DNA damage. UCH37 localizes to the promoters of E2F1 pro-apoptotic target genes such as caspase 3, caspase 7, PARP1, and Apaf-1 and activates their expression after DNA damage. Moreover, the expression of E2F1 proliferative and pro-apoptotic genes is correlated with the levels of UCH37 in many primary tumors. These results uncover a novel mechanism for E2F1 transcriptional activation through removal of its Lys-63-linked ubiquitination by UCH37.

Genotoxic stress triggers apoptosis through multiple signaling pathways. Recent studies have demonstrated a specific induction of E2F1 accumulation and a role for E2F1 in apoptosis upon DNA damage. Induction of E2F1 is mediated by phosphorylation events that are dependent on DNA damage-responsive protein kinases, such as ATM. How ATM phosphorylation leads to E2F1 stabilization is unknown. We now show that 14-3-3 tau, a phosphoserine-binding protein, mediates E2F1 stabilization. 14-3-3 tau interacts with ATM-phosphorylated E2F1 during DNA damage and inhibits E2F1 ubiquitination. Depletion of 14-3-3 tau or E2F1, but not E2F2 or E2F3, blocks adriamycin-induced apoptosis. 14-3-3 tau is also required for expression and induction of E2F1 apoptotic targets, such as p73, Apaf-1, and caspases, during DNA damage. Together, these data demonstrate a novel function for 14-3-3 tau in the regulation of E2F1 protein stability and apoptosis during DNA damage.

The progression of many solid tumors is driven by de-regulation of multiple common pathways, particularly Rb, PI (3) K/Akt and p53. Prior studies identified TopBP1as a key mediator for the oncogenic gain-of-function activities of mutant p53 (mutp53) in cancer. In Akt-hyperactive cancer, TopBP1 forms oligomers and represses E2F1-dependent apoptosis. Here we perform a molecular docking screening and identify a lead compound, calcein, capable of blocking TopBP1 oligomerization and p53 binding, resulting in re-activation of E2F1-dependent apoptosis and blockade of mutp53 gain-of-function. Calcein AM, the cell permeable derivative of calcein, shows significant anti-tumor activity in a wide-spectrum of cultured cancer cells harboring high TopBP1 levels. These biochemical findings are recapitulated in breast cancer xenograft models. Thus, our study provides proof-of-concept evidence for targeting TopBP1, a convergent point of multiple pathways, as a cancer therapy.

Yang Xiao, Vivian Y. Lin, Shi Ke, Gregory E. Lin, Fang-Tsyr Lin, Weei-Chin Lin Section of Hematology/Oncology, Departments of Medicine and Molecular and Cellular Biology, and Department of Radiology, Baylor College of Medicine, Houston, Texas, USA; Department of Computer Science, Rice University, Houston, Texas, USA Volume 34, no. 14, p. 2635–2649, 2014. Page 2640, column 1, bottom line: “(RGSYSIKS)” should read “(RGSYSIKS).”

Several ring between ring fingers (RBR) -domain proteins, such as Parkin and Parc, have been shown to be E3 ligases involved in important biological processes. Here, we identify a poorly characterized RBR protein, Ring Finger protein 144A (RNF144A), as the first, to our knowledge, mammalian E3 ubiquitin ligase for DNA-PKcs. We show that DNA damage induces RNF144A expression in a p53-dependent manner. RNF144A is mainly localized in the cytoplasmic vesicles and plasma membrane and interacts with cytoplasmic DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). DNA-PKcs plays a critical role in the nonhomologous end-joining DNA repair pathway and provides prosurvival signaling during DNA damage. We show that RNF144A induces ubiquitination of DNA-PKcs in vitro and in vivo and promotes its degradation. Depletion of RNF144A leads to an increased level of DNA-PKcs and resistance to DNA damaging agents, which is reversed by a DNA-PK inhibitor. Taken together, our data suggest that RNF144A may be involved in p53-mediated apoptosis through down-regulation of DNA-PKcs when cells suffer from persistent or severe DNA damage insults.

14-3-3τ is frequently overexpressed in breast cancer; however, whether it contributes to breast cancer progression remains undetermined. Here, we identify a critical role for 14-3-3τ in promoting breast cancer metastasis, in part through binding to and inhibition of RhoGDIα, a negative regulator of Rho GTPases and a metastasis suppressor. 14-3-3τ binds Ser174-phosphorylated RhoGDIα and blocks its association with Rho GTPases, thereby promoting epidermal growth factor (EGF)-induced RhoA, Rac1, and Cdc42 activation. When 14-3-3τ is overexpressed in MCF7 breast cancer cells that express 14-3-3τ at low levels, it increases motility, reduces adhesion, and promotes metastasis in mammary fat pad xenografts. On the other hand, depletion of 14-3-3τ in MCF7 cells and in an invasive cell line, MDA-MB231, inhibits Rho GTPase activation and blocks breast cancer migration and invasion. Moreover, 14-3-3τ overexpression in human breast tumors is associated with the activation of ROCK (a Rho GTPase effector), high metastatic rate, and shorter survival, underscoring a clinically significant role for 14-3-3τ in breast cancer progression. Our work indicates that 14-3-3τ is a novel therapeutic target to prevent breast cancer metastasis.

Our previous study showed that Akt phosphorylates TopBP1 at the Ser-1159 residue and induces its oligomerization. Oligomerization is required for TopBP1 to bind and repress E2F1 activity. However, the mechanism through which phosphorylation of TopBP1 by Akt leads to its oligomerization remains to be determined. Here, we demonstrate that binding between the phosphorylated Ser-1159 (pS1159) residue and the 7th and 8th BRCT domains of TopBP1 mediates TopBP1 oligomerization. Mutations within the 7th and 8th BRCT domains of TopBP1 that block binding to a pS1159-containing peptide block TopBP1 oligomerization and its ability to bind and repress E2F1 activities. The Akt-induced TopBP1 oligomerization is also directly demonstrated in vitro by size exclusion chromatography. Importantly, oligomerization perturbs the checkpoint-activating function of TopBP1 by preventing its recruitment to chromatin and ATR binding upon replicative stress. Hyperactivation of Akt inhibits Chk1 phosphorylation after hydroxyurea treatment, and this effect is dependent on TopBP1 phosphorylation at Ser-1159. Thus, Akt can switch the TopBP1 function from checkpoint activation to transcriptional regulation by regulating its quaternary structure. This pathway of regulation is clinically significant, since treatment of a specific Akt inhibitor in PTEN-mutated cancer cells inhibits TopBP1 oligomerization and causes its function to revert from promoting survival to checkpoint activation.

V(D)J recombination is a major source of antigen receptor diversity and represents the only known form of site-specific DNA rearrangement in vertebrates. V(D)J recombination is initiated by specific DNA cleavage at recombinational signal sequences and requires components of the general machinery used for double-strand (DS)-break repair. The involvement of DS cleavage and repair mechanisms suggests that V(D)J recombination might be coupled to the cell cycle, as introduction or persistence of DS breaks during DNA replication or mitosis could interfere with faithful transmission of genetic information to daughter cells. Here, Weei-Chin Lin and Stephen Desiderio review recent evidence indicating that this is indeed the case and consider some biological implications of this linkage.

This is a response to a letter by Watts and Saunders (1) The nice study by Saunders and colleagues does indicate that the role of EDD in extraembryonic development is p53-independent (2). However, we want to draw attention to a new study, which might explain the seemingly conflicting results from HeLa cells (3) compared with those from other cell lines and non-transformed fibroblasts (4). A recent paper by Tomaic et al. (5) demonstrates that EDD directly binds HPV-18 E6/E6AP and inhibits E6 degradation activity. As a result, EDD knockdown in HeLa cells (harboring HPV-18) enhances E6 activity and leads to a striking decrease in the levels of p53 expression and correspondingly a decrease in etoposide-induced apoptosis. Thus, the E6-mediated effects could, at least in part, be responsible for the different outcomes upon EDD depletion in HeLa cells. With the EDD-E6/E6AP regulation, some changes on cell cycle/DNA damage checkpoints following EDD depletion in HeLa cells might be mediated by E6 activation, rather than reflect the physiological function of EDD in normal cells. It is also worth mentioning that we have not been able to observe a consistent change of E2F1 levels following EDD depletion in our cell lines, suggesting that E2F1 may not be regulated directly by EDD.

Antigen receptor genes are assembled by site-specific DNA rearrangement. The recombination activator genes RAG-1 and RAG-2 are essential for this process, termed V(D)J rearrangement. The activity and stability of the RAG-2 protein have now been shown to be regulated by phosphorylation. In fibroblasts RAG-2 was phosphorylated predominantly at two serine residues, one of which affected RAG-2 activity in vivo. The threonine at residue 490 was phosphorylated by p34cdc2 kinase in vitro; phosphorylation at this site in vivo was associated with rapid degradation of RAG-2. Instability was transferred to chimeric proteins by a 90-residue portion of RAG-2. Mutation of the p34cdc2 phosphorylation site of the tumor suppressor protein p53 conferred a similar phenotype, suggesting that this association between phosphorylation and degradation is a general mechanism.

The role of the mismatch repair (MMR) system in correcting base-base mismatches is well established; its involvement in the response to DNA double strand breaks, however, is less clear. We investigated the influence of the essential component of MMR, the hMLH1 protein, on the cellular response to DNA-double strand breaks induced by treatment with SN-38, the active metabolite of topoisomerase I inhibitor irinotecan, in a strictly isogenic cell system (p53(wt), hMLH1(+)/p53(wt), hMLH1(-)). By using hMLH1 expressing clones or cells transduced with the hMLH1-expressing adenovirus as well as siRNA technology, we show that in response to SN-38-induced DNA damage the MMR proficient (MMR(+)) cells make: (i) a stronger G2/M arrest, (ii) a subsequent longer tetraploid G1 arrest, (iii) a stronger activation of Chk1 and Chk2 kinases than the MMR deficient (MMR(-)) counterparts. Both Cdk2 and Cdk4 kinases contribute to the basal tetraploid G1 arrest in MMR(+) and MMR(-) cells. Although the Chk1 kinase is involved in the G2/M arrest, neither Chk1 nor Chk2 are involved in the enhancement of the tetraploid G1 arrest. The long-lasting tetraploid G1 arrest of MMR(+) cells is associated with their lower clonogenic survival after SN-38 treatment, the abrogation of the tetraploid G1 arrest resulted in their better clonogenic survival. These data show that the stabilization of the tetraploid G1 arrest in response to double strand breaks is a novel function of the MMR system that contributes to the lesser survival of MMR(+) cells.

Regulation of the E2F family of transcription factors is important in control of cellular proliferation; dysregulation of the E2Fs is a hallmark of many cancers. One member of the E2F family, E2F1, also has the paradoxical ability to induce apoptosis; however, the mechanisms underlying this selectivity are not fully understood. We now identify a nucleolar protein, RRP1B, as an E2F1-specific transcriptional target. We characterize the RRP1B promoter and demonstrate its selective response to E2F1. Consistent with the activation of E2F1 activity upon DNA damage, RRP1B is induced by several DNA-damaging agents. Importantly, RRP1B is required for the expression of certain E2F1 proapoptotic target genes and the induction of apoptosis by DNA-damaging agents. This activity is mediated in part by complex formation between RRP1B and E2F1 on selective E2F1 target gene promoters. Interaction between RRP1B and E2F1 can be found inside the nucleolus and diffuse nucleoplasmic punctates. Thus, E2F1 makes use of its transcriptional target RRP1B to activate other genes directly involved in apoptosis. Our data also suggest an underappreciated role for nucleolar proteins in transcriptional regulation.

Proper control of the G(1)/S checkpoint is essential for normal proliferation. The activity of p53 must be kept at a very low level under unstressed conditions to allow growth. Here we provide evidence supporting a crucial role for TopBP1 in actively repressing p53. Depletion of TopBP1 upregulates p53 target genes involved in cell cycle arrest and apoptosis and enhances DNA damage-induced apoptosis. The regulation is mediated by an interaction between the seventh and eighth BRCT domains of TopBP1 and the DNA-binding domain of p53, leading to inhibition of p53 promoter binding activity. Importantly, TopBP1 overexpression is found in 46 of 79 primary breast cancer tissues and is associated with high tumor grade and shorter patient survival time. Overexpression of TopBP1 to a level comparable to that seen in breast tumors leads to inhibition of p53 target gene expression and DNA damage-induced apoptosis and G(1) arrest. Thus, a physiological level of TopBP1 is essential for normal G(1)/S transition, but a pathological level of TopBP1 in cancer may perturb p53 function and contribute to an aggressive tumor behavior.

Anemia is prevalent in 30% to 90% of patients with cancer. Anemia can be corrected through either treating the underlying cause or providing supportive care through transfusion with packed red blood cells or administration of erythropoiesis-stimulating agents (ESAs), with or without iron supplementation. Recent studies showing detrimental health effects of ESAs sparked a series of FDA label revisions and a sea change in the perception of these once commonly used agents. In light of this, the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Cancer- and Chemotherapy-Induced Anemia underwent substantial revisions this year. The purpose of these NCCN Guidelines is twofold: 1) to operationalize the evaluation and treatment of anemia in adult cancer patients, with an emphasis on those who are receiving concomitant chemotherapy, and 2) to enable patients and clinicians to individualize anemia treatment options based on patient condition.

Microcephalin (MCPH1) has a crucial role in the DNA damage response by promoting the expression of Checkpoint kinase 1 (CHK1) and Breast cancer susceptibility gene 1 (BRCA1); however, the mechanism of this regulation remains unclear. Here, we show that MCPH1 regulates CHK1 and BRCA1 through the interaction with E2F1 on the promoters of both genes. MCPH1 also regulates other E2F target genes involved in DNA repair and apoptosis such as RAD51, DDB2, TOPBP1, p73 and caspases. MCPH1 interacts with E2F1 on the p73 promoter, and regulates p73 induction and E2F1-induced apoptosis as a result of DNA damage. MCPH1 forms oligomers through the second and third BRCT domains. An MCPH1 mutant containing only its oligomerization domain has a dominant-negative role by blocking MCPH1 binding to E2F1. It also inhibits p73 induction in DNA damage and E2F1-dependent apoptosis. Taken together, MCPH1 cooperates with E2F1 to regulate genes involved in DNA repair, checkpoint and apoptosis, and might participate in the maintenance of genomic integrity.

Although the nuclear p27KIP1 functions as a negative regulator of G1/S cell cycle progression by binding to and inhibiting cyclin-CDK complexes, the cytosolic p27KIP1 has been shown to promote adhesion disassembly and tumor invasion. Loss of nuclear p27KIP1 and cytosolic mislocalization of p27KIP1 are frequently found during cancer progression, and these events correlate with poor clinical outcome in many types of cancers, including ovarian cancer. However, the mechanisms underlying this dysregulation are not yet fully understood. TRIP6 is an adaptor protein that is highly expressed in ovarian serous cystadenocarcinoma. It serves as a platform to recruit a number of molecules, such as NF-κB p65, c-Src, p130cas, Crk, LPA2 and Fas/CD95 receptors, to promote cell motility and antiapoptotic signaling. Using an ovarian cancer xenograft mouse model, we find that TRIP6 knockdown significantly reduces ovarian tumor proliferation. We determine that this effect is in part through the regulation of p27KIP1. Our data show that TRIP6 serves as a bridge to promote the recruitment of p27KIP1 to AKT in the cytosol. TRIP6 regulates the membrane translocation and activation of AKT, and facilitates AKT-mediated recognition and phosphorylation of p27KIP1 specifically at T157, thereby promoting the cytosolic mislocalization of p27KIP1. This is required for p27KIP1 to enhance LPA-induced ovarian cancer cell migration. TRIP6 also promotes serum-induced reduction of nuclear p27KIP1 expression through Skp2-dependent and -independent mechanisms. Consequently, knockdown of TRIP6 in ovarian cancer xenografts restores nuclear p27KIP1 expression and impairs tumor proliferation. Since the expression of TRIP6 and the activity of AKT are significantly higher in ovarian cancer, restoring the expression of nuclear p27KIP1 and inhibiting the migratory effect of phospho-T157-p27KIP1 by targeting TRIP6 may prove to be an effective approach to reduce ovarian cancer progression. Citation Format: Fang-Tsyr Lin, Victor T. G. Lin, Vivian Y. Lin, Yun-Ju Lai, Chen-Shan Chen, Kang Liu, Weei-Chin Lin. TRIP6 regulates p27KIP1 to promote ovarian tumorigenesis [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1333.

Regulation of E2F1-mediated apoptosis is essential for proper cellular growth. This control requires TopBP1, a BRCT (BRCA1 carboxyl-terminal) domain-containing protein, which interacts with E2F1 but not other E2Fs and represses its proapoptotic activity. We now show that the regulation of E2F1 by TopBP1 involves the phosphoinositide 3-kinase (PI3K)–Akt signaling pathway, and is independent of pocket proteins. Akt phosphorylates TopBP1 in vitro and in vivo. Phosphorylation by Akt induces oligomerization of TopBP1 through its seventh and eighth BRCT domains. The Akt-dependent oligomerization is crucial for TopBP1 to interact with and repress E2F1. Akt phosphorylation is also required for interaction between TopBP1 and Miz1 or HPV16 E2, and repression of Miz1 transcriptional activity, suggesting a general role for TopBP1 oligomerization in the control of transcription factors. Together, this study defines a novel pathway involving PI3K–Akt–TopBP1 for specific control of E2F1 apoptosis, in parallel with cyclin–Cdk–Rb for general control of E2F activities.

TRIP6 (thyroid receptor-interacting protein 6), also known as ZRP-1 (zyxin-related protein 1), is a member of the zyxin family that has been implicated in cell motility. Previously we have shown that TRIP6 binds to the LPA2 receptor and associates with several components of focal complexes in an agonist-dependent manner and, thus, enhances lysophosphatidic acid (LPA)-induced cell migration. Here we further report that the function of TRIP6 in LPA signaling is regulated by c-Src-mediated phosphorylation of TRIP6 at the Tyr-55 residue. LPA stimulation induces tyrosine phosphorylation of endogenous TRIP6 in NIH 3T3 cells and c-Src-expressing fibroblasts, which is virtually eliminated in Src-null fibroblasts. Strikingly, both phosphotyrosine-55 and proline-58 residues of TRIP6 are required for Crk binding in vitro and in cells. Mutation of Tyr-55 to Phe does not alter the ability of TRIP6 to localize at focal adhesions or associate with actin. However, it abolishes the association of TRIP6 with Crk and p130cas in cells and significantly reduces the function of TRIP6 to promote LPA-induced ERK activation. Ultimately, these signaling events control TRIP6 function in promoting LPA-induced morphological changes and cell migration.

Human DNA mismatch repair (MMR) proteins correct DNA errors and regulate cellular response to DNA damage by signaling apoptosis. Mutations of MMR genes result in genomic instability and cancer development. Nonetheless, how MMR proteins are regulated has not yet been determined. While hMLH1, hPMS2, and hMLH3 are known to participate in MMR, the function of another member of MutL-related proteins, hPMS1, remains unclear. Here we show that DNA damage induces the accumulation of hPMS1, hPMS2, and hMLH1 through ataxia-telangiectasia-mutated (ATM)-mediated protein stabilization. The subcellular localization of PMS proteins is also regulated during DNA damage, which induces nuclear localization of hPMS1 and hPMS2 in an hMLH1-dependent manner. The induced levels of hMLH1 and hPMS1 are important for the augmentation of p53 phosphorylation by ATM in response to DNA damage. These observations identify hMutL proteins as regulators of p53 response and demonstrate for the first time a function of hMLH1-hPMS1 complex in controlling the DNA damage response.

TopBP1 (DNA topoisomerase IIβ binding protein I) contains multiple BRCT domains and is involved in replication and the DNA damage checkpoint. Through its BRCT domain, TopBP1 interacts with and represses exclusively E2F1 but not other E2F factors. This regulation of E2F1 transcriptional activity is mediated by a pRb-independent, but Brg1/Brm-dependent mechanism. TopBP1 recruits Brg1/Brm, a central component of the SWI/SNF chromatin-remodeling complex, to E2F1-responsive promoters and represses the activities of E2F1, but not E2F2 or E2F3. This regulation is crucial in the control of E2F1-dependent apoptosis during normal cell growth and DNA damage. Interestingly, TopBP1 is induced by E2F and interacts with E2F1 during G1/S transition. Thus, TopBP1 functions as a critical modulator and serves as a negative feedback regulator of E2F1 by inhibiting E2F1-dependent apoptosis during G1/S transition as well as DNA damage to promote cell survival.