Alexey S. Revenko

Androgen-induced coactivator ANCCA mediates specific androgen receptor signaling in prostate cancer.

Androgen receptor (AR) plays a pivotal role in prostate cancer, primarily by regulating different gene expression programs elicited by androgen, which is important for cancer cell proliferation, survival, and differentiation. It is believed that the transcriptional function of AR is mediated largely by distinct nuclear coregulators. We report here the identification of ANCCA (also known as ATAD2), a new member of the AAA+ ATPase family proteins, as a novel AR coactivator. ANCCA interacts directly with AR and enhances its transcriptional activity, and is required for androgen-stimulated expression of a specific subgroup of genes including IGF1R, IRS-2, SGK1, and survivin. Upon androgen stimulation, ANCCA together with AR is recruited to the specific AR target genes. Suppression of ANCCA expression strongly inhibited the proliferation of androgen-responsive or androgen-independent, AR-positive prostate cancer cells and caused a significant increase of cellular apoptosis. Strikingly, the ANCCA gene itself, located at chromosome 8q24, is highly induced by androgen in androgen-dependent prostate cancer cells and xenograft tumors. Although ANCCA is hardly detected in normal human prostate tissue, high levels of ANCCA are found in hormone-independent prostate cancer cell lines, xenograft tumor, and a subset of prostate cancers with high Gleason scores. Together, these findings suggest that ANCCA plays an important role in prostate cancer by mediating specific AR functions in cancer cell survival and proliferation. The possession of ATPase and bromodomain by ANCCA makes it an attractive target for the development of therapeutics for the disease.

ANCCA/ATAD2 Overexpression Identifies Breast Cancer Patients with Poor Prognosis, Acting to Drive Proliferation and Survival of Triple-Negative Cells through Control of B-Myb and EZH2

Chromatin coregulators are important factors in tumorigenesis and cancer progression. ANCCA is an AAA+ ATPase and a bromodomain-containing nuclear coactivator for the estrogen and androgen receptors that is crucial for assembly of chromatin-modifying complexes and proliferation of hormone-responsive cancer cells. In this study, we show that ANCCA is overexpressed in >70% of breast tumors and that its high protein level correlates well with tumor histologic grades (P<0.0001), highlighting ANCCA as a prognostic factor for poor overall survival and disease recurrence. Strikingly, high-level ANCCA correlated with triple-negative tumors that represent highly aggressive disease. Analysis of ANCCA transcript levels in multiple expression profiles of breast cancer identified ANCCA as a common signature gene, indicating that elevated transcripts also strongly correlate with tumor metastasis and poor survival. Biological and mechanistic investigations revealed that ANCCA is crucial for proliferation and survival of triple-negative/basal-like cancer cells and that it controls the expression of B-Myb, histone methyltransferase EZH2, and an Rb-E2F core program for proliferation, along with a subset of key mitotic kinesins and cell survival genes (IRS2, VEGF, and Akt1). In particular, ANCCA overexpression correlated strongly with EZH2 in tumors. Our results suggest that ANCCA may integrate multiple oncogenic programs in breast cancer, serving in particular as a prognostic marker and a therapeutic target for triple-negative cancers.

ANCCA, an estrogen-regulated AAA+ ATPase coactivator for ERα, is required for coregulator occupancy and chromatin modification

AAA+ proteins play crucial roles in diverse biological processes via their ATPase-driven remodeling of macromolecular complexes. Here we report our identification of an evolutionarily conserved AAA+ protein, ANCCA/pro2000, endowed with a bromodomain that is strongly induced by estrogen in human breast cancer cells and is a direct target of protooncogene ACTR/AIB1/SRC-3. We found that ANCCA associates directly with estrogen-bound estrogen receptor (ER) α and ACTR. It is selectively recruited, upon estrogen stimulation, to a subset of ERα target genes including cyclin D1, c-myc, and E2F1 and is required for their estrogen-induced expression as well as breast cancer cell proliferation. Further studies indicate that ANCCA binds and hydrolyzes ATP and is critical for recruitment of coregulator CBP and histone hyperacetylation at the ER target chromatin. Moreover, mutations at the ATP binding motifs rendered ANCCA defective as a coactivator in mediating estrogen induction of gene expression. Together, our findings reveal an unexpected layer of regulatory mechanism in hormone signaling mediated by ANCCA and suggest that hormone-induced assembly of transcriptional coregulator complexes at chromatin is a process facilitated by AAA+ ATPase proteins.

Chromatin Loading of E2F-MLL Complex by Cancer-Associated Coregulator ANCCA via Reading a Specific Histone Mark

ABSTRACT Histone modifications are regarded as the carrier of epigenetic memory through cell divisions. How the marks facilitate cell cycle-dependent gene expression is poorly understood. The evolutionarily conserved AAA ATPase ANCCA (AAA nuclear coregulator cancer-associated protein)/ATAD2 was identified as a direct target of oncogene AIB1/ACTR/SRC-3 and a transcriptional coregulator for estrogen and androgen receptors and is strongly implicated in tumorigenesis. We report here that ANCCA directly interacts with E2F1 to E2F3 and that its N terminus interacts with both the N and C termini of E2F1. ANCCA preferentially associates via its bromodomain with H3 acetylated at lysine 14 (H3K14ac) and is required for key cell cycle gene expression and cancer cell proliferation. ANCCA associates with chromosomes at late mitosis, and its occupancy at E2F targets peaks at the G1-to-S transition. Strikingly, ANCCA is required for recruitment of specific E2Fs to their targets and chromatin assembly of the host cell factor 1 (HCF-1)-MLL histone methyltransferase complex. ANCCA depletion results in a marked decrease of the gene activation-linked H3K4me3 mark. Bromodomain mutations disable ANCCA function as an E2F coactivator and its ability to promote cancer cell proliferation, while ANCCA overexpression in tumors correlates with tumor growth. Together, these results suggest that ANCCA acts as a pioneer factor in E2F-dependent gene activation and that a novel mechanism involving ANCCA bromodomain may contribute to cancer cell proliferation.

Direct control of cell cycle gene expression by proto-oncogene product ACTR, and its autoregulation underlies its transforming activity.

ABSTRACT ACTR (also called AIB1 and SRC-3) was identified as a coactivator for nuclear receptors and is linked to multiple types of human cancer due to its frequent overexpression. However, the molecular mechanism of ACTR oncogenicity and its function independent of nuclear receptors remain to be defined. We demonstrate here that ACTR is required for both normal and malignant human cells to effectively enter S phase. RNA interference-mediated depletion and chromatin immunoprecipitation assays show that endogenous ACTR directly controls the expression of genes important for initiation of DNA replication, which include cdc6, cdc25A, MCM7, cyclin E, and Cdk2. Moreover, consistent with its critical role in cell cycle control, ACTR expression appears to be cell cycle regulated, which involves E2F. Interestingly, ACTR is recruited to its own promoter at the G1/S transition and activates its own expression, suggesting a positive feedback mechanism for ACTR action in the control of cell cycle progression and for its aberrant expression in cancers. Importantly, overexpression of ACTR alone transforms human mammary epithelial cells, which requires its association with E2F. These findings reveal a novel role for ACTR in cell cycle control and support the notion that the ability of aberrant ACTR to deregulate the cell cycle through E2F underlies its oncogenicity in human cancers.

Deregulated E2F and the AAA+ coregulator ANCCA drive proto-oncogene ACTR/AIB1 overexpression in breast cancer

The proto-oncogene ACTR/AIB1, a coactivator for transcription factors such as the nuclear receptors and E2Fs, is frequently overexpressed in various cancers including breast cancers. However, the underlying mechanism is poorly understood. Here, we identified several functional, noncanonical E2F binding sites in the ACTR first exon and intron that are critical for ACTR gene activation. We also found that the newly identified AAA+ coregulator AAA+ nuclear coregulator cancer associated (ANCCA) is recruited to the ACTR promoter and directly controls ACTR expression in breast cancer cells. Importantly, immunohistochemistry analysis indicated that ACTR overexpression is highly correlated with the expression of E2F1 and ANCCA in a cohort of human primary and lymph node–metastasized breast cancer specimens. Along with previous findings from us and others that ACTR is involved in its own gene regulation, these results suggest that one major mechanism of ACTR overexpression in cancer is the concerted, aberrant function of the nuclear coregulators such as ANCCA and ACTR, and they point to therapeutic strategies that target the Rb-E2F axis and/or the coregulator ANCCA for ACTR-overexpressing cancers. Mol Cancer Res; 8(2); 183–93

Targeting KRAS-dependent tumors with AZD4785, a high-affinity therapeutic antisense oligonucleotide inhibitor of KRAS

AZD4785 is a therapeutic antisense oligonucleotide targeting KRAS mRNA with promising preclinical antitumor activity and a favorable safety profile. An antisensible approach to targeting KRAS Mutations that cause activation of the KRAS oncogene are common in human cancer, including treatment-resistant tumor types such as lung and pancreatic cancer. KRAS has also proven to be notoriously difficult to target with small molecules. To overcome this issue, Ross et al. have turned to genetic technology, demonstrating an antisense oligonucleotide–based therapy for inhibiting KRAS. The antisense oligonucleotide used in this study was chemically modified, allowing systemic delivery through subcutaneous injection and avoiding the need for a specialized delivery vehicle. The authors tested the efficacy of this therapy in multiple mouse models of non–small cell lung cancer and evaluated its safety in primates, demonstrating its potential suitability for translation to humans. Activating mutations in KRAS underlie the pathogenesis of up to 20% of human tumors, and KRAS is one of the most frequently mutated genes in cancer. Developing therapeutics to block KRAS activity has proven difficult, and no direct inhibitor of KRAS function has entered clinical trials. We describe the preclinical evaluation of AZD4785, a high-affinity constrained ethyl–containing therapeutic antisense oligonucleotide (ASO) targeting KRAS mRNA. AZD4785 potently and selectively depleted cellular KRAS mRNA and protein, resulting in inhibition of downstream effector pathways and antiproliferative effects selectively in KRAS mutant cells. AZD4785-mediated depletion of KRAS was not associated with feedback activation of the mitogen-activated protein kinase (MAPK) pathway, which is seen with RAS-MAPK pathway inhibitors. Systemic delivery of AZD4785 to mice bearing KRAS mutant non–small cell lung cancer cell line xenografts or patient-derived xenografts resulted in inhibition of KRAS expression in tumors and antitumor activity. The safety of this approach was demonstrated in mice and monkeys with KRAS ASOs that produced robust target knockdown in a broad set of tissues without any adverse effects. Together, these data suggest that AZD4785 is an attractive therapeutic for the treatment of KRAS-driven human cancers and warrants further development.

Depletion of coagulation factor XII ameliorates brain pathology and cognitive impairment in Alzheimer disease mice

Vascular abnormalities and inflammation are found in many Alzheimer disease (AD) patients, but whether these changes play a causative role in AD is not clear. The factor XII (FXII) -initiated contact system can trigger both vascular pathology and inflammation and is activated in AD patients and AD mice. We have investigated the role of the contact system in AD pathogenesis. Cleavage of high-molecular-weight kininogen (HK), a marker for activation of the inflammatory arm of the contact system, is increased in a mouse model of AD, and this cleavage is temporally correlated with the onset of brain inflammation. Depletion of FXII in AD mice inhibited HK cleavage in plasma and reduced neuroinflammation, fibrinogen deposition, and neurodegeneration in the brain. Moreover, FXII-depleted AD mice showed better cognitive function than untreated AD mice. These results indicate that FXII-mediated contact system activation contributes to AD pathogenesis, and therefore this system may offer novel targets for AD treatment.

Knockdown of endogenous circulating C1 inhibitor induces neurovascular impairment, neuroinflammation and cognitive decline

Plasma proteins and activated immune cells are known contributors of vascular brain disorders. However, the mechanisms and routes involved are still unclear. In order to understand the cross-talk between plasma proteins and the brain, we knocked down circulating C1 inhibitor (C1INH) in wild-type (WT) mice using antisense-oligonucleotide (ASO) technique and examined the brain. C1INH is a plasma protein inhibitor of vascular inflammation induced by activation of the kallikrein-kinin system (KKS) and the complement system. This knockdown induced the activation of the KKS but spared the activation of the classical complement system. Activation of the KKS induced an upregulation of the bradykinin pathway in the periphery and the brain, resulting in hypotension. Blood-brain barrier (BBB) permeability, plasma protein extravasations, activated glial cells and elevated levels of IL-1beta, IL-6, TNF-alpha, and iNOS were detected in brains of C1INH ASO treated mice. Infiltrating innate immune cells were evident, entering the brain through the lateral ventricle walls and the neurovascular units. The mice showed normal motor functions, however, cognition was impaired. Altogether, our results highlight the important role of regulated plasma-C1INH as a gatekeeper of the neurovascular system. Thus, manipulation of C1INH in neurovascular disorders might be therapeutically beneficial.Activation of the innate immune system, mediated by the complement and the contact systems, induces inflammation. The inhibition of these pathways by C1 inhibitor (C1INH) has been shown to decrease pro-inflammatory response and mediate vascular permeability in the periphery. However, the role of peripheral C1INH has not been associated with brain function. Using an antisense oligonucleotide (ASO) to deplete circulating liver-derived C1INH in wildtype (WT) mice, we induced the activation of the kallikrein-kinin system (KKS), which produced bradykinin in the plasma, resulting in hypotension. Interestingly, the complement system was quiescent. Depletion of liver-derived C1INH increased blood-brain barrier (BBB) permeability, increased expression of bradykinin 2 receptor, activated resident glial cells to secrete pro-inflammatory mediators such as Il-1beta, Il-6, TNF-alpha and iNOS, and induced cognitive decline. The results of this study emphasize the important role of circulating C1INH in mediating brain function through the activation of the KKS. Thus, manipulation of C1INH in neuro-vascular disorders might be therapeutically beneficial.

Knock-down of endogenous circulating C1 inhibitor induces neurovascular impairment and neuroinflammation

Plasma proteins and activated immune cells are known contributors of vascular brain disorders. However, the mechanisms and routes involved are still unclear. In order to understand the cross-talk between plasma proteins and the brain, we knocked down circulating C1 inhibitor (C1INH) in wild-type (WT) mice using antisense-oligonucleotide (ASO) technique and examined the brain. C1INH is a plasma protein inhibitor of vascular inflammation induced by activation of the kallikrein-kinin system (KKS) and the complement system. This knockdown induced the activation of the KKS but spared the activation of the classical complement system. Activation of the KKS induced an upregulation of the bradykinin pathway in the periphery and the brain, resulting in hypotension. Blood-brain barrier (BBB) permeability, plasma protein extravasations, activated glial cells and elevated levels of IL-1beta, IL-6, TNF-alpha, and iNOS were detected in brains of C1INH ASO treated mice. Infiltrating innate immune cells were evident, entering the brain through the lateral ventricle walls and the neurovascular units. The mice showed normal motor functions, however, cognition was impaired. Altogether, our results highlight the important role of regulated plasma-C1INH as a gatekeeper of the neurovascular system. Thus, manipulation of C1INH in neurovascular disorders might be therapeutically beneficial.Activation of the innate immune system, mediated by the complement and the contact systems, induces inflammation. The inhibition of these pathways by C1 inhibitor (C1INH) has been shown to decrease pro-inflammatory response and mediate vascular permeability in the periphery. However, the role of peripheral C1INH has not been associated with brain function. Using an antisense oligonucleotide (ASO) to deplete circulating liver-derived C1INH in wildtype (WT) mice, we induced the activation of the kallikrein-kinin system (KKS), which produced bradykinin in the plasma, resulting in hypotension. Interestingly, the complement system was quiescent. Depletion of liver-derived C1INH increased blood-brain barrier (BBB) permeability, increased expression of bradykinin 2 receptor, activated resident glial cells to secrete pro-inflammatory mediators such as Il-1beta, Il-6, TNF-alpha and iNOS, and induced cognitive decline. The results of this study emphasize the important role of circulating C1INH in mediating brain function through the activation of the KKS. Thus, manipulation of C1INH in neuro-vascular disorders might be therapeutically beneficial.