Adenylate Kinase 4 Expression Research Articles

Association of AK4 Protein From Stem Cell-Derived Neurons With Cognitive Reserve: An Autopsy Study.

Identifying protein targets that provide cognitive reserve is a strategy to prevent and treat Alzheimer disease and Alzheimer disease related dementias (AD/ADRD). Previous studies using bulk human brain tissue reported 12 proteins associated with cognitive reserve. This study examined whether the same proteins from induced neurons (iNs) are associated with cognitive reserve of their human donors. Induced pluripotent stem cell (iPSC) lines were generated from cryopreserved peripheral blood mononuclear cells of older adults who were autopsied as part of the Religious Orders Study or Rush Memory and Aging Project. Neurons were induced from iPSCs using a standard neurogenin2 protocol. Tandem mass tag proteomics analyses were conducted on iNs day 21. Cognitive reserve of their human donors was measured as person-specific slopes of cognitive change not accounted for by common neuropathologies. The 53 human donors died at a mean age of 91 years, all were non-Latino White, and 36 (67.9%) were female. Eighteen were diagnosed with Alzheimer dementia proximate to death, and 34 had pathologic AD diagnosis at autopsy. Approximately 60% of the donors had above-average cognitive reserve such that their cognition declined slower than an average person with comparable burdens of neuropathologies. Eight of the 12 candidate proteins were quantified in iNs proteomics analyses. Higher adenylate kinase 4 (AK4) expression in iNs was associated with lower cognitive reserve, consistent with the previous report for brain AK4 expression. By replicating cortical protein associations with cognitive reserve in human iNs, these data provide a valuable molecular readout for studying complex clinical phenotypes such as cognitive reserve in a dish.

Overexpression of integrin alpha 2 (ITGA2) correlates with poor survival in patients with pancreatic ductal adenocarcinoma

AimsDue to the known malignant potential and the poor overall prognosis of pancreatic ductal adenocarcinoma (PDAC), the identification of new biomarkers is of utmost importance. It has been reported that...

MiR-3666 serves as a tumor suppressor in ovarian carcinoma by down-regulating AK4 via targeting STAT3.

As a result of metastasis and high recurrence, ovarian carcinoma (OC) is one of the most frequent gynecological carcinomas affecting women up to now. In spite of advances in OC treatments, the molecular mechanisms underlying OC progression are still needed to be deeply understood. MicroRNAs (miRNAs) with aberrant expressions are widely known to regulate target genes so as to mediate diverse biological activities of tumor cells. In the present study, we inspected the expression profile and latent mechanism of miR-3666 in OC. First of all, our research revealed the down-regulated miR-3666 in OC cells. Furthermore, miR-3666 up-regulation could repress cell proliferation and migration as well as induce cell apoptosis in OC. In addition, we unmasked that miR-3666 targeted STAT3 (signal transducer and activator of transcription 3) and further down-regulated STAT3 expression. Moreover, adenylate kinase 4 (AK4) was transcriptionally enhanced by STAT3, and then miR-3666 restrained AK4 expression by mediating STAT3. In the end, rescue experiments depicted that miR-3666 suppressed the development of OC via STAT3-mediated AK4. We uncovered that miR-3666 inhibited the tumorigenesis and even development of OC via suppressing STAT3/AK4 axis, offering a novel biomarker and therapeutic target for OC.

TRIM29 alters bioenergetics of pancreatic cancer cells via cooperation of miR-2355-3p and DDX3X recruitment to AK4 transcript

TRIM29 is dysregulated in pancreatic cancer and implicated in maintenance of stem-cell-like characters of pancreatic cancer cells. However, the exact mechanisms underlying oncogenic function of TRIM29 in pancreatic cancer cells remain largely unclarified. Using a global screening procedure, the current study found that adenylate kinase 4 (AK4) was profoundly reduced by TRIM29 knockdown. In addition, our data demonstrated that TRIM29 knockdown altered bioenergetics and suppressed proliferation and invasion of pancreatic cancer cells via downregulation of AK4 at the posttranscriptional level. The current study demonstrated that upregulation of microRNA-2355-3p (miR-2355-3p) upregulated AK4 expression via facilitating DDX3X recruitment to the AK4 transcript, and TRIM29 knockdown thereby destabilized the AK4 transcript via miR-2355-3p downregulation. Collectively, our study uncovers posttranscriptional stabilization of the AK4 transcript by miR-2355-3p interaction to facilitate DDX3X recruitment. Regulation of AK4 by TRIM29 via miR-2355-3p thereby provides additional information for further identification of attractive targets for therapy with pancreatic cancer.

Adenylate Kinase 4 Modulates the Resistance of Breast Cancer Cells to Tamoxifen through an m6A-Based Epitranscriptomic Mechanism

N6-methyladenosine (m6A) is the most abundant internal modification in mRNA and this methylation constitutes an important regulatory mechanism for the stability and translational efficiency of mRNA. In this study, we found that the protein levels of adenylate kinase 4 (AK4) and m6A writer METTL3 are significantly higher in tamoxifen-resistant (TamR) MCF-7 cells than in parental cells. The TamR MCF-7 cells also exhibit increased methylation at multiple m6A consensus motif sites in the 5' untranslated region (5' UTR) of AK4 mRNA, and genetic depletion of METTL3 in TamR MCF-7 cells led to a diminished AK4 protein level and attenuated resistance to tamoxifen. In addition, we observed augmented levels of reactive oxygen species (ROS) and p38 activity in TamR MCF-7 cells, and both are diminished upon genetic depletion of AK4. Reciprocally, overexpression of AK4 in MCF-7 cells stimulates ROS and p38 phosphorylation levels, and it suppresses mitochondrial apoptosis. Moreover, scavenging of intracellular ROS leads to reduced p38 activity and re-sensitizes TamR MCF-7 cells to tamoxifen. Thus, our results uncover a novel m6A-mediated epitranscriptomic mechanism for the regulation of AK4, illustrate the cellular pathways through which increased AK4 expression contributes to tamoxifen resistance, and reveal AK4 as a potential therapeutic target for overcoming tamoxifen resistance.

AK4 Promotes the Progression of HER2-Positive Breast Cancer by Facilitating Cell Proliferation and Invasion.

Breast cancer (BC) is a type of malignant tumor originating from the epithelial tissue of the mammary gland, and about 20% of breast cancers are human epidermal growth factor receptor 2 positive (HER2+), which is a subtype with more aggression. Recently, HER2-positive breast cancer is often accompanied by poor prognosis of patients, and targeted therapy showed a promising prospect. To combat this disease, novel therapeutic targets are still needed. Adenylate kinase 4 (AK4) is a member of the adenylate kinase family and is expressed in the mitochondrial matrix. AK4 is involved in multiple cellular functions such as energy metabolism homeostasis. Interestingly, AK4 was observed highly expressed in several tumor tissues, and the involvement of AK4 in cancer development was generally revealed. However, the possible role of AK4 on the growth and development of breast cancer is still unclear. Here, we investigated the possible functions of AK4 on the progression of HER2-positive breast cancer. We found the high expression of AK4 in HER2-positive breast cancer tissues from patients who received surgical treatment. Additionally, AK4 expression levels were obviously correlated with clinical-pathological features, including pTNM stage (P = 0.017) and lymph node metastasis (P = 0.046). We mechanically confirmed that AK4 depletion showed the obvious impairment of cell proliferation and invasion in MCF7 and MDA-MB-231 cells. AK4 also facilitates tumor growth and metastasis of HER2-positive breast cancer in vivo. In conclusion, we identified and mechanically confirmed that AK4 is a novel therapeutic target of HER2-positive breast cancer.

Adenylate kinase 4 promotes bladder cancer cell proliferation and invasion.

Bladder cancer is the second most common urological cancer worldwide with low early diagnosis and high mortality. Since the time of diagnosis directly affects survival rate, early detection and precise biomarkers of bladder cancer are very important. Adenylate kinase 4 (AK4) is a key enzyme involved in cellular metabolism and multiple cancer development; however, the potential role of AK4 in bladder tumorigenesis is still unclear. Immunohistochemistry assay was conducted to evaluate the expression level of AK4 in 107 human bladder cancer tissues. Overall survival and recurrence-free survival were used to assess the prognosis of patients. Colony formation and MTT assays [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] were performed to measure the proliferation capacity of tumor cells. Cell scratch assays and transwell assays were performed to measure the invasion capacity of tumor cells. The expression level of involved genes was measured by reverse transcription-polymerase chain reaction and western blot assays. The animal model was used to examine the effects of indicated protein on tumorigenesis and invasion in vivo. Herein, our study demonstrated that increased AK4 expression in patients with bladder cancer was associated with a poor prognosis. We further found that inhibition of AK4 in bladder cancer cell line T24 and 5637 can obviously inhibit the proliferation of cancer cells. Transwell assay results showed that down-regulated AK4 was related to the decreased metastasis of T24 and 5637 cells. In addition, AK4-shRNA transfected obviously inhibited tumor growth and metastasis in mice compared with the scramble group. Taken together, the results provide strong evidence of the involvement of AK4 in the progression of bladder cancer and suggest that it could have high potential as a therapeutic target of disease.

Expression of adenylate kinase 4 in prostate cancer and its clinical significance

Objective To investigate the expression of adenylate kinase 4 (AK4) in prostate cancer and its clinical significance. Methods The clinical data of 85 patients with prostate cancer who have underwent radical surgery were retrospectively analyzed. The expression of AK4 in prostate cancer tissues and corresponding paracancerous tissues was detected by immunohistochemistry to analyze the relationship with clinicopathological features. Results The positive rates of AK4 in prostate cancer and paracancerous tissues were 85.9% and 38.8% respectively, and the difference was statistically significant (P<0.05). The abnormal expression of AK4 was correlated with recurrence and distant metastasis (all P<0.05). The survival time of AK4-overexpressing patients with prostate cancer was significantly lower than that of AK4 low-expression patients, and the difference was statistically significant (P<0.05). Conclusions The expression of AK4 in prostate cancer tissues is up-regulated, which is related to the distant metastasis of carcinoma and vascular tumor thrombus, which suggests that the expression of AK4 may indicate the poor prognosis of patients with prostate cancer. Key words: Prostate cancer; Immunohistochemistry; Adenylate kinase 4; Prognosis

Glioma Cell Proliferation Promoted by AK4 Regulated mTORC1 Pathway Activation

A hallmark of cancer cell biology is the dysregulation of signaling pathways that manage cancer‐associated phenotypes such as proliferation and survival. One dysfunctional pathway in cancer cells is the mechanistic target of rapamycin (mTOR). The mTOR pathway, involving both mTOR Complex 1 (mTORC1) and mTORC2, functions as a central regulator of cell metabolism, growth, proliferation, and survival. mTORC1 kinase signaling allows for transitions between metabolic states by responding to environmental signals and controlling processes that regulate energy. Recently, the expression of an Adenylate Kinase 4 (AK4) protein has been identified as modulating cellular bioenergetics through the activation of pathways such as mTORC1, resulting in both enhanced proliferation and activation of stress responses. Additionally, reduced AK4 expression has been observed in primary human glioma cells, while the mTORC1 signaling pathway is highly activated in identical primary glioma cells. We hypothesize that AK4 expression regulates mTORC1 signaling and induces downstream effects that promote glioma cell biology. To determine if AK4 expression affects mTORC1 signaling, we knocked down AK4 expression using siRNA in U87‐MG glioma cell lines. Phospho‐specific modifications of activators and inhibitors upstream of the mTORC1 pathway and its downstream targets were assessed using Western blot (WB). AK4 knockdown (KD) and WB analysis revealed over‐active mTORC1 phosphorylation and inactivation of the downstream translational repressor, 4E‐BP1, compared to control glioma cell lines. Likewise, WB analysis displayed increased mTORC1 phosphorylation and activation of the downstream protein ribosomal S6, a regulator of protein translation. Reduced phosphorylation of upstream inhibitors and regulatory proteins of mTORC1, Tuberous Sclerosis Complex 2 (TSC2) and Regulatory‐associated protein of mTOR (RPTOR), was observed when compared to controls. Additionally, reduced mTORC1 phosphorylation of the autophagy inducing serine/threonine protein kinase ULK1 was evident by WB. Furthermore, bioluminescence analysis of AK4 KD U87‐MG glioma cells shows an increase in cell proliferation under nutrient stressed conditions compared to control. Overall, these data suggest that AK4 reduction may contribute to over‐active mTORC1 kinase signaling in glioma cells. Studies characterizing the role of AK4 reduction on glioma cell proliferation could further explain the contribution of endogenously reduced AK4 expression in glioma cell biology. By identifying the regulatory role of AK4, we hope to elucidate the therapeutic potential of modulating AK4 levels in cancer cells.Support or Funding InformationNSF# HRD‐1463889.

Modulation of anti-cancer drug sensitivity through the regulation of mitochondrial activity by adenylate kinase 4

BackgroundAdenylate kinase is a key enzyme in the high-energy phosphoryl transfer reaction in living cells. An isoform of this enzyme, adenylate kinase 4 (AK4), is localized in the mitochondrial matrix and is believed to be involved in stress, drug resistance, malignant transformation in cancer, and ATP regulation. However, the molecular basis for the AK4 functions remained to be determined.MethodsHeLa cells were transiently transfected with an AK4 small interfering RNA (siRNA), an AK4 short hairpin RNA (shRNA) plasmid, a control shRNA plasmid, an AK4 expression vector, and a control expression vector to examine the effect of the AK4 expression on cell proliferation, sensitivity to anti-cancer drug, metabolome, gene expression, and mitochondrial activity.ResultsAK4 knockdown cells treated with short hairpin RNA increased ATP production and showed greater sensitivity to hypoxia and anti-cancer drug, cis-diamminedichloro-platinum (II) (CDDP). Subcutaneous grafting AK4 knockdown cells into nude mice revealed that the grafted cells exhibited both slower proliferation and reduced the tumor sizes in response to CDDP. AK4 knockdown cell showed a increased oxygen consumption rate with FCCP treatment, while AK4 overexpression lowered it. Metabolome analysis showed the increased levels of the tricarboxylic acid cycle intermediates, fumarate and malate in AK4 knockdown cells, while AK4 overexpression lowered them. Electron microscopy detected the increased mitochondrial numbers in AK4 knockdown cells. Microarray analysis detected the increased gene expression of two key enzymes in TCA cycle, succinate dehydrogenase A (SDHA) and oxoglutarate dehydrogenease L (OGDHL), which are components of SDH complex and OGDH complex, supporting the metabolomic results.ConclusionsWe found that AK4 was involved in hypoxia tolerance, resistance to anti-tumor drug, and the regulation of mitochondrial activity. These findings provide a new potential target for efficient anticancer therapies by controlling AK4 expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0322-2) contains supplementary material, which is available to authorized users.

Abstract 1129: Adenylate kinase 4 promotes metabolic reprogramming and induces EMT in lung cancer through ROS-dependent stabilization of HIF1-α

Abstract The metabolic alterations that are essential for cancer progression remain largely unknown. Previously, we have identified adenylate kinase 4 (AK4) as a marker for poor clinical outcome that promotes metastasis of lung cancer. However, the molecular mechanism of AK4-induced phenotype remains elusive. By microarray analysis, we found downstream targets of HIF1-α were differentially regulated upon AK4 overexpression in CL1-0 lung cancer cells. Furthermore, AK4 overexpression induces glycolysis and results in accelerated glucose consumption, ATP utilization and lactate production. Overexpression of AK4 also increases the levels of reactive oxygen species (ROS), which exaggerates HIF1-α protein expression under hypoxia and concurrently induces epithelial-to-mesenchymal transition (EMT) in a HIF1-α-dependent manner. IHC analysis showed an inverse correlation between AK4 and E-cadherin and a positive correlation between AK4 and nuclear HIF1-α in lung cancer patients. Moreover, patients with AK4 high/E-Cadherin low showed worse outcome compared to patients with AK4 low/E-cadherin high. Our study indicates that enhanced expression of AK4 is a critical factor to trigger aerobic glycolysis and induced HIF1-α-mediated EMT in lung cancer. Citation Format: Yi-Hua Jan, Michael Hsiao. Adenylate kinase 4 promotes metabolic reprogramming and induces EMT in lung cancer through ROS-dependent stabilization of HIF1-α. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1129. doi:10.1158/1538-7445.AM2014-1129

Adenylate Kinase-4 Is a Marker of Poor Clinical Outcomes That Promotes Metastasis of Lung Cancer by Downregulating the Transcription Factor ATF3

Biomarkers predicting metastatic capacity might assist the development of better therapeutic strategies for aggressive cancers such as lung cancer. In this study, we show that adenylate kinase-4 (AK4) is a progression-associated gene in human lung cancer that promotes metastasis. Analysis of published microarray data showed that AK4 was upregulated in lung adenocarcinoma compared with normal cells. High AK4 expression was associated with advanced stage, disease recurrence and poor prognosis. Loss of AK4 expression suppressed the invasive potential of lung cancer cell lines, whereas AK4 overexpression promoted invasion in vitro and in vivo. Mechanistically, the transcription factor ATF3 was identified as a pivotal regulatory target of AK4. Simultaneous reduction in AK4 and ATF3 expression abolished the inhibitory effects of ATF3 on invasion. ATF3 overexpression in AK4-overexpressing cells limits invasion activity. Furthermore, patients with high AK4 and low ATF3 expression showed unfavorable outcomes compared with patients with low AK4 and high ATF3 expression. Taken together, our findings indicated that AK4 promotes malignant progression and recurrence by promoting metastasis in an ATF3-dependent manner.

Evidence of an intact N-terminal translocation sequence of human mitochondrial adenylate kinase 4

Adenylate kinases are abundant nucleoside monophosphate kinases, which catalyze the phosphorylation of AMP by using ATP or GTP as phosphate donors. A previously cloned cDNA was named adenylate kinase 4 (AK4) based on its sequence similarity with known AKs but with no confirmed AK enzyme activity. In the present study the AK4 cDNA was expressed in Escherichia coli and the substrate specificity and kinetic properties of the recombinant protein were characterized. The enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP or GTP as phosphate donors and AK4 also phosphorylated AMP with UTP as phosphate donor. The kinetic parameters of the enzyme were determined for AMP and dAMP with ATP as phosphate donor and for AMP with GTP as phosphate donor. AK4 showed its highest efficiency when phosphorylating AMP with GTP and a slightly lower efficiency for the phosphorylation of AMP with ATP. Among the three reactions for which kinetics were performed, dAMP was the poorest substrate. The AK4 mitochondrial localization was confirmed by expression of AK4 as a fusion protein with GFP in HeLa cells. The mitochondrial import sequence was shown to be located within the first N-terminal 11 amino acid residues, very close to the ATP-binding region of the enzyme. Import analysis suggested that the mitochondrial import sequence was not cleaved and thus the enzyme retained its activity upon entering the mitochondria. Site directed mutagenesis of amino acids Lys 4 and Arg 7 showed that these two residues were essential for mitochondrial import.