Adenylate Kinase 4 Research Articles

Adenylate kinase 4 promotes neuronal energy metabolism and mitophagy in early cerebral ischemia via Parkin/PKM2 pathway

Mitochondrial dysfunction is closely related to brain injury and neurological dysfunction in ischemic stroke. Adenylate kinase 4 (AK4) plays a critical role in energy metabolism and mitochondrial homeostasis. However, the underlying mechanisms remain unclear. In the present study, we demonstrated an important role of AK4 in mitochondrial dysfunction in the early cerebral ischemia. Early focal cerebral ischemia induced decrease of AK4 protein expression in ischemic hemispheric brain tissue in mice. Exposure of cultured primary neuron to oxygen-glucose deprivation (OGD) also induced AK4 downregulation. Overexpression of AK4 in neuron using adeno-associated virus (AAV-AK4) in mice promoted neuronal survival reflected by decreased infarction volume and TUNEL staining. AK4 overexpression inhibited mitochondrial decline and downregulation of energy metabolism-associated proteins (p-AMPK and ATP1A3) induced by MCAO. Moreover, AK4 knock-in using lentivirus carried AK4 vector (LV-AK4) induced energy metabolism shift from glycolysis to oxidation in neuron. Using transmission electron microscope and western blot, we revealed that AK4 overexpression promoted mitophagy and mitophagy-associated proteins expression PINK1 and Parkin after MCAO. Mass spectrometry and co-immunoprecipitation revealed an interaction between AK4 and PKM2. Mechanistically, AK4 indirectly decreased PKM2 expression via enhancing its ubiquitination by increasing the interaction between PKM2 and its ubiquitin E3 ligase Parkin, and inhibits Parkin downregulation. In conclusion, our data demonstrate that AK4/ Parkin /PKM axis prevents cerebral ischemia damage via regulation of neuronal energy metabolism model and mitophagy. AK4 was a new target for intervention of early ischemic neuron injury.

In-depth proteomics reveals the characteristic developmental profiles of early lung adenocarcinoma with epidermal growth factor receptor mutation.

Lung adenocarcinoma progresses stepwise from atypical adenomatous hyperplasia to adenocarcinoma in situ (AIS), followed by minimally invasive adenocarcinoma (MIA), and then obvious invasive adenocarcinoma. In this study, we examined the protein expression profiles of early and epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinomas. Fifteen cases of small and EGFR mutation-positive adenocarcinomas were collected, including AIS, MIA, and small invasive adenocarcinoma (SIA). We examined their protein expression profiles by tandem mass tag (TMT)-labeling liquid chromatography-mass spectrometry (LC-MS/MS) and compared the results between AIS and MIA versus SIA. The differentially expressed proteins were then verified by Western blot analysis and immunohistochemistry (IHC). The clinicopathological implications of the proteins were also examined by IHC. A total of 4220 proteins were identified by LC-MS/MS analysis. Pathway analysis of the differentially expressed proteins revealed that pathways related to interferon α/β signaling, glutamate and glutamine metabolism, and gluconeogenesis were upregulated in SIA relative to AIS. Among the 13 differentially expressed proteins, cellular retinoic acid binding protein 2 (CRABP2), delta(24)-sterol reductase (DHCR24), and adenylate kinase 4 (AK4) were expressed significantly more strongly in SIA than in AIS. Patients with high expression of CRABP2, DHCR24, and AK4 showed a significantly poorer outcome than those with low expression. In comparison with AIS, SIA shows differences in several different protein expression pathways. Furthermore, CRABP2, DHCR24, and AK4 are useful IHC markers for diagnosis of lung adenocarcinoma invasiveness and may be associated with malignant progression of AIS.

SPOC domain-containing protein 1 regulates the proliferation and apoptosis of human spermatogonial stem cells through adenylate kinase 4

Spermatogonial stem cells (SSCs) are the origin of male spermatogenesis, which can reconstruct germ cell lineage in mice. However, the application of SSCs for male fertility restoration is hindered due to the unclear mechanisms of proliferation and self-renewal in humans. To investigate the role and mechanism of SPOC domain-containing protein 1 (SPOCD1) in human SSC proliferation. We analyzed publicly available human testis single-cell RNA sequencing (RNA-seq) data and found that SPOCD1 is predominantly expressed in SSCs in the early developmental stages. Small interfering RNA was applied to suppress SPOCD1 expression to detect the impacts of SPOCD1 inhibition on SSC proliferation and apoptosis. Subsequently, we explored the target genes of SPOCD1 using RNA-seq and confirmed their role by restoring the expression of the target genes. In addition, we examined SPOCD1 expression in some non-obstructive azoospermia (NOA) patients to explore the correlation between SPOCD1 and NOA. The uniform manifold approximation and projection clustering and pseudotime analysis showed that SPOCD1 was highly expressed in the early stages of SSC, and immunohistological results showed that SPOCD1 was mainly localized in glial cell line-derived neurotrophic factor family receptor alpha-1 positive SSCs. SPOCD1 knockdown significantly inhibited cell proliferation and promoted apoptosis. RNA-seq results showed that SPOCD1 knockdown significantly downregulated genes such as adenylate kinase 4 (AK4). Overexpression of AK4 in SPOCD1 knockdown cells partially reversed the phenotypic changes, indicating that AK4 is a functional target gene of SPOCD1. In addition, we found a significant downregulation of SPOCD1 expression in some NOA patients, suggesting that the downregulation of SPOCD1 may be relevant for NOA. Our study broadens the understanding of human SSC fate determination and may offer new theories on the etiology of male infertility.

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...

AK4P1 is a cancer-promoting pseudogene in pancreatic adenocarcinoma cells whose transcripts can be transmitted by exosomes.

Adenylate kinase 4 pseudogene 1 (AK4P1) is a processed pseudogene whose function in cancer biology remains largely underexplored. Bioinformatics analysis suggested an association between the expression levels of adenylate kinase 4 (AK4) gene and AK4P1, as well as a clinical significance in relation to the increased transcription levels of AK4P1 in pancreatic adenocarcinoma (PAAD). In the present study, the expression levels of AK4P1 and AK4 were compared by RT-qPCR and western blotting between PAAD tissue and paired adjacent tissue. The level of AK4P1 transcript was compared between the circulating exosomes derived from patients with PAAD and those derived from healthy donors. Overall survival of the patients with PAAD with high or low expression levels of AK4P1 or AK4 was compared. AK4 gene expression level, in vitro cell viability and gemcitabine-induced apoptosis in PAAD cells with or without AK4P1 overexpression were also assessed using Cell Counting Kit-8 and TUNEL assays. It was identified that the transcription level of AK4P1 and the expression level of AK4 in PAAD tissue were significantly higher compared with those in paired non-cancerous tissue specimens. Transcription levels of AK4P1 and AK4 showed a significant relationship in PAAD. Circulating exosomes derived from patients with PAAD showed significantly higher level of AK4P1 transcript compared with that from circulating exosomes derived from blood samples of healthy donors. Patients with high expression of AK4P1 or AK4 exhibited significantly reduced overall survival compared with those with low expression. AK4P1 overexpression significantly upregulated the expression levels of AK4 in PAAD cells and rescued the viability and survival under gemcitabine challenge decreased by AK4 knockdown but not that by AK4 knockout. Treatment with exosomes secreted by AK4P1-overexpressing PAAD cells but not with those secreted by wild-type PAAD cells significantly increased the viability and survival under gemcitabine challenge of the recipient cells. These results suggested that AK4P1 affects the cellular biological functions of PAAD cells in vitro by upregulating the expression level of AK4. AK4P1 transcripts with elevated expression levels can be transmitted between PAAD cells through exosomes and exert pro-oncogenic effects in recipient cells.

Adenylate Kinase 4-A Key Regulator of Proliferation and Metabolic Shift in Human Pulmonary Arterial Smooth Muscle Cells via Akt and HIF-1α Signaling Pathways.

Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.

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.

Identification of AK4 as a novel therapeutic target for serous ovarian cancer

The present study aimed to assess the expression level of adenylate kinase 4 (AK4) in human serous ovarian cancer (SOC) tissues and investigate the possible involvement of AK4 in SOC progression. Bioinformatics analysis based on The Cancer Genome Atlas (TCGA) database and immunohistochemical (IHC) assays were performed to assess the expression level of AK4 in human SOC tissues. Clinical pathological features of patients with SOC were also evaluated. Colony formation, MTT, wound healing and Transwell assays were conducted to investigate the effects of AK4 on the proliferation, migration, and invasion of SOC cells in vitro. Mouse xenograft and lung metastasis models were developed to evaluate the effects of AK4 on tumor growth and metastasis in vivo. High expression levels of AK4 were identified in human SOC tissues compared with in normal tissues according to TCGA database and the results of IHC assays. A contribution of AK4 to tumor growth and metastasis of SOC cells in vivo was also shown. The present study confirmed the involvement of AK4 in the progression of SOC, and the results indicated that AK4 could serve as a novel therapeutic target for SOC treatment.

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.

MiR-199a-3p affects the multi-chemoresistance of osteosarcoma through targeting AK4

BackgroundMicroRNAs (miRNAs) play vital roles in regulating various biological processes. The dysregulations of miRNAs may result in severe human diseases, including cancer.MethodsWe performed the qRT-PCR, western blot and the luciferase reporter assays to test whether Adenylate Kinase 4 (AK4) is the target of miR-199a-3p. Up- or down-regulation of miR-199a-3p and/or the AK4 gene was done to detect their roles in OS multi-drug resistance using drug resistance profiling assays. We further predicted the putative signal pathway involved in the miR-199a-3p-mediated OS drug-resistance.ResultsThe AK4 gene is one of the targets of miR-199a-3p and negatively correlates with the effect of miR-199a-3p on OS drug-resistance. In addition, the activity of the NF-кB signaling pathway was drastically altered by the forced changes of the miR-199a-3p level in OS cells.ConclusionsOur data revealed that both miR-199a-3p and its target gene AK4 are reversely correlated with the OS drug resistance.

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.

Abstract A29: Targeting adenylate kinase-4 modulation of HIF-1α: Stability identifies Withaferin-A suppresses lung cancer metastasis

Abstract Aberrant activation of hypoxia inducible factor (HIF-1α) often plays a crucial role in promoting metabolic reprogramming and metastatic dissemination. Here we report functional evidences of adenylate kinase 4 (AK4), one of the hypoxia-responsive genes, in enhancing HIF-1α; transcriptional activity to drive epithelial-to-mesenchymal transition (EMT). By analyzing lung adenocarcinoma microarray datasets, we identified AK4-associated metabolic gene signature and revealed HIF-1α; was transcriptionally activated and associated with poor prognosis. Immunohistochemistry analysis confirms the positive correlation between AK4 and HIF-1α; in non-small cell lung (NSCLC) cancer patients. Overexpression of AK4 induces metabolic shift towards aerobic glycolysis and increases intracellular reactive oxygen species (ROS), which exaggerates HIF-1a; protein expression under hypoxia and concurrently induces EMT in a HIF-1α-dependent manner. Furthermore, overexpression of AK4 reduces hypoxic necrosis in tumors and promotes liver metastasis in vivo. Connectivity map analysis of AK4 gene signature identifies Withaferin-A as a potential inhibitor to inverse AK4 signature and proven to be a potent anti-metastatic agent in orthotopic xenograft model of lung cancer. Our findings offer an alternative strategy to treat metastatic lung cancer via targeting AK4-HIF-1α axis.α Citation Format: Michael Hsiao. Targeting adenylate kinase-4 modulation of HIF-1α: Stability identifies Withaferin-A suppresses lung cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A29.

Pharmacogenomic Analysis of Adenylate Kinase‐4 Gene Expression Signature Identifies Withaferin‐A as an Anti‐Metastatic Agent in Non‐Small Cell Lung Cancer

Lung cancer remains the leading cause of cancer death in the world due to metastatic disseminations. So far, there are no effective therapies available to fight this fatal process. Previously, we identified adenylate kinase 4 (AK4) as a lung cancer progression marker that enhances the invasion and metastasis ability of lung cancer cells. Therefore, we queried connectivity map profiles of AK4‐associated gene expression signature and identified Withaferin‐A (WFA) as a potential agent to treat metastatic lung cancer. We found WFA potently inhibited invasion and migration of CL1‐5 and A549 cells up to 50 percent at low‐cytotoxic doses in vitro. WFA treatment also abolished the invasion activity induced by AK4 ectopic overexpression in CL1‐0 cells. Microarray analysis further revealed that differentially regulated genes upon WFA treatment were significantly enriched in gene sets annotated with cell movement and invasion of cancer cells. WFA treatment resulted in activation of stress response pathways as canonical pathway analysis indicated genes involved in ER and oxidative stress were also significantly enriched. In addition, upstream analysis showed AMPK was activated upon WFA treatment. We then validated AMPK activation was sufficient to suppress TGF‐beta‐mediated epithelial‐to‐mesenchymal transition through knockdown of AK4. Furthermore, in vivo therapeutic model showed that WFA administration significantly decreased lung orthotopic xenograft tumor growth and liver and kidney metastasis compared to the control group. These results identify WFA can be applied to treat metastatic lung cancer through repurposing approved drugs that reverse AK4‐associated gene expression signature.

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.

Inhibition of AMPK by Adenylate Kinase 4 Promotes EMT in Lung Cancer through ROS‐dependent Stabilization of HIF1‐α

The field of cancer metabolism has largely focused on the metabolic reprogramming in supporting cancer proliferation. However, the metabolic alterations that are critical for cancer progression remain largely unknown. In this study, we analyzed lung cancer microarray datasets and found a set of metabolic genes that is positively correlated with mesenchymal signature in lung cancer patients. Upstream kinase analysis of EMT‐associated metabolic gene signature predicted AMP‐activated protein kinase (AMPK) inactivation by adenylate kinase may up‐regulate NADPH oxidase which leads to the production of intracellular reactive oxygen species (ROS). We next show TGF‐β‐induced EMT is accompanied by reduced AMPK activity and concurrent up‐regulation of adenylate kinase 4 (AK4) and NADPH oxidase. Knockdown of AK4 was sufficient to suppress TGF‐β‐induced EMT through AMPK activation. On the other hand, overexpression of AK4 inhibits AMPK activation, leading to NADPH oxidase‐mediated production of ROS, which in turn exaggerates HIF1‐α protein expression and induces EMT. IHC analysis showed an inverse correlation between AK4 and phosphorylated AMPK and a positive correlation between AK4 and nuclear HIF1‐α in lung cancer patients. Moreover, patients with high AK4 and low phosphorylated AMPK showed worse outcome compared to patients with low AK4 and high phosphorylated AMPK. Taken together, we identified overexpression of AK4 is critical for lung cancer cells to avoid AMPK‐mediated metabolic checkpoint and undergo EMT.