Knockdown of A-kinase anchor protein 4 inhibits proliferation of triple-negative breast cancer cells <i>in vitro</i> and <i>in vivo</i>

Jagadish, Nirmala; Devi, Sonika; Gupta, Namita; Suri, Vitusha; Suri, Anil

doi:10.1177/1010428320914477

Cited by 5 publications

(4 citation statements)

References 48 publications

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“…To further understand the impact of p.S152P mutation on AKAP4 function, we detected the cell apoptosis of GC2‐spd cells by transfection of pEGFP‐AKAP4 p.S152P mutant plasmid. Our study showed that AKAP4 p.S152P mutant protein‐induced cell apoptosis in GC2‐spd cells, which was consistent with results of AKAP4 knockdown reported in colorectal cancer and triple‐negative breast cancer cells (Jagadish et al, 2015; Jagadish et al, 2020).…”

Section: Discussionsupporting

confidence: 91%

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A new hemizygous missense mutation, c.454T＞C (p.S152P), in AKAP4 gene is associated with asthenozoospermia

Liu

Yang

et al. 2021

Molecular Reproduction Devel

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Asthenozoospermia (ASZ) is a condition characterized by reduced forward motility of spermatozoa affecting approximately 19% of infertile men. A kinase anchor protein 4 (AKAP4) is an X‐linked testis‐specific gene and plays a major role in sperm motility and flagella formation. However, few studies have reported its association with ASZ. Here, we sequenced for exonic mutations of human AKAP4 gene by high‐fidelity PCR/Sanger sequencing in peripheral blood samples from 150 ASZ patients and 150 fertile men. We reported the identification of three novel hemizygous mutations unique to four ASZ patients, including one patient carrying missense mutation c.454T>C (p.S152P), two patient carrying synonymous mutation c.1173T>C (p.H391H), and one patient carrying synonymous mutation c.2007 A>G (p.R669R). The p.S152P mutation was located in a precursor pro‐polypeptide domain of AKAP4 protein, which was predicted to be damaging by SIFT and PolyPhen‐2 and could cause the protein accumulation in the cytoplasm of COS‐7 cells. The mature protein of AKAP4 was absent in spermatozoa of ASZ patient harboring AKAP4 p.S152P mutation. Further in vitro cellular assays showed that reactive oxygen species (ROS), malondialdehyde (MDA), myeloperoxidase (MPO) levels, and apoptotic cells were increased in GC2‐spd cells by AKAP4 p.S152P mutant protein, whereas superoxide dismutase (SOD) level was decreased. AKAP4 p.H391H and p.R669R mutant proteins were coimmunoprecipitated with ribonuclease T2 (RNASET2) protein in GC2‐spd cells, whereas no interaction between the AKAP4 p.S152P mutant protein and RNASET2 protein was observed. In addition, AKAP4 p.S152P mutant protein could decrease the activity of PKA/PI3K signaling. Overall, our study identifies a novel AKAP4 p.S152P mutation is associated with ASZ probably through affecting oxidative stress and cell apoptosis by regulating the interaction with RNASET2 and the activity of the PKA/PI3K signaling pathway.

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Section: Discussionsupporting

confidence: 91%

“…AKAP4 has been reported to act as a regulator of cell apoptosis in several kinds of human cancers (Jagadish et al, 2015; Jagadish et al, 2020). To further explore the impact of p.S152P mutation on AKAP4 function, cell apoptosis in GC2‐spd cells was determined by using flow cytometry and caspase‐3 activity assays.…”

Section: Resultsmentioning

confidence: 99%

A new hemizygous missense mutation, c.454T＞C (p.S152P), in AKAP4 gene is associated with asthenozoospermia

Liu

Yang

et al. 2021

Molecular Reproduction Devel

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“…Gene knockdown experiments in model TNBC cell lines are a useful tool to identify the roles played by a gene on processes that involve the cancer cells. Thus, using such experiments it has been found that TNBC cell proliferation inhibition could occur upon knockdown of G-protein–coupled receptor 161 (GPR161) ( 73 ), pyruvate kinase muscle (PKM) ( 74 ), A-kinase anchor protein 4 (AKAP4) ( 75 ), ARHGAP9 ( 76 ), and EP300 ( 77 ), where promotion/inhibition of other processes has also been identified, e.g., promotion of apoptosis ( 75 , 76 ) and inhibitions of cell viability ( 75 ), cell migration/metastasis ability ( 75 – 77 ), cell invasiveness ability and cell colony forming ability ( 75 , 76 ). Also, for a model TNBC cell line, knockdown of arylamine N-acetyltransferase I (NAT1) enzyme resulted in a reduced cell invasiveness ability ( 78 ).…”

Section: Discussionmentioning

confidence: 99%

B4GALNT2 Gene Promotes Proliferation, and Invasiveness and Migration Abilities of Model Triple Negative Breast Cancer (TNBC) Cells by Interacting With HLA-B Protein

et al. 2021

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B4GALNT2 gene encodes the enzyme β1,4-N-acetylgalactosaminyltransferase 2 that biosynthesizes the histo-blood group antigen Sda, which is expressed on the surface of erythrocytes and in body secretions. Analysis of The Cancer Genome Atlas (TCGA) database revealed that this gene was highly expressed in breast cancer tissues in comparison with adjacent healthy ones. In-vitro lentivirus-assisted B4GALNT2 gene knockdown experiments in model triple negative breast cancer (TNBC) cell lines (HCC1937 and MDA-MB-231) showed inhibition in cell proliferation, decrease in cell viability, promotion of cell apoptosis and inhibitions in cell migration and invasiveness abilities in comparison with empty lentivirus transfectant controls. Also, in cell cycle tests, the number of cells in the G1 phase increased, in the S phase decreased and did not change in the G2/M phase (indicative of the presence of a block in the G1 phase). In-vivo tumor formation experiments in mice revealed that knockdown of the B4GALNT2 gene in MDA-MB-231 cells inhibited their proliferation. Using co-immunoprecipitation (Co-IP) mass spectroscopy-assisted analysis, it was found that HLA-B protein [a product of the human leukocyte antigen (HLA) class I gene] interacts with B4GALNT2 protein. In-vitro overexpression of HLA-B in B4GALNT2-knocked down MDA-MB-231 cell lines significantly recovered the cell proliferation, viability and migration ability of B4GALNT2 gene. These indicate that HLA-B is one of the interaction proteins in the downstream pathway of the B4GALNT2 gene.

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“…Heat treatment induces sperm DNA fragmentation significantly increasing and spermatozoa apoptosis [ 25 , 36 , 37 ]. Furthermore, knockdown of AKAP4 led to increased DNA fragmentation and apoptosis [ 38 ]. That indicated AKAP4 downregulation is associated with human sperm damage after the transient scrotal hyperthermia.…”

Section: Discussionmentioning

confidence: 99%

Effect of transient scrotal hyperthermia on human sperm: an iTRAQ-based proteomic analysis

Rao

et al. 2020

Reprod Biol Endocrinol

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Background Through this prospective study, we aimed to explore the change of molecular modification after the transient scrotal hyperthermia on human sperm. Methods Ten healthy subjects selected with strict screening criteria underwent testicular warming in a 43 °C water bath for 30 min a day for 10 consecutive days. Semen samples were collected 2 weeks before the first heat treatment and 6 weeks after the first heat treatment. Proteins from the samples were labeled with isobaric tags for relative and absolute quantitation and analyzed by two-dimensional liquid chromatography–tandem mass spectrometry. Results In contrast to the control, of the 3446 proteins identified, 61 proteins were deregulated: 28 were up-regulated and 33 were down-regulated. Approximately 95% of the differentially expressed proteins were found to participate in spermatogenesis, fertilization, or other aspects of reproduction. In particular, the expression of sperm motility and energy metabolism-related proteins AKAP4, SPESP1, ODF1, ODF2, GAPDHS, and ACTRT2, validated by western blotting of the proteins obtained from human and mouse samples, tended to be reduced under scrotal hyperthermia. Conclusions The results indicated that the proteins AKAP4, ODF1, ODF2, GAPDHS, SPESP1, and ACTRT2, play an important role in the heat-induced reversible reduction in sperm concentration and motility and have the potential to be the biomarkers and clinical targets for scrotal heat treatment induced male infertility.

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Knockdown of A-kinase anchor protein 4 inhibits proliferation of triple-negative breast cancer cells in vitro and in vivo

Cited by 5 publications

References 48 publications

A new hemizygous missense mutation, c.454T＞C (p.S152P), in AKAP4 gene is associated with asthenozoospermia

A new hemizygous missense mutation, c.454T＞C (p.S152P), in AKAP4 gene is associated with asthenozoospermia

B4GALNT2 Gene Promotes Proliferation, and Invasiveness and Migration Abilities of Model Triple Negative Breast Cancer (TNBC) Cells by Interacting With HLA-B Protein

Effect of transient scrotal hyperthermia on human sperm: an iTRAQ-based proteomic analysis

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