Downregulation of the PHLDA1 gene in IMR-32 neuroblastoma cells increases levels of Aurora A, TRKB and affects proteins involved in apoptosis and autophagy pathways

Durbas, Małgorzata; Horwacik, Irena; Boratyn, Elżbieta; Rokita, Hanna

doi:10.3892/ijo.2016.3572

Cited by 18 publications

(18 citation statements)

References 47 publications

(55 reference statements)

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“…Our results showed that PHLDA1 is a positive modulator of autophagy and both processes exist in a mutually exclusive manner in the PHLDA1 -silenced CHP-134 cells. These results are contrary to our previous results in IMR-32 cells, where the function of PHLDA1 pointed to inhibition of autophagosomes formation and higher susceptibility to apoptosis [ 21 ]. Further studies are warranted to determine the perplexing role of PHLDA1 which, as it is implicated in many studies, relies on cell-specific environment [ 33 ].…”

Section: Discussioncontrasting

confidence: 99%

“…We have previously implicated that the PHLDA1 protein level is significantly increased after the 14G2a mAb treatment, as compared to the control IMR-32 cells [ 11 ]. In addition, we have previously shown that silencing of the PHLDA1 gene results in induction of autophagic activity and inhibits apoptotic characteristics in IMR-32 cells when treated with the 14G2a mAb, thus contributing to apoptosis resistance [ 21 ]. To explain the observed difference between IMR-32 and CHP-134 in autophagy regulation, with no autophagy stimulation upon the 14G2a mAb treatment in the latter, we further elaborated on the potential role of PHLDA1 in autophagy modulation in the CHP-134 cell line.…”

Section: Resultsmentioning

confidence: 99%

“…All samples were run in triplicate. Primers used for qPCR reaction were designed using Primer-BLAST ( http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ), unless otherwise stated, and were as follows: LC3B (F: GATGTCCGACTTATTCGAGAGC, R: TTGAGCTGTAAGCGCCTTCTA); ATG7 (F: ACCCAGAAGAAGCTGAACGA, R: CTCATTTGCTGCTTGTTCCA); BCN1 (F: AGGATGATGTCCACAGAAAGTGC, R: AGTGACCTTCAGTCTTCGGCTG); ATG12 (F: GCGAACACGAACCATCCAAG, R: CCATCACTGCCAAAACACTCAT); ATG5 (F: GGTGAAGGTGGTTCCTCCG, R: AGCCAAACTTAGTAAGCAACAGAC), ATG16L (F: GCATGACGTACCAAACAGGC, R: ATCACCAGTTGAGCTCCCCA), RPS13 (F: TCGGCTTTACCCTATCGACGCAG, R: ACGTACTTGTGCAACACCATGTGA) [ 20 ] and PHLDA1 (F: TGCCTGAAAGGGGCAGCTCC, R: TGATCTGGTGCGGGGCGGA) as described in [ 21 ].…”

Section: Methodsmentioning

confidence: 99%

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GD2 ganglioside-binding antibody 14G2a and specific aurora A kinase inhibitor MK-5108 induce autophagy in IMR-32 neuroblastoma cells

et al. 2018

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The process of autophagy and its role in survival of human neuroblastoma cell cultures was studied upon addition of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (14G2a mAb) and an aurora A kinase specific inhibitor, MK-5108. It was recently shown that combination of these agents significantly potentiates cytotoxicity against IMR-32 and CHP-134 neuroblastoma cells in vitro, as compared to the inhibitor used alone. In this study we gained mechanistic insights on autophagy in the observed cytotoxic effects exerted by both agents using cytotoxicity assays, RT-qPCR, immunoblotting, and autophagy detection methods. Enhancement of the autophagy process in the 14G2a mAb- and MK-5108-treated IMR-32 cells was documented by assessing autophagic flux. Application of a lysosomotropic agent—chloroquine (CQ) affected the 14G2a mAb- and MK-5108-stimulated autophagic flux. It is our conclusion that the 14G2a mAb (40 μg/ml) and MK-5108 inhibitor (0.1 μM) induce autophagy in IMR-32 cells. Moreover, the combinatorial treatment of IMR-32 cells with the 14G2a mAb and CQ significantly potentiates cytotoxic effect, as compared to CQ used alone. Most importantly, we showed that interfering with autophagy at its early and late step augments the 14G2a mAb-induced apoptosis, therefore we can conclude that inhibition of autophagy is the primary mechanism of the CQ-mediated sensitization to the 14G2a mAb-induced apoptosis. Although, there was no virtual stimulation of autophagy in the 14G2a mAb-treated CHP-134 neuroblastoma cells, we were able to show that PHLDA1 protein positively regulates autophagy and this process exists in a mutually exclusive manner with apoptosis in PHLDA1-silenced CHP-134 cells.Electronic supplementary materialThe online version of this article (10.1007/s10495-018-1472-9) contains supplementary material, which is available to authorized users.

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Section: Discussioncontrasting

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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GD2 ganglioside-binding antibody 14G2a and specific aurora A kinase inhibitor MK-5108 induce autophagy in IMR-32 neuroblastoma cells

et al. 2018

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“…We next sought to determine whether PHLDA1 could regulate the activity of Akt, as has been previously implicated ( Durbas et al., 2016 , Li et al., 2014 ), thus providing a link between our proteomic and microarray datasets. Expression of a GFP-tagged PHLDA1 construct in the breast cancer cell line HCC1954 reduced the levels of pAkt (S473), suggesting negative regulation of Akt activation ( Figure 3 E).…”

Section: Resultsmentioning

confidence: 99%

PHLDA1 Mediates Drug Resistance in Receptor Tyrosine Kinase-Driven Cancer

et al. 2018

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SummaryDevelopment of resistance causes failure of drugs targeting receptor tyrosine kinase (RTK) networks and represents a critical challenge for precision medicine. Here, we show that PHLDA1 downregulation is critical to acquisition and maintenance of drug resistance in RTK-driven cancer. Using fibroblast growth factor receptor (FGFR) inhibition in endometrial cancer cells, we identify an Akt-driven compensatory mechanism underpinned by downregulation of PHLDA1. We demonstrate broad clinical relevance of our findings, showing that PHLDA1 downregulation also occurs in response to RTK-targeted therapy in breast and renal cancer patients, as well as following trastuzumab treatment in HER2+ breast cancer cells. Crucially, knockdown of PHLDA1 alone was sufficient to confer de novo resistance to RTK inhibitors and induction of PHLDA1 expression re-sensitized drug-resistant cancer cells to targeted therapies, identifying PHLDA1 as a biomarker for drug response and highlighting the potential of PHLDA1 reactivation as a means of circumventing drug resistance.

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“…For RT‐PCR and qRT‐PCR, the previously described primers were used for the following genes: ACTB [Kowalczyk et al, ], RPS13 (40S ribosomal protein S13), and GAPDH (glyceraldehyde‐3‐phosphate dehydrogenase) [Dupasquier et al, ], c‐MYC [Wang et al, ]. MYCN , ID1 , ID2 , DKK3 , NTRK1 , and BDNF were listed with references where appropriate in our earlier studies [Durbas et al, ]. ID3 primers were designed using Primer‐BLAST [http://www.ncbi.nlm.nih.gov/tools/primer-blast/] (F: 5′‐CTTCCCATCCAGACACAGCCGA‐3′, R: 5′‐TCACAGTCCTTCGCTCCTGA‐3′).…”

Section: Methodsmentioning

confidence: 99%

MCPIP1 Exogenous Overexpression Inhibits Pathways Regulating MYCN Oncoprotein Stability in Neuroblastoma

Boratyn

Nowak

Durbas

et al. 2017

J of Cellular Biochemistry

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The main physiological function of MCPIP1 (regnase-1) is negative regulation of inflammation. Moreover, roles of regnase-1 in apoptosis and differentiation have also been described, but its involvement in cancer is yet to be fully recognized. Earlier, we showed a lack of expression of MCPIP1 in both primary tumors and several neuroblastoma cell lines. Additionally, we reported that levels of MCPIP1 and the key neuroblastoma oncoprotein-MYCN were inversely correlated in BE(2)-C clones overexpressing the MCPIP1 gene. Here, we show that exogenous expression of the MCPIP1 protein decreases MYCN mRNA and protein levels without changing the MYCN mRNA half-life. Furthermore, it was shown that MCPIP1-wt exogenous expression affects levels and phosphorylation of MYCN partners such as Aurora A (Thr288), CDC2 (Tyr15 and Thr161), GSK3β (Ser9), and key cellular components of Akt/mTOR signaling, which regulate MYCN stability and activation. In accordance with the obtained results, we found increased phosphorylation of MYCN protein at Thr58 that causes destabilization of the oncoprotein. Moreover, it is shown that exogenous expression of MCPIP1 does not cause apoptosis. Our data extend knowledge on roles of MCPIP1 in our model and link the protein to regulation of expression and stability of MYCN through decrease of signaling via Akt/mTOR pathway. J. Cell. Biochem. 118: 1741-1755, 2017. © 2016 Wiley Periodicals, Inc.

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Downregulation of the PHLDA1 gene in IMR-32 neuroblastoma cells increases levels of Aurora A, TRKB and affects proteins involved in apoptosis and autophagy pathways

Cited by 18 publications

References 47 publications

GD2 ganglioside-binding antibody 14G2a and specific aurora A kinase inhibitor MK-5108 induce autophagy in IMR-32 neuroblastoma cells

GD2 ganglioside-binding antibody 14G2a and specific aurora A kinase inhibitor MK-5108 induce autophagy in IMR-32 neuroblastoma cells

PHLDA1 Mediates Drug Resistance in Receptor Tyrosine Kinase-Driven Cancer

MCPIP1 Exogenous Overexpression Inhibits Pathways Regulating MYCN Oncoprotein Stability in Neuroblastoma

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