ZNF322A-mediated protein phosphorylation induces autophagosome formation through modulation of IRS1-AKT glucose uptake and HSP-elicited UPR in lung cancer

Abstract

Background: ZNF322A is an oncogenic transcription factor that belongs to the Cys2His2-type zinc-finger protein family. Accumulating evidence suggests that ZNF322A may contribute to the tumorigenesis of lung cancer, however, the ZNF322A-mediated downstream signaling pathways remain unknown.

Methods: To uncover ZNF322A-mediated functional network, we applied phosphopeptide enrichment and isobaric labeling strategies with mass spectrometry-based proteomics using A549 lung cancer cells, and analyzed the differentially expressed proteins of phosphoproteomic and proteomic profiles to determine ZNF322A-modulated pathways.

Results: ZNF322A highlighted a previously unidentified insulin signaling, heat stress, and signal attenuation at the post-translational level. Consistently, protein-phosphoprotein-kinase interaction network analysis revealed phosphorylation of IRS1 and HSP27 were altered upon ZNF322A-silenced lung cancer cells. Thus, we further investigated the molecular regulation of ZNF322A, and found the inhibitory transcriptional regulation of ZNF322A on PIM3, which was able to phosphorylate IRS1 at serine¹¹⁰¹ in order to manipulate glucose uptake via the PI3K/AKT/mTOR signaling pathway. Moreover, ZNF322A also affects the unfolded protein response by phosphorylation of HSP27^S82 and eIF2a^S51, and triggers autophagosome formation in lung cancer cells.

Conclusions: These findings not only give new information about the molecular regulation of the cellular proteins through ZNF322A at the post-translational level, but also provides a resource for the study of lung cancer therapy.

Keywords: Autophagy; Glucose starvation; Heat stress; Lung cancer; Phosphoproteomics; Proteomics; Zinc-finger protein.

Fig. 1

Overall workflow for the analyses of the proteome and phosphoproteome regulated by ZNF322A in lung cancer cells. a Experimental strategy for quantitative global proteomic profiling in response to silencing of ZNF322A (siZNF322A) in A549 cells. Protein extracts obtained from the transfected cells were digested, iTRAQ labeled, and analyzed with mass spectrometry. b Experimental strategy for quantitative phosphoproteomic profiling in response to siZNF322A in A549 cells. Protein extracts obtained from the transfected cells were digested, dimethyl labeled, phosphopeptide enriched, SCX fractionated, and analyzed with mass spectrometry. Construction of ZNF322A-perturbed functional network, and protein-protein interaction and the biological processes in lung cancer cells were validated by functional assays

Fig. 2

Phosphoproteomic profile of siZNF322A in A549 lung cancer cells. a Number of identified phosphoproteins, phosphopeptides, and phosphosites of the phosphoproteomics of ZNF322A-silenced A549 cells. b Number of singly, doubly, triply and quadruply phosphorylated peptides. c Localization probability of identified phosphorylated sites. Bar plot showed the percentages of localization probability, of which the majority of localization probability > 0.75, in ZNF322A-silencing phosphoproteome. d Distribution of phosphorylated serine, threonine, and tyrosine sites. Pie chart showed the distribution of phosphorylation sites on Ser (88%) Thr (10%) and Tyr (2%) residues

Fig. 3

ZNF322A-mediated biological processes in A549 lung cancer cells. a Differentially regulated proteins from proteome and phosphoproteome profiles (normalized ratio ≥ 1.5 or ≤ 0.67) were annotated with GO terms. Each node represents an enriched GO term (corrected p-value < 0.05), and the size of nodes represents the total number of genes in each GO term. Pie chart in a node represents the proportion of significantly regulated proteins derived from proteomics and phosphoprotemics (red: protein expression; orange: protein phosphorylation). The edge represents gene overlap score between nodes over a given threshold (0.5). Functionally related GO terms are manually grouped and labeled. b Pathway analysis of ZNF322A-regulated phosphoproteins. Top 15 enriched pathways of the phosphoproteomics with the lowest p-value. The number of entities is shown as bar label

Fig. 4

ZNF322A modulates glucose uptake via the IRS1/PI3K/AKT pathway in A549 lung cancer cells. a Schematic representation of the inhibitory transcription regulation of ZNF322A on PIM3. b Relative mRNA levels of ZNF322A and PIM3 were examined by RT-qPCR analysis, and normalized by GAPDH. c-d Proteins were extracted from ZNF322A-silenced (siZNF322A) A549 cells after 48 h transfection. Protein expressions of ZNF322A and PIM3 were examined by immunoblot analysis. Representative western blot (c) and associated densitometric analysis (d) for ZNF322A and PIM3 expressions in A549 lung cancer cells. e-f Protein expressions of IRS1^S1101 and AKT ^S473 phosphorylation were examined by immunoblot analysis. Phosphorylation levels were determined and normalized to the level of total proteins. The normalized values were compared between siRNA control (siControl) and siZNF322A groups. g-h Effect of 2-NBDG uptake upon ZNF322A perturbation in A549 lung cancer cells. Representative fluorescence microscopy images of 2-NBDG uptake ability in siRNA control (upper) and siZNF322A-silenced (bottom) A549 cells. 2-NBDG: green; DAPI: blue; scale bar: 10 μm (g). Mean fluorescence intensity (left) and number (right) of 2-NBDG in siControl and siZNF322A cells (h). (i) Schematic representation of the depletion of ZNF322A by siRNAs in A549 lung cancer cells transcriptionally regulates PIM3 kinase to induces IRS1^Ser1101 phosphorylation, which attenuates PI3K/AKT signaling pathway and inhibits AKT^S473, leading to glucose uptake blockade. β-actin is the internal control to normalize protein expression. The bars represent densitometric analysis of three biological replicates and the data are shown as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 5

ZNF322A mediates heat shock protein 27 phosphorylation and elicits unfolded protein response. a The protein expressions of eIF2α in UPR signaling pathway were analysis by immnoblotting. b Phosphorylation of eIF2α^S51 was determined and normalized to the level of total eIF2α. The normalized values were then compared between siControl and siZNF322A. c Enriched consensus motifs of MAPKAPK2 on HSP27^S82 were predicted and identified in ZNF322A-silenced phosphoproteome. MS/MS spectrum for HSP27 (QIpSSGVSEIR), and fragment ions in the MS/MS spectrum localize at serine⁸² in HSP27. d Validation of HSPB1^S82 phosphorylation using immunoblot analysis. e Phosphorylation of HSP27 was determined and normalized to the level of total HSP27. The normalized values were compared between siControl and siZNF322A groups. f Schematic representation of the heat stress responses of HSP27^S82 via UPR pathway by silencing of ZNF322A in A549 lung cancer. β-actin is the internal control to normalize protein expression. The bars represent densitometric analysis of three biological replicates and the data are shown as mean ± SD. * p < 0.05

Fig. 6

Silencing of ZNF322A induces autophagosome formation in lung cancer cells. a-b Protein expression of mTOR and p-mTOR^S2448 was determined and normalized by immunoblotting. c-d Induction of autophagy related proteins including LC3B-I, LC3B-II, and SQSTM1 were analyzed and quantified by immunoblotting. Earle’s balanced salts solution (EBSS) was used as positive control to induce autophagy. e-f Electron microscopy of A549 lung cancer cells after 24 or 48 h of siControl or ZNF322A siRNA transfection was analyzed. Double- and multi-membrane autophagosomes were clearly observed in ZNF322A-silenced cells. Final stages of autophagic cell death were accompanied by ballooning of the perinuclear space and disappearance of cellular organelles such as autophagosomes, autolysosomes, and ER. Arrows: diverse autophagosomes, N: nuclear. β-actin is the internal control to normalize protein expression. The bars represent densitometric analysis of three biological replicates and the data are shown as mean ± SD. * p < 0.05; ** p < 0.01

Fig. 7

Silencing of ZNF322A was attributed to its induction of autophagy via protein phosphorylation. Silencing of ZNF322A transcriptionally regulates PIM3 to phosphorylate IRS1^S1101 and further inhibits the PI3K/AKT/mTOR pathways, limiting access of lung cancer cell to glucose. Silencing of ZNF322A induces phosphorylation of HSP27^S82 and leads to activation of the unfolded protein response (UPR). Glucose starvation and heat stress-elicited activation trigger autophagosome formation in ZNF322A-silenced A549 lung cancer cells