ER chaperone GRP78/BiP translocates to the nucleus under stress and acts as a transcriptional regulator

Significance Endoplasmic reticulum (ER) stress-mediated relocalization of ER chaperones to other cellular compartments allows cells to expand their functionality beyond the ER. Our finding that the ER luminal chaperone GRP78/BiP, commonly overexpressed in cancer cells, can translocate to the nucleus represents a paradigm shift about its role in regulating homeostasis and tumorigenesis. This study uncovers a molecular mechanism by which cancer cells respond to stress through nuclear translocation of GRP78/BiP, which assumes a role as a transcriptional regulator, allowing cells to adopt an invasive phenotype and impacting other pathways. Our study further suggests that GRP78/BiP inhibitors may offer a therapeutic approach to suppress EGFR in various human lung cancer cells without the limitations of targeting specific mutations.


RT-qPCR
For RNA extraction, cells were washed three times with PBS and lysed directly with 1 ml of TRI Reagent (MilliporeSigma) following the manufacturer's instructions. Two micrograms of purified RNA were reverse transcribed using qScript XLT cDNA SuperMix (Quantabio) following the manufacturer's instructions. One microliter of the resulting cDNA was used in real-time PCR (35 cycles: 30 s at 95°C, 30 s at 58°C, 45 s at 72°C). Samples were tested in triplicate using the SYBR Green Super mix (BioRad) on the Strata gene MX3000P Real-Time QPCR System (Agilent). The primers used for each gene are as follows:
Reverse transcription and real-time PCR analysis were performed as described above.

Luciferase reporter assay
HEK293AD cells were seeded on 6-well plates and transfected as described. After 24 hr, cell extracts were prepared using Dual-Luciferase® Reporter Assay System (Promega) according to manufacturer's instructions. Luciferase activity was detected by a luminometer (BMG LabTech FLUOstar OPTIMA) when Luciferase Assay Buffer (Promega) was added. All experiments were performed in triplicate.

Live cell imaging
Wildtype or NLS mutant GRP78-GFP transfected HEK293AD cells were cultured with 10 μM Hoechst 33342 solution (Thermo-Fisher Scientific, 62249) for 1 hr at 37°C, and then imaged in phenol red-free RPMI-1640 media supplemented with 10 mM HEPES, 5 mM glutamine and 10% FBS. Live cells images were acquired with the Leica SP8 LIGHTNING Confocal Microscope with 63× oil objective.

Subcellular fractionation
H1838 cells were seeded at a density of 5 × 10 6 cells in a 10 cm dish 24 hr before collection in 500 µl cell fractionation buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 2 mM MgCl2, 2 mM EDTA, 1 mM dithiothreitol (DTT) and protease inhibitor). 100 µl was taken as whole cell lysate. The remaining cells were passed ten times through a 27-gauge needle. Nuclei and cytoplasm were separated by centrifugation at 750g for 5 min. The supernatant was taken as cytoplasmic fraction and the nuclear pellet washed three times with cell fractionation buffer. Cold lysis buffer (0.5% NP-40, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 10% glycerol and 1 mM dithiothreitol) was added to all fractions. Complete lysis was achieved by freezing and thawing.
Lysates were spun down at 14,000g for 20 min to remove insoluble components and sodium dodecyl sulfate (SDS) loading buffer was added before Western blot analysis.

GRP78 protein refolding assay
HEK293AD cells were seeded on 6-well plate and allowed to attach overnight. Next day, the cells were transfected with Renilla Luciferase expressing construct and pcDNA3 empty vector or vectors expressing the F-78(WT), F-78(NLS Mut), or F-78(G227D). Cell lysates were collected 48 hr after transfection and subjected to heat-shock at 40°C for 1 hr. Renilla Luciferase activity was measure at 1-hr intervals for the next 4 hr. All assays were performed in the presence of an ATP-regenerating system (3 mM phosphoenol pyruvate, 20 μg/ml pyruvate kinase, 2 mM ATP).

Luciferase activity was detected by a luminometer (BMG LabTech FLUOstar OPTIMA) when
Luciferase Assay Buffer (Promega) was added. All experiments were performed in triplicate.

Co-immunoprecipitation
HEK293AD cells were transfected with ID2-Myc-FLAG or co-transfected with ID2-Myc-FLAG and F-78 constructs. After 24 hr of transfection, cells were lysed by sonication in IP buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1% Triton X-100) supplemented with protease inhibitor cocktail (Roche). Cell lysates were centrifuged at 13,000 RPM for 30 minutes at 4°C and the clarified supernatant was saved. Immunoprecipitation was carried out by incubating the supernatants with anti-FLAG antibody or anti-Myc antibody (Thermo-Fisher Scientific, 9E10) for 1 hr at room temperature, followed by incubation with protein A agarose magnetic beads (Thermo-Fisher Scientific) at 4°C overnight. The beads were wash three times with IP buffer, and the immunoprecipitated proteins were eluted in 2X SDS sample buffer and detected by Western blot.

RNA-Seq analysis
H1975 cells were seeded on 6cm dishes and allowed to attached overnight. Next day, the cells were transfected with si78(3'UTR) for 16 hr. Cells were then transfected with F-78(WT) or F-78(NLS Mut) for 48 hr. Total RNA was extracted by TRI Reagent (MilliporeSigma). RNA purity was measured by the NanoDrop 1000 (Thermo Fisher Scientific). Library preparation and RNA-Seq were performed by Novogene (Sacramento, CA) using an Illumina HiSeq 2500 System (2x150bp configuration, single index, per lane).

Bioinformatics analysis
Reads were mapped to the full human genome (GrCh38.p14) using STAR (v 2.7.9a) to generate gene counts. Counts were passed into DESeq2 (v 1.36.0) to perform differential expression analysis, removing genes with fewer than 10 counts. Heatmaps were generated using the pheatmap package (v 1.0.12). Gene ontology analysis of enriched pathways of differentially expressed genes was performed using clusterProfiler (v 4.6.0).

Cell invasion transwell assay
Transwell invasion assay was performed using the BD BioCoat Matrigel invasion chambers (BD Biosciences, #354480), according to the manufacturer's protocol. H1975 cells were seeded on 6well plate and allowed to attached overnight. Next day, the cells were transfected with si78(3'UTR)