Targeting HSPA1A in ARID2-deficient lung adenocarcinoma

Abstract Somatic mutations of the chromatin remodeling gene ARID2 are observed in ∼7% of human lung adenocarcinomas (LUADs). However, the role of ARID2 in the pathogenesis of LUADs remains largely unknown. Here we find that ARID2 expression is decreased during the malignant progression of both human and mice LUADs. Using two KrasG12D-based genetically engineered murine models, we demonstrate that ARID2 knockout significantly promotes lung cancer malignant progression and shortens overall survival. Consistently, ARID2 knockdown significantly promotes cell proliferation in human and mice lung cancer cells. Through integrative analyses of ChIP-Seq and RNA-Seq data, we find that Hspa1a is up-regulated by Arid2 loss. Knockdown of Hspa1a specifically inhibits malignant progression of Arid2-deficient but not Arid2-wt lung cancers in both cell lines as well as animal models. Treatment with an HSPA1A inhibitor could significantly inhibit the malignant progression of lung cancer with ARID2 deficiency. Together, our findings establish ARID2 as an important tumor suppressor in LUADs with novel mechanistic insights, and further identify HSPA1A as a potential therapeutic target in ARID2-deficient LUADs.

into two groups treated with 200 mg/kg KNK437 or with equal volume of oil daily via intra-peritoneal injection.

Gene knockdown and overexpression
For gene knockdown study, lentivirus-mediated delivery of shRNAs targeting ARID2 and HSPA1A were performed as described previously (2). The shRNAs were packaged in lentiviral particles by co-transfection with packaging plasmids into HEK-293T cells and the filtered cell culture supernatant was then used to infect cells.
The shRNA sequences are listed in the Table S2.
For gene overexpression study, the pCDH vector containing the target gene and packaging plasmids were introduced into HEK-293T cells for the production of lentivirus. The filtered cell culture supernatant was then used to infect target cells.
The transfected cells were expanded for follow-up experiments.

Transwell migration assay
For cell migration, 1 × 10 5 cells were plated onto Transwell filters with 8-mm pores, a 24-well plate chamber insert (Corning, NY, USA) The top of the insert was supplemented with serum-free medium, while the bottom was supplemented with DMEM with 8% FBS. Cells were incubated for 12 hours and fixed with 4% paraformaldehyde for 15 minutes. After washing with PBS, cells at the top of the insert were scraped with a cotton swab. Cells adherent to the bottom were stained with hematoxylin for 1 minute and then washed 3 times with double-distilled H 2 O.
The positively stained cells on the underside of the filters were photographed and examined under the microscope.

ChIP-seq and ChIP-qPCR
Cells were cross-linked with 1% formaldehyde for 5 min at room temperature, lysed by SDS Lysis buffer and sonicated to generate DNA fragments with an average size of 500 bp. After pre-clearing with Protein G beads, antibodies against ARID2 (SC 98299X, Santa Cruz), RNA polymerase II (SC 900, Santa Cruz) or control IgG was added to cell lysate and incubated at 4℃ overnight. DNA crosslinked with ARID2 was pulled down with Protein G beads, washed and purified with minElute PCR purification kit (NO. 28004, QIANGEN, Duesseldorf, Germany). Aliquots of ChIP-enriched DNA and whole-cell lysate DNA were subjected to sequencing and qPCR analyses.

RNA sequencing and data analysis
Arid2 loss and control RNA samples were obtained from both K and KL mice tumors.
The numbers of replicates were one and three for K and KL backgrounds, respectively.
The library preparation and sequencing were performed according to the standard Illumina RNA-seq protocol (NovaSeq 6000, Berry Genomics, Inc.). Raw sequencing reads were first quality filtered with the following criteria: N% > 3%, or fraction of low quality bases (< 20) above 50%, or matching of known adapter sequences (>= 8bp) in any reads. RNA-seq reads were mapped to the current mouse genome (mm10) with the STAR aligner (v2.6.0c) and counted with featureCounts (v1.6.4) for each gene. Reads per million (RPM) values after log2 transformation were used for downstream analysis. For K background data with a single replicate, log2 fold changes was used to determine differential expression. For KL background data with three replicates, differential expression was evaluated with the Student's t test (two-sided). Multiple testing P values were then corrected with the widely used 'q value' method (v2.10.0) (5).

ChIP-seq data analysis
ChIP-seq reads were aligned to the mm10 genome with bwa (v0.7.15). The uniquely and properly mapped read pairs were retained after removal of duplicated reads.

Human LUAD dataset analysis
Survival analysis of the full spectrum of patients with LUAD was performed based on ARID2 expression status using the online Kaplan Meier-plotter analysis tool (8). We performed survival analysis using the Affymetrix ID 225486_at for ARID2 gene symbol. The median value was chosen as the cut-off for high and low ARID2 6 expression. The TCGA-LUAD dataset was employed to screen genes with an increased risk on patient survival (HR>1 and FDR<0.1).
Tumor samples with ARID2 mutations including both point mutations and fragment deletions were compared with other ARID2 wild-type samples for differential expression gene analysis. Since the expression of downstream genes could be affected by other mechanisms such as copy number variations (9), we restricted our analysis on samples with a neutral copy number of these downstream genes. For HSPA1A, 257 lung adenocarcinoma samples are with a neutral copy number, among which 221 are ARID2 wild-type and 36 are ARID2 mutant. To evaluate differential expression, Wilcoxon rank sum test was used with a P value threshold at 0.05.

Human lung cancer specimen analysis
The study cohort containing 63 LUAD tumor samples from Shanghai Cancer Center of Fudan University was collected and used for immunostaining of ARID2 and HSPA1A for correlation analysis. Patients were excluded from the study cohort with the following exclusion criteria: previously received any anticancer therapy; impaired heart, lung, liver or kidney function; previous malignant disease. All