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. 2020 Aug 27;39(1):170.
doi: 10.1186/s13046-020-01684-x.

KIAA1522 potentiates TNFα-NFκB signaling to antagonize platinum-based chemotherapy in lung adenocarcinoma

Boshi Wang  1 , Tiantian Jing  2 , Weilin Jin  3 , Jinnan Chen  4 , Chengsi Wu  2 , Mingrong Wang  5 , Yizhen Liu  6   7
Affiliations

KIAA1522 potentiates TNFα-NFκB signaling to antagonize platinum-based chemotherapy in lung adenocarcinoma

Boshi Wang et al. J Exp Clin Cancer Res. .

Abstract

Background: The platinum-based chemotherapy is the first-line regimen for the treatment of Non-small cell lung cancer (NSCLC). However, the therapeutic efficiency is largely limited by tenacious chemo-insensitivity that results in inferior prognosis in a cohort of patients. It has been known that KIAA1522 is aberrantly expressed and implicated in several types of solid tumors including NSCLC. Nowadays, knowledge about this gene is quite limited. Here, we aimed to identify the role of KIAA1522 in lung adenocarcinomas, and the molecular events that underlie KIAA1522-mediated chemoresistance to the platinum.

Methods: Immunohistochemistry were used to detect KIAA1522 expression in clinical NSCLC samples. Then, the survival analyses were performed to assess the link between KIAA1522 expression and overall survival or therapeutic outcome. In vivo depletion of KIAA1522 in adenocarcinoma cells were achieved by adeno-associated virus-mediated sgRNA/Cre delivery into the conditional KrasG12D/Cas9 expressed mice, which were designated to identify the roles of KIAA1522 in tumorigenesis and/or chemotherapy responses. The effects of KIAA1522 and downstream molecular events were studied by pharmacology in mice model and assays using in vitro cultured cells. The clinical relevance of our findings was examined by data-mining of online datasets from multiple cohorts.

Results: The clinical evidences reveal that KIAA1522 independently predicts both the overall survival and the outcome of platinum-based chemotherapy in lung adenocarcinomas. By using a KrasG12D-driven murine lung adenocarcinoma model and performing in vitro assays, we demonstrated that KIAA1522 is a critical positive regulator of lung adenocarcinoma and a modulator of cisplatin response. KIAA1522 potentiates the TNFα-TNFR2-NFκB signaling which in turn intensifies recalcitrance to cisplatin treatment. These results were further manifested by integrative bioinformatic analyses of independent datasets, in which KIAA1522 is tightly associated with the activity of TNFα-NFκB pathway and the cisplatin-resistant gene signatures. More strikingly, overexpression of KIAA1522 counteracts the cisplatin-induced tumor growth arrest in vivo, and this effect can be remarkably diminished by the disruption of NFκB activity.

Conclusion: High expression of KIAA1522 is turned out to be an indicator of dismal effectiveness of platinum-based therapy in lung adenocarcinomas. KIAA1522 hyperactivates TNFα-NFκB signaling to facilitate resistance to platinum reagents. Targeting NFκB signaling through small molecule inhibitors may be a rational strategy to conquer chemoresistance and synergize platinum-based chemotherapy in KIAA1522 overexpressed lung adenocarcinomas.

Keywords: Chemoresistance; KIAA1522; Lung adenocarcinoma; NFκB; Platinum-based chemotherapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
KIAA1522 expression predicts platinum-based therapeutic effectiveness in lung adenocarcinomas. a Forest plot shows the hazard ratio with 95% confidence interval of each tested protein. The hazard ratios are estimated by univariate cox analyses in all NSCLC patients (blue) or the patients receiving post-surgery platinum-based chemotherapy (red). The proteins were ranked by the p-values in the chemotherapy group. b-f Immunohistochemical analyses of KIAA1522 expression in tissue samples from NSCLC patients. b Representative images of KIAA1522 immunohistochemical staining in NSCLC specimens and non-tumoral normal lung tissues. Bar = 100 μm. c Paired comparisons of KIAA1522 expression in non-tumoral and tumor samples in either lung adenocarcinomas (ADC) or squamous cell carcinomas (SCC), paired t-test, ****P < 0.0001. d In ADC and SCC patients, multivariate cox analyses of prognostic values of KIAA1522 levels excluding age, sex, stage, grade and post-surgical chemotherapy. e Kaplan–Meier curves show the overall survival of NSCLC patients expressing high/low levels of KIAA1522 protein with or without platinum-based post-surgical adjuvant chemotherapy. f Comparisons of prognostic effects of platinum-based chemotherapy in KIAA1522 high expression or low expression groups respectively
Fig. 2
Fig. 2
Genetically depletion of kiaa1522 gene impairs KrasG12D-induced lung adenocarcinoma and sensitizes tumors to cisplatin. a Schematic showing of adenovirus associated virus (AAV)-mediated kiaa1522 depletion in KrasG12D-induced lung. The Kras(LSL-G12D), LSL-Cas9 mice were intratracheal administrated by Cre- and control sgRNA or kiaa1522 sgRNAs-expressing-AAVs. Then, the mice were subjected to i.p. injections of vehicle/cisplatin. b The comparison of lung weights in different groups. t-test, **P < 0.01, ***P < 0.001. c-d Representative image of the lung after Bouin′s staining (c) and HE staining (d) in each group. e The comparison of tumor area in different groups. t-test, *P < 0.05, ****P < 0.0001. Bar = 2 mm. f Kaplan-Meier curves show the role of KIAA1522-depletion and/or effect of cisplatin on the outcome of lung cancer in mice. The survival rates were compared between each group. Log-rank test, *P < 0.05, **P < 0.01. g Immunohistochemical staining of cleaved-caspase 3 in the lung tumor samples from the indicated mice. t-test, ***P < 0.001. Bar = 100 μm
Fig. 3
Fig. 3
Depletion of KIAA1522 in lung adenocarcinoma cells reduces tumor growth and resistance to cisplatin. a Murine cell line 889 with the sgRNA-mediated depletion of KIAA1522 were injected into C57BL/6 J mice subcutaneously. One month later, the mice were sacrificed and the tumor weights were quantified. t-test, **P < 0.01. b Human adenocarcinoma cell A549 expressing KIAA1522-shRNA were injected into nude mice subcutaneously. After two months, the mice were subjected to tumor weights quantification. t-test, **P < 0.01. c Immunohistochemical staining of Ki67 in control sgRNA/Cas9 or kiaa1522-sgRNA/Cas9 expressing 889 cells. Bar = 100 μm. d IC50 of cisplatin in both 889 cells and A549 cells with down-regulated KIAA1522 were analyzed by CCK8 assays. e-f The inhibitory effects of cisplatin on 889 cells expressing kiaa1522-sgRNA/Cas9 (e) or A549 cells overexpressing KIAA1522 (f) by colony formation assays
Fig. 4
Fig. 4
KIAA1522 regulates gene expression signatures associated with TNF-NFκB pathway and cisplatin resistance. a-e RNA-sequencing experiments were performed to detect the expression profiling of control and 889 cells depleting of KIAA1522. Each group includes three replicates. a volcano plot shows the different expressed genes of KIAA1522 depleted 889 cells compared to the control cells. The genes with both |log2FC| > 2 and p-value< 0.05 were considered as significantly differentiated genes. b Enrichment analysis of KIAA1522 positively regulated genes in HALLMARK gene signatures, the significantly enriched signatures were ranked by q-values, the bar plot shows the top 20 signatures. c Gene sets enrichment analysis (GSEA) of genes by ranked by log2FC (sgkiaa1522/ sgcontrol) values using a set of TNF-NFκB pathway related signatures. d-e The GSVA score of the genesets related to the TNF-NFκB pathway (d) and experiments derived cisplatin-resistant gene signatures (e) were determined by GSVA algorithm. The heatmaps show the distribution of GSVA values between the indicated group of cells. f The patients in TCGA-LUAD cohort were classified by both KIAA1522 expression and NFκB activity into four groups and subjected to Kaplan–Meier overall survival analysis. The activity of NFκB signaling was determined by the GSVA score of the geneset JAIN_NFKB_SIGNALING. g The genes were ranked by the Pearson correlation coefficient with KIAA1522 expression in TCGA-LUAD datasets. Then, the GSEA assays were performed using a set of NFκB activity positively associated signatures to evaluate the correlation of KIAA1522 expression level with NFκB signaling activity
Fig. 5
Fig. 5
KIAA1522 positively regulates TNFα-NFκB signaling in lung adenocarcinoma cells. a Immunoblotting assays of Phospho-NFκB p65 (Ser536) in the control and KIAA1522 down-regulated cells. b-c The indicated group of A549 cells (b) or 889 cells (c) were deprived of serum for 20 h, then treated with TNFα for 5 min. Then the cells were tested by Western blotting assay to detect the phosphorylation of p65, IKK and IκB proteins. d H1299 cells overexpressing Flag tagged KIAA1522 protein were cultured in serum-free condition for 20 h, then treated by TNFα and harvest at the indicated time point. The harvested cells were subjected to Western blotting assay. e Western blotting assays show the expression of TNFR2 and TNFR1 in KIAA1522 down-regulated A549 cells. f Western blotting assays show the expression of TNFR2 and TNFR1 in KIAA1522-overexpressed cells. g Immunohistochemical staining of TNFR2, phosphorylated IKKα/β and KIAA1522 in Kras-induced murine lung tumor samples with or without KIAA1522 depletion. Bar = 100 μm
Fig. 6
Fig. 6
KIAA1522 signature correlates with TNF-NFκB-Cisplatin resistance in independent cohorts of adenocarcinomas. The KIAA1522 signature was generated by the collection of KIAA1522 down-stream genes and transformed to KIAA1522 signature score through GSVA algorithm. a Single-cell sequencing data from E-MTAB-6149 dataset was subjected to tSNE dimension reduction and clustering the NAPSA positive clusters were shown. b The KIAA1522 signature scores in KIAA1522 mRNA positive and negative adenocarcinoma single cells were compared, t-test, ****P < 0.0001. c 3D plot shows the distributions of KIAA1522 score levels according to TNFα signaling score, NFκB activity score and cisplatin resistance score. d Heatmap shows the scaled GSVA score of a collection of TNF-NFκB-Cisplatin resistance signatures in adenocarcinoma single-cells within E-MTAB-6149 dataset in parallel with KIAA1522 mRNA expression and KIAA1522 signature score. e In multi-central ADC samples, meta-analytical evaluation of the correlation between KIAA1522 signature score with TNF signaling, NFκB activity and cisplatin resistance signatures. Forest plots show the correlations together with the 95% confidence intervals, and the fixed-model estimated correlation in each geneset. f Systematical univariate cox analyses estimate the Hazard Ratio of KIAA1522 signature score in multiple datasets from TCGA or GEO databases. The meta-analysis was performed by fixed model to estimate the prognostic effect of KIAA1522 signature score. g-h In GSE14814 datasets, adenocarcinoma patients receiving platinum-based chemotherapy were grouped according to KIAA1522 signature score alone (g) or the combination of KIAA1522 signature score and NFκB activity score (h), then survival rates of each group were revealed by Kaplan–Meier survival analyses. i Alluvial diagram of KIAA1522 signature score, NFκB activity, combined KIAA1522_NFκB score and the survival outcome
Fig. 7
Fig. 7
Inhibition of NFκB signaling counteracts cisplatin insensitivity in KIAA1522 hyperactivated cells. a Inhibitory effect of QNZ on NFκB signaling in KIAA1522-overexpressed A549 cells was tested by immunoblotting assay. b The control and KIAA1522-overexpressed A549 cells were treated by indicated concentration of cisplatin with or without QNZ (500 nM). Cell viability were analyzed by CCK8 assays. c Clonal formation assays measuring the effect of QNZ on cisplatin-induced growth inhibition in the control and KIAA1522-overexpressed A549 and H1299 cells. The cells were treated with 500 nM QNZ and the indicated concentration of cisplatin for 48 h before crystal violet staining. d The control and KIAA1522 depleted 889 cells were treated by different concentrations of cisplatin with or without QNZ, then analyzed by CCK8 assays to determine cell viability. e Clonal formation assays of the control-sgRNA/Cas9 and kiaa1522-sgRNA/Cas9 expressing 889 cells treated by QNZ and cisplatin for 48 h. f the control and KIAA1522-overexpressed A549 cells were subcutaneously injected into nude mice. From 14 days after cell injection to the 50th day, the mice were treated by cisplatin (7 mg/kg B.W.) once a week and QNZ (1 mg/kg B.W.) twice a week via intraperitoneal injection. Tumor volumes in the last day were compared using t-test, **P < 0.01, ****P < 0.0001
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