Re-expression of epigenetically silenced PTPRR by histone acetylation sensitizes RAS-mutant lung adenocarcinoma to SHP2 inhibition

Abstract

Silenced protein tyrosine phosphatase receptor type R (PTPRR) participates in mitogen-activated protein kinase (MAPK) signaling cascades during the genesis and development of tumors. Rat sarcoma virus (Ras) genes are frequently mutated in lung adenocarcinoma, thereby resulting in hyperactivation of downstream MAPK signaling. However, the molecular mechanism manipulating the regulation and function of PTPRR in RAS-mutant lung adenocarcinoma is not known. Patient records collected from the Cancer Genome Atlas and Gene Expression Omnibus showed that silenced PTPRR was positively correlated with the prognosis. Exogenous expression of PTPRR suppressed the proliferation and migration of lung cancer cells. PTPRR expression and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibition acted synergistically to control ERK1/2 phosphorylation in RAS-driven lung cancer cells. Chromatin immunoprecipitation assay revealed that HDAC inhibition induced enriched histone acetylation in the promoter region of PTPRR and recovered PTPRR transcription. The combination of the HDAC inhibitor SAHA and SHP2 inhibitor SHP099 suppressed the progression of lung cancer markedly in vitro and in vivo. Therefore, we revealed the epigenetic silencing mechanism of PTPRR and demonstrated that combination therapy targeting HDAC and SHP2 might represent a novel strategy to treat RAS-mutant lung cancer.

Keywords: Anti-cancer drugs; Drug combination; Epigenetics; Signaling pathway; Targeted therapy.

Fig. 1

PTPRR was underexpressed in lung cancer. a Diminished expression of PTPRR was found in lung adenocarcinoma tissues (n = 57) versus adjacent normal lung tissues (n = 57) by TMNplot. b Tissue sections documented on the Human Protein Atlas indicated low expression of PTPRR in malignant lung carcinoma. Scale bar, 200 μm c PTPRR expression in the immortalized human normal lung cell line BEAS-2B and three lung cancer-derived cell lines were examined by immunoblotting. d Kaplan–Meier survival analysis of GSE30219 data set from GEO showed positive correlation of PTPRR (Affymetrix ID: 206084_at) expression with overall survival. e Growth curve of NCI-H1299 cells and NCI-H2087 cells infected with pCDH or pCDH-PTPRR lentivirus. f and g Motility and migration capabilities of PTPRR-expressing cells were assessed by wound-healing (f) and Transwell™ assays (g). Scale bar, 100 μm. Data are shown as mean ± SD. n = 3, two-tailed unpaired Student’s t-test was used for statistical analysis. *p < 0.05 and ***p < 0.001

Fig. 2

ERK1/2 activation in lung cancer cells was regulated by PTPRR and SHP2. a PTPRR inhibited ERK1/2 phosphorylation in NCI-H1299 cells and NCI-H2087 cells, but not in A549 cells or NCI-H358 cells. b Fra1 (downstream target of the MAPK pathway) expression was reduced by PTPRR expression in NCI-H1299 cells and NCI-H2087 cells. c ERK1/2 phosphorylation in lung cancer cells after SHP099 (50 μM) treatment for 24 h. d PTPRR downregulated ERK1/2 activation in SHP2-deficient cells significantly. e Western immunoblots in PTPRR-expressing NCI-H1299 cells and A549 cells treated with SHP099 (50 μM). f PTPRR-expressing NCI-H1299 cells and A549 cells were subjected to viability assessment with increasing concentrations of SHP099 for 72 h. Data are shown as mean ± SD. n = 3, two-tailed unpaired Student’s t-test was used for statistical analysis. *p < 0.05, **p < 0.01, and ***p < 0.001. n.s., no significance

Fig. 3

Upregulated H3K9ac level in the promoter region initiates PTPRR transcription. a DNMT3B expression in NCI-H1299 cells was knocked down using the lentiviral vector pKO.1 expressing shDNMT3B. The change in PTPRR mRNA expression was measured by RT-qPCR. b NCI-H1299 cells were treated with decitabine for 24 h and the transcription of PTPRR was measured. c NCI-H1299 cells were infected by lentivirus containing the empty pLKO.1 vector or pLKO.1-shHDAC1, HDAC2, and HDAC3. d Lung cancer cells were treated with SAHA (0, 2.5, 5, 10, or 20 μM) or TSA (2 μM) for 12 h and the acetylation level of histone H3K9 was determined. e After treating NCI-H1299 cells and NCI-H2087 cells with TSA (2 μM) for 12 h, PTPRR expression was detected by real-time RT-qPCR. f Expression of PTPRR mRNA was detected in SAHA (0, 10, or 20 μM)-treated lung cancer cells. g ChIP-qPCR of H3K9ac enrichment around the promoter region of PTPRR in SAHA-treated NCI-H1299 cells. h Higher acetylation level of histone H3K9 was found in BEAS-2B cells than NCI-H1299, NCI-H2087, and A549 cells. Data are shown as mean ± SD. n = 3, two-tailed unpaired Student’s t-test was used for statistical analysis. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 4

HDAC inhibition restrains ERK1/2 signaling. a Lentivirus packaging empty pLKO.1 vector or shRNAs targeting HDAC1, HDAC2, and HDAC3 were used to infect NCI-H1299 cells. b Lung cancer cells were treated with SAHA (0, 5, 10, or 20 μM) for 24 h. c NCI-1299 cells were treated with SAHA (10 μM) for different times to assess expression of PTPRR and ERK1/2 phosphorylation. d NCI-H1299 and NCI-H2087 cells were treated with SAHA for 12 h and subjected to real-time RT-qPCR to determine fra-1 mRNA expression. ***p < 0.001. n.s., no significance

Fig. 5

SAHA treatment sensitizes RAS-mutant lung cancer cells to SHP2 inhibition. a Lentivirus containing shRNAs targeting PTPRR were used to infect NCI-H1299 cells with or without SAHA treatment (10 μM). b siRNAs were transfected to NCI-H1299 cells and A549 cells for 48 h, SAHA (10 μM) was added and incubated for 12 h. c Silencing of SHP2 expression in NCI-H1299 and A549 cells in combination with SAHA treatment (10 μM) restrain cell proliferation. Relative cell growth was determined by crystal violet staining and solubilized in 10% acetic acid. Data are shown as mean ± SD. n = 3, two-tailed unpaired Student’s t-test was used for statistical analysis. **p < 0.01 and ***p < 0.001

Fig. 6

Combination therapy of SAHA and SHP099 strongly impairs the proliferation of lung cancer cells. a Increasing concentrations of SHP099 plus SAHA (1.25 μM) were added to a panel of RAS-mutant lung cancer cells. Cell viabilities were assessed by Cell-titer Glo at 72 h post-treatment. b Fraction affected (Fa)-combination index (CI) plots were obtained by determining the combination of a series concentrations of SAHA and SHP099. c Single and combination treatment of SAHA (10 μM) and SHP099 (25 μM) were added to four RAS-mutant lung cancer cell lines and western immunoblots obtained. d EdU staining of NCI-H1299 cells and A549 cells treated with SAHA (10 μM), SHP099 (50 μM), or their combination for 24 h. Scale bar, 100 μm. e NCI-H1299 cells and A549 cells were treated with SAHA (1.25 μM), SHP099 (50 μM for NCI-H1299, 10 μM for A549) or their combinations for 7 days and subjected to crystal-violet staining. Data are the mean ± SD. n = 3, two-tailed unpaired Student’s t-test was used for statistical analysis. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 7

Dual inhibition of HDAC and SHP2 reduced the tumor burden in RAS-mutant NCI-H1299 and A549 lung cancer xenografts. a NCI-H1299 cells expressing empty pCDH or ectopic PTPRR were mixed with Matrigel™ and transplanted to the left flank and right flank of male Balb/c nude mice, respectively. b Tumor volumes of NCI-H1299 pCDH and PTPRR were tracked for 30 days (n = 4). c Tumor weight of xenografts were obtained at the endpoint (n = 4). d Representative image of NCI-H1299 pCDH and PTPRR tumor xenografts (n = 4). e Tumor volume of NCI-H1299 xenografts treated with SAHA (75 mg/kg, p.o.), SHP099 (75 mg/kg, p.o.), or their combination (n = 5). f Tumor weight of NCI-H1299 xenografts (n = 5). g Representative image of NCI-H1299 tumor xenografts (n = 5). h Body weight change of mice bearing NCI-H1299 xenografts (n = 5). i Tumor volume of A549 xenografts treated with SAHA (75 mg/kg, p.o.), SHP099 (75 mg/kg, p.o.), or their combination (n = 5). j Tumor weight of A549 xenografts (n = 5). k Representative image of A549 tumor xenografts (n = 5). l Body weight change of mice bearing A549 xenografts (n = 5). m and n Expression of phosphorylated ERK1/2 and total ERK1/2 in NCI-H1299 (m) and A549 (n) tumor lysates was measured by western blotting. Data are presented as the mean ± SEM. One-way ANOVA was used for statistical analysis. *p < 0.05, **p < 0.01, and ***p < 0.001. n.s., no significance

Fig. 8

Graphical summary of suppressing RAS-MAPK pathway by regulating dysfunctional phosphatase PTPRR and SHP2. Histone acetylation facilitates PTPRR expression in lung adenocarcinoma. A combination of the HDAC inhibitor (SAHA) and SHP2 inhibitor (SHP099) suppressed RAS-mutant lung cancer progression markedly through regulation of ERK1/2 signaling. The figure was created with BioRender.com