A prognostic model of drug tolerant persister-related genes in lung adenocarcinoma based on single cell and bulk RNA sequencing data

Background: Acquired resistance to targeted drugs is a major challenge in cancer. The drug-tolerant state has been proposed to be an initial step towards acquisition of real drug-resistance. Drug tolerant persister (DTP) cells are purported to survive during treatment and stay dormant for several years. Single cell sequencing can provide a comprehensive landscape of gene expression in DTP cells, which can facilitate investigation of heterogeneity of a drug tolerant state and identification of new anticancer targets. Methods: The genetic profiling of DTPs was explored by integrating Gene Expression Omnibus (GEO) datasets, and a prognostic signature of DTP-related genes (DTPRGs) in lung adenocarcinoma of TCGA LUAD cohort was constructed. The scores of infiltrating immune cells were calculated and activity of immune-related pathways was evaluated by single-sample gene set enrichment analysis (ssGSEA). Functional enrichment analysis of the DTPRGs between low- and high-risk groups was performed. Immune cell subtypes and immune-related pathways were analyzed. Results: An 11-gene panel (MT2A, UBE2S, CLTB, KRT7, IGFBP3, CTSH, NPC2, HMGA1, HNRNPAB, DTYMK, and IHNA) was established. DTPRGs were mainly correlated with nuclear division, chromosome segregation, and cell cycle pathways. Infiltration of immune cells was lower in the high-risk group while the inflammation-promoting and MCH-class I response pathway had higher activity in the high-risk group. A nomogram was generated with prognostic accuracy, further validated using clinical outcomes following therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). Discussion: A prognostic model of lung adenocarcinoma based on DTPRGs was constructed. Targeting DTP cells is a potential therapeutic approach to prevent a drug tolerant state

Progression of cells through the cell cycle following flow cytometry analysis.

<p>The graph shows the percentage of Mero-14 cells in phase S+G2+M treated with 40 nM of the siCtrl or siMSLN-1. The S+G2+M phase of the cell cycle was slightly reduced following the treatment with siMSLN-1 (*P =  0.033). Error bars represent SEM of six independent experiments.</p

Role of MSLN in cellular migration and invasion.

<p><b><i>A</i>.</b> No effects observed in the wound-healing assay, following siRNA transfections. Confluent monolayers of Mero-14 cells transfected with 40 nM of siCtrl, or siMSLN-1, respectively. Two different experiments were carried out, each performed in triplicate. <b><i>B</i></b><b>.</b> Trans-well cell invasion assay on Mero-14 cells transfected with 40 nM of the siCtrl (top), or siMSLN-1 (bottom). Pictures were taken using a fluorescence microscope at 10X magnification and are reported as negative of the originals to enhance the contrast between the background and the DAPI-stained cells. The bar chart shows the average of invasive cells (error bars represent SEM of two independent experiments, each done in triplicate, *P  =  0.0044).</p

Expression levels of <i>MSLN</i> in human MPM cell lines and Met5A.

<p><b><i>A.</i></b> RT-qPCR showing the mRNA expression levels of <i>MSLN</i> measured on MPM cell lines and related to Met5A cells (set to 1). <i>RPLP0</i>, <i>HPRT</i>, and <i>TBP</i> were used for normalization. Error bars show the standard error of the mean (SEM) from three independent experiments, each performed in triplicate. Mero-14 cells showed the highest expression levels of <i>MSLN</i> (P = 0.02). <b><i>B.</i></b> The protein levels of MSLN in Met5A, Mero-14, IstMes2, and NCI-H28 cells. β-actin was used as reference. The protein levels were confirmed by two independent experiments. MSLN is shown as a band at 40 kDa. <b><i>C.</i></b> RT-qPCR showing the endogenous mRNA expression levels of <i>MSLN</i> in Mero-14 cells, related to their own siCtrl (set to 1). <i>RPLP0</i>, <i>HPRT</i>, and <i>TBP</i> were used for normalization. Error bars are SEM, from three independent experiments, each performed in triplicate. The siRNA chosen for the analysis is: siMSLN-1 (40 nM; *P = 0.002) active on Mero-14 cells. <b><i>D.</i></b> Protein levels of MSLN (shown as a band at 40 kDa) after depletion with siMSLN-1 and -2 (40 nM). β-actin was used as reference. The protein levels were confirmed by three independent experiments.</p

Role of MSLN in cellular growth, cell cycle progression and apoptosis, following treatment with chemotherapeutic drugs.

<p><i>A</i>. Proliferation assay in Mero-14 cells. The graph shows the effect of the treatments with 5 µM cisplatin and 40 nM siMSLN-1, used as single agents or in combination. On day 6, MANOVA shows a statistically significant effect both for cisplatin (P = 0.0168) and siMSLN-1 (P<10⁻⁴) in reducing proliferation. However, the interaction term for the effect of both agents in combination is not statistically significant (P = 0.145). Error bars represent SEM of three independent experiments, each performed in quadruplicate. <i>B</i>. Flow cytometry analysis. The graph shows the percentage of cells in phase S+G2+M in Mero-14 cells treated with 40 nM of the siCtrl or siMSLN-1 in combination with imatinib (25 µM) or gemcitabine (1 µM) (alone) or imatinib+gemcitabine (10 µM and 1 µM, respectively). The transfection with siMSLN-1 was accompanied with a marked decrease of cells in S+G2+M phase, as compared with the respective cultures transfected with siCtrl, irrespectively of the drugs employed (P =  0.00033). Error bars represent SEM of two independent experiments. <i>C</i>. Caspase activity measured on Mero-14 cells transfected with 40 nM of siCtrl, or siMSLN-1, with or without cisplatin 5 µM. A marked increase in apoptosis is observed when siMSLN-1 and cisplatin are administered together, compared to cultures treated with cisplatin and transfected with siCtrl (*P = 0.018), suggesting a synergistic effect. Error bars represent SEM of three independent experiments, each performed in triplicate. <i>D</i>. Western blotting analysis of MSLN, p53, and PARP under different combinations of siRNAs and cisplatin (at 5, 10 and 20 µM). β-actin was used as reference. The protein levels were confirmed with three independent experiments. Legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085935#pone-0085935-g005" target="_blank">figure 5:</a> Dark line: cells trated with siCtrl; gray line and triangles: cells treated with siCtrl plus cisplatin; gray line and dark spots: cells treated with siMSLN plus cisplatin; dark line and white spots: cells treated with siMSLN-1.</p

RT-qPCR of selected miRNAs performed on fresh tissues.

<p>(A) <i>miR-21</i>, (B) <i>miR-126</i> and (C) <i>miR-16</i> were measured using RT-qPCR in order to compare PDAC to normal pancreas for the miRNAs of interest. <i>MiR-21</i> is overexpressed in PDAC compared to normal pancreas tissue (*** <i>P</i><0.001). <i>MiR-126</i> and <i>miR-16</i> expression levels were not significantly different between PDAC and normal pancreas tissue. Samples included: normal pancreas (n = 9) and PDAC (n = 15). Box and Whiskers indicate median, minimum and maximum.</p

RT-qPCR confirmed <i>miR-21</i> overexpression in PDAC and SMCA compared to normal pancreas.

<p>This suggests that <i>miR-21</i> overexpression may be an early event in the formation of pancreatic BCT from normal pancreas. <i>MiR-21</i> was unable to differentiate PDAC from SMCA and therefore it may be questionable as a future biomarker of PDAC. RNA was isolated from FFPE samples for all 3 tissue types. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032068#s3" target="_blank">Results</a> presented as mean±SEM).</p

A subset of miRNAs are down-regulated in PDAC compared to Benign Cystic Tumors (BCT).

<p>(A) Hierarchical Clustering Heatmap was created to detect possible clusters in rows (transcripts) and columns (samples) of the normalized expression matrix. For this analysis we used the 35 miRNAs with highest overall variability. As the heatmap, with its dendrogram on top and the contingency table at the bottom, shows we detect three clusters indicated by the solid blue lines. The first cluster consists of 80% PDAC, 20% CEI and no BCT samples and thus contains predominantly PDAC samples. The second cluster contains 41% PDAC, 41% BCT and 18% CEI samples. Subdividing it into two additional clusters, as indicated by the dashed blue lines, we see that the left part consists predominantly of CEI, while the right part entails a slight enrichment for BCT samples. Finally, the third cluster contains 14% PDAC, 24% CEI and 62% BCT samples, thus consists predominantly of BCT samples (<i>P</i> = 0.034). (Red indicates high intensity; green indicates low intensity; PDAC, Pancreatic Ductal Adenocarcinoma; CEI, Carcinoma-Ex-IPMN; BCT, Benign-Cystic-Tumors). (B) <i>miR-21</i> (C) <i>miR-126</i> and (D) <i>miR-16</i> were measured using RT-qPCR, performed on the 43 FFPE tissues in order to validate the microarray data. Samples included: SMCA (n = 7), MCN (n = 6), IPMN (n = 7), and CEI (n = 9) and PDAC (n = 14). (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032068#s3" target="_blank">Results</a> presented as mean±SEM; *** <i>P</i> = 0.003, ** <i>P</i> = 0.02 and * <i>P</i> = 0.05 respectively).</p

KRAS is experimentally validated as a direct target of <i>miR-126</i> in pancreatic cancer cells.

<p>(A) Putative <i>miR-126</i> binding sequences in the 3′-UTR of KRAS mRNA. Two different fragments from the 3′-UTR region of KRAS were cloned downstream of the luciferase reporters and named as wild-type (KRAS_A_WT and KRAS_B_WT). Two mutated versions of the <i>miR-126</i> binding site were also generated (KRAS_A_MUT and KRAS_B_MUT); the mutated nucleotides of the <i>miR-126</i> binding site are underlined. Boxed areas represent conserved complementary nucleotides of the <i>miR-126</i> seed sequence in various species (Hsa, human; Ptr, chimpanzee; Mml, rhesus; Mmu, mouse; Laf, elephant; Gga, chicken). *indicates that KRAS_B_WT is conserved in 16 species. (B) Luciferase reporter assay. Each of the 4 plasmids (150 ng) and a <i>Renilla</i> luciferase reporter (50 ng) were co-transfected into MIA PaCa-2 cells with precursor miR-126 (100 nM). Luciferase activity was assayed 48 hours after transfection. All experiments were independently repeated at least three times; the results are presented as mean±SD (**<i>P</i><0.01).</p

Flow chart of miRNA expression profiling in pancreatic BCTs and miRNA target acquisition.

<p>(FFPE, formalin-fixed paraffin embedded tissue; **indicates that RNA could not be isolated from 1 sample; RT- qPCR, quantitative reverse transcription polymerase chain reaction).</p