FAM117A Is a New Prognostic Marker of Lung Adenocarcinoma and Predicts Sensitivity to PD0332991

Abstract

Lung cancer is the second most common cancer and the leading cause for cancer mortality worldwide. Accelerated cell cycle progression is a well-characterized hallmark for cancer. The present study aims to identify biomarkers for clinical outcomes of lung cancer patients and their sensitivity to CDK inhibitors. To this end, bioinformatics analysis of transcriptome datasets from the Cancer Genome Atlas (TCGA) was first performed to identify survival-related genes; cell proliferation assay, colony formation assay, flow cell cytometry, western blot, EDU labelling, and xenograft models were then used to confirm the potential roles of the identified factors. Our results identified the decreased FAM117A expression as the most significant survival related factor for poor outcome. The cell cycle transition from G1 to S phase was suppressed upon FAM117A overexpression and was promoted upon FAM117A knockdown. Accordingly, the tumor cell growth induced by FAM117A depletion was completely blocked by treatment with PD0332991, which has been approved for cancer therapy. In summary, our work identified FAM117A as a new prognostic marker for poor outcomes of lung cancer patients, predicting sensitivity to PD0332991 treatment.

Figure 1

FAM117A expression is decreased in lung cancer patients. (a) Kaplan–Meier overall survival curve comparing the high (50%–100%, N = 267) and low (0%–50%, N = 268) expression value of FAM117A (determined by the quantile value) for the TCGA LUAD patient cohort. (b) Kaplan–Meier overall survival curve comparing the high (50%–100%, N = 134) and low (0%–50%, N = 135) expression value of FAM117A (determined by the quantile value) for the TCGA LUAD patient cohort. (c) Dot plot of the FAM117A mRNA expression between lung cancer tissue (N = 535) and adjacent normal tissue (N = 59). (d) Dot plot of the FAM117A mRNA expression between paired lung cancer tissue (N = 59) and adjacent normal tissue (N = 59). (e) Dot plot of the FAM117A protein expression between lung cancer tissue (N = 111) and adjacent normal tissue (N = 102). (f) Dot plot of the FAM117A mRNA expression between paired lung cancer tissue (N = 102) and adjacent normal tissue (N = 102). (g) Box plot of FAM117A expression among patients of different T stages (T1: N = 175; T2: N = 289; T3: N = 49; T4: N = 19). (h) Box plot of FAM117A expression among patients of different N stages (Normal: N = 59; N0: N = 348; N1: N = 95; N2: N = 74).

Figure 2

Identification of differentially upregulated expressed genes. (a) Volcano plot of mRNA expression changes between LUAD samples harboring FAM117A high- and low- expression value. The X-axis specifies the fold-change (FC) and the Y-axis specifies the negative logarithm to the base 10 of the adjusted p values. (b) Heatmap of differentially expressed genes between LUAD samples harboring FAM117A high- and low-expression values. (c, d) KEGG Gene Ontology enrichment analysis for upregulated (c) and downregulated (d) genes, respectively. (e, f) KEGG enrichment analysis for upregulated (E) and downregulated (f) genes, respectively. (g, i) Gene set enrichment analysis between FAM117A high- and low-expression lung cancer samples. (j, k) Protein-protein interaction network and MCODE components identified in upregulated genes with low FAM117A expression.

Figure 3

Effects of FAM117A expression in lung cancer cells. (a) Images of western blot results of FAM117A expression in lung cancer cell lines (NCI-H1975 and A549) upon transfection with short hairpin RNA oligos targeting the FAM117A transcript, β-actin was used as an internal control. (b) Quantitative analysis of the grey scale of FAM117A expression by western blot. (c) Quantitative real-time RT-PCR of FAM117A expression in lung cancer cells transfected with shRNA oligo targeting its transcript. (d, e) Images of colony formation for lung cancer cells knockdown with FAM117A. (f, g) Quantitative results of the colony formation assay in lung cancer cells. (h, i) Lung cancer cell proliferation rate as confirmed by CCK-8 based assay for 3 days post seeding in 96-well plate. (j) Images of western blot results of FAM117A overexpression in lung cancer cell lines (NCI-H1975 and A549) upon transfection with Flag-FAM117A, β-actin was used as an internal control. (k, l) Lung cancer cell proliferation rate as confirmed by CCK-8 based assay for 3 days postseeding in 96-well plate. (m, o) Images of colony formation and quantitative results for lung cancer cells overexpressed with the empty vector of Flag-FAM117A.

Figure 4

FAM117A regulates the cell cycle progress of lung cancer cells. (a–c) Representative images of the cell cycle profile determined by flow cell cytometry in lung cancer cells transfected with negative control oligo or shRNA oligos targeting the FAM117A transcript. Quantitative analysis results were shown in B (NCI-H1975) and C (A549). (d–f) Representative images of the cell cycle profile determined by flow cell cytometry in lung cancer cells transfected with an empty vector or Flag-FAM117A transcript. Quantitative analysis results were shown in B (NCI-H1975) and C (A549). (g–h) Representative images of EDU labelling lung cancer cells captured by an immunofluorescence microscope. (h, i) Quantitative analysis results of western blot results of FAM117A expression in lung cancer cell lines (NCI-H1975 and A549) upon transfection with Flag-Vector or Flag-FAM117A. Cell cycle-related markers were detected as indicated, and β-actin was used as an internal control. (j, k) Quantitative analysis result of the grey scale of the western blot result.

Figure 5

PD0332991 rescued the accelerated cell growth induced by FAM117A knockdown. (a) Representative images of the cell cycle profile determined by flow cell cytometry in lung cancer cells transfected with negative control oligo or shRNA oligos targeting the FAM117A transcript or combined treated with PD0332991. (b) Quantitative analysis results of cell cycle analysis. (c, d) Representative images of EDU labelling lung cancer cells (NCI-H1975 and A549 treated with PD0332991 at 100 nM or DMSO) captured by immunofluorescence microscope. (d) Quantitative analysis results.(e, f) Images of colony formation and quantitative results for lung cancer cells (NCI-H1975 and A549 treated with PD0332991 at 100 nM.