FAM83A and FAM83B as Prognostic Biomarkers and Potential New Therapeutic Targets in NSCLC

Abstract

Although targeted therapy has improved the survival rates in the last decade, non-small-cell lung cancer (NSCLC) is still the most common cause of cancer-related death. The challenge of identifying new targets for further effective therapies still remains. The FAMily with sequence similarity 83 (FAM83) members have recently been described as novel oncogenes in numerous human cancer specimens and shown to be involved in epidermal growth factor receptor (EGFR) signaling. Here, gene expression of FAM83A and B was analyzed in a cohort of 362 NSCLC patients using qPCR. We further investigated relations in expression and their prognostic value. Functional assays in NSCLC cell lines were performed to evaluate FAM83A and B involvement in proliferation, anchorage-independent growth, migration, and the EGFR pathway. We observed a highly increased gene expression level of FAM83A (ø = 68-fold) and FAM83B (ø = 20-fold) which resulted in poor survival prognosis (p < 0.0001 and p = 0.002). Their expression was influenced by EGFR levels, pathway signaling, and mutation status. Both genes affected cell proliferation, and FAM83A depletion resulted in reduced migration and anchorage-independent growth. The results support the hypothesis that FAM83A and B have different functions in different histological subtypes of NSCLC and might be new therapeutic targets.

Keywords: EGFR-TKI; FAM83A; FAM83B; NSCLC; biomarker.

Figure 1

The FAMily with sequence similarity 83 (FAM83) genes FAM83A and FAM83B are highly overexpressed in non-small-cell lung cancer (NSCLC). The waterfall plots depict the x-fold change expression of FAM83A and FAM83B in tumor vs. normal tissue. (A) FAM83A expression in all patient samples, (B) separated by histology, (C) and furthermore by gender; (D–F) corresponding plots for FAM83B. Equal expression of the genes in tumor and adjacent non-neoplastic tissue was set to 1 (dotted line); Ø = median overexpression.

Figure 2

FAM83A and FAM83B expression has a prognostic value in NSCLC patients. Effect of (A) FAM83A and (B) FAM83B tumor expression on the overall survival of all patients, (C,D) of patients with ADC and (E,F) SQCC, and (G,H) of female and (I,J) male patients.; p < 0.05 was considered significant.

Figure 3

Relation between FAM83A and B expression and EGFR status and expression. (A) Relative expression of FAM83A in patients with wild-type EGFR and mutational variants normalized to the housekeeper genes ESD and RPS18. As relative expression is depicted (∆C_t), lower values correspond to a higher expression. (B) Correlation plots between FAM83A and EGFR expression in the tumor tissue. (C,D) represent the respective results for FAM83B. (E) Absolute copy number of FAM83A, FAM83B, and EGFR in ADC and SQCC cell lines. The correlations were estimated using the Spearman’s rank correlation coefficient. For statistical analysis, Mann–Whitney U test was performed (** p < 0.005, ns = not significant).

Figure 4

Investigation of the role of FAM83A and B in NSCLC cell lines. (A) FAM83A depletion efficiency. (B–D) Effect of decreased gene expression in ADC cell lines on (B) proliferation, (C) migration, and (D) ability of anchorage-independent growth (AIG). Relative cell or spheroid number of cells after FAM83A knockdown (kd) compared to control (c). (E–H) Corresponding results for FAM83B. At least three independent biological replicates were used. Significance was determined using the unpaired t-test (*** p < 0.0001, ** p < 0.005, * p < 0.05).

Figure 5

Relation between FAM83A and B and the EGFR pathway in ADC cell lines. (A) Gene expression of FAM83A and B after treatment with the tyrosine kinase inhibitors (TKIs) AZD9291 and erlotinib in H1975 and (B) in HCC827. The TKIs were applied at concentrations of 0.1 µM, 1 µM, and 10 µM for 24 h. (C) Effect of TKI treatment on cell viability in H1975 and (D) HCC827 cells. (E) siRNA-mediated depletion of FAM83A and B and its impact on cell proliferation in H1975 and (F) HCC827 (Relative cell number after FAM83 knockdown (kd) compared to control (c)). (G) Influence of FAM83A and B depletion on TKI treatment in H1975 and (H) HCC827 cells. The dotted line at 1 represents either the expression (A,B,E,F,) or the cell number (C,D,G,H) in control-treated cells. At least three independent biological replicates were used. Significance of expression and cell viability variation were estimated using unpaired t-test (*** p < 0.0001; ** p < 0.005; * p < 0.05).