FAM83A protein upregulation in breast cancer
FAM83A was first discovered for its ability to induce cell transformation in breast epithelial cells (6). The FAM83A locus is located on chromosome 8q24.13, which is frequently amplified in a number of human cancers (23). Analysis of FAM83A genomic aberrations in either the METABRIC or TCGA dataset revealed FAM83A locus amplification in 23% or 15% of BC, respectively (Additional File 2: Supplementary Fig. 1A). Overexpression of FAM83A protein level in Her2 positive (HER2+) BC was previously reported (8), and FAM83A mRNA upregulation in HER2+ BC was also detected in TCGA and METABRIC databases (Additional File 2: Supplementary Fig. 1B-C).
Here, we examined the expression of FAM83A in both normal breast tissues (N=411) and breast cancer (BC) cases (N=349) (Figure 1A and B, Additional File 1: Table S1 and S2). Immunohistochemical analysis revealed a 1.5-fold increase in FAM83A protein levels in breast tumors as compared with the normal breasts (p=5E-13). In our TMA analysis we observed an increase in FAM83A protein in estrogen and progesterone receptor (ER and PR) positive BC as well as in HER2+ BC as compared with normal breasts (Figure 1C, p=3E-11, p=1E-08 and p=0.002, respectively for Positivity analysis, and p=1E-11, p=1E-08, and p=0.002, respectively, for H-score analysis). FAM83A was overexpressed in early stages of BC (T1, p=1.8E-12 and T2, p=6E-07 for both Positivity and H-score analyses) more than in the later stages (T3, p=0.001 and T4, p=0.01 for Positivity; T3, p=0.001 and T4, p=0.008 for H-score, Figure 1D). No differences in FAM83A levels in relation with lymph node positivity (N) and metastatic disease (M) were observed (Additional File 2: Supplementary Fig 1D and E). Moreover, Kaplan-Meier curves indicated that the overall survival was significantly poor in high FAM83A expression BC cases as compared with the cases with low FAM83A expression (p=0.02 for positivity data; p=0.04 for H-score data; Figure 1E).
FAM83A expression increased in early phase of BC development
We recently reported FAM83A upregulation in normal breasts from women at high risk of developing breast cancer as compared with breasts from average-risk women, according to the Tyrer-Cuzick risk estimation model (5). The correlation of FAM83A protein level with the estimated risk score was confirmed by using an independent immunostaining of our large sample cohort (Additional File 2: Supplementary Fig. 2A). Then, we examined FAM83A expression in histologically normal breast tissues from either healthy women (healthy control, HC) or women who had later a diagnosis of breast cancer (susceptible normal, Susc) retrieved from the GSE141828 dataset (Figure 2) (15). Only data points with >10 reads were included into the analysis. FAM83A showed a 3.9-fold increased expression in Susc (N=4) as compared with the HC (N=8) (p=0.02) (Figure 2A). To confirm these data, we performed a transcriptomic analysis of whole breast tissue from an independent cohort of either HC or Susc (Additional File 1: Table S3). FAM83A expression showed a 1.9-fold increase in Susc (N=3) breasts as compared with HC samples (N=8) (p=0.03; Figure 2B), suggesting an upregulation of FAM83A in early phase of breast cancer development. To verify our observation, we analyzed the dataset generated by Aran et al (24) including transcriptome profiling of normal breast (from the GTEx database), normal adjacent to tumor (NAT, from the TCGA database) and breast tumors (from TCGA). FAM83A expression increased in the (NAT) compared with the normal breast (logFoldChange: 3.96, p=9.9E-20, and also compared with the tumor (logFoldChange: 2.69, p=1.0E-18) (24). Next, we examined FAM83A mRNA expression in primary epithelial cells generated from breast tissue biopsies of healthy women, and isogenic hTERT-immortalized and hTERT/RAS-transformed cells described in Kumar et al (25). FAM83A expression increased 6.2-fold in the immortalized cells (p=0.01), while only 4.3-fold in the transformed cells (p=0.03) (Additional File 2: Supplementary Fig. 2B). Both our findings and the data from Aran et al suggest that FAM83A is specifically activated in the early/intermediate phase of breast cancer development but has limited impact on the later phases of cancer progression.
FAM83A expression correlates with EGFR level
FAM83A protein has been reported as a possible key regulator in the EGFR pathway in breast cancer cells, leading to the development of resistance to tyrosine kinase inhibitors (TKIs) (6, 8). A recent report revealed a direct correlation between EGFR and FAM83A expression in NSCLC (12). We examined EGFR expression in both tumor and normal breast tissues and its correlation with FAM83A protein level (Figure 3). As previously reported (26), and in accordance with what observed in the METABRIC and TCGA cancer datasets (Additional File 2: Supplementary Fig. 3), EGFR expression is downregulated in tumors as compared with the normal breasts (2.4-fold change, p<0.0001 for positivity, 2.6-fold, p<0.0001 for H-score; Figure 3A). Nevertheless, EGFR expression in BC is directly correlated with FAM83A levels (r=0.28 for positivity and r=0.25 for H-score, both at p<0.0001) (Figure 3B).
A 1.2-fold overexpression of EGFR protein was also detected in tissues from women at high risk for BC as compared to the average-risk group (Figure 3C and D). However, the difference in EGFR expression between the two groups was significant only for the positivity analysis (p=0.01) and not for the H-score analysis (p=0.11). Pearson’s correlation analysis revealed a direct correlation between the EGFR and FAM83A expression levels in normal breast tissues (r=0.40 for positivity and r=0.33 for H-score, both at p<0.0001) (Figure 3E). Data suggest that FAM83A may be involved in the EGFR signaling in both normal and cancer cells.
FAM83A overexpression promotes metabolic activation and cell proliferation of primary breast epithelial cells
To investigate the functional role of FAM83A in normal breasts as well as in cell transformation, we employed mGFP tagged-lentivirus infection to manipulate FAM83A expression in both primary and hTERT-immortalized epithelial cells (Figure 4A, B and C). Primary breast epithelial cells were isolated as previously described (15) from the cryopreserved breast tissue cores of average-risk women, and then infected with hTERT-expressing lentivirus. We next sought to determine whether FAM83A overexpression or loss impacted proliferation capacity or survival. First, cell viability and proliferation were monitored using Sulforhodamine B and bromodeoxyuridine (BrdU) incorporation assays, respectively (Figure 4). Compared with control cells, both cell viability (up to 90% at 48h and 3.4-fold at 72h, p>0.05) and proliferation rate (up to 47%, p<0.05) of FAM83A-overexpressing primary epithelial cells were significantly increased (Figure 4D and E). However, after downregulating the expression of FAM83A in primary epithelial cells, a reduction in cell viability was detected (22% and 31%, p=0.003 and p=0.04 respectively at 48hr; 25% and 26%. p=0.03 and p=0.04 respectively at 72h), whereas no change in cell proliferation was observed (Figure 4D and E). In the immortalized cells, the overexpression of FAM83A induced a significant increase in both cell viability and proliferation as compared with the control cells (p<0.05, Figure 4F and G). In contrast, when the expression of FAM83A was downregulated the cell viability and proliferation rate of immortalized breast epithelial cells significantly decreased compared with those of control cells (p<0.05; Figure 4F and G).
Next we assessed how FAM83A overexpression impacted the expression of survival- and proliferation-related genes in primary epithelial cells (Figure 4H). Because of the limited growth rate of the shFAM83A-primary cells and limited RNA yield, the transcriptome profiling generated a small number of reads (<15,000), below the appropriate threshold, and therefore the data were not included in any further analysis. FAM83A overexpression in primary epithelial cells induced the expression of Bcl2; however, no other apoptosis-related genes were affected. Cell proliferation genes such as MKI67, PCNA, BUB1 and the cell cycle regulator CCNB1 were also induced by FAM83A overexpression, thus reflecting the increase in cell viability and proliferation observed in these cells when compared with the control cells.
Transcriptome profiling of either FAM83A- or mock (control)-overexpressing primary cells was performed followed by differential expression analysis using DeSeq2 (Additional File 1: Table S4). Upon FAM83A overexpression in primary epithelial cells, 234 genes transcripts were downregulated, whereas 125 genes were upregulated compared with the control (mock)-expressing cells (fold change:2, p<0.05). The majority of the genes affected by FAM83A overexpression were involved in cell adhesion (p=0.0003), epithelial mesenchymal transition (p=0.0003), metabolism (p=0.005) and estrogen biosynthesis (p=0.0003. The latter included downregulated genes only (Figure 5A). Molecular networks including the differentially expressed genes between FAM83A-overexpression and control and with a score >30 were the following: Cancer and endocrine system disorders (score 45), Cancer and cell morphology (score 38), and Carbohydrate metabolism (score 31) (Figure 5B).
FAM83A shows unique interaction partners in primary and immortalized epithelial cells.
To determine the mechanism of action of FAM83A in epithelial cells, protein interaction partners were also investigated. GFP-tagged FAM83A was overexpressed in either primary or hTERT-immortalized breast epithelial cells. Affinity purification with anti-GFP antibody combined with mass spectrometry (AP-MS) was used for protein–protein interactions mapping (27). Only targets associated with more than 20 peptides detected in the FAM83A overexpressing samples were considered for the analysis. Moreover, because a large number of nonspecific interactors, also contaminants, are co-purified with bait proteins and identified by MS, previously identified contaminants were selected and removed from the analysis (28, 29). In primary epithelial cells two proteins were found interacting with FAM83A: the helicase DDX3X and laminin subunit beta-3, LAMB3 (Table 1 and Additional File 1: Table S5). While the first is involved in post-transcriptional regulation (30), LAMB3 plays a role in cell growth and cell adhesion (31). In the hTERT-immortalized cells FAM83A interacts with four molecules including LIMA1, PLEC, MYL6 and MYH10, involved in cytoskeleton reorganization.