FAM83A is a potential biomarker for breast cancer initiation

Abstract

Background: Family with sequence similarity 83 member A (FAM83A) presents oncogenic properties in several cancers including breast cancer. Recently, we reported FAM83A overexpression in normal breast tissues from women at high risk of breast cancer. We now hypothesize that FAM83A is a key factor in breast cancer initiation.

Methods: Immunohistochemical staining was used to evaluate FAM83A protein levels in both a normal breast tissue microarray (TMA, N = 411) and a breast tumor TMA (N = 349). EGFR staining and its correlation with FAM83A expression were also assessed. Lentivirus-mediated manipulation of FAM83A expression in primary and hTERT-immortalized breast epithelial cells was employed. Biological and molecular alterations upon FAM83A overexpression/downregulation and FAM83A's interaction partners were investigated.

Results: TMA analysis revealed a 1.5-fold increase in FAM83A expression level in breast cancer cases as compared with normal breast tissues (p < 0.0001). FAM83A protein expression was directly correlated with EGFR level in both normal and breast cancer tissues. In in vitro assays, exogenous expression of FAM83A in either primary or immortalized breast epithelial cells promoted cell viability and proliferation. Additionally, Ingenuity Pathway Analysis (IPA) revealed that FAM83A overexpression in primary cells affected the expression of genes involved in cellular morphology and metabolism. Mass spectrometry analysis identified DDX3X and LAMB3 as potential FAM83A interaction partners in primary cells, while we detected FAM83A interaction with cytoskeleton reorganization factors, including LIMA1, MYH10, PLEC, MYL6 in the immortalized cells.

Conclusions: This study shows that FAM83A promotes metabolic activation in primary breast epithelial cells and cell proliferation in both primary and immortalized cells. These findings support its role in early breast oncogenesis.

Keywords: Breast cancer; Cell transformation; FAM83A; Normal breast.

Fig. 1

FAM83A expression in breast tumors. A Representative images at 20 X magnification and B quantification of FAM83A staining of either normal or tumor breast tissue sections. Data are shown as box and whisker at 5th-95th percentile. C FAM83A staining quantification in different breast cancer hormone receptor-based subtypes or D tumor stage (T1-T4) as compared with normal breast. Each dot represents a single subject data point. Data are shown as average ± standard error of the mean. E Kaplan–Meier survival curves of breast cancer patients based on FAM83A expression status for both positivity and H-score analyses (blue lines indicate patients with high FAM83A level; red lines indicate patients with low FAM83A level). ER: estrogen receptor, PR: progesterone receptor, Her2: HER2 gene amplification, TN: triple negative. *p < 0.05.**p < 0.001, ***p < 0.0001

Fig. 2

FAM83A expression in breast prior to cancer diagnosis. A FAM83A expression in microdissected-epithelial breast tissues from either women prior their breast cancer diagnosis (Susc) or healthy women (healthy controls, HC). Data were retrieved from GEO, GSE141828. B FAM83A expression in breast tissues from either women prior their breast cancer diagnosis (Susc) or healthy women (HC). *p < 0.05

Fig. 3

EGFR and FAM83A correlation in cancerous and normal breast. A EGFR staining quantification in normal breast (N) and Tumor (T) tissues. Staining quantification is expressed as positivity and H-score. Data are shown as mean ± standard error of the mean. B Pearson’s correlation between FAM83A and EGFR level in breast tumors. C Representative images of EGFR staining of normal breast tissues from women at either average or high risk for breast cancer. 20X magnification is shown. D Quantification of EGFR staining of normal breast expressed as positivity and H-score. E Pearson’s correlation between FAM83A and EGFR level in normal breast tissues. *p < 0.05, ***p < 0.0001

Fig. 4

FAM83A overexpression promotes cell proliferation. A Representative images of either mGFP-positive primary epithelial cells (N = 3) or paired hTERT-immortalized cells after lentiviral infection to express either control mock (CTR), FAM83A (FAM83A), pLKO control (shCTR), or shRNA for FAM83A (shFAM83A). 20 × magnification images are shown and mGFP is in green. Level of FAM83A expression in either primary (B) or immortalized (C) cells infected with either control, FAM83A-overexpression, or shFAM83A lentivirus particles. Cell viability and cell proliferation of primary (D and E) or immortalized (F and G) cells measured with SRB assay or BrdU assay, respectively. Data are shown as percentage of either cell viability or cell growth of the FAM83A overexpressing/downregulating cells versus the control cells. H Expression of cell proliferation-, apoptosis-, differentiation-, and stemness-related genes in primary epithelial cells overexpressing FAM83A (n = 3) as compared to mock-expressing cells (n = 3). Data are shown as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.001

Fig. 5

FAM83A overexpression induced metabolic pathway activation in primary epithelial cells. A Ingenuity pathway analysis (IPA) revealed the canonical pathways linked with the genes differentially expressed between FAM83A-overexpressing and control cells. Downregulated genes are in green and upregulated genes are in red. B Molecular networks linking the differentially expressed genes were obtained using IPA. Upregulated genes are shown in red while downregulated genes are in green and connecting molecules are white. The three networks shown in the figure include: Cancer and endocrine system disorders (left); Cancer and cell morphology (center); and Carbohydrate metabolism (right)