The emerging role of FAM46C as a biomarker and therapeutic target in gastric adenocarcinoma

Abstract

On a global scale, gastric adenocarcinoma (GCa) accounts for a large burden of death from cancer. Despite advances in systemic therapy and surgical technique, the fatality rate for GCa remains unacceptably high in Europe and North America, where diagnosis is typically made at an advanced stage. Biomarkers that can accurately predict response to new therapies and provide novel therapeutic strategies are urgently sought. FAM46C, a putative noncanonical nucleotidyltransferase, has garnered interest for its tumor suppressor function in multiple myeloma. A frequent and profound depletion of FAM46C has been described in GCa patients from China, Japan and now Canada. Furthermore, the degree of FAM46C depletion meaningfully portends cancer recurrence following resection, and death from GCa. In this review, we provide an updated summary of the literature regarding FAM46C as a biomarker in GCa and explore the potential mechanism(s) through which FAM46C depletion promotes GCa progression, including dis-inhibition of oncogenic Plk4 kinase activity. We highlight the potential for restoration of FAM46C levels as a therapeutic strategy. Norcantharidin, a synthetic analogue of the traditional Chinese medicine cantharidin derived from the blister beetle, is the only bio-available compound presently known to upregulate FAM46C expression and is under investigation in phase one trials in cancer patients.

Keywords: FAM46C; TENT5C; biomarker; gastric cancer; norcantharidin; tumor suppressor.

Figure 1

Depletion of FAM46C in patients with gastric cancer and rescue by NCTD. (A) FAM46C expression is reduced in tumor versus paired normal mucosa (logT/NM <0) in 90% of patients who underwent curative-intent resection for gastric adenocarcinoma. Each patient is represented by a single vertical unit (n=94). (B) Loss of FAM46C expression is associated with inferior DSS in patients with gastric adenocarcinoma (n=94). High and low FAM46C expression were defined using median FAM46C T/NM mRNA expression (0.26) of the entire cohort as a cut-point. (C) Loss of FAM46C expression is associated with inferior disease-specific survival in the subset of patients with node-positive gastric adenocarcinoma (n=60). (D) FAM46C expression varies significantly between cell lines, with aggressive GCa line MKN-45 showing markedly lower FAM46C mRNA levels than GCa line AGS, cervical Ca line HeLa, and osteosarcoma line U2OS. mRNA expression was measured by real time RT-PCR, and calculated by the ΔΔCt method, with all lines normalized to expression in MKN-45 cells. N=3–4 biologic replicates per cell line. (E) NCTD treatment for 48 hours resulted in a dose-dependent increase in FAM46C expression, particularly notable in MKN-45 GCa cells. The inflection point was between 50 and 100 µM NCTD for all lines. FAM46C mRNA was normalized to GAPDH expression, as measured by real time RT-PCR. *, P<0.05 vs. 0.1% DMSO-treated control for each cell line, t-test with Welch’s correction, n=3–4. (F) NCTD treatment for 48 hours resulted in loss of cell viability, with the extent of viability loss being exaggerated in MKN-45 vs. AGS, HeLa and U2OS cells. The inflection point was between 50 and 100 µM NCTD for all lines. Viability was assessed by staining cells with 0.8 µM calcein AM (484/517 nm) and 0.8 µM ethidium homodimer-1 (528/617 nm) for 1 h and calculated as the number of viable cells divided by the total number of cells per well and is expressed relative to 0.1% DMSO-treated control for each line, n=6. All data are expressed as means ± SEM. NCTD, norcantharidin; DSS, disease-specific survival; GCa, gastric adenocarcinoma; RT-PCR, reverse transcription polymerase chain reaction; DMSO, dimethyl sulfoxide; SEM, standard error of the mean.

Figure 2

NCTD kills gastric cancer cells in a FAM46C-dependent manner. (A) FAM46C mRNA expression is reduced by 45–55% in two independent shFAM46C AGS lines, compared with shGFP control (left panel). mRNA expression was measured using real time RT-PCR, calculated by the ΔΔCt method, and normalized to GAPDH. *, P<0.05 t-test with Welch’s correction, n=4 experiments. Reduced FAM46C expression was also seen at the protein level in AGS cell pellets, composed of 2 independent shFAM46C lines vs. shGFP control, assessed by immunohistochemistry using 1/600 rabbit polyclonal FAM46C antibody (Abcam ab222808) with BDDAB chromogen; 40× magnification, scale bar, 1,500 µm (right panel). (B) Viability of AGS cells over 48 hours in culture is not affected by a 45–55% reduction in FAM46C mRNA expression, either without (left panel) or with 0.1% DMSO (right panel), n=6 experiments for each condition. Viability was measured as the number of viable cells divided by the total number of cells per well. (C) Induction of FAM46C mRNA expression by NCTD treatment (for 48 hours) is suppressed in shFAM46C vs. shGFP AGS cells, n=4 experiments. (D) AGS cells depleted of FAM46C are resistant to NCTD-induced toxicity. NCTD treatment for 48 hours caused loss of cell viability in shGFP control AGS cells; this effect was blunted in shFAM46C cells (left panel), particularly at lower doses of NCTD (right panel). Viability is expressed relative to 0.1% DMSO treatment for each line. *, P<0.05, **, P<0.01 vs. shGFP control, t-test with Welch’s correction, n=6 independent experiments. All data are expressed as means ± SEM. NCTD, norcantharidin; RT-PCR, reverse transcription polymerase chain reaction; DMSO, dimethyl sulfoxide; SEM, standard error of the mean.

Figure 3

Plk4 kinase inhibition suppresses AGS gastric cancer cell migration without effect on viability, proliferation, or Plk4 expression. (A) Viability of AGS cells treated with varying doses of centrinone-B (Cent) ×24 hours prior to assay, relative to untreated cells (0.1% DMSO control). n=3 individual experiments. Relative viability was determined as the number of alive/total number of cells in experimental wells divided by the number of alive/total number of cells in corresponding cell DMSO controls. (B) Viability (left panel) and proliferation (right panel) of centrinone-B treated and untreated siFAM46C or si-luciferase AGS cells. Absolute viability defined as total number of alive cells divided by total number of cells in well. P=NS vs. si-luciferase control for all time points. (C) Quantification of scratch wound assay demonstrates treatment of AGS gastric cancer cells with centrinone-B (1 mM) impairs wound healing. Percentage of original wound area healed at indicated time point, n=4. **, P<0.01, ***, P<0.001 vs. DMSO control, unpaired t-test with Welch’s correction. (D) mRNA expression (ΔΔCt) of FAM46C and PLK4 in siFAM46C-transfected (pool of 4) AGS cells treated with 1 µM centrinone-B or DMSO ×16 h, relative to luciferase siRNA control (=1), normalized to GAPDH. P=NS siFAM46C-pool DMSO vs. centrinone-B, n=3. All data are means ± SEM. DMSO, dimethyl sulfoxide; NS, not significant; SEM, standard error of the mean.