Prognostic role of NF-YA splicing isoforms and Lamin A status in low grade endometrial cancer

Abstract

Although most cases of low grade (G1) endometrial cancer (EC) do not behave aggressively, in rare instances, can progress in a highly aggressive manner. In this study we analyzed formalin-fixed, paraffin-embedded (FFPE) EC tissues to find novel clinical and biological features to help diagnosis and treatment of G1 ECs s in order to better stratify patient risk of recurrence. A retrospective cohort of FFPE specimens from patients with EC (n=87) and benign tissue specimens (NE) from patients who underwent a hysterectomy to treat other benign disease (n = 13) were collected. Total RNA and proteins were extracted and analyzed, respectively, by quantitative PCR and western blotting. NF-YAs is expressed and lamin A is down-modulated in all high grade (G2 and G3) ECs. In G1 ECs, NF-YAs expression is heterogeneous being expressed only in a subset of these tumours. Interestingly, the G1 ECs that express NF-YAs display low levels of lamin A similar to those present in G2 and G3 ECs. Of note, this pattern of NF-YAs and lamin A expression correlates with tumor aggressiveness assessed by comparative analysis with estrogen receptor (ER) status and epithelial-mesenchymal transition (EMT) markers thus suggesting its potential role as biomarker of tumour aggressiveness in G1 EC. In all grade ECs, lamin A is strongly downmodulated, being its expression inversely correlated with tumor aggressiveness and its loss of expression. We identified NF-YAs and lamin A expression levels as novel potential biomarkers useful to identify G1 ECs patients with risk of recurrence.

Keywords: NF-Y; endometrial cancer; estrogen receptor; lamin A; miR-200 family.

Figure 1. Analysis of NF-YA isoforms and lamin A protein expression in benign and EC tissues

A. Representative immunoblottings of proteins extracted from benign (NE), low grade G1 (G1) and high grade endomentrial endometrioid cancer (G2-G3) and non endometrioid (NEM) EC FFPE tissues with anti NF-YA, anti-NF-YB, anti-Lamin A antibodies. The dashed lines separate the different groups. Anti-β actin was used as loading control. B. Average expression of the ratio NF-YAs to NF-YAl mRNA expression examined by qRT-PCR±SD in G1 EEC tissues. mRNA expression was normalized for 18S rRNA levels. The error bars indicate the standard error.

Figure 2. ESR1 decreased mRNA levels are associated with increase CDH2 expression

Average of expression of the ESR-1 mRNA A., and of CDH2 B. mRNA in EC tissues examined by qRT-PCR±SD. mRNA expression was normalized for 18S rRNA levels as endogenous control. Statistical significance: *P≤0.05, **P≤0.01, ***P≤0.001 vs NE. The error bars indicate the standard error.

Figure 3. miR-200s and ZEBs expression in EC FFPE tissues

Average of miR-200 family members (miR-200a, miR-200b, miR-200c, and miR-141) expression A, of ZEB1 and ZEB2 B, mRNA in benign (NE), G1 EEC, G2-G3 EEC, and NEM FFPE tissues. Average of miR-200 family members (miR-200a, miR-200b, miR-200c, and miR-141) expression C, and of ZEB1 and ZEB2 D, mRNA in benign (NE), NF-YAs positive (NF-YA+) and negative (NF-YAs-) G1 subsets of EEC FFPE tissues. Analysis was performed by qRT-PCR±SD. miRNA expression was normalized using small nuclear RNA U6 as endogenous control. mRNA expression was normalized for 18S rRNA levels as endogenous control. Statistical significance: *P≤0.05, **P≤0.01, ***P≤0.001, The error bars indicate the standard error.

Figure 4. Evaluation of lamin A and mir-9 expression in ECs

Average of expression of LMNA mRNA A. and miR-9 B. in benign (NE), in the two subsets of FFPE G1 EEC tissues (NF-YAs- and NF-YAs+) G1 EEC, G2-G3 EEC, and NEM FFPE tissues examined by qRT-PCR±SD. mRNA expression was normalized for 18S rRNA levels. miRNA expression was normalized using small nuclear RNA U6 as endogenous control. Statistical significance: *P≤0.05, **P≤0.01, ***P≤0.001. The error bars indicate the standard error.