Differential Expression of IR-A, IR-B and IGF-1R in Endometrial Physiology and Distinct Signature in Adenocarcinoma

Abstract

Context: Type 2 diabetes and obesity are risk factors for endometrial hyperplasia and cancer, suggesting that hyperinsulinemia contributes to pathogenesis. Insulin action through insulin receptor (IR) splice variants IR-A and IR-B regulates cellular mitogenesis and metabolism, respectively.

Objective: We hypothesized that IR-A and IR-B are differentially regulated in normal endometrium, according to mitogenic and metabolic requirements through the menstrual cycle, as well as in endometrial hyperplasia and cancer.

Design: IR-A, IR-B, and IGF-1 receptor (IGF-1R) mRNA was quantified in endometrium, endometrial epithelial and stromal cells, and in vitro after hormone stimulation.

Setting: Academic center.

Patients: Endometrium was collected from women with regular cycles (n = 71), complex hyperplasia (n = 5), or endometrioid adenocarcinoma (n = 11).

Intervention(s): In vitro sex-steroid treatment.

Main outcome measure(s): IR-A and IR-B expression Results: IR-A increased dramatically during the early proliferative phase, 20-fold more than IR-B. In early secretory phase, IR-B and IGF-1R expression increased, reaching maximal expression, whereas IR-A decreased. In adenocarcinoma, IR-B and IGF-1R expression was 5- to 6-fold higher than normal endometrium, whereas IR-A expression was similar to IR-B. Receptor expression was unrelated to body mass index.

Conclusion: IR-A was elevated during the normal proliferative phase, and in endometrial hyperplasia and adenocarcinoma. The dramatic early rise of IR-A in normal endometrium indicates IR-A is the predominant isoform responsible for initial estrogen-independent endometrial proliferation as well as that of cancer. IR-B is elevated during the normal secretory phase when glucose uptake and glycogen synthesis support embryo development. Differing from other cancers, IR-B expression equals mitogenic IR-A in endometrial adenocarcinoma. Differential IR isoform expression suggests a distinct role for each in endometrial physiology and cancer.

Figure 1.

IR-A, IR-B, and IGF-1R mRNA levels across the menstrual cycle in normal endometrial tissue from 45 women. Graphs (A) IR-A (ο), (C) IR-B (□), and (E) IGF-1R (Δ) show individual tissue levels by day of the menstrual cycle. Graphs (B) IR-A, (D) IR-B, and (F) IGF-1R are mean receptor levels ± SEM for each phase of the menstrual cycle: EP (d 1–7), LP (d 8–14), ES (d 15–21), and LS (d 22–28). The y-axis for IR-A graphs are scaled differently due to higher levels in EP. The significance is defined as follows: *, P ≤ .05 vs EP; **, P ≤ .001 vs EP; ***, P ≤ .0001 vs EP; +, P ≤ .05 vs LP; #, P ≤ .05 vs IR-A; ##P < .005 vs IR-A.

Figure 2.

Distribution of IR-A and IR-B mRNA and Ki67 protein in endometrial epithelial and stromal cells. Mean IR-A and IR-B expression from each cell type isolated from normal endometrial tissues in EP (A), LP (B), ES (C), and LS (D) phases. *, P ≤ .03, epithelial vs stromal. Ki67 immunohistochemistry of benign endometrium from cycle days 2 to 25, magnification ×900. E, Cycle day 2 endometrium reveals mixture of menstrual debris and gland fragments, one of which has 3 Ki67 positive nuclei (arrows). F, Cycle day 4 endometrium showing glands with many darkly stained positive nuclei. G, Cycle day 8 endometrium with Ki67 positive glands and intact stroma, containing scattered Ki67 positive nuclei (arrows). H, Cycle day 14 endometrium exhibiting many Ki67 positive glands and stromal cells. I, Cycle day 17.5 endometrium reveals large glands with sub- and supranuclear vacuoles, and few Ki67 positive glandular nuclei (arrows). J, Cycle day 25 endometrium has complex glands without Ki67 positive nuclei; however, several stromal nuclei are Ki67 positive (arrows).

Figure 3.

In vitro hormone assay to determine whether IR-A, IR-B, and IGF-1R are regulated by E₂ and/or P₄. Isolated epithelial (n = 4) and stromal cells (n = 5) from endometrial tissues were treated separately for 6 hours with E₂ 10–8M (E), P₄ 10–6M (P), combined E + P, or vehicle (V). Mean ± SEM mRNA levels are shown for epithelial cell IR-A (A), IR-B (B), IGF-1R (C), and stromal cell IR-A (D), IR-B (E), and IGF-1R (F). *, P ≤ .05 vs V; **, P ≤ .01 vs V.

Figure 4.

IR-A (A) and IR-B (B) levels from normal endometrial tissue do not correlate with BMI.

Figure 5.

Mean mRNA levels ± SEM of IR-A (A), IR-B (B), IGF-1R (C) in complex hyperplasia (n = 5) and type 1 endometrioid adenocarcinoma (n = 11), as compared with normal endometrial tissue in the EP and LP phases previously represented in Figure 2. The significance is defined as follows: *, P ≤ .05 vs EP; **, P ≤ .01 vs EP; ***, P ≤ .001 vs EP; ****, P ≤ .0001 vs EP; +, P ≤ .05 vs LP; ++, P ≤ .001 vs LP; +++, P ≤ .0001 vs LP; #, P ≤ .05 vs adenocarcinoma.