Estropipate

Endometrial cancer (EC) is the most common gynecological malignancy in the Western world. The molecular basis and effects of various agents are frequently studied in model EC cell lines, but the most commonly used cell lines Ishikawa, HEC-1-A, RL95-2 and KLE have not been thoroughly and systematically investigated. We characterized EC cell lines of different grades by reassessing the expression of estrogen receptors ERα, ERβ, and GPER by qPCR and Western blot and investigated the effects of estrogens, estrone-sulfate, estrone and estradiol on their proliferation, migration, and clonogenicity. Estradiol promoted the proliferation of grade 1 Ishikawa EC cells and grade 2 RL95-2 cells. Estrone and estrone sulfate also stimulated the proliferation of Ishikawa, showed a tendency to increase the proliferation of HEC-1-A and RL95-2 cells, but decreased the proliferation of KLE. Estrogens had no effect on the migration and clonogenicity of these four EC cell lines, however, there was a trend toward a smaller colony area for cells incubated with higher estrogen concentrations. We have previously shown that in EC estradiol forms from inactive estrone sulfate via the sulfatase pathway. This study showed that estrogens significantly promote the proliferation of grade 1 Ishikawa EC cells, and grade 2 RL95-2 and decrease the proliferation of grade 3 KLE cells. These differences in proliferation were associated with ERα positivity of Ishikawa cells and GPER expression in other cells.

Recent modifications to glucagon-like peptide 1 (GLP-1), known for its insulinotropic and satiety-inducing effects, have focused on conjugating small molecules to enable selective delivery into GLP-1R+ tissues to achieve targeted synergy and improved metabolic outcomes. Despite continued advancements in GLP-1/small molecule conjugate strategies, the intracellular mechanisms facilitating concurrent GLP-1R signaling and small molecule cargo release remain poorly understood.

We evaluate an estradiol (E2)-conjugated GLP-1 (GLP-1-CEX/E2) for relative differences in GLP-1R signaling and trafficking, and elucidate endolysosomal dynamics that lead to estrogenic activity using various live-cell, reporter, imaging, and mass-spectrometry techniques.

We find GLP-1-CEX/E2 does not differentially activate or traffic the GLP-1R relative to its unconjugated GLP-1 backbone (GLP-1-CEX), but uniquely internalizes the E2 moiety and stimulates estrogenic signaling. Endolysosomal pH-dependent proteolytic activity likely mediates E2 moiety liberation, as evidenced by clear amplification in estrogenic activity following co-administration with lysosomal VATPase activator EN6. The hypothesized liberated metabolite from GLP-1-CEX/E2, E2-3-ether, exhibits partial estrogenic efficacy through ERα, and is predisposed toward estrone-3-sulfate conversion. Finally, we identify relative increases in intracellular E2, estrone, and estrone-3-sulfate following GLP-1-CEX/E2 incubation in GLP-1R+ cells, demonstrating proof-of-principle for desired cargo release.

Together, our data suggest that GLP-1-CEX/E2 depends on GLP-1R trafficking and lysosome acidification for estrogenic efficacy, with a likely conversion of the liberated E2-3-ether metabolite into estrone-3-sulfate, resulting in a residual downstream flux into active estradiol. Our current findings aim to improve the understanding of small molecule targeting and the efficacy behind GLP-1/small molecule conjugates.