Estrogen receptor β stimulation as a possible novel therapeutic target for cutaneous T-cell lymphoma

Abstract

Cutaneous T-cell lymphomas (CTCLs), including mycosis fungoides (MF) and Sézary syndrome (SS), are rare hematological malignancies with limited curative treatment options. Despite early-stage responsiveness, these malignancies often relapse in advanced stages, highlighting the need for novel, durable therapies. Similar to other non-Hodgkin lymphomas (NHLs), MF and SS have a greater incidence rate in males than females. The endocrine contribution to this sex difference is unknown. Although several studies could show a potential role of estrogen receptor β (ERβ) on NHL lymphomagenesis, its impact on CTCL development is unknown. In this study, we investigated LY500307, a selective ERβ agonist, as a potential treatment for CTCL. Our results show that LY500307 selectively reduced the viability of CTCL cells, sparing noncancerous skin cells. Liquid chromatography with tandem mass spectrometry analysis revealed that CTCL cells accumulated significantly higher concentrations of LY500307 than normal skin cells, likely contributing to its selective cytotoxicity. Mechanistically, LY500307 induced apoptosis, G2/M cell cycle arrest, and increased sensitivity to chemotherapeutic agents, particularly Monomethyl auristatin E. Furthermore, LY500307 treatment significantly reduced tumor growth in a CTCL xenograft mouse model without notable toxicity. These findings suggest LY500307 as a promising therapeutic agent for CTCL, warranting further clinical investigation, including the potential for topical applications.

Graphical abstract

Figure 1.

Selective ERβ agonist LY500307 enhances ERβ signaling and reduces cell viability in CTCL cells, sparing noncancerous skin cells. (A) ERβ protein expression in CTCL cells was determined by performing western blots; 50 μg of protein was loaded for each sample, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control (Ctrl). The data shown are from 3 independent experiments. (B) CTCL cells were transfected with ERE-luc plasmid. After 6 hours, the cells were treated with either vehicle or LY500307 for additional 24 hours, and then reporter activity was measured. (C) Antiproliferative effect of LY500307 compared with placebo in CTCL cells, determined by performing MTS assays. Cells were treated for 48 hours with increasing compound concentrations, from which 50% inhibitory concentration (IC₅₀) values and Hill slopes were calculated. Cell growth was normalized to cells treated with dimethyl sulfoxide (DMSO; untreated Ctrl). Mean values of minimum 3 independent experiments with standard deviation (SD) are plotted. (D) Isolated malignant cells from samples of patients (Pts) with SS (n = 5; left) and CD4⁺ T cells isolated from peripheral blood of healthy volunteers (n = 4; middle) were incubated with increasing concentrations of LY500307 for 72 hours, and cell viability was assessed using MTS assays. Cell growth was normalized to cells treated with DMSO (untreated Ctrl). Mean values of at least 3 replicates with SD are plotted. A comparison is presented of the average cell viability between malignant and healthy cells across the tested LY500307 concentrations (right). (E) Antiproliferative effect of LY500307 compared with placebo in HaCaT cells, primary human keratinocytes, and fibroblasts, determined by performing MTS assays. Cells were treated for 48 hours with increasing compound concentrations. Cell growth was normalized to cells treated with DMSO (untreated Ctrl). Mean values of a minimum of 3 independent experiments with SD are plotted. ∗P < .5; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 2.

LY500307 induces apoptosis and G2/M cell cycle arrest in CTCL cells. (A) CTCL cells were treated with 10-μM LY500307 or DMSO for 48 hours. Cells were then stained for 20 minutes with annexin V fluorescein isothiocyanate and 7AAD. Annexin V–positive cells were determined by flow cytometry. Representative data shown from 3 independent experiments (left) and mean value of 3 independent experiments with SD (right). Mean value of 3 independent experiments. (B) LY500307 activates caspase 3/7, which was determined using Caspase-Glo 3/7 assay. Cells were treated for 48 hours with 10 μM of LY500307. Activity was normalized to cells treated with DMSO (untreated Ctrl). Mean values of 3 independent experiments with SD are plotted. (C) Protein expression in CTCL cells, determined by performing western blots; 30 μg of protein was loaded for each sample, and GAPDH served as a loading Ctrl. The data shown are from 3 independent experiments. (D) LY500307 induces G2/M arrest of CTCL cells. Hut, MyLa, SeAx, and HH cells were either treated with vehicle or LY500307 for 24 hours. Cells were then fixed with 70% ethanol, and PI staining was performed for 30 minutes. Cell cycle distribution was analyzed using flow cytometry. Mean value of 3 independent experiments with SD is plotted. ∗P < .5. 7AAD, 7-aminoactinomycin; Ly, LY500307; PE-A, phycoerythrin-A.

Figure 3.

LY500307 sensitizes CTCL cells to chemotherapeutic agents. Antiproliferative effect of LY500307 and/or chemotherapeutic agent compared with placebo in CTCL cells, determined by performing MTS assays. Cells were treated with either vehicle or LY500307 and chemotherapeutics at different concentrations for 48 hours. The degree of synergy was quantified as CI using the Chou-Talalay method, from dose-response curves with constant ratios of agents tested (CI = 1 indicates a purely additive effect; CI < 1 reveals synergy). Mean value of 3 independent experiments with SD is plotted.

Figure 4.

Metabolism of LY500307 in fibroblasts, HaCaT keratinocytes, and CTCL cell lines. CTCL cells accumulate significantly higher concentrations of LY500307 than normal skin cells. Fibroblasts and HaCaT keratinocytes catabolize intracellular LY-500307 faster than CTCL cell lines. SeAx cells, MyLa cells, HaCaT keratinocytes, and fibroblasts were treated with 10-μM LY-500307 for 0.5, 24, and 48 hours. The concentrations of LY-500307 were measured by LC-MS/MS in the cell pellet (A) and supernatant (B). Data are shown as means with SD (n = 3-4). ∗P < .05; ∗∗P < .01; # indicates significant difference compared with 24 hours.

Figure 5.

LY500307 inhibits tumor growth progression in a CTCL xenograft mouse model. (A) NSG mice were xenografted with MyLa cells subcutaneously and treated once daily with either 10 mg/kg body weight of LY500307 per os or placebo (n = 12 each); mean tumor volume ± SD over time of subcutaneous MyLa xenografts. (B) Tumor volume growth curves for individual mice in each treatment group. (C) Final tumor volumes for individual mice on day 17. Data are shown both as individual values and as mean ± SD. (D) Area under the curve (AUC) values reflecting the entire tumor growth curve for individual mice in each treatment group. Data are shown both as individual AUC values and as mean ± SD. ∗∗∗P < .001; ∗∗∗∗P < .0001.