Targeted destruction of follicle stimulating hormone receptor-positive cancer cells in vitro and in vivo by a lytic peptide Phor21-FSHβ conjugate

Abstract

Background: Extragonadal follicle-stimulating hormone (FSH) receptor (FSHR) expression in various cancers and their endothelial vessel cells has highlighted novel opportunities for targeted FSHR therapy.

Methods: We investigated the specificity/cytotoxicity of Phor21 fusion lytic peptide, conjugated to 12 different FSHβ-chain fragments to ablate FSHR-expressing cancer cells in vitro and in vivo. Additionally, the use of the gonadotropin-releasing hormone (GnRH) antagonist cetrorelix (CTX) alone or with the Phor21-FSHβ33-53 C/S conjugate for anticancer therapy was analyzed.

Results: Phor21 linked to the FSHβ33–53 fragment with cysteine (Cys) replaced by serine (Ser) (Phor21-FSHβ33-53 C/S) demonstrated the highest specific cytotoxicity towards FSHR possessing cancer cells vs. other compounds. Recombinant human FSH treatment significantly decreased the cytotoxicity of Phor21-FSHβ33-53 C/S conjugate in FSHR-positive cancer cells. Phor21-FSHβ33-53 C/S (further addressed as Phor21-FSHβ) treatment in vivo significantly inhibited the growth of FSHR-positive cancer xenografts, resulting in necrosis. The efficacy of the Phor21-FSHβ was enhanced by co-treatment with the gonadotropin-releasing hormone (GnRH) antagonist cetrorelix (CTX). CTX alone exerted pro-apoptotic effects. CTX significantly inhibited the growth of prostate cancer LNCaP cell xenografts. Although FSHR-positive tumor vessel endothelial cells were previously reported in LNCaP cell xenografts, we were unable to reproduce FSHR expression. Consequently, Phor21-FSHβ had no effect on tumor destruction because of the lack of Fshr transcripts in the endothelium of these tumor vessel cells.

Conclusion: This novel functional evidence shows that any cancer cell expressing FSHR can be specifically targeted and destroyed by the conjugated lytic peptide Phor21-FSHβ33–53 (Phor21-FSHβ). FSHR expression was not detected in the tumor vessel endothelial cells, which needs further re-evaluation.

Graphical Abstract: Schematic overview of the Phor21-FSHb33-53C/S (Phor21-FSHβ) conjugate or CTX specifically targeted to kill FSHR-positive cancer cells. (Figure created using BioRender.com). Phor21-FSHb33-53C/S conjugate, Phor21 lytic backbone conjugated with a native or modified fragment of the FSHb subunit (FSHb33-53); CTX, GnRH antagonist cetrorelix

Supplementary Information: The online version contains supplementary material available at 10.1186/s10020-025-01292-5.

Keywords: Cancer; FSHR; FSHβ; Lytic peptide; Phor21.

Fig. 1

Functional characterization of the HEK293-FSHR cells. A qPCR relative expression of FSHR over 15 passages in HEK293-FSHR cells. HEK293 cells were used as a negative control, whereas the human ovary (OV) was used as a positive control for FSHR expression. Each bar represents the mean ±SEM of relative gene expression run in triplicate. B Immunofluorescence colocalization of human FSHR and FLAG in HEK293-FSHR cells. C Kinetics of rhFSH-stimulated cAMP production in HEK293-FSHR and control HEK293 cells, as determined by the CANDLES assay. D Effect of rhFSH on HEK293-FSHR cell proliferation. Fetal calf serum (FCS 2.5%) was used as a positive control stimulant for proliferation. Each bar represents the mean ±SEM of three independent experiments (n = 8/experiment). Asterisks indicate significant differences (**P ≤ 0.01) between control and stimulated cells. CANDLES, Cyclic AMP iNdirect detection by light emission from sensor cell assay; FLAG, polypeptide protein tag; FSHR, follicle-stimulating hormone receptor; HEK293, human embryonic kidney 293 cell line; HEK293-FSHR, HEK293 cells stably transfected with FLAG-hFSHR/pcDNA3.1; OV, ovary; rhFSH, recombinant human follicle-stimulating hormone; FCS, fetal calf serum; FRK, forskolin

Fig. 2

Characterization of Phor21-FSHβ33–53 C/S (further mentioned as Phor21-FSHβ) conjugate cytotoxicity and specificity in vitro determined by the release of lactose dehydrogenase (LDH) into the culture supernatant (A-D) and fluorescent probe imaging (E). A Dose-dependent Phor21-FSHβ33–53 C/S (Phor21-FSHβ) –induced cytotoxicity in HEK293-FSHR cells. B Comparison of Phor21 and Phor21-SHβ33–53 C/S (Phor21-FSHβ) cytotoxicity in HEK293-FSHR cells. C Comparison of the sensitivity of HEK293 and HEK293-FSHR cells to Phor21-FSHβ33–53 C/S (Phor21-FSHβ) cytotoxicity. D Effects of pre- and co-treatment with rhFSH (100 IU/L) on Phor21-FSHβ33–53 C/S (Phor21-FSHβ) -mediated cytotoxicity in HEK293-FSHR cells. The values are the means ±SEMs of three independent experiments (n = 8/experiment) in three different passages of the cell line. Bars with different superscript letters differ significantly from each other (P ≤ 0.05). Asterisks indicate significant differences (*P ≤ 0.05; ***P ≤ 0.001) between the indicated groups. (E) Live/dead viability test of Phor21-FSHβ33–53 C/S (Phor21-FSHβ) -treated HEK293-FSHR cells discriminating live from dead cells by simultaneous staining with green-fluorescent calcein-AM (live) and red-fluorescent propidium iodide (dead). HEK293, human embryonic kidney 293 cell line; HEK293-FSHR, HEK293 cells stably transfected with FLAG-hFSHR/pcDNA3.1; rhFSH, recombinant human follicle-stimulating hormone

Fig. 3

Effects of Phor21-FSHβ33–53 C/S (Phor21-FSHβ) and cetrorelix (CTX) treatments on HEK293-FSHR xenograft growth. A Total xenograft volume at necropsy. B Plasma FSH levels. C Representative images of HEK293-FHSR xenografts that developed subcutaneously in the interscapular area of nude mice. D Histology and immunohistochemical staining for nuclear Ki-67 and cytoplasmic active caspase 3/7 apoptosis markers in xenografts from control and Phor21-FSHβ or CTX-treated mice. The arrows indicated shrunken cells with condensed cytoplasm and pyknotic and fragmented nuclei. The values are the means ±SEMs (n = 10–12). Asterisks/hashtags indicate significant differences between the CTR group and the treatments or indicated groups (*P ≤ 0.05; ***P ≤ 0.001) Cetrorelix, CTX; CTR, control treated with vehicle; HEK293, human embryonic kidney 293 cell line; HEK293-FSHR, HEK293 cells stably transfected with the FLAG-hFSHR/pcDNA3.1 expression plasmid

Fig. 4

Effect of Phor21-FSHb33-53 C/S (Phor21-FSHβ) treatment on HEK-293 xenograft growth. Each bar represents the mean total xenograft volume measured by calipers at necropsy (n = 8–12)

Fig. 5

The mechanism of action of the GnRH antagonist cetrorelix (CTX). A GNRHR expression in HEK-293, HEK293-FSHR and prostate cancer LNCaP cells. B-D Effects of CTX on the proliferation of HEK-293, HEK293-FSHR and LNCaP cells. Fetal calf serum (2.5%) was used as a positive control stimulant. (E-G) Effects of CTX (20 µM) on caspase 3/7 activity. Staurosporin (STS, 0.5 µM) was used as a positive inducer of caspase3/7-mediated apoptosis. Each bar represents the mean ±SEM of three independent experiments (n = 8/experiment). Asterisks indicate significant differences (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.01) between control and stimulated cells. Cetrorelix, CTX; GnRH, gonadotropin-releasing hormone; HEK293, human embryonic kidney 293 cell line; HEK293-FSHR, HEK293 cells stably transfected with FLAG-hFSHR/pcDNA3.1; LNCaP, human prostate cancer cell line; STS, staurosporin

Fig. 6

Effects of Phor21-FSHb33-53 C/S (Phor21-FSHβ) and cetrorelix (CTX) treatments on xenograft growth in LNCaP cells. A Total xenograft volume at necropsy. B Histology of LNCaP xenografts from control and Phor21-FSHb33-53 C/S (Phor21-FSHβ)- or CTX-treated mice. C Plasma FSH levels. The values are the means ±SEMs (n = 10–12). Asterisks indicate significant differences between the CTR and the treatments or indicated groups (*P ≤ 0.05; ***P ≤ 0.001). D RNA scope in situ hybridization of adjacent sections of LNCaP cell xenografts. Lack of Fshr (arrows) in Vwf-positive murine xenograft vessel endothelial cells (arrowheads). A mouse ovary was used as a positive control tissue for the Fshr and Vwf probes, while the Pol2a probe was used as a housekeeping gene (arrow heads). Cetrorelix, CTX; CTR, control treated with vehicle; GC, granulosa cells; LNCaP, human prostate cancer cell line; O, oocyte; SC, stroma cells; STS, staurosporine; TC, theca cells; V, vessel

Fig. 7

Graphical Abstract Schematic overview of the Phor21-FSHb33-53 C/S (Phor21-FSHβ) conjugate or CTX specifically targeted to kill FSHR-positive cancer cells. (Figure created using BioRender.com). Phor21-FSHb33-53 C/S conjugate, Phor21 lytic backbone conjugated with a native or modified fragment of the FSHβ subunit (FSHβ33–53); CTX, GnRH antagonist cetrorelix