Inhibiting HSD17B8 suppresses the cell proliferation caused by PTEN failure

Abstract

Loss of the tumor suppressor PTEN homolog daf-18 in Caenorhabditis elegans (C. elegans) triggers diapause cell division during L1 arrest. While prior studies have delved into established pathways, our investigation takes an innovative route. Through forward genetic screening in C. elegans, we pinpoint a new player, F12E12.11, regulated by daf-18, impacting cell proliferation independently of PTEN's typical phosphatase activity. F12E12.11 is an ortholog of human estradiol 17-beta-dehydrogenase 8 (HSD17B8), which converts estradiol to estrone through its NAD-dependent 17-beta-hydroxysteroid dehydrogenase activity. We found that PTEN engages in a physical interplay with HSD17B8, introducing a distinctive suppression mechanism. The reduction in estrone levels and accumulation of estradiol may arrest tumor cells in the G2/M phase of the cell cycle through MAPK/ERK. Our study illuminates an unconventional protein interplay, providing insights into how PTEN modulates tumor suppression by restraining cell division through intricate molecular interactions.

Keywords: C. elegans; Daf-18; F12E12.11; Cell proliferation; MCF-7; PTEN.

Figure 1

F12E12.11 plays a role in Q cell proliferation in L1-arrested worms. (a) Whole-genome sequencing revealed that the “TCAA” sequence in the second exon of F12E12.11 was deleted in F12E12.11 mutants generated by EMS mutagenesis. The deletion was a frame-shift deletion that resulted in an early stop codon. (b) Q cell proliferation is suppressed by F12E12.11 mutation. (c) The gene expression level of F12E12.11 in F12E12.11 mutants generated by EMS mutagenesis. The test was repeated at least 3 times, error bar: mean ± SEM. (d–e) Q cell proliferation was tested by knocking down F12E12.11 in daf-18(−) mutants. (f) Q cell proliferation caused by overexpression of F12E12.11 in wild-type and daf-18(−); F12E12.11(−) worms. (g) Effects of F12E12.11, E1, E2, and NMN on Q cell proliferation. (h) Overexpression of daf-18 in neurons suppressed Q cell proliferation caused by F12.E12.11. N.S.: no significant difference. ***: P < 0.001, t-test. (b, d–h) The test was repeated at least 3 times, with a sample size larger than 60 in each repeat, error bar: mean ± SEM.

Figure 2

F12E12.11/HSD17B8 works through MAPK/ERK. (a) Effects of knocking down F12E12.11 on Q cell proliferation in daf-18 and daf-18;mpk-1 worms. (b) F12E12.11 overexpression caused Q cell proliferation to be suppressed by mpk-1. (c) Q cell proliferation in daf-18 and daf-18;mpk-1 worms treated with E2. (d) Q cell proliferation in daf-18 and daf-18;mpk-1 worms treated with NMN. (a–d) The test was repeated at least 3 times, with a sample size larger than 60 in each repeat, error bar: mean ± SEM. (e) Knockdown of the F12E12.11 homolog gene HSD17B8 in MCF-7 cells. (f) Knockdown of HSD17B8 reduced the phosphorylation level of ERK. The phosphorylation of ERK was reduced by treatment with NMN (g) or E2 (h) in a dose-dependent manner. (g–h) The test was repeated 3 times, error bar: mean ± SEM. N.S.: no significant difference. *: P < 0.05, **: P < 0.01, ***: P < 0.001, t-test.

Figure 3

Cell proliferation and the cell cycle are affected by HSD17B8. (a) Treatment with E1 promoted the growth of MCF-7 cells. (b) Treatment with E2 suppressed the growth of MCF-7 cells. (c) Knocking down HSD17B8 suppressed the growth of MCF-7 cells. The colony formation assay confirmed the effect of E1 (d), E2 (e), and HSD17B8 knockdown (f) on MCF-7 cell growth. The cell cycle of MCF-7 cells was analyzed by using a flow cytometer after treatment with E1 (g) or E2 (h) or HSD17B8 knockdown (i). For all experiments, at least 3 biological replicates were performed. *P < 0.05; **P < 0.01; ***P < 0.001, two-sided Student’s t test. N.S.: no significant difference.

Figure 4

DAF-18/PTEN does not affect the translation, transcription, or phosphorylation of F12E12.11/HSD17B8. (a) Expression of F12E12.11 in worms when daf-18 was knocked out or overexpressed. (b) Expression of HSD17B8 in MCF-7 cells when PTEN was knocked down or overexpressed. (a–b) The test was repeated 3 times, error bar: mean ± SEM. (c–d) HSD17B8 protein expression in MCF-7 cells when PTEN was knocked down or overexpressed. (e) Phosphorylation of HSD17B8 when TEN was knocked down or overexpressed was assessed by using a Phos-tag gel. Vector 1: empty knockdown vector. Vector 2: empty overexpression vector. N.S.: no significant difference. α-Casein and phosphorylated α-casein (P-α-casein) were used as positive controls. N.S.: no significant difference. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

Figure 5

PTEN interacts with HSD17B8. (a) Serum-starved MCF-7 cells were treated with 1 mM H2O2. All samples were alkylated with 10 mM NEM and subjected to nonreducing or reducing SDS‒PAGE. Reduced and oxidized PTEN levels were measured. (b) Interaction between HSD17B8 and PTEN was tested by using native PAGE. (c) Co-IP was used to test the physical interaction between PTEN and HSD17B8. PTEN(−): MCF-7 cell line with the strongest PTEN knockdown achieved using RNAi. (d) Proposed mechanism of how PTEN regulates HSD17B8 to affect cell proliferation. PTEN does not affect HSD17B8 transcription, translation, or phosphorylation. PTEN can physically interact with HSD17B8, which may affect its function in producing estrone from estradiol. Estradiol can block cell proliferation. Loss of estradiol and accumulation of estrone can induce the phosphorylation of ERK to promote cell proliferation.