Inhibiting HSD17B8 suppresses the cell proliferation caused by PTEN failure

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.

The F12E12.11 gene is predicted to encode an ortholog of human NAD-dependent 17-beta-hydroxysteroid dehydrogenase, the primary function of which is converting E2-E1 9 .Reduction of E2-E1 by 17-betahydroxysteroid dehydrogenase was reported to increase breast cancer cell growth 12 .We treated daf-18(−) worms with E2, and our results showed that Q cell proliferation was significantly suppressed by 15 µM E2 treatment (Fig. 2c).The protein sequence of F12E12.11 was blasted using BLASTP (NCBI), and the best match in H. sapiens was found to be estradiol 17-beta-dehydrogenase 8 (HSD17B8).HSD17B8 is an NAD-dependent dehydrogenase, and the cytosolic NAD + is converted from nicotinamide mononucleotide [20][21][22] .NMN was also administered to daf-18(−) mutants, and we found that 1 mM NMN significantly suppressed Q cell proliferation in daf-18-deficient L1-arrested worms (Fig. 2d).
Sex hormones, including testosterone and E2, originate from pregnenolone, a precursor to steroid hormones 23 .Enzymes in various branches of steroid hormone biosynthesis determine the conversion of pregnenolone into its derivatives.The conversion to any one of its derivatives is determined by the specific enzymes that act in different carbon arms of steroid hormone biosynthesis.Specifically, 17β-hydroxysteroid dehydrogenase/reductases play a crucial role in the biosynthesis of 19-carbon steroids, such as testosterone and E2 23,24 .Research has demonstrated that F12E12.11 in the intestine regulates the biosynthesis of 17β-diol, impacting learning and feeding behaviors 24 .However, conversion of dehydroepiandrosterone into 17β-diol in the nervous system cannot be ruled out, as F12E12.11also may be expressed in neurons.Our study revealed that F12E12.11 in the Q neuron cell lineage facilitates the conversion of E2 to E1. Notably, 17β-diol acts as a ligand for estrogen receptor-β (ER-β), a nuclear hormone receptor crucial for the actions of estrogen, particularly 17β-estradiol.Given that ER-β possesses antitumor properties, as evidenced by previous research 25 , our findings align with this by demonstrating that reducing E1 levels through knocking down F12E12.11suppresses the proliferation of ER-positive breast cancer cells.There are many genes encoding the large 17-beta-hydroxysteroid dehydrogenase family in C. elegans and humans; however, not all of them play the same role in animals.Some previous studies also showed that individual HSD17 has a specific role in enzyme activity and regulating the function of cell proliferation 12,24 .
According to our study, MPK-1/ERK works downstream of DAF-18 and HDS17B8, so loss of mpk-1 should abolish the suppressive effects of E2 and NMN treatment on cell proliferation.Next, we treated daf-18(−); mpk-1(−) double mutants with E2 and NMN.Our results showed that E2 and NMN treatment indeed failed to further suppress Q cell proliferation (Fig. 2c-d), suggesting that the effects of E2 and NMN were abolished by loss of mpk-1.We predicted that the role of F12E12.11 in cell proliferation may involve mpk-1, which encodes an ERK ortholog mitogen-activated protein (MAP) kinase 26,27 .Estrogen might be involved in cell proliferation in daf-18/ PTEN-deficient animals, so ER + breast cancer cell lines 28,29 were used in this study.The ER + breast cancer cell www.nature.com/scientificreports/lines were screened, and MCF-7 cells (Michigan Cancer Foundation-7, MCF-7, human breast cancer cell line) were found to possess high levels of PTEN and HSD17B8 proteins.We knocked down the expression of HSD17B8 in MCF-7 cells using RNAi to assess the activation of ERK1/2.We found that the phosphorylated ERK level was significantly reduced by siHSD17B8 in the MCF-7 breast cancer cell line (Fig. 2e-f).To further confirm that the effect of HSD17B8 on Q cell proliferation is dependent on MPK-1/ERK, we also tested the phosphorylation levels of ERK in MCF-7 cells treated with E2 or NMN.Our results showed that activated ERK levels were significantly reduced in 1 mM NMM-or 15 μM E2-treated MCF-7 cells (Fig. 2g-h).Together, our results suggest that the roles of F12E12.11 and its ortholog HSD17B8 in cell proliferation are dependent on MPK-1/ERK.

E2 treatment and HSD17B8 knockdown suppress MCF-7 cell growth
By analyzing patient tissues, we evaluated the roles of HSD17B family members in human breast cancers 12,30,31 .Another 17-beta-hydroxysteroid dehydrogenase, HSD17B14, was reported to be involved in breast cancer cell growth 12 .HSD17B family dehydrogenases oxidize E2 to E1.While E1 stimulates breast cancer cell proliferation, E2 has the opposite effect.To further test the effect of HSD17B8 on tumor cell growth, we treated MCF-7 cells with E1 and E2.The cell growth assay results showed that the growth of MCF-7 cells treated with E1 was promoted (Fig. 3a), while E2 inhibited the growth of MCF-7 cells (Fig. 3b).Our results showed that HSD17B8 knockdown significantly suppressed the growth of MCF-7 cells (Fig. 3c).We also performed a cell colony formation assay, and our results further confirmed that E1 increased the growth of MCF-7 cells (Fig. 3d) and that E2 supplementation suppressed the growth of MCF-7 cells (Fig. 3e).Knocking down HSD17B8 in MCF-7 cells could suppress the cell growth of MCF-7 cells (Fig. 3f).E2 was reported to arrest cells in G2/M phase to suppress cell growth 32 .We treated MCF-7 cells with E1 and E2 and used a flow cytometer to analyze the cell cycle.The results showed that the number of E1-treated MCF-7 cells in S phase was increased (Fig. 3g).E2 treatment (Fig. 3h) and HSD17B8 knockdown (Fig. 3i) decreased the percentage of cells in S phase and increased the percentage of cells in G2/M phase.These results suggest that HSD17B8 knockdown and the consequent accumulation of E2 can arrest tumor cells at G2/M phase.This is consistent with previous reports that PTEN promotes G2/M arrest in vitro [33][34][35] .We previously reported that loss of daf-18/PTEN can arrest somatic Q neuroblasts in G1/S phase of the cell cycle during L1 arrest in vivo 6 .PTEN has also been reported to control G1/S cell cycle arrest in breast cancer cells 36 .However, PTEN is known to regulate multiple cell cycle checkpoints, including the G1, S, G2, and M checkpoints [37][38][39][40][41] .These results suggest that the function of PTEN in cell cycle regulation is based on the cell type, culture conditions, and specific regulation mode.In our study, we found that PTEN physically interacts with HSD17B8 to inhibit the conversion of E2-E1 by HSD17B8; thus, the inhibitory effect of HSD17B8 blocking on cell proliferation is mainly achieved through the accumulation of E2 and a reduction in E1 levels.This may explain why knocking down HSD17B8 arrests MCF-7 cells in G2/M phase, which is also consistent with previous reports that E2 can arrest tumor cells in G2/M phase.We speculate that E2 or HSD17B8 knockdown can arrest MCF-7 cells in G2/M phase, at least in vitro.

PTEN interacts with and impairs the function of HSD17B8
Next, we wanted to determine how daf-18 affects the activity of F12E12.11.First, we tested whether the expression of F12E12.11 is regulated by daf-18.The expression levels of F12E12.11were tested in daf-18(−) and daf-18(oe) worms, and we found that F12E12.11expression levels were not significantly different between these worms (Fig. 4a).We also tested the expression levels of the F12E12.11homolog HSD17B8 in MCF-7 breast cancer cells with knockdown or overexpression of PTEN, a daf-18 homolog.Our results showed that the expression levels of HSD17B8 were not significantly changed in PTEN-knockdown or PTEN-overexpressing MCF-7 cells (Fig. 4b).These results suggested that daf-18/PTEN does not affect F12E12.11/HSD17B8at the transcriptional level.Next, we also evaluated the protein level of HSD17B8.Our results showed that the protein level of HSD17B8 was not changed in either PTEN-knockdown or PTEN-overexpressing MCF-7 cells (Fig. 4c-d).PTEN is widely known as a potent tumor suppressor, and its main function is as a phosphatase 13,[42][43][44] .As a dehydrogenase enzyme 9,45 , HSD17B8 has not been reported to be regulated by a kinase or phosphatase.PTEN was also reported to have protein phosphatase activity 42,46 .To test whether the phosphatase PTEN can regulate the phosphorylation of HSD17B8, phosphorylated HSD17B8 levels were analyzed using a published standard method (Phos-tag gels, a gel copolymerized with Phos-tag™ acrylamide for phosphorylated protein analysis) 47 .Our results showed that no form of phosphorylated HSD17B8 was detected in control, PTEN-knockdown, or PTEN-overexpressing MCF-7 cells (Fig. 4e), suggesting that PTEN does not affect the phosphorylation of HSD17B8 or that HSD17B8 is not activated through phosphorylation.All these results show that PTEN does not affect the activity of HSD17B8 in a conventional manner.We speculate that PTEN may interact with HSD17B8 to inactivate it.The PTEN active-site cysteine can be oxidized by cellular reactive oxygen species (ROS) to restrain its phosphatase activity [48][49][50] .An in vitro protein binding affinity analysis was performed to identify the binding partners of the purified H 2 O 2 -oxidized form PTEN, and HSD17B8 was one of the 97 potential protein interactors identified by using LC-MS (Liquid Chromatograph Mass Spectrometer) in that study 48 .This suggested that PTEN oxidation, which abolishes the phosphatase activity of PTEN, may also control the other functions of PTEN.Additionally, considering that L1-arrested worms also have higher oxidation levels than normally cultured worms 2,51 , we tested whether oxidized PTEN interacts with HSD17B8.H 2 O 2 was used to generate oxidized PTEN, and we found that MCF-7 cells treated with 1 mM H 2 O 2 for 5 to 15 min exhibited high levels of oxidized PTEN (Fig. 5a).Protein was extracted from H 2 O 2 -treated MCF-7 cells, and to maintain both the conformation and biological activity of the proteins, native PAGE 52 was used to test the interaction of oxidized PTEN with HSD17B8.Interestingly, our results showed that PTEN interacted with HSD17B8 in MCF-3 cells regardless of whether it was oxidized by H 2 O 2 (Fig. 5b), indicating that PTEN endogenously interacts with HSD17B8 to inactivate it.Furthermore, we used coimmunoprecipitation (Co-IP) to confirm that the protein PTEN physically interacts with HSD17B8 (Fig. 5c).
Vol:.( 1234567890 The results suggested that PTEN acts as a tumor suppressor through its nonphosphatase activity and interacts with HSD17B8.This interaction may affect the function of HSD17B8 on preventing E2 from being converted to E1.Thus, a change in the E2/E1 ratio may play an important role in controlling cell proliferation (Fig. 5d).

EMS (ethyl methane sulfonate) mutagenesis and mutation identification
rdvIs1; daf-18(ok480) worms were used for genome-wide EMS mutagenesis for forward genetic screening 16 .Four screens were performed to identify mutants with no Q cell proliferation.In short, more than 800 synchronized L4 stage worms were incubated in a total volume of 4 mL of 50 mM EMS (Sigma) in M9 buffer for 4 h at 20 °C, and approximately 20,000 F3 generation embryos were placed onto NGM plates.The progenies of these worms were cultured in M9 buffer to generate L1-arrested worms, and mutants with no Q cell proliferation were identified.www.nature.com/scientificreports/ The selected mutants were crossed with the rdvIs1; daf-18 (ok480) strain 3 times.The mutations of interest were identified using the recently published Sibling Subtraction method 54 .

qRT-PCR (real-time quantitative reverse transcription PCR)
To analyze the expression level of F12E12.11,worms of various stages were collected.HSD17B8 expression was tested in PTEN-knockdown and PTEN-overexpressing MCF-7 cells.Total RNA was extracted using a High Pure RNA Isolation Kit (Roche).Five micrograms of total RNA was used to synthesize cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).qRT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) and an ABI 7500 system.The relative expression levels of the genes were determined using the 2 −△△CT method and normalized to cdc-42 expression.

RNAi (RNA interference) in C. elegans
Bacteria (HT115) were transformed with a double-stranded target RNA vector (L4440) carrying RNAi to inactivate the target gene.RNAi clones from the Horizon Discovery Library were grown according to the manufacturer's protocol and then seeded onto NGM plates containing 1 mM isopropyl-β-Dthiogalactopyranoside (IPTG) for the formation of double-stranded RNA.The knockdown efficiencies of the RNAis were measured using qPCR.Eggs from RNAi-treated worms were transferred to M9 buffer at 20 °C for 2-3 days to generate L1-arrested worms.The percentage of worms with proliferating Q cells was determined under a fluorescence microscope (Nikon, SMZ18).

Transgenic worm strains
To prepare F12E12.11-overexpressingstrains, the gene sequence was amplified from C. elegans genomic DNA and placed under the control of its own promoter.The sequence was cloned and inserted into the L2528 plasmid

CO-IP (coimmunoprecipitation)
For Co-IP and western blotting, cells were lysed in 2 ml of RIPA buffer supplemented with 0.5 mM PMSF and protease inhibitors (Sigma).The cleared lysate was incubated with a specific antibody overnight at 4 °C on a rotating shaker, followed by incubation with protein A-Sepharose beads for 2 h.The samples were boiled for 5 min, and the denatured samples were electrophoresed on 7.5% Criterion SDS-PAGE gels (Bio-Rad, Germany) and transferred to nitrocellulose membranes (Whatman GmbH, Germany).Immunoprecipitated proteins were detected with either an anti-PTEN or anti-HSD17B8 primary antibody and secondary antibodies (anti-mouse HRP 31,450, Pierce).

Analysis of cell proliferation in C. elegans
In brief, embryos were maintained and hatched in sterile M9 buffer and incubated at 20 °C to initiate L1 arrest.The final Q cell descendants (A/PVM) were observed under an Axioplan fluorescence microscope (Zeiss, Germany) after 2-3 days in L1 arrest.Fifty microliters of M9 buffer containing more than 50 L1-arrested worms were collected.The total number of worms and the number of worms with proliferating A/PVM cells were counted.

Figure 1 .
Figure 1.F12E12.11plays 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 F12E12.11mutants 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 F12E12.11mutation.(c) The gene expression level of F12E12.11 in F12E12.11mutants 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, dh) The test was repeated at least 3 times, with a sample size larger than 60 in each repeat, error bar: mean ± SEM.

Figure 3 .
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 5 .
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.