SREBF1-mediated SND1 transcriptional activation promotes prostate cancer progression via MTDH interaction through the SESN2/AMPK/mTOR axis

Abstract

Background: Prostate cancer (PCa) is a prevalent cancer and a major cause of cancer-related deaths in men worldwide. Growing evidence indicates that Staphylococcal nuclease and Tudor domain containing 1 (SND1) is a multifunctional protein extensively involved in transcriptional regulation, RNA maturation, post-transcriptional modifications, and other processes. However, previous studies have rarely investigated the function of SND1 as an RNA-binding protein in PCa tumorigenesis.

Methods: The Cancer Genome Atlas and NCBI Gene Expression Omnibus (GEO) databases were used to evaluate SND1 expression levels in PCa. We conducted a series of in vitro and in vivo functional experiments to assess the biological functions of SND1, including cell counting kit-8, colony formation, Transwell and wound-healing assays, and animal experiments in nude mice. Chromatin immunoprecipitation, dual-luciferase reporter assay, and DNA pull-down assay were performed to validate the association between the upstream transcription factor and SND1. Based on mass spectrometry, RNA-seq, and RNA immunoprecipitation (RIP)-seq, we identified the downstream targets of SND1- Sestrin 2 (SESN2), which were validated through qRT-PCR, Western blotting, RIP-qPCR, dual-luciferase reporter assay, and RNA pull-down assay. Finally, a series of functional assays and Western blotting analyses confirmed SESN2 as a downstream target of SND1.

Results: Our research identified that SND1 was significantly elevated in PCa, and knocking down SND1 repressed PCa multiplication and migration. Mechanistically, sterol regulatory element binding transcription factor 1 (SREBF1) bound to the promoter of the SND1 gene and activated its transcription, which subsequently formed a complex with metadherin (MTDH). This complex is directly bound to and degraded SESN2 mRNA, and disruption of this interaction with C26-A6 inhibited MTDH-SND1-mediated SESN2 degradation. Notably, SESN2 expression was inhibited in PCa and may exert tumor-suppressive effects by affecting the AMPK/mTOR signaling pathway. Rescue experiments indicated that knocking down SND1 or MTDH significantly inhibited PCa proliferation and migration, and knocking down SESN2 partially reversed this effect.

Conclusions: Our study reveals SND1 overexpression in PCa, which is transcriptionally activated by SREBF1. Mechanistically, SND1 interacts with MTDH and promotes SESN2 mRNA degradation, modulating PCa progression through the AMPK/mTOR pathway.

Keywords: MTDH; Prostate cancer; SESN2; SND1; mTOR.

Fig. 1

The expression of SND1 is elevated in PCa clinical samples and cell lines. A The Venn diagram shows the overlap of upregulated genes across TCGA, GSE21034, and GSE6919. B The expression mode of SND1 was analyzed between 497 PCa tissues and 53 adjacent normal prostate tissues (TCGA database). C, D The data from GSE21034 and GSE6919 revealed higher expression of SND1 in PCa tissues in comparison to benign tissues. E Single-cell sequencing data from GSE141445 showed that the relative expression level of SND1 in different types of PCa cells. E In our analysis of 32 clinical PCa samples, along with their paired normal tissues, SND1 levels were increased in 22 cases (68.75%). G Fourteen pairs of PCa samples were subject to western blot analysis of SND1. H Expression level of SND1 protein in PCa cell lines and RWPE-1 were analyzed. β-actin acted as internal reference

Fig. 2

Knockdown of SND1 inhibits proliferation and migration of PCa cells in vitro. A Knockdown of SND1 was verified by western blot. B-D Knockdown of SND1 suppressed PCa proliferation evaluated by CCK-8, EdU and colony formation assay. E Cell cycle analysis indicated a notable increase of cells arrested in G1 phase in SND1 KD cells. F Overexpression of SND1 was verified by western blot. G-I Overexpression of SND1 promoted PCa proliferation and migration. J The expression of CDK2, CDK4, CCND1 was detected by western blot upon SND1 KD. K, L Knockdown of SND1 repressed PCa migration evaluated by trans-well assay and wound-healing assay (scale bar = 250 μm). M Overexpressing SND1 with plasmids promoted PCa migration evaluated by trans-well assay. N The expression of E-cadherin, N-cadherin and vimentin were detected by western blot upon SND1 KD. β-actin was the internal reference

Fig. 3

Knockdown of SND1 inhibits tumor growth and metastasis in vivo. A SND1 was stably knocked down using lentivirus-based shRNA technique in continuously expressing luciferase PC-3 cell lines (PC3-luci). B The growth curve of tumors was plotted (n = 5 each group) by measuring the tumor size with electronic vernier caliper every 4 days. C Subcutaneous xenografts were photographed and recorded at the end of observation. D The nude mice were first anesthetized and then subjected to dissection to obtain subcutaneous tumors, which were measured for size. E The dissected tumors were weighed in each group. F, G IHC staining of SND1 and Ki-67 in tumors were conducted (scale bar = 100 μm). H Intravenous tail vein tumor metastasis models in vivo were imaged at 6th weeks. I All imaged metastatic organs were anatomized and imaged again to confirm the metastatic sites. J H&E staining of metastasis (lung) were made to validate the metastatic tumor tissues. β-actin was the internal reference

Fig. 4

SREBF1 activates the transcription of SND1 in Pca A JASPAR, hTFtarget, TFDB and GeneCards were used to predict potential TFs of SND1. B Three TFs were screened out according to data from Linkedomics combined with online websites. C Correlation analysis was made between SND1 and SREBF1. D, E Expression level of SND1 mRNA was evaluated with qRT-PCR after knocking down SREBF1 or ZBTB7B with siRNAs. F Expression level of SND1 was evaluated with western blot after knocking down SREBF1 with siRNAs. G-I Knockdown of SREBF1 suppressed PCa proliferation and migration ability evaluated by CCK-8, colony formation assay and trans-well assay. J The motif of binding sites was downloaded from JASPAR. K ChIP-qPCR was performed and analyzed using SREBF1 antibody. L Dual luciferase reporter assay was conducted driven by SREBF1 plasmid and the SND1 promoters. M DNA pull-down assay was performed using biotin-labeled probe. β-actin was the internal reference

Fig. 5

SND1 regulates the expression level of SESN2 through MTDH interaction A IF showed the subcellular localization of SND1. B Isolation of nuclear and cytoplasmic extract showed the localization of SND1 protein in PCa cells. C Proteins with the highest binding abundance to SND1 identified by LC/MS were listed. D Co-IP was performed to validate the interaction between MTDH and SND1. E IF was performed to validate the interaction between MTDH and SND1. F The diagram of truncated plasmids was as presented. G Co-IP was performed to validated the region of SND1 binding to MTDH. H Venn diagram suggested the number of overlapping genes preliminarily screened by eCLIP-seq, RNA-seq and RIP-seq (48 up). I Correlation analysis was made between SND1 and SESN2. J The expression of potential target mRNAs was detected by qRT-PCR upon SND1 KD. K, L The mRNA level of SESN2 was detected by qRT-PCR upon SND1 KD or MTDH KD. M The protein level of SESN2 was detected by western blot upon SND1 KD or MTDH KD. β-actin was the internal reference

Fig. 6

MTDH-SND1 complex promotes the decay of SESN2 mRNA while C26-A6 suppresses this effect A, B C26-A6 inhibited the proliferation and migration of PCa in vitro. C-E C26-A6 inhibited the proliferation and migration of PCa in vivo.F, G C26-A6 significantly enhanced SESN2 mRNA and protein levels. H RIP-PCR assay clarified the association of MTDH, SND1 and SESN2 mRNA. I RIP-PCR assay clarified the regulation of MTDH to SESN2 depended on SND1. J RIP-PCR assay clarified the regulation of SND1 to SESN2 depended on MTDH. K RNA pull-down assay confirmed the interaction between SND1 and SESN2 mRNA. L Relative luciferase activities of SESN2-WT and SESN2-Mut in SND1 KD or MTDH KD cells were detected. M-P RNA stability assays revealed that SND1 KD, or MTDH KD, or C26-A6 prolonged SESN2 mRNA half-life. β-actin was the internal reference

Fig. 7

SESN2 are responsible for SND1 and MTDH induced regulation of PCa progression A The expression pattern of SESN2 was plotted in 497 PCa tissues and 52 normal prostate tissues using data downloaded from TCGA database. B Three pairs of PCa samples were subject to western blot analysis of SND1. C-E Overexpression of SESN2 inhibited PCa tumor growth and metastasis accessed by colony formation assay and trans-well assay (scale bar = 250 μm). F-I Knockdown of SESN2 promoted PCa tumor growth and metastasis accessed by CCK-8, colony formation assay and trans-well assay (scale bar = 250 μm). J The expression of CDK2, CCND1, E-cadherin, N-cadherin and vimentin was detected by western blot after overexpressing SESN2. K The expression of AMPK, p-AMPK, mTOR and p-mTOR were detected by western blot after overexpressing SESN2. L, M Western blot showed knockdown of SESN2 partly reversed the increase of SESN2 expression induced by SND1 KD or MTDH KD. N, O CCK-8 and trans-well assay after SND1 knockdown alone or co-transfected with siSESN2 (scale bar = 250 μm). P The expression of p-AMPK after knocking down SND1 was detected. β-actin was the internal reference