c-Jun Transcriptional Regulates NPRL2 to Promote the Proliferation Activity of Prostate Cancer Cells via AKT/MDM2/p53 Signaling Pathway

doi:10.21203/rs.3.rs-108065/v1

Download PDF

Research

c-Jun Transcriptional Regulates NPRL2 to Promote the Proliferation Activity of Prostate Cancer Cells via AKT/MDM2/p53 Signaling Pathway

https://doi.org/10.21203/rs.3.rs-108065/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

High NPRL2 (Nitrogen permease regulator-like 2) expression is a prognostic marker for poor clinical outcomes in prostate cancer (PCa). However, the regulatory mechanisms of NPRL2 in PCa remain unknown.

Methods

The expression level of NPRL2 in prostate cancer tissues were verified through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The effect of NPRL2 gene in promoting the proliferation of prostate cancer was determined by CCK8 and clone formation assays. The apoptosis rate and cell cycle analysis were tested by ﬂow cytometry. Luciferase reporter gene and ChIP were used to verify the binding relationship between transcription factor c-Jun and the NPRL2 5’ region. The effect of NPRL2 and the MK2206 on the AKT/MDM2/p53 signaling pathway was verified by western blotting.

Results

NPRL2 expression level was significantly increased in prostate cancer tissues than normal tissues base on TCGA and GEO database. We investigated that transcription factor c-Jun can bind to the NPRL2 promoter and regulated NPRL2 transcription directly. Then, we revealed that NPRL2 significantly promotes proliferation and reduces apoptosis in PCa cells. Further mechanistic investigations revealed that NPRL2 mediated proliferation promotion effect is associated with the AKT/MDM2/p53 pathway's participation. Besides, CDK2 might serve as an intermediate effector for NPRL2 to regulate the AKT pathway.

Conclusion

Taken together, our study identiﬁed that c-Jun contributes to transcriptional control of NPRL2 and NPRL2 can promote prostate cancer proliferation by activating AKT/MDM2/p53 signaling.

Cancer Biology

Prostate cancer

NPRL2

Proliferation

Transcription

Prostate cancer (PCa) is a common male urinary system malignant tumor in western developed countries¹. The annual number of new cases of PCa were more than 170000, and more than 31000 patient die from prostate cancer each year in 2019 in the USA². There are many treatments strategies for prostate cancer, including surgery, chemotherapy, radiation therapy, and hormone therapy³. Androgen-deprivation therapy (ADT) is an effective treatment for locally advanced or metastatic PCa⁴. However, a large number of prostate cancer patients gradually resist ADT treatment and become lethal castration-resistant PCa (CRPC) inevitably, which results in a poor prognosis⁵. Thus, novel diagnostic and therapeutic approaches need to be developed based on the biological and molecular mechanisms underlying the progression of PCa.

Nitrogen permease regulator-like 2 (NPRL2) is located on chromosome 3p21.3, which was discovered as a tumor suppressor gene by bioinformatics analysis^6,7. NPRL2 expresses in many normal tissues, a tumor suppressor deregulated in various tumors, including breast cancer⁸, colon cancer⁹, and kidney cancer¹⁰, and is involved in DNA mismatch repair and cell-cycle checkpoint signaling^11,12. Our previous studies indicated that the expression level of NPRL2 was significantly higher in prostate cancer tissues and cells than normal tissues and cells, which related to the poor prognosis of prostate cancer patients¹³. Besides, the high expression of NPRL2 promotes the resistance to everolimus by enhancing autophagy in CRPC¹⁴. Our previous results indicated that silencing NPRL2 gene enhances sensitive to Olaparib treatment in prostate cancer cells, and NPRL2 gene may also be the target of therapy and the key molecule of Olaparib resistance in PCa¹⁵. These findings suggest NPRL2 can act as an oncogene in prostate cancer.

Here, we verified that the NPRL2 gene is highly expressed in prostate cancer tissues by bioinformatics analysis, which has a molecular role in promoting cell proliferation and reducing apoptosis in prostate cancer cells. Subsequently, we investigated the characterization of the NPRL2 upstream sequence of transcription start, which contains a c-Jun transcription factor binding site. The data showed that the high expression of NPRL2 in PCa cells is partly due to the transcription factor c-Jun, providing a mechanism for NPRL2 to appear as an oncogene in PCa. The results of our study demonstrated that NPRL2 promotes proliferation by regulating AKT/MDM2/p53 pathways. CDK2 might serve as an intermediate effector for NPRL2 to regulate the AKT pathway. Collectively, NPRL2 might act as an oncogene in PCa and promotes prostate cancer cell progression, which may be an important factor for predicting the prognosis and therapeutic target in prostate cancer.

Cell culture

The human PC3 and LNCaP cell were purchased from Shanghai Life Academy of Sciences Cell Library (Shanghai, China). RPMI1640 medium (C11875500BT; Gibco, Beijing, China) and F12K medium (PYG0036, BOSTER; Beijing, China) was used to culture LNCaP and PC3 cells, respectively, with 10% of fetal bovine serum (FBS) (ST190210, ST30-3302p, PAN, Germany) and 1% of penicillin‐streptomycin at 37℃, in humidified air containing 5% of CO₂.

Bioinformatics mining

RNA-seq data were obtained from the Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) datasets (GSE6956 and GSE68882). The sequence of the 2300 bp 5′ regulatory region of NPRL2 was downloaded from the National Center for Biotechnology Information. PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3) software was used to find the binding sites of transcription factors of NPRL2. Interrelationships between protein molecules were analyzed by Gene Mania (https://genemania.org).

Cell proliferation, apoptosis, and cell cycle assays

Cell Counting Kit-8 (MedchemExpress, Monmouth Junction, NJ, USA) and colony formation detected cell proliferation activity and cloning capability, respectively. The apoptosis rate and cell cycle analysis were tested by ﬂow cytometry (NovoCyte, ACEA Biosciences, San Diego, CA, USA) and FlowJo performed data analyses.

Animal experiments

All animal studies were approved by the Ethics Committee of Chongqing Medical University and performed in accordance with the guidelines of the National Institutes of Health. Five‐week‐old male athymic nude mice were subcutaneously injected with 2 × 10⁶PC3 or LNCaP cells in 100 μL of a 1:1 Matrigel and PBS mixture in the dorsal region. Tumor volume and body weight of nude mice were measured periodically in the next three weeks after cells injection. Tumor volume was estimated: V = A × B²/2 (V, volume; A, length; B, width). After 3‐weeks, the mice were sacrificed, and the tumors were dissected and imaged.

RNA extraction, reverse transcription, and qRT-PCR

RNA was isolated from PCa cell lines by the Trizol reagent (TAKARA, Shiga, Japan) and used for reverse transcription and qRT-PCR. CFX Connect Real-time System (BIO-RAD, CA, USA) calculated the relative expression of genes mRNA levels using SYBR Green (TAKARA, Shiga, Japan). Gene expression levels were evaluated with the 2^-ΔΔCT method. Speciﬁc primers for NPRL2, c-Jun, CDK2, and GAPDH are shown in Supplementary Table 1.

Western blot analysis

Total proteins of cells were extracted by radioimmunoprecipitation assay (RIPA) buffer (Bosterbio) with 1% PMSF. Cell protein lysates were separated by 10% or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto 0.22-μm polyvinylidene ﬂuoride (PVDF) membranes (Sigma). Bovine serum albumin (BSA) was used to block the PVDF membranes for 2 h at room temperature. Primary antibodies, shown in supplementary table 2, incubated overnight. Secondary antibodies (Cell Signaling Technology, Beverly, MA, USA) incubated at room temperature for 30 minutes. Densitometry (Quantity One software; Bio-Rad) was used to quantify the autoradiograms.

NPRL2 5′ Regulatory Region Reporter Construct Cloning Strategies

NPRL2 5′ regulatory region (-2000 bp tp +300 bp) was generated by PCR ampliﬁcation of human genomic DNA that was isolated from HEK 293. Raw materials for reaction included dNTP, DMSO, Betaine, and iProof DNA Polymerase (Bio-Rad, Hercules, CA, USA). The order of PCR cycling was initial denaturation 98℃ for 30s, denaturation 98℃ for 10s, annealing 55℃–65℃ for 30s, extension at 72℃ for 75 s for 35 cycles and ﬁnal extension 72℃ for 10 min. The ampliﬁed 2300 bp fragment was cloned into the luciferase reporter pGL3 basic vector (Promega). Using the full-length upstream sequence of NPRL2 5′ regulatory region, we subsequently constructed deletion sequences of different lengths. All primers used in this step are listed in Supplementary Table 1.

Vector construction and cell transfection

The full-length cDNA of NPRL2 was amplified in 293T cells and then cloned into overexpression vector pcDNA-NPRL2 (Genscript, Jiangsu, China), while the mock vector with no NPRL2 sequence served as a control. siRNA targeting NPRL2 and siRNA-NC were synthesized by Genscript (Jiangsu, China) to knock down NPRL2 expression. The sequence of siRNA-NC and NPRL2 siRNA and the transfection procedure is described in our previous publication¹⁴. siRNA-NC and NPRL2 siRNA were cloned into the plasmid vector called sh-NPRL2 and sh-NC, respectively. Commercialized si-c-Jun, si-CDK2, pcDNA3.1-c-Jun plasmid, pcDNA3.1-CDK2 plasmid, and negative-control siRNA were purchased from Genscript (Jiangsu, China) (Supplementary Table 1). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to conduct cell transfections. AKT inhibitor, MK-2206 were purchased from MedchemExpress (Monmouth Junction, NJ, USA).

Microarray of mRNA

Total RNA was extracted from PC3 cells treated with sh-NC and sh-NPRL2. Expression proﬁling of mRNAs was performed using the MGISEQ-2000 FAST sequencing platform (Sangon Biotech, Shanghai, China). The microarray was used to performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.

Luciferase activity report assays and Chromatin immunoprecipitation (ChIP) analysis

The LNCaP and PC3 cells were plated into 24-well plates (6 × 10⁴ cells/well). Transfections were performed using prime reagent (Polyplus) with the pGL3 basic-NPRL2 (0.5 µg) luciferase construct together with 100 ng of renilla. Dual-Luciferase Reporter Assay System (Promega) was used to detect luciferase activity 48 h after transfection.

Formaldehyde (final concentration 1%) was used to incubate PC3 and LNCaP cells for 10 min at room temperature, followed by 0.2 M glycine for 5 min. Removed the medium, wash the cells twice with 20 ml ice PBS. After adding ice PBS + PIC (Protease Inhibitor Cocktail), the cells were scraped into the cold buffer. Centrifuge at 2000 x g for 5 min at 4°C. Micrococcal Nuclease was used to digest the DNA to approximately 150-900 bp in length. The lysate was sonicated with several pulses to destroy the nuclear membrane. The chromatin was immunoprecipitated by incubating with antibodies and Magnetic beads (Invitrogen, Waltham, MA, USA). The chromatin was eluted from the antibody/ Magnetic beads and de-crosslinked. Purify DNA using spin columns, followed analyze by the Quantitative PCR. The reaction mixture (20µL) consisted of 2µL of DNA sample, 1µL of forward and 1µL of reverse primer, 6µL DEPC and 10 µL of Syber Green Mastermix (Bio-Rad).

Statistical analysis

Statistical analyses were performed with the SPSS 25.0 and GraphPad Prism 7. Overall survival and disease-free survival analysis based on TCGA and GEO datasets were performed using the Kaplan–Meier method. All analyses performed were two-sided, and P-values < 0.05 indicated statistical difference. The data are displayed as mean ± SD of at least three independent experiments. Throughout this study: *p < 0.05; **p < 0.01; and *** p < 0.001.

NPRL2 is up-regulated and associated with poor prognosis in PCa based on TCGA and GEO datasets

The expression profile of NPRL2 was depicted by heatmap based on TCGA database (Fig.1a). The expression of NPRL2 in PCa was significantly higher than the adjacent samples (Fig.1b). Among the patients who died, the NPRL2 gene was also significantly higher than that of the surviving patients (Fig.1c). In addition, NPRL2 was significantly higher in tumor tissues than normal prostate tissues based on the GEO datasets (GSE6956 and GSE68882) (Fig.1d, e). Kaplan–Meier curves demonstrated that the high NPRL2 expression group had a worse overall survival (OS) than the low NPRL2 expression group, although there was no statistical difference (Fig.1f). The NPRL2 high expression group was significantly associated with worse disease-free survival (DFS) (Fig.1g). The expression level of NPRL2 was higher in PCa tissues than that in normal tissues in immunohistochemical staining (Fig.1h, i). Hence, we focused our attention on NPRL2 in tumorigenesis and progression of PCa.

NPRL2 promotes PCa cell proliferation and modulates cell cycle and apoptosis

We first constructed stably overexpression vector and RNAi vector of NPRL2 in PC3 and LNCaP cells (Fig. S1a-d). Overexpression of NPRL2 enhances cell proliferation viability, while silencing NPRL2 inhibited cell proliferation viability in CCK8 assays (Fig. 2a) and colony formation assays in PCa cells (Fig. 2b, c). Knockdown of NPRL2 gene led to higher percentages of PCa cells in the G0/G1 phase in cell cycle analysis (Fig. 2d, e). Meanwhile, the upregulation of NPRL2 led to lower percentages of PC3 and LNCaP cells in the G0/G1 phase, and more cells in the S phase compared with the vector group. Flow cytometry analysis showed that knockdown of NPRL2 gene increases prostate cancer cell apoptosis rate (Fig. 2f, g). The apoptosis rate of NPRL2 overexpression PC3 and LNCaP cells was slightly lower than that of the control group. Furthermore, western blot was performed to detect apoptosis-related protein levels. The results found that the downregulation of NPRL2 in PC3 and LNCaP cells had higher levels of BAX and cleaved caspase-3 and lower levels of BCL2 compared with the NC group (Fig. 2h, i). The levels of BAX and cleaved caspase-3 were lower and BCL2 was higher in NPRL2 overexpression PC3 and LNCaP cells than the control group. To determine the effects of NPRL2 on tumor growth in vivo, PC3 and LNCaP cells were stably transfected with knocked down or sh-NC vector and then injected into male nude mice. the tumors of NPRL2 knockdown PC3 and LNCaP cells had smaller sizes and lower weights than those of the control group (Fig. 2j-m).

NPRL2 expression levels in PCa and detection of NPRL2 gene promoter activity

As shown in figure 3a, b, LNCaP cell showed a lower protein level of NPRL2 than the PC3 cell line. The difference of NPRL2 mRNA expression level between PC3 and LNCaP cells is almost the same as the protein level (Fig. 3c). These pieces of evidence demonstrated that NPRL2 expression levels are controlled at the transcriptional level in prostate cancer cell lines. We constructed the 2300bp sequence from -2000bp to +300 of the 5′ regulatory regions of NPRL2 gene and then cloned into the vector to create pGL3 basic-NPRL2 and a series of 5′ deletion constructs (Fig. 3d). Luciferase reporter detection found the most active region of NPRL2 promoter may be located in the sequence from -1100 bp to −1500 bp (Fig. 3e, f).

The transcription factor c- Jun regulates the expression of NPRL2 gene

Promo software analyzed the 5′ region from -1100 to -1500bp of the transcription start site of NPRL2, suggesting that c-Jun may be a transcription factor regulating NPRL2. Silencing c-Jun significantly down-regulated the mRNA and protein expression of NPRL2 in PC3 (Fig. 4a, b) and LNCaP cell lines (Fig. S3a, b). Silencing c-Jun also significantly reduced the luciferin activity of the NPRL2 upstream promoter in PC3 (Fig. 4c) and LNCaP cell lines (Fig. S3c). Furthermore, the chip assay confirmed that the transcription factor c-Jun directly bind to the upstream sequence of the NPRL2 gene in PC3 and LNCaP cell lines (Fig. 4d), with an IgG-precipitated sample used as a negative control. Collectively, the transcription factor c-Jun regulates the expression of NPRL2 gene at the transcriptional level, thereby affecting its biological function. The expression of c-Jun was significantly higher in PCa tissues than in normal prostate tissues based on the GSE6956 dataset (Fig. 4e). Besides, the expression levels of NPRL2 was significantly and positively correlated with c-Jun (Fig. 4f). According to the expression median value of the NPRL2 and c-Jun, the high expression level of NPRL2 and c-Jun group were significantly associated with worse DFS and OS (Fig. 4g, h).

Silencing and overexpression of c-Jun impacts NPRL2-dependent PCa cell proliferation

The overexpression vector of c-Jun and the siRNA against c-Jun were constructed. c-Jun overexpression and silence in PC3 and LNCaP cells were verified by western blot and qRT-PCR (Fig. S2a-d). Simultaneous up-regulation of NPRL2 and c-Jun significantly increased the proliferation viability of prostate cancer cells by CCK8 assays, whereas the upregulation of NPRL2 and the downregulation of c-Jun inhibited cell growth (Fig. 5a). Additionally, downregulation of NPRL2 and c-Jun at the same time significantly inhibited the proliferation viability of PC3 and LNCaP cells, whereas downregulation of NPRL2 and upregulation of c-Jun enhanced cell growth. Furthermore, the upregulation of NPRL2 and c-Jun group had lower apoptotic cells than the group upregulation of NPRL2 and downregulation of c-Jun in PC3 and LNCaP cells. Meanwhile, the downregulation of NPRL2 and c-Jun group had significantly higher apoptotic cells than the group downregulation of NPRL2 and upregulation of c-Jun (Fig. 5b). Western blotting results showed that the upregulation of NPRL2 and c-Jun suppressed the expression level of cleaved caspase-3 and BAX and induced the expression of BCL2. On the contrary, simultaneous down-regulation of NPRL2 and c-Jun significantly increased the expression level of cleaved caspase-3 and BAX, while a reducing the expression of BCL2 (Fig. 5c).

NPRL2 promotes proliferation via activating AKT/MDM2/p53 axis

Western blot analysis found that NPRL2 overexpression enhanced the expression of p-AKT and p-MDM2 and reduced the expression of p53, while the knockdown of NPRL2 had an opposite effect in the LNCaP cell. Overexpression of NPRL2 resulted in the increased expression of the p-AKT and p-MDM2 protein levels in the PC3 cell (Fig. 6a). However, the expression level of p53 was not changed significantly in knockdown or overexpression of NPRL2 in the p53 null PC3 cell. To examine the regulation of AKT/MDM2/p53 pathways mediated by NPRL2, PC3 and LNCaP cells were treated with a p-AKT speciﬁc inhibitor (MK2206). We tested the inhibitory effect of MK2206 at 5 μM concentrations for 24 h by western blotting. As a result, MK2206 block the expression of p-AKT and p-MDM2 and increase the expression of p53 in the LNCaP cell. However, the expression level of p53 was not changed significantly treated with MK2206 in the p53 null PC3 cell (Fig. 6b). Western blot experiment found that MK2206 significantly up-regulated the expression of cleaved caspase-3 and BAX, while down-regulating the expression of BCL2 even if the NPRL2 gene was overexpressed (Fig. 6c, d). We found that MK2206 signiﬁcantly suppressed the viability of PC3 and LNCaP cells even if the NPRL2 gene was overexpressed (Fig. 6e).

CDK2 serves as an intermediate effector for NPRL2 to regulate the AKT pathway

CDK2 overexpression and silence in PC3 and LNCaP cells were verified by western blot and qRT-PCR (Fig. S5a-d). The expression levels of NPRL2 was significantly and positively correlated with CDK2 in GSE68882 (Fig. 7a). According to the expression of the NPRL2 and CDK2, the patients were divided into high and low groups, the patients with high expression of NPRL2 and CDK2 was significantly associated with worse DFS and OS in Kaplan–Meier curves (Fig. 7b, c). NPRL2 overexpression upregulated CDK2 mRNA expression, and NPRL2 knockdown suppressed CDK2 mRNA expression in PC3 (Fig. 7d, e) and LNCaP cell line (Fig. S5e, f). CDK2 was significantly higher in tumor tissues than normal prostate tissues based on GSE6956 and GSE68882 (Fig. 7f, g). Bioinformatics prediction indicates that there may be four key molecules between NPRL2 and CDK2, including CDKN1A, CCNB2, CKS1B, and CDKN1B (Fig. S5g). The silencing or overexpression of NPRL2 may significantly affect the expression of p-AKT, but this effect will be reversed by the overexpression and silence of CDK2, respectively (Fig. 7h). Therefore, CDK2 might act as a key intermediate effector for NPRL2 to regulate the AKT pathway.

In our previous reports, NPRL2 was studied and identiﬁed as an oncogene in PCa¹⁴. Here, we validated the expression level of NPRL2 in TCGA and GEO datasets. NPRL2 was upregulated in PCa tissues and significantly correlated with poor DFS and OS. The NPRL2 gene promote the proliferation activity of prostate cancer cells in vivo and in vitro, while silencing NPRL2 significantly reduced the proliferation activity and increased apoptosis rate. Previous researches have shown that NPRL2 is closely related to cell cycle¹⁶. We found silencing NPRL2 caused a large number of PC3 and LNCaP cells to be arrested in the G1 stage.

Much evidence has shown that NPRL2 acts as a tumor suppressor in colon cancer, glioma, lung cancer, and breast cancer^17-20. The high expression of the NPRL2 gene in prostate cancer and the oncogenic characteristics has aroused great interest in our team. We hope to explore the reasons for its high expression different from other cancers. Protein expression is regulated by transcription and post-transcriptional processes. This study focused on the transcription level of the NPRL2 gene and found that c-Jun can directly bind to the NPRL2 promoter and regulate NPRL2 transcription.

c-Jun is an important member of the AP-1 transcription factor family, which acts as a cancer-promoting transcription factor contribiuted in differentiation, cell cycle, and apoptosis²¹. In addition, c-Jun also participates in stress response, and the function is conserved in yeast and mammals²². Thakur et al. demonstrated that c-Jun is activated by TGF-b, leading to invasion of prostate cancer cells, which has high expression levels in aggressive prostate cancer tissues with poor prognosis^23,24. The transcription regulation of the NPRL2 by c-Jun may be one of the reasons that NPRL2 gene appears as an oncogene in PCa.

KEGG pathway analysis suggested that the PI3K/AKT pathway is a hub differential pathway between NPRL2 knockdown group and NC group (Fig. S4a, b). PI3K/AKT is an important pathway that participates in the multiple biological activities, including cell proliferation, cell repair, cell growth, cell migration, and angiogenesis, and is constitutively activated in nearly all cancer types²⁵. AKT is a major effector in the PI3K/AKT signaling axis, one of the essential kinase signaling molecules in prostate cancer development and treatment²⁶. Increased murine double minute 2 (MDM2) expression is associated with increased the risk for recurrence, metastasis, and cancer-specific mortality with PCa treated with radiotherapy²⁷. p53 is a classic tumor suppressor gene and also an important downstream effector of AKT. Quinn et al. reported that p53 aberrations in radical prostatectomy tissue could predict clinically relevant endpoints of metastatic relapse and prostate cancer-specific mortality²⁸. We observed that NPRL2 regulated the AKT/MDM2/p53 signaling in PCa, which serves as an AKT coactivator to stimulate the AKT/MDM2/p53 pathway mediating the proliferation promoting effect.

Gene Ontology (GO) enrichment analysis revealed that the top five enrichment of NPRL2 involved are protein kinase activity, GTPase activator activity, lysosomal membrane, cellular response to nitrogen starvation, and positive regulation of autophagy (Fig. S4c). Liu et al. reported that cyclin-dependent kinase 2 (CDK2), an important protein kinase, promotes AKT phosphorylation activation through facilitating or functionally compensating²⁹. CDK2 was significantly upregulated in PCa tissues and was positively correlated with the expression level of NPRL2 in the GEO dataset. Therefore, we inferred that there migut be a connection between NPRL2 and CDK2. We found that NPRL2 overexpression upregulated CDK2 mRNA expression and NPRL2 knockdown suppressed CDK2 mRNA expression in PC3 and LNCaP cell lines. Furthermore, western blot demonstrated that the p-AKT levels caused by overexpressing or silencing NPRL2 could be reversed by CDK2 silencing or overexpressing, respectively. In summary, these data suggested that CDK2 might serve as a downstream molecule and regulated by NPRL2, thereby affecting the phosphorylation of AKT, which acts as an intermediate effector for NPRL2 to regulate the AKT/MDM2/p53 pathway.

In this study, we provided a novel mechanism to address how NPRL2 promotes tumor proliferation in PCa. Our data showed that the NPRL2 acts as an oncogene in PCa, which is positively regulated by c-Jun to coregulate the AKT/MDM2/p53 signaling. In addition, the effect of NPRL2 gene on the AKT MDM2/p53 pathway may be mediated through CDK2. Accordingly, NPRL2 may be a novel molecular therapeutic target for developing a new treatment approach for prostate cancer.

NPRL2: Nitrogen permease regulator-like 2; PCa: prostate cancer; ADT: Androgen-deprivation therapy; TCGA: The Cancer Genome Atlas; GEO: Gene Expression Omnibus; KEGG: Kyoto Encyclopedia of Genes and Genomes; ChIP: Chromatin immunoprecipitation; OS: overall survival; DFS: disease-free survival.

Ethics approval

All studies were approved by the Ethics Committee of Chongqing Medical University.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and analyzed in the present study are available from the corresponding author on reasonable request.

Conﬂict of interest

The authors declare no potential conﬂicts of interest.

Funding

This work was supported by the Foundation for Science and Technology Research Project of Chongqing (CSTC2019jcyj-msxmX0708).

Author contributions

D.H., L.J., and W.T. conceived and designed the study; D.H., X.Z., Y.T., G.Z., and X.D. conducted experiments and collected data; D.H., X.Z., Y.T., and L.J. analyzed and interpreted data; D.H. and X.Z. drafted the paper; L.J. and S.L. provided technical or material support; W.T. provided valuable comments and suggestions to improve the paper and data presentation; L.J. and W.T. supervised the whole study; all authors read and approved the ﬁnal version of the paper.

Acknowledgements

We would like to acknowledge the GEO database and the TCGA databases for free use. The authors would like to thank the team of the Institute of Life Sciences, Chongqing Medical University.

1 Ferlay, J. et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. International journal of cancer144, 1941-1953, doi:10.1002/ijc.31937 (2019).

2 Siegel, R., Miller, K. & Jemal, A. Cancer statistics, 2019. CA: a cancer journal for clinicians69, 7-34, doi:10.3322/caac.21551 (2019).

3 Seikkula, H. A. et al. The impact of socioeconomic status on stage specific prostate cancer survival and mortality before and after introduction of PSA test in Finland. International journal of cancer142, 891-898, doi:10.1002/ijc.31109 (2018).

4 Madan, R. A. & Arlen, P. M. Recent advances revolutionize treatment of metastatic prostate cancer. Future Oncol9, 1133-1144, doi:10.2217/fon.13.65 (2013).

5 Small, E. J. et al. Apalutamide and overall survival in non-metastatic castration-resistant prostate cancer. Annals of oncology : official journal of the European Society for Medical Oncology30, 1813-1820, doi:10.1093/annonc/mdz397 (2019).

6 Lerman, M. I. & Minna, J. D. The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer research60, 6116-6133 (2000).

7 Ueda, K. et al. The 3p21.3 tumor suppressor NPRL2 plays an important role in cisplatin-induced resistance in human non-small-cell lung cancer cells. Cancer research66, 9682-9690, doi:10.1158/0008-5472.Can-06-1483 (2006).

8 Peng, Y. et al. TUSC4 functions as a tumor suppressor by regulating BRCA1 stability. Cancer research75, 378-386, doi:10.1158/0008-5472.Can-14-2315 (2015).

9 Liu, M. N. et al. Functional mechanism of the enhancement of 5-fluorouracil sensitivity by TUSC4 in colon cancer cells. Oncol Lett10, 3682-3688, doi:10.3892/ol.2015.3801 (2015).

10 Tang, Y., Jiang, L. & Tang, W. Decreased expression of NPRL2 in renal cancer cells is associated with unfavourable pathological, proliferation and apoptotic features. Pathol Oncol Res20, 829-837, doi:10.1007/s12253-014-9761-2 (2014).

11 Ji, L. et al. Expression of several genes in the human chromosome 3p21.3 homozygous deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo. Cancer research62, 2715-2720 (2002).

12 Shen, K. et al. Architecture of the human GATOR1 and GATOR1-Rag GTPases complexes. Nature556, 64-69, doi:10.1038/nature26158 (2018).

13 Chen, Z. et al. High expression of NPRL2 is linked to poor prognosis in patients with prostate cancer. Human pathology76, 141-148, doi:10.1016/j.humpath.2018.02.011 (2018).

14 Chen, Z. et al. NPRL2 enhances autophagy and the resistance to Everolimus in castration-resistant prostate cancer. The Prostate79, 44-53, doi:10.1002/pros.23709 (2019).

15 Chen, X., Chen, Z., Zheng, B. & Tang, W. Targeting NPRL2 to enhance the efficacy of Olaparib in castration-resistant prostate cancer. Biochemical and biophysical research communications508, 620-625, doi:10.1016/j.bbrc.2018.11.062 (2019).

16 Ma, Y., Silveri, L., LaCava, J. & Dokudovskaya, S. Tumor suppressor NPRL2 induces ROS production and DNA damage response. Scientific reports7, 15311, doi:10.1038/s41598-017-15497-0 (2017).

17 Huang, N. et al. Downregulation of nitrogen permease regulator like-2 activates PDK1-AKT1 and contributes to the malignant growth of glioma cells. Mol Carcinog55, 1613-1626, doi:10.1002/mc.22413 (2016).

18 Jayachandran, G., Ueda, K., Wang, B., Roth, J. A. & Ji, L. NPRL2 sensitizes human non-small cell lung cancer (NSCLC) cells to cisplatin treatment by regulating key components in the DNA repair pathway. PloS one5, e11994, doi:10.1371/journal.pone.0011994 (2010).

19 Liu, S. & Liu, B. Overexpression of Nitrogen Permease Regulator Like-2 (NPRL2) Enhances Sensitivity to Irinotecan (CPT-11) in Colon Cancer Cells by Activating the DNA Damage Checkpoint Pathway. Medical science monitor : international medical journal of experimental and clinical research24, 1424-1433, doi:10.12659/msm.909186 (2018).

20 da Costa Prando, E., Cavalli, L. & Rainho, C. Evidence of epigenetic regulation of the tumor suppressor gene cluster flanking RASSF1 in breast cancer cell lines. Epigenetics6, 1413-1424, doi:10.4161/epi.6.12.18271 (2011).

21 Shaulian, E. & Karin, M. AP-1 in cell proliferation and survival. Oncogene20, 2390-2400, doi:10.1038/sj.onc.1204383 (2001).

22 Xiang, D. M. et al. Oncofetal HLF transactivates c-Jun to promote hepatocellular carcinoma development and sorafenib resistance. Gut68, 1858-1871, doi:10.1136/gutjnl-2018-317440 (2019).

23 Thakur, N. et al. Smad7 Enhances TGF-β-Induced Transcription of c-Jun and HDAC6 Promoting Invasion of Prostate Cancer Cells. iScience23, 101470, doi:10.1016/j.isci.2020.101470 (2020).

24 Thakur, N. et al. TGFβ-induced invasion of prostate cancer cells is promoted by c-Jun-dependent transcriptional activation of Snail1. Cell Cycle13, 2400-2414, doi:10.4161/cc.29339 (2014).

25 Zhao, H. F., Wang, J. & Tony To, S. S. The phosphatidylinositol 3-kinase/Akt and c-Jun N-terminal kinase signaling in cancer: Alliance or contradiction? (Review). International journal of oncology47, 429-436, doi:10.3892/ijo.2015.3052 (2015).

26 Amirani, E., Hallajzadeh, J., Asemi, Z., Mansournia, M. & Yousefi, B. Effects of chitosan and oligochitosans on the phosphatidylinositol 3-kinase-AKT pathway in cancer therapy. International journal of biological macromolecules164, 456-467, doi:10.1016/j.ijbiomac.2020.07.137 (2020).

27 Feng, F. et al. MDM2 Inhibition Sensitizes Prostate Cancer Cells to Androgen Ablation and Radiotherapy in a p53-Dependent Manner. Neoplasia (New York, N.Y.)18, 213-222, doi:10.1016/j.neo.2016.01.006 (2016).

28 Quinn, D. I. et al. p53 nuclear accumulation as an early indicator of lethal prostate cancer. Br J Cancer121, 578-583, doi:10.1038/s41416-019-0549-8 (2019).

29 Liu, P. et al. Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus. Nature508, 541-545, doi:10.1038/nature13079 (2014).

Download PDF

Version 1

posted

You are reading this latest preprint version

c-Jun Transcriptional Regulates NPRL2 to Promote the Proliferation Activity of Prostate Cancer Cells via AKT/MDM2/p53 Signaling Pathway

Status:

Version 1

Abstract

Figures

Background

Methods

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1