Tumor protein D52 (isoform 3) contributes to prostate cancer cell growth via targeting nuclear factor-κB transactivation in LNCaP cells

Abstract

Our previous study showed that TPD52 overexpression could increase migration and proliferation of LNCaP cells contributing to the development of prostate cancer. However, mechanism of TPD52 in prostate cancer initiation and progression remains elusive. In this study, we investigated the possible underlying mechanism of TPD52 in prostate cancer progression. In LNCaP cells, TPD52 expression was altered by transfecting with either EGFP-TPD52 or specific short hairpin RNA. Overexpression of TPD52 protected LNCaP cells from apoptosis through elevated anti-apoptotic proteins XIAP, Bcl-2, and Cyclin D1, whereas Bax was downregulated. Mechanistically, we found that TPD52 confers transactivation of nuclear factor-κB, thereby enhancing its target gene expression in LNCaP cells. TPD52 promotes LNCaP cell invasion probably via increased matrix metalloproteinase 9 expression and its activity while tissue inhibitor of metalloproteinase expression is significantly downregulated. Notably, TPD52 might be involved in cell adhesion, promoting tumor metastasis by inducing loss of E-cadherin, expression of vimentin and vascular cell adhesion molecule, and additionally activation of focal adhesion kinase. Furthermore, TPD52 directly interacts with nuclear factor-κB p65 (RelA) and promotes accumulation of phosphorylated nuclear factor-κB (p65)^S536 that is directly linked with nuclear factor-κB transactivation. Indeed, depletion of TPD52 or inhibition of nuclear factor-κB in TPD52-positive cells inhibited secretion of tumor-related cytokines and contributes to the activation of STAT3, nuclear factor-κB, and Akt. Interestingly, in TPD52 overexpressing LNCaP cells, nuclear factor-κB inhibition prevented the autocrine/paracrine activation of STAT3. TPD52 activates STAT3 through ascertaining a cross talk between the nuclear factor-κB and the STAT3 signaling systems. Collectively, these results reveal mechanism by which TPD52 is associated with prostate cancer progression and highlight the approach for therapeutic targeting of TPD52 in prostate cancer.

Introduction

Prostate cancer (PCa) is the second most prevalent cancer in men after lung cancer.¹ It is the fifth leading cause of cancer deaths in men worldwide with an estimated 307,500 deaths in 2012.² Early diagnosis and effective treatment are the options to cure PCa. In PCa, molecular events including genetic alterations such as the gain or loss of chromosome 8 at 8q21 region are significantly associated with its pathogenesis and progression.^3,4 Furthermore, genomic and proteomic analyses have discovered the differential expression tumor protein D52 (TPD52) with strong correlation to breast and PCa progression. The gene encoding TPD52 is mapped to 8q21 locus on chromosome 8, which is susceptible for occurrence of PCa.⁵ It is also denoted as PC-1, a prostate Leucin Zipper gene (PrLZ). Its gene expresses three isoforms of the TPD52 family by splicing and was designated as a proto-oncogene.⁶ A recent study on PCa patients using integrative analysis of copy number alterations (CNA) and array transcriptomics confirmed that TPD52 is one of the six oncogenes associated with PCa.² It is involved in vesicle trafficking, cell survival, proliferation, DNA repair, exocytosis, migration, and invasion.^{7 –13} It has been reported already that TPD52 is overexpressed in different cancers such as breast,¹⁴ prostate,¹⁵ and ovarian cancer¹³ due to gene amplification. In LNCaP cells, expression of TPD52 family proteins is controlled by androgens and interleukin (IL)-6, thereby controlling cell proliferation and apoptosis.^15–17 Recent studies have demonstrated that TPD52 promotes metastasis; its expression increases with advancement of cancer and regulates apoptosis of cancer cells.^18,19 There was ample of interest given to TPD52 as an oncogene, due to its multifaceted functions in various cellular mechanisms and multiple carcinomas.^20,21 It is also proved that TPD52 increases lipid metabolism by facilitating de novo synthesis, exogenous uptake, and intracellular lipid storage.^22,23 Suppression of TPD52 using miR-34a inhibits migratory and invasive capacity of breast cancer cells,²⁴ and miR-218 represses cell growth and promotes apoptosis in PCa cells.²⁵ A recent study on PC-1, isoform-1 of TPD52, shows the correlation between TPD52 expression and transdifferentiation of LNCaP cells upon treatment with IL-6.¹⁷ Overexpression of PrLZ in PCa cells mediates increased tumor formation followed by bone metastasis in vivo.^18,26 Since the specific mechanism through which TPD52 is involved in PCa progression is still under investigation, this study is aimed to understand the effect of TPD52 expression on different cellular signaling pathways in the androgen-responsive PCa cell line LNCaP.

Materials and methods

Cell lines and chemicals

LNCaP cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS; Invitrogen, Waltham, MA, USA) and 100 units/mL penicillin–streptomycin antibiotics (Sigma Aldrich, Bangalore, India). To avoid contamination of cells with mycoplasma, cells were regularly tested with the MycoAlert Kit (Cambrex Bio Science, Rockland, ME, USA). All chemicals are purchased from Sigma-Aldrich, Bangalore, India.

Protein network analysis of differentially regulated proteins in PCa

To predict the novel functions and molecular partners and/or pathways of TPD52 associated to PCa, the gene and protein expression data have been analyzed using a systematic web-based tool GeneMANIA (http://www.genemania.org).^27–29 It is a web-based tool that generates protein networks from gene/protein expression data according to published literature presenting regulation of gene expression and activation of proteins along with their interacting proteins. First, a global network of all differentially regulated proteins and genes in PCa was generated. Then, further subnetworks were created based on the observed central hubs in master network focusing on activation of pathways and protein–protein interactions. From all the subnetworks, the most significant networks including expression and interactions among the input objects are considered to determine their association with PCa initiation and progression.

Overexpression or downregulation of TPD52 in LNCaP cells

Recombinant vectors for enhanced green fluorescent protein (EGFP)-TPD52 and FLAG-TPD52 fusion protein expression were generated by cloning the coding region of the human TPD52 isoform 3 (accession number: NM 001001875) into pEGFP-N3 and p3×Flag-CMV vectors (Clontech Laboratories, Palo Alto, CA, USA). To downregulate TPD52, LNCaP cells were transfected with previously validated vectors producing short hairpin RNA (shRNA) against TPD52. The cloning of TPD52 and shRNA was carried out as reported in Ummanni et al.⁷

RNA isolation and measurements of gene transcripts of interests by semi-quantitative reverse transcription polymerase chain reaction

Quantitative reverse transcription polymerase chain reaction (RT-PCR) for determining the expression of genes of interest was performed using standard RT-PCR. RNA isolation and complementary DNA (cDNA) synthesis by reverse transcription using oligo(dT) primer and Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) were performed as reported earlier.⁷ The primer sets for amplifying known size of amplicons from genes of interest were verified at suitable thermal cycling conditions. The corresponding gene IDs and primer sequences used in this study are mentioned in supplementary information (Table S1). Followed by RT-PCR, relative expression changes were determined by normalization to housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/Actin) expression.

Western blotting

For western blotting, the cells were directly collected and lysed in two-dimensional (2D) lysis buffer (8 M urea, 2 M thiourea, 4% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), 40 mM Tris-base, and 65 mM dithiothreitol (DTT)) containing inhibitors against proteases and phosphatases. The protein concentration was estimated by modified Bradford assay.³⁰ The protein lysates were separated on 8% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred on to a nitrocellulose membrane. The membrane with proteins was incubated in blocking solution (3% bovine serum albumin (BSA)) for 1 h at room temperature. The membrane was further incubated with primary antibody (all antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA) for overnight at 4°C. Then, the membrane was washed three times with Tris buffered saline with Tween 20 (TBST) buffer (20 mM Tris, 138 mM NaCl (pH 7.6), and 0.1% Tween 20) to remove excess unbound antibody. The blots were incubated with secondary antibody conjugated to either horseradish peroxidase anti-mouse IgG (1:5000 in TBST) or anti-rabbit IgG (1:5000) for 1 h at room temperature. After washing three times to remove excess secondary antibody, the blots were developed by chemiluminescence using an enhanced chemiluminescence (ECL; Bio-Rad, Hercules, CA, USA) substrate followed by capturing chemiluminescence signal using XRL+ ChemiDoc (Bio-Rad).

Immunoprecipitation

To determine interaction between nuclear factor-κB (NF-κB) and TPD52, FLAG-TPD52 or FLAG-tag alone was transfected separately into LNCaP cells and cultured for 24 h for expression of fusion proteins. Immunoprecipitation was performed 24 h after transfection as reported previously.³¹ Briefly, cells were washed with cold phosphate-buffered saline (PBS) and collected using cell scrapers directly in freshly prepared co-IP buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.5), 120 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1% Triton X-100, 5 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium orthovandate, and protease inhibitor cocktail (Invitrogen)). Then, the cells were lysed by passing through a syringe with fine needle for 5–10 times. To remove debris, lysate was centrifuged at 14,000g for 10 min. The supernatant was used for immunoprecipitation using 1 µg of each antibody (anti-FLAG or anti-NF-κB p65) from 200 µg LNCaP cell lysate. Immunoprecipitation was performed at 4°C by overnight incubation of cell lysates with antibodies on rotary shaker for constant mixing. The antigen and antibody complexes were isolated by incubating with 25 µL of IgG beads for each sample for 2 h at 4°C. Beads were collected by centrifugation at 500g for 5 min at 4°C. The beads were washed thrice in co-IP buffer and then directly mixed with 2 × SDS sample buffer. To resolve precipitates on SDS-PAGE followed by western blotting, samples were heated to 95°C for 5 min before electrophoresis.

Immunofluorescence

To observe co-localization of TPD52 and NF-κB p65 in LNCaP cells, cells were seeded on sterile cover slips placed in six-well plate. After 18 h, cells were transfected with either pFlag-CMV or pFlag-CMV-TPD52 vectors. Cells were washed twice with cold PBS and fixed with 4% paraformaldehyde in PBS 24 h after transfection. After washing thrice with PBS (each 5 min), cells were permeabilized by incubation in 0.25% Triton X-100 in TBST (50 mM Tris, 150 mM NaCl (pH 7.4), and 0.1% Tween 20) for 10 min. Followed by washing thrice with PBS, to reduce non-specific binding of antibodies, cells were incubated in 2% BSA dissolved in TBST solution for 2 h. The cells were incubated with required primary antibody (1:100) for overnight at 4°C. After washing thrice with TBST to remove excess primary antibody, cells were incubated with secondary antibody (1:500; cy3 or cy5 labeled) for 2 h in dark. The unbound secondary antibodies were washed with TBST, and the cover slips were mounted on glass slide using mounting medium containing anti-fade 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). To visualize localization of proteins, images were collected using confocal microscope (Olympus FV1000 FluoView, Tokyo, Japan) and images were superimposed to show co-localization of target proteins.

Gelatin zymography for matrix metalloproteinase activity

To determine activity of matrix metalloproteinases (MMPs) in LNCaP cells with altered TPD52 expression, 3 × 10⁵ cells/mL in RPMI 1640 medium were transfected with suitable vectors (pEGFP or pEGFP-TPD52 or shRNA specific to TPD52 or scrambled shRNA encoding vectors); 24 h after transfection, cells were washed with PBS and allowed to grow in the serum-free RPMI 1640 for 24 h. The supernatant medium was separated on SDS-PAGE containing 0.1% (w/v) gelatin as a substrate at 4°C. Then, the gel was washed with 2.5% Triton X-100 three times (each 10 min interval) at room temperature and subsequently incubated for 24 h in activation buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 35.5 mM CaCl₂, and 0.2% Brij-35) at 37°C. Subsequent to activation of proteases, the gel was stained for 3 h in Coomassie staining solution and then destained with 40% methanol/10% acetic acid until the areas of protease activity appeared as clear bands. Protein estimation was performed using the Bradford reagent to ensure equal loading of conditioned medium for each sample.³⁰

NF-κB reporter activity assay

To determine NF-κB reporter activation in TPD52 overexpressing LNCaP cells, cells were seeded in triplicates in 24-well plate and co-transfected with pGL3-NFκB-RE along with pEGFP or pEGFP-TPD52. After 24 h of transfection, cells were directly lysed in cell lysis buffer, and then, the luciferase activity was measured by using a kit (Promega-E4530) according to manufacturer’s protocol (Promega Corporations, Fitchburg, WI). Briefly, the cells were washed with PBS and lysed directly in 1× cell culture lysis reagent (CCLR) provided by the manufacturer. The lysate was centrifuged at 12,500 r/min for 2 min at 4°C to remove insoluble portion, and supernatant was collected for measuring luciferase activity. A volume of 10 µL of sample was mixed with 50 µL of substrate solution in a microtiter plate (MTP), and luminescence was measured using multi-mode reader (Tecan Pro200, Maennedorf, Switzerland) within 2 min. The luminescence observed is directly proportional to NF-κB reporter activity. The observed luminescence is normalized to total protein concentration of the samples. All the experiments were performed in triplicates and the results are presented as ±SD.

Nuclear and cytosolic fractionation

To determine enrichment of NF-κB in nucleus of LNCaP cells with TPD52 overexpression, nuclear and cytosolic fractions were prepared as reported previously.³² LNCaP cells were rinsed with PBS and scraped using cell scraper directly in buffer A (50 mM HEPES (pH 7.4), 19 mM KCl, 1 mM EDTA, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM DTT, and 0.025% NP40 along with protease inhibitor cocktail (10 µL/mL)). For complete cell lysis, the lysate was incubated with constant mixing for 30 min at 4°C. Then, the lysate was centrifuged at 4000g for 10 min to collect cytosolic fraction in supernatant. The remaining pellet was washed four times with buffer A followed by centrifugation at 4000g for 5 min each at 4°C. The pellet was resuspended in Buffer B (50 mM HEPES (pH 7.4), 400 mM KCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT containing protease inhibitor cocktail (10 µL/mL)) and incubated on shaking rotor for 30 min at 4°C. The nuclear fraction was collected by centrifugation at 14,000g for 30 min. To determine quality of fractionation, individual fractions were analyzed in western blot for detection of GAPDH (cytosolic protein) and H3 histone (nuclear marker).

Enzyme-linked immunosorbent assay for measurement of cytokines (IL-6, IL-8, and TNF-α)

To determine the effect of TPD52 on expression of cytokines IL-6, IL-8, and TNF-α, conditioned medium was collected and centrifuged to remove debris for 10 min at 12,000 r/min. Supernatant was separated carefully and stored at −30°C until further use. The amount of cytokines secreted into medium was estimated using specific enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer’s protocol (ELISA MAX Deluxe Set Human Kits: IL-6: 430505, IL-8: 431505, and TNF-α: 430205, BioLegend, San Diego, CA). Briefly, 96-well plates were coated with capture antibody overnight at 4°C and then blocked with 1 × assay diluent A for 1 h at room temperature with constant shaking at 200 r/min. Then, media supernatant and cytokine standards (1000–7.8 pg in 1× assay diluent) were added to designated wells along with suitable controls. Plates were incubated in room temperature for 2 h with constant shaking. After capturing the analytes, samples were replaced by respective detection antibodies and incubated for 1 h to form a sandwich complex. Followed by washing to remove excess antibodies, all the wells were filled with Avidin–HRP solution allowing binding with detection antibody for 30 min. To detect complexes, specific substrate solution was added which turns yellow to blue. The reaction was stopped by 2 N H₂SO₄ for 15 min. The absorbance was measured at 570 nm with reference to 450 nm. A standard graph was plotted from absorbance values of standards. The absorbance of unknown samples was interpolated with standard graph to determine concentration of cytokines in culture media. All measurements were performed in triplicates; results are expressed as ±SD.

Statistics

All cell-based assays were carried out in triplicates, and each assay was repeated three times. The data presented are the mean of triplicates and median of three repeated experiments. Statistics program, GraphPad Prism V5, was used to test significance of the results with t-test and calculate p values with 95% confidence interval. The p values ≤0.05 are considered as significant. In RT-PCR and western blotting experiments, densitometry was performed. The relative expression of genes or proteins was determined by normalizing to expression of housekeeping genes or proteins, respectively. The fold change in expression is presented on the respective images in figures.

Results

TPD52 positively regulated NF-κB pathway in PCa cells

Previously, we have identified a list of differentially regulated proteins in PCa patients compared to benign prostate hyperplasia.³³ To identify potential candidate proteins associated with PCa initiation and its progression, we have made an attempt to map all the proteins to cancer-relevant pathways and networks that involved deregulated proteins. The global protein network generated showed a significant interconnectivity between proteins indicating their co-regulation and/or interaction with cancer-relevant signaling pathways. However, the network architecture is more complex to determine the nodes (proteins) joined. Therefore, we have checked significant subnetworks including expression and protein-to-protein interactions for altered proteins specifically in PCa patients. In the network analysis, the proteins (nodes) with high degrees of connectivity are considered to be the central regulators of a network (Figure 1). Interestingly, one of the most significant subnetworks is enriched with proteins from NF-κB pathway. In this subnetwork, TPD52 family proteins are interconnected very well with NF-κB pathway proteins highlighting their role in regulation of the NF-κB signaling and its associated pathways.

Figure 1.

Protein network analysis of differentially expressed protein in prostate cancer patients. Protein–protein physical and functional interaction networks were generated. One of the significant subnetworks displayed TPD52 along with its regulatory protein network.

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To validate whether TPD52 has any role in activation status of NF-κB pathway, we performed NF-κB-specific luciferase reporter assay in LNCaP cells with altered TPD52 levels upon both overexpression and downregulation (Figure 2(a)). In LNCaP cells, luciferase activity is significantly increased with overexpression of TPD52 confirming that the NF-κB activation is regulated by TPD52. In contrast, downregulation of TPD52 by shRNA has clearly reduced the NF-κB reporter activity without affecting RelA and RelB expression. Notably, this result excludes the possibility that NF-κB activation is not due to the altered expression of both RelA and RelB proteins and highlights that TPD52 has an important role in NF-κB activation (Figure 2(a) and (d)). Conversely, overexpression of TPD52 in the presence of parthenolide, an inhibitor against NF-κB, did not show any effect on activation of NF-κB promoter (Figure 2(b)). Moreover, in addition to the basal activity of NF-κB, we also demonstrate that the NF-κB activation by TNF-α is also stopped by TPD52 knockdown in LNCaP cells (Figure 2(c)). Together, these experiments draw attention to the fact that TPD52 has an effect on NF-κB activation.

Figure 2.

Reporter assay for determining activation of NF-κB in LNCaP cells with altered TPD52 expression. (a) NF-κB luciferase reporter activity in LNCaP cells upon overexpression and downregulation of TPD52. (b) NF-κB luciferase reporter activity in LNCaP cells upon TPD52 overexpression and NF-κB inhibition using parthenolide. (c) NF-κB luciferase reporter activity in LNCaP cells upon TPD52 overexpression and TNFα treatment. The results are the mean ± SD of three independent experiments (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001). (d) Expression of NF-κB subunits (RelA and RelB) upon altered expression of TPD52 in LNCaP cells.

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Altered expression of TPD52 affects the proliferation of LNCaP cells

To determine physiological role of TPD52 in LNCaP cells, we analyzed whether overexpression or downregulation of TPD52 has any effect on LNCaP cell proliferation. To modulate TPD52 expression in LNCaP cells, a recombinant vector expressing EGFP-TPD52 fusion protein and a vector expressing shRNA against TPD52 were generated. LNCaP cells were transfected with respective vectors either for overexpression or downregulation of TPD52 or control vectors. The stable cell lines selected were assessed for altered TPD52 expression. LNCaP cells with EGFP-TPD52 or EGFP alone as control have confirmed increased expression of TPD52 at both messenger RNA (mRNA) and protein levels. In cells transfected with shRNA producing vectors, TPD52 is significantly downregulated compared to control cells expressing scrambled shRNA (Figure 3(a)). To determine the effect of TPD52 expression on cell proliferation, sulforhodamine B (SRB) assays were performed after overexpression or downregulation of TPD52 in LNCaP cells. The proliferation assays showed a significant increase in proliferation of LNCaP cells with exogenous overexpression of EGFP-TPD52, whereas knockdown of TPD52 led to decreased cell proliferation (Figure 3(a)).

Figure 3.

Modulation of TPD52 expression in androgen-dependent prostate cancer cells (LNCaP). (a) Overexpression and downregulation of TPD52 by pEGFP-TPD52 fusion protein expression and gene-specific shRNA, respectively. TPD52 mRNA expression was assessed by semi-quantitative RT-PCR and protein levels measured by western blot. EGFP (left panel) or EGFP-TPD52 (right panel) fusion protein produces recombinant vectors transfected into LNCaP cells, and expression of EGFP tag alone or fusion proteins was confirmed. (b) LNCaP cell proliferation after overexpression of TPD52 and downregulation of TPD52; results are the mean ± SD of three independent experiments (*p ≤ 0.05 and **p ≤ 0.01). (c) Expression of anti-apoptotic proteins Bcl-2 and XIAP and cell-cycle regulator cyclin D1 in LNcaP cells with altered TPD52 expression. (d) Expression of pro-apoptotic proteins PARP and Bax and cell-cycle inhibitor p27kip1 in LNcaP cells with altered TPD52 expression. Only representative blots are shown.

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Previously, it has been shown that TPD52 regulates apoptosis and migration of PCa cells. To determine whether LNCaP cells’ growth arrest was mediated via the effect of TPD52 on regulation of pro- and anti-apoptotic proteins, we have measured expression levels of key apoptosis regulating proteins in cells with altered TPD52 expression. Western blotting with cell lysate collected from cells in which TPD52 expression is altered showed that cyclin D1, B-cell lymphoma 2 (Bcl-2), and X-linked inhibitor of apoptosis protein (XIAP; anti-apoptotic) protein levels were substantially higher as a result of TPD52 overexpression in LNCaP cells (Figure 3(c)). In LNCaP cells with depleted TPD52 expression, these anti-apoptotic proteins are downregulated (Figure 3(c)). In support of TPD52’s role in growth arrest and apoptosis, we extended to analyze pro-apoptotic proteins upon TPD52 knockdown in LNCaP cells. As expected, our results clearly show that Bax (a pro-apoptotic protein) and p27Kip1 (a cell-cycle inhibitor) are accumulated in LNCaP cells with TPD52 downregulation (Figure 3(d)). Also, activation of poly(ADP-ribose) polymerase (PARP) is evident by appearance of cleaved PARP after TPD52 knockdown (Figure 3(d)). In line with these results, due to overexpression of TPD52, both Bax and p27kip1 levels are decreased compared to control cells. No PARP cleavage is observed with TPD52 overexpression in LNCaP cells. These results together suggest the PARP cleavage and accumulation of Bax and p27Kip1 as a result of TPD52 downregulation which might interfere with cell-cycle progression and thus influence cell growth and survival.

TPD52’s role in expression of pro-inflammatory genes may confer its pro-tumorigenic potential

Promoter-specific reporter assays have confirmed that the altered TPD52 expression leads to the activation of NF-κB in LNCaP cells. NF-κB activation involves its increased transcriptional potential and nuclear localization of its p65 subunit, thereby increasing the expression of its target genes. A big set of genes for expression of chemokines, cytokines, cell survival proteins, and cell adhesion molecules are transcriptionally controlled by NF-κB. With overexpression of TPD52 in LNCaP cells, we detected an increase in phosphorylation of NF-κB p65 subunit compared to cells with EGFP expression (Figure 4(a)). As NF-κB plays a key role in the regulation of expression of different cytokines, we next investigated the role of NF-κB in the release of cytokines relevant to cancer progression. Indeed, in agreement with the increased NF-κB reporter activity by overexpression of TPD52, mRNA levels of NF-κB targets such as TNF-α and IL-8 were significantly increased in LNCaP cells. Contrarily, after downregulation of TPD52 with reduced NF-κB reporter activity, TNF-α and IL-8 were significantly downregulated in LNCaP cells (Figure 4(b)). Additionally, we have also measured IL-6, IL-8, and TNFα secreted into culture media by LNCaP cells with or without TPD52 overexpression. The obtained results clearly show that there is a significant increase in IL-6, IL-8, and TNF-α secreted by cells with EGFP-TPD52 expression compared to control cells with EGFP alone (Figure 4(c)). Taken together, these results substantiate an affirmative role of TPD52 in conferring the NF-κB-dependent transcription of pro-proliferative proteins in PCa cells.

Figure 4.

(a) Overexpression of TPD52 led to NF-κB activation by increasing NF-κB p65 (Ser536) phosphorylation, whereas its depletion led to inactivation due to significant reduction in phosphorylated NF-κB p65 while its total protein remains unaffected. (b) RT-PCR analysis of IL-8, TNFα, and NF-κB target genes in LNCaP cells upon TPD52 overexpression and downregulation. (c) Analysis of cytokines secreted IL-6, IL-8, and TNFα upon TPD52 overexpression in LNCaP cells. The results are the mean ± SD of three independent experiments (**p ≤ 0.01).

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TPD52 induces expression and activation of extracellular matrix proteins and adhesion molecules

Previously, it has been reported that TPD52 stimulates LNCaP cell migration through αVβ3 integrin via activation of protein kinase B (PKB)/Akt pathway.⁷ Conversely, NF-κB controls the expression of adhesion molecules involved in cellular contacts and stimulating cell migration. Considering that TPD52 may be involved in regulation of cell migration through NF-κB-targeted proteins, we next studied whether altered expression of TPD52 has any influence on expression of extracellular matrix (ECM) proteins and adhesion molecules involved in cell migration, thereby promoting metastatic potential of cancer cells. In this study, RT-PCR results revealed that both MMP-9 and MMP-2 are upregulated significantly in LNCaP cells with overexpression of TPD52 (Figure 5(a)). Furthermore, western blotting results showed that MMP-9 and MMP-2 protein levels were notably higher as a consequence of TPD52 overexpression in LNCaP cells (Figure 5(b)). To determine the consequence of MMP expression, we have measured the activity of MMPs with altered TPD52 expression in LNCaP cells. It is clear that MMP-9 activity is increased in LNCaP cells with TPD52 overexpression (Figure 5(b)). Contrary to this, MMP-9 activity is significantly decreased with shRNA-mediated downregulation of TPD52. Activity of MMPs is regulated and controlled by the homeostasis between MMP inhibitors such as tissue inhibitor of metalloproteinases (TIMPs) and RECK. Therefore, we measured the expression of key inhibitors TIMP-1, TIMP-2, and RECK proteins in TPD52-positive and depleted LNCaP cells. From the results, it is clear that the overexpression of TPD52 showed positive regulation on TIMP-1 expression but a significant negative regulation on TIMP-2 and RECK expression in LNCaP cells. In line with these observations, downregulation of TPD52 reverted expression of TIMPs and RECK protein in PCa cells (Figure 5(a)).

Figure 5.

(a) RT-PCR analysis of MMP and TIMP expression in LNCaP cells with altered TPD52 expression. (b) Western blots analysis for protein level expression of MMPs and gelatin zymography for activation of MMPs upon modulation of TPD52 expression in LNCaP cells. (c) Western blot analysis showed altered expression of E-Cadherin, Vimentin and V-Cam in LNCaP cells with overexpression of TPD52 and the same was reversed with NF-κB. (d) Activation of FAK in TPD52 overexpressing LNCaP cells is NF-κB dependent.

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Plethora of studies has shown a positive correlation between cancer cell metastasis and elevated MMP levels and activity. It is known that TPD52 promotes tumor cell invasion and migration, thereby disseminating tumor cells toward metastasis. Metastasis is a multistep process involving many different regulatory proteins. As we observe changes in the expression of MMPs and TIMPs with altered TPD52 in cancer cells, TPD52-induced invasive potential of tumor cells may be supported by MMPs acting on cell adhesion molecules. Since several molecules including receptors expressed on tumor cell surface control cell adhesion, we have measured expression of key adhesion molecules in TPD52-positive LNCaP cells. Our results show that the overexpression of TPD52 led to a decreased E-cadherin and increased expression of vimentin and vascular cell adhesion molecule (VCAM) in LNCaP cells, and their expression is reversed by NF-κB inhibition by parthenolide, a specific inhibitor against NF-κB pathway activation (Figure 5(c)). Furthermore, focal adhesion kinase (FAK) signaling pathway controlled by cell surface integrins associated with cell adhesion is activated in TPD52-positive LNCaP cells by increased phosphorylation of FAK compared to control cells (Figure 5(d)).

TPD52 promotes nuclear translocation of NF-κB and interacts with p65 NF-κB subunit

As we observed that the altered expression of TPD52 in LNCaP cells varies NF-κB promoter activity, and we have analyzed whether TPD52 directly affects NF-κB subunit, thereby regulating expression of its target genes in LNCaP cells. First, we studied whether TPD52 has an effect on localization of NF-κB p65 subunit in LNCaP cells. The nuclear and cytosolic fractions were prepared from the LNCaP cells expressing EGFP-TPD52 or EGFP alone. The fractions were analyzed in western blotting for detection of NF-κB p65 along with GAPDH and histone H3 as cytosolic and nuclear reference markers, respectively. Both detected markers confirmed the purity of prepared cellular fractions. Normalization of detected NF-κB levels to the corresponding markers GAPDH or histone H3 shows that p65 subunit levels are higher in the nuclear fraction compared to the cytosolic fraction (Figure 6(a)). To further confirm translocation of NF-κB in TPD52-positive LNCaP cells, immunocytochemistry has been performed with anti NF-κB p65 antibody. Direct immunofluorescence for detection of NF-κB shows that there is an increase in red fluorescence for NF-κB in the nucleus of LNCaP cells with FLAG-TPD52 expression compared to control cells with FLAG alone (Figure 6(b)). This result clearly indicates that TPD52 promotes nuclear translocation of NF-κB p65 subunit, thus promoting its transcriptional activity for expression of cell survival–related proteins.

Figure 6.

(a) Nuclear and cytosolic fractionation of LNCaP cells expressing EGFP alone or EGFP-TPD52. H3 histone and GAPDH are used as markers to confirm purity of nuclear and cytosolic fractions, respectively. (b) Co-localization of Flag-TPD52 with NF-κB p65. LNCaP cells were transfected with FLAG or FLAG-TPD52. After 24 h, immunofluorescence was performed with either anti-Flag or anti-NF-κB p65 antibody. In LNCaP cells, co-localization of FLAG-TPD52 (green) and NF-κB p65 (red) results yellow (merged). (c) Immunoprecipitation with anti-FLAG antibody. From FLAG-TPD52-positive LNCaP cells, immunoprecipitation was performed with anti–FLAG antibody and NF-κB p65 was detected with anti-NF-κB p65 antibody. (d) Immunoprecipitation with anti-NF-κB p65 antibody. From FLAG-TPD52-positive LNCaP cells, immunoprecipitation was performed with anti-NF-κB p65 antibody, and FLAG-TPD52 and IKBα were detected with anti-FLAG and anti-IKBα antibodies. IgG was used as isotype control in both experiments.

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To establish how TPD52 promotes NF-κB translocation, we tested whether TPD52 interacts with NF-κB in LNCaP cells. To evaluate this, co-immunoprecipitation was carried out from FLAG-TPD52 expressing LNCaP cell extracts with anti-FLAG antibody, followed by detection of NF-κB p65 in precipitates (Figure 6(c)). Our results from co-immunoprecipitation experiments show that NF-κB p65 is detected with FLAG-TPD52 precipitation but not with FLAG alone. IgG antibody was used as isotype control for immunoprecipitation. To verify specificity of this interaction alternatively, co-immunoprecipitation was carried out from same lysates with anti-NF-κB antibody followed by FLAG-TPD52 detection with anti-FLAG (Figure 6(d)). As expected, we also could detect IkBα as part of NF-κB complex along with TPD52. These experiments confirmed an interaction between TPD52 and NF-κB p65 subunit.

TPD52-dependent NF-κB activation promotes autocrine or paracrine activation of signal transducer and activator of transcription 3

It has been reported that TPD52 promotes phosphorylation of signal transducer and activator of transcription 3 (STAT3) in tyrosine705 through the activation of tyrosine kinase Janus kinase 2 (JAK2).³⁴ In search of the TPD52 mechanism in PCa, we observed that the overexpression of TPD52 increased the release of IL-6 through activation of NF-κB in LNCaP cells. The significant effect of TPD52 on the activation of NF-κB followed by release of IL-6 and other cytokines in LNCaP cells prompted us to investigate the role of TPD52 in the activation of STAT3 signaling pathway via NF-κB pathway. Overexpression of TPD52 led to activation of STAT3 (pSTAT3^Y705) in LNCaP cells, but active STAT3 levels are reversed significantly when the cells were treated with NF-κB inhibitor. In the same experiment, NF-κB activation is also observed, whereas its expression is not affected by TPD52 overexpression (Figure 7(a)). Furthermore, when STAT3 inhibitor (stattic) is added to TPD52-positive LNCaP cells, NF-κB p65 is inhibited by decreased phosphorylation (Figure 7(b)). As NF-κB plays a major role in the expression of different cytokines and cytokines activate STAT3 signaling pathway, we investigated the contribution of NF-κB in the expression of cytokines and its potential role in autocrine/paracrine activation of STAT3 in TPD52 expressing LNCaP cells. Indeed, with inhibition of NF-κB, we observed a significant decrease in the release of IL-6, IL-8, and TNF-α from TPD52-positive LNCaP cells (Figure 7(c)). Furthermore, it prompted us to explore whether TPD52 contributes to the activation of STAT3 through NF-κB that is known for autocrine/paracrine activation of STAT3 signaling pathway in LNCaP cells. As revealed in Figure 7(a), inhibition of NF-κB diminished pSTAT3^Y705 (activated STAT3), but STAT3 expression remains unaffected in LNCaP cells with TPD52 overexpression. Furthermore, conditioned media (CM) from TPD52-positive LNCaP cells led to elevated levels of pSTAT3^Y705 when added to wild-type LNCaP cells. We also found that the inhibition of NF-κB in TPD52-positive LNCaP cells resulted in a decrease in secretion of IL-6, and other cytokines decreased the capability of their conditioned medium to activate STAT3 (Figure 7(d)).

Figure 7.

Inhibition of NF-κB restrains autocrine/paracrine activation of STAT3 in LNCaP cells. (a) Western blot analysis shows an increased level of active STAT3^Y705 and NF-κB p65^S536 in LNCaP cells expressing TPD52. NF-κB inhibitor parthenolide attenuated the activation of STAT3. (b) A STAT3-specific inhibitor stattic also attenuated activation of NF-κB in LNCaP cells. (c) NF-κB inhibitor parthenolide diminished elevated secretion of cytokines by TPD52 expression in LNCaP cells (*p ≤ 0.05 and **p ≤ 0.01). (d) LNCaP cells were stimulated with IL-6 or conditioned medium (CM) from LNCaP cells expressing TPD52 or EGFP as a control. The IL-6 and CM from TPD52-positive LNCaP cells induce STAT3 activation (pSTAT3^Y705). The effect of CM on LNCaP cells was inhibited by NF-κB and STAT3 inhibition.

[Figure omitted. See PDF]

Discussion

Despite the advancements in early diagnosis and efficient treatment, PCa causes significant deaths per year in Western countries next to lung and colorectal cancers. Highest number of PCa patients die due to advanced PCa. It is still a challenge to treat effectively for saving patients’ life. Therefore, understanding molecular mechanisms involved in PCa progression allows developing new therapeutic approaches. TPD52 is an oncogene, which is overexpressed in ovarian, breast, and PCas due to gene amplification.^15,18,35 TPD52 gene is located at the chromosome 8q21.1, which is most frequently amplified in PCa. TPD52 expression in different cancer cell types is linked to various cellular functions including proliferation, and its expression is regulated by testosterone in hormone-responsive PCa cells. Previously, we showed that TPD52 contributes to increased PCa cell proliferation and migration phenotypes, and its expression stimulates phosphorylation of the PKB/Akt PCa cells.⁷ TPD52 promotes cell migration through activation of the αVβ3 signaling pathway in PCa cells. Also, TPD52 contributes to cancer cells evading apoptosis that is a remarkable feature of cancer cells. A tumor cell evades apoptosis through downregulation of pro-apoptotic proteins and upregulation of anti-apoptotic proteins.³⁶ A TPD52 family protein interacts with the apoptosis signal–regulating kinase 1 to control apoptosis of tumor cells.³⁷ In our previous studies, results from proteomic analysis followed by functional characterization showed the role of TPD52 in PCa progression. Another study also confirmed that overexpression of TPD52 family proteins in PCa cells promotes their metastatic potential in vivo and promotes castration-resistant PCa progression.^19,38,39 However, molecular-level mechanisms regulated by TPD52 toward cancer progression are still under investigation. These observations inspired us to explore the type of the molecular mechanism regulated by TPD52 to influence tumor initiation and dissemination.

In order to predict possible cancer pathways regulated by TPD52 proteins in cancer cells, we used protein network analysis, a system biology approach which connects specific disease-related proteins and builds networks based on the available databases as a rational strategy to proceed further. The system biology platform is a well-recognized application tool for identification of diagnostic and prognostic biomarkers and new drug targets specific to diseases.⁴⁰ We used GeneMANIA^™, which creates an interaction network with proteins showing physical or functional interactions such as pathway activation, inhibition, and protein modifications. From all the networks built for TPD52 family proteins, the protein–protein interaction network showed high number of interactions with activation and inactivation of NF-κB pathway. Remarkably, all three isoforms of TPD52 formed a single node along with MAL2, a known interaction partner for TPD52. This significant node showed a valid interaction with NF-κB pathway proteins. Therefore, we further investigated the effect of TPD52 expression on NF-κB activity in vitro for better understanding of TPD52 role in affecting essential mechanisms involved in tumor progression. The NF-κB-specific reporter assay results have confirmed that the overexpression of TPD52 in LNCaP cells promoted transactivation of NF-κB. Activation of NF-κB implicates its localization in nucleus leading to the expression of its targets that play important role in cancer progression.⁴¹ NF-κB is the key regulator in many inflammation-linked cancers like hepatocellular cancer (HCC) and colitis-associated cancer (CAC).⁴² In particular, NF-κB plays a vital role in controlling the expression of cell adhesion molecules, cytokines, and other mediators that can promote tumor initiation and progression.⁴¹ Though progression of PCa may involve some other driver factors for tumorigenesis, activation of NF-κB pathway is a central feature that contributes to PCa progression.⁴³ However, the biochemical basis of NF-κB activation or inactivation is still largely unknown. In this study, our results have identified a role of TPD52 in transactivation of NF-κB. TPD52 confers transactivation of NF-κB on expression of its target genes such as IL-6, IL-8, and TNF-α that are important in PCa progression. These findings ascertain TPD52 as a modulator of NF-κB pathway that plays a significant role in the progression of castrate-resistant and metastatic PCa.^{42,44 –47} Therefore, in this study, we sought to investigate how TPD52 promotes PCa initiation and progression in detail. As reported earlier, altered expression of TPD52 showed effect on rate of LNCaP cell proliferation in vitro. Exogenous expression of TPD52 in LNCaP cells induced expression of gene encoding anti-apoptotic proteins, whereas expression of pro-apoptotic proteins is significantly suppressed, promoting cell survival. Conversely, depletion of TPD52 in LNCaP cells showed increased expression of genes involved in induction of apoptosis and inhibition of cell-cycle progression. As suggested that the expression of these cell survival–related genes is under NF-κB transcriptional regulation, to evade apoptosis of cancer cells, TPD52 interferes with NF-κB signaling activation contributing to cellular growth and proliferation.

It has been proposed that cancer is attributed to alteration of several molecular signaling pathways controlling normal cell transformation to tumor cells, further progressing to metastasis involving dissemination of tumor cells into the surrounding tissues.³⁶ The expression of several genes, including ECM proteins for cell attachment, matrix metalloproteinase, and surface proteins such as integrins is important in determining the formation of metastatic cells.⁴⁸ Gene expression studies have revealed that the murine TPD52 induces metastasis-related genes in NIH3T3 fibroblasts.⁴⁹ We, and others, have shown that overexpression of TPD52 promotes LNCaP cell migration envisaging its role in dissemination of tumor cells for metastasis.⁷

Activated NF-κB is associated with PCa progression through MMP expression and/or activation or inducing androgen independence.⁵⁰ Interestingly, our results in this study implicate that overexpression of TPD52 induces expression and activity of MMP-9 in LNCaP cells. TIMPs will regulate intracellular activity of MMPs, and there are four TIMP family members, including TIMP-1, TIMP-2, TIMP-3, and TIMP-4.⁵¹ In LNCaP cells, TPD52 expression altered the expression of TIMP-1 and TIMP-2 enabling increased MMP-9 activity. Typically, TIMP-2 functions both as inhibitor and activator of MMPs and also induces expression of RECK, resulting in inhibition of cell migration.⁵² Overexpression of TPD52 led to decreased expression of RECK as reported, thereby promoting cancer cell migration. RECK is a plasma membrane protein, a known inhibitor of MMPs including MMP-2 and MMP-9. In invasive cancers, MMP-9 interacts with αVβ3 integrin, thereby promoting cancer cell migration.⁵³ Previously, we have shown systematically that TPD52 promotes migration and attachment of LNCaP cells to vitronectin through activation of αVβ3 signaling pathway.⁷ These results are further supported by this study indicating that the overexpression of TPD52 activates MMPs, which in turn activate αVβ3 signaling and consequently increase migration of LNCaP cells. MMPs play a significant role in tumor cell invasion and metastasis through their overexpression and/or increased activation.^54,55 Active MMPs target proteolysis of ECM proteins and cell adhesion receptors such as integrins and E-cadherin, thus disrupting cell-to-matrix and cell-to-cell adhesion properties.⁵⁶ Results from this study show that with overexpression of TPD52, E-cadherin levels are significantly reduced compared to control LNCaP cells. This may be due to the fact that activated MMPs targeting E-cadherin leads to disruption of cell adhesion, invasion of tumor cells, and ultimately initiation of epithelial–mesenchymal transition (EMT). In support of this statement, we also found vimentin, an EMT marker significantly increased in LNCaP cell with TPD52 expression. Interestingly, NF-κB inhibition did not allow positive regulation of vimentin expression by TPD52 in LNCaP cells. Conclusively, overexpression of TPD52 induced expression and activation of MMPs or downregulation of endogenous MMPs inhibitors (TIMPs), which affects the ratio of MMPs and TIMPs in tumor cells and ultimately leads to PCa progression.

Furthermore, dissemination of tumor cells is dependent on cell adhesion properties regulated by cell surface receptors and ECM proteins. For instance, ECM proteins such as fibronectin and vitronectin directly bind to their specific integrins (αVβ1, αVβ3, αVβ1, αVβ5, αVβ6, and αVβ8) and activate cell signaling through cytoskeletal rearrangement for cell adhesion.⁵⁷ Ligation of integrins with ECM ligands activates FAK signaling pathway regulating cellular functions, including cell survival, proliferation, migration, and adhesion. Already it has been reported that FAK pathway is activated in PCa leading to increased tumor cell growth.⁵⁸ From our results, we found that overexpression of TPD52 in LNCaP cells activated FAK, as shown by its increased phosphorylation. However, overexpression of TPD52 along with inhibition of NF-κB activity did not show significant increase in activation of FAK. These results suggest that the activation of NF-κB is required for TPD52 to activate FAK pathway promoting LNCaP cell migration.

It has been described that NF-κB is implicated in many cancers including PCa, but so far understanding molecular-level factors responsible for NF-κB activation is continuously under investigation. Often, NF-κB activation in cancers is due to inflammation through overexpression of tumor-promoting cytokines (IL-6 or TNF-α) and cell survival factors including Bcl-2 family proteins.⁴³ In this study, it was observed that the expression of TPD52 in LNCaP cells has increased phosphorylation of NF-κB (p65) at serine 536 that is associated with transactivation of NF-κB p65 subunit. Furthermore, we also observed a significant increase in NF-κB p65 in the nuclear fraction of LNCaP cells with TPD52 expression. This suggests that TPD52 promotes activation of NF-κB in LNCaP cells. However, the defined mechanisms of TPD52 followed for NF-κB activation are appealing to be determined. In cancers, NF-κB can be activated by diverse factors associated with tumor progression. In this study, we found increased secretion of IL-6 and TNFα by TPD52-positive LNCaP cells. These cytokines may be involved in NF-κB activation. Alternatively, TPD52 may be activating NF-κB directly and NF-κB induces expression of cytokines in LNCaP cells. Previous studies have shown that PKB/Akt can activate NF-κB in castration-resistant PCa.^42,50,59 Therefore, we made an attempt to elucidate detailed mechanism and found that TPD52 interacts with p65 subunit of NF-κB that can regulate expression of genes associated with cell growth. From these results, it appears that upon phosphorylation of IkB after being dissociated from IkB, NF-κB p65 interacts with TPD52 in cytoplasm. This interaction further may assist in transactivation of NF-κB by its nuclear translocation. Activated NF-κB can in turn induce the expression of its target genes including cytokines and adhesion molecules involved in tumor cell survival.

Formerly, Zhang et al.³⁴ reported that PrLZ protects cells from apoptosis by activation of AKT and STAT3 signaling pathways in LNCaP cells. PrLZ gene belongs to TPD52 family, and its C-terminal protein sequence shares homology with TPD52. Hitherto, we also reported that TPD52 promotes cell migration through activation of PKB/Akt pathway. STAT3 activation by phosphorylation on tyrosine⁷⁰⁵ residue has been shown to play a vital role in survival of PCa cells.^60,61 From this study, it is clear that TPD52 contributes to the activation of NF-κB signaling. NF-κB is progressively acknowledged as a central signaling pathway in multiple stages of cancer progression. In cancers, NF-κB assists and cross talk with multiple signaling pathways through kinases, STAT3, and p53 that regulate NF-κB activity or its related signaling pathways.⁶² Latest observations indicate that TPD52 family proteins activate STAT3 in PCa cells. In solid tumors, a cross talk between NF-κB and STAT3 pathways through elevated secretion of IL-6 contributes to cancer initiation and progression.⁶³ Therefore, we speculate that TPD52 proteins activate STAT3 via activation of NF-κB, and this cross talk may regulate expression of target genes involved in cell proliferation and anti-apoptosis properties of tumor cells. In this study, we could identify a role of TPD52 in regulation of NF-κB-dependent transcription of its target genes such as TNF-α, IL-6, and IL-8. Moreover, secreted IL-6 is involved in activation of STAT3 which is supported by rescue experiments presenting that the inhibition of NF-κB by its specific inhibitor could inverse STAT3 phosphorylation in TPD52 expressing cells, whereas blocking STAT3 activity could not change NF-κB p65 phosphorylation. From these results, we conclude that activated NF-κB acts as an upstream regulator of STAT3 activation in TPD52 expressing cells. Thus, this study proposes existence of cross talk between NF-κB and STAT3 signaling pathways through IL-6 and other cytokine secretion in LNCaP cells with increased expression of TPD52. Therefore, NF-κB and STAT3 together regulate expression of cell cycle–related and anti-apoptotic genes to promote tumor cell proliferation.

Authors acknowledge Dr Rajesh Chandra for careful reading of the manuscript for improvement and language corrections. R.U. contributed to the concept and design and overall supervision of the study. C.D., D.P.Y. and R.U. did the experiments. The write-up was done by R.U. and R.W. All authors contributed for reviewing and editing of the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by SMILE (CSC0111) project under 12th FYP to Chemical Biology, CSIR-IICT, Hyderabad from CSIR, India.