Prostate cancer remains a major public health problem in developed countries with an estimated 181,000 new cases in 2016 in the United States [
]. The disease can progress from a hormone sensitive to castrate-resistant phenotype and eventually metastasize [
]. Multiple factors, including screening using prostate specific antigen (PSA) levels, and an aging population have resulted in increased frequency of diagnosis of early stage prostate tumors, most of which do not require immediate therapeutic intervention [
]. However, a small number of high-grade tumors are underdiagnosed and undertreated.
Therapies for cancer including that of the prostate have shifted from administering broadly acting cytotoxic drugs to specific therapies targeted to each tumor. In order to facilitate the shift, a “precision-medicine” approach where tests that predict the clinical outcome of patients on the basis of genes expressed by their tumors are likely to influence patient management and drug development. Molecular signatures will have utility both in clinical management of disease and in elucidating the mechanism involved, thereby providing insight into potentially novel therapies [
Testis specific Y-like-5 (
) is a member of testis-specific protein Y-encoded-like (TSPY-L) family of genes, whose functions are currently unknown [
]. Testis specific Y-like (TSPYL) proteins are members of the nucleosome assembly protein (NAP) superfamily [
]. TSPYL proteins show high sequence homology to NAP’s which possess a highly conserved NAP domain (~180 amino acids) that participates in histone binding. In general the NAP proteins participate in transcriptional regulation [
] and in regulation of the cell cycle [
]. Also, NAP-1 shuttles histones between the cytoplasm and nucleus, assembles nucleosomes and affects transcription of many genes by promoting chromatin fluidity [
Silencing of tumor suppressor genes (TSG’s) by aberrant DNA methylation at critical gene control regions plays a central role in the development of cancers [
]. Alternately, a decrease in methylation at specific sequences could increase the expression of cancer-promoting genes [
gene is of particular interest because, apart from the documented role as a putative TSG in glioblastoma and gastric cancer [
], it has been implicated in cancer signaling pathways involving CDKN1A (p21, WAF1/Cip 1) and pAKT in lung carcinoma cells [
]. CDKN1A has been implicated in both anti-proliferative, pro-proliferative and survival roles [
]. Moreover, AKT activation increases cell survival and proliferation [
]. It is likely that TSPYL5 could participate in more than one function, depending on the cell type and its epigenetic modulation. Overall, little is known about the definite role of this gene in carcinomas including that of the prostate.
It is hypothesized that more advanced prostate tumors will have low
TSPYL5 gene and protein expression compared to moderately advanced or normal phenotype, and such differential expression of TSPYL5 is due to epigenetic modulation of this gene. To gain insight into the role of
TSPYL5 in prostate cancer, we investigated its expression, methylation pattern, its role in signaling pathways and drug sensitivity and presence of its protein with respect to disease severity. In this study we report that
TSPYL5 gene and protein expression varied in prostate adenocarcinoma (PC) cells and human benign and prostate tumor tissues as analyzed by qRT-PCR and immunoblotting. Consistent with variable TSPYL5 expression in cells and tissues, more advanced tumor tissues had an inverse correlation between methylation and gene or protein expression as studied by methyl-specific PCR (MSP), pyrosequencing (PSQ) and immunohistochemistry (IHC) analysis. We also report that in low TSPYL5 protein expressing PC cells, varied expression of proteins such as pAKT was observed. Moreover, TSPYL5 may play a role in sensitivity to chemotherapy likely by modulating pleiotropic protein such as CDKN1A.
Chemicals and antibodies
Demethylating agent 5-aza-2′-deoxycytidine (Decitabine, DT) was from (Sigma Chemical Company, St Louis, MO). Antibodies used were rabbit anti-TSPYL5 (Immunoblot), rabbit anti- CDKN1A (Thr-145) (Santa Cruz Biotechnology. Santa Cruz, CA), rabbit anti-TSPYL5 (Sigma, Immunohistochemistry), rabbit anti-AKT, mouse anti-DNMT3B (Novus Biologicals, Littleton, CO), anti-DNMT1, anti-PTEN, anti-β-actin, anti-Histone-H3, anti- p-CDKN1A (T-145), anti-pAKT (Ser- 473) (rabbit), including secondary HRP-conjugated anti-rabbit and mouse (Cell Signaling Technology, Danvers, MA). Chemotherapy drugs paclitaxel (px) and docetaxel (dtx) were procured from the local veterinary pharmacy.
Cells and patient tumor specimens
The PC cell lines, DU145, LNCaP and non-tumorigenic (NT) prostate epithelial cells RWPE-1 were purchased from ATCC (Manassas, VA). All of the carcinoma cells were maintained in custom RPMI or DMEM/F12 media with 10% FBS and Gentamycin. The RWPE-1 cells were maintained in a keratinocyte serum free media with growth factor supplements. The cells were tested routinely for mycoplasma contamination with the MycoAlert luciferase kit (Lonza, Allendale, NJ). Archival formalin fixed paraffin embedded (FFPE) tumor specimens from normal, benign or prostate carcinoma patients were obtained from the Pathology department at the University of Missouri Hospital after institutional IRB approval.
Demethylation of TSPYL5 in PC cells
The PC cells DU145 and LNCaP were treated with a demethylation drug DT (0.5 μM) for 4 days with fresh addition of DT every 12 h. Subsequently, total RNA was isolated and reverse transcribed to cDNA. qRT-PCR was performed to analyze
TSPYL5 gene expression in drug treated and untreated samples.
cDNA synthesis and PCR amplification
Total RNA from prostate carcinoma cells (DU145 and LNCaP), epithelial cells (RWPE-1) and FFPE prostate tissues was extracted using RNeasy or RNeasy FFPE kits (Qiagen, Valencia, CA), respectively. cDNA was generated from total RNA using a cDNA synthesis kit (Bio-rad, Hercules, CA). PCR was performed with
TSPYL5 primers. β-
actin was used as a housekeeping gene. The PCR conditions were as follows: denaturation at 98 °C for 1 min, followed by 28 cycles at 95 °C for 30 s, 55 °C for 30 s and 70 °C for 30 s, with a final extension at 70 °C for 8 min. The amplified PCR products were analyzed by 2% agarose gel electrophoresis containing Gel Red (Biotium, Hayward, CA). Quantitative real-time PCR (qRT-PCR), was performed with CFX Connect and a Sybr Green reaction (Biorad). The following primers were used for
TSPYL5 PCR: Forward, 5′-TGGGCCCTTCTACTGGTGAACTTT-3′; Reverse, 5′- TCACCTGGAGCCACAGCATAATGA-3′. The mRNA expression in tissues was analyzed and the relative cumulative density was calculated by measuring area under curve (AUC) for each sample using an image processing and analysis program (Image J, NIH). Percentage average was obtained for each group and an arbitrary number of 1 was assigned for highest percentage group and subsequent groups were assigned numbers relative to 1 for graphical representation.
Genomic DNA isolation and bisulfite conversion
The genomic DNA isolated from PC cells using DNeasy Blood & Tissue Kit or tumor tissues using QIAamp DNA FFPE Tissue Kit (Qiagen) was bisulfite-modified with EZ-DNA Methylation-Gold Kit (Zymo Research, Irvine, CA) according to manufacturer’s instructions. The bisulfite reaction was carried out with 500 ng genomic DNA. Bisulfite converted DNA samples were stored at −20 °C until further use.
Methyl specific PCR and pyrosequencing analysis
Methyl specific PCR (MSP) was performed in PC cells as well as FFPE tumor tissues using bisulfite-converted DNA with primers designed to include two CpG dinucleotides in each forward and reverse primer. Two sets of primers (CpG island) were designed; one, for methylated sequence (which retains CpG complementarity); 5′-GAGGTTATAGTTTAGGGGGAGTTG-3′; R- 5′- CCAAACAACACAAATACAAACTAAC-3′. For unmethylated sequences (complimentary to TpG sequence), the primers F- 5′-GAGAAATTTGTTGAGATTTAAAGTGA-3′; R- 5′CCATCACAAAAAAACATAATA-CACC-3′ were used. The presence of a methylated band in PCR is indicative of methylation in the original sequence [
]. Primers were designed using MethPrimer program [
]. The MSP and unmethylated sequence (USP) PCR bands in tissues upon gel electrophoresis (2% agarose) were analyzed for AUC using the Image J program. The percent methylation for each sample was calculated using AUC of methylated A (M) and unmethylated bands A (U) as follows: Percentage = A (M) ×100/A (M) + A (U).
Pyrosequencing (PSQ) of genomic DNA to quantitate the methylation of individual cytosine residues was performed as described earlier [
]. PSQ is a fast, reliable and quantitative method for analysis of CpG methylation [
]. Methylation analysis of DU145, LNCaP and RWPE-1 cells was performed with
specific primers (CpG island shores) which consisted of a forward (5′- AGAGAAAGTAAAGGTGGATGTTATAATGT-3′), biotinylated reverse (5′-Biosg/ATACTTCCATCCCTTACTATATAACCTA-3′) and sequencing primers (5′-AAAGGAGGTGTTGATAT-3′) designed for a
promoter sequence, followed by DNA sequencing in a Pyro Mark ID system by employing the Pyro Gold reagents kit (Life Technologies, Grand Island, NY). The primers were designed using a PSQ assay design program. The average degree of methylation at four CpG sites was analyzed using Pyro Mark ID software and results are depicted as percentage methylation.
Patient samples and IHC analyses
IHC studies were performed as described previously [
] to identify the protein expression levels and cellular localization of TSPYL5 in non-malignant and malignant FFPE human prostate tissues using intelliPATH FLX (Biocare Medical). The analyzed tissue specimens included core tissue from patients with prostate adenocarcinoma (Gleason scores (GS) ranging from 6 to 9), normal and benign prostate tissues. Human testis tissue was used as positive control to detect TSPYL5 protein expression.
Immunoreactivity was scored by a board-certified pathologist (ME) in at least five random fields at 400× magnification in each section and the intensity of protein staining was scored on a 0–3+ scale (0 = no staining, 1 + = weak staining, 2 + = moderate staining, and 3 + = strong staining). The percentage of cells staining positive was scored on 1 - 4 scale (1 = 0–25% positive PC cells, 2 = 26–50% positive cells, 3 = 51–75% positive cells, and 4 = 76–100% positive cells). Composite score (CS) (0–12) was obtained by multiplying the staining intensity and percent of immunoreactive cells. Statistical significance was evaluated by the Mann–Whitney test.
< 0.05 was considered significant. H & E staining was performed according to standard procedures described in literature. Grading is assigned according to 2005 International Society of Urological Pathology Consensus Statement on Gleason Grading of Prostate Cancer (Epstein JI, Allsbrook WC Jr, Amin MB, Egevad LL; ISUP Grading Committee. The 2005 International Society of Urological Pathology (ISUP) Consensus Conference on Gleason Grading of Prostatic Carcinoma [
TSPYL5 overexpression in LNCaP cells
For overexpression of TSPYL5, LNCaP cells were plated in 6 well plates (0.3-1 ×10
6/well) and allowed to grow to 70–80% confluency at 37 °C. The mammalian expression vector TSPYL5/pCMV6-AN-GFP (PV-TSPYL5) or pCMV6-AN-GFP (PV) (Origene, Rockville, MD) with N-terminal tGFP tag was transiently transfected into LNCaP cells using Lipofectamine 3000 (ThermoFisher Scientific, Walthem, MA) according to the manufacturer’s protocol and were allowed to grow for 72 h, harvested and subsequently used for further studies.
A cell viability assay was performed as described previously [
] using a WST-1 assay (Roche Applied Science, Indianapolis, IN) with or without 10 nM of chemotherapy drugs px or dtx. The results are expressed as percent viable cells after respective analysis. All experiments were performed in triplicate.
Protein was extracted from whole cell lysates using the M-PER mammalian protein extraction reagent (Thermo Scientific), and the concentrations were estimated by the Bradford method. Equal amounts of protein (35 μg) were loaded on to the gel. Subsequently, the proteins were blotted on to a nitrocellulose membrane. The membrane was probed separately with primary antibodies for TSPYL5, CDKN1A, and AKT including P-CDKN1A (Thr 145), pAKT (S-473), β-actin, histone H3, PTEN, DNMT-1 and DNMT3b. Following incubation with the primary antibody at 4 °C overnight, the membrane was incubated with a horseradish peroxidase-labeled secondary antibody and visualized with Luminate Forte Western HRP substrate (Millipore, Billerica, MA). The blot was imaged in a Kodak imaging station (Carestream Health). The protein band ratios were calculated from the protein band intensities obtained using Image J program.
Independent experiments were performed a minimum of three times. Statistical analyses on experiments were performed by unpaired two-tailed Student’s t-test for protein expression evaluations, one-way analysis of variance (ANOVA) for RT-PCR and Mann–Whitney U test for immunohistochemical analysis. The graphs were generated using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA).
P ≤ 0.05 was considered significant.
In this study we have demonstrated that the presence of DNA methylation in the 5′ region of the gene is negatively associated with expression of
mRNA and protein in PC cells, NT cells and clinical prostate tissue samples. Methylation induced TSPYL5 gene silencing was previously reported in glioma and gastric cancer types [
]. The TSPYL5 protein expression mirrored the expression pattern of mRNA in the cells (DU145, LNCaP or RWPE-1). The TSPYL proteins are members of the NAP superfamily of proteins [
] that have been shown to bind to proteins involved in transcription, cell cycle regulation [
], and shuttling histones between nucleus and cytoplasm [
]. However, it is not clear whether such a function for TSPYL5 exists in PC cells.
Previous studies with colorectal HCT116 cells indicated that both DNMT1 and DNMT3B enzymes were essential to methylate
gene promoter regions [
]. While one or the other enzyme was observed in the cells tested in this study, we observed only DNMT3B protein was predominantly expressed in more advanced PC tissues in which TSPYL5 was absent. Earlier studies in prostate cancer have analyzed various methyltransferases and found that DNMT1 expression was found to be lower than DNMT3b. Further, de novo methylation remains in DNMT1 knockout embryonic stem cells and the role of DNMT1 in tumor methylation remains ambiguous [
]. Depending on the cellular context, the TSPYL5 gene might be differentially targeted for methylation by methyltransferases.
Previous studies have shown a correlation between methylation in chromosome 8 region (Chr 8: 97278129–97278175) and loss of
gene expression in lung carcinoma cells, although, no tissue studies or normal cell studies have been done [
]. While CpG islands are important to regulate gene expression [
], previous studies suggest that the lower density CpG shores of islands may also be important [
]. Our studies with MSP analysis of the CpG island identified methylation of the
gene in PC cells and tissues. As anticipated, PSQ analysis of CpG dinucleotides on the 5′ shore of the CpG island revealed higher methylation of the four cytosine residues (Chr 8: 97278367–97278417) in DU145 cells relative to other cell lines. Only a subtle difference was observed in individual cytosine methylation between, LNCaP and RWPE-1. This is in keeping with our observations that DU145 had the least
expression due to methylation-induced gene silencing.
Planning treatment for prostate cancer patients relies on histopathological grading by GS [
] which currently lacks a precise molecular correlate [
]. There is a critical need to identify companion biomoleules that distinguish more advanced phenotype tumors within intermediate GS-7 specimens. Our studies identified
mRNA and protein expression in benign and tumor tissues with a GS-6 or-7. High grade tumors with GS ≥ 8 had the least expression of TSPYL5, likely due to DNA methylation. Interestingly, a few GS-7 tumor samples with Gleason pattern (4 + 3) had no message for
. At this time, it is not clear whether the absence of
mRNA expression in tissues with GS 7 (4 + 3) would indicate any undetected higher-grade disease. Further studies with more tissues are needed to assess this possibility. Taken together, these data suggest that the absence of TSPYL5 may be an indicator of more advanced prostate cancer disease.
MSP analysis of the
gene indicated a mixture of methylated and unmethylated bands in benign and intermediate-grade tumors with GS-6 or −7, while GS-8 tumors had predominantly methylated bands suggesting a methylation induced
silencing in these tumors. Previous studies indicated that
could be an independent marker of poor outcome in breast cancer based on their high expression in aggressive basal-like breast cancers [
]. On the contrary, we observed both by mRNA expression and IHC that TSPYL5 expression diminishes in high grade tumors. Such a difference in TSPYL5 expression could be exploited to identify the clinical behavior of intermediate grade prostate tumors (GS-7). A recent report suggested the use of higher levels of SNPs-rs2735839 to stratify patients with GS-7 because of the association with aggressive PC [
]. However, to classify GS-7 patients based on diminished TSPYL5, large cohorts of prostate tumor samples will need to be investigated. Studies along this direction are in progress in our laboratory.
In addition to its anti-proliferative role, CDKN1A is also vital to proliferation and survival. A previous study reported that knockdown of
increased the endogenous expression of p53 and its downstream target CDKN1A in MCF7 breast carcinoma cells [
]. It has been reported that in lung carcinoma cells, TSPYL5 was able to suppress CDKN1A by modulating PTEN/AKT pathway [
gene silencing increased the CDKN1A protein expression and caused growth reduction in cells [
]. However, we observed that TSPYL5 gene silenced cells (DU145) exhibited very minimal expression of CDKN1A. Conversely, low or moderately TSPYL5 expressing LNCaP and RWPE-1 cells showed high and relatively low CDKN1A expression. We also observed a decrease in CDKN1Aprotein expression in TSPYL5 overexpressing LNCaP cells. However, in contrast to lung carcinoma cells [
] LNCaP cells lack
, so any involvement of TSPYL5 in modulating CDKN1A must work by a PTEN-independent mechanism [
Our observations identified that even low
expressing cells (eg: LNCaP) had higher pAKT. Also, TSPYL5 expressing RWPE-1 cells expressed basal pAKT, albeit low levels compared to LNCaP cells. This in sharp contrast to the observation made in lung carcinoma cells [
] that high TSPLY-5 expression can activate AKT. The exact role of
is not clear in modulating AKT expression in PC cells and could vary depending on the cellular phenotype.
Mounting evidence suggests that TSG’s play an important role in the response to a variety of chemotherapeutic drugs such as px, 5-Fluorouracil, cisplatin and trastuzumab [
]. It was reported that a decrease in retinoblastoma (Rb) protein, a TSP, in sarcoma cells conferred resistance to doxorubicin and cisplatin [
]. PC cells and glioblastoma cells deficient in Rb were resistant to cisplatin and doxorubicin, respectively [
]. Similarly, p53 inactivation resulted in reduced sensitivity to cisplatin but not px in ovarian carcinoma cells, suggesting that the role of p53 in response to chemotherapy depends on both cellular context as well as the class of chemotherapeutic compounds [
]. Increased expression of CDKN1A leads to chemoresistance, and its loss sensitizes the cells to chemotherapy response [
]. Interestingly, it was reported earlier that LNCaP cells were resistant for dtx and knockdown of p53 protein increases its sensitivity to the drug by decreasing CDKN1A [
]. Previous studies had shown that increase in TSPYL5 can compete with USP7, a deubiqutinylating protein thereby decreasing p53 in MCF 7 cells [
]. Our studies show that reduction in CDKN1A in TSPYL5 overexpressing LNCaP cells exhibit more sensitivity for dtx and px compared to WT cells. All the above studies highlight the possible roles of TSP’s in chemotherapy response. Our studies suggest that increased TSPYL5 enhances the sensitivity of the cells to chemotherapy drugs, likely by downregulating CDKN1A. While it is tempting to suggest that
status in PC cells could be indicative of predicting chemotherapy response, further studies are needed to substantiate this notion. In keeping with the previous studies regarding the role of TSP’s in chemosensitivity [
], we speculate that the response to chemotherapy drugs in
expressing PC cells likely may vary depending upon the cellular context and the type of chemotherapy drug.
Equipment for this research was supported in part by an award from the Jay Dix Race for the Cure Fund from Ellis Fischel Cancer Center.
Not applicable. No funding from any major external public or private agencies has been obtained for this study. This work was supported by a faculty development award (SRK) from the College of Veterinary Medicine.
Availability of data and materials
Not applicable. No major genomic data to report.
SRK contributed for conception, design of the experiments, analysis and interpretation of the data, including writing of the manuscript. JNB has contributed for inputs and suggestions for the design of experiments, methylation analysis and critical reading of the manuscript. EM has made contributions in data interpretation and IHC analysis of tissue samples and suggestions for data involving pathology of the tumor samples. JA-L contributed to pyrosequencing analysis for methylation studies and data interpretation. TJM made substantial contribution in performing majority of the experiments and acquisition of data. All authors have given final approval of the version to be published.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Archival formalin fixed paraffin embedded (FFPE) tumor specimens from normal, benign or prostate carcinoma patients were obtained from the Pathology department, School of Medicine, University of Missouri. This study was approved by the ethics committee (IRB), University of Missouri. No human subjects were involved in this study and the consent for the use of de-identified patient data has been waived by the IRB committee, University of Missouri.