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The Y-linked proto-oncogene TSPY contributes to poor prognosis of the male hepatocellular carcinoma patients by promoting the pro-oncogenic and suppressing the anti-oncogenic gene expression
© The Author(s) 2019
- Received: 25 November 2018
- Accepted: 27 February 2019
- Published: 4 March 2019
Liver cancer is one of the major causes of cancer death worldwide, with significantly higher incidence and mortality among the male patients. Although sex hormones and their receptors could contribute to such sex differences, the story is incomplete. Genes on the male-specific region of the Y chromosome could play a role(s) in this cancer. TSPY is the putative gene for the gonadoblastoma locus on the Y chromosome (GBY) that is ectopically expressed in a subset of male hepatocellular carcinomas (HCCs). Although various studies showed that TSPY expression is associated with poor prognosis in the patients and its overexpression promotes cell proliferation of various cancer cell lines, it remains unclear how TSPY contributes to the clinical outcomes of the HCC patients. Identifying the downstream genes and pathways of TSPY actions would provide novel insights on its contribution(s) to male predominance in this deadly cancer.
To determine the effects of TSPY on HCC, a TSPY transgene was introduced to the HCC cell line, HuH-7, and studied with RNA-Seq transcriptome analysis. The results showed that TSPY upregulates various genes associated with cell-cycle and cell-viability, and suppresses cell-death related genes. To correlate the experimental observations with those of clinical specimens, transcriptomes of male HCCs with high TSPY expression were analyzed with reference to those with silent TSPY expression from the Cancer Genome Atlas (TCGA). The comparative analysis identified 49 genes, which showed parallel expression patterns between HuH-7 cells overexpressing TSPY and clinical specimens with high TSPY expression. Among these 49 genes, 16 likely downstream genes could be associated with survival rates in HCC patients. The major upregulated targets were cell-cycle related genes and growth factor receptor genes, including CDC25B and HMMR, whose expression levels are negatively correlated with the patient survival rates. In contrast, PPARGC1A, SLC25A25 and SOCS2 were downregulated with TSPY expression, and possess favorable prognoses for HCC patients.
We demonstrate that TSPY could exacerbate the oncogenesis of HCC by differentially upregulate the expression of pro-oncogenic genes and downregulate those of anti-oncogenic genes in male HCC patients, thereby contributing to the male predominance in this deadly cancer.
- Hepatocellular carcinoma
- Male predominance
- Transcriptome analysis
- TCGA dataset
The testis specific protein Y-encoded (TSPY) is a tandemly repeated gene (> 30 times) located within the gonadoblastoma locus on the Y chromosome (GBY), which predisposes XY patients with disorders of sexual development (DSDs) to gonadoblastoma development [1–3]. TSPY encodes a protein harboring a SET/NAP domain, initially identified in the SET oncoprotein and nucleosome assembly proteins (NAPs) [4, 5]. SET/NAP proteins play important roles in transcriptional regulation, chromatin assembly, histone chaperone, and cell-cycle regulation [6–10]. Previously, we demonstrated that TSPY interacts with the cyclin-B/CDK1 complex, eukaryotic translation elongation factor 1A (EEF1A), and androgen receptor (AR) via its SET/NAP domain, and enhances the respective functions of these binding partners [11–13]. Overexpression of TSPY accelerates cell proliferation in various cancer cell lines [11, 13, 14]. Further, TSPY directly binds to the chromatin at its own exon-1 region and amplifies its own expression in a positive-feedback manner , suggesting the potential role of TSPY as a transcriptional regulator. Others showed that TSPY could suppress TP53 functions and enhance cell proliferation by inhibiting USP7, a protective deubiquitinase for TP53 . TSPY is predominantly expressed at the early-stages of male germ cells, i.e. gonocytes and prespermatogonia at fetal testis  and spermatogonia and spermatocytes in adult testis . It is postulated to participate in mitotic proliferation and meiotic division of male germ cells . Importantly, TSPY is frequently expressed in germ cell tumors including gonadoblastoma, seminoma, and the carcinoma-in situ/intratubular germ cell neoplasia unclassified (CIS/ITHCNU), the precursor for all germ cell tumors [20–24]. Since TSPY is the primary gene located within GBY locus and is expressed in gonadoblastoma, TSPY is the putative gene for this oncogenic locus and could predispose dysfunctional germ cells to tumor development in dysgenetic gonads. Indeed, transgenic mouse studies showed that ectopic expression of TSPY in ovaries resulted in gonadoblastoma-like structures in female mice . Significantly, TSPY is also expressed in various types of somatic cancer; including prostate cancer, lung cancer, and hepatocellular carcinoma (HCC) [26–30]. Accordingly, TSPY could also promote oncogenic initiation and/or progression of somatic cancers in male patients.
Liver cancer causes more than 700,000 cancer deaths each year worldwide [31, 32]. Significantly the incidence and mortality of HCC is much higher in males than females, with greater than threefold difference [32–34]. Both sex hormones and/or their receptors and the sex chromosome genes have been postulated to contribute to such sex differences, the mechanisms of which, however, could be quite complex [35–40]. As a proto-oncogene on the Y chromosome, TSPY is expressed in a subclass of male HCC cases (30–40%), frequently associated with the global hypomethylation of the genomic DNA, including its own promoter sequence [27, 28]. However, beside its general functions in cell proliferation and growth, the exact mechanism(s) by which TSPY exerts on the oncogenic processes in HCC patients remains unknown.
In order to determine the effects of TSPY expression on HCC, we overexpressed TSPY in the HCC cell line, HuH-7, and analyzed the differential gene expression patterns between HuH-7 cells expressing and non-expressing the TSPY transgene using transcriptome analysis, and correlated the results with those derived from comparative study between the male HCC cases with high TSPY expression and non-expression (silent) from the Cancer Genome Atlas (TCGA) [27, 41]. We identified 16 genes that were potentially regulated by TSPY and their differential expression could be correlated with the prognoses of the HCC patients. Among these genes, TSPY upregulates cell-cycle regulators and components of cell-division machinery, e.g. BUB1, CDC25B, CDC45, CENPA, PRC1, PRIM1, RRM2, and SPC24, as well as growth factor receptors, e.g. ADGRD1 and HMMR, which are associated with unfavorable prognosis and poor survivals of the patients while the expression of PPARGC1A, SLC25A25 and SOCS2 are inversely correlated with TSPY expression but are associated with favorable prognosis and better survival of the patients. Our results suggest that ectopic expression of the Y-located TSPY could promote the expression of pro-oncogenic genes and suppression of anti-oncogenic genes in HCC, thereby contributing to the male predominance of this deadly cancer.
Cell culture and lentiviruses
Western-blot was performed as described previously , using anti-TSPY mouse monoclonal antibody (clone 17, generated in our laboratory) and anti-β-actin mouse monoclonal antibody (clone AC-15, Sigma-Aldrich). Immunoreactive signals were visualized by IRDye680RD conjugated anti-mouse IgG antibody, and recorded by the Odyssey system (LI-COR, Lincoln, NE).
Immunofluorescence was performed as described previously . Briefly, cells were fixed with 4% paraformaldehyde-PBS solution for 5 min, and permeabilized by methanol treatment. After blocking with 3% bovine serum albumin (Sigma)-PBS solution for 1 h, the slides were incubated with primary antibodies at 4 °C overnight. The primary antibodies used for immunofluorescence were anti-GFP goat IgG (Abcam) and anti-TSPY mouse monoclonal antibody (clone 17). The immunoreactive signals were visualized by Alexa Fluor 488 conjugated anti-goat IgG antibody (Invitrogen) and Alexa Fluor 594 conjugated anti-mouse IgG antibody respectively. Nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche Applied Science). Fluorescent images were recorded with a Nikon Eclipse Ti fluorescence microscope and image acquisition workstation (Nikon instrument Inc., Melville, NY).
RNA preparation and RNA-Seq transcriptome analysis of the transduced HuH-7 cells
Total RNA was isolated from the cells cultured in a 6-well plate (9.6 cm2 surface area) at 24 h after Dox-induction using TRIZOL-Plus RNA purification kit (ThermoFisher Scientific, NY). One μg total RNA from each triplicate sample was used for the library preparation using KAPA Stranded mRNA-Seq kit (Kapa Biosystems, MA). Libraries were indexed (bar-coded) with NEBNext multiplex primers for Illumina (New England Biolabs, CA). The libraries were subjected to 75 single-end read cycles of sequencing on the NextSeq 500 (Illumina, CA). All procedures were performed according to the manufacturer’s instructions.
The sequence reads were mapped onto the Ensembl GRCh37 (hg19) human reference genome using TopHat (version 1.0.1) , after quality assessment by FastQC program (version 0.11.4) . The mapped reads were summarized and calculated to the count reads, which were then quantitated as the expression levels using the featureCounts (version 1.5.1) program . Normalization and differential gene expression analysis were performed using an R package TCC .
Gene expression enrichment analysis, functional network analyses and pathway analyses were performed with Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, build version 463341M) and DAVID bioinformatics resources . A schematic diagram of cell cycle pathway was downloaded from KEGG website (https://www.genome.jp/kegg/) with permission .
Data set and data mining analysis of HCC specimens from TCGA
The RNA-Seq gene expression data and associated clinical information of HCC cases at the Cancer Genome Atlas (TCGA) data portal were downloaded from the UCSC Xena Browser . The datasets included 50 non-tumor samples (28 male cases and 22 female cases) and 371 tumor samples (250 male cases and 121 female cases). The expression levels were calculated as RSEM normalized read count . The survival information of the respective HCC patients was obtained from the Human Protein Atlas (HPA) data portal , except for classification of the high TSPY-expressing patients and the TSPY-silent patients. Statistical analyses were performed with the Prism6 program (GraphPad Software, Inc., La Jolla, CA).
TSPY-overexpression enhances cell proliferation in HuH-7 cells
The effects of TSPY in HCC were examined in the HCC HuH-7 cells, which were transduced with Tet-On lentiviral vectors overexpressing TSPY-ires-EGFP and EGFP alone under doxycycline-induced conditions, hereby designated as HuH-7-tetON-TSPY and HuH-7-tetON-EGFP respectively (Fig. 1a). HuH-7-tetON-EGFP cells served as a control for all experiments in the study. Western-blot analyses confirmed the expression of TSPY and EGFP being appropriately induced by Dox in the culture media (Fig. 1b). Immunocytochemical analyses showed that the TSPY protein was localized in both cytoplasm and nuclei (Fig. 1c), consistently with the observations in clinical HCC samples . Cell proliferation assays showed that overexpression of TSPY promoted cell proliferation in HuH-7 cells (Fig. 1d), as previously reported on other cancer cell types [13, 14, 16].
TSPY upregulated various cell-cycle related genes and suppressed tumor suppressor genes in HuH-7 cells
Using a set of DEGs derived from a more stringent expression level, we further analyzed the TSPY effects with the Ingenuity Pathways Analysis (IPA) program [53, 54]. Our results showed that the biological functions remarkably affected by TSPY overexpression were pathways associated with cell-cycle and cell viability (Additional file 2: Figure S1a and S1b). Noticeably, the pathways of necrosis and cell-death were inhibited (Additional file 2: Figure S1b, blue columns) while the pathways associated with cellular interphase processes and cell-survival were enhanced (Additional file 2: Figure S1b, red columns), as indicated by the |z-scores of − and + |> 2 for significance in inhibition and activation respectively. The IPA hepatotoxicity analysis demonstrated that the TSPY-induced DEGs were significantly associated with liver cancer development (Additional file 2: Figure S1c). Altogether, both gene annotation/ontology programs consistently suggested that the pathways of cell-cycle and cell viability could be mostly affected by TSPY overexpression in HuH-7 cells. To further illustrate the various cell cycle genes being affected by TSPY, we mapped the associated DEGs onto the KEGG cell-cycle pathway [48, 49]. Our results showed that the tumor suppressors CDKN1A (also known as Cip1 and p21) and GADD45 were downregulated while various positive cell-cycle regulators and components for DNA replication, including cyclin-B, cyclin-D, CDK1, CDC25B, and MCMs, were upregulated by TSPY-overexpression (Fig. 2c).
Overall, the transcriptome analysis of the HuH-7 cells overexpressing TSPY showed that TSPY could promote cell proliferation and viability by directly or indirectly augmenting a pro-oncogenic gene expression pattern consistent with the results of cell-proliferation assay (Fig. 1d).
High expression of TSPY is associated with poor survival rate in male HCC patients
Correlation of the differentially expressed genes between the TSPY overexpression in HuH-7 cells and TSPY-high group male HCC patients
Sixteen downstream genes potentially regulated by TSPY and associated with prognosis in HCC patients
Gene symbol (alias)
Effect by TSPY
Effect to HCC survival
An initiator for chromosomal DNA replication by recruiting DNA polymerase to chromatin
A component of the DNA polymerase synthesizing RNA primers for the Okazaki fragments
A ribonucleotide reductase catalyzing the biosynthesis of deoxyribonucleotides from ribonucleotide
A Ser/Thr protein kinase playing a central role in the spindle checkpoint.
A Thr protein phosphatase activating CDC2 and CDK1, playing key roles in initiation of G2/M phase
A centromere protein required for the recruitment of kinetochore proteins to centromere
A key regulator of cytokinesis by forming spatiotemporal midzone with KIF14, central spindle and midbody
A component of NDC80 complex organizing the stable microtubule binding sites of the kinetochore
A G-protein-coupled membrane receptor
A receptor of hyaluronic acid (HA); intracellular HMMR plays a role in cytokinesis
DNA repair and recombination
A DNA exonuclease functioning in DNA mismatch repair
A component of the DNA repairing machinery in homologous recombination
A prolyl hydroxylase regulating the activities of HIFs
A transcriptional coactivator of PPARgammma
A calcium-dependent mitochondrial solute carrier
A negative regulator of cytokine receptor signaling
TSPY promotes pro-oncogenic gene expression and poor survival of male HCC patients
Comparison of the gene expression levels among respective TSPY-related groups showed that the expression levels of the unfavorable/pro-oncogenic genes, e.g. ADGRD1, CDC25B, CDC45, EXO1, HMMR, PRIM1, RAD51, RRM2, and SPC24, were significantly higher in the male TSPY-high group HCCs than female HCCs, while the expression level of the favorable/anti-oncogenic gene, e.g. PPARGC1A, was significantly lower in the male TSPY-high group HCCs than the female HCCs (Fig. 4 and Additional file 6: Figure S3). However, there was no significant difference in the expression levels of the 16 downstream genes between the male TSPY-silent group HCCs and female HCCs, except EGLN3 and SPC24 (Fig. 4 and Additional file 6: Figure S3). These results suggest that TSPY could differentially affect the expression levels of pro-oncogenic and unfavorable genes in male HCCs, as compared to those in female HCCs. Indeed, as described above, the survival data indicate that, while there was no significant difference between female HCC group and the male TSPY-silent HCC group, the survival ratio of the male TSPY-high HCC group was significantly lower than that of female HCC group (Log-rank test P-value = 0.0164) with 3 years survival ratio of 35% in the male TSPY-high HCC group and 60–70% in female HCC and the male TSPY-silent HCC groups respectively (Fig. 3b). These observations support the hypothesis that TSPY could be a male-specific genetic risk factor and its aberrant and epigenetic activation contributes to the oncogenic processes and the male predominance of HCC.
Recent studies have provided significant evidence demonstrating the sex differences in various diseases, including cardiovascular, inflammatory and neurodegenerative diseases, as well as numerous types of cancer [38, 63–69]. Currently, the mechanisms contributing to such sex differences are still largely unknown. Liver cancer is one of the most male predominant cancers with men having approximately three to sixfolds higher incidence and mortality than women, depending on the geographic locations [32–34]. The sex hormones and their receptors have been postulated to play key roles in such sex difference in HCC [39, 40, 70, 71]. Female hormones, i.e. estrogen and prolactin, have been proposed to suppress HCC development by reducing the activities of inflammatory pathways, such as IL-6 and TNF signaling cascades in liver [36, 37, 72]. In contrast, male hormones, i.e. androgens, could stimulate hepatitis B virus replication and expression of the HBV viral oncoprotein HBx via androgen receptor actions, resulting in exacerbation of HCC development [73–77]. Since it is estimated that 50–80% of HCC patients could be associated with HBV-infection, the androgen-dependent upregulation of the viral oncoprotein HBx might contribute to the male predominance of HCC. Notably, sex differences in HCC still observed between men and women after menopause age, and various clinical trials targeting androgen receptor showed limited effects on HCC progression [33, 78]. Hence, in addition to sex hormones and their receptors, genetic factors could also be important for the male predominance of HCC. Such genetic factors include single nucleotide polymorphisms (SNPs) on autosomal genes correlating with the sex difference of HCC [79, 80]. Further, we demonstrated that an X-encoded tumor suppressor TSPX, the homologue of TSPY on the X chromosome, could bind to and promote the proteasomal degradation of the viral oncoprotein HBx . Since men have only one X-chromosome, inactivation and/or mutation of such tumor suppressor gene could increase the risk of HBx-medicated HCC in men [38, 81].
HMMR was originally identified as the receptor for hyaluronan (HA), a component of extracellular matrix, and it is frequently upregulated in various cancer types, including colon cancer, bladder cancer, prostate cancer, and liver cancer [83, 92–95]. The high HMMR expression correlates with cancer growth, metastasis, and poor prognosis in these types of cancer [83, 92–95]. The level of extracellular HA is elevated according to the progression of liver fibrosis, which is a pathological precondition for HCC . The intracellular HMMR participates in mitotic spindle pole formation and cytokinesis . The identification of HMMR as a downstream gene upregulated by TSPY suggests that it could be involved in the initiation and progression of HCC via its stimulation of the HA-HMMR signaling cascade, in addition to oncogenic activities as noted above.
The present study shows that TSPY is a male-specific genetic risk factor, whose ectopic expression could contribute partially to the male predominance of HCC. The ectopically expressed TSPY upregulates the expression of genes involved in cell-cycle progression, especially G2/M-phase, in HCC. In addition, TSPY may promote liver fibrosis and expedite an oncogenic transition to HCC via activation of the HA-HMMR signaling cascade, among others. Future studies on the mechanisms of TSPY-mediated differential regulation of its downstream genes will provide novel insights on its oncogenic actions, prognostic values and potential therapeutic targets for effective clinical treatments of HCC.
TK and Y-FCL conceived the idea of TSPY as an oncogene in liver cancer. TK performed the experimental and data analyses; Y-FCL and TK co-wrote the manuscript. Both authors read and approved the final manuscript.
We thank Dr. Yunmin Li for technical assistance.
The authors declare that they have no competing interests.
Availability of data and materials
Transcriptome data of HuH-7 cells will be submitted to the Gene Expression Omnibus database, and the accession numbers posted upon acceptance of the manuscript. All other reagents will be available upon request from the authors after the manuscript is published.
Consent for publication
All authors have read and consented for the publication of this manuscript.
Ethics approval and consent to participate
This work is partially supported by a grant from the Department of Defense Peer-Reviewed Cancer Program to YFCL (CA150248) and a Merit-Review Grant IO1 BX004446 from the Department of Veterans Affairs. YFCL is a Research Career Scientist of the Department of Veterans Affairs.
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