TSPY as a Proto-Oncogene on the Human Y Chromosome

The Y chromosome is critical for male development and physiology. The involvement of the Y chromosome in human cancers has been highlighted by the gonadoblastoma locus (GBY), initially proposed to explain the significantly high incidence of gonadoblastoma, a benign tumor cell tumor, among patients of disorders of sex development (DSD) harboring residual Y chromosome sequences. The GBY gene(s) is postulated to serve a normal function(s) in the testis, but exert oncogenic activities in dysfunctional gonadal environments, such as streaked dysgenetic gonads. Numerous studies, by our laboratory and others, suggest that the testis-specific protein Y-encoded (TSPY) gene is the putative gene for GBY. Indeed, TSPY is abundantly expressed in gonadoblastoma, testicular germ cell tumors (TGCTs), seminomas, selected nonseminomas, intracranial germ cell tumors of male origin, and somatic cancers including prostate cancer, hepatocellular carcinoma (HCC), head and neck cancer, lung cancer and melanoma. High levels of TSPY expression in HCC is associated with poor survival of the patients.

TSPY is tandemly repeated 20-60 times and constitutes the largest block (up to 1 MB of DNA) of functional gene array in the human genome. Variations of its gene-copy number have been observed among the normal populations, and could be associated with male fertility. TSPY gene array is a hot spot for genetic variations on the human Y chromosome. We demonstrated that in a humanized mouse strain, TgTSPY9, in which the human TSPY is tandemly integrated on the mouse Y chromosome, the TSPY transgene is aberrantly activated in the LADY mouse model of prostate cancer, suggesting its genetic/epigenetic instability during oncogenesis.

Over-expression of TSPY accelerates cell proliferation, promotes tumorigenicity in mice, up-regulates oncogenic genes and pathways, and represses tumor suppressors. Transgenic mouse modeling demonstrated that TSPY is capable of expressing in female germ cells and promotes gonadoblastoma-like structure in female transgenic mice, suggesting that TSPY is a Y-located proto-oncogene exerting cell proliferative properties and oncogenic functions when inappropriately expressed in dysfunctional germ cells and/or somatic cells incapable of entering male meiosis.

TSPY and cancer stem cells. TSPY is expressed at high levels in the premalignant precursor, carcinoma-in-situ(CIS)/germ cell neoplasia in situ (GCNIS), gonadoblastoma and testicular germ cell tumors. Its expression is closely correlated with cancer stem cell marker, i.e. CD33, and specific germ cell tumor markers, i.e. placental alkaline phosphatase, c-Kit and Oct4. Since CIS/GCNIS has been considered to be a cancer stem cell for gonadoblastoma and all testicular germ cell tumors, TSPY could potentially be important for either the maintenance or oncogenic differentiation of CIS/GCNIS in germ cell tumorigenesis.

Functions of TSPY Gene in Normal Physiology and Diseases

Our studies suggest that TSPY serves normal functions in male stem germ cell propagation and meiotic division. It is expressed in gonocytes of embryonic testis and co-expressed with Oct4 and c-Kit in spermatogonia and spermatocytes of the adult testis. It interacts with the type B cyclins and stimulates the cyclin B-CDK1 phosphorylation activities, potentially critical for spermatogonial renewal and male meiosis. TSPY and cyclin B1 are co-expressed tightly during spermatocyte differentiation. We hypothesize that TSPY stimulation of cyclin B-CDK1 phosphorylation could be important for the two rounds of meiotic divisions without an intermediate interphase during spermatogenesis, a unique feature for male meiosis.

TSPY co-localizes with cyclin B1 during interphase and preferentially at the spindles at various stages of mitosis of somatic cells (Figure 1). TSPY stimulates the cyclin B-CDK1 phosphorylation activities and promotes a rapid transition of the G₂/M phase. Aberrant and high-level expressions of TSPY in incompatible cells, such as dysfunctional germ cells and somatic cells incapable of entering male meiosis, stimulates cell proliferation, disrupts the G₂/M checkpoints in the cell cycle, and potentially induces genome instability, thereby promoting oncogenesis of the affected cells. Hence, TSPY is a male-specific proto-oncogene on the Y chromosome, contributing to the oncogenesis in male-specific/related cancers, such as gonadoblastoma, testicular germ cell tumors and prostate cancer, and male biases in somatic cancers, such as hepatocellular carcinoma, head and neck and lung cancers.

Figure 1. Co-localization of TSPY and Cyclin B1 during mitosis of somatic cells. A. TSPY distributions during various mitotic phases. B. TSPY and C. Cyclin B1 are preferentially localized in the mitotic spindles in metaphase cells, D. Co-localization of TSPY and Cyclin B1 in a metaphase cell.

TSPY Forms a Positive Feedback Loop with Androgen Receptor

As a proto-oncogene on the Y chromosome, TSPY plays key roles in male-specific/biased cancers. Further, its expression is regulated by the male sex hormone androgen via binding of the androgen receptor (AR) on its promoter. Significantly, we further demonstrated that TSPY physically interacts with AR and its constitutively active variants, such as AR-V7, and stimulates the AR and AR-V7 transactivation of responsive genes in ligand dependent and independent manners respectively. These findings suggest the existence of a positive feedback loop between the TSPY and AR/AR-V7, in which TSPY is transcriptionally up-regulated by AR/AR-V7 and its protein, in turn, binds to AR/AR-V7 and exacerbates the AR/AR-V7 transactivation of its own gene as well as other AR/AR-V7 responsive targets (Figure 2). Accordingly, a Y-located oncogene amplifies its own expression through exacerbating the transcriptional activities of the male sex hormone receptors, and promotes oncogenesis in specific cancers, such as prostate cancer, in which the AR and AR variants play key roles in the initiation and progression to metastatic castration resistant prostate cancer (CRPC). Indeed, TSPY expression has been detected in various stages of prostate cancer and is associated with poor prognosis of the patients. Further, TSPY expression has also been detected in liver cancer, head and neck cancer, lung cancer and melanoma, some of which also show male biases in the incidence and/or pathogenesis and/or are subjected to sex hormone/receptor modulations. Hence, the TSPY-AR/AR-V7 positive feedback loop could play key roles in the oncogenic processes of these male-biased cancers.

Figure 2. A positive feedback loop between TSPY and AR/AR-V7, in which AR/AR-V7 bind to the TSPY promoter and stimulate its expression in a ligand-dependent and independent manners, the TSPY protein, in turn, interacts and exacerbates AR/AR-V7 transactivation of its own gene (A) as well as AR/AR-V7 target genes (B).