Piwil2 is expressed specifically in testis and in a wide variety of tumors

We showed that Piwil2 is specifically expressed in testis of mouse and human, in most tumors and in testis of mutant mice, except W/W^V. By immunohistochemical analysis we demonstrated that Piwil2 is expressed specifically in spermatogonia and spermatocytes, but not in somatic cells. This expression pattern resembles expression of testis-cancer antigens, which are expressed in some tumor cells, tumor tissues and testis.

Therefore, Piwil2 appears to fall in category of testis-cancer antigens (CTAs). Testis-cancer antigens were the first human tumor-associated antigens characterised at the molecular level (Scanlan et al., 2004). In testis, CTAs are exclusively present in the germ cell lineage, although there is a lot of variation in the expression profile during different stages of germ cell development (Zendman et al., 2003). CTAs are normally expressed by gametes and trophoblasts and are aberrantly expressed in a range of human cancers (Fig. 4.1). So far, 44 distinct CTAs families, some of which have multiple members, have been identified. CTAs are immunogenic and, as a result, have the potential to be used as tumor vaccines. CTAs can be divided into those that are encoded on the X chromosome (CT-X antigens) and those that are not (non-X CT antigens). CT-X antigens tend to form recently expanded gene families that are usually highly expressed in the spermatogonia (mitotically proliferating germ cells). The CT-X genes are frequently co-expressed in cancer cells which tend to express several CT antigens. The genes for the Non-X CT antigens are distributed throughout the genome. In testis, they are usually expressed in the spermatocytes and many have roles in meiosis. Their aberrant expression in cancer cells might cause abnormal chromosome segregation and aneuploidy. Methylated CpG islands associated with the

CT-X genes in normal somatic cells become demethylated in cancer cells, indicating activation of their expression in somatic cells (Simpson et al., 2005).

Figure 4.1: Shared characteristics of germ cells and cancer cells. Activation of the gametogenic programme (shown by brown cells) might contribute to properties of tumor formation and progression (shown by blue cells). Corresponding features between cancer cells and those in the germ cell/gamete/trophoblast differentiation pathways include: immortalization (involved in transformation), invasion, induction of meiosis (can lead to aneuploidy) and migration (contributes to metastasis).

Shared phenotypes between germ cells and cancer cells include demethylation, angiogenesis induction, downregulation of the major histocompatibility complex (immune evasion) and expression of chorionic gonadotropin. The numbers (1–9) indicate gametogenesis- and tumorigenesis-related phenotypic traits and the stages at which these events occur (adapted by Simpson et al., 2005).

As germline stem cells and their trophoblastic derivatives share many characteristics with tumor cells, the activation of normally silent germline-specific genes in cancer stem cells (gametic recapitulation) could mediate the malignant phenotype in the absence of mutations in known oncogenes and tumor-suppressor genes.

However, literature regarding the biological properties of Piwil2 is very limited and, furthermore, the role of Piwil2 in solid cancer is completely unknown. The aim of our present work was to investigate Piwil2 expression in human and mouse cancer and to identify potential roles for Piwil2 in the genesis of cancer. We screened 10 human cancer cell lines and 8 mouse cancer cell lines for Piwil2 expression. 9 of 10 human cancer cell lines and 7 of 7 mouse cancer cell lines showed Piwil2 mRNA expression. In addition, three types of tumors with their corresponding normal tissues were examined in mouse.

Whereas in breast tumor, rhabdomyosarcoma and medulloblastoma expression of Piwil2 was detected, no expression was observed in normal breast, muscle and cerebellum tissue.

In human, in different tumor types expression of Piwil2 was detectable by using RT-PCR.

In 3 of 4 different ovarian tumors, in 4 of 4 prostate carcinomas, in 4 of 4 tumors in lymphatic gland and in 7 of 7 breast tumors, expression of Piwil2 was detected by RT-PCR analysis. These data suggest that Piwil2 is expressed in most human and mouse cancer tissues and it may play an important role in cancer development.

In order to investigate expression of Piwil2 on protein level, we generated a rabbit polyclonal antibody against mouse Piwil2 and a human monoclonal antibody against human PIWIL2. The specificity of the antibodies was examined with Piwil2 gene

transfection in NIH3T3 cells and immunostaining on paraffin-embedded tissue sections of each human and mouse testis. In mouse, we used this specific antibody for immunohistochemistry to examine the expression of Piwil2. In mouse and human testis, the Piwil2 protein was detected in spermatogonia and early spermatocytes. While expression of Piwil2 was not detected in normal skeletal muscle and cerebellar tissues, Piwil2 was detectable in corresponding tumor tissues, rhabdomyosarcoma and medulloblastoma. In human, Piwil2 was found in cytoplasm of breast tumor cell line MDA-MB-231. In all seven breast tumor tissues from different patients, expression of Piwil2 was observed. No expression was detectable in normal breast tissue. Furthermore, immunohistochemical analysis showed expression in other tumors either in dispersed or in clonal form. These results indicate that Piwil2 is specifically expressed in spermatogonia of testis and ectopically in most tumor cell lines and tumor tissues.

Our immunohistochemical results indicate that Piwil2 is a potential marker for cancer cell proliferation. Likewise, we used proliferation assay and soft agar assay in Piwil2 expressing cell line in order to estimate the proliferation status of the cells in this study.

In human, an elevated expression of Piwil2 was observed in testicular germ cell tumors (Fig. 3.11). This expression pattern mimics expression pattern of Piwil1 in testicular germ cell tumors. In normal human testis, Piwil1 (hiwi) is specifically expressed in germ cells, with its expression detectable in spermatocytes and round spermatids during spermatogenesis (Qiao et al., 2002). Enhanced expression of Piwil1 was found in 12 out of 19 sampled testicular seminomas originating from embryonic germ cells with retention of germ cell phenotype. In contrast, no enhanced expression was detected in 10 nonseminomatous testicular tumors that originate from the same precursor cells as seminomas (Qiao et al., 2002).

Moreover, the specific correlation between Piwil2 overexpression and seminomas but not nonseminomas suggests that Piwil2 can function in spermatogonia or their precursors.

Seminomas could be caused by the ectopic expression of Piwil2 in spermatogonia. Both possibilities may reflect a functional evolution of the piwi family genes from Drosophila to mammalian systems. In this context it is also interesting to note that there are multiple human homologs of piwi. It is therefore possible that the various Piwil2 homologs became specialized in subsets of the piwi functions, with Piwil2 in particular concentrating on and

further enhancing the cell-autonomous function of piwi in promoting the division of stem cells and/or their differentiating daughter cells in the germline.

Nonseminomas do not show enhanced Piwil2 expression, likely because these tumors have lost their germline properties, even though they also originate from the same precursor cells as seminomas, i.e. CIS (Carcinoma In Situ). The results presented in this study allow us to conclude that the overexpression of Piwil2 probably does not lead to the development of spermatocytic seminomas but might be involved in the formation of TGCTs (Testicular Germ Cell Tumor). Several studies have identified genetic components in the development of TGCTs. It has been suggested that up to 30% of TGCTs are affected by genetic predisposition (Nicholson and Harland, 1995). In fact, the development of TGCTs has been found to show linkage to a number of chromosomal regions, suggesting the involvement of more than one gene, for which both a dominant and a recessive model might be applicable (Bishop, 1998). The specific correlation between Piwil2 overexpression and seminomas is likely due to the fact that these cells retain the phenotype of CIS cells. Yet, varied levels of Piwil2 expression in different seminomas or even within the same tumor might be related to the heterogeneity of the tumor cells. This heterogeneity is not correlated to the mitotic or apoptotic frequency of these cells. It thus remains to be elucidated whether it might be explained by the difference in other aspects of cellular metabolism or the heterogeneity of tumor cells in activating various Piwil2 homologies. More specific antibodies against PIWIL2 and its Piwil2 homologs of human could be informative to investigate this hypothesis. This intriguing expression pattern of Piwil2, as well as its expression in cancer cell lines and tissues, offer novel opportunities for studying the mechanisms of stem cell divisions and oncogenesis.

These results show a possible synergistic effect of piwi genes on initiation and progression of testicular germ cell tumors.

Therefore, the wide range of tumors in which Piwil2 has been detected urges further efforts to develop effective specific immunotherapeutic procedures.

Based on these results in cancer samples, it would be interesting to analyse the relationship between Piwil2 expression and clinical parameters. It is possible that the number of samples used in this study may not be large enough to evaluate the link between Piwil2 expression and clinical parameters.