Testicular germ cell tumors (TGCT) of adolescents and adults are the most frequent carcinomas of Caucasian males ages 15–40.1 TGCTs are classified into either nonseminomas, which include embryonal carcinoma, yolk sac, teratomas, and choriocarcinoma or seminomas, which morphologically resemble transformed primordial germ cells (PGCs).2 Seminomas are the most common form of TGCT, accounting for 50% of cases, whereas nonseminomas are identified in 40% and mixed forms in 10% of cases (for review, see Ref. 3.)
Despite the common occurrence of TGCTs in young men, the underlying genetic mechanisms leading to their development are unknown. It is currently supposed that seminomas develop from carcinoma in situ (CIS) within the seminiferous tubules.4–6 CIS are microscopically distinct cells that reside on the basement membrane of the seminiferous tubules of the testis and have morphological features more similar to embryonic germ cells than spermatogonial stem cells. The evidence for CIS being the precursor of seminomas arises from the knowledge that they both histologically resemble PGCs and gonocytes and are characterized by positive staining for glycogen, stem cell factor receptor (c-KIT), and germ cell/placental alkaline phosphatase.7–10 However, at the genomic level seminomas are characterized by the presence of extra 12p genomic material, often appearing as an isochromosome (i12p) or as 12p11.2–p12.1 and 12p13.3 amplifications, whereas CIS does not have this characteristic amplification.11–13 Therefore, 12p duplications are hypothesized to be a later event in TGCT malignant transformation.14 Although it is clear that 12p duplications are a hallmark of invasive seminoma, to date no genes have been pathogenically linked to this malignancy.
We recently demonstrated that three genes on human chromosome 12p were elevated in two seminoma samples.15 These genes included STELLAR, also known as developmental pluripotency associated-3 (DPPA3), NANOG, and GDF3 (growth and differentiation factor 3). Interestingly, all three genes are normally expressed in fetal germ cells and human embryonic stem cells (hESCs),15 with expression of all three 12p genes decreasing with hESC differentiation.15 The finding that these three genes are located on human chromosome 12p suggests a potential association with the malignancy of seminoma relative to CIS. However, a systematic study of the expression of these three 12p genes has never been reported. In the current study, we examined expression of NANOG, STELLAR, and GDF3 in seminomas, as well as additional marker genes of hESCs, PGCs, gonocytes, and adult germ cells, to gain more information as to the identity of this carcinoma. Then, we also examined expression of the basic stem cell profile of OCT4, STELLAR, NANOG, and GDF3 in tumors other than TGCTs. The results suggest that stem cell genes may either play a direct role in different types of carcinoma progression or serve as valuable markers of tumorigenesis.
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We hypothesized that NANOG, STELLAR, and GDF3 would be up-regulated in seminomas given that these stem cell genes map to 12p and that all seminomas are reported to have amplifications at 12p being either isochromosomes i(12p) or 12p11.2–12p11.1 and 12p12.2–12p13.3 amplifications.13, 28 Our results demonstrated that NANOG and STELLAR were elevated in several seminoma samples; however, only GDF3 was elevated in almost all seminomas relative to normal testis. GDF3 is a secreted TGF-beta superfamily ligand that interacts with unknown receptors. In humans, GDF3 expression has been documented in human embryonic carcinoma cells (hECCs), hESCs, and fetal gonads; it is not expressed at high levels in other human somatic tissues.15, 25 Although this pattern of expression is intriguing, little is known of GDF3 function, including the identity of the receptors to which it binds.
In this study, we observed the highest expression of NANOG and OCT4 in seminoma compared with all other genes analyzed. Several recent studies have addressed gene expression of NANOG or OCT4 in both CIS and seminomas using gene chip expression profiling.28, 29 In particular, Almstrup et al.29 compared one CIS sample directly to normal testis and identified several genes that were up-regulated in CIS, including NANOG and OCT4. In comparison, Sperger et al.28 compared various pluripotent cell populations including hESCs, seminomas, nonseminomas, and hECCs to normal testis and somatic tumor cell lines. That study determined that OCT4 was highly up-regulated in hECCs, hESCs, and seminomas and that NANOG mRNA expression was highly associated with hECCs, but did not report the levels of NANOG expression in seminomas.28 In our study, elevated expression of NANOG and OCT4 were not identified in all seminomas compared with normal testis; however, these two genes were expressed at the highest levels in all seminomas compared with all the other genes in this analysis. Such variability in gene expression was also found by Almstrup et al.29 during PCR validation of CIS, therefore suggesting that variability in NANOG and OCT4 expression is found not only in the seminoma samples examined in our study, but also in the putative precursor CIS cells. Despite this variability, our data demonstrate that OCT4 and NANOG were consistently the highest-expressed mRNAs compared with all other genes analyzed in our study. Although more samples would reinforce our current data, we expect that this trend will continue in future studies.
With regard to protein, OCT4 expression has previously been examined in human seminomas using multitissue microarray immunohistochemistry.30 In this multitissue microarray study, 4 of 4 seminomas were positive for OCT4. NANOG protein expression has never been reported in seminomas. Therefore, in the current study we examined NANOG protein in both seminomas and hESCs. We found that NANOG protein was expressed exclusively in the nucleus of hESCs and seminomas, suggesting that in both cell types NANOG's main site of activity is in this cellular compartment. Furthermore, it is known that both mouse and human NANOG proteins contain a DNA recognition sequence, implying that NANOG may regulate transcription. However, the exact mechanism of NANOG activity in seminoma remains to be identified.
Although seminomas clearly express hESC and embryonic germ cell markers, we also examined the presence of additional germ cell markers in an attempt to further characterize the genetic identify of the seminoma cell with regard to temporal development of the germ cell lineage. Notably, the adult germ cell genes that mark meiotic and postmeiotic cells, BOULE and TEKT1, were not expressed in seminomas. This lends further genetic support to the finding that seminomas are not composed of meiotic or postmeiotic germ cells. Furthermore, given the complete absence of BOULE and TEKT1 in the seminomas, this also demonstrates that the carcinoma specimens were not contaminated with normal testis. Interestingly, both DAZL and VASA expression were decreased in all seminomas relative to normal testis. Previous work has examined DAZL protein expression in CIS and seminomas and shown that all CIS cells express DAZL,31 but DAZL expression in pure seminomas is rare and focal in nature.31 This is consistent with our findings that DAZL expression is not elevated in seminoma relative to normal testis.
Similar to DAZL expression, VASA expression in the current study was also uniformly low in seminomas. VASA protein expression has previously been examined in CIS and seminoma, being highly expressed in CIS cells, but having relatively weak expression in seminomas.32 These two genes, DAZL and VASA, are classic markers of gonocytes and spermatogonia23,33; therefore, their reduced expression in seminomas would suggest that seminomas are less similar to CIS and more closely resemble a different cell type that is yet to be fully characterized.
In addition to this work on seminomas, we also learned that hESC marker expression is not limited to germ cell tumors. We identified elevated expression levels of OCT4, together with NANOG, STELLAR, and GDF3 mRNA, in both breast carcinoma tissue and the breast carcinoma cell line MCF7. This suggests that breast carcinomas, and possibly other human malignancies, may contain cells reminiscent of embryonic-like stem cells. Furthermore, the absence of the germ cell-specific genes DAZL and VASA in the breast carcinoma would suggest that the OCT4-expressing breast carcinoma cells identified in the current study are distinct from germline stem cells that may have metastasized to the breast. In particular, a previous study using OCT4 protein alone as a marker of germ cell tumor metastasis to the breast argued that OCT4 is a useful marker of germ cell metastasis to extragonadal tissues.34 Our results would argue that OCT4 expression should be analyzed in the context of additional germ cell-specific markers including DAZL and/or VASA to identify germ cell metastatic origin versus an alternate origin of the OCT4-positive cells in this invasive carcinoma.
A model for the development of seminoma and reacquisition of markers of pluripotency in nongerm-cell tumors is illustrated in Figure 7. This model shows that PGCs are specified at a similar time to the somatic lineages from the epiblast of the embryo at the time of implantation. The specified PGCs subsequently retain the expression of many markers associated with pluripotency that are normally expressed by cells of the inner cell mass (ICM)16,18–21 (in red, Fig. 7). Somatic cells (green box, Fig. 7), by comparison, do not express pluripotent genes, including NANOG, OCT4, STELLAR, and GDF3.15, 18 The current hypothesis (pathway A, Fig. 7) suggests that seminoma is derived from CIS, and that CIS is derived from a PGC/gonocyte-type cell that accumulates chromosomal abnormalities.35 Our data supports this hypothesis, particularly through the elevated expression of the ESC- and PGC-expressed genes NANOG and OCT4. However, our data also reveal that in 100% of seminomas analyzed DAZL and VASA mRNA levels are reduced, whereas it has been reported that CIS express abundant levels of these genes.31, 32 This would suggest that in addition to chromosomal differences observed between CIS and seminoma, there is a reduction in germ cell marker expression in favor of a more robust pluripotent stem cell profile. Recently, a new cell type was identified in normal neonatal rodent testis that has hallmarks of embryonic stem cells as well as PGCs.36 This cell type is called a multipotent germline stem cell (mGSC) and, given its hallmark characteristics of pluripotency, it should now also be considered as a potential precursor to either CIS (B) or potentially seminoma directly (C) (Fig. 7). Furthermore, isolation of this cell type is enhanced in genetic backgrounds predisposed to germ cell tumors, suggesting a common link between this cell type and germ cell tumor.36 In addition, this cell type is also associated with elevated levels of NANOG.36 Although our data do not unequivocally support that seminomas arise from the mGSC, they do not refute this alternate pathway either, as currently there is no clear marker that would distinguish the mGSC from the PGC/gonocyte. Finally, our data also show that acquisition of markers at both the mRNA and protein level of pluripotency-associated genes such as NANOG can be identified in breast carcinomas but not normal breast tissue (red cells; Fig. 7). Therefore, acquisition of these pluripotent genes could be a marker of carcinoma progression from somatic tissues.
Figure 7. Model for pluripotent gene expression in seminoma and breast carcinoma. Once specified, PGCs (red) express many pluripotency-associated genes found exclusively in the inner cell mass (ICM) (red) such as OCT4, NANOG, STELLAR, and GDF3. Somatic cells do not express these pluripotency- associated genes (green). PGCs migrate to the gonad, where they form gonocytes and/or multipotent germ cells (mGSC) (red) followed by spermatogonia (pink). In germ cell tumor, the current hypothesis suggests that seminoma is formed from CIS, which is reported to develop from misregulated PGCs/gonocytes. Our data suggest that seminoma, in addition to developing from CIS, could also develop from misregulated mGSC, given that both cell types express high levels of pluripotent genes, whereas CIS and gonocytes express high levels of DAZL and VASA. In certain carcinomas, such as breast, progression of the carcinoma may be associated with expression of pluripotent genes such as NANOG (red).
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In conclusion, our results confirm and extend recent work suggesting that seminomas are not genetically like CIS with regard to the expression of the germ cell-specific genes DAZL and VASA. Our results also reveal that seminomas have elevated expression of GDF3 compared with normal testis, together with high expression levels of NANOG and OCT4 compared with additional germ cell and stem cell markers, in particular DAZL. We also identified a similar stem cell program (minus DAZL and VASA) and expression of NANOG protein in breast carcinoma, but not in normal breast tissue. This suggests that either amplification of a resident stem cell population or a reversion of somatic breast cells to a stem cell-like state may be associated with the progression of this and possibly other carcinomas. Certainly, the presence or activation of a stem cell-like program should be further investigated in these and other human malignancies.