The findings in this study indicate that in addition to TP53, several genes including Rsf-1, cyclin E, p16, and FASN that are amplified or upregulated in high-grade ovarian serous carcinoma are also overexpressed in the majority of STICs. In contrast, mucin-4 expression was either down regulated or showed no change in most STICs. Approximately 90% of STICs displayed intense or virtually no p53 immunoreactivity. Both of these immunostaining patterns are highly associated with TP53 mutations. A complete absence of p53 immunoreactivity has been shown in these lesions and in high-grade serous carcinoma to be associated with nonsense TP53 mutations that result in a truncated protein which is not recognized by the p53 antibody.
Rsf-1, also known as HBXAP, was upregulated in 63% of STICs. It encodes a nuclear protein 14
which is a subunit in a chromatin assembly factor, called ISWI-containing factor RSF. 15
Rsf-1 has been shown to function as a histone chaperone while its binding partner, hSNF2H, possesses nucleosome-dependent ATPase activity. 16
The Rsf-1/hSNF2H complex (RSF complex) mediates ATP-dependent chromatin remodeling by mobilizing nucleosomes, a process that is essential for transcriptional activation or repression,17
DNA replication 18
and cell cycle progression. 19
In ovarian cancer, Rsf-1
amplification has been detected in approximately 15% and Rsf-1 overexpression in the majority of high-grade serous carcinomas. 8, 12, 20
It appears that in these serous carcinomas cellular proliferation and survival depend on the expression of Rsf-1, especially in the presence of paclitaxel. 8, 21, 22
In addition, Rsf-1 amplification or upregulation is associated with shorter overall survival in patients with high-grade serous carcinoma patients as compared to tumors in which Rsf-1 is not amplified or upregulated. 8
Cyclin E was expressed in 77% of STICs. 20 (83%) of 24 STICs that expressed cyclin E showed either intense or no p53 staining. Cyclin E which is encoded by CCNE1
is a well established cell cycle protein that complexes with Cdk2 to mediate the transition from G1 to S phase by inactivating the retinoblastoma protein. Cyclin E expression is upregulated in a variety of neoplasms through amplification of the CCNE1
or through its transcriptional activation. Among amplicons in high-grade serous carcinomas, the CCNE1
locus is the most common, occurring in approximately 36% of specimens. 20
Cyclin E has been demonstrated to play a role in the pathogenesis of ovarian carcinoma 9, 23-25
and therefore the high proportion of STICs expressing cyclin E underscores the important role of cyclin E in the early events of high-grade serous carcinogenesis.
Fatty acid synthase was expressed in 62% of STICs. 19 (86%) of 22 STICs that overexpressed fatty acid synthase showed either intense or no p53 staining. Fatty acid synthase (FASN)
encodes a cytoplasmic enzyme that is responsible for biochemical processes involving de novo
fatty acid synthesis. 26
Since normal adult tissues contain abundant dietary lipids, only a minimal amount of FASN is expressed. In contrast, in a variety of neoplastic diseases including ovarian serous carcinomas 13, 26, 27
elevated FASN levels are detected. In mouse xenograft ovarian tumor models that overexpress FASN, inhibition of the enzyme activity led to a reduction in tumor volume.10
Although the mechanism by which FASN participates in tumor progression remains unclear, it has been proposed that de novo synthesis of long chain fatty acid is important to promote tumor growth and survival. In addition, FASN may act in concert with other signaling pathways including AMP-activated kinase, AKT and ErbB2 28-31
to mediate tumor initiation and progression.
In this study 89% of STICs demonstrated immunostaining patterns consistent with mutations of TP53
confirming that TP53
mutations are highly prevalent in STICs with a mutation frequency similar to high-grade serous carcinomas. Our data also demonstrate that these p53 staining patterns were substantially higher than the other markers examined in this study (). In other words, several STICs showing either intense or no p53 staining but did not show upregulation of p16, Rsf-1, cyclin E and FASN (). Moreover, analysis of eight STICs with very low proliferation indices (Ki-67 labeling <10%) showed that most of these lesions displayed p53 immunostaining patterns consistent with TP53
mutations, whereas 43% expressed increased Rsf-1 immunoreactivity. All of the associated carcinomas in this study expressed increased levels all of the markers including Rsf-1, cyclin E and FASN. These findings indicate that TP53
mutations probably represent a very early molecular genetic change in the development of high-grade serous carcinoma. It is likely that TP53
mutation, since it is a gatekeeper that regulates transformation, initiates a cascade of molecular changes that allow cells to survive in the presence of DNA damage and oncogenic activation without undergoing p53-dependent senescence or apoptosis.32
The newly proposed hypothesis of the tubal as opposed to the ovarian surface epithelial (OSE) origin of pelvic serous carcinomas challenges many of the previous reports demonstrating “overexpressed” ovarian cancer-associated genes in which the expression levels in the carcinomas were almost always compared to ovarian surface epithelium. Low-molecular-weight forms of cyclin E have been reported to be reliable markers for the distinction of ovarian carcinoma from benign and malignant mesothelial lesions.9
Accordingly, the high frequency of cyclin E expression in STICs and high-grade ovarian serous carcinoma casts further doubt on the OSE as the site of origin of these tumors. Furthermore, as the gene expression profiles in ovarian surface epithelium, which is of mesothelial origin, are different from the mullerian-derived fallopian tube epithelium it will be important to validate whether these previously reported overexpressed genes are indeed upregulated using fallopian tube epithelium rather than OSE as a control.33
For example, mucin-4 is regarded as a biomarker of ovarian cancer but in the present study we found that mucin-4 unlike Rsf-1, cyclin E and FASN, mucin-4 was not overexpressed in the majority of STICs as compared to the adjacent normal-appearing tubal epithelium. In fact, 50% of STICs either retained the same level of mucin-4 immunoreactivity as the adjacent normal-appearing tubal epithelium or exhibited decreased mucin-4 expression, suggesting that mucin-4 expression is tissue lineage-specific rather than tumor-specific. These findings argue against the role of mucin-4 in the pathogenesis of early high-grade serous carcinogenesis.
In conclusion, this report demonstrates that in addition to p53 several other oncogenic proteins associated with high-grade serous carcinomas are upregulated in STICs. In this study, 95% of STICs that overexpressed Rsf-1 showed either intense or no p53 staining, suggesting that TP53 mutations are required for Rsf-1 upregulation. The upregulation of Rsf-1 in STICs compared to the adjacent normal-appearing tubal epithelium suggests that excessive RSF (Rsf)-mediated chromatin remodeling occurs early in the development of high-grade serous carcinoma. Thus, it appears that overexpression of Rsf-1, cyclin E and FASN acts in concert with TP53 mutations to propel tumor development. We also found that STICs rarely demonstrated upregulation of mucin-4, which has been reported to be a tumor-associated protein in ovarian cancer, arguing against the role of mucin-4 in the pathogenesis of high-grade serous carcinoma. This finding underscores the importance of reassessing the validity of other previously reported overexpressed genes in ovarian high-grade cancer-associated proteins in which ovarian surface epithelium was used as a control. This has important clinical implications for the identification of candidate biomarkers for the development of screening tests and novel prevention strategies. Given the possibility that STICs that are detected in advanced cases of high-grade serous carcinoma may be different from those discovered incidentally, such as during risk reducing procedures in women with inherited BRCA1 or BRCA2 mutations, future studies should analyze the expression patterns of Rsf-1, cyclin E, FASN and mucin-4 in the STICs not associated with invasive carcinomas.