Clear cell foci (CCF) of the liver are considered to be pre-neoplastic lesions of hepatocellular adenomas and carcinomas. They are hallmarked by glycogen overload and activation of AKT (v-akt murine thymoma viral oncogene homolog)/mTOR (mammalian target of rapamycin)-signaling. Here, we report the transcriptome and proteome of CCF extracted from human liver biopsies by laser capture microdissection. We found 14 genes and 22 proteins differentially expressed in CCF and the majority of these were expressed at lower levels in CCF. Using immunohistochemistry, the reduced expressions of STBD1 (starch-binding domain-containing protein 1), USP28 (ubiquitin-specific peptidase 28), monad/WDR92 (WD repeat domain 92), CYB5B (Cytochrome b5 type B), and HSPE1 (10 kDa heat shock protein, mitochondrial) were validated in CCF in independent specimens. Knockout of Stbd1, the gene coding for Starch-binding domain-containing protein 1, in mice did not have a significant effect on liver glycogen levels, indicating that additional factors are required for glycogen overload in CCF. Usp28 knockout mice did not show changes in glycogen storage in diethylnitrosamine-induced liver carcinoma, demonstrating that CCF are distinct from this type of cancer model, despite the decreased USP28 expression. Moreover, our data indicates that decreased USP28 expression is a novel factor contributing to the pre-neoplastic character of CCF. In summary, our work identifies several novel and unexpected candidates that are differentially expressed in CCF and that have functions in glycogen metabolism and tumorigenesis.

Clear cell foci (CCF) of the liver are considered to be pre-neoplastic lesions of hepatocellular adenomas and carcinomas. They are hallmarked by glycogen-overload and activation of AKT/mTOR-signaling. Here we report the transcriptome and proteome of CCF extracted from human liver biopsies by laser capture microdissection. We mainly found genes and proteins with lower expression in CCF compared to control samples. Of 14 differentially expressed transcripts only 3 were overexpressed and belong to previously uncharacterized long non-coding RNAs. At the protein level, 3 proteins were at least 1.5-fold higher expressed in CCF than in control samples, while 22 proteins were at least 1.5-fold less abundant. Using immunohistochemistry, the reduced expressions of STBD1, USP28, monad/WDR92, CYB5B and HSPE1 were validated in CCF. Knockout of STBD1, the gene coding for Starch-binding domain-containing protein 1, in mice did not have an effect on liver glycogen levels. Similiarly, Usp28 knockout in mice did not affect glycogen storage in diethylnitrosamine-induced liver carcinoma. Thus, these data indicate that reduced expression of STBD1 or USP28 in CCF is unlikely to explain glycogen overload. Our study revealed several novel proteins and RNAs that are differentially expressed in CCF and have functions relevant for carcinogenesis or the glycogen metabolism.

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