Here we report the concurrent sequencing of human tumor genomes, exomes, transcriptomes, and cytosine methylomes, the results of which were integrated to yield a picture of genomic alterations in a primary and a locally recurrent liposarcoma. While these tumors harbored abnormalities at all levels, the structural remodeling of liposarcoma genomes was profound and had varied effects on their transcriptomes. Exome sequencing revealed a modest point mutation rate in these tumors, yet uncovered for the first time genes recurrently mutated in this subtype of soft-tissue sarcoma (HDAC1, MAPKAP1, PTPN9, and DAZAP2). Also, this first portrait of liposarcoma methylomes indicates that aberrant methylation has broad effects tied closely to differentiation phenotypes and suggests a role for demethylating agents in the treatment of DLPS tumors.
The prevalence of CEBPA
methylation found here, as well as its specificity and clonality in DLPS, raises interesting questions about the dysregulation of adipogenesis in these tumors. The C/EBPβ-C/EBPα transcriptional network is complex, regulating multiple stages of the commitment of cells to terminal differentiation. C/EBPβ directly induces the expression of CEBPA
, a process disrupted by JUN
amplification in a subset of undifferentiated tumors (17
). While the adipogenic block is common to all DLPS tumors, we and others observed JUN
genomic amplification and over-expression in only ~24% of cases (3
) (though in the absence of amplification, JUN
may also be over-expressed via upstream kinase-driven activation). Moreover, recent evidence indicates that activated JUN, while oncogenic in DLPS cells, is not sufficient to block adipocytic differentiation, implying alternative abnormalities are necessary (24
). We speculate that the CEBPA
promoter methylation identified here may uncouple CEBPA
expression from C/EBPβ-mediated induction and therefore may at least partially contribute to the unexplained adipogenic block in DLPS.
The relationship between CEBPA
methylation and the HDAC1
mutations discovered here in adipogenesis is unknown. Generally, the histone deacetylase activity of HDAC1 targets promoters and blocks transcription, and although the loss of HDACs can enhance adipogenesis (25
), this is not true in all contexts (27
). HDAC1 specifically sequesters and represses the C/EBPα promoter, and PPARγ is necessary to release C/EBPα from HDAC1-mediated repression (28
). These data, in combination with our observation of KLF4
methylation (data not shown), suggests the possibility of a series of alterations necessary to mediate different aspects of the adipogenic block in these tumors.
Other pathways and non-adipogenic phenotypes are likely also involved in the progression of well-differentiated liposarcomas to dedifferentiated disease, and our finding of specific miR-193b methylation and silencing in aggressive disease implies an important role for aberrant expression of small RNAs in liposarcomagenesis. A role for miR-193b in cancer has been proposed based on its reduced expression in both hepatocellular carcinoma and melanoma (29
), and recent evidence suggests miR-193b is epigenetically silenced in prostate cancer (31
). While the validated miR-193b targets CCND1
were over-expressed in some of these malignancies (29
), expression analysis of liposarcomas with and without miR-193b methylation indicated that neither gene is elevated in a DLPS-specific manner, as might be expected for miR-193b loss. However, because CCND1 regulates CDK4 activity, and CDK4
is amplified in ~90% of DLPSs, upregulation of CCND1 due to miR-193b loss might be functionally redundant. This is confirmed by its lack of induction in DLPS tumors harboring miR-193b loss. Nevertheless, other predicted miR-193b targets, including KRAS
, and RAD51
, were preferentially upregulated in miR-193b-silenced tumors, in not only the original tumor in which we identified miR-193b methylation (DLPS1), but also in the larger panel of dedifferentiated tumors. These data raise the possibility that the effects of miR-193b silencing are lineage-and context-specific.
This work represents an initial integrated sequence analysis of liposarcoma, and while most non-coding sequence is insufficiently covered by these experiments, the depth and breadth of alterations revealed here indicates the somatic complexity of cancers is still under-appreciated. These data also reveal an unanticipated role for epigenetic abnormalities in dedifferentiated liposarcoma and the potential therapeutic importance of demethylating agents for treating liposarcomas.