The goal of this study was to use an integrated genome-wide approach to identify genes which play an important role in sporadic ccRCC and two individual ccRCC subtypes H1H2 and H2. Copy number analysis was integrated with gene expression data for 54 sporadic ccRCCs to find genes that were either concordantly gained and overexpressed, or lost and underexpressed. The same analysis was also performed on a publicly available dataset (Broad Data) (16
), and the results compared to find overlapping targets. This process led to the identification of 72 gained-overexpressed and 187 lost-underexpressed genes.
We also used the integrated genomic approach to study the differences between the H1H2 and H2 sub-sets. Although H1H2 and H2 tumors share some overlapping copy number and expression changes, the integrated genomic analysis revealed that there are genes involved in tumorigenesis that are unique and specific to each subtype. Using copy number analysis, we found that the genome of the H1H2 group is on average more aberrant than the H2 group. This difference may be because H2 tumors express DNA damage response genes, particularly those involved in double strand break repair, at a higher level than the H1H2 group (11
). The genomic differences in H1H2 and H2 tumors may have clinical implications. GISTIC analysis revealed that copy number losses in 9p and 14q are more significant in the H2 group compared to the H1H2 group. However, at the level of gene expression we only see significant changes in the genes from 14q in the H2 subtype. Intriguingly, losses of 14q have been independently associated with a decrease in disease-specific survival in ccRCC (12
). The data presented herein support the postulate that ccRCC can be subtyped based on HIFα expression; the survival data from Klatte et al. indicate that the H2 subtype may be potentially linked to clinical outcome (12
). Together, these findings potentially suggest that different therapeutic regimens may need to be employed to treat patients with H1H2 and H2 tumors.
In order to validate the integrated genomic approach, we chose to study three of the identified targets using cell culture studies. Genome-wide studies of ccRCC to-date have primarily revealed genes which are inactivated during tumorigenesis. Thus, we focused on the concordantly gained and overexpressed genes. Three gained-overexpressed genes, EZH2
(5q35.2), and VCAN
(5q14.3) were chosen for study in cell culture experiments. EZH2
is thought to promote cell proliferation and inhibit cell differentiation by silencing tumor suppressors (25
), and known to be activated in breast (27
) and prostate cancers (28
). It also has been implicated in ccRCC (20
). It is interesting that while EZH2
, a histone methyltransferase, is gained, KDM6A
, a histone demethylase, is mutated in ccRCC (14
, a member of the PcG family, is believed to play a role in cancer by silencing tumor suppressors, such as ARF
). KDM6A has been shown to demethylate many RB binding proteins leading to their activation and subsequent cell cycle arrest (30
). Thus both KDM6A
mutation and EZH2
gain will lead to increased cell cycle progression. STC2
has been shown to be overexpressed in prostate (31
), breast (32
), and colorectal cancers (33
), and has been linked to ccRCC (22
), but its functional role has not yet been determined. STC2
is upregulated under hypoxia, and thought to help cells adapt to the stress of the tumor microenvironment (33
is involved in cell adhesion, proliferation, migration, and angiogenesis, and thought to promote cell proliferation by increasing the propagation of signals from mitogens such as platelet-derived growth factor
) and transforming growth factor beta 1
has been linked to prostate, breast, and ovarian cancers (23
), but not yet to ccRCC. Using siRNA experiments, we demonstrated that EZH2, STC2
, and VCAN
promote tumor growth by inhibiting cell death in ccRCC cells. These results strongly suggest that EZH2, STC2
, and VCAN
play roles in ccRCC and validate the integrated approach employed to identify them.
While the experiments described here examine EZH2, STC2, and VCAN individually, it is evident from the copy number data that the genes are gained simultaneously. In tumors that show a gain of STC2, there is a 29% and 48% chance that EZH2 or VCAN will also be gained, respectively. In ~4% of the tumors all three targets are gained. In order to test whether STC2 and VCAN have an additive effect on cell numbers and cell death, we simultaneously silenced them. We did not detect any additive or synergistic effects. More studies will be required to better understand the detailed functions of EZH2, STC2, and VCAN and whether they interact in vivo.
EZH2, STC2, and VCAN are commonly gained in both the H1H2 and H2 subtypes of ccRCC. EZH2, STC2 and VCAN are gained in 7%, 31%, and 24% of H1H2 samples respectively, and in 11%, 32%, and 11% of H2 samples respectively. In addition to these common targets, the individual subtype analysis has revealed that there are targets that are unique to each subtype. EZH2, STC2, and VCAN may be responsible for the earlier steps in tumorigenesis in both subtypes, whereas the targets that are specific to each subtype may be more important in the later steps of tumorigenesis. More work is needed to understand the different roles played by the common targets and roles played by the targets specific to each subgroup. It is also important to note that most (46%) of the tumors in this study are Stage I tumors, and that it is possible that alternative targets may be important during different tumor stages.
In summary, we are the first to identify and functionally validate two potentially important targets on 5q (STC2 and VCAN), a region gained in more than 30% of ccRCC samples. We also have further established that ccRCC can be classified into subtypes based on HIFα expression with each group having its own specific pattern of copy number alterations.