It is now well established that loss of proper gene function by gene silencing contributes heavily to CRC initiation and progression. In this regard, several important Wnt signaling inhibitors such as SFRPs, WIF-1
, and DKK-1
have been previously reported to be frequently hypermethylated in primary colorectal tumors (14
). Our results suggest that epigenetic silencing of SOX17
is another important step in activation or amplification of aberrant Wnt signaling in CRC. This conclusion is supported by our observations that (a
) methylation of SOX17
is strongly associated with the loss of gene expression in seven CRC cell lines; (b
is unmethylated in normal colon but methylated with high frequency in a variety of human primary tumors including CRC; (c
) methylation of SOX17
occurs frequently in premalignant colonic neoplasms; (d
) restoration of SOX17 function reduces colony formation in colon cancer cells; and (e
) SOX17 can efficiently suppress both wild-type and mutant β-catenin–mediated transcriptional activity. These findings suggest that SOX17
hypermethylation may play an important role in the early steps of cancer formation.
Our current findings for SOX17
make yet another addition of epigenetic inactivation events to the growing list of DNA hypermethylated Wnt antagonist genes in colon and other cancers. The question arises as to why so many potentially redundant steps for Wnt pathway activation would simultaneously be present in a single tumor. The answer may be 2-fold, as we and others have postulated (14
). First, that each of these individual gene epigenetic inactivation steps alone may be less potent than single mutations for driving the Wnt pathway and that they are required to summate to yield the full epigenetic drive for tumorigenesis. Second, this summation may be additionally necessary to give Wnt pathway mutations their full effect to help drive abnormal activation of the Wnt pathway. In this regard, silenced Wnt antagonist genes can be divided into three broad classes, each contributing to individual steps in amplifying the effects of increasing nuclear β-catenin function as the final readout for the active Wnt pathway. The first class, including the secreted SFRPs, WIF-1, and DKK, acts at the level of the cell membrane to prevent ligand-receptor interactions. The inactivation of this class, as we and others have shown for SFRPs (14
), can up-regulate the Wnt pathway at the cell membrane and this leads to increased cellular levels of β-catenin. When these increases meet a crippled cytoplasmic degradation complex for this protein, such as in colon cancer cells with APC mutations, or when this leads to increased levels of mutant β-catenin, which can evade the complex, then more β-catenin reaches the nucleus to transcriptionally drive Wnt pathway target genes (23
). The second class comprises certain members of the cytoplasmic degradation complex for β-catenin and the example here is the APC
gene. Thus, APC
promoter hypermethylation is an alternative mechanism to mutations for inactivation of this key gene in colon cancer development (29
) and can, especially in the setting of inactivation of the above membrane Wnt antagonists, result in Wnt nuclear activation. Finally, we now show epigenetic inactivation of another class of Wnt antagonist. Certain nuclear proteins, including SOX17, inhibit Wnt signaling at the level of the nuclear complex between β-catenin and TCF. Inactivation of SOX17 is then added to abnormal activation of the pathway by stabilizing and/or facilitating this key Wnt-driven transcription complex. In summary, we hypothesize that simultaneous epigenetic down-regulation of SFRPs, WIF-1, DKK, and SOX17, especially in the setting of key pathway mutations, act in a complementary manner for the constitutive activation of Wnt signaling, which can drive tumor initiation and progression.
Our detection of SOX17
hypermethylation now adds this gene to the repertoire of DNA hypermethylated genes that may potentially be used as new molecular markers for cancer detection and/or risk. The high frequency of SOX17
methylation in 50% of non–small cell lung cancers and nearly 90% of esophageal squamous cancers further supports this possibility and is consistent with the known role of aberrant activation of Wnt signaling during tumorigenesis for multiple cancer types (30
). Given that APC inactivation or β-catenin mutations rarely occurs in these tumor types other than CRC (32
), our findings suggest that SOX17 maybe have wide-ranging importance for tumor suppression.
Our present results and those from other groups all indicate that negative regulation of β-catenin/TCF transcription activity is an important function of Sox17 (10
). However, how the full range of mechanisms underlying SOX17 repression of Wnt activity in CRC and other cancers must still be determined. Three Sox proteins in Xenopus
embryos (Sox17α, Sox17β, and Sox3) have been reported to inhibit Wnt signaling by directly competing with TCF/LEF for β-catenin binding (10
). Most recently, another model based on studies mainly in SW480 colon cancer cells suggests that mouse Sox17 antagonizes Wnt signaling by physically associating with TCF/LEF (via the NH2
-terminal HMG) and β-catenin (via the COOH terminus) to promote the protein degradation of both TCF/LEF and β-catenin (12
). In contrast to the two models discussed above, our structure-based deletion analysis data have clearly shown that only the NH2
-terminal HMG box of SOX17 is required for inhibition of TCF/β-catenin transcriptional activity, whereas the COOH-terminal β-catenin binding domain may not be heavily required for this inhibition. The discrepancy between our data and other studies may be due to different Sox17
genes from different species used in the experimental systems.
Although the HMG box of Sox family proteins is a conserved motif for minor-groove DNA recognition, accumulating studies suggest that it is also an important domain involved in protein-protein interaction. For instance, HBP1 can down-regulate Wnt signaling by blocking the DNA-binding ability of TCF4 protein via its HMG box (34
). The HMG domain of SOX8 and SOX10 can interact with numerous transcription factors through the COOH-terminal part of the HMG box, including homeodomain proteins Meox1 and Pax6 and the zinc-finger protein Hivep1 (35
). Importantly, structural biology studies show that SOX2 and Oct1 bind to adjacent sites on DNA, and interact with each other through the COOH-terminal region of the HMG box in SOX2 and the POU domain in Oct1 to regulate transcription synergistically (36
). Furthermore, mouse Sox17 can physically interact with TCF/LEF family members, including TCF3, TCF4, and LEF1, and this interaction is mediated by their respective HMG domains (12
). In light of all of the above results, we speculate that SOX17 could physically interact with TCF/LEF or yet to be identified protein(s) via its HMG box, which can affect TCF/LEF DNA binding–dependent transcription, thereby repressing Wnt signaling target transcription in vivo
. Thus, an important aspect of future studies is to investigate the DNA elements on endogenous SOX17 target gene promoters through which SOX17 can repress transcription. Understanding the molecular mechanisms underlying tumor growth control by SOX17 may improve our ability to provide diagnostic, prognostic, and treatment paradigms for cancer patients.