In our study, we sought to identify signatures of metastasis embedded in a subset of primary tumors, which might predict clinically-aggressive behavior 
. Using supervised SAM analysis, we identified 963 unique genes that are significantly overexpressed in CLM vs. primary CRC (and potentially contaminating normal liver tissue). In an independent set of tissue microarrays, we examined two highly-ranked genes (LEF1 and SPP1) as surrogate biomarkers for the CLM signature, and demonstrated that overexpression of LEF1, but not SPP1, in the primary CRC tissues correlates with a statistically significant increased risk of CLM, albeit its sensitivity and specificity in predicting liver metastasis were modest. In addition, independent of tumor stage, overexpression of LEF1, not SPP1, denotes a poor prognosis for survival.
LEF1 was initially identified as a pre-B and T-lymphoid-specific gene encoding a DNA-binding protein of high mobility group (HMG) proteins.
It is a member of the T-cell factor/lymphoid-enhancing factor (TCF/LEF) family of transcription factors, which acts through the Wnt signaling pathway
to regulate gene expression and coordinate many cellular processes in normal development and tissue homeostasis, and, when deregulated, in colonic tumorogenesis and metastasis. Upon Wnt stimulation, LEF1 or other TCF/LEF-family transcription factors associate with β-catenin, a key cytoplasmic/nuclear mediator of Wnt pathway, and activate Wnt-responsive target genes. In contrast, without Wnt stimulation, glycogen synthase kinase (GSK)-3 (in a complex with APC) constitutively phosphorylates β-catenin, resulting in its proteasome-dependent degradation.
Although genetic and epigenetic changes have been documented in several targets throughout the pathway, mutation in either APC or β-catenin appears to be a crucial element in CRC carcinogenesis.
The LEF1 gene itself is not normally expressed in the adult intestinal epithelium, but only observed in the embryos while development is in progress. However, its overexpression has been well documented in CRC tumorigenesis
, and denotes aberrant activation of the Wnt/β-catenin pathway via
stabilization of LEF1/β-catenin complex. Indeed, LEF1 is a direct transcriptional target of the LEF1/β-catenin complex
, indicating a positive feedback loop for Wnt signaling, and suggesting LEF1 might be useful as a prognostic biomarker of Wnt pathway activation. Our findings are consistent with a prior study showing nuclear (active) β-catenin staining to be prognostic in colon cancer,
and highlight a role of Wnt signaling in colon cancer progression and liver metastasis.
As the prefix "osteo" suggests, osteopontin (or OPN, also known as secreted phosphoprotein 1 [SPP1], bone sialoprotein I, early T-lymphocyte activation 1) was initially recognized as an important glycosylated, adhesive phosphoprotein in bone.
Since then, several lines of evidence have shown its role in controlling tumorigenicity, progression and metastasis via
its diverse ability as a cell-matrix mediator to interact with a variety of factors such as cell surface receptors (integrins and CD44), secreted proteases (matrix metalloproteinases and urokinase plasminogen activator), and growth factor/receptor pathways (TGF/EGFR and HGF/Met).
Overexpression of SPP1 has been reported in several human cancers, including lung, breast and colon cancers. In a gene-expression profiling study, Agrawal et al
identified SPP1 as a lead marker correlating with CRC progression, and strongly expressed in CLM.
Most recently, Rohde et al
observed that overexpression of SPP1 is indicative of poor survival in CRC and is significantly correlated with CLM.
In addition, overexpression of SPP1 correlates with increased immunohistochemical staining of β-catenin and, in an in vivo
model, with Wnt activating mutations. These data suggest a crucial role of SPP1 in CRC progression and metastasis likely via
molecular cross-talk with the Wnt pathway. Our results partially corroborate with published data in that we observe a trend towards poorer survival and CLM with SPP1 overexpression, though it is not statistically significant. This difference may be due to a smaller sample size and/or shorter follow-up time. Nonetheless, our data suggest that the overexpression of LEF1 is a stronger prognostic factor than SPP1 in correlating overall survival and CLM.
Though our analysis focused primarily on LEF1 and SPP1, other highly-ranked signature genes with increased expression in CLM compared to primary CRC also have biological functions consistent with roles in tumor progression, and might have prognostic utility. For example, CXCR4 (Chemokine (C-X-C motif) receptor 4) has been implicated in breast 
and colon cancer metastasis.
LOX (lysyl oxidase) was shown to be associated with hypoxia where it functions in metastasis and predicts poor outcome in breast cancer.
SPARC (secreted protein, acidic, cysteine-rich; osteonectin) was identified among genes that mark and mediate breast cancer metastasis.
Further studies are needed to characterize these and other signature genes in CRC progression.
Much of the published data comparing gene expression profiles from primary CRC vs. CLM have reported the differences in up- or down-regulated genes.
To the best of our knowledge, our data are the first to identify the expression of LEF1 as a predictor of overall survival, and an indicator of CLM. Comparisons of primary CRC vs. CLM, or of primary CRC associated with or without CLM, have identified gene signatures with relevance to colorectal cancer progression.
However, there are minimal overlaps between our CLM signature genes and the top-ranking CLM/progression-associated genes reported in these studies (). Several issues may explain the discordance among studies, such as: variability in patient cohorts, technical differences (the composition of the microarrays used, study design, statistical methodologies), and variability in the use of independent cohorts of patients to validate candidate prognostic genes.
Overlap of CLM genes with top-ranking CLM/progression genes from other published studies.
In conclusion, our study shows that overexpression of LEF1 in primary CRC correlates with a higher risk of CLM and denotes poor overall survival. It is a stronger predictor than SPP1, a marker reported in previous transcriptome studies. High-throughput gene expression profiling technology has revealed new insights into the molecular heterogeneity of CRC and identified new and better molecular markers for risk stratification. This holds promise for personalized medicine and improved targeted therapy. To achieve these goals, further studies are needed to understand the functional roles and clinical implications of LEF1, SPP1 and other signature genes for CLM.