Abstract
BackgroundIn colorectal cancer, CDX2 expression is lost in approximately 20% of cases and associated with poor outcome. Here, we aim to validate the clinical impact of CDX2 and investigate the role of promoter methylation and histone deacetylation in CDX2 repression and restoration.MethodsCDX2 immunohistochemistry was performed on multi-punch tissue microarrays (n = 637 patients). Promoter methylation and protein expression investigated on 11 colorectal cancer cell lines identified two CDX2 low expressors (SW620, COLO205) for treatment with decitabine (DNA methyltransferase inhibitor), trichostatin A (TSA) (general HDAC inhibitor), and LMK-235 (specific HDAC4 and HDAC5 inhibitor). RNA and protein levels were assessed. HDAC5 recruitment to the CDX2 gene promoter region was tested by chromatin immunoprecipitation.ResultsSixty percent of tumors showed focal CDX2 loss; 5% were negative. Reduced CDX2 was associated with lymph node metastasis (p = 0.0167), distant metastasis (p = 0.0123), and unfavorable survival (multivariate analysis: p = 0.0008; HR (95%CI) 0.922 (0.988–0.997)) as well as BRAFV600E, mismatch repair deficiency, and CpG island methylator phenotype. Decitabine treatment alone induced CDX2 RNA and protein with values from 2- to 25-fold. TSA treatment ± decitabine also led to successful restoration of RNA and/or protein. Treatment with LMK-235 alone had marked effects on RNA and protein levels, mainly in COLO205 cells that responded less to decitabine. Lastly, decitabine co-treatment was more effective than LMK-235 alone at restoring CDX2.ConclusionCDX2 loss is an adverse prognostic factor and linked to molecular features of the serrated pathway. RNA/protein expression is restored in CDX2 low-expressing CRC cell lines by demethylation and HDAC inhibition. Importantly, our data underline HDAC4 and HDAC5 as new epigenetic CDX2 regulators that warrant further investigation.
Highlights
In colorectal cancer, CDX2 expression is lost in approximately 20% of cases and associated with poor outcome
CDX2 has been linked to colorectal cancer (CRC) progression, with reduced expression of the protein associated with more advanced tumor stage, vessel invasion, and metastasis [3,4,5,6,7]
Previous work by our group and others shows that most sporadic microsatellite unstable (MSI) cancers show some degree of loss of the protein in the tumor, whether in a small or substantial percentage of cells [3, 5, 9]
Summary
CDX2 expression is lost in approximately 20% of cases and associated with poor outcome. Many studies, including the work by Dalerba et al, underline the unfavorable survival time in patients with a complete absence of CDX2 in the tumor [8], a feature that occurs in approximately 5% of patients [7, 8] They demonstrate that CDX2-negative CRC patients may benefit from chemotherapy, in a stage II setting [8]. Previous work by our group and others shows that most sporadic microsatellite unstable (MSI) cancers show some degree of loss of the protein in the tumor, whether in a small or substantial percentage of cells [3, 5, 9] This loss is not limited to MSI-high cancers, but is found in microsatellite stable (MSS) tumors with BRAF mutation and high-level CpG island methylator phenotype (CIMP), in other words, in cancers deriving from the so-called serrated pathway [10]. More than 20% of CRCs show some degree (or complete loss) of CDX2 protein in the tumor, which is often reported along with a preponderance for female gender and right-sided tumor location, two features frequently associated with serrated lesions [11]
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