Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter.
Key molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) are well described. However, the mechanisms through which clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we used an integrative genomics approach to examine CRC progression. We used laser capture microdissection to isolate colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas and metastases, and used gene expression profiling to identify pathways that were differentially expressed between the different cell types. We identified a number of potentially important transcriptional changes in developmental and oncogenic pathways, and noted a marked upregulation of EREG in primary and metastatic cancer cells. We confirmed this pattern of gene expression by in situ hybridization and observed staining consistent with autocrine expression in the tumor cells. Upregulation of EREG during the adenoma-carcinoma transition was associated with demethylation of two key sites within its promoter, and this was accompanied by an increase in the levels of epidermal growth factor receptor (EGFR) phosphorylation, as assessed by reverse-phase protein analysis. In CRC cell lines, we demonstrated that EREG demethylation led to its transcriptional upregulation, higher levels of EGFR phosphorylation, and sensitization to EGFR inhibitors. Low levels of EREG methylation in patients who received cetuximab as part of a phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers, including those of the head and neck, lung and bladder. Therefore, we propose that upregulation of EREG expression through promoter demethylation might be an important means of activating the EGFR pathway during the genesis of CRC and potentially other cancers.
Oncology Biomarker Development, Genentech Inc., South San Francisco, CA, USA.
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X
Qu
Bioinformatics and Computational Biology, Genentech Inc., South San Francisco, CA, USA.
T
T
Sandmann
Department of Pathology, University of Virginia Health System, Charlottesville, VA, USA.
H
H
Frierson
Oncology Biomarker Development, Genentech Inc., South San Francisco, CA, USA.
L
L
Fu
Department of Pathology, Genentech Inc., South San Francisco, CA, USA.
E
E
Fuentes
Oncology Biomarker Development, Genentech Inc., South San Francisco, CA, USA.
K
K
Walter
Department of Biostatistics, Genentech Inc., South San Francisco, CA, USA.
K
K
Okrah
Department of Pathology, University of Virginia Health System, Charlottesville, VA, USA.
C
C
Rumpel
Department of Pathology, University of Virginia Health System, Charlottesville, VA, USA.
C
C
Moskaluk
Oncology Biomarker Development, Genentech Inc., South San Francisco, CA, USA.
S
S
Lu
Oncology Biomarker Development, Genentech Inc., South San Francisco, CA, USA.
Y
Y
Wang
Bioinformatics and Computational Biology, Genentech Inc., South San Francisco, CA, USA.
R
R
Bourgon
Oncology Biomarker Development, Genentech Inc., South San Francisco, CA, USA.
E
E
Penuel
Department of Clinical Sciences, Genentech Inc., South San Francisco, CA, USA.
A
A
Pirzkall
Oncology Biomarker Development, Genentech Inc., South San Francisco, CA, USA.
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L
Amler
Oncology Biomarker Development, Genentech Inc., South San Francisco, CA, USA.
M R
MR
Lackner
Department of Medical Oncology, Vall d'Hebron University Hospital, Barcelona, Spain.
J
J
Tabernero
Oncology Biomarker Development, Genentech Inc., South San Francisco, CA, USA.
G M
GM
Hampton
Oncology Biomarker Development, Genentech Inc., South San Francisco, CA, USA.
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Kabbarah
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics*; D019175:DNA Methylation; D000077209:Decitabine; D018450:Disease Progression; D066260:Epiregulin / Q000235:genetics*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D011401:Promoter Regions, Genetic
C587880:EREG protein, human; D066260:Epiregulin; D000077209:Decitabine; D066246:ErbB Receptors; D001374:Azacitidine