Poster No. 71
Title:
The Tumor Microenvironment Meets Epigenetics: Regulation of E-cadherin Expression in Squamous Cell
Carcinoma Progression through DNA Methylation
Authors:
Teresa DesRochers, Yasusei Kudo, Takashi Takata, Jonathan Garlick
Presented by:
Teresa DesRochers
Departments:
Department of Anatomy and Cellular Biology, Tufts University School of Medicine; Department of Oral and
Maxillofacial Pathology, Tufts University School of Dental Medicine; Department of Oral Maxillofacial
Pathobiology, Hiroshima University
Abstract:
Epigenetic silencing of E-cadherin through DNA hypermethylation of the promoter has been well documented
in many cancer types. The observed pattern of gene silencing is heterogeneous throughout the tumor and
changes during cancer progression indicating a possible role for the tumor microenvironment in the regulation
of epigenetic-mediated silencing. As it is well established that the tumor microenvironment has a significant
impact on the phenotypic properties of a broad spectrum of cancers, we propose that the microenvironment
modifies epigenetic control of gene expression, thereby regulating transient changes in the expression of key
proteins during tumor progression. We have studied whether the tumor microenvironment plays a role in the
epigenetic control of E-cadherin expression during the progression of oral squamous cell carcinoma (OSCC).
We have used bio-engineered, 3D human tissue models that mimic various stages of OSCC progression to
elucidate if methylation-mediated gene silencing of E-cadherin is regulated by the tissue microenvironment.
Tumor cells were derived from a lymph node metastasis that originated as an OSCC and a clonal isolate
(C1-Inv-1) was characterized in 2D, monolayer culture and in 3D, engineered tissues. E-cadherin expression
was analyzed by Western blot, immunofluorescence, and RT-PCR while the methylation status of the
E-cadherin promoter was examined by both methylation-specific PCR (MSP) and bisulfite sequencing (BSP). In
2D culture, C1-Inv-1 cells demonstrated complete loss of E-cadherin expression due to promoter
hypermethylation.
We observed that methylation was limited to only a few distinct CpGs rather than being widespread throughout
the CpG island of the E-cadherin promoter, suggesting regulated DNA methylation in these cells. When
C1-Inv-1 cells were grown as a 3D tissue at an air-liquid interface in order to induce homotypic cell-cell
interactions in a tissue that mimicked the premalignant stage of tumor progression, E-cadherin expression and
localization were restored in cells in the suprabasal layers of the tissue. In addition, tumor cells manifested their
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Poster No. 71
in vivo behavior and spread as single cells into the underlying stroma. These invasive cells maintained loss of
E-cadherin expression, suggesting that continued suppression of E-cadherin and the acquisition of invasive
properties were dependent upon contact with the stromal microenvironment that sustained promoter
hypermethylation.
Currently, we are examining the methylation state of the E-cadherin promoter in those cells that maintained
E-cadherin loss as they invaded into the underlying stroma in comparison to those cells that re-expressed
E-cadherin as they stratified above the epithelial-stromal interface. Initial work has revealed an increase in
methylation in those cells grown in 3D tissues compared to 2D monolayer culture, and a significant change in
the methylation of specific CpGs in the E-cadherin promoter between those cells that maintained loss of
E-cadherin and invaded into the stroma and those that re-expressed E-cadherin and stratified above the
epithelial-stromal interface. We propose a model in which transient and heterogeneous patterns of E-cadherin
expression during cancer progression are due to epigenetic control that is dynamically sensitive to regulation by
the microenvironment and results in the unstable and reversible tumor cell phenotypes seen in invasive and
metastatic human cancers.
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