Overall, our results reveal a novel tumor invasive signature derived from invading transformed cells compared to control noninvading normal and dysplastic cells in organotypic (3D) culture. This signature is one that annotates primary invasive human esophageal squamous cell cancer as distinctive from adjacent normal human esophageal mucosa in two independent cohorts of tumors, underscoring the fidelity and utility of this tumor invasive signature. It is conceivable that this molecular signature might be informative in the future for other squamous cell cancers arising in different tissues.
Gene ontology analysis (DAVID databases) of specific biological processes that are believed to be involved in the tumor microenvironment reveals the consistent and unique upregulation of periostin, suggesting its critical functional role. First, periostin was found to be upregulated in invading transformed cells and in primary esophageal squamous cell cancers based upon immunohistochemical and Western blot analysis. Second, periostin’s direct functional role is underscored by overexpression and knockdown experiments in which genetic manipulation of periostin dramatically influences the degree of tumor invasion in organotypic culture. Third, EGFR signaling and p53 mutation, both canonical genetic alterations in ESCC, appear to converge upon periostin based upon luciferase reporter gene assays as well as inhibition of EGFR signaling and restoration of wild type p53 function. In aggregate, these novel results underscore the utility of the organotypic culture model for the discovery of direct biological effectors of tumor invasion into the microenvironment, which has been largely elusive to date. Local tumor invasion in the mesenchymal stromal compartment is important given that it temporally precedes tumor dissemination in the lymphatic and blood vessels for tumor metastasis.
Various avenues of investigation have highlighted the important role of the microenvironment in enhancing the initial dissemination of malignant tumor cells. Dynamic interactions between the epithelium and mesenchymal stroma contribute to boosting the invasive phenotype of tumor cells by activating a variety of genes facilitating cell proliferation, de-differentiation, migration and invasion (23
). Significant changes such as loss of cell-cell contacts, disruptions in cell adhesion junctions and altered cell-extracellular matrix interactions within the tumor microenvironment converge to increase the ultimate metastatic potential of tumor cells (25
). Therefore, identification of gene expression pattern changes during initial stages of tumor progression within the tumor microenvironment is crucial to understanding the causes of tumor invasion, and ultimately, tumor metastasis to distant organ sites. Thus, our results provide new platforms into the investigation of tumor invasion.
Periostin has been shown also to have a role in bone, tooth and heart formation during development (26
) and is only re-expressed and upregulated in adult tissue after vascular, skeletal or bone injuries (28
).Periostin is similarly overexpressed in human cancers (14
). Our results highlight that a tumor invasive signature defines genetically engineered ESCC that is reproduced in human ESCC. Our studies also suggest that induction of periostin, a secreted protein with a long half-life (Supplementary Figure S4
and data not shown), may alter the tumor microenvironment by accumulating in the stroma and facilitating invasion through matrix remodeling. This may be achieved through regulation of collagen I fibrillogenesis (31
), serving as a bridge between tenascin C and the extracellular matrix, as well as through interaction with αV
integrins. Furthermore, periostin may promote tumor cell survival in the matrix of the microenvironment by activating the Akt/PI3K pathway. Lack of periostin may lead to suppression of Notch1 signaling (32
), conversely, it is conceivable that overexpression of periostin could activate Notch1 signaling in cancers. Indeed, we have evidence of activated Notch signaling in invasive tumor cells grown in organotypic culture (data not shown). Future translational and clinical investigations might seek to exploit periostin as an attractive therapeutic target, especially in a combinatorial fashion, for example, with inhibitors of receptor tryosine kinases.