Abstract
Epithelial-mesenchymal transition (EMT) is a key contributor in tumor progression and metastasis. EMT produces cellular heterogeneity within head and neck squamous cell carcinomas (HNSCC) by creating a phenotypically distinct mesenchymal subpopulation that is resistant to conventional therapies. In this study, we systematically characterized differences in the secretomes of E-cadherin high epithelial-like and E-cadherin low mesenchymal-like subpopulations using unbiased and targeted proteomics. A total 1765 proteins showed significant changes with 177 elevated in the epithelial subpopulation and 173 elevated in the mesenchymal cells. Key nodes in affected networks included NFκB, Akt, and ERK, and most implicated cellular components involved various aspects of the extracellular matrix. In particular, large changes were observed in multiple collagens with most affected collagens at much higher abundance levels in the mesenchymal subpopulation. These cells also exhibited a secretome profile resembling that of cancer-associated fibroblastic cells (CAF). S100A4, a commonly used marker for cancer-associated fibroblastic cells, was elevated more than 20-fold in the mesenchymal cells and this increase was further verified at the transcriptome level. S100A4 is a known mediator of EMT, leading to metastasis and EMT has been proposed as a potential source of cancer-associated fibroblastic cells in solid tumors. S100A4 knockdown by small interfering RNA led to decreased expression, secretion and activity of matrix metalloproteinase 2, as verified by quantitative PCR, multiple reaction monitoring and zymography analyses, and reduced invasion in collagen-embedded spheroids. Further confirmation in three-dimensional organotypic reconstructs showed less invasion and advanced differentiation in the S100A4 RNA interference samples. Orthotopic metastasis model, developed to validate the findings in vivo, demonstrated a decrease in spontaneous metastasis and augmented differentiation in the primary tumor in siS100A4 xenografts. These results demonstrate the value of secretome profiling to evaluate phenotypic conversion and identify potential novel therapeutic targets such as S100A4.
Highlights
Head and neck squamous cell carcinoma (HNSCC)1 is the sixth most common cancer worldwide with high morbidity and mortality
Since epithelial-mesenchymal transition (EMT) is proposed as a possible origin for cancerassociated fibroblastic cells, we wanted to investigate whether the mesenchymal subpopulation (MSP) of heterogeneous HNSCCs resembles CAFs
Functional Characterization of Epithelial and Mesenchymal HNSCC Subpopulations—Considering the concept that carcinomas initially depend on local fibroblasts for activation of EMT, and later lose this dependence [29], we investigated the role of fibroblasts in affecting the epithelial and mesenchymal subpopulations of the heterogeneous OCTT2 cell line
Summary
We have previously shown that an E-cadherin low mesenchymal subpopulation (MSP) within a predominantly epithelial carcinoma confers resistance to both conventional chemotherapy and cetuximab treatment [3, 4]. CAFs secrete a variety of factors that promote both tumor growth and progression, such as growth factors, cytokines, extracellular matrix (ECM) components and matrix metalloproteinases (MMP) [9] They are highly heterogeneous, no single marker can distinguish them. Since EMT is proposed as a possible origin for cancerassociated fibroblastic cells, we wanted to investigate whether the MSP of heterogeneous HNSCCs resembles CAFs. As the first step we characterized the phenotypic properties of the epithelial and mesenchymal subpopulations in vitro and in vivo. Liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) was used to analyze the secretomes of the epithelial and mesenchymal subpopulations in an unbiased manner Following this approach we further validated the expression of several CAF markers at the transcriptome level and identified S100A4 as an important mediator of the phenotypic conversion. Knockdown of S100A4 by small interfering RNA (siRNA), verified by targeted proteomics using multiple reaction monitoring (MRM), led to reduced metastasis burden, and to advanced differentiation of the primary tumor, demonstrating a novel finding with potential implications for therapeutic strategy
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