Identifying Cancer-Stem-Cell-Invadopodia-Related Prognostic Biomarkers in Gastric Cancer

A prevailing new paradigm of tumorigenesis proposes that only a subset of tumor cells with stem-like properties, termed “cancer stem cells (CSCs)”, has the ability to self-renew and sustain tumorigenesis. The discovery of CSCs supports that there is an organizational hierarchy in tumors. Interestingly, organizational hierarchy exists not only in tumors but also within CSCs, and different subpopulations of CSCs may differ considerably with each other with respect to their abilities to initiate and maintain tumorigenesis. Along this line, the concept of metastatic CSCs (mCSCs) has begun to emerge with increasing data supporting their existence in solid tumors. Presumably, the opportunity to detect mCSCs in tumors and understand their ability to promote cancer metastasis may suggest a new avenue for preventing cancer dissemination and metastasization. Invadopodia are transient actin-based protrusions in invasive cancer cells that mediate focal degradation of extracellular matrix (ECM) by the localized proteolytic activity of proteases. Cancer cells use invadopodia during mesenchymal-type migration to degrade and invade ECM structures. Our group has recently developed an invadopodia-protein enrichment assay, which enabled us to isolate and compare the proteins from the invadopodia of CD90+ gastric CSCs and their non-stem like cancer cell (NSCC) counterparts. In our preliminary research, we identified that the invadopodia of gastric CSCs contain higher amounts of the pro-invasive and stemness protein CD44 than NSCCs, supporting the existence of CSC-invadopodia-specific components that may contribute to their invasive properties. In this study, we will seek to further expand our assay to establish a high throughput “Invadopodia Proteomic Screening” (IPS) system to identify novel and functionally important CSC-specific invadopodia proteins that may serve as novel biomarkers for mCSCs that can be used to predict cancer metastasis in as gastric cancer. To this end, we will isolate highly invasive CSCs (hiCSCs), which may functionally resemble mCSCs, from gastric cancer using FACS and in vitro cell invasion assays (Aim 1). We will then induce invadopodia formation in hiCSCs, CSCs and NSCCs, isolate the invadopodia-enriched fractions, and subject them to Difference Gel Electrophoresis followed by mass spectrometric analysis (Aim 2). Once we identified the invadopodia markers specific for hiCSCs, we will verify the high invasive capacity of CSCs carrying these markers using a series of in vitro and in vivo tumor invasion and metastasis models (Aim 3). We will then genetically knockdown the expressions of the individual hiCSC-invadopodia proteins to explore their functional importance (Aim 4). To explore the potential clinical applicability, we will examine the prognostic significance of these novel biomarkers by immunostaining analyses on tissue microarrays of human gastric cancer tissues (Aim 5). Through these high innovative and clinically translatable approaches, we envision that identifying the existence of mCSC-specific invadopodia markers using the IPS will enable us to distinguish mCSCs from other populations of cancer cells, which may not only lead to a more in-depth understanding of the role of CSCs in cancer metastasis but also may help a more rationalized and personalized strategies for the prognostic prediction and the treatment of patients with gastric cancer.