Reliable approaches to identify stem cell mechanisms that mediate aggressive cancer could have great therapeutic value, based on the growing evidence of embryonic signatures in metastatic cancers. However, how to best identify and target stem-like mechanisms aberrantly acquired by cancer cells has been challenging. We harnessed the power of reprogramming to examine GRP78, a chaperone protein generally restricted to the endoplasmic reticulum in normal tissues, but which is expressed on the cell surface of human embryonic stem cells and many cancer types. We have discovered that (1) cell surface GRP78 (sGRP78) is expressed on iPSCs and is important in reprogramming, (2) sGRP78 promotes cellular functions in both pluripotent and breast cancer cells (3) overexpression of GRP78 in breast cancer cells leads to an induction of a CD24−/CD44+ tumor initiating cell (TIC) population (4) sGRP78+ breast cancer cells are enriched for stemness genes and appear to be a subset of TICs (5) sGRP78+ breast cancer cells show an enhanced ability to seed metastatic organ sites in vivo. These collective findings show that GRP78 has important functions in regulating both pluripotency and oncogenesis, and suggest that sGRP78 marks a stem-like population in breast cancer cells that has increased metastatic potential in vivo.
The heat shock protein GRP78 typically resides in the endoplasmic reticulum in normal tissues, but it has been shown to be expressed on the cell surface of several cancer cells, and some stem cells, where it can act as a signaling molecule by not-yet-fully defined mechanisms. Although cell surface GRP78 (sGRP78) has emerged as an attractive chemotherapeutic target, understanding how sGRP78 is functioning in cancer has been complicated by the fact that sGRP78 can function in a cell-context dependent manner, with a diverse array of reported binding partners, to regulate a variety of cellular responses. We had previously shown that sGRP78 was important in regulating pluripotent stem cell (PSC) functions, and hypothesized that embryonic-like mechanisms of GRP78 were critical to regulating aggressive breast cancer cell functions. Here, using proteomics we identify Dermcidin (DCD) as a novel sGRP78 binding partner common to both PSCs and breast cancer cells. We show that GRP78 and DCD cooperate to regulate stem cell and cancer cell migration that is dependent on the cell surface functions of these proteins. Finally, we identify Wnt/β-catenin signaling, a critical pathway in stem cell and cancer cell biology, as an important downstream intermediate in regulating this migration phenotype.
Over 30% of ovarian cancer (OvCa) patients present with thrombocytosis (elevated plasma platelet count above 450,000 per cubic millimeter) at the time of diagnosis. These patients exhibit shorter survival times and a higher likelihood of advanced stage disease. Platelets have also been shown to enhance metastasis and promote tumor cell survival by coating tumor cells which provides immune escape and protection from chemotherapeutic agents. While previous studies have shown that platelets exhibit differential OvCa cell binding and facilitate cancer cell growth, the mechanism and molecules involved have yet to be elucidated. SUSD2 (SUShi Domain containing 2), an 822 amino acid type I transmembrane protein, is highly abundant in ovarian tumors and normal endothelial cells that line the blood vessels. Endothelial cells do not normally adhere to platelets; therefore, we hypothesized that SUSD2 inhibits the binding of platelets to cancer cells, which subsequently decreases platelet activation. To investigate the role of SUSD2 in platelet binding, SUSD2 knock-down (SUSD2neg) and non-targeting (SUSD2pos) OVCAR3 cell lines were generated. SUSD2pos and SUSD2neg OVCAR3 cell lines were cultured and grown to confluency. Platelets were isolated, dyed with Calcein-AM, washed and added to the cancer cells. After a 15-minute incubation, the co-culture was washed to remove non-adherent platelets. We demonstrated that platelets were bound to the OVCAR3-SUSD2neg an average of 35% more compared to OVCAR3-SUSD2pos cells. In order to identify the receptor on the platelets mediating adhesion to the tumor cells, we used the drug eptifibatide to block GPIIb/IIIa, an integrin receptor on the platelet surface that primarily binds to fibrinogen. Using the same methods described above, platelets were incubated with the inhibitor before being co-incubated with the cancer cells. Treating the platelets with eptifibatide decreased platelet binding by an average of 35% to both cancer cell lines, indicating that platelet binding to tumor cells is partially, yet equally, mediated by GPIIb/IIIa regardless of the presence of SUSD2. The role of this receptor was further explored by performing flow cytometry on the supernatant collected from co-culturing platelets with the cancer cells. The conditioned media contains the platelets that were not bound to the cancer cells. We measured the activation of the unbound platelets using the anti-PAC-1 antibody that binds exclusively to the activated conformation of GPIIb/IIIa. Incubation with the OVCAR-3-SUSD2neg cells activated the unbound platelets three-fold more compared to the OVCAR3-SUSD2pos cells. These data indicate that SUSD2 expression may mediate the activation state of GPIIb/IIIa, and this, in turn, may affect adhesion mediated by other factors. Citation Format: Tyson Lager, Megan Thacker, Charissa Etrheim, Kristi A. Egland, Mark K. Larson, Jennifer A. A. Gubbels. A novel epithelial ovarian cancer protein, SUSD2, inhibits platelet activation and binding to tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 301A. doi:10.1158/1538-7445.AM2015-301A
Reliable approaches to identify and target stem-cell mechanisms that mediate aggressive cancer could have great therapeutic value, based on the growing evidence of embryonic signatures in metastatic cancers. However, how to best identify and target stem-like mechanisms aberrantly utilized by cancer cells has been challenging. We harnessed the power of induced pluripotent stem cells (iPSCs) to identify embryonic mechanisms exploited by cancer. A screen comparing the cell surface proteome of iPSCs and breast cancer cells identified GRP78, a heat shock protein that is normally ER-restricted, but has been shown to be aberrantly expressed on the cell surface of several cancers, where it can act as a signaling molecule by poorly understood mechanisms. Although cell surface GRP78 (sGRP78) has emerged as an attractive chemotherapeutic target, understanding how sGRP78 is functioning in cancer has been complicated by the fact that GRP78 can function to regulate a variety of cellular responses, using a diverse array of reported binding partners, which can vary by cell type. Therefore, without insight into the specific GRP78-dependent mechanisms that are responsible for mediating aggressive cancer, it will be difficult to determine how to best target GRP78. We have discovered that (1) sGRP78 is expressed on iPSCs (but not their somatic parental populations) and plays an important role in reprogramming, (2) sGRP78 promotes cellular functions such as proliferation/survival and migration in both stem cells and breast cancer cells (3) overexpression of GRP78 in breast cancer cells leads to an induction of a previously established CD24-/CD44+ 'cancer stem cell' (CSC) population (4) sGRP78+ breast cancer cell populations are enriched for genes involved in stemness and appear to be a subset of previously established CSCs (5) sGRP78+ breast cancer cell populations show a significantly enhanced ability to seed metastatic organ sites in vivo (6) GRP78 interacts with Dermcidin (DCD) at the cell surface of cancer cells and iPSCs, where it is important in regulating stem cell and cancer cell migration and survival/proliferation. These collective findings suggest that sGRP78 marks a stem-like population in breast cancer cells that has increased metastatic potential in vivo, and that sGRP78 and DCD cooperate to regulate key cellular functions important in mediating tumorigenesis. Overall, this work has implications for understanding how cancer cells exploit embryonic-like mechanisms, which could provide novel strategies for chemotherapeutic targeting of aggressive breast cancer cell populations. Citation Format: Tyson W. Lager, Henry C. Conner, Ian H. Guldner, Michael Z. Wu, Yuriko Hishida, Tomoaki Hishida, Sergio Ruiz, Amanda E. Yamasaki, Juan Carlos Izpisua Belmonte, Peter C. Gray, Jonathan A. Kelber, Siyuan Zhang, Athanasia D. Panopoulos. Aberrant cell surface expression of GRP78 in breast cancer cells marks a stem-like population that has increased metastatic potential in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1990.
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