Thromboembolism (TE) is a common complication in cancer patients and the second leading cause of cancer-related deaths. The incidence of TE varies in different cancer types, with the highest risk in pancreatic ductal adenocarcinoma (PDAC) and in advanced-stage and metastatic cancers. Despite the benefits associated with anti-coagulant therapy for symptomatic TE, the prevention of TE still remains an unmet clinical need due to lack of biomarkers predictive of TE risk and the bleeding risk associated with the routine use of anti-coagulants. Small extracellular vesicles (sEV) mediate cell-to-cell communication. Cancer cells and the tumor microenvironment release large numbers of sEV into the blood circulation and sEVs have displayed a therapeutic and predictive value in systemic diseases. However, the role of sEVs in cancer-associated TE remains to be investigated. Here we show that sEVs from (pre)metastatic lungs of mice with melanoma, breast, lung, and PDAC induce TE in mice and express high levels of integrin beta 2 (ITGB2), while sEVs from tumor cell lines, primary tumors, or other metastasis-bearing organs did not show any pro-thrombotic properties. A specific subtype of interstitial macrophages infiltrating (pre-)metastatic lungs were the main source of ITGB2+ pro-thrombotic sEVs. Blockade of ITGB2 on lung-derived sEVs, or systemically in mice, prevented EV-induced platelet aggregation and TE, and reduced metastasis. Examination of the mechanisms of ITGB2-induced TE showed that EV-associated ITGB2 interacts directly or through fibrin with different binding partners on platelets, and induce their activation and aggregation. Importantly, we found that levels of ITGB2 on sEVs are elevated in the plasma of PDAC patients prior (<30 days) to TE events in comparison to PDAC patients with no history of TE, and thus might serve as prognostic biomarker of TE. Together, our results provide the first evidence of the establishment of a pro-thrombotic lung niche in PDAC as well as other cancer types. Moreover, we identify EV-associated ITGB2 as a new target for the prevention and/or treatment of TE, as well as a potential “liquid biopsy” analyte for the early stratification of patients at high risk of TE. Citation Format: Serena Lucotti, Yusuke Ogitani, Candia M. Kenific, Linda Bojmar, Michele Cioffi, Pernille Lauritzen, Henrik Molina, Soeren Heissel, Henry B. Lengel, Xiaohong Jing, Haiying Zhang, Irina Matei, Eileen M. O'Reilly, William R. Jarnagin, David Jones, James B. Bussel, David Kelsen, Jacqueline F. Bromberg, Diane M. Simeone, David Lyden. The lung pro-thrombotic niche drives cancer-associated thromboembolism and metastasis via extracellular vesicle ITGB2 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C028.
Fibrolamellar hepatocellular carcinoma, FLC, is a usually lethal cancer affecting children, and young adults. We have shown that all patients have a disruption in the ecology of protein kinase A activity. Hundreds of patients tested have a fusion of the first exon of DNABJ1, a heat shock protein cofactor, to PRKACA, the catalytic subunit of protein kinase A and expression of this DNAJB1-PRKACA fusion oncokinase is sufficient to produce the tumor. One patient is missing the regulatory subunit of protein kinase A and three patients have a fusion of the first exon of a different protein, ATP1B1, to the catalytic subunit of protein kinase A. We characterized the proteome and phosphoproteome of FLC cells relative to adjacent normal tissue. The changes were sufficiently characteristic to identify cell extracts from FLC, based solely on the proteome or phosphoproteome, and distinguishable from other tumor or normal liver. By calibrating protein level we found that tumor cells have an increase of catalytic subunit to such an extent as to exceed the capacity of protein kinase A regulatory subunits. This has two implications. First, there is free catalytic subunit that is unregulated, creating a high basal level of kinase activity. Second, the catalytic subunit is no longer localized by tethering to the regulatory subunit. Thus, the catalytic subunit has access to novel substrates. As an independent confirmation, we found the free basal kinase activity was higher in FLC cells. As a further test, we showed free catalytic subunit, that is not conjugated to regulatory subunit, is higher in FLC cells. We next tested whether these changes were solely due to overexpression of the catalytic subunit or if there was some additional change in the intrinsic activity of the kinase as a result of the fusion. We purified, without using an affinity tag, either PRKACA (which is myristoylated), DNAJB1-PRKACA, and PRKACA which was not myristoylated (the DNAJB1-PRKACA is not myristoylated). As a further control we also used the PRKACAL206R mutation, which does not engage regulatory subunits and is present in some forms of Cushing’s disease. We purified cytosol from human liver, blocked activity of all the endogenous kinases, and added our purified kinase. Mass spectrometry was then used to identify all newly phosphorylated proteins. While many proteins were equally phosphorylated by all four kinases (PRKACA, DNAJB1-PRKACA, PRKACA not myristoylated, PRKACAL206R) there were some distinct differences. From an analysis of these we found alterations in the optimal recognition sequence for phosphorylation for each variant of the kinase. Significantly, when we examined the human tissue data, we found many proteins that were phosphorylated by the DNAJB1-PRKACA in vitro were also phosphorylated in patient tumor tissue, but not in adjacent non-transformed tissue. This validated these observations of altered phosphorylation from the in vitro assays. Citation Format: Solomon Levin, Mahsa Shironi, Melissa Jarmel, Michael Tomasini, Bassem Shebl, Tova Finkelstein, David Requena, Soeren Heissel, Henrik Molina, Philip Coffino, Barbara Lyons, Sanford M. Simon. Pathogenesis of fibrolamellar: Proteome and phosphome of an oncokinase driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2494.
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