Identification and characterization of calreticulin as a novel plasminogen receptor.

Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular functions, including roles in calcium homeostasis and protein folding. Recently, several extracellular functions have been identified for CRT, including roles in cancer cell invasion and phagocytosis of apoptotic and cancer cells by macrophages. In the current report, we uncover a novel function for extracellular CRT and report that CRT functions as a plasminogen-binding receptor that regulates the conversion of plasminogen to plasmin. We show that human recombinant or bovine tissue-derived CRT dramatically stimulated the conversion of plasminogen to plasmin by tissue plasminogen activator or urokinase-type plasminogen activator. Surface plasmon resonance analysis revealed that CRT-bound plasminogen (KD = 1.8 µM) with moderate affinity. Plasminogen binding and activation by CRT were inhibited by ε-aminocaproic acid, suggesting that an internal lysine residue of CRT interacts with plasminogen. We subsequently show that clinically relevant CRT variants (lacking four or eight lysines in carboxyl-terminal region) exhibited decreased plasminogen activation. Furthermore, CRT-deficient fibroblasts generated 90% less plasmin and CRT-depleted MDA MB 231 cells also demonstrated a significant reduction in plasmin generation. Moreover, treatment of fibroblasts with mitoxantrone dramatically stimulated plasmin generation by WT but not CRT-deficient fibroblasts. Our results suggest that CRT is an important cellular plasminogen regulatory protein. Given that CRT can empower cells with plasmin proteolytic activity, this discovery may provide new mechanistic insight into the established role of CRT in cancer.

Quantitative Proteome Responses to Oncolytic Reovirus in GM-CSF- and M-CSF-Differentiated Bone Marrow-Derived Cells.

The efficacy of oncolytic viruses (OVs), such as reovirus, is dictated by host immune responses, including those mediated by the pro- versus anti-inflammatory macrophages. As such, a detailed understanding of the interaction between reovirus and different macrophage types is critical for therapeutic efficacy. To explore reovirus-macrophage interactions, we performed tandem mass tag (TMT)-based quantitative temporal proteomics on mouse bone marrow-derived macrophages (BMMs) generated with two cytokines, macrophage colony stimulating factor (M-CSF) and granulocytic-macrophage colony stimulating factor (GM-CSF), representing anti- and proinflammatory macrophages, respectively. We quantified 6863 proteins across five time points in duplicate, comparing M-CSF (M-BMM) and GM-CSF (GM-BMM) in response to OV. We find that GM-BMMs have lower expression of key intrinsic proteins that facilitate an antiviral immune response, express higher levels of reovirus receptor protein JAM-A, and are more susceptible to oncolytic reovirus infection compared to M-BMMs. Interestingly, although M-BMMs are less susceptible to reovirus infection and subsequent cell death, they initiate an antireovirus adaptive T cell immune response comparable to that of GM-BMMs. Taken together, these data describe distinct proteome differences between these two macrophage populations in terms of their ability to mount antiviral immune responses.

On the contribution of S100A10 and annexin A2 to plasminogen activation and oncogenesis: an enduring ambiguity.

Plasminogen receptors are becoming increasingly relevant in regulating many diseases such as cancer, stroke and inflammation. However, controversy has emerged concerning the putative role of some receptors, in particular annexin A2, in binding plasminogen. Several reports failed to account for the effects of annexin A2 on the stability and conformation of its binding partner S100A10. This has created an enduring ambiguity as to the actual function of annexin A2 in plasmin regulation. Supported by a long line of evidence, we conclude that S100A10, and not annexin A2, is the primary plasminogen receptor within the annexin A2-S100A10 complex and contributes to the plasmin-mediated effects that were originally ascribed to annexin A2.

ALDH1A3 promotes invasion and metastasis in triple-negative breast cancer by regulating the plasminogen activation pathway.

Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell marker that promotes metastasis. Triple-negative breast cancer (TNBC) progression has been linked to ALDH1A3-induced gene expression changes. To investigate the mechanism of ALDH1A3-mediated breast cancer metastasis, we assessed the effect of ALDH1A3 on the expression of proteases and the regulators of proteases that degrade the extracellular matrix, a process that is essential for invasion and metastasis. This revealed that ALDH1A3 regulates the plasminogen activation pathway; it increased the levels and activity of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). This resulted in a corresponding increase in the activity of serine protease plasmin, the enzymatic product of tPA and uPA. The ALDH1A3 product all-trans-retinoic acid similarly increased tPA and plasmin activity. The increased invasion of TNBC cells by ALDH1A3 was plasminogen-dependent. In patient tumours, ALDH1A3 and tPA are co-expressed and their combined expression correlated with the TNBC subtype, high tumour grade and recurrent metastatic disease. Knockdown of tPA in TNBC cells inhibited plasmin generation and lymph node metastasis. These results identify the ALDH1A3-tPA-plasmin axis as a key contributor to breast cancer progression.

Regulation of S100A10 by the PML-RAR-α oncoprotein.

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia that results from the expression of the promyelocytic leukemia-retinoic acid receptor α (PML-RAR-α) oncoprotein. It is characterized by severe hemorrhagic complications due in part to excessive fibrinolysis, resulting from the excessive generation of the fibrinolytic enzyme, plasmin, at the cell surface of the PML cells. The treatment of patients with all-trans retinoic acid (ATRA) effectively ameliorates the disease by promoting the destruction of the PML-RAR-α oncoprotein. In the present study we show for the first time that the plasminogen receptor, S100A10, is present on the extracellular surface of APL cells and is rapidly down-regulated in response to all-trans retinoic acid. The loss of S100A10 is concomitant with a loss in fibrinolytic activity. Furthermore, the induced expression of the PML-RAR-α oncoprotein increased the expression of cell surface S100A10 and also caused a dramatic increase in fibrinolytic activity. Depletion of S100A10 by RNA interference effectively blocked the enhanced fibrinolytic activity observed after induction of the PML-RAR-α oncoprotein. These experiments show that S100A10 plays a crucial role in the generation of plasmin leading to fibrinolysis, thus providing a link to the clinical hemorrhagic phenotype of APL.

Regulation of fibrinolysis by S100A10 in vivo.

Endothelial cells form the inner lining of vascular networks and maintain blood fluidity by inhibiting blood coagulation and promoting blood clot dissolution (fibrinolysis). Plasmin, the primary fibrinolytic enzyme, is generated by the cleavage of the plasma protein, plasminogen, by its activator, tissue plasminogen activator. This reaction is regulated by plasminogen receptors at the surface of the vascular endothelial cells. Previous studies have identified the plasminogen receptor protein S100A10 as a key regulator of plasmin generation by cancer cells and macrophages. Here we examine the role of S100A10 and its annexin A2 binding partner in endothelial cell function using a homozygous S100A10-null mouse. Compared with wild-type mice, S100A10-null mice displayed increased deposition of fibrin in the vasculature and reduced clearance of batroxobin-induced vascular thrombi, suggesting a role for S100A10 in fibrinolysis in vivo. Compared with wild-type cells, endothelial cells from S100A10-null mice demonstrated a 40% reduction in plasminogen binding and plasmin generation in vitro. Furthermore, S100A10-deficient endothelial cells demonstrated impaired neovascularization of Matrigel plugs in vivo, suggesting a role for S100A10 in angiogenesis. These results establish an important role for S100A10 in the regulation of fibrinolysis and angiogenesis in vivo, suggesting S100A10 plays a critical role in endothelial cell function.

The role of the annexin A2 heterotetramer in vascular fibrinolysis.

The vascular endothelial cells line the inner surface of blood vessels and function to maintain blood fluidity by producing the protease plasmin that removes blood clots from the vasculature, a process called fibrinolysis. Plasminogen receptors play a central role in the regulation of plasmin activity. The protein complex annexin A2 heterotetramer (AIIt) is an important plasminogen receptor at the surface of the endothelial cell. AIIt is composed of 2 molecules of annexin A2 (ANXA2) bound together by a dimer of the protein S100A10. Recent work performed by our laboratory allowed us to clarify the specific roles played by ANXA2 and S100A10 subunits within the AIIt complex, which has been the subject of debate for many years. The ANXA2 subunit of AIIt functions to stabilize and anchor S100A10 to the plasma membrane, whereas the S100A10 subunit initiates the fibrinolytic cascade by colocalizing with the urokinase type plasminogen activator and receptor complex and also providing a common binding site for both tissue-type plasminogen activator and plasminogen via its C-terminal lysine residue. The AIIt mediated colocalization of the plasminogen activators with plasminogen results in the rapid and localized generation of plasmin to the endothelial cell surface, thereby regulating fibrinolysis.

Annexin A2: the importance of being redox sensitive.

Hydrogen peroxide (H(2)O(2)) is an important second messenger in cellular signal transduction. H(2)O(2)-dependent signalling regulates many cellular processes, such as proliferation, differentiation, migration and apoptosis. Nevertheless, H(2)O(2) is an oxidant and a major contributor to DNA damage, protein oxidation and lipid peroxidation, which can ultimately result in cell death and/or tumourigenesis. For this reason, cells have developed complex antioxidant systems to scavenge ROS. Recently, our laboratory identified the protein, annexin A2, as a novel cellular redox regulatory protein. Annexin A2 possesses a reactive cysteine residue (Cys-8) that is readily oxidized by H(2)O(2) and subsequently reduced by the thioredoxin system, thereby enabling annexin A2 to participate in multiple redox cycles. Thus, a single molecule of annexin A2 can inactivate several molecules of H(2)O(2). In this report, we will review the studies detailing the reactivity of annexin A2 thiols and the importance of these reactive cysteine(s) in regulating annexin A2 structure and function. We will also focus on the recent reports that establish novel functions for annexin A2, namely as a protein reductase and as a cellular redox regulatory protein. We will further discuss the importance of annexin A2 redox regulatory function in disease, with a particular focus on tumour progression.

Recent Advances in Molecular and Cellular Functions of S100A10.

S100A10 (p11, annexin II light chain, calpactin light chain) is a multifunctional protein with a wide range of physiological activity. S100A10 is unique among the S100 family members of proteins since it does not bind to Ca2+, despite its sequence and structural similarity. This review focuses on studies highlighting the structure, regulation, and binding partners of S100A10. The binding partners of S100A10 were collated and summarized.

S100A10 regulates plasminogen-dependent macrophage invasion.

The plasminogen activation system plays an integral role in the migration of macrophages in response to an inflammatory stimulus, and the binding of plasminogen to its cell-surface receptor initiates this process. Although previous studies from our laboratory have shown the importance of the plasminogen receptor S100A10 in cancer cell plasmin production, the potential role of this protein in macrophage migration has not been investigated. Using thioglycollate to induce a peritoneal inflammatory response, we demonstrate, for the first time, that compared with wild-type (WT) mice, macrophage migration across the peritoneal membrane into the peritoneal cavity in S100A10-deficient (S100A10(-/-)) mice was decreased by up to 53% at 24, 48, and 72 hours. Furthermore, the number of S100A10-deficient macrophages that infiltrated Matrigel plugs was reduced by 8-fold compared with their WT counterpart in vivo. Compared with WT macrophages, macrophages from S100A10(-/-) mice demonstrated a 50% reduction in plasmin-dependent invasion across a Matrigel barrier and a 45% reduction in plasmin generation in vitro. This loss in plasmin-dependent invasion was in part the result of a decreased generation of plasmin and a decreased activation of pro-MMP-9 by S100A10-deficient macrophages. This study establishes a direct involvement of S100A10 in macrophage recruitment in response to inflammatory stimuli.

The biochemistry and regulation of S100A10: a multifunctional plasminogen receptor involved in oncogenesis.

The plasminogen receptors mediate the production and localization to the cell surface of the broad spectrum proteinase, plasmin. S100A10 is a key regulator of cellular plasmin production and may account for as much as 50% of cellular plasmin generation. In parallel to plasminogen, the plasminogen-binding site on S100A10 is highly conserved from mammals to fish. S100A10 is constitutively expressed in many cells and is also induced by many diverse factors and physiological stimuli including dexamethasone, epidermal growth factor, transforming growth factor-α, interferon-γ, nerve growth factor, keratinocyte growth factor, retinoic acid, and thrombin. Therefore, S100A10 is utilized by cells to regulate plasmin proteolytic activity in response to a wide diversity of physiological stimuli. The expression of the oncogenes, PML-RARα and KRas, also stimulates the levels of S100A10, suggesting a role for S100A10 in pathophysiological processes such as in the oncogenic-mediated increases in plasmin production. The S100A10-null mouse model system has established the critical role that S100A10 plays as a regulator of fibrinolysis and oncogenesis. S100A10 plays two major roles in oncogenesis, first as a regulator of cancer cell invasion and metastasis and secondly as a regulator of the recruitment of tumor-associated cells, such as macrophages, to the tumor site.

The ANXA2/S100A10 Complex-Regulation of the Oncogenic Plasminogen Receptor.

The generation of the serine protease plasmin is initiated by the binding of its zymogenic precursor, plasminogen, to cell surface receptors. The proteolytic activity of plasmin, generated at the cell surface, plays a crucial role in several physiological processes, including fibrinolysis, angiogenesis, wound healing, and the invasion of cells through both the basement membrane and extracellular matrix. The seminal observation by Albert Fischer that cancer cells, but not normal cells in culture, produce large amounts of plasmin formed the basis of current-day observations that plasmin generation can be hijacked by cancer cells to allow tumor development, progression, and metastasis. Thus, the cell surface plasminogen-binding receptor proteins are critical to generating plasmin proteolytic activity at the cell surface. This review focuses on one of the twelve well-described plasminogen receptors, S100A10, which, when in complex with its regulatory partner, annexin A2 (ANXA2), forms the ANXA2/S100A10 heterotetrameric complex referred to as AIIt. We present the theme that AIIt is the quintessential cellular plasminogen receptor since it regulates the formation and the destruction of plasmin. We also introduce the term oncogenic plasminogen receptor to define those plasminogen receptors directly activated during cancer progression. We then discuss the research establishing AIIt as an oncogenic plasminogen receptor-regulated during EMT and activated by oncogenes such as SRC, RAS, HIF1α, and PML-RAR and epigenetically by DNA methylation. We further discuss the evidence derived from animal models supporting the role of S100A10 in tumor progression and oncogenesis. Lastly, we describe the potential of S100A10 as a biomarker for cancer diagnosis and prognosis.

Plasmin and Plasminogen System in the Tumor Microenvironment: Implications for Cancer Diagnosis, Prognosis, and Therapy.

The tumor microenvironment (TME) is now being widely accepted as the key contributor to a range of processes involved in cancer progression from tumor growth to metastasis and chemoresistance. The extracellular matrix (ECM) and the proteases that mediate the remodeling of the ECM form an integral part of the TME. Plasmin is a broad-spectrum, highly potent, serine protease whose activation from its precursor plasminogen is tightly regulated by the activators (uPA, uPAR, and tPA), the inhibitors (PAI-1, PAI-2), and plasminogen receptors. Collectively, this system is called the plasminogen activation system. The expression of the components of the plasminogen activation system by malignant cells and the surrounding stromal cells modulates the TME resulting in sustained cancer progression signals. In this review, we provide a detailed discussion of the roles of plasminogen activation system in tumor growth, invasion, metastasis, and chemoresistance with specific emphasis on their role in the TME. We particularly review the recent highlights of the plasminogen receptor S100A10 (p11), which is a pivotal component of the plasminogen activation system.

Author Correction: A novel mechanism of plasminogen activation in epithelial and mesenchymal cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

S100A10 Has a Critical Regulatory Function in Mammary Tumor Growth and Metastasis: Insights Using MMTV-PyMT Oncomice and Clinical Patient Sample Analysis.

S100A10 (p11) is a plasminogen receptor that regulates cellular plasmin generation by cancer cells. In the current study, we used the MMTV-PyMT mouse breast cancer model, patient tumor microarray, and immunohistochemical (IHC) analysis to investigate the role of p11 in oncogenesis. The genetic deletion of p11 resulted in significantly decreased tumor onset, growth rate, and spontaneous pulmonary metastatic burden in the PyMT/p11-KO (knock-out) mice. This phenotype was accompanied by substantial reduction in Ki67 positivity, macrophage infiltration, decreased vascular density in the primary tumors, and decrease in invasive carcinoma and pulmonary metastasis. Surprisingly, IHC analysis of wild-type MMTV-PyMT mice failed to detect p11 expression in the tumors or metastatic tumor cells and loss of p11 did not decrease plasmin generation in the PyMT tumors and cells. Furthermore, tumor cells expressing p11 displayed dramatically reduced lung metastasis when injected into p11-depleted mice, further strengthening the stromal role of p11 in tumor growth and metastasis. Transcriptome analysis of the PyMT tumors from p11-KO mice showed marked reduction in genes such as Areg, Muc1, and S100a8 involved in breast cancer development, progression, and inflammation. The PyMT/p11-KO tumors displayed a remarkable increase in inflammatory cytokines such as interleukin (Il)-6, Il-10, and interferon (Ifn)-γ. Gene expression profiling and IHC of primary breast cancer samples showed that p11 mRNA and protein levels were significantly higher in tumor tissues compared to normal mammary tissue. P11 mRNA expression was significantly associated with poor patient prognosis and significantly elevated in high grade, triple negative (TN) tumors, and tumors with high proliferative index. This is the first study examining the crucial role of p11 in breast tumor development and metastasis, thus emphasizing its potential as a diagnostic and prognostic biomarker in breast cancer.

Decitabine Response in Breast Cancer Requires Efficient Drug Processing and Is Not Limited by Multidrug Resistance.

Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBC) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naïve breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in patients with TNBC treated with decitabine as a second-line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene-specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways. Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxol-resistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.

Chromium-mediated apoptosis: involvement of DNA-dependent protein kinase (DNA-PK) and differential induction of p53 target genes.

Cellular stress and DNA damage up-regulate and activate p53, fundamental for cell cycle control, senescence, DNA repair and apoptosis. The specific mechanism(s) that determine whether p53-dependent cell cycle arrest or p53-dependent apoptosis prevails in response to specific DNA damage are poorly understood. In this study, we investigated two types of DNA damage, chromium treatment and gamma irradiation (IR) that induced similar levels of p53, but that mediated two distinct p53-dependent cell fates. Chromium exposure induced a robust DNA-dependent protein kinase (DNA-PK)-mediated apoptotic response that was accompanied by the rapid loss of the cyclin-dependent kinase inhibitor 1A (p21) protein, whereas IR treatment-induced cell cycle arrests that was supported by the rapid induction of p21. Inhibition of DNA-PK effectively blocked chromium-, but not IR-induced p53 stabilization and activation. In contrast, inhibition of ATM and ATR by caffeine had the inverse effect of blocking IR-, but not chromium-induced p53 stabilization and activation. Chromium exposure ablated p21 transcription but PUMA and Bax transcription was significantly enhanced compared to non-damaged cells. In contrast, IR treatment triggered significant p21 mRNA synthesis in addition to PUMA and Bax mRNA production. While chromium treatment enhanced the binding of p53 and RNA polymerase II (RNA Pol II) to both the p21 and PUMA promoters, RNA Pol II elongation was only observed along the PUMA gene and not the p21 gene. In contrast, following IR treatment, RNA Pol II elongation was observed on both p21 and PUMA. Chromium-induced apoptosis therefore involves DNA-PK-mediated p53 activation followed by preferential transcription of pro-apoptotic PUMA over anti-apoptotic p21 genes.

Hypersensitivity to chromium-induced DNA damage correlates with constitutive deregulation of upstream p53 kinases in p21-/- HCT116 colon cancer cells.

The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) is a key component in cell cycle control and apoptosis, directing an anti-apoptotic response following DNA damage. Chromium exposure resulted in a 500-1000 fold increase in apoptosis-induced cell death in p21-/- HCT116 cells compared to wild-type or p53-/- cells. p53 shRNA (or transient p53 siRNA) into p21-/- HCT116 cells reduced Cr(VI) sensitivity, suggesting the enhanced apoptosis in p21-/- cells is p53-dependent. Under non-DNA damage conditions, the p53 level in p21-/- cells was significantly higher than in wild-type cells, due to enhanced p53 phosphorylation and stabilization rather than elevated p53 transcription. Wild-type cells showed significant p53 protein induction upon DNA damage whereas p21-/- cells showed no p53 increase. p21-/- cells display the constitutive activation of upstream p53 kinases (ATM, DNA-PK, ATR, AKT and p38). 2D gel analysis revealed p53 patterns in p21-/- cells were distinct from those in wild-type cells before and after chromium exposure. Our results suggest that p21 has an important role in the cellular response to normal replicative stress and its absence leads to a "chronic DNA damage" state that primes the cell for p53-dependent apoptosis.

Regulation of the low-dose radiation paracrine-specific anchorage-independent growth response by annexin A2.

Here we identify the release of annexin A2 into the culture medium in response to low-dose X-radiation exposure and establish functional linkages to an established paracrine factor-mediated anchorage-independent growth response. Using a standard bicameral coculture model, we demonstrate that annexin A2 is secreted into the medium by irradiated cells (seeded in upper chamber) and is capable of binding to nonirradiated neighboring cells (seeded in lower chamber). The paracrine factor-mediated anchorage-independent growth response to low-dose X irradiation is reduced when irradiated annexin A2-silenced (shRNA) JB6 cells are co-cultured with nonirradiated cells relative to co-culture with irradiated annexin A2-competent vector control cells. Consistent with this observation, purified bovine annexin A2 tetramer induces anchorage-independent growth. These observations suggest that annexin A2 regulates, in part, the radiation paracrine factor-specific anchorage-independent growth response in JB6 cells.

Retraction: DNA-PKCS binding to p53 on the p21WAF1/CIP1 promoter blocks transcription resulting in cell death.

[This retracts the article DOI: 10.18632/oncotarget.378.].