Cerebral cavernous malformations (CCMs) is a microvascular disorder in the central nervous system. Despite tremendous efforts, the causal genetic mutation in some CCM patients has not be identified, raising the possibility of an unknown CCM locus. The CCM2/MGC4607 gene has been identified as one of three known genes causing CCMs. In this report, we defined a total of 29 novel exons and 4 novel promoters in CCM2 genomic structure and subsequently identified a total of 50 new alternative spliced isoforms of CCM2 which eventually generated 22 novel protein isoforms. Genetic analysis of CCM2 isoforms revealed that the CCM2 isoforms can be classified into two groups based on their alternative promoters and alternative start codon exons. Our data demonstrated that CCM2 isoforms not only are specific in their subcellular compartmentation but also have distinct cellular expression patterns among various tissues and cells, indicating the pleiotropic cellular roles of CCM2 through their multiple isoforms. In fact, the complexity of the CCM2 genomic structure was reflected by the multiple layers of regulation of CCM2 expression patterns. At the transcriptional level, it is accomplished by alternative promoters, alternative splicing, and multiple transcriptional start sites and termination sites; while at the translational level, it is carried out with various cellular functions with a distinguishable CCM2 protein group pattern, specified abundance and composition of selective isoforms in a cell and tissue specific fashion. Through experimentation, we discovered a unique phosphotyrosine binding (PTB) domain, namely atypical phosphotyrosine binding (aPTB) domain. Some long CCM2 isoform proteins contain both classes of PTB domains, making them a dual PTB domain-containing protein. Both CCM1 and CCM3 can bind competitively to this aPTB domain, indicating CCM2 as the cornerstone for CCM signaling complex (CSC).

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Breast cancer is the most diagnosed cancer worldwide and remains the second leading cause of cancer death. While breast cancer mortality has steadily declined over the past decades through medical advances, an alarming disparity in breast cancer mortality has emerged between African American women (AAW) and Caucasian American women (CAW). New evidence suggests more aggressive behavior of triple-negative breast cancer (TNBC) in AAW may contribute to racial differences in tumor biology and mortality. Progesterone (PRG) can exert its cellular effects through either its classic, non-classic, or combined responses through binding to either classic nuclear PRG receptors (nPRs) or non-classic membrane PRG receptors (mPRs), warranting both pathways equally important in PRG-mediated signaling. In our previous report, we demonstrated that the CCM signaling complex (CSC) consisting of CCM1, CCM2, and CCM3 can couple both nPRs and mPRs signaling cascades to form a CSC-mPRs-PRG-nPRs (CmPn) signaling network in nPR positive(+) breast cancer cells. In this report, we furthered our research by establishing the CSC-mPRs-PRG (CmP) signaling network in nPR(-) breast cancer cells, demonstrating that a common core mechanism exists, regardless of nPR(+⁣/⁣-) status. This is the first report stating that inducible expression patterns exist between CCMs and major mPRs in TNBC cells. Furthermore, we firstly show mPRs in TNBC cells are localized in the nucleus and participate in nucleocytoplasmic shuttling in a coordinately synchronized fashion with CCMs under steroid actions, following the same cellular distribution as other well-defined steroid hormone receptors. Finally, for the first time, we deconvoluted the CmP signalosome by using systems biology and TNBC clinical data, which helped us understand key factors within the CmP network and identify 6 specific biomarkers with potential clinical applications associated with AAW-TNBC tumorigenesis. These novel biomarkers could have immediate clinical implications to dramatically improve health disparities among AAW-TNBCs.

Cerebral cavernous malformations (CCMs) are microvascular anomalies in the brain that result in increased susceptibility to stroke. Three genes have been identified as causes of CCMs: cerebral cavernous malformations 1 [(CCM1) also termed Krev interaction trapped 1 (KRIT1)], cerebral cavernous malformation 2 [(CCM2) also termed MGC4607] and cerebral cavernous malformation 3 [(CCM3) also termed programmed cell death 10 (PDCD10)]. It has been demonstrated that both CCM1 and CCM3 bind to CCM2 to form a CCM signaling complex (CSC) with which to modulate multiple signaling cascades. CCM proteins have been reported to play major roles in microvascular angiogenesis in human and animal models. However, CCM proteins are ubiquitously expressed in all major tissues, suggesting an unseen broader role of the CSC in biogenesis. Recent evidence suggests the possible involvement of the CSC complex during tumorigenesis; however, studies concerning this aspect are limited. This is the first report to systematically investigate the expression patterns of CCM proteins in major human tumors using real-time quantitative PCR, RNA-fluorescence in situ hybridization, immunohistochemistry and multicolor immunofluorescence imaging. Our data demonstrated that differential expression patterns of the CSC complex are correlated with certain types and grades of major human cancers, indicating the potential application of CCM genes as molecular biomarkers for clinical oncology. Our data strongly suggest that more efforts are needed to elucidate the role of the CSC complex in tumorigenesis, which may have enormous clinical potential for cancer diagnostic, prognostic and therapeutic applications.

Background Breast cancer, the most diagnosed cancer, remains the second leading cause of cancer death in the United States, and excessive Progesterone (PRG) or Mifepristone (MIF) exposure may be at an increased risk for developing breast cancer. PRG exerts its cellular responses through signaling cascades involving classic, non-classic, or combined responses by binding to either classic nuclear PRG receptors (nPRs) or non-classic membrane PRG receptors (mPRs). Currently, the intricate balance and switch mechanisms between these two signaling cascades remain elusive. Three genes, CCM1-3, form the CCM signaling complex (CSC) which mediates multiple signaling cascades. Methods Utilizing molecular, cellular, Omics, and systems biology approaches, we analyzed the relationship among the CSC, PRG, and nPRs/mPRs during breast cancer tumorigenesis. Results We discovered that the CSC plays an essential role in coupling both classic and non-classic PRG signaling pathways by mediating crosstalk between them, forming the CmPn (CSC-mPRs-PRG-nPRs) signaling network. We found that mPR-specific PRG actions (PRG + MIF) play an essential role in this CmPn network during breast cancer tumorigenesis. Additionally, we have identified 4 categories of candidate biomarkers (9 intrinsic, 2 PRG-inducible, 1 PRG-repressive, 1 mPR-specific PRG-repressive, and 2 mPR-responsive) for Luminal-A breast cancers during tumorigenesis and have confirmed the prognostic application of RPL13 and RPL38 as intrinsic biomarkers using a dual validation method. Conclusions We have discovered that the CSC plays an essential role in the CmPn signaling network for Luminal-A breast cancers with identification of two intrinsic biomarkers.

A B S T R A C TCerebral cavernous malformations (CCMs) are characterized by abnormally dilated intracranial capillaries that result in increased susceptibility to stroke. Three genes have been identified as causes of CCMs; KRIT1 (CCM1), MGC4607 (CCM2) and PDCD10 (CCM3); one of them is disrupted in most CCM cases. It was demonstrated that both CCM1 and CCM3 bind to CCM2 to form a CCM signaling complex (CSC) to modulate angiogenesis. In this report, we deployed both RNA-seq and proteomic analysis of perturbed CSC after depletion of one of three CCM genes to generate interactomes for system-wide studies. Our results demonstrated a unique portrait detailing alterations in angiogenesis and vascular integrity. Interestingly, only in-direct overlapped alterations between RNA and protein levels were detected, supporting the existence of multiple layers of regulation in CSC cascades. Notably, this is the first report identifying that both β4 integrin and CAV1 signaling are downstream of CSC, conveying the angiogenic signaling. Our results provide a global view of signal transduction modulated by the CSC, identifies novel regulatory signaling networks and key cellular factors associated with CSC.Heliyon 5 (2019) e02899 Protein extraction for HBMVEC cells and zebrafish embryosHBMVEC cells and zebrafish embryos were lysed using a digital sonifier cell disruptor (Branson model 450 with model 102C converter and double step microtip) in ice cold lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40 (Sigma), 50 mM sodium fluoride (Sigma), 1 mM PMSF (Sigma), 1 mM dithiothreitol (Invitrogen) and EDTA-free complete protease inhibitor (Roche). The concentration of protein lysates were measure by Qubit assay (Invitrogen) before analysis.

We demonstrate that a novel signaling network among the CSC and mPRS is dynamically modulated and fine-tuned with intricate feedback regulations in PR negative cells, especially endothelial cells (ECs). Depletion of any of three CCMs (1, 2, 3) genes results in the disruption of non-classic mPRs-mediated signaling in-vitro as well as defective homeostasis of PRG in-vivo. Therefore, we propose the CSC is a master regulator of homeostasis of PRG and its associated classic and non-classic signaling cascades. Assisted with omic approaches, we identified signaling pathways involved and specific biomarkers associated with hemorrhagic events during CCM pathogenesis in-vitro and in-vivo. To our knowledge, this is the first report detailing etiology to predict the occurrence of early hemorrhagic events with a set of serum biomarkers.

Cerebral cavernous malformations (CCMs) are characterized by abnormally dilated intracranial microvascular sinusoids that result in increased susceptibility to hemorrhagic stroke. It has been demonstrated that three CCM proteins (CCM1, CCM2, and CCM3) form the CCM signaling complex (CSC) to mediate angiogenic signaling. Disruption of the CSC will result in hemorrhagic CCMs, a consequence of compromised blood–brain barrier (BBB) integrity. Due to their characteristically incomplete penetrance, the majority of CCM mutation carriers (presumed CCM patients) are largely asymptomatic, but when symptoms occur, the disease has typically reached a clinical stage of focal hemorrhage with irreversible brain damage. We recently reported that the CSC couples both classic (nuclear; nPRs) and nonclassic (membrane; mPRs) progesterone (PRG)-receptors-mediated signaling within the CSC-mPRs-PRG (CmP) signaling network in nPR(−) breast cancer cells. In this report, we demonstrate that depletion of any of the three CCM genes or treatment with mPR-specific PRG actions (PRG/mifepristone) results in the disruption of the CmP signaling network, leading to increased permeability in the nPR(−) endothelial cells (ECs) monolayer in vitro. Finally, utilizing our in vivo hemizygous Ccm mutant mice models, we demonstrate that depletion of any of the three CCM genes, in combination with mPR-specific PRG actions, is also capable of leading to defective homeostasis of PRG in vivo and subsequent BBB disruption, allowing us to identify a specific panel of etiological blood biomarkers associated with BBB disruption. To our knowledge, this is the first report detailing the etiology to predict the occurrence of a disrupted BBB, an indication of early hemorrhagic events.