Proteomic Profiling of Serum Extracellular Vesicles Identifies Diagnostic Signatures and Therapeutic Targets in Breast Cancer.

Analysis of extracellular vesicles (EVs) is a promising noninvasive liquid biopsy approach for breast cancer (BC) detection, prognosis, and therapeutic monitoring. A comprehensive understanding of the characteristics and proteomic composition of BC-specific EVs from human samples is required to realize the potential of this strategy. In this study, we applied a mass spectrometry-based, data-independent acquisition (DIA) proteomic approach to characterize human serum EVs derived from patients with BC (n = 126) and healthy donors (HDs, n = 70) in a discovery cohort and validated the findings in five independent cohorts. Examination of the EV proteomes enabled construction of specific EV protein classifiers for diagnosing BC and distinguishing patients with metastatic disease. Of note, TALDO1 was found to be an EV biomarker of distant metastasis of BC. In vitro and in vivo analysis confirmed the role of TALDO1 in stimulating BC invasion and metastasis. Finally, high-throughput molecular docking and virtual screening of a library consisting of 271,380 small molecules identified a potent TALDO1 allosteric inhibitor, AO-022, which could inhibit BC migration in vitro and tumor progression in vivo. Together, this work elucidates the proteomic alterations in the serum EVs of BC patients to guide development of improved diagnosis, monitoring, and treatment strategies.

The Correct Localization of Borealin in Midbody during Cytokinesis Depends on IQGAP1.

Borealin is a key component of chromosomal passenger complex, which is vital in cytokinesis. IQ domain-containing GTPase-activating protein 1 (IQGAP1) also participates in cytokinesis. The correlation between Borealin and IQGAP1 during cytokinesis is not yet clear. Here, we used mass spectrometry and endogenous coimmunoprecipitation experiments to investigate the interaction between IQGAP1 and Borealin. Results of the current study showed that Borealin interacted directly with IQGAP1 both in vitro and in vivo. Knockdown of IQGAP1 resulted in an abnormal location of Borealin in the midbody. Knocking down Borealin alone, IQGAP1 alone, or Borealin and IQGAP1 at the same time inhibited the completion of cytokinesis and formed multinucleated cells. Our results indicated that IQGAP1 interacts with Borealin during cytokinesis, and the correct localization of Borealin in the midbody during cytokinesis is determined by IQGAP1, and IQGAP1 may play an important role in regulating Borealin function in cytokinesis.

Anatomic evidence shows that lymphatic drainage exists in the pituitary to loop the cerebral lymphatic circulation.

Respiratory infections can result in intracranial infections and unknown neurological symptoms. The central nervous system lacks classical meningeal lymphatic (circulation) drainage, and the exact underlying mechanisms of how immune cells from the peripheral lymphatic system enter the central nervous system (CNS) remain unknown. To determine whether the perinasal lymphatic system or lymphatic vessels are involved in cerebral immune defence and play a role in causing CNS infections (especially respiratory tract-related infections), we performed an anatomic study to investigate the drainage differences between the perinasal and intracerebral lymphatic systems by using injection of Evans blue and anatomic surgery, together with immunohistochemistry and immunofluorescence assays. Surprisingly, we found that (1) the pituitary (adenohypophysis) is involved and is rich in lymphatic vessels and (2) perinasal tissue could communicate with central pituitary lymphatic vessels in a specific and unidirectional manner. Taken together, our study may be the first to anatomically demonstrate the existence of novel lymphatic vessel structures in the pituitary, as well as their communication with the perinasal (lymphatic) tissue. Our findings suggest the existence of an ultimate loop for "classical" meningeal lymphatic drainage and are relevant to cerebral infection and immune defence.

HEY2 acting as a co-repressor with smad3 and smad4 interferes with the response of TGF-beta in hepatocellular carcinoma.

The HEY2 (hairy and enhancer of split-related with YRPW motif 2) is reported to play potential roles in tumorigenesis. However, the underlying mechanism in tumorigenesis is remain elusive. The present study aims to investigate the molecular mechanism of biological function of HEY2 in hepatocellular carcinoma (HCC). Dysfunction of the transforming growth factor-beta (TGF-ß) pathway plays a critical role in HCC pathogenesis. Here, we identified HEY2 as a suppressor for TGF-ß biological response. We demonstrated that HEY2 protein in tumor cytoplasm was up-regulated in HCC. Further, HEY2 overexpression inhibited TGF-ß-induced growth arrest of HCC cells and inhibited TGF-ß-induced downregulation of c-Myc, both in mRNA and in protein levels. While knockdown of HEY2, by small interfering RNA, was shown to enhance the TGF-ß-mediated biological response of HCC cells. Moreover, HEY2 could form complexes with Smad3 and Smad4 and repress Smad3/Smad4 transcriptional activity. In conclusion, our findings indicate a novel role of HEY2 in mediating the TGF-ß/Smad signaling pathway in HCC tumorigenesis.

Molecular chaperone CCT3 supports proper mitotic progression and cell proliferation in hepatocellular carcinoma cells.

CCT3 was one of the subunits of molecular chaperone CCT/TRiC complex, which plays a central role in maintaining cellular proteostasis. We demonstrated that expressions of CCT3 mRNA and protein are highly up-regulated in hepatocellular carcinoma (HCC) tissues, and high level of CCT3 is correlated with poor survival in cancer patients. In HCC cell lines, CCT3 depletion suppresses cell proliferation by inducing mitotic arrest at prometaphase and apoptosis eventually. We also identified CCT3 as a novel regulator of spindle integrity and as a requirement for proper kinetochore-microtubule attachment during mitosis. Moreover, we found that CCT3 depletion sensitizes HCC cells to microtubule destabilizing drug Vincristine. Collectively, our study suggests that CCT3 is indispensible for HCC cell proliferation, and provides a potential drug target for treatment of HCC.

ASPP1/2-PP1 complexes are required for chromosome segregation and kinetochore-microtubule attachments.

Regulated interactions between kinetochores and spindle microtubules are critical for maintaining genomic stability during chromosome segregation. Defects in chromosome segregation are widespread phenomenon in human cancers that are thought to serve as the fuel for tumorigenic progression. Tumor suppressor proteins ASPP1 and ASPP2, two members of the apoptosis stimulating proteins of p53 (ASPP) family, are frequently down-regulated in human cancers. Here we report that ASPP1/2 are required for proper mitotic progression. In ASPP1/2 co-depleted cells, the persistence of unaligned chromosomes and the reduction of tension across sister kinetochores on aligned chromosomes resulted in persistent spindle assembly checkpoint (SAC) activation. Using protein affinity purification methods, we searched for functional partners of ASPP1/2, and found that ASPP1/2 were associated with a subset of kinetochore proteins (Hec1, KNL-1, and CENP-F). It was found that ASPP1/2 act as PP1-targeting subunits to facilitate the interaction between PP1 and Hec1, and catalyze Hec1 (Ser165) dephosphorylation during late mitosis. These observations revealed a previously unrecognized function of ASPP1/2 in chromosome segregation and kinetochore-microtubule attachments that likely contributes to their roles in chromosome stability and tumor suppression.

The tumor suppressor proteins ASPP1 and ASPP2 interact with C-Nap1 and regulate centrosome linker reassembly.

Centrosome linker tethers interphase centrosomes together allowing them to function as a single microtubule organization center. The centrosome linker is disrupted at the onset of mitosis to ensure timely centrosome disjunction and bipolar spindle formation and is reassembled at the end of mitosis. While the mechanism controlling centrosome linker disassembly at early mitosis has been well explored, little is known about how the linker is subsequently reassembled before mitotic exit. Here we report that ASPP1 and ASPP2, two members of the apoptosis stimulating proteins of p53 (ASPP) family, are involved in centrosome linker reassembly. We showed that ASPP1/2 interacted with centrosome linker protein C-Nap1. Co-depletion of ASPP1 and ASPP2 inhibited re-association of C-Nap1 with centrosome at the end of mitosis. Moreover, ASPP1/2 facilitated the interaction between C-Nap1 and PP1α, and this interaction was significantly reduced by co-depletion of ASPP1/2. ASPP1/2 antagonized the NEK2A-mediated C-Nap1 Ser2417/2421 phosphorylation in a PP1-dependent manner. Co-depletion of ASPP1 and ASPP2 inhibited dephosphorylation of C-Nap1 (Ser2417/2421) at the end of mitosis. Based on these findings, we propose that ASPP1/2 act as PP1-targeting subunits to facilitate C-Nap1 dephosphorylation and centrosome linker reassembly at the end of mitosis.

HDGF-related protein-2 (HRP-2) acts as an oncogene to promote cell growth in hepatocellular carcinoma.

HDGFRP2 (HRP-2) belongs to the Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) family, which are characterized by a conserved HATH/PWWP domain at a well-conserved region of the N-terminus. However, the cellular function of HRP-2 remains unknown. In this study, we showed for the first time that HRP-2 is frequently overexpressed in human HCC tissues at mRNA and protein levels. We further showed that HRP-2 can promote HCC cells growth in vitro and xenograft tumors in vivo. Using protein affinity purification methods, we searched for functional partners of HRP-2, and found that HRP-2 interacts with various proteins known to be involved in transcription elongation and processing. Furthermore, we demonstrate HRP-2 interacts and co-localizes with RNA processing regulator IWS1, and positively regulated the mRNA level of Cyclin D1. Together, our study suggests HRP-2 may act as an mRNA processing co-factor to promote cells growth by regulating the mRNA of key oncogenes, which can be explored further for cancer treatment.