Tumor suppressor SCUBE2 inhibits breast-cancer cell migration and invasion through the reversal of epithelial-mesenchymal transition.

Signal peptide-CUB-EGF domain-containing protein 2 (SCUBE2) belongs to a secreted and membrane-associated multi-domain SCUBE protein family. We previously demonstrated that SCUBE2 is a novel breast-tumor suppressor and could be a useful prognostic marker. However, the role of SCUBE2 in breast-cancer cell migration and invasion and how it is regulated during the epithelial-mesenchymal transition (EMT) remain undefined. In this study, we showed that ectopic SCUBE2 overexpression could enhance the formation of E-cadherin-containing adherens junctions by ß-catenin-SOX-mediated induction of forkhead box A1 (a positive regulator of E-cadherin) and upregulation of E-cadherin, which in turn led to epithelial transition and inhibited migration and invasion of aggressive MDA-MB-231 breast-carcinoma cells. SCUBE2 expression was repressed together with that of E-cadherin in TGF-ß-induced EMT; direct expression of SCUBE2 alone was sufficient to inhibit the TGF-ß-induced EMT. Furthermore, quantitative DNA methylation, methylation-specific PCR, and chromatin immunoprecipitation analyses revealed that SCUBE2 expression was inactivated by DNA hypermethylation at the CpG islands by recruiting and binding DNA methyltransferase 1 during TGF-ß-induced EMT. Together, our results suggest that SCUBE2 plays a key role in suppressing breast-carcinoma-cell mobility and invasiveness by increasing the formation of the epithelial E-cadherin-containing adherens junctions to promote epithelial differentiation and drive the reversal of EMT.

Domain and functional analysis of a novel breast tumor suppressor protein, SCUBE2.

Signal peptide CUB (complement proteins C1r/C1s, Uegf, and Bmp 1)-EGF domain-containing protein 2 (SCUBE2) is a secreted, membrane-associated multidomain protein composed of five recognizable motifs: an NH(2)-terminal signal peptide sequence, nine copies of epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeats, and one CUB domain at the COOH terminus. Our previous clinical study showed that SCUBE2 may act as a novel breast tumor suppressor gene and serve as a useful prognostic marker. However, the specific domain responsible for its tumor suppressor activity and the precise mechanisms of its anti-tumor effect remain unknown. Using a combination of biochemical, molecular, and cell biology techniques, we further dissected the molecular functions and signal pathways mediated by the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain of SCUBE2. Independent overexpression of the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH-terminal CUB domain could directly bind to and antagonize bone morphogenetic protein activity in an autocrine manner, whereas the NH(2)-terminal EGF-like repeats could mediate cell-cell homophilic adhesions in a calcium-dependent fashion, interact with E-cadherin (a master tumor suppressor), and decrease the ß-catenin signaling pathway. Together, our data demonstrate that SCUBE2 has growth inhibitory effects through a coordinated regulation of two distinct mechanisms: antagonizing bone morphogenetic protein and suppressing the ß-catenin pathway in breast cancer cells.

Association of plasma concentration of small heat shock protein B7 with acute coronary syndrome.

BACKGROUND: Heat shock proteins (HSPs) act as chaperones and have a protective function in cardiovascular diseases. The clinical association of a novel small HSPB7 with cardiovascular disease, however, has not been reported. The aim of this study was to investigate the potential biological functions of HSPB7 and its relationship with acute coronary syndrome (ACS). METHODS AND RESULTS: A mouse myocardial infarction (MI) model and samples from clinical human subjects were used to determine plasma HSPB7 concentration after acute MI. The associations of plasma HSPB7 concentration with ACS and other risk factors of coronary artery disease were analyzed. Plasma HSPB7 concentration was found to be rapidly elevated in mice after coronary artery ligation. In addition, plasma HSPB7 concentration was significantly higher in patients with ACS than in control patients with non-cardiac chest pain (5.1 ng/ml vs. 2.9 ng/ml, P<0.001). Plasma HSPB7 was detected as early as 1-3 h after the onset of symptoms and remained detectable up to 24h. Furthermore, in patients presenting to the emergency department with acute chest pain, HSPB7 level was an independent risk factor of ACS (adjusted odds ratio, 7.44; 95% confidence interval: 1.91-28.93, P<0.01). CONCLUSIONS: HSPB7 is a potential early biomarker after MI and serves as an independent risk factor of ACS in patients with acute chest pain.

Androgen depletion up-regulates cadherin-11 expression in prostate cancer.

Men with castration-resistant prostate cancer (PCa) frequently develop metastasis in bone. The reason for this association is unclear. We have previously shown that cadherin-11 (also known as OB-cadherin), a homophilic cell adhesion molecule that mediates osteoblast adhesion, plays a role in the metastasis of PCa to bone. Here, we report that androgen-deprivation therapy up-regulates cadherin-11 expression in PCa. In human PCa specimens, immunohistochemical staining showed that 22/26 (85%) primary PCa tumours from men with castration-resistant PCa expressed cadherin-11. In contrast, only 7/50 (14%) androgen-dependent PCa tumours expressed cadherin-11. In the MDA-PCa-2b xenograft animal model, cadherin-11 was expressed in the recurrent tumours following castration. In the PCa cell lines, there is an inverse correlation between expression of cadherin-11 and androgen receptor (AR), and cadherin-11 is expressed in very low levels or not expressed in AR-positive cell lines, including LNCaP, C4-2B4 and VCaP cells. We showed that AR likely regulates cadherin-11 expression in PCa through an indirect mechanism. Although re-expression of AR in the AR-negative PC3 cells led to the inhibition of cadherin-11 expression, depletion of androgen from the culture medium or down-regulation of AR by RNA interference in the C4-2B4 cells or VCaP cells only produced a modest increase of cadherin-11 expression. Promoter analysis indicated that cadherin-11 promoter does not contain a typical AR-binding element, and AR elicits a modest inhibition of cadherin-11 promoter activity, suggesting that AR does not regulate cadherin-11 expression directly. Together, these results suggest that androgen deprivation up-regulates cadherin-11 expression in prostate cancer, and this may contribute to the metastasis of PCa to bone. Our study suggests that therapeutic strategies that block cadherin-11 expression or function may be considered when applying androgen-ablation therapy.

Guanylate cyclase-G, expressed in the Grueneberg ganglion olfactory subsystem, is activated by bicarbonate.

GC (guanylate cyclase)-G is the most recently identified member of the receptor GC family. However, the regulation of its activity and protein expression in the mammalian olfactory system remains unclear. In the present study, we used a GC-G-specific antibody to validate that the GC-G protein is expressed in Grueneberg ganglion neurons, a newly recognized olfactory subsystem co-expressing other cGMP signalling components such as the cGMP-regulated PDE2A (phosphodiesterase 2A) and the cGMP-gated ion channel CNGA3 (cyclic nucleotide-gated cation channel α-3). Further molecular and biochemical analyses showed that heterologously expressed GC-G protein, specifically the C-terminal cyclase domain, was directly stimulated by bicarbonate in both in vivo cellular cGMP accumulation assays in human embryonic kidney-293T cells and in vitro GC assays with a purified recombinant protein containing the GC domain. In addition, overexpression of GC-G in NG108 neuronal cells resulted in a CO2-dependent increase in cellular cGMP level that could be blocked by treatment with acetazolamide, an inhibitor of carbonic anhydrases, which implies that the stimulatory effect of CO2 requires its conversion to bicarbonate. Together, our data demonstrate a novel CO2/bicarbonate-dependent activation mechanism for GC-G and suggest that GC-G may be involved in a wide variety of CO2/bicarbonate-regulated biological processes such as the chemosensory function in Grueneberg ganglion neurons.

Isolation and characterization of a secreted, cell-surface glycoprotein SCUBE2 from humans.

SCUBE2 [signal peptide, CUB domain, EGF (epidermal growth factor)-like protein 2] belongs to an evolutionarily conserved SCUBE protein family, which possesses domain organization characteristic of an N-terminal signal peptide sequence followed by nine EGF-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C-terminus. Despite several genetic analyses suggesting that the zebrafish orthologue of the mammalian SCUBE2 gene participates in HH (Hedgehog) signalling, the complete full-length cDNA and biochemical function for mammalian SCUBE2 on HH signalling remains uninvestigated. In the present study, we isolated the full-length cDNA and studied the role of human SCUBE2 in the HH signalling cascade. When overexpressed, recombinant human SCUBE2 manifests as a secreted surface-anchored glycoprotein. Deletion mapping analysis defines the critical role of the spacer region and/or cysteine-rich repeats for membrane association. Further biochemical analyses and functional reporter assays demonstrated that human SCUBE2 can specifically interact with SHH (Sonic Hedgehog) and SHH receptor PTCH1 (Patched-1), and enhance the SHH signalling activity within the cholesterol-rich raft microdomains of the plasma membranes. Together, our results reveal that human SCUBE2 is a novel positive component of the HH signal, acting upstream of ligand binding at the plasma membrane. Thus human SCUBE2 could play important roles in HH-related biology and pathology, such as during organ development and tumour progression.

Cadherin-11 promotes the metastasis of prostate cancer cells to bone.

Bone is the most common site of metastases from prostate cancer. The mechanism by which prostate cancer cells metastasize to bone is not fully understood, but interactions between prostate cancer cells and bone cells are thought to initiate the colonization of metastatic cells at that site. Here, we show that cadherin-11 (also known as osteoblast-cadherin) was highly expressed in prostate cancer cell line derived from bone metastases and had strong homophilic binding to recombinant cadherin-11 in vitro. Down-regulation of cadherin-11 in bone metastasis-derived PC3 cells with cadherin-11-specific short hairpin RNA (PC3-shCad-11) significantly decreased the adhesion of those cells to cadherin-11 in vitro. In a mouse model of metastasis, intracardiac injection of PC3 cells led to metastasis of those cells to bone. However, the incidence of PC3 metastasis to bone in this model was reduced greatly when the expression of cadherin-11 by those cells was silenced. The clinical relevance of cadherin-11 in prostate cancer metastases was further studied by examining the expression of cadherin-11 in human prostate cancer specimens. Cadherin-11 was not expressed by normal prostate epithelial cells but was detected in prostate cancer, with its expression increasing from primary to metastatic disease in lymph nodes and especially bone. Cadherin-11 expression was not detected in metastatic lesions that occur in other organs. Collectively, these findings suggest that cadherin-11 is involved in the metastasis of prostate cancer cells to bone.

Disruption of guanylyl cyclase-G protects against acute renal injury.

The membrane forms of guanylyl cyclase (GC) serve as cell-surface receptors that synthesize the second messenger cGMP, which mediates diverse cellular processes. Rat kidney contains mRNA for the GC-G isoform, but the role of this receptor in health and disease has not been characterized. It was found that mouse kidney also contains GC-G mRNA, and immunohistochemistry identified GC-G protein in the epithelial cells of the proximal tubule and collecting ducts. Six hours after ischemia-reperfusion (I/R) injury, GC-G mRNA and protein expression increased three-fold and remained upregulated at 24 h. For determination of whether GC-G mediates I/R injury, a mutant mouse with a targeted disruption of the GC-G gene (Gucy2g) was created. At baseline, no histologic abnormalities were observed in GC-G(-/-) mice. After I/R injury, elevations in serum creatinine and urea were attenuated in GC-G(-/-) mice compared with wild-type controls, and this correlated with less tubular disruption, less tubular cell apoptosis, and less caspase-3 activation. Measures of inflammation (number of infiltrating neutrophils, myeloperoxidase activity, and induction of IL-6 and P-selectin) and activation of NF-kappaB were lower in GC-G(-/-) mice compared with wild-type mice. Direct transfer of a GC-G expression plasmid to the kidneys of GC-G(-/-) mice resulted in a dramatically higher mortality after renal I/R injury, further supporting a role for GC-G in mediating injury. In summary, GC-G may act as an early signaling molecule that promotes apoptotic and inflammatory responses in I/R-induced acute renal injury.

Enhanced Efficiencies of Perovskite Solar Cells by Incorporating Silver Nanowires into the Hole Transport Layer.

In this study, we incorporated silver nanowires (AgNWs) into poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) as a hole transport layer (HTL) for inverted perovskite solar cells (PVSCs). The effect of AgNW incorporation on the perovskite crystallization, charge transfer, and power conversion efficiency (PCE) of PVSCs were analyzed and discussed. Compared with neat PEDOT:PSS HTL, incorporation of few AgNWs into PEDOT:PSS can significantly enhance the PCE by 25%. However, the AgNW incorporation may result in performance overestimation due to the lateral charge transfer. The corrosion of AgNWs with a perovskite layer was discussed. Too much AgNW incorporation may lead to defects on the interface between the HTL and the perovskite layer. An extra PEDOT:PSS layer over the pristine PEDOT:PSS-AgNW layer can prevent AgNWs from corrosion by iodide ions.

Primary peritoneal carcinosarcoma: A report of two cases.

OBJECTIVE: Carcinosarcomas also known as malignant mixed mullerian tumors (MMMTs) contain both carcinomatous and sarcomatous elements. Most MMMTs are arising from female genital tract, including ovaries, uterus and fallopian tubes. Extragenital carcinosarcomas are extremely rare, with an estimation less than 40 cases so far. CASE REPORT: We report two cases of primary peritoneal carcinosarcomas. An 81-year-old woman with pelvic peritoneal carcinosarcoma, heterologous type, was treated with incomplete surgery without further chemotherapy, and died of disease soon. The other one was a 76 year-old woman with abdominal peritoneal carcinosarcoma, homologous type. After optimal debulking surgery and subsequent 6 cycles of combination of paclitaxel and carboplatin chemotherapy, the patient is free of tumor half of year. CONCLUSION: Active therapy, including complete cytoreduction surgery and carboplatin-paclitaxel chemotherapy might offer a chance of disease control for these unusual primary peritoneal carcinosarcomas.

Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis.

INTRODUCTION: Estrogen receptors (ERs) play key roles in breast cancer development and influence treatment outcome in breast cancer patients. Identification of molecules that regulate ER function may facilitate development of breast cancer treatment strategies. The forkhead box class O (FOXO) transcription factor FOXO3a has been suggested to function as a tumor suppressor in breast cancer. Using protein-protein interaction screening, we found that FOXO3a interacted with ER-alpha and ER-beta proteins in the human breast carcinoma cell line MCF-7, suggesting that there exists a crosstalk between the FOXO3a and ER signaling pathways in estrogen-dependent breast cancer cells. METHODS: The interaction between FOXO3a and ER was investigated by using co-immunoprecipitation and immunoblotting assays. Inhibition of ER-alpha and ER-beta transactivation activity by FOXO was determined by luciferase reporter assays. Cell proliferation in culture was evaluated by counting cell numbers. Tumorigenesis was assessed in athymic mice that were injected with MCF-7 cell lines over-expressing FOXO3a. Protein expression levels of cyclin-dependent kinase inhibitors, cyclins, ERs, FOXM1, and the proteins encoded by ER-regulated genes in MCF-7 cell lines and breast tumors were examined by immunoblotting analysis and immunohistochemical staining. RESULTS: We found that FOXO3a interacted with ER-alpha and ER-beta proteins and inhibited 17beta-estradiol (E2)-dependent, ER-regulated transcriptional activities. Consistent with these observations, expression of FOXO3a in the ER-positive MCF-7 cells decreased the expression of several ER-regulated genes, some of which play important roles in cell proliferation. Moreover, we found that FOXO3a upregulated the expression of the cyclin-dependent kinase inhibitors p21Cip1, p27Kip1, and p57Kip2. These findings suggest that FOXO3a induces cell growth arrest to effect tumor suppression. FOXO3a repressed the growth and survival of MCF-7 cells in cell culture. In an orthotopic breast cancer xenograft model in athymic mice, over-expression of FOXO3a in MCF-7 cells suppressed their E2-induced tumorigenesis, whereas knockdown of FOXO3a in MCF-7 resulted in the E2-independent growth. CONCLUSION: Functional interaction between FOXO3a and ER plays a critical role in suppressing estrogen-dependent breast cancer cell growth and tumorigenesis in vivo. This suggests that agents that activate FOXO3a may be novel therapeutic agents that can inhibit and prevent tumor proliferation and development in breast cancer.

Bone microenvironment and androgen status modulate subcellular localization of ErbB3 in prostate cancer cells.

ErbB-3, an ErbB receptor tyrosine kinase, has been implicated in the pathogenesis of several malignancies, including prostate cancer. We found that ErbB-3 expression was up-regulated in prostate cancer cells within lymph node and bone metastases. Despite being a plasma membrane protein, ErbB-3 was also detected in the nuclei of the prostate cancer cells in the metastatic specimens. Because most metastatic specimens were from men who had undergone androgen ablation, we examined the primary tumors from patients who have undergone hormone deprivation therapy and found that a significant fraction of these specimens showed nuclear localization of ErbB3. We thus assessed the effect of androgens and the bone microenvironment on the nuclear translocation of ErbB-3 by using xenograft tumor models generated from bone-derived prostate cancer cell lines, MDA PCa 2b, and PC-3. In subcutaneous tumors, ErbB-3 was predominantly in the membrane/cytoplasm; however, it was present in the nuclei of the tumor cells in the femur. Castration of mice bearing subcutaneous MDA PCa 2b tumors induced a transient nuclear translocation of ErbB-3, with relocalization to the membrane/cytoplasm upon tumor recurrence. These findings suggest that the bone microenvironment and androgen status influence the subcellular localization of ErbB-3 in prostate cancer cells. We speculate that nuclear localization of ErbB-3 may aid prostate cancer cell survival during androgen ablation and progression of prostate cancer in bone.

Transgenic overexpression of the secreted, extracellular EGF-CUB domain-containing protein SCUBE3 induces cardiac hypertrophy in mice.

OBJECTIVE: The aim of this study was to investigate in a transgenic animal model the cardiac expression and function of a novel extracellular protein SCUBE3 [signal peptide-CUB (complement proteins C1r/C1s, Uegf, and Bmp1)-EGF (epidermal growth factor)-like domain-containing protein 3]. METHODS AND RESULTS: Real-time quantitative reverse transcriptase (RT)-PCR analysis showed that SCUBE3 is expressed in the ventricular myocardium. Male transgenic (TG) mice overexpressing SCUBE3 appeared normal during development, from birth to adulthood. However, echocardiography and histopathological examination revealed significant cardiac hypertrophy in TG animals as they aged, at 8 months. Interestingly, left-ventricle hypertrophy occurred more rapidly and more severely under pressure overload in TG mice, as compared to age-matched wild-type littermates. Induced SCUBE3 expression, together with elevated transforming growth factor (TGF)-beta1 level under pressure overload, may account for the accelerated onset and progression of cardiac hypertrophy in TG mice. Furthermore, biochemical and molecular studies revealed that the carboxyl-terminal portion of SCUBE3 could physically interact with TGF-beta1 and promote the TGF-beta1-mediated transcriptional activation. CONCLUSION: This report is the first demonstration that SCUBE3 may play a role in the regulation of cardiac growth and remodeling responses, possibly through the stabilization of the TGF-beta1 signaling pathway.

Organelle-Derived Acetyl-CoA Promotes Prostate Cancer Cell Survival, Migration, and Metastasis via Activation of Calmodulin Kinase II.

Although emerging evidence suggests a potential role of calcium/calmodulin-dependent kinase II (CaMKII) in prostate cancer, its role in prostate cancer tumorigenesis is largely unknown. Here, we examine whether the acetyl CoA-CaMKII pathway, first described in frog oocytes, promotes prostate cancer tumorigenesis. In human prostate cancer specimens, metastatic prostate cancer expressed higher levels of active CaMKII compared with localized prostate cancer. Correspondingly, basal CaMKII activity was significantly higher in the more tumorigenic PC3 and PC3-mm2 cells relative to the less tumorigenic LNCaP and C4-2B4 cells. Deletion of CaMKII by CRISPR/Cas9 in PC3-mm2 cells abrogated cell survival under low-serum conditions, anchorage-independent growth and cell migration; overexpression of constitutively active CaMKII in C4-2B4 cells promoted these phenotypes. In an animal model of prostate cancer metastasis, genetic ablation of CaMKII reduced PC3-mm2 cell metastasis from the prostate to the lymph nodes. Knockdown of the acetyl-CoA transporter carnitine acetyltransferase abolished CaMKII activation, providing evidence that acetyl-CoA generated from organelles is a major activator of CaMKII. Genetic deletion of the ß-oxidation rate-limiting enzyme ACOX family proteins decreased CaMKII activation, whereas overexpression of ACOXI increased CaMKII activation. Overall, our studies identify active CaMKII as a novel connection between organelle ß-oxidation and acetyl-CoA transport with cell survival, migration, and prostate cancer metastasis.Significance: This study identifies a cell metabolic pathway that promotes prostate cancer metastasis and suggests prostate cancer may be susceptible to ß-oxidation inhibitors. Cancer Res; 78(10); 2490-502. ©2018 AACR.

Bullous amyloidosis in a hemodialysis patient is myeloma-associated rather than hemodialysis-associated amyloidosis.

We report a 78-year-old woman on hemodialysis who presented with refractory multiple pruritic vesicles and bullae on her trunk and extremities for 2 months. Histopathologic examination of skin biopsy specimen showed subepidermal bullae with many amyloid deposits in the papillary dermis. No evidence of systemic amyloidosis could be found on physical examination. While the initial clinical diagnosis was bullous pemphigoid, the histopathology and direct immunofluorescence result favored hemodialysis-associated amyloidosis. However, immunochemical study for beta(2)-microglobulin was negative. Further hematologic and immunologic work-up revealed the presence of multiple myeloma and that the deposit was AL amyloid. This is the first case of bullous amyloidosis in a hemodialysis patient and should remind dermatologists that bullous amyloidosis should be considered in addition to the usual presentation of porphyria cutanea tarda and pseudoporphyria for bullous dermatosis in the hemodialysis patient. We also suggest that hemodialysis-associated amyloidosis should not be taken for granted in the hemodialysis patient with cutaneous amyloidosis without systemic signs and symptoms. Further testing for other types of amyloid should be performed.

Localization and characterization of a novel secreted protein SCUBE1 in human platelets.

OBJECTIVE: The aim of the study was to investigate the protein expression and function of a novel secreted protein in the vascular system, named SCUBE1 for signal peptide, CUB (Complement proteins C1r/C1s, Uegf, and Bmp1) and epidermal growth factor-like (EGF)-like domain containing protein 1. METHODS AND RESULTS: Immunohistochemical analysis demonstrated that the SCUBE1 staining is mainly confined to the intravascular platelet-rich thrombus in vascular tissue samples. While quantitative real-time RT-PCR verified that the SCUBE1 mRNA is expressed in human platelets, numerous immunolocalization techniques revealed that the preformed SCUBE1 protein is stored in the alpha-granules and translocated to the surface upon platelet stimulation. A smaller SCUBE1 fragment, possibly formed by limited proteolysis after being released from the storage granules, was detected in thrombus lysate by Western blot analysis. Interestingly, deposition of SCUBE1 into the subendothelial matrix of the atherosclerotic plaques was evidenced by immunohistochemistry. In addition, studies of platelet adhesion and ristocetin-induced platelet agglutination showed that fragments containing the amino-terminal EGF-like repeats were able to support platelet adhesion and enhance the ristocetin-induced platelet agglutination, respectively. CONCLUSION: These data suggest that platelet-derived SCUBE1 could function as a novel adhesive molecule and its matrix-bound and soluble fragments may play critical (patho)physiological roles in cardiovascular biology.