Castration-Induced Downregulation of SPARC in Stromal Cells Drives Neuroendocrine Differentiation of Prostate Cancer.

Fatal neuroendocrine differentiation (NED) of castration-resistant prostate cancer is a recurrent mechanism of resistance to androgen deprivation therapies (ADT) and antiandrogen receptor pathway inhibitors (ARPI) in patients. The design of effective therapies for neuroendocrine prostate cancer (NEPC) is complicated by limited knowledge of the molecular mechanisms governing NED. The paucity of acquired genomic alterations and the deregulation of epigenetic and transcription factors suggest a potential contribution from the microenvironment. In this context, whether ADT/ARPI induces stromal cells to release NED-promoting molecules and the underlying molecular networks are unestablished. Here, we utilized transgenic and transplantable mouse models and coculture experiments to unveil a novel tumor-stroma cross-talk that is able to induce NED under the pressure of androgen deprivation. Castration induced upregulation of GRP78 in tumor cells, which triggers miR29-b-mediated downregulation of the matricellular protein SPARC in the nearby stroma. SPARC downregulation enabled stromal cells to release IL6, a known inducer of NED. A drug that targets GRP78 blocked NED in castrated mice. A public, human NEPC gene expression dataset showed that Hspa5 (encoding for GRP78) positively correlates with hallmarks of NED. Finally, prostate cancer specimens from patients developing local NED after ADT showed GRP78 upregulation in tumor cells and SPARC downregulation in the stroma. These results point to GRP78 as a potential therapeutic target and to SPARC downregulation in stromal cells as a potential early biomarker of tumors undergoing NED. SIGNIFICANCE: Tumor-stroma cross-talk promotes neuroendocrine differentiation in prostate cancer in response to hormone therapy via a GRP78/SPARC/IL6 axis, providing potential therapeutic targets and biomarkers for neuroendocrine prostate cancer.

Myokine Responses to Exercise in a Rat Model of Low/High Adaptive Potential.

Introduction: Assuming myokines underlie some of the health benefits of exercise, we hypothesised that 'high responder trainer' (HRT) rats would exhibit distinct myokine profiles to 'low responder trainers' (LRT), reflecting distinct health and adaptive traits. Methods: Blood was collected from LRT and HRT (N=8) rats at baseline (BL), immediately (0h), 1h, and 3h after running; repeated after 3-wks training. Myokines were analysed by ELISA (i.e. BDNF/Fractalkine/SPARC/Irisin/FGF21/Musclin/IL-6). Results: At baseline, Musclin (LRT: 84 ± 24 vs HRT: 26 ± 3 pg/ml, P=0.05) and FGF21 (LRT: 133 ± 34 vs HRT: 63.5 ± 13 pg/ml, P=0.08) were higher in LRT than HRT. Training increased Musclin in HRT (26 ± 3 to 54 ± 9 pg/ml, P<0.05) and decreased FGF21 in LRT (133 ± 34 to 60 ± 28 pg/ml, P<0.05). Training increased SPARC (LRT: 0.8 ± 0.1 to 2.1 ± 0.6 ng/ml, P<0.05; HRT: 0.7 ± 0.06 to 1.8 ± 0.3 ng/ml, P=0.06) and Irisin (LRT 0.62 ± 0.1 to 2.6 ± 0.4 ng/ml, P<0.01; HRT 0.53 ± 0.1 to 2.8 ± 0.7 ng/ml, P<0.01) while decreasing BDNF (LRT: 2747 ± 293 to 1081 ± 330 pg/ml, P<0.01; HRT: 1976 ± 328 to 797 ± 160 pg/ml, P<0.05). Acute exercise response of Musclin (AUC) was higher in LRT vs HRT (306 ± 74 vs. 88 ± 12 pg/ml×3h-1, P<0.01) and elevated in HRT after training (221 ± 31 pg/ml×3h-1, P<0.01). Training elevated SPARC (LRT: 2.4 ± 0.1 to 7.7 ± 1.3 ng/ml×3h-1, P<0.05; HRT: 2.5 ± 0.13 to 11.2 ± 2.2 ng/ml×3h-1, P<0.001) and Irisin (LRT: 1.34 ± 0.3 to 9.6 ± 1.7 ng/ml×3h-1, P<0.001; HRT: 1.5 ± 0.5 to 12.1 ± 1.9 ng/ml×3h-1, P<0.0001). Conclusion: Exercise training alters how myokines are secreted in response to acute exercise. Myokine responses were not robustly linked to adaptive potential in aerobic capacity, making them an unlikely regulator of adaptive traits.

Intestinal production of secreted protein acidic and rich in cysteine (SPARC) in patients with ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory disease of the intestine. The genetics factors play an important role in the pathogenesis of UC. SPARC exacerbates colonic inflammatory symptoms in dextran sodium sulphate-induced murine colitis. The aim of the study was to measure the gene expression and intestinal production of SPARC in patients with UC and controls as well as, to determine its correlation with histological activity. METHODS: We included 40 patients with confirmed diagnosis of UC, and 20 controls without endoscopic evidence of any type of colitis or neoplasia. The relative quantification of the gene expression was performed by real time PCR. GAPDH was used as housekeeping gene for normalization purposes and quality controls. Protein expression was determined by immunohistochemistry. RESULTS: The gene expression of SPARC was increased in patients with active UC vs in remission UC and vs. controls (P = 0.005). There was no significant difference between patients with remission UC and controls. The overexpression of SPARC in patients with active UC correlated significantly with mild histological activity (P = 0.06, OR = 7.77, IC = 0.77-77.9) moderate (P = 0.06, OR = 8.1, IC 95%=0.79-82.73), and severe (P = 0.03, OR = 6.5, IC 95%=1.09-38.6). Double positive SPARC+/CD16+ cells were localized mainly in submucosa, muscular layer, and adventitia, and in perivascular inflammatory infiltrates in patients with active UC. CONCLUSION: The gene and protein expression of SPARC is increased in active UC. SPARC could be a marker of intestinal inflammation and its expression correlates with histological activity.

Roles of the synaptic molecules Hevin and SPARC in mouse neuromuscular junction development and repair.

Hevin and secreted protein acidic and rich in cysteine (SPARC) are highly homologous matricellular proteins that function in concert to guide the formation of brain synapses. Here, we investigated the role of these glycoproteins in neuromuscular junction (NMJ) maturation, stability, and repair following injury. Hevin and SPARC mRNA levels in developing (postnatal day 9), adult (postnatal days 90 and 120), and injured (fibular nerve crush) skeletal muscles were assessed with qPCR. Muscle fiber size was analyzed in developing (P9) mice lacking SPARC, Hevin, and both SPARC and Hevin. NMJ morphology was assessed in developing (P9), adult (P90) and injured (fibular nerve crush) mice lacking SPARC, Hevin, and both SPARC and Hevin skeletal muscle. Hevin and SPARC are expressed in skeletal muscles and are upregulated following nerve injury. Hevin-/- mice exhibited delayed NMJ and muscle fiber development but displayed normal NMJ morphology in adulthood and accelerated NMJ reinnervation following nerve injury. Mice lacking SPARC displayed normal NMJ and muscle fiber development but exhibited smaller NMJs with fewer acetylcholine receptor islands in adulthood. Further, SPARC deletion did not result in overt changes in NMJ reformation following nerve injury. The combined deletion of Hevin and SPARC had little effect on NMJ phenotypes observed in single knockouts, however deletion of SPARC in combination with Hevin reversed deficiencies in muscle fiber maturation observed in Hevin-/- muscle. These results identify SPARC and Hevin as extracellular matrix proteins with roles in NMJ development and repair.

Lack of Correlation Between Immunohistochemical Expression of SPARC and Invasion in Different Grades of Meningiomas.

BACKGROUND: Grade I meningiomas are generally benign and non-invasive whereas Grade II (atypical) and Grade III (malignant) meningiomas tend to be invasive with a high risk of recurrence. SPARC, secreted protein, acidic and rich in cysteine, is a multifunctional glycoprotein which has been proposed to be a potential diagnostic marker of invasive meningiomas. There has been increased reporting of atypical meningiomas since the current World Health Organization (WHO) included brain invasion as a grading criterion for classification of these particular meningiomas. MATERIALS AND METHODS: The aim of this study was to re-evaluate any correlation between immunohistochemical expression of SPARC in 34 meningiomas of various grades using the current classification (2016). We had previously classified these cases using the 2002 WHO criteria. RESULTS: There is no correlation between expression of SPARC and invasion in different grades of meningioma. CONCLUSION: SPARC does not appear to be a good predictor of invasion in meningiomas.

RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas.

Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.

Modulation of SPARC/Hevin Proteins in Alzheimer's Disease Brain Injury.

Alzheimer's disease (AD) is an age-related progressive form of dementia that features neuronal loss, intracellular tau, and extracellular amyloid-ß (Aß) protein deposition. Neurodegeneration is accompanied by neuroinflammation mainly involving microglia, the resident innate immune cell population of the brain. During AD progression, microglia shift their phenotype, and it has been suggested that they express matricellular proteins such as secreted protein acidic and rich in cysteine (SPARC) and Hevin protein, which facilitate the migration of other immune cells, such as blood-derived dendritic cells. We have detected both SPARC and Hevin in postmortem AD brain tissues and confirmed significant alterations in transcript expression using real-time qPCR. We suggest that an infiltration of myeloid-derived immune cells occurs in the areas of diseased tissue. SPARC is highly expressed in AD brain and collocates to Aß protein deposits, thus contributing actively to cerebral inflammation and subsequent tissue repair, and Hevin may be downregulated in the diseased state. However, further research is needed to reveal the exact roles of SPARC and Hevin proteins and associated signaling pathways in AD-related neuroinflammation. Nevertheless, normalizing SPARC/Hevin protein expression such as interdicting heightened SPARC protein expression may confer a novel therapeutic opportunity for modulating AD progression.

Vimentin, osteocalcin and osteonectin expression in canine primary bone tumors: diagnostic and prognostic implications.

Canine primary bone tumors have a plastic radiographic image, demanding histopathological confirmation. Bone tumors are characterized by the type and amount of extracellular matrix produced what cannot be easily recognized, especially in biopsy samples. Identifying cellular markers that could aid diagnosis has supported various studies in oncological pathology. This study aimed to evaluate 22 canine primary bone neoplasms, establishing their histopathological diagnosis and evaluated vimentin, osteonectin and osteocalcin expression and their implication in diagnosis and prognosis. There were 12 productive osteoblastic osteosarcomas, six minimally productive osteoblastic osteosarcoma, two chondrosarcomas, one fibrosarcoma and one hemangiosarcoma. Immunostaining was cytoplasmatic in all cases, with average percentage of 87.9% for vimentin, 98.0% for osteonectin and 99.9% for osteocalcin. In this last case, only osteosarcomas were considered. Intensity was higher in vimentin labeling (+++), followed by osteonectin (++) and osteocalcin (+). One osteosarcoma showed negative immunostaining for vimentin and of samples submitted to anti-osteocalcin immunostaining, three osteosarcomas and one fibrosarcoma had negative staining. Besides identifying mesenchymal origin, vimentin elevated expression in canine bone tumors can be related to epithelial-mesenchymal transition, leading to more aggressive tumoral phenotypes and metastasis development. Similarly, high osteonectin expression is implicated in neoplastic cell invasion and is also related to metastasis spread. Decreased osteocalcin expression was found in some osteosarcoma samples and can be related to poor prognosis, as in human osteosarcomas. Our findings suggest that vimentin, osteonectin and osteocalcin not only aid diagnosis but can be related to prognosis in canine primary bone tumors, especially osteosarcomas and its osteoblastic subtype.

Targeted therapy for the post-operative conjunctiva: SPARC silencing reduces collagen deposition.

BACKGROUND: To develop targeted antifibrotic therapy for glaucoma filtration surgery; this study determines the effectiveness of small interfering RNA (siRNA) to reduce in vivo secreted protein acidic and rich in cysteine (SPARC) expression using the mouse model of conjunctival scarring. METHODS: Experimental surgery was performed as described for the mouse model of conjunctival scarring. Scrambled (siScram) or Sparc (siSparc) siRNAs, loaded on layer-by-layer (LbL) nanoparticles, were injected into the conjunctiva immediately after surgery. Expression of Sparc, Col1a1, Fn1 and Mmp14 was measured by real-time PCR and immunoblotting on days 7 and 14 postsurgery. Live imaging of the operated eyes was performed using slit lamp, anterior segment-optical coherence tomography and confocal microscopy. Tissue pathology was evaluated by histochemical and immunofluorescent analyses of operated conjunctival cryosections. Tissue apoptosis was quantitated by annexin V assay. RESULTS : siSparc, delivered via expanded LbL nanoparticles, significantly inhibited Sparc transcription in both day 7 (2.04-fold) and day 14 (1.39-fold) treated tissues. Sparc suppression on day 7 was associated with a significant reduction of Col1a1 (2.52-fold), Fn1 (2.89-fold) and Mmp14 (2.23-fold) mRNAs. At the protein level, both SPARC and collagen 1A1 (COL1A1) were significantly reduced at both time points with siSparc treatment. Nanoparticles were visualised within cell-like structures by confocal microscopy, while overt tissue response or apoptosis was not observed. CONCLUSIONS : SPARC targeted therapy effectively reduced both SPARC and collagen production in the operated mouse conjunctiva. This proof-of-concept study suggests that targeted treatment of fibrosis in glaucoma surgery is safe and feasible, with the potential to extend to a range of potential genes associated with fibrosis.

The prognostic significance of cancer-associated fibroblasts in pancreatic ductal adenocarcinoma.

Cancer-associated fibroblasts are abundant in the desmoplastic stroma of pancreatic ductal adenocarcinomas and are considered to play important roles in tumor progression. In this study, we investigated the expression status of secreted protein acidic and rich in cysteine, periostin, fibroblast-activated protein, and the newly developed proCOL11A1 antibody in the stroma of surgically resected pancreatic ductal adenocarcinomas and their prognostic implications. Tissue microarrays were constructed from 155 surgically resected pancreatic ductal adenocarcinomas and paired non-neoplastic pancreata and from another independent set of 48 normal/benign pancreata, and immunohistochemical stains were performed for proCOL11A1, fibroblast-activated protein, secreted protein acidic and rich in cysteine, and periostin. The immunohistochemical stain results were correlated with clinicopathological features and survival data. proCOL11A1, fibroblast-activated protein, secreted protein acidic and rich in cysteine, and periostin expression was significantly increased in the intratumoral stroma of pancreatic ductal adenocarcinomas compared to paired non-neoplastic pancreata (proCOL11A1: 145/155 (93.5%) vs 26/154 (16.9%); fibroblast-activated protein: 139/143 (97.2%) vs 82/132 (62.1%); secreted protein acidic and rich in cysteine: 113/150 (75.3%) vs 49/132 (37.1%); periostin: 135/151 (89.4%) vs 45/135 (33.3%); p < 0.001, all). While the four markers were expressed at lower levels in normal/benign pancreata, there were no significant differences in the expression frequencies among normal pancreas, acute pancreatitis, and chronic pancreatitis. Interestingly, on survival analysis, low intratumoral fibroblast-activated protein+ cancer-associated fibroblast counts (<100/high-power field) were associated with a significantly reduced overall survival compared to those with high fibroblast-activated protein+ cancer-associated fibroblast counts (p = 0.010; hazard ratio 5.2 (95% confidence interval 1.3-21.3)). Similar patterns were seen for proCOL11A and secreted protein acidic and rich in cysteine and overall and disease-free survival, although not statistically significant. In conclusion, we demonstrate that the presence of cancer-associated fibroblasts in the tumor stroma may not always be associated with a poor prognosis as suggested in many studies; on the contrary, it may even be associated with prolonged survival, supporting the recent experimental findings that tumor stroma may have a protective role rather than enhance aggressive behavior.

Altered Anterior Segment Biometric Parameters in Mice Deficient in SPARC.

Purpose: Secreted protein acidic and rich in cysteine (SPARC) and Hevin are structurally related matricellular proteins involved in extracellular matrix assembly. In this study, we compared the anterior chamber biometric parameters and iris collagen properties in SPARC-, Hevin- and SPARC-/Hevin-null with wild-type (WT) mice. Methods: The right eyes of 53 WT, 35 SPARC-, 56 Hevin-, and 63 SPARC-/Hevin-null mice were imaged using the RTVue-100 Fourier-domain optical coherence tomography system. The parameters measured were anterior chamber depth (ACD), trabecular-iris space area (TISA), angle opening distance (AOD), and pupil diameter. Biometric data were analyzed using analysis of covariance and adjusted for age, sex, and pupil diameter. Expression of Col1a1, Col8a1, and Col8a2 transcripts in the irises was measured by quantitative polymerase chain reaction. Collagen fibril thickness was evaluated by transmission electron microscopy. Results: Mice that were SPARC- and SPARC-/Hevin-null had 1.28- and 1.25-fold deeper ACD, 1.45- and 1.53-fold larger TISA, as well as 1.42- and 1.51-fold wider AOD than WT, respectively. These measurements were not significantly different between SPARC- and SPARC-/Hevin-null mice. The SPARC-null iris expressed lower Col1a1, but higher Col8a1 and Col8a2 transcripts compared with WT. Collagen fibrils in the SPARC- and SPARC-/Hevin-null irises were 1.5- and 1.7-fold thinner than WT, respectively. The Hevin-null iris did not differ from WT in these collagen properties. Conclusions: SPARC-null mice have deeper anterior chamber as well as wider drainage angles compared with WT. Therefore, SPARC plays a key role in influencing the spatial organization of the anterior segment, potentially via modulation of collagen properties, while Hevin is not likely to be involved.

SPARC expression by cerebral microvascular endothelial cells in vitro and its influence on blood-brain barrier properties.

BACKGROUND: SPARC (secreted protein acidic and rich in cysteine) is a nonstructural, cell-matrix modulating protein involved in angiogenesis and endothelial barrier function, yet its potential role in cerebrovascular development, inflammation, and repair in the central nervous system (CNS) remains undetermined. METHODS: This study examines SPARC expression in cultured human cerebral microvascular endothelial cells (hCMEC/D3)-an in vitro model of the blood-brain barrier (BBB)-as they transition between proliferative and barrier phenotypes and encounter pro-inflammatory stimuli. SPARC protein levels were quantified by Western blotting and immunocytochemistry and messenger RNA (mRNA) by RT-PCR. RESULTS: Constitutive SPARC expression by proliferating hCMEC/D3s is reduced as cells mature and establish a confluent monolayer. SPARC expression positively correlated with the proliferation marker Ki-67 suggesting a role for SPARC in cerebrovascular development. The pro-inflammatory molecules tumor necrosis factor-α (TNF-α) and endotoxin lipopolysaccharide (LPS) increased SPARC expression in cerebral endothelia. Interferon gamma (IFN-γ) abrogated SPARC induction observed with TNF-α alone. Barrier function assays show recombinant human (rh)-SPARC increased paracellular permeability and decreased transendothelial electrical resistance (TEER). This was paralleled by reduced zonula occludens-1 (ZO-1) and occludin expression in hCMEC/D3s exposed to rh-SPARC (1-10 µg/ml) compared with cells in media containing a physiological dose of SPARC. CONCLUSIONS: Together, these findings define a role for SPARC in influencing cerebral microvascular properties and function during development and inflammation at the BBB such that it may mediate processes of CNS inflammation and repair.

Osteonectin (SPARC) Expression in Vascular Calcification: In Vitro and Ex Vivo Studies.

Osteonectin, also termed SPARC, is a noncollagenous protein of bone matrix. Since there are controversial results regarding its role during the process of vascular calcification, we investigated osteonectin expression in our in vitro calcification model. Rat vascular smooth muscle cells (VSMCs) were challenged with high phosphate (5 mmol/L Pi) and analyzed quantifying calcium levels, through immunohistochemical studies, and studying gene expression. We detected a peak of osteonectin expression at day 7 in cell treated with high phosphate. The time course of calcium deposition, reflected the expression of osteonectin, resulting extensively present at day 7. On the contrary, the expression of the mitotic marker Ki-67 had a peak at day 4, showing no correlation between osteonectin and cell proliferation. Moreover, 7 days was the time point in which Cbfα1/RUNX-2 had its maximal expression. Furthermore, ascorbic acid increased osteonectin expression, supporting a procalcifying role for this protein. Next we decided to study osteonectin expression ex vivo in fetal, adult not calcified, and adult calcific vessels. Immunohistochemical studies demonstrated a spread and strong reactivity in VSMCs of a 20-week fetus, confirming that osteonectin may have a potential role in regulation of mitosis and in cell differentiation. In adult not calcified arteries, osteonectin was constitutively expressed and its levels increased in atherosclerotic and in calcified plaques, where it could have a regulatory role in the calcification process. Our in vitro and ex vivo data show osteonectin expression during the calcification process and suggest its potential role as procalcifying factor.

MiRNA-211 suppresses cell proliferation, migration and invasion by targeting SPARC in human hepatocellular carcinoma.

Previous studies have shown that the expression of miR-211 was downregulated in hepatocellular carcinoma (HCC). However, the molecular function and mechanism of miR-211 in HCC growth and invasion are largely unclear. We found that miR-211 is downregulated in HCC tissues and cell lines, respectively. Further results showed that low miR-211 associated with TNM stage, vein invasion status, and poor prognosis. Ectopic expression of miR-211 effectively suppressed HCC cell proliferation, migration and invasion both in vitro and in vivo. We identified SPARC as a bona fide target of miR-211, and overexpression of miR-211 decreased the mRNA and protein expression of SPARC. Finally, we confirmed that the overexpression of SPARC in miR-211-expressing HCC cells can partially restore the inhibitory effect of miR-211. Taken together, our results demonstrated that loss of miR-211 expression and thus uncontrolled SPARC overexpression might drive progression of HCC, which may provide a novel therapeutic strategy for the treatment of HCC.

Heterozygous PTCH1 Mutations Impact the Bone Metabolism in Patients With Nevoid Basal Cell Carcinoma Syndrome Likely by Regulating SPARC Expression.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by bone and skin abnormalities and a predisposition to various tumors. Keratocystic odontogenic tumors (KCOTs), which are common tumors of the jaw that cause extensive damage to the jawbone, are usually accompanied with NBCCS. Germline PTCH1 mutations in NBCCS tumorigenesis have been frequently studied; however, little is known regarding the pathogenesis of bone abnormalities in this disease. This study sought to investigate the mechanism underlying heterozygous PTCH1 mutation-mediated abnormal bone metabolism in patients with NBCCS. Stromal cells were isolated from the fibrous capsules of patients with NBCCS-associated or non-syndromic keratocystic odontogenic tumors and non-syndromic tumor stromal cells without PTCH1 mutations served as controls. Germline PTCH1 heterozygous mutations were confirmed in all NBCCS samples and differential protein expression was identified using tandem mass tag-labeled proteomics analysis. Our findings revealed that osteonectin/SPARC expression was significantly downregulated in syndromic stromal cells compared with non-syndromic stromal cells. SPARC expression was even lower in stromal cells carrying PTCH1 protein truncation mutations. PTCH1 siRNA transfection demonstrated that SPARC downregulation correlates with decreased PTCH1 expression. Furthermore, exogenous SPARC promoted osteogenic differentiation of syndromic stromal cells with enhanced development of calcium nodules. In addition, bone mineral density tests showed that patients with NBCCS exhibit weak bone mass compared with sex- and age-matched controls. This study indicates that germline PTCH1 heterozygous mutations play a major role in bone metabolism in patients with NBCCS, in particular in those with PTCH1 protein truncation mutations. SPARC may represent an important downstream modulator of PTCH1 mediation of bone metabolism. Thus, bone mineral density monitoring is critical for patients with NBCCS for prevention of osteoporosis. In addition, surgical procedures on syndromic-associated KCOTs should be performed with consideration of the weaker bone mass in such patients. © 2016 American Society for Bone and Mineral Research.

Expression of secreted protein acidic and rich in cysteine is an independent prognostic indicator of a poor clinical outcome in oropharyngeal carcinoma patients.

CONCLUSION: SPARC-expression is an indicator of the prognosis in terms of OS, independent of HPV-infection. HPV-negative patients with SPARC-Low show survival as favorable as HPV-positive patients, probably because of their higher salvage rate after relapse than SPARC-High patients. OBJECTIVE: The objectives of the study were to clarify the correlation between the expression of secreted protein acidic and rich in cysteine (SPARC) and HPV-status, and to determine the prognostic value of SPARC-expression in oropharyngeal squamous cell carcinoma (OPSCC) patients. METHODS: Fifty-three formalin-fixed and paraffin-embedded tissues were obtained from patients with OPSCC who underwent curative treatment. The SPARC protein was detected by immunohistochemistry. SPARC-expression level was divided into two categories, SPARC-High and SPARC-Low, according to the staining index. RESULTS: Twenty-two out of the 53 OPSCC patients were HPV-positive. There was no significant correlation between the HPV-status and SPARC-expression level. Multivariate Cox proportional hazards regression analysis revealed that the HPV-status and SPARC-expression are independent prognostic indicators of favorable and unfavorable overall survival (OS) (p = 0.021 and p = 0.012), respectively. For disease-free survival, the HPV-status was the only predictive factor (p = 0.022). After stratification by the HPV-status, high SPARC-expression was a significant predictor of poor OS in HPV-negative OPSCC patients using Kaplan-Meier analysis and the log-rank test (p = 0.014). Ten out of 28 SPARC-Low patients relapsed, among which six patients (60%) were salvaged. However, 14 out of 25 SPARC-High patients relapsed, and only three patients (21.4%) were salvaged.

Coordinate regulation of microenvironmental stimuli and role of methylation in bone metastasis from breast carcinoma.

The pathogenesis of bone metastasis is unclear, and much focus in metastatic biology and therapy relays on epigenetic alterations. Since DNA-methyltransferase blockade with 5-aza-2'-deoxycytidine (dAza) counteracts tumour growth, here we utilized dAza to clarify whether molecular events undergoing epigenetic control were critical for bone metastatization. In particular, we investigated the patterns of secreted-protein acidic and rich in cysteine (SPARC) and of Endothelin 1, affected by DNA methyltransferases in tumours, with the hypothesis that in bone metastasis a coordinate function of SPARC and Endothelin 1, if any occurs, was orchestrated by DNA methylation. To this purpose, we prepared a xenograft model with the clone 1833, derived from human-MDA-MB231 cells, and dAza administration slowed-down metastasis outgrowth. This seemed consequent to the reductions of SPARC and Endothelin 1 at invasive front and in the bone marrow, mostly due to loss of Twist. In the metastasis bulk Snail, partly reduced by dAza, might sustain Endothelin 1-SPARC cooperativity. Both SPARC and Endothelin 1 underwent post-translational control by miRNAs, a molecular mechanism that might explain the in vivo data. Ectopic miR29a reduced SPARC expression also under long-term dAza exposure, while Endothelin 1 down-regulation occurred in the presence of endogenous-miR98 expression. Notably, dAza effects differed depending on in vivo and in vitro conditions. In 1833 cells exposed to 30-days dAza, SPARC-protein level was practically unaffected, while Endothelin 1 induction depended on the 3'-UTR functionality. The blockade of methyltransferases leading to SPARC reduction in vivo, might represent a promising strategy to hamper early steps of the metastatic process affecting the osteogenic niche.

SPARC ectopic overexpression inhibits growth and promotes programmed cell death in acute myeloid leukemia transformed from myelodysplastic syndrome cells, alone and in combination with Ara-C treatment.

Secreted protein acidic and rich in cysteine (SPARC) has a complex and pleiotropic biological role in cell life during disease. The role of SPARC in myelodysplastic syndrome (MDS) is not yet fully understood. In the present study, we investigated the role of SPARC protein overproduction in the proliferation and apoptosis of SKM-1 cells, an acute myeloid leukemia cell line transformed from MDS. SKM-1 cells were infected with the pGC-GV-SPARC vector. The cells were then assessed for proliferation and cell death following treatment with low-dose cytosine arabinoside (Ara­C). The microarray analysis results revealed that samples from SPARC­overexpressed cells compared to SPARC protein, in SKM-1 cells led to proliferation inhibition and promoted programmed cell death and these effects were greater when treated with Ara-C. The mRNA and protein expression levels of SPARC were detected by SPARC overexpression in cells treated with Ara-C resulting in a significant upregulation of the mixed lineage kinase domain-like (MLKL) gene expression and five other genes. The results showed that the necrotic signaling pathway may play a role when the two conditions were combined via the upregulation of the MLKL protein. MLKL upregulation in SPARC overexpressed cells treated with Ara-C, indicates necrosis as a possible cell death process for the SKM-1 cells under these stringent conditions.

Secreted protein acidic and rich in cysteine expression in human colorectal cancer predicts postoperative prognosis.

OBJECTIVE: Secreted protein acidic and rich in cysteine (SPARC) is an extracellular matrix glycoprotein involved in cell proliferation, migration and angiogenesis. The aim of this study was to assess its expression in colorectal cancer, see whether and how it correlates with clinicopathological features, and evaluate its potential prognostic significance. PATIENTS AND METHODS: SPARC expression was detected by microarrays containing 847 immunohistochemically stained specimens, and further correlated with the clinicopathological and prognostic data. The prognostic significance of its expression was assessed using Kaplan-Meier survival with log-rank tests. Multivariate regression utilizing Cox's proportional hazard model was used to evaluate prognostic factors. RESULTS: SPARC expression in the normal colorectal mucosa and colorectal cancer tissue was significantly different (p < 0.001). Low SPARC expression was found to be associated with poor prognosis, and it was unfavorably correlated with overall survival and disease-free survival in colorectal cancer patients. In addition, SPARC expression in surrounding mesenchymal and stromal cells, bowel wall invasion, lymph node metastasis, and distant metastasis were independent prognostic factors for overall survival and disease-free survival. CONCLUSIONS: Reduced expression of SPARC in colorectal cancer tissue is associated with poor prognosis and aggressive clinicopathological features. Therefore, SPARC expression could potentially be used as a prognostic predictor for colorectal cancer patients.

Matricellular protein SPARC/osteonectin expression is regulated by DNA methylation in its core promoter region.

BACKGROUND: SPARC/osteonectin is an evolutionarily conserved matricellular protein that modulates cell-matrix interaction and cell function. In all vertebrates, SPARC is dynamically expressed during embryogenesis. However, the precise function of SPARC and the regulatory elements required for its expression in particular during early embryogenesis are largely unknown. RESULTS: The present study was undertaken to explore the molecular mechanisms that regulate sparc gene expression by in vivo functional characterization of the sparc promoter and identification of possible putative regulatory elements that govern basal promoter activity. We report here transient expression analyses of eGFP expression from transgenic zebrafish containing a Sparc-iTol2-eGFP-BAC and/or 7.25 kb-sparc-Tol2-eGFP constructs. eGFP expression was specifically found in the notochord, otic vesicle, fin fold, intermediate cell mass, and olfactory placode of BAC and Tol2 transposon vectors injected embryos. Deletion analysis revealed that promoter activity resides in the unique 5'-untranslated intronic region. Computer-based analysis revealed a putative CpG island immediately proximal to the translation start site within the intron sequence. Global inhibition of methylation with 5-Aza-2-deoxycytidine promoted sparc expression in association with decreasing CpG methylation. CONCLUSIONS: Taken together, these data identify a contributory role for DNA methylation in regulating sparc expression in zebrafish embryogenesis.