Secreted Protein Acidic Rich In Cysteine Research Articles

Effect of nab-paclitaxel combination chemotherapy on response and survival in pancreatic cancer.

e14718 Background: Pancreatic cancer has traditionally had few effective chemotherapy options. Recent discovery of the over-expression of SPARC (Secreted Protein Acidic Rich in Cysteine) on the majority of pancreatic cancers suggests possible utility of nab-paclitaxel, (a novel albumin-bound form of paclitaxel) as a treatment option. A small prospective study (ASCO 2009) supported this theory. We sought to retrospectively evaluate the treatment efficacy of nab-paclitaxel with gemcitabine or carboplatin in patients with pancreatic cancer to assess concordance with the previous small prospective study. Methods: Data was collected retrospectively from the medical records of pancreatic cancer patients who presented to the private clinic of two treating oncologists (n = 63) from June 2009 to December 2011 and were treated with nab- paclitaxel in combination with either gemcitabine or carboplatin. Patients were assessed on the basis of changes in serum tumour marker (CA19-9, CEA or CA15-3) and changes in tumour size on radiological imaging. Results: 39 (70%) patients had a biochemical response with a greater than 20% drop in tumour markers. Of these, 30 (94%) showed greater than 70% drop with all responders showing reduction in markers within 6 weeks of starting. The number of patients with biochemically defined disease control was 48 (86%). 36 patients were deceased by the end of the series with median overall survival being 12.9 months. The median survival time from starting nab-paclitaxel for these patients was 7.9 months. Very long survival times (up to 28.1 months following commencement nab-paclitaxel) were also observed in 2 patients at the time of data analysis. The overall disease control rate radiologically was 76.79% (43 of 56). Of the 43 responders, 10 were complete responders, 22 showed partial radiological response and 11 had stable disease. Conclusions: This retrospective case series demonstrates a near doubling of overall median survival time and also an improved response rate in patients on nab-paclitaxel treatment as compared to historical data of other treatment options for pancreatic carcinoma. It further supports Von Hoff’s data from ASCO 2010. Further prospective data is eagerly awaited.

Lumbar intervertebral disc degeneration associated with axial and radiating low back pain in ageing SPARC-null mice

Chronic low back pain (LBP) is a complex, multifactorial disorder with unclear underlying mechanisms. In humans and rodents, decreased expression of secreted protein acidic rich in cysteine (SPARC) is associated with intervertebral disc (IVD) degeneration and signs of LBP. The current study investigates the hypothesis that IVD degeneration is a risk factor for chronic LBP. SPARC-null and age-matched control mice ranging from 6 to 78weeks of age were evaluated in this study. X-ray and histologic analysis revealed reduced IVD height, increased wedging, and signs of degeneration (bulging and herniation). Cutaneous sensitivity to cold, heat, and mechanical stimuli were used as measures of referred (low back and tail) and radiating pain (hind paw). Region specificity was assessed by measuring icilin- and capsaicin-evoked behaviour after subcutaneous injection into the hind paw or upper lip. Axial discomfort was measured by the tail suspension and grip force assays. Motor impairment was determined by the accelerating rotarod. Physical function was evaluated by voluntary activity after axial strain or during ambulation with forced lateral flexion. SPARC-null mice developed (1) region-specific, age-dependent hypersensitivity to cold, icilin, and capsaicin (hind paw only), (2) axial discomfort, (3) motor impairment, and (4) reduced physical function. Morphine (6mg/kg, i.p.) reduced cutaneous sensitivity and alleviated axial discomfort in SPARC-null mice. Ageing SPARC-null mice mirror many aspects of the complex and challenging nature of LBP in humans and incorporate both anatomic and functional components of the disease. The current study supports the hypothesis that IVD degeneration is a risk factor for chronic LBP.

Autocrine control of glioma cells adhesion/migration through Inositol Requiring enzyme 1α (IRE1α)-mediated cleavage of Secreted Protein Acidic Rich in Cysteine (SPARC) mRNA

The endoplasmic reticulum (ER) is an organelle specialized for the folding and assembly of secretory and transmembrane proteins. ER homeostasis is often perturbed in tumor cells because of dramatic changes in the microenvironment of solid tumors, thereby leading to the activation of an adaptive mechanism named the unfolded protein response (UPR). The activation of the UPR sensor IRE1α has been described to play an important role in tumor progression. However, the molecular events associated with this phenotype remain poorly characterized. In the present study, we examined the effects of IRE1α signaling on the adaptation of glioma cells to their microenvironment. We show that the characteristics of U87 cell migration are modified under conditions where IRE1α activity is impaired (DN_IRE1). This is linked to increased stress fiber formation and enhanced RhoA activity. Gene expression profiling also revealed that loss of functional IRE1α signaling mostly resulted in the upregulation of genes encoding extracellular matrix proteins. Among these genes, Sparc, whose mRNA is a direct target of IRE1α endoribonuclease activity, was in part responsible for the phenotypic changes associated with IRE1α inactivation. Hence, our data demonstrate that IRE1α is a key regulator of SPARC expression in vitro in a glioma model. Our results also further support the crucial contribution of IRE1α to tumor growth, infiltration and invasion and extend the paradigm of secretome control in tumor microenvironment conditioning.

Preclinical Investigation of Nanoparticle Albumin-Bound Paclitaxel as a Potential Treatment for Adrenocortical Cancer

Traditional drug discovery methods have a limited role in rare cancers. We hypothesized that molecular technology including gene expression profiling could expose novel targets for therapy in adrenocortical carcinoma (ACC), a rare and lethal cancer. SPARC (secreted protein acidic rich in cysteine) is an albumin-binding matrix-associated protein that is proposed to act as a mechanism for the increased efficacy of a nanoparticle albumin-bound preparation of the antimicrotubular drug Paclitaxel (nab-paclitaxel). The transcriptomes of 19 ACC tumors and 4 normal adrenal glands were profiled on Affymetrix U133 Plus2 expression microarrays to identify genes representing potential therapeutic targets. Immunohistochemical analysis for target proteins was performed on 10 ACC, 6 benign adenomas, and 1 normal adrenal gland. Agents known to inhibit selected targets were tested in comparison with mitotane in the 2 ACC cell lines (H295R and SW-13) in vitro and in mouse xenografts. SPARC expression is increased in ACC samples by 1.56 ± 0.44 (μ ± SD) fold. Paclitaxel and nab-paclitaxel show in vitro inhibition of H295R and SW-13 cells at IC50 concentrations of 0.33 μM and 0.0078 μM for paclitaxel and 0.35 μM and 0.0087 μM for nab-paclitaxel compared with mitotane concentrations of 15.9 μM and 46.4 μM. In vivo nab-paclitaxel treatment shows a greater decrease in tumor weight in both xenograft models than mitotane. Biological insights garnered through expression profiling of ACC tumors suggest further investigation into the use of nab-paclitaxel for the treatment of ACC.

DNA Methylation of SPARC and Chronic Low Back Pain

BackgroundThe extracellular matrix protein SPARC (Secreted Protein, Acidic, Rich in Cysteine) has been linked to degeneration of the intervertebral discs and chronic low back pain (LBP). In humans, SPARC protein expression is decreased as a function of age and disc degeneration. In mice, inactivation of the SPARC gene results in the development of accelerated age-dependent disc degeneration concurrent with age-dependent behavioral signs of chronic LBP.DNA methylation is the covalent modification of DNA by addition of methyl moieties to cytosines in DNA. DNA methylation plays an important role in programming of gene expression, including in the dynamic regulation of changes in gene expression in response to aging and environmental signals.We tested the hypothesis that DNA methylation down-regulates SPARC expression in chronic LBP in pre-clinical models and in patients with chronic LBP.ResultsOur data shows that aging mice develop anatomical and behavioral signs of disc degeneration and back pain, decreased SPARC expression and increased methylation of the SPARC promoter. In parallel, we show that human subjects with back pain exhibit signs of disc degeneration and increased methylation of the SPARC promoter. Methylation of either the human or mouse SPARC promoter silences its activity in transient transfection assays.ConclusionsThis study provides the first evidence that DNA methylation of a single gene plays a role in chronic pain in humans and animal models. This has important implications for understanding the mechanisms involved in chronic pain and for pain therapy.

Gemcitabine and nab-paclitaxel in patients with unresectable/borderline resectable pancreatic cancer.

e14675 Background: Targeting the tumor stroma has evolved as a promising therapeutic strategy. Secreted protein acidic rich in cysteine (SPARC) which binds albumin has been putatively linked to enhanced drug delivery of nab-paclitaxel (ABX) to pancreatic tumor stroma. A recent phase I/II study demonstrated a 44% response in patients with metastatic pancreatic cancer treated with gemcitabine (gem) and ABX (Von Hoff et al, ASCO 2009). Based on these encouraging results, a pilot investigation was conducted in patients with unresectable (UR)/borderline resectable (BR) disease. Methods: A retrospective review was done on UR/BR pancreatic ca patients treated at Mayo Clinic Arizona from 3/09-12/09. Patients received gemcitabine 1,000 mg/m2 and nab-paclitaxel 100-125 mg/m2 neoadjuvantly. Demographics, staging, RECIST response, CA19-9 response (defined as > 50% maximal decline), EORTC PET response, post-treatment resectabilty and treatment related toxicities were assessed. Results: 13 patients were treated with this combination (age: median 69, range 47-86 years; sex: male 6, female 7, ECOG: 0-10, 1-3; location: head 8, body 5, median baseline CA19-9: 397 u/mL). A median of 3 cycles of therapy was delivered (range: 3-6). 3/13 (23%) underwent surgical resection with curative intent. CA19-9 response was 13/13 (100%), RECIST response was: PR-9/13 (69%), SD-3/13 (23%) and PD-1/13 (8%). EORTC PET response was: PR-7/10 (70%), SD3/10 (30%). No patients encountered drug related grade 4 or higher toxicities. There were 2 cases of grade 3 neutropenia,1 case of grade 3 anemia and no cases of grade 3 or higher thrombocytopenia; 4 patients had grade 2 neuropathy. Conclusions: Gemcitabine and nab-paclitaxel has a very promising antitumor activity in unresectable and borderline resectable pancreatic cancer patients. Investigation in a larger cohort of patients is ongoing. No significant financial relationships to disclose.

18 Novel agents for the treatment of multiple myeloma

Breast cancer deaths in western countries are falling due to screening and adjuvant therapy, but the treatment of metastatic breast cancer (MBC) has not shown comparable advances. The most active single agents are taxanes, which extend both disease-free and overall survival. However, opportunities remain for improving outcome. Nanoparticle technology is proving a valuable addition to the pharmaceutical armamentarium, particularly in oncology. Its use to bind paclitaxel to human albumin (nanoparticle albumin-bound paclitaxel; nab-paclitaxel; Abraxane®) ensures solubility of the taxane without the use of solvents and minimizes the risk of hypersensitivity reactions without premedication. The homogeneous colloidal suspension created allows rapid dispersal of unbound drug and linear pharmacokinetics. Albumin-mediated transport of paclitaxel across the endothelium facilitates uptake of drug, and a degree of tumour selectivity is achieved by the albumin-binding propensity of SPARC (Secreted Protein Acidic Rich in Cysteine), a substance expressed on and around many breast tumours. Clinical trials in first- and second-line MBC show that nab-paclitaxel is both more effective than solvent-based taxanes and associated with less severe neutropenia. Sensory neuropathy occurs but improves rapidly when compared with that caused by conventional taxanes. A clinical development programme is investigating nab-paclitaxel in the adjuvant and neoadjuvant settings. The low incidence of neutropenia makes nab-paclitaxel a good candidate for combination with other cytotoxics. It is also being assessed when given with biologic agents such as trastuzumab and bevacizumab.

Interaction of Pax6 with SPARC and p53 in Brain of Mice Indicates Smad3 Dependent Auto-regulation

Pax6 regulates formation of cerebral cortex, axon guidance, differentiation of neurons from glia and neuronal migration in the cerebellum. Although Pax6 is known to be a nuclear protein, it is presumed that it may interact with matricelluar proteins like SPARC during transport and processing of Pax6. The proteins involved in cell survival and cell proliferation can also not be ignored. The present study demonstrates co-localization of Pax6, secreted protein acidic rich in cysteine (SPARC), and p53 in astrocytes of primary culture, cerebellum and cortex region of mouse brain. The physical interaction of Pax6 with SPARC and p53 in brain of mice is also evident. The Pax6 probably participates with p53 to regulate neuronal survival. It indicates that the interaction of Pax6, SPARC and p53 may influence Smad3-dependent auto-regulation of Pax6.

Nanoparticle albumin-bound ( nab™)-paclitaxel: improving efficacy and tolerability by targeted drug delivery in metastatic breast cancer

Breast cancer deaths in western countries are falling due to screening and adjuvant therapy, but the treatment of metastatic breast cancer (MBC) has not shown comparable advances. The most active single agents are taxanes, which extend both disease-free and overall survival. However, opportunities remain for improving outcome. Nanoparticle technology is proving a valuable addition to the pharmaceutical armamentarium, particularly in oncology. Its use to bind paclitaxel to human albumin (nanoparticle albumin-bound paclitaxel; nab-paclitaxel; Abraxane®) ensures solubility of the taxane without the use of solvents and minimizes the risk of hypersensitivity reactions without premedication. The homogeneous colloidal suspension created allows rapid dispersal of unbound drug and linear pharmacokinetics. Albumin-mediated transport of paclitaxel across the endothelium facilitates uptake of drug, and a degree of tumour selectivity is achieved by the albumin-binding propensity of SPARC (Secreted Protein Acidic Rich in Cysteine), a substance expressed on and around many breast tumours. Clinical trials in first- and second-line MBC show that nab-paclitaxel is both more effective than solvent-based taxanes and associated with less severe neutropenia. Sensory neuropathy occurs but improves rapidly when compared with that caused by conventional taxanes. A clinical development programme is investigating nab-paclitaxel in the adjuvant and neoadjuvant settings. The low incidence of neutropenia makes nab-paclitaxel a good candidate for combination with other cytotoxics. It is also being assessed when given with biologic agents such as trastuzumab and bevacizumab.

Correlation of SPARC, ER, PR, and HER2 tumor with progression-free survival from a phase II neoadjuvant trial of gemcitabine, epirubicin, and nab-paclitaxel

618 Background: Neoadjuvant combinations of gemcitabine (G), anthracyclines and taxanes have demonstrated substantial activity with pCR rates of 20–25%. Secreted protein acidic rich in cysteine (SPARC) is an albumin binding protein that mediates intratumoral accumulation of nab-paclitaxel (nab-P) via a SPARC-albumin binding activity and is a poor prognostic factor for survival. This study was designed to evaluate the feasibility and safety of a biweekly schedule of neoadjuvant nab paclitaxel (nab-P) with G and epirubicin (E) with SPARC tumor assessments performed in consenting patients (pts). Methods: Eligibility: Clinical T1c-T4d and/or N0–3, M0 breast cancer (T1N0M0 excluded), normal LVEF. ER/PR/HER-2 obtained for all pts. Treatment: neoadjuvant G 2,000 mg/m2, E 50 mg/m2, and nab-P 175 mg/m2 q14 days x 6 cycles followed by surgery. Post operative therapy: G 2,000 mg/m2 and nab-P 220 mg/m2 q14 days x 4 cycles. Myeloid growth factors were mandated with all cycles. Two different antibody reagents were used to probe for SPARC expression of tumoral SPARC and stromal SPARC; level 3 immunohistochemical (IHC) SPARC staining was considered positive. Results: 123 pts enrolled. Pathologic responses are available for 112 pts with 11 unevaluable. 82 pts consented to SPARC tumor testing. Median age 51 (29–72). Median tumor size 4.5 cm. 42% ER-/PR -. 55% clinical T3/T4 and 66% node positive. Pathologic complete responses (pCR) were noted in 22 pts (18%) with PRs in 84 pts (68%), and 6 SD (5%). SPARC IHC staining was available in 77 tumor samples; SPARC positivity was noted in 89% of evaluated tumors achieving pCR. Triple negative, ER negative or PR negative tumors were associated with worse PFS. SPARC + tumors showed a trend to improved PFS that was strongly associated with tumoral SPARC but not stromal SPARC. Conclusions: Dose dense neoadjuvant G, E, and nab-P is active and well tolerated with a favorable pCR rate of 20%. ER neg, PR neg, and triple negative tumors were associated with worse PFS. Despite literature reports of SPARC positivity associated with poor prognosis, SPARC+ tumors in this study showed a high concordance with tumor response and a trend to improved PFS with tumoral SPARC. [Table: see text]

Results of a multicenter pilot study of weekly nab-paclitaxel, carboplatin with bevacizumab, and trastuzumab as neoadjuvant therapy in HER2+ locally advanced breast cancer with SPARC correlatives

527 Background: The addition of targeted therapies to standard chemotherapy has resulted in improved outcomes in metastatic, neoadjuvant and early stage breast cancer. A phase III study in MBC demonstrated improved efficacy with nab paclitaxel (nab-P) compared with standard solvent-based paclitaxel. The secreted protein acidic rich in cysteine (SPARC) is a poor prognostic factor for survival and may mediate enhanced intratumoral accumulation of nab-P via an interaction with albumin. This multicenter phase II pilot study was designed to evaluate the feasibility, safety, and preliminary efficacy of dual VEGF and HER-2 monoclonal antibodies of bevacizumab (B) and trastuzumab (T) administered in with neoadjuvant nab-P and carboplatin (C) with SPARC tumor correlatives. Methods: Eligibility: clinical T1c-T4d and/or N0–3, M0 (T1N0M0 excluded) chemo naive, ECOG PS 0–2, normal LVEF, adequate organ function. Treatment: nab-P 125 mg/m2 D1, 8, 15 with C AUC 6 D1 q28 days plus T 4 mg/kg load followed by 2 mg/kg/wk and B 5 mg/kg/wk x 6 cycles followed by surgery. Post surgery T and B continued for 52 wks. SPARC tumor expression was measured by immunohistochemistry. Results: 29 pts are enrolled and evaluable for safety. Median age: 52 years (29–76), ECOG PS 0–93%, median tumor size 3.2 cm, 54% ER-/PR-, 72% node positive. G3/4 neutropenia was 38% with no febrile neutropenia. Nonheme toxicity: G3/4 hyperglycemia 10%, proteinuria and hypertension, each in 7%. 8 pts were hospitalized (infection-4, ↓LVEF-1, wound dehiscence-1 pt, other-2). 5 pts did not complete neoadjuvant therapy (↓LVEF-1, noncompliance-1, pt request-3) and 10 pts did not complete post op therapy ((pt request-2, toxicity-3, wound-2, unk-3). Pathologic responses are available for 20 pts. pCR was noted in 13/20 pts (65%) and PR was noted in 7/20 pts (35%). 77% of the pCR pts (10/13) and 86% of the PR pts (6/7) were positive for SPARC. Conclusions: Neoadjuvant B, T with nab-P and C is feasible and highly active with a remarkable pCR of 65%. SPARC tumor correlations with pathologic response data reveal a concordance between the high response rate and high incidence of SPARC positivity. [Table: see text]

Preliminary progression free survival and SPARC tumor correlatives from a phase II neoadjuvant trial of gemcitabine, epirubicin, and nab paclitaxel.

Abstract Abstract #5116 Background: Neoadjuvant combinations of gemcitabine (G), anthracyclines and taxanes have demonstrated significant activity with pCR rates of 20-25% albeit with significant myelosuppression. Secreted protein acidic rich in cysteine (SPARC) is an albumin binding protein that mediates intratumoral accumulation of nab-paclitaxel (nab-P) via a SPARC-albumin specific binding activity. This study was designed to evaluate a biweekly schedule of neoadjuvant nab paclitaxel (nab-P) with gemcitabine and epirubicin (E). SPARC tumor assessments were performed in consenting patients (pts) with subsequent pathologic and survival correlatives.
 Methods: Eligibility: Clinical T1c-T4d and/or N0-3, M0 breast cancer (T1N0M0 excluded), ECOG PS 0-2, normal LVEF. ER/PR/HER2 obtained for all pts. Treatment: 6 cycles neoadjuvant G 2000 mg/m2, E 50 mg/m2, and nab-P 175 mg/m2 q14 days followed by surgery. Post operative therapy: 4 cycles G 2000 mg/m2 and nab-P 220 mg/m2 q14 days. Myeloid growth factors were mandated with all cycles. 2 antibody reagents were used to probe for SPARC expressions. For this study, level 3 immunohistochemical (IHC) SPARC staining was considered positive. Results: 123 pts have enrolled with pathologic responses available for 106. 82 pts consented to SPARC testing. Median age 51 (29-72). Median tumor size 4.5 cm. Histology: 81% ductal, 10% lobular, 9% other. 42% ER and PR negative. 55% clinical T3/T4 and 66% lymph node positive at presentation. G3/4 toxicity present in >5% of pts: neutropenia 16% (febrile neutropenia 1 pt), thrombocytopenia 9% with arthralgias 11%, fatigue 10%, and infection 7%. 20 pts did not complete study treatment for the following reasons: disease progression 7, toxicity 3, pt/MD request 5, and other 5. pCR was noted in 23 pts (22%) with PRs in 76 pts (71%), and 6 SD. SPARC IHC staining was available for 76 tumors. SPARC level 3 IHC staining was noted in 65 tumors (86%); 65% were associated with a PR or pCR. Median PFS was 23 months and overall survival at 24 months is 77%. Pts who relapsed ≤16 months from diagnosis were more likely to be triple negative (p = 0.04), or ER negative (p = 0.001), or PR negative (p = 0.0006). Conclusions: Neoadjuvant dose dense GEA is active and better tolerated than other gemcitabine/anthracycline/taxane combinations. The pCR rate of 22% compares favorably with other comparable neoadjuvant regimens albeit with minimal toxicity. Pts with early relapse defined as ≤16 months from diagnosis were more likely to demonstrate a triple negative phenotype or demonstrate estrogen or progesterone receptor negativity. SPARC tumor correlatives with progression free and overall survival are on ongoing and will be presented. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5116.

Functional role of periostin in development and wound repair: implications for connective tissue disease

Integrity of the extracellular matrix (ECM) is essential for maintaining the normal structure and function of connective tissues. ECM is secreted locally by cells and organized into a complex meshwork providing physical support to cells, tissues, and organs. Initially thought to act only as a scaffold, the ECM is now known to provide a myriad of signals to cells regulating all aspects of their phenotype from morphology to differentiation. Matricellular proteins are a class of ECM related molecules defined through their ability to modulate cell-matrix interactions. Matricellular proteins are expressed at high levels during development, but typically only appear in postnatal tissue in wound repair or disease, where their levels increase substantially. Members of the CCN family, tenascin-C, osteopontin, secreted protein acidic rich in cysteine (SPARC), bone sialoprotein, thrombospondins, and galectins have all been classed as matricellular proteins. Periostin, a 90 kDa secreted homophilic cell adhesion protein, was recently added to matricellular class of proteins based on its expression pattern and function during development as well as in wound repair. Periostin is expressed in connective tissues including the periodontal ligament, tendons, skin and bone, and is also prominent in neoplastic tissues, cardiovascular disease, as well as in connective tissue wound repair. This review will focus on the functional role of periostin in tissue physiology. Fundamentally, it appears that periostin influences cell behaviour as well as collagen fibrillogenesis, and therefore exerts control over the structural and functional properties of connective tissues in both health and disease. Periostin is a novel matricellular protein with close homology to Drosophila fasciclin 1. In this review, the functional role of periostin is discussed in the context of connective tissue physiology, in development, disease, and wound repair.

Pre-clinical evidence for nab-paclitaxel efficacy in the treatment of adrenocortical cancer

22070 Background: Adrenocortical carcinoma (ACC) is a rare and lethal cancer with an average incidence of 0.7 in 1 million. Response to current standard therapy including mitotane is approximately 22%. Expression profiling analysis of human tumors has identified potential therapeutic targets which were then tested in in vitro and in vivo systems. Methods: RNA samples from 25 ACC tumors and 4 normal adrenal glands were profiled on both the Affymetrix Human Genome U133 Plus 2 and Agilent 22K element expression arrays. Novel therapeutic targets identified were tested in comparison with mitotane in two ACC cell lines (H295R and SW-13) in vitro and in vivo in a Scid mouse xenograft system. Protein levels of selected genes were subsequently assayed by IHC of a xenograft tumor TMA. Results: Expression of SPARC (secreted protein acidic rich in cysteine), an albumin binding matrix protein involved in cell structure and cell cycle progression, is upregulated within the ACC tumor population 1.50 + 0.81 (μ+SD) and 3.54+ 2.37 (μ+SD) fold on the Affymetrix and Agilent platforms, respectively. The anti-microtubular drugs paclitaxel and nanoparticle albumin bound paclitaxel (ABRAXANE®) exhibited in vitro inhibition of H295R and SW-13 cells at IC50 concentrations of 0.33 μM and 0.0078 μM for paclitaxel and 0.35 μM and 0.0087 μM for nab-paclitaxel compared to mitotane concentrations of 15.9 μM and 46.4 μM. IHC staining of treated xenograft tumors shows no induction of MDR1 with nab-paclitaxel treatment. Conclusions: Biological insight garnered through expression profiling of ACC tumors supports the clinical investigation of nab-paclitaxel use in the treatment of ACC in which there is over-expression of SPARC. No significant financial relationships to disclose.

Examination of Oral Cancer Biomarkers by Tissue Microarray Analysis

To validate the DNA microarray results on a subset of genes that could potentially serve as biomarkers of oral squamous cell carcinoma (OSCC) by examining their expression with an alternate quantitative method and by assessing their protein levels. Based on DNA microarray data from our laboratory and data reported in the literature, we identified 6 potential biomarkers of OSCC to investigate further. We used quantitative real-time polymerase chain reaction to examine expression changes of CDH11, MMP3, SPARC, POSTN, TNC, and TGM3 in OSCC and histologically normal control tissues. We further examined validated markers at the protein level by immunohistochemical analysis of OSCC tissue microarray sections. Quantitative real-time polymerase chain reaction analysis revealed upregulation of CDH11, SPARC, POSTN, and TNC gene expression and decreased TGM3 expression in OSCC tissue compared with control tissue; MMP3 was not found to be differentially expressed. In tissue microarray immunohistochemical analyses, SPARC (secreted protein, acidic, rich in cysteine), periostin, and tenascin C exhibited increased protein expression in tumor tissue compared with control tissue, and their expression was primarily localized within tumor-associated stroma rather than tumor epithelium. Conversely, transglutaminase 3 protein expression was found only within keratinocytes in control tissue and was significantly downregulated in cancer cells. Of 6 potential gene markers of OSCC, initially identified by DNA microarray analyses, differential expression of CDH11, SPARC, POSTN, TNC, and TGM3 were validated by quantitative real-time polymerase chain reaction. Differential expression and localization of proteins encoded by SPARC, POSTN, TNC, and TGM3 were clearly shown by tissue microarray immunohistochemical analysis.

Secreted protein acidic, rich in cysteine induces pulp cell migration via αvβ3 integrin and extracellular signal‐regulated kinase

The aim of this study was to investigate the influence of secreted protein acidic, rich in cysteine (SPARC) on the migration of human dental pulp (HDP) cells. Secreted protein acidic, rich in cysteine was applied in the lower chamber of the chemotaxis apparatus and migration was determined by counting the cells that migrated through the membrane. To determine the signaling pathway involved, cells were incubated with inhibitors for 30 min prior to the migration assay. The results indicated that SPARC induced HDP cell migration in a dose-dependent manner via extracellular signal-regulated kinase (ERK). The migration could be inhibited both by the anti-alphavbeta3 integrin antibody and by suramin, a non-selective growth factor receptor and G-protein coupled receptor antagonists. The anti-alphavbeta3 integrin antibody could also inhibit ERK activation, suggesting the possible role of alphavbeta3 integrin on the regulation of ERK and cell migration. Interestingly, both suramin and SB225002, another G-protein coupled receptor antagonist, suppressed ERK activation. Secreted protein acidic, rich in cysteine could act as a chemotactic factor and facilitate migration, possibly through the G-protein coupled receptor, alphavbeta3 integrin and ERK. The data support that SPARC could play a crucial role in dental pulp tissue repair by inducing dental pulp cell migration.

Schwann cells in neuroblastoma express erythropoietin

The glycoprotein erythropoietin (Epo) and its cognate receptor EpoR are expressed both in the central nervous system (CNS) and the peripheral nervous system (PNS) [1–3]. The importance of EpoR in the CNS was demonstrated in studies of EpoR-null mice, which shows significantly reduced number of neuronal progenitor cells, extensive apoptosis in embryonic brain, and increased sensitivity to low oxygen tension [4]. Li et al. [5] demonstrated that Epo and EpoR are expressed by Schwann cells in rat sciatic nerve and that Epo is substantially upregulated in response to chronic constriction injury. Moreover, exogenously administered Epo increases the number of Schwann cells present at chronic constriction injury site in vivo and also promotes Schwann cell proliferation in vitro. Keswani et al. [6] showed that in response to axonal injury, periaxonal Schwann cells release Epo, which via EpoR binding on neurons, prevents axonal degeneration. Moreover, in an animal model of distal axonopathy, systemic Epo administration prevents axonal degeneration. Campana and Myers [7] reported localized Epo and EpoR in the sciatic nerve and dorse root ganglion of adult rats. Their data indicated that Epo is produced in the cell bodies and axons in normal root ganglion and is up-regulated in Schwann cells after painful chronic constriction injury. Neuroblastoma (NB) is an heterogenous tumor exhibiting a large spectrum of histological features ranging from immature NBs, composed of sheets of small undifferentiated neuroblasts with scarce Schwannian stroma, to progressively mature NBs composed of larger neuroblastic/ganglion-like cells with abundant Schwannian stroma [8]. The increasing presence of Schwannian stroma and differentiating/ differentiated neuroblasts that biologically resemble ganglion cells directly correlates with tumor maturation and better prognosis. In addition to a differentiated phenotype, Schwannian stroma rich tumors also have low vascularity [9]. This association between abundant Schwannian stroma, neuronal differentiation, and decreased vascularity has led to the hypothesis that Schwann cells may influence the biology of NB tumors by producing soluble factors that are capable of inducing neuroblast differentiation and inhibiting angiogenesis [10]. It has been demonstrated that Schwann cells secrete several potent angiogenesis inhibitors, such as secreted protein acidic rich in cysteine (SPARC) and pigment epithelium-derived factor [11, 12]. Here, we demonstrated a strong cytoplasmic immunoreactivity for Epo (1:200 in TBS, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in Schwann cells of surgical specimens obtained form five cases of Schwannian stroma rich NBs (ganglioneuroblastomas, intermixed by Shimada classification) (Fig. 1). Several studies have suggested that Epo might stimulate angiogenesis by acting to promote proliferation and/ or migration of endothelial cells [13, 14], inhibiting D. Ribatti (&) V. Longo Department of Human Anatomy and Histology, University of Bari Medical School, Piazza Giulio Cesare, 11 – Policlinico, Bari 70124, Italy e-mail: ribatti@anatomia.uniba.it

110 POSTER Synergistic effect of nab-paclitaxel and anti-VEGF-A antibody (bevacizumab) against the metastasis of breast tumor xenografts

Breast cancer deaths in western countries are falling due to screening and adjuvant therapy, but the treatment of metastatic breast cancer (MBC) has not shown comparable advances. The most active single agents are taxanes, which extend both disease-free and overall survival. However, opportunities remain for improving outcome. Nanoparticle technology is proving a valuable addition to the pharmaceutical armamentarium, particularly in oncology. Its use to bind paclitaxel to human albumin (nanoparticle albumin-bound paclitaxel; nab-paclitaxel; Abraxane®) ensures solubility of the taxane without the use of solvents and minimizes the risk of hypersensitivity reactions without premedication. The homogeneous colloidal suspension created allows rapid dispersal of unbound drug and linear pharmacokinetics. Albumin-mediated transport of paclitaxel across the endothelium facilitates uptake of drug, and a degree of tumour selectivity is achieved by the albumin-binding propensity of SPARC (Secreted Protein Acidic Rich in Cysteine), a substance expressed on and around many breast tumours. Clinical trials in first- and second-line MBC show that nab-paclitaxel is both more effective than solvent-based taxanes and associated with less severe neutropenia. Sensory neuropathy occurs but improves rapidly when compared with that caused by conventional taxanes. A clinical development programme is investigating nab-paclitaxel in the adjuvant and neoadjuvant settings. The low incidence of neutropenia makes nab-paclitaxel a good candidate for combination with other cytotoxics. It is also being assessed when given with biologic agents such as trastuzumab and bevacizumab.

Effects of hyaluronan and SPARC on fibroproliferative events assessed in an in vitro bladder acellular matrix model

Bladder acellular matrix (BAM) is a promising candidate for urinary biomaterials development. In the current work we have modified the BAM construct to include two biologically active components; hyaluronan (HA) and a peptide (SP 4.2) derived from secreted protein, acidic, rich in cysteine (SPARC), a matricellular glycoprotein. In order to assess the potential of an HA/SP 4.2 modified BAM to influence cellular functions associated with bladder healing, experiments were conducted to evaluate the individual and combined effects of these molecules on in vitro fibroproliferative endpoints within a co-culture model. Thiol-modified HA (246 kDa, 15 mg/ml)±SP 4.2 (200 μ m) was incorporated and cross-linked into BAM disks through disulfide bond formation. The following scaffolds compositions were then evaluated in a bladder smooth muscle cell (SMC)–urothelial (UEC) cell co-culture model: BAM unmodified; BAM+HA, BAM+SP 4.2 (media addition); BAM+HA+SP 4.2 (media addition); BAM+HA+SP 4.2 (matrix incorporated). At 3, 7 and 14 days post-seeding, SMC-mediated matrix contraction and gelatinolytic activity were evaluated. HA-modified BAM exhibited a significantly higher degree of contraction and gelatinase activity compared to unmodified BAM. In contrast, addition of SP 4.2 to BAM produced a negligible effect on contraction, while significantly reducing gelatinase activity. Matrices containing both molecules displayed significant increases in contraction, while gelatinase activity was dependent upon the method of peptide delivery. These results demonstrate that both HA and SP 4.2 have significant, yet distinct effects on the contractile and proteolytic activity of bladder SMCs and suggest that a modified BAM may be capable of modulating processes associated with post-surgical graft contracture and scar formation.

Secreted Protein Acidic, Rich in Cysteine (SPARC), Mediates Cellular Survival of Gliomas through AKT Activation

Secreted protein acidic, rich in cysteine (SPARC), is an extracellular matrix protein expressed in many advanced cancers, including malignant gliomas. We and others have previously shown that human glioma cell lines engineered to overexpress SPARC adopt an invasive phenotype. We now show that SPARC expression increases cell survival under stress initiated by serum withdrawal through a decrease in apoptosis. Phosphatidylinositol 3-OH kinase/AKT is a potent pro-survival pathway that contributes to the malignancy of gliomas. Cells expressing SPARC display increased AKT activation with decreased caspase 3/7 activity. Exogenous SPARC rapidly induces AKT phosphorylation, an effect that is blocked by a neutralizing SPARC antibody. Furthermore, AKT activation is essential for the anti-apoptotic effects of SPARC as the decreased apoptosis and caspase activity associated with SPARC expression can be blocked with dominant-negative AKT or a specific AKT inhibitor. As tumor cells face stressful microenvironments particularly during the process of invasion, these results suggest that SPARC functions, in part, to promote tumor progression by enabling tumor cells to survive under stressful conditions.